CN110835605A - Method for culturing and observing peronospora parasitica - Google Patents
Method for culturing and observing peronospora parasitica Download PDFInfo
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Abstract
The invention relates to the technical field of biology, in particular to a method for culturing and observing peronospora parasitica. Collecting downy mildew; preparing a sporangium suspension; and (5) culturing the sporangium suspension. Repeated tests are carried out to obtain the technology which can induce cucumber downy mildew sporangium to germinate and release zoospores, the zoospores form stationary spores through movement and morphological change, the stationary spores germinate to form attachment cells, and the attachment cells germinate to generate invasive filaments. The technology can provide an effective technology for researchers to carry out deep research on the biological basis, disease resistance identification, bactericide screening and the like of the peronospora parasitica. By the method, researchers can realize quantitative statistics and monitoring on the quantity and morphological change of the zoospores of downy mildew and the quantity of formed infectants, and can also realize the research on the action mechanism of the bactericide. Clearly observed are the several developmental stages that the peronospora has to undergo before invading the host. The systematic observation method can provide first-hand data for the prevention and treatment of the symptomatic medicine of the pathogenic bacteria.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for culturing and observing peronospora parasitica.
Background
Cucumber downy mildew is an important disease in cucumber production, the spreading speed is high, and scabs formed on leaves often cause leaf tissue withering, so that the cucumber production is seriously influenced. A great deal of research is carried out at home and abroad on the field prevalence of cucumber downy mildew, the correlation with the environment temperature and humidity, the establishment of an early warning model and the like. However, the research on the biological basis of the Pseudoperonospora cubensis is relatively weak, and the important reason is that the research on the biological basis of the Pseudoperonospora cubensis is restricted due to the lack of related observation and test technologies. The cucumber downy mildew is used as oomycetes, the research on the biological basis of pathogenic bacteria is limited to a certain extent due to the fact that the cucumber downy mildew cannot be artificially cultured, and meanwhile, the asexual propagation spores of the downy mildew are zoospores which can successfully invade a host after a plurality of development stages, so that the links of formation, movement regulation, rest, germination and the like of the zoospores become essential observation contents for deep research. Due to the lack of related observation methods, the observation of the process is restricted, and an ideal test result and an optimal temperature range capable of reflecting the growth and development of the peronospora parasitica cannot be obtained. No system report of relevant documents is found at present. A large number of correlation analysis results depend on artificial inoculation of leaves to promote the onset of diseases to obtain the temperature and humidity range from infection to manifestation at different times. The disease needs to go through several stages and is not observed as expected. The establishment of the observation method of the system is a key problem which needs to be solved urgently at present. Through reference to the literature, the research on the biological basis of the peronospora parasitica is deficient, and the important reason is the lack of support of observation technology. However, in the prior art, the propagules of pathogenic bacteria are obtained by collecting diseased leaves in the field, the leaves are artificially inoculated, appropriate disease conditions are given to promote the disease occurrence, the optimal temperature and humidity required by the disease occurrence are obtained, and the temperature range suitable for the growth and development of the downy mildew is deduced. No detailed report is found on 5 key development links before invading hosts, such as zoospores released by the cysts of the peronospora parasitica and zoospores, zoospores are migrated and form resting spores, germinate and form attachment cells, and the like. Further limits the observation of the activity process before the peronospora destructor invades the host, and leads to slow progress of related research.
Disclosure of Invention
The purpose of the invention is as follows: in order to provide a method for culturing and observing peronospora parasitica with better effect, the specific purpose is seen in a plurality of substantial technical effects of the concrete implementation part.
In order to achieve the purpose, the invention adopts the following technical scheme:
the method for culturing and observing peronospora parasitica is characterized by comprising the following steps of:
collecting peronospora parasitica: collecting fresh downy mildew disease spots in a field, and observing gray-white and gray-black mildew layers growing on the back of the disease spots, namely sporangium and sporangium of the downy mildew; putting into a clean plastic bag, and taking back to a laboratory for later use;
preparation of sporangia suspension: the method comprises the following steps of placing downy mildew diseased leaves collected in the field on a clean workbench, carefully observing with naked eyes, taking a mildew layer generated by the back of a bright yellow scab leaf as a strain, brushing the mildew layer on the back of the leaf with a clean writing brush gently, pushing the mildew layer into a centrifugal tube or a finger-shaped bottle filled with sterilized or distilled water, repeatedly brushing for multiple times, shaking the centrifugal tube or the finger-shaped bottle uniformly, simultaneously sucking a suspension containing sporangium, dripping the suspension on a concave glass sheet, covering a glass slide, placing the glass slide under a microscope for observation, and observing the number and the development condition of the sporangium;
culturing of sporangia suspension: placing the centrifuge tube or finger-shaped bottle containing sporangium suspension in an incubator with illumination and temperature of 15-20 ℃ for pre-culture for 1 h.
The further technical proposal of the invention is that the method also comprises the following steps after the culture of the sporangium suspension, the sporangium suspension is pre-cultured for 1 hour at low temperature, and then the sporangium suspension is cultured for 1 hour at room temperature of 23-26 ℃.
The invention further adopts the technical scheme that the glass slide is taken out and placed at normal temperature, a concave glass slide hanging drop method is adopted to prepare the glass slide, and the glass slide is placed under a microscope for observation: firstly, the field of view needed to be observed is found by observation under a low-power lens, and then observation is carried out under an objective lens with the power of 20 times or 40 times.
The invention further adopts the technical scheme that the method comprises the following steps,
preparation work before the experiment: cleaning centrifuge tube or finger-shaped bottle, sterilizing, drying at 60 deg.C,
preparation of sterilized water: filling distilled water into a triangular flask for sterilizing a sterilization pot;
collecting downy mildew diseased leaves: keeping 3-5cm of petiole during collection; and (3) acquisition standard: collecting the polygonal scab with bright yellow on the front of the leaf and the frosty mildew layer with gray to gray black or black on the back of the leaf, namely the sporangium peduncle or sporangium of the downy mildew, quickly filling into a holding bag, and preventing the water loss of the leaf and the influence on the sporangium; or collected in the afternoon before sunset, and quickly filled into a moisture-keeping bag to prevent the leaves from losing water; taking back to the laboratory, wrapping petiole with cotton soaked with distilled water, placing into a culture dish with filter paper with saturated water laid at the bottom of 15cm for moisture-keeping culture, and placing into a 20-degree incubator for 12 h;
preparation of sporangia suspension: taking a disposable glove, brushing the sporangium gently by using a clean writing brush to avoid re-brushing mesophyll tissues by hands, and preparing sporangium suspension for later use;
pre-culture of sporangia suspension: adjusting the incubator at 15-20 deg.C under moderate illumination; putting a centrifuge tube or a finger-shaped bottle containing sporangium suspension into an incubator, and taking out after culturing for 1 h;
temperature change treatment: taking out the centrifuge tube or finger-shaped bottle containing the sporangium suspension, and placing the centrifuge tube or finger-shaped bottle on a test platform for standing culture for 1h at the temperature of 23-26 ℃;
and (4) microscopic observation: the sporangium suspension is absorbed and dropped on a concave glass sheet and is placed under a microscope for observation under a low power microscope, the zoospores with violent activities and the sporangium which is germinating and releasing the zoospores can be obviously observed, the zoospores stop moving to form stationary spores, the stationary spores germinate to form attachment cells, and the attachment cells further germinate to form the infected silks. The stage can complete the statistics of zoospore number; calculating the release rate of zoospores; calculating the release rate according to the empty capsule rate, counting the stationary spores, and calculating the formation rate of the stationary spores and the germination rate of anchorage zone; calculating an infection point or an infection rate;
after 2h, observing by using a pendant drop method: sucking the sporangium suspension, dropping the sporangium suspension into a concave glass slide, covering a cover glass, and placing under a microscope for observation, so that stationary spores can be obviously observed, and the stationary spores germinate to generate adherent cells;
after 3h, observing by using a pendant drop method: the sporangium suspension is sucked, dropped into a concave slide, covered with a cover glass and placed under a microscope for observation, and the invasion of the attached cells generated by the germination of the resting spores can be obviously observed to generate the infected silks.
The technical scheme is that the culture dish is placed into a culture dish of filter paper with the bottom of 15cm paved with saturated moisture for moisturizing culture, and the moisturizing culture is finished by adopting special design equipment, wherein the special design equipment is a moisturizing culture structure, the moisturizing culture structure comprises a culture dish lower part 1 and a culture dish upper part 2 which are mutually buckled, a cubic stainless steel filter screen 3 is downwards thermally bonded on the culture dish lower part 1, a sponge 4 is placed in the stainless steel filter screen 3, a top cover hole 5 is formed in the position, corresponding to the sponge, of the culture dish upper part 2, the top cover hole comprises a top cover 8 which can be lifted, and distilled water can be poured towards the sponge through the top cover after the top cover is opened;
because the maximum water holding capacity of the sponge is fixed, water in the sponge can downwards pass through the stainless steel filter screen 3 and drip on the moisturizing culture structure 6; therefore, water can be added without opening the lower part 1 of the culture dish and the upper part 2 of the culture dish to avoid the entry of mixed bacteria in the air; and the sponge is intermittently and continuously dripped;
the moisturizing culture structure 6 comprises a coating structure, the middle part of the coating structure comprises a clamping space 7, and the clamping space 7 is a water holding material;
the downy mildew blades can be placed in the holding space 7 such that both sides of the downy mildew blades are in contact with the water holding material.
The invention further adopts the technical scheme that the water holding material is filter paper.
Compared with the prior art, the invention adopting the technical scheme has the following beneficial effects: repeated tests are carried out to obtain the technology which can induce cucumber downy mildew sporangium to release zoospores, the zoospores form stationary spores through movement and morphological change, the stationary spores germinate to form attachment cells, and the attachment cells germinate to produce invasive filaments. The technology can provide an effective technology for researchers to carry out deep research on the peronospora parasitica. By this method it is clearly observed that the downy mildew fungus has to go through several stages of development before invading the host. The systematic observation method can provide first-hand data for the prevention and treatment of the symptomatic medicine of the pathogenic bacteria.
Drawings
To further illustrate the present invention, further description is provided below with reference to the accompanying drawings:
FIG. 1 is a flow chart of the process of inducing the release of the zoospores of downy mildew to develop into adherent cells; FIG. 2 is an example of a series of views of an infected silk formed by resting spores, zoospores, sporangium, released zoospores, resting spore germination, attached cells, attached cell germination; FIG. 3 is a moisturizing culture structure specifically designed in this patent; FIG. 4 is a top view of FIG. 3; wherein: 1. below the culture dish; 2. above the culture dish; 3. a stainless steel filter screen; 4. a sponge water holding space; 5. a cap hole; 6. a moisture-retaining culture structure; 7. a clamping space; 8. and a top cover.
Detailed Description
The invention is further illustrated below with the understanding that the following detailed description is illustrative of the invention only and is not intended to limit the scope of the invention. The patent provides a plurality of parallel schemes, and different descriptions belong to an improved scheme based on a basic scheme or a parallel scheme. Each solution has its own unique features.
The technology comprises the following elements: collecting fresh downy mildew in the field (collected in the morning and can be directly used, and collected in the afternoon and needs to be subjected to moisturizing culture treatment), wherein the judgment standard is that the blades have bright yellow irregular spots (mostly polygonal spots due to the limitation of veins), and observing gray-white-gray black mildew layers growing on the back of the spots, namely sporangium and sporangium of the downy mildew; put into a clean plastic bag and taken back to the laboratory for standby. Collecting in the morning as far as possible and completing before 10 points; preparation of sporangia suspension: the method comprises the steps of placing downy mildew diseased leaves collected in the field on a clean workbench, carefully observing with naked eyes, using a mildew layer generated on the back of bright yellow diseased leaves as a strain, gently brushing the mildew layer (sporangium peduncle and sporangium) on the back of the leaves by using a clean writing brush, pushing the mildew layer into a centrifugal tube or a finger-shaped bottle filled with sterilized or distilled water in a proper manner, repeatedly brushing for multiple times, shaking the centrifugal tube or the finger-shaped bottle uniformly, simultaneously sucking a suspending liquid containing sporangium, dripping the suspending liquid on a concave glass sheet, covering the concave glass sheet with the centrifugal tube or the finger-shaped bottle, observing the number and the development condition of the sporangium under a microscope. Placing a centrifuge tube or a finger-shaped bottle containing sporangium suspension in an incubator with illumination and a temperature of 15-20 ℃ for pre-culturing for 1h, taking out and placing at normal temperature, preparing a slide by a concave slide hanging drop method, and placing under a microscope for observation: after the visual field needing to be observed is found by observing under a low-power lens, the visual field is observed under a 20-time or 40-time objective lens.
The more specific implementation mode is as follows:
brief description of the drawings
1. Preparation work before the experiment: cleaning centrifugal tube or finger-shaped bottle, sterilizing, drying at 60 deg.C,
2. preparation of sterilized water: putting distilled water into a triangular flask, and putting the triangular flask into a sterilizing pot for sterilizing.
3. Collecting downy mildew diseased leaves: the leaf stalks of 3-5cm are kept during collection. Collecting a standard: collecting fresh yellow polygonal scab on the front of the leaf, gray to gray black or black frost mildew layer (sporangium peduncle or sporangium of peronospora parasitica) on the back of the leaf, and quickly filling into a holding bag to prevent the water loss of the leaf and influence on the sporangium; or collected in the afternoon before sunset, and quickly packed into a moisture-keeping bag to prevent the leaves from losing water. Taking back to the laboratory, wrapping petiole with cotton soaked with distilled water, placing into a culture dish with 15cm bottom and filter paper with saturated water for moisture-keeping culture, and placing into a 20 deg.C incubator for 12 hr.
4. Preparation of sporangia suspension: the disposable glove is worn, and the sporangium is lightly brushed by using a clean brush pen, so that the mesophyll tissue is prevented from being brushed by the hand again. Preparing sporangium suspension for use.
5. Pre-culture of sporangia suspension: adjusting the temperature of the incubator to 15-20 deg.C, and illuminating properly. Putting the centrifuge tube or the finger-shaped bottle containing the sporangium suspension into an incubator, and taking out the sporangium suspension after culturing for 1 h.
6. Temperature change treatment: the centrifuge tube or the finger-shaped bottle containing the sporangium suspension is taken out and placed on a test platform for standing and culturing for 1h (23-26 degrees).
7. And (4) microscopic observation: the sporangium suspension is absorbed and dropped on a concave glass slide, and the drop is observed under a low power microscope under a microscope, so that the zoospores with violent activities and the sporangium which germinates and releases the zoospores can be obviously observed, and the number of the released zoospores is counted. The release rate and the number of zoospores released were calculated. Calculating the release rate according to the empty capsule rate, calculating the number of zoospores according to the number of the zoospores, observing the stationary spores, germinating the stationary spores to form attachment cells, calculating the formation rate and the germination rate of the stationary spores, calculating the germination rate according to the attachment cells in a transparent leaf state, and calculating the infection point or the infection rate.
8. After 2h, observing by using a pendant drop method: the sporangium suspension is sucked, dropped into a concave slide, covered with a cover glass and placed under a microscope for observation, so that stationary spores can be obviously observed, and the stationary spores germinate to generate adherent cells.
9. After 3h, observing by using a pendant drop method: the sporangium suspension is sucked, dropped into a concave glass slide, covered with a cover glass and placed under a microscope for observation, and the invasion of the attached cells generated by the germination of the stationary spores can be obviously observed to generate the infected silks.
By utilizing the technology, the process that 5 different development stages are formed before the cucumber downy mildew bacteria invade the host can be clearly observed: namely 5 processes that the sporangium releases zoospores, the zoospores move and form change to form resting spores, the resting spores germinate to generate anchorage-dependent cells, and the anchorage-dependent cells germinate to generate invasive filaments. The observation of all key links that the peronospora sporangium germinates and releases zoospores to form stationary spores and then the stationary spores germinate and germinate attached cells to continue to grow and form infected silks and then successfully invade a host is realized. The technical bottleneck that zoospores are unstable in release, difficult to observe and count and difficult to observe the formation of attachment cells is broken through. However, the observation of the process in the prior art methods and techniques is incomplete and discontinuous. The systematic observation of 5 development stages before the invasion of the peronospora destructor is difficult to carry out, so that an accurate and ideal research result cannot be obtained, the systematic research around the peronospora destructor is restricted, such as tests of the biological basis of the peronospora destructor, an interaction mechanism with a host, an infection process, the screening of an antibacterial agent and the like, and the research result tends to be consistent or has large difference due to the lack of an operable research method. Therefore, the research results obtained by the technical observation strongly support the relevant research results of the biological basis of the peronospora parasitica, and draw the conclusion according with the fact. However, the existing research methods only have the germination test results of sporangium at different temperatures, and most of documents infer the optimal temperature condition for the growth and development of pathogenic bacteria through the incidence of leaf inoculation. There is a lack of experimental techniques that allow complete and systematic observation of the 5 development stages that peroid peronospora species before invading the host. According to the existing experimental methods in the literature, the observation purpose cannot be achieved. Therefore, the system and the deep research of the peronospora parasitica by people are restricted. However, the experimental technique not only overcomes the problem of difficult observation, but also provides convenience for counting the number of zoospores and changing the form. The technical method is characterized in that the zoospore germination can be greatly promoted to release the zoospores through the temperature conversion treatment of low-temperature induced culture and high-temperature promoted culture, the zoospores move and develop to form stationary spores, the stationary spores germinate to generate adherent cells, the adherent cells germinate to generate invasive silks, and the like. Therefore, compared with the existing research technology, the technical method overcomes the restriction factors that the zoospore is released unstably or not released, the zoospore quantity cannot be counted, the attachment cells are difficult to observe and the like, breaks through the bottleneck of the existing technology, and has substantial progress. Meanwhile, the method has the obvious advantages of simple operation, no need of large-scale equipment and instruments, no limitation of test conditions and the like, and realizes breakthrough of the technical method in the aspect of the experimental technology of the peronospora parasitica. The observation of the whole process of forming the infective silks from the zoospores to the anchorage zone is effectively solved, and the support is provided for the quantitative statistics and monitoring of the statistics of the number of the zoospores and the number of the infective bodies. Provides a feasible technical method for people to develop and deeply research the pathogenic mechanism and the infection process of the peronospora parasitica, the identification of disease resistance and the screening of bactericide. This is therefore a substantial technical breakthrough.
The technology is simple to operate, special equipment is not needed, the zoospore release rate is high, the zoospore quantity statistics can be carried out, the zoospore is observed to move to form stationary spores, the germination rate of the stationary spores reaches 100%, typical attachment cells are generated, and the attachment cells generate obvious infective hyphae. The correlation between the growth and development of pathogenic bacteria and the temperature and humidity is more accurate compared with the prior leaf inoculation. And 3, the method is more intuitive.
In general terms: the method solves the problem of observing the whole development process before the propamocarb asexual propagule of cucumber invades the mesophyll tissue of cucumber. The method comprises the steps of releasing zoospores, swimming and developing the zoospores, forming resting spores, germinating the resting spores to generate anchorage zone, generating invasion hyphae by the anchorage zone and other key development links, and establishes an induction technology for observing the whole process. The technology provides a feasible test technology for deeply researching the biological foundation of the cucumber downy mildew pathogen, the pathogen invasion mechanism, the invasion process, the disease resistance identification, the bactericide screening test and the like. The problem of relevant observation result difference caused by the problems of release, difficult statistics and observation, inaccurate quantification and the like of the cucumber downy mildew zoospores is effectively solved.
The technical scheme is that the culture dish is placed into a culture dish of filter paper with the bottom of 15cm paved with saturated moisture for moisturizing culture, and the moisturizing culture is finished by adopting special design equipment, wherein the special design equipment is a moisturizing culture structure, the moisturizing culture structure comprises a culture dish lower part 1 and a culture dish upper part 2 which are mutually buckled, a cubic stainless steel filter screen 3 is downwards thermally bonded on the culture dish lower part 1, a sponge 4 is placed in the stainless steel filter screen 3, a top cover hole 5 is formed in the position, corresponding to the sponge, of the culture dish upper part 2, the top cover hole comprises a top cover 8 which can be lifted, and distilled water can be poured towards the sponge through the top cover after the top cover is opened; because the maximum water holding capacity of the sponge is fixed, water in the sponge can downwards pass through the stainless steel filter screen 3 and drip on the moisturizing culture structure 6; therefore, water can be added without opening the lower part 1 of the culture dish and the upper part 2 of the culture dish to avoid the entry of mixed bacteria in the air; and the sponge is intermittently and continuously dripped;
the moisturizing culture structure 6 comprises a coating structure, the middle part of the coating structure comprises a clamping space 7, and the clamping space 7 is a water holding material;
the downy mildew blades can be placed in the holding space 7 such that both sides of the downy mildew blades are in contact with the water holding material. The technical scheme of the invention has the following substantial technical effects and the realization process: the defects of the prior art are that when the culture dish is required to be opened continuously and water is required to be added continuously during cultivation, the culture effect is poor if water is forgotten to be added; this patent directly adopts the mode that does not open culture dish below 1 to add water, avoids the miscellaneous fungus in the air to get into.
In addition, the existence of the centre gripping space 4 of this patent makes the leaf both sides can both hold water and cultivate, has avoided the problem that makes a confused which side of leaf has the bacterial of cultivateing sometimes and has caused the cultivation mistake in the time of original cultivation. The water holding material is filter paper. Of course, other materials can be used.
The foregoing shows and describes the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are intended to illustrate the principles of the invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, and the invention is intended to be protected by the following claims.
Claims (6)
1. The method for culturing and observing peronospora parasitica is characterized by comprising the following steps of:
collecting peronospora parasitica: collecting fresh downy mildew disease spots in a field, and observing gray-white and gray-black mildew layers growing on the back of the disease spots, namely sporangium and sporangium of the downy mildew; putting into a clean plastic bag, and taking back to a laboratory for later use;
preparation of sporangia suspension: the method comprises the following steps of placing downy mildew diseased leaves collected in the field on a clean workbench, carefully observing with naked eyes, taking a mildew layer generated by a bright yellow diseased leaf back as a strain, gently brushing the mildew layer on the leaf back by using a clean writing brush, pushing the mildew layer in a centrifugal tube or a finger-shaped bottle filled with sterilized or distilled water in a proper manner, repeatedly brushing for multiple times, shaking the centrifugal tube or the finger-shaped bottle uniformly, simultaneously sucking a suspension containing sporangium, dripping the suspension on a concave glass sheet, covering a glass slide, placing under a microscope for observation, and observing the number and the development condition of the sporangium;
culturing of sporangia suspension: placing the centrifuge tube or finger-shaped bottle containing the sporangium suspension into an incubator with illumination and the temperature of 15-20 ℃ for pre-culture for 1 h.
2. A method for downy mildew growth and observation according to claim 1, further comprising the step after the cultivation of the sporangium suspension, of subjecting the sporangium suspension to a low temperature pre-cultivation for 1 hour, and then subjecting the sporangium suspension to a room temperature of 23-26 ℃ for cultivation for 1 hour.
3. The method for culturing and observing peronospora parasitica according to claim 2, wherein the slide is prepared by the concave glass hanging drop method after being taken out and placed at normal temperature, and is observed under a microscope: firstly, the field of view needed to be observed is found by observation under a low-power lens, and then observation is carried out under an objective lens with the power of 20 times or 40 times.
4. A method for downy mildew cultivation and observation according to claim 2, comprising the steps of,
preparation work before the experiment: cleaning centrifuge tube or finger-shaped bottle, sterilizing, drying at 60 deg.C,
preparation of sterilized water: filling distilled water into a triangular flask for sterilizing a sterilization pot;
collecting downy mildew diseased leaves: keeping 3-5cm of petiole during collection; and (3) acquisition standard: collecting the polygonal scab with bright yellow on the front of the leaf and the frosty mildew layer with gray to gray black or black on the back of the leaf, namely the sporangium peduncle or sporangium of the downy mildew, quickly filling into a holding bag, and preventing the water loss of the leaf and the influence on the sporangium; or collected in the afternoon before sunset, and quickly filled into a moisture-keeping bag to prevent the leaves from losing water; taking back to the laboratory, wrapping petiole with cotton soaked with distilled water, placing into a culture dish with 15cm bottom and filter paper with saturated water for moisture-keeping culture, and placing into a 20 deg.C incubator for 12 hr;
preparation of sporangia suspension: taking a disposable glove, brushing the sporangium gently by using a clean writing brush to avoid re-brushing mesophyll tissues by hands, and preparing sporangium suspension for later use;
pre-culture of sporangia suspension: adjusting the incubator at 15-20 deg.C under moderate illumination; putting a centrifuge tube or a finger-shaped bottle containing sporangium suspension into an incubator, and taking out after culturing for 1 h;
temperature change treatment: taking out the centrifuge tube or finger-shaped bottle containing the sporangium suspension, and placing the centrifuge tube or finger-shaped bottle on a test platform for standing culture for 1h at the temperature of 23-26 ℃;
and (4) microscopic observation: the sporangium suspension is absorbed and dropped on a concave glass sheet, and is observed under a microscope under a low power microscope, so that the zoospores with violent activities and the sporangium which is germinating and releasing the zoospores can be obviously observed, the zoospores stop moving to form stationary spores, the stationary spores germinate to form attachment cells, and the attachment cells further germinate to form invasive filaments; the stage can complete the statistics of zoospore number; calculating the release rate of zoospores; calculating the release rate according to the empty capsule rate, counting the resting spores, and calculating the formation rate of the resting spores and the germination rate of anchorage zone; calculating an infection point or an infection rate;
after 2h, observing by using a pendant drop method: sucking the sporangium suspension, dropping the sporangium suspension into a concave glass slide, covering a cover glass, and observing under a microscope, wherein stationary spores can be obviously observed, and the stationary spores germinate to generate adherent cells;
after 3h, observing by using a pendant drop method: the sporangium suspension is sucked, dropped into a concave glass slide, covered with a cover glass and placed under a microscope for observation, and the invasion of the attached cells generated by the germination of the stationary spores can be obviously observed to generate the infected silks.
5. The method for downy mildew cultivation and observation according to claim 4, wherein the cultivation of keeping moisture in a petri dish with a filter paper with saturated water laid on the bottom of 15cm is performed by a special design equipment, wherein the special design equipment is a moisture keeping cultivation structure, the moisture keeping cultivation structure comprises a lower part (1) of the petri dish and an upper part (2) of the petri dish which are mutually buckled, a cubic stainless steel filter screen (3) is downwards thermally sealed on the lower part (1) of the petri dish, sponge (4) is placed in the stainless steel filter screen (3), a top cover hole (5) is formed in the upper part (2) of the petri dish corresponding to the sponge, the top cover hole comprises a top cover (8) which can be lifted, and distilled water can be poured into the sponge through the top cover after the top cover is opened; because the maximum water holding capacity of the sponge is fixed, water in the sponge can downwards pass through the stainless steel filter screen (3) and drip on the moisturizing culture structure (6); therefore, water can be added without opening the lower part (1) and the upper part (2) of the culture dish to avoid the entry of the mixed bacteria in the air; and the sponge is intermittently and continuously dripped;
the moisturizing culture structure (6) comprises a coating structure, the middle part of the coating structure comprises a clamping space (7), and the clamping space (7) is made of a water holding material;
the downy mildew blades can be placed in the clamping space (7) so that both sides of the downy mildew blades can be contacted with the water holding material.
6. A method for downy mildew growth and observation according to claim 5, wherein said water holding material is filter paper.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110564667A (en) * | 2019-09-25 | 2019-12-13 | 云南省烟草农业科学研究院 | method for efficiently suspending and concentrating zoospores of plant pathogenic oomycetes and application of zoospores |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104396597A (en) * | 2014-12-04 | 2015-03-11 | 云南省烟草农业科学研究院 | Real-time observation method for phytophthora nicotianae infection process |
| CN204550630U (en) * | 2015-03-30 | 2015-08-12 | 贵州大学 | A kind of modified version culture dish |
| CN206940875U (en) * | 2017-04-16 | 2018-01-30 | 中国热带农业科学院热带作物品种资源研究所 | A kind of plant disease moisturizing device |
| CN208079940U (en) * | 2017-11-29 | 2018-11-13 | 长江大学 | A kind of dandelion breeding apparatus |
-
2019
- 2019-08-27 CN CN201910794854.4A patent/CN110835605A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104396597A (en) * | 2014-12-04 | 2015-03-11 | 云南省烟草农业科学研究院 | Real-time observation method for phytophthora nicotianae infection process |
| CN204550630U (en) * | 2015-03-30 | 2015-08-12 | 贵州大学 | A kind of modified version culture dish |
| CN206940875U (en) * | 2017-04-16 | 2018-01-30 | 中国热带农业科学院热带作物品种资源研究所 | A kind of plant disease moisturizing device |
| CN208079940U (en) * | 2017-11-29 | 2018-11-13 | 长江大学 | A kind of dandelion breeding apparatus |
Non-Patent Citations (2)
| Title |
|---|
| SUN ET AL.: "Effects of Temperature and Moisture on Sporulation and Infection by Pseudoperonospora cubensis", 《PLANT DISEASE》 * |
| 虞皓等: "温度对黄瓜霜霉病菌产孢及孢子囊萌发的影响研究", 《广东农业科学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110564667A (en) * | 2019-09-25 | 2019-12-13 | 云南省烟草农业科学研究院 | method for efficiently suspending and concentrating zoospores of plant pathogenic oomycetes and application of zoospores |
| CN110564667B (en) * | 2019-09-25 | 2023-02-28 | 云南省烟草农业科学研究院 | Method for efficiently suspending and concentrating zoospores of plant pathogenic oomycetes and application of zoospores |
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