Streptomyces complex, microbial seedling culture substrate thereof, and application thereof

Abstract

To provide a fungal complex for preventing and treating strawberry anthracnose using biological control bacteria, a microbial seedling substrate thereof, and applications thereof.SOLUTION: The complex comprises Streptomyces CMB19 and Streptomyces CMB20, wherein the storage number of Streptomyces CMB19 is CGMCCNo. 30422, and the storage number of Streptomyces CMB20 is CGMCCNo. 30423. The fungal complex composed of Strepomyces CMB19 and Strepomyces CMB20 provided by the present invention can effectively inhibit the growth of strawberry anthracnose, and the microbial seedling culture substrate prepared therefrom can effectively prevent and treat the occurrence and spread of strawberry anthracnose. In the present invention, the growth of strawberry seedlings can be greatly promoted through the streptomyces CMB19 and the microbial seedling substrate prepared by the streptomyces CMB20.SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] The present invention relates to the technical field of microorganisms, and more particularly to a Streptomyces complex agent, its microbial propagation substrate, and its applications. [Background technology]

[0002] In the production of greenhouse crops, the seedling substrate is the basic material for producing seedlings in factories. Currently, the problem of continuous crop damage in greenhouse soil is becoming increasingly serious, resulting in a growing demand for healthy, disease-resistant seedlings. Researchers have been preparing microbial seedling substrates that have positive effects on seedling disease resistance and growth promotion by adding functional bacteria with biological control and growth-promoting effects to the seedling substrate. However, the persistence of the activity of the functional bacteria in the substrate and their ability to colonize the target crop affect the effectiveness and persistence of the microbial seedling substrate on cultivated crops. Therefore, to fully utilize the effects of the functional bacteria, the composition of conventional seedling substrates needs to be improved.

[0003] The strawberry industry has become one of the pillars of modern agriculture due to the beauty of its fruit, its high nutritional value, its short production cycle, and its high economic efficiency. However, hot and humid weather during the strawberry growing season can cause very serious diseases at the seedling stage, with strawberry anthracnose being one of the most common. Anthracnose harms strawberries throughout their entire growth period, particularly the seedling and transplanting stages, and in severe cases can even lead to the death of the entire plant. Currently, chemical fungicides such as methoxyacrylates and triazoles are primarily used in production to prevent and treat strawberry anthracnose. However, there is currently little research on using biological control bacteria to combat strawberry anthracnose.

[0004] Actinomycetes are a type of Gram-positive, unicellular, prokaryotic microorganism that grows in a mycelial form and reproduces primarily by spores. Streptomyces, a member of the higher actinomycetes family, is capable of producing a variety of active substances, including antibacterial compounds and plant hormones, which have beneficial effects such as inhibiting the growth of pathogenic bacteria, enhancing plant disease resistance, and promoting plant growth. These substances have great potential for the prevention and treatment of various plant diseases. To date, there have been no reports that a microbial seedling substrate containing Streptomyces as an active ingredient can simultaneously prevent and treat strawberry anthracnose and promote the growth of strawberry seedlings. Summary of the Invention [Problem to be solved by the invention]

[0005] In view of the problems in the prior art, the present invention provides a Streptomyces complex comprising Streptomyces CMB19 and Streptomyces CMB20. A seedling substrate prepared using the complex of the present invention as an active ingredient can simultaneously prevent and treat strawberry anthracnose and promote the growth of strawberry seedlings.

[0006] Another object of the present invention is to provide a microbial seedling substrate prepared with the above Streptomyces complex.

[0007] The present invention further provides the application of the above microbial seedling raising substrate in preventing and treating strawberry anthracnose and promoting the growth of strawberry seedlings.

[0008] To achieve the above objectives, the technical solutions adopted by the present invention are as follows:

[0009] The present invention provides a Streptomyces sp. composite fungus preparation comprising Streptomyces CMB19 and Streptomyces CMB20. The Streptomyces CMB19 is preserved at the Comprehensive Center of Microbiology, China National Commission for the Preservation of Microorganisms, with the preservation number CGMCC No. 30422 and the preservation date April 24, 2024, and is classified as Streptomyces sp. The Streptomyces CMB20 is preserved at the Comprehensive Center of Microbiology, China National Commission for the Preservation of Microorganisms, with the preservation number CGMCC No. 30423 and the preservation date April 24, 2024, and is classified as Streptomyces sp.

[0010] The present invention further provides a microbial seedling substrate prepared using the above-mentioned Streptomyces complex fungus agent, which is composed of a mixture of CMB19 fermentation liquid, CMB20 fermentation liquid, and seedling substrate supplement in a weight ratio of 1:1:20.

[0011] In the microbial seedling substrate provided by the present invention, the mixture of seedling substrate supplements is composed, in parts by weight, of 2 to 3 parts fermented chili pepper straw, 1 to 2 parts fermented eggplant straw, 1 to 2 parts fermented tomato straw, 3 to 6 parts vermiculite, 3 to 6 parts perlite, and 0.5 to 1 part inorganic salt solution.

[0012] The fermented chili straw, eggplant straw, and tomato straw are Trichoderma fermented chili straw, eggplant straw, and tomato straw, respectively. The specific preparation method is to mechanically crush the chili straw, eggplant straw, or tomato straw, respectively, and then add 100 ml of PDB fermented Trichoderma harzianum Ta97 fermented water to the crushed chili straw, eggplant straw, or tomato straw. 9 The straws were mixed in at a concentration of cfu / kg, fermented at room temperature for 30 days, autoclaved, dried, and crushed to a diameter of 3-6 mm to obtain fermented chili straw, fermented eggplant straw, or fermented tomato straw. The inorganic salt solution contained 3 g / L of (NH4)3PO4 and 0.5 g / L of MgSO4·7H2O.

[0013] The specific methods for preparing the fermentation liquid of CMB19 and the fermentation liquid of CMB20 are as follows: Step (1) Streptomyces CMB19 and Streptomyces CMB20 are scribed onto Takashi No. 1 medium, respectively, and purified single colonies are obtained. Step (2) inoculating the single colonies into seed media and culturing them to obtain CMB19 seed liquid and CMB20 seed liquid, respectively; Step (3) is to add the CMB19 seed liquid and the CMB20 seed liquid to a fermentation medium, respectively, and ferment and culture the mixture to obtain a CMB19 fermentation liquid and a CMB20 fermentation liquid.

[0014] In step (1), the formulation of the Gaoji No. 1 medium is 1 g of KNO3, 0.5 g of KH2PO4, 0.5 g of MgSO4·7H2O, 0.5 g of NaCl, 0.01 g of FeSO4·7H2O, 20 g of soluble starch, 15 g of agar, and 1000 mL of deionized water, adjusted to pH 7.2-7.4, sterilized at 121°C for 20 minutes, and cooled to 50-55°C, and then 1 mL of 3% potassium dichromate is added for every 300 mL of the Gaoji No. 1 medium. In step (2), the seed medium is Gaoji No. 1 medium, and its formulation is 1 g of KNO3, 0.5 g of KH2PO4, 0.5 g of MgSO4·7H2O, 0.5 g of NaCl, 0.01 g of FeSO4·7H2O, 20 g of soluble starch, 15 g of agar, and 1000 mL of deionized water. In step (3), the fermentation medium is Gaozi No. 1 medium, and the blending method is as follows: 1 g KNO3, 0.5 g KH2PO4, 0.5 g MgSO4 7H2O, 0.5 g NaCl, 0.01 g FeSO4 7H2O, 20 g soluble starch, and 1000 mL deionized water are adjusted to pH 7.2-7.4, sterilized at 121°C for 20 minutes, and cooled to 50-55°C, and then 1 mL of 3% potassium dichromate is added to each 300 mL of Gaozi No. 1 medium.

[0015] In step (2), the culture is carried out at 28° C. and 160 to 180 rpm for 72 hours.

[0016] In step (3), the weight ratio of the CMB19 seed liquid to the fermentation medium is 1:50, and the weight ratio of the CMB20 seed liquid to the fermentation medium is 1:50.

[0017] In step (3), the fermentation culture is carried out at 28°C and 160 to 180 rpm for 120 hours.

[0018] The present invention further provides the application of the microbial seedling substrate prepared with the Streptomyces complex in preventing and treating strawberry anthracnose and promoting the growth of strawberry seedlings.

[0019] The two Streptomyces strains provided by the present invention were isolated and selected from the rhizosphere of strawberry in a greenhouse and cultured in Gaoshi Medium No. 1. The 16S rDNA sequences of CMB19 and CMB20 were determined and compared with GenBank by Blast. Results showed that CMB19 shares 98.69% identity with Streptomyces caeruleatus LB27 (GenBank accession number: KU870780.1) (query coverage: 100%, E value: 0), and that CMB20 shares 99.93% identity with Streptomyces canus (GenBank accession number: MT083986.1) (query coverage: 100%, E value: 0). The inhibition rates of Streptomyces CMB19 and Streptomyces CMB20 against strawberry anthracnose (Colletotrichum siamense) were 54% and 82.4%, respectively. Streptomyces CMB19 has phosphorus removal activity and ACC deaminase activity, and Streptomyces CMB20 has phosphorus removal activity and iron carrier activity.

[0020] The viable cell counts of the fermentation broths of Streptomyces CMB19 and Streptomyces CMB20 prepared according to the present invention were both 10 9 More than CFU / mL.

[0021] The viable cell counts of Streptomyces CMB19 and Streptomyces CMB20 in the microbial seedling substrate provided by the present invention were both 10 8 The seedling substrate should have an organic matter content of 25% or more, a total nutrient content (N + P2O5 + K2O) of 2% to 5%, a moisture content (free water) of 35% or less, a pH of 6 to 7, and an EC of 2ms / cm or less.

[0022] The beneficial effects of the present invention are as follows:

[0023] (1) The Streptomyces CMB19 and Streptomyces CMB20 provided by the present invention can highly efficiently inhibit the growth of strawberry anthracnose fungus, and the microbial seedling growing substrate prepared therefrom can effectively prevent and treat the occurrence and spread of strawberry anthracnose. The microbial seedling growing substrate prepared by the Streptomyces CMB19 and Streptomyces CMB20 of the present invention can significantly promote the growth of strawberry seedlings.

[0024] (2) The microbial seedling substrate provided by this invention uses fermented vegetable straw from greenhouses instead of conventional peat, which has two advantages. First, in major greenhouse vegetable production areas, large amounts of vegetable straw are simply discarded, causing agricultural land pollution and resource waste. Peat is an important land resource, providing a good habitat and breeding ground for several rare plants and animals and occupying an important position in the natural balance. Therefore, straw fermentation products are a good substitute for peat. This invention allows for the efficient use of discarded vegetable straw in the local greenhouse vegetable production area, which has the effect of promoting environmental protection. Second, Streptomyces CMB19 and Streptomyces CMB20 were collected from strawberry greenhouses. They are naturally adapted to the nutrients in the dead humus of greenhouse-grown crops and have a good digestibility of fermented vegetable straw. These bacteria can survive stably in the substrate product for a long time, with the viable bacterial count after 180 days being more than 85% of the original bacterial count. They also have good colonization effects in the rhizosphere of strawberries, preventing disease and promoting growth.

[0025] Save information 1 Strain name: CMB19 Save date: April 24, 2024 Storage location: Comprehensive Microbiology Center, China Committee for the Preservation and Management of Microbial Species Storage number: CGMCC No.30422 Storage address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, No. 1, Chaoyang District, Beijing Postal code: 100101 Classification name: Streptomyces sp. Save information 2 Strain name:CMB20 Save date: April 24, 2024 Storage location: Comprehensive Microbiology Center, China Committee for the Preservation and Management of Microbial Species Storage number: CGMCC No. 30423 Storage address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, No. 1, Chaoyang District, Beijing Postal code: 100101 Classification name: Streptomyces sp. Save information 3 Strain name: Trichoderma harzianum Ta97 Save date: July 14, 2020 Storage location: Comprehensive Microbiology Center, China Committee for the Preservation and Management of Microbial Species Storage number: CGMCC No. 19930 Storage address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, No. 1, Chaoyang District, Beijing Postal code: 100101 Classification name: Trichoderma harzianum [Brief explanation of the drawings]

[0026] [Figure 1] FIG. 1 shows the colony morphology of Streptomyces CMB19. [Figure 2] FIG. 1 shows the colony morphology of Streptomyces CMB20. [Figure 3]FIG. 1 shows the clearing zone formed by Streptomyces CMB19 on inorganic phosphorus medium. [Figure 4] FIG. 1 shows the clearing zone formed by Streptomyces CMB20 on inorganic phosphorus medium. [Figure 5] FIG. 1 shows the detection of iron carrier activity in Streptomyces CMB20. DETAILED DESCRIPTION OF THE INVENTION

[0027] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. It is obvious that the described embodiments are only a part of the embodiments, not all of the embodiments of the present invention. All other embodiments that can be obtained by those skilled in the art based on the embodiments of the present invention without any inventive ideas fall within the protection scope of the present invention.

[0028] In the microbial seedling substrate prepared using the Streptomyces complex fungus preparation of the present invention, the mixture of seedling substrate supplements is composed of, by weight, 2 to 3 parts fermented chili pepper straw, 1 to 2 parts fermented eggplant straw, 1 to 2 parts fermented tomato straw, 3 to 6 parts vermiculite, 3 to 6 parts perlite, and 0.5 to 1 part inorganic salt solution.

[0029] In some embodiments, the vermiculite has a diameter of 3 to 6 mm.

[0030] In some embodiments, the pearlite has a diameter of 3 to 6 mm.

[0031] Trichoderma harzianum Ta97, which is used in the preparation of the fermented chili straw, fermented eggplant straw, and fermented tomato straw in the present invention, is described in a patent publication with the reserve number CGMCC No. 19930 and application number 202011309961.2.

[0032] In some examples, the specific preparation method for the PDB fermentation liquid of Trichoderma harzianum Ta97 used is as follows: Trichoderma harzianum Ta97 stored at -80°C is inoculated onto a PDA solid plate (200g potato, 20g glucose, 15g agar, 1000mL water, natural pH), activated at 25°C for 3 days, mycelium is collected from the edge of the colony and inoculated onto a PDA solid plate again, cultured at 27°C for 3 days, mycelium is collected again, i.e., the activated strain is cultured. The activated strain is cultured on a PDA solid plate at 27°C under 12 hours of light and 12 hours of darkness for 10 days, and then the 10 7 A conidial suspension of 1 × 10 conidia / mL was prepared and inoculated into PDB culture medium (300 g of potato (to extract the leachate powder), 20 g of glucose, 1000 mL of water, natural pH) at a volume ratio of 1:100. The mixture was then shaken at 27°C and 180 rpm for 40 hours until the viable cell count reached 1 × 10 4 Cultivate the seed solution for cfu / mL.

[0033] Example 1 The present invention provides two Streptomyces strains, Streptomyces CMB19 and Streptomyces CMB20, which were deposited at the Comprehensive Microbiology Center of the China Microorganism Species Collection, No. 3, Courtyard No. 1, Beichen West Road, Chaoyang District, Beijing, on April 24, 2024, with storage numbers CGMCC No. 30422 and CGMCC No. 30423, respectively. Streptomyces CMB19 and Streptomyces CMB20 were isolated and selected from the strawberry rhizosphere in a greenhouse and can be cultured on Gaoshi No. 1 medium. Specific details are as follows:

[0034] 1.1 Isolation of strains Fresh strawberry root systems were collected from a greenhouse facility (collection location: Tangwang Town, Licheng District, Jinan City, Shandong Province) and washed with sterile water until no visible soil particles were attached. The root systems were transferred to a new 50 mL Falcon centrifuge tube, 30 mL of sterile 1x PBS (pH = 7) was added, and the tube was shaken at 180 rpm at room temperature for 15 minutes. The PBS wash was repeated three times. The washed root systems were removed and placed on sterile filter paper to absorb the PBS from the surface of the root systems. The root systems were cut into small pieces approximately 2 mm thick and mixed. 1 g of root tissue was weighed and transferred to a 50 mL centrifuge tube. The roots were resuspended in 10 mL of 10 mM sterile MgCl2 solution in a sterile environment and then ground to a homogeneous consistency with a sterile pestle. The roots were then spun in sterile water in a sterile test tube for 10 minutes. -2 double, 10 -3 double, 10 -4 double, 10 -5 double, 10 -6 double, 10 -7 Double and 10 -8 Dilute the gradient 2x and evenly spread 100 μL from each gradient onto a Gaozzi No. 1 plate. Invert the plate and incubate it in a microbial incubator at 30°C for 5-7 days, observing the colony growth. According to the colony morphology (as shown in Figures 1 and 2), scribe differential colonies onto new Gaozzi No. 1 plates to obtain single colonies. Repeat this single colony scribing procedure three times to purify the strain.

[0035] 1.2 Screening of strains for biocontrol and growth-promoting properties The strawberry anthracnose pathogen was isolated and purified from the root system of diseased strawberry plants and identified as Colletotrichum siamense by ITS sequencing. Strains were screened for their ability to inhibit the growth of the strawberry anthracnose pathogen using a classical paired plate assay. Among them, strains CMB19 and CMB20 showed 54% and 82.4% inhibition, respectively. Furthermore, strains were screened for phosphorus removal ability using inorganic phosphorus medium. Both strains CMB19 and CMB20 were able to produce phosphorus-removing transparent zones (Figures 3 and 4). ACC deaminase activity of the strains was qualitatively detected using a unique carbon source method. Strain CMB19 showed a positive reaction, indicating that it possessed ACC deaminase activity. The iron carrier production ability of the strains was measured using the CAS plate coating method. Strain CMB20 possessed iron carrier activity (Figure 5).

[0036] The inorganic phosphorus medium is composed of 10.0 g / L glucose, 0.5 g / L ammonium sulfate, 0.5 g / L yeast extract powder, 0.3 g / L sodium chloride, 0.3 g / L potassium chloride, 0.3 g / L magnesium sulfate, 0.03 g / L ferrous sulfate, 0.03 g / L manganese sulfate, 5.0 g / L tricalcium phosphate, 12 g agar, and a pH value of 7.0 to 7.5.

[0037] 1.3 Strain identification The 16S rDNA sequences of the CMB19 and CMB20 strains are as follows: CMB19 strain (as shown in SEQ ID NO. 1): GGGGGTTGGGCCACCGGCTTCGGGTGTTACCGACTTTCGTGACGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCAGCACTGCTGATCTG CGATTACTAGCAACTCCGACTTCATGGGGTCGAGTTGCAGACCCCAATCC GAACTGAGACCGGCTTTTTGAGATTCGCTCCACCTCACGGTATCGCAGCT CATTGTACCGGCCATTGTAGCACGTGTGCAGCCCAAGACATAAGGGGCAT GATGACTTGACGTCGTCCCCACCTTCCTCCGAGTTGACCCCGGCGGTCTC CTGTGAGTCCCCGTCACCCCGAAAGGCACGCTGGCAACACAGGACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTACACCGACCACAAGGGGGCGACCATCTCTGGCCGTTTCCGGTGTATGTCAAGCCTTGGTAAGGTTCTTCGCGTTGCGTCGAA TTAAGCCACATGCTCCGCTGCTTGTGCGGGCCCCCGTCAATTCCTTTGAG TTTTAGCCTTGCGGCCGTACTCCCCAGGCGGGGAACTTAATGCGTTAGCT GCGGCACCGACGACGTGGAATGTCGCCAACACCTAGTTCCCACCGTTTAC GGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCT CCTCAGCGTCAGTAATGGCCCAGAGATCCGCCTTCGCCACCGGTGTTCCT CCTGATATCTGCGCATTTCACCGCTACACCAGGAATTCCGATCTCCCCTA CCACACTCTAGCTAGCCCGTATCGAATGCAGACCCGGGGTTAAGCCCCGG GCTTTCACACCCGACGTGACAAGCCGCCTACGAGCTCTTTACGCCCAATA ATTCCGGACAACGCTCGCGCCCTACGTATTACCGCGGCTGCTGGCACGTA GTTAGCCGGCGCTTCTTCTGCAGGTACCGTCACTTGCGCTTCTTCCCTGC TGAAAGAGGTTTACAACCCGAAGGCCGTCATCCCTCACGCGGCGTCGCTG CATCAGGCTTTCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAG GAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGGTCGCCCTCTCAGGCC GGCTACCCGTCGTCGCCTTGGTGAGCCATTACCTCACCAACAAGCTGATA GGCCGCGGGCTCATCCTTCACCGCCGGAGCTTTCAACCACAGACCATGAG ACCCGCAGTATTATCCGGTATTAGACCCCGTTTCCAGGGCTTGTCCCAGA GTGAAGGGCAGATTGCCCACGTGTTACTCACCCGTTCGCCACTAATCCAC CCCGAAGGGCTTCATCGTTCGACTGC Strain CMB20 (as shown in SEQ ID NO.2): GGGGTTGGGCCACCGGCTTCGGGTGTTACCGACTTTCGTGACGTGACGGG CGGTGTGTACAAGGCCCGGGAACGTATTCACCGCAGCAATGCTGATCTGC GATTACTAGCAACTCCGACTTCATGGGGTCGAGTTGCAGACCCCAATCCG AACTGAGACAGGCTTTTTGAGATTCGCTCCACCTCACGGTATCGCAGCTC ATTGTACCTGCCATTGTAGCACGTGTGCAGCCCAAGACATAAGGGGCATG ATGACTTGACGTCGTCCCCACCTTCCTCCGAGTTGACCCCGGCGGTCTCC TGTGAGTCCCCATCACCCCGAAGGGCATGCTGGCAACACAGGACAAGGGT TGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGAC AGCCATGCACCACCTGTACACCGACCACAAGGGGGGCACCATCTCTGATG CTTTCCGGCGTATGTCAAGCCTTGGTAAGGTTCTTCGCGTTGCGTCGAAT TAAGCCACATGCTCCGCTGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGT TTTAGCCTTGCGGCCGTACTCCCCAGGCGGGGAACTTAATGCGTTAGCTG CGGCACCGACGACGTGGAATGTCGCCAACACCTAGTTCCCACCGTTTACG GCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTC CTCAGCGTCAGTAATGGCCCAGAGATCCGCCTTCGCCACCGGTGTTCCTC CTGATATCTGCGCATTTCACCGCTACACCAGGAATTCCGATCTCCCCTAC CACACTCTAGCTAGCCCGTATCGAATGCAGACCCGGGGTTAAGCCCCGGG CTTTCACACCCGACGTGACAAGCCGCCTACGAGCTCTTTACGCCCAATAA TTCCGGACAACGCTTGCGCCCTACGTATTACCGCGGCTGCTGGCACGTAG TTAGCCGGCGCTTCTTCTGCAGGTACCGTCACTTTCGCTTCTTCCCTGCT GAAAGAGGTTTACAACCCGAAGGCCGTCATCCCTCACGCGGCGTCGCTGC ATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGG AGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGGTCGCCCTCTCAGGCCG GCTACCCGTCGTCGCCTTGGTGAGCCATTACCTCACCAAACAAGCTGATAG GCCCGGGCTCATCCTTCACCGCCGGAGCTTTCAACCACCACAGATGCCT GCGGTAGTGGTATCCGGTATTAGACCCCGTTTCCAGGGCTTGTCCCAGAG TGAAGGGCAGATTGCCCACGTGTTACTCACCCGTTCGCCACTAATCCCCA CCGAAGTGGTTCATCGTTCGAC

[0038] Blast comparison results on GenBank showed that the homology between the CMB19 strain and Streptomyces caeruleatus LB27 (GenBank accession number: KU870780.1) reached 98.69% (query coverage: 100%, E value: 0), and the homology between the CMB20 strain and Streptomyces canus (GenBank accession number: MT083986.1) reached 99.93% (query coverage: 100%, E value: 0).

[0039] <Example 2> Preparation of fermentation broth of Streptomyces CMB19 and Streptomyces CMB20 Streptomyces CMB19 and Streptomyces CMB20 were each scribed three times onto Gaoshi No. 1 medium to obtain purified single colonies. The single colonies were then inoculated into seed medium and cultured at 28°C and 160-180 rpm for 72 hours to obtain seed broth. The seed broth was then added to fermentation medium at a weight ratio of 1:50 and cultured at 28°C and 160-180 rpm for 120 hours to obtain fermentation broths of CMB19 and CMB20.

[0040] The formulation of Gaozi No. 1 medium is as follows: 1g KNO3, 0.5g KH2PO4, 0.5g MgSO4·7H2O, 0.5g NaCl, 0.01g FeSO4·7H2O, 20g soluble starch, 15g agar, and 1000mL deionized water are adjusted to a pH of 7.2-7.4, sterilized at 121°C for 20 minutes, and cooled to 50-55°C. 1mL of 3% potassium dichromate is added for every 300mL of Gaozi No. 1 medium. The seed medium is Gaozi No. 1 medium, and its formulation is as follows: 1g KNO3, 0.5g KH2PO4, 0.5g MgSO4·7H2O, 0.5g NaCl, 0.01g FeSO4·7H2, 20g soluble starch, and 1000mL deionized water are adjusted to a pH of 7.2-7.4, sterilized at 121°C for 20 minutes, and cooled to 50-55°C. After this, 1mL of 3% potassium dichromate is added for every 300mL of Gaozi No. 1 medium. The fermentation medium is Gaozi No. 1 medium, and its formulation method is as follows: 1g KNO3, 0.5g KH2PO4, 0.5g MgSO4·7H2O, 0.5g NaCl, 0.01g FeSO4·7H2O, 20g soluble starch, and 1000mL deionized water are adjusted to pH 7.2-7.4, sterilized at 121°C for 20 minutes, and cooled to 50-55°C. Then, 1mL of 3% potassium dichromate is added for every 300mL of Gaozi No. 1 medium.

[0041] As a result of the detection, the viable cell counts of Streptomyces CMB19 and Streptomyces CMB20 in the fermentation broth were both 10 9 CFU / mL.

[0042] Example 3 The microbial seedling substrate containing Streptomyces CMB19 and Streptomyces CMB20 contains a mixture of the fermentation broth of CMB19 and CMB20 prepared in Example 2 and a seedling substrate supplement, with the weight ratio of the fermentation broth of CMB19, the fermentation broth of CMB20, and the seedling substrate supplement being 1:1:20.

[0043] The seedling substrate supplement contains the following components by weight: 3 parts fermented chili straw, 1 part fermented eggplant straw, 1 part fermented tomato straw, 5 parts vermiculite (diameter 3-6 mm), 5 parts perlite (diameter 3-6 mm), and 0.8 parts inorganic salt solution.

[0044] The fermented chili straw, eggplant straw, and tomato straw are fermented chili straw, eggplant straw, and tomato straw, respectively, by Trichoderma fermentation. The specific preparation method is to mechanically crush the chili straw, eggplant straw, and tomato straw into short straws of about 1 cm each, and then add 10 ml of Trichoderma harzianum Ta97 seed juice to the crushed chili straw, eggplant straw, and tomato straw. 9 The straw is mixed at a concentration of cfu / kg, fermented at room temperature for 30 days, autoclaved, dried, and crushed to a diameter of 3-6 mm to obtain fermented chili straw, fermented eggplant straw, or fermented tomato straw.

[0045] The inorganic salt solution contains 3 g / L of (NH4)3PO4 and 0.5 g / L of MgSO4·7H2O.

[0046] The fermentation broth of CMB19, the fermentation broth of CMB20, and the microbial seedling substrate supplement were uniformly mixed in a weight ratio of 1:1:20 to obtain a microbial seedling substrate containing Streptomyces CMB19 and CMB20. As a result of the detection, the viable counts of CMB19 and CMB20 in the microbial seedling substrate were 10 8 CFU / g.

[0047] Example 4 The microbial seedling substrate containing Streptomyces CMB19 and Streptomyces CMB20 contains a mixture of the fermentation broth of CMB19 and CMB20 prepared in Example 2 and a seedling substrate supplement, with the weight ratio of the fermentation broth of CMB19, the fermentation broth of CMB20, and the seedling substrate supplement being 1:1:20.

[0048] The seedling substrate supplement contains the following ingredients in parts by weight: 2 parts fermented chili straw, 2 parts fermented eggplant straw, 1 part fermented tomato straw, 6 parts vermiculite (diameter 3-6 mm), 3 parts perlite (diameter 3-6 mm), and 1 part inorganic salt solution.

[0049] The preparation methods of the fermented chili straw, fermented eggplant straw and fermented tomato straw are the same as those in Example 3.

[0050] The components of the inorganic salt solution are the same as those in Example 3.

[0051] The fermentation broth of CMB19, the fermentation broth of CMB20, and the microbial seedling substrate supplement were uniformly mixed in a weight ratio of 1:1:20 to obtain a microbial seedling substrate containing Streptomyces CMB19 and CMB20. As a result of the detection, the viable cell counts of CMB19 and CMB20 in the microbial seedling substrate were 10 8 CFU / g.

[0052] <Example 5> Taking the microbial seedling substrate prepared in Example 3 as an example, the effects of different seedling substrates on preventing the occurrence of strawberry anthracnose were tested and compared.

[0053] A seedling substrate supplement containing no microbial agent (2 parts fermented chili straw, 2 parts fermented eggplant straw, 1 part fermented tomato straw, 6 parts vermiculite (3-6 mm diameter), 3 parts perlite (3-6 mm diameter), and 1 part inorganic salt solution) was selected and used as control group 1. A microbial seedling substrate containing only CMB19 fermentation liquid (CMB19 fermentation liquid and seedling substrate supplement (same ingredients as in Example 3) in a weight ratio of 1:15) was selected and used as control group 2. A microbial seedling substrate containing only CMB20 fermentation liquid (CMB20 fermentation liquid and seedling substrate supplement (same ingredients as in Example 3) in a weight ratio of 1:15) was selected and used as control group 3.

[0054] The strawberry cultivar used was Akihime strawberry. Different seedling substrates were placed in seedling pots in advance, and one white seed was sown in each pot. After the four true leaves had fully developed, 20 complete seedlings were selected from each treatment group as replicates. Then, 2 mL of a Bacillus anthracis spore suspension (1 × 10 8CFU / mL) around the roots, and then irrigate as usual. After 14 days of cultivation, the disease index is recorded. The classification criteria for strawberry anthracnose were established by referring to the "Pesticide Field Efficacy Testing Guidelines" compiled by the Pesticide Inspection Service of the Ministry of Agriculture, and incorporating actual conditions.

[0055] Level 0: The entire plant is healthy and disease-free. Level 1: Less than 1 / 5 of the leaves (petioles) on the entire plant are affected, black lesions have appeared on the leaves, and the area of ​​the lesions is less than 5% of the leaf area. Level 3: 1 / 5 to 2 / 5 of the leaves (petioles) on the entire plant are affected, the leaf lesions occupy 6% to 10% of the total leaf area, and black-brown depressions are beginning to appear on the stems. Level 5: 2 / 5 to 3 / 5 of the leaves (petioles) on the entire plant are affected, the leaf lesions occupy 11% to 25% of the total leaf area, or one leaf is wilted, but the wilting status of the entire plant is not clear. Level 7: 3 / 5 to 4 / 5 of the leaves (petioles) on the entire plant are affected, the leaf lesions occupy 26% to 50% of the total leaf area, and more than half of the leaves on the entire plant are wilted, but the leaves are not completely dead. Level 9: Leaf (petiole) lesions cover more than 50% of the total leaf area, or the leaves wither and the plant completely wilts or dies. Disease index = [Σ(number of diseased leaves at each level × disease level) / (total number of inspected leaves × 9)] × 100.

[0056] Using the above test conditions, the effectiveness of the microbial seedling substrate prepared in Example 3 in preventing and treating strawberry anthracnose was tested. Compared to Control Group 1, which did not contain Streptomyces CMB19 or Streptomyces CMB20, the microbial seedling substrate provided in Example 3 of the present invention reduced the disease index by 71.4%, and compared to Control Groups 2 and 3, which contained single strains, the disease index reduced by 14.1% and 23.4%, respectively.

[0057] Example 6 Taking the microbial seedling substrate prepared in Example 3 as an example, the effects of different seedling substrates on promoting the growth of strawberry seedlings were tested and compared.

[0058] A seedling substrate supplement containing no microbial agent (2 parts fermented chili straw, 2 parts fermented eggplant straw, 1 part fermented tomato straw, 6 parts vermiculite (3-6 mm diameter), 3 parts perlite (3-6 mm diameter), and 1 part inorganic salt solution) was selected and used as control group 1. A microbial seedling substrate containing only CMB19 fermentation liquid (CMB19 fermentation liquid and seedling substrate supplement (same ingredients as in Example 3) in a weight ratio of 1:15) was selected and used as control group 2. A microbial seedling substrate containing only CMB20 fermentation liquid (CMB20 fermentation liquid and seedling substrate supplement (same ingredients as in Example 3) in a weight ratio of 1:15) was selected and used as control group 3.

[0059] The strawberry variety used was Akihime strawberry. Different seedling substrates were placed in seedling pots in advance, and one white seed was sown in each seedling pot. After the cotyledons had fully expanded, 20 complete seedlings were selected from each treatment group as duplicates. After that, irrigation management was normal.

[0060] Under the above experimental conditions, plant height, stem thickness, and dry weights of above-ground and below-ground parts were measured 28 days after seedling emergence, and the seedling viability index was calculated as (stem thickness / plant height + below-ground dry weight / above-ground dry weight) x plant dry weight. When the microbial seedling substrate provided in Example 3 of the present invention was used, strawberry biomass increased by 33.2% and the seedling viability index increased by 22.4% compared to control group 1, which was not supplemented with Streptomyces CMB19 or CMB20. Compared to control groups 2 and 3, which contained single strains, strawberry biomass increased by 7.3% and 11.9%, respectively.

[0061] Although the present invention has been described in detail by combining preferred embodiments with reference to the accompanying drawings, the present invention is not limited thereto. Those skilled in the art may make various equivalent modifications or substitutions to the embodiments of the present invention without departing from the spirit and essence of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention. Those skilled in the art may easily think of modifications or substitutions within the technical scope disclosed by the present invention, and all such modifications or substitutions should be covered within the scope of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection set forth in the claims.

Claims

1. A Streptomyces complex fungus preparation, A Streptomyces complex fungus preparation comprising Streptomyces CMB19 and Streptomyces CMB20, wherein the Streptomyces CMB19 is preserved at the Comprehensive Center of Microbiology of the Chinese Commission for the Preservation of Microorganisms, with the preservation number CGMCC No. 30422, the preservation date of April 24, 2024, and the taxonomic name Streptomyces sp., and the Streptomyces CMB20 is preserved at the Comprehensive Center of Microbiology of the Chinese Commission for the Preservation of Microorganisms, with the preservation number CGMCC No. 30423, the preservation date of April 24, 2024, and the taxonomic name Streptomyces sp.

2. A microbial seedling substrate prepared using the Streptomyces complex fungus preparation according to claim 1, The microbial seedling substrate is characterized in that it is composed of a mixture of a fermentation liquid of CMB19, a fermentation liquid of CMB20, and a seedling substrate supplement in a weight ratio of 1:1:

20.

3. The microbial seedling raising substrate according to claim 2, characterized in that the mixture of seedling raising substrate supplements is composed of, in parts by weight, 2 to 3 parts fermented chili pepper straw, 1 to 2 parts fermented eggplant straw, 1 to 2 parts fermented tomato straw, 3 to 6 parts vermiculite, 3 to 6 parts perlite, and 0.5 to 1 part inorganic salt solution.

4. The fermented chili straw, eggplant straw, and tomato straw are Trichoderma fermented chili straw, eggplant straw, and tomato straw, respectively. The specific preparation method is to mechanically crush chili straw, eggplant straw, and tomato straw, respectively, and then add 100 ml of PDB fermentation liquid of Trichoderma harzianum Ta97 to the crushed chili straw, eggplant straw, and tomato straw. ９ cfu / kg, fermented at room temperature for 30 days, autoclaved, dried, and crushed to a diameter of 3-6 mm, thus obtaining fermented chili straw, fermented eggplant straw, or fermented tomato straw. The inorganic salt solution contained 3 g / L of (NH ４ ) ３ P.O. ４ , 0.5 g / L MgSO ４ ・7H ２ The microbial seedling growing substrate according to claim 3, characterized in that it contains an O component.

5. The specific methods for preparing the fermentation broth of CMB19 and the fermentation broth of CMB20 are as follows: Step (1) Streptomyces CMB19 and Streptomyces CMB20 are scribbled onto Takashi No. 1 medium, respectively, to obtain purified single colonies; Step (2) inoculating the single colonies into seed media and culturing them to obtain CMB19 seed liquid and CMB20 seed liquid, respectively; The microbial seedling substrate according to claim 2, wherein in step (3), the CMB19 seed liquid and the CMB20 seed liquid are added to a fermentation medium, respectively, and then fermented and cultured to obtain a CMB19 fermentation liquid and a CMB20 fermentation liquid.

6. In step (1), the formulation of the Gaoji No. 1 medium is as follows: ３ , 0.5g KH ２ P.O. ４ , 0.5 g MgSO ４ ・7H ２ 0, 0.5 g NaCl, 0.01 g FeSO ４ ・7H ２ 0, 20 g of soluble starch, 15 g of agar, and 1000 mL of deionized water are adjusted to pH 7.2-7.4, sterilized at 121°C for 20 minutes, and cooled to 50-55°C. Then, 1 mL of 3% potassium dichromate is added to every 300 mL of Gaozi No. 1 medium. In step (2), the seed medium is Gaozi No. 1 medium, and the compounding method is as follows: 1 g of KNO ３ , 0.5g KH ２ P.O. ４ , 0.5 g MgSO ４ ・7H ２ 0, 0.5 g NaCl, 0.01 g FeSO ４ ・7H ２ 0, 20 g of soluble starch, 1000 mL of deionized water are adjusted to pH 7.2-7.4, sterilized under the condition of 121 ° C for 20 minutes, and cooled to 50-55 ° C., and then mixed by adding 1 mL of 3% potassium dichromate for every 300 mL of Gao Zhi No. 1 medium; in step (3), the fermentation medium is Gao Zhi No. 1 medium, and the mixing method is as follows: 1 g of KNO ３ , 0.5g KH ２ P.O. ４ , 0.5 g MgSO ４ ・7H ２ 0, 0.5 g NaCl, 0.01 g FeSO ４ ・7H ２ 20 g of soluble starch and 1000 mL of deionized water are adjusted to pH 7.2-7.4, sterilized at 121°C for 20 minutes, and cooled to 50-55°C. The microbial seedling substrate of claim 5 is prepared by adding 1 mL of 3% potassium dichromate to every 300 mL of Gaozi No. 1 medium.

7. The microbial seedling substrate according to claim 6, wherein in step (2), the culturing is carried out under conditions of 28°C and 160-180 rpm for 72 hours.

8. The microbial seedling substrate of claim 5, wherein in step (3), the weight ratio of the CMB19 seed liquid to the fermentation medium is 1:50, and the weight ratio of the CMB20 seed liquid to the fermentation medium is 1:

50.

9. The microbial seedling substrate according to claim 8, wherein in step (3), the fermentation culture is carried out under conditions of 28°C and 160-180 rpm for 120 hours.

10. 10. The use of the microbial seedling raising substrate according to any one of claims 2 to 9 in the prevention and treatment of strawberry anthracnose and the promotion of strawberry seedling growth.

Images