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Council Directive 98/57/EC of 20 July 1998 on the control of Ralstonia solanacearum (Smith) Yabuuchi et al.
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Note: Preliminary testing with this method should permit reproducible detection of 10 3 to 10 4 colony-forming units of R. solanacearum per ml added to sample extracts that previously tested negative (preparation see Appendix 3). U.K.
Highest sensitivity of detection can be expected when using freshly prepared sample extract and optimal growth conditions. However, the method can be successfully applied to extracts that have been stored under glycerol at -68 to -86 °C.
The following protocol is based upon Janse (1988):
Distribute 100 µl of sample extract between the test plants. U.K.
Inoculate the plant stems just above the cotyledons using a syringe fitted with a hypodermic needle (not less than 23G). Distribute the sample between the test plants.
Holding the plant between two fingers, pipette a drop (approximately 5 - 10 µl) of the suspended pellet on the stem between the cotyledons and the first leaf.
Using a sterile scalpel, make a diagonal slit, about 1.0 cm long and approximately 2/3 of the stem thickness deep, starting the cut from the pellet drop.
Seal the cut with sterile vaseline from a syringe.
Observe for symptoms of wilting, epinasty, chlorosis and/or stunting.
Valid Bioassay test results are obtained when plants of the positive control show typical symptoms, the bacteria can be reisolated from these plants and no symptoms are found on the negative controls.
The bioassay test is negative if test plants are not infected by R. solanacearum , and provided that R. solanacearum is detected in positive controls.
The bioassay test is positive if the test plants are infected by R. solanacearum .]
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