CN102925367B - Trichoderma viride strain and application thereof - Google Patents
Trichoderma viride strain and application thereof Download PDFInfo
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- CN102925367B CN102925367B CN201210411827.2A CN201210411827A CN102925367B CN 102925367 B CN102925367 B CN 102925367B CN 201210411827 A CN201210411827 A CN 201210411827A CN 102925367 B CN102925367 B CN 102925367B
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Abstract
The invention discloses a Trichoderma viride strain and application thereof. The Trichoderma viride stain is WLTR35, and the collection number is CGMCC No.6673. The invention also discloses application of the strain or biological control preparation comprising the strain in controlling plant soil-borne disease. The experiment indicates that the Trichoderma viride strain WLTR35 has the characteristics of fast growth, large spore production quantity, wide action spectrum and the like, can quickly and massively implement colonization, and therefore, has wide application prospects. The biological control preparation prepared from the Trichoderma viride WLTR35 can efficiently control the plant soil-borne disease and effectively enhance the crop yield.
Description
Technical field
The invention belongs to biological control Plant diseases field.Particularly, the present invention relates to strain viride and an application thereof, and the biological control preparation that adopts this trichoderma viride strain to prepare.
Background technology
Plant diseases has a strong impact on farm-forestry crop growth, even causes crop death, causes great financial loss.For the control of Plant diseases, the conventional method adopting comprises traditional chemical prevention, cultivates disease-resistant variety and biological control.The disease-resistant variety cycle is long, and resistance is single.Chemical prevention makes that the with medicament time is long, usage quantity is large, causes pesticide residue, environmental pollution; Meanwhile, the life-time service of chemical pesticide, makes pathogenic bacteria develop immunity to drugs to it, causes preventive effect to decline even failed.Utilize beneficial microorganism controlling plant diseases to have broad prospects.
Wood mould (Trichoderma spp.) belongs to hyphomycetes, Moniliales, the Moniliaceae of imperfect fungi, be that a class is distributed widely in the fungi 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 produces the mechanism such as resistance by producing microbiotic, nutrient competition, superparasitism, cell wall degrading enzyme and inducing plant, there is antagonistic action for various plants pathogenic bacteria, can suppress plurality of plant diseases, be research one of bacterial strain the most widely in current biocontrol strain.The mould biological control mechanism to phytopathogen of wood is various and complicated, it is usually the coefficient result of number of mechanisms, the special emphasis difference of different Biocontrol Activity mechanism, type, agrotype and the envrionment conditions of its Biocontrol Effect effect and Strain type, pathogenic fungi are closely related.
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---viride (Trichoderma viride) WLTR35.Trichoderma viride strain of the present invention has fast growth, sporulation quantity is large, action spectrum is wide and the feature such as can rapid, high volume surely growing, therefore have a good application prospect.
Another object of the present invention be to provide a kind of adopt prepared by viride WLTR35 can high-efficiency prevention and control Plant diseases biological control preparation.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
The invention provides a kind of viride (Trichoderma viride) bacterial strain WLTR35, the deposit number of described bacterial strain is CGMCC No.6673.
Bacterial strain provided by the present invention is viride WLTR35, separate and obtain from Wulian County mushroom, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 12nd, 2012 and (be called for short CGMCC, address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), preserving number is CGMCC No.6673.It has following biological characteristics: on PDA nutrient agar, growth soon, is cultivated 3d under 30 DEG C of dark conditions, colony diameter 70mm.Bacterium colony initial stage white is sparse, and the later stage forms yellow-green colour Chan Bao district.Reverse side is colourless.Mycelia is poly-every, branch.Conidiophore forms the branch profile of pine and cypress formula, the stigma Shu Sheng of branch end, to life, alternate or Dan Sheng, doleiform, 4.5-10 × 2.5-3.5 μ m.Conidium elliposoidal, single, closely colourless, when gathering, be light green, wall is slightly coarse, 3.5-5 × 3-4 μ m.The rRNA gene sequencing result (ITS-5.8S-ITS2 district) of this bacterial strain is as shown in SEQ-1.
Cultural method or the propagation method of trichoderma viride WLTR35 of the present invention comprise:
(1) common cultivation 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 ratio of 1:1.8 preparation in mass ratio.Corn cob and wheat bran that wherein said solid material is 60:40 by mass ratio form; By mass percentage, described inorganic salt solution comprises 3.5% potassium primary phosphate, 0.04% magnesium sulfate, and 4% ammonium sulfate, remains as water.
(4) bulk fermentation is cultivated formula: with solid culture based formulas in (3)
It is a kind of for preventing and treating the biological control preparation of plant soil-borne diseases that the present invention also provides, and described biological control preparation comprises described viride WLTR35.
The preparation method of biological control preparation of the present invention comprises the following steps:
(1) prepare the seed liquor of viride WLTR35;
(2) seed liquor of being prepared by step (1) is inoculated into constant temperature culture in solid medium;
(3) culture of step (2) being cultivated dilutes with sterilized water, filters, and filtrate is seeded to and in bulk fermentation substratum, carries out fermentation culture.
In a specific embodiments of the present invention, described preparation method comprises the following steps:
(1) spore of viride WLTR35 is transplanted in PDB liquid nutrient medium, 28 DEG C of shaking table shaking culture 3～5d obtain seed liquor;
(2) seed liquor of being prepared by step (1) in mass ratio 10% ratio is inoculated in solid medium, shaking culture 3～5d at 28 DEG C;
(3) the sterilized water ratio mixing of 1:15 in mass ratio for the culture of step (2) being cultivated, filter, by filtrate by volume the ratio of 1:6 be seeded to bulk fermentation substratum, fermentation culture 8～9d in 28 DEG C of room temperatures, more than 85% proving room of relative humidity.
It is a kind of for preventing and treating the biological control method of plant soil-borne diseases that the present invention also provides, and described biological control method comprises to the plant with soil-borne disease uses above-mentioned viride WLTR35 or above-mentioned biological control preparation.
The present invention also provides above-mentioned trichoderma viride WLTR35 or the purposes of above-mentioned biological control preparation in control plant soil-borne diseases.
With regard to above-mentioned biological control preparation, biological control method and purposes, described plant soil-borne diseases can be selected from cucumber anthracnose, walnut anthrax, apple anthracnose, bitter rot or anthracnose of grape, fruit white rot of grape, watermelon anthrax, winter jujube black spot, Cure of Coryneum kunzei, pomegranate dry rot etc. one or more.
Experiment shows, trichoderma viride strain WLTR35 of the present invention has fast growth, sporulation quantity is large, action spectrum is wide and the feature such as can rapid, high volume surely growing, therefore have a good application prospect.The biological control preparation of being prepared by this viride WLTR35, not only can prevent and treat plant soil-borne diseases efficiently, can also effectively improve crop yield, is a kind of biological control preparation that has application prospect.This biological control preparation can be used as biological pesticide or bio-feritlizer, prevent and treat multiple soil and pass Plant diseases, comprise cucumber anthracnose, walnut anthrax, apple anthracnose, bitter rot or anthracnose of grape, fruit white rot of grape, watermelon anthrax, winter jujube black spot, Cure of Coryneum kunzei, pomegranate dry rot etc. one or more.
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 embodiment are only exemplary, scope of the present invention are not formed to any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can the details of technical solution of the present invention and form be modified or be replaced, but these amendments and replacement all fall within the scope of protection of the present invention.
Embodiment 1
1, trichoderma viride (T.viride) WLTR35(CGMCC No.6673) isolation and purification
Viride of the present invention (T.viride) WLTR35(CGMCC No.6673) adopt plate streak separation to obtain from Wulian County mushroom, separation method is:
(1) the separation of trichoderma strain: get the mushroom bacteria bag of being injured serious, first use 75% alcohol Local treatment 1min, on Bechtop, process after 20min with ultraviolet lamp again, by sterilizing blade incision treatment sites, with a small amount of Trichoderma spore of inoculating needle picking, at the flat lining out of PDA, colony growth situation is observed in timing.Then adopt plate streak, purifying trichoderma strain, goes to PDA test tube slant and saves backup.
(2) the screening of the efficient antagonistic Trichoderma bacterial strain of cucumber anthracnose
1. primary dcreening operation: adopt face-off culture method, preparation PDA flat board, buys at Trichoderma, cucumber anthracnose, walnut anthrax bacteria edge the bacterium cake that cut-off footpath is 5mm with punch tool, is implanted in respectively dull and stereotyped relative both sides central authorities, 25 DEG C of constant temperature culture, observe the restraining effect of Trichoderma to pathogenic bacteria day by day.
2. sieve again: the trichoderma strain with efficient antagonistic activity screening is carried out to multiple sieve, is mainly through temperature tolerance, resistance to acids and bases, drug-resistant test, screens the good trichoderma strain of patience, carries out potted plant control test and field test.
Wherein, PDA culture medium prescription: potato 200g(peeling), glucose 20g, agar 12g, distilled water 1000mL.
The inventor obtains by a large amount of screening operations trichoderma viride (T.viride) the WLTR35(CGMCC No.6673 that a strain can high-efficiency prevention and control plurality of plant diseases).Experiment showed, that the former powder of this viride all demonstrates prevention effect very efficiently in control cucumber anthracnose, walnut anthrax, significantly increases production farm crop.Thereby viride of the present invention is the new bacterial strain of viride with wide application prospect, can be for the preparation of the biological control preparation of preventing and treating plurality of plant diseases.
2, identification of strains
(1) Microbiological Characteristics: growth soon, is cultivated 3d under 30 DEG C of dark conditions, colony diameter 70mm on PDA nutrient agar.Bacterium colony initial stage white is sparse, and the later stage forms yellow-green colour Chan Bao district.Reverse side is colourless.Mycelia is poly-every, branch.Conidiophore forms the branch profile of pine and cypress formula, the stigma Shu Sheng of branch end, to life, alternate or Dan Sheng, doleiform, 4.5-10 × 2.5-3.5 μ m.Conidium elliposoidal, single, closely colourless, when gathering, be light green, wall is slightly coarse, 3.5-5 × 3-4 μ m.
(2) molecular biological characteristic
The rRNA gene sequencing result (ITS-5.8S-ITS2 district) following (SEQ-1) of this bacterial strain:
CTTTCTTGGCACAATCGTGTCCGACAATCCTGTTCTCAGTCTTGCCAATTT
TTCTCGCGTCGTCACACCCCGCTCTACCTGTCTACCCCTCCTTTGGCACA
GCAGAAATTTTCTGGCTGCCTTGTTTGGCTTTTAGTGGGGTGCCAATTTTT
TTTTGGCAACAACCCCGCTATCGCCGTTGTCCCTCATCCATCGTCCCAAC
AATTTGATCTCACTCAATCACATCGTCTTCTGGTTCATTGTGTCAAAGATT
CGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCG
TTCTTCATCGATGCCAGAACCAAGAGATCCGTTGTTGAATGGGTTCTTGA
CAAGCTCAAGGCCGGCGTGAGCGTGGTATCACCATCGACATTGCCCTCTG
GAAGTTCGAGACTCCCAAGTACTATGTCACCGTCATTGGTATGTTTTGTG
TTTCCTCATTGACCCCCGGAATCCTTATTCTAACGTGCCGCTCTACAGACG
CTCCCGGTCACGTGATTTCATCAAGAACATGATCACTGGTACTTCCCAGG
CTGACTGCGTATCCTGATTATCGCTGCCGGTACTGGTGAGTTCGAGGCTG
GTATCTCCAAGGATGGCCAGACCCGTGAGCACGCTCTGCTCGCCTACACC
CTGGGTGTCAAGCAGCTCATCGTTGCCATCAACAAGATGGACACTGCCA
ACTGGGCCGAGGCTCGTTACCTTGAGATCATCAAGGAGACTCCAACTTCA
TCAAGAAGGTCGGCTTCAACCCCAAGTCCGTCGCCGCGCTCCCGGTGCG
AGTGTGCAACTACTGCGCAGGAGAGGCTGCGGCGAGACCAACCCCTGG
AAGGGCT?GGGAGAAGGAGACCAAGGCT?GGCAAGAGCA?GCG
Embodiment 2
1, trichoderma viride fermenting process
PDB culture medium prescription: potato 200g(peeling), glucose 20g, distilled water 1000mL.
A large amount of solid fermentation culture medium prescriptions (quality percentage composition):
Gu material: corn cob 60%, wheat bran 40%
Inorganic salt solution: potassium primary phosphate 3.5%, magnesium sulfate 0.04%, ammonium sulfate 4%, remains as water.
Solid-to-liquid ratio is 1:1.8(mass ratio)
Viride (T.viride) WLTR35(CGMCC No.6673) a large amount of solid fermentation process:
1. bacterial classification seed liquor is cultivated: by a small amount of spore of trichoderma viride (T.viride) WLTR35CGMCC No.6673 picking from test tube slant, move in PDB liquid nutrient medium, and 28 DEG C of shaking table shaking culture 3～5d, this is seed liquor.
2. the cultivation of solids manufacture bacterial classification: seed liquor is inoculated in solid medium (500mL triangular flask) to 28 DEG C of constant temperature culture 3～5d, middle multiple oscillation by 10% mass ratio.
3. a large amount of solid fermentations: the culture of solid culture in 2. press to 1:15 dilution proportion with sterilized water, and filter with sterile gauze, go out thick slag, be production bacterium liquid, inoculative proportion is inoculated in bulk fermentation substratum by 1:6.The raw material of inoculation is placed in to proving room (28 DEG C and relative humidity more than 85%) fermentation culture 8～9d, can obtains the former powder of Trichoderma.
Embodiment 3
The present embodiment provides viride (T.viride) WLTR35(CGMCC No.6673) related experiment of former powder to cucumber anthracnose disease-preventing and yield-increasing.
1, reagent agent
The former powder of viride WLTR35 (preparation in embodiment 2), 70% mancozeb wettable powder (commercially available).
2, for studying thing and controlling object
Be cucumber for studying thing, kind is Chang Chun Mi Ci;
Controlling object is anthrax.
3, situation experimental field
Experimental field be located at cucumber greenhouse gardening base, Shouguang, Shandong Province, this base plastic tent cucumber anthrax occurred seriously in recent years, and control difficulty, test is in 600 square metres of (0.9 mu) cucumber booths, soil property is light loam, and organic content is that 1.09%, pH value is 6.7, all experimental plots cultivation condition and control measures are consistent, and when dispenser, cucumber anthracnose is in their early stage.
4, test design and arrangement
This test is established 200 times, 400 times, 600 times, the former powder of trichoderma viride WLTR35,800 times of liquid of 70% mancozeb wettable powder and not dispenser clear water and is compared totally 5 processing, repeats 4 times, and Gong20Ge community, each community random alignment, community area is 20m
2.In each spray of on June 9th, 2012 and June 16 medicine once, by plant leaf spray up and down evenly, thoughtful, when spray medicine, block with plastic cloth, mu using liquid 60kg, medicinal sprayer tool is workers and peasants-16 type atomizers.
5, pilot survey and method of calculation
(1) meteorological conditions
The 1st dispenser (on June 9th, 2012) was fine the same day, 2 grades of wind-force, and the highest temperature is 29 DEG C, and the lowest temperature is 15 DEG C, and relative humidity is 65%.The 2nd dispenser (on June 16th, 2012), the same day is fine, 3 grades of wind-force, the highest temperature is 25 DEG C, and the lowest temperature is 18 DEG C, and relative humidity is 75%; Because of plastic greenhouse envrionment conditions relatively stable, so this weather does not affect drug effect.
(2) drug effect and security survey
Efficacy survey: after the front and last dispenser of the 1st dispenser, 14d investigates.Every community adopts 4 point samplings, looks into 2 strains at every, investigates whole blades, and Investigate incidence records disease index and calculates disease index.
Security survey: 7d and 14d observe the security of cucumber after dispenser for the first time, occurs if any poisoning, describes symptom of chemical damage in detail and presses poisoning grading standard poisoning degree.
Stage division (taking blade as unit):
0 grade: without scab
1 grade: lesion area accounts for below 5% of whole leaf area;
3 grades: lesion area accounts for the 6%-10% of whole leaf area;
5 grades: lesion area accounts for the 11%-25% of whole leaf area;
7 grades: lesion area accounts for the 26%-30% of whole leaf area;
9 grades: lesion area accounts for the more than 50% of whole leaf area;
(3) control time and number of times
Investigation state of an illness radix before dispenser, 14d investigation prevention result before next dispenser and after last dispenser.
(4) drug effect method of calculation
Drug effect is calculated by formula (1), (2):
(5) the direct impact on crop
Observe medicament crop is had or not to poisoning, record type and the degree of poisoning.
6, result
(1) prevention effect of reagent agent to cucumber anthracnose
Table 1 result shows, after 200 times, 400 times, 600 times processing of the former powder of trichoderma viride WLTR35, disease index rate of increase proportion by subtraction is 188.68%, 229.50%, 278.71%, the disease index rate of increase of contrast medicament is 306.56%, all lower than the disease index rate of increase 1155.05% contrasting.The correcting controling effect that the former powder of trichoderma viride WLTR35 is 200 times, 400 times is respectively 83.66%, 80.13%, and there was no significant difference in P<0.05 level, all higher than the correcting controling effect of other processing.600 times, the former powder of trichoderma viride WLTR35 to the prevention effect of cucumber anthracnose there was no significant difference in P<0.05 level, is respectively 75.87%, 73.46% with 70% mancozeb wettable powder.The former powder of trichoderma viride WLTR35 can effectively be controlled the harm of cucumber anthracnose as can be seen here.
Disease index and the prevention effect of cucumber anthracnose after each processing of table 1
(2) cucumber security survey: observe through dispenser 7d and 14d, each chemicals treatment district is compared with check plot, and cucumber growth is normal, produces without poisoning, illustrates that the former powder of trichoderma viride WLTR35 is supplying examination concentration to cucumber safety.
(3) the former powder of viride is to cucumber production promoting effect
The former powder of table 2 trichoderma viride WLTR35 is to cucumber production promoting effect analysis table
Note: community area 33.4m
2; Processing 1 is 200 times, the former powder of trichoderma viride WLTR35, and processing 2 is 400 times, the former powder of trichoderma viride WLTR35, and processing 3 is 600 times, the former powder of trichoderma viride WLTR35, and processing 4 is control group.Different letter representation significant difference (P<0.01) after Duncan multiple comparisons after data in table.
As seen from the results in Table 2, the former powder of trichoderma viride WLTR35 of spraying on cucumber is respectively processed contrast check plot, and stimulation ratio is respectively 27.24%, 25.52% and 20.44%; Effect is fairly obvious.
Therefore, from disease index rate of increase and prevention effect, the former powder of trichoderma viride WLTR35 has good prevention effect to cucumber anthracnose, and prevention effect can reach more than 75%, significantly better than contrast medicament, significant difference.From cucumber production promoting effect, the former powder of trichoderma viride WLTR35 has obvious growth-promoting effect of increasing production to cucumber, and after 2 dispensers, stimulation ratio reaches more than 20%.
Embodiment 4
The present embodiment provides the related experiment of the former powder of trichoderma viride WLTR35 to walnut anthrax prevention effect.
1) reagent agent
The former powder of trichoderma viride WLTR35 (preparation in embodiment 2); 70% mancozeb wettable powder (commercially available).
2) for studying thing and controlling object:
Be walnut for studying thing, kind is the fragrant tinkling of pieces of jade;
Controlling object: anthrax.
3) experimental field situation, test design and arrangement
Experimental field be located at walnut plantation, Tai’an, Shandong Province, soil is sand ground.Walnut is 10 years fragrant tinkling of pieces of jade of life, spacing in the rows 3m × 4m, and tree vigo(u)r is medium on the weak side, and disease occurs serious.
200 times, 400 times, 600 times, the former powder of trichoderma viride WLTR35 is established in this test, and 70% 800 times of mancozeb wettable powders liquid and not dispenser clear water compare totally 5 processing, repeats 4 times.Walnut bloom after medication in 3 weeks, every 15d dispenser 1 time, amount to 7 times.5d investigation before gathering, 300 fruits of each processing random searching, calculate diseased fruit rate, prevention effect, whole pilot survey 1 time.By plant leaf spray up and down evenly, thoughtful, when spray medicine, block with plastic cloth, it is workers and peasants-16 type atomizers that mu is used liquid 60kg, medicinal sprayer tool.
5) pilot survey and method of calculation
(1) meteorological conditions
The 1st dispenser (on April 19th, 2012) was fine the same day, gentle breeze, and the highest temperature is 23 DEG C, and the lowest temperature is 12 DEG C, and relative humidity is 75%; The 2nd dispenser (on May 4th, 2011), the same day is fine, 2 grades of wind-force, the highest temperature is 29 DEG C, and the lowest temperature is 15 DEG C, and relative humidity is 70%; The 3rd dispenser (on May 19th, 2011), the same day is fine, 2 grades of wind-force, the highest temperature is 30 DEG C, and the lowest temperature is 17 DEG C, and relative humidity is 65%; The 4th dispenser (on June 3rd, 2011), the same day is fine, 2 grades of wind-force, the highest temperature is 30 DEG C, and the lowest temperature is 19 DEG C, and relative humidity is 60%; The 5th dispenser (on June 18th, 2011), the same day is clear to cloudy, 4 grades of wind-force, the highest temperature is 34 DEG C, and the lowest temperature is 21 DEG C, and relative humidity is 75%; The 6th dispenser (on July 3rd, 2012), the same day is fine, 2 grades of wind-force, the highest temperature is 34 DEG C, and the lowest temperature is 24 DEG C, and relative humidity is 85%; The 7th dispenser (on July 18th, 2011), the same day is overcast to cloudy, 4 grades of wind-force, the highest temperature is 33 DEG C, and the lowest temperature is 24 DEG C, and relative humidity is 85%.
(2) drug effect and security survey
Efficacy survey: 5d investigation before gathering, 300 fruits of each processing random searching, calculate diseased fruit rate, prevention effect
Security survey: 7d observes the security of walnut after each dispenser.
(3) drug effect method of calculation
Drug effect is calculated by formula (1), (2):
(4) the direct impact on crop
Observe medicament crop is had or not to poisoning, record type and the degree of poisoning.And the other influences of record to crop.
6) result
(1) prevention effect of reagent agent to walnut anthrax
5d investigation diseased fruit rate before gathering, the demonstration of table 3 result, the diseased fruit rate of clear water contrast is up to 5.366%, and the diseased fruit rate of each processing all contrasts lower than clear water.200 times, 400 times, 600 times diseased fruit rates after treatment of the former powder of trichoderma viride WLTR35 are respectively 8.63%, 9.98%, 12.36%, all lower than the diseased fruit rate that contrasts medicament 18.69%; The prevention effect of former each concentration of powder of trichoderma viride WLTR35 is respectively 83.92%, 81.40%, 76.97%, wherein prevention effect there was no significant difference in P<0.05 level of 200 times and 400 times, 70% mancozeb wettable powder is 65.17% to the prevention effect of walnut anthrax.The former powder of trichoderma viride WLTR35 can effectively be controlled the harm of walnut anthrax.
Sickness rate and the prevention effect of walnut anthrax after each processing of table 3
Note: with there being same letter to represent not remarkable (P<0.05) of difference after Duncan multiple comparisons after data line.
(2) walnut security survey: through observing, each chemicals treatment district is compared with check plot, and walnut growth is normal, produces without poisoning, illustrates that the former powder of trichoderma viride WLTR35 is supplying examination concentration to walnut safety.
Therefore, from sickness rate and prevention effect, trichoderma viride WLTR35 has good prevention effect to walnut anthrax, and after dispenser, prevention effect can reach more than 75%, significantly better than contrast medicament, significant difference.
Claims (7)
- A viride ( trichoderma viride) bacterial strain WLTR35, the deposit number of described bacterial strain is CGMCC No. 6673.
- 2. the purposes of trichoderma viride strain WLTR35 claimed in claim 1 in control plant soil-borne diseases; Described plant soil-borne diseases is cucumber anthracnose or walnut anthrax.
- 3. a biological control preparation, comprises trichoderma viride strain WLTR35 claimed in claim 1.
- 4. a method of preparing biological control preparation claimed in claim 3, is characterized in that, comprises the following steps:(1) prepare the seed liquor of trichoderma viride strain WLTR35;(2) seed liquor of being prepared by step (1) is inoculated into constant temperature culture in solid medium;(3) culture of step (2) being cultivated dilutes with sterilized water, filters, and filtrate is seeded to and in bulk fermentation substratum, carries out fermentation culture.
- 5. preparation method as claimed in claim 4, is characterized in that,(1) spore of trichoderma viride strain WLTR35 is transplanted in PDB liquid nutrient medium, 28 DEG C of shaking table shaking culture 3 ~ 5 d obtain seed liquor;(2) seed liquor of being prepared by step (1) in mass ratio 10% ratio is inoculated in solid medium, shaking culture 3 ~ 5 d at 28 DEG C;(3) the sterilized water ratio mixing of 1:15 in mass ratio for the culture of step (2) being cultivated, filter, by filtrate by volume the ratio of 1:6 be seeded to bulk fermentation substratum, fermentation culture 8 ~ 9 d in 28 DEG C of room temperatures, more than 85% proving room of relative humidity;Described PDB substratum is: potato 200g, glucose 20g, distilled water 1000mL; Described solid medium is: Gu material and inorganic salt solution, the ratio of 1:1.8 preparation in mass ratio; Corn cob and wheat bran that wherein said solid material is 60:40 by mass ratio form; Described inorganic salt solution by mass percentage, comprises 3.5% potassium primary phosphate, 0.04% magnesium sulfate, and 4% ammonium sulfate, remains as water; The same solid medium of described bulk fermentation substratum.
- 6. the purposes of biological control preparation claimed in claim 3 in control plant soil-borne diseases, described plant soil-borne diseases is cucumber anthracnose or walnut anthrax.
- 7. a biological control method for plant soil-borne diseases, is characterized in that, uses the trichoderma viride strain WLTR35 of claim 1 or the biological control preparation of claim 3 to the plant with soil-borne disease; Described plant soil-borne diseases is cucumber anthracnose or walnut anthrax.
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| CN105567573A (en) * | 2016-01-13 | 2016-05-11 | 西南林业大学 | Biocontrol strain for preventing and treating colletotrichum gloeosporium penz and screening method and application thereof |
| CN106857682A (en) * | 2017-01-26 | 2017-06-20 | 新疆农垦科学院 | A kind of biological control method of dense planting jujube garden black spot |
| CN107118971A (en) * | 2017-06-08 | 2017-09-01 | 黄河三角洲京博化工研究院有限公司 | A kind of Trichoderma viride and its cultural method |
| CN108587922B (en) * | 2018-04-17 | 2020-03-27 | 山东省林业科学研究院 | Trichoderma aureoviride and application thereof in preventing and treating winter jujube anthracnose |
| CN109042742A (en) * | 2018-08-18 | 2018-12-21 | 杭州渔森农业技术开发有限公司 | Compound formulation is used to prevent and treat soil and passes the purposes fallen ill |
| CN113881578B (en) * | 2021-11-15 | 2022-12-16 | 中国农业科学院特产研究所 | Trichoderma viride and application thereof as soil conditioner for panax plants |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101485336A (en) * | 2009-03-04 | 2009-07-22 | 四川省农业科学院经济作物育种栽培研究所 | Trichoderma sp. preparation for preventing and treating soil-borne disease of crop |
| CN102071145A (en) * | 2010-09-09 | 2011-05-25 | 河南省农业科学院 | Trichoderma viride fungi and preparation and application of fungicide thereof |
| CN102604842A (en) * | 2012-03-15 | 2012-07-25 | 江苏省农业科学院 | Trichoderma atroviride strain for producing myrosase and application thereof |
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| CN101485336A (en) * | 2009-03-04 | 2009-07-22 | 四川省农业科学院经济作物育种栽培研究所 | Trichoderma sp. preparation for preventing and treating soil-borne disease of crop |
| CN102071145A (en) * | 2010-09-09 | 2011-05-25 | 河南省农业科学院 | Trichoderma viride fungi and preparation and application of fungicide thereof |
| CN102604842A (en) * | 2012-03-15 | 2012-07-25 | 江苏省农业科学院 | Trichoderma atroviride strain for producing myrosase and application thereof |
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