Burkholderia G1 and application thereof

Abstract

The invention discloses a Burkholderia sp. G1. The Burkholderia sp. G1 has the preservation number of GDMCC No: 64694 in the Guangdong Microbial Culture Collection Center, and the preservation number of the Burkholderia sp. G1 in the Guangdong Microbial Culture Collection Center is GDMCC No: 64694. The bacterial strain is applied to degradation of pyraclostrobin, and experimental results show that the bacterial strain not only can effectively degrade pyraclostrobin in a wide pH and temperature range, but also shows certain plant growth promoting characteristics; the strain can be used as an excellent pesticide degradation and plant growth promotion bifunctional bacterium to be applied to bioremediation of polluted environments such as water, soil or crops; the discovery of the strain provides dual solutions for treating pesticide residues and promoting plant growth, further expands the application range of a bioremediation technology, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial degradation, and specifically relates to a strain of Burkholderia ( Burkholderia sp.) G1 and its application in degrading pyraclostrobin and promoting plant growth. Background Art

[0002] Pyraclostrobin, molecular formula: C 19 H 18 N3O4Cl, also known as pyraclostrobin and pyraclostrobin, belongs to the strobilurins fungicide class. It was discovered by BASF in Germany in 1993 and launched on the market in 2001. Pyraclostrobin is a fungicide that inhibits the growth of cytochromes B and C. l Pyraclostrobin inhibits mitochondrial respiration by inhibiting electron transfer, making mitochondria unable to produce and provide energy (ATP) required for normal cell metabolism, ultimately leading to cell death. It is a type of fungicide widely used to prevent and control fungal diseases in crops, and can prevent and control Ascomycetes, Basidiomycetes, Deuteromycetes and Oomycetes. Pyraclostrobin has the characteristics of broad spectrum, high efficiency and low toxicity, and is used in crops such as rice, corn, wheat, peanuts, vegetables, bayberry, tea trees, etc.

[0003] Because pyraclostrobin has the characteristics of wide range and high efficacy, as a good fungicide, as people understand it, the application areas, populations, targets and dosages are constantly increasing. Pyraclostrobin spreads through irrigation and surface flow, causing serious pollution to soil and water ecosystems and endangering the environment. The harm of pyraclostrobin to non-target organisms is becoming increasingly prominent, especially to fish, invertebrates and amphibians in aquatic ecosystems. It is relatively safe for mammals and birds, but it can also cause certain chronic diseases under long-term exposure. The mechanism of action of pyraclostrobin itself is to inhibit mitochondrial respiration and damage DNA. The dual effects lead to excessive production of cellular ROS and cell apoptosis. It also has genetic toxicity to human liver cells and potential risks to the human body.

[0004] The accumulation of toxic and carcinogenic environmental pollutants such as pesticides, plastics, and dyes containing hydrocarbons has caused serious harm to the ecosystem. Therefore, it is necessary to develop a rapid, easy-to-use, and socially acceptable environmentally friendly method to solve the problem of pyraclostrobin pollution. The research on microbial degradation of pesticides began in the late 1940s. Among the existing methods for treating pesticide residues, microbial degradation has attracted much attention from researchers at home and abroad due to its advantages of safety, low cost, convenient operation, and no secondary pollution. However, the specificity of microbial degradation is relatively obvious, so it is necessary to screen for efficient degradation strains. At present, there are very few studies on pyraclostrobin-degrading bacteria, and there is no report on pyraclostrobin bioremediation. Summary of the invention

[0005] In view of the shortcomings of the degradation and remediation technology of pyraclostrobin fungicide pesticide residues, the present invention provides a Burkholderia species ( Burkholderia sp.) G1, which was deposited in Guangdong Microbiological Culture Collection Center on May 27, 2024, with the deposit number GDMCC No: 64694, and the deposit address is: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0006] Another object of the present invention is to Burkholderia sp. G1 is used in the degradation of pyraclostrobin and the promotion of plant growth.

[0007] In order to achieve the above purpose, the present invention adopts the following technical solutions: 1. Screening and enrichment of strain G1 The present invention collects soil from a Panax notoginseng plantation in Guangnan County, Wenshan Zhuang and Miao Autonomous Prefecture, Yunnan Province, adds the soil to an MSM liquid culture medium containing pyraclostrobin for continuous enrichment culture, separates and purifies multiple strains capable of degrading pyraclostrobin, and then uses high performance liquid chromatography (HPLC) to detect their degradation abilities, and finally screens out the strain G1 with the strongest degradation ability.

[0008] 2. Identification of strain G1 (1) Morphological characteristics: Strain G1 forms round, small and raised milky white colonies on LB medium. The surface is smooth, moist and opaque, with neat edges. Scanning electron microscopy showed that its cells were rod-shaped and single, and Gram staining was negative.

[0009] (2) Molecular identification: The genomic DNA of strain G1 was extracted using a bacterial genomic DNA extraction kit. The extracted genome was used as a template and 16S rDNA bacterial universal primers were used for PCR amplification. After the PCR product was sequenced, the sequencing results were compared with the sequences on NCBI. Combined with the morphological characteristics and molecular identification results, the strain was finally identified as Burkholderia ( Burkholderia sp.).

[0010] 3. Degradation performance and optimal conditions The strain G1 was applied to degrade pyraclostrobin, and the experimental results showed Burkholderia sp The degradation rate of G1 on pyraclostrobin was as high as 83.22% within 7 days, and its optimal degradation conditions were: pH 7, temperature 30℃, and inoculation amount 10%; the results showed that strain G1 had significant efficiency and stability in degrading pyraclostrobin, and its optimal degradation conditions were relatively mild and easy to achieve in practical applications; 4. Strain G1 was applied to promote plant growth. The results showed that Burkholderia G1 has the ability to fix nitrogen, dissolve phosphorus and potassium, and can promote plant growth.

[0011] The present invention has the following beneficial effects: The present invention screened Burkholderia sp . The G1 strain showed significant effects in remediating water and soil contaminated by pyraclostrobin. The strain can not only effectively degrade pyraclostrobin, but also has the ability to fix nitrogen, dissolve phosphorus and potassium, promote plant growth, and has multiple environmental restoration functions. Burkholderia sp. The discovery of G1 further enriched the germplasm resource library of pesticide-degrading bacteria and provided a new technical means for the bioremediation of pyraclostrobin contamination. It is expected to break through the current difficulties in pesticide residue management and open up new application directions. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The colony morphology of strain G1 cultured on LB solid medium for 1 day; Figure 2 is the scanning electron micrograph of strain G1; Figure 3 is the phylogenetic tree of 16S rDNA of strain G1; Figure 4 This is the standard curve diagram for HPLC detection of pyraclostrobin; Figure 5 The dynamic relationship of pyraclostrobin degradation by strain G1; Figure 6 The degradation effect of strain G1 on pyraclostrobin under different pH conditions; Figure 7The degradation effect of strain G1 on pyraclostrobin at different temperatures; Figure 8 The degradation effect of strain G1 on different concentrations of pyraclostrobin; Fig. 9 The growth of strain G1 on Ashby nitrogen-free medium; Fig.10 This is the dynamic process of the phosphorus dissolving effect of strain G1; Fig.11 This is the potassium-solubilizing effect of strain G1 on improved Aleksandrov solid medium. DETAILED DESCRIPTION

[0013] The technical scheme of the present invention will be described in detail by specific embodiments and accompanying drawings, but it should be clear that the present invention is not limited to the following technical scheme. In the present invention, unless otherwise specified, the reagents, methods and equipment used are reagents, methods and equipment commonly used in the art. Those of ordinary skill in the art can implement and apply the present invention according to the relevant reference books, scientific and technological literature or instructions, manuals, etc. before the application date of the present invention, in combination with their own experience and professional knowledge.

[0014] The culture medium formulations in the following examples are as follows: MSM liquid medium (1L): (NH4)2SO4 2.0g, CaCl2·2H2O 0.01g, FeSO4·7H2O 0.001g, Na2HPO4·12H2O 1.5g, MgSO4·7H2O 0.2g, KH2PO41.5g, pH 7.0; MSM solid medium is prepared by adding 17-20g agar powder to every 1L of liquid medium; LB liquid medium: 5.0 g yeast extract, 10.0 g peptone, 10.0 g sodium chloride, 1000 mL deionized water, pH 7.2, sterilization at 121°C for 20 min; LB solid medium: add 17-20 g agar powder per 1 L of liquid medium.

[0015] Modified Aleksandrov solid medium: glucose 5g, MgSO4 0.5g, FeCl3 0.005g, CaCO3 0.1g, Ca3(PO4)2 2g, potassium-containing mineral 3g, agar 20g, bromothymol blue 0.1g, deionized water 1000mL; Ashby nitrogen-free solid medium: sucrose 10 g, NaCl 0.12 g, K2HPO4·3H2O 0.5 g, CaCO3 1 g, MgSO4·7H2O 0.2 g, agar 20 g, deionized water 1000 mL; NBRIP liquid culture medium: glucose 10 g, Ca2(PO4)3 5 g, MgCl2 5 g, MgSO4·7H2O 0.25 g, KCl 0.2 g, (NH4)2SO4 0.1 g, deionized water 1000 mL; Example 1: Isolation and identification of strain G1 1. Screening, enrichment and purification of pyraclostrobin-degrading strains Soil was collected from the Panax notoginseng plantation in Guangnan County, Wenshan Zhuang and Miao Autonomous Prefecture, Yunnan Province. 1 g of soil sample was weighed and added to 100 mL of MSM liquid culture medium containing 20 mg / L of pyraclostrobin. After culturing at 30 °C and 180 rpm for 7 days, the culture solution was taken at a 10% inoculation rate and inoculated into MSM liquid culture medium containing 40 mg / L of pyraclostrobin and cultured for 7 days. This was repeated to increase the concentration of pyraclostrobin in the MSM liquid culture medium to 60 mg / L and 100 mg / L in turn. The culture solution of the last concentration was taken for preservation. The stored culture medium was diluted 10-fold to 1×10 -5 Then spread it on MSM solid medium containing 25 mg / L pyraclostrobin and culture it upside down at 30°C for 2 days. After a single colony grows on the plate, pick a single colony and streak it multiple times to purify it, and separate and obtain multiple purified strains. The culture fluid of the purified strain was added to 50 mL MSM liquid medium containing pyraclostrobin (25 mg / L) and cultured for 3 days. The concentration of pyraclostrobin in the culture fluid was detected by high performance liquid chromatography (HPLC). The concentration of pyraclostrobin was determined according to the size of the HPLC peak area. At the same time, the culture medium without culture fluid was used as the control group. The strain G1 with the strongest degradation ability was obtained, and its degradation rate was 65.85%, while the degradation rate of the blank control was 44.12%; 2. Identification of strain G1 (1) Morphological identification The strain G1 was inoculated on a solid LB plate and inverted for 1 day at 30°C to observe the colony morphology; the colonies cultured on the LB plate for 1 day were round, small, and convex milky white colonies with a smooth, moist, opaque surface and neat edges (see Figure 1 Scanning electron microscopy showed that the cells were rod-shaped, single bacteria, and Gram staining was negative (see Figure 2 ).

[0016] (2) 16S rDNA molecular biological identification The bacterial genomic DNA extraction kit (Solebo) was used to extract the genomic DNA of strain G1. The genomic DNA of strain G1 was extracted according to the operating instructions of the kit. The extracted genome was used as a template and 16S rDNA bacterial universal primers (27F: 5'-AGAGTTGATCCTGGCTCAG-3'; 1429R: 5'-GGTTACCTTGTTACGACTT-3') were used for PCR amplification. The PCR product was entrusted to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The 16S rDNA sequence measured by the strain was compared and analyzed in the GenBank database using BLAST, and related sequences with higher homology were selected to construct a phylogenetic tree and analyze the evolutionary relationship using MAGE 5.0 software. As a result, the 16S rDNA sequence of strain G1 separated and purified by the present invention was consistent with that of Burkholderia sp strain MMS16-CNU072 has a homology of 77% and is the closest in evolution; its morphological characteristics are also most similar to those of Burkholderia sp.

[0017] Example 2: Degradation experiment of pyraclostrobin by strain G1 (1) Seed solution preparation The purified strain G1 was inoculated into 100 mL LB liquid medium and placed at 30°C and 150 rpm for overnight activation culture until the bacteria entered the logarithmic growth phase. Subsequently, 30 mL of the culture solution was transferred to a 50 mL sterilized centrifuge tube and centrifuged at 4000 rpm for 10 min at 4°C. The bacteria were collected and rinsed with sterile water, and 30 mL of sterile water was added to prepare the seed solution of strain G1.

[0018] (2) Degradation performance determination At an inoculation rate of 10%, G1 seed solution was inoculated into 50mL of MSM liquid medium containing 25mg / L pyraclostrobin, and a medium without inoculation of bacteria was set as a control group, with three replicates in each group. Culture was carried out in a constant temperature shaker at 30℃ and 150 rpm for 7 days, and samples were taken once a day. When sampling, 1mL was taken out from the MSM medium, 1mL of acetone was added, and ultrasonic treatment was performed for 20 min, followed by 1mL of ethyl acetate, shaking for 1min, and the lower layer was removed; after repeating the above extraction process twice, rotary evaporation was performed, and finally, the amount of pyraclostrobin in the MSM liquid medium was determined by HPLC every day.

[0019] Chromatographic conditions: Shimadzu LC-2030 high performance liquid chromatograph was used; the chromatographic column was Elite C 18Reversed phase column (Supersil AQ-C18, 250 nm×4.60 mm, 5 μm), injection temperature 30°C, injection volume 10 μL, flow rate 1.000 mL / min, mobile phase: V (acetonitrile) ∶ V (water) = 80 ∶ 20, detection wavelength 276 nm; Quality control: The standard curve of pyraclostrobin was prepared by calibrating the standard substance with external standard method according to the conventional method (see Figure 4 ); According to the degradation rate formula, the degradation rate of pyraclostrobin in the 7-day sample was calculated. Pyraclostrobin degradation rate (%) = [1-(A1 / A0)] × 100%, A1 is the concentration of pyraclostrobin in the culture medium after strain G1 treatment for 7 days, A0 is the initial concentration of pyraclostrobin in the culture medium; Results Figure 5 The degradation rate of pyraclostrobin by strain G1 was the fastest in the first day of culture. With the extension of culture time, the degradation rate of pyraclostrobin decreased. When pyraclostrobin was used as the only carbon source, the degradation rates reached 50.46% and 58.38% after 1 and 2 days of culture, respectively, and the degradation rate slowed down after the fourth day.

[0020] The results showed that strain G1 could use pyraclostrobin as the sole carbon source and energy source for growth and reproduction. When the concentration of pyraclostrobin was 25 mg / L, the degradation rate was 50.46% after 1 day of cultivation, and 83.22% after 7 days of cultivation, indicating that the strain has the ability to efficiently and quickly degrade pyraclostrobin.

[0021] Example 3 Study on the degradation characteristics of strain G1 on pyraclostrobin 1. Effect of pH value on the degradation of pyraclostrobin by strain G1 Into 50mL MSM liquid medium with different pH values ​​(5.0, 6.0, 7.0, 8.0, 9.0), G1 seed solution was inoculated at 10% inoculation rate, and pyraclostrobin was added to a final concentration of 25mg / L. Three replicates were added to each group. After 4 days of culture in a constant temperature shaker at 30℃ and 150 rpm, HPLC was used to determine the degradation of pyraclostrobin by strain G1 under different pH conditions. Results Figure 6 From the figure, it can be seen that the bacteria can degrade pyraclostrobin in the pH range of 5.0 to 9.0, and the degradation effect is best at pH 7.0. The acidic or alkaline environment affects the enzyme activity in G1, reducing the degradation efficiency of pyraclostrobin.

[0022] 2. Effect of temperature on the degradation of pyraclostrobin by strain G1 In 50mL MSM culture medium, G1 seed solution was inoculated at 10% inoculation volume, and pyraclostrobin was added to a final concentration of 50mg / L. The culture was placed in a constant temperature shaker at 20℃, 25℃, 30℃, 35℃, and 40℃, 150rpm, with three replicates in each group. After 4 days of culture, HPLC was used to determine the degradation of pyraclostrobin by strain G1 under different culture temperature conditions. Results Figure 7 From the figure, it can be seen that strain G1 can degrade pyraclostrobin in the range of 20-40℃, and the degradation rate of pyraclostrobin reaches the highest at 30℃, which is about 70.82%; when the temperature is higher than 30℃, the degradation ability decreases.

[0023] 3. Effect of initial concentration of pyraclostrobin on the degradation performance of strain G1 G1 seed solution was inoculated at 20% into 50 mL of MSM liquid culture medium containing different initial concentrations of pyraclostrobin (20 mg / L, 40 mg / L, 60 mg / L, 80 mg / L, and 100 mg / L), with three replicates in each group. After culturing at 30°C and 150 rpm for 4 d, HPLC was used to determine the degradation of pyraclostrobin by the strain at different initial concentrations.

[0024] Results Figure 8 From the figure, it can be seen that the strain can degrade pyraclostrobin in the range of 20-100 mg / L of initial pyraclostrobin concentration. When the initial concentration of pyraclostrobin is 40 mg / L, the degradation performance of strain G1 is the best; when the initial concentration increases to 60 mg / L, the degradation rate decreases.

[0025] Example 3: Study on the plant growth-promoting properties of strain G1 1. Nitrogen fixation effect of strain G1 Pour the frozen strain G1 into LB liquid medium for revival and enrichment. Pipette 1 mL of the enriched bacterial solution into 9 mL of sterile distilled water, repeat 4 times, and dilute to 10 -5 Pipette 100 μL of the dilution onto the Ashby nitrogen-free medium, seal it, invert it and place it in a 30°C constant temperature incubator for 3 days.

[0026] Results Fig. 9 ,It can be seen from the figure that after 3 days of cultivation, colonies grew on Ashby nitrogen-free medium, indicating that strain G1 can absorb and utilize nitrogen in the air as a nitrogen source and convert it into the energy needed for self-growth.

[0027] 2. Phosphate dissolving effect of strain G1 The frozen strain G1 was poured into LB liquid medium for resurrection and enrichment, the bacteria were collected, washed with sterile water, added to NBPIR liquid medium at a 2% inoculation rate, and cultured at 30°C and 150rpm for 4 days. Samples were taken every 1 day, and the phosphorus content in the NBPIR medium was determined by the molybdenum antimony colorimetric method; Results Fig.10 ,It can be seen from the figure that after 4 days of cultivation, the increase of available phosphorus in the culture medium is 14.36 mg / L, and it has the ability to convert insoluble phosphorus into soluble phosphorus.

[0028] 3. Potassium-dissolving ability of strain G1 In the clean bench, add 5 μL of G1 bacterial suspension to the prepared modified Aleksandrov solid medium plate, and use 5 μL of potassium-solubilized solid medium with sterile water as a blank control; place the plate in a 30°C constant temperature incubator for 3 days, and observe whether there is a yellow halo near the colony.

[0029] Results Fig.11 ,It can be seen from the figure that after 3 days of cultivation, a yellow halo appeared on the culture medium, indicating the ability to dissolve potassium.

Claims

1. A strain of Burkholderia Burkholderia sp.) G1, whose deposit number in Guangdong Microbiological Culture Collection Center is GDMCC No:64694.

2. Use of the Burkholderia G1 according to claim 1 in degrading pyraclostrobin.

3. The effect of Burkholderia G1 in promoting plant growth according to claim 1, characterized in that: Burkholderia G1 has the ability to fix nitrogen, dissolve phosphorus and dissolve potassium.

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