Microbiome dynamics during rust fungal infections

Abstract

Plants don't exist naturally in isolation. They are surrounded by, colonised by, and interact with, microorganisms. Plant microbiomes have many reported benefits to their hosts, from directly suppressing pathogen infection to improving nutrient availability. While leveraging these benefits for agricultural benefit is of great research interest, we lack fundamental understanding of the composition and diversity of plant microbiomes in non-model systems. Environmental DNA (eDNA) sequencing has opened new doors for profiling microbial communities, enabling molecular delineation of species with morphological similarities, or for which pure in vitro culture is not possible. Metabarcoding, using the 16S region for bacteria, or the internal transcribed spacer (ITS) region for fungi, can increase sample throughput, and identify many organisms simultaneously from a single sample. Using long-read sequencing to encompass entire metabarcode sequences can give insight at the species level, but this technology has not been thoroughly benchmarked for applications in microbial metabarcoding. In this thesis, I benchmark and optimise a wet lab and bioinformatics workflow for using Oxford Nanopore Technologies long-read sequencing to profile microbial communities with metabarcodes. I present a novel multiplexing strategy to improve cost-effectiveness, and use an in silico mock community to demonstrate robust, species-level identification of fungi using long-read ITS sequences. I have then applied my knowledge from this benchmarking work to two leaf microbiome datasets: a sample set of rust fungus-infected wild grasses, and a sample set encompassing three spore stages of the wheat stripe rust fungus (Puccinia striiformis f. sp. tritici) across the sexual and asexual phases of its life cycle. I compare fungal and bacterial community composition and abundance across levels of infection status and host plant, to better understand how rust fungal infection influences microbiome community dynamics in the phyllosphere.