PUMA2—grid-based high-throughput analysis of genomes and metabolic pathways

The PUMA2 system (available at ) is an interactive, integrated bioinformatics environment for high-throughput genetic sequence analysis and metabolic reconstructions from sequence data. PUMA2 provides a framework for comparative and evolutionary analysis of genomic data and metabolic networks in the context of taxonomic and phenotypic information. Grid infrastructure is used to perform computationally intensive tasks. PUMA2 currently contains precomputed analysis of 213 prokaryotic, 22 eukaryotic, 650 mitochondrial and 1493 viral genomes and automated metabolic reconstructions for >200 organisms. Genomic data is annotated with information integrated from >20 sequence, structural and metabolic databases and ontologies. PUMA2 supports both automated and interactive expert-driven annotation of genomes, using a variety of publicly available bioinformatics tools. It also contains a suite of unique PUMA2 tools for automated assignment of gene function, evolutionary analysis of protein families and comparative analysis of metabolic pathways. PUMA2 allows users to submit batch sequence data for automated functional analysis and construction of metabolic models. The results of these analyses are made available to the users in the PUMA2 environment for further interactive sequence analysis and annotation

Global meta-analysis and metagenomics approach on the soil microbiome associated with cover cropping

Soil nutrient loss is one of the major causes of soil degradation that threatens future global food security. Cover cropping is a promising sustainable agricultural method with the potential to enhance soil health and mitigate consequences of soil degradation. As one of the agricultural practices that can affect cover cropping, effects of tillage on cover cropping have been widely researched as well. Because cover cropping and tillage can form an agroecosystem distinct from that of bare fallow, the soil microbiome is hypothesized to respond to the altered environmental circumstances. Therefore, studying their impact on the soil microbiome is necessary because the soil microbes are important drivers of soil processes including those relevant to soil health. The objectives of this MS research were i) estimate the baseline effect size of cover cropping on soil microbial abundance, activity, and diversity, ii) identify environmental and agricultural factors that affect the cover crop effects sizes on the soil microbiome, iii) further understand the cover crop effects on the soil microbial diversity by investigating the shifts in the soil microbial compositions, and iv) contribute to understanding how the relationship between cover cropping and the soil microbiome may affect the soil health. A meta-analysis was conducted to estimate the global average effects of cover cropping on the soil microbiome. This study compiled the results of 60 relevant studies reporting cover cropping effects on soil microbial properties to estimate global effect sizes and explore the current landscape of this topic. Overall, cover cropping significantly increased parameters of soil microbial abundance, activity, and diversity by 27%, 22%, and 2.5% respectively, compared to those of bare fallow. Moreover, cover cropping effect sizes varied by agricultural covariates like cover crop termination or tillage methods. Notably, cover cropping effects were less pronounced under conditions like continental climate, chemical cover crop termination, and conservation tillage. This meta-analysis showed that the soil microbiome can become more robust under cover cropping when properly managed with other agricultural practices. However, more primary research is still needed to control between-study heterogeneity and to more elaborately assess the relationships between cover cropping and the soil microbiome. This meta-analysis revealed that cover cropping affect the overall soil microbial diversity and that tillage is a major cofactor that affect this relationship. To further investigate the cover cropping and tillage effects on the soil microbial diversity, a metagenomics study was conducted. This second part of the study was to observe compositional changes in the soil microbiome in response to cover cropping and tillage. Also, this study sought to identify microbial indicators that can be used to gauge responses of microbial guilds with functions relevant to soil health. This study used soil DNA data from a long-term cover cropping and tillage experiment on corn and soybean rotation in Illinois, USA. This study found that copiotrophic bacterial decomposers increased with legume cover crops and tillage, while oligotrophic and stress tolerant bacteria did so with bare fallow and no-till. Fungal groups responded to cover cropping and tillage based on their physiology, interaction with plant hosts, and nutrient strategies. This study also found an ammonia-oxidizing archaea species that increased with bare fallow. The consistent patterns that the microbial groups in this study display make them potential microbial indicators. Also, grass cover crops with no-till showed most potential for soil nutrient loss. Overall, this MS research found that cover cropping significantly enriches the soil microbiome. However, cover cropping effects may apply differential pressures on microbial groups with different adaptations so that the overall diversity is not changed significantly. This research suggests that timing and other agricultural practices like tillage need to be carefully considered to direct the changes in the soil microbiome to benefit the soil health

Diversity of cultured isolates and field populations of the arbuscular mycorrhizal fungus "Glomus intraradices" : development and application of molecular detection methods for mitochondrial haplotypes

Today’s plant communities have evolved together with arbuscular mycorrhizal fungi (AMF, Glomeromycota) for millions of years. In “arbuscular mycorrhiza”, a mutualistic symbiosis, plants provide carbohydrates to the fungi, which in turn make mineral nutrients like phosphate or nitrogen available to the plants. AMF species diversity is generally higher in natural sites than in agroecosystems, where it can be strongly reduced. The detection of AMF is either based on morphotyping of soil-borne spores or on molecular markers, which can be directly applied using colonized roots of the host plant. Until recently, studies of AMF diversity on the population level were impossible, as no suitable marker genes were available. The first population studies on AMF had to rely on DNA from spores or root organ cultures (ROCs) and the molecular markers used could not be applied for the detection of AMF genotypes directly in colonized plant roots from the field. Previous work from our laboratory had shown that mitochondrial ribosomal RNA large subunit gene (mtLSU) sequences are homogeneous within several isolates of Glomus species and that the mitochondrial gene region is a promising marker for distinguishing strains of G. intraradices. The phylotype GLOM A-1 of this morphospecies which was defined in previous studies of our laboratory based on nuclear-encoded rDNA internal transcribed spacers (ITS) sequences seems to occur ubiquitously, showing a high ecological versatility. It is frequently used as model organism and its genome is being sequenced. The aim of this thesis was to develop and apply detection methods based on the mtLSU in order to investigate the diversity of G. intraradices isolates and field populations. The main question was whether this marker is suitable to resolve the genetic structure of this morphospecies which might allow shedding light on the ecological role of strains within the species. In the first part of this thesis, the diversity of the mtLSU was investigated in a set of 16 G. intraradices isolates originating from five continents, either obtained as soil inoculum or as ROC. Among these isolates, 12 different mtLSU haplotypes could be distinguished, whereas homogeneity of the marker within the isolates was confirmed. Several mtLSU haplotypes were already distinguishable by size differences of the PCR products, mainly based on the presence or absence of length-variable introns. The reliability of the marker is dependent on evolutionary intron stability, which was confirmed for some introns by comparisons of multiple culture lineages of the same isolate obtained from different culture collections. In phylogenetic analyses of mtLSU exon sequences from isolates and root-colonizing G. intraradices, several clades could be distinguished. Comparison with ITS sequences from the isolates showed a higher resolution of mtLSU exon sequences which was increased by intron sequences. In order to increase the specificity for G. intraradices and to optimize amplification of the mtLSU fragment from colonized plant roots, a new nested PCR approach was developed and tested using field root samples from a semi-natural grassland and a mine spoil in Hungary. A RFLP approach was developed to reduce time-consuming and expensive cloning and sequencing procedures. In the second part of this thesis, the population structure of an AMF in roots from the environment was analyzed for the first time. Two agricultural field experiments in Switzerland, including different tillage treatments, and two semi-natural grasslands in Switzerland and France were chosen for the investigation of the genetic structure of G. intraradices phylotype GLOM A-1 using the PCR-RFLP approach. Each field site was dominated by one or two frequently found RFLP patterns of G. intraradices GLOM A-1, which were defined as Intra types. The composition of Intra types differed strongly between the agricultural sites and the semi-natural grasslands, but also between the two agricultural sites. In contrast to the situation often found in AMF species community studies, RFLP type richness was higher in the agricultural sites compared to the grasslands. Four Intra types, shared by different sites, were further resolved by sequence analyses, but only the two grasslands were found to share mtLSU sequence haplotypes. In phylogenetic analyses of completely sequenced examples of each Intra type, almost all haplotypes from the grassland sites fell within a separate “grassland clade”. If a single mtLSU haplotype could be specifically detected in a pool of others, such a molecular tool could be used for tracing single strains inoculated in a field site. Nested PCR primers were developed specifically for one single mtLSU haplotype, which dominated one of the agricultural sites and was known from previous studies analyzing ROCs. By applying this approach to all samples from the four study sites, it could be shown that the respective haplotype was only detected in samples previously tested positive for this type using the general approach. In other words, both methods confirmed each other. Two further specific nested PCR approaches were developed for two mtLSU haplotypes representing the G. intraradices isolate BEG140. These approaches were designed to be applied for tracing this isolate inoculated in a field experiment performed in a mine spoil bank of the Czech Republic in the context of a reclamation project. Besides the considerable genetic structure of this fungus among the isolates studied and in the roots of the field sites, evidence of specialized mtLSU haplotypes was reported, which might represent ecotypes or even different (“cryptic”) species. It could be shown that world-wide mtLSU haplotype diversity of G. intraradices is considerably higher than previously assumed. More investigations of different ecosystems are required for the determination of adapted ecotypes. The approaches developed here will be furthermore useful for instance in inoculation experiments and functional tests, e.g. in greenhouse experiments. By presenting first insights into the genetic structure of the most widespread species of arbuscular mycorrhizal fungi, the findings presented here will have major implications on our views of processes of adaptation and specialization in these plant ⁄ fungus associations

Quantification of a sensitive soil carbon constituent as affected by soil type, tillage system, and crop rotation

Differences in tillage intensity and crop rotation management practices have been shown to lead to significant changes in the chemical, physical, and biological partitioning of soil carbon over various periods of time. Although many studies have focused on the roles of microbial biomass or specific enzymes in transformative processes within soils, the underlying net potential metabolic activity of a soil system remains to be addressed in terms of a quantitatively feasible constituent of the soil carbon pool. As carbohydrates are the primary source of carbon input into soil systems and one of the degradation products of carbohydrates is reducing sugars, analysis of a soil system\u27s ability to either accumulate or release reducing sugars stands as a reasonable assessment of a soil\u27s metabolic index of soil carbon. Therefore, the objectives of this research were to develop a method by which to assess the total potential release of reducing sugars from soils and then to use that method to assess the effects of soil type, tillage system, crop rotation, time, and physio-spatial distribution on that soil organic carbon pool. The potential release of total reducing sugars from soil was quantified by incubation of surface soil (0-15 cm) in 60% methanol solution by volume at 30°C for time spans increasing in 2 h increments up to 24 h. The supernatant of eight distinct, uncultivated Iowa soils from under similar vegetation (grass areas predominated by Bromus inermis Leyss.) was colorimetrically analyzed by Somogyi-Nelson method. Release of reducing sugars from soils with respect to time fit a parabolic curve, and a double inverse transformation was done in order to calculate a total releasable reducing sugar pool (Rr) and the time it would take to release one-half of that pool (Kt). The Rr values ranged from 39 to 152 mg kg-1 field-moist soils and from 11 to 98 mg kg-1 in air-dry soils. The Kt values ranged from 3.9 to 16 h in field-moist soils and from 1.6 to 12 h in air-dry soils. Further findings of this research demonstrated that the decrease in rate of production of reducing sugars as the value of total reducing sugars released reached Rr was due to a decrease in the substrate pool from which these monosaccharides were nascent. After five days of incubation, concentrations of total reducing sugars released matched calculated Rr values. Therefore, incubation of field-moist surface soil (0-15 cm) for five days at 30°C in 60% methanol solution was used to estimate Rr and to assess the impacts of soil type at different locations, tillage system, and crop rotation on the total potential reducing sugar carbon pool. Tillage, crop rotation, and location all significantly impacted the concentration of releasable reducing sugar in soils. On average, soil from the no-till system exhibited mean concentrations of 7.5 and 19.9 mg kg-1 soil more releasable reducing sugars than chisel-plow and moldboard plow tillage systems, respectively. The chisel-plow tillage system had concentrations that were, on average, 12.3 mg kg-1 soil greater than the moldboard plow tillage system. In general, releasable reducing sugar concentrations were 2.4 mg kg-1 soil greater in continuous corn than corn-soybean rotation. Although of different magnitude, the trends of these management effects were the same regardless of location. The effects of a temporal variable on these releasable reducing sugar concentrations were significant; the impact of one single spring secondary tillage treatment was assessed and was found to be significant. Regardless of tillage management system, results show that releasable reducing sugar concentrations in soils had, on average, significantly decreased the following spring when compared to concentrations analyzed from samples that were collected the previous fall. Furthermore, when soil reducing sugar concentrations of spring baseline (prior to spring secondary tillage), were compared to concentrations after a single secondary tillage pass, concentrations averaged 18% lower in the moldboard plow tillage system, 6.9% lower in the chisel plow tillage system, and 9% greater in the no-till system (which was used as a control). Changes in reducing sugar concentrations during this six-day time period in the corn-soybean rotation, were as follows: decreases of 18.7% in reducing sugar concentration with moldboard plow tillage system, 8.3% with the chisel plow tillage system, and an increase of 11% with no-till were noted compared to decreases of 17.4%, 5.4%, and an increase of 6.9% for the same tillage treatments, respectively, in the continuous corn cropping system. Analysis of the physio-spatial distribution of releasable reducing sugars in field-moist soil aggregates from no-till, chisel plow, and moldboard plow surface (0-7.5 cm) and subsurface (7.5-15 cm) soil samples demonstrated that soil aggregates of size fractions 1-2 and 2-4 mm held the greatest concentrations of releasable reducing sugars. A stratification effect was noted in the no-till system, where the average concentration of releasable reducing sugars from all aggregate fractions was 63.9 mg kg-1 in the top 0-7.5 cm surface soil and 33.4 mg kg-1 in the 7.5-15 cm subsurface soil depth. Average concentrations were more homogenized in the other tillage systems with greater concentrations in subsurface soil (7.5-15 cm), and significantly greater concentrations in chisel plow subsurface soil depth than in the moldboard plow subsurface soil depth. Greater potential reducing sugar values in no-till tillage systems lend support to the hypotheses of increased carbon sequestration and organic matter resilience associated with decreased disturbance of the soil. Overall, these findings provided evidence that the method developed for analysis of total releasable reducing sugars is a sensitive method for detecting and quantifying impacts of land-use, management practices, and crop rotations on soil carbon stocks, and should be useful in further study of mechanisms that regulate the transformative processes of soil carbon

Using Growing Self-Organising Maps to Improve the Binning Process in Environmental Whole-Genome Shotgun Sequencing

Metagenomic projects using whole-genome shotgun (WGS) sequencing produces many unassembled DNA sequences and small contigs. The step of clustering these sequences, based on biological and molecular features, is called binning. A reported strategy for binning that combines oligonucleotide frequency and self-organising maps (SOM) shows high potential. We improve this strategy by identifying suitable training features, implementing a better clustering algorithm, and defining quantitative measures for assessing results. We investigated the suitability of each of di-, tri-, tetra-, and pentanucleotide frequencies. The results show that dinucleotide frequency is not a sufficiently strong signature for binning 10 kb long DNA sequences, compared to the other three. Furthermore, we observed that increased order of oligonucleotide frequency may deteriorate the assignment result in some cases, which indicates the possible existence of optimal species-specific oligonucleotide frequency. We replaced SOM with growing self-organising map (GSOM) where comparable results are obtained while gaining 7%–15% speed improvement

Climate change promotes parasitism in a coral symbiosis.

Coastal oceans are increasingly eutrophic, warm and acidic through the addition of anthropogenic nitrogen and carbon, respectively. Among the most sensitive taxa to these changes are scleractinian corals, which engineer the most biodiverse ecosystems on Earth. Corals' sensitivity is a consequence of their evolutionary investment in symbiosis with the dinoflagellate alga, Symbiodinium. Together, the coral holobiont has dominated oligotrophic tropical marine habitats. However, warming destabilizes this association and reduces coral fitness. It has been theorized that, when reefs become warm and eutrophic, mutualistic Symbiodinium sequester more resources for their own growth, thus parasitizing their hosts of nutrition. Here, we tested the hypothesis that sub-bleaching temperature and excess nitrogen promotes symbiont parasitism by measuring respiration (costs) and the assimilation and translocation of both carbon (energy) and nitrogen (growth; both benefits) within Orbicella faveolata hosting one of two Symbiodinium phylotypes using a dual stable isotope tracer incubation at ambient (26 °C) and sub-bleaching (31 °C) temperatures under elevated nitrate. Warming to 31 °C reduced holobiont net primary productivity (NPP) by 60% due to increased respiration which decreased host %carbon by 15% with no apparent cost to the symbiont. Concurrently, Symbiodinium carbon and nitrogen assimilation increased by 14 and 32%, respectively while increasing their mitotic index by 15%, whereas hosts did not gain a proportional increase in translocated photosynthates. We conclude that the disparity in benefits and costs to both partners is evidence of symbiont parasitism in the coral symbiosis and has major implications for the resilience of coral reefs under threat of global change

Genomic Reconstruction of an Uncultured Hydrothermal Vent Gammaproteobacterial Methanotroph (Family Methylothermaceae) Indicates Multiple Adaptations to Oxygen Limitation

Hydrothermal vents are an important contributor to marine biogeochemistry, producing large volumes of reduced fluids, gasses, and metals and housing unique, productive microbial and animal communities fueled by chemosynthesis. Methane is a common constituent of hydrothermal vent fluid and is frequently consumed at vent sites by methanotrophic bacteria that serve to control escape of this greenhouse gas into the atmosphere. Despite their ecological and geochemical importance, little is known about the ecophysiology of uncultured hydrothermal vent-associated methanotrophic bacteria. Using metagenomic binning techniques, we recovered and analyzed a near-complete genome from a novel gammaproteobacterial methanotroph (B42) associated with a white smoker chimney in the Southern Lau basin. B42 was the dominant methanotroph in the community, at ∼80x coverage, with only four others detected in the metagenome, all on low coverage contigs (7x–12x). Phylogenetic placement of B42 showed it is a member of the Methylothermaceae, a family currently represented by only one sequenced genome. Metabolic inferences based on the presence of known pathways in the genome showed that B42 possesses a branched respiratory chain with A- and B-family heme copper oxidases, cytochrome bd oxidase and a partial denitrification pathway. These genes could allow B42 to respire over a wide range of oxygen concentrations within the highly dynamic vent environment. Phylogenies of the denitrification genes revealed they are the result of separate horizontal gene transfer from other Proteobacteria and suggest that denitrification is a selective advantage in conditions where extremely low oxygen concentrations require all oxygen to be used for methane activation

Assessing the agronomic and ecological relevance of mineral-associated organic matter

As the largest terrestrial sink for carbon (C) and a critical source of nitrogen (N) for plants, soil organic matter (SOM) is a major driver of ecosystem function. It is critical to understand the mechanistic controls on SOM in order to improve models of global C cycling and to develop accurate measures of soil fertility. SOM consists of a wide spectrum of compounds, varying in chemical characteristics and function. The chemical and physical fractionation of SOM is a valuable tool for distilling this complexity into meaningful and distinct pools: detrital or particulate organic matter (POM), which contains mostly recent litter inputs at early stages of decomposition, and mineral-associated organic matter (MAOM), which is far more processed, consisting of small organic compounds bound to reactive mineral surfaces. For decades, MAOM has been studied primarily for its capacity to sequester soil C and N. In this dissertation, my research reveals the under-appreciated role of clay minerals in mediating the short-term accrual and turnover of SOM. I examine the mechanistic controls on MAOM and specifically, how agricultural management and plant-microbe interactions influence C and N within MAOM. Agricultural practices can directly impact the capacity for soils to store MAOM. Approaches that minimize soil disturbance, such as conservation tillage, and those that increase crop residue input and diversity, such as cover cropping, can facilitate the rapid accrual of N within MAOM (Chapter 1). Through this research, I found that MAOM N may also be an important, but overlooked, source of N for crops. This work led me to develop a conceptual framework in which I synthesized literature from the fields of geochemistry and soil biology to investigate the potential mechanisms that drive MAOM turnover. Although this conceptual work stands alone (Chapter 2), the hypotheses and ideas therein form the basis for my experimental work. My overarching hypothesis addresses the biochemical strategies that plants employ to disrupt mineral-organic interactions and release both C and N from MAOM. Specifically, I examine two mechanisms by which plant root inputs may stimulate the destabilization and turnover of both C and N within MAOM: belowground root C inputs, specifically in the form of sugars and organic acids, can stimulate MAOM decomposition through indirect and direct mechanisms, respectively. Through a series of laboratory incubations, I demonstrate that simulated root exudates can stimulate the mobilization of both C and N from MAOM through microbial and non-microbial pathways (Chapter 3). Additions of a sugar substrate, glucose, were associated with the microbial-mediated mineralization of C and N from MAOM. The organic acid substrate, oxalic acid, was associated with the direct and concomitant mobilization of DON and metals into exchangeable and soluble pools. Most notably, both substrates stimulated the respiration of MAOM-C (i.e., positive priming), with total increases ranging from 35–89%. Our results provide evidence for pathways of MAOM destabilization, and more generally reveal that a pool of soil nutrients generally considered passive or inert has the potential to function as a significant source of C and N

Community Ecology and Sirex noctilio: Interactions with Microbial Symbionts and Native Insects

Sirex noctilio is an invasive woodwasp with a global distribution that feeds on the sapwood of pine trees. Wood-feeding in the basal Hymenoptera (sawflies) arose out of sequential adaptations to feeding on nutrient poor and digestively refractive internal plant organs (xylem). Symbiotic association with White-rot fungi are thought to aid overcoming nutritional and digestive barriers, including exceedingly low nitrogen (N) and refractory lignocellulosic polymers. In this dissertation I evaluate wood-feeding relative to nutrition, symbiosis and biotic resistance to invasion of exotic North American habitats in Sirex noctilio [Hymenoptera: Siricidae]. I evaluated nutrient relations within fungal mutualism using: 1) functional morphological analysis of insect feeding, 2) sterol molecules to determine diet sources and 3) metagenomic and isotopic analyses for discovery of novel microbial associates and their associated nutrient pathways. Nutritional constraints of wood feeding are potentially compounded by the presence of diverse fungal and insect communities as they divide the tree resource. I examined the role biotic resistance to Sirex and its fungal mutualist, Amylostereum, in North America using field and laboratory experiments. Morphological evidence supported a role for Amylostereum in external digestion of wood. Observational evidence confirmed Sirex larvae did not ingest wood biomass but preferentially extracted liquid substances via specialized structures of mandibles. Sterol analysis indicated plant compounds as the primary constituent of the diet, while metagenomic analysis of bacteria and their metabolic pathways showed a bacterial microbiome adapted to short chain plant polymers, starch and sugar metabolism. Stable isotopes suggested an additional symbiotic association with nitrogen fixing bacteria enriched the nitrogen deficient food substrate. These studies point toward herbivory with microbial supplementation of nutrients as a tri-partite relationship, pending conclusive identification of the bacterial symbiont for Sirex. Specific constraints of wood feeding by the Sirex-Amylostereum symbiotic complex were antagonized by intraguild predation and fungal competition in North America. Competition interfered with Amylostereum, while intraguild predation accounted for an additional 15% mortality of larval populations. This research describes the evolutionary role of microbial symbionts in wood-feeding in the Hymenoptera and the internal and external constraints to foraging this ubiquitous, yet nutrient poor food resource