CN102060914A - Lipopeptide compound produced by bacillus marinus B-9987 and preparation and application thereof - Google Patents
Lipopeptide compound produced by bacillus marinus B-9987 and preparation and application thereof Download PDFInfo
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Abstract
The invention provides a novel lipopeptide compound produced by bacillus marinus B-9987. The invention also relates to a preparation method of the compound and application of the compound as a pesticide.
Description
Technical field
The present invention relates to come from that bacillus marinus (B.marinus) B-9987 of tideland, Bohai Sea plant Suaeda salsa (Suaeda salsa) produces has active new cyclic lipopeptide compound Marihysin A of antagonism phytopathogen and preparation method thereof and the application aspect agricultural chemicals.
Background technology
Because the singularity of ocean environment, marine microorganism has some unique pathways metabolisms that are different from the land microorganism, thereby can produce the active metabolite of a large amount of novel structures.As first ocean new antibiotic---the cynnematin that from thalassiomycetes cephalosporium acremonium (Cephalosporiumacremonium), separates and success is gone on the market, started the antibiotic beginning in exploitation ocean.
Bacillus marinus can produce many valuable materials.Carisse etc. (2003) (comprise compositions such as paper-mill sludge, plant residue) and isolate bacillus marinus that dull and stereotyped antagonistic experiment and pot experiment show that all it is to the cucumber da mping-off fungi from compost---the growth of ultimate corruption mould (Pythium ultimum) has strong restraining effect.Zhang Hailongs etc. (2004) have been found 3 new cyclic lipopeptide compound Mixirin A-C from bacillus marinus, they have cytotoxicity.Ashish etc. (2006) find that cellulase that bacillus marinus produces is better active when pH 6 and 50 ℃.Noureddin etc. (2006) find that bacillus marinus can malaga sugar-6-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase.Tian Li etc. (2007) have reported 3 new macrolides compound Macrolactin O, P and Q that a strain bacillus marinus B.marinus B-9987 produces, discover that these 3 new compounds have a good restraining activity to interlinkage spore and rice blast are mould.Find that in addition the bacillus marinus B.marinus B-9987 fermentation concentrated solution that contains such material can effectively suppress the growth of phytopathogen.Bacillus marinus very is suitable for being developed to microbial pesticide because of it has that bacteriostatic action is strong, strong stress resistance and be easy to be processed into many advantages such as formulation.The 1000000000 CFU/g sea bacillus wettable powders that contriver place seminar successfully researches and develops are as safe as a house as agricultural chemicals, belong to the micro-virus kind agricultural chemicals, preliminary potted plant and field plot trial result shows that this wettable powder has better prevention effect (Chinese patent application publication number: CN101331881) to gray mold, Powdery Mildew, bacterial wilt, soft rot, early blight, oidium.
Lipopeptid class material is mainly derived from the secondary metabolite of microorganism, is made up of hydrophilic peptide bond and lipophilic fat hydrocarbon chain two portions, is divided into linear lipopeptid and ring-type lipopeptid two big classes.Because its special amphiphile, amphiphilic molecule structure has brought up the microorganism lipopeptid and has had good surfactivity, in fields such as medicine, food, makeup, biological control, environmental improvement and microbe oil productions important application prospects is arranged.
The cyclic lipopeptide compound majority has anti-microbial activity, wherein successful example is a daptomycin, it is that first is applied to clinical cyclic lipopeptide microbiotic, be used for the treatment of by concurrency skin and skin texture due to the gram positive organism infection and infect, comprise that methicillin resistance bacterium gold Portugal bacterium reaches the enterococcus faecalis to the vancomycin resistance, mechanism of action uniqueness.
Subtilis (Bacillus subtilis) is the important rhizospheric microorganism of a class, can promote infecting of plant opposing pathogenic bacteria, thereby be widely used in the biological control of Plant diseases.B.subtilis can produce the microbiotic of tens kinds of different structures, and wherein non-ribosomal synthetic lipopeptide antibiotic is a wherein modal class, mainly comprises iturin, a surfactin and fengycin3 family.Lipopeptide antibiotic is made up of amino acid chain and fatty acid side chain, and good stability is free from environmental pollution to the person poultry harmless, is to have the new bio source pesticide that important exploitation is worth.There is report iturin to be successfully applied to the example (Klich MA, 1994) of biological control abroad.
Catherine etc. (calendar year 2001) discovery lipopeptid type biological surfactant energy selectivity is removed the metal ions such as Pb, Zn, Cu and Cd in the soil, wherein Cu
2+The easiest removal.
Huang Xianqing etc. (2006) discover, the antimicrobial lipopeptid that subtilis fmbJ strain produces can directly act on external anti-pseudorabies virus (Pseudorabies viru) and pig parvoviral (Porcine parvoviru) Nanjing strain, thereby suppresses its infection effect to pig kidney (Porcine Kidney) cell.
Kim etc. (2007) discover that cyclic lipopeptide Surfactin (MW 1036) can suppress the propagation of Lovo cell, cell death inducing, and its mechanism of action may be for suppressing cells survival conditioning signal path ERK and PI3K/Akt.
Wang Dawei etc. (2008) discover, the lipopeptid type biological surfactant that a bacillus subtilis (Bacillussubtilis) ZW-3 that is separated to from Daqing oil field produces has good emulsification and reduces the ability of oil water interfacial tension, and can adapt to complex environment in the oil reservoir, can improve recovery ratio 9.2%, in microbe oil production, have extraordinary application prospect.
Xue Chunmei etc. (2008) report separates from bacillus marinus and has obtained Macrolactin T and U and known structure Macrolactin A, B, D, O, S, and wherein Macrolactin T, B, D have antagonistic action to Pyricularia oryzae, tomato early blight bacterium and streptococcus aureus.
Summary of the invention
The present inventor is through discovering, 1 the new cyclic lipopeptide compound Marihysin A that is produced by bacillus marinus (B.marinus) B-9987 all has the good restraining activity to eggplant interlinkage spore (Alternaria solani) and Fusarium oxysporum phytopathogens such as (Fusarium oxysporum f.sp.cuberse), therefore can prevent and treat by the microbial Plant diseases of these cause of diseases with used as pesticides.
The object of the present invention is to provide of deriving from that bacillus marinus (B.marinus) B-9987 produces new have cyclic lipopeptide compound of using value and its production and application.Specifically, first aspect present invention provides has new texture cyclic lipopeptide compound Marihysin A, and described compound structure is as shown below:
When being used for when of the present invention, the implication of term " compound of the present invention " or similar terms has not only comprised the defined compound of said structure formula, has also comprised their salt.Described salt can be form water-soluble, that liposoluble maybe can disperse product, and it can pass through and mineral acid or organic acid or alkali reaction formation.The example of these acid salt comprises acetate, adipate, alginate, benzoate, benzene sulfonate, hydrosulfate, butyrates, Citrate trianion, dodecane sulfonate, hydrochloride, oxalate, propionic salt, succinate, tartrate.Basic salt comprises ammonium salt, an alkali metal salt, and as sodium salt and sylvite, alkaline earth salt, as calcium salt and magnesium salts, the salt of organic bases is as dicyclohexyl amine salt etc.
Compound of the present invention also can its tautomeric form exist.Although do not spell out these form in the compound of describing here, they are also contained within the scope of the present invention.Except as otherwise noted, the chemical name of compound described here comprises the mixture of the stereochemistry heterogeneous forms that all possible, described compound can comprise.Described mixture can contain all diastereomers and/or the enantiomer of described compound basis molecular structure.The all stereochemistry heterogeneous forms of The compounds of this invention can be purified forms, or mutual blended form, and this is included within the scope of the invention.
Second aspect present invention provides a kind of method for preparing above-claimed cpd, it is characterized in that, this method may further comprise the steps:
(1) cultivates bacillus marinus (Bacillus marinus) B-9987, obtain fermented liquid;
(2) from described fermented liquid, separate the described compound of acquisition with one or more methods that are selected from organic solvent extraction, Acid precipitation, lixiviate or chromatography.
The related bacterial strain of the inventive method is bacillus marinus (B.marinus) B-9987, this bacterial strain separates from the plant Suaeda salsa of tideland, the Bohai Sea and obtains, this bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) (No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City Institute of Microorganism, Academia Sinica) on June 18th, 2007, preserving number is CGMCC No.2095, and is disclosed among the Chinese patent application CN 101333206A.
Term used herein " fermentation " or " cultivation " have the implication that those skilled in the art know usually and admit.Described " fermented liquid " or " nutrient solution " can make it grow to certain bacterial concentration and obtain by cultivate bacillus marinus B.marinusB-9987 of the present invention under the condition that is fit to growth.
The nutrition source that is used for cultivating the substratum of bacterial strain of the present invention has no particular limits.Those skilled in the art can select suitable carbon source, nitrogenous source and other nutrition source according to technique known.For example, carbon source can be starch, dextrin, glycerine, glucose, sucrose, inositol, N.F,USP MANNITOL etc.Nitrogenous source can be peptone, soyflour, protein powder, meat extract, rice sugar, wheat skin, yeast powder, corn steep liquor, ammonium salt and other organic or inorganic nitrogenous compound.In addition, also can suitably add some inorganic salts in the substratum, as metal-salts such as sodium-chlor, phosphoric acid salt (as dipotassium hydrogen phosphate and potassium primary phosphate etc.), ammonium sulfate, manganous sulfate, sal epsom, lime carbonate.Usually can adopt various known conventional substratum, as LB nutrient agar, nutrient agar, glucose yeast cream nutrient agar and ox meat extract nutrient agar etc.In a specific embodiments, the substratum that is used to cultivate bacterial strain of the present invention has following composition (% represents mass/volume): glucose 0.1%～1%, sucrose 0.1%～2%, yeast powder 0.01%～1.5%, protein powder 0.1%～1%, MgCl2 0.001%～0.1%, KCl 0.01%～0.5%, KH2PO4 0.01%～0.5% and NaCl 0.1%～6%, pH6.0～7.0.Yet, it will be appreciated by those skilled in the art that these concrete culture medium prescriptions that the present invention is not limited to enumerate herein.
Conditions such as the temperature of the bacterial strain among cultivation the present invention, pH, vapour-liquid ratio, tank pressure, rotating speed do not have strict especially restriction, as long as this condition is fit to the growth of this bacterium.
When cultivating, can adopt defoamers such as soya-bean oil, bubble enemy to carry out froth breaking.In some preferable embodiments, pH should be controlled between 5.5～8.0, and culture temperature should be between 20～35 ℃.Between 12h～200h, final bacteria concentration usually can be up to 5 * 10 usually for incubation time
7Cfu/ml～1 * 10
11Cfu/ml.
In a preferable embodiment of the present invention, cultivate described bacillus marinus by the following method and obtain fermented liquid: containing carbon source, nitrogenous source, in the substratum of inorganic salt, being controlled at vapour-liquid ratio at air flow is 0.2: 1～2: 1, rotating speed 100r/min or more than, cultivate described bacillus marinus more than 24 hours, wherein said carbon source is selected from glucose, sucrose, in starch and the rice meal a kind or several, described nitrogenous source is selected from protein powder, yeast powder, in groundnut meal and the soyflour a kind or several, described inorganic salt comprise that conventional inorganic salt are (as MgSO
4, (NH
4)
2SO
4, MgCl
2, KCl, KH
2PO
4, NaCl, K
2HPO
4And CaCO
3).
Above-mentioned these parameters of enumerating just realize preferred version of the present invention.Therefore, those skilled in the art select suitable culture condition also can obtain fermented liquid of the present invention beyond above-mentioned scope.
After having obtained above-mentioned fermented liquid, from fermented liquid, separate the acquisition target compound with one or more methods that are selected from organic solvent extraction, Acid precipitation, lixiviate or chromatography.Compound of the present invention can separate from the fermentation clear liquid of fermented liquid and obtains, and also can the thalline from fermented liquid separates to obtain.In preferred embodiments, also can be respectively from fermentation clear liquid (also deserving to be called clear liquid or fermented supernatant fluid) and thalline, separate crude product or the vat liquor that obtains containing compound of the present invention, carry out further separation and purification after the mixing again.
Therefore, a preferred version as the inventive method, at first separate and obtain supernatant liquor from described fermented liquid, with the solvent extraction that is selected from ethyl acetate, chloroform, N-BUTYL ACETATE or propyl carbinol, use column chromatography with the chloroform/methanol gradient elution then, the collection chloroform/methanol is 2: 1 to 0: 1 wash-out position, prepares compound of the present invention with HPLC.
Come separation of supernatant can adopt conventional separation means such as centrifuging etc. from fermented liquid.Then, after obtaining supernatant liquor, a kind of feasible mode is to extract with organic solvent (for example, ethyl acetate, chloroform, N-BUTYL ACETATE or propyl carbinol).Preferred scheme is to adopt the combination of organic solvent to carry out described extraction, for example, adopts ethyl acetate and n-butanol extraction successively; Adopt chloroform, ethyl acetate and propyl carbinol to extract successively; Adopt that successively N-BUTYL ACETATE and propyl carbinol extract etc.
Another feasible scheme is, obtains supernatant liquor as mentioned above earlier, makes the supernatant liquor acid precipitation then, with anhydrous methanol to the acid precipitation lixiviate.Specifically, in supernatant liquor, add acid (example hydrochloric acid), make solution left standstill, centrifugal then acid precipitation and the acidifying supernatant liquor of obtaining.In the application's a scheme, adopt the 5N hydrochloric acid soln to carry out acidifying.Those skilled in the art also can suitably adjust concentration of hydrochloric acid can obtain acid precipitation equally.Subsequently, the pH of acid precipitation is transferred to neutral postlyophilization, with anhydrous methanol lixiviate one or many, each extraction time is 6-12 hour.
Also having a kind of feasible mode is to adopt such as conventional meanses such as centrifugations to obtain thalline from the fermented liquid separation.Then, with the described thalline one or many of anhydrous methanol lixiviate, each extraction time 6-12 hour.
Subsequently, carry out n-butanol extraction reactive site or the anhydrous methanol vat liquor that chloroform/methanol gradient elution abovementioned steps obtains with column chromatography, the collection chloroform/methanol is 2: 1 to 0: 1 wash-out position (preferred 1: 1 wash-out position of chloroform/methanol).Used column chromatography for example can be a silica gel column chromatography.At last, prepare described compound with Sephadex LH-20 and/or HPLC (analysis and preparation condition are 70% methanol aqueous solution) etc.
Yet, it will be appreciated by those skilled in the art that can adopt other separation means, solvent or eluent to separate according to routine techniques fully obtains compound of the present invention.Therefore, the scope of the inventive method is not limited to concrete grammar used among the specification sheets embodiment.
The present invention provides a kind of pesticide composition on the other hand, it is characterized in that, described composition comprises compound of the present invention as active ingredient, and acceptable carrier on the Pesticide Science.Pesticide composition of the present invention can be used for suppressing to be selected from tomato early blight bacterium (A.solani), muskmelon fruit rot bacterium (F.oxysporum), verticillium dahliae (V.alboatrum), fusarium graminearum (F.graminearum), bighead atractylodes rhizome southern blight bacterium (Sclerotium sp.), mould germ (Penicillium sp.), botrytis cinerea pers (B.cinerea), botrytis cinerea (B.cinerea), southern corn leaf blight (D.turcica), Rhizoctonia solani Kuhn (R.solani), melon anthrax bacteria (Colletotrichum sp.), the phytopathogen of banana blight bacteria (F.oxysporum f.sp.cuberse).
Compound of the present invention or Pestcidal compositions can directly be used with the fermented liquid form, also it suitably can be diluted (for example diluting 10 times, 100 times, 1000 times or higher) and use with the diluent form.Compound of the present invention or its composition can be applied on the positions such as the root, leaf, stem of plant.Applying method is the routine techniques in this area, for example can be to soak seed at seeding time, before transplanting plant root is immersed in fermented liquid or its diluent, perhaps directly with fermented liquid or diluent is bold and vigorous waters on the seedbed.But irritate root during field planting, also can in growing process, irritate root.If described pesticide composition is preserved with carrier format, then can after facing, apply again with preceding dilute with water.As for the suitableeest dispenser dosage of the present invention, those skilled in the art need not can determine through too much test.
Embodiment
Embodiment 1 utilizes acid precipitation method to separate from bacillus marinus (Bacillus marinus) B-9987 fermented liquid and obtains new cyclic lipopeptide compound Marihysin A
Present embodiment has obtained new cyclic lipopeptide compound Marihysin A by separation means from the fermented liquid of bacillus marinus B.marinus B-9987.Concrete purification procedures is as follows:
(1) bacillus marinus (B.marinus) B-9987 is cultivated under appropriate condition obtains the 30L fermented liquid.
Bacillus marinus B-9987 bacterial classification as first order seed, is inoculated in secondary seed with this first order seed and shakes bottle after being inoculated in first order seed shake-flask culture 12-24h after the flat board activation, is inoculated in the 50L fermentor tank behind the cultivation 12-24h.Under 28 ℃, 300rpm, cultivate about 20h and can finish.
(2) 30L fermented liquid 4000rpm obtains fermented supernatant fluid and wet thallus after centrifugal.Fermented supernatant fluid add the acidifying of 5N hydrochloric acid soln and staticly settle the back centrifugal (4000rpm) obtain acid precipitation and acidifying supernatant liquor.Acid precipitation pH is transferred to 7.0 postlyophilizations, obtain freeze-drying precipitation 174.8g, with anhydrous methanol (440mL, 220mL, 220mL) lixiviate 3 times successively, each extraction time 12 hours, vat liquor merged be evaporated to driedly, obtain acid precipitation lixiviate crude product 10.2g altogether.With acid precipitation lixiviate crude product through silica gel column chromatography with 10: 1 → 0: 1 gradient elution (500mL/ flow point) of chloroform/methanol, flow point to wash-out carries out biological activity assay, indicator is eggplant interlinkage spore (Alternaria solani), the result shows that the bacteriostatic activity material mainly concentrates on chloroform/methanol 1: 1 and methanol-eluted fractions position, and stronger with 1: 1 wash-out position of chloroform/methanol activity, above-mentioned active flow point after detecting, TLC is merged.With the active flow point after merging through Sephadex LH-20 column chromatography with methanol-eluted fractions, the wash-out flow point is carried out biological activity assay, indicator is eggplant interlinkage spore (Alternaria solani), the active flow point behind the biological activity assay detects the back merging through TLC.Active flow point after merging is analyzed and preparation (analysis and preparation condition are 70% methanol aqueous solution) through HPLC, finally prepared compound Marihysin A.
Embodiment 2 utilizes the organic solvent extraction method to separate from bacillus marinus (Bacillus marinus) B-9987 fermented liquid and obtains new cyclic lipopeptide compound Marihysin A
Present embodiment has obtained new cyclic lipopeptide compound Marihysin A by the separation means of routine from the fermented liquid of bacillus marinus B.marinus B-9987.Concrete purification procedures is as follows:
(1) bacillus marinus (B.marinus) B-9987 is cultivated under appropriate condition obtains 30L fermented liquid (concrete culture condition and step are with embodiment 1).
(2) 30L fermented liquid 4000rpm obtains fermented supernatant fluid and wet thallus after centrifugal.Wet thallus obtains dry mycelium 92.0g through the centrifugal postlyophilization of washing, with anhydrous methanol (600mL, 300mL, 300mL) lixiviate 3 times successively, each extraction time 12 hours merges vat liquor and is evaporated to the dried thalline lixiviate crude product 17.3g altogether that obtains.With thalline lixiviate crude product through silica gel column chromatography with 10: 1 → 0: 1 gradient elution (500mL/ flow point) of chloroform/methanol, flow point to wash-out carries out biological activity assay, indicator is eggplant interlinkage spore (Alternaria solani), the result shows that the bacteriostatic activity material mainly concentrates on chloroform/methanol 1: 1 and methanol-eluted fractions position, and stronger with 1: 1 wash-out position of chloroform/methanol activity, above-mentioned active flow point after detecting, TLC is merged.With the active flow point after merging through Sephadex LH-20 column chromatography with methanol-eluted fractions, the wash-out flow point is carried out biological activity assay, indicator is an eggplant interlinkage spore (Alternariasolani), the active flow point behind the biological activity assay detects the back merging through TLC.Active flow point after merging is analyzed and preparation (analysis and preparation condition are 70% methanol aqueous solution) through HPLC, finally prepared compound Marihysin A.
Embodiment 3 organic solvent extractionprocesses separate from bacillus marinus (Bacillus marinus) B-9987 fermented liquid and obtain new cyclic lipopeptide compound Marihysin A
Present embodiment has obtained new cyclic lipopeptide compound Marihysin A by the separation means of routine from the fermented liquid of bacillus marinus B.marinus B-9987.Concrete purification procedures is as follows:
(1) bacillus marinus (B.marinus) B-9987 is cultivated under appropriate condition obtains the 10L fermented liquid.Bacillus marinus B-9987 bacterial classification as first order seed, is inoculated in secondary seed with this first order seed and shakes bottle after being inoculated in first order seed shake-flask culture 12-24h after the flat board activation, is inoculated in the 5L fermentor tank behind the cultivation 12-24h.Under 28 ℃, 300rpm, cultivate about 20h and can finish.3 the 5L jars that ferment altogether obtain the 10L fermented liquid.
(2) 10L fermented liquid 4000rpm obtains fermented supernatant fluid and wet thallus after centrifugal.Fermented supernatant fluid extracts respectively 3 times with the equal-volume ethyl acetate, extracts 12 hours time at every turn, ethyl acetate extraction is merged mutually be evaporated to the original fermented solution volume.With the equal-volume propyl carbinol ethyl acetate extracting phase is extracted respectively 3 times again, extract 12 hours time at every turn, n-butanol extraction is merged mutually be evaporated to the original fermented solution volume.Above-mentioned ethyl acetate extraction is carried out biological activity assay mutually with n-butanol extraction mutually, and indicator is eggplant interlinkage spore (Alternaria solani), determines that n-butanol portion is main bacteriostatic activity position.N-butanol extraction merged mutually be evaporated to driedly, obtain altogether 13.8g of n-butyl alcohol extract crude product.With the n-butyl alcohol extract crude product through silica gel column chromatography with 10: 1 → 0: 1 gradient elution (500mL/ flow point) of chloroform/methanol, flow point to wash-out carries out biological activity assay, indicator is an eggplant interlinkage spore (Alternariasolani), the result shows that the bacteriostatic activity material mainly concentrates on chloroform/methanol 1: 1 and methanol-eluted fractions position, and stronger with 1: 1 wash-out position of chloroform/methanol activity, above-mentioned active flow point after detecting, TLC is merged.With the active flow point after merging through Sephadex LH-20 column chromatography with methanol-eluted fractions, the wash-out flow point is carried out biological activity assay, indicator is eggplant interlinkage spore (Alternaria solani), the active flow point behind the biological activity assay detects the back merging through TLC.Active flow point after merging is analyzed and preparation (analysis and preparation condition are 70% methanol aqueous solution) through HPLC, finally prepared compound Marihysin A.
The structure of embodiment 4 new cyclic lipopeptide compound Marihysin A is identified and is analyzed
(1) Marihysin A structure is identified and is analyzed
The new cyclic lipopeptide compound Marihysin A that embodiment 1,2 or 3 is obtained analyze and identify that its result is as follows:
Marihysin A chemical structure is as follows
It is the colorless solid powder, under the UV254nm blackening is arranged, and is yellow under the iodo steam displaing color.mp＞250℃；IR(KBr)ν
max(cm
-1)：3333，2929，2864，1659，1545，1449，1423，1247，1128。HRFAB-MS?m/z1065.6308[M+Na]
+(calcd?for?C
48H
74N
12O
14)。
1H,
13C NMR data,
1H-
1H COSY and HMBC see Table 1.
The structure appraising datum table (DMSO-d of table 1 compound Marihysin A
6)
Embodiment 5Marihysin A is to the restraining effect of phytopathogen
Present embodiment has been investigated the restraining effect of the compound Marihysin A of embodiment 1 acquisition to phytopathogen.
Adopt dull and stereotyped antagonism method to investigate Marihysin A to tomato early blight bacterium (A.solani), muskmelon fruit rot bacterium (F.oxysporum), verticillium dahliae (V.alboatrum), fusarium graminearum (F.graminearum), bighead atractylodes rhizome southern blight bacterium (Sclerotium sp.), mould germ (Penicillium sp.), botrytis cinerea pers (B.cinerea), botrytis cinerea (B.cinerea), southern corn leaf blight (D.turcica), Rhizoctonia solani Kuhn (R.solani), melon anthrax bacteria (Colletotrichumsp.), the restraining effect of banana blight bacteria phytopathogens such as (F.oxysporum f.sp.cuberse).
Marihysin A is to the MIC such as the following table 2 of phytopathogen.
Table 2Marihysin A is to the restraining effect of phytopathogen
Marihysin A has stronger bacteriostatic activity to bighead atractylodes rhizome southern blight bacterium (Sclerotium sp.), muskmelon fruit rot bacterium (F.oxysporum), mould germ (Penicillium sp.) and melon anthrax bacteria phytopathogens such as (Colletotrichum sp.) as can be seen from Table 2.
Embodiment 6 contains the inhibition activity of bacillus marinus (Bacillus marinus) the B-9987 fermented liquid of Marihysin A to phytopathogen
Present embodiment has been investigated the growth that bacillus marinus (B.marinus) the B-9987 fermented liquid that contains compound Marihysin A of the present invention can effectively suppress phytopathogen.
Present embodiment adopts dull and stereotyped antagonism method investigation to contain the restraining effect of bacillus marinus (B.marinus) the B-9987 fermented liquid of Marihysin A to phytopathogen.
The mensuration of pathogenic fungi antimicrobial spectrum: will in 4 ℃ of inclined-planes, preserve phytopathogenic fungi be connected to an about week of PDA flat board activation culture under 28 ℃ condition, treat to finish to cultivate after fungi is covered with whole flat board, the punch tool that with sterilized diameter is 14.50mm is bought a bacterium cake in activatory pathogenic fungi bacterium colony outer rim, with the bacterium cake face up be positioned over the face-off culture plate a side, near the about 1cm of ware wall, get 1 ring with inoculating needle and cultivate the B-9987 fermented liquid that obtains, in the face-off flat board, be parallel to standardized bacterium line of bacterium piece, the line distance is decided on the speed of growth of pathogenic fungi, and the culture plate that will stand facing each other writes down antibacterial bandwidth after putting into 28 ℃ of cultivation 5d.
The mensuration of pathogenetic bacteria antimicrobial spectrum: the pathogenic bacteria liquid that will cultivate 24h is sneaked in the corresponding semisolid medium and (to be made and to contain the bacterium flat board and reach 10
9Cfu/mL), be poured on then on the water agar plate for preparing, cool off the back and beat the hole that diameter is 14.00mm, then the B-9987 fermented liquid is added in the hand-hole, every hole 150 μ L, record inhibition zone size behind 30 ℃ of cultivation 24h in semi-solid dull and stereotyped central authorities.
Its result is as shown in table 3 below.
Table 3 bacillus marinus B-9987 fermented liquid is to the restraining effect of phytopathogen
| Pathogenic bacteria | Antibacterial band (circle)/mm |
| Southern corn leaf blight (C.lunata) | 11.00 |
| Botrytis cinerea pers (B.cinerea) | 12.00 |
| Melon anthrax bacteria (Colletotrichum sp.) | 6.50 |
| Tomato early blight bacterium (Alternaria sp.) | 6.40 |
| Tobacco brown spot pathogen (A.alternate) | 6.30 |
| Pyricularia oryzae (P.grisea) | 5.67 |
| Banana blight bacteria (F.oxysporum f.sp.cuberse) | 5.60 |
| Muskmelon wilt (F.oxysporum) | 3.10 |
| Fusarium graminearum (F.graminearum) | 3.06 |
| Bacterial wilt of tomato bacterium (R.solanacearum) | 23.0 |
| Soft rot of cabbage bacterium (E.carotovora var.carotovora) | 18.8 |
By the inhibition activity of table 3 as can be known, bacillus marinus (B.marinus) the B-9987 fermented liquid that contains compound Marihysin A can suppress the growth of phytopathogen effectively.
Although object lesson of the present invention described above, having a bit is significantly to those skilled in the art, promptly can do various variations and change to the present invention under the premise without departing from the spirit and scope of the present invention.Therefore, claims have covered all these changes within the scope of the present invention.
Claims (10)
1. lipopeptide compound, described compound has following structure:
2. a method for preparing the described compound of claim 1 is characterized in that, this method may further comprise the steps:
(1) cultivates bacillus marinus (Bacillus marinus) B-9987, obtain fermented liquid;
(2) from described fermented liquid, separate the described compound of acquisition with one or more methods that are selected from organic solvent extraction, Acid precipitation, lixiviate or chromatography.
3. method as claimed in claim 2 is characterized in that, described step (2) comprises following one or more step:
(a) obtain supernatant liquor from described fermented liquid separation, with the solvent extraction that is selected from ethyl acetate, chloroform, N-BUTYL ACETATE or propyl carbinol, use column chromatography with the chloroform/methanol gradient elution then, the collection chloroform/methanol is 2: 1 to 0: 1 wash-out position, prepares the described compound of claim 1 with Sephadex LH-20 and/or HPLC;
(b) obtain supernatant liquor from described fermented liquid separation, make the supernatant liquor acid precipitation, with anhydrous methanol to the acid precipitation lixiviate, vat liquor uses column chromatography with the chloroform/methanol gradient elution, the collection chloroform/methanol is 2: 1 to 0: 1 wash-out position, prepares the described compound of claim 1 with SephadexLH-20 and/or HPLC;
(c) obtain thalline from described fermented liquid separation, use the anhydrous methanol lixiviate, with the chloroform/methanol gradient elution, the collection chloroform/methanol is 2: 1 to 0: 1 wash-out position to vat liquor, prepares the described compound of claim 1 with Sephadex LH-20 and/or HPLC with column chromatography.
4. method as claimed in claim 3 is characterized in that described column chromatography is a silica gel column chromatography.
5. method as claimed in claim 3 is characterized in that the preparation condition of described HPLC is 70% methanol solution.
6. a pesticide composition is characterized in that, described composition comprises the described compound of claim 1 as active ingredient, and acceptable carrier on the Pesticide Science.
7. the described compound of claim 1 is as the application in the agricultural chemicals.
8. application as claimed in claim 7, it is characterized in that described agricultural chemicals is used for suppressing to be selected from tomato early blight bacterium (A.solani), muskmelon fruit rot bacterium (F.oxysporum), verticillium dahliae (V.alboatrum), fusarium graminearum (F.graminearum), bighead atractylodes rhizome southern blight bacterium (Sclerotium sp.), mould germ (Penicillium sp.), botrytis cinerea pers (B.cinerea), botrytis cinerea (B.cinerea), southern corn leaf blight (D.turcica), Rhizoctonia solani Kuhn (R.solani), melon anthrax bacteria (Colletotrichum sp.), the phytopathogen of banana blight bacteria (F.oxysporum f.sp.cuberse).
9. the method for a controlling plant diseases is characterized in that, described compound of claim 1 or the described pesticide composition of claim 6 are applied on the described plant.
10. method as claimed in claim 9, it is characterized in that the pathogenic bacteria of described Plant diseases is selected from tomato early blight bacterium (A.solani), muskmelon fruit rot bacterium (F.oxysporum), verticillium dahliae (V.alboatrum), fusarium graminearum (F.graminearum), bighead atractylodes rhizome southern blight bacterium (Sclerotium sp.), mould germ (Penicillium sp.), botrytis cinerea pers (B.cinerea), botrytis cinerea (B.cinerea), southern corn leaf blight (D.turcica), Rhizoctonia solani Kuhn (R.solani), melon anthrax bacteria (Colletotrichum sp.), banana blight bacteria (F.oxysporum f.sp.cuberse), bacterial wilt of tomato bacterium (R.solanacearum), soft rot of cabbage bacterium (E.carotovora var.carotovora).
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| CN101838314A (en) * | 2009-11-16 | 2010-09-22 | 华东理工大学 | Preparation of cyclic lipopeptide compound and application thereof |
| CN102845475A (en) * | 2012-09-11 | 2013-01-02 | 倪烈 | Compound pesticide used for controlling paddy rice sheath blight |
| CN103627643A (en) * | 2013-12-16 | 2014-03-12 | 中国农业科学院棉花研究所 | Cotton endophytic fungus CEF-818 and application thereof in control of cotton verticillium wilt |
| CN103687589A (en) * | 2011-05-26 | 2014-03-26 | 丘比斯特药物股份有限公司 | Cb-183,315 compositions and related methods |
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| CN101838314B (en) * | 2009-11-16 | 2014-11-05 | 华东理工大学 | Preparation of cyclic lipopeptide compound and application thereof |
| CN103687589A (en) * | 2011-05-26 | 2014-03-26 | 丘比斯特药物股份有限公司 | Cb-183,315 compositions and related methods |
| CN102845475A (en) * | 2012-09-11 | 2013-01-02 | 倪烈 | Compound pesticide used for controlling paddy rice sheath blight |
| CN102845475B (en) * | 2012-09-11 | 2014-09-17 | 倪烈 | Compound pesticide used for controlling paddy rice sheath blight |
| CN103627643B (en) * | 2013-12-16 | 2015-11-11 | 中国农业科学院棉花研究所 | Cotton endogenetic fungus CEF-818 and the application in cotton verticillium wilt control thereof |
| CN103627643A (en) * | 2013-12-16 | 2014-03-12 | 中国农业科学院棉花研究所 | Cotton endophytic fungus CEF-818 and application thereof in control of cotton verticillium wilt |
| CN103952347B (en) * | 2014-04-22 | 2016-01-06 | 四川大学 | One strain moderate salt tolerant bacillus marinus and application thereof |
| CN103952347A (en) * | 2014-04-22 | 2014-07-30 | 四川大学 | Moderate salt-resisting bacillus marinus and application thereof |
| CN105753936A (en) * | 2016-03-28 | 2016-07-13 | 福建省微生物研究所 | Rakicidins compounds Rakicidin B1 and preparation method thereof |
| CN105753936B (en) * | 2016-03-28 | 2019-01-11 | 福建省微生物研究所 | A kind of Rakicidins class compound R akicidin B1 and preparation method thereof |
| CN108271776A (en) * | 2017-12-25 | 2018-07-13 | 武汉科诺生物科技股份有限公司 | Bacillus lipopeptid class metabolite is used as the purposes of microbial bacteria agent aid |
| CN108271776B (en) * | 2017-12-25 | 2021-06-11 | 武汉科诺生物科技股份有限公司 | Use of bacillus lipopeptide metabolite as microbial agent aid |
| CN110452848A (en) * | 2019-08-20 | 2019-11-15 | 昆明理工大学 | One plant of Bei Laisi bacillus and its application |
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| CN111349146A (en) * | 2020-01-20 | 2020-06-30 | 复旦大学 | Cyclic lipopeptide derivative and preparation and application thereof |
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