CN105018354B - One plant of pick up the ears trichoderma and its application - Google Patents
One plant of pick up the ears trichoderma and its application Download PDFInfo
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- 241000223259 Trichoderma Species 0.000 title claims abstract description 62
- 210000005069 ears Anatomy 0.000 title claims abstract description 50
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- 241000346878 Trichoderma pleuroticola Species 0.000 claims abstract description 4
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- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 4
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 4
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- 238000012545 processing Methods 0.000 description 5
- 239000005842 Thiophanate-methyl Substances 0.000 description 4
- 230000003042 antagnostic effect Effects 0.000 description 4
- 229930187593 rose bengal Natural products 0.000 description 4
- AZJPTIGZZTZIDR-UHFFFAOYSA-L rose bengal Chemical compound [K+].[K+].[O-]C(=O)C1=C(Cl)C(Cl)=C(Cl)C(Cl)=C1C1=C2C=C(I)C(=O)C(I)=C2OC2=C(I)C([O-])=C(I)C=C21 AZJPTIGZZTZIDR-UHFFFAOYSA-L 0.000 description 4
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Abstract
The invention discloses one plant of pick up the ears trichoderma and its application.The bacterial strain is trichoderma (Trichoderma pleuroticola) the bacterial strain QZ 001H that pick up the ears, and deposit number is CGMCC No.11049.The invention also discloses including described biological control agent for the trichoderma strain QZ 001H that pick up the ears and preparation method thereof.Experiment shows, trichoderma strain of picking up the ears of the invention has that the speed of growth is fast, sporulation quantity is big, action spectrum is wide, strong stress resistance, plant rhizosphere can rapid, high volume colonize the features such as, therefore have a good application prospect.By the biological control agent of the trichoderma strain preparation of picking up the ears, a variety of soilborne fungal pathogens can be prevented and treated, include the one or more of capsicum wilt, Cure of Coryneum kunzei, pomegranate dry rot etc., are a kind of biological control agents of great application prospect.
Description
Technical field
The invention belongs to biological control plant disease field.Specifically, the present invention relates to one kind can efficiently prevent and treat plant
Trichoderma and application thereof of picking up the ears of thing disease, and the biological control agent using the Trichoderma preparation of picking up the ears.
Background technology
Trichoderma (Trichoderma spp.), belong to the Hyphomycetes, Moniliales, Moniliaceae of Fungi Imperfecti, be a kind of
The fungi being distributed widely on soil, air, dry branches and fallen leaves and various fermentates, from plant roots circle, blade and seed, bulb table
Face can be separated to.Trichoderma is nature generally existing and has the antagonistic microbe of affluent resources.Trichoderma growth and breeding speed
Degree is fast, can capture Vegetation space rapidly, can secrete generation antibiotic and suppress other growth of pathogenic bacteria, can also be competing by nutrition
Strive, hyperparasite, cell wall degrading enzyme and induction plant produce the mechanism of action such as resistance, reach the purpose for suppressing disease, be raw
One of most commonly used bacterial strain is studied in anti-bacterial strain.Many in more than 30 kinds of the Trichoderma having now been found that is dived with biological and ecological methods to prevent plant disease, pests, and erosion
Power, such as Trichoderma viride, Trichoderma harzianum, trichodermaharzianum, thorn spore trichoderma and trichoderma of picking up the ears, to 18 category, 29 kinds of phytopathogen tables
Reveal antagonistic activity.Biological control mechanism of the trichoderma to phytopathogen is various and complicated, and often number of mechanisms is common
The result of effect, the emphasis of different Biocontrol Activity mechanism is different, its Biocontrol Effect effect and Strain type, disease fungus
Type, agrotype and environmental condition are closely related.
Inventor was once separated to a kind of salt tolerant of efficient phosphate-solubilizing and picked up the ears Trichoderma (number of patent application：
201310442613.6).But trichoderma of picking up the ears is used for the soil-borne diseases such as mixed with rice bran, Cure of Coryneum kunzei, pomegranate dry rot
Evil, has not been reported.
The content of the invention
It is an object of the invention to provide a kind of new trichoderma strain for being used to efficiently prevent and treat plurality of plant diseases --- pick up the ears
Trichoderma strain QZ-001H.The trichoderma strain of picking up the ears of the present invention has that the speed of growth is fast, sporulation quantity is big, action spectrum is wide, resistance
By force, plant rhizosphere can rapid, high volume colonize the features such as, therefore have a good application prospect.
The above-mentioned purpose of the present invention is achieved through the following technical solutions：Trichoderma provided by the present invention of picking up the ears
(Trichoderma pleuroticola) bacterial strain QZ-001H, (obtained on top layer from the marine alga of Qingdao City Huangdao District shallow sea reef
) middle separating acquisition, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms on July 6th, 2015
Center (abbreviation CGMCC, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica), preservation
Numbering is CGMCC No.11049.It has following biological characteristics：In PDA culture medium, 7 are cultivated under 25 DEG C of dark conditions
My god, colony diameter extension 80mm, initial stage, white was sparse, after be changed into celadon, it is cotton-shaped.Reverse side is colourless.Mycelia has every branch.Point
Raw sporophore just forms the branch profile of pine and cypress formula, the stigma Shu Sheng of branch ends, to life, alternate or Dan Sheng, doleiform, and 6-10 ×
3-4.5μm.It is conidium elliposoidal, single, near colourless, it is in pistac during aggregation, wall is smooth or slightly coarse, 4-5.5 × 3-4
μm。
The present invention picks up the ears trichoderma strain QZ-001H cultural method or propagation method includes：
(1) Nostoc commune Vanch preserves and uses PDA culture medium, is formulated and is：Potato 200g, glucose 20g, agar 12g, distillation
Water 1000mL；
(2) laboratory fluids culture uses PDB culture mediums, is formulated and is：Potato 200g, glucose 20g, distilled water
1000mL。
(3) solid culture based formulas：Gu material and inorganic salt solution, in mass ratio 1：1.8 ratio is prepared.It is wherein described solid
Material is 50 by mass ratio:40:10 corncob, wheat bran and rice husk composition；By mass percentage, the group of the inorganic salt solution
Divide and content is：3.5% potassium dihydrogen phosphate, 0.04% magnesium sulfate, 4% ammonium sulfate, remaining as water.
(4) bulk fermentation culture formula：With solid culture based formulas in (3).
The present invention also provides a kind of biological control agent for including the described trichoderma strain QZ-001H that picks up the ears.
Present invention also offers the preparation method of above-mentioned biological control agent, comprise the following steps：
(1) seed liquor for the trichoderma strain QZ-001H that picks up the ears is prepared；
(2) seed liquor prepared by step (1) is inoculated into solid medium, incubated 3~5 days at 28-30 DEG C；
(3) culture of step (2) culture is added into sterilized water to mix, filtering, filtrate is seeded to bulk fermentation culture
Base, 28-30 DEG C of room temperature, relative humidity more than 85% fermenting cellar in carry out fermented and cultured.
Wherein, the solid medium in step (2) is made up of solid material and inorganic salt solution, and in mass ratio 1：1.8 ratio
Prepare；Wherein described solid material is 50 by mass ratio:40:10 corncob, wheat bran and rice husk composition；By mass percentage, institute
The component and content for stating inorganic salt solution be：3.5% potassium dihydrogen phosphate, 0.04% magnesium sulfate, 4% ammonium sulfate, remaining as water.
Also, the same solid medium of bulk fermentation culture medium in step (3).
In one embodiment of the invention, the preparation method comprises the following steps：
(1) spore for the trichoderma strain QZ-001H that picks up the ears is transplanted in PDB fluid nutrient mediums, 28 DEG C of shaking table shaken cultivations 3
Obtain seed liquor within~5 days；
(2) ratio for the seed liquor in mass ratio 10% for preparing step (1) is inoculated into solid medium, is shaken at 28 DEG C
Swing culture 3~5 days；
(3) culture by step (2) culture adds sterilized water in mass ratio 1：15 ratio mixing, filtering, by filtrate
By volume 1：6 ratio is seeded to fermentation medium, 28 DEG C of room temperature, relative humidity more than 85% fermenting cellar in fermented and cultured
8~9 days.
Present invention also offers above-mentioned trichoderma strain QZ-001H or the above-mentioned biological control agent of picking up the ears in preventing and treating plant soil
Pass the purposes in disease, the plant disease can be selected from capsicum wilt, Cure of Coryneum kunzei, pomegranate dry rot etc. one kind or
It is several.
Present invention also offers a kind of biological control method for being used to prevent and treat plant soil-borne diseases, the biological control method
Including applying above-mentioned trichoderma strain QZ-001H or the above-mentioned biological control agent of picking up the ears to the plant with soil-borne disease.
Experiment shows, trichoderma strain of picking up the ears of the invention has that the speed of growth is fast, sporulation quantity is big, action spectrum is wide, resistance
By force, plant rhizosphere can rapid, high volume colonize the features such as, therefore have a good application prospect.By the trichoderma strain system of picking up the ears
Standby biological control agent, a variety of soilborne fungal pathogens, including capsicum wilt, Cure of Coryneum kunzei, pomegranate dry rot can be prevented and treated
The one or more of disease etc., it is a kind of biological control agent of great application prospect.
Embodiment
The invention will now be further described with reference to specific embodiments, advantages of the present invention and feature will be with description and
It is apparent.But these embodiments are only exemplary, do not form any restrictions to the scope of the present invention.People in the art
Member to the details and form of technical solution of the present invention it should be understood that can enter without departing from the spirit and scope of the invention
Row modifications or substitutions, but these modifications and replacement are each fallen within protection scope of the present invention.
Embodiment 1
1st, pick up the ears trichoderma strain QZ-001H isolation and purification
The trichoderma strain QZ-001H that picks up the ears of the present invention is to gather marine alga from the reef of Qingdao City Huangdao District shallow sea, and use is dilute
Release flat band method and plate streak separation obtains, separation method is：
(1) separate：The marine alga (top layer acquisition) of Qingdao City Huangdao District shallow sea reef is gathered, the sample of collection is maintained in
In aseptic bottle, collecting location, time and collection people are indicated.Now by marine alga aseptic water washing, then pulverize, claim in sterile crucible
1g algae clays are taken in 100mL sterilized waters, 150rpm in 30 DEG C of shaking tables is placed in and shakes 10min, be subsequently placed in 60 DEG C of water-baths
30min is incubated, takes 100 μ L 10-2、10-3、10-4Dilution is coated on rose bengal medium flat board, each gradient coating three
Individual parallel, the microbial bacteria of the different shape after being cultivated 3 days at 30 DEG C on picking rose bengal medium falls within rose-bengal training
Support and rule on base flat board, colony growth situation is observed in timing.Then plate streak is used, purifies trichoderma strain, respectively
Numbering preserves.
(2) screening of the efficient antagonistic Trichoderma bacterial strain of capsicum wilt
1. primary dcreening operation：Using opposite culture method, PDA plate is prepared, is beaten with card punch at Trichoderma, capsicum wilt bacterium edge
A diameter of 5mm bacteria cake is taken, is implanted in the relative both sides center of flat board respectively, 25 DEG C incubated, observes Trichoderma pair day by day
The inhibitory action of pathogen.
2. secondary screening：The trichoderma strain with efficient antagonistic activity that will be screened carries out secondary screening, mainly by temperature tolerance,
Resistance to acids and bases, drug-resistant test, the preferable trichoderma strain of patience is screened, carries out potted plant controlling experiment and field test.
The present inventor obtains one plant of Trichoderma of picking up the ears that can efficiently prevent and treat plurality of plant diseases by a large amount of screening operations
(T.pleuroticola)QZ-001H.It is demonstrated experimentally that this is picked up the ears, trichoderma original powder is shown very high in mixed with rice bran
The prevention effect of effect so that crop is significantly increased production.Thus, trichoderma of picking up the ears of the invention is the wood of picking up the ears for having wide application prospect
Mould new strains, it can be used for the biological control agent for preparing preventing and treating plurality of plant diseases.
2nd, bacterial strain is identified
(1) Microbiological Characteristics：In PDA culture medium, cultivated 7 days under 25 DEG C of dark conditions, colony diameter extension 80mm,
Initial stage, white was sparse, after be changed into celadon, it is cotton-shaped.Reverse side is colourless.Mycelia has every branch.Conidiophore just forms pine and cypress formula
Branch profile, the stigma Shu Sheng of branch ends, to life, alternate or Dan Sheng, doleiform, 6-10 × 3-4.5 μm.Conidium ellipsoid
It is shape, single, near colourless, it is in pistac during aggregation, wall is smooth or slightly coarse, 4-5.5 × 3-4 μm.
(2) molecular biological characteristic
The rRNA gene sequencings result (ITS-5.8S-ITS2 areas) of the bacterial strain is following (SEQ-1)：
TCGGAAGTAAAAAGTCGTAACAAGGTCTCCGTTGGTGAACCAGCGGAGGGATCATTACCGAGTTTACAA
CTCCCAAACCCAATGTGAACGTTACCAAACTGTTGCCTCGGCGGGATCTCTGCCCCGGGTGCGTCGCAGCCCCGGAC
CAAGGCGCCCGCCGGAGGACCAACCAAAACTCTTATTGTATACCCCCTCGCGGGTTTTTTTATAATCTGAGCCTTCT
CGGCGCCCCTCGTGGGCGTTTCGAAAATGAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGA
ACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCG
CCAGTATTCTGGCGGGCATGCCTGTCCGAGCGTCATTTCAACCCTCGAACCCCTCCCGGGGGTCGGCGTTGGGGATC
GGCCCTCCCTCTGCGGGGGCCGTCTCCGAAATACAGTGGCGGTCTCGCCGCAGCCTCTCCTGCGCAGTAGTTTGCAC
ACTCGCACCGGGAGCGCGGCGCGTCCACAGCCGTTAAACACCCAACTTTCTGAAATGTGACCTCGATCAGGTAGAAT
CCCGC
Wherein, PDA culture medium formula：Potato 200g (peeling), glucose 20g, agar 12g, distilled water 1000mL.Meng
Add and draw red culture medium prescription：Potato 200g (peeling), glucose 20g, 1% rose-bengal solution 3.3mL, sodium propionate 3.5g,
Streptomysin 0.03g, water 1000mL, pH are natural.
Embodiment 2
1st, pick up the ears trichoderma strain QZ-001H fermentation process
PDB culture medium prescriptions：Potato 200g (peeling), glucose 20g, distilled water 1000mL.
A large amount of solid fermentation culture medium prescriptions (weight/mass percentage composition)：Gu material：Corncob 50%, wheat bran 40%, rice husk
10%；Inorganic salt solution：Potassium dihydrogen phosphate 3.5%, magnesium sulfate 0.04%, ammonium sulfate 4%, remaining as water.Solid-to-liquid ratio is 1:1.8
(mass ratio).
Pick up the ears trichoderma strain QZ-001H a large amount of solid fermentation process：
1. strain seed liquor culture：To be picked up the ears a small amount of spore of trichoderma strain QZ-001H pickings from test tube slant, be moved to
In PDB fluid nutrient mediums, 28 DEG C of shaking table shaken cultivations 3~5 days, this is seed liquor, and it is 0.15-0.30cfu/ml that bacterium, which lives,；
2. the culture of solids manufacture strain：Seed liquor is inoculated into solid medium (500mL triangles in 10% ratio
Bottle) in, 28 DEG C are incubated 3~5 days, middle multiple oscillation；
3. a large amount of solid fermentations：2. the culture of middle solid culture is pressed 1 with sterilized water:15 dilution proportions, and with sterilizing
Filtered through gauze, thick slag of going out as produce bacterium solution, and inoculative proportion presses 1:6 are inoculated in bulk fermentation culture medium.By the raw material of inoculation
Fermented and cultured 8~9 days in fermenting cellar (28 DEG C and relative humidity more than 85%) are placed in, zymotic fluid can obtain wood of picking up the ears after drying
Mould original powder, viable count be 40-50 hundred million/gram.
Embodiment 3
Present embodiments provide related experiment of the trichoderma strain QZ-001H originals powder to capsicum wilt prevention effect of picking up the ears.
1) reagent agent
Trichoderma strain QZ-001H originals of picking up the ears powder (being prepared in embodiment 2)；70% thiophanate methyl wettable powder (city
Sell).
2) for studying thing and controlling object：
It is capsicum for studying thing；Kind is middle capsicum No. 1；
Controlling object：Droop.
3) experimental field situation, experimental design and arrangement
With being experimental field located at the remaining village capsicum of Jimo City of Shandong Province Nan Quan towns celebrating, former years droop occurs soil for sand
It is heavier.Experimental field cultivation condition uniformity.
This experiment sets trichoderma strain QZ-001H original powder of picking up the ears and is watered pouring root；70% thiophanate methyl wettable powder dilution agent
1000 times；Clear water compares totally 5 processing, each 500 grams of capsicum hole irrigation decoction or water, is repeated 4 times.Each random district's groups row of cell
Row.Earthing after dispenser, each cell arrange nutritive cube 300, per alms bowl 2 chilli seeds of point.
4) pilot survey and computational methods
(1) meteorological condition
The dispenser same day is fine, gentle breeze, and the highest temperature is 30 DEG C, and the lowest temperature is 26 DEG C, relative humidity 65%
(2) drug effect and security survey
Efficacy survey：Investigation 1 time altogether, carried out within 30 days in emergence, record 300 nutritive cube kind capsicum wilt morbidities
Number, calculate diseased plant rate and prevention effect.
(3) drug effect computational methods
Drug effect following formula calculates：
5) result
(1) prevention effect of the reagent agent to capsicum wilt
30 days investigation diseased plant numbers, the result of table 1 are shown after dispenser, and the diseased plant rate of clear water control is up to 32.01%, each processing
Diseased plant rate be below clear water control.Trichoderma strain QZ-001H originals powder of picking up the ears using multiple is respectively at 250,500 and 1000 times
Diseased plant rate after reason is respectively 7.85%, 9.88%, 12.19%, is below the diseased plant rate of comparison medicament 12.39%；Pick up the ears wood
The prevention effect of each processing of trichoderma strain QZ-001H original powder is respectively 75.48%, 69.14%, 61.94%, wherein 1:250
Prevention effect is best, with other processing in P<There is significant difference, 70% thiophanate methyl is withered to capsicum in 0.05 level
The prevention effect of disease is 61.31%.The trichoderma strain QZ-001H originals powder that proves to pick up the ears can effectively control the harm of capsicum wilt.
The incidence of disease and prevention effect of capsicum wilt after 1 each processing of table
| Handle multiple | Diseased plant rate (%) | Prevention effect (%) | The significance of difference |
| Trichoderma QZ-001H originals of picking up the ears powder 250 | 7.85 | 75.48 | a |
| Trichoderma QZ-001H originals of picking up the ears powder 500 | 9.88 | 69.14 | b |
| Trichoderma QZ-001H originals of picking up the ears powder 1000 | 12.19 | 61.94 | c |
| 70% thiophanate methyl 500 | 12.39 | 61.31 | c |
| CK | 32.01 | - | - |
Note：Not notable (the P of difference after Duncan Multiple range tests is represented with there is same letter after data line<0.05).
(2) capsicum security survey：It is observed that each chemicals treatment district, compared with check plot, chili growth is normal, no poisoning
Produce, the Trichoderma QZ-001H originals powder that illustrates to pick up the ears is safe to capsicum for examination concentration.
Therefore, from the point of view of the incidence of disease and prevention effect, trichoderma strain of picking up the ears QZ-001H has preferable to capsicum wilt
Prevention effect, prevention effect is up to more than 75% after dispenser, hence it is evident that is better than comparison medicament, significant difference.
Claims (9)
- Trichoderma of picking up the ears 1. (Trichoderma pleuroticola) bacterial strain QZ-001H, the deposit number of the bacterial strain are CGMCCNo.11049。
- 2. purposes of the trichoderma strain QZ-001H in plant soil-borne diseases are prevented and treated of picking up the ears described in claim 1.
- 3. purposes as claimed in claim 2, it is characterized in that, the plant disease is selected from capsicum wilt, Cure of Coryneum kunzei, stone One or more in pomegranate dry rot.
- A kind of 4. biological control agent of the trichoderma strain QZ-001H that picks up the ears including described in claim 1.
- 5. the preparation method of biological control agent as claimed in claim 4, comprises the following steps：(1) seed liquor for the trichoderma strain QZ-001H that picks up the ears is prepared；(2) seed liquor prepared by step (1) is inoculated into solid medium, incubated 3~5 days at 28-30 DEG C；(3) culture of step (2) culture is added into sterilized water to mix, filtering, filtrate is seeded to bulk fermentation culture medium, 28-30 DEG C of room temperature, relative humidity more than 85% fermenting cellar in carry out fermented and cultured；Wherein, the solid medium in step (2) is made up of solid material and inorganic salt solution, and the two mass ratio is 1：1.8；Wherein institute It is 50 that solid material, which is stated, by mass ratio:40:10 corncob, wheat bran and rice husk composition；By mass percentage, the inorganic salt solution Component and content be：3.5% potassium dihydrogen phosphate, 0.04% magnesium sulfate, 4% ammonium sulfate, remaining as water；The same solid medium of bulk fermentation culture medium in the step (3).
- 6. the preparation method of biological control agent as claimed in claim 5, it is characterized in that, comprise the following steps：(1) spore for the trichoderma strain QZ-001H that picks up the ears is transplanted in PDB fluid nutrient mediums, 28 DEG C of shaking table shaken cultivations 3~5 It obtains seed liquor；(2) ratio for the seed liquor in mass ratio 10% for preparing step (1) is inoculated into solid medium, and training is vibrated at 28 DEG C Support 3~5 days；(3) culture by step (2) culture adds sterilized water in mass ratio 1：15 ratio mixing, filtering, body is pressed by filtrate Product ratio 1：6 ratio is seeded to fermentation medium, 28 DEG C of room temperature, relative humidity more than 85% fermenting cellar in fermented and cultured 8~9 My god.
- 7. purposes of the biological control agent in plant soil-borne diseases are prevented and treated described in claim 4.
- 8. purposes as claimed in claim 7, it is characterized in that, the plant disease is selected from capsicum wilt, Cure of Coryneum kunzei, stone One or more in pomegranate dry rot.
- 9. a kind of be used to prevent and treat the biological control methods of plant soil-borne diseases, it is characterized in that, including to the plant with soil-borne disease Thing applies pick up the ears trichoderma strain QZ-001H or the biological control agent described in claim 4 described in claim 1.
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