Application of pseudopenicillin variotii in preparation of resistance inducer for preventing and treating plant diseases

Abstract

The invention discloses application of pseudopenicillin variotii in preparation of a resistance inducer for preventing and treating plant diseases, and belongs to the technical field of plant protection. The invention provides an application of Panax variotii in prevention and treatment of plant diseases, the application concentration of the Panax variotii is 0.1 microgram / mL-1 microgram / mL, a plant resistance inducer containing the Panax variotii can be sprayed on plant leaves or fruit surfaces and has a remarkable prevention effect on anthrax leaf blight, and the higher the concentration is, the better the prevention effect is; the pseudopenicillin variotii can promote synthesis or activity improvement of catalase, peroxidase, polyphenol oxidase, glutathione S-transferase and superoxide dismutase in apple bodies, and also can remarkably induce up-regulation expression of disease-resistant related genes such as MdPR2 and MdPR5, so that the disease resistance, the antioxidant level and the plant immunity of apples are enhanced; a novel and efficient plant resistance inducer application scheme is provided for apple disease control and physiological regulation.

Description

Technical Field

[0001] This invention belongs to the field of plant protection technology, specifically relating to the application of Wan's penicillin in the preparation of inducers for the prevention and control of plant diseases. Background Technology

[0002] As an important economic crop in my country, the development of apple industry has long been affected by anthracnose leaf blight (… Glomerella cingulata Anthracnose poses a serious threat to plants and fruits. It mainly infects leaves and fruits, causing premature leaf drop and sunken spots on fruits, and has shown an explosive epidemic trend in recent years.

[0003] Currently, the control of these two diseases still relies mainly on chemical fungicides. For example, benzimidazole and triazole fungicides are used to control ring rot, while mancozeb and pyraclostrobin are used to control anthracnose leaf blight. However, long-term use of chemical agents alone has led to significant drug resistance in pathogens, resulting in a gradual decline in control efficacy. At the same time, pesticide residues and environmental pollution problems are becoming increasingly prominent, seriously hindering the sustainable development of the apple industry. In addition, fungicides such as chitosan and oligosaccharides are used on some crops, but they still suffer from unstable efficacy and short-lasting effects in apple disease control.

[0004] Given the current challenges in apple disease control and the current state of research on plant inhibitors, it is urgent to develop a new type of formulation that is green, environmentally friendly, highly effective in controlling apple diseases, and effectively improves the antioxidant capacity of apples. Summary of the Invention

[0005] Based on the above shortcomings, the purpose of this invention is to provide the application of *Vanillarin* in the preparation of inducers for controlling plant diseases. The *Vanillarin* provided by this invention has the molecular formula C0.05. 12 H 22 N2O2, with a relative molecular weight of 226.32, the described *Penicillium wani* can inhibit *Amanita muscaria*, enhance the synthesis and activity of important enzymes in plants, and induce the upregulation of disease resistance genes, effectively improving the antioxidant capacity and disease resistance of apples. This provides a novel and efficient plant inducer pathway and method for plant pest control, including apple disease control and physiological regulation.

[0006] To achieve the above objectives, the present invention is implemented through the following solution: This invention provides the application of Wan's penicillin in the preparation of an inducer for the prevention and control of plant diseases.

[0007] Furthermore, the chemical name of the aforementioned penicillin is 3,6-bis(1-methylpropyl)-2,5-piperazinedione, and its chemical formula is C. 12 H 22 N2O2.

[0008] Furthermore, the plant diseases mentioned include anthracnose leaf blight.

[0009] Furthermore, the inducer contains a concentration of 0.1 μg / mL to 1 μg / mL of *Vincilia wansii*.

[0010] Furthermore, the described penicillin can induce the upregulation of plant disease resistance genes.

[0011] Furthermore, the plant disease resistance gene includes MdPR2 and / or MdPR5 .

[0012] Furthermore, the aforementioned *Penicillium wansii* can increase the content of at least one of catalase, peroxidase, polyphenol oxidase, glutathione S-transferase, and superoxide dismutase in plants.

[0013] Furthermore, the method for preventing and controlling plant diseases includes spraying an inducer onto the surface of plant leaves or fruits.

[0014] Furthermore, the inducing agent is applied over a period of 2 to 6 days.

[0015] Furthermore, the plant includes an apple tree.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention is the first systematic study of the inducing resistance effect of *Pseudomonas wani* on anthracnose leaf blight. *Pseudomonas wani*, chemically named 3,6-bis(1-methylpropyl)-2,5-piperazindione, is the same substance (compound). The common name of 3,6-bis(1-methylpropyl)-2,5-piperazindione is *Pseudomonas wani*, as published in the National Pesticide Standardization Technical Committee document

[2025] No. 4. This invention provides a novel, highly efficient, and safe plant inducer by deeply analyzing its molecular mechanism of activating plant defense responses, offering an innovative solution to the problem of disease control in apple production.

[0017] 2. The inducer containing penicillin wansi provided by this invention can significantly improve the control effect against anthracnose leaf blight. By activating the plant's own immune system, it induces the plant to produce disease resistance, improves the antioxidant capacity and disease resistance of plants, including apple trees, reduces production costs, and has a simple application method, making it easy to promote and use.

[0018] 3. Compared with chemical pesticides, the plant inducer containing penicillin wansi described in this invention has low biological pesticide residues, can effectively reduce the amount of chemical pesticides used, and is environmentally friendly, meeting the development needs of "green prevention and control" in modern agriculture, and has become an important research direction for replacing chemical pesticides. Attached Figure Description

[0019] Figure 1 shows the protective effect of Wan's penicillin on apple anthracnose leaf blight pathogens on the leaf surface, where a is an image of apple leaf appearance and b is a bar chart of lesion data.

[0020] Figure 2 shows the protective effect of Wan's penicillin against apple anthracnose leaf blight on fruit, where (a) is an appearance of the young fruit of Meiyue, (b) is an appearance of the fruit of Fuji, (c) is an analysis diagram of the diameter of lesions on the fruit of Fuji, and (d) is an analysis diagram of the diameter of lesions on the young fruit of Meiyue.

[0021] Figure 3 shows the catalase content measured after apple leaves were treated with Wan's penicillin.

[0022] Figure 4 shows the peroxidase content of apple leaves after treatment with penicillin.

[0023] Figure 5 shows the polyphenol oxidase content measured after apple leaves were treated with penicillin.

[0024] Figure 6 shows the glutathione S-transferase content measured after apple leaves were treated with penicillin.

[0025] Figure 7 shows the superoxide dismutase content measured in apple leaves after treatment with penicillin.

[0026] Figure 8 shows the expression level of disease resistance genes in apple leaves after treatment with penicillin. Detailed Implementation

[0027] 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 some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0028] To ensure a complete and unambiguous understanding of the technical solution of this invention, the term "apple" as used in this invention refers to an apple tree (sapling). Malus domestica This refers to tissue culture seedlings obtained through tissue culture, rather than simply edible apple fruits. Of course, those skilled in the art can clearly and completely understand the meaning and description of the relevant genes and their encoded proteins based on the description in this invention.

[0029] The penicillin described in this invention, namely 3,6-bis(1-methylpropyl)-2,5-piperazinedione, has the chemical formula C. 12 H 22 N2O2, with a relative molecular weight of 226.32, has the following structural formula: .

[0030] Example 1: Investigating the efficacy of *Penicillium wani* on apple leaves against *Amyloliquefaciens*, the causal agent of 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 a 0.1 μg / mL solution of penicillin, while the control group was sprayed with an equal volume of sterile water as a blank control (CK). 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 Add 0.1% Tween 20 to each leaf, spray evenly onto the detached fresh leaves, keep moist, and count the number of lesions after 4-6 days and take photos.

[0031] Experimental results are as follows Figure 1 As shown, the number of lesions on apple leaves treated with different concentrations of *Penicillium wani* significantly decreased with increasing concentration. The water control group (CK) had the most lesions, exceeding 150; while the 0.1 μg / mL treatment group (W) had very few lesions, almost none. This indicates that *Penicillium wani* has a significant inhibitory effect on *A. wani*, 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.

[0032] Example 2: Investigating the inhibitory effect of Wan's penicillin solution on apple fruit against *Amygdalinia rubra*, the causal agent of apple anthracnose leaf blight. Young 'Mingyue' apples and mature 'Fuji' apples were thoroughly rinsed with sterile water and then air-dried. For the experimental group, fruits were punctured with a 200 μL pipette tip, and then 0.1 μg / mL of penicillin was added. After air-drying, the fruits were placed in a sterile, humidified culture box for 48 h. Fungal cakes were collected from the edge of wild-type *Anthracnose leaf blight* colonies using a 5 mm diameter punch and inoculated onto the punctured sites. The sites were kept moist, and the diameter of the lesions was measured and photographed after 5-7 days.

[0033] Experimental results are as follows Figure 2As shown, the diameter of lesions on leaves treated with 0.1 μg / mL penicillin solution (W) was significantly smaller than that on young and mature apple fruits treated with sterile water (CK). This indicates that penicillin solution has a significant inhibitory effect on apple anthracnose leaf blight pathogens on fruit.

[0034] Example 3: Investigating the changes in catalase (CAT) content in apples after treatment with *Vincilia wani*. Following the procedure described in Example 1, fresh 'Gala' apple leaves were treated with 0.1 μg / mL Wan's penicillin solution for 48 h before sampling. The catalase content in the apple leaves was detected using Solarbio (commercially available, Cat: BC1335).

[0035] Data such as Figure 3 The results showed that the flavonoid content in apple leaves of the treatment group (W) was significantly higher than that of the control group (CK, sterile water treatment). Overexpression of catalase in transgenic crops can improve seedling survival rate. In agriculture, its biostimulant treatment of seeds can enhance low temperature tolerance. As an important stress-resistance component in plants, its increased content helps to improve the disease resistance and antioxidant level of apples.

[0036] Example 4: Changes in peroxidase (POD) content in apples after treatment with *Vincilia wans* Following the procedure described in Example 1, fresh 'Gala' apple leaves were treated with 0.1 μg / mL of Wan's penicillin solution for 48 h before sampling. The peroxidase content in the apple leaves was detected using Solarbio (commercially available, Cat: BC0090). Data are as follows: Figure 4 The results showed that the content of plant peroxidase in apple leaves of the treatment group (W) was significantly higher than that of the control group (CK, sterile water treatment). Plant peroxidase is involved in key processes such as reactive oxygen scavenging and cell wall lignification in plants. Increased activity of plant peroxidase can enhance the antioxidant capacity and structural defense capabilities of apples, helping them resist pathogen invasion.

[0037] Example 5: Changes in apple polyphenol oxidase (PPO) content after treatment with Wan's penicillin Following the procedure described in Example 1, fresh 'Gala' apple leaves were treated with 0.1 μg / mL of valerate solution for 48 h, and samples were taken. The polyphenol oxidase content in the apple leaves was detected using Solarbio (commercially available, Cat: BC0190). Data are as follows: Figure 5 The results showed that the content of polyphenol oxidase in apple leaves of the treatment group (W) was significantly higher than that of the control group (CK, sterile water treatment). Polyphenol oxidase plays an important role in the oxidation of phenolic substances in plants, disease resistance response, and wound healing. Its increased activity can enhance the defense response of apples against pathogens.

[0038] Example 6: Changes in glutathione S-transferase (GST) content in apples after treatment with *Vincilia wans* Following the procedure in Example 1, fresh 'Gala' apple leaves were treated with 0.1 μg / mL of Wan's penicillin solution for 48 h, and samples were taken. The glutathione S-transferase content in the apple leaves was detected using Solarbio (commercially available, Cat: BC0350). Data are as follows: Figure 6 The results showed that the content of glutathione S-transferase in apple leaves of the treatment group (W) was significantly higher than that of the control group (CK, sterile water treatment). Glutathione S-transferase is involved in plant detoxification metabolism, antioxidant responses, and signal transduction processes. Its enhanced activity can improve the tolerance of apples to adverse environments and pathogen toxins.

[0039] Example 7: Changes in superoxide dismutase (SOD) content in apples after treatment with *Vincilia wans* Following the procedure in Example 1, fresh 'Gala' apple leaves were treated with 0.1 μg / mL of Wan's penicillin solution for 48 h, and samples were taken. The superoxide dismutase content in the apple leaves was detected using Solarbio (commercially available, Cat: BC0170). Data are as follows: Figure 7 The results showed that the content of superoxide dismutase in apple leaves of the treatment group (W) was significantly higher than that of the control group (CK, sterile water treatment). Superoxide dismutase is a core enzyme in the plant's antioxidant defense system. It reduces oxidative damage by scavenging superoxide anion free radicals, enhances the plant's resistance to abiotic stresses such as drought, salinity, and low temperature, as well as pests and diseases, and promotes growth and improves apple quality.

[0040] Example 8: Treatment with *Vincilia wani* induces upregulation of disease resistance-related genes in apples. Following the procedure described in Example 1, fresh 'Gala' apple leaves were treated with 0.1 μg / mL penicillin 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 using the Novizan HiScript® III RT SuperMix reverse transcription kit.

[0041] 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 *Penicillium wani* treatment on the expression levels of a series of disease resistance-related pathological genes (PR) in apple leaves were systematically analyzed using the SYBR Green qPCR method, including... MdPR2 (β-1,3-glucanase) MdPR5 (Sweet protein). Three technical replicates were set up for each reaction to ensure data reliability.

[0042] Use 2 –ΔΔCT The method involves quantitative analysis of relative gene expression levels using qRT-PCR results. The results are as follows: Figure 8 As shown, compared with the sterile water control group, treatment with 0.1 μg / mL of *Vancirum vinifera* solution significantly induced related [conditions / conditions]. PR Gene expression is upregulated. Among them, MdPR2 As a marker gene for the salicylic acid signaling pathway, its strong activation indicates that systemic acquired resistance (SAR) is effectively triggered; MdPR5 Increased expression of (sweet protein) indicates that the plant's molecular defense against pathogen infection has been further enhanced.

[0043] Combination Figure 8 This indicates that Wan's penicillin can significantly improve... MdPR2 and MdPR5 Gene expression was significantly increased by 500-700% in the treatment group (W) compared to the control group (CK), further demonstrating that Wan's penicillin can be used as a novel plant immune inducer.

[0044] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. Application of Wan's penicillin in the preparation of inducers for the prevention and control of plant diseases.

2. The application according to claim 1, characterized in that, The chemical name of the aforementioned penicillin is 3,6-bis(1-methylpropyl)-2,5-piperazindione, and its chemical formula is C. 12 H 22 N2O2.

3. The application according to claim 1, characterized in that, The plant diseases mentioned include anthracnose leaf blight.

4. The application according to claim 1, characterized in that, The concentration of the inducer containing *Vanillaris* is 0.1 μg / mL to 1 μg / mL.

5. The application according to claim 1, characterized in that, The described penicillin can induce the upregulation of plant disease resistance genes.

6. The application according to claim 5, characterized in that, The plant disease resistance genes include MdPR2 and / or MdPR5 .

7. The application according to claim 1, characterized in that, The aforementioned penicillin can increase the content of at least one of catalase, peroxidase, polyphenol oxidase, glutathione S-transferase, and superoxide dismutase in plants.

8. The application according to any one of claims 1-7, characterized in that, The method for preventing and controlling plant diseases includes spraying an inducer onto the surface of plant leaves or fruits.

9. The application according to claim 8, characterized in that, The inducing agent is applied over a period of 2 to 6 days.

10. The application according to any one of claims 1-7, characterized in that, The plants mentioned include apple trees.

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