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.