Strain Aureimonas ureilyticaL12 and application thereof

Abstract

The invention discloses a strain Areimonas ureilytica L12 and an application of the strain Areimonas ureilytica L12. According to the invention, the strain, namely the Aureimonas ureilytica L12, capable of degrading the glyphosate is separated from fresh leaves of male plants of the glyphosate-resistant amaranth, and the degradation rate of the strain on the glyphosate is high. The strain L12 provided by the invention enriches a germplasm resource library of pesticide degrading bacteria, and by using the strain, the environmental residue of glyphosate can be remarkably reduced, and the harm of glyphosate to an ecological system and human health is reduced.

Description

Technical Field

[0001] The present invention belongs to the field of microbial degradation, and in particular, relates to a strain Aureimonas ureilytica L12 and its applications. Background Art

[0002] Glyphosate was chemically synthesized in 1959 and started to be used as an agricultural herbicide in 1971. It is widely used to remove weeds in crops due to its high efficiency, broad spectrum and low cost. It has been found that it can remove more than 100 annual weeds and 60 perennial weeds, becoming the most widely used and largest-yield pesticide in the world, and its annual sales volume has always been the first among pesticides. However, with the promotion and use of glyphosate around the world, some negative effects have become increasingly obvious. Large amounts of glyphosate used will remain in the environment, and glyphosate remaining in the environment will not only affect the ecosystem and destroy the ecological balance, but may also have adverse effects on animals, plants and microorganisms. Glyphosate remaining in the food chain enters the human body through bioaccumulation, causing the concentration in the human body to exceed the concentration in the environment, and even causing systemic poisoning in the human body, threatening human health. Therefore, how to eliminate glyphosate residues in the soil has become an urgent problem for humans to solve.

[0003] There are three methods for degrading residual pesticides: physical degradation, chemical degradation, and biological degradation. Compared with physical degradation and chemical degradation, microbial degradation has the advantages of high degradation efficiency, multiple metabolic pathways, and no secondary pollution. It is currently the main means of removing residual pesticides in soil and water environments, and can effectively reduce the hazards of residual pesticides. There are many microorganisms that degrade organophosphorus pesticides, including bacteria, fungi, actinomycetes, and algae. Among them, bacteria occupy an important position in the microorganisms that degrade organophosphorus pesticides due to their multiple physiological and biochemical adaptability and the easy induction of mutant strains. The use of microorganisms and the degradation enzymes they produce to degrade organophosphorus pesticide residues in the environment has shown good results and is the main direction of research on organophosphorus pesticide degradation in recent years.

[0004] At present, many microbial strains that are resistant to glyphosate or can degrade and utilize glyphosate have been obtained at home and abroad, which play an important role in eliminating the enrichment of glyphosate in nature and preventing ecological damage. However, the degradation of glyphosate by microorganisms also has different effects due to different strains, and the current strains have degradation problems and few species, which seriously restricts the research on glyphosate-degrading microorganisms. This study isolated endophytes from the glyphosate-resistant population of Amaranthus serrata and determined its glyphosate-degrading ability, aiming to screen strains that can degrade glyphosate to enrich the bacterial library. Summary of the invention

[0005] The purpose of the present invention is to provide a bacterium that can degrade glyphosate - a strain Aureimonas ureilytica L12 and its applications.

[0006] In order to achieve the purpose of the present invention, in the first aspect, the present invention provides a glyphosate-degrading strain, Auromonas frostii L12, which is isolated from fresh leaves of glyphosate-resistant male plants of Palmer amaranth and is classified and named Aureimonas Ureilytica The strain has been deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101, with deposit number CGMCC No. 33169 and deposit date December 23, 2024.

[0007] In a second aspect, the present invention provides a microbial preparation comprising one or more selected from bacterial bodies, bacterial powder, bacterial suspension or bacterial liquid extract of the strain L12.

[0008] In a third aspect, the present invention provides a method for preparing the microbial preparation, which comprises the step of culturing the strain L12.

[0009] Furthermore, the strain L12 was cultured under the following conditions: 28° C., 180 rpm shaking in the dark.

[0010] Furthermore, the strain L12 is cultured using an improved enrichment medium.

[0011] The formula of the improved enrichment medium is: glucose 1g / L, peptone 5g / L, potassium nitrate 1g / L and sodium chloride 2g / L, glyphosate (added to a concentration of 100mg / L); pH 6.6-7.6.

[0012] In a fourth aspect, the present invention provides the use of the strain L12 or the microbial preparation in degrading glyphosate.

[0013] In a fifth aspect, the present invention provides a method for degrading glyphosate, the method comprising: inoculating the strain L12 into a modified enrichment medium containing glyphosate.

[0014] In one embodiment of the present invention, the method for degrading glyphosate comprises: 600 The value of the bacterial suspension is 0.5 (the content of live bacteria in the bacterial suspension is about 1.67×10 7 CFU / mL) was inoculated into 30 mL of modified enrichment medium containing 100 mg / L glyphosate and cultured at 28°C in a shaking incubator at 180 rpm in the dark for 5 days.

[0015] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: The present invention provides a bacterium capable of degrading glyphosate - a strain Aureimonas ureilyticaL12, OD 600 1 mL of bacterial suspension with a value of 0.5 was inoculated into 30 mL of improved enrichment medium containing 100 mg / L glyphosate, and after culturing at 180 rpm and 28°C for 5 days, the glyphosate content in the culture supernatant (0.5 mL) was detected, and the results showed that the degradation rate of glyphosate by the bacteria could reach 85.44%. The strain L12 provided by the present invention enriches the germplasm resource library of pesticide-degrading bacteria, and the use of the strain can significantly reduce the environmental residue of glyphosate and reduce its harm to the ecosystem and human health. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The figure shows the growth and colony morphology distribution of strain L12 on LB solid plate in a preferred embodiment of the present invention.

[0017] Figure 2 This is a phylogenetic tree diagram of the 16S rRNA of strain L12 in a preferred embodiment of the present invention.

[0018] Figure 3 It is a scatter plot of the relationship between glyphosate and peak area of ​​strain L12 in a preferred embodiment of the present invention.

[0019] Figure 4 This is a bar graph of the degradation rate of glyphosate by strain L12 in a preferred embodiment of the present invention.

[0020] Figure 5 The morphology of the colonies of strain L12 in a preferred embodiment of the present invention on LB medium with pH values ​​of 5, 5.3, 5.4, 7, 8, 9, and 10 (from left to right) is shown. DETAILED DESCRIPTION

[0021] The present invention is based on the background that glyphosate remains in the soil and causes harm to the human body. Starting from the field of microbial degradation, a strain capable of degrading glyphosate is isolated from fresh leaves of glyphosate-resistant male Palmer amaranth plants. The details are as follows: The fresh ground liquid of Palmer amaranth leaves was diluted and inoculated on TSA / R2A / LB / NA solid culture media by coating plates. After cultivation, the strains were purified and identified. The strains significantly enriched in the isolated resistant population were inoculated into LB culture medium and cultured for 24 hours. The cultured bacterial liquid was centrifuged at 3800 rpm and the supernatant was discarded. The bacterial cells were washed twice with sterile water and the concentration of OD was 0.04. 600 The value was 0.5 and inoculated into 30 mL of improved enrichment medium containing 100 mg / L glyphosate. After 5 days, samples were collected and the glyphosate residue was determined, thus obtaining a strain that can degrade glyphosate - Auromonas frostii L12.

[0022] The 16S rRNA of the bacterium was amplified using bacterial primers (27F and 1492R), and the PCR product fragment was sequenced by a bioengineering sequencing company (SEQ ID NO: 1). The spliced ​​sequence was uploaded to the NCBI website for sequence alignment, and phylogenetic analysis was performed using MEGA (version 7.0) software. Finally, the strain was determined to be Aureimonas Ureilytica .

[0023] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0024] Example 1 Classification, purification and identification of strain L12 1. Sample collection The seeds of Amaranthus palmensis were collected from Jilin, China, and fresh leaves were collected from the plants after planting.

[0025] 2. Isolation and identification of endophytes from Amaranthus avicularis leaves Collect fresh leaves of resistant Palmer amaranth plants and rinse them with running water. Transfer about 0.05g of leaves to a 50mL centrifuge tube for surface disinfection, rinse with sterile water for 30s, treat with 75% alcohol for 1min, treat with 2% NaClO for 3min, and finally rinse with sterile water for 30s. Add 10mL of sterile water to grind into a suspension, and dilute it into different multiples, and finally select a 10-fold plate for purification. Pipette 100μL of the dilution and inoculate it on TSA / R2A / LB / NA solid culture medium by plate coating. After drying, place it at 28℃ for 2d (the culture conditions will be changed according to the specific situation), and pick a single colony with a sterile pipette tip for streak purification. The growth of strain L12 on LB solid plates and the distribution of colony morphology are shown in Figure 1 Finally, the DNA of the purified strains was observed and extracted, and the 16S rRNA gene of the isolated and cultured bacteria was identified by high-throughput sequencing using double-sided tag PCR amplification to determine the taxonomic status of the bacteria.

[0026] The 16S rRNA gene was amplified using bacterial primers (27F and 1492R). The PCR fragment was sequenced by Bioengineering Sequencing Company. The spliced ​​sequence was uploaded to the NCBI website for sequence alignment, and phylogenetic analysis was performed using MEGA (version 7.0) software. The phylogenetic tree constructed is shown in the figure. Figure 2 Finally, strain L12 was determined to be Aureimonas ureilytica .

[0027] 3. Microbiological characteristics of colonies The colony morphology of colony L12 cultured on LB solid plate for 2 days is as follows Figure 1 As shown, the colonies are round, yellow, with smooth edges, shiny surfaces, a raised center, and opaque. Strain L12 is Gram-negative.

[0028] Example 2 Determination of glyphosate degradation by bacterial strains The strains significantly enriched in the isolated resistant population were inoculated into LB liquid culture medium and cultured in a shaker at 180 rpm and 28°C. The culture medium was centrifuged to obtain the bacterial cells, which were washed twice with sterile water and then measured at a concentration of OD 600 The value of the bacterial suspension is 0.5 (the content of live bacteria in the bacterial suspension is about 1.67×10 7 CFU / mL) was inoculated into 30 mL of modified enrichment medium containing 100 mg / L glyphosate. The sample was cultured at 180 rpm and 28°C in the dark for 5 days. The residual amount of glyphosate in the culture medium was determined by pre-column derivatization-high performance liquid chromatography. Each treatment was repeated three times, and the sample without the strain was the control group.

[0029] The formula of the improved enrichment medium is: glucose 1g / L, peptone 5g / L, potassium nitrate 1g / L, sodium chloride 2g / L, glyphosate (added to a concentration of 100mg / L); pH value 6.6-7.6. The method for determining glyphosate residue is as follows: Preparation of blank solution: Accurately measure 7mL of water in a 50mL volumetric flask, add 0.5mL of 50% sulfuric acid solution, 0.1mL of 250g / L potassium bromide solution, and 0.5mL of 14g / L sodium nitrite solution in sequence, plug the stopper tightly and shake well. After the total system reacts for 20min (the reaction temperature is not less than 15℃), dilute to the scale with pure water and shake well, and let it stand for 15min. Preparation of standard solution: Take 10mg of glyphosate (glyphosate purity is 95%) and add it to 100mL of improved enrichment liquid culture medium containing glyphosate to prepare a mother solution with a concentration of 100mg / L. Take 5 50mL volumetric flasks and number them, add 0.05mL, 0.25mL, 0.5mL, 1mL, 2mL glyphosate mother solution and 6.95mL, 6.75mL, 6.5mL, 6mL, 5mL water, then add 0.5mL sulfuric acid solution, 0.1mL potassium bromide solution, 0.5mL sodium nitrite solution, plug the stopper, shake well, react for 20min, dilute to the scale with pure water, shake well, and let stand for 15min to obtain glyphosate standard solutions with concentrations of 0.1mg / L, 0.5mg / L, 1.0mg / L, 2.0mg / L and 4mg / L. Preparation of sample solution: After the strain is cultured in the improved enrichment medium for 5 days, centrifuge and take the supernatant to carry out nitrosation reaction according to the method of treating the standard solution. Take the blank solution as the control, record the absorbance value at a wavelength of 242 nm, and fit the standard curve.

[0030] Calculate the degradation rate of glyphosate by the bacterial community: Degradation rate (%) = (C1-C2) / C2×100% Where: C1 is the original concentration of glyphosate; C2 is the glyphosate concentration obtained from the standard curve. The unit is mg / L.

[0031] After culturing in 100 mg / L improved enrichment medium for 5 days, the degradation rate of glyphosate by the strain was 85.44% ( Figure 3 and Figure 4 ).

[0032] Example 3 Stress resistance experiment of strain L12 In order to explore the acid and alkali resistance of strain L12, the normal growth pH condition of strain L12 is 7. In this experiment, single colonies were transferred to LB culture medium with pH values ​​of 5, 5.3, 5.4, 7, 8, 9, and 10 (pH was adjusted using hydrochloric acid and sodium hydroxide solution), and the growth of the strain was observed after culturing at 28°C for 2 days.

[0033] Figure 5 The results showed that as the alkalinity of the culture medium increased, the growth of the strain gradually deteriorated, and the alkalinity resistance could reach a pH of 10; while acidity had a greater impact on the strain, and the strain grew extremely poorly at a pH of 5. As the pH rose, the strain's growth ability gradually became stronger.

[0034] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. Strains Aureimonas ureilytica L12, the deposit number is CGMCC No. 33169.

2. A microbial preparation, characterized in that: It comprises one or more selected from the bacterial bodies, bacterial powders, bacterial suspensions or bacterial liquid extracts of the strain L12 according to claim 1.

3. The method for preparing the microbial preparation according to claim 2, characterized in that: The preparation method comprises the step of culturing the strain L12.

4. The preparation method according to claim 3, characterized in that: The strain L12 was cultured at 28°C and 180 rpm in a shaking in the dark.

5. The preparation method according to claim 3 or 4, characterized in that: Cultivating the strain L12 using a modified enrichment medium; The formula of the improved enrichment medium is: 1 g / L glucose, 5 g / L peptone, 1 g / L potassium nitrate and 2 g / L sodium chloride, glyphosate is added to a concentration of 100 mg / L; pH value is 6.6-7.

6.

6. Use of the strain L12 according to claim 1 or the microbial preparation according to claim 2 in degrading glyphosate.

7. A method for degrading glyphosate, characterized in that: The method comprises: inoculating the strain L12 of claim 1 into a modified enrichment medium containing glyphosate; The improved enriched culture medium is the same as the culture medium described in claim 5.

8. The method according to claim 7, characterized in that OD 600 1 mL of the bacterial suspension with a value of 0.5 was inoculated into 30 mL of the improved enrichment medium containing 100 mg / L glyphosate and cultured in a shaking incubator at 28°C and 180 rpm in the dark for 5 days.

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