CN110408570A - It is a kind of prevent and treat Pattern of Asarum Leaf Blight Antagonistic bacteria strains and its application - Google Patents
It is a kind of prevent and treat Pattern of Asarum Leaf Blight Antagonistic bacteria strains and its application Download PDFInfo
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- CN110408570A CN110408570A CN201910742201.1A CN201910742201A CN110408570A CN 110408570 A CN110408570 A CN 110408570A CN 201910742201 A CN201910742201 A CN 201910742201A CN 110408570 A CN110408570 A CN 110408570A
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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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- 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
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Abstract
The invention discloses a kind of Antagonistic bacteria strains for preventing and treating Pattern of Asarum Leaf Blight and its applications, and the Antagonistic bacteria strains are isolated from healthy asarum plant rhizosphere soil, are used for antagonism Pattern of Asarum Leaf Blight pathogen；Classification naming are as follows: Brevibacillus laterosporus (Brevibacillus laterosporus)；Deposit number: CGMCC No.18015；The method of the present invention from asarum healthy plant rhizosphere soil by isolating the Antagonistic bacteria strains-Brevibacillus laterosporus for having biocontrol effect to Pattern of Asarum Leaf Blight, then fungistatic effect test and the test of living body antagonistic effect in plate are carried out, show that Antagonistic bacteria strains-Brevibacillus laterosporus preventive effect is 79.87%, Antagonistic bacteria strains-Brevibacillus laterosporus has preferable control action to the generation of Pattern of Asarum Leaf Blight, and the raising of Disease Resistance-Related Enzymes in asarum plant body can be induced, to resist infecting for disease.
Description
Technical field
Microbiology engineering and technical field of the present invention more particularly to a kind of Antagonistic bacteria strains for preventing and treating Pattern of Asarum Leaf Blight
And its application.
Background technique
Asarum also known as thin ginseng, bowl of a pipe flower, are pungent the categorys perennial grass undergraduate course plant of Aristolochiaceae, are important medicinal of one kind
Plant, asarum is warm-natured, acrid flavour, has warming channel and expelling cold, wind-expelling pain-stopping, sensible, relieving cough and asthma, promoting blood circulation effect, for treating
The diseases such as anemofrigid headache, phlegm retention cough and asthma, arthralgia, dentalgia, nasosinusitis.Pattern of Asarum Leaf Blight is typical polycyclic disease, pathogen
For M. acerina (Mycocentrospora acerina (Hartig) Deihton), conidium is main source of infection, can
It is propagated by splash gentle stream of rainwater, main harm blade, scab subcircular, with wheel line around sepia, serious disease occurs
Mottled rot is rotten, and perforation, entire blade is withered, seriously affects the yield and quality of asarum.
Traditional chemical control method has the features such as quick, wide sterilization spectrum, but caused by the unreasonable use of chemical pesticide
The problems such as medicament residue, food safety and environmental pollution, increasingly shows, and even results in pest resurgence and disease drug resistance is passed
Phenomena such as increasing, and biological control can prevent and treat plant pest under conditions of free from environmental pollution, have safety, noresidue, specifically
Property it is strong the advantages that, using biocontrol bacterial strain prevention and treatment asarum disease facilitate medicinal material quality, improve medicinal material yield, have ecology and
Economic double benefit, less to the research of Pattern of Asarum Leaf Blight biological control both at home and abroad at present, chemical pesticide is to Pattern of Asarum Leaf Blight
Prevention and treatment will cause medicament residue to influence the quality and quality of medicinal material, therefore, the present invention proposes that a kind of prevention and treatment asarum leaf is withered
The Antagonistic bacteria strains of disease and its application, to solve shortcoming in the prior art.
Summary of the invention
In view of the above-mentioned problems, the present invention propose it is a kind of prevent and treat Pattern of Asarum Leaf Blight Antagonistic bacteria strains and its application, pass through
Antagonistic bacteria strains-short bud of side spore that there is biocontrol effect to Pattern of Asarum Leaf Blight is isolated from asarum healthy plant rhizosphere soil
Then spore bacillus carries out fungistatic effect test and living body antagonistic effect in plate to the Antagonistic bacteria strains filtered out and tests, obtains
Antagonistic bacteria strains-Brevibacillus laterosporus preventive effect is 79.87% out, and Antagonistic bacteria strains-Brevibacillus laterosporus is to thin
The generation of pungent leaf blight has preferable control action, and can induce the liter of Disease Resistance-Related Enzymes in asarum plant body
Height, to resist infecting for disease.
The present invention proposes a kind of Antagonistic bacteria strains for preventing and treating Pattern of Asarum Leaf Blight, and the Antagonistic bacteria strains are from healthy asarum
It is isolated in plant rhizosphere soil, it is used for antagonism Pattern of Asarum Leaf Blight pathogen；Classification naming are as follows: Brevibacillus laterosporus
(Brevibacillus laterosporus)；Deposit number: CGMCC No.18015；The preservation time: on June 21st, 2019；
Preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica；Depositary institution: China is micro-
Biological inoculum preservation administration committee common micro-organisms center.
Further improvement lies in that: the filtering of the strain culture, fermentation liquid or fermentation culture of the Antagonistic bacteria strains
Liquid.
Further improvement lies in that: the Antagonistic bacteria strains are the bacteria agent of active constituent in Pattern of Asarum Leaf Blight prevention and treatment
Application.
Further improvement lies in that: the occurrence and development of the Antagonistic bacteria strains control Pattern of Asarum Leaf Blight have prevention thin
The effect that pungent leaf blight occurs.
It is a kind of prevent and treat Pattern of Asarum Leaf Blight Antagonistic bacteria strains prevention and treatment the application of pathogen-M. acerina.
The invention has the benefit that the method for the present invention from asarum healthy plant rhizosphere soil by isolating to asarum
Leaf blight has Antagonistic bacteria strains-Brevibacillus laterosporus of biocontrol effect, using Pattern of Asarum Leaf Blight bacterium as target bacterium, by flat
Plate face-off method screens Antagonistic bacteria strains, then carries out fungistatic effect in plate to the Antagonistic bacteria strains filtered out and tries
It tests and is tested with living body antagonistic effect, show that Antagonistic bacteria strains-Brevibacillus laterosporus preventive effect is 79.87%, antagonistic bacterium
Bacterial strain-Brevibacillus laterosporus has preferable control action to the generation of Pattern of Asarum Leaf Blight, and can induce asarum plant
The raising of internal Disease Resistance-Related Enzymes provides to resist infecting for disease for the biological control of Pattern of Asarum Leaf Blight
Science data are supported, the development of Pattern of Asarum Leaf Blight biological control can be effectively facilitated.
Detailed description of the invention
Fig. 1 is that constant temperature opposite culture of the present invention inhibits Pattern of Asarum Leaf Blight pathogen effect diagram.
Fig. 2 is the hypha form schematic diagram of Pattern of Asarum Leaf Blight pathogen after Antagonistic bacteria strains of the present invention processing.
Fig. 3 is Antagonistic bacteria strains Immune effect schematic diagram of the present invention.
Fig. 4 is that Antagonistic bacteria strains of the present invention induce the active schematic diagram of polyphenol oxidase (PPO) in asarum plant body.
Fig. 5 is that Antagonistic bacteria strains of the present invention induce the active schematic diagram of phenylalanine lyase (PAL) in asarum plant body.
Fig. 6 is that Antagonistic bacteria strains of the present invention induce the active schematic diagram of peroxidase (POD) in asarum plant body.
Fig. 7 is that Antagonistic bacteria strains of the present invention induce the active schematic diagram of superoxide dismutase (SOD) in asarum plant body.
Specific embodiment
In order to deepen the understanding of the present invention, the present invention is further described below in conjunction with embodiment, the present embodiment
For explaining only the invention, it is not intended to limit the scope of the present invention..
Embodiment one
The present embodiment proposes Antagonistic bacteria strains-Brevibacillus laterosporus separation screening and identification: planting from asarum health
A variety of bacterial strains are isolated by gradient dilution method in strain rhizosphere soil, using Pattern of Asarum Leaf Blight bacterium as target bacterium, are stood facing each other using plate
Method filter out one plant number be S2-31 Antagonistic bacteria strains, through morphology and molecular biology identification, classification naming
For Brevibacillus laterosporus (Brevibacillus laterosporus)；
The separation screening detailed processes of Antagonistic bacteria strains the following steps are included:
Step 1: Antagonistic bacteria strains are separated using soil dilution partition method, 10g asarum healthy plant rhizosphere soil is taken to add
Enter into the triangular flask of 90mL sterile water, triangular flask is then subjected to concussion processing 20min, the speed for shaking processing is 160r/
Min is mixed evenly to prepare soil supension, then soil supension is diluted to 10-4、10-5、10-6Three gradients；
Step 2: respectively from 10-4、10-5、10-6100uL even spread is drawn in the dilution soil supension of three gradients to arrive
In NA plate；Each gradient is coated with 3 plates as repeating, obtain 9 even spreads the NA plate of dilution soil supension；
Step 4: the NA plate of 9 even spreads dilution soil supension is subjected to constant temperature incubation, constant temperature incubation temperature is
28 DEG C, the constant temperature incubation time is 2 days, carries out scribing line purifying according to colonial morphology, and obtained variety classes bacterial strain is sterile at 4 DEG C
It is saved backup under environment；
Step 5: using Pattern of Asarum Leaf Blight bacterium as target bacterium, Antagonistic Fungi is screened using the method for plate face-off, it is thin to obtain antagonism
Then bacteria strain carries out strain idenfication further according to the method for morphology and molecular biology, qualification result indicates, antagonistic bacterium bacterium
Strain culture forms more typical bacterium colony for 24 hours, and bacterium colony is in yellow-white on NA culture medium, and late stage of culture colony edge is in yellowish-brown
Color, the smooth of the edge, flat, center projections, Gram's staining result is variable, and gemma side is raw；
16SrDNA Sequence Identification is carried out to Antagonistic bacteria strains, nucleic acid sequence BLAST is carried out in GenBank database
Analysis, the results showed that the homology of Antagonistic bacteria strains and Brevibacillus laterosporus are 99%, are determined as side
Spore bacillus brevis (Brevibacillus laterosporus).
Embodiment two
According to Fig. 1,2, the present embodiment proposes Antagonistic bacteria strains-Brevibacillus laterosporus to Pattern of Asarum Leaf Blight bacterium bacterium
The inhibiting effect of silk growth: antagonistic bacterium-Brevibacillus laterosporus and Pattern of Asarum Leaf Blight pathogen-M. acerina are subjected to constant temperature
Opposite culture, constant temperature opposite culture temperature are 25 DEG C, and the constant temperature incubation time is 7 days, and observes the pathogen edge of opposite culture
Hypha form；
Through Antagonistic bacteria strains-Brevibacillus laterosporus treated Pattern of Asarum Leaf Blight pathogen-M. acerina (such as Fig. 1
It is shown), under the microscope it has been observed that pathogen colony edge mycelia often has greatly fracture, distorts, contracting of hanging, chlamydospore increasing
More phenomenons (as shown in Figure 2) shows Antagonistic bacteria strains-Brevibacillus laterosporus to Pattern of Asarum Leaf Blight pathogen-maple bacteria thorn
The growth of spore significantly inhibits.
Embodiment three
According to Fig. 3,4, the present embodiment proposes Antagonistic bacteria strains-Brevibacillus laterosporus to Pattern of Asarum Leaf Blight potting
The measurement of living body antagonistic effect, comprising the following steps:
Step 1: the Antagonistic bacteria strains-Brevibacillus laterosporus of activation for 24 hours is seeded in the LB equipped with 150mL sterilizing
In fluid nutrient medium, concussion 48h is carried out in the case where revolving speed is 180r/min, temperature is 32 DEG C, obtains thallus fermentation liquid, then by thallus
It is 1 × 10 that fermentation liquid, which is adjusted to concentration,8The bacteria suspension of a/mL；
Step 2: being divided into 2 processing for the measurement of antagonistic effect, is respectively first inoculated with Antagonistic bacteria strains-short bud of side spore
Spore bacillus inoculates leaf blight pathogen processing group and only inoculation leaf blight pathogen-M. acerina sprinkling sterile water control group
(CK group), then from health potting asarum in choose the consistent asarum blade of 200 growing ways, each processing group 100, and
6 circular holes are respectively pricked on each blade；
Step 3: immunization campaign processing group first uniformly sprays bacteria suspension, is inoculated with Pattern of Asarum Leaf Blight pathogen-maple bacteria thorn after 3h
Spore, control group first spray sterile water, leaf spoting bacteria are inoculated with after 3h, and then moisturizing culture started to observe incidence after 3 days, go forward side by side
Row control efficiency counts (as shown in Figure 3), and morbidity classification standard is as shown in table 1, disease index calculation formula such as formula (1) institute
Show, shown in preventive effect calculation formula such as formula (2):
The morbidity classification standard of table 1
| Grade | Standard |
| 0 grade | Disease-free spot |
| 1 grade | Lesion area accounts for 5% or less entire leaf area |
| 3 grades | Lesion area accounts for the 6%-10% of entire leaf area |
| 5 grades | Lesion area accounts for the 11%-25% of entire leaf area |
| 7 grades | Lesion area accounts for the 26%-50% of entire leaf area |
| 9 grades | Lesion area accounts for 50% or more of entire leaf area |
Control efficiency statistical results show only to be inoculated with Pattern of Asarum Leaf Blight pathogen-M. acerina processing group blade
Disease index is 30.67, and antibacterium bacterial strain-Brevibacillus laterosporus processing disease index is 6.17, and prevention preventive effect is
79.87%.
Example IV
According to Fig. 4,5,6,7, the present embodiment proposes antibacterium bacterial strain-Brevibacillus laterosporus in asarum plant body
The measurement of Disease Resistance-Related Enzymes: the 0th, 2,4,6,8 day progress asarum herb resistance-related enzymes activity after immunization campaign processing
Measurement, including to polyphenol oxidase (PPO), phenylalanine lyase (PAL), peroxidase (POD) and superoxides discrimination
Change the enzyme assay of enzyme (SOD)；
Shown after being inoculated with antibacterium bacterial strain-Brevibacillus laterosporus by the detection of Disease Resistance-Related Enzymes, asarum blade
The activity of interior PPO, PAL, POD, SOD is not compared with being inoculated with Antagonistic bacteria strains-Brevibacillus laterosporus asarum blade, enzyme activity
Property significantly rise, show that antibacterium bacterial strain-Brevibacillus laterosporus can induce in asarum plant body disease resistance related enzymes living
The raising (as shown in FIG. 4,5,6, 7) of property, to resist infecting for disease.
The method of the present invention has biocontrol effect to Pattern of Asarum Leaf Blight by isolating from asarum healthy plant rhizosphere soil
Antagonistic bacteria strains-Brevibacillus laterosporus, using Pattern of Asarum Leaf Blight bacterium as target bacterium, by tablet face-off method to antagonistic bacterium
Bacterial strain is screened, and is then carried out fungistatic effect test and living body antagonistic effect in plate to the Antagonistic bacteria strains filtered out and is tried
It tests, show that Antagonistic bacteria strains-Brevibacillus laterosporus preventive effect is 79.87%, Antagonistic bacteria strains-Brevibacillus laterosporus
There is preferable control action to the generation of Pattern of Asarum Leaf Blight, and Disease Resistance-Related Enzymes in asarum plant body can be induced
Raising provide science data for the biological control of Pattern of Asarum Leaf Blight and support, can effectively promote to resist infecting for disease
Into the development of Pattern of Asarum Leaf Blight biological control.
The basic principles, main features and advantages of the invention have been shown and described above.The technical staff of the industry should
Understand, the present invention is not limited to the above embodiments, and the above embodiments and description only describe originals of the invention
Reason, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these changes and improvements
It all fall within the protetion scope of the claimed invention.The claimed scope of the invention is by appended claims and its equivalent circle
It is fixed.
Claims (5)
1. a kind of Antagonistic bacteria strains for preventing and treating Pattern of Asarum Leaf Blight, it is characterised in that: the Antagonistic bacteria strains are from healthy asarum
It is isolated in plant rhizosphere soil, it is used for antagonism Pattern of Asarum Leaf Blight pathogen；Classification naming are as follows: Brevibacillus laterosporus
(Brevibacillus laterosporus)；Deposit number: CGMCC No.18015；The preservation time: on June 21st, 2019；
Preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica；Depositary institution: China is micro-
Biological inoculum preservation administration committee common micro-organisms center.
2. a kind of Antagonistic bacteria strains for preventing and treating Pattern of Asarum Leaf Blight according to claim 1, it is characterised in that: the antagonism
The filtered fluid of the strain culture of bacterium bacterial strain, fermentation liquid or fermentation culture.
3. a kind of Antagonistic bacteria strains for preventing and treating Pattern of Asarum Leaf Blight according to claim 1, it is characterised in that: the antagonism
Bacterium bacterial strain is application of the bacteria agent of active constituent in Pattern of Asarum Leaf Blight prevention and treatment.
4. a kind of Antagonistic bacteria strains for preventing and treating Pattern of Asarum Leaf Blight according to claim 1, it is characterised in that: the antagonism
Bacterium bacterial strain controls the occurrence and development of Pattern of Asarum Leaf Blight, has the effect of preventing Pattern of Asarum Leaf Blight.
5. a kind of Antagonistic bacteria strains for preventing and treating Pattern of Asarum Leaf Blight as described in claim 1 are in prevention and treatment pathogen-M. acerina
Application.
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