Application of cedrol in inhibiting the development of camellia ring spot disease

Abstract

The application discloses application of cembrene in inhibition of a disease development process of a tea tree annular spot, and belongs to the technical field of tea tree annular spot prevention and treatment. In vitro, standard cembrene and Pestalotiopsis funerea mycelium blocks are co-cultured, the MIC50 value is calculated to measure the bacteriostatic effect, and when the cembrene is co-cultured with a colletotrichum genus fungus, it is also found that the cembrene has a good inhibitory effect on mycelium growth. Meanwhile, it is also found that the cembrene can damage Pestalotiopsis funerea spore cells and affect the pathogenicity of the Pestalotiopsis funerea to tea tree leaves. Compared with traditional bacteriostatic agents, the application provides a more effective and environment-friendly bacteriostatic agent for inhibiting plant annular spot in the market.

Description

TECHNICAL FIELD

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

[0002] Tea tree is a perennial evergreen woody plant, which is mostly grown in tropical and subtropical regions, and these regions are also conducive to the growth and reproduction of pathogenic bacteria. Tea tree annular spot disease is caused by Pestalotiopsis-like fungi, and belongs to a high-temperature and high-humidity type of disease, which is distributed in all tea areas in China and is one of the most destructive leaf diseases of tea trees worldwide. The disease first appears on the edge of the leaf, and then expands inward, and in severe cases, it becomes a necrotic spot with a dark brown edge, which eventually leads to the leaf falling off the tea tree. Worldwide, Pestalotiopsis-like fungi can infect many plants and cause various plant diseases. For example, Pestalotiopsis (P.) apiculatus can cause top dieback disease of Douglas fir, leading to the death of the main shoot of the tree, which is one of the main diseases that harm the growth and development of plants.

[0003] Coniferyl alcohol (CA), also known as 4-hydroxy-3-methoxycinnamyl alcohol, is a natural metabolite widely present in plant bodies. When plants are infected by pathogens, the content of coniferyl alcohol increases significantly. It not only participates in the composition of lignin, and its derivative coniferin has a direct inhibitory effect on pathogens, but the inhibitory effect of coniferyl alcohol itself is less studied. Currently, the main methods for preventing and treating annular 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, it is difficult to breed 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 reduce the use of chemical pesticides, but there is still a risk of pesticide residue exceeding the standard, so we need to invent natural bacteriostatic agents to prevent and treat annular spot disease.

[0004] The Chinese patent application document with the publication number CN110237114A discloses the application of dehydrodiconiferyl alcohol in the preparation of a drug for resisting liver damage. The extraction method is as follows: after the powder of Trichosanthes kirilowii Maxim is crushed, it is extracted with ethanol, and the extract is extracted with ethyl acetate, and concentrated into an extract under reduced pressure. The patent finds through experiments that dehydrodiconiferyl alcohol can significantly reverse the increase of ALT and AST liver function levels, the decrease of bile flow, and liver lesions in ANIT-induced cholestasis SD rats, and has a significant liver damage protection effect; and mainly plays a role based on activating the FXR target point to regulate the expression of downstream genes. The patent broadens the application range of dehydrodiconiferyl alcohol and ethyl acetate extract of Trichosanthes kirilowii Maxim, and has important application value and economic benefits for developing FXR agonist drugs or liver damage protection drugs. However, the patent does not disclose the application of coniferyl alcohol in inhibiting the occurrence of tea tree leaf spot disease. SUMMARY

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

[0006] Firstly, the content of coniferyl alcohol in tea tree leaves before and after being infected by Pestalotiopsis funerea is detected by QQQ, then coniferyl alcohol standard is co-cultured with Pestalotiopsis funerea mycelium block, and the MIC50 value is calculated to measure the antibacterial effect, and when coniferyl alcohol is co-cultured with Colletotrichum fungi mycelium, it is also found that it has good antibacterial effect. Finally, coniferyl alcohol is co-cultured with Pestalotiopsis funerea spores, the health status of the spores is detected by PI staining, and further, the spores co-cultured with coniferyl alcohol are used to infect tea tree leaves, and the pathogenicity of the spores to tea tree leaves is observed.

[0007] The present application solves the above technical problems by the following technical means:

[0008] The present application proposes the application of coniferyl alcohol in preventing and treating tea tree leaf spot disease.

[0009] Preferably, the tea tree leaf spot disease is caused by Pestalotiopsis funerea.

[0010] Preferably, the MIC50 value of coniferyl alcohol to Pestalotiopsis funerea is 0.409 mg / mL.

[0011] The present application also proposes an antibacterial agent for preventing and treating tea tree leaf spot disease, and the main active ingredient includes coniferyl alcohol.

[0012] The present application has the following advantages:

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

[0014] (2) The bacteriostatic agent used in the present application has more significant inhibiting effect on diseases compared with other traditional natural metabolite bacteriostatic agents.

[0015] (3) The bacteriostatic agent used in the present application not only can inhibit Pestalotiopsis fungi, but also has inhibiting effect on Colletotrichum fungi mycelium, and has certain extensive inhibiting effect on fungi. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is a graph showing the content change of cedrol in tea tree leaves after being infected by Pestalotiopsis fungi according to Example 2 of the present application;

[0017] Figure 2 is a graph showing the determination of MIC50 value of cedrol on Pestalotiopsis mycelium according to Example 3 of the present application, wherein A is a mycelium block area phenotype graph after cedrol with different concentration gradients is co-cultured with Pestalotiopsis mycelium block, B is a mycelium growth area statistical graph under different concentration gradients of cedrol, and C is a nonlinear fitting curve graph of mycelium growth inhibition rate under different concentration gradients of cedrol.

[0018] Figure 3 is a graph showing the inhibiting effect of cedrol on Colletotrichum mycelium according to Example 3 of the present application, wherein A is a mycelium block area phenotype graph after 0.1 mg / ml cedrol is co-cultured with Colletotrichum mycelium, and B is a mycelium growth area statistical graph under 0.1 mg / ml cedrol;

[0019] Figure 4 is a graph showing the influence of cedrol on Pestalotiopsis spore cells by PI staining according to Example 4 of the present application, wherein A is a fluorescence brightness graph after 0.1 mg / ml cedrol is co-cultured with Pestalotiopsis spores, and B is a fluorescence intensity statistical graph;

[0020] Figure 5 is a graph showing the pathogenicity analysis of tea tree leaves after cedrol is co-cultured with Pestalotiopsis spores according to Example 4 of the present application, wherein A is a lesion area phenotype graph of tea tree leaves infected by 0.1 mg / ml cedrol co-cultured with Pestalotiopsis spores, and B is a lesion area statistical graph of tea tree leaves. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0022] The test materials and reagents used in the following examples, and the like, can be obtained from commercial channels unless otherwise specified.

[0023] The specific techniques or conditions not specified in the examples 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: Pestalotiopsis infection of tea plant leaves

[0025] (1) Select tea plant samples: Shuchazao. The material is from the tea germplasm resource garden of Central Anhui Comprehensive Experimental Station.

[0026] (2) Pestalotiopsis spore suspension infection of tea plant two leaves

[0027] ① In a 28℃ constant temperature incubator, the PDA plate inoculated with Pestalotiopsis mycelium was inverted, and after 14 days, a large number of black spores were produced in the PDA plate.

[0028] ② The spores were transferred from the PDA plate to a 50mL centrifuge tube using double distilled water, centrifuged at 6000rpm for 10min, and then resuspended and adjusted to a concentration of 10 7 individuals / mL using double distilled water.

[0029] ③ Take mature two leaves of tea plant, make a clover hole on each side of the leaf, apply 50μL of spore suspension to the clover hole, and use plastic wrap to wrap the tea plant leaves containing the suspension; double distilled water treatment as control.

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

[0031] (1) Use tea plant leaves treated with double distilled water and spore suspension as material, the specific implementation process is

[0032] ① Freeze-drying of fresh tea leaves for 72h

[0033] ② Take 20mg of freeze-dried tea leaves and place them in a 2mL centrifuge tube, add 2 steel balls to the tube, and use a ball mill to grind the tea leaves. After grinding, add 1.5mL of 80% methanol solution to the tube, and sonicate in an ice bath for 30min, shaking every 5min.

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

[0035] (2) Determine the content of coniferyl alcohol by QQQ (Triple Quad 5500+), the specific implementation process is

[0036] ① Dilute the coniferyl alcohol standard to 5 μg / mL with 80% methanol solution, then dilute it 12 times 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), with a flow rate of 0.3 ml / min and an elution gradient of: 92% A (0-2 min), 75% A (2-15 min), 53% A (15-20 min), 92% A (20-25 min), and 92% A (25-28 min).

[0038] Example 3: Determination of MIC50 value of coniferol against *Polytrichum gloeosporioides* hyphae and its inhibitory effect on *Anthracis* fungi hyphae.

[0039] (1) Prepare coniferyl alcohol solutions of different concentration gradients and co-culture them with *Pseudomonas sylvestris* hyphae. The specific implementation process is as follows:

[0040] ① Dilute the coniferyl alcohol standard to 35.2 mg / mL with 60% methanol solution, and then dilute it in 2 / 3 times to obtain four concentration gradients of 17.6, 8.8, 4.4 and 2.2 mg / mL.

[0041] ② After heating and melting solid PDA, cool it to about 60°C. In a clean bench, measure 11 mL of PDA and add 1 mL of coniferol standard solution with different concentration gradients to make the final concentrations 3.2, 1.6, 0.8, 0.4, and 0.2 mg / mL, respectively. Mix well and pour into a plate to cool and solidify. Then, invert a block of *Plasmodium spp.* mycelium into the center of the solidified PDA plate. Repeat the experiment three times for each gradient. Use 60% methanol solution as a control group and mix it with PDA.

[0042] (2) Prepare a 0.1 mg / ml coniferol solution and co-culture it with anthracnose fungal hyphae. 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 heating and melting solid PDA, cool it to about 60°C. In a clean bench, measure 11 mL of PDA and add 1 mL of 1.2 mg / mL coniferyl alcohol standard solution to make the final concentration 0.1 mg / mL. Mix well and pour into a plate to cool and solidify. Then, invert the solidified PDA plate and place an anthracnose fungal hyphae block in the center. Set up three experimental replicates. Use 60% methanol solution as a control group and mix it with PDA.

[0045] Example 4: PI-stained suspension of coniferyl alcohol and spore co-culture

[0046] A spore suspension containing coniferyl alcohol was prepared, and PI staining reagent was added. The concentrations of coniferyl alcohol and PI dye in the solution were 0.1 mg / mL and 40 μg / mL, respectively. The suspension was incubated at 28℃ for 30 min, and then the spore fluorescence intensity was observed using laser confocal microscopy at 488 nm.

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

[0048] (1) Prepare 1 mg / mL DAB and 2 mg / mL NBT solutions using phosphate buffer solution (pH = 7.5) 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 staining solutions respectively. Vacuum for 30 minutes, then stain with DAB for 6 hours and NBT for 3 hours in the dark.

[0051] ② After staining, rinse the leaves three times with clean water, then immerse them in 95% alcohol to decolorize. After decolorization, soak the leaves in clean water for 10 minutes, observe the phenotype, and take photos.

[0052] result

[0053] First, the changes in coniferol content in tea leaves at different time points after infection with *Pseudomonas spores* were determined using QQQ. The results showed that the coniferol content was significantly increased on days 10 and 13 after infection (e.g., ...). Figure 1 As shown in the figure, the results indicate that coniferol may be involved in the resistance of tea trees to ring spot disease.

[0054] Then, the MIC50 value of coniferol against *Plasmodium spp.* hyphae was determined. The results showed that the higher the concentration of coniferol, the stronger the inhibitory effect on hyphal growth. The MIC50 value of coniferol was calculated to be 0.409 mg / mL using nonlinear fitting. Figure 2 (As shown). Simultaneously, co-culturing coniferol with anthracnose fungal hyphae also inhibited hyphal growth (e.g., ...). Figure 3 (As shown in the image). The results indicate that coniferol not only has a good inhibitory effect on ring spot disease, but also has a good inhibitory effect on other plant fungi.

[0055] Finally, PI staining was used to detect the effect of coniferol on *Polytrichum gloeosporioides* spores. Fluorescence results showed that the fluorescence intensity of spores treated with coniferol was significantly higher than that of the control group (treated with 80% methanol), indicating that coniferol can damage the cells of *Polytrichum gloeosporioides* spores. Furthermore, when spores co-cultured with coniferol were used to infect tea leaves, the lesion area was significantly reduced compared to the control group, indicating that coniferol can affect the activity of *Polytrichum gloeosporioides* spores.

[0056] from Figure 4 The results show that co-culturing coniferol with *Plasmodium spp.* significantly increased the fluorescence intensity of PI staining, indicating that coniferol can damage the cellular structure of spores.

[0057] from Figure 5 The results show that when coniferol was co-cultured with Polychaete spores and then infecting tea leaves, the lesion area was significantly reduced compared with the control group. The results indicate that coniferol 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, and are not intended to limit it. 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. Such 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 coniferol in the control of tea leaf spot disease, characterized in that, The tea tree ring spot disease is caused by *Plasmodium spp.* 2. The application according to claim 1, characterized in that, The MIC50 value of the coniferol against Polytrichum candida was 0.409 mg / mL.

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