Phylogenetic and chemical diversity of fungal endophytes isolated fromSilybum marianum(L) Gaertn. (milk thistle)

Abstract

Use of the herb milk thistle (Silybum marianum) is widespread, and its chemistry has been studied for over 50 years. However, milk thistle endophytes have not been studied previously for their fungal and chemical diversity. We examined the fungal endophytes inhabiting this medicinal herb to determine: (1) species composition and phylogenetic diversity of fungal endophytes; (2) chemical diversity of secondary metabolites produced by these organisms; and (3) cytotoxicity of the pure compounds against the human prostate carcinoma (PC-3) cell line. Forty-one fungal isolates were identified from milk thistle comprising 25 operational taxonomic units based on BLAST search via GenBank using published authentic sequences from nuclear ribosomal internal transcribed spacer sequence data. Maximum likelihood analyses of partial 28S rRNA gene showed that these endophytes had phylogenetic affinities to four major classes of Ascomycota, the Dothideomycetes, Sordariomycetes, Eurotiomycetes, and Leotiomycetes. Chemical studies of solid–substrate fermentation cultures led to the isolation of four new natural products. In addition, 58 known secondary metabolites, representing diverse biosynthetic classes, were isolated and characterized using a suite of nuclear magnetic resonance and mass spectrometry techniques. Selected pure compounds were tested against the PC-3 cell line, where six compounds displayed cytotoxicity.

Biscogniauxone, a New Isopyrrolonaphthoquinone Compound from the Fungus Biscogniauxia mediterranea Isolated from Deep-Sea Sediments

“…Two new azaphilone derivatives, biscogniazaphilones A and B, with antimycobacterial activity were produced by Biscogniauxia formosana together with nine known compounds [16]. In addition, two new substances, biscognin A and B, were found in extracts of B. mediterranea [17]. The new guaiane sesquiterpene xylaranone and the known terpenoid xylaranol B (together with mellein derivatives) were isolated from Biscogniauxia nummularia .…”

Antimicrobial fungal endophytes from the botanical medicine goldenseal ( Hydrastis canadensis )

“…Fresh plant samples were surface sterilized as described previously (Raja et al, 2015). A total of 320 segments were plated, which included 100 stem segments; 100 leaf segments; 70 root segments; and 50 seeds.…”

“…All fungal endophyte cultures that emerged from goldenseal plant parts are maintained on Potato Dextrose Agar; Difco (PDA) agar slants at 9 C at the University of North Carolina at Greensboro, Department of Chemistry and Biochemistry Fungal Culture Collection. A total of 23 fungi were cultured from tissues of H. canadensis (collected on July 11, 2011) and were grown on a solid-state rice fermentation medium, as previously described (Raja et al, 2015). All rice cultures were allowed to grow for 14–21 days prior to chemical extraction.…”

Nomenclatural and identification pitfalls of endophytic mycota based on DNA sequence analyses of ribosomal and protein genes phylogenetic markers: A taxonomic dead end?

“…In addition, we also noted that limited or inappropriate sampling as well as the poorly resolved phylogenetic relationships makes it difficult to confidently establish an endophyte in a specific genus or species or even family. This taxonomic scenario is also common to many other endophytes and uncultured environmental samples, and even a number of them have been referred to as uncultured Pleosporales (Herrera et al 2011).Of particular interest in this study are the ancestral relationships of this endophyte to other bitunicates fungi which are poorly documented (Slippers & Wingfield 2007, Tao et al 2008,Sunayana et al 2014, Raja et al 2015, Cosoveanu et al 2016). …”

“…Despite having recovered some morphological characters from cultures and analysed nine different gene datasets here, we still refrain from naming or associate the isolate to any known species. It is common practice that phylogeneticists perform a blast search, rely on sequence similarity and capitalise on gene trees (especially on ribosomal DNA sequences) for species identity (e.g Sunayana et al 2014, Raja et al 2015. This can be quite erroneous as not all sequences from known species have been deposited in GenBank or some species could have been wrongly named.…”