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- Materials and Methods
- Supporting Information
An estimated 20–30% of all grass species host systemic endophytes (Leuchtmann, 1992), which makes these associations of wide interest to studies of plant–fungal interactions. The largest plant family hosting these endophytes is the Poaceae and this contains a number of significant agricultural species which are often hosts of systemic endophytic fungi of the Clavicipitaceae family (Clay & Schardl, 2002). Lolium perenne–Neotyphodium lolii associations are the most common in temperate grasslands in Europe and Australasia, and L. arundinaceum (=Festuca arundinacea)–N. coenophialum associations in North America (Christensen et al., 1993). Hyphae grow in the intercellular spaces of above-ground organs and rely on nutrients produced by the plant (Clay, 1990; Clay & Schardl, 2002). Endophytic fungi have been demonstrated to confer benefits to their grass hosts, partly through altered physiology but notably through insect resistance/repellence because of the production of alkaloids (see review by Malinowski & Belesky, 2000; Schardl et al., 2004). These associations have widely been regarded as mutualistic, but there is growing evidence of negative interactions between host and endophyte under certain circumstances (Cheplick, 2004; Müller & Krauss, 2005; Malinowski & Belesky, 2006) and a recently published meta-analysis of grass/endophyte literature suggests that the degree of mutual benefit is conditional on environmental factors such as nutrient availability (Saikkonen et al., 2006). Changes in the balance of grass and endophyte can alter the fitness of both species and can have implications for wider ecosystem function (Clay & Holah, 1999; Müller, 2003; Rudgers et al., 2004; Clay et al., 2005; Malinowski & Belesky, 2006).
The common strain (CS) of the endophyte N. lolii in ryegrass produces three known alkaloids: lolitrem B, peramine, and ergovaline. Lolitrem B is the putative cause of ryegrass staggers in sheep (Gallagher et al., 1984), while ergovaline (the cause of fescue toxicosis; Lyons et al., 1986) and peramine provide the host plant with protection against herbivorous insects such as the Argentine stem weevil (Prestidge & Gallagher, 1988). For use in pasture grazing systems, novel associations between naturally occurring endophyte strains and high-yielding grass cultivars have been developed to retain insect deterrent properties but to have much reduced detrimental impact on grazing herbivores (Fletcher & Easton, 1997; but see Hunt & Newman, 2005); for example, the N. lolii strain ‘AR1’ has peramine only and ‘AR37’ lacks all three major alkaloids but produces janthitrems.
The production and concentration of endophyte alkaloids in plant tissues vary considerably in the field with season, weather and management (for L. perenne see Keogh, 1983; Ball et al., 1995; Salminen & Grewal, 2002; Hume & Barker, 2005; for L. arundinaceum see Lyons et al., 1986; Belesky et al., 1988; Arechevaleta et al., 1992; Malinowski et al., 1998). But there have been surprisingly few controlled studies of the effects of individual environmental components on alkaloid accumulation, sufficient to predict the consequences of environments on alkaloids. Few reports have been published on interactions between nitrogen (N) availability and endophytic alkaloid production, with sometimes contrasting results. In tall fescue, Lyons & Bacon (1984) and Belesky et al. (1988) showed increased alkaloids at elevated N availability, especially under moderate water stress (Arechevaleta et al., 1992), but Faeth et al. (2002) found no such increase in Arizona fescue. In L. perenne, ‘no consistent pattern has been observed’ (Lane et al., 1997), although Hunt et al. (2005) found N decreased alkaloid concentrations. We are not aware of any studies of the impact of new grass cultivars, bred for altered sugar metabolism, on alkaloid production, although sugar content has been proposed to alter function in a root endophyte (Peucedanum spec.) (Hadacek & Kraus, 2002).
One of the major difficulties encountered in previous studies has been in assessing the concentration of the fungus itself, as is necessary to address whether an environmental factor alters alkaloids by altering metabolism (e.g. alkaloid production per unit fungus) or by altering the concentration of the fungus. Although real-time PCR (qPCR) has been used previously to quantify, for example, arbuscular mycorrhizal fungi (Alkan et al., 2004), this method has only rarely been used in grass–Neotyphodium associations (Young et al., 2005). We present here an extension of this technique, for this purpose, which also has implications for understanding the scale of the contribution of the fungal genome to the expression and regulation of the metabolism of the association. Previous techniques based on hyphal counts, genetically modified reporter genes (Spiering et al., 2005) or ELISA (Stewart, 1986) have not led to widespread quantification of endophyte content in experimentation or practice in the productive pasture context.
The N. lolii/ryegrass association prevails in some of the most fertile, productive temperate grassland regions typified by New Zealand. It is important to confirm whether recent trends for greatly increased use of N fertilizer, and the introduction of novel traits such as ‘high sugar’ grass cultivars, will lead to sufficiently retained toxicity to insects, and yet remain safe for grazing mammals. However, the insights gained from relatively controlled manipulations of nutrient and energy supply to the host and fungus can have wider significance to understanding the nature of grass/endophyte associations, in general. Notably, more controlled manipulations are required to test the extent to which the association is truly mutualistic, or whether changes in resource supply in the host plant, to its direct advantage, may have adverse impacts on fungal presence, as would be implied by more recent hypotheses of the nature of plant/endophyte associations in natural as well as cultivated grassland ecosystems (Faeth & Fagan, 2002; Saikkonen et al., 2006).
Here, we report an environmental study of how N fertilization interacts with a selected ‘high sugar’ trait in ryegrass and how both features interact with abundance and alkaloid production of three N. lolii strains.