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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 in95% 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.

Several studies have shown that nectar-inhabiting bacteria and fungi influence plant and pollinator fitness by modulating their mutualistic interactions. However, little is known about the nectar microbe communities in African agricultural crops, and how visitations by different Afrotropical bee species shape floral nectar microbial community, chemistry, and plant reproductive fitness. In this study, we explored these interactions focusing on Afrotropical pollinator bee species found in Kenya. We found that the stingless bee species Hypotrigona gribodoi and Dactylurina schmidti, and the African savannah honey bee Apis mellifera scutellata introduce only bacteria into the floral nectar of the yellow bell pepper plant Capsicum annum cultivated in a greenhouse, which differed in their volatile emission profiles. Additionally, nectar probing by these bee species shaped the microbiome of C. annum, and significantly affected nectar volume and sugar composition. These findings lay the foundation for further exploration of the microbial influence into plant-pollinator interactions in this country.

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.

SummaryEpiphytic microbes frequently impact plant phenotype and fitness, but effects depend on microbe community composition. Deterministic filtering by plant traits and dispersal-mediated processes can affect microbiome assembly yet their relative contribution is poorly understood.We tested the impact of host-plant filtering and dispersal limitation on nectar microbiome abundance and composition. We inoculated bacteria and yeast into 30 plants across 4 phenotypically distinct cultivars ofEpilobium canum. We compared the growth of inoculated communities to openly visited flowers from a subset of the same plants.The abundance and composition of microbial communities differed among plant individuals and cultivars in both inoculated and open flowers. However, plants hosting the highest microbial abundance when inoculated did not have the highest abundances when openly visited. Rather microbial density among open flowers was correlated with pollen receipt, a proxy for animal visitation, suggesting a primary role of deterministic dispersal in floral microbiome assembly despite variation in host-quality.While host-quality can affect microbiome assembly, variation in dispersal was more important here. Host quality could drive microbial community assembly in plant tissues where species pools are large and dispersal is consistent, but dispersal may be more important when microbial dispersal is limited, or arrival order is important.

Prolonged water stress can reduce plant growth and fitness and may shift rhizoplane microbial communities. Although drought affects microbial structure of many plant species, it is still poorly understood whether closely related plant species host distinct microbial communities or respond similarly to drought. To explore this question, eight members of the Streptanthus clade with varying affinity to serpentine were subjected to three watering regimes. Rhizoplane bacterial communities were described using 16S rRNA gene amplicon sequencing and we explored the impact of watering treatment and plant species on alpha diversity, differential abundance, and beta diversity of soil bacterial communities. Among most plant species, reduced watering also reduced alpha diversity, but plant species did not strongly impact alpha diversity. Drought treatment reduced microbial community dissimilarity among samples: bacterial communities were more similar when plants received less water. Watering altered the relative abundance of bacterial genera within Proteobacteria, Firmicutes, Bacteroidetes, Planctomycetes, and Acidobacteria, which responded similarly in the rhizoplane of most plant species. These results suggest that prolonged water stress in Streptanthus spp. can result in convergent microbial communities among plant species. We suggest the functional consequences of these shifts be examined to assess effects on plant and microbial fitness under drought conditions.

Due to the difficulty of tracking microbial dispersal, it is rarely possible to disentangle the relative importance of dispersal and species sorting for microbial community assembly. Here, we leverage a detailed multilevel network to examine drivers of bacterial community assembly within flowers. We observed flower visitors to 20 focal plant species in a coflowering community in the Sierra Nevada, revealing 289 species of arthropods. We also analyzed bacterial communities on flowers of each species. We found that plant species with similar visitor communities tend to have similar bacterial communities, and visitor identity to be more important than plant relatedness in structuring floral bacterial communities. However, plant species that were hubs of arthropod visitation were not necessarily hubs of floral bacteria, suggesting an important role for species sorting. Across plant species, the composition of flower‐visiting Diptera (flies), bees and non‐bee Hymenoptera best predicted bacterial species composition on flowers. Taken together, our analyses suggest dispersal is important in determining similarity in microbial communities across plant species, but not as important in determining the overall macrostructure (nestedness, modularity) and microstructure (connectedness based on shared interactors) of the floral bacterial network. A multilevel network approach thus allows us to address features of community assembly that cannot be considered when viewing networks as separate entities.

Flowers at times host abundant and specialized communities of bacteria and fungi that influence floral phenotypes and interactions with pollinators. Ecological processes drive variation in microbial abundance and composition at multiple scales, including among plant species, among flower tissues, and among flowers on the same plant. Variation in microbial effects on floral phenotype suggests that microbial metabolites could cue the presence or quality of rewards for pollinators, but most plants are unlikely to rely on microbes for pollinator attraction or reproduction. From a microbial perspective, flowers offer opportunities to disperse between habitats, but microbial species differ in requirements for and benefits received from such dispersal. The extent to which floral microbes shape the evolution of floral traits, influence fitness of floral visitors, and respond to anthropogenic change is unclear. A deeper understanding of these phenomena could illuminate the ecological and evolutionary importance of floral microbiomes and their role in the conservation of plant–pollinator interactions.

Reduced insect pest populations found on long-term organic farms have mostly been attributed to increased biodiversity and abundance of beneficial predators, as well as to changes in plant nutrient content. However, the role of plant resistance has largely been ignored. Here, we determine whether host plant resistance mediates decreased pest populations in organic systems and identify potential underpinning mechanisms. We demonstrate that fewer numbers of leafhoppers (Circulifer tenellus) settle on tomatoes (Solanum lycopersicum) grown using organic management as compared to conventional. We present multiple lines of evidence, including rhizosphere soil microbiome sequencing, chemical analysis and transgenic approaches, to demonstrate that changes in leafhopper settling between organically and conventionally grown tomatoes are dependent on salicylic acid accumulation in plants and mediated by rhizosphere microbial communities. These results suggest that organically managed soils and microbial communities may play an unappreciated role in reducing plant attractiveness to pests by increasing plant resistance.

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.

Floral nectar is frequently colonized by fungi and bacteria. However, within individual flowers, nectar microbial communities are typically species-poor and dominated by few cosmopolitan genera. One hypothesis is that nectar constituents may act as a strong environmental filter. Non-sugar constituents in nectar could affect species composition via broad antimicrobial activity or differential effects on nectar microbial species. Here, we tested how five non-sugar nectar compounds as well as elevated sugar impacted the growth of 12 fungal and bacterial species isolated from flowers, pollinators, and the environment. We hypothesized that microbes isolated from nectar would be better able to grow in the presence of these compounds. Additionally, to test if nectar compounds could affect the outcome of competition among microbial taxa, we grew a subset of microbes in co-culture assays across a subset of treatments.We found that some compounds such as H2O2 broadly suppressed microbial growth across many but not all microbes tested. Other tested compounds were more specialized in the microbes they impacted. As hypothesized, the nectar specialist Metschnikowia reukaufii was unaffected by most nectar compounds assayed. However, many non-nectar specialist microbes remained unaffected by compounds thought to reduce microbial growth in nectar. Our results show that nectar chemistry can influence nectar microbial communities but that microbe-specific responses to nectar compounds are common. Nectar chemistry also affected the outcome of species interactions among microbial taxa, suggesting that non-sugar compounds in nectar can affect microbial community assembly and abundance in flowers.

Microorganisms within ectotherms must withstand the variable body temperatures of their hosts. Shifts in host body temperature resulting from climate change have the potential to shape ectotherm microbiome composition. Microbiome compositional changes occurring in response to temperature in nature have not been frequently examined, restricting our ability to predict microbe-mediated ectotherm responses to climate change. In a set of field-based observations, we characterized gut bacterial communities and thermal exposure across a population of desert arboreal ants (Cephalotes rohweri). In a paired growth chamber experiment, we exposed ant colonies to variable temperature regimes differing by 5°C for three months. We found that the abundance and composition of ant-associated bacteria were sensitive to elevated temperatures in both field and laboratory experiments. We observed a subset of taxa that responded similarly to temperature in the experimental and observational study, suggesting a role of seasonal temperature and local temperature differences amongst nests in shaping microbiomes within the ant population. Bacterial mutualists in the genus Cephaloticoccus (Opitutales: Opitutaceae) were especially sensitive to change in temperature—decreasing in abundance in naturally warm summer nests and warm growth chambers. We also report the discovery of a member of the Candidate Phlya Radiation (Phylum: Gracilibacteria), a suspected epibiont, found in low abundance within the guts of this ant species.

Microorganisms within ectotherms must withstand the variable body temperatures of their hosts. Shifts in host body temperature resulting from climate change have the potential to shape ectotherm microbiome composition. Microbiome compositional changes occurring in response to temperature in nature have not been frequently examined, restricting our ability to predict microbe-mediated ectotherm responses to climate change. In a set of field-based observations, we characterized gut bacterial communities and thermal exposure across a population of desert arboreal ants (Cephalotes rohweri). In a paired growth chamber experiment, we exposed ant colonies to variable temperature regimes differing by 5 °C for three months. We found that the abundance and composition of ant-associated bacteria were sensitive to elevated temperatures in both field and laboratory experiments. We observed a subset of taxa that responded similarly to temperature in the experimental and observational study, suggesting a role of seasonal temperature and local temperature differences amongst nests in shaping microbiomes within the ant population. Bacterial mutualists in the genus Cephalotococcus (Opitutales: Opitutaceae) were especially sensitive to change in temperature—decreasing in abundance in naturally warm summer nests and warm growth chambers. We also report the discovery of a member of the Candidate Phlya Radiation (Phylum: Gracilibacteria), a suspected epibiont, found in low abundance within the guts of this ant species.

Variation in dispersal ability among taxa affects community assembly and biodiversity maintenance within metacommunities. Although fungi and bacteria frequently coexist, their relative dispersal abilities are poorly understood. Nectar-inhabiting microbial communities affect plant reproduction and pollinator behavior, and are excellent models for studying dispersal of bacteria and fungi in a metacommunity framework. Here, we assay dispersal ability of common nectar bacteria and fungi in an insect-based dispersal experiment. We then compare these results with the incidence and abundance of culturable flower-inhabiting bacteria and fungi within naturally occurring flowers across two coflowering communities in California across two flowering seasons. Our microbial dispersal experiment demonstrates that bacteria disperse via thrips among artificial habitat patches more readily than fungi. In the field, incidence and abundance of culturable bacteria and fungi were positively correlated, but bacteria were much more widespread. These patterns suggest shared dispersal routes or habitat requirements among culturable bacteria and fungi, but differences in dispersal or colonization frequency by thrips, common flower visitors. The finding that culturable bacteria are more common among nectar sampled here, in part due to superior thrips-mediated dispersal, may have relevance for microbial life history, community assembly of microbes, and plant–pollinator interactions.

Monarch butterflies are one of the preeminent examples of a toxin-sequestering animal, gaining protection against predators via cardenolides obtained from their milkweed host plants. Although cardenolide sequestration by monarchs has been studied in ecological, physiological, and phylogenetic contexts, relatively little research has surveyed genetic variation in the ability to sequester, nor has monarch sequestration been studied in relation to divergent host plant assemblages or variation in exposure to predation. Here, we use the monarch9s recent global range expansion to test hypotheses about how cardenolide sequestration evolves over relatively contemporary time scales. First, we test for whether sympatric monarch/milkweed combinations have a sequestration advantage by rearing six geographically disparate monarch populations on six associated milkweed host species and measuring levels of sequestered cardenolides in a set of 440 adult butterflies. Second, we use monarchs from Guam - an oceanic island where birds have been functionally extirpated for approximately 40 years - to test hypotheses about how exposure to avian predation affects cardenolide sequestration. We find little overall evidence for increased sequestration on sympatric hosts. However, one monarch population (Puerto Rico) shows strong support for cross-host tradeoffs in sequestration ability, primarily driven by limited sequestration of polar cardenolides from two temperate North American milkweeds (Asclepias syriaca and A. speciosa). Monarchs from Guam show some evidence for reduced cardenolide sequestration in both a cross-island comparison of wild-caught butterflies as well as population-level comparisons of greenhouse-reared butterflies. Our results suggest that there is substantial genetic variation in sequestration ability (both within and between monarch populations) and that evolutionary history and contemporary species interactions may influence patterns of cardenolide sequestration.

Bumble bees are important pollinators that face threats from multiple sources, including agrochemical application. Declining bumble bee populations have been linked to fungicide application, which could directly affect the fungi often found in the stored food and GI tract of healthy bumble bees. Here, we test the hypothesis that fungicides impact bee health by disrupting bee-fungi interactions. We examine the interactive effects of the fungicide propiconazole and fungal supplementation on the survival, reproduction, and microbiome composition of microcolonies (queenless colonies) using two species,Bombus vosnesenskiiandB. impatiens. We found that both bee species benefitted from fungi, but were differentially affected by fungicides. InB. vosnesenskii, fungicide exposure decreased survival while fungal supplementation mitigated fungicide effects. ForB. impatiens, fungicide application had no effect, but fungal supplementation improved survival and offspring production. Fungicides reduced fungal abundance inB. vosnesenskiimicrocolonies, but not inB. impatiens, where instead fungal addition decreased fungal abundance. InB. vosnesenskii, the abundance of the pathogenAscosphaerawas negatively associated with survival, while the yeastZygosaccharomyceswas positively associated with survival. Our results highlight species-specific differences in response to fungicides and the nature of bee-fungi associations, and caution the use of results obtained using one species to predict responses of other species. These results demonstrate that fungicides can alter bee-fungi interactions with consequences for bee survival and reproduction, and suggest that exploring the mechanisms of such interactions, including interactions among fungi in the bee GI tract, may offer insights into bumble bee biology and conservation strategies.Significance statementWild bumble bee populations are declining globally, and a major predictor of these declines is agricultural fungicide application. We test the hypothesis that bee-associated fungi mediate fungicide effects on bees, examining how fungicide exposure and subsequent fungal supplementation impact bumble bee survival and microbiome. Fungal supplementation enhanced survival in both bumble bee species tested here, with fungi mediating effects of fungicide exposure in one species. Differences in bee survival were associated with changes in the gut mycobiome: negatively with the pathogenAscosphaeraand positively withZygosaccharomycesyeasts. This study highlights the importance of the mycobiome in bumble bee health both as a mechanism by which fungicides impact bumble bees and as an avenue of further research on beneficial bee-associated fungi.

Background and aim: Root-associated microbial communities influence plant phenotype, growth and local abundance, yet the factors that structure these microbial communities are still poorly understood. California landscapes contain serpentine soils, which are nutrient-poor and high in heavy metals, and distinct from neighboring soils making them ideal for studying the factors that structure root microbiomes and their functions. Method: Here, we surveyed the rhizoplane of serpentine-indifferent plants, which grow on and off serpentine soil, to determine the relative influence of plant identity and soil chemistry on rhizoplane microbial community structure using 16S rRNA metabarcoding. Additionally, we experimentally examined if serpentine vs. non-serpentine microorganisms differentially affected plant growth in serpentine soil. Results: Rhizoplane bacterial communities differed among plant species, soil types, and the interaction between them in both the field and experimental soils. In the experiment, soil microbial community source influenced seedling survival, but plant growth phenotypes were largely invariant to microbial community with a few exceptions. Conclusions: Rhizosplane bacterial species composition differed between plant species and soil types, and Amplicon Sequence Variants (ASVs) from the phyla Acidobacteria and Proteobacteria (Genus: Microvirga) were characteristic of serpentine soils. While soil microbial community composition influenced seedling survival in the current study, further study is required to disentangle the role of microbial associations and plant tolerance to serpentine.

Management of

Crop-associated microbiota are key factors 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, with organic and bio-based IPM best explaining 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 taxa-related turnover 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.In tree fruits, proper crop management during bloom is essential for producing disease-free 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, USA, where major production occurs in inter-mountain valleys and basins with variable elevation and microclimates. Our results show that both local- (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.

Serpentine soils are drought-prone and rich in heavy metals, and plants growing on serpentine soils host distinct microbial communities that may affect plant survival and phenotype. However, whether the rhizosphere communities of plants from different soil chemistries are initially distinct or diverge over time may help us understand drivers of microbial community structure and function in stressful soils. Here, we test the hypothesis that rhizosphere microbial communities will converge over time (plant development), independent of soil chemistry and microbial source. We grewPlantago erectain serpentine or nonserpentine soil, with serpentine or nonserpentine microbes and tracked plant growth and root phenotypes. We used 16S rRNA barcoding to compare bacterial species composition at seedling, vegetative, early-, and late-flowering phases. Plant phenotype and rhizosphere bacterial communities were mainly structured by soil type, with minor contributions by plant development, microbe source and their interactions. Serpentine microorganisms promoted early flowering in plants on non-serpentine soils. Despite strong effects of soil chemistry, the convergence in bacterial community composition across development demonstrates the importance of the plant-microbe interactions in shaping microbial assembly processes across soil types.

Summary Many organisms consume pollen, yet mechanisms of its digestion remain a fundamental enigma in pollination biology,1–3 as pollen is protected by a recalcitrant outer shell.4–8 Pollen is commonly found in floral nectar,9,10 as are nectar microbes, which are nearly ubiquitous among flowers.11–13 Nectar specialist bacteria, like Acinetobacter, can reach high densities (up to 109 cells/mL), despite the fact that floral nectar is nitrogen poor.14–17 Here, we show evidence that the genus Acinetobacter, prevalent nectar- and bee-associated bacteria,12,18–20 can induce pollen germination and bursting, gain access to protoplasm nutrients, and thereby grow to higher densities. Although induced germination had been suggested as a potential method in macroscopic pollen consumers,2,21–23 and fungal inhibition of pollen germination has been shown,24–27 direct biological induction of germination has not been empirically documented outside of plants.28–32 Acinetobacter pollinis SCC47719 induced over 5× greater pollen germination and 20× greater pollen bursting than that of uninoculated pollen by 45 min. When provided with germinable pollen, A. pollinis stimulates protein release and grows to nearly twice the density compared to growth with ungerminable pollen, indicating that stimulation of germination benefits bacterial fitness. In contrast, a common nectar-inhabiting yeast (Metschnikowia)33 neither induced nor benefited from pollen germination. We conclude that Acinetobacter both specifically causes and benefits from inducing pollen germination and bursting. Further study of microbe-pollen interactions may inform many aspects of pollination ecology, including floral microbial ecology,34,35 pollinator nutrient acquisition from pollen,2,3,21,36 and cues of pollen germination for plant reproduction.37–39

For many flower visitors, pollen is the primary source of non-carbon nutrition, but pollen has physical defenses that make it difficult for consumers to access nutrients. Nectar-dwelling microbes are nearly ubiquitous among flowers and can reach high densities, despite the fact that floral nectar is nitrogen limited, containing only very low concentrations of non-carbon nutrients. Pollen contains trace micronutrients and high protein content but is protected by a recalcitrant outer shell. Here, we report that a common genus of nectar-dwelling bacteria, Acinetobacter, exploits pollen nutrition by inducing pollen germination and bursting. We use time course germination assays to quantify the effect of Acinetobacter species on pollen germination and pollen bursting. Inoculation with Acinetobacter species resulted in increased germination rates within 15 minutes, and bursting by 45 minutes, as compared to uninoculated pollen. The pollen germination and bursting phenotype is density-dependent, with lower concentrations of A. pollinis SCC477 resulting in a longer lag time before the spike in germination, which is then closely followed by a spike in bursting. Lastly, A. pollinis grows to nearly twice the density with germinable pollen vs ungerminable pollen, indicating that their ability to induce and exploit germination plays an important role in rapid growth. To our knowledge, this is the first direct test of non-plant biological induction of pollen germination, as well as the first evidence of induced germination as a method of nutrient procurement, as the microbes appear to hijack the pollen’s normally tightly controlled germination mechanisms for their benefit. Our results suggest that further study of microbe-pollen interactions may inform many aspects of pollination ecology, including microbial ecology in flowers, the mechanisms of pollinator nutrient acquisition from pollen, and cues of pollen germination for plant reproduction.

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 ( 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).

Crop-associated microbiota are key factors 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, with organic and bio-based IPM best explaining 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 taxa-related turnover 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.IMPORTANCEIn tree fruits, proper crop management during bloom is essential for producing disease-free 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, USA, where major production occurs in inter-mountain valleys and basins with variable elevation and microclimates. Our results show that both local (crop management) and landscape (habitat types and climate) level factors affect floral microbiota, but in disparate ways for each kingdom, suggesting a need for unique management strategies for each group. More broadly, these findings can potentially inform microbiome management in orchards for promotion of host health and high-quality yields.

Crop tissues harbor microbiomes that can affect host health and yield. However, processes driving microbiome assembly, and resulting effects on ecosystem services, remain poorly understood. This is particularly true of flowering crops that rely on pollinators for yield.We assessed effects of orchard management tactics and landscape context on the flower microbiome in almond, Prunus dulcis. Fourteen orchards (5 conventional, 4 organic, 5 habitat augmentation) were sampled at two bloom stages to characterize bacterial and fungal communities associated with floral tissues. The surveys were complemented by in vitro experiments to assess effects of arrival order and fungicides on nectar microbial communities, and effects of fungicides and microbes on honey bee foraging. Finally, a field trial was conducted to test effects of fungicides and microbes on pollination.As bloom progressed, bacterial and fungal abundance and diversity increased, across all floral tissue types and management strategies. The magnitude by which microbial abundance and diversity were affected varied, with host proximity to apiaries and orchard management having notable effects on bacteria and fungi, respectively.Experiments showed immigration history and fungicides affected the composition of nectar microbial communities, but only fungicides affected pollinator foraging through reduced nectar removal. Neither treatment affected pollination services.Synthesis and applications. Our results shed light on routes through which management practices can shape microbiota associated with flowers of a pollinator-dependent crop. With growing appreciation for the role of floral-associated microbes in affecting biotic interactions at the floral interface, understanding such drivers can potentially inform microbial-derived ecosystem services in agricultural landscapes, including pollination and biocontrol.

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Flowers provide resources for pollinators, and can also be transmission venues for beneficial or pathogenic pollinator-associated microbes. Floral traits could mediate transmission similarly for beneficial and pathogenic microbes, although some beneficial microbes can grow in flowers while pathogenic microbes may only survive until acquired by a new host. In spite of conceptual similarities, research on beneficial and pathogenic pollinator-associated microbes has progressed mostly independently. Recent advances demonstrate that floral traits are associated with transmission of beneficial and pathogenic microbes, with consequences for pollinator populations and communities. However, there is a near-absence of experimental manipulations of floral traits to determine causal effects on transmission, and a need to understand how floral, microbe and host traits interact to mediate transmission.

Many plant-associated microbial communities produce volatile signals that influence insect responses, yet the impact of floral microorganisms has received less attention than other plant microbiomes. Floral microorganisms alter plant and floral odors by adding their own emissions or modifying plant volatiles. These contextual and microbe species-specific changes in floral signaling are detectable by insects and can modify their behavior. Opportunities for future work in floral systems include identifying specific microbial semiochemicals that underlie insect behavioral responses and examining if insect species vary in their responses to microbial volatiles. Examining if documented patterns are consistent across diverse plant–microbe–insect interactions and in realistic plant-based studies will improve our understanding of how microbes mediate pollination interactions in complex system.

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.

Variation in dispersal ability among taxa affects community assembly and biodiversity maintenance within metacommunities. Although fungi and bacteria frequently coexist, their relative dispersal abilities are poorly understood. Nectar-inhabiting microbial communities affect plant reproduction and pollinator behavior, and are excellent models for studying dispersal of bacteria and fungi in a metacommunity framework. Here, we assay dispersal ability of common nectar bacteria and fungi in an insect-based dispersal experiment. We then compare these results to the incidence and abundance of culturable flower-inhabiting bacteria and fungi within naturally occurring flowers across two coflowering communities in California across two flowering seasons. Our microbial dispersal experiment demonstrates that bacteria disperse among habitat patches more readily than fungi via thrips. Across all flowers, bacterial and fungal incidence and abundance were positively correlated but bacteria were much more widespread, suggesting shared dispersal routes or habitat requirements but differences in dispersal and colonization frequency. The finding that bacteria are more common among flowers sampled here, in part due to superior insect-mediated dispersal, may have broad relevance for microbial life-history, community assembly of microbes and plant-pollinator interactions.

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.

Author(s): Toju, H; Vannette, RL; Gauthier, MPL; Dhami, MK; Fukami, T | Abstract: The order of species arrival can influence how species interact with one another and, consequently, which species may coexist in local communities. This phenomenon, called priority effects, has been observed in various types of communities, but it remains unclear whether priority effects persist over the long term spanning multiple generations of local communities in metacommunities. Focusing on bacteria and yeasts that colonize floral nectar of the sticky monkey flower, Mimulus aurantiacus, via hummingbirds and other flower-visiting animals, we experimentally manipulated initial microbial dominance on plants (regarded as metacommunities) to examine whether its effects persisted across multiple generations of flowers (regarded as local microbial habitats). The experimental introduction of Neokomagataea (= Gluconobacter) bacteria and Metschnikowia yeasts into wild flowers showed that the effects of initial dominance were observable across multiple floral generations. Three weeks after introduction, corresponding approximately to three floral generations, Neokomagataea introduction led to exclusion of yeasts, whereas Metschnikowia introduction did not result in the exclusion of Neokomagataea. Our results suggest that, even when local habitats are ephemeral, priority effects may influence multiple generations of local communities within metacommunities.

Lower insect pest populations found on long-term organic farms have largely been attributed to increased biodiversity and abundance of beneficial predators. However, potential induction of plant defenses has largely been ignored. This study aims to determine whether host plant resistance mediates decreased pest populations in organic systems, and to identify the underpinning mechanisms. We demonstrate that greater numbers of leafhoppers (Circulifer tenellus) settle on tomatoes (Solanum lycopersicum) grown using conventional management as compared to organic. Soil microbiome sequencing, chemical analysis, and transgenic approaches, coupled with multi-model inference, suggest that changes in leafhopper settling between organically and conventionally-grown tomatoes are dependent on salicylic acid accumulation in the plant, likely mediated by rhizosphere microbial communities. These results suggest that organically-managed soils and microbial communities may play an unappreciated role in reducing plant attractiveness to pests by increasing plant resistance.

Reduced insect pest populations found on long-term organic farms have mostly been attributed to increased biodiversity and abundance of beneficial predators, as well as to changes in plant nutrient content. However, the role of plant resistance has largely been ignored. Here, we determine whether host plant resistance mediates decreased pest populations in organic systems and identify potential underpinning mechanisms. We demonstrate that fewer numbers of leafhoppers (Circulifer tenellus) settle on tomatoes (Solanum lycopersicum) grown using organic management as compared to conventional. We present multiple lines of evidence, including rhizosphere soil microbiome sequencing, chemical analysis and transgenic approaches, to demonstrate that changes in leafhopper settling between organically and conventionally grown tomatoes are dependent on salicylic acid accumulation in plants and mediated by rhizosphere microbial communities. These results suggest that organically managed soils and microbial communities may play an unappreciated role in reducing plant attractiveness to pests by increasing plant resistance.

Microbial dispersal is essential for establishment in new habitats, but the role of vector identity is poorly understood in community assembly and function. Here, we compared microbial assembly and function in floral nectar visited by legitimate pollinators (hummingbirds) and nectar robbers (carpenter bees). We assessed effects of visitation on the abundance and composition of culturable bacteria and fungi and their taxonomy and function using shotgun metagenomics and nectar chemistry. We also compared metagenome-assembled genomes (MAGs) of Acinetobacter, a common and highly abundant nectar bacterium, among visitor treatments. Visitation increased microbial abundance, but robbing resulted in 10× higher microbial abundance than pollination. Microbial communities differed among visitor treatments: robbed flowers were characterized by predominant nectar specialists within Acetobacteraceae and Metschnikowiaceae, with a concurrent loss of rare taxa, and these resulting communities harbored genes relating to osmotic stress, saccharide metabolism and specialized transporters. Gene differences were mirrored in function: robbed nectar contained a higher percentage of monosaccharides. Draft genomes of Acinetobacter revealed distinct amino acid and saccharide utilization pathways in strains isolated from robbed versus pollinated flowers. Our results suggest an unrecognized cost of nectar robbing for pollination and distinct effects of visitor type on interactions between plants and pollinators. Overall, these results suggest vector identity is an underappreciated factor structuring microbial community assembly and function.

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 NO(3)-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.

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.

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.

Nectar mediates interactions between plants and pollinators in natural and agricultural systems. Specialized microorganisms are common nectar inhabitants, and potentially important mediators of plant-pollinator interactions. However, their diversity and role in mediating pollination services in agricultural systems are poorly characterized. Moreover, agrochemicals are commonly applied to minimize crop damage, but may present ecological consequences for non-target organisms. Assessment of ecological risk has tended to focus on beneficial macroorganisms such as pollinators, with less attention paid to microorganisms. Here, using culture-independent methods, we assess the impact of two widely-used fungicides on nectar microbial community structure in the mass-flowering crop almond (Prunus dulcis). We predicted that fungicide application would reduce fungal richness and diversity, whereas competing bacterial richness would increase, benefitting from negative effects on fungi. We found that fungicides reduced fungal richness and diversity in exposed flowers, but did not significantly affect bacterial richness, diversity, or community composition. The relative abundance of Metschnikowia OTUs, nectar specialists that can impact pollination, was reduced by both fungicides. Given growing recognition of the importance of nectar microorganisms as mediators of plant-pollinator mutualisms, future research should consider the impact of management practices on plant-associated microorganisms and consequences for pollination services in agricultural landscapes.

Insect pests cause serious economic, yield, and food safety problems to managed crops worldwide. Compounding these problems, insect pests often vector pathogenic or toxigenic microbes to plants. Previous work has considered plant-insect and plant-microbe interactions separately. Although insects are well-understood to use plant volatiles to locate hosts, microorganisms can produce distinct and abundant volatile compounds that in some cases strongly attract insects. In this paper, we focus on the microbial contribution to plant volatile blends, highlighting the compounds emitted and the potential for variation in microbial emission. We suggest that these aspects of microbial volatile emission may make these compounds ideal for use in agricultural applications, as they may be more specific or enhance methods currently used in insect control or monitoring. Our survey of microbial volatiles in insect-plant interactions suggests that these emissions not only signal host suitability but may indicate a distinctive time frame for optimal conditions for both insect and microbe. Exploitation of these host-specific microbe semiochemicals may provide important microbe- and host-based attractants and a basis for future plant-insect-microbe chemical ecology investigations.

Animals such as bumble bees use chemosensory cues to localize and evaluate essential resources. Increasingly, it is recognized that microbes can alter the quality of foraged resources and produce metabolites that act as foraging cues. The distinct nature of these sensory 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 in nectar. We examined electrophysiological potential of bumble bee antennae to respond to volatile microbial metabolites, followed by behavioral responses using choice assays. We assessed gustatory responses through both no-choice and choice feeding assays. Antennae responded to some microbial volatiles, and bees chose Asaia bacterial solutions compared to Metschnikowia yeast based on volatiles alone. However, B. impatiens consumed significantly more Metschnikowia-inoculated nectar, suggesting distinct roles for volatile and non-volatile microbial metabolites in mediating feeding decisions, with potential to affect associative learning and future foraging. Our results suggest that microbial metabolites may represent non-reinforcing cues with potential consequences for forager learning, economics and floral host reproduction.

Root-associated microbial communities influence plant phenotype, growth and local abundance, yet the factors that structure these microbial communities are still poorly understood. California landscapes contain serpentine soils, which are nutrient-poor and high in heavy metals, and distinct from neighboring soils. Here, we surveyed the rhizoplane of serpentine-indifferent plants species growing on serpentine and non-serpentine soils to determine the relative influence of plant identity and soil chemistry on rhizoplane microbial community structure using 16S rRNA metabarcoding. Additionally, we experimentally examined if locally adapted microorganisms enhance plant growth in serpentine soil. Plant species, soil chemistry, and the interaction between them were important in structuring rhizoplane bacterial communities in both the field and experimental soils. In the experiment, rhizoplane microbial community source influenced seedling survival, but plant growth phenotypes measured were largely invariant to microbial community with a few exceptions. Results from the field sampling suggest that plant species associate with specific microbial communities even across chemically distinct soils, and that microbial communities can differentially influence seedling survival on harsh serpentine soils.