Nontarget impacts of neonicotinoids on nectar-inhabiting microbes.

Plant-systemic neonicotinoid (NN) insecticides can exert non-target impacts on organisms like beneficial insects and soil microbes. NNs can affect plant microbiomes, but we know little about their effects on microbial communities that mediate plant-insect interactions, including nectar-inhabiting microbes (NIMs). Here we employed two approaches to assess the impacts of NN exposure on several NIM taxa. First, we assayed the in vitro effects of six NN compounds on NIM growth using plate assays. Second, we inoculated a standardised NIM community into the nectar of NN-treated canola (Brassica napus) and assessed microbial survival and growth after 24 h. With few exceptions, in vitro NN exposure tended to decrease bacterial growth metrics. However, the magnitude of the decrease and the NN concentrations at which effects were observed varied substantially across bacteria. Yeasts showed no consistent in vitro response to NNs. In nectar, we saw no effects of NN treatment on NIM community metrics. Rather, NIM abundance and diversity responded to inherent plant qualities like nectar volume. In conclusion, we found no evidence that NIMs respond to field-relevant NN levels in nectar within 24 h, but our study suggests that context, specifically assay methods, time and plant traits, is important in assaying the effects of NNs on microbial communities.

Intraspecific variation in realized dispersal probability and host quality shape nectar microbiomes.

Epiphytic microbes frequently affect plant phenotype and fitness, but their effects depend on microbe abundance and community composition. Filtering by plant traits and deterministic dispersal-mediated processes can affect microbiome assembly, yet their relative contribution to predictable variation in microbiome is poorly understood. We compared the effects of host-plant filtering and dispersal on nectar microbiome presence, abundance, and composition. We inoculated representative bacteria and yeast into 30 plants across four phenotypically distinct cultivars of Epilobium canum. We compared the growth of inoculated communities to openly visited flowers from a subset of the same plants. There was clear evidence of host selection when we inoculated flowers with synthetic communities. However, plants with the highest microbial densities when inoculated did not have the highest microbial densities when openly visited. Instead, plants predictably varied in the presence of bacteria, which was correlated with pollen receipt and floral traits, suggesting a role for deterministic dispersal. These findings suggest that host filtering could drive plant microbiome assembly in tissues where species pools are large and dispersal is high. However, deterministic differences in microbial dispersal to hosts may be equally or more important when microbes rely on an animal vector, dispersal is low, or arrival order is important.

Incipiently social carpenter bees (Xylocopa) host distinctive gut bacterial communities and display geographical structure as revealed by full-length PacBio 16S rRNA sequencing.

The gut microbiota of bees affects nutrition, immunity and host fitness, yet the roles of diet, sociality and geographical variation in determining microbiome structure, including variant-level diversity and relatedness, remain poorly understood. Here, we use full-length 16S rRNA amplicon sequencing to compare the crop and gut microbiomes of two incipiently social carpenter bee species, Xylocopa sonorina and Xylocopa tabaniformis, from multiple geographical sites within each species' range. We found that Xylocopa species share a set of core taxa consisting of Bombilactobacillus, Bombiscardovia and Lactobacillus, found in >95% of all individual bees sampled, and Gilliamella and Apibacter were also detected in the gut of both species with high frequency. The crop bacterial community of X. sonorina comprised nearly entirely Apilactobacillus with occasionally abundant nectar bacteria. Despite sharing core taxa, Xylocopa species' microbiomes were distinguished by multiple bacterial lineages, including species-specific variants of core taxa. The use of long-read amplicons revealed otherwise cryptic species and population-level differentiation in core microbiome members, which was masked when a shorter fragment of the 16S rRNA (V4) was considered. Of the core taxa, Bombilactobacillus and Bombiscardovia exhibited differentiation in amplicon sequence variants among bee populations, but this was lacking in Lactobacillus, suggesting that some bacterial genera in the gut may be structured by different processes. We conclude that these Xylocopa species host a distinctive microbiome, similar to that of previously characterized social corbiculate apids, which suggests that further investigation to understand the evolution of the bee microbiome and its drivers is warranted.

Floral Microbes Suppress Growth of Monilinia laxa with Minimal Effects on Honey Bee Feeding.

Management of Monilinia laxa, the causal agent of brown rot blossom blight in almond (Prunus dulcis), relies heavily on the use of chemical fungicides during bloom. However, chemical fungicides can have nontarget effects on beneficial arthropods, including pollinators, and select for resistance in the pathogen of concern. Almond yield is heavily reliant on successful pollination by healthy honey bees (Apis mellifera); thus, identifying sustainable, effective, and pollinator-friendly control methods for blossom blight during bloom is desirable. Flower-inhabiting microbes could provide a natural, sustainable form of biocontrol for M. laxa, while potentially minimizing costly nontarget effects on almond pollinators and the services they provide. As pollinators are sensitive to floral microbes and their associated taste and scent cues, assessing effects of prospective biocontrol species on pollinator attraction is also necessary. Here, our objective was to isolate and identify potential biocontrol microbes from an array of agricultural and natural flowering hosts and test their efficacy in suppressing M. laxa growth in culture. Out of an initial 287 bacterial and fungal isolates identified, 56 were screened using a dual culture plate assay. Most strains reduced M. laxa growth in vitro. Ten particularly effective candidate microbes were further screened for their effect on honey bee feeding. Of the 10, nine were found to both strongly suppress M. laxa growth in culture and not reduce honey bee feeding. These promising results suggest a number of strong candidates for augmentative microbial biocontrol of brown rot blossom blight in almond with potentially minimal effects on honey bee pollination.

Orchard Management and Landscape Context Mediate the Pear Floral Microbiome.

Crop-associated microbiota are a key factor affecting host health and productivity. Most crops are grown within heterogeneous landscapes, and interactions between management practices and landscape context often affect plant and animal biodiversity in agroecosystems. However, whether these same factors typically affect crop-associated microbiota is less clear. Here, we assessed whether orchard management strategies and landscape context affected bacterial and fungal communities in pear (Pyrus communis) flowers. We found that bacteria and fungi responded differently to management schemes. Organically certified orchards had higher fungal diversity in flowers than conventional or bio-based integrated pest management (IPM) orchards, but organic orchards had the lowest bacterial diversity. Orchard management scheme also best predicted the distribution of several important bacterial and fungal genera that either cause or suppress disease; organic and bio-based IPM best explained the distributions of bacterial and fungal genera, respectively. Moreover, patterns of bacterial and fungal diversity were affected by interactions between management, landscape context, and climate. When examining the similarity of bacterial and fungal communities across sites, both abundance- and taxon-related turnovers were mediated primarily by orchard management scheme and landscape context and, specifically, the amount of land in cultivation. Our study reveals local- and landscape-level drivers of floral microbiome structure in a major fruit crop, providing insights that can inform microbiome management to promote host health and high-yielding quality fruit. IMPORTANCE Proper crop management during bloom is essential for producing disease-free tree fruit. Tree fruits are often grown in heterogeneous landscapes; however, few studies have assessed whether landscape context and crop management affect the floral microbiome, which plays a critical role in shaping plant health and disease tolerance. Such work is key for identification of tactics and/or contexts where beneficial microbes proliferate and pathogenic microbes are limited. Here, we characterize the floral microbiome of pear crops in Washington State, where major production occurs in intermountain valleys and basins with variable elevation and microclimates. Our results show that both local-level (crop management) and landscape-level (habitat types and climate) factors affect floral microbiota but in disparate ways for each kingdom. More broadly, these findings can potentially inform microbiome management in orchards for promotion of host health and high-quality yields.

Acinetobacter pollinis sp. nov., Acinetobacter baretiae sp. nov. and Acinetobacter rathckeae sp. nov., isolated from floral nectar and honey bees.

A detailed evaluation of eight bacterial isolates from floral nectar and animal visitors to flowers shows evidence that they represent three novel species in the genus Acinetobacter. Phylogenomic analysis shows the closest relatives of these new isolates are Acinetobacter apis, Acinetobacter boissieri and Acinetobacter nectaris, previously described species associated with floral nectar and bees, but high genome-wide sequence divergence defines these isolates as novel species. Pairwise comparisons of the average nucleotide identity of the new isolates compared to known species is extremely low (<83 %), thus confirming that these samples are representative of three novel Acinetobacter species, for which the names Acinetobacter pollinis sp. nov., Acinetobacter baretiae sp. nov. and Acinetobacter rathckeae sp. nov. are proposed. The respective type strains are SCC477T (=TSD-214T=LMG 31655T), B10AT (=TSD-213T=LMG 31702T) and EC24T (=TSD-215T=LMG 31703T=DSM 111781T).

Nitrogen Assimilation Varies Among Clades of Nectar- and Insect-Associated Acinetobacters.

Floral nectar is commonly colonized by yeasts and bacteria, whose growth largely depends on their capacity to assimilate nutrient resources, withstand high osmotic pressures, and cope with unbalanced carbon-to-nitrogen ratios. Although the basis of the ecological success of these microbes in the harsh environment of nectar is still poorly understood, it is reasonable to assume that they are efficient nitrogen scavengers that can consume a wide range of nitrogen sources in nectar. Furthermore, it can be hypothesized that phylogenetically closely related strains have more similar phenotypic characteristics than distant relatives. We tested these hypotheses by investigating the growth performance on different nitrogen-rich substrates of a collection of 82 acinetobacters isolated from nectar and honeybees, representing members of five species (Acinetobacter nectaris, A. boissieri, A. apis, and the recently described taxa A. bareti and A. pollinis). We also analyzed possible links between growth performance and phylogenetic affiliation of the isolates, while taking into account their geographical origin. Results demonstrated that the studied isolates could utilize a wide variety of nitrogen sources, including common metabolic by-products of yeasts (e.g., ammonium and urea), and that phylogenetic relatedness was associated with the variation in nitrogen assimilation among the studied acinetobacters. Finally, nutrient source and the origin (sample type and country) of isolates also predicted the ability of the acinetobacters to assimilate nitrogen-rich compounds. Overall, these results demonstrate inter-clade variation in the potential of the acinetobacters as nitrogen scavengers and suggest that nutritional dependences might influence interactions between bacteria and yeasts in floral nectar.

Microbial Co-Occurrence in Floral Nectar Affects Metabolites and Attractiveness to a Generalist Pollinator.

Microbial metabolism can shape cues important for animal attraction in service-resource mutualisms. Resources are frequently colonized by microbial communities, but experimental assessment of animal-microbial interactions often focus on microbial monocultures. Such an approach likely fails to predict effects of microbial assemblages, as microbe-microbe interactions may affect in a non-additive manner microbial metabolism and resulting chemosensory cues. Here, we compared effects of microbial mono- and cocultures on growth of constituent microbes, volatile metabolite production, sugar catabolism, and effects on pollinator foraging across two nectar environments that differed in sugar concentration. Growth in co-culture decreased the abundance of the yeast Metschnikowia reukaufii, but not the bacterium Asaia astilbes. Volatile emissions differed significantly between microbial treatments and with nectar concentration, while sugar concentration was relatively similar among mono- and cocultures. Coculture volatile emission closely resembled an additive combination of monoculture volatiles. Despite differences in microbial growth and chemosensory cues, honey bee feeding did not differ between microbial monocultures and assemblages. Taken together, our results suggest that in some cases, chemical and ecological effects of microbial assemblages are largely predictable from those of component species, but caution that more work is necessary to predict under what circumstances non-additive effects are important.

Microbial communities in hummingbird feeders are distinct from floral nectar and influenced by bird visitation.

Human provisioning can shape resource availability for wildlife, but consequences for microbiota availability and exchange remain relatively unexplored. Here, we characterized microbial communities on bills and faecal material of hummingbirds and their food resources, including feeders and floral nectar. We experimentally manipulated bird visitation to feeders and examined effects on sucrose solution microbial communities. Birds, feeders and flowers hosted distinct bacterial and fungal communities. Proteobacteria comprised over 80% of nectar bacteria but feeder solutions contained a high relative abundance of Proteobacteria, Firmicutes and Actinobacteria. Hummingbirds hosted bacterial taxa commonly found in other birds and novel genera including Zymobacter [Proteobacteria] and Ascomycete fungi. For feeders, bird-visited and unvisited solutions both accumulated abundant microbial populations that changed solution pH, but microbial composition was largely determined by visitation treatment. Our results reveal that feeders host abundant microbial populations, including some bird-associated microbial taxa. Microbial taxa in feeders were primarily non-pathogenic bacteria and fungi but differed substantially from those in floral nectar. These results demonstrate that human provisioning influences microbial intake by free-ranging hummingbirds; however, it is unknown how these changes impact hummingbird gastrointestinal flora or health.

Effects of Agricultural Management on Rhizosphere Microbial Structure and Function in Processing Tomato Plants.

Agricultural management practices affect bulk soil microbial communities and the functions they carry out, but it remains unclear how these effects extend to the rhizosphere in different agroecosystem contexts. Given close linkages between rhizosphere processes and plant nutrition and productivity, understanding how management practices impact this critical zone is of great importance to optimize plant-soil interactions for agricultural sustainability. A comparison of six paired conventional-organic processing tomato farms was conducted to investigate relationships between management, soil physicochemical parameters, and rhizosphere microbial community composition and functions. Organically managed fields were higher in soil total N and NO3-N, total and labile C, plant Ca, S, and Cu, and other essential nutrients, while soil pH was higher in conventionally managed fields. Differential abundance, indicator species, and random forest analyses of rhizosphere communities revealed compositional differences between organic and conventional systems and identified management-specific microbial taxa. Phylogeny-based trait prediction showed that these differences translated into more abundant pathogenesis-related gene functions in conventional systems. Structural equation modeling revealed a greater effect of soil biological communities than physicochemical parameters on plant outcomes. These results highlight the importance of rhizosphere-specific studies, as plant selection likely interacts with management in regulating microbial communities and functions that impact agricultural productivity.IMPORTANCE Agriculture relies, in part, on close linkages between plants and the microorganisms that live in association with plant roots. These rhizosphere bacteria and fungi are distinct from microbial communities found in the rest of the soil and are even more important to plant nutrient uptake and health. Evidence from field studies shows that agricultural management practices such as fertilization and tillage shape microbial communities in bulk soil, but little is known about how these practices affect the rhizosphere. We investigated how agricultural management affects plant-soil-microbe interactions by comparing soil physical and chemical properties, plant nutrients, and rhizosphere microbial communities from paired fields under organic and conventional management. Our results show that human management effects extend even to microorganisms living in close association with plant roots and highlight the importance of these bacteria and fungi to crop nutrition and productivity.

Microbial metabolites elicit distinct olfactory and gustatory preferences in bumblebees.

Animals such as bumblebees use chemosensory cues to both locate and evaluate essential resources. Increasingly, it is recognized that microbes can alter the quality of foraged resources and produce metabolites that may act as foraging cues. The distinct nature of these chemosensory cues however and their use in animal foraging remain poorly understood. Here, we test the hypothesis that species of nectar-inhabiting microbes differentially influence pollinator attraction and feeding via microbial metabolites produced in nectar. We first examined the electrophysiological potential for bumblebee (Bombus impatiens) antennal olfactory neurons to respond to microbial volatile organic compounds (mVOCs), followed by an olfactory preference test. We also assessed gustatory preferences for microbial-altered nectar through both no-choice and choice feeding assays. Antennal olfactory neurons responded to some mVOCs, and bees preferred nectar solutions inoculated with the bacterium Asaia astilbes over the yeast Metschnikowia reukaufii based on volatiles alone. However, B. impatiens foragers consumed significantly more Metschnikowia-inoculated nectar, suggesting distinct roles for mVOCs and non-volatile metabolites in mediating both attraction and feeding decisions. Collectively, our results suggest that microbial metabolites have significant potential to shape interspecific, plant-pollinator signalling, with consequences for forager learning, economics and floral host reproduction.

Nectar-inhabiting microorganisms influence nectar volatile composition and attractiveness to a generalist pollinator.

The plant microbiome can influence plant phenotype in diverse ways, yet microbial contribution to plant volatile phenotype remains poorly understood. We examine the presence of fungi and bacteria in the nectar of a coflowering plant community, characterize the volatiles produced by common nectar microbes and examine their influence on pollinator preference. Nectar was sampled for the presence of nectar-inhabiting microbes. We characterized the headspace of four common fungi and bacteria in a nectar analog. We examined electrophysiological and behavioral responses of honey bees to microbial volatiles. Floral headspace samples collected in the field were surveyed for the presence of microbial volatiles. Microbes commonly inhabit floral nectar and the common species differ in volatile profiles. Honey bees detected most microbial volatiles tested and distinguished among solutions based on volatiles only. Floral headspace samples contained microbial-associated volatiles, with 2-ethyl-1-hexanol and 2-nonanone - both detected by bees - more often detected when fungi were abundant. Nectar-inhabiting microorganisms produce volatile compounds, which can differentially affect honey bee preference. The yeast Metschnikowia reukaufii produced distinctive compounds and was the most attractive of all microbes compared. The variable presence of microbes may provide volatile cues that influence plant-pollinator interactions.

Contrasting effects of yeasts and bacteria on floral nectar traits.

Background and Aims: Flowers can be highly variable in nectar volume and chemical composition, even within the same plant, but the causes of this variation are not fully understood. One potential cause is nectar-colonizing bacteria and yeasts, but experimental tests isolating their effects on wildflowers are largely lacking. This study examines the effects of dominant species of yeasts and bacteria on the hummingbird-pollinated shrub, Mimulus aurantiacus, in California. Methods: Wildflowers were inoculated with field-relevant titres of either the yeast Metschnikowia reukaufii or the bacterium Neokomagataea sp. (formerly Gluconobacter sp.), both isolated from M. aurantiacus nectar. Newly opened flowers were bagged, inoculated, harvested after 3 d and analysed for microbial abundance, nectar volume, and sugar and amino acid concentration and composition. Key Results: Yeast inoculation reduced amino acid concentration and altered amino acid composition, but had no significant effect on nectar volume or sugar composition. In contrast, bacterial inoculation increased amino acid concentration, enhanced the proportion of nectar sugars comprised by monosaccharides, and reduced nectar volume. Conclusions: The results presented suggest that microbial inhabitants of floral nectar can make nectar characteristics variable among flowers through divergent effects of yeasts and bacteria on nectar chemistry and availability, probably modifying plant-pollinator interactions.

Dispersal enhances beta diversity in nectar microbes.

Dispersal is considered a key driver of beta diversity, the variation in species composition among local communities, but empirical tests remain limited. We manipulated dispersal of nectar-inhabiting bacteria and yeasts via flower-visiting animals to examine how dispersal influenced microbial beta diversity among flowers. Contrary to the prevailing view that dispersal lowers beta diversity, we found beta diversity was highest when dispersal was least limited. Our analysis suggested that this unexpected pattern might have resulted from stronger priority effects under increased dispersal. Dispersal is highly stochastic, generating variability in species arrival history and consequently the potential for community divergence via priority effects, in these and likely many other microbial, plant, and animal communities. Yet most previous experiments eliminated this possibility. We suggest that the positive effects of dispersal on beta diversity, like the one we report here, may have been greatly underappreciated.

Orthogonal fitness benefits of nitrogen and ants for nitrogen-limited plants in the presence of herbivores.

Predictable effects of resource availability on plant growth-defense strategies provide a unifying theme in theories of direct anti-herbivore defense, but it is less clear how resource availability modulates plant indirect defense. Ant-plant-hemipteran interactions produce mutualistic trophic cascades when hemipteran-tending ants reduce total herbivory, and these interactions are a key component of plant indirect defense in most terrestrial ecosystems. Here we conducted an experiment to test how ant-plant-hemipteran interactions depend on nitrogen (N) availability by manipulating the presence of ants and aphids under different N fertilization treatments. Ants increased plant flowering success by decreasing the densities of herbivores, and the effects of ants on folivores were positively related to the density of aphids. Unexpectedly, N fertilization produced no changes in plant N concentrations. Plants grown in higher N grew and flowered more, but aphid honeydew chemistry stayed the same, and neither the density of aphids nor the rate of ant attraction per aphid changed with N addition. The positive effects of ants and N addition on plant fitness were thus independent of one another. We conclude that N was the plant's limiting nutrient and propose that addition of the limiting nutrient is unlikely to alter the strength of mutualistic trophic cascades.

Nectar microbes can reduce secondary metabolites in nectar and alter effects on nectar consumption by pollinators.

Secondary metabolites that are present in floral nectar have been hypothesized to enhance specificity in plant-pollinator mutualism by reducing larceny by non-pollinators, including microorganisms that colonize nectar. However, few studies have tested this hypothesis. Using synthetic nectar, we conducted laboratory and field experiments to examine the effects of five chemical compounds found in nectar on the growth and metabolism of nectar-colonizing yeasts and bacteria, and the interactive effects of these compounds and nectar microbes on the consumption of nectar by pollinators. In most cases, focal compounds inhibited microbial growth, but the extent of these effects depended on compound identity, concentration, and microbial species. Moreover, most compounds did not substantially decrease sugar metabolism by microbes, and microbes reduced the concentration of some compounds in nectar. Using artificial flowers in the field, we also found that the common nectar yeast Metschnikowia reukaufii altered nectar consumption by small floral visitors, but only in nectar containing catalpol. This effect was likely mediated by a mechanism independent of catalpol metabolism. Despite strong compound-specific effects on microbial growth, our results suggest that the secondary metabolites tested here are unlikely to be an effective general defense mechanism for preserving nectar sugars for pollinators. Instead, our results indicate that microbial colonization of nectar could reduce the concentration of secondary compounds in nectar and, in some cases, reduce deterrence to pollinators.

Forest area and connectivity influence root-associated fungal communities in a fragmented landscape.

Habitat fragmentation is well known to affect plant and animal diversity as a result of reduced habitat area and connectivity, but its effects on microorganisms are poorly understood. Using high-throughput sequencing of two regions of the rRNA gene, we studied the effects of forest area and connectivity on the diversity and composition of fungi associated with the roots of the dominant tree, Metrosideros polymorpha, in a lava-fragmented landscape on the Island of Hawaii. We found that local fungal diversity increased with forest area, whereas fungal species composition was correlated with fragment connectivity. Fragment size and connectivity were significant predictors even when we included environmental covariates, which were also associated with fungal diversity and composition. Fungal species composition was more similar among highly connected fragments than among poorly connected ones. We also identified individual taxa that varied in abundance with connectivity. Taken together, our results show that habitat fragmentation can alter microbial diversity and composition via differential response among fungal phyla and individual taxa to habitat connectivity.

Historical contingency in species interactions: towards niche-based predictions.

The way species affect one another in ecological communities often depends on the order of species arrival. The magnitude of such historical contingency, known as priority effects, varies across species and environments, but this variation has proven difficult to predict, presenting a major challenge in understanding species interactions and consequences for community structure and function. Here, we argue that improved predictions can be achieved by decomposing species' niches into three components: overlap, impact and requirement. Based on classic theories of community assembly, three hypotheses that emphasise related, but distinct influences of the niche components are proposed: priority effects are stronger among species with higher resource use overlap; species that impact the environment to a greater extent exert stronger priority effects; and species whose growth rate is more sensitive to changes in the environment experience stronger priority effects. Using nectar-inhabiting microorganisms as a model system, we present evidence that these hypotheses complement the conventional hypothesis that focuses on the role of environmental harshness, and show that niches can be twice as predictive when separated into components. Taken together, our hypotheses provide a basis for developing a general framework within which the magnitude of historical contingency in species interactions can be predicted.