Rhizobia-legume symbiosis mediates direct and indirect interactions between plants, herbivores and their parasitoids.

Microorganisms associated with plant roots significantly impact the quality and quantity of plant defences. However, the bottom-up effects of soil microbes on the aboveground multitrophic interactions remain largely under studied. To address this gap, we investigated the chemically-mediated effects of nitrogen-fixing rhizobia on legume-herbivore-parasitoid multitrophic interactions. To address this, we initially examined the cascading effects of the rhizobia bean association on herbivore caterpillars, their parasitoids, and subsequently investigated how rhizobia influence on plant volatiles and extrafloral nectar. Our goal was to understand how these plant-mediated effects can affect parasitoids. Lima bean plants (Phaseoulus lunatus) inoculated with rhizobia exhibited better growth, and the number of root nodules positively correlated with defensive cyanogenic compounds. Despite increase of these chemical defences, Spodoptera latifascia caterpillars preferred to feed and grew faster on rhizobia-inoculated plants. Moreover, the emission of plant volatiles after leaf damage showed distinct patterns between inoculation treatments, with inoculated plants producing more sesquiterpenes and benzyl nitrile than non-inoculated plants. Despite these differences, Euplectrus platyhypenae parasitoid wasps were similarly attracted to rhizobia- or no rhizobia-treated plants. Yet, the oviposition and offspring development of E. platyhypenae was better on caterpillars fed with rhizobia-inoculated plants. We additionally show that rhizobia-inoculated common bean plants (Phaseolus vulgaris) produced more extrafloral nectar, with higher hydrocarbon concentration, than non-inoculated plants. Consequently, parasitoids performed better when fed with extrafloral nectar from rhizobia-inoculated plants. While the overall effects of bean-rhizobia symbiosis on caterpillars were positive, rhizobia also indirectly benefited parasitoids through the caterpillar host, and directly through the improved production of high quality extrafloral nectar. This study underscores the importance of exploring diverse facets and chemical mechanisms that influence the dynamics between herbivores and predators. This knowledge is crucial for gaining a comprehensive understanding of the ecological implications of rhizobia symbiosis on these interactions.

Transcriptomic analysis of deceptively pollinated Arum maculatum (Araceae) reveals association between terpene synthase expression in floral trap chamber and species-specific pollinator attraction.

Deceptive pollination often involves volatile organic compound emissions that mislead insects into performing nonrewarding pollination. Among deceptively pollinated plants, Arum maculatum is particularly well-known for its potent dung-like volatile organic compound emissions and specialized floral chamber, which traps pollinators-mainly Psychoda phalaenoides and Psychoda grisescens-overnight. However, little is known about the genes underlying the production of many Arum maculatum volatile organic compounds, and their influence on variation in pollinator attraction rates. Therefore, we performed de novo transcriptome sequencing of Arum maculatum appendix and male floret tissue collected during anthesis and postanthesis, from 10 natural populations across Europe. These RNA-seq data were paired with gas chromatography-mass spectrometry analyses of floral scent composition and pollinator data collected from the same inflorescences. Differential expression analyses revealed candidate transcripts in appendix tissue linked to malodourous volatile organic compounds including indole, p-cresol, and 2-heptanone. In addition, we found that terpene synthase expression in male floret tissue during anthesis significantly covaried with sex- and species-specific attraction of Psychoda phalaenoides and Psychoda grisescens. Taken together, our results provide the first insights into molecular mechanisms underlying pollinator attraction patterns in Arum maculatum and highlight floral chamber sesquiterpene (e.g. bicyclogermacrene) synthases as interesting candidate genes for further study.

Bioturbation by endogeic earthworms facilitates entomopathogenic nematode movement toward herbivore-damaged maize roots.

Entomopathogenic nematodes (EPNs) have been extensively studied as potential biological control agents against root-feeding crop pests. Maize roots under rootworm attack have been shown to release volatile organic compounds, such as (E)-ß-caryophyllene (Eßc) that guide EPNs toward the damaging larvae. As yet, it is unknown how belowground ecosystems engineers, such as earthworms, affect the biological control capacity of EPNs by altering the root Eßc-mediated tritrophic interactions. We here asked whether and how, the presence of endogeic earthworms affects the ability of EPNs to find root-feeding larvae of the beetle Diabrotica balteata. First, we performed a field mesocosm experiment with two diverse cropping systems, and revealed that the presence of earthworms increased the EPN infection potential of larvae near maize roots. Subsequently, using climate-controlled, olfactometer-based bioassays, we confirmed that EPNs response to Eßc alone (released from dispensers) was two-fold higher in earthworm-worked soil than in earthworm-free soil. Together our results indicate that endogeic earthworms, through burrowing and casting activities, not only change soil properties in a way that improves soil fertility but may also enhance the biocontrol potential of EPNs against root feeding pests. For an ecologically-sound pest reduction in crop fields, we advocate agricultural practices that favour earthworm community structure and diversity.

Contribution of different predator guilds to tritrophic interactions along ecological clines.

The strengths of interactions between plants, herbivores, and predators are predicted to relax with elevation. Despite the fundamental role predators play in tritrophic interactions, high-resolution experimental evidence describing predation across habitat gradients is still scarce in the literature and varies by predator. With this opinion paper, we look at how tritrophic strength of systems including different vertebrate and invertebrate predator guilds changes with elevation. Specifically, we focus on how birds, ants, parasitoids, and nematodes exert top-down pressure as predators and propose ways, in which each group could be better understood through elevational gradient studies. We hope to enrich future perspectives for disentangling the different biotic and abiotic factors underlying predator-mediated trophic interactions in a diversity of habitats.

Is protection against florivory consistent with the optimal defense hypothesis?

BACKGROUND: Plant defense traits require resources and energy that plants may otherwise use for growth and reproduction. In order to most efficiently protect plant tissues from herbivory, one widely accepted assumption of the optimal defense hypothesis states that plants protect tissues most relevant to fitness. Reproductive organs directly determining plant fitness, including flowers and immature fruit, as well as young, productive leaf tissue thus should be particularly well-defended. To test this hypothesis, we quantified the cyanogenic potential (HCNp)-a direct, chemical defense-systemically expressed in vegetative and reproductive organs in lima bean (Phaseolus lunatus), and we tested susceptibility of these organs in bioassays with a generalist insect herbivore, the Large Yellow Underwing (Noctuidae: Noctua pronuba). To determine the actual impact of either florivory (herbivory on flowers) or folivory on seed production as a measure of maternal fitness, we removed varying percentages of total flowers or young leaf tissue and quantified developing fruit, seeds, and seed viability. RESULTS: We found extremely low HCNp in flowers (8.66 ± 2.19 µmol CN(-) g(-1) FW in young, white flowers, 6.23 ± 1.25 µmol CN(-) g(-1) FW in mature, yellow flowers) and in pods (ranging from 32.05 ± 7.08 to 0.09 ± 0.08 µmol CN(-) g(-1) FW in young to mature pods, respectively) whereas young leaves showed high levels of defense (67.35 ± 3.15 µmol CN(-) g(-1) FW). Correspondingly, herbivores consumed more flowers than any other tissue, which, when taken alone, appears to contradict the optimal defense hypothesis. However, experimentally removing flowers did not significantly impact fitness, while leaf tissue removal significantly reduced production of viable seeds. CONCLUSIONS: Even though flowers were the least defended and most consumed, our results support the optimal defense hypothesis due to i) the lack of flower removal effects on fitness and ii) the high defense investment in young leaves, which have high consequences for fitness. These data highlight the importance of considering plant defense interactions from multiple angles; interpreting where empirical data fit within any plant defense hypothesis requires understanding the fitness consequences associated with the observed defense pattern.

Ants are less attracted to the extrafloral nectar of plants with symbiotic, nitrogen-fixing rhizobia.

Plants simultaneously maintain mutualistic relationships with different partners that are connected through the same host, but do not interact directly. One or more participating mutualists may alter their host's phenotype, resulting in a shift in the host's ecological interactions with all other mutualists involved. Understanding the functional interplay of mutualists associated with the same host remains an important challenge in biology. Here, we show belowground nitrogen-fixing rhizobia on lima bean (Phaseolus lunatus) alter their host plant's defensive mutualism with aboveground ants. We induced extrafloral nectar (EFN), an indirect defense acting through ant attraction. We also measured various nutritive and defensive plant traits, biomass, and counted ants on rhizobial and rhizobia-free plants. Rhizobia increased plant protein as well as cyanogenesis, a direct chemical defense against herbivores, but decreased EFN. Ants were significantly more attracted to rhizobia-free plants, and our structural equation model shows a strong link between rhizobia and reduced EFN as well as between EFN and ants: the sole path to ant recruitment. The rhizobia-mediated effects on simultaneously expressed defensive plant traits indicate rhizobia can have significant bottom-up effects on higher trophic levels. Our results show belowground symbionts play a critical and underestimated role in determining aboveground mutualistic interactions.

Chemical defense lowers plant competitiveness.

Both plant competition and plant defense affect biodiversity and food web dynamics and are central themes in ecology research. The evolutionary pressures determining plant allocation toward defense or competition are not well understood. According to the growth-differentiation balance hypothesis (GDB), the relative importance of herbivory and competition have led to the evolution of plant allocation patterns, with herbivore pressure leading to increased differentiated tissues (defensive traits), and competition pressure leading to resource investment towards cellular division and elongation (growth-related traits). Here, we tested the GDB hypothesis by assessing the competitive response of lima bean (Phaseolus lunatus) plants with quantitatively different levels of cyanogenesis-a constitutive direct, nitrogen-based defense against herbivores. We used high (HC) and low cyanogenic (LC) genotypes in different competition treatments (intra-genotypic, inter-genotypic, interspecific), and in the presence or absence of insect herbivores (Mexican bean beetle, Epilachna varivestis) to quantify vegetative and generative plant parameters (above and belowground biomass as well as seed production). Highly defended HC-plants had significantly lower aboveground biomass and seed production than LC-plants when grown in the absence of herbivores implying significant intrinsic costs of plant cyanogenesis. However, the reduced performance of HC- compared to LC-plants was mitigated in the presence of herbivores. The two plant genotypes exhibited fundamentally different responses to various stresses (competition, herbivory). Our study supports the GDB hypothesis by demonstrating that competition and herbivory affect different plant genotypes differentially and contributes to understanding the causes of variation in defense within a single plant species.

Co-variation of chemical and mechanical defenses in lima bean (Phaseolus lunatus L.).

Plants usually express multiple chemical and mechanical defenses simultaneously. The interplay of these defenses is still poorly understood, as predictions range from negative associations such as allocation tradeoffs to positive correlations forming synergistic defense syndromes. Surprisingly, little empirical evidence exists on the co-variation of multiple plant defenses. In the present study, we analyzed different genotypes of lima bean (Phaseolus lunatus L.) for the expression of two direct chemical defenses [cyanogenic potential (constitutive), polyphenol oxidase activity (inducible)], two indirect chemical defenses [volatiles (VOCs) and extrafloral nectar (EFN; both inducible)] and a constitutive mechanical defense (hook-shaped trichomes). While the occurrence of trichomes was positively correlated with cyanogenesis, these traits showed a tradeoff with polyphenol oxidase activity, release of VOCs, and secretion of EFN. Hook-shaped trichomes were abundantly present in four of 14 genotypes investigated, and were found only in one monophyletic group of an AFLP-based tree, thus indicating a single evolutionary origin within the species. Our findings show that different lima bean genotypes express either one of two defense systems: 1) high constitutive defense via cyanogenesis and trichomes or 2) high inducible defense via VOCs, EFN, and PPO activity.