CN112941008A

CN112941008A - Separation method of botrytis cinerea

Info

Publication number: CN112941008A
Application number: CN202110451421.6A
Authority: CN
Inventors: 张容博; 张君成; 王忠文
Original assignee: Guangxi University
Current assignee: Guangxi University
Priority date: 2021-04-19
Filing date: 2021-04-19
Publication date: 2021-06-11
Anticipated expiration: 2041-04-19
Also published as: CN112941008B

Abstract

The invention discloses a separation method of botrytis cinerea. The method adopts a fine, soft and elastic spore trailing and dispersing filament, directly separates out a single conidium from a tomato gray mold standard sample, and cultures to form a tomato gray mold bacterial strain with single genetic composition. The operation steps of the isolated culture are as follows: 1) collecting a tomato gray mold standard sample; 2) firing the spore dispersion silk; 3) preparing a spore dispersing plate; 4) dragging conidia; 5) cutting the monospore small square; 6) formation of a monospore isolate. The invention has the following advantages: 1) the key apparatus for separating the spores is simple and easy to manufacture, and the separation operation is simple and easy to implement; 2) separating and obtaining the single spore strain in one time; 3) the separation work consumes less work and short time; 4) can obviously improve the working efficiency of the separation of the botrytis cinerea and the reliability of the separation result.

Description

Separation method of botrytis cinerea

Technical Field

The present invention relates to the field of plant pathology. In particular to a method for separating botrytis cinerea.

Background

The tomato gray mold is a serious disease of tomato which is a large vegetable in China and is caused by infection of Botrytis cinerea (Botrytis cinerea). The development of the biological research on the botrytis cinerea is an important basis for preventing and controlling the botrytis cinerea. On the premise of developing the biological research of botrytis cinerea, the botrytis cinerea material is required to be possessed. In general, laboratories often need to separate and obtain botrytis cinerea from disease standard samples.

The genetic composition of the botrytis cinerea population is complex, and the biological characteristics and pathogenicity of different strains are often greatly different. Normal research usually requires the acquisition of botrytis cinerea material, or monoclonal strain material, with a single genetic make-up. It is generally accepted in the art that bacterial strain material formed by the growth of a single spore is genetically composed of a single pathogen material. Therefore, the strain material for biological research of botrytis cinerea generally needs to obtain single spore isolation strain material.

The method for separating botrytis cinerea disclosed so far mainly comprises a conventional tissue isolation culture method and a conidium dilution isolation culture method.

The tissue isolation culture method is mainly technically characterized in that the surface of a tissue with bacteria needs to be disinfected, and then a disinfected tissue block is transferred to a culture medium for culture. The method is easily polluted by saprophytic bacteria, and importantly, the pure culture bacteria obtained by separation generally do not belong to botrytis cinerea with single genetic composition.

The conidium dilution separation culture method is mainly technically characterized in that conidia at a diseased part are washed in an aqueous medium, a conidium suspension is diluted and dispersed appropriately, then spores in the suspension are separated through a micro liquid transfer gun, a capillary tube, a transplantation needle or other instruments, and a single conidium is picked for culture. The method needs to implement the spore quantification and dilution operation process of the spore liquid, is relatively troublesome, and is easy to cause the mixed bacteria pollution to influence the efficiency and the effect of the separation work.

Disclosure of Invention

The invention aims to provide a separation method for obtaining pure culture of botrytis cinerea with single genetic composition by separation culture.

The technical scheme for solving the technical problems is as follows:

a method for separating botrytis cinerea adopts a fine, soft and elastic spore trailing and dispersing filament, directly separates out a single conidium from a tomato gray mold standard sample, and cultures to form a tomato gray mold bacterial strain with single genetic composition, and the operation steps of separation culture are as follows:

1. collecting tomato gray mold standard samples: in the tomato planting season, typical tomato gray mold standard samples are collected in the field and taken back to a laboratory to be used as standard samples of the tomato gray mold to be separated.

2. Firing spore dispersion silk: taking out a 1.5ml common centrifugal tube, putting the bottom end of the centrifugal tube on flame for ignition, melting the bottom end of the centrifugal tube at the ignition position, after safety flameout, clamping a little melted centrifugal tube by using a pointed-end forceps, slightly elongating until a filamentous filament is formed, cooling, and cutting off redundant filaments to form a spore drag-off filament used for separating conidia of botrytis cinerea, wherein the drag-off filament comprises a sliding filament a1 and a silk handle a 2; a section of common penholder is firmly sleeved in the centrifugal tube, so that the length of the screw handle a2 is increased; .

3. Preparing a spore dispersing plate:

preparation of agar plate substrate: preparing agar matrix containing conventional nutrition according to the mixture ratio of agar 15g and water 1000ml, and sterilizing at high temperature and high pressure to obtain agar plate matrix for use.

Pouring agar plate and purifying plate surface layer: heating the agar plate substrate prepared in the first operation to fully melt, pouring the agar plate substrate into a culture dish to prepare a thin-layer plate while the agar plate substrate is hot, placing the hot liquid-shaped plate into a heat preservation device capable of continuously keeping hot liquid, preserving heat and standing for more than 20 minutes, then smoothly transferring the liquid-shaped thin-layer plate which is kept standing to a sterile working table, and forming the agar plate with a purified surface layer after the liquid-shaped thin-layer plate is condensed.

Cutting and loading the spore dispersing plate: cutting the agar plate prepared by the operation II into long strip-shaped agar blocks by using a sterilization blade, wherein the agar blocks are the trawling plates for dispersing spores; the swab was picked up with a sterile blade and placed on a sterile slide to form a swab b1 for spores to be dispersed.

4. Dragging conidia: and (3) carrying out conventional surface disinfection and cleaning on the spore dispersing silk prepared in the step 2, then carrying out manual handling on a silk handle a2 of the spore dispersing silk, lightly touching the end part of the sliding silk a1 on the mildew layer on the surface of the standard scab prepared in the step 1, adhering conidia, transferring the sliding silk a1 adhered with spores to one end of the spore dispersing plate b1 prepared in the step 3, attaching the sliding silk a1 to the surface of the dispersing plate, and relatively sliding the sliding silk a1 on the surface of the dispersing plate to cause the spores adhered to the sliding silk a1 to be separated and dispersed on the surface of the dispersing plate.

5. Cutting single spore dices: observing the trawling plate b1 carrying the conidia obtained in the step 4 under a microscope, searching for discrete spores along the sliding track of the sliding silk a1, finding out single spores, selecting single spores far away from the left and right adjacent spores, cutting off and removing the trawling plate b1 on the left and right of the single spores by using a sterile blade to obtain a small square carrying only 1 single conidia.

6. Formation of monospore isolates: and (3) transferring the small square with the single conidium cut out in the step (5) to a conventional culture medium of botrytis cinerea by using a blade, and performing conventional culture until the spore germinates and grows to form a single spore colony. And (4) transplanting hypha of the single spore colony to expand and propagate to form the single spore isolation strain of botrytis cinerea.

The invention has the advantages that:

1) the key apparatus (spore dragging silk) for separating the spores is simple and easy to manufacture, the separation operation is simple and easy to implement, and the difficult micro-operation technical training is not needed.

2) The separation and the acquisition of the monoclonal strain can be achieved at one time, and a special monospore separation procedure is not needed.

3) The aim of separating botrytis cinerea is achieved, labor and time are saved.

4) The plate surface bearing conidia is purified through the surface layer, the effective visual field observed by a microscope is uniform and clean, the interference of non-fused impurities is not easy to be caused in the operation process of finding and identifying the conidia of botrytis cinerea, and the efficiency of separation work and the reliability of separation results can be obviously improved.

Drawings

FIG. 1 is a schematic drawing of spore streaks produced using a 1.5ml centrifuge tube. In the figure, the slide wire a1 and the wire handle a2 are shown. Fig. 1-1 is the original spore dispersion silk, and fig. 1-2 is the spore dispersion silk sleeved with a penholder.

FIG. 2 is a schematic view of a spore dispersion plate placed on a slide. In the figure, there is a drag plate b1, slide b 2. Fig. 2-1 illustrates a front view and fig. 2-2 illustrates a side view.

FIG. 3 is a schematic diagram of spore blooming operation. In the figure, a slide wire a1, a wire handle a2, a distraction plate b1, a slide b2, and a slide direction c1 of the slide wire.

FIG. 4 is a drawing of the surface of a die plate viewed microscopically. In the figure, conidia a, impurity b. FIG. 4-1 is a top-cleaned mop plate, and FIG. 4-2 is a top-unpurified mop plate. The black bars in the figure are 50 μm long.

FIG. 5 is a process of spreading conidia on a surface of a scattering plate by sliding on the scattering plate after the conidia are adhered to the sliding silk of the scattering wire. In the figure, conidia a. FIG. 5-1 shows the initial contact of the sliding strand with the distribution plate, where many spores are scattered off the sliding strand onto the distribution plate. FIG. 5-2 shows the gradual reduction of spore dispersion in the sliding trajectory. Fig. 5-3 shows the appearance of discrete, independent monospores in a gliding trace. FIGS. 5-4 are isolated single spores that are relatively distant from the left and right adjacent spores. The black bars in the figure are 50 μm long.

FIG. 6 shows the process and results of isolating a single spore colony of Botrytis cinerea from the sample 1 using the method of the present invention. FIG. 6-1 is Botrytis cinerea on the tomato petioles of Standard 1. FIG. 6-2 shows 3 cubes placed on a petri dish plate, each cube carrying a single conidia isolated from the tomato gray mold standard of FIG. 6-1. FIG. 6-3 shows that the conidia of FIG. 6-2 germinate and grow to form distinct microcolonies after being cultured at 28 ℃ for 2 days. FIGS. 6-4 show that the microcolonies of FIGS. 6-3 grow into larger colonies after further incubation at 28 ℃ for 1 day. The black bars in the figure are 5mm long.

FIG. 7 shows the process and results of isolating a single spore colony of Botrytis cinerea from the sample 1 using the method of the present invention. FIG. 7-1 is Botrytis cinerea on tomato fruit of Standard 2. FIG. 7-2 shows 3 cubes on a petri dish plate, each of which carries a single conidium isolated from the tomato gray mold standard of FIG. 7-1. FIG. 7-3 shows that the conidia of FIG. 7-2 germinate and grow to form distinct microcolonies after being cultured at 28 ℃ for 2 days. FIGS. 7-4 show that the microcolonies of FIGS. 7-3 grow into larger colonies after further incubation at 28 ℃ for 1 day. The black bars in the figure are 5mm long.

Detailed Description

The invention is further described with reference to the following figures and examples.

The botrytis cinerea can infect leaves, petioles and fruits of tomatoes, and conidia of the botrytis cinerea can be formed at the diseased parts. The invention adopts an ingenious means to separate a single conidium from a standard sample of the botrytis cinerea, and the pure culture of the botrytis cinerea with a single genetic composition is formed by culture. The specific embodiment is as follows.

1. Collection of tomato gray mold standard sample

Generally, tomato gray mold occurs commonly in tomatoes planted in production places, and the disease usually has a typical symptom at the diseased part, namely, a gray mold layer which generates a large number of conidia is formed on the surface of a diseased spot. The tomato gray mold standard sample with typical symptoms can be selectively collected in the field, put into a clean appliance and taken back to a laboratory, and stored in a refrigerated container at 4 ℃ for separation.

2. Firing of spore dispersion silk

Taking out a 1.5ml common centrifugal tube, putting the bottom end of the centrifugal tube on flame for burning, melting the burning part at the bottom end of the centrifugal tube, safely extinguishing the burning on the centrifugal tube by using an alcohol lamp fire extinguishing cover and other devices, clamping a little melted centrifugal tube by using a pointed forceps, slightly elongating to form a hairline-shaped filament, cutting off redundant filaments after cooling, and keeping the filament with the length of about 3cm connected on the centrifugal tube, namely forming a spore dispersing filament used for separating botrytis cinerea spores of tomatoes, wherein the filament dispersing tool comprises a sliding filament a1 and a screw handle a2, and the two parts are naturally connected into a whole as shown in a figure 1-1, and the key technical function is the sliding filament a 1. The sliding wire a1 is thin, soft and elastic.

The screw handle a2 is actually the tube body of the centrifuge tube, which is not convenient for subsequent handling operation due to its short length, and a section of ordinary pen holder or pencil can be sleeved and screwed into the centrifuge tube through the tube mouth of the centrifuge tube to form a long screw rod convenient for manual handling operation, as shown in fig. 1-2.

When separating different specimens for use, the sliding wire a1 can be wiped by a 75% alcohol cotton ball for surface sterilization, and the above operations can be repeated to burn and stretch new sliding wire.

3. Preparation of spore dispersing plate

The preparation method is as follows.

Firstly, preparing an agar plate substrate:

agar matrix containing conventional nutrients is prepared according to the mixture ratio of 15.0g of agar and 1000.0ml of water. The conventional nutrition of the botrytis cinerea can be added into an agar substrate by using a Potato Dextrose (PD) culture medium according to a conventional dosage, and the agar substrate containing the nutrition is obtained after conventional high-temperature and high-pressure sterilization. In practical application, the usage amount of the agar can be determined according to the quality of the commercially available agar, the usage amount of the agar per liter of the matrix can be changed from 10.0g to 15.0g, and in principle, the usage amount is small, and when the agar is cut into small agar strips after condensation, the small agar strips still have good elasticity.

The surface-clarified agar plates were then inverted:

the agar content of a conventional agar medium plate was 2.0 g/liter, and non-melting impurities in the surface layer of the agar plate were easily seen under a microscope in a plate poured in a conventional manner. The inventor finds that the non-melting impurities in the surface layer of the agar plate can be effectively reduced by adopting the following preparation technology: the flat plate melted and poured at high temperature is kept in a hot melting state for more than 20 minutes and then is condensed. The method specifically comprises the following steps:

the heat-insulating device can be assembled by assembling common appliances and materials, for example, a common food packaging iron box is covered on a common small steel basin for containing hot water, namely, a hot iron box panel (a culture dish is placed by using the iron box panel) is obtained, then a plastic basin is turned upside down to cover the iron box and the hot water steel basin, and heat-insulating materials such as towels are laid on the plastic basin.

When the hot liquid type culture dish is in work, a common small steel basin is placed on a sterile workbench, hot water which is just boiled is poured into the small steel basin, an iron box is covered, a flat-bottom culture dish is placed on a panel of the iron box, the culture dish is preheated for 5 minutes by covering a plastic basin, then the plastic basin with the covering is opened, an agar plate substrate which is fully melted by heating is poured into the preheated culture dish while the agar plate substrate is hot, a thin plate with the thickness of about 1.0mm is formed, the plastic basin is covered and laid with a heat insulation material, the thin plate is kept in a hot liquid state for heat insulation and standing for more than 20 minutes, non-molten impurities on the surface layer of the hot liquid plate can be slowly settled into the lower layer, excessive evaporation of water of the hot liquid plate due to long-time high temperature is considered, the solidification performance of agar is influenced, and the heat insulation. And opening the plastic basin with the cover, stably transferring the liquid thin-layer agar plate to a sterile working table for cooling, and condensing to obtain the agar plate with a purified surface layer.

In the subsequent step, the agar plate is used to carry conidia of the tomato gray mold standard, and antibacterial drugs such as streptomycin and the like can be added to the agar matrix in a conventional manner before the plate is poured in consideration of possible bacterial contamination.

Cutting and loading the agar plate to form a spore dispersion plate:

the prepared agar plate is cut into long and narrow agar strips by a sterile surgical blade, the agar strips can be cut into long agar blocks with the width of 3mm and the length of 4-5 cm, the long agar blocks are picked and transferred onto a sterile glass slide by the surgical blade, the long agar blocks are arranged along the length direction of the glass slide, and the arranged long agar blocks are used as a smearing plate b1 for smearing spores as shown in figure 2.

4. Medicinal powder of conidia

Cleaning and sterilizing the surface of the prepared spore mop with alcohol and sterile water in a conventional manner, holding the mop handle a2 of the mop by hand, lightly touching the slide silk a1 on the mildew layer on the surface of the prepared tomato gray mold standard scab, adhering a plurality of conidia on the surface of the slide silk a1, transferring the slide silk a1 adhered with spores to the prepared spore mop b1 and attaching the slide silk to one end of the mop plate, then dragging the slide silk a1 along the mop plate surface and sliding the slide silk to the other end of the mop plate b1, as shown in fig. 3, the contact sliding of the slide silk a1 on the mop plate surface causes the spores adhered on the slide silk a1 to fall off and be scattered on the mop plate surface.

5. Cutting of single spore dices

Observing the sporulated distraction plate b1 under a common microscope, the surface impurities of the purified distraction plate b1 are obviously reduced, as shown in figure 4, so that the accuracy of identifying and judging the botrytis cinerea spores and the mixed spores of the tomato can be obviously improved.

Usually, many conidia fall off from the place where the slide wire a1 first contacts the trawl board, and the slide track of the slide wire a1 is observed, so that the spores falling off the trawl board gradually become sparse and less, discrete single spores appear, the search is continued along the slide wire track, and independent single spores far away from the left and right adjacent spores can be found, as shown in fig. 5. Usually, 1-5 single spores which are independent and far away can be dragged out on one dragging and scattering plate.

When the microscope stage is moved and it is confirmed that the single spore is far from the adjacent spores on the left and right sides, the left and right dispersion plates of the single spore are cut off and removed by a sterile scalpel blade to obtain a small square carrying only one single conidium.

The microscopic observation process mainly uses a 10-time objective lens, and if necessary, the objective lens is switched to a 40-time objective lens to observe fine characteristics, so that the single botrytis cinerea conidia belong to. The cutting operation can leave the eyepiece of the microscope and be directly observed by naked eyes without micromanipulation. The operation is as follows: the method is characterized in that a light-gathering diaphragm under a microscope stage is reduced, a light-gathering module is adjusted up and down until a visible light source of naked eyes gathers into a small light spot which penetrates through a diffusion plate on a slide, the diameter of the small light spot is about 2mm, the diameter of a real object covered by a 10-time objective field of a common microscope is about 2mm, the lower knife position when the spore diffusion plate is cut is judged by referring to the two parameters, the distance between a lens of the 10-time objective and an observed object is usually 1cm, and the method is suitable for cutting operation with a sharp-pointed blade (such as a No. 23 surgical blade). After cutting, the cutting result can be observed by returning to the microscope eyepiece, and the cutting result comprises whether the cutting position of the lower knife is proper or not and the existence condition of spores on the smearing plate before and after cutting.

6. Formation of monospore isolates

The cut cubes carrying a single conidium are transferred with a scalpel blade to a conventional plate (e.g. potato dextrose agar PDA) medium for conventional cultivation of Botrytis cinerea. When the culture medium plate is poured, antibacterial drugs (such as streptomycin and the like) can be added according to the conventional method in consideration of the possibility of bacterial contamination. Conidia normally germinate and grow on the diamonds, and can form obvious microcolonies after being cultured for 2 days at the temperature of 23 ℃ generally, and the microcolonies are continuously cultured and expanded to form larger colonies. The single colony is transplanted with hypha for propagation to obtain the isolating strain of botrytis cinerea.

Example 1

By adopting the separation method of the botrytis cinerea, the botrytis cinerea of the tomato gray mold standard sample 1 is separated, and the separation culture result is shown in fig. 6.

Example 2

By adopting the separation method of the botrytis cinerea, the botrytis cinerea of the tomato botrytis cinerea standard sample 2 is separated, and the separation culture result is shown in fig. 7.

Claims

1. A method for separating botrytis cinerea is characterized in that a fine, soft and elastic spore trailing and dispersing filament is adopted, a single conidium is directly separated from a tomato gray mold standard sample, a tomato gray mold bacterial strain with single genetic composition is formed by culture, and the operation steps of separation culture are as follows:

1) collecting tomato gray mold standard samples: collecting a typical tomato gray mold standard sample in the field in the tomato planting season, and taking the standard sample back to a laboratory as a standard sample for separating the tomato gray mold germs;

2) firing spore dispersion silk: taking out a 1.5ml common centrifugal tube, putting the bottom end of the centrifugal tube on flame for ignition, melting the bottom end of the centrifugal tube at the ignition position, after safety flameout, clamping a little melted centrifugal tube by using a pointed-end forceps, slightly elongating until a filamentous filament is formed, cooling, and cutting off redundant filaments to form a spore drag-off filament used for separating conidia of botrytis cinerea, wherein the drag-off filament comprises a sliding filament (a1) and a silk handle (a 2); a section of common penholder is firmly sleeved in the centrifugal tube, so that the length of the screw handle (a2) is increased;

3) preparing a spore dispersing plate:

preparation of agar plate substrate: preparing agar matrix containing conventional nutrition according to the mixture ratio of 15g of agar and 1000ml of water, and sterilizing at high temperature and high pressure to obtain agar plate matrix for later use;

pouring agar plate and purifying plate surface layer: heating the agar plate substrate prepared in the first operation to fully melt, pouring the agar plate substrate into a culture dish to prepare a thin-layer plate while the agar plate substrate is hot, placing the hot liquid-shaped plate into a heat preservation device capable of continuously keeping hot liquid, preserving heat and standing for more than 20 minutes, then stably transferring the liquid-shaped thin-layer plate which is kept standing to a sterile working table, and forming the agar plate with a purified surface layer after the liquid-shaped thin-layer plate is condensed;

cutting and loading the spore dispersing plate: cutting the agar plate prepared by the operation II into long strip-shaped agar blocks by using a sterilization blade, wherein the agar blocks are the trawling plates for dispersing spores; picking up the smear with a sterilizing blade and placing the smear on a sterilizing glass slide to form a smear for spore dispersion (b 1);

4) dragging conidia: carrying out conventional surface disinfection and cleaning on the spore dispersing silk prepared in the step 2), then carrying out manual handling on a silk handle (a2) of the spore dispersing silk, lightly touching the end part of the sliding silk (a1) on the mildew layer on the surface of the standard scab prepared in the step 1), adhering conidia, transferring the sliding silk (a1) adhered with spores to one end of the spore dispersing plate (b1) prepared in the step 3), attaching the sliding silk to the surface of the dispersing plate, dragging the sliding silk (a1) along the surface of the dispersing plate, and relatively sliding the sliding silk (a1) on the surface of the dispersing plate to cause the spores adhered to the sliding silk (a1) to be separated and scattered on the surface of the dispersing plate;

5) cutting single spore dices: observing the trawl board (b1) carrying the conidia obtained in the step 4) under a microscope, searching for discrete spores along the sliding track of the sliding silk (a1), finding out single spores, selecting single spores far away from the left and right adjacent spores, cutting and removing the trawl board (b1) on the left and right of the single spores by using a sterilizing blade to obtain a small square carrying only 1 single conidia;

6) formation of monospore isolates: transferring the small cubes loaded with the single conidia cut out in the step 5) to a conventional culture medium of botrytis cinerea by using a blade, and performing conventional culture until the spores germinate and grow to form single spore colonies; and (4) transplanting hypha of the single spore colony to expand and propagate to form the single spore isolation strain of botrytis cinerea.