Discussion
This is the first experimental demonstration of seasonal changes in both
carbohydrates and carbon-dependent defense metabolites through mortality
of any conifer species during drought and bark beetle attacks in the
field. Here, root trenching increased tree water stress which was
manifested in reductions in both xylem water potential and leaf gas
exchange, in agreement with previous studies with induced water stress
(McCullough & Wagner 1987; Woodruff & Meinzer 2011; Hartmann 2015;
Arango-Velez et al. 2016). We showed that trenching did not
influence NSCs whereas both biotic challenge treatments reduced amounts
of starch and sugars of trees. Furthermore, live trees had higher NSCs
than dying trees, but the terpene concentrations did not vary between
them. Only the trenched-beetle attacked trees depleted carbohydrates and
died within the first year of bark beetle attacks.
We developed a new schematic representation of how mild drought alone or
in combination with biotic stress has influenced NSCs (Fig. 8). Overall,
our results show the importance of cumulative stress in tree
carbohydrate depletion and mortality during drought, especially when
considering that the effect of trenching alone on tree NSCs was
negligible prior to bark beetle attacks or microbial inoculations (Fig.
8, Box 1). In fact, six of eight trees in the trench-attacked treatment
died within 2-3 months of bark beetle attacks (2014) and at the time of
death they had less than 10% of NSCs found in live trees. Since
increased water stress following trenching had no impact on tree NSC
amounts, our results suggest that trees stressed only by moderate
drought may recover by compensating for carbohydrate loss (Gaylordet al. 2015; Galiano et al. 2017; Trugman et al .
2018; Gessler et al. 2020; He et al. 2020). However, if
such trees are further stressed by biotic agents, the cumulative
stressors (drought plus biotic agents) can lead to tree death (Fig. 8,
Boxes 2 & 3) (Anderegg et al. 2015; Camarero et al. 2015;
Gaylord et al. 2015).
When bark beetles successfully enter the host, they consume phloem
tissue to excavate oviposition and larval galleries, resulting in
girdling and thus disrupting carbon transport in the phloem within the
tree (Fig. 8, Box 3) (Paine et al. 1997; Wiley et al.2016). Bark beetles also carry propagules of a diverse community of
fungal species (Frago et al . 2012); some of them have been shown
to be at least moderately phytopathogenic (Krokene 2015). Once inside
the tree, fungal propagules germinate and fungal hyphae spread and
penetrate water conducting tissues in the xylem, blocking water
conduction from the soil to the canopy and thus reducing photosynthesis,
carbon assimilation, and NSC storage (Fig. 8, Box 2) (Lahr & Krokene
2013; Arango-Velez et al. 2016; Wiley et al. 2016; Adamset al. 2017). Furthermore, hyphal growth and expansion inside the
tree can be a significant carbon sink as fungal growth requires carbon
(Fig. 8, Box 2) (Cale et al. 2019b). This may explain why both
microbial inoculation and bark beetle attackswithout trenchingled to
roughly similar depletion levels of NSCs in trees. However, inoculation
of trenched trees did not result in any tree mortality as the bark
beetle attacks on the trenched trees did, suggesting that the degree of
stress imposed on trees appears to be proportional to the presence of
both bark beetles and associated microbes. It is noteworthy for future
assessments of tree defense capacity that using crushed beetles as the
inoculum source generates higher variance to mean ratios of host
chemistry than using standardized fungal inoculum (e.g., Keefover-Ringet al . 2016; Raffa et al . 2017). This result also suggests
that single inoculation treatments, while useful for simulating beetle
entry to access tree defensive capacity, cannot fully generate the
effects of bark beetle mass attacks on declining tree health and
mortality.
The current study suggested two mechanisms to support our main
hypothesis, that continuous bark beetle attacks during tree water stress
can deplete NSCs in tree stems relative to the trenching or bark
beetle-alone treatments, and lead to tree mortality. Firstly, dying
trees had much lower NSCs than live trees. In addition to our above
explanation on the role of bark beetle-fungal activities in carbon
biosynthesis and transport, bark beetle attacks created stronger sinks
for carbohydrates than the trenching or inoculation alone treatments
(Fig. 8, Box 3). Consequently, the amounts of terpenes produced in dying
trees were similar to or higher than live trees (depending on the
comparison and sampling date). Particularly, while NSC amounts were
relatively stable in dying trees from July to November (2014), diterpene
amounts increased over time particularly from July to September. In
contrast, NSCs steadily increased in live trees but diterpenes sharply
declined. In fact, at the time of death (October-November 2014), the
dying trees had similar or higher concentrations of total diterpenes in
the xylem and total monoterpenes in the phloem than live trees in the
untrenched-control treatment despite having only 5.8% of the total NSCs
present in the live trees. These results are expected as conifer trees
with low NSCs have been found to prioritize chemical defenses over
growth and respiration (Huang et al . 2019). Our results suggest
that terpene production in dying trees acted as a carbon sink because of
the continuous allocation and remobilization of NSCs from storage to
terpene biosynthesis (Goodsman et al. 2013; Roth et al .
2018; Huang et al. 2020). This likely reduced the NSC storage
pool in the tree and altered carbon allocation to other tree functions
(Sapes et al. 2021) (Fig. 8, Box 3), potentially resulting in
carbon starvation in the tree stem (McDowell et al. 2008;
McDowell & Sevanto 2010).
Furthermore, lower NSC amounts within the trenched-attacked treatment
for dying trees compared to live trees further supports a role of
carbohydrates in tree survival (Fig. 8, Box 3). While six out of eight
trees in the trenched-attacked treatment died, the remaining two trees
were still alive at the end of the experiment. Although induced terpene
amounts did not vary between dying and live trees in the
trenched-attacked treatment, dying trees had considerably less sugars
and starch than live trees. Thus, the differences between dying and live
trees may be explained by the higher amounts of NSCs in the latter
(Galiano et al. 2011; Poyatos et al. 2013; Dietze et
al. 2014; Camarero et al. 2015). Similar to our results, Wileyet al. (2016) reported that phloem and xylem NSCs of lodgepole
pine (P. contorta var. latifolia ) trees were affected byD. ponderosae attacks, with attacked trees having lower NSC
amounts in both phloem and xylem than trees protected from such attacks
(via experimental exclusion).
In conclusion, tree NSCs are critical for understanding the impacts of
drought and bark beetles on conifers because of the importance of
carbon-dependent terpenes as defenses against bark beetles. Using a
multi-year field experiment, we showed that bark beetle colonization on
drought-stressed trees reduced not only local carbohydrate availability
for essential tree functions, but also inhibited tree’s ability to
replenish carbohydrate reserves. Furthermore, carbon-dependent terpenes
and spreading fungal biomass in tree tissues become sinks for NSCs.
Overall, this study enhances mechanistic understanding of how
drought-mediated bark beetle attacks kill trees and underscores the
importance of multiple stressors in altering tree carbohydrate
source-sink relationships.
Acknowledgements – This field work was supported by
McIntire-Stennis Program project accession no. 230732 from the USDA
National Institute of Food and Agriculture. John Kaplan, Ansley Roberts,
Teresa Reyes, and Patrick Dunn provided valuable help in the field; A
Roberts and T Reyes were supported by the Hooper Undergraduate Research
Program at Northern Arizona University and the National Science
Foundation Research Experience for Undergraduates Program, respectively.
The Northern Arizona University Centennial Forest provided the study
site. NSERC to NE supported all the work at the University of Alberta.
Non-structural carbohydrate and diterpene resin acid analyses were
conducted in Erbilgin lab (https://sites.ualberta.ca/~erbilgin/).
Authors Contribution – SJB, MG, RH, and TK designed and
implemented the field experiment and collected phloem and increment core
samples; KK-R and KFR measured phloem monoterpenes; LZ, SZ, GI, S-HC
prepared increment cores and conducted chemical analysis; SZ, JGK, and
NE conducted statistical analyses; JGK, NE and S-HC prepared figures and
tables; NE wrote the entire manuscript; all authors contributed to
writing.
Data Availability – Upon acceptance of this manuscript, data
will be posted at the University of Alberta Dataverse
(https://dataverse.org/)