KR101499759B1 - Large Scale Screening Method for the Evaluation of Resistance against Bakanae diseases - Google Patents

Abstract

The present invention provides a method for massively assaying for two or more crop seeds at the same time to determine whether or not seed-borne resistant cultivars are cultivated.
The assay method according to the present invention comprises the steps of: (a) preparing a second container having two or more compartments through which the treatment liquid can pass, or two or more compartments through which the treatment liquid can pass; (b) chewing two or more crop seeds in the container, wherein different seeds are in different compartments of different first or second containers; (c) simultaneously dipping the container (s) in contact with two or more crop seeds in a bath in which the seed infectious agent pathogen suspension is housed; And (d) separately seeding the seeds inoculated in the previous stage for each compartment of the first vessel or the second vessel in a seedling box.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0002] The present invention relates to a large scale screening method for a rice-

The present invention relates to a method for massively assaying for two or more crop seeds at the same time to determine whether or not seed resistant to an infectious disease.

Breeding is a technique to nurture new or new varieties of crops that have a higher value for human life by improving or altering the hereditary quality of living things. New varieties must have a certain degree of excellence, uniformity and permanence, which is beneficial to agricultural production as crops, even if their traits are somewhat different from existing ones. On the other hand, the cultivation safety can be increased by cultivating and promoting new varieties that have improved resistance to cold weather, disease and insect damage. In particular, the spread of disease resistance and insect resistance cultivars makes it possible to economize agricultural management, such as saving pesticides.

As an example, rice gut disease is caused by Gibberella fujikuroi, or Fusarium moniliforme . It is a disease that occurs from the time of seedling to the date of seedling. It is named Bakanae disease due to abnormal growth of the seedlings. It was first reported in Japan in 1898. In Korea, it was a serious problem in some farms in the 1960s. In recent years, severe infections in rice seeds and the emergence of resistant bacteria to medicines have led to a rapid increase in the incidence, It is a tendency to develop. In addition, since rice varieties with high resistance to rice seedling disease have not been developed, enormous pesticides and labor force are consumed for spraying pesticides for control, which is a serious obstacle to production cost reduction and production of environmentally friendly agricultural products.

These rice seed disinfection methods include (1) hot water bathing method, which is a method of sterilizing and preventing pathogens and harmful microorganisms by making a hot water hot water at an appropriate temperature and immersing sowing seeds for a certain period of time, (2) hexaconazole, tebuconazole However, there is a limitation in that it is difficult to treat a large amount of seeds at a uniform temperature, and the chemical treatment such as seed disinfectant has a problem that the occurrence of drug resistant bacteria is worried have. Therefore, it is necessary to cultivate resistant varieties against rice pathogens in order to solve the root disease of rice.

As a conventional assay method for evaluation of rice hind resistance, there are a method of i) seeding rice seeds in a spore culture of a pathogenic bacterium and seeding the seedlings, A method for investigating the onset of Kidari disease in a seedling by sowing seeds on a soil mixed with a pathogenic bacterium cultivated in a medium, iii) an indoor test method using a test tube culture, iv) a method in which rice seeds are immersed in a spore culture medium, And the like. However, these methods have a limitation in that accuracy is decreased according to the method, a large area is required, and a long time is required for the verification.

In order to develop and produce new varieties, it usually takes 8 to 12 years or longer, and a wide cultivation area is required for system development and selection. Most of the rice cultivars cultivated in Korea are weak against rice pandemic, so the development of resistant cultivars is very demanding. Therefore, technology to improve breeding efficiency is required by developing new technology that can obtain precise and objective test results in order to save the time and cost required for selection of resistance resources and lines for rice genetic resources and mating groups.

Therefore, the inventors of the present invention have found that the use of a tissue embedding cassette such as a tissue embedding cassette for cultivating a variety of cultivars can be carried out in a cultivating process as a result of testing the resistant cultivars against seed infectious diseases, The present inventors have confirmed that a large number of resistant breeds can be tested against seed infectious diseases and completed the present invention.

It is an object of the present invention to provide a method for quantitatively testing whether two or more kinds of crop seeds are simultaneously seed-resistant to an infectious disease.

In order to achieve the above object, the present invention provides a method for massively testing seed-borne resistant crop varieties for two or more crop seeds at the same time, comprising the steps of: (a) Preparing a second container having two or more compartments through which the liquid can pass; (b) chewing two or more crop seeds in the container, wherein different seeds are in different compartments of different first or second containers; (c) simultaneously dipping the container (s) in contact with two or more crop seeds in a bath in which the seed infectious agent pathogen suspension is housed; And (d) separately seeding the seeds inoculated in the previous stage for each compartment of the first vessel or the second vessel in a seedling box.

Hereinafter, the present invention will be described in detail.

The present invention is based on the finding that two or more first vessels or treatment liquids through which a treatment liquid can pass such as a tissue embedding cassette can pass therethrough in order to massively test whether two or more kinds of crop seeds are simultaneously seed- Using a second vessel having two or more compartments, the different seeds are dipped into different compartments of different first vessels or second vessels, and then simultaneously immersed in a bath in which the seed infectious agent pathogen suspension is housed . This makes it possible to inoculate homogeneous concentrations of pathogens simultaneously for many crop genetic resources or breeding lines.

In addition, seeds infected with seed pathogens in the same environment are inoculated by using such containers, seeds inoculated from the previous stage for each of the compartments of the first container or the second container are sown and seeded in a box for seedling breeding, From this, it is possible to quickly, accurately and easily confirm the ratio of normal plants (dry matter) among the total germinated plants according to the kinds of cultivars, so that it is possible to objectively and quickly evaluate resistance to many kinds of crop genetic resources or breeding lines.

In the present invention, the term " crops " refers collectively to edible crops and artificial crops cultivated in a field of rice, and refers to a plant that cultivates rice fields or fields by farming. The crops include: i) food crops such as burdock, barley, beer barley, rice, bean, red bean, mung bean, corn, sweet potato and potato; ii) cabbage, cabbage, spinach, lettuce, buttercup, watermelon, melon, Tomato, strawberry, eggplant, radish, carrot, burdock, taro, red pepper, garlic, onion, ginger etc .; iii) fruit, apple, pear, peach, grape, iv) persimmon, )) Special crops such as cotton, rapeseed, sesame, perilla, peanut, hops, etc. Ⅵ) Gugija, Gyungyang, Angelica, Peony, Hwanggi, Medicinal crops such as mushrooms, mushrooms, and medicinal crops such as mushrooms, tobacco plants, tobacco plants such as tobacco, ginseng, etc.,

Preferably, the crops may be food crops such as burdock, barley, beer barley, rice, bean, bean, mung bean, corn, sweet potato, potato and the like, more preferably rice.

The term " seed infectious disease " in the present invention refers to a disease caused by seed mediated by a pathogen. The mode of seed transmission varies depending on the type of plant or pathogen, but is largely due to: i) the spread of the pathogens when they are mixed with the pathogens and ii) the pathogens adhering to the surface of the seeds or infiltrating into extremely shallow places of the seed tissues And iii) seeds can be divided into internal tissues, particularly those infiltrating deep into the stomach and the endosperm.

Specific examples thereof include rice blast, corn bacterium, poultry bacterium, bacterial egg blight, chimeric bottle, ear broth, barley bloom, streak bottle, anthracnose of bean, mummified bottle, Spotty disease, mosaic disease, and the like, and are not limited to those corresponding to diseases that are transmitted to the next generation of milk plants by seeds.

Preferably, the seed infectious disease may be a rice hind leg disease, wherein the rice hind leg disease is Gibberella fujikuroi or Fusarium moniliforme, etc., and the leaf becomes pale yellow immediately after the transplanting of the seedling of the rice or the transplantation of the rice, and the length of the grass is about twice that of the healthy one. In the pathogenesis of seed transmission, the fungus of the pathogen formed on the fungus after the flowering of the rice is scattered and becomes infected. In addition, new infections occur when the seeds are mixed when immersed in water to pre-sow. If pathogens are invading, they do not germinate or die soon. The untreated is painted as described above and exhibits typical symptoms, but is terminated at the end. Gibberella , belonging to the Acanthopanax senticosus, fujikuroi, whose incomplete generation is Fusarium moniliforme .

The term "resistant cultivar" in the present invention refers to a cultivar having high resistance to climate, disease, chemical substances and the like. "Resistance" is also referred to as disease resistance, which means that plants do not get sick when they are attacked by pathogens. In general, tolerance refers to the structural characteristics of plants, biochemical reactions of plants, for example, when plants produce substances that inhibit toxic substances or pathogen development to pathogens. Can be exercised. The intensity of the resistance exhibited varies from host-pathogen system to host system-pathogen system, and may vary depending on host stage, host or host system, host nutrition, weather conditions, etc. . Specifically, the host in the present invention may be a crop or a crop seed, and the pathogen may be a pathogen of a seed infectious disease. More specifically, the host is rice or rice seed, and the pathogen is a pathogen .

The " assaying method " of the present invention may include screening of varieties resistant to the disease under the same seeding conditions and the same cultivation environment. Therefore, in the present invention, it is possible to more objectively select resistant cultivars by inoculating a large number of crop cultivars in a small area and cultivating them at the same time. Selection of resistant breeds for seed infectious diseases can be made by visual observation or statistical calculation as described below.

In the method of the present invention, the step (a) is a step of preparing two or more first vessels through which the treatment liquid can pass, or a second vessel having two or more segments through which the treatment liquid can pass.

In the present invention, the term "treatment liquid" is a generic term for a liquid substance to be treated on a sample. In the present invention, a suspension of seed infectious pathogens necessary for inoculating a seed- It can be a treatment liquid. More specifically, it may be a spore suspension of a rice seedling pathogen for inoculating a rice seedling with a rice seedling pathogen.

In the present invention, the term "container" refers collectively to a container for holding articles, which means a container having a container for holding a black crop. It is not limited to the size, shape, shape, color, material, volume, etc. of the container. The container may be manufactured directly or may be a container already on the market.

The "first container " referred to in the present invention means a container having a form in which a liquid or the like such as a treatment liquid can pass. Specifically, it means a container having a form in which the liquid can freely move into and out of the container, and is not limited to the size, shape, material, color, volume and the like of the container. Preferably, there is a cover which can be opened in the first container. The same inoculation condition among cultivars can be continuously maintained in inoculating a seed infectious agent pathogen suspension into crop seeds or the like which have passed through a liquid such as a treatment liquid and floated in the first container.

In one embodiment of the present invention, a resistance embryo resistance against rice tiller disease was massively assayed using a tissue embedding cassette as a first container. In the case of a tissue embedding cassette, it has a structure of a porous structure on the side and / or bottom surface so that a liquid type processing solution can freely pass therethrough. In this case, There may also be covers that can be opened and closed.

Preferably, the first vessel has the same size and shape, and several first vessels can be arranged in an in-line arrangement in the following baths. Specifically, several of the first vessels have the same size and the same shape, so that the crop seeds can be impregnated under the same conditions. By seeding the same number of crop seeds of different varieties into each first container having the same size and shape, the seeding conditions such as the seeding conditions such as the degree of exposure to the treatment liquid during the germination period, More objective sorting of resistant varieties against seed infectious diseases can be made.

Preferably, the first vessels may be housed in another vessel through which the process liquid may pass. In the pathogenic infection of seed infectious diseases of crop seeds dipped in the first container, two or more first containers are accommodated in another container and are inoculated at the same time, whereby the inoculation conditions such as exposure time to pathogens can be made the same , Which makes it possible to more objectively select resistant cultivars. The container for accommodating two or more first containers is not limited in size, shape, material, color, volume and the like as long as the container has a structure in which the process liquid can pass therethrough like the first container. The rice seedling box can also be used as another container as described above.

In the case of the tissue embedding cassette proposed as one embodiment of the present invention, as a container for accommodating the cassette, about 280 cassettes can be accommodated per box for seedling breeding, so that a large number of test materials are simultaneously inoculated into pathogenic bacteria (Example 1-2 and Fig. 1-E). ≪ tb >< TABLE >

The "second container " referred to in the present invention means a container having two or more compartments in one container while allowing the treatment liquid to pass through.

In the present invention, the term "compartment" refers to a structure in which a certain place is bound to be distinguished or bounded. For example, a second container can be manufactured by dividing one container into several spaces, such as a partition, by having two or more compartments, but the method is not limited thereto. The container may be manufactured directly or may be a container already on the market.

The second container is divided into at least two compartments, but the processing liquid and the like can freely pass between the compartments. Specifically, the partition for dividing the container has a porous structure, or a gap is present in a part of the divided partition or the like, so that the treatment liquid or the like can move freely, so that the seed of the crop, The same inoculation condition among varieties can be continuously maintained.

Preferably, the compartments of the second container may be of the same size and shape. By taking the same size and shape of the compartments of the second container, it is possible to cultivate the same conditions in the cultivation of crop seeds. Specifically, the same number of different kinds of crop seeds of different varieties are confined to the respective compartments, so that more objective selection can be made by making the cultivation environments such as the degree of exposure of the treatment liquid and the cultivation area per seed during the germination period the same. The size and shape of the compartment are the same, and the shape, material and the like of the compartment are not limited. There may be a cover for each compartment, and a cover for the entire second container may be present.

In the present invention, the step (b) is a step of impregnating two or more kinds of crop seeds into the container, wherein different seeds are confronted with each other in different compartments of the first container or the second container.

In the present invention, the term "seed" means seed which is a basic substance necessary for propagation of an organism. The period of time when the seed has germination is called the lifetime of the seed, which greatly depends on the type of the crop and the storage conditions. Seeds obtained directly from each crop or commercially available seeds can be used.

Preferably, the seeds of the present invention can be rice seeds, which can be stored or stored in a generally dry and cold state for use in assay methods. More preferably, a field inspection conducted according to the " Major Crop Seed Act " and a good seed tested by indoor inspection can be used for the assay.

In the present invention, the term "bed" means that the seed is placed on a flat surface, and means that the seed of the crop according to the present invention is contained in the first container or the second container.

In the present invention, when seeding a test crop to the first container or the second container, the different seeds are confined within different compartments of the first container or the second container. Specifically, in order to select resistant cultivars against seed infectious diseases, seeds of different cultivars may be confined to other containers or other compartments to prevent mixing among the cultivars. In addition, resistance evaluation stages can be evaluated by containers or compartments, so that resistant cultivars can be easily selected visually.

As one experimental example of the present invention, 10 different rice varieties were subjected to a tissue embedding cassette for each variety (Experimental Examples 1-2 and 2-2), thereby evaluating the degree of resistance for each tissue embedding cassette, Resistant varieties were identified (Fig. 1-D).

In the present invention, the step (c) is a step of simultaneously dipping the container (s) in which two or more kinds of crop seeds are stuck into a bath containing a pathogen suspension, and inoculating the same.

In the present invention, the term "pathogen" means a microorganism causing seed infectious diseases and the like. Therefore, the pathogen in the present invention is, for example, Pyricularia oryzae , Achlya prolifera , Burkholderia glumae , Fusarium moniliforme , Gibberellia fujikuroi , Cochliobolus myobanus , Ustilaginoidea virens , Tuburcinia cepulae , Ustilago nuda , Ustilago hordei , Ustilago zeae , Rice stripe virus , Pyrenophora graminea , Colletotrichum truncatum , Glomerella glycines , Colletotrichum gloeosporioides , Diaporthe sojae , Diaporthe phaseolorum , Cercospora kikuchii , Soybean yellow common mosaic virus , Xanthomonas campestris , Pseudomonas savastanoi , Alfalfa mosaic virus, and the like.

Preferably, the pathogenic microorganism refers to a microorganism that causes rice seedling disease, and more specifically, Fusarium < RTI ID = 0.0 > moniliforme or Gibberellia fujikuroi , but is not limited thereto and may include various other pathogens known in the art. Gibberella , known as the pathogen of rice seedlings While fujikuroi is a complete generation of ovarian gonadal generation, Fusarium Moniliforme is an incomplete generation, an asexual generation. Usually damage is known to occur by incomplete genera , Fusarium In the case of moniliforme , it forms large and small conidiospore and forms thick-membrane spores. Large - sized follicles have 3 to 5 septa, and occasionally large conidia with 6 to 7 septa. It is characterized by the fact that the small-sized granules are in the form of a chain.

And pathogens in rice tall bottle used as an experimental example of the present invention corresponds to the Fusarium moniliforme, more specifically, was used as the CF283 of Fusarium moniliforme mycelial received pre-sale at the National Academy of Agricultural Science (Example 1-1). In addition, the Fusarium species The moniliforme strains were also assigned to the National Institute of Agricultural Science and Technology (KACC45819, 46888, 44008, 44014, 45818 and 44021, respectively).

The term in the present invention. &Quot; Suspension " refers to a suspended system in which solid fine particles are dispersed in a liquid, and is also referred to as a suspension. In general, the suspension refers to the extent to which the particles are visually or microscopically visible, but may also include suspension colloids in a broad sense. Specifically, the pathogen suspension in the present invention refers to a liquid substance in which seed pathogenic pathogens are dispersed, and is not limited to the type and concentration of the suspension and the type of pathogen.

Preferably, in the pathogen suspension, the pathogens may be present in the form of spores.

Thus, spore suspension means that spores of pathogens are suspended in water. Spore is a reproductive cell formed by fungi or plants as a means of asexual reproduction, and can be a new individual by itself, unlike a spouse. When plant pathogens are inoculated, water is added to the culture medium of the pathogens and the spores are rubbed with sterilized brush or platinum ears, etc., and the spores are dropped by filtration with gauze to make a spore suspension, and one drop is taken with a microscope And diluted with water to adjust the spore concentration to an appropriate value. The spore suspension thus prepared can be used for spraying, inoculation, needle, and immersion in a plant. Spore suspension can also be widely used for plant pathology studies, such as the effects of various chemicals such as bactericides, sugars and amino acids on spore germination, or penetration of pathogens into the host body and observation of infectious processes.

As an example of the present invention, the spores of Fusarium moniliforme were cultivated in PDA (potato dextrose agar) medium for 3 weeks (FIG. 1-A) A suspension was prepared (Fig. 1-B).

The concentration of the spore suspension of the pathogen of the present invention may preferably be 1 × 10 ⁴ spore / ml to 1 × 10 ⁶ spore / ml, more preferably 1 × 10 ⁶ spore / ml.

As an embodiment of the present invention, an analysis was made on the morbidity rate of Kidari disease according to the cultivar spore concentration. The concentration of pathogen spores was adjusted to 1 × 10 ² spore / mL, 1 × 10 ⁴ spore / mL, and 1 × 10 ⁶ spore / mL using a microscope (FIG. 1-C) , It was found that the higher the pathogen spore concentration was, the lower the mean value of the dry matter ratio, the lower the standard error, the lower the coefficient of variation, and the more statistically significant results were obtained (Experimental Example 1 -4). Therefore, the concentration of the suspension of the pathogen inoculated to the rice seedlings is preferably 1 × 10 ⁴ spore / ml to 1 × 10 ⁶ spore / ml, more preferably 1 × 10 ⁶ spore / ml. < / RTI >

As used herein, the term "bath" means a container capable of containing a pathogen suspension. Specifically, the bathtub according to the present invention is used to inoculate a pathological suspension of a test agricultural product seeded on a first container or a second container, and is capable of receiving several first or second containers or several first containers A container capable of accommodating another container is sufficient, and is not limited to the size, shape, shape, color, and material of the bathtub.

In the present invention, the term "immersion" refers to wetting a material in a liquid such as water. Specifically, in the present invention, the seeds for test harvesting in the first container or the second container are immersed in the pathogen suspension.

The term "inoculation " in the present invention means injecting pathogens into the body of a human body, a medium or other animals or plants for the prevention, diagnosis, treatment, or biological experiment of diseases. Specifically, the present invention means injecting pathogens of seed infectious diseases into the seeds of the test crops for the screening and selection of resistant varieties against seed infectious diseases. Preferably, pathogens of rice hind legs can be injected into the seeds of the assay rice.

As an experimental example of the present invention, the inoculation of the pathogen of rice seedlings was carried out by immersing the seeds of probiotics dipped in the first or second containers in a spore suspension of rice seedlings. More specifically, the seeds in the tissue embedding cassette were inoculated in a diluted spore suspension of Fusarium moniliforme for 3 days at 26 ° C. At this time, the spore suspension was shaken more than four times a day for the uniform inoculation of pathogenic bacteria (Experimental Examples 1-3 and 2-3, Fig. 1-G).

In addition, the method may further comprise disinfecting the seeds before the step (c) of the present invention. The physical or chemical treatment applied to seeds to eliminate germs or nematodes of seed transmission is called seed disinfection. Conventionally, in general, chemical disinfection of seeds with internal adhesion bacteria is performed using chemical agents, and physical disinfection . Among them, typical examples of chemical disinfection include immersion disinfection in which seeds are immersed in an aqueous solution of agrochemicals for a certain period of time, or disinfection of disinfection methods in which seeds of pesticides are applied to seeds. On the other hand, as a physical disinfection method, there is a warm water immersion method for eradicating the germs and nematodes in the seeds with hot water. In particular, in this method, the seeds are immersed in hot water at about 60 ° C. for 10 minutes, There is a cold water warming method in which water is soaked for 10 minutes in a cold water container and dried in the shade. In addition, methods such as thermal sterilization, fermentation disinfection, fumigation disinfection, aging disinfection, and seed disinfection using supercritical fluid are also available, and the present invention is not limited thereto as long as the seeds for assay are disinfected. As one embodiment of the present invention, a method of immersing a seed in a rice seedling box for 15 minutes in a water bath at 57 ° C was used (Experimental Examples 1-2 and 2-2, FIG. 1-F)

In the present invention, the step (d) is a step of seeding the seeds inoculated in the previous stage for each compartment of the first container or the second container separately in the seedling box.

The term "box for seedling exploitation " in the present invention refers to a box for raising a flat box for cultivating the rice husks of rice cultivated rice or a standard rice for gardening. Sowing seedlings by sowing rice seeds in a seedling box is a common method of rice cultivation. It is possible to place a box for seedling in a greenhouse or a protected place, usually until the seedling reaches a certain height, And so on.

Specifically, the box for rice seedling cultivation may be a rectangular container or tray having an open top and several centimeters in height, but the size, shape, shape, color, material, volume and the like of the seedling box are not limited in the present invention. Preferably, there are several openings for drainage of surplus irrigation water at the lower end or side of the seedling box, and the openings may be of a structure capable of opening and closing. The box may include a growth medium in which the crop seeds are seeded and grown, and the growth medium may be an artificial solid growth medium, but preferably it may be soil.

The term in the present invention. The term "planting" refers to the planting of seeds used for the propagation of crops in a broad sense, but usually means planting seeds or similar seeds. The seedling box can be used to grow young crops until the crop seeds have buds and reach a predetermined size. The predetermined size generally refers to the size at which each plant is usually transferred to the open field, which can be attributed to a number of factors, such as plant species and breeds, the climate of the area in which the plant is subsequently transplanted, the size of the box, Density, and the like. The crop seeds inoculated with the pathogens can be sown on a seedling box and cultivated until reaching a predetermined size, so that resistant cultivars against seed infectious diseases can be selected.

Preferably, the rice seeds inoculated into rice hind legs can be sown and cultivated to select resistant cultivars against rice hind legs.

More preferably, in sowing the rice seedlings into a seedling box, i) seeding the seedlings in the case of the first container, and ii) sowing the seedlings in the second container by dividing the seedlings into a predetermined size The cultivation of the cultivated young rice plants can further facilitate the resistance test against rice seedlings (Fig. 1-H).

The step (d) may further include the step of evaluating the pruning strain and the non-pruning strain for the germinating plant after sowing.

Preferably, the pruning ratio is calculated and evaluated, and the pruning ratio can be calculated by calculating the ratio of the normal plants among the total germinated plants by type.

In the present invention, the term "prunus tract" refers to a plant in a healthy state in which disease has not progressed at all, and a plant which is not severe enough to cause an onset but recovered after a slight coating is observed.

On the other hand, in the present invention, "vein electrophoresis" means a plant that has been severely painted, a plant that has spread 45 degrees or more between the new leaf and the lower leaf, a plant that has been exhumed by the organism, a plant that has been immediately after germination, .

The difference between the dry and the non-dry state of rice among the crops is as shown in Fig.

The assay method of the present invention can be used to select a crop genetic resource, or a resistant resource or lineage for a crossing population. Specifically, it is possible to find a crop genetic resource for resistance to seed infectious disease through genetic analysis among the seed breeding disease resistant varieties of the present invention. Also, through crosses between resistant cultivars, the present invention can be used to select cultivars that are more resistant to seed infectious diseases, i. E. Mating resistance resources or lines.

In accordance with the present invention, a method of massively assaying for two or more kinds of crop seeds at the same time by using a container through which a treatment liquid such as a tissue embedding cassette can pass can be used for a variety of crop genetic resources In addition to being able to inoculate homogenous concentrations of pathogens simultaneously in the growing system, objective and rapid resistance evaluation is possible.

FIG. 1 is a photograph of the inoculation method according to the test for rice hind resistance.
Figure 1-A shows Fusarium Moniliforme spores were cultured on PDA (potato dextrose agar) medium for 3 weeks.
Fig. 1-B is a photograph of suspension of cultured spores in water.
Fig. 1-C is a photograph of diluting the spores suspended in water to a proper concentration using a microscope.
1-D is a photograph showing seeds in a Tissue Embedding Cassette.
FIG. 1-E is a photograph showing a seeded embedding cassette arranged in a rice seedling box.
1-F is a photograph of sterilizing the seeds contained in the rice seedling box for 15 minutes in a 57 ° C water bath.
Fig. 1-G is a photograph showing that seeds contained in rice seedling boxes are immersed in a diluted spore suspension to inoculate the bacterium at 26 ° C for 3 days.
FIG. 1-H is a photograph of sowing seeds in a rice seedling box.
Figure 1-I is a photograph of the point at which the bottle developed.
FIG. 2 is a graph showing the distinction between a dry cell and a non-dry cell when evaluating the resistance.
FIG. 3 shows the results of Fusarium The difference in onset of spore concentration of moniliforme and the change in height of kidney per gibberellin (GA) concentration are examples.
Fig. 4 shows the results of various Fusarium < RTI ID = 0.0 > moniliforme strains. < / RTI >

Hereinafter, the present invention will be described in more detail in the following Examples and Experimental Examples. However, these examples are provided only to aid understanding of the present invention, and the present invention is not limited thereto.

Example  1: Cultivation and dilution of rice cultivars and pathogens

Example  1-1: Screening of rice cultivars

Ten varieties known to have different degrees of resistance were selected and used as a test for the difference in onset of spore concentration of Fusarium moniliforme against rice varieties. The selected varieties are Weonseadaesoo, Hawn, Nep Huong, Nanareum2, Nampyeong, Goaminybyeo, Samgang, Gayanybyeo, Junam and Hopum.

Example  1-2: Pathogen culture and dilution

Fusarium , a pathogenic bacterium of rice Moniliforme spores were cultured on a PDA (potato dextrose agar) medium for 3 weeks. The cultured spores were suspended in water and observed with a microscope to determine the appropriate concentration (1 × 10 ² spores / mL, 1 × 10 ⁴ spores / mL , 1 × 10 ⁶ spores / mL).

Experimental Example  One: Fusarium moniliforme Difference in onset of spore concentration

Experimental Example  1-1: Fusarium moniliforme Fungi  And Gibberellin  density

Fusarium used in this test The moniliforme strains were obtained from the National Institute of Agricultural Science and Technology, Korea. Fusarium used for inoculation The spore concentration of moniliforme was adjusted to 1 × 10 ² spore / mL, 1 × 10 ⁴ spore / mL, and 1 × 10 ⁶ spore / mL as in Example 1-2.

Experimental Example  1-2: Tissue Embedding Cassette Seedling and disinfection of seeds

Tissue Embedding Cassette was seeded with rice seeds and arranged in a rice seedling box. In order to remove various germs including Kideri disease causing bacteria that were inoculated during the previous year 's cultivation process, the seeds contained in the rice seedling box were sterilized for 15 minutes at 57 ℃ water bath.

Experimental Example  1-3: Inoculation of pathogens and Gibberellin  Cultivation after treatment

After sterilization of the seeds, the seeds in the rice seedling box were collected by Fusarium moniliforme diluted spore suspension and inoculated at 26 ° C for 3 days. At this time, spore suspension was shaken more than 4 times a day for uniform inoculation of pathogenic bacteria.

Fusarium Gibberellin was treated with 50ppm of gibberellin in order to compare the results with the gibberellin (GA) treatments and the mating rate of Kidney Disease according to the concentration of moniliforme spores.

Fusarium The seeds inoculated with the spores of moniliforme were arranged in a rice seedling box and cultivated from the greenhouse to the point where the disease was sufficiently developed. The degree of resistance was evaluated.

Experimental Example  1-4: Fusarium moniliforme  According to the spore concentration, Kidari bottle At the rate of  Analysis of variance

Spore concentration
(spore / mL) Average Standard error Coefficient of variation Sum of squares Mean squared F-value 1 x 10 ² 74.0 20.0 27.1 9513.98 1057.10 2.63 ^* 1 x 10 ⁴ 69.0 14.0 20.4 19094.81 2121.64 10.69 ^** 1 x 10 ⁶ 60.9 8.89 14.6 24190.89 2687.87 33.96 ^**

*: Significant at 5% level; **: Remark at 1% level.

In the distinction of chickenpox and vigorous chickenpox according to the rice plant variety, i) the healthy poultry is in a state of healthy condition in which the disease has not progressed at all, (Ii) plants that have been severely impregnated with plants that have been severely painted, plants with an angle of more than 45 ° between the apexes of the young and the young, plants that have contracted by germination, plants that have undergone atrophy, It says. Furthermore, the dry matter ratio of the inoculated cultivars is calculated as the proportion of the total dry matter germinated by each cultivar.

Fusarium Table 1 shows the results of variance analysis of the dry matter ratio according to the onset of Kidari disease according to the spore concentration of moniliforme . The higher the concentration, the lower the mean value of the dry cell ratio, but the lower the standard error, the lower the coefficient of variation.

Experimental Example  1-5: Fusarium moniliforme  According to the spore concentration, Kidari bottle this Percentage and Gibberellin  Depending on the treatment Extension length Correlation analysis

process Rate of dry matter ^{1 )} Plant height
Elongation rate ^{2 )} 1 x 10 ²
spore / mL 1 x 10 ⁴
spore / mL 1 x 10 ⁶
spore / mL 50 ppm
F. moniliforme
Spore concentration 1 x 10 ² 1,000 0.361 ^ns 0.586 ^** -0.171 ^ns 1 x 10 ⁴ - 1,000 0.852 ^** -0.390 ^ns 1 x 10 ⁶ - - 1,000 -0.442 ^* GA ₃ density 50 ppm - - - 1,000

¹⁾ Fusarium Rate of dry matter following moniliforme treatment.

²⁾ Shoot increment with GA ₃ .

*: Significant at 5% level; **: Remark at 1% level.

For the 10 rice varieties, Fusarium according to Rice Varieties tall disease onset of the spore concentration of moniliforme gun correlation between plant height extension ratio of the pole ratio and GA 50ppm processing results 1 × 10 ⁶ spore / mL treatment is ^{1 × 10 2 spore / mL,} 1 × 10 4 spore / mL and gibberellin treatment, whereas 1 × 10 ² spore / mL and 1 × 10 ⁴ spore / mL treatments were not correlated with each other and were not significantly correlated with gibberellin treatment .

Experimental Example  1-6: by rice variety Fusarium moniliforme  According to the spore concentration, key Leg bottle Disease rate  And Gibberellin  Depending on the treatment Extension length  And rank

Breed name Rate of dry matter ^{1 )} Plant height
Elongation rate ^{2 )} 1 x 10 < ² > spores / mL 1 x 10 ⁴ spore / mL 1 x 10 < ⁶ > spores / mL 50 ppm Average
(%) ³⁾ ranking Average
(%) ³⁾ ranking Average
(%) ³⁾ ranking Average
(%) ³⁾ ranking Weonseadaesoo 97.4 ^a One 100.0 ^a One 98.7 ^a One 8.7 ^bc 2 Hawn 90.4 ^ab 3 87.1 ^ab 4 90.9 ^ab 2 4.0 ^c One Nep Huong 82.8 ^ab 4 96.7 ^ab 2 86.6 ^ab 3 17.2 ^abc 4 Hanareum2 80.9 ^ab 5 87.1 ^ab 5 78.2 ^bc 4 31.6 ^a 10 Nampyeong 74.3 ^abc 6 71.4 ^bc 6 68.2 ^c 5 17.7 ^abc 5 Goamibyeo 63.9 ^abc 7 89.7 ^ab 3 67.4 ^c 6 14.0 ^abc 3 Samgang 91.9 ^a 2 29.8 ^d 10 44.0 ^d 7 23.0 ^abc 7 Gayabyeo 62.8 ^abc 8 55.9 ^cd 7 37.7 ^de 8 21.3 ^abc 6 Junam 53.5 ^bc 9 39.2 ^d 8 23.1 ^ef 9 30.4 ^ab 9 Hopum 39.4 ^c 10 35.8 ^d 9 12.5 ^f 10 24.7 ^abc 8

¹⁾ Fusarium Rate of dry matter following moniliforme treatment.

²⁾ Shoot increment with GA ₃ .

³⁾ Other characters in the same column for the mean mean that there is a significant difference.

Fusarium by Rice Varieties The results of the statistical analysis of the growth rate and rank order of Kideri disease according to the concentration of moniliforme spores and the growth rate and the ranking according to the treatment with gibberellin were found to be divided into 6 groups according to treatment of Fusarium moniliforme 1 × 10 ⁶ spore / 1 × 10 ² spore / mL and 1 × 10 ⁴ spore / mL treatments were divided into 3 groups and 4 groups, respectively. In the 1 × 10 ⁶ spore / mL treatment, the cultivars corresponding to group ab in group a and the cultivars corresponding to group f in group d tended to be classified into similar groups in other treatments. Especially, in 1 × 10 ⁶ spore / mL and 1 × 10 ⁴ spore / mL treatments, the mating status of the test varieties was highly consistent.

In general, breeds containing the interrupted gene, sd1, are very sensitive to gibberellin and are also susceptible to Kidari disease, while varieties containing the sdq (t) or sdg gene are highly sensitive to gibberellin but are known to be somewhat strong for Kidari disease (Ma, 2008 ). In this patent, Hanareum2 in rice varieties was relatively strong in the case of Kidari disease, but the result of gibberellin treatment was the highest among the tested varieties. Therefore, the most suitable Fusarium The concentration of moniliforme spores was 1 × 10 ⁶ spores / mL, and the growth rate of gibberellin-treated plants was correlated with Kidari disease, but it was found to be inadequate as a direct evaluation criterion for resistance to Kidari disease.

Experimental Example  2: Fusarium moniliforme In the species  Of rice varieties Kidari bottle  Resistance comparison

Experimental Example  2-1: Fusarium moniliforme Fungi

Fusarium used in this test The moniliforme strains were selected from 6 species (KACC45819, 46888, 44008, 44014, 45818 and 44021) of the National Institute of Agricultural Science and Technology (RDA). In this test, the spore concentration of Fusarium moniliforme used for inoculation was adjusted to 1 × 10 ² spores / mL.

Experimental Example  2-2: Tissue Embedding Cassette Seedling and disinfection of seeds

Tissue Embedding Cassette was seeded with rice seeds and arranged in a rice seedling box. In order to remove various germs including Kideri disease causing bacteria that were inoculated during the previous year 's cultivation process, the seeds contained in the rice seedling box were sterilized for 15 minutes at 57 ℃ water bath.

Experimental Example  2-3: Inoculation of pathogens and Gibberellin  Cultivation after treatment

After sterilization of the seeds, the seeds in the rice seedling box were collected by Fusarium were immersed in 6 different diluted spore suspensions of moniliforme and inoculated at 26 ° C for 3 days. At this time, spore suspension was shaken more than 4 times a day for uniform inoculation of pathogenic bacteria.

Fusarium seeds inoculated with six different spores of moniliforme were arranged in a box for seedling breeding, and after sowing, cultivated in a greenhouse until the disease was sufficiently advanced, the degree of resistance was evaluated.

Experimental Example  2-4: Fusarium moniliforme In the species  Of rice varieties Kidari bottle Disease rate  black

Variety The ratio of dry matter to dry weight of Kidari disease strain ^{1 )} KCC44008 KCC45818 Average (%) ^{2 )} ranking Average (%) ^{2 )} ranking Weonseadaesoo 78.8 ^ab 2 100.0 ^a One Hawn 63.7 ^b 4 98.5 ^a 6 Nep Huong 87.5 ^a One 100.0 ^a One Hanareum2 8.3 ^cd 5 98.6 ^a 5 Nampyeong 70.0 ^ab 3 96.3 ^ab 9 Goamibyeo 31.7 ^c 6 98.6 ^a 5 Samgang 1.8 ^d 10 100.0 ^a One Gayabyeo 7.4 ^d 7 98.7 ^a 4 Junam 7.4 ^d 7 89.3 ^b 10 Hopum 7.1 ^d 9 97.2 ^ab 7

^One) Fusarium moniliforme Proportion ratio by treatment.

²⁾ Other letters in the same column mean that there is a significant difference.

In the distinction of chickenpox and vigorous chickenpox according to the rice plant variety, i) the healthy poultry is in a state of healthy condition in which the disease has not progressed at all, (Ii) plants that have been severely impregnated with plants that have been severely painted, plants with an angle of more than 45 ° between the apexes of the young and the young, plants that have contracted by germination, plants that have undergone atrophy, It says. Furthermore, the dry matter ratio of the inoculated cultivars is calculated as the proportion of the total dry matter germinated by each cultivar.

Different Fusarium species for 10 rice varieties Table 4 shows the results of analyzing the dry cell ratios of the rice varieties according to the treatments of the six moniliforme strains.

Fusarium Among the moniliforme strains, KCC44008 showed the symptoms of Kidari disease, but the rest of the strains (KACC45818, 45819, 46888, 44014 and 44021) had little white or very weak symptoms And it was difficult to distinguish the difference in resistance between cultivars.

The results of KCC44008 treatment were highly correlated with CF283 (R = 0.814) as shown in Example 1, but there were also differences in response between the two strains. Among the tested varieties, weonseadaesoo, Hawn and Nep Huong were resistant to CF283 and KCC44008 treatments, and were classified as stable resistant cultivars. Ⅱ. Samgang, Gayabyeo, Junam and Hopum were sensitive to both strains . On the other hand, iii) Hanareum2 showed moderate resistance in treatment with CF283, but in KCC44008 treatment, some varieties showed Fusarium moniliforme fungi were different from each other.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments and experiments are illustrative in all aspects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, and all changes or modifications derived from the equivalents thereof will be included in the scope of the present invention.

Claims (11)

As a method for massively testing whether two or more kinds of crop seeds are simultaneously seed-resistant to an infectious disease,
(a) preparing a second container having two or more compartments through which at least two first containers or treatment liquids capable of passing the treatment liquid can pass;
(b) chewing two or more crop seeds in the container, wherein different seeds are in different compartments of different first or second containers;
(c) simultaneously dipping a container in which two or more crop seeds have been dipped into a bath containing a seed infectious agent pathogen suspension;
(d) separately seeding the seeds inoculated in the previous stage for each compartment of the first vessel or the second vessel in a seedling box; And
(e) evaluating the prion and the vein pole for the germinated plant after sowing.
The method according to claim 1, wherein the crop is rice, and the seed infectious disease is rice pandemic.
The method of claim 1, wherein said pathogen suspension is a spore suspension of pathogenic bacteria.
The method of claim 1, wherein the two or more first containers are arranged in the bathtub in the form of checkers of the same size and shape, or wherein the at least two compartments in the second container are of the same size and shape.
The method of claim 1, wherein in step (a), the two or more first containers are housed in another container through which the process liquid can pass.
The method of claim 1, further comprising disinfecting seeds prior to step (c).
delete The method according to claim 1, wherein the dry matter ratio is calculated as a ratio of normal plants among total germinated plants per cultivar.
The method according to claim 2, wherein the rice pathogen pathogen is Fusarium moniliforme or Gibberellia fujikuroi .
The method according to claim 2, wherein the concentration of the spore suspension of the pathogen is 1 x 10 ⁴ spore / ml to 1 x 10 ⁶ spore / ml.
10. The method according to any one of claims 1 to 6 and 8 to 9, wherein the assay method is used to select a crop genetic resource, or a resistant resource or lineage for a crossing population.

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