CN117210361A - Preparation method and application of alternaria alternate and weeding active substances thereof - Google Patents

Abstract

The invention discloses a preparation method and application of a alternaria alternate and a weeding active substance thereof, belonging to the technical field of microbial weeding. The alternaria alternata (Alternaria alternata) HY-02 provided by the invention has herbicidal activity, and is preserved in China general microbiological culture Collection center (CGMCC No. 40750) at the date of 08/04 of 2023. And (3) separating and purifying the weeding active substances in the strain by adopting column chromatography and thin-layer chromatography, and determining that the weeding active substances comprise 5-hydroxybenzoate. The invention discloses that the butyl5-hydroxybenzoate has herbicidal activity for the first time, which lays a foundation for the development and large-scale production of novel herbicides.

Description

Preparation method and application of alternaria alternate and weeding active substances thereof

Technical Field

The invention relates to the technical field of microbial weeding, in particular to a preparation method and application of alternaria alternate and a weeding active substance thereof.

Background

Weeds in farmland influence the yield and quality of crops in the farmland production process, so that the yield and quality of crops are seriously damaged. The most common weeding modes adopted at present are artificial mechanical weeding and chemical pesticide weeding. The manual and mechanical weeding is high in cost, and time-consuming and labor-consuming. The advent and use of chemical herbicides has played an important role in reducing weed damage, releasing agricultural labor, and promoting agricultural modernization, largely alleviating crop losses. The active ingredients of the domestic chemical herbicide are more than 100, and the maximum use amount comprises: imazethapyr, clomazone, mesosulfuron, atrazine, thidiazuron, and amicarbazone. Because of the use of chemical herbicides, the drug resistance of weeds is generally enhanced, and meanwhile, the long-term use of chemical herbicides can aggravate pesticide residues in soil, so that the ecological influence is extremely large.

The microbial herbicide is used as a novel microbial pesticide, has the characteristics of easy degradation, safety to people and livestock, strong selectivity and the like, can effectively reduce the use of the pesticide, and is increasingly focused on development. Among them, the use of secondary metabolites produced by microorganisms and having biological activity to prepare formulations for weed control is a relatively green, environmentally friendly and safe way. Therefore, more weeding microorganisms are explored, and the weeding active substances are intensively researched, so that the method has important significance for the development and large-scale production of novel biological herbicides.

Disclosure of Invention

The invention aims to provide a preparation method and application of alternaria alternate and a weeding active substance thereof, so as to solve the problems in the prior art. The alternaria alternate provided by the invention has herbicidal activity, and the herbicidal active substances comprise 5-hydroxybenzoate, so that a foundation is laid for the development and large-scale production of novel herbicides.

In order to achieve the above object, the present invention provides the following solutions:

the invention provides a strain of alternaria alternata HY-02 with herbicidal activity, which is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of 40750 in the year 08 and 04 of 2023.

The invention also provides application of the alternaria alternata HY-02 in production of butyl 5-hydroxybenzoate.

The invention also provides a preparation method of the butyl5-hydroxybenzoate, which comprises the steps of fermenting and culturing the alternaria alternate HY-02, performing organic extraction on fermentation liquor obtained by fermenting and culturing, and performing chromatographic separation on a solution obtained by extraction to obtain the butyl 5-hydroxybenzoate.

Further, in the organic extraction, the volume ratio of the extractant to the fermentation broth is 1:1, and the extractant is ethyl acetate.

Further, the chromatography includes column chromatography and thin layer chromatography.

Further, in the column chromatography, the eluent is a mixed solution of dichloromethane and methanol, and the volume ratio of the dichloromethane to the methanol in the mixed solution is 5:1.

Further, in the thin layer chromatography, the developing agent is a mixed solution of dichloromethane and methanol, and the volume ratio of the dichloromethane to the methanol in the mixed solution is 20:1.

The invention also provides application of the butyl5-hydroxybenzoate in preparing herbicides.

Further, the herbicide is applied to a subject comprising quinoa.

The invention also provides a herbicide, and the effective substances comprise 5-hydroxy butyl benzoate.

The invention discloses the following technical effects:

the bacterial strain HY-02 with herbicidal activity is separated from natural disease-sensing papyrifera leaves, so that the microbial species with herbicidal activity are enriched, and a theoretical basis is provided for developing novel microbial herbicides.

According to the invention, the weeding active substances in the HY-02 strain are separated and purified by adopting column chromatography and thin layer chromatography, the components with obvious pathogenic effects on target weed quinoa leaves are found through biological activity measurement, the components 1 with the largest absorption peak area are obtained through continuous separation and purification of high performance liquid chromatography, and the components are finally determined to be the butyl 5-hydroxybenzoate. The invention discloses that the butyl5-hydroxybenzoate has herbicidal activity for the first time, which lays a foundation for the development and large-scale production of novel herbicides.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

Fig. 1 is the pathogenic effects of 4 extractants treating target weed quinoa leaves, wherein a and e: ethyl acetate organic and aqueous phases, b and f: n-butanol organic and aqueous phases, c and g: dichloromethane organic and aqueous phases, d and h: petroleum ether organic and aqueous phases;

FIG. 2 shows a thin layer chromatography fraction developing band;

FIG. 3 is the pathogenicity of herbicidally active fractions Fr.9 (a) and Fr.10 (b) against target weed quinoa leaves;

FIG. 4 is the pathogenicity of component Fr.9-1 (a) and component Fr.10-1 (b) against target weed quinoa leaves;

FIG. 5 is an HPLC chromatogram of fraction Fr.9-1;

FIG. 6 is an HPLC chromatogram of fraction Fr.10-1;

fig. 7 is the pathogenicity of component 1 against target weed quinoa leaf.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

The main reagents used in the invention include:

petroleum ether, ethyl acetate, methylene chloride, n-butanol, absolute ethanol and methanol (analytically pure, fuyu fine chemical Co., tianjin); glucose and sucrose (analytically pure, red rock reagent factory in eastern region of the Tianjin city); agar powder (analytically pure, beijing obo star biotechnology limited company); silica gel powder (Qingdao ocean chemical plant).

The experimental instrument mainly used in the invention comprises:

hirayama HVA-110 autoclave of Japan (Shanghai Ten. Of science and technology Co., ltd.), SPX-100SM microorganism incubator (Kang Heng Instrument Co., ltd.), SW-CJ-2D vertical double super clean bench (Shaoxing city Jingmei instrument Co., ltd.), high capacity high-speed centrifuge (GT-19 Shanghai Lu Xiangyi centrifuge instruments Co., ltd.), TS2112B double-layer high capacity thermostatic shaker (Shanghai Shunkan instruments Co., ltd.), horizontal conversion refrigerator (BC-300 KHAT Henan New Country refrigerator Co., ltd.), 101A-2E electrothermal blast drying oven (52 83 Instrument Co., ltd.) TGL-50 high capacity high speed centrifuge (Shanghai Shun instruments Co., ltd.), XDSY-2000A rotary evaporator (Shanghai Yi Heng instruments Co., ltd.), silica gel plate for HSGF254 preparation (200 mm. Times.200 mm Qingdao ocean chemical Co., ltd.), glass spreading jar (35 mm. Times.70 mm Shanghai Dafeng glass instruments Co., ltd.), UV-IE ultraviolet analyzer, HT-300 type ultrasonic cleaner (Shanghai Heng instruments Co., ltd.), SHB-I circulating water type multipurpose vacuum pump (Shen Guang instrument Co., ltd.).

Example 1

1. Isolation and selection of strains

34 fungi were isolated from diseased leaf tissue of Rumex madaio (harvested from the peasant home in Bovoxiang county, west Ning, qinghai) by tissue isolation. These isolated strains were used for the primary screening of in vitro pathogenicity of weed leaves, and finally a strain with the strongest pathogenicity was screened out and designated as strain HY-02.

2. Preservation of strains

Through identification, the classification of the strain HY-02 is named as Alternaria alternata (Alternaria alternata) HY-02, and the strain is preserved in China general microbiological culture Collection center (China Committee) at the date of 08, 2023, with a preservation address of Beijing, chaoyang, national institute 1, no. 3 and a preservation number of CGMCC No.40750.

Example 2

1. Method of

1. Preparation of bacterial strain HY-02 fermentation liquor

After strain HY-02 was cultured on PDA medium plates for 7d, bacterial blocks were taken at the edges of the colonies with a hole puncher of phi=8mm, inoculated into PDB culture fluid (250 mL/bottle), inoculated with 5 blocks per bottle, and cultured for 5-7d at 25℃with a shaking table rotation speed of 180 r/min. Filtering the fermentation broth with a centrifuge to remove mycelium, and keeping the supernatant for later use.

2. Selection of crude extract extractant of Strain HY-02

Ethyl acetate, petroleum ether, n-butanol and methylene dichloride are respectively selected as extraction agents, 4 organic solvents with different polarities are respectively mixed with fermentation filtrate according to the volume ratio of 1:1, and then the mixture is stood for 12 hours, and an organic phase is obtained by collecting. Concentrating the crude extract under reduced pressure at 45deg.C with rotary evaporator.

3. Crude extract Activity test

Selecting weed quinoa leaves as a target, washing the leaves, draining the water, spreading the leaves in a 9cm culture dish with filter paper, dissolving the organic phase crude extract with methanol, dripping the dissolved organic phase crude extract on the weed leaves, and repeating each treatment for three times by taking methanol as a reference. It was incubated at room temperature and the extent of infection was observed by leaf symptoms. Degree of infestation grading: -is asymptomatic; + is slight, with lesions accounting for 15% of leaf area; ++ is moderate, and the disease spots occupy 16% -59% of the leaf area; ++ is heavy, and the disease spots occupy 60-80% of the leaf area.

4. Large scale preparation of crude extract

And (3) performing equal-volume extraction on the fermentation broth by using an organic solvent with optimal activity test as an extractant, and performing mass preparation of the crude extract. Dissolving the crude extract with methanol, filtering to remove insoluble substances, and concentrating under reduced pressure to obtain crude product.

5. Column chromatography

(1) Filling the column, pouring 200-300 mesh silica gel powder into a beaker, adding a proper amount of petroleum ether with low polarity, and adding silica gel while stirring until the mixture is in a transparent paste without bubbles. The stirred pasty liquid is added into a glass column (column volume: 2355 mL), the column is filled with 120cm in height, and bubbles generated in the column filling process are removed by lightly knocking the column with an ear washing ball. After standing for 24 hours, the column is punched by petroleum ether until the silica gel in the column is stationary, and the column punching is stopped.

(2) The crude active substance is dissolved by methanol, and silica gel powder is added to ensure that the sample and the silica gel powder are fully and evenly mixed (methanol: silica gel powder=2:1 (W/W)), and then the mixture is concentrated under reduced pressure (the sample is fully adsorbed on the silica gel powder). And (3) placing the silica gel powder adhered with the sample in a mortar for full grinding, adding a silica gel column, and slowly adding to ensure that the surface of the sample is kept planar.

(3) The elution uses organic solvent mixed solution with different proportions as eluent, and the elution flow rate is set as follows: 1-2 drops/s. The elution gradient sequence is: petroleum ether: ethyl acetate (20:1), petroleum ether: ethyl acetate (10:1), petroleum ether: ethyl acetate (5:1), petroleum ether: ethyl acetate (3:1), dichloromethane: methanol (5:1) and dichloromethane: methanol (3:1), dichloromethane: methanol (1:1) 7 mixed solutions were eluted and 200mL was collected per bottle.

6. Thin layer chromatography

(1) Sample application and combination the prepared silica gel plates were cut into small plates (2.5 cm. Times.5 cm) with a glass knife, and a line was drawn at 0.5cm from the bottom and top of each small plate as the base line and top line. Spotting was repeated multiple times with capillaries at baseline until color developed under 254nm uv light, and then fractions of the same band were pooled.

(2) The expanding step is carried out in a closed chromatographic cylinder, and the corresponding chromatographic cylinder is selected according to the cut silica gel plate. Organic solvent mixed liquor with different proportions is selected as developing agent, and the proportion of the mixed liquor is slightly adjusted according to the color development condition.

(3) Evaporating and concentrating the combined fractions, spreading the fractions again by using organic solvent mixed solution with different proportions, developing the color under an ultraviolet lamp, and taking different strips as different fractions.

(4) And (3) quantitatively analyzing different strips on the silica gel plate, which are subjected to color development, together with silica gel powder, and then dissolving the strips with methanol for later use.

7. Herbicidal active ingredient bioassays

Fresh healthy target quinoa leaves are taken, washed with sterile water, dried in the air, and the leaves are spread in a sterile culture dish (phi=9 cm) in which wet filter paper is spread. Each fraction was dissolved in methanol and diluted to 100. Mu.g/mL, 15-20. Mu.L of each leaf was dropped, and each treatment was repeated three times with methanol treatment as a control. The cells were incubated at 25℃and the degree of infection was observed (method 3).

8. High Performance Liquid Chromatography (HPLC) condition exploration

The separation conditions were explored using a C18 reverse analytical column (4.6 mm×250 mm) with an initial ratio of mobile phases of methanol: water (95:5, v/v) was the exploration condition. The ratios of methanol and water were adjusted sequentially (90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, 50:50, 45:55, and 40:60), with detection wavelengths of 254nm until the optimal peak pattern was explored. And finally, selecting the optimal peak type ratio as the optimal mobile phase ratio to separate and purify the components. Then, the C18 reverse direction preparation column (30 mm. Times.250 mm) was used to continuously explore the optimal conditions for separation based on the optimal ratio, and the obtained chromatographic peaks were collected and their activities were detected.

9. Pure product structure identification

Nuclear magnetic resonance analysis: the monomer compound is dissolved by a proper deuterated solvent and transferred into a nuclear magnetic resonance tube for nuclear magnetic resonance detection. Obtaining ¹ HNMR、 ¹³ CNMR nuclear magnetic resonance spectroscopy by ¹³ C nuclear and hydrogen nuclear chemical shift values, inquiring micro-spectrum data, and primarily judging the structure of the compound through comparison.

Mass spectrometry of molecular weight of compounds: dissolving a sample to be detected by using chromatographic methanol, injecting the dissolved sample into a mass spectrometer, detecting mass spectrum by using an electrospray ion source (ESI), and calculating the molecular weight of the compound.

Nuclear Magnetic (NMR) analysis conditions: bruk ADVANCEIII MHz, deuterated methanol was used as the solvent, hydrogen spectrum resonance frequency 400MHz, resolution 0.244532Hz, carbon spectrum resonance frequency 100MHz, and carbon spectrum resolution 0.733596Hz.

And analyzing spectrograms such as a compound hydrogen spectrum, a carbon spectrum, a mass spectrum and the like, and determining the structure of the compound by combining literature investigation. Structure mapping was performed using chemdraw 12.0.

2. Results

1. Screening of crude extract extractant of bacterial strain HY-02

The pathogenicity of the 4 polar organic solvent crude extracts on the target quinoa leaf is shown in fig. 1 and table 1.

Table 14 pathogenicity of crude polar organic solvent extracts on target quinoa leaves

Note that: -is asymptomatic; + is slight, with lesions accounting for 15% of leaf area; ++ is moderate, and the disease spots occupy 16% -59% of the leaf area; ++ is heavy, and the disease spots occupy 60-80% of the leaf area.

Dissolving four organic phase (dichloromethane, petroleum ether, ethyl acetate and n-butanol) extracts with methanol, treating in vitro quinoa with them and water phase, and showing that dichloromethane organic phase produces small lesions with respect to quinoa, and water phase has no pathogenic symptoms with respect to quinoa, indicating that dichloromethane is used as extractant, and active substance is in organic phase; the petroleum ether organic phase has a large number of lesions relative to the quinoa leaves, which are shown as moderate symptoms, while the petroleum ether water phase has no pathogenic symptoms relative to the quinoa leaves, which indicates that the active substances are in the organic phase; the organic phase of the n-butanol and the water have no pathogenic effect on the quinoa leaves, and are asymptomatic, which indicates that the n-butanol can not extract the weeding active substance of the strain HY-02; the organic phase of ethyl acetate causes large lesions to develop on the quinoa leaves, which are manifested as severe symptoms. The ethyl acetate water did not produce pathogenic symptoms relative to the quinoa leaves, indicating that the active was in the organic phase. Thus, ethyl acetate was chosen as the optimal extractant.

2. Large-scale preparation of crude extract of bacterial strain HY-02

After the strain is cultured on a PDA culture medium plate for 7d, the strain is inoculated into PDB culture solution after punching, and the rotation speed of a shaking table is adjusted to 180r/min at 25 ℃ for 6d of culture. Filtering the fermentation broth with a centrifuge to remove mycelium, and keeping the supernatant for later use. The supernatant was extracted three times with ethyl acetate in equal volume, and the resulting organic phase was concentrated under reduced pressure to give 21.08g of crude product.

3. Elution ratio by column chromatography

Four organic solvents of different polarities (petroleum ether, ethyl acetate, methylene chloride and methanol) were selected, and the active substances were separated by column chromatography after mixing them in different proportions (Table 2).

TABLE 2 column chromatography elution ratio System

4. Thin layer chromatography

After developing the 7 different samples obtained by thin layer chromatography, 10 different fractions (Fr.1, fr.2, fr.3, fr.4, fr.5, fr.6, fr.7, fr.8, fr.9 and Fr.10) were obtained in total, color was developed under an ultraviolet lamp, and R of each developed substance was calculated as shown in FIG. 2 _f Values (0.2-0.8) and the results are shown in Table 3.

TABLE 3R for the respective band components _f Value of

5. Fraction bioactivity assay

The activity of 10 fractions was tested and the results showed that only two fractions could cause disease in the target weed leaves. As shown in fig. 3, fraction fr.9 was able to turn the quinoa leaves out green over a large area, causing the leaves to develop a large area of yellow lesions, which are manifested as severe symptoms. Fraction fr.10 causes the leaf portion to lose green, the leaf surface has a small number of lesions and the symptoms appear moderate. While none of the other fractions were pathogenic to leaves.

Fractions fr.9 and fr.10 were developed with a developing agent, quantitatively analyzed, the portion with the band on the silica gel plate was scraped off together with silica gel, dissolved with methanol, and concentrated to prepare a 100 μg/mL solution, and the pathogenicity against the leaf was measured. The results show that Fr.9-1 also has pathogenic effects on the quinoa leaves, and leaf symptoms are shown by yellow and black lesions on the leaf surfaces, and the leaves are green. Fr.10-1 also had a pathogenic effect on quinoa leaves, which exhibited yellowing of the leaves and the generation of lesions (FIG. 4).

6. High performance liquid chromatography

High performance liquid chromatography (P230P Dalian Lite analytical instrument Co., ltd.) is used, a C18 reverse analytical column (4.6 mm. Times.250 mm) is used, methanol and water are used as mobile phases, the sample injection amount is 20 mu L, the flow rate is 1mL/min, the detection wavelength is 254nm, the ratio of the two is continuously regulated, and the optimal peak of Fr.9-1 is explored. The results are shown in FIGS. 5 to 6, in methanol: under the condition of water=65:35 (v/v), 30 peaks with different sizes are obtained, the signal value of the 24 th peak is the highest and is 2674.687mAU, the peak area is the largest and is 119488.934mAU.s, and the peak is named as component 1 for structure detection.

The fraction Fr.10-1 was analyzed by high performance liquid chromatography on methanol: under the condition of water=75:25 (v/v), the detection wavelength is 254nm, 16 peaks with different sizes are obtained, the 10 th peak signal value is highest and is 2929.779mAU, the peak area is largest and is 37311.732mAU.s, and the peak is named as component 2 for structure detection.

Component 1 and component 2 were diluted to a concentration of 100 μg/mL and tested for biological activity against target weeds, as shown in fig. 7 and table 4, component 1 had a pathogenic effect on the target weeds in vitro leaves, leaves had lesions produced, leaves yellow, and the degree of pathogenicity could reach "++". While component 2 has a slightly pathogenic effect on the leaves.

TABLE 4 pathogenic effects of active ingredients on target weed quinoa leaves

Note that: -is asymptomatic; + is slight, with lesions accounting for 15% of leaf area; ++ is moderate, and the disease spots occupy 16% -59% of the leaf area; ++ is heavy, and the disease spots occupy 60-80% of the leaf area.

7. Structure identification of monomeric compounds

The molecular formula of the carbon spectrum data can be determined to be C ₁₁ H ₁₄ O ₃ The molecular weight was 194.2.

¹ HNMR(CDCl ₃ ,400MHz),δ7.47(2H,dd,H-3,H-7),7.25(2H,dd,H-4,H-6),4.07(2H,t,H-1’),1.47(2H,m,H-2’),1.20(2H,m,H-3’),0.71(3H,t,H-4’)。

¹³ CNMR(CDCl ₃ ,100MHz),δ166.8(C,C-1,C-5),131.9(CH,C-3,C-7),130.3(CH,C-4,C-6),128.2(C,C-2),64.7(CH ₂ ,C-1’),30.0(CH ₂ ,C-2’),18.6(CH ₂ ,C-3’),13.1(CH ₃ C-4'). The compound name is determined to be 5-hydroxybenzoic acid butyl5-hydroxy benzoate, and the molecular structural formula is as follows:

the above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims (10)

1. A strain of alternaria alternata (Alternaria alternata) HY-02 with herbicidal activity is characterized in that the strain is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of 40750 in the year 08 and 04 of 2023.

2. The use of alternaria alternata HY-02 as defined in claim 1 for producing butyl 5-hydroxybenzoate.

3. A preparation method of butyl5-hydroxybenzoate is characterized by comprising the steps of fermenting and culturing the alternaria alternate HY-02 according to claim 1, performing organic extraction on fermentation liquor obtained by the fermenting and culturing, and performing chromatographic separation on a solution obtained by the extraction to obtain butyl 5-hydroxybenzoate.

4. A method according to claim 3, wherein the volume ratio of extractant to fermentation broth is 1:1 during organic extraction, and the extractant is ethyl acetate.

5. A method of preparation according to claim 3, wherein the chromatography comprises column chromatography and thin layer chromatography.

6. The method according to claim 5, wherein in the column chromatography, the eluent is a mixed solution of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol in the mixed solution is 5:1.

7. The method according to claim 5, wherein in the thin layer chromatography, the developing agent is a mixed solution of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol in the mixed solution is 20:1.

Application of butyl 8.5-hydroxybenzoate in preparing herbicide.

9. The use according to claim 8, wherein the herbicide is applied to a subject comprising quinoa.

10. A herbicide characterized in that the active substance comprises butyl 5-hydroxybenzoate.