Gamma Rays and CarbonIon-Beams Irradiation for Mutation ...

and transferred to a 50 mL Erlenmeyer containing 20 mL of distilled water.
A vortex was used to promote fast release of conidia into the solution.
Once homogenized, the solution was filtered with 4 layers of gauze for
collecting the filtrates in a flask (Fig 29, B). The solution was adjusted to
60 mL and then a concentration of 1.5 x 10 6 /mL was obtained using a
hemacytometer. Prior to inoculation, the sprayer was calibrated to
determine the amount of solution to use. It was found that 45 mL of
solution was enough to inoculate 87 plants, meaning that each plant was
inoculated by about 0.5 mL of solution.
Before applying the inoculum to the plants, the old leaves were
removed and the newly expanded leaf was selected and marked to be
inoculated. Also the abaxial side of the marked leaf was cleaned using a
soft cotton tissue. Using a small sprayer, the abaxial part of the leaf was
sprayed with the solution in both sides (Fig 29, C). Immediately after the
inoculation, the plants were kept in a dark incubation room for 48 hours
adequate for the normal development of the fungus (Fig 29, D). The room
provided continuous water aspersions to maintain a high relative humidity.
The room temperature was 26 o C. After incubation, the plants were kept in a
nursery plot for one week before transplanting to the field.
Due to the lack of monosporic cultures of Mycosphaerella
fijiensis Morelet, the method to inoculate the second and third leaves was
changed. The method consisted of applying completely diseased leaves
fragments among the plantlets to produce high inoculum pressure. The
leaves were collected from a banana collection located in the Estación
Experimental Tropical Pichilingue, which have been kept without any
chemical control for this disease (Fig. 30, A). Therefore, those leaves are
considered as a potential natural inoculum capable to produce efficient
sporulation pressure to inoculate the plantlets. Leaf-fragments were cut
around 15 x 20 cm (Fig. 30, B) and they were sprayed with water prior to
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