Alternaria alternata metabolite and application thereof
By preparing polyketide compounds of Alternaria alternata, the problems of drug resistance and environmental pollution of chemical fungicides in the prevention and control of plant pathogenic bacteria are solved, providing an efficient and low-cost biopesticide solution.
Patent Information
- Application Number
- CN202510817490.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-18
- Publication Date
- 2025-09-19
AI Technical Summary
Existing chemical fungicides have problems with drug resistance, environmental pollution and high costs in controlling plant pathogenic bacteria, making it difficult to effectively control plant diseases.
Polyketide compounds of Alternaria alternata are used to prepare antimicrobial agents through liquid fermentation and separation technology, which are used to inhibit the growth of various plant pathogenic bacteria.
It provides a broad-spectrum antibacterial activity, is low-cost, and environmentally friendly antibacterial agent that can effectively inhibit a variety of plant pathogenic bacteria and has the potential to develop new biopesticides.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, in particular to metabolites extracted from liquid fermentation products of Alternaria alternata and applications thereof. Background Art
[0002] Plant pathogenic bacteria have become the second most common plant pathogen group in global agricultural production, second only to fungi, causing annual crop yield losses of up to 20%-50%. Their damage is characterized by rapid outbreaks, rapid spread, and difficulty in prevention and control. Internationally quarantined diseases such as citrus canker, rice bacterial leaf blight, and bacterial leaf streak are particularly challenging, and existing methods are difficult to eradicate.
[0003] The current control model, which relies on chemical fungicides, faces three challenges: increasing resistance, with detection rates exceeding 60% for pathogen resistance genes to mainstream fungicides like thiophanate-copper and kasugamycin; increasing residue and contamination, with copper ion accumulation leading to soil ecological degradation and excessive pesticide residues causing trade barriers; and rising control costs, with resistance management requiring rotation of pesticides, increasing per-acre investment by over 35%. Against this backdrop, microbial biopesticides, with their core advantages of good environmental compatibility, high target specificity, and resistance resistance, are widely recognized as a strategic alternative to chemical pesticides.
[0004] Fungal secondary metabolites, a treasure trove of natural antimicrobial compounds, offer a new path forward in addressing the challenges of controlling plant pathogens due to their novel structures and diverse mechanisms of action. Alternaria, a widely distributed filamentous fungus, possesses structurally diverse metabolites with significant biological activity, particularly in the field of antimicrobial research. Their rich secondary metabolites provide a core foundation for the development of novel antimicrobial compounds. Summary of the Invention
[0005] The technical problem to be solved by the present invention is to provide a preparation method of a metabolite of Alternaria alternata and an application thereof in terms of its antibacterial activity.
[0006] In order to solve the above technical problems, the present invention provides two polyketide compounds of Alternaria alternata, whose structural formula is:
[0007]
[0008] The Alternaria alternata described in the present invention is specifically a strain preserved in the China Center for Type Culture Collection with a preservation number of CCTCCAF 2020021.
[0009] The present invention also provides a method for preparing the polyketone compound, comprising the following steps:
[0010] 1) Pick the preserved Alternaria mycelium, inoculate it into PDA plate culture medium, and activate and culture it at 28℃ for 3-4 days;
[0011] 2) Fresh mycelium was picked from the vial culture and inoculated into 100 mL of autoclaved liquid PDA medium. The culture was carried out on a shaker at 28 ± 0.5 °C and 180 rpm for 3 days to obtain the seed solution.
[0012] 3) Transfer the seed solution to the fermentation medium at an inoculum rate of 10%-15%, and ferment and culture for 7 days under the shaking conditions in step 1;
[0013] 4) Filter the fermentation broth obtained in step 3 through 4 layers of gauze to separate the bacterial cells, extract the filtrate three times with ethyl acetate, and concentrate the ethyl acetate component under reduced pressure to obtain a crude extract;
[0014] 5) Using the crude extract of step 4, a silica gel column was prepared with 10 times the mass of silica gel. The column was initially eluted with pure dichloromethane, and then eluted with dichloromethane and methanol at a ratio of 100:1, 100:2, and 100:4 to obtain different elution fractions;
[0015] 6) The eluted portion in step 5 is dried and separated by gel column chromatography using petroleum ether as eluent, and the resulting precipitate is recrystallized from petroleum ether to obtain a polyketone compound.
[0016] The present invention also provides the use of the above-mentioned polyketide compounds in antibacterial agents, specifically for inhibiting the growth of pathogenic bacteria, including oxysporum leaf streak pathogenic bacteria, oxysporum leaf streak pathogenic bacteria, tetragenous cocci, Staphylococcus aureus, Escherichia coli and Pseudomonas syringae pv. kiwifruit.
[0017] The present invention has the following beneficial effects:
[0018] 1. The compounds of the present invention have broad-spectrum antibacterial activity, can effectively inhibit a variety of plant pathogenic bacteria, and have the potential to be developed into new antibacterial agents.
[0019] 2. The strain and its metabolites of the present invention can be preserved and have clear sources.
[0020] 3. The present invention is based on the metabolite production technology of deep liquid fermentation, which has the core advantages of short cycle, low cost and controllable process parameters.
[0021] 4. The present invention utilizes biological methods to synthesize compounds without causing pollution to the environment. DETAILED DESCRIPTION
[0022] The present invention will be further explained below with reference to specific implementation cases.
[0023] Example 1: Large-scale fermentation of Alternaria alternata
[0024] Alternaria alternata (accession number CCTCC AF 2020021) was obtained from the China Center for Type Culture Collection. The strain was inoculated on PDA plate culture medium and cultured in a constant temperature incubator at 28±0.5°C for 3 days. Three pieces of bacterial cakes were picked from the vial culture and inoculated into a 250-mL Erlenmeyer flask (containing 150 mL of liquid culture medium). The culture was shaken at 28°C and 180 rpm for 3 days as a seed solution, which was then inoculated into a 1000-mL Erlenmeyer flask (containing 400 mL of liquid culture medium) at a volume ratio of 1:20.
[0025] The obtained fermentation broth was filtered through four layers of gauze to separate the bacterial cells to obtain a fermentation broth, which was extracted three times with ethyl acetate (v / v=1:1), and the ethyl acetate component was concentrated under reduced pressure to obtain an extract.
[0026] Example 2: Extraction and separation of polyketides from Alternaria alternata
[0027] The extract of Example 1 was mixed with an equal mass of 100-200 mesh silica gel, dried in an oven at 37°C, and further ground into a powder for later use. A silica gel column was prepared using 10 times the mass of the crude extract, and eluted with dichloromethane and methanol in different ratios, with polarity from low to high. Initially, pure dichloromethane was used for elution, followed by elution with dichloromethane and methanol ratios of 100:1, 100:2, and 100:4 (each eluent was used in an amount of about 10 column volumes). After drying, the precipitate was separated by gel column chromatography using petroleum ether as the eluent. The structural formula of the resulting precipitate was:
[0028]
[0029] Example 3: Structural Identification of Alternaria polyketide compounds
[0030] The structures of the polyketides of Alternaria alternata were determined based on their properties and analysis of nuclear magnetic resonance spectroscopy data.
[0031] The properties and spectral data are as follows:
[0032] Compound 1: white crystals. HR-ESI-MS: m / z: 257.0455 [MH] - ; 1 H NMR (600 MHz, Methanol-d4) δ7.17 (s, 1H), 6.56 (s, 1H), 6.56 (s, 1H), 6.28 (s, 1H), 2.73 (s, 3H). The compound was identified as Alternariol.
[0033] Compound 2: yellow-green solid. HR-ESI-MS: m / z: 287.0563 [MH] - ;1 H NMR (600MHz, CDCL3) δ11.29(s,OH-3),6,73(s,H-6),6.96(s,H-7),6.63(s,H-4),6.40(s,OH-9),6.28(s,H-10),3.91(s,OCH3-5),1.73(s,OCH3-1a); 13 C NMR (150 MHz, CDCL3) δ180.73 (C-8), 167.28 (C-2), 166.32 (C-5), 164.71 (C-3), 153.08 (C-7a), 146.13 (C-9), 134.99 (C-6a), 120.75 (C-7), 116.08 (C-10), 104.27 (C-6), 103.66 (C-4), 99.84 (C-2a), 55.92 (OCH3-5), 29.42 (CH3-1a). The compound was identified as dehydroaltenusin.
[0034] Example 4: Inhibitory Effects of Polyketides of Alternaria on Pathogens
[0035] The activity of polyketides from Alternaria alternata against bacteria was tested using the filter paper diffusion method. The test bacteria were inoculated into LB liquid medium and activated at 37°C for 24 hours. A small amount of bacteria was collected using an inoculating loop and mixed evenly with the sterile water to create a bacterial suspension. The concentration of the bacterial suspension was adjusted to approximately 10% by comparing it with the turbidity control tube. 6 cFU / mL. Dissolve each compound in acetone to 6 mg / mL. Use gentamicin sulfate as a positive control and acetone as a blank control. Incubate in liquid culture at 28°C, 180 rpm until logarithmic phase. Then dilute and plate, then allow to condense. Use a pipette to aspirate 5 μL of each solution and dropwise onto a prepared 5 mm sterile filter paper. After drying, place on a solid culture medium containing bacteria, attaching three filter paper sheets to each plate as replicates. Incubate at 37°C for 24 hours, and measure colony diameters using the cross-hatch method.
[0036] The growth inhibition effects of the two compounds on pathogenic bacteria are shown in Table 1 below. As shown in the table, at a test concentration of 30 μg / filter paper, compared to the positive control gentamicin sulfate, the compound Alternariol exhibited significant inhibitory activity against bacterial leaf blight pathogens of rice and Pseudomonas syringae pv. kiwifruit, with inhibition zone diameters of 11.7 mm and 17.0 mm, respectively. It also exhibited moderate inhibitory activity against Staphylococcus aureus and Tetracoccus sp., with inhibition zone diameters of 9.7 mm and 8.8 mm, respectively. Dehydroaltenusin, on the other hand, exhibited significant inhibitory activity against bacterial leaf blight pathogens of rice and bacterial leaf streak pathogens of rice, with inhibition zone diameters of 12.2 mm and 12.3 mm, respectively. It also exhibited moderate inhibitory activity against Tetracoccus sp., Staphylococcus aureus sp., and Escherichia coli, with inhibition zone diameters of 9.3 mm, 8.3 mm, and 11.8 mm, respectively.
[0037] Table 1. Inhibitory effect of polyketides of Alternaria alternata on the growth of pathogenic bacteria (mm)
[0038]
[0039] Note: The results are expressed as the standard of the diameter of the inhibition zone ± 3 parallel measurements; gentamicin sulfate is the positive control for pathogenic bacteria; NI means no activity; the test concentration is 30 μg / filter paper.
Claims
1. Alternaria metabolites and their applications, characterized in that Provided are a polyketide compound of Alternaria alternata, a preparation method thereof, and a use thereof as an antibacterial agent.
2. The method for preparing the polyketide compound of Alternaria alternata according to claim 1, characterized by the following steps: 1) Inoculate Alternaria alternata on PDA plate medium and culture in a constant temperature incubator at 28±0.5℃ for 7 days; 2) Then take 3 pieces of cake and inoculate them into 100mL of autoclaved liquid PDA culture medium, at a temperature of 28±0.5℃ and a speed of 180 Culture on a shaker at rpm for 3 days to obtain seed solution; 3) Transfer the seed solution to the fermentation medium at an inoculum rate of 10%-15%, and ferment and culture for 7 days under the shaking conditions in step 2; 4) Filter the fermentation broth obtained in step 3, extract the filtrate with ethyl acetate, and vacuum concentrate and dry to obtain a crude extract; 5) Using the crude extract of step 4, a silica gel column was prepared with 10 times the mass of silica gel, initially eluted with pure dichloromethane, and then eluted with dichloromethane and methanol ratios of 100:1, 100:2, and 100:4 to obtain different elution fractions; 6) The obtained fractions are repeatedly passed through a silica gel column, a gel column, a reverse column, etc. to further purify the monomer compound, and finally obtain the monomer compound.
3. The polyketide compound of Alternaria according to claim 1 has antagonistic effects on bacterial leaf blight of rice, bacterial leaf streak of rice, Tetragenococcus, Staphylococcus aureus, Escherichia coli and Pseudomonas syringae pv. kiwifruit, and can be used to develop fungicides.