CN103952320A - Trichoderma harzianum and application thereof in preventing and controlling lawn fusarium blight - Google Patents
Trichoderma harzianum and application thereof in preventing and controlling lawn fusarium blight Download PDFInfo
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Abstract
The invention discloses trichoderma harzianum and an application thereof in preventing and controlling lawn fusarium blight. The trichoderma harzianum JTM55 provided by the invention, with a Preservation No. of CGMCCNo.8916, is named JTM55 for short. According to the invention, fermented substances of JTM55 strains are also protected. JTM55 strains have the properties such as strong survival ability, wide adaptability, no environment pollution, and the like, and as a biocontrol bacterium, have a broad application prospect. Indoor confrontation and antagonism tests show that JTM55 strains have a good antibacterial effect on fusarium oxysporum. Furthermore, JTM55 strains are used for preparing solid fermented substances, and then the solid fermented substances are subjected to a potting control test and a field control test, so that an effect of preventing and treating fusarium oxysporum is good and stable. The trichoderma harzianum disclosed by the invention has an important application value on the prevention and control on lawn fusarium blight.
Description
Technical field
The present invention relates to a strain trichoderma harziarum and the application in the reaping hook blight of prevention and control lawn thereof.
Background technology
Lawn reaping hook blight causes, occurs in the crushing fungal disease of one on lawn by Fusarium oxysporum.This disease also can cause different areas various crop morbidity, causes a large amount of financial losses.
Lawn dogstail plant is mostly perennial, this accumulation that is Fusarium oxysporum, lawn reaping hook blight suitable condition is provided.To be lawn dogstail just can be caused root, stem and leaf portion tissue to rot to the lawn typical symptom characteristic of reaping hook blight in a short time after infecting or the death of a large amount of plant, up to the present the good disease-resistant variety of appearance effect not also aborning.
At present, in to the control of lawn reaping hook blight, chemical prevention occupies consequence.Chemical prevention not only cost is high, and easy contaminate environment, and in prevention and control pathogenic bacteria, pathogenic bacteria also easily produces even resistance of resistance to chemical agent.
Summary of the invention
The object of this invention is to provide a strain trichoderma harziarum and the application in the reaping hook blight of prevention and control lawn thereof.
Trichoderma harziarum provided by the invention (Trichoderma harzianum) JTM55, be called for short bacterial strain JTM55, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 03 13rd, 2014 and (be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), deposit number is CGMCC No.8916.
The present invention also protects the fermented product of described JTM55 bacterial strain.
The preparation method of described fermented product is specific as follows:
(1) by the spore inoculating of JTM55 bacterial strain, to seed culture medium, (starting point concentration specifically can be 10
6cFU/mL), cultivate, obtain seed liquor;
(2) seed liquor step (1) being obtained is mixed with fermention medium, ferments, and obtains fermented product.
In step (1), the condition of described cultivation specifically can be: dissolved oxygen concentration 20%, temperature 28-30 DEG C, stirring velocity 200-250rpm (specifically can be 200rpm), air flow 10-15L/min (specifically can be 15L/min), 3 days.Described seed culture medium 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 primary phosphate 0.3% (mass ratio), calcium chloride 0.3% (mass ratio).
In step (2), the condition of described fermentation specifically can be: temperature 28-30 DEG C, relative air humidity 95-100%, substratum thickness 5-7cm, cultivating 5-7 days (specifically can be 7 days), is then 7%-10% (specifically can be 10%) by culture natural air drying to water content.Described fermention medium is specific as follows: fermention medium (pH6.5): 7 mass parts wheat brans are mixed with 3 mass parts wheat wheat straw powder, add water, the water content that makes substratum is 70% (volume ratio).
In step (2), the culture that specifically 1 parts by volume step (1) can be obtained mixes with 9 parts by volume fermention mediums.
The present invention also protects the fermented product of JTM55 bacterial strain or described JTM55 bacterial strain in the application suppressing in Fusarium oxysporum.
The application of the fermented product that the present invention also protects JTM55 bacterial strain or described JTM55 bacterial strain in the reaping hook blight of prevention and control lawn.
The present invention also protects a kind of for suppressing the preparation of Fusarium oxysporum, comprises the fermented product of JTM55 bacterial strain or described JTM55 bacterial strain.
The present invention also protects the preparation of a kind of prevention and control lawn reaping hook blight, comprises the fermented product of JTM55 bacterial strain or described JTM55 bacterial strain.
The present invention also protects the fermented product of JTM55 bacterial strain or described JTM55 bacterial strain, prevention and control annual bluegrass, the application in the reaping hook blight of lawn occurs.
The present invention also protects the preparation of a kind of prevention and control annual bluegrass generation lawn reaping hook blight, comprises the fermented product of JTM55 bacterial strain or described JTM55 bacterial strain.
As Trichoderma, JTM55 bacterial strain has that viability 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, JTM55 bacterial strain has good fungistatic effect to Fusarium oxysporum.Further prepare solid fermentation thing with JTM55 bacterial strain, then carry out potted plant control experiment and daejeon prevention test with solid fermentation thing, good and stable to Fusarium oxysporum prevention effect.The present invention has great using value for the prevention and control of lawn reaping hook blight.
Brief description of the drawings
Fig. 1 is the bacterium colony photo of JTM55 bacterial strain 20 DEG C of cultivation 5d on PDA substratum.
Fig. 2 is conidiophore and conidial photo of JTM55 bacterial strain.
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 1 of embodiment 5.
Fig. 6 is the result photo in the step 2 of embodiment 5.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment, results averaged for three times.70% thiophanate methyl wettable powder: Lintong District, Xi'an insecticide factory.Annual bluegrass: Sheng Hua development in science and technology company limited of Beijing Cathay.
Fusarium oxysporum (Fusarium oxysporum), claims again sharp Fusariumsp: mould antagonism and the biocontrol effect research to sharp Fusariumsp of field adhesion .2001. wood. plant protection, 27 (4): 47-48.
The acquisition of embodiment 1, bacterial strain, preservation
PDA substratum: potato 200g, glucose 15g, agar 20g, distilled water 1000ml, 121 DEG C of sterilizing 20min.
TSM substratum: MgSO
47H
200.2g, K
2hPO
40.9g, KCl0.15g, NH
4nO
31.0g, glucose 3.0g, rose-red 0.15g, 60% fenaminosulf wettable powder 0.3g, PCNB0.2g, agar 20g, distilled water 950ml, 121 DEG C of sterilizing 20min.
One, the acquisition of JTM55 bacterial strain
Sampling time: in February, 2008.
Sampling position: Beijing golf course.
Push the veneer of soil of 3-5cm aside, then the root system of plant is dug out, together with the soil of rhizosphere, install in polyethylene plastic bag, take back laboratory.By slightly air-dry the root system that is stained with soil, pat root system, make to adhere to superincumbent soil and come off, use sterilized water gradient dilution, draw respectively 0.1mL diluent and splash on TSM culture medium flat plate, even with the L shaped spreading rod coating of sterilizing, the constant incubator that is placed in 25-28 DEG C is cultivated 3-4 days.Picking form is transferred on PDA culture medium flat plate and carries out purifying cultivation like single bacterium colony of Trichoderma, numbers, and be saved in PDA medium slant after mirror mirror is tentatively regarded as Trichoderma.
By above-mentioned steps, obtain the bacterial strain of some pure cultures, a wherein strain called after JTM55 bacterial strain.
Two, the morphological specificity of JTM55 bacterial strain
On PDA substratum, Fig. 1 is shown in by 20 DEG C of bacterium colony photos of cultivating 5d.Diameter 9.0cm, the speed of growth is very fast, and 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, and the later stage is because sporulation quantity ambassador bacterium colony is slightly green.
Fig. 2 is shown in by conidiophore and conidial photo.The tree-shaped conidiophore of multi-branched forms quite loose flora.The wide 4-5 μ of major branch m, side shoot is many, forms pyramid.Adnation bottle stalk can reach 5, becomes to intend colyliform arrangement or single along little side shoot irregular alignment.The contracting of slightly hanging of adnation bottle metulae portion, expand at middle part, upwards narrow one-tenth bottle stalk gradually, 5-7 × 3-3.5 μ m.Push up the relative long and very thin with atypical bottle of stalk of life, 13-18 × 2.5-3.5 μ m.Bottle stalk greatly mainly with wide-angle on its carrier raw, sometimes slightly bend towards top.Phialospore is spheric conidium head, the subsphaeroidal or obovate of conidium, and wall is smooth, light green, 2.8-3.2 × 2.5-2.8 μ m.
Three, the Molecular Identification of JTM55 bacterial strain
JTM55 bacterial strain ITS district is as shown in the sequence 1 of sequence table.
Four, the preservation of JTM55 bacterial strain
Based on morphological specificity and Molecular Identification result, JTM55 bacterial strain belongs to trichoderma harziarum (Trichoderma harzianum).Trichoderma harziarum (Trichoderma harzianum) JTM55, be called for short bacterial strain JTM55, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 03 13rd, 2014 and (be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), deposit number is CGMCC No.8916.
Embodiment 2, the restraining effect of JTM55 bacterial strain to pathogenic bacteria
1, by JTM55 bacterial strain list colony inoculation to PDA culture medium flat plate, 20 DEG C cultivate 5d.
2, get the PDA culture medium flat plate of completing steps 1, buy the bacterium cake that cut-off footpath is 5mm (biocontrol microorganisms cake) in bacterium colony forward position with punch tool.
3, by single Fusarium oxysporum colony inoculation to PDA culture medium flat plate, cultivate 5d for 20 DEG C.
4, get the PDA culture medium flat plate of completing steps 3, buy the bacterium cake that cut-off footpath is 5mm (pathogenic bacterium cake) in bacterium colony forward position with punch tool.
5, get new PDA culture medium flat plate, beating cut-off footpath with punch tool is 5mm, in contrast thing.
6, packet transaction
Test group: get new PDA culture medium flat plate, place 1 biocontrol microorganisms cake and 1 pathogenic bacterium cake in the above, the slant range at Liang Gejunbing center is 3cm, 25 DEG C of cultivations (12h illumination/12h dark);
Control group: get new PDA culture medium flat plate, place 1 contrast and 1 pathogenic bacterium cake in the above, the slant range at both centers is 3cm, 25 DEG C of cultivations (12h illumination/12h dark).
Cultivate the colony diameter of measuring afterwards pathogenic bacterium for 3 days, calculate inhibiting rate.Colony diameter × 100% of inhibiting rate=(colony diameters of colony diameter-test group pathogenic bacterium of control group pathogenic bacterium) ÷ control group pathogenic bacterium.JTM55 bacterial strain is 71.4% (mean value repeating for three times) to the inhibiting rate of Fusarium oxysporum.
Cultivate photo after 6 days see Fig. 3 (left side be biocontrol microorganisms cake, the right be pathogenic bacterium cake), can observe, Fusarium oxysporum is covered by JTM55 bacterial strain completely.
Embodiment 3, the superparasitism ability of JTM55 bacterial strain to pathogenic bacteria
1, by JTM55 bacterial strain list colony inoculation to PDA culture medium flat plate, 20 DEG C cultivate 5d.
2, get the PDA culture medium flat plate of completing steps 1, buy the bacterium cake that cut-off footpath is 5mm (biocontrol microorganisms cake) in bacterium colony forward position with punch tool.
3, by single Fusarium oxysporum colony inoculation to PDA culture medium flat plate, cultivate 5d for 20 DEG C.
4, get the PDA culture medium flat plate of completing steps 3, buy the bacterium cake that cut-off footpath is 5mm (pathogenic bacterium cake) in bacterium colony forward position with punch tool.
5, get new PDA culture medium flat plate, place the celloyarn of a lcm × 3cm in the above, then place 1 biocontrol microorganisms cake, 1 pathogenic bacterium cake of the other end placement, 25 DEG C of cultivations (12h illumination/12h dark) in one end of celloyarn length.After the mycelia of observation pathogenic bacterium and biocontrol microorganisms contacts (approximately cultivating 2 days), hyperparasitism at electric Microscopic observation biocontrol microorganisms to pathogenic bacterium mycelia, the results are shown in Figure 4, can observe JTM55 bacterial strain parasitizes on Fusarium oxysporum mycelia in modes such as winding or Parallel Growths, Fusarium oxysporum mycelial growth is restricted, mycelia distortion and distortion.Cultivate after 1 week, the bacterium colony of JTM55 bacterial strain can cover Fusarium oxysporum bacterium colony completely, and produces in a large number green spores.
Preparation and the application of the solid fermentation thing of embodiment 4, JTM55 bacterial strain
One, the preparation of the solid fermentation thing of JTM55 bacterial strain
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 primary phosphate 0.3% (mass ratio), calcium chloride 0.3% (mass ratio); 121 DEG C of sterilizing 30min.
Fermention medium (pH6.5): 7 mass parts wheat brans are mixed with 3 mass parts wheat wheat straw powder, add water, the water content that makes substratum is 70% (volume ratio); 121 DEG C of sterilizing 30min.
1, by the spore inoculating of JTM55 bacterial strain, to seed culture medium, (starting point concentration is 10
6cFU/mL), keep dissolved oxygen concentration 20%, temperature 28-30 DEG C, stirring velocity 200rpm (in practical application, 200-250rpm all can), air flow 15L/min (in practical application, 10-15L/min all can), cultivate 3 days.
2, the culture 1 parts by volume step 1 being obtained mixes with 9 parts by volume solid mediums, keep 28-30 DEG C of temperature, relative air humidity 95-100%, substratum thickness 5-7cm, cultivate 7 days (in practical application, 5-7 days all can), then be 10% (in practical application by culture natural air drying to water content, 7%-10% all can), be the solid fermentation thing (claiming again biocontrol microorganisms solid fermentation thing) of JTM55 bacterial strain.
Two, the preparation of the solid fermentation thing of Fusarium oxysporum
By Fusarium oxysporum replacement JTM55 bacterial strain, other same step 1, obtain the solid fermentation thing (claiming again pathogenic bacterium solid fermentation thing) of Fusarium oxysporum.
Three, the greenhouse biocontrol effect of biocontrol microorganisms solid fermentation thing (inoculation pathogenic bacterium)
1, experiment the 1st day, by the annual bluegrass seedling replanting with 4 true leaves, to nutrition pot (each nutrition pot is equipped with the culture medium that 250g is mixed to get by the 7 mass parts peats composed of rotten mosses and 3 mass parts vermiculites), each nutrition pot is transplanted 10 strain seedling.
2, packet transaction
Test group: test the 8th day, (add-on of wheat bran powder is 1% of culture medium quality to add wheat bran powder in nutrition pot; Object is enrichment fungal colonization and the generation pressure that increases lawn reaping hook blight), then in nutrition pot, seedling root adds the pathogenic bacterium solid fermentation thing that the biocontrol microorganisms solid fermentation thing that step 1 obtains (add-on of biocontrol microorganisms solid fermentation thing be culture medium quality 1%) and step 2 obtain (add-on of pathogenic bacterium solid fermentation thing be culture medium quality 1%), then at surface coverage one deck sterilizing fine earth, water;
Control group: do not add biocontrol microorganisms solid fermentation thing, other same test group.
3, test the 33rd day, investigate as follows the incidence of lawn reaping hook blight: the matrix that is attached to seedling root is rinsed well, then investigated the degree of damage of root.
The grade scale of the degree 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%.
When statistics sickness rate, carry out repeating for three times experiment, repeat every group of processing in experiment at every turn and get at random 5 strain plant.
When statistics disease index, carry out repeating for three times experiment, repeat every group of processing in experiment at every turn and get at random 10 strain plant.
The sickness rate of control group is 100%.The sickness rate of experimental group is 15.0%, and disease index is 2.3.
Four, field biocontrol effect (natural occurrence)
Field test is divided and is carried out for 2 years, Random Design, every group of 5 re-treatments, the lawn (plant variety of planting lawn is annual bluegrass) that each re-treatment is 15m × 3m.
Experimental group: the biocontrol microorganisms solid fermentation thing that step 1 is obtained is mixed with appropriate fine earth, evenly applies 2～5cm soil layer on lawn in the 3 leaf phases of plant, and the applied amount of biocontrol microorganisms solid fermentation thing is 8g/m2;
Positive controls: 70% thiophanate methyl wettable powder and appropriate fine earth are mixed, evenly apply 2～5cm soil layer on lawn in the 3 leaf phases of plant, the applied amount of 70% thiophanate methyl wettable powder is 10g/m2;
Negative control group: do not carry out any processing.
Every 7 days Investigate incidences (DI), while investigation at every turn, 4 points of each repetition random searching, the diameter of every is 0.15m.According to the formula statistics disease index (DS) of step 3.The results are shown in Table 1.
Table 1 sickness rate and disease index result
The bacteriostatic activity of the meta-bolites of embodiment 5, JTM55 bacterial strain
Test group: the mycelia piece of JTM55 bacterial strain (activation 3d diameter 5mm) is inoculated in to the PDA culture medium flat plate central authorities that diameter is 9cm, 25 DEG C are cultured to colony diameter and reach 5cm, cover with above it double-deck slightly larger in diameter in the aseptic glassine paper diaphragm of circle of 10cm, the mycelia piece (activation 3d diameter 5mm) of a Fusarium oxysporum of aseptic glassine paper diaphragm top make-up, with after scotch tape sealing, 25 DEG C of cultivations.
Control group: the PDA culture medium flat plate that cut-off footpath is 9cm, cover with above it double-deck slightly larger in diameter in the aseptic glassine paper diaphragm of circle of 10cm, the corresponding PDA culture medium flat plate of aseptic glassine paper diaphragm central authorities buckle the mycelia piece (activation 3d diameter 5mm) of a Fusarium oxysporum, with after scotch tape sealing, 25 DEG C of cultivations.
Every group arranges 5 re-treatments.From placing the mycelia BOB(beginning of block) timing of Fusarium oxysporum, after cultivating 24h, 48h and 72h, detect inhibiting rate, the results are shown in Table 2.
Fig. 5 is shown in by the photo of cultivating after 72h.
Result shows, the volatile compound that JTM55 bacterial strain produces is inhibited to Fusarium oxysporum.
Two, non-volatile compounds
Test group: the PDA culture medium flat plate that cut-off footpath is 9cm, spread double-deck slightly larger in diameter in the aseptic glassine paper diaphragm of circle of 10cm; Then on glassine paper diaphragm, (corresponding PDA culture medium flat plate central authorities) places the mycelia piece of the JTM55 bacterial strain that a diameter is 5mm, and 25 DEG C are cultured to colony diameter and reach 5cm; Then throw off glassine paper diaphragm, at PDA culture medium flat plate, the mycelia piece of the Fusarium oxysporum that a diameter is 5mm, 25 DEG C of cultivations are placed by central authorities;
Control group: the PDA culture medium flat plate that cut-off footpath is 9cm, place the mycelia piece of the Fusarium oxysporum that a diameter is 5mm, 25 DEG C of cultivations in dull and stereotyped central authorities.
Every group arranges 5 re-treatments, results averaged.
From the mycelia BOB(beginning of block) timing of Fusarium oxysporum, after cultivating 24h, 48h and 72h, detect inhibiting rate, the results are shown in Table 2.
Fig. 6 is shown in by the photo of cultivating after 72h.
Result shows, the non-volatile compounds that JTM55 bacterial strain produces is inhibited to Fusarium oxysporum.
Table 2 inhibiting rate result
Claims (9)
1. trichoderma harziarum (Trichoderma harzianum) JTM55, its deposit number is CGMCC No.8916.
2. the fermented product of trichoderma harziarum described in claim 1.
3. fermented product as claimed in claim 2, is characterized in that: the preparation method of described fermented product is as follows:
(1) by the spore inoculating of trichoderma harziarum described in claim 1 to seed culture medium, cultivate, obtain seed liquor;
(2) seed liquor step (1) being obtained is mixed with fermention medium, ferments, and obtains fermented product.
Described in claim 1 described in trichoderma harziarum, claim 2 described in fermented product or claim 3 fermented product in the application suppressing in Fusarium oxysporum.
5. the application of fermented product in the reaping hook blight of prevention and control lawn described in fermented product or claim 3 described in trichoderma harziarum, claim 2 described in claim 1.
6. for suppressing a preparation for Fusarium oxysporum, comprise described in trichoderma harziarum described in claim 1, claim 2 fermented product described in fermented product or claim 3.
7. a preparation for prevention and control lawn reaping hook blight, comprises described in trichoderma harziarum described in claim 1, claim 2 fermented product described in fermented product or claim 3.
Described in claim 1 described in trichoderma harziarum, claim 2 described in fermented product or claim 3 fermented product there is the application in the reaping hook blight of lawn prevention and control annual bluegrass.
9. there is a preparation for lawn reaping hook blight in prevention and control annual bluegrass, comprises described in trichoderma harziarum described in claim 1, claim 2 fermented product described in fermented product or claim 3.
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Cited By (5)
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| CN105754870A (en) * | 2016-03-22 | 2016-07-13 | 山东省科学院生物研究所 | Trichoderma cyanodichotomus and application thereof |
| CN112955538A (en) * | 2018-07-19 | 2021-06-11 | 巴黎-萨克雷大学 | Method for biologically controlling fusarium ear blight of grain |
| WO2022110848A1 (en) * | 2020-11-26 | 2022-06-02 | 南京思农生物有机肥研究院有限公司 | Evaluation and analysis method for preventing and controlling fusarium verticillioides of maize by means of chitin-enhanced trichoderma |
| CN114836329A (en) * | 2022-05-18 | 2022-08-02 | 上海市农业科学院 | Trichoderma harzianum HB40609 and application thereof |
| CN114907986A (en) * | 2022-04-28 | 2022-08-16 | 云南省农业科学院生物技术与种质资源研究所 | Trichoderma harzianum and application thereof in preparation for preventing and treating root rot of panax notoginseng |
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| CN108342330B (en) * | 2018-05-09 | 2020-08-28 | 中国科学院天津工业生物技术研究所 | Trichoderma longibrachiatum with broad-spectrum antibacterial performance and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105754870A (en) * | 2016-03-22 | 2016-07-13 | 山东省科学院生物研究所 | Trichoderma cyanodichotomus and application thereof |
| CN112955538A (en) * | 2018-07-19 | 2021-06-11 | 巴黎-萨克雷大学 | Method for biologically controlling fusarium ear blight of grain |
| WO2022110848A1 (en) * | 2020-11-26 | 2022-06-02 | 南京思农生物有机肥研究院有限公司 | Evaluation and analysis method for preventing and controlling fusarium verticillioides of maize by means of chitin-enhanced trichoderma |
| CN114907986A (en) * | 2022-04-28 | 2022-08-16 | 云南省农业科学院生物技术与种质资源研究所 | Trichoderma harzianum and application thereof in preparation for preventing and treating root rot of panax notoginseng |
| CN114907986B (en) * | 2022-04-28 | 2022-11-18 | 云南省农业科学院生物技术与种质资源研究所 | Trichoderma harzianum and application thereof in preparation for preventing and treating root rot of panax notoginseng |
| CN114836329A (en) * | 2022-05-18 | 2022-08-02 | 上海市农业科学院 | Trichoderma harzianum HB40609 and application thereof |
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