Application of aldehyde compound 3-methylorcinaldehyde in controlling soybean root rot

Abstract

The present invention belongs to the field of fungicides, and discloses an application of an aldehyde compound 3‑methylorcinaldehyde in preventing and treating soybean root rot. The aldehyde compound is named 3‑methylorcinaldehyde, which is isolated from the secondary metabolite of Talaromyces adpressus PK‑1. The Talaromyces adpressus PK‑1 strain has been deposited in the General Microbiology Center of the China Culture Collection of Microorganisms, with the registration number CGMCC No.40517. The specific preparation method of the PK‑1 metabolite 3‑methylorcinaldehyde disclosed in the present invention includes the steps of strain liquid fermentation, extract concentration, column chromatography, high performance liquid chromatography purification, and the like. The compound 3‑methylorcinaldehyde has strong antibacterial activity against soybean root rot pathogens, and can be used as a new fungicide or lead compound for preventing and treating soybean root rot.

Description

Technical Field

[0001] The invention belongs to the field of fungicides, and particularly relates to application of an aldehyde compound 3-methylorcinaldehyde in preventing and controlling soybean root rot. Background Art

[0002] Root rot is a common soil-borne disease in agricultural production, occurring at all stages of plant development. Root rot can cause underdeveloped and stunted root systems, dwarfed and emaciated above-ground parts, chlorotic leaves, and a significant reduction in branching and fruiting. Current marketed root rot control agents, due to prolonged and intensive use, are increasingly becoming resistant to these agents. Therefore, identifying and developing new agricultural fungicides is a key approach to addressing this resistance.

[0003] The natural environment surrounding plants is rich in microorganisms, some of which can promote plant growth and help plants resist infection by pathogens. They can directly antagonize pathogens or induce plant resistance to pathogens. These microorganisms can produce abundant secondary metabolites and have good biological activity. Therefore, screening for compounds with antibacterial activity from soil microbial metabolites is an effective strategy for finding new green pesticides. Basilisk is a type of fungus that is widely present in the soil. Studies have shown that saccharomyces and their metabolites have antibacterial, antifungal, antitumor activities, and enzyme production, and are widely used in food, environment, agriculture, and medicine.

[0004] However, there are no reports on screening compounds with antibacterial activity from soybean rhizospheric fungi, especially Talaromyces, as fungicides. Summary of the Invention

[0005] In response to the above-mentioned problems, the present invention provides an application of the aldehyde compound 3-methylorcinaldehyde in preventing and controlling soybean root rot, and a method for preparing the aldehyde compound 3-methylorcinaldehyde using Bacillaceae.

[0006] In a first aspect, the present invention provides an application of the aldehyde compound 3-methylorcinaldehyde, which includes one or more of the following: A) inhibiting the growth of soybean root rot pathogens; B) preventing and controlling soybean root rot; C) preparing a composition that inhibits the growth of soybean root rot pathogens and / or prevents and controls soybean root rot.

[0007] The structural formula of the aldehyde compound 3-methylorcinaldehyde is as follows:

[0008]

[0009] Furthermore, the soybean root rot pathogens include, but are not limited to, Phytophthora sojae, Pythium ultimum, Fusarium oxysporum, Fusarium solani, Fusarium graminearum, Fusarium equiseti, Rhizoctonia solani, Phomopsis longicolla and Macrophomina phaseolina.

[0010] On the other hand, the present invention also provides a method for preparing the aldehyde compound 3-methylorcinaldehyde.

[0011] The aldehyde compound 3-methylorcinaldehyde can be obtained from the fermentation broth of Talaromyces adpressus PK-1 fungus from the soybean rhizosphere through a variety of separation and purification methods.

[0012] Furthermore, the preparation method comprises the following steps:

[0013] (1) Inoculate the compacted tularensis PK-1 with the deposit number CGMCC No. 40517 onto a PDA plate, incubate the plate upside down at 23-28°C for 2-5 days, take the bacterial block and incubate it in a PDA liquid medium at 23-28°C and 130-160 rpm for 5-8 days to obtain a fermentation broth;

[0014] (2) filtering the fermentation broth obtained in step (1), extracting the filtrate with ethyl acetate, and concentrating under reduced pressure to obtain a crude extract of the fermentation product;

[0015] (3) subjecting the crude fermentation extract obtained in step (2) to silica gel column chromatography and gel column chromatography, and combining the components containing 3-methylorcinaldehyde by TLC detection;

[0016] (4) The component containing 3-methylorcinaldehyde in step (3) is separated and purified by high performance liquid chromatography to obtain the aldehyde compound 3-methylorcinaldehyde.

[0017] Furthermore, the compact basket fungus PK-1, taxonomically named Talaromyces adpressus, was registered and deposited in the General Microbiology Center of the China Culture Collection Administration (CGMCC) on March 6, 2023, with the deposit number CGMCC NO.40517, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0018] On the other hand, the present invention also provides a composition for inhibiting the growth of soybean root rot pathogens and / or preventing and treating soybean root rot, wherein the main active ingredient of the composition is an aldehyde compound.

[0019] 3-methylorcinaldehyde.

[0020] Furthermore, the composition is an emulsifiable concentrate, an aqueous emulsion, a microemulsion, a wettable powder, a water-dispersible granule, or a suspension.

[0021] On the other hand, the present invention also provides the use of the composition in inhibiting the growth of soybean root rot pathogens and / or preventing and treating soybean root rot.

[0022] Furthermore, the soybean root rot pathogens include, but are not limited to, Phytophthora sojae, Pythium ultimum, Fusarium oxysporum, Fusarium solani, Fusarium graminearum, Fusarium equiseti, Rhizoctonia solani, Phomopsis longicolla and Macrophomina phaseolina.

[0023] On the other hand, the present invention also provides a method for preventing and controlling soybean root rot, comprising applying the composition to the soybean growth environment.

[0024] Compared with the prior art, the present invention determines the in vitro antibacterial activity of the compound 3-methylorcinaldehyde against various soybean root rot pathogens, including Phytophthora sojae, Pythium ultimum, Fusarium oxysporum, Fusarium solani, Fusarium graminearum, Fusarium equiseti, Rhizoctonia solani, Phomopsis longicolla and Macrophomina phaseolina, by using a mycelium growth rate method. The results showed that 3-methylorcinaldehyde exhibited strong antibacterial activity against the aforementioned pathogens. The inhibitory rate of 3-methylorcinaldehyde (100 μg / mL) against Fusarium graminearum, Phytophthora sojae, and Rhizoctonia solani was significantly superior to that of pyraclostrobin (100 μg / mL). This indicates that 3-methylorcinaldehyde has strong inhibitory activity against multiple soybean root rot pathogens and has great potential for development as a new fungicide or lead compound for the prevention and treatment of soybean root rot. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the structural diagram of the compound 3-methylorcinaldehyde.

[0026] Figure 2 For the compound 3-methylorcinaldehyde 1 H-NMR spectrum.

[0027] Figure 3 For the compound 3-methylorcinaldehyde 13 C-NMR spectrum.

[0028] Figure 4 This is a graph showing the antibacterial activity of the compound 3-methylorcinaldehyde against soybean root rot pathogens. DETAILED DESCRIPTION

[0029] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores.

[0030] Example 1 Preparation and structural analysis of the compound

[0031] 1. Preparation of compounds:

[0032] (1) Fermentation of Talaromyces adpressus PK-1

[0033] Talaromyces adpressus PK-1 was inoculated onto a PDA plate and incubated upside down at 25°C for 3 days. The bacterial mass was then placed in a PDA liquid medium and incubated at 25°C and 150 rpm for 7 days.

[0034] (2) Extraction of secondary metabolites from Talaromyces adpressus PK-1

[0035] The fermentation product after fermentation was filtered through two layers of gauze to obtain a fermentation broth, which was extracted three times with equal volumes of ethyl acetate, and the organic layers were combined, concentrated under reduced pressure to obtain a crude extract;

[0036] (3) Isolation and purification of secondary metabolites (3-methylorcinaldehyde) from bacterial extracts

[0037] The crude extract was subjected to silica gel column chromatography and gel column chromatography, and the same components were combined by TLC detection. The column chromatography was repeated multiple times until the compounds contained in the components were relatively simple. Finally, the product was purified by high performance liquid chromatography to obtain the aldehyde compound 3-methylorcinaldehyde.

[0038] 2. Structural identification of compounds:

[0039] The structural formula of the compound is Figure 1 As shown, 1 H-NMR spectra and 13 The C-NMR spectra are as follows Figure 2-3 The compound was analyzed to be colorless needle-shaped crystals, HR-ESI-MS. m / z: 165.055 [MH]-. 1 H-NMR(500MHz,Dimethylsulfoxide-d6)δ H 6.28(1H,s),9.99(1H,s),1.91(3H,s),2.44(3H,s). 13C-NMR(150MHz,Dimethyl sulfoxide-d6)δ C 142.08(C-1),163.78(C-2),110.39(C-3),163.88(C-4),108.47(C-5),112.50(C-6),194.43(C-7),7.61(C-8),18.19(C-9).

[0040] The above data are consistent with the reported data of 3-methylorcinaldehyde, thus confirming that they are the same compound.

[0041] Example 2 Inhibition experiment of compound 3-methylorcinaldehyde on soybean root rot pathogens

[0042] The antibacterial activity of the compound 3-methylorcinaldehyde against soybean root rot pathogens was determined using a hyphal growth rate assay, with pyraclostrobin used as a control. The specific steps were as follows: Preserved soybean root rot pathogens were activated on plates and incubated in a darkroom at 25°C. After 3-5 days, colonies emerged. The activated strains were transferred to plates containing the corresponding culture medium and allowed to grow until hyphae completely covered the plates. 3-methylorcinaldehyde was diluted with DMSO to prepare stock solutions of 2 mg / mL, 10 mg / mL, and 20 mg / mL. Pyraclostrobin was also diluted with DMSO to prepare a stock solution of 20 mg / mL. Melt the culture medium in a microwave oven. When the temperature drops to around 50 degrees, add the mother solution of each compound at a volume ratio of 200:1. Mix well and pour onto plates. Cool and blow dry to make plates containing 10μg / mL, 50μg / mL, and 100μg / mL of the compound 3-methylorcinaldehyde, as well as 100μg / mL of pyraclostrobin. At the same time, use plates with the corresponding volume of DMSO as a control. Use a 5mm punch to punch out bacterial cakes on each pathogen plate, inoculate them onto the corresponding medium-containing plate, and culture in a dark box in the greenhouse. Each treatment was repeated three times, and the colony diameter was measured using the cross-hatch method to calculate the inhibition rate.

[0043] Inhibition rate calculation formula:

[0044]

[0045] The results of the antibacterial activity of compound 3-methylorcinaldehyde against soybean root rot pathogens are shown in Tables 1 and Figure 4 shown.

[0046] Table 1 Inhibition rate of compound 3-methylorcinaldehyde on soybean root rot pathogens

[0047]

[0048] As can be seen from Table 1, compound 3-methylorcinaldehyde has strong antibacterial activity against the above-mentioned pathogens. The inhibition rate of compound 3-methylorcinaldehyde (100 μg / mL) against Fusarium graminearum, Phytophthora sojae and Rhizoctonia solani is significantly better than that of pyraclostrobin (100 μg / mL).

[0049] Example 3 Experiment on the Effect of Compound 3-methylorcinaldehyde on Soybean Growth

[0050] Soybean growth was measured on water agar. The specific steps were as follows: An appropriate amount of soybeans was spread onto a 15-cm petri dish and placed in a sealed container. Meanwhile, 4 mL of concentrated hydrochloric acid and 96 mL of sodium hypochlorite were prepared and mixed in a beaker in the same sealed container. The container was immediately sealed. After 14 hours of chlorine sterilization, the soybeans were removed and, under a clean bench, chlorine residue on the surface was removed and set aside. 3-methylorcinaldehyde was diluted with DMSO to prepare stock solutions of 2 mg / mL, 10 mg / mL, and 20 mg / mL. The water agar was melted in a microwave oven. When the temperature dropped to approximately 50°C, the stock solutions of each compound were added at a ratio of 200:1 by volume. After mixing, the stock solutions were poured onto plates, cooled, and air-dried. Plates containing 10 μg / mL, 50 μg / mL, and 100 μg / mL of 3-methylorcinaldehyde were prepared. A control plate containing the corresponding volume of DMSO was also prepared. Surface-sterilized soybeans were gently placed on the surface of the culture medium, with 5 soybeans per dish and three replicates per treatment. The dishes were covered but not sealed. Incubated in a 25°C incubator for 7 days, the length of the soybean rhizomes was counted.

[0051] The effects of the compound 3-methylorcinaldehyde on soybean growth are shown in Table 2:

[0052] Table 2 Root and stem length of soybean under the influence of different concentrations of compound 3-methylorcinaldehyde

[0053]

[0054] The experimental results show that the compound 3-methylorcinaldehyde has no significant effect on soybean growth. The compound 3-methylorcinaldehyde can be developed as a new fungicide or lead compound for the prevention and control of soybean root rot.

[0055] While the specific embodiments of the present invention have been described in detail above, these are intended to be exemplary only, and the present invention is not limited thereto. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, any equivalent changes and modifications made without departing from the spirit and scope of the present invention are intended to be encompassed within the scope of the present invention.

Claims

1. The use of the aldehyde compound 3-methylorcinaldehyde, characterized in that: The application is one or more of the following, A) Inhibit the growth of soybean root rot pathogens; B) Control soybean root rot; C) preparing a composition capable of inhibiting the growth of soybean root rot pathogens and / or preventing soybean root rot; The soybean root rot pathogen is selected from Phytophthora sojae ( Phytophthora sojae ultimum ( Pythium ultimum ), Fusarium oxysporum ( Fusarium oxysporum ), Fusarium solani ( Fusarium solani graminearum ( Fusarium graminearum ), Fusarium equisetum ( Fusarium equiseti ) Rhizoctonia solani ( Rhizoctonia solani )、Phomopsis ( Phomopsis longicolla ) and Phaseolus vulgaris ( Macrophomina phaseolina ); The structural formula of the aldehyde compound 3-methylorcinaldehyde is .

2. A method for preparing the aldehyde compound 3-methylorcinaldehyde according to claim 1, characterized in that: The preparation method The following steps are included: (1) Inoculate the compacted tularensis PK-1 with the deposit number CGMCC No. 40517 onto a PDA plate, invert and culture at 23-28°C for 2-5 days, take the bacterial block and culture it in a PDA liquid medium at 23-28°C and 130-160 rpm for 5-8 days to obtain a fermentation broth; (2) filtering the fermentation broth obtained in step (1), extracting the filtrate with ethyl acetate, and concentrating under reduced pressure to obtain a crude extract of the fermentation product; (3) The crude extract of the fermentation product obtained in step (2) was subjected to silica gel column chromatography and gel column chromatography, and the components containing 3-methylorcinaldehyde were combined by TLC detection; (4) The component containing 3-methylorcinaldehyde in step (3) is separated and purified by high performance liquid chromatography to obtain the aldehyde compound 3-methylorcinaldehyde.

3. Use of the composition in inhibiting the growth of soybean root rot pathogens and / or preventing and treating soybean root rot, characterized in that: The active ingredient of the composition is the aldehyde compound 3-methylorcinaldehyde described in claim 1, and the pathogen of soybean root rot is selected from soybean phytophthora ( Phytophthora sojae ultimum ( Pythium ultimum ), Fusarium oxysporum ( Fusarium oxysporum ), Fusarium solani ( Fusarium solani graminearum ( Fusarium graminearum ), Fusarium equisetum ( Fusarium equiseti ) Rhizoctonia solani ( Rhizoctonia solani )、Phomopsis ( Phomopsis longicolla ) and Phaseolus vulgaris ( Macrophomina phaseolina ).

4. The use according to claim 3, characterized in that The composition is emulsifiable concentrate, aqueous emulsion, microemulsion, wettable powder, water-dispersible granule or suspension.

5. A method for preventing and treating soybean root rot, characterized in that: The method comprises applying the aldehyde compound 3-methylorcinaldehyde described in claim 1 or the composition described in claim 4 to the soybean growth environment, wherein the soybean root rot pathogen is selected from Phytophthora sojae ( Phytophthora sojae ultimum ( Pythium ultimum ), Fusarium oxysporum ( Fusarium oxysporum ), Fusarium solani ( Fusarium solani graminearum ( Fusarium graminearum ), Fusarium equisetum ( Fusarium equiseti ) Rhizoctonia solani ( Rhizoctonia solani )、Phomopsis ( Phomopsis longicolla ) and Phaseolus vulgaris ( Macrophomina phaseolina ).

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