Mycorrhizal fungi compromise production of endophytic alkaloids, increasing plant susceptibility to an aphid herbivore

1 INTRODUCTION

Symbiosis plays a key role in plant biology affecting growth, adaptation, and speciation (Uroz et al., 2019). The plant and fungal kingdoms engage in symbiotic relationships such as mycorrhizae and endophytic associations, supporting nutrient uptake and stress tolerance. Fungi also play key roles in decomposition, nutrient cycling, and disease dynamics, underscoring their importance in ecosystem health and agriculture. For example, some temperate grasses within the Poaceae family, establish symbiotic associations with a myriad of microbes including asexual Epichloë endophytes (Ascomycota: Clavicipitaceae) and arbuscular mycorrhizal (AM) fungi (Glomeromycotina) in shoots and roots, respectively. These symbionts often occur simultaneously and influence enemies of the plant, including herbivorous animals, via changes in plant nutritional and defensive traits (Casas et al., 2022; Omacini et al., 2006; Perez et al., 2021). However, predicting the impact of simultaneous symbioses on plant–herbivore interactions remains challenging since they are usually studied separately.

As protective mutualists, endophyte anti-herbivore defences mainly operate directly via the production of toxic alkaloids (Bastías et al., 2017). In terms of alkaloids, four major groups of endophyte alkaloids have been reported including ergot alkaloids, indole-diterpenes, pyrrolizidines (e.g. loline) and pyrrolopyrazines (e.g. peramine) (Berry et al., 2019; Schardl, Florea, et al., 2013; Schardl, Young, et al., 2013). Nonetheless, only grasses harbouring endophytes that putatively produce peramine (e.g. anti-chewing insect defences) and loline (e.g. anti-aphid and anti-chewing defences) are desirable in pastures because they confer herbivorous insect resistance without affecting grazing mammals (Young et al., 2013). While lolines are strongly insecticidal (i.e. toxic), peramine often acts as a potent insect-feeding deterrent (Saikkonen et al., 2016). Additionally, endophytes can have a detrimental effect on herbivores by stimulating the increased production of host plant defences (Bastías et al., 2017).

As nutritional mutualists, AM fungi often enhance plant nutrient supply (mainly phosphorous and nitrogen) through their highly specialized intracellular structures (i.e. arbuscules and vesicles) and their extensive hyphal networks that capture soil nutrients otherwise unavailable for plants (Lanfranco et al., 2018). In addition to improving plant nutritional quality, a growing number of reports suggest that AM fungi also improve host resistance to both herbivorous insects (Koricheva et al., 2009) and pathogens (Gernns et al., 2001). Specifically, for piercing-sucking aphids, AM fungi have been reported to have positive (Hartley & Gange, 2009; Simon et al., 2017), negative (Guerrieri et al., 2004) and neutral (Williams et al., 2014) impacts on aphid performance. Positive effects of AM fungi on aphid performance can be mediated by their effects on plant N uptake and the increase of tissue quality (Wilkinson et al., 2019). Nitrogen is often a limiting factor in insect herbivore diets, thus, nitrogen acquisition by insects is strongly correlated with higher food utilization (Mattson, 1980). AM fungi-mediated increases in plant growth may result in increased plant tolerance to herbivorous pests (Bennett et al., 2006) or AM fungi may negatively impact herbivores via increased production of host plant defences (e.g. defence priming, Pozo & Azcón-Aguilar, 2007). However, the exact mechanism for the influence of AM fungi on anti-herbivore defence still remains highly speculative (Biere & Bennett, 2013; Pozo & Azcón-Aguilar, 2007).

Both symbionts act as carbon sinks thus, dual infection, at least in some environments, may result in competition for photosynthates. This competition may result in excessive costs to the plant (e.g. non-additive dynamics), potentially negating any benefits derived from the symbiosis (Omacini et al., 2006). In this sense, endophytes might have both spatial and temporal priority over AM fungi with respect to plant carbon, since they are located within the leaf sheaths where carbon is fixed; thus, presumably they have greater access to carbon compared with fungi in roots. with regards to temporal advantages, endophytes are vertically transmitted through the host seed, therefore, they are associated with the host even before germination (Mack & Rudgers, 2008). Previous studies show that endophytes and AM fungi affect one another in terms of colonization and functionality, ranging from being (antagonistic Li, Guo, et al., 2018; Müller, 2003; Omacini et al., 2006) to mutually beneficial (Novas et al., 2010; Vignale et al., 2018). The underlying mechanism for antagonism includes the indirect effects of endophytes on the soil environment that reduce sporulation and colonization of AM fungi, mainly via the release of endophyte alkaloids into the soil (Omacini et al., 2006; Vignale et al., 2016). In cases of facilitation, endophytic exudates in the soil have been proven to stimulate AM colonization and even lengthen extraradical mycelia (Novas et al., 2010; Vignale et al., 2018).

Another highly effective anti-herbivore mechanism in grasses is their ability to accumulate large amounts of silicon (Si) from the soil (Alhousari & Greger, 2018). Si anti-herbivore defences operate through several mechanisms, including strengthened plant tissues that can wear down herbivore mouthparts, impede tissue penetration, and interfere with digestion and nutrient acquisition (Andama et al., 2020; Cibils-Stewart et al., 2023; Massey & Hartley, 2009), or alter herbivore immunity (Cibils-Stewart et al., 2023). Si accumulation can also affect plant allocation to a range of secondary metabolites (Hall et al., 2019), including alkaloids (Hall et al., 2021). It has been suggested that Si may act as a metabolically cheaper herbivore defence (i.e. physical) than secondary metabolite production (i.e. carbon-based compounds), a hypothesis supported by the negative relationship between Si and both phenolics and tannins reported for many plant taxa (Cooke & Leishman, 2012; Frew et al., 2016; Moles et al., 2013).

There is evidence that Si accumulation in plants increases when colonized by either Epichloë endophytes (Cibils-Stewart et al., 2020, 2021, 2023; Huitu et al., 2014) or AM fungi (Frew et al., 2017), although not universal (AM: Johnson et al., 2022; Vega et al., 2021; endophytes: Johnson et al., 2023). However, despite their co-occurrence in nature, no studies have yet addressed whether the two symbionts interact to affect Si accumulation in plants and associated herbivore resistance, or whether Si supply affects symbiont-mediated herbivore defences. It is currently unknown how endophytes and AM fungi interact, and if any potential competition for carbon, affects the accumulation of Si and the production of alkaloid defences. Furthermore, there is still uncertainty about the effectiveness of Si defences against piercing-sucking insects such as aphids (Massey et al., 2006; Johnson et al., 2020; but see Reynolds et al., 2009). However, since symbiont-produced metabolites (i.e. endophytic alkaloids) generally come at an energetic cost (i.e. carbon) to the plant (Bastías et al., 2017), there is potential for Si supply to affect their production.

The objective of this study was to investigate the interactions between Si supply, multisymbiotic relationships (endophytes and AM fungi), and the presence/absence of aphid herbivory in the production and effectiveness of physical (Si) or symbiont-mediated (endophyte alkaloids) anti-aphid defences in grasses. For this, we determined whether Si supply, Epichloë endophyte, AM fungi, and herbivory by the global pest Rhopalosiphum padi altered (i) grass physical defences (i.e. foliar Si concentrations), as well as (ii) plant growth, primary chemistry and physiology. Moreover, we determined if these factors singly or in combination are additively or non-additively (i.e. antagonistically, additively, or synergistically) altered, (iii) symbiotic traits including AM fungi colonization and endophytic alkaloid production. While positive relationships between concentrations of hyphae and alkaloids have been demonstrated in some plant–endophyte symbioses, it is important to note that alkaloid production may not always be a performance variable for Epichloë fungal endophytes. Finally, the consequence of the interactions between Si supply, Epichloë endophyte, and AM fungi presence was evaluated for (iv) population and individual performance of the aphid pest.