Bacillus strains capable of degrading prometryn and / or acetochlor and uses thereof

Abstract

The present application relates to a bacillus strain capable of degrading prometryn and / or acetochlor. The bacillus strain is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 30590, which can be used for degrading prometryn and / or acetochlor.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a Bacillus strain capable of degrading prometryn and / or acetochlor. Background Art

[0002] Prometryn is structurally stable and remains stable for 12 to 18 months after application. Its low mobility in soil allows it to adsorb onto solid surfaces or suspended sediments in water. Therefore, long-term and excessive use of Prometryn can cause serious soil contamination, posing toxicological risks to plants and the land ecosystem.

[0003] Acetochlor is generally stable in the environment and is not easily hydrolyzed or photolyzed.

[0004] Large-scale application of cypermethrin, sethoxydim and their combination will cause obvious phytotoxicity to crops and bring serious pollution to the ecological environment of farmland.

[0005] The residues of acetochlor and prometryn herbicides in soil generally disappear through physical, chemical, and biological processes. Using microorganisms to degrade prometryn and acetochlor has the beneficial properties of being inexpensive, effective, safe, and clean. Summary of the Invention

[0006] One of the present invention provides a Bacillus arachidis The strain NM-1 was deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number CGMCC No. 30590.

[0007] The second aspect of the present invention provides an engineered bacterium obtained by genetically improving the strain NM-1 described in one of the present inventions; the engineered bacterium obtained after the genetic improvement is an engineered strain obtained by introducing a plasmid carrying a functional gene, or an engineered strain obtained by recombining a functional gene into the genome of a wild strain; the functional gene is at least one of a gene for controlling plant pests, a gene for controlling plant pathogenic microorganisms, and a gene that enhances the effect of the strain NM-1 in degrading prometryn and / or acetochlor.

[0008] The third aspect of the present invention provides a composition comprising the strain NM-1 as described in one of the present inventions or the engineered bacteria as described in the second aspect of the present invention.

[0009] In one embodiment, the composition further comprises an acceptable carrier.

[0010] In a specific embodiment, the dosage form of the composition is one of a suspension, a powder and a granule.

[0011] In one embodiment, the composition is in the form of an oil suspension or a wettable powder.

[0012] The fourth aspect of the present invention provides the use of the strain NM-1 according to one of the present inventions, the engineered bacteria according to the second aspect of the present invention, or the composition according to any one of the third aspects of the present invention in the degradation of prometryn and / or acetochlor.

[0013] Beneficial effects of the present invention: The present invention found that the Bacillus strain NM-1 can efficiently degrade prometryn or acetochlor.

[0014] Strain Deposit: The strain screened in the present invention that has a degrading effect on prometryn or acetochlor is named NM-1. The strain is deposited in the General Microbiology Center of the China Microorganism Culture Collection Committee with a deposit number of CGMCC No. 30590 and a deposit date of May 10, 2024. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Its system classification is Bacillus arachidis . BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The degradation rate curves of prometryn and acetochlor by the NM-1 isolate in 1 / 2 LB medium are shown.

[0016] Figure 2 The degradation rates of prometryn in each treatment group of Example 3 are shown.

[0017] Figure 3 The graph shows the degradation rate of acetochlor by each treatment group in Example 3.

[0018] Figure 4 The soybean root lengths for each treatment at various time points in Example 4 are shown.

[0019] Figure 5 The soybean plant heights for each treatment at various time points in Example 4 are shown.

[0020] Figure 6 The moisture content of soybeans in each treatment at various time points in Example 4 is shown.

[0021] Figure 7 The soybean root volumes for each treatment at various time points in Example 4 are shown.

[0022] Figure 8 The chlorophyll content in soybean leaves of each treatment at each time point in Example 4 is shown.

[0023] Figure 9 The carotenoid content in soybean leaves of each treatment at each time point in Example 4 is shown.

[0024] Figure 10 The root activity of each treatment at each time point in Example 4 is shown.

[0025] Figure 11 The colony morphology and color of the NM-1 isolate are shown.

[0026] Figure 12 The isolated Gram results for the NM-1 isolate are shown.

[0027] Figure 13 The results of physiological and biochemical characterization of the NM-1 isolate are shown.

[0028] Figure 14 The phylogenetic tree of the NM-1 strain is shown.

[0029] Figure 15 Shown is the growth curve of the NM-1 strain cultured in 1 / 2 LB medium containing 100 mg / L each of prometryn and acetochlor. DETAILED DESCRIPTION

[0030] The above contents of the present invention are further described in detail below in the form of preferred implementation cases, but they do not constitute a limitation of the present invention.

[0031] Unless otherwise specified, the reagents in the embodiments of the present invention can be purchased through commercial channels.

[0032] LB liquid medium: 1% peptone, 0.5% yeast extract, 1% NaCl, sterilized at 121°C for 20 min.

[0033] LB solid medium: 1% peptone, 0.5% yeast powder, 1% NaCl, 1.4% agar powder, sterilized at 121°C for 20 min.

[0034] Inorganic salt culture medium containing prometryn and acetochlor: distilled water, 0.5 g / L K2HPO4, 0.8 g / L KH2PO4, 0.2 g / L MgSO4·H2O, 0.1 g / L CaCl2·H2O, 0.005 g / L FeSO4, 0.0033 g / L Na2MoO4, 50 mg / L prometryn (or gradually increasing the concentration from 50 mg / L to 100 mg / L, 150 mg / L, and 200 mg / L), 50 mg / L acetochlor (or gradually increasing the concentration from 50 mg / L to 100 mg / L, 150 mg / L, and 200 mg / L). The mass ratio of prometryn to acetochlor in the culture medium is 1:1.

[0035] Example 1: Isolation of strains.

[0036] 5 g of the collected soil sample was put into 100 mL of an inorganic salt medium containing prometryn and acetochlor (wherein the initial concentrations of prometryn and acetochlor were 50 mg / L, respectively), mixed evenly, and cultured aerobically at 30°C and 180 r / min for 7 days to obtain a first enrichment culture; then, the first enrichment culture was inoculated into a new inorganic salt medium containing prometryn and acetochlor (wherein the concentrations of prometryn and acetochlor were 100 mg / L, respectively) at an inoculum size of 5% (v / v), mixed evenly, and cultured aerobically at 30°C and 180 r / min for 7 days to obtain a second enrichment culture; then, the second enrichment culture was inoculated into a new inorganic salt medium containing prometryn and acetochlor (wherein the concentrations of prometryn and acetochlor were 150 mg / L) at an inoculum size of 5% (v / v), mixed evenly, and cultured aerobically at 30°C and 180 r / min for 7 days. d, obtaining the third enrichment culture; repeat this process three times until the prometryn and acetochlor concentrations reach 200 mg / L, ultimately obtaining the fourth enrichment culture. The fourth enrichment culture was diluted, plated onto a solid LB plate, and incubated at 30°C. Colonies with distinct morphologies were selected and streaked onto the LB plate to isolate and purify different strains. Each isolate was numbered.

[0037] Example 2: Degradation analysis of prometryn or acetochlor.

[0038] Single colonies of each isolated strain growing on LB solid medium were picked and inoculated into LB liquid medium. The culture was shaken at 30°C and 180 rpm for 24 h, and the cells were collected by centrifugation at 8000 rpm for 5 min. Sterile water was added to the culture solution until the concentration reached OD 600 =1.0, and obtain bacterial suspensions of each isolated strain.

[0039] Inoculate 100 mL of 1 / 2LB medium (with prometryn and acetochlor added to an initial concentration of 100 mg / L each) with a 5% (v / v) suspension of each isolate. Incubate in a constant-temperature shaker at 30°C, 180 rpm, protected from light, to obtain culture fluid at various time points. Sample 15 mL of culture fluid starting on day 1, and then at the same time daily until day 7.

[0040] Extract residual prometryn and acetochlor from the culture medium: Add 15 mL of dichloromethane to 15 mL of culture medium sampled at each time point. Shake vigorously for 10 minutes, then let stand at room temperature for 20 minutes. After the sample is separated, discard the supernatant with a pipette. Pour the lower layer into a round-bottom flask and evaporate to dryness using a rotary evaporator. Add chromatography-grade methanol to dissolve the residue to obtain a methanol solution. Filter the methanol solution through a 0.22-μm organic phase microporous filter to obtain a filtrate, which is then placed into a sample bottle.

[0041] Liquid chromatography detection conditions for prometryn and acetochlor in the filtrate:

[0042] The chromatographic column was a C18 reversed-phase column (4.6 mm × 250 mm, 5 μm), with a mobile phase of (acetonitrile, volume:water = 60:40), a flow rate of 1 ml / min, a column temperature of 35°C, a detection wavelength of 215 nm, an injection volume of 10 μl, and retention times of approximately 9.24 minutes for prometryn and approximately 11.13 minutes for acetochlor. The degradation rate of prometryn or acetochlor was calculated based on the residual amount of prometryn or acetochlor. The calculation formula for the degradation rate was: X = (CCK-CX) × 100% / CCK. Where: X is the degradation rate of prometryn or acetochlor; CCK is the initial concentration of prometryn or acetochlor in 1 / 2 LB culture medium; and CX is the residual concentration of prometryn or acetochlor in the culture medium.

[0043] The degradation rate curve was drawn based on the degradation rate at each time point. Figure 1 Specifically, isolate NM-1 was able to degrade 64% of prometryn and 91% of acetochlor within 7 days. The degradation rates increased over time.

[0044] Example 3: Remediation of herbicide-contaminated soil.

[0045] The experimental soil was divided into two parts. In the first part, prometryn and acetochlor were artificially added to fresh, non-sterile soil (prometryn and acetochlor were dissolved in methanol and evenly sprayed onto the soil surface, followed by stirring). The concentration of prometryn and acetochlor in the soil was 100 mg / kg, respectively, to produce a herbicide-non-sterile soil mixture. The herbicide-soil mixture was then dried in a fume hood until the methanol completely evaporated. In the second part, the soil was sterilized (121°C for 50 minutes) and all other conditions were the same as in the first part. Finally, a herbicide-sterile soil mixture was prepared.

[0046] The strain NM-1 was inoculated into LB medium containing 100 mg / L of prometryn and acetochlor for enrichment culture. 600 = 1.0, and then the cells were collected and washed 3 to 5 times with sterile water. The cells were then resuspended in sterile water to a concentration of 1 × 108 CFU / mL, and obtain NM-1 bacterial suspension.

[0047] Experimental treatments: (A) Weigh 200 g of the herbicide-unsterilized soil mixture, add 10 mL of sterile water, and stir evenly to obtain the herbicide-unsterilized soil negative control group; (B) Weigh 200 g of the herbicide-unsterilized soil mixture, add 10 mL of NM-1 bacterial suspension, and stir evenly to obtain the herbicide-unsterilized soil treatment group; (C) Weigh 200 g of the herbicide-sterilized soil mixture, add 10 mL of sterile water, and stir evenly to obtain the herbicide-sterilized soil negative control group; (D) Weigh 200 g of the herbicide-sterilized soil mixture, add 10 mL of NM-1 bacterial suspension, and stir evenly to obtain the herbicide-sterilized soil treatment group. Each treatment group was replicated three times. The experiment was conducted at ambient temperature, with water sprayed daily to ensure that the soil moisture was around 70%. Samples were taken every three days, with 10 g of sample taken each time, until the 15th day of treatment to detect the residual amounts of cypermethrin and acetochlor in each group of soil, and then to evaluate the remediation effect of NM-1 on soil contaminated with cypermethrin and acetochlor.

[0048] Extraction of promethazine and acetochlor in soil: 20 mL of dichloromethane was added to 10 g of the test soil sample collected at each time point for each treatment. The sample was vigorously shaken in a shaker for 1 h, left to rest for 1 h, and centrifuged at 12,000 r / min for 15 min. All the supernatant was aspirated and concentrated using a rotary evaporator. The dichloromethane was evaporated and the remaining promethazine and acetochlor were dissolved in 2 mL of chromatographic grade methanol. The sample was filtered through a 0.22 μm organic phase filter membrane to obtain a filtrate. The filtrate was stored in a liquid phase bottle and the content of promethazine and acetochlor was finally determined by HPLC.

[0049] The residues of prometryn and acetochlor in the soil of each treatment group were determined using a high-performance liquid chromatography (HPLC) instrument (Agilent 1260 Infinity II) equipped with a reversed-phase C18 column (4.6 × 100 mm, 5 μm). The HPLC detection conditions for prometryn and acetochlor in the filtrate were: 10 μL injection volume, 1.0 mL / min flow rate, 35°C column temperature, detection wavelength at 215 nm, and a mobile phase of acetonitrile:water (60:40 v:v).

[0050] The degradation rate of prometryn or acetochlor is calculated based on the residual amount of prometryn or acetochlor. The degradation rate calculation formula is: X = (CCK - CX) × 100% / CCK. Where: X is the degradation rate of prometryn or acetochlor; CCK is the initial concentration of prometryn or acetochlor in the soil; CX is the residual concentration of prometryn or acetochlor in the soil.

[0051] The degradation rates of prometryn on the 7th, 14th and 21st days were shown in Table 2. Figure 2 The degradation rate of acetochlor is shown in Figure 3 . Figure 2 and Figure 3 The results show the residual dynamics of prometryn or acetochlor in four different soil treatments. After the addition of strain NM-1, the degradation rates of prometryn in the herbicide-unsterilized soil treatment group and the herbicide-sterilized soil treatment group were 90.7% and 84.8% respectively after 21 days. Furthermore, the degradation rates of acetochlor in the herbicide-unsterilized soil treatment group and the herbicide-sterilized soil treatment group were 93.9% and 86.5% respectively after 21 days. This indicates that strain NM-1 significantly improved the degradation efficiency of prometryn and acetochlor in soil. Therefore, strain NM-1 can be used to remediate soils contaminated with prometryn and / or acetochlor.

[0052] Example 4: Potted plant experiment.

[0053] The seedling raising soil was dry soil that passed through a 30-mesh sieve and was not contaminated by cypermethrin and acetochlor.

[0054] Preparation of pesticides: Prepare mother solutions of 100 mg / L each of prometryn and acetochlor.

[0055] Seedling cultivation: Soak the soybean seeds for germination, and culture them in an artificial incubator until they reach the two-leaf and one-heart stage. Select the seedlings with uniform growth and strong growth and transplant them into pots with a diameter of 7.5 cm. Put 0.5 kg of sieved dry soil in each pot.

[0056] Three treatments were designed for the experiment: control group (CK), in which the soil in the potting bucket was not inoculated with microorganisms and no herbicide was sprayed; treatment group 1 (NM), on the day of seedling transplanting, the soil in the potting bucket was evenly sprayed with the mother solutions of prometryn and acetochlor, and stirred until the final concentrations of prometryn and acetochlor in the soil were both 50 mg / kg; treatment group 2 (NMJ), on the day of seedling transplanting, the mother solutions of prometryn and acetochlor were evenly sprayed with the soil in the potting bucket until the final concentrations of prometryn and acetochlor in the soil were both 50 mg / kg, and the herbicide was sprayed at a concentration of 1×10 8 CFU / mL of NM-1 strain bacterial suspension, stir evenly.

[0057] Each pot was irrigated with water, ensuring the same soil moisture across treatments. Thirty replicates were used for each treatment. Soil samples were collected 7, 14, 21, and 28 days after application (random multi-point sampling). The collected soil samples were divided into two parts: one subsample was used to analyze soil enzyme activities, and the other subsample was used to analyze changes in microbial community structure. Additionally, soybean plant root length, plant height, water content, root volume, chlorophyll content, carotenoid content, and root activity were measured 7, 14, and 21 days after application. Soil sucrase, cellulase, urease, and catalase activities were measured using the 3,5-dinitrosalicylic acid colorimetric method, the anthrone colorimetric method, the sodium phenolate-sodium hypochlorite colorimetric method, and ultraviolet absorption spectrophotometry. Among them, the root length measurement method is: dig the plant out of the soil completely, clean the root system, and then use a ruler or tape measure to directly measure the longest length of the root; the plant height measurement method is: use a ruler or measuring tool to measure from the base of the plant (usually the root neck) to the top of the plant (the top of the main stem or the growth point); water content: select fresh, disease-free leaves, clean them to remove surface impurities, put the leaves in an oven, and dry them at a constant temperature of 105 degrees Celsius for 30 minutes to constant weight, record the leaf dry weight, and then place the dried leaves in a known volume of water, ensuring that the leaves are completely sunk and in contact with the water surface, allowing them to fully absorb water until they no longer absorb water, take out the water-absorbing leaves, gently absorb excess water with filter paper, and then quickly weigh them under dry conditions to obtain the saturated water leaf weight, and calculate the leaf saturated water content by the formula: Leaf saturated water content = (saturated leaf weight - Leaf dry weight) × 100%‌ / leaf dry weight; Root volume (drainage method): Carefully dig out the roots, gently rinse with water until there is no sand or soil, keeping the roots intact, dry them with absorbent paper, immerse the roots in a volume meter, and record the reading of the rising water level. Remove the roots, add water again to the initial water level, and record the amount of water added, which is the root volume; Chlorophyll content and carotenoid content (spectrophotometric method): Take fresh plant leaves, wipe off the surface dirt, cut them into pieces, weigh a certain amount of sample and put it into a mortar. Add an appropriate amount of 95% ethanol or 80% acetone, grind into a homogenous slurry, and then let it stand for a while (to fully extract the chlorophyll), filter into a volumetric flask, rinse the mortar and filter paper with ethanol to ensure that all the chlorophyll is completely extracted, and finally dilute to volume with ethanol to obtain an extract. Use a spectrophotometer to measure the absorbance A of the extract at wavelengths of 665nm, 649nm, and 470nm. 665 、A 649 and A 470 , chlorophyll a content is calculated based on the formula: Ca 叶绿素a =13.95A 665 -6.88A 649 ; Chlorophyll b content: Cb 叶绿素b =24.96A 649 -7.32A665 ; and carotenoid content: Cx 类胡萝卜素 =(1000A 470 -2.05Ca-114.8Cb) / 245); Root activity (TTC): Weigh 0.5g of the root tip and place it in a 25ml beaker (for the control experiment, sulfuric acid was added first, followed by the root sample; all other procedures were the same). Add 10ml of a mixture of equal parts 0.4% TTC solution and Tris-HCl buffer, pH 8.4, and fully immerse the roots in the mixture. Incubate at 37°C in the dark for 2 hours. Afterward, add 2ml of 1mol / l sulfuric acid (except for the control) to stop the reaction. Remove the roots, blot dry with absorbent paper, transfer to the original beaker, and add 6ml of 95% ethanol. Extract for 30 minutes until the red substance is completely extracted. The extract was transferred to a 10ml centrifuge tube, washed repeatedly with ethanol 2 to 3 times, and all the extracts were transferred to a centrifuge tube. Finally, 95% ethanol was added to make the volume up to 10ml. Centrifuged at 4000 rpm for 10min, cooled, and colorimetrically measured at 485nm using a spectrophotometer. The root activity was calculated based on the measured absorbance value.

[0058] See the results Figures 4 to 10 .

[0059] The results showed that after being stressed by prometryn and acetochlor, the plant height, root length, chlorophyll concentration and root activity of soybean were inhibited, while inoculation with strain NM-1 alleviated the stress of prometryn and / or acetochlor on soybean.

[0060] Prometryn and / or acetochlor inhibited soybean root length, plant height, water content, root volume, chlorophyll content, carotenoid content, and root activity. Strain NM-1 alleviated the inhibitory effects of prometryn and / or acetochlor on soybean root length. Root length and root activity in the NMJ treatment were lower than those in the CK treatment, but higher than those in the NM treatment. At 21 days, chlorophyll and carotenoid concentrations in the NMJ treatment were higher than those in the CK treatment. Chlorophyll and carotenoids are associated with plant photosynthesis, and the presence of strain NM-1 promoted the accumulation of organic matter in soybeans.

[0061] Example 5: Taxonomic identification of isolated strain NM-1.

[0062] Colony morphology of NM-1 isolate: NM-1 isolate was cultured in an inverted manner on LB solid medium at 30°C for 24 h. The colony morphology was observed and photographed. Figure 11 .

[0063] according to Figure 11 It can be seen that the colonies of the NM-1 isolate are white, round, smooth and opaque.

[0064] Gram identification of NM-1 isolates: drop a drop of sterile water onto the slide, gently pick up the NM-1 bacterial moss with an inoculating loop and place it in sterile water, gently stir to make the bacterial solution uniform, stain and decolorize according to the Gram staining solution operation method, and then observe under an oil immersion lens. The staining results of NM-1 are all blue-purple, indicating that it is a Gram-positive bacterium. The staining results are shown in Figure 12 .

[0065] Physiological and biochemical identification of NM-1 isolates: The biochemical index identification tube kit was purchased from Guangdong Huankai Microbiology Technology Co., Ltd. The kit was used to perform physiological and biochemical identification of NM-1 isolates. The results are shown in Figure 13 .

[0066] According to Bergey's Manual of Systematic Bacteriology, the strain was preliminarily identified as Bacillus by observing its morphology and performing physiological and biochemical tests. Bacillus .

[0067] Total DNA from each isolate was extracted according to the bacterial DNA genomic kit protocol. 16S rDNA was amplified by PCR using primers 27F (shown in SEQ ID No. 1) and 1492R (shown in SEQ ID No. 2) and total DNA from the NM-1 isolate as a template. The amplified PCR products were analyzed by gel electrophoresis and then sent to Shanghai Sangon Technology Co., Ltd. for sequencing. The 16S rDNA sequence for the NM-1 strain is shown in SEQ ID No. 3.

[0068] The 16S rDNA sequence results were compared with the data in the EZ BioCloud nucleotide database. The appropriate species sequence was selected based on the comparison results. The phylogenetic tree of the NM-1 strain was constructed using MEGA-7 for comparison analysis. Figure 14 .according to Figure 14 The phylogenetic tree shows that the NM-1 isolate is Bacillus arachidis Located in the same branch.

[0069] In summary, the systematic classification of NM-1 isolates is Bacillus arachidis .

[0070] The NM-1 isolate was deposited in the General Microbiology Center of the China Culture Collection Administration under the deposit number CGMCC No. 30590, on May 10, 2024, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Bacillus arachidis .

[0071] Example 6: Growth curve of NM-1 isolation.

[0072] The activated bacterial suspension of NM-1 isolate (1×10 8 CFU / mL) was inoculated into 1 / 2LB medium containing 100 mg / L of prometryn and 100 mg / L of acetochlor at 5% of the culture volume (volume / volume: v / v). The culture was carried out under aerobic conditions, darkness, temperature of 30°C, and rotation speed of 180 r / min to determine the growth curve. During the culture process, the OD was measured every 2 h. 600 The experiment was repeated three times. Figure 15 As can be seen from the figure, the growth of the NM-1 isolate is in the lag phase from 0h to 2h, the logarithmic growth phase from 2h to 20h, the stable phase from 20h to 36h and then the decline phase.

Claims

1. One plant Bacillus arachidis The strain NM-1 was deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number CGMCC No. 30590.

2. An engineered bacterium obtained by genetically modifying the strain NM-1 according to claim 1; The engineered bacteria obtained after genetic improvement are engineered strains obtained by introducing plasmids carrying functional genes, or engineered strains obtained by recombining functional genes into the genome of wild strains; The functional gene is at least one of a gene for preventing and controlling plant pests, a gene for preventing and controlling plant pathogenic microorganisms, and a gene for enhancing the effect of the strain NM-1 in degrading prometryn and / or acetochlor.

3. A composition comprising the strain NM-1 according to claim 1 or the engineered bacteria according to claim 2.

4. The composition according to claim 3, characterized in that The composition also includes an acceptable carrier.

5. The composition according to claim 3, characterized in that The dosage form of the composition is one of a suspension, a powder and a granule.

6. The composition according to claim 3, characterized in that The composition is in the form of an oil suspension or a wettable powder.

7. Use of the strain NM-1 according to claim 1, the engineered bacterium according to claim 2, or the composition according to any one of claims 3 to 6 for degrading prometryn and / or acetochlor.

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