Direct Defense Research Articles

Systematic analysis of rice (Oryza sativa) metabolic responses to herbivory.

Plants defend against attack from herbivores by direct and indirect defence mechanisms mediated by the accumulation of phytoalexins and release of volatile signals, respectively. While the defensive arsenals of some plants, such as tobacco and Arabidopsis are well known, most of rice's (Oryza sativa) defence metabolites and their effectiveness against herbivores remain uncharacterized. Here, we used a non-biassed metabolomics approach to identify many novel herbivory-regulated metabolic signatures in rice. Most were up-regulated by herbivore attack while only a few were suppressed. Two of the most prominent up-regulated signatures were characterized as phenolamides (PAs), p-coumaroylputrescine and feruloylputrescine. PAs accumulated in response to attack by both chewing insects, i.e. feeding of the lawn armyworm (Spodoptera mauritia) and the rice skipper (Parnara guttata) larvae, and the attack of the sucking insect, the brown planthopper (Nilaparvata lugens, BPH). In bioassays, BPH insects feeding on 15% sugar solution containing p-coumaroylputrescine or feruloylputrescine, at concentrations similar to those elicited by heavy BPH attack in rice, had a higher mortality compared to those feeding on sugar diet alone. Our results highlight PAs as a rapidly expanding new group of plant defence metabolites that are elicited by herbivore attack, and deter herbivores in rice and other plants.

Elm defence against herbivores and pathogens: morphological, chemical and molecular regulation aspects

Elms (Ulmus spp.) have long been appreciated for their environmental tolerance, landscape and ornamental value, and the quality of their wood. Although elm trees are extremely hardy against abiotic stresses such as wind and pollution, they are susceptible to attacks of biotic stressors. Over 100 phytopathogens and invertebrate pests are associated with elms: fungi, bacteria and insects like beetles and moths, and to a lesser extent aphids, mites, viruses and nematodes. While the biology of the pathogen and insect vector of the Dutch elm disease has been intensively studied, less attention has been paid so far to the defence mechanisms of elms to other biotic stressors. This review highlights knowledge of direct and indirect elm defences against biotic stressors focusing on morphological, chemical and gene regulation aspects. First, we report how morphological defence mechanisms via barrier formation and vessel occlusion prevent colonisation and spread of wood- and bark-inhabiting fungi and bacteria. Second, we outline how secondary metabolites such as terpenoids (volatile terpenoids, mansonones and triterpenoids) and phenolics (lignans, coumarins, flavonoids) in leaves and bark are involved in constitutive and induced chemical defence mechanisms of elms. Third, we address knowledge on how the molecular regulation of elm defence is orchestrated through the interaction of a huge variety of stress- and defence-related genes. We conclude by pointing to the gaps of knowledge on the chemical and molecular mechanisms of elm defence against pest insects and diseases. An in-depth understanding of defence mechanisms of elms will support the development of sustainable integrated management of pests and diseases attacking elms.

Optimizing Crops for Biocontrol of Pests and Disease.

Volatile compounds and extrafloral nectar are common defenses of wild plants; however, in crops they bear an as-yet underused potential for biological control of pests and diseases. Odor emission and nectar secretion are multigene traits in wild plants, and thus form difficult targets for breeding. Furthermore, domestication has changed the capacity of crops to express these traits. We propose that breeding crops for an enhanced capacity for tritrophic interactions and volatile-mediated direct resistance to herbivores and pathogens can contribute to environmentally-friendly and sustainable agriculture. Natural plant volatiles with antifungal or repellent properties can serve as direct resistance agents. In addition, volatiles mediating tritrophic interactions can be combined with nectar-based food rewards for carnivores to boost indirect plant defense.

Salinity stress effects on direct and indirect defence metabolites in maize

In nature, plants are often exposed to multiple stress factors at the same time. The effects of single biotic or abiotic stresses on plant metabolism are well documented but how plants respond to a combination of these is little researched. Here we studied the effects of high salinity and herbivory on levels of secondary compounds and gene expression associated with defences against insects. Hydroponically grown maize plants were subjected to sodium chloride (1, 50, 100mM NaCl) and/or damage by caterpillars of Spodoptera exigua. Salt-stressed plants showed stunted growth, reduced chlorophyll fluorescence and enhanced levels of reactive oxygen species and 1,4-benzoxazin-3-one aglycones (aBX). Herbivory induced higher transcript levels of the Zm-Bx1 gene involved in aBX biosynthesis and of the Zm-SerPIN gene coding for a serine proteinase inhibitor which might affect plant feeding insects. Herbivory also triggered the emission of volatile organic compounds (VOCs) that are attractive signals for parasitoids and predators and thus regarded as an indirect defence. Herbivore-induced metabolites were differentially affected in salt-stressed plants. High salinity resulted in transient priming of jasmonic acid while aBX levels were reduced in double-stressed plants. Salt stress led to lower herbivore-induced VOC emission per plant but not per unit biomass. However, quantitative shifts in individual compounds were found in both cases. Our study confirms the notion that combined stresses produce a unique phenotype that cannot be derived from single-stress effects. The ecological implications of these changes for organisms from different trophic levels and for plant fitness remain to be tested.

Bone diseases: Targeting myostatin for direct joint defence.

Bone diseases: Targeting myostatin for direct joint defence.

From induced resistance to defence in plant‐insect interactions

AbstractPlant–insect interactions typically take place in complex settings of interactions among multiple trophic levels as well as multiple species in each trophic level. The complex interaction network may strongly impact on extrapolations of resistance traits to have a defensive function. For example, the induced response plants express to their current attacker often enhances resistance to that attacker, but may make a plant more susceptible to attack by another herbivore. Hence, the defensive function or plant fitness benefit of the response to a single attacker may be misinterpreted from pairwise interactions. Moreover, plant physiological responses to a first stress by herbivory may hamper the response to a second stress and lead to conclusions of maladaptation in plant defence responses. In light of the entire community of attackers and beneficial organisms the plant interacts with, the susceptibility to some attackers may be a consequence of adaptations that reduce fitness costs of herbivory when considering the full sweep of species that affect plant fitness. A similar argumentation may apply for indirect resistance in which predators or parasitoids dampen the effect of herbivores on plants. Plant volatiles that attract third trophic level organisms such as parasitoids may at the same time attract enemies of the parasitoids in the fourth trophic level, hyperparasitoids, which again dampen the effect of parasitoids on herbivores. In addition, the effectiveness of predators and parasitoids may be dependent on habitat complexity. Here, I plea for studies on the full plant‐associated community to understand the fitness outcome of an (induced) plant trait and hence coin it induced direct or indirect plant defence.

Trade-off among different anti-herbivore defence strategies along an altitudinal gradient.

The type and intensity of plant-herbivore interactions are likely to be altered under climate change as a consequence of differential dispersal rates of plants and their herbivores. Here, we studied variation in herbivore damage on Salvia nubicola in the field and compared its growth and defence strategies against herbivores in controlled conditions using seeds from populations along a broad altitudinal gradient. Our work is one of the first studies to simultaneously measure complex intraspecific variation in plant growth, direct and indirect defences as well as plant tolerance (ability to regrow) as a consequence of herbivore attack simulated by clipping. In the field, we found that plants experienced higher herbivore pressure in lower altitudes. In the greenhouse, plants grown from seeds collected in lower-altitude populations grew better and produced a higher content of phenolic compounds (direct defence) and volatile organic compounds (indirect defence) in response to simulated herbivory. However, there were no differences in tolerance and effect of S. nubicola extracts on the model generalist herbivore Spodoptera littoralis (direct defence) along the altitudinal gradient. Although we found that S. nubicola developed a range of defence strategies, the strategies do not seem to be used simultaneously in all populations even though most of them are correlated with altitudinal gradient. Our finding is in agreement with the current knowledge that co-expression of multiple defences might be costly for a plant, since investment in defensive traits is assumed to reduce the resource availability for growth and reproduction. Our study thus shows the importance of simultaneous study of different defence strategies since understanding these trade-offs could be necessary for detecting the mechanisms by which plants are able to cope with future climate change.

The equal effectiveness of different defensive strategies.

Plants have evolved a variety of defensive strategies to resist herbivory, but at the interspecific level, the relative effectiveness of these strategies has been poorly evaluated. In this study, we compared the level of herbivory between species that depend on ants as indirect defenders and species that rely primarily on their own direct defenses. Using a dataset of 871 species and 1,405 data points, we found that in general, ant-associated species had levels of herbivory equal to those of species that are unattractive to ants; the pattern was unaffected by plant life form, climate and phylogenetic relationships between species. Interestingly, species that offer both food and nesting spaces for ants suffered significantly lower herbivory compared to species that offer either food or nesting spaces only or no reward for ants. A negative relationship between herbivory and latitude was detected, but the pattern can be changed by ants. These findings suggest that, at the interspecific level, the effectiveness of different defensive strategies may be equal. Considering the effects of herbivory on plant performance and fitness, the equal effectiveness of different defensive strategies may play an important role in the coexistence of various species at the community scale.

Application of chemical elicitor (Z)-3-hexenol enhances direct and indirect plant defenses against tea geometrid Ectropis obliqua

Plants can respond to herbivorous attack with induced defense mechanisms. Plant elicitors play vital roles in inducing the plant defense response against herbivores. In this study, we investigated the effect of (Z)-3-hexenol (z3HOL), an important component of green leaf volatiles (GLVs), on the induced defense against a tea geometrid (TG) Ectropis obliqua Prout in tea plants. We showed that treatment with z3HOL elicites increased levels of jasmonic acid (JA) and ethylene (ET) as well as of expression of a lipoxygenase gene CsiLOX1 and a putative ACC synthase gene CsiACS1. Such reactions resulted in a marked increase in polyphenol oxidase (PPO) activity and volatile production. The induced tea plants reduced the performance of TG and became highly attractive to the main parasitoid wasp, Apanteles sp. These findings suggest that z3HOL can serve as an elicitor that triggers direct and indirect defense responses against TG by modulating signaling pathways in tea and provide a friendly strategy for biological control of pests.

(E)-β-farnesene gene reduces Lipaphis erysimi colonization in transgenic Brassica juncea lines

Aphids are the major concern that significantly reduces the yield of crops. (E)-β-farnesene (Eβf) is the principal component of the alarm pheromone of many aphids. The results of current research support the direct defense response of (E)-β-farnesene (Eβf) against aphid Lipaphis erysimi (L.) Kaltenbach in Brassica juncea. Eβf gene was isolated from Mentha arvensis and transformed into B. juncea, showed direct repellent against aphid colonization. The seasonal mean population (SMP) recorded under field condition showed significantly higher aphid colonization in wild type in comparison to most of the transgenic lines, and shows positive correlation with the repellency of transgenic plant expressing (E)-β-farnesene. The current research investigation provides direct evidence for aphid control in B. juncea using Eβf, a non-toxic mode of action.

Leaf-mining by Phyllonorycter blancardella reprograms the host-leaf transcriptome to modulate phytohormones associated with nutrient mobilization and plant defense

Phytohormones have long been hypothesized to play a key role in the interactions between plant-manipulating organisms and their host–plants such as insect–plant interactions that lead to gall or ‘green-islands’ induction. However, mechanistic understanding of how phytohormones operate in these plant reconfigurations is lacking due to limited information on the molecular and biochemical phytohormonal modulation following attack by plant-manipulating insects. In an attempt to fill this gap, the present study provides an extensive characterization of how the leaf-miner Phyllonorycter blancardella modulates the major phytohormones and the transcriptional activity of plant cells in leaves of Malus domestica. We show here, that cytokinins strongly accumulate in mined tissues despite a weak expression of plant cytokinin-related genes. Leaf-mining is also associated with enhanced biosynthesis of jasmonic acid precursors but not the active form, a weak alteration of the salicylic acid pathway and a clear inhibition of the abscisic acid pathway. Our study consolidates previous results suggesting that insects may produce and deliver cytokinins to the plant as a strategy to manipulate the physiology of the leaf to create a favorable nutritional environment. We also demonstrate that leaf-mining by P. blancardella leads to a strong reprogramming of the plant phytohormonal balance associated with increased nutrient mobilization, inhibition of leaf senescence and mitigation of plant direct and indirect defense.

Floral volatiles interfere with plant attraction of parasitoids: ontogeny-dependent infochemical dynamics in Brassica rapa.

BackgroundThe role of plant ontogeny on investment in direct defense against herbivores is well accepted, but the transition from the vegetative to the reproductive stage can also affect indirect resistance traits (i.e. attraction of the natural enemies of plant attackers). Here, we conducted behavioral bioassays in olfactometers to determine whether the developmental stage (vegetative, pre-flowering, and flowering) of Brassica rapa plants affects attraction of Cotesiaglomerata, a parasitoid of the herbivore Pieris brassicae, and examined the blends of volatile compounds emitted by plants at each developmental stage.ResultsPieris-infested plants were always more attractive to parasitoids than control plants and plants infested by a non-host herbivore, independently of plant developmental stage. On the other hand, the relative attractiveness of Pieris-infested plants was ontogeny dependent: Pieris-infested plants were more attractive at the pre-flowering stage than at the vegetative stage, and more attractive at the vegetative stage than at the flowering stage. Chemical analyses revealed that the induction of leaf volatiles after herbivory is strongly reduced in flowering plants. The addition of synthetic floral volatiles to infested vegetative plants decreased their attractiveness to parasitoids, suggesting a trade-off between signaling to pollinators and parasitoids.ConclusionOur results show that putative indirect resistance traits are affected by plant development, and are reduced during B. rapa reproductive stage. The effects of ontogenetic shifts in resource allocation on the behavior of members of the third trophic level may have important implications for the evolution of plant defense strategies against herbivores.Electronic supplementary materialThe online version of this article (doi:10.1186/s12898-015-0047-7) contains supplementary material, which is available to authorized users.

The maize cytochrome P450 CYP79A61 produces phenylacetaldoxime and indole-3-acetaldoxime in heterologous systems and might contribute to plant defense and auxin formation.

BackgroundPlants produce a group of aldoxime metabolites that are well known as volatiles and as intermediates in cyanogenic glycoside and glucosinolate biosynthesis in particular plant families. Recently it has been demonstrated that aldoximes can also accumulate as part of direct plant defense in poplar. Cytochrome P450 enzymes of the CYP79 family were shown to be responsible for the formation of aldoximes from their amino acid precursors.ResultsHere we describe the identification and characterization of maize CYP79A61 which was heterologously expressed in yeast and Nicotiana benthamiana and shown to catalyze the formation of (E/Z)-phenylacetaldoxime and (E/Z)-indole-3-acetaldoxime from L-phenylalanine and L-tryptophan, respectively. Simulated herbivory on maize leaves resulted in an increased CYP79A61 transcript accumulation and in elevated levels of L-phenylalanine and (E/Z)-phenylacetaldoxime. Although L-tryptophan levels were also increased after the treatment, (E/Z)-indole-3-acetaldoxime could not be detected in the damaged leaves. However, simulated herbivory caused a significant increase in auxin concentration.ConclusionsOur data suggest that CYP79A61 might contribute to the formation of (E/Z)-phenylacetaldoxime in maize. Since aldoximes have been described as toxic compounds for insect herbivores and pathogens, the increased accumulation of (E/Z)-phenylacetaldoxime after simulated herbivory indicates that this compound plays a role in plant defense. In addition, it is conceivable that (E/Z)-indole-3-acetaldoxime produced by recombinant CYP79A61 could be further converted into the plant hormone indole-3-acetic acid after herbivore feeding in maize.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0526-1) contains supplementary material, which is available to authorized users.

Defence against vertebrate herbivores trades off into architectural and low nutrient strategies amongst savanna Fabaceae species

Herbivory contributes substantially to plant functional diversity and in ways that move far beyond direct defence trait patterns, as effective growth strategies under herbivory require modification of multiple functional traits that are indirectly related to defence. In order to understand how herbivory has shaped plant functional diversity, we need to consider the physiology and architecture of the herbivores and how this constrains effective defence strategies. Here we consider herbivory by mammals in savanna communities that range from semi‐arid to humid conditions. We posited that the saplings of savanna trees can be grouped into two contrasting defence strategies against mammals, namely architectural defence versus low nutrient defence. We provide a mechanistic explanation for these different strategies based on the fact that plants are under competing selection pressures to limit herbivore damage and outcompete neighbouring plants. Plant competitiveness depends on growth rate, itself a function of leaf mass fraction (LMF) and leaf nitrogen per unit mass (Nm). Architectural defence against vertebrates (which includes spinescence) limits herbivore access to plant leaf materials, and partly depends on leaf‐size reduction, thereby compromising LMF. Low nutrient defence requires that leaf material is of insufficient nutrient value to support vertebrate metabolic requirements, which depends on low Nm. Thus there is an enforced tradeoff between LMF and Nm, leading to distinct trait suites for each defence strategy. We demonstrate this tradeoff by showing that numerous traits can be distinguished between 28 spinescent (architectural defenders) and non‐spinescent (low nutrient defenders) Fabaceae tree species from savannas, where mammalian herbivory is an important constraint on plant growth. Distributions of the strategies along an LMF‐Nm tradeoff further provides a predictive and parsimonious explanation for the uneven distribution of spinescent and non‐spinescent species across water and nutrient gradients.

The effect of symbiotic ant colonies on plant growth: a test using an Azteca-Cecropia system.

In studies of ant-plant mutualisms, the role that ants play in increasing the growth rates of their plant partners is potentially a key beneficial service. In the field, we measured the growth of Cecropia glaziovii saplings and compared individuals that were naturally colonized by Azteca muelleri ants with uncolonized plants in different seasons (wet and dry). We also measured light availability as well as attributes that could be influenced by the presence of Azteca colonies, such as herbivory, leaf nutrients (total nitrogen and δ15N), and investments in defense (total phenolics and leaf mass per area). We found that colonized plants grew faster than uncolonized plants and experienced a lower level of herbivory in both the wet and dry seasons. Colonized plants had higher nitrogen content than uncolonized plants, although the δ15N, light environment, total phenolics and leaf mass per area, did not differ between colonized and uncolonized plants. Since colonized and uncolonized plants did not differ in the direct defenses that we evaluated, yet herbivory was lower in colonized plants, we conclude that biotic defenses were the most effective protection against herbivores in our system. This result supports the hypothesis that protection provided by ants is an important factor promoting plant growth. Since C. glaziovii is widely distributed among a variety of forests and ecotones, and since we demonstrated a strong relationship with their ant partners, this system can be useful for comparative studies of ant-plant interactions in different habitats. Also, given this study was carried out near the transition to the subtropics, these results help generalize the geographic distribution of this mutualism and may shed light on the persistence of the interactions in the face of climate change.

Rhizobacterial colonization of roots modulates plant volatile emission and enhances the attraction of a parasitoid wasp to host-infested plants.

Beneficial root-associated microbes modify the physiological status of their host plants and affect direct and indirect plant defense against insect herbivores. While the effects of these microbes on direct plant defense against insect herbivores are well described, knowledge of the effect of the microbes on indirect plant defense against insect herbivores is still limited. In this study, we evaluate the role of the rhizobacterium Pseudomonas fluorescens WCS417r in indirect plant defense against the generalist leaf-chewing insect Mamestra brassicae through a combination of behavioral, chemical, and gene-transcriptional approaches. We show that rhizobacterial colonization of Arabidopsis thaliana roots results in an increased attraction of the parasitoid Microplitis mediator to caterpillar-infested plants. Volatile analysis revealed that rhizobacterial colonization suppressed the emission of the terpene (E)-α-bergamotene and the aromatics methyl salicylate and lilial in response to caterpillar feeding. Rhizobacterial colonization decreased the caterpillar-induced transcription of the terpene synthase genes TPS03 and TPS04. Rhizobacteria enhanced both the growth and the indirect defense of plants under caterpillar attack. This study shows that rhizobacteria have a high potential to enhance the biocontrol of leaf-chewing herbivores based on enhanced attraction of parasitoids.Electronic supplementary materialThe online version of this article (doi:10.1007/s00442-015-3277-7) contains supplementary material, which is available to authorized users.

Strong Induction of Minor Terpenes in Italian Cypress, Cupressus sempervirens, in Response to Infection by the Fungus Seiridium cardinale.

Seiridium cardinale, the main fungal pathogen responsible for cypress bark canker, is the largest threat to cypresses worldwide. The terpene response of canker-resistant clones of Italian cypress, Cupressus sempervirens, to two differently aggressive isolates of S. cardinale was studied. Phloem terpene concentrations, foliar terpene concentrations, as well as foliar terpene emission rates were analyzed 1, 10, 30, and 90 days after artificial inoculation with fungal isolates. The phloem surrounding the inoculation point exhibited de novo production of four oxygenated monoterpenes and two unidentified terpenes. The concentrations of several constitutive mono- and diterpenes increased strongly (especially α-thujene, sabinene, terpinolene, terpinen-4-ol, oxygenated monoterpenes, manool, and two unidentified diterpenes) as the infection progressed. The proportion of minor terpenes in the infected cypresses increased markedly from the first day after inoculation (from 10% in the control to 30-50% in the infected treatments). Foliar concentrations showed no clear trend, but emission rates peaked at day 10 in infected trees, with higher δ-3-carene (15-fold) and total monoterpene (10-fold) emissions than the control. No substantial differences were found among cypresses infected by the two fungal isolates. These results suggest that cypresses activate several direct and indirect chemical defense mechanisms after infection by S. cardinale.

IP 스푸핑을 통한 DDoS 공격 탐지 방안에 대한 연구

서비스거부공격은 여전히 많은 웹 서비스 사이트에서 중요한 취약 요소이기에 공공기관을 포함한 사이트에서는 방어체계 구축에 많은 노력을 기울여야 한다. 최근에는 NTP의 monlist 기능을 이용한 대량의 네트워크 트래픽을 유발시켜 분산 서비스 거부 공격을 일으키거나 직접적인 방어가 불가능한 DNS 인프라에 대한 서비스거부 공격 등 형태로 이동하고 있다. 일례로 2013년 6월에 발생한 정부통합전산센터 DNS 서버를 대상으로 발생한 DNS 어플리케이션 서비스거부공격은 공공기관의 정보시스템들에 대한 정상적 서비스를 불가능하게 하는 것이 목적이었다. 이와 같이 분산 서비스거부공격은 특정 정보시스템에 대한 피해 뿐 아니라, 불특정 정보서비스에 대한 광범위한 피해로 이어질 수 있기 때문에 사이버위협을 최소화 하는 노력을 기울여야 한다. 본 논문에서는 해외정보시스템과 국내정보시스템 사이에 정해진 규약에 의해 자료를 전송하는 경우에 발생하는 IP 스푸핑을 통한 DDoS 공격에 대해 IP 헤더의 TTL(Time To Live) 값을 활용하여 스푸핑 여부를 탐지하는 방법을 제안한다. Since the DoS(Denial of Service) attack is still an important vulnerable element in many web service sites, sites including public institution should try their best in constructing defensive systems. Recently, DDoS(Distributed Denial of Service) has been raised by prompting mass network traffic that uses NTP's monlist function or DoS attack has been made related to the DNS infrastructure which is impossible for direct defense. For instance, in June 2013, there has been an outbreak of an infringement accident where Computing and Information Agency was the target. There was a DNS application DoS attack which made the public institution's Information System impossible to run its normal services. Like this, since there is a high possibility in having an extensive damage due to the characteristics of DDoS in attacking unspecific information service and not being limited to a particular information system, efforts have to be made in order to minimize cyber threats. This thesis proposes a method for using TTL (Time To Live) value in IP header to detect DDoS attack with IP spoofing, which occurs when data is transmitted under the agreed regulation between the international and domestic information system.

Ant-Attendance in Extrafloral Nectar-Bearing Plants Promotes Growth and Decreases the Expression of Traits Related to Direct Defenses

In addition to direct defenses, some plant species provide extrafloral nectar (EF-nectar) and/or food bodies (lipid-rich particles) to attract ants for their own indirect defenses. To ascertain why such plants use indirect defenses, we investigated the respective costs of direct and indirect defenses of Mallotus japonicus seedlings grown with and without ants present. Mallotus japonicus plants growing with ants present (ant-present) secreted larger volumes of EF-nectar, containing greater amounts of sugars, as an indirect defense trait. These plants also showed chemical defensive traits, such as the number of pellucid dots and the amount of accumulated phenolics, to a lesser degree than plants without ants (ant-absent) did. Moreover, the ant-present plants grew faster. The estimated amounts of EF-nectar sugars and food bodies were small compared to the amount of phenolics. Plant biomass was correlated negatively with pellucid dot density and phenolic concentration. Plant height was correlated negatively with phenolic concentration. Moreover, leaf biomass was correlated negatively with trichome density. Taken together, these results suggest a tradeoff between the expression of direct defense traits and plant growth. Mallotus japonicus achieves more rapid growth with ants present. We propose that this occurs because these ants provide low-cost indirect defenses allowing plants to re-allocate their energy from direct defenses to growth instead. This mutual benefit apparently facilitates ant–plant defensive mutualism.

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.