Application of ceramide in the control of rice blast fungus

Abstract

The application of ceramide in the control of rice blast fungus falls within the field of biological control. This invention relates to the application of ceramide in the control of rice blast fungus: a combination of ceramide and lipopeptides is used for the control of rice blast fungus. Exogenous addition of ceramide significantly enhances the stress effect of lipopeptides on rice blast fungus. It also enhances the abnormal accumulation of glycogen and lipid droplets in conidia, promotes the bursting of ROS, and increases intracellular ceramide and calcium... 2+ The invention increases the content of malondialdehyde (MDA) in mycelium, enhances membrane lipid peroxidation, and decreases ergosterol content. Furthermore, it inhibits the expression of ergosterol synthesis genes, UPR target genes, and sphingolipid metabolism-related genes. This invention develops a highly efficient natural agent against rice blast by combining exogenous Cer with lipopeptides. This agent effectively reduces the pathogenicity of rice blast fungus and significantly inhibits its infection of rice, laying the foundation for the development of novel biological control agents.

Description

Technical Field

[0001] This invention relates to the field of biological control, specifically to the application of ceramide in the control of rice blast fungus. Background Technology

[0002] Rice blast disease, caused by Magnaporthe oryzae, severely impacts rice yields, resulting in global yield losses of up to 6% in normal years and exceeding 30% in severe cases, posing a serious threat to global food security. Therefore, the control of rice blast has always been a topic of great global concern. Although integrated management strategies based on the use of resistant varieties, strengthened cultivation management, and appropriate use of chemical control have been implemented, pathogen mutations often lead to the loss of resistance in resistant rice varieties. The direct or indirect potential harm of synthetic chemical agents to the ecological environment and human health drives the development of new, highly efficient, and environmentally friendly biological control agents to ensure the safe control of rice production from source to table.

[0003] Lipopeptides are microbial secondary metabolites composed of a hydrophilic polypeptide and a hydrophobic long-chain carboxylic acid. They mainly belong to three families: surfactins, iturins, and fungycins. They possess good potential as biocontrol agents against plant fungal diseases, but their role in this process remains unclear, and research on their combined use with other agents is extremely limited.

[0004] Sphingolipids are a class of amphipathic lipids containing a sphingosine backbone. They are structurally complex, functionally diverse, and widely distributed in the membranes of eukaryotic cells and some bacteria, typically accounting for about 15% of cellular lipids. Sphingolipids are important regulators of cell signal transduction and homeostasis, participating in various biological processes such as polar growth, energy supply, proliferation, and differentiation. Current research largely focuses on inhibiting ceramide production in fungal cells to control disease occurrence. For example, ceramide synthase (CerS) is a crucial regulator of fungal pathogenicity; CerS inhibitors can deplete complex sphingolipid pools and lead to the accumulation of toxic intermediates. Inositol phosphorylated ceramide (IPC) is an acidic glycosphingolipid essential for fungal growth; inhibiting its synthesis leads to fungal cell death. Aureobasidin A (AbA) was the first developed inhibitor of IPC synthase. AbA binds to the hydrophobic site of IPC synthase, thereby inhibiting enzyme activity and reducing IPC synthesis. It has a strong inhibitory effect on fungi such as Candida albicans, Cryptococcus neoformans, and Ustilago maydis. Treatment with Myriocin, a specific inhibitor of serine palmitoyltransferase (SPT), inhibits ceramide production, causing abnormal appressorium development and impaired penetrating nail formation in rice blast fungus, thus reducing pathogenicity. However, no studies have been found on the control effects of exogenous application of sphingolipids such as ceramides on plant fungal diseases. Summary of the Invention

[0005] This invention provides an application of ceramide (Cer) in the control of rice blast fungus, which exerts a strong inhibitory effect on the growth of rice blast fungus by exogenous application of ceramide.

[0006] The present invention relates to the application of ceramide in the control of rice blast fungus: a combination of ceramide and lipopeptide is used for the control of rice blast fungus.

[0007] Furthermore, ceramides and lipopeptides are dissolved in methanol to prepare a spray solution for use.

[0008] Furthermore, the concentration of lipopeptides in the spray solution is the half-maximal effective concentration against rice blast fungus.

[0009] Furthermore, the concentration of ceramide in the spray solution is 1.25 mM to 5 mM.

[0010] Furthermore, ceramides include glucosylceramide and galactosylceramide; lipopeptides include surfactants, iturins, and fengycins.

[0011] Beneficial effects of this invention:

[0012] Exogenous addition of ceramides significantly enhanced the stress effect of lipopeptides on rice blast fungus. It increased the abnormal accumulation of glycogen and lipid droplets in conidia, promoted the bursting of ROS, and increased intracellular ceramide and calcium levels. 2+ The invention increases the content of malondialdehyde (MDA) in mycelium, enhances membrane lipid peroxidation, and decreases ergosterol content. It also inhibits the expression of ergosterol synthesis genes, UPR target genes (OST1, SCJ1, PPS1, WBP1, HAC1, ERV29, SEC61, PDI1, KAR2, HRD1, PMT2, PMT4, PMT1), and sphingolipid metabolism-related genes. This invention develops a highly effective natural agent against rice blast by combining exogenous Cer with lipopeptides. This agent effectively reduces the pathogenicity of rice blast fungus and significantly inhibits its infection of rice, laying the foundation for the development of novel biological control agents. Attached Figure Description

[0013] Figure 1 This is a diagram showing the effects of lipopeptides and exogenous addition of ceramide and cAMP on conidial germination and appressorium formation of rice blast fungus in Example 1.

[0014] Figure 2 This is a graph showing the effect of 6 hours of culture treatment on the malondialdehyde content of mycelium in each treatment group in Example 2.

[0015] Figure 3 This is a graph showing the effects of lipopeptides, exogenously added ceramides, and serine on glycogen transfer in conidia of rice blast fungus in each treatment group in Example 3.

[0016] Figure 4 This is a graph showing the effects of lipopeptides and exogenous addition of ceramide and serine on lipid droplet transfer in conidia of rice blast fungus in each treatment group in Example 4.

[0017] Figure 5 This is a graph showing the effects of lipopeptides and exogenous addition of ceramide and serine on ROS formation of conidia of rice blast fungus in each treatment group in Example 5.

[0018] Figure 6 This is a graph showing the effect of lipopeptides and exogenous addition of ceramide and serine on the formation of ROS in rice blast fungus mycelium in each treatment group in Example 5.

[0019] Figure 7 This is a graph showing the effect of lipopeptides and exogenous addition of ceramide and serine on the formation of ROS in rice blast fungus mycelium in each treatment group in Example 5.

[0020] Figure 8 This is a graph showing the effects of lipopeptides and exogenous addition of ceramide and serine on calcium ion levels in the mycelium of rice blast fungus in each treatment group in Example 6.

[0021] Figure 9 This is a graph showing the effect of lipopeptides and exogenously added ceramides on the ergosterol content of rice blast fungus mycelium in each treatment group in Example 7.

[0022] Figure 10 This is a graph showing the effects of lipopeptides and exogenous ceramide addition on the expression of ergosterol-related genes in the mycelium of rice blast fungus in Example 8.

[0023] Figure 11 This is a graph from Example 9 showing the effect of qPCR analysis of different concentrations of lipopeptide treatment for 96 h on the UPR target gene of rice blast fungus mycelium;

[0024] Figure 12 This is a diagram illustrating the effects of lipopeptides and exogenous Cer treatment on the expression of mycelial UPR-related genes in Example 9; where... Figure 12 (A) 3 h; (B) 6 h;

[0025] Figure 13 This is a diagram illustrating the effects of lipopeptides and exogenous Cer treatment on the expression of mycelial sphingolipid metabolism-related genes in Example 10; where... Figure 13 (A) 3 h; (B) 6 h. Detailed Implementation

[0026] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. 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.

[0027] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0028] Explanation:

[0029] Gao's No. 1 medium: 20 g soluble starch, 1 g KNO3, 0.5 g NaCl, 0.5 g K2HPO4, 0.5 g MgSO4, 0.01 g FeSO4, diluted to 1 L with distilled water, pH adjusted to 7.2-7.4, and autoclaved at 121 ℃ for 15 min. Mainly used for the activation of *S. bikiniensis* HD-087 and the cultivation of seed culture.

[0030] (2) DBY fermentation medium: 20 g glucose, 5 g soybean powder, 4 g yeast powder, 5 g (NH4)2SO4, 1 g NaCl, 0.05 g K2HPO4, diluted to 1 L with distilled water, pH set to natural, autoclaved at 121 ℃ for 15 min. Mainly used for the fermentation culture of S. bikiniensis HD-087.

[0031] (3) CM medium: 10 g glucose, 1 g yeast extract, 2 g peptone, 1 g casein acid hydrolysate, 6 g NaNO3, 1.52 g KH2PO4, 0.52 g KCl, 0.52 g MgSO4·7H2O, 1 mL vitamin solution, 1 mL trace elements, diluted to 1 L with distilled water, pH adjusted to 6.5, and autoclaved at 121 ℃ for 15 min. Mainly used for sporulation culture of rice blast fungus.

[0032] Vitamin solution: Biotin 0.01 g, Vitamin B 0.01 g, Thiamine 0.01 g, Riboflavin 0.01 g, Para-aminobenzoic acid 0.01 g, Nicotinic acid 0.01 g, distilled water to a final volume of 100 mL.

[0033] The "lipopeptide" mentioned in the specific embodiments of this invention is a crude lipopeptide extract. The crude lipopeptide extract was obtained using Streptomyces bikiniensis HD-087; LC-MS analysis showed that it contained 2 surfactants, 3 iturins, and 2 fengycins.

[0034] The "ceramide" mentioned in the specific embodiments of this invention is Ceramide, which was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0035] Unless otherwise specified, the testing materials and reagents used in the specific implementation methods described in this manual are all products that can be obtained through conventional sales channels.

[0036] Unless otherwise specified in the specific implementation methods described in this manual, all testing methods follow the conventional testing procedures.

[0037] In the specific implementation method described in this manual, all experiments were repeated three times. Relevant data are presented as the mean and standard deviation of three independent samples. The significance of differences was determined using a t-test, with P < 0.05 indicating a significant difference and P < 0.01 indicating a highly significant difference. Data statistical analysis and graphical analysis were performed using Origin 2022, IBM SPSS Statistics 26, and TBtools software.

[0038] Example 1

[0039] In this embodiment, the rice blast fungus conidial suspensions were configured into the following treatment groups:

[0040] CK—Blank group: Rice blast fungus conidia suspension with added distilled water;

[0041] EC 50 —Control group EC 50 Lipopeptides were added to a suspension of rice blast fungus conidia, with the lipopeptide concentration in the suspension being the half-maximal effective concentration (MCC) against rice blast fungus. 50 =6.24 μg / mL;

[0042] EC 50 +1.25 mM Cer——Experimental group EC 50 +1.25 mM Cer (ceramide): Lipopeptides and ceramides were added to the conidial suspension of rice blast fungus. The lipopeptide concentration in the suspension was the half-maximal effective concentration (MCC) against rice blast fungus. 50 =6.24 μg / mL; the concentration of ceramide in the suspension was 1.25 mM;

[0043] EC 50 +2.5 mM Cer——Experimental group EC 50 +2.5 mM Cer (ceramide): Lipopeptides and ceramides were added to the conidial suspension of rice blast fungus. The lipopeptide concentration in the suspension was the half-maximal effective concentration (MCC) against rice blast fungus. 50 =6.24 μg / mL; the concentration of ceramide in the suspension was 2.5 mM;

[0044] EC 50 +5 mM Cer——Experimental group EC 50 +5 mM Cer (ceramide): Lipopeptides and ceramides were added to the conidial suspension of rice blast fungus. The lipopeptide concentration in the suspension was the half-maximal effective concentration (MCC) against rice blast fungus. 50 =6.24 μg / mL; the concentration of ceramide in the suspension was 5 mM;

[0045] EC 50 +1.25 mM cAMP — Experimental group EC 50 +1.25 mM cAMP (cyclic adenosine monophosphate): Lipopeptides and cAMP were added to a suspension of rice blast fungus conidia. The lipopeptide concentration in the suspension was the half-maximal effective concentration (MCP) against rice blast fungus. 50 =6.24 μg / mL; the concentration of cAMP in the suspension was 1.25 mM;

[0046] EC 50 +2.5 mM cAMP — Experimental group EC50 +2.5 mM cAMP (cyclic adenosine monophosphate): Lipopeptides and cAMP were added to a suspension of rice blast fungus conidia. The lipopeptide concentration in the suspension was the half-maximal effective concentration (MCP) against rice blast fungus. 50 =6.24 μg / mL; the concentration of cAMP in the suspension was 2.5 mM;

[0047] EC 50 +5 mM cAMP — Experimental group EC 50 +5 mM cAMP (cyclic adenosine monophosphate): Lipopeptides and cAMP were added to the conidial suspension of rice blast fungus. The lipopeptide concentration in the suspension was the half-maximal effective concentration (MCP) against rice blast fungus. 50 =6.24 μg / mL; the concentration of cAMP in the suspension was 5 mM;

[0048] In each of the above treatment groups, the number of conidia of rice blast fungus in the suspension was 10. 6 per mL.

[0049] Mix the above-mentioned CK and EC evenly. 50 EC 50 +1.25 mM Cer, EC 50 +2.5 mM Cer, EC 50 +5 mM Cer, EC 50 +1.25 mM cAMP, EC 50 +2.5 mM cAMP, EC 50 Eight suspensions of +5 mM cAMP were dropped onto hydrophobic glass slides, 40 μL per drop, and incubated at 28°C under humidity. After 6 h of incubation, spore germination and appressorium formation were observed under an optical microscope. Figure 1 As shown.

[0050] Figure 1 In (A), almost all the spores of rice blast fungus germinated and formed appressoria. The appressoria were dark in color and had clear cell walls, indicating that the formation of the infection structure was normal and the melanin accumulation in the appressoria was successful. Figure 1 In (B), only a small number of conidia were able to form appressoria, and the appressoria were slightly lighter in color than those in (A), indicating that melanin synthesis was inhibited by lipopeptides, resulting in insufficient turgor pressure within the appressoria to reach the level required for infection.

[0051] Joining EC 50 Adding Cer along with a high concentration of lipopeptides did not improve conidial germination and appressorium formation; in fact, it enhanced the inhibitory effect. The toxicity of Cer to rice blast fungus spores exhibited a dose-dependent effect. Figure 1In (C) no normal appressoria formed; only small, light-colored appressoria formed, or only the end of the germ tube swelled slightly. In (D) no appressoria were observed; only a small number of spores germinated and produced germ tubes. In (E) neither appressoria nor germ tubes were formed; spore germination was completely inhibited, and the volume of the spore vacuoles was significantly reduced, indicating a dead state. This suggests that lipopeptides can induce apoptosis of rice blast fungus by inducing Cerebrolysin (Cer) production. Furthermore, exogenous Cerebrolysin can be combined with lipopeptides to develop highly effective natural agents against rice blast.

[0052] Joining EC 50 When cAMP was added along with lipopeptides, the results were the opposite of those when Cer was added. cAMP showed a certain ability to restore the growth and development of rice blast fungus conidia under lipopeptide stress. Figure 1 (F) Conidial germination rate compared to EC 50 The group showed improvement; the appressorium formation rate in (G) was significantly higher than that in EC. 50 In group (H), conidial germination and appressorium formation were essentially restored to the levels of group CK, but the appressorium color was very light, indicating that no melanin was formed. This suggests that cAMP can attenuate the toxicity of lipopeptides in a dose-dependent manner, demonstrating that lipopeptides can inhibit the growth of rice blast fungus by reducing the concentration of the second messenger cAMP in cells. It also demonstrates that 5 mM cAMP cannot reverse the melanin reduction caused by lipopeptides, and melanin is an essential factor for the formation of appressorium turgor pressure. Therefore, another target of lipopeptides is to inhibit melanin synthesis and reduce the pathogenicity of rice blast fungus.

[0053] A method for extracting crude lipopeptides using *Streptomyces bikiniensis* HD-087: One loop of activated *S. bikiniensis* HD-087 was picked from Gao's No. 1 slant agar and inoculated into 50 mL of Gao's No. 1 liquid medium. The mixture was incubated at 28 ℃ with shaking at 180 r / min for 36 h to obtain the seed culture of *S. bikiniensis* HD-087. 2 mL of the *S. bikiniensis* HD-087 seed culture was inoculated into an Erlenmeyer flask containing 60 mL of DBY liquid medium and cultured with shaking at the same parameters (28 ℃, 180 r / min) for 96 h to obtain the fermentation broth. The broth was centrifuged at 10,000 r / min for 20 min, the precipitate was removed, and the supernatant was collected. The supernatant was concentrated under reduced pressure by rotary evaporation at 50 ℃, and the pH was adjusted to 2.0 with HCl. A large amount of flocculent material precipitated out. The supernatant was then placed in a refrigerator at 4 ℃ overnight. Centrifuge at 10,000 r / min for 20 min, collect the precipitate, add 1 / 4 volume of methanol (equivalent to 1 / 4 volume of the total fermentation supernatant), and stir thoroughly with a magnetic stirrer for at least 2 h to ensure complete dissolution (incubate overnight at 4 ℃). Centrifuge at 10,000 r / min for 20 min, discard the precipitate, and collect the supernatant (methanol extract). Adjust the pH to 7.0 with NaOH. Remove the methanol by rotary evaporation at 50 ℃ to obtain the lipopeptide.

[0054] The microorganisms used in this embodiment are: Streptomyces bikiniensis HD-087 (Optimization of antibacterial substance production conditions and preliminary study on antibacterial effect of cucumber wilt biocontrol bacterium HD-087), provided by the Key Laboratory of Microbiology of Heilongjiang Province, Heilongjiang University; and Magnaphalthe oryzae, the blast fungus of rice, provided by the Key Laboratory of Microbiology of Heilongjiang Province, Heilongjiang University.

[0055] Example 2

[0056] In this embodiment, 5 mL of fresh rice blast fungus mycelial suspension was added to 50 mL / 250 mL CM liquid culture medium to prepare mycelial solution. The rice blast fungus mycelial solution was set up in the following treatment groups:

[0057] CK – Control group: Rice blast fungus mycelium solution with added distilled water;

[0058] EC 50 —Control group EC 50 Lipopeptides were added to the mycelial solution of *Oryza sativa*, with the lipopeptide concentration in the mycelial solution reaching the half-maximal effective concentration (MCC) against *Oryza sativa*. 50 =8.41 μg / mL;

[0059] EC50 +1.25 mM Cer——Experimental group EC 50 +1.25 mM Cer: Lipopeptides and ceramides were added to the mycelial solution of *Oryza sativa* blast fungus. The lipopeptide concentration in the mycelial solution was the half-maximal effective concentration (MCC) against *Oryza sativa* blast fungus. 50 =8.41 μg / mL; the concentration of ceramide in the mycelial solution was 1.25 mM;

[0060] EC 50 +2.5 mM Cer——Experimental group EC 50 +2.5 mM Cer: Lipopeptides and ceramides were added to the mycelial solution of *Oryza sativa* blast fungus. The lipopeptide concentration in the mycelial solution was the half-maximal effective concentration (MCC) against *Oryza sativa* blast fungus. 50 =8.41 μg / mL; the concentration of ceramide in the mycelial solution was 2.5 mM;

[0061] EC 50 +5 mM Cer——Experimental group EC 50 +5 mM Cer: Lipopeptides and ceramides are added to the mycelial solution of *Oryza sativa* blast fungus. The lipopeptide concentration in the mycelial solution is the half-maximal effective concentration (MCC) against *Oryza sativa* blast fungus. 50 =8.41 μg / mL; the concentration of ceramide in the mycelial solution was 5 mM;

[0062] The mycelial concentration in the mycelial solution was the same in all the above treatment groups. After culturing the mycelial solutions of each treatment group for 6 h at 28℃ and 180 r / min, centrifuging at 7500 r / min for 5 min, discarding the supernatant and collecting the mycelial precipitate, the MDA (malondialdehyde) content in the rice blast fungus mycelium was determined. The determination method is as follows: 1 mL of 10... 5 Rice blast fungus conidia suspension (number of spores / mL) was inoculated into 50 mL / 250 mL CM liquid medium, and lipopeptides were added to a final concentration of EC50, with an equal volume of sterile water as a control. The mixture was cultured at 28 ℃ with shaking at 180 r / min. Mycelia of rice blast fungus were collected at 0 h, 6 h, 12 h, 24 h, 48 h, and 96 h, washed three times with sterile water, and 0.5 g of mycelia were added to 1 mL of extraction solution. The mixture was homogenized in an ice bath and centrifuged at 12000 r / min for 10 min at 4 ℃. The supernatant was collected, and the MDA content in the mycelia was determined using a malondialdehyde (MDA) content test kit.

[0063] MDA content measurement results are as follows: Figure 2 As shown, EC after 6 h of culture 50 There was no significant difference in malondialdehyde (MDA) content in the mycelium within the groups, indicating that short-term action of lipopeptides does not cause significant peroxidation of membrane lipids. (The addition of EC...) 50When Cer was added concurrently with lipopeptides, the malondialdehyde (MDA) content increased significantly in a dose-dependent manner. In the experimental group with 5 mM Cer, the MDA content reached 6.46 nmol / g, which was 3.28 times that of the CK group and significantly higher than that of the control group EC. 50 It is 2.74 times that of the previous generation. Therefore, it is speculated that exogenous Cer can insert into the membrane structure and change the composition of lipid rafts, causing a serious change in membrane fluidity. The PUFAs (polyunsaturated fatty acids) in the lipid bilayer are exposed to the oxidative environment and are more susceptible to ROS (reactive oxygen species) attack, resulting in peroxidation.

[0064] Example 3

[0065] In this embodiment, the rice blast fungus conidial suspensions were configured into the following treatment groups:

[0066] CK—Blank group: Rice blast fungus conidia suspension with added distilled water;

[0067] EC 50 —Control group EC 50 Lipopeptides were added to a suspension of rice blast fungus conidia, with the lipopeptide concentration in the suspension being the half-maximal effective concentration (MCC) against rice blast fungus. 50 =6.24 μg / mL;

[0068] EC 50 +1.25 mM Cer——Experimental group EC 50 +1.25 mM Cer: Lipopeptides and ceramides were added to a suspension of rice blast fungus conidia. The lipopeptide concentration in the suspension was the half-maximal effective concentration (MCP) against rice blast fungus. 50 =6.24 μg / mL; the concentration of ceramide in the suspension was 1.25 mM;

[0069] EC 50 +2.5 mM Cer——Experimental group EC 50 +2.5 mM Cer: Lipopeptides and ceramides were added to a suspension of rice blast fungus conidia. The lipopeptide concentration in the suspension was the half-maximal effective concentration (MCP) against rice blast fungus. 50 =6.24 μg / mL; the concentration of ceramide in the suspension was 2.5 mM;

[0070] EC 50 +5 mM Cer——Experimental group EC 50 +5 mM Cer: Lipopeptides and ceramides were added to a suspension of rice blast fungus conidia. The lipopeptide concentration in the suspension was the half-maximal effective concentration (MCC) against rice blast fungus. 50 =6.24 μg / mL; the concentration of ceramide in the suspension was 5 mM;

[0071] EC50 +1.25 mM Ser——Experimental group EC 50 +1.25 mM Ser: Lipopeptides and Ser were added to a suspension of rice blast fungus conidia. The lipopeptide concentration in the suspension was the half-maximal effective concentration (MCC) against rice blast fungus. 50 =6.24 μg / mL; the concentration of Ser in the suspension was 1.25 mM;

[0072] EC 50 +2.5 mM Ser——Experimental group EC 50 +2.5 mM Ser: Lipopeptides and Ser were added to a suspension of rice blast fungus conidia. The lipopeptide concentration in the suspension was the half-maximal effective concentration (MCP) against rice blast fungus. 50 =6.24 μg / mL; the concentration of Ser in the suspension was 2.5 mM;

[0073] EC 50 +5 mM Ser——Experimental group EC 50 +5 mM Ser: Lipopeptides and Ser were added to a suspension of rice blast fungus conidia. The lipopeptide concentration in the suspension was the half-maximal effective concentration (MCC) against rice blast fungus. 50 =6.24 μg / mL; the concentration of Ser in the suspension was 5 mM;

[0074] In each of the above treatment groups, the number of conidia of rice blast fungus in the suspension was 10. 6 per mL.

[0075] Mix the above-mentioned CK and EC evenly. 50 EC 50 +1.25 mM Cer, EC 50 +2.5 mM Cer, EC 50 +5 mM Cer, EC 50 +1.25 mM Ser, EC 50 +2.5 mM Ser, EC 50 Eight suspensions of +5 mM Ser were dropped onto hydrophobic glass slides, 40 μL per drop, and incubated at 28°C under humidification. After 3 h and 6 h of incubation, the liquid on the slides was carefully aspirated with a pipette, and the conidia were found attached to the slide surface. After staining with 60 mg / mL KI + 10 mg / mL I2 solution for 3 min, the slides were observed under an optical microscope.

[0076] The results of staining the glycogen of the spores of each experimental group are as follows: Figure 3 As shown. At 3 hours, Figure 3(A) In the conidia, no germ tubes or appressoria have formed. The glycogen in the conidia is light in color, scarce, and evenly dispersed throughout the conidia, indicating that glycogen has not yet been mobilized in large quantities at this time; at 6 h, Figure 3 (I) In the formation of germ tubes and appressoria by conidia, the color of glycogen deepens, and evenly distributed glycogen can also be observed in the appressoria. This proves that glycogen can be transferred from the germ tube to the appressoria, and the remaining glycogen is mainly concentrated in the conidial septum or single cells. The larger appressoria are darker red, indicating that the appressoria enlarges as the amount of glycogen transferred increases.

[0077] like Figure 3 As shown in (B) and (J), in EC 50 After treatment with concentrated lipopeptides, glycogen was mainly concentrated near the conidial septum, and the glycogen in the appressorium was lighter in color, indicating reduced glycogen transfer. This may be one reason why lipopeptides inhibit the formation of germ tubes and appressoriums. Glycogen is a key energy source for conidial germination. After lipopeptide treatment, glycogen was retained in the three cells of the conidium and could not be successfully transferred to the germ tube. This leads to a lack of energy for subsequent appressorium formation, hindering the formation and maturation of appressoriums, thereby inhibiting the infection of rice by rice blast fungus.

[0078] Joining EC 50 Different concentrations of Cerebrolysin (Cer) and Ser (Ser) were added simultaneously with the lipopeptide, such as... Figure 3 As shown in (M) and (P), the red color deepened upon the addition of 5 mM Cer or Ser, indicating that glycogen in the conidia was not transferred and utilized. Furthermore, with increasing Cer concentration, the ratio of germ tubes to appressoria formed by conidia decreased compared to EC. 50 The number of groups decreased further. Relatively speaking, the addition of Ser could restore the formation of germ tubes and appressorium to some extent, but the appressorium was smaller and the glycogen staining was lighter, indicating that Ser could not completely restore the inhibitory effect caused by lipopeptides.

[0079] Example 4

[0080] In this embodiment, the rice blast fungus conidial suspensions were configured into the following treatment groups:

[0081] CK—Blank group: Rice blast fungus conidia suspension with added distilled water;

[0082] EC 50 —Control group EC 50 Lipopeptides were added to a suspension of rice blast fungus conidia, with the lipopeptide concentration in the suspension being the half-maximal effective concentration (MCC) against rice blast fungus. 50 =6.24 μg / mL;

[0083] EC 50 +1.25 mM Cer——Experimental group EC 50+1.25 mM Cer: Lipopeptides and ceramides were added to a suspension of rice blast fungus conidia. The lipopeptide concentration in the suspension was the half-maximal effective concentration (MCP) against rice blast fungus. 50 =6.24 μg / mL; the concentration of ceramide in the suspension was 1.25 mM;

[0084] EC 50 +2.5 mM Cer——Experimental group EC 50 +2.5 mM Cer: Lipopeptides and ceramides were added to a suspension of rice blast fungus conidia. The lipopeptide concentration in the suspension was the half-maximal effective concentration (MCP) against rice blast fungus. 50 =6.24 μg / mL; the concentration of ceramide in the suspension was 2.5 mM;

[0085] EC 50 +5 mM Cer——Experimental group EC 50 +5 mM Cer: Lipopeptides and ceramides were added to a suspension of rice blast fungus conidia. The lipopeptide concentration in the suspension was the half-maximal effective concentration (MCC) against rice blast fungus. 50 =6.24 μg / mL; the concentration of ceramide in the suspension was 5 mM;

[0086] EC 50 +1.25 mM Ser——Experimental group EC 50 +1.25 mM Ser: Lipopeptides and Ser were added to a suspension of rice blast fungus conidia. The lipopeptide concentration in the suspension was the half-maximal effective concentration (MCC) against rice blast fungus. 50 =6.24 μg / mL; the concentration of Ser in the suspension was 1.25 mM;

[0087] EC 50 +2.5 mM Ser——Experimental group EC 50 +2.5 mM Ser: Lipopeptides and Ser were added to a suspension of rice blast fungus conidia. The lipopeptide concentration in the suspension was the half-maximal effective concentration (MCP) against rice blast fungus. 50 =6.24 μg / mL; the concentration of Ser in the suspension was 2.5 mM;

[0088] EC 50 +5 mM Ser——Experimental group EC 50 +5 mM Ser: Lipopeptides and Ser were added to a suspension of rice blast fungus conidia. The lipopeptide concentration in the suspension was the half-maximal effective concentration (MCC) against rice blast fungus. 50 =6.24 μg / mL; the concentration of Ser in the suspension was 5 mM;

[0089] In each of the above treatment groups, the number of conidia of rice blast fungus in the suspension was 10. 6per mL.

[0090] Mix the above-mentioned CK and EC evenly. 50 EC 50 +1.25 mM Cer, EC 50 +2.5 mM Cer, EC 50 +5 mM Cer, EC 50 +1.25 mM Ser, EC 50 +2.5 mM Ser, EC 50 Eight suspensions of +5 mM Ser were dropped onto hydrophobic glass slides, 40 μL each, and incubated at 28°C under humidification. After 6 h of incubation, the slides were stained with 250 μg / mL Nile Red in the dark for 3 min, followed by gentle washing away the dye with sterile water. 40 μL of sterile water was added again, and the fluorescence was observed using a confocal microscope with radiofrequency fluorescence (RFP).

[0091] Lipid droplets are rich in lipids such as triglycerides (TG), serving as an energy storage structure besides glycogen. They provide sufficient turgor pressure for attaching cells, providing energy for the formation of infection spikes and penetration of the host cell wall. In this example, lipid droplet staining is as follows... Figure 4 As shown. From Figure 4 In (A), it can be observed that the fluorescence of lipid droplets in the CK group protospores is almost invisible at 6 h, indicating that they have been completely transferred into the appressorium and the fluorescence intensity is low. This suggests that the lipid droplet transport mechanism and appressorium development are normal and that the lipid droplets have been partially consumed.

[0092] Figure 4 The fluorescence of conidia in group (B) was stronger than that in group (A), and they mainly remained at the conidial septum. Stronger fluorescence was also observed in the appressorium than in group CK. The retention of lipid droplets inside the conidia indicates that their decomposition or utilization was hindered. This suggests that lipopeptide treatment inhibited the transfer and utilization of lipid droplets.

[0093] No fluorescence was observed in the appressorium after adding different concentrations of Cer and Ser along with the lipopeptide. Figure 4 In (D), (E), and (H), strong fluorescence is concentrated near the conidial septum or evenly dispersed throughout the conidia, while only a very small amount of fluorescence is observed in the germ tube near the conidia. This indicates that lipid droplet transfer is hindered, suggesting that both Cer and Ser can exacerbate lipid droplet retention within the conidia. Higher concentrations of Cer and Ser have a more pronounced inhibitory effect on lipid droplet transfer, possibly by disrupting lipid metabolism homeostasis, thereby inhibiting lipid droplet transfer and utilization.

[0094] The results of lipopeptide inhibition of glycogen transfer in Example 3 indicate that lipopeptides simultaneously target the pathways of both glycogen and lipid droplets, two types of energy storage substances, to disrupt the energy homeostasis of *Strombus haemolyticus*. Furthermore, the ability of Cer and Ser to inhibit lipid droplet transfer and utilization can enhance their potential as active ingredients in the development of antifungal compound natural agents. In addition, lipid droplet retention may also lead to the accumulation of free fatty acids, which, through acetyl-CoA feedback, inhibit glycogen phosphorylase, thereby inhibiting glycogen breakdown and disrupting the homeostasis of intracellular energy flow.

[0095] Example 5

[0096] In this embodiment, the rice blast fungus conidial suspensions were configured into the following treatment groups:

[0097] CK—Blank group: Rice blast fungus conidia suspension with added distilled water;

[0098] EC 50 —Control group EC 50 Lipopeptides were added to a suspension of rice blast fungus conidia, with the lipopeptide concentration in the suspension being the half-maximal effective concentration (MCC) against rice blast fungus. 50 =6.24 μg / mL;

[0099] EC 50 +1.25 mM Cer——Experimental group EC 50 +1.25 mM Cer: Lipopeptides and ceramides were added to a suspension of rice blast fungus conidia. The lipopeptide concentration in the suspension was the half-maximal effective concentration (MCP) against rice blast fungus. 50 =6.24 μg / mL; the concentration of ceramide in the suspension was 1.25 mM;

[0100] EC 50 +2.5 mM Cer——Experimental group EC 50 +2.5 mM Cer: Lipopeptides and ceramides were added to a suspension of rice blast fungus conidia. The lipopeptide concentration in the suspension was the half-maximal effective concentration (MCP) against rice blast fungus. 50 =6.24 μg / mL; the concentration of ceramide in the suspension was 2.5 mM;

[0101] EC 50 +5 mM Cer——Experimental group EC 50 +5 mM Cer: Lipopeptides and ceramides were added to a suspension of rice blast fungus conidia. The lipopeptide concentration in the suspension was the half-maximal effective concentration (MCC) against rice blast fungus. 50 =6.24 μg / mL; the concentration of ceramide in the suspension was 5 mM;

[0102] EC 50 +1.25 mM Ser——Experimental group EC 50+1.25 mM Ser: Lipopeptides and Ser were added to a suspension of rice blast fungus conidia. The lipopeptide concentration in the suspension was the half-maximal effective concentration (MCC) against rice blast fungus. 50 =6.24 μg / mL; the concentration of Ser in the suspension was 1.25 mM;

[0103] EC 50 +2.5 mM Ser——Experimental group EC 50 +2.5 mM Ser: Lipopeptides and Ser were added to a suspension of rice blast fungus conidia. The lipopeptide concentration in the suspension was the half-maximal effective concentration (MCP) against rice blast fungus. 50 =6.24 μg / mL; the concentration of Ser in the suspension was 2.5 mM;

[0104] EC 50 +5 mM Ser——Experimental group EC 50 +5 mM Ser: Lipopeptides and Ser were added to a suspension of rice blast fungus conidia. The lipopeptide concentration in the suspension was the half-maximal effective concentration (MCC) against rice blast fungus. 50 =6.24 μg / mL; the concentration of Ser in the suspension was 5 mM;

[0105] In each of the above treatment groups, the number of conidia of rice blast fungus in the suspension was 10. 6 per mL.

[0106] Mix the above-mentioned CK and EC evenly. 50 EC 50 +1.25 mM Cer, EC 50 +2.5 mM Cer, EC 50 +5 mM Cer, EC 50 +1.25 mM Ser, EC 50 +2.5 mM Ser, EC 50 Eight suspensions of +5 mM Ser were dropped onto hydrophobic glass slides, 40 μL each, and incubated at 28°C under humidification. After 6 h of incubation, the slides were stained with a 10 μmol / L DCFH-DA fluorescent probe, incubated at 37 °C for 20 min, and then the DCFH-DA dye was gently washed off with sterile water. Fluorescence was observed under a confocal microscope using an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0107] The results of ROS staining of rice blast fungus conidia are as follows: Figure 5 .from Figure 5As observed in (A), the conidia in the CK group showed no obvious fluorescence, and their growth was normal. During normal conidial germination, the conidia maintained redox homeostasis, producing extremely low levels of ROS, which is beneficial for maintaining cell integrity and the normal development of infection structures. Figure 5 (B) A small amount of green fluorescence was observed in conidia and appressoria, indicating that lipopeptide stress led to ROS accumulation. When different concentrations of Cer and Ser were added simultaneously with the lipopeptide, the fluorescence intensity in conidia increased in a dose-dependent manner, with fluorescence mainly concentrated on the conidial septum. The fluorescence intensity in appressoria was similar to or slightly lower than that in the EC50 group. 50 The results indicate that Cer and Ser primarily lead to the accumulation and explosive growth of ROS in conidia, suggesting this is the main early reason for their inhibition of conidial pathogenicity development. Cer may exacerbate overload in the mitochondrial electron transport chain by activating the mitochondrial apoptosis pathway, strongly inhibiting germ tube and appressorium formation, reducing aerobic metabolism within appressorium, downregulating the expression of antioxidant enzymes, and weakening the cell's ability to scavenge ROS, thus leading to the explosive accumulation of ROS in conidia. Ser, as a one-carbon unit donor, can promote glutathione synthesis and enhance antioxidant capacity at low concentrations, possibly partially offsetting lipopeptide-induced ROS; while high concentrations of Ser may indirectly promote ROS generation by competitively inhibiting the metabolism of other amino acids, increasing metabolic stress.

[0108] In this embodiment, 5 mL of fresh rice blast fungus mycelial suspension was added to 50 mL / 250 mL CM liquid culture medium to prepare mycelial solution. The rice blast fungus mycelial solution was set up in the following treatment groups:

[0109] CK – Control group: Rice blast fungus mycelium solution with added distilled water;

[0110] EC 50 —Control group EC 50 Lipopeptides were added to the mycelial solution of *Oryza sativa*, with the lipopeptide concentration in the mycelial solution reaching the half-maximal effective concentration (MCC) against *Oryza sativa*. 50 =8.41 μg / mL;

[0111] EC 50 +1.25 mM Cer——Experimental group EC 50 +1.25 mM Cer: Lipopeptides and ceramides were added to the mycelial solution of *Oryza sativa* blast fungus. The lipopeptide concentration in the mycelial solution was the half-maximal effective concentration (MCC) against *Oryza sativa* blast fungus. 50 =8.41 μg / mL; the concentration of ceramide in the mycelial solution was 1.25 mM;

[0112] EC 50 +2.5 mM Cer——Experimental group EC50 +2.5 mM Cer: Lipopeptides and ceramides were added to the mycelial solution of *Oryza sativa* blast fungus. The lipopeptide concentration in the mycelial solution was the half-maximal effective concentration (MCC) against *Oryza sativa* blast fungus. 50 =8.41 μg / mL; the concentration of ceramide in the mycelial solution was 2.5 mM;

[0113] EC 50 +5 mM Cer——Experimental group EC 50 +5 mM Cer: Lipopeptides and ceramides are added to the mycelial solution of *Oryza sativa* blast fungus. The lipopeptide concentration in the mycelial solution is the half-maximal effective concentration (MCC) against *Oryza sativa* blast fungus. 50 =8.41 μg / mL; the concentration of ceramide in the mycelial solution was 5 mM;

[0114] EC 50 +1.25 mM Ser——Experimental group EC 50 +1.25 mM Cer: Lipopeptides and ceramides were added to the mycelial solution of *Oryza sativa* blast fungus. The lipopeptide concentration in the mycelial solution was the half-maximal effective concentration (MCC) against *Oryza sativa* blast fungus. 50 =8.41 μg / mL; the concentration of ceramide in the mycelial solution was 1.25 mM;

[0115] EC 50 +2.5 mM Ser——Experimental group EC 50 +2.5 mM Cer: Lipopeptides and ceramides were added to the mycelial solution of *Oryza sativa* blast fungus. The lipopeptide concentration in the mycelial solution was the half-maximal effective concentration (MCC) against *Oryza sativa* blast fungus. 50 =8.41 μg / mL; the concentration of ceramide in the mycelial solution was 2.5 mM;

[0116] EC 50 +5 mM Ser——Experimental group EC 50 +5 mM Cer: Lipopeptides and ceramides are added to the mycelial solution of *Oryza sativa* blast fungus. The lipopeptide concentration in the mycelial solution is the half-maximal effective concentration (MCC) against *Oryza sativa* blast fungus. 50 =8.41 μg / mL; the concentration of ceramide in the mycelial solution was 5 mM;

[0117] The mycelial concentration in the mycelial solution was the same in all the above treatment groups. The mycelial solutions in each of the above treatment groups were cultured at 28°C and 180 r / min. After 3 h and 6 h of culture, the mycelial solutions were stained with a DCFH-DA fluorescent probe, and the fluorescence was observed under a confocal microscope.

[0118] The ROS staining results of the mycelium in this embodiment are as follows: Figure 6 and Figure 7As shown in the figure. No obvious fluorescence was observed in the CK group, indicating that the mycelium grew normally and maintained a good redox balance. EC 50 The control group showed significant green fluorescence, indicating that lipopeptide-triggered ROS accumulation in mycelia was the early cause of its inhibition of mycelial growth. When different concentrations of Cer and Ser were added simultaneously with the lipopeptide, the fluorescence intensity in the mycelia increased in a dose-dependent manner at 3 h, leading to a ROS burst. At 6 h, the addition of 2.5 mM Cer... Figure 7 (D) The fluorescence burst was more significant, while the addition of 5 mM Cer... Figure 7 (E) The fluorescence intensity decreased, possibly due to the death of some bacteria. At 6 h, the group supplemented with 5 mM Ser... Figure 7 (H) Fluorescence intensity is lower than that of the sample containing 2.5 mM Ser. Figure 7 (G) This may be because the buffering effect of Ser metabolism slows down ROS synthesis. ROS can induce apoptosis by activating the caspase pathway, thereby inhibiting the infection of rice blast fungus.

[0119] Example 6

[0120] In this embodiment, 5 mL of fresh rice blast fungus mycelial suspension was added to 50 mL / 250 mL CM liquid culture medium to prepare mycelial solution. The rice blast fungus mycelial solution was set up in the following treatment groups:

[0121] CK – Control group: Rice blast fungus mycelium solution with added distilled water;

[0122] EC 50 —Control group EC 50 Lipopeptides were added to the mycelial solution of *Oryza sativa*, with the lipopeptide concentration in the mycelial solution reaching the half-maximal effective concentration (MCC) against *Oryza sativa*. 50 =8.41 μg / mL;

[0123] EC 50 +1.25 mM Cer——Experimental group EC 50 +1.25 mM Cer: Lipopeptides and ceramides were added to the mycelial solution of *Oryza sativa* blast fungus. The lipopeptide concentration in the mycelial solution was the half-maximal effective concentration (MCC) against *Oryza sativa* blast fungus. 50 =8.41 μg / mL; the concentration of ceramide in the mycelial solution was 1.25 mM;

[0124] EC 50 +2.5 mM Cer——Experimental group EC 50 +2.5 mM Cer: Lipopeptides and ceramides were added to the mycelial solution of *Oryza sativa* blast fungus. The lipopeptide concentration in the mycelial solution was the half-maximal effective concentration (MCC) against *Oryza sativa* blast fungus. 50=8.41 μg / mL; the concentration of ceramide in the mycelial solution was 2.5 mM;

[0125] EC 50 +5 mM Cer——Experimental group EC 50 +5 mM Cer: Lipopeptides and ceramides are added to the mycelial solution of *Oryza sativa* blast fungus. The lipopeptide concentration in the mycelial solution is the half-maximal effective concentration (MCC) against *Oryza sativa* blast fungus. 50 =8.41 μg / mL; the concentration of ceramide in the mycelial solution was 5 mM;

[0126] EC 50 +1.25 mM Ser——Experimental group EC 50 +1.25 mM Cer: Lipopeptides and ceramides were added to the mycelial solution of *Oryza sativa* blast fungus. The lipopeptide concentration in the mycelial solution was the half-maximal effective concentration (MCC) against *Oryza sativa* blast fungus. 50 =8.41 μg / mL; the concentration of ceramide in the mycelial solution was 1.25 mM;

[0127] EC 50 +2.5 mM Ser——Experimental group EC 50 +2.5 mM Cer: Lipopeptides and ceramides were added to the mycelial solution of *Oryza sativa* blast fungus. The lipopeptide concentration in the mycelial solution was the half-maximal effective concentration (MCC) against *Oryza sativa* blast fungus. 50 =8.41 μg / mL; the concentration of ceramide in the mycelial solution was 2.5 mM;

[0128] EC 50 +5 mM Ser——Experimental group EC 50 +5 mM Cer: Lipopeptides and ceramides are added to the mycelial solution of *Oryza sativa* blast fungus. The lipopeptide concentration in the mycelial solution is the half-maximal effective concentration (MCC) against *Oryza sativa* blast fungus. 50 =8.41 μg / mL; the concentration of ceramide in the mycelial solution was 5 mM;

[0129] The mycelial concentration in the mycelial solution was the same in all the above treatment groups. The mycelial solutions in each treatment group were cultured at 28°C and 180 r / min. After culturing for 3 h and 6 h respectively, the mycelial precipitate was washed three times with sterile water, centrifuged, and the changes in calcium ion levels in each sample were then determined using a calcium content colorimetric detection kit.

[0130] Mitochondrial Ca 2+ Overload is an early cause of cell apoptosis. From Figure 8 It can be seen that EC 50 Ca in the experimental group 2+The content was significantly higher than that in the control group. When Cer and Ser were added along with lipopeptides, it was found that the Ca2+ content in the 1.25 mM Cer and Ser groups was significantly higher than that in the control group. 2+ The levels were significantly reduced. The 2.5 mM and 5 mM Cer groups showed significantly lower levels of Ca. 2+ The level increased significantly at 3 h, and the 2.5 mM Cere group reached the highest value, indicating that Ca... 2+ Overload is a key early cause of apoptosis induced by Cer and Ser. At 6 h, Ca2+ in both groups... 2+ The levels all rapidly decreased to their lowest values. This may be due to apoptosis caused by prolonged treatment, Ca... 2+ Leakage. Ca in the 2.5 mM Ser group. 2+ The content is slightly lower than EC. 50 The experimental group showed that Ser's metabolic buffering capacity was insufficient to fully restore calcium homeostasis, and the ER remained in a state of mild stress. Meanwhile, the 5 mM Ser group showed a slightly lower EC at 3 h. 50 The experimental group, but significantly higher than EC at 6 h. 50 The experimental group demonstrates that high concentrations of Ser lead to Ca... 2+ Overload can also induce apoptosis.

[0131] Example 7

[0132] In this embodiment, 5 mL of fresh rice blast fungus mycelial suspension was added to 50 mL / 250 mL CM liquid culture medium to prepare mycelial solution. The rice blast fungus mycelial solution was set up in the following treatment groups:

[0133] CK – Control group: Rice blast fungus mycelium solution with added distilled water;

[0134] EC 50 —Control group EC 50 Lipopeptides were added to the mycelial solution of *Oryza sativa*, with the lipopeptide concentration in the mycelial solution reaching the half-maximal effective concentration (MCC) against *Oryza sativa*. 50 =8.41 μg / mL;

[0135] EC 50 +1.25 mM Cer——Experimental group EC 50 +1.25 mM Cer: Lipopeptides and ceramides were added to the mycelial solution of *Oryza sativa* blast fungus. The lipopeptide concentration in the mycelial solution was the half-maximal effective concentration (MCC) against *Oryza sativa* blast fungus. 50 =8.41 μg / mL; the concentration of ceramide in the mycelial solution was 1.25 mM;

[0136] EC 50 +2.5 mM Cer——Experimental group EC 50+2.5 mM Cer: Lipopeptides and ceramides were added to the mycelial solution of *Oryza sativa* blast fungus. The lipopeptide concentration in the mycelial solution was the half-maximal effective concentration (MCC) against *Oryza sativa* blast fungus. 50 =8.41 μg / mL; the concentration of ceramide in the mycelial solution was 2.5 mM;

[0137] EC 50 +5 mM Cer——Experimental group EC 50 +5 mM Cer: Lipopeptides and ceramides are added to the mycelial solution of *Oryza sativa* blast fungus. The lipopeptide concentration in the mycelial solution is the half-maximal effective concentration (MCC) against *Oryza sativa* blast fungus. 50 =8.41 μg / mL; the concentration of ceramide in the mycelial solution was 5 mM;

[0138] The mycelial concentration in the mycelial solution was the same in all the above treatment groups. After incubating the mycelial solution at 28 ℃ and 180 r / min for 6 h, the mycelial solution in each treatment group was centrifuged at 7500 r / min for 5 min. The supernatant was discarded, and the mycelial precipitate was collected. The ergosterol content in the mycelium of *Strombus haematocephala* was determined using the following method: Mycelia were filtered at 0 h, 6 h, 12 h, 24 h, 48 h, and 96 h, washed three times with sterile water, and then dried. 0.5 g of dried mycelia were weighed, flash-frozen in liquid nitrogen, ground into powder, and extracted with 10 mL of anhydrous ethanol using ultrasonic oscillation for 15 min. The precipitate was allowed to stand. After centrifugation at 4000 r / min for 15 min, 2 mL of the supernatant was filtered through a 0.22 μm microporous membrane, and the absorbance was measured at 292 nm. The ergosterol content was determined using anhydrous ethanol as a control.

[0139] The measurement results are as follows Figure 9 As shown, EC after 6 h of lipopeptide treatment 50 The ergosterol content in the mycelium of the experimental group was significantly upregulated compared to the control group. This trend was reversed after the addition of 1.25 mM Cer under lipopeptide stress, and the ergosterol content was significantly higher than that of the EC group. 50 The experimental group showed a decrease, and when the Cerebroside concentration reached 2.5 mM and 5 mM, the ergosterol content further decreased, falling below the ergosterol level in the control group. This indicates that lipopeptides can activate the ergosterol synthesis pathway in *Blastomyces oryzae*, while Cerebroside inhibits ergosterol synthesis. This suggests that the mechanisms by which Cerebroside and lipopeptides inhibit mycelial growth and development differ, although the initial causes are similar, such as ROS production. The addition of Cerebroside competes with ergosterol for lipid raft regions, inhibiting ergosterol synthesis, ultimately leading to membrane structural remodeling and apoptosis.

[0140] Example 8

[0141] In this embodiment, 5 mL of fresh rice blast fungus mycelial suspension was added to 50 mL / 250 mL CM liquid culture medium to prepare mycelial solution. The rice blast fungus mycelial solution was set up in the following treatment groups:

[0142] CK – Control group: Rice blast fungus mycelium solution with added distilled water;

[0143] EC 50 —Control group EC 50 Lipopeptides were added to the mycelial solution of *Oryza sativa*, with the lipopeptide concentration in the mycelial solution reaching the half-maximal effective concentration (MCC) against *Oryza sativa*. 50 =8.41 μg / mL;

[0144] EC 50 +1.25 mM Cer——Experimental group EC 50 +1.25 mM Cer: Lipopeptides and ceramides were added to the mycelial solution of *Oryza sativa* blast fungus. The lipopeptide concentration in the mycelial solution was the half-maximal effective concentration (MCC) against *Oryza sativa* blast fungus. 50 =8.41 μg / mL; the concentration of ceramide in the mycelial solution was 1.25 mM;

[0145] EC 50 +2.5 mM Cer——Experimental group EC 50 +2.5 mM Cer: Lipopeptides and ceramides were added to the mycelial solution of *Oryza sativa* blast fungus. The lipopeptide concentration in the mycelial solution was the half-maximal effective concentration (MCC) against *Oryza sativa* blast fungus. 50 =8.41 μg / mL; the concentration of ceramide in the mycelial solution was 2.5 mM;

[0146] EC 50 +5 mM Cer——Experimental group EC 50 +5 mM Cer: Lipopeptides and ceramides are added to the mycelial solution of *Oryza sativa* blast fungus. The lipopeptide concentration in the mycelial solution is the half-maximal effective concentration (MCC) against *Oryza sativa* blast fungus. 50 =8.41 μg / mL; the concentration of ceramide in the mycelial solution was 5 mM;

[0147] The mycelial concentration in the mycelial solution was the same in all the above treatment groups. The mycelial solutions in each treatment group were cultured at 28 ℃ and 180 r / min for 3 h and 6 h, respectively. Total RNA was extracted, cDNA was prepared, and the relative expression levels of ergosterol synthesis-related genes (ERG2, ERG3, ERG4, ERG5, HMG1, IDI) were measured and analyzed. The primer sequences used are shown in Table 1 below.

[0148] Table 1

[0149]

[0150] The qPCR results of this embodiment ( Figure 10 This indicates that EC 50 In the experimental group, the expression of several ergosterol synthesis-related genes was significantly upregulated, with ERG2 showing the largest upregulation, reaching 12.42-fold and 2.51-fold higher than the CK group at 3 h and 6 h, respectively. At 6 h, the expression of ERG5, HMG1, IDI, and ERG2 was upregulated, while ERG3 was significantly downregulated. The downregulation of some genes at 3 h may be due to the acute feedback inhibition of ergosterol synthesis by Cer. Both Cer and ergosterol are important components of the cell membrane; an increase in one leads to the inhibition of the other. Exogenous Cer inserts into the cell membrane, disrupting lipid raft homeostasis and interfering with sterol synthesis. At 6 h, the upregulated HMG1 and IDI are located upstream of the ergosterol synthesis pathway; the downregulated ERG2, ERG3, and ERG4 are located downstream. Their inhibition leads to the accumulation of a large amount of intermediate products. The upregulation of ERG5, as the final step in synthesis, may compensate for the accumulation of intermediate products caused by the inhibition of ERG3.

[0151] Example 9

[0152] In this embodiment, 5 mL of fresh rice blast fungus mycelial suspension was added to 50 mL / 250 mL CM liquid culture medium to prepare mycelial solution. The rice blast fungus mycelial solution was set up in the following treatment groups:

[0153] CK – Control group: Rice blast fungus mycelium solution with added distilled water;

[0154] EC 50 —Control group EC 50 Lipopeptides were added to the mycelial solution of *Oryza sativa*, with the lipopeptide concentration in the mycelial solution reaching the half-maximal effective concentration (MCC) against *Oryza sativa*. 50 =8.41 μg / mL;

[0155] EC 50 +1.25 mM Cer——Experimental group EC 50 +1.25 mM Cer: Lipopeptides and ceramides were added to the mycelial solution of *Oryza sativa* blast fungus. The lipopeptide concentration in the mycelial solution was the half-maximal effective concentration (MCC) against *Oryza sativa* blast fungus. 50 =8.41 μg / mL; the concentration of ceramide in the mycelial solution was 1.25 mM;

[0156] EC 50 +2.5 mM Cer——Experimental group EC 50 +2.5 mM Cer: Lipopeptides and ceramides were added to the mycelial solution of *Oryza sativa* blast fungus. The lipopeptide concentration in the mycelial solution was the half-maximal effective concentration (MCC) against *Oryza sativa* blast fungus. 50=8.41 μg / mL; the concentration of ceramide in the mycelial solution was 2.5 mM;

[0157] EC 50 +5 mM Cer——Experimental group EC 50 +5 mM Cer: Lipopeptides and ceramides are added to the mycelial solution of *Oryza sativa* blast fungus. The lipopeptide concentration in the mycelial solution is the half-maximal effective concentration (MCC) against *Oryza sativa* blast fungus. 50 =8.41 μg / mL; the concentration of ceramide in the mycelial solution was 5 mM;

[0158] The mycelial concentration in the mycelial solution was the same in all the above treatment groups. 1 mL of a 10... 5 Rice blast fungus conidia suspension (number of spores / mL) were inoculated into 50 mL / 250 mL CM liquid medium, and different concentrations of [unspecified ingredient] were added. Lipopeptides were cultured at 28 ℃ with shaking at 180 r / min for 4 days, with an equal volume of sterile water as a control. Mycelia were collected, and total RNA was extracted from the mycelial liquid of each treatment group, cDNA was prepared, and the relative expression levels of UPR target genes (ERG2, ERG3, ERG4, ERG5, HMG1, IDI) were determined and analyzed. The primer sequences used are shown in Table 2 below.

[0159] Table 2

[0160]

[0161] Lipopeptides significantly affect the expression of UPR target genes in rice blast fungus mycelium (e.g., Figure 11 Cluster analysis revealed that after treatment with different concentrations of lipopeptides, the expression of OST1, SCJ1, and PPS1 was significantly downregulated, while the expression of HAC1, ERV29, SEC61, PDI1, KAR2, HRD1, PMT2, PMT4, and PMT1 was significantly upregulated. The expression of WBP1 fluctuated without a clear up- or down-regulation trend. These genes are all regulators of the UPR response pathway, and their instability indicates that lipopeptides disrupt the homeostasis of the rice blast fungus's ER.

[0162] SCJ1 (Suppressor of CbpJ1) is a molecular chaperone of the Hsp40 / DnaJ family in the endoepithelial process (ER), working synergistically with Hsp70 (such as KAR2 / BiP) to assist in the proper folding or targeted degradation of unfolded proteins. It may participate in quality control by recognizing exposed hydrophobic domains. Downregulation of SCJ1 gene expression following lipopeptide treatment has a negative effect on the proper folding or degradation of unfolded proteins, potentially leading to the accumulation of unfolded proteins and inducing an unstable state in the ER.

[0163] PPS1 belongs to the serine / threonine phosphatase family and may regulate signaling pathways in the unfolded protein response (UPR) through dephosphorylation, such as reversing kinase activity to maintain signal homeostasis. Downregulation of PPS1 gene expression after lipopeptide action may disrupt this homeostasis, exacerbating the accumulation of unfolded proteins.

[0164] In yeast, HAC1 is a core transcription factor in the unfolded protein response (UPR). During ER stress, IRE1 kinase splices HAC1 mRNA to generate the active protein Hac1p, which in turn activates the expression of UPR-related genes (such as KAR2 and PDI1), enhancing ER folding capacity. Upregulation of HAC1 gene expression following lipopeptide action leads to increased protein folding frequency in the ER, exacerbating the accumulation of unfolded proteins. KAR2, a member of the Hsp70 family in the ER, assists in the folding of unfolded proteins or their targeting to the ERAD pathway by binding to the hydrophobic regions of unfolded proteins. KAR2 expression is regulated by HAC1 and fluctuates with HAC1 gene expression.

[0165] PDI1 catalyzes the formation and isomerization of disulfide bonds, ensuring correct protein folding. Furthermore, PDI1 may act as a molecular chaperone, directly binding to unfolded proteins to prevent aggregation. ERV29 participates in vesicle transport from the ER to the Golgi apparatus, potentially retaining unfolded or misfolded proteins in the ER for refolding or degradation via ERAD (ER-associated degradation). HRD1 is a core component of the E3 ubiquitin ligase complex on the ER membrane, responsible for recognizing and ubiquitinizing misfolded proteins, guiding their degradation via the proteasome (ERAD pathway). The upregulation of these three genes is likely more of a remedial measure. Lipopeptide stress causes protein misfolding in the ER, while upregulating PDI1 reduces the presence of unfolded proteins, and upregulating ERV29 accelerates the transfer of misfolded proteins to the Golgi apparatus for degradation, thus alleviating lipopeptide stress.

[0166] SEC61, as a protein transport channel on the ER membrane, is responsible for the co-translational transport of nascent polypeptide chains into the ER lumen. During the accumulation of unfolded proteins, SEC61 may participate in the retrograde transport of misfolded proteins into the cytoplasm for ubiquitination and degradation. Compared to PDI1, ERV29, and HRD1, the upregulation of the SEC61 gene is both a cause and an effect; under lipopeptide stress, it provides a large amount of raw materials for unfolded proteins and can also promote the transport of unfolded proteins out of the ER.

[0167] The PMT family, including PMT1, PMT2, and PMT4, encodes protein O-mannosyltransferases, which catalyze O-mannosylation of proteins in the endoplasmic reticulum (ER). Glycosylation affects protein stability, folding, and quality control; PMT deficiency leads to the accumulation of unfolded proteins and activation of the endoplasmic reticulum (UPR). WBP1, a key subunit of the oligosaccharide transferase complex, performs N-glycosylation in the ER. Glycosylation modification is crucial for protein folding and ER quality control. Upregulation of these four glycosylation-related genes (PMT1, PMT2, PMT4, and WBP1) may have caused disordered glycoprotein encoding in *Blastoma oryzae*, resulting in misfolding of proteins that should have been modified into normal glycoproteins.

[0168] In each of the above treatment groups, the mycelial broth was cultured at 28 ℃ and 180 r / min for 3 h and 6 h, respectively, for total RNA extraction, cDNA preparation, and determination of the relative expression levels of UPR-related genes.

[0169] Changes in UPR target gene expression after Cer addition, such as Figure 12 As shown, when Cer was added along with lipopeptides, the expression of UPR-related genes showed a significant downregulation trend with increasing Cer concentration and treatment time. At the addition of 5 mM Cer, the expression of almost all UPR-related genes showed a significant downregulation.

[0170] After inserting into the ER membrane, Cer increases membrane rigidity and promotes lipid raft formation, which may hinder the oligomerization of UPR sensors. As an apoptosis signaling molecule, Cer may also directly inhibit UPR transcription factors, blocking the repair capacity of ER stress, leading to ER dysfunction and cell death.

[0171] Example 10

[0172] In this embodiment, 5 mL of fresh rice blast fungus mycelial suspension was added to 50 mL / 250 mL CM liquid culture medium to prepare mycelial solution. The rice blast fungus mycelial solution was set up in the following treatment groups:

[0173] CK – Control group: Rice blast fungus mycelium solution with added distilled water;

[0174] EC 50 —Control group EC 50 Lipopeptides were added to the mycelial solution of *Oryza sativa*, with the lipopeptide concentration in the mycelial solution reaching the half-maximal effective concentration (MCC) against *Oryza sativa*. 50 =8.41 μg / mL;

[0175] EC 50 +1.25 mM Cer——Experimental group EC50 +1.25 mM Cer: Lipopeptides and ceramides were added to the mycelial solution of *Oryza sativa* blast fungus. The lipopeptide concentration in the mycelial solution was the half-maximal effective concentration (MCC) against *Oryza sativa* blast fungus. 50 =8.41 μg / mL; the concentration of ceramide in the mycelial solution was 1.25 mM;

[0176] EC 50 +2.5 mM Cer——Experimental group EC 50 +2.5 mM Cer: Lipopeptides and ceramides were added to the mycelial solution of *Oryza sativa* blast fungus. The lipopeptide concentration in the mycelial solution was the half-maximal effective concentration (MCC) against *Oryza sativa* blast fungus. 50 =8.41 μg / mL; the concentration of ceramide in the mycelial solution was 2.5 mM;

[0177] EC 50 +5 mM Cer——Experimental group EC 50 +5 mM Cer: Lipopeptides and ceramides are added to the mycelial solution of *Oryza sativa* blast fungus. The lipopeptide concentration in the mycelial solution is the half-maximal effective concentration (MCC) against *Oryza sativa* blast fungus. 50 =8.41 μg / mL; the concentration of ceramide in the mycelial solution was 5 mM;

[0178] In each of the above treatment groups, the mycelial broth was cultured at 28 ℃ and 180 r / min for 3 h and 6 h, respectively. Total RNA was extracted, cDNA was prepared, and the relative expression levels of genes related to the sphingolipid metabolism pathway (CGT1, TSC10, SUR2, LAG1, LCB1, LCB2, LAC1, MGG_11535) were measured and analyzed.

[0179] The measurement results in this embodiment are as follows: Figure 13 Seven genes related to sphingolipid metabolism in EC 50 The experimental groups all showed significant upregulation. In EC 50 In the +1.25 mM Cer group, the expression of each gene showed slight fluctuations compared to the CK group, but was significantly lower than that in the EC group. 50 Expression levels in the experimental group. Low concentrations of Cer were sufficient to trigger negative feedback inhibition, significantly interfering with sphingolipid metabolism. Meanwhile, in EC... 50 +2.5 mMCer and EC 50In the +5 mM Cer group, this interference was more pronounced, resulting in gene expression levels at 3 h being 21% lower than in the CK group, and at 6 h being 80% lower than in the CK group. This indicates that high concentrations of exogenous Cer can directly shut down the sphingolipid synthesis pathway by inhibiting the activity of the SPT complex, leading to the obstruction of energy-dependent transcription. At 6 h, cells may have attempted to restart sphingolipid metabolism to cope with the continuous stress, showing a slight increase in expression levels compared to 3 h, but still experiencing severe inhibition and unable to reverse the overall cell death trend.

Claims

1. The application of ceramide in the control of rice blast fungus, characterized in that, A combination of ceramides and lipopeptides is used for the control of rice blast fungus.

2. The application of ceramide in the control of rice blast fungus according to claim 1, characterized in that, Ceramides and lipopeptides are dissolved in methanol to make a spray solution for use.

3. The application of ceramide in the control of rice blast fungus according to claim 2, characterized in that, The concentration of lipopeptides in the spray solution is the half-maximal effective concentration against rice blast fungus.

4. The application of ceramide in the control of rice blast fungus according to claim 3, characterized in that, The concentration of ceramide in the spray solution is 1.25 mM to 5 mM.

5. The application of ceramide in the control of rice blast fungus according to claim 1, 2, 3 or 4, characterized in that, Ceramides include glucosylceramide and galactosylceramide; lipopeptides include surfactants, itursin, and fenestratorin.

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