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.