CN113214241A - Compound for detecting sclerotinia sclerotiorum of cruciferous vegetables and application thereof - Google Patents

Compound for detecting sclerotinia sclerotiorum of cruciferous vegetables and application thereof Download PDF

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CN113214241A
CN113214241A CN202110471715.5A CN202110471715A CN113214241A CN 113214241 A CN113214241 A CN 113214241A CN 202110471715 A CN202110471715 A CN 202110471715A CN 113214241 A CN113214241 A CN 113214241A
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sclerotinia sclerotiorum
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徐文平
鲁剑
陶黎明
张阳
陈文涛
胡浩富
王鑫
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East China University of Science and Technology
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Abstract

The invention discloses a compound for detecting sclerotinia sclerotiorum of cruciferous vegetables, which has the following structure:
Figure DDA0003045687570000011
wherein R is1Selected from hydrogen, halogen; r2Selected from hydrogen, halogen; r3Selected from hydrogen, halogen; r4Selected from hydrogen, halogen; r5Selected from hydrogen and halogen. The compound disclosed by the invention can realize rapid detection of sclerotinia sclerotiorum of cruciferous vegetables, has the characteristics of high accuracy, low operation difficulty, short time consumption and the like, and is worthy of popularization and application.

Description

Compound for detecting sclerotinia sclerotiorum of cruciferous vegetables and application thereof
Technical Field
The invention belongs to the technical field of rapid detection of plant pathogenic bacteria, and particularly relates to a compound for detecting cruciferous vegetable Sclerotinia sclerotiorum (sclerotiorum) and application thereof.
Background
Sclerotinia sclerotiorum belongs to the subgenus of ascomycetes, is an important soil-borne disease, can infect more than 400 kinds of plants, common important hosts comprise plants such as sunflower, lettuce, peanut, broad bean and the like, especially cruciferous vegetables, become one of main diseases of the cruciferous vegetables, and seriously affect the production of the cruciferous vegetables in China.
The primary source of infection for sclerotinia sclerotiorum is sclerotia that is dormant in soil. Under appropriate conditions, the sclerotium germinates, producing ascospores in the air, which usually land on the surface of cruciferous vegetable petals and infect the petals. Sclerotinia can infect all parts of crucifer plants, including stems, leaves, flowers and pods. During the onset, the pathogenic bacteria mainly infect the stems and leaves of cruciferous plants. After the disease, the leaves at the bottom of the plant usually begin to age and yellow, and the back of the leaves with the disease spots turns yellow brown from green. The infected stem turns light brown, initially with water-stained lesions, which then expand into depressions in the form of long spindle shapes with a white middle and brown edges. The stem surface of the diseased plant becomes dry and fragile, the vascular bundle is exposed, the stem begins to decay and erode, and a large amount of rat manure-like sclerotia is formed. Sclerotia on the stem surface can also be seen at high humidity.
The traditional method for separately culturing the pathogenic bacteria consumes long time, pathogenic bacteria are identified by observing the ascospore form of sclerotinia sclerotiorum of cruciferae, the identification result is influenced by personal subjectivity, and the accuracy is unreliable; the method for detecting the phytopathogens by the ELISA method has the limitations of low immunogenicity of the phytopathogens, interference of inhibitory substances in plant extracts and the detection cost of phenanthrene; the commonly used quantitative PCR assays such as PCR-RFLP and PCR-SSCP also have the disadvantages of high assay cost and applicability to only a few pathogenic bacteria. At present, no reagent for specifically detecting and diagnosing sclerotinia sclerotiorum of cruciferous vegetables exists in the field of plant protection.
Therefore, a technical support which can quickly identify the cruciferae, diagnose, detect and identify the fusion group in the early disease stage, effectively prevent and treat the diseases in time, reduce the economic loss and have important field disease prevention and treatment is urgently needed.
Disclosure of Invention
Aiming at the technical problems that the detection of sclerotinia sclerotiorum of cruciferous vegetables in the prior art method consumes long time, has complex detection conditions and inaccurate test results, and the prediction, prevention and control of sclerotinia sclerotiorum of cruciferous vegetables are difficult to solve, the invention aims to provide the compound for detecting the sclerotinia sclerotiorum of cruciferous vegetables.
The second object of the present invention is to provide a kit prepared from the compound for detecting sclerotinia sclerotiorum of brassicaceous vegetables.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the invention provides a compound for detecting sclerotinia sclerotiorum of cruciferous vegetables, which has the following structure:
Figure BDA0003045687550000021
wherein R is1Selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine); r2Selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine); r3Selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine); r4Selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine); r5Selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine).
Preferably, the compound for detecting sclerotinia sclerotiorum of cruciferous vegetables has the following structure:
Figure BDA0003045687550000022
the second aspect of the present invention provides a preparation method of the compound for detecting sclerotinia sclerotiorum of brassicaceous vegetables, comprising the steps of:
Figure BDA0003045687550000023
dissolving ethanolamine and 2-pyridine formaldehyde with a molar ratio of 1:1 in methanol, stirring at room temperature for 1-48 h, adding sodium borohydride into the reaction solution in batches under an ice bath condition, stirring for 0.5 h, stirring in an oil bath at 45-55 ℃ for 1-12 h, tracking by TLC (thin layer chromatography), cooling to room temperature, quenching the redundant sodium borohydride with hydrochloric acid under the ice bath condition, neutralizing the reaction solution with 40% NaOH aqueous solution, adjusting the pH to about 10, extracting with dichloromethane, concentrating to obtain a crude product, and performing column chromatography (dichloromethane/methanol/triethylamine is 150:20:1) to obtain a compound 1;
Figure BDA0003045687550000024
dissolving a compound 1 in absolute ethyl alcohol, slowly adding a compound 2 within 1-10 min, wherein the molar ratio of the compound 1 to the compound 2 is 1:1, stirring at room temperature for 10-60 min, performing TLC (thin layer chromatography) tracking reaction until the reaction is finished, and removing the solvent under reduced pressure to obtain a compound 3;
Figure BDA0003045687550000031
dropwise adding excessive concentrated hydrochloric acid into the compound 3, refluxing for 10-60 min at 85-95 ℃, tracking and reacting by TLC (thin layer chromatography), cooling the reaction solution to room temperature, adjusting the pH to 10-11 by using 40% NaOH solution under ice bath, and using CH (CH)2Cl2Extracting, washing organic phase with saturated saline solution, and removing anhydrous Na2SO4Drying, removing the solvent under reduced pressure to obtain a crude product, and purifying by column chromatography (ethyl acetate: V petroleum ether ═ 1:3) or recrystallization from ethanol to obtain compound 4.
The third aspect of the present invention provides a kit prepared from the compound for detecting sclerotinia sclerotiorum of brassicaceous vegetables.
The kit comprises a kit main body A, a detection reagent B and an auxiliary reagent C;
the kit main body A: at least one of potato dextrose broth, PDB medium or PDA solid medium, in a volume of 1000 mL;
and (3) detection reagent B: compound 4, sealed at 4 ℃ in the dark;
and (5) storing an auxiliary reagent C and streptomycin in a dark and sealed manner at normal temperature.
The preparation method of the potato glucose broth culture medium comprises the following steps: cleaning and peeling potatoes, cutting the potatoes into small pieces, adding water, boiling until the small pieces of the potatoes can be slightly punctured, filtering potato residues by using thick gauze, adding glucose into filtrate, wherein the mass ratio of the glucose to the potatoes is 1:10, stirring for dissolving, adding water to a constant volume of 1000mL, cooling and packaging the filtrate, sterilizing, taking out and cooling, and storing for later use.
The sclerotinia sclerotiorum of cruciferous vegetables is obtained by separating and purifying plant cotyledons, stems or soil containing the sclerotinia sclerotiorum of cruciferous vegetables, and is stored in a laboratory for later use.
The fourth aspect of the invention provides an application of the compound for detecting sclerotinia sclerotiorum of cruciferous vegetables in preparing a detection reagent for sclerotinia sclerotiorum of cruciferous vegetables.
The fifth aspect of the invention provides a method for rapidly detecting sclerotinia sclerotiorum of cruciferous vegetables by using the kit, which comprises the following steps:
weighing a detection reagent B, namely a compound 4, adding the detection reagent B into a hot-melt 1000mL kit main body A (at least one of a potato glucose broth culture medium, a PDB culture medium or a PDA solid culture medium) to prepare a liquid medicine with the concentration of 20-50 mu g/mL (preferably 30 mu g/mL), adding an auxiliary reagent C, namely streptomycin, which accounts for 0.1% of the mass percent of the compound 4, shaking up, flatly pouring the liquid medicine on a flat plate, and cooling the liquid medicine for use;
weighing 2g of a sample to be detected, inoculating the sample to the culture system, culturing the system in a microorganism constant-temperature shaking incubator at 200rpm and 28 ℃ for 24-72 h, and observing the color change of hyphae.
The sample to be detected is a plant cotyledon, a stem or soil containing sclerotinia sclerotiorum of vegetables of cruciferae.
The observation of the color change of the mycelia refers to the observation of the color of the mycelia in the culture medium by naked eyes or a microscope, and if green mycelia are observed, the sample contains sclerotinia sclerotiorum of vegetables of Brassicaceae.
Due to the adoption of the technical scheme, the invention has the following advantages and beneficial effects:
the invention provides that the compound 4 prepared in the example 1 has the color of hyphae changed to green when only acting with sclerotinia sclerotiorum of cruciferous vegetables among 9 different plant pathogenic bacteria, and the hyphae of the other 8 plant pathogenic bacteria of botrytis cinerea, peanut brown spot pathogen, rice blast pathogen, wheat scab pathogen, apple ring rot pathogen, potato late blight pathogen, banana leaf spot pathogen and banana vascular wilt pathogen do not have green change, so that the compound can be made into a kit by utilizing the characteristic of the specific change of the compound on the sclerotinia sclerotiorum of cruciferous vegetables.
The invention relates to a detection method based on the sclerotinia sclerotiorum of cruciferous vegetables and the color change of a culture system of a kit, which can quickly detect whether sclerotinia sclerotiorum of cruciferous vegetables exists in a planting environment before the cruciferous vegetables are planted, thereby preventing pathogenic bacteria. The sclerotinia sclerotiorum of the cruciferous vegetables is distinguished and eliminated according to the change of the color, the result is more visual and accurate, and the accuracy rate reaches one hundred percent. Meanwhile, the simple experiment operation method and the requirement for operation are low, and the kit is more favorable for popularization. Compared with the traditional detection method, the kit is more suitable for detecting the sclerotinia sclerotiorum of the cruciferous vegetables.
The invention can realize the rapid detection of the sclerotinia sclerotiorum of the cruciferous vegetables, has the characteristics of high accuracy, low operation difficulty, short time consumption and the like, and is worthy of popularization and application.
Drawings
Fig. 1 is a schematic view of a kit body a.
FIG. 2 is a schematic diagram containing detection reagent B at a concentration of 30. mu.g/mL.
FIG. 3 is a schematic diagram of auxiliary reagent C.
FIG. 4 is a NMR spectrum of a hydrogen nucleus of the compound 4 as a detection reagent.
FIG. 5 shows the carbon nuclear magnetic resonance spectrum of the detection reagent Compound 4.
FIG. 6 is an EI-MS spectrum of detection reagent Compound 4.
FIG. 7 is a schematic diagram showing the color change of specific hyphae of a detection reagent compound 4 to cruciferous vegetables.
FIG. 8 is a schematic diagram showing the color change of the detection reagent compound 4 on sclerotinia sclerotiorum hyphae of vegetables of Brassicaceae.
FIG. 9 is a schematic diagram showing the color change of the detection reagent compound 4 on the hyphae of Botrytis cinerea.
FIG. 10 is a schematic diagram showing the color change of the hypha of the brown spot pathogen in the detection reagent compound 4.
FIG. 11 is a schematic diagram showing the color change of the hypha of Pyricularia oryzae by the detection reagent compound 4.
FIG. 12 is a schematic diagram showing the color change of the detection reagent compound 4 to hyphae of Fusarium graminearum.
FIG. 13 is a schematic diagram showing the color change of hyphae of ring rot apple with the detection reagent compound 4.
FIG. 14 is a schematic diagram showing the color change of the detection reagent compound 4 on the hyphae of potato late blight bacterium.
FIG. 15 is a schematic diagram showing the color change of the detection reagent compound 4 to the mycelial filament of sigatoka.
FIG. 16 is a schematic view showing the color change of the hyphae of banana vascular wilt bacteria by the detection reagent compound 4.
FIG. 17 is a diagram showing the specific reaction of the detection reagent compound 4 with different concentrations to sclerotinia sclerotiorum of cruciferous vegetables.
FIG. 18 is a diagram showing the specific reaction of the detection reagent compound 4 with different concentrations to sclerotinia sclerotiorum of cruciferous vegetables.
FIG. 19 is a diagram showing the specific reaction of the detection reagent compound 4 with different concentrations to sclerotinia sclerotiorum of cruciferous vegetables.
FIG. 20 is a diagram showing the specific reaction of the detection reagent compound 4 with different concentrations to sclerotinia sclerotiorum of cruciferous vegetables.
FIG. 21 is a schematic diagram showing the growth of cells on four PDA plates.
FIG. 22 is a schematic diagram showing the growth of cells on four PDA plates.
FIG. 23 is a schematic diagram showing the growth of cells on four PDA plates.
FIG. 24 is a schematic view showing the growth of cells on four PDA plates.
Detailed Description
In order to more clearly illustrate the invention, the invention is further described below in connection with preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
Example 1
Synthesis of detection reagent 2- (4-bromophenylimino) -3- (pyridine-2-methyl) thiazolidine
Figure BDA0003045687550000051
In a 250mL round-bottom flask, ethanolamine (1.22g, 20mmol), 2-pyridinecarboxaldehyde (2.14g, 20mmol) and 50mL of methanol were added in this order, and the mixture was stirred at room temperature for 36 hours. Sodium borohydride (1.14g, 30mmol) was added to the reaction in portions under ice bath, stirred for 0.5 h, stirred in 50 ℃ oil bath for 4h, TLC followed the end of the reaction, cooled to room temperature. Under the ice bath condition, excess sodium borohydride is quenched by hydrochloric acid, a 40% NaOH aqueous solution is used for neutralizing the reaction liquid, the pH value is adjusted to be about 10, dichloromethane (3 × 40mL) is used for extraction, a crude product is obtained by concentration, and 3.12g of yellow oily liquid, namely the compound 1, is obtained by column chromatography (dichloromethane/methanol/triethylamine is 150:20:1), and the yield is 93%.
Figure BDA0003045687550000061
In a 50mL round bottom flask, compound 1(0.336g, 2mmol) was dissolved in 5mL absolute ethanol, compound 2(0.27g, 2mmol) was added slowly over 5min, stirred at room temperature for 45min, TLC tracked the reaction to completion, and the solvent was removed under reduced pressure to give 0.51g of a white solid, compound 3, 89% yield.
Figure BDA0003045687550000062
Adding the compound 3(0.287g and 1mmol) into a 25mL round-bottom flask, dropwise adding 5mL concentrated hydrochloric acid, refluxing for 45min at 90 ℃, tracking and reacting by TLC until the reaction is finished, cooling the reaction solution to room temperature after the reaction is finished, and adjusting the pH to 10-11 by using 40% NaOH solution under ice bath to obtain turbid liquid. By CH2Cl2Extracting, washing organic phase with saturated saline solution, and removing anhydrous Na2SO4Drying, removing the solvent under reduced pressure to obtain a crude product, purifying by column chromatography (ethyl acetate: V petroleum ether ═ 1:3) or recrystallization from ethanol to obtain 0.271g of a white solid, i.e., compound 4, in 78% yield, melting point: 73.7-74.9 ℃.
FIG. 4 is a NMR spectrum of a hydrogen nucleus of the compound 4 as a detection reagent. FIG. 5 shows the carbon nuclear magnetic resonance spectrum of the detection reagent Compound 4. FIG. 6 is an EI-MS spectrum of detection reagent Compound 4.1H NMR(400MHz,CDCl3):δ3.09(t,J＝6.80Hz,2H),3.59(t,J＝6.80Hz,2H),4.76(s,2H),δ＝6.76(d,J＝8.80Hz,2H),δ＝7.13(dd,J＝7.32,4.96Hz，1H),δ＝7.29(d,J＝8.64Hz,2H),δ＝7.34(d,J＝7.84Hz,1H),δ＝7.61(td,J＝7.68,1.76Hz，1H),δ＝8.49(d,J＝4.84Hz,1H)ppm；13C NMR(100MHz,CDCl3):δ27.02,50.87,52.00,115.84,122.45,122.52,123.85,131.78,136.83,149.26,151.06,157.06,159.19ppm；HRMS(EI)calculated for C15H14 79BrN3S[M+]347.0092,found 347.0091,calculated for C15H14 79BrN3S[M+]349.0071,found 349.0077。
Example 2
Compound 4 prepared in example 1 reacts specifically
In the first step, 9 fungi: the cruciferous vegetable sclerotinia sclerotiorum, the botrytis cinerea, the peanut brown spot pathogen, the rice blast pathogen, the wheat scab pathogen, the apple ring rot pathogen, the potato late blight pathogen, the banana leaf spot pathogen and the banana vascular wilt pathogen are inoculated on a PDA culture medium flat plate, cultured for three days at 28 ℃, and a bacteria block at the edge of the flat plate is taken by a puncher with the diameter of 9mm to be used as a bacteria cake.
And secondly, preparing a compound 4 solution with the concentration of 30mg/mL by using dimethyl sulfoxide, sucking 500uL by using a pipette gun, directly adding the solution into 500mL of PDB culture medium, and shaking up to obtain a culture system with the concentration of 30 mug/mL. The culture system was inoculated with 9 test bacteria: the fungus cakes of cruciferous vegetable sclerotinia sclerotiorum, botrytis cinerea, peanut brown spot pathogen, rice blast pathogen, wheat scab pathogen, apple ring rot pathogen, potato late blight pathogen, banana leaf spot pathogen and banana wilt pathogen are cultured for 48 hours in a microorganism constant-temperature oscillation incubator at 200rpm and 28 ℃, and the color change and the growth condition of hyphae are observed during the culture period.
And thirdly, in order to facilitate the color change of the hyphae to be observed by naked eyes, after culturing for 48 hours, taking out the fungus cakes, placing the fungus cakes in a clean test tube, and adding distilled water.
The fourth step, the results are shown in fig. 8 to 16: FIG. 8 is a schematic view showing the color change of the hyphae of sclerotinia sclerotiorum of cruciferous vegetables by the detection reagent compound 4, wherein the hyphae of sclerotinia sclerotiorum of cruciferous vegetables are changed into green. FIG. 9 is a schematic diagram showing the color change of the detection reagent compound 4 on the botrytis cinerea hyphae, which is not green. FIG. 10 is a schematic diagram showing the color change of the mycelium of peanut brown spot pathogen by the detection reagent compound 4, and the mycelium of peanut brown spot pathogen is not changed into green. FIG. 11 is a schematic view showing the color change of the hypha of Pyricularia oryzae by the detecting reagent compound 4, and the hypha of Pyricularia oryzae is not changed to green. FIG. 12 is a schematic diagram showing the color change of the compound 4 in the mycelial color of Fusarium graminearum, which is not changed to green. FIG. 13 is a schematic diagram showing the color change of the compound 4 as a detection reagent to the hyphae of ring rot of apple, which is not changed to green. FIG. 14 is a schematic diagram showing the color change of the detection reagent compound 4 to the hyphae of potato late blight bacteria, which is not green. FIG. 15 is a schematic diagram showing the color change of the detection reagent compound 4 to the hyphae of sigatoka bacteria, and the hyphae of sigatoka bacteria are not changed into green. FIG. 16 is a schematic view showing the color change of the detection reagent compound 4 to the mycelia of banana vascular wilt bacteria, and the mycelia of banana vascular wilt bacteria are not changed into green.
The compound 4 acts on the common vegetable pathogenic bacteria in the 9, the hypha of the sclerotinia sclerotiorum of the cruciferous vegetables turns green, and the hypha of the pathogenic bacteria of other vegetables does not change, so that the compound 4 has specific reaction on the sclerotinia sclerotiorum of the cruciferous vegetables.
Example 3
Kit detection reagent Compound 4 concentration selection
Firstly, preparing four PDB culture mediums with the concentration of 20, 30, 40 and 50 mug/mL and containing the compound 4, and respectively inoculating sclerotinia sclerotiorum cakes of cruciferous vegetables.
And secondly, culturing in a microorganism constant-temperature shaking incubator at 200rpm and 28 ℃, and observing hypha change every 12 hours.
The results are shown in FIGS. 17 to 20, and FIG. 17 is a schematic diagram showing the specific reaction of the detection reagent compound 4 at different concentrations to sclerotinia sclerotiorum of cruciferous vegetables. FIG. 18 is a diagram showing the specific reaction of the detection reagent compound 4 with different concentrations to sclerotinia sclerotiorum of cruciferous vegetables. FIG. 19 is a diagram showing the specific reaction of the detection reagent compound 4 with different concentrations to sclerotinia sclerotiorum of cruciferous vegetables. FIG. 20 is a diagram showing the specific reaction of the detection reagent compound 4 with different concentrations to sclerotinia sclerotiorum of cruciferous vegetables.
As can be seen from FIG. 17, when Sclerotinia sclerotiorum of Brassicaceae grows for 36 hours in PDB medium with compound 4 concentration of 20. mu.g/mL under the culture conditions of 200rpm and 28 ℃ in the microorganism constant temperature shaking incubator, and part of green color appears on the surface of hypha, the PDB medium containing compound 4 with concentration of 20. mu.g/mL shows that Sclerotinia sclerotiorum of Brassicaceae can be detected, and the effect is not significant.
As can be seen from FIG. 18, under the culture conditions of 200rpm and 28 ℃ of the microbial constant temperature shaking incubator, Sclerotinia sclerotiorum of Cruciferae vegetables grows for 36 hours in the PDB culture medium with the concentration of the compound 4 being 30 mug/mL, and obvious green color appears on the surface of hyphae, which indicates that the PDB culture medium with the concentration of the compound 4 being 30 mug/mL can detect the Sclerotinia sclerotiorum of Cruciferae vegetables, and the effect is obvious.
As can be seen from FIG. 19, when Sclerotinia sclerotiorum of Brassicaceae grows for 36 hours in PDB medium with compound 4 concentration of 40 μ g/mL under the culture conditions of 200rpm and 28 ℃ in the microorganism constant temperature shaking incubator, and part of green color appears on the surface of hypha, the PDB medium containing compound 4 with concentration of 40 μ g/mL shows that Sclerotinia sclerotiorum of Brassicaceae can be detected, and the effect is not significant.
As can be seen from FIG. 20, when Sclerotinia sclerotiorum of Brassicaceae grows for 36 hours in PDB medium with compound 4 concentration of 50 μ g/mL under the culture conditions of 200rpm and 28 ℃ in the microorganism constant temperature shaking incubator, and part of green color appears on the surface of hypha, the PDB medium containing compound 4 with concentration of 50 μ g/mL shows that Sclerotinia sclerotiorum of Brassicaceae can be detected, and the effect is not significant.
From this, it can be seen that the detection effect is most remarkable when the concentration of compound 4 is 30. mu.g/mL.
Example 4
Pouring the soil with the sclerotinia sclerotiorum into a clean conical flask filled with 50mL of sterile water, and oscillating for 5min to fully disperse the soil sample to obtain a soil suspension. Absorbing 0.5mL of soil suspension by using a liquid transfer gun, and adding the soil suspension into a centrifuge tube containing 4.5mL of sterile water to obtain 10-2The dilution is repeated in this way, in turn 10 is obtained-3-10-5The diluent (2).
Compound 4 solution with concentration of 30mg/mL is prepared by dimethyl sulfoxide, 500uL is absorbed by a pipette gun, and is directly added into 500mL PDA culture medium, and a culture system with concentration of 30 mug/mL is obtained after shaking up. Suction with pipette 10-50.5mL of the dilution suspension is evenly smeared in a culture system with the concentration of 30 mu g/mL to obtain a soil dilution culture system.
Placing the PDA plate containing the compound 4 with the concentration of 30 mug/mL at the constant temperature of 28 ℃ for culturing for 48 hours, and growing a green colony on the PDA plate, namely the sclerotinia sclerotiorum of the cruciferous vegetables.
Example 5
Accurately weighing 300mg of compound 4, dissolving in 1mL of DMSO, sucking 500uL of the solution by using a pipette gun, adding the solution into 500mL of PDB culture medium to prepare 30 mu g/mL of liquid medicine, and adding 500uL of the liquid medicine (an auxiliary reagent streptomycin (used for preventing bacterial contamination)) to obtain a detection system.
Taking plant tissue samples such as stems, cotyledons and true leaves suspected of having sclerotinia sclerotiorum of cruciferous vegetables, inoculating the plant tissue samples into a PDB culture medium, and culturing for 36h at 28 ℃ and 200rpm of a microbial constant-temperature shaking incubator.
And observing the color of the mycelium in the culture medium by naked eyes or a microscope, wherein if green mycelium is observed, the plant tissue sample contains the sclerotinia sclerotiorum of the cruciferous vegetables, and if the green mycelium is not observed, the plant tissue sample does not contain the sclerotinia sclerotiorum of the cruciferous vegetables.
Example 6
Taking 1g soil sample, dividing into two parts of 0.5g, and adding black brown sclerotium of Brassicaceae vegetable crushed into fine particles into one part. And respectively pouring the two soil samples into a clean conical flask filled with 50mL of sterile water, and oscillating for 5min to fully disperse the soil samples to obtain two soil suspensions.
0.5mL of the two kinds of soil suspensions are respectively absorbed by a liquid transfer gun and added into a centrifuge tube with 4.5mL of sterile water to obtain 10-2Diluting the solution to obtain 10-3-10-5The diluent (2). Two kinds are taken 10-50.5mL of the dilution suspension is respectively and uniformly coated on the surfaces of two PDA culture media (one contains a compound 4, the concentration is 30ug/mL, and the other does not contain the compound 4) plates to obtain four soil dilution culture systems: a. adding no compound 4 and no sclerotium, b adding no compound 4 and no sclerotium, c adding compound 4 and no sclerotium, d adding compound 4 and sclerotium.
The four PDA plates are placed at the constant temperature of 28 ℃ for culturing for 48h, and green colonies growing on the plates are sclerotinia sclerotiorum of cruciferous vegetables.
As shown in FIGS. 21 to 24, FIG. 21 shows a medium in which compound 4 was not added and sclerotia was not added, and it was judged that there were some yellow colonies as Staphylococcus aureus in the soil. FIG. 22 is a graph of b medium without compound 4 plus sclerotia, with yellow colonies and off-white hyphae, which is judged to be Sclerotinia sclerotiorum under normal growth conditions. FIG. 23 shows a case where the bacterium was judged as a bacterium when compound 4 was added to a medium without adding sclerotia. FIG. 24 shows a case of a culture medium containing compound 4 and sclerotium, in which a green colony is observed and it is judged that hypha of sclerotium bacteria of cruciferous vegetables turns green.
Example 7
A kit suitable for rapid detection of sclerotinia sclerotiorum of cruciferous vegetables comprises a kit main body A, a detection reagent B and an auxiliary reagent C;
the kit main body A: at least one of potato dextrose broth, PDB medium, or PDA solid medium, in a volume of 1000 mL. Fig. 1 is a schematic view of a kit body a.
And (3) detection reagent B: compound 4 prepared in example 1, stored sealed at 4 ℃ in the dark;
and (5) storing an auxiliary reagent C and streptomycin in a dark and sealed manner at normal temperature. FIG. 3 is a schematic diagram of auxiliary reagent C.
The use method of the kit suitable for rapidly detecting sclerotinia sclerotiorum of cruciferous vegetables comprises the following steps: weighing the compound 4, adding the compound 4 into a hot-melt 1000mL kit body A (at least one of a potato dextrose broth culture medium, a PDB culture medium or a PDA solid culture medium) to prepare a liquid medicine with the concentration of 20-50 μ g/mL (preferably 30 μ g/mL, and the diagram of figure 2 is a schematic diagram containing a detection reagent B with the concentration of 30 μ g/mL), adding streptomycin accounting for 0.1% of the mass of the compound 4 into the liquid medicine, enabling the concentration of the streptomycin to be preferably 30ug/L, shaking up, flatly pouring the liquid medicine on a flat plate, and cooling the liquid medicine to be used.
The preparation method of the potato glucose broth culture medium comprises the following steps: cleaning potato, peeling, weighing 200g, cutting into small pieces, placing into a fine steel pot, adding water, boiling until the small pieces can be slightly punctured by a glass rod, and filtering with thick gauze to remove potato residue. Then adding 20g of glucose into the filtrate, stirring and dissolving, and adding water to a constant volume of 1000 mL; cooling the filtrate, subpackaging in conical bottles, plugging, sealing, sterilizing in a sterilizing pot, taking out, cooling, and storing for later use.
Inoculating plant tissue sample or soil containing Sclerotinia sclerotiorum of Brassicaceae vegetables into culture medium, and culturing at 28 deg.C in microorganism biochemical incubator for 36 hr. And observing the color of the mycelium in the culture medium by naked eyes or a microscope, wherein if green mycelium or colony is observed, the sample contains the sclerotinia sclerotiorum of the cruciferous vegetables.
The kit has the characteristics of high sensitivity, strong specificity, stability and effectiveness, low manufacturing cost and strong applicability, and stalks, cotyledons, true leaves or soil of agricultural and forestry plants and field water can be used as detection samples.
The detection method is simple and easy to operate, and the whole operation process does not need to use complex and expensive advanced instruments or technologies such as a PCR (polymerase chain reaction) amplification instrument, a photosynthetic measurement instrument, an enzyme-linked immunosorbent assay technology, thermal infrared imaging and the like.
The traditional detection method has long detection period, for example, the PCR detection technology needs at least 3-4 days, the detection kit can have the effect within 48 hours, and the obvious detection effect within 36 hours.
The traditional detection method needs to process plant tissue samples, the operation of RNA extraction and the like is too high for farmers, the detection result is easy to be inaccurate, and the detection kit does not need to process the samples and can be directly used.
Therefore, in practical application, the kit can be conveniently used by farmers to detect the occurrence density of diseases in the environment and timely take corresponding control measures.
Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (10)

1. A compound for detecting sclerotinia sclerotiorum of cruciferous vegetables is characterized by having the following structure:
Figure FDA0003045687540000011
wherein R is1Selected from hydrogen, halogen; r2Selected from hydrogen, halogen; r3Selected from hydrogen, halogen; r4Selected from hydrogen, halogen; r5Selected from hydrogen and halogen.
2. The compound for detecting sclerotinia on brassicaceae vegetables according to claim 1, characterized by the following structure:
Figure FDA0003045687540000012
3. the method of claim 2, wherein the method comprises the steps of:
Figure FDA0003045687540000013
dissolving ethanolamine and 2-pyridine formaldehyde with a molar ratio of 1:1 in methanol, stirring for 1-48 h at room temperature, adding sodium borohydride into the reaction solution in batches under an ice bath condition, stirring for 0.5 h, stirring for 1-12 h in an oil bath at 45-55 ℃, tracking by TLC (thin layer chromatography), cooling to room temperature, quenching redundant sodium borohydride with hydrochloric acid under the ice bath condition, neutralizing the reaction solution with 40% NaOH aqueous solution, adjusting the pH to about 10, extracting with dichloromethane, concentrating to obtain a crude product, and performing column chromatography to obtain a compound 1;
Figure FDA0003045687540000014
dissolving a compound 1 in absolute ethyl alcohol, slowly adding a compound 2 within 1-10 min, wherein the molar ratio of the compound 1 to the compound 2 is 1:1, stirring at room temperature for 10-60 min, performing TLC (thin layer chromatography) tracking reaction until the reaction is finished, and removing the solvent under reduced pressure to obtain a compound 3;
Figure FDA0003045687540000021
dropwise adding excessive concentrated hydrochloric acid into the compound 3, refluxing for 10-60 min at 85-95 ℃, tracking and reacting by TLC (thin layer chromatography), cooling the reaction solution to room temperature, adjusting the pH to 10-11 by using 40% NaOH solution under ice bath, and using CH (CH)2Cl2Extracting, washing organic phase with saturated saline solution, and removing anhydrous Na2SO4Drying, removing solvent under reduced pressure to obtain crude product, and purifying by column chromatography or ethanol recrystallization to obtain compound 4.
4. A kit prepared from the compound for detecting sclerotinia sclerotiorum of brassicaceous vegetables of claim 1 or 2.
5. The kit according to claim 4, wherein the kit comprises a kit body A, a detection reagent B and an auxiliary reagent C;
the kit main body A: at least one of potato dextrose broth, PDB medium or PDA solid medium, in a volume of 1000 mL;
and (3) detection reagent B: compound 4, sealed at 4 ℃ in the dark;
and (5) storing an auxiliary reagent C and streptomycin in a dark and sealed manner at normal temperature.
6. The kit of claim 5, wherein the potato dextrose broth is prepared by the method of: cleaning and peeling potatoes, cutting the potatoes into small pieces, adding water, boiling until the small pieces of the potatoes can be slightly punctured, filtering potato residues by using thick gauze, adding glucose into filtrate, wherein the mass ratio of the glucose to the potatoes is 1:10, stirring for dissolving, adding water to a constant volume of 1000mL, cooling and packaging the filtrate, sterilizing, taking out and cooling, and storing for later use.
7. Use of the compound for detecting sclerotinia sclerotiorum of brassicaceae vegetables according to claim 1 or 2 in preparing a detection reagent for sclerotinia sclerotiorum of brassicaceae vegetables.
8. A method for rapidly detecting sclerotinia sclerotiorum of cruciferous vegetables by using the kit as claimed in any one of claims 4 to 6, which is characterized by comprising the following steps:
weighing a detection reagent B, namely a compound 4, adding the detection reagent B into a hot-melt 1000mL kit main body A to prepare a liquid medicine with the concentration of 20-50 mug/mL, adding an auxiliary reagent C, namely streptomycin, which accounts for 0.1% of the mass percent of the compound 4, shaking up, flatly pouring the liquid medicine on a flat plate, and cooling the liquid medicine for use;
weighing 2g of a sample to be detected, inoculating the sample to the culture system, culturing the system in a microorganism constant-temperature shaking incubator at 200rpm and 28 ℃ for 24-72 h, and observing the color change of hyphae.
9. The method for rapidly detecting sclerotinia sclerotiorum of brassicaceae vegetables by using the kit according to claim 8, wherein the sample to be detected is plant cotyledon, stem or soil containing sclerotinia sclerotiorum of brassicaceae vegetables.
10. The kit for rapidly detecting sclerotinia sclerotiorum of brassicaceous vegetables according to claim 8, wherein said observing color change of hyphae means observing color of mycelium in culture medium by naked eye or microscope, if green mycelium is observed, it indicates that sample contains sclerotinia sclerotiorum of brassicaceous vegetables.
CN202110471715.5A 2021-04-29 2021-04-29 Compound for detecting sclerotinia sclerotiorum of cruciferous vegetables and application thereof Pending CN113214241A (en)

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