CN116574640A - Pseudomonas syriacus CN-1 and screening method and application thereof - Google Patents
Pseudomonas syriacus CN-1 and screening method and application thereof Download PDFInfo
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Abstract
The invention provides a strain CN-1 of pseudolaris (pseudomonads) which is preserved in China general microbiological culture Collection center, accession number: CGMCCNO.26191, preservation address: no.1 and No. 3 of the north cinquefoil of the morning sun area of beijing city. Based on the same inventive concept, the invention also provides a screening method of the pseudomonas syriacus strain CN-1, which adopts a step-by-step domestication mode for screening. Based on the same inventive concept, the invention also provides an application of the pseudomonas syriacus strain CN-1 in the degradation of the wilt. The invention also provides a degradation microbial inoculum capable of degrading the cotton wilt and a preparation method thereof. The invention provides a strain CN-1 of pseudolariella syriacus, which accelerates the degradation of gossypium in soil, and has the effects of repairing soil polluted by gossypium and protecting ecological environment. The microbial inoculum of the strain has the advantages of simple preparation process, high efficiency, low cost, no secondary pollution and good application prospect.
Description
Technical Field
The invention belongs to the technical field of plant protection, and particularly relates to a pseudolarix himalayan CN-1, a screening method and application thereof.
Background
Gossypium hirsutum (MBC) is a high-efficiency low-toxicity systemic broad-spectrum bactericide of benzimidazole, also called captan, molbanmide and benzimidazole No. 44, and has the chemical name of N- (2-benzimidazolyl) -carbamic acid methyl ester and the molecular formula of C 9 H 9 N 3 O 2 . The gossypium wilt is used as systemic bactericide to kill pathogenic fungi by inhibiting the growth of mycelium, bud tube or aspirator, and is used in preventing and controlling most of pathogenic fungi in ascomycetes. The sterilization molecular mechanism is mainly divided into two aspects, namely, the formation of nucleoside is blocked, and the synthesis of nucleic acid is interfered; secondly, the formation of the spindle body is blocked, and the mitosis of cells is inhibited. The cotton wilt agent is widely applied to the prevention and treatment of diseases of field crops, vegetables, fruit trees and cash crops in the agricultural production of China. Has good effect in preventing and treating wheat scab, rice blast, banded sclerotial blight, sclerotial blight of small grain, sclerotial blight of colza, early blight of tomato and the like.
In recent years, the capacity of cotton wilt has reached 67,000T. Due to the long-term use of the pesticide in large quantities, gossypol and metabolites have been detected in various crops, which affects human health. Studies show that the cotton wilt is stable in soil, the retention time can be as long as 2 years, the cotton wilt remained in the soil can enter the whole plant through plant root systems, and the cotton wilt is transmitted to consumers at all levels through food chains to cause toxic and harmful effects. At present, a great deal of research reports that long-term exposure to gossypium wilt environment can produce adverse effects such as endocrine disrupting effects, reproductive toxicity, immune toxicity and the like on non-target organisms (fishes, mice, birds and the like). Wilt can enter human tissue through the food chain leading to the development of lesions. A large amount of pesticide residues are remained in soil and water environments, and potential harm is caused to various organisms and even human health. Therefore, how to efficiently and environmentally friendly accelerate the degradation of the cotton wilt in the environment and reduce the bioavailability thereof is significant.
Pesticide residues are degraded mainly by microorganisms in soil, but the natural degradation process is very slow. Therefore, the high-efficiency degradation strain can be screened out in a targeted way, which is helpful for solving the problem of serious pollution of soil pesticides. So far, many reports show that rhodococcus, klebsiella, flavobacterium, stenotrophomonas maltophilia and the like have better degradation effect on cotton wilt. The screened pseudomonas syriacus is a strain which is found to have high-efficiency degradation capability on the gossypium hirsutum for the first time, and has important significance for reducing the pollution of pesticides formed by surface runoff or farmland leakage to water environment, improving the quality safety of agricultural products and promoting the sustainable development of agriculture in China.
Disclosure of Invention
The invention aims at overcoming the defects of the prior art and provides a pseudomonas syriacus CN-1, a screening method and application thereof.
The invention provides a strain of pseudolarix sinensis CN-1, enriches strain resources for degrading the wilting, lays a foundation for solving the problem of pesticide residue of the wilting, and provides materials for preparing the immobilized microbial agent for degrading the wilting. The inventor finally separates a novel degradation strain with high-efficiency degradation efficiency on the cotton wilt from soil continuously applied for many years through a large number of experiments, carries out homology comparison according to GenBank sequences through 16SrDNA molecular identification, combines the morphological characteristics, physiological and biochemical characteristics and biological characteristics of the strain to carry out experiments, and is identified to be primarily identified as the pseudolarix (Pseudomonas hibiscus) and named as CN-1. The strain has been uploaded to GenBank, the gene number is OP288122, and the strain has been preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at 12 months 14 of 2022, and the preservation address is: beijing, chaoyang area, north Chen Xi Lu 1, 3, china academy of sciences microbiological institute, deposit number: cgmccno.26191. In the present invention, pseudomonas stutzeri (Pseudomonas stutzeri) strain CN-1 or simply referred to as strain CN-1.
The second object of the invention is to provide a screening method of the Pseudomonas syriacus strain CN-1.
A third object of the present invention is to provide the use of Pseudomonas stutzeri strain CN-1 for degrading gossypium.
The fourth object of the invention is to provide a degradation microbial inoculum capable of degrading the cotton wilt.
The fifth aim of the invention is to provide a preparation method of a degradation microbial inoculum capable of degrading the gossypium barbasum.
The sixth object of the invention is to provide an application method of the pseudomonas syriacus strain CN-1 in preparing a wilting degradation microbial inoculum.
The seventh object of the invention is to provide an application method of the pseudomonas syriacus strain CN-1 in repairing the natural environment polluted by gossypium hirsutum.
An eighth object of the present invention is to provide a method of degrading or repairing wilt's pollution.
The above object of the present invention is achieved by the following technical scheme:
pseudomonas syriacus (Pseudomonas hibiscus) strain CN-1, which was deposited at the China general microbiological culture Collection center, accession number: CGMCCNO.26191, preservation address: no.1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
The nucleotide sequence of the 16SrDNA of the Pseudomonas syriacus strain CN-1 is shown as SEQ ID NO. 1.
Based on the same inventive concept, the invention also provides a screening method of the pseudolarix syriacus strain CN-1, which adopts a step-by-step domestication mode to screen until the concentration of the gossypium hirsutum reaches 500mg/L, and performs gradient dilution to separate and purify bacteria to obtain the pseudolarix syriacus strain CN-1.
Based on the same inventive concept, the invention also provides an application of the pseudomonas syriacus strain CN-1 in the degradation of the wilt.
For the application described above, the conditions of degradation are: the degradation temperature is 30-32 ℃, the pH is 6-7, and the inoculation amount is 6-7%. More preferably, the optimal degradation conditions are obtained by a response surface method: the temperature was 31.13 ℃, the pH was 6.6, the inoculum size was 6.5%, and the optimum conditions obtained were modified for ease of handling: degradation temperature is 31 ℃, pH is 6.6, inoculation amount is 6.5%, degradation rate predicted value is 67.7%, and OD is carried out under revised optimal degradation condition 600 The bacterial liquid with the concentration of=1 is added into a culture medium containing 50mg/L of the inorganic salt of the gossypol, and the degradation rate of the gossypol is about 70 percent.
Based on the same inventive concept, the invention also provides a degradation microbial inoculum capable of degrading the cotton wilt, which comprises the pseudomonas syriacus strain CN-1. Preferably, the degradation microbial inoculum is a bacterial suspension formed by amplifying and culturing a bacterial suspension of a pseudomonas syriacus strain CN-1 in a nutrient medium.
Based on the same inventive concept, the invention also provides a preparation method of the degradable bacterial agent capable of degrading the gossypium hirsutum, which comprises the preparation of seed liquid, wherein the preparation steps of the seed liquid are as follows: picking up thallus of Pseudomonas stutzeri strain CN-1, inoculating to liquid culture medium containing LB, shake culturing at 30-40deg.C for 24 hr at 150-160 r/min, transferring the bacterial liquid to centrifugal tube, centrifuging at 4-6deg.C at 4000-5000 r/min for 5-10 min, washing twice with sterilized inorganic salt culture medium, and re-suspending to regulate OD 600 =1, and stored for later use. Preferably, the seed solution is prepared by the following steps: picking up thallus of Pseudomonas pseudolaricis strain CN-1, inoculating into liquid culture medium containing LBShake culturing at 30deg.C and 150r/min for 24 hr, transferring bacterial liquid into centrifuge tube, centrifuging at 4deg.C and 4000r/min for 5min, washing twice with sterilized inorganic salt culture medium, and re-suspending to adjust OD 600 =1, and stored for later use.
Based on the same inventive concept, the invention also provides an application of the pseudomonas syriacus strain CN-1 in preparing a wilting degradation microbial inoculum.
Based on the same inventive concept, the invention also provides application of the pseudomonas syriacus strain CN-1 in repairing the natural environment polluted by gossypium hirsutum, wherein the natural environment is water or soil.
Based on the same inventive concept, the invention also provides a method for degrading gossypol or repairing the pollution of gossypol, which is to add the strain CN-1 of the pseudolariella syriacus directly or after dilution into an object to be treated.
The beneficial effects of the invention are as follows:
1. the invention provides a strain CN-1 of pseudolariella syriacus, which can degrade the gossypium in the soil by directly adding the strain CN-1 into the soil after preparing a bacterial suspension, so that the residual gossypium in the soil can be rapidly degraded, and compared with a non-inoculation control, the degradation half-life of the gossypium is shortened from 30.97d to 19.46d, the degradation of the gossypium in the soil is quickened, and the effects of repairing the contaminated soil of the gossypium and protecting the ecological environment are achieved.
2. The microbial inoculum of the strain has the advantages of simple preparation process, high efficiency, low cost, no secondary pollution and good application prospect.
Drawings
FIG. 1 is a liquid chromatogram of cotton wilt (black solid lines in the figure are control chromatogram, black dotted lines are chromatogram of Pseudomonas stutzeri CN-1 after degradation);
FIG. 2 is a colony morphology of Pseudomonas syriacus CN-1;
FIG. 3 is a P.hibiscus CN-1 phylogenetic tree;
FIG. 4 is a response surface plot;
FIG. 5 is a graph showing the correspondence of bacterial growth and degradation rate under optimal degradation conditions;
FIG. 6 is a mass spectrum of a gossypium degradation product;
FIG. 7 shows a possible simple metabolic pathway of gossypii;
FIG. 8 shows the degradation rate of gossypium in various treated soils.
Detailed Description
The following description of the embodiments of the present invention will clearly and fully describe the technical solutions of the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, 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.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Example 1 screening and identification of Cotton wilting degrading bacteria
Taking 5g of soil which is applied with the gossypium in 100mL of inorganic salt culture medium throughout the year, wherein the formula of the inorganic salt culture medium is as follows: 1g NaCl,0.5g KH 2 PO 4 ，1.5gK 2 HPO 4 ，2g(NH 4 ) 2 SO 4 ，0.2gMgSO 4 ·7H 2 O, 1000mL of distilled water. Adding the sole carbon source gossypium wilt to be 100mg/L, culturing for 5d at 30 ℃ at 150r/min, taking out, standing for 30min, sucking the upper layer fungus suspension by a sterilized gun head, inoculating to an inorganic salt culture medium with an inoculum size of 5% by volume, increasing the concentration of the gossypium wilt in the inoculated inorganic salt culture medium to 200mg/L, culturing for 5 periods under the same culture conditions, and culturing until the concentration of the gossypium wilt in the culture medium is 500mg/L. Taking and standing the final domestication enrichment culture solution, and sequentially mixing the culture solution with the culture solution according to 10 -4 ～10 -8 Each concentration of the dilution was applied to a separation and purification medium (same as an inorganic salt medium, 1.5% agar was added) containing 100mg/L of gossypium, and incubated at 30℃with 3 replicates for each gradient. Picking up colony with good growth state, different morphology and good colony quality in separation and purification culture mediumAnd (3) a large single colony strain is further separated and purified. And (3) determining the degradation capability of the purified strain to the gossypium by using a High Performance Liquid Chromatography (HPLC) with non-inoculation as a control.
HPLC assay conditions:
HPLC model: shimadzu, chromatographic column: c18 column (4.6 mm. Times.250 mm. Times.5 μm), column temperature: 30 ℃, flow rate: 1mL/min, sample injection amount: 20. Mu.L; mobile phase: methanol: water = 1:1 (volume ratio), the detection wavelength is 281nm, and under this condition the peak time of the gossypium is 8.87min. The chromatogram is shown in FIG. 1.
The degradation rate is calculated as follows:
the method is used for successfully separating and obtaining a strain of the wilting degradation bacterium, named CN-1, from the soil sample, and the degradation rate of the strain after 3d can reach about 70 percent under the optimal degradation condition when the initial concentration of the wilting is 50 mg/L.
The bacterial morphology was observed, the surface of strain CN-1 was smooth and moist, the colony was yellow and raised, and the edges were neat as shown in FIG. 2. The physiological and biochemical characteristics of the strain are measured, and the strain belongs to gram-negative bacteria, wherein oxidase, contact enzyme and nitrate reductase are positive, indole and gelatin are liquefied, the test is negative, and the strain is preserved in China general microbiological culture Collection center (CGMCC NO. 26191).
The bacterial 16SrDNA identification method is utilized, the total DNA of the bacterial strain CN-1 is used as a template, and the bacterial 16SrDNA universal primer is utilized: 27F (AGAGTTTGATCCTGGCTCAG) and 1492R: 5'-GGTTACCTTGTTACGACTT-3'), PCR amplification was performed to obtain an amplified product of about 1.5kb, sequencing was performed by Shanghai Biotechnology Co., ltd, and the sequence was uploaded to GenBank under the gene number OP288122, and FIG. 3 is a phylogenetic tree made by MEGA. By combining morphological characteristics, physiological and biochemical characteristics and gene sequence comparison results of the strain, the strain CN-1 is finally identified as Pseudomonas syriacus (Pseudomonas hibiscus).
EXAMPLE 2 optimization of degradation conditions of Cotton wilt by degrading bacteria CN-1
(1) Preparation of seed liquid
Selecting a ring of thallus, inoculating into conical flask containing LB liquid culture medium (formula: 5g yeast extract, 10g peptone, 10g NaCl,1000mL distilled water), shake culturing at 30deg.C for 24 hr/min, transferring the bacterial liquid into 50mL centrifuge tube, centrifuging at 4deg.C at 4000r/min for 5min, washing twice with sterilized inorganic salt culture medium, and re-suspending to adjust OD 600 =1, and stored for later use.
(2) Optimization of degradation conditions of degrading bacteria CN-1
Important factors affecting strain degradation include temperature, pH and inoculum size. Temperature (a) range used in this experiment: 20-40 ℃; pH (B): 5.0 to 9.0; inoculation amount (C): 2-10% (OD) 600 =1). The test design method of Box-Behnken statistics test is adopted to carry out test design on the 3 influencing factors, the test design and the results are shown in table 1, and the test result is the degradation rate (R) of the strain CN-1 on the 3 rd day of growth in the culture medium containing 50mg/L of the gossypium hirsutum inorganic salt. And (3) analyzing the results of the table 1, and simultaneously drawing a three-dimensional response surface graph, so as to obtain the theoretical value of the optimal degradation condition.
Gossypol degradation rate (%) =67.03+2.31a-3.69b+2.86c+0.8254ab+2.37ac+1.28bc-11.02A 2 -8.55B 2 -10.10C 2
TABLE 1Box-Behnken test design and results
The analysis of the results shows that three factors have obvious influence on the degradation of the cotton wilt by the strain, and the influence sequence of each factor on the degradation rate is C & gtB & gtA, namely the inoculation quantity is more than pH & gttemperature. The analysis result is combined with a response surface curve (figure 4) to obtain the optimal condition of CN-1 on the degradation of the gossypium, wherein the degradation temperature is 31.13 ℃, the pH is 6.6, the inoculation amount is 6.5 percent, and the obtained optimal condition is corrected for convenient operation: the degradation temperature is 31 ℃, the pH is 6.6, the inoculation amount is 6.5%, and the degradation rate predicted value is 67.7%.
The embodiment provides theoretical optimal degradation conditions for the cotton wilt degrading bacteria CN-1 and guarantees the best performance of the cotton wilt degrading bacteria CN-1 in practical application.
Example 3 determination of growth, degradation Curve and degradation products under optimal degradation conditions
Inoculating strain CN-1 into inorganic salt culture medium with initial concentration of 50mg/L under the conditions of optimal pH, temperature and inoculation amount, and measuring OD of bacterial liquid at different times 600 Values and gossypium degradation rate, time on abscissa, OD 600 Values and gossypol degradation rate are on the ordinate, and are based on time and OD 600 And drawing a growth curve and a degradation curve of the degradation bacteria according to the corresponding relation between the value and the degradation rate of the gossypium barbarum.
The growth and degradation curves of strain CN-1 are shown in FIG. 5. Strain CN-1 grows rapidly within 20h in inorganic salt culture solution with concentration of gossypium wilting of 50mg/L, OD at 12h 600 The value reached a maximum of 0.85. With the presence of the strain CN-1, the degradation rate of the gossypium is gradually increased, and after 120 hours, the degradation rate is 68.29 percent, and the deviation from the predicted value is 0.59 percent.
To further investigate the degradation pathway of the degrading bacterium CN-1 on gossypium, the degradation products were determined by HPLC-MS, as shown in FIG. 6, m/z178[ m+H ]] + And m/z135[ m+H] + The fragment ion peaks at these sites are identical to those of benzimidazole-2-carbamic acid and 2-hydroxy benzimidazole (2-HB). Based on the analysis of the intermediates, a simple MBC metabolic pathway was proposed, as shown in fig. 7. The inventor speculates that the gossypium wilt firstly removes methyl to generate benzimidazole-2-carbamic acid with poor stability, then generates 2-hydroxy benzimidazole, and the degradation of the strain CN-1 reduces the toxicity of the gossypium wilt, thereby having practical significance for bioremediation of the pollution of the gossypium wilt.
EXAMPLE 4 Effect of Strain CN-1 on the degradation of Cotton wilt in soil
The soil is obtained from a peony garden at Qingdao agricultural university, and is brown soilThe soil has the physical and chemical properties that: total organic carbon: 18.2g/kg; total nitrogen: 1.43g/kg; total phosphorus: 1.05g/kg; pH:6.89. three treatments were set up in this experiment: natural soil + cotton wilt, sterilized soil + cotton wilt, natural soil + cotton wilt + strain CN-1. 3 replicates per treatment set were set with an initial content of 10mg/kg of gossypium in the soil. The amount of the pseudomonas syriacus strain CN-1 added into the fungus adding soil is 10 7 cfu/g soil. All the soil is adjusted to the water content of about 60% of the maximum water holding capacity, and the soil is placed in a biochemical incubator with constant temperature of 30 ℃ for dark culture, and the treated soil is sampled and detected for the residual quantity of the wilt in the soil at 0, 1, 3, 7, 14, 21, 28, 45 and 60d respectively.
The degradation rate of gossypol in the various treated soils is shown in figure 8. Over time, the degradation rate of the gossypium in the soil is gradually increased, and the residual concentration is gradually reduced. At 60d, the residual amounts of the natural soil, the cotton wilt, the sterilized soil and the cotton wilt under three different treatments of the natural soil, the cotton wilt and the strain CN-1 are respectively 2.0mg/kg, 3.3mg/kg and 0.82mg/kg, and the degradation rates are respectively 79.78%, 67.48% and 91.88%. The half-life of the soil inoculated with the cotton wilt strain CN-1 bacterial liquid is shortened from 30.97d to 19.46d, wherein the half-life of the sterilized soil is up to 42.37d, which indicates that the original microorganism of the soil can promote the degradation of the cotton wilt. Therefore, the addition of the wilt degrading bacteria CN-1 can degrade the wilt in the soil more quickly, and has good repairing effect on the soil polluted by the wilt.
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.
Claims (10)
1. A strain of pseudomonas syriacus (pseudomonas hibiscus) CN-1, wherein the strain is deposited at the China general microbiological culture Collection center, accession number: CGMCCNO.26191, preservation address: no.1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
2. The pseudomonas syriacus strain CN-1 according to claim 1, wherein the nucleotide sequence of the 16SrDNA of the pseudomonas syriacus strain CN-1 is shown in SEQ ID No. 1.
3. A screening method of a Pseudomonas stutzeri strain CN-1 according to claim 1, wherein screening is performed by adopting a gradual domestication mode until the concentration of gossypium is 500mg/L, and separating and purifying the strain by gradient dilution to obtain the Pseudomonas stutzeri strain CN-1.
4. Use of a strain of pseudomonas syriacus CN-1 according to claim 1 for degrading gossypium.
5. The use according to claim 4, wherein the degradation conditions are: the degradation temperature is 30-32 ℃, the pH is 6-7, and the inoculation amount is 6-7%.
6. A degradation microbial agent capable of degrading cotton wilt, which is characterized by comprising a pseudomonas syriacus strain CN-1.
7. The preparation method of the degradable bacterial agent for the degradable cotton wilt is characterized by comprising the preparation of seed liquid, wherein the preparation steps of the seed liquid are as follows: picking up thallus of Pseudomonas stutzeri strain CN-1, inoculating to liquid culture medium containing LB, shake culturing at 30-40deg.C for 24 hr at 150-160 r/min, transferring the bacterial liquid to centrifugal tube, centrifuging at 4-6deg.C at 4000-5000 r/min for 5-10 min, washing twice with sterilized inorganic salt culture medium, and re-suspending to regulate OD 600 =1, and stored for later use.
8. Use of a strain of pseudomonas syriacus CN-1 according to claim 1 for the preparation of a wilting degradation inoculant.
9. Use of a strain CN-1 of pseudomonas syriacus according to claim 1 for restoring the natural environment contaminated by gossypium, wherein said natural environment is a body of water or soil.
10. A method for degrading or repairing wilt's pollution, characterized in that the strain CN-1 of pseudolarix is added directly or after dilution to the object to be treated.
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| CN106754582A (en) * | 2017-03-21 | 2017-05-31 | 青岛农业大学 | Pseudomonas putida RXX 01 and its application in soil phthalic acid ester of degrading |
| CN108034624A (en) * | 2018-02-05 | 2018-05-15 | 厦门理工学院 | It is a kind of to be used to handle biological agent of high-concentration ammonia nitrogenous wastewater and preparation method thereof |
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| CN106754582A (en) * | 2017-03-21 | 2017-05-31 | 青岛农业大学 | Pseudomonas putida RXX 01 and its application in soil phthalic acid ester of degrading |
| CN108034624A (en) * | 2018-02-05 | 2018-05-15 | 厦门理工学院 | It is a kind of to be used to handle biological agent of high-concentration ammonia nitrogenous wastewater and preparation method thereof |
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