Application of apigenin and cosmosiin in prevention and treatment of pepper phytophthora blight

Abstract

According to the application of the apigenin and the cosmosiin in prevention and treatment of the pepper phytophthora blight, the apigenin and the cosmosiin can effectively inhibit vegetative growth and sporangia formation of phytophthora capsici and are expected to be used for preparing novel green pesticides for resisting the pepper phytophthora blight, pesticide residues and resistance are reduced, and the prevention and treatment effect on the pepper phytophthora blight is improved.

Description

Technical Field

[0001] The invention relates to the technical field of pepper blight prevention and treatment, and in particular to application of apigenin and cosmos glycoside in preventing and treating pepper blight. Background Art

[0002] Phytophthora capsici is a widely destructive oomycete pathogen that can infect 78 plant species from 18 families, including Cucurbitaceae, Solanaceae, and Leguminosae. Under natural conditions, high temperature and high humidity are conducive to the production of three types of spores by Phytophthora capsici, including oospores, sporangia, and zoospores. Oospores are the main inoculum for the initiation of pepper blight in the field. Sporangia can be spread through soil, air flow, rainwater, and other channels. In addition to infecting host plants and causing large-scale seedling death, they can also release a large number of zoospores to infect the stems, vines, leaves, and fruits of plants, causing leaf wilting, fruit rot, stem necrosis, and wilting and death, leading to the occurrence of diseases such as leaf blight and stem blight, causing immeasurable losses. Due to the multi-host characteristics of Phytophthora capsici, it poses a huge threat to the safety of fruit and vegetable production in my country and around the world. Although traditional chemical control can effectively control diseases, the pathogens are very likely to develop drug resistance, which leads to increasing control costs; the irregular use of chemical pesticides will also cause pesticide residue problems and damage the ecological environment on which we depend for survival. Therefore, finding a new natural and effective control drug is of great significance to reduce the harm caused by diseases to crops. Summary of the invention

[0003] In view of the shortcomings of the prior art, the purpose of the present invention is to propose the application of apigenin and cosmos glycoside in the prevention and treatment of pepper blight. Apigenin and cosmos glycoside have the function of inhibiting the vegetative growth and sporangium formation of pepper Phytophthora, and are expected to be used to prepare a new green biological pesticide against pepper Phytophthora, reduce pesticide residues and resistance, and improve the prevention and treatment effect of pepper blight.

[0004] In order to achieve the above purpose, the scheme of the present invention is as follows:

[0005] The invention provides application of apigenin and / or cosmosin in preventing and treating pepper blight.

[0006] Preferably, the pepper blight is pepper blight caused by Phytophthora capsici.

[0007] The structural formulas of apigenin and apigenin-7-ο-β-d-glucoside are as follows:

[0008]

[0009] Preferably, the application includes:

[0010] A. Inhibit the vegetative growth of pepper phytophthora;

[0011] B. Inhibit the sporangium formation of pepper phytophthora;

[0012] C. Improve the resistance of pepper to pepper blight;

[0013] D. Inhibit the pathogenicity of pepper phytophthora.

[0014] Preferably, for Application A, apigenin is used alone at a concentration of at least 15 μM, cosmosin is used alone at a concentration of at least 30 μM, and the mixture of apigenin and cosmosin is used at a concentration of at least 5 μM.

[0015] Preferably, for application B, apigenin or cosmosin alone is used at a concentration of at least 100 μM, and the mixture of apigenin and cosmosin is used at a concentration of at least 50 μM.

[0016] Preferably, for application D, apigenin is used alone at a concentration of at least 126.9 μM, cosmosin is used alone at a concentration of at least 110.6 μM, and the mixture of apigenin and cosmosin is used at a concentration of at least 35.63 μM.

[0017] Preferably, in the mixture, the molar ratio of apigenin to cosmosin is 1:1.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention proposes the application of apigenin and cosmos glycoside in preventing and controlling pepper blight. Apigenin and cosmos glycoside can effectively inhibit the vegetative growth of pepper blight fungus, and are expected to be used in the preparation of a new green biological pesticide resistant to pepper blight, reduce pesticide residues and resistance, and improve the prevention and control effect of pepper blight.

[0020] The application of apigenin and cosmos glycoside proposed by the present invention in preventing and controlling pepper blight has an inhibitory effect on the vegetative growth and sporangium formation of pepper Phytophthora. Apigenin at a concentration of 15 μM or more, cosmos glycoside at a concentration of 30 μM or more, and a 1:1 mixture of apigenin and cosmos glycoside at a concentration of 5 μM or more can significantly inhibit the vegetative growth of pepper Phytophthora to varying degrees. Apigenin and cosmos glycoside at a concentration of 100 μM or more, and a 1:1 mixture of apigenin and cosmos glycoside at 50 μM can effectively inhibit the sporangium formation of pepper Phytophthora.

[0021] The use of apigenin and cosmos glycosides helps to improve the disease resistance of susceptible pepper varieties to pepper blight. It is simple, effective, green and environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Comparison of the abundance of Apigenin and Apigenin-7-ο-β-d-glucoside in pepper varieties with different resistance during infection with Phytophthora capsici at different stages. A, B: Comparison of the abundance of Apigenin and Cosmoside produced by pepper varieties with different resistance during infection. C, D: Chromatographic quantitative results of pepper leaf extract.

[0023] Figure 2 The contents of Apigenin and Apigenin-7-ο-β-d-glucoside in pepper varieties with different resistance to Phytophthora capsici without and with Phytophthora capsici. A, B: Comparison of the contents of apigenin and cosmosin in pepper varieties with different resistance to Phytophthora capsici. C, D: Chromatographic quantitative results of pepper leaf extract.

[0024] Figure 3A to Figure 3I This is the effect of Apigenin, Apigenin-7-ο-β-d-glucoside alone and a 1:1 mixture of the two on the nutritional growth of pepper Phytophthora. Figure 3A-3C : The nutritional growth of pepper phytophthora strains in solid culture medium supplemented with different concentrations of apigenin, cosmosin and a 1:1 mixture of the two. Figure 3D-Figure 3F : The bar graph shows the statistical calculation of the inhibitory effect of exogenously added apigenin, cosmosin at different concentrations and a 1:1 mixture of the two on pepper phytophthora. Figure 3G-Figure 3I The exogenous addition of different concentrations of apigenin, cosmosin and a 1:1 mixture of the two could inhibit the growth of Phytophthora capsici by 50% of the half inhibitory concentration (IC 50 Note: Ap is Apigenin, Apg is Apigenin-7-ο-β-d-glucoside; Statistical calculations were performed using nonparametric one-way analysis of variance.

[0025] Figure 4A to Figure 4I The results show the effects of Apigenin, Apigenin-7-ο-β-d-glucoside alone and a 1:1 mixture of the two on the sporangium formation of Phytophthora capsici. Figure 4A-4F :The effects of exogenous addition of different concentrations of apigenin, cosmosin and a 1:1 mixture of the two on the sporangium formation of Phytophthora capsici were observed under a microscope. Figure 4G-Figure 4I :The bar graph shows the statistical calculation of the effects of apigenin, cosmosin and a 1:1 mixture of the two on the sporangium yield of pepper phytophthora. Note: Ap is Apigenin, Apg is Apigenin-7-ο-β-d-glucoside; non-parametric one-way ANOVA was used for statistical calculation. Scale bar = 100 μm.

[0026] Figure 5Apigenin, Apigenin-7-ο-β-d-glucoside alone or a 1:1 mixture of the two enhanced the resistance of susceptible pepper varieties to pepper blight. Figure 5 A shows the effects of different concentrations of Apigenin, Apigenin-7-ο-β-d-glucoside alone and a 1:1 mixture of the two on the pathogenicity of pepper Phytophthora. Figure 5 B: The bar graph shows the statistical calculation of lesions after different concentrations of Apigenin, Apigenin-7-ο-β-d-glucoside alone and a 1:1 mixture of the two. Note: Ap is Apigenin, and Apg is Apigenin-7-ο-β-d-glucoside. DETAILED DESCRIPTION

[0027] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0028] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0029] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0030] Example 1: Pepper metabolomics experiment

[0031] Pepper Phytophthora was inoculated on V8 medium (formula: take V8 juice and filter it with 4 layers of medical gauze to obtain 300 mL of filtrate, add 3.5 g of calcium carbonate and stir to mix, then use dH2O to make the volume to 3 L (the volume ratio of filtrate to dH2O is 1:10), and dispense it into 500 mL conical flasks while stirring, wherein each 500 mL conical flask contains 300 mL of solution, and finally, add 4.5 g of agar powder to each 300 mL of solution, seal it with sealing film and 4 layers of newspaper, and sterilize it by high pressure at 121°C for 20 min. Note: V8 juice was purchased from Taobao: American Jinbao V8 100% mixed vegetable juice original flavor) and cultured for four days, and then light-induced sporulation for three days, then the sporangia were collected, washed with sterile water and prepared into a concentration of about 5×10 5100 μL of sporangium suspension was then inoculated on the leaves of 6-week-old susceptible pepper varieties (HNUCB0081), medium-resistant pepper varieties (HNUCB0226) and high-resistant pepper varieties (XIAO ZHOU PI). Different pepper varieties inoculated with sterile water were used as the control group. The inoculated peppers were first cultured in a dark environment at 25°C for 24 hours and then transferred to natural light conditions for another 24 hours. During this period, the pepper leaves of each group were collected 1, 3, 6, 12, 24, and 48 hours after inoculation, first frozen with liquid nitrogen and then stored at -80°C. After all samples were collected, they were ground into powder with liquid nitrogen, and then 0.1g of powder was weighed in a 2mL centrifuge tube. 0.8mL of pre-cooled 70% methanol solution was added to each tube, and two small steel balls were added. The samples were ground in a TissueLyser at 50Hz for 5 minutes. After the end, the sample was placed in a 4°C refrigerator for precipitation for 12 hours, centrifuged at 4°C, 25000g for 15 minutes, 550 μL of supernatant was aspirated into a new 2mL centrifuge tube, and finally 50 μL was taken for non-targeted metabolomics analysis. The metabolomics experiment was detected and analyzed by ultra-high performance liquid chromatography tandem mass spectrometry. The liquid chromatography used a 2777C UPLC system (Waters, UK), and the mass spectrometry used a Xevo G2-XS QTOF system (Waters, UK). The chromatographic column used was a Hypersil GOLD aQ Dim column (1.9μm 1.2*100mm, Thermo Fisher Scientific, USA), and the mobile phase was an aqueous solution of 0.1% formic acid (liquid A) and acetonitrile containing 0.1% formic acid (liquid B). The following gradient was used for elution: 0-2 min, 5% B solution; 2-22 min, 5%-95% B solution; 22-27 min, 95% B solution; 27-27.1 min, 95% B solution-5% B solution; 27.1-30 min, 5% B solution. The flow rate was 0.3 mL / min, the column temperature was 40°C, and the injection volume was 5 μL. The results are shown in Figure 1 .

[0032] Depend on Figure 1 It can be seen that compared with other pepper varieties, the pepper leaves of the disease-resistant variety (XIAO ZHOU PI) at all infection periods accumulated a large amount of secondary metabolites apigenin and apigenin-7-ο-β-d-glucoside.

[0033] Example 2: Contents of Apigenin and Apigenin-7-ο-β-d-glucoside in Different Resistant Pepper Varieties Not Inoculated and Inoculated with Phytophthora Capsici

[0034] As in the previous steps, 40 μL of sporangium suspension was inoculated on the leaves of 6-week-old susceptible pepper varieties (HNUCB0081) and medium-resistant pepper varieties (HNUCB0226). The control group did not receive any treatment. The inoculated peppers were first cultured in a dark environment at 25°C for 24 hours and then transferred to natural light conditions for further culture for 48 hours. The leaves were collected, ground into powder with liquid nitrogen, and freeze-dried for 48 hours. 30 mg of each sample was weighed, 1 mL of 70% methanol was added, vortexed, room temperature, 100% power ultrasound for 20 minutes, 4°C, 12000 rpm, centrifuged for 10 minutes, and the supernatant was transferred to a new centrifuge tube. 100 μL of the original supernatant was taken, 900 μL of 70% methanol was added, vortexed to mix, 4°C, 12000 rpm, centrifuged for 10 minutes, and finally 100 μL of the supernatant was taken for quantitative metabolomics analysis. Metabolomics experiments were performed using ultra-high performance liquid chromatography tandem mass spectrometry for detection and analysis. The instrument used was a triple quadrupole liquid-mass spectrometer (Waters ACQUITY UPLC-XEVO TQ-S MS). The mass spectrometer used MRM (Multiple Reaction Monitor) technology to collect data. The chromatographic column model used for liquid chromatography was ACQUITY UPLC BEH C18, specification: 1.7μm 100mm×2.1mm, mobile phase A: water + 5mM ammonium acetate, mobile phase B: methanol. The following gradient was used for elution: 0-4min, 95% A solution, 5% B solution; 4-8min, 80% A solution, 20% B solution; 8-9min, 100% B solution; 9-9.1min, 100% B solution; 9.1-12min, 95% A solution, 5% B solution. The flow rate was 0.3 mL / min, the column temperature was 40 °C, and the injection volume was 2 μL. The peaks were integrated using Waters' Targetlynx software, and the concentrations were calculated by constructing a standard curve. The results are shown in Figure 2 .

[0035] Depend on Figure 2 It can be seen that the level of apigenin increased significantly in HNUCB0081 and HNUCB0226 varieties infected with P. capsici. Contrary to expectations, we observed that the level of cosmosin in XIAO ZHOU PI cells before inoculation was significantly higher than that in the inoculated and control groups HNUCB0081 and HNUCB0226. At the same time, during the infection of P. capsici, the level of cosmosin accumulated in XIAO ZHOU PI was significantly higher than that in HNUCB0081 and HNUCB0226 varieties under the same treatment conditions. Based on this, we speculate that the production and accumulation of apigenin and cosmosin in pepper may directly or indirectly affect the resistance of pepper seedlings to biotic and abiotic stresses.

[0036] Example 3: Effects of Apigenin, Apigenin-7-ο-β-d-glucoside alone and a mixture of the two in a molar ratio of 1:1 on the vegetative growth of Phytophthora capsici

[0037] Mycelial blocks of the same size were cut from the edge of the 5-day-old colony of Phytophthora capsici LT1534 and inoculated into the culture medium with different concentrations of Apigenin, Apigenin-7-ο-β-d-glucoside, and Apigenin and Apigenin-7-ο-β-d-glucoside added exogenously. 1:1 mixture of TA medium (formula: take tomato juice and filter it with 4 layers of medical gauze to obtain 300mL of filtrate, add 3.5g of calcium carbonate and stir to mix, then use dH2O to make the volume to 3L (the volume ratio of filtrate to dH2O is 1:10), and divide it into 500mL conical flasks while stirring, where each 500mL conical flask contains 300mL of solution. Finally, add 4.5g of agar powder to every 300mL of solution, seal it with sealing film and 4 layers of newspaper, and autoclave at 121℃ for 20min. Note: Tomato juice was purchased from Taobao: Zhenfu 100% tomato juice from Tmall Zhenfu flagship store), cultured in the dark at 25℃ for 4 days, measure and record the growth diameter and take pictures. Results are shown in Figure 3A to Figure 3I .

[0038] Depend on Figure 3A to Figure 3I It can be seen that Apigenin at a concentration of 15 μM and above, Apigenin-7-ο-β-d-glucoside at a concentration of 30 μM and above, and a 1:1 mixture of Apigenin and Apigenin-7-ο-β-d-glucoside at a concentration of 5 μM and above can effectively inhibit the vegetative growth of pepper phytophthora, while pepper phytophthora treated with 0.2% v / v DMSO (dimethyl sulfoxide) had no inhibition. The results show that Apigenin, Apigenin-7-ο-β-d-glucoside alone, and Apigenin and Apigenin-7-ο-β-d-glucoside 1:1 mixture all have a significant inhibitory effect on the vegetative growth of pepper phytophthora.

[0039] Example 4: Effects of Apigenin, Apigenin-7-ο-β-d-glucoside, and a 1:1 mixture of the two on sporangium formation of Phytophthora capsici

[0040] As in the previous steps, the pepper phytophthora after measuring the growth diameter and taking pictures was transferred to a 25°C incubator for light-induced sporulation for 1 to 2 days. After taking pictures under a microscope, the sporangium was washed with 2 mL of sterile water, and 10 μL of the sporangium suspension was aspirated each time and the number of sporangium was counted under a microscope using a hemocytometer. The results are shown in Figure 4A to Figure 4I .

[0041] Depend on Figure 4A to Figure 4I It can be seen that Apigenin, Apigenin-7-ο-β-d-glucoside at concentrations of 100 μM and above and a 1:1 mixture of the two at 50 μM can effectively inhibit the sporangium formation of pepper Phytophthora, while 0.2% v / v DMSO cannot inhibit the spore formation of pepper Phytophthora. The results showed that Apigenin, Apigenin-7-ο-β-d-glucoside alone and a 1:1 mixture of Apigenin and Apigenin-7-ο-β-d-glucoside had a significant inhibitory effect on the sporangium formation of pepper Phytophthora.

[0042] Example 5: Effects of Apigenin, Apigenin-7-ο-β-d-glucoside, and a 1:1 mixture of the two on the pathogenicity of Phytophthora capsici

[0043] The pepper phytophthora was inoculated on TA medium and cultured at 25°C for four days, then transferred to 25°C for light induction for three days. Sporangia were collected, washed with sterile water and prepared into a concentration of about 5×10 3 / mL of sporangium suspension, and then prepared different concentrations of Apigenin, Apigenin-7-ο-β-d-glucoside and Apigenin and Apigenin-7-ο-β-d-glucoside 1:1 mixture and sprayed on the susceptible pepper leaves. After standing in the dark for 30 minutes, 20μL of pepper phytophthora sporangium suspension was inoculated into the susceptible pepper leaves. The inoculated peppers were first cultured in the dark at 25℃ for 24 hours and then transferred to natural light conditions for continued moisturizing culture for 48 hours. The results are shown in Figure 2. Figure 5 .

[0044] Depend on Figure 5 It can be seen that Apigenin and Apigenin-7-ο-β-d-glucoside can inhibit the infection ability of pepper cultivars by enhancing the resistance of susceptible pepper varieties. 50 Apigenin with an IC value of 126.9 μM or above 50 Apigenin-7-ο-β-d-glucoside at concentrations of 110.6 μM and above, as well as IC 50Apigenin and Apigenin-7-ο-β-d-glucoside 1:1 mixture at concentrations of 35.63 μM and above can effectively inhibit pepper phytophthora, while 0.2% v / v DMSO cannot resist the infection of pepper phytophthora. The results showed that Apigenin, Apigenin-7-ο-β-d-glucoside alone and Apigenin and Apigenin-7-ο-β-d-glucoside 1:1 mixture had a significant inhibitory effect on the pathogenicity of pepper phytophthora.

[0045] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of apigenin and / or cosmosin in preventing and controlling pepper blight.

2. The use according to claim 1, characterized in that: The pepper blight is caused by Phytophthora capsici.

3. The use according to claim 1, characterized in that: Include at least one of the following applications A to D: A. Inhibit the vegetative growth of pepper phytophthora; B. Inhibit the sporangium formation of pepper phytophthora; C. Improve the resistance of pepper to pepper blight; D. Inhibit the pathogenicity of pepper phytophthora.

4. The use according to claim 3, characterized in that: For Application A, apigenin alone is used at a concentration of at least 15 μM, cosmosin alone is used at a concentration of at least 30 μM, and the mixture of apigenin and cosmosin is used at a concentration of at least 5 μM.

5. The use according to claim 3, characterized in that: For application B, apigenin or cosmosin alone is used at a concentration of at least 100 μM, and the mixture of apigenin and cosmosin is used at a concentration of at least 50 μM.

6. The use according to claim 3, characterized in that: For Application D, apigenin alone was used at a concentration of at least 126.9 μM, cosmosin alone was used at a concentration of at least 110.6 μM, and the mixture of apigenin and cosmosin was used at a concentration of at least 35.63 μM.

7. The use according to any one of claims 4 to 6, characterized in that: In the mixture, the molar ratio of apigenin to cosmosin is 1:1.

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