CN114214236B - Streptomyces scorpion LSS-F01 and application thereof in crop fungal disease control - Google Patents
Streptomyces scorpion LSS-F01 and application thereof in crop fungal disease control Download PDFInfo
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- CN114214236B CN114214236B CN202111578126.3A CN202111578126A CN114214236B CN 114214236 B CN114214236 B CN 114214236B CN 202111578126 A CN202111578126 A CN 202111578126A CN 114214236 B CN114214236 B CN 114214236B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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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Abstract
The invention belongs to the technical field of microbial control, and particularly relates to streptomyces scorpion and application thereof in crop fungal disease control. The preservation number of the Streptomyces scorpion (Streptomyces scopuliridis) LSS-F01 is CCTCC NO: M20211361, and the preservation date is 2021, 11 and 4. The streptomyces scorpion disclosed by the invention has a good inhibition effect on fusarium wilt bacteria, rice sheath blight bacteria, rice bakanae disease bacteria, sclerotinia rot bacteria, lily root rot bacteria and lily fusarium, and has a good application prospect.
Description
Technical Field
The invention relates to a novel microorganism strain and application thereof, in particular to a streptomyces scorpion strain and application thereof in biological disease control.
Background
With the development of the planting industry, a large amount of agricultural products are used, diseases of soil are increased, such as fungal diseases, bacterial disease virus infection and the like, the soil is acidified and hardened, and symptoms such as root rot, stem rot, fruit rot and the like of plants are caused, wherein the symptoms are mainly caused by serious inundation of soil-borne diseases, particularly fungal diseases which spread faster, including watermelon fusarium wilt, rice sheath blight, rice bakanae disease, rape sclerotium, lily root rot, lily fusarium and the like. In order to reduce this type of soil-borne disease, subsoil, plant rotation, rational fertilization, crop variety, etc. are relatively effective physical means.
The microbial pesticide is more and more widely paid attention to and applied to the aspect of preventing and controlling crop diseases and insect pests due to the characteristics of environmental protection, difficult generation of drug resistance and the like. The popularization and application technology of microbial pesticides are gradually becoming mature, the policy support force of the country in the field of biological control is increased year by year, the use of microbial pesticides can overcome the pollution of chemical pesticides to the ecological environment and reduce the pesticide residues in agricultural and sideline products, and the quality and price of the agricultural and sideline products are improved, so that the microbial pesticide has environmental benefits and economic benefits.
With the improvement of ecological consciousness of people, the development of ecological agriculture, green agriculture and organic agriculture is greatly focused, especially green ecological agriculture, such as biological prevention and control, and actinomycetes are also gradually put into the field of view of scientists as main production strains of antibiotics, and streptomyces is a kind of microorganism bacteria capable of producing antibiotics, and the streptomyces is hopeful to become another kind of microorganism pesticide widely applied in the field of biological prevention and control after bacillus and pseudomonas.
Disclosure of Invention
The invention aims to provide streptomyces scorpion (Streptomyces scopuliridis).
The invention also aims to provide the application of the streptomyces scorpion in crop disease control.
In order to achieve the above purpose, the invention adopts the following technical scheme:
streptomyces scorpion (Streptomyces scopuliridis) LSS-F01 is isolated from Hunan Xiangxidan lily soil actinomycetes and subjected to antagonistic screening of partial fungal diseases; the culture medium is preserved in China center for type culture Collection, the preservation address is the university of Wuhan, china, the preservation date is 2021, 11 and 4 days, and the preservation number is CCTCCNO: M20211361.
Further, the antagonistic streptomyces scorpion is obtained by screening by the following method: by adopting a flat plate facing method, pathogenic bacteria such as watermelon fusarium wilt bacteria, rice sheath blight bacteria, rice bakanae disease bacteria and sclerotinia rot bacteria are inoculated on a PDA culture medium for activation. On a sterile operation table, a hole is punched in the center of a PDA flat plate by a puncher, then four holes with the same size are uniformly punched at the periphery of a middle hole, actinomycetes and pathogenic fungi can be respectively put into the holes according to antagonistic capability of strains, and the radius of a bacteria inhibition zone is calculated. 3 times of each group are placed in a constant temperature incubator at 28 ℃ for culture, the growth vigor and the growth direction of fungus hyphae are observed, and the strain with antagonistic effect is preserved at 4 ℃ for molecular identification.
Further, the streptomyces scorpion has the following biological characteristics: the morphological characteristics of the Streptomyces scorpion (Streptomyces scopuliridis) LSS-F01 strain on a solid culture medium of Gaoshi No. one are as follows: aerial hyphae are grey-white to grey, and vegetative hyphae are pale yellow to yellow, resulting in a small amount of pale yellow soluble pigments.
Further, the Streptomyces scorpion LSS-F01 can be used for the control of pathogenic fungi of crops including, but not limited to, crop blight, banded sclerotial blight, bakanae disease, sclerotial blight.
Furthermore, the streptomyces scorpion LSS-F01 can be prepared into a pesticide preparation suitable for agriculture.
Compared with the prior art, the invention has the beneficial effects that: the streptomyces scorpion disclosed by the invention can effectively inhibit the growth of bacteria such as fusarium wilt bacteria, rice sheath blight bacteria, bakanae disease bacteria and sclerotinia rot of rice, is more environment-friendly and free of pesticide residues and is not easy to generate drug resistance compared with other traditional chemical control methods.
Drawings
FIG. 1 shows colony morphology of Streptomyces scorpion LSS-F01 strain on a medium;
FIG. 2 shows a spore pattern of Streptomyces scorpion LSS-F01 strain;
FIG. 3 shows antagonism of Streptomyces scorpion LSS-F01 strain against 7 different pathogenic bacteria;
FIG. 4 shows a phylogenetic tree of Streptomyces scorpion LSS-F01 strain.
Detailed Description
The following examples further illustrate the invention but are not to be construed as limiting the invention and modifications or alternatives to the methods, steps or fields of the invention are within the scope of the invention without departing from the spirit and nature of the invention.
The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
1. Isolation and screening of strains
50 soil actinomycetes of Hunan Xylon lily are obtained by separation through a plate dilution method, and are subjected to antagonistic test screening of partial fungal diseases.
Classification and purification of actinomycetes: taking 10.0g of soil sample, drying in a drying oven at 80 ℃ for 2 hours, taking 1.0g of dried soil, dissolving the dried soil into 30mL of sterile water, processing the dried soil for 30 minutes at 180rpm of a shaking table, taking out the dried soil and standing the dried soil, taking supernatant liquid as 10-1 initial liquid, sequentially obtaining 10-2, 10-3, 10-4, 10-5 and 10-6 gradient dilutions (1 mL of soil suspension is added into 9 mL of sterile water), respectively coating 300 mu L of each of 10-2, 10-4 and 10-6 onto a first Gao's and modified first Gao's separation medium, culturing the dried soil at 28 ℃ for 3-4 days, growing single bacterial colonies on a flat plate, picking single bacterial colonies to purify the culture medium for 3 times, and finally obtaining purified bacterial strains, identifying and storing the purified bacterial strains.
The formula of the Gaoshi No. 1 culture medium is as follows: soluble starch 20.0g, KNO 3 1.0g， NaCl 0.5g，K 2 HPO 4 ·3H 2 O 0.5g，MgSO 4 ·7H 2 O 0.5g，FeSO 4 ·7H 2 O0.01 g, agar18.0g, 1000mL of distilled water, pH7.4-7.6.
The formula of the modified Gaoshi No. 1 culture medium is as follows: soluble starch 10.0g, KNO 3 0.5g， NaCl 0.25g，K 2 HPO 4 ·3H 2 O 0.25g，MgSO 4 ·7H 2 O 0.25g，FeSO 4 ·7H 2 O0.01 g, agar 18.0g, distilled water 1000mL, pH7.4-7.6.
Screening of antagonistic actinomycetes: by adopting a flat plate facing method, watermelon fusarium wilt, rice sheath blight, bakanae disease, sclerotinia rot of colza, root rot of lily and fusarium lily are inoculated on a PDA culture medium for activation. On a sterile operation table, a hole is punched in the center of a PDA flat plate by a puncher, then four holes with the same size are uniformly punched at the periphery of a middle hole, and actinomycetes and pathogenic fungi can be respectively put into the holes according to antagonistic capability of the strain, so that the antibacterial radius is measured. 3 times of each group are placed in a constant temperature incubator at 28 ℃ for culture, the growth vigor and the growth direction of fungus hyphae are observed, and the strain with antagonistic effect is preserved at 4 ℃ for molecular identification.
2. Identification of actinomycetes
1. Morphological identification
The morphological characteristics of the streptomyces scorpion (Streptomyces scopuliridis) strain on a solid culture medium of Gao's No. one are as follows: aerial hyphae are grey-white to grey, and vegetative hyphae are pale yellow to yellow, resulting in a small amount of pale yellow soluble pigments.
Inoculating Streptomyces scorpion strain to a culture medium of Gao's first, culturing for 14-21d at 28 ℃ by adopting an inserting method, observing individual forms of the strain, and observing mycelium forms of the strain under an optical microscope: the spore chain morphology and spore shape are shown in FIG. 2.
2. Physiological and biochemical identification
The physiological and biochemical characteristics of the LSS-F01 strain were determined by reference to ISP series media and methods recommended in the handbook of the identification of common bacterial systems and the handbook of the identification of Berger systems, as shown in tables 1 and 2.
TABLE 1 characterization of Strain culture (ISP 2-7 is a Medium specific for actinomycetes identification)
TABLE 2 physiological and biochemical characteristics of strains
Remarks: positive for "+"; "-" is negative;
3. molecular biological identification
(1) Scraping aerial hyphae rich in the surface of a culture medium from solid culture, and placing the aerial hyphae in a mortar frozen in advance;
(2) adding appropriate amount of 2×CTAB solution and appropriate amount of sterilized and dried fine sand, rapidly grinding in mortar (frozen for 48 hr after autoclaving), or grinding in liquid nitrogen;
(3) after grinding, rapidly transferring into an EP tube of 1.5mL, carrying out water bath at 65 ℃ for 40min, and gently reversing every 10min;
(4) taking out, standing, cooling, and adding an equal volume of chloroform: the isoamyl alcohol mixed solution (24:1) is fully and evenly mixed, and is centrifuged for 10min at the temperature of 4 ℃ and at the speed of 11000 rpm/min;
(5) aspirate supernatant into new EP tube;
(6) purification was performed using a gel recovery kit manufactured by TOYOBO company, the procedure of which is described in the specification;
(7) add 30. Mu.L sterile water to dissolve DNA,1.2% agarose gel electrophoresis.
Identification of Strain molecular biology Universal primers synthesized by Shanghai Biotechnology Co., ltd
27F：5’-AGAGTTTGATCCTGGCTCAG-3’
1492R：5’-ACGGTTACCTTGTTACGACTT-3’
And amplifying to obtain a PCR product, sequencing and correcting to obtain a partial 16S rRNA gene sequence of LSS-F01 for molecular taxonomic identification of actinomycetes. The PCR amplification system is adopted: 25. Mu.L of reaction system (1. Mu.L of template DNA,1. Mu.L of forward and reverse primers, 2X Taq PCR Master Mix. Mu.L each), actinomycete reaction conditions: pre-denaturation at 94℃for 3min, denaturation at 94℃for 30s, annealing at 55℃for 1min, extension at 72℃for 2min,32 cycles; finally, the mixture is extended for 10min at 72 ℃ and stored at 4 ℃. The identification result shows that the strain belongs to Streptomyces and is classified and named as Streptomyces scorpion (Streptomyces scopuliridis).
16S rRNA gene sequence and phylogenetic analysis of actinomycete strains: BLAST comparison analysis is carried out on the obtained strain 16S rRNA gene sequence and known sequences in GenBank database, and a system evolutionary tree LSS-F01 strain constructed by adopting MEGA 5.0 is Streptomyces scopuliridis which belongs to the same branch, and the sequence similarity is 99.79%. The final product is named as Streptomyces scorpion (Streptomyces scopuliridis) through morphological characteristics and molecular biological identification results.
3. Determination of bacteriostatic activity of streptomyces scorpion on pathogenic bacteria
By adopting a flat plate facing method, watermelon fusarium wilt bacteria, rice sheath blight bacteria, rice bakanae disease bacteria, sclerotinia rot bacteria, lily root rot bacteria and lily fusarium are inoculated on a PDA culture medium for activation. On a sterile operation table, a hole is punched in the center of a PDA flat plate by using a puncher, then four holes with the same size are uniformly punched at the periphery of a middle hole, actinomycetes and pathogenic fungi can be respectively put into the holes according to antagonistic capability of the strain, and the antibacterial radius is measured, and the antagonistic effect is shown in table 3.
TABLE 3 radius of inhibition (cm) of Streptomyces scorpions against pathogenic fungi
As shown in Table 3, the Streptomyces scorpion strain has stronger antagonism to fusarium wilt bacteria, rice sheath blight bacteria, bakanae disease bacteria, sclerotinia rot bacteria, root rot bacteria and fusarium wilt bacteria of lily, and utilizes the means of biological control, namely the biological or metabolic products thereof to effectively control plant diseases, thereby being more environment-friendly, free of pesticide residues and not easy to generate drug resistance compared with other traditional chemical control.
The above embodiments are only for illustrating the present invention, and are not limiting of the present invention. While the invention has been described in detail with reference to the embodiments, those skilled in the art will appreciate that various combinations, modifications, and substitutions can be made thereto without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
<110> institute of agricultural biotechnology in Hunan province
<120> Streptomyces scorpion LSS-F01 and application thereof in crop fungal disease control
<130> 1
<141> 2021-12-22
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1396
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
tacacatgca gtcgaacgat gaagcctttc ggggtggatt agtggcgaac gggtgagtaa 60
cacgtgggca atctgccctt cactctggga caagccctgg aaacggggtc taataccgga 120
taatacttct gcctgcatgg gcgggggttg aaagctccgg cggtgaagga tgagcccgcg 180
gcctatcagc ttgttggtgg ggtgatggcc taccaaggcg acgacgggta gccggcctga 240
gagggcgacc ggccacactg ggactgagac acggcccaga ctcctacggg aggcagcagt 300
ggggaatatt gcacaatggg cgaaagcctg atgcagcgac gccgcgtgag ggatgacggc 360
cttcgggttg taaacctctt tcagcaggga agaagcgaaa gtgacggtac ctgcagaaga 420
agcgccggct aactacgtgc cagcagccgc ggtaatacgt agggcgcaag cgttgtccgg 480
aattattggg cgtaaagagc tcgtaggcgg cttgtcgcgt cggatgtgaa agcccggggc 540
ttaaccccgg gtctgcattc gatacgggca ggctagagtg tggtagggga gatcggaatt 600
cctggtgtag cggtgaaatg cgcagatatc aggaggaaca ccggtggcga aggcggatct 660
ctgggccatt actgacgctg aggagcgaaa gcgtggggag cgaacaggat tagataccct 720
ggtagtccac gccgtaaacg ttgggaacta ggtgttggcg acattccacg tcgtcggtgc 780
cgcagctaac gcattaagtt ccccgcctgg ggagtacggc cgcaaggcta aaactcaaag 840
gaattgacgg gggcccgcac aagcagcgga gcatgtggct taattcgacg caacgcgaag 900
aaccttacca aggcttgaca tacaccggaa acggccagag atggtcgccc ccttgtggtc 960
ggtgtacagg tggtgcatgg ctgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc 1020
gcaacgagcg caacccttgt tctgtgttgc cagcatgcct ttcggggtga tggggactca 1080
caggagactg ccggggtcaa ctcggaggaa ggtggggacg acgtcaagtc atcatgcccc 1140
ttatgtcttg ggctgcacac gtgctacaat ggccggtaca atgagctgcg atgccgtgag 1200
gcggagcgaa tctcaaaaag ccggtctcag ttcggattgg ggtctgcaac tcgaccccat 1260
gaagtcggag ttgctagtaa tcgcagatca gcattgctgc ggtgaatacg ttcccgggcc 1320
ttgtacacac cgcccgtcac gtcacgaaag tcggtaacac ccgaagccgg tggcccaacc 1380
ccttgtggga gggagc 1396
Claims (3)
1. Streptomyces scorpion (Streptomyces scopuliridis) LSS-F01 has a preservation date of 2021, 11 and 4 days and a preservation number of CCTCCM 20211361.
2. A microbial agent comprising the streptomyces scorpion LSS-F01 of claim 1.
3. Use of streptomyces scorpion (Streptomyces scopuliridis) LSS-F01 as claimed in claim 1 for controlling crop diseases, characterized in that the crop diseases are watermelon fusarium wilt, rice sheath blight, rice bakanae disease, sclerotinia rot, lily root rot and lily sickle.
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| CN103114064A (en) * | 2013-03-08 | 2013-05-22 | 浙江省柑桔研究所 | Marine actinomycete with antibacterial activity to multiple plant pathogens |
| CN106635878A (en) * | 2016-10-25 | 2017-05-10 | 扬州大学 | Streptomyces sp. 5X4 and applications thereof as biocontrol bacterium in preventing and treating plant diseases |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103114064A (en) * | 2013-03-08 | 2013-05-22 | 浙江省柑桔研究所 | Marine actinomycete with antibacterial activity to multiple plant pathogens |
| CN106635878A (en) * | 2016-10-25 | 2017-05-10 | 扬州大学 | Streptomyces sp. 5X4 and applications thereof as biocontrol bacterium in preventing and treating plant diseases |
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