Application of methyl ferulate in prevention and treatment of apple anthrax leaf blight

Abstract

The invention discloses an application of methyl ferulate in prevention and treatment of apple anthrax leaf blight, which is characterized in that the methyl ferulate is prepared into a solution with the concentration of 0.05 microgram / mL-0. 5 microgram / mL, and the solution is uniformly sprayed on the surfaces of apple leaves or fruits. The methyl ferulate can simultaneously induce the activity of key disease-resistant enzymes in apple bodies to be remarkably improved, promote accumulation of secondary metabolites, namely flavonoid substances, and regulate and control synthesis and signal channels of various defense-related hormones, so that a multi-layer and systematic plant immune defense network is constructed. Expression of multiple disease course related protein genes of apples is remarkably up-regulated, a salicylic acid signal channel is activated, a system obtains resistance, and the effectiveness of the compound as a plant immune resistance inducer is proved from the molecular level. The methyl ferulate provided by the invention is derived from a natural metabolite of a plant, is low in use concentration and small in residual risk, realizes disease prevention and control by inducing the resistance of the plant, does not easily cause pathogenic bacteria to generate drug resistance, meets the requirements of green agriculture and sustainable development, and has important ecological and economic values.

Description

Technical Field

[0001] This invention relates to the fields of agricultural biotechnology and plant protection technology, and in particular to the application of methyl ferulic acid in the control of apple anthracnose leaf blight. Background Technology

[0002] As an important economic crop in my country, apple production has long been severely threatened by diseases such as anthracnose leaf blight (Glomerella cingulata). Anthracnose leaf blight mainly infects leaves and fruits, leading to premature leaf drop and sunken fruit spots, and has shown an explosive epidemic trend in recent years. Currently, the control of anthracnose leaf blight still relies mainly on chemical fungicides, such as benzimidazole and triazole fungicides used to control ring rot, and mancozeb and pyraclostrobin used to control anthracnose leaf blight. However, the long-term use of chemical agents alone has led to significant drug resistance in pathogens, and the control effect has declined year by year. At the same time, pesticide residues and environmental pollution problems are becoming increasingly prominent, seriously restricting the sustainable development of the apple industry.

[0003] Plant resistance inducers have become an important research direction for replacing chemical pesticides due to their environmental friendliness and low likelihood of inducing resistance. These substances activate the plant's own immune system, inducing systemic resistance to resist pathogen infection. Currently, chitosan, oligosaccharides, and other inducers have been applied to some crops, but they still suffer from unstable efficacy and short-lasting effects in apple disease control. Methyl ferulic acid, a naturally occurring phenylpropanoid metabolite in plants, has significant antibacterial activity and the potential to induce disease resistance. Studies have shown that methyl ferulic acid can activate the expression of plant defense-related genes and enhance the activity of enzymes related to apple disease resistance.

[0004] Based on the current challenges in apple disease control and the current state of research on plant inducers, this invention is the first to systematically study the inducing effect of methyl ferulic acid on apple anthracnose leaf blight. Through in-depth analysis of its molecular mechanism for activating plant defense responses, a novel, highly efficient, and safe plant inducer has been developed, providing an innovative solution to the challenges of disease control in apple production. This technology not only effectively reduces the use of chemical pesticides and alleviates environmental pressure, but also improves the quality and safety of apple fruits, meeting the requirements of modern green agricultural development and possessing significant value for widespread application. Summary of the Invention

[0005] The present invention aims to solve the above problems and provides the application of methyl ferulic acid in the prevention and control of apple anthracnose leaf blight. Methyl ferulic acid is prepared into a solution with a concentration of 0.05 μg / mL to 0.5 μg / mL and sprayed evenly on the surface of apple leaves or fruits.

[0006] More preferably, the concentration of the methyl ferulic acid solution is 0.1 μg / mL.

[0007] Furthermore, the apple varieties mentioned include 'Gala' and 'Bright Moon'.

[0008] Furthermore, the methyl ferulic acid ester enhances the disease resistance and antioxidant level of apples by promoting the synthesis or increasing the activity of flavonoids, peroxidase, polyphenol oxidase, and glutathione S-transferase in apples.

[0009] Furthermore, the methyl ferulic acid ester promotes the synthesis of IAA, ACC, SA, SAG, and OPDA in apples and inhibits the accumulation of JA.

[0010] Furthermore, the methyl ferulic acid significantly induced apple MdPR1 , MdPR2 , MdPR4 , MdPR5 , MdPR8 and MdPR10 Gene expression.

[0011] Based on the same invention, this invention also provides a plant immune inducer, wherein the active ingredient of the plant immune inducer is methyl ferulic acid, and the effective concentration of methyl ferulic acid is 0.05 μg / mL to 0.5 μg / mL.

[0012] Furthermore, the effective concentration of the methyl ferulic acid is 0.1 μg / mL.

[0013] Furthermore, the effective concentration of the methyl ferulic acid is 0.5 μg / mL.

[0014] This invention also provides the application of the plant immune inducer described above in the control of apple anthracnose leaf blight.

[0015] The present invention has the following beneficial effects: 1. This invention clarifies for the first time that methyl ferulic acid has significant preventive effects against apple anthracnose leaf blight and fruit anthracnose within a specific concentration range (0.05 μg / mL to 0.5 μg / mL). Among them, 0.1 μg / mL is the optimal cost-effectiveness concentration, which can significantly reduce the number of leaf lesions and inhibit the expansion of fruit lesions, showing good protective effects.

[0016] 2. Methyl ferulic acid can simultaneously induce a significant increase in the activity of key disease-resistant enzymes (POD, PPO, GST) in apples, promote the accumulation of secondary metabolites such as flavonoids, and regulate the synthesis and signaling pathways of various defense-related hormones (IAA, ACC, SA, SAG, OPDA), thereby constructing a multi-level, systematic plant immune defense network.

[0017] 3. Methyl ferulic acid can significantly upregulate multiple disease-related protein genes in apples (including...) MdPR1 , MdPR2 , MdPR4 , MdPR5 , MdPR8 and MdPR10 The expression of salicylic acid activates the salicylic acid signaling pathway and systemic acquisition of resistance, confirming its effectiveness as a plant immune inducer at the molecular level.

[0018] 4. The methyl ferulic acid provided by this invention is derived from a natural plant metabolite. It is used at low concentrations with minimal residue risk. It controls diseases by inducing plant resistance, and is unlikely to cause pathogens to develop resistance. This aligns with the requirements of green agriculture and sustainable development, and thus possesses significant ecological and economic value. Attached Figure Description To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only one embodiment of the present invention. For those skilled in the art, other embodiments can be derived from the provided drawings without creative effort.

[0019] Figure 1 Example 1: The protective effect of methyl ferulic acid against apple anthracnose leaf blight pathogens on the leaf surface; Figure 2 Example 2: The protective effect of methyl ferulic acid against apple anthracnose leaf blight pathogens on apple fruit; Figure 3 Example 3: Determination of total flavonoid content after treatment of apple leaves with methyl ferulic acid; Figure 4 Example 4: Determination of the content of various disease resistance-related enzymes after apple leaves were treated with methyl ferulic acid; Figure 5 Example 5: Determination of hormone content in apple leaves after treatment with methyl ferulic acid; Figure 6 Example 6: Expression levels of disease resistance-related genes in apple leaves after treatment with methyl ferulic acid. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. The following embodiments are only for illustrative purposes and are not intended to limit the scope of the present invention in any way. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the present invention are conventional methods. Unless otherwise specified, the materials and reagents used in the present invention are commercially available. Furthermore, other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those skilled in the art.

[0021] Example 1: The efficacy of methyl ferulic acid against apple anthracnose leaf blight Fresh Gala apple leaves were collected, thoroughly rinsed with sterile water, and then air-dried. Using an in vitro leaf culture method, the petioles were wrapped in moistened absorbent cotton and placed in a sterile, humidified culture box. The experimental group leaves were sprayed evenly with methyl ferulic acid solutions at concentrations of 0.05 μg / mL, 0.1 μg / mL, 0.25 μg / mL, and 0.5 μg / mL, respectively, while the control group was sprayed with an equal volume of sterile water as a blank control. All treatments were cultured under the same humidified conditions for 48 h. The strain of *Anthracnose leaf blight* was cultured on liquid Czapek's medium for 3 days. After filtration through two layers of lens paper to obtain a spore suspension, the spores were collected by centrifugation at 5000 rpm for 10 minutes, rinsed with sterile water, and the spore concentration was adjusted to 1×10⁻⁶. 6 The concentration of lesions was increased to 0.1% Tween 20, and the solution was sprayed evenly onto the detached fresh leaves. The leaves were kept moist, and the number of lesions was counted and photographed after 4-6 days. The methyl ferulic acid used was a 95% purity technical grade, purchased from Maclean's Reagents.

[0022] Experimental results are as follows Figure 1 As shown, the number of lesions on apple leaves treated with different concentrations of methyl ferulic acid decreased significantly with increasing concentration. The water control group (CK) had the most lesions, exceeding 150; the 0.1 μg / mL treatment group had fewer than 50 lesions, while the 0.5 μg / mL treatment group had very few lesions, almost none. This indicates that methyl ferulic acid has a significant inhibitory effect on *Hydrocotyle erythrorhizon*, the causal agent of apple anthracnose leaf blight, and the higher the concentration, the better the control effect, effectively reducing the formation of leaf lesions caused by pathogen infection.

[0023] Therefore, considering both the prevention and control effect and the economic cost, although the 0.1 μg / mL concentration has moderate efficacy, it has a high cost-effectiveness. Therefore, this concentration was used for further research in subsequent experiments.

[0024] Example 2: The efficacy of methyl ferulic acid against anthracnose in apple fruit Young fruits of the "Mingyue" variety were collected, thoroughly rinsed with sterile water, and then air-dried. For the experimental group, fruits were treated by puncturing them with a 200 μL pipette tip, followed by the addition of 50 μL of 0.1 μg / mL methyl ferulic acid. After air-drying, the fruits were placed in a sterile, humidified culture box for 48 h. Fungal cakes were collected from the edge of fresh wild-type *Anthracnose leaf blight* colonies using a 5 mm diameter punch and inoculated onto the puncture sites. The sites were then kept moist. The diameter of the lesions was measured and photographed after 5-7 days.

[0025] Experimental results are as follows Figure 2 As shown, fruit treated with 0.1 μg / mL methyl ferulic acid solution exhibited significantly inhibited lesion expansion, with lesion diameters significantly smaller than the control group (treated with sterile water). This result indicates that methyl ferulic acid possesses significant antifungal activity against *A. anthracnose*, the causal agent of apple leaf blight, on apple fruit, effectively delaying or inhibiting pathogen infection and spread. The images further visually demonstrate the differences in lesion size and morphology between the treated and control groups, confirming the potential application of methyl ferulic acid as a potential disease resistance inducer or antifungal agent in postharvest disease control.

[0026] Example 3: Changes in total flavonoid content in apples after treatment with methyl ferulic acid Following the procedure described in Example 1, fresh 'Gala' apple leaves were treated with 0.1 μg / mL methyl ferulic acid solution for 48 h before sampling. The flavonoid content in the apple leaves was detected using Solarbio (Cat: BC1335).

[0027] Experimental data such as Figure 3 As shown, the flavonoid content in apple leaves treated with methyl ferulic acid was significantly higher than that in the control group (sterile water treatment). Flavonoids, as important secondary metabolites in plants, play a crucial role in plant stress response. Their accumulation not only strengthens cell wall structure and inhibits pathogen infection, but also reduces oxidative damage by scavenging reactive oxygen species and regulating antioxidant enzyme activity, thereby systematically improving the disease resistance and antioxidant levels of apple plants. This result suggests that methyl ferulic acid may indirectly enhance the physiological resistance of apple leaves to diseases such as anthracnose by activating the biosynthetic pathway of flavonoids.

[0028] Example 4: Methyl ferulic acid induced the activity of apple disease resistance-related enzymes Fresh apple leaves were collected, thoroughly rinsed with sterile water, and then air-dried. An in vitro leaf culture method was used, with the petiole wrapped in moistened absorbent cotton and placed in a sterile, humidified culture box. The experimental group leaves were sprayed evenly with a 0.1 μg / mL methyl ferulic acid solution on both sides, while the control group was sprayed with an equal volume of sterile water as a blank control. All treatments were cultured under the same humidified conditions for 48 h before sampling. The peroxidase content in apple leaves was detected using Solarbio (Cat: BC0090), the polyphenol oxidase content using Solarbio (Cat: BC0190), and the glutathione S-transferase content using Solarbio (Cat: BC0350).

[0029] Experimental data such as Figure 4 As shown, after treatment with methyl ferulic acid, the activities of three key defense enzymes in apple leaves—peroxidase (POD), polyphenol oxidase (PPO), and glutathione S-transferase (GST)—were significantly increased compared with the control group (sterile water treatment), indicating that methyl ferulic acid can systematically activate the physiological defense network of apple leaves, thereby enhancing their overall disease resistance.

[0030] Specifically, increased peroxidase (POD) activity directly promotes the scavenging of reactive oxygen species (ROS), alleviating oxidative stress caused by pathogen infection. Simultaneously, POD participates in the polymerization of lignin and polyphenols in the cell wall, enhancing its structural strength and forming an effective physical barrier to hinder pathogen spread. Increased polyphenol oxidase (PPO) activity accelerates the conversion of phenolic substances into quinones. These quinones not only possess direct antibacterial activity but also form a browning isolation zone around the infection site, limiting disease spread and participating in wound healing. Increased glutathione S-transferase (GST) activity, on the one hand, enhances the plant's detoxification and antioxidant capacity by catalyzing the binding reaction of glutathione with harmful substances such as lipid peroxides and exogenous toxins. On the other hand, GST also participates in the metabolic regulation of defense signaling molecules such as jasmonic acid and salicylic acid, potentially playing an important role in the systemic acquired resistance (SAR) signaling network.

[0031] In summary, methyl ferulic acid may significantly enhance the overall resistance of apples to anthracnose and other pathogens by synergistically inducing three complementary defense enzymes: POD, PPO, and GST.

[0032] Example 5: Changes in hormone levels in apples after treatment with methyl ferulic acid Using the procedure described in Example 1, fresh 'Gala' apple leaves were treated with 0.1 μg / mL methyl ferulic acid solution for 48 h before being sampled. The samples were then immediately flash-frozen in liquid nitrogen and sent to Suzhou Panomike Biotechnology Co., Ltd. for analysis of their hormone levels.

[0033] The results are as follows Figure 5 The results showed significant differences in the levels of several key hormones and related substances between the treatment group and the control group, and these changes were closely related to the functions of each hormone: Indoleacetic acid (IAA), as an auxin-like hormone, mainly regulates plant growth and development, such as cell elongation and tissue differentiation. Its content was significantly higher than that of the control group, indicating that methyl ferulic acid can promote the accumulation of IAA and thus affect the growth process of apple plants. 1-Aminocyclopropane-1-carboxylic acid is an ethylene precursor (ACC), and ethylene plays a key role in plant senescence and stress response. Its content was much higher than that of the control group, which shows that methyl ferulic acid can strongly promote ACC synthesis, and thus may enhance the ethylene signaling pathway, helping apples to cope with stress such as pathogens. Salicylic acid (SA) is an important disease resistance signaling molecule that can activate the systemic acquired resistance of plants. Salicylic acid glycoside (SAG) is the storage form of salicylic acid. The contents of both SA and SAG were significantly higher than those of the control group, indicating that methyl ferulic acid can not only promote SAG synthesis, but also promote SA synthesis. This change may activate salicylic acid-related defense pathways and enhance plant disease resistance or stress response. 12-O-phytodienoic acid, a precursor of jasmonic acid (OPDA), plays a role in the upstream stage of jasmonic acid biosynthesis. Its content was significantly higher than that of the control group, suggesting that the agent can promote OPDA synthesis. Its effect may be in the upstream link of the jasmonic acid pathway, thereby indirectly affecting the physiological processes mediated by jasmonic acid.

[0034] Methyl ferulate treatment simultaneously induced the accumulation of IAA, ACC / ethylene pathways, SA, and OPDA / JA-related substances, exhibiting synergistic regulation of growth, stress, and defense signaling. Auxin may provide metabolic resources for defense; activation of the SA and ethylene / JA pathways respectively enhanced systemic resistance to both live and necrotrophic pathogens; and the increase in OPDA further enriched the layers of jasmonic acid-related signaling. This integrated response of a multi-hormonal network is likely an important physiological mechanism by which methyl ferulate enhances the overall resistance of apples to diseases such as anthracnose.

[0035] Example 6: Methyl ferulic acid treatment induces upregulation of apple disease resistance-related genes. Following the procedure described in Example 1, fresh 'Gala' apple leaves were treated with 0.1 μg / mL methyl ferulic acid solution for 48 h before sampling. The samples were immediately flash-frozen in liquid nitrogen and stored at -80°C for subsequent RNA extraction. Total RNA was extracted from the apple leaves using a plant total RNA extraction kit manufactured by Acrel Biotech Co., Ltd. The integrity and purity of the RNA were assessed by agarose gel electrophoresis and a nucleic acid concentration analyzer. One μg of high-quality total RNA was used to synthesize first-strand cDNA according to the Novizan HiScript® III RT SuperMix reverse transcription kit.

[0036] Using the above cDNA as a template, the QuantStudio® series real-time quantitative PCR system was used to perform the assay on apples. MdEFa1 Using genes as internal controls, the effects of methyl ferulic acid treatment on the expression levels of a series of disease resistance-related pathological genes (PR) family members in apple leaves were systematically analyzed using the SYBR Green qPCR method, including... MdPR1 , MdPR2 , MdPR4 , MdPR5 , MdPR8 and MdPR10 Three technical replicates were set up for each reaction to ensure data reliability.

[0037] Use 2 –ΔΔCT The method involves quantitative analysis of relative gene expression levels using qRT-PCR results. The results are as follows: Figure 6 As shown, compared with the sterile water control group, treatment with 0.1 μg / mL methyl ferulic acid solution significantly induced all assays. PR Gene expression is upregulated. Among them, MdPR1 , MdPR2 As a marker gene for the salicylic acid signaling pathway, its strong activation indicates that systemic acquired resistance (SAR) is effectively triggered; MdPR4 , MdPR8 It belongs to the chitinase class, and its upregulation indicates enhanced cell wall degradation resistance mechanisms; MdPR5 (sweetenedin) and MdPR10 Increased expression of (ribonuclease analogs) further enhances the plant's molecular defense against pathogen infection. This suggests that methyl ferulic acid can be used as a novel plant immune inducer.

[0038] It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solutions and concepts of this invention, and all such substitutions or modifications should fall within the protection scope of the appended claims.

Claims

1. The application of methyl ferulic acid in the control of apple anthracnose leaf blight, characterized in that, Methyl ferulic acid was prepared into a solution with a concentration of 0.05 μg / mL to 0.5 μg / mL and sprayed evenly on the surface of apple leaves or fruits.

2. The application of methyl ferulic acid according to claim 1 in the control of apple anthracnose leaf blight, characterized in that, The concentration of the methyl ferulic acid solution is 0.1 μg / mL.

3. The application of methyl ferulic acid according to claim 1 in the control of apple anthracnose leaf blight, characterized in that, The apple varieties mentioned include 'Gala', 'Bright Moon', and 'Golden Delicious'.

4. The application of methyl ferulic acid according to claim 2 in the control of apple anthracnose leaf blight, characterized in that, The methyl ferulic acid ester enhances the disease resistance and antioxidant level of apples by promoting the synthesis or activity of flavonoids, peroxidase, polyphenol oxidase, and glutathione S-transferase in the apple body.

5. The application of methyl ferulic acid according to claim 2 in the control of apple anthracnose leaf blight, characterized in that, The methyl ferulic acid ester promotes the synthesis of IAA, ACC, SA, SAG, and OPDA in apples and inhibits the accumulation of JA.

6. The application of methyl ferulic acid according to claim 2 in the control of apple anthracnose leaf blight, characterized in that, The methyl ferulic acid significantly induced apple MdPR1 , MdPR2 , MdPR4 , MdPR5 , MdPR8 and MdPR10 Gene expression.

7. A plant immune inducer, characterized in that, The active ingredient of the plant immune inducer is methyl ferulic acid, and the effective concentration of methyl ferulic acid is 0.05 μg / mL to 0.5 μg / mL.

8. A plant immune inducer according to claim 7, characterized in that, The effective concentration of the methyl ferulic acid is 0.1 μg / mL.

9. A plant immune inducer according to claim 7, characterized in that, The effective concentration of the methyl ferulic acid is 0.5 μg / mL.

10. The application of a plant immune inducer as described in any one of claims 7-9 in the control of apple anthracnose leaf blight.

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