阅读说明：本技术 一种具有除草作用的短梗霉素及其制备方法、测定方法 (Aureobasidin with weeding effect and preparation method and determination method thereof ) 是由 程亮 魏有海 郭青云 朱海霞 翁华 郭良芝 于 2021-01-11 设计创作，主要内容包括：本发明属于除草剂制备技术领域,公开了一种具有除草作用的短梗霉素及其制备方法,利用出芽短梗霉菌PA-2制备孢子悬浮液,接种于种子培养基中进行活化,转入到发酵罐中进行发酵；从温度、装液量、培养时间和转速4个方面对培养条件进行优化；取发酵液,弃去菌体,利用大孔树脂进行振荡吸附,对吸附后的树脂进行清洗,洗脱后减压干燥,即为出芽短梗霉菌PA-2的短梗霉素粗提物；短梗霉素的分离纯化。本发明提供的具有除草作用的短梗霉素具有较强的耐热性,耐受pH值广泛,对蛋白酶不敏感,该化合物抑制种子萌发时期α-淀粉酶活性,同时抑制乙酰乳酸酶活性,进而抑制种子的萌发和杂草的生长,达到除草的目的。(The invention belongs to the technical field of herbicide preparation, and discloses aureobasidin with a weeding effect and a preparation method thereof, wherein aureobasidin PA-2 is used for preparing a spore suspension, inoculated in a seed culture medium for activation, and transferred into a fermentation tank for fermentation; optimizing culture conditions in 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, eluting, and drying under reduced pressure to obtain an aureobasidin crude extract of aureobasidium pullulans PA-2; and (5) separating and purifying aureobasidin. The aureobasidin with the weeding effect provided by the invention has stronger heat resistance, wide tolerance pH value and insensitivity to protease, and the compound inhibits the activity of alpha-amylase in the seed germination period and the activity of acetolactate enzyme, so that the germination of seeds and the growth of weeds are inhibited, and the weeding purpose is achieved.)

1. A method for preparing herbicidal aureobasidin, characterized in that it 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 activated spore suspension into a fermentation tank for fermentation;

step two, fermentation production optimization: optimizing culture conditions in 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, eluting, and drying under reduced pressure to obtain an aureobasidin crude extract of aureobasidium pullulans PA-2;

step four, separating and purifying aureobasidin: and (3) carrying out primary separation by thin-layer chromatography to obtain an aureobasidin product sample, and then processing the sample by high performance liquid chromatography to obtain an aureobasidin pure product.

2. The method for preparing herbicidally active aureobasidin according to claim 1, wherein in step one, the strain activation and fermentation specifically comprises:

(1) placing a-80 ℃ glycerol preservation strain PA-2 in an ice bath device, pouring off glycerol in a super clean workbench when the glycerol is in a molten state, digging a lawn, placing the lawn in a potato glucose solid culture medium, scraping the surface of the colony for 3-5 days at 25 ℃, and preparing a spore suspension;

(2) inoculating 5mL of spore suspension into 150mL of seed culture medium, and performing shaking culture at 25 ℃ and 180r/m for 2d for activation;

(3) transferring 150mL of seed culture medium into a 30L fermentation tank containing 15L of fermentation medium A, performing shake culture at 25 deg.C and 180r/m for 56h, adding 2L of fermentation medium B, and performing shake culture at 25 deg.C and 180r/m for 88h to obtain fermentation liquid.

3. The method for preparing herbicidally effective aureobasidin according to claim 2, wherein the seed culture medium comprises 0.67% yeast reagent base and 2% glucose, the potato dextrose solids medium comprises 2% potato, 2% glucose and 1.5% agar;

the fermentation medium A comprises 4% glucose, 3% skim mil, 3% soybean flours and 0.5% (NH)₄)₂SO₄、0.15％KH₂PO₄、0.05％MgSO₄·7H₂O、0.01％CaCl₂·2H₂O、0.01％NaCl、0.5μg/mL FeCl₃·6H₂O and 0.5. mu.g/mL ZnSO₄·7H₂O; the fermentation medium B comprises 10% glucose, 5% polypepton and 0.75% KH₂PO₄、0.25％MgSO₄·7H₂O、0.05％CaCl₂·2H₂O、0.05％NaCl、2.5μg/mL FeCl₃·6H₂O and 2.5. mu.g/mL ZnSO₄·7H₂O。

4. The method for preparing aureobasidin with herbicidal effect as claimed in claim 1, wherein in step three, the preparation of the crude extract of aureobasidin specifically comprises:

centrifuging 30L of fermentation liquid at 10000r/m, discarding thallus, adding 3% macroporous resin XAD-16, shaking at room temperature in a shaking table for 24h at 120 r/m; and (3) cleaning the adsorbed resin with distilled water until no fermentation liquor residue exists 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 at room temperature for elution for 24h, and then drying the resin under reduced pressure to obtain the aureobasidin crude extract of the aureobasidium pullulans PA-2.

5. The method for producing an herbicidal aureobasidin according to claim 1, wherein in step four, the isolation and purification of the aureobasidin specifically comprises:

(1) primary separation by thin layer chromatography: adopting a silica gel plate as a chromatographic stationary phase, dissolving a crude extract and an aureobasidin standard sample in a small amount of methanol respectively, spotting the crude extract and the aureobasidin standard sample on the silica gel plate to carry out chromatography, spreading the crude extract on two plates at each point in a chromatographic system for 2 hours, volatilizing the dry reagents, developing the color of one of the crude extract and the aureobasidin standard sample by ninhydrin, putting the other one of the crude extract and the aureobasidin standard sample in a high-temperature resistant closed container, holding about 2mL of concentrated hydrochloric acid by a small cup, putting the small cup into a 110 ℃ oven to fumigate for 3 hours for in-situ hydrolysis, cooling, blowing off hydrochloric acid, developing the color by ninhydrin, fumigating for 1 hour in the 110 ℃ oven to observe the result, and confirming the position of aureobasidin; taking an analysis plate after the thin-layer chromatography under the same condition, scraping silica gel from a corresponding color development site, collecting silica gel powder, soaking, eluting and concentrating the silica gel powder by using methanol to obtain an aureobasidin product sample, and performing high performance liquid chromatography analysis;

(2) high performance liquid chromatography analysis: dissolving the crude extract sample and standard substance with mobile phase, filtering with 0.22 μm pinhole organic filter membrane for removing bacteria and impurities, balancing chromatographic column, adding sample, gradient eluting to separate sample compounds, and repeatedly collecting eluate for 10 times according to peak time of standard sample and sample;

(3) identifying the structure of the aureobasidin substance: measuring the molecular weight of the aureobasidin by adopting a mass spectrometer; weighing sample 5mg dissolved in 0.5mL CD₃In OD¹H-NMR detection; sample was weighed 15mg dissolved in 0.5mL CD₃In OD¹³C-NMR detection.

6. A herbicidal aureobasidin prepared by the method for preparing a herbicidal aureobasidin according to claims 1 to 5.

7. An assay method for the herbicidally effective aureobasidin according to claim 6, wherein the assay method for the herbicidally effective aureobasidin comprises an assay of herbicidal activity of the aureobasidin compound, an assay of physicochemical properties of the aureobasidin compound and an assay of herbicidal mechanism of the aureobasidin compound.

8. The method for assaying herbicidally-effective aureobasidin according to claim 7, wherein the aureobasidin compound herbicidal activity assay comprises:

1) a seed germination method:

preparing aureobasidin into 3 solutions with concentrations of 100, 50 and 10 mu g/mL by using dimethyl sulfoxide, respectively uniformly adding 1mL of solution to be detected into a 12-hole culture plate paved with double-layer filter paper, after the solvent is dried, selecting 10 weed seeds subjected to germination acceleration to be uniformly dispersed in holes of the treated culture plate, using clear water as a contrast, repeating the treatment for 4 times, placing the 12-hole culture plate into a constant-temperature culture box with the temperature of 25 ℃ and the relative humidity of 70 percent for culture, measuring the length of radicle and germ of the weed seeds after 3 days, calculating the inhibition rate of the radicle and the germ by using the following formula,

2) and (3) determining the weeding effect of the pot culture:

respectively sowing the test weed seeds in small pots, wherein each pot is 20-30 plants/pot; when various plants grow to 10cm high, spraying aureobasidin with concentration of 100, 50 and 10 mug/mL on the leaf surfaces of the weed plants, and spraying clear water in contrast to the condition that the leaf surfaces are wet but the liquid medicine does not drip; repeating the treatment for 4 times, measuring plant height, plant number and fresh weight 7 days after application, calculating plant height inhibition rate, plant control effect and fresh weight effect, observing weed poisoning symptoms,

3) weeding test in field plot:

the field test is carried out in a test field, the test field is randomly arranged in blocks, and the area of each block is 8m²Treating with the aureobasidin concentration of 100 mu g/mL, taking clear water as a reference, repeating the treatment for 4 times each time, spraying when the weeds grow 3-5 leaves, measuring the plant number and fresh weight of the weeds 15 days after application, and calculating the plant control effect and the fresh weight effect.

9. The method for assaying herbicidally effective aureobasidin according to claim 7, wherein the determination of the physicochemical properties of the aureobasidin compound comprises:

1) determination of the sensitivity to temperature:

respectively treating aureobasidin at 25 deg.C, 35 deg.C, 55 deg.C, 65 deg.C, 85 deg.C, 100 deg.C and 121 deg.C for 30min, cooling to room temperature, detecting its herbicidal activity by seed germination method, and observing its herbicidal activity change with sterile water treated at corresponding temperature as control;

2) determination of sensitivity to acid and base:

respectively adjusting the 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 by using HCI or NaOH with the concentration of 6M, standing for 15min, centrifuging at 10000r/min for 10min, reserving supernate, detecting the herbicidal activity of the aureobasidin by adopting a seed germination method, and observing the change of the herbicidal activity by using sterile water with the corresponding pH value as a reference;

3) determination of the sensitivity to proteases:

respectively treating aureobasidin with trypsin, pepsin and proteinase K at 37 deg.C for 1h, and treating at 94 deg.C for 10min to stop enzyme reaction, wherein the enzyme reaction concentration is 500 μ g/mL; centrifuging at 12000r/m for 5min to retain supernatant, detecting its herbicidal activity, and observing its herbicidal activity change with sterile water treated with various proteases as control.

10. The method for assaying aureobasidin with herbicidal action according to claim 7, wherein the assaying of the herbicidal mechanism of the aureobasidin compound comprises assaying the influence of the aureobasidin compound on the activity of α -amylase and assaying the influence of the aureobasidin compound on the activity of acetolactate synthase.

Technical Field

The invention belongs to the technical field of herbicide preparation, and particularly relates to aureobasidin with a weeding effect, and a preparation method and a determination method thereof.

Background

At present: weeds are an important factor that compromises agricultural production, and crop yield and quality are severely affected by competing with the crop for essential conditions for growth such as moisture, nutrients and light. At present, chemical weeding is mainly used for farmland weed control in China, the annual input cost of control is 235 million yuan, the yield of grains is reduced by 5000 million tons, and billions of yuan is directly lost economically. The increase of agricultural efficiency still depends on the application of chemical herbicide, so that the development of novel efficient and environment-friendly green herbicide has important practical significance. In recent years, the microbial herbicide gradually enters the sight of people due to the advantages of small side effect and easy degradation on the environment, and becomes a main trend in green agriculture and the idea of reassuring agricultural products. The aureobasidin is a cyclic peptide antibiotic produced by aureobasidium pullulans, shows strong antibacterial activity, and mainly can inhibit the activity of inositol phosphatidyl ceramide synthetase of fungi strongly, so that sphingomyelin substances are insufficiently synthesized, a cell membrane structure is damaged, and intracellular substances are leaked out to cause the death of the fungi. Therefore, the antibiotic is often used as a biocontrol agent and a preservative in agriculture and food. However, studies on the application of aureobasidin to the herbicidal activity and the herbicidal mechanism thereof have not been reported so far. The invention aims to expand the application of an aureobasidin compound and provide a new application of the aureobasidin compound as an agricultural herbicide.

Through the above analysis, the problems and defects of 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 the herbicidal activity.

The difficulty in solving the above problems and defects is: the inventor carries out intensive research on the preparation method and the bioassay method of the compound and the activity of various weeds, and the experimental result shows that the aureobasidin has better control effect on the weeds, thereby providing a new thought for developing the compound into a novel herbicide.

Disclosure of Invention

Aiming at the problems 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 the weeding effect and the preparation method thereof comprise the following steps:

step one, strain activation and fermentation: utilizing aureobasidium pullulans PA-2 (with the preservation number of CGMCC No.8413, preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms No. 3 of Xilu No. 1 Hospital of North Chen in the south-oriented region in China, Beijing city for 28 days in 2013 and 10 months), preparing a spore suspension, inoculating the spore suspension into a seed culture medium for activation, and then transferring the activated spore suspension into a fermentation tank for fermentation;

step two, fermentation production optimization: optimizing culture conditions from 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, performing oscillation adsorption by using macroporous resin, cleaning the adsorbed resin, and performing reduced pressure drying after elution to obtain an aureobasidin crude extract of aureobasidium pullulans PA-2;

step four, separating and purifying aureobasidin: obtaining an aureobasidin product sample through thin-layer chromatography initial separation, 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 comprise:

(1) placing a-80 ℃ glycerol preservation strain PA-2 in an ice bath device, pouring off glycerol in a super clean workbench when the glycerol is in a molten state, digging a lawn, placing the lawn in a potato glucose solid culture medium, scraping the surface of the colony for 3-5 days at 25 ℃, and preparing a spore suspension;

(2) inoculating 5mL of spore suspension into 150mL of seed culture medium, and performing shaking culture at 25 ℃ and 180r/m for 2d for activation;

(3) transferring 150mL of seed culture medium into a 30L fermentation tank containing 15L of fermentation medium A, performing shake culture at 25 deg.C and 180r/m for 56h, adding 2L of fermentation medium B, and performing shake culture at 25 deg.C and 180r/m for 88h to obtain fermentation liquid.

Further, the seed medium comprises 0.67% yeast reagent base and 2% glucose, the PDA solid medium comprises 2% potato, 2% glucose and 1.5% agar;

the fermentation medium A comprises 4% glucose, 3% skim mil, 3% soybean flours and 0.5% (NH)₄)₂SO₄、0.15％KH₂PO₄、0.05％MgSO₄·7H₂O、0.01％CaCl₂·2H₂O、0.01％NaCl、 0.5μg/mL FeCl₃·6H₂O and 0.5. mu.g/mL ZnSO₄·7H₂O; the fermentation medium B comprises 10% glucose, 5% polypepton and 0.75% KH₂PO₄、0.25％MgSO₄·7H₂O、0.05％CaCl₂·2H₂O、 0.05％NaCl、2.5μg/mL FeCl₃·6H₂O and 2.5. mu.g/mL ZnSO₄·7H₂O。

Further, in the third step, the preparation of the crude extract of aureobasidin specifically comprises:

centrifuging 30L of fermentation liquid at 10000r/m, discarding thallus, adding 3% macroporous resin XAD-16, shaking at room temperature in a shaking table for 24h at 120 r/m; and (3) cleaning the adsorbed resin with distilled water until no fermentation liquor residue exists 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 at room temperature for elution for 24h, and then drying the resin under reduced pressure to obtain the aureobasidin crude extract of the aureobasidium pullulans PA-2.

Further, in the fourth step, the separation and purification of the aureobasidin specifically comprises:

(1) primary separation by thin layer chromatography: adopting a silica gel plate as a chromatographic stationary phase, dissolving a crude extract and an aureobasidin standard sample in a small amount of methanol respectively, spotting the crude extract and the aureobasidin standard sample on the silica gel plate for chromatography, spreading two plates of each point of the crude extract in a chromatographic system for 2h, volatilizing the reagents, then developing the color of one plate by ninhydrin, putting the other plate in a high-temperature-resistant closed container, holding about 2mL of concentrated hydrochloric acid in a small cup, fumigating the solution in an oven at 110 ℃ for 3h for in-situ hydrolysis, cooling, blowing off hydrochloric acid, developing the color by ninhydrin, fumigating the solution in the oven at 110 ℃ for 1h for observation, and confirming the position of aureobasidin; scraping silica gel from the corresponding color development site and collecting silica gel powder, soaking in methanol, eluting, and concentrating to obtain Aureobasidium pullulans sample for high performance liquid chromatography analysis;

(2) high performance liquid chromatography analysis: dissolving an aureobasidin sample and a standard substance by using a mobile phase, then sterilizing and removing impurities by passing through a 0.22 mu m pinhole type organic filter membrane, balancing a chromatographic column, then adding a sample and the like for gradient elution to separate a sample compound, and repeatedly collecting sample peaks for 10 times according to the peak emergence time of the standard sample and a crude extraction sample;

(3) identifying the structure of aureobasidin: measuring the molecular weight of the aureobasidin by adopting a mass spectrometer; weighing sample 5mg dissolved in 0.5mL CD₃In OD¹H-NMR detection; sample was weighed 15mg dissolved in 0.5mL CD₃In OD¹³C-NMR detection.

Another object of the present invention is to provide a method for measuring herbicidal aureobasidin, which comprises measuring herbicidal activity of an aureobasidin compound, measuring physicochemical properties of the aureobasidin compound, and measuring a herbicidal mechanism of the aureobasidin compound.

Further, the determination of the herbicidal activity of the aureobasidin compound comprises:

1) a seed germination method:

preparing aureobasidin into 3 solutions with concentrations of 100, 50 and 10 μ g/mL with dimethyl sulfoxide, respectively adding 1mL of solution to be tested into a 12-hole culture plate paved with double-layer filter paper, after the solvent is dried, selecting 10 weed seeds subjected to germination acceleration, uniformly dispersing in the holes of the treated culture plate, performing treatment for 4 times by using clear water as a contrast, culturing the 12-hole culture plate in a constant temperature incubator with the temperature of 25 ℃ and the relative humidity of 70%, measuring the length of the radicle and the germ of the weed seeds after 3 days, calculating the inhibition rate of the radicle (bud) by using the following formula,

2) and (3) determining the weeding effect of the pot culture:

respectively sowing the test weed seeds in small pots, wherein each pot is 20-30 plants/pot; when various plants grow to 3-5 leaf stages, respectively spraying aureobasidin leaf surfaces with the concentrations of 100, 50 and 10 mu g/mL to weed plants, and spraying clear water in contrast at the degree that the leaf surfaces are wet but liquid medicine does not drip; repeating the treatment for 4 times, measuring plant height, plant number and fresh weight 7 days after the application of the herbicide, calculating the inhibition rate of plant height, the prevention effect of plant and the fresh weight effect, observing the toxic symptoms of weeds,

3) weeding test in field plot:

the field test is carried out in a test field, the test field is randomly arranged in blocks, and the area of each block is 8m²Treating with the aureobasidin at the concentration of 100 mu g/mL, taking clear water as a reference, repeating the treatment for 4 times each time, spraying when the weeds grow 3-5 leaves, measuring the plant number and fresh weight of the weeds 15 days after application, and calculating the plant control effect and the fresh weight effect.

Further, the determination of the physicochemical properties of the aureobasidin compound comprises:

1) determination of the sensitivity to temperature:

respectively treating aureobasidin at 25 deg.C, 35 deg.C, 55 deg.C, 65 deg.C, 85 deg.C, 100 deg.C and 121 deg.C for 30min, cooling to room temperature, detecting its herbicidal activity by seed germination method, and observing its herbicidal activity change with sterile water treated at corresponding temperature as control;

2) determination of sensitivity to acid and base:

respectively adjusting the 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 by using HCI or NaOH with the concentration of 6M, standing for 15min, centrifuging at 10000r/min for 10min, reserving supernate, detecting the herbicidal activity of the aureobasidin by adopting a seed germination method, and observing the change of the herbicidal activity by using sterile water with the corresponding pH value as a reference;

3) determination of the sensitivity to proteases:

respectively treating aureobasidin with trypsin, pepsin and proteinase K at 37 deg.C for 1h, and treating at 94 deg.C for 10min to stop enzyme reaction, wherein the enzyme reaction concentration is 500 μ g/mL; centrifuging at 12000r/m for 5min, collecting supernatant, detecting herbicidal activity by seed germination method, and observing herbicidal activity change with sterile water treated with protease as control.

Further, the determination of the herbicidal mechanism of the aureobasidin compound includes determining the influence of the aureobasidin compound on the activity of the α -amylase and determining the influence of the aureobasidin compound on the activity of the acetolactate synthase.

By combining all the technical schemes, the invention has the advantages and positive effects that:

the aureobasidin with the weeding effect provided by the invention has stronger heat resistance, wide tolerance pH value and insensitivity to protease, and the compound inhibits the activity of alpha-amylase in the seed germination period and the activity of acetolactate enzyme, so that the germination of seeds and the growth of weeds 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 on the gramineous weeds at the concentration of 100 mu g/mL reaches more than 88%, the cultivation inhibition rate on the broadleaf weeds is only about 73%, and similarly, the germ inhibition rate of the compound on the gramineous weeds reaches more than 80%, and the radicle inhibition rate on the broadleaf weeds reaches less than 80%. The potted plant weeding activity shows that, under the concentration of 100 mug/mL, the investigation 5 days after the application shows that the aureobasidin has different degrees of inhibition effects on the plant height, the plant control effect and the fresh weight effect of different weeds, the highest plant height inhibition rate reaches 59%, the plant control effect and the fresh weight effect of the gramineous weeds reach more than 85% and 81%, and the plant control effect and the fresh weight effect of the broadleaf weeds reach more than 73% and 71%, respectively.

The test result of the field plot shows that under the concentration of 100 mu g/mL, 15 days after spraying, the weeds in the treated plot are killed except for the growth of the plants in the Zenita, the weeds in the control plot grow vigorously, and the leaves are dark green. The grass weeds are found to have better plant control effect on the weeds, which is 90.6 percent, and the control effect on the chenopodium album is the lowest and is only 76.3 percent; similarly, the fresh weight efficiency of wild oat is the highest and reaches 88.2%, and the fresh weight efficiency of elsholtzia densa is the lowest and reaches 72.9%. In general, the weeding composition has good weed control effect under the concentration of 100 mu g/mL, and can be subjected to the next formulation development.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings used in the embodiments of the present application will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present application, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a flow chart of a method for preparing aureobasidin with herbicidal effect according to the embodiment of the present invention.

FIG. 2 is a schematic diagram showing the effect of various conditions on the yield of aureobasidin, which is provided in the examples of the present invention.

FIG. 3 is a UV absorption spectrum of aureobasidin according to the present invention.

FIG. 4 is a mass spectrum of an aureobasidin compound provided in the examples of the present invention.

FIG. 5 is a nuclear magnetic map of an aureobasidin compound provided in an example of the present invention.

FIG. 6 is a schematic structural diagram of aureobasidin provided in the examples of the present invention.

FIG. 7 is a schematic representation of the inhibition of germination of germs of weed seeds by aureobasidin at various concentrations provided by the embodiments of the present invention.

FIG. 8 is a schematic diagram of the inhibition effect of aureobasidin at different concentrations on the radicle germination of weed seeds provided by the embodiment of the present invention.

FIG. 9 is a schematic diagram showing the effect of spraying aureobasidin at a concentration of 100. mu.g/mL on weed plants, as provided in the examples of the present invention.

FIG. 10 is a graph showing the effect of spraying aureobasidin at a concentration of 100. mu.g/mL on the number of weed plants and fresh weight, as provided in the examples of the present invention.

FIG. 11 is a schematic diagram of the weeding effect of the sesamomycin sprayed on the field plot.

FIG. 12 is a schematic illustration of the effect of temperature on the biological activity of aureobasidin as provided in the examples of the present invention.

FIG. 13 is a graphical representation of the effect of pH on the biological activity of aureobasidin as provided in the examples of the present invention.

FIG. 14 is a schematic diagram showing the effect of protease on the biological activity of aureobasidin according to the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Aiming at the problems in the prior art, the invention provides aureobasidin with weeding effect and a preparation method thereof, and the invention is described in detail with reference to the attached drawings.

As shown in fig. 1, the preparation method of aureobasidin with herbicidal effect provided by the embodiment of the present invention comprises:

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 activated spore suspension into a fermentation tank for fermentation;

s102, fermentation production optimization: optimizing culture conditions in 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, eluting, and drying under reduced pressure to obtain an aureobasidin crude extract of aureobasidium pullulans PA-2;

s104, separating and purifying aureobasidin: obtaining a crude aureobasidin sample through thin-layer chromatography primary separation, and then analyzing and processing the sample through high performance liquid chromatography to obtain a pure aureobasidin product.

The invention is further described with reference to specific examples.

1. Test strain and culture medium

Test strains: aureobasidium pullulans PA-2 is separated and stored in a key laboratory of the integrated control of agricultural harmful organisms in Qinghai province.

Target grass: wild oat, sparrow, barnyard grass, green bristlegrass, chenopodium album, wild rape, wild carrot, purpleflower herb, elsholtzia herb and cleavers.

PDA culture medium: 2% potato, 2% glucose and 1.5% agar.

Seed culture medium: 0.67% Yeast Nitrogen Base and 2% glucose.

Fermentation medium a: 4% glucose, 3% skim mil, 3% soybean flow, 0.5% (NH)₄)₂SO₄、 0.15％KH₂PO₄、0.05％MgSO₄·7H₂O、0.01％CaCl₂·2H₂O、0.01％NaCl、0.5μg/mL FeCl₃·6H₂O and 0.5. mu.g/mL ZnSO₄·7H₂O。

Fermentation medium B: 10% glucose, 5% polypepton, 0.75% KH₂PO₄、0.25％ MgSO₄·7H₂O、0.05％CaCl₂·2H₂O、0.05％NaCl、2.5μg/mL FeCl₃·6H₂O and 2.5. mu.g/mL ZnSO₄·7H₂O。

2. Method and step

2.1 Strain PA-2 activation and fermentation

(1) Placing a-80 ℃ glycerol preservation strain PA-2 in an ice bath device, pouring off glycerol in a super clean workbench when the glycerol is in a molten state, digging a lawn, placing the lawn in a potato glucose solid culture medium, scraping the surface of the colony for 3-5 days at 25 ℃, and preparing a spore suspension.

(2) 5mL of the spore suspension was inoculated into 150mL of seed medium and cultured at 25 ℃ under shaking at 180r/m for 2 days for activation.

(3) 150mL of the seed culture was transferred to a 30L fermentor containing 15L of fermentation medium A and cultured with shaking at 25 ℃ and 180r/m for 56 hours. Then 2L of fermentation medium B is added, and the shaking culture is continued for 88h under the conditions of 25 ℃ and 180r/m, thus obtaining the fermentation liquor.

2.2 fermentation production optimization

The culture conditions were optimized in 4 aspects of temperature, liquid loading amount, culture time and rotation speed. A single-factor test was conducted by setting the temperature (23 ℃, 25 ℃, 27 ℃, 30 ℃), the liquid loading (150mL, 200mL, 250mL, 300mL), the culture time (5d, 7d, 9d, 11d), and the rotational speed of the shaker (140r/m, 160r/m, 180r/m, 200r/m, 220r/m), and the influence of each factor on the yield of the aureobasidin substance was examined.

2.3 aureobasidin crude extract preparation and yield calculation

Centrifuging 30L fermentation liquid at 10000r/m, discarding thallus, adding 3% macroporous resin XAD-16, shaking at room temperature in shaking table at 120r/m for 24 hr. And (3) cleaning the adsorbed resin with distilled water until no fermentation liquor residue exists 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 at room temperature for elution for 24h, and then drying the resin under reduced pressure to obtain the aureobasidin crude extract of the aureobasidium pullulans PA-2. Weighing an empty sample bottle with a label, and recording the mass M of the sample bottle on the label₁(ii) a Placing the crude AbA extract in a sample bottle, and marking M₂。PA-2, obtaining the yield formula of the fermentation crude extract aureobasidin as M ═ M₂-M₁(ii) a Yield Y-M/L (L is the total volume of the fermentation broth).

2.4 isolation and purification of aureobasidin

(1) Thin layer chromatography preliminary separation

Using a Silica Gel plate (Silica Gel GF)₂₅₄20cm × 5cm) as stationary chromatographic phase, and the chromatographic reagent is dichloromethane: methanol: water 65: 25: 4 (v: v). Firstly, respectively dissolving an aureobasidin crude extract and an aureobasidin standard sample in a small amount of methanol, spotting on a silica gel plate for chromatography, spotting two plates on each crude extract, developing the crude extract in a chromatography system for 2 hours, volatilizing a dry reagent, then putting one of the crude extract and the standard sample into a high-temperature-resistant closed container, holding about 2mL of concentrated hydrochloric acid in a small cup, fumigating the concentrated hydrochloric acid in an oven at 110 ℃ for 3 hours for in-situ hydrolysis, then cooling, blowing off hydrochloric acid, developing the color with the ninhydrin, fumigating the mixture in the oven at 110 ℃ for 1 hour for observation, and confirming the position of aureobasidin. Taking an analysis plate subjected to thin-layer chromatography under the same condition, scraping silica gel from the corresponding chromogenic site, collecting silica gel powder, soaking in methanol, eluting, and concentrating to obtain an aureobasidin sample, and performing high performance liquid chromatography analysis.

(2) High performance liquid chromatography

Chromatographic conditions are as follows: ODS-silica C₁₈A chromatographic column (4.6mm × 250mm, 5 μ L, pH 1.0-14.0);

mobile phase: a: 70% -95%, B: 30 to 5 percent

A: water (containing 0.1% trifluoroacetic acid (TFA))

B: methanol; 0-30 min;

flow rate: 1 mL/min; column temperature: 30 ℃; sample introduction volume: 10 μ L

Detection wavelength: 210 nm.

Sample treatment: dissolving the crude aureobasidin sample and the standard substance by using a mobile phase, then passing through a 0.22 mu m pinhole type organic filter membrane for degerming and impurity removal, balancing a chromatographic column, then adding the sample for gradient elution and separation, and repeatedly collecting sample peaks for 10 times according to the peak emergence time of the standard sample and the sample.

(4) Identification of aureobasidin substance structure

The molecular weight of aureobasidin (AbA) was determined using a Mariner System 5304 mass spectrometer. Adopts Bruker UltraShied of Germany^TMAnd detecting by a 400Plus nuclear magnetic resonance spectrometer (TMS is an internal standard).¹H-NMR detection, 5mg of the sample was weighed and dissolved in 0.5mL of CD₃In OD.¹³For C-NMR detection, 15mg of a sample was weighed out and dissolved in 0.5mL of CD₃Detection in OD.

2.5 determination of herbicidal Activity of aureobasidin

2.5.1 seed Germination method

Preparing aureobasidin into 3 solutions with concentrations of 100, 50 and 10 mu g/mL by using dimethyl sulfoxide (DMSO), respectively and uniformly adding 1mL of solution to be detected into a 12-hole culture plate paved with double-layer filter paper, after the solvent is volatilized, selecting 10 weed seeds subjected to germination acceleration to uniformly disperse in the well-treated culture plate holes, and repeating the treatment for 4 times by using clear water as a control. Culturing 12-hole culture plate in a constant temperature incubator with 25 deg.C and 70% relative humidity, measuring the length of the radicle and embryo of the weed seed after 3 days, and calculating the inhibition rate of the radicle (bud).

2.5.2 weeding effect of potted plant

Respectively sowing the test weed seeds in small pots, wherein each pot is 20-30 plants/pot; when various weeds grow to 3-5 leaf stages, respectively spraying aureobasidin leaf surfaces with the concentrations of 100, 50 and 10 mu g/mL to weed plants, and spraying clear water in contrast to the spraying degree of the liquid medicine without dripping when the leaf surfaces are wet. Each treatment was repeated 4 times. And measuring the plant height, the plant number and the fresh weight 7 days after the application of the herbicide, calculating the inhibition rate of the plant height, the control effect of the plant and the fresh weight effect, and observing the toxicity symptoms of the weeds.

2.5.3 field plot weeding test

The field test is carried out in a test field of a plant protection institute of an agricultural academy, and is randomly arranged in blocks, and the area of each block is 8m²Treating with the aureobasidin at the concentration of 100 mu g/mL, taking clear water as a reference, repeating the treatment for 4 times each time, spraying when the weeds grow 3-5 leaves, measuring the plant number and fresh weight of the weeds 15 days after application, and calculating the plant control effect and the fresh weight effect.

2.6 determination of physicochemical Properties of aureobasidin

(1) Sensitivity to temperature

Treating aureobasidin at 25 deg.C, 35 deg.C, 55 deg.C, 65 deg.C, 85 deg.C, 100 deg.C and 121 deg.C for 30min, cooling to room temperature, detecting its herbicidal activity by seed germination method, and observing its herbicidal activity change with sterile water treated at corresponding temperature as control.

(2) Sensitivity to acid and alkali

Respectively adjusting the 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 by using HCI or NaOH with the concentration of 6M, standing for 15min, centrifuging at 10000r/min for 10min, reserving supernate, detecting the herbicidal activity by adopting a seed germination method, and observing the change of the herbicidal activity by using sterile water with the corresponding pH value as a reference.

(3) Sensitivity to proteases

Respectively treating aureobasidin with trypsin, pepsin and proteinase K at 37 deg.C for 1h, and treating at 94 deg.C for 10min to stop enzyme reaction, wherein the enzyme reaction concentration is 500 μ g/mL. Centrifuging at 12000r/m for 5min, collecting supernatant, detecting herbicidal activity by seed germination method, and observing herbicidal activity change with sterile water treated with protease as control.

2.7 determination of the weeding mechanism of aureobasidin

2.7.1 Effect of aureobasidin on alpha-amylase Activity

(1) And (3) treating the weed seeds with the aureobasidin diluent, and culturing for 3-8 days by using sterile water as a reference, wherein the alpha-amylase is extracted every day.

(2) Weighing about 0.1g of sample, adding 0.8mL of distilled water, and homogenizing; homogenizing, standing at room temperature for 15min, and oscillating for 1 time every 5min to extract; 6000g, centrifuging for 10min at normal temperature, sucking supernatant, adding distilled water to a constant volume of 10mL, and shaking up to obtain the amylase stock solution.

(3) The alpha-amylase activity was measured by taking 12 tubes, 6 for the treatment group and 6 for the control group, and labeling each. 1mL of crude enzyme solution is absorbed into a test tube, the test tube is placed in a water bath at 70 ℃ for 15min to ensure that alpha-amylase is heated to generate passivation, and the test tube is quickly taken out after the water bath and is cooled in tap water; sucking 4mL of citric acid buffer solution with the pH value of 5.6 and adding the citric acid buffer solution into a test tube; sucking 4mL of 0.4M NaOH solution into two groups of test tubes; inactivating the activity of the enzyme, and sucking 2mL of 1% starch into a test tube; the test tubes of the treatment group are placed in a water bath at 40 ℃ for 15min, then are added into 2mL (40 ℃) of preheated 1% starch, are uniformly mixed, are subjected to a water bath at 40 ℃ for 13-15 min, and finally are added with 4mL of 0.4M NaOH solution to stop the activity of the enzyme.

(4) A standard curve was prepared. Marking 5 numbers on 10mL test tubes, respectively adding 0, 0.5, 1.0, 1.5 and 2.0mL of maltose standard solution with the concentration of 1mg/mL, adding 2mL of distilled water into each test tube to obtain a solution, adding 2mL of 3, 5-dinitrosalicylic acid solution, accurately boiling for 5min in a boiling water bath, taking out, cooling, diluting to 10mL with distilled water, mixing, measuring the absorbance value of the mixed solution at the wavelength of 520nm by using a UV-7500 spectrophotometer, recording the optical density reading, and drawing a curve by using the optical density reading as a total coordinate and the maltose content as an abscissa.

(5) And (4) sample determination. Sucking 2mL of reaction solution in the two groups of treatment test tubes and the control test tube, respectively adding the reaction solution 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. And (3) carrying out color comparison by using a UV-7500 type spectrophotometer at the wavelength of 520nm, recording optical density reading, and calculating the maltose content through the standard curve so as to express the enzyme activity.

(6) And (6) calculating a result. A: maltose content (mg) produced by alpha-amylase hydrolysis of starch; b: maltose content (mg) in control tubes of alpha-amylase; c: the sample solution is used for colorimetric analysis.

α -amylase activity [ maltose (mg)/fresh weight (g)/5 min ] [ [ (a-B) × total product of sample dilutions ]/sample weight (g) × C ] × 100

2.7.2 Effect of aureobasidin on acetolactate synthase (ALS) Activity

(1) And (6) performing pretreatment. When the tested weeds are cultured to the 3-4 leaf stage, prepared medium-concentration aureobasidin is sprayed on the liquid surface of the tested weeds, clear water is sprayed on a control group until the leaf surfaces do not drip, the control group is placed in a light incubator for culture, and the overground parts are respectively cut at the 3 rd, 5 th, 7 th, 9 th and 11 th days after treatment for ALS activity measurement.

(2) ALS extraction. Cutting leaf tissues of the treated group and the control group, placing the cut leaf tissues into a mortar which is pre-cooled, adding 10mmol/L potassium phosphate buffer solution (according to the weight of the leaf, the buffer solution is 1: 1), grinding the leaf tissues until the leaf tissues are homogenized, centrifuging the mixture at 4 ℃ for 30min, obtaining supernatant which is extracted crude enzyme solution, and storing the supernatant at 4 ℃ for later use, wherein the operations are all carried out at the temperature below 4 ℃.

(3) ALS activity assay. 6 tubes of 10mL were taken, 3 for the treatment group and 3 for the control group. 0.8mL of the enzymatic reaction buffer and 0.4mL of the crude enzyme solution were added to the tube, the tube was placed in a 37 ℃ water bath for 60min, and 50. mu.L of 3mol/L H was added₂SO₄The reaction was stopped (50. mu.L of 3mol/L H was added to the control before the water bath₂SO₄To terminate the reaction), then carrying out water bath at 60 ℃ for 15min to carry out decarboxylation, then sequentially adding 0.5mL of 0.5 creatine (dissolved in distilled water) and 0.5mL of 5% alpha-naphthol (dissolved in 2.5mol/L NaOH), carrying out color development reaction at 60 ℃ for 15min, then moving out and cooling to room temperature, carrying out color comparison at 520nm by using an ultraviolet spectrophotometer, and directly using A to carry out ALS activity_520nmAnd (4) showing.

3. Test results

3.1 fermentation production optimization

The effect on the yield of aureobasidin was tested by 4 factors from temperature, liquid loading, incubation time and shaker rotation speed (FIG. 2). The yield of the aureobasidin increases and then decreases with the increase of the liquid loading amount, and the yield is maximum when the liquid loading amount is 200 mL. The yield of the aureobasidin is increased firstly and then tends to be unchanged along with the increase of the culture days, and the yield of the aureobasidin is hardly increased when the culture reaches the 7 th day. The yield of the aureobasidin also has the phenomenon of increasing firstly and then decreasing along with the increase of the temperature, the yield is the highest when the temperature is 25 ℃, and the influence of the temperature around 25 ℃ on the yield of the aureobasidin is large, which indicates that the yield of the product is sensitive to the temperature. When the rotating speed of the shaking table is 200r/m, the yield of the aureobasidin reaches 0.65g/L at most, the yield is not obviously different from the yield of the aureobasidin (0.64g/L) when the rotating speed is 180r/m, and the yield slowly decreases when the rotating speed is higher than 220 r/m. Thus, the optimal culture conditions were determined as: the temperature is 25 ℃, the liquid loading amount is 200mL, the culture time is 7d, the rotating speed of a shaking table is 180r/m, and the yield of the aureobasidin is between 0.60g/L and 0.65 g/L.

3.2 isolation and purification of aureobasidin

3.2.1 Aureomycin Compound ultraviolet absorption Spectrum

An ultraviolet spectrophotometer is used for scanning the active band with strong biological activity in a full wavelength range of 200-490 nm in an ultraviolet spectrum region to determine the maximum absorption wavelength of the band, as can be seen from figure 3, a larger absorption peak exists at the position of 220nm in wavelength, and no secondary absorption exists, so that 220nm is selected as the analysis wavelength in the following high performance liquid chromatography.

3.2.2 identification of aureobasidin Structure

The structure of aureobasidin was resolved by mass spectrometry and nuclear magnetic resonance spectroscopy (fig. 4, 5, table 1), the spectral data are as follows:

TABLE 1 Hydrogen and carbon spectra data of aureobasidin

MS m/z 1100.694(M+H),123.696[M+Na]⁺,1099.694[M-H]^-,1253.696 [M+C₇H₇NO₃]⁺,¹H-NMR(CDCI₃)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-CH₃),1.39(s,3H,γ-CH₃ HOMeVal),1.19(s,3H,γ-CH₃HOMeVal). Molecular formula C₆₀H₉₂N₈O₁₁Molecular weight is 1100, colorless rod-shaped crystal, soluble in ethanol, chloroform and ether, insoluble in water, melting point 155-]_D245.3 ° (c 1.0, MeOH). Aureobasidin (AbA) is a cyclic peptide consisting of 8 alpha-amino acid units and 1 hydroxy acid. Respectively 2(R) -hydroxy-3 (R) -methylvaleric 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) (FIG. 6)

3.2.3 determination of herbicidal Activity of aureobasidin

3.2.3.1 seed Germination method

The aureobasidin with different concentrations has higher effect of inhibiting the growth of embryo of gramineous weeds, wild oat, sparrow, barnyard grass and green bristlegrass than that of broadleaf weeds (figure 7), wherein the effect of inhibiting the growth of embryo of the wild oat reaches 90.98 percent under the concentration of 100 mu g/mL, the effect of inhibiting the growth of embryo of the barnyard grass is 90.58 percent, and the effect of inhibiting the growth of the embryo of the wild oat is 88.2 percent; the inhibition effect on the germ growth of Chenopodium quinoa with the same concentration reaches 73.67%, while the inhibition effect on the germ growth of Chenopodium quinoa with the concentration of 10 mug/mL is only 28.02%. The rape with the best growth and germination inhibiting effect on broadleaf weeds has the inhibiting effect of 84.44% under the concentration of 100 mu g/mL, and the growth inhibiting effect on other weeds is below 80%. Similarly, the effect of inhibiting the growth of radicles of Avena sativa, Spinosa, Echinochloa crusgalli and Setaria viridis of the Gramineae family is higher than that of broadleaf weeds (FIG. 8), in which the effect of inhibiting the growth of radicles of Spinosa viridis is 86.64%, the effect of inhibiting the growth of radicles of Echinochloa crusgalli is 81.05%, the effect of inhibiting the growth of radicles of Setaria viridis 80.62% and the effect of inhibiting the growth of radicles of Avena sativa is 79.28% at a concentration of 100. mu.g/mL; the wild rape with the best effect of inhibiting the growth of the radicles of broadleaf weeds is 85.92% under the concentration of 100 mu g/mL, and the radicles of quinoa, wild carrot, Thalasia sessilifolia, Elsholtzia densa and cleavers are all under 80%, wherein the inhibition effect on the growth of the radicles of quinoa is the worst, and the inhibition effects under the concentrations of 100 and 10 mu g/mL are 74.33% and 37.95% respectively. In general, the inhibition effect on the germ of the weeds is better than that on the radicle.

3.2.3.2 weeding effect of potted plants

As can be seen from Table 2, after aureobasidin with a concentration of 100. mu.g/mL is sprayed, the weeds begin to appear symptoms within 2-3 days, and the grassy weeds can be completely killed within 7 days. Wild rape and cleavers can be completely killed within 7-10 days, but some broadleaf weeds (quinoa, wild carrot, Thalasia repens and Elsholtzia ciliata) can be completely killed within 15 days. It shows that the aureobasidin has certain selectivity on weed control.

TABLE 2 weed poisoning symptoms after Aureomycin spray application

In addition, after the aureobasidin with different concentrations is sprayed, the time for the weeds to have symptoms is different, the weeds begin to have symptoms in 3-5 days under the concentration of 100 mug/mL, the weeds are completely killed in about 7-15 days, and simultaneously, the victims of the grassy weeds are 3-5 days earlier than the victims of the broadleaf weeds. Similarly, at concentrations of 50. mu.g/mL and 10. mu.g/mL, the weed development was delayed by a corresponding 2-3 days.

The potted weeds were sprayed with aureobasidin at a concentration of 100. mu.g/mL, and the plant height, the number of plants and the fresh weight of the weeds were investigated 5 days after application to evaluate the weed control effect. As can be seen from FIG. 9, aureobasidin has different degrees of inhibition on the plant heights of different weeds, wherein the inhibition on the wild oat plant is the largest and reaches 59.1%, the plant height of sparassis crispa is 50.7%, and the plant heights of other weeds are less than 50%; in the plant height inhibition rate of broad-leaved weeds, the highest inhibition rate of the wild rape is 49.7%, and the lowest inhibition rate of the wild rape is only 30.3%. As can be seen from FIG. 10, the control effect on the gramineous weeds is more than 85%, wherein the control effect on the barnyard grass is 91.9% at most, and the control effect on the sparassis crispa is 90% secondly; the control effect on broadleaf weed Chenopodium album L is 80.5%, and the control effect on dense-flowered elsholtzia herb is 73.6%. But from the fresh weight effect, the fresh weight effect on wild oat is the highest, 83.6%, the fresh weight effect is barnyard grass for the second time, 81.8%, but the fresh weight effect on chenopodium is the lowest, only 62.8%, and in broad-leaved weeds, the fresh weight effect on wild carrot is the highest, reaching 71%.

3.2.3.3 herbicidal effect in field plot

Investigation is carried out 15 days after spraying, except for sporadic plant growth, weeds in a treatment plot are killed, weeds in a control plot grow vigorously, and leaves are dark green. By investigating the plant number and fresh weight of a field plot (figure 11), the weed control effect is better, the control effect on wild oat is the highest and is 90.6%, and the control effect on chenopodium quinoa is the lowest and is only 76.3%; similarly, the fresh weight efficiency of wild oat is the highest and reaches 88.2%, and the fresh weight efficiency of elsholtzia densa is the lowest and reaches 72.9%. In general, the weeding composition has good weed control effect under the concentration of 100 mu g/mL, and can be subjected to the next formulation development.

3.3 physicochemical Properties of aureobasidin

3.3.1 sensitivity to temperature

After the aureobasidin is treated at different temperatures, the inhibition rate of the aureobasidin on the growth of weeds is between 72.1% and 82.4%, no obvious difference exists among the temperature treatments, and the inhibition rate of the aureobasidin treated at 121 ℃ reaches 72.1% (figure 12), so that the aureobasidin has stronger heat resistance.

3.3.2 sensitivity to acids, bases

The inhibition of weed growth was maximal at pH7.0, 82.5%. When the pH was 4.0 to 11.0, the weed inhibition rate was not greatly changed and the difference was not significant (fig. 13). The weed inhibition rate of the aureobasidin is slowly reduced along with the reduction or the increase of the pH, which shows that the biological activity of the aureobasidin is reduced under the condition of extreme acid or extreme alkali, but the pH range which can be tolerated by the whole body is wider.

3.3.3 sensitivity to proteases

As can be seen from FIG. 14, after the aureobasidin is treated by 3 enzymes, the weed growth inhibition rate is not changed greatly, is between 78.9% and 79.3%, and no significant difference exists between the treatments, which indicates 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

Carrying out a plate seed germination test on weeds by using aureobasidin, and extracting alpha-amylase of seeds and measuring the activity of the alpha-amylase at 3 rd, 4 th, 5 th, 6 th and 7 th days after treatment. As shown in Table 3, the activity of the alpha-amylase in the treated group was significantly inhibited compared to the control, and the inhibition rate was higher than 93%, and it was found that the alpha-amylase activity gradually decreased from the germination stage at 3d to the 1 st leaf and 1 st heart stage at 7d, indicating that the alpha-amylase was mainly present in the germination stage of the seeds. The above results indicate that the compound inhibits alpha-amylase activity upon seed germination, and thus inhibits the germination of weed seeds.

TABLE 3 Effect of aureobasidin on alpha-amylase activity during germination of weed seeds

Note: + -, standard deviation; different column letters represent significance of difference (P < 0.05).

3.4.2 Effect of aureobasidin on acetolactate synthase Activity

And (3) culturing the weed seeds to 3-leaf stage, spraying with aureobasidin, respectively, cutting aerial parts of weeds at 3,5, 7, 9 and 11d, extracting acetolactate synthase (ALS), and measuring the activity of the acetolactate synthase. The results are shown in Table 4. ALS activity in the treatment group decreased, and the inhibition rate of ALS enzyme activity gradually increased with the number of treatment days. The results show that the compound can inhibit ALS activity in the weed plants, further inhibit the biosynthesis of the weed plants, and finally cause the death of weeds.

TABLE 4 Effect of aureobasidin on ALS Activity during weed seed growth

Note: + -, standard deviation; different column letters represent significance of difference (P < 0.05).

4. Conclusion

(1) Fermentation optimization production tests show that the optimal culture 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 rotating speed of a shaking table is 180r/m, and the yield of the aureobasidin is between 0.60g/L and 0.65 g/L. The substance is a cyclic lipopeptide 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. Research experiments on weeding mechanism show that the compound inhibits the activity of alpha-amylase in the seed germination period and the activity of acetolactate enzyme, so that the germination of seeds and the growth of weeds are inhibited, and the aim of weeding is fulfilled.

(3) The weeding activity plate test shows that the seed germination inhibition rate of the aureobasidin on the grassy weeds is higher than that of the broad-leaved weeds, the germ inhibition on the grassy weeds under the concentration of 100 mu g/mL reaches more than 88%, the culture inhibition rate on the broad-leaved weeds is only about 73%, and similarly, the germ inhibition rate on the grassy weeds reaches more than 80%, and the radicle inhibition rate on the broad-leaved weeds reaches less than 80%. The potted plant weeding activity shows that, at the concentration of 100 mug/mL, the investigation 5 days after the application shows that the aureobasidin has different degrees of inhibition effects on the plant height, the plant control effect and the fresh weight effect of different weeds, the highest plant height inhibition rate reaches 59%, the plant control effect and the fresh weight effect on the grass weeds respectively reach more than 85% and 81%, and the plant control effect and the fresh weight effect on the broadleaf weeds respectively reach more than 73% and 71%.

(4) The test result of the field plot shows that under the concentration of 100 mu g/mL, 15 days after spraying, the weeds in the treated plot are killed except for sporadic plant growth, the weeds in the control plot grow vigorously, and the leaves are dark green. The grass weeds are found to have better plant control effect on the weeds, which reaches 90.6 percent, and the control effect on the chenopodium album is the lowest and is only 76.3 percent; similarly, the fresh weight efficiency of wild oat is the highest and reaches 88.2%, and the fresh weight efficiency of elsholtzia densa is the lowest and reaches 72.9%. In general, the weeding composition has good weed control effect under the concentration of 100 mu g/mL, and can be subjected to the next formulation development.

In the description of the present invention, "a plurality" means two or more unless otherwise specified; the terms "upper", "lower", "left", "right", "inner", "outer", "front", "rear", "head", "tail", and the like, indicate orientations or positional relationships that are based on the orientations or positional relationships shown in the drawings, are merely for convenience in describing and simplifying the description, and do not indicate or imply that the referenced devices or elements must have a particular orientation, be constructed and operated in a particular orientation, and thus, should not be construed as limiting the present 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 above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any modification, equivalent replacement, and improvement made by those skilled in the art within the technical scope of the present invention disclosed in the present invention should be covered within the scope of the present invention.