Fungal endophytes in potato roots studied by traditional isolation and cultivation-independent DNA-based methods

The composition and relative abundance of endophytic fungi in roots of field-grown transgenic T4-lysozyme producing potatoes and the parental line were assessed by classical isolation from root segments and cultivation-independent techniques to test the hypothesis that endophytic fungi are affected by T4-lysozyme. Fungi were isolated from the majority of root segments of both lines and at least 63 morphological groups were obtained with Verticillium dahliae, Cylindrocarpon destructans, Colletotrichum coccodes and Plectosporium tabacinum as the most frequently isolated species. Dominant bands in the fungal fingerprints obtained by denaturing gradient gel electrophoresis analysis of 18S rRNA gene fragments amplified from total community DNA corresponded to the electrophoretic mobility of the 18S rRNA gene fragments of the three most abundant fungal isolates, V. dahliae, C. destructans and Col. coccodes, but not to P. tabacinum. The assignment of the bands to these isolates was confirmed for V. dahliae and Col. coccodes by sequencing of clones. Verticillium dahliae was the most abundant endophytic fungus in the roots of healthy potato plants. Differences in the relative abundance of endophytic fungi colonizing the roots of T4-lysozyme producing potatoes and the parental line could be detected by both methods.