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- Materials and methods
Most plants have microbial associates (Clay 2004) and such associations may alter processes of plant succession (Clay & Holah 1999), general relationships between plant species diversity and productivity (Rudgers, Koslow & Clay 2004), and insect food web interactions (Omacini et al. 2001). Endophytic fungi of cool season grasses are often seen as mutualistic symbionts, with the fungi receiving shelter, nutrients and transmission to the next generation via grass seeds, and host grasses having higher stress tolerance and herbivore resistance (Schardl, Leuchtmann & Spiering 2004; Müller & Krauss 2005). Cereal aphids, which are common grass herbivores, often show strong negative responses when feeding on endophyte-infected agronomic grass species (Breen 1994; Hunt & Newman 2005; Meister et al. 2006). The grass–fungus association produces a cocktail of alkaloids and, in the grass Lolium perenne, the main insect toxic substance is peramine. The alkaloids ergovaline and lolitrem B are also found in L. perenne (Spiering et al. 2002; Schardl et al. 2004), the latter being responsible for ryegrass staggers in sheep (Schardl et al. 2004). All alkaloids vary in concentration and distribution within a single host plant (Fannin, Bush & Siegel 1990; Ball, Prestidge & Sprosen 1995; Keogh, Tapper & Fletcher 1996; Ball et al. 1997; Spiering et al. 2002) and toxic effects depend on environmental conditions (Faeth, Bush & Sullivan 2002) and the genetic backgrounds of fungus and grass host (Roylance, Hill & Agee 1994; Faeth et al. 2002). As nitrogen is a key component of alkaloids, it could be expected that nitrogen addition will increase the alkaloid concentration in infected grasses (Lyons, Plattner & Bacon 1986; Marks, Clay & Cheplick 1991; Latch 1993; Faeth & Fagan 2002). Indeed, concentrations of lolitrem B and peramine have been shown to be higher in well fertilized ryegrass compared with poorly fertilized plants (Latch 1993). However, even though plant nitrogen concentrations typically increase in response to fertilization (Davidson & Potter 1995), Faeth et al. (2002) found that the peramine concentration of Arizona Fescue was not altered by fertilizer treatment.
Generally, aphid densities are enhanced when plants are grown with additional fertilizer (Honek 1991; Davidson & Potter 1995); this could result in a conflicting situation for aphids on endophyte-infected plants where insect growth rates are enhanced by fertilization, but reduced through higher concentrations of toxic alkaloids. The aphid Rhopalosiphum padi benefits from fertilizer addition, showing higher growth rates on fertilized plants of Lolium (formerly Festuca) arundinacea. However, when the grass is infected with the endophyte Neotyphodium coenophialum, the positive effect of fertilizer is counteracted and aphid population densities decrease (Davidson & Potter 1995). In this latter study, effects on the population densities of natural enemies of aphids were not considered. It is, however, conceivable that not only herbivores, but also their natural enemies are affected by both endophyte presence and fertilizer addition, with further feedbacks on herbivore densities. Flying predators (Müller & Godfray 1999) and particularly parasitoids (Schmidt et al. 2003) can have strong negative effects on aphid colony growth. Several laboratory studies on endophytes have found that predators (de Sassi, Müller & Krauss 2006) and parasitoids (Barker & Addison 1996; Bultman et al. 1997; Bultman, McNeil & Goldson 2003) are negatively affected by the presence of endophytic fungi. However, these studies were conducted under laboratory conditions, with insects being fed on endophyte-infected food. Providing natural enemies with a choice, under field conditions, might result in less distinct fitness losses.
As with most studies on the effects of endophytes on herbivores and predators, effects on plant life-history traits are often measured only in greenhouse experiments and only during the first few months of the lifespan of grasses (e.g. Cheplick 1998, 2004; Cheplick & Cho 2003). Field conditions may alter these results, because more species, at different trophic levels, will interact in the field, potentially resulting in higher order interactions (Wootton 1994; van Veen, Morris & Godfray 2006). In addition, all endophyte-mediated effects on plant life-history, alkaloid concentration, density of herbivores and natural enemies may be influenced by the plant's genotype or cultivar (Cheplick 1998, 2004; Faeth et al. 2002; Cheplick & Cho 2003; Meister et al. 2006). Here we present data from four agronomically important cultivars of Lolium perenne L., with the asexually transmitted endophyte, Neotyphodium lolii Glenn, Bacon and Hanlin, which relies entirely on seed production of the host plant to pass to the next generation. It would be expected that such an endosymbiont would manipulate its host plant to allocate more resources to reproduction, compared with uninfected plants.
The main aim of this study was to understand the relationships between fertilizer treatment, endophyte infection and plant cultivar on plant life-history of L. perenne and the associated insect population densities in the field. This was achieved by a full factorial outdoor experiment, in which insects were left to colonize the plants naturally. The main predictions addressed were that: (1) endophyte infection alters plant performance, especially the allocation of resources to reproduction; (2) fertilizer addition and grass cultivar affect plant life-history traits and these may interact with endophyte infection; (3) peramine and nitrogen concentrations are enhanced after fertilizer addition; and (4) endophyte infection decreases aphid and parasitoid abundances, but grass cultivar and fertilizer treatment modify this effect.
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- Materials and methods
In our fully factorial field experiment, fertilizer addition strongly enhanced the abundances of naturally colonizing aphids and parasitoids on agricultural grasses. Plant cultivar had a small effect on insect species abundance, while endophyte infection of the resource plant had no negative effect on insect abundances in this study. The absence of an effect of endophyte infection is in contrast to short-term laboratory trials. For example, clear negative effects of the endophyte N. lolii have been shown for herbivores and predators associated with L. perenne (Meister et al. 2006; de Sassi et al. 2006).
The aphid R. padi is known to be negatively affected by the presence of N. coenophialum in Tall Fescue (L. arundinacea), with the associated insect-toxic loline group of compounds (Davidson & Potter 1995; Hunt & Newman 2005), and by N. lolii and associated peramine production in L. perenne (Meister et al. 2006). It is surprising, therefore, that there was a trend towards higher densities of this aphid species on infected unfertilized plants, compared with uninfected and fertilized plants in our experiment. In another field experiment conducted in 2005, R. padi was also more abundant on infected L. perenne (Jochen Krauss, unpublished data). We currently have no explanation for why this aphid species shows such contrasting results. Endophyte effects on the aphids M. festucae and S. avenae could not be detected in our study; this supports data from laboratory studies that show that M. festucae has no clear negative response to endophyte infection (Simone Härri, unpublished data). Sitobion avenae colonizes ears of the grass and therefore depends on ear rather than leaf quality (Honek 1991). The concentration of peramine in ears is, however, unknown and was not measured separately in our study. Ear biomass, number of ears, ear length and number of spikelets all differed between plant cultivars; cultivar also significantly affected the abundance of S. avenae. In the absence of a fertilizer-related increase in foliar nitrogen concentration, the increase in abundance of S. avenae and M. festucae is likely to be linked to the overall increase in above-ground plant biomass following fertilizer treatment. Growth dilution of foliar N concentrations is a common phenomenon (Johnson, Ball & Walker 1997) and is likely to explain the lack of concentration increase observed in this study.
The increase in parasitoid numbers associated with fertilizer treatment appears to be a direct result of increased aphid availability resulting from the treatment-related increase in plant biomass. Correlations between the four trophic levels – plants, aphids, primary and secondary parasitoids – make this interpretation plausible. Such cascading trophic interactions are common in food webs and have frequently been described for terrestrial webs (e.g. Schmitz 1993; Dyer & Stireman 2003).
Endophyte infection did not provide any significant defence against the aphid herbivores in our study. Similarly, neither the treatment-related increase in peramine production nor the effect of plant cultivar affected the level of herbivore protection offered by the endophyte. Elsewhere, a further peramine producing Neotyphodium species has also been shown to provide no protection against a grasshopper species feeding on infected Arizona Fescue (Saikkonen et al. 1999). The relatively small effects of endophytes on aphids and parasitoids in our study might be explained by the relatively low concentrations of peramine found in our L. perenne plants (unfertilized: 5·5 µg g−1, fertilized 8·0 µg g−1). Other studies have reported concentrations in excess of 10·0 µg g−1 (e.g. Ball et al. 1995; Spiering et al. 2002), which is also the threshold level for feeding deterrence for the Argentine Stem Weevil (Keogh et al. 1996). Peramine concentrations below 3·0 µg g−1 are generally considered nontoxic for invertebrate herbivores (Siegel & Bush 1996).
Another reason for the relatively small effect of endophytes on insect herbivores and their parasitoids might be as a result of the experiment being conducted under field conditions, with numerous indirect interactions between species and insects having a wide choice of plants on which to feed and oviposit, in contrast to more controlled laboratory conditions. Furthermore, the clear effect of endophytes on plant performance in our 2-week growth room experiment disappeared 8 months later under field conditions. The main driver for plant performance in the field was fertilization and, to a lesser degree, plant cultivar. In the growth room study, endophyte infection and plant cultivar showed significant interactions in terms of germination success and plant height. These findings are consistent with other laboratory studies where plant performance is often affected by interactions between endophyte infection and host-plant genotype (Cheplick 1998, 2004; Cheplick & Cho 2003).
In conclusion, our study showed that under field conditions endophyte effects on plant performance, herbivores and natural enemies are less consistent than laboratory studies suggest. For aphid populations and their parasitoids, fertilizer addition at agricultural rates has much stronger effects on abundance than endophyte and alkaloid presence. The increase in peramine concentrations associated with fertilizer addition was not sufficient to decrease aphid population sizes. Overall, in this study system with four endophyte-infected agronomic grass cultivars and trophic interactions based on aphids and their parasitoids, we found that the effect of fertilizer on aphid and parasitoid abundance was greater than the effect of plant cultivar on L. perenne, and the effect of endophyte infection by N. lolii was minimal.