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Since the 1970s, over 6,500 articles have been published about microbial biocontrols and over 200 microbial isolates have been registered for commercial use. However, many of these solutions have seen limited use due to limitations with their in-field efficacy. Even when multiple biocontrol agents are combined to create multistrain biocontrols, the resulting combinations can be less effective than the individual agents. One likely contributor is due to how multistrain microbial biocontrols are created. Multistrain microbial biocontrols are generally produced under controlled settings that are divorced from the ecological conditions they will need to function under. Traditionally, researchers culture, identify, and screen isolates for pathogen suppression traits. Then these researchers will combine the most promising isolates in an attempt to create more effective solutions. This approach, while effective for identifying suppressive isolates and determining the mechanisms of pathogen suppression, does not take into consideration the variability of natural environments, nor the complex ecological interactions that occur between plant hosts, pathogens, and component biocontrol agents, thus limiting the range of circumstances that these multistrain solutions can reliably succeed. To address these limitations, we suggest the application of relevant ecological theory to determine which isolates should be combined to create more reliable multistrain biocontrols. In this synthesis, we build on prior work focused on addressing plant pathogens through the use of multistrain microbial biocontrols, but we argue that viewing this work through the lens of ecology reveals key “design principles” from natural communities that are stable, functioning, and comprise multiple species.

While a plant's microbiome can facilitate adaptive phenotypes, the plant's role in selecting for these microbes is unclear. Do plants actively recruit microbes beneficial to their current environment, or are beneficial microbes only an incidental by-product of microbial adaptation? We addressed these questions through a multigeneration greenhouse experiment, selecting for either dry- or wet-adapted soil microbial communities, either with or without plants. After three plant generations, we conducted a full reciprocal transplant of each soil community onto wet- and dry-treated plants. We found that plants generally benefited from soil microbes, and this benefit was greater whenever their current watering conditions matched the microbes' historical watering conditions. Principally, the plant's presence was not necessary in the historical treatments for this environmental matching benefit to emerge. Moreover, we found microbes from droughted soils could better tolerate drought stress. Taken together, these results suggest that the moisture environment selects for microbes that benefit plants under those specific moisture conditions, and that these beneficial properties arise as a by-product of microbial adaptation to the watering environment and not as a co-adapting plant–microbe system. This work highlights that understanding the selective agents on these plant-associated microbes will lead to a better understanding of plant adaptation.

IntroductionAlliaria petiolata (garlic mustard), an invasive forest herb in North America, often alters nutrient availability in its non-native ecosystems, but the mechanisms driving these changes have yet to be determined. We hypothesized three potential mechanisms through which garlic mustard could directly influence soil nitrogen (N) cycling: by increasing soil pH, by modifying soil microbial community composition, and by altering nutrient availability through litter inputs.Materials and methodsTo test these hypotheses, we evaluated garlic mustard effects on soil pH and other soil properties; fungal and prokaryotic (bacterial and archaeal) community composition; and soil N cycling rates (gross N mineralization and nitrification rates, microbial N assimilation rates, and nitrification- versus denitrification-derived nitrous oxide fluxes); and we assessed correlations among these variables. We collected soil samples from garlic mustard present, absent, and removed treatments in eight forests in central Illinois, United States, during the rosette, flowering, and senescence phenological stages of garlic mustard life cycle.ResultsWe found that garlic mustard increased soil pH, altered fungal and prokaryotic communities, and increased rates of N mineralization, nitrification, nitrification-derived net N2O emission. Significant correlations between soil pH and microbial community composition suggest that garlic mustard effects on soil pH could both directly and indirectly influence soil N cycling rates.DiscussionCorrespondence of gross rates of N mineralization and nitrification with microbial community composition suggest that garlic mustard modification of soil microbial communities could directly lead to changes in soil N cycling. We had expected that early season, nutrient-rich litter inputs from mortality of young garlic mustard could accelerate gross N mineralization and microbial N assimilation whereas late season, nutrient poorer litter inputs from senesced garlic mustard could suppress N mineralization, but we did not observe these patterns in support of the litter input mechanism. Together, our results elucidate how garlic mustard effects on soil pH and microbial community composition can accelerate soil N cycling to potentially contribute to the invasion success of garlic mustard.

Understanding the major drivers that influence soil bacterial and fungal communities is essential to mitigate the impacts of human activity on vulnerable ecosystems, like those found on the Galápagos Islands. Located ~1000 km off the coast of Ecuador, the volcanically formed islands are situated within distinct oceanic currents, which provide seasonal weather patterns and unique microclimates within small spatial scales across the islands. Although much is known about the impacts of human activity, such as climate change and invasive plant species, on above ground biodiversity of the Galápagos Islands, little is known about the resident soil microbial communities and the drivers that shape these communities. Here, our goal was to investigate the bacterial and fungal communities found in soil located in three distinct microclimates: Mirador (arid), Cerro Alto (transition zone), and El Junco (humid), and associated with native and invasive plant types. At each site, we collected soil at three depths (rhizosphere, 5 cm, and 15 cm) associated with the invasive plant, Psidium guajava (guava), and native plant types. We determined that the sampling location (microclimate) was the strongest driver of both bacterial and fungal communities (74 and 38%, respectively), with additional minor but significant impacts from plant type and soil depth. This study highlights the continued need to explore microbial communities across diverse environments and demonstrates the weight of different abiotic and biotic factors impacting soil microbial communities across San Cristóbal Island in the Galápagos archipelago.IMPORTANCE/SIGNIFICANCEHuman activity such as climate change, pollution, introduction of invasive species, and deforestation, poses a huge threat to biodiverse environments. Soil microbiota are an essential component to maintaining healthy ecosystems. However, a greater understanding of factors that alter these microbial communities is needed in order to find ways to mitigate and reverse the impacts imposed by human activity. The Galápagos Islands are a unique real-world laboratory, in that the islands’ biogeography and physical locations in the Pacific Ocean provide distinct microclimates within small geographic distances. Harnessing these distinct environments allowed us to investigate the influence of microclimates, soil depth, and vegetation cover on bacterial and fungal community composition.

Bacillus subtilis is a soil bacterium that can form biofilms, which are communities of cells encased by an extracellular matrix. In these complex communities, cells perform numerous metabolic processes and undergo differentiation into functionally distinct phenotypes as a survival strategy. Because biofilms are often studied in bulk, it remains unclear how metabolite production spatially correlates with B. subtilis phenotypes within biofilm structures. In many cases, we still do not know where these biological processes are occurring in the biofilm. Here, we developed a method to analyze the localization of molecules within sagittal thin sections of B. subtilis biofilms using high-resolution mass spectrometry imaging. We correlated the organization of specific molecules to the localization of well-studied B. subtilis phenotypic reporters determined by confocal laser scanning fluorescence microscopy within analogous biofilm thin sections. The correlations between these two data sets suggest the role of surfactin as a signal for extracellular matrix gene expression in the biofilm periphery and the role of bacillibactin as an iron-scavenging molecule. Taken together, this method will help us generate hypotheses to discover relationships between metabolites and phenotypic cell states in B. subtilis and other biofilm-forming bacteria. IMPORTANCE Bacterial biofilms are complex and heterogeneous structures. Cells within biofilms carry out numerous metabolic processes in a nuanced and organized manner, details of which are still being discovered. Here, we used multimodal imaging to analyze B. subtilis biofilm processes at the metabolic and gene expression levels in biofilm sagittal thin sections. Often, imaging techniques analyze only the top of the surface of the biofilm and miss the multifaceted interactions that occur deep within the biofilm. Our analysis of the sagittal planes of B. subtilis biofilms revealed the distributions of metabolic processes throughout the depths of these structures and allowed us to draw correlations between metabolites and phenotypically important subpopulations of B. subtilis cells. This technique provides a platform to generate hypotheses about the role of specific molecules and their relationships to B. subtilis subpopulations of cells.

New, non-chemical weed suppression methods are needed to support multi-tactic integrated weed management (IWM) programs that reduce reliance on herbicides. One such approach is to manage arbuscular mycorrhizal (AM) fungal communities to enhance their weed biocontrol. Successful implementation will require a better understanding of AM fungi-crop-weed relationships under field conditions, especially in the face of soil disturbances imposed by agricultural practices. We examined root AM fungal communities of four weed species and one model crop species (i.e., corn) in a field experiment with contrasting tillage and cover cropping treatments in Illinois, USA. Our objectives were to quantify the impact of AM fungi on the growth of corn and weed species, to assess influences of plant species, tillage, and cover cropping on AM fungal community structure, and to characterize the co-occurrence patterns of AM fungal taxa in weed and corn roots. Our results showed that effects of AM fungal taxa on corn and weed growth varied widely depending on plant species, and several AM fungal taxa had species-specific negative effects on weeds but not on corn. In addition, plant identity was the primary factor affecting root AM fungal community structure and co-occurrence patterns, whereas effects of tillage and cover cropping were relatively limited. We propose that AM fungal taxa that have selectively suppressive effects on weeds but not on corn are candidates for developing new strategies for weed biocontrol as part of IWM. Future approaches to weed management based on direct inoculation with AM fungal taxa or manipulation of AM fungal communities through specific management practices, are promising opportunities to increase the density of weed-suppressive AM fungal taxa in agroecosystems, and our methods provide important insights for AM fungal taxa selection.

Cover crops can supply and recycle soil nitrogen (N) in agroecosystems. With increasing interest in cover crop mixtures, farmers have questions about how N cycling is affected by individual species when planted alone or in mixtures. Field trials were conducted on two organic vegetable farms during the spring and summer of 2014–2016 to quantify biomass tissue N provisioned, soil nitrate (NO3-N) scavenged, and soil NO3-N mineralized from 12 species of cover crops (six spring and six summer species) planted in monoculture or five-way mixtures. All cover crops were compared to weedy and weeded (no cover crops or weeds) controls. Cover crops provisioned 28–33% more biomass tissue N than fallow weeds. Among spring cover crops, field pea produced the most biomass tissue N (131 ± 13 kg N ha−1; mean ± 1 SE) but that benefit disappeared when pea abundance was reduced in mixtures. Soil NO3-N to a depth of 0.2 m was reduced by 50–67% during cover crop and weed growth, regardless of treatment. Soil NO3-N availability approximately 1 month after incorporation of cover crop and weedy residues varied by treatment. Post-incorporation soil NO3-N was 17–19% greater in pea and soybean monocultures compared to mixtures; conversely, soil NO3-N was 17–19% lower following incorporation of buckwheat and sudangrass monocultures compared to mixtures. Results of this study demonstrate that cover crop N recycling services in near-surface soil are largely independent of species or mixture, but N provisioning services require careful management and species selection to ensure maximum biomass tissue N and timely N mineralization.

Mass spectrometry imaging (MSI) is becoming an increasingly popular analytical technique to investigate microbial systems. However, differences in the ionization efficiencies of distinct MSI methods lead to biases in terms of what types and classes of molecules can be detected. Here, we sought to increase the molecular coverage of microbial colonies by employing metal-assisted laser desorption/ionization (MetA-LDI) MSI, and we compared our results to more commonly utilized matrix-assisted laser desorption/ionization MALDI MSI. We found substantial (∼67%) overlap in the molecules detected in our analysis of Bacillus subtilis colony biofilms using both methods, but each ionization technique did lead to the identification of a unique subset of molecular species. MetA-LDI MSI tended to identify more small molecules and neutral lipids, whereas MALDI MSI more readily detected other lipids and surfactin species. Putative annotations were made using METASPACE, Metlin, and the BsubCyc database. These annotations were then confirmed from analyses of replicate bacterial colonies using liquid extraction surface analysis tandem mass spectrometry. Additionally, we analyzed B. subtilis biofilms in a polymer-based emulated soil micromodel using MetA-LDI MSI to better understand bacterial processes and metabolism in a native, soil-like environment. We were able to detect different molecular signatures within the micropore regions of the micromodel. We also show that MetA-LDI MSI can be used to analyze microbial biofilms from electrically insulating material. Overall, this study expands the molecular universe of microbial metabolism that can be visualized by MSI. IMPORTANCE Matrix-assisted laser desorption/ionization mass spectrometry imaging is becoming an important technique to investigate molecular processes within microbial colonies and microbiomes under different environmental conditions. However, this method is limited in terms of the types and classes of molecules that can be detected. In this study, we utilized metal-assisted laser desorption/ionization mass spectrometry imaging, which expanded the range of molecules that could be imaged from microbial samples. One advantage of this technique is that the addition of a metal helps facilitate ionization from electrically nonconductive substrates, which allows for the investigation of biofilms grown in polymer-based devices, like soil-emulating micromodels.

The invasive forest plant garlic mustard (Alliaria petiolata) has been shown to alter soil microbial communities in the northeastern part of its invaded range in the United States, and this disruption of soil communities may contribute to its invasion success. However, garlic mustard allelochemistry can vary with invasion age, and it is not clear whether garlic mustard’s impacts on soil microbes are consistent over its invaded range. Here, we compare the composition and diversity of soil fungal, bacterial, and archaeal communities among garlic mustard present, absent, and removed treatments in replicated blocks across five forests in the midwestern United States with relatively young garlic mustard invasions (approximately 17-26 years old, with consistent management). We collected samples in May and August, corresponding to garlic mustard active and senescent life history stages. While soil fungal and bacterial/ archaeal communities (based on ITS2 region and 16S rRNA gene DNA sequencing, respectively) differed significantly between different blocks/ forests and over time, we found no significant effect of garlic mustard treatment on soil microbial community composition or the relative abundance of mycorrhizal, saprotrophic, or pathogenic fungal guilds. The lack of garlic mustard impacts on the soil microbial community in recently invaded central Illinois forests suggests that these well-documented impacts in the northeastern United States and in older invasions cannot necessarily be generalized across all environmental contexts.

Here, we report the complete genome sequence of Bacillus velezensis strain S4, which was isolated from biochar-amended agricultural soil collected in Smyrna, Delaware. The genome is 4.07 Mbp, encodes 3,918 predicted proteins, and has a GC content of 46.4%.

Bacillus subtilis is a soil-dwelling bacterium that forms highly structured microbial communities called biofilms. Biofilm formation is important for bacterial survival, as biofilms are highly tolerant to environmental stresses. In B. subtilis, the formation of biofilms facilitates important interactions with plants. While the genetic regulation of biofilm formation is highly studied in B. subtilis, little is known regarding the molecular details of how signaling molecules feed into the biofilm regulatory network of this bacterium. Recent studies found that the second messenger cyclic di-adenylate monophosphate (cyclic di-AMP) plays an important role in B. subtilis biofilm formation and plant attachment. B. subtilis secretes cyclic di-AMP via three putative cyclic di-AMP transporters, suggesting that cyclic di-AMP can act as an extracellular signal for biofilm formation and plant attachment. Here, we discuss how cyclic di-AMP metabolism and secretion impact colony biofilm architecture, biofilm gene expression, and plant attachment in B. subtilis and speculate on future directions for the field.

Integrated weed management (IWM) relies upon multiple chemical, physical, or biological weed management techniques to achieve an acceptable level of weed control. Agents that selectively suppress weeds but not crops and that can be manipulated in agriculture will be promising components for inclusion in IWM. We used a meta-analytic approach to investigate the potential of arbuscular mycorrhizal fungi (AMF) to contribute to IWM. We quantified the effect of crop and weed host status (strong and weak AMF hosts are divided in this study by a 10% root length colonization threshold), AMF diversity (single vs. mixed), and soil N and P fertility management on plant mycorrhizal growth responses (MGRs). Our results indicated that weak host weeds had consistently lower MGRs than strong host crops in both controlled and field conditions. Moreover, these differences in MGRs between weak host weeds and strong host crops were more pronounced under mixed AMF inoculum and low N and P nutrient availability. In contrast, MGR of strong host weeds was not different from strong host crops in general. However, we observed a wide range of MGRs among strong host weeds, some of which had much lower MGRs than strong host crops. In addition, in the presence of N and P fertilizers, strong host crops had a stronger positive response to AMF than strong host weeds. Thus, our meta-analysis indicates that AMF have potential to contribute to weed control by direct and indirect pathways: directly suppress weak host weeds, and indirectly suppress some strong host weeds mediating by competitive effects exerted by strong host crops. We suggest that management practices affecting AMF diversity and crop and weed mycorrhizal responses could be chosen to improve the contribution of AMF to IWM. Better understanding is needed of crop–weed–AMF interactions and management practices that enhance this form of weed management.

Managing the soil nitrogen (N) cycle is a major component of agricultural sustainability. Soil functional zone management (zonal management) is a novel agroecological strategy for managing row-crop agroecosystems. It may improve the efficiency of soil N cycling compared with conventional and no-tillage approaches, by managing the timing and location (crop row vs inter-row) of key soil N cycling processes. We compared N mineralization and availability during the period of maize peak N demand in crop rows and inter-rows in zonal management and conventional chisel plow tillage systems at four sites across the US Corn Belt over three growing seasons. Under zonal management, potential N mineralization and N availability during crop peak N demand were significantly greater in crop rows, where the majority of crop roots are found, compared with inter-rows. Averaged across all site-years, plant-available N in zonal management crop rows was 46 mg kg −1 compared with 21 mg kg −1 in inter-rows. In contrast, in conventional tillage, potential N mineralization and N availability were greater in inter-rows compared with crop rows; averaged across all site-years, plant-available N in conventional tillage crop rows was 24 mg kg −1 compared with 51 mg kg −1 in inter-rows. The results demonstrate that the active management of crop residues under zonal management can enhance the spatiotemporal efficiency of soil N cycling processes, by concentrating N mineralization and availability close to crop roots in synchrony with crop developmental needs. Zonal management therefore has potential to increase crop N-use efficiency compared with conventional tillage, and thereby reduce the impacts of row-crop agricultural production on water resources and greenhouse gas emissions that result from N leaching and denitrification.

Zonal tillage (e.g. ridge tillage, RT) separates management of row and inter-row positions, while non-zonal tillage (e.g. chisel plough, CP) applies management uniformly across a field. This may have large effects on soil hydrothermal properties, affecting soil processes and crop development. We examined the effects of RT versus CP on soil hydrothermal conditions under maize (Zea mays L.) at four sites spanning the US Corn Belt over two growing seasons (2012–2013). We also investigated whether RT, as a result of changes in hydrothermal conditions, could stimulate greater soil nitrogen (N) availability during peak maize N demand. We captured wide variation in soil types and climates, allowing us to generalise tillage effects across a large environmental gradient. Continuous hydrothermal measurements were taken in the centre of row and inter-row positions. Soil cores collected shortly after maize six leaf stage (V6) were analysed for plant-available N and potentially mineralisable N (PMN). We hypothesised: 1) in spring CP and RT both produce warm, dry seedbeds with equivalent accumulations of growing degree days (GDD), but later in season RT holds greater soil moisture, providing better conditions for cover or relay crop establishment; 2) Hydrothermal properties of RT rows are distinct from RT inter-rows, while CP rows and inter-rows are indistinguishable; 3) RT promotes greater soil N mineralisation and availability in crop rows compared with CP. Results largely confirmed all hypotheses. In early spring, rows were drier in RT than CP, and both were similar in warmth (i.e. in accumulated GDD). From V6 to tasselling, CP accumulated more GDD than RT in inter-rows, while row positions remained similar; RT maintained greater soil moisture across both positions. From tasselling to harvest, RT inter-rows held greater soil moisture than CP, but accumulated fewer GDD. Both tillage systems showed zonation of soil moisture between planting and harvest (inter-rows moister than rows); the magnitude of zonation was greatest in RT. Plant-available N and PMN were greater in RT compared with CP at V6, suggesting RT increases synchrony of soil N availability with crop requirements. The results demonstrate that zonal tillage can integrate the seedbed benefits of conventional tillage with increased soil moisture retention across a wide range of climates and soil types. Increased moisture retention may help buffer agricultural systems against drought, and improve seedbed conditions for cover and relay crops in late summer and early autumn, thus potentially improving both sustainability and production in these systems.

Many microbes coexist within biofilms, or multispecies communities of cells encased in an extracellular matrix. However, little is known about the microbe-microbe interactions relevant for creating these structures. In this study, we explored a striking dual-species biofilm between Bacillus subtilis and Pantoea agglomerans that exhibited characteristics that were not predictable from previous work examining monoculture biofilms. Coculture wrinkle formation required a P. agglomerans exopolysaccharide as well as the B. subtilis amyloid-like protein TasA. Unexpectedly, other B. subtilis matrix components essential for monoculture biofilm formation were not necessary for coculture wrinkling (e.g., the exopolysaccharide EPS, the hydrophobin BslA, and cell chaining). In addition, B. subtilis cell chaining prevented coculture wrinkling, even though chaining was previously associated with more robust monoculture biofilms. We also observed that increasing the relative proportion of P. agglomerans (which forms completely featureless monoculture colonies) increased coculture wrinkling. Using microscopy and rheology, we observed that these two bacteria assemble into an organized layered structure that reflects the physical properties of both monocultures. This partitioning into distinct regions negatively affected the survival of P. agglomerans while also serving as a protective mechanism in the presence of antibiotic stress. Taken together, these data indicate that studying cocultures is a productive avenue to identify novel mechanisms that drive the formation of structured microbial communities. IMPORTANCE In the environment, many microbes form biofilms. However, the interspecies interactions underlying bacterial coexistence within these biofilms remain understudied. Here, we mimic environmentally relevant biofilms by studying a dual-species biofilm formed between Bacillus subtilis and Pantoea agglomerans and subjecting the coculture to chemical and physical stressors that it may experience in the natural world. We determined that both bacteria contribute structural elements to the coculture, which is reflected in its overall viscoelastic behavior. Existence within the coculture can be either beneficial or detrimental depending on the context. Many of the features and determinants of the coculture biofilm appear distinct from those identified in monoculture biofilm studies, highlighting the importance of characterizing multispecies consortia to understand naturally occurring bacterial interactions.

There is a growing appreciation for the impact that bacteria have on higher organisms. Plant roots often harbor beneficial microbes, such as the Gram-positive rhizobacterium Bacillus subtilis, that influence their growth and susceptibility to disease. The ability to form surface-attached microbial communities called biofilms is crucial for the ability of B. subtilis to adhere to and protect plant roots. In this study, strains harboring deletions of the B. subtilis genes known to synthesize and degrade the second messenger cyclic di-adenylate monophosphate (c-di-AMP) were examined for their involvement in biofilm formation and plant attachment. We found that intracellular production of c-di-AMP impacts colony biofilm architecture, biofilm gene expression, and plant attachment in B. subtilis. We also show that B. subtilis secretes c-di-AMP and that putative c-di-AMP transporters impact biofilm formation and plant root colonization. Taken together, our data describe a new role for c-di-AMP as a chemical signal that affects important cellular processes in the environmentally and agriculturally important soil bacterium B. subtilis. These results suggest that the “intracellular” signaling molecule c-di-AMP may also play a previously unappreciated role in interbacterial cell-cell communication within plant microbiomes., IMPORTANCE Plants harbor bacterial communities on their roots that can significantly impact their growth and pathogen resistance. In most cases, however, the signals that mediate host-microbe and microbe-microbe interactions within these communities are unknown. A detailed understanding of these interaction mechanisms could facilitate the manipulation of these communities for agricultural or environmental purposes. Bacillus subtilis is a plant-growth-promoting bacterium that adheres to roots by forming biofilms. We therefore began by exploring signals that might impact its biofilm formation. We found that B. subtilis secretes c-di-AMP and that the ability to produce, degrade, or transport cyclic di-adenylate monophosphate (c-di-AMP; a common bacterial second messenger) affects B. subtilis biofilm gene expression and plant attachment. To our knowledge, this is the first demonstration of c-di-AMP impacting a mutualist host-microbe association and suggests that c-di-AMP may function as a previously unappreciated extracellular signal able to mediate interactions within plant microbiomes.

Lespedeza cuneata is a non-native invasive legume that alters the soil bacterial community, associates promiscuously with rhizobia, and benefits more from rhizobial interactions compared to coexisting native Lespedeza in North America. We tested the hypothesis that native congeners differ in their nodule bacteria associations compared to L. cuneata. Plots with high L. cuneata invasion, low L. cuneata invasion with native Lespedeza species present, and uninvaded plots where native Lespedeza species existed without L. cuneata were sampled. Nodules were collected from all Lespedeza species present, and Chamaecrista fasciculata, a common native annual legume. Bacterial DNA from nodules was isolated and sequenced. Nodule bacterial composition differed significantly between hosts. L. cuneata nodules contained high frequencies of rhizobial DNA and low bacterial diversity, while native Lespedeza nodules contained lower rhizobial frequencies and higher non-rhizobial bacterial diversity. Specific non-rhizobial bacterial groups exhibited strong associations with native legumes and uninvaded sites. Significant differences exist in the nodule bacterial composition between native legumes and an introduced congener. The mechanism(s) and ecological importance of these differences remain unknown. These differences in bacterial associations could influence not only the competitive ability of the invader, but recovery of invaded sites as well.

Invasive plants often benefit from changes that they impose on soil microbes via positive plant–soil feedback, but the mechanisms that underlie these changes, and the legacy of their effects, remain poorly quantified. We investigated the impacts of an invasive annual grass, Microstegium vimineum, on the structure and functioning of soil microbial communities in a multi-year, field-based common garden experiment. Given previous reports that M. vimineum can both elevate nitrification rates in soil and benefit from enhanced nitrate availability, we sought to answer the following questions: 1) Does M. vimineum alter the abundance or composition of soil nitrifying microbial communities (ammonia oxidizing archaea and bacteria, AOA and AOB, respectively)? 2) Are such effects reversible or do soil legacy effects persist after M. vimineum is no longer present? After three years, invaded plots had greater AOA abundances than uninvaded native dominated plots, as well as different AOA community structure. However, after seven years, and following a period of M. vimineum replacement by native plants in the invaded plots, AOA abundances and nitrification rates declined toward levels found in uninvaded plots. Collectively, our results suggest that while the impacts of M. vimineum invasions on nitrogen cycling likely relate to their association with AOA, these effects may not persist if M. vimineum declines over time and native plants and their associated microbes are able to re-establish.

Lespedeza cuneata is an introduced legume that is invasive in the tallgrass prairie system and open woodlands of North America. This system includes native Lespedeza species that coexist with L. cuneata, including L. capitata and L. virginica. Previous research has indicated that L. cuneata exudates have profound influences on soil biological functions. The goal of this current research was to identify and then compare exudate chemicals from L. cuneata, L. capitata, L. virginica, and common tallgrass prairie grasses. Exudates from Lespedeza species were found to be distinct from the grasses. In particular, L. cuneata and, to a lesser extent, a subset of L. virginica could be differentiated based on the presence of five unique compounds. Two low molecular weight compounds were identified via gas chromatography/mass spectrometry (GC/MS) and tentatively identified as benzophenone and 1,4-diacetylbenzene. Three higher molecular weight compounds were identified by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS), with possible relationships to a 3 sterol, phosphatidylglycerol, and the 19-nor analogues of vitamin D. The relative abundance of these compounds were found to be greater in the invasive species, L. cuneata, than in the native noninvasive species, L. virginica. These results are an integral step in further understanding through chemical exudation how L. cuneata can be benefiting from greater rhizobial associations.

Particulate organic matter (POM) is considered an “active” source of nitrogen (N) in cultivated soils, responding readily to management and being more physically accessible to decomposers than mineral-associated forms of organic matter. However, there is increasing evidence that mineral-associated organic matter (MAOM) can also exhibit short-term changes to management that may impact plant and microbial N dynamics. In this study, we investigated how N within soil organic matter fractions responded to three years of tillage and cover crop treatments. We collected soils from a row-crop (maize-soybean rotation) field experiment replicated across three sites in the north central and mid-Atlantic United States: a high-soil organic matter site (3.1% soil organic carbon) in Illinois (IL) and two sites in Michigan (MI) and Pennsylvania (PA) with lower soil organic matter content (1.0% and 1.4% soil organic carbon, respectively). Management treatments included two levels of tillage (chisel plow and ridge tillage) and two levels of cover crop (with and without rye cover crop). Using an optimized sonication method coupled with particle size separation, we isolated and analyzed for N content free POM, occluded POM, a coarse silt fraction, and MAOM. Using partial least squares regression, we also explored broad cross-site relationships between soil organic matter (SOM) fractions, soil N availability, and crop performance. Both particulate and fine fractions responded to tillage and cover crop treatments, but patterns varied by site and fraction. In the low-SOM MI and PA soils, ridge tillage and cover cropping both increased N within POM fractions. The response to ridge tillage was most pronounced, with a 76% and 24% increase in occluded POM N content in MI and PA, respectively. In contrast, at the IL site (high-SOM), the inclusion of cover crops led to higher N, specifically within the fine fractions (coarse silt and MAOM). Cover cropping increased MAOM N content in IL by 24%. When analyzing all sites together, variables associated with fine fractions were more closely associated with N mineralization and crop performance. MAOM can be responsive to short-term management practices and, along with POM, may also be potential sources of N for crops.

Identification of microbes that actively utilize root exudates is essential to understand plant-microbe interactions. To identify active root exudate-utilizing microorganisms associated with switchgrass - a potential bioenergy crop - plants were labelled in situ with (13) CO2 , and 16S and 18S rRNA genes in the (13) C-labelled rhizosphere DNA were pyrosequenced. Multi-pulse labelling for 5 days produced detectable (13) C-DNA, which was well separated from unlabelled DNA. Methylibium from the order Burkholderiales were the most heavily labelled bacteria. Pythium, Auricularia and Galerina were the most heavily labelled eukaryotic microbes. We also identified a Glomus intraradices-like species; Glomus members are arbuscular mycorrhizal fungi that are able to colonize the switchgrass root. All of these heavily labelled microorganisms were also among the most abundant species in the rhizosphere. Species belonging to Methylibium and Pythium were the most heavily labelled and the most abundant bacteria and eukaryotes in the rhizosphere of switchgrass. Our results revealed that nearly all of the dominant rhizosphere bacterial and eukaryotic microbes were able to utilize root exudates. The enrichment of microbial species in the rhizosphere is selective and mostly due to root exudation, which functions as a nutrition source, promoting the growth of these microbes.

Increasingly, funding agencies are investing in integrated and transdisciplinary research to tackle “grand challenge” priority areas, critical for sustaining agriculture and protecting the envi -ronment. Coordinating multidisciplinary research teams capable of addressing these priority areas, however, presents its own unique set of chal-lenges, ranging from bridging across multiple dis -ciplinary perspectives to achieve common ques-tions and methods to facilitating engagement in holistic and integrative thinking that promotes linkages from scholarship to societal needs. We propose that structural equation modeling (SEM) can provide a powerful framework for synergizing multidisciplinary research teams around grand challenge issues. Structural equation modeling can integrate both visual and statistical expres-sion of complex hypotheses at all stages of the research process, from planning to analysis. Three elements of the SEM framework are par-ticularly beneficial to multidisciplinary research teams; these include (i) a common graphical lan-guage that transcends disciplinary boundaries, (ii) iterative, critical evaluation of complex hypoth -eses involving manifest and latent variables and direct and indirect interactions, and (iii) enhanced opportunities to discover unanticipated interac-tions or causal pathways as empirical data are tested statistically against the model. Using our ongoing multidisciplinary, multisite field inves-tigation of climate change adaptation and miti-gation in annual row crop agroecosystems as a case study, we demonstrate the value of the SEM framework for project design, coordination, and implementation and provide recommendations for its broader application as a means to more effectively engage and address issues of critical societal concern.R.G. Smith, L.W. Atwood, A.B. Daly, and A.S. Grandy, Dep. of Natural Resources and the Environment, Univ. of New Hampshire, Durham, NH 03824; A.S. Davis, USDA-ARS Global Change and Photosynthesis Research Unit, Urbana, IL 61801; N.R. Jordan and P. Ewing, Dep. of Agronomy and Plant Genetics, Univ. of Minnesota, St. Paul, MN 55108; M.C. Hunter, and D.A. Mortensen, Dep. of Plant Science, The Pennsylvania State Univ., University Park, PA 16802; R.T. Koide, Dep. of Biology, Brigham Young Univ., Provo, UT 84602; D. Kane and S.S. Snapp, Dep. of Plant, Soil and Microbial Sciences, Michigan State Univ., East Lansing, MI 48824; M. Li, Dep. of Crop Sciences, Univ. of Illinois, Urbana, IL 61801; Y. Lou and A.C. Yannarell, Dep. of Natural Resources and Environmental Sciences, Univ. of Illinois, Urbana, IL 61801; K.A. Spokas, USDA-ARS Soil and Water Management Research Unit, St. Paul, MN 55108. Received 19 July 2013. *Corresponding author (richard.smith@unh.edu).

Rhizosphere bacteria have significant contributions to crop health, productivity and carbon sequestration. As maize (Zea mays) is an important economic crop, its rhizosphere bacterial communities have been intensively investigated using various approaches. However, low-resolution profiling methods often make it difficult to understand the complicated rhizosphere bacterial communities and their dynamics. In this study, we analyzed growth-stage related dynamics of bacterial community structures in the rhizosphere of maize using the pyrosequencing method, which revealed an assembly of bacteria enriched in the rhizosphere. Our results revealed that the rhizosphere of maize was preferentially colonized by Proteobacteria, Bacteroidetes and Actinobacteria, and each bacterial phylum was represented by one or two dominating subsets of bacterial groups. Dominant genera enriched in the rhizosphere included Massilia, Burkholderia, Ralstonia, Dyella, Chitinophaga and Sphingobium. Rhizosphere bacterial community structures significantly changed through different growth stages at lower taxonomic ranks (family, genus and OTU levels). Genera Massilia, Flavobacterium, Arenimonas and Ohtaekwangia were relatively abundant at early growth stages, while genera Burkholderia, Ralstonia, Dyella, Chitinophaga, Sphingobium, Bradyrhizobium and Variovorax populations were dominant at later stages. Comparisons of pyrosequencing data collected in Illinois, USA in this study with the available data from Braunschweig, Germany indicated many common bacterial inhabitants but also many differences in the structure of bacterial communities, implying that some site-specific factors, such as soil properties, may play important roles in shaping the structure of rhizosphere bacterial community.

A low-diversity microbial community, dominated by the γ-proteobacterium Halomonas sulfidaeris, was detected in samples of warm saline formation porewater collected from the Cambrian Mt. Simon Sandstone in the Illinois Basin of the North American Midcontinent (1.8 km/5872 ft burial depth, 50°C, pH 8, 181 bars pressure). These highly porous and permeable quartz arenite sandstones are directly analogous to reservoirs around the world targeted for large-scale hydrocarbon extraction, as well as subsurface gas and carbon storage. A new downhole low-contamination subsurface sampling probe was used to collect in situ formation water samples for microbial environmental metagenomic analyses. Multiple lines of evidence suggest that this H. sulfidaeris-dominated subsurface microbial community is indigenous and not derived from drilling mud microbial contamination. Data to support this includes V1-V3 pyrosequencing of formation water and drilling mud, as well as comparison with previously published microbial analyses of drilling muds in other sites. Metabolic pathway reconstruction, constrained by the geology, geochemistry and present-day environmental conditions of the Mt. Simon Sandstone, implies that H. sulfidaeris-dominated subsurface microbial community may utilize iron and nitrogen metabolisms and extensively recycle indigenous nutrients and substrates. The presence of aromatic compound metabolic pathways suggests this microbial community can readily adapt to and survive subsurface hydrocarbon migration.

This study aimed to determine if biotic contaminants originating from pig production farms are disseminated into soil and groundwater microbial communities. A spatial and temporal sampling of soil and groundwater in proximity to pig production farms was conducted, and quantitative PCR (Q-PCR) was utilized to determine the abundances of tetracycline resistance genes (i.e., tetQ and tetZ ) and integrase genes (i.e., intI1 and intI2 ). We observed that the abundances of tetZ , tetQ , intI1 , and intI2 in the soils increased at least 6-fold after manure application, and their abundances remained elevated above the background for up to 16 months. Q-PCR further determined total abundances of up to 5.88 × 10 9 copies/ng DNA for tetZ , tetQ , intI1 , and intI2 in some of the groundwater wells that were situated next to the manure lagoon and in the facility well used to supply water for one of the farms. We further utilized 16S rRNA-based pyrosequencing to assess the microbial communities, and our comparative analyses suggest that most of the soil samples collected before and after manure application did not change significantly, sharing a high Bray-Curtis similarity of 78.5%. In contrast, an increase in Bacteroidetes and sulfur-oxidizing bacterial populations was observed in the groundwaters collected from lagoon-associated groundwater wells. Genera associated with opportunistic human and animal pathogens, such as Acinetobacter , Arcobacter , Yersinia , and Coxiella , were detected in some of the manure-treated soils and affected groundwater wells. Feces-associated bacteria such as Streptococcus , Erysipelothrix , and Bacteroides were detected in the manure, soil, and groundwater ecosystems, suggesting a perturbation of the soil and groundwater environments by invader species from pig production activities.

Summary Biomass production for bioenergy may change soil microbes and influence ecosystem properties. To explore the impact of different bioenergy cropping systems on soil microorganisms, the compositions and quantities of soil microbial communities (16S rRNA gene) and N-cycling functional groups (nifH, bacterial amoA, archaeal amoA and nosZ genes) were assessed under maize, switchgrass and Miscanthus x giganteus at seven sites representing a climate gradient (precipitation and temperature) in Illinois, USA. Overall, the site-to-site variation in community composition surpassed the variation due to plant type, and microbial communities under each crop did not converge on a ‘typical’ species assemblage. Fewer than 5% of archaeal amoA, bacterial amoA, nifH and nosZ OTUs were significantly different among these crops, but the largest differences observed at each site were found between maize and the two perennial grasses. Quantitative PCR revealed that the abundance of the nifH gene was significantly higher in the perennial grasses than in maize, and we also found significantly higher total N in the perennial grass soils than in maize. Thus, we conclude that cultivation of these perennial grasses, instead of maize, as bioenergy feedstocks can improve soil ecosystem nitrogen sustainability by increasing the population size of N-fixing bacteria.

A combination of culture-dependent and culture-independent methods was used to assess bacterial diversity at different depths within a former solid waste dump in Medellin, Colombia. Sampling sites included a densely populated area, which is built upon 40 m of solid waste (domestic, industrial, agricultural, and medical). The soil and leachate contain high levels of contaminants and the natural soil is highly disturbed with solid anthropogenic materials, disrupting natural aggregation and resulting in a loose, porous matrix with irregular aggregate structure. The unusual physical structure and contaminant levels at the site made it unclear if the indigenous bacterial community would possess the complexity commonly observed for natural soils, and thus may limit potential for remediating the site using monitored natural attenuation. Bacterial diversity patterns were determined through 16S-TTGE and T-RFLP at depths of 0, 10, 20 and 30 m. Abundance and diversity patterns, as estimated by number and intensity of terminal restriction fragments and TTGE bands, varied among the 4 different depths, showing more complex patterns in deeper samples (20 and 30 m), which also contained greater concentrations of organic carbon. General diversity patterns were dominated by the phylum Proteobacteria (λ, β, and α divisions). These findings were reinforced by analysis of the culturable fraction able to use n-hexadecane as sole carbon source, in which the genera Acinetobacter sp. (λ-Proteobacteria) was dominant. This research offers new clues regarding bacterial diversity patterns through different depths in polluted environments with unique physicochemical conditions, suggesting that bacterial diversity profiles may be highly influenced by the nature of pollutants present. Additionally, results imply that the culturable fraction at the site has a very important role in the community.

Reverse complementary DNA sequences - sequences that are inadvertently given backwards with all purines and pyrimidines transposed - can affect sequence analysis detrimentally unless taken into account. We present an open-source, high-throughput software tool -v-revcomp (http://www.cmde.science.ubc.ca/mohn/software.html) - to detect and reorient reverse complementary entries of the small-subunit rRNA (16S) gene from sequencing datasets, particularly from environmental sources. The software supports sequence lengths ranging from full length down to the short reads that are characteristic of next-generation sequencing technologies. We evaluated the reliability of v-revcomp by screening all 406 781 16S sequences deposited in release 102 of the curated SILVA database and demonstrated that the tool has a detection accuracy of virtually 100%. We subsequently used v-revcomp to analyse 1 171 646 16S sequences deposited in the International Nucleotide Sequence Databases and found that about 1% of these user-submitted sequences were reverse complementary. In addition, a nontrivial proportion of the entries were otherwise anomalous, including reverse complementary chimeras, sequences associated with wrong taxa, nonribosomal genes, sequences of poor quality or otherwise erroneous sequences without a reasonable match to any other entry in the database. Thus, v-revcomp is highly efficient in detecting and reorienting reverse complementary 16S sequences of almost any length and can be used to detect various sequence anomalies.

In this study, we evaluated the specificity, distribution, and sensitivity of Prevotella strain-based (PF163 and PigBac1) and methanogen-based (P23-2) PCR assays proposed to detect swine fecal pollution in environmental waters. The assays were tested against 222 fecal DNA extracts derived from target and nontarget animal hosts and against 34 groundwater and 15 surface water samples from five different sites. We also investigated the phylogenetic diversity of 1,340 “ Bacteroidales ” 16S rRNA gene sequences derived from swine feces, swine waste lagoons, swine manure pits, and waters adjacent to swine operations. Most swine fecal samples were positive for the host-specific Prevotella- based PCR assays (80 to 87%), while fewer were positive with the methanogen-targeted PCR assay (53%). Similarly, the Prevotella markers were detected more frequently than the methanogen-targeted assay markers in waters historically impacted with swine fecal contamination. However, the PF163 PCR assay cross-reacted with 23% of nontarget fecal DNA extracts, although Bayesian statistics suggested that it yielded the highest probability of detecting pig fecal contamination in a given water sample. Phylogenetic analyses revealed previously unknown swine-associated clades comprised of clones from geographically diverse swine sources and from water samples adjacent to swine operations that are not targeted by the Prevotella assays. While deeper sequencing coverage might be necessary to better understand the molecular diversity of fecal Bacteroidales species, results of sequence analyses supported the presence of swine fecal pollution in the studied watersheds. Overall, due to nontarget cross amplification and poor geographic stability of currently available host-specific PCR assays, development of additional assays is necessary to accurately detect sources of swine fecal pollution.

Antibiotics are used in animal livestock production for therapeutic treatment of disease and at subtherapeutic levels for growth promotion and improvement of feed efficiency. It is estimated that approximately 75% of antibiotics are not absorbed by animals and are excreted in waste. Antibiotic resistance selection occurs among gastrointestinal bacteria, which are also excreted in manure and stored in waste holding systems. Land application of animal waste is a common disposal method used in the United States and is a means for environmental entry of both antibiotics and genetic resistance determinants. Concerns for bacterial resistance gene selection and dissemination of resistance genes have prompted interest about the concentrations and biological activity of drug residues and break-down metabolites, and their fate and transport. Fecal bacteria can survive for weeks to months in the environment, depending on species and temperature, however, genetic elements can persist regardless of cell viability. Phylogenetic analyses indicate antibiotic resistance genes have evolved, although some genes have been maintained in bacteria before the modern antibiotic era. Quantitative measurements of drug residues and levels of resistance genes are needed, in addition to understanding the environmental mechanisms of genetic selection, gene acquisition, and the spatiotemporal dynamics of these resistance genes and their bacterial hosts. This review article discusses an accumulation of findings that address aspects of the fate, transport, and persistence of antibiotics and antibiotic resistance genes in natural environments, with emphasis on mechanisms pertaining to soil environments following land application of animal waste effluent.

Utilization of dissolved organic matter (DOM) is thought to be the purview of heterotrophic microorganisms, but photoautotrophs can take up dissolved organic nitrogen (DON) and dissolved organic carbon (DOC). This study investigated DOC and DON uptake in a laminated cyanobacterial mat community from hypersaline Salt Pond (San Salvador, Bahamas). The total community uptake of 3H-labeled substrates was measured in the light and in the dark and under conditions of high and low salinity. Salinity was the primary control of DOM uptake, with increased uptake occurring under low-salinity, ‘freshened’ conditions. DOC uptake was also enhanced in the light as compared with the dark and in samples incubated with the photosystem II inhibitor 3(3,4-dichlorophenyl)-1, 1-dimethylurea, suggesting a positive association between photosynthetic activity and DOC uptake. Microautoradiography revealed that some DOM uptake was attributed to cyanobacteria. Cyanobacteria DOM uptake was negatively correlated with that of smaller filamentous microorganisms, and DOM uptake by individual coccoid cells was negatively correlated with uptake by colonial coccoids. These patterns of activity suggest that Salt Pond microorganisms are engaged in resource partitioning, and DOM utilization may provide a metabolic boost to both heterotrophs and photoautrophs during periods of lowered salinity.

To monitor the dissemination of resistance genes into the environment, we determined the occurrence of tetracycline resistance (Tc r ) genes in groundwater underlying two swine confinement operations. Monitoring well networks (16 wells at site A and 6 wells at site C) were established around the lagoons at each facility. Groundwater ( n = 124) and lagoon ( n = 12) samples were collected from the two sites at six sampling times from 2000 through 2003. Total DNA was extracted, and PCR was used to detect seven Tc r genes [ tet (M), tet (O), tet (Q), tet (W), tet (C), tet (H), and tet (Z)]. The concentration of Tc r genes was quantified by real-time quantitative PCR. To confirm the Tc r gene source in groundwater, comparative analysis of tet (W) gene sequences was performed on groundwater and lagoon samples. All seven Tc r genes were continually detected in groundwater during the 3-year monitoring period at both sites. At site A, elevated detection frequency and concentration of Tc r genes were observed in the wells located down-gradient of the lagoon. Comparative analysis of tet (W) sequences revealed that the impacted groundwater contained gene sequences almost identical (99.8% identity) to those in the lagoon, but these genes were not found in background libraries. Novel sequence clusters and unique indigenous resistance gene pools were also found in the groundwater. Thus, antibiotic resistance genes in groundwater are affected by swine manure, but they are also part of the indigenous gene pool.

Trees that fall into lakes from riparian forest become habitat for aquatic organisms, and are potentially an important link between terrestrial and aquatic ecosystems. Coarse woody habitat (CWH) promotes the production of benthic invertebrate prey and offers refuge for prey fishes, which are in turn consumed by piscivorous fishes. We used a simulation model to explore responses of an aquatic food web to changes in CWH caused by lakeshore residential development and windstorm, as well as to harvest of adult piscivores. Residential development had a negative effect on fishes, and could lead to extirpation of benthivorous prey fish species. In contrast, pulsed addition of CWH following a windstorm had little effect on the aquatic food web. Our results suggest that CWH is more important as shelter for prey fishes than as a substrate for benthic invertebrate production. However, piscivore harvest can supersede the role of CWH as prey shelter, leading to piscivore collapse and prey persistence even when CWH levels are low enough to promote piscivore dominance. Thus, the effects of lakeshore residential development on fishes can be masked by angler harvest of top piscivores.

Disturbance and recovery influence microbial community structure and ecosystem functions in most natural environments. This study from a hypersaline Bahamian lagoon details the response of a benthic cyanobacterial mat to disturbance by Hurricane Frances, a category-4 storm. Clone libraries of cyanobacterial small subunit r-RNA genes and nitrogenase genes revealed significant shifts in cyanobacterial and diazotroph community composition following the hurricane. Post-hurricane clone libraries were dominated by sequences that had been rare in pre-hurricane communities. In spite of this dominance shift, re-colonizing mat communities performed nitrogen fixation and photosynthesis at rates within the normal range of variation measured in the mat at similar salinities. There was a tendency for nitrogen fixation rates from mats re-colonizing sites with hurricane-related sand deposition to be higher than those from mats re-colonizing sites without significant sand deposition. This suggests that the altered communities responded to a carbon : nitrogen imbalance that was particularly pronounced in areas subjected to disturbance by sand burial. The post-hurricane dominance of organisms that had been previously rare suggests that pre-hurricane diversity and functional redundancy contributed to the rapid recovery of ecosystem function in the post-disturbance environment.

There is increasing global demand for food, bioenergy feedstocks and a wide variety of bio-based products. In response, agriculture has advanced production, but is increasingly depleting soil regulating and supporting ecosystem services. New production systems have emerged, such as no-tillage, that can enhance soil services but may limit yields. Moving forward, agricultural systems must reduce trade-offs between production and soil services. Soil functional zone management (SFZM) is a novel strategy for developing sustainable production systems that attempts to integrate the benefits of conventional, intensive agriculture and no-tillage. SFZM creates distinct functional zones within crop row and inter-row spaces. By incorporating decimetre-scale spatial and temporal heterogeneity, SFZM attempts to foster greater soil biodiversity and integrate complementary soil processes at the sub-field level. Such integration maximizes soil services by creating zones of ‘active turnover’, optimized for crop growth and yield (provisioning services); and adjacent zones of ‘soil building’, that promote soil structure development, carbon storage and moisture regulation (regulating and supporting services). These zones allow SFZM to secure existing agricultural productivity while avoiding or minimizing trade-offs with soil ecosystem services. Moreover, the specific properties of SFZM may enable sustainable increases in provisioning services via temporal intensification (expanding the portion of the year during which harvestable crops are grown). We present a conceptual model of ‘virtuous cycles’, illustrating how increases in crop yields within SFZM systems could create self-reinforcing feedback processes with desirable effects, including mitigation of trade-offs between yield maximization and soil ecosystem services. Through the creation of functionally distinct but interacting zones, SFZM may provide a vehicle for optimizing the delivery of multiple goods and services in agricultural systems, allowing sustainable temporal intensification while protecting and enhancing soil functioning.

Examination of variation in ecological communities can lead to an understanding of the forces that structure communities, the consequences of change at the ecosystem level, and the relevant scales involved. This study details spatial and seasonal variability in the composition of nitrogen-fixing and cyanobacterial (i.e., oxygenic photosynthetic) functional groups of a benthic, hypersaline microbial mat from Salt Pond, San Salvador Island, Bahamas. This system shows extreme annual variability in the salinity of the overlying water and the extent of water coverage. Analysis of molecular variance and F ST tests of genetic differentiation of nifH and cyanobacterial 16S rRNA gene clone libraries allowed for changes at multiple taxonomic levels (i.e., above, below, and at the species level) to inform the conclusions regarding these functional groups. Composition of the nitrogen-fixing community showed significant seasonal changes related to salinity, while cyanobacterial composition showed no consistent seasonal pattern. Both functional groups exhibited significant spatial variation, changing with depth in the mat and horizontally with distance from the shoreline. The patterns of change suggest that cyanobacterial composition was more insensitive to water stress, and consequently, cyanobacteria dominated the nitrogen-fixing community during dry months but gave way to a more diverse community of diazotrophs in wet months. This seasonal pattern may allow the mat community to respond quickly to water-freshening events after prolonged dry conditions (system recovery) and maintain ecosystem function in the face of disturbance during the wet season (system resilience).

This study used a genetic fingerprinting technique (automated ribosomal intergenic spacer analysis [ARISA]) to characterize microbial communities from a culture-independent perspective and to identify those environmental factors that influence the diversity of bacterial assemblages in Wisconsin lakes. The relationships between bacterial community composition and 11 environmental variables for a suite of 30 lakes from northern and southern Wisconsin were explored by canonical correspondence analysis (CCA). In addition, the study assessed the influences of ARISA fragment detection threshold (sensitivity) and the quantitative, semiquantitative, and binary (presence-absence) use of ARISA data. It was determined that the sensitivity of ARISA was influential only when presence-absence-transformed data were used. The outcomes of analyses depended somewhat on the data transformation applied to ARISA data, but there were some features common to all of the CCA models. These commonalities indicated that differences in bacterial communities were best explained by regional (i.e., northern versus southern Wisconsin lakes) and landscape level (i.e., seepage lakes versus drainage lakes) factors. ARISA profiles from May samples were consistently different from those collected in other months. In addition, communities varied along gradients of pH and water clarity (Secchi depth) both within and among regions. The results demonstrate that environmental, temporal, regional, and landscape level features interact to determine the makeup of bacterial assemblages in northern temperate lakes.

Species diversity and richness, and seasonal population dynamics of phytoplankton, planktonic protozoa, and bacterioplankton sampled from the epilimnion of Crystal Bog in 2000, were examined in order to test the hypothesis that these groups' diversity and abundance patterns might be linked. Crystal Bog, a humic lake in Vilas County, Wisconsin, is part of the North Temperate Lakes Long-Term Ecological Research Site. Phytoplankton and planktonic protozoa were identified and enumerated in a settling chamber with an inverted microscope. Bacterial cells were enumerated with the use of fluorescence 4', 6'-diamidino-2-phenylindole (DAPI)-staining procedures, and automated ribosomal intergenic spacer analysis (ARISA) was used to assess bacterioplankton diversity. Bacterial cell counts showed little seasonal variation and averaged 2.6 x 10(6) cells/mL over the ice-free season. Phytoplankton and planktonic protozoan numbers varied by up to two orders of magnitude and were most numerous in late spring and summer. Dinoflagellates largely dominated Crystal Bog throughout the ice-free period, specifically Peridiniopsis quadridens in the spring, Peridinium limbatum in summer, and Gymnodinium fuscum and P. quadridens in fall. Brief blooms of Cryptomonas, Dinobryon, and Synura occurred between periods of dinoflagellate domination. The dominant dinoflagellate, Peridinium limbatum, was calculated to have a growth rate of 0.065 day(-1) and a doubling time of 10.7 days. Heterotrophic nanoflagellates (HNFs) were a consistent component of the planktonic protozoa; seasonal patterns were determined for three genera of HNFs (Monosiga, Bicosoeca, and Desmarella moniliformis). Three genera of ciliates (Coleps, Strobilidium, and Strombidium) comprised the greater part of the planktonic protozoa in Crystal Bog. The number of species of planktonic protozoa was too low to calculate a diversity index. Shannon-Weaver diversity indices for phytoplankton and bacterioplankton in the epilimnion followed very similar seasonal patterns in this lake, supporting the hypothesis that in freshwaters, diversity patterns of these groups are linked.

Despite considerable attention in recent years, the composition and dynamics of lake bacterial communities over annual time scales are poorly understood. This study used automated ribosomal intergenic spacer analysis (ARISA) to explore the patterns of change in lake bacterial communities in three temperate lakes over 2 consecutive years. The study lakes included a humic lake, an oligotrophic lake, and a eutrophic lake, and the epilimnetic bacterial communities were sampled every 2 weeks. The patterns of change in bacterial communities indicated that seasonal forces were important in structuring the behavior of the bacterial communities in each lake. All three lakes had relatively stable community composition in spring and fall, but summer changes were dramatic. Summertime variability was often characterized by recurrent drops in bacterial diversity. Specific ARISA fragments derived from these lakes were not constant among lakes or from year to year, and those fragments that did recur in lakes in different years did not exhibit the same seasonal pattern of recurrence. Nonetheless, seasonal patterns observed in 2000 were fairly successful predictors of the rate of change in bacterial communities and in the degree of autocorrelation of bacterial communities in 2001. Thus, seasonal forces may be important structuring elements of these systems as a whole even if they are uncoupled from the dynamics of the individual system components.

Various diluents, stabilizers, buffers, and storage conditions were assessed for their efficacy in stabilizing cold-adapted influenza virus vaccine. Frozen liquid vaccine formulations, comprised of a normal uninfected allantoic fluid diluent and an SPG (sucrose-phosphate-glutamate) stabilizer, generated complete stability of H1N1, H3N2, and Type B strains for at least 1 year of storage at -20 degrees C. The ability to store live influenza virus frozen liquid vaccines, at the moderate temperature of -20 degrees C, has not been demonstrated previously. This significant advance could facilitate influenza vaccine storage and administration in the clinic, and subsequently increase marketability. The stability of lyophilized formulations was also augmented by the addition of 2% Casitone and the control of pH with 0.066 M phosphate in the SPG stabilizer. This alternative formulation may be useful in markets where freezing is not feasible or short-term room temperature storage is necessary.

This chapter provides a survey of established molecular approaches as well as emerging ones for studying microbial communities in gastrointestinal systems. Whole-genome typing methods are cost-effective alternatives for assessing genetic relatedness. These molecular techniques provide more specificity than phenotypic identification of culturable microbes and include pulsed-field gel electrophoresis (PFGE), rep-PCR, multilocus sequence typing (MLST), and octamer-based genome scanning (OGBS). The genotyping method used in recent years for Enterobacteriaceae has been a PCR-based technique using primers targeting repetitive sequences (rep-PCR), which also produces numerous DNA fragments that are separable by electrophoresis. MLST is another PCR-enabled genotyping method that has found widespread use for subtyping a variety of pathogens. The technique is an extension of multilocus enzyme electrophoresis and was first described in 1998 using Neisseria meningitidis as an example. OBGS, a powerful method enabled by genome sequence information, was developed as a result of determining the complete genome sequence of Escherichia coli. Researchers using molecular community fingerprinting methods can rapidly infer the genetic composition or community structure following the analysis of a region of amplified DNA. All community fingerprinting methods begin with extraction of total genomic DNA from the sample. Direct DNA extraction followed by PCR amplification and subsequent restriction digestion is the basis for (terminal restriction fragment length polymorphism (T-RFLP). The greatest challenge to microbial community analysis is linking the community structure at the phylogenetic level with its metabolic potential and subsequent functionality in the environment being studied.

Various manipulations to production procedures have been investigated in order to discover methods to attain adequate or augmented titers of cold-adapted influenza virus (CAIV) vaccine. The methods modified include those used for reassortant selection and the determination of virus growth parameters. Increased infectivity titers were achieved through selection of high-yielding mutants by isolating multiple plaques during plaque purification of reassortant clones, as well as through optimiztion of egg incubation times, age, and lot for individual strains. Up to 6-fold increases in virus yield were obtained by selecting high yielding mutants, up to 9-fold increases were achieved by modifying egg incubation times, and a nearly 1 log increase was realized by determining the ideal egg age for individual strains.

Hill prairies are remnant grasslands perched on the bluffs of major river valleys, and because their steep slopes make them unsuitable for traditional row crop agriculture, they have some of the lowest levels of anthropogenic disturbance of any prairie ecosystems in the Midwestern USA. However, many decades of fire suppression have allowed for shrub encroachment from the surrounding forests. While shrub encroachment of grasslands can modify soil respiration rates and nutrient storage, it is not known whether shrubs also alter the community composition of soil microorganisms. We conducted transect sampling of nine different hill prairie remnants showing varying degrees of shrub encroachment, and we used DNA-based community profiling (automated ribosomal intergenic spacer analysis) to characterize the composition of bacterial and fungal communities in the open prairie habitat, the shrub-encroached border, and the surrounding forest. While both bacterial and fungal communities showed statistically significant variation across these habitats, their predominant patterns were different. Bacterial communities of forest soils were distinct from those of the open prairie and the shrub-encroached areas, while fungal communities of the open prairie were distinct from those of the forest and the shrub-encroached border. Shrub encroachment significantly altered the community composition of soil fungal communities. Furthermore, fungal communities of heavily encroached prairie remnants more closely resembled those of the surrounding forest than those of lightly encroached prairies. Thus, shrub encroachment can cause soil fungi to shift from a “grassland” community to a “woody” community, with potential consequences for soil processes and plant-microbe interactions.

Identification of microbes that actively utilize root exudates is essential to understand plant-microbe interactions. To identify active root exudate-utilizing microorganisms associated with switchgrass - a potential bioenergy crop - plants were labelled in situ with (13) CO2 , and 16S and 18S rRNA genes in the (13) C-labelled rhizosphere DNA were pyrosequenced. Multi-pulse labelling for 5 days produced detectable (13) C-DNA, which was well separated from unlabelled DNA. Methylibium from the order Burkholderiales were the most heavily labelled bacteria. Pythium, Auricularia and Galerina were the most heavily labelled eukaryotic microbes. We also identified a Glomus intraradices-like species; Glomus members are arbuscular mycorrhizal fungi that are able to colonize the switchgrass root. All of these heavily labelled microorganisms were also among the most abundant species in the rhizosphere. Species belonging to Methylibium and Pythium were the most heavily labelled and the most abundant bacteria and eukaryotes in the rhizosphere of switchgrass. Our results revealed that nearly all of the dominant rhizosphere bacterial and eukaryotic microbes were able to utilize root exudates. The enrichment of microbial species in the rhizosphere is selective and mostly due to root exudation, which functions as a nutrition source, promoting the growth of these microbes.

Biomass production for bioenergy may change soil microbes and influence ecosystem properties. To explore the impact of different bioenergy cropping systems on soil microorganisms, the compositions and quantities of soil microbial communities (16S rRNA gene) and N-cycling functional groups (nifH, bacterial amoA, archaeal amoA and nosZ genes) were assessed under maize, switchgrass and Miscanthus x giganteus at seven sites representing a climate gradient (precipitation and temperature) in Illinois, USA. Overall, the site-to-site variation in community composition surpassed the variation due to plant type, and microbial communities under each crop did not converge on a 'typical' species assemblage. Fewer than 5% of archaeal amoA, bacterial amoA, nifH and nosZ OTUs were significantly different among these crops, but the largest differences observed at each site were found between maize and the two perennial grasses. Quantitative PCR revealed that the abundance of the nifH gene was significantly higher in the perennial grasses than in maize, and we also found significantly higher total N in the perennial grass soils than in maize. Thus, we conclude that cultivation of these perennial grasses, instead of maize, as bioenergy feedstocks can improve soil ecosystem nitrogen sustainability by increasing the population size of N-fixing bacteria.