CN114350565A - A multifunctional strain of Brevibacterium cold-resistant and its application - Google Patents

Abstract

The invention discloses a multifunctional strain of Brevibacterium cold-resistant and its application, belonging to the technical field of agricultural microorganisms. Described Brevibacterium frigoritolerans (Brevibacterium frigoritolerans) CW-2, its deposit number is: CCTCC NO: M 20211598, this Brevibacterium frigoritolerans (Brevibacterium frigoritolerans) can be resistant to organic substances such as chlorpyrifos, profenofos, oxytetracycline and tylosin It has a certain degradation ability, has a strong function of producing biofilm, has a biocontrol effect on some soil-borne pathogens, and also has a certain ability to dissolve phosphorus and promote plant growth. Therefore, the bacteria can be used in soil, In order to achieve the purpose of remediating polluted soil, improving soil quality, preventing soil-borne diseases and promoting growth.

Description

A multifunctional strain of Brevibacterium cold-resistant and its application

technical field

The invention relates to the technical field of agricultural microorganisms, in particular to a multifunctional strain of Brevibacterium cold-resistant and its application.

Background technique

Chlorpyrifos, also known as chlorpyrifos, is a broad-spectrum insecticide and acaricide. As one of the most widely produced and sold insecticides in the world, it has been used in more than 14 countries including China, EU, US, etc. for registration and use. Profenofos (Profenofos), also known as bromochlorophos, polyphosphorus, has contact and stomach poisoning effects, and is still effective against other organophosphorus and pyrethroid-resistant pests. The long-term use of chlorpyrifos and profenofos in large quantities has led to the problem of excessive residues in agricultural products, which has attracted widespread attention. And remaining in the environment, especially the chlorpyrifos and profenofos in the soil environment have a significant impact on the safety of the soil ecological environment and the quality of agricultural products, therefore, the method for effectively removing the chlorpyrifos and profenofos in the soil environment is in urgent need of research and development, and The use of microorganisms to degrade chlorpyrifos or profenofos is an environmentally friendly method.

The demand for antibiotics in disease treatment and livestock and poultry breeding is very large. Therefore, the amount of antibiotics that directly or indirectly enter the environment is also increasing year by year. Antibiotics entering the environment bring a certain degree of pollution to the water environment and soil environment, and the more serious consequence is to promote a large number of bacteria to develop drug resistance. Some studies have found that after animals ingest veterinary antibiotics, most of the antibiotics will be excreted in the form of original drugs or metabolites with the feces of livestock and poultry. Drugs that are excluded from the body enter the environment in different ways, such as migration, leaching, or infiltration. They can accumulate in soil and water bodies, and then enter the human body through the food chain, causing potential health hazards to the human body. The tetracycline and macrolide antibiotics tylosin are the most widely used antibiotics in the world, and oxytetracycline or tylosin can be detected in different environments.

Bacterial biofilm is a highly organized and systematic microbial film polymer attached to the surface of the carrier or the interface of different media. It is a life phenomenon that is conducive to the survival of bacteria in order to adapt to the natural environment. . Film-forming bacteria can firmly colonize the surface of plant roots and even enter the interior of plant tissues to symbiotic with plants, assisting plants to resist the adverse external environment and promote plant growth. At the same time, bacterial biofilms can help microorganisms to obtain ecological advantages, drive the formation of soil microaggregates, affect soil structure, and are also the key to regulating soil organic carbon turnover. Biofilms can promote soil microbial diversity and metabolic activity, which is of great significance for in-depth understanding of the nature of soil biological processes and better regulation of nutrient cycling, pollutant degradation and soil health in which organisms are involved.

Phosphorus plays an important role in agriculture. The lack of soil available phosphorus will inhibit the formation of new cells, make the root system stunted, plant growth stagnation, and the phenomenon of "stiff seedlings" often encountered in growth, resulting in plant yield reduction. Most of the phosphorus in the soil is fixed by calcium, iron, aluminum and other ions and soil clay, forming ineffective phosphorus, which cannot be directly absorbed and utilized by plants. Some microorganisms have the ability to convert phosphorus that is difficult for plants to absorb into a usable state. Such phosphorus-solubilizing microorganisms can activate insoluble phosphorus in soil and promote plant growth.

Soil-borne diseases Under normal circumstances, soil pathogens can produce a large number of bacteria. As long as the conditions are favorable for the growth and development of the bacteria and the host is infected, the bacteria can multiply and infect the host. In the presence of the susceptible host, these pathogens can enter a persistent pathogenic period, multiply and spread in large numbers with the continuous cropping of crops, but after the nutrients are consumed or the soil conditions such as temperature and humidity are unfavorable to the bacteria, the bacteria can be Entering the dormant period, when the conditions are suitable, the disease will re-emerge, and it is difficult to kill. Once a disease occurs in the early stage of crop growth, the root of the seedling rots or the stem rots and collapses, and the seedling will die soon, seriously affecting the crop production. Diseases occur in the late stage of crop growth, which can reduce production by 20%-30% in normal years, 50%-60% in severe years, or even fail to harvest.

There are pesticides, antibiotic residues and soil-borne diseases in most natural soil environments. The use of microorganisms to remediate contaminated soils has attracted much attention. However, most of the currently isolated microbial strains have single functions, which are difficult to meet the needs of agricultural production.

SUMMARY OF THE INVENTION

In view of the above-mentioned prior art, the purpose of the present invention is to provide a multifunctional strain of Brevibacterium cold-resistant and its application. The multifunctional strain of Brevibacterium cold-resistant in the present invention integrates the functions of degrading pesticides and antibiotics, producing biofilm, dissolving phosphorus, biocontrol, and promoting growth, and is of great significance for improving soil quality and protecting soil environment.

To achieve the above object, the present invention adopts the following technical solutions:

The first aspect of the present invention provides a strain of Brevibacterium frigoritolerans CW-2, which has been deposited in the China Center for Type Culture Collection (CCTCC for short) on December 13, 2021, and the address is: Wuhan, China, Wuhan University), its deposit number is: CCTCC NO: M 20211598.

Described Brevibacterium frigoritolerans (Brevibacterium frigoritolerans) CW-2 is isolated from the farmland soil of long-term use of chlorpyrifos, and it has the following characteristics:

The colonies were off-white, opaque, and had a smooth surface. The colonies were round with regular circumferences. The cells were rod-shaped when observed under a microscope; Gram staining was positive.

The bacterial agent containing the above-mentioned Brevibacterium frigoritolerans CW-2 also belongs to the protection scope of the present invention.

In the above inoculants, the Brevibacterium frigoritolerans CW-2 may exist in the form of cultured live bacteria, fermentation broth of live bacteria or filtrate of strain culture.

The above-mentioned inoculum may also contain other active ingredients, and those skilled in the art can determine the composition of other active ingredients according to the use effect of the inoculum.

A carrier may also be included in the above-mentioned bacterial agent. The carrier can be a solid carrier or a liquid carrier; wherein the solid carrier can be peat, clay, kaolin, diatomaceous earth, straw, corn meal, soybean meal, starch and animal manure, etc.; the liquid carrier can be water.

The dosage form of the bacterial agent can be liquid, emulsion, suspension, powder, granule, wettable powder, water-dispersible granule and the like.

The second aspect of the present invention provides the application of the above-mentioned Brevibacterium frigoritolerans CW-2 or a bacterial agent containing Brevibacterium frigoritolerans CW-2 in at least one of the following (1)-(10):

(1) Degrade organophosphorus pesticides;

(2) Preparation of products for degrading organophosphorus pesticides;

(3) Degradation of antibiotics;

(4) Preparation of products that degrade antibiotics;

(5) Form biofilm and improve soil quality;

(6) Inhibit soil-borne pathogens;

(7) Preparation of pharmaceutical preparations for preventing and treating soil-borne diseases;

(8) Ammonia production;

(9) Produce IAA;

(10) Activation of insoluble phosphorus in 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 Bipolaris sorokinlana (Sacc) Shoem, Fusarium graminearum, Fusarium solani (Mart.) App.et Wollenw) and one or more of Rhizoctonia cereadis Vander Hoeven.

In the above application, preferably, the soil-borne disease is wheat root rot, wheat stem rot, pepper root rot and/or wheat sheath blight.

A third aspect of the present invention provides a method for simultaneously degrading organophosphorus pesticides and antibiotics in soil, improving soil quality and promoting crop growth, comprising the following steps:

The above-mentioned Brevibacterium frigoritolerans CW-2 or an inoculant containing Brevibacterium frigoritolerans CW-2 was applied to the soil to be treated.

Beneficial effects of the present invention:

The cold-resistant Brevibacterium frigoritolerans CW-2 of the present invention can efficiently degrade chlorpyrifos, and has degradation effects on profenofos, oxytetracycline and tylosin, and the degradation rate of 50 mg/L chlorpyrifos within 2 days reaches 68.9% and tended to be stable; the degradation rate of 25mg/L profenofos reached more than 20%; the degradation rate of 10mg/L oxytetracycline reached 32%; the degradation rate of 10mg/L tylosin reached more than 26%. Meanwhile, the Brevibacterium frigoritolerans CW-2 of the present invention has a strong ability to generate biofilms at 48 hours. Moreover, the cold-tolerant Brevibacterium frigoritolerans CW-2 of the present invention has biocontrol effects on wheat root rot, wheat stem base rot, pepper root rot and wheat sheath blight, and the antibacterial rate is 33.34%, respectively, 16.67%, 13.68% and 11.29%; in addition, the Brevibacterium frigoritolerans CW-2 of the present invention also has a strong ability to produce ammonia and a strong ability to produce auxin. In addition, the Brevibacterium frigoritolerans CW-2 of the present invention also has a certain ability to dissolve phosphorus.

In conclusion, the Brevibacterium frigoritolerans CW-2 of the present invention integrates the functions of pesticide and antibiotic degradation, soil remediation, soil-borne disease control and growth promotion, and is a multifunctional strain with broad application prospects.

Description of drawings

Fig. 1 is the microscopic examination picture of the strain CW-2 of the present invention.

Figure 2 is a morphological diagram of the colony of the strain CW-2 of the present invention.

Figure 3 is a phylogenetic tree diagram of the strain CW-2 of the present invention.

Figure 4 shows the effect of time on the degradation rate of chlorpyrifos degraded by strain CW-2;

The figure shows that the degradation rate of chlorpyrifos by strain CW-2 can reach more than 68% in 2d.

Figure 5 shows the effect of time on the degradation rate of profenofos degraded by strain CW-2;

The figure illustrates that the degradation rate of profenofos by strain CW-2 reached more than 20% at 2d.

Figure 6 is the determination of the degradation effect of strain CW-2 on oxytetracycline;

The figure illustrates that the degradation rate of oxytetracycline by strain CW-2 in carbon source medium reached 37% at 2d.

Figure 7 is the determination of the degradation effect of strain CW-2 on tylosin;

The figure illustrates that the degradation rate of tylosin by strain CW-2 in carbon source medium reached 28% at 2d.

Fig. 8 is the change of bacterial biofilm amount of the strain CW-2 of the present invention cultivated for 24h and 48h.

The figure shows that the strain CW-2 produces a medium level of biofilm at 24h, and a high level of biofilm production at 48h

FIG. 9 is a diagram of the confrontation test between strain CW-2 and wheat root rot fungus.

Figure 10 is a diagram of the confrontation test between strain CW-2 and wheat stem rot.

Figure 11 is a diagram of the confrontation test between strain CW-2 and pepper root rot fungus.

Figure 12 is a diagram of the confrontation test between strain CW-2 and Rhizoctonia solani.

Figure 13 is a graph showing the results of ammonia production of strain CW-2.

Figure 14 shows the results of the ability of strain CW-2 to produce auxin.

Figure 15 shows the results of phosphorus transformation ability of strain CW-2.

Detailed ways

It should be noted that the following detailed description is exemplary and intended to provide further explanation of the application. Unless otherwise defined, 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 mentioned above, pesticides, antibiotic residues and soil-borne diseases coexist in most natural soil environments, and the use of microorganisms to remediate contaminated soils has attracted much attention.

The inventor of this patent has been working in the field of agricultural microorganisms for many years. In view of the current problem of single function of microbial strains, the inventor isolated a cold-tolerant Brevibacterium frigoritolerans CW-2 that can degrade chlorpyrifos from the farmland soil where chlorpyrifos has been used for a long time; Further research on the function of the Brevibacterium frigoritolerans CW-2 showed that the CW-2 strain is significantly different from the existing reported Brevibacterium frigoritolerans, and the CW-2 strain also has the following functions:

(1) degrade chlorpyrifos, profenofos, oxytetracycline and tylosin;

(2) It has a strong ability to produce biofilm and can improve soil quality;

(3) It has a significant inhibitory effect on soil-borne pathogens such as wheat root rot, wheat stem rot, pepper root rot and wheat sheath blight;

(4) It has the ability to convert phosphorus and improve the conversion of phosphorus available state;

(5) It has the ability to produce ammonia and auxin to promote crop growth.

Therefore, the Brevibacterium frigoritolerans CW-2 integrates the functions of pesticide and antibiotic degradation, soil remediation, soil-borne disease control, phosphorus dissolution and growth promotion, which is of great significance for improving soil quality and protecting soil environment. The present invention has thus been proposed.

In order to enable those skilled in the art to understand the technical solutions of the present application more clearly, the technical solutions of the present application will be described in detail below with reference to specific embodiments. If the specific experimental conditions are not specified in the examples, generally follow the conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples can be obtained through commercial channels unless otherwise specified. Wherein, the composition of the medium used is as follows:

Enrichment medium: NaCl 10.0g, peptone 10.0g, yeast powder 5.0g, distilled water 1000mL, adjust pH to about 7.0, and autoclave at 121°C for 20min.

Inorganic salt medium: K ₂ HPO ₄ 5.8 g, KH ₂ PO ₄ 4.5 g, (NH ₄ ) ₂ SO ₄ 2.0 g, MgSO ₄ 0.16 g, CaCl ₂ 0.02 g, Na ₂ MoO ₄ 0.002 g, FeSO ₄ 0.001 g , MnCl ₂ 0.001g, distilled water 1000mL, adjust pH to about 7.0, autoclave at 121℃ for 20min.

Carbon source medium: 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 0.001 g, MnCl ₂ 0.001 g, sucrose 5.0 g, distilled water 1000 mL, adjusted pH to about 7.0, and autoclaved at 121° C. for 20 min.

LB medium: NaCl 10.0g, peptone 10.0g, yeast powder 5.0g, agar powder 15g, distilled water 1000mL, adjust pH to about 7.0, and autoclave at 121°C for 20min.

PDA medium: potato (peeled) 200g, sucrose 20g, agar 20g, distilled water 1000mL, no need to adjust pH, autoclave at 121°C for 20min.

Example 1: Screening and identification of strain CW-2

1. Strain enrichment and isolation:

Chlorpyrifos-degrading bacteria were isolated from farmland soil where chlorpyrifos was used for a long time in Tai'an City, Shandong Province. The collected farmland soil was cultivated by the method of liquid enrichment culture.

Liquid enrichment culture: The collected farmland soil was inoculated into the enrichment medium containing 50mg/L chlorpyrifos according to the ratio of 10% (mass ratio), and was shaken at 30°C and 120r/min for one week. 10% (mass ratio) of the inoculum was inoculated into a new enriched medium containing 50 mg/L chlorpyrifos, followed by repeated domestication, and the last culture solution was separated and purified by plate culture, and numbered and stored.

Gently scrape the colony in the petri dish that has been cultivated for 72 hours on the sterile operating table with a bacterial ring, transfer it to a sterilized triangular flask, add an appropriate amount of physiological saline to obtain a certain concentration of bacteria, and put it in a constant temperature Incubate in a shaking box for 3 hours, and then add sterile water to adjust the concentration, so that the OD _400nm of the bacteria is about 1.0, and it is ready for use.

An appropriate amount of the bacterial suspension was added to the enriched medium containing 50 mg/L chlorpyrifos, and the culture was shaken at 30 °C and 120 r/min. At the same time, no bacterial suspension was set as a control. Timed incubation sampling, chromatographic conditions and temperature conditions: the injection port is 250 °C; the column temperature is programmed temperature, the initial temperature is 140 °C and the temperature is increased to 200 °C at 15 °C/min, and maintained for 1 min; the detector temperature is 250 °C; the carrier gas flow rate is 13mL/min; pressure is 311.04KPa, constant pressure mode; air flow is 300mL/min, _H2 flow rate is 40mL/min; makeup is 40mL/min; The degradation rate of chlorpyrifos was calculated, the degradation ability of different degrading bacteria to chlorpyrifos was determined, and the strain with higher degradation rate was selected and named as CW-2.

2. Identification of strains:

(1) Colony morphology and physiological and biochemical identification:

The CW-2 strain obtained above was identified by colony morphology, physiology and biochemistry, and the microscope 100*10 times photo of the strain is shown in Figure 1. The main biological characteristics of this strain are: the colony is off-white, opaque, and has a smooth surface. The optimum growth conditions of this strain were pH 8.0 and temperature 30℃.

(2) 16S rDNA molecular identification:

1) Extraction of bacterial DNA:

A single colony of strain CW-2 was picked and inoculated into LB medium under aseptic conditions, and incubated at 30°C and 120 r/min for 24 hours with constant temperature shaking until use.

Take 1 mL of the culture solution of strain CW-2 into a sterilized EP tube containing 100 μL of sterile water, place it in a boiling water bath at 80 °C for 5 min after vortexing, and then centrifuge it with a speed centrifuge at 12,000 rpm for 5 min to obtain the upper layer. The liquid is the genomic DNA of strain CW-2.

2) 16S rDNA sequence analysis of strain CW-2:

Using the genomic DNA of strain CW-2 as a template, PCR amplification was performed using bacterial 16S rDNA universal primers. The primer sequences were 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:

Template 0.5 μL

Forward primer 0.5μL

Reverse primer 0.5 μL

2×Taq Master Mix 12.5μL

Sterile dd _{H2O 11} μL

Total volume 25 μL

PCR reaction program: pre-denaturation: 94°C, 5min; cycle conditions: 94°C, 1min, 55°C, 1min, 72°C, 1min, 30 cycles; post-extension: 72°C, 10mim. Agarose gel electrophoresis was performed after the reaction.

Detection and sequencing of PCR amplification reaction products:

The sequencing of the PCR amplification product was completed by Sangon Bioengineering (Shanghai) Co., Ltd., and its nucleotide sequence is shown in SEQ ID NO 1. The sequencing results were uploaded to Genbank in NCBI for sequence comparison and analysis to determine the type of strain CW-2.

According to the Gene Bank sequence homology comparison, strain CW-2 and Brevibacterium frigoritolerans belong to the same clade, and their homology is as high as 100% (Fig. 3). Combined with the physiological and biochemical characteristics of the strain, the strain CW-2 was identified as Brevibacterium frigoritolerans. And the strain is biologically preserved, and the preservation information is as follows:

Strain name: Brevibacterium cold-resistant CW-2

Latin name: Brevibacterium frigoritolerans

Depository institution: China Center for Type Culture Collection

Abbreviation of depositary institution: CCTCC

Address: Wuhan University, Wuhan, China

Deposit date: December 13, 2021

The registration number of the collection center: CCTCC NO: M 20211598.

Example 2: Degradation characteristics of chlorpyrifos by Brevibacterium cold-resistant CW-2

Extraction of chlorpyrifos in solution: After the degradation experiment culture period is over, add petroleum ether and an appropriate amount of NaCl, fully shake on a vortex shaker for 2 min, and let stand for stratification, and take the upper petroleum ether solution into a sample bottle for testing.

Determination of chlorpyrifos: Agilent 7890B high performance gas chromatograph, FID detector, temperature conditions: the injection port is 250 °C; the column temperature is programmed temperature, the initial temperature is 140 °C, 15 °C/min to 200 °C, and keep for 1min; The temperature is 250°C; the carrier gas flow is 13mL/min; the pressure is 311.04KPa, constant pressure mode; the air flow is 300mL/min, the H ₂ flow is 40mL/min; the makeup is 40mL/min; the injection volume is 1μL, Retention time 2.5min.

Add recovery rate calculation formula:

Calculation of chlorpyrifos degradation rate:

In the formula: x: the degradation rate of chlorpyrifos by Brevibacterium cold-resistant CW-2 (%); C _X : the concentration of chlorpyrifos in the inoculated culture solution; C _CK : the concentration of chlorpyrifos in the uninoculated control culture solution.

Using petroleum ether as solvent, prepare standard solutions of chlorpyrifos with gradient concentrations of 5, 10, 20, 50, 100, and 200 mg/L, respectively, and inject samples under the selected gas chromatographic conditions. Taking the concentration of chlorpyrifos as the abscissa, the peak area As the ordinate, draw the standard curve of chlorpyrifos, its linear equation is y=4.1633x+1.5234, and the correlation coefficient is 0.9998, which indicates that the concentration of chlorpyrifos has a good linear relationship with its peak area.

Using petroleum ether as the extraction solvent, the same volume of petroleum ether was added to the chlorpyrifos medium with concentrations of 5.15, 10.38, 51.55, and 103.09 mg/L for extraction, and repeated three times for each concentration. The samples were injected under the selected gas chromatographic conditions. The recovery rate of chlorpyrifos addition ranged from 96.9% to 110.8%, and the coefficient of variation was from 0.7 to 3.4. Using petroleum ether as the extractant, the gas chromatography method could meet the requirements of the experiment.

The effect of time on the degradation rate of the degrading bacteria: the initial pH was 7.0, and the inoculation amount of 3% (volume ratio) in the inorganic salt culture with the concentration of chlorpyrifos was 50 mg/L. The samples were cultured under shaking conditions, and then samples were taken at 12h, 24h, 48h, 72h, and 96h to measure and calculate the degradation rate of chlorpyrifos. The results are shown in Figure 4. With the passage of time, the degradation rate of Brevibacterium cold-resistant CW-2 to chlorpyrifos continued to increase. At the 48th hour, the degradation had reached a stable state, and the degradation rate reached more than 68%. It can be said that this degrading bacteria has a high-efficiency degradation effect on chlorpyrifos, and the degradation rate reaches 70% at the 96th hour.

The above experiments proved that: the degradation rate of chlorpyrifos by the multifunctional strain CW-2 reached a steady state at 48h, and the degradation rate reached more than 68%.

Example 3: Study on the degradation of profenofos by Brevibacterium cold-resistant CW-2

Extraction of profenofos from the solution: After the incubation period of the degradation experiment, 0.2 mL of dilute hydrochloric acid with a concentration of 5 M was added for denaturation, and then excess NaCl (1.5 g) was added to form a saturated NaCl solution. Ethyl acetate was added at a ratio of 1:1, vortexed and mixed for 2 min, then centrifuged at 3000 r/min for 20 min, and the upper organic phase was taken and extracted twice in total. Take 3 mL of the two mixed extracts and pass through anhydrous _Na2SO4 to remove water. After standing for stratification, take the upper layer liquid and put it into a brown vial, then dry it with a nitrogen purger, and then redissolve it in acetonitrile. Its absorbance was measured using a UV spectrophotometer.

Add recovery rate calculation formula:

Calculation of the degradation rate of profenofos:

In the formula: x: the degradation rate of profenofos by Brevibacterium cold-resistant CW-2 (%); C _X : the concentration of profenofos in the inoculated culture medium; C _CK : profenofos in the uninoculated control medium concentration.

Using acetonitrile as a solvent, prepare standard solutions of profenofos with gradient concentrations of 1, 5, 10, 20, and 50 mg/L, respectively, and measure the absorbance using an ultraviolet spectrophotometer at OD ₂₀₀ , with the concentration of profenofos as the abscissa , the absorbance is the ordinate, and the standard curve of profenofos is drawn. Its linear equation is y=0.0655x-0.007, and the correlation coefficient is 0.9999, which indicates that the concentration of profenofos has a good linear relationship with the absorbance.

The effect of time on the degradation rate of the degrading bacteria: the initial pH was 7.0, and the inoculation amount of 3% (volume ratio) in the inorganic salt culture with the concentration of profenofos was 25 mg/L. 12h, 24h and 48h were sampled and measured and the degradation rate of profenofos was calculated. The results are shown in Figure 5. With the passage of time, the degradation rate of Profenofos by Brevibacterium cold resistant CW-2 slightly increased, and the degradation rate reached more than 20% at the 48th hour. Bromophosphine has a certain ability to degrade

The above experiments proved that the degradation rate of profenofos by Brevibacterium cold-resistant CW-2 was already stable at 48h, and the degradation rate reached more than 20%.

Example 4: Study on the degradation of oxytetracycline by Brevibacterium cold-resistant CW-2

Determination of oxytetracycline: The conditions of high performance liquid chromatography are: column temperature 25°C, wavelength 350nm, 1mL/min, injection volume 10μL, the aqueous phase of the mobile phase is 0.05mol/L phosphoric acid, the organic phase is chromatographically pure acetonitrile, The mobile phase ratio was 87:13 (volume ratio).

Using methanol as a solvent, a series of oxytetracycline standard solutions with different gradient concentrations of 500, 250, 125, 50, 10, and 5 mg/L were prepared. Each sample was injected three times, and the average peak area was taken as the ordinate and the concentration of oxytetracycline as the abscissa, and a standard curve was drawn. The correlation linear equation is y=15.153x-46.636, and the correlation coefficient is 0.9991, which indicates that the concentration of oxytetracycline has a good linear relationship with its peak area.

Determination of oxytetracycline degradation effect:

The initial pH was 7.0, respectively in the high-temperature sterilized oxytetracycline concentration of 10mg/L inorganic salt medium and carbon source medium were inoculated according to 3% (volume ratio) inoculation amount of Brevibacterium cold-resistant CW- 2. After shaking and culturing for 3 days at 30°C and 120 r/min, the degraded culture medium was filtered through a 0.45 μm microporous membrane and tested on the machine. The results are shown in Figure 6. In the inorganic salt medium, the degradation effect of the multifunctional degrading bacteria CW-2 on tylosin was not obvious, but after adding an external carbon source to the culture medium, its effect on oxytetracycline was significantly reduced. The degradation rate has a certain increase, reaching 32%.

Add the formula for calculating the recovery rate:

Calculation of oxytetracycline degradation rate:

In the formula: x: the degradation rate of oxytetracycline by Brevibacterium cold-resistant CW-2 (%); C _X : the concentration of oxytetracycline in the inoculated culture medium; C _CK : oxytetracycline in the uninoculated control medium concentration.

The above experiments proved that the degradation rate of the antibiotic oxytetracycline by Brevibacterium cold tolerance CW-2 reached 32%, indicating that Brevibacterium cold tolerance CW-2 is a strain capable of degrading oxytetracycline.

Example 5: Study on the degradation of tylosin by Brevibacterium cold resistant CW-2

Determination of tylosin: the conditions of high performance liquid chromatography are column temperature 25°C, wavelength 275nm, 1mL/min, injection volume 10μL, the aqueous phase of the mobile phase is 0.05mol/L phosphoric acid, the organic phase is chromatographically pure acetonitrile, The mobile phase ratio was 73:27 (volume ratio).

Using methanol as solvent, prepare 500, 250, 125, 50, 10, 5 mg/L tylosin standard solutions with different gradient concentrations. Under the selected liquid chromatography conditions, each sample was injected three times, and the average peak area was taken as the ordinate and the concentration of tylosin as the abscissa, and a standard curve was drawn. The correlation linear equation is y=7.5443x+0.0517 and the correlation coefficient is 0.9993, which indicates that the concentration of tylosin has a good linear relationship with its peak area.

Determination of tylosin degradation effect:

The initial pH is 7.0, respectively in the inorganic salt medium and the carbon source medium with a tylosin concentration of 10 mg/L that have been sterilized by high temperature, inoculate the Brevibacterium cold hardy CW according to the inoculation amount of 3% (volume ratio) -2, placed at 30°C and incubated with shaking at 120 r/min for 3 days, the degraded culture medium was filtered through a 0.45 μm microporous membrane and put on the machine for testing. The results are shown in Figure 7. In the inorganic salt medium, the multifunctional degrading bacteria CW-2 has almost no degradation effect on tylosin, but after adding an external carbon source to the culture medium, it can degrade tylosin. The rate has increased to a certain extent, reaching more than 26%.

Add the formula for calculating the recovery rate:

Calculation of tylosin degradation rate:

In the formula: x: the degradation rate of tylosin by Brevibacterium cold-resistant CW-2 (%); C _X : the concentration of tylosin in the inoculated culture solution; C _CK : the tylosin in the uninoculated control culture solution The concentration of lecithin.

The above experiments proved that the degradation rate of the antibiotic tylosin by Brevibacterium cold resistance CW-2 reached more than 26%, indicating that Brevibacterium cold resistance CW-2 is a strain capable of degrading tylosin.

Example 6: Study on the function of biofilm produced by Brevibacterium cold-resistant CW-2

Determination of biofilm: Brevibacterium hardy CW-2 was cultured in LB medium overnight, diluted with LB liquid medium (1:100), and 200 μL per well was added to a 96-well plate. The cells were cultured at 30°C for 2 days, washed twice with PBS to remove unadsorbed bacteria, and air-dried naturally. 100 μL of 0.1% crystal violet staining solution was added to each well, and the staining was performed for 30 min. Aspirate the crystal violet staining solution, wash the surface floating color with PBS, and air dry. Then add 200 μL of 95% ethanol, let stand for 15 min to dissolve crystal violet, and analyze the content of biofilm by detecting the absorbance value (As) at 590 nm of the sample, while taking LB (Ac) as a control.

Based on the OD value of bacterial biofilm at 590nm, the ability of bacteria to form biofilm was evaluated: if As≤Ac, there is no biofilm production function; if Ac<As≤(2×Ac), there is a lower biofilm If (2×Ac)<As≤(4×Ac), there is medium biofilm generation ability, and if (4×Ac)<As, then there is strong biofilm generation ability.

The results are shown in Fig. 8. Brevibacterium cold-resistant CW-2 has a moderate biofilm-producing ability at 24 hours, and a strong biofilm-producing ability at 48 hours.

Example 7: Study on the control effect of Brevibacterium cold-resistant CW-2 on soil-borne pathogens

Plate confrontation method: take soil-borne pathogens as the target bacteria, inoculate the target bacteria cake with a diameter of 7 mm on the center of the PDA plate, and connect Brevibacterium hardy CW-2 on two symmetrical points 2.5 cm from the center of the plate. The plate not inoculated with strain CW-2 was used as the control, and each treatment was repeated 3 times, and then placed upside down in a 28±2°C incubator for cultivation.

The soil-borne pathogenic bacteria used in this example are wheat root rot (Bipolarissorokinlana (Sacc) Shoem), wheat stem rot (Fusarium graminearum), pepper root rot (Bipolarissorokinlana (Sacc) Shoem) Fusarium solani (Mart.) App.et Wollenw) and wheat sheath blight (Rhizoctonia cereadis Vander Hoeven) were provided by the Plant Pathology Laboratory, School of Plant Protection, Shandong Agricultural University.

The results are shown in Figures 9, 10, 11, 12 and Table 1, the multifunctional strain CW-2 has a certain biocontrol effect on wheat root rot, wheat stem rot, pepper root rot and wheat sheath blight , the antibacterial rates were 33.34%, 16.67%, 13.68% and 11.29%, respectively.

Table 1: Results of confrontation test between CW-2 strain and soil-borne pathogens

Example 8: Study on the growth-promoting potential of Brevibacterium cold-resistant CW-2

Salkowski's developer: 1 mL of 0.5mol/L FeCl ₃ , 50 mL of 35% perchloric acid solution.

Peptone ammoniated medium: 5 g of peptone, 1000 mL of deionized water, pH 7.2, sterilized at 121 °C for 20 min.

(1) Determination of ammonia production capacity:

The screened Brevibacterium cold-resistant CW-2 was inoculated into LB liquid medium for activation, and the bacterial liquid of the strain was diluted to OD ₆₀₀ ≈ 1.0 with sterile water, and 10 μL of the bacterial liquid was inoculated into peptone ammoniated medium. The medium of antagonistic bacteria was the control, and each treatment was repeated 3 times, 28±2℃, 180rpm shaking culture for 5d. After the incubation, centrifuge at 10,000 rpm for 10 min, add 1 mL of Nessler's reagent to the supernatant, and observe the change of the solution. If an orange or yellow precipitate is produced, it means that the strain has ammonia-producing ability. The more the precipitate, the stronger the ammonia-producing ability.

The results are shown in Figure 13 and Table 2. Brevibacterium cold resistance CW-2 has strong ammonia production ability, indicating that Brevibacterium cold resistance CW-2 has deaminase, which can deaminate amino acids to generate ammonia and various acids, thereby Conducive to plant growth and development.

(2) Determination of the ability to produce auxin:

The screened Brevibacterium cold-resistant CW-2 was inoculated into LB liquid medium for activation and culture, and the bacterial liquid of each strain was diluted to OD ₆₀₀ ≈ 1.0 with sterile water, and 10 μL of bacterial liquid was inoculated into LB liquid culture containing tryptophan. In the base (1 liter of LB liquid medium contains 5 mmol tryptophan), the medium without antagonistic bacteria was used as the control, and each treatment was repeated 3 times, 28 ± 2 ° C, 180 rpm shaking culture for 2 d. After culturing, centrifuge at 10000rpm for 10min, take 1mL of supernatant, add an equal amount of Salkowski's chromogenic reagent, let stand in the dark for 30min to make the culture solution develop color, then observe the color change of the solution, if the solution turns pink, it means that the antagonistic bacteria It has the ability to produce auxin IAA, and the darker the color, the stronger the ability to produce IAA.

The results are shown in Figure 14 and Table 2. Brevibacterium cold-resistant CW-2 has a strong ability to produce auxin, which is beneficial to promote plant growth and development.

Table 2: Results of CW-2 strain growth promotion test

Note: "+" means positive reaction, "-" means negative reaction, the more "+", the stronger the ability to produce ammonia or auxin.

Example 9: Study on Phosphorus Dissolving Ability of Brevibacterium Cold-resistant Brevibacterium CW-2

Pikovaskain's medium: glucose 10g, 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 0.02 g, FeSO ₄ ·7H ₂ O 0.02 g, agar 20 g, deionized water 1000 mL, pH 7.0, sterilized at 121° C. for 20 min.

The screened Brevibacterium cold-resistant CW-2 was inoculated into LB liquid medium for activation, and the bacterial solution of each strain was diluted with sterile water to OD ₆₀₀ ≈ 1.0, and 10 μL of the bacterial solution was inoculated into the center of Pikovaskain's medium plate. The medium of antagonistic bacteria is the control, and each treatment is repeated 3 times. The plate is placed upside down at 28 ± 2 °C for 7 days, and then the edge of the strain is observed. If a transparent circle appears, it means that the strain has the ability to dissolve phosphorus. The stronger the phosphorus ability.

The results are shown in Figure 15, indicating that Brevibacterium hardy CW-2 has a certain ability to dissolve phosphorus, and can convert organic phosphorus compounds in the soil into phosphates or convert insoluble phosphorus in the soil into soluble phosphorus, which can increase the amount of available phosphorus in the soil. Phosphorus content is beneficial to plant growth.

In summary, Brevibacterium hardy CW-2 is a strain that can efficiently degrade chlorpyrifos, and has a certain degradation effect on profenofos, oxytetracycline and tylosin, and has a strong function of producing biofilms. The pathogens wheat root rot, wheat stem rot, pepper root rot and wheat sheath blight have a certain biocontrol effect, and have the ability to promote growth of ammonia and auxin, and have a certain ability to dissolve phosphorus. It can be used in water and soil to achieve the purpose of remediating chlorpyrifos, oxytetracycline and tylosin polluted soil, preventing and controlling soil-borne diseases and promoting growth.

The above descriptions are only preferred embodiments of the present application, and are not intended to limit the present application. For those skilled in the art, the present application may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of this application shall be included within the protection scope of this application.

SEQUENCE LISTING

<110> Shandong Agricultural University

<120> A multifunctional strain of Brevibacterium cold-resistant and its application

<130> 2022

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Claims (10)

1. Brevibacterium frigoritolerans (Brevibacterium frigoritolerans) CW-2 has a preservation number of: CCTCC NO: m20211598.

2. A microbial preparation comprising CW-2 of Brevibacterium frigoritolerans (Brevibacterium frigoritolerans) according to claim 1.

3. The microbial preparation according to claim 2, wherein the Brevibacterium frigoritolerans (CW-2) is present in the form of a cultured viable bacterium, a fermentation broth of a viable bacterium, or a filtrate of a culture of a strain.

4. The microbial inoculum according to claim 3, wherein the microbial inoculum is in the form of a liquid, an emulsion, a suspension, a powder, a granule, a wettable powder or a water dispersible granule.

5. Use of Brevibacterium frigoritolerans (Brevibacterium frigoritolerans) CW-2 according to claim 1 or Brevibacterium frigoritolerans (Brevibacterium frigoritolerans) CW-2-containing microbial agent according to any one of claims 2 to 4 for at least one of the following (1) to (10):

(1) degrading organophosphorus pesticide;

(2) preparing a product for degrading organophosphorus pesticide;

(3) degrading antibiotics;

(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 insoluble phosphorus in the soil.

6. Use according to claim 5, characterized in that the organophosphorus pesticide is chlorpyrifos and/or profenofos.

7. Use according to claim 5, characterized in that the antibiotic is oxytetracycline and/or tylosin.

8. The use according to claim 5, wherein the soil-borne disease bacteria are one or more of Helminthosporium putrescentii (Bipolar is sorokinana (Sacc) Shoem), Fusarium graminearum (Fusarium graminearum), Fusarium solani (Mart.) App.et Wellenw), and Rhizoctonia cerealis Vander Ho even.

9. The use according to claim 5, wherein the soil-borne disease is wheat root rot, wheat stalk rot, pepper root rot and/or wheat sharp eyespot.

10. A method for simultaneously degrading organophosphorus pesticide and antibiotics in soil, improving soil quality and promoting crop growth is characterized by comprising the following steps:

brevibacterium frigoritolerans (Brevibacterium frigoritolerans) CW-2 according to claim 1 or Brevibacterium frigoritolerans (Brevibacterium frigoritolerans) CW-2 according to any one of claims 2 to 4 is applied to soil to be treated.

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