Bacillus strain capable of degrading prometryn and / or acetochlor and application

Abstract

The present invention 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 (CGMCC), the preservation number is CGMCC No.30590, and the bacillus strain 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] Promethazine has a stable structure and can remain stable for 12 to 18 months after application. It has a low mobility in the soil and can be adsorbed onto solid surfaces or suspended sediments in water. Therefore, long-term and excessive use of Promethazine will cause serious soil pollution, and further cause toxicological hazards to plants and the land ecological environment.

[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 promethazine herbicides in the soil generally disappear through physical, chemical and biological processes. Using microorganisms to degrade promethazine or acetochlor has the beneficial characteristics of being cheap, effective, safe and clean. Summary of the invention

[0006] One of the present invention provides a Bacillus arachidis The strain NM-1 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration 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 invention; the engineered bacterium obtained after 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 promethazine and / or sethoxydim.

[0008] The third aspect of the present invention provides a composition, which includes 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 a specific 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 aspect of the present invention in the degradation of promethazine 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 preservation: The strain screened in the present invention that has a degradation effect on promethazine or acetochlor is named NM-1, and the strain is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee, with a deposit number of CGMCC No.30590, a deposit date of May 10, 2024, and a deposit address of: 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 / 2LB medium are shown.

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

[0017] Figure 3 The degradation rate of acetochlor by each treatment group in Example 3 is shown.

[0018] Figure 4 The soybean root lengths of each treatment at each time point in Example 4 are shown.

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

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

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

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

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

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

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

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

[0029] Fig.15 The growth curve of the NM-1 strain cultured in 1 / 2LB medium containing 100 mg / L each of prometryn and acetochlor is shown. 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 to 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 powder, 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, K2HPO4 0.5g / L, KH2PO4 0.8g / L, MgSO4·H2O 0.2g / L, CaCl2·H2O 0.1g / L, FeSO4 0.005g / L, Na2MoO4 0.0033g / L, prometryn 50mg / L (or the concentration is gradually increased from 50mg / L to 100mg / L, 150mg / L and 200mg / L), acetochlor 50mg / L (or the concentration is gradually increased from 50mg / L to 100mg / L, 150mg / L and 200mg / L). The mass ratio of prometryn and 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 promethazine and acetochlor (wherein the initial concentrations of promethazine and acetochlor were 50 mg / L, respectively), mixed evenly, and cultured aerobically at 30°C and 180 r / min for 7 d to obtain the first enrichment culture; then the first enrichment culture was inoculated into a new inorganic salt medium containing promethazine and acetochlor (wherein the concentrations of promethazine 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 d to obtain the second enrichment culture; then the second enrichment culture was inoculated into a new inorganic salt medium containing promethazine and acetochlor (wherein the concentrations of promethazine 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 d, and the third enrichment culture was obtained; the whole process was repeated 3 times until the concentration of promethazine and acetochlor was 200 mg / L, and the fourth enrichment culture was finally obtained. The fourth enrichment culture was diluted, spread on an LB solid plate, and cultured at 30°C. Colonies with different morphologies were picked, and different isolated strains were obtained by streaking and purification on the LB plate. Each isolated strain was numbered.

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

[0038] Pick a single colony of each isolated strain growing on LB solid medium, inoculate it into LB liquid medium, shake and culture it in a shaker at 30°C and 180 r / min for 24 h, centrifuge at 8000 r / min for 5 min to collect the bacteria, and add sterile water to the bacterial solution until the concentration is OD 600 =1.0, and obtain bacterial suspension of each isolated strain.

[0039] Inoculate 100 mL 1 / 2LB medium (add promethazine and acetochlor to make the initial concentrations of promethazine and acetochlor both 100 mg / L) with 5% (v / v) of the bacterial suspension of each isolated strain. Cultivate in a constant temperature shaking incubator at 30°C and 180 r / min in the dark to obtain the culture solution at each culture time point. Among them, sample 15 mL of the culture solution from the first day of culture, and then sample at the same time point every day until the 7th day of culture.

[0040] Extract the residual promethazine and acetochlor in the culture medium: add 15 mL of dichloromethane to 15 mL of the culture medium sampled at each time point, shake vigorously for 10 minutes, and then stand at room temperature for 20 minutes. After the sample is layered, discard the supernatant with a pipette, pour the lower layer liquid into a round-bottom flask, evaporate the liquid to dryness with a rotary evaporator, add chromatographic grade methanol to dissolve the residue, and obtain a methanol solution. Filter the methanol solution through a 0.22 micron organic phase microporous filter to obtain a filtrate, which is placed in a sample bottle.

[0041] Liquid chromatography detection conditions of prometryn and acetochlor in the filtrate: The chromatographic column is a C18 reverse phase column (4.6 mm × 250 mm, 5 μm, mobile phase (acetonitrile volume: water volume = 60:40), flow rate: 1 ml / min, column temperature: 35 degrees Celsius, detection wavelength: 215 nm, injection volume: 10 μl, retention time: about 9.24 minutes for promethazine and about 11.13 minutes for acetochlor. The degradation rate of promethazine or acetochlor was calculated based on the residual amount of promethazine or acetochlor, and the calculation formula of the degradation rate was: X=(CCK-CX)×100% / CCK. In the formula: X is the degradation rate of promethazine or acetochlor; CCK is the initial concentration of promethazine or acetochlor in 1 / 2LB culture medium; CX is the residual concentration of promethazine or acetochlor in the culture medium.

[0042] The degradation rate curve was drawn according to the degradation rate at each time point. Figure 1 Specifically, isolate NM-1 was able to degrade 64% of promethazine and 91% of acetochlor within 7 days. The degradation rates increased with time.

[0043] Example 3: Remediation of herbicide-contaminated soil.

[0044] The experimental soil was divided into two parts. In the first part, promethazine and acetochlor were artificially added to the fresh non-sterilized soil (promethazine and acetochlor were dissolved in methanol, then evenly sprayed on the soil surface, and then stirred evenly), so that the concentrations of promethazine and acetochlor in the soil were 100 mg / kg, respectively, to obtain a herbicide-non-sterile soil mixture, and the herbicide-soil mixture was dried in a fume hood until the methanol was completely volatilized. In the second part, except that the soil was sterilized (121°C, 50min), the rest was the same as the first part, and finally a herbicide-sterilized soil mixture was obtained.

[0045] The strain NM-1 was inoculated into LB medium containing 100 mg / L of promethazine and acetochlor for enrichment culture. 600 =1.0, collect the cells, wash them 3 to 5 times with sterile water, and resuspend them in sterile water to a bacterial solution concentration of 1×10 8CFU / mL, and obtain NM-1 bacterial suspension.

[0046] Experimental treatment: (A) Weigh 200 g of herbicide-unsterilized soil mixture, add 10 mL of sterile water, stir evenly, and obtain the herbicide-unsterilized soil negative control group; (B) Weigh 200 g of herbicide-unsterilized soil mixture, add 10 mL of NM-1 bacterial suspension, stir evenly, and obtain the herbicide-unsterilized soil treatment group; (C) Weigh 200 g of herbicide-sterilized soil mixture, add 10 mL of sterile water, stir evenly, and obtain the herbicide-sterilized soil negative control group; (D) Weigh 200 g of herbicide-sterilized soil mixture, add 10 mL of NM-1 bacterial suspension, stir evenly, and obtain the herbicide-sterilized soil treatment group. Each treatment group was repeated three times. The experiment was carried out at ambient temperature, with water sprayed every day to ensure that the soil moisture was around 70%. Samples were taken every 3 days, with 10 g sampled each time, until the 15th day of treatment to detect the residual amounts of cypermethrin and setochlor in each group of soil, and then to evaluate the remediation effect of NM-1 on soil contaminated by cypermethrin and setochlor.

[0047] Extraction of promethazine and setochlor in soil: 20 mL of dichloromethane was added to 10 g of the soil sample to be tested collected at each time point in each treatment. The sample was shaken vigorously in a shaker for 1 h, then left to stand 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 setochlor were dissolved with 2 mL of chromatographic grade methanol. The sample was filtered with 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 setochlor was determined by HPLC.

[0048] The residues of promethazine and acetochlor in the soil of each treatment group were detected by HPLC (Agilent 1260 Infinity II) equipped with a reversed phase C18 column (4.6 × 100 mm, 5 μm). The HPLC detection conditions of promethazine and acetochlor in the filtrate were as follows: injection volume of 10 μL, flow rate of 1.0 mL / min, column temperature of 35 °C, detection wavelength of 215 nm, and mobile phase of acetonitrile: water = 60:40 (v:v).

[0049] The degradation rate of promethazine or acetochlor is calculated according to the residual amount of promethazine or acetochlor, and the calculation formula of the degradation rate is: X=(CCK-CX)×100% / CCK. In the formula: X is the degradation rate of promethazine or acetochlor; CCK is the initial concentration of promethazine or acetochlor in the soil; CX is the residual concentration of promethazine or acetochlor in the soil.

[0050] 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 residual dynamics of promethazine or acetochlor in four different soil treatments were shown. Among them, after adding strain NM-1, the degradation rate of promethazine in the herbicide-unsterilized soil treatment group was 90.7% at 21 days, and the degradation rate of promethazine in the herbicide-sterilized soil treatment group was 84.8% at 21 days; the degradation rate of acetochlor in the herbicide-unsterilized soil treatment group was 93.9% at 21 days, and the degradation rate of acetochlor in the herbicide-sterilized soil treatment group was 86.5% at 21 days. It can be seen that strain NM-1 significantly improved the degradation efficiency of promethazine and acetochlor in soil. Therefore, strain NM-1 can be used to remediate soil contaminated with promethazine and / or acetochlor.

[0051] Example 4: Potted plant experiment.

[0052] The soil for raising seedlings was dry soil that passed through a 30-mesh sieve and was not contaminated by cypermethrin and acetochlor.

[0053] Preparation of pesticides: prepare stock solutions of 100 mg / L each of promethazine and sethoxydim.

[0054] 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 potting barrels with a diameter of 7.5 cm. Put 0.5 kg of sieved dry soil in each potting barrel.

[0055] The experiment set up three treatments: control group (CK), 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 solution of promethazine and acetochlor, and stirred evenly until the final concentration of promethazine and acetochlor in the soil was 50 mg / kg; treatment group 2 (NMJ), on the day of seedling transplanting, the mother solution of promethazine and acetochlor was evenly sprayed on the soil in the potting bucket until the final concentration of promethazine and acetochlor in the soil was 50 mg / kg, and the spraying concentration was 1×10 8 CFU / mL of NM-1 strain bacterial suspension was stirred evenly.

[0056] Each potting barrel was irrigated with water to ensure that the soil moisture of each treatment group was the same, and each treatment was replicated 30 times. Soil samples were collected on the 7th, 14th, 21st and 28th day after application (random multi-point sampling method). The collected soil samples were divided into two parts, one of which was used to analyze the activity of soil enzymes and the other was used to analyze the changes in microbial community structure. In addition, the changes in root length, plant height, water content, root volume, chlorophyll content, carotenoid content and root activity of soybean plants were measured on the 7th, 14th and 21st day after application. The activities of soil sucrase, cellulase, urease and catalase were determined by 3,5-dinitrosalicylic acid colorimetry, anthrone colorimetry, sodium phenol-sodium hypochlorite colorimetry and ultraviolet absorption method, respectively. 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, dry them at a constant temperature of 105 degrees Celsius for 30 minutes to constant weight, record the dry weight of the leaves, and then place the dried leaves in a known volume of water to ensure 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 absorbed leaves, gently absorb excess water with filter paper, and then quickly weigh them under dry conditions to obtain the saturated water-containing 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, rinse gently with water until there is no sand, keep the roots intact, use absorbent paper to absorb the water, immerse the roots in a volume meter, and record the reading of the rising water level. Take out the roots, add water to the initial water level again, 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 and 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 homogenate, and 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 chlorophyll is completely extracted, and finally use ethanol to make up the volume to obtain the 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 sample root tip and put it into a 25ml small beaker (for the control experiment, sulfuric acid was added first and then the root sample, and other operations were the same), add 10ml of an equal amount of a mixture of 0.4% TTC solution and PH8.4 Tris-HCL buffer, fully immerse the roots in the mixture, keep it in the dark at 37℃ for 2h, and then add 2ml of 1mol / l sulfuric acid (except for the control) to stop the reaction. Take out the roots, use absorbent paper to absorb the water on the surface of the material, transfer it to the original small beaker, add 6ml of 95% ethanol first, and extract for 30min until the red substance is completely extracted. The extract was transferred to a 10 ml centrifuge tube, washed repeatedly with ethanol for 2 to 3 times, and all transferred to a centrifuge tube. Finally, 95% ethanol was added to make the volume up to 10 ml. Centrifuged at 4000 rpm for 10 min, cooled, and colorimetrically compared at 485 nm using a spectrophotometer. The root activity was calculated based on the measured absorbance value.

[0057] Results Figures 4 to 10 .

[0058] The results showed that after being stressed by promethazine and setochlor, the plant height, root length, chlorophyll concentration and root activity of soybean were inhibited, while the stress of promethazine and / or setochlor on soybean was alleviated after inoculation with strain NM-1.

[0059] Promethazine and / or acetochlor had an inhibitory effect on soybean root length, plant height, water content, root volume, chlorophyll content, carotenoid content and root activity. Strain NM-1 alleviated the inhibitory effect of promethazine and / or acetochlor on soybean. The root length and root activity of treatment group NMJ were lower than those of CK group, but higher than those of treatment group NM. At 21 days, the concentrations of chlorophyll and carotenoids in treatment group NMJ were higher than those in CK group. Chlorophyll and carotenoids are related to plant photosynthesis. The presence of strain NM-1 promoted the accumulation of organic matter in soybean.

[0060] Example 5: Taxonomic identification of isolated strain NM-1.

[0061] Colony morphology of NM-1 isolate: NM-1 isolate was cultured inverted on LB solid medium at 30°C for 24 h, and the colony morphology was observed and photographed. Fig.11 .

[0062] according to Fig.11 It can be seen that the colonies of the NM-1 isolate are white, round, smooth and opaque.

[0063] Gram identification of NM-1 isolates: drop a drop of sterile water on the slide, use an inoculation loop to gently pick up the NM-1 bacterial moss and put it into the 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 microscope. The staining results of NM-1 are all blue-purple, indicating that it is a Gram-positive bacterium. The staining results are shown in Fig.12 .

[0064] Physiological and biochemical identification of NM-1 isolates: The biochemical index identification tube kit was purchased from Guangdong Huankai Microbiological Technology Co., Ltd. The kit was used to perform physiological and biochemical identification of NM-1 isolates. The results are shown in Fig.13 .

[0065] According to Bergey's Manual of Systematic Bacteriology, the strain was preliminarily identified as Bacillus by observing its morphology and conducting physiological and biochemical tests. Bacillus .

[0066] According to the operation steps of the bacterial DNA genome kit, the total DNA of each isolate was extracted, and 27F (as shown in SEQ ID No. 1) / 1492R (as shown in SEQ ID No. 2) was used as primers, and the total DNA of the NM-1 isolate was used as a template to perform PCR amplification of 16S rDNA. After the amplified PCR product was detected by gel electrophoresis, the amplified product was sent to Shanghai Biotech Co., Ltd. for sequencing. Among them, the sequence of 16S rDNA measured by the NM-1 strain is (as shown in SEQ ID No. 3).

[0067] The sequence results of the 16S rDNA were compared with the data in the EZ BioCloud nucleotide database. The appropriate species sequence was selected according to the comparison results. The phylogenetic tree of the NM-1 strain was constructed using MEGA-7 for comparison analysis. Fig.14 .according to Fig.14 The phylogenetic tree shows that the NM-1 isolate is Bacillus arachidis Located in the same branch.

[0068] In summary, the systematic classification of NM-1 isolates is Bacillus arachidis .

[0069] The NM-1 isolate was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration with the deposit number CGMCC No. 30590 and the deposit date May 10, 2024 at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Its system classification is Bacillus arachidis .

[0070] Example 6: Growth curve of NM-1 isolation.

[0071] The activated NM-1 isolate bacterial suspension (1×10 8 CFU / mL) was inoculated into 1 / 2LB medium containing 100 mg / L of promethazine and 100 mg / L of acetochlor at 5% of the culture volume (volume / volume: v / v), and cultured in aerobic, dark, 30°C, and 180 r / min conditions to determine the growth curve. During the culture process, OD was measured every 2 h. 600 The experiment was repeated three times. The growth curve was drawn as shown in Fig.15 As can be seen from the figure, the growth of the NM-1 isolate is in the lag phase from 0h to 2h, in the logarithmic growth phase from 2h to 20h, and enters the stable phase and then the decay phase after 20h to 36h.

Claims

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

2. An engineered bacterium obtained by genetically modifying the strain NM-1 as claimed in 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 controlling plant pests, a gene for 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 as claimed in claim 1 or the engineered bacteria as claimed in 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 dosage form of the composition is oil suspension or wettable powder.

7. Use of the strain NM-1 according to claim 1, the engineered bacteria 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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