Application of pear short peptide PbeRALF317 in plant disease prevention and control

Abstract

The application provides application of a pear short peptide PbeRALF317 in plant disease prevention and control, and is application of a Pyrus betulifolia short peptide PbeRALF317 in pear fire blight, Sclerotinia sclerotiorum of rape, and grape and watermelon Botrytis blight prevention and control. The PbeRALF317 short peptide is artificially synthesized, the excitation effect of the short peptide on the above diseases is clarified, and the pear, rape, watermelon and grape are treated by spraying, so that the resistance to fire blight, sclerotinia and Botrytis blight is excited, and the above diseases are prevented and controlled. The short peptide provided by the application is a product synthesized by plants themselves, and the short peptide is used to excite immunity to prevent and control diseases, and has the advantages of natural green, environment-friendly, no induction of pathogen resistance and the like. The short peptide is easy to artificially synthesize, is easy to obtain, has strong disease resistance excitation effect, and has low required concentration, and is a new type of ecological and environment-friendly crop disease control agent.

Description

Technical Field

[0001] The present invention belongs to the field of plant immunity and biotechnology, and relates to the application of pear short peptide PbeRALF317 in plant disease prevention and control. It is an application of the immunity and disease resistance stimulating function of the pear short peptide PbeRALF317, and is an application of the pear short peptide PbeRALF317 in the green prevention and control of various crop diseases such as amylopectin, rapeseed sclerotinia rot, watermelon gray mold and grape gray mold. Background Art

[0002] 1. Plant immune stimulation technology

[0003] Plant immunity is a defensive response activated by plant receptors recognizing pathogens or plant molecules. The plant immune system has multiple layers, the first of which is pattern-triggered immunity (PTI), 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 reactive oxygen species (ROS) release, activation of kinase cascades, callose deposition, and expression of defense-related genes, ultimately resulting in resistance to pathogens. PTI plays an important role in nonhost resistance, which prevents nonadapted microorganisms from infecting plants, and in basal resistance, which limits the infectivity of adaptive pathogens in susceptible host plants. Immunity stimulated by DAMPs / phytocytokines has the advantages of being broad-spectrum, stable, and long-lasting. Furthermore, since DAMPs / phytocytokines are natural products of plants, using them to stimulate immunity to prevent or control diseases represents a new, environmentally friendly, and green disease control approach. RALF (Rapid Alkalization Factor) is a class of plant cytokines whose role in plant immunity and disease resistance has been reported in Arabidopsis thaliana and several other crops. However, the role of RALF peptides as plant immunity elicitors in disease resistance in fruit trees, such as the Rosaceae family, remains unclear. Furthermore, it remains unclear whether a single RALF peptide can induce broad-spectrum disease resistance against multiple pathogens in crops from different families.

[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. Because some pesticides cause ecological pollution, harm to humans and animals, and easily cause pathogens to develop drug resistance, long-term excessive use has exacerbated the severity of these problems. New green broad-spectrum control preparations are urgently needed to be developed. Summary of the Invention

[0006] The present invention aims to provide a pear short peptide, PbeRALF317, for use in plant disease prevention and control. The present invention discloses the immune and disease resistance stimulating functions of the pear (Pyrus betulifolia) short peptide, PbeRALF317, and its application in the green prevention and control of various crop diseases, including fire blight, rapeseed sclerotinia, watermelon gray mold, and grape gray mold. The amino acid sequence of the pear short peptide, PbeRALF317, used in the present invention is shown in SEQ ID NO. 1, representing the C-terminal 49 amino acids of the full-length protein. The full-length protein sequence is shown in SEQ ID NO. 2.

[0007] The present invention utilizes a pear short peptide PbeRALF317 to stimulate the production of resistance to fire blight (Erwinia amylovora) in the Rosaceae crop Pyrus betulifolia, to sclerotinia sclerotiorum (Sclerotinia sclerotiorum) in the Cruciferae crop Rapeseed (Brassica napus), and to gray mold (Botrytis cinerea) in the Cucurbitaceae crop Watermelon (Citrullus lanatus) and the Vitaceae crop Grape (Vitis vinifera), thereby achieving green prevention and control of apricot fire blight, rapeseed sclerotinia, watermelon and grape gray mold.

[0008] Prior to the present invention, there were no public reports on the function of the short peptide PbeRALF317. This present invention, for the first time, artificially synthesizes the short peptide PbeRALF317, demonstrating its stimulating effect on resistance to fire blight, sclerotinia rot in rapeseed, and gray mold in watermelon and grapes. Furthermore, this short peptide is used to stimulate resistance to fire blight, sclerotinia rot in pear, rapeseed, watermelon, and grapes, respectively, thereby controlling these important crop diseases. This application is achieved by the following steps:

[0009] (1) Artificial synthesis or biological expression of PbeRALF317 short peptide

[0010] The short peptide PbeRALF317 can be synthesized by entrusting a professional peptide synthesis company, or obtained through biological expression: the corresponding nucleotide sequence of PbeRALF317 is cloned into an expression vector, transformed into a eukaryotic or prokaryotic microorganism, microbial propagation, extraction and purification of the target short peptide.

[0011] (2) Plant treatment with PbeRALF317 short peptide

[0012] Plant leaves were treated with an appropriate concentration of a short peptide PbeRALF317 aqueous solution (containing 0.1% lauryl glucoside) to stimulate plant immunity. Spraying can be used for large-scale plant treatment.

[0013] (3) PbeRALF317 short peptide stimulates plant resistance to pathogens

[0014] Treatment with the short peptide PbeRALF317 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 PbeRALF317 than by water controls.

[0015] Advantages of the present invention: (1) The PbeRALF317 short peptide provided by the present invention is a product synthesized by the plant itself. The use of the PbeRALF317 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) Plant cytokines stimulating plant immunity is a new achievement in theoretical research. The application of PbeRALF317 as a plant cytokine 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 PbeRALF317 short peptide has only 49 amino acids, is easy to synthesize artificially, and is simple and easy to obtain. (4) The PbeRALF317 short peptide has a strong disease resistance stimulating effect and requires a low concentration, so the cost of disease prevention and control is low. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The results showed that spraying PbeRALF317 short peptide aqueous solution on Pyrus betulifolia leaves could induce the resistance of Pyrus betulifolia to fire blight. The PbeRALF317 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 PbeRALF317 short peptide (containing 0.1% lauryl glucoside) at a concentration of 1 μM. The ... 600 Leaves were inoculated using a toothpick soaked in a 0.7% 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 28°C to maintain moisture. The figure shows the disease progression four days after inoculation. Results showed that compared with the mock treatment with sterile double-distilled water (containing 0.1% lauryl glucoside), leaves treated with PbeRALF317 developed significantly smaller lesions and significantly inhibited lesion expansion. This suggests that PbeRALF317 treatment strongly stimulates resistance to fire blight in pear trees. The inoculation assay was repeated three times, and the results were analyzed statistically using Student's t-tests in GraphPad Prism. Data are presented as mean ± SD. Significant differences are indicated by asterisks (**, P < 0.01). Scale bar, 0.5 cm.

[0017] Figure 2 This study demonstrates that spraying rapeseed (Brassica napus) leaves with an aqueous solution of the short peptide PbeRALF317 stimulates resistance to Sclerotinia sclerotiorum. Rapeseed leaves were sprayed with a 1 μM aqueous solution of the short peptide PbeRALF317 (containing 0.1% lauryl glucoside). One day later, the leaves were inoculated with 3 mm diameter pieces of Sclerotinia sclerotiorum mycelium and incubated at 23°C covered with a film to maintain moisture retention. The figure shows lesion size one day after inoculation. Results showed that compared with treatment with sterile double-distilled water (containing 0.1% lauryl glucoside) (mock), lesions formed on leaves treated with PbeRALF317 were significantly smaller and their expansion was severely inhibited. This indicates that PbeRALF317 treatment strongly stimulates resistance to Sclerotinia sclerotiorum in rapeseed. The inoculation assay was repeated three times, and the results were analyzed using the Student's t-test in GraphPad Prism. Data are presented as mean ± SD. Significant differences are indicated by * (****, P < 0.0001). Scale bar is 2 cm.

[0018] Figure 3This study demonstrates that spraying watermelon (Citrullus lanatus) leaves with an aqueous solution of the short peptide PbeRALF317 stimulates resistance to gray mold in watermelon. Watermelon leaves were sprayed with a 1 μM aqueous solution of the short peptide PbeRALF317 (containing 0.1% lauryl glucoside). One day later, the leaves were inoculated with 3 mm diameter mycelial pieces of Botrytis cinerea and incubated at 23°C covered with a film to maintain moisture. The figure shows the disease progression two days after inoculation. 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 PbeRALF317 were significantly smaller and their expansion was significantly inhibited. This indicates that PbeRALF317 treatment significantly stimulates resistance to gray mold in watermelon. The inoculation assay was repeated three times, and the results were analyzed using GraphPad Prism using a Student's t-test. Data are presented as mean ± SD. Significant differences are indicated by asterisks (**, P < 0.01). Scale bar is 1 cm.

[0019] Figure 4 This study demonstrates that spraying grape (Vitis vinifera) leaves with an aqueous solution of the short peptide PbeRALF317 stimulates resistance to gray mold. Grape leaves were sprayed with a 1 μM aqueous solution of the short peptide PbeRALF317 (containing 0.1% lauryl glucoside). One day later, the leaves were inoculated with 3 mm diameter pieces of Botrytis cinerea mycelium and incubated at 23°C covered with a film to maintain moisture. The figure shows the disease progression three days after inoculation. Results show that compared with the mock treatment with sterile double-distilled water (containing 0.1% lauryl glucoside), the lesions formed on leaves treated with PbeRALF317 were significantly smaller and their expansion was significantly inhibited. This indicates that PbeRALF317 treatment significantly stimulates resistance to gray mold in grapes. The inoculation assay was repeated three times, and the results were analyzed statistically using Student's t-tests in GraphPad Prism. Data are presented as mean ± SD. Significant differences are indicated by asterisks (***, P < 0.001). Scale bar, 2 cm. DETAILED DESCRIPTION

[0020] The present invention is further described with reference to the accompanying drawings and embodiments.

[0021] Example 1 Application of PbeRALF317 short peptide in fire blight resistance of Pyrus betulifolia

[0022] The present invention utilizes the synthetic short peptide PbeRALF317 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:

[0023] 1) Synthesis of PbeRALF317 short peptide

[0024] The PbeRALF317 short peptide sequence provided by the present invention is shown in SEQ ID NO. 1, which consists of 49 amino acids and is the C-terminal portion of the full-length protein (sequence shown in SEQ ID NO. 2).

[0025] In this example, a professional peptide synthesis company (Qiangyao Biotechnology Co., Ltd.) was commissioned to synthesize the PbeRALF317 short peptide for analysis and application of pear disease resistance stimulation and fire blight prevention and control functions.

[0026] 2) Treatment of Pyrus betula with PbeRALF317 short peptide

[0027] The plant treatment method of PbeRALF317 short peptide is spraying. Spraying the leaves of Pyrus betula with a 1 μM concentration of PbeRALF317 short peptide aqueous solution (containing 0.1% lauryl glucoside) can treat plants on a large scale.

[0028] 3) PbeRALF317 short peptide stimulates Pyrus betula to produce resistance to fire blight pathogen

[0029] Spraying pear leaves with an aqueous solution of PbeRALF317 (containing 0.1% lauryl glucoside) significantly stimulated resistance to fire blight in pear trees. Fire blight inoculation analysis showed that after one day of spraying with 1 μM PbeRALF317, the lesions formed on leaves inoculated with fire blight bacteria by piercing the main vein with a toothpick containing the bacteria were significantly smaller than those in the control treated with sterile double-distilled water four days after inoculation, and lesion expansion was significantly inhibited ( Figure 1 ). This indicates that spraying with PbeRALF317 can strongly stimulate the resistance of Pyrus betula to fire blight.

[0030] Example 2 Application of PbeRALF317 short peptide in the treatment of rapeseed (Brassica napus) against bacterial sclerotinia

[0031] The present invention utilizes the synthetic short peptide PbeRALF317 to treat rapeseed by spraying, thereby stimulating its resistance to Sclerotinia sclerotiorum, thereby controlling Sclerotinia sclerotiorum in rapeseed. The main steps include:

[0032] 1) Synthesis of PbeRALF317 short peptide

[0033] The PbeRALF317 short peptide sequence provided by the present invention is shown in SEQ ID NO. 1, which consists of 49 amino acids and is the C-terminal portion of the full-length protein (sequence shown in SEQ ID NO. 2).

[0034] In this example, a professional peptide synthesis company (Qiangyao Biotechnology Co., Ltd.) was commissioned to synthesize the PbeRALF317 short peptide for analysis and application of rapeseed disease resistance stimulation and Sclerotinia sclerotiorum prevention and control functions.

[0035] 2) Treatment of rapeseed with PbeRALF317 short peptide

[0036] The plant treatment method of PbeRALF317 short peptide is spraying. Spraying rapeseed plant leaves with a 1 μM concentration of PbeRALF317 short peptide aqueous solution (containing 0.1% lauryl glucoside) can treat plants on a large scale.

[0037] 3) PbeRALF317 short peptide stimulates rapeseed to produce resistance to Sclerotinia sclerotiorum

[0038] Spraying rapeseed leaves with an aqueous solution of the PbeRALF317 peptide significantly stimulated resistance to Sclerotinia sclerotiorum. Analysis of Sclerotinia sclerotiorum inoculation showed that after one day of spraying with 1 μM PbeRALF317 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 spraying with PbeRALF317 can rapidly and strongly stimulate rapeseed to develop resistance to Sclerotinia sclerotiorum.

[0039] Example 3 Application of PbeRALF317 short peptide in watermelon (Citrullus lanatus) resistance to gray mold

[0040] The present invention uses the artificially synthesized PbeRALF317 short peptide to treat watermelon by spraying, stimulating its resistance to gray mold, thereby controlling gray mold in watermelon. The main steps include:

[0041] 1) Synthesis of PbeRALF317 short peptide

[0042] The PbeRALF317 short peptide sequence provided by the present invention is shown in SEQ ID NO. 1, which consists of 49 amino acids and is the C-terminal portion of the full-length protein (sequence shown in SEQ ID NO. 2).

[0043] In this example, a professional peptide synthesis company (Qiangyao Biotechnology Co., Ltd.) was commissioned to synthesize the PbeRALF317 short peptide for analysis and application of watermelon disease resistance stimulation and gray mold prevention and control functions.

[0044] 2) Treatment of watermelon with PbeRALF317 short peptide

[0045] The plant treatment method of PbeRALF317 short peptide is spraying. Spraying the leaves of watermelon plants with a 1 μM concentration of PbeRALF317 short peptide aqueous solution (containing 0.1% lauryl glucoside) can treat plants on a large scale.

[0046] 3) PbeRALF317 short peptide stimulates watermelon to produce resistance to gray mold

[0047] Spraying watermelon leaves with an aqueous solution of the short peptide PbeRALF317 significantly stimulated resistance to Botrytis cinerea. Botrytis cinerea inoculation analysis showed that after one day of spraying with 1 μM PbeRALF317, leaves inoculated with 3 mm diameter Botrytis cinerea mycelium had significantly smaller lesions than the control treated with sterile double-distilled water 48 hours after inoculation, and lesion expansion was significantly inhibited. Figure 3 ). This indicates that spraying with PbeRALF317 can significantly stimulate watermelon's resistance to gray mold.

[0048] Example 4 Application of PbeRALF317 short peptide in resistance to gray mold in grapes (Vitis vinifera)

[0049] The present invention utilizes the synthetic short peptide PbeRALF317 to treat grape leaves by spraying, thereby stimulating their resistance to gray mold, thereby controlling gray mold in grapes. The main steps include:

[0050] 1) Synthesis of PbeRALF317 short peptide

[0051] The PbeRALF317 short peptide sequence provided by the present invention is shown in SEQ ID NO. 1, which consists of 49 amino acids and is the C-terminal portion of the full-length protein (sequence shown in SEQ ID NO. 2).

[0052] In this example, a professional peptide synthesis company (Qiangyao Biotechnology Co., Ltd.) was commissioned to synthesize the PbeRALF317 short peptide for analysis and application of grape disease resistance stimulation and gray mold prevention and control functions.

[0053] 2) Treatment of grapes with PbeRALF317 short peptide

[0054] The plant treatment method of PbeRALF317 short peptide is spraying. Spraying grape plant leaves with a 1 μM concentration of PbeRALF317 short peptide aqueous solution (containing 0.1% lauryl glucoside) can treat plants on a large scale.

[0055] 3) PbeRALF317 short peptide stimulates grape resistance to gray mold

[0056] Spraying grape leaves with an aqueous solution of the PbeRALF317 peptide significantly stimulated resistance to Botrytis cinerea. Botrytis cinerea inoculation analysis showed that after one day of spraying with 1 μM PbeRALF317 peptide, leaves inoculated with 3 mm diameter Botrytis cinerea mycelium had significantly smaller lesions than the control treated with sterile double-distilled water 72 hours after inoculation, and lesion expansion was significantly inhibited. Figure 3 ). This indicates that spraying with PbeRALF317 can significantly stimulate grape resistance to gray mold.

Claims

1. An application of a pear short peptide PbeRALF317 in plant disease prevention and control, characterized in that: It is Duli ( Pyrus betulifolia ) Application of the short peptide PbeRALF317 in the prevention and control of apricot fire blight, rapeseed sclerotinia, and watermelon and grape gray mold, the amino acid sequence of the pear short peptide PbeRALF317 is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that The application is to use the short peptide PbeRALF317 of Pyrus betulus to stimulate the Rosaceae crop Pyrus betulus ( Pyrus betulifolia ) produces fire blight ( Erwinia amylovora ), cruciferous crops rapeseed ( Brassica napus ) produces resistance to Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum ), Cucurbitaceae crops watermelon ( Citrullus lanatus ) and grapes from the Vitaceae family ( Vitis vinifera L.) produces resistance to Botrytis cinerea ( Botrytis cinerea ) resistance, thereby achieving green control of pear fire blight, rapeseed sclerotinia, watermelon and grape gray mold.

3. The use according to claim 1, characterized in that The application is implemented by the following steps: (1) Artificial synthesis or biological expression of PbeRALF317 short peptide Entrust a professional peptide synthesis company to synthesize, or obtain the short peptide PbeRALF317 through biological expression: clone the corresponding nucleotide sequence of PbeRALF317 into an expression vector, transform it into a eukaryotic or prokaryotic microorganism, propagate the microorganism, and extract and purify the target short peptide; (2) Plant treatment with PbeRALF317 short peptide Spraying plant leaves with an appropriate concentration of a short peptide PbeRALF317 aqueous solution stimulates plant immunity and can treat plants on a large scale; (3) PbeRALF317 short peptide stimulates plant resistance to pathogens The short peptide PbeRALF317 stimulates plants to produce immune and resistance responses to pathogens after treatment. The intensity of the stimulation can be detected through pathogen inoculation analysis. The immune response of plants to pathogens is manifested in that the short peptide PbeRALF317 treatment makes the necrotic lesions produced after inoculation smaller than those in the water control treatment.

4. The use according to claim 3, characterized in that The short peptide PbeRALF317 aqueous solution in step (2) contains 0.1% lauryl glucoside.

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