Streptomyces rochei and application thereof

Abstract

The invention discloses streptomyces rochei and application thereof, and relates to the field of microorganisms. The streptomyces rochei YHLC-1 is preserved in the China General Microbiological Culture Collection Center on August 1, 2025, and the preservation number of the streptomyces rochei YHLC-1 is CGMCC (China General Microbiological Culture Collection Center) No. 35491. According to the invention, a strain of streptomyces rochei YHLC-1 is separated from rhizosphere soil of cucumbers, and a fermentation product of the streptomyces rochei has an obvious inhibition effect on main pathogenic bacteria of cucumber root rot and can obviously promote growth of cucumbers.

Description

Technical Field

[0001] This application relates to the field of microbiology, and in particular to a Streptomyces loucherii and its applications. Background Technology

[0002] Cucumber root rot is a common soil-borne fungal disease affecting both greenhouse and open-field cucumbers. After infection, it can lead to root rot, plant wilting, and in severe cases, plant death, resulting in yield losses of 5%-45%. Current main control measures for cucumber root rot include crop rotation, soil improvement, and chemical control.

[0003] Crop rotation: Rotate with non-cucurbitaceous crops (such as corn, beans, etc.) for more than 3 years. Crop rotation plans must be formulated in advance. If sudden market price fluctuations or climate changes occur, the room for adjustment is limited, which can easily lead to a decrease in yield or income. If the crop rotation arrangement is unreasonable, pathogens or pests can still remain in the soil, and there may even be a phenomenon of "crop rotation curing disease," which can exacerbate problems such as root rot.

[0004] Soil improvement involves deep tilling, sun-drying, adding organic matter, improving drainage, and preventing waterlogging in low-lying areas to prevent root rot. Soil improvement often requires prior drainage systems, infrastructure construction, and regular application of chemical amendments, resulting in significant investment. Furthermore, the formulation and application methods of the soil amendment substrate require professional knowledge and technical training; inexperienced operators can easily lead to poor improvement results or secondary pollution.

[0005] Chemical control: At the initial stage of disease, apply a 300-800 times dilution of chlorothalonil or carbendazim to the roots for drenching, once every 7-10 days, for 2-3 times. Chemical control relies on large amounts of chemical agents, which easily leads to pesticide residues. These residues can enter the soil, water bodies, and food chain, causing imbalances in the soil microbial community and eutrophication of water bodies, thereby affecting crop growth and the balance and health of the ecosystem. Furthermore, for cucumber root rot, frequent use of commonly used chemical agents such as chlorothalonil can easily lead to the development of drug-resistant strains, rendering control ineffective. While chemical control can quickly suppress cucumber root rot, its drawbacks, such as environmental pollution, drug resistance, and soil degradation, cannot be ignored. Summary of the Invention

[0006] The main purpose of this application is to propose a Streptomyces loucheri and its application, aiming to solve the problems of high cost, poor control effect and easy development of drug resistance in the existing technology for controlling cucumber root rot.

[0007] Firstly, this application provides a strain of Streptomyces loucherii ( Streptomyces rochei YHLC-1 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35491.

[0008] By adopting the above technical solution, Streptomyces, as an important biocontrol microbial resource, can produce a variety of antibacterial active substances. This invention isolated a Streptomyces loucherei strain YHLC-1 from cucumber rhizosphere soil. Streptomyces rochei The fermentation product of this strain showed a significant inhibitory effect on the main pathogens causing cucumber root rot and significantly promoted cucumber growth. The discovery of this strain provides an important resource for the development of novel biological pesticides, offering advantages such as being green and safe, less prone to developing resistance, and low in cost, making it significant for achieving green cucumber cultivation.

[0009] Secondly, this application provides a bactericide, wherein the active ingredient of the bactericide is the aforementioned Streptomyces louchei (Streptomyces louchei). Streptomyces rochei YHLC-1 and its fermentation products.

[0010] By adopting the above technical solution, using Streptomyces loucheri ( Streptomyces rochei YHLC-1 and its fermentation products, as active ingredients in bactericides, can effectively control fungicides caused by Fusarium oxysporum (…). Fusarium oxysporum Rhizoctonia solani ( ) Rhizoctonia solani ) and Phytophthora indicum ( Phytophthora nicotianae This method effectively controls cucumber root rot caused by multiple infections, and its biocontrol effect is significant in practice.

[0011] Optionally, the method for preparing the bactericide includes the following steps: S1. The preserved Streptomyces louchei ( Streptomyces rochei YHLC-1 strain was inoculated into seed culture medium and cultured to obtain seed liquid; S2. Mix wheat bran and rice husks to obtain a solid matrix. Add glucose, ammonium sulfate, potassium dihydrogen phosphate, and magnesium sulfate to the solid matrix to obtain a mixed matrix. Adjust the moisture content of the mixed matrix to 60-65 wt% with water. S3. Spray the seed liquid obtained in step S1 onto the mixed substrate with a water content of 60-65 wt% obtained in step S2, stir thoroughly, and place in an incubator for fermentation to obtain the bactericide.

[0012] Using the above-mentioned technical solution, the seed liquid is sprayed into a mixed substrate with a water content of 60-65 wt%, stirred evenly, and then placed in an incubator at 30℃ for fermentation for 10 days. During fermentation, days 2-3 are the stage of abundant mycelial growth, and days 4-10 are the stage of abundant spore formation. During this period, the material changes from white to grayish-white or gray, accompanied by a typical actinomycete spore aroma, indicating that fermentation is complete. After fermentation, a large number of *Streptomyces louchei* spores are obtained.

[0013] Optionally, in step S3, the ratio of the inoculation volume of the seed solution to the mass of the mixed substrate is 0.2~0.4 mL / g.

[0014] Preferably, in step S1, the seed culture medium consists of: 25 g / L glucose, 15 g / L medium-temperature soybean meal, 2.5 g / L yeast powder, 1 g / L calcium carbonate, and pH 7.5.

[0015] Preferably, in step S2, wheat bran and rice husk are mixed in a mass ratio of 8:2 to obtain a solid matrix. Glucose, ammonium sulfate, potassium dihydrogen phosphate and magnesium sulfate are added to the solid matrix to obtain a mixed matrix. The mass ratio of glucose, ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate and the solid matrix is ​​1:0.5:0.1:0.05:100.

[0016] Optionally, the method for preparing the bactericide includes the following steps: (1) The preserved Streptomyces louchei ( Streptomyces rochei YHLC-1 strain was inoculated into seed culture medium and cultured to obtain seed liquid; (2) The seed liquid obtained in step (1) is inoculated into a fermentation culture medium and fermented to obtain a fermentation broth. The fermentation broth is centrifuged to obtain a supernatant. The supernatant is filtered through a filter membrane, and the resulting filtrate is the bactericide.

[0017] By adopting the above technical solution, the bactericide is a liquid bactericide, which can easily meet the needs of different application scenarios.

[0018] Preferably, in step (2), the seed liquid obtained in step (1) is inoculated into the fermentation medium at an inoculation amount of 4% (v / v, mL seed liquid / mL fermentation medium volume) and cultured in a shaking incubator at 30℃ and 200rpm for 72h to obtain the fermentation broth.

[0019] It should be noted that the fermentation medium and the seed culture medium have the same composition.

[0020] Optionally, the fungicide is effective against Fusarium oxysporum (Fusarium oxysporum) Fusarium oxysporum ), Tobacco Phytophthora ( Phytophthora nicotianae ) and Rhizoctonia solani ( Rhizoctonia solani It has antibacterial activity.

[0021] Optionally, the heat resistance temperature of the bactericide is 40~100℃.

[0022] Optionally, the bactericide has a pH tolerance of 2 to 12.

[0023] Optionally, the bactericide has UV resistance and room temperature storage stability.

[0024] By adopting the above technical solution, Streptomyces loucheri (Streptomyces loucheri) Streptomyces rochei YHLC-1 active material has acid-base stability, thermal stability, ultraviolet stability and room temperature storage stability, and has strong environmental adaptability, making it valuable for industrial development.

[0025] Thirdly, this application also provides the application of the above-mentioned fungicide in the prevention and control of cucumber root rot.

[0026] By adopting the above technical solution, the fungicide containing the antibacterial active ingredient of Streptomyces loucheri YHLC-1 has a significant control effect on cucumber root rot, and its control effect is better than that of conventional chemical agents such as carbendazim.

[0027] In summary, this application includes at least the following beneficial technical effects: Streptomyces, as an important biocontrol microbial resource, can produce a variety of antibacterial active substances. This invention isolated a Streptomyces loucherei strain YHLC-1 from cucumber rhizosphere soil. Streptomyces rochei The fermentation product of this strain showed a significant inhibitory effect on the main pathogens causing cucumber root rot and significantly promoted cucumber growth. The discovery of this strain provides an important resource for the development of novel biological pesticides, offering advantages such as being green and safe, less prone to developing resistance, and low in cost, making it significant for achieving green cucumber cultivation.

[0028] Microbial strain preservation information Streptomyces loucheri ( Streptomyces rochei YHLC-1 was deposited on August 1, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 35491. Attached Figure Description

[0029] Figure 1 Photographs showing the colony morphology of strain YHLC-1; Figure 2 A comparative graph showing the inhibitory effect of strain YHLC-1 on Fusarium oxysporum; Figure 3 A comparative diagram showing the inhibitory effects of strain YHLC-1 on Phytophthora indicata; Figure 4 A comparative diagram showing the inhibitory effect of strain YHLC-1 on Rhizoctonia solani; Figure 5 A comparative graph showing the inhibitory effect of fermentation supernatant filtrate of strain YHLC-1 on Fusarium oxysporum; Figure 6 A comparative graph showing the inhibitory effect of fermentation supernatant filtrate of strain YHLC-1 on Phytophthora indicum; Figure 7A comparative graph showing the inhibitory effect of fermentation supernatant filtrate of strain YHLC-1 on Rhizoctonia solani. Detailed Implementation

[0030] The present application will be further described in detail below with reference to the accompanying drawings and embodiments.

[0031] Culture medium instructions: Gao's No. 1 solid culture medium: soluble starch 20g / L, potassium nitrate 1g / L, ferrous sulfate heptahydrate 0.01g / L, magnesium sulfate heptahydrate 0.5g / L, dipotassium hydrogen phosphate trihydrate 0.5g / L, sodium chloride 0.5g / L, agar 20g / L, balance deionized water.

[0032] Example 1: Isolation and identification of Streptomyces loucherei YHLC-1 (1) Isolation of YHLC-1 bacteria 1g of rhizosphere soil was taken from a cucumber greenhouse in Shuainan Village, Daotian Town, Shouguang City, Shandong Province, and added to a 250mL Erlenmeyer flask containing 99mL of sterile water (containing 10 glass beads) to obtain a soil suspension. The soil suspension was heat-treated in a 55℃ water bath for 15 minutes to kill vegetative bacteria and fungal spores. 1mL of the water-treated soil suspension was diluted with sterile water to a final concentration of 10. -1 10 -3 10 -4 10 -5 Four gradients were prepared, with 100 μL of each gradient spread onto Gao's No. 1 solid medium. The medium was then incubated at 28°C for 7 days. A single colony was picked and streaked onto Gao's No. 1 solid medium to obtain pure colony YHLC-1.

[0033] (2) Morphological characteristics of the strain Figure 1 The image shows the morphological characteristics of the YHLC-1 colonies obtained in step (1). Figure 1 It can be seen that the strain grows well on Gao's No. 1 solid medium. The aerial mycelium is white to grayish-white, while the substrate mycelium is yellow. The colony center is raised, and dew is produced in the later stage. Under the microscope, the spore filaments are straight or flexible, and the spores are elliptical.

[0034] (3) Identification of strain YHLC-1 Genomic DNA was extracted from strain YHLC-1 obtained in step (1), and the 16S rDNA sequence was amplified using universal primers 27F and 1492R, where 27F: 5′-AGA GTT TGA TCC TGG CTC AG-3′; 1492R: 5′-TAC GGC TAC CTTGTT ACGACT T-3′. Sequencing was performed, and the sequence is shown in SEQ NO. 1. BLAST alignment of this sequence with the NCBI database showed that it was similar to *Streptomyces louchei* (…). Streptomyces rochei The highest homology (>99.5%) was found in *Streptomyces loucheri*. Based on this molecular biological identification result, the YHLC-1 strain obtained by isolation and purification in step (1) was identified as *Streptomyces loucheri*. Streptomyces rochei Streptomyces loucherii ( Streptomyces rochei YHLC-1 was deposited on August 1, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 35491.

[0035] (4) Streptomyces louchei ( Streptomyces rochei YHLC-1 storage Add 0.5 mL of 30 wt% sterile glycerol to a 2 mL sterile cryopreservation tube, scrape a loop of Streptomyces louchei spores obtained in step (1) into the tube with an inoculation loop, and transfer it to an ultra-low temperature freezer at -80℃ for long-term storage.

[0036] Example 2: Activation and fermentation of Streptomyces loucheri YHLC-1 (1) Activation of Streptomyces louchei YHLC-1 Take the bacterial strain from the glycerol preservation tube obtained in step (4) of Example 1, dip a loop of spore liquid into it with an inoculation loop and streak it on Gao's No. 1 solid medium to activate it. Place it in a constant temperature incubator at 30°C for 7 days to obtain activated Streptomyces loucheri single colony.

[0037] (2) Preparation of seed liquid Select a single colony of activated Streptomyces loucherii obtained in step (1) above and inoculate it into a 250 mL seed bottle containing 50 mL of seed culture medium (25 g / L glucose, 15 g / L medium-temperature soybean meal, 2.5 g / L yeast powder, 1 g / L calcium carbonate, pH 7.5). Incubate in a shaking incubator at 30℃ and 200 rpm for 40 h to obtain seed liquid.

[0038] (3) Preparation of fermentation supernatant filtrate The seed culture obtained in step (2) above was inoculated into a 250 mL fermentation flask containing 50 mL of fermentation medium at an inoculation rate of 4% (v / v, mL seed culture / mL fermentation medium volume). The flask was then incubated in a shaking incubator at 30℃ and 200 rpm for 72 h to obtain the fermentation broth. The fermentation broth was centrifuged at 8000 rpm for 10 min to obtain the supernatant. The supernatant was filtered through a 0.22 μm filter membrane, and the resulting filtrate was the fermentation supernatant. The composition of the fermentation medium was the same as that of the seed culture medium in step (2) above.

[0039] (4) Solid substrate fermentation Wheat bran (60 mesh) and rice husks (60 mesh) were mixed at a mass ratio of 8:2 and stirred thoroughly to obtain a solid matrix. Glucose, ammonium sulfate, potassium dihydrogen phosphate, and magnesium sulfate heptahydrate were added to the solid matrix and stirred thoroughly to obtain a mixed matrix. The mass ratio of glucose, ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, and the solid matrix was 1:0.5:0.1:0.05:100. Deionized water was added to the mixed matrix to achieve a moisture content of 60 wt%. The 60 wt% moisture mixed matrix was then dispensed into solid fermentation trays, with a filling thickness of 3 mm. The sample was autoclaved at 121℃ for 30 minutes and cooled to room temperature (25℃). Seed culture was obtained on a sterile operating table according to step (2) above. The seed culture was then evenly sprayed onto a mixed substrate with a water content of 60wt% at an inoculation rate of 30% (v / w, mL seed culture / g mixed substrate). The substrate was thoroughly stirred and placed in a 30℃ incubator for 10 days of fermentation. Days 2-3 of fermentation were the stage of abundant mycelial growth, and days 4-10 were the stage of abundant spore formation. During this period, the material changed from white to grayish-white or gray, accompanied by a typical actinomycete spore aroma, indicating mature fermentation. After fermentation, the number of spores of *Streptomyces louchei* was 2.6 × 10⁻⁶. 10 spores / g.

[0040] Example 3: Antibacterial spectrum of Streptomyces loucherei YHLC-1 Fusarium oxysporum (SAH) specimens with a diameter of 5 mm were inoculated onto PDA solid medium (12 g / L potato extract, 20 g / L glucose, 15 g / L agar, with the remainder being distilled water). Fusarium oxysporum Silk blocks, tobacco phytoestrogens ( Phytophthora nicotianae ) silk blocks and Rhizoctonia solani ( Rhizoctonia solani Silk blocks were cultured in a 26℃ incubator for 5 days to obtain activated Fusarium oxysporum ( ). Fusarium oxysporum ), Tobacco Phytophthora ( Phytophthora nicotianae ) and Rhizoctonia solani ( Rhizoctonia solani ).

[0041] Antibacterial spectrum test was conducted using the plate confrontation method: Use a punch to cut activated Fusarium oxysporum to a diameter of 7 mm. Fusarium oxysporum ), Tobacco Phytophthora ( Phytophthora nicotianae ) and Rhizoctonia solani ( Rhizoctonia solani The mycelium cake was inoculated into the center of the corresponding PDA solid medium (potato extract powder 12g / L, glucose 20g / L, agar 15g / L, the remainder being distilled water); the spores of Streptomyces loucherii obtained by fermentation according to step (4) of Example 2 were scraped off and streaked on both sides 3cm from the edge of the mycelium cake to obtain the antibacterial group.

[0042] Use a punch to cut activated Fusarium oxysporum to a diameter of 7 mm. Fusarium oxysporum ), Tobacco Phytophthora ( Phytophthora nicotianae ) and Rhizoctonia solani ( Rhizoctonia solani The mycelium cakes were inoculated into the center of PDA solid medium (12 g / L potato extract powder, 20 g / L glucose, 15 g / L agar, and the remainder distilled water) to obtain the control group.

[0043] The antibacterial group and the control group were incubated in a 28℃ constant temperature incubator. When the diameter of the pathogen in the control group reached 3 / 4 of the plate diameter, the radius of the pathogen in both groups was counted, and the inhibition rate was calculated. The experimental results are shown in Table 1 below. Figures 2 - 4 As shown, Figures 2 - 4 The image on the left shows the antibacterial effect of the control group.

[0044] Wherein, the inhibition rate = (radius of colonies in the control group - radius of colonies in the inhibition group) / (radius of colonies in the control group - radius of the initial bacterial cake) × 100%.

[0045] Table 1. Antimicrobial activity of Streptomyces louchei YHLC-1 against pathogens.

[0046] Figure 2 This is a comparison of the inhibitory effects of Streptomyces loucherei YHLC-1 on Fusarium oxysporum. Figure 3 This is a comparison of the inhibitory effects of Streptomyces loucheri YHLC-1 on Phytophthora tobaccoii. Figure 4 A comparative diagram showing the inhibitory effects of Streptomyces loucheri YHLC-1 on Rhizoctonia solani.

[0047] Antibacterial tests showed that Streptomyces loucherii YHLC-1 has good antibacterial activity against pathogens such as Fusarium oxysporum, Phytophthora tobaccoii, and Rhizoctonia solani.

[0048] Example 4: Antibacterial rate and stability of antibacterial active substances from Streptomyces louchei YHLC-1 (1) Antibacterial activity rate of Streptomyces louchei YHLC-1 The antibacterial rate of the antibacterial active substance of Streptomyces louchei YHLC-1 was detected by the mycelial growth rate method: PDA solid medium (potato extract powder 12g / L, glucose 20g / L, agar 15g / L, the remainder being distilled water) was melted and cooled to 55℃. The fermentation supernatant filtrate was obtained according to step (3) of Example 2. The fermentation supernatant filtrate was mixed with the melted PDA medium at a volume ratio of 4:100, shaken well, and poured into a plate. After solidification, the pathogenic fungal cake (7 mm in diameter) was inoculated in the center of the plate according to the method of pathogen inoculation in Example 3 as the antibacterial group.

[0049] The PDA solid medium (12 g / L potato extract powder, 20 g / L glucose, 15 g / L agar, and the remainder distilled water) was melted and cooled to 55°C. Sterile water was mixed with the melted PDA medium at a volume ratio of 4:100, shaken well, and poured onto a plate. After cooling, the pathogenic fungal cake (7 mm in diameter) was inoculated in the center of the plate as a control group, following the method of inoculating pathogenic bacteria in Example 3.

[0050] The antibacterial group and the control group were incubated in a 28℃ constant temperature incubator. When the diameter of the pathogen in the control group reached 3 / 4 of the plate diameter, the diameter of the pathogen in both groups was counted, and the inhibition rate was calculated. The experimental results are shown in Table 2 below. Figures 5 - 7 As shown, Figures 5 - 7 The image on the left shows the antibacterial effect of the control group.

[0051] The inhibition rate is calculated as follows: (diameter of control group colonies - diameter of inhibited group colonies) / (diameter of control group colonies - diameter of initial bacterial cake) × 100%.

[0052] Table 2 Antibacterial activity of Streptomyces louchei YHLC-1 supernatant

[0053] Figure 5 This is a comparative graph showing the inhibitory effect of the fermentation supernatant filtrate of Streptomyces loucheri YHLC-1 on Fusarium oxysporum. Figure 6 This is a comparative graph showing the inhibitory effect of the fermentation supernatant filtrate of Streptomyces loucheri YHLC-1 on Phytophthora tobaccoii. Figure 7 A comparative graph showing the inhibitory effect of fermentation supernatant filtrate of Streptomyces louchei YHLC-1 on Rhizoctonia solani.

[0054] Antibacterial tests showed that the fermentation supernatant filtrate of Streptomyces louchei YHLC-1 had good antibacterial activity against pathogens such as Fusarium oxysporum, Phytophthora tobaccoii, and Rhizoctonia solani.

[0055] (2) Acid-base stability test of antibacterial active substances of Streptomyces louchei YHLC-1 The fermentation supernatant filtrate was obtained according to step (3) of Example 2. Eleven 10 mL portions of fermentation supernatant filtrate were taken and their pH values ​​were adjusted to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 respectively with 1 mol / L HCl solution and 1 mol / L NaOH solution. The pH values ​​were adjusted to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12. The supernatant after pH adjustment was placed at 25°C and allowed to stand for 30 min. The pH value of the fermentation supernatant filtrate of each group was then adjusted to 7.0. The supernatant was centrifuged at 8000 rpm for 10 min to obtain the supernatant. The supernatant was filtered through a 0.22 μm filter membrane. The filtrate obtained by filtration was the sterile supernatant filtrate.

[0056] The PDA solid medium (12 g / L potato extract powder, 20 g / L glucose, 15 g / L agar, and the remainder being distilled water) was melted and cooled to 55°C. The sterile supernatant filtrate of each group was mixed with the melted PDA medium at a volume ratio of 4:100, shaken well, and poured into plates. After cooling, the Fusarium oxysporum mycelium (7 mm in diameter) was inoculated in the center of the plate as a control group, following the method for inoculating pathogens in Example 3.

[0057] The PDA solid medium (12 g / L potato extract powder, 20 g / L glucose, 15 g / L agar, and the remainder distilled water) was melted and cooled to 55°C. Sterile water was mixed with the melted PDA medium at a volume ratio of 4:100, shaken well, and poured onto a plate. After cooling, Fusarium oxysporum mycelium (7 mm in diameter) was inoculated in the center of the plate as a control group, following the method for inoculating pathogens in Example 3.

[0058] The inhibition group and the control group were placed in a constant temperature incubator at 28℃. When the diameter of the pathogen in the control group reached 3 / 4 of the plate diameter, the diameters of the pathogens in the control group and the inhibition group were counted, and the inhibition rate was calculated. The inhibition rate was calculated as follows: (diameter of colonies in the control group - diameter of colonies in the inhibition group) / (diameter of colonies in the control group - initial diameter of the mycelial cake) × 100%.

[0059] The experimental results showed that the fermentation supernatant filtrate had the highest antibacterial activity at pH 8, with an inhibition rate of 64.15%. However, the inhibition rate dropped to 53.32% and 50.60% at pH 4 and pH 10, respectively. In environments with pH 2 and pH 12, the inhibition rate remained at 45.56% and 44.18%, respectively. This indicates that the antibacterial active substances of the strain have good stability in a weakly alkaline environment and also have strong acid and alkali tolerance.

[0060] (3) Thermostability test of antibacterial active substances of Streptomyces loucheri YHLC-1 The fermentation supernatant filtrate was obtained according to step (3) of Example 2, and 9 portions of 10 mL fermentation supernatant filtrate were taken. 8 portions of fermentation supernatant filtrate were treated at 40℃, 50℃, 60℃, 70℃, 80℃, 90℃, 100℃ and 121℃ for 30 min respectively. The remaining portion was not heated and served as a control. The antibacterial rate against Fusarium oxysporum was tested according to the antibacterial method described in (2) above.

[0061] Experimental results showed that the antibacterial rate of the fermentation supernatant filtrate remained above 60% after heat treatment at 40-100℃.

[0062] (4) UV stability test of antibacterial active substances of Streptomyces louchei YHLC-1 The fermentation supernatant filtrate was obtained according to step (3) of Example 2. Ten 10 mL portions of the fermentation supernatant were placed under an 8W UV lamp at a distance of 20 cm for 0 min, 1 min, 2 min, 3 min, 4 min, 5 min, 10 min, 15 min, 20 min and 30 min respectively. The fermentation supernatant filtrate without UV irradiation was used as a control. The antibacterial rate against Fusarium oxysporum was tested according to the antibacterial method described in (2) above.

[0063] Experimental results showed that the antibacterial rate of the fermentation supernatant filtrate remained at 55-60% after 1-30 minutes of ultraviolet irradiation.

[0064] (5) Room temperature storage stability test of antibacterial active substances of Streptomyces louchei YHLC-1 The fermentation supernatant filtrate was obtained according to step (3) of Example 2, and four 10 mL portions of fermentation supernatant filtrate were taken. Three portions were placed at room temperature (25°C) for 15 days, 30 days and 45 days respectively, and the remaining portion was placed at 4°C as a control. The antibacterial rate against Fusarium oxysporum was tested according to the antibacterial method described in (2) above.

[0065] Experimental results showed that the antibacterial rate of the fermentation supernatant filtrate remained above 60% after being left at room temperature for 15, 30, and 45 days.

[0066] Example 5: Effect of Streptomyces loucheri YHLC-1 on the control of cucumber root rot Fusarium oxysporum (SAH) specimens with a diameter of 5 mm were inoculated onto PDA solid medium (12 g / L potato extract, 20 g / L glucose, 15 g / L agar, with the remainder being distilled water). Fusarium oxysporum Silk blocks, tobacco phytoestrogens ( Phytophthora nicotianae ) silk blocks and Rhizoctonia solani ( Rhizoctonia solani Silk blocks were cultured in a 28°C incubator for 5 days. Spores or hyphae on each PDA solid medium were washed off with sterile water, and the concentration was adjusted to 1×10⁻⁶ using a hemocytometer.6 CFU / mL (spores or hyphal fragments).

[0067] Using the greenhouse pot cultivation method, cucumber seeds were sown in sterilized nutrient soil, with 3 seedlings of uniform growth retained in each pot (15cm in diameter). When the seedlings reached the two-leaf-one-heart stage, the following treatment was performed: Streptomyces loucherei YHLC-1 spores were obtained according to step (4) of Example 2 and diluted with sterile water to a viable count of 1×10⁻⁶. 8 A bacterial suspension of CFU / mL was used to drench the roots of cucumber seedlings at a rate of 20 ml per pot. The above concentration was 1×10⁻⁶. 6 CFU / mL *Fusarium oxysporum* suspension, *Phytophthora indicum* suspension, and *Rhizoctonia solani* suspension were mixed at a volume ratio of 1:1:1 to obtain a mixed suspension. Twenty-four hours after drenching the roots with *Streptomyces louchei* suspension, the mixed suspension of the three pathogens was used for root drenching, with a drenching volume of 20 ml / pot, serving as the YHLC-1 experimental group. A water control and a chemical control (800-fold dilution of 50% carbendazim wettable powder) were also included, with the same drenching volume as the YHLC-1 experimental group. Each treatment was replicated in 5 pots, and the disease index and control effect were statistically analyzed after 15 days.

[0068] Record the disease level according to the following grading standards, and calculate the disease severity index and prevention and control effect.

[0069] Grade 0: Healthy root system, no symptoms; Grade 1: The roots are slightly browned, or the leaves above ground are slightly yellowed; Grade 3: The roots are obviously brown and rotten, less than 1 / 3 of the root system is rotten, and the above-ground parts of the plant are stunted and wilted; Level 5: The roots are severely rotten, with 1 / 3 to 2 / 3 of the root system rotten, and the above-ground parts are severely wilted; Level 7: The entire root system is rotten, and the plant is dead.

[0070] Disease index = [100 × Σ(number of disease-grade plants × corresponding grade value)] / (total number of plants surveyed × 7) Prevention and control efficacy (%) = (Disease index of water control - Disease index of treated control) / Disease index of water control × 100% Table 3. Efficacy of Streptomyces loucheri YHLC-1 in controlling cucumber root rot

[0071] The experimental results show that Streptomyces loucheri YHLC-1 has a significant control effect on cucumber root rot, and its control effect is better than that of conventional chemical agents such as carbendazim.

[0072] Example 6: Colonization ability of Streptomyces loucherei YHLC-1 in cucumber roots Take the preserved bacterial strain from the glycerol tube in step (4) of Example 1, dip a loopful of the inoculation loop into the Gao's No. 1 solid medium containing 100 μg / mL rifampin, and culture it at 30°C for 7 days. Pick the single colony that grows, and obtain the genetically stable resistant mutant strain YHLC-1-Rif through purification and subculture.

[0073] The resistant mutant strain YHLC-1-Rif was fermented according to step (4) of Example 2 to obtain spores of the resistant mutant strain YHLC-1-Rif, which were then diluted with sterile water to a viable count of 1×10⁻⁶. 8 A bacterial suspension of CFU / mL was prepared. Cucumber seeds were disinfected and sown in sterilized seedling substrate. When seedlings reached two leaves and a central bud, the roots were drenched with the bacterial suspension, 10 mL per seedling (experimental group). A control group was prepared using plain water. Each treatment was replicated three times, with 10 seedlings per replicate. After 15 days, five seedlings were randomly selected from each treatment, and their roots were cut and treated with 75wt% ethanol solution for 30 seconds to kill root surface microorganisms. The roots were then rinsed three times with sterile water. The disinfected roots were thoroughly ground in a sterile mortar and diluted with sterile water to release the YHLC-1-Rif bacteria colonizing the roots. The ground suspension was diluted with sterile water to a concentration of 10 CFU / mL. -2 10 -3 10 -4 10 -5 Four gradients were used, with 100 μL of each dilution spread onto Gao's No. 1 solid medium containing 50 μg / mL potassium dichromate. The medium was incubated at 30℃ for 6 days. Based on the colony morphology of YHLC-1-Rif, the colonies in the plates were counted and the number of viable Streptomyces colonized per gram of root was calculated.

[0074] The experimental results showed that *Streptomyces loucherei* YHLC-1 had good colonization ability in cucumber roots, achieving a colonization rate of 1 × 10⁶ cells / day in 15 days. 4 CFU / g, while no corresponding colonies were detected in the water control group.

[0075] The embodiments described in this specific implementation are preferred embodiments of this application and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the principles of this application should be covered within the scope of protection of this application.

Claims

1. A strain of Streptomyces louchei ( Streptomyces rochei YHLC-1 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35491.

2. A bactericide characterized by, The active ingredient of the bactericide is Streptomyces rochei (ATCC 27414) Streptomyces rochei ) YHLC-1 and a fermentation product thereof.

3. The sterilant of claim 2, wherein, The preparation method of the bactericide comprises the following steps: S1, inoculate the preserved Streptomyces roubuchii (S. roubuchii) YHLC-1 strain into a seed culture medium, and obtain a seed liquid after cultivation; Streptomyces rochei ) YHLC-1 strain into a seed culture medium, and obtain a seed liquid after cultivation; S2, mixing wheat bran and rice husk to obtain a solid substrate, adding glucose, ammonium sulfate, potassium dihydrogen phosphate and magnesium sulfate into the solid substrate to obtain a mixed substrate, and adjusting the water content of the mixed substrate to 60-65 wt% by water; S3, spraying the seed liquid obtained in the step S1 into the mixed substrate with the water content of 60-65 wt% obtained in the step S2, fully stirring and mixing, and placing in a culture box for fermentation to obtain the bactericide.

4. The sterilant of claim 3, wherein, In the step S3, the ratio of the inoculation volume of the seed liquid to the mass of the mixed substrate is 0.2-0.4 mL / g.

5. The sterilant of claim 2, wherein, The preparation method of the bactericide comprises the following steps: (1) inoculate the preserved Streptomyces roubuchii (S. roubuchii) YHLC-1 strain into a seed culture medium, and obtain a seed liquid after cultivation; Streptomyces rochei ) (2) inoculating the seed liquid obtained in the step (1) into a fermentation culture medium, obtaining a fermentation liquid through fermentation culture, centrifuging the fermentation liquid to obtain a supernatant, and filtering the supernatant through a filter membrane, so that the obtained filtrate is the bactericide.

6. The sterilant of claim 5, wherein, The heat-resistant temperature of the bactericide is 40-100℃.

7. The sterilant of claim 5, wherein, The pH value resistant temperature of the bactericide is 2-12.

8. The sterilant of claim 5, wherein, The bactericide has ultraviolet resistance and room temperature storage stability.

9. The sterilant of claim 2, wherein The fungicide has fungicidal activity against Fusarium oxysporum Fusarium oxysporum ), Phytophthora nicotianae (P. parasitica) Phytophthora nicotianae ) and Rhizoctonia solani (R. solani) Rhizoctonia solani ).

10. The bactericide according to claim 2 is used for preventing and treating cucumber root rot.

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