Silicon accumulation suppresses arbuscular mycorrhizal fungal colonisation in the model grass Brachypodium distachyon

Purpose Silicon (Si) accumulation by grasses alleviates diverse biotic and abiotic stresses. Despite this important functional role, we have limited understanding of how root microbial symbionts, such as arbuscular mycorrhizal (AM) fungi, affect Si uptake and even less about how Si supply and accumulation affect AM fungal colonisation. Our objective was to determine the nature of this two–way interaction in the model grass, Brachypodium distachyon. Methods We grew B. distachyon with five levels of Si supplementation using wild-type plants and a mutant (Bdlsi1-1) that has little capacity for Si uptake. Half of the plants were colonised by AM fungi; half were free of AM fungi. We measured Si accumulation, AM fungal colonisation, leaf carbon (C), nitrogen (N) and phosphorus (P) concentrations. Results AM fungi did not affect Si accumulation, although small increases occurred when root mass was included as a covariate. Si supplemented soil promoted plant growth and P uptake. Si accumulation suppressed colonisation by AM fungi and C concentrations in wild type but not in Bdlsi1-1 plants. Si concentrations were negatively correlated with C and N concentrations, with correlations being stronger in wild-type plants than Bdlsi1-1 plants. Conclusions Our results indicate that Si accumulation in the plant, rather than Si availability in the soil, underpinned reduced AMF colonisation. We propose that Si accumulation is unlikely to be impacted by AM fungi in plants with inherently high Si accumulation, but Si accumulation may suppress AM fungal colonisation in such plants

Reciprocal effects of silicon supply and endophytes on silicon accumulation and Epichloë colonization in grasses

Cool season grasses associate asymptomatically with foliar Epichloë endophytic fungi in a symbiosis where Epichloë spp. protects the plant from a number of biotic and abiotic stresses. Furthermore, many grass species can accumulate large quantities of silicon (Si), which also alleviates a similar range of stresses. While Epichloë endophytes may improve uptake of minerals and nutrients, their impact on Si is largely unknown. Likewise, the effect of Si availability on Epichloë colonization remains untested. To assess the bidirectional relationship, we grew tall fescue (Festuca arundinacea) and perennial ryegrass (Lolium perenne) hydroponically with or without Si. Grasses were associated with five different Epichloë endophyte strains [tall fescue: AR584 or wild type (WT); perennial ryegrass: AR37, AR1, or WT] or as Epichloë-free controls. Reciprocally beneficial effects were observed for tall fescue associations. Specifically, Epichloë presence increased Si concentration in the foliage of tall fescue by at least 31%, regardless of endophyte strain. In perennial ryegrass, an increase in foliar Si was observed only for plants associated with the AR37. Epichloë promotion of Si was (i) independent of responses in plant growth, and (ii) positively correlated with endophyte colonization, which lends support to an endophyte effect independent of their impacts on root growth. Moreover, Epichloë colonization in tall fescue increased by more than 60% in the presence of silicon; however, this was not observed in perennial ryegrass. The reciprocal benefits of Epichloë-endophytes and foliar Si accumulation reported here, especially for tall fescue, might further increase grass tolerance to stress

Elevated atmospheric CO2 suppresses silicon accumulation and exacerbates endophyte reductions in plant phosphorus

Many temperate grasses are both hyper-accumulators of silicon (Si) and hosts of Epichloë fungal endophytes, functional traits which may alleviate environmental stresses such as herbivore attack. Si accumulation and endophyte infection may operate synergistically, but this has not been tested in a field setting, nor in the context of changing environmental conditions. Predicted increases in atmospheric CO2 concentrations can affect both Si accumulation and endophyte function, but these have not been studied in combination. We investigated how elevated atmospheric CO2 (eCO2), Si supplementation, endophyte-presence and insect herbivory impacted plant growth, stoichiometry (C, N, P and Si), leaf gas exchange (rates of photosynthesis, stomatal conductance, transpiration rates) and endophyte production of anti-herbivore defences (alkaloids) of an important pasture grass (tall fescue; Lolium arundinaceum) in the field. eCO2 and Si supplementation increased shoot biomass (+52% and +31%, respectively), whereas herbivory reduced shoot biomass by at least 35% and induced Si accumulation by 24%. Shoot Si concentrations, in contrast, decreased by 17%–21% under eCO2. Si supplementation and herbivory reduced shoot C concentrations. eCO2 reduced shoot N concentrations which led to increased shoot C:N ratios. Overall, shoot P concentrations were 26% lower in endophytic plants compared to non-endophytic plants, potentially due to decreased mass flow (i.e. observed reductions in stomatal conductance and transpiration). Alkaloid production was not discernibly affected by any experimental treatment. The negative impacts of endophytes on P uptake were particularly strong under eCO2. We show that eCO2 and insect herbivory reduce and promote Si accumulation, respectively, incorporating some field conditions for the first time. This indicates that these drivers operate in a more realistic ecological context than previously demonstrated. Reduced uptake of P in endophytic plants may adversely affect plant productivity in the future, particularly if increased demand for P due to improved plant growth under eCO2 cannot be met. Read the free Plain Language Summary for this article on the Journal blog. © 2023 The Authors. Functional Ecology published by John Wiley & Sons Ltd on behalf of British Ecological Society

Advances in understanding plant root responses to root-feeding insects

In terms of the number of animal and plant species that occupy our planet, around 26% are herbivorous insects (Strong et al., 1984). They represent enormous diversity, particularly in terms of how they consume and exploit plant resources. A comparatively small proportion of herbivorous insects, limited to six orders, feed belowground on plant roots (Brown and Gange, 1990). While some root herbivores spend their entire lifecycle belowground (e.g. grape phylloxera), it is likely that the majority of species have aboveground life stages (usually adult) and it is the juvenile (larval) life-stages that attack roots. Rootfeeding insects differ markedly from shoot-feeding insects in terms of their ecology and life-history traits. While there are many exceptions to the rule, these differences can be broadly generalized (see Table 1) (Johnson et al., 2016b). In particular, root herbivores are probably represented in just 17% of families: they are predominantly chewers, have relatively long lifespans, live in highly aggregated populations and are in constant contact with the immense microbial communities found in the soil (Johnson et al., 2016b). As a group, there are still major gaps in our knowledge about how they interact with roots compared with our understanding of aboveground insect herbivore interactions (Hunter, 2001)

Short-term exposure to silicon rapidly enhances plant resistance to herbivory

Silicon (Si) can adversely affect insect herbivores, particularly in plants that evolved the ability to accumulate large quantities of Si. Very rapid herbivore-induced accumulation of Si has recently been demonstrated, but the level of protection against herbivory this affords plants remains unknown. Brachypodium distachyon, a model Si hyperaccumulating grass, was exposed to the chewing herbivore, Helicoverpa armigera, and grown under three conditions: supplied Si over 34 days (+Si), not supplied Si (-Si), or supplied Si once herbivory began (-Si+Si). We evaluated the effectiveness of each Si treatment at reducing herbivore performance and measured Si-based defenses and phenolics (another form of defense often reduced by Si). Although Si concentrations remained lower, within 72 hr of exposure to Si, -Si+Si plants were as resistant to herbivory as +Si plants. Both +Si and -Si+Si treatments reduced herbivore damage and growth, and increased mandible wear compared to -Si. After 6 hr, herbivory increased filled Si cell density in -Si+Si plants, and within 24 hr, -Si+Si plants reached similar filled Si cell densities to +Si plants, although decreased phenolics only occurred in +Si plants. We demonstrate that plants with short-term Si exposure can rapidly accumulate Si-based anti-herbivore defenses as effectively as plants with long-term exposure

Elevated atmospheric CO2 changes defence allocation in wheat but herbivore resistance persists

Predicting how plants allocate to different anti-herbivore defences in response to elevated carbon dioxide (CO2) concentrations is important for understanding future patterns of crop susceptibility to herbivory. Theories of defence allocation, especially in the context of environmental change, largely overlook the role of silicon (Si), despite it being the major anti-herbivore defence in the Poaceae. We demonstrated that elevated levels of atmospheric CO2 (e[CO2]) promoted plant growth by 33% and caused wheat (Triticum aestivum) to switch from Si (–19%) to phenolic (+44%) defences. Despite the lower levels of Si under e[CO2], resistance to the global pest Helicoverpa armigera persisted; relative growth rates (RGRs) were reduced by at least 33% on Si-supplied plants, irrespective of CO2 levels. RGR was negatively correlated with leaf Si concentrations. Mandible wear was c. 30% higher when feeding on Si-supplemented plants compared to those feeding on plants with no Si supply. We conclude that higher carbon availability under e[CO2] reduces silicification and causes wheat to increase concentrations of phenolics. However, Si supply, at all levels, suppressed the growth of H. armigera under both CO2 regimes, suggesting that shifts in defence allocation under future climate change may not compromise herbivore resistance in wheat

Leaf silicification provides herbivore defence regardless of the extensive impacts of water stress

1. Altered precipitation patterns due to climate change are likely to impose water-deficit stress in plants resulting in changes to specific leaf mass, leaf water content and chemical defences that may impact herbivorous arthropods. Grasses, in particular, accumulate large concentrations of silicon (Si) which provides physical defence against herbivores. Although Si uptake by plants may be affected by water availability, very few studies have investigated the combined effect of water-deficit stress and Si on insect herbivore performance. 2. We grew tall fescue (Festuca arundinacea Schreb.) hydroponically, with and without Si, and half of the plants were treated with 20% polyethylene glycol (PEG) to impose osmotic stress. Eleven leaf traits (physiological, chemical and structural) were measured, silicified phytoliths on the leaf surface were visualised using scanning electron microscopy (SEM) in conjunction with X‐ray mapping, and plants were exposed to a chewing insect herbivore (Helicoverpa armigera Hübner (Lepidoptera: Noctuidae)). 3. Although osmotic stress was associated with changes to leaf physiological and chemical traits, including increased specific leaf mass, decreased leaf relative water content and increased leaf nitrogen (N), there was no significant effect on H. armigera relative growth rate (RGR). However, Si reduced RGR of H. armigera by 80‐98%, while generating few changes to physiological and chemical leaf traits. Instead, the decline in RGR with Si was associated with changes to leaf structural traits, in particular, a greater density of silicified phytoliths on the leaf surface. 4. Comparison of effect sizes indicated that leaf traits were primarily affected by osmotic stress but not Si, and that herbivore RGR was strongly negatively affected by Si but not osmotic stress. There was no interactive effect between the osmotic stress and Si treatments on H. armigera RGR or plant traits except for leaf nitrogen and phenolic concentrations. This study provides further support that Si may prove to be beneficial to plants against chewing insect pests and remains robust regardless of water‐deficit stress conditions

Anti-herbivore silicon defences in a model grass are greatest under Miocene levels of atmospheric CO2

Silicon (Si) has an important role in mitigating diverse biotic and abiotic stresses in plants, mainly via the silicification of plant tissues. Environmental changes such as atmospheric CO2 concentrations may affect grass Si concentrations which, in turn, can alter herbivore performance. We recently demonstrated that pre‐industrial atmospheric CO2 increased Si accumulation in Brachypodium distachyon grass, yet the patterns of Si deposition in leaves and whether this affects insect herbivores performance remains unknown. Moreover, it is unclear whether CO2‐driven changes in Si accumulation are linked to changes in gas exchange (e.g. transpiration rates). We therefore investigated how pre‐industrial (reduced) (rCO2, 200 ppm), ambient (aCO2, 410 ppm) and elevated (eCO2, 640 ppm) CO2 concentrations, in combination with Si‐treatment (Si+ or Si‐), affected Si accumulation in B. distachyon and its subsequent effect on the performance of the global insect pest, Helicoverpa armigera. rCO2 increased Si concentrations by 29% and 36% compared to aCO2 and eCO2, respectively. These changes were not related to observed changes in gas exchange under different CO2 regimes, however. The increased Si accumulation under rCO2 decreased herbivore relative growth rate (RGR) by 120% relative to eCO2, whereas rCO2 caused herbivore RGR to decrease by 26% compared to eCO2. Si supplementation also increased the density of macrohairs, silica and prickle cells, which was associated with reduced herbivore performance. There was a negative correlation between macrohair density, silica cell density, prickle cell density and herbivore RGR under rCO2 suggesting that these changes in leaf surface morphology were linked to reduced performance under this CO2 regime. To our knowledge, this is the first study to demonstrate that increased Si accumulation under pre‐industrial CO2 reduces insect herbivore performance. Contrastingly, we found reduced Si accumulation under higher CO2, which suggests that some grasses may become more susceptible to insect herbivores under projected climate change scenarios

[In Press] Siliceous and non-nutritious : nitrogen limitation increases anti-herbivore silicon defences in a model grass

1. Silicon (Si) accumulation alleviates a diverse array of environmental stresses in many plants, including conferring physical resistance against insect herbivores. It has been hypothesised that grasses, in particular, utilise ‘low metabolic cost’ Si for structural and defensive roles under nutrient limitation. While carbon (C) concentrations often negatively correlate with Si concentrations, the relationship between nitrogen (N) status and Si is more variable. Moreover, the impacts of N limitation on constitutive physical Si defences (e.g. silica and prickle cells) against herbivores are unknown. 2. We determined how N limitation affected Si deposition in the model grass Brachypodium distachyon and how changes in these constitutive defences impacted insect herbivore (Helicoverpa armigera) growth rates. We used scanning electron microscopy (SEM) and energy dispersive X-ray spectrometry in conjunction with X-ray mapping (XRM) to quantify physical structures on leaves and determine Si deposition patterns. We also determined how N limitation and Si supply impacted the jasmonic acid (JA) pathway, the master-regulator of induced defences against arthropod herbivores. 3. N limitation reduced shoot growth by over 40%, but increased root mass (+21%), leaf Si concentrations (+50%) and the density of silica (+28%) and flattened prickle (+76%) cells. EDS and XRM established that Si was being deposited in these structures, together with hooked prickle cells and macro-hairs. Herbivore relative growth rates (RGR) were more than 115% lower in Si supplied plants compared to plants without Si supply and negatively correlated with leaf Si concentration and silica cell density. RGR was further reduced by N limitation and positively correlated with leaf N concentrations. Increases in JA concentrations following induction of the JA pathway were at least doubled by N limitation. 4. Synthesis: Si accumulation and deposition were highly regulated by N availability, with N limitation promoting both constitutive Si physical defences and induction of the JA defensive pathway, in line with the resource availability hypothesis. These results indicate that grasses use ‘low cost Si’ when resources are limited and suggests that plant productivity may benefit from optimising conventional fertilisers and Si fertilisation

Interactions between silicon and alkaloid defences in endophyte-infected grasses and the consequences for a folivore

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, whether this impacts insect herbivores is unknown. 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 remainsuntested. 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 (RGRs) 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