Use of a pachouli ketone in the preparation of a plant-derived fungicide for preventing and treating peanut pellicularia ssp. through multiple pathways

Abstract

This invention relates to the application of plant-derived fungicides in the preparation of multi-pathway control of peanut white mold disease. It reveals for the first time the specific mechanisms by which patchouli inhibits the pathogen causing peanut white mold, including disrupting the ultrastructure of pathogen cells, interfering with energy metabolism, and inhibiting sclerotium germination. The EC50 of patchouli inhibits the mycelial growth of this pathogen. 50 The concentration was 3.59 mg / L. At a concentration of 20 mg / L, the inhibition rate of mycelial growth reached 89.52%, and at a concentration of 150 mg / L, sclerotium germination was completely inhibited. After treatment, the mycelia of the pathogen showed swelling, apical deformity, separation of the cell wall from the cell membrane, and cytoplasmic vacuolation. The ATP content and enzyme activities, succinate dehydrogenase, and NADP-malate dehydrogenase in the mycelia were significantly reduced, while the malondialdehyde content increased. The content of reducing sugar, soluble protein, pyruvate, oxalate, and polygalacturonase activity decreased. It has the advantages of multi-target and low resistance.

Description

Technical Field

[0001] This invention relates to the field of pesticide technology, and in particular to the application of patchouli ketone in the preparation of a plant-derived fungicide that controls peanut white mold through multiple pathways. Background Technology

[0002] Peanuts are one of my country's important oilseed crops, but white mold disease caused by *Sclerotium rolfsii* has become a serious soil-borne disease restricting the sustainable development of the peanut industry. This pathogen has an extremely wide host range, infecting over 500 plant species, and is characterized by its strong persistence and difficulty in control. Currently, production mainly relies on chemical pesticides for control, but long-term and excessive use of chemical agents has not only led to the emergence of pesticide-resistant strains but also caused problems such as pesticide residues and soil ecological damage. Biological control technology has attracted much attention due to its environmentally friendly characteristics. Existing research shows that microbial resources such as *Bacillus*, *Pseudomonas*, *Streptomyces*, and *Trichoderma* have potential control value. However, biocontrol agents generally suffer from technical bottlenecks such as large fluctuations in field efficacy and long lag periods, which seriously restrict their industrial application.

[0003] Plant-derived natural products, due to their diverse structures, novel mechanisms of action, easy degradation, and safety for non-target organisms, show broad application prospects in the biocontrol of plant diseases. Patchouli, a key active component of patchouli essential oil (a plant in the Lamiaceae family), has currently seen its activity research primarily focused on the medical field, with existing literature reporting its antioxidant, anti-inflammatory, anti-photoaging, and antibacterial effects. In agricultural applications, this compound has shown some toxicity against the beet armyworm (Spodoptera exigua) and the cotton bollworm (S. exigua). However, the antagonistic effect of patchouli on peanut white mold pathogens has not yet been reported. Summary of the Invention

[0004] Based on the above technical background, the purpose of this invention is to provide an application of patchouli ketone in the preparation of a plant-derived fungicide for the prevention and control of peanut white mold through multiple pathways. It reveals for the first time the specific mechanisms by which patchouli ketone inhibits peanut white mold pathogens, including destroying the ultrastructure of pathogen cells, interfering with energy metabolism, and inhibiting sclerotium germination, providing a new technical solution for the green control of peanut white mold.

[0005] The technical solution provided by this invention is the application of patchouli ketone in the preparation of a plant-derived fungicide for controlling peanut white mold through multiple pathways, wherein the peanut white mold pathogen is *Sclerotium rolfsii*; the patchouli ketone inhibits the peanut white mold pathogen through at least one of the following pathways: disrupting the ultrastructure of the pathogen's mycelial cells, interfering with the pathogen's energy metabolism, and inhibiting sclerotium germination.

[0006] Furthermore, the destruction of the cell's ultrastructure manifests as swelling and thickening of the fungal hyphae, cross-entanglement, and swollen and deformed tips, as well as separation of the cell wall from the cell membrane and the appearance of vacuolar structures in the cytoplasm.

[0007] Furthermore, the disruption of energy metabolism manifests as a significant decrease in ATP content within the mycelium. The activities of enzymes, succinate dehydrogenase, and NADP-malate dehydrogenase were all inhibited. In addition, patchouli ketone could increase the malondialdehyde content in the mycelium, decrease the content of reducing sugar, soluble protein, and pyruvate, and at the same time reduce the oxalic acid content and polygalacturonase activity.

[0008] Furthermore, the patchouli ketone inhibits the mycelial growth of peanut white mold fungus (EC). 50 The concentration was 3.59 mg / L. At a concentration of 20 mg / L, the inhibition rate of mycelial growth reached 89.52%, and at a concentration of 150 mg / L, the inhibition rate of sclerotium germination reached 100%.

[0009] The present invention also provides a plant-derived fungicide for controlling peanut white mold disease, the active ingredient of which is patchouli ketone, and may contain agriculturally acceptable adjuvants, solvents or auxiliaries, wherein the peanut white mold pathogen is Sclerotium rolfsii.

[0010] The beneficial technical effects of this invention are as follows:

[0011] This invention systematically reveals for the first time the multi-pathway antibacterial mechanism of patchouli against *Sclerotium affine*, the causal agent of peanut white mold. This multi-target action mode is beneficial in delaying the development of drug resistance in the pathogen. Sclerotia are the main overwintering stage and reinfection source of this pathogen in the soil. This invention demonstrates that patchouli can completely inhibit sclerotia germination, which is of great significance for blocking the disease cycle and reducing the initial source of infection the following year. Simultaneously, oxalic acid and polygalacturonase are key factors in the pathogenicity of necrotic fungi. This invention demonstrates that patchouli can significantly reduce the levels of both, thereby weakening the pathogen's infectivity. Compared with existing technologies, this invention reveals for the first time the multi-pathway antibacterial mechanism of patchouli and comprehensive physiological and biochemical effect data, representing a substantial improvement and breakthrough to existing technologies. Patchouli is a natural plant-derived compound, characterized by easy degradation and safety for non-target organisms, aligning with the development direction of green pesticides. Attached Figure Description

[0012] Figure 1 This invention demonstrates the inhibitory effect of different concentrations of patchouli ketone on the mycelial growth of peanut white mold disease.

[0013] Figure 2 This invention relates to the effect of patchouli ketone on the mycelial growth of peanut white mold causal agent;

[0014] Figure 3 This invention relates to the inhibitory effect of patchouli ketone on the sclerotium germination of peanut white mold fungus;

[0015] Figure 4 This invention relates to the effect of patchouli ketone on the mycelial morphology of peanut white mold fungus;

[0016] Figure 5 This invention relates to the antagonistic effect of patchouli ketone on peanut white mold cells. Detailed Implementation

[0017] The specific implementation of the present invention will be described in detail below with reference to examples and specific circumstances.

[0018] Example 1: Virulence determination of patchouli against peanut white mold pathogen

[0019] The inhibitory effect of patchouli on the mycelial growth of *Sclerotium arachnoides*, the causal agent of peanut white mold, was determined using the mycelial growth rate method. Patchouli was dissolved in a sterile aqueous solution containing 1% DMSO and 1% Tween-20 to prepare a stock solution. The stock solution was mixed with PDA medium at a ratio of 1:9 to prepare plates with concentrations of 1, 2.5, 5, 10, and 20 mg / L, which were then poured into 90 mm diameter Petri dishes. PDA medium containing the same volume of DMSO and Tween-20 served as a control plate. A 5 mm mycelial cake was taken from the edge of a 5-day-old colony and placed in the center of the drug-containing plate. After incubation at 30 ℃ in the dark for 5 days, the diameter of each colony was measured using the cross-crossing method, and the mycelial growth inhibition rate of each compound against *Sclerotium arachnoides* was calculated. Mycelial growth inhibition rate (%) = (Control colony diameter - Treated colony diameter) / (Control colony diameter - Mycelial cake diameter) × 100. Using the fungicide concentration after log10 transformation as the x-axis and the probability value corresponding to the mycelial growth inhibition rate as the y-axis, the toxicity regression equation of patchouli ketone was obtained, and EC was calculated. 50 value.

[0020] Based on the antibacterial effect of different concentrations of patchouli ketone on peanut white mold ( Figure 1 The virulence regression equation was obtained as y = 1.16 + 2.08x. Calculations showed that patchouli ketone inhibited the mycelial growth of *Sclerotium arachnoides* var. *arachnoides*. 50 The concentration was 3.59 mg / L, indicating strong antibacterial activity.

[0021] Example 2: Effects of patchouli ketone on mycelial growth and sclerotium germination of *Peanut white mold*.

[0022] PDB media containing patchouli at final concentrations of 1, 2.5, 5, 10, and 20 mg / L were prepared. Five mycelial cakes were taken from the edge of *Sclerotium arachnoideum* and inoculated into these media, with the treatment without patchouli serving as a blank control. The media were cultured at 30 °C and 160 r / min with shaking for 4 days. Mycelia were then collected and dried in an oven at 60 °C for 24 h, and weighed. Each treatment was repeated in triplicate. Mature sclerotia were immersed in 75% ethanol solution for 2 min, followed by rinsing three times with sterile water for surface disinfection. After surface disinfection, the sclerotia were placed on PDB plates containing patchouli at concentrations of 50, 100, and 150 mg / L and cultured at 25 °C, with PDB plates containing an equal volume of solvent serving as a control. Three plates were used for each treatment, with 16 sclerotia placed on each plate. The germination of the sclerotia was observed after 72 h, and the inhibition rate of sclerotia germination by different treatments was calculated. Sclerotia germination inhibition rate (%) = (number of ungerminated sclerotia / total number of sclerotia) × 100.

[0023] The effect of patchouli ketone on the mycelial growth of peanut white mold causal agent is as follows: Figure 2 As shown, patchouli significantly inhibited the mycelial growth of *Sclerotium arachnoides*, the causal agent of peanut white mold, with the inhibitory effect gradually increasing with increasing concentration. At a concentration of 20 mg / L, patchouli inhibited the mycelial growth of *Sclerotium arachnoides* by 89.52%. Figure 3 It was found that patchouli ketone also had a strong inhibitory effect on the sclerotium germination of *Sclerotium affine*. On the control PDA plate without patchouli ketone, all sclerotia germinated. The inhibition rate of sclerotium germination gradually increased with increasing patchouli ketone concentration. On the PDA plate containing 50 mg / L patchouli ketone, the inhibition rate of sclerotium germination was 2.50%. When the patchouli ketone concentration increased to 150 mg / L, sclerotium germination was completely inhibited, and the proportion of ungerminated sclerotia reached 100%. These results indicate that patchouli ketone has a strong inhibitory effect on both mycelial growth and sclerotium germination of *Sclerotium affine*.

[0024] Example 3: Effects of patchouli ketone on the ultrastructure of peanut white mold mycelium

[0025] The ultrastructural changes of *Phyllostachys chinensis* hyphae before and after treatment with patchouli were observed using optical microscopy and transmission electron microscopy (TEM). 4 mg / L patchouli (EC) was used. 50Peanut white mold pathogen was treated, with white mold mycelia treated with the corresponding solvent as controls. The mycelia of the control group and the treatment group were placed on glass slides and the phenotypic changes of the mycelia were observed directly under an upright microscope (ZEISS AxioImager. M2). The specific pretreatment method for TEM samples is as follows: The samples were fixed overnight with 2.5% glutaraldehyde at 4℃, rinsed with phosphate buffer, fixed with 1% osmium tetroxide at room temperature for 2 h, and rinsed again; then dehydrated with gradient acetone (50%–90%), and treated twice with 100% acetone; gradient permeation with a mixture of 812R embedding agent and acetone (1:3, 1:1, 3:1), and then soaked in pure 812R embedding agent; after embedding, the samples were polymerized at 35℃, 65℃, and 70℃ to obtain resin embedding blocks; 70–90 nm sections were cut using an ultramicrotome (Leica UC7), stained with uranium acetate and lead citrate, and observed under a transmission electron microscope (Hitachi HT 7800).

[0026] The results of optical microscopy observation show that ( Figure 4 After treatment with patchouli ketone, the hyphae of *Sclerotium affine* exhibited abnormal morphology, characterized by swollen and thickened hyphae, intertwining, and deformed hyphal tips. Transmission electron microscopy revealed (…). Figure 5 In the control group, the mycelial cell walls of *Sclerotium affine* were intact, the plasma membrane was uniform, and there was abundant cytoplasm and complete organelles. The cell wall thickness was visible, the layers were distinct, and the plasma membranes had clear boundaries. After treatment with patchouli ketone, the mycelial cell structure of *Sclerotium affine* was destroyed, mainly manifested in the separation of the cell membrane and cell wall, and significant changes in the cytoplasmic structure, including the appearance of vacuolar structures.

[0027] Example 4: Effects of patchouli ketone on oxalic acid and polygalacturonase in *Sclerotium affine*

[0028] The formulation contains patchouli at a concentration of 4 mg / L (EC). 50 100 mL of PDB medium was used, with an equal volume of PDB solvent added as a control. Five *Sclerotium arachnoides* mycelial cakes were taken using a 5 mm diameter punch and placed in the above PDB medium, and cultured at 30 ℃ and 160 r / min on a shaker for 4 days. The culture solution was centrifuged at 4 ℃ and 8000 r / min for 10 min, and the supernatant was collected. The oxalic acid content and polygalacturonase activity in the supernatant of different treatments were determined according to the Solarbio kit requirements. Polygalacturonase is defined as: at 40 ℃ and pH 6.0, the production of 1 μmol of galacturonic acid per milliliter of sample per hour is defined as one unit of enzyme activity.

[0029] Oxalic acid and polygalacturonase are key factors in the pathogenicity of necrotic fungal diseases. Plants resist pathogens by producing bursts of reactive oxygen species (ROS), which oxalic acid can inhibit. Polygalacturonase is a key cell wall degrading enzyme secreted by plant pathogens, which degrades pectin and disrupts cell wall structure, promoting pathogen infection and disease occurrence. This study found that patchouli significantly reduced oxalic acid content and polygalacturonase activity in *Sclerotium affine* (the causal agent of peanut white mold) (Table 1), thereby reducing the pathogenicity of the fungus.

[0030] Table 1. Effects of patchouli ketone on oxalic acid content and polygalacturonase activity of *Sclerotium affine* in peanuts.

[0031]

[0032] Example 5: Determination of mycelial physiological and biochemical indicators and ATP metabolism-related enzyme activities

[0033] Five peanut white mold cakes were collected using a 5 mm diameter punch and placed in 100 mL of PDB. After incubation at 30 ℃ and 160 r / min for 3 days, patchouli ketone was added to the treatment group to a concentration of 4 mg / L (EC). 50 The control group was treated with an equal volume of solvent. After culturing for another day, fresh mycelia from different treatments were collected by centrifugation and ground into powder using liquid nitrogen. The contents of malondialdehyde, reducing sugar, soluble protein, and pyruvate in the mycelia were determined according to the Solarbio reagent kit method; simultaneously, the ATP content and Na+ content in the mycelia were also determined. + -K + -Activities of ATPase, succinate dehydrogenase (SDH), and NADP-malate dehydrogenase (NADP-MDH).

[0034] Under adverse conditions, cell membrane lipid peroxidation generally occurs, with malondialdehyde (MDA) being one of the products, serving as an important indicator of cell membrane damage. Table 2 shows that treatment with patchouli significantly increased the MDA content in *Sclerotium arachnoides*, indicating that membrane peroxidation occurred in the treated group. Simultaneously, the levels of reducing sugars, soluble proteins, and pyruvate within the mycelium decreased significantly, possibly due to extravasation of these substances or inhibition of their synthesis, thus affecting mycelial growth. ATP is the direct energy source for cellular life activities; impaired synthesis directly impacts fungal growth and proliferation. The tricarboxylic acid (TCA) cycle within mitochondria provides the energy needed for cellular life activities and reduces reducing forces; disruption of the TCA cycle leads to cell death. + -K +-ATPase, SDH, and NADP-MDH are all enzymes that play important roles in the tricarboxylic acid cycle and mitochondrial function. As shown in Table 2, the ATP content and Na+ content of peanut white mold fungus after treatment with 4 mg / L patchouli ketone... + -K + - The activities of ATPase, SDH, and NADP-MDH were significantly reduced, indicating that patchouli ketone can affect the normal function of mitochondria in peanut white mold by reducing the activity of key enzymes in mitochondrial function, thereby inhibiting the activity of the bacteria.

[0035] Table 2. Effects of patchouli ketone on biochemical indicators of peanut white mold pathogen.

[0036]

[0037] In summary, this invention discloses the application of patchouli ketone in inhibiting peanut white mold pathogens through multiple pathways, including at least one of disrupting the ultrastructure of pathogen cells, interfering with energy metabolism, and inhibiting sclerotium germination. Patchouli ketone inhibits the mycelial growth of this pathogen via EC50. 50 The concentration was 3.59 mg / L. At a concentration of 20 mg / L, the inhibition rate of mycelial growth reached 89.52%, and at a concentration of 150 mg / L, sclerotium germination was completely inhibited. After treatment, the mycelium of the pathogen showed swelling, apical deformity, separation of cell wall from cell membrane, and cytoplasmic vacuolation. The ATP content and activities of Na⁺-K⁺-ATPase, succinate dehydrogenase, and NADP-malate dehydrogenase in the mycelium were significantly reduced, while the malondialdehyde content increased. The content of reducing sugar, soluble protein, pyruvate, oxalate, and polygalacturonase activity decreased. This invention reveals for the first time the multi-pathway antibacterial mechanism of patchouli against peanut white mold pathogen, which has the advantages of multi-target and low resistance. Moreover, patchouli is a natural plant-derived compound, which is environmentally friendly and provides a new technical solution for the green control of peanut white mold.

[0038] It should be noted that the above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Any modifications or alterations made by those skilled in the art without departing from the scope of the present invention to create equivalent embodiments based on the disclosed technical content shall fall within the protection scope of the present invention.

Claims

1. The application of patchouli ketone in the preparation of a plant-derived fungicide for controlling peanut white mold through multiple pathways, characterized in that, The peanut white mold pathogen is *Sclerotium rolfsii*, and the patchouli ketone inhibits the peanut white mold pathogen through at least one of the following pathways: disrupting the ultrastructure of the pathogen's hyphal cells, interfering with the pathogen's energy metabolism, and inhibiting sclerotium germination.

2. The application of patchouli ketone according to claim 1 in the preparation of a plant-derived fungicide for controlling peanut white mold through multiple pathways, characterized in that, The damage to the ultrastructure of cells is manifested as swelling and thickening of the fungal hyphae, cross-entanglement, and swollen and deformed tips, as well as separation of the cell wall from the cell membrane and the appearance of vacuolar structures in the cytoplasm.

3. The application of patchouli ketone according to claim 1 in the preparation of a plant-derived fungicide for controlling peanut white mold through multiple pathways, characterized in that, The disruption of energy metabolism is manifested by a significant decrease in ATP content within the mycelium. The activities of enzymes, succinate dehydrogenase, and NADP-malate dehydrogenase were all inhibited.

4. The application of patchouli ketone according to claim 1 in the preparation of a plant-derived fungicide for controlling peanut white mold through multiple pathways, characterized in that, The patchouli ketone can also increase the malondialdehyde content in the mycelium, decrease the content of reducing sugar, soluble protein and pyruvate, and at the same time reduce the oxalic acid content and polygalacturonase activity.

5. The application of patchouli ketone according to claim 1 in the preparation of a plant-derived fungicide for controlling peanut white mold through multiple pathways, characterized in that, The patchouli ketone inhibits the mycelial growth of peanut white mold fungus. 50 The concentration was 3.59 mg / L. At a concentration of 20 mg / L, the inhibition rate of mycelial growth reached 89.52%, and at a concentration of 150 mg / L, the inhibition rate of sclerotium germination reached 100%.

6. A plant-derived fungicide for controlling peanut white mold disease, wherein the active ingredient is patchouli ketone as described in claim 1, and may contain agriculturally acceptable adjuvants, solvents or auxiliaries.

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