Plants, similar to all higher organisms, interact with an abundant and diverse microbiota, which is discovered to affect plant fitness (Zilber-Rosenberg and Rosenberg, 2008; Vandenkoornhuyse et al., 2015; Roughgarden et al., 2018; Enebe and Babalola, 2019; Saikkonen et al., 2020). Microbes, such as mycorrhizae, nitrogen-fixing bacteria, and asymptomatic endophytes, live symbiotically with their host plant. These symbionts can, for instance, affect the nutrient acquisition, phytohormone regulation, abiotic stress tolerance, and herbivory and pathogen resistance of their host (Ortíz-Castro et al., 2009; Berendsen et al., 2012; Dupont et al., 2015; Hassani et al., 2018; Nelson, 2018; Compant et al., 2019). Accordingly, these symbiotic microbes are commonly regarded as defensive plant mutualists, contrary to antagonistic parasitic and pathogenic microbes that have negative effects on host fitness (Clay, 2009).

However, the ecological roles of microbes in plant–microbe interactions are often complex, diverse, and prone to change from mutualistic to antagonistic and vice versa, depending on the abiotic and biotic conditions (Hayward, 1974; Carroll, 1988; Ahlholm et al., 2002; Partida-Martinez and Heil, 2011; Marsberg et al., 2017; Marchetto and Power, 2018; Shaffer et al., 2018; Meier and Hunter, 2019; Afkhami et al., 2020; Franklin et al., 2020; Laihonen et al., 2020; Petipas et al., 2020; Saikkonen et al., 2020). This continuum of antagonistic to mutualistic interactions in space and time (Saikkonen et al., 1998; Hirsch, 2004; Drew et al., 2021) must be considered in studies on plant microbe interactions. Furthermore, plants interact with myriads of organisms that probably interact not only with plants but also with each other (Lamichhane and Venturi, 2015; Bass et al., 2019). Despite the increasing number of studies characterizing plant microbiomes, ecological studies on the functional role of plant-associated microbes have only rarely examined the joint effects of the co-occurring microbes on the host plant. All this complexity of plant microbiomes shows the importance of comprehensive studies on reputed plant mutualistic or antagonistic microbes.

In this study, we examine the four-way interaction between red fescue (Festuca rubra L.), two fungal species, a vertically transmitted endophyte (Epichloë festucae Leuchtm., Schardl & M.R. Siegel) and a grass seed pathogen (Claviceps purpurea [Fr.] Tul.), and a phloem-feeding aphid (Sitobion sp.). F. rubra is a fine-leaved perennial grass growing in a wide range of habitats in the northern temperate zone. Some Epichloë species are common mutualistic symbionts of cool-season grasses. These systemic fungi grow asymptomatically in the aboveground parts of their hosts and reproduce asexually via host plant seeds (Schardl, 1996; Saikkonen et al., 2016). They often enhance their host's ability to endure abiotic stress, such as salinity or drought, or increase resistance against herbivores and pathogens (Schardl, 1996; Saikkonen et al., 1998, 2010). By contrast, C. purpurea, the ergot fungus, is widely recognized as a plant pathogen that infects the inflorescences in the Poaceae family. The fungus sterilizes the ovaries in flowers and utilizes the resources of its host to develop a sclerotium, preventing the development of a host plant seed. However, the pathogen usually castrates only a few flowers in a host plant inflorescence (Luttrell, 1980). Even though ecologically different, both Epichloë and Claviceps belong to the same fungal family, Clavicipitaceae.

Plant interactions with Epichloë and Claviceps have been thoroughly studied, but separately. Existing studies show that the presence of Epichloë occultans in Lolium multiflorum and Epichloë gansuensis in Achnatherum inebrians is correlated with a decreased frequency of C. purpurea infection, indicating that Epichloë mediates protection against C. purpurea (Pérez et al., 2013, 2017; Zhang et al., 2021). The increased pathogen resistance may result from the Epichloë-increased immunocompetence of the host, or from a direct competition between the invading microbe and the endophytic Epichloë (Saikkonen et al., 2016; Malinowski and Belesky, 2019). However, four-way interactions, including herbivores, have not been studied in this context. Both Epichloë and Claviceps produce animal-toxic alkaloids (Saikkonen et al., 2013; Miedaner and Geiger, 2015; Florea et al., 2017) that may synergistically increase herbivore resistance. The Epichloë-increased anti-herbivory defenses are primarily accounted for fungal-origin alkaloids including pyrrolizidines (lolines), ergot alkaloids, indolediterpenoids (lolitrems), and pyrrolopyrazines (peramine) (Saikkonen et al., 2013, 2016; Schardl et al., 2013). The sclerotia of C. purpurea contain high concentrations of ergot alkaloids that deter herbivores and fungivores (Miedaner and Geiger, 2015). Vertebrate herbivores avoid seed sets with only a few C. purpurea sclerotia which can increase host plant fitness in highly grazed environments. This attribute questions the strict pathogenic nature of C. purpurea (Wäli et al., 2013).

Here, in a four-way interaction, we examine how symbiotic E. festucae affects the natural colonization of C. purpurea and how these two fungi jointly affect herbivores in their common host plant in a semi-natural subarctic setting. We investigated in particular, whether:

All three species compete for the host plant's resources; however, we assume their ecological interactions with the host are as follows: Epichloë commonly forms mutualistic interaction with the host plant; C. purpurea is mainly antagonistic but potentially becomes beneficial under high herbivory pressure; and aphids as herbivores are pure antagonists to their host. Based on existing literature, we hypothesize that symbiosis with Epichloë decreases Claviceps infection frequency, and both fungal species contribute to plant defense, negatively affecting aphid performance. We discuss our findings regarding multiple contexts and defensive mutualism hypotheses.

Our results do not support the hypothesis that defensive mutualism against invertebrate herbivores and pathogens drives endophyte–plant symbiosis. Contrary to our predictions that E. festucae should increase the resistance of its host grass, F. rubra, the aphid numbers were similar on Epichloë-symbiotic (E+) and Epichloë-free (E–) plants, and the infection frequencies of pathogenic Claviceps were higher in E+ plants than in E– ones. However, as expected, Claviceps provided notable protection against aphid herbivory, suggesting that the net effects on the host can remain positive as the infection causes only minor seed loss (Luttrell, 1980; Wäli et al., 2013). These results emphasize the importance of understanding the structure and functional complexity of plant microbiomes by demonstrating that plant–pathogen interactions are context dependent, ranging from antagonistic to mutualistic, rather than always disadvantageous to the host plant (Saikkonen et al., 1998, 2020; Vázquez de Aldana et al., 2010; Wäli et al., 2013; Rybakova et al., 2016; Selosse et al., 2018).

As the defensive mutualism between grasses and Epichloë species is primarily attributable to fungal origin alkaloids (Saikkonen et al., 2010; Schardl et al., 2013), our results suggest that the fungal genotypes associated with the plants could not provide protection against aphids by producing a sufficient amount of alkaloids. Our previous transplant study with F. rubra plants collected from the same wild populations revealed that E. festucae can produce peramine and ergovaline; however, alkaloid profiles in plants varied among the geographic origin and growth conditions of the symbiotum (Vázquez de Aldana et al., 2020). Peramine was only produced by the Spanish genotypes. Ergovaline was detected in E+ plants across Europe, but the concentrations varied among fungal strains, and the profiles changed when the symbiotum was transplanted to new environments. For example, none of the Spanish E+ plants contained detectable amounts of ergovaline in Northern Finland. In line with our previous study, ergot alkaloid concentrations varied considerably among E+ plants in the present study. Similarly, many other studies with other grass species have revealed that the quantities of Epichloë alkaloids vary among geographic origin and genetic plant–fungus combinations and can be plastic during the growing season depending on environmental conditions (Siegel et al., 1990; Thom et al., 2014; Helander et al., 2016; König et al., 2018; Fuchs et al., 2020).

In this study, we focused on the putative anti-herbivore properties of ergot alkaloids, which are a diverse family of mycotoxin compounds with a common origin (Panaccione, 2005). Unlike the majority of past studies treating ergot alkaloids as a pooled ensemble, we identified seven different ergot alkaloids from plant extracts (ergovaline, ergonovine, ergosine, ergotamine, ergocornine, α-ergocryptine, and ergocristine) and examined their potential in modulating host plant quality to aphids. Ergovaline is the main ergot alkaloid produced by Epichloë in most host grasses and a minor component of C. purpurea (Garner et al., 1993); the other six compounds are the main alkaloids of C. purpurea (Miedaner and Geiger, 2015). Our alkaloid results are in line with this literature, but ergonovine was also present in E+ plants, and we were not able to clearly associate presence of ergotamine with Claviceps infection only. The few earlier studies on individual alkaloids have shown variable effects on herbivores. For example, ergonovine is responsible for aphid mortality on sleepygrass (Achnatherum robustum) (Shymanovich et al., 2015), and similarly ergocryptine markedly explains the variation in fall armyworm dry weight on perennial ryegrass (Lolium perenne) (Salminen et al., 2005). By contrast, ergovaline appears to be insignificant for the performance of root or shoot aphids (Siegel et al., 1990; Popay et al., 2021). The issue becomes even more complicated when other trophic layers are involved. For example, Kunkel et al. (2004) showed that ergot alkaloids can mediate cascading effects in food webs, thereby indirectly benefiting herbivores due to their toxic effects on their natural enemies. In concordance with these studies, our results demonstrate dissimilar effects of individual ergot alkaloids on herbivores. Overall, the concentrations of all examined alkaloids were highest in Claviceps-infected E+ plants, suggesting the synergistic effect of both fungi on the alkaloid profile of the host grass. Concentrations of three out of the seven examined alkaloids (ergovaline, ergotamine, and ergocristine) were negatively correlated with the number of aphids on the plants. Epichloë symbiosis explained only the presence of ergovaline and ergonovine, whereas Claviceps explained the concentrations of ergosine, ergocornine, α-ergocryptine, and ergocristine. Furthermore, ergocristine was associated with both Claviceps infection and the low number of aphids on the plants. We acknowledge that the chemical ecology underlying these results should be interpreted cautiously, since we studied only ergot alkaloids, and the analyses do not allow us to distinguish other Epichloë- and Claviceps-origin alkaloids in the samples. Still, since we found no compelling evidence that Epichloë symbiosis confers protection against aphids, we suggest that Claviceps is primarily responsible for the production of anti-herbivore compounds and for the reduction of aphid infestation. However, we acknowledge that additional chemical changes may occur (volatiles, etc.), which when combined with the examined alkaloids might reduce the aphid infestation in C+ plants.

The lack of Epichloë-enhanced host grass resistance to aphids can be partly explained by the high variation among the examined fungal lineages in their ability to produce alkaloids due to their genetic and chemotypic differentiation in Spanish and Finnish plants under different selection pressures. Empirical evidence supports the idea that post-glacial colonization history and contrasting climatic environments have resulted in local adaptations and genetic differentiation in symbiotum across its range in Europe (Dirihan et al., 2016; Leinonen et al., 2019; von Cräutlein et al., 2019, 2021). Strong seasonal changes in temperature, including short growing seasons and long winters, and variation in day length and light quality characterize environments at high latitudes, whereas plants in Spanish semiarid grasslands must adapt to seasonal drought (Zabalgogeazcoa et al., 2006; Leinonen et al., 2019). Genetic potential for diverse alkaloid production is remarkable in southern populations due to the prevalence of sexual reproduction in Spanish Epichloë populations (von Cräutlein et al., 2019, 2021). Although the northern populations have not adapted to strong invertebrate herbivory, the typically high Epichloë frequencies in wild F. rubra populations across its range in Europe are likely to be attributable to other benefits associated with Epichloë. Our previous transplant experiment with plants collected from the same geographic regions suggests that abiotic factors have not played a significant role in maintaining Epichloë symbiosis (Dirihan et al., 2016; Leinonen et al., 2019). Many examined European F. rubra populations with high frequencies of E+ commonly show strong vertebrate grazing—for example, by sheep in the Faroe Islands and Iceland and large ungulates in Spain, Switzerland, and northern Finland (Dirihan et al., 2016). This finding suggests that vertebrate grazing has been among the main selective forces driving the coevolution of E. festucae and F. rubra in Europe.

Increased susceptibility of E+ plants to the pathogenic Claviceps fungus compared with their E-conspecifics suggests that reciprocal changes during the long coevolutionary history of Epichloë-species and their host grasses involve the loss of host traits that prevent microbial invasions. These changes may result from modulated recognition, signaling, and defense responses (Saikkonen et al., 2013; van Overbeek and Saikkonen, 2016; Schmid et al., 2017; Bastías et al., 2018; Compant et al., 2019; Nissinen et al., 2019). Our results demonstrate that the consequences of such a predisposition to pathogens can be advantageous to the host plant in environments where a pathogen with only marginal damage to the host provides reinforced protection against pests. This finding questions whether these changes in host traits resulted from tripartite coevolution with reciprocal changes in the partners.

We propose that the independent coevolution of these two closely related fungi with their shared host plant might have resulted in the detection of complementary protection against herbivores and other benefits to each other as by-products without reciprocity and cooperation (Leimar and Connor, 2003). Vertical transmission and alkaloid production ability of Epichloë-species have selected benign symbiosis with the host grass, particularly in environments driven by strong herbivory pressure. The fitness of Claviceps is similarly highly dependent on the protection of host inflorescences to ensure successful sclerotium development to complete its life cycle. However, the occurrence and distribution of Claviceps is primarily dependent on the presence of the host and favorable weather and climatic conditions, whereas the heritable infections of Epichloë species depend on the host fitness. Although host protection can be regarded as an indirect by-product of mutualism, our results are consistent with a few other recent findings, suggesting that Epichloë species must be considered keystone species in shaping the microbial communities of their shared hosts (Nissinen et al., 2019).

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ML and BF originally formulated the idea, performed the experiments, and analyzed the data. ML, BF, KS, and MH conceived and designed the experiments. BV and IZ performed the chemical analyses. ML, KS, MH, BV, IZ, and BF wrote the manuscript. All authors contributed to the article and approved the submitted version.

This work was supported by the Academy of Finland (KS, Grant Nos. 295976 and 326226; MH, Grant No. 311077; BF, Grant No. 324523), the Spanish Ministry of Science and Innovation and FEDER grant PID2019-109133RB-I00, and from the project CLU-2019-05—IRNASA/CSIC Unit of Excellence funded by Junta de Castilla y León and co-financed by the European Union (ERDF Europe drives our growth).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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