Molecular sequencing and bioinformatics
DNA was extracted from a total of 491 roots harvested from individual plants from all 80 mesocosms at the end of the experiment, using MoBio PowerSoil extraction kits. We characterized the fungal communities by amplifying the internal transcribed spacer (ITS) of the ribosomal RNA (rRNA) operon using polymerase chain reaction (PCR) with the barcoded primers fITS7/ITS4 (Ihrmark et al. 2012) (PCR conditions are described in Waller et al. 2020). Amplicons were sequenced on an Illumina MiSeq analyzer using the 600-cycle Reagent Kit V3, delivering 2 X 300 base pair reads/sequence.
Sequences were paired, putative chimeras removed, and clustered into operational taxonomic units (OTUs) at 97% sequence similarity using Vsearch (Rognes et al. 2016). Quality and barcode filtering resulted in 6,093,371 reads with a median length of 225 bases.
We assigned functional attributes to OTUs using FUNGuild (Nguyenet al. 2016) and retained only the taxa assigned as “probable” or “highly probable” plant pathogens for subsequent analyses (details about taxa from FUNGuild are listed in Table S2). We restricted our inclusion of taxa to those that receive the majority of their nutrients by harming host cells (defined as “pathotrophs” by FUNGuild), and excluded taxa with mixed strategies from our analyses (i.e. “pathotroph-saprotroph”), as many of these taxa receive most of their nutrients by breaking down dead host cells. We acknowledge that by limiting our pathogen assignment in this way we have likely excluded many taxa that may be pathogens in some environments, so our results represent a conservative analysis of the pathogen communities hosted by our plants. Moreover, the taxa listed here are putative pathogens (herein referred to simply as ”pathogens”), as we rely on commonly accepted life history descriptions rather than performing real-time functional assays on each taxon.