CN110592173A - Rapid identification kit for phytophthora sojae and preparation method thereof - Google Patents
Rapid identification kit for phytophthora sojae and preparation method thereof Download PDFInfo
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- CN110592173A CN110592173A CN201910806451.7A CN201910806451A CN110592173A CN 110592173 A CN110592173 A CN 110592173A CN 201910806451 A CN201910806451 A CN 201910806451A CN 110592173 A CN110592173 A CN 110592173A
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- epicatechin
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- 241000948155 Phytophthora sojae Species 0.000 title claims abstract description 26
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- XMOCLSLCDHWDHP-IUODEOHRSA-N epi-Gallocatechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@H]2O)=CC(O)=C(O)C(O)=C1 XMOCLSLCDHWDHP-IUODEOHRSA-N 0.000 claims abstract description 24
- 235000012734 epicatechin Nutrition 0.000 claims abstract description 22
- 239000000126 substance Substances 0.000 claims abstract description 22
- 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 21
- ADRVNXBAWSRFAJ-UHFFFAOYSA-N catechin Natural products OC1Cc2cc(O)cc(O)c2OC1c3ccc(O)c(O)c3 ADRVNXBAWSRFAJ-UHFFFAOYSA-N 0.000 claims abstract description 21
- 235000005487 catechin Nutrition 0.000 claims abstract description 21
- PFTAWBLQPZVEMU-DZGCQCFKSA-N (+)-catechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-DZGCQCFKSA-N 0.000 claims abstract description 20
- XMOCLSLCDHWDHP-UHFFFAOYSA-N L-Epigallocatechin Natural products OC1CC2=C(O)C=C(O)C=C2OC1C1=CC(O)=C(O)C(O)=C1 XMOCLSLCDHWDHP-UHFFFAOYSA-N 0.000 claims abstract description 20
- 229950001002 cianidanol Drugs 0.000 claims abstract description 20
- DZYNKLUGCOSVKS-UHFFFAOYSA-N epigallocatechin Natural products OC1Cc2cc(O)cc(O)c2OC1c3cc(O)c(O)c(O)c3 DZYNKLUGCOSVKS-UHFFFAOYSA-N 0.000 claims abstract description 20
- 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 19
- 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 19
- XMOCLSLCDHWDHP-SWLSCSKDSA-N (+)-Epigallocatechin Natural products C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@@H]2O)=CC(O)=C(O)C(O)=C1 XMOCLSLCDHWDHP-SWLSCSKDSA-N 0.000 claims abstract description 16
- 201000010099 disease Diseases 0.000 claims abstract description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 5
- 244000068988 Glycine max Species 0.000 claims description 12
- 235000010469 Glycine max Nutrition 0.000 claims description 12
- 235000013824 polyphenols Nutrition 0.000 claims description 12
- 239000001963 growth medium Substances 0.000 claims description 9
- 150000008442 polyphenolic compounds Chemical class 0.000 claims description 9
- 238000012360 testing method Methods 0.000 claims description 9
- VFPFQHQNJCMNBZ-UHFFFAOYSA-N ethyl gallate Chemical compound CCOC(=O)C1=CC(O)=C(O)C(O)=C1 VFPFQHQNJCMNBZ-UHFFFAOYSA-N 0.000 claims description 8
- LSHVYAFMTMFKBA-UHFFFAOYSA-N ECG Natural products C=1C=C(O)C(O)=CC=1C1OC2=CC(O)=CC(O)=C2CC1OC(=O)C1=CC(O)=C(O)C(O)=C1 LSHVYAFMTMFKBA-UHFFFAOYSA-N 0.000 claims description 6
- 239000002207 metabolite Substances 0.000 claims description 6
- 239000002244 precipitate Substances 0.000 claims description 6
- 238000012545 processing Methods 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- LSHVYAFMTMFKBA-TZIWHRDSSA-N (-)-epicatechin-3-O-gallate Chemical compound O([C@@H]1CC2=C(O)C=C(C=C2O[C@@H]1C=1C=C(O)C(O)=CC=1)O)C(=O)C1=CC(O)=C(O)C(O)=C1 LSHVYAFMTMFKBA-TZIWHRDSSA-N 0.000 claims description 4
- 239000004262 Ethyl gallate Substances 0.000 claims description 4
- WMBWREPUVVBILR-UHFFFAOYSA-N GCG Natural products C=1C(O)=C(O)C(O)=CC=1C1OC2=CC(O)=CC(O)=C2CC1OC(=O)C1=CC(O)=C(O)C(O)=C1 WMBWREPUVVBILR-UHFFFAOYSA-N 0.000 claims description 4
- 229940030275 epigallocatechin gallate Drugs 0.000 claims description 4
- 235000019277 ethyl gallate Nutrition 0.000 claims description 4
- 244000052769 pathogen Species 0.000 claims description 4
- 241000233614 Phytophthora Species 0.000 claims description 3
- 244000269722 Thea sinensis Species 0.000 claims description 3
- 238000013178 mathematical model Methods 0.000 claims description 3
- -1 polyphenols epigallocatechin gallate Chemical class 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 238000001514 detection method Methods 0.000 description 8
- 239000002689 soil Substances 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- 241000233629 Phytophthora parasitica Species 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 244000052616 bacterial pathogen Species 0.000 description 2
- 244000053095 fungal pathogen Species 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- WMBWREPUVVBILR-WIYYLYMNSA-N (-)-Epigallocatechin-3-o-gallate Chemical compound O([C@@H]1CC2=C(O)C=C(C=C2O[C@@H]1C=1C=C(O)C(O)=C(O)C=1)O)C(=O)C1=CC(O)=C(O)C(O)=C1 WMBWREPUVVBILR-WIYYLYMNSA-N 0.000 description 1
- XMOCLSLCDHWDHP-DOMZBBRYSA-N (-)-gallocatechin Chemical compound C1([C@@H]2OC3=CC(O)=CC(O)=C3C[C@H]2O)=CC(O)=C(O)C(O)=C1 XMOCLSLCDHWDHP-DOMZBBRYSA-N 0.000 description 1
- VYIRVAXUEZSDNC-TXDLOWMYSA-N (3R,3'S,5'R)-3,3'-dihydroxy-beta-kappa-caroten-6'-one Chemical compound C([C@H](O)CC=1C)C(C)(C)C=1/C=C/C(/C)=C/C=C/C(/C)=C/C=C/C=C(C)C=CC=C(C)C=CC(=O)[C@]1(C)C[C@@H](O)CC1(C)C VYIRVAXUEZSDNC-TXDLOWMYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- VYIRVAXUEZSDNC-LOFNIBRQSA-N Capsanthyn Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CC(O)CC1(C)C)C=CC=C(/C)C=CC(=O)C2(C)CC(O)CC2(C)C VYIRVAXUEZSDNC-LOFNIBRQSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- WRANYHFEXGNSND-LOFNIBRQSA-N capsanthin Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CC(O)CC1(C)C)C=CC=C(/C)C=CC(=O)C2(C)CCC(O)C2(C)C WRANYHFEXGNSND-LOFNIBRQSA-N 0.000 description 1
- 235000018889 capsanthin Nutrition 0.000 description 1
- 150000001765 catechin Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000005059 dormancy Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 235000012658 paprika extract Nutrition 0.000 description 1
- 239000001688 paprika extract Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Toxicology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
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Abstract
The invention provides a quick identification kit for phytophthora sojae and a preparation method thereof, and relates to the field of biological control of plant diseases.A kit comprises gallocatechin, catechin and epicatechin, which can generate orange substances with the phytophthora sojae, and the proportion of the catechin, the gallocatechin and the epicatechin is 3.8-4.5%, 12.0-15.5% and 80-84.2%.
Description
Technical Field
The invention relates to the field of biological control of plant diseases, in particular to a phytophthora sojae rapid identification kit and a preparation method thereof.
Background
The soybean phytophthora caused by phytophthora sojae is one of the devastating diseases in soybean production, and is one kind of plant quarantine dangerous pathogenic fungi published externally in China. The pathogen is mainly spread in soil. Currently, the major soybean exporting countries in the world, such as the united states, brazilian argentina, etc., have the occurrence and distribution of soybean blight, and the exported commercial soybeans often contain quantitative soil. In order to prevent the pathogen from entering China, the quarantine identification method of the soybean phytophthora parasitica needs to be standardized and correctly mastered when the imported plant is quarantined, the existing detection method determines various technical requirements of quarantine detection and identification of the soybean phytophthora parasitica according to the characteristics of soil spreading characteristics, biological characteristic morphological characteristics and the like of the soybean phytophthora parasitica, and the detection principle is as follows: the pathogenic fungi are typical soil inhabitation bacteria, and mainly live in the soil and host residues in the soil for a long time in the form of highly stress-resistant oospores. When the field temperature and humidity are proper, the oospores break dormancy to germinate to produce sporangia, when water is accumulated after rain, the sporangia quickly releases a large amount of zoospores, and the zoospores are splashed to the surface of the host soil by rain water or are attracted by host secretions. Move towards the root system and finally infect the host. When water exists, a large number of sporangia are formed on the outer surface of the root of a host, a large number of oospores are formed in the host, the characteristic of pathogenic bacteria is that soil is collected and cultured for a certain time under proper conditions, the oospores are germinated to generate sporangia, then water is added for soaking, the sporangia releases zoospores, then the zoospores are induced and collected by a leaf disc of a susceptible soybean variety, and when the zoospores infect the leaf disc and generate new sporangia at the edge of the leaf disc (occasionally on the surface of the leaf disc) and release the zoospores, the separation and identification of the pathogenic bacteria can be carried out.
Disclosure of Invention
The invention aims to provide a rapid identification kit for phytophthora sojae and a preparation method thereof, and aims to solve the technical problem of low detection speed of the phytophthora sojae.
In order to solve the technical problems, the invention adopts the following technical scheme: a kit for rapidly identifying Phytophthora sojae and its preparation method are provided, wherein the kit comprises gallocatechin, catechin and epicatechin, which are capable of generating orange-colored substances with Phytophthora sojae, and the ratio of catechin, gallocatechin and epicatechin is 3.8-4.5%, 12.0-15.5% and 80-84.2%.
The preparation of the kit comprises the following steps:
(1) adding polyphenol substances into the culture medium, wherein the polyphenol substances can form orange substances with phytophthora sojae metabolites, and the orange substances cannot be formed with other phytophthora sojae;
(2) dimox catechin, catechin and epicatechin were selected from seven kinds of tea polyphenols epigallocatechin gallate (EGCG), Gallocatechin (GC), Epigallocatechin (EGC), catechin (C), epicatechin gallate (ECG), Epicatechin (EC) and Ethyl Gallate (EG) and subjected to color chromaticity test.
(3) Optimally combining the proportions of the three components by a mathematical model, adding the mixture into a culture medium to obtain bright orange precipitates which are easy to identify, and analyzing and processing test data by using Design-expert7.0 to obtain the optimal proportion of gallocatechin, catechin and epicatechin;
(4) adding the mixed reagent into a culture medium according to the optimal ratio for preparation, sterilizing, pouring into a small sterile plate, and placing into a sterile container to obtain the rapid identification kit.
The regression equation for analyzing and processing the test data by using Design-expert7.0 is as follows: y ═ 90.6-0.25A +3.25B-0.75C-0.25AB-2.25AC +3.75BC-3.42A2-3.92B2-5.43C2。
The invention has the beneficial effects that:
experiments show that several polyphenol substances can react with phytophthora sojae metabolites to generate orange precipitates, gallocatechin, catechin and epicatechin, which only react with the phytophthora sojae metabolites, are screened out in a comparison mode, Design-expert7.0 is used for analyzing experimental data to obtain the optimal proportion of the polyphenol substances, and a mixture prepared according to the proportion is placed in a sterile container to obtain an identification reagent which can react with phytophthora sojae surrogate products to generate the orange precipitates.
Drawings
FIG. 1 is a graph showing the effect of the medium supplemented with a polyphenol substance according to the present invention.
Detailed Description
The present invention will be further described with reference to specific embodiments for the purpose of facilitating an understanding of technical means, characteristics of creation, objectives and functions realized by the present invention, but the following embodiments are only preferred embodiments of the present invention, and are not intended to be exhaustive. Based on the embodiments in the implementation, other embodiments obtained by those skilled in the art without any creative efforts belong to the protection scope of the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified, and materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
As shown in figure 1, the kit comprises gallocatechin, catechin and epicatechin, and the components in the kit can generate orange red substances with the phytophthora sojae, the proportion of the catechin, the gallocatechin and the epicatechin is 4.5%, 15.5% and 80%, the adding concentration is 2mmol/L, the rightmost side in figure 1 is the result of the phytophthora sojae culture under the condition, and the color of the generated red substances is deepest.
The preparation of the kit comprises the following steps:
as shown in Table 1, the addition of polyphenol substances to the culture medium can form orange-colored substances with phytophthora sojae metabolites, but the orange-colored substances cannot be formed with other phytophthora sojae and common soybean disease pathogens;
TABLE 1
In table 1, "+" represents the formation of an orange-colored substance and "-" represents no color; more "+" indicates more obvious color and easier recognition.
As shown in table 2, catechins and epicatechins were selected from seven kinds of tea polyphenols epigallocatechin gallate, gallocatechin, epigallocatechin, catechin, epicatechin gallate, epicatechin and ethyl gallate, and subjected to a color chromaticity test.
TABLE 2
Taking 0-50% of capsanthin as a reference color for contrast and scoring, multiplying a scoring result by 2 times to obtain color chromaticity, and optimally combining the three proportions, wherein in the table 2, "+ 1, -1 and 0" respectively represent the concentrations of three levels of polyphenol substances; wherein the numbers in () each represent the ratio of polyphenols in the polyphenolic substance experiment.
Combining the experimental results in the table 2, optimally combining the proportions of the three by a mathematical model, adding the mixture into a culture medium to obtain bright orange-colored precipitates which are easy to identify, analyzing and processing test data by using Design-expert7.0 to obtain the optimal proportion of the gallocatechin, the catechin and the epicatechin, and finally obtaining the optimal composition proportions of the gallocatechin, the catechin and the epicatechin of 3.8-4.5%, 12.0-15.5% and 80-84.2%;
adding the mixed reagent into a culture medium according to the optimal ratio for preparation, sterilizing, pouring into a small sterile plate, and placing into a sterile container to obtain the rapid identification kit.
The regression equation for analyzing and processing the test data by using Design-expert7.0 is as follows:
Y＝90.6-0.25A+3.25B-0.75C-0.25AB-2.25AC+3.75BC-3.42A2-3.92B2-5.43C2wherein A, B, C, Y are catechin, gallocatechin, epicatechin, and color shade, respectively.
The detection kit can react with the phytophthora sojae metabolite to generate the orange precipitate substance through the steps, so that whether the phytophthora sojae exists or not is judged, the detection kit can be used for detecting the phytophthora sojae at any time through advanced preparation, the detection speed is high, the detection result is obvious, and the observation is convenient.
The foregoing shows and describes the general principles, essential 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 (3)
1. A rapid identification kit for phytophthora sojae and a preparation method thereof are characterized in that: the components in the kit comprise gallocatechin, catechin and epicatechin, which can generate orange red substance with Phytophthora sojae, and the ratio of catechin, gallocatechin and epicatechin is 3.8-4.5%, 12.0-15.5% and 80-84.2%.
2. The kit for rapidly identifying phytophthora sojae according to claim 1 and the preparation method thereof, wherein the kit comprises: the preparation of the kit comprises the following steps:
(1) the culture medium is added with polyphenol substances, so that orange substances can be formed with soybean phytophthora metabolites, and the orange substances cannot be formed with other phytophthora and common soybean disease pathogens;
(2) selecting epicatechin, catechin and epicatechin from seven tea polyphenols epigallocatechin gallate, gallocatechin, epigallocatechin, catechin, epicatechin gallate, epicatechin and ethyl gallate, and performing color chromaticity test;
(3) optimally combining the proportions of the three components by a mathematical model, adding the mixture into a culture medium to obtain bright orange precipitates which are easy to identify, and analyzing and processing test data by using Design-expert7.0 to obtain the optimal proportion of gallocatechin, catechin and epicatechin;
(4) adding the mixed reagent into a culture medium according to the optimal ratio for preparation, sterilizing, pouring into a small sterile plate, and placing into a sterile container to obtain the rapid identification kit.
3. The kit for rapidly identifying phytophthora sojae according to claim 2 and the preparation method thereof, wherein the kit comprises: the regression equation for analyzing and processing the test data by using Design-expert7.0 is as follows: y ═ 90.6-0.25A +3.25B-0.75C-0.25AB-2.25AC +3.75BC-3.42A2-3.92B2-5.43C2。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115873418A (en) * | 2022-11-24 | 2023-03-31 | 吕新霞 | Preparation method and application of phytophthora oospore coloring agent |
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| US20150245620A1 (en) * | 2012-09-11 | 2015-09-03 | Erik Andreasson | Plant extracts comprising at least a phenolic compound, for inducing the natural defence of a plant against a pathogen, such as phytophthora infestans |
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2019
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| US20150245620A1 (en) * | 2012-09-11 | 2015-09-03 | Erik Andreasson | Plant extracts comprising at least a phenolic compound, for inducing the natural defence of a plant against a pathogen, such as phytophthora infestans |
| CN103014152A (en) * | 2012-11-21 | 2013-04-03 | 安徽省农业科学院植物保护与农产品质量安全研究所 | LAMP (loop-mediated isothermal amplification) primer, kit and detecting method for detecting phytophthora sojae |
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| Title |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115873418A (en) * | 2022-11-24 | 2023-03-31 | 吕新霞 | Preparation method and application of phytophthora oospore coloring agent |
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