WO1990011522A1

WO1990011522A1 - Direct soil immunoassay

Info

Publication number: WO1990011522A1
Application number: PCT/US1990/001615
Authority: WO
Inventors: Suzanne M. Miller; Frank P. Petersen
Original assignee: Miller Suzanne M; Petersen Frank P
Priority date: 1989-03-27
Filing date: 1990-03-26
Publication date: 1990-10-04
Also published as: CA2049992A1; JPH04506401A; EP0466765A4; EP0466765A1

Abstract

The present invention provides a method and kit for determining the presence or absence of plant pathogens in a soil sample. The method comprises concentrating a pathogen component from the sample, disrupting the components to release an antigen of the pathogen, contacting the disrupted sample with an antibody having specificity for the pathogen and observing the presence or absence of a reaction between the antigen and the antibody.

Description

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DIRECT SOIL I MUNOASSAY

1. FIELD OF THE INVENTION The present invention relates to a method for detecting the presence of plant pathogens in soil. More

5 . . specifically, the invention relates to a method for direct i munoassay of soil to detail the presence of antigens of a plant pathogen of interest.

2. BACKGROUND OF THE INVENTION 0 a. PLANT DISEASE The human race depends almost completely on plants for its ultimate survival. In fact, most of the world's food supply is derived from ten major crop types, namely: rice, corn, potato, wheat, sweet potato, cassava, soybean, peas and beans, sugar cane, and millet and sorghum. In addition to providing a direct source of nutrition, plants also provide nutrition for the animals we eat, and are, further, the basis for many fibers, paper, lumber and 0 pharmaceuticals. Perhaps most importantly, plants also provide the very oxygen we breathe.

Man throughout history has learned, through selective breeding of wild plant species, to cultivate many important crops. However, despite the numerous advances in agriculture in recent years, man has still not been able to eradicate many of the plant diseases which plague the world's crops. Human history is rife with examples of the devastating effect a plant pathogen can have on the human

-_npopulation; although the Irish potato blight of the late 19th century is one of the most dramatic and sweeping examples of such an effect, even now localized infestations of a particular crop can cause severe hardship, or famine, for the human population depending on the affected crop.

32For example, it is estimated that as much as 15% of the total agricultural production of the United States, worth more than 15 billion dollars, and 60 billion dollars worldwide, is lost each year to plant disease.

b. PATHOGEN MANAGEMENT . . ^~

In view of the significant losses routinely experienced as a result of the action of plant pathogens, new and more effective methods of pest management are constantly being sought. There currently exist upward of

50,000 diseases of economically important plants, and control is complicated by the fact that they are caused by a variety of different types of organisms, such as fungi, bacteria, viruses, mycoplasmas, nematodes, and parasitic plants. Each type of pathogen has its own particular habit and means for attacking the host. The pathogens can be spread from plant to plant by wind, water, soil, in plant parts, or by vectors. Although most plant pests can never be fully eradicated, careful monitoring of the crops or soil in which they are grown for the presence of pathogens provides a first step in the efficient control of disease outbreaks. Plant tissue which shows no external symptoms of disease may frequently harbor small quantities of disease- causing organisms, which, if detected early, may be more readily controlled. Similarly, soil may contain small quantities of both germinative and dormant organisms which, when activated, may severely injure the plant life there; again, early recognition of the presence of such organisms in soil can prevent a serious outbreak of disease in a valuable crop. Prevention can often be achieved by either application of chemical pesticides to existing plants, by the use of disease-resistant varieties or by crop rotation. i. SOIL-BORNE PATHOGENS AND PESTS Some of the most damaging of the plant pests are soil inhabitants. Among those which are capable of causing significant plant damage are soilborne fungi, insects, nematodes and bacteria. Each of these groups has members which are capable of overwintering or oversummering in soil when their usual plant hosts are not not available. For example, nematodes usually overwinter or oversummer as eggs, or first stage larvae. The eggs of many species can remain dormant in the soil for several months, or even years, with no ill effects. The adult nematode commonly feeds on roots of plants in relatively shallow soil, causing symptoms such as root knots, or root galls, excessive root branching, injured root tips and, when accompanied by bacterial or fungal infection, root rot. Among the important nematode parasites in soil are root knot nematodes, such as

Meloidogyne, which can attack almost all cultivated plants, cyst nematodes, such as Heterodera and Globodera and other genera such as Tylenchus and Pratylenchus.

Similarly, many insects, particularly insect larvae, are responsible for causing significant damage to a large number of different crop plants. A number of species deposit their eggs in the soil around the bases of the plants that the larvae eventually attack. The larvae of some species, too, may spend considerable periods of time in the soil. Among the important plant insect pests are corn root worm (Diabrotica spp) , which has been found to attack a number of cultivated crops; cutworms (various genera of the lepidopteran family Noctuidae) , which are particularly damaging to corn, beans cabbage, cotton and tobacco; wire worms (Agriotes sp.) which cause injury to wheat and corn; and root maggots (various genera of the dipteran family

Anthomyiidae) , which feed on the roots of a wide variety of vegetables. Several bacterial species are also known to be phytopathogens. Many of these are true soil inhabitants which, although their populations build up in the host plant, only gradually decline when released into soil. In some cases, if the host plants are grown from year to year in the same soil, there can potentially be a net increase of the populations in the soil. Among the important bacterial pathogens are Agrobacterium, which causes crown, twig and cane galls; Pseudomonas, which causes wilts, blights and soft rots; Streptomyces, which causes potato scab; Erwinia, . . which causes soft rots; and Xanthomonas, which causes bacterial spots and rots. Many of these pathogenic species can overwinter in diseased plants, organic debris, and seeds, or even directly in the soil.

Particularly important pathogens are the soilborne fungi. A number of different genera are found throughout the world, wherever crops are grown. Various types of diseases are caused by these fungi. For example, species of the genus Phytophthora are responsible for causing root, crown, and stalk rot in many important crops such as tobacco, soybeans, and strawberries, and ornamentals such as azalea, rhododendron and camellia. Members of the genus

Pythium cause a damping-off disease of seedlings which affects flowering plants, vegetables and row crops world- wide. The species Rhizoctonia solani causes more different 5 types of diseases, among a wider variety of plants, than any other plant-pathogenic fungus. Among the diseases caused by this destructive pathogen are seed rot, damping-off, root and crown rot, affecting virtually all stages of plant _Qdevelopment. The genera Fusarium and Verticillium have many species which cause seed rots, root rots, stalk rots, foot rots, wilts, and ear and kernel rots, in almost all cultivated crops. The many species of Sclerotinia are causative agents of watery soft rots, and crown and stem 5 rots in beans, crucifers, soybean, tobacco, turfgrasses and ornamentals. Thielaviopsis is responsible for causing black root rot and damping off in many vegetables and flowers.

Many, if not all, of these fungi, and others like them, have a long-lived life cycle stage which permits them to survive prolonged periods without their natural host, and then, under the proper conditions, to become infective again. Among the mechanisms by which fungi overwinter are sclerotia (compact masses of hyphae, with or without host tissue) , oospores (produced by water molds) , chlamydospores . . . . and mycelia in organic debris. The more resistant of these structures may exist almost indefinitely, visually undetectable, in soil until an appropriate host is provided.

Even small numbers of these propagules can have a devastating effect on the hapless crop ultimately planted in such infected soil.

ii. DISEASE PREVENTION

There are a number of potential ways of preventing substantial losses due to the activity of such pathogens.

Many fungicides, bactericides and nematicides are available for treatment of seed or plants, and preventive measures, such as proper soil preparation and sanitation techniques are also known. The most effective prevention, however, depends on recognition of the potential risk for infection in soil of a given area, by identifying at an early stage any latent pathogens in the soil. Unlike the situation with fresh plant tissue, however, the detection and identification of pathogens in soil, or in decayed or dried plant tissue, is far more elusive.

One of the primary laboratory techniques currently employed is dilution plating. This method is particularly common in the detection of bacteria in soil. The problem with this technique arises in large part from the presence of faster-growing secondary micro-flora in the same sample, which may interfere with the recognition of the primary pathogen of interest; this is particularly a problem when the pathogen is in a dormant stage, and therefore is slow to emerge. Similarly, if the number of propagules is low in a given sample, they may be difficult, if not impossible, to detect in the presence of other organisms. Further, simple detection, in the qualitative sense, may not be an adequate indication of the problems; quantitation is always preferable, and may be essential, in defining the type of treatment or preventive measures to be taken. .

Although isolation, identification, and quantitative analysis of soilborne bacterial pathogens is difficult and prone to variability, suitable methods have been developed in a limited number of instances. However, application of the same techniques to soilborne fungi still lag far behind. 5

The plating-out technique which is routinely used for bacteria is not readily applicable to a number of different fungal pathogens, and is, further, a very time consuming process, requiring identification of all colonies which develop. 0

Immunoassay offers many prospective advantages for pathogen detection in that it is rapid and quantitative, as well as potentially highly specific; however, this technique has not been widely applied to fungal pathogen detection, and moreover has never been used to directly detect overwintering propagules in soil samples. This is in large part due to the difficulties encountered in processing soil samples in a simple and efficient matter. Interference in the effective use of immunoassay arises from soil colloids _Q and organic matter, and the wide variation encountered in physical and chemical properties of different soils. Methods of immunoassay in which the soil is suspended and particles removed by centrifugation have not been successful in accurately representing the level of infestation in a 5particular soil sample. Even at this date, there is no truly convenient method for the testing of soil for the presence of pathogenic fungi, and the more traditional methods continue to be used.

For example, Sutton and Barron (Can. J. Bot. 50:1909-

1914, 1972) described the use of adhesion-flotation to . isolate Endogone spores: spores are floated in aqueous solutions, by agitating a soil sample in the solution, and allowing the suspension to settle out. The sample is decanted into a separatory funnel, and that material which adheres is washed onto paper discs and counted. Furlan et — al. (Trans. Br. Mycol Soc. 75:336-338, 1980) recommend a method for density gradient extraction of endomycorrhizal spores, in which the soil samples are first processed to allow flotation of the spores, as described above, and then the supernatant subjected to centrifugation. Wet-sieving is also still a commonly used method to extract very large spores from soil. In this process, soil samples are mixed with water and, after sedimentation, decanted through sieves of progressively smaller sizes; spores are presumably retained on the finer mesh sieves. Abd-Elrazik and Lorbeer

(Phytopathology 7^:892-894, 1980) disclose a method which combines wet sieving and flotation of sclerotia in glycerol, followed by counting of the sclerotia recovered. Even more recently, Ianson and Allen reexa ined the use of wet sieving, flotation-adhesion, and density-gradient centrifugation, noting that certain of these techniques are better than others depending on the particular type of soil to be tested.

In all the methods described above, i.e., all methods currently in use for soil pathogen detection, quantitation depends upon visual inspection with a microscope and actual counting, by a highly trained individual, of the number of spores, sclerotia (or organic debris) retained by the technique in question. While such methods may be adequate for a situation in which mere collection of propagules is desired, or in which the soil sample is already known to contain the propagules of interest, these methods are unacceptable for the purposes of an initial determination of the presence of a particular pathogen, or for an accurate estimate of the level of infestation of a known pathogen. . . .

Further, the processing time required by the aforementioned techniques is far too long, and often requires the use of complex equipment, rendering the use impractical for efficient large-scale testing outside the laboratory environment.

There exists a need for a method of detecting plant pathogens directly in soil which is both rapid and quantitatively accurate. This result is provided by the method of the present invention, which can provide an accurate evaluation of soil infestation by a variety of plant pathogens on numerous samples in as little time as twenty to forty-five minutes.

3. SUMMARY OF THE INVENTION

The present invention provides a method for determining the presence or absence of a plant pathogen in a soil sample which comprises the steps of treating the soil sample so as to isolate pathogen components, if present, into a concentrated unit; in a fluid medium, treating the concentrated unit so as to disrupt any pathogen components 5 and to expose an antigen of the pathogen; contacting the medium with an antibody having specificity for an antigen of the pathogen, and observing the presence or absence of a reaction between the antigen and the antibody. _Q As used throughout the specification and claims, the term "pathogen" is to be understood in a broad sense, to encompass true pathogens, such as bacteria and fungi, as well as plant pests, such as nematodes or insects. A "pathogen component" refers to both propagules (i.e., part 5of an organism that may be disseminated and reproduce the pathogen) as well as non-propagative fragments or portions of the pathogen, such as mycelia or organic debris containing the pathogen, which would bear identifying antigens of the pathogen. The terms "concentrating" or "isolating in a concentrated unit" is meant to convey that the pathogen components are effectively removed from the soil and isolated in a smaller unit capable of being tested immunologically.

In a preferred embodiment of the present invention, the components are concentrated by flotation. This requires that the components to be detected are buoyant, and is particularly well adapted to the detection of fungal components such as oospores, sclerotia, chlamydospores or mycelia. In a particularly preferred embodiment, the pathogen component, in combination with flotation, is captured on an adherent surface by adhesion of the component, or a water droplet containing the component, to the surface.

The present method is very adaptable, and under certain conditions, a single sample can be processed in as little as twenty minutes, if the infestation is a high positive. In cases in which infestation is at a lower level, testing times may be longer, but seldom more than

1.5-2 hours for a single sample. This is in comparison with plating out, which typically requires 3-5 days, and which 5 cannot be used for certain purposes, such as detection of dormant propagules that do not germinate under lab conditions. Because the method does not require the use of any complex machinery or equipment, unlike, for example, _Qdensity gradient centrifugation, it is readily adaptable for use either partially or totally in a field environment. The invention also provides a kit specifically adapted for use with the present method, which kit comprises means for concentrating a pathogen component, means for disrupting the 5pathogen component, and at least one antibody having specificity for an antigen of the pathogen to be detected. In a preferred embodiment, the kit contains two antibodies, one immobilized, and another labelled, for the performance of a sandwich immunoassay.

4. DETAILED DESCRIPTION OF THE INVENTION

a. CONCENTRATING THE PATHOGEN COMPONENT The present method may utilize any method of concentrating the components to be detected from the soil, provided that the associated soil can be adequately removed to eliminate any possible interference from soil components. Some of the techniques originally developed as improvements over dilution-plating, such as wet-seiving or density- gradient centrifugation, may provide a useful first step in 5 the performance of the present method, in the ability to isolate spores or other components to be tested from soil.

However, the benefit of the present method is not fully achieved in utilizing these techniques, because of the complexity and time involved in each; thus, although they 0 can indeed be employed as a means of concentrating the pathogen component, they are not particularly preferred.

The preferred method employs a type of flotation-adhesion as a first step, and is described in detail below. 5 i. FLOTATION AND CAPTURE

In a preferred embodiment, the present method utilizes a method of flotation and capture to concentrate the propagules or pathogen components in nearly simultaneous _Qsteps. Soil is collected from the areas of interest; to facilitate processing, the soil can be air dried, and large clumps broken up with mortar and pestle. If desired, the soil can be further processed by passage through sieve, sifter, or filter. A measured amount of soil is then placed 5in an appropriate container, and water added to it. The a ount of water added is not critical, but should be adequate to permit agitation and bubble formation in the suspension. Typically, about twice the volume of soil is sufficient water for this purpose. The container holding soil and water is capped, and vigorously agitated for several seconds. This agitation will cause any buoyant material in the soil to float to the surface. In one embodiment of the present method, the flotation alone is adequate to effectively concentrate the component of interest. This is the case, for example, with components which can be simply lifted off the water surface; one such example is citrus-infecting Phytophthora, which is associated with organic debris. In another embodiment of the method, however, capture of the pathogen components in most cases requires additional measures. The components are preferably captured on a surface capable of achieving adhesion of the components, or which permit adequate surface tension for water droplets containing the components to adhere to the surface. As used in the specification and claims, the term "adherent surface" is used to encompass both types of surfaces. In general terms, the useful adherent surfaces are smooth, rigid, and do not absorb water. Among materials appropriate for this purpose are polyethylene, polypropylene, polystyrene, or glass.

Preferred surfaces for adhesion are glass or polypropylene.

After agitation, the container is then filled to the brim with water and a coverslip, glass slide, or other adherent surface placed directly in contact with the surface liquid, in such a manner as to avoid air pockets and to prevent overflow of liquid from the brim of the container. The cover is allowed to remain for a short period of time, i.e., from about 5 minutes to about 30 minutes, during which time the buoyant material at the water's surface adheres to the adherent surface. The cover is then lifted straight up, so that the adhering droplets of water do not drip off. The hanging drop, and any adhering solid particles, are transferred to a second container. The surface is washed thoroughly; preferably this is done with water from the top of the original extraction container. Some buoyant particles, particularly oospores, can cling tenaciously to certain adherent surfaces.

b. DISRUPTING THE COMPONENTS In order to render the pathogen components suitable for testing, it is not necessary to completely dissolve them; rather, it is only necessary to disrupt the integrity in such a manner as to permit release of one or more antigens, which themselves may be solubilized. Disruption of the components may be achieved by any known means. This may be done mechanically, for example, by means of a mortar and pestle type grinder, or other grinding device.

Alternately, electric shock can be employed on the collected fluid sample to break up components such as oospores.

Additional methods of disruption include solubilizing chemical treatment, or enzymatic treatment. Variations on these methods will be apparent to those skilled in the art.

The sample, after grinding, can then be used for testing directly in an immunoassay. However, depending on the amount of insoluble debris in the sample, it may be preferable to remove any solid matter before testing. A brief centrifugation may serve to conveniently remove such debris and the supernatant used for testing; alternately, the sample may be filtered, and the filtrate employed in the immunoassay.

c. IMMUNOASSAY

The methods of immunological testing are well known, and the present method is not necessarily limited to any particular format of assay. Such methods include agglutination reactions, precipitation reactions, im unoelectrophoresis, radioimmunoassays, fluorescent-linked im unosorbent assays, and enzyme linked immunosorbent assays, in solid or liquid phase. However, for ease of performance, the preferred format is a solid phase ELISA.

In such an assay, an antibody having specificity for an . . . antigen of the pathogen of interest is immobilized on a solid substrate, by art-recognized methods. The sample to be tested is contacted with the bound antibody forming an antigen-antibody complex; the complex is then contacted with a second pathogen-specific antibody which is detectably labelled. The labelled antibody binds to the antigen, and an antibody-antigen-antibody complex is formed, the presence of which is confirmed by observation of the detectable label. Variations on this general scheme are well known to those skilled in the art. In a preferred embodiment, the detectable label is an enzyme; the presence of the enzyme label is shown by addition of the appropriate enzyme substrate, which upon hydrolysis produces an analytically detectable change in the medium.

The type of antibodies employed depends upon the identity of the pathogen to be determined. There are currently available a number of antibodies of varying specificities for several different fungal pathogens. Among these are antibodies which react with Pythium or Phytophthora (EP Publ. No. 222 998), Sclerotinia (EP Publ. No. 234 501), or Rhizoctonia (EP Appl. No. 88 106 775.5). Schots, "A Serological Approach to the Identification of Potato Cyst Nematodes.", p. 118, Agricultural University, Wageningen, The Netherlands, 1988, has described anti- nematode antibodies. The antibodies may be monoclonal or polyclonal, or a combination of monoclonal and polyclonal. Labelling of the "reporter" antibody can theoretically be done with any type of molecule which is analytically detectable, e.g., a radioisotope, a chemiluminescent molecule, a fluorescent molecule or a bioluminescent molecule. Particularly preferred, however, for simplicity and cost, are enzyme labels. A variety of enzymes are available for this purpose, e.g. , alkaline phosphatase, β- galactosidase, or horseradish peroxidase. The skilled artisan will readily recognize the possible variations in the recognized ELISA technique.

d. DIAGNOSTIC KIT In one embodiment, the present method can be practiced with a test kit adaptable for laboratory or field use. The 0 test kit contains as its essential elements a means for flotation of the components in a soil sample, a means for capture of components, a means for disrupting components, and an antibody having specificity for the pathogen of interest. The flotation means may be any type of container suitable for holding a soil and water sample, which is further capable of agitation; this may be a vial, bottle, test tube, flask or the like. The capture means, as noted above, should have a surface to which the components or water droplets containing same can adhere. This can be in the form of a cap which fits over the surface of the container, or may be a simple glass cover slip or slide. The means for disrupting can be any convenient mechanical grinding implement, such as a mortar and pestle, or maybe a chemical or enzymatic solubilizer. The solid phase antibody may be immobilized on, for example, a glass slide, a multiwell plate, a test tube, a dipstick, a flow-through device, or any other solid phase known to those skilled in the art. The labelled antibody will be in solution, and for g-_jβase of observation outside a laboratory will be, preferably, enzyme labelled, although any other type of detectable label may also be employed. In the case in which an enzyme label is employed, the kit also contains a

35 substrate for the enzyme. Preferably the kit contains a filter capable of removing insoluble debris from the solubilized sample.

The present method, and the diagnostic kit, can be used to detect the presence of a variety of plant pathogens, , in a number of forms. The types of components detectable include, but are not limited to, nematode eggs or larvae, insect eggs or larvae, fungal oospores, sclerotia, teliospores, sporangia, zygospores, chlamydospores, zoospores, or mycelia, and bacterial cells. The adaptations which can be made in the method in order to detect other types of organisms will be apparent to one skilled in the art.

The following non-limiting example is illustrative of the method of the present invention.

5. EXAMPLES

a. DETECTION OF FUNGAL PATHOGENS IN SOIL

A. Preparation of Soil Samples

Soil samples were air dried at room temperature; large clumps were broken with a mortar and pestle. The soils were passed through a flour sifter and mixed thoroughly. Four

(10 g) subsamples per soil were weighed into 20 cc scintillation vials with screw top caps.

B. Flotation Method of Extracting Phytophthora From the Soil

Each soil sample was processed sequentially as follows: the subsample vials were filled to approximately three quarters capacity with distilled deionized water. The vials were capped and shaken vigorously for 20 seconds. Water was added to the brim of the vials and coverslips wer placed on top of each so that they were in contact with the liquid. The water level was high enough to prevent air pockets beneath the coverslips yet not allow overflow of liquid. After 30 minutes the coverslips were removed one at a time by lifting straight up, without tipping. The hanging drops and solid particles adhering to the coverslip were transferred to microcentrifuge tubes which are made for use with the Kontes Disposable Pellet Pestle. The coverslips were washed with 50 μl from the top of each vial into the microcentrifuge tubes. One extract per subsample was collected; subsamples were later pooled.

C. Preparation of the Soil Extracts for Immunoassay

The extracts were ground for one minute at 2,500 rpm using a Kontes Pellet Pestle driven by a Talboys Laboratory Stirrer (Model 134-2) . 300 μl of extract diluent was added to each of the tubes and mixed by vortexing briefly. The four subsamples per soil sample were pooled, centrifuged one minute at 10,000 rpm and filtered through an .08 μm filter. The filtered extract was collected in a clean microcentrifuge tube.

D. Immunoassay for Phytophthora

The soil extracts were tested for the presence of Phytophthora using Agri-Diagnostics' Phytophthora "E" Multiwell Kit. The kit was a double antibody ELISA which uses affinity-purified sheep anti-Phytophthora megasperma antibody at 5 μg/ml as the primary antibody which was immobilized on the surface of a microtiter plate. A series of laboratory-prepared Phytophthora megasperma oospore -standards at concentrations of 0/ml, 50/ml, 100/ml and 200/ml was run with each microtiter plate. 100 μl of prepared soil extract or standard was pipeted to each of two antibody-sensitized wells and one nonsensitized well. The plate was incubated 20 minutes at room temperature on a 5Titertek plate shaker, washed six times and incubated an additional 20 minutes with 100 μl/well conjugate. The conjugate was a mixture of two monoclonal anti-Phytophthora megasperma antibodies at a concentration of 7 μg/ml coupled to horseradish peroxidase. After washing the plate, 100 μl/well ABTS substrate was added and incubated 10 minutes with shaking. The color reaction of the substrate was stopped with 50 μl/well 1.5% NaF; the absorbance 405 nm of each well is read using a Dynatech MiniReader II. For each test and standard the absorbance of the nonsensitized well is subtracted from mean absorbance of the sensitized wells.

The relative level of Phytophthora was estimated by comparing the "corrected" mean absorbance of the soil extracts to the corrected readings of the laboratory prepared oospore standards.

The results of various tests using the present method are shown in Tables la-d. These data show that the present method is effective in detection of at least four different crop/pathogen systems.

TABLE 1

Multiwell Absorbance Readings (O.D. 405 nm sensitized-nonsensitized) Phytophthora Level

Absorbance of 0 std +.15 no Phytophthora detected

Absorbance of 0 std +.15 -> 50 ml std low level of Phytophthora

Absorbance of 50 ml std -> 100 ml std medium level of Phytophthora

> Absorbance of 100 ml std high level of Phytophthora

a. Phytophthora Detection in Soybean Soil Extracts Tested by the Direct Soil Immunoassay

Sample # MW Absorbance Pmg Level

A-3 .03 Not Detected ^A~⁴ .01 Not Detected A-26 .09 Not Detected

A-5 .30 Low A-25 .43 Low A-12 .88 Medium A-22 .74 Medium A-18 1.31 High A-24 1.48 High A-27 1.65 High

θospore Standards (oospores/ml) 0 0.06

50 0.47

100 0.92

200 1.97 b. Phytophthora Detection in Citrus Soil Extracts Tested by the Direct Soil Immunoassay

Sample # MW Absorbance Pmg Level

JB 19-1 .11 Not Detected

JB 19-8 .10 Not Detected

JB 14-3 .36 Low JB 14-8 .40 Low

JB 8-3 .73 Medium

JB 13-6 .61 Medium

JB 13-1 1.05 High

JB BSO-27 1.63 High

Oospore Standards (oospores/ml)

0 0.05

50 0.47

100 0.96 200 1.91

c. Rhizoctonia Detection in Results of Soybean Soil Extracts Tested by Agri-Diagnostics' Rhizoctonia

Immunoassay

Sample # MW Absorbance Result

8000 >2.00 Positive 8001 1.38 Positive 8002 1.37 Positive 8003 >2.00 Positive 8009 0.28 Positive Buffer 0.03 Not Detected d. Pythium Detection in Soybean Soil Extracts Tested by Agri-Diagnostics ' Pythium C Multiwell Kit

Sample # MW Absorbance Result

A-4 1.35 Positive A-26 0.00 Not Detected

A-27 0.57 Positive

K-BUC 2-6 1.77 Positive

PNO 2-6 0.55 Positive

Watson 0.23 Positive Buffer 0.00 Not Detected

Claims

WHAT IS CLAIMED IS:

1. A method for determining the presence or absence, and level, if present, of a plant pathogen in a soil sample suspected of containing the pathogen, which comprises

(a) treating the soil sample so as to isolate pathogen components into a concentrated unit;

(b) treating the concentrated unit in a fluid medium so as to disrupt any pathogen components and to expose an antigen of the pathogen;

(c) contacting the medium of step (b) with at least one antibody specific for the pathogen; and

(d) observing the presence or absence of a reaction between antigen and antibody.

2. The method of Claim 1 wherein the components are concentrated by flotation, centrifugation, or wet-sieving.

3. The method of Claim 2 wherein the components are concentrated by flotation.

4. The method of Claim 3 wherein the components are further concentrated by capture on an adherent surface.

5. The method of Claim 4 wherein the adherent surfac is glass, polyethylene, polypropylene or polystyrene.

6. The method of Claim 1 wherein the antibody is detectably labelled.

7. The method of Claim 6 wherein the antibody is labelled with an enzyme.

8. The method of Claim 1 wherein the antigen is contacted with two antibodies, one being immobilized and the other being detectably labelled.

9. The method of Claim 8 wherein the detectably labelled antibody is labelled with an enzyme.

10. The method of any one of Claims 1 to 9 wherein the components are disrupted mechanically, chemically, or enzymatically.

11. The method of any one of Claims 1 to 9 wherein the pathogen is a nematode, a fungus, a bacterium or an insect.

12. The method of any one of Claims 1-9 wherein the pathogen is a fungus.

13. The method of Claim 12 wherein the fungus is Pythium, Rhizoctonia, Sclerotinia, Phytophthora, Fusarium, Verticillium, or Thielaviopsis.

14. The method of Claim 12 wherein the components are oospores, sporangia, chlamydospores, zoospores, sclerotia, or mycelia. 5

15. The method of any one of Claims 1-9 wherein the pathogen is a nematode.

16. The method of Claim 15 wherein the nematode is _QMeloidogyne, Heterodera, Globodera, Tylenchus, or

Pratylenchus.

17. The method of any one of Claims 1-9 wherein the pathogen is a bacterium. 5

18. The method of Claim 17 wherein the bacterium is Erwinia, Agrobac erium, Pseudomonas, Streptomyces or Xanthomonas.

19. The method of any one of Claims 1-9 wherein the pathogen is an insect.

20. The method of Claim 19 wherein the insect is corn root worm (Diabrotica spp), cut worms, wire worms, or root maggots.

21. A method for determining the presence or absence of a plant pathogen in a soil sample suspected of containing the pathogen which comprises

(a) agitating the soil sample in a fluid medium to float any buoyant pathogen components on the fluid surface;

(b) isolating a portion of the fluid expected to contain components, if present, so as to concentrate the components;

(c) treating the isolated portion of the medium so as to disrupt any pathogen components therein and expose an antigen of the pathogen;

(d) contacting the treated portion of step (c) with an antibody specific for the pathogen; and

(e) observing the presence or absence of a reaction between antigen and antibody.

22. The method of Claim 1 wherein isolation is achieved by contacting the fluid surface with an adherent surface.

23. The method of Claim 22 wherein the adherent surface is glass, polyethylene, polypropylene or polystyrene.

24. The method of Claim 23 wherein the antibody is detectably labelled.

25. The method of Claim 24 wherein the antibody is labelled with an enzyme.

26. The method of Claim 23 wherein in step (d), the treated portion is contacted with two antibodies, one being immobilized and the other being detectably labelled.

27. The method of Claim 26 wherein the labelled antibody is labelled with an enzyme.

28. The method of any one of Claims 21 to 27 wherein the pathogen is a nematode, a fungus, a bacterium or an insect.

29. The method of any one of Claims 21 to 27 wherein the pathogen is a fungus.

30. The method of Claim 29 wherein the pathogen is Pythium, Rhizoctonia, Sclerotinia, Phytophthora, Fusarium, Verticillium, or Thielaviopsis.

31. The method of Claim 29 wherein the components ar oospores, sporangia, chlamydospores, zoospores, sclerotia, or mycelia.

32. The method of any one of Claims 21 to 27 wherein the pathogen is a nematode.

33. The method of Claim 32 wherein the nematode is Meloidogyne, Heterodera, Globodera, Tylenchus, or Pratylenchus.

34. The method of any one of Claims 21 to 27 wherein the pathogen is a bacterium.

35. The method of Claim 34 wherein the bacterium is Agrobacterium, Pseudomonas, Streptomyces, Erwinia or Xanthomonas.

36. The method of any one of Claims 21 to 27 wherein the pathogen is an insect.

37. The method of Claim 36 wherein the insect is cor root worm (Diabrotica spp), cut worms, wire worms, or root maggots.

38. A diagnostic test kit for detection of a plant pathogen in soil which comprises

(a) means for concentrating pathogen component from the soil;

(b) means for disrupting pathogen components;

(c) an immobilized antibody having specificity for the pathogen; and

(d) a labelled antibody having specificity for the pathogen.

39. The kit of Claim 38 wherein the means for concentrating the pathogen comprises

(i) a flotation means, and (ii) a capture means.

40. The kit of Claim 39 wherein the capture means is an adherent surface.

41. The kit of Claim 40 wherein the surface is glass, polyethylene, polypropylene or polystyrene.

42. The kit of any one of Claims 38 to 41 wherein the labelled antibody is labelled with an enzyme.

43. The kit of Claim 42 which also comprises a substrate for the enzyme.

44. The kit of any one of Claims 38-41 wherein the pathogen is a nematode, a fungus, a bacterium, or an insect,

45. The kit of any one of Claims 38-41 wherein the pathogen is a fungus. 5

46. The kit of Claim 45 wherein the pathogen is

Pythium, Rhizoctonia, Sclerotinia, Phytophthora, Fusarium or Verticillium, or Thielaviopsis.

47. The kit of any one of Claims 38-41 wherein the 0 pathogen is a nematode.

48. The kit of Claim 47 wherein the nematode is Meloidogyne, Heterodera, Globodera, Tylenchus or Pratylenchus. 5

49. The kit of any one of Claims 38-41 wherein the pathogen is a bacterium.

_Q 50. The kit of Claim 49 wherein the bacterium is Erwinia, Agrobacterium, Pseudomonas, Streptomyces, or Xanthomonas.

51. The kit of any one of Claims 38-41 wherein the 5pathogen is an insect.

52. The kit of Claim 51 wherein the insect is corn root worm (Diabrotica spp), cut worms, wire worms, or root maggots.

53. The kit -of Claim 45 wherein the labelled antibody is labelled with an enzyme.

54. The kit of claim 53 which also comprises a substrate for the enzyme.

55. The kit of Claim 47 wherein the labelled antibody is labelled with an enzyme.

56. The kit of Claim 55 which also comprises a substrate for the enzyme.

57. The kit of Claim 49 wherein the labelled antibody is labelled with an enzyme.

58. The kit of Claim 51 which also comprises a substrate for the enzyme.

59. The kit of Claim 51 wherein the labelled antibody is labelled with an enzyme.

60. The kit of Claim 59 which also comprises a substrate for the enzyme.