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Endophytic fungi from the genus Neotyphodium, formerly Acremonium (Glenn et al. 1996), infect a variety of cool season grasses (Schardl et al. 1997). Although derived from the sexually reproducing pathogenic Epichloë (Clay 1990), Neotyphodium species lack a sexual phase, and do not produce spores (however, see White, Martin & Cabral 1996). They have no free-living stage and are thus ‘trapped’ in their host plant, never moving to uninfected hosts (i.e. there is no horizontal transmission). It has been argued that such an intimate relationship should select for evolution away from parasitism and toward mutualism (Ewald 1987). Nevertheless, the exact nature of the relationship between the grass and its endophytic fungus has been hotly debated in the literature (Faeth 2002; Saikkonen et al. 2004; Faeth & Hamilton 2006).
Two commonly studied Neotyphodium species are N. lolii (Latch, Christensen & Samuels) Glenn, Bacon & Hanlin, which infects Lolium perenne L. (perennial ryegrass), and Neotyphodium coenophialum (Morgan-Jones & Gams) Glenn, Bacon & Hanlin, which infects Schedonorus phoenix (Scop.) Holub (tall fescue), formerly known as Lolium arundinaceum (Schreb.) Darbysh. The relationship between these pairs of interacting species has frequently been described as a mutualism. The fungi benefit by obtaining nutrients from the host plant. The grasses benefit in several ways. Faeth et al. (2002) summarize the benefits as follows: (i) enhanced resistance of seeds and seedlings to predators and pathogens; (ii) reduced herbivory of mature plants; (iii) increased drought tolerance; (iv) increased resistance to other abiotic factors such as fire; and (v) increased intra- and interspecific competitive abilities of the host plant. While the mechanisms are not all known for each of these benefits, certainly some of them derive, at least in part, from the production of secondary metabolites, in planta, by the grass and fungus together (but see Rasmussen et al. 2007, 2008 for additional details on the biochemical implications of the grass-endophyte relationship).
Arbuscular mycorrhizal (AM) fungi form symbiotic associations with the roots of a wide variety of plant species, including a variety of grass species (Smith & Read 1997). In exchange for photosynthates, AM fungi can provide their host plants with increased access to nutrients and water and enhanced protection against pathogens (Newsham, Fitter & Watkinson 1995). Similarly to Neotyphodium species, AM fungi are obligate symbionts, fully reliant on their plant hosts for fixed carbon.
It has been known since the early 1990s that endophytes can affect AM fungi in the soil. These studies can be divided into two types: those that study the effects of the endophyte on AM fungal colonization of the endophyte's host plant; and those that study the effect of the endophyte on AM fungal colonization of non-endophytic plants or of AM fungal abundance in the soil in general. We will refer to the former studies as ‘direct effects’ of the endophyte, and the latter as ‘indirect effects’ of the endophyte.
A number of studies have examined the direct effects of endophyte-infection on AM fungal colonization. Several host plant – endophyte combinations were investigated using a variety of AM fungal species, primarily from the Glomus genus. In general, these studies show that AM fungal root colonization is reduced when the host plant is infected with the endophyte (e.g. Chu-Chou et al. 1992; Guo et al. 1992; Omacini et al. 2006; Mack & Rudgers 2008). Nevertheless, Novas, Cabral & Godeas (2005) observed an increase in colonization in their study. While the possible mechanism(s) by which the endophyte reduces colonization have not been investigated, the general assumption seems to be that endophyte-produced allelochemicals are likely responsible for the effect. Studies showing negative effects are from agronomic cultivars and the only study involving a native grass in its native habitat showed positive effects (Novas et al. 2005).
Indirect effects have been hypothesized based on the results from experiments on the direct effects of endophytes on AM fungi, and on the basis of the few studies that have specifically investigated indirect effects (Chu-Chou et al. 1992; Guo et al. 1993; Matthews & Clay 2001). In general, the idea is that endophytes alter the AM fungal community, either via allelopathic effects of the endophyte, or via the effect of the endophyte on the plant community and hence on the AM fungal community that might subsequently develop. In addition to studying the direct effects of N. coenophialum on AM fungal colonization, Chu-Chou et al. (1992) also reported that propagule densities of total Glomus spp. (i.e. all species combined), and specifically of G. fecundisporum and G. monosporum, were smaller in soil from endophyte-infected S. phoenix plots than from endophyte-free plots. In contrast, even though Matthews & Clay (2001) did not look at AM fungi directly, their data provide no evidence that endophyte induced changes in AM fungal communities directly affect the growth of subsequent plants over a large spatial scale.
Allelopathy is one possible mechanism by which endophyte-infected plants may exhibit reduced AM fungal colonization. Such potential effects have been reported several times on plant species (Preece et al. 1991; Malinowski, Belesky & Fedders 1999; Bertin et al. 2003; Orr, Rudgers & Clay 2005), but only rarely on AM fungal species (see above). While these previous studies demonstrate the potential for endophytes to produce allelopathic effects, they do not isolate the putative compounds involved, nor do they have high external validity. That is, these experiments show the potential for allelopathic effects, but are largely divorced from an appropriate ecological context, so it is unclear whether such effects are present in nature. Furthermore, there is a natural tendency to assume that alkaloids are the putative allelochemicals, for both plants and AM fungi. However, as Rasmussen et al. (2007, 2008) recently showed, endophyte-infected and endophyte-free grasses differ in the concentrations of many biochemicals, not just alkaloids. It is entirely possible that other compounds could be involved, in addition to, or instead of alkaloids.
Both Orr et al. (2005) and Preece et al. (1991) point out that the method of extraction of allelopathic compounds strongly influences the reported effects and interpretations of endophyte effects. Some of these methods are clearly not ecologically relevant. However, there are at least three ecologically relevant mechanisms by which putative allelopathic compounds might enter the soil and influence AM fungi. Koulman et al. (2007) demonstrated that alkaloids are at least sometimes mobilized and translocated in S. phoenix and L. perenne. They were able to detect alkaloids in both the guttation fluid and ‘cut leaf fluid’ (i.e. the fluid that flows out of tissue damaged by, for example, grazing mammals) of endophyte-infected L. perenne. Another possible pathway is via root exudation. Although these exudates have not, to our knowledge, been tested for alkaloid presence, Koulman et al.'s demonstration that alkaloids can be mobilized and translocated makes this route a viable possibility. Finally, potentially allelopathic compounds can enter the soil via the processes of leaching and decomposition, but little is known about these pathways’ potential role in the reported allelopathic effects of endophyte infection.
In this study, we use two strains of N. coenophialum (common and AR542) infecting the same cultivar of S. phoenix and known to produce different profiles of putative allelochemicals (i.e. alkaloids) to test the hypotheses that (i) allelopathic effects of the endophyte reduce AM fungal spore germination; and (ii) the allelochemical compound(s) are leached into the soil even after the death of S. phoenix, where they reduce AM fungal colonization of other plants.
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We identified a mode of entry into soil of chemicals possibly associated with reductions in AM fungal colonization. This was done in an ecological context, using a plant that co-exists with S. phoenix and soil that was supplied with its native microbial community. Moreover, given that the common strain of N. coenophialum is known to produce different putative allelochemicals (i.e. alkaloids) than AR542, specific compounds may potentially be responsible for those reductions. Ergovaline, peramine, N-formyl loline, N-acetyl loline and N-acetyl norloline are produced by the common endophyte, while AR542-infected plants produce only peramine and N-acetyl norloline (see Hunt & Newman 2005 for more details). Therefore, the fact that AR542-infected thatch did not affect the symbiosis suggests that ergovaline, N-formyl loline, N-acetyl loline, or some combination of these alkaloids could potentially be the cause of allelopathy.
Although the current results are consistent with the hypothesis that one or more of the alkaloids that are absent from AR542 are responsible for the reduced AM colonization, reasonable alternative hypotheses exist. First, Rasmussen et al. (2007) have shown that different strains of endophyte achieve different fungal densities when grown in hosts from the same genetic background (cultivar). They also showed that alkaloid concentration is linearly related to fungal concentration. Mack & Rudgers (2008) showed that AM fungal colonization was linearly correlated with endophyte hyphal density. Taken together, these results suggest the hypothesis that AR542 does not produce as dense a population of hyphae as the common strain of the endophyte, and as a result does not produce sufficient quantities of alkaloids to impact the AM fungal colonization. A second reasonable alternative hypothesis is that alkaloids have nothing to do with the reduction in AM colonization. Rasmussen et al. (2007, 2008) have shown that, within the same cultivar of ryegrass, plants infected with different strains of the endophyte were biochemically very different from each other, beyond differences in alkaloid production. This suggests that the difference between AR542 and common strain infected thatch, in terms of the AM colonization, might be due to compounds other than alkaloids. Until someone does a full metabolomics screening of these grass-endophyte combinations (cf. Rasmussen et al. 2008) little can be said about the likelihood of this hypothesis. Thus, differences in AM fungal response cannot be unequivocally attributed either to specific alkaloids, or even specifically to alkaloids. Nevertheless, differences in AM fungal responses to these two strains of endophyte offer a tool, albeit crude, for the preliminary investigation of mechanisms and compounds that may have adverse effects on AM fungi.
It is interesting that the ‘leached thatch’ treatment produced results similar to those seen with the common strain endophyte-infection. Even tough we cannot provide a definite explanation for this result, since the leached treatment consisted of a 1 : 1 mix of common strain-infected and AR542-infected thatch, and this mixture was leached by soaking it in water for 96 h, the fact that it did not differ substantially from the common strain treatment suggests that: (i) at least 50% less common strain-infected thatch is needed to produce the same result; and (ii) perhaps the compound(s) responsible is sparingly water soluble and effective at low concentrations. Many secondary metabolites, including alkaloids, are typically extracted by organic solvents under basic conditions (Schardl et al. 2007). So it would seem that our protocol based on leaching is unlikely to have freed much of the alkaloid content of the thatch, if indeed these were to be the chemicals responsible for the observed effects.
While there was undoubtedly some decomposition of the grass thatch, it would have been relatively low over the time course of this experiment (120 days). Moreover, the endophyte-infected thatch would decompose more slowly than the endophyte-free thatch (Omacini et al. 2004). Therefore, since watering was done through thatch laid on the soil surface of the pots, we presume that the mode of action was long-term leaching. A 96 h of leaching and half the biomass were insufficient to reduce the effectiveness of the common strain-endophyte in interfering with the B. inermis–G. intraradices relationship hence the long-term.
In contrast to AM fungal root colonization, the endophyte-effect on spore germination was comparable between the common and AR542 strains. It is possible that allelopathy produced by the common strain endophyte on the AM fungal symbiosis, is stronger on AM fungal growth or colonization, than on spore germination end points. A potential explanation for this is that starting with the germination of AM fungal spores, hyphal growth through the soil, host recognition and root colonization, each step may be influenced by different signalling compounds (Douds, Nagahashi & Podila 2000). Alternatively, it is possible that the differential patterns between endophytes observed in the second experiment are due to interactions between edaphic factors, biotic and/or abiotic, and the different varieties of thatch.
Certainly further work is needed to explain the phenomenon of reduced colonization by AM fungi in the presence of endophytes. We need to know whether the effects are indeed the result of alkaloid allelopathy, and if so what compound(s) is responsible, what concentrations are necessary and the mode of action on AM fungi. To further clarify these questions, we may be able to follow the same simple and low-cost approach used in this study to exploit other endophyte strains that produce even different profiles of secondary metabolites. For example, Lp19, AR1 and AR37 have been used previously with L. perenne. Lp19 produces ergovaline, lolitrem B and peramine. AR1 produces only peramine, and AR37 produces only a class of alkaloids called janthitrems (Rasmussen et al. 2007). From our study, we would predict that L. perenne infected with AR1 would not show decreased AM fungal colonization. However, we can say nothing about the potential of janthitrems to influence colonization.
Further work also needs to be conducted on the ecological implications of endophyte-mediated changes on the AM fungal symbiosis. This is a component that has been largely ignored in previous studies. If endophyte-allelochemicals inhibit AM colonization of competing plants, does this increase competitive abilities of S. phoenix? Alternatively, direct endophyte effects may negate any beneficial effects of inhibiting AM fungal colonization on competing plants. On one hand, this is supported by studies showing endophyte-AM fungal interactions which result in reductions of the beneficial effect of the endophyte on the host plant, in terms of insect resistance (Vicari, Hatcher & Ayres 2002). On the other hand, absence and sometimes even reduction of growth responses in plants colonized by AM fungi have been shown to be a common phenomenon, although the mechanisms responsible are not clear (Klironomos 2003; Jones & Smith 2004). This is consistent for example with our results for B. inermis and also with those of Mack & Rudgers (2008) for S. phoenix. Nevertheless, caution should be taken in generalizing that certain endophyte strains have negative impacts on the AM fungal symbiosis. As mentioned earlier, there is only a reasonable degree of support for this in agronomic cultivars.
In conclusion, specific characteristics of different Neotyphodium endophytes of S. phoenix may be responsible for negative effects on the AM fungal colonization of roots of other neighboring plant species, even upon the death of S. phoenix, through long-term leaching into soil. Such possibility may have important implications for natural ecosystem functioning, the management of pastures or forage crops and conservation efforts.