Validation and application of a metagenomics protocol to the root-associated fungal communities of Northern wild rice (Zizania palustris)

Abstract

The occurrence of root-associated fungi in wetland plants is not yet well-understood, despite recent indications that some contain diverse mycobiomes, including potential plant mutualists. Although arbuscular mycorrhizal fungi (AMF) can be neutral and even parasitic, emerging evidence suggests that AMF provide benefits to many wetland plants, as in terrestrial soils. Dark septate endophytes (DSEs) occur in wetland plants at similar or greater frequencies than AMF and may also confer benefits to host plants. Zizania palustris (Northern wild rice, manômin) is an emergent annual macrophyte found in shallow lakes and slow-moving rivers in central and eastern North America, where it is harvested from both wild stands and cultivated paddies. Despite the significance of this plant to Indigenous peoples and emerging agricultural interests, its fungal diversity in traditional rice lakes or wild stands is currently unknown. To identify the root-associated fungal communities of Z. palustris collected from wild stands across Manitoba and determine its mycorrhizal status, I undertook ITS and 18S targeted amplicon sequencing of the rhizosphere soil and determined the incidence of fungal structures within roots via microscopy. Since wetland soils contain contaminants that inhibit metabarcoding, I optimized DNA extraction and amplification protocols prior to their application to these samples. The ITS data suggested that the rhizosphere is primarily basidiomycotan and ascomycotan. The 18S data identified three genera of AMF available in the surrounding rhizosphere for association with Z. palustris: Glomus, Acaulospora, and Claroideoglomus. The root incidence data showed consistent fungal colonization by aseptate and dematiaceous hyphae, with somewhat lower incidence of hyaline hyphae and AMF-like structures, despite complete inundation of all plants. While confirmation of Z. palustris as a mycorrhizal plant requires sequencing of the root-endogenous fungi, this thesis presents preliminary data confirming the presence of AMF in the rhizosphere and AMF-resembling structures within the roots.