In forest ecosystems, habitat fragmentation negatively impacts stand structure and biodiversity; the resulting fragmented patches of forest have distinct, disturbed edge habitats that experience different environmental conditions than the interiors of the fragments. In southwest Western Australia, there is a large-scale decline of the keystone tree species Corymbia calophylla following fragmentation and land use change. These changes have altered stand structure and increased their susceptibility to an endemic fungal pathogen, Quambalaria coyrecup, which causes chronic canker disease especially along disturbed forest habitats. However, the impacts of fragmentation on belowground processes in this system are not well understood. We examined the effects of fragmentation on abiotic soil properties and ectomycorrhizal (ECM) and arbuscular mycorrhizal fungal (AMF) communities, and whether these belowground changes were drivers of disease incidence. We collected soil from 17 sites across the distribution range of C. calophylla. Soils were collected across a gradient from disturbed, diseased areas to undisturbed, disease-free areas. We analysed soil nutrients and grew C. calophylla plants as a bioassay host. Seedlings were harvested and roots collected after six months of growth. DNA was extracted from the roots, amplified using fungal specific primers and sequenced using Illumina MiSeq. Concentrations of key soil nutrients such as nitrogen, phosphorus and potassium were much higher along the disturbed, diseased edges in comparison to undisturbed areas. Disturbance altered the community composition of ECM and AMF; however, only ECM communities had lower rarefied richness and diversity along the disturbed, diseased areas compared to undisturbed areas. Accounting for effects of disturbance, ECM diversity and leaf litter depth were highly correlated with increased disease incidence in C. calophylla. In the face of global change, increased virulence of an endemic pathogen has emerged in this Mediterranean-type forest.

Fifty-one soil samples were collected across seventeen sites in southwest Western Australia. Soils were used in a bioassy experiment with Corymbia calophylla as the host. Seedlings were harvested after six months of growth and the roots were rinsed and soil and stripped of fine roots. DNA was extracted from 50mg of fine roots using DNeasy PowerPlant Pro kit as per manufacturer's protocol. Amplicon library preparation was preformed using the Illumina Demonstrated Protocol with exceptions. eDNA was amplified using fungal specific primers fITS7 and ITS4 which amplifies the ITS2 region, the universal genetic barcode for fungi. Uniquely indexed libraries (265) were pooled for the sequencing run preformed on an Illumina MiSeq using 600-cycle V3 chemistry.

Paired-end reads were merged using USEARCH v8.0.1623 with a minimum overlap of 50bp with no gaps allowed in the merged alignments. Sequence deconvolution was carried out using the bioinformatics platform Sequence Clustering and Analysis of Tagged Amplicons (SCATA;  scata.mykopat.slu.se), developed and maintained by the Swedish University of Agricultural Sciences in Uppsala. Reads with low mean quality (<20) and sequences with missing primers were removed. Primer sequences were removed and sequences passing quality control were then clustered into operational taxonomic units (OTUs). The threshold distance for clustering was set at 0.015 (corresponding approximately to species level for ITS) and minimum alignment for clusters (i.e., minimum length of pairwise alignment in the clustering process required to consider a sequence pair for clustering) was 0.85.

Sample IDs, OTU table (produced through SCATA) and sample information are included in the attached SCATAResults.xlsx.