CN116590199B - Paenibacillus piri and application thereof in prevention and treatment of corn ear rot - Google Patents
Paenibacillus piri and application thereof in prevention and treatment of corn ear rot Download PDFInfo
- Publication number
- CN116590199B CN116590199B CN202310761574.XA CN202310761574A CN116590199B CN 116590199 B CN116590199 B CN 116590199B CN 202310761574 A CN202310761574 A CN 202310761574A CN 116590199 B CN116590199 B CN 116590199B
- Authority
- CN
- China
- Prior art keywords
- paenibacillus
- piri
- corn
- ear rot
- corn ear
- 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
- 235000002017 Zea mays subsp mays Nutrition 0.000 title claims abstract description 70
- 240000008042 Zea mays Species 0.000 title claims abstract description 69
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 title claims abstract description 65
- 235000005822 corn Nutrition 0.000 title claims abstract description 65
- 241000179039 Paenibacillus Species 0.000 title claims abstract description 41
- 238000011282 treatment Methods 0.000 title claims description 12
- 230000002265 prevention Effects 0.000 title claims description 6
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 claims abstract description 14
- 241000223218 Fusarium Species 0.000 claims abstract description 14
- 241000082085 Verticillium <Phyllachorales> Species 0.000 claims abstract description 14
- 238000004321 preservation Methods 0.000 claims abstract description 5
- 230000001580 bacterial effect Effects 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 14
- 241000894006 Bacteria Species 0.000 claims description 13
- 241000196324 Embryophyta Species 0.000 claims description 10
- 241000611735 Paenibacillus peoriae Species 0.000 claims description 5
- 239000002068 microbial inoculum Substances 0.000 claims description 5
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 2
- 230000000813 microbial effect Effects 0.000 claims 1
- 230000003042 antagnostic effect Effects 0.000 abstract description 15
- 238000002474 experimental method Methods 0.000 abstract description 6
- 238000000338 in vitro Methods 0.000 abstract description 5
- 239000000463 material Substances 0.000 abstract description 3
- 238000012404 In vitro experiment Methods 0.000 abstract description 2
- 238000001727 in vivo Methods 0.000 abstract description 2
- 201000010099 disease Diseases 0.000 description 13
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- 108020004465 16S ribosomal RNA Proteins 0.000 description 9
- 244000052616 bacterial pathogen Species 0.000 description 9
- 239000000203 mixture Substances 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 239000008223 sterile water Substances 0.000 description 8
- 230000003321 amplification Effects 0.000 description 7
- 238000003199 nucleic acid amplification method Methods 0.000 description 7
- 239000000499 gel Substances 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 101150013736 gyrB gene Proteins 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 4
- 230000000443 biocontrol Effects 0.000 description 4
- 235000009973 maize Nutrition 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 230000035882 stress Effects 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 238000003794 Gram staining Methods 0.000 description 3
- 241000209140 Triticum Species 0.000 description 3
- 235000021307 Triticum Nutrition 0.000 description 3
- 230000000844 anti-bacterial effect Effects 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000008635 plant growth Effects 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000002791 soaking Methods 0.000 description 3
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 230000004544 DNA amplification Effects 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 231100000678 Mycotoxin Toxicity 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 241000520272 Pantoea Species 0.000 description 2
- 239000005708 Sodium hypochlorite Substances 0.000 description 2
- 235000013339 cereals Nutrition 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 238000009533 lab test Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000002636 mycotoxin Substances 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 101150012629 parE gene Proteins 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000005507 spraying Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 241001660259 Cereus <cactus> Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 241001057636 Dracaena deremensis Species 0.000 description 1
- 241000588914 Enterobacter Species 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- 241000233732 Fusarium verticillioides Species 0.000 description 1
- 208000031888 Mycoses Diseases 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 241000992035 Scopulibacillus daqui Species 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 240000006394 Sorghum bicolor Species 0.000 description 1
- 235000011684 Sorghum saccharatum Nutrition 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 241000482268 Zea mays subsp. mays Species 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000000853 biopesticidal effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000013043 chemical agent Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 230000008641 drought stress Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 210000005069 ears Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 239000007952 growth promoter Substances 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000006241 metabolic reaction Methods 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000003641 microbiacidal effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 238000004161 plant tissue culture Methods 0.000 description 1
- 238000004382 potting Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 238000004080 punching Methods 0.000 description 1
- 230000009933 reproductive health Effects 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229910021654 trace metal Inorganic materials 0.000 description 1
- 239000003039 volatile agent 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
- A01G13/00—Protecting plants
-
- 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
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/20—Cereals
-
- 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
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
-
- 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
- A01N63/25—Paenibacillus
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Environmental Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Microbiology (AREA)
- Virology (AREA)
- Plant Pathology (AREA)
- Medicinal Chemistry (AREA)
- Pest Control & Pesticides (AREA)
- General Engineering & Computer Science (AREA)
- Biodiversity & Conservation Biology (AREA)
- Ecology (AREA)
- Forests & Forestry (AREA)
- Botany (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Chemical & Material Sciences (AREA)
- Agronomy & Crop Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Toxicology (AREA)
- Dentistry (AREA)
- Mycology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses paenibacillus piri which is paenibacillus piri SAAS-3-B4 and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M20221703 and application thereof in corn ear rot. The in vitro antagonistic flat plate counter experiment and in vivo antagonistic leaf in vitro experiments prove that the strain can better prevent and treat corn ear rot caused by Fusarium verticillium. As a biological control material for corn ear rot, the strain has good application prospect.
Description
Technical Field
The invention relates to the field of biological control of plant diseases, in particular to paenibacillus piri and application thereof in control of corn ear rot.
Background
Endophyte is a microorganism that survives within plants but does not affect the host, and that is living in the plant cell environment to perform symbiotic specific functions, such as the synthesis of secondary metabolites or signal molecules as internal and external stimuli in the reciprocal process. Endophytes are a source of novel biomolecules for the biochemical and pharmaceutical industries. They produce bioactive metabolites including immunosuppressive compounds, anticancer agents, plant growth promoters, antibacterial volatiles, pesticides, antioxidants and antibiotics, and have great application potential in medicine, pharmaceutical industry. In addition, the endophyte can promote the growth of plants under severe conditions such as nutritional stress, temperature stress, salinity stress, trace metal stress or drought stress, and the growth of plants in polluted environments is assisted by degrading harmful compounds, so that the endophyte is also an important research object and resource treasury in the agricultural field. In addition, the endophytes are also an important treasury of plant disease biocontrol bacteria resources. In recent years, scientists have also screened a plurality of biocontrol bacteria for dealing with important plant diseases, so that the biocontrol technology is gradually paid attention to.
Corn is one of the main food crops in china and is one of the crops whose global production is currently in the first place. The total amount of planting area of the corn is only lower than that of wheat and rice. The corn ear rot is an infectious disease which occurs in the middle and later stages of the growth and development of corn, and is found in all large corn producing areas in the world, the loss rate in the field is generally 5% -10%, and the serious loss rate can reach more than 50%. These fungal diseases not only directly cause significant production and economic losses of corn and wheat crops, but also the mycotoxin products and many secondary metabolic reaction products caused by the infection of these pathogenic bacteria seriously jeopardize the safety quality of corn grain production, and long-term eating of these corn grains polluted and deteriorated by mycotoxins also seriously jeopardize the reproductive health of human life and domestic fowl and livestock, and even cause death.
The damage caused by corn ear rot to corn yield is increasing, and selecting corn disease-resistant varieties and using chemical microbiocides are common effective means for preventing corn ear rot. However, since selecting disease-resistant varieties consumes huge time and energy and a large amount of chemical agents are adopted to prevent the problems of pollution, ecological imbalance and the like caused by the corn ear rot, an effective and safe corn ear rot control scheme must be searched.
In view of this, the present invention has been made.
Disclosure of Invention
Aiming at the problems, the invention provides paenibacillus piri and application thereof in preventing and controlling corn ear rot.
Research has found that Fusarium verticillium (Fusarium verticillioides) is one of the most well known diseases during the growth and development of corn, which is often affected by a variety of pathogenic bacteria. Fusarium verticillium can infect multiple crops such as corn, sorghum, wheat and the like, causing a plurality of epidemic diseases such as seedling blight, stem rot, ear rot, seed rot and the like of corn.
Wherein, the corn ear rot caused by the fusarium verticillium is changed into: during the corn kernel filling period, fusarium verticillium can infect corn kernels or cobs, causing cobs and kernels to mould and rot, thus severely affecting corn yield. Based on the above, the present invention screens out the above paenibacillus pierce from a plurality of strains and uses it for controlling corn ear rot caused by fusarium verticillium.
In a first aspect, the present invention provides a paenibacillus piri, named paenibacillus piri SAAS-3-B4, deposited in the chinese collection of typical cultures; the preservation unit address is Chinese, wuhan, university of Wuhan; the preservation number is CCTCC NO, M20221703; the preservation time is 2022, 10 and 31 days; the taxonomical name of this strain is Paenibacillus peoriae.
According to the invention, more than one hundred strains of strains are obtained from a corn sample collected from a farm laboratory test station in Shanghai city, 1 antagonistic strain for better preventing and treating corn ear rot is obtained through screening, and through further research, the 16S rDNA sequence of the antagonistic strain is shown as SEQ ID NO:1, belonging to Paenibacillus peoriae strains, which were designated as Paenibacillus piri SAAS-3-B4.
In a second aspect, the invention also provides application of the paenibacillus piri in corn disease control.
In a third aspect, the invention also provides an application of the paenibacillus piri in controlling plant diseases caused by fusarium verticillium.
In a fourth aspect, the present invention also provides a Fusarium verticillium inhibitor comprising the Paenibacillus piri described above; the live bacteria content of the Paenibacillus aurei in the Fusarium verticillium inhibitor is 10 10~1011 CFU/Kg.
In a fifth aspect, the invention also provides a microbial inoculum for preventing and treating corn ear rot, the microbial inoculum comprises the paenibacillus piri, and the living bacterial content of the paenibacillus piri in the microbial inoculum is 10 10~1011 CFU/Kg.
In a sixth aspect, the invention also provides a method for preventing and treating corn ear rot, which comprises applying the bacterial liquid of paenibacillus piri or the bacterial agent to diseased parts of plants.
The invention has the following beneficial effects:
The invention separates and identifies endophytic bacteria of corn plants, screens an antagonistic strain capable of better preventing and controlling corn ear rot caused by fusarium verticillium through an in vitro antagonistic flat plate counter experiment and an in vivo antagonistic leaf in vitro experiment, and further, has good application prospect no matter developing new biopesticide or biocontrol microbial inoculum as a biological prevention and control material for corn ear rot.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the result of the facing culture of Paenibacillus cereus SAAS-3-B4 in example 1;
FIG. 2 is a colony morphology and gram stain of Paenibacillus piri SAAS-3-B4 of example 1;
FIG. 3 is a phylogenetic tree of Paenibacillus piri SAAS-3-B4 in example 1;
FIG. 4 is a graph showing the effect of Paenibacillus piri SAAS-3-B4 in example 2 on controlling maize leaves;
FIG. 5 is a graph showing the leaf area statistics under different experimental conditions in example 2;
FIG. 6 is a comparison of potting results in example 2.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
This example is the isolation, screening and identification of Paenibacillus piri SAAS-3-B4
1. Material acquisition:
(1) Corn samples collected at a laboratory test station of Shanghai national academy of agricultural sciences are placed in sterile bags and stored in a 4 ℃ refrigerator of a timely transportation laboratory.
(2) Classifying and marking according to the sample collecting parts, respectively washing corn samples with sterile water for 5min, washing for 2 times, soaking with 75% alcohol solution for 3min, and washing with sterile water for 3 times; then, the mixture was immersed in a 3% sodium hypochlorite solution for 3min, and finally, the mixture was rinsed with sterile water for 4 times.
2. Isolation of endophyte:
(1) The method for separating the crop endophytes comprises the following steps: according to different sampling positions, the corn samples are divided into 6 types, namely sweet corn leaves, waxy corn leaves, corn ears, corn aerial roots, corn stems and corn roots. The sterilized corn samples were cut into small pieces by a sterile surgical scissors and put into a mortar for grinding to obtain a ground sample suspension, the sample suspension was diluted to 10 -1、10-2、10-3 times of dilution, 200 μl of each of the different dilutions was sequentially extracted and respectively applied to three solid media (NB, PDA and YPD), and each treatment was repeated three times. During the cultivation, colonies and mycelia growing on the plates were observed daily, and the grown colonies and mycelia were transferred to a new plate medium in time for the next step of isolation and purification.
(2) And (3) surface disinfection effect inspection: in the process of sterilizing the corn surface, a set of blank control experiments are required to check whether the sterilization is thorough or not so as to ensure that the separated endophytes are not polluted by microorganisms attached to the corn surface. The specific method comprises the following steps: the wash solution from the last sterile water wash of corn surface was collected and spread on NB planar medium and co-cultured with the experimental group.
If no colony grows in the blank control plate, the corn surface is thoroughly disinfected and sterilized, and the colony grown by the experimental group is corn endophyte. If colonies grow out in the blank control plate, the surface of the corn is proved to be not thoroughly disinfected and sterilized, and non-corn endophytes are mixed in.
3. Purification of endophyte:
Colonies growing in the three media were observed and colonies were selected for further culture on new solid media. Purified maize endophyte was stored in a refrigerator at 4℃and-80℃respectively.
4. Extraction of genomic DNA:
(1) Performing amplification culture on the purified single colony, picking 1-2-ring thalli from an amplification culture plate, uniformly mixing in 200 mu L of sterile water, adding a proper amount of 0.5mm glass beads, adopting a DNA rapid preparation system, extracting DNA by shaking to obtain crude extract of corn endophytic bacteria DNA, centrifuging, and taking 1 mu L of supernatant as template DNA for subsequent PCR amplification.
(2) 16S rDNA Gene amplification: 1 PCR tube (200. Mu.L) was used, and the mixture was sequentially added according to the composition shown in Table 5, and the mixture was divided into 2 PCR tubes (25. Mu.L). The general primers for bacterial 16S rDNA were used, and the primer sequences are shown in Table 1.
TABLE 1 primers for identifying species classifications
(3) Detecting PCR products by gel electrophoresis: ① Agarose gel was prepared at a concentration of 1%. ② Dye (20 ml plus 1 μl) was added, and the mixture was poured into a gel making tank into which a comb was inserted. ③ And (5) gelling and fixing, pulling out the comb, and putting the gel making groove into the electrophoresis groove. ④ The corresponding substances were added 5. Mu.L each to each well. ⑤ And taking out the gel after electrophoresis, and putting the gel into a gel imaging analyzer. ⑥ The electrophoresis results were observed with a gel imaging analyzer.
(4) 16S rDNA sequence determination: and (3) sending the PCR amplified product to Shanghai biological company for sequencing, and completing sequence splicing by using DNAMAN software according to the obtained sequencing result. The spliced sequences were aligned using the NCBI BLAST tool.
Through analysis, 148 bacterial strains were obtained in this example, and these 148 bacterial strains were distributed among 16 families and 26 genera, wherein the genera with higher abundance were Bacillus (Bacillus), escherichia (Enterobacter), and Pantoea (Pantoea), respectively.
5. Screening of strains
(1) Primary screen
Screening antagonistic bacteria of 148 corn endophytes by a plate counter experiment: inoculating Fusarium verticillium serving as a pathogenic bacterium to a PDA culture medium for activation, taking a bacterial cake from a pathogenic bacterium flat plate by using a puncher with the aperture of 5mm, placing the bacterial cake in the center position of a new PDA flat plate, inoculating 4 different corn endophytic bacteria in the cross direction of the bacterial cake, culturing at the constant temperature of 28 ℃ for 5 days, and checking whether a bacteriostasis ring appears.
(2) Double screen
Antagonistic strains obtained in Primary Inspection screens were subjected to a plate counter method to determine the in vitro antagonistic capacity: the bacterial cake is obtained from the pathogenic bacteria flat plate by using a puncher with the aperture of 5mm, the bacterial cake is placed at the center of a new PDA flat plate, antagonistic bacteria are inoculated at the position 5cm away from the bacterial cake in the cross direction of the center of the flat plate, the antagonistic bacteria at four inoculated positions are connected into a square shape, each treatment is repeated three times, the constant temperature culture is carried out at 28 ℃, the diameter of bacterial colony is measured when the pathogenic bacteria hypha of a control group is fully paved in a culture dish, and the bacteriostasis rate of different antagonistic bacteria is calculated.
Inhibition (%) = (control colony diameter-experimental colony diameter)/control colony diameter×100
The experimental results are shown in FIG. 1. According to statistics, the paenibacillus piri SAAS-3-B4 has the best antibacterial effect, and the average antibacterial rate is 64%.
6. Identification of strains
(1) Morphological observation
Endophyte colony morphology: after 24h incubation of the coated medium plates, single colonies were observed and the colony size, color, appearance, boundary, permeability, etc. were recorded. Endophytic bacteria were stained by gram staining and observed with an optical microscope.
Endophytic fungus mycelium morphology: the mycelia of the pathogenic B17-1 strain were observed in morphology after 5d culture on PDA medium. Inoculating endophytic fungus spores, shake culturing at 28deg.C and 200rpm in sterile liquid culture medium for 3d, and observing the characteristics such as spore shape by using an optical microscope.
According to observation, the paenibacillus piri SAAS-3-B4 is in a rod shape, so that spores can be generated, and single bacterial colonies of the paenibacillus piri SAAS-3-B are irregularly round, convex on the surface, fine and opaque, pale yellow in bacterial colony color and complete in boundary. The result of the gram staining is red purple, and the identification of the Paenibacillus piri SAAS-3-B4 as gram negative bacteria is proved. As shown in fig. 2, the left side is a colony morphology; on the right are gram staining charts.
And (3) analyzing the evolutionary tree: 16S rDNA and gyrB gene sequence analysis of Paenibacillus piri SAAS-3-B4 and phylogenetic tree establishment: the PCR amplification of the 16S rDNA gene fragment and the gyrB gene was completed using the antagonistic strain genomic DNA as a template, the amplification system and the procedure for the 16S rDNA gene fragment amplification were as shown in Table 2, and the amplification system and the procedure for the gyrB gene amplification were as shown in Table 3.
TABLE 2 System and procedure for 16S rDNA amplification of Gene
TABLE 3 procedure for gene gyrB amplification
The amplified products were sent to Shanghai Biotechnology Inc. for sequencing, and the resulting 16S rDNA and gyrB sequencing results were aligned in NCBI, from which 8 Paenibacillus species and corresponding genomes were selected as the outer population, scopulibacillus daqui. And (3) comparing by using a ClustalW method in MEGA 7 to construct a phylogenetic tree.
Phylogenetic tree As shown in FIG. 3, paenibacillus piri SAAS-3-B4 (3-B4 for short) is in the same branch as Paenibacillus piri (Paenibacillus peoriae), so 3-B4 is considered Paenibacillus piri (Paenibacillus peoriae).
Example 2
The embodiment is the indoor disease prevention effect verification of paenibacillus piri SAAS-3-B4
1. Cultivation of maize seedlings
Taking Shen Kenuo corn seeds, washing with sterile water for about 5min, washing for 2 times, soaking with 75% alcohol solution for 3min, and washing with sterile water for 3 times; then, the mixture was immersed in a 3% sodium hypochlorite solution for 3min, and finally, the mixture was rinsed with sterile water for 4 times.
60 Corn seeds are selected and soaked in warm water with the water temperature of 35 ℃ for 4 hours, two layers of sterilized filter paper are paved on 4 large flat plates, water is added for soaking, the corn seeds are divided into 4 parts, and every 15 corn seeds are evenly paved in the flat plates. Placing the plant into a plant tissue culture chamber for culture.
2. Determination of biological control effect of antagonistic strain on corn ear rot
In order to further verify the resistance function of Paenibacillus piri SAAS-3-B4 to corn ear rot, corn leaf in vitro verification experiments are carried out, the experiments are divided into 4 groups, and the leaves are inoculated with pathogenic bacteria Fusarium verticillium (B17-1) by a needle-punching inoculation method. The treatment method of the experimental group is shown in table 4:
TABLE 4 in vitro blade test treatments
The experimental treatments were divided into 4 groups according to the above table, and leaf spot sizes were measured after 5d of indoor culture to observe the disease conditions of maize leaves. Wherein the experimental group 3 (the pinhole inoculation pathogenic bacteria B17-1, spraying 3-B4 suspension after 12 hours) is a treatment group; experimental group 4 (spraying 3-B4 suspension, pinhole inoculation of pathogenic bacteria B17-1 after 12 h) was a prophylactic group. The plaque area size of each experimental group was measured and statistically analyzed using ImageJ software, and the experimental results are shown in fig. 4.
As shown in fig. 5, the control treatment of the single inoculated pathogen in the experimental group 2 had an average plaque area of 0.8cm 2, the treatment group had an average plaque area of 0.29cm 2, and the prevention group had an average plaque area of 0.40cm 2, both of which had an average plaque area smaller than that in the experimental group 2.
As shown in the experimental results of the potted plant in FIG. 6, the group A is a control group without SAAS-3-B4 sprayed thereon, the disease spots are in an extended and enlarged shape, the leaves are curled and rotten, the group B is an experimental group, the disease spots have obvious limits, and the area of the disease spots is far smaller than that of the control group, which indicates that the paenibacillus piri SAAS-3-B4 has obvious inhibition effect in the treatment and prevention of the spike rot.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (4)
1. The paenibacillus piri is characterized in that the paenibacillus piri (Paenibacillus peoriae) is paenibacillus piri SAAS-3-B4, and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M20221703.
2. Use of paenibacillus piri according to claim 1 for the treatment and prevention of ear rot of corn caused by fusarium verticillium.
3. A microbial agent for preventing and treating corn ear rot, which is characterized by comprising the paenibacillus pierce according to claim 1;
the live bacteria content of Paenibacillus piri in the microbial inoculum is 10 10~1011 CFU/Kg.
4. A method for controlling corn ear rot caused by fusarium verticillium, characterized in that the bacterial liquid of paenibacillus piri according to claim 1 or the bacterial agent according to claim 3 is applied to diseased parts of plants.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310761574.XA CN116590199B (en) | 2023-06-26 | 2023-06-26 | Paenibacillus piri and application thereof in prevention and treatment of corn ear rot |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310761574.XA CN116590199B (en) | 2023-06-26 | 2023-06-26 | Paenibacillus piri and application thereof in prevention and treatment of corn ear rot |
Publications (2)
Publication Number | Publication Date |
---|---|
CN116590199A CN116590199A (en) | 2023-08-15 |
CN116590199B true CN116590199B (en) | 2024-07-05 |
Family
ID=87601010
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310761574.XA Active CN116590199B (en) | 2023-06-26 | 2023-06-26 | Paenibacillus piri and application thereof in prevention and treatment of corn ear rot |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116590199B (en) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114085793A (en) * | 2021-11-18 | 2022-02-25 | 河南省科学院生物研究所有限责任公司 | Production method and application of Paenibacillus epidermidis microbial inoculum for preventing and treating plant fungal diseases |
CN115895940A (en) * | 2022-09-15 | 2023-04-04 | 广西民族大学 | Application of paenibacillus dermorhinus volatile secondary metabolite in agricultural production |
Family Cites Families (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102776147B (en) * | 2012-04-16 | 2017-03-08 | 杨凌绿都生物科技有限公司 | Gene recombinaton Pierre auspicious Russia series bacillus and its construction method and application |
CA2956880C (en) * | 2014-08-04 | 2023-04-25 | Basf Se | Antifungal paenibacillus strains, fusaricidin-type compounds, and their use |
CN104498041B (en) * | 2014-11-10 | 2018-06-05 | 杨凌绿都生物科技有限公司 | Application of the auspicious Russia's series bacillus of Pierre as soil ecology improvement renovation agent |
CN104789490B (en) * | 2015-03-18 | 2018-05-18 | 杨凌绿都生物科技有限公司 | A kind of complex microorganism decomposing microbial inoculum, preparation method and application |
WO2016187703A1 (en) * | 2015-05-22 | 2016-12-01 | Institut National De La Recherche Scientifique | Bacterial and fungal metabolites possessing anti-microbial activity against xanthomonas species, compositions, methods, kits and uses relating to same |
CN110423712B (en) * | 2019-08-05 | 2022-12-23 | 慕恩(广州)生物科技有限公司 | Paenibacillus porrigens and application thereof |
US20230016040A1 (en) * | 2019-11-22 | 2023-01-19 | Novozymes Bioag A/S | Paenibacillus isolates and uses thereof |
RU2724464C1 (en) * | 2019-12-02 | 2020-06-23 | Общество с ограниченной ответственностью «Агрофирма «Урожайная» | Strains, biopreparation, biopreparation production method and method for biological protection of crops against fusariosis |
RU2751487C1 (en) * | 2020-07-03 | 2021-07-14 | Общество с ограниченной ответственностью «Агрофирма «Урожайная» | Method for producing liquid nutrition medium and method for obtaining liguid microbiological agent based on strains mixture of spore-forming bacteria-antagonists of phytopathogenic fungi of g. fusarium |
CN112760258B (en) * | 2021-01-27 | 2022-02-01 | 中国农业科学院蔬菜花卉研究所 | Paenibacillus piricola and application thereof in preventing and treating cucumber bacterial soft rot |
CN116144522A (en) * | 2022-08-11 | 2023-05-23 | 山东省葡萄研究院 | Paenibacillus pumilus and application thereof in preventing and treating white rot of grape |
-
2023
- 2023-06-26 CN CN202310761574.XA patent/CN116590199B/en active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114085793A (en) * | 2021-11-18 | 2022-02-25 | 河南省科学院生物研究所有限责任公司 | Production method and application of Paenibacillus epidermidis microbial inoculum for preventing and treating plant fungal diseases |
CN115895940A (en) * | 2022-09-15 | 2023-04-04 | 广西民族大学 | Application of paenibacillus dermorhinus volatile secondary metabolite in agricultural production |
Also Published As
Publication number | Publication date |
---|---|
CN116590199A (en) | 2023-08-15 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN102433282B (en) | Bacillus subtilis NB12, as well as culture method and application thereof | |
CN105543132B (en) | A kind of Methylotrophic bacillus YB-F7 and its application in controlling plant diseases | |
CN111778195B (en) | Bacillus aryabhattai, microbial inoculum, preparation method and application | |
CN112280716B (en) | Bacillus altitudinis YG045 and application thereof | |
CN112143686B (en) | Bacillus altitudinis ST15 for antagonizing xanthomonas oryzae and application thereof | |
CN109504639A (en) | Bacillus amyloliquefaciens GUMT319 and its application | |
CN108641989A (en) | One plant of Methylotrophic bacillus and its application | |
CN108913622B (en) | Bacillus megaterium BM22 and preparation and application of spore powder thereof | |
CN110317747A (en) | A kind of bacillus amyloliquefaciens JT68 and its application in prevention and treatment tea anthracnose | |
CN110184224A (en) | One staphylococcus epidermidis and its application | |
CN111778174B (en) | Bacillus subtilis with inhibiting effect on citrus sand skin disease and screening method thereof | |
CN107164233B (en) | Chaetomium globosum LJ-S2L1 strain and application thereof | |
CN109735457B (en) | Mutant eurotium cristatum and application thereof in preventing and controlling barnyard grass | |
CN114836329B (en) | Trichoderma harzianum HB40609 and application thereof | |
CN116590199B (en) | Paenibacillus piri and application thereof in prevention and treatment of corn ear rot | |
CN115197853A (en) | Endophyte Epicoccum thailandicumLF-28 strain and application thereof | |
CN111454848B (en) | Actinomucor elegans GD48, fermentation product, microbial inoculum and application thereof | |
CN109055236B (en) | Isaria pinicola WSWM1171 and application thereof in prevention and control of potato corm moth pupae | |
CN116179640B (en) | Method for efficiently screening antagonistic bacteria of banana vascular wilt | |
CN113925064B (en) | Application of B-lysine-resistant bacillus in inhibition of growth of pitaya soft rot germs | |
CN114891702B (en) | Bacillus amyloliquefaciens L19 with biocontrol effect, microbial inoculum and application thereof | |
CN114437943B (en) | High-efficiency pathogenic biocontrol strain Aspergillus fijiensis and application thereof | |
CN112646733B (en) | Tamarix chinensis endophytic antagonistic fungus as well as separation method and application thereof | |
CN114540213B (en) | Actinomycetes with antibacterial activity and application thereof | |
CN112646734B (en) | Orchid mycorrhizal fungus PF06 and application thereof |
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 |