Spatial and temporal variations of microorganisms in grassland soils : influences of land-use intensity, plants and soil properties

Grassland ecosystems provide a wide range of services to human societies (Allan et al., 2015) and plants and soil microorganisms have been identified as key drivers of ecosystem functioning (Soliveres et al., 2016). Therefore, understanding soil microbial distributions and processes in agricultural grassland soils is crucial for characterizing these ecosystems and for predicting how they may shift in a changing environment. Yet we are only beginning to understand these complex ecosystems, which account for about 26% of the worlds terrestrial surface (FAOSTATS, 2018), making it especially urgent to gain better insights into the effects of land-use intensity on soil microbial properties and plant-microbe interactions. This thesis was conducted to evaluate the impact land-use intensity has on soil microbial biogeography of grasslands with respect to both spatial patterns and temporal changes in soil microbial abundance, function (in terms of enzyme activities), and community composition. It also investigated the relationships between plants and the spatial and temporal distributions of soil microorganisms. Thereby both, land-use intensity effects and plant-microbe interactions, were assessed in light of ecological niche and neutral theory. This thesis is based on three observational studies conducted on from one to 150 continuously farmed, un-manipulated grassland sites in three regions of Germany within the Biodiversity Exploratories project (DFG priority program 1374). The first study assessed the effects of land-use intensity and physico-chemical soil properties on the spatial biogeography of soil microbial abundance and function in 18 grasslands sites from two of the three regions, sampled at one time point. The second study analyzed spatial and temporal distributions of alpha- and beta-diversity of arbuscular mycorrhizal fungi in a low land-use intensity grassland with six sampling time points across one season. The third study investigated both legacy and short-term change effects of land-use intensity, soil physico-chemical properties, plant functional traits, and plant biomass properties on temporal changes in soil microbial abundance, function, and community composition in 150 grassland sites across three regions, with particular regard to direct and indirect land-use intensity effects. Although the three studies used different approaches and assessed different soil microbial properties, general patterns were detectable. Abiotic soil properties, namely pH, nitrogen content, texture, and bulk density played fundamental roles for spatial and temporal microbial biogeography. Since these factors were specific and unique for each investigated site, they formed the background based on which other processes occurred. In addition to abiotic soil properties, impacts of land-use intensity and plants were detected, though to various degrees in the three studies. Land-use intensity played a much smaller role than anticipated in the first and third study. No influence on the spatial distribution of soil microbial abundance and function could be detected in the first study. In the third study, short-term changes in and legacy effects of land-use intensity played a minor role with respect to short-term changes in soil microbial abundance, function, and community composition. Where detected, changes in land-use intensity had a direct and negative effect on soil microbial properties in structural equation modelling; i.e., increases in land-use intensity reduced, e.g., soil microbial enzyme activities, while legacy effects of land-use intensity were shown to act both directly and indirectly on soil microbial properties. Thereby indirect legacy effects were mediated via plant functional traits. Only one of the three studies detected minor plant diversity effects on soil microbial properties. Instead, functional properties of the plant communities, i.e., plant functional traits, biomass, and nutritional quality, were significantly related to spatial and temporal distributions of soil microorganisms. Finally, the findings of the three studies suggest that processes related to niche and neutral theory both drive spatial and temporal patterns of soil microbial properties at the investigated plot scale (up to 50 m × 50 m). This thesis concluded that in order to gain deeper insights into the complex functions and processes occurring in grassland ecosystems, a multidisciplinary approach investigating fundamental physico-chemical site characteristics, microbial soil properties, and plants is necessary. The results of the thesis suggest that focus be turned to functional properties of plant and microbial communities, as they are closely intermingled, provide more detailed insights into plant-microbe interactions, and are able to reflect effects of human impacts on grassland soils better than diversity measures.Die vorliegende Dissertation diente dem Zweck, die Einflüsse der Landnutzungsintensität von Grünländern auf die Biogeographie von Bodenmikroorganismen in Bezug auf die räumliche Verteilung und zeitliche Veränderung von mikrobieller Biomasse, Funktion und Gemeinschaftsstrukturen aufzudecken und die Beziehungen zwischen Pflanzen und den räumlichen und zeitlichen Verteilungsmustern von Bodenmikroorganismen zu untersuchen. Dabei wurde auch betrachtet, inwieweit die Verteilung der Mikroorganismen den Konzepten der ökologischen Nischen- und Neutraltheorie entspricht. Die Dissertation basiert auf drei beobachtenden Studien, die auf einer bis 150 dauerhaft bewirtschafteten und nicht manipulierten Grünlandflächen durchgeführt wurden. Die Flächen sind verteilt auf drei Regionen in Deutschland und Teil des Schwerpunktforschungsprogramms Biodiversitäts-Exploratorien der Deutschen Forschungsgemeinschaft (DFG 1374). Die erste Studie untersuchte die Effekte von Landnutzungsintensität und physikalisch-chemischen Bodeneigenschaften auf die räumliche Biogeographie von mikrobieller Biomasse und Funktion zu einem einzelnen Zeitpunkt in 18 Grünlandflächen, die auf zwei Regionen verteilt sind. Die zweite Studie analysierte die räumliche und zeitliche Verteilung der Alpha- und Betadiversität arbuskulärer Mykorrhizapilze (AMF) in einem extensiv genutzten Grünland mittels sechs Probennahmezeitpunkten verteilt über die Vegetationsperiode eines Jahres. Die dritte Studie untersuchte sogenannte Legacy und kurzfristige Effekte von veränderter Landnutzungsintensität, physikalisch-chemischen Bodeneigenschaften, funktionellen Pflanzeneigenschaften und Charakteristika der pflanzlichen Biomasse auf zeitliche Veränderungen von mikrobieller Biomasse, Funktion und Gemeinschaftsstruktur in 150 Grünlandböden verteilt auf drei Regionen, sowie direkte und indirekte Effekte der Landnutzungsintensität. Obwohl alle drei Studien unterschiedliche Ansätze verfolgten und verschiedene bodenmikrobielle Eigenschaften untersuchten, sind generelle Muster erkennbar: abiotische Bodeneigenschaften, namentlich pH-Wert, Stickstoffgehalt, Textur und Lagerungsdichte, waren für die räumliche und zeitliche Biogeographie der Bodenmikroorganismen von fundamentaler Bedeutung. Sie waren spezifisch für jede der untersuchten Flächen und bildeten den Hintergrund, vor dem sich andere Prozesse abspielten. Zusätzlich zu den abiotischen Bodeneigenschaften wurden, wenn auch in unterschiedlichem Maße, Einflüsse von Landnutzungsintensität und Pflanzen in den drei Studien detektiert. Die Landnutzungsintensität spielte dabei eine wesentlich geringere Rolle als ursprünglich angenommen. Sie hatte keinen Einfluss auf die räumliche Verteilung der bodenmikrobiellen Biomasse oder Funktion in der ersten Studie. In der dritten Studie waren kurzfristige Veränderungen und Legacy-Effekte nur in geringem Maße mit kurzfristigen Veränderungen von bodenmikrobieller Biomasse, Funktion und Gemeinschaftsstruktur assoziiert. Die Strukturgleichungsmodelle zeigen, dass sich die Effekte von kurzfristigen Veränderung der Landnutzungsintensität, dort wo sie auftraten, direkt und negative auf die kurzfristigen Veränderungen bodenmikrobieller Eigenschaften auswirkten. Dahingegen wirkten sich Legacy-Effekte der Landnutzungsintensität sowohl direkt als auch indirekt auf bodenmikrobielle Eigenschaften aus. Die indirekten Legacy-Effekte wurden dabei über funktionelle Pflanzeneigenschaften auf die Mikroorganismen übertragen. Nur eine der drei Studien fand einen marginalen Einfluss der Pflanzendiversität auf die bodenmikrobiellen Eigenschaften. Stattdessen waren funktionelle Eigenschaften der Pflanzengemeinschaften sowie deren Biomasse und Futterqualität signifikant mit der räumlichen und zeitlichen Verteilung von Bodenmikroorganismen verbunden. In Bezug auf die Nischen- und Neutraltheorie sprechen die Ergebnisse der drei Studien dafür, dass sowohl Prozesse, die mit der Nischentheorie zusammenhängen, als auch solche, die mit der Neutraltheorie in Verbindung stehen, die räumliche und zeitliche Verteilung von bodenmikrobiellen Eigenschaften auf der untersuchten Plotskala (bis 50 m × 50 m) steuern. Diese Dissertation zieht die Schlussfolgerung, dass multidisziplinäre Forschung notwendig ist, um die komplexen Funktionen und Prozesse von Grünlandökosystemen zu erforschen. Diese müssen sowohl die fundamentalen physikalischen und chemischen Eigenschaften der Böden, als auch die Eigenschaften der Bodenmikroorganismen und Pflanzengemeinschaften umfassen. Dabei sprechen die Ergebnisse dieser Arbeit dafür, dass ein besonderes Augenmerk auf die funktionellen Eigenschaften von Pflanzen- und Mikrobengemeinschaften gelegt werden sollte, da diese eng miteinander verflochten sind und bessere Einblicke in die Interaktionen zwischen Pflanzen und Mikroorganismen gewähren sowie besser in der Lage sind die Effekte von menschlichen Einflüssen auf Grünlandböden wieder zu spiegeln, als die bisher oft üblichen Diversitätsmessungen

Unraveling spatiotemporal variability of arbuscular mycorrhizal fungi in a temperate grassland plot

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Goldmann, K., Boeddinghaus, R. S., Klemmer, S., Regan, K. M., Heintz-Buschart, A., Fischer, M., Prati, D., Piepho, H., Berner, D., Marhan, S., Kandeler, E., Buscot, F., & Wubet, T. Unraveling spatiotemporal variability of arbuscular mycorrhizal fungi in a temperate grassland plot. Environmental Microbiology, 22(3),(2020): 873-888, doi:10.1111/1462-2920.14653.Soils provide a heterogeneous environment varying in space and time; consequently, the biodiversity of soil microorganisms also differs spatially and temporally. For soil microbes tightly associated with plant roots, such as arbuscular mycorrhizal fungi (AMF), the diversity of plant partners and seasonal variability in trophic exchanges between the symbionts introduce additional heterogeneity. To clarify the impact of such heterogeneity, we investigated spatiotemporal variation in AMF diversity on a plot scale (10 × 10 m) in a grassland managed at low intensity in southwest Germany. AMF diversity was determined using 18S rDNA pyrosequencing analysis of 360 soil samples taken at six time points within a year. We observed high AMF alpha‐ and beta‐diversity across the plot and at all investigated time points. Relationships were detected between spatiotemporal variation in AMF OTU richness and plant species richness, root biomass, minimal changes in soil texture and pH. The plot was characterized by high AMF turnover rates with a positive spatiotemporal relationship for AMF beta‐diversity. However, environmental variables explained only ≈20% of the variation in AMF communities. This indicates that the observed spatiotemporal richness and community variability of AMF was largely independent of the abiotic environment, but related to plant properties and the cooccurring microbiome.We thank the managers of the three Exploratories, Kirsten Reichel‐Jung, Swen Renner, Katrin Hartwich, Sonja Gockel, Kerstin Wiesner, and Martin Gorke for their work in maintaining the plot and project infrastructure; Christiane Fischer and Simone Pfeiffer for giving support through the central office, Michael Owonibi and Andreas Ostrowski for managing the central data base, and Eduard Linsenmair, Dominik Hessenmöller, Jens Nieschulze, Ernst‐Detlef Schulze, Wolfgang W. Weisser and the late Elisabeth Kalko for their role in setting up the Biodiversity Exploratories project. The work has been funded by the DFG Priority Program 1374 ‘Infrastructure‐Biodiversity‐Exploratories’ (BU 941/22‐1, BU 941/22‐3, KA 1590/8‐2, KA 1590/8‐3). Field work permits were issued by the responsible state environmental office of Baden‐Württemberg (according to § 72 BbgNatSchG). Likewise, we kindly thank Beatrix Schnabel, Melanie Günther and Sigrid Härtling for 454 sequencing in Halle. AHB gratefully acknowledges the support of the German Centre for Integrative Biodiversity Research (iDiv) Halle‐Jena‐Leipzig funded by the German Research Foundation (FZT 118). Authors declare no conflict of interests

Effects of warming and drought on potential N2O emissions and denitrifying bacteria abundance in grasslands with different land-use

Increased warming in spring and prolonged summer drought may alter soil microbial denitrification. We measured potential denitrification activity and denitrifier marker gene abundances (nirK, nirS, nosZ) in grasslands soils in three geographic regions characterized by site-specific land-use indices (LUI) after warming in spring, at an intermediate sampling and after summer drought. Potential denitrification was significantly increased by warming, but did not persist over the intermediate sampling. At the intermediate sampling, the relevance of grassland land-use intensity was reflected by increased potential N2O production at sites with higher LUI. Abundances of total bacteria did not respond to experimental warming or drought treatments, displaying resilience to minor and short-term effects of climate change. In contrast, nirS- and nirK-type denitrifiers were more influenced by drought in combination with LUI and pH, while the nosZ abundance responded to the summer drought manipulation. Land-use was a strong driver for potential denitrification as grasslands with higher LUI also had greater potentials for N2O emissions. We conclude that both warming and drought affected the denitrifying communities and the potential denitrification in grassland soils. However, these effects are overruled by regional and site-specific differences in soil chemical and physical properties which are also related to grassland land-use intensit

Temporal and small-scale spatial variation in grassland productivity, biomass quality, and nutrient limitation

Characterization of spatial and temporal variation in grassland productivity and nutrition is crucial for a comprehensive understanding of ecosystem function. Although within-site heterogeneity in soil and plant properties has been shown to be relevant for plant community stability, spatiotemporal variability in these factors is still understudied in temperate grasslands. Our study aimed to detect if soil characteristics and plant diversity could explain observed small-scale spatial and temporal variability in grassland productivity, biomass nutrient concentrations, and nutrient limitation. Therefore, we sampled 360 plots of 20 cm × 20 cm each at six consecutive dates in an unfertilized grassland in Southern Germany. Nutrient limitation was estimated using nutrient ratios in plant biomass. Absolute values of, and spatial variability in, productivity, biomass nutrient concentrations, and nutrient limitation were strongly associated with sampling date. In April, spatial heterogeneity was high and most plots showed phosphorous deficiency, while later in the season nitrogen was the major limiting nutrient. Additionally, a small significant positive association between plant diversity and biomass phosphorus concentrations was observed, but should be tested in more detail. We discuss how low biological activity e.g., of soil microbial organisms might have influenced observed heterogeneity of plant nutrition in early spring in combination with reduced active acquisition of soil resources by plants. These early-season conditions are particularly relevant for future studies as they differ substantially from more thoroughly studied later season conditions. Our study underlines the importance of considering small spatial scales and temporal variability to better elucidate mechanisms of ecosystem functioning and plant community assembly

Stochastic Dispersal Rather Than Deterministic Selection Explains the Spatio-Temporal Distribution of Soil Bacteria in a Temperate Grassland

Spatial and temporal processes shaping microbial communities are inseparably linked but rarely studied together. By Illumina 16S rRNA sequencing, we monitored soil bacteria in 360 stations on a 100 square meter plot distributed across six intra-annual samplings in a rarely managed, temperate grassland. Using a multi-tiered approach, we tested the extent to which stochastic or deterministic processes influenced the composition of local communities. A combination of phylogenetic turnover analysis and null modeling demonstrated that either homogenization by unlimited stochastic dispersal or scenarios, in which neither stochastic processes nor deterministic forces dominated, explained local assembly processes. Thus, the majority of all sampled communities (82%) was rather homogeneous with no significant changes in abundance-weighted composition. However, we detected strong and uniform taxonomic shifts within just nine samples in early summer. Thus, community snapshots sampled from single points in time or space do not necessarily reflect a representative community state. The potential for change despite the overall homogeneity was further demonstrated when the focus shifted to the rare biosphere. Rare OTU turnover, rather than nestedness, characterized abundance-independent β-diversity. Accordingly, boosted generalized additive models encompassing spatial, temporal and environmental variables revealed strong and highly diverse effects of space on OTU abundance, even within the same genus. This pure spatial effect increased with decreasing OTU abundance and frequency, whereas soil moisture – the most important environmental variable – had an opposite effect by impacting abundant OTUs more than the rare ones. These results indicate that – despite considerable oscillation in space and time – the abundant and resident OTUs provide a community backbone that supports much higher β-diversity of a dynamic rare biosphere. Our findings reveal complex interactions among space, time, and environmental filters within bacterial communities in a long-established temperate grassland

Direct and plant community mediated effects of management intensity on annual nutrient leaching risk in temperate grasslands

Grassland management intensity influences nutrient cycling both directly, by changing nutrient inputs and outputs from the ecosystem, and indirectly, by altering the nutrient content, and the diversity and functional composition of plant and microbial communities. However, the relative importance of these direct and indirect processes for the leaching of multiple nutrients is poorly studied. We measured the annual leaching of nitrate, ammonium, phosphate and sulphate at a depth of 10 cm in 150 temperate managed grasslands using a resin method. Using Structural Equation Modeling, we distinguished between various direct and indirect effects of management intensity (i.e. grazing and fertilization) on nutrient leaching. We found that management intensity was positively associated with nitrate, ammonium and phosphate leaching risk both directly (i.e. via increased nutrient inputs) and indirectly, by changing the stoichiometry of soils, plants and microbes. In contrast, sulphate leaching risk was negatively associated with management intensity, presumably due to increased outputs with mowing and grazing. In addition, management intensification shifted plant communities towards an exploitative functional composition (characterized by high tissue turnover rates) and, thus, further promoted the leaching risk of inorganic nitrogen. Plant species richness was associated with lower inorganic nitrogen leaching risk, but most of its effects were mediated by stoichiometry and plant community functional traits. Maintaining and restoring diverse plant communities may therefore mitigate the increased leaching risk that management intensity imposes upon grasslands

Above- and belowground biodiversity jointly tighten the P cycle in agricultural grasslands

Experiments showed that biodiversity increases grassland productivity and nutrient exploitation, potentially reducing fertiliser needs. Enhancing biodiversity could improve P-use efficiency of grasslands, which is beneficial given that rock-derived P fertilisers are expected to become scarce in the future. Here, we show in a biodiversity experiment that more diverse plant communities were able to exploit P resources more completely than less diverse ones. In the agricultural grasslands that we studied, management effects either overruled or modified the driving role of plant diversity observed in the biodiversity experiment. Nevertheless, we show that greater above- (plants) and belowground (mycorrhizal fungi) biodiversity contributed to tightening the P cycle in agricultural grasslands, as reduced management intensity and the associated increased biodiversity fostered the exploitation of P resources. Our results demonstrate that promoting a high above- and belowground biodiversity has ecological (biodiversity protection) and economical (fertiliser savings) benefits. Such win-win situations for farmers and biodiversity are crucial to convince farmers of the benefits of biodiversity and thus counteract global biodiversity loss

The Evolution of Ecological Diversity in Acidobacteria

Acidobacteria occur in a large variety of ecosystems worldwide and are particularly abundant and highly diverse in soils. In spite of their diversity, only few species have been characterized to date which makes Acidobacteria one of the most poorly understood phyla among the domain Bacteria. We used a culture-independent niche modeling approach to elucidate ecological adaptations and their evolution for 4,154 operational taxonomic units (OTUs) of Acidobacteria across 150 different, comprehensively characterized grassland soils in Germany. Using the relative abundances of their 16S rRNA gene transcripts, the responses of active OTUs along gradients of 41 environmental variables were modeled using hierarchical logistic regression (HOF), which allowed to determine values for optimum activity for each variable (niche optima). By linking 16S rRNA transcripts to the phylogeny of full 16S rRNA gene sequences, we could trace the evolution of the different ecological adaptations during the diversification of Acidobacteria. This approach revealed a pronounced ecological diversification even among acidobacterial sister clades. Although the evolution of habitat adaptation was mainly cladogenic, it was disrupted by recurrent events of convergent evolution that resulted in frequent habitat switching within individual clades. Our findings indicate that the high diversity of soil acidobacterial communities is largely sustained by differential habitat adaptation even at the level of closely related species. A comparison of niche optima of individual OTUs with the phenotypic properties of their cultivated representatives showed that our niche modeling approach (1) correctly predicts those physiological properties that have been determined for cultivated species of Acidobacteria but (2) also provides ample information on ecological adaptations that cannot be inferred from standard taxonomic descriptions of bacterial isolates. These novel information on specific adaptations of not-yet-cultivated Acidobacteria can therefore guide future cultivation trials and likely will increase their cultivation success

The mineralo-sphere – Succession and physiology of bacteria and fungi colonising pristine minerals in grassland soils under different land-use intensities

The mineralo-sphere is an important micro habitat in terrestrial ecosystems. How different groups of microorganisms colonise mineral surfaces and whether the level of grassland land-use intensity (LUI) modifies this micro-habitat is not well known, however. We exposed mesh containers filled with pristine soil minerals (illite/goethite) mixed with 13C labelled root litter of Dactylis glomerata and Lolium perenne in grassland soils of the “Schwäbische Alb” (Germany) to characterise the succession of different microbial properties in the mineralo-sphere. The use of sites within the Biodiversity Exploratories made it possible to select five sites of low LUI and five sites of high LUI. After 1, 2, 7, 12 and 31 months of exposure in the grassland soils, we used physiological, microbiological and isotopic methods to elucidate in situ colonisation patterns, carbon use and levels of extracellular enzyme expression by soil microorganisms associated with mineral surfaces. Microorganisms slowly colonised pristine mineral surfaces and established functionally distinct communities over time. Fungi colonised mineral surfaces to a greater extent than bacteria, reaching 13.2% of control soils compared to 3.2% by bacteria after 31 months. Fungi also reached pristine mineral surfaces earlier than bacteria, probably due to their hyphal growth strategies, and made immediate use of the added complex root litter substrate. This result is evident by the incorporation of up to 74% root litter-derived C into the fungus-specific PLFA (18:2ω6,9) compared to 51% root litter-derived C in the bacteria-specific PLFAs. Both bacteria and fungi associated with minerals remained in an active state (high biomass-specific respiration, high bacterial and fungal growth rates) throughout the experimental period. Grassland LUI and physico-chemical properties of the adjacent soil modified both quantity and quality of substrates available to soil microorganisms in the mineralo-sphere. Since 13C incorporation into microbial biomass was greater under low LUI than under high LUI, we conclude that microorganisms in low LUI sites had to rely on the added root material, while the carbon signal in microorganisms in the high LUI sites was diluted by alternative sources resulting from transport of dissolved organic carbon into the mineralo-sphere

Spatial and temporal dynamics of nitrogen fixing, nitrifying and denitrifying microbes in an unfertilized grassland soil

The microbial groups of nitrogen fixers, ammonia oxidizers, and denitrifiers largely drive the inorganic nitrogen cycle in temperate terrestrial ecosystems. Their spatial and temporal dynamics, however, vary depending on the studied scale. The present study aimed to fill a knowledge gap by providing an explicit picture of spatial and temporal dynamics of a subset of these soil microorganisms at the plot scale. We selected an unfertilized perennial grassland, where nitrogen cycling is considered to be efficient and tightly coupled to plant growth. At six times over one growing season 60 soil samples were taken from a 10 m × 10 m area and abundances of marker genes for total archaea and bacteria (16S rRNA), nitrogen fixing bacteria (nifH), ammonia oxidizing archaea (amoA AOA) and bacteria (amoA AOB), and denitrifying bacteria (nirS, nirK and nosZ) were determined by qPCR. Potential nitrification activity (PNA) and denitrifying enzyme activity (DEA) were determined. Seasonal changes in abundance patterns of marker genes were detected, and were associated with changes in substrate availability associated with plant growth stages. Potential nitrification and denitrification enzyme activities were strongly spatially structured at the studied scale, corresponding to periods of rapid plant growth, June and October, and their spatial distributions were similar, providing visual evidence of highly localized spatial and temporal conditions at this scale. Temporal variability in the N-cycling communities versus the stability of their respective potential activities provided evidence of both short-lived temporal niche partitioning and a degree of microbial functional redundancy. Our results indicate that in an unfertilized grassland, at the meter scale, abundances of microbial N-cycling organisms can exhibit transient changes, while nitrogen cycling processes remain stable