Author's personal copy JChemEcol DOI 10.1007/s10886-015-0588-4 Contrasting Patterns of Diterpene Acid Induction by Red Pine and White Spruce to Simulated Bark Beetle Attack, and Interspecific Differences in Sensitivity Among Fungal Associates Charles J. Mason1,2 & Kier D. Klepzig3 & Brian J. Kopper1,4 & Philip J. Kersten5 & Barbara L. Illman5 & Kenneth F. Raffa1,6 Received: 26 January 2015 /Revised: 5 April 2015 /Accepted: 4 May 2015 # Springer Science+Business Media New York (outside the USA) 2015 Abstract Conifers possess a suite of physiochemical de- during induction than white spruce. In red pine,fungal inocu- fenses that protect their subcortical tissues from bark beetle - lation accompanying wounding elicited greater increases than fungal complexes. These defenses include rapid induction of wounding alone, but in white spruce total concentrations were terpenoids and phenolics at the site of attack. Studies of the higher following wounding alone. Spruce beetle fungal sym- distribution, induction, and bioactivity of conifer terpenoids biont growth varied among species and compounds. Some have focused heavily on monoterpenes. We assessed induc- diterpenes elicited both stimulatory and inhibitory effects on tion of diterpene acids in white spruce (Picea glauca) and red fungi, depending on concentration. All four fungi exhibited pine (Pinus resinosa) to fungal associates of two bark beetles, higher tolerances compared to those associated with pine bark and the responses of four spruce beetle (Dendroctonus beetles in previous studies. Variation in tolerances to, and rufipennis)—associated fungi to three diterpene acids. Consti- potentially metabolism of, diterpene acids by symbionts may tutive phloem contents differed between species, in that red reflect differences in constitutive levels between spruce and pine had extremely low concentrations of diterpene acids, pine, and partially explain differences in concentrations whereas white spruce had substantial constitutive levels. In- achieved during induction. duction differed quantitatively. Both red pine and white spruce exhibited marked increases, but red pine underwent greater Keywords Plant defense . Terpenoids . Ophiostoma . increases and achieved higher concentrations than white Leptographium . Ips . Dendroctonus spruce. Induction also differed qualitatively in that red pine showed lower diversity and fewer compositional changes Introduction * Kenneth F. Raffa Conifers possess sophisticated, multicomponent defenses that [email protected] can protect trees from attack by insects, pathogens, and insect- microbial complexes. Several species can sometimes over- 1 Department of Entomology, University of Wisconsin-Madison, 1630 come these defenses, particularly subcortically feeding bark Linden Dr., Madison, WI 53706, USA beetles and associated microbes (Carroll et al. 2006;Smith 2 Department of Entomology, The Pennsylvania State University, et al. 2012). Trees resist bark beetle attacks through integrated University Park, PA 16802, USA physical and chemical defenses, which include autonecrosis, 3 Southern Research Station, USDA Forest Service, 200 WT Weaver biosynthesis of secondary chemicals, and resins that flow Blvd, Asheville, NC 28804, USA from storage sites in ducts and glands to the point of attack 4 USDA APHIS PPQ, Raleigh, NC 27606, USA (Franceschi et al. 2005; Kane and And Kolb 2010;Lewinsohn 5 Forest Products Laboratory, USDA Forest Service, et al. 1993; Lieutier et al. 1992; Rosner and Hannrup 2004). Madison, WI 53726, USA These highly viscous resins can physically delay, expel or 6 345 Russell Laboratories, University of Wisconsin-Madison, 1630 entomb beetles, are rich in compounds that can repel host- Linden Dr., Madison, WI 53706, USA searching adults, intoxicate adults and their brood, inhibit Author's personal copy JChemEcol growth and germination of microbial associates, and interfere development (Larsson et al. 2000; Powell and Raffa 1999; with the pheromone signaling that beetles need to overcome Schmelz et al. 2011; Wagner et al. 1983). Their effects on tree defenses through synchronous attacks (Erbilgin et al. subcortical insects, however, are less clear. Sitka and white 2006; Everaerts et al. 1988; Raffa et al. 2005). spruce resistance to white pine weevil has been correlated with The effects of compounds in conifer resin and phloem often higher diterpene acid concentrations (Tomlin et al. 1996). In are dose-dependent, both in the magnitude and direction of contrast, diterpene acids exert minor effects on the pine en- their impacts on beetles and associated microbes. For exam- graver, Ips pini (Say). Instead, they greatly reduced growth ple, low concentrations often benefit beetles by stimulating and germination of I. pini’s fungal symbiont Ophiostoma ips entry, synergizing the attractiveness of their pheromones, or (Rumbold), exhibiting higher activity than any monoterpene stimulating pheromone synthesis. High concentrations of the or phenolic previously tested (Kopper et al. 2005). Further- same chemicals, however, typically deter entry, reduce attrac- more, growth of the mountain pine beetle (Dendroctonus tion to pheromones, and are lethal (Bohlmann 2012;Erbilgin ponderosae Hopkins) fungal associates Ophiostoma montium et al. 2006; Seybold et al. 2006; Wallin and Raffa 2000). The (Rumbold) and Grosmannia clavigera (Robinson-Jeffrey & flow of resin from a wound, and the composition and concen- R.W. Davidson) are greatly reduced by diterpene acids trations of its chemical constituents, are highly inducible, par- (Boone et al. 2013). These fungal associates are critical to ticularly in response to biotic agents (Clark et al. 2014). beetle success by helping to overcome tree defenses (Kim Conifers contain a diversity of secondary chemicals, with et al. 2008; Lieutier et al. 2009) and contributing to beetle the predominant compounds being monoterpenes, diterpene nutrition (Bleiker and Six 2007). acids, and phenolics (Franceschi et al. 2005; Kersten et al. Red pine (Pinus resinosa Aiton) is distributed throughout 2006; Raffa et al. 2005). Of these, monoterpenes and pheno- much of the Great Lakes region of North America. The major lics have received the most attention. Monoterpenes are highly insect attacking mature trees is I. pini. White spruce (Picea inducible, and undergo rapid biosynthesis within just a few glauca Moench) is widely distributed across North America. days (Huber et al. 2004;Zhaoetal.2011; Zulak and Its major tree killing insect is the spruce beetle, Dendroctonus Bohlmann 2010). Monoterpene proportions and concentra- rufipennis. The predominant symbiotic fungus of I. pini is tions can greatly alter in response to stimuli, with the extents O. ips (Klepzig et al. 1991). Dendroctonus rufipennis varying among conifer species (Raffa et al. 2005). Monoter- (Kirby) is associated with several symbiotic fungi, of which penes can exert behavioral aversive, physiologically sublethal, Leptographium abietinum (Peck) is most prominent (Aukema and toxic effects on bark beetles (Manning and Reid 2013; et al. 2005;BentzandSix2006; Haberkern et al. 2002). The Raffa and Smalley 1995; Wallin and Raffa 2000), and inhibit constitutive and induced monoterpene and phenolic composi- the beetles’ symbiotic fungi (Klepzig et al. 1996). They also tions of red pine and white spruce have been reported in sev- may be exploited by bark beetles as host recognition cues eral previous studies, (Klepzig et al. 1996; Raffa and Smalley (Elkinton et al. 1981), and as components in pheromone bio- 1995; Werner and Illman 1994). Diterpene acid contents of synthesis (Blomquist et al. 2010). white spruce in Wisconsin were reported by Kersten et al. Phenolics, likewise, are present in constitutive phloem tis- (2006), but there is no information on whether, and if so sue and undergo induction upon attack, although to a lesser how, they are induced by attack. Likewise, we have no infor- extent than monoterpenes (Hammerbacher et al. 2011; mation on how fungi associated with D. rufipennis respond to Klepzig et al. 1996). Phenolics contribute to tree defense diterpene acids. We assessed induction of diterpene acids in (Faccoli and And Schlyter 2007), and may function alongside red pine and white spruce, and responses of D. rufipennis - monoterpenes in a complementary manner, with monoter- associated fungi to three diterpene acids to complement prior penes typically having greater activity against the beetles, work on sensitivity of O. ips. and phenolics typically having greater activity against the beetles’ fungal symbionts (Klepzig et al. 1996). Conifer diterpene acids have received less attention in Methods terms of their concentrations, inducibilities, sources of varia- tion, and bioactivities (Erbilgin et al. 2006; Villari et al. 2012; Site Selection and Defense Induction Ten mature red pine Zhao et al. 2010). Diterpene acids are synthesized from and white spruce trees were selected from two planted stands isopentenyl diphosphate produced via the mevalonate- in Dane County, WI, USA. Experiments were conducted in independent pathway in plastids (Keeling and Bohlmann late June 2004, as previous work demonstrates this is within 2006; Trapp and Croteau 2001). Biosynthesis is regulated by the period of strong induction of defense chemicals to simu- several terpene synthases whose transcriptions can be elicited lated beetle attack (Raffa and Smalley 1988). Constitutive by biotic stimuli (Hall et al. 2013; Hamberger et al. 2011). phloem samples were removed from bark and placed