Council Directive 93/85/EECDangos y teitl llawn

6. Eggplant test

For cultural details, see Appendix 5.

6.1. Distribute the pellet from 3.3 between at least 25 eggplants at leaf stage 3 (Appendix 5) by one of the methods given below (6.2, 6.3 or 6.4).

6.2. Slit inoculation I

6.2.1. Support each pot horizontally (a block of expanded polystyrene with a piece 5 cm deep x 10 cm wide x 15 cm long, removed from one surface (Figure 3) is adequate for a 10 cm pot). A strip of sterile aluminium foil should be placed between the stem and the block for each sample tested. The plant may be held in place by a rubber band around the block.

6.2.2. Using a scalpel, make a longitudinal or slightly diagonal cut 0,5 to 1,0 cm long and approximately three quarters of the stem diameter deep, between the cotyledons and the first leaf.

6.2.3. Hold the slit open with the scalpel blade point and paint the inoculum into it using an eyeliner or fine artist's brush charged with the pellet. Distribute the remainder of the pellet between the eggplants.

6.2.4. Seal the cut with sterile vaseline from a 2 ml syringe barrel.

Figure 3

6.3. Slit inoculation II

6.3.1. Holding the plant between two fingers, pipette a drop (approximately 5 to 10 µl) of the suspended pellet on the stem between the cotyledons and the first leaf.

6.3.2. Using a sterile scalpel, make a diagonal (at an angle of approximately 5^o) slit, 1,0 cm long and approximately 2/3 of the stem thickness deep, starting the cut from the pellet drop.

6.3.3. Seal the cut with sterile vaseline from a syringe barrel.

6.4. Syringe inoculation

6.4.1. Do not water eggplants for one day prior to inoculation to reduce turgor pressure.

6.4.2. Inoculate the eggplant stems just above the cotyledons using a syringe fitted with a hypodermic needle (not less than 23G). Distribute the pellet between the eggplants.

6.5. Inoculate 25 plants with a known C. sepedonicum culture and, where possible, naturally infected tuber tissue (5.1) by the same inoculation method (6.2, 6.3 or 6.4).

6.6. Inoculate 25 plants with sterile 0,05 M PBS by the same inoculation method (6.2, 6.3 or 6.4).

6.7. Incubate the plants in appropriate conditions (Appendix 5) for 40 days. Examine regularly for symptoms after eight days. Count the number of plants showing symptoms. C. sepedonicum causes leaf wilting in eggplants which may commence as a marginal or interveinal flaccidity. Wilted tissue may initially appear dark green or mottled but turns paler before becoming necrotic. Interveinal wilts often have a greasy water-soaked appearance. Necrotic tissue often has a bright yellow margin. Plants are not necessarily killed; the longer the period before symptoms develop, the greater the chance of survival. Plants may outgrow the infection. Susceptible young eggplants are much more sensitive to low populations of C. sepedonicum than are older plants, hence the necessity to use plants at or just before leaf stage 3.

Wilts may also be induced by populations of other bacteria or fungi present in the tuber tissue pellet. These include Erwinia carotovora, subsp. carotovora and E. carotovora subsp. atroseptica, Phoma exigua var. foveata, as well as large populations of saprophytic bacteria. Such wilts can be distinguished from those caused by C. sepedonicum since whole leaves or whole plants wilt rapidly.

6.8. Prepare a Gram stain (4) for all batches of eggplants showing symptoms, using sections of wilted leaf tissue and stem tissue from plants and isolate on to suitable nutrient media (7). Surface disinfect the eggplant leaves and sterns by wiping with 70 % ethanol.

6.9. Under certain circumstances, in particular where growing conditions are not optimal, it may be possible for C. sepedonicum to exist as a latent infection within eggplants even after incubation for 40 days. Such infections may possibly result in stunting and lack of vigour in the inoculated plants. If the IF test is considered positive, it may be considered necessary to test further. It is, therefore, essential to compare the growth rates of all eggplant test plants with the sterile 0,05 M PBS inoculated controls and to monitor the environmental conditions of the glasshouse.

Recommendations for further testing are as follows:

6.9.1.

excise the stems above the inoculation site and remove the leaves;

6.9.2.

macerate the stems in 0,05 M PBS pH 7,0, as in 3.1 to 3.2;

6.9.3.

use half of the pellet to perform a Gram stain (4) and an IF test (5);

6.9.4.

use the other half to perform a further eggplant test (6) if the Gram stain and/or IF tests are positive. Use a known C. sepedonicum culture and sterile 0,05M PBS controls. If symptoms are not observed in the subsequent test, the sample must be considered negative.