Methods

Isolates of MPB-associated fungi (G. clavigera (Gc) and O. montium (Om)) were exposed to artificial amendments mimicking lodgepole pine chemistries of Oregon (OR), British Columbia (BC), and Alberta (AB). I used a full-factorial design comprising of five amendments (three lodgepole pine chemistries, one ethanol-only control, and one no-amendment control) crossed with three fungal cultures (two single-species inoculations and one mixed-species inoculation), resulting in 15 treatment combinations. Each treatment was replicated 10 times (Fig S1).

Figure S1. Full-factorial design.

Monoterpene amendments

Monoterpene laboratory standards were dissolved in ethanol in proportions reflecting the average phloem concentrations across lodgepole pine trees from each location. An ethanol-only control was included to assess any baseline effects of ethanol on fungal growth. A no-amendment control was also included to determine whether fungal growth differed from the ethanol treatment.

See Table S1 and S2 for detailed monoterpene phloem concentrations and volumes for artificial amendments.  

Experimental setup

The following procedure was derived from Cale et al. (2019). Two fungal inoculates were added to monoterpene-amended liquid media. The flasks were sealed and incubated in darkness at 22 °C for 8 days (Fig S2). Fungal growth was quantified by filtering, freeze-drying, and weighing the mycelial biomass, reported in milligrams. Additionally, fungal volatiles were measured to assess the biochemical effects of monoterpenes. Volatiles were extracted from the post-growth liquid broth and analyzed using gas chromatography–mass spectrometry (GC-MS).

Figure S2. Experimental setup for measuring fungal growth in monoterpene-amended growth media. Potato dextrose broth (PDB) was used as growth media.

Statistical Analysis

All statistical analysis and graphic visuals in this study were performed in Rstudio (v. 5.1.513 (R Core Team, 2025, RStudio: Integrated Development Environment for R; Boston, MA, USA) under macOS. Because the raw data were not normally distributed (see Data section), differences in fungal biomass and volatile concentrations among treatment groups were assessed using Kruskal–Wallis tests, followed by Dunn’s post-hoc tests for pairwise comparisons. An alpha level of 0.05 was used to evaluate statistical significance.