Litchi endogenous polypeptide and application thereof in controlling plant diseases

Abstract

The present application provides an effective and safe litchi endogenous polypeptide and its application in controlling plant diseases. The amino acid sequence of the litchi endogenous polypeptide P2 is shown as SEQ ID NO. 1. After the litchi endogenous polypeptide P2 is used to treat litchi, the resistance of litchi fruits to litchi blight Peronophythora litchii can be improved. Therefore, the litchi endogenous polypeptide P2 can be used to prepare a novel preservative and a plant disease resistance inducer against fungal diseases, and is applied in the field of plant fungal disease prevention and control.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to an endogenous polypeptide of litchi and its application, specifically an endogenous polypeptide of litchi and its application in controlling plant diseases. Background Technology

[0002] Downy mildew of litchi is one of the most important fungal diseases affecting litchi, causing a significant decline in the nutritional value and quality of litchi before and after harvest, resulting in substantial economic losses. Due to the lack of effective control methods, downy mildew has caused considerable difficulties for my country's litchi industry and has gradually become one of the major diseases affecting litchi. *Peronophythora litchii* is one of the main pathogens causing downy mildew. Currently, chemical agents are mainly used for control, which have certain impacts on fruit quality and are detrimental to the health of consumers; moreover, they easily lead to drug resistance. Therefore, screening for harmless, effective, and safe green fungicides to improve fruit resistance and control disease occurrence is of great significance for maintaining litchi quality and extending its storage period.

[0003] Plant endogenous peptides are a novel type of plant growth regulator. Related research has mainly focused on the model plant Arabidopsis thaliana. The functions of endogenous peptides in other species, especially the role and application of endogenous peptides in controlling postharvest diseases in litchi, have not yet been reported. Summary of the Invention

[0004] Based on the problems existing in the background art, the purpose of this invention is to provide a litchi endogenous polypeptide and its application in controlling plant diseases, specifically, its application in controlling litchi downy mildew or fruit diseases caused by *Peronophythoralitchii*. Using the aforementioned litchi endogenous polypeptide to control plant diseases has advantages such as being environmentally friendly, highly safe, and able to stimulate systemic resistance in plants, making it a green and sustainable control method.

[0005] On one hand, the present invention provides a litchi endogenous polypeptide P2, characterized in that: the amino acid sequence of the litchi endogenous polypeptide P2 is shown in SEQ ID NO.1.

[0006] The present invention also provides a plant disease resistance inducer, characterized in that: the plant disease resistance inducer includes the aforementioned litchi endogenous polypeptide P2.

[0007] The main active ingredient of the plant disease resistance inducer includes litchi endogenous polypeptide P2. Applying this plant disease resistance inducer to plants can improve their resistance to diseases.

[0008] This invention also provides a plant preservative, characterized in that: the plant preservative includes the aforementioned litchi endogenous polypeptide P2. The main active ingredient of the plant preservative includes litchi endogenous polypeptide P2.

[0009] In a preferred embodiment, the plant disease resistance inducer or plant preservative further contains a surfactant to enhance the adhesion of the plant disease resistance inducer or plant preservative to the plant surface.

[0010] Furthermore, the plant disease resistance inducer or plant preservative described in this invention may also contain at least one of the following adjuvants: thickener, stabilizer, solubilizer, wetting agent, etc., to optimize the physicochemical properties of litchi endogenous polypeptide P2 solution and improve the efficacy of litchi endogenous polypeptide P2.

[0011] On the other hand, the present invention also provides the application of the aforementioned litchi endogenous polypeptide P2 or the aforementioned plant disease resistance inducer in the prevention and control of plant diseases.

[0012] In the preferred embodiment, the plant disease is litchi downy mildew.

[0013] Alternatively, the plant disease mentioned may be a litchi disease caused by the pathogen Peronophythora litchii.

[0014] In a preferred embodiment, the concentration of litchi endogenous polypeptide P2 in the plant disease resistance inducer is less than 20 μM, for example, 1 μM, 5 μM, or 10 μM, preferably 5-10 μM; other concentrations may also be used.

[0015] On the other hand, the present invention also provides the application of the aforementioned litchi endogenous polypeptide P2 or the aforementioned plant preservative in plant preservation.

[0016] In the preferred embodiment, the plant preservation is achieved by exogenously applying litchi endogenous polypeptide P2 to enhance the plant's resistance to diseases.

[0017] Furthermore, the exogenous application of litchi endogenous polypeptide P2 improved the plant's resistance to litchi downy mildew.

[0018] In the preferred embodiment, the plant preservation method is the preservation of plants in the genus *Lychee*.

[0019] Furthermore, the plant preservation mentioned is lychee preservation.

[0020] In a preferred embodiment, the concentration of litchi endogenous polypeptide P2 in the plant preservative is less than 20 μM, for example, 1 μM, 5 μM, or 10 μM, preferably 5-10 μM; other concentrations may also be used.

[0021] In the aforementioned scheme, the litchi endogenous polypeptide P2, the plant preservative, or the plant disease resistance inducer can be applied to the plant by soaking, spraying, smearing, or injection.

[0022] In the aforementioned scheme, "plant" can refer to a growing plant or an detached organ of a plant.

[0023] Applying the aforementioned litchi endogenous polypeptide P2, plant preservative, or plant disease resistance inducer to growing plants or detached plant organs can inhibit the growth of *Peronophythoralitchii* by enhancing the plant's disease resistance.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] (1) The present invention uses biological methods to obtain a litchi endogenous polypeptide P2. This litchi endogenous polypeptide is environmentally friendly, harmless to the human body, and has high safety. Moreover, it can stimulate plant systemic resistance, thereby improving the plant's ability to resist diseases.

[0026] (2) This invention provides a new use for the above-mentioned litchi endogenous polypeptide P2, namely, for the preparation of plant disease resistance inducers or plant preservatives that are harmless to the human body and effective and safe.

[0027] (3) This invention provides for the first time the application of the above-mentioned litchi endogenous polypeptide P2 in the control of fruit diseases. By applying the litchi endogenous polypeptide P2, plant diseases caused by litchi downy mildew (Peronophythora litchii) can be effectively controlled; fruit quality can be maintained and shelf life can be extended. By applying it in the forest, not only can plant diseases be effectively controlled, but crop yield can also be increased. It is a green and sustainable means of plant disease prevention and control. Attached Figure Description

[0028] The method of the present invention and its beneficial effects will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0029] Figure 1 Effect of litchi endogenous peptide P2 treatment on the growth of *Phytophthora litica* on PDA plates. (A) Phenotypic characteristics of *Phytophthora litica* treated with different concentrations of P2; (B) Diameter of *Phytophthora litica* plaques treated with different concentrations of P2, p<0.05.

[0030] Figure 2 Effect of litchi endogenous peptide P3 treatment on the growth of *Phytophthora licheniformis* on PDA plates. (A) Phenotypic characteristics of *Phytophthora licheniformis* treated with different concentrations of P3; (B) Plaque diameter of *Phytophthora licheniformis* treated with different concentrations of P3, p<0.05.

[0031] Figure 3Effects of litchi endogenous peptide P2 treatment (5 μM) on litchi fruit infected with Phytophthora downycium. (A) Phenotype of infected litchi; (B) Histogram of plaque diameter, where different letters on the bars represent significant differences (p < 0.05). Detailed Implementation

[0032] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0033] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0034] The following examples involve two polypeptides, both endogenous polypeptides of litchi. These polypeptides were obtained through bioinformatics analysis of the litchi genome, predicting potential functional sequences, and were synthesized artificially (Shanghai Xinhao Biotechnology Co., Ltd.). One of them is the litchi endogenous polypeptide P2, which is claimed in this invention and is abbreviated as P2. The amino acid sequence of the P2 peptide is SEQ ID NO.1: LASGPSPKGPGT. The other litchi endogenous polypeptide is P3, abbreviated as P3. The amino acid sequence of the P3 peptide is SEQ ID NO.2: LGGIKESGPSPRGEGH.

[0035] Example 1: Antibacterial effect of litchi endogenous peptides on culture medium

[0036] Effects of litchi endogenous peptide (P2 and P3) treatment on the growth of *Phytophthora infestans* on litchi

[0037] Phytophthora lichee was cultured on PDA plates at 28°C for 7 days. The spores were washed with sterile water and filtered through gauze. The filtrate was collected, and the spore concentration was adjusted to 1×10⁻⁶ using a spore counter. 6 10 μl of the above spore solution was dropped into the middle of PDA medium containing 0 μM P2 (CK) and PDA medium containing 5 μM, 10 μM, and 20 μM P2, respectively. Simultaneously, 10 μl of the above spore solution was dropped into the middle of PDA medium containing 0 μM P3 (CK) and PDA medium containing 5 μM, 10 μM, and 20 μM P3, respectively. After 11 days of incubation, the effects of P2 and P3 on the growth of *Phytophthora lichei* were compared. Figure 1 As shown in Figure A, treatment with endogenous polypeptides (P2) in litchi had no significant effect on the growth of *Phytophthora indicum*; except for a slight increase in colony diameter at 20 μM treatment, there were no significant differences among the other groups. Figure 1B).

[0038] Similarly, treatment with litchi endogenous peptides (P3) had no significant effect on the growth of *Phytophthora infestans*. Figure 2 A); the colony diameter was also slightly higher in the 20μM treatment group than in other groups, while there was no significant difference among the other groups. Figure 2 B).

[0039] Example 2: Control of natural disease in litchi fruit by endogenous polypeptides (P2 and P3)

[0040] Lychee fruits harvested on sunny days, at 80% maturity, without mechanical damage or visible signs of disease or pests, were randomly divided into three groups of 100 fruits each, with three replicates per treatment group. The control and experimental groups were immersed in water, 5 μM P2 solution, and 5 μM P3 solution, respectively, for 10 minutes. The treated lychee fruits were then air-dried. All lychee fruits were stored in an incubator at 25℃ and 85% relative humidity, and disease incidence was observed. After 3 days of storage, as shown in Table 1, the incidence of downy mildew in the CK group was 95%, in the P2 group it was 43%, and in the P3 group it was 85%. This indicates that treatment with the endogenous peptide P2 in lychee can significantly control the occurrence of downy mildew in lychee. Therefore, the endogenous peptide P2 in lychee can be used as an effective preservative for lychee preservation.

[0041] Table 1. Effects of endogenous polypeptide treatment on the natural incidence of downy mildew in litchi.

[0042] deal with CK P2 P3 Average natural incidence rate 95% （a） 43% （b） 85% （a）

[0043] Note: Different letters indicate significant differences (p<0.05), while the same letter indicates no significant differences.

[0044] After in-depth research, the inventors discovered that although P2 and P3 had no effect on the growth of *Phytophthora indicum*, P2 treatment significantly controlled the occurrence of *Phytophthora indicum*, while other endogenous peptides in litchi (P3) had no significant effect on controlling litchi diseases. This indicates that the endogenous peptide P2 in litchi can improve fruit resistance, and that different endogenous peptides have certain differences in their effects on fruit resistance.

[0045] Example 3: Control of disease in lychee fruit by endogenous polypeptide (P2)

[0046] Litchi fruits were further treated with endogenous litchi peptides (P2). Fruits harvested on sunny days at 80% maturity, without mechanical damage or pests, were randomly divided into two groups of 30 fruits each, with three replicates of 10 fruits per replicate. The control and experimental groups were immersed in water and a 5 μM P2 solution, respectively, for 5 minutes. The treated litchi fruits were then air-dried. The litchi fruits were then inoculated with *Phytophthora litchiensis* (a type of downy mildew) using a needle-pricking method. Peronophythora litchii Take 10 μl of Phytophthora spores from litchi (1×10⁻⁶) 6 (Spores / ml) was dripped onto the puncture site of the litchi fruit. All litchi fruits were stored in an incubator at 25℃ and 85% relative humidity, and the disease incidence was observed. Three days after inoculation, it was first observed by the naked eye that the litchi peel treated with P2 had less disease ( Figure 3 A) and the diameter of fungal patches on the litchi peel treated with P2 was significantly smaller than that of the control group CK (p<0.05). Figure 3 B). This indicates that the P2 treatment can effectively control the growth of Phytophthora licheniformis on litchi fruits and delay the occurrence of diseases caused by Phytophthora licheniformis.

[0047] Based on the inventors' research results, the endogenous polypeptide P2 in litchi has no effect on the growth of litchi downy mildew, but it can specifically enhance the plant host's resistance, achieve effective control of the disease, and effectively prevent the emergence of drug resistance in pathogens.

[0048] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to the above embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of an endogenous litchi polypeptide P2 in the control of litchi diseases, characterized in that, The disease of litchi is downy mildew of litchi, and the amino acid sequence of the endogenous polypeptide is shown in SEQ ID NO.

1.

2. The application of a litchi endogenous polypeptide P2 in litchi preservation, characterized in that, The preservation of litchi is achieved by exogenously applying litchi endogenous polypeptide P2, the amino acid sequence of which is shown in SEQ ID NO.1.

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