Geneticin (G418) resistance and electroporation-mediated transformation of Fusarium graminearum and F. culmorum

Abstract

Fusarium graminearum and F. culmorum are phytopathogenic species causing scab and root rot diseases in all small grain cereals worldwide including Turkey. In this study, resistance levels to geneticin (G418) of 14 F. graminearum and 24 F. culmorum isolates collected from cereals were determined. Fungal cultures were grown on potato dextrose agar medium supplemented with 0, 25, 50, 75 and 100 mu g/mL of G418. Minimum inhibitory concentration was determined as 25 mu g/mL. As a result, it was concluded that all isolates were highly sensitive to G418. Plasmid pFA6-kanmx4 containing geneticin resistance gene (kanmx) was introduced singly or co-electroporated with pEGFP75 plasmid, containing GFP gene, into fungal protoplast cultures obtained with lytic enzyme. Transformants were grown in media including 25 mu g/mL G418. Transformation frequencies were 2.8 and 1.8 transformant per mu g plasmid for F. graminearum and F. culmorum isolates, respectively. Transformation process was also confirmed by spectrofluorimetric assay. Relative fluorescence unit values in co-transformants were calculated as 1.87 +/- 0.04 for F. graminearum and 2.26 +/- 0.08 for F. culmorum. The results obtained from the study gave information about antibiotic resistance levels of two Fusarium species in Turkey. Moreover, it was shown that pFA6-kanmx4 plasmid was a suitable vector, which can be used in genetic manipulation studies of these two fungal species in particular suppression of endogenous and/or the expression of exogenous genes.