Group of novel compounds separated from magnaporthe oryzae antagonistic bacteria ZY66 and application thereof

Abstract

The invention discloses a group of novel compounds separated from a magnaporthe oryzae antagonistic bacterium ZY66 and application of the novel compounds. The group of substances comprises the following raw materials: the compounds comprise compounds 1 to 4; the compound 1 is 2-acetyl-3, 8, 10-trihydroxy-1, 7, 10-trimethylanthracene-9 (10H)-ketone, the chemical formula of the compound 1 is C19H18O5, and the relative molecular mass of the compound 1 is 326.12; the compound 2 is 3, 8, 10-trihydroxy-1, 7, 10-trimethyl-9-oxo-9, 10-dihydroanthracene-2-formic acid, the chemical formula of the compound 2 is C18H16O6, and the relative molecular mass of the compound 2 is 328.09; the compound 3 is 3, 6-dihydroxy-1-methyl anthracene-9, 10-diketone, the chemical formula of the compound 3 is C15H10O4, and the relative molecular mass of the compound 3 is 254.06; the compound 4 is 3, 6, 10-trihydroxy-1, 10-dimethyl anthracene-9 (10H)-ketone, the chemical formula of the compound 4 is C16H14O4, and the relative molecular mass of the compound 4 is 270.09; through comparison with an SCI Finder database, the compounds 1-4 are all new compounds.

Description

Technical Field

[0001] The invention belongs to the field of creating new plant protection products, and particularly relates to a group of compounds separated from rice blast antagonistic bacteria ZY66 and uses thereof. Background Art

[0002] Rice is one of the most important food crops in the world today, and more than half of the world's population relies on rice as their staple food. Rice blast (Pyricularia oryzae) is an important disease on rice, and is known as the three major rice diseases together with sheath blight and bacterial blight. Rice blast can occur in all rice-growing areas around the world. In my country, it mainly harms rice-growing areas such as the northeast, southwest, and the middle and lower reaches of the Yangtze River. Rice blast seriously harms rice yield and affects the quality of rice. The rice yield reduction caused by rice blast is generally around 10%, but when the disease is severe, it can reduce yield by 50%, and even no grain can be harvested in some areas. At present, there are many methods for preventing and controlling rice blast in production, such as cultivation management, water and fertilizer management, physical control, ecological regulation, chemical control, etc. Among them, chemical control is still the main means, but long-term single application of a certain agent will lead to a series of problems such as bacterial resistance, reduced prevention effect, and environmental pollution. At the same time, pathogens are prone to mutation, resulting in disease prevention work not keeping up with mutations, causing difficulties in disease control. In addition, the damage caused by rice blast is getting worse year by year, and the development of new fungicides for rice blast fungus is slow. Therefore, it is urgent to actively find new fungicides to replace the main agents. Biological control has the advantages of low cost and no pollution compared with chemical control. It is a pollution-free control method that uses organisms and their metabolites to control pests and diseases. Actinomycetes are the largest natural resource for producing secondary metabolites, and there is a great possibility of discovering new compounds with novel structures for the prevention and treatment of rice blast. In the early stage of the present invention, an antagonistic bacterium ZY66 with antibacterial activity against rice blast fungus was discovered (a Streptomyces acidiscabies was deposited in the Institute of Microbiology, Chinese Academy of Sciences, General Microbiology Center, China Microbiological Culture Collection Administration Committee on April 1, 2024, with a deposit number of CGMCC No. 30225) (referred to as strain ZY66), but its type and active ingredients are unknown, which has attracted everyone's attention. Its type was identified through colony observation, morphological identification, molecular detection, etc. At the same time, its secondary metabolites were separated, purified and identified by means of various technologies such as TLC, column chromatography, NMR, etc., and the inhibitory effect of pure compounds on rice blast fungi and the influence on rice seed germination were detected. In summary, the present invention is not only conducive to enriching the control agents for controlling rice blast fungi, but also helps to provide a theoretical basis for the development of subsequent lead compounds. Summary of the invention

[0003] The present invention aims to overcome the deficiencies of the prior art and provides a group of compounds separated from the rice blast antagonistic bacteria ZY66 and uses thereof.

[0004] In order to achieve the above object, the technical solution provided by the present invention is:

[0005] The present invention evaluates the antibacterial activity of strain ZY66 and its ester extract on rice blast fungus by confrontation test and growth rate method. The test shows that strain ZY66 and its ester extract can produce strong inhibitory effect on rice blast fungus, and when the concentration of ester extract is 6.25 μg / mL, the inhibition rate can reach 52.61%. Spore germination test shows that ester extract has no obvious inhibitory effect on the germination of rice blast spores, but has obvious inhibitory effect on the growth of spores. Through morphological observation of strain ZY66 on four different culture media, it is judged that it belongs to Streptomyces; through molecular biological identification methods such as DNA extraction, PCR amplification, gel electrophoresis and phylogenetic analysis, it is found that the homology of strain ZY66 with model strain ATCC 49003 (NR_116534.1) is 99.86%, and it is identified as Streptomyces acidiscabies (Streptomyces acidiscabies) (preservation number: CGMCC No.30225). The strain is easy to culture, ferments quickly and has strong stress resistance.

[0006] The compounds isolated from the rice blast antagonist ZY66 include compounds 1 to 4; the compound 1 is 2-acetyl-3,8,10-trihydroxy-1,7,10-trimethylanthracene-9(10H)-one, with a chemical formula of C 19 H 18 O5, relative molecular mass 326.12, structural formula as shown in formula (Ⅰ);

[0007]

[0008] The compound 2 is 3,8,10-trihydroxy-1,7,10-trimethyl-9-oxy-9,10-dihydroanthracene-2-carboxylic acid, and its chemical formula is C 18 H 16 O6, relative molecular mass 328.09, structural formula as shown in formula (II):

[0009]

[0010] The compound 3 is 3,6-dihydroxy-1-methylanthracene-9,10-dione, and its chemical formula is C 15 H 10 O4, relative molecular mass 254.06, structural formula as shown in formula (III):

[0011]

[0012] The compound 4 is 3,6,10-trihydroxy-1,10-dimethylanthracen-9(10H)-one, and its chemical formula is C16 H 14 O4, relative molecular mass 270.09, structural formula as shown in formula (IV):

[0013]

[0014] By comparing with the SCI Finder database, compounds 1-4 are all new compounds.

[0015] The present invention uses a growth rate method to evaluate the activity of the new compound, and the results show that compounds 1, 2, 3 and 4 show obvious inhibitory effects on rice blast fungi. The activity ranking of the four antibacterial compounds is: compound 3> compound 4> compound 2> compound 1. The 96-well plate method was further used to determine the effects of the four compounds on rice blast fungi spores. The results showed that when the concentration of compound 4 was 125 μg / mL, the physiological structure of the rice blast fungi spores was destroyed, the intracellular diaphragm was dissolved, the cytoplasm overflowed, and the spore morphology was abnormal.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. For the first time, the antibacterial effect of Streptomyces acidiscabies strain and its secondary metabolites on rice blast fungus was discovered and evaluated. At present, there are few studies on the evaluation of the antibacterial effect of Streptomyces acidiscabies (preservation number: CGMCC No.30225) and the mining of its secondary metabolites, and its secondary metabolites are currently reported to be developed as herbicides. The present invention found that Streptomyces acidiscabies and its secondary metabolites have a good antibacterial effect on rice blast, which provides a theoretical basis for the future development and utilization of the agricultural value of Streptomyces acidiscabies.

[0018] 2. Four new anthraquinone compounds were discovered. The present invention separates and purifies the ester extract of strain ZY66 by multiple column chromatography techniques, and successfully analyzes five compounds by nuclear magnetic resonance technology, of which four compounds are new anthraquinone compounds, compound 1 is 2-acetyl-3,8,10-trihydroxy-1,7,10-trimethylanthracene-9(10H)-one, compound 2 is 3,8,10-trihydroxy-1,7,10-trimethyl-9-oxy-9,10-dihydroanthracene-2-carboxylic acid, compound 3 is 3,6-dihydroxy-1-methylanthracene-9,10-dione, and compound 4 is 3,6,10-trihydroxy-1,10-dimethylanthracene-9(10H)-one.

[0019] 3. Four new compounds with antibacterial activity against rice blast fungus were evaluated. In the indoor toxicity plate test, compounds 1, 2, 3 and 4 of the four isolated compounds showed obvious inhibitory effects on rice blast fungus. The activity ranking of the four antibacterial compounds was: compound 3> compound 4> compound 2> compound 1. Compound 4 had a certain effect on rice blast spores. The rice blast spores treated with compound 4 solution showed physiological structure destruction, intracellular septa dissolution, cytoplasm overflow, abnormal spore morphology and other phenomena. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The colony morphology of strain ZY66 on different culture media; in the figure: (A) Gao's medium No. 1; (B) potato dextrose agar medium; (C) potato sucrose agar medium; (D) beef extract peptone agar medium;

[0021] Figure 2 The results of the electrophoresis test of strain ZY6616S rRNA; in the figure: (A) strain ZY6616S rRNA gene amplification product; (M) molecular weight standard 3000bp;

[0022] Figure 3 is the phylogenetic tree of strain ZY66;

[0023] Figure 4 The inhibitory effect of antagonistic bacteria on rice blast fungi; in the figure: (A) one bacterial filter paper piece confronts with rice blast fungi; (B) two bacterial filter paper pieces confront with rice blast fungi; (C) three bacterial filter paper pieces confront with rice blast fungi; (D) four bacterial filter paper pieces confront with rice blast fungi; (E): control group;

[0024] Figure 5 The inhibitory effects of ester extracts of strain ZY66 at different concentrations on rice blast fungus; in the figure: (A) 100 μg / mL; (B) 50 μg / mL; (C) 25 μg / mL; (D) 12.5 μg / mL; (E) 6.25 μg / mL; (F) CK;

[0025] Figure 6 The inhibitory effects of different concentrations of strain ZY66 ester extract on rice blast spore germination; In the figure: the lowercase letters in the figure indicate whether there are significant differences at the 0.05 level after Duncan's new multiple range method multiple test;

[0026] Figure 7 are the structural formulas of Huanglongmycin N and compound 1; in the figure: (A) Huanglongmycin N; (B) compound 1;

[0027] Figure 8The data and spectra related to the structural identification of compound 1; in the figure: (A) structural formula; (B) thin layer chromatography detection, B1254 nm ultraviolet irradiation, B2365 nm ultraviolet irradiation, B3 iodine vapor fumigation, B4 sulfuric acid staining; (C) full wavelength scanning; (D) high performance liquid chromatography; (E) 1H NMR; (F) 13C NMR; (G) HSQC; (H) HMBC; (I) HH COSY; (J) mass spectrum;

[0028] Fig. 9 is the structural formula of compound 2;

[0029] Fig.10 The data and spectra related to the structural identification of compound 2; in the figure: (A) structural formula; (B) thin layer chromatography detection, B1254 nm ultraviolet irradiation, B2365 nm ultraviolet irradiation, B3 iodine vapor fumigation, B4 sulfuric acid staining; (C) full wavelength scanning; (D) high performance liquid chromatography; (E) 1H NMR;

[0030] Fig.11 Are the structural formulas of Aloesaponarin II and compound 3; In the figure: (A) Aloesaponarin II (B) Compound 3;

[0031] Fig.12 The data and spectra related to the structural identification of compound 3; in the figure: (A) structural formula; (B) thin layer chromatography detection, B1254 nm ultraviolet irradiation, B2365 nm ultraviolet irradiation, B3 iodine vapor fumigation, B4 sulfuric acid staining; (C) full wavelength scanning; (D) high performance liquid chromatography; (E) 1H NMR; (F) 13C NMR; (G) HSQC; (H) HMBC; (I) HH COSY

[0032] Fig.13 The structural formulas of WS9761 A and compound 4 are shown in the figure: (A) WS9761 A (B) compound 4;

[0033] Fig.14 The data and spectra related to the structural identification of compound 4; in the figure: (A) structural formula; (B) thin layer chromatography detection, B1254 nm ultraviolet irradiation, B2365 nm ultraviolet irradiation, B3 iodine vapor fumigation, B4 sulfuric acid staining; (C) full wavelength scanning; (D) high performance liquid chromatography; (E) 1H NMR; (F) 13C NMR; (G) HSQC; (H) HMBC; (I) HH COSY;

[0034] Fig.15The inhibitory effects of four compounds on rice blast; in the figure: (A) 150μg / mL carbendazim; (B) 500μg / mL compound 1; (C) 500μg / mL compound 2; (D) 500μg / mL compound 3; (E) 500μg / mL compound 4; the upper holes are positive control and test group, and the lower holes are negative control;

[0035] Fig.16 The effect of compound 4 on the spores of rice blast fungus; in the figure: (A), (B) spore morphology of the control group; (C), (D), (E), (F) spore morphology of 125 μg / mL compound 4. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is further described below in conjunction with specific tests and embodiments. The percentages involved in the tests and embodiments of the present invention are all mass percentages.

[0037] Embodiment 1:

[0038] (1) Test materials

[0039] Huang Huazhan conventional rice (Hunan Golden Nongfeng Seed Co., Ltd.); Pyricularia oryzae was provided by the Plant Pathology Laboratory of Hunan Agricultural University. The test culture media mainly include Potato Dextrose Agar (PDA), Potato Dextrose Broth (PDB), Potato Sucrose Agar (PSA), Nutrient Agar (NA), GAUZEˊs Medium, Tomato Oatmeal Agar, and Luria-Bertani (LB). The test reagents are mainly common reagents such as anhydrous ethanol, chloroform, acetone, petroleum ether, ninhydrin, copper sulfate, and sodium hydroxide. The test instruments mainly include clean bench, high-pressure steam sterilizer, mold constant temperature incubator, biochemical incubator, high-power magnetic stirrer, rotary evaporator, ultraviolet spectrophotometer and other commonly used laboratory instruments.

[0040] (2) Identification and antibacterial effect of strain ZY66

[0041] 2.1 Activation of strain ZY66 and preparation of extracts

[0042] Take the strain ZY66 stored at -20℃ and restore it from the refrigerated state to room temperature. Use a sterilized inoculation loop to pick the bacteria and inoculate them into a new PDA medium for cultivation. Repeat this operation 2 to 3 times, and culture continuously for 3 generations at 26℃ to obtain strains with excellent activity. Inoculate strain ZY66 into sterilized PDB medium, culture at 26℃ and 150r / min for 5 days, and remove the bacteria and precipitate by negative pressure filtration. Pour the treated fermentation broth and an equal volume of ethyl acetate into the separatory funnel, shake and mix thoroughly, let stand for 30 minutes, collect the upper organic phase, and extract each batch of fermentation broth with ethyl acetate three times. Pour the collected organic phase into a rotary evaporator, vacuum concentrate it to a paste using a rotary evaporator, and store it in a 4℃ refrigerator for storage.

[0043] 2.2 Inhibitory effects of strain ZY66 and its extracts on rice blast

[0044] iThe plate confrontation method was used to detect the inhibitory effect of strain ZY66 on rice blast fungus. The rice blast fungus was punched into a 10 mm round cake with a sterile hole puncher and then inoculated on the PDA medium. A small amount of mycelium of strain ZY66 was scraped with a sterile inoculation loop and dispersed in 1 mL of sterile water. A sterile filter paper (diameter: 0.5 cm) was immersed in the bacterial suspension, taken out, and the excess water was drained and attached to the PDA medium. The PDA medium inoculated with rice blast alone was used as the control. Each group was repeated 3 times and cultured at a constant temperature of 26°C for 9 days.

[0045] ii The growth rate method was used to determine the inhibitory effect of the ester extract of strain ZY66 on rice blast fungus. Take 2 mg of ester extract and dissolve it in 2 mL of methanol, then use sterile water to prepare a series of ester extract solutions by gradient dilution, the concentrations are 1,000 μg / mL, 500 μg / mL, 250 μg / mL, 125 μg / mL and 62.5 μg / mL. Mix each concentration solution with the heated and melted PDA medium at a ratio of 9:1 to obtain a drug-containing medium with a final concentration of 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, and 6.25 μg / mL. The rice blast fungus was inoculated on the drug-containing medium, and the PDA medium with sterile water added at a ratio of 9:1 was used as the control group. Each group was repeated 3 times and cultured at a constant temperature of 26°C for 9 days. The inhibition rate was calculated according to formula 1.

[0046] 2.3 Preparation of Rice Blast Spore Suspension

[0047] Inoculate the rice blast fungus onto the PDA medium. After it has grown all over the culture dish, add 1-2 mL of sterile water, apply it on the surface of the medium with a sterile cotton swab, use a pipette to suck out the water containing the mycelium of the rice blast fungus on the surface of the medium, and add an equal volume of sterile water to obtain a mycelium suspension. Drop 0.2 mL of mycelium suspension on the surface of the solidified tomato oatmeal agar medium, spread it evenly with a triangular device, and culture it continuously in a light incubator for 5 days. Use the same method as above, drop sterile water, apply it with a sterile cotton swab, and filter it with three layers of gauze to obtain a spore suspension. The spore germination rate is calculated according to Formula 2.

[0048] 2.4 Effects of strain ZY66 extract on spore germination of rice blast fungus

[0049] The inhibitory effect of ester extract on spore germination of rice blast fungus was determined by 2-fold dilution method in 96-well plates. The concentrations of the compounds in each test group were set to 50 μg / mL, 25 μg / mL, 12.5 μg / mL, and 6.25 μg / mL, respectively. Each treatment was repeated 10 times, and PDB medium and sterile water were mixed in equal proportions as the control group. 50 μL of spore suspension was added to each well and placed in a 26°C constant temperature incubator for 8 hours. The total number of spores and the number of germinated spores in each lens were recorded by five-point sampling method under a microscope, and the germination rate was calculated.

[0050] 2.5 Colony morphology observation and molecular identification of strain ZY66

[0051] The plate streak method was used to activate the antagonistic bacterial strain ZY66 on potato dextrose agar medium, potato sucrose agar medium, beef extract peptone agar medium, and Gao's medium No. 1. After culturing at 26°C for 5 days, the morphological characteristics such as colony color, shape, and texture were observed. The purified strains were inoculated into 30 mL of PDB medium, cultured at 26°C and 150 r / min for 72 hours, and then the supernatant was removed by centrifugation to collect the bacteria. The DNA of the bacteria was extracted using the Accurate Biotechnol DNA extraction kit, and the DNA concentration and purity were detected using an ultra-micro nucleic acid analyzer and agarose electrophoresis, respectively. The universal primers 27F and 1492R were used to amplify the strain 16S rRNA sequence, and the reaction solution was configured according to the reaction system shown in Table 1:

[0052] Table 1

[0053]

[0054]

[0055] PCR reaction conditions are shown in Table 2:

[0056] Table 2

[0057]

[0058] The PCR product was subjected to agarose gel electrophoresis to recover a 1500 bp fragment, and the fragment was purified using a DNA purification kit. All 16S rRNA genes were cloned into the pMD19-T vector and transformed into competent cells E. coli DH5α. Single colonies were picked into LB medium, and plasmids were extracted after overnight culture and sent to Sangon Biotech Co., Ltd. for sequencing. The sequencing results were compared and analyzed in NCBI (https: / / www.ncbi.nlm.nih.gov / ), and 16S rRNA sequences with high similarity were selected. Multiple sequence comparison analysis was performed using the neighbor-joining method in MEGA 11 software, and a phylogenetic tree was constructed and 1000 bootstraps tests were performed.

[0059] (3) Results and analysis

[0060]

[0061] Evenly evaporate all the methanol and then perform dry loading. Put a layer of cotton wool under the loading column, evenly add the sample-mixed silica gel to the reverse phase chromatography column, and then put another layer of cotton wool. Then elute in the order of mobile phase (30% methanol, 40% methanol, 50% methanol, 60% methanol, 70% methanol, 80% methanol, 90% methanol and pure methanol). Rinse 1000mL of each eluent and collect components every 80mL. Each collected component must be concentrated by rotary evaporation and then tested by thin layer chromatography for material spots and similar components are combined and stored at -20℃ for later use.

[0062] (2) Silica gel column chromatography

[0063] First, the eluent is screened, and then the stationary phase is prepared. The components to be separated are dissolved in methanol and mixed with twice the weight of silica gel, and then ground and mixed in a 60°C water bath. After the solvent evaporates, it is evenly added to the silica gel column, and gently tapped with an ear bulb to make the silica gel surface level, and then a ball of absorbent cotton is plugged on the silica gel to prepare for the next elution. The selected eluent ratio is gradient eluted in order of polarity from small to large, and 4 mL is collected once with a thumb tube. The collected fractions are tested for material spots by thin layer chromatography, and the components with the same spots are merged.

[0064] (3) Thin layer chromatography separation

[0065] Use methanol or chloroform to fully dissolve the sample. There should be no crystals or precipitation in the sample. Choose a thin layer chromatography silica gel plate (10×20cm), use a pencil to gently draw a straight line 1cm from the bottom of the thin layer chromatography plate as the starting line, and then gently draw a straight line 1cm from the top of the thin layer chromatography plate as the end line. Use a capillary glass tube to absorb an appropriate amount of sample, and evenly point points of the same size on the starting line of the silica gel plate from left to right. Pour the screened eluent (about 20mL) into the chromatography cylinder, and then gently put the loaded silica gel plate into the cylinder, take it out when the solvent runs to the end line, dry it at room temperature, and then perform ultraviolet detection. Under the ultraviolet detector, outline the individual bands with a pencil, and then scrape the silica gel with a scalpel according to the outline, soak the scraped silica gel with methanol, shake it on a shaker at 150r / min for 20min, then pour out the solution, and repeat 3 times to fully dissolve the components.

[0066] The methanol solution was centrifuged at 9000 r / min for 5 min to remove the silica gel powder, the supernatant was concentrated under reduced pressure until the methanol was completely volatilized, acetone was added in small amounts several times to dissolve it, and the silica gel adhesive was removed by filtering through a 0.22 μm microporous filter membrane.

[0067] (4) Sample testing method

[0068] The purity of the collected samples is detected by thin layer chromatography. After the different components are developed on the thin layer chromatography plate, it is usually difficult to directly observe the separation of each substance with the naked eye. In the present invention, the purity of the sample is detected mainly by means of three detection methods: optical detection method, iodine vapor colorimetric method, and reagent colorimetric method. After the sample is dissolved with methanol, the sample is filtered through a 0.22μm microporous filter membrane to remove impurities in the sample. Methanol and ultrapure water are selected to be configured as a mobile phase. The mobile phase needs to be negatively pressure filtered 2-3 times before use, and then ultrasonically debubbled for 30 minutes. The present invention selects a mobile phase ratio of methanol / water: 30%-100%, a flow rate of 1mL / min, a detection wavelength of 254nm, a detection time of 15min, and an injection volume of 20μL. Detect whether there is only one symmetrical peak on the spectrum after the later observation.

[0069] (5) Separation and purification process of active components of strain ZY66

[0070] 3 g of ester extract of strain ZY66 was purified by C 18Reverse phase column chromatography separation, mobile phase selected 30%, 40%, 50%, 60%, 70%, 80%, 90% and pure methanol, every 80mL collect a component, use thin layer chromatography to detect the spot situation, and merge the components with the same spot, a total of 4 components are obtained, namely components 20-33, 34-45, 46-48 and 60-68. Component 20-33 (A) is further separated by silica gel column chromatography, the mobile phase is selected methanol: chloroform (8: 2, 7: 3, 6: 4, 5: 5), every 5mL collect a component, thin layer chromatography detection again, merge similar components, obtain components A20-53 and A54-86. The five components 20-33, 34-45, 46-48, 60-68, A20-53 and A54-86 obtained are respectively subjected to thin layer chromatography preparative separation to obtain compounds 1-5, wherein compound 5 is a known compound and discarded.

[0071] (6) Compound structure identification

[0072] The compound dissolved in methanol was scanned at full wavelength using a photodiode array detector, with a measurement range of 190-1100nm, a scanning interval of 1nm, and a scanning frequency of 3 times. The pure compound to be tested was vacuum dried to remove the residual organic solvent and water in the sample. The sample was completely dissolved in deuterated methanol or deuterated dimethyl sulfoxide (about 500-600μL), and scanned on a BrukerAvance 500MHz to measure the hydrogen and carbon spectra. The test temperature was 297.3K, and tetramethylsilane (TMS) was used as the internal standard for both hydrogen and carbon spectra. According to the one-dimensional spectrum data, two-dimensional spectra such as HSQC, HMBC and HH COSY were detected. Ion source parameters: spray voltage 3.5kV; sheath gas flowrate 45Arb; aux gas flowrate10Arb; sweep gas flowrate 0Arb; Ion tube temperature 320℃; S-lens RF level 60; ion source temperature 350℃. Resolution: 70000FWHM; AGC: 3e6; Maximum IT: 250ms; scanned area: 300-1200Da. Resolution: 17500FWHM; AGC: le5; Maximum IT: 64ms; Loopcount: 3; Isolation Width: 1.2Da; collisionparameter: 30, 45; scanned area: 200-2000Da; Spectrum datatype: profile; Minimum response value: 1.3e4; Excludeisotope: on; Dynamic exclusion: 8.0s.

[0073] (7) Results and analysis

[0074] 7.1 Structural identification of compound 1

[0075] Compound 1 is a yellow powder, which is light yellow when dissolved in methanol. Thin layer chromatography has obvious absorption under 254nm ultraviolet light and orange-red fluorescence under 356nm ultraviolet light. There is no obvious band after iodine vapor treatment, and the band disappears after being placed in the air for a period of time. A light yellow band appears after sulfuric acid color development ( Figure 8 -B). The results of the full wavelength scan from 190 to 1100 nm showed that the substance had a maximum absorption wavelength at 224 nm and absorption peaks at 259 nm and 326 nm ( Figure 8 -D).

[0076] pass 1 H NMR spectrum ( Figure 8 -E), a signal peak containing 7 hydrogen signals was obtained: 1 H NMR (800MHz, Methanol-d4)δ H 7.36 (d, J = 7.8 Hz, 1H), 7.28 (s, 1H), 7.22 (d, J = 7.8 Hz, 1H), 2.96 (s, 3H), 2.23 (s, 3H), 1.95 (s, 3H), 1.52 (s, 3H). H =1.52, 1.95, 2.23, 2.96 are hydrogen signals on the methyl group, δ H =7.36, 7.28, 7.22 are hydrogen signals on conjugated olefins. 13 C NMR spectrum ( Figure 8 -F), a signal peak containing 19 carbon signals was obtained: 13 C NMR (151MHz, Methanol-d4)δ191.66,177.77,164.10,161.19,155.73,148.49,145.88,136.82,126.53,125.77,121.45,116.25,115.52,111.72,71.52,39.30,22.43,21.46,15.44. C =39.30, 22.43, 21.46 and 15.44 carbon signals are methyl carbon signals, δ C =71.52 may be the oxygen-carbon signal, δ C =155.73, 148.49, 145.88, 136.82, 126.53, 125.77, 121.45, 116.25, 115.52 and 111.72 are carbon signals on conjugated olefins, δ C =191.66 may be the carbon signal on the carbonyl group. According to the HSQC spectrum ( Figure 8 -G) we can see that δ C =191.66, 177.77, 164.10, 155.73, 148.49, 145.88, 126.53, 125.77, 121.45, 116.25, 71.52 are quaternary carbons, and the remaining atoms are assigned to: δ H =7.36 and δ C =136.82 has signal, δ H =7.28 and δ C =111.72 has signal, δ H =7.22 and δ C =115.52 has signal, δH =2.96 and δ C =21.46 has signal, δ H =2.23 and δ C =15.44 has signal, δ H =1.95 and δ C =22.43 has signal, δ H =1.53 and δ C =39.3 has a signal. According to the HMBC spectrum ( Figure 8 -H) we can see that δ H =7.36 and δ C =15.44, 115.52, 148.49 and 161.19 are associated, δ H =7.28 and δ C =71.52, 121.45, 126.53 and 164.10 are associated, δ H =7.22 and δ C =71.52, 116.25 and 125.77 are associated, δ H =2.96 and δ C =121.45, 126.53 and 145.88 are associated, δ H =2.23 and δ C =125.77, 136.82 and 161.19 are associated, δ H =1.95 and δ C =177.77, δ H =1.52 and δ C =71.53, 148.48 and 155.75. According to the HH COSY spectrum ( Figure 8 -I) we can see that δ H =2.26 and δ H =7.38 two hydrogen signals coupled to each other. According to high resolution mass spectrometry ( Figure 8 -I) we can know the quasi-molecular ion m / s of the compound: [M+H] + =327.0859.

[0077] After consulting relevant literature, it was found that the structure of this compound is similar to that of Huanglongmycin N ( Figure 7 -A), compound 1 is similar to Huanglongmycin N, the double bond oxygen at position 10 is replaced by a hydroxyl group and a methyl group, the hydroxyl group at position 1 is replaced by a methyl group, and the methyl group at position 3 is replaced by a hydroxyl group. The above data are analyzed to determine its structure ( Figure 7-B), through the search and comparison of relevant literature in databases such as SCI Finder, no compound with the same structure was found, and it was identified as a new anthraquinone compound - 2-acetyl-3,8,10-trihydroxy-1,7,10-trimethylanthracen-9(10H)-one. The chemical formula of compound 1 is C 19 H 18 O5, relative molecular mass is 326.12. .

[0078] 7.2 Structural identification of compound 2

[0079] Compound 2 is a yellow powder, which is light yellow when dissolved in methanol. Thin layer chromatography has obvious absorption under 254nm ultraviolet light and orange-red fluorescence under 356nm ultraviolet light. There is no obvious band after iodine vapor treatment, and yellow bands appear after sulfuric acid color development ( Fig.10 -B). The results of the full wavelength scan from 190 to 1100 nm showed that the substance had a maximum absorption wavelength at 222 nm and absorption peaks at 259 nm and 327 nm ( Fig.10 -C). By 1 H NMR spectrum ( Fig.10 -E), a signal peak containing 6 hydrogen signals was obtained: 1 H NMR (800MHz, Methanol-d4)δ H 7.37 (d, J = 7.8 Hz, 1H), 7.29 (s, 1H), 7.23 (d, J = 7.8 Hz, 1H), 2.98 (s, 3H), 2.25 (s, 3H), 1.54 (s, 3H). H =1.54, 2.25, 2.98 are hydrogen signals on the methyl group, δ H =7.37, 7.29, 7.23 are hydrogen signals on conjugated olefins.

[0080] Comparison of the hydrogen spectrum of compound 1 revealed that in addition to the lack of δ H =1.95, the other signals are exactly the same, so the structure of compound 2 is basically the same as compound 1, except that the carbon number 2 is not acetyl but carboxyl ( Fig. 9 Compound 2 was identified as a new anthraquinone compound - 3,8,10-trihydroxy-1,7,10-trimethyl-9-oxo-9,10-dihydroanthracene-2-carboxylic acid. The chemical formula of compound 2 is C18 H 16 O6, relative molecular mass is 328.09.

[0081] 7.3 Structural identification of compound 3

[0082] Compound 3 is a yellow powder, which is light yellow when dissolved in methanol. Thin layer chromatography has obvious absorption under 254nm ultraviolet light and obvious orange-yellow fluorescence under 356nm ultraviolet light. There is no obvious band after iodine vapor treatment, and an orange-yellow band appears after sulfuric acid color development ( Fig.12 -B). The results of the full wavelength scan from 190 to 1100 nm showed that the substance had a maximum absorption wavelength at 218 nm and absorption peaks at 281 nm and 417 nm ( Fig.12 -C).

[0083] pass 1 H NMR spectrum ( Fig.12 -E), a signal peak containing 6 hydrogen signals was obtained: 1 H NMR (800MHz, Methanol-d6)δ H 14.44 (s, 1H), 7.55–7.46 (m, 2H), 7.15 (dd, J = 8.0, 1.3 Hz, 1H), 6.83 (d, J = 2.5 Hz, 1H), 6.29 (s, 1H), 2.53 (s, 3H). H =2.53 is the hydrogen signal on the methyl group, δ H =6.29, 6.83, 7.15, 7.55-7.46 are hydrogen signals on the benzene ring or unsaturated carbon, δ H =14.44 is the active hydrogen signal. 13 C NMR spectrum ( Fig.12 -F), a signal peak containing 15 carbon signals was obtained: 13 C NMR (151MHz, Methanol-d6)δ C 184.82,184.77,175.59,161.63,145.06,136.94,133.84,132.89,127.59,123.60,117.61,117.43,117.02,113.00,24.87. Among them, δ C =24.87 is the methyl carbon signal, δ C =145.06, 136.94, 133.84, 132.89, 127.59, 123.60, 117.61, 117.43, 117.02 and 113.00 are carbon signals on conjugated olefins, δ C=184.82 and 184.77 may be carbon on the carbonyl group. According to the HSQC spectrum ( Fig.12 -G) we can see that δ C =184.82, 184.77, 175.59, 161.63, 145.06, 136.94, 132.89, 117.61, 113.00 are quaternary carbons, and the remaining atoms are assigned to: δ H =2.53 and δ C =24.87 has signal, δ H =6.29 and δ C =127.59 has signal, δ H =6.83 and δ C =117.43 has signal, δ H =7.15 and δ C =123.60 has signal, δ H =7.50 and δ C =117.02 has signal, δ H =7.536 and δ C =133.84 has a signal. According to the HMBC spectrum (based on 12-H), δ H =2.53 and δ C =113.84, 127.59, 145.06 are associated, δ H =6.29 and δ C =24.87, 113.00, 117.43 are associated, δ H =6.83 and δ C =113.00, 127.59, 184.77 are associated, δ H =7.18 and δ C =117.02, δ H =7.50 and δ C =117.61, 123.60, 184.77 are associated, δ H =7.54 and δ C =132.89, 161.6. According to the HH COSY spectrum (according to 12-I), δ H The hydrogen signals at 2.56 and 6.32 are coupled to each other.

[0084] After consulting relevant literature, it was found that the structure of this compound is similar to that of Aloesaponarin II ( Fig.11 -A), but when assigning atoms according to the HMBC spectrum, it was found that the chemical shifts of some carbon atoms were abnormal. After analyzing the above data, it was determined that the hydroxyl group at position 8 was translocated to position 6, thus determining its structure ( Fig.11-B), through the search and comparison of relevant literature in databases such as SCI Finder, no compound with exactly the same structure was found, and it was identified as a new anthraquinone compound - 3,6-dihydroxy-1-methylanthracene-9,10-dione. The chemical formula of compound 3 is C 15 H 10 O4, relative molecular mass is 254.06.

[0085] 7.4 Structural identification of compound 4

[0086] Compound 4 is a yellow powder, orange-red when dissolved in methanol, and purple-red when dissolved in dimethyl sulfoxide. Thin layer chromatography has obvious absorption under 254nm ultraviolet light and bright yellow fluorescence under 356nm ultraviolet light. Yellow strips appear after iodine vapor treatment, and the strips disappear after being placed in the air for a period of time. After sulfuric acid color development, khaki strips appear ( Fig.14 -B). The results of the full wavelength scan from 190 to 1100 nm showed that the substance had a maximum absorption wavelength at 208 nm and absorption peaks at 220 nm and 319 nm ( Fig.14 -D).

[0087] pass 1 H NMR spectrum ( Fig.14 -E), a signal peak containing 7 hydrogen signals was obtained: 1 HNMR (800MHz, DMSO-d4)δ H 7.50(t,J=7.9Hz,1H),7.35(d,J=7.2Hz,1H),7.27(d,J=2.5Hz,1H),6.83(d,J=7.9Hz,1H),6.68(d,J=2.0Hz,1H),2.73(s,3H),1.55(s,3H). where δ H =2.73 and 1.55 are hydrogen signals on the methyl group, δ H =7.50, 7.35, 7.27, 6.83 and 6.68 are hydrogen signals on the benzene ring or unsaturated carbon. 13 C NMR spectrum ( Fig.14 -F) showed some impurity carbon signals. Comprehensive analysis 13 C NMR spectrum, HSQC spectrum ( Fig.14 -G) and HMBC( Fig.14 -H) spectrum, and obtained signal peaks containing 17 target carbon signals: 13 C NMR (151 MHz, DMSO-d6)δ C190.94,163.89,163.37,156.45,151.66,136.21,132.36,129.87,120.83,119.91,116.87,116.61,116.28,111.54,71.65,39.40,24.67. C =39.40 and 24.67 are methyl carbons, δ C =71.65 may be oxygen-linked carbon, δ C =156.45, 151.66, 136.21, 132.36, 129.87, 120.83, 119.91, 116.87, 116.61, 116.28 and 111.54 may be carbon atoms on conjugated olefins, δ C =190.94 may be the carbon on the carbonyl group. According to the HSQC spectrum, δ C =190.94, 163.89, 163.37, 156.45, 151.66, 132.36, 129.87, 120.83, 116.28, 71.65 are quaternary carbons, and the remaining atoms are assigned to: δ H =7.50 and δ C =136.21 has signal, δ H =7.35 and δ C =116.61 has signal, δ H =7.27 and δ C =111.54 has signal, δ H =6.83 and δ C =116.87 has signal, δ H =6.68 and δ C =119.91 has signal, δ H =2.73 and δ C =24.67 has signal, δ H =1.56 and δ C =39.40 has a signal. According to the HMBC spectrum, δ H =7.50 and δ C =151.36, 163.08 are associated, δ H =7.35 and δ C =71.65, 116.84 are associated, δ H =7.27 and δ C =71.65, 120.54 are associated, δ H =6.84 and δ C =116.17 is associated with δ H =6.68 and δ C =24.30, 111.33, 120.54 are associated, δH =2.73 and δ C =119.37, 145.99 are associated, δ H =1.56 and δ C =71.65, 156.66, 156.45. According to the HH COSY spectrum ( Fig.14 -I) we can see that δ H The hydrogen signals at 6.70 and 2.75 are coupled to each other.

[0088] After reviewing relevant literature, it was found that the structure of this compound is similar to that of compound WS9761 A ( Fig.13 -A). Compared with compound 4, the hydroxyl group at position 8 is shifted to position 6 and there is no methyl group at position 7. The chemical structure of WS9761 A was determined ( Fig.13 -B), through the search and comparison of relevant literature in databases such as SCI Finder, no compound with exactly the same structure was found, and it was identified as a new anthraquinone compound - 3,6,10-trihydroxy-1,10-dimethylanthracen-9(10H)-one. The chemical formula of compound 4 is C 16 H 14 O4, relative molecular mass is 270.09.

[0089] Embodiment 3:

[0090] (1) Inhibitory effects of compounds on rice blast fungus

[0091] iUsing rice blast fungus as the target bacteria, the isolated and purified compounds were tested for indoor toxicity using the indoor plate inhibition zone method. Pour 6 mL PDA into a culture dish, wait for the culture medium to solidify, and symmetrically place two Oxford cups with a diameter of 6 mm on the PDA plate. Then pour 6 mL PDA and take out the Oxford cups after solidification. Place a rice blast fungus cake in the center of the culture dish, add 60 μL of compound methanol solution to the first hole, and add an equal volume of methanol solution to the second hole. Use carbendazim as a positive control, repeat 3 times, and culture at 26°C for 5 days.

[0092] ii The inhibitory effect of each pure compound on the germination of rice blast fungus spores was determined by the 2-fold dilution method of 96-well plates. The concentrations of the compounds in each test group were set to 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.5 μg / mL, and 31.25 μg / mL, respectively. Each treatment was repeated 3 times, and sterile water was set as the control group. After adding 50 μL of spore suspension to each well, it was placed in a constant temperature incubator at 26°C for 8 hours, and the spore growth was observed under a microscope.

[0093] (2) Effects of compounds on rice seed germination

[0094] The indoor germination method was used to determine the effect of the compound on the germination of rice seeds. The compound was prepared into 50μg / mL, 25μg / mL, 12.5μg / mL, 6.25μg / mL, 3.13μg / mL aqueous solutions, and the control group was deionized water. Two layers of filter paper were laid on the bottom of a 90mm diameter culture dish, and 20 rice seeds were placed in each dish. The rice seeds needed to be soaked at 30℃ for 24h in advance, and 10mL of the prepared solution was evenly dripped into the bottom of the dish. The treated culture dish was placed in a light incubator and cultured for 6 days. The length of the upper and lower hypocotyls and the weight of the rice seedlings were measured.

[0095]

[0096]

[0097] (3) Results and analysis

[0098] 3.1 Inhibitory effect of compounds on mycelium

[0099] Among the four compounds isolated, compounds 1, 2, 3 and 4 showed significant inhibitory effects on rice blast fungus. The activity ranking of the four antibacterial active compounds was: compound 3 > compound 4 > compound 2 > compound 1 ( Fig.15 3.2 Effect of compound 4 on rice blast spores

[0100] When the concentration of compound 4 was greater than or equal to 125 μg / mL, the rice blast spores showed obvious abnormalities, and small bubbles appeared inside some spores ( Fig.16 -C); some spores have completely lost their internal protoplasm and become empty shells ( Fig.16 -D); some spores germinate, and the spores are thicker and shorter than normal spores ( Fig.16 -E); some spores have abnormal structures, the septa are dissolved, and the spores are round ( Fig.16 -F).

Claims

1. A group of compounds isolated from the antagonistic bacteria ZY66 of rice blast fungus, characterized in that: The compounds include compounds 1 to 4; the compound 1 is 2-acetyl-3,8,10-trihydroxy-1,7,10-trimethylanthracene-9(10H)-one, the chemical formula of which is C 18 H 16 O5, relative molecular mass 326.12, structural formula as shown in formula (Ⅰ); Formula (I); The compound 2 is 3,8,10-trihydroxy-1,7,10-trimethyl-9-oxy-9,10-dihydroanthracene-2-carboxylic acid, and its chemical formula is C 18 H 16 O6, relative molecular mass 328.09, structural formula as shown in formula (II): Formula (II); The compound 3 is 3,6-dihydroxy-1-methylanthracene-9,10-dione, and its chemical formula is C 15 H 10 O4, relative molecular mass 254.06, structural formula as shown in formula (III): Formula (III); The compound 4 is 3,6,10-trihydroxy-1,10-dimethylanthracen-9(10H)-one, and its chemical formula is C 16 H 14 O4, relative molecular mass 270.09, structural formula as shown in formula (IV): Formula (IV); The deposit number of the rice blast antagonistic bacterium ZY66 is: CGMCC No.30225.

2. Use of the compound according to claim 1 in the preparation of an inhibitor of rice blast fungus.

3. A strain for producing the compound according to claim 1, characterized in that: The strain is Streptomyces acidiscabies ZY66, which is deposited in the Institute of Microbiology, Chinese Academy of Sciences, General Microbiology Center of China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.30225.

4. Use of the strain according to claim 3 in preparing an inhibitor of rice blast fungus.

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