Tobacco hypersensitivity reaction

Purpose

This test is useful for identifying Xanthomonas spp., the Pseudomonas syringae group, and Erwinia amylovora. For a more detailed explanation of the general procedure, see the Hypersensitivity Reaction (HR) procedure.

Materials

Syringe with 25-gauge needle
~10⁹ cfu/ml bacterial suspension
Tobacco plant

Instructions

With a syringe and 25-gauge needle, inject a fresh, very cloudy bacterial suspension (~10⁹ cfu/ml) into interveinal tissue of a tobacco leaf.
Second Step
Third Step
Fourth Step

Expected results

Injected tissue that starts to collapse and turn necrotic in 24 hours indicates a positive (hypersensitive) reaction.

Notes

Injecting the tobacco leaf takes a lot of practice.
It is easiest to inject near a major vein.
Be careful not to stab your finger while injecting.
It is only necessary to inject an area of about 1 cm².
You can tell you are doing it correctly when the tissue around the injection gets a water-soaked look.
The hypersensitivity reaction is variable depending upon temperature, light, bacterial concentration, and cultivar of tobacco used.
A delayed necrosis (30-192 hours) may occur if an insufficient number of bacterial cells is present, or if environmental conditions are unfavorable for hypersensitivity reaction.
The affected tissue may turn chlorotic before dying.
Be sure to use positive and negative controls.

References

Fahy, P. C., and G. J. Persley. 1983. Plant Bacterial Diseases, A Diagnostic Guide. (p.356). Academic Press, New York. 393 pp.
Thornley, M. J. 1960. The differentiation of Pseudomonas from other Gram negative bacteria on the basis of arginine metabolism. Journal of Applied Bacteriology 23:37 52.

Contributed by

From the Virginia Polytechnic Institute and State University Mediabook; Orignially created by Robert Wick; contributed by Mary Ann Hansen.