DOI: 10.5061/dryad.d7wm37q29
title: Interactions between silicon and alkaloid defences in endophyte-infected grasses and the consequences for a folivore

description: 1. Grasses have developed a wide range of morphological and physiological
 mechanisms to resist herbivory. For instance, they accumulate silicon (Si)
 in tissue, as physical defence, and associate symbiotically with foliar
 Epichloë-endophytes that provide chemical defence via antiherbivore
 alkaloids. Recent evidence showed that some Epichloë-endophytes increase
 foliar Si in forage grasses; however, it is unknown whether this impacts
 insect herbivores. Furthermore, while Si is primarily a physical defence,
 it also affects production of plant defensive secondary metabolites; Si
 supply might therefore affect Epichloë-alkaloids, although this remains
 untested. 2. We grew endophyte-free (Nil) and Epichloë-infected tall
 fescue and perennial ryegrass in a factorial combination with or without
 Si supplementation, in the absence or presence of Helicoverpa armigera.
 Epichloë-endophyte strains were AR584 for tall fescue, and AR37, AR1 or
 Wild-type (WT) for perennial ryegrass. We assessed how Si supply and
 Epichloë-endophytes in interaction with herbivory affected foliar Si and
 mutualist-derived alkaloid concentrations. Subsequently, their effects on
 H. armigera relative growth rates (RGR) were evaluated.  3.
 Endophytes generally increased Si concentrations in Si supplied plants. In
 tall fescue-AR584 and perennial ryegrass-AR37, endophytes increased
 constitutive (herbivore-free) and induced (herbivore-inoculated) Si
 concentrations by at least 25%; in contrast, in perennial ryegrass the AR1
 endophyte only increased constitutive levels. Si supply did not affect
 alkaloids produced by AR584- or AR1/WT-endophytes; however, in the
 presence of herbivory, Si supply decreased the induction of alkaloids
 produced by AR37 endophytes by 33%. For tall fescue, Si supply reduced H.
 armigera RGR by at least 76%, regardless of endophytic status, whereas,
 endophyte-alkaloids played a secondary role only reducing herbivore growth
 in the absence of Si supply. Conversely, in perennial ryegrass both Si and
 endophyte-alkaloids (regardless of Si supply) reduced herbivore RGR
 although not synergised.  4. Novel interactions between
 constitutive and induced Si- and alkaloid-based antiherbivore defences in
 grasses were observed. Overall, Si had a greater effect on the folivore
 than endophytes in both grasses. Endophyte-defences contributed more to
 herbivore resistance in perennial ryegrass than tall fescue. We
 demonstrate that Si and endophytes were not antagonistic and highlight
 that the protective nature of their interaction varies with the
 grass-endophyte species tested.
description: Dataset description <strong>ALL
 DATA.cvs</strong>- metadata utilized for tall fescue and perennial
 ryegrass respectively; This metadata was subsetted using R for analysis
 as described in the methods of the manuscript:
 "To determine individual treatment
 effects, both plant growth traits and primary chemistry
 were analyzed for each species
 separately, using a two-way ANOVA with Si supply and endophyte status as
 fixed effects." Here we utilized herbivore-free plants
 only.  "Likewise, shoot Si
 concentration was analyzed for each grass species separately, with a
 three-way ANOVA using Si supply, endophyte status, and herbivory as fixed
 effects." Here we used the full data set for each grass
 species.  "A
 multivariate analysis of variance (permutational MANOVA) using Si supply
 and herbivory treatments as fixed effect factors was utilized to determine
 differences in overall strain-specific alkaloid profiles (sum of all
 alkaloids produced by each endophyte-strain), using the ‘vegan’ package
 (Oksanen et al.,
 2019). The MANOVA function further
 provided results for univariate ANOVAs for treatment effects on individual
 alkaloids at the strain-specific level. " Here we
 utilized endophyte-infected plants only as endophyte-free plants do not
 produce alkaloids.  "Herbivore RGR were analyzed
 for each species separately, utilizing herbivore-inoculated plants only
 with a two-way ANOVA with Si supply and endophyte status as fixed
 effects." Here we utilized herbivore-inoculated plants only as HGR is
 a herbivore parameter.
 <strong>FES.ES.cvs &
 RYE.ES.cvs-</strong> data sets utilized to perform the effect
 size for tall fescue and perennial ryegrass respectively; as described in
 the methods of the manuscript: "...to compare
 the magnitude of the effect of Si supply and herbivory on strain-specific
 alkaloid production the standardised effect sizes (SES;
 Hedges’ d) was
 calculated for each grass species separately<strong> using only
 endophyte-infected plants</strong> as a
 standardised mean difference ((SMD) = (<i>µ1 – µ2)/s</i>,
 where <i>µ1 and µ2</i> are the mean trait value of
 <i>µ1</i> = herbivore-inoculated and <i>µ2</i> =
 herbivore-free, and <i>s</i> is the pooled standard
 deviation) using the
 ‘<i>effsize</i>’ package (Torchiano,
 2020). A 95% confidence interval bar
 that does not overlap zero indicates a significant effect; negative effect
 size indicates the effect decreases mean alkaloid production, and a
 positive effect size indicates that the effect increases mean alkaloid
 production. The SES was utilized to compare each strain-specific alkaloid
 production regardless of the amount produced (remove units) to describe
 the size of the effect"