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The pathogenicity test must be performed as final confirmation of a diagnosis of R. solanacearum and for assessment of virulence of cultures identified as R. solanacearum .
Prepare an inoculum of approximately 10 6 cells per ml from a 24 to 48 hour culture of the isolate to be tested and an appropriate positive control strain of R. solanacearum (e.g. NCPPB 4156 = PD 2762 = CFBP 3857; see Appendix 3).
Inoculate 5 to 10 susceptible tomato or eggplant seedlings at the third true leaf stage (see Section VI.A.9).
Incubate for up to two weeks at 25 to 28 °C and high relative humidity with appropriate watering to avoid waterlogging or drought stress. With pure cultures typical wilting should be obtained within 14 days. If after this period symptoms are not present, the culture cannot be confirmed as being a pathogenic form of R. solanacearum .
Observe for symptoms of wilting and/or epinasty, chlorosis and stunting.
Isolate from symptomatic plants by removing a section of stem about 2 cm above the inoculation point. Comminute and suspend in a small volume of sterile distilled water or 50 mM phosphate buffer (Appendix 4). Isolate from the suspension by dilution spreading or streaking on a suitable medium, preferably onto a selective medium (Appendix 2), incubate for 48 to 72 hours at 28 °C and observe the formation of colonies typical of R. solanacearum .]