Application of coniferyl alcohol in inhibition of pathogenesis process of physalospora piricola of tea trees

Abstract

The invention discloses an application of coniferyl alcohol in inhibiting the pathogenesis process of tea tree physalospora piricola, and belongs to the technical field of prevention and treatment of tea tree physalospora piricola. The coniferyl alcohol standard substance and pestalotiopsis hypha blocks are co-cultured in vitro, the MIC50 value is calculated to measure the antibacterial effect, and when the coniferyl alcohol standard substance and the pestalotiopsis hypha blocks are co-cultured with anthracnose fungi, the coniferyl alcohol standard substance and the pestalotiopsis hypha blocks also have a good inhibition effect on hypha growth. Meanwhile, coniferyl alcohol can damage cells of pestalotiopsis spores and influence the pathogenicity of the pestalotiopsis spores on tea tree leaves. Compared with a traditional bacteriostatic agent, the invention provides a more effective and environment-friendly bacteriostatic agent for inhibiting plant zonate spot on the market.

Description

Technical Field

[0001] The invention belongs to the technical field of tea tree ring spot disease prevention and treatment, and particularly relates to application of coniferyl alcohol in inhibiting the occurrence of tea tree ring spot disease. Background Art

[0002] Tea tree is a perennial evergreen woody plant that grows mostly in tropical and subtropical regions, which are also conducive to the growth and reproduction of pathogens. Tea tree ring spot disease is caused by Pestalotiopsis-like fungi, which is a high temperature and high humidity disease. It is distributed in various tea-growing areas in my country and is also one of the most destructive leaf diseases to tea trees worldwide. Tea ring spot disease symptoms first appear on the edge of the leaves, then expand inward. In severe cases, it will become necrotic spots with dark brown edges, eventually causing the leaves to fall off the tea tree. Worldwide, Pestalotiopsis-like fungi can infect many plants and cause various plant diseases. For example, Pestalotiopsis (P.) apiculatus can cause dieback of Chinese fir, causing the main branches of trees to die. It is one of the main diseases that endanger plant growth and development.

[0003] Coniferyl alcohol (CA), also known as 4-hydroxy-3-methoxycinnamyl alcohol, is a natural metabolite widely found in plants. When plants are infected by pathogens, the content of coniferyl alcohol will increase significantly. It not only participates in the formation of lignin, but its derivative coniferin also has a direct inhibitory effect on pathogens. However, there are few studies on the antibacterial effect of coniferyl alcohol itself. At present, the main methods for preventing and controlling wheel spot disease in tea gardens are breeding disease-resistant varieties, strengthening field management, and spraying fungicides. However, due to the genetic complexity of tea trees, there are relatively large obstacles to breeding resistant varieties. In recent years, it has been reported that Bacillus subtilis can be used together with fungicides to enhance the efficacy of fungicides and thus reduce the use of chemical pesticides, but there is still a risk of excessive pesticide residues. Therefore, we need to invent natural antibacterial agents to prevent and control wheel spot disease.

[0004] The Chinese patent application document with publication number CN110237114A discloses the use of dehydrodisinconiferyl alcohol in the preparation of drugs against liver damage. The extraction method is as follows: the seeds of the melon are crushed and extracted with ethanol, the extract is extracted with ethyl acetate, and the extract is concentrated under reduced pressure to form an extract. The patent found through experiments that dehydrodisinconiferyl alcohol can significantly reverse the increased liver function levels such as ALT and AST in the serum of SD rats with cholestasis induced by ANIT, the decreased bile flow, and liver lesions, and has a significant protective effect against liver damage; and it mainly acts based on activating the FXR target to regulate its downstream gene expression. The patent broadens the scope of application of dehydrodisinconiferyl alcohol and ethyl acetate extract of melon seeds, and has important application value and economic benefits for the development of FXR agonist drugs or liver damage protection drugs. However, the patent does not disclose the use of coniferyl alcohol in inhibiting the pathogenesis of tea tree ring spot disease. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to provide a method for inhibiting ring spot disease by utilizing natural metabolites in plants.

[0006] First, QQQ was used to detect the content of coniferyl alcohol in tea leaves before and after infection with Pseudomonas spp., and then coniferyl alcohol standards were co-cultured with Pseudomonas spp. hyphae to calculate the MIC50 value to measure the antibacterial effect. When coniferyl alcohol was co-cultured with Colletotrichum spp. hyphae, it was also found to have a good antibacterial effect. Finally, coniferyl alcohol was co-cultured with Pseudomonas spp. spores, and the health status of the spores was detected by PI staining. The spores co-cultured with coniferyl alcohol were further infecting tea leaves to observe their pathogenicity to tea leaves.

[0007] The present invention solves the above technical problems through the following technical means:

[0008] The invention provides application of coniferyl alcohol in preventing and treating tea tree ring spot disease.

[0009] Preferably, the tea tree ring spot disease is caused by Trichosporon spp.

[0010] Preferably, the MIC50 value of the coniferyl alcohol against Polytrichomonas pseudodiscus is 0.409 mg / mL.

[0011] The invention also provides an antibacterial agent for preventing and treating tea tree ring spot disease, wherein the main active ingredient comprises coniferyl alcohol.

[0012] The beneficial effects of the present invention are:

[0013] (1) The antibacterial agent coniferyl alcohol used in the present invention is a secondary metabolite widely present in plants and has little impact on organisms and the environment.

[0014] (2) Compared with other traditional natural metabolite antibacterial agents, the antibacterial agent used in the present invention has a more significant effect in inhibiting diseases.

[0015] (3) The antibacterial agent used in the present invention can not only inhibit the fungi of the genus Polytrichomoniasis, but also has an inhibitory effect on the hyphae of the fungi of the genus Anthracnose, and has a certain broad inhibitory effect on fungi. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is a graph showing changes in the content of coniferyl alcohol in tea leaves infected by the fungus Pseudomonas spp. provided in Example 2 of the present invention;

[0017] Figure 2 It is a determination graph of the MIC50 value of coniferyl alcohol on Pseudomonas mycelium provided in Example 3 of the present invention, wherein A is a phenotype graph of the mycelium block area after co-cultivation of coniferyl alcohol with Pseudomonas mycelium blocks at different concentration gradients, B is a statistical graph of mycelium growth area under coniferyl alcohol at different concentration gradients, and C is a nonlinear fitting curve graph of the inhibition rate of mycelium growth under coniferyl alcohol at different concentration gradients.

[0018] Figure 3 3 is a graph showing the inhibitory effect of coniferyl alcohol on the hyphae of Anthracnose fungi provided in Example 3 of the present invention, wherein A is a phenotype graph of the hyphae block area after co-cultivation of 0.1 mg / ml coniferyl alcohol and the hyphae of Anthracnose fungi, and B is a statistical graph of the hyphae growth area of ​​Anthracnose fungi under 0.1 mg / ml coniferyl alcohol;

[0019] Figure 4 PI staining is provided in Example 4 of the present invention to detect the effect of coniferyl alcohol on the spore cells of Pseudomonas spp., wherein A is a fluorescence brightness diagram after co-culture of 0.1 mg / ml coniferyl alcohol and Pseudomonas spores, and B is a fluorescence intensity statistical diagram;

[0020] Figure 5 It is a pathogenicity analysis diagram of tea leaves after co-cultivation of coniferyl alcohol and Pseudomonas spores provided in Example 4 of the present invention, wherein A is a phenotypic diagram of the lesion area of ​​tea leaves infected by co-cultivation of 0.1 mg / ml coniferyl alcohol and Pseudomonas spores, and B is a statistical diagram of the lesion area of ​​tea leaves. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0023] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0024] Example 1: Pseudodiscus polytrichosporium infects tea leaves

[0025] (1) Tea tree sample: Shucha Zao. The material comes from the tea tree germplasm resource garden of the Central Anhui Comprehensive Experiment Station.

[0026] (2) The spore suspension of Pseudodiscus hirsutus infects the second leaf of tea tree

[0027] ① In a 28℃ constant temperature incubator, turn the PDA plate with the hyphae of Pseudodiscotrichum upside down. After 14 days, a large number of black spores will appear on the PDA plate.

[0028] ② Use double distilled water to transfer the spores from the PDA plate to a 50 mL centrifuge tube, centrifuge at 6000 rpm for 10 min, and then resuspend the spores in double distilled water and adjust the concentration to 10 7 Pieces / mL.

[0029] ③Take two mature leaves of tea tree, poke a plum blossom hole on each side of the leaf, apply 50μL of spore suspension on the plum blossom hole, and wrap the tea leaf containing the suspension with plastic wrap; double distilled water treatment was used as a control.

[0030] Example 2: Determination of coniferyl alcohol content in tea leaves

[0031] (1) Tea tree leaves treated with double distilled water and spore suspension were selected as materials. The specific implementation process is as follows:

[0032] ① Freeze-dry fresh tea leaves for 72 hours

[0033] ② Take 20 mg of freeze-dried tea leaves and put them into a 2 mL centrifuge tube. Add 2 steel balls into the tube and grind the tea leaves using a ball mill. After grinding, add 1.5 mL of 80% methanol solution into the tube and ultrasonicate it in an ice bath for 30 minutes, shaking it every 5 minutes.

[0034] ③After ultrasonic extraction, centrifuge at 12000rpm for 10min, aspirate the supernatant, filter through 0.22μm oil filter, and store in -20℃ refrigerator

[0035] (2) QQQ (Triple Quad 5500+) was used to determine the content of coniferyl alcohol. The specific implementation process is as follows:

[0036] ① Use 80% methanol solution to dilute the coniferyl alcohol standard to 5μg / mL, and then dilute 12 concentration gradients with 80% methanol solution to establish a standard curve

[0037] ② The mobile phase solvents include A (0.1% acetic acid water) and B (pure acetonitrile), the flow rate is 0.3 ml / min, and the elution gradient is: 92% A (0-2 min), 75% A (2-15 min), 53% A (15-20 min), 92% A (20-25 min), 92% A (25-28 min).

[0038] Example 3: Determination of MIC50 value of coniferyl alcohol on mycelium of Pseudomonas spp. and study on its inhibitory effect on mycelium of Anthracnose fungi

[0039] (1) preparing coniferyl alcohol solutions of different concentration gradients and co-culturing with Pseudomonas mycelium, the specific implementation process is as follows:

[0040] ① Use 60% methanol solution to dilute the coniferyl alcohol standard to 35.2 mg / mL, and then dilute it by half to four concentration gradients of 17.6, 8.8, 4.4, and 2.2 mg / mL.

[0041] ② After the solid PDA is heated and melted, it is cooled to about 60°C. 11mL PDA is measured in the clean bench and 1mL of coniferyl alcohol standard solution with different concentration gradients is added to it to make the final concentrations of 3.2, 1.6, 0.8, 0.4, and 0.2 mg / mL respectively. After mixing, pour it into a plate and wait for it to cool and solidify. Then, place the Pseudodiscus mycelium block upside down in the center of the solidified PDA plate. Three experiments are repeated for each gradient. 60% methanol solution is mixed with PDA as the control group.

[0042] (2) preparing a 0.1 mg / ml coniferyl alcohol solution and co-culturing it with the mycelium of the genus Anthracnose fungus. The specific implementation process is as follows:

[0043] ① Dilute the coniferyl alcohol standard to 1.2 mg / mL using 60% methanol solution.

[0044] ② After the solid PDA is heated and melted, it is cooled to about 60°C. 11mL PDA is measured in the clean bench and 1mL of 1.2mg / mL coniferyl alcohol standard solution is added to it to make the final concentration 0.1mg / mL. After mixing, pour it into a plate and wait for it to cool and solidify. Then, place the anthracnose fungus hyphae block upside down in the center of the solidified PDA plate. Set up three experimental replicates and mix 60% methanol solution with PDA as the control group.

[0045] Example 4: PI staining of suspensions of coniferyl alcohol and spore co-culture

[0046] Prepare a spore suspension mixed with coniferyl alcohol and add PI staining reagent. The concentrations of coniferyl alcohol and PI dye in the solution are 0.1 mg / mL and 40 μg / mL, respectively. Incubate in a 28°C incubator for 30 minutes, and then observe the spore fluorescence intensity at 488 nm using a laser confocal microscope.

[0047] Example 5: DAB and NBT staining to detect hydrogen peroxide and superoxide anions around the lesion. The specific implementation process is as follows:

[0048] (1) Use phosphate buffer solution (pH=7.5) to prepare 1 mg / mL DAB and 2 mg / mL NBT solutions respectively and store them in the dark.

[0049] (2) DAB and NBT staining

[0050] ① Take fresh plant leaves and immerse them in DAB and NBT dye solutions respectively, evacuate for 30 minutes, and dye with DAB for 6 hours and NBT for 3 hours in dark conditions.

[0051] ②After staining, rinse the leaves with clean water three times, and then place the leaves in 95% alcohol for decolorization. After decolorization, place the leaves in clean water to absorb water for 10 minutes, observe the phenotype and take pictures.

[0052] result

[0053] Firstly, QQQ was used to determine the changes in the content of coniferyl alcohol in tea leaves at different time points after the tea leaves were infected by Pseudodiscotrichum spores. The results showed that the content of coniferyl alcohol was significantly increased on the 10th and 13th days after the tea leaves were infected (such as Figure 1 The results showed that coniferyl alcohol may be involved in the tea plant's resistance to ring spot disease.

[0054] Then the MIC50 value of coniferyl alcohol on the hyphae of Pseudomonas spp. was determined. The results showed that the higher the concentration of coniferyl alcohol, the stronger the inhibitory effect on the growth of hyphae. The MIC50 value of coniferyl alcohol was calculated by nonlinear fitting to be 0.409 mg / mL ( Figure 2 At the same time, coniferyl alcohol was co-cultured with the mycelium of the genus Anthracnose fungus, and it was found that the mycelium growth was also inhibited (as shown in Figure 3 The results showed that coniferyl alcohol not only had a good inhibitory effect on wheel spot disease, but also had a good inhibitory effect on other plant fungi.

[0055] Finally, PI staining was used to detect the effect of coniferyl alcohol on the spores of Pseudodiscopolysporus. The fluorescence results showed that the fluorescence intensity of the spores treated with coniferyl alcohol was significantly higher than that of the control group (80% methanol treatment), indicating that coniferyl alcohol can damage the cells of Pseudodiscopolysporus spores. And after the spores co-cultured with coniferyl alcohol were infecting tea leaves, the lesion area was significantly reduced compared with the control group. The results showed that coniferyl alcohol can affect the activity of Pseudodiscopolysporus spores.

[0056] from Figure 4 It can be seen that after co-culture of coniferyl alcohol and Pseudodisco hirsutei, the fluorescence intensity of PI staining was significantly increased, and the results showed that coniferyl alcohol could damage the cell structure of spores.

[0057] from Figure 5 It can be seen that after co-culture of coniferyl alcohol and Pseudodiscotrichum spores and then infecting tea leaves, the lesion area was significantly reduced compared with the control group. The results showed that coniferyl alcohol can significantly reduce the pathogenicity of spores to tea leaves.

[0058] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of coniferyl alcohol in preventing and controlling tea tree ring spot disease.

2. The use according to claim 1, characterized in that: The tea tree ring spot disease is caused by Trichosporon pseudodiscus.

3. The use according to claim 2, characterized in that: The MIC50 value of the coniferyl alcohol against Trichosporon pseudodiscus is 0.409 mg / mL.

4. An antibacterial agent for preventing and treating tea tree ring spot disease, characterized in that: The main active ingredients include coniferyl alcohol.

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