View source for 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
*~10<sup>9</sup> cfu/ml bacterial suspension
*Tobacco plant

==Instructions==
#With a syringe and 25-gauge needle, inject a fresh, very cloudy bacterial suspension (~10<sup>9</sup> 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 cm<sup>2</sup>. 
*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 Tech]] Mediabook; Orignially created by Robert Wick; contributed by Mary Ann Hansen.

[[Category:Bacterial Diagnostic Tests]]