WO2011095082A1 - 环脂肽化合物及其制备和应用 - Google Patents

环脂肽化合物及其制备和应用 Download PDF

Info

Publication number
WO2011095082A1
WO2011095082A1 PCT/CN2011/070507 CN2011070507W WO2011095082A1 WO 2011095082 A1 WO2011095082 A1 WO 2011095082A1 CN 2011070507 W CN2011070507 W CN 2011070507W WO 2011095082 A1 WO2011095082 A1 WO 2011095082A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
methanol
fermentation broth
chloroform
group
Prior art date
Application number
PCT/CN2011/070507
Other languages
English (en)
French (fr)
Inventor
李元广
张道敬
刘荣峰
魏鸿刚
罗远婵
李淑兰
沈国敏
陶黎明
田黎
Original Assignee
华东理工大学
上海泽元海洋生物技术有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 华东理工大学, 上海泽元海洋生物技术有限公司 filed Critical 华东理工大学
Publication of WO2011095082A1 publication Critical patent/WO2011095082A1/zh

Links

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/90—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having two or more relevant hetero rings, condensed among themselves or with a common carbocyclic ring system
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/22—Bacillus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links

Definitions

  • the present invention relates to a novel structural cyclolipopeptide compound Marihysin A, maribasin B produced by Bacillus mariacas ⁇ . mari ⁇ -9987, which is derived from the tidal zone of the Bohai Sea intertidal plant, Suaeda salsa, and the preparation method thereof And in the application of pesticides. Background technique
  • Marine organisms are capable of producing a large number of structurally active active metabolites, many of which have been developed into products.
  • the silkworm toxin is an insecticide invented by the Japanese from the animal silkworm.
  • Bacillus marineus produces many valuable substances.
  • Carisse et al. (2003) isolated marine bacillus from compost (including paper mill sludge, plant residues, etc.), and plate antagonism experiments and pot experiments showed that it was resistant to cucumber, the ultimate Pythium (l thium M) /tTMTM) has a strong inhibitory effect on growth.
  • Zhang Hailong et al. (2004) found three new cyclolipopeptide compounds, Mixirin A-C, from cyanobacteria, which have cytotoxic activity.
  • Ashish et al. (2006) found that cellulase produced by Bacillus mariterium was more active at pH 6 and 50 °C. Noureddin et al.
  • Bacillus marine bacilli can produce glucose-6-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase.
  • Tian Li et al. (2007) reported three new macrolide compounds, Macrollactin O, P and Q, produced by B. marinus B-9987. The study found that these three new compounds are interlinked. Spores and Magnaporthe oryzae have good inhibitory activity.
  • the marine bacillus A ar m ⁇ B-9987 fermentation concentrate containing such a substance can effectively inhibit the growth of plant pathogenic bacteria.
  • Bacillus marinus is very suitable for the development of microbial pesticides because of its strong antibacterial effect, strong resistance to stress and easy processing into dosage forms.
  • the 1 billion CFU/g marine bacillus wettable powder successfully developed by the inventor's research group is very safe as a pesticide. It is a micro-toxic pesticide.
  • the preliminary pot and field plot test results show that the wettable powder is against gray mold and white powder. Disease, bacterial wilt, soft rot, early blight, downy mildew have better control effect (Chinese Invention Patent Publication No.: CN101331881).
  • Lipopeptides are mainly derived from secondary metabolites of microorganisms. They are composed of hydrophilic peptide bonds and lipophilic aliphatic hydrocarbon chains. They are divided into linear lipopeptides and cyclic lipopeptides. Due to its special amphiphilic molecular structure, the microbial lipopeptide has excellent surface activity and has important application prospects in the fields of medicine, food, cosmetics, biological control, environmental treatment and microbial oil recovery.
  • cyclolipopeptide compounds have antibacterial activity, the most successful example of which is daptomycin, which is the first clinically applied cyclolipopeptide antibiotic for the treatment of complicated skin caused by Gram-positive infections.
  • Skin infections including The methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis have a unique mechanism of action.
  • Bacillus subtilis ⁇ «7/ is an important rhizosphere microorganism that promotes plant resistance to pathogens and is therefore widely used for biological control of plant diseases.
  • B.TM / s can produce dozens of different structures of antibiotics, among which non-ribosomal synthetic lipopeptide antibiotics are the most common ones, mainly including three families of ituriiu surfactin and fengycin.
  • Lipopeptide antibiotics are composed of amino acid chains and fatty acid side chains. They have good stability, are harmless to humans and animals, and do not pollute the environment. They are new bio-sourced pesticides with important development value. There are reports of successful use of iturin in biological control (Klich MA, 1994).
  • lipopeptide biosurfactants selectively remove metal ions such as Pb, Zn, Cu and Cd from soils, with Cu 2+ being the most easily removed.
  • cycloactin (MW 1036) inhibits the proliferation of Lovo cells and induces apoptosis, which may be due to inhibition of cell survival regulatory signaling pathways ERK and PI3K/Akt.
  • the lipopeptide biosurfactant produced by ZW-3 has excellent emulsification and ability to reduce oil-water interfacial tension, and can adapt to the complex environment in the reservoir, which can increase oil recovery by 9.2%. It is very good in microbial oil recovery. Application prospects.
  • the present inventors have found through research that a new structural cyclolipopeptide compound produced by A (B-Blocked 04/7" ⁇ "so/"” and a stalked sclerotium (3 ⁇ 4 ⁇ "/ ⁇ / 7 Oxysporum f. sp. cuberse) ⁇ 3 ⁇ 4 3 ⁇ 4J pathogens have good inhibitory activity and can therefore be used as pesticides to control plant diseases caused by these pathogens.
  • a first aspect of the invention provides a cyclolipopeptide compound having the following structural formula (I):
  • R is selected from H or a C1-C20 branched or straight chain alkyl group.
  • R is a C1-C20 straight or branched alkyl group.
  • R is a C6-C15 straight or linear alkyl group.
  • R1 and R2 are each independently H or a C1-C3 linear or branched alkyl group.
  • the compound is Marihysin A, and the structural formula is as shown in the following formula (III): ( ⁇ ).
  • the compound is Maribasin B, and the structural formula is as shown in the following formula (IV):
  • structural formulae (I) and (II) do not include the following compounds:
  • the term "compound of the present invention” or like terms means not only the compounds defined by the above structural formula but also the salts thereof.
  • the salt may be in the form of a water soluble, fat soluble or dispersible product which may be formed by reaction with a mineral or organic acid or base.
  • acid addition salts include acetates, adipates, alginates, benzoates, besylate, hydrogen sulfate, butyrate, citrate, dodecylsulfonate , hydrochloride, oxalate, propionate, succinate, tartrate.
  • the basic salts include ammonium salts, alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, salts of organic bases such as dicyclohexylamine salts and the like.
  • the compounds of the invention may also exist in their tautomeric form. Although these forms are not explicitly indicated in the compounds described herein, they are also included in the scope of the present invention. Unless otherwise indicated, the chemical names of the compounds described herein include all possible mixtures of stereochemically isomeric forms that the compounds may comprise. The mixture may contain all diastereomers and/or enantiomers of the basic molecular structure of the compound. All stereochemically isomeric forms of the compounds of the invention may be in purified form or in admixture with each other, and are intended to be included within the scope of the invention.
  • a second aspect of the invention provides a method of preparing the above compound, characterized in that the method comprises the following steps:
  • the compound is isolated from the fermentation broth by a method selected from the group consisting of organic solvent extraction, acid precipitation, leaching or chromatography.
  • the strain involved in the method of the present invention is Bacillus marinus, B-9987, which is isolated from the intertidal plant of the Bohai Sea, which has been preserved in Chinese microbes on June 18, 2007.
  • the term "fermentation” or “cultivation” as used herein has the meaning commonly known and recognized by those skilled in the art.
  • the "fermentation broth” or “culture broth” can be obtained by culturing the Bacillus marinus B-9987 of the present invention under conditions suitable for growth to grow to a certain bacterial concentration.
  • the nutrient source in the medium for cultivating the strain of the present invention is not particularly limited.
  • One skilled in the art can select a suitable carbon source, nitrogen source, and other nutrient source in accordance with well known techniques.
  • the carbon source may be starch, dextrin, glycerin, glucose, sucrose, inositol, mannitol, and the like.
  • the nitrogen source may be strontium, soy flour, protein powder, meat paste, rice sugar, wheat husk, yeast powder, corn syrup, ammonium salt, and other organic or inorganic nitrogen-containing compounds.
  • inorganic salts such as sodium chloride, phosphate (such as dipotassium hydrogen phosphate and potassium dihydrogen phosphate), ammonium sulfate, manganese sulfate, magnesium sulfate, calcium carbonate and the like may be appropriately added to the medium.
  • phosphate such as dipotassium hydrogen phosphate and potassium dihydrogen phosphate
  • ammonium sulfate manganese sulfate
  • magnesium sulfate calcium carbonate and the like
  • calcium carbonate and the like
  • Various known conventional media such as LB agar medium, nutrient agar medium, glucose yeast agar medium, and beef dip agar medium can be usually used.
  • the medium used to culture the strain of the present invention has the following composition (% means mass/volume): glucose 0.1% to 1%, sucrose 0.1% to 2%, yeast powder 0.01% to 1.5%, protein Powder 0.1% ⁇ 1%, MgCl 2 0.001% ⁇ 0.1%, KC1 0.01% ⁇ 0.5%, KH 2 PO 4 0.01% ⁇ 0.5% and NaC1 0.1% ⁇ 6%, pH 6.0 7.0.
  • composition % means mass/volume
  • glucose 0.1% to 1% sucrose 0.1% to 2%
  • protein Powder 0.1% ⁇ 1% MgCl 2 0.001% ⁇ 0.1%
  • KC1 0.01% ⁇ 0.5% KH 2 PO 4 0.01% ⁇ 0.5%
  • NaC1 0.1% ⁇ 6% pH 6.0 7.0.
  • the conditions for cultivating the strain of the present invention such as temperature, pH, gas-liquid ratio, tank pressure, and rotation speed are not particularly limited. As long as the conditions are suitable for the growth of the bacteria.
  • defoaming can be carried out by using an antifoaming agent such as soybean oil or an enemy.
  • the pH is preferably controlled between 5.5 and 8.0, and the culture temperature is preferably between 20 and 35 °C.
  • the training time is usually at 121! Between ⁇ 200h, the final bacterial concentration can usually be as high as 5 ⁇ 10 7 cfh / ml ⁇ 1 X lOUcfh / mL
  • the marine bacillus is cultured by the following method to obtain a fermentation broth: in a medium containing a carbon source, a nitrogen source, or an inorganic salt, the aeration amount is controlled at a gas-liquid ratio 0.2: 1 ⁇ 2: 1.
  • the Bacillus marinum is cultured for more than 24 hours at a rotation speed of 100 rpm or more, wherein the carbon source is one or more selected from the group consisting of glucose, sucrose, starch, and rice flour.
  • the nitrogen source is selected from one or more of protein powder, yeast powder, peanut cake powder and soybean powder
  • the inorganic salt includes a conventional inorganic salt (such as MgS0 4 , ( H 4 ) 2 S0 4 , MgCl 2 , KC1, KH 2 P0 4 , NaCK K 2 HP0 4 and CaC0 3 ).
  • the target compound is isolated from the fermentation broth by a method selected from the group consisting of organic solvent extraction, acid precipitation, leaching or chromatography.
  • the compound of the present invention can be isolated from the fermentation supernatant of the fermentation broth or can be isolated from the bacterial cells in the fermentation broth.
  • the crude product or extract containing the compound of the invention may also be isolated from the fermentation supernatant (also referred to as the supernatant or fermentation supernatant) and the bacterial cells, respectively, and then further separated. purification.
  • the supernatant is first separated from the fermentation broth, and extracted with a solvent selected from chloroform, ethyl acetate, butyl acetate or n-butanol, followed by column chromatography to chloroform. /Methanol gradient elution, the elution site of chloroform/methanol was collected from 2:1 to 0:1, and the compound of the present invention was obtained by HPLC.
  • the supernatant can be separated from the fermentation broth by a conventional separation means such as centrifugal filtration or the like. Then, after obtaining the supernatant, one possible method is to carry out extraction with an organic solvent (for example, chloroform, ethyl acetate, butyl acetate or n-butanol).
  • an organic solvent for example, chloroform, ethyl acetate, butyl acetate or n-butanol.
  • the extraction is carried out by using a combination of organic solvents, for example, extraction with ethyl acetate and n-butanol in sequence; extraction with chloroform, ethyl acetate and n-butanol in sequence; extraction with butyl acetate and n-butanol in sequence, etc. .
  • Another possible solution is to first obtain the supernatant as described above, then acidify the supernatant and extract the acidified precipitate with anhydrous methanol. Specifically, an acid (e.g., hydrochloric acid) is added to the supernatant to allow the solution to stand, and then centrifuged to obtain an acidified precipitate and an acidified supernatant.
  • acidification is carried out using a 5N hydrochloric acid solution.
  • the acidified precipitate can also be obtained by a person skilled in the art by appropriately adjusting the concentration of hydrochloric acid.
  • the pH of the acidified precipitate is adjusted to neutrality, lyophilized, and leached one or more times with anhydrous methanol for a leaching time of 6-12 hours.
  • Still another feasible method is to separate the cells from the fermentation broth by conventional means such as centrifugation. Then, the cells are leached with anhydrous methanol one or more times, each leaching time of 6-12 hours.
  • column chromatography is carried out by chloroform/methanol gradient elution of the n-butanol extraction active site or anhydrous methanol extract obtained in the previous step, and the elution site of chloroform/methanol is selected from 2:1 to 0:1 (preferably chloroform). /Methanol 1: 1 elution site).
  • the column chromatography used may be, for example, silica gel column chromatography.
  • the compound was obtained by Sephadex LH-20 and/or HPLC (analysis and preparation conditions were 70% aqueous methanol solution) or the like.
  • the compounds of the present invention can be prepared by chemical synthesis. Asn, Ser, Tyr, Pro and Gln 5 amino acids can be used as the basic raw materials. Pro-Gln is synthesized first by Pro and Gin, Pro-Gin is synthesized by di-peptide and then synthesized by Asn to form tripeptide Pr-Gln-Asn. After synthesizing linear heptapeptide with Ser, Asn, Tyr, Asn, marihysin A or maribaxin B is synthesized by cyclization with amino fatty acid ⁇ -aminotetradecanoic acid or ⁇ -amino-anti-hepentadecanoic acid. Other compounds in the formula can be synthesized in a similar manner and starting materials.
  • compositions characterized in that the composition comprises the compound of the present invention as an active ingredient, and a physiologically acceptable carrier.
  • the compound as an active ingredient may be contained in the agricultural chemical composition of the present invention in an amount of from 0.1% to 90% by weight, for example, from 1 to 50% by weight and from 10 to 40% by weight.
  • Those skilled in the art can formulate a suitable amount of the pesticide composition according to actual needs.
  • the compound or agrochemical composition of the present invention may be administered directly as a fermentation broth, or it may be suitably diluted (e.g., diluted 10-fold, 100-fold, 1000-fold or higher) as a diluent.
  • the compound of the present invention or a composition thereof can be applied to a root, a leaf, a stem or the like of a plant.
  • the application method is a conventional technique in the art, and may be, for example, soaking seeds at the time of sowing, immersing the roots of the plants in the fermentation broth or a diluent thereof before transplanting, or directly pouring the fermentation broth or the diluent onto the seedbed. Rooting can be carried out at the time of planting, and rooting can also be carried out during plant growth. If the pesticide composition is stored as a carrier, it can be applied after dilution with water immediately before use. As far as the optimum dosage of the present invention is concerned, those skilled in the art can determine it without undue experimentation.
  • the compounds and pesticidal compositions of the present invention are useful for inhibiting a variety of plant pathogenic bacteria including, but not limited to, A. so! anoporosis ox 3 ⁇ 4pon, Verticillium dahliae (J! a oatn ),
  • Botrytis cinerea S. cinerea tur turcica, R. solani ⁇ ⁇ &' (Colle totrichum sp.), Fusarium oxysporum f. sp.cuberse) ttiiS 3 ⁇ 4f(R- solanacearum) ⁇ soft rot (£. carotovora var. carotovora) and powder
  • Pathogenic bacteria can cause tomato early blight, melon fruit rot, cotton verticillium, wheat scab, Atractylodes chinensis, cucumber gray mold, tomato gray mold, corn leaf spot, rice stalk, melon anthracnose , banana wilt, tomato bacterial wilt, cabbage soft rot and cucumber powdery mildew.
  • a further aspect of the present invention provides a method of controlling a plant disease, the method comprising applying a compound pesticide composition of the present invention to the plant.
  • Plants suitable for use in the control of the methods of the invention include, but are not limited to, wheat, corn, rice, cotton, tomato, banana, cabbage, melon, watermelon, cucumber, and atractylodes.
  • the method of the present invention can be used for controlling plant diseases caused by the following pathogens: early blight (A so/a « ), fruit rot ox 3 ⁇ 4wn ), verticillium ( a»oatn ), scab graminearum Pathogen (c/erot w sp.), Penicillium sp. (3 ⁇ 4" c ⁇ w sp.), S. cinered) ⁇ turcica, R.
  • pathogens early blight (A so/a « ), fruit rot ox 3 ⁇ 4wn ), verticillium ( a»oatn ), scab graminearum Pathogen (c/erot w sp.), Penicillium sp. (3 ⁇ 4" c ⁇ w sp.
  • the method of the present invention can be used to control the following plant diseases: tomato early blight, melon fruit rot, cotton verticillium, wheat scab, atractylodes sinensis, cucumber gray mold, tomato ash Mildew, corn leaf spot, rice stalk, melon anthracnose, banana wilt, tomato bacterial wilt, cabbage soft rot and cucumber powdery mildew.
  • plant diseases tomato early blight, melon fruit rot, cotton verticillium, wheat scab, atractylodes sinensis, cucumber gray mold, tomato ash Mildew, corn leaf spot, rice stalk, melon anthracnose, banana wilt, tomato bacterial wilt, cabbage soft rot and cucumber powdery mildew.
  • Example 1 The new cyclolipopeptide compound Marihysin A was isolated from the fermentation broth of Bacillus marineus ( ⁇ z «7/ «s marinus) -99S7 by acid precipitation.
  • Marihysin A a novel cyclolipopeptide compound Marihysin A was obtained from the fermentation broth of Bacillus marinus A marinus B-9987 by separation means.
  • the specific separation and purification steps are as follows:
  • Bacillus marinum (A mari painting) B-9987 was cultured under appropriate conditions to obtain 30 L of fermentation broth.
  • the Bacillus marinum B-9987 strain was inoculated on the plate and inoculated in the first-stage seed shake flask for 12-24h to be used as the first-class seed.
  • the first-stage seed was inoculated into the second-stage seed shake flask, and cultured for 12-24h, then inoculated into 50L. In the fermenter. It can be completed by culturing at 28 °C and 300 rpm for about 20 hours.
  • the combined active fractions were separated by methanol on Sephadex LH-20 column chromatography, and the eluted fractions were tested for biological activity.
  • the indicator bacteria were Ahemaria so.
  • the active fraction after bioactivity detection was subjected to TLC.
  • the combined active fractions were analyzed by HPLC and prepared (analytical and preparative conditions were 70% aqueous methanol solution), and finally the compound Marihysin A was prepared.
  • Example 2 was extracted from marine bacillus using organic solvent extraction method ( ⁇ z «7/ «s marinus) New cyclolipopeptide compound Marihysin A isolated from -9987 fermentation broth
  • Marihysin A a novel cyclolipopeptide compound Marihysin A was obtained from a fermentation broth of Bacillus marinus A marinus B-9987 by conventional separation means.
  • the specific separation and purification steps are as follows:
  • Bacillus marinum (A mctri) B-9987 was cultured under suitable conditions to obtain 30 L of fermentation broth (specific culture conditions and procedures are the same as in Example 1).
  • the fermentation supernatant and the wet cells were obtained by centrifugation of 30 L of the fermentation broth at 4000 rpm. Wet cells are washed and centrifuged, then lyophilized Drying, 92.0 g of dried cells were obtained, and leaching three times with anhydrous methanol (600 mL, 300 mL, 300 mL), and the extraction time was 12 hours each time, and the extract was combined under reduced pressure to dryness to obtain crude leaching of the cells. A total of 17.3g.
  • the crude extract was extracted by silica gel column chromatography with a gradient of chloroform/methanol 10:1-0:1 (500 mL/min), and the eluted fraction was subjected to biological activity detection.
  • the results showed that the antibacterial active substances were mainly concentrated in the chloroform/methanol 1:1 and methanol elution sites, and the chloroform/methanol 1:1 elution sites were more active, and the above active fractions were combined by TLC detection.
  • the combined active fractions were separated into methanol by Sephadex LH-20 column chromatography, and the eluted fractions were tested for biological activity.
  • Marihysin A a novel structural cyclolipopeptide compound Marihysin A was obtained from a fermentation broth of Bacillus marinus A marinus B-9987 by conventional separation means.
  • the specific separation and purification steps are as follows:
  • Bacillus marinus S. mari B-9987 was cultured under suitable conditions to obtain 10 L of fermentation broth.
  • the Bacillus marinum B-9987 strain was inoculated on the plate and inoculated in the first-stage seed shake flask for 12-24h to be used as the first-class seed.
  • the first-stage seed was inoculated into the second-stage seed shake flask, and cultured for 12-24 hours, then inoculated into 5L.
  • In the fermenter It can be completed by culturing at 28 °C and 300 rpm for about 20 hours. Co-fermenting 3 5L tanks to obtain 10L fermentation broth. (2) 10 L of fermentation broth was centrifuged at 4000 rpm to obtain a fermentation supernatant and wet cells.
  • the fermentation supernatant was extracted three times with an equal volume of ethyl acetate, and each extraction time was 12 hours.
  • the ethyl acetate extracts were combined and concentrated under reduced pressure to a volume of the original fermentation broth.
  • the ethyl acetate raffinate phase was separately extracted three times with an equal volume of n-butanol, and the n-butanol extract phase was combined under reduced pressure to a volume of the original fermentation broth at a time of extraction for 12 hours.
  • the above-mentioned ethyl acetate extract phase and the n-butanol extract phase were subjected to biological activity detection, and the indicator strain was Solanum solani, and the n-butanol moiety was determined to be the main antibacterial active site.
  • the n-butanol extracts were combined and concentrated to dryness under reduced pressure to give a crude crude ethyl acetate.
  • the crude n-butanol extract was subjected to silica gel column chromatography with a gradient of chloroform/methanol 10:1 - 0:1 (500 mL / fraction), and the eluted fraction was subjected to biological activity detection.
  • Marihysin A The chemical structure of Marihysin A is as follows
  • This example examines the inhibitory effect of the compound Marihysin A obtained in Example 1 on plant pathogenic bacteria.
  • Marihysin A against A solani, melon rots (M. ⁇ Sclerotium sp.), Penicillium sp. (3 ⁇ 4"c ⁇ w sp.), S. cinered, S. cinerea, tur turcica, Inhibition of plant pathogens such as R. solani, Co ⁇ etotn'c/z sp., and oxysporum f. sp fer.
  • Marihysin A The MIC of Marihysin A against plant pathogens is shown in Table 2 below.
  • Marihysin A (Sclerotium sp.) oxysporum, blue fungus (3 ⁇ 4 c ⁇ / w sp.) and oletotric humilis sp. (Co lletotrichum sp.; ) Plant pathogens have strong antibacterial activity.
  • Example 6 Inhibitory activity of Bacillus marinum CBaci7/iis / narinus B-99S7 containing Marihysin A against plant pathogenic bacteria
  • the phytopathogenic fungi stored in the 4 °C slope were activated on a PDA plate and incubated at 28 °C for about one week. After the fungus had grown over the whole plate, the culture was terminated. A sterilized punch with a diameter of 14.50 mm is used to make a cake on the outer edge of the activated pathogenic fungus colony. The front side of the fungus cake is placed on the side of the sputum culture plate, approximately 1 cm near the wall of the dish, and 1 ring is taken with the inoculation needle. The obtained B-9987 fermentation broth was cultured, and a strain line was drawn parallel to the bacterium in the sputum plate. The scribe distance was determined according to the growth rate of the pathogenic fungus, and the sputum culture plate was placed at 28 ° C for 5 days, and the bacteriostatic record was recorded. With width.
  • pathogenic bacteria inhibition spectrum The pathogen bacteria cultured for 24 hours were mixed into the corresponding semi-solid medium (so that the bacteria-containing plate reached 10 9 C fij/mL), then poured onto the prepared water agar plate, and cooled. A semi-solid plate was punched with a hole having a diameter of 14.00 mm, and then B-9987 fermentation broth was added to the well, and 150 ⁇ g per well was cultured at 30 ° C for 24 h, and the size of the inhibition zone was recorded.
  • Maribasin B a novel cyclolipopeptide compound Maribasin B was obtained from the fermentation broth of Bacillus marinus A marinus B-9987 by conventional separation means.
  • the specific separation and purification steps are as follows:
  • Bacillus mariacas (A mari B-9987 was cultured under suitable conditions to obtain 30 L of fermentation broth. Bacillus marinum B-9987 was activated by plate and inoculated in a first-stage seed shake flask for 12-24 h as a The seed is inoculated into the secondary seed shake flask, cultured for 12-24 hours, and then inoculated into a 50 L fermenter. The culture is completed at 28 ° C, 300 rpm for about 20 h.
  • the fermentation supernatant and the wet cells were obtained by centrifugation of 30 L of the fermentation broth at 4000 rpm.
  • the fermentation supernatant was acidified with a 5N hydrochloric acid solution and allowed to stand for precipitation, and then centrifuged (4000 rpm) to obtain an acidified precipitate and an acidified supernatant.
  • the pH of the acidified precipitate was adjusted to 7.0, and then freeze-dried to obtain 174.8 g of a lyophilized precipitate, which was successively leached three times with anhydrous methanol (440 mL, 220 mL, 220 mL), and the extract was combined for 12 hours each time. Concentrated to dryness, a total of 10.2 g of crude acidified extract was obtained.
  • the acidified precipitated extract was subjected to silica gel column chromatography and eluted with a gradient of chloroform/methanol 10:1 ⁇ 0:1 (500 mL/min).
  • the eluted fraction was subjected to biological activity detection, and the indicator was tomato Phytophthora infestans. (A solani), the results showed that the antibacterial active substances were mainly concentrated in the chloroform/methanol 1:1 and methanol elution sites, and the chloroform/methanol 1:1 elution sites were more active, and the above active fractions were detected by TLC. merge.
  • the combined active fractions were subjected to Sephadex LH-20 column chromatography and eluted with methanol.
  • Maribasin B a novel cyclolipopeptide compound Maribasin B was obtained from Bacillus marinus A marinus B-9987 by conventional separation means.
  • the specific separation and purification steps are as follows:
  • Bacillus lentus (A mari painting) B-9987 was cultured under appropriate conditions to obtain 30 L of fermentation broth (specific culture conditions and procedures are the same as in Example 1).
  • the indicator was tomato Phytophthora infestans. 4.
  • the results showed that the antibacterial active substances were mainly concentrated in the chloroform/methanol 1: 1 and methanol elution sites, and the chloroform/methanol 1:1 elution sites were more active.
  • the above active fractions were combined by TLC and combined.
  • the combined active fractions were separated by methanol on Sephadex LH-20 column chromatography, and the biological activity of the eluted fractions was detected.
  • the indicator bacteria were tomato early blight. 4.
  • the active fractions after bioactivity detection were detected by TLC. After the merger.
  • the combined active fractions were analyzed by HPLC and prepared (analytical and preparative conditions were 70% aqueous methanol), and finally the compound Maribasin B was prepared.
  • Example 9 Separation of a new cyclolipopeptide compound from the fermentation supernatant of Bacillus marineus ( ⁇ z «7/ «s marinus)-9987 by organic solvent extraction Mari
  • Maribasin B a novel cyclolipopeptide compound Maribasin B was obtained from the fermentation supernatant of B. marinus A marinus B-9987 by conventional separation means.
  • the specific separation and purification steps are as follows:
  • Bacillus marinum (A mari painting) B-9987 was cultured under appropriate conditions to obtain 10 L of fermentation broth.
  • the Bacillus marinum B-9987 strain was inoculated on the plate and inoculated in the first-stage seed shake flask for 12-24h to be used as the first-class seed.
  • the first-stage seed was inoculated into the second-stage seed shake flask, and cultured for 12-24h, then inoculated into 5L.
  • the fermenter It can be completed by culturing at 28 °C and 300 rpm for about 20 hours. Co-fermenting 3 5L tanks to obtain 10L fermentation broth.
  • the fermentation supernatant and the wet cells were obtained by centrifugation of 10 L of the fermentation broth at 4000 rpm.
  • the fermentation supernatant was extracted three times with an equal volume of ethyl acetate, and each extraction time was 12 hours.
  • the ethyl acetate extracts were combined and concentrated under reduced pressure to a volume of the original fermentation broth.
  • the ethyl acetate raffinate phase was extracted three times with an equal volume of n-butanol, and the extraction time was 12 hours, and the n-butanol extract phase was combined and concentrated under reduced pressure to the original fermentation broth volume.
  • the above-mentioned ethyl acetate extract phase, n-butanol extract phase and raffinate phase were tested for biological activity, and the indicator strain was A solani, and the n-butanol site was determined to be the main antibacterial active site.
  • the n-butanol extract phase was combined and concentrated to dryness under reduced pressure to give a crude n-butanol extract (13.8 g).
  • the crude n-butanol extract was subjected to silica gel column chromatography and eluted with a gradient of chloroform/methanol 10:1-0:1 (500 mL/min). The eluted fraction was subjected to biological activity detection, and the indicator was tomato early blight.
  • This example examines the inhibitory effect of the compound Maribasin B obtained in Example 7 on plant pathogenic bacteria.
  • Maribasin B has strong antibacterial activity against plant pathogenic bacteria such as S. c «erea and Colletotrichum sp.
  • Example 12 Inhibitory activity of Bacillus marinum CBaci7/iis marinus) B-99S7 containing Maribasin B against plant pathogenic bacteria
  • the phytopathogenic fungi stored in the 4 °C slope were activated on a PDA plate and incubated at 28 °C for about one week. After the fungus had grown over the whole plate, the culture was terminated. A sterilized punch with a diameter of 14.50 mm is used to make a cake on the outer edge of the activated pathogenic fungus colony. The front side of the fungus cake is placed on the side of the sputum culture plate, approximately 1 cm near the wall of the dish, and 1 ring is taken with the inoculation needle. The obtained B-9987 fermentation broth was cultured, and a strain line was drawn parallel to the bacterium in the sputum plate. The scribe distance was determined according to the growth rate of the pathogenic fungus, and the sputum culture plate was placed at 28 ° C for 5 days, and the bacteriostatic record was recorded. With width.
  • Determination of the antibacterial spectrum of pathogenic bacteria Mix the pathogens cultured for 24 h into the corresponding semi-solid medium (to make the bacteria-containing The plate reached 10 9 C fij/mL), and then poured onto the prepared water agar plate. After cooling, a hole having a diameter of 14.00 mm was punched in the center of the semi-solid plate, and then B-9987 fermentation broth was added to the hole, 150 ⁇ per hole. The inhibition zone size was recorded after incubation at 30 ° C for 24 h. The results are shown in Table 6.
  • the potting test method was used to examine the control effect of the compound Maribasin B of the present invention on plant diseases in a potted living test.
  • Potted test method 1. Preparation of host plants (select cucumber seedlings with consistent growth in cotyledon stage), 2. Application method (testing materials in stems and leaves after spray treatment in a fume hood or room temperature for 3 hours), 3. Inoculation (in 2 hours after the treatment of the drug, for the gray mold model, in the plate of the plant pathogen, Sotr_yto c rea, the perforator with a diameter of 5 mm was used to cut the colony round at the edge of the colony, and then attached to the drug treatment.
  • the plant pathogen powdery mildew ( ⁇ a e rot1 ⁇ 2 Ca / M / ⁇ m ⁇ ) is formulated into a bacterial suspension, and then directly sprayed on various parts of the plant), 4, culture (The sample after inoculation is moved to the humidifier) Culture, temperature 19 ° C, light 2000 Lux, observe the growth of cucumber seedlings every day, investigate the control effect when the blank control is fully affected), 5.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Agronomy & Crop Science (AREA)
  • Pest Control & Pesticides (AREA)
  • Plant Pathology (AREA)
  • Dentistry (AREA)
  • Wood Science & Technology (AREA)
  • Environmental Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Virology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Peptides Or Proteins (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Description

环脂肽化合物及其制备和应用 技术领域
本发明涉及源于渤海潮间带植物盐地碱蓬 Suaeda salsa)的海洋芽孢杆菌 φ. mari Β-9987 产生的具有拮抗植物病原菌活性的新结构环脂肽化合物 Marihysin A、 maribasin B及其制备方法和在农药方面的应用。 背景技术
由于海洋生物生活在一定的水压、 高盐度、 小温差、有限的溶解氧、有限的光照及化 学缓冲海水体系， 故其新陈代谢、生存繁殖方式、适应机制具有不同于陆地生物的显著特 性， 海洋生物能够产生大量的结构新颖的活性代谢产物， 其中许多已经开发成产品。如从 海洋真菌顶头孢霉 (Cep/ra/o^or w acremonium)中分离并成功上市的第一个海洋新抗生 素一头孢菌素。 沙蚕毒素是日本人从动物沙蚕的提取物发明的一种杀虫剂。
海洋芽孢杆菌能产生许多有价值的物质。 Carisse等 (2003年)从堆肥中 (包括造纸厂淤 泥、 植物残体等成分)分离出海洋芽孢杆菌， 平板拮抗实验和盆栽试验均表明其对黄瓜猝 倒病菌——终极腐霉 (l thium M/t™™)的生长有较强抑制作用。 张海龙等 (2004年)从海洋 芽孢杆菌中发现了 3个新的环脂肽化合物 Mixirin A-C， 它们具有细胞毒活性。 Ashish等 (2006年)发现海洋芽孢杆菌产的纤维素酶在 pH 6和 50°C时活性较好。 Noureddin等 (2006 年)发现海洋芽孢杆菌可以产葡萄糖 -6-磷酸脱氢酶和 6-磷酸葡萄糖脱氢酶。 田黎等 (2007 年)报道了一株海洋芽孢杆菌 B. marinus B-9987 产生的 3 个新结构大环内酯类化合物 Macrolactin O、 P和 Q， 研究发现， 这 3个新化合物对交链孢和稻瘟霉具有很好的抑制活 性。 此外， 还发现含有该类物质的海洋芽孢杆菌 A ar m^ B-9987发酵浓缩液可有效地 抑制植物病原菌的生长。海洋芽孢杆菌因其具有抑菌作用强、抗逆性强及易于加工成剂型 等诸多优势，十分适合于开发成微生物农药。发明人所在课题组已成功研发的 10亿 CFU/g 海洋芽孢杆菌可湿性粉剂作为农药非常安全，属微毒类农药，初步的盆栽和田间小区试验 结果表明该可湿性粉剂对灰霉病、 白粉病、 青枯病、 软腐病、 早疫病、 霜霉病具有较好的 防治效果 (中国发明专利公开号： CN101331881)。
脂肽类物质主要来源于微生物的次级代谢产物，由亲水的肽键和亲油的脂肪烃链两部 分组成，分为线性脂肽和环状脂肽两大类。由于其特殊的两亲分子结构造就了微生物脂肽 具有优良的表面活性， 在医药、 食品、 化妆品、 生物防治、 环境治理及微生物采油等领域 具有重要的应用前景。
环脂肽化合物多数具有抗菌活性，其中最为成功的例子是达托霉素，它是第一个应用 于临床的环脂肽抗生素，用于治疗由革兰阳性菌感染所致的并发性皮肤及皮肤感染，包括 甲氧西林耐药菌金葡菌及对万古霉素抗性的粪肠球菌， 作用机制独特。
枯草芽孢杆菌 α«7/ 是一类重要的根际微生物， 能够促进植物抵抗病原菌 的侵染， 因而被广泛应用于植物病害的生物防治。 B.™ / s能够产生几十种不同结构的抗 生素， 其中非核糖体合成的脂肽类抗生素是其中最常见的一类， 主要包括 ituriiu surfactin 和 fengycin 3个家族。 脂肽类抗生素由氨基酸链和脂肪酸侧链组成， 稳定性好， 对人畜无 害， 不污染环境， 是具有重要开发价值的新型生物源农药。 国外有报道 iturin成功应用于 生物防治的例子 (Klich MA, 1994年)。
Catherine等 (2001年)发现脂肽类生物表面活性剂能选择性除去土壤中的 Pb、 Zn、 Cu 和 Cd等金属离子， 其中 Cu2+最易去除。
黄现青等 (2006年)研究发现， 枯草芽孢杆菌 fmbJ株产生的抗微生物脂肽可以直接作
Figure imgf000003_0001
v ra)禾口猪细小病毒 (Porc we parvo ra)南京株，从而 抑制其对猪肾 Por«'«e 细胞的感染作用。
Kim等 (2007年)研究发现， 环脂肽 Surfactin (MW 1036)能够抑制 Lovo细胞的增殖， 诱导细胞凋亡， 其作用机制可能为抑制细胞生存调节信号通路 ERK和 PI3K/Akt。
王大威等 (2008 年)研究发现， 从大庆油田中分离到的一株枯草芽孢杆菌 ( subtilis)
ZW-3产生的脂肽类生物表面活性剂具有优良的乳化和降低油水界面张力的能力， 并可以 适应油藏中复杂的环境， 可提高采收率 9.2 %， 在微生物采油中具有非常好的应用前景。
薛春美等 (2008年)报道从海洋芽孢杆菌中分离得到了 Macrolactin T和 U以及已知结 构 Macrolactin A、 B、 D、 0、 S， 其中 Macrolactin T、 B、 D对稻瘟病菌、 番茄早疫病菌 及金黄色葡萄球菌具有拮抗作用。 发明内容
本发明者经过研究发现， 由海洋芽孢杆菌 (A 产生的新结构环脂肽化 合物对 交链抱 04/ 7" ·" so/"" )禾口尖抱缠刀菌 ( ¾ν"/ · / 7 oxysporum f. sp . cuberse) ^ ¾ ¾J 病原菌均具有很好的抑制活性， 因此可以用作农药来防治由这些病原菌引起的植物病害。
本发明的目的在于提供来源于海洋芽孢杆菌 (A man'm«)B-9987产生的具有应用价值 的环脂肽化合物及其制备方法和应用。具体地说，本发明第一方面提供具有如下结构式 ( I) 的环脂肽化合物：
Figure imgf000004_0001
其中， R选自 H或 C1-C20支链或直链烷基。
在优选的实施例中， R为 C1-C20直链或支链烷基。
在更优选的实施例中， R为 C6-C15直链或直链烷基。
在一优 合物:
Figure imgf000004_0002
(11)， 式中， Rl、 R2各自独立为 H或 C1-C3直链或支链烷基。
在一优选实施例中， 所述化合物为 Marihysin A, 其结构式如下式 （III) 所示: (πι)。
在另一优选实施例中， 所述化合物为 Maribasin B, 其结构式如下式 （IV) 所示:
Figure imgf000005_0001
在一优选实施例中， 结构式 （I) 和 （II) 不包括以下的化合物:
Figure imgf000005_0002
当用于本发明时，术语"本发明的化合物"或类似术语的含义不仅包括了上述结构式所 定义的化合物， 还包括了它们的盐。所述盐可以是水溶、脂溶或可分散产物的形式， 其可 通过和无机酸或有机酸或碱反应形成。这些酸加成盐的例子包括乙酸盐、 己二酸盐、藻酸 盐、 苯甲酸盐、 苯磺酸盐、 硫酸氢盐、 丁酸盐、 柠檬酸盐、 十二烷基磺酸盐、 盐酸盐、 草 酸盐、 丙酸盐、 琥珀酸盐、 酒石酸盐。 碱性盐包括铵盐， 碱金属盐， 如钠盐和钾盐， 碱土 金属盐， 如钙盐和镁盐， 有机碱的盐， 如二环己胺盐等。
本发明的化合物也可以其互变异构形式存在。尽管未在这里描述的化合物中明确指出 这些形式， 但它们也包含在本发明的范围之内。 除非另有说明， 这里所述化合物的化学名 包括所有可能的、所述化合物可包含的立体化学异构形式的混合物。所述混合物可含有所 述化合物基础分子结构的所有非对映异构体和 /或对映异构体。 本发明化合物所有的立体 化学异构形式可以是纯化形式， 或者是相互混合的形式， 这都包含在本发明范围之内。
本发明第二方面提供了一种制备上述化合物的方法，其特征在于，该方法包括以下步 骤：
(1)培养海洋芽孢杆菌 ( α«7/^ marinus) -99S7 , 得到发酵液；
(2)用选自有机溶剂萃取、 酸沉淀、 浸提或层析的方法从所述发酵液中分离获得所述 化合物。
本发明方法所涉及的菌株为海洋芽孢杆菌 隱 i画) B-9987, 该菌株从渤海潮间带 植物盐地碱蓬中分离得到，该菌株已于 2007年 6月 18日保藏于中国微生物菌种保藏管理 委员会普通微生物中心 (CGMCC) (北京市海淀区中关村北一条 13 号中国科学院微生物研 究所；)， 保藏号为 CGMCC No. 2095， 并公开于中国专利申请 CN 101333206A中。
本发明所用的术语"发酵"或"培养"具有本领域技术人员通常熟知且承认的含义。所述 "发酵液"或"培养液"可通过在适合生长的条件下培养本发明的海洋芽孢杆菌 B. marinus B-9987, 使其生长至一定的细菌浓度来获得。
用于培养本发明菌株的培养基中的营养源没有特别的限制。本领域技术人员可以根据 公知的技术来选择合适的碳源、氮源和其它营养源。例如，碳源可以是淀粉、糊精、甘油、 葡萄糖、 蔗糖、 肌醇、 甘露醇等。 氮源可以是胨、 大豆粉、 蛋白粉、 肉膏、 米糖、 麦皮、 酵母粉、 玉米浆、铵盐以及其它有机或无机含氮化合物。 另外， 培养基中还可适当加入一 些无机盐类， 如氯化钠、 磷酸盐 （如磷酸氢二钾和磷酸二氢钾等）、 硫酸铵、 硫酸锰、 硫 酸镁、 碳酸钙等金属盐。 通常可采用各种已知的常规培养基， 如 LB琼脂培养基、 营养琼 脂培养基、 葡萄糖酵母膏琼脂培养基和牛肉浸汁琼脂培养基等。 在一个具体实施方案中， 用于培养本发明菌株的培养基具有以下组成(％表示质量 /体积)： 葡萄糖 0.1%~1%、 蔗糖 0.1%~2%、酵母粉 0.01%~1.5%、蛋白粉 0.1%~1%、 MgCl2 0.001%~0.1%、 KC1 0.01%~0.5%、 KH2PO4 0.01%~0.5%和 NaC1 0.1%~6%、 pH 6.0 7.0。 然而， 本领域技术人员应当理解， 本 发明并不局限于本文中列举的这些具体培养基配方。
培养本发明中的菌株的温度、 pH、 气液比、 罐压、 转速等条件没有特别严格的限制， 只要该条件适合该菌的生长即可。
在培养时可采用豆油、 泡敌等消泡剂进行消泡。 在一些较佳的实施方案中， pH宜控 制在 5.5〜8.0之间， 培养温度宜在 20〜35 °C之间。 培养时间通常在 121！〜 200h之间， 最 终的菌浓度通常可高达 5 χ 107cfh/ml〜 1 X lOUcfh/mL
在本发明的一个较佳的实施方案中，通过以下方法培养所述海洋芽孢杆菌来得到发酵 液： 在含有碳源、 氮源、 无机盐的培养基中， 在通气量控制在气液比为 0.2: 1〜2: 1、 转速 在 lOOr/min或以上， 培养所述海洋芽孢杆菌 24小时以上， 其中所述碳源选自葡萄糖、 蔗 糖、 淀粉和大米粉中的 1种或几种， 所述氮源选自蛋白粉、酵母粉、花生饼粉和大豆粉中 的 1种或几种， 所述无机盐包括常规的无机盐 (如 MgS04、 ( H4) 2S04、 MgCl2、 KC1、 KH2P04、 NaCK K2HP04和 CaC03)。
上述列举的这些参数只是实现本发明的较佳方案。因此，本领域技术人员在上述范围 以外选择合适的培养条件也能获得本发明的发酵液。
在获得了上述发酵液后， 用选自有机溶剂萃取、酸沉淀、浸提或层析的方法从发酵液 中分离获得目标化合物。本发明的化合物可以从发酵液的发酵清液中分离得到，也可以从 发酵液中的菌体分离得到。 在优选的实施方案中， 也可分别从发酵清液 (也称上清液或发 酵上清液)和菌体中分离得到含本发明的化合物的粗品或浸提液， 然后再进行进一步的分 离纯化。
因此， 作为本发明方法的一个优选方案， 首先从所述发酵液分离得到上清液， 用选自 氯仿、 乙酸乙酯、 醋酸丁酯或正丁醇的溶剂萃取， 然后用柱层析以氯仿 /甲醇梯度洗脱， 收集氯仿 /甲醇为 2: 1至 0: 1的洗脱部位， 用 HPLC制备得到本发明所述的化合物。
从发酵液来分离上清液可采用常规的分离手段如离心过滤等。然后，在获得上清液后， 一种可行的方式是用有机溶剂 (例如， 氯仿、 乙酸乙酯、 醋酸丁酯或正丁醇)进行萃取。 优 选的方案是采用有机溶剂的组合进行所述萃取， 例如， 依次采用乙酸乙酯和正丁醇萃取； 依次采用氯仿、 乙酸乙酯和正丁醇进行萃取； 依次采用醋酸丁酯和正丁醇进行萃取等。
另一个可行的方案是， 先如上所述获得上清液， 然后使上清液酸化沉淀， 用无水甲醇 对酸化沉淀浸提。 具体地说， 在上清液中加入酸 (如盐酸)， 使溶液静置， 然后离心得到酸 化沉淀和酸化上清液。 在本申请的一个方案中， 采用 5N盐酸溶液进行酸化。 本领域技术 人员也可以适当地调整盐酸浓度同样可以得到酸化沉淀。 随后， 将酸化沉淀的 pH调至中 性后冷冻干燥， 用无水甲醇浸提一次或多次， 每次浸提时间为 6-12小时。
还有一种可行的方式是采用诸如离心分离等常规手段从发酵液分离得到菌体。 然后， 用无水甲醇浸提所述菌体一次或多次， 每次浸提时间 6-12小时。
随后， 用柱层析进行氯仿 /甲醇梯度洗脱前述步骤得到的正丁醇萃取活性部位或无水 甲醇浸提物， 收集氯仿 /甲醇为 2: 1至 0: 1的洗脱部位 (优选氯仿 /甲醇 1 : 1的洗脱部位)。 所 用的柱层析例如可以是硅胶柱层析。 最后， 用 Sephadex LH-20和 /或 HPLC (分析和制备条 件均为 70%甲醇水溶液)等制备获得所述化合物。 然而， 本领域技术人员应当理解， 完全能够根据常规技术采用其它的分离手段、溶剂 或洗脱剂来分离得到本发明的化合物。因此，本发明方法的范围并不局限于说明书实施例 中所用的具体方法。
例如， 可采用化学合成的方法制备本发明的化合物。 可使用 Asn、 Ser、 Tyr、 Pro和 Gln5种氨基酸作为基本原料， 用 Pro和 Gin先合成二肽 Pro-Gln， 二肽 Pro-Gin再和 Asn 合成， 形成三肽 Pr-Gln-Asn， 再依次与 Ser、 Asn、 Tyr、 Asn合成线性七肽后， 再与氨基 脂肪酸 β -氨基十四烷酸或 β -氨基反异十五烷酸环化合成 marihysin A或 maribasin B。 通 式中的其它化合物可采用类似的方法和原料合成。
本发明另一方面提供了一种农药组合物， 其特征在于， 所述组合物包含本发明所述 的化合物作为活性组分， 以及农药学上可接受的载体。
作为活性成分的所述化合物在本发明农药组合物中的含量可以为 0.1%-90重量％， 例如， 1-50重量％、 10-40重量％不等。 本领域技术人员可根据实际需要配制含有适当量 的农药组合物。
本发明的化合物或农药组合物可以直接以发酵液形式施用， 也可将其适当稀释 (例如 稀释 10倍、 100倍、 1000倍或更高)以稀释液形式施用。 本发明的化合物或其组合物可以 施加到植物的根、 叶、茎等部位上。施加方法是本领域中的常规技术， 例如可以是在播种 时浸种，移栽前将植物根部浸在发酵液或其稀释液中，或者直接将发酵液或稀释液泼浇在 苗床上。可定植时进行灌根， 也可在植物生长过程中灌根。如果所述农药组合物是以载体 形式保存的， 则可在临用前用水稀释后再进行施加。至于本发明的最适施药剂量， 本领域 技术人员无需经过过多试验即可确定。
本发明的化合物和农药组合物可用于抑制各种植物病原菌， 包括但不限于早疫病菌 (A. so!ani 果腐病菌 ox ¾pon )、 黄萎病菌 (J !" a oatn )、
Figure imgf000008_0001
白绢病菌 OS /erot w sp.)、 青霉病菌 ( ¾" c〃w sp.)、 灰霉病菌 (S. cinerea 小斑病菌 (Ζ turcica)、 纹枯病菌 (R. solani) ι^ί&' (Colle totrichum sp.)、 枯萎病菌 oxysporum f. sp.cuberse)^ ttiiS ¾f(R- solanacearum)^软腐病菌 (£. carotovora var. carotovora)禾口白粉病 iSph theca fuligi a)。这些植物病原菌可导致番茄早疫病、甜瓜果腐病、棉花黄萎病、 小麦赤霉病、 白术白絹病、黄瓜灰霉病、番茄灰霉病、玉米小斑病、水稻纹枯、瓜炭疽病、 香蕉枯萎病、 番茄青枯病菌、 白菜软腐病和黄瓜白粉病。
因此， 本发明再一方面提供一种防治植物病害的方法， 所述方法包括将本发明的化 合物农药组合物施加到所述植物上。
适用于本发明方法防治的植物包括但不限于小麦、 玉米、 水稻、 棉花、 番茄、 香蕉、 白菜、 甜瓜、 西瓜、 黄瓜和白术等。 具体是， 本发明方法可用于防治由下列病原菌引起的 植物病害： 早疫病菌 (A so/a« )、 果腐病菌 ox ¾wn )、 黄萎病菌 ( a»oatn )、 赤霉 病菌 graminearum 白绢病菌 ( c/erot w sp.)、 青霉病菌 ( ¾" c〃w sp.)、 灰霉病菌 S. cinered)^ 小斑病菌 (Ζ turcica)、 纹枯病菌 (R. solani)、 炭疽病菌 (Co〃etotr c ra sp.)、 枯萎 病菌 oxysporum f. sp.cuberse) ^ 青枯病菌 ( i solanacearum) ^ 软腐病菌 (£. carotovora var. c"ro ovora)禾口白
Figure imgf000009_0001
在一优选实施例中， 可采用本发明方法防治以下的植物病害： 番茄早疫病、 甜瓜果 腐病、 棉花黄萎病、 小麦赤霉病、 白术白絹病、 黄瓜灰霉病、 番茄灰霉病、 玉米小斑病、 水稻纹枯、 瓜炭疽病、 香蕉枯萎病、 番茄青枯病菌、 白菜软腐病和黄瓜白粉病。 具体实施方式
实施例 1利用酸沉淀法从海洋芽孢杆菌 (^z«7/«s marinus) -99S7发酵液中分离得到新环 脂肽化合物 Marihysin A
本实施例通过分离手段从海洋芽孢杆菌 A marinus B-9987的发酵液中获得了新的环 脂肽化合物 Marihysin A。 具体分离纯化步骤如下：
(1)将海洋芽孢杆菌 (A mari画) B-9987在合适的条件下培养得到 30L发酵液。
海洋芽孢杆菌 B-9987菌种经平板活化后接种于一级种子摇瓶培养 12-24h后作为一级 种子，将该一级种子接种于二级种子摇瓶，培养 12-24h后接种于 50L发酵罐中。在 28 °C、 300rpm下培养 20h左右即可结束。
(2)30L发酵液 4000rpm离心后得到发酵上清液和湿菌体。 发酵上清液加 5N盐酸溶液酸 化并静置沉淀后离心 (4000rpm)得到酸化沉淀和酸化上清液。将酸化沉淀 pH调至 7.0 后冷冻干燥， 得到冻干沉淀 174.8g， 用无水甲醇 (440mL， 220mL, 220mLM衣次浸提 3次， 每次浸提时间 12小时， 将浸提液合并减压浓缩至干， 得到酸化沉淀浸提粗品 共 10.2g。将酸化沉淀浸提粗品经硅胶柱层析以氯仿 /甲醇 10: 1→0: 1梯度洗脱 (500mL/ 流分)， 对洗脱的流分进行生物活性检测， 指示菌为茄交链孢 solani) , 结 果显示抑菌活性物质主要集中在氯仿 /甲醇 1 : 1和甲醇洗脱部位， 且以氯仿 /甲醇 1 : 1 洗脱部位活性更强， 将上述活性流分经 TLC 检测后合并。 将合并后的活性流分经 Sephadex LH-20柱层析以甲醇洗脱， 对洗脱流分进行生物活性检测， 指示菌为茄交 観 (Ahemaria so ， 生物活性检测后的活性流分经 TLC检测后合并。 将合并后 的活性流分经 HPLC分析并制备 (分析和制备条件均为 70%甲醇水溶液)， 最终制备 得到化合物 Marihysin A。 实施例 2利用有机溶剂提取法从海洋芽孢杆菌 (^z«7/«s marinus) -9987发酵液中分离得 到新环脂肽化合物 Marihysin A
本实施例通过常规的分离手段从海洋芽孢杆菌 A marinus B-9987的发酵液中获得了 新的环脂肽化合物 Marihysin A。 具体分离纯化步骤如下：
(1)将海洋芽孢杆菌 (A mctri画) B-9987在合适的条件下培养得到 30L发酵液 (具体的培养 条件和步骤同实施例 1)。
(2) 30L发酵液 4000rpm离心后得到发酵上清液和湿菌体。 湿菌体经过水洗离心后冷冻干 燥， 得到干菌体 92.0g， 用无水甲醇 (600mL， 300mL, 300mL)依次浸提 3次， 每次 浸提时间 12小时，将浸提液合并减压浓缩至干得到菌体浸提粗品共 17.3g。将菌体 浸提粗品经硅胶柱层析以氯仿 /甲醇 10: 1—0: 1梯度洗脱 (500mL/流分)， 对洗脱的流 分进行生物活性检测， 指示菌为茄交链孢 solani), 结果显示抑菌活性物 质主要集中在氯仿 /甲醇 1 : 1和甲醇洗脱部位， 且以氯仿 /甲醇 1 : 1洗脱部位活性更 强， 将上述活性流分经 TLC检测后合并。 将合并后的活性流分经 Sephadex LH-20 柱层析以甲醇洗脱， 对洗脱流分进行生物活性检测， 指示菌为茄交链孢
solani), 生物活性检测后的活性流分经 TLC检测后合并。 将合并后的活性流分经 HPLC 分析并制备 (分析和制备条件均为 70%甲醇水溶液)， 最终制备得到化合物 Marihysin A。 实施例 3有机溶剂萃取法从海洋芽孢杆菌 (^z«7/«s »«m'««S)B-9987发酵液中分离得到新 环脂肽化合物 Marihysin A
本实施例通过常规的分离手段从海洋芽孢杆菌 A marinus B-9987的发酵液中获得了 新结构环脂肽化合物 Marihysin A。 具体分离纯化步骤如下：
(1)将海洋芽孢杆菌 S. mari B-9987在合适的条件下培养得到 10L发酵液。
海洋芽孢杆菌 B-9987菌种经平板活化后接种于一级种子摇瓶培养 12-24h后作为 一级种子，将该一级种子接种于二级种子摇瓶，培养 12-24h后接种于 5L发酵罐中。 在 28 °C、 300rpm下培养 20h左右即可结束。 共发酵 3个 5L罐获得 10L发酵液。 (2) 10L发酵液 4000rpm离心后得到发酵上清液和湿菌体。发酵上清液用等体积乙酸乙酯 分别萃取 3次， 每次萃取时间 12小时， 将乙酸乙酯萃取相合并减压浓缩至原发酵 液体积。 再用等体积正丁醇对乙酸乙酯萃余相分别萃取 3次， 每次萃取时间 12小 时，将正丁醇萃取相合并减压浓缩至原发酵液体积。对上述乙酸乙酯萃取相和正丁 醇萃取相进行生物活性检测， 指示菌为茄交链孢 solani), 确定正丁醇部 位为主要抑菌活性部位。将正丁醇萃取相合并减压浓缩至干，得到正丁醇萃取物粗 品共 13.8g。 将正丁醇萃取物粗品经硅胶柱层析以氯仿 /甲醇 10: 1— 0: 1 梯度洗脱 (500mL/流分）， 对洗脱的流分进行生物活性检测， 指示菌为茄交链孢
solani), 结果显示抑菌活性物质主要集中在氯仿 /甲醇 1 : 1和甲醇洗脱部位， 且以氯 仿 /甲醇 1 : 1洗脱部位活性更强， 将上述活性流分经 TLC检测后合并。 将合并后的 活性流分经 Sephadex LH-20柱层析以甲醇洗脱， 对洗脱流分进行生物活性检测， 指示菌为茄交链孢 so/a ')，生物活性检测后的活性流分经 TLC检测后合 并。 将合并后的活性流分经 HPLC分析并制备 (分析和制备条件均为 70%甲醇水溶 液)， 最终制备得到化合物 Marihysin A。 实施例 4新环脂肽化合物 Marihysin A的结构鉴定和分析 (l)Marihysin A结构鉴定和分析
将实施例 1、 2或 3获得的新的环脂肽化合物 Marihysin A进行分析和鉴定， 其结果 均为如下：
Marihysin A化学结构如下
Figure imgf000011_0001
其为无色固体粉末， UV254nm下有暗斑， 碘蒸汽显色下呈黄色。 mp>250°C ; IR (KBr) Vmax icm"1): 3333 , 2929, 2864, 1659， 1545， 1449， 1423 , 1247, 1128。 HRFAB-MS m/z 1065.6308 [M+Na] + (calcd for C48H74N12014)。 1H、 13C MR数据见表 1。
Figure imgf000011_0002
Figure imgf000012_0001
实施例 5 Marihysin A对植物病原菌的抑制作用
本实施例考察了实施例 1获得的化合物 Marihysin A对植物病原菌的抑制作用。
采用平板拮抗法考察 Marihysin A 对番茄早疫病菌 (A solani), 甜瓜果腐病菌 (^： oxysporum)^ 棉花黄萎病菌 (J!" a oatn )、 小麦赤霉病菌 gra wran )、 白术白绢病菌 {Sclerotium sp.)、 青霉病菌 ( ¾"c〃w sp.)、 黄瓜灰霉病菌 (S. cinered)^ 番 灰霉病菌 (S. cinerea), 玉米小斑病菌 (Ζ turcica), 水稻纹枯病菌 (R. solani), 瓜炭疽病菌 (Co〃etotn'c/z謹 sp.)、 香蕉枯萎病菌 oxysporum f. sp fer )等植物病原菌的抑制作用。
Marihysin A对植物病原菌的 MIC如下表 2。
表 2 Marihysin A对植物病原菌的抑制作用
Figure imgf000013_0001
从表 2中可以看出 Marihysin A对白术白绢病菌 {Sclerotium sp.) 甜瓜果腐病菌 oxysporum)、青霉病菌 ( ¾ c〃 / w sp.)禾口瓜炭疽病菌 (Co lletotrichum sp.；)等植物病原菌具有 较强的抑菌活性。 实施例 6 含有 Marihysin A的海洋芽孢杆菌 CBaci7/iis / narinus)B-99S7发酵液对植物病原 菌的抑制活性
本实施例考察了含有本发明所述的化合物 A larihysin A 的海洋芽孢杆菌 (S. marinus)B-9987发酵液可有效抑制植物病原菌的生长。
本实施例采用平板拮抗法考察含有 Marihysin A的海洋芽孢杆菌 S. marin s)B-9987 发酵液对植物病原菌的抑制作用。
病原真菌抑菌谱的测定： 将于 4 °C斜面中保存的的植物致病真菌接于 PDA平板在 28 °C的条件下活化培养大约一周， 待真菌长满整个平板后结束培养， 用已灭菌的直径为 14.50mm 的打孔器在活化的病原真菌菌落外缘打一菌饼， 将菌饼正面向上放置于对峙培 养平板的一侧， 靠近皿壁约 lcm， 用接种针取 1环培养获得的 B-9987发酵液， 在对峙平 板内平行于菌块划一条菌线，划线距离视病原真菌的生长速度而定，将对峙培养平板放入 28 °C培养 5 d后记录抑菌带宽度。
病原细菌抑菌谱的测定：将培养 24 h的病原菌液混入相应半固体培养基中 (使含菌平 板达到 109 Cfij/mL)， 然后倒在制备好的水琼脂平板上， 冷却后在半固体平板中央打直径为 14.00 mm的孔， 然后将 B-9987发酵液加入孔中， 每孔 150μί， 30°C培养 24 h后记录抑菌 圈大小。
其结果如下表 3所示。
表 3海洋芽孢杆菌 B-9987发酵液对植物病原菌的抑制作用 病原菌 抑菌带 (圈) /mm 玉米小斑病菌 (C. lunatd) 11.00 黄瓜灰霉病菌 δ· cinered) 12.00 瓜炭疽病菌 (Co〃etotr c ra sp. ) 6.50 番 早疫病菌 sp. ) 6.40 烟草赤星病菌 4. alternate) 6.30
稻痕病菌 ( ! grised) 5.67 香蕉枯萎病菌 oxysporum f. sp. cuberse) 5.60
舌甘瓜枯萎病菌 oxysporum) 3.10 小麦赤霉病菌 ( . graminearum) 3.06 番 青枯病菌 (R. solanacearum) 23.0 白菜软腐病菌 E. carotovora var. carotovora) 18.8 由表 3的抑制活性可知，含有化合物 Marihysin A的海洋芽孢杆菌 S. marin s)B-9987 发酵液可有效地抑制植物病原菌的生长。 实施例 7利用酸沉淀法从海洋芽孢杆菌 (^z«7/«s marinus)B-99S7发酵清液中分离得到新 环脂肽化合物 Maribasin B
本实施例通过常规的分离手段从海洋芽孢杆菌 A marinus B-9987的发酵液中获得了 新的环脂肽化合物 Maribasin B。 具体分离纯化步骤如下：
(1) 将海洋芽孢杆菌 (A mari B-9987在合适的条件下培养得到 30L发酵液。 海洋芽孢 杆菌 B-9987菌种经平板活化后接种于一级种子摇瓶培养 12-24h后作为一级种子，将该一 级种子接种于二级种子摇瓶， 培养 12-24h后接种于 50L发酵罐中。 在 28 °C、 300rpm下 培养 20h左右即可结束。
(2) 30L发酵液 4000rpm离心后得到发酵上清液和湿菌体。 发酵上清液加 5N盐酸溶液酸 化并静置沉淀后离心 (4000rpm)得到酸化沉淀和酸化上清液。将酸化沉淀 pH调至 7.0后冷 冻干燥， 得到冻干沉淀 174.8g， 用无水甲醇 (440mL， 220mL, 220mL)依次浸提 3次， 每 次浸提时间 12小时， 将浸提液合并减压浓缩至干， 得到酸化沉淀浸提粗品共 10.2g。将酸 化沉淀浸提粗品经硅胶柱层析以氯仿 /甲醇 10: 1→0: 1梯度洗脱 (500mL/流分)， 对洗脱的流 分进行生物活性检测， 指示菌为番茄早疫病菌 (A solani), 结果显示抑菌活性物质主要集 中在氯仿 /甲醇 1 : 1和甲醇洗脱部位， 且以氯仿 /甲醇 1 : 1洗脱部位活性更强， 将上述活性 流分经 TLC检测后合并。 将合并后的活性流分经 Sephadex LH-20柱层析以甲醇洗脱， 对 洗脱流分进行如上的生物活性检测， 生物活性检测后的活性流分经 TLC检测后合并。 将 合并后的活性流分经 HPLC分析并制备 (分析和制备条件均为 70%甲醇水溶液)，最终制备 得到化合物 Maribasin B。 实施例 8利用有机溶剂提取法从海洋芽孢杆菌 (^z«7/«s marinus)B-99S7菌体中分离得到 新环脂肽化合物 Maribasin B
本实施例通过常规的分离手段从海洋芽孢杆菌 A marinus B-9987菌体中获得了新的 环脂肽化合物 Maribasin B。 具体分离纯化步骤如下：
(1) 将海洋芽孢杆菌 (A mari画) B-9987在合适的条件下培养得到 30L发酵液 (具体的 培养条件和步骤同实施例 1)。
(2) 30L发酵液 4000rpm离心后得到发酵上清液和湿菌体。 湿菌体经过水洗离心后冷 冻干燥， 得到干菌体 92.0g， 用无水甲醇 (600mL， 300mL， 300mL)依次浸提 3次， 每次浸 提时间 12小时，将浸提液合并减压浓缩至干得到菌体浸提粗品共 17.3g。将菌体浸提粗品 经硅胶柱层析以氯仿 /甲醇 10: 1→0: 1梯度洗脱 (500mL/流分)， 对洗脱的流分进行生物活性 检测，指示菌为番茄早疫病菌 4. 结果显示抑菌活性物质主要集中在氯仿 /甲醇 1 : 1 和甲醇洗脱部位， 且以氯仿 /甲醇 1 : 1洗脱部位活性更强， 将上述活性流分经 TLC检测后 合并。 将合并后的活性流分经 Sephadex LH-20柱层析以甲醇洗脱， 对洗脱流分进行生物 活性检测， 指示菌为番茄早疫病菌 4. 生物活性检测后的活性流分经 TLC检测后 合并。 将合并后的活性流分经 HPLC分析并制备 (分析和制备条件均为 70%甲醇水溶液)， 最终制备得到化合物 Maribasin B。 实施例 9利用有机溶剂萃取法从海洋芽孢杆菌 (^z«7/«s marinus) -9987发酵清液中分离 得到新环脂肽化合物 Maribasin B
本实施例通过常规的分离手段从海洋芽孢杆菌 A marinus B-9987的发酵清液中获得 了新的环脂肽化合物 Maribasin B。 具体分离纯化步骤如下：
(1) 将海洋芽孢杆菌 (A mari画) B-9987在合适的条件下培养得到 10L发酵液。 海洋 芽孢杆菌 B-9987菌种经平板活化后接种于一级种子摇瓶培养 12-24h后作为一级种子，将 该一级种子接种于二级种子摇瓶， 培养 12-24h后接种于 5L发酵罐中。 在 28 °C、 300rpm 下培养 20h左右即可结束。 共发酵 3个 5L罐获得 10L发酵液。
(2) 10L发酵液 4000rpm离心后得到发酵上清液和湿菌体。 发酵上清液用等体积乙酸 乙酯分别萃取 3次， 每次萃取时间 12小时， 将乙酸乙酯萃取相合并减压浓缩至原发酵液 体积。 再用等体积正丁醇对乙酸乙酯萃余相分别萃取 3次， 每次萃取时间 12小时， 将正 丁醇萃取相合并减压浓缩至原发酵液体积。对上述乙酸乙酯萃取相、正丁醇萃取相与萃余 相进行生物活性检测， 指示菌为番茄早疫病菌 (A solani), 确定正丁醇部位为主要抑菌活 性部位。 将正丁醇萃取相合并减压浓缩至干， 得到正丁醇萃取物粗品共 13.8g。 将正丁醇 萃取物粗品经硅胶柱层析以氯仿 /甲醇 10: 1—0: 1梯度洗脱 (500mL/流分)， 对洗脱的流分进 行生物活性检测， 指示菌为番茄早疫病菌 (A solani), 结果显示抑菌活性物质主要集中在 氯仿 /甲醇 1 : 1和甲醇洗脱部位， 且以氯仿 /甲醇 1 : 1洗脱部位活性更强， 将上述活性流分 经 TLC检测后合并。 将合并后的活性流分经 Sephadex LH-20柱层析以甲醇洗脱， 对洗脱 流分进行生物活性检测， 指示菌为番茄早疫病菌 (A solani), 生物活性检测后的活性流分 经 TLC检测后合并。将合并后的活性流分经 HPLC分析并制备 (分析和制备条件均为 70% 甲醇水溶液)， 最终制备得到化合物 Maribasin B。 实施例 10 新环脂肽化合物 Maribasin B的结构鉴定和分析
(1) Maribasin B结构鉴定和分析
将实施例 7获得的新的环脂肽化合物 Maribasin B进行分析和鉴定， 其结果如下: Maribasin B化
Figure imgf000016_0001
其为无色固体粉末， UV254nm下有暗斑， 碘蒸汽显色下呈黄色。 mp>250°C ; IR (KBr) Vmax icm"1) : 3333 , 2929, 2864, 1659， 1545， 1449， 1423 , 1247, 1128。 HRFAB-MS m/z 1079.5507 [M+Na] + (calcd for C49H76N12014)。 1H、 13C MR数据见表 4。
Figure imgf000016_0002
Figure imgf000017_0001
实施例 11 Maribasin B对植物病原菌的抑制作用
本实施例考察了实施例 7获得的化合物 Maribasin B对植物病原菌的抑制作用。 采用平板拮抗法考察 Maribasin B 对番茄早疫病菌 (A solani) , 甜瓜果腐病菌 oxysporum)^ 棉花黄萎病菌 (J !" a oatn )、 小麦赤霉病菌 gra wran )、 白术白绢病菌 Sclerotium sp.)、 青霉病菌 ( ¾" c〃w sp.)、 黄瓜灰霉病菌 (S. cinered)^ 番 灰霉病菌 (S. cinerea), 玉米小斑病菌 (Ζ turcica), 水稻纹枯病菌 (R. solani), 瓜炭疽病菌 (Co〃etotn'c/z謹 sp.)和香蕉枯萎病菌 oxysporum f. sp.cMfer )等植物病原菌的抑制作用。
Maribasin B对植物病原菌的 MIC如表 5所示。
表 5 Maribasin B对植物病原菌的抑制作用 ^g/ml)
Figure imgf000018_0001
从表 5 中可以看出 Maribasin B 对黄瓜灰霉病菌 S. c «erea)和瓜炭疽病菌 (Colletotrichum sp. )等植物病原菌具有较强的抑菌活性。 实施例 12含有 Maribasin B的海洋芽孢杆菌 CBaci7/iis marinus)B-99S7发酵液对植物病原 菌的抑制活性
本实施例考察了含有本发明所述的化合物 Maribasin B 的海洋芽孢杆菌 S. mari B-9987发酵液可有效抑制植物病原菌的生长。
本实施例采用平板拮抗法考察含有 Maribasin B的海洋芽孢杆菌 S. marinns)B-99S7 发酵液对植物病原菌的抑制作用。
病原真菌抑菌谱的测定： 将于 4 °C斜面中保存的的植物致病真菌接于 PDA平板在 28 °C的条件下活化培养大约一周， 待真菌长满整个平板后结束培养， 用已灭菌的直径为 14.50mm 的打孔器在活化的病原真菌菌落外缘打一菌饼， 将菌饼正面向上放置于对峙培 养平板的一侧， 靠近皿壁约 lcm， 用接种针取 1环培养获得的 B-9987发酵液， 在对峙平 板内平行于菌块划一条菌线，划线距离视病原真菌的生长速度而定，将对峙培养平板放入 28 °C培养 5 d后记录抑菌带宽度。
病原细菌抑菌谱的测定：将培养 24 h的病原菌液混入相应半固体培养基中 (使含菌平 板达到 109 Cfij/mL)， 然后倒在制备好的水琼脂平板上， 冷却后在半固体平板中央打直径为 14.00 mm的孔， 然后将 B-9987发酵液加入孔中， 每孔 150μί， 30°C培养 24 h后记录抑菌 圈大小。 其结果如表 6所示。
表 6海洋芽孢杆菌 B-9987发酵液对植物病原菌的抑制作用
Figure imgf000019_0001
由表 6的抑制活性可知，含有化合物 Maribasin B的海洋芽孢杆菌 S. marin s)B-9987 发酵液可有效地抑制植物病原菌的生长。 实施例 13 Maribasin B在盆栽活体试验中对植物病害的防治效果
本实施例采用盆栽试验方法考察了本发明所述的化合物 Maribasin B在盆栽活体试 验中对植物病害的防治效果。
盆栽试验方法： 1、 寄主植物的准备（选取子叶期长势一致的黄瓜苗）， 2、 施药方式 (茎叶喷雾处理后将试材在通风橱或室温中阴干 3h)， 3、 接种 （在药剂处理后 2h接种， 对于灰霉病模型， 是在已长好植物病原菌灰霉病菌 Sotr_yto c rea)平皿中， 用直径 5mm 的打孔器， 在菌落边缘切取菌落圆块， 然后贴到药剂处理过的苗叶片中央； 对于白粉病模 型， 首先将植物病原菌白粉病菌 (^^aerot½Ca/M/^ m^)落配成菌悬液，然后直接喷雾在植 株各部位）， 4、 培养 （接种后的试材移至保湿箱中） 培养， 温度 19°C， 光照 2000Lux， 每天观察黄瓜苗生长状况， 待空白对照充分发病时调查防治效果）， 5、 调查 （分级标准 0 级： 无病； 1 级： 轻微发病， 发病面积占叶面积的 5%以下； 3级： 轻度发病， 发病面积 占叶面积的 6%-15%； 5级： 中度发病， 发病面积占叶面积的 16%-30%； 7级： 高度发病， 发病面积占叶面积的 31%-50%； 9级： 严重发病， 发病面积占叶面积的 50%以上； 防治 效果（％) = (对照病情指数-处理病情指数） /对照病情指数 * 100)， 5、 结果与分析（用邓 肯氏新复极差（DMRT )法对试验数据进行统计分析，特殊情况用相应的生物统计学方法。 并对实验结果加以分析、 评价）。 表 7黄瓜灰霉病盆栽防效试验结果
Figure imgf000020_0001
另外： 当农用抗生素 Maribasin B浓度为 250、 125 g/ml时， 其对黄瓜白粉病的盆栽防效 分别为 91.0%禾口 85.6%。 尽管上面已经描述了本发明的具体例子， 但是有一点对于本领域技术人员来说是明 显的， 即在不脱离本发明的精神和范围的前提下可对本发明作各种变化和改动。 因此， 所 附权利要求覆盖了所有这些在本发明范围内的变动。

Claims

权 利 要 求 书
1. 脂肽类化合物， 所述化合物具有以下结构:
Figure imgf000021_0001
其中， R选自 H或 C1-C20直链或支链烷基。
2. 如权利要求 1所述的化合物， 其特征在于， 所述化合物选自
Figure imgf000021_0002
式中， 和 R2各自独立地选自 H和 C 1 -C3直链或支链烷基。
3. 如权利要求 1所述的化合物， 其特征在于， 所述化合物选自: 和
Figure imgf000022_0001
4. 一种制备权利要求 1一 3中任一项所述的化合物的方法， 其特征在于， 该方法包括 以下步骤：
(1)培养海洋芽孢杆菌 ( α«7/^ marinus) -99S7 , 得到发酵液；
(2)用选自有机溶剂萃取、 浸提或层析的方法从所述发酵液中分离获得所述化合物。
5. 如权利要求 4所述的方法， 其特征在于， 所述步骤 (2)包括以下一个或多个步骤：
(a) 从所述发酵液分离得到上清液， 用选自氯仿、 乙酸乙酯、 醋酸丁酯或正丁醇的溶 剂萃取， 然后用柱层析以氯仿 /甲醇梯度洗脱， 收集氯仿 /甲醇为 2: 1至 0: 1 的洗脱部位， 用 Sephadex LH-20和 /或 HPLC制备得到权利要求 1所述的化合物；
(b) 从所述发酵液分离得到上清液，使上清液酸化沉淀，用无水甲醇对酸化沉淀浸提， 浸提液用柱层析以氯仿 /甲醇梯度洗脱， 收集氯仿 /甲醇为 2: 1 至 0: 1 的洗脱部位， 用 Sephadex LH-20和 /或 HPLC制备得到权利要求 1所述的化合物；
(c) 从所述发酵液分离得到菌体， 用无水甲醇浸提， 浸提液用柱层析以氯仿 /甲醇梯度 洗脱， 收集氯仿 /甲醇为 2: 1至 0: 1的洗脱部位， 用 Sephadex LH-20和 /或 HPLC制备得到 权利要求 1所述的化合物。
6. 如权利要求 5所述的方法， 其特征在于， 所述 HPLC的制备条件均为 70%甲醇水 溶液。
7. 一种农药组合物， 其特征在于， 所述组合物包含权利要求 1一 3中任一项所述的化 合物作为活性组分， 以及农药学上可接受的载体。
8. 权利要求 1一 3中任一项所述的化合物在作为农药中的应用。
9. 如权利要求 8所述的应用， 其特征在于， 所述农药用于抑制选自以下的植物病原 菌： 早疫病菌 (A solanf)、 果腐病菌 oxysporum)^ 黄萎病菌 cdboatrum)、 赤霉病菌 graminearum)^ 白绢病菌 ( c/erot w sp.)、青霉病菌 ( ¾" c〃w sp.)、灰霉病菌 (S. cinered)^ 小斑病菌 (Ζ turcica) 纹枯病菌 (R. solani) ^' ' (Colletotrichum sp.)、 白粉病菌 (Sphaerotheca fuliginea)、 枯萎病菌 ox ¾pon / sp.cuberse)^ 青枯病菌 (R. solanacearum) 禾口软腐病菌 (£. carotovora var. carotovora) 0
10. 一种防治植物病害的方法， 其特征在于， 所述方法包括将权利要求 1一 3中任一 项所述的化合物或权利要求 7所述的农药组合物施加到所述植物上。
11 . 如权利要求 10所述的方法， 其特征在于， 所述植物病害的病原菌选自早疫病菌
(A. solani)、 果腐病菌 ox ¾pon )、 黄萎病菌 (J !" a oatn )、
Figure imgf000023_0001
白绢病菌 OS /erot w sp.)、 青霉病菌 ( ¾" c〃w sp.)、 灰霉病菌 (S. cinerea)、 小斑病菌 (Ζ turcica)、 纹枯病菌 (R. solani)、 炭疽病菌 {Colletotrichum sp.) 白粉病菌 {Sphaerotheca fuliginea)、枯萎病菌 oxysporum f. sp.cuberse)^青枯病菌 (R. so/awacran )禾口软腐病菌 (£. carotovora var. carotovora)。
12. 如权利要求 10或 11所述的方法，其特征在于，所述植物选自小麦、玉米、水稻、 棉花、 番茄、 香蕉、 白菜、 甜瓜、 西瓜、 黄瓜和白术。
13 . 如权利要求 10所述的方法， 其特征在于， 所述植物病害选自番茄早疫病、 甜瓜 果腐病、棉花黄萎病、小麦赤霉病、 白术白絹病、黄瓜灰霉病、番茄灰霉病、玉米小斑病、 水稻纹枯、 瓜炭疽病、 香蕉枯萎病、 番茄青枯病菌、 白菜软腐病和黄瓜白粉病。
PCT/CN2011/070507 2010-02-08 2011-01-24 环脂肽化合物及其制备和应用 WO2011095082A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201019063051.8 2010-02-08
CN201019063051.8A CN101899098B (zh) 2010-02-08 2010-02-08 一种环脂肽类化合物Maribasin B制备及其应用

Publications (1)

Publication Number Publication Date
WO2011095082A1 true WO2011095082A1 (zh) 2011-08-11

Family

ID=43225052

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2011/070507 WO2011095082A1 (zh) 2010-02-08 2011-01-24 环脂肽化合物及其制备和应用

Country Status (2)

Country Link
CN (1) CN101899098B (zh)
WO (1) WO2011095082A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114957401A (zh) * 2021-01-28 2022-08-30 广东轻工职业技术学院 一种海洋真菌来源的肽类化合物及其制备方法与应用

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101838314B (zh) * 2009-11-16 2014-11-05 华东理工大学 一种环脂肽类化合物制备及其应用
CN101899098B (zh) * 2010-02-08 2014-03-05 华东理工大学 一种环脂肽类化合物Maribasin B制备及其应用
CN105859447A (zh) * 2016-06-13 2016-08-17 镇江贝思特有机活性肥料有限公司 一种抗烟草青枯病的生物肥料
CN109749954B (zh) * 2018-12-04 2020-09-01 青岛农业大学 一株变异链霉菌、其菌剂及其制备方法和应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101838314A (zh) * 2009-11-16 2010-09-22 华东理工大学 一种环脂肽类化合物制备及其应用
CN101899098A (zh) * 2010-02-08 2010-12-01 华东理工大学 一种环脂肽类化合物Maribasin B制备及其应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101331881A (zh) * 2007-06-27 2008-12-31 国家海洋局第一海洋研究所 海洋芽孢杆菌可湿性粉剂、及其制备和应用
CN101333206B (zh) * 2007-06-27 2012-12-12 国家海洋局第一海洋研究所 大环内酯类化合物及其制备和应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101838314A (zh) * 2009-11-16 2010-09-22 华东理工大学 一种环脂肽类化合物制备及其应用
CN101899098A (zh) * 2010-02-08 2010-12-01 华东理工大学 一种环脂肽类化合物Maribasin B制备及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TANG JINSHAN ET AL.: "Progress on the studies of cyclic lipopeptides", ACTA PHARMACEUTICA SINICA, vol. 43, no. 9, 2008, pages 873 - 883 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114957401A (zh) * 2021-01-28 2022-08-30 广东轻工职业技术学院 一种海洋真菌来源的肽类化合物及其制备方法与应用
CN114957401B (zh) * 2021-01-28 2023-04-28 广东轻工职业技术学院 一种海洋真菌来源的肽类化合物及其制备方法与应用

Also Published As

Publication number Publication date
CN101899098B (zh) 2014-03-05
CN101899098A (zh) 2010-12-01

Similar Documents

Publication Publication Date Title
Khan et al. The Endophytic Bacteria Bacillus velezensisLle-9, Isolated from Lilium leucanthum, Harbors Antifungal Activity and PlantGrowth-Promoting Effects
EP2694535B1 (de) Mittel zur behandlung von mikrobiellen erkrankungen bei kulturpflanzen
KR101291915B1 (ko) 식물병원성 진균에 항균 활성을 갖는 바실러스 아밀로리쿼파시엔스 ｇｙｌ４ 및 이를 이용한 항생물질의 제조방법
CN102060914B (zh) 海洋芽孢杆菌b-9987产脂肽类化合物及其制备和应用
KR101313900B1 (ko) 푸자리시딘을 이용한 식물 보호 방법
WO2011095082A1 (zh) 环脂肽化合物及其制备和应用
KR20150071011A (ko) 스트렙토미세스 미크로플라부스 균주를 사용하여 고제로틴을 생산하는 방법
Chowdhury et al. Bacterial endophytes isolated from mountain-cultivated ginseng (Panax ginseng Mayer) have biocontrol potential against ginseng pathogens
JP2001172112A (ja) 植物の活性付与剤、その製造方法と、活性付与方法及び活性促進剤並びにその施用方法
JPH0551305A (ja) バチルス属に属するｓｃ−３菌株による植物病害防除方法および使用される細菌
WO2010137404A1 (ja) 細菌のバイオフィルムを接種した種子
JPH11196862A (ja) ストレプトミセスの新規株及びその利用
KR101660229B1 (ko) 트라이코데르마 하지아눔 mpa167 및 이의 용도
KR102670172B1 (ko) 트리코더마 롱기브라키아툼 균주로부터 유래된 살균 화합물을 포함하는 식물병 방제용 조성물 및 이를 이용한 식물병 방제 방법
KR102604427B1 (ko) 트리코더마 롱기브라키아툼 균주를 포함하는 식물병 방제용 조성물 및 이를 이용한 식물병 방제 방법
CN109880746B (zh) 一种座坚壳属真菌菌株及其应用
US10980242B2 (en) Xylaria grammica EL 000614 strain having nematicidal activity against root knot nematode and uses thereof
CN101838314B (zh) 一种环脂肽类化合物制备及其应用
KR100612041B1 (ko) 식물병원균에 항균활성을 가지는 신규 스트렙토마이세스고양엔시스 브이케이-에이60 균주 및 이 균주로부터생산되는 살균제
EA039448B1 (ru) Штамм микроорганизма clonostachys rosea f. catenulata в качестве биофунгицида, стимулятора роста растений и продуцента метаболитов для сельскохозяйственного применения
US5773263A (en) Production aflastatin A from streptomyces sp., a pharmaceutical composition and methods of use
Baskaran et al. Screening of microbial metabolites and bioactive components
CN110093383A (zh) 一种苯并吡喃酮类化合物作为农用杀菌剂的应用
CN115873758B (zh) 一种高效拮抗禾谷镰刀菌的海洋放线菌及其筛选方法、应用
JP5578649B2 (ja) アクチノマデュラ属に属する新規微生物、その微生物が産生する新規化合物、及びその化合物を有効成分とする医薬

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11739361

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11739361

Country of ref document: EP

Kind code of ref document: A1