Results
Overall, eight of the 48 total trees died during the two-year
experiment. Bark beetle attacks occurred over 10 weeks, starting early
June and ending in middle of August. Attacks peaked in late June and
declined through August. All eight of the dead trees were attacked by
bark beetles, with six of them being in the trenched-attacked treatment
and two in the untrenched-attacked treatment. Trees in the combined
treatment died sooner, with all of the mortality to trenched-attacked
trees occurring in 2014 and all of the mortality to untrenched-attacked
trees occurring in 2015.
As reported by Kolb et al . (2019), predawn water potential over
the summer of 2013 averaged -0.97 MPa for trenched trees and -0.83 MPa
for untrenched trees. In the second year of the experiment (2014),
predawn water potential was approximately 0.5 MPa more negative for
trenched trees than untrenched trees between late May and early July
during the typical spring drought period of the region (Fig. 1 in Kolbet al . 2019). Predawn water potential of trees in the
trenched-attacked trees became much more negative than trees in all
other treatments for the remainder of summer in 2014, reaching an
average value of -2.5 MPa by middle of September compared with -0.5 MPa
in other treatments. Differences in light saturated net photosynthetic
rate among treatments in 2014 paralleled differences in predawn water
potential, with values of trenched trees 14% lower than untrenched
trees in May (mean of 6.1 and 5.3 umol m−2s−1, respectively) and 90% lower in late June (mean
of 0.4 and 0.04 umolm−2 s−1,
respectively). Trenching continued to cause water stress during dry
periods in the third year of the experiment (2015), with values of
trenched trees 0.2 MPa more negative than untrenched trees in late June.
In general, chemical profiles of ponderosa pine trees changed with
treatment (Fig. 1, Fig. S1, Tables S1 & S2), year (PerManova:
F(1.313)=4.45, P=0.009) and month (PerManova:
F(8.313)=7.11, P<0.001). Therefore, we tested
whether the NCSs differed among treatments for each month and each year.
The results showed that with the exception of September 2015, chemical
profiles varied among treatments in the remaining sampling times (Fig.
1, Figs. S1 & S2, Table S3). Note that terpenes of trees challenged
with either bark beetle attacks or inoculated with crushed beetles
represent “induced” concentrations, whereas terpenes of trees without
any biological challenge treatments represent “constitutive”
concentrations. Thus, the results should not be applied to define the
question of whether terpene concentrations predict which trees live or
die.
Do treatments influence NSCs of ponderosa pine trees?Overall, results show the importance of cumulative stresses in tree NSCs
reduction in the xylem and mortality during drought as the effects of
trenching on NSCs were negligible in trees without any additional
biological treatment especially in the trenched-attacked treatment (Fig.
1, Table S2, Fig. S1). For instance, bark beetle attacks with trenching
significantly lowered the total NSCs of trees relative to the trenching
without such attacks (Fig. 1).
Effects of trenching on NSCs. Overall, chemical profiles did not
differ between trenched-control and untrenched-control trees (Fig 2a,
Table S4). We further tested if the NSCs were affected by trenching in
each month. The difference was only significant in October 2014 (Fig. 3,
Table S5) when starch, total sugars and total NSCs were higher in the
trenched-control than the untrenched control.
Effects of bark beetle attacks on NSCs. For sugars, starch and
total NSCs, treatments and month were significant, but treatment-month
interaction was only significant for starch (Fig. 2b, Table S4). We then
tested if NSCs were a function of beetle attacks in each month and found
that bark beetle attacks significantly lowered sugars, starch, and total
NSCs in July, August, September, October, November in 2014 and in May,
June, August in 2015 (Fig. 3, Table S5).
Effects of crushed beetle inoculations on NSCs . Although
treatment and month were significant for starch, total sugars, and total
NSCs, the interaction was not (Fig. 2c, Table S4). We then tested
whether NSCs were affected by inoculation in each month and found that
inoculation significantly reduced total sugars and total NSCs in July,
August, September, October in 2014 and in May, June, July, and August in
2015 and starch in July, August, and September in 2014 and in May, June,
July, and August in 2015 (Fig. 3, Table S5).
Trench-biotic stress interactions. We did not find significant
interaction between trenching and biotic (beetle attacks or
inoculations) treatments for NSCs and diterpenes (p>0.05).
However, trenching did impact monoterpene concentrations in response to
inoculation (F(1,,21)=4.69, P=0.042).
Trenched-inoculated trees had significantly greater monoterpene
concentration (8.85±1.89 µg mg-1) than
trenched-control (2.80±0.60 µg mg-1) or
untrenched-control (3.82±0.82 µg mg-1) trees. Means of
trenched-inoculated and untrenched-inoculated (5.42±1.16 µg
mg-1) were statistically similar. Please note that
monoterpenes
NSCs and defense metabolite interactions. Relationship between
NSCs and diterpenes. There was a
significant negative relationship between diterpenes and NSCs (Total
NSCs: F (1,267)=36.22, P< 0.001, Fig. 4a; Total Sugars:
F(1,270) =34.41, P<0.001, Fig. 4b; Starch: F (1,271) =25.49,
P<0.001, Fig. 4c). However, treatments did not affect these
relationships (Fig. 4a).
Relationship between NSCs and monoterpenes . We did not find any
relationship between monoterpenes and NSCs (Total NSCs: F(1,291)=0.082,
P=0.774, Fig. 4d; Total Sugars: F(1,293) =0.080, P=0.783, Fig. 4e;
Starch: F(1,265)=0.001, P=0.969, Fig. 4f). However, the relationship
between starch and monoterpenes varied by treatments (Fig. 4f). Trees in
the untrenched-attacked treatment had a significant negative
relationship between monoterpenes and starch; in contrast, we found a
significant positive relationship in the untrenched-inoculated and
trenched-inoculated trees (t=-4.54, P<0.001, and t=-3.75,
P=0.003, respectively). Moreover, although there was a marginal effect
of treatment on the relationship between total NSCs and monoterpenes, we
did not find a significant interaction for total sugars (significance of
interactions are provided in Fig. 4 caption).
Concentrations of NSCs and terpenes between dying and live trees
(2014 data only). Treatment, month, and their interaction significantly
affected sugars, starch, or total NSCs (Suppl. Table S6). We then
compared the dying and live trees in the same trenched-attacked category
for each month and found that live trees had significantly higher total
sugars, starch, and total NSCs than dying trees in August, September,
October, and November in 2014 (Fig. 5).
We investigated similar relationships for diterpenes and monoterpenes of
dying and live trees in the same treatment category. Treatment, time and
their interaction were not significant effects for either terpenes
(Table S6), showing that the amount of diterpenes or monoterpenes
produced in the dying versus live trees were statistically similar (Fig.
5).
Comparisons of carbohydrates between the six dying trees in the
trenched-attacked treatment and live trees in the untrenched-control
treatment showed a significant effect of month (Table S7). In a pairwise
comparison between treatments for each month, we found that live trees
had significantly higher starch, total sugars, and total NSCs than dying
trees starting from July until November in 2014 (Fig. 6). We only used
the data in 2014 because all dying trees in the attacked-trenched
category died in 2014.
We investigated relationships between the six dying trees in the
trenched-attacked treatment and live trees in the untrenched-control
treatment for diterpenes and monoterpenes. Although the two treatments
did not vary significantly, the monthly comparisons showed that
constitutive tissue of live trees had significantly lower diterpenes in
August, September, October, and November than tissues of attacked trees
(Fig. 6, Table S7). Likewise, for monoterpenes, the overall interaction
was not significant but the monthly comparisons showed that live trees
had significantly lower monoterpenes in July, August, September, and
October than dying trees (Fig. 6).
Explanation of tree mortality by NSCs-terpene interactions. We
investigated the relationship between NSCs and terpenes from 2014 for
dying trees in the trenched-attacked treatment and all treatments of
live trees averaged over months. We found significant negative
relationships of NSCs with either with diterpenes or monoterpenes (Fig.
7). For live trees, diterpenes or monoterpenes had a significant
negative relationship with starch, total sugars, or total NSCs. For
dying trees, only monoterpenes had a significant relationship with
starch (P < 0.05). Other relationships between carbohydrates
and terpenes were not significant. The low sample size for dying trees
may have impacted the lack of significance in correlations compared to
live trees.