CN117568229B - Bacillus simplex CYG04 and application thereof - Google Patents
Bacillus simplex CYG04 and application thereof Download PDFInfo
- Publication number
- CN117568229B CN117568229B CN202311644621.9A CN202311644621A CN117568229B CN 117568229 B CN117568229 B CN 117568229B CN 202311644621 A CN202311644621 A CN 202311644621A CN 117568229 B CN117568229 B CN 117568229B
- Authority
- CN
- China
- Prior art keywords
- bacillus simplex
- tea
- bacillus
- cyg04
- simplex
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 241000193400 Bacillus simplex Species 0.000 title claims abstract description 34
- 201000010099 disease Diseases 0.000 claims abstract description 48
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 48
- 230000012010 growth Effects 0.000 claims abstract description 15
- 230000001737 promoting effect Effects 0.000 claims abstract description 9
- 238000003973 irrigation Methods 0.000 claims abstract description 5
- 230000002262 irrigation Effects 0.000 claims abstract description 5
- 238000005507 spraying Methods 0.000 claims abstract description 3
- 241001122767 Theaceae Species 0.000 claims description 55
- 238000000855 fermentation Methods 0.000 claims description 38
- 230000004151 fermentation Effects 0.000 claims description 38
- 241000123650 Botrytis cinerea Species 0.000 claims description 13
- 241000223221 Fusarium oxysporum Species 0.000 claims description 11
- 241000894006 Bacteria Species 0.000 claims description 8
- 241000813090 Rhizoctonia solani Species 0.000 claims description 6
- 244000000005 bacterial plant pathogen Species 0.000 claims description 6
- 239000012681 biocontrol agent Substances 0.000 claims description 6
- 238000004321 preservation Methods 0.000 claims description 6
- 241000221696 Sclerotinia sclerotiorum Species 0.000 claims description 5
- 241000235395 Mucor Species 0.000 claims description 3
- 239000000022 bacteriostatic agent Substances 0.000 claims description 3
- 230000005764 inhibitory process Effects 0.000 claims description 3
- 238000000034 method Methods 0.000 claims description 2
- 241000308375 Graminicola Species 0.000 claims 2
- 241000233614 Phytophthora Species 0.000 claims 2
- 239000003112 inhibitor Substances 0.000 claims 2
- 230000003385 bacteriostatic effect Effects 0.000 claims 1
- 230000002401 inhibitory effect Effects 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 26
- 240000003553 Leptospermum scoparium Species 0.000 abstract description 22
- 235000009024 Ceanothus sanguineus Nutrition 0.000 abstract description 21
- 235000015459 Lycium barbarum Nutrition 0.000 abstract description 21
- 230000001965 increasing effect Effects 0.000 abstract description 5
- 230000003405 preventing effect Effects 0.000 abstract description 5
- 244000269722 Thea sinensis Species 0.000 abstract 1
- 238000012271 agricultural production Methods 0.000 abstract 1
- 244000005700 microbiome Species 0.000 abstract 1
- 239000001963 growth medium Substances 0.000 description 16
- 241000193830 Bacillus <bacterium> Species 0.000 description 12
- 230000000844 anti-bacterial effect Effects 0.000 description 11
- 239000002689 soil Substances 0.000 description 11
- 244000052616 bacterial pathogen Species 0.000 description 10
- 230000002265 prevention Effects 0.000 description 8
- 238000011218 seed culture Methods 0.000 description 8
- 235000010633 broth Nutrition 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 5
- 239000003337 fertilizer Substances 0.000 description 5
- 241000196324 Embryophyta Species 0.000 description 4
- 241000233616 Phytophthora capsici Species 0.000 description 4
- 230000035784 germination Effects 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 229960001225 rifampicin Drugs 0.000 description 4
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 3
- 241000589540 Pseudomonas fluorescens Species 0.000 description 3
- 240000003768 Solanum lycopersicum Species 0.000 description 3
- 240000008042 Zea mays Species 0.000 description 3
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 3
- 239000003899 bactericide agent Substances 0.000 description 3
- 235000005822 corn Nutrition 0.000 description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 3
- 235000019341 magnesium sulphate Nutrition 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 description 3
- 244000052769 pathogen Species 0.000 description 3
- 230000001717 pathogenic effect Effects 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000008223 sterile water Substances 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- 238000004659 sterilization and disinfection Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- 241000223600 Alternaria Species 0.000 description 2
- 241000223602 Alternaria alternata Species 0.000 description 2
- 244000063299 Bacillus subtilis Species 0.000 description 2
- 241001661371 Exobasidium vexans Species 0.000 description 2
- 241000896533 Gliocladium Species 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 241001523629 Pestalotiopsis Species 0.000 description 2
- 241000233622 Phytophthora infestans Species 0.000 description 2
- 241001624808 Pseudopestalotiopsis theae Species 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 235000019764 Soybean Meal Nutrition 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 238000005273 aeration Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 230000006806 disease prevention Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 235000013312 flour Nutrition 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 239000000575 pesticide Substances 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000004455 soybean meal Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241000221198 Basidiomycota Species 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 241001429695 Colletotrichum graminicola Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 235000019733 Fish meal Nutrition 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 208000031888 Mycoses Diseases 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 206010034133 Pathogen resistance Diseases 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 229930182764 Polyoxin Natural products 0.000 description 1
- 241000588769 Proteus <enterobacteria> Species 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 206010039509 Scab Diseases 0.000 description 1
- 241000221662 Sclerotinia Species 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 241000223260 Trichoderma harzianum Species 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 230000009604 anaerobic growth Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000000443 biocontrol Effects 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 239000004467 fishmeal Substances 0.000 description 1
- 244000053095 fungal pathogen Species 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000020477 pH reduction Effects 0.000 description 1
- 230000003071 parasitic effect Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000000447 pesticide residue Substances 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- YEBIHIICWDDQOL-YBHNRIQQSA-N polyoxin Polymers O[C@@H]1[C@H](O)[C@@H](C(C=O)N)O[C@H]1N1C(=O)NC(=O)C(C(O)=O)=C1 YEBIHIICWDDQOL-YBHNRIQQSA-N 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G17/00—Cultivation of hops, vines, fruit trees, or like trees
- A01G17/005—Cultivation methods
-
- 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
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P21/00—Plant growth regulators
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P3/00—Fungicides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Environmental Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Virology (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Botany (AREA)
- Agronomy & Crop Science (AREA)
- Dentistry (AREA)
- Mycology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the field of agricultural microorganisms, and discloses a bacillus simplex CYG04 and application thereof, wherein the bacillus simplex is bacillus simplex @, and the bacillus simplex is bacillus simplex @Bacillus simplex) CYG04, accession number: CCTCC M20231901, the strain is reported to be used for preventing and controlling tea cake diseases, round spot diseases and gray mold of tea trees through leaf surface spraying for the first time, has the effect of promoting growth and increasing yield of tea trees through root irrigation, and has wide application prospect in agricultural production.
Description
Technical Field
The invention belongs to the field of microbial biocontrol bacteria, and particularly relates to bacillus simplex CYG04 and application thereof.
Background
Tea trees grow mostly in warm, moist tropical and subtropical areas, and the climate in these areas is also conducive to the development of tea tree diseases. Tea cake disease and round spot disease are two important diseases of tea, are reported in tea planting areas all over the world, and also commonly occur in tea areas in China, can cause great yield reduction of tea, quality reduction and serious influence on tea production. Wherein, the pathogenic bacteria of the tea cake disease are damage external basidiomycetes Exobasidium vexans (introducing pathogenic bacteria of the tea cake disease and the round spot disease), belong to obligate parasitic bacteria, and can not be cultured in vitro, which brings difficulty to the research of the disease. At present, pseudomonas fluorescens Pf has a certain prevention and control effect on tea cake disease fields, but the biological control bacteria such as Pseudomonas exist in a nutritional form, no spores exist, the shelf life is short, great difficulty exists in popularization and application, and bacillus subtilis Bacillus subtilis B-9 has a certain antibacterial activity on tea cake disease pathogenic bacteria. At present, only 3 pesticides of polyoxin, bacillus subtilis and fructus psoraleae seed extracts are registered in tea cake diseases in China, and the prevention and control medicament is relatively lacking. The tea leaf spot is mainly caused by Pestalotiopsis fungi (Pestalotiopsis spp.) and the like, ha Cimu mould (Trichoderma harzianum), gliocladium viridis (Gliocladium virena) and Pseudomonas fluorescens (Pseudomonas fluorescens) are separated from tea garden soil by Sanjay and the like, and the field control effect is below 40%, so that the effect is not ideal. In addition, bacillus belicus CY30 has been reported to have a certain prevention effect on tea leaf spot, and no registered medicament for tea leaf spot is available at present. The use of chemical bactericides is still the main means for preventing and treating tea cake diseases and round spot diseases, but the application of chemical bactericides is extremely easy to cause pathogen resistance, and simultaneously causes pesticide residues and environmental pollution.
The fertilizer is one of key agricultural measures affecting the quality and yield of tea and sustainable utilization of soil, and has remarkable quality improvement and synergy effects on tea. The traditional tea garden mainly uses the constant-speed effective fertilizers such as urea and compound fertilizer, and the fertilizer application amount is large, so that the problems of tea quality reduction and the like caused by soil acidification and hardening are solved in production.
At present, the report of simple bacillus in disease prevention and control is that the P10 strain has good antibacterial effect on phytophthora infestans of potato late blight pathogenic bacteria, the STY005 strain has good prevention effect on potato scab, the FJAT-47158 strain has good antibacterial effect on fusarium oxysporum, and the tea cake pathogenic bacteria belong to basidiomycetes and have a difference from the plant pathogenic bacteria taxonomies. In addition, none of the strains is isolated from the rhizosphere soil of tea trees, is not indigenous to the tea garden, and has differences in root and tea garden soil colonization ability and indigenous to the microbes, thereby affecting the growth-promoting and yield-increasing effects of tea trees.
Aiming at the problems, the invention separates and screens a bacillus simplex CYG04 strain from the rhizosphere soil of tea trees, researches the prevention and control effects of tea cake diseases, round spot diseases and gray mold, the rhizosphere colonization capability of the tea trees and the growth promotion effect of the tea trees, and aims to lay a foundation for the application of the CYG04 in the tea trees in the later period.
Disclosure of Invention
The invention aims at providing an isolated Bacillus simplex, which is named as Bacillus simplex (CYG 04), and has the preservation number of: cctccc No. M20231901.
It is another object of the present invention to provide the use of an isolated Bacillus simplex strain useful for preparing a microbial preparation having disease prevention and/or growth promotion properties for tea leaves, including for controlling tea cake disease, round spot and gray mold, for promoting tea growth.
In order to achieve the above object, the present invention adopts the following technical measures:
isolated Bacillus simplex is obtained by screening tea leaf spot and gray mold disease, and is determined to be Bacillus simplex by comparing 16s rDNA with physiological and biochemical identification, and the strain is sent to China center for type culture collection for preservation at 10 months and 16 days in 2023, and is named after classification: bacillus simplex CYG04; the preservation number is: cctccc No. M20231901; location: chinese, university of martial arts, martial arts.
The colony of the strain grows well in NA culture medium, and the colony is light yellow (figure 1).
The scope of the invention includes fermentation broths of bacillus simplex CYG04, and culture media used in obtaining fermentation broths include, but are not limited to, LB medium, or culture media: 10-12g/L corn flour, 6-8g/L soybean meal, 10-12g/L yeast powder, 0.2-0.5g/L monopotassium phosphate, 0.4-0.6g/L magnesium sulfate and pH value of 6-7.
The protection scope of the invention also comprises: the application of the simple bacillus or the fermentation liquor containing the simple bacillus viable bacteria in preparing plant pathogenic bacteria bacteriostat;
preferably, the inhibition object of the bacteriostatic agent is: mucor pulmonale (Pestalotiopsis theae), botrytis cinerea (Botrytis cinerea), proteus putida (Exobasidium vexans), phytophthora capsici (Phytophthora capsici), sclerotinia sclerotiorum (Sclerotinia sclerotiorum), rhizoctonia solani (Rhizoctonia solani), po Gu Tanju (Colletotrichum graminicola), fusarium oxysporum (Fusarium oxysporum) and/or Alternaria alternata.
The application of the simple bacillus or the fermentation liquor containing the simple bacillus viable bacteria in preparing the tea biocontrol agent;
preferably, the control object of the biocontrol agent is tea cake disease, round spot disease and/or gray mold;
the application of the simple bacillus or the fermentation liquor containing the simple bacillus viable bacteria in promoting the growth of tea.
In the application, the bacteriostatic agent or the biocontrol agent is applied in a spraying mode;
in the application, the mode of promoting the growth of the tea leaves is root irrigation.
Compared with the prior art, the invention has the following advantages:
(1) The invention provides a simple bacillus strain which has strong stress resistance and is easy to ferment in a large amount, and can prevent and control tea cake diseases and round spot diseases for the first time.
(2) The strain has the activity of promoting growth of tea trees, is convenient to use, has no residue and pollution, and can effectively reduce the application of chemical fertilizers and pesticides.
(3) The simple bacillus of the invention is separated from the rhizosphere of tea trees, can be colonized on tea tree plants, and is also the simple bacillus which can be colonized on tea trees after first separation.
Drawings
FIG. 1 schematic plate colony morphology of strain CYG04.
Figure 2 bacterial strain CYG04 has antibacterial effect on tea leaf spot and gray mold pathogenic bacteria.
FIG. 3 schematic diagram of the control effect of 10-fold dilution of CYG04 fermentation broth on tea gray mold.
Detailed Description
For a better explanation of the present invention, the main content of the present invention is further elucidated below in conjunction with the specific examples, but the content of the present invention is not limited to the following examples only. The technical scheme of the invention is conventional technology in the field unless specifically stated, and the reagents or materials are commercially available unless specifically stated.
Example 1:
acquisition and identification of Bacillus simplex CYG 04:
bacillus simplex CYG04 is isolated from the rhizosphere soil (figure 1) of Mao Bazhen tea trees in Enshichun, hubei province, and is obtained by screening the antibacterial activity of pathogenic bacteria of tea leaf spot and gray mold. The strain is further determined by 16s rDNA and physiological and biochemical identification (table 1), and is sent to China center for type culture collection for preservation in 2023, 10 months and 16 days, and is named after classification: bacillus simplex CYG04; the preservation number is CCTCC NO 20231901; location: chinese, university of martial arts, martial arts.
In the present invention, bacillus simplex (CYG 04 or simply CYG 04).
TABLE 1 physiological and biochemical characterization of CGY04
| Characteristics of | Results |
| Gram reaction | + |
| Anaerobic growth | — |
| Growth at 4 DEG C | — |
| Growth at 40 DEG C | — |
| Growth of 5% NaCl | + |
| Growth of 7% NaCl | — |
| Hydrolyzed starch | + |
| Hydrolyzed gelatin | + |
| Nitrate reduction | + |
| V-P test | — |
| Contact enzyme | + |
| Hydrolyzed casein | + |
| Oxidase enzyme | — |
Fermentation of Bacillus simplex CYG04 (the percentages in the following culture medium formula are mass percentages):
1) Primary seed culture: 100ml of primary seed culture medium is filled in a 500ml triangular flask, wet heat sterilization is carried out for 30min at 121 ℃, a CYG04 freeze-dried tube strain is inoculated in the primary seed culture medium, and shaking culture is carried out for 10h at 35 ℃ and 180 rpm; the culture medium used for the primary seed culture comprises the following raw materials in percentage by weight: glucose 4.0%, beef extract 2%, peptone 1.5%, sodium chloride 0.5% and pH of the culture medium 7;
2) Secondary seed culture: 200L of secondary seed culture medium is filled in a 500L fermentation tank, wet heat sterilization is carried out for 30min at 121 ℃, and 200ml of primary seeds are inoculated in the secondary seed culture medium; culturing at 35 ℃ for 12h; the tank pressure is controlled to be 0.05Mpa, the stirring rotation speed is 200rpm, the aeration ratio is 1:0.5, and the raw materials and the consumption of the culture medium used for the secondary seed culture are as follows: glucose 0.5%, yeast extract 05%, fish meal 2%, potassium dihydrogen phosphate 0.001%, magnesium sulfate 0.01%, and culture medium pH7;
3) Fermentation: a 10 ton fermentation tank is filled with 5 tons of CYG04 fermentation culture medium, wet heat sterilization is carried out for 30min at 121 ℃, 200L of secondary seed liquid is transplanted into the fermentation culture medium, the tank pressure is controlled to be 0.02Mpa, the stirring rotation speed is 150rpm, the aeration ratio is 0.5-1.0, and the culture is carried out for 24h at 35 ℃; the culture medium used for fermentation comprises the following raw materials in percentage by weight: 11g/L corn flour, 6g/L soybean meal, 10g/L yeast powder, 0.3g/L monopotassium phosphate, 0.5g/L magnesium sulfate and pH7 of a culture medium;
4) Ending fermentation when 20-40% of spores fall off, and placing the fermentation liquor in a tank to obtain the fermentation liquor with the number of 120 hundred million cfu/mL. For the following examples.
Example 3:
the application of the in vitro antibacterial activity of the bacillus simplex CYG04 in the prevention and treatment of tea cake diseases, round spot diseases and gray mold:
test alternaria leaf spot (Pestalotiopsis theae) and Botrytis cinerea (Botrytis cinerea) were isolated from alternaria leaf spot and Botrytis cinerea leaves and stored on PDA inclined planes at 4 ℃. Other pathogenic fungi are phytophthora capsici (Phytophthora capsici), sclerotinia rot pathogen (Sclerotinia sclerotiorum), rhizoctonia solani (Rhizoctonia solani) corn anthracnose pathogen grass Gu Tanju, fusarium oxysporum (Fusarium oxysporum) and tobacco brown spot pathogen chain lattice cells (Alternaria alternata), which are all stored on a PDA inclined plane at 4 ℃.
Transferring the bevel strain into PDA plate for activation, and culturing at 30deg.C for 6 d. The sterilized filter paper was placed at both ends of the PDA plate, followed by dropping 7. Mu.L of CYG04 fermentation broth. After 24 hours, the fungal disease pathogenic bacteria dish is accessed by a 4mm puncher, sterile water is used as a blank control, and the distance between pathogenic bacteria and a filter paper sheet is 25mm. Three replicates were set for each trial, and after the plates were full with control, the diameters of each treated and control colony were counted and the rate of inhibition was calculated.
Antibacterial ratio = [ (control colony diameter-treated colony diameter)/(control colony diameter-bacterial cake diameter) ] ×100%
In vitro bacteriostasis experiments show that CYG04 has good bacteriostasis activity on various plant pathogenic bacteria, wherein the bacteriostasis rate on the trichomadzus tea and the botrytis cinerea is 88.21% and 85.37% (figure 2, table 2).
TABLE 2 antibacterial Activity of CYG04 against pathogenic bacteria
After activation of Mucor pulmonale and Botrytis cinerea to PDA plate culture for 5d, tea leaves (middle tea 108) with consistent size and without bactericide are collected for in vitro leaf test. CYG04 fermentation broth was prepared according to example 2, with a spore count of about 120 hundred million cfu.mL -1 The fermentation broth was diluted 10-fold and 20-fold with sterile water, respectively, for use. The fermentation broth was sprayed evenly on leaf leaves using an electric sprayer, 2mL per leaf, and sterile water was sprayed in a blank. After each treatment was air-dried, tea leaf spot and gray mold cake were inoculated using a needle pricking inoculation method, and the culture was kept wet at 28℃for 12 days, and the control and treatment spot diameters were counted and the control effect was calculated, and each treatment was repeated 3 times, 15 leaves each, and the control effect= (control spot diameter-treatment spot diameter/control spot diameter) ×100.
Experiments show that the simple bacillus CYG04 has good control effects on tea leaf spot and gray mold, the diameter of the leaf spot after treatment by 10 times and 20 times of fermentation liquor is obviously lower than that of a control, the control effects on shift are 75.93% and 56.19%, and the control effects on gray mold are 70.93% and 56.83% (table 3, table 4 and figure 3).
TABLE 3 prevention and treatment effects of CYG04 fermentation liquor on tea leaf spot
| Treatment of | Diameter of disease spot (mm) | Preventing effect (%) |
| CYG04 times fermentation liquor | 7.11 | 75.93 |
| CYG04 20 times fermentation liquor | 12.94 | 56.19 |
| Control | 29.54 | - |
TABLE 4 control effect of CYG04 fermentation liquor on tea gray mold
| Treatment of | Diameter of disease spot (mm) | Preventing effect (%) |
| CYG04 times fermentation liquor | 6.82 | 70.93 |
| CYG04 20 times fermentation liquor | 10.17 | 56.83 |
| Control | 23.56 | - |
The tea cake disease field efficacy test is located in the Hefeng county of Enshi, and the CYG04 fermentation liquor diluted by 100 times is sprayed before the disease occurs, and the tea cake disease field efficacy test is sprayed for 3 times every 7 days, and the clear water is sprayed in a comparison mode. The test was run with a total of 4 replicates of 32m each 2 After the disease is fully developed by contrast, the disease index is counted and compared, 5 points are taken from each cell, 50 leaves are used for investigating the disease index, and the control effect is calculated. Disease index grading standard: level 0: no disease spots; stage 1: the area of the disease spots accounts for less than 1% of the area of the blade; 3 stages: the area of the disease spots accounts for 2% -10% of the area of the leaf blades; 5 stages: the area of the lesion accounts for 11% -25% of the area of the leaf; 7 stages: the area of the disease spots accounts for 26% -50% of the leaf area; stage 9: the area of the disease spots occupies more than 50 percent of the leaf area;
disease index = Σ (number of plants at each stage x grade)/(total number of plants investigated x highest representative grade) ×100;
control effect = (control disease index-treatment disease index)/control disease index x 100%
Experiments show that the CYG04 has good field control effect on tea cake diseases, and the control effect of 100 times of fermentation liquor reaches 65.59 percent (table 5).
TABLE 5 prevention and treatment effects of CYG04 fermentation liquor on tea cake diseases
| Treatment of | Index of disease condition | Preventing effect (%) |
| CYG04 100-fold fermentation liquor | 14.65 | 65.59 |
| Control | 42.57 | - |
Example 4:
growth promoting and yield increasing effects of bacillus simplex CYG04 on tea trees
The test field is located in Hefeng county of Enshi in Hubei province, and CYG04 fermentation stock solution (120 hundred million cfu.mL) -1 ) Diluting 20 times, root irrigation treatment is carried out on tea trees, the water consumption per mu is 600 jin, and water irrigation is carried out in comparison. Once every 7 days, three total applications. And counting the germination density in the period of one bud and two leaves after 20d, investigating the germination density (33.3 cm multiplied by 33.3cm square), and measuring the weight of hundred buds.
Experiments show that CYG04 can obviously improve the germination density of tea trees, and the hundred-bud weight is reduced, but the quality of one-bud two-leaf tea leaves per unit area is increased, so that the tea has obvious yield increasing effect (Table 6).
Table 6CYG04 has effect of promoting growth and increasing yield of tea tree
| Treatment of | Germination density (individual) | Hundred buds weight (g) |
| CYG04 | 143.61 | 38.64 |
| CK | 111.32 | 39.28 |
Example 5:
bacillus simplex CYG04 ability to colonize rhizosphere soil of tea tree
Screening single colony of Bacillus simplex CYG04 in medium containing different concentrations of rifampicin to obtain anti-rifampicin mutant strain (which has no significant difference between physiological and biochemical characteristics except rifampicin resistance and original strain), and can be used in medium containing 300 mug.mL -1 Is grown normally in LB of rifampicin.
The resistant mutation CYG04 is fermented according to the example 2, the number of spores of the fermentation liquor is regulated to 1 hundred million cfu/mL by using clean water, the fermentation liquor is sprayed on the roots of tea trees, the application amount is 2L/plant, and tomatoes are sprayed on control plants. Obtaining rhizosphere soil after 30d by shaking off, diluting by gradient dilution method, and coating on a substrate containing 300 mug.mL -1 After 24h incubation at 37 ℃ and determining the ability of CYG04 to colonize the soil of the rhizosphere of tea leaves and tomatoes.
Experiments show that the CYG04 has stronger colonization capability in the rhizosphere soil of tea trees, has specificity, and the colonization amount is 4.16X10 6 cfu/g, while at tomato colonization only 4.23×10 4 cfu/g。
Claims (8)
1. Isolated simple bacillusBacillus simplex) The bacillus simplex is bacillus simplex CYG04, and the preservation number is: cctccc No. M20231901.
2. The use of bacillus simplex or a fermentation broth containing live bacillus simplex as claimed in claim 1 for preparing a plant pathogenic bacteria inhibitor, wherein the inhibition object of the inhibitor is: tea trayRadix seu cortex Heteropanacis PubescentisPestalotiopsis theae) Botrytis cinerea (Botrytis cinerea)Botrytis cinerea) The damage to the external bacteriaExobasidium vexans) Phytophthora capsiciPhytophthora capsici) Sclerotinia sclerotiorum (L.) KuntzeSclerotinia sclerotiorum) Rhizoctonia solani (wall.) kuntzeRhizoctonia solani) Gu Tanju bacteria of He typeColletotrichum graminicola) Fusarium oxysporum (F.oxysporum)Fusarium oxysporum) And/or chain lattice cellAlternaria alternata）。
3. Use of bacillus simplex or a fermentation broth containing a live bacillus simplex strain according to claim 1 for inhibiting plant pathogenic bacteria, said plant pathogenic bacteria being: radix et rhizoma Rhei-like Mucor pulmonalePestalotiopsis theae) Botrytis cinerea (Botrytis cinerea)Botrytis cinerea) The damage to the external bacteriaExobasidium vexans) Phytophthora capsiciPhytophthora capsici) Sclerotinia sclerotiorum (L.) KuntzeSclerotinia sclerotiorum) Rhizoctonia solani (wall.) kuntzeRhizoctonia solani) Gu Tanju bacteria of He typeColletotrichum graminicola) Fusarium oxysporum (F.oxysporum)Fusarium oxysporum) And/or chain lattice cellAlternaria alternata）。
4. The use of bacillus simplex or a fermentation broth containing live bacillus simplex as claimed in claim 1 for preparing a tea biocontrol agent.
5. The method according to claim 4, wherein the control object of the biocontrol agent is tea cake disease, round spot disease and/or gray mold.
6. Use of bacillus simplex or a fermentation broth containing a viable bacillus simplex as claimed in claim 1 for promoting the growth of tea leaves.
7. The use according to claim 2 or 4, wherein the bacteriostatic or biocontrol agent is applied by spraying.
8. The use according to claim 6, wherein the means for promoting the growth of tea leaves is root irrigation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311644621.9A CN117568229B (en) | 2023-12-04 | 2023-12-04 | Bacillus simplex CYG04 and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311644621.9A CN117568229B (en) | 2023-12-04 | 2023-12-04 | Bacillus simplex CYG04 and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117568229A CN117568229A (en) | 2024-02-20 |
| CN117568229B true CN117568229B (en) | 2024-04-12 |
Family
ID=89889878
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311644621.9A Active CN117568229B (en) | 2023-12-04 | 2023-12-04 | Bacillus simplex CYG04 and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117568229B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105002121A (en) * | 2015-08-14 | 2015-10-28 | 山东泰诺药业有限公司 | Bacillus simplex and application thereof |
| CN109957535A (en) * | 2019-05-08 | 2019-07-02 | 河北省农林科学院遗传生理研究所（河北省农林科学院农产品质量安全研究中心） | Simple bacillus, microbial bacterial agent, bio-fertilizer and the application prepared using it |
| WO2023168448A1 (en) * | 2022-03-03 | 2023-09-07 | Indigo Ag, Inc. | Endophyte compositions and methods for improved plant health |
-
2023
- 2023-12-04 CN CN202311644621.9A patent/CN117568229B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105002121A (en) * | 2015-08-14 | 2015-10-28 | 山东泰诺药业有限公司 | Bacillus simplex and application thereof |
| CN109957535A (en) * | 2019-05-08 | 2019-07-02 | 河北省农林科学院遗传生理研究所（河北省农林科学院农产品质量安全研究中心） | Simple bacillus, microbial bacterial agent, bio-fertilizer and the application prepared using it |
| WO2023168448A1 (en) * | 2022-03-03 | 2023-09-07 | Indigo Ag, Inc. | Endophyte compositions and methods for improved plant health |
Non-Patent Citations (1)
| Title |
|---|
| 简单芽孢杆菌产高效微生物絮凝剂;薛冬等;环境工程学报;20120805;6(8);2897-2902 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117568229A (en) | 2024-02-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113969247B (en) | Bacterium for inhibiting tobacco disease pathogenic bacteria and application thereof | |
| CN110564646B (en) | Bacillus belgii CY30 and application thereof in prevention and treatment of tea round spot | |
| CN111944716B (en) | Special compound microbial agent for tobacco seedling culture and preparation method and application thereof | |
| CN111073825B (en) | Bacterium with plant soil-borne disease resistance effect and application thereof | |
| CN112725218B (en) | Bacillus belgii and culture method and application thereof | |
| CN111172063A (en) | Bacillus beleisi MES861 and application and product thereof | |
| CN101503708A (en) | Cultivation fermentation method of Bacillus coagulans antimycotics active substance | |
| CN113755393A (en) | Bacillus beilesensis HP-24 and application thereof in preparation of bacterial liquid for preventing and treating bacterial fruit blotch of melons | |
| CN103224897A (en) | Bacillussubtilis for tobacco black shank prevention and control | |
| CN107099475B (en) | Bacillus methylotrophicus and preparation and application of microbial inoculum thereof | |
| CN114231442B (en) | Parlerian Long Nishi pseudomonas and application thereof in prevention and treatment of oomycete pathogenic bacteria soil-borne diseases | |
| CN117305149B (en) | Bacillus HBTG1666 in strain table and application thereof in biocontrol of tea tree | |
| CN111748496B (en) | Application of Brevibacillus laterosporus MES818 in tomato cultivation | |
| CN114085797B (en) | Bacillus complex inoculant and application thereof to rice | |
| CN104152372A (en) | Biocontrol strain G68 for preventing and treating plant diseases, and preparation method and application of microbial inoculant thereof | |
| CN102586136B (en) | Microbial inoculant for improving disease resistance and promoting growth of plants | |
| CN103333844A (en) | Strain of antagonistic poplar colletotrichumgloeosporioides and application thereof | |
| CN114456973B (en) | Streptomyces rochei in tobacco and application thereof in prevention and control of tobacco diseases | |
| CN103122330A (en) | Alpine grassland pasture endogenous serratia plymuthica strain GH010 and application thereof as well as microbial agent prepared from same and preparation method | |
| CN114350552B (en) | Bacillus pumilus and application thereof in control of medlar diseases | |
| CN117568229B (en) | Bacillus simplex CYG04 and application thereof | |
| CN110240528A (en) | A kind of microbial organic fertilizer and its application | |
| CN110373362A (en) | One plant of Bacillus cercus and its application in prevention and treatment bacterial ring rot o potato | |
| CN116004419A (en) | Bacillus atrophaeus CY-2, microbial inoculum, preparation method and application thereof | |
| CN114128724A (en) | Application of bacillus amyloliquefaciens in tomato cultivation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |