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Root colonizing Trichoderma fungi can stimulate plant immunity, but net effects are strain × cultivar-specific and changing ambient conditions further contribute to variable outcomes. Here, we used four Trichoderma spp. to inoculate seeds of four common bean (Phaseolus vulgaris) cultivars and explored in three different experimental setups the effects on fungal anthracnose after leaf inoculation with Colletotrichum lindemuthianum. Plants growing in pots with field soil under greenhouse conditions exhibited the highest and those in the open field the lowest overall levels of disease. Among 48 Trichoderma strain × bean cultivar × setup combinations, Trichoderma-inoculation enhanced disease in six and decreased disease in ten cases, but with the exception of T. asperellum B6-inoculated Negro San Luis beans, the strain × cultivar-specific effects on anthracnose severity differed among the setups, and anthracnose severity did not predict seed yield in the open field. In the case of Flor de Mayo beans, Trichoderma even reduced yield in anthracnose-free field plots, although this effect was counterbalanced in anthracnose-infected plots. We consider our work as a case study that calls for stronger emphasis on field experiments in the early phases of screenings of Trichoderma inoculants as plant biostimulants.

Root colonizing Trichoderma fungi can stimulate plant immunity, but net effects are strain × cultivar-specific and changing ambient conditions further contribute to variable outcomes. Here, we used four Trichoderma spp. to inoculate seeds of four common bean (Phaseolus vulgaris) cultivars and explored in three different experimental setups the effects on fungal anthracnose after leaf inoculation with Colletotrichum lindemuthianum. Plants growing in pots with field soil under greenhouse conditions exhibited the highest and those in the open field the lowest overall levels of disease. Among 48 Trichoderma strain × bean cultivar × setup combinations, Trichoderma-inoculation enhanced disease in six and decreased disease in ten cases, but with the exception of T. asperellum B6-inoculated Negro San Luis beans, the strain × cultivar-specific effects on anthracnose severity differed among the setups, and anthracnose severity did not predict seed yield in the open field. In the case of Flor de Mayo beans, Trichoderma even reduced yield in anthracnose-free field plots, although this effect was counterbalanced in anthracnose-infected plots. We consider our work as a case study that calls for stronger emphasis on field experiments in the early phases of screenings of Trichoderma inoculants as plant biostimulants.

Numerous plant-derived volatile organic compounds (VOCs) induce the expression of resistance-related genes and thereby cause an “associational resistance” in neighbouring plants. However, VOCs can also be sequestered by plant cuticular waxes. In case that they maintain their biological activity, such sequestered VOCs could generate a “passive” associational resistance that is independent of any gene expression in the receiver. As a proof of concept, we used major components of the cuticular wax layers of the tree, Parkinsonia praecox, and conidia of Colletotrichum lindemuthianum, a fungal pathogen that has not been reported to infect P. praecox. Wax layers were re-constituted on glass slides and exposed to each of 20 pure VOCs for 1 d and then to ambient air for 1 d or 15 d. Gas chromatography-mass spectrometry (GC-MS) analyses showed that all 20 VOCs were sequestered by the re-constituted wax layers. Exposure to 18 of the VOCs significantly inhibited the germination of C. lindemuthianum conidia on these wax layers after 1 day of exposure to ambient air. Four of the VOCs: 4Z-heptenol, farnesene, limonene, and 2E-decenal, inhibited germination rates to less than 25% of the controls. After 15 d, all VOCs were still detectable, although at strongly reduced concentrations, and no significant inhibition of conidial germination could be detected anymore. Exogenous VOCs can be sequestered by the components of plant cuticular waxes and maintain their biological activity, at least over a certain time span: an effect that could generate a transient “passive associational resistance” to pathogens.

Domestication reduced the resistance of most crops which, therefore, require an ever-increasing application of pesticides. Nevertheless, in most domesticated species, the resistance against herbivores and disease agents can be activated by so-called elicitors. Here, we review various strategies of biological pest control and particularly focus on damage-associated molecular patterns (DAMPs) as a potential novel class of plant vaccines: plant-derived molecules that trigger immunity because they indicate damage when they appear in the extracellular space or, in the case of damage-induced volatile compounds, in the aerial space around the plant. The application of plant or algal extracts, which arguably contain many DAMPs, enhanced the resistance against herbivory in cabbage, tomato and maize and to fungal or bacterial disease in all major grain crops and in banana, apple, grapevine, melon, tomato, cucumber or carrot. Similarly, purified DAMPs such as ATP, fragmented DNA, peptides, cell-wall fragments, or volatiles, elicited resistance-related responses in species such wheat, corn, soybean, grapevine, bean, eggplant, cucumber, potato or tomato. In conclusion, plant-based preparations with different degrees of purity can improve the health of multiple crops and horticultural plants. Commercial products based on plant extracts are available, and leaf extracts can even be home-made, e.g., by low-income farmers. We conclude that plant extracts and their derivatives bear an as-yet underestimated potential to serve as environmentally friendly vaccines of cultivated plants.

Fungi emit a diverse blend of volatile organic compounds (VOCs) that mediate multiple fungus-fungus interactions. Plants emit VOCs as well, which can serve as resistance-inducing signals but might also act as direct resistance agents. We screened 22 VOCs that are emitted from infected plants for putative inhibitory effects on three fungal phytopathogens: Colletotrichum lindemuthianum, Fusarium oxysporum and Botrytis cinerea. The growth of all three fungi was significantly inhibited when the mycelia were exposed to an atmosphere containing nonanal, (+)-carvone, citral, trans-2-decenal, L-linalool, or nerolidol. Eugenol completely inhibited the growth of all three fungi, and 1-octen-3-ol, nonanal, 2,6-dimethyl-2,4,6-octatriene, citral, α-terpineol and trans-2-decanal inhibited at least one fungus completely. Most of these VOCs are also emitted from fungi. We conclude that antagonistic fungus-fungus and plant-fungus interactions might share common mechanisms and that plant VOCs can function as resistance agents that fungal pathogens must overcome for successful infection.

Summary 1.Host manipulation refers to the expression of a host phenotype that is partly under the genetic control of a parasite. This phenomenon can enhance parasite transmission rates and is responsible for biological marvels such as ‘Zombie-ants’ and the ‘fatal attraction’ of Toxoplasma-infected rodents to their feline predators. Such host manipulation has evolved in all major phylogenetic lineages of parasites and is assumed to enhance the fitness of the parasite. 2.However, the capacity to manipulate is not ubiquitous; that is, many clades of parasites comprise manipulating and non-manipulating species. This pattern leads to the prediction of costs that select against the evolution of manipulation, but this has been difficult to show empirically. 3.In the present study, we used a tripartitate system consisting of chili (Capsicum annuum) plants infected with Pepper golden mosaic virus and colonized by non-vector whiteflies (Trialeurodes vaporariorum), to study the effects on viral load when a non-vector herbivore feeds on the infected plants. 4.We observed that virus-infected plants emitted odours that attracted adult whiteflies, contained three times more amino acids in the phloem than mock-inoculated controls and supported higher whitefly reproduction as compared to controls. However, viral load decreased almost 100-fold in whitefly-carrying plants, which was associated with a depletion of phloem amino acids. 5.Synthesis. We show that a plant virus can suffer from a reduced within-host reproduction rate when virus-induced alterations of the plant cause a ‘fatal attraction’ of a non-vector insect that exploits the altered plant phenotype at its own benefits. The resulting fitness costs might represent a force that can select against the evolution of host manipulation by parasites. This article is protected by copyright. All rights reserved.

Establishing a horizontally transmitted mutualism is a critical step for many organisms, particularly when an individual can engage with only one partner over its lifetime. Ant foundresses shed their wings before they start to lay eggs, which makes host choice particularly critical for plant-ants that exclusively colonize myrmecophytes. Volatile organic compounds (VOCs) can indicate the identity and physiological condition of an individual and frequently serve as signals or cues that facilitate partner choice. Thus, we hypothesized that obligate plant-ants use VOCs to determine the quality of potential host plants. Indeed, winged females of the plant-ant, Pseudomyrmex ferrugineus, used plant odours to identify high-quality acacia hosts among different species and among individuals of the same species. The VOC blend of a branch was related to reward production and, interestingly, the least attractive blend contained the highest number of compounds. Although only seven different VOCs could be detected in the blend of the high-reward host species, the low-reward host emitted 13 different compounds, among which (S)-(-)-limonene and β-linalool were the dominant ones. Complementing the odour of the high-reward host with (S)-(-)-limonene, β-linalool or α-terpinene reduced its attractiveness. Strikingly, these compounds inhibited bacterial pathogens of the host plant. Synthesis. Plant-ants can use host plant odours that contain resistance-related VOCs to judge on host quality, likely because of negative crosstalk between direct resistance to disease and investment in ant-mediated defence. VOCs can serve as cues that provide reliable information for partner choice, particularly when their emission results from a physiological process that causally relates to the quality of an individual as a mutualist.

Phaseolus lunatus (lima bean) has been a model organism to understand direct and indirect defenses against herbivores. We investigated the identity and diversity of endophytic fungi associated with young, mature and old leaves of lima bean in its natural environment. We recovered 758 isolates that were identified and classified in 142 fungal species from 66 genera, with Curvularia , Fusarium , Phomopsis , Alternaria and Colletotrichum being the most abundant and diverse Ascomycota genera. Our data revealed that species richness and diversity increased with leaf age, this factor being also important for the structure of the endophytic community. The occurrence patterns of fungal species was highly heterogeneous among leaves, and only fourteen species were recovered from all three foliar ages investigated. In vitro dual assays between endophytes and the plant pathogen Colletotrichum lindemuthianum revealed that most endophytes tested can inhibit the growth of this pathogen, suggesting their potential as biocontrol agents.

The 'adaptive host manipulation' hypothesis predicts that parasites can enhance their transmission rates via manipulation of their host's phenotype. For example, many plant pathogens alter the nutritional quality of their host for herbivores that serve as their vectors. However, herbivores, including non-vectors, might cause additional alterations in the plant phenotype. Here, we studied changes in the amino acid (AA) content in the phloem of chilli (Capsicum annuum) plants infected with Pepper golden mosaic virus (PepGMV) upon subsequent colonization with a non-vector, the phloem-feeding whitefly (Trialeurodes vaporariorum). Virus infection alone caused an almost 30-fold increase in overall phloem AAs, but colonization by T. vaporariorum completely reversed this effect. At the level of individual AAs, contents of proline, tyrosine, and valine increased, and histidine and alanine decreased in PepGMV -infected as compared to control plants, whereas colonization by T. vaporariorum caused decreased contents of proline, tyrosine, and valine, and increased contents of histidine and alanine. Overall, the colonization by the whitefly had much stronger effects on phloem AA composition than virus infection. We conclude that the phloem composition of a virus-infected host plant can rapidly change upon arrival of an herbivore and that these changes need to be monitored to predict the nutritional quality of the plant in the long run.

Plants in more than 100 families secrete extrafloral nectar (EFN) to establish food-for-protection mutualisms with ants. Facultative ant-plants secrete EFN as a jasmonic acid (JA)-dependent response to attract generalist ants. In contrast, obligate ant-plants like the Central American “Swollen-Thorn Acacias” are colonized by specialized ants, although an individual host can carry ant colonies from different species that differ in the degree of protection they provide. We hypothesized that hosts that associate simultaneously with various partners should produce rewards in a modular manner to preferentially reward high quality partners. To test this hypothesis, we applied JA to distinct leaves and quantified cell wall invertase activity (CWIN; a regulator of nectar secretion) and EFN secretion by these “local” (i.e., treated) and the “systemic” (i.e., non-treated) leaves of the same branch. Both CWIN activity and EFN secretion increased in local and systemic leaves of the facultative ant-plant Acacia cochliacantha, but only in the local leaves of the obligate ant-plant, A. cornigera. The systemic EFN secretion in A. cochliacantha was associated with an enhanced emission of volatile organic compounds (VOCs). Such VOCs function as “external signals” that control systemic defense responses in diverse plant species. Indeed, the headspace of JA-treated branches of A. cochliacantha induced EFN secretion in both plant species, whereas the headspace of A. cornigera caused no detectable induction effect. Analyses of the headspace using GC-MS identified six VOCs in the headspace of A. cochliacantha that were not emitted by A. cornigera. Among these VOCs, β-caryophyllene and (cis)-hexenyl isovalerate have already been reported in other plant species to induce defense traits, including EFN secretion. Our observations underline the importance of VOCs as systemic within-plant signals and show that the modular rewarding in A. cornigera is likely to result from a reduced emission of the systemic signal, rather than from a reduced responsiveness to the signal. We suggest that modular rewarding allows hosts to restrict the metabolic investment to specific partners and to efficiently sanction potential exploiters.

Psittacanthus calyculatus is a hemiparasitic plant that infects a wide range of trees. Mainly the biology reproduction of this mistletoe lies in bright colored flower development. Furthermore, it uses the nectar secretion as the only reward to engage different flower visitors. We investigated the physiological mechanisms of the flower phenology per hour and per day to analyze the spatial-temporal patterns of the nectar secretion, Cell Wall Invertase Activity (key enzyme in the quality of nectar), nectar chemistry, volatile organic compounds (VOCs) emission, synthesis of carotenoids and frequency of floral visitors. Flowers lasted 4 days, total nectar was loaded just before the anthesis and the secretion was maintained over day 1 and 2, decreased on day 3, and stopped on day 4. The diurnal nectar secretion dynamic per hour on day 1 and 2 showed similar patterns with high production on the morning and a decrease in the afternoon, the secretion declined on day 3 and ceased on day 4. On the other hand, CWIN activity per day was less before the anthesis and increased on day 1 and 2, this enzymatic activity decreased on the old flower phenology. Moreover, diurnal CWIN activities showed different patterns in the morning, noon, and lastly in the afternoon. Nectar chemistry varied significantly throughout of the flower lifetime, sucrose decreased along the flower phenology increasing glucose and fructose. Amino acids showed the prevalence of proline and oxo-proline, both increased on the day 1 and diminished in subsequent old flower stages. The spatial VOCs emission showed the presence of 11 compounds being β-ocimene the main volatile; its release increased on day 1 and remained constant in the flower lifetime. Lutein, lycopene, and β-carotene were concentrated in old stages of the flowers. In field, the most frequent flower visitors were the hummingbirds that usually foraging in all phenologic flower stage and their foraging events decreased with the phenological flower lifetimes. The results showed that these traits presented by P. calyculatus flowers are able to engage and manipulate the behavior of flower visitors and contribute to the reproduction of the parasitic plant.

Summary Plants that express resistance to herbivores emit volatile organic compounds (VOCs) that can trigger resistance responses in undamaged neighbours. Recent reports indicate that VOCs can also trigger the resistance to pathogens, an effect that might be due to different mechanisms: the priming of an induced expression of resistance genes in the receiver or direct inhibitory effects on microbial pathogens that cause a passive ‘associational’ resistance in the VOC-exposed plant. We investigated whether VOCs emitted from a resistant common bean (Phaseolus vulgaris) cultivar enhance the resistance to the fungus Colletotrichum lindemuthianum in a susceptible cultivar and analysed whether specific VOCs are likely to directly affect the pathogen. We found that susceptible plants exposed to the headspace of resistance-expressing plants over 6 h became phenotypically as resistant as the resistant cultivar. Several resistance marker genes (PATHOGENESIS-RELATED [PR] 1, 2 and 4) were primed in VOC-exposed susceptible plants. After challenging, these genes reached expression levels at least as high as in the resistant cultivar. Additionally, individual VOCs such as limonene, linalool, nonanal, methyl salicylate and methyl jasmonate at natural concentrations directly inhibited the germination of conidia as did also the headspace of a resistance-expressing plant. This inhibition of conidial germination was dosage-dependent and irreversible. Synthesis. We conclude that VOCs are involved in the resistance of bean to fungal pathogens. They can contribute to the direct resistance in the emitter itself, and resistance phenotypes of neighbouring receiver plants can result from induced as well as associational resistance. Plant VOCs play multiple roles in the resistance of plants to microbial pathogens.

Plants secrete extrafloral nectar (EFN) that attracts predators. The efficiency of the resulting anti-herbivore defense depends on the quantity and spatial distribution of EFN. Thus, according to the optimal defense hypothesis (ODH), plants should secrete EFN on the most valuable organs and when herbivore pressure is high. Ricinus communis plants secreted most EFN on the youngest (i.e., most valuable) leaves and after the simulation of herbivory via the application of jasmonic acid (JA). Here, we investigated the physiological mechanisms that might produce these seemingly adaptive spatiotemporal patterns. Cell wall invertase (CWIN; EC 3.2.1.26) was most active in the hours before peak EFN secretion, its decrease preceded the decrease in EFN secretion, and CWIN activity was inducible by JA. Thus, CWIN appears to be a central player in EFN secretion: its activation by JA is likely to cause the induction of EFN secretion after herbivory. Shading individual leaves decreased EFN secretion locally on these leaves with no effect on CWIN activity in the nectaries, which is likely to be because it decreased the content of sucrose, the substrate of CWIN, in the phloem. Our results demonstrate how the interplay of two physiological processes can cause ecologically relevant spatiotemporal patterns in a plant defense trait.

Endophytic fungi colonize plants without causing symptoms of disease and can enhance the resistance of their host to pathogens. We cultivated 53 fungal strains from wild lima bean ( Phaseolus lunatus ) and investigated their effects on pathogens using in vitro assays and experiments in planta . Most strains were annotated as Rhizopus, Fusarium, Penicillium, Cochliobolus, and Artomyces spp. by the sequence of their 18S rRNA gene. In vitro confrontation assays between endophytes and three pathogens (the bacteria Pseudomonas syringae pv. syringae and Enterobacter sp. strain FCB1, and the fungus Colletotrichum lindemuthianum ) revealed strong and mainly symmetric reciprocal effects: endophyte and pathogen either mutually inhibited (mainly Enterobacter FCB1 and Colletotrichum ) or facilitated ( P. syringae ) the growth of each other. In planta , the endophytes had a strong inhibitory effect on P. syringae when they colonized the plant before the bacterium, whereas infection was facilitated when P. syringae colonized the plant before the endophyte. Infection with Enterobacter FCB1 was facilitated when the bacterium colonized the plant before or on the same day with the endophyte, but not when the endophyte was present before the bacterium. The order of arrival determines whether fungal endophytes enhance plant resistance to bacterial pathogens or facilitate disease.

A recent study reports partner manipulation for an interaction that was considered a reward-for-defence mutualism. Secretions of lycaenid caterpillars altered ant locomotion and aggressiveness, likely by manipulating dopaminergic signalling. This study opens the question whether such manipulation is common and whether manipulation necessarily characterises an interaction as parasitism.

Myrmecophytic Acacia species produce food bodies (FBs) to nourish ants of the Pseudomyrmex ferrugineus group, with which they live in an obligate mutualism. We investigated how the FBs are protected from exploiting nonmutualists. Two-dimensional gel electrophoresis of the FB proteomes and consecutive protein sequencing indicated the presence of several Kunitz-type protease inhibitors (PIs). PIs extracted from Acacia FBs were biologically active, as they effectively reduced the trypsin-like and elastase-like proteolytic activity in the guts of seed-feeding beetles (Prostephanus truncatus and Zabrotes subfasciatus), which were used as nonadapted herbivores representing potential exploiters. By contrast, the legitimate mutualistic consumers maintained high proteolytic activity dominated by chymotrypsin 1, which was insensitive to the FB PIs. Larvae of an exploiter ant (Pseudomyrmex gracilis) taken from Acacia hosts exhibited lower overall proteolytic activity than the mutualists. The proteases of this exploiter exhibited mainly elastase-like and to a lower degree chymotrypsin 1-like activity. We conclude that the mutualist ants possess specifically those proteases that are least sensitive to the PIs in their specific food source, whereas the congeneric exploiter ant appears partly, but not completely, adapted to consume Acacia FBs. By contrast, any consumption of the FBs by nonadapted exploiters would effectively inhibit their digestive capacities. We suggest that the term 'exclusive rewards' can be used to describe situations similar to the one that has evolved in myrmecophytic Acacia species, which reward mutualists with FBs but safeguard the reward from exploitation by generalists by making the FBs difficult for the nonadapted consumer to use.

Summary 1. Multiple plant species are engaged in defensive mutualisms with members of the third trophic level. However, mutualisms are prone to exploitation by low-quality symbionts that do not provide the adequate service to their host. Can mutualisms proceed only when hosts identify their symbionts in advance or continuously monitor their activity, or are there other mechanisms to avoid the invasion of mutualisms by exploiters? 2. High-reward species amongst Mesoamerican Acacia myrmecophytes are dominantly colonized by defending mutualistic ants, whereas about 50% of the low-reward hosts are inhabited by non-defending exploiters. I followed the development of recently founded ant colonies on a high-reward and a low-reward Acacia host species over 7 months, to investigate whether reward production correlates with a preferred maintenance of defending ants on the respective hosts. 3. Ant diversity decreased sooner on high-reward than on low-reward hosts, and mutualistic ants were more likely to finally dominate the high-reward hosts. I observed an increased frequency of mutualists replacing parasites at high initial rates of reward production. Apparently, higher nectar provisioning by the host plants shifted the competitive balance between mutualistic and parasitic ants. Independently of the causal reason for the different secretion rates, producing more nectar thereby favours the maintenance of defending mutualists on high-reward hosts. 4. Synthesis. The aggressiveness that enables ants to outcompete other ants also underlies their defensive effect against herbivores. I conclude that hosts can preferably associate with high-quality mutualists without measuring their effectiveness. Mutualisms remain stable when partner screening is based on traits that are relevant for the mutualistic interaction, with no need for the host to have information on the quality or identity of the symbiont.

Summary 1. Above-ground (AG) and below-ground (BG) biota show little overlap and are usually investigated independently. However, both communities are connected by a group of organisms that intrinsically belong to both worlds: plants. The responses of plants to environmental stresses often move from the aerial compartments to the roots and vice versa and thereby enable interactions among species that never come into direct physical contact. 2. Plant-mediated AG–BG interactions can have positive or negative effects on organisms from all phyla. For example, damage inflicted on roots or root symbioses with mycorrhizal fungi and rhizobia can affect the interactions between the aerial compartment and herbivores, pollinators, predators and parasitoids, whereas resistance expression in leaves can affect root herbivores as well as mutualistic or parasitic soil microorganisms. Given the tremendous diversity in the interacting organisms and also in the outcomes of the interactions, we are only just beginning to identify general patterns in the net effects of plant-mediated interactions between AG and BG communities. 3. Synthesis. The papers in this Special Feature reveal an important problem that remains to be solved: for most AG–BG interactions, the driving evolutionary forces remain to be identified. Because several plant responses in the context of AG–BG interactions ultimately result in negative effects on the plant itself, it appears obvious that in some cases the interacting partners manipulate the plant for their own benefit. In the future we will need to combine knowledge on the underlying physiological mechanisms with data on fitness effects. ‘Negative’ results, which demonstrate that the other compartment does NOT respond to a certain stress, or which demonstrate no or negative fitness effects on the involved species, are as important as reports on successful systemic resistance responses. Integrating knowledge on the physiological and genetic mechanisms with data on the fitness effects on all interacting partners will help to determine whether a certain AG–BG interaction is adaptive from the perspective of the plant, or the interacting organism, or both.

Summary 1. Plants possess numerous mechanisms to control infections by deleterious organisms. Unspecific resistance mechanisms may, however, also exert ecological costs when they have a negative effect on beneficial plant–microbe interactions. Such negative effects may even cross the border between a plant’s aerial parts and its roots and then affect very central functions such as nutrient uptake and root resistance to micro-organisms. Whereas an impaired nodulation indeed appears common after resistance expression in the leaves, contradictory results have been published for the case of arbuscular mycorrhizal (AM) fungi. 2. We analysed the effect of induction of resistance mechanisms in foliar tissues on AM colonization in soybean plants, using acibenzolar-S-methyl (ASM) as chemical elicitor. By determining different physiological and biochemical parameters, we assessed whether the effects are related to the activation of the plant defence mechanisms or rather to the re-allocation of primary metabolites. 3. Colonization with AM fungi transiently decreased after pathogen resistance mechanisms were elicited in the aerial parts of the plant. The induction with ASM led to a significant, yet moderate, defence response in the roots, which was modulated in mycorrhizal plants. No allocation or fitness costs associated with the induction of resistance were detected in this study. 4. Synthesis. Our study confirms a transient negative impact of the elicitation of foliar defences on root–AM interactions. The results show that induced resistance to foliar pathogens can (i) move from the above-ground to the below-ground compartment and (ii) affect mutualistic micro-organisms as well as plant pathogens. We also conclude that (iii) the negative effect is likely linked to changes in the defence status of the plant rather than to changes in resource allocation patterns and (iv) the AM association can modulate the activation of the plant defence mechanisms and overcome such effects.

Summary 1. Plants express multiple defensive traits, but little is known about the genetic stability and phenotypic plasticity of these traits in nature. To investigate sources of variation and their potential ecological consequences for herbivores, we combined field observations of cyanogenic lima bean with laboratory experiments. 2. Field studies in South Mexico revealed a distinct variability of cyanogenic traits within and among wild lima bean populations. To differentiate among genetic variation and the impact of ambient conditions on plant phenotypes, we used seed-grown plants as well as clones propagated from high- (HC) and low-cyanogenic (LC) wild type plants. 3. In growth chamber experiments, we cultivated plants under three intensities each of drought and salt stress, nutrient supply, and light availability. We consecutively quantified cyanogenesis and total phenolics as chemical defences, soluble proteins as measure of leaf quality, and leaf mass per area and tissue hydration as physical defence-associated traits. 4. Plant traits were genetically stable, as clones propagated from the HC- and LC-mother plants maintained their cyanogenic features and also showed constancy of other leaf parameters tested. 5. In contrast to genetically heterogeneous seed-grown plants, cyanogenesis and soluble protein concentration in clonal plants showed significant variation in response to reduced water supply, increased soil salinity, and fertilization. The other leaf traits, however, showed no significant phenotypic plasticity depending on treatment. 6. Among all traits analysed, genetic and phenotypic variation in cyanogenesis proved the best predictor of herbivore behaviour and development, as LC-plants were preferred by adult Mexican bean beetles and allowed for faster larval development. 7. Synthesis. We demonstrate that (i) functional analyses of plant responses to abiotic factors require methodical separation of genotypic variability and phenotypic plasticity, (ii) different abiotic parameters quantitatively affect the plants’ chemical phenotype and that (iii) changes of plant phenotype can have strong impacts on natural herbivores. Our results suggest that genetic variability and phenotypic plasticity of plant anti-herbivore defences allow plant populations to rapidly respond to changing environmental conditions.

Plants require protection against a wide range of attackers such as insects and pathogens. The adequate plant defense responses are regulated via sophisticated signal cascades, which are activated following the perception of specific cues of the attackers. Plants might, however, gain a significant fitness advantage when pre-empting enemy attack before it actually occurs. Monitoring cues from attacked neighbors can permit plants to reach this goal. We have recently found airborne disease resistance against a bacterial pathogen in uninfected lima bean plants when these were located close to conspecific, resistance-expressing neighbors. The emitters could be chemically induced with benzothiadiazole or biologically with an avirulent pathogen. Unexpectedly, receiver plants, although expressing a functioning resistance, did not show reduced growth rates, which represent a common side-effect of directly induced pathogen resistance. Nonanal was identified as an active volatile and, rather than directly inducing full resistance, primed defense gene expression, which became fully activated only when the plants were subsequently challenged by a virulent pathogen. Priming by airborne signals allows for a more efficient and less costly preparation of plants for future attack and airborne signaling can affect resistance against both major groups of plant enemies: herbivores and pathogens.

Phenotypic plasticity is the ability of an organism to express different phenotypes in response to changing environments and becomes particularly obvious when plants alter their transcriptome after enemy attack. The resulting alterations affect the metabolic, chemical and morphological phenotype and cause resistance or tolerance phenomena, which allow plants to main high fitness in the presence of enemies. Volatiles released from damaged plants can be received by their neighbours or undamaged parts of the same plant to mount an adequate level of resistance and thereby add a further level of phenotypic plasticity. The induced defence responses also include attraction of the third trophic level and, thus, dramatic changes of the ‘extended phenotype’ of the plant, that is, its surrounding fauna. The underlying interactions are, at least partly, under the control of the plant and, thus, subject to co-evolutionary processes. Fitness costs are a common explanation for the evolution of inducible resistance expression. However, variability in the resistance phenotype can per se be beneficial, because it makes counter-adaptations by the plants’ enemies more difficult. In the case of indirect defences, which are mediated by plant-carnivore mutualisms, signal reliability and reciprocal responses among phenotypically plastic partners appear necessary prerequisites for their evolutionary stabilisation. The expression of resistance and tolerance is induced by enemy attack but is also under control by abiotic factors, such as resource supply, and by biotic parameters, such as current and anticipated competition, efficiency of the expressed resistance and ontogenetic stage. All these levels of plasticity help plants to survive as sessile organisms in a rapidly changing environment and in the presence of mobile enemies.

Protective ant–plant interactions provide valuable model systems to study mutualisms. Here, we summarise our recent research on chemical and physiological adaptations that contribute to the stabilisation of the mutualism between Mesoamerican Acacia host plants and their Pseudomyrmex ant inhabitants against exploiters, that is, species using host-derived rewards without rendering a service. Acacia hosts produce food bodies (FBs) and extrafloral nectar (EFN). Both types of reward are chemically adapted to their specific function as ant food and protected from different exploiters. FBs contained higher amounts of specific proteins than the leaves from which they originate. EFN possessed amino acids making it attractive for the mutualist ants and an invertase making its carbohydrate composition nutritionally suitable for the mutualists but unattractive for generalists. Moreover, pathogenesis-related proteins such as glucanases, chitinases and peroxidases were found in EFN, which likely serve as protection from microorganisms. Digestive adaptations were found that make workers of the ant mutualists dependent on the host-derived food sources, a mechanism that likely counteracts the evolution of cheaters. The ants also possessed a high diversity of bacterial associates, several of which appeared involved in nitrogen fixation, thus contributing to the nutrition of these ‘vegetarian’ ants. By contrast, a non-defending ant species that parasitises the host plants appeared physiologically less adapted to the host-derived food rewards; this species, thus, likely is competitively inferior when colony growth is limited by plant-derived rewards. In summary, several physiological adaptations of both host plants and ants stabilise the Acacia–Pseudomyrmex mutualism against exploitation.

In ant-plant protection mutualisms, plants provide nesting space and nutrition to defending ants. Several plant-ants are polygynous. Possessing more than one queen per colony can reduce nestmate relatedness and consequently the inclusive fitness of workers. Here, we investigated the colony structure of the obligate acacia-ant Pseudomyrmex peperi, which competes for nesting space with several congeneric and sympatric species. Pseudomyrmex peperi had a lower colony founding success than its congeners and thus, appears to be competitively inferior during the early stages of colony development. Aggression assays showed that P. peperi establishes distinct, but highly polygynous supercolonies, which can inhabit large clusters of host trees. Analysing queens, workers, males and virgin queens from two supercolonies with eight polymorphic microsatellite markers revealed a maximum of three alleles per locus within a colony and, thus, high relatedness among nestmates. Colonies had probably been founded by one singly mated queen and supercolonies resulted from intranidal mating among colony-derived males and daughter queens. This strategy allows colonies to grow by budding and to occupy individual plant clusters for time spans that are longer than an individual queen's life. Ancestral states reconstruction indicated that polygyny represents the derived state within obligate acacia-ants. We suggest that the extreme polygyny of Pseudomyrmex peperi, which is achieved by intranidal mating and thereby maintains high nestmate relatedness, might play an important role for species coexistence in a dynamic and competitive habitat.

Herbivore-induced plant volatiles affect the systemic response of plants to local damage and hence represent potential plant hormones. These signals can also lead to “plant-plant communication,” a defense induction in yet undamaged plants growing close to damaged neighbors. We observed this phenomenon in the context of disease resistance. Lima bean (Phaseolus lunatus) plants in a natural population became more resistant against a bacterial pathogen, Pseudomonas syringae pv syringae, when located close to conspecific neighbors in which systemic acquired resistance to pathogens had been chemically induced with benzothiadiazole (BTH). Airborne disease resistance induction could also be triggered biologically by infection with avirulent P. syringae. Challenge inoculation after exposure to induced and noninduced plants revealed that the air coming from induced plants mainly primed resistance, since expression of PATHOGENESIS-RELATED PROTEIN2 (PR-2) was significantly stronger in exposed than in nonexposed individuals when the plants were subsequently challenged by P. syringae. Among others, the plant-derived volatile nonanal was present in the headspace of BTH-treated plants and significantly enhanced PR-2 expression in the exposed plants, resulting in reduced symptom appearance. Negative effects on growth of BTH-treated plants, which usually occur as a consequence of the high costs of direct resistance induction, were not observed in volatile organic compound-exposed plants. Volatile-mediated priming appears to be a highly attractive means for the tailoring of systemic acquired resistance against plant pathogens.

Feeding by herbivores induces plant defences, but we still do not know all the signals that mediate this response. Here, I argue that a general principle in this mediation is 'damaged-self recognition', that is, the perception of motifs by the plant that indicate disintegrated plant cells. Most defence-inducing molecules are (or contain) plant-derived motifs or disintegrate plant cells and thereby release defence elicitors. By perceiving the 'damaged self', plants can retain evolutionary control over their interactions with herbivores rather than allowing herbivores to dominate the interaction. The concept of 'damaged-self recognition' provides a paradigm for plant responses to herbivory and helps the search for the currently unknown elicitors of those defence responses, which have so far only been described at the phenotypic level.

Plants use inducible defence mechanisms to fend off harmful organisms. Resistance that is induced in response to local attack is often expressed systemically, that is, in organs that are not yet damaged. In the search for translocated defence signals, biochemical studies follow the physical movement of putative signals, and grafting experiments use mutants that are impaired in the production or perception of these signals. Long-distance signals can directly activate defence or can prime for the stronger and faster induction of defence. Historically, research has focused on the vascular transport of signalling metabolites, but volatiles can play a crucial role as well. We compare the advantages and constraints of vascular and airborne signals for the plant, and discuss how they can act in synergy to achieve optimised resistance in distal plant parts.

Many plants interact with carnivores as an indirect defence against herbivores. The release of volatile organic compounds (VOCs) and the secretion of extrafloral nectar (EFN) are induced by insect feeding, a response that is mediated by the plant hormone, jasmonic acid. Although VOCs mainly attract predatory mites and parasitic wasps, while EFN mainly attracts ants, many more animal-plant interactions are influenced by these two traits. Other traits involved in defensive tritrophic interactions are cellular food bodies and domatia, which serve the nutrition and housing of predators. They are not known to respond to herbivory, while food body production can be induced by the presence of the mutualists. Interactions among the different defensive traits, and between them and other biotic and abiotic factors exist on the genetic, physiological, and ecological levels, but so far remain understudied. Indirect defences are increasingly being discussed as an environmentally-friendly crop protection strategy, but much more knowledge on their fitness effects under certain environmental conditions is required before we can understand their ecological and evolutionary relevance, and before tritrophic interactions can serve as a reliable tool in agronomy.

Plants resist attack by pathogens and herbivorous insects through constitutive and inducible defences. Based on differences in signalling pathways and spectra of effectiveness, different types of induced resistance have been defined. Systemic acquired resistance (SAR) occurs in distal plant parts following localized infection by a necrotizing pathogen. It is controlled by a signalling pathway that depends upon the accumulation of salicylic acid (SA) and the regulatory protein NPR1. In contrast, induced systemic resistance (ISR) is induced by selected strains of non-pathogenic plant growth promoting bacteria (PGPR). ISR functions independently of SA, but requires NPR1 and is regulated by jasmonic acid (JA) and ethylene (ET). It is generally believed that induced resistance evolved to save energy under pathogen or insect-free conditions, although costs still arise when defences are activated following attack. Costs can arise from the allocation of resources to defence and away from plant growth and development, and there are also ecological costs which result from trade-offs between induced resistance and the plant's interaction with beneficial organisms e.g. mycorrhizal fungi. To date, few studies have examined the costs and trade-offs associated with induced resistance to pathogens. There is a clear need for long-term studies of costs and trade-offs associated with induced resistance in crops under commercial conditions. Without such information, the potential offered by induced resistance is unlikely to be realized.

Plants respond to herbivore attack with the release of volatile organic compounds (VOCs), which can attract predatory arthropods and/or repel herbivores and thus serve as a means of defense against herbivores. Such VOCs might also be perceived by neighboring plants to adjust their defensive phenotype according to the present risk of attack. We exposed lima bean plants at their natural growing site to volatiles of beetle-damaged conspecific shoots. This reduced herbivore damage and increased the growth rate of the exposed plants. To investigate whether VOCs also can serve in signaling processes within the same individual plant we focused on undamaged “receiver” leaves that were either exposed or not exposed to VOCs released by induced “emitter” leaves. Extrafloral nectar secretion by receiver leaves increased when they were exposed to VOCs of induced emitters of neighboring plants or of the same shoot, yet not when VOCs were removed from the system. Extrafloral nectar attracts predatory arthropods and represents an induced defense mechanism. The volatiles also primed extrafloral nectar secretion to show an augmented response to subsequent damage. Herbivore-induced VOCs elicit a defensive response in undamaged plants (or parts of plants) under natural conditions, and they function as external signal for within-plant communication, thus serving also a physiological role in the systemic response of a plant to local damage.

Nitrogen plays a central role in most metabolic processes and therefore represents a critical element in the growth of all organisms (Mattson, 1980). However, whereas many prokaryotes can assimilate atmospheric N2 and then are autotrophs for this nutrient, the higher organisms such as plants, animals, and fungi do not harbor this important physiological process. Although gaseous N2 covers each centimeter of the land and N cycles through the ecosystems, organically bound nitrogen represents a limiting factor in many ecosystems and for many species. The main source of an additional supply of organic N to the ecosystems is the biological fixation of N from the atmosphere by a selected group of prokaryotes (i.e., Rhizobiales and green sulfur bacteria) (Young, 1992) which possess the complex enzymatic nitrogenase machinery (LeBauer and Treseder, 2008). These prokaryotes reduce N2 and convert it into forms that can be utilized by animals, plants, and fungi (Nardi et al., 2002). The global biological nitrogen fixation amounts to ≈ 110 and 140 teragrams in terrestrial and oceanic ecosystems, respectively (Fowler et al., 2013; Gruber and Galloway, 2008; Nardi et al., 2002; Vitousek et al., 2013). Plants like the legumes establish nutritive mutualisms with prokaryotes (rhizobia), which allow them to have direct access to the de novo fixed N. By contrast, the survival, development, maintenance, and growth of arthropods are believed to depend on organic diets with their content of

Plants secrete extrafloral nectar (EFN) as an induced defense against herbivores. EFN contains not only carbohydrates and amino acids but also pathogenesis-related proteins and other protective enzymes, making EFN an exclusive reward. EFN secretion is commonly induced after wounding, likely owing to a jasmonic acid–induced cell wall invertase, and is limited by phloem sucrose availability: Both factors control EFN secretion according to the optimal defense hypothesis. Non-ant EFN consumers include parasitoids, wasps, spiders, mites, bugs, and predatory beetles. Little is known about the relevance of EFN to the nutrition of its consumers and, hence, to the structuring of arthropod communities. The mutualism can be established quickly among noncoevolved (e.g., invasive) species, indicating its easy assembly is due to ecological fitting. Therefore, increasing efforts are directed toward using EFN in biocontrol. However, documentation of the importance of EFN for the communities of plants and arthropods in natural, invasive, and agricultural ecosystems is still limited.

Leaf damage induces in many plant species the secretion of extrafloral nectar (EFN) and/or the release of specific odours (volatile organic compounds, VOCs). Both traits attract carnivorous arthropods and function as indirect plant defences by increasing the predation pressure on attacking herbivores. We have conducted field experiments in Mexico and found that plants that had been exposed to VOCs thereafter responded to subsequent leaf damage with an increased EFN secretion. VOCs 'primed' the plants to respond more efficiently once they were attacked themselves. Such priming effects are well known for induced resistance to pathogens, yet this is the first description of VOCs priming EFN secretion, a taxonomically widespread anti-herbivore defence. Plants can use chemical signals in their environment to assess the risk of herbivory and make use of this information to adjust their own defensive strategy accordingly.

Summary 1 Many plant species respond to herbivory with increased emission of volatile organic compounds (VOCs): these attract carnivorous arthropods and thereby function as an indirect defence mechanism. Whether neighbouring plants can ‘eavesdrop’ on such airborne cues and tailor their defences accordingly, remains controversial. 2 We used Lima bean plants (Phaseolus lunatus) to investigate whether herbivore-induced VOCs induce another indirect defence strategy, i.e. the secretion of extrafloral nectar (EFN) in conspecific plant neighbours, and whether this enhances the defence status of the receiving plant under natural conditions. 3 EFN secretion was induced by VOCs released from herbivore-damaged bean tendrils as well as by a synthetic VOC mixture resembling the natural one. One constituent of the herbivore-induced blend – the green leaf volatile (3Z)-hex-3-enyl acetate – was sufficient to elicit the defence reaction. 4 A long-term experiment comparing the defensive effect of EFN alone with the VOC-mediated effect (EFN induction plus attraction of plant defenders) suggested that Lima bean benefits from both indirect defences. Repeated treatment of tendrils with either an artificial blend of VOCs or with EFN led to the attraction of a higher cumulative number of predatory and parasitoid insects (i.e. ants and wasps) as well as to less herbivore damage and an increased production of inflorescences and leaves. 5 Our results demonstrate that one indirect defence mechanism can induce another one in conspecific plants, and that Lima bean plants can benefit from this VOC-induced EFN secretion under natural conditions. Both extrafloral nectaries and the capability to release VOCs upon herbivory are present in many plant taxa and airborne signalling may thus represent a common mechanism for regulating the secretion of EFN in plant parts which face an increased risk of herbivory.

The optimal defence hypothesis predicts that plant parts characterized by a high value and/or a high risk of being attacked should exhibit the highest level of defence. We tested this hypothesis with Macaranga bancana ant-plants, which are protected efficiently by resident, mutualistic ants from herbivores, parasites and encroaching vegetation. Because cost-effective defence of the host by ants increases ant fitness, selection should act on ant behaviour to produce patterns of distribution of defence as predicted for direct chemical defence traits. Termites and pieces of tape were equally distributed over the uppermost ten leaves and over the leaf-bearing part of the stems (with termites mimicking a transient herbivore, while tape mimics a long-term stress caused by a climber or plant parasite). This arrangement allowed a separation of putative coevolutionary adaptations in the ants' behaviour from other potential sources of spatial patterns in ant defence, such as differences in herbivore pressure, in the vulnerability of different herbivores, or in direct plant defences. Ant activity dropped rapidly at termite baits, but remained high at tapes for at least 5 h, thereby demonstrating adaptive differential responses to the differences between the two stressors. Most importantly, ants preferentially defended young leaves and shoot parts. The temporal and spatial patterns of ant attention to exogenous stressors thus were clearly adaptive, varying with likely costs and benefits of defence as formulated in the optimal defence theory.

Myrmecophytic plants use obligate ant mutualists as a constitutive indirect defence mechanism. These plants often produce cellular food bodies (FBs) to nourish their resident ants. Lipids, proteins, and even highly specialised compounds such as glycogen have been reported from FBs, but detailed chemical analyses of FB composition have so far been presented only for Southeast Asian Macaranga and Central American Piper myrmecophytes. Here we report the chemical composition of FBs of five myrmecophytic Acacia (Fabaceae) species from Mexico using HPLC (carbohydrates and proteins) and GC-MS (lipids). Feeding experiments revealed no hints on any use of external food sources by the inhabiting Pseudomyrmex ants. These ants obviously rely completely on FBs and extrafloral nectar provided by their hosts. The total content of nutrients in Acacia FBs was 15-25% of FB dry mass, being much lower than in Macaranga or Piper FBs. Proteins were dominating (8-14 % dm) in Acacia FBs and thus were present in higher amounts than in Macaranga FBs, yet in lower amounts than in Piper. Lipids contributed 1-9 % of dry mass, showing a lower proportion than in FBs of Macaranga or Piper. Carbohydrates made up 3-11 % dm, reaching in most Acacia species the same range as observed in Macaranga and in Piper FBs. Water content was 18-24 % of FB fresh mass, and structural tissue obviously made up a much higher proportion in Acacia FBs than in Macaranga or Piper FBs. Both characters might represent an adaptation to producing FBs unprotected at the leaf tips under dry conditions. Acacia FBs contain all amino acids and all fatty acids that are considered essential for insects, and their contents of lipids and proteins are higher than in the leaves from which they are ontogenetically derived. This indicates a putatively adaptive enrichment of nutritionally valuable compounds in structures functioning as ant-food.

Contents of phenolic compounds in leaf extracts often serve as a measure of plant anti-herbivore defence. This method suffers from the multifunctionality of phenolics and from problems with their colorimetric quantification. Here we present further evidence for the pertinence of these problems. Contents of condensed tannins (CCT) were spectrophotometrically quantified in leaf extracts of 11 closely related mimosoid species, and Spodoptera littoralis caterpillars were reared on artificial diet containing these extracts. The relationship of CCT with caterpillar growth differed considerably among plant species, since both positive and negative correlations were detected. There was, however, a negative correlation of CCT with fungal spore germination, indicating a role of these compounds in resistance to fungi. Detailed knowledge on the structure and biological function of defensive compounds and on the overall composition of leaves is required to estimate a plant's defensive efficacy against a particular group of enemies.

There has been rapid progress in detecting the genetic or allocation costs of induced resistance. In addition to these 'internal' costs, ecological costs may result from external mechanisms, that is, from the detrimental effects of resistance on the plant's interactions with its environment. All evolutionarily relevant costs affect a plant's ability to perform under natural conditions. The conceptual separation of different forms of resistance costs simplifies the study of mechanisms by which these costs arise. Yet, integrative measures of fitness must be applied under natural conditions so that researchers can fully understand the costs and benefits of induced resistance.

Summary 1. Allocation patterns are an important aspect of plant life strategies. In particular, many hypotheses on plant antiherbivore defence ‐ such as the growth differentiation balance hypothesis and the resource availability hypothesis ‐ concern constraints on resource allocation to defence, and assume defence to be limited by nutrient supply under most, yet not all, conditions. 2. Field-grown Macaranga triloba saplings were fertilized, and temporal and spatial patterns in the response of growth, photosynthesis and investment in biotic defence against herbivores and pathogens were measured simultaneously. Macaranga triloba produces food bodies to nourish mutualistic ants which protect their hosts against herbivores and pathogens. Food body production rate is quantitatively related to ant colony structure, and the latter is correlated with defensive efficacy. Food body production can therefore serve as a measure of the plant’s investment in defence. 3. Food body production responded quickly, and on all food body-producing stipules, to increased nutrient supply. In contrast, photosynthetic capacity responded much more slowly, to a smaller degree, and only in leaves that emerged after the onset of fertilization. No significant effects on plant growth were seen. 4. Our results show that food body production by M. triloba is regulated directly by nutrient availability. There was no evidence that increased nutrient supply was preferably allocated to growth and/or photosynthesis, which forms a central assumption of most hypotheses on plant antiherbivore defence. 5. Phenotypic plasticity and the ability to make use of short pulses in nutrient supply are of particular importance in pioneer plants inhabiting rapidly changing habitats. New theoretical frameworks are required for the inclusion of phenotypically plastic defence traits in the theory of plant antiherbivore defence.

Symptomless ‘type II’ fungal endophytes colonize their plant host horizontally and exert diverse effects on its resistance phenotype. Here, we used wild Lima bean (Phaseolus lunatus) plants that were experimentally colonized with one of three strains of natural endophytes (Bartalinia pondoensis, Fusarium sp., or Cochliobolus lunatus) to investigate the effects of fungal colonization on the endogenous levels of salicylic acid (SA) and jasmonic acid (JA) and on two JA-dependent indirect defense traits. Colonization with Fusarium sp. enhanced JA levels in intact leaves, whereas B. pondoensis suppressed the induction of endogenous JA in mechanically damaged leaves. Endogenous SA levels in intact leaves were significantly decreased by all strains and B. pondoensis and Fusarium sp. decreased SA levels after mechanical damage. Colonization with Fusarium sp. or C. lunatus enhanced the number of detectable volatile organic compounds (VOCs) emitted from intact leaves, and all three strains enhanced the relative amount of several VOCs emitted from intact leaves as well as the number of detectable VOCs emitted from slightly damaged leaves. All three strains completely suppressed the induced secretion of extrafloral nectar (EFN) after the exogenous application of JA. Symptomless endophytes interact in complex and strain-specific ways with the endogenous levels of SA and JA and with the defense traits that are controlled by these hormones. These interactions can occur both upstream and downstream of the defense hormones.

Nectar is a rich source of sugars that serves the attraction of pollinators (floral nectar) or predatory arthropods (extrafloral nectar). We just begin to understand the similarities and differences that underlie the secretory control of these two important types of plant secretions. Jasmonates are phytohormones, which are well documented to be involved in plant developmental processes and plant defence responses against herbivores, including the secretion of extrafloral nectar. Recently, jasmonates have also been implicated in the regulation of floral nectar secretion in Brassica napus. Due to a trade-off between reproduction and defence, however, plants need to functionally separate the regulation of these two secretory processes. In line with this prediction, externally applying jasmonates to leaves did indeed not affect floral nectar secretion. Here we compare the current knowledge on the regulation of floral and extrafloral nectar secretion to understand similarities and dissimilarities between these two secretory processes and highlight future research directions in this context.

Summary 1. Many plants produce extrafloral nectar (EFN) to nourish ants and other animals which defend them against herbivores. We aimed to find reasons for the high variability in amounts of EFN produced by most plant species. We investigated the influence of several biotic and abiotic factors (time of day, leaf age, nectar removal and leaf damage) on secretion rates of EFN in the common south-east Asian pioneer tree species, Macaranga tanarius (L.) Muell. Arg. 2. In most experiments leaves were washed with pure water and bagged in nets to protect them against nectar-collecting insects, and nectar was collected and quantified 24 h later. Six soluble sugars and up to eight amino acids were detected in nectar samples derived from untreated, field-grown plants. Total amounts of soluble substances varied more than the relative composition of EFN. 3. Nectar secretion rates were highest on young, expanded leaves. A diurnal pattern with a secretion peak in the first 2 h after dusk was detected in the field. Nectar removal had a positive effect and its accumulation a negative effect on further EFN production. Artificial leaf damage (punching leaves with a needle or removing parts of the leaf blade with scissors) led to a significant induction of EFN production for the next 3 days. 4. Extrafloral nectar of M. tanarius was secreted in complex patterns influenced by different biotic and abiotic factors; its production appeared to be adapted temporally and spatially in order to ensure optimal use of invested resources.

Summary 1 Although most theories on plant defence assume that costs will result from the production and maintenance of defensive traits, studies on the costs of induced defence against pathogens are comparatively rare. 2 We focus on fitness costs resulting from the chemical induction of systemic acquired resistance (SAR), a rather unspecific form of defence, which can be induced by and is effective against a broad spectrum of bacteria, fungi and viruses. 3 We used a model system in which we treated wheat plants that were protected against fungi by ‘traditional’ fungicides with BION® (a benzothiadiazole which induces pathogen resistance). Treated plants were therefore compelled to invest in defence without gaining any profit from the induction. 4 Treated plants achieved lower biomass than untreated controls, and developed fewer shoots and ears and therefore produced fewer seeds. The effects were most pronounced in plants that suffered from a shortage of nitrogen, and were observed only when pathogen resistance was induced during lateral shoot production. Later treatment revealed no significant effects. 5 We discuss whether the differences between treated and control plants can be interpreted as a consequence of allocation costs. Such costs could result from metabolic competition between processes involved in plant growth and the synthesis of defence-related compounds.

Many plant species have evolved mutualistic associations with ants, protecting their host against detrimental influences such as herbivorous insects. Letourneau (1998) reported in the case of Piper that ants defend their plants principally against stem-boring insects and also reduce fungal infections on inflorescences. Macaranga plants that were experimentally deprived of their symbiotic Crematogaster ants suffered heavily from shoot borers and pathogenic fungi (Heil 1998). Here we report that ants seem to reduce fungal infections actively in the obligate myrmecophyte Macarangatriloba (Euphorbiaceae), while ant-free plants can be easily infected. We also found extremely low chitinase activity in Macaranga plants. The plants' own biochemical defense seems to be reduced, and low chitinase activity perhaps may represent a predisposition for the evolution of myrmecophytism. These plants are therefore highly dependent on their ants, which obviously function not only as an antiherbivore defense but also as an effective agent against fungal pathogens.

Plants respond to local herbivory or pathogen infection with phenotypic changes, which reduce the danger of future attack. This so-called induced resistance is usually not restricted to the attacked plant organ but is also expressed in distant, so far undamaged parts of the plant. Signaling compounds such as jasmonic acid and salicylic acid have been discovered that move within the plant via the xylem or the phloem and elicit the resistance, thus acting as plant hormones. We now found that volatiles released in response to herbivore damage are required to elicit extrafloral nectar secretion in other parts of the same plant. Extrafloral nectar attracts ants and other carnivorous arthropods and serves as an effective indirect defense against herbivores. So called green leaf volatiles are released within minutes in response to tissue damage and were among the compounds that induced nectar secretion in yet undamaged parts of the damaged plant, but also in neighboring plants. Being gaseous and transported via the air, green leaf volatiles can serve a rapid within-plant communication, which moves much faster from one plant organ to the other than any plant-internal compound.

Summary 1. Macaranga (Euphorbiaceae) is a paleotropical tree genus comprising myrmecophytic and non-myrmecophytic species. All species are presumed to possess food bodies (FBs) to maintain or attract ants as anti-herbivore defence. 2. The hypothesis was tested that Macaranga species differing in their mode of association with ants would produce FBs differing in their chemical composition. We investigated contents of carbohydrates, proteins and lipids in FBs of four myrmecophytic and one non-myrmecophytic Macaranga as well as one Parthenocissus (Vitaceae) species. 3. On a dry weight basis, FBs of myrmecophytes contained relatively higher amounts of proteins compared to carbohydrates than those of non-myrmecophytes. Soluble carbohydrates showed species-specific patterns and were found in especially high amounts in both non-myrmecophytes. Furthermore, Parthenocissus FBs contained higher amounts of soluble compared to polymerous substances not only in carbohydrates but also in proteins. 4. FBs seem to be specifically adapted to their respective role in ant attraction and nutrition, with myrmecophytes providing ants with high amounts of lipids and proteins and non-myrmecophytes mainly offering carbohydrates in the form of common soluble sugars.

Plant pathogens affect the fitness of their hosts and maintain biodiversity. However, we lack theories to predict the type and intensity of infections in wild plants. Here we demonstrate using fungal pathogens of tropical plants that an examination of the life histories of hosts and pathogens can reveal general patterns in their interactions. Fungal infections were more commonly reported for light-demanding than for shade-tolerant species and for evergreen rather than for deciduous hosts. Both patterns are consistent with classical defence theory, which predicts lower resistance in fast-growing species and suggests that the deciduous habit can reduce enemy populations. In our literature survey, necrotrophs were found mainly to infect shade-tolerant woody species whereas biotrophs dominated in light-demanding herbaceous hosts. Far-red signalling and its inhibitory effects on jasmonic acid signalling are likely to explain this phenomenon. Multiple changes between the necrotrophic and the symptomless endophytic lifestyle at the ecological and evolutionary scale indicate that endophytes should be considered when trying to understand large-scale patterns in the fungal infections of plants. Combining knowledge about the molecular mechanisms of pathogen resistance with classical defence theory enables the formulation of testable predictions concerning general patterns in the infections of wild plants by fungal pathogens.

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. © 2013 Ballhorn et al.

BackgroundAnimal-derived elicitors can be used by plants to detect herbivory but they function only in specific insect-plant interactions. How can plants generally perceive damage caused by herbivores? Damaged-self recognition occurs when plants perceive molecular signals of damage: degraded plant molecules or molecules localized outside their original compartment.Methodology/principal findingsFlame wounding or applying leaf extract or solutions of sucrose or ATP to slightly wounded lima bean (Phaseolus lunatus) leaves induced the secretion of extrafloral nectar, an indirect defense mechanism. Chemically related molecules that would not be released in high concentrations from damaged plant cells (glucose, fructose, salt, and sorbitol) did not elicit a detectable response, excluding osmotic shock as an alternative explanation. Treatments inducing extrafloral nectar secretion also enhanced endogenous concentrations of the defense hormone jasmonic acid (JA). Endogenous JA was also induced by mechanically damaging leaves of lima bean, Arabidopsis, maize, strawberry, sesame and tomato. In lima bean, tomato and sesame, the application of leaf extract further increased endogenous JA content, indicating that damaged-self recognition is taxonomically widely distributed. Transcriptomic patterns obtained with untargeted 454 pyrosequencing of lima bean in response to flame wounding or the application of leaf extract or JA were highly similar to each other, but differed from the response to mere mechanical damage. We conclude that the amount or concentration of damaged-self signals can quantitatively determine the intensity of the wound response and that the full damaged-self response requires the disruption of many cells.Conclusions/significanceNumerous compounds function as JA-inducing elicitors in different plant species. Most of them are, contain, or release, plant-derived molecular motifs. Damaged-self recognition represents a taxonomically widespread mechanism that contributes to the perception of herbivore feeding by plants. This strategy is independent of insect-derived elicitors and, therefore, allows plants to maintain evolutionary control over their interaction with herbivores.

Plant–microbe interactions are ubiquitous. Plants are threatened by pathogens, but they are even more commonly engaged in neutral or mutualistic interactions with microbes: belowground microbial plant associates are mycorrhizal fungi, Rhizobia, and plant-growth promoting rhizosphere bacteria, aboveground plant parts are colonized by internally living bacteria and fungi (endophytes) and by microbes in the phyllosphere (epiphytes). We emphasize here that a completely microbe-free plant is an exotic exception rather than the biologically relevant rule. The complex interplay of such microbial communities with the host–plant affects multiple vital parameters such as plant nutrition, growth rate, resistance to biotic and abiotic stressors, and plant survival and distribution. The mechanisms involved reach from direct ones such as nutrient acquisition, the production of plant hormones, or direct antibiosis, to indirect ones that are mediated by effects on host resistance genes or via interactions at higher trophic levels. Plant-associated microbes are heterotrophic and cause costs to their host plant, whereas the benefits depend on the current environment. Thus, the outcome of the interaction for the plant host is highly context dependent. We argue that considering the microbe-free plant as the “normal” or control stage significantly impairs research into important phenomena such as (1) phenotypic and epigenetic plasticity, (2) the “normal” ecological outcome of a given interaction, and (3) the evolution of plants. For the future, we suggest cultivation-independent screening methods using direct PCR from plant tissue of more than one fungal and bacterial gene to collect data on the true microbial diversity in wild plants. The patterns found could be correlated to host species and environmental conditions, in order to formulate testable hypotheses on the biological roles of plant endophytes in nature. Experimental approaches should compare different host–endophyte combinations under various relevant environmental conditions and study at the genetic, epigenetic, transcriptional, and physiological level the parameters that cause the interaction to shift along the mutualism–parasitism continuum.