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Plant Disease Diagnosis: Moving Out of the Lab and Into the Field
ELISA Diagnostic Kits
Direct Tissue Blotting
Nucleic Acid Probes
Summary

Plant Disease Diagnosis: Moving Out of the Lab and Into the Field

ELISA Diagnostic Kits

A number of disease detection kits have been developed for use at the site where a disease is suspected. These kits, which in most cases do not require laboratory equipment, are especially useful to growers. Some tests only take five minutes to perform.

The diagnostic kits are based on a method that uses proteins called antibodies to detect disease-causing organisms of plants (plant pathogens). The technique used is called ELISA (enzyme-linked immunosorbent assay). This assay is based on the ability of an antibody to recognize and bind to a specific antigen, a substance associated with a plant pathogen. The process is illustrated in figure 1.

The antibodies used in the diagnostic kits are highly purified proteins produced by injecting a warm-blooded animal (like a rabbit) with an antigen associated with one particular plant disease (1). The animal reacts to the antigen and produces antibodies (2). The antibodies produced recognize and react only with the proteins associated with the causal agent of that plant disease.

These antibodies are bound to a plastic plate or similar detector unit in a test kit. The individual running the test prepares the plant sample by grinding it between two pieces of abrasive paper. The ground plant sample is placed in a bottle filled with an extraction liquid. This liquid is then placed in wells in the plastic plate (3). If the disease-causing organism (pathogen) is present in the sample, the specific antibodies in the plastic plate will bind to pathogen-associated proteins and adhere to the unit (4a). A second antibody is added that also reacts with the pathogen-associated proteins. This antibody is special because it can react with color-producing chemicals called reagents (5a). Color changes on the unit's surface indicate a positive (disease present) reaction. If no pathogen-associated proteins are present (4b), the detector antibodies cannot bind to the color carrying reagents and are washed away (5b).

At the present time, a limited number of these onsite disease detection kits are available. Many ELISA diagnostic kits are available for use in the laboratory, however, and can test for a wide range of plant pathogens. Examples of diseases ELISA kits can detect include bacterial canker of tomato, soybean root rot, and bacterial blight of geranium.

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Direct Tissue Blotting

Another diagnostic assay that also uses specific antibodies as a detection tool is known as direct tissue blotting. With this technique, the location of a disease-causing pathogen within the host plant can be determined, allowing earlier detection and a better understanding of how a disease progresses through a plant.

Host plant tissue is pressed onto a special piece of paper. Antibodies that bind only to the disease-causing pathogen in question are then introduced to this paper. A color change indicates a positive result and shows the location of the pathogen in the host tissue (figure 2).

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Nucleic Acid Probes

Squash Blot Method

A method that uses nucleic acid probe technology is known as the squash blot method (figure 3).This technology is similar to direct tissue blotting. Tissue from a plant that is suspected to be diseased is "squashed" onto a special piece of paper, called a membrane (1). This membrane is then treated with a probe (2) that can bind or hybridize with nucleic acid of the plant pathogen suspected to be in the plant tissue. Hybridization or binding will occur when like sequences are present (3). After adding several more substances to the membrane, a color reaction (4) indicates that the probe and pathogen nucleic acid sequences have hybridized and the disease is present. No color reaction means the test for the disease is negative.

PCR

Summary

References

North Central Regional
Extension Publication
NCR#492