Plant Diversity and Fertilizer Management Shape the Belowground Microbiome of Native Grass Bioenergy Feedstocks.

Plants may actively cultivate microorganisms in their roots and rhizosphere that enhance their nutrition. To develop cropping strategies that substitute mineral fertilizers for beneficial root symbioses, we must first understand how microbial communities associated with plant roots differ among plant taxa and how they respond to fertilization. Arbuscular mycorrhizal (AM) fungi and rhizobacteria are of particular interest because they enhance nutrient availability to plants and perform a suite of nutrient cycling functions. The purpose of this experiment is to examine the root and soil microbiome in a long-term switchgrass (Panicum virgatum) biofuel feedstock experiment and determine how AM fungi and rhizobacteria respond to plant diversity and soil fertility. We hypothesize that intra- and interspecific plant diversity, nitrogen fertilization (+N), and their interaction will influence the biomass and community composition of AM fungi and rhizobacteria. We further hypothesize that +N will reduce the abundance of nitrogenase-encoding nifH genes on the rhizoplane. Roots and soils were sampled from three switchgrass cultivars (Cave-in-Rock, Kanlow, Southlow) grown in monoculture, intraspecific mixture, and interspecific planting mixtures with either Andropogon gerardii or diverse native tallgrass prairie species. Molecular sequencing was performed on root and soil samples, fatty acid extractions were assessed to determine microbial biomass, and quantitative polymerase chain reaction (qPCR) was performed on nifH genes from the rhizoplane. Sequence data determined core AM fungal and bacterial microbiomes and indicator taxa for plant diversity and +N treatments. We found that plant diversity and +N influenced AM fungal biomass and community structure. Across all plant diversity treatments, +N reduced the biomass of AM fungi and nifH gene abundance by more than 40%. The AM fungal genus Scutellospora was an indicator for +N, with relative abundance significantly greater under +N and in monoculture treatments. Community composition of rhizobacteria was influenced by plant diversity but not by +N. Verrucomicrobia and Proteobacteria were the dominant bacterial phyla in both roots and soils. Our findings provide evidence that soil fertility and plant diversity structure the root and soil microbiome. Optimization of soil communities for switchgrass production must take into account differences among cultivars and their unique responses to shifts in soil fertility.

Genotypic diversity effects on biomass production in native perennial bioenergy cropping systems.

The perennial grass species that are being developed as biomass feedstock crops harbor extensive genotypic diversity, but the effects of this diversity on biomass production are not well understood. We investigated the effects of genotypic diversity in switchgrass (Panicum virgatum) and big bluestem (Andropogon gerardii) on perennial biomass cropping systems in two experiments conducted over 2008-2014 at a 5.4-ha fertile field site in northeastern Illinois, USA. We varied levels of switchgrass and big bluestem genotypic diversity using various local and nonlocal cultivars - under low or high species diversity, with or without nitrogen inputs - and quantified establishment, biomass yield, and biomass composition. In one experiment ('agronomic trial'), we compared three switchgrass cultivars in monoculture to a switchgrass cultivar mixture and three different species mixtures, with or without N fertilization. In another experiment ('diversity gradient'), we varied diversity levels in switchgrass and big bluestem (1, 2, 4, or 6 cultivars per plot), with one or two species per plot. In both experiments, cultivar mixtures produced yields equivalent to or greater than the best cultivars. In the agronomic trial, the three switchgrass mixture showed the highest production overall, though not significantly different than best cultivar monoculture. In the diversity gradient, genotypic mixtures had one-third higher biomass production than the average monoculture, and none of the monocultures were significantly higher yielding than the average mixture. Year-to-year variation in yields was lowest in the three-cultivar switchgrass mixtures and Cave-In-Rock (the southern Illinois cultivar) and also reduced in the mixture of switchgrass and big bluestem relative to the species monocultures. The effects of genotypic diversity on biomass composition were modest relative to the differences among species and genotypes. Our findings suggest that local genotypes can be included in biomass cropping systems without compromising yields and that genotypic mixtures could help provide high, stable yields of high-quality biomass feedstocks.

Fungi in the future: interannual variation and effects of atmospheric change on arbuscular mycorrhizal fungal communities.

Understanding the natural dynamics of arbuscular mycorrhizal (AM) fungi and their response to global environmental change is essential for the prediction of future plant growth and ecosystem functions. We investigated the long-term temporal dynamics and effect of elevated atmospheric carbon dioxide (CO2 ) and ozone (O3 ) concentrations on AM fungal communities. Molecular methods were used to characterize the AM fungal communities of soybean (Glycine max) grown under elevated and ambient atmospheric concentrations of both CO2 and O3 within a free air concentration enrichment experiment in three growing seasons over 5 yr. Elevated CO2 altered the community composition of AM fungi, increasing the ratio of Glomeraceae to Gigasporaceae. By contrast, no effect of elevated O3 on AM fungal communities was detected. However, the greatest compositional differences detected were between years, suggesting that, at least in the short term, large-scale interannual temporal dynamics are stronger mediators than atmospheric CO2 concentrations of AM fungal communities. We conclude that, although atmospheric change may significantly alter AM fungal communities, this effect may be masked by the influences of natural changes and successional patterns through time. We suggest that changes in carbon availability are important determinants of the community dynamics of AM fungi.

Mycorrhizal phenotypes and the Law of the Minimum.

Mycorrhizal phenotypes arise from interactions among plant and fungal genotypes and the environment. Differences in the stoichiometry and uptake capacity of fungi and plants make arbuscular mycorrhizal (AM) fungi inherently more nitrogen (N) limited and less phosphorus (P) limited than their host plants. Mutualistic phenotypes are most likely in P-limited systems and commensal or parasitic phenotypes in N-limited systems. Carbon (C) limitation is expected to cause phenotypes to shift from mutualism to commensalism and even parasitism. Two experiments compared the influence of fertilizer and shade on mycorrhizas in Andropogon gerardii across three naturally N-limited or P-limited grasslands. A third experiment examined the interactive effects of N and P enrichment and shade on A. gerardii mycorrhizas. Our experiments generated the full spectrum of mycorrhizal phenotypes. These findings support the hypothesis that mutualism is likely in P-limited systems and commensalism or parasitism is likely in N-limited systems. Furthermore, shade decreased C-assimilation and generated less mutualistic mycorrhizal phenotypes with reduced plant and fungal biomass. Soil fertility is a key controller of mycorrhizal costs and benefits and the Law of the Minimum is a useful predictor of mycorrhizal phenotype. In our experimental grasslands arbuscular mycorrhizas can ameliorate P-limitation but not N-limitation.

Modeling forest ecosystem responses to elevated carbon dioxide and ozone using artificial neural networks.

Rising atmospheric levels of carbon dioxide and ozone will impact productivity and carbon sequestration in forest ecosystems. The scale of this process and the potential economic consequences provide an incentive for the development of models to predict the types and rates of ecosystem responses and feedbacks that result from and influence of climate change. In this paper, we use phenotypic and molecular data derived from the Aspen Free Air CO2 Enrichment site (Aspen-FACE) to evaluate modeling approaches for ecosystem responses to changing conditions. At FACE, it was observed that different aspen clones exhibit clone-specific responses to elevated atmospheric levels of carbon dioxide and ozone. To identify the molecular basis for these observations, we used artificial neural networks (ANN) to examine above and below-ground community phenotype responses to elevated carbon dioxide, elevated ozone and gene expression profiles. The aspen community models generated using this approach identified specific genes and subnetworks of genes associated with variable sensitivities for aspen clones. The ANN model also predicts specific co-regulated gene clusters associated with differential sensitivity to elevated carbon dioxide and ozone in aspen species. The results suggest ANN is an effective approach to predict relevant gene expression changes resulting from environmental perturbation and provides useful information for the rational design of future biological experiments.

Resource limitation is a driver of local adaptation in mycorrhizal symbioses.

Symbioses may be important mechanisms of plant adaptation to their environment. We conducted a reciprocal inoculation experiment to test the hypothesis that soil fertility is a key driver of local adaptation in arbuscular mycorrhizal (AM) symbioses. Ecotypes of Andropogon gerardii from phosphorus-limited and nitrogen-limited grasslands were grown with all possible "home and away" combinations of soils and AM fungal communities. Our results indicate that Andropogon ecotypes adapt to their local soil and indigenous AM fungal communities such that mycorrhizal exchange of the most limiting resource is maximized. Grasses grown in home soil and inoculated with home AM fungi produced more arbuscules (symbiotic exchange structures) in their roots than those grown in away combinations. Also, regardless of the host ecotype, AM fungi produced more extraradical hyphae in their home soil, and locally adapted AM fungi were, therefore, able to sequester more carbon compared with nonlocal fungi. Locally adapted mycorrhizal associations were more mutualistic in the two phosphorus-limited sites and less parasitic at the nitrogen-limited site compared with novel combinations of plants, fungi, and soils. To our knowledge, these findings provide the strongest evidence to date that resource availability generates evolved geographic structure in symbioses among plants and soil organisms. Thus, edaphic origin of AM fungi should be considered when managing for their benefits in agriculture, ecosystem restoration, and soil-carbon sequestration.

The importance of soil microorganisms for maintaining diverse plant communities in tallgrass prairie.

PREMISE OF THE STUDY: According to the "Janzen-Connell hypothesis," soil microorganisms have the potential to increase plant community diversity by mediating negative feedback on plant growth. Evidence for such microbe-driven negative feedback has been found in a variety of terrestrial systems. However, it is currently unknown how general this phenomenon is within most plant communities. Also unknown is the role of mutualists in generating such feedback: do they decrease the influence of soil-mediated negative feedback on plant fitness or do they increase its effect by proliferating with plant hosts to which they give the least benefit? • METHODS: We investigated soil-microbe-mediated feedback via a series of reciprocal transplant experiments in the greenhouse using soil from a restored tallgrass prairie and native tallgrass prairie plant species. • KEY RESULTS: We found that negative feedback was very common but that mutualists (arbuscular mycorrhizal fungi) influence plant growth in opposition to the overall negative feedback trend. • CONCLUSIONS: Widespread microbially mediated negative feedback indicates that plant community diversity and composition in tallgrass prairie are dependent on soil microorganisms. Native soil microorganisms should be considered in restoration efforts of tallgrass prairie and, potentially, other native plant communities.

The GAAS metagenomic tool and its estimations of viral and microbial average genome size in four major biomes.

Metagenomic studies characterize both the composition and diversity of uncultured viral and microbial communities. BLAST-based comparisons have typically been used for such analyses; however, sampling biases, high percentages of unknown sequences, and the use of arbitrary thresholds to find significant similarities can decrease the accuracy and validity of estimates. Here, we present Genome relative Abundance and Average Size (GAAS), a complete software package that provides improved estimates of community composition and average genome length for metagenomes in both textual and graphical formats. GAAS implements a novel methodology to control for sampling bias via length normalization, to adjust for multiple BLAST similarities by similarity weighting, and to select significant similarities using relative alignment lengths. In benchmark tests, the GAAS method was robust to both high percentages of unknown sequences and to variations in metagenomic sequence read lengths. Re-analysis of the Sargasso Sea virome using GAAS indicated that standard methodologies for metagenomic analysis may dramatically underestimate the abundance and importance of organisms with small genomes in environmental systems. Using GAAS, we conducted a meta-analysis of microbial and viral average genome lengths in over 150 metagenomes from four biomes to determine whether genome lengths vary consistently between and within biomes, and between microbial and viral communities from the same environment. Significant differences between biomes and within aquatic sub-biomes (oceans, hypersaline systems, freshwater, and microbialites) suggested that average genome length is a fundamental property of environments driven by factors at the sub-biome level. The behavior of paired viral and microbial metagenomes from the same environment indicated that microbial and viral average genome sizes are independent of each other, but indicative of community responses to stressors and environmental conditions.

Scale-dependent niche axes of arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal fungi (AMF) are mutualistic with most species of plants and are known to influence plant community diversity and composition. To better understand natural plant communities and the ecological processes they control it is important to understand what determines the distribution and diversity of AMF. We tested three putative niche axes: plant species composition, disturbance history, and soil chemistry against AMF species composition to determine which axis correlated most strongly with a changing AMF community. Due to a scale dependency we were not able to absolutely rank their importance, but we did find that each correlated significantly with AMF community change at our site. Among soil properties, pH and NO(3) were found to be especially good predictors of AMF community change. In a similar analysis of the plant community we found that time since disturbance had by far the largest impact on community composition.

Decline of arbuscular mycorrhizal fungi in northern hardwood forests exposed to chronic nitrogen additions.

Arbuscular mycorrhizal (AM) fungi are important below-ground carbon (C) sinks that can be sensitive to increased nitrogen (N) availability. The abundance of AM fungi (AMF) was estimated in maple (Acer spp.) fine roots following more than a decade of experimental additions of N designed to simulate chronic atmospheric N deposition. Abundance of AMF was measured by staining and ocular estimation, as well as by analyzing for the AMF indicator fatty acid 16:1omega5c in phospholipid (biomass indicator) and neutral lipid (lipid storage indicator) fractions. Arbuscular mycorrhizal fungal biomass, storage structures and lipid storage declined in response to N addition measured by both methods. This pattern was found when AM response was characterized as colonization intensity, on an areal basis and in proportion to maple above-ground biomass. The phospholipid fraction of the fatty acid 16:1omega5c was positively correlated with total AMF colonization and the neutral lipid fraction with vesicle colonization. Decreased AMF abundance with simulated N deposition suggests reduced C allocation to these fungi or a direct soil N-mediated decline. The fatty acid (phospholipid and neutral lipid fractions) 16:1omega5c was found to be a good indicator for AMF active biomass and stored energy, respectively.