CN110093383B - Application of benzopyrone compound as agricultural bactericide - Google Patents
Application of benzopyrone compound as agricultural bactericide Download PDFInfo
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Abstract
The invention discloses a preparation method of benzopyrone compounds and application of the benzopyrone compounds as agricultural fungicides in plant disease control. Said benzopyranonesThe compound is named alterlactone, and the structural formula is as follows:
Description
Technical Field
The invention belongs to the field of new pesticide creation, and particularly relates to a preparation method of a benzopyrone compound and application of the benzopyrone compound as an agricultural bactericide to control plant diseases.
Background
Bacterial diseases of plants are caused by the infection of pathogenic bacteria of plants, and are easy to attack under the conditions of high temperature and high humidity, and the bacterial diseases often cause the symptoms of plant decay, wilting, fading, spots and the like, so that the yield and the quality of crops are reduced, and great economic loss is caused to agricultural production. At present, the varieties of medicaments for preventing and treating plant bacterial diseases in the market are few, and the medicament resistance problem is increasingly serious due to long-term unreasonable use. Therefore, the search and development of novel agricultural fungicides for controlling plant bacterial diseases are important subjects of current pesticide formulation research.
Because the secondary metabolites of endophytes of plants have rich structural diversity, the secondary metabolites are important sources for finding bioactive substances, and the screening of compounds with obvious inhibiting effect on plant pathogenic bacteria is an effective strategy for researching and developing novel green agricultural fungicides. Alternaria fungus is a common endophytic fungus widely existing in nature, and produces various metabolites which have biological activities such as insecticidal activity, bactericidal activity, herbicidal activity, stress resistance and the like during the interaction with host plants, and has strong development potential (Lou JF, Fu LY, Peng YL, et al. The Alternaria alternata (Alternaria alternata) ZHJG5 is a plant endophyte separated from healthy cercis chinensis leaves and is stored in China general microbiological culture collection center (collection number: CGMCC No. 17062). The strain shows stronger antagonistic activity to the growth of plant pathogenic bacteria, a metabolite showing stronger bacteriostatic activity to the plant pathogenic bacteria is obtained based on activity tracing separation, and is identified as a benzopyrone compound alterlactone (Aly AH, Edradaebel RA, Indani ID, et al. Cytoxic metabolites from the fungal end isomer sp. and the sub-sequence detection in host strain Polygonum senegalesense.J. Nat Prod, 2008, 71, 972-980).
Disclosure of Invention
The invention aims to provide a preparation method of a benzopyrone compound alterlactone and application of the benzopyrone compound alterlactone as an agricultural bactericide to control plant diseases.
The structural formula of the benzopyrone compound alterlactone is as follows:
the benzopyrone compound alterlactone can be obtained by separating the metabolite of alternaria alternata ZHJG5 from a plant endophytic strain through the preparation method disclosed by the invention. The strain is preserved in the China general microbiological culture Collection center, the preservation date is 2018, 12 and 19 months, and the preservation address is as follows: no. 3 of Xilu No.1 of Beijing Kogyang area, registration number is CGMCC No.17062, and is classified and named as Alternaria alternata.
The preparation method comprises the following steps:
(1) liquid fermentation of alternaria alternata ZHJG 5;
(2) extracting the fermentation liquor prepared in the step (1) to obtain a crude extract of the fermented product of alternaria alternata ZHJG 5;
(3) and (3) separating and purifying the crude extract of the fermentation product obtained in the step (2) to obtain a secondary metabolite alterlactone.
Preferably, the fermentation liquor obtained in the step (1) is extracted by ethyl acetate in the step (2).
Preferably, in the step (3), the crude extract of the fermentation product of the alternaria alternata ZHJG5 obtained in the step (2) is separated and purified by silica gel column chromatography, ODS reverse silica gel column chromatography and high performance liquid chromatography in sequence, so as to obtain the pyrone compound alterlactone.
The invention determines the in-vitro bacteriostatic activity of the compound alterlactone on rice bacterial blight (Xanthomonas oryzae pv. oryzae, Xoo) and rice bacterial streak germ (Xanthomonas oryzae pv. oryzae, Xoc) by an MTT micropore dilution method, and determines the in-vivo protection and prevention effect of the compound on rice bacterial blight by a leaf cutting method. The result shows that the compound alterlactone has stronger bacteriostatic activity to two pathogenic bacteria, and the Minimum Inhibitory Concentrations (MIC) of the compound alterlactone are respectively 16 mu g/mL and 32 mu g/mL; in the test of the in-vivo control effect on rice bacterial blight, the compound alterlactone has 82.6 percent of protection and control effect on rice bacterial blight diseases at 200 mu g/mL, and is better than 78.5 percent of protection and control effect of a positive control drug streptomycin sulfate at 200 mu g/mL. The compound alterlactone has stronger inhibitory activity to plant bacterial pathogenic bacteria, has development potential as a novel agricultural bactericide or a lead compound, and is applied to prevention and control of plant diseases.
Detailed Description
The following examples illustrate specific steps of the present invention, but are not intended to limit the invention.
Terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified.
The invention is described in further detail below with reference to specific examples and data, it being understood that these examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.
The following will explain in detail the preparation of benzopyrone fungal secondary metabolite alterlactone, structure analysis and bacteriostatic activity experiment according to the present invention with reference to the specific examples.
Example 1: preparation and structural analysis of Compounds
1. Preparation of compound alterlactone:
(1) fermentation of alternaria alternata ZHJG5
a. Strain activation: inoculating alternaria alternate ZHJG5 strain with the number of CGMCC No.17062 to a flat plate of PDA solid culture medium in an ultra-clean workbench, and culturing in a constant temperature incubator at 25 ℃ in dark for 3 days;
b. large-batch fermentation of the strains: preparing a PDB liquid culture medium accounting for 80L, sterilizing, and cooling the culture medium to room temperature after sterilization is finished; preparing activated strain into fungus cake with diameter of 0.5mm, inoculating into 1000mL conical flask containing 400mL PDB liquid culture medium, maintaining 8 fungus cakes in each flask at 25 deg.C and 150rpm, and shake-culturing for 12 days to obtain bacterial liquid.
(2) Extraction of alternaria alternata ZHJG5 secondary metabolite
After the fermentation is finished, filtering the obtained fermentation product by two layers of 100-mesh nylon nets to obtain thalli and fermentation liquor. Extracting the fermentation liquor with ethyl acetate of equal volume for three times, combining the organic layers, and concentrating under reduced pressure to obtain a crude extract of the fermentation product;
(3) separation and purification of secondary metabolite alterlactone
a. Performing column chromatography separation on the bacterial liquid extract by using 200-mesh 300-mesh silica gel, respectively preparing eluents with volume ratios of dichloromethane to methanol of 100: 0, 100: 1, 100: 2, 100: 4, 100: 8, 100: 16, 100: 32, 100: 50 and 0: 100, loading the sample by a dry method, sequentially performing gradient elution on the sample, and performing TLC detection and combination;
b. performing column chromatography separation on the component sample of 100: 2 in the step a by using ODS reverse silica gel, preparing eluent with the volume ratio of methanol to water being 20: 80, 30: 70, 40: 60, 50: 50, 60: 40, 70: 30, 80: 20 and 100: 0, loading the sample by using a wet method, sequentially performing gradient elution on the sample, and performing TLC detection and merging;
c. separating the eluate of methanol and water 40: 60 in step b by high performance liquid chromatography (Agilent 1200; Agilent Eclipse XDB-C18, 250mm × 4.6mm, 5 μm; mobile phase: methanol/water 70: 30 (v/v); flow rate: 1mL/min, detection wavelength: 254nm), and collecting tRThe compound alterlactone was obtained from the peak at 3.206 min.
2. Structural identification of compound alterlactone:
white feather-like crystals, HR-ESI-MS M/z 311.05086[ M + Na ]]+.1H-NMR(600MHz，acetone-d6)H 3.93(s，10-OMe)，4.91(s，H-5)，5.10(s，H-5)，6.54(d，J＝2.5，H-9)，6.61(d，J＝2.5，H-11)，7.03(s，H- 4)，7.12(s，H-1)，8.42(s，2-OH)，8.42(s，3-OH)，10.57(s，10-OH).13C-NMR(150MHz，acetone-d6) C56.1(10-OMe), 69.8(C-5), 101.3(C-9), 107.5(C-11), 108.3(C-7a), 115.9(C-4), 116.9(C-1), 127.7(C-11a), 131.9(C-11b), 142.4(C-4a), 146.8(C-3), 147.3(C-2), 164.0(C-8), 164.8(C-10), 173.4 (C-7). The above data are consistent with reported data for alterlactone (Aly AH, Edradaebel RA, Indani ID, et al. cytotoxic metabolites from the fungal end of alkaline Alternaria sp. and the sub-sequence detection in host plant polymeric seneganse. J Nat Prod, 2008, 71, 972-) and thereby identify the compounds as identical.
Example 2: determination of the bacteriostatic Activity of Compounds
The compound alterlactone is used for inhibiting the growth of plant pathogenic bacteria and preventing and treating rice bacterial blight:
(1) experiment for inhibiting growth of plant pathogenic bacteria by using compound alterlactone
The MTT microplate dilution method is adopted to determine the bacteriostatic activity of the compound alterlactone on rice bacterial blight disease (Xoo) and rice bacterial streak disease (Xoc), streptomycin sulfate is used as a positive control, and the specific implementation steps are as follows:
the pathogenic bacteria Xoo and Xoc stored in refrigerator at-80 deg.C are streaked and activated on a plate, and cultured in dark room at 28 deg.C for 1-2 days until bacterial colony grows out. Picking single colony and shake-culturing in 20mL NB medium for 1-2 days until colony concentration reaches about 108Removed at cfu/mL, and diluted to 10 colony concentration5cfu/mL for standby. Taking 100 μ L of 105Adding cfu/mL bacterial solution into 96-well cell culture plate, adding a series of concentrations of liquid medicine (dissolved by DMSO, concentrated by DMSO) of test compound alterlactone and streptomycin sulfate (98% biotechnology grade, Shanghai Michelin Biochemical technology Co., Ltd.)Degree should not exceed 1%), and finally 200. mu.L of the corresponding medium is made up. And (3) fully and uniformly mixing the liquid medicine in each hole with the culture medium, and sealing a 96-hole bacterial culture plate. After incubation at 28 ℃ for 16-24h, 20mM PBS-solubilized MTT (final concentration 100. mu.g/mL) was added to a 96-well cell culture plate, and after standing at room temperature for 4-5h, the results were compared to a blank control and observed, with 3 replicates per concentration setting.
(2) Experiment for preventing and treating bacterial leaf blight of rice by using compound alterlactone
The method for determining the protection and control effect of the compound alterlactone on the rice bacterial blight living body by adopting a leaf-cutting method comprises the following specific implementation steps:
selecting potted rice seedlings with long and consistent tillering prosperity period, removing lower old leaves, and 5 leaves in each pot for later use. Preparing solutions (containing 0.5% DMSO) with the concentrations of 100 μ g/mL and 200 μ g/mL for compound alterlactone, respectively, and using 0.5% aqueous solution containing DMSO as control, and setting clear water control; positive control streptomycin sulfate was prepared as solutions at concentrations of 100. mu.g/mL and 200. mu.g/mL, respectively (with DMSO at 0.5%). The method of stem and leaf spraying is adopted to apply the pesticide to the rice seedlings, 10mL of pesticide is sprayed to each pot, the rice seedlings are placed in a ventilation place to be dried in the shade after spraying treatment, and 3 times of concentration setting are carried out. The Xoo single colonies were picked and shake-cultured in 20mL NB medium for 2 days to a colony concentration of about 108Removed at cfu/mL. Inoculating after spraying for 24 hours, dipping bacterial liquid with scissors, cutting off the leaf tips of about 1cm of rice, inoculating 2 leaves to each piece of rice, and inoculating a sterile NB culture medium in a blank control manner. And after culturing for 15 days, counting the length of the disease spots and calculating the prevention and treatment effect.
The results of the experiment for inhibiting the growth of plant pathogenic bacteria and the experiment for preventing and treating bacterial blight of rice by using the compound alterlactone are shown in tables 1 and 2 respectively:
TABLE 1 bacteriostatic Activity of the Compound alterlactone against plant pathogenic bacteria
TABLE 2 protective and preventive effects of alterlactone on bacterial blight of rice
Experimental results show that MIC of the compound alterlactone on the inhibition activity of rice bacterial leaf blight germ and rice bacterial leaf streak germ respectively reach 16 mug/mL and 32 mug/mL; in the test of the in-vivo control effect on the bacterial leaf blight of rice, the compound alterlactone has 82.6 percent of protection and control effect on the bacterial leaf blight of rice at 200 mu g/mL and is better than 78.5 percent of protection and control effect of a positive control drug, namely the streptomycin sulfate at 200 mu g/mL. This shows that the compound alterlactone has strong inhibitory activity to plant pathogenic bacteria, can be developed as a novel agricultural bactericide or a lead compound, and is applied to the prevention and control of plant diseases.
Claims (5)
1. The application of Alternaria alternata (Alternaria alternata) ZHJG5 for producing benzopyrone compounds alterlactone in preventing and treating agricultural plant diseases is characterized in that the Alternaria alternata (Alternaria alternata) ZHJG5 is deposited in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, and the registration number is as follows: CGMCC No. 17062; the agricultural diseases are rice bacterial leaf blight and rice bacterial leaf streak.
2. The use according to claim 1, wherein the benzopyrone compound alterlactone is prepared by a method comprising the following steps:
(1) liquid fermentation of alternaria alternata ZHJG 5;
(2) extracting the fermentation liquor prepared in the step (1) to obtain a crude extract of the fermented product of alternaria alternata ZHJG 5;
(3) and (3) separating and purifying the crude extract of the fermentation product obtained in the step (2) to obtain a secondary metabolite alterlactone.
3. The use according to claim 2, wherein the fermentation liquid obtained in step (1) is extracted by using ethyl acetate in step (2).
4. The use of claim 2, wherein in the step (3), the crude extract of the fermented product of the alternaria alternata ZHJG5 obtained in the step (2) is separated and purified by sequentially performing silica gel column chromatography, ODS reverse silica gel column chromatography and high performance liquid chromatography to obtain the benzopyrone compound alterlactone.
5. The use according to claim 1, wherein the benzopyrone compound alterlactone is processed into missible oil, aqueous emulsion, microemulsion, wettable powder, water dispersible granule and suspending agent.
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