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Agriculture faces many emerging challenges to sustainability, including limited nutrient resources, losses from diseases caused by current and emerging pathogens and environmental degradation. Microorganisms have great importance for plant growth and performance, including the potential to increase yields, nutrient uptake and pathogen resistance. An urgent need is therefore to understand and engineer plants and their associated microbial communities. Recent massive genomic sequencing of host plants and associated microbes offers resources to identify novel mechanisms of communal assembly mediated by the host. For example, host–microbe and microbe–microbe interactions are involved in niche formation, thereby contributing to colonization. By leveraging genomic resources, genetic traits underlying those mechanisms will become important resources to design plants selecting and hosting beneficial microbial communities. [ABSTRACT FROM AUTHOR]

Plant-associated microorganisms have been shown to critically affect host physiology and performance, suggesting that evolution and ecology of plants and animals can only be understood in a holobiont (host and its associated organisms) context. Host-associated microbial community structures are affected by abiotic and host factors, and increased attention is given to the role of the microbiome in interactions such as pathogen inhibition. However, little is known about how these factors act on the microbial community, and especially what role microbe–microbe interaction dynamics play. We have begun to address this knowledge gap for phyllosphere microbiomes of plants by simultaneously studying three major groups of Arabidopsis thaliana symbionts (bacteria, fungi and oomycetes) using a systems biology approach. We evaluated multiple potential factors of microbial community control: we sampled various wild A. thaliana populations at different times, performed field plantings with different host genotypes, and implemented successive host colonization experiments under lab conditions where abiotic factors, host genotype, and pathogen colonization was manipulated. Our results indicate that both abiotic factors and host genotype interact to affect plant colonization by all three groups of microbes. Considering microbe–microbe interactions, however, uncovered a network of interkingdom interactions with significant contributions to community structure. As in other scale-free networks, a small number of taxa, which we call microbial “hubs,” are strongly interconnected and have a severe effect on communities. By documenting these microbe–microbe interactions, we uncover an important mechanism explaining how abiotic factors and host genotypic signatures control microbial communities. In short, they act directly on “hub” microbes, which, via microbe–microbe interactions, transmit the effects to the microbial community. We analyzed two “hub” microbes (the obligate biotrophic oomycete pathogen Albugo and the basidiomycete yeast fungus Dioszegia) more closely. Albugo had strong effects on epiphytic and endophytic bacterial colonization. Specifically, alpha diversity decreased and beta diversity stabilized in the presence of Albugo infection, whereas they otherwise varied between plants. Dioszegia, on the other hand, provided evidence for direct hub interaction with phyllosphere bacteria. The identification of microbial “hubs” and their importance in phyllosphere microbiome structuring has crucial implications for plant–pathogen and microbe–microbe research and opens new entry points for ecosystem management and future targeted biocontrol. The revelation that effects can cascade through communities via “hub” microbes is important to understand community structure perturbations in parallel fields including human microbiomes and bioprocesses. In particular, parallels to human microbiome “keystone” pathogens and microbes open new avenues of interdisciplinary research that promise to better our understanding of functions of host-associated microbiomes. [ABSTRACT FROM AUTHOR]

1207I.1208II.1209III.1211IV.1217V.1218VI.1219VII.1221VIII.12241224References1224 Summary: Research on obligate biotrophic plant parasites, which reproduce only on living hosts, has revealed a broad diversity of filamentous microbes that have independently acquired complex morphological structures, such as haustoria. Genome studies have also demonstrated a concerted loss of genes for metabolism and lytic enzymes, and gain of diversity of genes coding for effectors involved in host defense suppression. So far, these traits converge in all known obligate biotrophic parasites, but unexpected genome plasticity remains. This plasticity is manifested as transposable element (TE)‐driven increases in genome size, observed to be associated with the diversification of virulence genes under selection pressure. Genome expansion could result from the governing of the pathogen response to ecological selection pressures, such as host or nutrient availability, or to microbial interactions, such as competition, hyperparasitism and beneficial cooperations. Expansion is balanced by alternating sexual and asexual cycles, as well as selfing and outcrossing, which operate to control transposon activity in populations. In turn, the prevalence of these balancing mechanisms seems to be correlated with external biotic factors, suggesting a complex, interconnected evolutionary network in host–pathogen–microbe interactions. Therefore, the next phase of obligate biotrophic pathogen research will need to uncover how this network, including multitrophic interactions, shapes the evolution and diversity of pathogens. [ABSTRACT FROM AUTHOR]

To maximize the production of carboxylic acids with open cultures of microbial consortia (reactor microbiomes), we performed experiments to understand which factors affect the community dynamics and performance parameters. We operated six thermophilic (55 °C) bioreactors to test how the factors: (i) biomass pretreatment; (ii) bioreactor operating conditions; and (iii) bioreactor history (after perturbations during the operating period) affected total fermentation product and n-butyrate performance parameters with corn fiber as the cellulosic biomass waste. We observed a maximum total fermentation product yield of 39%, a n-butyrate yield of 23% (both on a COD basis), a maximum total fermentation production rate of 0.74 g COD l-1 d-1 and n-butyrate production rate of 0.47 g COD l-1 d-1 in bioreactors that were fed with dilute-acid pretreated corn fiber at a pH of 5.5. Pyrosequencing of 16S rRNA genes with constrained ordination and other statistical methods showed that changes in operating conditions to enable dilution of toxic carboxylic acid products, which lead to these maximum performance parameters, also altered the composition of the microbiome, and that the microbiome, in turn, affected the performance. Operating conditions are an important factor (tool for operators) to shape reactor microbiomes, but other factors, such as substrate composition after biomass pretreatment and bioreactor history are also important. Further optimization of operating conditions must relieve the toxicity of carboxylic acids at acidic bioreactor pH levels even more, and this can, for example, be accomplished by extracting the product from the bioreactor solutions. [ABSTRACT FROM AUTHOR]

Our societies generate increasing volumes of organic wastes. Considering that we also need alternatives to oil, an opportunity exists to extract liquid fuels or even industrial solvents from these abundant wastes. Anaerobic undefined mixed cultures can handle the complexity and variability of organic wastes, which produces carboxylates that can be efficiently converted to useful bioproducts. However, to date, barriers, such as inefficient liquid product separation and persistence of methanogens, have prevented the production of bioproducts other than methane. Here, we discuss combinations of biological and chemical pathways that comprise the ‘carboxylate platform’, which is used to convert waste to bioproducts. To develop the carboxylate platform into an important system within biorefineries, we must understand the kinetic and thermodynamic possibilities of anaerobic pathways, understand the ecological principles underlying pathway alternatives, and develop superior separation technologies. [ABSTRACT FROM AUTHOR]

Abstract: Anaerobic digestion of brewery wastewater solids in the form of primary sludge was investigated for its potential as a source of energy (methane). We operated a low-rate (hydraulic retention time (HRT)=solids retention time (SRT)) continuously stirred anaerobic digester (CSAD) and a high-rate (SRT>HRT) anaerobic sequencing batch reactor (ASBR) in parallel for 250days. We found that high-rate anaerobic digestion was beneficial for solids-rich waste flows even during a long-term operating period that included a shock load of nonbiodegradable total solids. The ASBR biomass achieved a higher specific methanogenic activity compared to the CSAD biomass (0.257±0.043 vs. 0.088±0.008g CH4-CODg−1 VSSd−1), which aided in stability during the shock load with total solids. The methane yield for the ASBR was 40–34% higher than for the CSAD (0.306 vs. 0.219l CH4 gVS−1 fed for days 1–183 and 0.174 vs. 0.130l CH4 gVS−1 fed for days 184–250, respectively). Finally, we operated an ASBR for an additional 295days to evaluate the effect of temperature variation on system stability. A stable performance was achieved between the operating temperatures of 22–41°C. [Copyright &y& Elsevier]

U.S. production of fuel ethanol from corn grain has increased considerably over the last 10 years. Intense debate regarding the true environmental impact of the overall production process has been ongoing. The present study evaluated the utilization of thin stillage (a major byproduct of the dry-mill corn grain- to-ethanol process) in laboratory-scale thermophilic anaerobic sequencing batch reactors for conversion to methane. We found that augmentation of cobalt as a growth factor to the thermophilic anaerobic digestion process is required. After reaching sustainable operating performances, the methane potential in the reactors was 0.254 L CH4/g total chemical oxygen demand (TCOD) fed. Together with a reduction in the mass of solids that needs drying, methane generation translates to a 51% reduction of natural gas consumption at a conventional dry mill, which improves the net energy balance ratio from 1.26 to 1.70. At the design hydraulic retention time of 10 days, the digesters achieved TCOD, biodegradable COD, volatile solids, and total solids removal efficiencies of 90%, 75%, 89%, and 81%, respectively. We also found that struvite precipitation occurred in the thermophilic digesters during the course of the study, resulting in possibilities for nutrient recovery. [ABSTRACT FROM AUTHOR]

Many beer breweries use high-rate anaerobic digestion (AD) systems to treat their soluble high-strength wastewater. Biogas from these AD systems is used to offset nonrenewable energy utilization in the brewery. With increasing nonrenewable energy costs, interest has mounted to also digest secondary residuals from the high-rate digester effluent, which consists of yeast cells, bacteria, methanogens, and small (hemi)cellulosic particles. Mesophilic (37 °C) and thermophilic (55 °C) lab-scale, low-rate continuously-stirred anaerobic digestion (CSAD) bioreactors were operated for 258 days by feeding secondary residuals at a volatile solids (VS) concentration of ∼40 g l−1. At a hydraulic retention time (HRT) of 15 days and a VS loading rate of 2.7 g VS l−1 day−1, the mesophilic bioreactor showed an average specific volumetric biogas production rate of 0.88 l CH4 l−1 day−1 and an effluent VS concentration of 22.2 g VS l−1 (43.0% VS removal efficiency) while the thermophilic bioreactor displayed similar performances. The overall methane yield for both systems was 0.21 l CH4 g−1 VS fed and 0.47–0.48 l CH4 g−1 VS removed. A primary limitation of thermophilic digestion of this protein-rich waste is the inhibition of methanogens due to higher nondissociated (free) ammonia (NH3) concentrations under similar total ammonium (NH4 +) concentrations at equilibrium. Since thermophilic AD did not result in advantageous methane production rates or yields, mesophilic AD was, therefore, superior in treating secondary residuals from high-rate AD effluent. An additional digester to convert secondary residuals to methane may increase the total biogas generation at the brewery by 8% compared to just conventional high-rate digestion of brewery wastewater alone. [ABSTRACT FROM AUTHOR]

Recent field and laboratory experiments with perennial Boechera stricta and annual Arabidopsis thaliana suggest that the root microbiota influences flowering time. Here we examined in long-term time-course experiments the bacterial root microbiota of the arctic-alpine perennial Arabis alpina in natural and controlled environments by 16S rRNA gene profiling. We identified soil type and residence time of plants in soil as major determinants explaining up to 15% of root microbiota variation, whereas environmental conditions and host genotype explain maximally 11% of variation. When grown in the same soil, the root microbiota composition of perennial A. alpina is largely similar to those of its annual relatives A. thaliana and Cardamine hirsuta. Non-flowering wild-type A. alpina and flowering pep1 mutant plants assemble an essentially indistinguishable root microbiota, thereby uncoupling flowering time from plant residence time-dependent microbiota changes. This reveals the robustness of the root microbiota against the onset and perpetual flowering of A. alpina. Together with previous studies, this implies a model in which parts of the root microbiota modulate flowering time, whereas, after microbiota acquisition during vegetative growth, the established root-associated bacterial assemblage is structurally robust to perturbations caused by flowering and drastic changes in plant stature. [ABSTRACT FROM AUTHOR]

Leaf microbiomes play crucial roles in plant health, making it important to understand the origins and functional relevance of their diversity. High strain-level leaf bacterial genetic diversity is known to be relevant for interactions with hosts, but little is known about its relevance for interactions with the multitude of diverse co-colonizing microorganisms. In leaves, nutrients like amino acids are major regulators of microbial growth and activity. Using metabolomics of leaf apoplast fluid, we found that different species of the plant genus Flaveria considerably differ in the concentrations of high-cost amino acids. We investigated how these differences affect bacterial community diversity and assembly by enriching leaf bacteria in vitro with only sucrose or sucrose + amino acids as possible carbon sources. Enrichments from F. robusta were dominated by Pantoea sp. and Pseudomonas sp., regardless of carbon source. The latter was unable to grow on sucrose alone but persisted in the sucrose-only enrichment thanks to exchange of diverse metabolites from Pantoea sp. Individual Pseudomonas strains in the enrichments had high genetic similarity but still displayed clear niche partitioning, enabling distinct strains to cross-feed in parallel. Pantoea strains were also closely related, but individuals enriched from F. trinervia fed Pseudomonas more poorly than those from F. robusta. This can be explained in part by the plant environment, since some cross-feeding interactions were selected for, when experimentally evolved in a poor (sucrose-only) environment but selected against in a rich (sucrose + amino acids) one. Together, our work shows that leaf bacterial diversity is functionally relevant in cross-feeding interactions and strongly suggests that the leaf resource environment can shape these interactions and thereby indirectly drive bacterial diversity. [ABSTRACT FROM AUTHOR]

Highlights: [•] Substrate type drives variation of reactor microbiomes. [•] Continuum exists and co-digestion microbiomes fall between two mixed substrate types. [•] To link other environmental factors, many more samples must be included. [ABSTRACT FROM AUTHOR]

Profiling diverse microbiomes is revolutionizing our understanding of biological mechanisms and ecologically relevant problems, including metaorganism (host + microbiome) assembly, functions and adaptation. Amplicon sequencing of multiple conserved, phylogenetically informative loci has therefore become an instrumental tool for many researchers. Investigations in many systems are hindered, however, since essential sequencing depth can be lost by amplification of nontarget DNA from hosts or overabundant microorganisms. Here, we introduce "blocking oligos", a low‐cost and flexible method using standard oligonucleotides to block amplification of diverse nontargets and software to aid their design. We apply them primarily in leaves, where exceptional challenges with host amplification prevail. A. thaliana‐specific blocking oligos applied in eight different target loci reduce undesirable host amplification by up to 90%. To expand applicability, we designed universal 16S and 18S rRNA gene plant blocking oligos for targets that are conserved in diverse plant species and demonstrate that they efficiently block five plant species from five orders spanning monocots and dicots (Bromuserectus, Plantagolanceolata, Lotuscorniculatus, Amaranth sp., Arabidopsisthaliana). These can increase alpha diversity discovery without biasing beta diversity patterns and do not compromise microbial load information inherent to plant‐derived 16S rRNA gene amplicon sequencing data. Finally, we designed and tested blocking oligos to avoid amplification of 18S rRNA genes of a sporulating oomycete pathogen, demonstrating their effectiveness in applications well beyond plants. Using these tools, we generated a survey of the A. thaliana leaf microbiome based on eight loci targeting bacterial, fungal, oomycete and other eukaryotic microorganisms and discuss complementarity of commonly used amplicon sequencing regions for describing leaf microbiota. This approach has potential to make questions in a variety of study systems more tractable by making amplicon sequencing more targeted, leading to deeper, systems‐based insights into microbial discovery. For fast and easy design for blocking oligos for any nontarget DNA in other study systems, we developed a publicly available R package. [ABSTRACT FROM AUTHOR]