Introduction
Climate change-induced dieback is a global problem affecting forest
ecosystems worldwide (Allen et al. 2010; Anderegg et al.2012; Young et al. 2017; Hartmann et al. 2018). The causal
factors underlying forest dieback have been commonly attributed to
complex interactions between abiotic and biotic factors
(Simler-Williamson et al. 2019). For instance, drought is often
viewed as a predisposing factor responsible for promoting outbreaks by
some insect species such as bark beetles (Coleoptera: Curculionidae,
Scolytinae) that attack the main stems of mature trees (Gaylord et
al. 2013; Anderegg et al. 2015; Netherer et al. 2019;
Gely et al. 2020; Öhrn et al . 2021). Although there are
proposed mechanisms underlying drought-induced tree mortality, i.e., a
failure of the plant’s water transport-hydraulic-system and carbon
starvation due to prolonged stomatal closure and reduced photosynthesis
(McDowell et al. 2008; Anderegg et al. 2012; Meir et
al. 2015; Adams et al. 2017; Choat et al. 2018; Hartmannet al. 2018), we have less understanding of the combined effects
of drought and insect attacks in the field (Kolb et al. 2016;
Stephenson et al. 2019; Huang et al. 2020). Significant
challenges in conducting field experiments include experimental control
of both water stress and bark beetle attacks on the mature trees, and
measurement of the cascading physiological changes through tree death
(Kolb et al. 2016; Choat et al. 2018).
Most studies focusing on tree mortality were either conducted during or
after observed mortality (retrospectively) (e.g., Camarero et al.2015; Gaylord et al. 2013; 2015; Kolb et al. 2016; Öhrnet al . 2021). Moreover, those studies primarily examined the role
of a single stress agent such as drought or herbivory (e.g., Meddenset al. 2014; Erbilgin et al. 2017) although their effects
can be interacting. Likewise, some studies utilized seedlings under
controlled conditions to improve standardization (i.e., Turtola et
al. 2003; Lusebrink et al. 2011) but it is well recognized that
plant ontogeny strongly affects many responses and thus results obtained
with seedlings may not be applicable to mature trees (Boege & Marquis,
2005; Erbilgin & Colgan, 2012; Moreira et al. 2017). As such,
experimental demonstrations of both predisposing and biotic factors
leading toand measuring the physiological mechanisms responsible
fortree mortality in the field are sparse (Kolb et al. 2016;
Huang et al. 2020). This knowledge gap hinders our ability to
accurately understand the mechanism of tree mortality from multiple and
sometimes synergistic factors.
Drought can have profound impacts on tree responses to subsequent insect
attacks. These responses vary by feeding guild and are most impactful
with bark and wood boring insects (reviewed by Kolb et al. 2016).
Briefly, prolonged droughts induce stomatal closure, which in turn
reduces photosynthesis, potentially leading to depletion of carbohydrate
reserves, i.e., non-structural carbohydrates (NSCs), comprised of
soluble sugars and starch (McDowell et al. 2008; Anderegget al. 2012; Mitchell et al. 2013; Choat et al.2018). Hydraulic failure occurs due to disruption of water movement in
the xylem due to formation of air bubbles (embolism) during desiccation
(Adams et al. 2017). Furthermore, NSC depletion may also reduce
water movement and retention, promoting cellular dehydration (Salaet al. 2012; Deans et al. 2020). Reductions in
carbohydrate production and depletion of NSCs due to drought can
influence many functions in plants including biosynthesis of
carbon-dependent terpenes that serve antidessication and antiherbivory
functions (McDowell et al. 2011; Huang et al. 2020;
Hussain et al. 2020).
Ponderosa pine (Pinus ponderosa ) is one of the most widely
distributed conifer species in western North America, ranging from
southern Canada to Mexico, and from the Plains States to the Pacific
Coast. Two major threats to ponderosa pines, and other co-occurring pine
species, are drought and bark beetles (Negrón et al. 2009; Bentzet al. 2010; Raffa et al. 2013; Creeden et al.2014). Over recent decades, several million ponderosa pine trees were
killed by a combination of these two stressors in various parts of the
species’ range (Savage et al. 1994; USDA Forest Service 2002;
Breshears et al. 2005; Allen et al. 2010; Pile et
al. 2019).
Bark beetles’ reproduction involves offspring completing their
development within the phloem layer beneath the outer bark of their host
trees. Pine phloem contains toxic carbon-dependent secondary metabolites
including monoterpenes, diterpene resin acids, and phenolics (Francheshiet al. 2005; Keeling & Bohlmann 2006; Celedon & Bohlmann 2019;
Erbilgin, 2019). Studies have reported that monoterpenes and diterpenes
are toxic to the bark beetles and their mutualistic fungi (Kopperet al. 2005; Chiu et al. 2017; Ullah et al. 2021).
Numerous studies with simulated or actual herbivore attacks have
extensively shown mobilization and transport of stored NSCs in support
of the synthesis of defense metabolites in plants including conifers
(Goodsman et al . 2013; Dietze et al. 2014; Hartmann &
Trumbore 2016; Adams et al . 2017; Li et al . 2018; Rothet al . 2018; Hussain et al . 2020). Effects of drought on
bark beetles can be positive or negative depending on drought intensity
and duration (Lombardero et al. 2000; Gaylord et al. 2013,
2015). Currently, we lack a clear understanding about the importance of
combined drought and bark beetle attacks on NSCs, carbon-based defense
metabolites, and their interactions in tree mortality (Kolb et
al. 2016; Huang et al. 2020).
An earlier study suggested a relationship between drought and ponderosa
pine terpene defenses (Kolb et al. 2019), but how drought and
biotic challenges interact to influence NSCs and their relationships
with carbon-dependent defense metabolites (terpenes) has not been
jointly investigated and remains poorly understood. Thus, our primary
objective was to characterize the changes in NSC and terpene
concentrations in ponderosa pine trees that were experimentally exposed
to a factorial combination of drought stress (induced via root
trenching), pheromone-induced bark beetle attacks, and crushing beetles
onto phloem/xylem interface in the experiment described in Kolb et
al . (2019). We hypothesized that the combination of drought and bark
beetle attacks more rapidly deplete NSCs in tree stems that either
stress agent alone, thereby eliciting tree mortality. We addressed the
following five research questions: (1) Do drought stress, bark beetle
attacks, and the beetle crushing influence NSCs of ponderosa pine trees?
(2) Does drought impact the effect of biotic stressors on NSCs and
terpenes? (3) Do changes in NSCs result in changes in defense
metabolites? (4) Do concentrations of NSCs and defense metabolites vary
between live and dying ponderosa pine trees? (5) Can interactions
between NSCs and defense metabolites in dying ponderosa pine trees
explain their mortality?