Molecular characterisation of Phaeomoniella chlamydospora isolated from grapevines in Castilla y León (Spain)

One of the major causal organisms of grapevine decline is the plant pathogenic fungus Phaeomoniella chlamydospora. Sequencing of the internal transcribed spacer (ITS) regions has been useful to classify this species. So far the fungus has shown only slight genetic variation. The aim of the present work was to determine the degree of genetic variation of Pa. chlamydospora isolates mainly from grapevines grown in Castilla y León, Spain. Thirty-five isolates of Pa. chlamydospora were subjected to PCR in order to amplify the nuclear 5.8S ribosomal RNA gene and its flanking ITS regions (529 bp fragment), the 5’ end of the b-tubulin gene (550 bp fragment) and the partial 5’end of the translation elongation factor 1-a gene (337 bp fragment). Nine different RAPD patterns were obtained, two of which were much more common than the others. Despite the great number of RAPD primers screened, only a few reproducible polymorphic fragments were obtained. The 35 isolates fell into two groups depending on two bases at nucleotides 389 and 438 of the ITS4-ITS5 fragment. Sequences of the 5’ end of the b-tubulin gene were 100% homologous across all 35 isolates. The 337 bp fragment of the elongation factor 1-a was also 100% homologous across the isolates. These results confirmed the low genetic variation shown by Pa. chlamydospora. However, RAPD pattern H was typical of aging grapevines. When two or three isolates were obtained from a single vine, two different Pa.chlamydospora genotypes were sometimes found on the same vine