Application of phosphatidyl ethanolamine in improving white mold resistance of morchella esculenta

Abstract

The invention provides application of phosphatidyl ethanolamine in improving resistance of morchella to white mold. Experimental verification shows that exogenous addition of the phosphatidyl ethanolamine can enhance the resistance of the morchella to pseudomonas longispora, so that the resistance of the morchella to the white mold is improved, the development of the white mold is inhibited, the growth of a morchella sporocarp is promoted, and the application possibly has huge potential in the aspect of development of morchella white mold prevention and treatment products.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to the application of phosphatidylethanolamine in enhancing the resistance of Morchella to Sclerotinia sclerotiorum Background Art

[0002] Morchella is famous for its unique flavor and health-beneficial properties. The consumption of Morchella has expanded rapidly due to its various biological functions and rich nutritional value. However, the wild Morchella resources are insufficient to meet the growing commercial demand. The artificial cultivation of Morchella has attracted extensive attention worldwide due to its economic value. Currently, the growth and development of Morchella are affected by various factors, among which diseases are the main constraints on yield and quality. The pathogens causing Morchella diseases are diverse, including fungi, bacteria, and viruses. Especially fungi are the main pathogens of most Morchella diseases.

[0003] White mold disease is the most serious disease in currently cultivated Morchella, which severely restricts the healthy development of the Morchella industry. Pseudoseptoria longispora longispora is the main pathogen of Morchella white mold disease recognized in recent years. The occurrence of white mold disease will seriously reduce the yield of Morchella, damage the commercial traits, and even cause the complete necrosis of the fruiting body in severe infections. The disease has a persistent characteristic and may be infected from the mycelium development stage to the mature harvest stage, and there is even a risk of secondary infection during the post-harvest storage and transportation. The typical symptom is that the surface of the fruiting body is covered with a white flocculent mycelium layer. In a hot and humid environment where the temperature is higher than 25 °C and the air humidity exceeds 90%, the pathogen proliferates rapidly and causes a series of lesions, including tissue wilting, soft rot, morphological distortion, and structural damage to the cap, etc., ultimately leading to the complete inactivation of the fungus. Therefore, how to improve the resistance of Morchella to Pseudoseptoria longispora is one of the hot topics concerned by those skilled in the art. Summary of the Invention

[0004] The present invention provides the application of phosphatidylethanolamine in enhancing the resistance of Morchella to Pseudoseptoria longispora.

[0005] In the first aspect, the present invention provides the application of phosphatidylethanolamine, and the application is selected from at least one of A1)-A10):

[0006] A1) Application in alleviating the damage of Pseudoseptoria longispora to Morchella;

[0007] A2) Application in preparing a product for alleviating the damage of Pseudoseptoria longispora to Morchella;

[0008] A3) Application in enhancing the resistance of Morchella to Pseudoseptoria longispora;

[0009] A4) Application in preparing a product for enhancing the resistance of Morchella to Pseudoseptoria longispora;

[0010] A5) Application in enhancing the resistance of morel mushrooms to white mold;

[0011] A6) Application in the preparation of products that enhance the resistance of morel mushrooms to white mold;

[0012] A7) Application in promoting the growth of morel mushrooms;

[0013] A8) Application in the preparation of products that promote the growth of morel mushrooms;

[0014] A9) Application in the prevention and control of white mold;

[0015] Application of A10 in the preparation of products for the prevention and control of white mold.

[0016] As described above, the CAS number of the phosphatidylethanolamine is 39382-08-6.

[0017] As described above, the morel mushroom is selected from at least one of the following: morel mushrooms: morel mushrooms of the sixth rank, morel mushrooms of the seventh rank, and morel mushrooms of the tiered ridge.

[0018] The application described above includes applying a solution containing phosphatidylethanolamine to the surface of morel mushrooms, wherein the concentration of phosphatidylethanolamine is not less than 80 μg / mL.

[0019] In the applications described above, the solvent used to dissolve the phosphatidylethanolamine can be anhydrous ethanol and sterile water.

[0020] In a second aspect, the present invention provides a method for mitigating damage to morel mushrooms by *Pseudomonas longissima*, comprising applying a solution containing phosphatidylethanolamine to the surface of the morel mushrooms.

[0021] Thirdly, the present invention provides a method for enhancing the resistance of morel mushrooms to Pseudomonas longisporus, comprising applying a solution containing phosphatidylethanolamine to the surface of morel mushrooms.

[0022] Fourthly, the present invention provides a method for enhancing the resistance of morel mushrooms to white mold, comprising applying a solution containing phosphatidylethanolamine to the surface of morel mushrooms.

[0023] Fifthly, the present invention provides a method for promoting the growth of morel mushrooms, comprising applying a solution containing phosphatidylethanolamine to the surface of the morel mushrooms.

[0024] In the method described above, the morel mushroom is selected from at least one of the following: morel mushroom 'Six Sister', morel mushroom 'Seven Sister', and morel mushroom 'Tiele'.

[0025] In the method described above, the concentration of phosphatidylethanolamine in the solution is not less than 80 μg / mL.

[0026] In a sixth aspect, the present invention provides a product for mitigating damage to morel fungi caused by *Pseudomonas longissima*, said product comprising phosphatidylethanolamine.

[0027] The product described above can be a pesticide. It is understood that the product may also include other conventional active ingredients, such as nutrients that promote the growth of morel mushrooms.

[0028] This invention discovers that the exogenous addition of phosphatidylethanolamine can enhance the resistance of morel mushrooms to Pseudomonas longisporus, thereby improving their resistance to white mold, inhibiting the development of white mold, and promoting the growth of morel fruiting bodies. It may have great potential in the development of morel white mold control products. Attached Figure Description

[0029] Figure 1 The effects of Pseudomonas longissima fermentation filtrate on the hyphal morphology and hyphal vigor of Morchella morel; where A represents the effect of Pseudomonas longissima fermentation filtrate on hyphal morphology, and B represents the effect of Pseudomonas longissima fermentation filtrate on hyphal cell membrane integrity.

[0030] Figure 2 The values ​​represent the chitinase and β-1,3-glucanase activities of Morel mycelia after treatment with the pathogenic bacteria fermentation broth; where A represents chitinase activity and B represents β-1,3-glucanase activity.

[0031] Figure 3 This study analyzed the differential metabolites (DAMs) in response to P. longispora infection. A represents the KEGG pathway enrichment analysis of DAMs; B is a bar chart showing the types and quantities of differentially expressed metabolites in different samples; C is a heatmap of DAMs in significantly enriched pathways; and D is a heatmap of DAMs of different compound types. In this heatmap, all DAMs were selected using p < 0.05 and VIP > 1 as screening criteria, with red, blue, and yellow blocks representing significantly upregulated, significantly downregulated, and no significant difference, respectively.

[0032] Figure 4 The effect of exogenous addition of phosphatidylethanolamine to the culture medium on the mycelial growth of Morchella esculenta;

[0033] Figure 5 The effect of exogenous spraying of phosphatidylethanolamine solution on young mushrooms infected with white mold in the field;

[0034] Figure 6 The addition of exogenous phosphatidylethanolamine promotes cell wall remodeling and enhances the resistance of morel mushrooms to the pathogenic fungus *Alternaria alternata*. Among them, A represents the effect of exogenous PE on enhancing resistance to *Alternaria alternata* mycelium, and B represents the protective effect of PE on morel mushroom mycelium. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, embodiments of this invention, and should not be construed as limiting the invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention. In the description of this invention, it should be understood that the terminology used is for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0037] Example 1: Determination of key differential metabolites between *Morchella esculenta* and *Morchella esculenta* afflicted with white mold.

[0038] 1. Sample collection

[0039] Morel fruiting body samples exhibiting typical symptoms of white mold were collected from a morel cultivation base in Xiaobai Township, Taigu District, Jinzhong City, Shanxi Province using a sterile sampler. Each sample was uniquely numbered and individually sealed in a No. 9 self-sealing bag, with the geographical coordinates, collection date, and microenvironmental data (temperature, humidity, etc.) simultaneously labeled. All samples were transported to the laboratory under low-temperature preservation.

[0040] 2. Isolation and purification of pathogenic fungi

[0041] The collected infected morel fruiting bodies were immediately placed on a clean bench for pathogen isolation. First, the surface of the fruiting bodies was rinsed with sterile water. Then, white mycelial tissue from the boundary between the diseased and healthy areas was picked and placed on PDA solid medium. The culture conditions were set at a constant temperature of 25°C, relative humidity maintained at 70-75%, and cultured in the dark. After the initial colony formation (approximately 5-7 days), continuous purification culture was performed (3-4 generations). Newly formed mycelia (0.5 mm) from the colony edge were picked at each subculture. After microscopic identification and confirmation, the purified strain was transferred to PDA slant medium and stored at 4°C for later use. During storage, it was subcultured and activated every 180 days.

[0042] 3. Effects of pathogenic bacteria fermentation broth on morel hyphal morphology and hyphal cell membrane integrity

[0043] 3.1. Pathogenic fungal hyphae with a diameter of 6 mm were inoculated into 250 mL Erlenmeyer flasks containing 150 mL of potato dextrose broth (PDB) and cultured with shaking at 25 °C and 150 rpm / min for 6 days. The fermentation stock solution was first obtained using hyphal isolation technology, and then filtered twice using a sterile filter with a pore size of 0.22 μm to obtain a sterile fermentation broth. PDA solid medium was sterilized and cooled to approximately 55 °C. 5 mL of the above sterile fermentation broth was added to every 100 mL of PDA medium, mixed thoroughly, and poured into plates to obtain PDA plates containing 5% fermentation broth (designated as the MT group). Simultaneously, distilled water was used to prepare PDA plates instead of the fermentation broth as a control group (designated as the CK group). Activated morel mycelial blocks were inoculated in the center of the PDA plates of the MT and CK groups and cultured in the dark at 18 °C for 4 days. The colony edges were observed and photographed using a stereomicroscope.

[0044] like Figure 1 As shown in Figure A, the treatment with the fermentation broth of *Pseudomonas longispora* significantly affected the mycelial morphology of *Morchella esculenta*. In the CK group, the morel mycelia were dense, neatly arranged, and transparent, with virtually no branching. However, in the MT group, the mycelial growth rate was significantly inhibited, the colony edges were irregular, the mycelia were sparse, some were yellow, and microscopic observation revealed irregular protrusions on the mycelial surface and increased mycelial branching.

[0045] 3.2 Propidium iodide (PI) and fluorescein diacetate (FDA) have been widely used to detect cell membrane integrity. When the cell membrane is intact, PI enters the cell but is released with the help of transporters. At this time, FDA mainly binds to intracellular lipases to produce fluorescein, thus generating a green fluorescent signal. When the cell membrane integrity is compromised, PI penetrates the cell and binds to intracellular nucleic acid components (DNA and RNA), causing the fluorescence color to turn red, producing red fluorescence. Therefore, this method was used to investigate the effect of *P. longispora* fermentation broth on the cell membrane integrity of *Morchella esculenta* hyphae. Specifically, using the slide insertion method, a sterile coverslip was inserted obliquely into the culture medium at a 45-degree angle with clean forceps and cultured at 18°C ​​for 5 days. Once the hyphae had grown to two-thirds of the coverlip, the coverslip was removed, and hyphal samples were prepared for observing the cell membrane integrity of the hyphae. Mix fluorescein diacetate (FDA), propidium iodide (PI), and sterile water thoroughly to ensure a final FDA concentration of 100 μg / mL and a final PI concentration of 60 μg / mL to prepare the staining solution. Place 20 μL of the staining solution onto a glass slide, carefully remove the coverslip, and use tweezers to invert the coverslip onto the slide, gently pressing it down evenly. Incubate the sample at room temperature for 5 minutes to ensure complete staining, then observe using a laser confocal microscope. The excitation wavelength of the PI-DNA complex is 535 nm, and the emission wavelength is 615 nm. The excitation wavelength indicated by FDA is 488 nm, and the emission wavelength is 530 nm.

[0046] The results are as follows Figure 1 As shown in Figure B, compared with the CK group, the MT group showed increased red fluorescence and decreased green fluorescence, indicating that the fermentation broth would damage the cell wall of morel mycelium, leading to a significant decrease in cell membrane integrity.

[0047] 4. Determination of cell wall-related enzyme activity in *Morchella esculenta* hyphae

[0048] The structural integrity of fungal cell walls depends on the synergistic effect of two key polysaccharide components: chitin and glucan. Chitin provides structural support as a rigid framework, while glucan forms an elastic network through multi-directional cross-linking. This composite structure endows the cell wall with unique physical properties and protective functions. Chitinase, as a biocatalyst, catalyzes the hydrolysis of chitin into N-acetylglucosamine monomers. β-1,3-glucanase is a type of hydrolase that specifically acts on β-1,3-glucan molecules, capable of specifically cleaving β-1,3-glycosidic bonds. Its catalytic products can generate products of different molecular weights, ranging from oligosaccharides to monosaccharides. Transmission electron microscopy observations show that the infection process of pathogens leads to changes in the cell wall structure of morel fungi. Therefore, we conducted activity tests on two hydrolases related to the main components of the cell wall.

[0049] Specifically, healthy morel mycelia and mycelia treated with pathogenic bacterial fermentation broth were collected, rapidly preserved in liquid nitrogen, and ground using liquid nitrogen. The sample powder was then placed in pre-chilled centrifuge tubes. Protein extraction buffer was added and the tubes were incubated on ice for 20 minutes to ensure complete mycelial lysis while preserving protein activity. The tubes were centrifuged at 8000 rpm for 10 minutes at 4°C, and the supernatant was collected. The activities of chitinase and β-1,3-glucanase were measured according to the instructions of the enzyme activity assay kit.

[0050] The results are as follows Figure 2 As shown, the activities of chitinase and β-1,3-glucanase in mycelial samples treated with the pathogenic bacteria fermentation broth showed a significant upward trend. This dynamic change in enzyme activity suggests a structural adjustment in cell wall components. This physiological test result is consistent with the ultrastructural changes observed in Morel mycelia under transmission electron microscopy, jointly confirming that the fermentation broth of *Pseudomonas longissima* disrupts the cell wall of Morel mycelia.

[0051] 5. Metabolomics Sequencing Sample Preparation and Processing

[0052] Prepare PDA medium containing pathogenic bacteria fermentation broth and control PDA medium using the same method as in Part 3. Simultaneously inoculate morel mycelia onto both solid media and incubate in the dark at 18°C ​​for 9 days. Scrape mycelial samples from both groups (CK & MT) for metabolomics testing and analysis.

[0053] like Figure 3As shown, KEGG metabolic pathway enrichment analysis was performed on the identified differential metabolites. Among these DAMs, 51 KEGG pathways (p < 0.05) were significantly enriched. The pathways significantly enriched included "glycerophospholipid metabolism (map00564)," "nucleotide metabolism (map01232)," "purine metabolism (map00230)," "ascorbic acid and aldehyde metabolism (map00053)," "ABC transporters (map02010)," "tricarboxylic acid cycle (TCA cycle) (map00020)," "nicotinic acid and nicotinamide metabolism (map00760)," "pyrimidine metabolism (map00240)," and "autophagy (map04138)," with autophagy being the most significant. Based on this, it is speculated that autophagy may have occurred within the cells of *Morchella esculenta* hyphae after treatment with the fermentation filtrate of *Pseudomonas longissima*. The changes in DAMs within the significantly enriched KEGG pathways indicate a significant increase in the metabolite phosphatidylethanolamine (PE), which is involved in autophagy. Furthermore, compound classification analysis was performed on the identified DAMs, revealing changes in some DAMs across different compound types. Phospholipid DAMs were the most numerous, with PE being a type of phospholipid. Carbohydrates were the next most abundant. Monosaccharides and oligosaccharides are both carbohydrates. D-glucuronic acid is a component of cell wall polysaccharides (such as glucuronidan), for example, glucuronidan xylan in hemicellulose and ulva polysaccharide. In algae, it has a significant impact on cell wall stability and structure. The significantly reduced content of D-glucuronic acid indicates changes in the cell wall composition of Morel mycelia, consistent with TEM observations of cell wall morphology. In addition, seven fatty acid DAMs and five nucleotide and derivative DAMs were identified. Compared to the CK group, the MT group showed a significant reduction in the five fatty acids and three nucleotides and their derivatives, indicating that *Pseudomonas longissima* caused impaired primary metabolism in Morel.

[0054] Example 2: Effect of exogenous phosphatidylethanolamine treatment on the response of Morchella esculenta to disease.

[0055] This embodiment primarily investigates whether an increase in the key differential metabolite phosphatidylethanolamine (PE) can alleviate the damage of Pseudomonas longisporus to Morel mycelia.

[0056] 2.1 Preparation method of 80 μg / mL PE: Dissolve PE powder (CAS No. 39382-08-6) in anhydrous ethanol and use ultrasound to assist dissolution for 10-15 min until completely dissolved to prepare a 4 mg / mL stock solution. The working solution needs to be diluted 50 times with the stock solution before use, i.e., take 1 mL in 49 mL of sterile water; Control group: use sterile water to dilute 1 mL of anhydrous ethanol 50 times for later use.

[0057] 2.2. PE was exogenously added to PDA medium at a final concentration of 80 μg / mL. The control group was prepared using the method described in 2.1. First, a *Morchella esculenta* mycelial block was inoculated in the center of the plate and cultured in the dark at 18℃ for 3 days. The effect of phosphatidylethanolamine on the mycelial growth of *Morchella esculenta* was observed. The results are shown in […]. Figure 4 As shown in the first column on the left, adding a certain concentration of PE exogenously to PDA medium can promote the growth rate of morel mycelia.

[0058] 2.3. *Pseudomonas longissima* and *Morchella esculenta* mycelial blocks were inoculated on both sides of a PDA plate, respectively. The plates were incubated in the dark at 18°C ​​for 3 days, and mycelial growth was observed. Results are shown in […]. Figure 4 The middle column; PDA medium was simultaneously supplemented with 5% *Pseudomonas longissima* fermentation broth and PE, with 5% *Pseudomonas longissima* fermentation broth used as a control. Morel mycelial blocks were inoculated in the center of the plates and incubated in the dark at 18°C ​​for 4 days. Mycelial growth was observed, and the results are shown in the table below. Figure 4 The first column on the right shows that the addition of PE can enhance the resistance of *Morchella esculenta* mycelium to *Pseudomonas longissima*.

[0059] 2.4 To investigate whether exogenous phosphatidylethanolamine could alleviate disease incidence in the field, young mushrooms infected with *Pseudomonas longissima* were used as the research subject. They were externally sprayed with a PE solution at a concentration of 80 μg / mL, and the disease incidence was observed and recorded after 5 days of continued growth. The results are as follows: Figure 5 As shown, the fruiting body cap can continue to grow, and the white mycelium at the infection site of tip white mold is reduced. These phenomena further indicate that PE not only promotes the growth of morel mycelium on PDA plates, but also enhances the resistance of morel mycelium to white mold during fruiting body growth.

[0060] 2.5 Observation of the pathogen's effect on morel hyphae using transmission electron microscopy

[0061] Using the same method as in 2.1 and 2.2, phosphatidylethanolamine (PE) solutions were adjusted to concentrations of 1 mg / mL, 2 mg / mL, and 3 mg / mL, and the growth of morel mycelia was observed. The results are as follows: Figure 6 As shown in A, according to Figure 6 As shown in Figure A, the exogenous addition of PE significantly promotes the growth of morel mycelia. When the mycelia are affected by the fermentation broth of the white mold pathogen, the morel mycelia stop growing, but the addition of exogenous PE allows the mycelia to continue growing under the influence of the fermentation broth.

[0062] Sample (1-2mm) 3The samples were fixed in 2.5% glutaraldehyde at 4°C. The glutaraldehyde solution was rinsed three times with PBS for 15 min each time. The samples were fixed with 1% osmium tetroxide for 1-2 h, then rinsed three times with PBS. The samples were dehydrated with ethanol solution and finally treated with pure acetone for 20 min. The samples were treated with pure embedding medium overnight, and then embedded at 70°C overnight to obtain embedded samples. Sample sections were obtained at 70-90 nm using an ultramicrotome, stained with lead citrate solution and uranyl acetate in 50% ethanol saturated solution for 5-10 min, dried, and observed by transmission electron microscopy (TEM). The results are as follows: Figure 6 As shown in Figure B, it can be seen that under the treatment of pathogenic bacteria fermentation broth, the addition of PE can reshape the cell wall of morel mushrooms and maintain the structure and morphology of the cell wall of morel mycelium.

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of phosphatidylethanolamine, characterized in that, The application is selected from at least one of A1)-A10): A1) Application in alleviating the damage of Pseudomonas longissima to morels; A2) Application in the preparation of products that alleviate the damage of Pseudomonas longissima to Morel mushrooms; A3) Application in enhancing the resistance of morel mushrooms to Pseudomonas longisporus; A4) Application in the preparation of products that enhance the resistance of morel mushrooms to Pseudomonas longisporus; A5) Application in enhancing the resistance of morel mushrooms to white mold; A6) Application in the preparation of products that enhance the resistance of morel mushrooms to white mold; A7) Application in promoting the growth of morel mushrooms; A8) Application in the preparation of products that promote the growth of morel mushrooms; A9) Application in the prevention and control of white mold; Application of A10 in the preparation of products for the prevention and control of white mold.

2. The application according to claim 1, characterized in that, The morel mushrooms are selected from at least one of the following: morel mushrooms of the sixth rank, morel mushrooms of the seventh rank, and morel mushrooms of the tiered ridge.

3. The application according to claim 1, characterized in that, The application includes applying a solution containing phosphatidylethanolamine to the surface of morel mushrooms, wherein the concentration of phosphatidylethanolamine is not less than 80 μg / mL.

4. A method for mitigating the damage caused by *Pseudomonas longissima* to *Morchella*, characterized in that, This includes applying a solution containing phosphatidylethanolamine to the surface of morel mushrooms.

5. A method for enhancing the resistance of morel mushrooms to *Pseudomonas longissima*, characterized in that, This includes applying a solution containing phosphatidylethanolamine to the surface of morel mushrooms.

6. A method for enhancing the resistance of morel mushrooms to white mold, characterized in that, This includes applying a solution containing phosphatidylethanolamine to the surface of morel mushrooms.

7. A method for promoting the growth of morel mushrooms, characterized in that, This includes applying a solution containing phosphatidylethanolamine to the surface of morel mushrooms.

8. The method according to any one of claims 4-7, characterized in that, The morel mushrooms are selected from at least one of the following: morel mushrooms of the sixth rank, morel mushrooms of the seventh rank, and morel mushrooms of the tiered ridge.

9. The method according to any one of claims 4-7, characterized in that, In a solution containing phosphatidylethanolamine, the concentration of phosphatidylethanolamine is not less than 80 μg / mL.

10. A product for alleviating the damage caused by *Pseudomonas longissima* to *Morchella*, characterized in that, The product includes phosphatidylethanolamine.

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