CN105018354A - Trichoderma pleuroticola and application thereof - Google Patents
Trichoderma pleuroticola and application thereof Download PDFInfo
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- CN105018354A CN105018354A CN201510502268.XA CN201510502268A CN105018354A CN 105018354 A CN105018354 A CN 105018354A CN 201510502268 A CN201510502268 A CN 201510502268A CN 105018354 A CN105018354 A CN 105018354A
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Landscapes
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses trichoderma pleuroticola and application thereof. A strain is a trichoderma pleuroticola strain QZ-001H, and the preservation number is CGMCC No.11049. The invention further discloses a biological control preparation comprising the trichoderma pleuroticola strain QZ-001H, and a preparing method thereof. Experiments show that the trichoderma pleuroticola strain has the advantages of being high in growth speed, large in sporulation quantity, wide in action spectrum and high in stress resistance, a large number of plant roots can be colonized fast, and therefore the trichoderma pleuroticola has a good application prospect. The biological control preparation prepared from the trichoderma pleuroticola strain can prevent and control multiple soil-borne plant diseases including one or more of pepper blight, Chinese chestnut shoot blight, pomegranate dry rot and the like, and has an application prospect.
Description
Technical field
The invention belongs to biological control Plant diseases field.Specifically, the present invention relates to a kind of can Trichoderma and uses thereof of picking up the ears of high-efficiency prevention and control Plant diseases, and the biological control agent adopting this Trichoderma of picking up the ears to prepare.
Background technology
Wood mould (Trichoderma spp.), belong to the hyphomycetes of imperfect fungi, Moniliales, Moniliaceae, be the fungi that a class is distributed widely on soil, air, dry branches and fallen leaves and various fermented product, all can be separated to from roots of plants circle, blade and seed, bulb surface.Wood is mould to be nature ubiquity and to have the antagonistic microbe of affluent resources.Trichoderma growth and breeding speed is fast, Vegetation space can be captured rapidly, generation microbiotic can be secreted and suppress other growth of pathogenic bacteria, nutrient competition, superparasitism, cell wall degrading enzyme and inducing plant can also be passed through and produce the mechanism of action such as resistance, reaching the object suppressing disease, is study one of bacterial strain the most widely in biocontrol strain.The Trichoderma had been found that at present more than 30 plant in many there is Biocontrol Potential, as viride, trichoderma harziarum, healthy and free from worry wood is mould, thorn spore wood is mould and wood of picking up the ears is mould etc., belongs to 29 kind of plant pathogenic bacterias show antagonistic activity to 18.The mould mechanism of the biological control to phytopathogen of wood is various and complexity, it is usually the coefficient result of number of mechanisms, the special emphasis of different Biocontrol Activity mechanism is different, and the type of its Biocontrol Effect effect and Strain type, pathogenic fungi, agrotype and envrionment conditions are closely related.
The salt tolerant that inventor was once separated to a kind of efficient phosphate-solubilizing is picked up the ears Trichoderma (number of patent application: 201310442613.6).But wood of picking up the ears is mould for soil-borne diseases such as mixed with rice bran, Cure of Coryneum kunzei, pomegranate dry rots, have not been reported.
Summary of the invention
The object of this invention is to provide a kind of new trichoderma strain for high-efficiency prevention and control plurality of plant diseases---pick up the ears trichoderma strain QZ-001H.Trichoderma strain of picking up the ears of the present invention has fast growth, sporulation quantity is large, action spectrum is wide, strong stress resistance, the feature such as can surely to grow by rapid, high volume, therefore have a good application prospect at plant rhizosphere.
Above-mentioned purpose of the present invention is achieved through the following technical solutions: wood mould (Trichodermapleuroticola) bacterial strain QZ-001H that picks up the ears provided by the present invention, be separated to obtain from the marine alga (top layer acquisition) of shallow sea, Huangdao District, Qingdao City reef, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on July 6th, 2015 and (be called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), deposit number is CGMCCNo.11049.It has following biological characteristics: on PDA substratum, under 25 DEG C of dark conditions cultivate 7 days, colony diameter expansion 80mm, initial stage white is sparse, after become greyish-green, cotton-shaped.Reverse side is colourless.Mycelia tool is every, branch.Conidiophore just forms the branch profile of pine and cypress formula, the stigma Shu Sheng of branch ends, to life, alternate or Dan Sheng, doleiform, 6-10 × 3-4.5 μm.Conidium elliposoidal, single, closely colourless, be pistac during gathering, wall is smooth or slightly coarse, 4-5.5 × 3-4 μm.
The pick up the ears cultural method of trichoderma strain QZ-001H or propagation method of the present invention comprises:
(1) Nostoc commune Vanch is preserved and is adopted PDA substratum, and filling a prescription is: potato 200g, glucose 20g, agar 12g, distilled water 1000mL;
(2) laboratory fluids is cultivated and is adopted PDB substratum, and filling a prescription is: potato 200g, glucose 20g, distilled water 1000mL.
(3) solid culture based formulas: Gu material and inorganic salt solution, the in mass ratio proportions of 1:1.8.Wherein said solid material by mass ratio be the corn cob of 50:40:10, wheat bran and rice husk form; By mass percentage, the component of described inorganic salt solution and content are: 3.5% potassium primary phosphate, 0.04% magnesium sulfate, and 4% ammonium sulfate, remaining as water.
(4) bulk fermentation cultivates formula: with solid culture based formulas in (3).
The present invention also provides a kind of biological control agent comprising 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 of the trichoderma strain QZ-001H that picks up the ears is prepared;
(2) seed liquor prepared by step (1) is inoculated in solid medium, constant temperature culture 3 ~ 5 days at 28-30 DEG C;
(3) culture that step (2) is cultivated is added sterilized water mixing, filters, filtrate is seeded to bulk fermentation substratum, room temperature 28-30 DEG C, carry out fermentation culture in the proving room of relative humidity more than 85%.
Wherein, the solid medium in step (2) is made up of solid material and inorganic salt solution, in mass ratio the proportions of 1:1.8; Wherein said solid material by mass ratio be the corn cob of 50:40:10, wheat bran and rice husk form; By mass percentage, the component of described inorganic salt solution and content are: 3.5% potassium primary phosphate, 0.04% magnesium sulfate, and 4% ammonium sulfate, remaining as water.
Further, the same solid medium of bulk fermentation substratum in step (3).
In a specific embodiments of the present invention, described preparation method comprises the following steps:
(1) be transplanted in PDB liquid nutrient medium by the spore of the trichoderma strain QZ-001H that picks up the ears, 28 DEG C of shaking table shaking culture obtain seed liquor in 3 ~ 5 days;
(2) seed liquor prepared by step (1) in mass ratio 10% ratio be inoculated in solid medium, shaking culture 3 ~ 5 days at 28 DEG C;
(3) culture that step (2) is cultivated is added sterilized water in mass ratio 1:15 ratio mixing, filter, the ratio of filtrate 1:6 is by volume seeded to fermention medium, fermentation culture 8 ~ 9 days in the proving room of room temperature 28 DEG C, relative humidity more than 85%.
Present invention also offers above-mentioned trichoderma strain QZ-001H or the above-mentioned biological control agent purposes in control plant soil-borne diseases of picking up the ears, described Plant diseases can be selected from one or more of capsicum wilt, Cure of Coryneum kunzei, pomegranate dry rot etc.
Present invention also offers a kind of biological control method for preventing and treating plant soil-borne diseases, described biological control method comprises uses 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 present invention has fast growth, sporulation quantity is large, action spectrum is wide, strong stress resistance, the feature such as can surely to grow by rapid, high volume, therefore have a good application prospect at plant rhizosphere.The biological control agent prepared by this trichoderma strain of picking up the ears, can prevent and treat multiple soilborne fungal pathogens, and comprising capsicum wilt, Cure of Coryneum kunzei, pomegranate dry rot etc. one or more, is a kind of biological control agent having application prospect.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
Embodiment 1
1, to pick up the ears the isolation and purification of trichoderma strain QZ-001H
The trichoderma strain QZ-001H that picks up the ears of the present invention gathers marine alga from shallow sea, Huangdao District, Qingdao City reef, and adopt dilution-plate method to be separated acquisition with plate streak, separation method is:
(1) be separated: the marine alga (top layer acquisition) gathering shallow sea, Huangdao District, Qingdao City reef, is all kept at the sample of collection in aseptic bottle, indicates collecting location, time and collection people.Now by marine alga aseptic water washing, then pulverize in aseptic crucible, take 1g algae clay in 100mL sterilized water, be placed in 30 DEG C of shaking table 150rpm and shake 10min, be then placed in 60 DEG C of water-baths and hatch 30min, get 100 μ L 10
-2, 10
-3, 10
-4diluent is coated on rose bengal medium flat board, each gradient be coated with three parallel, the microbial bacteria of the different shape after cultivating 3 days at 30 DEG C on picking rose bengal medium falls within rose bengal medium flat board rules, and colony growth situation is observed in timing.Then plate streak is adopted, purifying trichoderma strain, numbering preservation respectively.
(2) screening of the efficient antagonistic Trichoderma bacterial strain of capsicum wilt
1. primary dcreening operation: adopt opposite culture method, preparation PDA is dull and stereotyped, buys the bacterium cake that cut-off footpath is 5mm with punch tool at Trichoderma, capsicum wilt bacterium edge, is implanted in dull and stereotyped relative both sides central authorities respectively, 25 DEG C of constant temperature culture, observe Trichoderma day by day to the restraining effect of pathogenic bacteria.
2. sieve again: the trichoderma strain with efficient antagonistic activity screened is carried out multiple sieve, mainly through temperature tolerance, resistance to acids and bases, drug-resistant test, screens the good trichoderma strain of patience, carry out potted plant controlling experiment and field test.
The present inventor obtains a strain by a large amount of screening operation can Trichoderma of picking up the ears (T.pleuroticola) QZ-001H of high-efficiency prevention and control plurality of plant diseases.Experiment proves, this mould former powder of wood of picking up the ears demonstrates prevention effect very efficiently in mixed with rice bran, and crop is significantly increased production.Thus, wood of picking up the ears of the present invention is mould is the wooden mould new strains of picking up the ears with wide application prospect, may be used for preparing the biological control agent preventing and treating plurality of plant diseases.
2, identification of strains
(1) Microbiological Characteristics: on PDA substratum, under 25 DEG C of dark conditions cultivate 7 days, colony diameter expansion 80mm, initial stage white is sparse, after become greyish-green, cotton-shaped.Reverse side is colourless.Mycelia tool is every, branch.Conidiophore just forms the branch profile of pine and cypress formula, the stigma Shu Sheng of branch ends, to life, alternate or Dan Sheng, doleiform, 6-10 × 3-4.5 μm.Conidium elliposoidal, single, closely colourless, be pistac during gathering, wall is smooth or slightly coarse, 4-5.5 × 3-4 μm.
(2) molecular biological characteristic
The rRNA gene sequencing result (ITS-5.8S-ITS2 district) following (SEQ-1) of this bacterial strain:
TCGGAAGTAAAAAGTCGTAACAAGGTCTCCGTTGGTGAACCAGCGGAGGGATCATTACCGAGTTTACAACTCCCAAACCCAATGTGAACGTTACCAAACTGTTGCCTCGGCGGGATCTCTGCCCCGGGTGCGTCGCAGCCCCGGACCAAGGCGCCCGCCGGAGGACCAACCAAAACTCTTATTGTATACCCCCTCGCGGGTTTTTTTATAATCTGAGCCTTCTCGGCGCCCCTCGTGGGCGTTTCGAAAATGAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTCCGAGCGTCATTTCAACCCTCGAACCCCTCCCGGGGGTCGGCGTTGGGGATCGGCCCTCCCTCTGCGGGGGCCGTCTCCGAAATACAGTGGCGGTCTCGCCGCAGCCTCTCCTGCGCAGTAGTTTGCACACTCGCACCGGGAGCGCGGCGCGTCCACAGCCGTTAAACACCCAACTTTCTGAAATGTGACCTCGATCAGGTAGAATCCCGC
Wherein, PDA culture medium prescription: potato 200g (peeling), glucose 20g, agar 12g, distilled water 1000mL.Rose bengal medium is filled a prescription: potato 200g (peeling), glucose 20g, 1% rose-bengal solution 3.3mL, Sodium Propionate 3.5g, Streptomycin sulphate 0.03g, water 1000mL, pH nature.
Embodiment 2
1, to pick up the ears trichoderma strain QZ-001H fermenting process
PDB culture medium prescription: potato 200g (peeling), glucose 20g, distilled water 1000mL.
A large amount of solid fermentation culture medium prescription (mass percentage): Gu material: corn cob 50%, wheat bran 40%, rice husk 10%; Inorganic salt solution: potassium primary phosphate 3.5%, magnesium sulfate 0.04%, ammonium sulfate 4%, remaining as water.Solid-to-liquid ratio is 1:1.8 (mass ratio).
To pick up the ears a large amount of solid fermentation process of trichoderma strain QZ-001H:
1. bacterial classification seed liquor is cultivated: a small amount of spore of trichoderma strain QZ-001H picking from test tube slant of will picking up the ears, moves in PDB liquid nutrient medium, 28 DEG C of shaking table shaking culture 3 ~ 5 days, and this be seed liquor, and bacterium work is 0.15-0.30cfu/ml;
2. the cultivation of solids manufacture bacterial classification: by seed liquor in 10% ratio be inoculated in solid medium (500mL triangular flask), 28 DEG C of constant temperature culture 3 ~ 5 days, middle multiple oscillation;
3. a large amount of solid fermentation: the culture of solid culture in 2. press 1:15 dilution proportion with sterilized water, and filters with sterile gauze, goes out thick slag, and be production bacterium liquid, inoculative proportion is inoculated in bulk fermentation substratum by 1:6.The raw material of inoculation is placed in proving room (28 DEG C and relative humidity more than 85%) fermentation culture 8 ~ 9 days, the former powder of Trichoderma of picking up the ears after fermented liquid drying, can be obtained, viable count be 40-50 hundred million/gram.
Embodiment 3
Present embodiments provide the former powder of trichoderma strain QZ-001H of picking up the ears to the related experiment of capsicum wilt prevention effect.
1) reagent agent
To pick up the ears the former powder of trichoderma strain QZ-001H (in embodiment 2 prepare); 70% thiophanate methyl wettable powder (commercially available).
2) for studying thing and controlling object:
Supplying to study thing is capsicum; Kind is middle capsicum No. 1;
Controlling object: blight.
3) experimental field situation, test design and arrangement
Experimental field be located at capsicum ground, celebrating remaining village, Nan Quan town, Jimo City of Shandong Province, soil is sand ground, and former years, blight occurred heavier.Experimental field cultivation condition uniformity.
This test establishes the former powder of trichoderma strain QZ-001H of picking up the ears to be watered filling root; 70% thiophanate methyl wettable powder dilution agent 1000 times; Clear water compares totally 5 process, and each capsicum hole irrigation liquid or 500 grams, water repeat 4 times.The group arrangement of random district, each community.Earthing after dispenser, each community arranges nutrition pot 300, every alms bowl point 2 chilli seeds.
4) pilot survey and method of calculation
(1) meteorological conditions
Dispenser was fine for the same day, gentle breeze, and the highest temperature is 30 DEG C, and the lowest temperature is 26 DEG C, and relative humidity is 65%
(2) drug effect and security survey
Efficacy survey: investigation 1 time altogether, carries out emerging for 30 days, records 300 nutrition pot kind capsicum wilt morbidity numbers, calculates diseased plant rate and prevention effect.
(3) drug effect method of calculation
Drug effect following formula calculates:
5) result
(1) reagent agent is to the prevention effect of capsicum wilt
30 days investigation diseased plant numbers after dispenser, table 1 result shows, and the diseased plant rate of clear water contrast is up to 32.01%, and the diseased plant rate of each process all contrasts lower than clear water.Pick up the ears the former powder of trichoderma strain QZ-001H use multiple to be respectively 250,500 and 1000 times of process after diseased plant rate be respectively 7.85%, 9.88%, 12.19%, all lower than the diseased plant rate of contrast medicament 12.39%; Each prevention effect processed of the former powder of trichoderma strain QZ-001H of picking up the ears is respectively 75.48%, 69.14%, 61.94%, wherein the prevention effect of 1:250 is best, process with other and have significant difference in P<0.05 level, 70% thiophanate methyl is 61.31% to the prevention effect of capsicum wilt.The former powder of trichoderma strain QZ-001H that proves to pick up the ears effectively can control the harm of capsicum wilt.
The sickness rate of the rear capsicum wilt of each process of table 1 and prevention effect
| Process multiple | Diseased plant rate (%) | Prevention effect (%) | The significance of difference |
| To pick up the ears the former powder 250 of Trichoderma QZ-001H | 7.85 | 75.48 | a |
| To pick up the ears the former powder 500 of Trichoderma QZ-001H | 9.88 | 69.14 | b |
| To pick up the ears the former powder 1000 of Trichoderma QZ-001H | 12.19 | 61.94 | c |
| 70% thiophanate methyl 500 | 12.39 | 61.31 | c |
| CK | 32.01 | - | - |
Note: represent that with there being same letter after data line difference is not significantly (P<0.05) after Duncan multiple comparisons.
(2) capsicum security survey: through observing, each chemicals treatment district is compared with check plot, and chili growth is normal, produces without poisoning, illustrates that the former powder of Trichoderma QZ-001H of picking up the ears is supplying examination concentration to capsicum safety.
Therefore, from sickness rate and prevention effect, the trichoderma strain QZ-001H that picks up the ears has good prevention effect to capsicum wilt, and after dispenser, prevention effect can reach more than 75%, significantly better than contrast medicament, and significant difference.
Claims (9)
1. pick up the ears wood mould (Trichoderma pleuroticola) bacterial strain QZ-001H, and the deposit number of described bacterial strain is CGMCCNo.11049.
2. the purposes of trichoderma strain QZ-001H in control plant soil-borne diseases of picking up the ears according to claim 1.
3. purposes as claimed in claim 2, it is characterized in that, described Plant diseases is selected from one or more in capsicum wilt, Cure of Coryneum kunzei, pomegranate dry rot.
4. one kind comprises the biological control agent of the trichoderma strain QZ-001H that picks up the ears according to claim 1.
5. the preparation method of biological control agent as claimed in claim 4, comprises the following steps:
(1) seed liquor of the trichoderma strain QZ-001H that picks up the ears is prepared;
(2) seed liquor prepared by step (1) is inoculated in solid medium, constant temperature culture 3 ~ 5 days at 28-30 DEG C;
(3) culture that step (2) is cultivated is added sterilized water mixing, filters, filtrate is seeded to bulk fermentation substratum, room temperature 28-30 DEG C, carry out fermentation culture in the proving room of relative humidity more than 85%;
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 said solid material by mass ratio be the corn cob of 50:40:10, wheat bran and rice husk form; By mass percentage, the component of described inorganic salt solution and content are: 3.5% potassium primary phosphate, 0.04% magnesium sulfate, and 4% ammonium sulfate, remaining as water;
The same solid medium of bulk fermentation substratum in described step (3).
6. the preparation method of biological control agent as claimed in claim 5, is characterized in that, comprise the following steps:
(1) be transplanted in PDB liquid nutrient medium by the spore of the trichoderma strain QZ-001H that picks up the ears, 28 DEG C of shaking table shaking culture obtain seed liquor in 3 ~ 5 days;
(2) seed liquor prepared by step (1) in mass ratio 10% ratio be inoculated in solid medium, shaking culture 3 ~ 5 days at 28 DEG C;
(3) culture that step (2) is cultivated is added sterilized water in mass ratio 1:15 ratio mixing, filter, the ratio of filtrate 1:6 is by volume seeded to fermention medium, fermentation culture 8 ~ 9 days in the proving room of room temperature 28 DEG C, relative humidity more than 85%.
7. the purposes of biological control agent according to claim 4 in control plant soil-borne diseases.
8. purposes as claimed in claim 7, it is characterized in that, described Plant diseases is selected from one or more in capsicum wilt, Cure of Coryneum kunzei, pomegranate dry rot.
9. for preventing and treating a biological control method for plant soil-borne diseases, it is characterized in that, comprising and using trichoderma strain QZ-001H or the biological control agent according to claim 4 of picking up the ears according to claim 1 to the plant with soil-borne disease.
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| CN110747135A (en) * | 2019-12-11 | 2020-02-04 | 齐齐哈尔大学 | Trichoderma aureoviride and application thereof |
| CN110747135B (en) * | 2019-12-11 | 2022-03-04 | 齐齐哈尔大学 | Trichoderma aureoviride and application thereof |
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