Amycolatopsis sp., amycolatopsis agent and application

Abstract

The present disclosure relates to a kind of Amycolatopsis, the taxonomic name of the Amycolatopsis is Amycolatopsis nivea, the preservation number of the Amycolatopsis is GDMCC NO:63776.The strain has been preserved in the preservation unit designated by the State Intellectual Property Office, Guangdong Provincial Microbial Culture Collection Center, the preservation date is September 8, 2023, and the preservation number is GDMCC NO:63776.The present disclosure also provides an Amycolatopsis agent, which contains a culture medium and a bacterial body, the bacterial body contains the Amycolatopsis with the preservation number GDMCC NO:63776.The strain has good ability to degrade herbicides, especially nitrosulfuron, nicosulfuron, chlorimuron-ethyl and ether sulfonate, and the degradation speed is relatively fast.

Description

Technical Field

[0001] This disclosure relates to the field of microbiology, specifically to a pseudo-amylopectin bacterium, a pseudo-amylopectin bacterium agent, and its application. Background Technology

[0002] Chemical pesticides are crucial for ensuring bountiful agricultural harvests today, with herbicides being particularly widespread and greatly contributing to crop yields. However, the extensive use of herbicides has caused significant harm to farmland and the surrounding ecological environment, seriously threatening human health and safety. Herbicide residue-related phytotoxicity has been reported worldwide, making the solution to the problem of environmental herbicide residues a subject of extensive research for scientists.

[0003] In the study of microbial degradation of herbicides, scholars both domestically and internationally have conducted extensive research on herbicide degradation. However, research on the microbial degradation of nicosulfuron is relatively scarce. To date, only a few nicosulfuron-degrading strains have been reported, but no microbial resources capable of completely mineralizing nicosulfuron have been found. Research on the degradation mechanism of nicosulfuron is still in its preliminary stages. The molecular responses within the microbial cells during the degradation process of nicosulfuron have not yet been studied, and there are problems such as limited strain diversity, a small number of degradation genes, and unclear degradation mechanisms. Therefore, continuously enriching the resources of nicosulfuron-degrading strains and developing highly efficient degrading agents are issues that deserve further attention. Summary of the Invention

[0004] The purpose of this disclosure is to provide a pseudo-amylopectin bacterium, a pseudo-amylopectin bacterium agent, and its application. This strain has a good ability to degrade herbicides, especially mesotrione, nicosulfuron, chlorpyrifos, and ethersulfuron, and the degradation rate is relatively fast.

[0005] To achieve the above objectives, the first aspect of this disclosure provides a pseudo-amycolatopsis bacterium, which is classified as Amycolatopsis nivea and has the accession number GDMCC NO: 63776.

[0006] Optionally, the 16S rRNA sequence of the pseudomycolic acid bacteria is shown in SEQ ID NO.1.

[0007] Optionally, the pseudoamycete is Gram-positive, round in shape with a raised center and wrinkled surface. The front of the colony is white, and the back is pale yellow, with white aerial hyphae and pale yellow matrix hyphae.

[0008] The second aspect of this disclosure provides a pseudo-amylopectin agent, the pseudo-amylopectin agent containing a culture medium and bacterial cells, the bacterial cells containing pseudo-amylopectin with the preservation number GDMCC NO: 63776.

[0009] Optionally, in each gram of the *Amylopectinus* agent, the viable count of *Amylopectinus* with accession number GDMCC NO: 63776 is (2.3-2.7) × 10⁻¹⁰. 8 CFU.

[0010] Optionally, the culture medium includes at least one of LB medium, ISP2 medium, GSM medium, Gao's No. 1 medium, and beef extract peptone medium.

[0011] The third aspect of this disclosure provides the application of the pseudo-amylopectin bacteria described in the first aspect or the pseudo-amylopectin bacteria agent described in the second aspect in the degradation of herbicides.

[0012] Optionally, the method includes the following step: applying the pseudo-amylopectin or pseudo-amylopectin inoculant to plant soil containing the herbicide.

[0013] Optionally, the herbicide includes triketone herbicides and / or sulfonylurea herbicides; preferably, the triketone herbicide includes mesotrione; and the sulfonylurea herbicide includes nicosulfuron, chlorpyrifos, and ethersulfuron.

[0014] Optionally, the amount of *Amylopectinobacterium tumefaciens* used relative to 1 mg of herbicide is (1.0-2.0) × 10⁻⁶. 4 CFU.

[0015] Through the above technical solution, this disclosure provides a pseudo-amylopectin bacterium, a pseudo-amylopectin bacterial agent, and its application. This strain has a good ability to degrade herbicides, especially mesotrione, nicosulfuron, chlorpyrifos, and fensulfuron-methyl, and the degradation rate is relatively fast, reducing the environmental impact of herbicides. In addition, this strain is also resistant to carbenicillin and ampicillin, and is not easily destroyed by carbenicillin and ampicillin.

[0016] Other features and advantages of this disclosure will be described in detail in the following detailed description section.

[0017] Information on the preservation of biological materials

[0018] The pseudo-amycolatopsis nivea is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on September 8, 2023, with accession number GDMCC NO: 63776. Attached Figure Description

[0019] The accompanying drawings are provided to further illustrate the present disclosure and form part of the specification. They are used together with the following detailed description to explain the present disclosure, but do not constitute a limitation thereof. In the drawings:

[0020] Figure 1 This is a colony morphology diagram of the disclosed *Amylopectinobacterium*.

[0021] Figure 2 This is a phylogenetic tree of the 16S rRNA gene of the disclosed pseudoamycetes.

[0022] Figure 3 This is a graph showing the degradation rate and absorbance of nicotinamide by the disclosed strain of *Amylopectin*.

[0023] Figure 4 This is a graph showing the degradation rate of sulfonylurea herbicides by the *Amylopectinobacterium* strain disclosed in this publication.

[0024] Figure 5 This is a pH value diagram of the degradation of sulfonylurea herbicides by the disclosed strain of *Amylopectinus*.

[0025] Figure 6 This is a plate graph for detecting antibiotic resistance in *Amylopectinobacterium* strains disclosed in this publication. Detailed Implementation

[0026] The specific embodiments of this disclosure will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit this disclosure.

[0027] The inventors of this disclosure isolated and cultured a new bacterial strain from the bottom sludge of a wastewater pond in a chemical plant in Beijing. The strain was identified as belonging to the genus *Amycolatopsis* and named *Amycolatopsis nivea*. This strain has been deposited at the Guangdong Provincial Center for Microbial Culture Collection, a designated depository of the State Intellectual Property Office, on September 8, 2023, with accession number GDMCC NO: 63776.

[0028] The first aspect of this disclosure provides a pseudo-amycolatopsis bacterium, which is classified as Amycolatopsis nivea and has the accession number GDMCC NO: 63776.

[0029] According to this disclosure, the 16S rRNA sequence of the pseudomycolic acid bacterium is shown in SEQ ID NO.1.

[0030] In one embodiment of this disclosure, the morphological identification of *Amylopectinobacterium* was performed, such as... Figure 1 As shown, the pseudoamycin bacterium is Gram-positive, round in shape with a raised center and wrinkled surface. The front of the colony is white, and the back is pale yellow. It has white aerial hyphae and light yellow matrix hyphae.

[0031] The second aspect of this disclosure provides a pseudo-amylopectin agent, the pseudo-amylopectin agent containing a culture medium and bacterial cells, the bacterial cells containing pseudo-amylopectin with the preservation number GDMCC NO: 63776.

[0032] According to this disclosure, the viable count of *Amylopectinobacterium pseudoformibrio* with accession number GDMCC NO: 63776 can vary within a wide range. In one embodiment of this disclosure, the viable count of *Amylopectinobacterium pseudoformibrio* with accession number GDMCC NO: 63776 per gram of the *Amylopectinobacterium pseudoformibrio* inoculum is (2.3-2.7) × 10⁻¹⁰. 8 CFU.

[0033] In one embodiment of this disclosure, the culture medium may include at least one selected from LB medium, ISP2 medium, GSM medium, Gao's No. 1 medium, and beef extract peptone medium. In this disclosure, the culture medium may be a liquid medium or a solid medium, preferably a liquid medium. In the above embodiments, the culture medium may be selected from any one of the ISP series of culture media, preferably ISP2 medium.

[0034] The third aspect of this disclosure provides the application of the pseudo-amylopectin bacteria described in the first aspect or the pseudo-amylopectin bacteria agent described in the second aspect in the degradation of herbicides.

[0035] In this disclosure, *Amylopectinus pseudobranchii* or *Amylopectinus pseudobranchii* inoculants have a good ability to degrade herbicides, thereby reducing the environmental impact of herbicides.

[0036] In one embodiment of this disclosure, the method includes the following steps: applying the pseudo-amylopectin or pseudo-amylopectin inoculant to plant soil containing the herbicide.

[0037] In this disclosure, *Amylopectinus pseudotruncatella* and its inoculant can be in liquid or solid form, preferably in liquid form. For example, it can be a bacterial solution containing *Amylopectinus pseudotruncatella* or a liquid inoculant containing *Amylopectinus pseudotruncatella*. The application can be carried out using methods conventionally employed by those skilled in the art, such as spraying the *Amylopectinus pseudotruncatella* or its inoculant onto the soil surface, or mixing the *Amylopectinus pseudotruncatella* or its inoculant with the soil until homogeneous.

[0038] In one embodiment of this disclosure, the herbicide includes triketone herbicides and / or sulfonylurea herbicides. In a preferred embodiment of this disclosure, the triketone herbicide includes mesotrione; the sulfonylurea herbicide includes nicosulfuron, chlorpyrifos, and ethersulfuron.

[0039] In one embodiment of this disclosure, the amount of *Amylopectinobacterium pseudobulb* used is (1.0-2.0) × 10⁻¹¹ mg relative to each 1 mg herbicide. 4CFU.

[0040] The present invention will be further described in detail below with reference to the embodiments, but the scope of the present invention is not limited to the following embodiments.

[0041] Unless otherwise specified, all materials used in this embodiment are commercially available products.

[0042] LB medium: 5g yeast extract, 10g tryptone, 10g NaCl, 20g agar, 1L H2O, adjust pH to 6.8-7.2, sterilize at 121℃ for 30min.

[0043] ISP2 medium: 10g malt extract, 4g yeast extract, 4g glucose, 20g agar, 1L H2O, adjust pH to 7.0-7.4, sterilize at 115℃ for 20min.

[0044] Gao's No. 1: 10g soluble starch, 1g KNO3, 0.5g K2HPO4, 0.5g MgSO4·7H2O, 0.5g NaCl, 0.01g FeSO4·7H2O, 20g agar, 1L H2O, adjust pH to 7.4-7.6, sterilize at 121℃ for 20min.

[0045] Beef extract peptone medium: 3g beef extract, 10g peptone, 5g NaCl, 20g agar, 1L H2O, adjust pH to 7.4-7.6, sterilize at 121℃ for 20min.

[0046] GSM medium: 5g glucose; 1g NH4Cl; 0.2g NaCl; 0.2g MgSO4·7H2O; 0.5g K2HPO4; 0.5g KH2PO4; adjust pH to 7.0±0.2, sterilize at 115℃ for 20min.

[0047] Physiological saline: 0.9% sodium chloride solution, sterilized at 115℃ for 20 min.

[0048] Example 1

[0049] This example illustrates the isolation and purification of pseudoamycetes strains.

[0050] (1) Isolation of pseudo-amycosis strains:

[0051] Sample collection: Take 10g of bottom sludge from the sewage pond of a chemical plant in Beijing and put it into an Erlenmeyer flask containing 100mL of sterile water and mix thoroughly.

[0052] Sample enrichment: Take 10 mL of the well-mixed suspension and add it to an Erlenmeyer flask containing 90 mL of GSM medium. Add herbicide at a concentration of 5 mg / L and incubate in the dark at 30°C with a constant speed of 160 rpm. Substitute into fresh medium every 7 days, adding herbicide at a concentration of 10 mg / L. Repeat this process every 7 days until the concentration reaches 50 mg / L, then substitute every 2 months, adding herbicide at a concentration of 50 mg / L. The herbicides included mesotrione, nicosulfuron, chlorpyrifos, and sulfadiazine, with a weight ratio of 1:1:1:1. Each subtraction is recorded as one generation, and the screening samples were taken from the 23rd generation.

[0053] (2) Purification of pseudo-amycosis strains:

[0054] Take the long-term enriched culture medium LAMC from the LAM laboratory 3+ The 23rd generation sample was serially diluted; 1 mL of the enrichment solution was added to 9 mL of sterile water, recorded as 10⁻¹. -1 After thorough mixing, add 1 mL of the diluted solution to 9 mL of fresh sterile water, mix thoroughly, and record as 10. -2 And so on, up to 10 -5 Select 10 -3 10 -4 10 -5 Three serial dilutions were used for plating, with 200 μL of each dilution applied to LB and GSM agar plates containing 50 mg / L nicosulfuron. The plates were incubated upside down at 30°C for 3-5 days. Afterward, as many different colony morphologies as possible were picked and streaked onto LB and GSM agar plates containing 50 mg / L nicosulfuron for isolation and purification. The resulting single colony, designated La24, was capable of tolerating or degrading nicosulfuron. The residual amount of nicosulfuron in the culture medium was detected using high-performance liquid chromatography (HPLC) to assess the ability of strain La24 to degrade nicosulfuron. Ultimately, La24 was identified as a highly efficient nicosulfuron-degrading strain.

[0055] Example 2

[0056] This example illustrates the morphological identification of *Amylopectinobacterium* strains.

[0057] (1) Select the purified strain La24 and culture it on ISP2 solid medium using the streak plating method for 3-4 days at 30℃. Then observe the colony morphology. The colony morphology of *Amylopectinobacterium* is as follows: Figure 1 As shown.

[0058] The colonies of *Amycium pseudoamycinum* with accession number GDMCC NO: 63776 are round with a raised center and wrinkled surface. The front of the colony is white and the back is pale yellow. It has white aerial hyphae and pale yellow matrix hyphae; the white aerial hyphae are in the form of white powder.

[0059] (2) It turns purple after Gram staining and is identified as a Gram-positive bacterium.

[0060] Example 3

[0061] This example illustrates the molecular biological identification of pseudomycolic acid bacteria strains:

[0062] Molecular biological identification was performed on the isolated and purified strain La24. The 16S rRNA gene sequence of strains resistant to or degrading mesotrione was amplified using the universal bacterial primers 27F and 1492R.

[0063] Amplification reaction system: 12.5 μL Mix; 8.5 μL ddH2O; 1 μL 27F; 1 μL 1492R; 2 μL bacterial extract.

[0064] Forward primer: 27F: 5'-AGAGTTTGATCCTGGCTCA-3' (SEQ ID NO.2);

[0065] Reverse primer: 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO.3).

[0066] The PCR product of the amplified 16S rRNA was sent to Beijing Bomeide Gene Technology Co., Ltd. for sequencing. The nucleotide sequence obtained by sequencing is shown in SEQ ID NO.1, and see the appendix sequence listing for details.

[0067] The nucleic acid sequences were compared using BLAST (Brain Search) in the GenBank database on NCBI. The comparison results are as follows: Figure 2 As shown, strain La24 and Amycolatopsis nivea CFH S0261 T The two strains are evolutionarily closest, sharing 99.35% similarity in their 16S rRNA gene sequences. This was confirmed by comparing strain La24 with *Amycolatopsis nivea* CFH S0261. T Genome alignment was performed, and DNA-DNA hybridization (dDDH) values ​​and average nucleotide identity (ANI) values ​​based on genomic sequences were calculated. La24 and Amycolatopsis nivea CFH S0261 were found to be... TThe dDDH and ANI values ​​were 91.79% and 97.86%, respectively, both higher than the critical values ​​(dDDH < 70%, ANI < 95-96%). Based on the morphological characteristics, phylogenetic tree, and genome comparison results of strain La24, it was identified as belonging to the genus *Amycolatopsis*. In systematic taxonomy, this strain belongs to the kingdom Bacteria, phylum Actinobacteria, class Actinobacteria, order Pseudocorales, family Pseudocorales, and genus *Amycolatopsis*.

[0068] Example 4

[0069] This example illustrates the ability of *Amylopectinobacterium* strains to degrade mesotrione.

[0070] The external standard method was used to quantitatively analyze nicosulfuron. A standard curve of nicosulfuron concentration was plotted with concentration x as the abscissa and peak area y as the ordinate. The fitted standard equation was y = 27.61x - 10.71, R0. 2 =0.9999.

[0071] This indicates that when the concentration of mesotrione is between 0-100 mg / L, its concentration and peak area show a good linear relationship.

[0072] Single colonies were picked and inoculated into ISP2 liquid medium. The culture was incubated at 30°C and 160 rpm until the logarithmic growth phase (35-37 h). The bacterial suspension was centrifuged at 8000 rpm for 5 min, the supernatant was removed, sterile physiological saline was added, and the suspension was resuspended and centrifuged again. This process was repeated three times. For the final incubation, an appropriate amount of physiological saline was added to adjust the OD of the bacterial suspension. 600 Approximately equal to 1.000, inoculate at a 5% (v / v) inoculation rate into Erlenmeyer flasks containing 50 mL of GSM liquid medium. Before adding the bacterial cells, add filtered and sterilized nicosulfuron stock solution to adjust the final concentration of nicosulfuron in the GSM system to 50 mg / L. The experiment was conducted in triplicate and incubated at 30°C and 160 rpm in a constant temperature shaker. Samples were taken at 0, 12, 24, 30, 36, 42, and 48 h.

[0073] The residual concentration of mesotrione was detected and calculated using HPLC, and the degradation rate (%) at each time point was calculated. The sampling time was plotted on the x-axis, and the degradation rate (%) and OD were plotted on the y-axis. 600 The vertical axis is used to plot the characteristic curve of strain La24 degrading mesotrione.

[0074] Where y is the peak area detected by HPLC.

[0075] Where C0 is the initial concentration (mg / L) of mesotrione, C m It is the residual concentration (mg / L) of mesotrione at m·hour after the addition of the strain.

[0076] The HPLC method was modified from the national standard (GB 29382-2012): [acetonitrile:water (5.5‰ acetic acid)] = 40:60, filtered through a membrane; flow rate: 1 mL / min; column temperature: room temperature (25℃); detection wavelength: 270 nm; injection volume: 10 μL; retention time: approximately 3.400 min. The chromatographic column was an Agilent C18 reversed-phase column (100 mm × 4.6 mm, id 5 μm); the UV detector was an Agilent 1260 Infinity.

[0077] The results are as follows Figure 3 As shown in Figure 1 and Table 1, Figure 3 CK was the blank control group (uninoculated with strain La24). Figure 3 As shown in Table 1, when strain La24 was inoculated into GSM medium containing 50 mg / L nicosulfuron, it underwent three stages: Pre-degradation stage (0-12 h): During this period, strain La24 rapidly absorbed nutrients such as glucose from the external environment for its own metabolism, growth, and reproduction. During this stage, the strain grew slowly and did not exhibit nicosulfuron degradation ability; Rapid degradation stage (24-42 h): During this stage, the strain multiplied rapidly, and the OD... 600 It reached its highest value (1.284) and exhibited a relatively fast degradation rate; the bacterial culture OD at the end of the degradation period (42-48h) was... 600 The value decreased, the degradation rate slowed down, and finally the 50 mg / L mesotrione was completely degraded in 48 hours.

[0078] Table 1

[0079] Remaining concentration of nicosulfuron (mg / L) 48.71 48.78 37.78 17.68 7.18 1.30 0.00

[0080] Example 5

[0081] This example illustrates the ability of the strain to degrade nicosulfuron, chlorsulfuron, and ethersulfuron.

[0082] Add 50 mL of GSM liquid culture medium to a 150 mL Erlenmeyer flask, adjust the pH to 7.0 ± 0.2 with NaOH and dilute HCl solution, and sterilize. Add filtered and sterilized stock solutions of nicosulfuron, chlorimuron, and sulfadiazine to different Erlenmeyer flasks to an initial concentration of 50 mg / L, respectively. Inoculate OD at a rate of 5% (v / v). 600 A bacterial culture of 1.000 was incubated in a constant temperature shaker at 30℃ and 160rpm, with three replicates for each treatment. Samples were taken for 7 consecutive days to determine the remaining concentrations of nicosulfuron, chlorimuron, and ethersulfuron, and the degradation rate was calculated.

[0083] The results are as follows Figure 4 , Figure 5 As shown in Table 2, Figure 4 and 5 Nicosulfuron-CK was the blank control group for nicosulfuron (without inoculation with strain La24), chlorimuron-CK was the blank control group for chlorimuron (without inoculation with strain La24), and ethersulfuron-CK was the blank control group for ethersulfuron (without inoculation with strain La24).

[0084] Depend on Figure 4 Table 2 shows that strain La24 achieved degradation rates of 100%, 100%, and 97.95% for 50 mg / L of the three herbicides (nicosulfuron, chlorimuron, and fensulfuron) within 7 days, respectively. Chlorimuron showed the fastest degradation rate, reaching 100% by day 6. Strain La24 may contain genes / enzymes capable of degrading these three herbicides, but it is also possible that La24's ability to utilize glucose to produce acidic substances in GSM led to an acidic degradation environment. Figure 5 This leads to the breakage of the urea bridge in sulfonylurea herbicides, thereby achieving a degradation effect.

[0085] Table 2

[0086] Remaining concentration of nicosulfuron (mg / L) 50.897 44.968 18.119 8.206 4.061 2.190 0.974 0.000 Remaining concentration of chlorpyrifos (mg / L) 51.800 44.821 24.039 13.584 6.205 1.413 0.000 0.000 Remaining concentration of sulfamethoxazole (mg / L) 52.423 44.450 23.016 12.388 6.323 3.222 1.892 1.075

[0087] Example 6

[0088] This example illustrates the antibiotic resistance of *Amylopectinobacterium*.

[0089] Antibiotic resistance of strain La24 was determined using the disk diffusion method. The antimicrobial susceptibility test for strain La24 was performed using bacterial susceptibility test strips (YY / T1191-2011), with six antibiotics used: ampicillin (10 μg / tablet), gentamicin (10 μg / tablet), neomycin (30 μg / tablet), streptomycin (10 μg / tablet), carbenicillin, and polymyxin B (300 μg / tablet). The results are as follows: Figure 6 As shown.

[0090] Depend on Figure 6 It can be seen that La24 is resistant to carbenicillin and ampicillin. La24 is very sensitive to streptomycin, neomycin, and gentamicin, but less sensitive to polymyxin.

[0091] The preferred embodiments of this disclosure have been described in detail above with reference to the accompanying drawings. However, this disclosure is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this disclosure, various simple modifications can be made to the technical solutions of this disclosure, and these simple modifications all fall within the protection scope of this disclosure.

[0092] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this disclosure will not describe the various possible combinations separately.

[0093] Furthermore, various different embodiments of this disclosure can be combined in any way, as long as they do not violate the spirit of this disclosure, they should also be regarded as the content disclosed in this disclosure.

Claims

1. A pseudo-amycin bacterium ( Amycolatopsis nivea La24, characterized in that, The preservation number of the pseudoamycete La24 is GDMCC NO: 63776.

2. An inoculum agent of the pseudo-amylopectin bacteria according to claim 1, characterized in that, The inoculum of the pseudoamycete contains a culture medium and bacterial cells.

3. The inoculum agent of pseudo-amylopectin according to claim 2, wherein, The viable count of *Amylopectinus pseudomylopectin* per gram of the bacterial agent is (2.3-2.7) × 10⁻⁶. 8 CFU.

4. The inoculum agent of pseudo-amylopectin according to claim 2, wherein, The culture medium includes at least one of LB medium, ISP2 medium, GSM medium, Gao's No. 1 medium, and beef extract peptone medium.

5. The application of the *Amylopectinus* strain of claim 1 or any one of claims 2-4 in the degradation of herbicides, wherein... The herbicides are mesotrione, nicosulfuron, chlorpyrifos, and ethersulfuron.

6. The application according to claim 5, wherein, Includes the following steps: The herbicide or its agent is applied to plant soil containing the herbicide.

7. The application according to claim 6, wherein, The dosage of the *Amylopectinobacterium* relative to 1 mg of herbicide is (1.0-2.0) × 10⁻⁶. 4 CFU.

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