WO2015066961A1

WO2015066961A1 - Activity evaluation of plant pathogens and high throughput screening method for microbicides and kit therefor

Info

Publication number: WO2015066961A1
Application number: PCT/CN2014/000090
Authority: WO
Inventors: 李宝聚; 柴阿丽; 石延霞; 谢学文; 李金萍
Original assignee: 中国农业科学院蔬菜花卉研究所
Priority date: 2013-11-08
Filing date: 2014-01-24
Publication date: 2015-05-14
Also published as: CN104634768A

Abstract

Disclosed are activity evaluation of plant pathogens and a high throughput screening method for microbicides based on double fluorescent staining and a kit therefor, which include using a microtiter plate as tool carrier, using two different fluorescent dyes, respectively fluorescently labelling spores or mycelia of plant pathogens having vitality and no vitality, and due to the different colouring of spores or mycelia having no vitality and vitality, quantitatively detecting the survival rate of plant pathogens through fluorescence microscopy or flow cytometry, and accordingly preparing the kit. The method and kit can be used in aspects such as microbicide screening, evaluation of food safety, and water quality evaluation using the plant pathogen survival rate as an indicator. The method and kit are rapid, objective, simple and easy in operation, and have a low cost, and both can be used in studying the mechanism of the action of microbicides and high-throughput screening and the toxicity evaluation of microbicides.

Description

TECHNICAL FIELD The present invention relates to a method for evaluating the activity of a plant pathogen based on double fluorescent dyeing and a high-throughput screening method and kit for a bactericide, and the method and kit can be The application of pathogen activity evaluation, fungicide screening, food safety evaluation, water quality evaluation, etc. belongs to the field of plant protection and biotechnology. BACKGROUND OF THE INVENTION Plant pathogenic bacteria are an important factor in the occurrence and prevalence of plant diseases, and the annual losses due to diseases in the world are in the hundreds of millions. The rapid and accurate detection of plant pathogens and their activities is the basis for effective prediction of the condition.

At present, the detection of plant pathogens mainly uses conventional pathogen isolation and culture techniques, which is time consuming, laborious, and cannot detect obligate parasites. Later developed immunological techniques and molecular biology methods, such as PCR, can accurately and sensitively detect plant pathogens qualitatively and quantitatively, but cannot detect their activity. However, the evaluation of the activity of plant pathogens is the basis for accurate prediction and prediction of diseases. There are few studies on the detection and analysis of plant pathogen activity at home and abroad, mainly using the traditional method of Colony Forming Units (CFU). Although the method is accurate and reliable, the detection time is longer.

Fluorescence staining is an important means of medically detecting apoptosis and cell necrosis. After apoptosis or necrosis, changes in cell volume, changes in cell membrane permeability, and degradation of nuclear DNA occur. Based on these characteristics, a suitable fluorescent dye can be selected to accurately detect the cell state. The phenotype and physiological characteristics of apoptosis of plant pathogens are similar to those of animal cells, and the cell structure and metabolic processes are simple. At present, there are a few reports on the pathogen research by fluorescent staining. The activity of soybean sudden death syndrome is detected by the specific fluorescent dye propidium iodide of necrotic cells. SYTOX Green is used to detect human pathogenic bacteria Escherichia coli and Salmonella in the environment and water. Activity.

High thro ghput screening (HTS) technology is one of the important technical means to discover innovative drugs, and has become an important tool in pharmaceutical and biotechnology research. Modern agriculture is increasingly dependent on fungicides, with increasing calls for environmental and consumer safety, and the emergence of pest resistance to shorten the life of fungicides. The creation and development of new pesticides is becoming more and more urgent and increasingly difficult. According to statistics, the successful development of a new high-efficiency, low-toxicity and environment-friendly new pesticide requires the synthesis and screening of 80,000 compounds, costing hundreds of millions of yuan. This poses new challenges for the synthesis and screening of new fungicides. At present, spore germination method and mycelial growth rate method are used in China to screen fungicides. With the rapid development of new fungicide creation technology, it is required to establish a simple and rapid method for screening new compounds.

It can be seen from the prior art analysis of plant pathogen activity evaluation and fungicide screening that the prior art is time consuming, laborious, and costly or requires expensive equipment, and is urgently needed, simple, low cost, and inexpensive. Instruments and equipment can be used to quickly quantify the activity of plant pathogens. Instruction manual

SUMMARY OF THE INVENTION The object of the present invention is to provide a method for evaluating the activity of plant pathogenic bacteria and high-throughput screening of fungicides based on double fluorescence dyeing for the current labor pathogen activity detection and fungicide screening labor, time-consuming, unstable detection results and high cost. The method and the kit determine the activity of the pathogenic bacteria by detecting the fluorescent signal of the viable and non-viable spore or mycelial cells of the pathogenic bacteria, and realize the efficacy evaluation and high-throughput screening of the fungicide. The method and the kit have the characteristics of simple operation, short detection time, large detection amount, low cost, and simultaneous evaluation of various bactericides, and can be widely applied to bactericide screening, toxicity evaluation, food safety evaluation, Water quality assessment and other fields. The details are as follows:

1. Activity evaluation of plant pathogenic bacteria based on double fluorescent dyeing and high-throughput screening method of fungicide

The invention discloses a plant pathogen activity evaluation and a high-throughput screening method for a fungicide. The microtiter plate is used as a tool carrier, and two different fluorescent dyes are used to respectively perform vigor and non-viable spore or mycelial cells of the plant pathogen. Fluorescent labeling, due to the different coloration of viable and non-viable spores or mycelial cells, fluorescence signal or flow cytometry for detecting fluorescent signals, and counting the survival rate of pathogens, can directly reflect the activity of plant pathogens, and achieve the efficacy evaluation of fungicides. High-throughput screening.

The first fluorescent dye used in the method allows the viable spore or mycelial cells to be colored to produce specific fluorescence, selected from the group consisting of acridine orange (AO), fluorescein diacetate (FDA), Hoechst 33258 4', 6-dioxin. One of phenyl-2-phenylindole (DAPI), Annexin V-FITC, etc., but is not limited thereto; the second fluorescent dye can color dead spore or mycelial cells to produce specific fluorescence, selected from the group consisting of propidium iodide One of pyridine (PI), ethidium bromide (EB), SYTOX, etc., but is not limited thereto.

Among them, the first fluorescent dye AO has a working concentration of 10-1000 g/mL, and the 480 nm blue light excitation makes the living cell nucleus green or yellow-green uniform fluorescence; the FDA working concentration is 100-1000 g/mL, 488 Under the excitation of nm blue light, green fluorescence is produced in living cells; Hoechst 33258 has a working concentration of 1-100 g/mL, and the living cells emit blue fluorescence under 350 nm ultraviolet excitation; DAPI has a working concentration of 5-500 g. /mL, 358 nm ultraviolet light, live cells emit bright blue fluorescence; Annexin V-FITC working concentration is 5-500 g mL, 480 nm blue light excitation, living cells emit green fluorescence; second fluorescent dye PI The working concentration is 5-1000 _M g/mL, and the 358 nm ultraviolet light stimulates the dead cells to produce red fluorescence; the EB working concentration is 10-1000 g/mL, which causes the dead cells to produce orange fluorescence; the working concentration of SYTOX is Ol-lg/mL, causing necrotic cells to glow green.

Among them, the first fluorescent dye AO is dyed for 10-25 min in the dark, the FDA staining time is 5-40 min, the Hoechst 33258 staining time is 15-50 min, and the DAPI staining time is 3-15 min. The staining time of Annexin V-FITC is 15-40 min in the dark; the staining time of the second fluorescent dye PI is 4-30 min in the dark, the staining time of EB is 20-40 min, and the staining time of SYTOX is 5-20 min.

In the method, the plant pathogenic bacteria are obtained by artificial pure culture, or are obtained by enrichment from diseased tissues. The test for the activity evaluation of plant pathogenic bacteria includes mycelial cells or spores of plant pathogenic fungi, spores of plant pathogenic bacteria, and dormant spores of Rhizoctonia solani.

The plant pathogen spore or mycelial suspension is added to the microtiter plate containing a plurality of wells in an equal amount, and the number of spores per hole is 10 ² Ί 0 ⁵ . Two fluorescent dyes were added in order, and the dyeing temperature was 4-35 °C. Finally, the fluorescence signal was detected by flow cytometry, fluorescence microscopy or fluorescence spectrometry to quantitatively evaluate the activity of plant pathogens. Instruction manual

~" This method can be widely used in new compound screening, fungicide screening, food safety evaluation and water quality evaluation.

2. A method for evaluating the activity of plant pathogens based on double fluorescent staining and a high-throughput screening kit for fungicides

The invention can be used to prepare a kit for rapid and quantitative evaluation of plant pathogen activity and high-throughput screening of fungicides, the kit containing one or more microtiter plates containing a plurality of wells, two fluorescent dyes, the first type Fluorescent dyes stain the viable plant pathogen spores or mycelial cells, and the second fluorescent dye stains the non-viable plant pathogen spores or mycelial cells.

Among them, one or more microtiter plates containing a plurality of wells are selected from 12-well, 24-well, 96-well, 384-well, and 1536-well microtiter plates, of which 96-well plates and 384-well plates are optimal. Per well microtiter plate containing one or more plant pathogen spores or hyphae, wherein for 96-well plates, each well of ^{^103-105} spores preferred; for 384-well plates, each well of ^{^102-103} spores optimal.

The first fluorescent dye contained in the kit is selected from the group consisting of acridine orange (AO), fluorescein diacetate (FDA), Hoechst 33258, 4',6-diamidino-2-phenylindole (DAPI), and the like. One of them, but not limited to, can produce specific fluorescence of viable spore or mycelial cells: The second fluorescent dye is selected from propidium iodide (PI), ethidium bromide (EB), SYT0X, etc. One, but not limited to, can cause specific fluorescence of dead spore or mycelial cells.

The kit may also include one or more plant pathogenic bacteria, and a culture medium and a cleaning medium for the pathogen, which may be in lyophilized form or in liquid form.

The kit may also include a high throughput screening protocol for bactericides, or instructions for the treatment of plant pathogens by compounds or other methods.

The kit may also include a control agent that is resistant to a particular pathogen.

3. There are many effective biological stains available in this technology. Different stains react or concentrate in different parts of the pathogen cells or tissues, and these characteristics can be used to reveal specific portions of the area. Useful biological stains include, Hoest 33342, Carmine, Coomassie blue, crystal violet, DAPI, eosin, ethidium bromide, magenta, hematoxylin (Haemat _0X ulin), Iodine, malachite green, methyl chloride, methylene blue, neutral red, Nile red, osmium tetroxide, rhodamine and safranin. The present invention has the beneficial effects compared to the background art:

(1) The present invention establishes a fluorescent staining method and working environment suitable for the evaluation of the activity of plant pathogenic bacteria, including: selection of fluorescent dye species, staining method, dyeing time, incubation environment, fluorescence detection method, etc.;

(2) The method and kit provided can directly detect the survival rate of plant pathogenic bacteria, and the operation is simple, the equipment and the cost are low, and can be realized in an ordinary laboratory;

(3) It takes only 30 minutes from sample preparation to completion of detection. It can directly judge the activity of individual spores or hyphae. It is fast, accurate, sensitive and reproducible. It is expected to replace traditional spore germination methods and hyphae. Growth rate method, suitable for use in agricultural research, plant protection and other departments;

(4) The method and kit for detecting the survival rate of plant pathogenic bacteria provided by the invention have the characteristics of rapid operation, simultaneous screening of multiple compounds, saving manpower, material resources and time, and can be widely applied to high-throughput screening of fungicides, food safety and Water quality assessment and other aspects. Instruction manual

DRAWINGS

Figure 1 shows the identification of the conidia of Botrytis cinerea by FDA and PI fluorescence staining. In the figure: A. Observed by green polarized light of fluorescence microscope, live spores emit green fluorescence after FDA staining; B. Observed by blue polarized light under fluorescence microscope, red stain of dead spores after PI staining; C. Living spores and The dead spores were mixed and stained with FDA and PI. Under the fluorescence microscope, the green spores showed green fluorescence and the dead spores showed red fluorescence.

Figure 2 shows the activity of FDA and PI fluorescent staining to identify conidia and hyphae of Fusarium solani (wrar/w^oto '). In the figure: A. Observed with green polarized light from a fluorescence microscope. After FDA staining, live spores and viable filaments emit green fluorescence; B. Observed by blue polarized light under a fluorescence microscope, after FI staining, dead spores and dead hyphae Red fluorescence; C. Vigorous and non-viable spores, hyphae mixed with FDA and PI staining, observed under green polarized light, live spores and live hyphae green fluorescence, dead spores and dead hyphae red fluorescence.

Figure 3 shows the FDA and PI fluorescence staining methods for identifying the spore activity of P. syringae puncture-causing species (P5 «o/omcwa^ «gae pv. lachrymans ). In the figure: A. Observed with green polarized light from a fluorescence microscope. After FDA staining, the live spores emit green fluorescence; B. Under the fluorescence microscope, the blue polarized light is observed, and after PI staining, the dead spores are red-fluorescent.

Figure 4 shows the activity of FDA and PI fluorescent staining for identifying spores of 芸薹Odiophora brassicae. In the figure: A. Observed with green polarized light from a fluorescence microscope, after FDA staining, live spores emit green fluorescence; B. Observed by blue polarized light under a fluorescence microscope, after staining with PI, dead spores emit red fluorescence; C. Vibrant and The non-viable spores and hyphae were mixed and stained with FDA and PI. Under the fluorescence microscope, the green spores showed green fluorescence and the dead spores showed red fluorescence.

Figure 5 shows the fluorescence intensity at different times after Hoest 33258 and PI stained for Botrytis cinerea spores. In the figure: A. Different time after staining, the % pore microtiter plate collects the change of PI fluorescence intensity; B. The 384-well microtiter plate collects the change of PI fluorescence intensity at different times after staining; C. 96-well micro titration at different times after staining Plates were collected for Hoest 33258 fluorescence intensity changes; D. At different times after staining, 384-well microtiter plates were used to collect Hoest 33258 fluorescence intensity changes.

Figure 6 shows the quantitative analysis of spore activity of I Colletotrichum orbiculare by flow cytometry. In the figure: A. Negative control tube; B. Annexin V-FITC staining single positive tube; C. PI staining single negative tube; D. Sample tube to be tested.

Figure 7 Flow cytometry to determine the linear relationship between the proportion of viable spores and the germination rate of anthrax.

Figure 8 shows the effect of fungicides on the viability of dormant spores of Magnaporthe grisea. In the figure: A. The killing effect of thiophanate-methyl on Rhizoctonia solani; B. The killing effect of fluazinam on Rhizoctonia solani.

Figure 9 shows the effect of 2 mg/mL fluazinam treatment on the viability of dormant spores of T Plasmodiophora bmssicae. In the figure: A. 2 mg/mL fluazinam treatment for 10 min, the viability state of the dormant spores of B. sphaeroides; B. 2 mg/mL fluazinam treatment for 30 min, the viability state of the dormant spores of P. solani; C 2 mg/mL fluazinam treatment for 1 h, the viability state of the dormant spores of P. solani; D. 2 tng/mL fluazinam treatment for 2 h. detailed description

The substantial contents and advantageous effects of the present invention will be described in detail below with reference to the accompanying drawings and embodiments. The following examples are only intended to further illustrate the invention and are not to be construed as limiting the invention. Instruction manual

Reagents:

Fluorescein diacetate (FDA): Sigma;

Propidium iodide (PI): Sigma;

Annexin V-FITC: Sigma;

Hoechst 33258: Sigma;

Other reagents are of domestic analytical purity.

Instrument:

Olympus Fluorescence Microscope ( OLYMPUS BX51);

Flow cytometry (BDFACSCalibur);

Electric thermostatic water bath (XMTD-700). Example 1 Plant pathogen activity and drug evaluation based on fluorescent staining target FDA (fluorescein diacetate) and PI (propidium iodide) were used as dyes for spores, hyphae, and phytopathogenic bacteria of plant pathogenic fungi. The spore viability was identified and the target of the plant pathogen identifiable by fluorescent double staining was screened.

Experimental method

The phytopathogenic fungi spores, hyphae and phytopathogenic bacterial spores were selected as targets (Table 1), and the phytopathogenic bacteria activity was identified by FDA/PI double staining.

Fluorescent staining solution: Weigh 0.05g FDA, dissolve in 1mL of acetone, force into 9mL PBS 7.4, prepare FDA stock solution with concentration of 5 mg/mL, store in brown bottle at -20 °C, use before use. Dilute to a final concentration of 100 g/mL in PBS for use. Weigh 0.005 gPI, add 10 mL PBS 7.4 buffer, prepare 500 μ ₈ /mL stock solution, store the brown bottle in the dark, and dilute it to PBS with a final concentration of 5 g/mL before use. .

Dyeing method: Take freshly prepared spore or mycelial suspension 2 mL, and pack them into two groups, one group is live spores or hyphae without water bath treatment, and the other group is dead after being treated for 10 min in 95 °C water bath. Spores or hyphae. Take the initial concentration of 100 gmL of FDA staining solution (dissolved in PBS) 10 Add 90 spores or mycelial suspension, mix, the final concentration is 10 g / mL, stain at room temperature for 15 min, centrifuge to remove the dye solution; then add 100 The PI staining solution with a concentration of 5 g/mL was stained with light for 15 min at room temperature. The spores or hyphae of the dyed tablets were observed by fluorescence microscope, and the images of the staining results of the pathogens were collected. The shooting parameters were: green channel (excitation light 450~490nm, emission light >510nm), and objective lens 40 times.

2. Experimental results

The floristic pathogen spores or hyphae were stained by FDA/PI double fluorescent staining method. Under the fluorescence microscope, the viable spores or hyphae were green-fluorescent after FDA/PI staining, while the non-viable spores or hyphae were observed. Red fluorescence. Based on this fluorescence characteristic, it was confirmed that the fluorescent double staining method can evaluate and identify the activity of spores or hyphae of various plant pathogenic fungi and bacteria (Table 1). It mainly includes: (1) monocytogenes conidia of plant pathogenic fungi: powdery mildew (« /^), downy mildew (Pseudoperonospora), Botrytis (Botrytis) ^ Penicillium ( Penicillium )

(.Phyllosticta) ^ Monocytogenes conidia of phytopathogenic fungi such as Verticillium, Anthracnose (CoUetotrkhum, Phytophthora Phytophtho) (Fig. 1); (2) Polygenic conidia of phytopathogenic fungi : Dicotyledonous conidia of plant pathogenic fungi; (3) Polygenic conidia of plant pathogenic fungi: Nail Description

Polyporus conidia of plant pathogenic fungi such as Cladosporium, Fus rium, iStagonospora (Fig. 2); (4) Phytopathogenic fungi Brick partitioned conidia: Corynebacterium

(Corynespora), a brick-shaped conidia of A. te ία plant pathogenic fungi such as Stemphyliun; (5) Linear conidia of plant pathogenic fungi: Cercospora (Ce ροηι shell) Linear conidia of plant pathogenic fungi such as Septoria; (6) Hyphae of plant pathogenic fungi: hyphae of plant pathogenic fungi such as Rhizoctonia genus Sckwtinia; (7) Phytopathogenic bacteria Monosomies, twins or sporozoites: Pseudomonas (Pseudomo Corynebacterium (C! avibacter L.) to X.

(Xanthomonas), somatic, twin or streptozoon of plant pathogenic bacteria such as Envinia (Fig. 3); (8) Monosporous spores of protozoa: Plasmodiophom, Fusarium Single spore-type spores of plant pathogens such as iSpongospom (Fig. 4).

Table 1 Plant pathogen activity and drug evaluation target screening based on fluorescence double staining

Plant pathogens Fluorescent characteristics Type Target genus Species Viable Non-viable Cucurbitaceae powdery mildew

Powdery mildew Erysiphe strong green fluorescence red fluorescence

E. c ucurb it ace arum

Downy Mildew

Cuban Downy Mildew _P cubensis Green Fluorescent Light Red Fluor

Pse udoperonospora

Botrytis Botryiis Botrytis cinerea cinerea pale green fluorescence red fluorescence Penicillium Penicillium extended Penicillium P. expansion Strong green fluorescence Red fluorescence Monocytogene

Pythium genus Pfiylhsticta, spotted mold, commonsii, green fluorescence, red fluorescence, conidia

Verticill im Dalbergia sp. · dahliae green fluorescent red fluorescent anthracnose genus anthracis C orbiculare light green fluorescent strong red fluorescent

Colletotrichum Chrysosporium C. capsici Strong green fluorescent red fluorescent Phytophthora Phytophthor Phytophthora capsici . capsici pale green fluorescence light red fluorescence Pythium Pythium Phytophthora genus. aphanidermatum green fluorescent red fluorescent double cell

Plant, genus Ascochyt, watermelon shell, two cucum A citrullina, green fluorescence, red fluorescence, conidia

Pathogen

Pseudomonas Passalora, S. serrata, strong green fluorescence, red fluorescence, fungus

Polyporus

Narcissus shells hold more S. cuitisii light green fluorescent red fluorescent Stagonospora

Multicellular

Fusarium oxysporum

Conidia green fluorescence red fluorescence

Cladosporium C. cucumerinum

Fusarium oxysporum F. oxyspor m green fluorescence light red fluorescence Fusarium Fusarium

Fusarium solani F. solani green fluorescence red fluorescence Corynebacterium

C. mazei strong green fluorescence red fluorescence Corynespora

Brick partition

Alternaria Alternaria Brass Chain Brass bras sic ae Green Fluorescent Red Fluorescent Conidia

Pythium

3⁄4fi3⁄4J, 5*. solani, pale green fluorescence, pale red fluorescence, Stemphylium

Cercospora Cercospora celery C. pii, green fluorescent strong red fluorescent conidia, genus Septoria, Septoria, snail, stalk, stalk, stalk, stalk, stalk, stalk, stalk, stalk, stalk, stalk, stalk, stalk, stalk, stalk, stalk, stalk, stalk, stalk, stalk, stalk, stalk, stalk, stalk, stalk, stalk Fluorescent red fluorescent hyphae

Sclerotiinum Sclerotiorum Green Fluorescent Red Fluorescence Description

Table 1 Plant pathogen activity and drug evaluation target screening based on fluorescence double staining (continued)

Hoechst 33258/PI staining method for determining the optimal fluorescence detection time. A key condition for the evaluation of plant pathogen activity by fluorescent double staining or for high-throughput screening of fungicides is to determine the optimal fluorescence detection time so that the method The result is the most repeatable. Here, the identification of spore activity of ci rea is taken as an example, and experiments were carried out using a 96-well microtiter plate and a 384-well microtiter plate.

Experimental method

The gram-negative bacteria were cultured in PDA medium for 10 days. The spore suspension was collected by brushing method and divided into three groups. The first group of spores were killed by 95 °C water bath for 10 min, and the second group was without any The viable spores were treated, and the third group was an equal amount of heat-treated lethal and fresh live spores, and each set of samples was set to three replicates. Three groups of samples were added to 96-well and 384-well microtiter plates for each experiment. Spore suspensions of 3×10 ⁶ spores/mL were added to each well of a 96-well plate at a concentration of 3 μg per well. X 10 ⁶ spores/mL spore suspension 50. Each group of samples was stained with PI (5 g/mL) and Hoechst 33258 (5 μg/mL), and the fluorescence intensity was measured every 10 min after staining until the end of 120 min. The fluorescence detection intensity is the average of three replicates.

2. Experimental results

The viability of Botrytis cinerea spores was evaluated by Hoechst 33258/PI double staining method. 6 x 10 ⁵ spores per well in 96-well plates and 1.5 x 10 ⁵ spores per well in 384-well plates. When 96-well and 384-microtiter plates were used, the fluorescence intensity of PI and Hoest 33258 gradually increased with time, eventually reaching a maximum value, and the relative fluorescence intensity was no longer increased and remained stable. The optimal time to detect PI is 4-30 min after staining, and the best time to detect Hoest 33258 is 15-50 min after staining (Fig. 5). Instruction manual

Example 3 Quantitative analysis of plant pathogen activity by flow cytometry Using C. orbiculare spores as targets, flow cytometry was used to detect the spore activity of anthrax spores infected with Annexin V-FITC/PI , Quantitative analysis of spore survival rate. At the same time, the spore germination method was used as a control, and the correlation between the fluorescence double staining method and the traditional spore germination method was compared.

Experimental method

Five strains of anthrax were used as experimental materials. Freshly prepared spore suspensions were treated at 30 °C, 40 °C, 50 °C, 60 °C for 3 min, ie 5 min, 10 min, 20 min, and all samples were divided into two groups, one After double staining with Annexin V-FITC/PI, the flow cytometer was loaded to analyze the spore activity of the pathogen; the other group was subjected to spore germination experiment, and the SAS software was used to test the flow cytometry results and spore germination rate. The relationship is statistically analyzed.

2. Experimental results

The spore samples of the genus Anthracnose were treated with different temperature and time, and the spore survival rate was detected by the combination of Annexin V-FITC/PI fluorescent double staining and flow cytometry (Fig. 6) and spore germination. The proportion of viable spores detected by flow cytometry was consistent with the detection results of spore germination rate, and the two were positively correlated. The ratio of viable spores y detected by flow cytometry to spore germination rate X was linear, and the correlation coefficient was R ² =0.9357. The regression equation was y=0.9380x+1.3192 (Fig. 7). After fluorescent double staining, the results of flow cytometry for the detection of plant pathogens were accurate and the detection speed was fast, which could be used for the quantitative analysis of plant pathogen activity. Example 4 Screening of the control agent for Rhizoctonia solani based on Hoechst 33258/PI double fluorescent staining. Targeting the dormant spores of Magnaporthe grisea with Hoechst 33258/PI double fluorescent staining method to identify dormant spores of Brassica sinensis Vitality, evaluation of the killing effect of different agents such as fluazinam and thiophanate on Rhizoctonia solani, and screening for effective control agents for Rhizoctonia solani.

Experimental method

A freshly prepared suspension of spore suspension of Rhizoctonia solani was inoculated onto a 96-well culture plate at a density of 3 X 10 ⁴ spores/well, 100 spore suspension per well. Different concentrations of fluazinam and thiophanate-methyl (0.1, 0.5, 1, 2 mg/mL) were added to the wells, and 4 parallel holes were set for each concentration, and 100 doses were added to each well. After 10 min, 30 min, 1 h, and 2 h, the supernatant was discarded. Spore staining buffer 50 μί, Hoechst 33258 staining solution 50 μί, PI staining solution 50 μί, 4 Ό incubation for 30 min per well, the spores were observed by fluorescence microscope, and the pathogen staining images were collected. The imaging parameters were: UV The light is excited, and the objective lens is 40 times.

2. Experimental results

The Hoechst 33258/PI double staining method was used to observe the spores in the ultraviolet excitation (40 times mirror). The viable spores showed blue fluorescence, and the non-viable spores showed red fluorescence. As the concentration of the agent increases, the killing effect on the swollen root bacteria is enhanced; the longer the treatment time, the better the killing effect (Fig. 8). Among them, the killing effect of fluazinam at a concentration of 0.1, 0.5, 1, 2 mg/mL for 1 h against root sputum was 60.8%, 68.7%, 93.3%, 95.8%, respectively. The killing effect of swollen bacteria was 72.9%, 78.4%, 95.4% and 99.5%, respectively. It can be seen that fluazinam 1 mg/mL treatment for 1 h, S. serrata Description

The child fatality rate is over 90% (Figure 9). The thiophanate-methyl was treated at a concentration of 1 mg/mL for 2 h or 2 mg/mL for 1 h, and the killing effect on the root sputum was over 80% (Table 2). The effect of fluazinam on the killing of Rhizoctonia solani is better than that of thiophanate-methyl, which can be used as an alternative agent for cruciferous vegetable clubroot disease in the field.

Table 2 Effect of different fungicides on the viability of dormant spores

Concentration Spore mortality at different treatment times (%)

Test agent average mortality rate (%)

(mg/mL) 10 min 30 min 1 h 2 h

0.1 3.8 19.4 50.8 70.5 36.1

50% thiophanate 0.5 5.2 24.8 58.2 76.9 41.3 thiophanate-methyl 1 7.3 38.4 75.4 82.6 50.9

2 15.5 48.4 80.7 85.8 57.6

0.1 4.8 20.1 60.8 72.9 39.7

50% fluazinam 0.5 6.4 33.4 68.7 78.4 46.7 fluazinam 1 12.7 40.6 93.3 95.4 60.5

2 22.4 54.8 95.8 99.5 68.1 Control control - 1.5 2.4 2.9 3.4 36.1

Claims

claims

1. A method for evaluating the activity of plant pathogenic bacteria based on dual fluorescent staining, including (1) providing a sample containing phytopathogenic fungal spores or hyphal cells; (2) using an appropriate concentration of the first fluorescent dye to make the sample contain viable spores Or mycelial cells produce detectable fluorescence, use a second fluorescent dye at an appropriate concentration to cause dead spores or hyphal cells in the sample to produce detectable fluorescence: (3) Detect the two fluorescences of plant pathogenic fungus spores or hyphal cells.

2. A high-throughput screening method for fungicides based on dual fluorescent staining, including (1) providing a sample containing spores or hyphal cells of plant pathogenic bacteria; (2) treating the plant pathogenic bacteria with one or more compounds or other methods ; (3) Use the first fluorescent dye at an appropriate concentration to produce detectable fluorescence from viable spores or hyphal cells in the sample, and use the second fluorescent dye at an appropriate concentration to produce detectable fluorescence from dead spores or hyphal cells in the sample. Fluorescence; (4) Detect two kinds of fluorescence of spores or hyphal cells of plant pathogenic fungi.

3. The method according to claims 1-2, characterized in that: the first fluorescent dye is selected from the group consisting of acridine orange (AO), fluorescein diacetate (FDA), and Hoechst 33258. 4',6-diamidine One of 2-phenylindole (DAPI), Annexin V-FITC, etc., but not limited to this, can cause viable spores or hyphal cells to produce specific fluorescence; the second fluorescent dye is selected from propyl iodide One of PI, ethidium bromide (EB), SYTOX, etc., but not limited to this, can cause dead spores or hyphal cells to produce specific fluorescence.

4. The method according to claims 1-3, characterized in that: the working concentration of the first fluorescent dye AO is 10-1000 Mg/mL, so that the nuclei of living cells are uniformly fluorescent in green or yellow-green; the working concentration of FDA is 100-1000 g/mL, which produces green fluorescence in living cells; the working concentration of Hoechst 33258 is 1-100 g/mL, which causes living cells to emit blue fluorescence; the working concentration of DAPI is 5-500 g/mL, which causes living cells to emit blue fluorescence Emits bright blue fluorescence; the working concentration of Annexin V-FITC is 5-500 Mg/mL, and when excited by 480nm blue light, living cells emit green fluorescence; the working concentration of the second fluorescent dye PI is 5-1000 μg/L. Make dead cells produce red fluorescence; The working concentration of EB is 10-1000 g/mL, making dead cells produce orange fluorescence: The working concentration of SYTOX is 0.1 -l g/niL, making necrotic cells produce green fluorescence.

5. The method according to claims 1-3, characterized in that: the dyeing time of the first fluorescent dye AO is 10-25 min in light protection, the dyeing time of FDA is 5-40 min, and the dyeing time of Hoechst 33258 The staining time of DAPI is 15-50 min, the staining time of DAPI is 3-15 min, the staining time of Annexin V-FITC is 15-40 min in the dark; the staining time of the second fluorescent dye PI is 4-30 min in the dark. The staining time for EB is 20-40 min, and the staining time for SYTOX is 5-20 min.

6. The method according to claims 1-2, characterized in that: the plant pathogenic bacteria are obtained by artificial culture or enriched from plant diseased tissues.

7. The method according to claims 1-2, characterized in that: phytopathogenic bacteria activity evaluation detection objects include: spores or hyphal cells of phytopathogenic fungi, spores of phytopathogenic bacteria, and dormant spores of Clubroot fungi. .

8. The method according to claims 1-2, characterized in that: Fluorescence detection can use any instrument that can detect fluorescence signals, such as a flow cytometer, a fluorescence microscope or a fluorescence spectrometer.

9. A kit for evaluating the activity of plant pathogenic bacteria or high-throughput screening of fungicides based on dual fluorescent staining, including (1) one or more microtiter plates containing multiple wells, (2) two fluorescent dyes, first The first fluorescent dye colors the viable plant pathogenic fungus spores or mycelial cells, and the second fluorescent dye colors the nonviable plant pathogenic fungus spores or mycelial cells.

10. The kit according to claim 9, characterized in that: the one or more microdroplets containing multiple holes Claims

For fixed plates, choose from 12-well, 24-well, 100-well, 384-well, and 1536-well microtiter plates, of which 96-well plates and 384-well plates are the best.

1 1. The kit according to claim 9, characterized in that: the first fluorescent dye is selected from the group consisting of acridine orange (AO), fluorescein diacetate (FDA), Hoechst 33258, 4',6-diamidine One of 2-phenylindole (DAPI), but not limited to this, can cause viable spores or hyphal cells to produce specific fluorescence; the second fluorescent dye is selected from propidium iodide (PI), One of ethidium bromide (EB), SYTOX, etc., but not limited to this, can cause dead spores or hyphal cells to produce specific fluorescence.

12. The kit according to claim 9, further comprising: one or more plant pathogenic bacteria, and an artificial culture medium for the pathogenic bacteria.

13. The kit according to claim 9, further comprising: a cleaning medium, which may be in freeze-dried form or liquid form.

14. The kit according to claim 9, characterized in that: it may also include instructions for high-throughput screening of fungicides, or instructions for treating plant pathogenic bacteria with compounds or other methods.

15. The kit according to claim 9, characterized in that: it also includes a control agent for preventing and treating a specific pathogenic bacteria.

16. The kit according to claim 9, characterized in that: each well of the microtiter plate contains one or more plant pathogenic fungus spores or hyphal cells, wherein for a 96-well plate, each well contains 10 ³ -10 ⁵ spores are optimal; for 384-well plates, 10 ² -10 ³ spores per well are optimal.

17. The method and kit according to claims 1-16, characterized in that: the plant pathogenic bacteria include Erysiphe > Peronospora (

Phytophthora i PhyUosticta), Verticillium (VerticiUi let), Colletotrichum), Phytophthora

Single-celled conidia of plant pathogenic fungi such as Phytophthora); bicellular conidia of plant-pathogenic fungi such as Phytophthora (^oc/^to); Passalora, Cladosporium, Cladosporium, etc. Polycellular conidia of plant pathogenic fungi such as Fusarium and Stagospora; Coryspora (Ahem kt), Stemphylium, etc. Brick-septate conidia of phytopathogenic fungi; Cercospora spp.

Linear conidia of plant pathogenic fungi such as Cercospora and Septoria; hyphal cells of plant pathogenic fungi such as Rhizoctonia and Sckrotinia; Pseudomonas iPseudomo brain , Corynebacterium (Cl vibacter)^ (CRalstonia)-Xanthomonas (iXanthomonas), Erwinia (Erwinia) and other plant pathogenic bacteria's single, twin or chain spores; Clubbacter spp. Plasmodiophom , Plasmodiophom spp.

Single-spored spores of plant pathogenic bacteria such as Spongospora.