CN113785765B - Kit for visually researching interaction relation between plant and nematode, method and application - Google Patents
Kit for visually researching interaction relation between plant and nematode, method and application Download PDFInfo
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Images
Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G31/00—Soilless cultivation, e.g. hydroponics
- A01G31/02—Special apparatus therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/10—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/20—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material
- A01G24/22—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material containing plant material
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/20—Reduction of greenhouse gas [GHG] emissions in agriculture, e.g. CO2
- Y02P60/21—Dinitrogen oxide [N2O], e.g. using aquaponics, hydroponics or efficiency measures
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Inorganic Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention relates to a kit for visually researching interaction relation between plants and nematodes, a method and application thereof, and belongs to the technical field of plant disease control. The invention provides application of a plant nutrient medium containing 0.6-0.8% of agar in percentage by mass in preparation of a kit for visually researching interaction relation between plants and nematodes. The plant nutrient medium containing agar with the mass percentage of 0.6-0.8% can provide basic nutrient elements required by normal growth of plants and can simulate the migration characteristic of nematodes in soil, ensure that the plant parasitic nematodes are easy to migrate in the gel and can infect root systems, realize statistics and visualization of nematode infection amount and statistics of formation and counting of root knots, and greatly facilitate the related research on the interaction between plants and nematodes.
Description
Technical Field
The invention relates to the technical field of plant disease control, in particular to a kit for visually researching interaction relation between plants and nematodes, a method and application thereof.
Background
Plant parasitic nematodes are one of the important pathogens causing crop diseases in China and all over the world, can generally cause 10 to 30 percent of yield reduction, sometimes even cause no grain harvest, and cause agricultural loss of hundreds of billions each year. The most harmful area and the most harmful degree of cyst nematodes and root-knot nematodes are particularly used in China, and for horticultural crops, particularly vegetables, the root-knot nematodes are mainly used, are mainly obligate living body nutrition parasitic nematodes, can only parasitize and feed living host plant tissues and cells, and cannot be cultured in vitro. The pathogenic nematodes induce the abnormal differentiation of plant cells mainly through secretions secreted by oral needles of the nematodes, form specific feeding sites for the nematodes, form giant cells, cause the growth disorder of plants, block the transportation and information exchange of substances above the ground and underground, and seriously affect the growth and development of the plants.
The root-knot nematode disease has been known for a long time, and the prevention and the treatment of the root-knot nematode mainly adopt methods of chemical agent prevention and treatment (such as dazomet and metam) and physical prevention and treatment (such as high-temperature greenhouse closing and large water flood irrigation) at present. However, chemical agents themselves have environmental pollution and human toxicity, and long-term application not only improves the self-resistance of nematodes to reduce the drug efficacy of the agents, but also easily causes toxicity to non-target objects (such as harmless soil microorganisms and animals). In addition, physical control not only disrupts the physicochemical properties of the soil (e.g., disrupts soil aggregate structure, causes nutrient imbalance), but also is detrimental to beneficial microbial communities and crop growth. In recent years, the methods for inhibiting the propagation of root-knot nematodes in soil and preventing and controlling the root-knot nematode diseases of crops by biological measures, such as the mode of preventing and controlling the root-knot nematodes by using biocontrol bacteria, are also gradually increased.
Until now, there is no practical, effective and environment-friendly measure to reduce the occurrence of root-knot nematode disease, and one of the main reasons is that the factors affecting root-knot nematode infection on plant root system are very many, and root-knot nematode itself, as a lower organism, has already formed a complete infection mode and parasitic mechanism in the long-term evolution process, and brings great research challenges to researchers in related fields.
The infection amount or the infection rate of the second instar larvae J2 of the root-knot nematodes is often a key index for preventing and treating diseases, and meanwhile, in the research on the prevention and treatment of the root-knot nematodes by the biocontrol bacteria, how to screen effective biocontrol bacteria in a short time and verify the influence of the biocontrol bacteria on the infection amount of the root-knot nematodes are also a breakthrough in the field. Therefore, how to simplify the test factors to the greatest extent under the laboratory condition and research the interaction of the root-knot nematode and the root system is a key point in the field; how to simplify the test conditions, accurately count and visualize the infection amount of the root-knot nematode and visualize the root knot in the later period is a difficult point of researchers.
Disclosure of Invention
The invention aims to provide a kit for visually researching interaction relation between plants and nematodes, a method and application thereof. The plant nutrient medium containing 0.6-0.8% of agar by mass percentage can provide basic nutrient elements required by normal growth of plants, can simulate the characteristic of migration of nematodes in soil, ensures easy migration of plant parasitic nematodes in gel and infection of root systems, realizes statistics and visualization of nematode infection amount, realizes statistics of formation and counting of root knots, and is greatly convenient for relevant research on interaction of plants and nematodes.
The invention provides application of a plant nutrient medium containing 0.6-0.8% of agar in percentage by mass in preparation of a kit for visually researching interaction relation between plants and nematodes.
The invention also provides a kit for visually researching the interaction relationship between the plant and the nematode, which comprises a device for visually researching the interaction relationship between the plant and the nematode and a culture medium; the device is a transparent plant culture device, and the culture medium comprises a plant nutrient culture medium containing agar with the mass percentage of 0.6-0.8%.
Preferably, the plant nutrient medium further comprises 800-950 mg/L of calcium nitrate except agar, 550-600 mg/L of potassium sulfate, 90-100 mg/L of ammonium dihydrogen phosphate and 450-493 mg/L of magnesium sulfate, and the pH is adjusted to 6.8-7.2.
Preferably, the device comprises a flat-bottomed transparent glass tube; the shape of the device includes a cylinder.
Preferably, the device further comprises a tube cover for shading light.
The invention also provides a method for visually researching the interaction relation between plants and nematodes based on the kit in the technical scheme, which comprises the following steps:
culturing the seed with radicle in a device containing culture medium with radicle facing downwards and shading the part of the device containing culture medium;
and (4) after the root system is obviously stretched, inoculating the nematodes, and performing visual observation after 60-72 hours of inoculation.
Preferably, the radicle length of the radicle-growing seed is 0.8-1.0 cm; the root system is obviously extended to the length of 4-5 cm.
Preferably, the nematodes are aseptically cultured prior to inoculation; the sterile culture method comprises the following steps:
picking up nematode egg masses from roots of plants infected with nematodes, soaking the egg masses in 0.6-0.8% sodium hypochlorite aqueous solution for oscillation, screening the egg masses through a 600-mesh screen after oscillation, washing the egg masses on the screen with sterile water, then washing the egg masses into a culture dish by using aqueous solution containing 1.0-1.5 mg/ml gentamicin and 0.04-0.05 mg/ml nystatin, incubating for 4-5 days under the dark condition to obtain hatching fluid containing second-instar nematodes, and filtering the hatching fluid containing the second-instar nematodes through a filter screen with the aperture of 40 mu m to obtain filtrate, namely sterile nematode suspension.
Preferably, the nematodes comprise root knot nematodes.
Preferably, the visualization comprises direct visual observation of root knots and/or observation of nematode infestation by acid fuchsin staining.
The invention provides application of a plant nutrient medium containing 0.8% of agar by mass in preparation of a kit for visually researching interaction relation between plants and nematodes. The plant nutrient medium containing 0.8% of agar by mass can provide basic nutrient elements required by normal growth of plants, can efficiently simulate the good environment that nematodes infect root systems in soil under the condition of ensuring normal growth of the plants, ensures that the plant parasitic nematodes are easy to migrate and can infect the root systems in gel, can rapidly realize statistics and visualization of nematode infection amount in a short time, realizes statistics of formation and counting of root knots, improves research efficiency, has the advantages of short period, obvious effect, easiness in infection and the like, and is greatly convenient for relevant research on interaction of the plants and the nematodes. The culture medium can also be used for researching the infection efficiency of the nematodes under the conditions of different seeds and different treated seeds, so that plant materials with certain resistance can be screened more efficiently.
Drawings
FIG. 1 is a schematic view of the structure of the apparatus according to the present invention;
FIG. 2 is an experimental flow chart of the plant-nematode interaction method and apparatus provided by the present invention;
FIG. 3 is the growing state soil of cucumber root system in glass tube; wherein, A is the initially placed germinated seed, B is the growth state of the root system of the cucumber before inoculation of the meloidogyne incognita, and the direction shown by the arrow in C is the position for inoculation of the meloidogyne incognita;
FIG. 4 is a visual result diagram of nematodes in root systems after the cucumber root systems are dyed with fuchsin according to the invention; wherein A, B is respectively in the state after the dyed root system is tabletted under an optical microscope at 40 times, and C, D is respectively in the state without tabletted under a body type microscope;
FIG. 5 is a visual result diagram of root knots formed after root-knot nematode infection in a self-made culture medium of the cucumber root line provided by the invention; wherein A is a root system diagram of root knots after the second-instar larvae are inoculated for 14 days, and B is a root system diagram of root knots after the second-instar larvae are inoculated for 24 days;
FIG. 6 is a graph showing the different conditions and visualization degrees of seed germination under different treatments provided by the present invention;
FIG. 7 is a graph showing the different conditions and visualization degree of rooting and growth of seeds under different treatments provided by the present invention.
Detailed Description
The invention provides application of a plant nutrient medium containing 0.6-0.8% of agar in percentage by mass in preparation of a kit for visually researching interaction relation between plants and nematodes. The invention preferably prepares a gel nutrient medium containing 0.6 to 0.8 percent of agar according to essential nutrient elements required by plant growth. According to the invention, the interaction relation between the plant and the nematode is researched by utilizing the agar-containing culture medium which is prepared in advance and contains the nutrient elements for plant growth, so that the plant growth is facilitated, the migration and efficient root system infection of the root-knot nematode can be ensured, and the root-knot nematode infection can be visualized and quantified within a period of time. In the present invention, the agar is more preferably contained in an amount of 0.8% by mass.
The invention also provides a kit for visually researching the interaction relationship between the plant and the nematode, wherein the kit comprises a device for visually researching the interaction relationship between the plant and the nematode and a culture medium; the device is a transparent plant culture device, and the culture medium comprises a plant nutrient culture medium containing agar with the mass percentage of 0.6-0.8%. In the present invention, the agar is more preferably contained in an amount of 0.8% by mass.
In the invention, the plant nutrient medium preferably also comprises 800-950 mg/L of calcium nitrate, 550-600 mg/L of potassium sulfate, 90-100 mg/L of ammonium dihydrogen phosphate and 450-493 mg/L of magnesium sulfate except agar, and the pH is adjusted to 6.8-7.2; more preferably, the calcium nitrate is 945mg/L, the potassium sulfate is 607mg/L, the ammonium dihydrogen phosphate is 96mg/L and the magnesium sulfate is 493mg/L, and the pH value is preferably 7.0. When the kit is used, a culture medium is preferably prepared firstly, the preparation method of the culture medium is not particularly limited, and the conventional culture medium preparation method well known to a person skilled in the art is adopted, and after the culture medium is prepared, the culture medium is sterilized by high pressure steam, cooled to 53-60 ℃, more preferably 55 ℃, and poured into a device for solidification to be used. In the present invention, the autoclaving condition is preferably autoclaving at 121 ℃ for 20min.
In the present invention, the device preferably comprises a flat bottom transparent glass tube; the shape of the device preferably comprises a cylinder. In the present invention, the device preferably further comprises a tube cover for shielding light. The pipe cover is preferably used for shading the part containing the culture medium in the device, so that the natural growth of the plant root system is ensured. The device according to the invention preferably also comprises a transparent cover, which ensures that the plants are not contaminated and can receive light during the cultivation process. When the device is a cylindrical flat-bottomed transparent glass tube, the specification of the device is preferably: the bottom surface is preferably 2.5cm in diameter and 13cm in height, and the device is schematically shown in FIG. 1. In the apparatus of the present invention, the culture medium is preferably poured in a volume of 1/2 to 3/5 of the volume of the apparatus. When the specification of the device is as described above, the culture medium is preferably added in an amount of 30mL, and after pouring the culture medium, the culture medium is preferably used after it has cooled sufficiently and solidified and the tube wall has no water drops. The culture medium is in a gel state, and can ensure that the root system can normally grow, and the nematodes are easy to migrate and infect. The device containing the culture medium is easy to infect and observe on the whole, the test period is short, and the nematode infection condition and the root knot visualization effect are obvious.
The invention also provides a method for visually researching the interaction relation between plants and nematodes based on the kit in the technical scheme, which comprises the following steps:
culturing the seed with radicle in a device containing culture medium with radicle facing downwards and shading the part of the device containing culture medium;
and (4) after the root system is obviously stretched, inoculating the nematodes, and performing visual observation after 60-72 hours of inoculation.
The invention cultures the seeds with radicle in the device with culture medium, the radicle is downward, and the part of the device with culture medium is shaded. In the present invention, the method for preparing the radicle-growing seed preferably comprises: plant seeds were cultured for germination in the dark at 28 ℃. In the present invention, the plant seeds are preferably cultured in a culture dish containing sterile water and sterile filter paper. In the present invention, the plant seeds are preferably subjected to a surface sterilization operation before germination. In the present invention, the method of surface sterilization preferably includes: sterilizing an ethanol aqueous solution with the volume percentage of 75 percent of ethanol for 30s, sterilizing a sodium hypochlorite aqueous solution with the mass percentage of 2 percent for 5min, and fully washing with sterile water for 4-5 times until no peculiar smell exists. In the present invention, the radicle length of the radicle-growing seed is preferably 0.8 to 1.0cm, more preferably 0.8cm, and the seed is usedThe radicles with the length are moved into a special device of the kit, so that the survival rate of the germinated seeds can be ensured, and the root system can be more easily and normally stretched. After the radicle is placed downwards, the matched pipe cover is preferably covered; meanwhile, a pipe cover is used for wrapping the part which is guaranteed to shield the culture medium, and the material of the pipe cover is preferably aluminum foil paper. In the present invention, the conditions for the culture are preferably 14 hours (28 ℃ C.) under light, 10 hours (20 ℃ C.) in the dark, and 396. Mu. Mol. M under light -2 ·s -1 . In the present invention, the time for the culture is preferably 4 to 5 days.
The invention inoculates the nematodes after the root system is obviously stretched, and performs visual observation after 60-72 hours, preferably 72 hours, of inoculation. In the invention, the root system is obviously stretched, preferably the length of the root system is 4-5 cm, so that a certain root system growth is ensured, and the subsequent nematode infection and result observation are facilitated. In the present invention, the nematodes preferably include root knot nematodes, more preferably, meloidogyne incognita. The invention preferably inoculates second-instar larvae of root-knot nematodes J2.
In the present invention, the nematodes are preferably aseptically cultured before inoculation; the sterile culture method comprises the following steps: picking up nematode egg masses from roots of plants infected with nematodes, soaking the egg masses in 0.6-0.8% sodium hypochlorite aqueous solution for oscillation, screening the egg masses through a 600-mesh screen after oscillation, washing the egg masses on the screen with sterile water, then washing the egg masses into a culture dish by using aqueous solution containing 1.0-1.5 mg/ml gentamicin and 0.04-0.05 mg/ml nystatin, incubating for 4-5 days under the dark condition to obtain hatching fluid containing second-instar nematodes, filtering the hatching fluid containing the second-instar nematodes through a filter screen with the aperture of 40 mu m, and finally obtaining filtrate which is sterile nematode suspension. The sterile culture method can ensure the sterility of the second-instar larvae. The sterile nematode suspension according to the invention is preferably inoculated with 180 to 200 head of second instar larvae per plant with a root length of 4 to 5cm, in the specific embodiment of the invention 200. Mu.l.
In the present invention, the visual observation preferably includes direct visual observation of root knot and/or observation of nematode infestation by acid fuchsin staining. The present invention preferably visually observes the formation and counting of root knots directly. In the present invention, the infestation of the nematodes preferably includes the infestation amount of the nematodes. According to the invention, acid fuchsin dyeing is preferably carried out on infected root systems, and the infection amount of the second-instar larvae is counted. Specifically, the invention preferably takes out the root system in the culture medium 72h after inoculation, washes the root system with distilled water, and performs acid fuchsin staining. The specific dyeing step is preferably as follows: soaking the taken root system in a sodium hypochlorite aqueous solution with the mass percentage of 2% of sodium hypochlorite for 2min, fully washing the root system with tap water, then soaking the root system in 30ml of distilled water dripped with 1ml of acid fuchsin prepared in advance, immediately taking out the root system after dyeing for about 40s, immediately washing the fuchsin remained on the surface with the tap water, then immediately placing the root system in acid glycerol for color fading on a high-temperature electric furnace, and taking down the glycerol after boiling for 5-10 s and naturally cooling the glycerol. In the present invention, the specific configuration of the acid fuchsin is preferably as follows: 3.5g fuchsin +250ml acetic acid +750ml distilled water and mixed well. For acid fuchsin-stained roots, the invention preferably observes under a microscope 40 times after the glass slide is pressed and counts the infection amount of the second-instar larvae.
The invention is based on a plant nutrient medium (total nutrient migratory gel) containing agar with the mass percentage of 0.8 percent, and has the culture effect of simultaneously meeting the normal growth of plants and the normal and efficient infection of nematodes on the basis of obtaining sterile seeds and sterile root-knot nematodes; the infection amount and the root knot number of the root-knot nematodes can be visually counted in the later period through dyeing of acid fuchsin and transparent visualization of agar, so that the related research of the interaction of the plant nematodes is greatly facilitated; in addition, the method can be used for further researching the infection efficiency of the nematodes under the conditions of different seeds and different treated seeds, so that plant materials with certain resistance to plant parasitic nematodes can be screened more efficiently.
The kit, the method and the application for visually studying interaction relationship between plants and nematodes are described in detail with reference to the following embodiments, and the technical solutions of the present invention include, but are not limited to, the following embodiments.
Example 1
1. Sterilization and pregermination of plant seed surfaces
The seeds used in the embodiment are 'Zhongnong No. 26', and the quality of the seeds is ensured to be between 0.025g and 0.033 g. The seeds are sterilized by 75 percent ethanol for 30s and 2 percent sodium hypochlorite for 5min in turn, the seeds are fully washed by sterile water for 4 to 5 times until no peculiar smell exists, and then the seeds are placed in a culture dish containing a proper amount of sterile water and sterile filter paper for germination acceleration in dark at the temperature of 28 ℃ until the radicles are about 0.8 cm.
2. Preparation and application of self-made agar gel
In order to meet the normal growth of plants and provide a proper environment for the migration of root-knot nematodes, an agar gel culture medium containing nutrient elements required by the growth of the plants is prepared, the prepared culture medium is fully and uniformly mixed, and then the mixture is sterilized by high-pressure steam at 121 ℃ for 20min; after the self-made culture medium is cooled to about 55 ℃, pouring the self-made culture medium into flat-bottom glass tubes (13 cm multiplied by 2.5 cm) prepared in advance, wherein each tube is about 30ml, and using the self-made culture medium after the self-made culture medium is fully cooled and solidified and the tube walls have no water drops.
The prepared water solution containing the plant nutrient elements comprises the following specific contents: 945mg/L calcium nitrate, 607mg/L potassium sulfate, 96mg/L ammonium dihydrogen phosphate and 493mg/L magnesium sulfate.
The agar concentration of the self-made culture medium is 0.8%, and under the concentration, the second-instar larvae of the meloidogyne incognita can normally and efficiently infect root systems.
Plant growth and quantification of second instar larvae J2 and visualization of root knots
And (4) placing the cucumber seeds germinated in the first step into the self-made agar gel culture medium prepared in the second step by using sterile forceps, enabling radicles to face downwards, and wrapping the periphery of the tube walls by using tin foil paper. And (4) placing the plant in a plant illumination incubator to grow for 4 days (the root system is about 4-5 cm long). After two symmetrical holes were made around the root system with a yellow tip, 100. Mu.l of surface-sterilized Meloidogyne incognita J2 suspension, prepared beforehand, was injected. And (4) after the nematode is inoculated for about 72 hours, taking out the root system in the glass tube, washing the root system by distilled water, and dyeing by acid fuchsin. The acid fuchsin-stained roots were removed, pressed through a glass slide, observed under a 40-fold microscope and counted for J2 number by a counter.
The plant illumination incubator is arranged as follows: the plant light incubator is set to light for 14h (28 deg.C) and dark for 10h (20 deg.C), and the light is 396 mu mol.m -2 ·s -1 。
The root-knot nematode used above is meloidogyne incognita (melodogyne) and the preparation method of the meloidogyne incognita J2 suspension is as follows: selecting fresh egg blocks from a root system of a water spinach infected with meloidogyne incognita, soaking the egg blocks in 0.8% sodium hypochlorite solution for sufficient oscillation, filtering the egg blocks by using a 600-mesh screen sieve to enrich the meloidogyne incognita on the 600-mesh screen sieve, sufficiently washing the egg blocks by using sterile water, finally washing the egg blocks in a culture dish by using a solution containing 1.5mg/ml gentamicin and 0.05mg/ml nystatin, incubating the egg blocks for 4 to 5 days under a dark condition, passing an incubation solution containing J2 through a purple cell filter screen (40 mu m), and finally obtaining a pure J2 suspension, wherein the concentration of the suspension is adjusted to be 100J2/100 mu l.
The specific process of dyeing the acid fuchsin comprises the following steps: taking out root systems of the cucumbers, soaking the root systems in 2% sodium hypochlorite for 2min, fully washing the root systems with tap water, soaking the root systems in 30ml of distilled water dripped with 1ml of acid fuchsin prepared in advance, immediately taking out the root systems after dyeing for about 40s, cooling the root systems to room temperature, immediately putting the root systems in acid glycerol after washing residual fuchsin on the surfaces with the tap water, performing color fading on the acid glycerol in a high-temperature electric furnace, and taking down and naturally cooling the glycerol after the glycerol boils for about 5-10 s.
Wherein the specific configuration of the acid fuchsin is as follows: 3.5g fuchsin +250ml acetic acid +750ml distilled water and mixed well.
Fig. 2 is a schematic diagram of the test flow of example 1, and fig. 3, fig. 4, and fig. 5 are diagrams showing the test results of this example at different operating points. Wherein A, B in fig. 3 respectively show the state that the germinated seeds are moved into the device and the state that the root systems grow to 4-5 cm and are suitable for infection, and an arrow head shown as C in fig. 3 is a symmetrical nematode inoculation position (a hole needs to be pricked by a gun head in advance). FIG. 4 is a visual result diagram of nematodes on root systems after the cucumber root systems are dyed with fuchsin; wherein A, B is respectively in the state after the dyed root system is tabletted under an optical microscope at 40 times, and C, D is respectively in the state without tabletted under a body type microscope; FIG. 5 is a visual result diagram of root knot formation of cucumber root lines in a self-made culture medium after root knot nematode infection; wherein A is a root system diagram of root knots after the second-instar larvae are inoculated for 14 days, and B is a root system diagram of root knots after the second-instar larvae are inoculated for 24 days; fig. 4 and 5 respectively show that the root-knot nematode J2 can migrate in the device and can successfully infect the root system, and the infected root system can form root knots.
Example 2
Two different agar concentrations (0.8%, 1.0%) and two solvents (distilled water, nutrient solution (i.e., the aqueous solution containing plant nutrient elements used in example 1)) were set for treatment, namely distilled water +0.8% agar, distilled water +1.0% agar, nutrient solution +0.8% agar, and nutrient solution +1.0% agar, respectively.
The procedure was as in example 1 except for the specific description.
After the treatment by the method of example 1, the fresh weight of root systems, the number of single-plant second-instar larvae and the number of second-instar larvae per gram fresh weight of the four treated plants were counted 72 hours after inoculation of meloidogyne incognita. The results show that the fresh weight of the roots is obviously increased after the nutrient solution is added into the gel, and the growth and development of the roots can be promoted; in addition, compared with 1.0% agar, under the condition of 0.8% agar, the infection number of the single-plant second-instar larvae and the infection number of the fresh weight of root systems per gram can be obviously increased;
fig. 6 and 7 show different conditions and visualization degrees of seed germination and rooting growth under different treatments, and it can be seen that 0.8% agar gel can provide higher visualization for root systems and later-stage root knots compared with 1.0% condition, indicating that the kit provided by the present invention can facilitate the related research of nematode and root system interaction in combination with the appropriate concentration of jon in the culture medium.
As can be seen from Table 1, the statistics of the fresh weight of the root system and the number of nematode infections under different treatments show that the nutrient solution and 0.8% agar gel environment can provide better visualization, and provide a suitable environment for migration and infection of the root-knot nematodes J2, so that the soil environment can be simulated efficiently, the plants can grow, the nematodes can be infected, and root knots can be generated, and a suitable method and a device for researching interaction of the root-knot nematodes and the root system are provided for related researches.
Table 1 verification of the infection efficiency of root-knot nematodes by using 4 gels of different configuration
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.
Claims (6)
1. A method for visually researching the interaction relationship between plants and nematodes is characterized by comprising the following steps:
culturing the seed with radicle in a device containing culture medium with radicle facing downwards and shading the part of the device containing culture medium;
after the root system is obviously stretched, inoculating the nematodes in the culture medium, and after 60-72 hours of inoculation, carrying out visual observation;
the device is a transparent plant culture device, and the culture medium comprises a plant nutrient culture medium containing agar with the mass percentage of 0.6-0.8%;
the plant is cucumber;
the radicle length of the seed with the radicle is 0.8-1.0 cm;
the root system obviously extends to the length of 4-5 cm;
the plant nutrient medium also comprises 800-950 mg/L of calcium nitrate except agar, 550-600 mg/L of potassium sulfate, 90-100 mg/L of ammonium dihydrogen phosphate and 450-493 mg/L of magnesium sulfate, and the pH is adjusted to 6.8-7.2.
2. The method for visually studying the interaction relationship between plants and nematodes of claim 1, wherein said device comprises a flat bottom transparent glass tube; the shape of the device includes a cylinder.
3. The method of visually investigating the interaction relationship between a plant and a nematode as claimed in claim 2 wherein said device further comprises a tube housing for shading.
4. The method for visually studying the interaction relationship between plants and nematodes according to claim 1, wherein said nematodes are aseptically cultured before inoculation; the sterile culture method comprises the following steps:
picking up nematode egg masses from roots of plants infected with nematodes, soaking the egg masses in 0.6-0.8% sodium hypochlorite aqueous solution for oscillation, screening the egg masses through a 600-mesh screen after oscillation, washing the egg masses on the screen with sterile water, then washing the egg masses into a culture dish by using aqueous solution containing 1.0-1.5 mg/ml gentamicin and 0.04-0.05 mg/ml nystatin, incubating for 4-5 days under the dark condition to obtain hatching fluid containing second-instar nematodes, and filtering the hatching fluid containing the second-instar nematodes through a filter screen with the aperture of 40 mu m to obtain filtrate, namely sterile nematode suspension.
5. The method for visualizing the interaction relationship between a plant and a nematode as in claim 1 or 4 wherein said nematode comprises a root-knot nematode.
6. The method for visually studying the interaction relationship between a plant and a nematode as claimed in claim 1, wherein said visual observation comprises direct visual observation of root knot and/or observation of nematode infestation by acid fuchsin staining.
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