Molecular divergence of fungal communities in soil, roots and hyphae highlight the importance of sampling strategies

Molecular ecology studies of fungi in roots and soils involve sampling DNA from heterogeneous environments. There is a wide range of fungal life strategies, including modes of sporulation, and interactions some fungi may have with roots of different plant species. Therefore, assessment of the relative abundance of fungi in these complex environments needs to take into account fungal life cycles, including sporulation, as well as potential interactions with roots, whether they are specific or non-specific. Such heterogeneity means that a significant factor in quantification of fungi from environmental samples based on DNA sequences is the sampling strategy employed. Therefore, we investigated fungal communities associated with soil, roots, and hyphae extracted from soil in a relatively homogeneous agricultural soil in which Pennisetum clandestinum (Whittet Kikuyu) was grown as a monoculture in a glasshouse experiment with the naturally occurring fungal community. Pennisetum clandestinum occurs in pasture monocultures in south-western Australia and this model was used to minimise potential variability in fungal occurrence associated with multiple plant species. The community structures of the fungi overall, and that of the subset of arbuscular mycorrhizal (AM) fungi, were assessed using ITS rRNA sequencing. For fungi overall, there were marked differences in the relative abundance of the dominant phyla Chytridiomycota and Zygomycota in soil, roots, and hyphae from soil. For AM fungi (Glomeromycota), there was greater diversity within soil and among hyphae from soil than in roots, with marked differences in relative abundance of two orders, Archeosporales and Diversisporales. Roots were dominated by Glomus spp. Dark septate endophyte hyphae were dominant in roots but not in soil according to DNA sequencing, although they were not dominant in roots based on microscopy. Sporulation could influence fungal dominance in fungal DNA analyses, and this may be the case for the dark septate hyphae observed here. The considerable diversity in fungal communities based on DNA sequences from the relatively homogeneous environmental samples investigated in our study highlights the need for appropriate sampling and interpretation based on fungal life strategies, especially in more heterogeneous environmental samples of roots and soil under field conditions. There is potential for multiple DNA copies of fungal sequences associated with spores either inside roots or in soil, in addition to heterogeneity associated with growth of fungal hyphae in roots and in the rhizosphere of different plant species.