CN114350565B - Multifunctional strain of cold-resistant Brevibacterium and application thereof - Google Patents
Multifunctional strain of cold-resistant Brevibacterium and application thereof Download PDFInfo
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Abstract
The invention discloses a multifunctional strain of cold-resistant Brevibacterium and application thereof, belonging to the technical field of agricultural microorganisms. The cold-resistant Brevibacterium (Brevibacterium frigoritolerans) CW-2 has the preservation number: cctccc NO: m20211598 the cold-resistant Brevibacterium (Brevibacterium frigoritolerans) can degrade organic matters such as chlorpyrifos, profenofos, terramycin and tylosin to a certain extent, has a function of generating a biological film, has a biocontrol effect on partial soil-borne pathogenic bacteria, and also has a certain phosphorus dissolving capacity and growth promoting effect on plants, so that the bacteria can be applied to soil to achieve the purposes of restoring polluted soil, improving the quality of the soil, preventing and treating soil-borne diseases and promoting growth.
Description
Technical Field
The invention relates to the technical field of agricultural microorganisms, in particular to a multifunctional strain of Brevibacterium cold-resistant and application thereof.
Background
Chlorpyrifos (Chlorpyrifos) is also known as Chlorpyrifos, a broad-spectrum insecticidal and acaricidal agent, and has been registered and used in more than 14 countries including china, the european union, the united states and the like as one of the most productive and sales insecticide worldwide. Profenofos (Profenofos), also known as bromclofos, duotong, has contact and stomach poisoning effects and is effective against other organophosphorus and pyrethroid-resistant pests. The problem of excessive residual chlorpyrifos and profenofos in agricultural products caused by the long-term and large-scale use of the chlorpyrifos and profenofos is widely focused. But also the chlorpyrifos and profenofos remained in the environment, especially in the soil environment, have great influence on the ecological safety of the soil and the quality of agricultural products, so that the development of a method for effectively removing the residual chlorpyrifos and profenofos in the soil environment is urgent, and the method for reducing the chlorpyrifos or profenofos by utilizing microorganisms is an environment-friendly method.
Antibiotics are very demanding in disease treatment and livestock breeding processes, and thus, the number of antibiotics that lead to direct or indirect access to the environment is also increasing year by year. Antibiotics entering the environment pollute the water environment and the soil environment to a certain extent, and the serious consequence is promotion of drug resistance of a large number of bacteria. It has been found that after animals ingest antibiotics for veterinary use, most of the antibiotics are removed from the body in the form of raw medicines or metabolites along with animal feces. The medicines which are discharged from the body enter the environment in different modes such as migration, leaching or infiltration, can be accumulated in soil and water, enter the human body in a food chain mode, and cause potential health hazards to the human body. Tetracyclines and the macrolide antibiotic tylosin are very widely used antibiotics worldwide, and oxytetracycline or tylosin can be detected in different environments.
Bacterial biofilm is a highly organized, systematic microbial membranous polymer attached to the surface of a carrier or at the interface of different media, is a life phenomenon that bacteria are beneficial to survival in adaptation to natural environments, and is formed by accumulation of microorganisms and secretions thereof. The film-forming bacteria can firmly colonize on the surface of plant root system and even enter the inside of plant tissue to form symbiosis with plants, so as to assist the plants to resist the external adverse environment and promote the plant growth. Meanwhile, the bacterial biomembrane can help microorganisms to obtain ecological advantages, drive the formation of soil micro-aggregates, influence the soil structure and are also key for regulating and controlling the turnover of organic carbon in the soil. The biological membrane can promote the diversity and metabolic activity of soil microorganisms, and has important significance for deeply understanding the essence of the soil biological process and better regulating and controlling the nutrient circulation, pollutant degradation and soil health of the organisms.
Phosphorus plays an important role in agriculture, and the lack of available phosphorus in soil can inhibit the formation of new cells, so that root system development is poor, plant growth is stagnated, the phenomenon of 'stiff seedling' frequently encountered in growth occurs, and plant yield is reduced. Most of phosphorus in the soil is fixed by calcium, iron, aluminum and other ions and soil grains to form invalid phosphorus which cannot be directly absorbed and utilized by plants. Some microorganisms have the ability to convert phosphorus which is difficult to be absorbed by plants into a usable state, and the phosphate-solubilizing microorganisms can activate insoluble phosphorus in soil and promote plant growth.
Soil-borne diseases can generate a large amount of thalli under the general condition that the conditions are favorable for the growth and development of pathogens and hosts are infected, and the pathogens can propagate in a large amount and infect the hosts. In the presence of a disease-sensitive host, the germs can enter a continuous pathogenic period and propagate and spread in a large quantity along with continuous cropping of crops, but after that, when nutrients are consumed or soil conditions such as temperature, humidity and the like are unfavorable for the germs, the germs can enter a dormant period again, and the germs are ill again after the conditions are proper, so that the germs are difficult to kill. Once diseases occur in the early growth period of crops, seedlings decay or stem rot suddenly fall, the seedlings die quickly, and the crop production is seriously affected. Diseases occur in the later growth period of crops, generally, the yield is reduced by 20% -30% in the year, and the yield is reduced by 50% -60% in the serious year, even the crop is in a dead state.
Pesticide, antibiotic residues and soil-borne diseases exist in most natural soil environments at the same time, and the use of microorganisms for restoring polluted soil is of great concern, but most of microbial strains separated at present are single in function and difficult to adapt to the demands of agricultural production.
Disclosure of Invention
Aiming at the prior art, the invention aims to provide a multifunctional strain of Brevibacterium cold-resistant and application thereof. The multifunctional strain of the cold-resistant Brevibacterium provided by the invention integrates functions of degrading pesticides and antibiotics, generating biological films, dissolving phosphorus, biocontrol and promoting growth, and has very important significance for improving soil quality and protecting soil environment.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided a strain of Brevibacterium cold-resistant (Brevibacterium frigoritolerans) CW-2, which has been deposited at China center for type culture Collection (CCTCC for short, address: university of Wuhan, china) at 12-13 days 2021, with a deposit number of: cctccc NO: m20211598.
The Brevibacterium cold-resistant (Brevibacterium frigoritolerans) CW-2 is separated from farmland soil using chlorpyrifos for a long time, and has the following characteristics:
the bacterial colony is off-white, opaque, smooth in surface, round and regular in periphery, and the cells are observed to be in a rod shape under a microscope; gram staining positive.
The microbial inoculum containing the cold-resistant Brevibacterium (Brevibacterium frigoritolerans) CW-2 also belongs to the protection scope of the invention.
In the above-mentioned microbial inoculum, the Brevibacterium cold-resistant (Brevibacterium frigoritolerans) CW-2 can be present in the form of a cultured living bacterium, a fermentation broth of a living bacterium or a filtrate of a strain culture.
The microbial inoculum may further contain other active ingredients, and the composition of the other active ingredients can be determined by a person skilled in the art according to the use effect of the microbial inoculum.
The microbial inoculum can also comprise a carrier. The carrier may be a solid carrier or a liquid carrier; wherein the solid carrier can be grass carbon, clay, kaolin, diatomite, straw, corn meal and bean powder, starch, animal feces, etc.; the liquid carrier may be water.
The preparation forms of the microbial inoculum can be liquid, emulsion, suspending agent, powder, granule, wettable powder, water dispersible granule and the like.
In a second aspect of the present invention, there is provided the use of Brevibacterium cold-resistant (Brevibacterium frigoritolerans) CW-2 or a microbial inoculum containing Brevibacterium cold-resistant (Brevibacterium frigoritolerans) CW-2 as described above in at least one of the following (1) to (10):
(1) Degrading organophosphorus pesticides;
(2) Preparing a product for degrading the organophosphorus pesticide;
(3) Degrading the antibiotic;
(4) Preparing a product for degrading antibiotics;
(5) Forming a biological film, and improving the soil quality;
(6) Inhibiting soil-borne pathogens;
(7) Preparing a pharmaceutical preparation for preventing and treating soil-borne diseases;
(8) Producing ammonia;
(9) Producing IAA;
(10) Activating the indissoluble phosphorus in the soil.
In the above application, preferably, the organophosphorus pesticide is chlorpyrifos and/or profenofos.
In the above application, preferably, the antibiotic is oxytetracycline and/or tylosin.
In the above application, preferably, the soil-borne pathogen is one or more of helminth (Bipolaris sorokinlana (Sacc) Shoem), fusarium graminearum (Fusarium graminearum), fusarium putrescens (Fusarium solani (Mart.) App.et Wollenw) and Rhizoctonia graminea (Rhizoctonia cereadis Vander Hoeven).
In the above application, preferably, the soil-borne disease is wheat root rot, wheat stem rot, capsicum root rot and/or wheat sheath blight.
In a third aspect of the present invention, there is provided a method for simultaneously degrading organophosphorus pesticides, antibiotics in soil, improving soil quality and promoting crop growth, comprising the steps of:
the above Brevibacterium cold (Brevibacterium frigoritolerans) CW-2 or a microbial inoculum containing Brevibacterium cold (Brevibacterium frigoritolerans) CW-2 is applied to the soil to be treated.
The invention has the beneficial effects that:
the cold-resistant Brevibacterium (Brevibacterium frigoritolerans) CW-2 can degrade chlorpyrifos with high efficiency, has degradation effect on profenofos, terramycin and tylosin, and has a degradation rate of 68.9% to 50mg/L chlorpyrifos within 2 days and tends to be stable; the degradation rate of 25mg/L profenofos reaches more than 20 percent; the degradation rate of the oxytetracycline to 10mg/L reaches 32%; the degradation rate of tylosin of 10mg/L reaches more than 26 percent. Meanwhile, the cold-resistant Brevibacterium (Brevibacterium frigoritolerans) CW-2 has strong capability of generating a biological film at 48 h. Moreover, the cold-resistant Brevibacterium (Brevibacterium frigoritolerans) CW-2 has biocontrol effects on wheat root rot, wheat stem-based rot, chilli root rot and wheat sheath blight, and the bacteriostasis rates are 33.34%,16.67%,13.68% and 11.29% respectively; in addition, the cold-resistant Brevibacterium (Brevibacterium frigoritolerans) CW-2 of the invention also has strong ammonia production capacity and strong auxin production capacity. In addition, the cold-resistant Brevibacterium (Brevibacterium frigoritolerans) CW-2 of the invention also has a certain phosphorus dissolving capacity.
In conclusion, the cold-resistant Brevibacterium (Brevibacterium frigoritolerans) CW-2 integrates the functions of pesticide and antibiotic degradation, soil remediation, soil-borne disease control and growth promotion, is a multifunctional strain, and has wide application prospect.
Drawings
FIG. 1 is a microscopic image of strain CW-2 of the present invention.
FIG. 2 is a colony morphology of strain CW-2 of the present invention.
FIG. 3 is a phylogenetic tree of strain CW-2 of the present invention.
FIG. 4 is a graph showing the effect of time on the degradation rate of bacterial strain CW-2 to degrade chlorpyrifos;
the figure shows that the degradation rate of the bacterial strain CW-2 to chlorpyrifos can reach more than 68% at 2d.
FIG. 5 shows the effect of time on the degradation rate of bacterial strain CW-2 to profenofos;
the figure shows that the degradation rate of the bacterial strain CW-2 to profenofos reaches more than 20% at 2d.
FIG. 6 shows the determination of the effect of strain CW-2 on oxytetracycline degradation;
the figure illustrates that strain CW-2 has a 37% degradation rate of oxytetracycline in carbon-containing medium at 2d.
FIG. 7 is a graph showing the determination of the effect of strain CW-2 on tylosin degradation;
the figure illustrates that strain CW-2 has a 28% degradation rate of tylosin in carbon-containing medium at 2d.
FIG. 8 shows the bacterial biofilm amount change of the strain CW-2 of the present invention after culturing for 24 hours and 48 hours.
The figure illustrates that strain CW-2 is produced at a mid-level at 24h biofilm and at a high-level at 48h biofilm
FIG. 9 is a diagram showing the test of the strain CW-2 against wheat root rot.
FIG. 10 is a diagram showing a test of the strain CW-2 against Pythium gracile.
FIG. 11 is a diagram showing the reaction of the strain CW-2 with the root rot of capsicum.
FIG. 12 is a diagram showing a test of the bacterial strain CW-2 against Rhizoctonia cerealis.
FIG. 13 is a graph showing the results of ammonia production capacity of strain CW-2.
FIG. 14 shows the results of the ability of strain CW-2 to produce auxin.
FIG. 15 shows the results of the conversion capacity of the strain CW-2 phosphorus.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. 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 application belongs.
As described above, in most natural soil environments, pesticide and antibiotic residues and soil-borne diseases exist at the same time, and the use of microorganisms to repair contaminated soil is of great concern, but most of the microorganism strains separated at present are single in function and difficult to meet the demands of agricultural production.
The inventor of the patent deeply ploughs in the field of agricultural microorganisms for many years, and aims at the problem of single function of the existing microorganism strain, and the inventor separates a strain of cold-resistant Brevibacterium (Brevibacterium frigoritolerans) CW-2 capable of degrading chlorpyrifos from farmland soil which uses chlorpyrifos for a long time; as a result of further study on the function of CW-2 of the Brevibacterium cold-resistant strain (Brevibacterium frigoritolerans), it was found that the CW-2 strain is significantly different from the previously reported Brevibacterium cold-resistant strain, and the CW-2 strain has the following functions:
(1) Reducing chlorpyrifos, profenofos, oxytetracycline and tylosin;
(2) The method has strong capability of generating a biological film and can improve the soil quality;
(3) Has remarkable inhibiting effect on soil-borne pathogenic bacteria such as wheat root rot, wheat stem basal rot, capsicum root rot, wheat sheath blight and the like;
(4) The method has the phosphorus conversion capability and improves the conversion of the effective state of phosphorus;
(5) Has the capability of producing ammonia and auxin, and can promote the growth of crops.
Therefore, the cold-resistant Brevibacterium (Brevibacterium frigoritolerans) CW-2 integrates the functions of pesticide and antibiotic degradation, soil restoration, soil-borne disease control, phosphorus dissolution and growth promotion, and has very important significance for improving the soil quality and protecting the soil environment, thereby providing the invention.
In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below with reference to specific embodiments. If experimental details are not specified in the examples, the conditions are generally conventional or recommended by the reagent company; reagents, consumables, etc. used in the examples described below are commercially available unless otherwise specified. Wherein the composition of the culture medium used is as follows:
enrichment medium: 10.0g of NaCl, 10.0g of peptone, 5.0g of yeast powder, 1000mL of distilled water, adjusting the pH to about 7.0, and autoclaving at 121 ℃ for 20min.
Inorganic salt culture medium: k (K) 2 HPO 4 5.8g，KH 2 PO 4 4.5g，(NH 4 ) 2 SO 4 2.0g，MgSO 4 0.16g，CaCl 2 0.02g，Na 2 MoO 4 0.002g，FeSO 4 0.001g，MnCl 2 0.001g of distilled water 1000mL, adjusting the pH to about 7.0, and autoclaving at 121 ℃ for 20min.
Carbon source-containing medium: k (K) 2 HPO 4 5.8g，KH 2 PO 4 4.5g，(NH 4 ) 2 SO 4 2.0g，MgSO 4 0.16g，CaCl 2 0.02g，Na 2 MoO 4 0.002g，FeSO 4 0.001g，MnCl 2 0.001g, 5.0g of sucrose, 1000mL of distilled water, adjusting the pH to about 7.0, and autoclaving at 121 ℃ for 20min.
LB medium: 10.0g of NaCl, 10.0g of peptone, 5.0g of yeast powder, 15g of agar powder and 1000mL of distilled water, adjusting the pH to about 7.0, and sterilizing at 121 ℃ for 20min.
PDA medium: 200g of potato (peeled), 20g of sucrose, 20g of agar and 1000mL of distilled water, and the pH is not required to be adjusted, and the potato is autoclaved for 20min at 121 ℃.
Example 1: screening and identification of Strain CW-2
1. Enrichment and separation of strains:
separating chlorpyrifos degrading bacteria from farmland soil in Taian city of Shandong province where chlorpyrifos is used for a long time. And culturing the collected farmland soil by adopting a liquid enrichment culture method.
Liquid enrichment culture: inoculating the collected farmland soil into an enrichment culture medium containing 50mg/L chlorpyrifos according to the proportion of 10% (mass ratio), performing shake culture at 30 ℃ and 120r/min for one week, inoculating the farmland soil into a new enrichment culture medium containing 50mg/L chlorpyrifos according to the inoculum size of 10% (mass ratio), repeatedly domesticating the farmland soil in sequence, separating and purifying the last culture solution by adopting a plate culture method, and numbering and storing the culture solution.
The bacterial colony in the culture dish which is cultivated for 72 hours is gently scraped by a bacteria-collecting ring on a sterile operation table, the bacterial colony is transferred into a sterilized triangular flask, a proper amount of physiological saline is added to obtain thalli with a certain concentration, the thalli is placed into a constant temperature shaking box for cultivation for 3 hours, and then sterile water is added to adjust the concentration, so that the OD of the thalli is adjusted 400nm About 1.0 for standby.
And adding a proper amount of bacterial suspension into an enrichment medium containing 50mg/L chlorpyrifos, performing shake culture at 30 ℃ and 120r/min, and setting a non-inoculated bacterial suspension control. Sampling by regular culture, chromatographic condition and temperature condition: the sample inlet is 250 ℃; the column temperature is programmed to rise to 200 ℃ at 15 ℃/min at 140 ℃ initially, and is kept for 1min; the detector temperature was 250 ℃; the carrier gas flow is 13mL/min; the pressure is 311.04KPa, and the constant pressure mode is adopted; the air flow rate is 300mL/min, H 2 The flow rate is 40mL/min; tail blowing is 40mL/min; the sample feeding amount is 1 mu L, the retention time is 2.5min, the chlorpyrifos residue is measured, the chlorpyrifos degradation rate is calculated, the degradation capacity of different degradation bacteria to chlorpyrifos is determined, and the strain with higher degradation rate is selected and named as CW-2.
2. Identification of strains:
(1) Colony morphology and physiological biochemical identification:
colony morphology and physiological and biochemical identification were performed on the CW-2 strain obtained above, and a 100 x 10 fold micrograph of the strain is shown in FIG. 1. The main biological characteristics of the strain are as follows: the colony is off-white, opaque, smooth in surface, round and regular in periphery, and the cells are observed under a microscope to be in a rod shape (figure 2); gram staining positive. The most suitable growth condition of the strain is pH value 8.0 and temperature 30 ℃.
(2) 16S rDNA molecular identification:
1) Extraction of bacterial group DNA:
the single colony of the bacterial strain CW-2 is picked up and inoculated in LB culture medium under the aseptic condition, and is subjected to constant temperature shake culture for 24 hours at 30 ℃ and 120r/min for later use.
1mL of culture solution of the strain CW-2 is added into a sterilized EP tube containing 100 mu L of sterile water, the mixture is placed into boiling water bath at 80 ℃ for 5min after vortex oscillation, and then centrifuged for 5min under the condition of 12000rpm of a quick centrifuge, and the obtained upper layer liquid is the genome DNA of the strain CW-2.
2) 16S rDNA sequence analysis of strain CW-2:
the genome DNA of the strain CW-2 is used as a template, and bacterial 16S rDNA universal primers are used for PCR amplification, and the sequences of the primers are as follows:
27F:5'-AGA GTT TGA TCC TGG CTC AG-3'；(SEQ ID NO.2)
1492R:5'-GGC TAC CTT GTT ACG ACT-3'。(SEQ ID NO.3)
the PCR reaction system is as follows:
template 0.5. Mu.L
Forward primer 0.5. Mu.L
Reverse primer 0.5. Mu.L
2×Taq Master Mix 12.5μL
Sterile dd H 2 O 11μL
PCR reaction procedure: pre-denaturation: 94 ℃ for 5min; circulation conditions: cycling at 94 ℃, 1min,55 ℃, 1min,72 ℃ for 1min for 30 times; rear extension: 72℃and 10 mm. Agarose gel electrophoresis was performed after the completion of the reaction.
Detection and sequencing of PCR amplification reaction products:
sequencing of PCR amplified products was performed by biological engineering (Shanghai) Inc., and the nucleotide sequence is shown in SEQ ID NO 1. The sequencing results were uploaded to Genbank in NCBI, subjected to sequence comparison and analysis, and the species of strain CW-2 was determined.
Based on the Gene Bank sequence homology comparison, the strains CW-2 and Brevibacterium frigoritolerans are in one branch and have homology as high as 100% (FIG. 3). The strain CW-2 was identified as Brevibacterium cold-resistant (Brevibacterium frigoritolerans) by combining the physiological and biochemical characteristics of the strain. And performing biological preservation on the strain, wherein the preservation information is as follows:
strain name: brevibacterium cold-resistant CW-2
Latin name: brevibacterium frigoritolerans
Preservation mechanism: china center for type culture Collection
The preservation organization is abbreviated as: CCTCC (cctccc)
Address: university of Chinese Wuhan
Preservation date: 2021, 12, 13
Accession numbers of the preservation center: cctccc NO: m20211598.
Example 2: degradation characteristic of cold-resistant Brevibacterium CW-2 on chlorpyrifos
Extraction of chlorpyrifos in solution: after the degradation experiment culture period is finished, petroleum ether and a proper amount of NaCl are added, the mixture is fully vibrated on a vortex oscillator for 2min, then the mixture is stood for layering, and an upper petroleum ether solution is taken to be tested in a sample injection bottle.
Determination of chlorpyrifos: agilent 7890B high performance gas chromatograph, FID detector, temperature conditions: the sample inlet is 250 ℃; the column temperature is programmed to rise to 200 ℃ at 15 ℃/min at 140 ℃ initially, and is kept for 1min; the detector temperature was 250 ℃; the carrier gas flow is 13mL/min; the pressure is 311.04KPa, and the constant pressure mode is adopted; the air flow rate is 300mL/min, H 2 The flow rate is 40mL/min; tail blowing is 40mL/min; the sample injection amount was 1. Mu.L and the retention time was 2.5min.
The calculation formula of the addition recovery rate is as follows:
and (3) calculating the chlorpyrifos degradation rate:
wherein: x: degradation rate (%) of cold-resistant Brevibacterium CW-2 to chlorpyrifos; c (C) X : inoculating bacteria to treat chlorpyrifos concentration in the culture solution; c (C) CK : chlorpyrifos concentration in the non-inoculated control medium.
Petroleum ether is used as a solvent to prepare chlorpyrifos standard solutions with gradient concentrations of 5, 10, 20, 50, 100 and 200mg/L respectively, sample injection is carried out under the selected gas chromatography condition, the chlorpyrifos concentration is used as an abscissa, the peak area is used as an ordinate, a chlorpyrifos standard curve is drawn, the linear equation of the chlorpyrifos standard curve is y=4.1633x+1.5234, and the correlation coefficient is 0.9998, so that the good linear relation between the concentration of chlorpyrifos and the peak area is shown.
Petroleum ether is used as an extraction solvent, the same volume of petroleum ether is added into chlorpyrifos culture medium with the concentration of 5.15, 10.38, 51.55 and 103.09mg/L for extraction, and each concentration is repeated three times. The sample is introduced under selected gas chromatographic conditions for the measurement. The adding recovery rate of chlorpyrifos is between 96.9% and 110.8%, the variation coefficient is between 0.7 and 3.4, petroleum ether is adopted as an extracting agent, and the gas chromatography determination method can meet the experimental requirements.
Time effect on degradation rate of degrading bacteria: the initial pH value is 7.0, 3% (volume ratio) of inoculation amount is inoculated into the Brevibacterium cold-resistant bacillus CW-2 in the inorganic salt culture with chlorpyrifos concentration of 50mg/L, shake culture is carried out at 30 ℃ and 120r/min, and then sampling measurement is carried out at 12h, 24h, 48h, 72h and 96h respectively, and chlorpyrifos degradation rate is calculated. As shown in FIG. 4, the degradation rate of the cold-resistant Brevibacterium CW-2 on chlorpyrifos is continuously increased along with the time, the degradation is already in a stable state at 48h, the degradation rate reaches more than 68%, so that the degradation bacteria has an efficient degradation effect on chlorpyrifos, and the degradation rate reaches 70% at 96 h.
The experiment proves that: the degradation rate of the multifunctional bacterial strain CW-2 on chlorpyrifos is in a stable state when the degradation rate is 48 hours, and the degradation rate reaches more than 68 percent.
Example 3: degradation research of cold-resistant Brevibacterium CW-2 on profenofos
Extracting profenofos in the solution: after the degradation experiment culture period was completed, 0.2mL of diluted hydrochloric acid at a concentration of 5M was added for denaturation, and then an excessive amount of NaCl (1.5 g) was added to form a saturated NaCl solution. Adding ethyl acetate according to the ratio of 1:1, mixing for 2min by vortex oscillation, centrifuging for 20min at 3000r/min, taking an upper organic phase, and extracting twice. Taking 3mL of twice mixed extract to pass through anhydrous Na2SO 4 Removing water, standing for layering, taking the upper liquid, filling into a brown small bottle, then drying by a nitrogen purging instrument, and then redissolving by acetonitrile. Make the following stepsThe absorbance was measured with an ultraviolet spectrophotometer.
The calculation formula of the addition recovery rate is as follows:
and (3) calculating the degradation rate of profenofos:
wherein: x: degradation rate (%) of cold-resistant Brevibacterium CW-2 on profenofos; c (C) X : the concentration of profenofos in the inoculation treatment culture solution; c (C) CK : concentration of profenofos in the non-inoculated control medium.
Acetonitrile is used as solvent to prepare profenofos standard solution with gradient concentration of 1, 5, 10, 20 and 50mg/L respectively, and the profenofos standard solution is dissolved in OD 200 The absorbance is measured by an ultraviolet spectrophotometer under the condition that the profenofos concentration is taken as an abscissa, the absorbance is taken as an ordinate, and a profenofos standard curve is drawn, wherein a linear equation of the profenofos standard curve is y=0.0655x-0.007, and a correlation coefficient of the profenofos standard curve is 0.9999, so that the profenofos concentration and the absorbance have good linear relation.
Time effect on degradation rate of degrading bacteria: the initial pH value is 7.0, 3% (volume ratio) of inoculation amount is inoculated into cold-resistant Brevibacterium CW-2 in inorganic salt culture with profenofos concentration of 25mg/L, shake culture is carried out at 25 ℃ under 150r/min, and then sampling measurement is carried out at 12h, 24h and 48h respectively, and the profenofos degradation rate is calculated. As shown in FIG. 5, the degradation rate of cold-resistant Brevibacterium CW-2 to profenofos is slightly increased with the lapse of time, and the degradation rate reaches more than 20% at 48h, so that the degradation bacteria has a certain degradation capacity to profenofos
The experiment proves that: the degradation rate of the cold-resistant Brevibacterium CW-2 on profenofos is in a stable state when the degradation rate is 48 hours, and the degradation rate reaches more than 20 percent.
Example 4: degradation research of cold-resistant Brevibacterium CW-2 on oxytetracycline
Determination of oxytetracycline: the conditions of the high performance liquid chromatography are as follows: the column temperature is 25 ℃, the wavelength is 350nm,1mL/min, the sample injection amount is 10 mu L, the water phase of the mobile phase is 0.05mol/L phosphoric acid, the organic phase is chromatographic pure acetonitrile, and the mobile phase proportion is 87:13 (volume ratio).
Methanol is used as solvent to prepare a series of oxytetracycline standard solutions with different gradient concentrations of 500, 250, 125, 50, 10 and 5 mg/L. Each sample was sampled three times, the average peak area was taken as ordinate, the oxytetracycline concentration as abscissa, and a standard curve was drawn. The correlation linear equation is y= 15.153x-46.636, and the correlation coefficient is 0.9991, so that the concentration of oxytetracycline has a good linear relation with the peak area.
Determination of oxytetracycline degradation effect:
inoculating cold-resistant Brevibacterium CW-2 with initial pH of 7.0 in an inorganic salt culture medium and a carbon source-containing culture medium which are sterilized at high temperature and have oxytetracycline concentration of 10mg/L respectively according to 3% (volume ratio), placing the culture medium at 30 ℃ and under the condition of 120r/min for shake culture for 3d, filtering the degraded culture solution with a microporous filter membrane of 0.45 mu m, and loading the culture solution to be tested. As shown in FIG. 6, in the inorganic salt culture medium, the degradation effect of the multifunctional degrading bacterium CW-2 on tylosin is not obvious, but after an additional carbon source is added into the culture solution, the degradation rate of the multifunctional degrading bacterium CW-2 on terramycin is raised to a certain extent, and reaches 32%.
The calculation formula of the addition recovery rate is as follows:
and (3) calculating the oxytetracycline degradation rate:
wherein: x: degradation rate (%) of cold-resistant Brevibacterium CW-2 on terramycin; c (C) X : the concentration of terramycin in the inoculation treatment culture solution; c (C) CK : concentration of oxytetracycline in the control culture medium without inoculation.
The experiment proves that: the degradation rate of the cold-resistant Brevibacterium CW-2 on the antibiotic oxytetracycline reaches 32%, which indicates that the cold-resistant Brevibacterium CW-2 is a strain capable of degrading oxytetracycline.
Example 5: degradation research of cold-resistant Brevibacterium CW-2 on tylosin
Determination of tylosin: the conditions of high performance liquid chromatography are column temperature 25 ℃, wavelength 275nm,1mL/min, sample injection amount 10 μL, water phase of mobile phase of 0.05mol/L phosphoric acid, organic phase of chromatographic pure acetonitrile, and mobile phase ratio of 73:27 (volume ratio).
Methanol is used as solvent to prepare tylosin standard solutions with different gradient concentrations of 500, 250, 125, 50, 10 and 5 mg/L. And (3) measuring under the selected liquid chromatography condition, sampling each sample three times, taking the average peak area as an ordinate and the tylosin concentration as an abscissa, and drawing a standard curve. The correlation linear equation is that y=7.5443x+0.0517 correlation coefficient is 0.9993, so that the concentration of tylosin has good linear relation with the peak area.
Determination of tylosin degradation effect:
inoculating the cold-resistant Brevibacterium CW-2 with an initial pH of 7.0 in an inorganic salt culture medium and a carbon source-containing culture medium which are sterilized at high temperature and have the concentration of 10mg/L respectively according to the inoculation amount of 3% (volume ratio), placing the culture medium at 30 ℃ and under the condition of 120r/min for shake culture for 3d, filtering the degraded culture solution by a microporous filter membrane of 0.45 mu m, and then loading the culture solution to be tested. As shown in FIG. 7, in the inorganic salt culture medium, the multifunctional degrading bacterium CW-2 has almost no degrading effect on tylosin, but after an additional carbon source is added into the culture solution, the degrading rate of tylosin is increased to a certain extent, and reaches more than 26%.
The calculation formula of the addition recovery rate is as follows:
calculating the tylosin degradation rate:
wherein: x: degradation rate (%) of cold-resistant Brevibacterium CW-2 on tylosin; c (C) X : concentration of tylosin in the inoculation treatment culture; c (C) CK : concentration of tylosin in the non-inoculated control culture.
The experiment proves that: the degradation rate of the cold-resistant Brevibacterium CW-2 against the tylosin reaches more than 26%, which indicates that the cold-resistant Brevibacterium CW-2 is a strain capable of degrading the tylosin.
Example 6: functional research of cold-resistant Brevibacterium CW-2 production biological membrane
Determination of biofilm: brevibacterium cold-resistant CW-2 was cultured overnight in LB medium and diluted with LB liquid medium (1:100) and added to a 96-well plate at 200. Mu.L per well. Standing at 30deg.C for 2d, washing with PBS for 2 times to remove non-adsorbed bacteria, and naturally air-drying. To each well, 100. Mu.L of 0.1% crystal violet staining solution was added, and the wells were stained for 30min. Sucking out the crystal violet dye solution, washing off the surface flooding with PBS and air-drying. Then 200. Mu.L of 95% ethanol was added, and the mixture was allowed to stand for 15 minutes to dissolve crystal violet, and the biofilm content was analyzed by detecting absorbance (As) at 590nm of the sample, while LB (Ac) was used As a control.
The ability of bacteria to form biofilms was assessed based on the OD value produced by the bacteria biofilm at 590 nm: if As is less than or equal to Ac, the function of biofilm production is not provided, if Ac < As is less than or equal to (2 xAc), the biofilm production capacity is lower, if (2 xAc) < As is less than or equal to (4 xAc), the biofilm production capacity is medium, and if (4 xAc) < As, the biofilm production capacity is stronger.
As shown in FIG. 8, the Brevibacterium cold-resistant CW-2 had a moderate biofilm-producing ability at 24 hours and a strong biofilm-producing ability at 48 hours.
Example 7: research on control effect of cold-resistant Brevibacterium CW-2 on soil-borne pathogenic bacteria
Plate facing method: inoculating 7 mm-diameter target bacteria cakes to the center of a PDA flat plate by taking soil-borne pathogenic bacteria as target bacteria, inoculating cold-resistant Brevibacterium CW-2 at two symmetrical points which are 2.5cm away from the center of the flat plate, repeating each treatment for 3 times by taking a flat plate without inoculating the strain CW-2 as a contrast, culturing in a culture box at 28+/-2 ℃ in an inverted manner, measuring the diameters of the opposite colonies when the contrast colonies are full, and calculating the bacteriostasis rate.
The soil borne pathogens used in this example were wheat root rot (root rot, navel vermicular spore Bipolaris sorokinlana (Sacc) Shoem), wheat stem rot (Fusarium graminearum Fusarium graminearum), pepper root rot (Fusarium solani (Mart.) App.et Wollenw) and wheat sheath blight (Rhizoctonia cerealis Rhizoctonia cereadis Vander Hoeven) respectively, which were supplied by the plant pathology laboratory of the Shandong university of agriculture, plant protection institute.
The results are shown in fig. 9, 10, 11 and 12 and table 1, the multifunctional strain CW-2 has certain biocontrol effects on wheat root rot, wheat stem rot, capsicum root rot and wheat sheath blight, and the bacteriostasis rates are 33.34%,16.67%,13.68% and 11.29% respectively.
Table 1: CW-2 strain and soil-borne pathogenic bacterium counter test results
Example 8: research on growth promoting potential of cold-resistant Brevibacterium CW-2
Salkowski's color developer: 1mL of 0.5mol/L FeCl 3 50mL of 35% perchloric acid solution.
Peptone ammoniation medium: 5g of peptone, 1000mL of deionized water, pH 7.2 and sterilization at 121 ℃ for 20min.
(1) Determination of Ammonia production Capacity:
inoculating the screened cold-resistant Brevibacterium CW-2 to LB liquid culture medium for activation, diluting the bacterial strain bacterial liquid to OD by using sterile water 600 About 1.0. Mu.L of bacterial liquid is inoculated into peptone ammoniation medium respectively, the medium without antagonistic bacteria is used as a control, each treatment is repeated for 3 times, and shaking culture is carried out at 28+/-2 ℃ and 180rpm for 5d. After the culture is finished, the culture is centrifuged for 10min at 10000rpm, 1mL of Nahner reagent is added into the supernatant, the solution change is observed, if orange or yellow sediment is generated, the strain has the ammonia production capacity, and the more the sediment is, the stronger the ammonia production capacity is.
As shown in FIG. 13 and Table 2, the Bacillus brevis CW-2 has strong ammonia production capacity, which indicates that the Bacillus brevis CW-2 has deaminase, and can deaminate amino acids to generate ammonia and various acids, thereby being beneficial to plant growth.
(2) Determination of auxin production ability:
inoculating screened cold-resistant Brevibacterium CW-2 into LB liquid culture medium for activating culture, diluting bacterial liquid of each strain to OD by using sterile water 600 About 1.0. Mu.L of the bacterial liquid is inoculated into LB liquid culture medium containing tryptophan (5 mmol of tryptophan is contained in each liter of LB liquid culture medium), and each treatment is repeated for 3 times, 28+/-2 ℃ and 180rpm shaking culture is carried out for 2d by taking the culture medium without antagonistic bacteria as a control. After the cultivation is finished, centrifuging at 10000rpm for 10min, taking 1mL of supernatant, adding an equal amount of Salkowski's color reagent, standing in a dark place for 30min to enable the culture solution to develop color, observing the color change of the solution, and if the solution turns into pink, indicating that the antagonistic bacteria has the capability of producing auxin IAA, and the darker the color, the stronger the capability of the strain to produce IAA.
As shown in FIG. 14 and Table 2, the cold-resistant Brevibacterium CW-2 has a strong ability to produce auxin, which is advantageous for promoting plant growth.
Table 2: results of CW-2 Strain growth-promoting test
Note that: "+" indicates a positive reaction, "-" indicates a negative reaction, and more "+" indicates a greater ability to produce ammonia or produce auxin.
Example 9: study on phosphorus dissolving capacity of cold-resistant Brevibacterium CW-2
Pikovansky's medium: 10g of glucose, caCO 3 5g, yeast powder 0.5g, (NH) 4 ) 2 SO 4 0.5g，MgSO 4 ·7H 2 O 0.3g，KCl 0.3g，NaCl 0.3g，MnSO 4 ·H 2 O 0.02g，FeSO 4 ·7H 2 0.02g of O, 20g of agar, 1000mL of deionized water, pH 7.0 and sterilization at 121 ℃ for 20min.
Inoculating the screened cold-resistant Brevibacterium CW-2 to LB liquid culture medium for activation, diluting bacterial liquid of each strain to OD by using sterile water 600 About 1.0, 10. Mu.L of bacterial liquid is respectively inoculated to PikoAnd (3) taking the culture medium which is not inoculated with antagonistic bacteria as a control in the center of a Vaskain's culture medium plate, repeating each treatment for 3 times, inversely placing the plate at 28+/-2 ℃ for 7d of culture, observing the edge of the strain, and if a transparent circle appears, indicating that the strain has phosphorus dissolving capability, and if the transparent circle appears, indicating that the strain has stronger phosphorus dissolving capability.
As shown in FIG. 15, the cold-resistant Brevibacterium CW-2 has a certain phosphorus dissolving capacity, can convert organic phosphorus compounds in soil into phosphate or convert insoluble phosphorus in soil into soluble phosphorus, can increase the content of available phosphorus in soil, and is beneficial to plant growth.
In conclusion, the cold-resistant Brevibacterium CW-2 is a strain which can efficiently degrade chlorpyrifos, has a certain degradation effect on profenofos, terramycin and tylosin, has a very strong function of generating a biological film, has a certain biocontrol effect on soil borne germ wheat root rot, wheat stem rot, chilli root rot and wheat sheath blight, has ammonia production and growth promotion capability of producing auxin, has a certain phosphorus dissolution capability, and can be applied to water and soil to achieve the purposes of repairing chlorpyrifos, terramycin and tylosin polluted soil, preventing and treating soil borne diseases and promoting growth.
The foregoing description is only of the preferred embodiments of the present application and is not intended to limit the same, but rather, various modifications and variations may be made by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.
SEQUENCE LISTING
<110> Shandong agricultural university
<120> a multifunctional strain of Brevibacterium cold-resistant strain and application thereof
<130> 2022
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 1396
<212> DNA
<213> Strain CW-2
<400> 1
gttacctcac cgacttcggg tgttacaaac tctcgtggtg tgacgggcgg tgtgtacaag 60
gcccgggaac gtattcaccg cggcatgctg atccgcgatt actagcgatt ccggcttcat 120
gcaggcgagt tgcagcctgc aatccgaact gagaatggct ttatgggatt cgcttacctt 180
cgcaggtttg cagccctttg taccatccat tgtagcacgt gtgtagccca ggtcataagg 240
ggcatgatga tttgacgtca tccccacctt cctccggttt gtcaccggca gtcaccttag 300
agtgcccaac tgaatgctgg caactaagat caagggttgc gctcgttgcg ggacttaacc 360
caacatctca cgacacgagc tgacgacaac catgcaccac ctgtcactct gtcccccgaa 420
ggggaaagcc ctatctctag ggttgtcaga ggatgtcaag acctggtaag gttcttcgcg 480
ttgcttcgaa ttaaaccaca tgctccaccg cttgtgcggg cccccgtcaa ttcctttgag 540
tttcagcctt gcggccgtac tccccaggcg gagtgcttaa tgcgttagct gcagcactaa 600
agggcggaaa ccctctaaca cttagcactc atcgtttacg gcgtggacta ccagggtatc 660
taatcctgtt tgctccccac gctttcgcgc ctcagtgtca gttacagacc agaaagtcgc 720
cttcgccact ggtgttcctc caaatctcta cgcatttcac cgctacactt ggaattccac 780
tttcctcttc tgcactcaag ttccccagtt tccaatgacc ctccacggtt gagccgtggg 840
ctttcacatc agacttaagg aaccacctgc gcgcgcttta cgcccaataa ttccggacaa 900
cgcttgccac ctacgtatta ccgcggctgc tggcacgtag ttagccgtgg ctttctggtt 960
aggtaccgtc aaggtaccag cagttactct ggtacttgtt cttccctaac aacagaactt 1020
tacgacccga aggccttctt cgttcacgcg gcgttgctcc gtcagacttt cgtccattgc 1080
ggaagattcc ctactgctgc ctcccgtagg agtctgggcc gtgtctcagt cccagtgtgg 1140
ccgatcaccc tctcaggtcg gctacgcatc gtcgccttgg tgagccatta cctcaccaac 1200
tagctaatgc gccgcgggcc catctataag tgacagcgta aaccgtcttt ccatcttctc 1260
tcatgcgaga aaagaacgta tccggtatta gctccggttt cccgaagtta tcccagtctt 1320
ataggcaggt tgcccacgtg ttactcaccc gtccgccgct aatctcaggg agcaagctcc 1380
catcgattcg ctcgac 1396
<210> 2
<211> 20
<212> DNA
<213> artificial sequence
<400> 2
<210> 3
<211> 18
<212> DNA
<213> artificial sequence
<400> 3
ggctaccttg ttacgact 18
Claims (7)
1. Cold-resistant Brevibacterium strainBrevibacterium frigoritolerans) CW-2, having a deposit number of: cctccc NO: m20211598.
2. A process comprising the step of preparing a composition comprising the bacterium breve-acting as defined in claim 1Brevibacterium frigoritolerans) CW-2 bacterial agent.
3. The microbial inoculum according to claim 2, characterized in that the Brevibacterium frigidum isBrevibacterium frigoritolerans) CW-2 is present as a live bacterium or a fermentation broth of a live bacterium to be cultured.
4. A bacterial agent according to claim 3 in the form of a liquid, emulsion, suspension, powder or granule.
5. A bacterial agent according to claim 3, in the form of a wettable powder or a water dispersible granule.
6. The cold-resistant Brevibacterium strain of claim 1Brevibacterium frigoritolerans) CW-2 or the strain of any one of claims 2-5 containing Brevibacterium cold-resistantBrevibacterium frigoritolerans) Use of a bacterial agent of CW-2 in at least one of the following (1) - (10):
(1) Degrading organophosphorus pesticide, wherein the organophosphorus pesticide is chlorpyrifos and/or profenofos;
(2) Preparing a product for degrading organophosphorus pesticide, wherein the organophosphorus pesticide is chlorpyrifos and/or profenofos;
(3) Degrading an antibiotic, wherein the antibiotic is oxytetracycline and/or tylosin;
(4) Preparing a product for degrading antibiotics, wherein the antibiotics are oxytetracycline and/or tylosin;
(5) Forming a biological film, and improving the soil quality;
(6) Inhibiting soil-borne pathogens, wherein the soil-borne pathogens are root rot vermicularia gramineaBipolaris sorokinlana (Sacc) Sheem, fusarium graminearumFusarium graminearumFusarium solani (L.) KuntzeFusarium solani(Mart.) App. et Wollenw and Rhizoctonia cerealisRhizoctonia cereadis One or more of Vander Hoeven;
(7) Preparing a pharmaceutical preparation for preventing and treating soil-borne diseases, wherein the soil-borne diseases are wheat root rot, wheat stem rot, chilli root rot and/or wheat sheath blight;
(8) Producing ammonia;
(9) Producing IAA;
(10) Activating the indissoluble phosphorus in the soil.
7. A method for simultaneously degrading organophosphorus pesticides and antibiotics in soil, improving soil quality and promoting crop growth, comprising the steps of:
a method of treating a bacterial strain according to claim 1Brevibacterium frigoritolerans) CW-2 or the strain of any one of claims 2-5 containing Brevibacterium cold-resistantBrevibacterium frigoritolerans) Bacterial agent application of CW-2Adding the soil into the soil to be treated;
the organophosphorus pesticide is chlorpyrifos and/or profenofos; the antibiotics are oxytetracycline and/or tylosin.
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| WO2016165037A1 (en) * | 2015-04-17 | 2016-10-20 | Universidad De Chile | Biological composition for controlling nematodes |
| CN106164247A (en) * | 2014-02-10 | 2016-11-23 | 拜奥菲尔微生物和基因技术及生物化学有限公司 | Inoculation microbial inoculum for stress soil |
| CN113373094A (en) * | 2021-07-16 | 2021-09-10 | 榆林市中泰农业科技有限公司 | Brevibacterium fritolerans SDB5 and application thereof in promoting plant growth |
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| CN106164247A (en) * | 2014-02-10 | 2016-11-23 | 拜奥菲尔微生物和基因技术及生物化学有限公司 | Inoculation microbial inoculum for stress soil |
| WO2016165037A1 (en) * | 2015-04-17 | 2016-10-20 | Universidad De Chile | Biological composition for controlling nematodes |
| CN113373094A (en) * | 2021-07-16 | 2021-09-10 | 榆林市中泰农业科技有限公司 | Brevibacterium fritolerans SDB5 and application thereof in promoting plant growth |
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| 番茄根内生芽孢杆菌的多样性和系统发育研究;程志强;雷少楠;熊娟;马荣琴;陈优优;李容丹;路晶晶;吴寒;龚玉杰;田宝玉;;中国农学通报(第08期);全文 * |
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