CN109112071A - A kind of Trichoderma and its application - Google Patents
A kind of Trichoderma and its application Download PDFInfo
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- CN109112071A CN109112071A CN201810935616.6A CN201810935616A CN109112071A CN 109112071 A CN109112071 A CN 109112071A CN 201810935616 A CN201810935616 A CN 201810935616A CN 109112071 A CN109112071 A CN 109112071A
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- 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- 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
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/885—Trichoderma
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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
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
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- 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
- C12N1/14—Fungi; Culture media therefor
Abstract
The invention discloses a kind of Trichoderma and its applications.Trichoderma of the invention is trichoderma asperellum (Trichoderma asperellum) bacterium, bacterial strain number: HTTA-Z0002, deposit number CGMCC No.15677.Trichoderma of the invention can apply in controlling plant diseases, antibacterial and pesticide.Biological Characteristics of Strain of the invention and pesticide activity are stablized, it is easy culture, cultivation cycle is short, operating method is simple and feasible, it produces in bacteriostatic activity, Liquid Culture and is greatly improved in the characteristics such as conidium compared with wild strain, the few disadvantage of trichoderma strain Liquid Culture sporulation quantity is overcome, agricultural product and environment are not polluted, has wide and strong inhibiting effect to a variety of pathogens of plant.
Description
Technical field
The invention belongs to microorganisms technical fields, and in particular to a kind of Trichoderma and its application in pesticide.
Background technique
Cultivated plant chemical pesticide pollution problem is the hot spot of a worldwide concern.With facility cultivation vegetable melon and fruit
Technology becomes increasingly popular, this problem seems more prominent.The high temperature in greenhouse, the environment of high humidity meet people to vegetable melon and fruit
The demand in the four seasons also results in disease regardless of the serious generation in season.The pesticide and hormone of excess cause sternly vegetables and melon and fruit
The pollution of weight, toxicity and potential carcinogenesis constitute very big danger to human health and coerce.Biological pesticide is with it to non-target
Biology relatively the good outstanding advantages of safety and Environmental compatibility and meet requirement of the modern society to agricultural production and pesticide.
In all living resources, microorganism is fast with its breeding, at low cost, and wild strain is easier to transformation to quickly mention
The characteristics of high yield, becomes the preferred resource of pesticide manufacturing.Microbial pesticide sales volume reaches multi-billion dollar at present, and annual value of production is every
Year rises 10%~20%.To solve the status that Lu Yuan microorganism obtains the increasingly reduction of novel pesticide character, extreme marine microorganism
New resources start to attract attention.The unique geographical location in polar region forms the natural environments such as with high salt, bitter cold, strong ultraviolet radioactive, makes
With regard to a variety of adaptable existings of biology, has unique the Molecular Biology Mechanism and physio-biochemical characteristics, generate novel generation
Thanking to substance can be utilized by the mankind.
China's soil salinization is more serious, and salinization soil accounts for about the 1/3 of arable land, the characteristics of in view of having a large population and a few land, these
Salinization soil still by people utilize plantation compared with salt tolerant crop, at present both at home and abroad exploitation microbial pesticide due to biocontrol microorganisms all
From land, it is difficult to survive in the higher soil of salt content, colonizes, it is poor in the control efficiency of salt lick soil-borne disease,
Land biocontrol microorganisms are limited it and are played a role due to habitat.Marine microorganism original habitat with high salt, is easier in salinized soil
Earth plays a role, and shows its habitat advantage in novel pesticide development field.
The active bacteria formulation of biological pesticide is that a kind of opposite development cycle is shorter, and credit requirement is relatively fewer, pesticide function compared with
Full product.Occupy biggish ratio in biological pesticide in the land microorganism live bacteria preparation for having obtained registration, and in phytopathy
Important function has been played in harmful prevention and treatment.Pesticide bacterial strain by the mankind in order to preferably be utilized and industrialization production, bacterial strain will meet
Pesticide activity and production fermentation performance condition up to standard, after microbial strains are obtained by various screening models, usually all into
Row mutation breeding, such as success of operation, economic value can improve several hundred or even thousands of times on the basis of wild strain.Micro- life
Object liquid fermentation is the basis for embodying low cost and easy industrialized production, but trichoderma is few in liquid culture sporulation quantity, solid-state training
It supports and constrains large-scale production again, and what the active bacteria formulation of trichoderma strain was applied is conidium, is i.e. conidium quantity and medicine
It imitates directly proportional.
Present invention is generally directed to the most important bacteriostatic activities of trichoderma pesticide bacterial strain and Liquid Culture sporulation quantity characteristic, mutagenesis to educate
The bacterial strain HTTA-Z0002 that kind obtains not only has excellent and wide spectrum antagonistic property to the biggish pathogen of plant hazard, also
The characteristic of production is cultivated with good artificial fermentation.
Summary of the invention
The purpose of the present invention is to provide a kind of Trichoderma and its applications.
To achieve the goals above, the present invention provides the following technical solutions.
A kind of Trichoderma, the Trichoderma be trichoderma asperellum (Trichoderma asperellum) bacterium, bacterial strain number:
HTTA-Z0002, deposit number CGMCC No.15677.
Trichoderma asperellum (Trichoderma asperellum) bacterium of the invention is preserved in Chinese microorganism strain preservation pipe
Reason committee common micro-organisms center (CGMCC), preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3；Preservation date
On May 17th, 2018.
Application of the above-mentioned Trichoderma in controlling plant diseases.
Further, the plant disease includes eggplant early blight (Alternaia solani), graw mold of tomato
(Botrytis cinerea), the late blight of potato (Phytophthora infestans), wheat scab (Fusarium
Graminearum), soybean rust (hakopsora pachyrhizi), speckled leaf blotch (Zymoseptoria tritci),
One of Muskmelon Fusarium wilt (Muskmelon blight), wild cabbage powdery mildew (Cabbage powdery mildew).This hair
Bright trichoderma strain has good wide spectrum control efficiency to above-mentioned disease, especially very good to wheat scab control efficiency to reach
100%, 80.1%-87.9% reached to graw mold of tomato preventive effect, is fallen ill than the therapeutic effect after more serious also 52.5%, it is right
Muskmelon Fusarium wilt, the prevention of wild cabbage powdery mildew can reach 68.87%-72.26% and 59.49%-60.76%, as a result more traditional
Chemical agent effect is good.
Application of the above-mentioned Trichoderma in antibacterial.
Further, the Trichoderma is to Rice Blast Fungus (Pyricularia oryzae), eggplant pink mold (Alternaria
Salani), Fusarium (Fusarium oxysporrum), the pathogen of Botrytis cinerea (Botrytis cinerea), verticillium dahliae
(Verticillium dahliae), bud branch mould (Cladosporium fulvum), phytophthora blight of pepper (Phytophthora
Capsici), rhizoctonia (Rhizoctonia solani), gibberella (Fusarium graminearum), sclerotium bacteria
It is one of (Sclerotina sclerotiorum) or a variety of inhibited.Trichoderma asperellum HTTA-Z0002 of the present invention
Strain gene detection prove, bacterial strain have chitinase and antibacterial peptide synthesis key gene, can by generate antibacterial peptide and
Chitinase inhibits disease fungus, and harm serious 10 kind plant pathogenic fungi common to above-mentioned agricultural has strong and wide spectrum
Bacteriostasis, inhibition zone all 30mm or more, bacteriostasis rate reach 79.0-95.4%.
A kind of pesticide includes above-mentioned Trichoderma.
The cultural method of above-mentioned Trichoderma, the bacterial strain is on solid MPDA culture medium, in 20 DEG C -23 DEG C of temperature, cultivates 3-5
It, mycelium is transferred to liquid MPDA culture medium shake culture 5-7 days.Shake culture 3-7 days.
Further, the solid MPDA culture medium is made of following raw material: potato leaching juice 200-250ml, peptone
1-2g, glucose 25-30g, NaCl 5-8g, MgCl2·6H2O 0.1-0.3g, KCl 0.1g, agar 18g, water 1000ml.This
The bacterial strain of invention is easy to manually cultivate, and the well-grown on MPDA culture medium, culture medium material price is cheap, is easily obtained；
Bacterial strain grows vigorous in MPDA artificial medium, produces spore layer and conidiophore intensively such as felted, dark green, or white, point
Raw sporophore has diaphragm, generates lateral branch, and for branch vertically to life, base portion is wider, and end is thinner, is in ampuliform, and conidium is single
Spore, it is spherical to oval, it is colourless, obvious conidium group is formed at tip.
The present invention has a characteristic that
1) Trichoderma asperellum (Trichoderma asperellum) artificial culture method that the present invention obtains is simple, culture
Material price is cheap, is easily obtained.
2) Trichoderma asperellum (Trichoderma asperellum) that the present invention obtains, in bacteriostatic activity, Liquid Culture
It produces and is greatly improved in the characteristics such as conidium compared with wild strain, overcome few bad of trichoderma strain Liquid Culture sporulation quantity
Gesture, maximum sporulation quantity reach 4.25 × 108cfu/cm2。
3) Trichoderma asperellum (Trichoderma asperellum) is common to agricultural and the serious pathogenic of harm is true
Bacterium has strong and wide spectrum bacteriostasis, while having good preventive and therapeutic effect to related plant disease, and control efficiency is substantially better than
Traditional chemical medicament.
Detailed description of the invention
Fig. 1 affiliation branch tree.
Specific embodiment
Following specific embodiments are the further explanations to method provided by the invention and technical solution, but are not construed as
Limitation of the present invention.
1. the taxonomic identification of trichoderma strain
Trichoderma asperellum HTTA-Z0002 bacterial strain, in solid MPDA culture medium (potato leaching juice 200-250ml, peptone 1-
2g, glucose 25-30g, NaCl 5-8g, MgCl26H2O 0.1-0.3g, KCl 0.1g, agar 18g water 1000ml), 20 DEG C-
It 23 DEG C, cultivates 3-5 days, the variations such as record colonial morphology, color is observed continuously, picking mycelia produces spore in optical microphotograph under the microscope
Structure.Bacterial strain grows vigorous in MPDA artificial medium, produces spore layer and conidiophore intensively such as felted, dark green, or it is white
Color, conidiophore have diaphragm, generate lateral branch, and for branch vertically to life, base portion is wider, and end is thinner, are in ampuliform, mitogenetic spore
Sub- monospore, it is spherical to oval, it is colourless, obvious conidium group is formed at tip.
Bacterial strain is through above method culture, and using the universal primer of fungi ITS gene, conventional method pcr amplification product is sequenced,
And compared by the blast program of NCBI, analytical sequence homology and do the closer edge type of affiliation branch tree (see
Fig. 1).In conjunction with the microstructure of biology colonial morphology, color and conidial fructification, the molecular biology information of bacterial strain, with reference to state
Inside and outside correlation trichoderma taxonomic identification monograph identifies that bacterial strain is trichoderma asperellum Trichoderma asperellum.
2. the mutation breeding of trichoderma strain
The trichoderma strain gone out through multiple pesticide activity test screen, using Conventional UV-microwave complex mutation, selection growth
Vigorous monoclonal colonies are tested using eggplant pink mold Alternaria salani as cause of disease target bacterium using opposite culture method
Bacteriostatic activity calculates bacteriostasis rate: inhibiting rate (%)=(control group target colony diameter-processing group target colony diameter)/control
Group target colony diameter × 100%, and be compared with the inhibiting rate of wild strain, select bacteriostasis rate to improve 25% or more bacterium
Strain, first use the culture of solid-state MPDA culture medium 5 days, eliminate growth failure bacterial strain, then access liquid MPDA, respectively 20 DEG C,
23, it cultivates 7 days for 25 DEG C, microscopic count simultaneously compares its production spore quantity, and selection produces the bacterial strain that spore quantity improves 50%, the bacterial strain selected
In 5 generation of continuous liquid culture, using above-mentioned same activity and produces spore quantity test method, observes its antibacterial and production spore stability,
The HTTA-Z0002 trichoderma strain finally selected, bacteriostasis rate improve 67.2% compared with wild mushroom, and maximum sporulation quantity is 4.25 ×
108cfu/cm2, 150.7% is improved compared with wild mushroom.
3. the test of laboratory plants Antifungal Activity in Vitro and gene validation test
Bacterial strain HTTA-Z0002, through MPDA culture medium 7 days, using above-mentioned same opposite culture method to agriculture common and danger
The serious 10 kinds of plant pathogenic fungis of evil: Rice Blast Fungus Pyricularia oryzae, eggplant pink mold Alternaria
Salani, Fusarium Fusarium oxysporrum, the pathogen of Botrytis cinerea Botrytis cinerea, verticillium dahliae
Verticillium dahliae, bud branch mould Cladosporium fulvum, phytophthora blight of pepper Phytophthora
Capsici, rhizoctonia Rhizoctonia solani gibberella Fusarium graminearum, sclerotium bacteria (Sclerotina
Sclerotiorum it) tests bacteriostatic activity and calculates bacteriostasis rate.On NCBI (http://www.ncbi.nlm.nih.gov/)
Trichoderma β-Isosorbide-5-Nitrae-acetyl glucosaminidase gene Nag1 and Peptaibols synthase gene Tex1 sequence are retrieved, is utilized
Premier 5.0 designs corresponding primer JDZ-F/JDZ-R, KJT-F/KJT-R, JDZ-F (5 '-
CAGAACCGAATCCAGAGC-3 ')/JDZ-R (5 '-GAAGGAGTGTAGACGAGC-3 '), PCR conventional method amplification, to bacterium
The characteristic that strain generates bacteriostatic activity is verified, as a result as follows:
1 HTTA-Z0002 of table is to phytopathogen bacteriostatic activity
Annotation: T1: opposite culture test indicates strain activity, inhibition zone 30mm inhibition indicated above with inhibition zone size
It is active strong；T2: inhibiting rate=(control colony radius-processing colony radius)/control colony radius × 100% of pathogen is planted
Object pathogen: Po:Pyricularia oryzae；As:Alternaria salani；Fo:Fusarium oxysporum；
Bc:Botrytis cinerea；Vd:Verticillium dahliae；Cf:Cladosporium fulvum；Pc:
Phytophthora capsici； Rs:Rhizoctonia solani；Fg:Fusarium graminearum；Ss:
Sclerotina sclerotiorum。
By upper table data it is found that trichoderma HTTA-Z0002 bacterial strain is common to 10 kinds of agriculturals and endanger serious disease fungus,
With good fungistatic effect, in opposite culture method, high inhibition effect, inhibition zone 30mm are reached to the activity of pathogen
More than, bacteriostasis rate reaches 79.0-95.4%, shows strong bacteriostatic activity, has the potentiality for developing novel germicide.From HTTA-Z0002 bacterium
Strain PCR amplification arrives Nag1 gene, Tex1 gene, and characteristic bands respectively may be about 800bp, 250bp, illustrates bacterial strain with chitinase
With the key gene of antibacterial peptide synthesis, disease fungus can be inhibited by generating antibacterial peptide and chitinase.
4. plant living body controlling disease test (green plant test and field plot trial)
The test of plant living body controlling disease is carried out using conventional method, potted plant seedling culture to 3-4 piece true leaf, each basin
It plants test and is no less than 60 with seedling, field plot trial plot area 25m2More than, field salt-affected soil cell controlling experiment, salt
The natural salt marsh arable land that stain soil selects Jimo close by sea, chloride salt about 0.2- 0.3%, potting salt-affected soil is using artificial
The simulation salinized soil of system is trained, each processing is no less than 3 repetitions, inoculation morbidity or natural occurrence, is diluted with trichoderma viable bacteria spore
Liquid is prevented and treated, and prevents and treats the common traditional pesticide of the disease as positive control, at the without fermented liquid of concentration with agricultural production
Reason investigates the state of an illness as blank control after 6-7 days, calculate control efficiency.
Control efficiency of the 1 HTTA-Z0002 bacterial strain of table to potted tomato gray mold
Control efficiency of the 2 HTTA-Z0002 bacterial strain of table to field plot graw mold of tomato
Note: 200 times of liquid of dilution.
Field plot drug effect of the 3 salinization soil HTTA-Z0002 of table to Muskmelon Fusarium wilt
Note: 300 times of liquid of dilution.
Field plot drug effect of the 4 salinization soil HTTA-Z0002 of table to wild cabbage powdery mildew
Note: 300 times of liquid of dilution.
Table 1-4 number it was demonstrated that trichoderma HTTA-Z0002 bacterial strain normal soil potting or field plot prevent and treat graw mold of tomato,
Control efficiency respectively reaches 80.1%-87.9%, falls ill than the therapeutic effect after more serious also 52.5%, salt-affected soil is anti-
Control chemical agent Muskmelon Fusarium wilt more rambunctious, the prevention of wild cabbage powdery mildew can reach 68.87%-72.26% and 59.49%-
60.76% therapeutic effect, as a result all more traditional chemical agent effect are good
5. third-party authentication result
In order to confirm the pesticide potentiality of bacterial strain through third party, during cooperating with German BASF, sign in both sides to bacterial strain
Under the premise of confidentiality agreement, BASF has carried out multiple plant prevention disease test verifying to above-mentioned 2 plants of bacterial strains in Germany and has provided
Prevent and treat result.The disease of BASF test includes: eggplant early blight Alternaia solani；Graw mold of tomato Botrytis
cinerea；The late blight of potato Phytophthora infestans, wheat scab Fusarium graminearum；Greatly
Soybean rust Phakopsora pachyrhizi；Speckled leaf blotch Zymoseptoria tritci.
5 BASF AG of table surveys result in the raw of Germany
It is good that the above results prove that HTTA-Z0002 trichoderma asperellum Trichoderma asperellum has above-mentioned disease
Good wide spectrum control efficiency is especially very good up to 100% to wheat scab Fusarium graminearum control efficiency.Mirror
In BASF Corp. of Germany in the popularity of world's pesticide manufacturing and development field, theirs the result is that HTTA-Z0002 Trichoderma
The best verifying of strain pesticide potentiality.
As a result, in various aspects such as Vitro Plant test, living body test, pot experiment, field plot trial, third-party authentications
Confirm the good pesticide potentiality of bacterial strain.
The method of the present invention that the above embodiments are only used to help understand and its core concept.It should be pointed out that for
For those skilled in the art, without departing from the principle of the present invention, if can also be carried out to the present invention
Dry improvement and modification, these improvement and modification are also fallen into the claims in the present invention protection scope.
Claims (8)
1. a kind of Trichoderma, which is characterized in that the Trichoderma is trichoderma asperellum (Trichoderma asperellum) bacterium, bacterium
Strain number: HTTA-Z0002, deposit number CGMCC No.15677.
2. the cultural method of Trichoderma according to claim 1, which is characterized in that train trichoderma strain in solid MPDA
It supports on base, in 20 DEG C -23 DEG C of temperature, cultivates 3-5 days, mycelium is transferred to liquid MPDA culture medium shake culture 5-7 days.
3. the cultural method of Trichoderma according to claim 2, which is characterized in that the solid MPDA culture medium is by following
Raw material composition: potato leaching juice 200-250ml, peptone 1-2g, glucose 25-30g, NaCl 5-8g, MgCl2·6H2O
0.1-0.3g, KCl 0.1g, agar 18g, water 1000ml.
4. application of the Trichoderma according to claim 1 in controlling plant diseases.
5. application of the Trichoderma according to claim 4 in controlling plant diseases, which is characterized in that the plant disease
It is withered including eggplant early blight, graw mold of tomato, the late blight of potato, wheat scab, soybean rust, speckled leaf blotch, muskmelon
One of disease, wild cabbage powdery mildew.
6. application of the Trichoderma according to claim 1 in antibacterial.
7. application of the Trichoderma according to claim 6 in antibacterial, the Trichoderma to Rice Blast Fungus, eggplant pink mold,
Fusarium, the pathogen of Botrytis cinerea, verticillium dahliae, bud branch mould, phytophthora blight of pepper, rhizoctonia, gibberella, one in sclerotium bacteria
Kind is a variety of inhibited.
8. a kind of pesticide includes Trichoderma described in claim 1.
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| CN114258923A (en) * | 2021-12-30 | 2022-04-01 | 南京工业大学 | Application of trichoderma asperellum microbial agent in prevention and treatment of crop diseases |
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| CN110272832B (en) * | 2019-08-05 | 2021-07-27 | 海南大学 | Trichoderma asperellum FJ069 and application thereof |
| CN114258923A (en) * | 2021-12-30 | 2022-04-01 | 南京工业大学 | Application of trichoderma asperellum microbial agent in prevention and treatment of crop diseases |
| CN114258923B (en) * | 2021-12-30 | 2022-10-14 | 南京工业大学 | Application of trichoderma asperellum microbial agent in prevention and treatment of crop diseases |
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