CN102329740B - Carbendazim degrading trichoderma sp. strain and preparation method thereof - Google Patents
Carbendazim degrading trichoderma sp. strain and preparation method thereof Download PDFInfo
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- 241001557886 Trichoderma sp. Species 0.000 title claims abstract description 16
- 239000006013 carbendazim Substances 0.000 title claims abstract description 12
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
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- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 description 3
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Abstract
The invention discloses a carbendazim degrading trichoderma sp. strain and a preparation method thereof. A biocontrol strain provided by the invention is trichoderma sp. Tr1; the strain is preserved in the China General Microbiological Culture Collection Center; the preservation number is CGMCC No. 5210; the strain is obtained through separating, sieving and culturing from soil polluted by carbendazim by utilizing an inorganic salt culture medium containing the carbendazim; and a microbial preparation prepared by applying a zymophyte solution of the strain can be used for preventing and treating the fungal diseases of a plant and biologically repairing the soil which is polluted by the carbendazim simultaneously.
Description
Technical field
The invention belongs to biological high-tech field, relate to a kind of derosal chemical residual degradation wood mould, is to utilize method of microorganism degraded chemical pesticide residual, is applicable to production and the processing of modern agricultural production Green non-polluted farm product.
Background technology
Derosal (Carbendazim, MBC) be a kind of systemic fungicide of broad-spectrum high-efficiency and low-toxicity, chemical name is N-(2-benzimidazolyl-) Urethylane, various farm crop, gourd, fruit and vegetable disease are had to good prevention effect, also be other sterilant, the such as metabolic intermediate of the imidazoles such as F-1991, thiophanate_methyl sterilant.According to statistics, China 2006 agricultural chemicals total demand (effective content) is 29.96 ten thousand tons, and wherein the demand of derosal is at 0.8～1.0 ten thousand ton.Derosal character in soil and water is highly stable, and degradation half life is longer, in vegetables, fruit and soil residual with accumulation can affect HUMAN HEALTH by food chain, for example cause chromosome aberration, produce disease of the liver etc.Within 2002, classified as environmental hormone class chemical pesticide by China.Therefore, the Study on degradation of derosal in environment more and more receives people's concern.
Biological degradation is to remove the residual main path of derosal in environment, and the carbendazim degrading bacterium of having reported is at present bacterium class.Existing Trichoderma class does not almost have degradation function to derosal, even in the with serious pollution soil of derosal, cannot survive and breed.
Summary of the invention
The present invention seeks to for the deficiencies in the prior art, provide biocontrol trichoderma bacterial strain that a strain has derosal Degradation and
Preparation and application.
The biocontrol strain of a strain degradable derosal provided by the invention be wood mould (
trichoderma sp.) Tr1, this bacterial strain is in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms; Preserving number is CGMCC No.5210, and the preservation time is on September 1st, 2011, and preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
The trichoderma strain of degradable derosal of the present invention (
trichoderma sp.) Tr1 utilizes the minimal medium that contains derosal, separate from be subject to derosal contaminated soil, screening, cultivate and obtain.This bacterial strain not only has good degradation effect to derosal, and various pathogenic bacterias are also had to certain fungistatic effect.This invention is that the Trichoderma goods that obtain degraded derosal are laid a good foundation.
The biocontrol strain wood of degradable derosal of the present invention mould (
trichoderma sp.) the separation and Culture condition of Tr1, comprise the following steps:
Gather the vineyard agric (10-30cm) of Ji Wa village, Donggang District San Zhuan at sunshine town long-term application derosal, take soil sample 10g, be added in the minimal medium that 100ml carbendazim concentration is 1000 μ g/ml, 28 ℃ of 150r/min shaking tables are cultivated 7d, drawing 10ml nutrient solution is transferred in same medium, cultivate 7d, transfer 5 times continuously altogether.Get 1ml nutrient solution, dilution 10
-5, get 200 μ l nutrient solutions and evenly coat solid inorganic salt culture medium (wherein adding 100 μ g/ml Streptomycin sulphates for anti-bacteria), 28 ℃ are cultured to dull and stereotyped upper single bacterium colony that occurs, bacterial strain is transferred to PDA flat board (wherein adding 100 μ g/ml Streptomycin sulphates for anti-bacteria), 28 ℃ of constant temperature culture, repeatedly be purified to after pure culture, be transferred to and in minimal medium, continue shaking table and cultivate, utilize HPLC to detect the degradation capability of bacterial strain to derosal, screening is carried out preservation to the strongest bacterial strain of derosal degradation capability, the wood that is preserving number and is CGMCC No.5210 mould (
trichoderma sp.) Tr1 bacterial strain.
The bacterial strain that above-mentioned cultivation screening is obtained is transferred on PDA flat board, finds this bacterial strain bacterium colony initial stage aerial hyphae fine and close clump pencil that is white in color on PDA flat board, and edge is loose, be divergent shape growth, the back side initial stage is colourless, and mycelia Chan Bao district appears in the later stage, color from light green gradually to deep green.Conidiophore bears from mycelia side shoot, is the verticillate arrangement of cross, acroconidium, and conidium is smooth, sub-spherical to avette (Fig. 1).By a series of cultivation shapes and observation by light microscope, through be accredited as Trichoderma (
trichoderma sp.).
Above-mentioned cultivation, screening wood mould (
trichoderma sp.) culture medium prescription of Tr1 is as follows:
Minimal medium (g/L): NaCl 1.0g, K
2hPO
41.5g, KH
2pO
40.5g, MgSO
47H
2o 0.2g, NH
4nO
31.0g, distilled water 1000mL, derosal (100mg/mL) 10ml.
Solid inorganic salt culture medium (g/L): add agar 10g in above-mentioned minimal medium.
PDA substratum: potato 200g, glucose 20g, agar 15g, distilled water 1000ml.
The fermentation method for producing of wood of the present invention mould (Trichoderma sp.) Tr1 is as follows:
Adopt solid PDA substratum, mould this wood Tr1 is seeded on test-tube culture medium, cultivate after 48h at 27-30 ℃, cultured Trichoderma kind is transferred in liquid PDA substratum, 28 ℃ of shaking tables are cultivated 72h.By trichoderma strain good liquid culture according to 1%(v/w) inoculum size be transferred in wheat broth, 27-30 ℃ of enlarged culturing 3d.Clean cultured trichoderma strain, make 7 × 10
9the mould spore suspension bacteria liquid of wood of individual/ml.
The mould spore suspension bacteria liquid of above-mentioned obtained wood can be made commodity microbial preparation through conventional technical process.
Above-mentioned wheat broth is for soaking, boil prepared liquid wheat broth by wheat with clear water.
Concrete formulation examples is as follows:
Take 1000g wheat, after cleaning up, add 10L clear water soaked overnight, within second day, boil to till not hardening, feel that by feel wheat is not clamminess, 40 bottles of packing wide-mouth vials, 121 ℃, 20min autoclaving, twice simultaneously.
Apply wood of the present invention mould (
trichoderma sp.) microbial preparation that Tr1 zymocyte liquid makes, can be used for preventing and treating fungal diseases of plants, carry out biological restoration to being subject to derosal contaminated soil simultaneously.
Accompanying drawing explanation
Fig. 1 is wooden mould Tr1 conidiophore and conidium
Fig. 2 is the fungistatic effect of carbendazim degrading bacterium Tr1 to various pathogenic bacterias.1：
Rhizoctonia?solani；2：
Phytophthora?capsici；3：
Verticillium?dahliae?Kleb；4：
Phoma?uvicola?Berk；5：
Bipolaris?sorokiniana（Sacc.）hoemaker；6：
Fusarium?moniliforme?Sheld
Fig. 3 is that wooden mould Tr1 is to the biological restoration containing derosal soil.
Embodiment
Wood mould (
trichoderma sp.) cultivation of Tr1 and derosal degradation capability detect
Mould wood Tr1 is transferred to PDA(and adds Streptomycin sulphate, 100 μ g/ml) on flat board, after cultivation 5D, use aseptic water washing spore, make 10
9individual/ml spore suspension, is transferred in minimal medium according to 1% inoculum size, and 28 ℃ of shaking tables are cultivated 14D, degrades substratum as contrast not connect the derosal of bacterium.Cultivate completely, the ratio that is 1:4 according to volume ratio is adding acetone, after mixing, adopts high performance liquid chromatography (HPLC) to analyze.Moving phase is acetonitrile: water=18:82.Flow velocity is 1ml/min, sample size 10 μ l, and the operation wavelength of variable-wavelenght detector is 270nm, adopts external standard method to press peak area quantification, calculates according to the following formula derosal degradation rate:
In formula, X is degradation rate, and A is not for connecing carbendazim concentration in bacterium treatment solution, and B is for connecing carbendazim concentration in bacterium treatment solution.
In this bacterial strain 14D, the degradation rate of derosal has been reached to 55.47%, the 14 days 554.7mg that degraded, through condition optimizing, the inorganic nitrogen-sourced organic nitrogen source (yeast powder, 1.0g/L) that replaces to, culture temperature is 28 ℃, initial pH6.5, degradation rate has reached 63%, i.e. derosal about 630mg that degraded.
The mould Tr1 bacteriostatic activity of wood detects
Various pathogenic bacterias and wooden mould Tr1 cultivated after three days on PDA substratum, get the collar plate shape culture block with mycelia with sterilizing punch tool, collar plate shape is put apart from the dull and stereotyped central 1.5cm of PDA place with the culture block of pathogenic bacteria, the substratum bacterium piece of the mould Tr1 of wood is placed on apart from pathogenic bacteria bacterium piece 3.0 cm places, cultivates the width that records antibacterial band after 3 days at 28 ℃.Calculate the bacteriostasis rate of wooden mould Tr1.In experiment with the wooden removing mildew LTR-2 wettable powder sold on market in contrast.
Above-mentioned pathogenic bacteria is:
rhizoctonia solani,
phytophthora capsici,
verticillium dahliaekleb,
phoma uvicolaberk,
bipolaris sorokiniana(Sacc.) hoemaker,
fusarium moniliformesheld
The results are shown in Figure 2, as shown in Figure 2, wooden mould Tr1 and LTR-2 all have restraining effect to a certain degree to various pathogenic bacterias,
rhizoctonia solani,
phytophthora capsici,
verticillium dahliaeseveral pathogenic bacterias of Kleb, the fungistatic effect of LTR-2 is better than Tr1, and right
phoma uvicolaberk,
bipolaris sorokiniana(Sacc.) hoemaker,
fusarium moniliformesheld, the fungistatic effect of wooden mould Tr1 is slightly better than wooden mould LTR-2, illustrates in this invention and to separate the trichoderma strain Tr1 the obtaining derosal of not only can degrading, and various plant pathogenic fungis are also had to certain inhibition.
The biological restoration test of derosal degraded Trichoderma to soil
Get Bio-Centers base, Shandong Scientific Research Academy soil, air-dry, sieve, detect the content of derosal in soil.Do not find to screen in the soil obtaining to contain derosal, packing, 40g/ bottle, 121 ℃ of sterilizing 2h.Carry out soil treatment according to table 2, after stirring, get 1g and detect derosal content.Detect every sampling in 7 days later, detect altogether 4 weeks.Wherein derosal adopts 50% wettable powder, and every processing in triplicate.Adopt HPLC to detect the derosal content in soil, not add wooden mould Tr1 as contrast.
Note: "+", the grams adding is 5g; "-", does not add any material, and final grams is 50g/ bottle.
The mould Tr1 of wood the results are shown in Figure 3 to the soil organisms repairing test that pollutes derosal.
Can be seen by Fig. 3, after mould wood Tr1 and sterile soil, derosal are mixed, find the prolongation along with the time, the derosal content in soil obviously reduces, after 28 days, detect derosal content 29.74% through the mould Tr1 of wood soil after treatment, degradation rate has reached 43.96%.Illustrate that the mould Tr1 of the wood obtaining in the present invention has played main effect in soil remediation test.
Claims (3)
1. a strain has biocontrol trichoderma bacterial strain (Trichoderma sp.) Tr1 of derosal Degradation, is to use the minimal medium that contains derosal, and screening from be subject to derosal contaminated soil, cultivation obtain; It is characterized in that used minimal medium consists of: NaCl1.0g/l, K
2hPO
41.5g/l, KH
2pO
40.5g/l, MgSO
47H
2o0.2g/l, NH
4nO
31.0g/l, distilled water 1000ml, the derosal 10ml that concentration is 100mg/ml; Described screening, cultural method are: take soil sample 10g, be added in the minimal medium that 100ml carbendazim concentration is 1000 μ g/ml, 28 ℃ of 150r/min shaking tables are cultivated 7d, draw 10ml nutrient solution and are transferred in same medium, cultivate 7d, transfer 5 times continuously altogether.Get 1ml nutrient solution, dilution 10
-5getting 200 μ l nutrient solutions evenly coats in solid inorganic salt culture medium, 28 ℃ are cultured to dull and stereotyped upper single bacterium colony that occurs, bacterial strain is transferred on PDA flat board, 28 ℃ of constant temperature culture, are purified to after pure culture repeatedly, are transferred in minimal medium, to continue shaking table and cultivate, utilize HPLC to detect the degradation capability of bacterial strain to derosal, screening is to the strongest bacterial strain of derosal degradation capability; This bacterial strain is in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, and preserving number is CGMCC No.5210.
2. the production method of the zymocyte liquid of trichoderma strain as claimed in claim 1 (Trichoderma sp.) Tr1, is characterized in that concrete steps and processing condition are as follows:
Adopt solid PDA substratum, mould this wood (Trichoderma sp.) Tr1 is seeded on test-tube culture medium, cultivate after 48h at 27-30 ℃, cultured Trichoderma kind is transferred in liquid PDA substratum, 28 ℃ of shaking tables are cultivated 72h; The inoculum size that is 1% according to v/w by trichoderma strain good liquid culture is transferred in wheat broth, 27-30 ℃ of enlarged culturing 3d; Clean cultured trichoderma strain, make 7 × 10
9the mould spore suspension bacteria liquid of wood of individual/ml; Wherein, the preparation method of the wheat broth using is: take 1000g wheat, after cleaning up, add 10L clear water soaked overnight, within second day, boil to till not hardening, and packing, at 121 ℃, autoclaving 20min.
3. the application of trichoderma strain (Trichoderma sp.) the Tr1 zymocyte liquid of producing as claim 2, it is characterized in that applying described zymocyte liquid for the preparation of control fungal diseases of plants, carry out the microbial preparation of biological restoration to being subject to derosal contaminated soil simultaneously.
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