Induction of pathogenicity mutants from Elsinoe fawcettii, the causal fungus of citrus scab by genetic transformation

Genetic transformation carried out to induce the pathogenicity mutants from the two isolates, Elsinoe fawcettii R-34 and MUD of citrus scab fungus to hygromycin resistant by transferring plasmides (pUCATPH) that contain hygB gene. We produced protoplast for transformation by using of combinations of available enzymes including ${\beta}$-D-glucanase, ${\beta}$ -glucuronidase, Iyticase and driselase. The protoplasts regenerated at 64 $\mu\textrm{g}$/ml of hygromycin B but not 128 $\mu\textrm{g}$ in sensitivity test to identify the concentration of useful marker for the selection of transformants. Approximately 1200 and 67 hygromycin resistant isolates from strain R-34 and strain MUD, respectively, were isolated on PDA added with 200 $\mu\textrm{g}$ /ml of hygromycun B. Fifty seven and 4 of hygromycin resistant isolates from strain R-34 and MUD, respectively, did not produce necrotic lesions on the leaf in detached-leaf assay. Finally, 9 isolates were isolated from strain R-34, and these Isolates produced non or very few symptoms on seedlings of citrus in greenhouse pathogenicity test. And it's very interesting that some isolates produced melanose-like symptom on very young leaves which it was not typical symptom and somtimes produced on only expanded leaf.