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EP0466765A1 - Immunoanalyse directe de terre - Google Patents

Immunoanalyse directe de terre

Info

Publication number
EP0466765A1
EP0466765A1 EP19900905765 EP90905765A EP0466765A1 EP 0466765 A1 EP0466765 A1 EP 0466765A1 EP 19900905765 EP19900905765 EP 19900905765 EP 90905765 A EP90905765 A EP 90905765A EP 0466765 A1 EP0466765 A1 EP 0466765A1
Authority
EP
European Patent Office
Prior art keywords
pathogen
kit
labelled
antibody
enzyme
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19900905765
Other languages
German (de)
English (en)
Other versions
EP0466765A4 (fr
Inventor
Suzanne M. Miller
Frank P. Petersen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Agri-Diagnostics Associates
Original Assignee
Agri-Diagnostics Associates
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Agri-Diagnostics Associates filed Critical Agri-Diagnostics Associates
Publication of EP0466765A4 publication Critical patent/EP0466765A4/fr
Publication of EP0466765A1 publication Critical patent/EP0466765A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56961—Plant cells or fungi

Definitions

  • the present invention relates to a method for detecting the presence of plant pathogens in soil. More
  • the invention relates to a method for direct i munoassay of soil to detail the presence of antigens of a plant pathogen of interest.
  • 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.
  • 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.
  • 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.
  • root knot nematodes such as
  • cyst nematodes such as Heterodera and Globodera
  • other genera such as Tylenchus and Pratylenchus.
  • insects particularly insect larvae
  • 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 may spend considerable periods of time in the soil.
  • 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.
  • 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.
  • 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.
  • ornamentals such as azalea, rhododendron and camellia.
  • Pythium cause a damping-off disease of seedlings which affects flowering plants, vegetables and row crops world- wide.
  • Rhizoctonia solani causes more different 5 types of diseases, among a wider variety of plants, than any other plant-pathogenic fungus.
  • the diseases caused by this destructive pathogen are seed rot, damping-off, root and crown rot, affecting virtually all stages of plant Q development.
  • 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.
  • Sclerotinia 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.
  • fungi overwinter 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.
  • 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.
  • 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.
  • 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.
  • 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. .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the components are concentrated by flotation.
  • 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.
  • 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
  • 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.
  • the kit contains two antibodies, one immobilized, and another labelled, for the performance of a sandwich immunoassay.
  • 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.
  • the preferred method employs a type of flotation-adhesion as a first step, and is described in detail below. 5 i. FLOTATION AND CAPTURE
  • the present method utilizes a method of flotation and capture to concentrate the propagules or pathogen components in nearly simultaneous Q steps.
  • 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.
  • 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.
  • 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.
  • adherent surfaces are smooth, rigid, and do not absorb water.
  • materials appropriate for this purpose are polyethylene, polypropylene, polystyrene, or glass.
  • Preferred surfaces for adhesion are glass or polypropylene.
  • 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.
  • 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.
  • 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.
  • 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.
  • the preferred format is a solid phase ELISA.
  • 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.
  • 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.
  • enzyme labels are available for this purpose, e.g. , alkaline phosphatase, ⁇ - galactosidase, or horseradish peroxidase. The skilled artisan will readily recognize the possible variations in the recognized ELISA technique.
  • 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.
  • 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- j ⁇ ase of observation outside a laboratory will be, preferably, enzyme labelled, although any other type of detectable label may also be employed.
  • the kit also contains a
  • 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.
  • 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.
  • 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 were 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 ⁇ l from the top of each vial into the microcentrifuge tubes. One extract per subsample was collected; subsamples were later pooled.
  • 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 ⁇ l 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 ⁇ m filter. The filtered extract was collected in a clean microcentrifuge tube.
  • 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 ⁇ g/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 ⁇ l 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 ⁇ l/well conjugate.
  • the conjugate was a mixture of two monoclonal anti-Phytophthora megasperma antibodies at a concentration of 7 ⁇ g/ml coupled to horseradish peroxidase.
  • 100 ⁇ l/well ABTS substrate was added and incubated 10 minutes with shaking.
  • the color reaction of the substrate was stopped with 50 ⁇ l/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.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Food Science & Technology (AREA)
  • Biochemistry (AREA)
  • Pathology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Virology (AREA)
  • Botany (AREA)
  • Mycology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé et un kit permettant de déterminer la présence ou l'absence de germes pathogènes de plantes dans un échantillon de terre. Ledit procédé consiste à concentrer un composant de germe pathogène obtenu à partir de l'échantillon, à scinder les composants afin de libérer un antigène dudit germe pathogène, à mettre l'échantillon scindé en contact avec un anticorps présentant une spécificité à l'égard dudit germe pathogène, et à observer la présence ou l'absence de réaction entre l'antigène et l'anticorps.
EP19900905765 1989-03-27 1990-03-26 Immunoanalyse directe de terre Withdrawn EP0466765A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US32882489A 1989-03-27 1989-03-27
US328824 1989-03-27

Publications (2)

Publication Number Publication Date
EP0466765A4 EP0466765A4 (fr) 1991-11-28
EP0466765A1 true EP0466765A1 (fr) 1992-01-22

Family

ID=23282602

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19900905765 Withdrawn EP0466765A1 (fr) 1989-03-27 1990-03-26 Immunoanalyse directe de terre

Country Status (4)

Country Link
EP (1) EP0466765A1 (fr)
JP (1) JPH04506401A (fr)
CA (1) CA2049992A1 (fr)
WO (1) WO1990011522A1 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FI93742C (fi) * 1992-10-23 1995-05-26 Elias Hakalehto Menetelmä ja laite solujen osoittamiseksi
GB2342992A (en) * 1998-10-22 2000-04-26 Horticulture Res Int Dipstick detection system
CN115060903A (zh) * 2022-07-06 2022-09-16 四川省烟草公司宜宾市公司 一种用于检测烟草根黑腐病菌的酶联免疫试剂盒及其应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4780408A (en) * 1986-03-26 1988-10-25 Colorado State University Research Foundation Antibody for detection and quantification of trifluralin
US4879217A (en) * 1987-04-28 1989-11-07 Agri-Diagnostics Associates Test for Rhizoctonia brown and yellow patch

Also Published As

Publication number Publication date
JPH04506401A (ja) 1992-11-05
EP0466765A4 (fr) 1991-11-28
WO1990011522A1 (fr) 1990-10-04
CA2049992A1 (fr) 1990-09-28

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