Polypeptide PP alpha for preventing and treating fungal diseases and application thereof

Abstract

The invention belongs to the technical field of plant disease prevention and treatment, and particularly relates to a polypeptide PP alpha for preventing and treating fungal diseases and application thereof. The invention discloses application of polypeptide PP alpha in inhibiting plant pathogenic fungi and preventing and treating plant diseases caused by the plant pathogenic fungi, the polypeptide PP alpha is a yeast alpha mating factor, and the amino acid sequence of the polypeptide PP alpha is shown as SEQ ID No.1. Experiments prove that the polypeptide PP alpha can significantly inhibit spore germination and appressorium formation of various plant pathogenic fungi such as pyricularia oryzae, cucurbitaceae colletotrichum spinosus, colletotrichum cryptum and botrytis cinerea, so that the infection process of the fungi is blocked. The polypeptide PP alpha provided by the invention has broad-spectrum antifungal activity, can be used as a novel green bactericide for preventing and treating fungal diseases of crops, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of plant disease control technology, specifically relating to a polypeptide PPα for controlling fungal diseases and its application. Background Technology

[0002] Anthracnose fungi, Botrytis cinerea, and other pathogens have a wide host range, capable of infecting various grain crops, fruits, vegetables, and cash crops, further expanding their scope of damage and increasing the difficulty of control. Rice blast is one of the typical diseases of rice worldwide, characterized by high outbreaks, widespread occurrences, and devastation. Rice blast fungus (… Magnaporthe oryzae The infection process is typical: conidia germinate to form germ tubes, the top of the germ tubes swells to form appressoriums, the appressoriums form infection nails and invade plant tissues, and finally, infectious hyphae form, causing disease. Gray mold is named for the large amount of gray mold that grows on the surface of diseased plant tissues caused by its pathogen, *Botrytis cinerea*. Botrytis cinerea Anthracnose is a common fungal disease caused by *Anthracnose fungus*, affecting the stems, leaves, flowers, fruits, and seeds of crops. It can infect over 1000 types of fruits and vegetables, such as strawberries, tomatoes, and grapes. Infected fruits and vegetables develop white or gray mold spots on their surface, and infected areas show soft rot and browning. It is particularly damaging to solanaceous vegetables and berries, and is an important postharvest pathogen. Colletotrichum This genus of fungi is highly diverse and can cause diseases on a variety of host plants, leading to crop yield reduction and varying degrees of impact on the agricultural economy. Among them, *Cryptospira* (… Colletotrichum aenigma ) can cause anthracnose in strawberries and grapes, etc.; *Colletotrichum candida* (Cucurbitaceae family) Colletotrichum orbiculare Anthracnose, on the other hand, can cause anthracnose in cucurbitaceous crops such as watermelon and cucumber. Currently, the main control methods for these fungal diseases include chemical control, physical control, and biological control. However, with the widespread use of chemical reagents, environmental pollution has increased, and pathogens have become increasingly resistant to pesticides. Physical control methods are not practical in production. When using microbial control, fungi and bacteria also develop corresponding biological protection mechanisms within the host, such as antagonism, competition, and induction. Therefore, developing new control methods is extremely important.

[0003] Currently, bio-based fungicides are gradually becoming a research hotspot due to their advantages such as good environmental compatibility, target specificity, and low likelihood of inducing drug resistance. Among them, polypeptide bioactive substances, with their small molecular weight, high activity, and high safety, show great application potential. Antimicrobial peptides, also known as peptide antibiotics, are important substances in the innate immune response of organisms and are widely present in nature. Antimicrobial peptides are characterized by small molecular weight, broad antimicrobial spectrum, low likelihood of inducing drug resistance, and strong activity against pathogens such as bacteria. Due to their broad-spectrum antibacterial activity, antimicrobial peptides are considered a potential plant protection agent to overcome crop diseases caused by fungal and bacterial plant pathogens. Antimicrobial peptides can rapidly kill multidrug-resistant bacteria, such as Escherichia coli and Staphylococcus aureus, at extremely low concentrations. Their mechanism of action differs from traditional antibiotics; they mainly work by disrupting the cell membrane of pathogens, leading to increased membrane permeability and depolarization, forming transmembrane ion channels on the membrane, disrupting membrane integrity, causing leakage of cell contents and cell lysis, thereby killing the cell. In addition to direct sterilization, antimicrobial peptides also have antiviral, antifungal, anti-biofilm formation, and immunomodulatory functions. They are unlikely to induce bacterial resistance, and most antimicrobial peptides cause minimal damage to host cells, making them highly safe.

[0004] Based on the above situation, the present invention aims to systematically clarify the broad-spectrum antifungal activity and mechanism of action of chemically synthesized polypeptide PPα, develop its application in the prevention and control of plant fungal diseases, and provide a new method for the research and development of novel green fungicides. Summary of the Invention

[0005] To address the problems existing in the prior art, the purpose of this invention is to design and provide a technical solution for the prevention and control of fungal diseases using a polypeptide PPα and its application.

[0006] This invention is specifically achieved through the following technical solutions: The first aspect of the present invention provides the application of polypeptide PPα in inhibiting plant pathogenic fungi, wherein the polypeptide PPα is a yeast α-mating factor and its amino acid sequence is shown in SEQ ID NO.1.

[0007] Furthermore, the plant pathogenic fungi include one or more of the following: rice blast fungus, botrytis cinerea, cucurbita spp., or cryptic spp.

[0008] The second aspect of the present invention provides the application of polypeptide PPα in inhibiting or preventing plant diseases caused by plant pathogenic fungi, wherein the polypeptide PPα is a yeast α-mating factor and its amino acid sequence is shown in SEQ ID NO.1.

[0009] Furthermore, the plant diseases mentioned include those caused by rice blast fungus (… Magnaporthe oryzae ), Botrytis cinerea ( Botrytis cinereais ), Cucurbitaceae family, Colletotrichum ( Colletotrichum orbiculare ) or Glomerulosa cryptotachys ( Colletotrichum aenigma Plant diseases caused by one or more of the following:

[0010] Furthermore, the rice blast fungus includes strains Guy11, 70-15, TH3, and TH16.

[0011] The third aspect of the present invention provides a drug for preventing and controlling fungal diseases, the active ingredient of which is a polypeptide PPα, wherein the polypeptide PPα is a yeast α-mating factor and its amino acid sequence is shown in SEQ ID NO.1.

[0012] Furthermore, the concentration of the polypeptide PPα is 75-600 μM.

[0013] The fourth aspect of this invention provides the application of the above-mentioned drug for preventing and controlling fungal diseases in the prevention and control of plant pathogenic fungal infections.

[0014] Furthermore, the plants include barley, rice, grapes, strawberries, and cucumbers.

[0015] Furthermore, the fungal disease control agent is applied by drip inoculation onto the leaves or fruit, or sprayed onto the seedlings.

[0016] The present invention has the following beneficial effects: The polypeptide PPα provided by this invention can significantly inhibit rice blast fungus ( Magnaporthe oryzae ), Cucurbitaceae family, Colletotrichum ( Colletotrichum orbiculare ), *Discocephalus cryptogams* ( Colletotrichum aenigma ), Botrytis cinerea ( Botrytis cinereais It inhibits the spore germination and appressorium formation of various plant pathogenic fungi, thereby blocking the fungal infection process. The polypeptide PPα possesses broad-spectrum antifungal activity and can be used as a novel green fungicide for the control of crop fungal diseases, showing great promise for application. Attached Figure Description

[0017] Figure 1 Effects of different concentrations of PPα on the growth of rice blast fungus, **** indicates p<0.0001; Figure 2 Effect of PPα on appressorium formation of rice blast fungus, scale bar = 5 μm; Figure 3 Effect of PPα on the formation rate of rice blast fungus appressorium, ** indicates p < 0.01, **** indicates p < 0.0001, ns indicates p > 0.05; Figure 4 Effects of PPα on spore growth of *Colletotrichum gloeosporioides*, *Colletotrichum cryptosporioides* and *Botrytis cinerea* (Cucurbitaceae). * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001, and ns indicates p > 0.05. Figure 5 The effect of PPα on the pathogenicity of rice blast fungus, scale bar = 1 cm; Figure 6 Effects of PPα on the pathogenicity of Botrytis cinerea and Colletotrichum cryptococcosis, scale bar = 2 cm; Figure 7 A: Effect of PPα on pathogenicity of *Colletotrichum gloeosporioides* in cucumber leaves (left side: PPα treatment, right side: control); B: Effect of PPα on pathogenicity of *Colletotrichum gloeosporioides* in strawberry leaves, scale bar = 2 cm. Detailed Implementation

[0018] The technical features and advantages of the invention will be described in more detail below with reference to the accompanying drawings.

[0019] Example 1: Sensitivity determination of PPα peptide to mycelial growth of rice blast fungus The polypeptide PPα used in this invention was obtained from QYAOBIO (China Peptides Co., Ltd.) through solid-phase synthesis, with a purity >98%. Its amino acid sequence is WHWLQLKPGQPMY (SEQ ID NO.1).

[0020] The wild-type strain Guy11 of rice blast fungus was used as the test object. Complete culture medium (CM) was prepared according to the method of Talbot NJ. The formula in the literature "Identification and characterization of MPG1, a gene involved in pathogenicity from the rice blast fungus Magnaporthe grisea, Plant Cell. 1993 Nov;5(11):1575-90" by Talbot NJ et al. was as follows: 10 g glucose, 2 g peptone, 1 g yeast extract, 1 g casein amino acids, 50 mL 20× nitrogen salt, 1 mL 1000× trace element solution, 1 mL 1000× vitamin solution, diluted to 1 L with pure water, pH adjusted to 6.5, 15 g / L agar was added and sterilized, and then injected into 2 cm twelve-well plates (2 mL / well). PPα peptide was dissolved in sodium acetate buffer to prepare a 20 mM stock solution, which was then diluted to 300 and 600 μm final concentrations in liquid CM (the control group used an equal volume of solvent). After solidification, the solution was used for later use. Colonies cultured for 7-10 days were collected from CM plates, and after spore washing with sterile water, filtration through three layers of sterile lens paper, and centrifugation at 7500 rpm for 5 min, the solution was reconstituted with sterile water to a concentration of 5 × 10⁻⁶ mM. 4Spores / mL suspension, 5 μL inoculated into the center of each 12-well plate, with 3 replicates per group. After 72 h of incubation at 25°C under light and dark cycles (16 h light / 8 h dark), colony diameter was measured and photographed. Results showed that PPα peptide significantly inhibited the mycelial growth of Guy11, with the inhibitory effect increasing with increasing concentration (62.5% inhibition rate at 300 μm, reaching 71.9% at 600 μm), particularly significantly inhibiting aerial mycelial formation. Figure 1 ).

[0021] Example 2: Determination of the inhibitory effect of polypeptide PPα on appressorium formation of rice blast fungus Spore suspensions (1×10⁻⁶) of wild-type rice blast fungus strains Guy11 and 70-15 were prepared according to the method described in Example 1. 5 (Spores / mL), add polypeptide PPα to a final concentration of 300 μm (control group is an equal volume of sodium acetate solvent), take 25 μL of the mixture and drop it onto a hydrophobic plastic coverslip, and incubate in a dark humidified chamber at 22℃. The formation rate of rice blast fungus appressoria was counted by optical microscopy at 4 hpi, 8 hpi, and 24 hpi, respectively. The experiment was repeated three times, and each sample was counted three times in each experiment.

[0022] The results showed that peptide PPα significantly inhibited the formation and development of appressorium of *Strombus haematobium*. At 4 hpi, compared with the wild-type control Guy11 which had an appressorium formation rate of over 82.0%, the spore germination rate of the strain with 300 μm peptide PPα was reduced, with an appressorium formation rate of only 22.0%, and the germ tubes were significantly elongated. With increasing induction time, the appressorium formation rate of the strain with 300 μm peptide PPα gradually increased to 54.0% at 8 hpi, and further increased to 65.0% at 24 hpi, still significantly different from the almost 100% appressorium formation rate of the wild-type strain, and the germ tube length was still abnormally elongated. The inhibitory effect of peptide PPα on the formation of 70-15 appressoriums was even more pronounced at 4 hpi. Figure 2 , 3 These results indicate that the polypeptide PPα can affect the morphology of conidia of rice blast fungus, slow down the spore germination rate, and inhibit the formation of appressorium.

[0023] Example 3: Determination of the inhibitory effect of PPα peptide on appressorium formation of *Botrytis cinerea*, *Colletotrichum gloeosporioides*, and *Colletotrichum cryptosporioides*. A spore suspension of *Colletotrichum spp.*, *Colletotrichum cryptospp.*, and *Botrytis cinerea* was prepared according to the method described in Example 1 (1×10⁻⁶). 5Spore density (spores / mL) was increased, and PPα peptide was added to a final concentration of 300 μm (the control group used an equal volume of sodium acetate solvent). 25 μL of the mixture was then added dropwise to a hydrophobic plastic coverslip and incubated in a dark, humidified chamber at 22°C. Spore germination and appressorium formation rates were statistically analyzed and photographed at 4 hpi, 8 hpi, and 24 hpi using an optical microscope. Each sample was counted three times in each experiment, and the experiment was repeated three times.

[0024] The results showed that diluting the spore solution with diluted CM liquid medium (CM: sterile water = 1:4) was effective in inducing appressorium formation in *Botrytis cinerea*. The experiment revealed that adding 300 μm of polypeptide PPα delayed spore germination. Compared with the control, the spore germination rate was significantly reduced after PPα treatment, and abnormal thickening of the spore wall was observed at 8 hpi and 24 hpi. Figure 4 ).

[0025] PPα peptide significantly inhibited appressorium formation and development in *Colletotrichum spp.* (Cucurbitaceae). At 4 hpi, compared to the control group's 17.0% appressorium formation rate, the spore germination rate of *Colletotrichum spp.* with the addition of 300 μm PPα peptide decreased, with an appressorium formation rate of 9.0%. Furthermore, with increasing induction time, the appressorium formation rate of *Colletotrichum spp.* with the addition of 300 μm PPα peptide gradually increased to 35.0% at 8 hpi, reaching 43.7% at 24 hpi, which was significantly different from the control group's 79.7% appressorium formation rate. Figure 4 Furthermore, the addition of 300 μm PPα peptide reduced the degree of melanization of the appressorium cell wall.

[0026] The addition of 300 μm peptide PPα significantly inhibited appressorium formation in *Colletotrichum cryptosporidis*. Conidia showed extremely low germination rates at 4 hpi, failing to form appressoriums. While the germination rate increased over time, appressorium formation remained low; at 24 hpi, the control group had a 12.0% appressorium formation rate, while the peptide-treated group only achieved a 1.7% appressorium formation rate. Figure 4 This indicates that the polypeptide PPα can affect the normal growth of conidia of *Cryptococcus pyogenes*, slow down the spore germination rate, and inhibit the formation of appressorium.

[0027] The above results confirm that the polypeptide PPα significantly weakens the early infection ability of *Botrytis cinerea*, *Colletotrichum gloeosporioides*, and *Colletotrichum cryptosporioides* by interfering with the normal differentiation and maturation of appressorium, a key stage of pathogenicity.

[0028] Example 4: Determination of the inhibitory effect of polypeptide PPα on the pathogenicity of rice blast fungus In vitro barley inoculation experiment: Isolated barley leaves (Zhejiang Beer 96) were placed in humidified petri dishes. Spore suspensions (1×10⁻⁶) of four wild-type strains of rice blast fungus (Guy11, 70-15, TH3, TH16) were prepared. 5 The concentration of PPα peptide was mixed with sodium acetate (10 μL / mL) to a final concentration of 300 μm. The solvent control was an equal volume of sodium acetate. Three drops (10 μL / drop) were applied to each leaf, with three leaves inoculated for each treatment. After 4 days of incubation at 25℃ under light, lesion development was observed. Results showed that the severity of lesions caused by four types of rice blast fungi on barley leaves was significantly reduced. PPα peptide significantly inhibited lesion formation; at 300 μm, almost no or only tiny lesions appeared on the surface of leaves 70-15. Figure 5 This study confirmed that the polypeptide PPα effectively inhibits the pathogenicity of rice blast fungus.

[0029] Experiment on pathogenicity of rice spray: Rice seeds of variety CO39 were selected, with 30 seeds sown per pot. Seedlings were cultured outdoors for 10-15 days to obtain seedlings for subsequent inoculation experiments. Spores of wild-type strains of rice blast fungus (Guy11, 70-15, TH3, TH16) were collected from CM plates and diluted to a final concentration of 5 × 10⁻⁶ using 0.2% gelatin (to increase spore adhesion). 4 The spores were mixed with PPα peptide at a concentration of 75 μm, and an equal volume of sodium acetate solvent was added as a control. The spore suspension was sprayed evenly using a small sprayer. Each pot of rice seedlings was inoculated with 2.5 mL of spore suspension, while the blank control group was sprayed with 2.5 mL of 0.2% gelatin solution. Each treatment was performed in triplicate. After inoculation, the seedlings were first incubated at 22℃ in the dark for 48 hours, and then transferred to a 25℃ light incubator for 3-4 days. Leaf disease incidence was observed and recorded. A 5 cm segment of a typical diseased leaf was photographed, and the lesion area was quantitatively analyzed using Photoshop software.

[0030] The results are shown in the figure. In the control group, strains Guy11, 70-15, TH3, and TH16 infected rice leaves and induced typical rice blast lesions, with lesion area proportions of 70.0%, 43.1%, 37.3%, and 37.2%, respectively. In the 75 μm polypeptide PPα treatment group, the disease severity on rice leaves was reduced, and lesion expansion was significantly limited, with lesion area proportions decreasing to 20.2%, 20.4%, 14.1%, and 18.0%, respectively. Figure 5 This indicates that the polypeptide PPα significantly inhibited the infection and lesion expansion of various rice blast fungus strains.

[0031] The above experimental results show that the polypeptide PPα provided by this invention exhibits broad-spectrum and highly efficient inhibitory activity against a variety of wild-type strains of rice blast fungus, providing experimental basis for the development of novel biological pesticides for the prevention and control of rice blast.

[0032] Example 5: Determination of the inhibitory effect of polypeptide PPα on the pathogenicity of *Botrytis cinerea*, *Colletotrichum gloeosporioides*, and *Colletotrichum cryptosporioides*. Preparation of spore suspension: Spores of wild-type strains of *Colletotrichum gloeosporioides*, *Colletotrichum cryptosporioides*, and *Botrytis cinerea* were collected from CM plates and diluted with sterile water to a final concentration of 5 × 10⁻⁶. 4 The concentration of the peptide was 300 μm after being mixed with the peptide PPα at a concentration of 1 / mL. The control was prepared by adding an equal volume of sodium acetate solvent.

[0033] Materials Culture: Mature Red Face strawberries and Sunshine Rose grapes were used for fruit inoculation. After alcohol disinfection, the fruits were air-dried. 10 μL of a *Botrytis cinerea* spore mixture was applied to each strawberry fruit, and 10 μL of a *Cryptospira* spore mixture was applied to each grape fruit. The control was an equal volume of sodium acetate solution. Three fruits were inoculated for each treatment. Leaves were selected from Red Face strawberry leaves and Jinyou No. 2 cucumber leaves to determine the inhibitory effects of *Cryptospira* and *Cryptospira* caulis, respectively. Three drops (10 μL / drop) were applied to each leaf, and three leaves were inoculated for each treatment.

[0034] Culture conditions: After inoculation, the culture should be placed in a dark environment at 22℃ for 48 hours, and then transferred to a light incubator at 25℃ for 3-6 days. The experimental results should be observed and recorded daily.

[0035] The results are as follows Figure 6 As shown, six days after inoculating strawberry fruits with *Botrytis cinerea* and grape fruits with *Colletotrichum cryptococcosis*, obvious lesions appeared on the fruits in the control group. However, the disease severity at the inoculation site was reduced and the lesion area was significantly smaller in the treatment group treated with added polypeptide PPα. No susceptible symptoms were observed in grapes or strawberries from any of the undamaged inoculation groups. Figure 6 This indicates that the polypeptide PPα can effectively inhibit the pathogenicity of these two plant pathogenic fungi on strawberry or grape fruits.

[0036] To further investigate whether peptide PPα affects the pathogenicity of *Colletotrichum cryptomerioides* and *Colletotrichum cucurbita* to leaves, spores were inoculated onto strawberry and cucumber leaves for three days. In the control group, typical lesions appeared at the inoculation sites, while the lesions in the peptide PPα-treated group were significantly reduced. These results indicate that peptide PPα not only inhibits the pathogenicity of *Colletotrichum cryptomerioides* and *Botrytis cinerea* to strawberry and grape fruits, but also inhibits the pathogenicity of *Colletotrichum cryptomerioides* and *Colletotrichum cucurbita* to leaves. Figure 7 ).

Claims

1. The application of a polypeptide PPα in inhibiting plant pathogenic fungi, characterized in that, The polypeptide PPα is a yeast α-mating factor, and its amino acid sequence is shown in SEQ ID NO.

1.

2. The application as described in claim 1, characterized in that, The plant pathogenic fungi include one or more of the following: rice blast fungus, Botrytis cinerea, Colletotrichum gloeosporioides, or Colletotrichum cryptosporioides.

3. The application of a polypeptide PPα in the prevention and control of plant diseases caused by plant pathogenic fungi, characterized in that, The polypeptide PPα is a yeast α-mating factor, and its amino acid sequence is shown in SEQ ID NO.

1.

4. The application as described in claim 3, characterized in that, The plant diseases mentioned include those caused by one or more of the following fungi: rice blast fungus, botrytis cinerea, cucurbitaceous spirochetes, or cryptosporidium.

5. A drug for preventing and controlling fungal diseases, characterized in that, Its active ingredient is polypeptide PPα, which is a yeast α-crossing factor, and its amino acid sequence is shown in SEQ ID NO.

1.

6. The drug as described in claim 5, characterized in that, The concentration of the polypeptide PPα is 75-600 μM.

7. The application of the drug for preventing and controlling fungal diseases as described in claim 5 or 6 in the prevention and control of plant pathogenic fungal infections.

8. The application as described in claim 7, characterized in that, The plants mentioned include barley, rice, grapes, strawberries, and cucumbers.

9. The application as described in claim 8, characterized in that, The fungal disease control agent is applied by drip inoculation onto leaves or fruits, or sprayed onto seedlings.

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