CN102181499B - New method for preparing hydroxamate siderophore - Google Patents
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Abstract
The invention relates to a new method for preparing siderophore. The method comprises the following steps of: preparing fermentation liquid; extracting an organic solvent; and separating by the conventional column chromatography and high performance liquid chromatography to obtain the siderophore. The siderophore has a certain effect of preventing and controlling agricultural diseases, so that the siderophore can be used for preparing a novel biological pesticide.
Description
Technical field
The present invention relates to a kind of new iron element of having a liking for, be specifically related to a kind of preparation method of hydroxamate siderophore and the application in cucumber fusarium axysporum thereof.
Background technology
Utilizing probiotics to carry out the prevention and control of plant diseases, pest control, has been a hot issue of studying at present, and existing multiple bacterial strain successfully is developed to product and is promoted so far.Their critical roles in the prevention and control of plant diseases, pest control are from their some intrinsic attributes.False pseudomonas bacillus (Peudomonas spp.) is the biocontrol bacteria that a class obtains broad research, this bacterioid can produce particular matter and have a liking for the iron element, and a kind of main mechanism that the iron element is its control fungal disease is had a liking in secretion, thereby becomes the focus object of researcher research.Have a liking for the iron element and be small molecule amount (500～1000Da) chelating Fe specifically
3+Chelating agent, most bacteriums and fungi can both one or more have a liking for the iron element by non-ribose approach synthesis secretion, its generation is the result that microorganism adapts to the low iron hoop border of earth's crust hyperoxia on the earth.Have a liking for the iron element and can be divided into three major types according to the chemical property of its chelation group: a class be hydroxamic acid class (hydroxamate) to have a liking for iron element, a class be that pyrocatechol (catechol) has a liking for the iron element, also has a class is that mixed type (mixed ligand) is had a liking for the iron element, the report that has is called alpha-hydroxy carboxylic acid compounds (α-hydroxycarboxylic) (be also referred to as the lemon acid type).About having a liking for the research of iron element, still be at present the basic research stage both at home and abroad.It is purified and the research starting of structural analysis aspect morning, and domestic rarely seen report, up to now, it has been found that 500 number of chemical structures different have a liking for the iron element.At present, the research of having a liking for the iron element is mainly concentrated on the biological and ecological methods to prevent plant disease, pests, and erosion activity that it participates in both at home and abroad.For example, Xu Yuquan (1999) has verified that to biocontrol microorganisms Peudomonas sp.JKD-2 suppresses the growth of rice blast fungus under low iron bar spare research having a liking for the iron plain sheet competes this disease resistance function.Mention the antibiosis of having a liking for the iron element in Chen Shuanya (2003) and Chen Xuan (2006) research, namely have a liking for the iron element and directly play control disease purpose by suppressing pathogenic bacteria.Marla Guineth (2000) also mentions the antibiosis of having a liking for the iron element.Ran Longxian (2005) has a liking for the iron element to the induction of resistance effect of disease by the research of having a liking for iron element deletion mutant control eucalyptus gray mold has been mentioned.Van peer R (1992), Leeman M (1996) and V.Ramamoorthy (2000) also mention the inducing action of having a liking for the iron element.In addition, having a liking for the iron element also explores in recent years and is applied to medicine and other fields
[18]In a word, the function of having a liking for the iron element is various, and it does not still rely on the iron transfer system of the high-affinity of acceptor, or somatomedin or the sprouting factor, also can be microbiotic or virulence factor.This shows that having a liking for the iron element is a kind of novel bioactive material that has very much development and utilization to be worth.
Summary of the invention
The objective of the invention is from pseudomonas putida HZ-2 bacterial strain (Pseudomonas putida) (bacterial strain HZ-2, being the inventor in March, 2008 separates from the Hangzhou, Zhejiang province city for many years other booth in prison, husky Qiaosi under Hangzhou of planting vegetable, belong to pseudomonas putida through evaluation) in separate the preparation method that practical value is had a liking for iron element and this compound that has make new advances, and in controlling plant diseases, particularly prevent and treat the application in the cucumber fusarium axysporum.The bacterial strain of adopting from region differently of pseudomonas putida also can obtain this type of novel substance.
On the one hand, the invention provides a kind of hydroxamic acid type and have a liking for the iron element, have following structure:
Hydroxamic acid type of the present invention is had a liking for the iron element and is separated from plant-growth promoting rhizobacteria pseudomonas putida HZ-2 bacterial strain (Pseudomonas putida) and to obtain, and its structural formula represents (such as figure below) by general formula:
On the other hand, the present invention also provides a kind of new hydroxamic acid type to have a liking for the preparation method of iron element, comprises the steps:
(1), the preparation of fermented liq: bacterial classification pseudomonas putida HZ-2 is behind solid K MB substratum activation 24h, in the picking list bacterium colony access 5mL liquid KMB substratum, place shaking table, 28 ℃, cultivate 24h under the 150r/ minute condition, then be inoculated among the solid seed culture medium KMB, at 28 ℃ of lower 24h that cultivate, obtain first order seed; Then with cultured first order seed picking one ring, in the MKB substratum, ferment, wherein above all use solid or liquid KMB substratum composition under 1000 ml volumes comprises: casamino acids 5g, MgSO
47H
2O 2.5g, K
2HPO
43H
2O 2.5g, Fe
3+Content be 0.1mg/L.
(2), collect that fermented liquid 1000mL rotary evaporation (43 ℃) is condensed into slightly thick liquid in the step (1), centrifugal removal white insolubles, supernatant liquor transfers to pH 4 for subsequent use with HCl;
(3), detecting fluid inspection with CAS after the liquid concentration behind the macroporous adsorptive resins on the supernatant samples that obtains in the step (2), do not show activity, the elutriant of using again opposed polarity is wash-out successively, after elutriant carried out concentrating under reduced pressure, measure activity, wherein the elutriant of the successively wash-out of opposed polarity is: ionized water, 0.2% hydrochloric acid soln, 10% methanol-water solution, 30% methanol-water solution, 50% methanol-water solution, 80% methanol-water solution, 100% methyl alcohol, 100% acetone soln wash-out.
Preferably, the sample of step (3) wash-out is separated with HPLC, the condition of separation is: material ODS-BP; Flow velocity: 1ml/ minute; Moving phase: 30% methanol-water eluant solution 30 minutes.
Condition preferred, that HPLC separates is: material: ODS-BP; Flow velocity: 1ml/ minute; Moving phase: the 5-60% methanol-water carried out gradient elution 30 minutes; Detector: UV210nm;
The separation condition of wherein testing is: material: ODS-BP; Flow velocity: 1ml/ minute; Moving phase: 10% methanol-water; 100% methyl alcohol; 5-60% methanol-water (30 minutes); 5-40% methanol-water (30 minutes); 10-30% methanol-water (60 minutes); 5-15% methanol-water (30 minutes); The 10-40% methanol-water; 10-30% methanol-water (30 minutes); 10-30% methanol-water (30 minutes); Detector: UV210nm.Finally choose suitable condition: material: ODS-BP; Flow velocity: 1ml/ minute; Moving phase: the lower active ingredient 20mg that obtains that separates of 30% methanol-water solution (30 minutes) further separates.
Preferably, also fermented liquid being carried out the CAS test fluid afterwards in step (1) detects.After fermented liquid was concentrated, the CAS that adds equal volume detected liquid, is obviously orange red, proves to be rich in the fermented liquid and has a liking for the iron element.Then carry out Arnow Assay test: analytical results solution displaing yellow therefore preliminary definite fermented liquid may the oxygen-containing nitrolic acid type be had a liking for the iron element.
Preferably, fermention medium will be processed with the 1g8-hydroxyquinoline first and remove Trace Iron in the method in step (1).
Preferably, in the method in step (6), high performance liquid chromatograph is that Dalian Yi Lite P230 type is equipped with the UV-230 UV-vis detector, ODS post model ODS-BP wherein, and size is 200 * 4.6mm, 5 μ m, Dalian Yilite Analytical Instrument Co., Ltd.
Best, the glassware that this test uses all soaks 1 round the clock with concentrated hydrochloric acid solution, then uses washed with de-ionized water.Avoid using iron apparatus.
On the other hand, the invention provides a kind of microbial pesticide reagent, comprise that a kind of hydroxamic acid type has a liking for the iron element, the structure that this hydroxamic acid type is had a liking for the iron element is as follows:
Beneficial effect
The present invention is simple to operate, and resulting new hydroxamic acid type is had a liking for iron element microorganism belonging to genus meta-bolites, and to have toxicity low, easily in advantages such as nature degradeds, can be used as the novel cpd of microbial pesticide exploitation, thereby have a good application prospect.Particularly for Plant diseases, the control agricultural chemicals of cucumber fusarium axysporum for example.
Description of drawings
Fig. 1 is purification procedures figure;
Fig. 2: for be separated in the active ingredient appearance time figure under the preliminary elution requirement with HPLC, wherein material is: ODS-BP; Column length: 200mm; Detector: UV 210nm; Post footpath: 4.6mm; Sample size: 100 μ l, flow velocity: 1ml/ minute; Moving phase: 5-60% methanol-water, gradient elution 30 minutes;
Fig. 3: for be separated in the active ingredient appearance time figure under the preliminary elution requirement with HPLC, wherein material is: material: ODS-BP; Column length: 200mm; Detector: UV 210nm; Post footpath: 4.6mm; Sample size: 100 μ l; Flow velocity: 1ml/ minute; Moving phase: 10% methyl alcohol, gradient elution 30 minutes;
Fig. 4: for be separated in the active ingredient appearance time figure that adjusts under the elution requirement, wherein material with HPLC: ODS-BP column length: 200mm detector: UV 210nm post footpath: 4.6mm sample size: 60 μ l; Flow velocity: 1ml/ minute; Moving phase: 10-40% methyl alcohol, gradient elution 30 minutes;
Fig. 5: for be separated in the active ingredient appearance time figure that adjusts under the elution requirement, wherein material material: ODS-BP with HPLC; Column length: 200mm; Detector: UV 210nm; Post footpath: 4.6mm sample size: 100 μ l; Flow velocity: 1ml/ minute; Moving phase: 5-30% methyl alcohol, gradient elution 30 minutes;
Fig. 6: HPLC separation and purification result's time diagram: material: ODS-BP; Column length: 200mm detector: UV 210nm; Post footpath: 4.6mm; Sample size: 40 μ l; Flow velocity: 1ml/ minute; Moving phase: 5-30% gradient elution 30 minutes.
Embodiment
Following examples are to further specify of the present invention, are not limitations of the present invention.
1. test all appts and equipment
UV:Jasco V550 ultraviolet spectrophotometer
The IR:JascoFT-IR4100 infrared spectrometer
Nuclear-magnetism: BRUCKER AVANCE DMX500 type NMR spectrometer with superconducting magnet
Mass spectrum: ESI-MS:HP5989B type mass spectrograph
APEX III 7.0 fourier transform ion cyclotron resonance mass spectrometers
Satorius BSA124S electronic balance Beijing Sai Duolisi balance company limited
Low-temperature and high-speed whizzer BECKMAN
High performance liquid chromatography Erie is special
Solvent: be forever large chemical reagent company limited of commercially available AR level Tianjin
Column chromatography: academy of agricultural sciences, LS610 macroporous adsorbent resin Zhejiang Province
ODS reverse phase silica gel Beijing intelligent De Yi Science and Technology Ltd.
Reversed phase high efficiency thin layer chromatography board Merk company
Developer: ultraviolet lamp, iodine cylinder, phospho-molybdic acid developer
The glassware that this test uses all soaks 1 round the clock with concentrated hydrochloric acid solution, then uses washed with de-ionized water.Avoid using iron apparatus.
Embodiment 1: have a liking for the preparation of iron element
Specifically referring to Figure of description 1.
(1), fermentation culture.Produce the fermention medium of having a liking for the iron element as richness with the MKB substratum, pseudomonas putida HZ-2 bacterial strain (Pseudomonas putida) is as fermented bacterium, and the fermentation culture Initial pH is transferred to 6.5, in shaking table 28 ℃, cultivate 48h under the 150r/ minute condition, obtain fermentation culture.Being fit to our optimal culture condition of having a liking for the secretion of iron element of used bacterial classification pseudomonas putida HZ-2 bacterial strain (Pseudomonas putida) is: the pH value is 6.5, and inoculum size is 2%, and fermentation time is 48h, and the bottling amount is 50/150mL.Wherein substratum is improved MKB substratum, and it comprises: casamino acids 5g, MgSO
47H
2O 2.5g, K
2HPO
43H
2O 2.5g, Fe
3+Content be 0.1mg/L, the distilled water constant volume is to 1000mL.
Wherein the basal component of MKB substratum commonly used is: casamino acids 5g, MgSO
47H
2O 2.5g, K
2HPO
43H
2O2.5g, glycerine 15mL, the distilled water constant volume is to 1000mL, pH value 7.2, but we find in the present invention, glycerol adding not in the substratum, the amount of having a liking for the iron element of producing is more than glycerol adding, and having a liking for iron element generation can increase by 10%, also adds in addition the Fe of trace in substratum
3+, and make its final dense 0.1mg/L that is, and the iron element is had a liking in easier like this generation, and having a liking for iron element generation increases by 17% (concrete experimental data is slightly).
(2), have a liking for front detection of iron element preparation.Carrying out first the CAS test fluid detects: get the 1mL fermented liquid and be concentrated into 100 μ L, add chrome azurol (CAS) (purchase of the evergreen chemical industry in Hangzhou company limited) and detect liquid 100 μ L, be obviously orange red, prove to be rich in the fermented liquid and have a liking for the iron element.Then carry out the Arnow analytical test: after getting the 1mL fermented liquid and being concentrated into 100 μ L, add successively 100 μ L0.5N hydrochloric acid solns, with 100 μ L Sodium Nitrite-Sodium orthomolybdate mixed solutions, the solution displaing yellow, continue to add the sodium hydroxide solution of 100 μ L 1N, it is yellow that color still is, and therefore preliminary definite fermented liquid may the oxygen-containing nitrolic acid type be had a liking for the iron element.
(3), the separation and purification of fermented liquid.Collect the 1000mL fermention medium, after rotary evaporation (43 ℃) is condensed into slightly thick liquid, centrifugal (10000rpm, 4 ℃, 15 minutes) remove white insolubles, supernatant liquor transfers to pH 4 for subsequent use with HCl, this extract is carried out macroporous adsorptive resins (LS610) chromatography, and (the LS610 macroporous adsorbent resin is used the saturated common salt water soaking in advance, the flushing of 5% hydrochloric acid, soaked overnight, deionized water rinsing pH7, the dehydrated alcohol flushing, deionized water rinsing is to distinguishing the flavor of without ethanol), detect fluid inspection with CAS after the liquid concentration behind the upper macroporous absorption post, do not show activity.With the elutriant of opposed polarity successively wash-out: use successively deionized water, 0.2% hydrochloric acid soln, 10% methanol-water solution, 30% methanol-water solution, 50% methanol-water solution, 80% methanol-water solution, 100% methyl alcohol, 100% acetone soln wash-out.Behind the concentrating under reduced pressure, obtain active ingredient (30% methanol-water solution position) 50mg, detect fluid inspection with CAS again, measure active.
According to the anti-phase plate layer chromatography result of ODS, active ingredient is further opened the ODS post separate, make filler with reverse phase silica gel.Active ingredient (methanol-water solution part) 50mg further separation obtains active ingredient 20mg.
(4), HPLC separates.At last, according to each active ingredient ultraviolet wavelength (200-600nm) length scanning result, HPLC separation case and CAS detected result, determine separation condition, choose active ingredient (30% methanol-water solution position) and separate active ingredient (the 20% methanol-water solution position) 20mg obtain and further separate.Its HPLC initial gross separation result is as shown in Figure 1:
At first, according to ultraviolet wavelength (200-600nm) length scanning result and inverse thin layer chromatography result, carry out wash-out (material: ODS-BP by different methanol-water eluent system; Flow velocity: 1ml/ minute; Moving phase: the 5-60% methanol-water carries out gradient elution (Fig. 2) and 10% methanol-water carries out gradient elution (Fig. 3); Detector: UV210nm), recovery sample is tentatively determined high performance liquid phase active ingredient appearance time, shown in arrow among Fig. 2, Fig. 3.
Secondly, according to the preliminary wash-out situation of high performance liquid phase active ingredient, elutriant methanol-water ratio has been carried out regulating (material: ODS-BP; Flow velocity: 1ml/ minute; Moving phase: the 10-40% methanol-water carries out gradient elution (Fig. 4), and the 5-30% methanol-water carries out gradient elution (Fig. 5); Detector: UV210nm), determined active ingredient high performance liquid phase separation eluent ratio.Active ingredient is more obviously separated with other components as shown by arrows.As shown in Figure 4 and Figure 5.
At last, according to determined best eluent ratio (the 5-30% methanol-water carries out gradient elution), employing analytical column repeatedly sample introduction accumulation recovery separates respectively accumulation to active ingredient, obtains at last pure active ingredient, goes out the peak such as Fig. 6 arrow indication active ingredient.
Embodiment 2: Structural Identification and the attribution data of having a liking for iron element compound
Compound is light yellow solid, Mp:239-240 ℃ (decomposition).The indissoluble solution is soluble in water in methyl alcohol, and the CAS CAS that detects liquid detection display and contrast detects liquid colour-change is arranged, and point sample launches on the RP-18 inverse thin layer chromatography plate, and the colour developing of inverse thin layer chromatography iodine cylinder is yellowish brown.Explanation may be hydroxymates type siderophore.
1The HNMR spectrum provides 9 groups of peaks, and its integration is 4: 2: 4 than (by hanging down the field to High-Field): 2: 1: 1: 1: 2: 2.
A δ 3.11,3.27 is the dd peak, is equivalent to respectively 2 protons, and the two has identical coupling constant (J=14.5), simultaneously
1H-
1H COSY stave bright the two have dependency, so δ 3.11, δ 3.27 hydrogen proton peak are attributed to 2 H-7 and 2 H-14.
B δ 3.53,3.63 is the dd peak, is equivalent to respectively 1 proton, and the two has identical coupling constant (J=11.5), simultaneously
1H-
1H COSY stave bright the two have dependency, so δ 3.53, δ 3.63 hydrogen proton peak are attributed to H-11 and H-11.
C δ 3.76 is multiplet, because the compound amount affects the hydrogen spectral integral less, is equivalent to approximately 1 proton, simultaneously
1H-
1H COSY stave is bright to have dependency with δ 3.53, δ 3.63, so δ 3.76 place's hydrogen proton peak can be attributed to H-10.
D δ 3.97 is quartet, is equivalent to respectively 2 protons, with δ 3.27, δ 3.11 identical coupling constant (J=5.0, J=8.0) is arranged, simultaneously
1H-
1The bright three of H COSY stave has dependency, so δ 3.97 place's hydrogen proton peak can be attributed to H-8 and H-13.
E δ 7.41,7.36 is triplet, and δ 7.31 is doublet, be equivalent to respectively 4 protons, 2 protons and 4 protons, and three's coupling constant is identical,
1H-
1H COSY stave bright δ 7.41 peaks and δ 7.36,7.31 place's hydrogen proton peak are relevant, therefore δ 7.41 hydrogen proton peak can be attributed to H-2, H-4, H-17, H-19, δ 7.36 hydrogen proton peak are attributed to H-3, H-18, δ 7.31 hydrogen proton peak are attributed to H-1, H-5, H-16, H-20.
The f sample
1The HNMR spectrum conforms to substantially with the structure of possibility compound.
Sample
13Have 9 peaks among the CNMR, may be a symmetrical configuration molecule, corresponding to 20 carbon atoms in the molecule, conform to carbonatoms in the molecular formula.
A δ 176.6 is the carbonylic carbon atom peak, and hsqc spectrum shows without relevant proton peak, so this peak can be attributed to C-9, carbonyl carbon peak, C-12 position.
B δ 137.8,132.0,131.8,130.4 may be the carbon atoms on a benzene ring peak, and hsqc spectrum shows δ 137.8 without relevant proton peak, and hsqc spectrum shows δ 132.0,131.8, and 130.4 follow respectively
1HNMR δ 7.41,7.31,7.36 proton peak are relevant, so δ 137.8,132.0,131.8,130.4 carbon atom peaks can be attributed to C-6 on the phenyl ring, C-15, C-2, C4, C-17, C-19, C-1, C5, C-16, C-20, C-3, C-18 carbon atom peak.
C δ 74.7 carbon atom DEPT135 ° spectrums are indicated as the tertiary carbon peak, and are relevant with δ 3.76 proton peak, the HMBC stave bright with
1HNMR3.63,3.53 proton peak are relevant, and with
1Other protons of HNMR H is without relevant peaks, so δ 74.7 carbon atom peaks can be attributed to C-10 carbon atom peak.
D δ 65.2 carbon atom hsqc spectrums show with
1HNMR3.63,3.53 proton peak are relevant, and 3.63,3.53 proton peak are the dd peak, and with
1Other protons of HNMR H is without relevant peaks, so δ 65.2 carbon atom peaks can be attributed to C-11 carbon atom peak.
E δ 58.7 may be the carbon atom peak that links to each other with amino, and hsqc spectrum shows that δ 58.7 follows
1HNMR δ 3.97 proton peak are relevant,
1HNMR δ 3.97 is quartet, the HMBC stave bright with
1HNMR δ 3.27,3.21 proton peak are relevant, so δ 58.7 carbon atom peaks can be attributed to C-8, C-13 carbon atom peak.
F δ 39.0 may be the carbon atom peak, benzyl position that links to each other with phenyl ring, and hsqc spectrum shows that δ 39.0 follows
1HNMR δ 3.27,3.21 proton peak are relevant,
1HNMR δ 3.27,3.21 is the dd peak, the HMBC stave bright with
1HNMR δ 3.97,7.31 proton peak are relevant, so δ 39.0 carbon atom peaks can be attributed to C-7, C-14 carbon atom peak.
The g basis
13Carbonatoms and type thereof that CNMR, HSQC, HMBC compose visible sample conform to substantially with the molecular structure of possibility compound, may be this compound.
Table 1 sample
1The HNMR determination data
(D
2O; Interior mark: 3-(trimethyl silicon based)-1-propanesulfonic acid sodium salt (4,4-dimethyl-4-sila penta-1-sodium sulfonate DSS)
Table 2 sample
13The CNMR determination data
(D
2O; Interior mark: 3-(trimethyl silicon based)-1-propanesulfonic acid sodium salt (4,4-dimethyl-4-sila penta-1-sodium sulfonate DSS)
ESI-Ms?m/z：401[M-1]，M＝402。
According to compound of red external spectrum: 3431cm
-1Be the stretching vibration peak of OH, and the peak molded breadth, illustrating has association type OH, 3406cm in the molecule
-1Absorption peak is arranged, be NH
2Stretching vibration peak, 3033cm
-1Absorption peak is arranged, be the stretching vibration peak of phenyl C-H, 2962cm
-1Absorption peak is arranged, be the stretching vibration peak of alkyl C-H, 1625cm
-1Absorption peak is arranged, be the stretching vibration peak of amidocarbonylation, 1564cm
-1Be the C=C stretching vibration peak of benzene ring structure, 1459cm
-1Absorption peak shows to be had-absorption of CH2-, and 747cm
-1, 700cm
-1Two absorption bands prove the monobasic substituted benzene.
Table 3 sample infrared measurement data
Mass spectrum possibility lytic pathway:
M/z:330 may generate for two stable phenylalanine molecular ion peak collisions.
Sample in neutral solution (water) uv-absorbing λ=254nm is the absorption band of phenylalanyl residue, λ=378nm is the absorption band of hydroxamic acid group, the ultra-violet absorption spectrum of show sample and predictive compound is basically identical.
Ultraviolet-the spectroscopic data of table 4 sample
Embodiment 3: the stench rhzomorph to cucumber fusarium axysporum disease resisting effect test
The structure of the compound of stench rhzomorph is (as follows)
This tests used cucumber seeds; process without any kind of clothing agent; after the sterilization; be seeded in the polypots that twice autoclaved fertile vegetable garden soil matrix is housed; the seedling alms bowl places the greenhouse of Zhejiang Academy of Agricultural Science plant protection and institute of microbiology; after cotyledon launches fully, induce inoculation and simultaneously challenge inoculation.
The greenhouse pot culture method is adopted in disease resisting effect test: the sample compound dissolved in distilled water is mixed with the aqueous solution of 50mg/L, blank usefulness clear water and positive control 50mg/L derosal.Induce inoculation to adopt root-pouring method, 50mg/L stench rhzomorph, clear water and the 50mg/L derosal that has prepared injected respectively the cucumber rhizosphere, every alms bowl 15mL.After three days, adopt soil inoculation method to carry out the challenge inoculation of cucumber fusarium axysporum.Concrete operations be the husky substratum of the wheat that covers with cucumber fusarium axysporum is air-dry, smash to pieces, kind be implanted with in the little alms bowl of cucumber seedling to be sprinkled into behind 1: 20 the abundant mixing of ratio with sterilization soil, cover again one deck mattress soil that goes out.Water in good time, make the suitable temperature and humidity of Soil conservation.Observe weekly and record upgrowth situation and seedling rate.At last the result is carried out statistical study.
Test according to the method described above and find that 50mg/L stench rhzomorph has preferably preventive effect to cucumber fusarium axysporum, the results are shown in Table 5.After processing 14d, variance analysis and DuncanShi test of significance, the strain rate contrasts obvious raising, and is significant difference (P=0.05).Still keep preferably relative control effect during 21d, reach 53.84%.Be significant difference (P=0.05) with contrast.
Table 5 stench rhzomorph is processed cucumber to the prevention effect of blight
Annotate: adopt multiple range test, different letter representation significant differences (P=0.05) behind the numerical value.
Claims (5)
1. a hydroxamic acid type is had a liking for the preparation method of iron element compound, comprising:
(1), the preparation of fermented liq: the bacterial classification pseudomonas putida is behind solid K MB substratum activation 24h, in the picking list bacterium colony access 5mL liquid KMB substratum, place shaking table, 28 ℃, cultivate 24h under the 150r/ minute condition, then be inoculated among the solid seed culture medium KMB, at 28 ℃ of lower 24h that cultivate, obtain first order seed; Then with cultured first order seed picking one ring, in the MKB substratum, ferment, wherein, fermentation condition is: the pH value is 6.5, inoculum size is 2%, and fermentation time is 48h, and the bottling amount is 50/150mL, wherein the composition of 1000 milliliters KMB solid, liquid and fermention medium mainly is comprised of following component: casamino acids 5g, MgSO
47H
2O2.5g, K
2HPO
43H
2O2.5g, Fe
3+Content be 0.1mg/L;
(2), collect in the step (1) fermented liquid 1000mL through rotary evaporation after being condensed into slightly thick liquid under 43 ℃, at 10000rpm, 4 ℃, removed white insolubles in centrifugal 15 minutes, supernatant liquor transfers to pH 4 for subsequent use with HCl;
(3), detecting fluid inspection with CAS after the liquid concentration behind the macroporous adsorptive resins on the supernatant samples that obtains in the step (2), do not show activity, the elutriant of using again opposed polarity is wash-out successively, elutriant is carried out obtaining active product behind the concentrating under reduced pressure, measure its activity, wherein the elutriant of the successively wash-out of opposed polarity is: ionized water, 0.2% hydrochloric acid soln, 10% methanol-water solution, 30% methanol-water solution, 50% methanol-water solution, 80% methanol-water solution, 100% methyl alcohol, 100% acetone soln.
2. according to claim 1 method, the structure that the hydroxamic acid type that wherein obtains is had a liking for iron element compound is as follows:
3. according to claim 1 method, wherein the sample with wash-out in the step (3) separates with HPLC again, and the condition of separation is: material ODS-BP; Flow velocity: 1ml/ minute; Moving phase: 30% methanol-water eluant solution 30 minutes.
4. according to claim 3 method, wherein the HPLC condition of separating is: material is ODS-BP; Flow velocity is 1ml/ minute; Moving phase is that the 5-60% methanol-water carried out gradient elution 30 minutes.
5. described method one of according to claim 1-4, wherein also fermented liquid is carried out following detection in turn afterwards in step (1): (1) CAS test fluid detects, and is obviously orange red, then proves to be rich in the fermented liquid and has a liking for the iron element; (2) carry out Arnow Assay test, if analytical results solution displaing yellow then think preliminary and determine that fermented liquid may the oxygen-containing nitrolic acid type have a liking for the iron element.
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| CN104232711B (en) * | 2014-07-23 | 2018-02-27 | 中国医学科学院医药生物技术研究所 | The isolation and purification method of iron chelate in a kind of natural products |
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