Slow soil enzyme recovery following invasive tree removal through gradual changes in bacterial and fungal communities

Biological invasions of plants have profound effects on ecosystem functioning by directly and indirectly altering soil microbiota, especially when invasive plants co-invade with their associated microbiomes. Ecosystem functions may recover slowly following invader removal, with implications for restoration.
We investigated the recovery of soil ecosystem function (measured as soil enzymes) following the removal, at different densities and times, of invasive Pinus spp. in New Zealand, and how different enzymatic activities responded to pine legacies.
Enzymatic activities were driven by pine legacies via both abiotic (soil nutrients) and biotic (fungi and bacteria) soil properties, with different enzymes showing distinct patterns. The activity of the enzymes cellobiohydrolase (cellulose degrading), β-glucosidase (cellulose degrading), N-acetyl-glucosaminidase (chitin degrading), laccase (lignin oxidising) and acid phosphatase (organic phosphate hydrolysing) were influenced by time since ..., At each site (22 sites in total), 5 soil samples were collected. From each sample, DNA was extracted for amplicon sequencing of fungal and bacterial communities. Soil nutrient concentrations and enzyme activities were also measured. Specific methodology is described below and in the manuscript.
DNA extraction
DNA was extracted from all soil samples using a DNeasyPowerSoil Pro Kit (Qiagen, Hilden, Germany) as per the manufacturer’s protocol. The fungal amplicon library was prepared in a one-step PCR using fungal specific primers fITS7 (5’-GTG ART CAT CGA ATC TTT G -3’) and ITS4 (5’-TCC TCC GCT TAT TGA TAT GC-3’) which amplify the ITS2 region (universal genetic barcode for fungi; Ihrmark et al. 2012). The ITS4 reverse primer included both Illumina adapted and index sequences (for identification of sequenced amplicons) and the fITS7 primer included Illumina adaptor sequences. The bacterial amplicon library was prepared in a one-step PCR using bacterial specific primers 515F (5’- GTG YCA G..., All analyses were conducted in R version 4.1.0 (R Core Team 2021) as described in the manuscript.