Application of pear oligopeptide PbeRALF317 in prevention and control of plant diseases

Abstract

The invention provides an application of a pear oligopeptide PbeRALF317 in prevention and control of plant diseases, in particular to an application of a pyrus betulifolia oligopeptide PbeRALF317 in prevention and control of pear fire blight, sclerotinia rot of colza and gray mold of watermelons and grapes. The PbeRALF317 oligopeptide is artificially synthesized, the excitation effect of the PbeRALF317 oligopeptide on the resistance of the diseases is illustrated, and pear, rape, watermelon and grape are sprayed to excite the pear, rape, watermelon and grape to generate resistance on fire blight, sclerotiniose and gray mold respectively, so that the application of the diseases is prevented and controlled. The oligopeptide provided by the invention is a product synthesized by plants, and the oligopeptide is used for stimulating immunity so as to prevent and treat diseases, so that the oligopeptide has the advantages of being natural, green, environment-friendly, free from inducing pathogens to generate drug resistance and the like. The oligopeptide is easy to artificially synthesize, simple and convenient to obtain, strong in disease-resistant excitation effect and low in required concentration, and is a novel ecological and environment-friendly crop disease prevention and control preparation.

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. The present invention is an application of the immunity and disease resistance stimulating function of pear short peptide PbeRALF317, and is an application of 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 Plant immunity is a defense response activated by plants through receptors recognizing pathogens or plant molecules. The plant immune system has multiple levels, the first of which is the immunity generated by pattern recognition receptors (PRRs) on the surface of plant cells recognizing conservative molecular patterns from pathogens and plants themselves, which is called pattern-triggered immunity (PTI). Plants can sense the damage to the integrity of plant cells caused by pathogen infection, etc., and produce damage-associated molecular patterns (DAMPs) or synthesize plant cytokines (phytocytokines). These DAMPs / phytocytokines stimulate plants to produce PTI immune responses through PRR receptor recognition, including reactive oxygen burst, activation of kinase cascades, callose deposition, expression of defense-related genes, etc., and finally show resistance to pathogens. PTI plays an important role in preventing non-adaptive microorganisms from infecting plants through nonhost resistance and limiting the basal resistance of adaptive pathogens to infect susceptible host plants. Immunity stimulated by DAMPs / phytocytokines has the advantages of broad spectrum, stability and persistence. In addition, since DAMPs / phytocytokines are natural products of plants themselves, using DAMPs / phytocytokines to stimulate immunity to prevent or control diseases is an environmentally friendly and green new method for disease prevention and control. RALF (Rapid alkalization factor) is a type of plant cytokine. Its role in plant immunity and disease resistance has been reported in Arabidopsis and some crops, but the role of RALF peptides as plant immunity elicitors in the disease resistance of fruit trees such as Rosaceae is still unclear. Moreover, it is not clear whether a RALF peptide can induce crops from different families to produce broad-spectrum disease resistance to multiple pathogens across families.

[0003] 2. Plant disease prevention and control technology Crop diseases usually cause 10-30% yield loss, and disease prevention and control is an important guarantee for food security. Crop disease prevention and control measures include plant quarantine, disease-resistant variety breeding and utilization, agricultural prevention, biological control, physical control and chemical control. Efficient green prevention and control and broad-spectrum comprehensive 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 and watermelon gray mold are all important crop diseases in agricultural production. Fire blight is caused by the bacterium Erwinia amylovora, which is a devastating disease of fruit trees such as pears and apples, and is also an important quarantine disease in my country. Sclerotinia is caused by the fungus Sclerotinia sclerotiorum, which is a major disease of oil crops such as rapeseed and vegetable crops. Gray mold is caused by the fungus Botrytis cinerea, which 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 prevention and control of these diseases. Because some pesticides cause ecological pollution, toxicity to humans and animals, and the easy development of drug resistance in pathogens, long-term excessive use has aggravated the severity of these problems. New green broad-spectrum control preparations are urgently needed to be developed. Summary of the invention

[0004] The purpose of the present invention is to provide an application of a pear short peptide PbeRALF317 in plant disease prevention and control, an immune and disease resistance stimulating function of a pear (Pyrus betulifolia) short peptide PbeRALF317 and its application, and an application of the short peptide in green prevention and control of various crop diseases such as amylopectin, 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, which is the 49 amino acids of the C-terminal part of its full-length protein, and its full-length protein sequence is shown in SEQ ID NO.2.

[0005] The present invention utilizes the pear short peptide PbeRALF317 to stimulate the Rosaceae crop Pyrus betulifolia to produce resistance to fire blight (Erwinia amylovora), the Cruciferae crop rapeseed (Brassica napus) to produce resistance to sclerotinia sclerotiorum, the Cucurbitaceae crop watermelon (Citrullus lanatus) and the Vitaceae crop grape (Vitis vinifera) to produce resistance to gray mold (Botrytis cinerea), thereby greenly preventing and controlling apricot fire blight, rapeseed sclerotinia, watermelon and grape gray mold.

[0006] Prior to the present invention, there was no public report on the function of the short peptide PbeRALF317. The present invention, for the first time, artificially synthesized the short peptide PbeRALF317, clarified the stimulating effect of the short peptide on fire blight of apricot, sclerotinia of rapeseed, and gray mold of watermelon and grape, and provided the application of the short peptide to stimulate the resistance of pear, rapeseed, watermelon and grape to fire blight, sclerotinia and gray mold respectively by treating pear, rapeseed, watermelon and grape, thereby preventing and controlling these important diseases of crops. The application of the present invention is achieved by the following steps:

[0007] (1) Artificial synthesis or biological expression of PbeRALF317 short peptide The short peptide PbeRALF317 can be obtained by entrusting a professional peptide synthesis company to synthesize it, or by biological expression: cloning the corresponding nucleotide sequence of PbeRALF317 into an expression vector, transforming it into a eukaryotic or prokaryotic microorganism, propagating the microorganism, extracting and purifying the target short peptide.

[0008] (2) Plant treatment with PbeRALF317 short peptide The plant leaves were sprayed with an appropriate concentration of short peptide PbeRALF317 aqueous solution (containing 0.1% lauryl glucoside) to stimulate plant immunity. Spray treatment can be used for large-scale plant treatment.

[0009] (3) PbeRALF317 short peptide stimulates plants to develop resistance to pathogens The short peptide PbeRALF317 rapidly stimulates the plant to produce immune and disease resistance responses to a variety of pathogens. The intensity of the stimulation can be detected by pathogen inoculation analysis. The plant's immune and disease resistance response 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.

[0010] 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 and green, not polluting the environment, harmless to humans and animals, and not inducing pathogens to produce drug resistance. (2) Plant cytokines stimulating plant immunity is a new achievement in theoretical research. PbeRALF317, as a plant cytokine, has a sufficient theoretical basis for use in crop immune stimulation and disease prevention and control, and is also an example of theory guiding practical application. (3) The PbeRALF317 short peptide has only 49 amino acids, which is easy to synthesize artificially 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

[0011] Figure 1The results showed that spraying PbeRALF317 short peptide aqueous solution on Pyrus betulifolia leaves could induce Pyrus betulifolia to develop resistance to fire blight. The PbeRALF317 short peptide aqueous solution (containing 0.1% lauryl glucoside) at a concentration of 1 μM was sprayed on the leaves of Pyrus betulifolia. After 1 day, the leaves were heated to 4 ℃ and 7 ℃, and the temperature was set to 40 ℃. 600 The leaves were inoculated with a toothpick soaked in a 0.7% solution of fire blight pathogen for half an hour and inserted into the eye of the main vein of the leaf formed by sterile needle puncture, and then cultured at 28°C to keep moist. The figure shows the disease situation 4 days after inoculation. The results showed that compared with the sterile double distilled water (containing 0.1% lauryl glucoside) treatment (Mock), the lesion area formed on the leaves treated with PbeRALF317 was significantly smaller, and the expansion of the lesions was strongly inhibited. This shows that PbeRALF317 treatment strongly stimulated Pyrus betulaeformis to produce resistance to fire blight pathogens. The inoculation analysis was repeated three times, and the results were statistically analyzed by Student's t-test using GraphPad Prism. The data are expressed as mean ± standard deviation. The significance of the difference is indicated by different numbers of * (**, P<0.01). The scale bar is 0.5 cm.

[0012] Figure 2 The results show that spraying the leaves of rapeseed (Brassica napus) with an aqueous solution of PbeRALF317 short peptide stimulates the resistance of rapeseed to Sclerotinia sclerotiorum. The rapeseed leaves were sprayed with an aqueous solution of PbeRALF317 short peptide (containing 0.1% lauryl glucoside) at a concentration of 1 μM. One day later, the leaves were inoculated with 3 mm diameter mycelium of Sclerotinia sclerotiorum and cultured at 23°C. The image shows the size of the lesions one day after inoculation. The results showed that compared with the treatment with sterile double distilled water (containing 0.1% lauryl glucoside) (Mock), the lesions formed on the leaves treated with PbeRALF317 were significantly smaller and the expansion of the lesions was severely inhibited. This indicates that the treatment with PbeRALF317 strongly stimulates the resistance of rapeseed to Sclerotinia sclerotiorum. The inoculation analysis was repeated three times, and the results were statistically analyzed by Student's t-test using GraphPad Prism. The data are expressed as mean ± standard deviation. The significant difference is indicated by different number of * (****, P<0.0001). Scale bar is 2 cm.

[0013] Figure 3The results show that spraying watermelon (Citrullus lanatus) leaves with PbeRALF317 short peptide aqueous solution (containing 0.1% lauryl glucoside) stimulated watermelon to produce resistance to gray mold. Watermelon leaves were sprayed with a 1μM PbeRALF317 short peptide aqueous solution (containing 0.1% lauryl glucoside). One day later, watermelon leaves were inoculated with gray mold mycelium blocks with a diameter of 3mm, and cultured at 23℃ with film covering to keep moist. The figure shows the disease situation 2 days after inoculation. The results showed that compared with the sterile double distilled water (containing 0.1% lauryl glucoside) treatment (Mock), the lesions formed on the leaves treated with PbeRALF317 were significantly smaller, and the expansion of the lesions was significantly inhibited. This shows that PbeRALF317 treatment significantly stimulated watermelon to produce resistance to gray mold. The inoculation analysis was repeated three times, and the results were statistically analyzed by Student's t-test using GraphPad Prism. The data are expressed as mean ± standard deviation. The significant difference is indicated by different number of * (**, P<0.01). Scale bar is 1 cm.

[0014] Figure 4 The results show that spraying grape (Vitis vinifera) leaves with an aqueous solution of PbeRALF317 short peptide stimulated the grape to produce resistance to gray mold. Grape leaves were sprayed with an aqueous solution of PbeRALF317 short peptide (containing 0.1% lauryl glucoside) at a concentration of 1 μM. One day later, the grape leaves were inoculated with gray mold mycelium blocks with a diameter of 3 mm, and the leaves were covered with a film to keep them moist. The figure shows the disease situation 3 days after inoculation. The results showed that compared with the treatment with sterile double distilled water (containing 0.1% lauryl glucoside) (Mock), the lesions formed on the leaves treated with PbeRALF317 were significantly smaller, and the expansion of the lesions was significantly inhibited. This shows that PbeRALF317 treatment significantly stimulated the grape to produce resistance to gray mold. The inoculation analysis was repeated three times, and the results were statistically analyzed by Student's t-test using GraphPad Prism. The data are expressed as mean ± standard deviation. The significant difference is indicated by different numbers of * (***, P<0.001). The scale bar is 2 cm. DETAILED DESCRIPTION

[0015] The present invention is further described in conjunction with the accompanying drawings and embodiments.

[0016] Example 1 Application of PbeRALF317 short peptide in fire blight resistance of Pyrus betulifolia The embodiment of the present invention uses the artificially synthesized PbeRALF317 short peptide to treat Pyrus betulaeformis by spraying to stimulate its resistance to fire blight pathogens, thereby being used for the prevention and control of fire blight. The main steps include:

[0017] 1) Artificial synthesis of PbeRALF317 short peptide 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 part of the full-length protein (the sequence is shown in SEQ ID NO.2).

[0018] In this example, a professional peptide synthesis company (Qiangyao Biotechnology Co., Ltd.) was commissioned to synthesize the PbeRALF317 short peptide for the analysis and application of pear disease resistance stimulation and fire blight prevention and control functions.

[0019] 2) Treatment of Pyrus betula with PbeRALF317 short peptide The plant treatment method of PbeRALF317 short peptide is spraying. Spraying the leaves of Pyrus betulaeformis plants with a 1 μM PbeRALF317 short peptide aqueous solution (containing 0.1% lauryl glucoside) can treat plants on a large scale.

[0020] 3) PbeRALF317 short peptide stimulates Pyrus betula to produce resistance to fire blight pathogen Spraying Pyrus betulus leaves with an aqueous solution of PbeRALF317 (containing 0.1% lauryl glucoside) can significantly stimulate Pyrus betulus to develop resistance to fire blight. The results of the fire blight inoculation analysis showed that after 1 μM PbeRALF317 short peptide spraying for 1 day, the lesions formed on the leaves inoculated with fire blight bacteria by piercing the main vein with a toothpick carrying the bacteria were significantly smaller than those in the control group treated with sterile double distilled water 4 days after inoculation, and the expansion of the lesions was severely inhibited ( Figure 1 ). This indicates that spraying with PbeRALF317 can strongly stimulate Pyrus betulaeformis to develop resistance to fire blight.

[0021] Example 2 Application of PbeRALF317 short peptide in the fight against sclerotinia sclerotiorum of rapeseed (Brassica napus) The embodiment of the present invention uses the artificially synthesized PbeRALF317 short peptide to treat rapeseed by spraying to stimulate its resistance to Sclerotinia sclerotiorum, thereby being used to control rapeseed sclerotinia sclerotiorum. The main steps include:

[0022] 1) Artificial synthesis of PbeRALF317 short peptide 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 part of the full-length protein (the sequence is shown in SEQ ID NO.2).

[0023] 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.

[0024] 2) Treatment of rapeseed with PbeRALF317 short peptide The plant treatment method of PbeRALF317 short peptide is spraying. Spraying rapeseed plant leaves with a 1 μM PbeRALF317 short peptide aqueous solution (containing 0.1% lauryl glucoside) can treat plants on a large scale.

[0025] 3) PbeRALF317 short peptide stimulates rapeseed to produce resistance to Sclerotinia sclerotiorum Spraying rapeseed leaves with an aqueous solution of PbeRALF317 peptide can significantly stimulate rapeseed to develop resistance to Sclerotinia. The results of Sclerotinia inoculation analysis showed that after 1 μM PbeRALF317 peptide spraying for 1 day, the lesions on leaves inoculated with Sclerotinia mycelium blocks were significantly smaller than those in the control group treated with sterile double distilled water, and the expansion of lesions was severely inhibited ( Figure 2 ). This indicates that spraying with PbeRALF317 can rapidly and strongly stimulate rapeseed to develop resistance to Sclerotinia sclerotiorum.

[0026] Example 3 Application of PbeRALF317 short peptide in watermelon (Citrullus lanatus) resistance to gray mold The embodiment of the present invention uses the artificially synthesized PbeRALF317 short peptide to treat watermelon by spraying to stimulate its resistance to gray mold fungus, thereby being used to control gray mold of watermelon. The main steps include:

[0027] 1) Artificial synthesis of PbeRALF317 short peptide 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 part of the full-length protein (the sequence is shown in SEQ ID NO.2).

[0028] In this example, a professional peptide synthesis company (Qiangyao Biotechnology Co., Ltd.) was commissioned to synthesize the PbeRALF317 short peptide for the analysis and application of watermelon disease resistance stimulation and gray mold prevention and control functions.

[0029] 2) Treatment of watermelon with PbeRALF317 short peptide 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.

[0030] 3) PbeRALF317 short peptide stimulates watermelon to produce resistance to Botrytis cinerea Spraying watermelon leaves with an aqueous solution of PbeRALF317 short peptide can significantly stimulate watermelon to develop resistance to Botrytis cinerea. The results of the Botrytis cinerea inoculation analysis showed that after 1 μM PbeRALF317 short peptide spray treatment for 1 day, the lesions on leaves inoculated with Botrytis cinerea mycelium blocks with a diameter of 3 mm were significantly smaller than those in the control treated with sterile double distilled water 48 hours after inoculation, and the expansion of lesions was significantly inhibited ( Figure 3 ). This indicates that spraying with PbeRALF317 can significantly stimulate watermelon's resistance to Botrytis cinerea.

[0031] Example 4 Application of PbeRALF317 short peptide in resistance to gray mold in grapes (Vitis vinifera) The embodiment of the present invention uses the artificially synthesized PbeRALF317 short peptide to treat grape leaves by spraying to stimulate their resistance to gray mold fungi, thereby being used to control gray mold in grapes. The main steps include:

[0032] 1) Artificial synthesis of PbeRALF317 short peptide 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 part of the full-length protein (the sequence is shown in SEQ ID NO.2).

[0033] In this example, a professional peptide synthesis company (Qiangyao Biotechnology Co., Ltd.) was commissioned to synthesize the PbeRALF317 short peptide for the analysis and application of grape disease resistance stimulation and gray mold prevention and control functions.

[0034] 2) Treatment of grapes with PbeRALF317 short peptide The plant treatment method of PbeRALF317 short peptide is spraying. Spraying grape plant leaves with a 1 μM PbeRALF317 short peptide aqueous solution (containing 0.1% lauryl glucoside) can treat plants on a large scale.

[0035] 3) PbeRALF317 short peptide stimulates grape resistance to Botrytis cinerea Spraying grape leaves with an aqueous solution of PbeRALF317 short peptide can significantly stimulate grapes to develop resistance to Botrytis cinerea. The results of the Botrytis cinerea inoculation analysis showed that after 1 μM PbeRALF317 short peptide spray treatment for 1 day, the lesions on leaves inoculated with Botrytis cinerea mycelium blocks with a diameter of 3 mm were significantly smaller than those in the control treated with sterile double distilled water 72 hours after inoculation, and the expansion of lesions was significantly inhibited ( Figure 3 ). This indicates that spraying with PbeRALF317 can significantly stimulate grape resistance to Botrytis cinerea.

Claims

1. An application of a pear short peptide PbeRALF317 in plant disease prevention and control, characterized in that: The invention relates to the application of a short peptide PbeRALF317 from Pyrus betulifolia in the prevention and control of fire blight of apricot, sclerotinia sclerotiorum of rapeseed, and gray mold of watermelon and grape. The amino acid sequence of the short peptide PbeRALF317 is shown in SEQ ID NO.1, and the protein sequence thereof is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: The application is to use the pear short peptide PbeRALF317 to stimulate the Rosaceae crop Pyrus betulifolia to produce resistance to fire blight (Erwinia amylovora), the Cruciferae crop rapeseed (Brassica napus) to sclerotinia sclerotiorum, the Cucurbitaceae crop watermelon (Citrullus lanatus) and the Vitaceae crop grape (Vitis vinifera L.) to gray mold (Botrytiscinerea), thereby greenly preventing and controlling apricot 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 the expression vector, transform it into eukaryotic or prokaryotic microorganisms, propagate the microorganisms, extract and purify the target short peptide; (2) Plant treatment with PbeRALF317 short peptide Spray the plant leaves with an appropriate concentration of short peptide PbeRALF317 aqueous solution to stimulate plant immunity. It can treat plants on a large scale; (3) PbeRALF317 short peptide stimulates plants to develop resistance to pathogens The short peptide PbeRALF317 treatment stimulated the plant to produce an immune and resistance response to the pathogen, and the intensity of the stimulation can be detected by pathogen inoculation analysis. The plant produced an immune response to the pathogen, which was manifested in that the short peptide PbeRALF317 treatment made the necrotic lesions produced after inoculation smaller than those in the water control treatment.

4. The use according to claim 1, characterized in that: The short peptide PbeRALF317 aqueous solution in step (2) contains 0.1% lauryl glucoside.

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