Identification of Rosellinia species as producers of cyclodepsipeptide PF1022 A and resurrection of the genus Dematophora as inferred from polythetic taxonomy.

Rosellinia (Xylariaceae) is a large, cosmopolitan genus comprising over 130 species that have been defined based mainly on the morphology of their sexual morphs. The genus comprises both lignicolous and saprotrophic species that are frequently isolated as endophytes from healthy host plants, and important plant pathogens. In order to evaluate the utility of molecular phylogeny and secondary metabolite profiling to achieve a better basis for their classification, a set of strains was selected for a multi-locus phylogeny inferred from a combination of the sequences of the internal transcribed spacer region (ITS), the large subunit (LSU) of the nuclear rDNA, beta-tubulin (TUB2) and the second largest subunit of the RNA polymerase II (RPB2). Concurrently, various strains were surveyed for production of secondary metabolites. Metabolite profiling relied on methods with high performance liquid chromatography with diode array and mass spectrometric detection (HPLC-DAD/MS) as well as preparative isolation of the major components after re-fermentation followed by structure elucidation using nuclear magnetic resonance (NMR) spectroscopy and high resolution mass spectrometry (HR-MS). Two new and nine known isopimarane diterpenoids were identified during our mycochemical studies of two selected Dematophora strains and the metabolites were tested for biological activity. In addition, the nematicidal cyclodepsipeptide PF1022 A was purified and identified from a culture of Rosellinia corticium, which is the first time that this endophyte-derived drug precursor has been identified unambiguously from an ascospore-derived isolate of a Rosellinia species. While the results of this first HPLC profiling were largely inconclusive regarding the utility of secondary metabolites as genus-specific chemotaxonomic markers, the phylogeny clearly showed that species featuring a dematophora-like asexual morph were included in a well-defined clade, for which the genus Dematophora is resurrected. Dematophora now comprises all previously known important plant pathogens in the genus such as D. arcuata, D. bunodes, D. necatrix and D. pepo, while Rosellinia s. str. comprises those species that are known to have a geniculosporium-like or nodulisporium-like asexual morph, or where the asexual morph remains unknown. The extensive morphological studies of L.E. Petrini served as a basis to transfer several further species from Rosellinia to Dematophora, based on the morphology of their asexual morphs. However, most species of Rosellinia and allies still need to be recollected in fresh state, cultured, and studied for their morphology and their phylogenetic affinities before the infrageneric relationships can be clarified.

Fungal endophytes from the leaves and twigs of Quercus ilex L. from England, Majorca and Switzerland.

Leaves of Quercus ilex taken from sites in England, Majorca and Switzerland have been studied to detect the influence of the geographic position of the host within and outside its native range on the composition of its endophytic fungal assemblages. Samples of stem tissue of Q. ilex collected from the Swiss trees were also studied to confirm tissue-specific differences. Sixty different fungal taxa were isolated, but only 28 were frequent. Of the total number of isolates from the leaves from the Swiss, British and Spanish sites 87%, 31% and 63%, respectively, were coelomycetes. Four species of Phomopsis, which includes Phyllosticta ilicina (=Phomopsis ilicina v. d. Aa, ined.), were the most frequent endophytes of leaves and were either absent or rare in the twig units. Two distinct kinds of sterile mycelia were common in twigs. Swiss and Spanish trees possessed fungal assemblages distinct from those present in Britain. Naturalized stands were distinguished from native stands by the presence of rather cosmopolitan and non-specific fungal taxa, rare or absent in the samples collected in the native stands. Samples derived from the native stands were colonized by more host-specific fungi. The relative frequency of two sterile mycelia in the Swiss and Spanish sites determined their separation. Phyllosticta (Phomopsis) ilicina, the most numerous leaf colonizer, was virtually absent from the bark and the xylem. The frequent occurrence of coelomycetes as endophytes of woody trees is briefly discussed.