CN101724572A - High yield cell wall degrading enzyme and Trichoderme viride of antibacterial peptide and biocontrol reagent thereof - Google Patents
High yield cell wall degrading enzyme and Trichoderme viride of antibacterial peptide and biocontrol reagent thereof Download PDFInfo
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Abstract
The invention discloses a high yield cell wall degrading enzyme and Trichoderme viride of antibacterial peptide, which is named Trichoderma viride TV 501, and has a preservation number reading: CCTCC No:M209321.The Trichoderme viride can produce cell wall degrading enzyme and antibacterial peptide in large quantities, the biocontrol reagent made from the Trichoderme viride have relatively good field prevention effect on vegetable and fruit diseases such as strawberry powdery mildew, grey mould fruit rot of strawberry, powdery mildew of cucumber and the like, obviously improves yield and quality of the vegetables and fruits, reduces use amount of chemical pesticide, improves the ecological environment, enhances food security, demonstrates vast potential for future development and is conducive to enhancing agricultural efficiency and increasing the income of the farmers.
Description
Technical field
The present invention relates to a kind of fungi, relate in particular to the green wood fungus strain of viride and the biocontrol reagent thereof of a kind of high yield cell-wall degrading enzyme and antibacterial peptide.
Background technology
Enter 21 century, along with improving constantly of expanding economy and living standards of the people, people's health perception and environmental consciousness are strengthened greatly.Reduce the use of chemical pesticide, adopt green bio pesticide control disease pest and weed to become the inexorable trend of agricultural chemicals development.China is a large agricultural country, annual crop diseases and pest crop smothering injured area about 200,000,000 hectares (inferior), annual production and the use agricultural chemicals about 250,000 tons (effective ingredients) of needing.In employed agricultural chemicals, chemical pesticide has accounted for sizable ratio, and the shared ratio of biological pesticide is relatively very little.Because the residual quantity of chemical pesticide is bigger, and there is toxic action in human body, so the use of chemical pesticide can be subjected to limiting significantly, biological pesticide is a kind of pollution of effective solution chemical pesticide and a kind of environment-friendly type public nuisance-free agricultural chemicals of residue problem.People are grown to even greater heights to pollution-free, pollution-free green food cry at present, and biological control has become the another important method of preventing and treating after chemical prevention, and many biological bactericides come into operation and obtained unusual effect.Research and employing biological control technology are to replace the needs that agricultural chemicals is agriculture sustainable development gradually.。
Some results of study show, utilize some beneficial microorganisms that the host of the specific pathogen bacterium in the soil is produced objectionable impurities, reduce the quantity of pathogenic bacteria by approach such as competition nutrition and spaces, take place thereby alleviate disease.Therefore, applying biological is prevented and treated a kind of efficient ways of controlling the vegetables disease and pest of can yet be regarded as.Wood is mould to be one of the most promising biocontrol microorganisms, has the control disease and promotes the double effects of plant growth.Trichoderma is mainly used to prevent and treat plurality of plant diseases, as the samping off of tomato, cucumber and cowpea, the black root rot of strawberry, the anbury of Chinese cabbage etc.
Trichoderma is in mycota, double-core bacterium door, Deuteromycotina, hyphomycetes, Moniliales, from stalk spore section, it is the important component part of soil microflora, its mycelial growth and spore germination can adapt to wider temperature humidity range and pH scope, and saprophytic property is strong, growth and breeding are fast, can utilize nutrition rapidly and takes up space.At present, in 9 kinds of known Trichoderma, having 5 kinds to have the biological and ecological methods to prevent plant disease, pests, and erosion potentiality, is respectively trichoderma harziarum (T.harzianum), viride (T.virid), koning trichoderma (T.koningii), hook wood mould (T.hamatum) and long shoot wood mould (T.longibrachiatum).What current use was maximum is trichoderma harziarum and viride.
Wood is mould to be a kind of broad spectrum antagonism bacterium, can the multiple pathogenic fungi of antagonism, especially soil-borne fungus there is significant antagonistic effect.Except that being the weak pathogenic bacteria individually, great majority have antagonistic action to plant pathogenic fungi, and its characteristics show three aspects.The first, Trichoderma has adaptability widely for the environment of growth, can utilize various simple or complicated carbon sources and nitrogenous source, even some abilities harmful or the lasting pollutent that exists that transform in addition and degrade, optimum growth temperature is 28～32 ℃, but also can grow and breeding under 5 ℃ or on 60 ℃ individually.The second, Trichoderma produces various antagonistic action, mainly contains four kinds: i.e. competition effect, hyperparasitism, generation antibiotics and bacteriolyze enzyme.The 3rd, Trichoderma has parasitic broad spectrum, to 18 genus, 29 kinds can parasitism.
The effective biological pesticide resource of the mould conduct of wood, strong with its wide adaptability, viability, to the broad spectrum of phytopathogen antagonistic action, can also produce plurality of enzymes simultaneously in diseases prevention and decompose plant residue, degraded macromolecular cpd in the soil and make plant can absorb, can gain great popularity with other beneficial bacterium harmonic growth in the soil, advantage such as free from environmental pollution.
Found that at present many microorganisms have the biological and ecological methods to prevent plant disease, pests, and erosion effect, Trichoderma (Trichoderma spp.) is exactly a class ubiquity and the biological and ecological methods to prevent plant disease, pests, and erosion with an important economic implications benefit bacterium, has been used for preventing and treating plurality of plant diseases.Before more than 60 years, people just recognize the antagonistic action of Trichoderma to phytopathogen.In recent years; people have carried out a large amount of research to the biological control mechanism of Trichoderma, think that its antagonistic action mainly shows the aspects such as resistant effect, toxic protein and joint action of antagonism of defense response, protective enzyme and the plant of metabolism extracellular enzyme, antibiotic class material, inducing plant resistance, lytic enzyme and plant.
Summary of the invention
The invention provides the viride bacterial strain of a kind of high yield cell-wall degrading enzyme and antibacterial peptide.
The viride bacterial strain of a kind of high yield cell-wall degrading enzyme and antibacterial peptide, called after viride (Trichoderma viride) TV501, be preserved in the Chinese typical culture collection center (CCTCC) that is positioned at Lopa Nationality an ancient woman's ornament mountain, Wuhan City Wuhan University, preservation date is on December 31st, 2009, and preserving number is CCTCC NO:M 209321.
This bacterial strain major physiological is characterized as: bacterium colony is very fast in the growth of PDA media surface, forms the white hypha thin layer in second day, and after the conidium maturation, bacterium colony to the edge, graduates into green bacterium colony in various degree in central authorities, and bacterium colony combines closely with substratum.At microscopically, conidiophore bears from the side shoot of mycelia, and the vertical branches sprig goes up living spore ball estranged often to life.The conidium sphere, light or colourless, mycelia has tabula and branch, and branch is crooked or in wave shape.
A kind of biocontrol reagent that utilizes above-mentioned viride TV501 to make, in 1000 parts of gross weights, raw material consists of:
480～970 parts of viride TV501 fermented liquids
0.01～30 part in tensio-active agent
Water surplus.
The mould fermented liquid of described wood makes by the following method:
Mould TV501 is inoculated in the seed culture medium with wood, and shaking culture 3d makes seed liquor under 30 ℃, 200rpm, and seed liquor is inoculated in the fermention medium, and oscillation and fermentation cultivation is 5～7 days under 28～30 ℃, 150～200rpm, makes wooden mould fermented liquid.
Above-mentioned fermentation also can be amplified at fermentor tank, and control fermented liquid pH is 5.8～6.2 during fermentation, 28～30 ℃ of temperature, dissolved oxygen amount 20～30%, stirring velocity 150～200rpm.
Preferably, with the 1L volumeter, described fermention medium is by 1.5-2.5g NaNO
3, 0.5-1.5gK
2HPO
4, 0.4-0.6g KCl, 0.4-0.6g MgSO
4, 0.009-0.015g FeSO
4, 25-35g sucrose, 350-450ml wheat bran extracting solution and surplus water form, described wheat bran extracting solution boiled 1 hour at 90 ℃ by the 50g wheat bran being placed 1.5L water, filtered the back water and was settled to 450ml and makes.
More preferably, with the 1L volumeter, described fermention medium is by 0.25-0.35gCa (NO
3)
24H
2O, 0.05-0.15g KNO
3, 0.05-0.1g KCl, 0.2-0.4g MgSO
47H2O, 0.01-0.03g NaH
2PO
4.2H
2O, 0.5-2.00g soya peptone, 4.00-6.00g winestone acid amide, 2～3mgFeCl
36H
2O, 4.0-6.0mg MnSO
4.H
2O, 2.0-3.0mg ZnSO
47H
2O, 1.0-2.5mgH
3BO
3, 0.5-1.0mg KI, 0.2-0.4g yeast extract, 350-450ml wheat bran extracting solution and surplus water form, described wheat bran extracting solution boiled 1 hour at 90 ℃ by the 1g wheat bran being placed 1.5L water, was settled to 450ml after the filtration and made.
The described tensio-active agent of described tensio-active agent is tween 20 (Tween-20), Triton (Triton-100) and Silwet L-77.In the mould fermented liquid of wood, add tensio-active agent, to accelerate the medicament infiltration, the breeding of controlling plant growth of pathogenic bacteria.
Viride bacterial strain of the present invention can high yield cell wall degrading enzyme and antibacterial peptide, the biocontrol reagent that is made by it has better field efficacy to vegetables, melon and fruit diseases such as powdery mildew of strawberry, grey mould fruit rot of strawberry, powderys mildew of cucumber, significantly improved the yield and quality of vegetables, melon and fruit, reduced the usage quantity of chemical pesticide, improve the ecological environment, improve edible safety, demonstrate vast potential for future development, help the growth of agricultural efficiency increasing peasant income.
Embodiment
Bacterial strain mutagenesis, separation and purifying
Open ultraviolet lamp (30W) preheating 15min, getting 5ml viride (T.virid) spore suspension (water is medium), to place diameter be the 6cm culture dish, and vertical the placement from ultraviolet lamp 30cm place shone 15min, uses magnetic stirrer during handling.Postradiation spore suspension is secretly cultivated to coat behind the 4h and is carried out primary dcreening operation on the PDA flat board.
Select dull and stereotyped going up and grow soon, the bacterium colony of transparent circle big (colony diameter is greater than 0.5cm) is as sieving bacterial strain again.Several strain bacterial strains of scalping are transferred to the PDA slant medium, in 25 ℃ of incubators, cultivate 6d, inoculate the triangular flask solid fermentation substratum (NaNO of 250ml
32g/L, K
2HPO
41g/L, KCl 0.5g/L, MgSO
40.5g/L, FeSO
40.01g/L, sucrose 30g/L, wheat bran extracting solution 350ml/L and agar 10g/L, the wheat bran extracting solution boiled 1 hour at 90 ℃ by the 50g wheat bran being placed 1.5L water, filtering the back water is settled to 450ml and makes) in carry out multiple sieve, select fast, the transparent circle big (colony diameter is greater than 0.5cm) of growing, mycelia is intensive, sporulation quantity is many, change a green fast Trichoderma falls, and is inoculated into and carries out preservation on the PDA inclined-plane.
Identification of strains
The bacterial strain bacterium colony of above-mentioned separation and purification is very fast in the growth of PDA media surface, forms the white hypha thin layer in second day, and after the conidium maturation, bacterium colony to the edge, graduates into green bacterium colony in various degree in central authorities, and bacterium colony combines closely with substratum.At microscopically, conidiophore bears from the side shoot of mycelia, and the vertical branches sprig goes up living spore ball estranged often to life.The conidium sphere, light or colourless, mycelia has tabula and branch, and branch is crooked or in wave shape.
The 18S rDNA of this bacterial strain and other sequences of Trichoderma are carried out the homology comparison, the result shows the sequence homology of the 18S rDNA of this bacterial strain and the many bacterial strains of Trichoderma more than 99%, and wherein the homology with viride (Trichoderma viride) reaches 100%.From shape and the comparative analysis of conidium form of colonial morphology, mycelia, all similar with viride.Therefore, this identification of strains is viride (Trichoderma viride), and strain number is TV501.
The preparation fermented liquid
Viride (Trichoderma viride) TV501 that above-mentioned separation and purification is obtained is inoculated in the seed culture fluid, and 200rpm shaking culture 3d makes seed liquor.
Shake flask fermentation is inoculated into seed liquor and contains fermention medium and shake in the bottle, makes viride TV501 fermented liquid at 28-30 ℃ of following 200rpm shaking culture 7d.
Also can carry out the fermentor tank enlarged culturing, be example with the 10L fermentor tank, and fermented liquid pH is 6.0, and temperature is 28～30 ℃, and oxygen level is controlled at 20～30% (passing through Ventilation Control), and stirring velocity is 200rpm, and fermentation time is 6 days.
Preparation biocontrol reagent and preventive effect test
Embodiment 1
Viride TV501 fermented liquid 975 weight parts, tween 20 25 weight parts are mixed, make biocontrol reagent, fermented liquid obtains by shaking bottle shaking culture, and wherein the prescription of fermention medium is:
NaNO
31.8g/L, K
2HPO
40.5g/L, KCl 0.5g/L, MgSO
40.4g/L, FeSO
40.01g/L, sucrose 30g/L, wheat bran extracting solution 380ml/L.The wheat bran extracting solution boiled 1 hour at 90 ℃ by the 50g wheat bran being placed 1.5L water, filtered back water constant volume to 450ml.After the fermentation, fermented liquid detects by filter paper enzyme activity mensuration and antibacterial peptide active immne, obtain the active 550.7U/ml of cell-wall degrading enzyme and (represent every milliliter of unit of enzyme activity, the enzyme amount that generates 1 μ mol glucose with the per minute catalyzing cellulose hydrolysis is that 1 enzyme activity unit is 1U), the active 500.5U/ml of antibacterial peptide (represent every milliliter of antibacterial peptide activity unit, represent that with the contained antimicrobial peptide protein amount 1 μ g of minimum inhibitory concentration in every milliliter 1 activity unit is 1U).
The crop of selecting for use is a strawberry, and concrete kind is red cheek.
The biocontrol reagent water is diluted with 1: 500 ratio, the strawberry field is sprayed, every mu of usage quantity is 50kg (diluent), every spraying in 1 week 1 time, sprays continuously 3 times.The influence situation of medicament to strawberry blade and young fruit etc. observed in range estimation in 3 days after each dispenser, and the examination medicament is to the security of strawberry.And before medicine, carry out disease index radix investigation, preventive effect of 7 days investigation statisticses after each medication.Field test is established 1000 times of liquid of 50% Sukeling wettable powder respectively and clear water compares.
The result shows that this biocontrol reagent is 57.8% to the preventive effect of grey mould fruit rot of strawberry, and 1000 times of liquid of chemical agent 50% Sukeling only are 49.4%.
Simultaneously the strawberry crop is added up, the result shows that the output that has sprayed 1000 times of liquid of strawberry yield increased 50% Sukeling wettable powder of biocontrol reagent of the present invention increases by 2.1%, increases by 15.8% than clear water contrast output.
Embodiment 2
Viride TV501 fermented liquid 487.5 weight parts, tween 20 25 weight parts and water 487.5 weight parts are mixed, stir, make biocontrol reagent, the fermented liquid employing is shaken a bottle oscillation and fermentation and is made, and fermentative medium formula is:
Ca (NO
3)
24H
2O 0.28g/L, KNO
30.08g/L, KCl 0.06g/L, MgSO
47H2O0.36g/L, NaH
2PO
4.2H
2O 0.02g/L, soya peptone 1g/L, winestone acid amide 5g/L, FeCl
36H
2O2mg/L, MnSO
4.H
2O 5mg/L, ZnSO
47H
2O 2.5mg/L, H
3BO
31.4mg/L, KI0.7mg/L, yeast extract 0.25g/L, wheat bran extracting solution 450ml/L, the wheat bran extracting solution boiled 1 hour at 90 ℃ by the 1g wheat bran being placed 1.5L water, filtered back water constant volume to 450ml.After the fermentation, fermented liquid detects by filter paper enzyme activity mensuration and antibacterial peptide active immne, obtain the active 580.5U/ml of cell-wall degrading enzyme and (represent every milliliter of unit of enzyme activity, the enzyme amount that generates 1 μ mol glucose with the per minute catalyzing cellulose hydrolysis is that 1 enzyme activity unit is 1U), the active 540.8U/ml of antibacterial peptide (represent every milliliter of antibacterial peptide activity unit, represent that with the contained antimicrobial peptide protein amount 1 μ g of minimum inhibitory concentration in every milliliter 1 activity unit is 1U).
The crop of selecting for use is strawberry and cucumber, and concrete kind is red cheek (strawberry) and Tianjin excellent No. 1 (cucumber).
The biocontrol reagent water is diluted with 1: 500 ratio, respectively strawberry and cucumber field are sprayed, every mu of usage quantity is 50kg (diluent), every spraying in 1 week 1 time, sprays continuously 3 times.The influence situation of medicament to strawberry, cucumber leaves and young fruit etc. observed in range estimation in 3 days after each dispenser, and the examination medicament is to the security of strawberry, cucumber.And before medicine, carry out disease index radix investigation, preventive effect of 7 days investigation statisticses after each medication.
For powdery mildew of strawberry, establish 50% emerald green shellfish WDG3000 times liquid and clear water respectively and compare; For grey mould fruit rot of strawberry, establish 1000 times of liquid of 50% Sukeling wettable powder and clear water respectively and compare; For powdery mildew of cucumber, establish 1000 times of liquid of 30% kresoxim-methyl wettable powder (hundred U.S.s) and clear water respectively and compare.
Field test results shows, biocontrol reagent has the better prevention effect to powdery mildew of strawberry, and relative control effect (relative comparison) is more than 80%, and is safe in utilization, the output of its yield increased 50% emerald green shellfish WDG3000 times liquid increases by 7.6%, increases by 27.5% than clear water contrast output.
This biocontrol reagent also has certain prevention effect to grey mould fruit rot of strawberry, relative control effect (relative comparison) is more than 80%, safe in utilization, the output of 1000 times of liquid of its yield increased 50% Sukeling wettable powder increases by 3.1%, increases by 25.8% than clear water contrast output.
This biocontrol reagent has the better prevention effect to powdery mildew of cucumber, and anti-(relative comparison) imitates more than 80% relatively, and be safe in utilization, and 1000 times of liquid of its output and contrast 30% kresoxim-methyl wettable powder (hundred U.S.s) maintain an equal level, but increases by 25% than clear water contrast output.
Embodiment 3
Viride TV501 fermented liquid 975 weight parts, Triton-10025 weight part are mixed, make biocontrol reagent, fermented liquid obtains by shaking bottle oscillation and fermentation, and wherein the culture medium prescription of viride TV501 fermented liquid is:
NaNO
31.5g/L, K
2HPO
41.0g/L, KCl 0.4g/L, MgSO
40.5g/L, FeSO
40.012g/L, sucrose 25g/L, wheat bran extracting solution 420ml/L.The wheat bran extracting solution boiled 1 hour at 90 ℃ by the 50g wheat bran being placed 1.5L water, filtered back water constant volume to 450ml.After the fermentation, fermented liquid measures by total enzyme activity and the antibacterial peptide active immne detects, obtain the active 551.2U/ml of cell-wall degrading enzyme and (represent every milliliter of unit of enzyme activity, the enzyme amount that generates 1 μ mol glucose with the per minute catalyzing cellulose hydrolysis is that 1 enzyme activity unit is 1U), the active 507.1U/ml of antibacterial peptide (represent every milliliter of antibacterial peptide activity unit, represent that with the contained antimicrobial peptide protein amount 1 μ g of minimum inhibitory concentration in every milliliter 1 activity unit is 1U).
The crop of selecting for use is a Chinese cabbage, and concrete kind is precocious 5
The biocontrol reagent water is diluted with 1: 500 ratio, the Chinese cabbage field is sprayed, every mu of usage quantity is 50kg (diluent), every spraying in 1 week 1 time, sprays continuously 2 times.The influence situation of medicament to Chinese cabbage oidium observed in range estimation in 3 days after each dispenser, and the examination medicament is to the security of Chinese cabbage.And before medicine, carry out disease index radix investigation, preventive effect of 7 days investigation statisticses after each medication.Field test is established 600 times of liquid of 58% metalaxyl-mn-zn respectively and clear water compares.
The result shows that control has unusual effect to biocontrol reagent to Chinese cabbage oidium.Preventive effect reaches 77.5% behind the medicine for the first time, with the no significance difference of chemical agent 58% first frost MnZn (contrast) of producing upward use; Behind the medicine, the prevention effect of biocontrol reagent contrasts a little less than chemical agent for the second time, and relative control effect is more than 80%, and is safe in utilization, and the output of its yield increased 58% first frost MnZn does not have significant difference, but increases by 19.5% than clear water contrast output.
Embodiment 4
Viride TV501 fermented liquid 487.5 weight parts, Triton-10025 weight part and water 487.5 weight parts are mixed, make biocontrol reagent, fermented liquid adopts fermentor tank to make, and fermentative medium formula is:
Ca (NO
3)
24H
2O 0.30g/L, KNO
30.05g/L, KCl 0.08g/L, MgSO
47H2O0.2g/L, NaH
2PO
4.2H
2O 0.01g/L, soya peptone 0.5g/L, winestone acid amide 4g/L, FeCl
36H
2O2.5mg/L, MnSO
4.H
2O 4mg/L, ZnSO
47H
2O 2mg/L, H
3BO
31.0mg/L, KI0.5mg/L, yeast extract 0.2g/L, wheat bran extracting solution 380ml/L, the wheat bran extracting solution boiled 1 hour at 90 ℃ by the 1g wheat bran being placed 1.5L water, filtered back water constant volume to 450ml.After the fermentation, fermented liquid is by total enzyme activity and antibacterial peptide determination of activity, obtain the active 576.4U/ml of cell-wall degrading enzyme and (represent every milliliter of unit of enzyme activity, the enzyme amount that generates 1 μ mol glucose with the per minute catalyzing cellulose hydrolysis is that 1 enzyme activity unit is 1U), the active 542.1U/ml of antibacterial peptide (represent every milliliter of antibacterial peptide activity unit, represent that with the contained antimicrobial peptide protein amount 1 μ g of minimum inhibitory concentration in every milliliter 1 activity unit is 1U).
The crop of selecting for use is a Chinese cabbage, and concrete kind is precocious No. 5.
The biocontrol reagent water is diluted with 1: 500 ratio, the Chinese cabbage field is sprayed, every mu of usage quantity is 50kg (diluent), every spraying in 1 week 1 time, sprays continuously 2 times.The influence situation of medicament to Chinese cabbage oidium observed in range estimation in 3 days after each dispenser, and the examination medicament is to the security of Chinese cabbage.And before medicine, carry out disease index radix investigation, preventive effect of 7 days investigation statisticses after each medication.Field test is established 600 times of liquid of 58% metalaxyl-mn-zn respectively and clear water compares.
The result shows that control has unusual effect to biocontrol reagent to Chinese cabbage oidium.Preventive effect reaches 75.7% behind the medicine for the first time, with the no significance difference of chemical agent 58% first frost MnZn (contrast) of producing upward use; Behind the medicine, the biocontrol reagent preventive effect is 82.1% for the second time, is significantly higher than the chemical agent contrast, and is safe in utilization, and the output of its yield increased 58% first frost MnZn does not have significant difference, but increases by 21.8% than clear water contrast output.
Embodiment 5
With viride TV501 fermented liquid 975 weight parts, Silwet L-77 (poly suboxygen alkyl modification polymethyl siloxane) 0.01 weight part and water 24.99 weight part mixing and stirring, adopt ferment tank, fermentative medium formula is:
NaNO
32.5g/L, K
2HPO
41.5g/L, KCl 0.6g/L, MgSO
40.6g/L, FeSO
40.015g/L, sucrose 35g/L, wheat bran extracting solution 450ml/L.The wheat bran extracting solution boiled 1 hour at 90 ℃ by the 50g wheat bran being placed 1.5L water, filtered back water constant volume to 450ml.After the fermentation, fermented liquid is by total enzyme activity and antibacterial peptide determination of activity, obtain the active 553.1U/ml of cell-wall degrading enzyme and (represent every milliliter of unit of enzyme activity, the enzyme amount that generates 1 μ mol glucose with the per minute catalyzing cellulose hydrolysis is that 1 enzyme activity unit is 1U), the active 502.5U/ml of antibacterial peptide (represent every milliliter of antibacterial peptide activity unit, represent that with the contained antimicrobial peptide protein amount 1 μ g of minimum inhibitory concentration in every milliliter 1 activity unit is 1U).
The crop of selecting for use is a cucumber, and concrete kind is excellent No. 1 of Tianjin.
The biocontrol reagent water is diluted with 1: 500 ratio, the cucumber field is sprayed, not having a mu spraying agent amount is 50kg (diluent), every spraying in 1 week 1 time, sprays continuously 3 times.The influence situation of medicament to cucumber leaves and young fruit etc. observed in range estimation in 3 days after each dispenser, and the examination medicament is to the security of cucumber.And before medicine, carry out disease index radix investigation, preventive effect of 7 days investigation statisticses after each medication.Field test is established 1000 times of liquid of 30% kresoxim-methyl wettable powder (hundred U.S.s) respectively and clear water compares.
The result shows that biocontrol reagent has the better prevention effect to powdery mildew of cucumber, and field efficacy is stable, substantially maintain about 50%, safe in utilization, 1000 times of liquid of its output and contrast 30% kresoxim-methyl wettable powder (hundred U.S.s) remain basically stable, but increase by 20% than clear water contrast output.
Embodiment 6
With viride TV501 fermented liquid 975 weight parts, Silwet L-770.01 weight part and water 512.49 weight part mixing and stirring, adopt ferment tank, fermentative medium formula is:
Ca (NO
3)
24H
2O 0.35g/L, KNO
30.15g/L, KCl 0.1g/L, MgSO
47H2O0.4g/L, NaH
2PO
4.2H
2O 0.03g/L, soya peptone 2g/L, winestone acid amide 6g/L, FeCl
36H
2O3mg/L, MnSO
4.H
2O 6mg/L, ZnSO
47H
2O 3.0mg/L, H
3BO
32.5mg/L, KI100mg/L, yeast extract 0.4g/L, wheat bran extracting solution 450ml/L, the wheat bran extracting solution boiled 1 hour at 90 ℃ by the 1g wheat bran being placed 1.5L water, filtered back water constant volume to 450ml.After the fermentation, fermented liquid is by total enzyme activity and antibacterial peptide determination of activity, obtain the active 583.5U/ml of cell-wall degrading enzyme and (represent every milliliter of unit of enzyme activity, the enzyme amount that generates 1 μ mol glucose with the per minute catalyzing cellulose hydrolysis is that 1 enzyme activity unit is 1U), the active 547.2U/ml of antibacterial peptide (represent every milliliter of antibacterial peptide activity unit, represent that with the contained antimicrobial peptide protein amount 1 μ g of minimum inhibitory concentration in every milliliter 1 activity unit is 1U).
Selecting crop for use is abavo, is specially the abavo strain.
The biocontrol reagent water with: 500 ratio is diluted, and the abavo field is sprayed, and every mu of usage quantity is 50kg (diluent), every spraying in 1 week 1 time, sprays continuously 3 times.The influence situation of medicament to abavo blade and young fruit etc. observed in range estimation in 3 days after each dispenser, and the examination medicament is to the security of abavo.And before medicine, carry out disease index radix investigation, preventive effect of 7 days investigation statisticses after each medication.Field test is established 3000 times of liquid of 50% emerald green shellfish respectively and clear water compares.
The result shows that biocontrol reagent has the better prevention effect to the abavo Powdery Mildew, and field efficacy is 76.6.0%, there is not significant difference with the preventive effect of contrast 50% emerald green shellfish, safe in utilization, 3000 times of liquid of its output and contrast 50% emerald green shellfish remain basically stable, but increase by 15% than clear water contrast output.
Claims (7)
1. viride bacterial strain, it is characterized in that: called after viride (Trichodermaviride) TV501, preserving number is CCTCC NO:M 209321.
2. a biocontrol reagent that utilizes the described viride of claim 1 to make is characterized in that, in 1000 parts of gross weights, raw material consists of:
480～9700 parts of viride TV501 fermented liquids
0.01～30 part in tensio-active agent
Water surplus.
3. biocontrol reagent according to claim 1 is characterized in that: described tensio-active agent is at least a in tween 20, Triton and the poly suboxygen alkyl modification polymethyl siloxane.
4. biocontrol reagent according to claim 1 is characterized in that, described viride TV501 fermented liquid prepares by the following method:
Viride TV501 is seeded in the seed culture medium, makes seed liquor, seed liquor is inoculated into contains the shaking in the bottle of fermention medium, shaking culture is 5～7 days under 28～30 ℃, the condition of 150～200rpm, makes viride TV501 fermented liquid.
5. biocontrol reagent according to claim 1 is characterized in that, described viride TV501 fermented liquid prepares by the following method:
Viride TV501 is seeded in the seed culture medium, make seed liquor, seed liquor is inoculated in the fermentor tank that contains fermention medium, fermented liquid pH is controlled at 5.8～6.2, oxygen level is controlled at 20～30%, stir speed (S.S.) is controlled at 150～200rpm, 28～30 ℃ of fermentations 5～7 days, makes viride TV501 fermented liquid.
6. according to claim 4 or 5 described preparations, it is characterized in that: with the 1L volumeter, described fermention medium is by 1.5-2.5g NaNO
3, 0.5-1.5g K
2HPO
4, 0.4-0.6g KCl, 0.4-0.6g MgSO
4, 0.009-0.015g FeSO
4, 25-35g sucrose, 350-450ml wheat bran extracting solution and surplus water form, described wheat bran extracting solution boiled 1 hour at 90 ℃ by the 50g wheat bran being placed 1.5L water, filtered the back water and was settled to 450ml and makes.
7. according to claim 4 or 5 described biocontrol reagents, it is characterized in that with the 1L volumeter, described fermention medium is by 0.25-0.35g Ca (NO
3)
24H
2O, 0.05-0.15g KNO
3, 0.05-0.1g KCl, 0.2-0.4g MgSO
47H2O, 0.01-0.03g NaH
2PO
4.2H
2O, 0.5-2.00g soya peptone, 4.00-6.00g winestone acid amide, 2～3mg FeCl
36H
2O, 4.0-6.0mg MnSO
4.H
2O, 2.0-3.0mg ZnSO
47H
2O, 1.0-2.5mg H
3BO
3, 0.5-1.0mg KI, 0.2-0.4g yeast extract, 350-450ml wheat bran extracting solution and surplus water form, described wheat bran extracting solution boiled 1 hour at 90 ℃ by the 1g wheat bran being placed 1.5L water, was settled to 450ml after the filtration and made.
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| CN 201010040036 CN101724572B (en) | 2010-01-19 | 2010-01-19 | High yield cell wall degrading enzyme and Trichoderme viride of antibacterial peptide and biocontrol reagent thereof |
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| CN 201010040036 CN101724572B (en) | 2010-01-19 | 2010-01-19 | High yield cell wall degrading enzyme and Trichoderme viride of antibacterial peptide and biocontrol reagent thereof |
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| CN101724572B CN101724572B (en) | 2011-09-28 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102919230A (en) * | 2012-10-25 | 2013-02-13 | 牛赡光 | Application of nucleoside derivative |
| CN102919246A (en) * | 2012-10-25 | 2013-02-13 | 牛赡光 | Use of diketopiperazine derivatives |
| CN105062897A (en) * | 2015-08-14 | 2015-11-18 | 潍坊万胜生物农药有限公司 | Thichoderma viride with high chlamydospore yield and application of thichoderma viride |
| CN108477223A (en) * | 2018-05-07 | 2018-09-04 | 东北林业大学 | The preparation process of the antibacterial metabolite biocontrol agent aquas of green trichoderma M43 and its application |
-
2010
- 2010-01-19 CN CN 201010040036 patent/CN101724572B/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102919230A (en) * | 2012-10-25 | 2013-02-13 | 牛赡光 | Application of nucleoside derivative |
| CN102919246A (en) * | 2012-10-25 | 2013-02-13 | 牛赡光 | Use of diketopiperazine derivatives |
| CN102919246B (en) * | 2012-10-25 | 2014-05-14 | 牛赡光 | Use of diketopiperazine derivatives |
| CN105062897A (en) * | 2015-08-14 | 2015-11-18 | 潍坊万胜生物农药有限公司 | Thichoderma viride with high chlamydospore yield and application of thichoderma viride |
| CN105062897B (en) * | 2015-08-14 | 2018-02-23 | 潍坊万胜生物农药有限公司 | The Trichoderma viride of one plant height production chlamydospore and its application |
| CN108477223A (en) * | 2018-05-07 | 2018-09-04 | 东北林业大学 | The preparation process of the antibacterial metabolite biocontrol agent aquas of green trichoderma M43 and its application |
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| Publication number | Publication date |
|---|---|
| CN101724572B (en) | 2011-09-28 |
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