Molecular phylogeny and paclitaxel screening of fungal endophytes from Taxus globosa.

We studied the endophytic mycoflora associated with Taxus globosa, the Mexican yew. The study localities; Las Avispas (LA), San Gaspar (SG), and La Mina (LM) were three segments of cloud forest within the range of Sierra Gorda Biosphere Reserve, México. Overall, 245 endophytes were isolated and 105 representative Ascomycota (morphotaxons) were chosen for phylogenetic and genotypic characterization. Maximum likelihood analyses of large subunit of ribosomal RNA (LSU) rDNA showed well-supported clades of Dothideomycetes, Eurotiomycetes, Leotiomycetes, Pezizomycetes, and Sordariomycetes. Analyses of ITS rDNA groups showed 57 genotypes (95% sequence similarity), in general consistent with the phylogenetically delimitated taxa based on LSU rDNA sequences. The endophyte diversity measured by Fisher's α, Shanonn, and Simpson indices was ca. three-fold and ca. two-fold greater in LM than in LA and SG respectively. A screening for paclitaxel using a competitive inhibition enzyme immunoassay showed 16 positive isolates producing between 65 and 250 ng l(-1). The isolates included Acremonium, Botryosphaeria, Fusarium, Gyromitra, Nigrospora, Penicillium, three novel Pleosporales, and Xylaria.

Stable genetic transformation of the ectomycorrhizal fungus Pisolithus tinctorius.

In the present work the genetic transformation and the expression of gene markers in transgenic Pisolithus tinctorius are reported. The ectomycorrhizae are facultative symbionts of plant roots, which are capable of affording mineral nutrients to its co-host in exchange of fixed carbon. Given the importance of this association (more than 80% of gymnosperms are associated with these fungi), its study from both basic and applied viewpoints is relevant. We have transformed this fungus with reporter genes and analyzed their expression in its saprophytic state. Genetic transformation was performed by microprojectile bombardment and Agrobacterium-mediated transformation. This last method proved to be the more efficient. Southern analysis of biolistic-transformed fungi revealed the random integration of the transgene into the genome. The accumulation of the transcript of the reporter gene was demonstrated by RT-PCR. The visualization of GFP-associated fluorescence in saprophytic mycelia confirmed the expression of the reporter gene. This is the first report on the stable transformation and expression of GFP in the ectomycorrhizal fungus P. tinctorius.