Vicente González

Design of a primer for ribosomal DNA internal transcribed spacer with enhanced specificity for ascomycetes

A primer able to amplify the internal transcribed spacers (ITS) of the ribosomal DNA (rDNA), having enhanced specificity for ascomycetes, was identified by reviewing fungal ribosomal DNA sequences deposited in GenBank. The specificity of the primer, named ITS4A, was tested with DNA extracted from several species of ascomycetes, basidiomycetes, zygomycetes, mastigomycetes and mitosporic fungi (formerly deuteromycetes) and also from plants. The PCR annealing temperature most specific for ascomycetes was found to be 62 degrees C and 64 degrees C for the primer pairs ITS5 + ITS4A and ITS1F + ITS4A, respectively. At these annealing temperatures, all ascomycetous DNA samples were amplified efficiently with the ITS4A primer. The sensitivity limit was in the range 10(-14) g of DNA. This primer could also provide useful tools in suggesting the affinities of many mitosporic fungi with their perfect states.

The endophytic mycota associated with Vitis vinifera in central Spain

This study investigates the diversity of fungal endophytes associated with several varieties of grapevines with differing modes of cultivation in the Madrid region (central Spain). Our goal is to screen for and identify new fungal biocontrol agents against vine diseases, especially those associated with young plants produced in nurseries. A total of 500 fungal strains representing 68 taxa from six locations were isolated and characterised. Differences regarding cultivar and plant part processed were analysed in terms of composition and relative abundance of species. Some of the more frequently isolated strains represented were Acremonium, Alternaria, Aureobasidium, Botryotinia, Cladosporium, Epicoccum, Fusarium, Gibberella, Nectria, Penicillium, Phoma and Trichoderma species. Botryosphaeria species and Phomopsis viticola were also frequently isolated and may be vine pathogens. Several species of Acremonium, Phoma (P. glomerata) and Chaetomium showed promising antagonistic activity at the laboratory scale.

Molecular phylogenetic studies on the diatrypaceae based on rDNA-ITS sequences

The order Diatrypales (Ascomycota) contains one single family, the Diatrypaceae. To obtain insight in the phylogenetic relationships within this family, the complete sequences of the ITS region (ITS1, 5.8S rRNA gene and ITS2) of 53 isolates from the five main genera in the family (Diatrype, Diatrypella, Cryptosphaeria, Eutypa and Eutypella) were determined and aligned for phylogenetic reconstruction. Sequence analysis revealed the presence of tandem repeated motifs 11 nucleotides-long, placed in homologous positions along the ITS1 region. Parsimony analysis established the existence of nine monophyletic groups and one branch with a single isolate of Eutypella quaternata. The phylogenetic relationships established by parsimony analysis did not correlate well with classical taxonomic schemes. None of the five genera included in this study was found to be monophyletic. The genera Diatrype, Eutypa and Cryptosphaeria each were divided into two groups. Isolates of Diatrype flavovirens appeared in a clade separated from the one that grouped Diatrype disciformis and the rest of Diatrype species. The Eutypa strains appeared distributed into two clades, one grouping Eutypa lata and related species (Eutypa armeniacae, Eutypa laevata, Eutypa petrakii), and another with the remaining species of the genus. Eutypella (excluding Eutypella quaternaria) appeared as an unstable monophyletic group, which was lost when the sequence alignment was subjected to neighbor-joining analysis. The genus Diatrypella was not associated with any monophyletic group, suggesting that the multisporate asci character has appeared several times during the evolution of the group. Overall, our study suggests the need to revise many of the concepts usually applied to the classification of members of the family.