Biological challenge treatments
We administered two biological challenge treatments and one control
treatment. The first treatment involved attaching bark beetle pheromones
(Dendroctonus brevicomis lures; Synergy Semiochemicals Corp.
Burnaby, BC CAN) on selected trees at 1.5 m height on June 2, 2014 (app.
one year after trenching). We baited 16 trees, with one tree in each of
the trenched and untrenched treatments across eight blocks. We monitored
baited trees twice weekly and considered trees with pitch tubes (small
bark beetle boring holes filled with boring dust, frass, and resin) as
attacked. We counted all beetle attacks on each tree up to 3.5 m height.
Once the number of attacks on a tree reached 150, we removed baits from
that tree. Overall, the mean number of attacks per baited tree was 90±9
m2 and did not vary between trenched and untrenched
trees (P=0.161).
The second treatment consisted of crushing beetles, in a 3:1 ratio ofD. brevicomis to D. frontalis , onto the phloem/xylem
interface to inoculate trees with the full complement of microbial
biota, primarily fungi but also including bacteria, mites, and nematodes
(Hofstetter et al. 2015), associated with D. brevicomis ,D. frontalis , and D. valens , the most dominant bark beetle
species affecting pine forests of the study region (Gaylord et
al. 2006). We captured beetles in traps baited with D.
brevicomis lure near the study site and stored beetles at -10°C until
use. We inoculated selected trees by chorusing recently captured bark
beetles into the phloem/xylem interface. Locations and densities of
inoculations mimic the location and number of attacks on a paired tree
in the attacked category within the same stress treatment and block. To
inoculate trees, we removed a plug of bark (4 mm dia.) and inserted one
beetle into the resulting cavity and crushed the beetle into the
phloem/xylem interface by replacing the bark plug. We conducted all
inoculations on the same day that attacks occurred on the attacked trees
within the same block (e.g., twice weekly). We inoculated a total of 16
trees, with one tree in each of the trenched and untrenched treatments,
across eight blocks from June 4 to August 5, 2014.
The control treatment had no manipulations. Furthermore, we took steps
to prevent bark beetle attacks on trees in the non-attacked treatments
(control, inoculated) by spraying the entire bole with 2.0% carbaryl on
May 17, 2014 (DeGomez et al. 2006). The carbaryl spray was
effective as we only observed a total of six attacks in 32 trees, and
these were unsuccessful (lack of pitch tubes, boring dust, and frass).
Every two weeks during the warm months (May to August) in 2014 and 2015,
we determined tree status as dead or alive; a tree was categorized as
dead when over 90% of its canopy turned brown or red.