Use of a natural metabolite, D-erythro-dihydrosphingosine, for the control of plant pathogenic fungi

Abstract

This invention discloses the application of the natural metabolite D-erythrosine in the control of plant pathogenic fungi, with an application concentration of 10-50 μM. This invention can effectively inhibit plant pathogenic fungi, reduce the use of chemical pesticides, decrease the development of pesticide resistance, and protect the ecological environment.

Description

Technical Field

[0001] This invention relates to the field of plant disease control technology, and in particular to the application of a natural metabolite, D-erythro-dihydrosphingosine, in the control of plant pathogenic fungi. Background Technology

[0002] Plant pathogenic fungi such as rice blast fungus ( Magnaporthe oryzae ), wheat scab ( Fusarium graminearum ), gray mold ( Botrytis cinerea These fungal diseases, such as those caused by various pathogens, seriously threaten global crop yields and quality. While traditional chemical pesticides are effective in controlling these fungal diseases, they also pose problems such as environmental pollution, damage to non-target organisms, and increased pesticide resistance in pathogens. Therefore, finding green, environmentally friendly, and highly efficient natural products to replace chemical pesticides has become a research hotspot.

[0003] D-erythro-dihydrosphingosine, an important sphingosine base, plays a crucial role in biochemistry and medical research. Its chemical name is 2-amino-1,3-octadecanediol, CAS registry number 764-22-7, and its molecular weight is approximately 301.5 g / mol. D-erythro-dihydrosphingosine is a white to off-white solid powder at room temperature, with a melting point of approximately 70-72℃. It needs to be stored at -20℃ in a sealed, moisture-proof environment. Its lipid-soluble nature means that it is mainly found in cell membrane structures, especially in sphingolipid-rich membrane microdomains. This physicochemical property also determines its intracellular distribution and metabolic pathways.

[0004] D-erythro-dihydrosphingosine is primarily produced in vivo via a conserved sphingolipid synthesis pathway. This process begins in the endoplasmic reticulum, where serine palmitoyltransferase (SPT) catalyzes the condensation reaction of palmitoyl-CoA with L-serine to generate 3-keto-dihydrosphingosine. Subsequently, with the participation of NADPH, 3-keto-dihydrosphingosine reductase reduces 3-keto-dihydrosphingosine to D-erythro-dihydrosphingosine. Besides endogenous synthesis, D-erythro-dihydrosphingosine can also enter the body through dietary intake. Naturally rich sphingolipid foods include dairy products, eggs, and legumes. The complex sphingolipids in these foods are hydrolyzed in the digestive tract, releasing sphingosine bases, including dihydrosphingosine, which are then absorbed by the intestines and participate in sphingolipid metabolism.

[0005] D-Erythrosine (D-dihydrosphingosine) plays a role in regulating cell signaling and inducing apoptosis in mammals. It also shows great promise for disease treatment. Preclinical studies have demonstrated that its dihydrosphingosine-1-phosphate form significantly improves wound healing in a diabetic mouse model. In a rat model of liver ischemia-reperfusion injury, D-dihydrosphingosine-1-phosphate selectively activates the S1P1 receptor and pertussis toxin-sensitive G protein, thereby activating the ERK and Akt signaling pathways and exerting a protective effect on the liver and kidneys. However, the application of D-erythrosine in the control of plant pathogenic fungi has not been fully explored. Summary of the Invention

[0006] The purpose of this invention is to provide an application of the natural metabolite D-erythro-dihydrosphingosine in the control of plant pathogenic fungi, which can effectively inhibit plant pathogenic fungi, reduce the use of chemical pesticides, reduce the development of pesticide resistance, and protect the ecological environment.

[0007] The technical solution adopted by this invention to solve its technical problem is:

[0008] Application of a natural metabolite, D-erythro-dihydrosphingosine, as a fungicide for controlling plant pathogenic fungi.

[0009] The plant pathogenic fungi are rice blast fungus, cucumber anthracnose fungus, or gray mold fungus.

[0010] A fungicide for controlling plant pathogenic fungi, the active ingredient of which is D-erythro-dihydrosphingosine. It has the advantages of being environmentally friendly, highly effective, and widely applicable, effectively combating crop diseases and ensuring the sustainable development of agricultural production.

[0011] The concentration of D-erythro-dihydrosphingosine was 50 mM. D-erythro-dihydrosphingosine was dissolved in dimethyl sulfoxide (DMSO) to prepare a 50 mM stock solution.

[0012] A method for controlling plant pathogenic fungi, achieved by spraying D-erythro-dihydrosphingosine onto the plant surface.

[0013] The application concentration of D-erythro-dihydrosphingosine is 10-50 μM.

[0014] The beneficial effects of this invention are:

[0015] 1. Experiments have shown that 10 μM D-erythro-dihydrosphingosine can significantly inhibit the growth, spore germination, and appressorium formation of various plant pathogenic fungi, including but not limited to rice blast fungus, cucumber anthracnose fungus, and gray mold fungus.

[0016] 2. This natural product can effectively reduce the incidence and severity of diseases after application, and has no negative impact on crop growth.

[0017] 3. In addition to killing pathogenic fungi at low concentrations, this natural product can also significantly induce plant immunity, playing a dual role in the prevention and control of plant diseases.

[0018] 4. This natural metabolite does not pollute the environment and will not have adverse effects on soil, water bodies, or non-target organisms, thus meeting the requirements of green and sustainable development in modern agriculture. Attached Figure Description

[0019] Figure 1 The graph shows the effect of different concentrations of D-erythro-dihydrosphingosine on the germination rate of rice blast fungus spores.

[0020] Figure 2 This is a graph showing the effect of different concentrations of D-erythrosine on the growth of Escherichia coli.

[0021] Figure 3 This is a graph showing the effect of different concentrations of D-erythrosine on the growth of Saccharomyces cerevisiae;

[0022] Figure 4 This is a graph showing the effect of 50 μM D-erythro-dihydrosphingosine on rice seed germination.

[0023] Figure 5 This is a comparison chart showing the reduction of rice blast fungus infection in barley leaves by different concentrations of D-erythro-dihydrosphingosine.

[0024] Figure 6 This is a cryo-scanning electron microscope image showing how 50 μM D-erythro-dihydrosphingosine reduces rice blast fungus infection. Detailed Implementation

[0025] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0026] In this invention, unless otherwise specified, all raw materials and equipment used are commercially available or commonly used in the field. The methods described in the following embodiments are conventional methods in the field, unless otherwise specified.

[0027] Example 1: D-erythrosine can inhibit the growth of rice blast fungus.

[0028] D-erythrosine dihydrosphingosine (purchased from Taoshu Biotechnology, catalog number: T13632) was dissolved in DMSO to prepare a 50 mM stock solution, which was then diluted to different concentrations for use. Rice blast fungus spores cultured on CM medium were washed off with sterile water and diluted to a concentration of 5 × 10⁻⁶. 4D-erythro-dihydrosphingosine was added at concentrations of 0, 5, 10, 50, and 100 μM, respectively. 25 µL of each solution was dropped onto a glass slide and incubated at 25 °C for 24 hours. The results showed that D-erythro-dihydrosphingosine inhibited spore germination by 71% at a concentration of 5 μM and by 100% at a concentration of 10 μM. Figure 1 ).

[0029] Example 2: D-erythrosine had no significant inhibitory effect on Escherichia coli, Saccharomyces cerevisiae, and rice.

[0030] (1) D-erythro-dihydrosphingosine at concentrations below 100 μM has no significant inhibitory effect on Escherichia coli.

[0031] Escherichia coli was cultured overnight in 2 mL of LB liquid medium. 10 μL of the overnight cultured E. coli was then inoculated into LB liquid medium containing 0, 5, 10, 50, and 100 μM of D-erythro-dihydrosphingosine, respectively, to achieve an OD value of approximately 0.02. The cultures were incubated at 37°C and 200 rpm for 18 hours. Turbidity was observed, and the OD600 value was measured. The results showed that the growth of E. coli was not significantly inhibited in LB liquid medium containing 5-100 μM D-erythro-dihydrosphingosine. Figure 2 ).

[0032] (2) D-erythro-dihydrosphingosine at concentrations below 100 μM had no significant inhibitory effect on the growth of Saccharomyces cerevisiae.

[0033] Saccharomyces cerevisiae was cultured overnight in 2 mL YPDA medium. 10 μL of the overnight cultured Saccharomyces cerevisiae was then inoculated into YPDA solutions containing 0, 5, 10, 50, and 100 μM D-erythro-dihydrosphingosine, respectively, to achieve an OD value of approximately 0.02. The cultures were incubated for 18 hours at 28℃ and 200 rpm in a shaker. Turbidity was observed and the OD600 value was measured. The results showed that the growth of Saccharomyces cerevisiae was not significantly inhibited in YPDA solutions containing 5-100 μM D-erythro-dihydrosphingosine. Figure 3 ).

[0034] (3) 50 μM D-erythro-dihydrosphingosine had no significant inhibitory effect on rice seed germination.

[0035] Forty seeds of Zhonghua 11 (ZH11) rice were taken, and after removing the seed coat, they were soaked in 30% sodium hypochlorite solution for 30 min. After rinsing twice with sterile water, each sample (20 seeds per sample) was placed in 5 mL of sterile water containing 0.50 μM D-erythrosine. The seeds were then incubated at 28℃ for 5 days to observe germination. The results showed that 50 μM D-erythrosine had no significant inhibitory effect on rice seed germination. Figure 4 DHS stands for D-erythro-dihydrosphingosine.

[0036] Example 3: D-erythro-dihydrosphingosine significantly reduced the infectivity of rice blast fungus.

[0037] (1) D-erythro-dihydrosphingosine inhibits the infection of rice blast fungus on the host.

[0038] The spore concentrations of *Guy11*, the rice blast fungus containing 0, 10, 20, and 50 μM D-erythrodihydrosphingosine, were diluted to 5 × 10⁻⁶. 4 For each milliliter of barley, 25 µl was dropped onto the leaves and incubated in a moist environment for 4 days. The disease incidence on the leaves was then observed. Leaf inoculation experiments showed that leaves treated with 10 μM D-erythro-dihydrosphingosine were immune to rice blast fungus infection. Figure 5 ).

[0039] (2) D-erythro-dihydrosphingosine inhibits rice blast fungus infection.

[0040] The concentration of rice blast fungus spores containing 0.50 μM D-erythrodihydrosphingosine was diluted to 5 × 10⁻⁶. 4 ① Spray 2 mL of rice blast fungus spores containing 50 μM D-erythro-dihydrosphingosine onto rice leaves that have been growing for 2 weeks, and incubate at 25℃ for 24 hours; ② Take 2 mL of rice blast fungus spore solution (5 × 10⁻⁶) 4 Spraying (per milliliter) onto rice leaves that were 2 weeks old, and then incubating at 25°C with humidity for 24 hours, followed by the addition of 50 μM D-erythro-dihydrosphingosine, and continuing incubation for another 2 hours, revealed the presence of rice blast fungus infection under a cryo-scanning electron microscope. The leaf inoculation experiment showed that the spores of rice blast fungus treated with 50 μM D-erythro-dihydrosphingosine ruptured and failed to germinate normally. 24 hours after spore germination, the appressorium of rice blast fungus treated with 50 μM D-erythro-dihydrosphingosine completely collapsed, preventing infection of rice. Figure 6 ).

[0041] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications are possible without departing from the technical solutions described in the claims.

Claims

1. The application of a natural metabolite, D-erythrosine, as a fungicide for controlling plant pathogenic fungi, characterized in that, The plant pathogenic fungus is rice blast fungus.

2. A method for controlling plant pathogenic fungi, characterized in that, This is achieved by spraying D-erythro-dihydrosphingosine onto the plant surface, wherein the plant pathogenic fungus is rice blast fungus.

3. The method according to claim 2, characterized in that, The application concentration of D-erythro-dihydrosphingosine is 10-50 μM.

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