CN113528352B - Trichoderma atroviride and application thereof in degrading chlorpyrifos - Google Patents

Abstract

The technical scheme of the invention discloses Trichoderma atroviride and application thereof in degrading chlorpyrifos, wherein the Trichoderma atroviride is Trichoderma atroviride (SG 3403), which is preserved in China general microbiological culture Collection center (CGMCC) No. 1 Hospital 3 of Shangyang district, beijing, in 8-28 days of 2012, and the preservation number is CGMCC No.6479. The trichoderma atroviride provided by the technical scheme of the invention can degrade chlorpyrifos in a water body, provides important trichoderma resources and application methods for biodegradation or bioremediation of chlorpyrifos in a water body around a farmland or a water body of a fishpond, and has important significance for developing a green aquaculture technology and protecting the water area environment of the farmland.

Description

Trichoderma dark green and its application in degrading chlorpyrifos

technical field

The invention belongs to the technical field of microbial fermentation, and in particular relates to Trichoderma dark green and its application in degrading chlorpyrifos.

Background technique

Pesticides are now widely used in modern agricultural production, among which organophosphorus pesticides are one of the most widely used pesticides in the world, and play an important role in the prevention and control of crop diseases and insect pests. However, organophosphorus pesticides are highly toxic and tend to remain in crops, field soil and water after long-term use in large quantities, causing food safety and environmental pollution problems.

Chlorpyrifos (CP) is a high-efficiency broad-spectrum organophosphate insecticide, which is widely used in the control of various crop pests. The biggest advantage of chlorpyrifos is that it has a high control effect on underground crop pests. At present, there is still no ideal pesticide that can replace chlorpyrifos in the world. On the other hand, this pesticide does not have the high toxicity of pesticides such as methamidophos and omethoate, and its production cost is relatively low, so it is still widely used in agricultural production. However, chlorpyrifos is a moderately toxic organophosphorus pesticide. Chlorpyrifos has high acute toxicity, affects the central nervous system, cardiovascular system and respiratory system, and can also cause skin and eye allergies. Unreasonable use for a long time will lead to its harmful effects in the environment. The accumulation of residues has caused great harm to the ecological environment.

The degradation of chemical pesticides mainly involves physical, chemical and microbial methods. Physical methods mainly include adsorption, light, ultrasonic and other methods, chemical methods mainly include oxidant oxidation and enzymatic degradation and other methods, and microbial methods mainly include degradation methods using bacteria and fungi. The use of microorganisms to degrade pesticide residues has the advantages of low cost, non-toxicity, and no secondary pollution, and is the most widely used bioremediation method.

Contents of the invention

The technical problem to be solved by the technical solution of the present invention is to provide a Trichoderma dark viride that degrades chlorpyrifos in water.

In order to solve the above technical problems, the technical scheme of the present invention provides a kind of Trichoderma atroviride, which is Trichoderma atroviride (Trichoderma atroviride) SG3403, which was preserved in Beichen, Chaoyang District, Beijing on August 28, 2012. The General Microbiology Center of China Committee for the Collection of Microbial Cultures, No. 3, No. 1 West Road, the preservation number is CGMCCNo.6479.

The trichoderma dark viride of the present invention can be applied to degrade chlorpyrifos.

Optionally, the chlorpyrifos tolerance concentration of the Trichoderma dark green is 40 mg/L-50 mg/L, and the spore-forming ability of the Trichoderma dark green is above 60%.

Optionally, when degrading the chlorpyrifos, the Trichoderma dark viridans produces paraoxonase, and the catalytic efficiency of the paraoxonase to the chlorpyrifos is 5.0×10 ⁶ kcat/Kms ^-1 M ^-1 - 6.05×10 ⁶ kcat/Kms ^-1 M ^-1 , and the enzyme activity against the chlorpyrifos is 2.85U/mL-3.60U/mL.

The present invention also provides a method for Trichoderma dark viridans to degrade chlorpyrifos, comprising: inoculating Trichoderma dark green SG3403 strain on potato dextrose agar medium, and activating it at 25°C-28°C; culturing on potato dextrose agar Propagating the spores on the base and washing the spores to prepare a spore suspension; adding the spore suspension to potato dextrose medium so that the concentration of the spores is 10 ⁵ CFU/mL to 10 ⁶ CFU/mL; The spores were shaken and cultured for 4 to 5 days, the rotation speed was 180rpm to 200rpm, the temperature was 25°C to 28°C, vacuum filtration, sterile water washing, and drying mycelia twice; ～0.5g was inserted into 100mL～110mL of deionized water, and then chlorpyrifos was added, the concentration of chlorpyrifos was 280μg/mL～320μg/mL, and cultured at 25℃～28℃ and 180rpm～200rpm for 120 hours～125 hours, Protect from light, collect 1.0mL-1.5mL of liquid every 24 hours to 30 hours for solid-phase microextraction-gas chromatography-mass spectrometry detection, and analyze the hydrolysis dynamics of chlorpyrifos.

Optionally, the method for preparing the potato dextrose agar medium comprises:

Weigh 190g-200g of potatoes, peel and cut into small pieces, add water and boil for 30min-35min, filter with four layers of gauze, add 15g-20g of anhydrous glucose and 15g-20g of agar powder, cool to 1L, in 120℃ Sterilize at ~121°C for 15-30 minutes.

Optionally, the spore suspension is inoculated in the potato dextrose medium with an inoculum amount of 3%-5%.

Optionally, the method for preparing the potato glucose medium includes: weighing 190g-200g of potatoes, peeling and cutting into small pieces, adding water to boil for 30min-35min, filtering with four layers of gauze, adding 15g-20g of anhydrous glucose, Cool down to 1L, and sterilize at 120°C to 121°C for 15min-30min.

Optionally, when the Trichoderma dark green is used to degrade chlorpyrifos, the mass concentration ratio of the Trichoderma dark green to the chlorpyrifos is (3.7-4.0):(0.3-0.35).

The technical scheme of the present invention obtains Trichoderma dark green SG3403 resistant to chlorpyrifos through screening, and the culture of Trichoderma dark green SG3403 has paraoxonase activity to degrade chlorpyrifos, and can be applied to farmland soil and natural water bodies polluted by the organophosphorus pesticide chlorpyrifos The Trichoderma dark green SG3403 has strong environmental adaptability, high activity of degrading chlorpyrifos, and can be used for the restoration of organophosphorus pesticide pollution in different ecological environments.

The method for degrading chlorpyrifos by the dark green Trichoderma in the technical scheme of the present invention can reach 36% in 48 hours to the degradation rate of chlorpyrifos in the fish pond water, which is the biodegradation or biodegradation of chlorpyrifos in the surrounding waters of the farmland or the water body of the fish pond. Restoration provides important Trichoderma resources and application methods, which is of great significance to the development of green aquaculture technology and the protection of farmland and water environment.

Description of drawings

Fig. 1 is the colony form and the corresponding conidiophore form of the Trichoderma dark green SG3403 of the embodiment of the present invention;

Fig. 2 is the ITS sequence of Trichoderma dark green SG3403 of the embodiment of the present invention;

Fig. 3 is the tef1 sequence of Trichoderma dark green SG3403 of the embodiment of the present invention;

Fig. 4 is the phylogenetic tree of the Trichoderma dark green SG3403 of the embodiment of the present invention;

Fig. 5 is the tolerance of Trichoderma dark green SG3403 degrading chlorpyrifos of the embodiment of the present invention;

Fig. 6 is the degradation product of Trichoderma dark green SG3403 to pure water chlorpyrifos of the embodiment of the present invention;

Fig. 7 is the degradation efficiency of Trichoderma dark green SG3403 to chlorpyrifos in fish pond water according to the embodiment of the present invention.

detailed description

In order to enable those skilled in the art to better understand the technical solutions in the application, the present invention will be further described below in conjunction with the following examples. Obviously, the described examples are only some of the examples of the application, not all of them. Example. Based on the embodiments in this application, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts shall fall within the scope of protection of this application.

The embodiments of the present invention provide a kind of Trichoderma atroviride, which is Trichoderma atroviride SG3403, which has been preserved on August 28, 2012 at No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing. General Microbiology Center of the Preservation Management Committee, the deposit number is CGMCC No.6479.

1, the Trichoderma dark green SG3403 grows rapidly on the potato dextrose agar medium, and after being cultured at 20° C. for 3 days, the colony radius on the MA medium is 5.5 cm to 8 cm (Figure A). After culturing at 25°C for 72 hours, the colony radius on the PDA medium was 42.8 mm to 60.5 mm (Figure B). No sterile pili. No diffusing pigment is produced, and there is a relatively dense disc-shaped structure in the center of the colony, which is the main spore-producing area of conidia, and there is a coconut smell on the PDA. The conidiophores are serrated with regular branches. Although paired branches are more common, the typical branching pattern of conidiophores is unilateral; the branching angle is approximately 90°. The length of bottle stem is 6.0μm～9.7μ., the aspect ratio is 1.8～3.5, straight or curved, sometimes hooked; 2～4 are arranged in spiral, usually solitary. Conidia green, subspherical to oval, smooth, (3.0-3.8)×(2.8-3.5 smooth). The ITS sequence and tef1 sequence of the Trichoderma dark green SG3403 are the main characteristic basis for identifying the strain, and its ITS sequence and tef1 sequence are shown in Figure 2 and Figure 3 respectively and T.hamatum GJS 05-334, Hypomyces perniciosus in the sequence list , T. asperellum CBS 433.97, T. atroviride CBS 142.95, T. atroviride CBS 119499, T. pubescens GJS 01-207, T. viride strain GJS 92-14. The strain phylogenetic tree constructed based on the ITS sequence is shown in Figure 4.

The dark green Trichoderma SG3403 provided by the embodiment of the present invention has obvious tolerance and surface adsorption ability to chlorpyrifos, and can produce paraoxonase that hydrolyzes the P-O bond of chlorpyrifos at the same time, and the dark green Trichoderma SG3403 is a kind of can absorb and degrade A functional strain of chlorpyrifos.

The tolerant concentration of the Trichoderma dark viride to the chlorpyrifos is 40 mg/L-50 mg/L, and the sporulation ability of the Trichoderma dark green is above 60%. When degrading the chlorpyrifos, the Trichoderma dark viridans produces paraoxonase, and the catalytic efficiency of the paraoxonase to the chlorpyrifos is 5.0×10 ⁶ kcat/Kms ^-1 M ^-1 -6.05×10 ⁶ kcat/Kms ^-1 M ^-1 , the enzymatic activity against the chlorpyrifos is 2.85U/mL-3.60U/mL.

Inoculate the Trichoderma dark green SG3403 strain on the potato dextrose agar medium (PDA), and activate it at 25°C-28°C; multiply the spores on the potato dextrose agar medium and wash the spores to make a spore suspension ; insert the spore suspension into potato dextrose medium (PD), so that the concentration of the spores is 10 ⁵ CFU/mL ~ 10 ⁶ CFU/mL; shake the spores for 4 days to 5 days, rotate The temperature is 180rpm-200rpm, the temperature is 25°C-28°C, and the mycelium culture can be obtained by vacuum filtration, rinsing with sterile water, and drying the mycelia twice.

The method for preparing the potato dextrose agar medium includes: weighing 190g-200g of potatoes, peeling them and cutting them into small pieces, adding water to boil for 30min-35min, filtering with four layers of gauze, adding 15g-20g of anhydrous glucose and 15g-20g of Agar powder, cooled to 1L, sterilized at 120℃～121℃ for 15min～30min.

The spore suspension is inoculated in the potato dextrose medium with an inoculum amount of 3%-5%. The preparation method of the potato dextrose medium includes: weighing 190g-200g of potatoes, peeling and cutting into small pieces, adding water to boil for 30min-35min, filtering with four layers of gauze, adding 15g-20g of anhydrous glucose, cooling to a constant volume to 1L, sterilize at 120°C to 121°C for 15min to 30min.

Embodiments of the present invention also provide a method for Trichoderma dark viridans to degrade chlorpyrifos, comprising:

Step 1: inoculate the Trichoderma dark green SG3403 strain on the potato dextrose agar medium, and activate it at 25°C-28°C;

Step 2: Propagate the spores on the potato dextrose agar medium and wash the spores to make a spore suspension;

Step 3: adding the spore suspension into potato dextrose medium so that the concentration of the spores is 10 ⁵ CFU/mL-10 ⁶ CFU/mL;

Step 4: Vibrating the spores for 4 to 5 days at a rotational speed of 180 rpm to 200 rpm and a temperature of 25°C to 28°C, vacuum filtration, washing with sterile water, and drying mycelia twice;

Step 5: Put 0.4g~0.5g of dried hyphae into 100mL~110mL of deionized water, then add chlorpyrifos, the concentration of chlorpyrifos is 280μg/mL~320μg/mL, at 25℃~28℃, 180rpm~200rpm Cultivate under certain conditions for 120-125 hours, avoid light, collect 1.0mL-1.5mL of liquid every 24-30 hours for solid-phase microextraction-gas chromatography-mass spectrometry, and analyze the hydrolysis dynamics of chlorpyrifos.

The preparation methods of the potato dextrose agar medium and the potato dextrose medium are as described above. When the Trichoderma dark green is used to degrade chlorpyrifos, the mass concentration ratio of the Trichoderma dark green to the chlorpyrifos is (3.7-4.0):(0.3-0.35).

The detection method of the degradation activity of described Trichoderma dark viridans comprises:

Prepare modified Burk medium: CaSO ₄ 2H ₂ O 100 mg, MgSO ₄ 7H ₂ O 200 mg, (NH ₄ ) ₂ SO ₄ 1 g, MnSO ₄ 10 mg, FeSO ₄ 7H ₂ O 5 mg, Na ₂ MoO ₄ 2H ₂ O 3.3mg, anhydrous glucose 10g, use 5mol/L KOH to adjust the pH value of the improved Burk medium to 6.0, use chlorpyrifos as the only phosphorus source Burk medium, pack in 200mL/250mL Erlenmeyer flasks, and store at 115 ℃ high pressure damp heat sterilization for 20min;

Transfer the mycelia preparation (2g wet mycelia) to 200mL of modified Burk medium for culture, and add chlorpyrifos so that the final concentration of chlorpyrifos in the modified Burk medium is 50mg/L, at 28°C, 180rpm The culture was carried out under the following conditions, and the residual chlorpyrifos content was detected by gas chromatography GC/FPD. The experimental results show that the degradation efficiency reaches over 40% within 48 hours.

In some embodiments, the chlorpyrifos contamination of fish pond water is treated with Trichoderma dark viridans. Trichoderma dark green SG3403 degrades chlorpyrifos in fish pond water containing 50mg/L-100mg/L chlorpyrifos, and the degradation rate reaches 36%-60% after 48 hours, which can effectively degrade the residue of chlorpyrifos in natural water.

Example 1

Test the tolerance of Trichoderma dark green SG3403 to chlorpyrifos, the method is as follows: activate Trichoderma dark green (T. On the PDA medium plate of 50mg/L chlorpyrifos, the colony diameter was measured every 12 hours, so as to detect the tolerance of Trichoderma dark virides SG3403 to chlorpyrifos. Fig. 5 is the growth situation of Trichoderma dark viridans SG3403 containing chlorpyrifos (50mg/L) and on the PDA medium (control) without chlorpyrifos, cultivate the 6th day on the bacterium colony diameter containing chlorpyrifos medium to be 7.50cm, and The colony diameter on the control medium without chlorpyrifos was 9.0 cm, and the growth inhibition rate of chlorpyrifos on Trichoderma aureus SG3403 colony was only 16.6%, so the strain met the requirements for drug resistance in application.

Example 2

Enzyme activity and product analysis of Trichoderma dark green SG3403 on chlorpyrifos degradation in pure water:

The Burk medium was divided into 200mL/250mL Erlenmeyer flasks, and sterilized at 115°C for 20 minutes. 2g mycelium (fresh weight) was transferred to 200mL Burk medium for cultivation, and chlorpyrifos was added to make the final concentration of chlorpyrifos 50mg/L, and cultured at 28°C and 180rpm. Gas chromatography GC/FPD was used to detect the products of residual chlorpyrifos degradation for 48 hours, mainly including 6-monochloro-2-pyridinol, 3,5,6-trichloro-2-pyridinol, pyrithion-oxygen, 5, 6-dichloro-2-pyridinol, etc., as shown in Figure 6. The degradative activity of the extracted Trichoderma dark green paraoxonase protein to chlorpyrifos in water was 5465.37U/mg.

Example 3

Degradation of Trichoderma dark green SG3403 on chlorpyrifos in fish pond water

1) The water in an aquaculture pond is a representative of natural water bodies. The collection site is Shunming Farm (120.7°E, 30.8°N) in Jiaxing City, Zhejiang Province, China. Three pond breeding areas were randomly selected to take two samples along the diagonal in the horizontal direction. points; in the vertical direction, the data were collected at two depths of 0.2m and 0.7m.

2) The culture conditions in the water environment refer to Burk medium, the culture container is 100mL brown vials, each bottle contains 50mL medium, add 50mg/L of chlorpyrifos and 1% (W/V) of Trichoderma dark green culture fungus, normal temperature (25° C.) and cultured at 2 days and 5 days respectively. GC-MS or SPME-GC-MS were used to identify the extracellular degradation products of chlorpyrifos in Trichoderma dark viridans. As shown in Figure 3, the degradation rate of Trichoderma dark viridans SG3403 to chlorpyrifos in fish pond water can reach 36% within 2 days, and it can degrade more than 70% on the 5th day. Compared with Trichoderma reesei (T. reesei), the degradation efficiency of Trichoderma dark viridans SG3403 was increased by 27% on the 2nd day and 18% on the 7th day ( FIG. 7 ). Although the control strain Trichoderma harzianum (T.harzianum) also had a comparatively degradative effect, this strain was inconvenient for further development because of its poor ability to produce spores.

Although the present invention has been disclosed as above with a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can use the methods and technical contents disclosed above to analyze the present invention without departing from the spirit and scope of the present invention. Therefore, any simple modification, equivalent change and modification made to the above implementation methods according to the technical essence of the present invention, which do not depart from the content of the technical solution of the present invention, all belong to the technical solution of the present invention. protected range.

sequence listing

<110> Shanghai Dajing Biological Engineering Co., Ltd. Shanghai Jiaotong University

<120> Trichoderma dark viridans and its application in degrading chlorpyrifos

<130> 1

<160> 7

<170> SIP Sequence Listing 1.0

<210> 1

<211> 604

<212>DNA

<213> Trichoderma atroviride

<400> 1

gagaagttcg agaaggtaag ctcatttcgc tgcttttttc attccttttg gacacaattg 60

tgccagacaa ttctgttctc agtcttgtca aatttttcc caccaagcat cgcaccccgc 120

tttgtctacc tacccctcct ttggcacagc aaaatttttc tggctgcctg ggttggtttt 180

tagtggggtg ccaacttttt ggcagtgacc ccgccatcgc cactgttcct catgcattac 240

ccaacatatt ctacagtcaa ctgcttggtt cattgtgcta atcatacttc aatcaatagg 300

aagccgccga actcggcaag ggttccttca agtatgcgtg ggttcttgac aagctcaagg 360

ccgagcgtga gcgtggtatc accatcgaca ttgccctgtg gaagttcgag actcccaagt 420

actatgtcac cgtcattggt atgttttcag tctgactgtt cactatccca acatcaccat 480

gctaacttgc gactacagac gctcccggtc accgtgattt catcaagaac atgatcactg 540

gtacctccca ggccgattgc gctatcctca ttatcgctgc cggtactggt gagttcgagg 600

ctgg 604

<210> 2

<211> 1063

<212>DNA

<213> Trichoderma atroviride

<400> 2

tcccaaaagc ctcacaacca accgccaaaa tggggtatgt caagctcttc cgacgctgct 60

cgccttgaga gtgcggggcat cttgtctagc ttcgatactg actgctttta tagtaaggac 120

gataagaagc acgtcaacgt cgtcgtcatc gtatgtaccc tttcccccct gattgacaat 180

gaaaacaaac agttttgctg aattccatc tctaggggta tgtgccaacg atctccccga 240

cgtcttcgat gataatactg acttgaacac aagccacgtc gactccggca agtccaccac 300

tgtgagtggc ctccctcttt ggcctccagt ttatctggtg gctctctacc ccgcctgtcc 360

tagtgacgcg gggtgttttg catatacaca aaactgacaa atttctctat agaccggtca 420

cttgatctac aagtgcggtg gtatcgacaa gcgtaccatt gagaagttcg agaaggtacg 480

gaattggctt ttcatttttc ttcattctac acggcttgga ctagactttg ggacgtttcg 540

tcaccatggc gctctctctt tctctctacc ccgctcgcat tggatctcga gaaaaaaatt 600

tctggcctgt gttttttttt ttggtggggt gctccattga gttaccccgc tgcacagtgg 660

cagtgcaatt ttttcttttc tttgtgatcg ctatctacct tcaccactca ctcatcttcg 720

ctcttcatac cccaattcta acgacgccct ataggaagcc gccgaactcg gcaagggttc 780

cttcaagtat gcttgggttc ttgacaagct caaggccgag cgtgagcgtg gtatcaccat 840

cgatattgcc ctctggaagt tcgagactcc caagtacgag gtcactgtca ttggtatgtt 900

tatctcacat ctcatctctt catctgatgc atctcaacta acatactata gacgctcccg 960

gtcaccgtga tttcatcaag aacatgatca ctggtacctc ccaggccgat tgcgctattc 1020

tcatcatcgc tgccggtact ggtgagttcg aggctggtat ctc 1063

<210> 3

<211> 553

<212>DNA

<213> Trichoderma atroviride

<400> 3

catcgagaag ttcgagaagg taagctcatt tcactgcttt tcccatcaat ttttggcaca 60

atcatatgcc cgacaattct gctctcagtt tttgtctttt ttttccagcg tcaccccgct 120

ttgccagtct acctacccct cctttggcac agcaaaaatt ttctggctgc cttgtttggc 180

ttttagtggg gtgtcaaatt ttttggcagc aaccccgcta tcgccactgc acctcttcca 240

tcacccacca catgctattt gctcaatcgc gtcgtctttt tttgttcatt atgctgatca 300

tgcttcaatc aataggaagc cgccgaactc ggcaagggtt ccttcaagta tgcgtgggtt 360

cttgacaagc tcaaggccga gcgtgagcgt ggtatcacca tcgacattgc cctctggaag 420

ttcgagactc ccaagtacta tgtcaccgtc attggtatgt tttggactct tctctctagc 480

tatcgacatt ccaagtccgc cattctaaca tgctcttccc acagacgctc ccggtcaccg 540

tgatttcatc aag 553

<210> 4

<211> 548

<212>DNA

<213> Trichoderma atroviride

<400> 4

catcgagaag ttcgagaagg taagctcatt tctgcttttt cactccgctc cctgagcaca 60

atcgtgtccg acaattctgt cctcagtctt gtcatttttt ttcctcgcag catcacaccc 120

cgctttacct gtctacccct cctttggcac agcaaaattt tctggctgcc ttgcttggct 180

tttagtgggg tgccaacttt tttttgtttg gctgcaaccc cgctatcgcc actgtcccgt 240

cccaacgaat tgtactcaat tgcatcgtct tctccatctc tgtgtggttc attgtgctaa 300

tcatgcttca atcaatagga agccgccgag ctcggcaagg gttctttcaa gtatgcgtgg 360

gttcttgaca agctcaaggc cgagcgtgag cgtggtatca ccatcgacat tgccctctgg 420

aagttcgaga ctcccaagta ctatgtcacc gtcattggta tgttttcgct tttcctcatt 480

gatacttgga gaccaagatt ctaacgtgcc gctctgtaga cgctcccggt caccgtgatt 540

tcatcaag 548

<210> 5

<211> 1291

<212>DNA

<213> Trichoderma atroviride

<400> 5

catttctgct ttttcactcc gctccctgag cacaatcgtg tccgacaatt ctgtcctcag 60

tcttgtcatt ttttttcctc gcagcatcac accccgcttt acctgtctac ccctcctttg 120

gcacagcaaa attttctggc tgccttgctt ggcttttagt ggggtgccaa cttttttttg 180

tttggctgca accccgctat cgccactgtc ccgtcccaac gaattgtact caattgcatc 240

gtcttctcca tctctgtgtg gttcattgtg ctaatcatgc ttcaatcaat aggaagccgc 300

cgagctcggc aagggttctt tcaagtatgc gtgggttctt gacaagctca aggccgagcg 360

tgagcgtggt atcaccatcg aattgccct ctggaagttc gagactccca agtactatgt 420

caccgtcatt ggtatgtttt cgcttttcct cattgatact tggagaccaa gattctaacg 480

tgccgctctg tagacgctcc cggtcaccgt gatttcatca agaacatgat cactggtact 540

tcccaggctg actgcgctat cctgattatc gctgccggta ctggtgagtt cgaggctggt 600

atctccaagg atggccagac ccgtgagcac gctctgctcg cttacaccct gggtgtcaag 660

cagctcatcg ttgccatcaa caagatggac actgccaact gggccgaggc tcgttacctt 720

gagatcatca aggagacctc caacttcatc aagaaggtcg gcttcaaccc caagaccgtt 780

gccttcgtcc ccatctccgg cttcaacggc gacaacatgt tggctgcctc caccaactgc 840

ccctggtaca agggctggga gaaggagacc aaggctggca agtccaccgg caagaccctt 900

ctcgaggcca ttgacgccat tgagcccccc aagcgtccca cagacaagcc cctccgtctt 960

ccccttcagg atgtttacaa gatcggtggt attggaacag tccctgtcgg ccgtatcgag 1020

actggtatcc tcaagcccgg tatggtcgtt accttcgctc cctccaacgt caccactgaa 1080

gtcaagtccg ttgagatgca ccacgagcag ctcgtcgagg gtgtccccgg tgacaacgtt 1140

ggattcaacg tcaagaacgt ctccgtcaag gatatccgcc gtggtaacgt tgccggtgac 1200

tccaagaacg acccccccat gggtgccgct tctttcaacg cccaggtcat cgtcatgaac 1260

caccctggcc aggtcggtgc cggatacgtc c 1291

<210> 6

<211>619

<212>DNA

<213> Trichoderma atroviride

<400> 6

gagaagttcg agaaggtaag ctcatttcat tgtttttctc attcattatg ggcacaattg 60

tgtccgacaa ttctgttctc agtcttgtca acacttttttttccaccaag cattgcaccc 120

cgctttgcct acctacccct cctttggcac agcaaaattt ttctggctgc cttgtttggt 180

ttttagtggg gtgtcaaatt tttggcagca accccgctat tgccactgtc cctcacacat 240

tgcccaacaa tattctactt caatcaattt ctatttggtt cattgtgcta atcatacttc 300

aatcaatagg aagccgccga actcggcaag ggttcccttca agtatgcgtg ggttcttgac 360

aagctcaagg ccgagcgtga gcgtggtatc accatcgaca ttgccctgtg gaagttcgag 420

actcccaagt actatgtcac cgtcattggt atgttttcag tccgactggt cggtattatc 480

ccaacatcat cattctaaca cgttacttta tagacgctcc cggtcaccgt gatttcatca 540

agaacatgat cactggtacc tcccaggccg attgcgctat cctcattatc gctgccggta 600

ctggtgagtt cgaggctgg 619

<210> 7

<211> 514

<212>DNA

<213> Trichoderma atroviride

<220>

<221> misc_feature

<222> (497)..(497)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (501)..(501)

<223> n is a, c, g, or t

<400> 7

tcgagaagtt cgagaaggta agctcatttc actgcttttt cactacgcgt tcctggtcca 60

atcgtgcccg acaattctgt tctcagtctt gtcaactttt ccctcgcagc atcacacccc 120

gctttgcctg cctctacccc tcattttgca cagcaaaaat tttctggcag tcttgtttgg 180

ctctgagtgg ggtgccaact tttgttggca gcgaccccgc tatcgccact gtccctcatc 240

catcgtccca aacacattgtg ctcattcaat cgcatcgtct tttgcctcaa ttcctttgtg 300

attcattgtg ctgatcatgt ttcaaccaat aggaagccgc cgaactcggc aagggttctt 360

tcaagtatgc gtgggttctt gacaagctca aggccgagcg tgagcgtggt atcaccatcg 420

acattgccct ctggaagttc gagactccca gatactatgt caccgtcatt ggtatgtttt 480

tggttcccta aatgacngaa ngccatcatc attc 514

Claims (8)

1. a method for degrading chlorpyrifos by Trichoderma dark viridans, is characterized in that, comprises: Inoculate Trichoderma dark viridans SG3403 strain on potato dextrose agar medium and activate at 25°C~28°C; Propagate spores on potato dextrose agar medium and wash said spores to make spore suspension; Introducing the spore suspension into potato dextrose medium so that the concentration of the spores is 10 ⁵ CFU/mL to 10 ⁶ CFU/mL; The spores were shaken and cultured for 4 to 5 days, the rotation speed was 180rpm to 200rpm, the temperature was 25°C to 28°C, vacuum filtration, sterile water washing, and drying of the hyphae were performed twice; Put 0.4g~0.5g of dried mycelium into 100mL~110mL of deionized water, then add chlorpyrifos, the concentration of chlorpyrifos is 280μg/mL~320μg/mL, under the conditions of 25℃~28℃, 180rpm~200rpm Cultivate for 120 hours to 125 hours, avoid light, collect 1.0mL~1.5mL of liquid every 24 hours to 30 hours for solid phase microextraction-gas chromatography-mass spectrometry detection, and analyze the hydrolysis dynamics of chlorpyrifos; The dark green Trichoderma is Trichoderma atroviride (Trichoderma atroviride) SG3403, which has been preserved on August 28, 2012 in the General Microbiology Center of China Microbiological Culture Collection Management Committee, No. 1, No. 1, Beichen West Road, Chaoyang District, Beijing , the deposit number is CGMCC No. 6479. 2. the method that trichoderma dark green as claimed in claim 1 is used for degrading chlorpyrifos, it is characterized in that, the method for preparing described potato dextrose agar medium comprises: the potato that takes 190g～200g is peeled and cut into fritters , add water and boil for 30min~35min, filter with four layers of gauze, add 15g~20g anhydrous glucose and 15g~20g agar powder, cool to 1L, and sterilize at 120℃~121℃ for 15min~30min. 3. the method that Trichoderma dark viridans is used for degrading chlorpyrifos as claimed in claim 1, is characterized in that, described spore suspension is inoculated in described potato dextrose medium with 3%～5% inoculum size. 4. the method that trichoderma dark green as claimed in claim 1 is used for degrading chlorpyrifos, it is characterized in that, the method for preparing described potato glucose culture medium comprises: take by weighing the potato peeling of 190g～200g and cut into fritters, Add water and boil for 30min~35min, filter with four layers of gauze, add 15g~20g anhydrous glucose, cool to 1L, and sterilize at 120℃~121℃ for 15min~30min. 5. the method that Trichoderma dark green as claimed in claim 1 is used for degrading chlorpyrifos, is characterized in that, when described Trichoderma dark green is used for degrading chlorpyrifos, the quality of described Trichoderma dark green mycelia and described chlorpyrifos The concentration ratio is (3.7 ~ 4.0): (0.3 ~ 0.35). 6. An application of Trichoderma atroviride in degrading chlorpyrifos, characterized in that, said Trichoderma atroviride is Trichoderma atroviride SG3403, which was preserved in Chaoyang District, Beijing on August 28, 2012 The General Microbiology Center of China Microbiological Culture Collection Management Committee, No. 3, No. 1 Yard, Beichen West Road, the preservation number is CGMCC No. 6479. 7. the application of Trichoderma dark green as claimed in claim 6 in degrading chlorpyrifos, it is characterized in that, described Trichoderma dark green is 40mg/L-50mg/L to the tolerance concentration of described chlorpyrifos, and described The sporulation ability of Trichoderma dark viridans is above 60%. 8. the application of Trichoderma dark green as claimed in claim 6 in degrading chlorpyrifos, it is characterized in that, when degrading described chlorpyrifos, described Trichoderma dark green produces paraoxonase, and described paraoxonase is to The catalytic efficiency of the chlorpyrifos is 5.0×10 ⁶ kcat/Kms ^-1 M ^-1 -6.05×10 ⁶ kcat/Kms ^-1 M ^-1 , and the enzyme activity to the chlorpyrifos is 2.85U/mL-3.60U/mL .

Images