Experimental Growth Conditions affect Direct and Indirect Defences in two Cotton Species.

Cotton has been used as a model plant to study direct and indirect plant defence against herbivorous insects. However, the plant growing conditions could have an important effect on the outcome of such plant defence studies. We examined how common experimental growth conditions influence constitutive and inducible defences in two species of cotton, Gossypium hirsutum and G. herbaceum. We induced plants by applying caterpillar regurgitant to mechanical wounds to compare the induction levels between plants of both species grown in greenhouse or phytotron conditions. For this we measured defence metabolites (gossypol and heliocides) and performance of Spodoptera frugiperda caterpillars on different leaves, the emission of plant volatiles, and their attractiveness to parasitic wasps. Induction increased the levels of defence metabolites, which in turn decreased the performance of S. frugiperda larvae. Constitutive and induced defence levels were the highest in plants grown in the phytotron (compared to greenhouse plants), G. hirsutum and young leaves. Defence induction was more pronounced in plants grown in the phytotron and in young leaves. Also, the differences between growing conditions were more evident for metabolites in the youngest leaves, indicating an interaction with plant ontogeny. The composition of emitted volatiles was different between plants from the two growth conditions, with greenhouse-grown plants showing more variation than phytotron-grown plants. Also, G. hirsutum released higher amounts of volatiles and attracted more parasitic wasps than G. herbaceum. Overall, these results highlight the importance of experimental abiotic factors in plant defence induction and ontogeny of defences. We therefore suggest careful consideration in selecting the appropriate experimental growing conditions for studies on plant defences.

Potent Ant Deterrents Emitted from Nematode-Infected Insect Cadavers.

Most known species of entomopathogenic nematodes (EPNs) are generalist obligate parasites of insects. They kill their hosts within days after infection and mortality is mainly caused by toxins produced by bacteria that co-infect the hosts and serve as food for the nematodes. EPNs can infect a very broad spectrum of insects and these insects can therefore be expected to have evolved strategies to avoid infection. Indeed, ants are known to avoid feeding on EPN-infected insect cadavers, most likely because they are repelled by semiochemicals that emanate from the cadavers. The source and nature of these repellents are so far unknown. In a series of behavioral and chemical analytical experiments we identified hexadecanal and 2-heptadecanone as two compounds that are emitted by insect larva that are infected by the EPN Steinernema feltiae, but not by uninfected larvae. When spiking honey water with the two semiochemicals, they were confirmed to be highly deterrent to the ant Lasius niger. The environmentally benign hexadecanal and 2-heptadecanone could be employed to ward off ants and possibly other pests. Additional experiments are needed to fully determine their application potential.

Molecular detection and quantification of slug parasitic nematodes from the soil and their hosts.

Terrestrial gastropod molluscs are widely distributed and are well known as pests of many types of plants that are notoriously difficult to control. Many species of nematodes are able to parasitize land snails and slugs, but few of them are lethal to their host. Species and/or populations of mollusc-parasitic nematodes (MPNs) that kill their hosts are promising for biological control purposes. The recent discovery of new nematode species of the genus Phasmarhabditis in Europe and the associations between Alloionema spp. and slugs are expanding the possibilities of using MPNs as control agents. However, very little is known about the distribution and ecology of these species. Using molecular techniques based on qPCR methods for quick identification and quantification of various species of MPN isolated directly from the soil or from infected hosts can assist in providing information on their presence and persistence, as well as the composition of natural assemblages. Here, we developed new primers and probes for five species of the genus Phasmarhabditis and one species of the genus Alloionema. We employed these novel molecular techniques and implemented a published molecular set to detect MPN presence in soil samples coming from natural and agricultural areas in Switzerland. We also developed a method that allows the detection and quantification of Phasmarhabditis hermaphrodita directly from the tissues of their slug host in a laboratory experiment. The new molecular approaches were optimized to a satisfactory limit of detection of the species, with only few cross-amplifications with closely related species in late cycles (>32). Using these tools, we detected MPNs in 7.5% of sampled sites, corresponding to forest areas (P. hermaphrodita and Alloionema appendiculatum) and wheat-oriented agricultural areas (Phasmarhabditis bohemica). Moreover, we confirmed that the method can be used to detect the presence of P. hermaphrodita inside slug hosts, with more detections in the susceptible slug Deroceras larvae compared to the resistant Arion vulgaris. These primers/probe sets provide a novel and quick tool to identify MPNs from soil samples and infected slugs without having to culture and retrieve all nematode life stages, as well as a new tool to unravel the ecology of nematode-slug complexes.

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.

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.

Unraveling the intraguild competition between Oscheius spp. nematodes and entomopathogenic nematodes: Implications for their natural distribution in Swiss agricultural soils.

Entomopathogenic nematodes (EPN) are excellent biological control agents to fight soil-dwelling insect pests. In a previous survey of agricultural soils of Switzerland, we found mixtures of free-living nematodes (FLN) in the genus Oscheius, which appeared to be in intense competition with EPN. As this may have important implications for the long-term persistence of EPN, we studied this intraguild competition in detail. We hypothesized that (i) Oscheius spp. isolates act as scavengers rather than entomopathogens, and (ii) cadavers with relatively small numbers of EPN are highly suitable resources for Oscheius spp. reproduction. To study this, we identified Oscheius spp. isolated from Swiss soils, quantified the outcome of EPN/Oscheius competition in laboratory experiments, developed species-specific primers and probe for quantitative real-time PCR, and evaluated their relative occurrence in the field in the context of the soil food web. Molecular analysis (ITS/D2D3) identified MG-67/MG-69 as Oscheius onirici and MG-68 as O. tipulae (Dolichura-group). Oscheius spp. indeed behaved as scavengers, reproducing in ∼64% of frozen-killed cadavers from controlled experiments. Mixed infection in the laboratory by Oscheius spp. with low (3 IJs) or high (20 IJs) initial EPN numbers revealed simultaneous reproduction in double-exposed cadavers which resulted in a substantial reduction in the number of EPN progeny from the cadaver. This effect depended on the number of EPN in the initial inoculum and differed by EPN species; Heterorhabditis megidis was better at overcoming competition. This study reveals Oscheius spp. as facultative kleptoparasites that compete with EPN for insect cadavers. Using real-time qPCR, we were able to accurately quantify this strong competition between FLN and EPN in cadavers that were recovered after soil baiting (∼86% cadavers with >50% FLN production). The severe competition within the host cadavers and the intense management of the soils in annual crops readily explain the low EPN numbers in Swiss field samples. The developed molecular tools can be used to elucidate the extent to which the competitive interactions affect EPN populations. This can help to develop strategies to achieve good persistence and natural EPN recycling, in particular in systems where native EPN levels are low, such as annual crops.

Genetic and phenotypic population divergence on a microgeographic scale in brown trout.

Salmonid populations of many rivers are rapidly declining. One possible explanation is that habitat fragmentation increases genetic drift and reduces the populations' potential to adapt to changing environmental conditions. We measured the genetic and eco-morphological diversity of brown trout (Salmo trutta) in a Swiss stream system, using multivariate statistics and Bayesian clustering. We found large genetic and phenotypic variation within only 40 km of stream length. Eighty-eight percent of all pairwise F(ST) comparisons and 50% of the population comparisons in body shape were significant. High success rates of population assignment tests confirmed the distinctiveness of populations in both genotype and phenotype. Spatial analysis revealed that divergence increased with waterway distance, the number of weirs, and stretches of poor habitat between sampling locations, but effects of isolation-by-distance and habitat fragmentation could not be fully disentangled. Stocking intensity varied between streams but did not appear to erode genetic diversity within populations. A lack of association between phenotypic and genetic divergence points to a role of local adaptation or phenotypically plastic responses to habitat heterogeneity. Indeed, body shape could be largely explained by topographic stream slope, and variation in overall phenotype matched the flow regimes of the respective habitats.

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.

Plant-associated CO2 mediates long-distance host location and foraging behaviour of a root herbivore.

Insect herbivores use different cues to locate host plants. The importance of CO2 in this context is not well understood. We manipulated CO2 perception in western corn rootworm (WCR) larvae through RNAi and studied how CO2 perception impacts their interaction with their host plant. The expression of a carbon dioxide receptor, DvvGr2, is specifically required for dose-dependent larval responses to CO2. Silencing CO2 perception or scrubbing plant-associated CO2 has no effect on the ability of WCR larvae to locate host plants at short distances (<9 cm), but impairs host location at greater distances. WCR larvae preferentially orient and prefer plants that grow in well-fertilized soils compared to plants that grow in nutrient-poor soils, a behaviour that has direct consequences for larval growth and depends on the ability of the larvae to perceive root-emitted CO2. This study unravels how CO2 can mediate plant-herbivore interactions by serving as a distance-dependent host location cue.

Living deep in the ground and surrounded by darkness, soil insects must rely on the chemicals released by plants to find the roots they feed on. Carbon dioxide, for example, is a by-product of plant respiration, which, above ground, is thought to attract moths to flowers and flies to apples; underground, however, its role is still unclear. This gaseous compound can travel through soil and potentially act as a compass for root-eating insects. Yet, it is also produced by decaying plants or animals, which are not edible. It is therefore possible that insects use this signal as a long-range cue to orient themselves, but then switch to another chemical when closer to their target to narrow in on an actual food source. To test this idea, Arce et al. investigated whether carbon dioxide guides the larvae of Western corn rootworm to maize roots. First, the rootworm genes responsible for sensing carbon dioxide were identified and switched off, making the larvae unable to detect this gas. When the genetically engineered rootworms were further than 9cm from maize roots, they were less able to locate that food source; closer to the roots, however, the insects could orient themselves towards the plant. This suggests that the insects use carbon dioxide at long distances but rely on another chemicals to narrow down their search at close range. To confirm this finding, Arce et al. tried absorbing the carbon dioxide using soda lime, leading to similar effects: carbon dioxide sensitive insects stopped detecting the roots at long but not short distances. Additional experiments then revealed that the compound could help insects find the best roots to feed on. Indeed, eating plants that grow on rich terrain ­ for instance, fertilized soils ­ helps insects to grow bigger and faster. These roots also release more carbon dioxide, in turn attracting rootworms more frequently. In the United States and Eastern Europe, Western corn rootworms inflict major damage to crops, highlighting the need to understand and manage the link between fertilization regimes, carbon dioxide release and how these pests find their food.

Encapsulated Entomopathogenic Nematodes Can Protect Maize Plants from Diabrotica balteata Larvae.

To face the environmental problems caused by chemical pesticides, more ecologically friendly alternative pest control strategies are needed. Entomopathogenic nematodes (EPN) have great potential to control soil-dwelling insects that cause critical damage to the roots of cultivated plants. EPN are normally suspended in water and then sprayed on plants or onto the soil, but the inconsistent efficiency of this application method has led to the development of new formulations. Among them is the use of alginate capsules or beads that encapsulate the EPN in favorable conditions for later application. In this study, we evaluated whether alginate beads containing EPN are able to kill larvae of the banded cumber beetle Diabrotica balteata LeConte and thereby protect maize plants from damage by these generalist rootworms. EPN formulated in beads were as effective as sprayed EPN at killing D. balteata. They were found to protect maize plants from D. balteata damage, but only if applied in time. The treatment failed when rootworm attack started a week before the EPN beads were applied. Hence, the well-timed application of EPN-containing alginate beads may be an effective way to control root herbivores.

Protecting maize from rootworm damage with the combined application of arbuscular mycorrhizal fungi, Pseudomonas bacteria and entomopathogenic nematodes.

Diabrotica virgifera virgifera LeConte, the western corn rootworm (WCR), is the most destructive pest of maize in North America, and has recently spread across central Europe. Its subterranean larval stages are hard to reach with pesticides and it has evolved resistance to conventional management practices. The application of beneficial soil organisms is being considered as a sustainable and environmental friendly alternative. In a previous study, the combined application in wheat fields of arbuscular mycorrhizal fungi, entomopathogenic Pseudomonas bacteria, and entomopathogenic nematodes was found to promote growth and protection against a natural pest infestation, without negative cross effects. Because of the insect-killing capacity of the bacteria and nematodes, we hypothesized that the application of these organisms would have similar or even greater beneficial effects in WCR-infested maize fields. During three consecutive years (2015-2017), we conducted trials in Missouri (USA) in which we applied the three organisms, alone or in combinations, in plots that were artificially infested with WCR and in non-infested control plots. For two of the three trials, we found that in plots treated with entomopathogenic nematodes and/or entomopathogenic Pseudomonas bacteria, roots were less damaged than the roots of plants in control plots. During one year, WCR survival was significantly lower in plots treated with Pseudomonas than in control plots, and the surviving larvae that were recovered from these plots were lighter. The bacterial and nematodes treatments also enhanced yield, assessed as total grain weight, in one of the trials. The effects of the treatments varied considerable among the three years, but they were always positive for the plants.

Improved separation by at-column dilution in preparative hydrophilic interaction chromatography.

We evaluated at-column dilution (ACD) as a possible approach to reduce peak distortion caused by the injection of strong solvents in preparative hydrophilic interaction chromatography (HILIC). Three model compounds, namely uridine, cytidine and guanosine were selected and injected either in conventional or ACD conditions, using various proportions of water and acetonitrile in the sample diluent. Plate number and peak capacity were systematically investigated under both isocratic and gradient elution conditions, respectively. In isocratic conditions, ACD was found to provide higher plate numbers with sample diluent containing more than 40% water, but lower plate numbers with injections of less than 40% water, in comparison to a conventional injection system at the preparative scale. These contrasting results were attributed to the fact that i) efficiency was fundamentally reduced in ACD, due to the low make-up flow rate that was used to flush the injection loop, but ii) the trend was reversed for highly aqueous injection thanks to the capacity of ACD to maintain similar efficiency regardless of the injection solvent. On the other hand, peak capacity was comparable between ACD and conventional systems in gradient elution for injections containing low amounts of water. However, ACD became increasingly advantageous as the proportion of water in the injection solvent and/or the injection volume increased. This was confirmed by the separation of a plant root aqueous extract. Altogether, our findings suggest that ACD is beneficial both in isocratic and gradient modes when strongly aqueous solutions are injected, offering an attractive approach to increase loadability, while preserving peak shapes in preparative HILIC.

Wood ants produce a potent antimicrobial agent by applying formic acid on tree-collected resin.

Wood ants fight pathogens by incorporating tree resin with antimicrobial properties into their nests. They also produce large quantities of formic acid in their venom gland, which they readily spray to defend or disinfect their nest. Mixing chemicals to produce powerful antibiotics is common practice in human medicine, yet evidence for the use of such "defensive cocktails" by animals remains scant. Here, we test the hypothesis that wood ants enhance the antifungal activity of tree resin by treating it with formic acid. In a series of experiments, we document that (i) tree resin had much higher inhibitory activity against the common entomopathogenic fungus Metarhizium brunneum after having been in contact with ants, while no such effect was detected for other nest materials; (ii) wood ants applied significant amounts of endogenous formic and succinic acid on resin and other nest materials; and (iii) the application of synthetic formic acid greatly increased the antifungal activity of resin, but had no such effect when applied to inert glass material. Together, these results demonstrate that wood ants obtain an effective protection against a detrimental microorganism by mixing endogenous and plant-acquired chemical defenses. In conclusion, the ability to synergistically combine antimicrobial substances of diverse origins is not restricted to humans and may play an important role in insect societies.

Combined Field Inoculations of Pseudomonas Bacteria, Arbuscular Mycorrhizal Fungi, and Entomopathogenic Nematodes and their Effects on Wheat Performance.

In agricultural ecosystems, pest insects, pathogens, and reduced soil fertility pose major challenges to crop productivity and are responsible for significant yield losses worldwide. Management of belowground pests and diseases remains particularly challenging due to the complex nature of the soil and the limited reach of conventional agrochemicals. Boosting the presence of beneficial rhizosphere organisms is a potentially sustainable alternative and may help to optimize crop health and productivity. Field application of single beneficial soil organisms has shown satisfactory results under optimal conditions. This might be further enhanced by combining multiple beneficial soil organisms, but this remains poorly investigated. Here, we inoculated wheat plots with combinations of three beneficial soil organisms that have different rhizosphere functions and studied their effects on crop performance. Plant beneficial Pseudomonas bacteria, arbuscular mycorrhizal fungi (AMF), and entomopathogenic nematodes (EPN), were inoculated individually or in combinations at seeding, and their effects on plant performance were evaluated throughout the season. We used traditional and molecular identification tools to monitor their persistence over the cropping season in augmented and control treatments, and to estimate the possible displacement of native populations. In three separate trials, beneficial soil organisms were successfully introduced into the native populations and readily survived the field conditions. Various Pseudomonas, mycorrhiza, and nematode treatments improved plant health and productivity, while their combinations provided no significant additive or synergistic benefits compared to when applied alone. EPN application temporarily displaced some of the native EPN, but had no significant long-term effect on the associated food web. The strongest positive effect on wheat survival was observed for Pseudomonas and AMF during a season with heavy natural infestation by the frit fly, Oscinella frit, a major pest of cereals. Hence, beneficial impacts differed between the beneficial soil organisms and were most evident for plants under biotic stress. Overall, our findings indicate that in wheat production under the test conditions the three beneficial soil organisms can establish nicely and are compatible, but their combined application provides no additional benefits. Further studies are required, also in other cropping systems, to fine-tune the functional interactions among beneficial soil organisms, crops, and the environment.

Ant brood function as life preservers during floods.

Social organisms can surmount many ecological challenges by working collectively. An impressive example of such collective behavior occurs when ants physically link together into floating 'rafts' to escape from flooded habitat. However, raft formation may represent a social dilemma, with some positions posing greater individual risks than others. Here, we investigate the position and function of different colony members, and the costs and benefits of this functional geometry in rafts of the floodplain-dwelling ant Formica selysi. By causing groups of ants to raft in the laboratory, we observe that workers are distributed throughout the raft, queens are always in the center, and 100% of brood items are placed on the base. Through a series of experiments, we show that workers and brood are extremely resistant to submersion. Both workers and brood exhibit high survival rates after they have rafted, suggesting that occupying the base of the raft is not as costly as expected. The placement of all brood on the base of one cohesive raft confers several benefits: it preserves colony integrity, takes advantage of brood buoyancy, and increases the proportion of workers that immediately recover after rafting.