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 attackswithout trenchingled 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/)