CN114009452B - Application of fermentation broth of alternaria alternata JTF001 in inhibition of germination of seeds of orobanum cucurbitacearum - Google Patents
Application of fermentation broth of alternaria alternata JTF001 in inhibition of germination of seeds of orobanum cucurbitacearum Download PDFInfo
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Abstract
The invention provides application of a fermentation broth of alternaria alternata JTF001 to inhibition of germination of seeds of cucurbitacinus meloidogyne, wherein the fermentation broth of alternaria alternata JTF001 comprises 3-acetyl-5-sec-butyl-4-hydroxy-3-pyrroline-2-ol and/or 2,3,4,5 '-tetrahydro-2,3,7-trihydroxy-9-methoxy-4' -methyl-dibenzo-6-pyrone. The invention discloses the effect of inhibiting the germination of the seed of the cucurbituril by 3-acetyl-5-sec-butyl-4-hydroxy-3-pyrroline-2-ol and 2,3,4,5 '-tetrahydro-2,3,7-trihydroxy-9-methoxy-4' -methyl-dibenzo-6-pyrone in the fermentation broth of alternaria cross JTF001 for the first time, and the two compounds can be directly used for preventing and treating the cucurbituril and have obvious inhibition effect on the cucurbituril.
Description
Technical Field
The invention relates to the technical field of application of agricultural microorganisms and biological control, in particular to application of fermentation liquor of alternaria alternata JTF001 in inhibition of germination of seeds of orobanum cucurbitacearum.
Background
Orobanche aegyptica (Orabanche aegyptica) is a highly parasitic plant widely distributed in Mediterranean areas, western Asia and eastern Europe, has a great impact on local crop production, and is considered to be the most harmful parasitic weed worldwide. Guarangdang in China is mainly distributed in multiple provinces such as Xinjiang, jilin, gansu, heilongjiang, hebei, shandong, shanxi, liaoning, qinghai, inner Mongolia and Sichuan. Xinjiang in China is an area with serious distribution and harm of melons and broomrape, and can reduce yield by more than half of the amount of melons, watermelons and tomatoes. Due to the parasitic characteristic of the root of orobanche cucurbitacearum, the harm to the host is mainly generated in the underground growth period before the emergence of seedlings, so that the common weeding modes such as manual removal (damaging the root system of the host) and pesticide and the like hardly work, and the effective prevention and control cannot be realized in the Xinjiang, so that the agricultural production is seriously influenced.
The microbial herbicide is a novel biological herbicide for weed control by using metabolites produced by microorganisms. Generally, most microorganisms such as bacteria, actinomycetes, saprophytic microorganisms, phytopathogenic fungi, algae and the like contain herbicidally active compounds, and are a natural treasure house for the development of herbicides. The control of cucurbiturils by natural compounds has become one of the hot spots of research in recent years. The research and development of natural herbicides with high activity, high selectivity and high safety by using microbial metabolites and artificial simulated compounds of the metabolites is one of important directions for the development of novel herbicides.
Our previous studies found that the crude extract of alternaria alternata has the effect of preventing germination of seeds of the citrullinia cupana, but the components in the fermentation liquid are many, and particularly which substance component plays a role is not clear. Therefore, it is necessary to obtain active monomeric compounds from crude extracts of alternaria alternata, so as to provide a microbial strain and active secondary metabolites thereof which are effective in controlling citrulline.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide an application of a fermentation broth of alternaria alternata JTF001 in inhibiting germination of seeds of citrulline, wherein the fermentation broth of alternaria alternata JTF001 contains 3-acetyl-5-sec-butyl-4-hydroxy-3-pyrrolin-2-ol and/or 2,3,4,5 '-tetrahydro-2,3,7-trihydroxy-9-methoxy-4' -methyl-dibenzo-6-pyrone, and a new path is provided for controlling citrulline.
The technical scheme adopted by the invention is as follows:
according to one aspect of the application, there is provided a use of a fermentation broth of alternaria alternata JTF001, the fermentation broth of alternaria alternata JTF001 comprising 3-acetyl-5-sec-butyl-4-hydroxy-3-pyrrolin-2-ol, and/or 2,3,4,5 '-tetrahydro-2,3,7-trihydroxy-9-methoxy-4' -methyl-dibenzo-6-pyrone, for inhibiting germination of seeds of citrulline.
In a specific embodiment of the present application, the method for obtaining the fermentation broth of alternaria alternata JTF001 comprises the following steps:
(1) Carrying out solid fermentation on alternaria alternata JTF001 to obtain a solid fermentation product;
(2) Soaking with ethyl acetate to obtain an extracting solution, and distilling the extracting solution under reduced pressure to obtain a crude extract;
(3) And dissolving the crude extract for silica gel column chromatography separation to obtain the fermentation liquor of alternaria alternata JTF 001.
In a specific embodiment of the present application, in step (1), alternaria alternata JTF001 is subjected to solid fermentation in a rice solid medium under conditions comprising: and (3) performing static culture for 10-25 days at the temperature of 25-30 ℃ in the dark.
In the specific embodiment of the application, in the step (2), ethyl acetate is used for soaking for 15-30 h, and then ultrasonic extraction is carried out for 0.5-1.5 h.
In a specific embodiment of the present application, in the step (3), the crude extract is obtained by using petroleum ether and ethyl acetate eluent, and methanol and ethyl acetate eluent in the silica gel column chromatography separation;
the volume ratio of petroleum ether to ethyl acetate in the petroleum ether and ethyl acetate eluent is 0-1: 0 to 1;
the volume ratio of methanol to ethyl acetate in the methanol and ethyl acetate eluent is 0.01-1: 0 to 0.99.
In a specific embodiment of the present application, in step (3), the crude extract is subjected to high performance liquid chromatography to obtain an active ingredient, wherein the active ingredient is 3-acetyl-5-sec-butyl-4-hydroxy-3-pyrroline-2-ol, or 2,3,4,5 '-tetrahydro-2,3,7-trihydroxy-9-methoxy-4' -methyl-dibenzo-6-pyrone.
In a particular embodiment of the present application, in step (4), the active ingredient is analyzed using mass spectrometry and nuclear magnetic resonance spectroscopy.
According to another aspect of the application, the application of the 3-acetyl-5-sec-butyl-4-hydroxy-3-pyrrolin-2-ol in inhibiting the germination of the seeds of the cucurbitacin is also provided.
According to another aspect of the application, there is also provided the use of 2,3,4,5 '-tetrahydro-2,3,7-trihydroxy-9-methoxy-4' -methyl-dibenzo-6-pyrone in inhibiting germination of seeds of citrulline.
The beneficial effects of the invention include but are not limited to:
(1) The invention discloses the effect of inhibiting the germination of the seed of the cucurbituril by 3-acetyl-5-sec-butyl-4-hydroxy-3-pyrroline-2-ol and 2,3,4,5 '-tetrahydro-2,3,7-trihydroxy-9-methoxy-4' -methyl-dibenzo-6-pyrone in the fermentation broth of alternaria cross JTF001 for the first time, and the two compounds can be directly used for preventing and treating the cucurbituril and have obvious inhibition effect on the cucurbituril.
(2) The invention relates to a method for obtaining fermentation liquor of alternaria alternata JTF001, which extracts an active substance for preventing and treating the cucurbituril from a crude extract of the alternaria alternata JTF001, determines an effective component in the crude extract of the alternaria alternata JTF001, obtains a natural active substance, and provides a new path for preventing and treating the cucurbituril.
(3) The invention relates to a method for obtaining fermentation liquor of alternaria alternata JTF001, which is characterized in that compounds 3-acetyl-5-sec-butyl-4-hydroxy-3-pyrroline-2-ol and 2,3,4,5 '-tetrahydro-2,3,7-trihydroxy-9-methoxy-4' -methyl-dibenzo-6-pyrone are obtained by first extraction, and a new thought is provided for the development and research of the two natural compounds.
Drawings
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of a compound LF6-2 in an example of the present invention;
FIG. 2 is a nuclear magnetic resonance carbon spectrum of compound LF6-2 in the example of the present invention;
FIG. 3 is a diagram of nuclear magnetic resonance HSQC of compound LF6-2 in an example of the present invention;
FIG. 4 is a nuclear magnetic resonance HMBC graph of compound LF6-2 in an example of the present invention;
FIG. 5 is a graph of mass spectrometry data of a positive ion source of compound LF6-2 in an example of the present invention;
FIG. 6 is a NMR chart of compound LF12-29-3 in example of the present invention;
FIG. 7 is a NMR carbon spectrum of compound LF12-29-3 in example of the present invention;
FIG. 8 is a diagram of the nuclear magnetic resonance HSQC of compound LF12-29-3 in an example of the present invention;
FIG. 9 shows the NMR HMBC chart of the compound LF12-29-3 in the example of the present invention;
FIG. 10 is a graph of mass spectrometry data of a positive ion source of compound LF12-29-3 in an example of the present invention.
Detailed Description
The present invention is described in detail with reference to specific examples, which are provided to facilitate the understanding of the technical solutions of the present invention by those skilled in the art, and the implementation or use of the present invention is not limited by the description of the present invention.
In the present invention, the raw materials and equipment used are commercially available or commonly used in the art, if not specified. The methods in the examples are conventional in the art unless otherwise specified.
The strain Alternaria alternata JTF001 used in the examples was isolated from the stem base of a naturally occurring Cissus liana plant in the tomato field, processed in Jimusalar county, xinjiang and deposited in the microbial cultures Collection of Guangdong province 22 months in 2021 (GDMCC, address: first Liehuo Zhonglu 100, guangdong province, microbial institute, postal code: 510070) with a deposit number of GDMCC No.61964, proposed taxonomic name: alternaria alternata.
EXAMPLE 1 method for extracting active substance
(1) Solid fermentation of alternaria alternata JTF001
The deposited strain of Acidocella was transferred to PDA medium and cultured at 28 ℃ for 5 days. Adding the fungus cake into a triangular flask containing semi-solid culture medium (formula: peptone 10g, maltose 40g, yeast extract powder 10g, agar 4g and distilled water 1000 mL) to obtain semi-solid seed solution, and culturing at 28 deg.C for 180r min -1 And carrying out shake culture for 48h. 5mL of the semi-solid seed solution was added to a rice solid medium (formula: 80g of rice, 1.2g of yeast powder and 120mL of distilled water in a 500mL Erlenmeyer flask, soaked overnight and sterilized at 121 ℃ for 30 min), and cultured at 28 ℃ for 15d.
(2) Extraction of crude extract
Mechanically stirring the rice solid culture medium, adding 240mL ethyl acetate into each bottle, soaking for 24h, performing ultrasonic treatment for 1h, and extracting for 2 times. And (3) carrying out rotary evaporation and reduced pressure distillation on the ethyl acetate soak solution to obtain 17g of crude extract.
(3) Separation and purification of compounds
Fully mixing 17g of crude extract with 20g of 200-300-mesh silica gel, naturally drying, loading the mixture into a column by using 300-400-mesh silica gel, performing silica gel column reduced pressure elution by using a petroleum ether-ethyl acetate-methanol solvent system, and dividing the crude extract into 17 fractions according to the sequence of the polarity from small to large, wherein the fractions are named as LF 1-17. Specifically, the method comprises the following steps: eluting with petroleum ether to obtain fraction LF1; eluting with petroleum ether and ethyl acetate at a volume ratio of 99% to 1% to obtain fraction LF2; eluting with 97% by volume and 3% by volume of petroleum ether and ethyl acetate to obtain fraction LF3; eluting with 95 vol% and 5 vol% petroleum ether and ethyl acetate to obtain fraction LF4; eluting with petroleum ether and ethyl acetate at a volume ratio of 90% to 10% to obtain fraction LF5; eluting with petroleum ether and ethyl acetate at a volume ratio of 85% to 15% to obtain fraction LF6; eluting with 20% petroleum ether and ethyl acetate at a volume ratio of 80% to obtain fraction LF7; eluting with 75% by volume and 25% by volume of petroleum ether and ethyl acetate to obtain fraction LF8; eluting with petroleum ether and ethyl acetate at a volume ratio of 70% to 30% to obtain fraction LF9; eluting with petroleum ether and ethyl acetate at a volume ratio of 60% to 40% to obtain fraction LF10; eluting with 50% by volume of 50% petroleum ether and 50% ethyl acetate to obtain fraction LF11; eluting with petroleum ether and ethyl acetate at a volume ratio of 30% to 70% to obtain fraction LF12; after elution with ethyl acetate, fraction LF13 was obtained. Eluting with 1 vol% to 99 vol% methanol and ethyl acetate to obtain fraction LF14; eluting with 3 vol% 97 vol% methanol and ethyl acetate to obtain fraction LF15; eluting with 5 vol% to 95 vol% methanol and ethyl acetate to obtain fraction LF16; after elution with methanol, fraction LF17 was obtained. The inhibitory activity of LF1 to LF17 on the germination of the seeds of cucurbituril was tested with a volume of 1L for each elution gradient, and the results are shown in table 1, from which it was shown that fractions LF6 (petroleum ether: ethyl acetate =85%: 15%) and LF12 (petroleum ether: ethyl acetate =30%: 70%) had better inhibitory activity on the germination of seeds of cucurbituril.
TABLE 1 inhibitory Effect of different fractions on the germination of seeds of Orobanches
And (4) carrying out HPLC (high performance liquid chromatography) semi-preparative liquid phase purification on the fraction LF6 to obtain a target compound LF6-2. An Agilent 1260 type semi-preparative high pressure liquid chromatograph is adopted, and the HPLC detection method and conditions are as follows: phase A is 0.1% formic acid solution in water, and phase B is acetonitrile. Using Kromasil 100-5-C 18 Separating with reversed phase semi-preparative chromatographic column (10 μm, 10X 250mm) at flow rate of 2.0mL/min under gradient elution conditions: 0.00-30.00min, 35% of phase B; 30.00-30.50min, 35-100% of phase B; 30.50-35.50min, 100% of phase B; 35.50-36.00min, B phase 100-35%. 20.12mg of compound LF6-2 were obtained (retention time tR = 26.68-28.20 min).
And (3) carrying out HPLC (high performance liquid chromatography) semi-preparative liquid phase purification on the fraction LF12 to obtain a target compound LF12-29-3. An Agilent 1260 type semi-preparative high pressure liquid chromatograph is adopted, and the HPLC detection method and conditions are as follows: phase A is 0.1% formic acid solution in water, and phase B is acetonitrile. Using Kromasil 100-5-C 18 The separation was performed by reverse phase semi-preparative chromatography (10 μm, 10X 250mm) at a flow rate of 2.0mL/min under gradient elution conditions: 0.00-35.00min, 35% of phase B; 35.00-35.50min, 35-100% of phase B; 35.50-46.00min, 100% of phase B; 46.00-46.50min, B phase 100-35%. 7.16mg of the compound LF12-29-3 are obtained (retention time t) R ＝16.95～17.47min)。
Fully dried compounds LF6-2 and LF12-29-3 are completely dissolved by 600uL of deuterated acetone and deuterated methanol respectively and then transferred into a nuclear magnetic tube for sealing for testing 1 H-NMR and 13 C-NMR nuclear magnetic resonance spectrum, wherein the measurement of the nuclear magnetic spectrum is completed by the institute of microbiology of Chinese academy of sciences.
EXAMPLE 2 structural characterization of Compounds LF6-2 and LF12-29-3
Compound LF6-2: pale yellow oily solid, readily soluble in methanol and deuterated acetone. Display of [ M-H ] of the Compound according to Mass Spectrometry] - The molecular ion peak was 197.00 (FIG. 5), which is inferred to be of formula C 10 H 15 NO 3 Further by its nuclear magnetic resonance hydrogen spectrum ( 1 H-NMR) and carbon Spectroscopy ( 13 C-NMR) was compared with a known compound Tenuazonic acid reported in the literature (T rosett.1957,67, 390-400), and the structural formula is shown in formula i, with the chemical name 3-acetyl-5-sec-butyl-4-hydroxy-3-pyrrolin-2-ol.
Compound LF12-29-3: pale yellow oily solid, readily soluble in methanol and deuterated acetone. Shows [ M + H ] of the Compound according to Mass Spectrometry]+ molecular ion split was 292.00 (FIG. 10), which was inferred to be of formula C 15 H 16 O 6 Further, it was confirmed that the structural formula is shown in the formula II, chemical name is 2,3,4,5 '-Tetrahydro-2,3,7-trihydroxy-9-methoxy-4' -methyl-dibenzo-6-pyrone (2, 3,4 a-tetrahedron-2,3,7-trihydroxy) by comparing its nuclear magnetic resonance hydrogen spectrum (1H-NMR) and carbon spectrum (13C-NMR) with the known compound Altenene reported in the literature (R.W.Pero.1971, 230, 170-179.)-9-methoxy-4a-methyl-6H-dibenzo[b,d]pyran-6-one)。
Hydrogen spectra of the compounds LF6-2 and LF12-29-3 prepared by the invention 1 H-NMR) and carbon Spectroscopy ( 13 C-NMR) is shown in FIGS. 1-4 and 6-8, and the mass spectrum data is shown in FIGS. 5 and 10, and the specific data is shown in tables 2 and 3.
TABLE 2 NMR data on carbon spectrum and hydrogen spectrum of the compound LF6-2 (acetone-d) 6 )
TABLE 3 NMR data on carbon and hydrogen spectra of the compound LF12-29-3 (methane-d) 4 )
In conclusion, natural compounds LF6-2 and LF12-29-3 are extracted and separated from alternaria alternata JTF001, and the structural characteristics of the compounds are analyzed and identified through further purification and combination of high performance liquid chromatography, mass spectrometry and nuclear magnetic resonance technology.
EXAMPLE 3 determination of the inhibitory Effect of Natural Compounds LF6-2 and LF12-29-3 on Germination of Orobanches
a. And (3) sterilizing the seeds of the orobanche cucurbitae: sterilizing the seed of herba Orobanches in 75% ethanol for 1.5min, transferring to 1% sodium hypochlorite for 12min, and air drying on sterile filter paper.
b. Solution preparation: and blowing the solution in the nuclear magnetic tube with nitrogen, and measuring the mass of LF6-2 and LF12-29-3 to be 20.00mg and 7.00mg respectively after blow-drying. 100. Mu.L of methanol was added to dissolve the resulting solutions, and sterile water was added to prepare 8mM stock solutions, which were diluted to 4mM, 2mM, and 1mM, respectively.
c. Determination of germination inhibition of seed of orobanche cucurbitae: approximately 40 sterile dried seeds of Guaranthus uratus were added to each well of a 24-well plate, 0.5mL of strigolactone (GR 24) and 0.5mL of 8mM, 4mM, 2mM, and 1mM compound solutions were added to the treatment groups, respectively, with the final concentrations of 4mM, 2mM, 1mM, and 0.5mM for LF6-2 and LF12-29-3 compounds in the treatment groups, and 0.5mL of strigolactone (GR 24) and 0.5mL of sterile water in the control groups, for 3 replicates per treatment. And (4) observing the germination quantity of the seed of the orobanche coerulescens in a body type microscope and recording after the seed is cultured for 5d in a shading way at the temperature of 25 ℃. Calculating the concentration of compound the germination inhibiting effect of the broomrape seeds, as shown in table 4.
TABLE 4 inhibitory Effect of different concentrations of Compounds on the germination of seeds of Orobanches
From the results in Table 4, it can be seen that the above compounds LF6-2 and LF12-29-3 both have significant inhibitory effects on the germination of the seeds of Orobanches, and the higher the concentration is, the better the inhibitory effects on the germination of the seeds of Orobanches, and the more 1mM or more the concentrations of LF6-2 and LF12-29-3 are, the inhibition rate on the germination of the seeds of Orobanches is 1000%.
The application of the fermentation broth of alternaria alternata JTF001 in inhibiting the germination of seeds of cucurbitaceae is described in detail above. The principles and embodiments of the present invention are explained herein using specific examples, which are presented only to assist in understanding the method and its core concepts. It should be noted that those skilled in the art can make various modifications and optimization to the present invention without departing from the principle of the present invention, and the modifications and optimization also fall into the protection scope of the claims of the present invention.
Claims (8)
1. Fermentation broth of alternaria alternata JTF001 in inhibiting citrullus broomrape: (Orabanche aegyptaica) Use in seed germination, characterized in that the fermentation broth of alternaria alternata JTF001 comprises 3-acetyl-5-sec-butyl-4-hydroxy-3-pyrrolin-2-ol, and 2,3,4,5 '-tetrahydro-2,3,7-trihydroxy-9-methoxy-4' -methyl-dibenzo-6-pyrone;
the alternaria alternata JTF001 is preserved in 22 months 10 and 2021 in Guangdong province collection center with the preservation number GDMCCNo.61964.
2. The use according to claim 1, wherein the fermentation broth of alternaria alternata JTF001 is obtained by a process comprising the following steps:
(1) Carrying out solid fermentation on alternaria alternata JTF001 to obtain a solid fermentation product;
(2) Soaking with ethyl acetate to obtain an extracting solution, and distilling the extracting solution under reduced pressure to obtain a crude extract;
(3) And dissolving the crude extract for silica gel column chromatography separation to obtain the fermentation liquor of alternaria alternata JTF 001.
3. The use according to claim 2, wherein in step (1), alternaria alternata JTF001 is subjected to solid fermentation in a rice solid medium under conditions comprising: and (3) performing static culture in the dark at the temperature of 25-30 ℃ for 10-25d.
4. The use of claim 2, wherein in the step (2), the mixture is soaked in ethyl acetate for 15 to 30h and then extracted by ultrasonic for 0.5 to 1.5h.
5. The use of claim 2, wherein in the step (3), the crude extract is obtained by using petroleum ether and ethyl acetate eluent and methanol and ethyl acetate eluent in the silica gel column chromatography separation;
the volume ratio of the petroleum ether to the ethyl acetate in the petroleum ether and ethyl acetate eluent is 0~1:0~1;
the volume ratio of methanol to ethyl acetate in the methanol to ethyl acetate eluent is 0.01 to 1:0 to 0.99.
6. The use according to claim 5, wherein in step (3), the crude extract is subjected to HPLC to obtain active ingredients, wherein the active ingredients are 3-acetyl-5-sec-butyl-4-hydroxy-3-pyrroline-2-ol, and 2,3,4,5 '-tetrahydro-2,3,7-trihydroxy-9-methoxy-4' -methyl-dibenzo-6-pyrone.
7. The use according to claim 6, wherein in step (4), the active ingredient is analyzed using mass spectrometry and nuclear magnetic resonance spectroscopy.
3238 Zxft 3238 '-tetrahydro-3262 Zxft 3262-trihydroxy-9-methoxy-4' -methyl-dibenzo-6-pyrone for inhibiting germination of seeds of Meldonia cucurbitacearum.
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