Phosphonic acid compounds derived from Streptomyces, their preparation methods and applications

Abstract

This invention belongs to the field of natural compound extraction technology, specifically relating to phosphonic acid compounds derived from Streptomyces, their preparation methods, and applications. The Streptomyces (… Streptomyces sp. G0098 was deposited on May 9, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 30537. The phosphonic acid compounds include phosphonic acid compound 1 and phosphonic acid compound 2, with the molecular formulas C5H5O7P and C5H5O6P, respectively. Experimental results show that phosphonic acid compound 1 and phosphonic acid compound 2 possess significant herbicidal activity.

Description

Technical Field

[0001] This invention belongs to the field of natural compound extraction technology, specifically relating to phosphonic acid compounds derived from Streptomyces, their preparation methods, and applications. Background Technology

[0002] Phosphonates are a collective term for compounds containing carbon-phosphorus bonds in their chemical structure. They are widely distributed in nature and possess a variety of activities, finding applications in pharmaceuticals (antibiotics, antimalarial drugs, etc.), agriculture (fertilizers and herbicides), synthetic chemistry (catalysts), industry (metal chelating agents, antifouling agents, water treatment), cosmetics, and many other fields. As structural analogs of phosphate esters and carboxylic acids, small phosphonate molecules can compete with substrates for enzyme active sites in biochemical reactions, thereby blocking specific biochemical reactions and inhibiting or killing target organisms. Furthermore, the carbon-phosphorus bonds in phosphonates endow them with extremely high structural stability. From 1959 to 2010, nearly 30 small phosphonate natural products with significant pharmacological activity have been successfully isolated from nature using activity-guided separation strategies. For example, the U.S. Food and Drug Administration (FDA) has approved Monourol (fosfomycin) for the treatment of urinary tract infections. This drug, by mimicking the structure of phosphoenolpyruvate, irreversibly inhibits the activity of UDP-nitro-acetylglucosamine-3-oxo-enolpyruvate transferase, thereby inhibiting the growth of bacteria in mammals and effectively treating urinary tract infections caused by bacteria. For example, the pharmacophore phosphatidylcholine in phosphatidylcholine (PPT or Bialaphos), as a structural analog of glutamate, competitively inhibits the activity of glutamine synthase, exhibiting broad-spectrum resistance to both bacteria and fungi, and is therefore widely used in the preparation of antibiotics. Furthermore, FR-900098 and Fosmidomycin possess strong antimalarial activity, Rhizocticin exhibits good antifungal activity, and K-26 shows potential for treating hypertension, and so on. It is evident that many phosphonic acid small molecule natural products have novel structures and rich biological activities, playing an important role in human production and life.

[0003] With the continuous development of global agriculture and horticulture, herbicides are widely used for weed control in fields, orchards, vegetable gardens, lawns, and public areas. Their high efficiency and convenience make them the preferred weed management tool for farmers and horticulturalists. Currently, common herbicide active ingredients include glyphosate, glufosinate, glufosinate-ammonium, chlorpyrifos, and pyrazoquinoline. Although herbicides play a vital role in agricultural production, long-term use of the same herbicides has led to herbicide resistance and a series of environmental and health problems. Furthermore, the misuse or improper use of herbicides also pollutes soil and water sources and negatively impacts non-target plants and biodiversity. Therefore, there is an urgent need to develop new herbicides to provide more effective and safer control methods, helping farmers protect crops from weeds, improve yield and quality, reduce herbicide resistance, and thus promote the sustainable development of agriculture and horticulture. Summary of the Invention

[0004] The purpose of this invention is to provide phosphonic acid compounds derived from Streptomyces, their preparation methods, and applications.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A Streptomyces sp. G0098 was deposited on May 9, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 30537.

[0007] The application of the aforementioned Streptomyces sp. G0098 in the preparation of phosphonic acid compounds 1-2, the structures of which are as follows:

[0008]

[0009] Phosphonic acid compounds 1-2 prepared from the aforementioned Streptomyces sp. G0098 have the following structures:

[0010]

[0011] The preparation methods of the above-mentioned phosphonic acid compounds 1-2 include the following steps:

[0012] S1: After activating Streptomyces sp. G0098, a single colony was picked and inoculated into a 250ml Erlenmeyer flask containing 100ml ATCC172 liquid medium. The culture was incubated at 28℃ and 130rpm for 3 days to obtain seed culture. 1ml of seed culture was inoculated into a 250ml Erlenmeyer flask containing 100ml R2A liquid medium. The culture was incubated at 28℃ and 130rpm for 3 days to obtain fermentation culture 1. 100ml of fermentation culture 1 was inoculated into a 5L Erlenmeyer flask containing 1L R2A liquid medium. The culture was incubated at 28℃ and 130rpm for 12 days to obtain fermentation culture 2. Fermentation culture 2 was vacuum filtered to separate the mycelium and fermentation broth.

[0013] S2: The fermentation broth obtained in step S1 is concentrated into an extract, and then subjected to HP20 macroporous resin adsorption column chromatography, A-107 strong basic anion exchange resin column chromatography, Sephadex LH-20 dextran gel column chromatography, and semi-preparative liquid chromatography to obtain phosphonic acid compounds 1-2.

[0014] Among them, Streptomyces sp. G0098 was deposited on May 9, 2024 at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 30537.

[0015] The formulation of the ATCC172 liquid culture medium is as follows: acid-hydrolyzed casein 5 g / L, yeast extract 5 g / L, glucose 10 g / L, soluble starch 20 g / L, calcium carbonate 1 g / L, with water as the solvent; the formulation of the R2A liquid culture medium is as follows: peptone 0.5 g / L, acid-hydrolyzed casein 0.5 g / L, yeast extract 0.5 g / L, glucose 0.5 g / L, soluble starch 0.5 g / L, potassium dihydrogen phosphate 0.3 g / L, magnesium sulfate heptahydrate 0.05 g / L, sodium pyruvate 0.3 g / L, with water as the solvent.

[0016] The HP20 macroporous resin adsorption column chromatography was eluted with 100% H2O, 50% H2O-MeOH, and 100% MeOH; the A-107 strong basic anion exchange resin column chromatography was eluted with 2% NaCl, 3% NaCl, and 4% NaCl; the Sephadex LH-20 dextran gel column chromatography was eluted with 80% MeOH-H2O; and the semi-preparative liquid chromatography separation was eluted with 90% H2O-MeOH.

[0017] The application of the aforementioned Streptomyces sp. G0098 and phosphonic acid compounds 1-2 in the preparation of herbicides.

[0018] The above-mentioned application of Streptomyces sp. G0098 and phosphonic acid compounds 1-2 in inhibiting Arabidopsis growth refers to reducing the fresh weight and root length of Arabidopsis.

[0019] The significant advantages of this invention are:

[0020] Streptomyces, as an important class of soil microorganisms, produce secondary metabolites with rich biological activities, including insecticidal, fungicidal, and plant growth-inhibiting effects. Among these, glufosinate, a broad-spectrum herbicide, is produced through a mechanism closely related to the metabolic activities of Streptomyces. Therefore, finding novel Streptomyces metabolites with similar or superior activities is of great significance for agricultural biological control. This invention successfully isolated a Streptomyces sp. G0098 from soil microorganisms through screening and identification. The fermentation broth of Streptomyces sp. G0098 contains herbicide-active metabolites. We used... 31 P-NMR spectroscopy was used to perform detailed chemical structural analysis of phosphonic acid compounds in the fermentation broth. The experiment revealed that the active small-molecule phosphonic acid substances produced by *Streptomyces sp.* G0098 exhibit significant differences in chemical structure compared to known herbicides such as glufosinate, glyphosate, and diammonium phosphate. Especially... 31 In the P-NMR spectrum, the chemical shifts of the phosphine atoms in the CP bonds of the phosphonic acid compounds produced by this strain were concentrated between 14 ppm and 18 ppm, which is significantly different from common phosphonic acid compounds. Based on these results, we infer that *Streptomyces sp.* G0098 produces novel phosphonic acid compounds through metabolism, and that these compounds possess significant herbicidal activity. This discovery not only enriches the variety and function of *Streptomyces* secondary metabolites but also provides new ideas and methods for the development of novel herbicides. Attached Figure Description

[0021] Figure 1 : Structural formulas of phosphonic acid compound 1 and phosphonic acid compound 2.

[0022] Figure 2 : Phosphonic acid compound 1 1 H- 31 PHMBC and 1 H- 13 C HMBC Relationship Diagram.

[0023] Figure 3 : Phosphonic acid compound 2 1 H- 31 PHMBC and 1 H- 13C HMBC Relationship Diagram.

[0024] Figure 4 The inhibitory effects of phosphonic acid compound 1 and phosphonic acid compound 2 on Arabidopsis thaliana growth.

[0025] Figure 5 : 400MHz of phosphonic acid compound 1 in D2O solvent 1 H-NMR spectrum.

[0026] Figure 6 : Phosphonic acid compound 1 in D2O solvent 31 P-NMR spectrum.

[0027] Figure 7 : 100MHz of phosphonic acid compound 1 in D2O solvent 13 C-NMR spectrum.

[0028] Figure 8 : Phosphonic acid compound 1 in D2O solvent 1 H- 13 C HMBC NMR spectrum.

[0029] Figure 9 : Phosphonic acid compound 1 in D2O solvent 1 H- 31 P HMBC NMR spectrum.

[0030] Figure 10 : Phosphonic acid compound 1 in D2O solvent 1 H- 13 C HMQC NMR spectrum.

[0031] Figure 11 : Phosphonic acid compound 1 in D2O solvent 1 H- 1 H COSYNMR spectrum.

[0032] Figure 12 DEPTNMR spectrum of phosphonic acid compound 1 in D2O solvent.

[0033] Figure 13 High-resolution mass spectrum of phosphonic acid compound 1.

[0034] Figure 14 : 400MHz of phosphonic acid compound 2 in D2O solvent 1 H-NMR spectrum.

[0035] Figure 15 : Phosphonic acid compound 2 in D2O solvent 31 P-NMR spectrum.

[0036] Figure 16: 100MHz of phosphonic acid compound 2 in D2O solvent 13 C-NMR spectrum.

[0037] Figure 17 : Phosphonic acid compound 2 in D2O solvent 1 H- 13 C HMBC NMR spectrum.

[0038] Figure 18 : Phosphonic acid compound 2 in D2O solvent 1 H- 31 P HMBC NMR spectrum.

[0039] Figure 19 : Phosphonic acid compound 2 in D2O solvent 1 H- 13 C HMQC NMR spectrum.

[0040] Figure 20 : Phosphonic acid compound 2 in D2O solvent 1 H- 1 H COSYNMR spectrum.

[0041] Figure 21 DEPTNMR spectrum of phosphonic acid compound 2 in D2O solvent.

[0042] Figure 22 High-resolution mass spectrum of phosphonic acid compound 2. Detailed Implementation

[0043] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0044] In the following examples, the culture medium formulation is as follows:

[0045] Gao's No. 1 Agar Medium: 20 g / L soluble starch, 1 g / L KNO3, 0.5 g / L NaCl, 0.5 g / L K2HPO4·3H2O, 0.5 g / L MgSO4·7H2O, 0.01 g / L FeSO4·7H2O, 20 g / L agar, solvent is water; pH = 7.4-7.6; use after high temperature steam sterilization at 121℃ for 15 min.

[0046] ATCC172 solid culture medium: acid-hydrolyzed casein 5 g / L, yeast extract 5 g / L, glucose 10 g / L, soluble starch 20 g / L, calcium carbonate 1 g / L, agar 15 g / L, solvent: water; pH = 7.0; sterilize at 121℃ for 15 min before use.

[0047] The ATCC172 liquid culture medium consists of: acid-hydrolyzed casein 5 g / L, yeast extract 5 g / L, glucose 10 g / L, soluble starch 20 g / L, calcium carbonate 1 g / L, and water as the solvent; pH = 7.0; and is used after being sterilized by steam at 121°C for 15 min.

[0048] The R2A liquid culture medium formula is as follows: peptone 0.5 g / L, acid-hydrolyzed casein 0.5 g / L, yeast extract 0.5 g / L, glucose 0.5 g / L, soluble starch 0.5 g / L, potassium dihydrogen phosphate 0.3 g / L, magnesium sulfate heptahydrate 0.05 g / L, sodium pyruvate 0.3 g / L, with water as the solvent; pH = 7.2; sterilized by steam at 121℃ for 15 min before use.

[0049] Example 1:

[0050] The soil samples were collected from Fujian Province. In a clean bench, the soil samples were added to 50ml centrifuge tubes containing sterile water and shaken thoroughly to create a turbidity, thus preparing a soil suspension. 1ml of the soil suspension was measured using a pipette and serially diluted at 1:10, 1:50, and 1:100. Different concentrations of soil suspension were then inoculated sequentially onto Gao's No. 1 agar plates, from low to high concentration, and evenly spread using a sterile spreader. To ensure full penetration of the bacterial suspension into the medium, the spread plates were first placed face up for 20–30 minutes, then inverted and incubated at 30℃ until single colonies appeared. The single colonies were then transferred to Gao's No. 1 agar plates and streaked to obtain a pure culture of strain G9008. A bacterial suspension was prepared using 20% ​​glycerol and stored. The culture characteristics of strain G9008 conform to the general characteristics of Streptomyces: the colonies are radial, with a rough, dry surface covered by a layer of dry, powdery spores that adhere tightly to the culture medium and are difficult to pick up; they are dark gray in color; and the center and edge of the colonies differ in color on the front and back sides. The 16S rRNA gene of strain G9008 was amplified by PCR and sequenced, the results of which are shown in SEQ ID NO.1. Sequence alignment showed the highest similarity to Streptomyces, confirming it as Streptomyces p.

[0051] Streptomyces sp. G0098 was deposited on May 9, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 30537.

[0052] Example 2:

[0053] Streptomyces sp. G0098 preserved in glycerol was streaked onto ATCC172 solid medium plates for activation. After incubation at 28°C for 3 days, a single colony was picked and inoculated into a 250ml Erlenmeyer flask containing 100ml of ATCC172 liquid medium for propagation. The culture was incubated at 28°C and 130rpm for 3 days to obtain a seed culture. 1ml of the seed culture was inoculated into a 250ml Erlenmeyer flask containing 100ml of R2A liquid medium for the first fermentation, incubated at 28°C and 130rpm for 3 days to obtain fermentation culture 1. 100ml of fermentation culture 1 was inoculated into a 5L Erlenmeyer flask containing 1L of R2A liquid medium for the second fermentation, incubated at 28°C and 130rpm for 12 days to obtain fermentation culture 2. Fermentation culture 2 was vacuum filtered to separate the mycelium and fermentation broth. Discard the mycelium, and concentrate the fermentation broth under reduced pressure at 28℃ and 0.01MPa using a rotary evaporator until no liquid flows out, obtaining extract a. Dissolve 20g of extract a in 20ml of H2O, and perform chromatography using an HP20 macroporous resin adsorption column (9cm outer diameter, 108cm height, 64cm stock height) at a flow rate of 1ml / min. Elute sequentially with 100% H2O, 50% H2O-MeOH, and 100% MeOH at a flow rate of 100ml / min, with each gradient elution volume being 5L. Collect the 100% H2O eluent and concentrate it under reduced pressure at 28℃ and 0.01Pa using a rotary evaporator until no liquid flows out, obtaining extract b. Dissolve 2g of extract b in 2ml of H2O. Perform chromatography using an A-107 strong basic anion exchange resin column (9cm outer diameter, 108cm height, 60cm stock height) at a flow rate of 1ml / min. Elute sequentially with 2% NaCl, 3% NaCl, and 4% NaCl at a flow rate of 2ml / min, with each elution volume being 500ml. Collect the eluent fractions to obtain three groups. Separately analyze the second group using Sephadex chromatography. LH-20 dextran gel column chromatography (outer diameter 5 cm, column height 230 cm, stock height 210 cm) was performed with 80% MeOH-H2O as the mobile phase and a flow rate controlled at 2 mL / min. The solution was then purified by semi-preparative liquid chromatography under the following conditions: ODS column (4.6*250 mm, 5 μm), mobile phase 90% H2O-MeOH, UV detector, dual wavelengths (220 nm and 254 nm). Compound 1 and compound 2 were obtained in amounts of 5.2 mg and 6.5 mg, respectively. The retention time of compound 1 was 14.2 min (0.026%), and the retention time of compound 2 was 20.3 min (0.0325%).

[0054] Compound 1 and compound 2 were dissolved separately in deuterated water (D2O), and then... 1 H-NMR,13 C-NMR, 31 P-NMR and corresponding two-dimensional NMR spectroscopy were used for determination; molecular weight was determined by UPLC-QTOF-MS using ESI as the ion source at an injection volume of 1 μL, and isoleucine enkephalin (200 pg / mL) as the calibration substance. Gradient elution of the sample was performed using a Waters-ODS C18 column (2.1 × 100 nm, 5 μm). The elution conditions and MS operating conditions for high-resolution mass spectrometry are shown in Tables 1 and 2. The detection results were analyzed and processed using Masslynx V4.1 software. Furthermore, the determination of the compound's stereochemistry was mainly achieved using polarimetry and quantum computing.

[0055] Table 1 Elution conditions

[0056]

[0057] Table 2 MS Operating Conditions

[0058]

[0059] The results showed that compounds 1 and 2, isolated from the fermentation metabolites of Streptomyces sp. G0098, were novel natural phosphonic acid compounds, namely 2-(hydroxy(phosphono)methyl)furan-2,5-dion (1) and 2-(phosphonomethyl)furan-2,5-dione (2), respectively. Their structures are shown in the figure below. Figure 1 .

[0060] Compound 1: A white amorphous powder. Its molecular formula, as observed by HR-ESI-MS in negative ion mode, shows a quasi-molecular ion peak at m / z 206.9711 (calcd 206.9695). 1 H NMR and 13 C10 NMR spectroscopy revealed that the compound has the molecular formula C5H5O7P and an unsaturation degree of 5. 1 The H-NMR spectrum showed two hydrogen signals at 4.62 (d, J = 17.8 Hz, 1H) and 6.21 (d, J = 4.5 Hz, 1H) ppm. 13 The C-NMR spectrum showed five carbon signals at shifts of 170.29, 169.32, 147.07, 122.72, and 69.44 ppm. 1 H-NMR spectral information predicts that the compound contains two amide carbons or ester carbons, two alkene carbons, and one tertiary carbon bonded to an oxygen atom. 31 The P-chromatogram showed one phosphine at 14.60 ppm. 1 H- 31In the P HMBC two-dimensional spectrum, both hydrogen atoms are associated with δ P The correlation with phosphine atoms at 14.60 ppm indicates that these hydrogen atoms are very close to phosphine atoms. Based on δ... C The large coupling constant J = 151.2 Hz at 69.44 ppm suggests that the carbon atom is directly bonded to the phosphine atom. According to the HSQC spectrum, the carbon atom at chemical shift 122.72 ppm is bonded to a hydrogen atom at 6.21 ppm, and the carbon atom at chemical shift 69.44 ppm is bonded to a hydrogen atom at 4.62 ppm. 1 H- 13 The C HMBC spectrum shows that δ H 6.21 ppm hydrogen and δ C Carbon concentrations of 69.44, 147.07, and 170.29 ppm are all correlated, δ H 4.62 ppm hydrogen and δ C Carbon concentrations of 147.07, 170.29, and 122.72 ppm are all relevant. Figure 2 ).comprehensive 1 H NMR, 13 C NMR, 31 p-NMR, DEPT135, 1 H- 1 HCOSY, 1 H- 31 P HMBC, 1 H- 13 C HMBC ultimately determined the structure of compound 1 to be 2-(hydroxy(phosphono)methyl)furan-2,5-dione. NMR data are shown in Table 3.

[0061] Table 3 Chemical shifts of compound 1 (in D2O)

[0062]

[0063] Compound 2: A white amorphous powder. Its molecular formula, as shown by HR-ESI-MS in negative ion mode, has a quasi-molecular ion peak at m / z 190.9767 (calcd 190.9745). 1 H-NMR and 13 C-NMR analysis revealed that the molecular formula of this compound is C5H5O6P, with an unsaturation degree of 5. The NMR spectrum of compound 2 is very similar to that of compound 1; the significant difference lies in the tertiary carbon (δ¹⁸) bonded to oxygen in compound 1. C 69.44 ppm) was replaced by secondary carbon in compound 2 (δ) C 33.65ppm). 1 H-NMR spectra showed δ HThere are two hydrogens at 2.72 ppm with a coupling constant J = 21.44 Hz, which is carbon coupling. There is one hydrogen at 6.07 ppm. 31 The p-chromatogram showed one phosphine at 18.42 ppm. According to the δ... C The high coupling constant J = 127.5 Hz of 33.65 ppm carbon suggests that the carbon atom is directly bonded to the phosphine atom. 1 H- 31 In the PHMBC two-dimensional spectrum, these hydrogen atoms are all associated with δ P The phosphine atom at 18.42 ppm is relevant. According to... 13 C-NMR, 1 H- 13 Based on the C HMQC and DEPT spectra, it can be deduced that the compound contains 5 carbon atoms with chemical shifts of δ... C 171.50, 169.15, 141.40, 125.30, 33.65 ppm. According to the HSQC spectrum, δ C 125.30 carbon and δ H 6.07 ppm hydrogen, 33.65 ppm carbon and δ H The two hydrogen atoms at 2.72 ppm are connected. 1 H- 13 The C HMBC spectrum shows that δ H 6.07 ppm hydrogen and δ C Carbon concentrations of 33.65, 141.40, and 171.50 ppm are all relevant, δ H 2.72 ppm hydrogen and δ C Carbon concentrations of 125.30, 141.40, and 171.5 ppm are all relevant. Figure 3 ).comprehensive 1 H NMR, 13 C NMR, 31 p-NMR, DEPT135, 1 H- 1 H COSY, 1 H- 31 P HMBC, 1 H- 13 C HMBC ultimately determined the structure of compound 2 to be 2-(phosphonomethyl)furan-2,5-dione. NMR data are shown in Table 4.

[0064] Table 4 Chemical shifts of compound 2 (in D2O)

[0065]

[0066] Example 3:

[0067] Staphylococcus aureus ATCC65381, Bacillus subtilis ATCC9372, Pseudomonas aeruginosa ATCC27853, and Escherichia coli ATCC25922, preserved in the laboratory, were selected as subjects for antibacterial activity testing. The antibacterial activity of two newly isolated phosphonic acid compounds was studied. The specific experimental procedures are as follows:

[0068] Staphylococcus aureus ATCC65381, Bacillus subtilis ATCC9372, Pseudomonas aeruginosa ATCC27853, and Escherichia coli ATCC25922 were taken from a -80℃ freezer, thawed at room temperature, and then inoculated into LB solid medium in a clean bench. After activation culture, the strains were placed in a 28℃ incubator. After the strains grew, they were transferred to LB liquid medium and cultured in a shaker at 28℃ and 130 rpm for 12 h to prepare bacterial suspension. Take 200 μL of Staphylococcus aureus ATCC65381, Bacillus subtilis ATCC9372, Pseudomonas aeruginosa ATCC27853, and Escherichia coli ATCC25922 suspensions respectively and add them to 50 mL of melted and cooled LB solid medium to a suitable temperature. (During this process, pay attention to the temperature of the medium to prevent the bacteria from being killed, and slowly shake to ensure that the bacterial suspension and medium are fully mixed to prevent the formation of air bubbles and uneven growth of the bacteria later.) After mixing, pour the mixture evenly into three 90 mm disposable petri dishes (the bottom of the plates has been marked with crosses beforehand). Pour 15 mL of the mixed liquid onto each plate, and set up three replicates. After the medium solidifies, spot the samples on top. Take 5 μL each of the test compound (1 mg / mL), sterile water, and ampicillin (1 mg / mL) and spot them onto the center of each equal part of the plate. Seal the plate and incubate it in a 28 °C incubator for 12 h to determine the antibacterial activity. The results showed that compounds 1 and 2 had no antibacterial activity against Staphylococcus aureus ATCC65381, Bacillus subtilis ATCC9372, Pseudomonas aeruginosa ATCC27853 and Escherichia coli ATCC25922.

[0069] Example 4:

[0070] In a clean bench, Arabidopsis seeds were placed in sterilized 2ml centrifuge tubes, washed with anhydrous ethanol for 5 minutes, and the ethanol was removed. Then, the seeds were washed with 75% ethanol solution for 5 minutes, and the 75% ethanol solution was removed. The seeds were then washed repeatedly with sterile water 6-7 times. MS solid medium was prepared, sterilized, and poured into square petri dishes (approximately 15ml / plate). After solidification, 1ml of 1% agarose solution was added to the washed Arabidopsis seeds. Seeds were aspirated with a 1ml pipette and sown onto MS solid medium plates. The plates were sealed and placed in a 4°C refrigerator for vernalization for 2-3 days. Then, the plates were cultured at 23°C under 16 / 8 hour light / dark conditions for 3 days, after which germination was observed. Prepare five 50ml MS solid medium bottles, sterilize them, and allow them to cool to a suitable temperature in a laminar flow hood. Add 500ul of sterile glyphosate, glufosinate, the phosphonic acid compound to be tested, and sterile water to prepare a 0.1mM MS medium mixture of glyphosate, glufosinate, and the phosphonic acid compound to be tested. (During this process, ensure that the medium temperature is not too high to prevent affecting the structure of the compounds, and slowly shake to ensure that the sample and medium are fully mixed to prevent air bubbles and uneven seedling growth later.) Pour 15ml of the mixture onto each plate. After solidification, gently lift the germinated seedlings with a sterile pipette tip and transfer them to their respective mediums. Seal the plates and incubate them at 23°C under 16 / 8-hour light / dark conditions for 7 days. Observe their growth. Finally, gently pull the Arabidopsis thaliana with tweezers to ensure that the plant is basically free of agar. Immediately measure its root length. Then place the Arabidopsis thaliana on a paper towel and gently press it to ensure that the roots are free of agar before measuring its fresh weight.

[0071] In the experiment on the inhibition of Arabidopsis growth, we used water as a blank control group, and the experimental groups were 0.1 mM of compound 1 and 0.1 mM of compound 2, as well as 0.1 mM of glyphosate and 0.1 mM of glufosinate. After 7 days of growth, the experimental groups and the blank control group were observed, and it was found that the root length and leaf growth of Arabidopsis in the four experimental groups were significantly inhibited compared with the blank group. Figure 4 A shows the phenotypic diagram of Arabidopsis seedlings after 7 days of growth. All Arabidopsis plants treated with compounds 1 and 2 were in a necrotic state, especially those in the medium containing compound 1, where root and leaf growth was absent. The Arabidopsis in the medium containing compound 2 also rapidly died after a short period of growth. Among the four experimental groups, compound 1 exhibited the strongest growth-inhibiting activity against Arabidopsis. Compared to glufosinate, compound 2 showed a more significant inhibitory activity on leaf growth in Arabidopsis. Figure 4B is a bar chart of the total fresh weight of five groups of Arabidopsis plants, with 13 plants counted in each group. All four phosphonic acid compounds at 0.1 mM significantly reduced the total fresh weight of Arabidopsis plants. Among them, compound 1 showed significantly stronger growth-inhibiting activity against Arabidopsis than glyphosate, glufosinate, and compound 2. Compound 2 showed growth-inhibiting activity comparable to glyphosate against Arabidopsis, but stronger than glufosinate. Figure 4 C is a statistical chart of root length in Arabidopsis thaliana. In the root length measurement, the root length of Arabidopsis thaliana did not grow after treatment with compound 1, while compound 2 and glyphosate had a similar inhibitory effect on root growth of Arabidopsis thaliana. Glufosinate had a weaker activity on root length compared to other phosphine-containing compounds.

[0072] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. Streptomyces ( Streptomyces sp. The application of G0098 in the preparation of phosphonic acid compounds 1-2 is characterized by: The structures of the phosphonic acid compounds 1-2 are as follows: The Streptomyces G0098 was deposited on May 9, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 30537.

2. By Streptomyces ( Streptomyces sp. Phosphonic acid compounds 1-2 prepared by G0098 are characterized by: The structures of the phosphonic acid compounds 1-2 are as follows: The Streptomyces G0098 was deposited on May 9, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 30537.

3. The method for preparing phosphonic acid compounds 1-2 as described in claim 2, characterized in that: Includes the following steps: S1: Streptomyces ( Streptomyces sp. After activation, G0098 was picked and a single colony was inoculated into a 250ml Erlenmeyer flask containing 100ml of ATCC172 liquid medium. The flask was incubated at 28℃ and 130rpm for 3 days to obtain the seed culture. 1ml of the seed culture was then inoculated into a flask containing... In a 250ml Erlenmeyer flask containing 100ml of R2A liquid medium, culture at 28℃ and 130rpm for 3 days to obtain fermentation culture 1; take 100ml of fermentation culture 1 and inoculate it into a 5L Erlenmeyer flask containing 1L of R2A liquid medium, and culture at 28℃ and 130rpm for 12 days to obtain fermentation culture 2; vacuum filter fermentation culture 2 to separate mycelium and fermentation broth; S2: The fermentation broth obtained in step S1 is concentrated into an extract, and then subjected to HP20 macroporous resin adsorption column chromatography, A-107 strong basic anion exchange resin column chromatography, Sephadex LH-20 dextran gel column chromatography, and semi-preparative liquid chromatography to obtain phosphonic acid compounds 1-2.

4. The preparation method according to claim 3, characterized in that: The formulation of the ATCC172 liquid culture medium is as follows: acid-hydrolyzed casein 5 g / L, yeast extract 5 g / L, glucose 10 g / L, soluble starch 20 g / L, calcium carbonate 1 g / L, with water as the solvent; the formulation of the R2A liquid culture medium is as follows: peptone 0.5 g / L, acid-hydrolyzed casein 0.5 g / L, yeast extract 0.5 g / L, glucose 0.5 g / L, soluble starch 0.5 g / L, potassium dihydrogen phosphate 0.3 g / L, magnesium sulfate heptahydrate 0.05 g / L, sodium pyruvate 0.3 g / L, with water as the solvent.

5. The preparation method according to claim 3, characterized in that: The HP20 macroporous resin adsorption column chromatography was performed using 100% [a specific method / technology]. The elution was performed using H2O, 50% H2O-MeOH, and 100% MeOH. The A-107 strong basic anion exchange resin column chromatography was eluted with 2% NaCl, 3% NaCl, and 4% NaCl. The Sephadex LH-20 dextran gel column chromatography was eluted with 80% MeOH-H2O. The semi-preparative liquid chromatography separation was eluted with 90% H2O-MeOH.

6. The use of the phosphonic acid compounds 1-2 as described in claim 2 in the preparation of herbicides.

7. The application of phosphonic acid compounds 1-2 as described in claim 2 in inhibiting Arabidopsis thaliana growth.

8. The application according to claim 7, characterized in that: Inhibiting Arabidopsis growth refers to reducing the fresh weight and root length of Arabidopsis.

Images