Bull Streptomyces 11-2-1 and its application

Abstract

The present invention relates to the field of microbial technology, and specifically discloses a bull Streptomyces 11-2-1 and its application. The bull Streptomyces 11-2-1 (CGMCC No. 32268) provided by the present invention is a natural strain isolated from the rhizosphere soil of a wheat field. It is safe and environmentally friendly, can effectively inhibit the growth of ginger rot pathogens, and does not affect the normal growth of ginger. The test results show that the ginger rot pathogen can be observed to be inhibited after 24 hours of cultivation using the inhibition zone method. The inhibition zone is more obvious after 48 hours of cultivation, and the diameter of the inhibition zone can reach 35 mm. The bull Streptomyces 11-2-1 provided by the present invention provides a reference for the later research of natural strains, and is of great value to the development of microbial agents for preventing and treating ginger rot.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, in particular to a bull Streptomyces 11-2-1 and an application thereof. Background Art

[0002] Ginger ( Zingiber icinale Roscoe) is a plant of the genus Zingiberaceae, Zingiberaceae, mainly distributed in central, southeastern, and southwestern China, as well as in the subtropical regions. Ginger rot is a major bacterial disease in ginger production, severely affecting the yield and quality of ginger and causing enormous economic losses to ginger production. Chemical agents are generally used to control ginger rot in production, but chemical agents pollute the environment, and long-term abuse can lead to resistance to chemical agents. Biological control has the advantages of being environmentally friendly, safe, and effective, but there are relatively few microbial agents used to control ginger rot in production. Therefore, it is important to obtain biocontrol strains that can effectively control ginger rot. Summary of the Invention

[0003] In response to the above problems, the present invention provides a bull Streptomyces 11-2-1 and its application. The bull Streptomyces 11-2-1 can effectively antagonize ginger rot pathogens, has no effect on plant growth, and is harmless to the environment.

[0004] In order to solve the above technical problems, the technical solution provided by the present invention is:

[0005] In a first aspect, the present invention provides a bull Streptomyces ( Streptomyces tauricus )11-2-1, its deposit number is CGMCC No. 32268.

[0006] Compared with the existing technology, the bull Streptomyces 11-2-1 provided by the present invention is a natural strain isolated from the rhizosphere soil of a wheat field. It belongs to the genus Streptomyces and is an antagonistic bacterium from the soil. It can effectively prevent and control ginger rot disease and has no effect on plant growth. It has the ability to fix nitrogen and produce IAA, is harmless to the environment, and has the potential to be developed and applied as a new microbial strain resource for preventing and controlling ginger rot disease.

[0007] The Streptomyces bullii 11-2-1 strain provided by the present invention can effectively inhibit the growth of ginger rot pathogens without affecting the normal growth of ginger. Experimental results showed that inhibition of the ginger rot pathogen was observed after 24 hours of incubation using the inhibition zone method. The inhibition zone was more pronounced after 48 hours of incubation, with a diameter of up to 35 mm. The Streptomyces bullii 11-2-1 strain provided by the present invention provides a reference for future research and is of great value in the development of microbial agents for the prevention and treatment of ginger rot.

[0008] In a second aspect, the present invention provides a use of the bull Streptomyces 11-2-1 in inhibiting the growth of Enterobacter cloacae.

[0009] In a third aspect, the present invention provides a use of the bull Streptomyces 11-2-1 in preventing and treating ginger rot disease.

[0010] In a fourth aspect, the present invention provides a method for preparing a fermentation broth of Streptomyces bulls, comprising the following steps:

[0011] S1, adding the bull Streptomyces 11-2-1 to a culture medium plate for a first culture; then taking a bacterial cake and inoculating it into a culture medium for a second culture to obtain a seed solution;

[0012] S2, inoculating the seed liquid into the culture liquid, performing a third culture, and performing solid-liquid separation to obtain a fermentation liquid of Streptomyces bulls.

[0013] Preferably, in S1, the culture medium plate comprises a Gao's medium No. 1 plate.

[0014] Preferably, in S1, the temperature of the first culture is 28° C. to 32° C., and the culture time is 6 d to 8 d.

[0015] Preferably, in S1, the diameter of the mushroom cake is 6 mm to 9 mm.

[0016] Preferably, in S1, the number of the mushroom cakes is 2 to 4.

[0017] Preferably, in S1, the culture medium includes TSB culture medium.

[0018] Preferably, in S1, the volume of the culture solution is 90 mL to 110 mL.

[0019] Preferably, in S1, the second culture is a shaking culture with a temperature of 28°C to 32°C, a rotation speed of 180 rpm to 220 rpm, and a culture time of 2.5 d to 4 d.

[0020] Preferably, in S2, the culture medium includes TSB culture medium.

[0021] Preferably, in S2, the volume ratio of the seed solution to the culture solution is 1:(90~110).

[0022] Preferably, in S2, the third culture is shaking culture, with a temperature of 28°C to 32°C, a rotation speed of 180 rpm to 220 rpm, and a culture time of 4.5 d to 6 d.

[0023] Preferably, in S2, the solid-liquid separation comprises the following steps:

[0024] The mixture was centrifuged at 3500 rpm to 4500 rpm for 15 min to 25 min, and the supernatant was taken for bacterial filtration to obtain the fermentation liquid of Streptomyces bulls.

[0025] In a fifth aspect, the present invention provides a fermentation broth of Streptomyces bovis, which is prepared by the preparation method of the fermentation broth of Streptomyces bovis.

[0026] In a sixth aspect, the present invention provides an application of a fermentation broth of Streptomyces bulls in preventing and treating ginger rot disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a graph showing the inhibitory effect of strain 11-2-1 on Enterobacter cloacae in Example 1 of the present invention;

[0028] Figure 2 This is a morphological diagram of the aerial hyphae of strain 11-2-1 in Example 1 of the present invention;

[0029] Figure 3 This is a morphological diagram of the spores of strain 11-2-1 in Example 1 of the present invention;

[0030] Figure 4 Figures 1 and 2 show the morphology of strain 11-2-1 in Example 1 of the present invention grown on different culture media. In the figure, a represents Gao's medium No. 1, b represents PDA medium, c represents calcium malate agar medium, d represents oatmeal medium, e represents ammonium starch medium, f represents nutrient agar medium, and g represents Tianmensu medium.

[0031] Figure 5 The utilization of different carbon sources in the basal medium of the carbon source utilization test by the strain 11-2-1 in Example 1 of the present invention is shown; in the figure, a indicates that the carbon source is inositol, b indicates that the carbon source is xylose, c indicates that the carbon source is glucose, d indicates that the carbon source is lactose, e indicates that the carbon source is fructose, f indicates that the carbon source is galactose, g indicates that the carbon source is sucrose, and h indicates that the carbon source is D-mannitol;

[0032] Figure 6 This is the phylogenetic tree of Streptomyces bulls 11-2-1 in Example 1 of the present invention;

[0033] Figure 7 This is a graph showing the growth-promoting ability test results of Streptomyces bulls 11-2-1 in Example 4 of the present invention; in the graph, a indicates amylase production, b indicates protease production, c indicates nitrogen fixation, and d indicates IAA production. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0035] Culture medium used in the examples:

[0036] Gao's medium No. 1: soluble starch 20 g, potassium nitrate 1 g, dipotassium hydrogen phosphate 0.5 g, magnesium sulfate heptahydrate 0.5 g, sodium chloride 0.5 g, ferrous sulfate heptahydrate 0.01 g, potassium dichromate 0.1 g, agar 20 g, distilled water 1 L, pH = 7.

[0037] Potato dextrose medium (PDA medium): 200 g potatoes, 20 g glucose, 20 g agar, 1000 mL distilled water, pH = 7.0~7.4.

[0038] LB medium: 10 g of tryptone, 5 g of yeast powder, 10 g of sodium chloride, and 1000 mL of distilled water.

[0039] Calcium malate agar medium: agar 17 g, calcium malate 10 g, glycerol 10 g, NH4Cl 0.5 g, K2HPO4 0.5 g, distilled water 1000 mL, pH = 7.2-7.4.

[0040] Oatmeal culture medium: 30 g oats, 1000 mL distilled water.

[0041] Ammonium starch medium: soluble starch 10 g, (NH4)2SO4 2 g, MgSO4·7H2O 1 g, trace element solution 1 mL, distilled water 1000 mL, pH = 6.5~7.0.

[0042] Nutrient agar medium: peptone 10g, beef extract 5g, NaCl 5g, agar 20g.

[0043] Asparagine medium: 10 g glycerol, 1 g L-aspartic acid, 1 g K2HPO4, 1000 mL distilled water, 20 g agar powder, pH = 6.5~7.0.

[0044] Starch hydrolysis agar medium: agar 17g, soluble starch 10g, K2HPO4 0.3g, KNO3 1g, MgCO3 1g, NaCl 0.5g, distilled water 1000mL, pH=7.2~7.4.

[0045] Cellulose hydrolysis medium: NaCl 0.5 g, K2HPO4 0.5 g, MgSO4 0.5 g, KNO3 1 g, distilled water 1000 mL, pH = 7.2, filter paper strip 5 cm long and 0.8 cm wide.

[0046] Gelatin liquefaction medium: 5 g peptone, 200 g gelatin, 20 g glucose, 1000 mL distilled water, pH = 7.2~7.4.

[0047] Tresner medium: agar 10.0 g, peptone 10.0 g, ferric citrate 0.5 g, distilled water 1000 mL, pH = 7.2.

[0048] Tyrosine medium (melanin production): L-tyrosine 1.0 g, yeast extract 1.0 g, NaCl 8.5 g, agar 15 g, distilled water 1000 mL, pH = 7.2.

[0049] Milk coagulation peptone medium: CaCO3 0.02 g, skimmed fresh milk 1000 mL.

[0050] Carbon source utilization test basal culture medium: (NH4)2SO42.64g, KH2PO42.38g, K2HPO4·3H2O 5.65g, MgSO4·7H2O 1g, and each carbon source was added at 0.5wt% of fructose, sucrose, inositol, D-mannitol, lactose, galactose, glucose and xylose.

[0051] Nitrogen source utilization test basal culture medium: glucose 1 g, MgSO4·7H2O 0.05 g, NaCl 0.05 g, FeSO4·7H2O 0.001 g, K2HPO4 0.01 g, and urea, potassium nitrate, calcium nitrate, and glycine were added at 0.5 wt% of each nitrogen source.

[0052] Amylase culture medium: 5 g beef extract, 10 g peptone, 5 g sodium chloride, 18 g agar, 1 L ddH2O.

[0053] Protease Agar (PA) medium: 20 g skim milk, 15 g agar, 1 L ddH2O.

[0054] Ashburn medium: malic acid 5.0 g, KOH 4.5 g, K2HPO4 0.5 g, CaCO3·2H2O 0.02 g, NaCl 0.1 g, NaMoO4·2H2O 0.002 g, FeSO4·7H2O 0.002 g, agar 18 g, ddH2O 1 L.

[0055] The Enterobacter cloacae in the embodiment of the present invention ( Enterobacter cloacae ) was isolated from diseased ginger in a ginger-growing area in Tangshan City.

[0056] Unless otherwise specified, the raw materials, reagents, etc. used in the examples of the present invention were obtained from commercial sources.

[0057] In order to better illustrate the present invention, further examples are given below.

[0058] Example 1

[0059] This example is used to illustrate the acquisition, identification and preservation of Streptomyces bulls 11-2-1.

[0060] 1. Strain screening

[0061] Wheat rhizosphere soil (depth 0-15 cm, 2 mm around the roots) was collected from a wheat field in Botou, Cangzhou City, Hebei Province, air-dried at room temperature, and crushed. In a clean bench, 10 g of rhizosphere soil was added to 90 mL of sterile water, shaken for 1 min, and allowed to stand for 30 min. 1 mL of the supernatant was taken and diluted to 10% with sterile water according to the gradient dilution method. -2 , 10 -3 , 10 -4 and 10 -5 Take 0.2mL of each and spread it on Gao's medium No. 1 plate, and culture at 28℃ for 4~7 days. -4 A potassium dichromate solution (w / v) was added to inhibit the growth of fungi and other bacteria. Potassium dichromate was added when the culture medium cooled to 60°C-70°C. Mix thoroughly and pour onto plates. An appropriate dilution was selected to select single colonies of varying color and morphology for purification and culture. Purified Streptomyces strains were numbered and stored at 4°C. Using the plate inhibition zone method, strain 11-2-1 ( Figure 1 ).

[0062] 2. Identification of strains

[0063] 2.1 Morphological identification

[0064] The morphological characteristics of strain 11-2-1 on different culture media and the morphology of aerial hyphae and spores are as follows Figures 2-4 As shown in Table 1. The colonies of this strain are round, convex, light pink, with a circle of hairs on the outside, the spores are spiral, and the spores are cylindrical; the culture characteristics and growth conditions of the strain on different culture media are different.

[0065] Table 1 Culture characteristics of strain 11-2-1 on different culture media

[0066]

[0067] Note: + indicates that the plant can grow, ++ indicates that the plant is growing well, and +++ indicates that the plant is growing excellently.

[0068] 2.2 Physiological and biochemical identification

[0069] The strain 11-2-1 was cultured on different media to verify its physiological and biochemical characteristics and identify the carbon and nitrogen sources it utilizes. The results showed that the strain could hydrolyze starch but not cellulose, could liquefy gelatin, did not produce hydrogen sulfide, could produce melanin, and could peptone milk; it could utilize glucose, lactose, fructose, sucrose, D-mannitol, galactose, inositol, and xylose ( Figure 5 ); As nitrogen sources, urea, potassium nitrate, calcium nitrate and glycine can be used.

[0070] 2.3 Molecular Biological Identification

[0071] (1) Extraction of strain DNA: Using the boiling method, inoculate 1 mL of strain 11-2-1 seed solution into 100 mL of TSB culture medium and culture at 30°C with shaking for 48 h. Take 1000 μL and add it to a 1.5 mL centrifuge tube. Centrifuge at 4°C and 12,000 rpm for 10 min. Discard the supernatant, add 30 μL of ultrapure water, resuspend, boil in a boiling water bath for 10 min, centrifuge at 4°C and 12,000 rpm for 10 min, and aspirate the supernatant into a new centrifuge tube.

[0072] (2) PCR amplification, gel electrophoresis detection and sequencing:

[0073] ① PCR amplification, gel electrophoresis detection and sequencing of 16S rDNA

[0074] DNA extracted from strain 11-2-1 by the boiling method was used as the template, with 27F (5'-AGAGTTTGATCCTGGCTCAG-3') as the upstream primer (primer sequence shown in SEQ ID NO. 1) and 1492R (5'-GGTTACCTTGTTACGACTT-3') as the downstream primer (primer sequence shown in SEQ ID NO. 2). The PCR reaction system consisted of 12.5µL of Mix, 9.5µL of ddH2O, 1µL of DNA template, 1µL of the upstream primer, and 1µL of the downstream primer. DNA amplification was performed using a pre-denaturation step at 95°C for 5 minutes, followed by denaturation at 94°C for 45 seconds, annealing at 55°C for 45 seconds, and extension at 72°C for 1.5 minutes for 35 cycles, followed by extension at 72°C for 10 minutes.

[0075] The amplified product was detected by gel electrophoresis and the result was correct. The PCR product was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The amplified result of 16S rDNA is shown in SEQ ID NO. 3:

[0076] CGCGT GGCAG CATGC TTACA CATGC AGTCG AACGA TGAAG CCCTT CGGGG TGGATTAGTG GCGAA CGGGT GAGTA ACACG TGGGC AATCT GCCCT TCACT CTGGG ACAAG CCCTG GAAACGGGGT CTAAT ACCGATG GACTTGAT AACTCAG CTCCG GCGGT GAAGGATGAG CCCGC GGCCT ATCAG CTTGT TGGTG AGGTA GTGGC TCACC AAGGC GACGA CGGGT AGCCGGCCTG AGAGAC GCGAC CGGCC ACACT GGGAC TGAGA CACGG CCCAG ACTCC TACGATG AAGGGAGC ATCAG GATGC AGCGA CGCCG CGTGA GGGAT GACGG CCTTCGGGTT GTAAA CCTCT TTCAG CAGGG AAGAA GCGAA AGTGA CGGTA CCTGC AGAAG AAGCG CCGGCTAACT ACGTG CCAGC AGCCG CGGTA ATACG CAGGTGAG GTCCGAA GTCCGAT GTCTG TCGGA TCGGA TGTGA AAGCC CGGGG CTTAA CCCCG GGTCT GCATTCGATA CGGGC AGACT AGAGT GTGGT AGGGG AGATC GGAAT TCCTG GTGTA GCGGT GAAAT GCGCAGATAT CAGGA GGAAC ACCGCT GGACCTCG T AAGCCAT GCGAAAGCGT GGGGA GCGAA CAGGA TTAGA TACCC TGGTA GTCCA CGCCG TAAAC GGTGG GAACT AGGTGTTGGC GACAT TCCAC GTCGT CGGTG CCGCA GCTAA CGCAT TAAGT TCCCC GCCTG GGGAG TACGGCCGCA AGGCTAAAAC TCAAA GGAAT TGACG GGGGC CCGCA CAAGC AGCGG AGCAT GTGGC TTAATTCGAC GCAAC GCGAA GAACC TTACC AAGGC TTGAC ATCGC CCGGA AAGCC GTAGA GATAC GGCCCCCCTT GTGGT CGGGT GACAG GTGGT GCATG GCTGT CGTCA GCTCG TGTCG TGAGA TGTTG GGTTAAGTCC CGCAA CGAGC GCAAC CCTTG TTCTG TGTTG CCAGC ATGCC CTTCG GGGTG ATGGG GACTCACAGG AGACT GCCGG GGTCA ACTCG GAGGA AGGTG GGGAC GACGT CAAGT CATCA TGCCC CTTATGTCTT GGGCT GCACA CGTGC TACAA TGGCA GGTAC AATGA GCTGC GATAC CGCAA GGTGG AGCGAATCTC AAAAA GCCTG TCTCA GTTCG GATTG GGGTC TGCAA CTCGA CCCCA TGAAG TCGGA GTTGCTAGTA ATCGC AGATC AGCAT TGCTG CGGTG AATAC GTTCC CGGGC CTTGT ACACA CCGCC CGTCACGTCA CGAAA GTCGG TAACA CCCGA AGCCG GTGGC CCAAC CCCCT TGTGG GGAGG GAGCT GTCGAAGTTG AACCG GCGTT T。

[0077] ②PCR amplification, gel electrophoresis detection and sequencing of RECA

[0078] DNA extracted from strain 11-2-1 by the boiling method was used as a template, with recAF (5'-CCGCRCTCGCACAGATTGAACGSCAATTC-3') as the upstream primer (primer sequence shown in SEQ ID NO. 4), and recAR (5'-GCSAGGTCGGGGTTGTCCTTSAGGAAGTTGCG-3') as the downstream primer (primer sequence shown in SEQ ID NO. 5). The PCR reaction system consisted of 12.5 µL of Mix, 9.5 µL of ddH2O, 1 µL of DNA template, 1 µL of the upstream primer, and 1 µL of the downstream primer. DNA amplification was performed using a pre-denaturation step at 95°C for 5 min, followed by denaturation at 94°C for 30 s, annealing at 63°C for 30 s, and extension at 72°C for 1.5 min for 34 cycles, followed by extension at 72°C for 10 min.

[0079] The amplified product was tested by gel electrophoresis and then sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The amplified result of RECA is shown in SEQ ID NO.6:

[0080] ATCGG TAGGG CGCGG TGATG CGCCT CGGCG AGCGG CCGAC GAGCC CATCG AGGTCATCCC CACCG GCTCG ACGGC CCTCG ACGTC GCCCT CGGCG TCGGC GGTCT GCCGC GTGGC CGAGTGGTGG AGGTG TACGG CCCGG AGTCC TCCGG CAAGA CGACC CTGAC CCTGC ACGCC GTGGC GAACGCGCAG AAGGC CGGCG GCCAG GTGGC CTTCG TGGAC GCGGA GCACG CCCTC GATCC CGAGT ACGCGAAGAA GCTCG GCGTC GACAT CGAGA ACCTC ATCCT GTCGC AGCCG GACAA CGGCG AGCAG GCTCTGGAGA TCGTG GACAT GCTGA TCCGC TCCGG CGCGC TCGAC CTCAT CGTCA TCGAC TCCGT CGCGGCCCTC GTGCC GCGCG CGGAG ATCGA GGGCG AGATG GGCGA CTCGC ACGTG GGTCT GCAGG CCCGTCTGAT GAGCC AGGCA CTGCG GAAGA TCACC AGCGC GCTCA ACCAG TCGAA GACCA CCGCG ATCTTCATCA ACCAG CTCCG CGAGA AGATC GGCGT GATGT TCGGC TCGCC GGAGA CCACG ACCGG TGGCCGGGCG CTGAA GTTCT ACGCC TCGGT GCGCC TCGAC ATCCG TCGTA TCGAG ACCCT GAAGG ACGGCACCGA CGCGG TCGGC AACCG CACCC GCGTC AAGGT CGTCA AGAAC AAGGT CGCGC CGCCC TTCAAGCAGG CCGAG TTCGA CATCC TCTAC GGGCA GGGCA TCAGC CGCGA GGGCG GCCTG ATCGA CATGGGCGTG GAGAA CGGCT TCGTC CGCAA GGCCG GCGCC TGGTA CACGT ACGAG GGCGA CCAGC TCGCCAGTCA TGAGAACCCC GG.

[0081] ③ PCR amplification, gel electrophoresis detection and sequencing of ROPB

[0082] DNA extracted from strain 11-2-1 by the boiling method was used as a template. ropBF (5'-GAGCGCATGACCACCCAGGACGTCGAGGC-3') was used as the upstream primer (primer sequence shown in SEQ ID NO. 7), and ropBR (5'-CCTCGTAGTTGTGACCCTCCCACGGCATGA-3') was used as the downstream primer (primer sequence shown in SEQ ID NO. 8). The PCR reaction system consisted of 12.5µL of Mix, 9.5µL of ddH2O, 1µL of DNA template, 1µL of the upstream primer, and 1µL of the downstream primer. DNA amplification was performed using 34 cycles of initial denaturation at 95°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 63°C for 30 seconds, extension at 72°C for 1.5 minutes, and finally extension at 72°C for 10 minutes.

[0083] The amplified product was tested by gel electrophoresis. If qualified, the PCR product was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The amplification result of ROPB is shown in SEQ ID NO.9:

[0084] GGATC GCCGC AGACC CTGAT CACAT CCGGC CGGTC GTCGC CTCCA TCAAG GAGTTCTTCG GCACC AGCCA GCTGT CGCAG TTCAT GGACC AGAAC AACCC GCTGT CGGGT CTCAC CCACAAGCGC CGTCT GTCGG CGCTC GGCCC GGGTG GTCTC TCCCG TGAGC GGGCC GGCTT CGAGG TCCGTGACGT GCACC CCTCG CACTA CGGCC GCATG TGTCC GATCG AGACC CCTGA CGAAC ATCGGCCTGA TCGGC TCGCT CGCCT CCTAC GGCCG GGTCA ACGCG TTCGG CTTCG TCGAG ACGCC CTACCGCCGG GTCAC CGACG GTGTC GTCAC CGACG AGGTC GACTA CCTGA CCGCC GACGA GGAGG ACCGCTTCGT CATCG CGCAG GCCAA CGCCG TGCTC AACGA CGACA TGCGG TTCAA CGAGG CCCGT GTCCTGGTCC GCCGC CGTGG CGGAG AGGTC GACTA CGTCC CCGGC GACGA CGTCG ACTAC ATGGA CGTCTCGCCG CGCCA GATGG TGTCC GTCGC GACCG CGATG ATCCC CTTCC TGGAG CACGA CGACG CCAACCGTGC CCTCA TGGGC GCGAA CATGA TGCGC CAGGC CGTGC CGCTG ATCAC CGCCG CGGCC CCCCTCGTCG GTACG GGCAT GGAGT ACCGC TGCGC CGTCG ACGCC GGTGA CGTCA TCAAG GCCGA GAAGACCGGT GTGGT CCAGG AGGTC TCCGC GGACT ACGTC ACGGT GGCCA ACGAC GACGG CACCT ACACCACCTA CCGGG TGGCC AAGTT CTCCC GCTCC AACCA GGGGA CCTCG GTCAA CCAGA AGGTC GTCGTCAACG AGGGCGACCG GGTCG TCGAG AACCA GGTTC TCGCC GACGG TCCGG CCACC CAGGA AGGCGAGATG GCGCT GGCAA GATCC TGCCC TCCCC C.

[0085] The bull Streptomyces DNA sequence was submitted to the NCBI website for Blast sequence alignment and homology analysis. Multiple sequence alignment was performed using MeGa software to construct a 16S rDNA-RECA-ROPB phylogenetic tree, as shown in Figure 7 shown.

[0086] 3. Preservation of strains.

[0087] The strain 11-2-1 obtained above was deposited on October 21, 2024 at the General Microbiology Center of the China Culture Collection Administration (CGMCC), with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCC No. 32268.

[0088] Example 2

[0089] This example is used to illustrate the biocontrol effect of Streptomyces bulls 11-2-1 on Enterobacter cloacae.

[0090] Activation and culture of Enterobacter cloacae: The plate streak method was used to transfer the Enterobacter cloacae stored in a 4°C refrigerator to an LB plate using an inoculation needle and cultured in a constant temperature incubator at 28°C for 24 hours; the activated Enterobacter cloacae was inoculated into a finger-shaped tube of LB liquid culture medium and cultured at 28°C with shaking for 12 hours for antagonism test.

[0091] Determination of biocontrol efficacy: Use a pipette to transfer 50 μL of the E. cloacae suspension onto an LB plate and gently spread evenly with a spreader. Culture Streptomyces 11-2-1 on a Gao's medium No. 1 plate for 7 days. Use a sterile borer to punch out a 7 mm cake and invert it onto the center of the LB plate (a control plate was smeared with only the E. cloacae suspension). Set up three replicates and incubate in a 28°C incubator for 48 hours. Observe the growth of the pathogen (E. cloacae) and measure the diameter of the inhibition zone.

[0092] The results showed that the antagonistic activity of Streptomyces bulli 11-2-1 was very strong (see Figure 1 ), the boundary of the inhibition zone is clear, and the inhibition zone for the pathogen of ginger rot is 35mm.

[0093] Example 3

[0094] This example is used to illustrate the control effect of the fermentation liquid of Streptomyces bullosa 11-2-1 on ginger rot.

[0095] Cultivate Streptomyces bullii 11-2-1 on Gao's medium No. 1 plates at 30°C for 7 days. Use a sterile microporator to punch out three 7mm cakes. Inoculate these three cakes into 100mL of TSB culture medium and culture at 30°C with shaking for 3 days to obtain seed liquid. Inoculate 1mL of the S. bullii 11-2-1 seed liquid into 100mL of TSB culture medium. Cultivate at 30°C and 200 rpm for 5 days. Centrifuge at 4000 rpm for 20 minutes. Collect the supernatant and filter it through a bacterial filter to obtain the S. bullii 11-2-1 fermentation liquid.

[0096] Wash the ginger, disinfect its surface with 75% alcohol, and air dry. Use a sterilized knife to cut the ginger into slices 3 mm thick with a cross-section ≥ 2 cm apart. Soak the slices in a fermentation broth of Streptomyces bulls 11-2-1 and sterile water (control group) for 30 min, remove them, and air dry. Inoculate with 50 μL of a suspension of Enterobacter cloacae and incubate at 28°C for 5 days. Rinse the affected and rotten parts with clean water, air dry, and weigh them. Loss rate and control efficacy were calculated. Each treatment was replicated three times, with five slices per replicate.

[0097] Loss rate (%) = (weight before cleaning - weight after cleaning) / weight before cleaning × 100%;

[0098] Prevention effect (%) = (loss rate of control - loss rate of treatment) / loss rate of control × 100%.

[0099] This example used the half-ginger inoculation method to determine the in vitro efficacy of a fermentation broth of Streptomyces bullosa 11-2-1 against ginger rot. The results showed that the loss rate of the Enterobacter cloacae suspension inoculated with the fermentation broth was 23.8%, a 15.4% decrease compared to the control group (39.2%), resulting in a control efficacy of 39.3%.

[0100] Example 4

[0101] This example is used to illustrate the growth-promoting ability of Streptomyces bulls 11-2-1 and the identification of antagonistic active components.

[0102] 1. Identification of amylase production

[0103] A 7mm cake of Streptomyces bulli 11-2-1 cultured on Gao's medium No. 1 for 7 days was punched out with a hole punch and inoculated onto amylase medium. The culture was incubated at 28℃ for 3 days. If a transparent circle appeared around the colony, it indicated that the bacteria produced amylase. Figure 7 As shown, it was shown that Streptomyces bovis 11-2-1 could produce amylase.

[0104] 2. Identification of Protease Production

[0105] A 7 mm cake of Streptomyces bulli 11-2-1 cultured on Gao's medium No. 1 was punched out with a hole punch and inoculated onto PA medium. The culture was incubated at 28°C for 3 days. If a transparent circle appeared around the colony, it indicated that the bacteria produced protease. Figure 7 As shown, it was shown that Streptomyces bovis 11-2-1 could produce protease.

[0106] 3. Nitrogen fixation ability

[0107] A 7 mm cake of Streptomyces bulli 11-2-1 cultured on Gao's medium No. 1 was punched out with a hole punch and inoculated onto Asu-Bai's medium. The cake was cultured at 28°C for 5 days and then observed for growth. Figure 7 As shown, it was shown that Bull Streptomyces 11-2-1 had the ability to fix nitrogen.

[0108] 4. Identification of IAA production

[0109] Take 1mL of bull Streptomyces 11-2-1 cultured in TSB medium for 5 days, transfer it to a 1.5mL tube, and centrifuge it at 4000rpm for 2 minutes; take 100μL of the supernatant and transfer it to a new 1.5mL tube, and add 100μL of Salkowski reagent (Salkowski reagent consists of 1mL 0.5mol / L FeCl3 solution and 49mL 35wt% HClO4 solution). Let it stand for 5 minutes. If the color in the tube changes from colorless to pink, it is positive, indicating that IAA is produced. TSB culture medium without bull Streptomyces 11-2-1 was used as the control group. The test results are as follows: Figure 7 As shown, it was shown that Streptomyces bovis 11-2-1 can produce IAA.

[0110] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A bull Streptomyces 11-2-1, characterized in that: Its deposit number is CGMCC No. 32268.

2. Use of the bull Streptomyces 11-2-1 according to claim 1 in preventing and treating ginger rot.

3. A method for preparing a fermentation broth of Streptomyces bulli, characterized in that: The following steps are involved: S1, adding the bull Streptomyces 11-2-1 described in claim 1 to a culture medium plate for a first culture; then taking a bacterial cake and inoculating it into a culture solution for a second culture to obtain a seed solution; S2, inoculating the seed liquid into the culture liquid, performing a third culture, and performing solid-liquid separation to obtain a fermentation liquid of Streptomyces bulls.

4. The method for preparing the bull Streptomyces fermentation liquid as claimed in claim 3, wherein In S1, the culture medium plate is Gao's medium No. 1 plate; In S1, the temperature of the first culture is 28° C. to 32° C., and the culture time is 6 d to 8 d.

5. The method for preparing the bull Streptomyces fermentation liquid as claimed in claim 3, wherein In S1, the diameter of the mushroom cake is 6 mm to 9 mm; In S1, the number of the mushroom cakes is 2 to 4; In S1, the culture medium includes TSB culture medium; In S1, the volume of the culture medium is 90 mL to 110 mL; In S1, the second culture is a shaking culture with a temperature of 28°C to 32°C, a rotation speed of 180 rpm to 220 rpm, and a culture time of 2.5 days to 4 days.

6. The method for preparing the bull Streptomyces fermentation liquid as claimed in claim 3, wherein In S2, the culture medium includes TSB culture medium; In S2, the volume ratio of the seed solution to the culture solution is 1:(90-110); In S2, the third culture is a shaking culture with a temperature of 28°C to 32°C, a rotation speed of 180 rpm to 220 rpm, and a culture time of 4.5 days to 6 days.

7. The method for preparing the bull Streptomyces fermentation liquid as claimed in claim 3, wherein In S2, the solid-liquid separation comprises the following steps: The mixture was centrifuged at 3500 rpm to 4500 rpm for 15 min to 25 min, and the supernatant was taken for bacterial filtration to obtain the fermentation liquid of Streptomyces bulls.

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