JPH04506401A - Direct soil immunoassay - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Direct soil immunoassay 16 Fields of invention The present invention relates to a method for detecting the presence of plant pathogens in soil.

More specifically, the present invention provides methods for detecting the presence of antigens of plant pathogens of interest in soil. A method of direct immunoassay.

2. Background of the invention a. Plant diseases Humankind is almost completely dependent on plants for its fundamental survival. fact, Most of the world's food supply comes from 10 major grains: rice, corn, and corn. Potatoes, wheat, sweet potatoes, cassava, soybeans, peas and kidney beans, sugar It is derived from millet, as well as millet and grain sorghum. Provides a direct source of nutrition In addition to providing food for the animals we eat, plants also provide food for the animals we eat, and even more. of textiles, paper, wood and pharmaceuticals. Perhaps most importantly, plants It also provides the very oxygen we breathe.

Throughout history, humans cultivated many important crops through selective breeding of wild plant species. learning to do. However, despite many advances in agriculture in recent years, However, humans are unable to eradicate many of the plant diseases that plague the world's crops. It's not done yet. Human history is an example of the devastating effects of plant pathogens on populations. rich in ! In the latter half of the 9th century, Irish potato body blight disease spread. Although this is one of the most dramatic and definitive examples of Diseases can spread throughout the country, attacking crops and causing extreme hardship or famine for the population. Ru. For example, as much as 15% of all agricultural production in the United States, worth more than $15 billion, is also Around the world, $60 billion is lost each year to plant diseases.

b. Pathogen management Due to the significant losses routinely experienced as a result of the activity of plant pathogens, new approaches to late blight management are needed. Newer and more effective methods are always required. 50 economically important plants ．． There are over 1,000 diseases currently in existence, including fungi, bacteria, viruses, and mycobacterium. The worm, a nematode, and has developed its own specific habits and methods. Pathogen The body is spread from plant to plant by wind, water, soil, plant parts, or vectors.

Although most plant pests are never completely eradicated, the presence of pathogens Careful monitoring of the crops and soil where pests grow can help prevent disease outbreaks. This is the first step towards effective control. No external signs of disease plant tissues that do not contain pathogenic organisms often harbor small amounts of pathogenic organisms and, if detected initially, They can be more easily controlled. Similarly, soil is germinative and Contains small amounts of dormant organisms that, when activated, can severely (damage) the plants there. cormorant. Again, early recognition of the presence of such organisms in the soil can provide value. Serious outbreaks of diseases in certain crops can be prevented. Prevention is based on existing vegetation. by applying chemical pesticides to crops, by using disease-resistant species, or by This is often accomplished by working.

i. Soil-borne pathogens and parasites The most harmful species of plant-damaging organisms Some are animals that live in the soil. Soil inheritance is one of the causes of significant damage to plants. There are disseminated fungi, insects, nematodes and bacteria. These groups are the usual plant inns. Each has members that can overwinter or summer in the soil in the absence of a master. . For example, nematodes typically overwinter or oversummer as eggs or first stage larvae. many The eggs of some species can remain dormant in the soil for months or years without any adverse effects. be able to. Adult nematodes feed on plant roots, usually in relatively shallow soil, forming root nodules or is associated with root knots, excessive root branching, root tip damage, and bacterial or fungal infections. If it grows, symptoms similar to root rot occur.

Some important nematode parasites in the soil attack almost all cultivated plants. Root-knot nematodes such as Meloidone, Heterodera erodera) and Globodera and other genera Like nnchus and Praty-Ienchus. I have a cyst nematode.

Similarly, many insects, especially insect larvae, cause serious damage to many different agricultural plants. This may cause Many species live in the soil around the plant trunk, where the larvae eventually attack. lay those eggs. Larvae of some species also spend significant periods of time in the soil. heavy Some of the key plant insect pests include cane, which is known to attack many cultivated crops. Sorghum root soil [Diabrotica 5pp]) The beetle beetle is particularly damaging to sorghum, bean cabbage, cotton and tobacco [ Family Lebidopteranidae Noctuidae and a wide variety of other species Maggots that grow on the roots that feed on the roots of rape [Family Anthomaidae] There are various genera of (Anthom) and some bacterial species are also plant pathogens. 1 Many of these are pure soil-dwelling organisms; Their populations converge on host plants, but only gradually when released into the soil. decreases to If the host plant is grown in the same soil year after year, the population in the soil There may be a net increase. Important bacterial pathogens include clubroot, twig clubroot, and Agrobacterium, which causes stem club disease, damping-off disease, leaf blight disease, and soft rot disease. Pseudomonas causes potato scab, Streptomyces causes potato scab, Erwinia (Erwinfa), which causes soft rot, and bacterial spot disease and There is a species called Xanthomonas that causes rot.

Many of these pathogenic species are found on diseased plants, organic debris, and seeds. can even overwinter directly in the soil.

Pathogens of particular importance are soil-borne fungi. where crops are grown Various genera of fungi are found throughout the world.Different types of diseases are caused by these fungi. arise. For example, species of the genus Phytophthora are tobacco, Many important crops like soybean and strawberry and azalea, rhodopuntron and causes root shoulder pain, crown shoulder pain, and stem shoulder pain in ornamental plants such as camellias. Become. Members of the genus Pythium are the world's angiosperms, vegetables and It causes damping-off of seedlings that affects row-planted crops.

The species Rhizoctonia 5olani is similar to any other plant. They cause more types of diseases on a wider variety of plants than pathogenic fungi. Ru. Among the diseases caused by this destructive pathogen is (4) has seed rot, damping-off, root-shoulder, and crown-shoulder, which affect almost all stages of plant growth. effect. Fusarium and Verticilium The ci1lic+m) genus causes seed shoulder pain, root shoulder pain, stem shoulder pain, We grow many species that cause blight, damping off, and smut, as well as pod shoulder blight. . Many species of Sclerotinia are found on common beans, curds, etc. crucifers, soybeans, tobacco, lawn and ornamental plants, watery (water) Main cause of soft rot and crown and stem shoulder pain. chelaviobsis (Thielaviopsis) causes chalkboard shoulder and damping-off disease on many vegetables and flowers. cause it to occur.

Many if not all of these fungi and others like them They can survive for long periods of time without a natural host and sometimes reproduce under suitable conditions. It has durable life cycle stages that allow it to become infected and infectious.

Among the mechanisms by which fungi overwinter are sclerotia (small hyphae with or without host tissue). clumps), oospores (produced by water mold), chlamydospores in organic debris There are children and mycelium. The more resistant of these structures can be found in suitable hosts. They remain invisible and undiscovered in the soil almost indefinitely until they are harvested. There will be. Even a small number of these embryos are found in such contaminated soil. It will have a destructive effect on the unlucky crop that is eventually planted.

■ Disease prevention There are many potential ways to prevent substantial damage from the activity of such pathogens. be. Many fungicides, fungicides, and nematicides are available for treating seeds or plants. Preventive methods such as proper soil preparation and sanitation are also known. deer However, the most effective prevention is the early identification of any latent pathogens in the soil. It is important to be aware that there may be a risk of contamination of the soil in a particular location. There is. However, unlike the situation with new plant tissue, Discovering and identifying pathogens in defeated or dried plant tissue is much more difficult. There's nothing wrong with that.

One of the main experimental methods currently used is dilution brating. This person The method is particularly popular for finding bacteria in soil. The problem with this method is that caused by the presence of a second fast-growing microflora in the same sample. live and interfere with recognition of the primary pathogen of interest. The pathogen is dormant and therefore This is especially a problem if the symptoms appear slowly. Similarly, the number of embryos If they are small in a given sample, it is impossible to find them in the presence of other organisms. Although it is not Noh, it is difficult. Furthermore, in a qualitative sense, simple detection is sufficient to solve the problem. Do not display in minutes. Quantification is helpful in specifying the type of treatment or prevention that should be taken. Always desirable and essential.

Isolation, identification, and quantitative analysis of soil-borne bacterial pathogens is difficult and variable However, preferred methods have been demonstrated in a limited number of examples. however However, the application of the same method to soil-borne fungi still lags behind. bacteria The commonly used brating-out method is used to treat a large number of different fungal pathogens. It is a very time-consuming process that cannot be easily applied to Colonies need to be identified.

Immunoassays are fast and quantitative, as well as being highly specific. It offers many promising advantages for conformal detection.

However, this method has not been widely applied to fungal pathogen detection, and even in soil It has never been used to directly detect overwintering embryos in soil samples.

This is mainly due to the lack of success in accurately representing soil samples. today There is no adequate and convenient way to test soil for the presence of pathogenic fungi, even in the case of more conventional methods. Traditional methods continue to be used.

For example, 5utton and Barron (Can, J, Rot, 50 :1909-1914゜1972) produced Endogone spores. attached for isolation. Pour the sample into the separation funnel and see what sticks there. Rinse and count the samples on paper discs.Furlan et al. (Trans, Br. , Mucol S.

c, 75:336-338.1981) recommends a density gradient extraction method for internal rhizospores. I'm doing it. In the method, a soil sample is first collected as described above. The sea urchin spores are treated to float, and the supernatant fluid is then centrifuged. Wet sieving also It is also still commonly used to extract large numbers of spores from soil. child In the process, the soil sample is mixed with water and, after settling, becomes smaller and smaller. Pour into a small sieve. That is, the spores are formed into finer-sized sieves. Abd-Elrazik and Lorbeer (Ph.D. ytopa-thology 70:892-894.1980) is wet sieving and flotation of the sclerotia in glycerol, followed by counting the recovered sclerotia. Discloses how to do so. More recently, Ianson and A11en The use of wet sieving, flotation-adhesion, and density gradient centrifugation has been revisited and the use of these methods It was noted that some are better than others depending on the particular type of soil being tested.

All methods mentioned above, i.e. all methods currently used for soil pathogen detection , quantification of spores, sclerotia (or organic debris) retained by the method (objects) visually inspected under a microscope and physically counted by highly trained individuals It depends on that. Such methods may be used to determine whether mere collection of embryos is desired or such as when you already know that the soil sample contains embryos of interest. Although sufficient, these methods are not intended for determining the presence of a particular pathogen for the first time. or is acceptable for accurately estimating the level of disease prevalence for known pathogens. do not have. Furthermore, the processing time required by the above method is too long. It requires the use of complex equipment, making it difficult to conduct efficient large-scale testing outside of the laboratory environment. impractical to use.

There is a need for a method to accurately and directly detect plant pathogens in soil, quickly and quantitatively. exist. This means that the present invention, i.e., a large number of provides an accurate assessment of soil disease prevalence caused by various plant pathogens on a large number of samples. Provided by a method that can provide.

3. Summary of the invention The present invention isolates the pathogen components, if present, so that they become concentrated units. Treat the soil to destroy any pathogen components and remove the pathogen in a liquid medium. The concentration unit is processed in such a way that the antigen is exposed and the antibody has specificity for the antigen of the pathogen. to observe the presence or absence of a reaction between the antigen and antibody. measuring the presence or absence of plant pathogens in a soil sample comprising the steps of: provide a method to do so.

Throughout the specification and claims, the term "pathogen" refers to pathogens such as bacteria and fungi. It is understood in a broad sense to include natural pathogens as well as plant pests such as nematodes or insects. should be understood. “Pathogen component” refers to the embryo (for example, parts of living organisms) as well as pathogens that may carry the identifying antigens of the pathogen. non-embryonic fragments of pathogens such as mycelium or organic debris containing the body or It means both parts. The term “enriched” or “isolated into concentrated units” refers to The components can be effectively extracted from the soil and tested immunologically. Used to mean isolated into small units.

In a preferred embodiment of the invention, the components are concentrated by flotation. this is , requires that the constituents detected be buoyant, and that oospores, sclerotia, and thick Particularly well applied to detect fungal components such as membrane spores or mycelium . In particularly preferred embodiments, the pathogen component is combined with levitation on the component. to the adhesive surface of the material due to its adhesive strength, or to that surface by water droplets containing the constituent components. Captured.

The present invention is highly applicable and under certain conditions, if disease prevalence is high and positive A single sample can be processed in 20 minutes. vine of disease If the spread is at a low level, the inspection time may be longer, but a single sample It is rarely more than 1.5-2 hours per hour. This is brating However, it usually requires 3-5 days and does not germinate under experimental conditions. It cannot be used for some purposes, such as the detection of dormant embryos. of the present invention The method does not require the use of any complex machinery or equipment, unlike e.g. density gradient centrifugation. It is easily adapted for partial or full use in outdoor environments as it does not cause any damage. Book The invention also provides a kit particularly adapted for use in the method, the kit means to concentrate drug components, destroy pathogen components, and detect The antibody comprises at least one antibody that develops specificity for the antigen of the pathogen. preferable In embodiments, the kit includes one kit for performing a sandwich immunoassay. contains two antibodies, one immobilized and the other labeled.

4. Detailed description of the invention a. Concentration of pathogenic components This method ensures that the soil concerned is suitable by removing any possible disturbances from the soil constituents. Any method of concentrating the constituents found in the soil should be can be used. Dilution-brake methods such as wet sieving or density gradient centrifugation Some of the methods originally developed as improvements over soil This method is carried out in the ability to isolate the spores or other constituents to be examined from provides a useful first step. However, the benefits of this method are This cannot be completely achieved using these methods. Because it's complicated and This is because of the time required for each. Therefore, they concentrate pathogen components Although they are certainly used as a means to do so, they are not particularly preferred. good The preferred method is to use a flotation-deposition method as a first step, which is described below. Describe the details.

i. Levitation and capture In a preferred embodiment, the method of the invention comprises To concentrate, flotation and capture methods are utilized in simultaneous steps. soil Collect from designated locations. Air dry the soil to facilitate processing; Grind any large lumps with a mortar and pestle. If necessary, sieve and sieve the soil. Further processing by passing through a lid or filter.

The measured amount of soil is then placed in a suitable container and water is added to it. added The amount of water is not critical, but should be sufficient to allow stirring and foam to form in the suspension. shall be. Usually twice the amount of water as soil is sufficient for this purpose. soil and Cover the container with water and stir vigorously for a few seconds. This agitation removes any particles in the soil. Buoyant substances float on surfaces. In one embodiment of the method, flotation alone is also of interest. is sufficient to effectively concentrate certain components.

For example, it may have constituents that can simply be taken up from the surface of the water. This is the case. One such example is citrus, which is associated with organic debris. It is a phytophthora. However, another method In embodiments, capture of pathogen components in most cases is accomplished by Requires a special method. The components are preferably Water droplets containing constituents are trapped on surfaces that can be Allows for appropriate surface tension to be applied.

As used in the specification and claims, the term "adhesive surface" includes both types of surfaces. used in In general terms, the attachment surface of a plant is smooth, hard, and absorbs water. Not collected. Among the materials suitable for this purpose are polyethylene, polypropylene Polystyrene, polystyrene, or glass. The preferred surface for attachment is glass Or polypropylene. After stirring, fill the container with water to the brim and cover. Avoid air pockets on bar slips, glass slides or other adhesive surfaces in containers. Make sure that the liquid does not overflow from the edges, and place it so that it is in direct contact with the liquid surface.

Leave the lid on for a short time. That is, for about 5 minutes to about 30 minutes, during that time buoyant substances on the water surface adhere to the surface. Next, lift the lid straight up and Prevent any water droplets from dripping. Drops of water and any adherent solid particles to a second container. Thoroughly clean surfaces. preferred Do this with water at the top of the first brewing vessel. some buoyant particles, In particular, oospores adhere firmly to several attachment surfaces.

b. Destruction of constituent components Complete lysis of pathogenic components is not necessary to make them suitable for testing. do not have. Rather, one or more antigens are released and themselves solubilized. It is only necessary to destroy the complete state. Destruction of components is optional This is achieved by known methods. This is for example a mortar and pestle type flour or mechanically with other sieving equipment. On the other hand, electric shock can be applied to oocytes. It can be used on collected liquid samples to destroy such constituents. Other methods of destruction Methods include solubilization chemical treatments or enzymatic treatments. Variants of these methods are It will be clear to those skilled in the art.

After trituration, the sample is subsequently used for direct testing in an immunoassay. However However, depending on the amount of insoluble slag in the sample, any solids should be removed before testing. It is preferable to remove the A short centrifugation is used for such debris and inspection. It is useful for conveniently removing items from the top. Filter and filter samples in turn The product is used in immunoassays.

C9 immunoassay Immunological testing methods are well known and our method is specifically applicable to any format of assay. It is not necessarily limited to. Such methods include solid phase or liquid phase phase agglutination reaction, precipitation reaction, immunoelectrophoresis, radioimmunoassay, fluorophore-conjugated immunological immunosorbent assays and enzyme-linked immunosorbent assays. However, the truth is Due to ease of implementation, the preferred format is solid phase ELISA. For such assays A person skilled in the art will recognize antibodies that have specificity for the antigen of the pathogen of interest. method to immobilize on a solid substrate. The sample to be tested is processed to form antigen-antibody complexes. contact with the bound antibody. The complex is then detected with a detectably labeled compound. contact with two pathogen-specific antibodies. The labeled antibody binds to the antigen, resulting in antibody-antigen-antibody A complex is formed and its presence is confirmed by observation of a detectable label. This one Variations on the general method are familiar to those skilled in the art. Preferred embodiment The detectable label is an enzyme. The presence of the enzyme label is necessary for the addition of the appropriate enzyme substrate. It has been shown that, after hydrolysis, it produces analytically detectable changes in the culture medium. Jiru.

The type of antibody used depends on the identity of the pathogen being measured. Currently several fungal pathogens A large number of antibodies are available with different specificities for the body. Among these are Pithi Pythium or Phytophthora ( EP Publ, No, 222998), Sclerotinia inia) (EP Publ, No, 234501) or diphtonia ( Rhizoctonia) (E!P Appl, No, 88106775. There is an antibody that reacts with 5). 5chots, “A Serological A Pproach to the Identification of Pota to Cyst Nematodes,’, p, 11B, Agricult Ural University.

Wageningen, The Netherlands, 198g is anti- Describes nematode antibodies. Antibodies can be monoclonal or polyclonal; Or a combination of monoclonal and polyclonal. “Reporter” Antibody labels can be analytically detectable, such as radioisotopes, chemiluminescence, etc. with any type of molecule, such as fluorescent, fluorescent or bioluminescent molecules. It can be done logically. However, it is particularly preferred due to its simplicity and cost. is an enzyme label. A variety of enzymes are available for this purpose. For example, alkali Phosphatase, beta-galactosidase or horseradish peroxidase -ze. Those skilled in the art will readily recognize variations that occur during recognized ELISA methods. will.

d0 diagnostic kit In one embodiment, the method includes a test that is amenable to laboratory or field use. It is carried out with a test kit. The basic element of the test kit is to detect Means for flotation of the constituents, means for capturing the constituents, means for destroying the constituents, and and antibodies with specificity for the pathogen of interest. Flotation means are soil and water Any type of container suitable for holding the sample and capable of further agitation It is. This can be a vial, bottle, test tube, flask or other. above As stated, the capture means may contain components or water droplets containing them. It should have a surface to adhere to. This is done so that the surface of the container is firmly on top. in the form of a cap, or just a glass coverslip or slide. It is. The means of destruction is any convenient mechanical sieve, such as a mortar and pestle. chemical or enzymatic solubilizers. Solid-phase antibodies can be used, for example, on glass slides. a well, a multi-well plate, a test tube, a dipstick, an outflow device or any other device known to those skilled in the art. immobilized on any other solid phase. Any other detectable marker may also be used. However, labeled antibodies are in solution, and enzymes are required to facilitate observation outside the laboratory. Labels are preferred. If an enzyme label is used, the kit also contains a substrate for the enzyme. do. Preferably, the kit includes filtration to remove undissolved particles from the solubilized sample. contains.

The method and diagnostic kit detect the presence of various plant pathogens in numerous forms. used for. The types of components that can be detected are nematode eggs or larvae, insect eggs or larvae, or larvae, fungal oospores, sclerotia, ~teliospores, sporangia, zygospores, chlamydospores, and fungal oospores. Including, but not limited to, spores, or mycelia and bacterial cells. other Applications for detecting types of organisms will be apparent to those skilled in the art.

The following non-limiting examples are illustrative of the method of the invention.

5. Examples Let the soil sample air dry at room temperature and grind any large clumps using a mortar and pestle. Ta. The soil was passed through a powder sifter to mix thoroughly. 4 subsample per soil Weigh the sample (10g) into a 20cc cinch with a screw-on top cap. in a ration vial.

B. Soil samples for each flotation method for extracting Phytophthora from soil were processed sequentially as follows: approximately three-quarters of the way through the subsample vial. Filled with distilled deionized water. Cap the vial and shake vigorously for 20 seconds. Last night. Add water to the rim of the vials and place a coverslip on top of each vial. and brought into contact with liquid. Water to prevent air pockets from forming under the coverslip. The surface was raised high enough to prevent liquid from spilling out. 30 minutes later, Kabag The lath was removed one at a time by lifting it straight up without tilting it. Tare Contes disposable Kontes Disposable Pe1le Transfer to a microcentrifuge tube designed for use in combination with Pe5-tle). Ta. Wash the coverslips 50 μm from the top of each vial and place in a microcentrifuge tube. I put it in. Prepare one extract per subsample and pool the subsamples later. I did it.

Preparation of soil extract for C9 immunoassay Tallboys Laboratory Sta. -Talboys' Labora-tory 5tirrer 13 Using a Comtesse Pellet Pestur driven in a 4-2 model, the extract was It was crushed for 1 minute at 0 rpm.

Add 300 μm extract dilution to each tube and briefly portex mix. It was. Pool 4 subsamples per soil sample, l　0000　r　 Centrifuged at pm for 1 minute and filtered through a 0.08 μm filter. The filtered extract is Collected into a clean microcentrifuge tube.

D. Immunoassay of Phytophthora sp. Tiger “E” Multiwell Kit (Agri-Diagnostics’ Phy Tophthora "E" Multiwell Kit) Soil extracts were examined for the presence of Tora spp. This kit is a microtiter Affinity purification of 5 μg/ml as primary antibody immobilized on the surface of the plate. This is a two-antibody ELISA using a sheep anti-Phytophthora megasperma antibody. It was. A series of laboratory-prepared Phytophthora megasperma oospore standards in O/mL 50/ml, 100/ml and 200/ml each microtitre It was used in the rate.

Add 100 μl of the prepared soil extract or standard to two antibody-sensitized wells and one non-antibody sensitized well. Pipette into each sensitized well. Tighter this plate on a Titertek plateshaker at room temperature. Incubate for 20 minutes, wash 6 times, then plate with 100 μm/well of complex. The cells were incubated for an additional 20 minutes. This complex is horseradish peroxidase Two monoclonal anti-Phytophthora membranes at a concentration of 7 μg/ml bound to It was a mixture of gasperma antibodies. After washing the plate, 100 μm/well of A BTS substrate was added and incubated for 10 minutes with shaking. 50μI/U The color reaction of the substrate was stopped by adding 1.5% NaF in each well: 405 nm in each well. The absorbance was measured using a Dynatech MiniReader. r)　II. For each test and standard, the sensitized well The absorbance of non-sensitized wells was subtracted from the average absorbance. The relative levels of Phytophthora are , the “corrected” average absorbance of soil extracts and the corrected mean absorbance of laboratory-prepared oospore standards. It was estimated by comparing with the reading value.

The results of various tests using this method are shown in Tables la-d. From these data , this method has been shown to be effective in detecting at least four different crop/pathogen systems. I understand.

(Margins below this page) Table 1 Absorbance of 0 standard + 915 Phytophthora not detected 0 standard +, 15 → 50 m1 Absorbance of low level Phytophthora 50ml standard → absorbance of 100ml medium level Phytophthora >100+++I standard absorbance High level of Phytophthora a, of Phytophthora sp. in soybean soil extracts tested by direct soil immunoassay. detection A-3, 03 not detected A-271, 65 high 100 0, 92 200 1, 97 b. Phytophthalmos in citrus soil extracts tested by direct soil immunoassay Detection of Tora spp. Sample II MY absorbance Pmg level JB 19-1. 11 Not detected IJB 19-8. 10 // JB14-3.36 low JB14-8.40 low JB8-3.73 medium JB13-6.61 JB13-11.05 high JBBSO-271,63 high Oospore standard (oospore/■l) 9 0, 05 5Q 0.47 100 0, 96 200 1.91 C. Tested by Agree Diagnosticus Rhizoctonia Immunoassay Detection results of Diphtonia spp. in soybean soil extract Sample l MW absorbance results 8000>2.00 positive 8001 1.38/1 8002 1, 37 N 8003>2.00” 8009 0, 28 // Buffer 0.03 Not detected d, tested by Agree Diagnosticus pythium C multiwell kit. Detection sample of Pythium genus bacteria in soybean soil extract MW absorbance results A-41,35 positive A-260,00 not detected A-270,57 positive ni-BUC2-61,77/' PNO2-60,55// Watson 0.23” Buffer 0.00 Not detected international search report PCr/UIS90701615 1X, WXKLm 1smCKED/BlkRCB? ERX8:5 oil/ Funq? ins@ct# 1^l--11 town-11t@-^@tha-denka""'1? er/US≦X)1 0161’i

Claims (60)

[Claims] 1. Presence and presence of plant pathogens in soil samples suspected of containing them If so, how to measure the level, (a) processing soil samples to separate pathogen components as concentrated units; and; (b) the concentration unit is adapted to disrupt pathogen components and expose antigens of the pathogen; (c) treating the medium of step (b) in a liquid medium specific for the pathogen; contacting with at least one type of antibody; and (d) observing the presence or absence of a reaction between the antigen and the antibody; 2. Claim 1: Concentrating pathogen components by flotation, centrifugation, or hot sieving. the method of. 3. 3. The method of claim 2, wherein said components are concentrated by flotation. 4. 4. The method of claim 3, wherein said component is further concentrated by capture on a deposition surface. 5. The attachment surface may be glass, polyethylene, polypropylene, or polystyrene. 5. The method of claim 4. 6. 2. The method of claim 1, wherein the antibody is detectably labeled. 7. 7. The method of claim 6, wherein the antibody is enzyme labeled. 8. The antigen is brought into contact with two types of antibodies, one is immobilized and the other is detectable. 2. The method of claim 1, wherein the method is labeled as . 9. 9. The method of claim 8, wherein the detectably labeled antibody is enzyme labeled. 10. Claim 1, wherein the constituent components are disrupted mechanically, chemically, or enzymatically. −9. The method according to any one of 9. 11. Any of claims 1-9, wherein the pathogen is a nematode, a fungus, a bacterium or an insect. Method in section 1. 12. 10. The method of any one of claims 1-9, wherein the pathogen is a fungus. 13. Fungi include Pythium and Rhizoctonia. nia), Sclerotinia, Phytophthora (Phytophthora) -toPhthora), Fusarium, Verticillium ( Ver-ticillium, or Thielaviopsis 13. The method according to claim 12, wherein the bacterium belongs to the genus Psis. 14. The constituents are oospores, sporangia, chlamydospores, zoospores, sclerotia or mycelium. 13. The method of claim 12. 15. 10. The method of any one of claims 1-9, wherein the pathogen is a nematode. 16. Nematodes include Meloidogyne and Heterodera. -rodera), Globodera, Tylenchus nchus), or of the genus Pratylenchus, 16. The method of claim 15. 17. 10. The method of any one of claims 1-9, wherein the pathogen is a bacterium. 18. Bacteria include Erwinia and Agrobacterium. -bacterium), Pseudomonas, Stre. Ptomyces (Strepto-myces) or Xanthomonas (Xanth 18. The method of claim 17, wherein the plant belongs to the genus O-monas. 19. 10. The method of any one of claims 1-9, wherein the pathogen is an insect. 20. Insects are corn root worms, diabr. oti-ca species), a fall armyworm, a cutworm, or a hair beetle. The method of Section 19. 21. Those who investigate the presence or absence of plant pathogens in soil samples suspected of containing them. The law is (a) A soil sample is stirred in a liquid medium to deposit floating pathogen components on the surface of the liquid. (b) floating a substance expected to contain said constituent in order to concentrate said constituent; separating the parts of the liquid medium that are (c) The separated medium portion is crushed to remove the pathogen components contained therein, and the pathogen is removed. (d) treating the treated portion of step (c) to expose the antigen of the pathogen; contacting with an antibody specific for; and (e) observing the presence or absence of a reaction between the antigen and the antibody; 22. 2. The method of claim 1, wherein the separation is achieved by contacting the liquid surface with the adhering surface. . 23. The attachment surface can be glass, polyethylene, polypropylene, or polystyrene. 23. The method of claim 22. 24. 24. The method of claim 23, wherein the antibody is detectably labeled. 25. 25. The method of claim 24, wherein the antibody is enzyme labeled. 26. In step (d), the treated part is contacted with two types of antibodies, one of which is immobilized. 24. The method of claim 23, wherein the second one is detectably labeled. 27. 27. The method of claim 26, wherein the labeled antibody is labeled with an enzyme. 28. Any of claims 21-27, wherein the pathogen is a nematode, a fungus, a bacterium or an insect. Method 1. 29. 28. The method of any one of claims 21-27, wherein the pathogen is a fungus. 30. The pathogens include Pythium, Rhizoctonia tonia), Sclerotinia, Phytophthora (P hy-toPhthora), Fusarium, Verticillium Verti-cillium, or Thielaviopsis 30. The method according to claim 29, wherein the bacterium is of the genus Iopsis. 31. The constituents are oospores, sporangia, chlamydospores, zoospores, sclerotia or mycelium. 30. The method of claim 29. 32. 28. The method of any one of claims 21-27, wherein the pathogen is a nematode. 33. Nematodes include Meloidogyne and Heterodera. -rodera), Globodera, Tylenchus nchus), or of the genus Pratylenchus, 33. The method of claim 32. 34. 28. The method of any one of claims 21-27, wherein the pathogen is a bacterium. 35. Bacteria are Agrobacterium, Pseudomonas Pseudomonas, Streptomyce s), Erwinia, or Xanthomonas 35. The method of claim 34, wherein the plant belongs to the genus Monas. 36. 28. The method of any one of claims 21-27, wherein the pathogen is an insect. 37. Insects are corn root worms, diabr. o-tica species), a fall armyworm, a cutworm, or a hair beetle. The method of Section 36. 38. A diagnostic test kit for detecting plant pathogens in soil, comprising: (a) means of concentrating pathogen components from soil; (b) crushing pathogen components; (c) an immobilized antibody having specificity for the pathogen; and (d) a means for treating the disease. A kit comprising: a labeled antibody having specificity for the drug substance; 39. The means for concentrating the pathogen is (i) flotation means; and (ii) Capture means 39. The kit of claim 38, comprising: 40. 40. The kit of claim 39, wherein the capture means is an attachment surface. 41. The attachment surface can be glass, polyethylene, polypropylene, or polystyrene. 41. The kit of claim 40. 42. 42. The kit of any one of claims 38-41, wherein the labeled antibody is labeled with an enzyme. t. 43. 43. The kit of claim 42, further comprising a substrate for said enzyme. 44. Any of claims 38-41, wherein the pathogen is a nematode, a fungus, a bacterium or an insect. This is the first kit. 45. 42. The kit of any one of claims 38-41, wherein the pathogen is a fungus. 46. The pathogens include Pythium, Rhizoctonia tonia), Sclerotinia, Phytophthora (P hy-tophthora), Fusarium, verticillium Vertici-llium, or Thielaviopsis 46. The kit of claim 45, wherein the kit is a bacterium of the genus P.iopsis. 47. 42. The kit of any one of claims 38-41, wherein the pathogen is a nematode. 48. Nematodes include Meloidogyne and Heterodera. -rodera), Globodera, TyIe nchus), or of the genus Pratylenchus, 48. The kit of claim 47. 49. 42. The kit of any one of claims 38-41, wherein the pathogen is a bacterium. 50. Bacteria include Erwinia and Agrobacterium. bacterium), Pseudomonas, Strep Streptomyces, or Xanthomonas 50. The kit of claim 49, wherein the kit is of the genus Mo-nas. 51. 42. The kit of any one of claims 38-41, wherein the pathogen is an insect. 52. Insects are corn root worms, diabr. oti-ca species), a fall armyworm, a cutworm, or a hair beetle. Item 51 kit. 53. 46. The kit of claim 45, wherein the labeled antibody is labeled with an enzyme. 54. 54. The kit of claim 53, further comprising a substrate for said enzyme. 55. 48. The kit of claim 47, wherein the labeled antibody is labeled with an enzyme. 56. 56. The kit of claim 55, further comprising a substrate for said enzyme. 57. 50. The kit of claim 49, wherein the labeled antibody is labeled with an enzyme. 58. 52. The kit of claim 51, further comprising a substrate for said enzyme. 59. 52. The kit of claim 51, wherein the labeled antibody is labeled with an enzyme. 60. 60. The kit of claim 59, further comprising a substrate for said enzyme.