Experimental approach
The study was conducted in a naturally regenerated ponderosa pine-dominated stand at the Northern Arizona University’s Centennial Forest (35° 11′ 9.65” N, 111° 45′ 38.25” W; elevation 2,250 m). We administered two levels of drought stress and two types of biological challenge treatments. Drought stress was administered by cutting tree roots beneath the drip line (hereafter trenched or untrenched). The biological treatments were (1) bark beetle attacks (hereafter attacked), (2) crushing beetles, including their microbial associates, onto the phloem/xylem interface (hereafter inoculated), and (3) controls which had neither bark beetle attacks nor inoculations.
The experiment was a randomized complete block with eight spatial blocks within the stand. In each of the eight 1,300 m2blocks, six trees were randomly assigned to one of the following six treatments (n=8 per treatment across eight blocks): (1) trenched-control, (2) trenched-inoculated, (3) trenched-attacked, (4) untrenched-control, (5) untrenched-inoculated, and (6) untrenched-attacked. The trees were approximately 60 years of old, with a mean height of 8.0±0.12 m (range 5.2-11.5 m) and a mean diameter at breast height of 24.2±0.5 cm (range 20.0-31.9 cm). Neither height nor diameter of trees varied among treatments (height: P=0.464; diameter: P=0.133).
Detailed descriptions of field experimental methods were provided in Kolb et al. (2019) which reported results for resin flow, monoterpenes and sesquiterpenes in the phloem, xylem water potential, and leaf gas exchange. In the current study, we focused on monthly analyses of NSCs and diterpenes of xylem and thus only reported methods relevant to these analyses. We also used the total monoterpene data from Kolb et al . (2019) in our analysis.