Novel pheromone-mediated reproductive behaviour in the stag beetle, Lucanus cervus.

The iconic European stag beetle (Lucanus cervus) (Coleoptera: Lucanidae) is one of the largest terrestrial beetles in Europe. Due to decreasing population numbers, thought to be a consequence of habitat loss, this beetle has become a near-threatened species across much of Europe, and a reliable monitoring system is required to measure its future population trends. As part of a programme aimed at conserving UK populations, we have investigated the chemical ecology of the beetle, with a view to developing an efficient semiochemical-based monitoring system. Such a scheme will be beneficial not only in the UK but across the European range of the species, where the beetle is of conservation concern. Here, we report on a surprising discovery of a male-produced pheromone, which provokes initial sexual receptivity in females, and which has not been previously identified in the animal kingdom. Furthermore, we assign sex pheromone function to a previously described female-specific compound.

The Invasive Plant Impatiens glandulifera Manipulates Microbial Associates of Competing Native Species.

Impatiens glandulifera or Himalayan balsam is one of the most invasive weeds across Europe and can seriously reduce native plant diversity. It often forms continuous monocultures along river banks, but the mechanisms of this arrested succession are largely unknown. Here, we investigated the effect of arbuscular mycorrhizal (AM) fungi on balsam competitive ability with two native plant species, Plantago lanceolata and Holcus lanatus. We also studied how competition with Impatiens affects colonisation by foliar endophytes and mycorrhizas of two other co-occurring native species, Urtica dioica and Cirsium arvense. Mycorrhizal colonisation reduced balsam growth when the plants were grown singly, but appeared to have little effect when balsam experienced intra- or interspecific competition. Competition with balsam together with the addition of mycorrhizas had no effect on P. lanceolata biomass, suggesting that the fungi were beneficial to the latter, enabling it to compete effectively with balsam. However, this was not so with H. lanatus. Meanwhile, competition with Impatiens reduced endophyte numbers and mycorrhizal colonisation in U. dioica and C. arvense, leading to enhanced susceptibility of these plants to insect attack. Himalayan balsam is known to degrade soil fungal populations and can also reduce foliar beneficial fungi in neighbouring plants. This allows the plant to compete effectively with itself and other native species, thereby leading to the continuous monocultures.

Addition of Arbuscular Mycorrhizal Fungi Enhances Terpene Synthase Expression in Salvia rosmarinus Cultivars.

Culinary herbs are commercially cultivated for their wide range of volatile compounds that give characteristic aromas and tastes. Rosemary (Salvia rosmarinus Spenn.) is an excellent model for assessment of methods improvement of volatile production as cultivars offer a wide variety of aromatic profiles due to the large family of terpene synthase genes. Arbuscular mycorrhizal fungi (AMF) associations have been shown to improve essential oil production in aromatic plants and offer one approach to enhance aroma in commercial herb production. Changes in the expression of seven different terpene synthases were compared in six rosemary cultivars in response to addition of AMF to a peat substrate. Addition of AMF profoundly influenced terpene synthase expression in all cultivars and did so without impacting the optimised plant size and uniformity achieved in these conditions. In addition, two methods for AMF application, developed with the horticultural industry in mind, were tested in this study. Uniform incorporation of AMF mixed into the growing substrate prior to planting of a root plug produced the most consistent root colonisation. Overall, our findings demonstrate the potential for the use of AMF in the improvement of aroma in culinary herbs within a commercial setting but show that outcomes are likely to greatly vary depending on variety.

Multitrophic Interactions Between Arbuscular Mycorrhizal Fungi, Foliar Endophytic Fungi and Aphids.

Almost all living plants can be simultaneously colonised by arbuscular mycorrhizal fungi in the roots and endophytes in the shoots, while also being attacked by insect herbivores. However, to date, no study has ever examined the multitrophic interactions between these two different fungal groups and insects on any species of forb. Here, we examined the effects of two commercial species mixtures of arbuscular mycorrhizal fungi (AMF) and two foliar endophytes (Colletotrichum acutatum and Cladosporium oxysporum) on the growth of an invasive weed, Impatiens glandulifera, and the aphids that attack it. AMF reduced plant biomass, which was most evident when C. oxysporum was inoculated. Mycorrhizal fungi had few effects on aphids, and these depended on the identity of the endophytes present. Meanwhile, endophytes tended to increase aphid numbers, but this depended on the identity of the AMF inoculum. Throughout, there were differences in the responses of the plant to the two mycorrhizal mixtures, demonstrating clear AMF specificity in this plant. These specific effects were also strongly affected by the endophytes, with a greater number of interactions found between the AMF and endophytes than between the endophytes themselves. In particular, AMF reduced infection levels by the endophytes, while some endophyte inoculations reduced mycorrhizal colonisation. We suggest that both AMF and endophytes could play an important part in future biological control programmes of weeds, but further multitrophic experiments are required to unravel the complexity of interactions between spatially separated parts of the plant microbiome.

Aphids and Mycorrhizal Fungi Shape Maternal Effects in Senecio vulgaris.

Plant performance in any one generation is affected not only by the prevailing environmental conditions, but also by the conditions experienced by the parental generation of those plants. These maternal effects have been recorded in a many plant species, but the influence of external biotic (as opposed to abiotic) factors on shaping maternal effects have been rarely examined. Furthermore, almost all previous studies have taken place over one plant generation, rather than across multiple generations. Here, we studied the influence of insect herbivory and arbuscular mycorrhizal (AM) fungal colonisation on the shaping of maternal effects in the annual forb Senecio vulgaris. We grew plants with and without aphids (Myzus persicae) and AM fungi (hereafter termed 'induction events') over four successive generations, wherein seeds from plants in any one treatment were used to grow plants of the same treatment in the next generation, all in identical environmental conditions. We found strong evidence of maternal effects in the second plant generation, i.e., after one induction event. These plants took longer to germinate, flowered in a shorter time, produced lighter seeds and were shorter and of lower biomass than their parents. Aphid attack tended to enhance these effects, whereas AM fungi had little influence. However, thereafter there was a gradual recovery in these parameters, so that plants experiencing three inductions showed similar life history parameters to those in the original generation. We conclude that experiments investigating maternal effects need to be performed over multiple plant generations and that biotic factors such as insects and mycorrhizas must also be taken into account, along with abiotic factors, such as nutrient and water availability.

Effect of weather conditions, substrate pH, biochar amendment and plant species on two plant growth-promoting microbes on vegetated roofs and facades.

Background: Vegetated building envelopes (VBEs), such as vegetated roofs and facades, are becoming more frequent in urban planning nowadays. However, harsh growing conditions restrain the application of VBEs. Plant growth-promoting microbes (PGPMs) might help ease the stresses, but first, it is necessary to investigate how to ensure their survival and growth under VBE conditions. Methods: We conducted three experiments to test the impact of various factors on the microbial populations of inoculated PGPMs in VBEs, a mycorrhizal fungus Rhizophagus irregularis and a bacterium Bacillus amyloliquefaciens. The first experiment was conducted by inoculating the two PGPMs separately in Sedum roof plots, and the microbial populations associated with Poa alpina was monitored for two consecutive years under local weather conditions. The second experiment was conducted in a laboratory testing the effect of substrate pH (substrates collected from balcony gardens) on R. irregularis population associated with Trifolium repens and Viola tricolor. The third experiment was conducted on a meadow roof testing the effect of biochar amendment on R. irregularis population associated with Thymus serpyllum and Fragaria vesca. Results: In the first experiment, Bacillus was found to associate with P. alpina, but Rhizophagus wasn't. Yet, the fungus induced high Bacillus population density in the Rhizophagus treated plots in the first year. In the second experiment, Rhizophagus abundance in T. repens was higher in the neutral substrate (6-6.5), while V. tricolor was more colonized in acidic substrate (5-5.5), suggesting an important interactive effect of substrate pH and plant species on Rhizophagus abundance. The third experiment suggested a negligible impact of biochar amendment on Rhizophagus abundance for both host plants. Conclusion: Three experiments demonstrate that PGPM inoculation on VBEs is feasible, and various factors and interactions affect the PGPM populations. This paper provides reference and inspiration for other VBE research involving substrate microbial manipulation.

Evaluating the use of seaweed extracts against root knot nematodes: A meta-analytic approach.

Plant parasitic nematode (PPN) control has historically relied on the use of synthetic chemical nematicides, however many are toxic to both human health and the environment. The withdrawal of the more harmful nematicides coupled with increases in soil temperatures and increased occurrence of pests and diseases associated with climate change, may enable PPN to increase in numbers and spread globally. The need for sustainable and environmentally friendly management options is necessary while facing future food security scares in order to feed the ever-growing population. Seaweed extracts have been used for decades in agriculture and horticulture as soil biostimulants, however there is a growing body of evidence to suggest that they could be used to reduce the occurrence of damaging PPN infections. Using meta-analysis, we investigated whether seaweed extracts applied to soil could reduce root knot nematode (RKN) abundance and whether there could be confounding factors that influence their efficacy. We found that seaweed extracts reduce RKN performance and that various factors affected the efficacy of seaweed, including the seaweed species itself and the crop the seaweed was applied to. Ascophyllum nodosum extracts were found to be the most effective. Particular RKN species were more sensitive than others to seaweed species used and, in some cases, specific seaweed species only affected particular RKN species. Different life cycle stages were also differentially susceptible to seaweed application, where both egg hatching and population abundance could be reduced via seaweed use. This research indicates that seaweed extracts could potentially be used to help reduce RKN attack on plants.

Apple endophyte community in relation to location, scion and rootstock genotypes and susceptibility to European canker.

European apple canker, caused by Neonectria ditissima, is a severe disease of apple. Achieving effective control is difficult with the currently available pesticides. Specific apple endophytes associated with cultivars may partially contribute to the cultivar response to the pathogen and thus could be used for disease management. We sought to determine whether the overall endophyte community differed among cultivars differing in their susceptibility to N. ditissima and to identify specific microbial groups associated with the susceptibility. Using Illumina MiSeq meta-barcoding, we profiled apple tree endophytes in 16 scion-rootstock combinations at two locations and quantified the relative contribution of scion, rootstock and location to the observed variability in the endophyte communities. Endophyte diversity was primarily affected by the orchard location (accounting for 29.4% and 85.9% of the total variation in the PC1 for bacteria and fungi, respectively), followed by the scion genotype (24.3% and 19.5% of PC2), whereas rootstock effects were small (<3% of PC1 and PC2). There were significant differences in the endophyte community between canker-resistant and -susceptible cultivars. Several bacterial and fungal endophyte groups had different relative abundance between susceptible and resistant cultivars. These endophyte groups included putative pathogen antagonists as well as plant pathogens. Their possible ecological roles in the N. ditissima pathosystem are discussed.

European mushroom assemblages are darker in cold climates.

Thermal melanism theory states that dark-colored ectotherm organisms are at an advantage at low temperature due to increased warming. This theory is generally supported for ectotherm animals, however, the function of colors in the fungal kingdom is largely unknown. Here, we test whether the color lightness of mushroom assemblages is related to climate using a dataset of 3.2 million observations of 3,054 species across Europe. Consistent with the thermal melanism theory, mushroom assemblages are significantly darker in areas with cold climates. We further show differences in color phenotype between fungal lifestyles and a lifestyle differentiated response to seasonality. These results indicate a more complex ecological role of mushroom colors and suggest functions beyond thermal adaption. Because fungi play a crucial role in terrestrial carbon and nutrient cycles, understanding the links between the thermal environment, functional coloration and species' geographical distributions will be critical in predicting ecosystem responses to global warming.

Open-source data reveal how collections-based fungal diversity is sensitive to global change.

PREMISE OF THE STUDY: Fungal diversity (richness) trends at large scales are in urgent need of investigation, especially through novel situations that combine long-term observational with environmental and remotely sensed open-source data. METHODS: We modeled fungal richness, with collections-based records of saprotrophic (decaying) and ectomycorrhizal (plant mutualistic) fungi, using an array of environmental variables across geographical gradients from northern to central Europe. Temporal differences in covariables granted insight into the impacts of the shorter- versus longer-term environment on fungal richness. RESULTS: Fungal richness varied significantly across different land-use types, with highest richness in forests and lowest in urban areas. Latitudinal trends supported a unimodal pattern in diversity across Europe. Temperature, both annual mean and range, was positively correlated with richness, indicating the importance of seasonality in increasing richness amounts. Precipitation seasonality notably affected saprotrophic fungal diversity (a unimodal relationship), as did daily precipitation of the collection day (negatively correlated). Ectomycorrhizal fungal richness differed from that of saprotrophs by being positively associated with tree species richness. DISCUSSION: Our results demonstrate that fungal richness is strongly correlated with land use and climate conditions, especially concerning seasonality, and that ongoing global change processes will affect fungal richness patterns at large scales.

Meta-analysis of the role of entomopathogenic and unspecialized fungal endophytes as plant bodyguards.

Herbaceous plants harbour species-rich communities of asymptomatic endophytic fungi. Although some of these endophytes are entomopathogenic, many are not, and remarkably little is known about how the presence of these fungi in plant tissues affects phytophagous insects. Here we show through a meta-analysis that both entomopathogenic and nonentomopathogenic endophytes have a negative effect on insect herbivores. Growth and performance of polyphagous and sucking insects are reduced by nonentomopathogenic endophytes, but monophages are unaffected, likely because the latter are better adapted to secondary metabolites produced or induced by the fungi. Furthermore, studies using excised leaves report weaker effects than those with intact plants, likely caused by chemical changes being masked by leaf excision. Most surprisingly, endophyte infection of seeds produces the greatest effect on insect herbivores in subsequent mature plants, even though the usual mode of fungal transmission is infection of leaves by airborne spores. We conclude that these ubiquitous hidden fungi may be important bodyguards of plants. However, in order to fully understand their roles in plant protection, we must be aware that minor differences in experimental design can lead to contradictory results.

Environmentally vulnerable noble chafers exhibit unusual pheromone-mediated behaviour.

Conserving populations of environmentally vulnerable insect species requires a greater understanding of the factors that determine their abundance and distribution, which requires detailed knowledge of their population and community ecology. Chemical ecological tools such as pheromones can be used for non-destructive monitoring of scarab beetle populations, enabling European countries to detect and, in some cases, map the range of some of these species, proving a valuable technique for monitoring elusive saproxylic beetles. In this paper, we investigated the behavioural and chemical ecology of the noble chafer, Gnorimus nobilis L., a model insect species of conservation concern across a Europe-wide distribution, and a red-listed UK Biodiversity Action Plan priority species. We identified a potential pheromone of adult beetles using electrophysiological recordings, behavioural measurements and field trials in the UK. Gnorimus nobilis is highly unusual in that although both sexes produce, at high metabolic cost, the natural product 2-propyl (E)-3-hexenoate, it only attracts males. This pattern of chemical signalling makes the classification of the compound, based on current semiochemical terminology, somewhat problematic, but in our view, it should be termed an aggregation pheromone as a consequence of the production pattern. Since both sexes emit it, but apparently only males respond positively to it, 2-propyl (E)-3-hexenoate may reflect an intermediate evolutionary stage towards developing into a sex-specific signal. From an applied perspective, our study provides a model for the non-invasive surveillance of cryptic vulnerable insect species, without the need for habitat searching or disturbance, and continuous human monitoring.

Plant growth-promoting rhizobacteria promote plant size inequality.

The uniformity of crop yield is extremely important for consumers and of as much relevance to the grower as overall yield. However, size inequality within a plant population is rarely measured and has never before been considered in relation to the use of beneficial microbes for yield enhancement. For the first time, we show that addition of soil bacteria to calabrese plants significantly increased size inequality. These effects were usually more apparent in above-ground biomass. This was caused by some (but not all) plants growing very large when inoculated with bacteria, while control plants were mostly small. We suggest that the main reason is the incompatibility of the inoculated bacteria with those already present in the rhizosphere. In some cases the inoculum matched the indigenous community, providing a benefit to plant growth, while often it did not and plants remained relatively small. We conclude that analyses of size inequality should be an integral part of experiments using microbial soil amendments. These analyses can help to inform the production of more effective microbial products and to ensure that the integration of beneficial microbes into sustainable production systems does not impair uniformity in yield.

Explaining European fungal fruiting phenology with climate variability.

Here we assess the impact of geographically dependent (latitude, longitude, and altitude) changes in bioclimatic (temperature, precipitation, and primary productivity) variability on fungal fruiting phenology across Europe. Two main nutritional guilds of fungi, saprotrophic and ectomycorrhizal, were further separated into spring and autumn fruiters. We used a path analysis to investigate how biogeographic patterns in fungal fruiting phenology coincided with seasonal changes in climate and primary production. Across central to northern Europe, mean fruiting varied by approximately 25 d, primarily with latitude. Altitude affected fruiting by up to 30 d, with spring delays and autumnal accelerations. Fruiting was as much explained by the effects of bioclimatic variability as by their large-scale spatial patterns. Temperature drove fruiting of autumnal ectomycorrhizal and saprotrophic groups as well as spring saprotrophic groups, while primary production and precipitation were major drivers for spring-fruiting ectomycorrhizal fungi. Species-specific phenology predictors were not stable, instead deviating from the overall mean. There is significant likelihood that further climatic change, especially in temperature, will impact fungal phenology patterns at large spatial scales. The ecological implications are diverse, potentially affecting food webs (asynchrony), nutrient cycling and the timing of nutrient availability in ecosystems.

Soil Inoculation with Bacillus spp. Modifies Root Endophytic Bacterial Diversity, Evenness, and Community Composition in a Context-Specific Manner.

The use of microbial inoculants containing plant growth-promoting rhizobacteria as a promoter of plant fitness and health is becoming increasingly popular in agriculture. However, whether and how these bacteria affect indigenous bacterial communities in field conditions is sparsely explored. We studied the effects of seed inoculation and field soil application of ubiquitous soil bacteria, B. cereus, B. subtilis, and B. amyloliquefaciens, on the diversity, evenness, and richness of endophytic bacterial communities in sprouting broccoli roots using high-throughput metagenome sequencing. The multiple operational taxonomic units (OTUs) assigned to different bacterial taxa clearly showed changes in ecological measures and relative abundances of certain taxa between control and treatment groups. The Bacillus inocula, themselves, failed to flourish as endophytes; however, the effects they extended on the endophytic bacterial community were both generic as well as species specific. In each case, Pseudomonadales, Rhizobiales, Xanthomonadales, and Burkholderiales were the most abundant orders in the endosphere. B. amyloliquefaciens drastically reduced the most abundant genus, Pseudomonas, while increasing the relative abundance of a range of minor taxa. The Shannon-Weiner diversity and Buzas and Gibson's evenness indices showed that the diversity and evenness were increased in both B. amyloliquefaciens and mixed treated plants. The UniFrac measurement of beta diversity showed that all treatments affected the specific composition of the endophytic bacterial community, with an apparent interspecies competition in the mixed treatment. Taken together, Bacillus species influenced the diversity, evenness, and composition of the endophytic bacterial community. However, these effects varied between different Bacillus spp. in a context-specific manner.

Unravelling mycorrhiza-induced wheat susceptibility to the English grain aphid Sitobion avenae.

Arbuscular mycorrhizal (AM) fungi are root symbionts that can increase or decrease aphid growth rates and reproduction, but the reason by which this happens is unknown. To investigate the underlying mechanisms of this interaction, we examined the effect of AM fungi on the English Grain aphid (Sitobion avenae) development, reproduction, attraction, settlement and feeding behaviour on two naturally susceptible varieties Triticum aestivum (L.) variety Solstice and T. monococcum MDR037, and two naturally resistant lines, T. monococcum MDR045 and MDR049. Mycorrhizal colonisation increased the attractiveness of T. aestivum var. Solstice to aphids, but there was no effect on aphid development on this variety. Using the Electrical Penetration Graph (EPG) technique, we found that mycorrhizal colonisation increased aphid phloem feeding on T. monococcum MDR037 and MDR045, colonisation also increased growth rate and reproductive success of S. avenae on these varieties. Mycorrhizas increased vascular bundle size, demonstrating that these fungi can influence plant anatomy. We discuss if and how this could be related to an enhanced success rate in phloem feeding in two varieties. Overall, we present and discuss how mycorrhizal fungi can affect the feeding behaviour of S. avenae in wheat, inducing susceptibility in a resistant variety.

Using soil microbial inoculations to enhance substrate performance on extensive green roofs.

Green roofs are increasing in popularity in the urban environment for their contribution to green infrastructure; but their role for biodiversity is not often a design priority. Maximising biodiversity will impact positively on ecosystem services and is therefore fundamental for achieving the greatest benefits from green roofs. Extensive green roofs are lightweight systems generally constructed with a specialised growing medium that tends to be biologically limited and as such can be a harsh habitat for plants to thrive in. Thus, this investigation aimed to enhance the soil functioning with inoculations of soil microbes to increase plant diversity, improve vegetation health/performance and maximise access to soil nutrients. Manipulations included the addition of mycorrhizal fungi and a microbial mixture ('compost tea') to green roof rootzones, composed mainly of crushed brick or crushed concrete. The study revealed that growing media type and depth play a vital role in the microbial ecology of green roofs, with complex relationships between depth and type of substrate and the type of microbial inoculant applied, with no clear pattern being observed. For bait plant measurements (heights, leaf numbers, root/shoot biomass, leaf nutrients), a compost tea may have positive effects on plant performance when grown in substrates of shallower depths (5.5cm), even one year after inoculums are applied. Results from the species richness surveys show that diversity was significantly increased with the application of an AM fungal treatment and that overall, results suggest that brick-based substrate blends are most effective for vegetation performance as are deeper depths (although this varied with time). Microbial inoculations of green roof habitats appeared to be sustainable; they need only be done once for benefits to still been seen in subsequent years where treatments are added independently (not in combination). They seem to be a novel and viable method of enhancing rooftop conditions.

Does the Slow-Growth, High-Mortality Hypothesis Apply Below Ground?

Belowground tri-trophic study systems present a challenging environment in which to study plant-herbivore-natural enemy interactions. For this reason, belowground examples are rarely available for testing general ecological theories. To redress this imbalance, we present, for the first time, data on a belowground tri-trophic system to test the slow growth, high mortality hypothesis. We investigated whether the differing performance of entomopathogenic nematodes (EPNs) in controlling the common pest black vine weevil Otiorhynchus sulcatus could be linked to differently resistant cultivars of the red raspberry Rubus idaeus. The O. sulcatus larvae recovered from R. idaeus plants showed significantly slower growth and higher mortality on the Glen Rosa cultivar, relative to the more commercially favored Glen Ample cultivar creating a convenient system for testing this hypothesis. Heterorhabditis megidis was found to be less effective at controlling O. sulcatus than Steinernema kraussei, but conformed to the hypothesis. However, S. kraussei maintained high levels of O. sulcatus mortality regardless of how larval growth was influenced by R. idaeus cultivar. We link this to direct effects that S. kraussei had on reducing O. sulcatus larval mass, indicating potential sub-lethal effects of S. kraussei, which the slow-growth, high-mortality hypothesis does not account for. Possible origins of these sub-lethal effects of EPN infection and how they may impact on a hypothesis designed and tested with aboveground predator and parasitoid systems are discussed.

Developing Soil Microbial Inoculants for Pest Management: Can One Have Too Much of a Good Thing?

Soil microbes present a novel and cost-effective method of increasing plant resistance to insect pests and thus create a sustainable opportunity to reduce current pesticide application. However, the use of microbes in integrated pest management programs is still in its infancy. This can be attributed primarily to the variations in microbial inoculum performance under laboratory and field conditions. Soil inoculants containing single, indigenous microbial species have shown promising results in increasing chemical defenses of plants against foliar feeding insects. Conversely, commercial inoculants containing multiple species tend to show no effects on herbivore infestation in the field. We present here a simple model that endeavours to explain how single and multiple species in microbial inoculants differentially govern insect population dynamics via changes in plant chemical profiles. We discuss further how this knowledge can be applied to manipulate soil microbial species and develop 'tailored' microbial inoculants that could be used in plant protection against antagonists.