Streptomyces with resistance to banana wilt and growth-promoting effects

Abstract

The present invention provides a Streptomyces, named: Streptomyces coffeae , preserved in Guangdong Province Microbial Culture Collection Center, with the preservation number of GDMCC No: 64532. The Streptomyces and its fermentation liquid of the present invention have good antibacterial properties against banana wilt No. 4 physiological subspecies, gloeosporium anthracnose, cucumber wilt, banana anthracnose, litchi anthracnose, mango anthracnose, strawberry anthracnose, banana leaf spot pathogen, rice blast pathogen, banana gray spot pathogen, mango leaf blight pathogen, etc., and the antibacterial rate reaches more than 50%. Moreover, the growth of banana seedlings can be effectively promoted under the attack of pathogens, and the product has resistance to multiple antibiotics. The product is a potential biological agent for preventing and treating anthracnose diseases, wilt diseases, etc. and promoting plant growth, etc., and has broad development space and good development and application prospects.

Description

Technical Field

[0001] The invention relates to a strain, in particular to a streptomyces with banana wilt resistance and growth promotion effects. Background Art

[0002] banana( Musa nana Lour ) is a large herbaceous plant of the genus Musa in the family Musaceae. Its main production areas are distributed between tropical and subtropical areas, and the harvest period is long. The fruit is a berry, rich in various minerals and vitamins.

[0003] Banana pests and diseases have severely restricted the sustainable development of bananas and related industries. At present, the disease that has the most serious impact on the development of the banana industry is banana wilt, and very effective prevention and control measures have not yet been established. Fusarium wilt of banana ) is caused by Fusarium oxysporum Cuban type ( Fusarium oxysporum f. sp. Cubense, Foc ) is a soil-borne disease caused by infection. Foc As a soil-borne fungal pathogen, it can survive in the soil for nearly ten years. This type of fungus first colonizes and invades the roots of the plant, then enters the stem of the plant and spreads along the vascular bundle to the aboveground part, forming toxins and gelatinous substances in the vascular bundle and xylem, causing the cells of the vascular bundle to brown, necrotize, and become diseased, blocking the transport channels for water and organic matter, and causing the plant to wilt and die as a whole. In addition, the toxin also has a significant impact on the normal physiological metabolic functions of plant-related defense enzymes. This impact leads to the yellowing of banana leaves.

[0004] At present, the prevention and control strategies of banana wilt include chemical control, disease-resistant breeding and biological control. By comparing different prevention and control strategies, the use of microorganisms for biological control is a relatively effective prevention and control strategy against banana wilt.

[0005] Biological control refers to the prevention and control measures that inhibit pathogen infection through specific antagonistic microorganisms, and changes the population structure by increasing the diversity of microbial populations in the soil; the secondary metabolites produced by soil microorganisms can effectively inhibit the growth, reproduction and infection process of pathogens, thereby achieving the purpose of disease prevention and control. Biological control has little pollution to the environment and agricultural products, and can increase fruit yields. However, due to the limited existing biological control bacteria resource library and the unstable control effect of biological control bacteria in different geographical environments, they often mutate and degenerate during actual application. Therefore, it is necessary to continuously discover new biological control bacteria to expand the resource library. Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a streptomyces with banana wilt resistance and growth-promoting effects.

[0007] The first aspect of the present invention is to provide a Streptomyces, named: Streptomyces coffeae , deposited in Guangdong Microbiological Culture Collection Center, with the deposit number GDMCC No: 64532.

[0008] The second aspect of the present invention is to provide the fermentation broth or the filtrate of the fermentation broth of Streptomyces as described in the first aspect of the present invention.

[0009] The third aspect of the present invention is to provide a preparation containing the Streptomyces described in the first aspect of the present invention or the fermentation broth described in the second aspect of the present invention.

[0010] The fourth aspect of the present invention is to provide the Streptomyces described in the first aspect of the present invention, or the fermentation liquid or the filtrate of the fermentation liquid described in the second aspect of the present invention, or the preparation described in the third aspect of the present invention for preparing a preparation for antagonizing banana wilt No. 4 physiological subspecies ( F. oxysporum f. sp. cubense Race 4) preparations.

[0011] The fifth aspect of the present invention is to provide the Streptomyces described in the first aspect of the present invention, or the fermentation liquid or the filtrate of the fermentation liquid described in the second aspect of the present invention, or the preparation described in the third aspect of the present invention for preparing a method for preventing and controlling banana wilt race 4 ( F. oxysporum f. sp. cubense Race 4) in the preparation of diseases caused by.

[0012] The sixth aspect of the present invention is to provide the Streptomyces as described in the first aspect of the present invention, or the fermentation broth as described in the second aspect of the present invention, or the preparation as described in the third aspect of the present invention for preparing an antagonistic agent against Colletotrichum gloeosporioides ( Colletotrichum gloeosporioides ), and / or Fusarium wilt ( Fusarium oxysporum sp. Cucumebrium ), and / or Colletotrichum spp. ( Colletotrichum musae ), and / or Colletrichum litchi Trag, and / or Colletrichum mangoe Colletotrichum acutatum , and / or Colletotrichum spp. ( Colletotrichum fragariae ), and / or banana leaf blight pathogen (Alternaria musae), and / or rice blast pathogen (Pyricularia oryae Cav), and / or banana gray spot pathogen (Curvularialunata), and / or mango leaf blight pathogen (Pestalogiopsis sp.) in a preparation.

[0013] The seventh aspect of the present invention is to provide the Streptomyces described in the first aspect of the present invention, or the fermentation liquid described in the second aspect of the present invention, or the preparation described in the third aspect of the present invention for preparing a method for preventing and controlling banana wilt race 4 ( F. oxysporum f. sp. cubense Race 4), and / or Colletotrichum gloeosporioides ( Colletotrichum gloeosporioides ), and / or Fusarium wilt ( Fusarium oxysporum sp. Cucumebrium ), and / or Colletotrichum spp. ( Colletotrichum musae ), and / or Colletrichum litchiTrag, and / or Colletrichum mangoescens Colletotrichum acutatum , and / or Colletotrichum spp. ( Colletotrichum fragariae ), and / or banana leaf blight pathogen (Alternaria musae), and / or rice blast pathogen (Pyricularia oryae Cav), and / or banana gray spot pathogen (Curvularia lunata), and / or mango leaf blight pathogen (Pestalogiopsis sp.) in preparations for treating diseases caused by.

[0014] The eighth aspect of the present invention is to provide the use of the Streptomyces described in the first aspect of the present invention, or the fermentation liquid described in the second aspect of the present invention, or the preparation described in the third aspect of the present invention in promoting the growth of banana seedlings.

[0015] The ninth aspect of the present invention provides the use of the Streptomyces described in the first aspect of the present invention, or the fermentation liquid described in the second aspect of the present invention, or the preparation described in the third aspect of the present invention in increasing the plant height, and / or stem diameter, and / or leaf thickness, and / or leaf area, and / or fresh weight of banana seedlings.

[0016] The tenth aspect of the present invention provides the use of the Streptomyces described in the first aspect of the present invention, or the fermentation broth described in the second aspect of the present invention, or the preparation described in the third aspect of the present invention in the preparation of a bacterial agent resistant to imipenem, and / or levofloxacin, and / or clindamycin, and / or chloramphenicol, and / or ciprofloxacin, and / or norfloxacin, and / or tetracycline, and / or streptomycin, and / or amikacin, and / or ceftriaxone, and / or cephalexin.

[0017] The Streptomyces and its fermentation liquid of the present invention have broad-spectrum antibacterial activity and are effective against banana wilt species IV ( F. oxysporum f. sp. cubense Race 4), Colletotrichum gloeosporioides ( Colletotrichum gloeosporioides)、Cucumber wilt pathogen ( Fusarium oxysporum sp. Cucumebrium )、Banana Anthracnose ( Colletotrichum musae )、Lychee Anthracnose (Colletrichum litchi Trag), Mango Anthracnose Colletotrichum acutatum 、Strawberry Anthracnose Colletotrichum fragariae ), banana leaf edge spot fungus (Alternaria musae), rice blast fungus (Pyricularia oryae Cav), banana large gray spot fungus (Curvularia lunata), mango leaf blight fungus (Pestalogiopsis sp.), etc. It has good antibacterial properties, and the inhibition rate is more than 50%, especially for banana anthracnose fungi and litchi anthracnose fungi, the inhibition rate is more than 80%, and it can effectively promote the growth of banana seedlings under the invasion of pathogens. It is resistant to multiple antibiotics. It is a potential biological agent for preventing and controlling anthracnose diseases, wilt diseases, etc. and promoting plant growth. It has broad development space and good development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 These are the screening results of antagonistic actinomycetes, where the upper figure is the initial screening result of antagonistic actinomycetes, and the lower figure is the re-screening result of antagonistic actinomycetes.

[0019] Figure 2 It has a broad-spectrum antibacterial activity against actinomycetes CG-S15.

[0020] Figure 3 These are some pictures of biochemical experiments of actinomycetes CG-S15.

[0021] Figure 4 Phylogenetic tree of actinomycete CG-S15 and other homologous strains retrieved from GenBank database based on 16S rRNA sequences. Bootstrap values ​​(%) on nodes were calculated from 1000 replicates.

[0022] Figure 5 ANI and DDH values ​​of the genomes of actinomycetes CG-S15 and five similar standard strains

[0023] Figure 6 The biocontrol and growth-promoting effects of actinomycete CG-S15 on potted banana seedlings.

[0024] Actinomycetes CG-S15 is named as: Streptomyces coffeae , deposited in Guangdong Microbiological Culture Collection Center, with the deposit number GDMCC No: 64532, the deposit date is April 22, 2024, and the deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. DETAILED DESCRIPTION

[0025] The present invention will be further described below with reference to the accompanying drawings in conjunction with specific embodiments to better understand the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. Where the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be obtained commercially.

[0026] 1 Experimental Materials

[0027] 1.1 Soil samples

[0028] The root soil of medicinal plants is a potential resource library, containing a large number of active microorganisms and a high richness of beneficial bacteria. Therefore, this experiment uses the root soil of the medicinal plant Melon fasciata for research. Calotropis gigantea L .Dryander ) Rhizosphere soil samples were collected from the Tropical Crop Genetic Resources Research and Experimental Base of the Institute of Tropical Crop Genetic Resources, Chinese Academy of Tropical Agricultural Sciences. After the collected rhizosphere soil samples were transported to the laboratory, they were cut into small pieces and transferred to a 15 mL tube containing 10 mL of sterile PBS buffer. The tube was shaken at 180 r / min for 20 min to remove the soil on the root surface. Actinomycetes were isolated using rhizosphere soil samples.

[0029] 1.2 Test culture medium

[0030] The main test culture media required for this test include culture media for isolating actinomycetes, culture media for morphological observation and culture media for physiological and biochemical characteristics. See Table 1 for details.

[0031] Table 1 Culture medium and its formula for observation of culture characteristics

[0032] Media Compositions (g / L) Yeast extract malt extract medium (ISP2 or YE) Yeast extract 4 g, malt extract 10 g, glucose 4 g, distilled water 1000 mL, agar 20 g, pH 7.3 Oatmeal agar medium (ISP3) Oatmeal 20 g (boil in 1000 mL water for 20 min, filter and make up to 1000 mL), trace salt solution 1 mL, agar 20 g, pH 7.2 Inorganic salt starch agar medium (ISP4) <![CDATA[10 g of soluble starch, 1 g of K2HPO4, 1 g of MgSO4·7H2O, 1 g of NaCl, 2 g of (NH4)2SO4, 2 g of CaCO3, 1 mL of trace salt solution, 1000 mL of distilled water, 20 g of agar, pH 7.0 - 7.4]]> Glycerol-asparagine medium (ISP5) <![CDATA[1 g of asparagine, 10 g of glycerol, 1 g of K2HPO 4 1 g, 1 mL of trace salt solution, 1000 mL of distilled water, 20 g of agar, pH 7.0 - 7.4 <!-- 3 -->]]> Peptone-yeast extract agar medium (ISP6) <![CDATA[15 g of peptone, 5 g of peptone shown, 0.5 g of ammonium ferric citrate, 1 g of K2HPO4, 0.08 g of sodium thiosulfate, 1 g of yeast extract, 1000 mL of distilled water, 20 g of agar, pH 7.0 - 7.4]]> Tyrosine agar medium (ISP7) <![CDATA[15 g of glycerol, 0.5 g of tyrosine, 1 g of asparagine, 0.5 g of K2HPO4, 0.5 g of MgSO4·7H2O, 0.5 g of NaCl, 0.01 g of FeSO4·7H2O, 1 mL of trace salt solution, 1000 mL of distilled water, 20 g of agar, pH 7.2 - 7.4]]> Potato Dextrose Agar (PDA) 200 g potatoes, 20 g glucose, 17-20 g agar, 1000 mL distilled water, pH 7.0 Urease medium <![CDATA[Peptone 1 g, NaCl 0.5 g, Glucose 1 g, KH2PO4 2 g, Phenol Red 0.012 g, Agar 20 g, Distilled water 1000 mL, pH 6.8 - 6.9]]> Gelatin liquefaction medium 5 g peptone, 3 g beef extract, 1000 mL distilled water, pH 7.0 Nitrate reduction medium <![CDATA[0.5 g of MgSO4·7H2O, 0.5 g of NaCl, 0.5 g of K2HPO4, 1 g of KNO3, 20 g of sucrose, 1000 mL of distilled water, pH 7.2]]> Purdue basal medium <![CDATA[(NH4)2HPO4 1 g, NaCl 1 g, MgSO4•7H2O 0.2 g, KH2PO4 0.5 g, agar 20 g, distilled water 1000 mL, pH 7.0 - 7.2]]> Nitrogen Utilization Basal Medium <![CDATA[10 g of D-glucose, 5 g of MgSO4•7H2O, 0.5 g of NaCl, 0.01 g of FeSO4•7H2O, 0.1 g of K2HPO4, 1000 mL of distilled water, pH 7.2]]> Hydrogen sulfide production medium Peptone 10 g, ferric citrate 0.5 g, distilled water 1000 mL, pH 7.2 Soybean powder fermentation medium Soluble starch 20 g, soybean powder 15 g, yeast powder 5 g, peptone 2 g, CaCO3 4 g, NaCl 4 g, distilled water 1000 mL, pH: 7.0

[0033] Note: Trace salt solution (FeSO4·7H2O 0.1 g, ZnSO4·7H2O 0.1 g, MnCl2·4H2O 0.1 g, distilled water 100 mL)

[0034] 1.3 Instruments and equipment used in the test

[0035] Table 2 Instruments and equipment

[0036] Name Model Manufacturer Biochemical incubator ZXSD-B1270 Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd. pH meter Delta 320 Merrler-Toledo Instruments Constant temperature water bath HHS-11-2 Hangzhou Huier Instrument Equipment Co., Ltd. High Pressure Steam Sterilizer MJ-54A Shidukai Instrument Equipment Co., Ltd. PCR Amplifier Biometra TAdvanced96G Hainan Junyu Technology Co., Ltd. Gel imaging analysis system UVP EC3 UVP, USA Horizontal electrophoresis apparatus HR1022 Beijing Hengrui Tianchuang Mechanical and Electrical Equipment Co., Ltd. Vertical Shaker BSD-YX2200 Shanghai Boxun Industrial Co., Ltd. Clean bench SW-CJ-2FD Suzhou Purification Equipment Co., Ltd. Electronic balance ME104E Mettler-Toledo International Trading Co., Ltd. Benchtop centrifuge 5430R Hainan Junyu Technology Co., Ltd. Medical low temperature refrigerator MDF-382 Panasonic Cold Chain (Dalian) Co., Ltd.

[0037] 1.4 Test pathogens

[0038] Banana Fusarium wilt race 4 F. oxysporum f. sp. cubenseRace 4 (ATCC 76255), Colletotrichum gloeosporioides Colletotrichum gloeosporioides (ATCC 58222), Fusarium wilt of cucumber Fusarium oxysporum sp. Cucumebrium (ACCC 30220), banana anthracnose Colletotrichum musae (ATCC96167), Colletrichum litchi Trag, Colletrichum mango Colletotrichum acutatum (ATCC 56815), Colletotrichum spp. Colletotrichum fragariae (ATCC 58718), Alternaria musae, Pyricularia oryae Cav, Curvularia lunata, Pestalogiopsis sp. The above strains are preserved in the Microbial Resources Laboratory of the Institute of Tropical Biotechnology, Chinese Academy of Tropical Agricultural Sciences.

[0039] 1.5 Main Reagents

[0040] Table 3 Main biochemical reagents and their sources

[0041] Name of the reagent Manufacturer Bacterial Genomic DNA Rapid Extraction Kit Hangzhou Microbiological Reagent Co., Ltd. Antibiotic sensitive filter paper Wuxi Biotech Biotechnology Co., Ltd. 2×Taq PCR MasterMix Biomed Biopharm DNA marker Beijing Kangwei Century Biotechnology Co., Ltd. Agarose Promega CO. Ltd

[0042] 1.6 Data Analysis Software

[0043] The analysis software used in this experiment are mainly Excel2022, Diamond, blast2go, etc.

[0044] 2 Experimental methods and results

[0045] 2.1 Isolation of actinomycetes

[0046] The rhizosphere soil samples of medicinal plants were collected by five-point sampling method. The soil samples were brought back to the laboratory and placed in a fume hood to air dry naturally. After air drying, the soil samples were fully mixed and ground, and sieved using a 60-mesh sieve. 5g of the sieved soil sample was taken out, dissolved in 45ml of sterile water, and placed in a 55°C water bath for 20min. Then it was placed in a shaker at 28°C and 180r / min for 1h. The treated soil liquid was diluted with sterile water to 10 -1 , 10 -2 , 10 - 3100 μg of soil suspension of different concentrations was spread on SCA medium and cultured in an incubator at 28°C for 7 days. The same concentration gradient was repeated 3 times. After 7 days, colonies of different shapes and colors were picked out from the culture dish with a sterile needle and inoculated into YE culture medium by streaking method, and then purified again. A total of 49 actinomycetes were isolated.

[0047] 2.2 Screening of actinomycetes

[0048] (1) Initial screening of actinomycetes

[0049] The isolated and purified strains were screened for antagonism to banana wilt pathogen Foc TR4 was used as the target bacteria, and the antibacterial activity of actinomycetes was determined by the plate confrontation method. Specifically, a dot was drawn in the center of the culture dish, and the isolated and purified actinomycetes were inoculated on four PDA culture media at a distance of 2.5 cm from the center, and cultured in a 28°C incubator for 2 days. A 5mm diameter puncher was used to take out the bacterial cakes from the mycelium of the Foc TR4 pathogen with uniform growth. Subsequently, these bacterial cakes were accurately inoculated in the center of each PDA culture medium. As a control, the Foc TR4 bacterial cake was inoculated only in the central position of the PDA culture medium. In order to ensure the reliability of the experimental results, each group of experiments was repeated three times. After the inoculation, the culture medium was placed in an incubator with a constant temperature of 28°C, cultured for 7 days, and the effect of antagonism was observed. Using a ruler, the diameter of the strain was accurately measured by the cross method, and the inhibition rate was accurately calculated according to the formula to evaluate the experimental effect.

[0050] Inhibition rate = [(colony diameter of control group - colony diameter of treatment group) / colony diameter of control group] × 100%

[0051] (2) Rescreening of actinomycetes

[0052] The strain obtained from the preliminary screening was inoculated into the sterilized soybean powder fermentation medium, and cultured on a shaker at 28° C. and 180 r / min for 7 days to obtain a fermentation liquid.

[0053] The activity of the strain was tested by agar diffusion method. A 0.5 cm Foc The TR4 bacterial cake was perforated at 4 positions 2.5 cm from the center of the plate (aperture diameter was 0.5 cm), and 100 μL of filtrate filtered by a sterile filter membrane was added to the holes; only pathogens were inoculated in the control plate. After inoculation, the plate was placed in an incubator at 28°C for 7 days. Then, the diameter of the obtained strain was measured using a ruler according to the cross method. Finally, the antibacterial rate was calculated according to the calculation formula given in "(1) Initial screening of actinomycetes" to ensure the accuracy and reliability of the experimental results.

[0054] by Foc TR4 was used as the target strain, and the plate confrontation method was used to identify the resistance of 49 strains. After observation and calculation, the actinomycete CG-S15 had the highest antibacterial activity, with an antibacterial rate of 52%, which was significantly higher than that of other strains ( Figure 1 The strain was then selected for rescreening of the filtrate of actinomycete fermentation broth. The inhibition rate of the filtrate of actinomycete CG-S15 was 46.56±0.52% ( Figure 1 ).

[0055] 2.3 Determination of broad-spectrum antibacterial activity of actinomycete CG-S15

[0056] In order to evaluate the inhibitory activity of actinomycetes against various common plant pathogens, ten common pathogens including Colletotrichum gloeosporioides (Penz) Saec, Fusarium oxysporum (Schl.) F.sp cucumerinum Owen.anamorph, Colletotrichum gloeosporioides, Colletotrichum litchi Trag, Colletotrichum acutatum, Colletotrichum fragariae, Alternaria musae, Pyricularia oryae Cav, Curvularia lunata and Pestalogiopsis sp. were selected as reference strains, and the broad-spectrum antibacterial activity was determined by the plate confrontation method.

[0057] Specifically, a dot was drawn in the center of the culture dish, and the fermentation liquid of actinomycetes CG-S15 was inoculated onto four PDA culture media at a distance of 2.5 cm from the center, and cultured in a 28°C incubator for 2 days. A 5mm diameter punch was used to take out the bacterial cakes from the mycelium of each pathogen with uniform growth. Subsequently, these bacterial cakes were precisely inoculated in the center of each PDA culture medium. As a control, each pathogenic bacterial cake was inoculated only in the central position of the PDA culture medium. In order to ensure the reliability of the experimental results, each group of experiments was repeated three times. After the inoculation was completed, the culture medium was placed in an incubator at a constant temperature of 28°C, cultured for 7 days, and the effect of antagonism was observed.

[0058] The method for preparing the fermentation liquid of actinomycetes CG-S15 is as follows: the actinomycetes CG-S15 is inoculated into the sterilized soybean powder fermentation medium, and the medium is cultured on a shaker at 28° C. and 180 r / min for 7 days to obtain the fermentation liquid.

[0059] The test results are as follows Figure 2 As shown in the results, the fermentation liquid of actinomycetes CG-S15 showed strong antagonism to these ten plant pathogenic fungi, and the antibacterial effect was very good. Among these ten plant pathogens, the best inhibitory activity was observed against banana anthracnose (Colletotrichum gloeosporioides), with an inhibition rate of 82%, and the inhibition rate against other pathogens reached more than 51%. This result shows that actinomycetes CG-S15 has an inhibitory effect on the mycelial growth of other pathogens and has a broad-spectrum antibacterial property.

[0060] 2.4 Antibiotic sensitivity test of actinomycete CG-S15

[0061] Prepare YE solid and liquid culture medium. Use liquid culture medium to culture a spore suspension of actinomycetes CG-S15. The spore suspension is prepared by inoculating the strain into the liquid culture medium, setting the shaker speed to 180r / min, and culturing at a constant 28°C for 7 days. Take 100μL of the diluted spore suspension and evenly spread it on the surface of the YE solid culture medium. Use a spreader to ensure uniform distribution, and place a filter paper containing a specific antibiotic on the coated culture medium. To ensure the reliability of the experimental results, each experiment was repeated three times. The culture medium was placed in an incubator at 28°C and continued to be cultured for 7 days. Observe and count whether a halo phenomenon appears around the antibiotic filter paper. If a clear halo is observed, it indicates that the antibiotic has a significant inhibitory effect on the strain.

[0062] The test results are shown in Table 4. Among the 30 antibiotics tested, actinomycetes CG-S15 showed resistance to 11 antibiotic tablets including imipenem, levofloxacin, and clindamycin.

[0063] Table 4 Antibiotic sensitivity test

[0064] Number Types of antibiotic Content (ug / slice) Result 1 Florfenicol 300 S 2 Doxycycline 30 S 3 Imipenem 10 R 4 Levofloxacin 5 R 5 Clindamycin 2 R 6 Chloramphenicol 30 R 7 Co-trimoxazole 25 S 8 Polymyxin B 300 (IU / tablet) S 9 Vancomycin 30 S 10 Lincomycin 2 S 11 Ciprofloxacin 5 R 12 Norfloxacin 10 R 13 Azithromycin 15 S 14 Erythromycin 15 S 15 Minocycline 30 S 16 tetracycline 30 R 17 Streptomycin 10 R 18 Kanamycin 30 S 19 Gentamycin 10 S 20 Amikacin 30 R 21 Cefoperazone 75 S 22 Ceftriaxone 30 R 23 Ceftazidime 30 S 24 Cefuroxime Sodium 30 S 25 Cefazolin 30 S 26 Cephalexin 30 R 27 Piperacillin 100 S 28 Ampicillin 100 S 29 Oxacillin 1 S 30 penicillin 10 S

[0065] Note: “S” stands for sensitive; “R” stands for resistant.

[0066] 2.5 Classification and identification of actinomycete CG-S15

[0067] 2.5.1 Cultivation characteristics of actinomycete CG-S15

[0068] In this experiment, different culture media such as ISP2, ISP4, ISP5, ISP6, ISP7 and PDA need to be prepared. Actinomycetes CG-S15 was inoculated into each culture medium and cultured at 28°C for 7 days. The growth of actinomycetes CG-S15 on different culture media was observed, including the morphological characteristics of single colonies, the color of aerial hyphae, the color of intrabasal hyphae, the growth state and the production of soluble pigments, and the colony colors were compared and determined using the ISCC-NBS color atlas.

[0069] As shown in Table 5, the actinomycetes CG-S15 was cultured in six different culture media, and its normal growth was observed. Among them, the growth of the strain on ISP2, ISP4 and ISP5 culture media was good. Except for ISP6 culture medium, CG-S15 produced purple soluble pigments in the other five culture media; in addition, the aerial hyphae of CG-S15 were mainly white and purple, while the basal hyphae were mainly purple and dark purple; in ISP6 culture medium, the colonies of CG-S15 showed brown-yellow characteristics. Most of the single colonies on the culture medium were round, and all colonies had a hard surface.

[0070] Table 5 Growth morphology of actinomycetes CG-S15 in different culture media

[0071] Culture medium Aerial hyphae Mycelium in the base Soluble pigments Growth status ISP2 Dark Purple Dark Purple Dark Purple +++ ISP4 White Purple Purple +++ ISP5 Purple Purple Purple +++ ISP6 White Brown none ++ ISP7 Purple Purple Purple ++ PDA White Purple Purple ++

[0072] Note: “+++” indicates good growth on the culture medium; “++” indicates average growth on the culture medium; “+” indicates weak growth on the culture medium.

[0073] 2.5.2 Physiological and biochemical characteristics of actinomycete CG-S15

[0074] 1. Growth characteristics

[0075] Streak inoculation was performed on YE solid medium and cultured at 30°C for 24 h before observing the colony morphology, including color and texture.

[0076] 2. Carbon source utilization experiment

[0077] In the process of actinomycete identification, one of the most important evaluation criteria is the utilization of carbon sources. Actinomycetes have different abilities to utilize carbon sources such as sugars, alcohols, acids and fatty acids. For this reason, this study used Pugo's basal medium as the basic medium and replaced the basic medium with various carbon sources. The carbon sources selected in this experiment include sucrose, xyloside, D-fructose, soluble starch, alkaline lignin, D-sorbitol, raffinose, D-mannitol, D-trehalose, inositol, mannose, D-galactose, glucose, salicin, dextrin, melezitose, rhamnose, D-mannitol, and L-arabinose. This experiment used a variety of carbon sources for cultivation, and set up a basic medium without carbon source as a blank control. After 7 days of constant temperature cultivation at 28°C, it was cultured with different carbon sources to observe its growth status. If a certain strain can grow under a specific carbon source and grow well, it means that the strain can utilize this carbon source; if it cannot grow, it means that the strain cannot effectively utilize the carbon source.

[0078] 3. Nitrogen source utilization experiment

[0079] A variety of single nitrogen sources were added to the basic culture medium, including phenylalanine, N-CK, arginine, cysteine, methionine, histidine, ascorbic acid, potassium nitrate, ammonium nitrate, anhydrous creatine, L-tyrosine, L-valine, tryptophan, L-glutamic acid, acid hydrolyzed white, urea, glycine, L-asparagine, L-methionine, and C-hydroxyproline. In order to explore the effect of different carbon sources on the growth of antagonistic strains, this experiment used a variety of carbon sources for cultivation, and set up a basic culture medium without carbon source as a blank control. The actinomycetes were inoculated in the culture medium and cultured at a constant temperature of 28°C for 7 days. The growth of actinomycetes CG-S15 was observed and recorded, and the statistical method was the same as that of carbon source utilization.

[0080] 4. Enzyme properties experiment

[0081] (1) Starch hydrolysis experiment

[0082] Inoculate actinomycetes into a starch-containing culture medium and place it in a 28°C incubator for 7 days. Stain the colonies with iodine solution. If there are clear transparent circles around the colonies, they are positive bacteria.

[0083] (2) Urease test

[0084] The strain was inoculated into urease medium by streak inoculation method, and a urease medium without strain was set up as control. If the inoculated medium showed pink color, it indicated that the strain had urease activity and could be judged as positive.

[0085] (3) Nitrate reduction

[0086] The strain was inoculated into a nitrate reduction liquid culture medium test tube and cultured at 28°C for 7 days. The nitrate reduction medium without the strain was set as the control group. After 7 days, 1 mL of A solution, 1 mL of B solution, and 1 mL of bacterial solution were added to the test tube respectively. The mixed liquid was allowed to stand for 30 minutes. If the mixed liquid was pink, it was positive, otherwise it was negative.

[0087] 5. pH tolerance test

[0088] Prepare liquid culture media with pH values ​​of 4, 5, 6, 7, 8, 9 and 10, and sterilize them. Inoculate the strain to be tested in each liquid culture medium. Place the culture medium on a shaker at 28°C and 180r / min for 14 days. Observe and count the growth of actinomycetes CG-S15 under different pH conditions to determine the optimal pH culture conditions. The experiment will be repeated three times.

[0089] 6.NaCl tolerance test

[0090] In order to explore the tolerance of actinomycetes CG-S15 to NaCl, liquid culture media containing different NaCl concentrations (1%, 3%, 5%, 7%, 9%, 11%, 13%) were prepared to ensure that other nutritional conditions were consistent. The strains were inoculated into the culture media and placed on a shaker at 28°C and 180r / min for 14 days. After the culture, the growth of the strains was observed and their growth ability at different NaCl concentrations was recorded to determine the upper and lower limits of the tolerance of actinomycetes CG-S15 to NaCl.

[0091] The growth characteristics and enzymatic characteristics of actinomycetes CG-S15 are shown in Table 6. Actinomycetes CG-S15 showed good growth ability on YE medium, its aerial hyphae showed dark purple, the base hyphae showed purple, and the spore surface was hard. In terms of physiological experiments, the pH tolerance range of actinomycetes CG-S15 was 6.0-8.0, and the most suitable pH for the growth of actinomycetes CG-S15 was 7.0; the salt concentration of the culture medium was not more than 5%, and the most suitable salt concentration for the growth of the strain was 1%; it could produce starch hydrolase and urease; nitrate reduction was positive.

[0092] In the carbon source utilization test, all 19 carbon sources involved can be utilized by actinomycetes CG-S15, indicating that the bacteria can provide carbon sources through a variety of common carbohydrates or alcohols; after 4 types (dextrin, soluble starch, D-sorbitol, D-mannitol) replaced the original carbon sources in the culture medium, the CG-S15 strain grew well; 5 types grew generally; 3 types showed weak growth; and it did not grow on the alkaline lignin carbon source. In nitrogen source utilization, actinomycetes CG-S15 grew generally in potassium nitrate nitrogen source; grew weakly in 3 nitrogen sources; and did not grow in 8 nitrogen sources. The results are shown in Table 7.

[0093] Table 6 Physiological and biochemical characteristics of actinomycete CG-S15

[0094] feature result morphology Aerial hyphae color Dark Purple Aerial hyphae morphology hard Mycelium color Purple Biochemical experiments Urease test + Starch hydrolysis + Nitrate reduction + Physiological experiments pH tolerance test 6-8 (7 is the best) NaCl tolerance test (%) 1-5 (1 is the best)

[0095] Note: “+” indicates a positive reaction; “-” indicates a negative reaction.

[0096] Table 7 Carbon and nitrogen source utilization by actinomycete CG-S15

[0097] Carbon source utilization result Nitrogen source utilization result C-CK ++ Phenylalanine - sucrose ++ N-CK - Chestnut candy ++ Arginine + D-Xylose ++ Cysteine - D-Fructose + Methionine + dextrin +++ Histidine + Soluble starch +++ ascorbic acid - Alkaline lignin - Potassium Nitrate ++ D-Galactose + Ammonium sulfate - D-Sorbitol +++ Creatine Anhydrous - Raffinose + L-Tyrosine - D-Mannitol +++ L-Valine - D-Trehalose ++ Tryptophan + Inositol ++ Acid hydrolysis white + Mannose ++ Urea - glucose +++ Glycine ++ Salicin - L-Methionine - Melezitose ++ L-Glutamic Acid - Rhamnose ++ C-Hydroxyproline + D-Mannitol ++ L-Asparagine +++ L-arabinose ++ Tyrosine -

[0098] Note: “+++” means good growth, “++” means average growth, “+” means weak growth, and “-” means no growth.

[0099] 2.5.3 Molecular identification of actinomycetes

[0100] (1) Construction of phylogenetic tree based on 16S rRNA sequence

[0101] The bacterial genome rapid DNA extraction kit was used to extract total actinomycete DNA.

[0102] PCR amplification of DNA from actinomycetes was performed using bacterial universal primers, upstream primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and downstream primer 1492R (5'-GGTTACCTTGTTACGACTT-3'). 50 μL PCR reaction system.

[0103] Table 8 PCR reaction system

[0104] Reaction system Volume Actinomycete DNA 1 μL 2×TapPCR MasterMix 12.5 μL 27 F 0.5 μL 1492 R 0.5 μL <![CDATA[ddH2O]]> 10.5 μL Total volume 25 μL

[0105] The PCR products obtained above were sent to Shanghai Shenggong Biotechnology Co., Ltd. for 16S rRNA sequencing. After sequencing, primer was used for sequence splicing, and the 16S rRNA gene sequences with high similarity were compared and searched in the EzBiocloud database. The strains were selected for multiple comparative analysis, and the MEGA7.0 software was used to build the tree. The gene sequences with high similarity were selected to construct the phylogenetic tree using the neighbor-joining method. The results are shown in Figure 4 As shown, actinomycete CG-S15 and strain Streptomyces coffeae CA1R205(MN116550) The strains gathered into one branch with a branching rate of 71%, and were preliminarily identified as belonging to the genus Streptomyces.

[0106] (4) Genome comparison of actinomycete CG-S15 and its closely related species

[0107] The genome data of related strains were downloaded from NCBI or EzBicloud, and the genomes of related strains and the genome of actinomycete CG-S15 were uploaded to EzBicloud for average nucleotide identity (ANI) calculation and uploaded to (https: / / ggdc.mz.de) for DNA-DNA hybridization (DDH) calculation.

[0108] The results showed that actinomycete CG-S15 and its closely related species Streptomyces caniferus, Streptomyces libani subsp.libani、Streptomyces platensis、Streptomyces hygroscopicus、 Streptomyces iranensis The ANI values ​​of the strains were 81.23%, 81.2%, 81.19%, 84.86% and 85.41%, all below the 95% threshold for new species, and the DDH values ​​were 23.1%, 22.7%, 22.8%, 26.4% and 27.2%, all below the 70% threshold for new species. Therefore, the actinomycete CG-S15 was identified as a new species of Streptomyces ( Figure 5 ). Actinomycetes CG-S15 was named as: Streptomyces coffeae , hereinafter also referred to as Streptomyces CG-S15, is deposited in Guangdong Microbiological Culture Collection Center, with the deposit number GDMCC No: 64532, the deposit date is April 22, 2024, and the deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0109] 2.6 Control effect of Streptomyces CG-S15 on banana seedling wilt

[0110] The pot experiment was conducted at the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences from June to August 2020. The plants used in this study were propagated by in vitro micropropagation procedures, and banana seedlings with uniform growth, 3-4 leaves and healthy roots were inoculated with GFP-Foc TR4 (Foc TR4 with green fluorescence). Healthy soil from banana orchards collected from Danzhou City, Hainan Province was sieved through 20 mesh for later use.

[0111] The root injury method was used to cut off part of the roots of the banana seedlings, and then the treated seedlings were planted in a plastic pot filled with 1 kg of soil. After the transplant survived, the control effect test was carried out. The test had two treatments: treatment group (inoculated with GFP-Foc TR4: 1.0×10 5 cfu / g soil, and inoculated with Streptomyces CG-S15, 1.0×10 7 cfu / g soil); control group (inoculated with GFP-Foc TR4 and sterile soybean powder fermentation medium).

[0112] GFP-Foc TR4 inoculation: freshly cultured GFP-Foc TR4 mycelium was inoculated on PDA solid medium and cultured at 28°C for 5 days. The spores were washed with sterile water and filtered through two layers of sterile Mira cloth to collect the pathogen spore suspension. After counting with a hemocytometer, the spore suspension was diluted with sterile water and 100 ml of the spore suspension was inoculated into the soil so that the number of GFP-Foc TR4 spores in the soil was 1.0×10 5 cfu / g soil.

[0113] Inoculation of Streptomyces in the treatment group: 5% of the freshly cultured Streptomyces CG-S15 seed solution was inoculated into the soybean powder fermentation medium and cultured at 28°C with shaking (150 rpm) for 7 days. After counting by dilution plate spreading method, the solution was diluted with sterile water and 100 ml of the fermentation solution of Streptomyces CG-S15 was inoculated into the soil to make the number of Streptomyces CG-S15 in the soil 1.0×10 7 cfu / g soil.

[0114] Banana seedlings were placed in a greenhouse with a relative humidity of about 70% and a temperature of 28°C. Ten seedlings were selected for each treatment. According to the banana seedling growth characteristics measurement method in Chen Zhijie's 2018 master's thesis "Construction of soil functional microbial communities based on disease prevention and growth promotion and optimization of culture conditions", the physiological and ecological characteristics of banana seedlings in each experimental group were measured 30 days after inoculation, including stem diameter, leaf area, plant height, leaf thickness and fresh weight. Figure 6 shown.

[0115] The disease incidence rate of the control group was 73.33% after 30 days, and the disease was serious, with obvious blackening of the bulbs; however, after the banana seedlings were treated with the fermentation liquid of Streptomyces CG-S15, the disease symptoms were significantly suppressed. Compared with the control group, the disease index was reduced to 33.33% after 30 days of treatment with the fermentation liquid of Streptomyces CG-S15, and the control effect on banana wilt reached 54.55%, indicating that Streptomyces CG-S15 has a good control effect on banana wilt and can inhibit the infection of banana wilt pathogens.

[0116] After 30 days of transplanting, the height, stem diameter, leaf thickness, leaf area and fresh weight of banana seedlings treated with Streptomyces CG-S15 were 11.06 cm, 10.24 mm, 0.29 mm, 70.15 cm, respectively. 2 and 18.38 g, which were significantly higher than those of the control group. The results showed that Streptomyces CG-S15 had a certain growth-promoting effect on banana seedlings.

[0117] Table 9 Growth-promoting effect of Streptomyces CG-S15 on banana seedlings

[0118] Treatment Plant height (cm) Stem diameter (mm) Blade thickness (mm) <![CDATA[Leaf area (cm 2 ).]]> Fresh weight (g) Treatment Group 11.06 10.24 0.29 70.15 18.38 Control group 8.47 9.06 0.23 55.72 13.15

[0119] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modification and substitution of the practical is also within the scope of the present invention. Therefore, the equalization transformation and modification made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. A Streptomyces, characterized in that Name: Streptomyces Streptomyces coffeae CG-S15 is deposited in Guangdong Microbiological Culture Collection Center with the deposit number GDMCC No: 64532.

2. The fermentation broth of Streptomyces as claimed in claim 1.

3. A preparation, characterized in that Contains the Streptomyces according to claim 1 or the fermentation liquid according to claim 2.

4. The Streptomyces according to claim 1, or the fermentation liquid according to claim 2, or the preparation according to claim 3 is used in the preparation of a compound antagonistic to banana wilt race 4 ( F. oxysporum f. sp. cubense Race 4) preparations.

5. The Streptomyces according to claim 1, or the fermentation liquid according to claim 2, or the preparation according to claim 3 is used in the preparation of the fourth physiological subspecies ( F. oxysporum f. sp. cubense Race 4) in the application of preparations for diseases caused by.

6. The Streptomyces according to claim 1, or the fermentation liquid according to claim 2, or the preparation according to claim 3 in the preparation of an antagonistic agent against Colletotrichum gloeosporioides ( Colletotrichum gloeosporioides ), and / or Fusarium wilt ( Fusarium oxysporum sp. Cucumebrium ), and / or Colletotrichum spp. ( Colletotrichum musae ), and / or Colletrichum litchi Trag, and / or Colletrichum mangoe Colletotrichum acutatum , and / or Colletotrichum spp. ( Colletotrichum fragariae ), and / or banana leaf blight pathogen (Alternaria musae), and / or rice blast pathogen (Pyricularia oryae Cav), and / or banana gray spot pathogen (Curvularia lunata), and / or mango leaf blight pathogen (Pestalogiopsis sp.) in a preparation.

7. The Streptomyces according to claim 1, or the fermentation liquid according to claim 2, or the preparation according to claim 3 in the preparation of a method for preventing and treating gloeosporium anthracnose ( Colletotrichum gloeosporioides ), and / or Fusarium wilt ( Fusarium oxysporum sp. Cucumebrium ), and / or Colletotrichum spp. ( Colletotrichum musae ), and / or Colletrichum litchi Trag, and / or Colletrichum mangoe Colletotrichum acutatum , and / or Colletotrichum spp. ( Colletotrichum fragariae ), and / or banana leaf blight pathogen (Alternaria musae), and / or rice blast pathogen (Pyricularia oryae Cav), and / or banana gray spot pathogen (Curvularia lunata), and / or mango leaf blight pathogen (Pestalogiopsis sp.) in preparations for treating diseases caused by.

8. Application of Streptomyces as claimed in claim 1, or the fermented liquid as claimed in claim 2, or the preparation as claimed in claim 3 in promoting the growth of banana seedlings.

9. Application of Streptomyces as claimed in claim 1, or the fermented liquid as claimed in claim 2, or the preparation as claimed in claim 3 in increasing the plant height, and / or stem thickness, and / or leaf thickness, and / or leaf area, and / or fresh weight of banana seedlings.

10. Use of the Streptomyces according to claim 1, or the fermentation broth according to claim 2, or the preparation according to claim 3 in preparing a bacterial agent resistant to imipenem, and / or levofloxacin, and / or clindamycin, and / or chloramphenicol, and / or ciprofloxacin, and / or norfloxacin, and / or tetracycline, and / or streptomycin, and / or amikacin, and / or ceftriaxone, and / or cephalexin.

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