Plant roots release recent photosynthates into the rhizosphere, accelerating decomposition of organic matter by saprotrophic soil microbes (“rhizosphere priming effect”) which consequently increases nutrient availability for plants. However, about 90% of all higher plant species are mycorrhizal, transferring a significant fraction of their photosynthates directly to their fungal partners. Whether mycorrhizal fungi pass on plant-derived carbon (C) to bacteria in root-distant soil areas, i.e., incite a “hyphosphere priming effect,” is not known. Experimental evidence for C transfer from mycorrhizal hyphae to soil bacteria is limited, especially for ectomycorrhizal systems. As ectomycorrhizal fungi possess enzymatic capabilities to degrade organic matter themselves, it remains unclear whether they cooperate with soil bacteria by providing photosynthates, or compete for available nutrients. To investigate a possible C transfer from ectomycorrhizal hyphae to soil bacteria, and its response to changing nutrient availability, we planted young beech trees ( Fagus sylvatica ) into “split-root” boxes, dividing their root systems into two disconnected soil compartments. Each of these compartments was separated from a litter compartment by a mesh penetrable for fungal hyphae, but not for roots. Plants were exposed to a 13 C-CO 2 -labeled atmosphere, while 15 N-labeled ammonium and amino acids were added to one side of the split-root system. We found a rapid transfer of recent photosynthates via ectomycorrhizal hyphae to bacteria in root-distant soil areas. Fungal and bacterial phospholipid fatty acid (PLFA) biomarkers were significantly enriched in hyphae-exclusive compartments 24 h after 13 C-CO 2 -labeling. Isotope imaging with nanometer-scale secondary ion mass spectrometry (NanoSIMS) allowed for the first time in situ visualization of plant-derived C and N taken up by an extraradical fungal hypha, and in microbial cells thriving on hyphal surfaces. When N was added to the litter compartments, bacterial biomass, and the amount of incorporated 13 C strongly declined. Interestingly, this effect was also observed in adjacent soil compartments where added N was only available for bacteria through hyphal transport, indicating that ectomycorrhizal fungi were acting on soil bacteria. Together, our results demonstrate that (i) ectomycorrhizal hyphae rapidly transfer plant-derived C to bacterial communities in root-distant areas, and (ii) this transfer promptly responds to changing soil nutrient conditions.

<p>Phospholipid fatty acids (PLFA) are widely used as biomarkers for soil microbial biomass. In more recent years, neutral lipid fatty acids (NLFA) have additionally been used as storage biomarkers. Both lipid classes are usually separated via silica solid phase extraction (SPE) after extraction with a mixture of chloroform, methanol and citric acid buffer. However, in recent years several studies reported incomplete or inconsistent separation of lipid classes, depending on minor differences in the polarity of the eluents used during the SPE. Moreover, while PLFA profiles have been tested on microbial pure cultures, the taxonomic specificity of NLFA is only assumed to equal that of PLFA.</p><p>Complementary to fatty acid based biomarkers, many studies quantify ergosterol as a reliable indicator for fungal biomass because the fungal-specific PLFA 18:1ω9 and 18:2ω6,9 also occur in plants, which compromises their use for detecting fungal biomass in plant tissue (for example mycorrhizal fungi in plant roots). Measuring ergosterol requires an additional extraction method, but existing protocols include silylation for further gas chromatography analysis and are thus not compatible with determining <sup>13</sup>C by IRMS.</p><p>Here, we aimed to quantify the recovery of polar and non-polar lipid classes as well as ergosterol following lipid extraction and silica SPE fractionation. We used pure standards of representative phospholipids, glycolipids and neutral lipids with unique fatty acid chain lengths for unambiguous identification of the lipid class after SPE. Lipid fractionation was tested on a 96-well SPE plate with different eluents. Subsequently, we applied the modified method to characterize lipid fractions in microbial pure cultures from bacteria (Proteobacteria, Firmicutes, Actinobacteria), and saprotrophic and ectomycorrhizal fungi (Ascomycota, Basidiomycota).</p><p>Separation of lipid classes was achieved by successively eluting NLFA and sterols with a mixture of chloroform and ethanol (v:v = 98:2), glycolipid fatty acids (GLFA) with acetone, and PLFA with a mixture of methanol, chloroform and water (v:v:v = 5:5:1). GLFA were partially recovered in the NLFA or PLFA fraction depending on the nature of the lipid, which should be considered when interpreting PLFA data. Ergosterol recovery was unaffected by subsequent mild alkaline methanolysis of the NLFA fraction in which it was collected, allowing further analysis of both lipid classes in the same mixture. The gas-chromatographic method may be extended to elute both NLFA and (non-silylated) sterols in one run, assuming that the concentration of ergosterol in soil samples is high enough. Therefore, the method can be optimized by using an internal standard added to the NLFA fraction and simultaneously quantify ergosterol. Finally, we show how different lipid classes and attached fatty acid chains distribute in pure cultures of soil micro-organisms.</p>

<p>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.</p> <p>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.</p> <p>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.</p> <p>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.</p>

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<p>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.</p><p>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).</p><p>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.</p><p>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.</p><p>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.</p>