Hypersensitivity Reaction (HR)

Purpose

Determines the Hypersensitivity of the plant to bacterial isolates

Obtain a pure bacterial isolate and streak on nutrient agar, incubate at 30°C for 48 hours
Warm-up spectrophotometer and zero with sterile DI water
Add some sterile DI water to test tube
Using a cotton-tip applicator, collect bacteria and swish around in test tube
Transfer some of bacterial suspension to a cuvette and read transmittance
If needed, adjust suspension so transmittance is between 40 and 50 (bacterial concentration of approximately 10⁸ cells/mL)
Repeat process for each isolate and positive control
Carefully label leaf sections with identifiers using a thick-tipped sharpie: on tobacco, keep isolates separated by larger veins; on tomato- separate leaflets
Label sections for a positive control and negative control (water blank)
Draw up into a syringe approximately 1cc of suspension
Carefully flip leaf/leaflet over to expose the lower leaf surface where you labeled for that isolate
Place tip of syringe flat against leaf surface using your finger for support underneath (Be careful not to tear the leaf tissue)
Gently, but with enough force, depress plunger until a sufficient amount of the leaf tissue is infused with the bacterial suspension
With tomato, more than 1 injection site may be required to get sufficient infusion
After 24 to 48 hours, record necrosis in infused area as a positive HR

It is important to wear PPE with this procedure (Labcoat, gloves and eye protection). There is a high risk of getting sprayed from the injection.
It is much easier to inject plants that have not been watered recently
If getting excessive spray, you may need to adjust the way you are holding the syringe against the plant or move to a new injection site
If necrosis is limited to the area immediately around the injection site, it may be from damage and not necessarily a positive HR
Not all plant pathogens cause HR, or they may only cause an HR on one of the plants
Pictures of an HR response on tobacco can be found in the Bauer paper or Plant Pathology textbook

Schaad, N.W. 2001. Laboratory Guide for the Identification of Plant Pathogenic Bacteria, 3rd Edition. The American Phytopathological Society, St. Paul, MN. (pp. 44-45)
Bauer DW, Wei ZM, Beer SV, Collmer A (1995) Erwinia chrysanthemi HarpinEch: An elicitor of the hypersensitive response that contributes to soft-rot pathogenesis. Molecular Plant-Microbe Interactions 8: 484-491
Agrios, G. N. 1997. PLANT PATHOLOGY, 4th Ed. Academic Press. p101

From the University of Florida Plant Disease Clinic-GNV Systems Manual: originally created by Anne Vitoreli; contributed by Anne Vitoreli.