Parasites can affect and be affected by the host's microbiome, with consequences for host susceptibility, parasite transmission, and host and parasite fitness. Yet, two aspects of the relationship between parasite infection and host microbiota remain little understood: the nature of the relationship under field conditions, and how the relationship varies among parasites. To overcome these limitations, we performed a field survey of the within-leaf fungal community in a tall fescue population. We investigated how diversity and composition of the fungal microbiome associate with natural infection by fungal parasites with different feeding strategies. A parasite's feeding strategy affects both parasite requirements of the host environment and parasite impacts on the host environment. We hypothesized that parasites that more strongly modify niches available within a host will be associated with greater changes in microbiome diversity and composition. Parasites with a feeding strategy that creates necrotic tissue to extract resources (necrotrophs) may not only have different niche requirements, but also act as particularly strong niche modifiers. Barcoded amplicon sequencing of the fungal ITS region revealed that leaf segments symptomatic of necrotrophs had lower fungal diversity and distinct composition compared to segments that were asymptomatic or symptomatic of other parasites. There were no clear differences in fungal diversity or composition between leaf segments that were asymptomatic and segments symptomatic of other parasite feeding strategies. Our results motivate future experimental work to test how the relationship between the microbiome and parasite infection is impacted by parasite feeding strategy and highlight the potential importance of parasite traits.

Leaf segments were collected in a grass-dominated field within the Blackwood Division of the Due Forest Teaching and Research Laboratory. Leaf segments were collected at a total of 36 sites across an old field and processed within four hours back in the laboratory. Leaves were washing under running DI water for 30 seconds to remove fungi that were on the surface of the leaf but not attached to the leaf. Following surface washing, leaf segments were stored in a -80C freezer. Leaf segments were ground under liquid nitrogen with a mortar and pestle and transferred to 96-well lates for DNA extraction. DNA extraction was performed with the DNEeast PowerSoil kit according to the manufacturer's protocol. We amplified the first part of the internal transcribed spacer (ITS1) with a version of the primer set ITS1F and ITS2 modified for parallel sequenceing on the Illumina MiSeq platform. We cleaned samples with AMPure beads, concentration-normalized, and pooled samples in one run on an Illumina MiSeq instrument using a paired-end 2 x 250 bp kit. A spike of 10% PhiX was added to the library to increase sample heterogeneity.