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Abstract Understanding the interactions between plants and microorganisms can inform microbiome management to enhance crop productivity and resilience to stress. Here, we apply a genome-centric approach to identify ecologically important leaf microbiome members on replicated plots of field-grown switchgrass and miscanthus, and to quantify their activities over two growing seasons for switchgrass. We use metagenome and metatranscriptome sequencing and curate 40 medium- and high-quality metagenome-assembled-genomes (MAGs). We find that classes represented by these MAGs (Actinomycetia, Alpha- and Gamma- Proteobacteria, and Bacteroidota) are active in the late season, and upregulate transcripts for short-chain dehydrogenase, molybdopterin oxidoreductase, and polyketide cyclase. Stress-associated pathways are expressed for most MAGs, suggesting engagement with the host environment. We also detect seasonally activated biosynthetic pathways for terpenes and various non-ribosomal peptide pathways that are poorly annotated. Our findings support that leaf-associated bacterial populations are seasonally dynamic and responsive to host cues. 

Phyllosphere exudates create specialized microhabitats that shape microbial community diversity. We explored the microbiome associated with two sorghum phyllosphere exudates, the epicuticular wax and aerial root mucilage. We assessed the microbiome associated with the wax from sorghum plants over two growth stages, and the root mucilage additionally from nitrogen-fertilized and nonfertilized plants. In parallel, we isolated and characterized hundreds of bacteria from wax and mucilage, and integrated data from cultivation-independent and cultivation-dependent approaches to gain insights into exudate diversity and bacterial phenotypes. We found that Sphingomonadaceae and Rhizobiaceae families were the major taxa in the wax regardless of water availability and plant developmental stage to plants. The cultivation-independent mucilage-associated bacterial microbiome contained the families Erwiniaceae, Flavobacteriaceae, Rhizobiaceae, Pseudomonadaceae, and Sphingomonadaceae, and its structure was strongly influenced by sorghum development but only modestly influenced by fertilization. In contrast, the fungal community structure of mucilage was strongly affected by the year of sampling but not by fertilization or plant developmental stage, suggesting a decoupling of fungal–bacterial dynamics in the mucilage. Our bacterial isolate collection from wax and mucilage had several isolates that matched 100% to detected amplicon sequence variants, and were enriched on media that selected for phenotypes that included phosphate solubilization, putative diazotrophy, resistance to desiccation, capability to grow on methanol as a carbon source, and ability to grow in the presence of linalool and β-caryophyllene (terpenes in sorghum wax). This work expands our understanding of the microbiome of phyllosphere exudates and supports our long-term goal to translate microbiome research to support sorghum cultivation. 

Terpenes are among the oldest and largest class of plant-specialized bioproducts that are known to affect plant development, adaptation, and biological interactions. While their biosynthesis, evolution, and function in aboveground interactions with insects and individual microbial species are well studied, how different terpenes impact plant microbiomes belowground is much less understood. Here we designed an experiment to assess how belowground exogenous applications of monoterpenes (1,8-cineole and linalool) and a sesquiterpene (nerolidol) delivered through an artificial root system impacted its belowground bacterial and fungal microbiome. We found that the terpene applications had significant and variable impacts on bacterial and fungal communities, depending on terpene class and concentration; however, these impacts were localized to the artificial root system and the fungal rhizosphere. We complemented this experiment with pure culture bioassays on responsive bacteria and fungi isolated from the sorghum rhizobiome. Overall, higher concentrations (200 µM) of nerolidol were inhibitory toFerrovibriumand tested Firmicutes. While fungal isolates ofPenicilliumandPericoniawere also more inhibited by higher concentrations (200 µM) of nerolidol,Clonostachyswas enhanced at this higher level and together withHumicolawas inhibited by the lower concentration tested (100 µM). On the other hand, 1,8-cineole had an inhibitory effect onOrbiliaat both tested concentrations but had a promotive effect at 100 µM onPenicilliumandPericonia. Similarly, linalool at 100 µM had significant growth promotion inMortierella, but an inhibitory effect forOrbilia. Together, these results highlight the variable direct effects of terpenes on single microbial isolates and demonstrate the complexity of microbe-terpene interactions in the rhizobiome.

Terpenes represent one of the largest and oldest classes of plant-specialized metabolism, but their role in the belowground microbiome is poorly understood. Here, we used a “rhizobox” mesocosm experimental set-up to supply different concentrations and classes of terpenes into the soil compartment with growing sorghum for 1 month to assess how these terpenes affect sorghum bacterial and fungal rhizobiome communities. Changes in bacterial and fungal communities between treatments belowground were characterized, followed by bioassays screening on bacterial and fungal isolates from the sorghum rhizosphere against terpenes to validate direct microbial responses. We found that microbial growth stimulatory and inhibitory effects were localized, terpene specific, dose dependent, and transient in time. This work paves the way for engineering terpene metabolisms in plant microbiomes for improved sustainable agriculture and bioenergy crop production.

 

One interference mechanism of bacterial competition is the production of antibiotics. Bacteria exposed to antibiotics can resist antibiotic inhibition through intrinsic or acquired mechanisms. Here, we performed a coevolution experiment to understand the long-term consequences of antibiotic production and antibiotic susceptibility for two environmental bacterial strains. We grew five independent lines of the antibiotic-producing environmental strain, Burkholderia thailandensis E264, and the antibiotic-inhibited environmental strain, Flavobacterium johnsoniae UW101, together and separately on agar plates for 7.5 months (1.5 month incubations), transferring each line five times to new agar plates. We observed that the F. johnsoniae ancestor could tolerate the B. thailandensis -produced antibiotic through efflux mechanisms, but that the coevolved lines had reduced susceptibility. We then sequenced genomes from the coevolved and monoculture F. johnsoniae lines, and uncovered mutational ramifications for the long-term antibiotic exposure. The coevolved genomes from F. johnsoniae revealed four potential mutational signatures of reduced antibiotic susceptibility that were not observed in the evolved monoculture lines. Two mutations were found in tolC : one corresponding to a 33 bp deletion and the other corresponding to a nonsynonymous mutation. A third mutation was observed as a 1 bp insertion coding for a RagB/SusD nutrient uptake protein. The last mutation was a G83R nonsynonymous mutation in acetyl-coA carboxylayse carboxyltransferase subunit alpha (AccA). Deleting the 33 bp from tolC in the F. johnsoniae ancestor reduced antibiotic susceptibility, but not to the degree observed in coevolved lines. Furthermore, the accA mutation matched a previously described mutation conferring resistance to B. thailandensis -produced thailandamide. Analysis of B. thailandensis transposon mutants for thailandamide production revealed that thailandamide was bioactive against F. johnsoniae, but also suggested that additional B. thailandensis -produced antibiotics were involved in the inhibition of F. johnsoniae . This study reveals how multi-generational interspecies interactions, mediated through chemical exchange, can result in novel interaction-specific mutations, some of which may contribute to reductions in antibiotic susceptibility. 

ABSTRACT Microbial exponential growth is expected to occur infrequently in environments that have long periods of nutrient starvation punctuated by short periods of high nutrient flux. These conditions likely impose nongrowth states for microbes. However, nongrowth states are uncharacterized for the majority of environmental bacteria, especially in regard to exometabolite production. We compared exometabolites produced over stationary phase across three environmental bacteria: Burkholderia thailandensis E264 (ATCC 700388), Chromobacterium violaceum ATCC 31532, and Pseudomonas syringae pv. tomato DC3000 (ATCC BAA-871). We grew each strain in monoculture and investigated exometabolite dynamics from mid-exponential to stationary phases. We focused on exometabolites that were released into the medium and accumulated over 45 h, including approximately 20 h of stationary phase. We also analyzed transcripts (transcriptome sequencing [RNA-seq]) to interpret exometabolite output. We found that the majority of exometabolites released were strain specific, with a subset of identified exometabolites involved in both central and secondary metabolism. Transcript analysis supported that exometabolites were released from intact cells, as various transporters had either increased or consistent transcripts through time. Interestingly, we found that succinate was one of the most abundant identifiable exometabolites for all strains and that each strain rerouted their metabolic pathways involved in succinate production during stationary phase. These results show that nongrowth states can be metabolically dynamic and that environmental bacteria can enrich a minimal environment with diverse chemical compounds as a consequence of growth and postgrowth maintenance in stationary phase. This work provides insights into microbial community interactions via exometabolites under conditions of growth cessation or limitation. IMPORTANCE Nongrowth states are common for bacteria that live in environments that are densely populated and predominantly nutrient exhausted, and yet these states remain largely uncharacterized in cellular metabolism and metabolite output. Here, we investigated and compared stationary-phase exometabolites and RNA transcripts for each of three environmental bacterial strains. We observed that diverse exometabolites were produced and provide evidence that these exometabolites accumulate over time through release by intact cells. Additionally, each bacterial strain had a characteristic exometabolite profile and exhibited dynamics in exometabolite composition. This work affirms that stationary phase is metabolically dynamic, with each strain tested creating a unique chemical signature in the extracellular space and altering metabolism in stationary phase. These findings set the stage for understanding how bacterial populations can support surrounding neighbors in environments with prolonged nutrient exhaustion through exometabolite-mediated interspecies interactions. 

Plant leaves harbor complex microbial communities that influence plant health and productivity. Nevertheless, a detailed understanding of phyllosphere community assembly and drivers is needed, particularly for phyllosphere fungi. Here, we investigated seasonal dynamics of epiphytic phyllosphere fungal communities in switchgrass (Panicum virgatum L.), a focal bioenergy crop. We also leverage previously published data on switchgrass phyllosphere bacterial communities from the same experimental plants, allowing us to compare fungal and bacterial dynamics and explore interdomain network associations in the switchgrass phyllosphere. Overall, we found a strong impact of sampling date on fungal community composition, with multiple taxonomic levels exhibiting clear temporal patterns in relative abundance. In addition, leaf nitrogen concentration, leaf dry matter content, plant height, and minimum daily air temperature explained significant variation in phyllosphere fungal communities, likely due to their correlation with sampling date. Finally, among the core taxa, fungi–bacteria network associations were much more common than bacteria–bacteria associations, suggesting the importance of interdomain phylogenetic diversity in microbiome assembly. Although our findings highlight the complexity of phyllosphere microbiome assembly, the clear temporal patterns in lineage-specific fungal abundances give promise to the potential for accurately predicting shifts in fungal phyllosphere communities throughout the growing season, a key research priority for sustainable agriculture. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license . 

The spread of an enteric pathogen in the human gut depends on many interacting factors, including pathogen exposure, diet, host gut environment, and host microbiota, but how these factors jointly influence infection outcomes remains poorly characterized. Here, we develop a model of host-mediated resource-competition between mutualistic and pathogenic taxa in the gut that aims to explain why similar hosts, exposed to the same pathogen, can have such different infection outcomes. Our model successfully reproduces several empirically observed phenomena related to transitions between healthy and infected states, including (1) the nonlinear relationship between pathogen inoculum size and infection persistence, (2) the elevated risk of chronic infection during or after treatment with broad-spectrum antibiotics, (3) the resolution of gut dysbiosis with fecal microbiota transplants, and (4) the potential protection from infection conferred by probiotics. We then use the model to explore how host-mediated interventions, namely shifts in the supply rates of electron donors (e.g., dietary fiber) and respiratory electron acceptors (e.g., oxygen), can potentially be used to direct gut community assembly. Our study demonstrates how resource competition and ecological feedbacks between the host and the gut microbiota can be critical determinants of human health outcomes. We identify several testable model predictions ready for experimental validation. 

In disturbance ecology, stability is composed of resistance to change and resilience towards recovery after the disturbance subsides. Two key microbial mechanisms that can support microbiome stability include dormancy and dispersal. Specifically, microbial populations that are sensitive to disturbance can be re-seeded by local dormant pools of viable and reactivated cells, or by immigrants dispersed from regional metacommunities. However, it is difficult to quantify the contributions of these mechanisms to stability without, first, distinguishing the active from inactive membership, and, second, distinguishing the populations recovered by local resuscitation from those recovered by dispersed immigrants. Here, we investigate the contributions of dormancy dynamics (activation and inactivation), and dispersal to soil microbial community resistance and resilience. We designed a replicated, 45-week time-series experiment to quantify the responses of the active soil microbial community to a thermal press disturbance, including unwarmed control mesocosms, disturbed mesocosms without dispersal, and disturbed mesocosms with dispersal after the release of the stressor. Communities changed in structure within one week of warming. Though the disturbed mesocosms did not fully recover within 29 weeks, resuscitation of thermotolerant taxa was key for community transition during the press, and both resuscitation of opportunistic taxa and immigration contributed to community resilience. Also, mesocosms with dispersal were more resilient than mesocosms without. This work advances the mechanistic understanding of how microbiomes respond to disturbances in their environment. This article is part of the theme issue ‘Conceptual challenges in microbial community ecology’.