We established 80 experimental ecosystems (mesocosms), manipulated interactions between plants and soil biota in a fully factorial design. Each mesocosm was grown in a 125 L pot (575 mm diameter), and comprised one of 20 unique, eight-species plant communities varying orthogonally in the proportion of exotic and woody shrub/tree species (0-100% and 0-63%, respectively). These plants were taken from a pool of 20 exotic and 19 native/endemic New Zealand plant species. Soil biota were manipulated using a modified plant-soil feedback approach, where each plant species was grown in monoculture in 10 L pots containing field-collected soil for 9-10 months, allowing the conditioning of typical associated soil biota for each of the plant species. We created ‘home’ soils by taking the conditioned soil from each of the eight representative species in a mesocosm and mixing it together to create a single inoculum. Each ‘home’ soil mixture was also used as an ‘away soil’ in a different mesocosm that did not contain any of the representative plants in that inoculum. These soils were intended to increase the relative biomass in inocula of specialized and preferred interaction partners of the resident (or non-resident) plant species. After approximately one year of growth, we harvested all plants from each mesocosm, took root samples from each individual plant (n=491), extracted DNA and sequenced the fungi in the roots. Fungal sequences were paired and clustered into operational taxonomic units (OTUs) at 97% similarity. We assigned functional attributes to fungal OTUs using the FUNGUILD database and retained only the taxa assigned as “probable” or “highly probable” plant pathogens.

DNA was extracted from a total of 491 root samples harvested from individual plants from all 80 mesocosms at the end of the experiment, using MoBio PowerSoil (QIAGEN) extraction kits. Root samples were taken from each individual plant using a sterile razor blade, cutting approximately 10-15 fine-root fragments from random places on the washed root ball, then bundling the roots together and slicing a 0.5-1.0 cm cross section piece from the bundle. Root samples were immediately placed into a 96 well plate from the extraction kit and frozen at -80^oC as soon as a plate was full. 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. 

PCR was run on each sample and controls using the following thermal cycler conditions: one cycle of initial denaturation at 94°C for 5 mins; 35 cycles of denaturation at 94°C for 30 secs; annealing at 57°C for 30 secs; extension at 72°C for 30 secs; final extension at 72°C for 7 min. PCR products were checked for adequate intensity single bands on a 1% agarose gel with RedSafe. PCR reactions were carried out in duplicates for each sample and pooled prior to library preparation using the 96 well SequalPrep Normalization Kit (Invitrogen). Once PCR products were cleaned and normalized with the SequalPrep Kit, all samples were pooled and sent to Massey Genome Services, (Palmerston North, New Zealand) to be sequenced. Amplicons were sequenced on an Illumina MiSeq analyzer using the 600-cycle Reagent Kit V3, delivering 2 X 300 base pair reads/sequence (Illumina, San Diego, California, USA).

Sequences were paired, putative chimeras removed, and clustered into operational taxonomic units (OTUs) at 97% sequence similarity using Vsearch. Quality and barcode filtering resulted in 6,093,371 reads with a median length of 225 bases.

We assigned functional attributes to OTUs using the FUNGuild database and retained only the taxa assigned as “probable” or “highly probable” plant pathogens for subsequent analyses. We restricted our inclusion of taxa to those that receive most 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 are primarily saprotrophs and only occasionally pathogens. 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 (hereafter referred to simply as ”pathogens”), as we rely on commonly accepted life history descriptions rather than performing real-time functional assays for each plant-fungal interaction.