Application of pear short peptide PbePEP4 in plant disease prevention and control

Abstract

The present invention provides the application of a pear short peptide PbePEP4 in plant disease prevention and control, specifically the application of a pear (Pyrus betulifolia) short peptide PbePEP4 in the prevention and control of apricot fire blight, rapeseed sclerotinia and watermelon gray mold. The present invention artificially synthesizes the short peptide PbePEP4 for the first time, clarifies its stimulating effect on the resistance to the above-mentioned diseases, and provides a method of spraying pears, rapeseed and watermelon to stimulate their resistance to fire blight, sclerotinia and gray mold, thereby preventing and controlling the above-mentioned diseases. The PbePEP4 short peptide provided by the present invention is a product synthesized by the plant itself. The use of the short peptide to stimulate immunity and thus prevent and control diseases has the advantages of being natural, green, environmentally friendly, and not inducing pathogens to develop drug resistance. In addition, the short peptide is easy to synthesize artificially, is easy to obtain, has a strong disease resistance stimulating effect, and requires a low concentration. It is a new type of ecological and environmentally friendly crop disease prevention and control preparation.

Description

Technical Field

[0001] The present invention belongs to the field of plant immune biotechnology, and relates to the application of pear short peptide PbePEP4 in plant disease prevention and control. It is an application of the immune and disease resistance stimulating functions of pear short peptide PbePEP4, specifically the application of pear short peptide PbePEP4 in the green prevention and control of various crop diseases such as pear fire blight, rapeseed sclerotinia rot and watermelon gray mold. Background Art

[0002] 1. Plant immune stimulation technology

[0003] Plant immunity is a defense response activated by plant receptors recognizing pathogen molecules. The plant immune system has multiple layers. The first layer is pattern-triggered immunity (PTI), which is generated by pattern recognition receptors (PRRs) on the surface of plant cells recognizing conserved molecular patterns from pathogens and the plant itself. Plants can sense damage to plant cell integrity caused by pathogen infection and produce damage-associated molecular patterns (DAMPs) or synthesize plant cytokines (phytocytokines). These DAMPs / phytocytokines, recognized by PRRs, trigger the PTI immune response, including the release of reactive oxygen species, activation of kinase cascades, callose deposition, and expression of defense-related genes, ultimately resulting in resistance to the pathogen. PTI plays an important role in nonhost resistance, which prevents infection by nonadapted microorganisms, and in basal resistance, which limits infection of susceptible host plants by adapted pathogens. Immunity stimulated by DAMPs / phytocytokines is broad-spectrum, strong, stable, and long-lasting. Furthermore, since DAMPs / phytocytokines are natural products of plants, using them to stimulate immunity to prevent or control diseases is a new, environmentally friendly, and green disease control method.

[0004] 2. Plant disease prevention and control technology

[0005] Crop diseases usually cause 10-30% yield losses, and disease prevention and control are important guarantees for food security. Crop disease prevention and control measures include plant quarantine, selection and utilization of disease-resistant varieties, agricultural prevention and control, biological control, physical control and chemical control. Efficient and green prevention and control, as well as broad-spectrum integrated prevention and control of multiple diseases at the same time, are the development trends of crop disease prevention and control. Pear fire blight, rapeseed sclerotinia rot and watermelon gray mold are all important crop diseases in agricultural production. Fire blight is caused by the bacterium Erwinia amylovora and is a devastating disease of fruit trees such as pears and apples. It is also an important quarantine disease in my country. Sclerotinia rot is caused by the fungus Sclerotinia sclerotiorum and is a major disease of oil crops such as rapeseed and vegetable crops. Gray mold is caused by the fungus Botrytis cinerea and is an important disease of crops such as vegetables, fruits and melons. These diseases cause huge economic losses every year. Chemical control remains an important means of preventing and controlling these diseases. Since some pesticides pose problems such as ecological pollution, toxicity to humans and animals, and the tendency for pathogens to develop drug resistance, new green broad-spectrum pesticides are in urgent need of development. Summary of the Invention

[0006] The present invention aims to provide a pear (Pyrus betulifolia) short peptide, PbePEP4, for use in plant disease prevention and control, specifically for the green prevention and control of various crop diseases, including fire blight, rapeseed sclerotinia, and watermelon gray mold. The amino acid sequence of the pear short peptide, PbePEP4, used in this invention is shown in SEQ ID: 1, representing the C-terminal portion of the full-length protein. The full-length protein sequence is shown in SEQ ID: 2. This invention utilizes the pear short peptide, PbePEP4, for its immune and disease resistance-stimulating properties.

[0007] The present invention utilizes a pear short peptide PbePEP4 to stimulate the Rosaceae crop Pyrus betulifolia to produce resistance to fire blight (Erwinia amylovora), the Cruciferae crop Brassica napus to produce resistance to sclerotinia sclerotiorum, and the Cucurbitaceae crop Watermelon (Citrullus lanatus) to produce resistance to gray mold (Botrytis cinerea), thereby achieving green prevention and control of apricot fire blight, rapeseed sclerotinia, and watermelon gray mold.

[0008] Prior to the present invention, there were no public reports on the function of the short peptide PbePEP4. This present invention, for the first time, artificially synthesized the short peptide PbePEP4, demonstrating its stimulating effect on resistance to fire blight, sclerotinia rot in rapeseed, and gray mold in watermelon. Furthermore, this short peptide is used to stimulate resistance to fire blight, sclerotinia rot in rapeseed, and gray mold in watermelon by treating pear, rapeseed, and watermelon, respectively, thereby preventing and controlling these important crop diseases. This application is achieved by the following steps:

[0009] (1) Artificial synthesis or biological expression of PbePEP4 short peptide

[0010] You can entrust a professional peptide synthesis company to synthesize it. Alternatively, you can obtain the short peptide PbePEP4 through biological expression: clone the corresponding nucleotide sequence of PbePEP4 into an expression vector, transform it into a eukaryotic or prokaryotic microorganism, propagate the microorganism, and extract and purify the target short peptide.

[0011] (2) Plant treatment with PbePEP4 short peptide

[0012] Plant leaves were treated with a suitable concentration of a short peptide PbePEP4 aqueous solution (containing 0.1% lauryl glucoside) to stimulate plant immunity. Spray treatment can be used for large-scale plant treatment.

[0013] (3) PbePEP4 short peptide stimulates plant resistance to pathogens

[0014] Treatment with the short peptide PbePEP4 rapidly stimulates plant immunity and resistance to a variety of pathogens. The intensity of this stimulation can be measured using pathogen inoculation assays. This immunity and resistance response is demonstrated by smaller necrotic lesions produced by treatment with the short peptide PbePEP4 than by water controls.

[0015] Advantages of the present invention: (1) The PbePEP4 short peptide provided by the present invention is a product synthesized by the plant itself. The use of the PbePEP4 short peptide to stimulate immunity and thus prevent and control crop diseases has the advantages of being natural, green, environmentally friendly, harmless to humans and animals, and not inducing pathogens to develop drug resistance. (2) DAMP / phytocytokine stimulation of plant immunity is the latest theoretical research result. The application of PbePEP4 as a DAMP / phytocytokine in crop immunity stimulation and disease prevention and control has sufficient theoretical basis and is also an example of theoretical guidance of practical application. (3) The PbePEP4 short peptide has only 28 amino acids, is easy to synthesize artificially, and is simple and easy to obtain. (4) The PbePEP4 short peptide has a strong disease resistance stimulation effect and requires a low concentration, so the cost of disease prevention and control is low. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1The results showed that spraying PbePEP4 short peptide aqueous solution on Pyrus betulifolia leaves could induce the resistance of Pyrus betulifolia to fire blight. The PbePEP4 short peptide aqueous solution (containing 0.1% lauryl glucoside) at a concentration of 1 μM was sprayed on the Pyrus betulifolia leaves. After 1 day, the leaves were treated with a solution of PbePEP4 short peptide (containing 0.1% lauryl glucoside) at a concentration of 1 μM. The ... 600 Leaves were inoculated with a toothpick soaked in a 0.5% solution of fire blight pathogenic bacteria for half an hour, inserted into a needle-pierced puncture site in the main vein. The leaves were then incubated at 27°C in a fresh-keeping bag to maintain moisture. The figure shows the disease progression 10 days after inoculation. The results showed that compared with the mock treatment with sterile double-distilled water (containing 0.1% lauryl glucoside), the lesions formed on leaves treated with PbePEP4 were significantly smaller in area and their expansion was significantly inhibited. This suggests that PbePEP4 treatment strongly stimulates resistance to fire blight in pear trees.

[0017] Figure 2 This study demonstrates that spraying rapeseed (Brassica napus) leaves with an aqueous solution of the short peptide PbePEP4 stimulates resistance to Sclerotinia sclerotiorum. Rapeseed leaves were sprayed with a 1 μM aqueous solution of the short peptide PbePEP4 (containing 0.1% lauryl glucoside). One day later, the leaves were inoculated with Sclerotinia mycelium and incubated at 23°C covered with a film to maintain moisture. The figure shows lesion size one day after inoculation. Results show that compared to treatment with sterile double-distilled water (containing 0.1% lauryl glucoside) (mock), lesions formed on leaves treated with PbePEP4 were significantly smaller, and lesion expansion was significantly inhibited. This indicates that PbePEP4 treatment strongly stimulates resistance to Sclerotinia sclerotiorum in rapeseed.

[0018] Figure 3 The results showed that spraying watermelon (Citrullus lanatus) leaves with a PbePEP4 short peptide aqueous solution stimulated watermelon resistance to gray mold. The watermelon leaves were sprayed with a 1 μM PbePEP4 short peptide aqueous solution (containing 0.1% lauryl glucoside), and 2.5 μL of the solution was applied to each point after 1 day at a concentration of 1×10 5 Leaves were inoculated with a conidial suspension containing 100 spores, four inoculations per leaf, and incubated at 23°C covered with film to maintain moisture. The figure shows the disease progression 65 hours after inoculation. Results showed that compared with the mock treatment with sterile double-distilled water (containing 0.1% lauryl glucoside), lesions formed on leaves treated with PbePEP4 were significantly smaller and their expansion was significantly inhibited. This suggests that PbePEP4 treatment strongly stimulates resistance to gray mold in watermelon. DETAILED DESCRIPTION

[0019] The present invention is further described with reference to the accompanying drawings and embodiments.

[0020] Example 1 Application of PbePEP4 short peptide in fire blight resistance of Pyrus betulifolia

[0021] The present invention utilizes the synthetic short peptide PbePEP4 to treat Pyrus betulae by spraying, thereby stimulating its resistance to the fire blight pathogen, thereby being used to prevent and control the fire blight. The main steps include:

[0022] 1) Artificial synthesis of PbePEP4 short peptide

[0023] The PbePEP4 short peptide sequence provided by the present invention is shown in SEQ ID: 1, which consists of 28 amino acids and is the C-terminal portion of the full-length protein (sequence shown in SEQ ID: 2). Prior to the present invention, there was no public report on the function of the short peptide PbePEP4.

[0024] In this example, a professional peptide synthesis company (Qiangyao Biotechnology Co., Ltd.) was commissioned to synthesize the PbePEP4 short peptide for analysis and application of pear disease resistance stimulation and fire blight prevention and control functions.

[0025] 2) Treatment of Pyrus betula with PbePEP4 short peptide

[0026] The plant treatment method of PbePEP4 short peptide is spraying. Spraying the leaves of Pyrus betula with a 1 μM concentration of PbePEP4 short peptide aqueous solution (containing 0.1% lauryl glucoside) can treat plants on a large scale.

[0027] 3) PbePEP4 short peptide stimulates Pyrus betula to produce resistance to fire blight pathogen

[0028] Spraying Pyrus betulus leaves with an aqueous solution of PbePEP4 (containing 0.1% lauryl glucoside) significantly stimulated resistance to fire blight. Analysis of fire blight inoculation showed that after one day of spraying with 1 μM PbePEP4, leaves inoculated with fire blight bacteria by piercing the main vein with a toothpick pierced with the bacteria formed significantly smaller lesions than the control treated with sterile double-distilled water 10 days after inoculation, and lesion expansion was significantly inhibited. Figure 1 ). This indicates that PbePEP4 spray treatment can strongly stimulate the resistance of Pyrus betula to fire blight.

[0029] Example 2 Application of PbePEP4 short peptide in the treatment of rapeseed (Brassica napus) against bacterial sclerotinia

[0030] The present invention utilizes the synthetic short peptide PbePEP4 to treat rapeseed by spraying, thereby stimulating its resistance to Sclerotinia sclerotiorum, thereby controlling Sclerotinia sclerotiorum in rapeseed. The main steps include:

[0031] 1) Artificial synthesis of PbePEP4 short peptide

[0032] The PbePEP4 short peptide sequence provided by the present invention is shown in SEQ ID: 1, which consists of 28 amino acids and is the C-terminal portion of the full-length protein (sequence shown in SEQ ID: 2). Prior to the present invention, there was no public report on the function of the short peptide PbePEP4.

[0033] In this example, a professional peptide synthesis company (Qiangyao Biotechnology Co., Ltd.) was commissioned to synthesize the PbePEP4 short peptide for analysis and application of rapeseed disease resistance stimulation and Sclerotinia disease prevention and control functions.

[0034] 2) Treatment of rapeseed with PbePEP4 short peptide

[0035] The plant treatment method of PbePEP4 short peptide is spraying. Spraying rapeseed plant leaves with a 1 μM concentration of PbePEP4 short peptide aqueous solution (containing 0.1% lauryl glucoside) can treat plants on a large scale.

[0036] 3) PbePEP4 short peptide stimulates rapeseed to produce resistance to Sclerotinia sclerotiorum

[0037] Spraying rapeseed leaves with an aqueous solution of the PbePEP4 short peptide significantly stimulated resistance to Sclerotinia sclerotiorum. Analysis of Sclerotinia sclerotiorum inoculation showed that after one day of spraying with 1 μM PbePEP4 short peptide, the lesions on leaves inoculated with Sclerotinia sclerotiorum mycelium were significantly smaller than those in the control treated with sterile double-distilled water, and lesion expansion was severely inhibited ( Figure 2 ). This indicates that PbePEP4 spray treatment can quickly and strongly stimulate rapeseed to develop resistance to Sclerotinia sclerotiorum.

[0038] Example 3 Application of PbePEP4 short peptide in watermelon (Citrullus lanatus) resistance to gray mold

[0039] The present invention utilizes the synthetic PbePEP4 short peptide to treat watermelon by spraying, stimulating its resistance to gray mold, thereby controlling gray mold in watermelon. The main steps include:

[0040] 1) Artificial synthesis of PbePEP4 short peptide

[0041] The PbePEP4 short peptide sequence provided by the present invention is shown in SEQ ID: 1, which consists of 28 amino acids and is the C-terminal portion of the full-length protein (sequence shown in SEQ ID: 2). Prior to the present invention, there was no public report on the function of the short peptide PbePEP4.

[0042] In this example, a professional peptide synthesis company (Qiangyao Biotechnology Co., Ltd.) was commissioned to synthesize the PbePEP4 short peptide for analysis and application of watermelon disease resistance stimulation and gray mold prevention and control functions.

[0043] 2) Treatment of watermelon with PbePEP4 short peptide

[0044] The plant treatment method of PbePEP4 short peptide is spraying. Spraying the leaves of watermelon plants with a 1 μM concentration of PbePEP4 short peptide aqueous solution (containing 0.1% lauryl glucoside) can treat plants on a large scale.

[0045] 3) PbePEP4 short peptide stimulates watermelon to produce resistance to gray mold

[0046] Spraying watermelon leaves with a PbePEP4 short peptide aqueous solution can significantly stimulate watermelon resistance to gray mold. The results of gray mold inoculation analysis showed that after 1 μM PbePEP4 short peptide spraying for 1 day, the conidia suspension of gray mold (concentration of 1×10 per ml) 5 The lesions of leaves inoculated with 2.5 μL of spores per point were significantly smaller than those of the control treated with sterile double-distilled water 65 hours after inoculation, and the expansion of lesions was severely inhibited ( Figure 3 ). This indicates that PbePEP4 spray treatment can strongly stimulate watermelon to develop resistance to gray mold.

Claims

1. An application of a pear short peptide PbePEP4 in plant disease prevention and control, characterized in that: The application is to utilize Pyrus betula ( Pyrus betulifolia ) The short peptide PbePEP4 stimulates the Rosaceae crop Pyrus betula ( Pyrus betulifolia ) produces fire blight ( Erwinia amylovora ), cruciferous crops rapeseed ( Brassica napus ) produces resistance to Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum ) and the cucurbitaceae crop watermelon ( Citrullus lanatus ) produces resistance to Botrytis cinerea ( Botrytis cinerea ) resistance, thereby green prevention and control of pear fire blight, rapeseed sclerotinia and watermelon gray mold. The amino acid sequence of the pear short peptide PbePEP4 is shown in SEQ ID: 1, which is the C-terminal part of its full-length protein, and its full-length protein sequence is shown in SEQ ID:

2.

2. The use according to claim 1, characterized in that Plant disease control can be achieved through the following steps: (1) Artificial synthesis or biological expression of PbePEP4 short peptide Obtaining the short peptide PbePEP4: cloning the corresponding nucleotide sequence of PbePEP4 into an expression vector, transforming into a eukaryotic or prokaryotic microorganism, propagating the microorganism, extracting and purifying the target short peptide; (2) Plant treatment with PbePEP4 short peptide Spraying plant leaves with an aqueous solution of a short peptide PbePEP4 containing wetting and penetration aids stimulates plant immunity and can treat plants on a large scale; (3) PbePEP4 short peptide stimulates plants to develop resistance to pathogens The short peptide PbePEP4 stimulated the plants to produce immune and resistance responses to pathogens after treatment. The intensity of the stimulation was detected by pathogen inoculation analysis. The plants produced an immune response to pathogens, which was manifested in that the short peptide PbePEP4 treatment made the necrotic lesions produced after inoculation smaller than those in the water control treatment.

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