CN115873418A - Preparation method and application of phytophthora oospore coloring agent - Google Patents
Preparation method and application of phytophthora oospore coloring agent Download PDFInfo
- Publication number
- CN115873418A CN115873418A CN202211483475.1A CN202211483475A CN115873418A CN 115873418 A CN115873418 A CN 115873418A CN 202211483475 A CN202211483475 A CN 202211483475A CN 115873418 A CN115873418 A CN 115873418A
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- China
- Prior art keywords
- phytophthora
- epicatechin
- oospore
- oxide
- dyeing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- 241000233614 Phytophthora Species 0.000 title claims abstract description 30
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 239000003086 colorant Substances 0.000 title claims description 4
- PFTAWBLQPZVEMU-UKRRQHHQSA-N (-)-epicatechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-UKRRQHHQSA-N 0.000 claims abstract description 18
- 238000004043 dyeing Methods 0.000 claims abstract description 18
- PFTAWBLQPZVEMU-ZFWWWQNUSA-N (+)-epicatechin Natural products C1([C@@H]2OC3=CC(O)=CC(O)=C3C[C@@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-ZFWWWQNUSA-N 0.000 claims abstract description 15
- LPTRNLNOHUVQMS-UHFFFAOYSA-N epicatechin Natural products Cc1cc(O)cc2OC(C(O)Cc12)c1ccc(O)c(O)c1 LPTRNLNOHUVQMS-UHFFFAOYSA-N 0.000 claims abstract description 15
- 235000012734 epicatechin Nutrition 0.000 claims abstract description 15
- 238000010186 staining Methods 0.000 claims abstract description 13
- 239000000126 substance Substances 0.000 claims abstract description 10
- 238000000034 method Methods 0.000 claims abstract description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 150000008442 polyphenolic compounds Chemical class 0.000 claims description 8
- 235000013824 polyphenols Nutrition 0.000 claims description 8
- 239000012452 mother liquor Substances 0.000 claims description 7
- 239000008367 deionised water Substances 0.000 claims description 6
- 229910021641 deionized water Inorganic materials 0.000 claims description 6
- 238000005481 NMR spectroscopy Methods 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- 241000948155 Phytophthora sojae Species 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 239000011347 resin Substances 0.000 claims description 3
- 229920005989 resin Polymers 0.000 claims description 3
- 241000736483 Phytophthora hibernalis Species 0.000 claims description 2
- 244000269722 Thea sinensis Species 0.000 claims description 2
- 235000006468 Thea sinensis Nutrition 0.000 claims description 2
- 235000020279 black tea Nutrition 0.000 claims description 2
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 claims description 2
- 229920000642 polymer Polymers 0.000 claims description 2
- 235000014347 soups Nutrition 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 11
- 229950001002 cianidanol Drugs 0.000 description 5
- 239000000975 dye Substances 0.000 description 5
- 230000003071 parasitic effect Effects 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 229920000742 Cotton Polymers 0.000 description 1
- 241000233679 Peronosporaceae Species 0.000 description 1
- 241000233639 Pythium Species 0.000 description 1
- 206010039509 Scab Diseases 0.000 description 1
- 208000031320 Teratogenesis Diseases 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- MYSWGUAQZAJSOK-UHFFFAOYSA-N ciprofloxacin Chemical compound C12=CC(N3CCNCC3)=C(F)C=C2C(=O)C(C(=O)O)=CN1C1CC1 MYSWGUAQZAJSOK-UHFFFAOYSA-N 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000010413 mother solution Substances 0.000 description 1
- 231100000378 teratogenic Toxicity 0.000 description 1
- 230000003390 teratogenic effect Effects 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
Images
Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a preparation method and application of a phytophthora oospore stain, relating to the field of phytophthora oospore staining and aiming to identify that an orange substance is an oxide of epicatechin; according to the invention, the epicatechin oxide is used as a dyeing substance, the dyeing substance is high in safety, the phytophthora oospore is dyed, the dyeing result shows that the dyeing effect is obvious, and the activity of the phytophthora oospore is not influenced in the dyeing process.
Description
Technical Field
The invention relates to the field of phytophthora oospore dyeing, and particularly relates to a preparation method and application of a phytophthora oospore dyeing agent.
Background
Phytophthora is parasitic mostly, and is easy to induce various complicated plant diseases in agricultural production.
Phytophthora is almost all phytopathogens, mostly facultative parasitic, parasitic from weak to close to obligate parasitic, wide in host range, capable of infecting the overground and underground parts of plants, surviving as chlamydospores or oospores in soil, weak in saprophytic capacity in soil, damaging plants to form white cotton floccules similar to pythium on the surfaces of scabs or frost mildew layers similar to downy mildew, generally called epidemic diseases, and characterized in that the produced sporangium is nearly spherical, oval or pear-shaped.
In recent years, phytophthora has been studied more and more intensively, and the study includes the staining study of phytophthora oospores.
The problems that the dyeing effect is poor, the dyeing agent is unsafe and extremely toxic, the human life is easily damaged and teratogenic, or the dyeing effect is excellent but the activity of the phytophthora oospore can be inhibited and the like are solved.
Disclosure of Invention
The invention aims to provide a preparation method and application of a phytophthora oospore stain, and aims to solve the technical problems that the stain is poor in effect, unsafe, extremely toxic and easy to damage human life and cause teratogenesis, or the stain is excellent in effect and can inhibit the activity of phytophthora oospore in the existing staining research.
In order to solve the technical problems, the invention adopts the following technical scheme:
a phytophthora oospore stain is characterized in that an orange substance is an oxide of epicatechin;
wherein, the molecular structural formula of the epicatechin oxide is as follows:
the method for preparing the phytophthora oospore coloring agent by using the epicatechin oxide comprises the following preparation steps of:
1) Preparing an epicatechin oxide into a dyeing mother solution of 10mmol/L by using deionized water;
2) The dyeing mother liquor of 10mmol/L is stored at 4-10 ℃ for standby.
Preferably, 10mmol/L of the dye mother liquor is diluted to a concentration of 1mmol/L with deionized water before hypha staining.
The phytophthora ovata spore stain is applied by taking a V8 culture medium to culture phytophthora sojae for 5-7 days, adding a proper volume of 1mmol/L epicatechin oxide, placing for 15-30 minutes under natural conditions, washing for 3-5 times by using sterile pure water, then making a glass slide, and observing by using a microscope, wherein the red-orange ovata spores are observed.
The preparation method of epicatechin oxide (dehydro-di-catechin) is as follows:
1) Extracting polyphenol substances from black tea soup;
2) Purifying the eluted and purified polyphenol by using macroporous resin;
3) The purified polyphenol is identified as dehydro-epicatechin by LC-MSMS and nuclear magnetic resonance, and the new polyphenol oxidized polymer is orange red in nature.
Dehydro-di-catechin, which is orange in water, was found to be safe by the MTT method.
The beneficial effects of the invention are:
in the invention, the oxide of epicatechin (dehydro-catechin) is used as a dyeing substance to dye phytophthora oospore, the dyeing result shows that the dyeing effect is obvious, and the activity of the phytophthora oospore is not influenced in the dyeing process.
Drawings
FIG. 1 is a schematic view of a control group according to the present invention;
FIG. 2 is a schematic view of the experimental group of the present invention when stained for 15 minutes;
FIG. 3 is a schematic view showing the staining time of the experimental group for 30 minutes in the present invention;
FIG. 4 is a schematic view showing the staining time of the experimental group of the present invention at 60 minutes;
FIG. 5 is a schematic view showing the staining time of 120 minutes in the experimental group of the present invention;
FIG. 6 is a schematic view showing the staining time of 180 minutes in the experimental group of the present invention;
FIG. 7 is a NMR chart of orange-colored substances in accordance with the invention;
FIG. 8 is a schematic representation of extracellular dehydroepicatechin measured at 390nm or 420nm in accordance with the present invention;
FIG. 9 is a schematic representation of the experimental group of the present invention before unstaining;
FIG. 10 is a schematic representation of the experimental group of the present invention after staining.
Detailed Description
The present invention will be further described with reference to the following detailed description and the accompanying drawings, which are provided for the purpose of illustrating the invention and are not intended to limit the invention to the particular embodiments disclosed. Other embodiments obtained by persons skilled in the art without making creative efforts based on the embodiments in the implementation belong to the protection scope of the invention.
Specific embodiments of the present invention are described below with reference to the accompanying drawings.
Example 1
In this embodiment, a preparation method of a phytophthora oospore stain is provided, which includes the following preparation steps:
1) Preparing epicatechin oxide into 10mmol/L dyeing mother liquor by using deionized water;
2) The dyeing mother liquor of 10mmol/L is stored at 4-10 ℃ for later use.
Note that 10mmol/L of the dye stock was diluted to a concentration of 1mmol/L with deionized water prior to hyphal staining.
Example 2
Using the phytophthora oospore stain prepared in example 1, the application of a phytophthora oospore stain is proposed in this example, which includes:
1) Taking a V8 culture medium to culture phytophthora such as phytophthora sojae and phytophthora hibernalis for 5-7 days in liquid, adding a proper volume of 1mmol/L epicatechin oxide (dehydro-catechin) to stand for 15-30 minutes under natural conditions, washing for 3-5 times by using sterile pure water, then making a glass slide, and observing by using a microscope to observe orange oospores;
2) Homogenizing the hyphae, washing with distilled water, filtering with quick filter paper, and collecting an orange liquid substance;
3) Filtering with ultrafiltration membrane 0.22, adsorbing and eluting with macroporous resin, and freeze drying.
Example 3
Based on example 2, a control group and an experimental group were set in this example;
wherein the control group is not added with dehydroepicatechin, and the experimental group is added with 1mol/L dehydroepicatechin;
the control combined experimental groups are respectively observed for 180 minutes;
see figure 1 for a schematic of the control experiment;
for a graphic representation of the experiments in the experimental group, reference is made to FIGS. 2-6.
Referring to FIG. 7, NMR shows that the exocarpium Citri rubrum in Phytophthora oospore is epicatechin oxide (11 delta type dehydro-catechin).
Referring to FIG. 8, extracellular dehydroepicatechin was obtained by measurement at 390 or 420nm, in oospores, and was actively transported.
Referring to FIGS. 9-10, staining did not affect activity and the culture was continued.
The foregoing shows and describes the general principles, principal features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and the preferred embodiments of the present invention are described in the above embodiments and the description, and are not intended to limit the present invention. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (5)
1. A phytophthora oospore stain is characterized in that an orange substance to be identified is an oxide of epicatechin;
wherein, the molecular structural formula of the epicatechin oxide is as follows:
2. a method for preparing an epicatechin oxide, comprising the steps of:
1) Extracting polyphenol substances from black tea soup;
2) Purifying the eluted purified polyphenols using a macroporous resin;
3) The purified polyphenol is identified as dehydro-epicatechin by LC-MSMS and nuclear magnetic resonance, and the new polyphenol oxidized polymer is orange red in nature.
3. The method for preparing the phytophthora oospore coloring agent by using the epicatechin oxide is characterized by comprising the following preparation steps of:
1) Preparing epicatechin oxide into 10mmol/L dyeing mother liquor by using deionized water;
2) The dyeing mother liquor of 10mmol/L is stored at 4-10 ℃ for later use.
4. The method for preparing a phytophthora oospore stain according to claim 3, wherein: before hypha staining, 10mmol/L of staining mother liquor is diluted to 1mmol/L by deionized water.
5. The application of the phytophthora oospore stain is characterized in that: taking V8 culture medium liquid to culture phytophthora such as phytophthora sojae and phytophthora hibernalis for 5-7 days, adding a proper volume of 1mmol/L epicatechin oxide, standing for 15-30 minutes under natural conditions, washing for 3-5 times by using sterile pure water, then making a glass slide, and observing by a microscope, wherein orange-red oospores are observed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211483475.1A CN115873418A (en) | 2022-11-24 | 2022-11-24 | Preparation method and application of phytophthora oospore coloring agent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211483475.1A CN115873418A (en) | 2022-11-24 | 2022-11-24 | Preparation method and application of phytophthora oospore coloring agent |
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| Publication Number | Publication Date |
|---|---|
| CN115873418A true CN115873418A (en) | 2023-03-31 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211483475.1A Pending CN115873418A (en) | 2022-11-24 | 2022-11-24 | Preparation method and application of phytophthora oospore coloring agent |
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| CN (1) | CN115873418A (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110592173A (en) * | 2019-08-29 | 2019-12-20 | 刘冬 | Rapid identification kit for phytophthora sojae and preparation method thereof |
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- 2022-11-24 CN CN202211483475.1A patent/CN115873418A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110592173A (en) * | 2019-08-29 | 2019-12-20 | 刘冬 | Rapid identification kit for phytophthora sojae and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| VERLOOP, ANNEWIEKE J. W.: ""Peroxidase Can Perform the Hydroxylation Step in the "Oxidative Cascade" d uring Oxidation of Tea Catechins"", 《JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY》, vol. 64, no. 42, pages 8002 - 8009 * |
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Application publication date: 20230331 |