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Microbial communities that live in association with animals and plants play a major role in driving the ecological and evolutionary patterns observed in nature. For example, nutrient acquisition and defense for most organisms of terrestrial and marine communities are strongly influenced by microorganisms (van der Heijden et al., 1998; Lee et al., 2001; Herre et al., 2007; McFall-Ngai, 2008; Brownlie & Johnson, 2009). Plants, in particular, host a vast diversity of fungal symbionts with interactions that range from parasitic (e.g. pathogens) to mutualistic (e.g. mycorrhizas) and which shape ecological patterns (e.g. Gilbert, 2002; Hart et al., 2003; Mangan et al., 2010).
Fungal endophytes are cryptic symbionts that live inside plant tissue without causing overt signs of disease (Petrini, 1991; Schulz & Boyle, 2006). Although their presence in some cases does not seem to affect the plant's ecological interactions (Saikkonen et al., 2010), endophytes can occasionally provide short-term benefits to their host. For example, they can influence the outcome of interactions between plants and their natural enemies by limiting the spread of pathogenic fungi and damage by herbivores (Clay, 1991; Arnold et al., 2003; Mejía et al., 2008; Van Bael et al., 2009a,b; Saikkonen et al., 2010). This wide variation in effects is not unique to endophytes but also occurs in fungus–plant–insect interactions involving mycorrhizas and pathogens (Hatcher, 1995; Stout et al., 2006; Gehring & Bennett, 2009). Elucidating the mechanisms underlying the defense (or lack thereof) in plant–endophyte interactions is critical for understanding the variation in endophyte effects, assessing the impact that endophytes can have on the dynamics of communities, and evaluating their potential as biological control tools (Herre et al., 2007).
Reduction in herbivory by plants hosting endophytes can result from directly decreasing survival rates of herbivores to indirectly affecting their developmental time, fecundity or foraging behaviors (Webber, 1981; Clark et al., 1989; McGee, 2002; Jallow et al., 2004; Van Bael et al., 2009b; Bittleston et al., 2011). Production of endophyte-specific toxins is one of the best-known examples of a defense mechanism against herbivores (Clay, 1991; Calhoun et al., 1992). Nevertheless, herbivores could also be affected by the presence of endophytes, if fungi can infect insects or affect their symbiotic microbiome (Marcelino et al., 2008), or by plant responses that are induced by fungal colonization such as production of secondary compounds and/or physical defenses (Heath, 2000; Van Loon, 2000; Stout et al., 2006). The wide range of fungal life history traits and plant responses to microbial infections suggests that the effect of endophytes on plant–herbivore interactions also may originate from a similar wide range of mechanisms.
Leaf-cutting ants (genera Atta and Acromyrmex, Myrmicinae) are one of the most important defoliators in the Neotropics (Cherrett et al., 1989; Herz et al., 2007; Costa et al., 2008) and responsible for an estimated one billion US dollars per year in damage to agriculture (Hölldobler & Wilson, 1990). They maintain an obligate symbiosis with their fungal cultivar (Leucocoprinus gongylophorus, Agaricaceae, Basidiomycota) (Weber, 1972) that digests ant-collected plant material and serves as the main source of nutrition for the ants and their larvae. Recent studies have shown that leaf-cutting ants (Atta colombica) prefer harvesting leaves from plants with relatively lower densities of endophyte infections, and that they clean leaves to reduce the amount of endophytes before using them as substrate for their symbiotic fungi (Van Bael et al., 2009a; Bittleston et al., 2011). This finding has been consistent among laboratory colonies tested with two species of plants artificially inoculated with one endophyte species or colonized by a natural range of fungal symbionts. Previous work on selection of substrate by leaf-cutting ants found that they were highly selective with respect to the plant species and even the individual plant and the leaves within a plant that they used (Cherrett, 2007; Rockwood, 1976). Several traits influence their foraging decisions but leaf toughness and plant secondary compounds, particularly terpenoids and cuticular waxes seem to consistently explain a large part of their selectivity (reviewed in Van Bael et al., 2011 2011). How and whether fungal endophytes affect these plant traits involved in ant selectivity is unknown. Moreover, it is possible that ants are not selecting against changes in leaf traits but preventing the introduction of foreign microorganisms to their fungal garden (Fernández-Marín et al., 2006).
In the present study, we exploited the ability of ants to discriminate between plants with high (Ehigh) and low (Elow) endophyte densities to narrow the search for key chemical and physical changes responsible for ant host preferences. Using an approach of combining bioassays and chemical analyses, we tested whether the presence of endophytes altered plant traits in ways detectable to ants. Specifically, we studied the interaction between cucumber (Cucumis sativus), one common endophyte (Colletotrichum tropicale) and the leaf-cutting ant species Atta colombica (hereafter ‘ants’). Plants manipulated to facilitate C. tropicale colonization were compared with untreated low endophyte control plants to measure whether endophyte presence affected (1) the plant's composition of high molecular weight compounds involved in ant choice, (2) the range of volatiles produced by leaves when ants were cutting them, (3) cuticular waxes on leaf surfaces, (4) leaf nutrient content and (5) leaf physical traits. We found evidence that the presence of endophytes affected leaf compounds with relatively high molecular weight, but not highly volatile compounds, cuticular waxes, leaf nutrient content or physical leaf traits. We discuss how endophyte-mediated changes in plant chemistry could result from plant synthesis, endophyte synthesis or their interaction.
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A previous study showed that leaf-cutting ants prefer to cut leaves with low rather than high colonization by endophytic fungi (Bittleston et al., 2011). Here we show that changes in the chemical characteristics of leaves resulting from their symbiosis with leaf endophytes are associated with most of the observed ant preferences. In our experiments ants demonstrated a preference for paper disks impregnated with leaf extracts from Elow cucumber plants that was similar in magnitude to the observed preference for Elow detached leaves relative with Ehigh treatments. Ants cut about one-third more area of Elow cucumber leaves and removed c. 20% more paper disks impregnated with Elow extracts compared with Ehigh leaves and disks, respectively.
Leaves from both plant treatments were often consumed but ants consistently cut and carried Elow leaves to their nests faster than Ehigh leaves. This overall difference in the speed in which leaves were cut can partly be explained by the difference in recruitment of ants to both types of leaves (Table 1, Bittleston et al., 2011). The number of ants recruited to a resource is dynamically modulated by individual workers through deposition of pheromones and correlates with food quality (Littledyke & Cherrett, 1978a,b; Jaffe & Howse, 1979; Roces, 1990). The chemical profile of leaves that ants use during host selection includes compounds from the cuticle cover, volatile compounds released to the surface, and chemicals that can be smelled or tasted while cutting (Van Bael et al., 2011 2011). Discerning minor differences of cucumber leaf quality by ants was done using components of the chemical phenotype that were included in leaf extracts added to paper disks. Leaf extracts contained compounds from a wide range of polarity and relatively high molecular weight (C. Estrada, S. A. Van Bael & W. T. Wcislo, unpublished). Variations in the chemical composition of leaf volatile compounds and cuticular waxes were independent of the degree of colonization by C. tropicale. Furthermore, observed colony preferences during tests with leaves typically happened after ants had started cutting both types of leaves. Thus, overall, our results suggest that compounds with relatively low volatility exposed during tissue wounding play major role in ants' selectivity. Alternatively, changes may have been subtle and went undetected in our chemical analyses.
Our results showed no consistent qualitative or quantitative differences in the composition of volatile compounds emitted by the control and endophyte-colonized cucumber. The few studies that have evaluated the volatile composition resulting from plant–endophyte symbioses have shown alteration of such mixtures compared with endophyte-free controls, although not with predictable patterns. For example, while emissions of most terpenoids from tomato decreased by half in plants hosting a root fungal endophyte compared with control plants (Jallow et al., 2008), the same group of chemicals increased significantly in peppermint colonized by a growth-promoting endophyte (Mucciarelli et al., 2007). However, in the latter study it is unclear whether the increase in emissions is caused by the generally larger size of infected plants and leaves. Volatile emissions from endophyte- and pathogen-infected plants have also shown compounds that presumably have a fungal origin (3-octanone, 1-octen-3-ol) (Yue et al., 2001; Cardoza et al., 2002). Several endophytes can release volatiles in culture and have been shown to have antibiotic and insecticidal effects (Strobel et al., 2001; Daisy et al., 2002; Mucciarelli et al., 2007). Colletotrichum tropicale, in particular, produced small quantities of > 15 volatile organic compounds in in vitro cultures (C. Estrada, S. A. Van Bael & W. T. Wcislo, unpublished), most of which are likely sesquiterpenoids, compounds typically associated with host rejection by leaf-cutting ants (Van Bael et al., 2011 2011). Nevertheless, neither those sesquiterpenoids nor the typical fungal compounds found in other studies were detected in volatile mixtures from Ehigh leaves. This is not surprising given that production of secondary compounds varies considerably with the medium where fungi grows (Ezra & Strobel, 2003) and that leaf endophyte biomass, usually restricted to intercellular spaces, is much lower than the biomass of in vitro cultures (Cabral et al., 1993; Schulz et al., 2002). This means that fungal emissions in planta may fall below the detection sensitivity of chemical instruments.
Volatile compounds emitted by leaves from both plant treatments were mixtures typically found in undamaged or mechanically wounded cucumber (Kemp et al., 1974; Takabayashi et al., 1994). This is a group of chemicals stored permanently inside leaves and released soon after tissue damage. Biotic and abiotic stresses also induce complex networks of signaling cascades in plants that result in emission of a different set of volatile compounds (Thomma et al., 2001; Kessler & Baldwin, 2002; Chisholm et al., 2006; Dudareva et al., 2006). Our results do not show evidence of activation of such defense biochemical pathways in cucumber in response to colonization by C. tropicale. First, samples lacked methyl salicylate, a volatile ester derived from the hormone salicylic acid (SA) that plants emit when they are infected by pathogenic fungi (Cardoza et al., 2002). Salicylic acid-dependent defenses are typically activated in plants in response to infections by biotrophic fungi (Thomma et al., 2001; Chisholm et al., 2006). Both SA and its airborne signal, methyl salicylate, also generate systemic acquired resistance (SAR), a long-lasting disease resistance induced by early microbial or viral pathogenic infections (Van Loon, 2000). Second, volatile terpenoids known to be regulated in cucumber by the jasmonic acid (JA) pathway, β-ocimene and 4,8-dimethyl-1,3,7-nonatriene (Takabayashi et al., 1994; Mercke et al., 2004), were detected in a few of the plants but their occurrence was independent of the degree of colonization by C. tropicale. Both JA and ethylene are hormones involved in the induced systemic resistance (IRS), disease resistance caused by nonpathogenic root-colonizing microorganisms (Van Loon, 2000; Shoresh et al., 2005). These signaling molecules also activate defense mechanisms against herbivores and infections by necrotrophic fungi (Thomma et al., 2001; Chisholm et al., 2006).
Most compounds found in our leaf surface chloroform extracts had been reported as cuticular waxes or components of the cell membrane of leaves, fruits and seeds of cucumber (Steinmüller & Tevini, 1985; Akihisa et al., 1986, 1988; Hartmann, 1998; Chun et al., 2006). Our results showed that high levels of C. tropicale colonization did not cause significant changes in the composition of cuticular waxes of leaves. However, while all major compounds were present in both treatments in about the same proportions, the content of individual chemicals tended to be higher in Elow leaves than in Ehigh leaves where only traces of some minor compounds were found (e.g. aldehydes and triterpene alcohols). Our results were unexpected, as changes in cuticular waxes after biotic and abiotic challenges have been reported from cucumber leaves. First, irradiation with enhanced UV-B levels caused an increase in total wax in cotyledons, particularly in aldehyde and alkane content (Steinmüller & Tevini, 1985). Second, resistance of cucumber to Colletotrichum largenarium owing to an early inoculation with the same pathogen (Kuć & Richmond, 1977) results in part from chemical changes on the leaf surface that repress fungal penetration to epidermal cells (Xuei et al., 1988; Kováts et al., 1991).
Overall, neither volatile compound nor cuticle chemical composition indicated that C. tropicale activated defense signaling cascades (JA, SA) in cucumber in the manner described for infections by pathogens or root-colonizing fungi and bacteria (SAR, IRS). However, this does not indicate that activation of plant defenses in these or alternative pathways does occur. A closer examination of hormone presence or gene expression downstream in defense signaling pathways is necessary to determine the extent of the involvement of plant defense mechanisms. Indeed, evidence exists for activation of defense genes in cacao plants colonized by C. tropicale (L. C. Mejía & E. A. Herre, pers. comm.). Changes in cucumber leaf chemistry are suggested by the preferences of our ant colonies for paper disks impregnated with Elow extracts rather than Ehigh extracts. We are currently investigating the identity of the chemicals influencing ants' foraging decisions. Qualitative or quantitative changes in leaf chemistry could be caused by enzymes or antibiotic compounds expressed by cucumber in response to C. tropicale or toxins from the endophyte that are constitutive or induced by cucumber defense responses. Some forms of resistance by plants toward fungal strains that cause diseases in genetic varieties of the same plant species or in other species (non-host resistance) include the accumulation of antimicrobial compounds induced by fungal colonization (Heath, 2000). Moreover, chemical warfare appears to be common among fungal root endophytes and their hosts, suggesting that many of these interactions are antagonistic, although maintained at equilibrium where none of the partners is favored (‘balanced antagonism’, Schulz et al., 1999, 2002; Schulz & Boyle, 2005).
Our results for the mineral content of leaves also suggest that a balanced antagonism could occur between C. tropicale and cucumber. Although most of the elements analysed are constituents of the fungal cell (Martin, 1979), and thus could increase with fungal biomass inside leaves, only the amounts of Ca, Fe and Al showed a positive correlation with the estimated degree of colonization by C. tropicale. Such relationships for the first two compounds could be linked to the plant responses to fungi and subsequent fungal resistance. Calcium is crucial for regulating many responses of plant cells to their environment (Dodd et al., 2010). In particular, calcium crosslinks pectin molecules in cell walls and thus its content in leaf tissue increases during reinforcement of the cell wall induced by fungal infections (Moerschbacher & Mendgen, 2000). By contrast, iron is essential for fungal growth and influential in the stability of plant–fungal interactions (Johnson, 2008). Furthermore, iron-containing antioxidant enzymes in fungi degrade antimicrobial active oxygen molecules produced by plants as an early response to halt fungal colonization (Mayer et al., 2001). However, fungal antioxidant enzymes that use Zn, Cu or Mn also exist, yet these elements did not vary with colonization of leaves by C. tropicale. Moreover, changes in content of Ca and Fe were not detected in leaves colonized by a natural community of endophytes relative to control plants (Van Bael et al., 2012a). Clearly, further investigation is necessary to explain the observed increase of both elements with the dynamics of cucumber–C. tropicale symbiosis. The increase of Al with C. tropicale colonization is puzzling as, to our knowledge, neither cucumber nor Colletotrichum accumulate this metal. Interestingly, the amount of Al could have negatively influenced the foraging preferences of ants in one study (Folgarait et al., 1996; but see Mundim et al., 2009); thus an increase of this element with C. tropicale colonization could have accounted for part of the observed ants' preference for Elow treatments.
Along with leaf chemistry, toughness is one of the more important traits that ants use during host selection (Van Bael et al., 2011 2011). Changes in the physical properties of leaves owing to endophyte colonization could also contribute to the observed foraging patterns in our arenas using detached leaves. It is known that a plant's typical response to detection of microbes consists of adding lignin, cellulose and other components of their cell walls as a mechanism to decrease the chance that the infection will reach the cell's cytoplasm (Heath, 2000; Moerschbacher & Mendgen, 2000; Chisholm et al., 2006). This effect, which has been documented for fungal pathogens in host and nonhost plants, also occurs after colonization of leaves of Theobroma cacao by C. tropicale (S. Maximova & E. A. Herre unpublished), and Juncus spp. by several species of fungal endophytes (Cabral et al., 1993). Although the reinforcement of the cell walls is confined to cells surrounding the point of fungal growth (Moerschbacher & Mendgen, 2000), this reinforcement in highly endophyte-colonized leaves could result in an overall increase in toughness, making leaves less appealing to ants. Nevertheless, several studies, including ours, have found no evidence that endophytes trigger leaves to become harder when conventional surrogate methods such as total carbon, water content and SLA have been used (Witkowski & Lamont, 1991; Bittleston et al., 2011). Similar results have been obtained when using more direct toughness measures such as resistance by puncturing and tearing (Bittleston et al., 2011). Experiments are thus necessary to determine whether deposition of cell wall components is a general response of plants to colonization by endophytic fungi and whether such reinforcements make leaf cutting more difficult for ants.
Each tropical plant can host dozens of endophyte species at any given time (Arnold et al., 2000; Suryanarayanan & Johnson, 2005; Van Bael et al., 2005). Our simplified system is therefore a first step to identify the mechanisms involved in endophyte-mediated protection of tropical plants. Colletotrichum tropicale influences leaf chemistry and makes leaves less appealing to leaf-cutting ants but likely offers little protection to individual plants as endophyte-colonized leaves are also consumed. Nevertheless, for ants, leaves hosting fungal endophytes take longer to process than those free of the symbiont (Van Bael et al., 2012b), and this additional cost decreases colony development, at least for young colonies whose risk of mortality is the greatest (Van Bael et al., 2012a). It is still an open question whether plant fitness is affected by short-term benefits resulting from ants' food selection or by changes in colony populations resulting from plant-endophyte symbiosis. More importantly, this study shows that fungal communities in leaf tissues can create a mosaic of palatability driven by leaf and endophyte chemistry within and between plants beyond the existing variance resulting from plant genotype, environment and leaf age or location. Such variability can be high if leaf responses are tuned to endophyte species and if the local diversity of fungal endophytes results in a similar diversity of fungal toxins. Symbiosis with fungi can then influence the ecological interactions of plants with their natural enemies by making leaf defenses less predictable and thus more difficult to adapt to by herbivores (Herre et al., 2007).