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[ANNEX II U.K. TEST SCHEME FOR DIAGNOSIS, DETECTION AND IDENTIFICATION OF RALSTONIA SOLANACEARUM (SMITH) YABUUCHI ET AL.
SECTION VI U.K. OPTIMISED PROTOCOLS FOR DETECTION AND IDENTIFICATION OF R. SOLANACEARUM
A. DIAGNOSTIC AND DETECTION TESTS U.K.
9. Bioassay test U.K.
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):
9.1. Use 10 test plants of a susceptible tomato cultivar (e.g. Moneymaker or cultivar with equivalent susceptibility as determined by the testing laboratory) at the third true leaf stage for each sample. For cultural details, see Appendix 8. Alternatively, use eggplants (e.g. cultivar Black Beauty or cultivars with equivalent susceptibility), use only plants at leaf stage two to three up to full expansion of the third true leaf. Symptoms have been show to be less severe and to develop more slowly in eggplant. Where possible, it is therefore recommended to use tomato seedlings. U.K.
9.2.
Distribute 100 µl of sample extract between the test plants. U.K.
9.2.1. Syringe inoculation 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.
9.2.2. Slit inoculation U.K.
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.
9.3. Inoculate by the same technique, five seedlings with an aqueous suspension of 10 5 to 10 6 cells per ml prepared from a 48 hr culture of a virulent biovar 2 strain of R. solanacearum as a positive control and with pellet buffer (Appendix 4) as negative control. Separate positive and negative control plants from the other plants to avoid cross-contamination. U.K.
9.4. Grow the test plants in quarantine facilities for up to four weeks at 25 to 30 °C and high relative humidity with appropriate watering to prevent waterlogging or wilting through water deficiency. To avoid contamination incubate positive control and negative control plants on clearly separated benches in a glasshouse or growth chamber or, in case space is limited, ensure strict separation between treatments. If plants for different samples must be incubated close together, separate them with appropriate screens. When fertilising, watering, inspecting and any other manipulations take great care to avoid cross-contamination. It is essential to keep glasshouses and growth chambers free of all insect pests since they may transmit the bacterium from sample to sample. U.K.
Observe for symptoms of wilting, epinasty, chlorosis and/or stunting.
9.5. Isolate from infected plants (Section II.3.) and identify purified cultures of presumptive R. solanacearum (Section VI.B.). U.K.
9.6. If no symptoms are observed after three weeks perform IF/PCR/Isolation on a composite sample of 1 cm stem sections of each test plant taken above the inoculation site. If the test is positive perform dilution plating (section 4.1). U.K.
9.7. Identify any purified cultures of presumptive R. solanacearum (Section VI.B.). U.K.
Interpretation of the bioassay test results U.K.
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 .]