Anti-herbivore Defense Research Articles

Induction of antiherbivore defense responses in poplars using a methyl jasmonate and mesoporous silica nanoparticle complex.

Poplar in China has long been plagued by the fall webworm Hyphantria cunea. Enhancing plant immunity using chemical elicitors is an environmentally friendly approach to pest control. The phytohormone methyl jasmonate (MeJA) can stimulate the chemical defenses of poplars against herbivores but has been shown to have limited efficacy in practice. Here, we studied the effects of a MeJA and mesoporous silica nanoparticle (MSN) complex (MeJA@MSN) regarding the induction of poplar resistance to H. cunea, which may provide strategies for the effective use of MeJA. The silicon-based phytohormone complex (MeJA@MSNs) exhibited excellent biological and physiochemical properties, such as excellent biocompatibility and plant tissue transportability. The changes in metabolites in poplar leaves induced by MeJA, MSNs, and MeJA@MSNs were investigated by metabolic analysis. MeJA@MSNs led to highly potent induced resistance along with elevated salicylaldehyde content, which increased with the dose administered. The salicylaldehyde metabolite showed a strong antifeedant effect on H. cunea larvae at a dosage of 1 μg, with the 50% lethal dose being 20.4 μg/mg. Furthermore, transcriptional analysis showed that MeJA@MSNs upregulated key genes in biosynthetic pathways more than MeJA and MSNs. Our results show that MeJA and MSNs interact positively in poplar, leading to salicylaldehyde accumulation and increased induced resistance to H. cunea, providing new insights into the underlying resistance mechanisms induced by MeJA@MSNs. © 2024 Society of Chemical Industry.

Butterfly eggs prime anti-herbivore defense in an annual but not perennial Arabidopsis species

Main conclusionUnlike Arabidopsis thaliana, defenses of Arabidopsis lyrata against Pieris brassicae larval feeding are not primable by P. brassicae eggs. Thus, egg primability of plant anti-herbivore defenses is not phylogenetically conserved in the genus Arabidopsis.While plant anti-herbivore defenses of the annual species Arabidopsis thaliana were shown to be primable by Pieris brassicae eggs, the primability of the phylogenetically closely related perennial Arabidopsis lyrata has not yet been investigated. Previous studies revealed that closely related wild Brassicaceae plant species, the annual Brassica nigra and the perennial B. oleracea, exhibit an egg-primable defense trait, even though they have different life spans. Here, we tested whether P. brassicae eggs prime anti-herbivore defenses of the perennial A. lyrata. We exposed A. lyrata to P. brassicae eggs and larval feeding and assessed their primability by (i) determining the biomass of P. brassicae larvae after feeding on plants with and without prior P. brassicae egg deposition and (ii) investigating the plant transcriptomic response after egg deposition and/or larval feeding. For comparison, these studies were also conducted with A. thaliana. Consistent with previous findings, A. thaliana’s response to prior P. brassicae egg deposition negatively affected conspecific larvae feeding upon A. thaliana. However, this was not observed in A. lyrata. Arabidopsis thaliana responded to P. brassicae eggs with strong transcriptional reprogramming, whereas A. lyrata responses to eggs were negligible. In response to larval feeding, A. lyrata exhibited a greater transcriptome change compared to A. thaliana. Among the strongly feeding-induced A. lyrata genes were those that are egg-primed in feeding-induced A. thaliana, i.e., CAX3, PR1, PR5, and PDF1.4. These results suggest that A. lyrata has evolved a robust feeding response that is independent from prior egg exposure.

Plant Silicon Defences Suppress Herbivore Performance, but Mode of Feeding Is Key.

The performance of herbivorous animals depends on the nutritional and defensive traits of the plants they consume. The uptake and deposition of biogenic silicon in plant tissues is arguably the most basic and ubiquitous anti-herbivore defence used by plants, especially grasses. We conducted meta-analyses of 150 studies reporting how vertebrate and invertebrate herbivores performed when feeding on silicon-rich plants relative to those feeding on low-silicon plants. Silicon levels were 52% higher and 32% more variable in silicon-rich plants compared to plants with low silicon, which resulted in an overall 33% decline in herbivore performance. Fluid-feeding herbivore performance was less adversely impacted (-14%) than tissue-chewing herbivores, including mammals (-45%), chewing arthropods (-33%) and plant-boring arthropods (-39%). Fluid-feeding arthropods with a wide diet breadth or those feeding on perennial plant species were mostly unaffected by silicon defences. Unlike many other plant defences, where diet specialisation often helps herbivores overcome their effects, silicon negatively impacts chewing herbivores regardless of diet breadth. We conclude that silicon defences primarily target chewing herbivores and impact vertebrate and invertebrate herbivores to a similar degree.

Challenging the value of a pseudoreplicated study: A comment to Liu et al. (2024)

The paper by Liu et al. (2024) presents measurements of herbivory and six plant traits considered potential antiherbivore defences in seven plant species across two study plots: one mined and one adjacent unmined plot. I argue that this experimental design is inadequate for testing the authors' hypothesis that differences in herbivory and plant traits between these two plots are due to pollution impact, as the study lacks replication of pollution levels. This issue, which could have been easily addressed when designing the study, reflects insufficient attention to experimental design and seriously compromises the validity of the research findings. I briefly reiterate what pseudoreplication is and which fundamental principle of statistical inference it violates.

Elevated atmospheric CO2 and silicon antagonistically regulate anti-herbivore phytohormone and defence gene expression levels in wheat

Silicon (Si) accumulation by grasses is a key mechanism for alleviating biotic and abiotic stresses, including insect herbivory. In addition to conferring physical resistance, tissue silicification may enhance anti-herbivore phytohormone production, such as the jasmonic and salicylic (JA and SA) acid pathways, and downstream regulation of defence genes, although this is poorly understood. Elevated atmospheric carbon dioxide (eCO2) concentrations predicted by climate models are reported to reduce Si accumulation in several plant taxa and may therefore compromise Si-augmented resistance. We investigated how Si enrichment and eCO2 regulate the JA and SA pathways and expression of defence genes in wheat (Triticum aestivum) challenged by a global insect pest (Helicoverpa armigera). Si treatments increased JA production and expression of β-1,3-ENDOGLUCANASE (GNS), and MITOGEN-ACTIVATED PROTEIN KINASE (MAPK; WCK-1) defence genes, while suppressing SA production, resulting in reduced feeding and growth of H. armigera. In contrast, under eCO2 conditions, Si accumulation was reduced, GNS downregulated, but SA production was upregulated. Despite compromised plant defences, H. armigera growth rates were reduced under eCO2. We conclude that eCO2 and Si supplementation contrastingly regulate anti-herbivore defences in wheat; these important drivers operate independently and may influence future patterns of pest resistance in wheat under projected rises in atmospheric CO2.

Changes in tomato plant anti-herbivore defences after soil application of a biofungicide containing Bacillus subtilis (Serenade ASO)

ABSTRACT Plants have limited resources to allocate to defences against infection and herbivory. While interactions between plant responses to microbial and herbivore attack are complex, it is often the case that the induction of one response will act antagonistically to the other. Recent studies have shown that plant growth promoting rhizobacteria, which improves overall plant health and general stress resistance, can enhance both anti-microbial and anti-herbivore defences. We investigated how soil application of the biofungicide Serenade ASO (Bacillus subtilis strain QST 713), which primes plant defences against fungal and bacterial infection and promotes plant growth, affects anti-herbivore defences by measuring both constitutive and induced defences. We applied Serenade one or two times to the soil of tomato plants and measured the numbers of type IV glandular trichomes on leaves, the weight gain of a generalist caterpillar (beet armyworms; BAW), and the activity of two enzymes associated with defence against insects (polyphenoloxidase and peroxidase). Serenade treated plants grew faster and foliage from treated plants had significantly higher numbers of glandular trichomes and higher polyphenoloxidase and peroxidase activities than untreated plants. However, Serenade treatment did not affect the degree of induction of plant defences when damaged by BAW feeding and did not slow the growth rate of BAW relative to plants not treated with Serenade. Therefore, the biofungicide Serenade increased plant growth and altered the densities of trichomes and the activities of two defensive enzymes in plants, but it did not affect overall susceptibility of the plants to a generalist herbivore.

Effects of individual traits vs. trait syndromes on assemblages of various herbivore guilds associated with central European Salix.

Plants employ diverse anti-herbivore defences that can covary to form syndromes consisting of multiple traits. Such syndromes are hypothesized to impact herbivores more than individual defences. We studied 16 species of lowland willows occurring in central Europe and explored if their chemical and physical traits form detectable syndromes. We tested for phylogenetic trends in the syndromes and explored whether three herbivore guilds (i.e., generalist leaf-chewers, specialist leaf-chewers, and gallers) are affected more by the detected syndromes or individual traits. The recovered syndromes showed low phylogenetic signal and were mainly defined by investment in concentration, richness, or uniqueness of structurally related phenolic metabolites. Resource acquisition traits or inducible volatile organic compounds exhibited a limited correlation with the syndromes. Individual traits composing the syndromes showed various correlations to the assemblages of herbivores from the three studied guilds. In turn, we found some support for the hypothesis that defence syndromes are composed of traits that provide defence against various herbivores. However, individual traits rather than trait syndromes explained more variation for all studied herbivore assemblages. The detected negative correlations between various phenolics suggest that investment trade-offs may occur primarily among plant metabolites with shared metabolic pathways that may compete for their precursors. Moreover, several traits characterizing the recovered syndromes play additional roles in willows other than defence from herbivory. Taken together, our findings suggest that the detected syndromes did not solely evolve as an anti-herbivore defence.

OsRCI-1-Mediated GLVs Enhance Rice Resistance to Brown Planthoppers.

Green leaf volatiles (GLVs) play pivotal roles in plant anti-herbivore defense. This study investigated whether the rice 13-lipoxygense gene OsRCI-1 is involved in GLV production and plant defense in rice. The overexpression of OsRCI-1 (oeRCI lines) in rice resulted in increased wound-induced levels of two prominent GLVs, cis-3-hexen-1-ol and cis-3-hexenal. In a previous study, we found that the overexpression of OsRCI-1 reduced the colonization by the rice brown planthopper (BPH, Nilaparvata lugens) but increased the attractiveness to the egg parasitoid Anagrus nilaparvatae compared to wild-type (WT) plants. This study found that when cis-3-hexen-1-ol, but not cis-3-hexenal, was added to WT plants, it could change the BPH's colonization preference, i.e., more BPHs preferred to colonize the oeRCI lines. The exogenous application of cis-3-hexen-1-ol or cis-3-hexenal to BPH-infested WT plants could weaken or overturn the preference of A. nilaparvatae for oeRCI lines. However, field experiments revealed that only cis-3-hexenal was attractive to the parasitoid and increased the parasitism rates of BPH eggs. These results indicate that OsRCI-1 is involved in rice GLV production and therefore modulates both direct and indirect defense in rice.

Windthrow causes declines in carbohydrate and phenolic concentrations and increased monoterpene emission in Norway spruce.

With the increasing frequencies of extreme weather events caused by climate change, the risk of forest damage from insect attacks grows. Storms and droughts can damage and weaken trees, reduce tree vigour and defence capacity and thus provide host trees that can be successfully attacked by damaging insects, as often observed in Norway spruce stands attacked by the Eurasian spruce bark beetle Ips typographus. Following storms, partially uprooted trees with grounded crowns suffer reduced water uptake and carbon assimilation, which may lower their vigour and decrease their ability to defend against insect attack. We conducted in situ measurements on windthrown and standing control trees to determine the concentrations of non-structural carbohydrates (NSCs), of phenolic defences and volatile monoterpene emissions. These are the main storage and defence compounds responsible for beetle´s pioneer success and host tree selection. Our results show that while sugar and phenolic concentrations of standing trees remained rather constant over a 4-month period, windthrown trees experienced a decrease of 78% and 37% of sugar and phenolic concentrations, respectively. This strong decline was especially pronounced for fructose (-83%) and glucose (-85%) and for taxifolin (-50.1%). Windthrown trees emitted 25 times greater monoterpene concentrations than standing trees, in particular alpha-pinene (23 times greater), beta-pinene (27 times greater) and 3-carene (90 times greater). We conclude that windthrown trees exhibited reduced resources of anti-herbivore and anti-pathogen defence compounds needed for the response to herbivore attack. The enhanced emission rates of volatile terpenes from windthrown trees may provide olfactory cues during bark beetle early swarming related to altered tree defences. Our results contribute to the knowledge of fallen trees vigour and their defence capacity during the first months after the wind-throw disturbance. Yet, the influence of different emission rates and profiles on bark beetle behaviour and host selection requires further investigation.

Does spatial variation in insect herbivory match variations in plant quality? A meta-analysis.

Variation in herbivore pressure has often been predicted from patterns in plant traits considered as antiherbivore defences. Here, we tested whether spatial variation in field insect herbivory is associated with the variation in plant quality by conducting a meta-analysis of 223 correlation coefficients between herbivory levels and the expression of selected plant traits. We found no overall correlation between herbivory and either concentrations of plant secondary metabolites or values of physical leaf traits. This result was due to both the large number of low correlations and the opposing directions of high correlations in individual studies. Field herbivory demonstrated a significant association only with nitrogen: herbivore pressure increased with an increase in nitrogen concentration in plant tissues. Thus, our meta-analysis does not support either theoretical prediction, i.e., that plants possess high antiherbivore defences in localities with high herbivore pressure or that herbivory is low in localities where plant defences are high. We conclude that information about putative plant defences is insufficient to predict plant losses to insects in field conditions and that the only bottom-up factor shaping spatial variation in insect herbivory is plant nutritive value. Our findings stress the need to improve a theory linking plant putative defences and herbivory.

Duplicated Copy Number Variant of the Maize 9-Lipoxygenase ZmLOX5 Improves 9,10-KODA-Mediated Resistance to Fall Armyworms.

Extensive genome structure variations, such as copy number variations (CNVs) and presence/absence variations, are the basis for the remarkable genetic diversity of maize; however, the effect of CNVs on maize herbivory defense remains largely underexplored. Here, we report that the naturally occurring duplication of the maize 9-lipoxygenase gene ZmLOX5 leads to increased resistance of maize to herbivory by fall armyworms (FAWs). Previously, we showed that ZmLOX5-derived oxylipins are required for defense against chewing insect herbivores and identified several inbred lines, including Yu796, that contained duplicated CNVs of ZmLOX5, referred to as Yu796-2×LOX5. To test whether introgression of the Yu796-2×LOX5 locus into a herbivore-susceptible B73 background that contains a single ZmLOX5 gene is a feasible approach to increase resistance, we generated a series of near-isogenic lines that contained either two, one, or zero copies of the Yu796-2×LOX5 locus in the B73 background via six backcrosses (BC6). Droplet digital PCR (ddPCR) confirmed the successful introgression of the Yu796-2×LOX5 locus in B73. The resulting B73-2×LOX5 inbred line displayed increased resistance against FAW, associated with increased expression of ZmLOX5, increased wound-induced production of its primary oxylipin product, the α-ketol, 9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid (9,10-KODA), and the downstream defense hormones regulated by this molecule, 12-oxo-phytodienoic acid (12-OPDA) and abscisic acid (ABA). Surprisingly, wound-induced JA-Ile production was not increased in B73-2×LOX5, resulting from the increased JA catabolism. Furthermore, B73-2×LOX5 displayed reduced water loss in response to drought stress, likely due to increased ABA and 12-OPDA content. Taken together, this study revealed that the duplicated CNV of ZmLOX5 quantitively contributes to maize antiherbivore defense and presents proof-of-concept evidence that the introgression of naturally occurring duplicated CNVs of a defensive gene into productive but susceptible crop varieties is a feasible breeding approach for enhancing plant resistance to herbivory and tolerance to abiotic stress.

Anti-herbivory defences delivered by Epichloë fungal endophytes: a quantitative review of alkaloid concentration variation among hosts and plant parts.

In the subfamily Poöideae (Poaceae), certain grass species possess anti-herbivore alkaloids synthesized by fungal endophytes that belong to the genus Epichloë (Clavicipitaceae). The protective role of these symbiotic endophytes can vary, depending on alkaloid concentrations within specific plant-endophyte associations and plant parts. We conducted a literature review to identify articles containing alkaloid concentration data for various plant parts in six important pasture species, Lolium arundinaceum, Lolium perenne, Lolium pratense, Lolium multiflorum|Lolium rigidum and Festuca rubra, associated with their common endophytes. We considered the alkaloids lolines (1-aminopyrrolizidines), peramine (pyrrolopyrazines), ergovaline (ergot alkaloids) and lolitrem B (indole-diterpenes). While all these alkaloids have shown bioactivity against insect herbivores, ergovaline and lolitrem B are harmful for mammals. Loline alkaloid levels were higher in the perennial grasses L. pratense and L. arundinaceum compared to the annual species L. multiflorum and L. rigidum, and higher in reproductive tissues than in vegetative structures. This is probably due to the greater biomass accumulation in perennial species that can result in higher endophyte mycelial biomass. Peramine concentrations were higher in L. perenne than in L. arundinaceum and not affected by plant part. This can be attributed to the high within-plant mobility of peramine. Ergovaline and lolitrem B, both hydrophobic compounds, were associated with plant parts where fungal mycelium is usually present, and their concentrations were higher in plant reproductive tissues. Only loline alkaloid data were sufficient for below-ground tissue analyses and concentrations were lower than in above-ground parts. Our study provides a comprehensive synthesis of fungal alkaloid variation across host grasses and plant parts, essential for understanding the endophyte-conferred defence extent. The patterns can be understood by considering endophyte growth within the plant and alkaloid mobility. Our study identifies research gaps, including the limited documentation of alkaloid presence in roots and the need to investigate the influence of different environmental conditions.

Endogenous rhythmic growth and ectomycorrhizal fungi modulate priming of antiherbivore defences in subsequently formed new leaves of oak trees

Abstract Priming of plant defences provides increased plant protection against herbivores and reduces the allocation costs of defence. Defence priming in woody plants remains obscure, in particular, due to plant development traits such as endogenous rhythmic growth. By using bioassays with oak microcuttings (Quercus robur) and combining transcriptomic and metabolomic analyses, we investigated how leaf herbivory by Lymantria dispar and root inoculation with the ectomycorrhizal fungus Piloderma croceum prime oak defences. We further investigated how defence priming is modulated by the rhythmic growth of the oaks. A first herbivory challenge in oak leaves primed newly grown leaves for enhanced induction of jasmonic acid (JA)‐related direct defences, or enhanced emission of volatiles, depending on the specific growth stage at which the plants were first challenged. Root inoculation with Piloderma abolished the enhanced induction of JA‐related defences and volatile emission. Synthesis: Our results indicate that a first herbivore attack primes direct and indirect defences of newly formed oak leaves and that the specific display of defence priming is regulated by rhythmic growth, and modulated by the interaction Piloderma. Our results show that the priming memory in oaks can be transmitted to the next growth cycle, to the leaves of the new shoot unit.

Drought reduces productivity and anti-herbivore defences, but not mycorrhizal associations, of perennial prairie forbs.

During drought, plants allocate resources to aboveground biomass production and belowground carbohydrate reserves, often at the expense of production of defence traits. Additionally, drought has been shown to alter floral resources, with potential implications for plant-pollinator interactions. Although soil symbionts, such as arbuscular mycorrhizal (AM) fungi, can alleviate drought stress in plants, certain levels of drought may negatively impact this relationship, with potential cascading effects. Because of their importance to plant and animal community diversity, we examined effects of drought on biomass production, physical defence properties, nectar production, and associated AM fungal abundance of five common prairie forb species in a greenhouse study. Reduced soil moisture decreased vegetative biomass production. Production of trichomes and latex decreased under drought, relative to well-watered conditions. Ruellia humilis flowers produced less nectar under drought, relative to well-watered conditions. Intra-radical AM fungal colonization was not significantly affected by drought, although extra-radical AM fungal biomass associated with S. azurea decreased following drought. Overall, grassland forb productivity, defence, and nectar production were negatively impacted by moderate drought, with possible negative implications for biotic interactions. Reduced flower and nectar production may lead to fewer pollinator visitors, which may contribute to seed limitation in forb species. Reduced physical defences increase the likelihood of herbivory, further decreasing the ability to store energy for essential functions, such as reproduction. Together, these results suggest drought can potentially impact biotic interactions between plants and herbivores, pollinators, and soil symbionts, and highlights the need for direct assessments of these relationships under climate change scenarios.

Ca2+/calmodulin-mediated desensitization of glutamate receptors shapes plant systemic wound signalling and anti-herbivore defence.

Plants rely on systemic signalling mechanisms to establish whole-body defence in response to insect and nematode attacks. GLUTAMATE RECEPTOR-LIKE (GLR) genes have been implicated in long-distance transmission of wound signals to initiate the accumulation of the defence hormone jasmonate (JA) at undamaged distal sites. The systemic signalling entails the activation of Ca2+-permeable GLR channels by wound-released glutamate, triggering membrane depolarization and cytosolic Ca2+ influx throughout the whole plant. The systemic electrical and calcium signals rapidly dissipate to restore the resting state, partially due to desensitization of the GLR channels. Here we report the discovery of calmodulin-mediated, Ca2+-dependent desensitization of GLR channels, revealing a negative feedback loop in the orchestration of plant systemic wound responses. A CRISPR-engineered GLR3.3 allele with impaired desensitization showed prolonged systemic electrical signalling and Ca2+ waves, leading to enhanced plant defence against herbivores. Moreover, this Ca2+/calmodulin-mediated desensitization of GLR channels is a highly conserved mechanism in plants, providing a potential target for engineering anti-herbivore defence in crops.

The role of the Bemisia tabaci and Trialeurodes vaporariorum cytochrome-P450 clade CYP6DPx in resistance to nicotine and neonicotinoids

The alkaloid, nicotine, produced by tobacco and other Solanaceae as an anti-herbivore defence chemical is one of the most toxic natural insecticides in nature. However, some insects, such as the whitefly species, Trialeurodes vaporariorum and Bemisia tabaci show strong tolerance to this allelochemical and can utilise tobacco as a host. Here, we used biological, molecular and functional approaches to investigate the role of cytochrome P450 enzymes in nicotine tolerance in T. vaporariorum and B. tabaci. Insecticide bioassays revealed that feeding on tobacco resulted in strong induced tolerance to nicotine in both species. Transcriptome profiling of both species reared on tobacco and bean hosts revealed profound differences in the transcriptional response these host plants. Interrogation of the expression of P450 genes in the host-adapted lines revealed that P450 genes belonging to the CYP6DP subfamily are strongly upregulated in lines reared on tobacco. Functional characterisation of these P450s revealed that CYP6DP1 and CYP6DP2 of T. vaporariorum and CYP6DP3 of B. tabaci confer resistance to nicotine in vivo. These three genes, in addition to the B. tabaci P450 CYP6DP5, were also found to confer resistance to the neonicotinoid imidacloprid. Our data provide new insight into the molecular basis of nicotine resistance in insects and illustrates how divergence in the evolution of P450 genes in this subfamily in whiteflies may have impacted the extent to which different species can tolerate a potent natural insecticide.

Urban spatial heterogeneity shapes the evolution of an antiherbivore defense trait and its genes in white clover

Urbanization is a global threat to biodiversity due to its large impact on environmental changes. Recently, urban environmental change has been shown to impact the evolution of many species. However, much remains unknown about how urban environments influence evolutionary processes and outcomes due to the non‐linearity and discontinuity of environmental variables along urban–rural gradients. Here, we focused on the evolution of hydrogen cyanide (HCN) production and its components (presence/absence of cyanogenic glycosides and the hydrolytic enzyme linamarase) in the herbaceous plant white clover Trifolium repens, which thrive in both urban and rural areas. To comprehensively elucidate how plants evolve and adapt to heterogenous urban environments, we collected 3299 white clover plants from 122 populations throughout Sapporo, Japan. We examined the spatial variation in environmental factors, such as herbivory, sky openness, impervious surface cover, snow depth, and temperature, and how variation in these factors was related to the production of HCN, cyanogenic glycosides, and linamarase. Environmental factors showed complex spatial variation due to the heterogeneity of the urban landscape. Among these factors, herbivory, sky openness, and impervious surface cover were highly related to the frequency of plants producing HCN in populations. We also found that impervious surface cover was related to the frequency of plants producing cyanogenic glycosides, while herbivory pressure was not. As a result, the cyanogenic glycoside frequency showed a clearer trend along urban–rural gradient rather than HCN frequency, and thus, the predicted spatial distributions of HCN and cyanogenic glycosides were inconsistent. These results suggest that urban landscape heterogeneity and trait multifunctionality determines mosaic‐like spatial distribution of evolutionary traits.

Feeding Assay to Study the Effect of Phytocytokines on Direct and Indirect Defense in Maize.

Phytocytokines mediate defense against pests and pathogens. Many methods have been developed to study the physiological responses triggered by phytocytokines in dicot plants. Here, we describe a detailed peptide feeding protocol to study the effect of phytocytokines on direct and indirect anti-herbivore defense in maize. This method relies on peptide uptake by the excised maize seedling or leaves via the transpiration stream. The headspace volatiles from plant samples are then analyzed by proton transfer reaction time-of-flight mass spectrometry (PTR-TOF-MS), or by gas chromatography-mass spectrometry (GC-MS). The samples can also be further processed to evaluate phytocytokine-induced defense gene expression or phytohormone production.

Silicon-based anti-herbivore defense in tropical tree seedlings.

Silicon-based defenses deter insect herbivores in many cultivated and wild grass species. Furthermore, in some of these species, silicon (Si) uptake and defense can be induced by herbivory. Tropical trees also take up Si and leaf Si concentrations vary greatly across and within species. As herbivory is a major driver of seedling mortality and niche differentiation of tropical tree species, understanding anti-herbivore defenses is pivotal. Yet, whether silicon is a constitutive and inducible herbivory defense in tropical forest tree species remains unknown. We grew seedlings of eight tropical tree species in a full factorial experiment, including two levels of plant-available soil Si concentrations (-Si/+Si) and a simulated herbivory treatment (-H/+H). The simulated herbivory treatment was a combination of clipping and application of methyl jasmonate. We then carried out multiple-choice feeding trials, separately for each tree species, in which leaves of each treatment combination were offered to a generalist caterpillar (Spodoptera frugiperda). Leaf damage was assessed. Three species showed a significant decrease in leaf damage under high compared to low Si conditions (by up to 72%), consistent with our expectation of Si-based defenses acting in tropical tree species. In one species, leaf damage was increased by increasing soil Si and in four species, no effect of soil Si on leaf damage was observed. Opposite to our expectation of Si uptake and defense being inducible by herbivory damage, simulated herbivory increased leaf damage in two species. Furthermore, simulated herbivory reduced Si concentrations in one species. Our results showed that tropical tree seedlings can be better defended when growing in Si-rich compared to Si-poor soils, and that the effects of Si on plant defense vary strongly across species. Furthermore, Si-based defenses may not be inducible in tropical tree species. Overall, constitutive Si-based defense should be considered part of the vast array of anti-herbivore defenses of tropical tree species. Our finding that Si-based defenses are highly species-specific combined with the fact that herbivory is a major driver of mortality in tropical tree seedling, suggests that variation in soil Si concentrations may have pervasive consequences for regeneration and performance across tropical tree species.

Herbivory and elevated levels of CO2 and nutrients separately, rather than synergistically, impacted biomass production and allocation in invasive and native plant species.

Large parts of the Earth are experiencing environmental change caused by alien plant invasions, rising atmospheric concentration of carbon dioxide (CO2 ), and nutrient enrichments. Elevated CO2 and nutrient concentrations can separately favour growth of invasive plants over that of natives but how herbivory may modulate the magnitude and direction of net responses by the two groups of plants to simultaneous CO2 and nutrient enrichments remains unknown. In line with the enemy release hypothesis, invasive plant species should reallocate metabolites from costly anti-herbivore defences into greater growth following escape from intense herbivory in the native range. Therefore, invasive plants should have greater growth than native plants under simultaneous CO2 and nutrient enrichments in the absence of herbivory. To test this prediction, we grew nine congeneric pairs of invasive and native plant species that naturally co-occurred in grasslands in China under two levels each of nutrient enrichment (low-nutrient vs. high-nutrient), herbivory (with herbivory vs. without herbivory) and under ambient (412.9 ± 0.6 ppm) and elevated (790.1 ± 6.2 ppm) levels of CO2 concentrations in open top chambers in a common garden. Elevated CO2 and nutrient enrichment separately increased total plant biomass, while herbivory reduced it regardless of the plant invasive status. High-nutrient treatment caused the plants to allocate a significantly lower proportion of total biomass to roots, while herbivory induced an opposite pattern. Herbivory suppressed total biomass production more strongly in native plants than invasive plants. The plants exhibited significant interspecific and intergeneric variation in their responses to the various treatment combinations. Overall, these results suggest that elevated CO2 and nutrients and herbivory may separately, rather than synergistically, impact productivity of the invasive and co-occurring native plant species in our study system. Moreover, interspecific variation in resource-use strategies was more important than invasive status in determining plant responses to the various treatment combinations.