Attraction of entomopathogenic nematodes to black truffle and its volatile organic compounds: A new approach for truffle beetle biocontrol.

The European truffle beetle, Leiodes cinnamomeus, is the most important pest in black truffle (Tuber melanosporum) plantations. Entomopathogenic nematodes (EPNs) are a promising biological control agents against L. cinnamomeus. EPNs may employ multiple sensory cues while seeking for hosts, such as volatile organic compounds (VOCs) and CO2 gradients. We report for the first time the attraction of EPNs to truffle fruitbodies, and identified some VOCs potentially playing a key role in this interaction. We conducted olfactometer assays to investigate the attraction behavior of Steinernema feltiae and Steinernema carpocapsae towards both T. melanosporum fruitbodies and larvae of L. cinnamomeus. Subsequently, a chemotaxis assay using agar plates was performed to determine which of the 14 of the main VOCs emitted by the fruitbodies attracted S. feltiae at low (0.1 %) and high (mg/100 g truffle) concentrations. Both EPN species were attracted to mature fruitbodies of T. melanosporum, which may enhance the likelihood of encountering L. cinnamomeus during field applications. L. cinnamomeus larvae in the presence of truffles did not significantly affect the behavior of EPNs 24 h after application, underscoring the importance of the chemical compounds emitted by truffles themselves. Chemotaxis assays showed that four long-chain alcohol compounds emitted by T. melanosporum fruitbodies attracted S. feltiae, especially at low concentration, providing a first hint in the chemical ecology of a little-studied ecological system of great economical value. Further studies should be conducted to gain a finer understanding of the tritrophic interactions between T. melanosporum, EPNs, and L. cinnamomeus, as this knowledge may have practical implications for the efficacy of EPNs in the biological control of this pest.

Root Exudation of Specialized Molecules for Plant-Environment Interaction.

It has been estimated that between 40 and 60 % of the assimilated carbon is diverted to the roots and released in the rhizosphere in form of root exudates. Root exudates thus define a complex mixture of low and high molecular weight compounds, including carbohydrates, amino acids, organic, and proteins, but also a broad spectrum of specialized molecules, such as flavonoids, glucosinolates, terpenoids, or alkaloids. Root exudates favour soil mineral nutrition, can bind to soil aggregate and in turn modify soil physico-chemical properties, but also mediate plant-plant, plant-microbe, and plant-animal interactions belowground. With this review, we aim to highlight how chemical ecologists have approached the study of root exudates-mediated interactions between plants and their biotic and abiotic surroundings. We do so by presenting a series of study cases for, on one hand, showcasing different methodologies that have been developed to test the activity of different root exudates, and, on the other hand, to show the broad array of interactions mediated by root exudates. Ultimately, we aim to spur further research and collaborations between chemists and ecologists studying belowground chemically-mediated interactions, so as to tackle essential challenges in terms of food security and climate change in the near future.

Calcium-alginate beads as a formulation for the application of entomopathogenic nematodes to control rootworms.

Entomopathogenic nematodes (EPN) have great potential as biological control agents against root-feeding insects. They have a rapid and long-lasting mode of action, minimal adverse effects on the environment and can be readily mass-produced. However, they have a relatively short shelf-life and are susceptible to desiccation and UV light. These shortcomings may be overcome by encapsulating EPN in Ca2+-alginate hydrogels, which have been shown to provide a humid and UV protective shelter. Yet, current Ca2+-alginate formulations do not keep EPN vigorous and infectious for a prolonged period of time and do not allow for their controlled release upon application. Here, we introduce solid Ca2+-alginate beads which we supplemented with glycerol to better retain the EPN during storage and to ensure a steady release when applied in soil. Glycerol-induced metabolic arrest in EPN (Heterorhabditis bacteriophora) resulting in quiescence and total retainment of EPN when added to beads made with 0.5% sodium alginate and 2% CaCl2·2H2O solutions. More than 4,000 EPN could be embedded in a single 4-5-mm diameter bead, and quiescence could be broken by adding water, after which the EPN readily emerged from the beads. In a field trial, the EPN beads were as effective in reducing root damage by the western corn rootworm (WCR, Diabrotica virgifera virgifera) as EPN that were applied in water. Although further improvements are desirable, we conclude that Ca2+-alginate beads can provide an effective and practical way to apply EPN for the control of WCR larvae. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10340-021-01349-4.

Detecting the Conspecific: Herbivory-Induced Olfactory Cues in the Fall Armyworm (Lepidoptera: Noctuidae).

The fall armyworm (FAW), Spodoptera frugiperda (Smith), is a polyphagous pest whose larval feeding threatens several economically important crops worldwide with especially severe damage to corn (Zea mays L.). Field-derived resistance to several conventional pesticides and Bt toxins have threatened the efficacy of current management strategies, necessitating the development of alternative pest management methods and technologies. One possible avenue is the use of volatile organic compounds (VOCs) and other secondary metabolites that are produced and sequestered by plants as a response to larval feeding. The effects of conspecific larval feeding on fall armyworm oviposition preferences and larval fitness were examined using two-choice oviposition experiments, larval feeding trials, targeted metabolomics, and VOC analyses. There was a significant preference for oviposition on corn plants that lacked larval feeding damage, and larvae fed tissue from damaged plants exhibited reduced weights and head capsule widths. All larval feeding promoted significantly increased metabolite and VOC concentrations compared to corn plants without any feeding. Metabolite differences were driven primarily by linoleic acid (which is directly toxic to fall armyworm) and tricarboxylic acids. Several VOCs with significantly increased concentrations in damaged corn plants were known oviposition deterrents that warrant further investigation in an integrated pest management context.

Indirect Root Defenses Cause Induced Fitness Costs in Bt-Resistant Western Corn Rootworm.

Plants genetically modified to produce insecticidal toxins from the bacterium Bacillus thuringiensis Berliner (Bt) have been extensively used to manage the western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) in the United States. Evolution of WCR resistance to Bt toxins has forced the consideration of alternative pest management and improved insect resistance management. Entomopathogenic nematodes (EPNs), obligate insect parasites, are attracted toward volatile organic compounds (VOCs) emitted by maize roots after WCR herbivory. The production of VOCs of two types of Bt maize (MON88017 and MIR604) and their near-isolines was evaluated after induction with Bt-susceptible and resistant WCR. The attraction of EPNs toward the Bt hybrids was tested in the laboratory and the field. Bt hybrids emitted VOCs when induced by Bt-resistant insects whereas induction by Bt-susceptible WCR did not elicit a plant response. Survival of Bt-resistant WCR was lower on the hybrid attracting EPNs and similar to the survival of Bt-susceptible WCR without EPNs. This trade-off of Bt-resistance is defined here as an induced fitness cost, and offers a viable tool to management of Bt-resistant WCR.

Birds Bug on Indirect Plant Defenses to Locate Insect Prey.

It has long been thought that most birds do not use volatile cues to perceive their environment. Aside from some scavenging birds, this large group of vertebrates was believed to mostly rely on highly developed vision while foraging and there are relatively few studies exploring bird response to volatile organic compounds. In response to insect herbivory, plants release volatile organic compounds to attract parasitoids and predators of the pests. To test if insectivorous birds use herbivore-induced plant volatiles (HIPV), dispensers emitting a synthetic blend of HIPV typically emitted after insect herbivory were deployed in a maize field along with imitation clay caterpillars. Significantly more imitation insects were attacked by birds when located close to dispensers releasing HIPV than close to dispenser with organic solvent only. Seven times more peck marks, an index of avian predation, were counted on caterpillars in the vicinity of the HIPV dispensers than on insects close to control dispensers. This is the first field demonstration that insectivorous birds cue on HIPV to locate prey in agricultural settings. These results support the growing evidence that foraging birds exploit volatile cues. This more accurate understanding of their behavior will be important when implementing pest management program involving insectivorous birds.

Novel In vitro Procedures for Rearing a Root-Feeding Pest (Heteronychus arator) of Grasslands.

Optimizing plant protection against insect herbivory relies on testing plant defense mechanisms and how the insect response to these defensive strategies. Such experiments benefit from using insects generated from standardized rearing protocols since this reduces stochastic variation. Such protocols can be challenging to devise, however, especially for root herbivores. These insects generally have complex and long life cycles, which are often only poorly described. Moreover, using field-captured root herbivores is often suboptimal because it involves extensive excavation from sites selected by chance (their location is not obvious) and larval stages are frequently indistinguishable beyond the family level. We investigated in vitro procedures to improve the availability of the African Black Beetle (ABB) Heteronychus arator, an invasive alien pest in both Australia and New Zealand. Native to Africa, this scarab beetle has established in Australian and New Zealand grasslands, pastures, and crops. Adults feed on the stem of young plants just beneath the soil surface. During the mating season, gravid females lay eggs in the soil, giving rise to larvae feeding on grass roots, causing severe damage, and impairing plant growth. Here, we propose laboratory approaches to collect eggs from field-captured adult beetles, to hatch eggs, and to rear neonate larvae to adults. We propose that these methods will provide plant scientists and entomologists with a better and more controlled supply of ABB larvae for laboratory and field assays. In turn, this will assist with the collection of important information for the management of this insect pest and enhanced protection of plants in crop and grassland ecosystems.

Highly Potent Extracts from Pea (Pisum sativum) and Maize (Zea mays) Roots Can Be Used to Induce Quiescence in Entomopathogenic Nematodes.

Root exudates can play an important role in plant-nematode interactions. Recent studies have shown that the root cap exudates obtained from several plant species trigger a state of dormancy or quiescence in various genera of nematodes. This phenomenon is not only of fundamental ecological interest, but also has application potential if the plant-produced compound(s) could be used to control harmful nematodes or help to prolong the shelf-life of beneficial entomopathogenic nematodes (EPNs). The identification of the compound(s) involved in quiescence induction has proven to be a major challenge and requires large amounts of active material. Here, we present a high-throughput method to obtain bioactive root extracts from flash-frozen root caps of green pea and maize. The root cap extract obtained via this method was considerably more potent in inducing quiescence than exudate obtained by a previously used method, and consistently induced quiescence in the EPN Heterorhabditis megidis, even after a 30-fold dilution. Extracts obtained from the rest of the root were equally effective in inducing quiescence. Infective juveniles (IJs) of H. megidis exposed to these extracts readily recovered from their quiescent state as soon as they were placed in moist soil, and they were at least as infectious as the IJs that had been stored in water. Excessive exposure of IJs to air interfered with the triggering of quiescence. The implications of these results and the next steps towards identification of the quiescence-inducing compound(s) are discussed from the perspective of applying EPN against soil-dwelling insect pests.

The dual effects of root-cap exudates on nematodes: from quiescence in plant-parasitic nematodes to frenzy in entomopathogenic nematodes.

To defend themselves against herbivores and pathogens, plants produce numerous secondary metabolites, either constitutively or de novo in response to attacks. An intriguing constitutive example is the exudate produced by certain root-cap cells that can induce a state of reversible quiescence in plant-parasitic nematodes, thereby providing protection against these antagonists. The effect of such root exudates on beneficial entomopathogenic nematodes (EPNs) remains unclear, but could potentially impair their use in pest management programmes. We therefore tested how the exudates secreted by green pea (Pisum sativum) root caps affect four commercial EPN species. The exudates induced reversible quiescence in all EPN species tested. Quiescence levels varied with the green pea cultivars tested. Notably, after storage in root exudate, EPN performance traits were maintained over time, whereas performances of EPNs stored in water rapidly declined. In sharp contrast to high concentrations, lower concentrations of the exudate resulted in a significant increase in EPN activity and infectiousness, but still reduced the activity of two plant-parasitic nematode species. Our study suggests a finely tuned dual bioactivity of the exudate from green pea root caps. Appropriately formulated, it can favour long-term storage of EPNs and boost their infectiousness, while it may also be used to protect plants from plant-parasitic nematodes.

The role of root architecture in foraging behavior of entomopathogenic nematodes.

As obligate parasites, entomopathogenic nematodes (EPN) rely on insect hosts to complete their development. In insect pest management, EPN infectiousness has varied a lot. A better understanding of their host-finding behavior in the rhizosphere is therefore crucial to enhance EPN potential in biological control. As previously demonstrated, roots can be used as a pathway to insect hosts by EPN, but this interaction and its impact on EPN foraging remain poorly documented. Three artificial model-roots with different degrees of complexity and connectivity were designed to investigate the impact of root architecture on foraging behavior of the EPN Heterorhabditis megidis. Insect baits were placed at the bottom of each model-root that was subsequently buried in moist sand. After injection of the EPN, the number of EPN-infected baits as well as the number of mature nematodes inside each individual carcass was recorded. The influence of insect-induced root volatiles was also evaluated by spiking the baits with a synthetic version of a natural insect-induced root cue. The ecological relevance of the results was tested in soil with two maize genotypes each exhibiting broadly different root architectures. H. megidi performed better in presence of model-roots. Foraging performances of H. megidis declined with the increasing model-root complexity. Adding the synthetic root volatile dramatically changed this pattern and favored the EPN on the most complex model-roots. H. megidis also moved in the vicinity of maize roots to find the insect baits in soil, and natural root architecture also tended to shape H. megidis foraging behavior. This study adds to the scarce body of literature characterizing physical and chemical interactions between EPN and roots. The present data illustrate that root architecture not only modifies plant quality but also shapes upper trophic levels' ecology.

Carbon isotope ratios document that the elytra of western corn rootworm (Coleoptera: Chrysomelidae) reflects adult versus larval feeding and later instar larvae prefer Bt corn to alternate hosts.

In much of the Corn Belt and parts of Europe, the western corn rootworm, Diabrotica virgifera virgifera LeConte, is the most important insect pest of maize. The need for additional basic knowledge of this pest has been highlighted while developing resistance management plans for insecticidal genetically modified crops. This study evaluated the possibility of tracking feeding habits of western corn rootworm larvae using stable carbon isotope signatures. Plants accumulate different ratios of (13)C:(12)C isotopes, usually expressed as δ(13)C, according to whether they use the C3 or C4 photosynthetic pathway. Herbivore biomass is expected to reflect the δ(13)C of the food they eat. For the current experiment, western corn rootworm larvae were grown on different species of plants exhibiting different δ(13)C values. The δ(13)C values were then measured in elytra of emerged beetles. When beetles were unfed, biomass reflected larval feeding. When beetles were fed for 31 d postemergence, δ(13)C values of elytra almost exclusively reflected adult feeding. These results suggest the use of caution in the interpretation of δ(13)C data aiming to document larval diet history when adult feeding history is unknown. The technique was also used to evaluate western corn rootworm larval choice between alternate hosts and maize with and without genetically modified (Bt) traits aimed at their control. Propensity for feeding on alternate hosts versus maize was biased toward feeding on maize regardless whether the maize had Bt or not, suggesting western corn rootworm larvae were not repelled by Bt. These data will be helpful for regulators in interpreting western corn rootworm feeding data on Bt maize.

Genetically engineered maize plants reveal distinct costs and benefits of constitutive volatile emissions in the field.

Genetic manipulation of plant volatile emissions is a promising tool to enhance plant defences against herbivores. However, the potential costs associated with the manipulation of specific volatile synthase genes are unknown. Therefore, we investigated the physiological and ecological effects of transforming a maize line with a terpene synthase gene in field and laboratory assays, both above- and below ground. The transformation, which resulted in the constitutive emission of (E)-ß-caryophyllene and α-humulene, was found to compromise seed germination, plant growth and yield. These physiological costs provide a possible explanation for the inducibility of an (E)-ß-caryophyllene-synthase gene in wild and cultivated maize. The overexpression of the terpene synthase gene did not impair plant resistance nor volatile emission. However, constitutive terpenoid emission increased plant apparency to herbivores, including adults and larvae of the above ground pest Spodoptera frugiperda, resulting in an increase in leaf damage. Although terpenoid overproducing lines were also attractive to the specialist root herbivore Diabrotica virgifera virgifera below ground, they did not suffer more root damage in the field, possibly because of the enhanced attraction of entomopathogenic nematodes. Furthermore, fewer adults of the root herbivore Diabrotica undecimpunctata howardii were found to emerge near plants that emitted (E)-ß-caryophyllene and α-humulene. Yet, overall, under the given field conditions, the costs of constitutive volatile production overshadowed its benefits. This study highlights the need for a thorough assessment of the physiological and ecological consequences of genetically engineering plant signals in the field to determine the potential of this approach for sustainable pest management strategies.

Manipulation of chemically mediated interactions in agricultural soils to enhance the control of crop pests and to improve crop yield.

In most agro-ecosystems the organisms that feed on plant roots have an important impact on crop yield and can impose tremendous costs to farmers. Similar to aboveground pests, they rely on a broad range of chemical cues to locate their host plant. In their turn, plants have co-evolved a large arsenal of direct and indirect defense to face these attacks. For instance, insect herbivory induces the synthesis and release of specific volatile compounds in plants. These volatiles have been shown to be highly attractive to natural enemies of the herbivores, such as parasitoids, predators, or entomopathogenic nematodes. So far few of the key compounds mediating these so-called tritrophic interactions have been identified and only few genes and biochemical pathways responsible for the production of the emitted volatiles have been elucidated and described. Roots also exude chemicals that directly impact belowground herbivores by altering their behavior or development. Many of these compounds remain unknown, but the identification of, for instance, a key compound that triggers nematode egg hatching to some plant parasitic nematodes has great potential for application in crop protection. These advances in understanding the chemical emissions and their role in ecological signaling open novel ways to manipulate plant exudates in order to enhance their natural defense properties. The potential of this approach is discussed, and we identify several gaps in our knowledge and steps that need to be taken to arrive at ecologically sound strategies for belowground pest management.

Systemic root signalling in a belowground, volatile-mediated tritrophic interaction.

Plants attacked by leaf herbivores release volatile organic compounds (VOCs) both locally from the wounded site and systemically from non-attacked tissues. These volatiles serve as attractants for predators and parasitoids. This phenomenon is well described for plant leaves, but systemic induction of VOCs in the roots has remained unstudied. We assessed the spatial and temporal activation of the synthesis and release of (E)-ß-caryophyllene (EßC) in maize roots upon feeding by larvae of Diabrotica virgifera virgifera, as well as the importance of systemically produced EßC for the attraction of the entomopathogenic nematode Heterorhabditis megidis. The production of EßC was found to be significantly stronger at the site of attack than in non-attacked tissues. A weak, but significant, increase in transcriptional activity of the EßC synthase gene tps23 and a corresponding increase in EßC content were observed in the roots above the feeding site and in adjacent roots, demonstrating for the first time that herbivory triggers systemic production of a volatile within root systems. In belowground olfactometers, the nematodes were significantly more attracted towards local feeding sites than systemically induced roots. The possible advantages and disadvantages of systemic volatile signalling in roots are discussed.

Selective breeding of entomopathogenic nematodes for enhanced attraction to a root signal did not reduce their establishment or persistence after field release.

We recently showed that the efficacy of an entomopathogenic nematode (EPN) as a biological control agent against a root pest could be enhanced through artificial selection. The EPN Heterorhabditis bacteriophora was selected for higher responsiveness towards (E)-ß-caryophyllene (EßC), a sesquiterpene that is emitted by maize roots in response to feeding damage by the western corn rootworm (WCR). EßC is normally only weakly attractive to H. bacteriophora, which is one of the most infectious nematodes against WCR. By selecting H. bacteriophora to move more readily along a EßC gradient we obtained a strain that was almost twice more efficient in controlling WCR population in fields planted with an EßC-producing maize variety. However, artificial selection for one trait may come at a cost for other important traits such as infectiousness, establishment and/or persistence in the field. Indeed, infectiousness was slightly but significantly reduced in the selected strain. Yet, this apparent cost was largely compensated for by the higher responsiveness to the root signal. Here we show that the selection process had no negative effect on establishment and persistence of field-released EPN. This knowledge, combined with the previously reported results, attest to the feasibility of manipulating key traits to improve the efficacy of beneficial organisms.

Selection of entomopathogenic nematodes for enhanced responsiveness to a volatile root signal helps to control a major root pest.

The efficacy of natural enemies as biological control agents against insect pests can theoretically be enhanced by artificial selection for high responsiveness to foraging cues. The recent discovery that maize roots damaged by the western corn rootworm (WCR) emit a key attractant for insect-killing nematodes has opened the way to explore whether a selection strategy can improve the control of root pests. The compound in question, (E)-beta-caryophyllene, is only weakly attractive to Heterorhabditis bacteriophora, one of the most infectious nematodes against WCR. To overcome this drawback, we used a six-arm below-ground olfactometer to select for a strain of H. bacteriophora that is more readily attracted to (E)-beta-caryophyllene. After six generations of selection, the selected strain responded considerably better and moved twice as rapidly towards a (E)-beta-caryophyllene source than the original strain. There was a minor trade-off between this enhanced responsiveness and nematode infectiveness. Yet, in subsequent field tests, the selected strain was significantly more effective than the original strain in reducing WCR populations in plots with a maize variety that releases (E)-beta-caryophyllene, but not in plots with a maize variety that does not emit this root signal. These results illustrate the great potential of manipulating natural enemies of herbivores to improve biological pest control.

Restoring a maize root signal that attracts insect-killing nematodes to control a major pest.

When attacked by herbivorous insects, plants emit volatile compounds that attract natural enemies of the insects. It has been proposed that these volatile signals can be manipulated to improve crop protection. Here, we demonstrate the full potential of this strategy by restoring the emission of a specific belowground signal emitted by insect-damaged maize roots. The western corn rootworm induces the roots of many maize varieties to emit (E)-beta-caryophyllene, which attracts entomopathogenic nematodes that infect and kill the voracious root pest. However, most North American maize varieties have lost the ability to emit (E)-beta-caryophyllene and may therefore receive little protection from the nematodes. To restore the signal, a nonemitting maize line was transformed with a (E)-beta-caryophyllene synthase gene from oregano, resulting in constitutive emissions of this sesquiterpene. In rootworm-infested field plots in which nematodes were released, the (E)-beta-caryophyllene-emitting plants suffered significantly less root damage and had 60% fewer adult beetles emerge than untransformed, nonemitting lines. This demonstration that plant volatile emissions can be manipulated to enhance the effectiveness of biological control agents opens the way for novel and ecologically sound strategies to fight a variety of insect pests.

Belowground chemical signaling in maize: when simplicity rhymes with efficiency.

Maize roots respond to feeding by larvae of the beetle Diabrotica virgifera virgifera by releasing (E)-beta-caryophyllene. This sesquiterpene, which is not found in healthy maize roots, attracts the entomopathogenic nematode Heterorhabditis megidis. In sharp contrast to the emission of virtually only this single compound by damaged roots, maize leaves emit a blend of numerous volatile organic compounds in response to herbivory. To try to explain this difference between roots and leaves, we studied the diffusion properties of various maize volatiles in sand and soil. The best diffusing compounds were found to be terpenes. Only one other sesquiterpene known for maize, alpha-copaene, diffused better than (E)-beta-caryophyllene, but biosynthesis of the former is far more costly for the plant than the latter. The diffusion of (E)-beta-caryophyllene occurs through the gaseous rather than the aqueous phase, as it was found to diffuse faster and further at low moisture level. However, a water layer is needed to prevent complete loss through vertical diffusion, as was found for totally dry sand. Hence, it appears that maize has adapted to emit a readily diffusing and cost-effective belowground signal from its insect-damaged roots.

A maize (E)-beta-caryophyllene synthase implicated in indirect defense responses against herbivores is not expressed in most American maize varieties.

The sesquiterpene (E)-beta-caryophyllene is emitted by maize (Zea mays) leaves in response to attack by lepidopteran larvae like Spodoptera littoralis and released from roots after damage by larvae of the coleopteran Diabrotica virgifera virgifera. We identified a maize terpene synthase, Terpene Synthase 23 (TPS23), that produces (E)-beta-caryophyllene from farnesyl diphosphate. The expression of TPS23 is controlled at the transcript level and induced independently by D. v. virgifera damage in roots and S. littoralis damage in leaves. We demonstrate that (E)-beta-caryophyllene can attract natural enemies of both herbivores: entomopathogenic nematodes below ground and parasitic wasps, after an initial learning experience, above ground. The biochemical properties of TPS23 are similar to those of (E)-beta-caryophyllene synthases from dicotyledons but are the result of repeated evolution. The sequence of TPS23 is maintained by positive selection in maize and its closest wild relatives, teosinte (Zea sp) species. The gene encoding TPS23 is active in teosinte species and European maize lines, but decreased transcription in most North American lines resulted in the loss of (E)-beta-caryophyllene production. We argue that the (E)-beta-caryophyllene defense signal was lost during breeding of the North American lines and that its restoration might help to increase the resistance of these lines against agronomically important pests.