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Arbuscular mycorrhizal fungi (AMF) form mutualistic associations with roughly 70% of vascular plant species, supporting the nutrient acquisition of their host plants and deriving carbon in return. AMF and plant communities are linked to each other by host-specificity and the ecological selection of favorable nutrient and carbon trading strategies. Changing soil nutrient availabilities can affect both plant and AMF communities directly and also indirectly via the response of their partners. We aimed to elucidate the combined response of AMF (belowground) and plant (aboveground) community compositions to changing soil nutrient availabilities.We sampled soil and roots from a long-term nutrient deficiency experimental grassland in Admont (Styria, Austria). The grassland plots have been fertilized with different combinations of nitrogen (N), phosphorus (P), and potassium (K) over 70 years. Aboveground biomass cuts were removed three times each year, leading to long-term deficiencies of nutrients not replaced by fertilizers. Soil and root AMF community compositions were measured by DNA and RNA amplicon sequencing of the 18S rRNA gene. In addition, we assessed the plant community composition of the sampled roots by amplicon sequencing of the chloroplast rbcL (RuBisCo large subunit) gene region, and visually recorded the plant community composition on each investigated plot.Our results demonstrate that N and P deficiencies influenced soil AMF community composition, whereas K deficiency had a major impact on root AMF community composition. Interestingly, the plant community composition was affected by N and P, similar to the soil AMF community composition. Both, soil and root AMF community compositions were significantly correlated to plant community composition across all treatments, the correlation was however stronger for soil AMF communities (R2 = 0.55, p< 0.001). By using bipartite network analysis, we identified several fungus-plant pairs that responded consistently to treatments.Our results indicate that the response of grasslands to nutrient deficiencies is potentially driven by strong feedbacks between plant and belowground AMF community compositions. We here demonstrate that the known interactions between grassland plants and AMF - which are often investigated from a single plant or monoculture perspective - are major drivers of how diverse plant community compositions will respond to environmental change, such as fertilization. In conclusion, considering the ecology of the subsurface AMF communities may strongly benefit our understanding of plant communities in a future environment.

Fertilization experiments provide insights into elemental imbalances in soil microbial communities and their consequences for soil nutrient cycling. By addition of selected nutrients, other nutrients become deficient and limiting for soil microorganisms as well as for plants. In this study we focused on microbial nitrogen (N) cycling in a long-term nutrient manipulation experiment. In many soils, the rate-limiting step in N cycling is depolymerization of high-molecular-weight nitrogen compounds (e.g., proteins) to oligomers (e.g., peptides) and monomers (e.g., amino acids) rather than the subsequent steps of mineralization (ammonification) and nitrification. The aim of our study was to determine whether nutrient deficiency directly or indirectly – via changes in plant carbon (C) inputs - affects soil microbial N processing.We collected soil samples from a fertilization experiment, established in 1946 on a hay meadow close to Admont (Styria, Austria). The field experiment consisted of a full factorial combination of inorganic N, P, and K fertilization and a control with no fertilizers. Furthermore, liming (Ca-addition) and organic fertilizer application treatments (solid manure and liquid slurry) were established. In the experiment, plant biomass is harvested three times per year, inducing strong nutrient limitation in plots that have not received nutrient additions (fully deficient or deficient in a single element). We determined gross rates of microbial protein depolymerization, N-mineralization and nitrification via isotope pool dilution assays with 15N-labeled amino acids, NH4+, and NO3-. We hypothesized that N deficiency (lack of N fertilization) would stimulate microbial N mining (depolymerization), and reduce subsequent N mineralization and nitrification. In contrast, we expected that organic fertilization would alleviate microbial C and N limitations, reducing N depolymerization rates and increasing mineralization and nitrification.Our results show that organically fertilized and limed soils have significantly lower gross protein depolymerization rates than plots receiving inorganic N. No significant differences were found comparing gross N-mineralization and gross nitrification rates across the different treatments. Given the higher rates of protein depolymerization in inorganically fertilized soils as compared to organically fertilized and limed soils, microbial N processes seem to be controlled by plant C input and/or soil pH rather than by direct soil nutrient availability. However, depolymerization of macromolecular N does not only supply N to the soil microbial community but also organic C. Thus, the reduced plant C input compared to fully fertilized soils may have caused microorganisms to increase their mining for a C-containing energy source, thereby increasing protein depolymerization rates. In summary, this study suggests that long term nutrient deficiency or nutrient imbalances may affect soil nutrient cycling indirectly by changing plant C inputs (via reduced primary production) and/or changing soil pH, rather than directly, by nutrient availability. This further indicates that soil microbial communities are rather C than nutrient limited.

Arbuscular mycorrhiza (AM) fungi are associated with almost all land plants and provide soil nutrients and other benefits to their plant hosts in exchange for photosynthetic products. While fertilization regimes in managed grasslands or agricultural systems are tailored for increasing plant biomass, their potential effects on AM fungi are rarely taken into account. Nutrient-driven changes in abundance and community composition of AM fungi, however, may feedback on ecosystem performance in the long term. Therefore, it is necessary to get a better understanding on how AM fungal communities respond to changes and imbalances in soil nutrient availabilities.Here, we evaluated how long-term nutrient deficiency of phosphorus (P), nitrogen (N) and potassium (K) affects the abundance and community composition of AM fungi in a mountainous grassland. In addition, we investigated how the responses of AM fungi to those deficiencies were modulated by liming and the type of fertilizer addition (inorganic versus organic).Our study was carried out on a long-term nutrient deficiency experimental grassland site in Admont (Styria, Austria), established in 1946. Different fertilization treatments were applied for more than 70 years in a randomized block design, including numerous combinations of inorganic (P, N, K with/without lime) and organic (solid manure and liquid slurry) fertilizers. The hay meadow at the site is cut three times per year and biomass is not returned to the system. Therefore, biomass and nutrients have been continuously removed for decades, leading to different types of soil nutrient deficiency. In this study, we collected both root and soil samples in July 2019 and quantified AM fungi and other microbial groups by measuring neutral fatty acid (NLFA) and phospholipid fatty acid (PLFA) biomarkers, respectively. Additionally, we applied DNA and RNA-based amplicon sequencing of the 18S rRNA gene to identify AM fungal community composition.Our data shows that deficiencies of one or more elements had a major impact on both AM fungal biomass and community composition. AM fungal biomass was higher in plots that received no fertilizers compared to inorganically fertilized plots, but lower in plots which were deficient only in certain single or multiple elements, specifically in plots fertilized with inorganic N only (i.e., deficient in P and K). Conversely, liming and organic fertilizer amendments increased AM fungal biomass compared to plots containing inorganic fertilizers without lime. Across all treatments, AM fungal biomass was positively correlated with pH and soil water content, and negatively with dissolved N compounds, indicating indirect effects via responses of other soil parameters to nutrient deficiency. Long-term nutrient deficiency also altered plant community composition, which may also have indirectly affected AM fungal communities.We conclude that long-term nutrient deficiency, and in particular the stoichiometry of available nutrients, strongly affects the abundance and community composition of AM fungi in grassland soil. This response may be linked to changes in plant community composition or soil chemistry both as a result and as a cause, emphasizing the complexity of feedbacks determining the response of grassland ecosystems to changing nutrient conditions.

Mycorrhizal fungi are an important partner of almost all land plants, who trade soil nutrients, such as Phosphorus or Nitrogen, for photosynthetic Carbon (C). Moreover, mycorrhizal fungi connect multiple plants with their mycelium in so called Common Mycorrhizal Networks (CMNs). CMNs formed by ectomycorrhizal (EM) fungi are an inherent part of boreal and temperate forests, often termed the ‘wood-wide web’. However, the role of these networks for plant belowground C allocation and distribution is not well known.Here, we examined how plant photosynthates are distributed within EM mycelium networks connecting pairs of young beech trees, addressing the following questions: (1) Is the total belowground C allocation of plant photosynthates influenced by the size of the mycorrhizal network and its access to resources? (2) Is the belowground C distribution within a CMN altered if trees have unequal access to C from photosynthesis? (3) Do CMNs amplify or alleviate competition for nutrients between connected trees?We planted young beech trees in pots in a special two-plant box set-up which allows to control the establishment of mycorrhizal networks between them. For this, two plant pots, penetrable by fungal hyphae but not by roots, were placed inside of plastic boxes and the interstitial space was filled with quartz sand. In addition, a hyphal-exclusive N source consisting of 15N labeled peat (‘peat bag’), was buried within each plant pot. Two treatments were applied in a fully factorial design: 1) Allowing/preventing the establishment of a CMN between the pots (some pots were turned around at a regular interval to prevent the establishment of CMNs) and 2) inequality of access to photoassimilated C (in part of the boxes one of the two plants was shaded). In a 13C-CO2 labeling approach, we traced 13C assimilated by one plant of each tree pair into belowground pools of both plants by isotope ratio mass spectrometry (EA-IRMS) and 13C phospholipid fatty acid (PLFAs) analysis (GC-IRMS). At the same time, we investigated plant uptake of 15N via mycorrhiza by EA-IRMS.Our results demonstrate that plants relied mostly on their fungal partners to acquire nutrients (63% of plant N was derived from mycorrhiza-exclusive peat bags), and also directed the majority of the C allocated belowground to their mycorrhizal partners. The presence of a larger mycorrhizal network connecting to another plant and an additional N source almost doubled photosynthetic CO2 assimilation and belowground C allocation by plants. Fungi translocated carbon via hyphal linkages preferentially into mycorrhiza-exclusive nutrient patches, even when they were located within the realm of a neighboring plant and this necessitates to cross a nutrient-poor zone of sand. Shading did not affect the belowground distribution of C.We conclude that belowground ectomycorrhizal networks represent a significant sink strength for plant photosynthates and may thus be a major driver of C sequestration in beech forest soils. The belowground distribution of C via fungal networks is mainly related to the distribution of nutrient-rich patches in the soil and less to differences in the photosynthetic capacity of the host plants.