CN112852908B - Aureobasidin with weeding effect, and preparation method and determination method thereof - Google Patents
Aureobasidin with weeding effect, and preparation method and determination method thereof Download PDFInfo
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- CN112852908B CN112852908B CN202110029146.9A CN202110029146A CN112852908B CN 112852908 B CN112852908 B CN 112852908B CN 202110029146 A CN202110029146 A CN 202110029146A CN 112852908 B CN112852908 B CN 112852908B
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- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
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Abstract
The invention belongs to the technical field of herbicide preparation, and discloses a aureobasidin with a weeding effect and a preparation method thereof, wherein a spore suspension is prepared by using Aureobasidium pullulans PA-2, inoculated into a seed culture medium for activation, and transferred into a fermentation tank for fermentation; optimizing culture conditions in terms of 4 aspects of temperature, liquid loading amount, culture time and rotating speed; taking fermentation liquor, discarding thalli, carrying out oscillation adsorption by using macroporous resin, cleaning the adsorbed resin, and drying under reduced pressure after elution to obtain a crude aureobasidin extract of Aureobasidium pullulans PA-2; and (5) separating and purifying aureobasidin. The aureobasidin with weeding effect provided by the invention has the advantages of strong heat resistance, wide tolerance pH value and insensitivity to protease, and the compound can inhibit the activity of alpha-amylase in the seed germination period and simultaneously inhibit the activity of acetolactate enzyme, so that the seed germination and weed growth are inhibited, and the weeding purpose is achieved.
Description
Technical Field
The invention belongs to the technical field of herbicide preparation, and particularly relates to aureobasidin with a weeding effect, a preparation method and a determination method thereof.
Background
At present: weeds are an important factor in jeopardizing agricultural production, and the yield and quality of crops are severely affected by competing with the crops for moisture, nutrients, and lighting, etc. growth requirements. At present, chemical weeding is still the main part for farmland weed control in China, and 235 hundred million yuan of control cost is input each year, so that 5000 ten thousand tons of grain yield is still reduced, and the economic loss is directly caused. The increase in agricultural efficiency is still largely dependent on the application of chemical herbicides, and therefore the development of novel efficient, environmentally friendly green herbicides is of great practical significance. In recent years, microbial herbicides gradually enter the line of sight of people due to the advantages of small side effect on the environment, easy degradation and the like, and become a main trend under the concepts of green agriculture and safe agricultural products. Aureobasidin is a cyclic peptide antibiotic produced by Aureobasidium pullulans, and has strong antibacterial activity, mainly because the antibiotic can strongly inhibit the activity of fungal inositol phosphatidyl ceramide synthase, the synthesis of sphingomyelin substances is insufficient, the cell membrane structure is destroyed, and intracellular substances are extravasated, so that fungi die. Therefore, the antibiotic is often used as a biocontrol agent and a preservative in agriculture and food. However, up to now, studies on the application of aureobasidin in terms of herbicidal activity and the herbicidal mechanism thereof have not been reported. The invention aims to expand the application of an aureobasidin compound and provide a novel application of the aureobasidin compound as an agricultural herbicide.
Through the above analysis, the problems and defects existing in the prior art are as follows: the prior art is that the application of aureobasidin is only in the field of bactericidal activity, and the aureobasidin is not applied to herbicidal activity.
The difficulty of solving the problems and the defects is as follows: the inventor has conducted intensive researches on the preparation method and the bioassay method of the compound and activities of various weeds, and experimental results show that aureobasidin has a good preventing and killing effect on the weeds, and a new thought is provided for developing the compound into a novel herbicide.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides aureobasidin with a weeding effect and a preparation method thereof.
The invention is realized in such a way that the aureobasidin with weeding effect and the preparation method thereof comprise the following steps:
step one, strain activation and fermentation: preparing spore suspension by using Aureobasidium pullulans PA-2 (with a preservation number of CGMCC No.8413, preserved in the North Chen Silu No. 1 and 3 China general microbiological culture Collection center of the Korean area of Beijing in the 10 month 28 days of 2013), inoculating into a seed culture medium for activation, and then transferring into a fermentation tank for fermentation;
Step two, fermentation production optimization: optimizing culture conditions in terms of 4 aspects of temperature, liquid loading amount, culture time and rotating speed;
step three, preparing a crude extract of aureobasidin: taking fermentation liquor, discarding thalli, carrying out oscillation adsorption by using macroporous resin, cleaning the adsorbed resin, and drying under reduced pressure after elution to obtain the aureobasidin crude extract of Aureobasidium pullulans PA-2;
step four, separating and purifying aureobasidin: and (3) carrying out primary separation through thin layer chromatography to obtain a aureobasidin product sample, and then preparing the sample through high performance liquid chromatography to obtain the separated and purified aureobasidin.
Further, in the first step, the strain activation and fermentation specifically includes:
(1) Placing glycerol preservation strain PA-2 at the temperature of minus 80 ℃ in an ice bath, pouring glycerol in an ultra-clean workbench when glycerol is molten, digging a lawn, placing the lawn in a potato glucose solid culture medium, scraping the surface of a bacterial colony at the temperature of 25 ℃ for 3-5 d, and preparing spore suspension;
(2) Inoculating 5mL of spore suspension into 150mL of seed culture medium, and performing shake culture at 25 ℃ and 180r/m for 2d for activation;
(3) 150mL of the seed culture medium is transferred into a 30L fermentation tank containing 15L of fermentation culture medium A, shake culture is carried out for 56h at 25 ℃ under 180r/m, then 2L of fermentation culture medium B is added, shake culture is continued for 88h at 25 ℃ under 180r/m, and fermentation liquor is obtained.
Further, the seed medium included 0.67%yeast nitrogen base and 2% glucose, and the PDA solid medium included 2% pototo, 2% glucose, and 1.5% agar;
the fermentation medium A comprises 4% glucose, 3% skip, 3% soybean fluorine, 0.5% (NH) 4 ) 2 SO 4 、0.15％KH 2 PO 4 、0.05％MgSO 4 ·7H 2 O、0.01％CaCl 2 ·2H 2 O、0.01％NaCl、 0.5μg/mL FeCl 3 ·6H 2 O and 0.5. Mu.g/mL ZnSO 4 ·7H 2 O; the fermentation medium B comprises 10% glucose, 5% polypepton and 0.75% KH 2 PO 4 、0.25％MgSO 4 ·7H 2 O、0.05％CaCl 2 ·2H 2 O、 0.05％NaCl、2.5μg/mL FeCl 3 ·6H 2 O and 2.5. Mu.g/mL ZnSO 4 ·7H 2 O。
Further, in the third step, the preparation of the crude aureobasidin extract specifically includes:
centrifuging 30L of fermentation broth with a speed of 10000r/m, removing thalli, adding 3% macroporous resin XAD-16, and shaking at room temperature in a shaking table for 120r/m for 24h; washing the adsorbed resin with distilled water until no fermentation liquid remains on the surface of the resin, then placing the resin in a 1000mL triangular flask with a plug, adding 300mL absolute ethyl alcohol, placing the resin in a shaking table for eluting for 24 hours at room temperature, and then drying under reduced pressure to obtain the aureobasidin crude extract of Aureobasidium pullulans PA-2.
In the fourth step, the separation and purification of aureobasidin specifically includes:
(1) Primary separation by thin layer chromatography: the method comprises the steps of adopting a silica gel plate as a chromatographic stationary phase, adopting a chromatographic reagent of dichloromethane, methanol and water=65:25:4 (v: v), respectively dissolving a crude extract and a standard sample of aureobasidin in a small amount of methanol, spotting the crude extract and the standard sample of aureobasidin on the silica gel plate for chromatography, spreading each two plates of the crude extract in a chromatography system for 2 hours, volatilizing the dry reagent, one piece of the compound is developed by ninhydrin, the other piece of the compound is placed in a high-temperature-resistant closed container, about 2mL of concentrated hydrochloric acid is filled in a small cup, the compound is placed in a 110 ℃ oven for fumigation for 3 hours to carry out in-situ hydrolysis, then cooling and blowing off the hydrochloric acid, the ninhydrin is developed, and the compound is placed in the 110 ℃ oven for fumigation for 1 hour to observe the result, so that the position of the aureobasidin is confirmed; taking an analysis plate subjected to thin layer chromatography under the same conditions, scraping silica gel at a position corresponding to the color development site, collecting silica gel powder, soaking, eluting and concentrating by using methanol to obtain an aureobasidin sample, and performing high performance liquid chromatography;
(2) High performance liquid chromatography: dissolving aureobasidin sample and standard substance with mobile phase, sterilizing with 0.22 μm pinhole organic filter membrane, removing impurities, balancing chromatographic column, adding sample, and performing gradient elution to separate sample compound, and repeatedly collecting sample peak for 10 times according to peak time of standard sample and crude extract sample;
(3) Identification of aureobasidin structure: measuring the molecular weight of the aureobasidin by a mass spectrometer; 5mg of the samples were weighed and dissolved in 0.5mL of CD, respectively 3 In OD to perform 1 H-NMR detection; 15mg of the sample was weighed and dissolved in 0.5mL of CD 3 In OD to perform 13 C-NMR detection.
It is another object of the present invention to provide a method for assaying aureobasidin having a herbicidal action, which comprises assaying the herbicidal activity of aureobasidin compound, assaying the physicochemical properties of aureobasidin compound and assaying the herbicidal mechanism of aureobasidin compound.
Further, the aureobasidin compound herbicidal activity assay comprises:
1) Seed germination:
preparing aureobasidin into 100, 50, 10 mug/mL 3 kinds of solutions by dimethyl sulfoxide, respectively taking 1mL of solution to be detected, uniformly adding into 12-hole culture plates paved with double-layer filter paper, after solvent volatilizes to dryness, selecting 10 pieces of weed seeds after germination accelerating to uniformly disperse in the treated culture plate holes, taking clear water as a contrast, repeating each treatment for 4 times, placing the 12-hole culture plates into a constant-temperature incubator with the temperature of 25 ℃ and the relative humidity of 70%, culturing, measuring the lengths of radicle and embryo of the weed seeds after 3 days, calculating the radicle (bud) inhibition rate by using the following formula,
2) And (3) measuring the weeding effect of the potted plants:
sowing the test weed seeds in small pots, wherein each pot is 20-30 plants/pot; when various plants grow to 3-5 leaf periods, respectively spraying the aureobasidin leaf surfaces with the concentration of 100, 50 and 10 mug/mL on weed plants, spraying the leaf surfaces wet without dropping the liquid medicine, and spraying clear water in contrast; the treatment is repeated for 4 times, the plant height, the plant number and the fresh weight are measured after 7 days of drug application, the plant height inhibition rate, the plant prevention effect and the fresh weight effect are calculated, and meanwhile, the weed poisoning is observed,
3) Field plot weeding test:
the field test is carried out in a test field, the random block arrangement is carried out, and the cell area is 8m 2 Setting the concentration of aureobasidin 100 mug/mL, taking clear water as a control, repeating the treatment for 4 times, spraying when the weeds grow 3-5 leaves, measuring the plant number and fresh weight of the weeds respectively 15 days after the application of the pesticide, and calculating the plant prevention effect and fresh weight effect.
Further, the determination of the physical properties of aureobasidin compounds comprises:
1) Sensitivity to temperature determination:
treating aureobasidin at 25deg.C, 35deg.C, 55deg.C, 65deg.C, 85deg.C, 100deg.C and 121deg.C for 30min, cooling to room temperature, detecting herbicidal activity by seed germination method, and observing herbicidal activity change with sterilized water treated at corresponding temperature;
2) Sensitivity to acid, base assay:
regulating pH value of aureobasidin to 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0 and 12.0 respectively by using 6M HCI or NaOH, standing for 15min, centrifuging for 10min at 10000r/min, retaining supernatant, detecting weeding activity by using a seed germination method, and observing weeding activity change by using sterile water with the corresponding pH value as a control;
3) Sensitivity assay to protease:
treating aureobasidin with trypsin, pepsin and proteinase K at 37deg.C for 1 hr, and treating at 94deg.C for 10min to stop enzyme reaction with concentration of 500 μg/mL; centrifuging at 12000r/m for 5min to obtain supernatant, detecting the herbicidal activity by seed germination, and observing the change of the herbicidal activity by using various protease-treated sterile water as control.
Further, the determination of the herbicidal mechanism of the aureobasidin compound includes determining the effect of the aureobasidin compound on the activity of alpha-amylase and determining the effect of the aureobasidin compound on the activity of acetolactate synthase.
By combining all the technical schemes, the invention has the advantages and positive effects that:
the aureobasidin with weeding effect provided by the invention has the advantages of strong heat resistance, wide tolerance pH value and insensitivity to protease, and the compound can inhibit the activity of alpha-amylase in the seed germination period and simultaneously inhibit the activity of oxalyl lactate, so that the seed germination and weed growth are inhibited, and the weeding purpose is achieved. The weeding activity plate test shows that the seed germination inhibition rate of the compound on the gramineous weeds is higher than that of the broadleaf weeds, the germ inhibition rate of the compound on the gramineous weeds reaches more than 88 percent at the concentration of 100 mug/mL, the culture inhibition rate of the compound on the broadleaf weeds is only about 73 percent, and the germ inhibition rate of the compound on the gramineous weeds reaches more than 80 percent and the radicle inhibition rate of the compound on the broadleaf weeds reaches less than 80 percent. The potted plant weeding activity shows that under the concentration of 100 mug/mL, 5 days after the drug is studied, the aureobasidin has different degrees of inhibition effects on plant heights, plant prevention effects and fresh weight effects of different weeds, the highest plant height inhibition rate reaches 59%, the plant prevention effects and fresh weight effects of gramineous weeds are distributed by more than 85% and 81%, and the plant prevention effects and fresh weight effects of broadleaf weeds are respectively more than 73% and 71%.
The field plot test result shows that, at the concentration of 100 mug/mL, after 15 days of spraying, weeds in the treated plot are killed except for the growth of plants of the zero star, weeds in the control plot grow vigorously, and the leaf color is dark green. The grass weeds are found to have good plant prevention effect on the weeds, the prevention effect on the chenopodium album is 90.6%, and the minimum prevention effect is only 76.3%; similarly, the fresh weight effect on wild oat is highest and reaches 88.2%, and the fresh weight effect on elsholtzia pachyrhizi is lowest and is 72.9%. In general, the weeding composition has good weed control effect at the concentration of 100 mug/mL, and can be used for further formulation development.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following description will briefly explain the drawings needed in the embodiments of the present application, and it is obvious that the drawings described below are only some embodiments of the present application, and that other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.
Fig. 1 is a flowchart of a preparation method of aureobasidin with a herbicidal effect provided by the embodiment of the invention.
FIG. 2 is a schematic representation of the effect of various conditions on aureobasidin production provided in the examples of the present invention.
Fig. 3 is an ultraviolet absorbance spectrum of aureobasidin provided in the example of the present invention.
Fig. 4 is a mass spectrum of an aureobasidin compound provided in the example of the present invention.
Fig. 5 is a nuclear magnetic pattern of an aureobasidin compound provided in the example of the present invention.
Fig. 6 is a schematic structural diagram of aureobasidin provided in an embodiment of the present invention.
FIG. 7 is a schematic illustration of the inhibition of germination of weed seed embryos by aureobasidin at different concentrations provided in the examples of the present invention.
Fig. 8 is a schematic diagram showing the inhibition of the radicle germination of weed seeds by aureobasidin with different concentrations provided in the example of the present invention.
FIG. 9 is a schematic diagram showing the effect on weed plant height of the aureobasidin spray applied at a concentration of 100. Mu.g/mL.
FIG. 10 is a graph showing the effect on weed plant number and fresh weight after spraying aureobasidin at a concentration of 100. Mu.g/mL provided in the example of the present invention.
Fig. 11 is a schematic diagram of a field plot weeding effect after spraying aureobasidin according to the embodiment of the invention.
FIG. 12 is a graph showing the effect of temperature on the biological activity of aureobasidin provided in the examples of the present invention.
FIG. 13 is a graph showing the effect of pH on the biological activity of aureobasidin according to an example of the present invention.
FIG. 14 is a schematic diagram showing the effect of protease provided in the examples of the present invention on the biological activity of aureobasidin.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Aiming at the problems existing in the prior art, the invention provides aureobasidin with a weeding effect and a preparation method thereof, and the invention is described in detail below with reference to the accompanying drawings.
As shown in fig. 1, the preparation method of aureobasidin with herbicidal effect provided by the embodiment of the invention comprises the following steps:
s101, strain activation and fermentation: preparing spore suspension by using Aureobasidium pullulans PA-2, inoculating the spore suspension into a seed culture medium for activation, and transferring the spore suspension into a fermentation tank for fermentation;
s102, fermentation production optimization: optimizing culture conditions in terms of 4 aspects of temperature, liquid loading amount, culture time and rotating speed;
s103, preparing a crude extract of aureobasidin: taking fermentation liquor, discarding thalli, carrying out oscillation adsorption by using macroporous resin, cleaning the adsorbed resin, and drying under reduced pressure after elution to obtain a crude aureobasidin extract of Aureobasidium pullulans PA-2;
S104, separating and purifying aureobasidin: and (3) carrying out primary separation through thin layer chromatography to obtain a crude aureobasidin sample, and then carrying out analysis treatment on the sample through high performance liquid chromatography to obtain a pure aureobasidin product.
The invention is further described below in connection with specific embodiments.
1. Test strain and culture medium
Test strain: aureobasidium pullulans (Aureobasidium pullulans) PA-2 is separated and stored by a comprehensive treatment key laboratory of agricultural pests in Qinghai province.
Target grass: wild oat, eclipta alba, barnyard grass, green bristlegrass, quinoa, wild rape, wild carrot, blue-green-grass, elsholtzia and cleavers.
PDA medium: 2% pototo, 2% glucose and 1.5% agar.
Seed culture medium: 0.67%Yeast Nitrogen Base and 2% glucose.
Fermentation medium a:4% glucose, 3% ski mill, 3% soybean fluorine, 0.5% (NH) 4 ) 2 SO 4 、 0.15％KH 2 PO 4 、0.05％MgSO 4 ·7H 2 O、0.01％CaCl 2 ·2H 2 O、0.01％NaCl、0.5μg/mL FeCl 3 ·6H 2 O and 0.5. Mu.g/mL ZnSO 4 ·7H 2 O。
Fermentation medium B:10% glucose, 5% polypepton, 0.75% KH 2 PO 4 、0.25％ MgSO 4 ·7H 2 O、0.05％CaCl 2 ·2H 2 O、0.05％NaCl、2.5μg/mL FeCl 3 ·6H 2 O and 2.5. Mu.g/mL ZnSO 4 ·7H 2 O。
2. Method and procedure
2.1 Strain PA-2 activation and fermentation
(1) Placing glycerol preservation strain PA-2 at-80 ℃ in an ice bath, pouring glycerol in an ultra-clean workbench when glycerol is molten, digging a lawn, placing the lawn in a potato glucose solid culture medium, and scraping the surface of a bacterial colony at 25 ℃ for 3-5 d to prepare spore suspension.
(2) 5mL of the spore suspension was inoculated into 150mL of a seed medium, and the mixture was subjected to shaking culture at 25℃and 180r/m for 2d for activation.
(3) 150mL of the seed medium was transferred to a 30L fermenter containing 15L of fermentation medium A, and cultured with shaking at 25℃and 180r/m for 56 hours. Then adding 2L of fermentation medium B, and continuously carrying out shaking culture for 88 hours at the temperature of 25 ℃ and under the condition of 180r/m to obtain fermentation liquor.
2.2 fermentation production optimization
The culture conditions were optimized in terms of 4 aspects of temperature, liquid loading amount, culture time and rotation speed. A single factor test was conducted at a predetermined temperature (23 ℃,25 ℃, 27 ℃, 30 ℃), a liquid loading amount (150 mL, 200mL, 250mL, 300 mL), a culturing time (5 d, 7d, 9d, 11 d), and a shaking table rotation speed (140 r/m, 160r/m, 180r/m, 200r/m, 220 r/m), and the effect of each factor on the production of aureobasidin substance was examined.
2.3 preparation of crude Aureobasidin extract and calculation of yield
Taking 30L of fermentation liquor, centrifuging at 10000r/m, discarding thalli, adding 3% macroporous resin XAD-16, and placing in a shaking table for shaking at room temperature for 120r/m and 24h. Washing the adsorbed resin with distilled water until no fermentation liquid remains on the surface of the resin, then placing the resin in a 1000mL triangular flask with a plug, adding 300mL absolute ethyl alcohol, placing the resin in a shaking table for eluting for 24 hours at room temperature, and then drying under reduced pressure to obtain the aureobasidin crude extract of Aureobasidium pullulans PA-2. Weighing an empty labeled sample bottle, and recording the mass M of the sample bottle on the label 1 The method comprises the steps of carrying out a first treatment on the surface of the The mass of the AbA crude extract placed in the sample bottle is marked M 2 . The yield formula of the PA-2 fermentation crude extract aureobasidin is M=M 2 -M 1 The method comprises the steps of carrying out a first treatment on the surface of the Yield y=m/L (L is total volume of fermentation broth).
2.4 separation and purification of Aureobasidin
(1) Preliminary separation by thin layer chromatography
By means of Silica Gel plates (Silica Gel GF) 254 20 cm. Times.5 cm) was used as the stationary phase for chromatography, and the chromatographic reagent was dichloromethane: methanol: water=65:25:4 (v: v). Firstly, respectively dissolving a crude aureobasidin extract and a standard aureobasidin sample in a small amount of methanol, spotting the crude aureobasidin extract and the standard aureobasidin sample on a silica gel plate for chromatography, developing two plates at each spot of the crude extract in a chromatography system for 2 hours, volatilizing dry reagents, developing one of the two plates with ninhydrin (0.4%), placing the other plate in a high-temperature-resistant sealed container, placing the other plate in a small cup for fumigation for 3 hours in a 110 ℃ oven for in-situ hydrolysis by using 2mL of concentrated hydrochloric acid, cooling, blowing hydrochloric acid, developing the colors with ninhydrin, fumigating the two plates in the 110 ℃ oven for 1 hour, and confirming the position of aureobasidin. And (3) taking an analysis plate subjected to thin layer chromatography under the same conditions, scraping silica gel at a corresponding color development site, collecting silica gel powder, soaking, eluting and concentrating by using methanol to obtain a aureobasidin sample, and performing high performance liquid chromatography.
(2) High performance liquid chromatography
Chromatographic conditions: ODS-silica C 18 Chromatographic column (4.6 mm. Times.250 mm, 5. Mu.L, pH 1.0-14.0);
mobile phase: a:70% -95%, B:30 to 5 percent
A: water (with 0.1% trifluoroacetic acid (TFA))
B: methanol; 0 to 30 minutes;
flow rate: 1mL/min; column temperature: 30 ℃; sample injection volume: 10 mu L
Detection wavelength: 210nm.
Sample treatment: and (3) performing sterilization and impurity removal on the aureobasidin crude extract sample and the standard substance by using a mobile phase dissolved and then passing through a needle-hole type organic filter membrane of 0.22 mu m, balancing a chromatographic column, adding a sample, performing gradient elution to separate the sample, and repeatedly collecting sample peaks for 10 times according to the peak time of the standard sample and the sample.
(4) Identification of aureobasidin substance structure
Molecular weight of aureobasidin (AbA) was determined using a Mariner System 5304 mass spectrometer. By German Bruker UltraShied TM 400Plus nuclear magnetic resonance spectrometer detection (TMS as internal standard). 1 H-NMR measurement, 5mg of the sample was dissolved in 0.5mL of CD, respectively 3 In OD. 13 C-NMR measurement, 15mg of the sample was dissolved in 0.5mL of CD 3 Detection in OD.
2.5 determination of herbicidal Activity of Aureobasidin
2.5.1 seed germination
Preparing aureobasidin into solutions with the concentration of 100, 50 and 10 mug/mL and 3 types by using dimethyl sulfoxide (DMSO), respectively taking 1mL of to-be-detected solution, uniformly adding the solutions into a 12-hole culture plate paved with double-layer filter paper, after the solvent volatilizes, selecting 10 pieces of weed seeds after germination acceleration to uniformly disperse in the holes of the culture plate, and using clear water as a control, wherein each treatment is repeated for 4 times. The 12-well culture plate was placed in a constant temperature incubator at 25℃and a relative humidity of 70%, and after 3 days, the lengths of radicle and embryo of the weed seed were measured, and the radicle (bud) inhibition rate was calculated.
2.5.2 herbicidal effect in potted plants
Sowing the test weed seeds in small pots, wherein each pot is 20-30 plants/pot; when various weeds grow to 3-5 leaf periods, the aureobasidin leaf surfaces with the concentration of 100, 50 and 10 mug/mL are respectively used for spraying weed plants, and the leaf surfaces are sprayed with wet liquid medicine without dropping, so that clear water is sprayed in a contrast mode. Each treatment was repeated 4 times. The plant height, plant number and fresh weight were measured 7 days after the application of the pesticide, and the plant height inhibition rate, plant control effect and fresh weight effect were calculated while the toxic symptoms in weeds were observed.
2.5.3 field plot weeding test
The field test is carried out in the field of plant protection institute of academy of agricultural sciences, the random block arrangement is carried out, and the cell area is 8m 2 Setting the concentration of aureobasidin 100 mug/mL, taking clear water as a control, repeating the treatment for 4 times, spraying when the weeds grow 3-5 leaves, measuring the plant number and fresh weight of the weeds respectively 15 days after the application of the pesticide, and calculating the plant prevention effect and fresh weight effect.
2.6 determination of physicochemical Properties of Aureobasidin
(1) Sensitivity to temperature
The aureobasidin (treated at 25deg.C, 35deg.C, 55deg.C, 65deg.C, 85deg.C, 100deg.C, 121deg.C respectively) is treated for 30min, cooled to room temperature, and subjected to seed germination to obtain the final product, and sterilized water treated at corresponding temperature is used as control to observe the change of herbicidal activity.
(2) Sensitivity to acids and bases
And 6M HCI or NaOH is used for respectively regulating the pH value of the aureobasidin to 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0 and 12.0, standing for 15min, centrifuging for 10min at 10000r/min, retaining supernatant, detecting the weeding activity by adopting a seed germination method, and observing the weeding activity change by taking sterile water with the corresponding pH value as a control.
(3) Sensitivity to proteases
Aureobasidin is treated with trypsin, pepsin and proteinase K at 37deg.C for 1 hr, and 94 deg.C for 10min to stop enzyme reaction, wherein the concentration of enzyme reaction is 500 μg/mL. Centrifuging at 12000r/m for 5min to obtain supernatant, detecting the herbicidal activity by seed germination, and observing the change of the herbicidal activity by using various protease-treated sterile water as control.
2.7 determination of herbicidal mechanism of Aureobasidin
2.7.1 Effect of Aureobasidin on alpha-amylase Activity
(1) The weed seeds are treated with the aureobasidin diluent and incubated for 3-8 days with sterile water for daily extraction of alpha-amylase.
(2) About 0.1g of the sample is weighed, and 0.8mL of distilled water is added for homogenate; homogenizing, standing at room temperature, extracting for 15min, oscillating for 1 time every 5min, and fully extracting; 6000g, centrifuging at normal temperature for 10min, sucking supernatant, adding distilled water to constant volume of 10mL, and shaking to obtain the amylase zymogen liquid.
(3) And (3) measuring the activity of the alpha-amylase, taking 12 test tubes, using 6 test tubes for a treatment group and using 6 test tubes for a control group, and respectively labeling. Sucking 1mL of crude enzyme liquid into a test tube, placing in a water bath at 70 ℃ for 15min to enable the alpha-amylase to be heated and passivated, and rapidly taking out the alpha-amylase from the water bath and cooling the alpha-amylase in tap water; 4mL of citric acid buffer solution with pH of 5.6 is sucked and added into a test tube; sucking 4mL of 0.4M NaOH solution into two groups of test tubes; inactivating the enzyme activity, and then sucking 2mL of 1% starch into a test tube; the test tube of the treatment group is placed in a water bath at 40 ℃ for 15min, then added into 2mL of preheated 1% starch (40 ℃), uniformly mixed, then subjected to a water bath at 40 ℃ for 13-15 min, and finally added with 4mL of 0.4M NaOH solution to stop the activity of enzyme.
(4) And (5) making a standard curve. Taking 10mL test tubes, labeling 5 numbers, respectively adding maltose standard solution 0, 0.5, 1.0, 1.5 and 2.0mL with the concentration of 1mg/mL, then adding distilled water into each test tube to enable the solution to be 2mL, respectively adding 3, 5-dinitrosalicylic acid solution 2mL, putting into boiling water bath to accurately boil for 5min, taking out, cooling, diluting to 10mL with distilled water, uniformly mixing, measuring the absorbance value of the mixed solution under the wavelength of 520nm by adopting a UV-7500 type spectrophotometer, recording optical density readings, taking the optical density readings as a total coordinate, and drawing a curve by taking the content of the maltose as an abscissa.
(5) And (5) measuring a sample. And (3) respectively adding 2mL of the reaction solution in the two groups of treatment test tubes and the control test tubes into a 10mL measuring flask, adding 2mL of 3, 5-dinitrosalicylic acid solution, uniformly mixing, boiling in boiling water for 5min, taking out, cooling, adding distilled water to dilute to 10mL, and uniformly mixing. The enzyme activity was expressed by performing colorimetry at a wavelength of 520nm using a UV-7500 spectrometer, recording optical density readings, and calculating maltose content by the standard curve.
(6) And (5) calculating results. A: maltose content (milligrams) produced by alpha-amylase hydrolysis of starch; b: maltose content (milligrams) in control tubes of alpha-amylase; c: milliliters of the sample solution are used in colorimetric.
Alpha-amylase activity [ maltose (mg)/fresh weight (g)/5 min ] = [ [ (A-B) x sample dilution total volume ]/sample weight (g) x C ]. Times.100
2.7.2 Effect of Aureobasidin on acetolactate synthase (ALS) Activity
(1) And (5) pre-treatment. When the tested weeds are cultivated to 3-4 leaf stage, the prepared aureobasidin with medium concentration inhibition is sprayed on the liquid surface, the control group is sprayed with clear water until the leaf surface is not dripped, and the control group is cultivated in an illumination incubator, and the aerial parts are cut off respectively at the 3 rd, 5 th, 7 th, 9 th and 11 th days after treatment, and ALS activity is measured.
(2) Extraction of ALS. Cutting leaf tissues of a treatment group and a control group, putting the leaf tissues into a pre-cooled mortar after cutting, adding 10mmol/L potassium phosphate buffer (according to the weight of the leaf: the buffer=1:1) into the mortar, grinding to be uniform, centrifuging at 4 ℃ for 30min, obtaining supernatant which is extracted crude enzyme liquid, and preserving at 4 ℃ for later use, wherein the above operations are all carried out at the temperature below 4 ℃.
(3) ALS activity assay. 6 10mL tubes were taken, 3 for treatment groups and 3 for control groups. Adding 0.8mL of the enzyme reaction buffer solution and 0.4mL of the crude enzyme solution into a test tube, placing the test tube into a water bath kettle at 37 ℃ for water bath for 60min, and adding 50 mu L of 3mol/L H 2 SO 4 Stop reaction (control group added 50. Mu.L 3mol/L H before water bath) 2 SO 4 To terminate the reaction), then carrying out water bath at 60 ℃ for 15min to carry out decarboxylation reaction, then sequentially adding 0.5mL0.5 creatine (dissolved in distilled water) and 0.5mL 5% alpha-naphthol (dissolved in 2.5mol/L NaOH), carrying out color development reaction at 60 ℃ for 15min, removing and cooling to room temperature, carrying out color comparison at 520nm by an ultraviolet spectrophotometer, and directly using A for ALS activity 520nm And (3) representing.
3. Test results
3.1 fermentation production optimization
The effect on aureobasidin production was tested by 4 factors from temperature, liquid loading, incubation time and shaking table rotation speed (FIG. 2). The increase and decrease of the aureobasidin yield occur with the increase of the liquid loading amount, and the yield is maximum when the liquid loading amount is 200 mL. The aureobasidin yield increased with the number of days of culture, and tended to be unchanged, and was hardly increased by the time of culture to 7 d. The phenomenon that the yield of the aureobasidin is increased and then decreased along with the increase of the temperature also appears, the yield is highest when the temperature is 25 ℃, and the influence of the temperature before and after the temperature is 25 ℃ on the yield of the aureobasidin is larger, which indicates that the yield of the aureobasidin is relatively sensitive to the temperature. The highest yield of aureobasidin reaches 0.65g/L when the rotation speed of the shaking table is 200r/m, no obvious difference exists between the yield of aureobasidin (0.64 g/L) when the rotation speed is 180r/m, and the yield slowly drops when the rotation speed is higher than 220 r/m. Thus, the optimal culture conditions were determined as follows: the temperature is 25 ℃, the liquid loading amount is 200mL, the culture time is 7d, the rotation speed of a shaking table is 180r/m, and the yield of aureobasidin is between 0.60g/L and 0.65 g/L.
3.2 separation and purification of Aureobasidin
3.2.1 ultraviolet absorption broad Spectrum of Aureobasidin Compounds
The active band with strong biological activity is scanned by ultraviolet spectrophotometer at the full wavelength of 200-490 nm in ultraviolet spectrum region to determine the maximum absorption wavelength of the band, and as can be seen from figure 3, there is a larger absorption peak at 220nm wavelength, and no secondary absorption, so 220nm is selected as the analysis wavelength in the following high performance liquid phase analysis.
3.2.2 structural identification of Aureobasidin
The structure of aureobasidin was resolved by mass spectrometry and nuclear magnetic resonance spectroscopy (fig. 4, 5, table 1), and the spectrum data were as follows:
TABLE 1 Hydrogen and carbon Spectrum data for Aureobasidin
MS m/z 1100.694(M+H),123.696[M+Na] + ,1099.694[M-H] - ,1253.696 [M+C 7 H 7 NO 3 ] + , 1 H-NMR(CDCI 3 )δ8.90～7.59(total 3H,NH),7.32～7.10(m,9H,ar. MePhe,Phe),6.52(d,1H,J＝7.5Hz,ar.MePhe),5.80(s)and 5.77(s)total 1H,α-CH Hmp),4.20(s,1H,HO HOMeVal),3.41(s,1H,α-CH HOMeVal),3.31(s),3,18(s), 3.16(s),3.07(s),2.67(s)and 2.50(s)(total 12H,N-CH 3 ),1.39(s,3H,γ-CH 3 HOMeVal),1.19(s,3H,γ-CH 3 HOMeVal). Molecular formula C 60 H 92 N 8 O 11 Molecular weight of 1100, colorless rod-like crystal, dissolving in ethanol, chloroform, diethyl ether, insoluble in water, melting point 155-157 deg.C, optical rotation [ alpha ]] D -245.3 ° (c 1.0, meoh). Aureobasidin (AbA) is a cyclic peptide consisting of 8 alpha-amino acid units and 1 hydroxy acid. 2 (R) -hydroxy-3 (R) -methylpentanoic acid (Hmp), beta-hydroxy-N-methyl-L-valine (beta HOMeVal), N-methyl-L-valine (MeVal), L-proline (Pro), isoleucine (aIle), N-methyl-L-phenylalanine (MePhe), L-leucine (Leu) and L-phenylalanine (Phe), respectively (FIG. 6)
3.2.3 determination of herbicidal Activity of Aureobasidin
3.2.3.1 seed germination
The effect of aureobasidin on inhibiting the growth of grass weeds, namely wild oat, eclipta alba, barnyard grass and green bristlegrass, is higher than that of broadleaf weeds (figure 7), wherein the effect of aureobasidin on inhibiting the growth of eclipta alba embryo buds reaches 90.98% at the concentration of 100 mug/mL, the effect of aureobasidin on inhibiting the growth of barnyard grass embryo buds is 90.58%, and the effect of inhibiting the growth of wild oat embryo buds is 88.2%; while the effect of inhibiting the growth of the embryo of the quinoa with broadleaf weeds reaches 73.67% at the same concentration, and the effect of inhibiting the growth of the embryo of the quinoa with the concentration of 10 mug/mL is only 28.02%. The most effective inhibition effect on broadleaf weeds is rape, the inhibition effect is 84.44% at the concentration of 100 mug/mL, and the inhibition effect on other weeds is below 80%. Also, the effect of inhibiting the growth of the radicle of grassy weed wild oat, eclipta alba, barnyard grass and green bristlegrass was higher than that of broadleaf weed (fig. 8), wherein the effect of inhibiting the growth of the radicle of eclipta alba was 86.64%, the effect of inhibiting the growth of the radicle of barnyard grass was 81.05%, the effect of inhibiting the growth of the radicle of green bristlegrass was 80.62%, and the effect of inhibiting the growth of the radicle of wild oat was 79.28% at a concentration of 100 μg/mL; the rape with the best effect of inhibiting the growth of the radicle of broadleaf weeds is 85.92% at the concentration of 100 mug/mL, and the radicle growth inhibition effects of quinoa, wild carrot, colza, elsholtzia and galium are all below 80%, wherein the inhibiting effect of the radicle growth inhibition effects of the quinoa is the worst, and the inhibiting effects of the radicle growth inhibition effects of the radicle of the chenopodium are 74.33% and 37.95% at the concentrations of 100 mug/mL and 10 mug/mL respectively. Overall, the inhibition effect on weed embryos is better than on radicles.
3.2.3.2 herbicidal effect of potted plants
As can be seen from Table 2, the potted plants showed symptoms after spraying aureobasidin at a concentration of 100. Mu.g/mL, and the grass became completely killed within 2-3 days. The wild rape and the cleavers are completely killed until about 7-10 days, but for some broadleaf weeds (quinoa, wild carrot, cole and elsholtzia) 15 days are needed for complete killing. The aureobasidin has certain selectivity on preventing and killing weeds.
TABLE 2 symptoms of weed poisoning after Aureobasidin spraying
In addition, after the aureobasidin with different concentrations is sprayed, the time for the occurrence of symptoms of the weeds is different, the weeds begin to generate symptoms at the concentration of 100 mug/mL for 3-5 days, the weeds are completely killed about 7-15 days, and meanwhile, the victims of the gramineous weeds are 3-5 days earlier than the victims of the broadleaf weeds. Likewise, weed emergence was correspondingly delayed for 2-3 days at concentrations of 50. Mu.g/mL and 10. Mu.g/mL.
The potted weeds were sprayed with aureobasidin at a concentration of 100. Mu.g/mL, and the plant height, plant number and fresh weight of the weeds were examined 5 days after the spraying, and the control effect on the weeds was evaluated. From fig. 9, it can be seen that aureobasidin has different degrees of inhibition on the plant height of different weeds, wherein the inhibition rate on the plant height of the wild oat is maximum and reaches 59.1%, the eclipta alba is 50.7%, and the plant height rate of other weeds is less than 50%; the highest inhibition rate of the strain height of the wild rape is 49.7%, and the lowest inhibition rate of the strain height of the quinoa is only 30.3%. As shown in FIG. 10, the plant control effect on grassy weeds is over 85%, wherein the plant control effect of barnyard grass is 91.9% at the highest, and the plant control effect of eclipta is 90% at the next; the plant control effect on broadleaf weeds, namely quinoa is 80.5%, and the plant control effect on elsholtzia rugulosa is 73.6%. However, from the viewpoint of fresh weight efficiency, the fresh weight efficiency of wild oat is highest and is 83.6%, the secondary fresh weight efficiency is barnyard grass, the fresh weight efficiency is 81.8%, the fresh weight efficiency of quinoa is lowest and is only 62.8%, and the fresh weight efficiency of wild carrot is highest in broadleaf weeds and reaches 71%.
3.2.3.3 field district weeding effect
Investigation is carried out 15 days after spraying, weeds in the treated cells are killed except for sporadic plant growth, weeds in the control area grow vigorously, and the leaf color is dark green. By investigating the plant number and fresh weight of a field district (figure 11), the plant control effect on weeds is found to be good, the plant control effect on wild oat is highest and is 90.6%, and the plant control effect on quinoa is lowest and is only 76.3%; similarly, the fresh weight effect on wild oat is highest and reaches 88.2%, and the fresh weight effect on elsholtzia pachyrhizi is lowest and is 72.9%. In general, the weeding composition has good weed control effect at the concentration of 100 mug/mL, and can be used for further formulation development.
3.3 physicochemical Properties of Aureobasidin
3.3.1 sensitivity to temperature
The inhibition rate of aureobasidin on weed growth after treatment at different temperatures is between 72.1% and 82.4%, no obvious difference exists between the treatments at different temperatures, and the inhibition rate of weed growth after treatment at 121 ℃ reaches 72.1% (figure 12), thus indicating that aureobasidin has stronger heat resistance.
3.3.2 sensitivity to acids and bases
The weed growth inhibition rate was at a maximum of 82.5% at pH 7.0. When the pH was 4.0 to 11.0, the weed control rate was not changed much, and the difference was not significant (FIG. 13). The slow decrease in weed inhibition rate with decreasing or increasing pH indicates that the biological activity of aureobasidin is decreased under polar acid or base conditions, but the overall tolerable pH range is broader.
3.3.3 sensitivity to proteases
From fig. 14, it can be seen that the change of weed growth inhibition rate is not great after the aureobasidin is treated by 3 enzymes, and there is no significant difference between treatments, indicating that the compound is insensitive to 3 proteases.
3.4 determination of the herbicidal mechanism of aureobasidin
3.4.1 Effect of Aureobasidin on alpha-amylase Activity
The germination test of the plate seeds is carried out on weeds by using aureobasidin, and the alpha-amylase of the seeds is respectively extracted and the activity of the alpha-amylase is measured at the 3 rd, the 4 th, the 5 th, the 6 th and the 7 th days after the treatment. As a result, as shown in table 3, the activity of alpha-amylase in the treated group was significantly inhibited compared to the control, and the inhibition rate was higher than 93%, while it was found that the alpha-amylase activity gradually decreased from the germination period of 3d to the 1 st leaf 1 heart period of 7d, indicating that the alpha-amylase was mainly present in the germination period of seeds. The above results indicate that the compound inhibits alpha-amylase activity upon seed germination, thus inhibiting weed seed germination.
TABLE 3 Effect of aureobasidin on alpha-amylase activity during weed seed germination
Note that: (+/-) standard deviation; different column letters represent the difference significance (P < 0.05).
Effects of 3.4.2 Aureobasidin on acetolactate synthase Activity
The weed seeds were cultured until the 3-leaf stage, the liquid surface spraying treatment was performed by using aureobasidin, and the aerial parts of the weeds were cut out at 3, 5, 7, 9, and 11d after the treatment, and acetolactate synthase (ALS) was extracted and the activity thereof was measured. The results are shown in Table 4. ALS activity in the treated group decreased, and the inhibition rate of ALS enzyme activity gradually increased with the increase of the number of treatment days. The results show that the compound can inhibit ALS activity in weed plants, inhibit biosynthesis thereof and finally lead to death of weeds.
TABLE 4 Effect of aureobasidin on ALS Activity during weed seed growth
Note that: (+/-) standard deviation; different column letters represent the difference significance (P < 0.05).
4. Conclusion(s)
(1) Fermentation optimization production tests show that the optimal cultivation conditions for producing aureobasidin by the aureobasidium pullulans strain PA-2 are as follows: the temperature is 25 ℃, the liquid loading amount is 200mL, the culture time is 7d, the rotation speed of a shaking table is 180r/m, and the yield of aureobasidin is between 0.60g/L and 0.65 g/L. The substance is a cyclic lipopeptid substance, consists of cyclic peptide consisting of 8 alpha-amino acid units and 1 hydroxy acid, and has strong absorption at 220 nm.
(2) The physical and chemical property analysis of the substance shows that the substance has stronger heat resistance, wide tolerance pH value and insensitivity to protease. The weeding mechanism research experiment shows that the compound inhibits the activity of alpha-amylase in the germination period of seeds, simultaneously inhibits the activity of acetolactate, further inhibits the germination of seeds and the growth of weeds, and achieves the weeding purpose.
(3) The weeding activity plate test shows that the seed germination inhibition rate of the aureobasidin on the gramineous weeds is higher than that of the broadleaf weeds, the germ inhibition rate of the aureobasidin on the gramineous weeds reaches more than 88 percent at the concentration of 100 mug/mL, the culture inhibition rate of the aureobasidin on the broadleaf weeds is only about 73 percent, and the germ inhibition rate of the aureobasidin on the gramineous weeds reaches more than 80 percent, and the radicle inhibition rate of the aureobasidin on the broadleaf weeds reaches less than 80 percent. The pot plant weeding activity shows that under the concentration of 100 mug/mL, 5 days after the drug, the investigation shows that the aureobasidin has different degrees of inhibition effects on plant heights, plant prevention effects and fresh weight effects of different weeds, the highest plant height inhibition rate reaches 59%, the plant prevention effects and fresh weight effects on gramineous weeds respectively reach more than 85% and 81%, and the plant prevention effects and fresh weight effects on broadleaf weeds respectively reach more than 73% and 71%.
(4) The field plot test result shows that, at the concentration of 100 mug/mL, after 15 days of spraying, weeds in the treated plot are killed except for sporadic plant growth, weeds in the control plot grow vigorously, and the leaf color is dark green. The grass weeds are found to have good plant prevention effect on the weeds, the prevention effect on the chenopodium album is 90.6%, and the prevention effect on the chenopodium album is the lowest and is only 76.3%; similarly, the fresh weight effect on wild oat is highest and reaches 88.2%, and the fresh weight effect on elsholtzia pachyrhizi is lowest and is 72.9%. In general, the weeding composition has good weed control effect at the concentration of 100 mug/mL, and can be used for further formulation development.
In the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more; the positional or positional relationships indicated by the terms "upper", "lower", "left", "right", "inner", "outer", "front", "rear", "head", "tail", etc., are based on the positional or positional relationships shown in the drawings, are merely for convenience in describing the invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as limiting the invention. Furthermore, the terms "first," "second," "third," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.
The foregoing is merely illustrative of specific embodiments of the present invention, and the scope of the invention is not limited thereto, but any modifications, equivalents, improvements and alternatives falling within the spirit and principles of the present invention will be apparent to those skilled in the art within the scope of the present invention.
Claims (10)
1. The application of aureobasidin in preparing herbicide is characterized in that aureobasidin is aureobasidin A.
2. Use of aureobasidin according to claim 1 for the preparation of herbicides, wherein the preparation process of aureobasidin a comprises:
step one, strain activation and fermentation: preparing spore suspension by using Aureobasidium pullulans PA-2, inoculating the spore suspension into a seed culture medium for activation, and transferring the spore suspension into a fermentation tank for fermentation;
step two, fermentation production optimization: optimizing culture conditions in terms of 4 aspects of temperature, liquid loading amount, culture time and rotating speed;
step three, preparing a crude extract of aureobasidin: taking fermentation liquor, discarding thalli, carrying out oscillation adsorption by using macroporous resin, cleaning the adsorbed resin, and drying under reduced pressure after elution to obtain a crude aureobasidin extract of Aureobasidium pullulans PA-2;
step four, separating and purifying aureobasidin: and (3) carrying out primary separation through thin layer chromatography to obtain a aureobasidin product sample, and then carrying out high performance liquid chromatography analysis on the sample to obtain a pure aureobasidin product.
3. Use of aureobasidin according to claim 2 for the preparation of herbicides, wherein in step one the strain activation and fermentation comprises in particular:
(1) Placing glycerol preservation strain PA-2 at the temperature of minus 80 ℃ in an ice bath, pouring glycerol in an ultra-clean workbench when glycerol is molten, digging a lawn, placing the lawn in a potato glucose solid culture medium, scraping the surface of a bacterial colony at the temperature of 25 ℃ for 3-5 d, and preparing spore suspension;
(2) Inoculating 5mL of spore suspension into 150mL of seed culture medium, and performing shake culture at 25 ℃ and 180r/min for 2d for activation;
(3) 150mL of the seed culture medium is transferred into a 30L fermentation tank containing 15L of fermentation culture medium A, shake culture is carried out for 56h at 25 ℃ under 180r/min, then 2L of fermentation culture medium B is added, shake culture is carried out for 88h at 25 ℃ under 180r/min, and fermentation liquor is obtained.
4. Use of aureobasidin according to claim 3 for the preparation of herbicides, wherein the seed medium comprises 0.67% yeastnitrogenbase and 2% glucose and the potato dextrose solid medium comprises 2% potto, 2% glucose and 1.5% agar;
the fermentation medium A comprises 4% glucose, 3% shimmilk, 3% soybeanflour, 0.5% (NH) 4 ) 2 SO 4 、0.15％KH 2 PO 4 、0.05％MgSO 4 ·7H 2 O、0.01％CaCl 2 ·2H 2 O、0.01％NaCl、0.5μg/mLFeCl 3 ·6H 2 O and 0.5. Mu.g/ml ZnSO 4 ·7H 2 O; the fermentation medium B comprises 10% glucose, 5% polypepton and 0.75% KH 2 PO 4 、0.25％MgSO 4 ·7H 2 O、0.05％CaCl 2 ·2H 2 O、0.05％NaCl、2.5μg/mLFeCl 3 ·6H 2 O and 2.5. Mu.g/ml ZnSO 4 ·7H 2 O。
5. Use of aureobasidin according to claim 2 for the preparation of herbicides, wherein in step three the preparation of a crude extract of aureobasidin comprises in particular:
centrifuging 30L of fermentation broth at 10000r/min, removing thallus, adding 3% macroporous resin XAD-16, shaking at room temperature in shaking table, and standing for 24 hr at 120 r/min; washing the adsorbed resin with distilled water until no fermentation liquor remains on the surface of the resin, then placing the resin in a 1000mL triangular flask with a plug, adding 300mL absolute ethyl alcohol, placing the resin in a shaking table for eluting for 24 hours at room temperature, and then drying the resin under reduced pressure to obtain the aureobasidin crude extract of aureobasidium pullulans PA-2.
6. The use of aureobasidin according to claim 2 for the preparation of herbicides, wherein in step four, the isolation and purification of aureobasidin specifically comprises:
(1) Primary separation by thin layer chromatography: the method comprises the steps of adopting a silica gel plate as a chromatographic stationary phase, dissolving a crude extract and a standard sample of aureobasidin in a small amount of methanol respectively, spotting the crude extract and the standard sample of aureobasidin on the silica gel plate for chromatography, spreading each two plates of the crude extract in a chromatography system for 2 hours, volatilizing the dry reagent, wherein one plate is developed by ninhydrin, the other plate is placed in a high-temperature resistant closed container, 2mL of concentrated hydrochloric acid is contained in a small cup, fumigating the other plate in a 110 ℃ oven for 3 hours for in-situ hydrolysis, cooling and blowing off the hydrochloric acid, developing the color by ninhydrin, fumigating the two plates in the 110 ℃ oven for 1 hour, and confirming the position of aureobasidin; taking an analysis plate subjected to thin layer chromatography under the same conditions, scraping silica gel at a corresponding color development site, collecting silica gel powder, soaking, eluting and concentrating by using methanol to obtain a aureobasidin product sample, and performing high performance liquid chromatography;
(2) High performance liquid chromatography: dissolving the crude extract sample and the standard substance with mobile phase, sterilizing with a pinhole organic filter membrane of 0.22 μm, removing impurities, balancing chromatographic column, adding sample, and performing gradient elution to separate sample compounds, and repeatedly collecting eluate for 10 times according to peak time of the standard sample and the sample;
(3) Identification of the aureobasidin substance structure: measuring the molecular weight of the aureobasidin by a mass spectrometer; 5mg of the sample was dissolved in 0.5mL CD3OD for 1H-NMR detection; 15mg of the sample was dissolved in 0.5mL CD3OD and detected by 13C-NMR.
7. Use of aureobasidin according to claim 1 for verifying the use of aureobasidin in the preparation of herbicides, characterized in that the method of verification of the use of aureobasidin in the preparation of herbicides comprises measurement of herbicidal activity of aureobasidin compounds, measurement of the physicochemical properties of aureobasidin compounds and measurement of the herbicidal mechanism of aureobasidin compounds.
8. The method for verifying the use of aureobasidin in the preparation of herbicides as claimed in claim 7, wherein the measurement of herbicidal activity of aureobasidin compound comprises:
1) Seed germination:
preparation of Aureobasidin into 100, 50, 10 μg/mL with dimethyl sulfoxide 3 1mL of solution with seed concentration is respectively and evenly added into a 12-hole culture plate paved with double-layer filter paper, after the solvent volatilizes, 10 weed seeds after germination acceleration are selected and evenly dispersed into the treated culture plate holes so as to obtain the plant seed culture plateFresh water was used as a control, each treatment was repeated 4 times, a 12-well culture plate was placed in a constant temperature incubator at 25℃and a relative humidity of 70% for cultivation, the lengths of radicle and embryo of weed seeds were measured after 3 days, the radicle and embryo inhibition rate was calculated using the following,
2) And (3) measuring the weeding effect of the potted plants:
sowing the test weed seeds in small pots, wherein each pot is 20-30 plants/pot; when various plants grow to 10cm high, using aureobasidin with the concentration of 100, 50 and 10 mug/mL to spray leaf surfaces of weed plants, and spraying clear water in a contrast mode when the leaf surfaces are wet and the liquid medicine is not dropped; the treatment is repeated for 4 times, the plant height, the plant number and the fresh weight are measured after 7 days of drug application, the plant height inhibition rate, the plant prevention effect and the fresh weight effect are calculated, and meanwhile, the symptom of weed poisoning is observed,
3) Field plot weeding test:
the field test is carried out in a test field, the random block arrangement is carried out, and the cell area is 8m 2 Treating with aureobasidin with concentration of 100 μg/mL, repeating the treatment with clear water for 4 times, spraying when weeds grow 3-5 leaves, and applyingAfter 15 days, the plant number and fresh weight of the weeds are measured respectively, and plant control efficiency and fresh weight efficiency are calculated.
9. The method for verifying the use of aureobasidin in the preparation of a herbicide according to claim 7, wherein the determination of the compound physicalization property of aureobasidin comprises:
1) Sensitivity to temperature determination:
treating aureobasidin at 25deg.C, 35deg.C, 55deg.C, 65deg.C, 85deg.C, 100deg.C and 121deg.C for 30min, cooling to room temperature, detecting herbicidal activity by seed germination method, and observing herbicidal activity change with sterilized water treated at corresponding temperature;
2) Sensitivity to acid, base assay:
respectively regulating pH values of aureobasidin to 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0 and 12.0 by using 6M HCl or NaOH, standing for 15min, centrifuging for 10min at 10000r/min, retaining supernatant, detecting weeding activity by using a seed germination method, and observing weeding activity change by using sterile water with the corresponding pH value as a reference;
3) Sensitivity assay to protease:
treating aureobasidin with trypsin, pepsin, proteinase K at 37deg.C for 1 hr, and 94 deg.C for 10min to stop enzyme reaction with concentration of 500 μg/mL; supernatant was retained by centrifugation at 12000r/min for 5min, the herbicidal activity was examined, and the change in herbicidal activity was observed by using various protease-treated sterile water as a control.
10. The method for validating the use of aureobasidin in the preparation of herbicides as claimed in claim 7, wherein the determination of the herbicidal mechanism of aureobasidin compound comprises determining the effect of aureobasidin compound on alpha-amylase activity and determining the effect of aureobasidin compound on acetolactate synthase activity.
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