CN102888350A - Trichoderma strain for antagonizing soil-borne disease - Google Patents
Trichoderma strain for antagonizing soil-borne disease Download PDFInfo
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Abstract
The invention relates to a Trichoderma strain for antagonizing soil-borne disease, belonging to the field of biological control. The Trichoderma strain is Trichoderma asperellum ZJSX5003 CGMCC No.6480. The Trichoderma strain provided by the invention has the characteristics of high growth speed and high spore production amount; the in-vitro standoff pathogen inhibition rates are respectively up to 79.79%, 73.68% and 72.25%, the pathogen spore survival rates are respectively 0.00%, the in-vivo preventive effects for cucumber fusarium wilt and maize stem rot are respectively 85.08% and 66.67%, the enzyme activity of chitinase is 4.0783U, the enzyme activity of beta-1,3-glucanase is 0.8050U, the enzyme activity of extracellular proteinase is 4.8503U, and the content of silicone, terpene and carboxylic acid having antagonistic actions in antibiotic secondary metabolites is 44.44%. The Trichoderma strain provided by the invention has the advantages of high antagonism and high specificity. The biological pesticide produced from the strain can effectively control the soil-borne disease, thereby ensuring the stable increase of crop and vegetable yields.
Description
Technical field
The invention belongs to the biological control field, relate to a strain Trichoderma bacterial strain, relate in particular to a kind of Trichoderma bacterial strain of antagonism soil-borne disease.
Background technology
Because over a long time a certain crop of year after year plantation, cause the aggravation of causing harm of the serious and soil-borne disease of soil secondary salinization, cause the crop quality reduction, production declining has now become the significant obstacle factor that restricts the industrialized agriculture sustainable development.Plant soil-borne diseases is the important Plant diseases of a class, causes the pathogen kind a lot (fungi, bacterium, nematode, virus) of soil-borne disease, and they infect plant root usually, cause the disease of plant root and even complete stool, cause great financial loss.Wherein, the soil-borne disease fungal pathogens has become one of main restricting factor of farm crop, vegetables stable and high yields at present.
Summarize its economic loss as an example of corn stalk rot disease (Corn Stalk Rot) example, claim again Corn Stalk Rot, it distributes extensively, harm is heavy, corn each producing region in the world's all generally occurs, general time sickness rate is 10～20%, can reach more than 50% when serious, cause the corn underproduction 20～30%.The main infective pathogen bacterium that causes corn stalk rot disease is pythium spp, sickle-like bacteria, dry rot fungus, anthrax-bacilus and bacterium etc.Take cucumber fusarium axysporum as example, being again dead arm, wilt disease again, is a kind of vascular bundle diseases that soil is propagated, root is invaded, general sickness rate 20%～30%, and serious plot reaches 80%～90%, even all ruins kind, has a strong impact on cucumber production output.Therefore, in order effectively to prevent and treat soil-borne disease, to guarantee China farm crop, vegetable crop steady growth, must need to carry out integrated control.Wherein, paid close attention to by government, farm and peasant household because the biological control green is harmless, the Biocontrol microorganism the Study on Resources is extremely urgent.And as the Trichoderma of internationally recognized biocontrol microorganisms, the effect of preferred strain is distinguished in recent years, and the trichoderma strain resource of seeking efficient biological and ecological methods to prevent plant disease, pests, and erosion ability is extremely important.
Summary of the invention
The objective of the invention is to cultivate by the face-off of exsomatizing the screening of mensuration, pathogenic bacteria Conidia persistence mensuration, potted plant inoculation test index, a kind of Trichoderma bacterial strain of novel antagonism soil-borne disease is provided, Trichoderma bacterial strain provided by the invention has the advantage of high Antagonism, strong specialization, use the biological pesticide that this bacterial strain is produced, can effectively prevent and treat soil-borne disease, guarantee the steady growth of farm crop, vegetable crop.
The present invention realizes by following technical scheme,
The present invention relates to a kind of Trichoderma bacterial strain of antagonism soil-borne disease, described Trichoderma bacterial strain is trichoderma asperellum (Trichoderma asperellum) Z JSX5003 CGMCC No.6480.
Preferably, the ITS sequence of described bacterial strain is shown in SEQ ID NO.1.
Preferably, the Tef1-α sequence of described bacterial strain is shown in SEQ ID NO.2.
Trichoderma bacterial strain provided by the invention is in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) preservation, and the address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101; The information of bacterial strain of the present invention is: trichoderma asperellum (Trichoderma asperellum) ZJSX5003; Preserving number is CGMCC No.6480; Preservation date is 2012.8.28.
The strain culturing feature comprises: the most suitable growth temperature on the PDA is 30 ℃, and 30 ℃ of growth 48h colony radius are about 31～47mm; Growing period gives of short duration fluorescence irradiation every 8h under 30 ℃ of dark conditions, and bacterium colony can form 5 concentric wheel stripe structures, is comprised of intensive conidium, and the conidium in close centre is blackish green, lacks aerial hyphae; Do not produce the diffustivity pigment, without obvious smell; The bacterium colony reverse side is cream-colored, gauffer is arranged or winding arrangement is arranged.
The strain morphology feature comprises: conidium bunch is cushion to hemispherical, and diameter is 0.5～2mm, and discrete distribution or converge intersperses among whole bacterium colony or is arranged as 2～3 concentric wheel stripes; The conidium polymer is deep green.In conidium bunch, the bottle stalk on the conidiophore presents symmetrical, and the top has two or more bottles of stalks, and a bottle stalk width is 2.8～5.4 μ m, and the first branch that bear below on the main shaft top often is life, with the angle of main shaft be nearly 90 °; That bottle stalk typically results from is first, the top of secondary, tertiary branching, lessly directly results from first and secondary branch middle part, and 2～3 bottles stalks are the arrangement of whirlpool shape; Upright, the ampoule shape of bottle, the only a little overstriking at the middle part, length is 7.1～11.3 μ m, and the middle part width is 3.0～5.5 μ m, and base widths is 2.0～3.5 μ m, and base portion has the contracting of hanging a little, is about 100 ° with its parent cell angle.Conidium is spherical, and size is 3.7～6.0 μ m * 3.0～5.0 μ m to inferior sphere or oval, does not have the visible base portion vestige that comes off, and the spinelet structure is arranged, but sometimes be difficult to observe.
The present invention gathers SOUTHERN CHINA area agricultural soil and connects the bacterium efficiency test in 4 ℃ of preservations, separation and purification bacterial strain, the face-off pathogenic bacteria inhibition test (Fusarium moniliforme, Fusarium graminearum, dry thread Pyrenomycetes) that exsomatizes, pathogenic bacteria (Fusarium moniliforme, Fusarium graminearum, dry thread Pyrenomycetes) Conidia persistence mensuration, live body, through above step repeated screening, finally select the Trichoderma bacterial strain that kills soil-borne disease of high Antagonism, strong specialization, be numbered ZJSX5003.By distinguishing determining nucleic acid sequence and analysis between the identification by morphological characters such as bacterium colony and conidiophore, bottle stalk, conidium and interior open gene, determined the classification position of this bacterial strain: Trichoderma (Trichoderma Pers.ex Fr.), trichoderma asperellum (Trichodermaasperellum).The present invention has also carried out the Physiology and biochemistry of this bacterial strain and has measured, and comprises that antagonism relevant enzyme enzyme (chitinase, beta-1,3-glucanase, extracellular protease) alive detects, antibiosis secondary metabolite extraction and analysis, better develops this bacterial strain.
The ITS sequence of Trichoderma bacterial strain provided by the invention shown in the sequence 1 in the sequence table (the Genebank sequence number: JQ617302), its Tef1-α sequence (Genebank sequence number: JQ617305) shown in the sequence 2 in the sequence table.
Beneficial effect of the present invention is as follows: the fast growth of Trichoderma bacterial strain provided by the invention, sporulation quantity is large, stripped face-off pathogenic bacteria (Fusarium oxysporum, Fusarium moniliforme, Fusarium graminearum) inhibiting rate reaches respectively 79.79%, 73.68%, 72.25%, pathogenic bacteria (Fusarium oxysporum, Fusarium moniliforme, Fusarium graminearum) Conidia persistence is 0.00%, for cucumber fusarium axysporum, the live body preventive effect of corn stalk rot disease is respectively 85.08%, 66.67%, the chitinase enzyme is lived and is 4.0783U, β-1,3-dextranase enzyme is lived and is 0.8050U, the extracellular protease enzyme is lived and is 4.8503U, plays the polyketone class of antagonistic action in the antibiosis secondary metabolite, terpenes and carboxylic-acid account for 44.44% of overall proportion.Trichoderma bacterial strain provided by the invention has the advantage of high Antagonism, strong specialization as from the foregoing, uses the biological pesticide of this bacterial strain production and can effectively prevent and treat soil-borne disease, guarantees the steady growth of farm crop, vegetable crop.
Description of drawings
By reading the detailed description of non-limiting example being done with reference to the following drawings, it is more obvious that other features, objects and advantages of the present invention will become:
Fig. 1 is antibiosis secondary metabolite partition ratio illustration dissimilar in the ZJSX5003 bacterial strain provided by the invention.
Embodiment
The present invention is described in detail below in conjunction with specific embodiment.Following examples will help those skilled in the art further to understand the present invention, but not limit in any form the present invention.Should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, can also make some distortion and improvement.These all belong to protection scope of the present invention.
The present invention is by separating the wild trichoderma strain of 886 strains of Shaoxing, Zhejiang Province city black salted turnip vegetable soil, choose exsomatize face-off Fusarium oxysporum, Fusarium graminearum, fusarium moniliforme as the primary dcreening operation index, then choose the pathogenic bacteria Conidia persistence as sieving index again, connect the bacterium efficiency test by live body at last and determine Trichoderma bacterial strain ZJSX5003.
Embodiment 1, the face-off bacteriostatic test exsomatizes
Dull and stereotyped face-off culture method: use the punch tool of the Φ 6mm of the bacterium of going out to play Trichoderma and pathogenic bacteria (Fusarium moniliforme, Fusarium graminearum, dry thread Pyrenomycetes) the bacterium dish of getting about cultivation 4d, pathogenic bacteria bacterium dish places respectively the dull and stereotyped side of the culture dish of diameter 90mm, opposite side inoculation Trichoderma bacterium dish, two bacterium dish are at a distance of 4cm, and take independent pathogenic fungi as contrast, 3 repetitions are in 28 ℃ of constant temperature culture.After cultivating 5d, adopt the right-angled intersection method to measure respectively the pathogenic fungi colony diameter for the treatment of group and control group, calculate respectively and cultivate inhibiting rate behind the 5d.
Colony growth diameter (mm)=colony diameter mean value-6.0mm;
Mycelial growth inhibition rate (%)=(contrast colony growth diameter-processing colony growth diameter)/contrast colony growth diameter * 100.
The mensuration of the stripped bacteriostasis rate of table 1 bacterial strain
As can be seen from Table 1, ZJSX5003 is better to the restraining effect of three pathogen strain bacterium (Fusarium oxysporum, Fusarium moniliforme, Fusarium graminearum), is respectively 79.79%, 73.68%, 72.25%.
Embodiment 2, the pathogenic bacteria Conidia persistence measures
By the abundant contact action of pathogenic bacteria (Fusarium oxysporum, Fusarium graminearum, dry thread Pyrenomycetes) and biocontrol microorganisms spore suspension, beat from the pathogenic bacteria edge of activation 5d and to get 4 diameter 5mm bacterium cakes, it is immersed Trichoderma spore suspension (10
6Individual/mL) in 30s, then be placed on incubated overnight in the culture dish that is covered with 3 layers of wet filter paper, replace the Trichoderma spore suspension as contrast take sterilized water, next day pathogenic bacteria bacterium cake is forwarded to and contains in the 1mg/L F-1991 selective medium, cultivate in 28 ℃ of constant incubators, repeat 3 times.The 5d log represents with pathogenic bacteria growth diameter and survival rate that finally its mean value is got in each processing.Thereby observe the preventive and therapeutic effect of the existence trend section of the declaring biocontrol microorganisms of pathogenic bacteria.
Table 2ZJSX5003 is on the impact of pathogenic bacteria Conidia persistence
As can be seen from Table 2, ZJSX5003 is 00.00% to three pathogen strain bacterium (Fusarium oxysporum, Fusarium moniliforme, Fusarium graminearum) Conidia persistence.
Embodiment 3, the control test of greenhouse live body
In the PDA substratum, activate respectively pathogenic bacteria and Trichoderma in 28 ℃ of lower 7d of cultivation, wash respectively pathogenic bacteria spore on the substratum with sterilized water, removing mycelia, pathogenic bacteria adds suitable distilled water diluting with filtrate, and to become spore concentration be 1 * 10 to bacterium liquid with sterilized filter paper filtering
6The suspension of individual/mL, Trichoderma adds suitable distilled water diluting with filtrate, and to become spore concentration be 1 * 10
7The suspension of individual/mL, for subsequent use.
(1) cucumber fusarium axysporum control experiment
With cucumber seeds vernalization in the culture dish of the wet filter paper of bilayer of surface sterilization, 6 in every ware repeats 3 times.The a few days is cultivated in the sterilized water moisturizing, after decorporating kind of skin, seed germination plants cucumber seedling at the indoor flowerpot of temperature, when treating that cucumber seedling grows 3～5 leaves, the spore suspension of the cucumber fusarium axysporum of sterilized water preparation is irrigated the root of cucumber seedling, every strain cucumber seedling is filled with root 15～20mL, observes incidence behind the moisturizing 24h; Again the Trichoderma spore suspension is irrigated the root of cucumber seedling, to fill with sterilized water as contrast, observe prevention effect behind the 7d.
Cucumber fusarium axysporum disease scale standard: 0 grade, without Visual symptoms; 1 grade, lesion area accounts for below 5% of whole leaf disc area; 2 grades, scab accounts for 5%～25% of whole leaf disc area; 3 grades, scab accounts for 25%～50% of whole leaf disc area; 4 grades, scab accounts for 50%～75% of whole leaf disc area; 5 grades, scab accounts for more than 75% of whole leaf disc area.
The prevention effect method of calculation of disease index and bacterial strain are as follows:
Disease index=∑ (each sick level strain number * typical value at different levels)/(investigating total strain number * typical value at the highest level) * 100
Preventive effect=(disease index before the rear disease index * check plot of control)/(disease index behind the front disease index * check plot of control) * 100.
(2) corn stalk rot disease control experiment
Pour into pathogenic bacteria spore suspension in the sterile soil that autoclaving is processed, airtight morbidity is until the appearance of red hair.Potted plant carrying out adopted in test, and flowerpot fills soil to 2/3 place that processed after with 70% alcohol disinfecting, and then every basin is broadcast the seed of crossing through alcohol disinfecting, covers the 2cm sterile soil, compacting again.The rear every basin of emerging is kept a full stand of seedings 2.Seedling management pouring is irrigated corn root with the Trichoderma spore suspension again to 3～4 leaves during the phase after the milpa morbidity, filling with sterilized water as contrast, and be injured affectionate person's condition and the plant height of 7d " Invest, Then Investigate " plant root, long, the radical (radicle ten secondary roots etc.) of root.
Corn seedling investigation severity Scaling standard: 0 grade, whole strain growth is normal, anosis; 1 grade, above and below ground section growth normal, the visible a small amount of scab of root, lesion area accounts for the root table total area below 1/4, has brown in the root mass white color; 2 grades, above and below ground growth obviously is obstructed, and the leaf look thin out, and plant height only reaches 3/4 of contrast, and lateral root is few and lack, need not root, scab in flakes, lesion area accounts for 1/4～1/2 of the root table total area, root mass white color, brown suitable; 3 grades, above and below ground section growth is abnormal, overground part visible blue or green withered-yellow withered shape, plant height only reaches 1/2 of contrast, lateral root is minimum, lesion area accounts for 1/2～3/4 of the root total area, the brown middle band of root mass color is white; 4 grades, germinate, but do not emerge, be almost stifled and dead, lesion area accounts for more than 3/4 of the root table total area, and root is brown.
Table 3 bacterial strain ZJSX5003 processes the prevention effect to soil-borne disease
ZJSX5003 has good promoter action to cucumber and corn growth, and plant height and well developed root system degree are all more remarkable.The preventive effect measurement result shows, is respectively 85.08%, 66.67% for the live body preventive effect of cucumber fusarium axysporum, corn stalk rot disease, significantly reduced rotten brown stain, the preventive effect highly significant.
Embodiment 4, the chitinase enzyme activity detects
Trichoderma strain is inoculated on the PDA culture medium flat plate (diameter 90mm), cultivates 5d in 25 ℃ of illumination fixed temperature and humidity incubators, 60% humidity.Soak 3～5min covering with the mould flat board of wood with aqua sterilisa, scrape gently with cotton swab and wash spore and mycelium, absorbent cotton or 4 layers of filtered through gauze are removed mycelium, and filtrate is spore suspension.Count according to the blood cell counting plate method, spore suspension concentration is 10 after the dilution
6Individual/mL spore amount.
Preparation 2-Acetamido-2-deoxy-D-glucose (NAG) (chromatographically pure, Sigma Chemical Co.P.O., Germany) standardized solution of 1mg/mL, get 7 25 * 250mm test tubes, press table 4 and add reagent, with each pipe mixing, in boiling water, accurately boil 5min, be cooled to immediately room temperature after the taking-up, add 21.5mL distilled water to each test tube again, shake all.The concentration of respectively managing NAG after the dilution is followed successively by 0,8,12,16,20,24,28(μ g/mL).First 7 pipe samples are carried out all-wave scanning by ultraviolet spectrophotometer in 200～700nm scope, obtain the wavelength of its obtained the maximum absorption.Again above-mentioned 6 arms are measured light absorption value from low to high by NAG concentration under the wavelength of maximum light absorption (OD) value, take NAG concentration (μ g/mL) as X-coordinate, the OD value is ordinate zou, draws out typical curve.
Table 4 is set up the reagent of NAG typical curve
| ? | 0(CK) | 1 | 2 | 3 | 4 | 5 |
| NAG reference liquid (mg/mL) | 0 | 0.1 | 0.2 | 0.3 | 0.4 | 0.5 |
| Be equivalent to NAG amount (mg) | 0 | 0.1 | 0.2 | 0.3 | 0.4 | 0.5 |
| Distilled water (mL) | 2.0 | 1.9 | 1.8 | 1.7 | 1.6 | 1.5 |
| DNS reagent (mL) | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 |
Every bottle is produced chitinase fermented liquid (nutrient media components: NH
4NO
33g/L, KH
2PO
43g/L, MgS0
47H
20 0.6g/L, FeS0
47H
20 0.1g/L, tobacco brown spot pathogen 5g/L powdery, pH 5～6,1000mL distilled water) inoculation 5mL spore suspension, place constant-temperature table with 180r/min, 28 ℃ of cultivations.Taking the DNS colorimetry to survey chitinase lives: get the 0.5mL fermented liquid, add 0.05M pH 6.0 phosphate buffered saline buffer 2mL, add again the 0.5mL tobacco brown spot pathogen, and three repetitions of every sample, a contrast, reaction is carried out in 25 * 250mm test tube; Then (37 ℃ of lower accurately insulation water-bath 1h more rapidly at 4 ℃ of centrifugal 10min of lower 11000g, get supernatant liquor 2mL, add DNS reagent 1.5mL, and boiling water bath accurately boils 10min, is cooled to immediately room temperature after the taking-up at thermostat water bath immediately.Contrast adds 0.05M pH6.0 phosphate buffered saline buffer 2mL for the 0.5mL fermented liquid, and mixed solution boils 10min in boiling water bath, is cooled to room temperature with cold water after taking out, and adds the 0.5mL tobacco brown spot pathogen again, the centrifugal 10min of 11000r/min then, and other operation is the same.
Above-mentioned reaction solution mixing, getting the 1mL reaction mixture measures respectively at 3d, 4d, 5d, 6d, 7d, 8d, 9d, 10d, measure photoabsorption (OD) value at the 530nm place by ultraviolet spectrophotometer, the OD value that records and the contrast of NAG typical curve, the quantitatively activity of chitinase.
The chitinase enzyme is lived: a chitinase enzyme unit alive (1U) is defined as under given conditions (37 ℃, pH6.0), and the reaction of per minute enzyme catalysis tobacco brown spot pathogen discharges the required enzyme amount of 1 μ mol 2-Acetamido-2-deoxy-D-glucose.
The chitinase enzyme of the different cultivated days of table 5 bacterial strain ZJSX5003 is lived
| Fate (d) | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Enzyme (U) alive | 0.5757 | 2.4799 | 4.0783 | 2.9416 | 2.2665 | 1.8462 | 1.1871 | 0.8305 |
As can be seen from Table 5, along with the increase of cultivated days, ZJSX5003 chitinase enzyme activity is the again downward trend that rises first; The chitinase enzyme activity is the highest when 5d, is 4.0783U.
Embodiment 5, the beta-1,3-glucanase enzyme activity detects
The making of glucose typical curve, with reference to the method for Yang Yanhong (Yang Yanhong. Pinus armandi Franch-P. Komavovii Lavl. blister rust former bacterium superparasitism screening and mechanism of action preliminary study [D] thereof. Kunming: Xi'nan College of Forestry, 2004), press table 6 and add reagent, get 1mg/mL standard glucose solution 0,0.1,0.2,0.3,0.4,0.5mL, respectively add again distilled water and complement to 2mL, then add respectively the abundant mixing of 0.75mL DNS solution, in boiling water bath behind the accurate response 15min, immediately taking-up is put into ice-water bath and is cooled to room temperature, adds the abundant mixing of 5mL distilled water again.Measure absorbance value in 722 type spectrophotometer 540nm places, take the concentration of standard glucose solution as X-coordinate, take the absorbance value of correspondence as ordinate zou drawing standard curve.
Table 6 is set up the reagent of glucose typical curve
| ? | 0(CK) | 1 | 2 | 3 | 4 | 5 |
| The glucose reference liquid | 0 | 0.1 | 0.2 | 0.3 | 0.4 | 0.5 |
| Be equivalent to glucose amount (mg) | 0 | 0.1 | 0.2 | 0.3 | 0.4 | 0.5 |
| Distilled water (mL) | 2.0 | 1.9 | 1.8 | 1.7 | 1.6 | 1.5 |
| DNS reagent (mL) | 0.75 | 0.75 | 0.75 | 0.75 | 0.75 | 0.75 |
Every bottle of TLE produces enzyme induction nutrient solution (composition: 1g bactopeptone, 0.3g urea, 2g KH
2PO
4, 1.4g (NH
4)
2SO
4, 0.3g MgSO
4.7H
2O, 0.3g glucose, 0.005g FeSO
4, 0.0017g MnSO
4, 0.0014g ZnSO
4, 0.002g CaCl
2, 1000ml distilled water) and inoculation 5mL spore suspension, place constant-temperature table with 180rmin
-128 ℃ of cultivations.Fermented liquid sample liquid 2mL filtered through gauze behind the cultivation 3d, centrifugal 20min under 4 ℃ of 5000r/min, supernatant liquor is crude enzyme liquid.Also changing a little the survey enzyme with reference to the method for Bara lives, draw crude enzyme liquid 0.5ml, place the laminarin solution 1mL that adds behind 40 ℃ of water-bath preheating 2min through the 0.1mg/mL of 40 ℃ of preheatings, 50mM sodium acetate buffer (pH 5.0) 0.5mL, mixing. behind 40 ℃ of lower accurate response 1h, add immediately 0.75mLDNS solution with termination reaction, mixing, accurate response 15min in boiling water bath again, immediately taking-up is put into ice-water bath and is cooled to room temperature, 25 ℃ of lower 2min that place add the abundant mixing of 5mL distilled water again.
Above-mentioned reaction solution mixing, get the 1mL reaction mixture and measure β-1 respectively at 3d, 4d, 5d, 6d, 7d, 8d, 9d, 10d, 3-dextranase enzyme is lived, measure photoabsorption (OD) value at the 540nm place by ultraviolet spectrophotometer, measured OD value and typical curve contrast, the quantitatively activity of beta-1,3-glucanase, obtain glucose content in the sample according to typical curve, each is processed three times and repeats.
The beta-1,3-glucanase enzyme is lived: beta-1,3-glucanase enzyme unit (1U) that lives is defined as, and under specific 40 ℃, pH5.0 condition, 1h catalytic decomposition laminarin produces the enzyme amount of l μ g glucose.
The beta-1,3-glucanase enzyme of the different cultivated days of table 7 bacterial strain is lived
| Fate (d) | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Enzyme (U) alive | 0.4517 | 0.8050 | 0.3312 | 0.1915 | 0.0847 | 0.0655 | 0.0490 | 0.0490 |
As can be seen from Table 7, along with the increase of cultivated days, ZJSX5003 beta-1,3-glucanase enzyme activity is the again downward trend that rises first; The beta-1,3-glucanase enzyme activity is the highest when 4d, is 0.8050U.
Embodiment 6, the extracellular protease enzyme activity detects
The making of bovine serum albumin (BSA) typical curve: press table 8 and add reagent, get 0.1mg/mL standard bovine serum solution 0,20,40,60,80,100 μ L respectively add distilled water again and complement to 20 μ L, then add respectively 0.02mL SK5031-1 solution A and the abundant mixing of 0.98mL SK5031-2 solution B, after leaving standstill accurate response 10min under the room temperature, add immediately the rapid mixing of 100 μ L Folin-phenol reagents, leave standstill 30min under the room temperature, measure absorbance value in ultraviolet spectrophotometer 750nm place, take the concentration of standard bovine serum albumin solution as X-coordinate, take the absorbance value of correspondence as ordinate zou drawing standard curve.
Table 8 is set up the reagent of BSA typical curve
| ? | 0（CK） | 1 | 2 | 3 | 4 | 5 |
| BSA standardized solution (μ L) | 0 | 20 | 40 | 60 | 80 | 100 |
| Distilled water (μ L) | 200 | 180 | 160 | 140 | 120 | 100 |
| BSA final concentration (mg/ μ L) | 0 | 0.01 | 0.02 | 0.03 | 0.04 | 0.05 |
Every bottle of MYG produces enzyme induction nutrient solution (composition: 10.0g glucose, 5.0g maltose, 5.0g yeast extract paste, 1000mL distilled water) inoculation 5mL spore suspension, places constant-temperature table with 180r/min, 28 ℃ of cultivations.Sampling fermented liquid every day, sample liquid 2mL through centrifugal (10000r/min, 4 ℃, 20min) after, supernatant liquor saves backup in-20 ℃.
Adopt method (the Lovrien RE of Lovrien, Gusek T, Hart B.Cellulase and proteasespecific activities of commercially available cellulase preparations.J ApplBiochem, 1985,7:258-272) carry out proteinase activity and measure.Get each sample protein enzyme solution 200 μ L, (this solution is that the routine of this area is selected to add respectively 0.02mL SK5031-1 solution A, coming from model is the Folin-Phenol Reagent forint phenol determination of protein concentration reagent of SK5031, available from Sangon Biotech (Shanghai) Co., Ltd., the SK5031-2 solution B, the Folin-phenol reagent is originated with it identical) and the abundant mixing of 0.98mL SK5031-2 solution B, after leaving standstill accurate response 10min under the room temperature, add immediately the rapid mixing of 100 μ L Folin phenol reagents, leave standstill 30min under the room temperature, get the 1mL reaction mixture and measure absorbance value in ultraviolet spectrophotometer 750nm place, respectively at 1d, 2d, 3d, 4d, 5d, 6d, 7d, 8d measures the extracellular protease enzyme and lives.Each processes three replications.
Extracellular protease enzyme activity: generate the enzyme amount of 1g bovine serum albumin as a unit of enzyme activity (Pu) take per minute catalytic decomposition protein.
The extracellular protease enzyme of the different cultivated days of table 9 bacterial strain is lived
| Fate (d) | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| Enzyme (U) alive | 4.7398 | 4.8503 | 1.7309 | 1.2574 | 0.9501 | 0.9758 | 1.2389 | 1.3216 |
As can be seen from Table 9, along with the increase of cultivated days, ZJSX5003 extracellular protease enzyme activity is the again downward trend that rises first; Proteinase activity power is the highest when 2d, is 4.8503U.
Embodiment 7, the extraction of antibiotics in the Trichoderma conidium
CH with 50 times of volumes
2C1
2In the conidium 2d of 4 ℃ of leach at low temperature 6g dryings, vat liquor is through 5% gac vibration absorption 2h, and with sterilized four layers of filtered through gauze removal of impurities, supernatant liquor adds isopyknic 3% sodium carbonate solution extraction, repeats once, leaves and takes CH after sodium carbonate solution is merged
2Cl
2Part is used a little CH
2C1
2After the washing and recycling and people's washings, washings adds anhydrous sodium sulfate dehydration, filters with quick filter paper again, and the washings that will dewater at last obtains the thick crude extract that contains antibiotics of 1mL behind 40 ℃ of lower vacuum-concentrcteds.
Embodiment 8, extract chemical ingredients GS-MS analyzes
Adopt gas chromatograph-mass spectrometer that extract component is analyzed, the instrument model is Agilent 6890-5973NGC-MS, and parameter is set to: ion source is EI, electron energy is 70eV, ion source temperature is 230 ℃, and interface temperature is 280 ℃, and the mass scanning scope is 290amu-500amu, 50 ℃ of initial column temperatures, be warmed up to 300 ℃ with 5 ℃/min behind the constant temperature 5min, temperature of vaporization chamber is 300 ℃, and carrier gas is helium, splitting ratio is 10:1, and sample size is 1 μ L.
Adopt gas chromatograph-mass spectrometer to carry out qualitative analysis, in conjunction with the computer search technology chemical ingredients is carried out isolation identification, use the relative content that the gas-chromatography areas of peak normalization method is measured each composition.
Table 10 bacterial strain antibiosis secondary metabolite analytical results
As shown in Table 10, Trichoderma bacterial strain ZJSX5003 antibiosis secondary metabolite material is more, detect altogether and analyze 78 cuts, antibiosis secondary metabolite classification of type gathers as shown in Figure 1, wherein terpenes proportion is maximum, next is that alkanes, carboxylic acid and derivative material thereof are more, and alcohols, aldehydes are minimum.All in all, the antibiosis secondary metabolite that plays main antagonistic action among the bacterial strain ZJSX5003 is that terpenes, polyketone class and carboxylic acid and derivatives class thereof account for 40.40% of total proportion altogether.
More than specific embodiments of the invention are described.It will be appreciated that the present invention is not limited to above-mentioned particular implementation, those skilled in the art can make various distortion or modification within the scope of the claims, and this does not affect flesh and blood of the present invention.
Claims (3)
1. the Trichoderma bacterial strain of an antagonism soil-borne disease is characterized in that, described Trichoderma bacterial strain is trichoderma asperellum (Trichoderma asperellum) ZJSX5003 CGMCC No.6480.
2. the Trichoderma bacterial strain of antagonism soil-borne disease as claimed in claim 1 is characterized in that, the ITS sequence of described bacterial strain is shown in SEQ ID NO.1.
3. the Trichoderma bacterial strain of antagonism soil-borne disease as claimed in claim 1 is characterized in that, the Tef1-α sequence of described bacterial strain is shown in SEQ ID NO.2.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103184162A (en) * | 2013-04-24 | 2013-07-03 | 牛赡光 | Trichoderma asperellum and applications thereof |
| CN103289902A (en) * | 2013-05-02 | 2013-09-11 | 华南农业大学 | Trichoderma asperellum and biological preparation and application in controlling and preventing cucumber phytophthora root rot |
| CN106754426A (en) * | 2017-01-17 | 2017-05-31 | 中国科学院烟台海岸带研究所 | A kind of trichoderma asperellum and its application |
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| CN102144642B (en) * | 2010-12-20 | 2013-02-20 | 惠州市南天生物科技有限公司 | Trichoderma pill as well as preparation method and application thereof |
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2012
- 2012-09-29 CN CN 201210378679 patent/CN102888350A/en active Pending
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| CN103184162A (en) * | 2013-04-24 | 2013-07-03 | 牛赡光 | Trichoderma asperellum and applications thereof |
| CN103184162B (en) * | 2013-04-24 | 2014-11-19 | 牛赡光 | Trichoderma asperellum and applications thereof |
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| CN106754426B (en) * | 2017-01-17 | 2020-05-08 | 中国科学院烟台海岸带研究所 | Trichoderma asperellum and application thereof |
| CN106754426A (en) * | 2017-01-17 | 2017-05-31 | 中国科学院烟台海岸带研究所 | A kind of trichoderma asperellum and its application |
| CN107058126A (en) * | 2017-03-28 | 2017-08-18 | 东北林业大学 | One plant of trichoderma asperellum and its application |
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| CN103484376B (en) | 2015-07-08 |
| CN103484376A (en) | 2014-01-01 |
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