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.

Stable coexistence or competitive exclusion? Fern endophytes demonstrate rapid turnover favouring a dominant fungus.

Fungal endophytes are critical members of the plant microbiome, but their community dynamics throughout an entire growing season are underexplored. Additionally, most fungal endophyte research has centred on seed-reproducing hosts, while spore-reproducing plants also host endophytes and may be colonized by unique community members. In order to examine annual fungal endophyte community dynamics in a spore-reproducing host, we explored endophytes in a single population of ferns, Polystichum munitum, in the Pacific Northwest. Through metabarcoding, we characterized the community assembly and temporal turnover of foliar endophytes throughout a growing season. From these results, we selected endophytes with outsized representations in sequence data and performed in vitro competition assays. Finally, we inoculated sterile fern gametophytes with dominant fungi observed in the field and determined their effects on host performance. Sequencing demonstrated that ferns were colonized by a diverse community of fungal endophytes in newly emerged tissue, but diversity decreased throughout the season leading to the preponderance of a single fungus in later sampling months. This previously undescribed endophyte appears to abundantly colonize the host to the detriment of other microfungi. Competition assays on a variety of media types failed to demonstrate that the dominant fungus was competitive against other fungi isolated from the same hosts, and inoculation onto sterile fern gametophytes did not alter growth compared to sterile controls, suggesting its effects are not antagonistic. The presence of this endophyte in the fern population probably demonstrates a case of repeated colonization driving competitive exclusion of other fungal community members.

Culturable fungal endophyte communities of primary successional plants on Mount St. Helens, WA, USA.

BACKGROUND: While a considerable amount of research has explored plant community composition in primary successional systems, little is known about the microbial communities inhabiting these pioneer plant species. Fungal endophytes are ubiquitous within plants, and may play major roles in early successional ecosystems. Specifically, endophytes have been shown to affect successional processes, as well as alter host stress tolerance and litter decomposition dynamics-both of which are important components in harsh environments where soil organic matter is still scarce. RESULTS: To determine possible contributions of fungal endophytes to plant colonization patterns, we surveyed six of the most common woody species on the Pumice Plain of Mount St. Helens (WA, USA; Lawetlat'la in the Cowlitz language; created during the 1980 eruption)-a model primary successional ecosystem-and found low colonization rates (< 15%), low species richness, and low diversity. Furthermore, while endophyte community composition did differ among woody species, we found only marginal evidence of temporal changes in community composition over a single field season (July-September). CONCLUSIONS: Our results indicate that even after a post-eruption period of 40 years, foliar endophyte communities still seem to be in the early stages of community development, and that the dominant pioneer riparian species Sitka alder (Alnus viridis ssp. sinuata) and Sitka willow (Salix sitchensis) may be serving as important microbial reservoirs for incoming plant colonizers.

Do Foliar Endophytes Matter in Litter Decomposition?

Litter decomposition rates are affected by a variety of abiotic and biotic factors, including the presence of fungal endophytes in host plant tissues. This review broadly analyzes the findings of 67 studies on the roles of foliar endophytes in litter decomposition, and their effects on decomposition rates. From 29 studies and 1 review, we compiled a comprehensive table of 710 leaf-associated fungal taxa, including the type of tissue these taxa were associated with and isolated from, whether they were reported as endo- or epiphytic, and whether they had reported saprophytic abilities. Aquatic (i.e., in-stream) decomposition studies of endophyte-affected litter were significantly under-represented in the search results (p < 0.0001). Indicator species analyses revealed that different groups of fungal endophytes were significantly associated with cool or tropical climates, as well as specific plant host genera (p < 0.05). Finally, we argue that host plant and endophyte interactions can significantly influence litter decomposition rates and should be considered when interpreting results from both terrestrial and in-stream litter decomposition experiments.

Fungal endophyte communities in the temperate fern Polystichum munitum show early colonization and extensive temporal turnover.

PREMISE OF THE STUDY: Fungal endophytes have been shown to colonize all land plants, yet surprisingly little attention has been given to their community composition in ferns. We examined the diversity and temporal turnover of fungal endophytes in foliar tissue of the temperate western sword fern, Polystichum munitum, comparing taxa in newly emerged leaflets and in the same fronds after 1 mo of exposure in the field. METHODS: Utilizing next-generation sequencing, we sampled pinnae from P. munitum in spring, 2-3 d after they emerged. We additionally sampled pinnae 30 d later from the same fern blades. From these samples, we sequenced fungal DNA to characterize the initial colonization and temporal turnover of endophytes in the host. KEY RESULTS: We demonstrate that P. munitum is abundantly colonized by endophytes in newly emerged foliar tissue. However, 1 mo later, the community composition undergoes a marked shift: the overall richness of endophytes increases, but the evenness of the community wanes as a single taxon, Flagellospora fusarioides, comes to dominate. CONCLUSIONS: We conclude that P. munitum hosts a variety of fungal endophyte taxa, similarly to other land plants. However, the rapid shift of the endophyte community we report is an unprecedented observation. Therefore, we further conclude that repeated sampling should be the standard in endophyte studies, because single sampling events are not sufficient to capture the dynamic nature of these cryptic microfungi.

Colonization by nitrogen-fixing Frankia bacteria causes short-term increases in herbivore susceptibility in red alder (Alnus rubra) seedlings.

Carbon allocation demands from root-nodulating nitrogen-fixing bacteria (NFB) can modulate the host plant's chemical phenotype, with strong bottom-up effects on herbivores. In contrast to well-studied rhizobia, the effects of other important NFB on plant chemistry and herbivory are much less understood. Here, combining field surveys in the Oregon Coast Range, USA with laboratory experiments, we analyzed how N2-fixing Frankia bacteria influenced plant growth, chemistry, and herbivory on Alnus rubra (red alder) seedlings. In the field, we quantified Frankia nodulation, herbivore damage, and plant size. In the laboratory, we grew seedlings with Frankia (F+), Frankia-free but nitrogen-fertilized (N+), or both uncolonized and unfertilized (F-N-) and assessed growth and leaf chemistry. We further conducted choice trials with black slugs, Arion rufus, a natural red alder herbivore. In the field, Frankia nodulation was significantly positively correlated with herbivory and negatively with seedling height. In contrast, in the lab, F+ as well as N+ seedlings were significantly taller than the F-N- controls. Seedlings from both treatments also had significantly increased leaf protein concentration compared to controls, whereas carbon-based nutritive compounds (carbohydrates) as well as leaf palatability-decreasing condensed tannins, lignin, and fiber were decreased in F+ but not in N+ treatments. In the choice assays, slugs preferred leaf material from F+ seedlings, but the effects were only significant in young leaves. Our study indicates that colonization by Frankia causes short-term ecological costs in terms of susceptibility to herbivory. However, the ubiquity of this symbiosis in natural settings suggests that these costs are outweighed by benefits beyond the seedling stage.

Is biomass a reliable estimate of plant fitness?

The measurement of fitness is critical to biological research. Although the determination of fitness for some organisms may be relatively straightforward under controlled conditions, it is often a difficult or nearly impossible task in nature. Plants are no exception. The potential for long-distance pollen dispersal, likelihood of multiple reproductive events per inflorescence, varying degrees of reproductive growth in perennials, and asexual reproduction all confound accurate fitness measurements. For these reasons, biomass is frequently used as a proxy for plant fitness. However, the suitability of indirect fitness measurements such as plant size is rarely evaluated. This review outlines the important associations between plant performance, fecundity, and fitness. We make a case for the reliability of biomass as an estimate of fitness when comparing conspecifics of the same age class. We reviewed 170 studies on plant fitness and discuss the metrics commonly employed for fitness estimations. We find that biomass or growth rate are frequently used and often positively associated with fecundity, which in turn suggests greater overall fitness. Our results support the utility of biomass as an appropriate surrogate for fitness under many circumstances, and suggest that additional fitness measures should be reported along with biomass or growth rate whenever possible.

Coevolution of Cyanogenic Bamboos and Bamboo Lemurs on Madagascar.

Feeding strategies of specialist herbivores often originate from the coevolutionary arms race of plant defenses and counter-adaptations of herbivores. The interaction between bamboo lemurs and cyanogenic bamboos on Madagascar represents a unique system to study diffuse coevolutionary processes between mammalian herbivores and plant defenses. Bamboo lemurs have different degrees of dietary specialization while bamboos show different levels of chemical defense. In this study, we found variation in cyanogenic potential (HCNp) and nutritive characteristics among five sympatric bamboo species in the Ranomafana area, southeastern Madagascar. The HCNp ranged from 209±72 µmol cyanide*g-1 dwt in Cathariostachys madagascariensis to no cyanide in Bambusa madagascariensis. Among three sympatric bamboo lemur species, the greater bamboo lemur (Prolemur simus) has the narrowest food range as it almost exclusively feeds on the highly cyanogenic C. madagascariensis. Our data suggest that high HCNp is the derived state in bamboos. The ancestral state of lemurs is most likely "generalist" while the ancestral state of bamboo lemurs was determined as equivocal. Nevertheless, as recent bamboo lemurs comprise several "facultative specialists" and only one "obligate specialist" adaptive radiation due to increased flexibility is likely. We propose that escaping a strict food plant specialization enabled facultative specialist bamboo lemurs to inhabit diverse geographical areas.

Friend or Foe-Light Availability Determines the Relationship between Mycorrhizal Fungi, Rhizobia and Lima Bean (Phaseolus lunatus L.).

Plant associations with root microbes represent some of the most important symbioses on earth. While often critically promoting plant fitness, nitrogen-fixing rhizobia and arbuscular mycorrhizal fungi (AMF) also demand significant carbohydrate allocation in exchange for key nutrients. Though plants may often compensate for carbon loss, constraints may arise under light limitation when plants cannot extensively increase photosynthesis. Under such conditions, costs for maintaining symbioses may outweigh benefits, turning mutualist microbes into parasites, resulting in reduced plant growth and reproduction. In natural systems plants commonly grow with different symbionts simultaneously which again may interact with each other. This might add complexity to the responses of such multipartite relationships. We experimented with lima bean (Phaseolus lunatus), which efficiently forms associations with both types of root symbionts. We applied full light and low-light to each of four treatments of microbial inoculation. After an incubation period of 14 weeks, we quantified vegetative aboveground and belowground biomass and number and viability of seeds to determine effects of combined inoculant and light treatment on plant fitness. Under light-limited conditions, vegetative and reproductive traits were inhibited in AMF and rhizobia inoculated lima bean plants relative to controls (un-colonized plants). Strikingly, reductions in seed production were most critical in combined treatments with rhizobia x AMF. Our findings suggest microbial root symbionts create additive costs resulting in decreased plant fitness under light-limited conditions.

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.

Jasmonic acid enhances plant cyanogenesis and resistance to herbivory in lima bean.

Jasmonic acid (JA) is a natural plant hormone ubiquitously distributed in plants and centrally involved in the induction of direct and indirect plant defenses. Defenses up-regulated by this hormone include trichomes--a direct, mechanical defense--and alkaloids--a direct chemical defense--as well as two indirect chemical defenses: volatile organic compounds (VOCs) and extrafloral nectar (EFN). Plant cyanogenesis--the release of toxic hydrogen cyanide (HCN) from preformed cyanogenic precursors in fruits, leaves, and seeds of many plants--is recognized as a direct, constitutive plant defensive trait, and is among the most widely distributed of all direct chemical plant defenses. The cyanogenic system in plants is composed of three parameters: The cyanogenic potential (HCNp; concentration of cyanogenic precursors), ß-glucosidase activity, and cyanogenic capacity (HCNc; release of gaseous hydrogen cyanide). Here, we demonstrated that experimental application of aqueous solutions of JA ranging from 0.001 to 1.0 mmol L(-1), as well as insect herbivory significantly enhanced HCNc via the induction of ß-glucosidase activity in wild lima bean (Phaseolus lunatus L.). In choice feeding trials with JA induced and damaged leaves, adult Mexican bean beetles--natural herbivores of lima bean--rejected leaves with enhanced ß-glucosidase activity and HCNc. Our findings suggest that jasmonic acid plays a critical role in regulating activity of ß-glucosidases, which determines the rate of cyanogenesis, and thus mediates direct plant defense against herbivores.

An aboveground pathogen inhibits belowground rhizobia and arbuscular mycorrhizal fungi in Phaseolus vulgaris.

BACKGROUND: Induced aboveground plant defenses against pathogens can have negative effects on belowground microbial symbionts. While a considerable number of studies have utilized chemical elicitors to experimentally induce such defenses, there is surprisingly little evidence that actual aboveground pathogens affect root-associated microbes. We report here that an aboveground fungal pathogen of common bean (Phaseolus vulgaris) induces a defense response that inhibits both the belowground formation of root nodules elicited by rhizobia and the colonization with arbuscular mycorrhizal fungi (AMF). RESULTS: Foliage of plants inoculated with either rhizobia or AMF was treated with both live Colletotrichum gloeosporioides-a generalist hemibiotrophic plant pathogen-and C. gloeosporioides fragments. Polyphenol oxidase (PPO), chitinase and ß-1,3-glucanase activity in leaves and roots, as well as the number of rhizobia nodules and the extent of AMF colonization, were measured after pathogen treatments. Both the live pathogen and pathogen fragments significantly increased PPO, chitinase and ß-1,3-glucanase activity in the leaves, but only PPO activity was increased in roots. The number of rhizobia nodules and the extent of AMF colonization was significantly reduced in treatment plants when compared to controls. CONCLUSION: We demonstrate that aboveground fungal pathogens can affect belowground mutualism with two very different types of microbial symbionts-rhizobia and AMF. Our results suggest that systemically induced PPO activity is functionally involved in this above-belowground interaction. We predict that the top-down effects we show here can drastically impact plant performance in soils with limited nutrients and water; abiotic stress conditions usually mitigated by microbial belowground mutualists.

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.

Salinity-mediated cyanogenesis in white clover (Trifolium repens) affects trophic interactions.

BACKGROUND AND AIMS: Increasing soil salinity poses a major plant stress in agro-ecosystems worldwide. Surprisingly little is known about the quantitative effect of elevated salinity on secondary metabolism in many agricultural crops. Such salt-mediated changes in defence-associated compounds may significantly alter the quality of food and forage plants as well as their resistance against pests. In the present study, the effects of soil salinity on cyanogenesis in white clover (Trifolium repens), a forage crop of international importance, are analysed. METHODS: Experimental clonal plants were exposed to five levels of soil salinity, and cyanogenic potential (HCNp, total amount of accumulated cyanide in a given plant tissue), ß-glucosidase activity, soluble protein concentration and biomass production were quantified. The attractiveness of plant material grown under the different salt treatments was tested using cafeteria-style feeding trials with a generalist (grey garden slug, Deroceras reticulatum) and a specialist (clover leaf weevil, Hypera punctata) herbivore. KEY RESULTS: Salt treatment resulted in an upregulation of HCNp, whereas ß-glucosidase activity and soluble protein concentration showed no significant variation among treatments. Leaf area consumption of both herbivore species was negatively correlated with HCNp, indicating bottom-up effects of salinity-mediated changes in HCNp on plant consumers. CONCLUSIONS: The results suggest that soil salinity leads to an upregulation of cyanogenesis in white clover, which results in enhanced resistance against two different natural herbivores. The potential implications for such salinity-mediated changes in plant defence for livestock grazing remain to be tested.

Quantitative effects of leaf area removal on indirect defense of lima bean (Phaseolus lunatus) in nature.

Plants employ a diverse array of defensive traits against multiple enemies. While many plant defenses are well-studied, quantitative feedback effects of leaf area loss on the expression of defensive traits remain little understood. Extrafloral nectar (EFN; an indirect defense acting via the attraction of carnivorous arthropods) is generally considered 'cheap' as it is composed mainly of photosynthates. However, to what extent EFN secretion is related to the amount of intact photosynthetic leaf area is unknown. In this study, we measured the production of EFN, ant attraction, and herbivore damage in response to a gradient of leaf area removal in wild lima bean (Phaseolus lunatus) under natural conditions in southern Mexico. EFN production and ant recruitment were significantly decreased with increasing leaf area removal. Consequently, EFN production was inversely correlated with leaf area loss, which suggests that EFN is metabolically more expensive than previously thought. Further, we found increased herbivory in plants with reduced EFN secretion indicating additive negative feedback effects of leaf area loss. Our study is one of the first showing a quantitative negative impact of leaf damage on EFN secretion-one of the most widely distributed defensive traits in the plant kingdom.

How useful are olfactometer experiments in chemical ecology research?

Olfactometer experiments, in which arthropods are given the choice between two or more odor sources to test behavioral preferences, are commonly used in chemical ecology research. Results of such often lead to conclusions on behavior in an ecologically relevant setting. However, it is widely unknown how well these experiments reflect actual behavior in nature. Recently, we used natural insect herbivores of wild lima bean plants to evaluate their behavior in Y-tube olfactometer experiments compared with feeding experiments. We demonstrated that depending on volatile concentration, insect sex significantly determined preference, and that independent of sex, the actual feeding choice of insects depended on defensive short-distance cues, which did not correlate with volatile cues emitted by the plants. Thus, our study shows that olfactory decisions do not reflect actual feeding choice and that olfactometer experiments may only provide a limited and simplified picture of actual decision making by insects.

Distance and sex determine host plant choice by herbivorous beetles.

BACKGROUND: Plants respond to herbivore damage with the release of volatile organic compounds (VOCs). This indirect defense can cause ecological costs when herbivores themselves use VOCs as cues to localize suitable host plants. Can VOCs reliably indicate food plant quality to herbivores? METHODOLOGY: We determined the choice behavior of herbivorous beetles (Chrysomelidae: Gynandrobrotica guerreroensis and Cerotoma ruficornis) when facing lima bean plants (Fabaceae: Phaseolus lunatus) with different cyanogenic potential, which is an important constitutive direct defense. Expression of inducible indirect defenses was experimentally manipulated by jasmonic acid treatment at different concentrations. The long-distance responses of male and female beetles to the resulting induced plant volatiles were investigated in olfactometer and free-flight experiments and compared to the short-distance decisions of the same beetles in feeding trials. CONCLUSION: Female beetles of both species were repelled by VOCs released from all induced plants independent of the level of induction. In contrast, male beetles were repelled by strongly induced plants, showed no significant differences in choice behavior towards moderately induced plants, but responded positively to VOCs released from little induced plants. Thus, beetle sex and plant VOCs had a significant effect on host searching behavior. By contrast, feeding behavior of both sexes was strongly determined by the cyanogenic potential of leaves, although females again responded more sensitively than males. Apparently, VOCs mainly provide information to these beetles that are not directly related to food quality. Being induced by herbivory and involved in indirect plant defense, such VOCs might indicate the presence of competitors and predators to herbivores. We conclude that plant quality as a food source and finding a potentially enemy-free space is more important for female than for male insect herbivores, whereas the presence of a slightly damaged plant can help males to localize putative mating partners.

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.

Induced plant defense via volatile production is dependent on rhizobial symbiosis.

Nitrogen-fixing rhizobia can substantially influence plant-herbivore interactions by altering plant chemical composition and food quality. However, the effects of rhizobia on plant volatiles, which serve as indirect and direct defenses against arthropod herbivores and as signals in defense-associated plant-plant and within-plant signaling, are still unstudied. We measured the release of jasmonic acid (JA)-induced volatiles of rhizobia-colonized and rhizobia-free lima bean plants (Fabaceae: Phaseolus lunatus L.) and tested effects of their respective bouquets of volatile organic compounds (VOCs) on a specialist insect herbivore (Mexican bean beetle; Coccinellidae: Epilachna varivestis Mulsant) in olfactometer choice trials. In a further experiment, we showed that VOC induction by JA reflects the plant responses to mechanical wounding and insect herbivory. Following induction with JA, rhizobia-colonized plants released significantly higher amounts of the shikimic acid-derived compounds, whereas the emission of compounds produced via the octadecanoid, mevalonate and non-mevalonate pathways was reduced. These changes affected the choice behavior of beetles as the preference of non-induced plants was much more pronounced for plants that were colonized by rhizobia. We showed that indole likely represents the causing agent for the observed repellent effects of jasmonic acid-induced VOCs of rhizobia-colonized lima bean plants. Our study demonstrates a rhizobia-triggered efficacy of induced plant defense via volatiles. Due to these findings, we interpret rhizobia as an integral part of legume defenses against herbivores.