CN111235056B - Rigid fungus BI-1 capable of degrading tribenuron-methyl, and culture method and application thereof - Google Patents
Rigid fungus BI-1 capable of degrading tribenuron-methyl, and culture method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of microorganisms, and particularly relates to rigid-forming bacterium BI-1 capable of degrading tribenuron-methyl, and a culture method and application thereof. The strain is named as rigid strain Chengangzhanglangella sp.BI-1, deposit number: CCTCC No. M2019586, when the concentration of the tribenuron-methyl Long Chushi is below 50mg/L, the tribenuron-methyl can be completely degraded after 48 hours. The discovery of the strain enriches the germplasm resources of degraded tribenuron-methyl, can also be used for repairing tribenuron-methyl polluted soil by preparing a degrading microbial inoculum and combining with plants, improves the repairing efficiency and has wide prospect.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to rigid-forming bacterium BI-1 capable of degrading tribenuron-methyl, and a culture method and application thereof.
Background
Sulfonylurea herbicides are the second largest herbicides used in the world at present, and the weeding mechanism is to inhibit the activity of acetolactate synthase (ALS) in plants, further block the synthesis of branched-chain amino acids, stop cells at the mitotic stage, and stop weeds from growing and dying. The tribenuron-methyl is a systemic conduction sulfonylurea herbicide developed by Dupont in the United states in the 80 th century, is mainly used for preventing and killing various annual broadleaf weeds, has the characteristics of high efficiency, spectrum, high selectivity and the like, has a wide application range in Yangtze river, yellow river and Huaihe river basin of China, but causes a large amount of pollution to soil, water body, organisms, atmosphere and other environmental factors due to the prolonged service life, increased application amount, improper application operation and the like, and influences farmland rotation and production. Therefore, the method for solving the residual problem of the tribenuron-methyl has important theoretical significance and practical value for improving the phytotoxicity of the herbicide to crops and repairing the land polluted by the herbicide.
In the ecological system, microorganisms are taken as decomposers, have the characteristics of various metabolic types and strong adaptability, and can be converted into non-toxic or low-toxic substances by metabolizing organic pollutants in soil. The microbial degradation has the characteristics of safety, rapidness, low cost and no secondary pollution, and a great deal of researches show that the microbial degradation is an effective method for restoring the pesticide polluted soil and has important significance for agricultural product safety and environmental protection. The study of tribenuron-methyl at home and abroad mainly focuses on the aspects of field efficacy, residue analysis method study, influence on plant physiological characteristics and the like, and the study of isolation and identification of tribenuron-methyl degradation strains is less.
At present, few strains of degradable tribenuron-methyl are disclosed, and the following is briefly described: chinese patent document CN102154141A discloses a Pseudomonas NyZ42 strain (with a preservation number of CCTCC No: M2010222) for degrading tribenuron-methyl, wherein the strain can degrade 200mg/L of tribenuron-methyl within 5 days, and the degradation efficiency reaches 80%; chinese patent document CN102321539A discloses a composite microbial agent which is obtained by respectively taking bacillus subtilis ACCC10244 and bacillus chitin-eating bacillus ACCC10227 as bacterial sources for production and fermentation, and can be used for degrading various sulfonylurea herbicides including tribenuron-methyl; chinese patent document CN106834149A discloses a Brevibacterium methyl-phages and application thereof in degrading sulfonylurea herbicides, wherein the Brevibacterium methyl-phages is separated from sludge in a sewage treatment tank of Nanjing pharmaceutical factory, and the preservation number is Brevibacterium methyl-phages CGMCC No.11649. Will contain 3.2X10 of viable bacteria number 9 The inoculum size of each/mL of the Brevibacterium methanotrophic CGMCC No.11649 is 1/10 (v/v) of that of the Bacillus stearothermophilusInoculating to inorganic salt culture medium with 50mg/L chlorimuron-ethyl, metsulfuron-methyl, tribenuron-methyl, cinosulfuron-methyl and chlorsulfuron-methyl as the only carbon sources, culturing at 30deg.C under 180r/min, sampling every 12h, and detecting residual amount of sulfonylurea herbicide such as chlorimuron-ethyl. Experimental results show that the strain shows stronger degradation activity on chlorimuron-ethyl, metsulfuron-methyl and tribenuron-methyl after the initial 20h adaptation period. The degradation rate of 3 herbicides reaches 90% after 60h reaction.
Currently, there are few microbial strains that degrade tribenuron-methyl, and in situ repair techniques are still immature and are under further investigation.
Disclosure of Invention
The invention provides a rigid fungus strain Chengangzhanghangella sp.BI-1 capable of degrading tribenuron-methyl, which can completely degrade tribenuron-methyl in 48 hours when the concentration of benzenesulfone Long Chushi is less than 50mg/L, has a better degradation effect on low-concentration tribenuron-methyl, and can provide new germplasm resources for repairing tribenuron-methyl pollution. The degradable rigid fungus Chengangzhanglangella sp.BI-1 for tribenuron-methyl is characterized by having a preservation number of CCTCC No. M2019586.
The second aim of the invention is to provide a method for culturing the rigid-forming fungus BI-1 of the degradable tribenuron-methyl, which adopts the following technical scheme: the strain BI-1 was cultured using ME medium.
The fermentation culture method of the rigid fungus BI-1 adopts the following technical scheme: ME medium (g/L, 1.0 NH) 4 NO 3 ，0.5NaCl，1.5K 2 HPO 4 ，0.5KH 2 PO 4 ，0.2MgSO 4 ·7H 2 O,1% methanol) to the logarithmic growth phase, and inoculating to a fermentation medium containing yeast powder 0.3-0.5%, peptone 0.4-0.6% and calcium chloride 0.001-0.002% for fermentation culture.
Preferably, during fermentation culture, the culture temperature in the fermentation tank is 30-35 ℃, the inoculation ratio is 15%, the ventilation is 1:0.8, the stirring speed is 200r/min, and the pH is 7.2.
Preferably, during the fermentation process, the method further comprises the step of feeding the fermentation tank withThe feeding operation, the fermentation culture medium is fed once every 32 hours, the fermentation time is 80 hours, and the number of viable bacteria reaches 10 after the fermentation is finished 9 And (3) the concentration of the active component is more than one/mL.
The third object of the invention is to provide a microbial inoculum for degrading tribenuron-methyl in soil, which adopts the following technical scheme: the microbial inoculum comprises the rigid fungus Chengangzhanghangella sp.BI-1 of the degradable tribenuron-methyl. In actual use, the strain BI-1 may be mixed with a suitable carrier (e.g., fertilizer, liquid, pelleting adjuvants, etc.) to prepare the microbial inoculum. The preparation form of the microbial inoculum can be liquid, dry powder (method of drying fermentation culture solution of strain BI-1 and the like), granule (mixed with common auxiliary materials) and the like.
Preferably, the microbial inoculum is in the form of a liquid, and is prepared by the fermentation method according to any one of the above, wherein the viable count of the strain BI-1 is 10 9 And (3) the concentration of the active component is more than one/mL.
The fourth object of the invention is to provide a method for degrading tribenuron-methyl, which adopts the following technical scheme: the strain BI-1 or the microbial inoculum prepared by taking the rigid forming fungus BI-1 as a raw material is adopted to degrade the tribenuron-methyl in the sample. For example, the microbial inoculum is used for degrading tribenuron-methyl in samples such as water, soil and the like.
Preferably, cucumber seedlings are planted, and a degradation microbial inoculum is inoculated in a root irrigation mode, so that the survival capability of Gao Junzhu BI-1 in soil is improved, and the degradation efficiency of tribenuron-methyl in rhizosphere soil is further improved.
The invention also provides a method for improving the survival ability of the strain BI-1 of the degradable tribenuron-methyl, which comprises the following steps: the rigid-forming bacteria BI-1 can colonize the root system of the cucumber, and grow by utilizing root secretion, so that the biomass is improved.
The beneficial effects of the invention are as follows: 1. the tribenuron-methyl degrading bacteria disclosed by the invention are separated from farmland soil for long-term application of sulfonylurea herbicides, and can be used for efficiently degrading tribenuron-methyl. Under the laboratory condition, the strain BI-1 is added to the culture medium for reaction for 2 days, 50mg/L tribenuron-methyl can be completely degraded, and new germplasm resources can be provided for repairing the soil polluted by the tribenuron-methyl;
2. the preparation process of the tribenuron-methyl degrading bacterial agent disclosed by the invention is simple, wide in raw material source, low in cost and easy to popularize on a large scale.
3. The strain BI-1 is separated from farmland soil, can colonize the rhizosphere of cucumbers, improves the survival capability, has strong adaptability to complex field restoration environments, and has no obvious phytotoxicity to the cucumbers due to the fact that the strain degrades tribenuron-methyl, so that the problem of pollution of the tribenuron-methyl in agricultural activities is solved, green agricultural products without pesticide toxicity can be produced, and restoration and planting are synchronously carried out.
4. Compared with the traditional physicochemical repair method, the strain BI-1 and cucumber combined repair provided by the invention can improve the field utilization rate of the degradation microbial inoculum, has quick response, does not have secondary pollution, and is more scientific and environment-friendly.
The strain of the invention has a preservation date of 2019, 7, 26 and a preservation number: cctccc No. m 2019586. The classification is named as rigid fungus Chengangzhanglangella sp.BI-1, the preservation unit is named as China center for type culture Collection, and the address is the university of Wuhan in Wuhan, china, post code 430072.
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In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.
FIG. 1 is a colony morphology of the rigid fungus Chengangzhangrangella sp.BI-1 of the present invention on ME medium;
FIG. 2 is a liquid chromatogram showing the degradation effect of the rigid fungus Chengangzhangrangella sp.BI-1 on tribenuron-methyl of the present invention, FIG. 2A is a liquid chromatogram after the reaction of the non-inoculated strain BI-1 containing tribenuron-methyl alone, and FIG. 2B is a liquid chromatogram of the rigid fungus Chengangzhangrangella sp.BI-1 containing tribenuron-methyl in the culture medium and inoculated with the rigid fungus Chengangzhangrangella sp.BI-1 of the present invention and cultured for 2 d.
FIG. 3A is a bar graph of the effect of temperature on the degradation of tribenuron-methyl by the rigid bacterium Chengangzhangrangella sp.BI-1 of the present invention, FIG. 3B is a bar graph of the effect of pH on the degradation of tribenuron-methyl by the rigid bacterium Chengangzhangrangella sp.BI-1 of the present invention, and FIG. 3C is a bar graph of the effect of concentration of benzenesulfon Long Chushi on the degradation of tribenuron-methyl by the rigid bacterium Chengangzhangrangella sp.BI-1 of the present invention;
in fig. 4: the left panel shows a cucumber root system of 5d cultured without inoculating the rigid fungus Chengangzhangrangella sp.BI-1 of the present invention, and the right panel shows a cucumber root system of 5d cultured with inoculating the rigid fungus Chengangzhangrangella sp.BI-1 of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 screening and identification of the rigid Achromobacter sp.BI-1
Taking 5g farmland soil sample (Jiang Ningou, 31.95 DEG N118.85 DEG E) with sulfonylurea herbicide for a long time, adding into 100mL basic salt culture medium containing 50mg/L tribenuron-methyl, shaking and culturing at 30 ℃ and 180rpm for 5d, transferring once every 5d, repeating 3 times, determining degradation effect of enrichment liquid on tribenuron-methyl, gradient diluting enrichment liquid with degradation effect, coating on 50mg/L solid culture medium plate of tribenuron-methyl, culturing at 30 ℃ until single colony is generated, picking single colony liquid and culturing to logarithmic phase, inoculating into 20mL basic salt culture medium containing 50mg/L tribenuron-methyl (g/L, 1.0 NH) 4 NO 3 ，0.5NaCl，1.5K 2 HPO 4 ，0.5KH 2 PO 4 ，0.2MgSO 4 ·7H 2 O), culturing for 4d, and detecting the degradation effect of single colony.
The strain is degraded by adopting High Performance Liquid Chromatography (HPLC): adding 20mL of dichloromethane into the culture medium after the reaction, carrying out vortex shaking for 1min, standing and layering to remove an upper water phase, adding anhydrous sodium sulfate into an organic phase, taking 2mL of organic phase liquid, naturally airing, re-suspending with 0.2mL of methanol, filtering with a 0.22 mu m nylon filter membrane, and detecting the content of tribenuron-methyl by HPLC (Shimadzu RID-10A). High performance liquid chromatography conditions: chromatographic columns, C18 reverse phase columns (250 mm. Times.4.6 mm. Times.5 μm, agilent Technologies, palo Alto, calif., USA); mobile phase, acetonitrile: water: acetic acid (60:40:0.5); column temperature, 40 ℃; a flow rate of 1.0mL/min; detection wavelength, 230nm; sample injection amount, 20. Mu.L.
Through the separation route, a strain of bacteria named as BI-1 is obtained by screening, and the strain is verified to have excellent degradation capability on tribenuron-methyl.
Example 2 identification of the rigid Acidovorax chenggangzhanghangella sp.BI-1
And (3) picking single colony with degradation capability, inoculating the single colony into an ME liquid culture medium, culturing the single colony to a logarithmic phase, and identifying the strain by using a conventional physiological and biochemical measurement method, wherein experimental steps are described in the specification. And simultaneously streaking the strain onto an ME solid medium, and observing morphological characteristics of colony morphology.
Morphological characteristics: after 3d of cultivation, the colony diameter is about 0.5-1.0mm, and the colony is white, round and convex (see figure 1 of the specification for details)
The physiological and biochemical identification results are as follows: the physiological and biochemical identification result shows that the strain BI-1 is a gram-negative bacterium, the strain BI-1 is strictly aerobic, the optimal growth temperature is 30 ℃, the optimal pH is 7.0, the optimal salt concentration is 2.0%, oxidase, catalase and urease are positive, nitrate is reduced, and indole is generated to be negative. Sucrose, maltose, L-aspartic acid, L-glutamic acid, D-gluconic acid, methyl pyruvate, DL-malic acid, acetic acid and formic acid can be grown by the strain but not inositol, dextrin, trehalose, cellobiose, gentiobiose.
The strain BI-1 genomic DNA was extracted and used as a template to PCR amplify the 16S rRNA gene sequence of the strain with universal primers 27f (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492r (5'-TACCTTGTTACGACTT-3'). The PCR reaction was as follows (30. Mu.L): 2X EasyTaq PCR SuperMix. Mu.L, 27f (25 pmol/. Mu.L) 1. Mu.L, 1492r (25 pmol/. Mu.L) 1. Mu.L, DNA 1. Mu.L, ddH 2 O12. Mu.L. Reaction stripPiece (2): 95 ℃ for 5min;94 ℃ for 30s;55 ℃,30s,72 ℃ for 1.5min;72 ℃,10min,10 ℃ and 5min, wherein the second step to the fourth step are circulated for 30 times. And (3) purifying and recovering the amplified fragment by using an OMEGA recovery kit, and sequencing after T/A cloning.
The 16S rRNA gene sequence (1451 bp) of strain BI-1 is as follows:
GAGTTTGATCCTGGCTCAGAACGAACGCTGGCGGCAGGCTTAACACATGCAAGTCGAGCGCATCCTTCGGGGTGAGCGGCAGACGGGTGAGTAACGCGTGGGGATGTGCCTGGTGGTACGGAACAACTCATGGAAACGTGAGCTAATACCGTATAAGCCCTTTTGGGGAAAGATTTATCGCCACCAGATCAACCCGCGTTGGATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATCCATAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGAGTGATGAAGGCCTTAGGGTTGTAAAGCTCTTTCACTGGGGAAGATAATGACGGTACCCAGAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACTAAGGGGGCTAGCGTTGTTCGGAATCACTGGGCGTAAAGCGCACGTAGGCGGAGTCTTAAGTCAGAGGTGAAATCCCAAGGCTCAACCTTGGAACTGCCTTTGATACTGGGTGTCTTGAGGTCGAGAGAGGTGAGTGGAACTGCGAGTGTAGAGGTGAAATTCGTAGATATTCGCAAGAACACCAGTGGCGAAGGCGGCTCACTGGCTCGATTCTGACGCTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTACCTGATACCCTGGTAGTCCACGCCGTAAACGATGGAAGCTAGCCGTTGGTCAGCATGCTGATCAGTGGCGCAGCTAACGCTTTAAGCTTCCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCAGCCTTTGACATCCTGTGCCACTCAGAGAGATTTGAGGTTCCCTTCGGGGACGCAGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCCTAGTTGCCAGCATTCAGTTGGGCACTCTAGGGGGACTGCCGGTGATAAGCCGAGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTACGGGCTGGGCTACACACGTGCTACAATGGCGGTGACAGTGGGCAGCGAAGGGGTGACCCGGAGCTAATCTCCAGAAGCCGTCTCAGTTCGGATTGCACTCTGCAACTCGAGTGCATGAAGTTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGGTTTTACCCGAAGGCGTTGCGCTAACCCGCAAGGGAGGCAGGCGACCACGGTAGGGTCAGCGACTGGGGTGAAGTCGTAACAAGGTAACCGTA
on-line analysis of the sequencing results (www.ezbiocloud.net), ezBioCloud database comparison showed that strain BI-1 was closest to Chenggangzhanghangella, which was associated with Chenggangzhangella methanolivorans CHL1 T 16S rRN of (2)The sequence similarity of the A gene reaches 99.79%, and the sequence similarity of the A gene and other strains is not more than 98%. Construction of a phylogenetic tree of the strain BI-1 by the Neighbor-Joining method, the strain BI-1 being located inside the phylogenetic tree of the genus Chengangzhanghangella and being associated with Chenggangzhangella methanolivorans CHL1 T Constitutes a sub-branch, and the strain BI-1 is identified as being different from CHL1 in the genus Chengangzhanghangella in combination with the physiological and biochemical characteristics of BI-1 T A strain of bacteria, accession number: cctccc No. m 2019586.
Example 3 culture condition screening of the Strain Chengangzhanglangella sp.BI-1
The effect of temperature, pH and benzenesulfonate Long Chushi concentration on strain BI-1 degradation was studied:
inoculating strain BI-1 single colony into ME liquid culture medium, culturing to logarithmic phase, and regulating to 10 8 CFU/mL is ready for use.
Inoculating BI-1 bacterial suspension to 50mg/L of tribenuron-methyl basic salt liquid culture medium at a ratio of 5% (v/v), shake culturing at 15 deg.C, 20 deg.C, 25 deg.C, 30 deg.C, 37 deg.C and 42 deg.C in a shaker at 160rpm for 2d, and calculating the concentration of tribenuron-methyl by HPLC; inoculating BI-1 bacterial suspension to 50mg/L of tribenuron-methyl basic salt liquid culture medium at a ratio of 5% (v/v), regulating pH to 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 30 ℃, shake culturing at 160rpm for 2d, and calculating the concentration of tribenuron-methyl by HPLC; the BI-1 bacterial suspension was inoculated at a ratio of 5% (v/v) to 10, 25, 50, 100, 200mg/L of tribenuron-methyl in a basic salt liquid medium, shake-cultured at 30℃and 160rpm for 2d, and a control group without inoculating the BI-1 bacterial suspension was set up, and the concentration of tribenuron-methyl was calculated by HPLC (HPLC detection method is the same as in example 1).
FIG. 2A is an HPLC detection chart of a basal salt liquid medium added with only 50mg/L tribenuron-methyl, and FIG. 2B is an HPLC detection chart of a basal salt liquid medium added with 50mg/L tribenuron-methyl and inoculated with BI-1 bacterial suspension and shake cultured for 2 d; as can be seen from FIG. 2, 50mg/L tribenuron-methyl can be completely degraded by adding the strain BI-1 to the culture medium for 2d, the discovery of the strain BI-1 of the invention enriches the germplasm resources of the tribenuron-methyl degrading strain, and the degradation efficiency of the rigid forming bacterium Chengangzhanglang hangella sp.BI-1 of the invention on the low-concentration tribenuron-methyl is higher.
As shown in FIG. 3A, strain BI-1 has an optimal degradation temperature of 30 ℃; as shown in FIG. 3B, the pH-optimum of strain BI-1 is 7.0; as shown in FIG. 3C, the concentration of benzenesulfonyl Long Chushi is below 50mg/L, and the reaction 2d can be completely degraded; the concentration reaches 100mg/L, and the degradation rate is 70.3%; when the concentration is increased to 200mg/L, the strain degradation effect is large, and the degradation rate is only 25.2%.
Example 4 fermentation of the Strain Chengangzhanglangella sp.BI-1 and preparation of the microbial inoculum
After activating strain BI-1 on ME solid medium, inoculating into 400mL ME liquid medium, and culturing to logarithmic phase to obtain seed solution for inoculating fermentation tank. 500L of fermentation tank, 420L of feeding amount and fermentation medium formula (%): yeast powder 0.3, peptone 0.5, calcium chloride 0.001. And (3) after the material feeding is finished, sterilizing at 121 ℃ under high pressure and moist heat, cooling to 30-40 ℃, and inoculating the logarithmic phase strain into a fermentation tank according to the proportion of 15% (v/v). The temperature is controlled at 30 ℃, and the ventilation of sterile air in the culture process is 1:0.8, the stirring speed is 200r/min, the pH is 7.2, the material is fed once every 32 hours, the culture time of the whole process flow is 80 hours, and the number of viable bacteria reaches 10 after the culture is finished 9 More than 25kg barreled liquid dosage form.
Example 5 use of the Strain Chengangzhanglangella sp.BI-1 for degrading tribenuron-methyl in soil
Placing cucumber seeds in a culture dish filled with sterile water, soaking overnight, transferring to 8% NaClO, shaking and sterilizing for 3min, repeatedly cleaning with sterile water for 5 times, placing on wet sterile gauze, germinating in the dark at 25 ℃ for 2d, selecting seeds with good germination, inoculating in sterile Hoagland semisolid culture solution, placing in a climatic incubator, and culturing for 3d with 25 ℃,16h illumination and 22 ℃ for a period of 8h darkness.
Collecting farmland soil (tribenuron-methyl is not detected), loading the farmland soil into pot bodies, adding 450g of tribenuron-methyl into each pot body to 3mg/kg, stirring uniformly, spraying water to enable the soil to keep a certain water content, planting 3 cucumber seedlings in each pot body, filling roots and inoculating the prepared microbial inoculum, injecting 10mL of microbial inoculum into the root of each seedling, adding only microbial inoculum and only tribenuron-methyl, adding tribenuron-methyl and microbial inoculum but not planting cucumbers, and taking untreated samples as blank control, wherein each treatment is set to be 3 parallels.
The pots arranged according to the above were placed in a climatic incubator (GXZ-type intelligent illumination incubator, ningbo Jiangnan Instrument Co., GXZ-500D) respectively, and cultured for 20D with 25℃for 16h illumination and 22℃for 8h darkness as a period. For cucumber planting treatment, rhizosphere soil is taken every 5d, the content of tribenuron-methyl and the number of bacterial strains BI-1 are measured, a small-section cucumber root system sample is taken, the colonization condition of the bacterial strains BI-1 on root systems is observed by using a laser confocal microscope (CLSM, leicaTCSSP 3), and the bacterial strains BI-1 are excited by green fluorescent protein genes gfp to emit green fluorescence under the excitation of blue light. For the treatment of the non-planted cucumber, a soil sample is randomly collected, the tribenuron-methyl content is measured, and the comparison is made with the tribenuron-methyl content in the rhizosphere soil sample of the planted cucumber. And 20d, measuring the real leaf area, root length, fresh weight of stems and leaves and fresh weight of root systems of the cucumber.
Determination of the concentration of tribenuron-methyl in the soil: 5g of the collected soil sample was weighed, placed in a 50mL centrifuge tube, 20mL of extract (PBS: acetonitrile=6:4) was added, shaken at 100rpm for 2h,12000g was centrifuged for 30min, and the supernatant was collected. The resulting precipitate was then resuspended in 20mL of extract, the above procedure repeated 3 times, the resulting supernatants combined and the extract purified by Cleanert HXN solid phase extraction column, the specific procedure being as described in the specification. N (N) 2 Blow-dried, resuspended in 1mL of methanol, filtered through a 0.22 μm nylon membrane, and the tribenuron-methyl content calculated by HPLC and the liquid chromatography conditions were as described above (same as in example 1).
Experimental results: (1) The effect of the strain Chengangzhangella sp.BI-1 on the removal of cucumber phytotoxicity is shown in Table 1 below:
TABLE 1 removal effect of strain BI-1 on cucumber phytotoxicity (20 days)
Note that: control: cucumber is planted, and strain BI-1 and tribenuron-methyl are not added; BI-1: cucumber is planted, and only strain BI-1 is added; tribenuron-methyl: planting cucumber, and adding only tribenuron-methyl; tribenuron-methyl + BI-1: cucumber is planted, and tribenuron-methyl and strain BI-1 are added. The different letters represent a significant difference at the P <0.05 level (duncan test)
As shown in Table 1, various indexes of the cucumber, such as true leaf area, root length and the like, are obviously reduced due to the phytotoxicity of the tribenuron-methyl, when the BI-1 bacterial suspension is inoculated simultaneously, various indexes of the cucumber are restored to the control level, and the independent inoculation of the BI-1 bacterial suspension has no influence on the growth of the cucumber, so that the strain BI-1 generates a product which has no phytotoxicity on the cucumber by metabolizing the tribenuron-methyl, and the harm to the environment is reduced.
(2) Degradation of tribenuron-methyl by the strain Chengangzhanglangella sp.BI-1 is shown in Table 2 below:
TABLE 2 degradation of tribenuron-methyl by strain BI-1 under different treatments (degradation rate,%)
Note that: tribenuron-methyl + BI-1+ cucumber: planting cucumber, and adding tribenuron-methyl and a strain BI-1; tribenuron-methyl + BI-1: cucumber is not planted, tribenuron-methyl and strain BI-1 are added
As can be seen from table 2 above: the strain BI-1 is used for independently repairing 3mg/kg of tribenuron-methyl polluted soil, the degradation rate of the tribenuron-methyl is 54.7% and 88.7% respectively when the strain BI-1 is cultured for 5d and 10d, and the presence of the tribenuron-methyl cannot be detected by 15 d; and cucumber seedlings are planted, and the BI-1 bacterial suspension is inoculated by root irrigation, so that the degradation rate is improved to 77.9% when the cultivation is carried out for 5 days, the existence of tribenuron-methyl cannot be detected in 10 days, the cucumber can play a role in promoting the degradation of the strain Chengangzhanglang lang lla sp.BI-1 to the tribenuron-methyl, and the degradation efficiency of the tribenuron-methyl is improved.
(3) The observation result of the laser confocal microscope shows that: the strain Chengangzhanglangella sp.BI-1 can be used for culturing cucumber root system colonisation (see figure 4 of the specification, the left graph in figure 4 shows cucumber root system diagram of 5d under the condition of not inoculating strain BI-1, the right graph shows cucumber root system diagram of 5d under the condition of inoculating strain BI-1), through interaction with cucumber root system, utilizing root secretion to raise its survival ability, and the number of 20d strains still has 2.21×10 6 CFU/g, which is far higher than the number of strains treated without cucumber seedlings (7.19X10) 4 CFU/g), thereby improving degradation efficiency.
The tribenuron-methyl high-efficiency degradation strain BI-1 obtained by separation is a strain in Chengangzhangchanella, so that the germplasm resources of tribenuron-methyl degradation are enriched, a culture method of the tribenuron-methyl high-efficiency degradation strain is provided, meanwhile, the tribenuron-methyl contaminated soil is repaired by combining the strain BI-1 with cucumbers, 3mg/kg of tribenuron-methyl in the rhizosphere soil of the cucumbers can be completely degraded after 10 days of inoculation, and degradation products are harmless to the cucumbers, so that the repair efficiency is improved, and the method has the advantages of high efficiency, low price, convenience and no secondary pollution. The invention has important significance for restoring the pollution environment of tribenuron-methyl and protecting the ecological environment.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Sequence listing
<110> Nanyang teaching and learning school
<120> a rigid fungus BI-1 of degradable tribenuron-methyl, and culture method and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1451
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
gagtttgatc ctggctcaga acgaacgctg gcggcaggct taacacatgc aagtcgagcg 60
catccttcgg ggtgagcggc agacgggtga gtaacgcgtg gggatgtgcc tggtggtacg 120
gaacaactca tggaaacgtg agctaatacc gtataagccc ttttggggaa agatttatcg 180
ccaccagatc aacccgcgtt ggattagcta gttggtgagg taacggctca ccaaggcgac 240
gatccatagc tggtctgaga ggatgatcag ccacactggg actgagacac ggcccagact 300
cctacgggag gcagcagtgg ggaatattgg acaatgggcg caagcctgat ccagccatgc 360
cgcgtgagtg atgaaggcct tagggttgta aagctctttc actggggaag ataatgacgg 420
tacccagaga agaagccccg gctaacttcg tgccagcagc cgcggtaata ctaagggggc 480
tagcgttgtt cggaatcact gggcgtaaag cgcacgtagg cggagtctta agtcagaggt 540
gaaatcccaa ggctcaacct tggaactgcc tttgatactg ggtgtcttga ggtcgagaga 600
ggtgagtgga actgcgagtg tagaggtgaa attcgtagat attcgcaaga acaccagtgg 660
cgaaggcggc tcactggctc gattctgacg ctgaggtgcg aaagcgtggg gagcaaacag 720
gattacctga taccctggta gtccacgccg taaacgatgg aagctagccg ttggtcagca 780
tgctgatcag tggcgcagct aacgctttaa gcttcccgcc tggggagtac ggtcgcaaga 840
ttaaaactca aaggaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg 900
aagcaacgcg cagaacctta ccagcctttg acatcctgtg ccactcagag agatttgagg 960
ttcccttcgg ggacgcagag acaggtgctg catggctgtc gtcagctcgt gtcgtgagat 1020
gttgggttaa gtcccgcaac gagcgcaacc ctcgccccta gttgccagca ttcagttggg 1080
cactctaggg ggactgccgg tgataagccg agaggaaggt ggggatgacg tcaagtcctc 1140
atggccctta cgggctgggc tacacacgtg ctacaatggc ggtgacagtg ggcagcgaag 1200
gggtgacccg gagctaatct ccagaagccg tctcagttcg gattgcactc tgcaactcga 1260
gtgcatgaag ttggaatcgc tagtaatcgc ggatcagcat gccgcggtga atacgttccc 1320
gggccttgta cacaccgccc gtcacaccat gggagttggt tttacccgaa ggcgttgcgc 1380
taacccgcaa gggaggcagg cgaccacggt agggtcagcg actggggtga agtcgtaaca 1440
aggtaaccgt a 1451
Claims (8)
1. Rigid fungus capable of degrading tribenuron-methylChenggangzhangella sp.) A fermentation culture method of the strain BI-1 is characterized in that an ME culture medium is adopted to culture the strain BI-1; the strain has a preservation number of CCTCC No. M2019586.
2. The fermentation culture method according to claim 1, wherein the rigid-forming bacterium BI-1 is cultured to the logarithmic phase using an ME medium, and inoculated into a fermentation medium containing yeast powder 0.3-0.5%, peptone 0.4-0.6% and calcium chloride 0.001-0.002% for fermentation culture.
3. The fermentation culture method according to claim 1, wherein the fermentation culture is performed at a culture temperature of 30-35 ℃, an inoculation ratio of 15%, a ventilation rate of 1:0.8, a stirring speed of 200r/min, and a pH of 7.2.
4. The fermentation culture method according to claim 2 or 3, further comprising the step of feeding the fermentation tank with a feed, wherein the fermentation medium is fed once every 32 hours for 80 hours, and the number of viable bacteria reaches 10 after the fermentation is completed 9 And (3) the concentration of the active component is more than one/mL.
5. A microbial inoculum for degrading tribenuron-methyl, wherein the microbial inoculum is in a liquid form, and is prepared from the fermentation culture solution prepared by the fermentation culture method according to any one of claims 1-4.
6. A method for degrading tribenuron-methyl is characterized in that the tribenuron-methyl is degraded by using the rigid-forming bacterium BI-1 or the microbial inoculum according to claim 5, wherein the collection number of the rigid-forming bacterium BI-1 is CCTCC No. M2019586.
7. The method for degrading tribenuron-methyl according to claim 6, wherein cucumber seedlings are planted and inoculated into just fungus BI-1 or said fungus agent by root irrigation.
8. A method for improving the survival capability of rigid fungus BI-1, which is characterized in that rigid fungus BI-1 is colonised in cucumber root systems, wherein the preservation number of rigid fungus BI-1 is CCTCC No. M2019586.
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