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Abstract BackgroundDespite being among the most abundant biological entities on earth, bacteriophage (phage) remain an understudied component of host-associated systems. One limitation to studying host-associated phage is the lack of consensus on methods for sampling phage communities. Here, we compare paired total metagenomes and viral size fraction metagenomes (viromes) as methods for investigating the dsDNA viral communities associated with the GI tract of two bee species: the European honey beeApis melliferaand the eastern bumble beeBombus impatiens. ResultsWe find that viromes successfully enriched for phage, thereby increasing phage recovery, but only in honey bees. In contrast, for bumble bees, total metagenomes recovered greater phage diversity. Across both bee species, viromes better sampled low occupancy phage, while total metagenomes were biased towards sampling temperate phage. Additionally, many of the phage captured by total metagenomes were absent altogether from viromes. Comparing between bees, we show that phage communities in commercially reared bumble bees are significantly reduced in diversity compared to honey bees, likely reflecting differences in bacterial titer and diversity. In a broader context, these results highlight the complementary nature of total metagenomes and targeted viromes, especially when applied to host-associated environments. ConclusionsOverall, we suggest that studies interested in assessing total communities of host-associated phage should consider using both approaches. However, given the constraints of virome sampling, total metagenomes may serve to sample phage communities with the understanding that they will preferentially sample dominant and temperate phage. 

Summary Flower-sourced assembly of seed microbiota remains an understudied ecological process. Here, we investigated the floral transmission pathway for bacterial acquisition by developing seeds of watermelon (Citrullus lanatus). Comparison of stigma- and seed-associated bacterial communities from field-grownC. lanatusrevealed significant overlap: up to 40% of the bacterial diversity that was detected in seed was also found on stigmas. In a field pollinator exclusion experiment, honeybee visitation to flower stigmas had no significant effect on bacterial community composition in seeds. Among a collection of bacterial isolates from stigmas and seeds in the field, more than half (57%) were able to transmit to seeds after inoculation onto stigmas under laboratory conditions. Interestingly, for most bacterial strains, fruit set rates increased after floral inoculation, and in some cases even in the absence of transmission to the seed. We also found that bacterial isolates from watermelon stigmas and seeds had variable (i.e. positive or negative) effects on seed germination and seedling emergence. Our findings highlight the contribution of floral transmission to seed microbiota assembly and its consequences for plant fitness. 

ABSTRACT Plant–microbe associations are ubiquitous, but parsing contributions of dispersal, host filtering, competition and temperature on microbial community composition is challenging. Floral nectar‐inhabiting microbes, which can influence flowering plant health and pollination, offer a tractable system to disentangle community assembly processes. We inoculated a synthetic community of yeasts and bacteria into nectars of 31 plant species while excluding pollinators. We monitored weather and, after 24 h, collected and cultured communities. We found a strong signature of plant species on resulting microbial abundance and community composition, in part explained by plant phylogeny and nectar peroxide content, but not floral morphology. Increasing temperature reduced microbial diversity, while higher minimum temperatures increased growth, suggesting complex ecological effects of temperature. Consistent nectar microbial communities within plant species could enable plant or pollinator adaptation. Our work supports the roles of host identity, traits and temperature in microbial community assembly, and indicates diversity–productivity relationships within host‐associated microbiomes. 

Abstract 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. 

Summary 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 ofEpilobium 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. 

Abstract Floral nectar is frequently colonised by microbes. However, nectar microbial communities are typically species‐poor and dominated by few cosmopolitan genera. One hypothesis is that nectar constituents may act as environmental filters. We tested how five non‐sugar nectar compounds as well as elevated sugar impacted the growth of 12 fungal and bacterial species isolated from nectar, pollinators, and the environment. We hypothesised that nectar isolated microbes would have the least growth suppression. Additionally, to test if nectar compounds could affect the outcome of competition between microbes, we grew a subset of microbes in co‐culture across a subset of treatments. We found that some compounds such as H₂O₂suppressed microbial growth across many but not all microbes tested. Other compounds were more specialised in the microbes they impacted. As hypothesised, the nectar specialist yeastMetschnikowia reukaufiiwas unaffected by most nectar compounds assayed. However, many non‐nectar specialist microbes remained unaffected by nectar compounds thought to reduce microbial growth. Our results show that nectar chemistry can influence 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 can affect microbial community assembly in flowers. 

ABSTRACT 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. 

Wild pollinator declines are increasingly linked to pesticide exposure, yet it is unclear how intraspecific differences contribute to observed variation in sensitivity, and the role gut microbes play in the sensitivity of wild bees is largely unexplored. Here, we investigate site-level differences in survival and microbiome structure of a wild bumble bee exposed to multiple pesticides, both individually and in combination. We collected wildBombus vosnesenskiiforagers (N= 175) from an alpine meadow, a valley lake shoreline and a suburban park and maintained them on a diet containing a herbicide (glyphosate), a fungicide (tebuconazole), an insecticide (imidacloprid) or a combination of these chemicals. Alpine bees had the highest overall survival, followed by shoreline bees then suburban bees. This was in part explained by body size differences across sites and the presence of conopid parasitoids at two of the sites. Notably, site of origin impacted bee survival on the herbicide, fungicide and combination treatment. We did not find evidence of gut microbiome differences across pesticide treatment, nor a site-by-treatment interaction. Regardless, the survival differences we observed emphasize the importance of considering population of origin when studying pesticide toxicity of wild bees. 

Nectar contains antimicrobial constituents including hydrogen peroxide, yet it is unclear how widespread nectar hydrogen peroxide might be among plant species or how effective it is against common nectar microbes.Here, we surveyed 45 flowering plant species across 23 families and reviewed the literature to assess the field‐realistic range of nectar hydrogen peroxide (Aim 1). We experimentally explored whether plant defense hormones increase nectar hydrogen peroxide (Aim 2). Further, we tested the hypotheses that variation in microbial tolerance to peroxide is predicted by the microbe isolation environment (Aim 3); increasing hydrogen peroxide in flowers alters microbial abundance and community assembly (Aim 4), and that the microbial community context affects microbial tolerance to peroxide (Aim 5).Peroxide in sampled plants ranged from undetectable toc3000 μM, with 50% of species containing less than 100 μM. Plant defensive hormones did not affect hydrogen peroxide in floral nectar, but enzymatically upregulated hydrogen peroxide significantly reduced microbial growth.Together, our results suggest that nectar peroxide is a common but not pervasive antimicrobial defense among nectar‐producing plants. Microbes vary in tolerance and detoxification ability, and co‐growth can facilitate the survival and growth of less tolerant species, suggesting a key role for community dynamics in the microbial colonization of nectar. 

Fire blight is a devastating disease affecting pome fruit trees that is caused by Erwinia amylovora and leads to substantial annual losses worldwide. While antibiotic-based management approaches like streptomycin can be effective, there are concerns over evolved resistance of the pathogen and non-target effects on beneficial microbes and insects. Using microbial biological control agents (mBCAs) to combat fire blight has promise, but variable performance necessitates the discovery of more effective solutions. Here we used a niche-based predictive framework to assess the strength of priority effects exerted by prospective mBCAs, and the mechanisms behind growth suppression in floral nectar. Through in vitro and in vivo assays, we show that antagonist impacts on nectar pH and sucrose concentration were the primary predictors of priority effects. Surprisingly, overlap in amino acid use, and the degree of phylogenetic relatedness between mBCA and Erwinia did not significantly predict pathogen suppression in vitro, suggesting that competition for limited shared resources played a lesser role than alterations in the chemical environment created by the initial colonizing species. We also failed to detect an association between our measures of in vitro and in vivo Erwinia suppression, suggesting other mechanisms may dictate mBCA establishment and efficacy in flowers, including priming of host defenses.