CN103952321B - One strain Trichoderma harzianum and the application in prevention and control Brown patch disease thereof - Google Patents
One strain Trichoderma harzianum and the application in prevention and control Brown patch disease thereof Download PDFInfo
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Abstract
The invention discloses a strain Trichoderma harzianum and the application in prevention and control Brown patch disease thereof.Trichoderma harzianum (Trichoderma harzianum) JTM53 that the present invention provides, its deposit number is CGMCC No.8914.The present invention also protects the fermentate of described JTM53 bacterial strain.JTM53 bacterial strain has that survival ability is strong, wide adaptability, the attribute such as free from environmental pollution, has broad application prospects as biocontrol microorganisms.Indoor face-off antagonistic effect shows, JTM53 bacterial strain Rhizoctonia solani has good fungistatic effect.Preparing solid fermentation thing with JTM53 bacterial strain further, then carry out potted plant preventing and treating experiment and daejeon prevention test with solid fermentation thing, Rhizoctonia solani prevention effect is good and stable.The present invention has great using value for the prevention and control of Brown patch disease.
Description
Technical field
The present invention relates to a strain Trichoderma harzianum and the application in prevention and control Brown patch disease thereof.
Background technology
Brown patch disease is the silborne fungal diseases that a class microbial by miliary damping-off, that generation is on lawn is important.
Generally occur on the coldest warm-season turf of this disease, the g and D on lawn is endangered serious, the time even having
Lawn is caused crushing harm.This disease can also cause the various crop of different regions to be fallen ill, and causes substantial amounts of economic damage
Lose.
Dogstail plant is mostly perennial on lawn, and this is that the accumulation of Rhizoctonia solani Kuhn, Brown patch disease provide
Suitable condition.The typical symptom characteristic of Brown patch disease is the most just to cause root, stem and leaf portion group after lawn dogstail is infected
Knit and rot or the death of a large amount of plant, the preferable disease-resistant variety of the most non-appearance effect.
At present in the preventing and treating to Brown patch disease, chemical prevention occupies consequence.Chemical prevention not only cost is high,
And easily pollute environment, while prevention and control pathogen, pathogen also easily produces the drug resistance even resistance to the action of a drug to chemical agent.
Summary of the invention
It is an object of the invention to provide a strain Trichoderma harzianum and the application in prevention and control Brown patch disease thereof.
The present invention provide Trichoderma harzianum (Trichoderma harzianum) JTM53, be called for short JTM53 bacterial strain, in
Within on 03 13rd, 2014, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (is called for short CGMCC, address
For: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), deposit number is CGMCC No.8914.
The present invention also protects the fermentate of described JTM53 bacterial strain.
The preparation method of described fermentate is specific as follows:
(1) by the spore inoculating of JTM53 bacterial strain to seed culture medium (initial concentration concretely 106CFU/mL), carry out
Cultivate, obtain seed liquor；
(2) seed liquor that step (1) obtains is mixed with fermentation medium, ferment, obtain fermentate.
In step (1), the condition of described cultivation concretely: dissolved oxygen concentration 20%, temperature 28-30 DEG C, mixing speed
200-250rpm (concretely 200rpm), throughput 10-15L/min (concretely 15L/min), 3 days.Described seed culture
Base specific as follows: seed culture medium (pH6.0-6.5): solvent is water, containing wheat bran 2% (mass ratio), glucose 1.0%
(mass ratio), magnesium sulfate 0.5% (mass ratio), potassium dihydrogen phosphate 0.3% (mass ratio), calcium chloride 0.3% (mass ratio).
In step (2), the condition of described fermentation concretely: temperature 28-30 DEG C, relative air humidity 95-100%, training
Support base thickness 5-7cm, cultivate 5-7 days (concretely 7 days), be then 7%-10% by culture natural air drying to water content
(concretely 10%).Described fermentation medium is specific as follows: fermentation medium (pH6.5): by 7 mass parts wheat brans and 3
Mass parts wheat wheat straw powder mixes, and adds water so that the water content of culture medium is 70% (volume ratio).
In step (2), the culture that specifically 1 parts by volume step (1) can be obtained and the mixing of 9 parts by volume fermentation mediums.
The present invention also protects the application in suppression Rhizoctonia solani Kuhn of the fermentate of JTM53 bacterial strain or described JTM53 bacterial strain.
The present invention also protects the application in prevention and control Brown patch disease of the fermentate of JTM53 bacterial strain or described JTM53 bacterial strain.
The present invention also protects the fermentate of JTM53 bacterial strain or described JTM53 bacterial strain, prevention and control annual bluegrass, lawn foxiness occurs
Application in disease.
The present invention also protects a kind of preparation for suppressing Rhizoctonia solani Kuhn, including JTM53 bacterial strain or described JTM53 bacterial strain
Fermentate.
The present invention also protects the preparation of a kind of prevention and control Brown patch disease, sending out including JTM53 bacterial strain or described JTM53 bacterial strain
Ferment thing.
The present invention also protects the preparation of a kind of prevention and control annual bluegrass generation Brown patch disease, including JTM53 bacterial strain or described
The fermentate of JTM53 bacterial strain.
As Trichoderma, JTM53 bacterial strain has that survival ability is strong, wide adaptability, the attribute such as free from environmental pollution, as biological and ecological methods to prevent plant disease, pests, and erosion
Bacterium has broad application prospects.Indoor face-off antagonistic effect shows, JTM53 bacterial strain Rhizoctonia solani has good antibacterial
Effect.Prepare solid fermentation thing with JTM53 bacterial strain further, then carry out potted plant preventing and treating experiment with solid fermentation thing and land for growing field crops is prevented
Controlling test, Rhizoctonia solani prevention effect is good and stable.The present invention has great answering for the prevention and control of Brown patch disease
By value.
Accompanying drawing explanation
Fig. 1 is JTM53 bacterial strain 20 DEG C of bacterium colony photos cultivating 5d on PDA culture medium.
Fig. 2 is the conidiophore of JTM53 bacterial strain and conidial photo.
Fig. 3 is the result photo in embodiment 2.
Fig. 4 is the result photo in embodiment 3.
Fig. 5 is the result photo in the step one of embodiment 5.
Fig. 6 is the result photo in the step 2 of embodiment 5.
Detailed description of the invention
Below example facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiment
Method, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions, is certainly
Routine biochemistry reagent shop is commercially available.Quantitative test in following example, is respectively provided with three times and repeats experiment, and result is made even
Average.70% thiophanate methyl wettable powder: Lintong District, Xi'an insecticide factory.Annual bluegrass: Beijing Cathay Sheng Hua development in science and technology
Co., Ltd.
Rhizoctonia solani Kuhn (Rhizoctonia solani): bibliography: Chao Longjun, Dan Xuemin, car lacks the .2000. such as minister
The research of Brown patch disease pathogen identification, regularty of epidemic and Comprehensive Control Technology thereof. Chinese Grassland, (4): 4247.
Embodiment 1, the acquisition of bacterial strain, qualification and preservation
PDA culture medium: potato 200g, glucose 15g, agar 20g, distilled water 1000ml, 121 DEG C of sterilizing 20min.
TSM culture medium: MgSO4·7H200.2g、K2HPO40.9g、KCl0.15g、NH4NO31.0g, glucose 3.0g, rose
Rare red 0.15g, 60% fenaminosulf wettable powder 0.3g, PCNB0.2g, agar 20g, distilled water 950ml, 121 DEG C of sterilizings
20min。
One, the acquisition of JTM53 bacterial strain
Sampling time: in February, 2008.
Sampling position: Beijing golf course.
Push the topsoil of 3-5cm aside, then the root system of plant is dug out, together with the soil of rhizosphere, install to polyethylene
In polybag, take back laboratory.The root system that is stained with soil is the most air-dried, pat root system, make the superincumbent soil of adhesion come off,
Use sterilized water gradient dilution, draw 0.1mL dilution respectively and instill on TSM culture medium flat plate, be coated with the L-shaped spreading rod of sterilizing
Uniformly, cultivation 3-4 days it is placed in the constant incubator of 25-28 DEG C.Picking form is transferred to PDA cultivation like single bacterium colony of Trichoderma
Being purified cultivation on base flat board, mirror mirror is numbered after tentatively regarding as Trichoderma, and is saved in PDA medium slant.
Pass through above-mentioned steps, it is thus achieved that the bacterial strain of some pure cultures, by a wherein strain named JTM53 bacterial strain.
Two, the morphological feature of JTM53 bacterial strain
On PDA culture medium, Fig. 1 is shown in by 20 DEG C of bacterium colony photos cultivating 5d.Diameter about 9.0cm, the speed of growth quickly, the spider's thread
Shape is to ulotrichy, and white, is green owing to producing spore surface under scattered light, and the back side is colourless, the later stage due to sporulation quantity ambassador's bacterium colony in
Slightly green.
Fig. 2 is shown in by conidiophore and conidial photo.The tree-shaped conidiophore of multi-branched is formed quite loose
Flora.Major branch width 4-5 μm, side shoot is many, forms pyramid.The raw bottle in side obstructs up to 5, becomes to intend colyliform arrangement or single along little side
Branch irregular alignment.Slightly hanging contracting in raw bottle metulae portion, side, middle part is expanded, the narrowest one-tenth bottle stalk, 5-7 × 3-3.5 μm.The sum that top is raw
Atypical bottle of stalk is relatively long and very thin, 13-18 × 2.5-3.5 μm.Bottle stalk mostly with wide-angle on its carrier raw, sometimes
Slightly curved to top.Phialospore is spheric conidium head, and conidium is subsphaeroidal or obovate, and wall is smooth, light green, 2.8-
3.2×2.5-2.8μm。
Three, the Molecular Identification of JTM53 bacterial strain
The ITS district of JTM53 bacterial strain is as shown in the sequence 1 of sequence table.
Four, the preservation of JTM53 bacterial strain
Based on morphological feature and Molecular Identification result, JTM53 bacterial strain belongs to Trichoderma harzianum (Trichoderma
harzianum).Trichoderma harzianum (Trichoderma harzianum) JTM53, is called for short JTM53 bacterial strain, in 2014 03 month
(being called for short CGMCC, address is: Beijing within 13rd, to be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center
North Star West Road, Chaoyang District 1 institute 3), deposit number is CGMCC No.8914.
Embodiment 2, the JTM53 bacterial strain inhibitory action to pathogen
1, by JTM53 bacterial strain list colony inoculation to PDA culture medium flat plate, 5d is cultivated for 20 DEG C.
2, take into the PDA culture medium flat plate of step 1, play the bacterium cake (life taking a diameter of 5mm at bacterium colony forward position card punch
Fungi-proofing cake).
3, by Rhizoctonia solani Kuhn list colony inoculation to PDA culture medium flat plate, 5d is cultivated for 20 DEG C.
4, take into the PDA culture medium flat plate of step 3, play the bacterium cake (cause taking a diameter of 5mm at bacterium colony forward position card punch
Germ cake).
5, take new PDA culture medium flat plate, beat with card punch and take a diameter of 5mm, as tester.
6, packet transaction
Test group: take new PDA culture medium flat plate, is being placed above 1 biocontrol microorganisms cake and 1 pathogenic bacteria cake, two bacterium
The air line distance at cake center is 3cm, 25 DEG C of cultivations (12h illumination/12h is dark)；
Control group: take new PDA culture medium flat plate, is being placed above 1 tester and 1 pathogenic bacteria cake, both centers
Air line distance be 3cm, 25 DEG C of cultivations (12h illumination/12h is dark).
Measure the colony diameter of pathogenic bacteria after cultivating 3 days, calculate inhibiting rate.Inhibiting rate=(bacterium colony of control group pathogenic bacteria
The colony diameter of diameter-test group pathogenic bacteria) colony diameter × 100% of ÷ control group pathogenic bacteria.The JTM53 bacterial strain withered silk of opposition
The inhibiting rate of pyrenomycetes is 68.7% (mean value that three times are repeated).
Photo after cultivating 6 days see Fig. 3 (left side for biocontrol microorganisms cake, the right for pathogenic bacteria cake), it can be observed that, vertical
Withered silk kernel fungus is covered by JTM53 bacterial strain the most completely.
Embodiment 3, the JTM53 bacterial strain hyperparasite ability to pathogen
1, by JTM53 bacterial strain list colony inoculation to PDA culture medium flat plate, 5d is cultivated for 20 DEG C.
2, take into the PDA culture medium flat plate of step 1, play the bacterium cake (life taking a diameter of 5mm at bacterium colony forward position card punch
Fungi-proofing cake).
3, by Rhizoctonia solani Kuhn list colony inoculation to PDA culture medium flat plate, 5d is cultivated for 20 DEG C.
4, take into the PDA culture medium flat plate of step 3, play the bacterium cake (cause taking a diameter of 5mm at bacterium colony forward position card punch
Germ cake).
5, take new PDA culture medium flat plate, the celloyarn of a lcm × 3cm is being placed above, then at celloyarn
1 biocontrol microorganisms cake is placed in one end of length, the other end places 1 pathogenic bacteria cake, 25 DEG C of cultivations (12h illumination/12h is dark).When
Observe (about cultivating 2 days) after pathogenic bacteria contact with the mycelia of biocontrol microorganisms, at the electricity Microscopic observation biocontrol microorganisms weight to pathogenic bacteria mycelia
Parasitization, result is shown in Fig. 4, it can be observed that JTM53 bacterial strain parasitizes Rhizoctonia solani Kuhn in modes such as winding or Parallel Growths
On mycelia, Rhizoctonia Solani growth is restricted, mycelia deformation and distortion.After cultivating 1 week, the bacterium colony energy of JTM53 bacterial strain
Rhizoctonia solani Kuhn bacterium colony is completely covered, and produces green spores in a large number.
Embodiment 4, the preparation of solid fermentation thing of JTM53 bacterial strain and application
One, the preparation of the solid fermentation thing of JTM53 bacterial strain
Seed culture medium (pH6.0-6.5): solvent is water, containing wheat bran 2% (mass ratio), glucose 1.0% (quality
Than), magnesium sulfate 0.5% (mass ratio), potassium dihydrogen phosphate 0.3% (mass ratio), calcium chloride 0.3% (mass ratio)；121 DEG C of sterilizings
30min。
Fermentation medium (pH6.5): 7 mass parts wheat brans and 3 mass parts wheat stalk powder are mixed, adds water,
The water content making culture medium is 70% (volume ratio)；121 DEG C of sterilizing 30min.
1, by the spore inoculating of JTM53 bacterial strain to seed culture medium (initial concentration is 106CFU/mL), keep dissolved oxygen dense
Degree 20%, temperature 28-30 DEG C, mixing speed 200rpm (in actual application, 200-250rpm), throughput 15L/min are (real
In the application of border, 10-15L/min), cultivate 3 days.
2, the culture and the 9 parts by volume solid mediums that 1 parts by volume step 1 are obtained mix, and keep temperature 28-30
DEG C, relative air humidity 95-100%, culture medium thickness 5-7cm, cultivate 7 days (actual application in, 5-7 days), then will
Culture natural air drying to water content is 10% (in actual application, 7%-10%), is the solid fermentation of JTM53 bacterial strain
Thing (also known as biocontrol microorganisms solid fermentation thing).
Two, the preparation of the solid fermentation thing of Rhizoctonia solani Kuhn
Replacing JTM53 bacterial strain with Rhizoctonia solani Kuhn, other, with step one, obtains the solid fermentation thing of Rhizoctonia solani Kuhn (again
Claim pathogenic bacteria solid fermentation thing).
Three, the greenhouse biocontrol effect (inoculation pathogenic bacteria) of biocontrol microorganisms solid fermentation thing
1, experiment the 1st day, by with the annual bluegrass seedling replanting of 4 true leaves, to nutritive cube, (each nutritive cube is equipped with 250g
The culture matrix being mixed to get by the 7 mass parts peats composed of rotten mosses and 3 mass parts vermiculites), each nutritive cube transplants 10 strain seedling.
2, packet transaction
Test group: test the 8th day, (addition of wheat bran powder is training to add wheat bran powder in nutritive cube
Support the 1% of matrix quality；Purpose is enrichment fungal colonization and the generation pressure increasing Brown patch disease), then in nutritive cube
(addition of biocontrol microorganisms solid fermentation thing is culture matrix matter to the biocontrol microorganisms solid fermentation thing that seedling root addition step one obtains
The 1% of amount) and the pathogenic bacteria solid fermentation thing that obtains of step 2 (addition of pathogenic bacteria solid fermentation thing is culture matrix quality
1%), then surface cover one layer of sterilizing fine earth, water；
Control group: be added without biocontrol microorganisms solid fermentation thing, other same test group.
3, experiment the 33rd day, investigates the incidence of Brown patch disease: as follows the base being attached to seedling root
Matter is rinsed well, then investigates the extent of damage of root.
The grade scale of the extent of damage of root:
0: butt rot degree < 1%；1: butt rot degree 1% 25%；
2: butt rot degree 26% 50%；3: butt rot degree 51% 75%；
4: butt rot degree 76% 89%；5: butt rot degree > 90%.
Carrying out three times repeating experiment during statistics disease index, in repeating to test, often group processes and takes 10 strain plant at random every time.
The incidence of disease of control group is 100%.The incidence of disease of experimental group is 18.0%, and disease index is 3.5.
Four, field biocontrol effect (natural occurrence)
Field test is divided 2 years and is carried out, Random Design, and often group 5 repeats to process, each grass repeating to be processed as 15m × 3m
Level ground (plant variety of planting lawn is annual bluegrass).
Experimental group: biocontrol microorganisms solid fermentation thing step one obtained is mixed with appropriate fine earth, uniformly executes in the plant 3 leaf phase
Entering the 2～5cm soil layers on lawn, the applied amount of biocontrol microorganisms solid fermentation thing is 8g/m2；
Positive controls: be mixed with appropriate fine earth by 70% thiophanate methyl wettable powder, uniformly executed in the plant 3 leaf phase
Entering the 2～5cm soil layers on lawn, the applied amount of 70% thiophanate methyl wettable powder is 10g/m2；
Negative control group: do not carry out any process.
Every 7 days Investigate incidences (DI), when investigating every time, 4 points of each repetition random searching, every a diameter of
0.15m.Disease index (DS) is added up according to the formula of step 3.The results are shown in Table 1.
Table 1 incidence of disease and disease index result
Embodiment 5, the bacteriostatic activity of metabolite of JTM53 bacterial strain
One, volatile compound
Test group: the PDA culture medium that the mycelia block (activation 3d diameter 5mm) of JTM53 bacterial strain is inoculated in a diameter of 9cm is put down
Plate central authorities, 25 DEG C of cultivations reach 5cm to colony diameter, cover with the double-deck diameter circular sterile glass paper slightly larger than 10cm above it
Diaphragm, above sterile glass paper membrane sheet, the mycelia block (activation 3d diameter 5mm) of one Rhizoctonia solani Kuhn of make-up, close with adhesive tape
It is honored as a queen, 25 DEG C of cultivations.
Control group: take the PDA culture medium flat plate of a diameter of 9cm, covers with the double-deck diameter circle slightly larger than 10cm above it
Shape sterile glass paper membrane sheet, the mycelia block of sterile glass paper membrane sheet correspondence PDA culture medium flat plate central authorities one Rhizoctonia solani Kuhn of button
(activation 3d diameter 5mm), after sealing with adhesive tape, 25 DEG C of cultivations.
Often group arrange 5 repeat process.From place Rhizoctonia solani Kuhn mycelia BOB(beginning of block) timing, respectively at cultivate 24h,
Detect inhibiting rate after 48h and 72h, the results are shown in Table 2.
Cultivate the photo after 72h and see Fig. 5.
Result shows, the volatile compound Rhizoctonia solani that JTM53 bacterial strain produces is inhibited.
Two, non-volatile compounds
Test group: take the PDA culture medium flat plate of a diameter of 9cm, spreads the double-deck diameter aseptic glass of circle slightly larger than 10cm
Glass paper membrane sheet；Then on glassine paper diaphragm, (corresponding PDA culture medium flat plate central authorities) place the JTM53 bacterium of an a diameter of 5mm
The mycelia block of strain, 25 DEG C of cultivation to colony diameters reach 5cm；Then throw off glassine paper diaphragm, put in PDA culture medium flat plate central authorities
Put the mycelia block of the Rhizoctonia solani Kuhn of an a diameter of 5mm, 25 DEG C of cultivations；
Control group: take the PDA culture medium flat plate of a diameter of 9cm, places the vertical withered silk of an a diameter of 5mm in flat board central authorities
The mycelia block of pyrenomycetes, 25 DEG C of cultivations.
Often group arrange 5 repeat process, results averaged.
From the mycelia BOB(beginning of block) timing of placement Rhizoctonia solani Kuhn, after cultivating 24h, 48h and 72h, detect inhibiting rate,
The results are shown in Table 2.
Cultivate the photo after 72h and see Fig. 6.
Result shows, the non-volatile compounds Rhizoctonia solani that JTM53 bacterial strain produces is inhibited.
Table 2 inhibiting rate result
Claims (5)
1. a fermentate, its preparation method is as follows:
(1) to seed culture medium and make it initial the spore inoculating of Trichoderma harzianum (Trichoderma harzianum) JTM53
Concentration is 106CFU/mL, cultivates, and obtains seed liquor；
(2) seed liquor that step (1) obtains is mixed with fermentation medium, ferment, obtain fermentate；
In step (1), the deposit number of described Trichoderma harzianum (Trichoderma harzianum) JTM53 is CGMCC
No.8914；In step (1), the condition of described cultivation is: dissolved oxygen concentration 20%, temperature 28-30 DEG C, mixing speed 200-
250rpm, throughput 10-15L/min, 3 days；In step (1), the solvent of described seed culture medium is water, containing weight/mass percentage composition
Be 2% wheat bran, weight/mass percentage composition be 1.0% glucose, weight/mass percentage composition be 0.5% magnesium sulfate, quality
Percentage composition be 0.3% potassium dihydrogen phosphate and weight/mass percentage composition be the calcium chloride of 0.3%；
In step (2), the seed liquor that 1 parts by volume step (1) obtains is mixed with 9 parts by volume fermentation mediums；In step (2),
The condition of described fermentation is: temperature 28-30 DEG C, relative air humidity 95-100%, culture medium thickness 5-7cm, cultivates 5-7 days,
Then it is 7%-10% by culture natural air drying to water content；In step (2), the preparation method of described fermentation medium is such as
Under: 7 mass parts wheat brans and 3 mass parts wheat wheat straw powder are mixed, adds water so that the volume basis of water in culture medium
Content is 70%.
2. the application in prevention and control Brown patch disease of the fermentate described in claim 1.
3. a preparation for prevention and control Brown patch disease, including fermentate described in claim 1.
4. the application in prevention and control annual bluegrass generation Brown patch disease of the fermentate described in claim 1.
5. a preparation for prevention and control annual bluegrass generation Brown patch disease, including fermentate described in claim 1.
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| CN105754875B (en) * | 2016-04-08 | 2019-04-19 | 广东海洋大学 | One plant of marine fungi Trichoderma harzianum DLEN2008005 and its application |
| CN109337829B (en) * | 2018-12-18 | 2020-11-03 | 山东五福生生态工程有限公司 | Trichoderma harzianum solid fermentation method, solid fermentation product, trichoderma harzianum microbial agent, and preparation method and application thereof |
| CN112266879B (en) * | 2020-09-23 | 2022-11-22 | 岭南师范学院 | Trichoderma harzianum SQ-18 strain and application thereof in preventing and treating turfgrass diseases |
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