Application of physalin F in prevention and treatment of large patch disease of corn

Abstract

The application discloses an application of physalin F in prevention and treatment of corn southern leaf blight, and belongs to the technical field of plant disease prevention and treatment. The application aims to break through the limitation of the prior art, and provides a novel prevention and treatment strategy of a small molecule compound physalin F. The strategy can not only directly inhibit spore germination of pathogenic bacteria, but also focuses on stimulating the immune capacity of crops, so as to provide an efficient, safe and novel mechanism solution for sustainable management of corn diseases.

Description

Technical Field

[0001] This invention relates to the field of plant disease control technology, specifically the application of physalin F in the control of maize leaf spot disease. Background Technology

[0002] Maize leaf spot ( Setosphaeriaturcica ) and corn leaf blight ( Cochliobolusheterostrophus Large and small leaf spot (LFS) is a significant foliar disease that has long hindered high and stable yields in maize. It causes premature leaf death, a sharp reduction in photosynthetic area, and in severe cases, insufficient grain filling, seriously impacting both yield and quality. Therefore, effectively controlling large and small leaf spot in maize is a crucial agricultural issue that urgently needs to be addressed.

[0003] Currently, disease control still heavily relies on chemical pesticides, such as triazoles and methoxyacrylates. While chemical control is fast-acting, long-term, single-use not only leads to the continuous accumulation of pathogen resistance but also causes problems such as pesticide residues, environmental pressure, and increased application costs. Breeding disease-resistant varieties is the fundamental approach, but the breeding cycle is long, and the rapid variation of pathogen physiological races often leads to the "ineffectiveness" of varietal resistance. Therefore, developing new, efficient, low-toxicity, and environmentally compatible green control technologies has become an urgent need for safe corn production. In recent years, the development of biopesticides based on natural products has received considerable attention. Plant-derived active substances, due to their wide availability, rapid degradation, diverse targets, and low likelihood of inducing resistance, show great potential. Physalis bitter extract F (… PhysalinF Steroids are a class of steroidal endoses derived from plants of the genus *Physalis* in the Solanaceae family. Early research focused primarily on their antitumor and anti-inflammatory medicinal activities, such as application number 2018101181445, entitled "Application of Steroidal Compounds in the Preparation of Drugs for the Prevention or Treatment of Gastric Ulcers and Their Complications," and application number 2018103508023, entitled "An Inhibitory Drug for Human Breast Cancer MDA-MB-231 Cells and Its Preparation Method." However, the potential of these compounds in plant protection, particularly in the control of important crop diseases, has not yet been fully explored.

[0004] In summary, the current control technology system for maize leaf spot and varicella-zoster virus (VZV) faces a common dilemma: it is difficult to balance effectiveness, safety, and sustainability. While widely used chemical fungicides offer direct results, their residue and resistance risks are increasingly prominent, contradicting the direction of green agriculture development. Meanwhile, the breeding cycle for disease-resistant varieties is long and prone to obsolescence, often failing to keep pace with the rapid mutation of pathogens in the field. Furthermore, the effectiveness of some biological control methods is not stable in actual field applications and is greatly affected by the environment, limiting their widespread adoption. These factors collectively constrain the level of green control of maize diseases. Summary of the Invention

[0005] To overcome the aforementioned problems, this invention provides an application of physalin in the control of maize leaf spot and varicella-zoster virus (VZV). By spraying small-molecule physalin F onto maize plants, it can effectively inhibit the germination of pathogenic spores, reduce their pathogenicity, and induce the upregulation of maize disease resistance-related genes, thereby achieving green control of maize VZV. This invention is the first to systematically reveal and verify the unique role and application value of physalin F in the control of maize VZV.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] The application of a bitter physalin F in the preparation of an application solution for the prevention and control of maize leaf spot or maize small leaf spot.

[0008] Furthermore, the concentration of physalin F in the applied solution ranged from 5.2653 µg / mL to 52.653 µg / mL.

[0009] Furthermore, the concentration of physalin F in the applied solution was 5.2653 µg / mL.

[0010] Furthermore, physalin F can inhibit the germination of pathogenic spores and reduce the pathogenicity of pathogens.

[0011] Furthermore, the pathogens are either corn leaf blight pathogens or corn spot pathogens.

[0012] Furthermore, physalin F can induce systemic resistance in maize plants against maize large leaf spot or small leaf spot.

[0013] Furthermore, systemic resistance is associated with the upregulation of one or more defense-related genes selected from ZmPR1, ZmPR5, ZmAOS, and ZmERF1α in maize plants.

[0014] A method for controlling maize large leaf spot or small leaf spot involves applying a solution of physalin F at a concentration of 5.2653 µg / mL to 52.653 µg / mL to the leaves of maize plants.

[0015] Furthermore, the preparation of the physalin F solution involves weighing physalin F, dissolving it in dimethyl sulfoxide to prepare a 10 mM / mL stock solution, then diluting and dissolving it with distilled water to achieve a physalin F concentration of 5.2653 µg / mL.

[0016] The beneficial effects of this invention are:

[0017] The physalin F provided by this invention exhibits significant advantages in the green control of maize leaf spot and gray leaf spot diseases. Experimental results show that this compound directly inhibits the spore germination of maize large leaf spot fungus and small leaf spot fungus, effectively blocking the initial infection of the pathogens. More importantly, physalin F can activate the systemic resistance of maize plants, significantly upregulating the expression levels of key disease resistance-related genes in leaves, thereby establishing a durable immune defense. As a plant-derived natural small molecule, it has good environmental compatibility and is easily degraded in nature, avoiding the potential risks of chemical pesticide residues, which meets the requirements of green agriculture development. In practical applications, physalin F is stable, easy to formulate into suitable field application formulations, and requires a low effective concentration, demonstrating good cost-effectiveness and application prospects. Attached Figure Description

[0018] Figure 1 The chemical structural formula of the physalin F of this invention is shown below;

[0019] Figure 2 Schematic diagram of the application mode of the present invention;

[0020] Figure 3 Schematic diagram I showing the inhibition of spore germination by physalin F, dimethyl sulfoxide, and the control group against maize leaf spot pathogens;

[0021] Figure 4 Schematic diagram II showing the inhibition of spore germination by physalin F, dimethyl sulfoxide, and the control group in maize leaf spot pathogens;

[0022] Figure 5 Schematic diagram I showing the inhibition of maize large leaf spot fungus spore germination by physalin F, dimethyl sulfoxide, and the control group;

[0023] Figure 6 Schematic diagram II showing the inhibition of maize large leaf spot fungus spore germination by physalin F, dimethyl sulfoxide, and the control group;

[0024] Figure 7 This is a schematic diagram illustrating the induction of maize resistance gene ZmERF1a expression by physalin F.

[0025] Figure 8 This is a schematic diagram illustrating the induction of maize resistance gene ZmPR1 expression by physalin F.

[0026] Figure 9 This is a schematic diagram illustrating the induction of maize resistance gene ZmAOS expression by physalin F.

[0027] Figure 10 This is a schematic diagram illustrating the induction of maize resistance gene ZmPR5 expression by physalin F.

[0028] Figure 11Schematic diagram I illustrating how physalin F reduces the pathogenicity of corn leaf blight pathogen;

[0029] Figure 12 Schematic diagram II showing how physalin F reduces the pathogenicity of corn leaf blight pathogen. Detailed Implementation

[0030] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0031] Evaluation of the activity of physalin F (inhibition of spore germination) → Evaluation of the induced disease resistance of maize plants, which helps in the green control of maize leaf spot and small leaf spot diseases.

[0032] Physalis bittering F ( PhysalinF The extract is an ethanolic extract of the plant *Solanum tuberosum* (Solanaceae family). The physalin F used in this invention was purchased from Proteintech, model number CM15245, weight: 1 mg, purity: 99.07%.

[0033] Functional definition: Physalis bitter extract F has an effect on maize leaf spot pathogen (… Setosphaeriaturcica ) and corn leaf blight fungus ( Cochliobolusheterostrophus It has a significant inhibitory effect on spore germination. Among them, physalin F induces disease resistance in maize plants by activating the plant defense system by inhibiting the germination, infectivity and activity of maize microspores.

[0034] See the chemical structural formula of physalin F. Figure 1 CAS No.: 57423-71-9.

[0035] Antibacterial activity testing procedure:

[0036] Physalis extract F was dissolved in DMSO to prepare a stock solution with a concentration of 10 mM / mL. The stock solution was then quantitatively diluted with sterile distilled water to prepare a working solution with a final concentration of 5.2653 µg / mL of physalis extract F. The prepared working solution was gently mixed in a vortex mixer to ensure complete dissolution, yielding the final working solution. The final working solution was then sprayed onto maize leaves to determine the maize's disease resistance. Alternatively, the final working solution was pre-sprayed onto maize leaves, followed by inoculation with maize large and small leaf spot fungi to evaluate the application of this agent in controlling maize large and small leaf spot diseases.

[0037] The direct inhibitory effect of physalis extract F on *Heterophyllum heliotropium* and *Heterophyllum oxypetalum* was evaluated using the in vitro spore germination method. In an in vitro leaf inoculation experiment, 10 µL of a *Heterophyllum oxypetalum* spore suspension (1×10⁻⁶) was inoculated onto detached maize leaves. 5 (Spores / mL), 24 hours after inoculation, the sample was inoculated with the final working solution prepared by physalin F, with a mixture of water and DMSO as a negative control. The lesion area was counted 3 days later. It was found that physalin F had a controlling effect on the spread of maize leaf spot, verifying the weakening effect of physalin F on the pathogenicity of the pathogen.

[0038] Physalicylic acid bitter extract F inhibits the germination of spores of maize large and small leaf spot pathogens.

[0039] To assess the effect of physalin F on conidial germination, conidia of wild-type maize leaf spot pathogens were collected from 9-day-old potato dextrose agar (PDA) and 7-day-old corn meal agar (CMA) media using sterile water (containing 0.05% Tween-20). Mycelial fragments were removed with gauze, and the conidial suspension was adjusted to 1×10⁻⁶ using a hemocytometer. 4 spores / mL. A mixture of physalin F stock solution (10 mM / mL) and *S. cornensis* conidia suspension was prepared to achieve a final concentration of physalin F of 5.2653 µg / mL. 20 μL of this mixture (final concentration 5.2653 µg / mL) was dropped onto a glass slide, and the germination rate was recorded after 2–6 hours. The control group was treated with distilled water and DMSO. In the spore germination inhibition experiment, the effective concentration of physalin F in the spore suspension ranged from 5.2653 µg / mL to 52.653 µg / mL. The same method was used to verify *S. cornensis*. Spore germination of both large and small leaf spot diseases in corn was inhibited. See [link to relevant documentation]. Figures 3-6 .

[0040] Physalis extract F, at a concentration of 5.2653 µg / mL, inhibited the germination of spores of *Heterophyllum heliotropium* and *Heterophyllum spp.* by 67.78% and 77.5%, respectively. At a concentration of 52.653 µg / mL, the inhibition rate against *Heterophyllum spp.* spore germination was 89.55%. Physalis extract F significantly inhibited the germination of conidia of *Heterophyllum heliotropium* and *Heterophyllum spp.*, thus demonstrating its potential for application in the control of these diseases. Treatment with the final working solution significantly reduced the spore germination rate of *Heterophyllum heliotropium* and *Heterophyllum spp.* spores. The germination rate increased slightly with time from 2 to 6 hours, but remained essentially unchanged after 6 hours. This indicates that physalis extract F inhibits the germination of spores of both *Heterophyllum heliotropium* and *Heterophyllum spp.*

[0041] Calculation formula: Inhibition rate (%) = (Germination rate of control group - Germination rate of treatment group) / Germination rate of control group × 100%

[0042] Application of physalin F in enhancing disease resistance in maize plants:

[0043] Accurately weigh pure physalin F powder, first dissolve and bring to volume with dimethyl sulfoxide (DMSO) to prepare a high-concentration stock solution, then perform serial dilution with sterile distilled water until completely dissolved to obtain the final working solution (5.2653 µg / mL). All solutions were prepared fresh before use. Spray the final working solution onto corn plants until the leaves are completely wet. Use a mixture of water and DMSO as a negative control for pretreatment of corn plants. For the pesticide application treatment: use the final working solution, water, and DMSO mixture to evenly spray the leaves of corn plants until the leaves are completely wet. Induction period culture: Transfer the sprayed plants to the same environmental conditions and incubate statically for 12 hours. After 12 hours, spray the leaves with a spore suspension of *Microsporum tobira* (1×10⁻⁶). 5 (spores / mL).

[0044] Maize leaves were collected after spraying with water and DMSO for 12 hours, spraying with the final working solution for 12 hours, spraying with water and DMSO + *Microsporum simulans* suspension for 12 hours, spraying with the final working solution + *Microsporum simulans* suspension for 12 hours, spraying with water and DMSO for 24 hours, spraying with the final working solution for 24 hours, spraying with water and DMSO + *Microsporum simulans* suspension for 24 hours, and spraying with the final working solution + *Microsporum simulans* suspension for 24 hours. RNA was extracted and reverse transcribed into cDNA. The expression levels of disease resistance-related genes in maize were detected using real-time quantitative PCR. The expression of disease resistance-related genes in maize is a well-known technique in this field. ZmPR1 , ZmPR5 , ZmAOS and ZmERF1α Gene expression was increased after pretreatment with physalin F, indicating that physalin F has the potential to enhance the disease resistance of maize plants. Figures 7-10 .

[0045] Application of physalin F in the control of maize leaf spot disease:

[0046] Accurately weigh pure physalin F powder, first dissolve and dilute it in dimethyl sulfoxide (DMSO) to prepare a high-concentration stock solution, then perform serial dilution with sterile distilled water until completely dissolved to obtain the final working solution (physalin F concentration of 5.2653 µg / mL). Apply the solution during the jointing stage of corn plants using a foliar spray method with a handheld small sprayer, ensuring the solution evenly covers both sides of the corn leaves until a uniform liquid film forms on the leaf surface without any droplets rolling off.

[0047] As a preventative treatment, a single foliar spray was applied 24 hours before pathogen inoculation. For corn plants, a pretreatment was performed, with water and DMSO used as negative controls. 24 hours later, leaves were inoculated with 10 µL of a spore suspension of *Sclerotium cerevisiae* (1×10⁻⁶). 5 (Spores / mL). The area of ​​lesions was counted 3 days after inoculation. After pretreatment with the final working solution, the area of ​​lesions decreased by 71.9%. It was found that pretreatment with the final working solution, i.e., physalin F, has a preventive effect on the occurrence of maize leaf spot disease.

[0048] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification.

Claims

1. The application of a bitter physalin F in the preparation of an application solution for the prevention and control of maize leaf spot or maize small leaf spot.

2. The application according to claim 1, characterized in that, The concentration of physalin F in the applied solution ranged from 5.2653 µg / mL to 52.653 µg / mL.

3. The application according to claim 2, characterized in that, The concentration of physalin F in the applied solution was 5.2653 µg / mL.

4. The application according to claim 1, characterized in that, Physalis extract F can inhibit the germination of pathogenic spores and reduce the pathogenicity of pathogens.

5. The application according to claim 4, characterized in that, The pathogens are either large leaf spot fungus or small leaf spot fungus of corn.

6. The application according to claim 1, characterized in that, Physalis extract F can induce systemic resistance in maize plants against maize leaf spot or maize scab.

7. The application according to claim 6, characterized in that, Systemic resistance is associated with the upregulation of one or more defense-related genes selected from ZmPR1, ZmPR5, ZmAOS and ZmERF1α in maize plants.

8. A method for controlling maize leaf spot or maize short leaf spot, characterized in that, A solution of physalin F with a concentration ranging from 5.2653 µg / mL to 52.653 µg / mL was applied to the leaves of maize plants.

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