The Effects of Arbuscular Mycorrhizal Fungi on Direct and Indirect Defense Metabolites of Plantago lanceolata L.

Anna Fontana 0 Michael Reichelt 0 Stefan Hempel 0 Jonathan Gershenzon 0 Sybille B. Unsicker 0 0 S. Hempel Department of Soil Ecology, UFZ - Helmholtzcentre for Environmental Research , Theodor-Lieser-Strae 4, 06120 Halle (Saale), Germany 1 ) Department of Biochemistry, Max Planck Institute for Chemical Ecology , Hans-Knll Strae 8, 07745 Jena, Germany Arbuscular mycorrhizal fungi can strongly influence the metabolism of their host plant, but their effect on plant defense mechanisms has not yet been thoroughly investigated. We studied how the principal direct defenses (iridoid glycosides) and indirect defenses (volatile organic compounds) of Plantago lanceolata L. are affected by insect herbivory and mechanical wounding. Volatile compounds were collected and quantified from mycorrhizal and non-mycorrhizal P. lanceolata plants that underwent three different treatments: 1) insect herbivory, 2) mechanical wounding, or 3) no damage. The iridoids aucubin and catalpol were extracted and quantified from the same plants. Emission of terpenoid volatiles was significantly higher after insect herbivory than after the other treatments. However, herbivore-damaged mycorrhizal plants emitted lower amounts of sesquiterpenes, but not monoterpenes, than herbivore-damaged non-mycorrhizal plants. In contrast, mycorrhizal infection increased the emission of the green leaf volatile (Z)-3-hexenyl acetate in untreated control plants, making it comparable to emission from mechanically wounded or herbivore-damaged plants whether or not they had mycorrhizal associates. Neither mycorrhization nor treatment had any influence on the levels of iridoid glycosides. Thus, mycorrhizal infection did not have any effect on the levels of direct defense compounds measured in P. lanceolata. However, the large decline in herbivoreinduced sesquiterpene emission may have important implications for the indirect defense potential of this species. - About 80% of all terrestrial plants form associations with arbuscular mycorrhizal fungi (AMF) (Wang and Qiu 2006). These are fungal symbionts that are well known to improve plant nutritional status by enhancing the uptake of essential nutrients such as phosphorous and nitrogen and by improving the water supply through an increase in root surface area (Smith and Read 1997). In return, fungi receive carbon in the form of photosynthates from the plant. For both the establishment and the maintenance of the symbiotic association between plants and AMF, it is essential that both partners recognize each other. These recognition processes are initiated via molecular cross-talk mediated by changes in the gene expression and production of signal compounds in both partners (Harrison 2005). On the plant side, altered gene expression in the presence of AMF can influence other aspects of metabolism and even result in the induction of chemical defenses (Gange et al. 2007). A number of studies in the recent past have reported the effects of AMF on plant defensive compounds, such as volatile terpenoids (Akiyama and Hayashi 2002; Rapparini et al. 2008), essential oils (Copetta et al. 2006; Khaosaad et al. 2006) and glucosinolates (Vierheilig et al. 2000). Furthermore, effects of AMF on salicylic acid (SA) and jasmonate dependent signaling pathways have been reported, suggesting that AMF modulate plant defenses (review by Pozo and Azcn-Aguilar 2007 and references therein). AMF invasion triggers a general plant response to pathogen attack (Dumas-Gaudot et al. 2000), causing a transient accumulation of SA and activation of the SAdependent signaling pathway at the early stages of the association. These responses then seem to be repressed once the compatibility of the symbionts is recognized (reviewed by Garca-Garrido and Ocampo 2002). Two major forms of plant anti-herbivore defenses can be distinguished: direct defenses, which are toxic to the herbivore or deter feeding, and indirect defenses, which protect the plant by attracting natural enemies of the herbivore, either parasitoids or predators. Direct and indirect defenses can be constitutively expressed or induced by mechanical damage or herbivore feeding. The literature on mycorrhizal influence on direct and indirect defenses and the consequences for insect herbivores is scarce (Gange et al. 2007; Hartley and Gange 2009), although some studies that show the effects of AMF on direct defenses have been published (e.g., Marak et al. 2002; Fuchs and Bowers 2004). A major group of indirect plant defenses are volatile organic compounds (VOCs) that consist principally of green leaf volatiles (GLVs) and mono- and sesquiterpenes (Pichersky and Gershenzon 2002; Degenhardt et al. 2003). Herbivore-induced VOCs play an important role in attracting natural enemies of insect herbivores (e.g., Dicke et al. 1990; Turlings et al. 1990; Kessler and Baldwin 2001). Furthermore, herbivore-induced VOCs act as both intra- and inter- plant signals, and can result in priming and induction of plant defenses (e.g. Kost and Heil 2006; Frost et al. 2007; Heil and Silva Bueno 2007). Naturally occurring VOC emissions have been compared in mycorrhizal vs. non-mycorrhizal plants. For example, mycorrhization of Artemisia annua L. with two AMF species did not affect the amount of total terpenes emitted, but there were slight changes in the relative quantities of single compounds (Rapparini et al. 2008). In addition, an unspecialized fungal root endophyte (Acremonium strictum) reduced terpene emission of tomato plants with consequences for insect oviposition preference (Jallow et al. 2008). However, no study to date has investigated how herbivore damage alters the production of defenses in AMF vs. nonAMF plants, even though herbivory is known to have marked effects on VOC emission profiles and the levels of other defense compounds. Plantago lanceolata L. is a perennial forb with a cosmopolitan distribution and commonly forms associations with a large number of AMF species (Johnson et al. 2004; Oehl et al. 2004). The main group of secondary metabolites in P. lanceolata is the iridoid glycosides, with two dominant compounds, namely aucubin and catalpol. These compounds function as feeding and oviposition stimulants for specialized insects, and as deterrents or toxins for generalist herbivores (e.g., Bowers and Puttick 1988, Biere et al. 2004). Antimicrobial functions of these monoterpene derivatives also have been documented (Marak et al. 2002). The association of P. lanceolata with AMF can modify plant defense properties. In a study by Gange and West, the levels of the two iridoid glycosides (IGs), aucubin and catalpol, increased when the plants were associated with AMF (Gange and West 1994). However, the effects of AMF on other groups of defensive compounds in P. lanceolata, such as green leaf volatiles or volatile terpenoids, have not yet been documented. In this study, we investigated the effects of the arbuscular mycorrhizal fungus Glomus intraradices (N.C. (...truncated)