Streptomyces bulbus 11-2-1 and application thereof

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses streptomyces bulbus 11-2-1 and application thereof. The streptomyces bulbus 11-2-1 (CGMCC No. 32268) provided by the invention is a natural strain separated from rhizosphere soil of a wheat field, is safe and environment-friendly, can effectively inhibit the growth of valsa zingiberensis, and does not affect the normal growth of ginger. The test result shows that the pathogenic bacteria of the ginger rot disease can be observed to be inhibited after the ginger rot disease is cultured for 24 hours by adopting an inhibition zone method, the inhibition zone is observed to be more obvious after the ginger rot disease is cultured for 48 hours, and the diameter of the inhibition zone can reach 35 mm. The streptomyces bulbus 11-2-1 provided by the invention provides reference for later research of natural strains, and has important value for development of a microbial agent for preventing and treating the ginger rot disease.

Description

Technical Field

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

[0002] Ginger ( Zingiber icinale Roscoe) is a plant of the genus Zingiber in the family Zingiberaceae, mainly distributed in central, southeastern to southwestern China, and subtropical regions. Ginger rot is an important bacterial disease in ginger production, seriously affecting the yield and quality of ginger, and causing huge economic losses to ginger production. Generally, chemical agents are used to control ginger rot in production, but chemical agents will cause pollution to the environment, and long-term abuse will lead to the resistance of pathogens to chemical agents. Biological control has the advantages of environmental friendliness, safety, effectiveness, etc., but there are few microbial agents used for the control of ginger rot in production. Therefore, it is of great significance to obtain biocontrol strains that can effectively control ginger rot. Summary of the Invention

[0003] In view of the above problems, the present invention provides a Streptomyces taurus 11-2-1 and its application. Streptomyces taurus 11-2-1 can effectively antagonize the ginger rot pathogen, has no influence on plant growth, and is harmless to the environment.

[0004] To solve the above technical problems, the technical solution provided by the present invention is: In the first aspect, the present invention provides a Streptomyces taurus ( Streptomyces tauricus ) 11-2-1, whose preservation number is CGMCC No. 32268.

[0005] Compared with the prior art, the Streptomyces taurus 11-2-1 provided by the present invention is a natural strain isolated from the rhizosphere soil of wheat fields, belonging to the genus Streptomyces, and is an antagonistic bacterium from the soil. It can effectively control ginger rot, has no influence on plant growth, has the ability of nitrogen fixation and IAA production, is harmless to the environment, and has the potential for development and application as a new microbial strain resource for controlling ginger rot.

[0006] The Streptomyces taurus 11-2-1 provided by the present invention can effectively inhibit the growth of the ginger rot pathogen and does not affect the normal growth of ginger. The test results show that the inhibition of the ginger rot pathogen can be observed by using the inhibition zone method after culturing for 24 h, and the inhibition zone is more obvious after culturing for 48 h, and the diameter of the inhibition zone can reach 35 mm. The Streptomyces taurus 11-2-1 provided by the present invention provides a reference for later research and has important value for the development of microbial agents for controlling ginger rot.

[0007] In the second aspect, the present invention provides an application of the above-mentioned Streptomyces taurus 11-2-1 in inhibiting the growth of Enterobacter cloacae.

[0008] Thirdly, the present invention provides an application of the Streptomyces taurus 11-2-1 in preventing and treating ginger rot disease.

[0009] Fourthly, the present invention provides a preparation method of a Streptomyces taurus fermentation broth, comprising the following steps: S1, adding the Streptomyces taurus 11-2-1 onto a culture medium plate for the first culture; then punching out a bacterial cake and inoculating it into a culture solution for the second culture to obtain a seed solution; S2, inoculating the seed solution into a culture solution for the third culture, and performing solid-liquid separation to obtain the Streptomyces taurus fermentation broth.

[0010] Preferably, in S1, the culture medium plate includes a Gao's No. 1 culture medium plate.

[0011] Preferably, in S1, the temperature of the first culture is 28°C to 32°C, and the culture time is 6d to 8d.

[0012] Preferably, in S1, the diameter of the bacterial cake is 6mm to 9mm.

[0013] Preferably, in S1, the number of the bacterial cakes is 2 to 4.

[0014] Preferably, in S1, the culture solution includes a TSB culture solution.

[0015] Preferably, in S1, the volume of the culture solution is 90mL to 110mL.

[0016] Preferably, in S1, the second culture is an oscillating culture, the temperature is 28°C to 32°C, the rotation speed is 180rpm to 220rpm, and the culture time is 2.5d to 4d.

[0017] Preferably, in S2, the culture solution includes a TSB culture solution.

[0018] Preferably, in S2, the volume ratio of the seed solution to the culture solution is 1:(90 to 110).

[0019] Preferably, in S2, the third culture is an oscillating culture, the temperature is 28°C to 32°C, the rotation speed is 180rpm to 220rpm, and the culture time is 4.5d to 6d.

[0020] Preferably, in S2, the solid-liquid separation includes the following steps: Centrifuging at 3500rpm to 4500rpm for 15min to 25min, taking the supernatant for bacterial filtration to obtain the Streptomyces taurus fermentation broth.

[0021] Fifth aspect, the present invention provides a Streptomyces taurus fermentation broth, which is prepared by the preparation method of the Streptomyces taurus fermentation broth described above.

[0022] Sixth aspect, the present invention provides an application of the Streptomyces taurus fermentation broth in preventing and treating ginger soft rot. Description of the Drawings

[0023] Figure 1 It is the inhibition effect diagram of strain 11-2-1 against Enterobacter cloacae in Example 1 of the present invention; Figure 2 It is the morphological diagram of the aerial mycelium of strain 11-2-1 in Example 1 of the present invention; Figure 3 It is the morphological diagram of the spores of strain 11-2-1 in Example 1 of the present invention; Figure 4 It is the morphological diagram of strain 11-2-1 on different culture media in Example 1 of the present invention; in the figure, a represents Gao's No. 1 medium, b represents PDA medium, c represents calcium malate agar medium, d represents oatmeal medium, e represents starch ammonium medium, f represents nutrient agar medium, g represents aspartic acid medium; Figure 5 It is the utilization situation of different carbon sources of the basic medium for the carbon source utilization test of strain 11-2-1 in Example 1 of the present invention; in the figure, a represents inositol as the carbon source, b represents xylose as the carbon source, c represents glucose as the carbon source, d represents lactose as the carbon source, e represents fructose as the carbon source, f represents galactose as the carbon source, g represents sucrose as the carbon source, h represents D-mannitol as the carbon source; Figure 6 It is the phylogenetic tree of Streptomyces taurus 11-2-1 in Example 1 of the present invention; Figure 7 It is the test result diagram of the growth-promoting ability of Streptomyces taurus 11-2-1 in Example 4 of the present invention; in the figure, a represents amylase production, b represents protease production, c represents nitrogen fixation, d represents IAA production. Detailed Embodiments

[0024] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the following further describes the present invention in detail with reference to examples. It should be understood that the specific examples described herein are only used to explain the present invention and are not used to limit the present invention.

[0025] Media used in the examples: Gao's No. 1 medium: soluble starch 20g, potassium nitrate 1g, dipotassium hydrogen phosphate 0.5g, magnesium sulfate heptahydrate 0.5g, sodium chloride 0.5g, ferrous sulfate heptahydrate 0.01g, potassium dichromate 0.1g, agar 20g, distilled water 1L, pH = 7.

[0026] Potato Dextrose Agar Medium (PDA Medium): 200 g of potatoes, 20 g of dextrose, 20 g of agar, 1000 mL of distilled water, pH = 7.0 - 7.4.

[0027] LB Medium: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, 1000 mL of distilled water.

[0028] Calcium Malate Agar Medium: 17 g of agar, 10 g of calcium malate, 10 g of glycerol, 0.5 g of NH 4 Cl, 0.5 g of K 2 HPO 4 0.5 g, 1000 mL of distilled water, pH = 7.2 - 7.4.

[0029] Oat Medium: 30 g of oats, 1000 mL of distilled water.

[0030] Starch Ammonium Medium: 10 g of soluble starch, (NH 4 ) 2 SO 4 2 g, 1 g of MgSO4·7H 2 O, 1 mL of trace element solution, 1000 mL of distilled water, pH = 6.5 - 7.0.

[0031] Nutrient Agar Medium: 10 g of peptone, 5 g of beef extract, 5 g of NaCl, 20 g of agar.

[0032] Asparagine Medium: 10 g of glycerol, 1 g of L - asparagine, 1 g of K 2 HPO 4 1 g, 1000 mL of distilled water, 20 g of agar powder, pH = 6.5 - 7.0.

[0033] Starch Hydrolysis Agar Medium: 17 g of agar, 10 g of soluble starch, 0.3 g of K 2 HPO 4 0.3 g, 1 g of KNO 3 1 g, 1 g of MgCO 3 1 g, 0.5 g of NaCl, 1000 mL of distilled water, pH = 7.2 - 7.4.

[0034] Cellulose Hydrolysis Medium: 0.5 g of NaCl, 0.5 g of K 2 HPO 4 0.5 g, 0.5 g of MgSO 4 0.5 g, 1 g of KNO 3 1 g, 1000 mL of distilled water, pH = 7.2, filter paper strip 5 cm long and 0.8 cm wide.

[0035] Gelatin liquefaction medium: 5 g of peptone, 200 g of gelatin, 20 g of glucose, 1000 mL of distilled water, pH = 7.2 - 7.4.

[0036] Tresner medium: 10.0 g of agar, 10.0 g of peptone, 0.5 g of ferric citrate, 1000 mL of distilled water, pH = 7.2.

[0037] Tyrosine medium (for melanin production): 1.0 g of L - tyrosine, 1.0 g of yeast extract, 8.5 g of NaCl, 15 g of agar, 1000 mL of distilled water, pH = 7.2.

[0038] Milk coagulation and peptonization medium: 0.02 g of CaCO 3 1000 mL of skim fresh milk.

[0039] Carbon source utilization test basal medium: (NH 4 ) 2 SO 4 2.64 g, KH 2 PO 4 2.38 g, K 2 HPO 4 ·3H 2 O 5.65 g, MgSO 4 ·7H2O 1 g. Each carbon source is added separately in an amount of 0.5 wt% of fructose, sucrose, inositol, D - mannitol, lactose, galactose, glucose, and xylose.

[0040] Nitrogen source utilization test basal medium: 1 g of glucose, 0.05 g of MgSO 4 ·7H 2 O 0.05 g, 0.05 g of NaCl, 0.001 g of FeSO 4 ·7H 2 O 0.001 g, K 2 HPO 4 0.01 g. Each nitrogen source is added separately in an amount of 0.5 wt% of urea, potassium nitrate, calcium nitrate, and glycine.

[0041] Amylase medium: 5 g of beef extract, 10 g of peptone, 5 g of sodium chloride, 18 g of agar, 1 L of ddH 2 O.

[0042] Proteinase (Proteinase Agar, PA) medium: 20 g of skim milk, 15 g of Agar, 1 L of ddH 2 O.

[0043] Ashby's medium: 5.0 g of malic acid, 4.5 g of KOH, K 2 HPO 40.5 g, CaCO 3 ·2H2O 0.02 g, NaCl 0.1 g, NaMoO 4 ·2H 2 O 0.002 g, FeSO 4 ·7H 2 O 0.002 g, agar 18 g, ddH 2 O 1 L.

[0044] The Enterobacter cloacae in the embodiments of the present invention ( Enterobacter cloacae ) was isolated from diseased ginger in the ginger planting area of Tangshan City.

[0045] The raw materials, reagents, etc. used in the embodiments of the present invention are all obtained from commercial channels unless otherwise specified.

[0046] To better illustrate the present invention, further examples are given below through embodiments.

[0047] Example 1 This example is used to illustrate the acquisition, identification, and preservation of Streptomyces tauricus 11-2-1.

[0048] 1. Strain screening Rhizosphere soil of wheat (depth 0 - 15 cm, 2 mm around the roots) was collected from a wheat field in Botou, Cangzhou City, Hebei Province, air-dried naturally at room temperature, and crushed. In a laminar flow hood, 10 g of rhizosphere soil was added to 90 mL of sterile water, shaken for 1 min, and left to stand for 30 min; 1 mL of the supernatant was taken and diluted to 10 -2 , 10 -3 , 10 -4 and 10 -5 with sterile water according to the gradient dilution method. 0.2 mL of each dilution was taken and spread on Gause's No. 1 medium plates, and incubated at 28 °C for 4 d - 7 d. Potassium dichromate solution with a final concentration of 10 -4 (w / v) was added to Gause's No. 1 medium to inhibit the growth of fungi and other bacteria. The potassium dichromate was added when the medium cooled to 60 °C - 70 °C, and after mixing, the plates were poured. Appropriate dilutions were selected to pick different-colored and -shaped single colonies for purification culture. The purified Streptomyces strains were numbered and stored at 4 °C. Using the plate inhibition zone method, strain 11-2-1 ( Figure 1 ) with a significant inhibitory effect on the ginger rot pathogen was screened.

[0049] 2. Identification of the strain 2.1 Morphological identification The morphological characteristics of strain 11-2-1 on different media, as well as the morphology of aerial hyphae and spores, are as Figures 2 - 4As shown in Table 1. The colonies of this strain are round, convex, light pink, with a tuft of hair around the periphery. The spore chains are spiral, and the spores are cylindrical; there are differences in the culture characteristics and growth conditions of the strain on different media.

[0050] Table 1 Culture characteristics of strain 11-2-1 on different media

[0051] Note: + indicates that it can grow, ++ indicates good growth, and +++ indicates excellent growth.

[0052] 2.2 Physiological and biochemical identification Strain 11-2-1 was cultured on different media respectively to verify its physiological and biochemical characteristics and identify the carbon sources and nitrogen sources it utilized. The results showed that this strain could hydrolyze starch, could not hydrolyze cellulose, could liquefy gelatin, did not produce hydrogen sulfide, could produce melanin, and peptonized milk; it could utilize glucose, lactose, fructose, sucrose, D-mannitol, galactose, inositol and xylose ( Figure 5 ); for nitrogen sources, it could utilize urea, potassium nitrate, calcium nitrate and glycine.

[0053] 2.3 Molecular biology identification (1) Extracting strain DNA: Using the boiling water method, inoculate 1 mL of the seed solution of strain 11-2-1 into 100 mL of TSB culture medium and culture it with shaking at 30 °C for 48 h. Take 1000 μL and add it to a 1.5 mL centrifuge tube, centrifuge at 4 °C and 12000 rpm for 10 min, discard the supernatant, add 30 μL of ultrapure water, suspend it, perform a boiling water bath for 10 min, centrifuge at 4 °C and 12000 rpm for 10 min, and aspirate the supernatant into a new centrifuge tube.

[0054] (2) PCR amplification, gel electrophoresis detection and sequencing: ① PCR amplification, gel electrophoresis detection and sequencing of 16S rDNA Using the DNA of strain 11-2-1 extracted by the boiling water method as a template, using 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) as the upstream primer (the primer sequence is shown in SEQ ID NO.1), and 1492R (5’-GGTTACCTTGTTACGACTT-3’) as the downstream primer (the primer sequence is shown in SEQ ID NO.2). Using 12.5 µL of Mix, 9.5 µL of ddH 2O, 1 µL of DNA template, 1 µL of upstream primer and 1 µL of downstream primer were used to form a PCR reaction system. The reaction program for DNA amplification was as follows: pre-denaturation at 95 °C for 5 min; denaturation at 94 °C for 45 s; annealing at 55 °C for 45 s; extension at 72 °C for 1.5 min, for 35 cycles; and extension at 72 °C for 10 min.

[0055] The amplified products were detected by gel electrophoresis and the results were correct. The PCR products were sent to Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. for sequencing. The amplification results of 16S rDNA were as shown in SEQ ID NO.3: CGCGT GGCAG CATGC TTACA CATGC AGTCG AACGA TGAAG CCCTT CGGGG TGGATTAGTG GCGAA CGGGT GAGTA ACACG TGGGC AATCT GCCCT TCACT CTGGG ACAAG CCCTG GAAACGGGGT CTAAT ACCGG ATGAT ACTTC CACTC GCATG GGTGG GGGTT GAAAG CTCCG GCGGT GAAGGATGAG CCCGC GGCCT ATCAG CTTGT TGGTG AGGTA GTGGC TCACC AAGGC GACGA CGGGT AGCCGGCCTG AGAGG GCGAC CGGCC ACACT GGGAC TGAGA CACGG CCCAG ACTCC TACGG GAGGC AGCAGTGGGG AATAT TGCAC AATGG GCGAA AGCCT GATGC AGCGA CGCCG CGTGA GGGAT GACGG CCTTCGGGTT GTAAA CCTCT TTCAG CAGGG AAGAA GCGAA AGTGA CGGTA CCTGC AGAAG AAGCG CCGGCTAACT ACGTG CCAGC AGCCG CGGTA ATACG TAGGG CGCAA GCGTT GTCCG GAATT ATTGG GCGTAAAGAG CTCGT AGGCG GTCTG TCGCG TCGGA TGTGA AAGCC CGGGG CTTAA CCCCG GGTCT GCATTCGATA CGGGC AGACT AGAGT GTGGT AGGGG AGATC GGAAT TCCTG GTGTA GCGGT GAAAT GCGCAGATAT CAGGA GGAAC ACCGG TGGCG AAGGC GGATC TCTGG GCCAT TACTG ACGCT GAGGA 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。

[0056] ②PCR amplification, gel electrophoresis detection and sequencing of RECA Using the DNA of strain 11-2-1 extracted by the water-boiling method as a template, recAF (5’-CCGCRCTCGCACAGATTGAACGSCAATTC-3’) was used as the upstream primer (the primer sequence is shown in SEQ ID NO.4), and recAR (5’-GCSAGGTCGGGGTTGTCCTTSAGGAAGTTGCG-3’) was used as the downstream primer (the primer sequence is shown in SEQ ID NO.5). A PCR reaction system was composed of 12.5µL Mix, 9.5µL ddH2O, 1µL DNA template, 1µL upstream primer, and 1µL downstream primer. The DNA amplification was carried out with the reaction program: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 30 s; annealing at 63°C for 30 s; extension at 72°C for 1.5 min, for 34 cycles; and extension at 72°C for 10 min.

[0057] After the amplification products were detected by gel electrophoresis for the amplification results, they were sent to Shanghai Sangon Biological Engineering Co., Ltd. for sequencing. The amplification result of RECA is shown in SEQ ID NO.6: 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。

[0058] ③PCR amplification, gel electrophoresis detection and sequencing of ROPB Using the DNA of strain 11-2-1 extracted by the boiling method as a template, ropBF (5'-GAGCGCATGACCACCCAGGACGTCGAGGC-3') was used as the upstream primer (the primer sequence is shown in SEQ ID NO.7), and ropBR (5'-CCTCGTAGTTGTGACCCTCCCACGGCATGA-3') was used as the downstream primer (the primer sequence is shown in SEQ ID NO.8). Using 12.5µL Mix, 9.5µL ddH 2 O, 1µL DNA template, 1µL upstream primer and 1µL downstream primer as the PCR reaction system. The reaction program for DNA amplification was as follows: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 30 s; annealing at 63°C for 30 s; extension at 72°C for 1.5 min, for 34 cycles; extension at 72°C for 10 min.

[0059] The amplified product was detected by gel electrophoresis. After passing the test, the PCR product was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The amplification result of ROPB is shown in SEQ ID NO.9: 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 AGGCC 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 AGGCC 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。

[0060] The DNA sequence of Streptomyces taurus was submitted to the NCBI website for Blast sequence alignment and homology analysis. Multiple sequence alignment was performed using the MeGa software, and a 16S rDNA-RECA-ROPB phylogenetic tree was constructed, as Figure 7 shown.

[0061] 3. Preservation of the strain.

[0062] The obtained strain 11-2-1 was preserved in the China General Microbiological Culture Collection Center (CGMCC) on October 21, 2024. The preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The preservation number is CGMCC No. 32268.

[0063] Example 2 This example is used to illustrate the biocontrol effect of Streptomyces taurus 11-2-1 against Enterobacter cloacae.

[0064] Activation and culture of Enterobacter cloacae: Using the streak plate method, the Enterobacter cloacae stored in a 4°C refrigerator was transferred to an LB plate with an inoculation needle and placed in an incubator at 28°C for 24 h; the activated Enterobacter cloacae was inoculated into a finger tube containing LB liquid medium and cultured with shaking at 28°C for 12 h for the antagonism test.

[0065] Determination of biocontrol effect: Using a pipette, 50 μL of the Enterobacter cloacae bacterial suspension was taken onto an LB plate and gently spread evenly with a spreader. Streptomyces taurus 11-2-1 was cultured on a Gause's No. 1 medium plate for 7 d, and a 7-mm bacterial cake was punched out with a sterile punch and placed upside down in the center of the LB plate (the control group was only coated with the Enterobacter cloacae bacterial suspension). Three replicates were set up and placed in an incubator at 28°C for 48 h. The growth of the pathogenic bacterium (Enterobacter cloacae) was observed, and the diameter of the inhibition zone was measured.

[0066] The results showed that the antagonistic activity of Streptomyces taurus 11-2-1 was very strong (see Figure 1 ), the boundary of the inhibition zone was clear, and the inhibition zone against the pathogen of ginger rot was 35 mm.

[0067] Example 3 This example is used to illustrate the control effect of the fermentation broth of Streptomyces taurus 11-2-1 against ginger rot.

[0068] Streptomyces taurus 11-2-1 was cultured on a Gao's No. 1 medium plate at 30 °C for 7 days. Three 7-mm bacterial cakes were punched out using a sterile puncher and inoculated into 100 mL of TSB culture medium. The mixture was cultured with shaking at 30 °C for 3 days to obtain a seed solution. 1 mL of the Streptomyces taurus 11-2-1 seed solution was inoculated into 100 mL of TSB culture medium and cultured at 30 °C and 200 rpm for 5 days. Then, it was centrifuged at 4000 rpm for 20 min. The supernatant was taken and filtered through a bacterial filter to obtain the fermentation broth of Streptomyces taurus 11-2-1.

[0069] The ginger was washed clean, surface disinfected with 75% alcohol, air-dried, and cut into ginger slices with a thickness of 3 mm and a cross-sectional area of ≥2 cm between the cross-sections using a sterilized knife. The ginger slices were respectively soaked in the fermentation broth of Streptomyces taurus 11-2-1 and sterile water (control group) for 30 min, taken out, and air-dried. 50 μL of Enterobacter cloacae bacterial suspension was inoculated, and after culturing at 28 °C for 5 days, the diseased and rotten parts were washed off with clean water, air-dried, weighed and recorded, and the loss rate and control efficacy were calculated. Each treatment was repeated 3 times, and each repetition had 5 ginger slices.

[0070] Loss rate (%) = (weight before cleaning - weight after cleaning) / weight before cleaning × 100%; Control efficacy (%) = (loss rate of control - loss rate of treatment) / loss rate of control × 100%.

[0071] In this example, the in vitro control effect of the fermentation broth of Streptomyces taurus 11-2-1 on ginger rot was determined by the semi-ginger inoculation method. The results showed that the loss rate of the ginger slices inoculated with Enterobacter cloacae bacterial suspension after treatment with the fermentation broth was 23.8%. Compared with the control group (39.2%), the loss rate decreased by 15.4%, and the control efficacy was 39.3%.

[0072] Example 4 This example is used to illustrate the growth-promoting ability of Streptomyces taurus 11-2-1 and the identification of antagonistic active components.

[0073] 1. Identification of amylase production A 7-mm bacterial cake of Streptomyces taurus 11-2-1 cultured on a Gao's No. 1 medium for 7 days was punched out using a puncher and inoculated onto an amylase medium. It was incubated at 28 °C for 3 days. If a clear zone appeared around the colony, it indicated that the bacterium produced amylase. The test results are as Figure 7 shown, indicating that Streptomyces taurus 11-2-1 can produce amylase.

[0074] 2. Identification of protease production The Streptomyces bovis 11-2-1 plate cultured on Gao's No. 1 medium for 7 days was punched with a puncher to obtain a 7-mm mycelial cake and inoculated on PA medium, and incubated at 28 °C for 3 days. If a clear zone appeared around the colony, it indicated that the bacterium produced protease. The test results are as Figure 7 shown, indicating that Streptomyces bovis 11-2-1 can produce protease.

[0075] 3. Nitrogen fixation ability The Streptomyces bovis 11-2-1 plate cultured on Gao's No. 1 medium for 7 days was punched with a puncher to obtain a 7-mm mycelial cake and inoculated on Ashby's medium, and cultured at 28 °C for 5 days to observe whether it grew. The test results are as Figure 7 shown, indicating that Streptomyces bovis 11-2-1 has nitrogen fixation ability.

[0076] 4. Identification of IAA production Absorb 1 mL of Streptomyces bovis 11-2-1 cultured on TSB culture medium for 5 days, transfer it to a 1.5-mL tube, and centrifuge it at 4000 rpm for 2 min; take 100 μL of the supernatant and transfer it to a new 1.5-mL tube, and add 100 μL of Salkowski reagent (Salkowski reagent consists of 1 mL of 0.5 mol / L FeCl 3 solution and 49 mL of 35 wt% HClO 4 solution), and let it stand for 5 min. If the color in the tube changes from colorless to pink, it is positive, indicating the production of IAA. Using the TSB culture medium without Streptomyces bovis 11-2-1 as a control group, the test results are as Figure 7 shown, indicating that Streptomyces bovis 11-2-1 can produce IAA.

[0077] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, or improvements made within the spirit and principle of the present invention shall be included within the protection scope 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 described in claim 1 in inhibiting the growth of Enterobacter cloacae.

3. Use of the bull Streptomyces 11-2-1 described in claim 1 in preventing and treating ginger rot disease.

4. 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, inoculating it into a culture solution, and performing a second culture to obtain a seed solution; S2, inoculating the seed liquid into the culture liquid, performing a third culture, and separating the solid and liquid to obtain a fermentation liquid of Streptomyces bulli.

5. The method for preparing the bull Streptomyces fermentation liquid as claimed in claim 4, characterized in that, 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 6d to 8d.

6. The method for preparing the bull Streptomyces fermentation liquid as claimed in claim 4, characterized in that: 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 solution is 90 mL to 110 mL; In S1, the second culture is shaking culture, the temperature is 28°C to 32°C, the rotation speed is 180rpm to 220rpm, and the culture time is 2.5d to 4d.

7. The method for preparing the bull Streptomyces fermentation liquid as claimed in claim 4, characterized in that: 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 shaking culture, the temperature is 28°C to 32°C, the rotation speed is 180rpm to 220rpm, and the culture time is 4.5d to 6d.

8. The method for preparing the bull Streptomyces fermentation liquid as claimed in claim 4, characterized in that: 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 bulli.

9. A fermentation broth of Streptomyces bulli, characterized in that: The fermentation liquid is prepared by the method for preparing the fermentation liquid of Streptomyces bulli according to any one of claims 4 to 8.

10. Use of the bull Streptomyces fermentation liquid according to claim 9 in preventing and treating ginger rot disease.

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