CN117903976A

CN117903976A - Amycolatopsis, amycolatopsis bacterial agent and application

Info

Publication number: CN117903976A
Application number: CN202311865179.2A
Authority: CN
Inventors: 阮志勇; 李情情; 江旭; 周义清; 马青云; 焦思雨
Original assignee: Institute of Agricultural Resources and Regional Planning of CAAS
Current assignee: Institute of Agricultural Resources and Regional Planning of CAAS
Priority date: 2023-12-29
Filing date: 2023-12-29
Publication date: 2024-04-19

Abstract

The present disclosure relates to amycolatopsis, the classification of which is named Amycolatopsis nivea, the preservation number of which is GDMCC NO:63776. the strain is preserved in the microorganism strain collection of Guangdong province of the preservation unit designated by the national intellectual property agency, the preservation date is 2023, 09 and 08, and the preservation number is GDMCC NO:63776. the disclosure also provides a amycolatopsis microbial inoculant, which comprises a culture medium and thalli, wherein the thalli comprises a preservation number GDMCC NO:63776 Amycolatopsis sp. The strain has better herbicide degradation capability, especially mesotrione, nicosulfuron, chlorimuron and cinosulfuron, and has higher degradation speed.

Description

Amycolatopsis, amycolatopsis bacterial agent and application

Technical Field

The present disclosure relates to the field of microorganisms, and in particular, to amycolatopsis, amycolatopsis bacterial agents and applications thereof.

Background

Chemical pesticides are an important guarantee for the harvest of agriculture nowadays, wherein herbicides are widely applied in agriculture, and the harvest of grains is greatly promoted. However, the use of herbicides in large quantities causes considerable harm to farmland and surrounding ecological environment, seriously threatens human health and life safety, and chemical injury caused by herbicide residue is reported worldwide, so how to solve the problem of environmental herbicide residue has become the content of extensive study by scientists.

Among studies on microbial degradation of herbicides, scholars at home and abroad have made many studies on herbicide degradation, but related studies on microbial degradation of mesotrione have been relatively poor. Only a few mesotrione degradation strains are reported at present, but microbial resources capable of completely mineralizing mesotrione are not found yet, the research on the degradation mechanism of mesotrione is still in a preliminary stage, the molecular response inside thalli in the process of degrading mesotrione by microorganisms is not yet researched, and the problems of single strain, fewer degradation genes, unclear degradation mechanism and the like exist, so that the mesotrione degradation strains are continuously enriched, and the development of efficient degradation microbial agents is a problem which should be further focused.

Disclosure of Invention

The invention aims to provide amycolatopsis, amycolatopsis microbial inoculum and application, and the strain has better herbicide degradation capability, especially mesotrione, nicosulfuron, chlorimuron-ethyl and cinosulfuron, and has higher degradation speed.

To achieve the above object, a first aspect of the present disclosure provides a amycolatopsis, the amycolatopsis being classified under the name Amycolatopsis nivea, the amycolatopsis having a deposit number of GDMCC NO:63776.

Alternatively, the 16S rRNA sequence of the amycolatopsis is shown as SEQ ID NO. 1.

Optionally, the amycolatopsis is gram positive, circular in shape, convex in the middle, provided with folds on the surface, white on the front surface of the colony, pale yellow on the back surface of the colony, and white aerial hyphae and pale yellow matrix hyphae.

A second aspect of the present disclosure provides a amycolatopsis microbial inoculant comprising a culture medium and a microbial inoculant comprising a cell having a accession number GDMCC NO:63776 Amycolatopsis sp.

Optionally, each gram of the amycolatopsis microbial inoculum has a preservation number of GDMCC NO:63776 the number of viable bacteria of Amycolatopsis was (2.3-2.7). Times.10 ⁸ CFU.

Optionally, the medium comprises at least one of LB medium, ISP2 medium, GSM medium, gao's first medium, and beef extract peptone medium.

A third aspect of the present disclosure provides the use of the amycolatopsis of the first aspect or the amycolatopsis of the second aspect for degrading herbicides.

Optionally, the method comprises the following steps: the amycolatopsis or amycolatopsis inoculant is applied to plant soil containing the herbicide.

Optionally, the herbicide comprises a triketone herbicide and/or a sulfonylurea herbicide; preferably, the trione herbicide comprises mesotrione; the sulfonylurea herbicide comprises nicosulfuron, chlorimuron and cinosulfuron.

Alternatively, the amycolatopsis is used in an amount of (1.0-2.0) x 10 ⁴ CFU per 1mg of herbicide.

Through the technical scheme, the invention provides amycolatopsis, amycolatopsis microbial inoculum and application, and the strain has better herbicide degradation capability, especially mesotrione, nicosulfuron, chlorimuron and cinosulfuron degradation, has higher degradation speed and reduces the influence of the herbicide on the environment. In addition, the strain has resistance to carbenicillin and ampicillin, and is not easily damaged by carbenicillin and ampicillin.

Additional features and advantages of the present disclosure will be set forth in the detailed description which follows.

Biological material preservation information

The amycolatopsis is classified and named Amycolatopsis nivea and is preserved in the collection of microorganism strains in Guangdong province, and the preservation address is: building 5, no. 59, no. 100 university, hibiscus sabdariffa, city martyrs, with a preservation date of 2023, 09, 08 and a preservation number of GDMCC NO:63776.

Drawings

The accompanying drawings are included to provide a further understanding of the disclosure, and are incorporated in and constitute a part of this specification, illustrate the disclosure and together with the description serve to explain, but do not limit the disclosure. In the drawings:

FIG. 1 is a colony morphology of amycolatopsis of the present disclosure.

FIG. 2 is a phylogenetic tree of the 16Sr RNA genes of amycolatopsis of the present disclosure.

Fig. 3 is a graph of degradation rate and absorbance of mesotrione by amycolatopsis strains of the present disclosure.

Fig. 4 is a graph of degradation rate of a amycolatopsis strain of the present disclosure to degrade sulfonylurea herbicides.

Fig. 5 is a graph of pH of a amycolatopsis strain of the present disclosure degrading sulfonylurea herbicides.

FIG. 6 is a panel of detection panels of antibiotic resistance of amycolatopsis strains of the present disclosure.

Detailed Description

Specific embodiments of the present disclosure are described in detail below with reference to the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating and illustrating the disclosure, are not intended to limit the disclosure.

The inventor of the present disclosure isolated and cultured a new strain from the bottom sludge of the sewage pool of Beijing certain chemical plant, and identified that the strain belongs to Amycolatopsis (Amycolatopsis), and named Amycolatopsis nivea. The strain is preserved in the microorganism strain preservation center of Guangdong province of the preservation unit designated by the national intellectual property agency, the preservation date is 2023, 09 and 08, and the preservation number is GDMCC NO:63776.

The first aspect of the present disclosure provides a amycolatopsis, the amycolatopsis being classified as Amycolatopsis nivea, the amycolatopsis having a deposit number of GDMCC NO:63776.

According to the present disclosure, the 16S rRNA sequence of the amycolatopsis is shown as SEQ ID NO. 1.

In one embodiment of the disclosure, the present disclosure morphologically identifies amycolatopsis, as shown in fig. 1, which is gram positive, circular in shape, with folds in the middle, white on the front of the colony, yellowish on the back of the colony, with white aerial hyphae and pale yellow matrix hyphae.

According to the present disclosure, the deposit number GDMCC NO: the number of viable bacteria of the amycolatopsis 63776 can vary within a wide range. In one embodiment of the present disclosure, the deposit number is GDMCC NO per gram of the amycolatopsis agent: 63776 the number of viable bacteria of Amycolatopsis was (2.3-2.7). Times.10 ⁸ CFU.

In one embodiment of the present disclosure, the medium may include at least one of LB medium, ISP2 medium, GSM medium, gao's first medium, and beef extract peptone medium. In the present disclosure, the medium may be a liquid medium, or may be a solid medium, preferably a liquid medium. In the above embodiment, the medium may be selected from any one of ISP series media, preferably ISP2 medium.

In the present disclosure, amycolatopsis or amycolatopsis bacteria have better herbicide degrading ability, and reduce the influence of herbicide on environment.

In one embodiment of the present disclosure, the method comprises the steps of: the amycolatopsis or amycolatopsis inoculant is applied to plant soil containing the herbicide.

In the present disclosure, the amycolatopsis and amycolatopsis inoculant may be in liquid form or solid form, preferably in liquid form, for example, may be a inoculant comprising amycolatopsis or a liquid inoculant comprising amycolatopsis. The application can be carried out by methods conventionally adopted by those skilled in the art, for example, spraying the amycolatopsis or amycolatopsis microbial inoculum on the soil surface, and stirring the amycolatopsis or amycolatopsis microbial inoculum and the soil uniformly.

In one embodiment of the present disclosure, the herbicide comprises a triketone herbicide and/or a sulfonylurea herbicide. In a preferred embodiment of the present disclosure, the trione herbicide comprises mesotrione; the sulfonylurea herbicide comprises nicosulfuron, chlorimuron and cinosulfuron.

In one embodiment of the present disclosure, the amycolatopsis is used in an amount of (1.0-2.0) x 10 ⁴ CFU per 1mg of herbicide.

The present invention will be described in further detail with reference to the following examples, but the scope of the present invention is not limited to the following examples.

The materials used in this example are all commercially available products unless otherwise specified.

LB medium: 5g of yeast extract, 10g of tryptone, 10g of NaCl,20g of agar, 1L H ₂ O, pH=6.8-7.2, 121 ℃ and sterilization for 30min.

ISP2 medium: 10g malt extract, 4g yeast extract, 4g glucose, 20g agar, 1L H ₂ O, pH=7.0-7.4, 115℃and 20min sterilization.

Gao's number one: 10g of soluble starch ,1g KNO₃,0.5g K₂HPO₄,0.5g MgSO₄·7H₂O,0.5g NaCl,0.01g FeSO₄·7H₂O,20g agar, 1L H ₂ O, adjusting the pH=7.4-7.6, sterilizing at 121 ℃ for 20min.

Beef extract peptone medium: 3g of beef extract, 10g of peptone, 5g of NaCl,20g of agar, 1L H ₂ O, pH=7.4-7.6, 121 ℃ and sterilization for 20min.

GSM medium: 5g glucose ;1g NH₄Cl;0.2g NaCl,0.2g MgSO₄·7H₂O;0.5gK₂HPO₄;0.5g KH₂PO₄; pH=7.0.+ -. 0.2, 115 ℃ and sterilized for 20min.

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

Example 1

This example is used to illustrate the isolation and purification of amycolatopsis strains.

(1) Isolation of amycolatopsis strains:

Sample collection: 10g of sediment in the sediment of the sewage pool of a Beijing certain chemical plant is taken and put into a triangular flask containing 100mL of sterile water, and the mixture is fully and uniformly mixed.

Sample enrichment: 10mL of the uniformly mixed suspension is taken, added into a triangular flask containing 90mL of GSM culture medium, herbicide is added in an amount of 5mg/L, and the mixture is placed in a constant temperature culture medium at 30 ℃ for dark culture at a constant speed of 160 rpm. The medium was transferred once for 7 days, and the herbicide was added in an amount of 10 mg/L. Similarly, the transfer was performed every 7 days until the concentration was increased to 50mg/L, and thereafter, the transfer was performed every 2 months, and the herbicide was added in an amount of 50 mg/L. Wherein the herbicide comprises mesotrione, nicosulfuron, chlorimuron and cinosulfuron, and the weight ratio of mesotrione, nicosulfuron, chlorimuron and cinosulfuron is 1:1:1: 1). Each transfer was counted as one generation and the screened samples were taken from the 23 rd generation.

(2) Purification of amycolatopsis strains:

Taking a 23 rd generation sample of a culture solution LAMC ³⁺ enriched for a long time in a LAM laboratory for gradient dilution, adding 1mL of enriched solution into 9mL of sterile water to be recorded as 10 ^-1, fully mixing, adding 1mL of diluted solution into new 9mL of sterile water after fully mixing, fully mixing and recording as 10 ^-2, and the like until 10 ^-5. Three gradient dilutions of 10 ^-3、10^-4、10^-5 are selected for dilution and coating, 200 mu L of each dilution is taken to LB and GSM solid plates containing 50mg/L mesotrione, the solid plates are inverted and cultured in a constant temperature incubator at 30 ℃, strains with different colony forms as much as possible are selected out after 3-5d, and streaked to LB and GSM solid culture media containing 50mg/L mesotrione for separation and purification, and finally single colony capable of tolerating or degrading mesotrione is obtained and marked as La24. The residual amount of mesotrione in the culture medium was measured by means of a high performance liquid chromatograph to evaluate the ability of the strain La24 to degrade mesotrione. Finally, the mesotrione high-efficiency degradation strain La24 is determined.

Example 2

This example is used to illustrate morphological identification of amycolatopsis strains.

(1) The purified strain La24 is selected, cultured in ISP2 solid medium by streaking method, and cultured for 3-4 days at 30 ℃. Then, the colony morphology was observed, and the colony morphology of amycolatopsis was as shown in FIG. 1.

Deposit No. GDMCC NO:63776 the bacterial colony of amycolatopsis is round, the middle is convex, the surface is provided with folds, the front surface of the bacterial colony is white, the back surface of the bacterial colony is yellowish, and the bacterial colony is provided with white aerial hyphae and yellowish matrix hyphae; and white aerial hyphae are white powder.

(2) After gram staining, the bacterial strain appeared purple, and was identified as gram-positive bacteria.

Example 3

This example is used to illustrate molecular biological identification of amycolatopsis strains:

The isolated and purified strain La24 was used for molecular biological identification, and the 16S rRNA gene sequences of the mesotrione-tolerant or mesotrione-degrading strain were amplified using primers 27F and 1492R common to the bacteria.

Amplification reaction system: 12.5. Mu.L Mix;8.5 μL ddH ₂ O;1 μL 27F;1 μL 1492R; 2. Mu.L of the strain extract.

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

Reverse primer: 14992R 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO. 3).

And (3) sending the PCR product of the amplified 16S rRNA to Beijing Bomaide gene technology Co., ltd for sequencing, wherein the nucleotide sequence obtained by sequencing is shown as SEQ ID NO.1, and the detail is shown in an annex sequence table.

The homology comparison (Blast) of nucleic acid sequences is carried out in GenBank database on NCBI, the comparison result is shown in figure 2, the evolution distance between the strain La24 and Amycolatopsis NIVEA CFH S0261 ^T is nearest, and the similarity of the 16S rRNA gene sequences of the two is 99.35%. By genomic alignment of strain La24 with Amycolatopsis NIVEA CFH S0261 ^T, DNA-DNA hybridization (dDDH) and Average Nucleotide Identity (ANI) values based on genomic sequence were calculated for both, found to be 91.79% and 97.86% respectively for dDDH and ANI values between La24 and Amycolatopsis NIVEA CFH S0261 ^T, both above the threshold (dDDH <70%, ANI < 95-96%). Analysis of phylogenetic tree and genome comparison results combined with morphological characteristics of strain La24, was judged as Amycolatopsis. The strain belongs to the bacterial kingdom in the system taxonomy, actinomycota, actinomycetes, pseudonocardia order, pseudonocardiaceae, amycolatopsis.

Example 4

This example is intended to illustrate the ability of amycolatopsis strains to degrade mesotrione.

The mesotrione is quantitatively analyzed by using an external standard method, a mesotrione concentration standard curve is drawn by taking the concentration x as an abscissa and the peak area y as an ordinate, and a fitted standard equation is y=27.61 x-10.71 and r ² =0.9999.

Indicating that the mesotrione concentration shows good linear relationship with peak area when the mesotrione concentration is between 0 and 100 mg/L.

Selecting single colony, inoculating into ISP2 liquid culture medium, culturing at 30deg.C at 160rpm for constant temperature to logarithmic phase (35-37 h), centrifuging at 8000rpm for 5min, removing supernatant culture medium, adding sterile physiological saline, suspending again, centrifuging, repeating for 3 times, adding appropriate amount of physiological saline for the last time, adjusting bacterial liquid OD ₆₀₀ to be equal to 1.000, inoculating into conical flask containing 50mL GSM liquid culture medium according to 5% (v/v) inoculum size, adding filtered sterilized mesotrione mother liquor before adding thallus, adjusting final concentration of mesotrione in GSM system to 50mg/L, setting 3 times, and culturing in shaking table at 30deg.C at 160rpm constant temperature. Samples were taken at 0, 12, 24, 30, 36, 42, 48 h.

Detecting and calculating the residual concentration of mesotrione by means of HPLC, calculating the degradation rate (%) of each time point, taking the sampling time as an abscissa, taking the degradation rate (%) and the OD ₆₀₀ value as an ordinate, and drawing a characteristic curve of the strain La24 for degrading mesotrione.

Where y is the peak area detected by HPLC.

Wherein C ₀ is the initial concentration (mg/L) of mesotrione, and C _m is the residual concentration (mg/L) of mesotrione at m.multidot.hour after addition of the strain.

The HPLC assay was partially modified with reference to the national standard (GB 29382-2012): [ acetonitrile: water (5.5%o acetic acid) ] = 40:60, filtering by a filter membrane; flow rate: 1mL/min; column temperature: room temperature (25 ℃); detection wavelength: 270nm; sample injection amount: 10. Mu.L; the retention time was around 3.400 min. The chromatographic column is an Agilent C18 reverse phase column (100 mm. Times.4.6 mm, i.d.5 μm); the ultraviolet detector is model Agilent 1260Infinity.

The results are shown in FIG. 3 and Table 1, where CK in FIG. 3 is a blank (unvaccinated strain La 24). As can be seen from FIG. 3 and Table 1, the bacterial solution of strain La24 was inoculated into a GSM medium containing 50mg/L mesotrione, and subjected to 3 stages: the strain La24 in the early degradation stage (0-12 h) can quickly absorb external glucose and other nutrient substances during the period so as to prepare for self metabolism, growth and reproduction, and the strain grows slowly and does not show mesotrione degradation capability in the stage; a rapid degradation period (24-42 h) which is propagated in mass by the strain, with OD ₆₀₀ reaching the highest (1.284) and exhibiting a faster degradation rate; the OD ₆₀₀ value of the bacterial liquid at the end of degradation (42-48 h) is reduced, the degradation rate is slowed down, and finally 50mg/L mesotrione is completely degraded at 48 h.

TABLE 1

Time (h)	0	12	24	20	36	42	48
								Mesotrione residual concentration (mg/L)	48.71	48.78	37.78	17.68	7.18	1.30	0.00

Example 5

This example is used to demonstrate the ability of the strain to degrade nicosulfuron, chlorimuron-ethyl and cinosulfuron.

50ML of GSM liquid medium was placed in a 150mL triangular flask, pH=7.0.+ -. 0.2 was adjusted with NaOH, dilute HCl solution, and sterilized. The stock solution of nicosulfuron, chlorimuron and cinosulfuron which were sterilized by filtration was added to each of the conical flasks to an initial concentration of 50mg/L, and the stock solution was inoculated with a bacterial solution of OD ₆₀₀ =1.000 in an inoculum size of 5% (v/v), placed in a constant temperature shaker at 30℃and 160rpm for cultivation, and 3 replicates were set for each treatment. Sampling is carried out for 7 days continuously, residual concentrations of nicosulfuron, chlorimuron and cinosulfuron are detected, and the degradation rate is calculated.

The results are shown in fig. 4, 5 and table 2, wherein nicosulfuron-CK in fig. 4 and 5 is nicosulfuron control group (unvaccinated strain La 24), chlorimuron-CK is chlorimuron control group (unvaccinated strain La 24), and cinosulfuron-CK is cinosulfuron control group (unvaccinated strain La 24).

As can be seen from FIG. 4 and Table 2, the degradation rate of the strain La24 to 50mg/L of the three herbicides (nicosulfuron, chlorimuron-ethyl and cinosulfuron) within 7d is 100%,100% and 97.95%, respectively, wherein the degradation rate to chlorimuron-ethyl is the fastest, and the degradation rate reaches 100% at 6 d. Genes/enzymes that can degrade these three herbicides may be present in strain La24, but may also be because La24 is able to utilize glucose to produce acidic substances in GSM, resulting in the entire degradation environment becoming acid (fig. 5), resulting in the urea bridge of sulfonylurea herbicides breaking to achieve the degradation effect.

TABLE 2

Time (d)	0	1	2	3	4	5	6	7
									Residual concentration of nicosulfuron (mg/L)	50.897	44.968	18.119	8.206	4.061	2.190	0.974	0.000
Residual concentration of chlorimuron-ethyl (mg/L)	51.800	44.821	24.039	13.584	6.205	1.413	0.000	0.000
									Residual concentration of cinosulfuron (mg/L)	52.423	44.450	23.016	12.388	6.323	3.222	1.892	1.075

Example 6

This example is intended to illustrate the resistance of amycolatopsis to antibiotics.

The antibiotic resistance of strain La24 was determined by the paper diffusion method. The drug-sensitive resistance test of strain La24 was bacterial drug-sensitive test paper (YY/T1191-2011), and the 6 antibiotics used were ampicillin (10. Mu.g/tablet), gentamicin (10. Mu.g/tablet), neomycin (30. Mu.g/tablet), streptomycin (10. Mu.g/tablet), carbenicillin and polymyxin B (300. Mu.g/tablet), respectively. The results are shown in FIG. 6.

As can be seen from FIG. 6, la24 is resistant to carbenicillin and ampicillin. La24 is very sensitive to streptomycin, neomycin and gentamicin, and less sensitive to polymyxin.

The preferred embodiments of the present disclosure have been described in detail above with reference to the accompanying drawings, but the present disclosure is not limited to the specific details of the embodiments described above, and various simple modifications may be made to the technical solutions of the present disclosure within the scope of the technical concept of the present disclosure, and all the simple modifications belong to the protection scope of the present disclosure.

In addition, the specific features described in the foregoing embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, the present disclosure does not further describe various possible combinations.

Moreover, any combination between the various embodiments of the present disclosure is possible as long as it does not depart from the spirit of the present disclosure, which should also be construed as the disclosure of the present disclosure.

Claims

1. A amycolatopsis characterized by a classification designated Amycolatopsis nivea for amycolatopsis, the amycolatopsis having a deposit number GDMCC NO:63776.

2. The amycolatopsis of claim 1, wherein the 16SrRNA sequence of the amycolatopsis is shown in SEQ ID No. 1.

3. The amycolatopsis of claim 1, wherein the amycolatopsis is gram positive, round in shape, convex in the middle, wrinkled on the surface, white on the front of the colony, pale yellow on the back of the colony, white aerial hyphae and pale yellow matrix hyphae.

4. A amycolatopsis microbial agent, comprising a culture medium and a bacterial cell, wherein the bacterial cell comprises a cell preservation number GDMCC NO:63776 Amycolatopsis sp.

5. The amycolatopsis microbial inoculant of claim 4, wherein the amycolatopsis microbial inoculant is deposited with accession number GDMCC NO per gram of: 63776 the number of viable bacteria of Amycolatopsis was (2.3-2.7). Times.10 ⁸ CFU.

6. The amycolatopsis microbial inoculant of claim 4, wherein the medium comprises at least one of LB medium, ISP2 medium, GSM medium, gao's first medium, and beef extract peptone medium.

7. Use of a amycolatopsis according to any one of claims 1-3 or an amycolatopsis inoculant according to any one of claims 4-6 for degrading herbicides.

8. The use according to claim 7, comprising the steps of: the amycolatopsis or amycolatopsis inoculant is applied to plant soil containing the herbicide.

9. The use according to claim 8, wherein the herbicide comprises a triketone herbicide and/or a sulfonylurea herbicide;

preferably, the trione herbicide comprises mesotrione; the sulfonylurea herbicide comprises nicosulfuron, chlorimuron and cinosulfuron.

10. The use according to claim 9, wherein the amycolatopsis is used in an amount of (1.0-2.0) x 10 ⁴ CFU per 1mg of herbicide.