Application of daidzin in preventing and controlling Phytophthora blight of pepper

Abstract

The present invention provides the application of daidzin in preventing and controlling Phytophthora capsici. Daidzin can effectively inhibit the vegetative growth of Phytophthora capsici, and is expected to be used for preparing a new type of fungicide against Phytophthora capsici, reducing pesticide resistance and improving the control effect on Phytophthora capsici. In the application of daidzin in preventing and controlling Phytophthora capsici provided by the present invention, it has a certain inhibitory effect on three types of spores produced by Phytophthora capsici. Daidzin at a concentration of 1 μM - 5 μM can induce apoptotic-like death of the spores of Phytophthora capsici, and daidzin at a concentration of 7 μM - 15 μM can inhibit the release of sporangia of Phytophthora capsici and effectively inhibit the germination of zoospores of Phytophthora capsici. Using daidzin also helps to improve the disease resistance of susceptible varieties to Phytophthora capsici, which is simple, effective, green and environmentally friendly.

Description

Technical Field

[0001] The present invention relates to the field of prevention and control of Phytophthora capsici, and particularly to the application of daidzin in the prevention and control of Phytophthora capsici. Background Art

[0002] Phytophthora capsici is a hemibiotrophic and filamentous fungus-like oomycete pathogen that causes Phytophthora blight and wilt diseases in 45 important economic fruit and vegetable crops globally. Except for roots, Phytophthora capsici, as a soil-borne pathogen, can infect all foliar tissues, including the stems / vines, leaves, fruits, etc. of host plants, resulting in root, crown, and fruit rot of crops, and severely leading to a 100% reduction in crop yield. Phytophthora capsici produces three types of spores, including oospores, sporangia, and zoospores. Oospores, as long-lived sexual spores, can be formed on the fruit surface and then fall into the soil, which is the main inoculum for the initiation of Phytophthora capsici in the field. Sporangia are short-lived spores that are water-borne and can directly induce wilt disease in susceptible hosts. Sporangia can also indirectly cause infection by producing zoospores, which can sense the soil and living plant parts on the soil surface and swim towards them. Once they come into contact with the host plant, zoospores can infect the roots and crowns of crops in the soil, or fall onto the leaves and fruit surfaces through splashing water for infection. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to propose the application of daidzin in the prevention and control of Phytophthora capsici. Daidzin has the function of inducing apoptosis of Phytophthora capsici zoospores, and is expected to be used to prepare a new fungicide against Phytophthora capsici, reduce pesticide resistance, and improve the prevention and control effect of Phytophthora capsici.

[0004] The technical solution of the present invention is realized as follows:

[0005] The present invention provides an application of daidzin in the prevention and control of plant diseases.

[0006] Preferably, the application of daidzin in the prevention and control of Phytophthora capsici.

[0007] Preferably, daidzin inhibits the growth of Phytophthora capsici.

[0008] Preferably, daidzin induces apoptotic-like death of Phytophthora capsici spores.

[0009] Preferably, daidzin inhibits the release of Phytophthora capsici sporangia.

[0010] Preferably, daidzin inhibits the germination of Phytophthora capsici zoospores.

[0011] The present invention also provides an application of daidzin in improving the disease resistance of susceptible pepper varieties.

[0012] Preferably, it includes disease resistance against Phytophthora capsici caused by Phytophthora capsici Leonian.

[0013] Preferably, the present invention provides a pesticide composition for controlling plant diseases, and the active ingredient of the pesticide composition includes daidzin.

[0014] Preferably, the plant is pepper.

[0015] It has been found through research that the extract of pepper leaves infected by Phytophthora capsici has the function of inducing the apoptosis of Phytophthora capsici zoospores, and it is expected to be used to prepare a new fungicide against Phytophthora capsici.

[0016] The secondary metabolite of pepper, Daidzin, has the following structural formula:

[0017]

[0018] Daidzin only appears in the metabolites of peppers inoculated with Phytophthora capsici, and the higher the disease resistance of the pepper, the higher the accumulation of Daidzin. That is, the inhibitory effect of the pepper leaf extract on the spore germination of Phytophthora capsici is related to the amount of the secondary metabolite Daidzin it contains.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] The present invention proposes the application of daidzin in controlling Phytophthora capsici. Daidzin can effectively inhibit the vegetative growth of Phytophthora capsici, and is expected to be used to prepare a new fungicide against Phytophthora capsici, reduce pesticide resistance, and improve the control effect on Phytophthora capsici.

[0021] In the application of daidzin proposed by the present invention in controlling Phytophthora capsici, it has an inhibitory effect on three types of spores produced by Phytophthora capsici. Daidzin at a concentration of 1 μM - 5 μM can induce apoptotic-like death of Phytophthora capsici spores, and daidzin at a concentration of 7 μM - 15 μM can inhibit the release of Phytophthora capsici sporangia and effectively inhibit the germination of Phytophthora capsici zoospores.

[0022] Using daidzin helps to improve the disease resistance of susceptible varieties to Phytophthora capsici, which is simple, effective, green and environmentally friendly. Description of the Drawings

[0023] Figure 1 is the content of Daidzin in different varieties of peppers infected by Phytophthora capsici;

[0024] Figure 2 is the effect of Daidzin on the vegetative growth of Phytophthora capsici;

[0025] Figure 3Effect of daidzin on sporangium release and zoospore germination of Phytophthora capsici. Among them, (A) confocal observation of the differences in sporangium release of Phytophthora capsici at different time points with different concentrations of daidzin added externally; (B-C) histograms showing the statistical effects of different concentrations of externally added daidzin on sporangium release of Phytophthora capsici; (D) effects of different concentrations of daidzin on zoospore germination and survival of Phytophthora capsici at different time points. (E-F) Histograms representing the statistical effects of different concentrations of externally added daidzin on zoospore germination and survival. Note: Non-parametric one-way ANOVA was used for statistical calculation, scale bar = 10 μm;

[0026] Figure 4 Qualitative and quantitative analysis of the resistance of different pepper varieties to Phytophthora capsici. Among them, (A-B) show the progress and quantitative analysis of leaf blight in leaf tissues of the sensitive pepper variety (HNUC00081) at different infection stages; (C-D) represent the progress and quantitative analysis of leaf blight in leaf tissues of the moderately resistant pepper variety (HNUC0226) at different infection stages; (E-F) show the progress and quantitative analysis of leaf blight lesions in leaf tissues of the highly resistant pepper variety (XIAO ZHOU PI) at different stages of Phytophthora blight infection. Note: Non-parametric one-way ANOVA was used for statistical calculation of lesion types. Detailed implementation mode

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

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

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

[0030] Example 1 - Pepper metabolomics experiment

[0031] Collect the sporangia of Phytophthora capsici grown on V8 medium for seven days, and wash and elute them with sterile water to prepare a concentration of about 5×10 5A sporangium suspension of Figure 1 CFU / mL was then prepared. Next, 40 μL of the suspension was inoculated onto 6-week-old susceptible pepper cultivars (HNUCB0081 and HNUCB0226) and resistant cultivar (XIAO ZHOU), and the control group was inoculated with sterilized water. After inoculation, the peppers were placed in a dark and humid environment at 25 °C for 24 hours and then transferred to natural light conditions for continued cultivation. Pepper leaves were collected at 1, 3, 6, 12, 24, and 48 hours after inoculation in each group, ground into fine powder with liquid nitrogen, and then 0.1 g of the powder was placed in an EP tube. 0.8 mL of frozen 70% methanol buffer solution was added, and two small steel beads were added to the EP tube. The sample was placed in a TissueLyser for grinding with the parameters set at 50 Hz for 5 minutes. After grinding, the steel beads were removed, and the centrifuge tube was placed in a -20 °C refrigerator for precipitation for 2 hours, centrifuged at 25000 g at 4 °C for 15 minutes, and 550 μL of the supernatant was taken and transferred to a new EP tube. 50 μL of the supernatant was taken for metabolomics analysis. The metabolomics experiment was performed using ultra-high performance liquid chromatography tandem mass spectrometry. The liquid chromatography system used was a 2777C UPLC system (Waters, UK), and the mass spectrometry system used was an Xevo G2-XS QTOF system (Waters, UK). The chromatographic column used was a Hypersil GOLD aQ Dim column (1.9 μm 1.2 * 100 mm, Thermo Fisher Scientific, USA), and the mobile phases were an aqueous solution of 0.1% formic acid (solution A) and acetonitrile containing 0.1% formic acid (solution B). The following gradient was used for elution: 0 - 2 min, 5% solution B; 2 - 22 min, 5% - 95% solution B; 22 - 27 min, 95% solution B; 27 - 27.1 min, 95% solution B - 5% solution B; 27.1 - 30 min, 5% solution B. 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

[0032] As Figure 1 can be seen, compared with other pepper cultivars, the secondary metabolite Daidzin accumulated significantly in the pepper leaves of the resistant cultivar (XIAO ZHOU) at each infection stage.

[0033] Example 2 - Effect of Daidzin on the vegetative growth of Phytophthora capsici

[0034] Equal-sized mycelial blocks were cut from the edge of a freshly grown Phytophthora capsici LT1534 colony and inoculated onto V8 medium containing different concentrations of Daidzin. After incubating in the dark at 25 °C for 5 days in an inverted position, the diameter was measured and recorded, and photos were taken. The results are shown in Figure 2 。

[0035] As Figure 2It can be seen that Daidzin at 10 μmol / L and above can effectively inhibit the vegetative growth of Phytophthora capsici, while 0.2% v / v DMSO (dimethyl sulfoxide) cannot inhibit the vegetative growth of Phytophthora capsici, indicating that Daidzin has a certain inhibitory effect on the vegetative growth of Phytophthora capsici.

[0036] Example 3 - Effect of Daidzin on Different Types of Spores of Phytophthora capsici

[0037] Inoculate Phytophthora capsici LT1534 on V8 medium. After incubating in an inverted position at 25 °C for 5 days, tear open the sealing film and induce sporangia for 3 days with a 12 h / 12 h light / dark cycle. Wash the sporangia of Phytophthora capsici with different concentrations of Daidzin, filter and collect, and then adjust the sporangia concentration to 1×10 5 cells / mL. After inducing for 30 min and 60 min at 4 °C, press the slices to observe the release of sporangia; Zoospore germination: Add an appropriate amount of sterilized water to the sporulation medium, induce the release of sporangia by treating at 4 °C for 30 min, filter the zoospores, and let them stand at room temperature for induction of germination. The results are shown in Figure 3 .

[0038] As Figure 3 can be seen, the Daidzin sporangia treatment solution at 1 μM - 5 μM will cause apoptotic-like death of the spores of Phytophthora capsici after the release of sporangia. As the concentration increases (7 μM - 15 μM), the Daidzin sporangia treatment solution will cause the rupture of the sporangia of Phytophthora capsici. Daidzin can effectively inhibit the germination of zoospores of Phytophthora capsici, while there is no inhibitory phenomenon with 0.2% v / v DMSO. This indicates that Daidzin has an obvious inhibitory effect on the spore release and germination of Phytophthora capsici.

[0039] Example 4 - Effect of Daidzin on the Pathogenicity and Virulence of Phytophthora capsici

[0040] As in the previous steps of Example 3, obtain the sporangia suspension of Phytophthora capsici using sterile water or different concentrations of Daidzin solution, adjust the sporangia concentration to 1×10 5 cells / mL and then inoculate different pepper varieties (HNUCB0226, HNUCB0081, and XIAO ZHOU PI). The results are shown in Figure 4 .

[0041] As Figure 4 can be seen, for different varieties of peppers, Daidzin can effectively inhibit the infection ability of Phytophthora capsici. This further indicates that Daidzin has an obvious inhibitory effect on the pathogenicity and virulence of Phytophthora capsici.

[0042] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. Application of daidzin in preventing and treating Phytophthora blight of pepper.

2. Use of daidzin in preventing and treating Phytophthora blight of pepper according to claim 1, characterized in that, Daidzin inhibits the growth of Phytophthora capsici.

3. Use of daidzin in preventing and treating Phytophthora blight of pepper according to claim 1 or 2, characterized in that, Daidzin induces apoptotic-like death of Phytophthora capsici spores.

4. Use of daidzin in preventing and treating Phytophthora blight of pepper according to claim 1 or 2, characterized in that, Daidzin inhibits the germination of Phytophthora capsici zoospores.

5. Application of daidzin in enhancing the disease resistance against Phytophthora blight of pepper caused by Phytophthora capsici.

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