Tobacco endophytic fungus metabolite, preparation method and application in prevention and treatment of tobacco alternaria alternata
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- INNER MONGOLIA KUNMING CIGARETTE CO LTD
- Filing Date
- 2026-02-03
- Publication Date
- 2026-05-29
Smart Images
Figure CN122102878A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial natural product pesticide technology, specifically to a metabolite of tobacco endophytic fungi, its preparation method, and its application in the control of tobacco scab. Background Technology
[0002] Tobacco star disease is caused by Alternaria alternata (Alternaria alternata). Alternaria alternata Tobacco leaf spot disease (Fries. Keisslar) is a fungal disease affecting tobacco leaves, prevalent in tobacco-growing regions. It primarily occurs in the later stages of tobacco growth, manifesting as brown to black necrotic spots on leaves, premature senescence, scorching, and even complete leaf death. This not only significantly reduces tobacco yield but also severely impacts the industrial usability and economic value of tobacco leaves. Currently, control of tobacco leaf spot still heavily relies on chemical pesticides such as mancozeb, sclerotinia oleifera, and azoxystrobin. However, the long-term and extensive use of chemical pesticides leads to pathogen resistance, pesticide residues and environmental pollution, and damage to non-target organisms, contradicting the current concepts of green agriculture and green development. Therefore, finding efficient, low-toxicity, and environmentally friendly new biological control methods has become a research hotspot in the field of agricultural disease control. Among these methods, plant endophytic fungi, due to their unique ecological niche and rich diversity of metabolites, exhibit enormous biocontrol potential.
[0003] Plant endophytic fungi have long resided within plant tissues, co-evolving with their host plants and producing a series of secondary metabolites with antibacterial, insecticidal, and growth-promoting activities. These metabolites typically possess novel structures and mechanisms of action, are readily degraded in the natural environment, and are safe for both the environment and non-target organisms. Currently, studies have reported antagonistic effects of endophytic fungi isolated from various plants against plant pathogens. CN 120665724 A discloses a species of endophytic fungus *Principia fragilis* found in the roots of *Polygonatum yunnanensis*. Prillingera fragicola DHJ068 (deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M2025748), its fermentation broth crude extract can effectively inhibit the growth of *Brassica napus*, the pathogen causing soft rot of Chinese cabbage, and *Brassica napus*, the pathogen causing bacterial angular leaf spot of cucumber, achieving a certain degree of biological control. CN 120442419 A discloses a type of endophytic fungus, *Vepithecus flabellulatum*, found in the roots of *Camellia yunnanensis*. Lecanicillium saksenae JHC098 (deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M2025749) showed that its crude extract from liquid fermentation broth effectively inhibited the growth of pathogenic bacteria causing citrus canker and tobacco bacterial wilt. However, systematic research and application of tobacco endophytic fungi, especially their specific metabolites, in the control of tobacco scab remains relatively limited. Summary of the Invention
[0004] This invention aims to develop a novel, efficient, and safe metabolite derived from tobacco endophytic fungi with biocontrol effects, thereby reducing the use of chemical pesticides, ensuring safe tobacco production, and promoting the sustainable development of tobacco agriculture. To this end, this invention provides a tobacco endophytic fungal metabolite, its preparation method, and its application in the control of tobacco scab.
[0005] In a first aspect, the present invention provides a metabolite of tobacco endophytic fungi, wherein the metabolite is a benzoquinone compound peniquinone L, with the following structural formula: .
[0006] A second aspect of the present invention provides a method for preparing the above-mentioned tobacco endophytic fungal metabolites, comprising: Fermentation culture: The endophytic fungus *Aspergillus echinospora* from tobacco... Aspergillus aculeatus Aa-1 was inoculated into potato glucose aqueous medium and placed in a static culture and fermented for 28-32 days under natural light at 26℃-28℃. Extraction: After fermentation, the fermentation broth was extracted with ethyl acetate, the extracts were combined and concentrated to obtain the crude fermentation extract; Separation and purification: The crude fermentation extract was separated by vacuum silica gel column chromatography. The eluent was a petroleum ether-ethyl acetate mixture. Five components with increasing polarity were obtained. The most polar component was purified by semi-preparative high performance liquid chromatography to obtain the benzoquinone compound peniquinone L.
[0007] Furthermore, the reduced-pressure silica gel column chromatography uses a mixed solvent of petroleum ether and ethyl acetate for gradient elution, and the volume ratio of petroleum ether to ethyl acetate used in the eluent is 20:1, 10:1, 5:1, 2:1 and 1:1, respectively.
[0008] Furthermore, the most polar component is obtained by elution with petroleum ether-ethyl acetate at a volume ratio of 1:1.
[0009] Furthermore, in the semi-preparative high-performance liquid chromatography purification, the mobile phase is an acetonitrile-water mixture with a volume ratio of 50:50.
[0010] Further, under the purification conditions, the retention time of the benzoquinone compound peniquinone L... t R It takes 8.7 minutes.
[0011] In a third aspect, the present invention provides the use of the above-described tobacco endophytic fungal metabolite or the benzoquinone compound peniquinone L prepared by the above-described preparation method in the preparation of a medicament for the prevention and control of plant pathogenic fungi.
[0012] Furthermore, the plant pathogenic fungus is selected from at least one of the following: *Botrytis cinerea* (tomato gray mold), *Phyllostachys rubrum* (apple rot fungus), and *Aureobasidium adolphii* (tobacco red spot fungus).
[0013] Furthermore, the plant pathogenic fungus is *Tobacco Star*.
[0014] Furthermore, the minimum inhibitory concentration of the benzoquinone compound peniquinone L against *Aureobasidium aizoon* is 2 μg / mL.
[0015] Compared with the prior art, the present invention has at least the following beneficial effects: This invention prepares a benzoquinone compound, peniquinone L, which is then processed by SciFinder. n A search of natural product databases revealed that this compound is a novel compound with a newly reported structure. This compound was derived from the tobacco endophytic fungus *Aspergillus echinococcosis*. Aspergillus aculeatus Aa-1 is produced through fermentation, and its fermentation and preparation process is simple and easy to scale up. Activity studies have shown that peniquinone L has inhibitory effects on various agricultural pathogens, particularly against *Tobacco Red Spot*, with a significant inhibitory activity. Its MIC value is 2 μg / mL, superior to the positive control carbendazim (MIC = 4 μg / mL). This compound is a naturally derived fungal metabolite, possessing both safety and high efficiency. It can serve as a lead compound or a novel microbial natural product pesticide for the control of *Tobacco Red Spot*, showing promising potential for biocontrol applications. Attached Figure Description
[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments recorded in the embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings.
[0017] Figure 1 The high-resolution mass spectrum (HRESIMS) of the compound peniquinone L provided in the embodiments of the present invention.
[0018] Figure 2 Superconducting nuclear magnetic resonance hydrogen spectrum (500 MHz, DMSO-) of compound peniquinone L provided in embodiments of the present invention d 6) Figure.
[0019] Figure 3 Superconducting carbon NMR spectrum (125 MHz, DMSO-) of compound peniquinone L provided in embodiments of the present invention. d 6) Figure. Detailed Implementation
[0020] To better understand the above technical solutions, the technical solutions of the embodiments of this application will be described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the embodiments of this application and the specific features in the embodiments are detailed descriptions of the technical solutions of the embodiments of this application, rather than limitations on the technical solutions of this application. In the absence of conflict, the embodiments of this application and the technical features in the embodiments can be combined with each other.
[0021] This invention provides a novel structural secondary metabolite derived from tobacco endophytic fungi, reports a rapid preparation method for this metabolite, and for the first time discovers its significant inhibitory activity against the growth of *Acer buergerianum*, the causal agent of tobacco star rot. This endophytic fungal metabolite has the potential to be developed into a novel microbial natural product pesticide.
[0022] A first aspect of this invention provides a metabolite from tobacco endophytic fungi, wherein the metabolite is a benzoquinone compound peniquinone L, with the following structural formula: .
[0023] The secondary metabolites derived from tobacco endophytic fungi provided in this embodiment of the invention are from a tobacco endophytic fungus, Aspergillus echinococcosis. Aspergillus aculeatus This secondary metabolite was obtained by purification from the crude extract of Aa-1 (deposited at the China General Microbiological Culture Collection Center, accession number CGMCC No. 40424) during liquid fermentation. Using modern spectroscopic techniques, high-resolution mass spectrometry (HRESIMS) and superconducting nuclear magnetic resonance (NMR) spectroscopy, the product was identified as a benzoquinone compound. Further analysis using the natural product SciFinder... n A database search revealed that this benzoquinone compound is a novel structural compound that has been discovered and reported for the first time internationally, and it has been named peniquinone L.
[0024] A second aspect of the present invention provides a method for preparing the above-mentioned tobacco endophytic fungal metabolites, comprising: Fermentation culture: The endophytic fungus *Aspergillus echinospora* from tobacco... Aspergillus aculeatus Aa-1 was inoculated into potato glucose aqueous medium and placed in a static culture and fermented for 28-32 days under natural light at 26℃-28℃. Extraction: After fermentation, the fermentation broth was extracted with ethyl acetate, the extracts were combined and concentrated to obtain the crude fermentation extract; Separation and purification: The crude fermentation extract was separated by vacuum silica gel column chromatography. The eluent was a mixture of petroleum ether and ethyl acetate. Five components with increasing polarity were obtained. The most polar component was purified by semi-preparative high performance liquid chromatography to obtain benzoquinone L.
[0025] The method for preparing metabolites of tobacco endophytic fungi provided in this embodiment of the invention first involves the preparation of metabolites of the tobacco endophytic fungus Aspergillus echinospora. Aspergillus aculeatus Aa-1 was used for fermentation culture; then the crude fermentation extract was extracted and purified, and the benzoquinone compound peniquinone L was further separated and prepared. The benzoquinone compound peniquinone L involved in the embodiments of this invention was prepared by the tobacco endophytic fungus *Aspergillus echinococcosis*. Aspergillus aculeatus Aa-1 is produced by fermentation, which is easy to carry out on a large scale for fermentation and preparation; the preparation process can quickly and accurately prepare benzoquinone compounds peniquinone L.
[0026] Preferably, the reduced pressure silica gel column chromatography uses a gradient elution with a mixed solvent of petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate used in the eluent is 20:1, 10:1, 5:1, 2:1 and 1:1, respectively.
[0027] Preferably, the most polar component is obtained by elution with petroleum ether-ethyl acetate at a volume ratio of 1:1; the most polar component is then purified using semi-preparative high-performance liquid chromatography to obtain the benzoquinone compound peniquinone L, with a retention time of [missing information]. t R The time is 8.7 min. In the semi-preparative high performance liquid chromatography purification, the preferred mobile phase is a 50:50 volume ratio acetonitrile-water mixed solution.
[0028] In a third aspect of the present invention, the application of the above-described tobacco endophytic fungal metabolite or the benzoquinone compound peniquinone L prepared by the above-described preparation method in the preparation of a drug for the prevention and control of plant pathogenic fungi is provided.
[0029] In this embodiment of the invention, the benzoquinone compound peniquinone L was screened for its activity against various plant pathogenic fungi. It was found that peniquinone L exhibited inhibitory activity against *Botrytis cinerea* (tomato rot fungus), *Pseudomonas aeruginosa* (apple rot fungus), and *Aureobasidium albivenis* (tobacco scab fungus), with MIC values of 16 μg / mL, 8 μg / mL, and 2 μg / mL, respectively. Among these, peniquinone L showed significant growth inhibition against *Aureobasidium albivenis*, with a minimum inhibitory concentration (MIC) of 2 μg / mL. Its activity was superior to the positive control carbendazim, whose MIC against *Aureobasidium albivenis* was 4 μg / mL.
[0030] In summary, the benzoquinone compound peniquinone L provided in the embodiments of the present invention is a compound derived from natural products, which has the advantages of safety and high efficiency. It can be used as a lead compound for the control of tobacco red spot disease or a new microbial natural product pesticide, and has certain application prospects in biological control.
[0031] Example 1: A metabolite of tobacco endophytic fungi and its preparation method (a) Metabolites of tobacco endophytic fungi The metabolite of tobacco endophytic fungi is a benzoquinone compound, peniquinone L, with the following structural formula: .
[0032] (II) Preparation method The specific preparation process for tobacco endophytic fungal metabolites is as follows: (1) Fermentation culture process of the strain The endophytic fungus Aspergillus echinospora in tobacco Aspergillus aculeatus Aa-1 was inoculated into potato dextrose water medium (Qingdao Haibo Biotechnology Co., Ltd.; product number: HB0233-4) and incubated statically for 30 days under room temperature (approximately 26℃-28℃) and natural light conditions (approximately 12 hours of light and 12 hours of darkness per day). The endophytic fungus *Aspergillus echinosporum* was also present. Aspergillus aculeatus Aa-1 is deposited by the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 40424 and is classified as *Aspergillus echinococcosis*. Aspergillus prickly Date of deposit: November 14, 2022; Location of deposit: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.
[0033] (2) Extraction process of fermented crude extract After fermentation, the liquid culture medium was extracted with ethyl acetate three times, 300 mL each time (the volume ratio of extract to fermentation broth was 1:1). After extraction, the three extracts were combined and concentrated under vacuum (0.09 MPa) to obtain the crude fermentation extract.
[0034] (3) Compound purification and preparation process The obtained crude fermentation extract was separated into components by vacuum silica gel column chromatography (a 50 mm (inner diameter) × 50 cm (length) glass column with a sand plate and nozzle). The eluent was a petroleum ether-ethyl acetate mixture with volume ratios of 20:1, 10:1, 5:1, 2:1, and 1:1, yielding five fractions (Fr.1) with increasing polarity. Fr.5). The fraction Fr.5 eluted from the petroleum ether-ethyl acetate 1:1 system was collected and purified using semi-preparative high-performance liquid chromatography (elution system: 50% acetonitrile-water) to finally prepare the compound peniquinone L (retention time). t R =8.7 min).
[0035] (III) Characterization of metabolites of tobacco endophytic fungi (compound peniquinone L) The chemical structure of the compound peniquinone L was determined using modern spectroscopic techniques, such as high-resolution mass spectrometry (HRESIMS) and superconducting nuclear magnetic resonance (NMR). The physicochemical properties of peniquinone L are as follows: Properties: Yellowish-brown oily substance; Solubility: Easily soluble in methanol, DMSO, and acetone; insoluble in petroleum ether and ethyl acetate; Ultraviolet absorption spectrum. λ max (log ε): 210 (3.45), 272 (3.29) nm; e.g. Figure 1 As shown, High Resolution Mass Spectrometry (HRESIMS): m / z 329.1391 [M – H] – (Theoretical value C) 19 H 21 O5 – (329.1394), indicating its molecular formula is C 19 H 22 O5; such as Figure 2 and Figure 3 As shown, superconducting nuclear magnetic resonance hydrogen spectrum ( 1 H NMR data and carbon spectra ( 13 The C NMR data are shown in Table 1.
[0036] Table 1. NMR data of compound peniquinone L ( 1 H NMR: 500 MHz; 13 C NMR: 125 MHz; Solvent used for NMR testing: DMSO- d 6)
[0037] Example 2: Inhibitory activity test of benzoquinone compound peniquinone L against six common agricultural pathogenic fungi. In this embodiment, the minimum inhibitory concentration (MIC) method was used to determine the inhibitory activity of the compound peniquinone L against six common agricultural pathogenic fungi, that is, the lowest drug concentration that can inhibit the growth of pathogenic fungi in vitro.
[0038] Six common agricultural pathogenic fungi were selected for antibacterial activity testing, namely *Botrytis cinerea* (tomato gray mold). Botrytis gray Pers, apple rot pathogen ( Valsamali Miyabe et Yamada), Tobacco Star Disease Bacterium ( Alternaria alternata (Fries) Keisslar), Penicillium citrus causal agent ( Penicillium italicum ), cucumber anthracnose bacteria ( Colletotrichum lagenarium ) and wheat sheath blight fungus ( Ceratobasidium horned (Borud.) Rogers). The above-mentioned agricultural pathogenic fungi were provided by the Tobacco Research Institute of the Chinese Academy of Agricultural Sciences.
[0039] Six agricultural pathogenic fungi were inoculated into potato dextrose water (PDW) medium and cultured at 28°C for 5 days. The bacterial concentration was then diluted with PDW medium to 10⁻⁶. 6 CFU / mL is used for the determination of minimum inhibitory concentration (MIC).
[0040] The compound peniquinone L was dissolved in DMSO to prepare a stock solution with a final concentration of 2560 μg / mL. A certain volume of the stock solution was then diluted with bacterial suspension to different concentrations: 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, and 1 μg / mL. A positive control was prepared using the fungicide carbendazim, also to the same concentration.
[0041] Different concentrations of the compound peniquinone L and the positive control carbendazim were added to the bacterial suspension of the pathogen. After incubation, the bacterial growth was observed. If the pathogen grew in a well, it indicated that the drug concentration in that well was insufficient to inhibit the growth of the bacteria, resulting in turbidity and decreased transmittance in the liquid of that well. Conversely, if the liquid in that well was clear and the decrease in transmittance was not significant, the lowest sample concentration that completely inhibited the growth of the pathogen in the well was defined as the MIC value of the compound.
[0042] The results of the activity tests of compound peniquinone L against six agricultural pathogenic fungi are shown in Table 2. The concentration corresponding to "clarified" is the MIC value of the treatment group. The results showed that compound peniquinone L exhibited inhibitory activity against *Botrytis cinerea* (tomato rot fungus), *Pseudomonas aeruginosa* (apple rot fungus), and *Aureobasidium aureum* (tobacco scab), with MIC values of 16 μg / mL, 8 μg / mL, and 2 μg / mL, respectively. Notably, the activity of compound peniquinone L against *Aureobasidium aureum* was superior to that of the positive control agent carbendazim (MIC = 4 μg / mL). This invention provides a benzoquinone compound derived from a natural product, offering a lead compound for the control of *Aureobasidium aureum* and a compound template for novel microbial natural product pesticides.
[0043] Table 2. Results of antibacterial activity experiments of compounds peniquinone L and carbendazim.
[0044] It will be readily understood by those skilled in the art that the above-described advantageous methods can be freely combined and superimposed without conflict. The above are merely preferred embodiments of this application and are not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application. The above are merely preferred embodiments of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of this application, and these improvements and modifications should also be considered within the protection scope of this application.
Claims
1. A metabolite of a tobacco endophytic fungus, characterized in that, The metabolite of the tobacco endophytic fungus is a benzoquinone compound, peniquinone L, with the following structural formula: 。 2. The method for preparing the tobacco endophytic fungal metabolites according to claim 1, characterized in that, include: Fermentation culture: The endophytic fungus *Aspergillus echinospora* from tobacco... Aspergillus aculeatus Aa-1 was inoculated into potato glucose aqueous medium and placed in a static culture and fermented for 28-32 days under natural light at 26℃-28℃. Extraction: After fermentation, the fermentation broth was extracted with ethyl acetate, the extracts were combined and concentrated to obtain the crude fermentation extract; Separation and purification: The crude fermentation extract was separated by vacuum silica gel column chromatography. The eluent was a petroleum ether-ethyl acetate mixture. Five components with increasing polarity were obtained. The most polar component was purified by semi-preparative high performance liquid chromatography to obtain the benzoquinone compound peniquinone L.
3. The preparation method according to claim 2, characterized in that, The reduced pressure silica gel column chromatography uses a gradient elution with a mixed solvent of petroleum ether and ethyl acetate. The volume ratios of petroleum ether to ethyl acetate used in the eluent are 20:1, 10:1, 5:1, 2:1 and 1:1, respectively.
4. The preparation method according to claim 3, characterized in that, The most polar component was obtained by elution with petroleum ether-ethyl acetate at a volume ratio of 1:
1.
5. The preparation method according to claim 2, characterized in that, In the semi-preparative high-performance liquid chromatography purification, the mobile phase is an acetonitrile-water mixture with a volume ratio of 50:
50.
6. The preparation method according to claim 5, characterized in that, Under the purification conditions, the retention time of the benzoquinone compound peniquinone L t R It takes 8.7 minutes.
7. The use of the tobacco endophytic fungal metabolite as described in claim 1 or the benzoquinone compound peniquinone L prepared by any one of claims 2-6 in the preparation of a drug for the prevention and control of plant pathogenic fungi.
8. The application according to claim 7, characterized in that, The plant pathogenic fungus is selected from at least one of the following: tomato gray mold, apple rot fungus, and tobacco red spot fungus.
9. The application according to claim 7, characterized in that, The plant pathogenic fungus is *Tobacco Star*.
10. The application according to claim 9, characterized in that, The minimum inhibitory concentration of the benzoquinone compound peniquinone L against *Aureobasidium aizoon* is 2 μg / mL.