CN110408559B - Streptomyces roseosporus and application thereof - Google Patents
Streptomyces roseosporus and application thereof Download PDFInfo
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/465—Streptomyces
Abstract
The invention discloses a Streptomyces roseoflavus strain LY-1, wherein the strain LY-1 is preserved in 5 months and 30 days in 2019: china general microbiological culture Collection center (CGMCC) with a collection number of CGMCC NO.17874 and a collection address of: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North. The invention successfully separates and screens a Streptomyces roseoflavus strain LY-1 from a soil sample in a cabbage field, and the strain LY-1 is preserved in: the China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC NO.17874 is used for preventing and treating vegetable diseases and cabbage black spot, has obvious prevention and treatment effect, is green and environment-friendly, and is environment-friendly.
Description
Technical Field
The invention relates to the technical field of microorganisms, and particularly relates to streptomyces roseosporus and application thereof.
Background
The Chinese cabbage black spot is also called black mold, and is a fungal disease which is commonly generated in Chinese cabbage production and seriously harmful. In recent years, the disease is more and more serious due to changes of environment and cultivation conditions, and the yield of the Chinese cabbage is greatly influenced. The chemical agent is still one of the main measures at present for preventing and treating the cabbage black spot disease, and the bactericides for preventing and treating the cabbage black spot disease in China comprise chlorothalonil, thiram, prochloraz, carbendazim, cyhalothrin, difenoconazole and the like. The chemical bactericide has high efficiency, low cost and simple operation, but has potential risks and influences on the environment and human health while preventing and treating diseases.
The biological agent is environment-friendly to prevent and treat vegetable diseases, and can avoid a series of problems caused by chemical prevention and treatment. The biological agent can reduce or replace the use of chemical agents, thereby achieving the purpose of preventing and treating vegetable diseases, and avoiding or reducing the 3R problem and the threat to human health caused by the use of chemical agents. In the prior art, the application of Streptomyces roseoviticus (Streptomyces roseoviticus) in the prevention and treatment of cabbage black spot has not been reported. How to solve the technical problems is a technical problem to be solved in the technical field of microorganisms at present.
Disclosure of Invention
The Streptomyces roseoflavus (Streptomyces roseovitericus) strain LY-1 provided by the invention is preserved in the culture medium in 5-30 days in 2019: china general microbiological culture Collection center (CGMCC) with a collection number of CGMCC NO.17874 and a collection address of: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.
The Streptomyces roseoalvestris (Streptomyces roseoviticus) strain LY-1 provided by the invention is used for controlling vegetable diseases.
The Streptomyces roseoalvestris (Streptomyces roseoviticus) strain LY-1 provided by the invention is used for preventing and treating cabbage black spot.
The effective active ingredient of the microbial agent for preventing and treating the cabbage black spot is fermentation supernatant of Streptomyces roseoviticus strain LY-1, and the fermentation supernatant is prepared by fermenting the fermentation supernatant with the concentration of 108CFU/mL of Streptomyces roseoalvens (Streptomyces roseoviticus) strain LY-1. The strain LY-1 fermentation broth under the concentration has an obvious effect of preventing and treating the cabbage black leaf spot, is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC NO. 17874.
The invention has the beneficial effects that: the invention successfully separates and screens a Streptomyces roseoflavus strain LY-1 from a soil sample in a cabbage field, and the strain LY-1 is preserved in: the China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC NO.17874 is used for preventing and treating vegetable diseases and cabbage black spot, has obvious prevention and treatment effect, is green and environment-friendly, and is environment-friendly.
Drawings
FIG. 1 shows the normal growth of Neurospora brassicae on the Gao's No. one medium;
FIG. 2 shows the inhibitory effect of Streptomyces roseoviticus strain LY-1 on alternaria solani.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Isolation, purification and preservation of Actinomycetes
And (3) separating actinomycetes by adopting a dilution coating plate method: taking soil samples in a Chinese cabbage field, preparing soil suspensions, diluting the soil suspensions with different concentrations respectively, and centrifuging for 30min in a shaking table at 200 r/min. And after the centrifugation is finished, taking out the soil suspensions with different concentrations, standing for 30min, respectively taking a proper amount of supernatant to coat on a Gao's first plate, and placing the plate in a constant-temperature incubator at 28 ℃ for culture for 5-7 d.
Observing the growth conditions of the bacterial colonies, respectively purifying the bacterial colonies, numbering different bacterial strains, and refrigerating and storing at 4 ℃, wherein the numbering of the different bacterial strains is as follows in sequence: AY-1, AY-2, LC-3, LR-1, LR-2, LR-3, LY-1, LY-2.
It should be noted that the medium for isolation and preservation is a culture medium of Gauss-I, which is a synthetic medium for culturing and observing the morphological characteristics of actinomycetes. The formula of the Gao's No. one culture medium used in the invention is as follows: 20g of soluble starch, 0.01g of FeSO4.7H2O, 31.0g of KNO31, 20.5g of K2HPO20, 0.5g of MgSO2.7H2O, 20g of agar, 1000mL of distilled water and pH of 7.4-7.6.
Example 2
Screening of actinomycetes with bacteriostatic activity
The actinomycetes with the bacteriostatic effect is obtained by measuring and separating by adopting a confrontation culture method:
experimental groups: the method comprises the steps of taking cabbage black spot bacteria as target bacteria, respectively beating different actinomycete strains AY-1, AY-2, LC-3, LR-1, LR-2, LR-3, LY-1, LY-2 and the target bacteria separated and purified in example 1 into bacterial cakes of 0.4cm, placing the target bacterial cakes in the center of a Gao's 1 plate of 9cm, dividing the different actinomycete strains AY-1, AY-2, LC-3, LR-1, LR-2, LR-3, LY-1 and LY-2 into 8 experimental groups, respectively placing the experimental groups on 4 symmetrical points 3cm away from the target bacterial cakes, and placing the experimental groups in a constant temperature incubator at 25 ℃ for culture for 5 d.
Blank control group: taking cabbage black spot pathogen as target bacteria, beating the target bacteria into a bacterial cake of 0.4cm, placing the target bacterial cake in the center of a Gao's No.1 plate of 9cm, and placing the plate in a constant temperature incubator at 25 ℃ for culture for 5 days.
And measuring the colony diameters of the alternaria alternata in the experimental group and the blank control group by adopting a cross method, measuring the size of an antibacterial zone, and calculating the antibacterial rate. Each experimental group and blank control group were treated 5 times in duplicate. The calculation formula is as follows: the bacteriostatic rate (%) was (blank group colony diameter-each experimental group colony diameter)/(blank group colony diameter-cake diameter) × 100.
The experimental result in the table 1 shows the inhibition effect of different actinomycete strains AY-1, AY-2, LC-3, LR-1, LR-2, LR-3, LY-1 and LY-2 on the black speck disease of the cabbage. FIG. 1 shows the normal growth of alternaria alternata as a blank control group on a culture medium of Homoku's disease, FIG. 2 shows a photograph of an experiment effect of Streptomyces roseoviticus strain LY-1 on alternaria alternata, and a comparison of the photograph with FIG. 1 and FIG. 2 shows that the inhibition effect of strain LY-1 on alternaria alternata is obvious.
TABLE 1 inhibition of cabbage black spot pathogen by Actinomycetes
As can be seen from the table 1, the inhibition rate of the strain LY-1 group on the cabbage black spot pathogen is the highest, and is 67.1%, and the inhibition zone is 7.0mm, so that the strain LY-1 has a remarkable inhibition effect on the cabbage black spot pathogen, and is the required biological bacterium for preventing and treating the cabbage black spot pathogen.
Example 3
Determination of the bacterial inhibition Spectrum of Strain LY-1
Determining the antibacterial spectrum of the strain LY-1 by adopting a confrontation culture method: the target bacteria include Botrytis cinerea, Colletotrichum viticola, Fusarium oxysporum and apple rot.
Experimental groups: respectively beating the strain LY-1 and the target bacteria into fungus cakes of 0.4cm, dividing 4 plant pathogenic bacteria of botrytis cinerea, colletotrichum botrytis, fusarium oxysporum and apple rot pathogen into 4 experimental groups, beating the 4 target bacteria into fungus cakes of 0.4cm, respectively placing the fungus cakes in the center of a 9cm Gao's No.1 flat plate, placing the strain LY-1 on 4 symmetrical points 3cm away from the target fungus cakes, and placing the fungus cakes in a constant-temperature incubator at 25 ℃ for culture for 5 days.
Blank control group: dividing 4 plant pathogenic bacteria of botrytis cinerea, colletotrichum gloeosporioides, fusarium oxysporum and apple rot pathogen into 4 blank control groups, beating 4 target bacteria into 0.4cm fungus cakes, respectively placing the fungus cakes in the center of a 9cm Gao's No.1 flat plate, and culturing in a constant temperature incubator at 25 ℃ for 5 days.
Each experimental group and blank control group were treated 5 times in duplicate. The calculation formula is as follows: the bacteriostatic rate (%) was (blank group colony diameter-each experimental group colony diameter)/(blank group colony diameter-cake diameter) × 100.
And (3) calculating the bacteriostasis rate after the experiment is finished, wherein the experiment result in the table 2 is the bacteriostasis effect of the strain LY-1 on 4 plant pathogenic bacteria.
TABLE 2 bacteriostatic effect of Strain LY-1 on 4 plant pathogens
As can be seen from Table 2, the strain LY-1 has a certain inhibition rate on Botrytis cinerea, Colletotrichum viticola, Fusarium oxysporum f.sp.cubense and apple canker, and therefore, the strain LY-1 has a control effect on most vegetable diseases.
Example 4
Protective effect and treatment effect of strain LY-1 fermentation supernatant on cabbage black spot
Preparing fermentation liquor: culturing strain LY-1 on Gao's No.1 plate for 5 days, and scraping mycelium and spore to concentration of 108And (3) inoculating the CFU/ml spore solution into a liquid culture medium according to the volume ratio of 1%, wherein the liquid culture medium is preferably 1/3-2/5 in volume of the fermentation container, preparing a label, placing the label into a constant-temperature oscillator at 28 ℃ for shaking fermentation at 160r/min for 5-7d, centrifuging the fermentation liquor at 4 ℃ and 10000r/min for 20min, and taking the supernatant for measuring the prevention effect of the black spot of the Chinese cabbage.
The formula of the liquid culture medium is as follows: 20g of soluble starch, 0.01g of FeSO4.7H2O, 31.0g of KNO31, 20.5g of K2HPO20.5g, 0.5g of MgSO2.7H2O and 1000mL of distilled water, wherein the pH value is 7.4-7.6.
The prevention and treatment effect of LY-1 strain fermentation supernatant on cabbage black spot is determined by adopting a leaf method: the experimental group for testing comprises a LY-1 strain fermentation supernatant stock solution, a diluted 5-fold solution and a 10-fold solution, and a control medicament 10% difenoconazole WG which is a 1000-fold diluted solution.
Protection effect experiment: taking LY-1 strain fermented supernatant stock solution, diluted 5-fold solution and 10-fold solution and a control medicament 10% difenoconazole WG as 1000-fold diluent, dividing into 4 experimental groups, respectively spraying for 1 time, and inoculating 0.4cm of cabbage black spot germ cake after 24 h; spraying clear water as a blank control group, treating 10 Chinese cabbage leaves per concentration, performing moisture preservation culture for 5-7 days, measuring the diameter of lesion spots by adopting a cross method, and calculating the control effect of different concentrations.
Therapeutic effect experiments: inoculating 0.4cm cabbage black spot germ fungus cake, taking LY-1 strain fermented supernatant stock solution, diluted 5-fold solution and 10-fold solution and a control medicament 10% difenoconazole WG as 1000-fold diluent to divide into 4 experimental groups, respectively spraying for 24h for 1 time, spraying clear water as a blank control group, treating 10 cabbage leaves per concentration, performing moisture preservation culture for 5-7d, measuring the diameter of a disease spot by adopting a cross method, and calculating the control effect of different concentrations.
The control effect (%) is [ (blank control group lesion diameter-experimental group lesion diameter)/blank control group lesion diameter ] X100%
TABLE 3 prevention and treatment effect of fermentation supernatant of strain LY-1 on cabbage black spot
As can be seen from Table 3, the protective effect of the stock solution of the fermentation supernatant of the strain LY-1 on the black spot of the Chinese cabbage is 64.1%, the therapeutic effect is 41.7%, the protective effect of the 1000-fold diluent of the control medicament 10% difenoconazole WG is 66.2%, and the therapeutic effect is 45.5%, so that the difference between the protective effect and the therapeutic effect of the stock solution of the fermentation supernatant of the strain LY-1 and the control medicament is not significant, the control effect equivalent to the control medicament is achieved, and the control effect of the fermentation supernatant of the strain LY-1 on the black spot of the Chinese cabbage is achieved under the test conditions.
Example 5
Classification and identification of Strain LY-1
And (3) morphological identification: bacterial colony of the strain LY-1 on a Gao's first culture medium is initially red, turns pink at the later stage, is velvet and wet on the surface, is circular in shape, and is raised and filamentous at the edge. The substrate hyphae are rich, the aerial hyphae grow well, and the mature spore filaments are broken to form spores. It was identified as Streptomyces (Streptomyces) according to morphological observation.
And (3) molecular identification: the strain LY-1 is sent to Shanghai bio-engineering company for molecular identification. The primers are universal primers 5 '-AGTTTGATCMTGGCTCAG-3' and 5'-GGTTACCTTGTTACGACTT-3' used for bacterial 16SrDNA, PCR amplification is carried out, an amplification band (1460bp, see a sequence table specifically) with the size of about 1.5kb is obtained, and the strain LY-1 is identified to be Streptomyces roseoflavoticus (Streptomyces roseoviticus) through sequence comparison.
The results of examples 1 to 5 show that the strain LY-1 of Streptomyces roseoflavus (Streptomyces roseoviticus) is successfully separated and screened from soil samples in a cabbage field, and the strain LY-1 is deposited in: the China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC NO.17874 is used for preventing and treating vegetable diseases and cabbage black spot, has obvious prevention and treatment effect, is green and environment-friendly, and is environment-friendly.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> Qingdao agricultural university
<120> Streptomyces roseosporus strain and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1460
<212> DNA
<213> 16S rDNA of Streptomyces roseosporus LY-1 (16S rDNA of Streptomyces roseoviticus)
<400> 1
ggctcaggac gaacgctggc ggcgtgctta acacatgcaa gtcgaacgat gaagcccttc 60
ggggtggatt agtggcgaac gggtgagtaa cacgtgggca atctgccctg cactctggga 120
caagccctgg aaacggggtc taataccgga tatgaccttc gagcgcatgc ttgaaggtgg 180
aaagctccgg cggtgcagga tgagcccgcg gcctatcagc ttgttggtgg ggtgatggcc 240
taccaaggcg acgacgggta gccggcctga gagggcgacc ggccacactg ggactgagac 300
acggcccaga ctcctacggg aggcagcagt ggggaatatt gcacaatggg cgaaagcctg 360
atgcagcgac gccgcgtgag ggatgacggc cttcgggttg taaacctctt tcagcaggga 420
agaagcgaaa gtgacggtac ctgcagaaga agcgccggct aactacgtgc cagcagccgc 480
ggtaatacgt agggcgcaag cgttgtccgg aattattggg cgtaaagagc tcgtaggcgg 540
cttgtcacgt cggatgtgaa agcccggggc ttaaccccgg gtctgcattc gatacgggca 600
ggctagagtt cggtagggga gatcggaatt cctggtgtag cggtgaaatg cgcagatatc 660
aggaggaaca ccggtggcga aggcggatct ctgggccgat actgacgctg aggagcgaaa 720
gcgtggggag cgaacaggat tagataccct ggtagtccac gccgtaaacg ttgggaacta 780
ggtgtgggcg acattccacg tcgtccgtgc cgcagctaac gcattaagtt ccccgcctgg 840
ggagtacggc cgcaaggcta aaactcaaag gaattgacgg gggcccgcac aagcagcgga 900
gcatgtggct taattcgacg caacgcgaag aaccttacca aggcttgaca tacaccggaa 960
acggccagag atggtcgccc ccttgtggtc ggtgtacagg tggtgcatgg ctgtcgtcag 1020
ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccttgt cctgtgttgc 1080
cagcatgccc ttcggggtga tggggactca caggagactg ccggggtcaa ctcggaggaa 1140
ggtggggacg acgtcaagtc atcatgcccc ttatgtcttg ggctgcacac gtgctacaat 1200
ggccggtaca atgagctgcg ataccgtgag gtggagcgaa tctcaaaaag ccggtctcag 1260
ttcggattgg ggtctgcaac tcgaccccat gaagttggag ttgctagtaa tcgcagatca 1320
gcattgctgc ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac gtcacgaaag 1380
tcggtaacac ccgaagccgg tggcccaacc cttgtggagg gagccgtcga aggtgggact 1440
ggcgattggg acgaagtcgt 1460
Claims (5)
1. A Streptomyces roseofilariae (Streptomyces roseoviteriticus) strain LY-1, wherein the strain LY-1 is preserved in: china general microbiological culture Collection center (CGMCC) with a collection number of CGMCC NO. 17874.
2. Use of the Streptomyces roseovitericus strain LY-1 according to claim 1, wherein: the Streptomyces roseoflavus strain LY-1 is used for preventing and treating tomato gray mold, grape anthracnose, cucumber fusarium wilt and apple rot.
3. The use of Streptomyces roseoalvestris (Streptomyces roseoviticus) strain LY-1 according to claim 2, characterized in that: the Streptomyces roseoarworticus (Streptomyces roseoviteriticus) strain LY-1 is used for preventing and treating cabbage black spot.
4. A microbial agent for preventing and treating cabbage black spot is characterized in that: the effective active ingredient is the fermentation supernatant of Streptomyces roseovitericus strain LY-1 as claimed in claim 1.
5. The microbial agent for preventing and treating cabbage black spot disease according to claim 4, which is characterized in that: the fermentation supernatant is prepared from the raw materials with the concentration of 108CFU/mL of Streptomyces roseoalvens (Streptomyces roseoviticus) strain LY-1.
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Citations (3)
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| CN85102956A (en) * | 1984-04-16 | 1987-01-21 | 盐野义制药株式会社 | The preparation process of antibiotic DO-248-A and DO-248-B |
| WO2012019054A2 (en) * | 2010-08-05 | 2012-02-09 | Hera Pharmaceuticals, Inc. | Expression of antibody or a fragment thereof in lactobacillus |
| CN108220194A (en) * | 2018-01-09 | 2018-06-29 | 中国农业科学院植物保护研究所 | One plant of streptomyces parvus for having disease prevention growth-promoting function and its application |
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| CN103805527B (en) * | 2012-11-06 | 2017-02-08 | 陈家任 | StreptomycesYunnanensis var daliensis and fermentation broth and application thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN85102956A (en) * | 1984-04-16 | 1987-01-21 | 盐野义制药株式会社 | The preparation process of antibiotic DO-248-A and DO-248-B |
| WO2012019054A2 (en) * | 2010-08-05 | 2012-02-09 | Hera Pharmaceuticals, Inc. | Expression of antibody or a fragment thereof in lactobacillus |
| CN108220194A (en) * | 2018-01-09 | 2018-06-29 | 中国农业科学院植物保护研究所 | One plant of streptomyces parvus for having disease prevention growth-promoting function and its application |
Non-Patent Citations (2)
| Title |
|---|
| Identification of Polyketide Gene from Streptomyces roseoverticillatus and Its Antimicrobial Properties against Pathogenic Bacteria and Fungi;Arasu, Mariadhas Valan等;《Journal of Pure and Applied Microbiology》;20141231;第8卷;第59-67页，参见全文 * |
| 两株海洋放线菌的筛选鉴定及其次级代谢产物研究;齐麟;《中国优秀硕士学位论文全文数据库 (基础科学辑)》;20180630;第A006-190页，参见全文 * |
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