Erich Inselsbacher

The below‐ground perspective of forest plants: soil provides mainly organic nitrogen for plants and mycorrhizal fungi

• Nitrogen (N) availability has a major impact on a wide range of biogeochemical processes in terrestrial ecosystems. Changes in N availability modify the capacity of plants to sequester carbon (C), but despite the crucial importance for our understanding of terrestrial ecosystems, the relative contribution of different N forms to plant N nutrition in the field is not known. Until now, reliably assessing the highly dynamic pool of plant-available N in soil microsites was virtually impossible, because of the lack of adequate sampling techniques. • For the first time we have applied a novel microdialysis technique for disturbance-free monitoring of diffusive fluxes of inorganic and organic N in 15 contrasting boreal forest soils in situ. • We found that amino acids accounted for 80% of the soil N supply, while ammonium and nitrate contributed only 10% each. In contrast to common soil extractions, microdialysis revealed that the majority of amino acids are available for plant and mycorrhizal uptake. • Our results suggest that the N supply of boreal forest soils is dominated by organic N as a major component of plant-available N and thus as a regulator of growth and C sequestration.

Dynamics of ammonia-oxidizing communities in barley-planted bulk soil and rhizosphere following nitrate and ammonium fertilizer amendment.

Oxidation of ammonia by nitrifying microorganisms is a major pathway that fertilizer nitrogen (N) may take upon application to agricultural soils, but the relative roles of bacterial (AOB) vs. archaeal (AOA) ammonia oxidizers are controversial. We explored the effects of various forms of mineral N fertilizer on the AOB and AOA community dynamics in two different soils planted with barley. Ammonia oxidizers were monitored via real-time PCR and terminal restriction fragment length polymorphism analysis of bacterial and archaeal amoA genes following the addition of either [NH₄]₂SO₄, NH₄NO₃ or KNO₃. AOB and AOA communities were also studied specifically in the rhizospheres of two different barley varieties upon [NH₄]₂SO₄ vs. KNO₃ addition. AOB changed in community composition and increased in abundance upon ammonium amendment in bulk soil and rhizosphere, with changes in bacterial amoA copy numbers lagging behind relative to changes in soil ammonium. In both soils, only T-RFs corresponding to phylotypes related to Nitrosospira clade 3a underwent significant community changes. Increases in AOB abundance were generally stronger in the bulk soil than in the rhizosphere, implying significant ammonia uptake by plant roots. AOA underwent shifts in the community composition over time and fluctuated in abundance in all treatments irrespective of ammonia availability. AOB were thus considered as the main agents responsible for fertilizer ammonium oxidation, while the functions of AOA in soil N cycling remain unresolved.

Molecular diversity of fungal communities in agricultural soils from Lower Austria

A culture-independent survey of fungal diversity in four arable soils and one grassland in Lower Austria was conducted by RFLP and sequence analysis of clone libraries of the partial ITS/LSU-region. All soils were dominated by the ascomycetous orders Sordariales, Hypocreales and Helotiales, taxa that are known from traditional cultivation approaches to occur in agricultural soils. The most abundant genus in the investigated soils was Tetracladium, a hyphomycete which has been described as occurring predominantly in aquatic habitats, but was also found in agricultural soils. Additionally, soil clone group I (SCGI), a subphylum at the base of the Ascomycota with so far no cultivated members, was identified at high frequency in the grassland soil but was below detection limit in the four arable fields. In addition to this striking difference, general fungal community parameters like richness, diversity and evenness were similar between cropland and grassland soils. The presented data provide a fungal community inventory of agricultural soils and reveal the most prominent species.

Community profiling and gene expression of fungal assimilatory nitrate reductases in agricultural soil

Although fungi contribute significantly to the microbial biomass in terrestrial ecosystems, little is known about their contribution to biogeochemical nitrogen cycles. Agricultural soils usually contain comparably high amounts of inorganic nitrogen, mainly in the form of nitrate. Many studies focused on bacterial and archaeal turnover of nitrate by nitrification, denitrification and assimilation, whereas the fungal role remained largely neglected. To enable research on the fungal contribution to the biogeochemical nitrogen cycle tools for monitoring the presence and expression of fungal assimilatory nitrate reductase genes were developed. To the ∼100 currently available fungal full-length gene sequences, another 109 partial sequences were added by amplification from individual culture isolates, representing all major orders occurring in agricultural soils. The extended database led to the discovery of new horizontal gene transfer events within the fungal kingdom. The newly developed PCR primers were used to study gene pools and gene expression of fungal nitrate reductases in agricultural soils. The availability of the extended database allowed affiliation of many sequences to known species, genera or families. Energy supply by a carbon source seems to be the major regulator of nitrate reductase gene expression for fungi in agricultural soils, which is in good agreement with the high energy demand of complete reduction of nitrate to ammonium.

Greenhouse gas fluxes respond to different N fertilizer types due to altered plant-soil-microbe interactions

The application of inorganic nitrogen (N) fertilizers strongly influences the contribution of agriculture to the greenhouse effect, especially by potentially increasing emissions of nitrous oxide (N2O), carbon dioxide (CO2) and methane (CH4) from soils. The present microcosm-study investigates the effect of different forms of inorganic N fertilizers on greenhouse gas (GHG) emissions from two different agricultural soils. The relationship between greenhouse gas emissions and soil microbial communities, N transformation rates and plant (Hordeum vulgare L. cv. Morex) growth were investigated. Repeated N fertilization led to increased N2O emissions. In a parallel survey of functional microbial population dynamics we observed a stimulation of bacterial and archaeal ammonia oxidisers accompanied with these N2O emissions. The ratio of archaeal to bacterial ammonium monooxygenase subunit A (amoA) gene copies (data obtained from Inselsbacher et al., 2010) correlated positively with N2O fluxes, which suggests a direct or indirect involvement of archaea in N2O fluxes. Repeated N fertilization also stimulated methane oxidation, which may also be related to a stimulation of ammonia oxidizers. The fertilizer effects differed between soil types: In the more organic Niederschleinz soil N-turnover rates increased more strongly after fertilization, while in the sandy Purkersdorf soil plant growth and soil respiration were accelerated depending on fertilizer N type. Compared to addition of NH4+ and NO3−, addition of NH4NO3 fertilizer resulted in the largest increase in global warming potential as a summary indicator of all GHG related effects. This effect resulted from the strongest increase of both N2O and CO2 emission while plant growth was not equally stimulated, compared to e.g. KNO3 fertilization. In order to decrease N losses from agricultural ecosystems and in order to minimize soil derived global warming potential, this study points to the need for interdisciplinary investigations of the highly complex interactions within plant-soil-microbe-atmosphere systems. By understanding the microbial processes underlying fertilizer effects on GHG emissions the N use efficiency of crops could be refined.

Nitrogen fluxes at the root-soil interface show a mismatch of nitrogen fertilizer supply and sugarcane root uptake capacity

Globally only ≈50% of applied nitrogen (N) fertilizer is captured by crops, and the remainder can cause pollution via runoff and gaseous emissions. Synchronizing soil N supply and crop demand will address this problem, however current soil analysis methods provide little insight into delivery and acquisition of N forms by roots. We used microdialysis, a novel technique for in situ quantification of soil nutrient fluxes, to measure N fluxes in sugarcane cropping soils receiving different fertilizer regimes, and compare these with N uptake capacities of sugarcane roots. We show that in fertilized sugarcane soils, fluxes of inorganic N exceed the uptake capacities of sugarcane roots by several orders of magnitude. Contrary, fluxes of organic N closely matched roots’ uptake capacity. These results indicate root uptake capacity constrains plant acquisition of inorganic N. This mismatch between soil N supply and root N uptake capacity is a likely key driver for low N efficiency in the studied crop system. Our results also suggest that (i) the relative contribution of inorganic N for plant nutrition may be overestimated when relying on soil extracts as indicators for root-available N, and (ii) organic N may contribute more to crop N supply than is currently assumed.

A cost-effective high-throughput microcosm system for studying nitrogen dynamics at the plant- microbe-soil interface

In the present study a new microcosm system was evaluated for its suitability to investigate nitrogen dynamics between soils, plants and microbes. Five different agricultural soils were homogenized and transferred in the test tubes, and kept under controlled conditions in a climate chamber for 4weeks. Soils differed clearly in nitrogen pools and microbial population structures but less in their activities. Bacterial and fungal community compositions and soil properties, except gross N transformation rates, remained stable and reproducible during the test period in all soils. 15N tracer studies showed that N uptake patterns of barley as well as plant growth were linear in the initial growth period. Overall, the presented microcosm system proved to be a powerful tool to elucidate N pathways in soil-plant-microbe systems. In future studies the microcosm system may greatly help generating new insights in the complex processes and controls of nitrogen biogeochemical cycle in agricultural systems.

Greater carbon allocation to mycorrhizal fungi reduces tree nitrogen uptake in a boreal forest

The central role that ectomycorrhizal (EM) symbioses play in the structure and function of boreal forests pivots around the common assumption that carbon (C) and nitrogen (N) are exchanged at rates favorable for plant growth. However, this may not always be the case. It has been hypothesized that the benefits mycorrhizal fungi convey to their host plants strongly depends upon the availability of C and N, both of which are rapidly changing as a result of intensified human land use and climate change. Using large-scale shading and N addition treatments, we assessed the independent and interactive effects of changes in C and N supply on the transfer of N in intact EM associations with -15 yr. old Scots pine trees. To assess the dynamics of N transfer in EM symbioses, we added trace amounts of highly enriched 5NO3(-) label to the EM-dominated mor-layer and followed the fate of the 15N label in tree foliage, fungal chitin on EM root tips, and EM sporocarps. Despite no change in leaf biomass, shading resulted in reduced tree C uptake, ca. 40% lower fungal biomass on EM root tips, and greater 15N label in tree foliage compared to unshaded control plots, where more 15N label was found in fungal biomass on EM colonized root tips. Short-term addition of N shifted the incorporation of 15N label from EM fungi to tree foliage, despite no significant changes in below-ground tree C allocation to EM fungi. Contrary to the common assumption that C and N are exchanged at rates favorable for plant growth, our results show for the first time that under N-limited conditions greater C allocation to EM fungi in the field results in reduced, not increased, N transfer to host trees. Moreover, given the ubiquitous nature of mycorrhizal symbioses, our results stress the need to incorporate mycorrhizal dynamics into process-based ecosystem models to better predict forest C and N cycles in light of global climate change.

A novel method to measure the effect of temperature on diffusion of plant-available nitrogen in soil

Background and aimsSoil temperature influences nitrogen (N) diffusion in soil but until now, such effects have been difficult to quantify. This study aimed at estimating the effect of temperature on the diffusive fluxes of plant-available N forms in two contrasting soils.MethodsUsing a novel technique based on micro-dialysis, we established relationships between diffusive fluxes and temperature in aqueous solutions and in soil samples.ResultsAveraged for all compounds, the decreases of diffusive fluxes from the soil to the microdialysis sampler were 3.8 and 4.7% per degree Celsius in an agricultural and a boreal forest soil, respectively. The temperature-related shift of diffusive flux was, however, significantly dependent on molecular weight of the N compound. In accordance with established functions for temperature effects on diffusive fluxes, the non-linearity of this relationship resulted in a greater temperature response for small N compounds compared to larger compounds.ConclusionsOur results show that, all other factors being equal, the relative contribution of smaller N compounds to the diffusive flux of total plant-available N increases with increasing soil temperatures.

Amino acid transporter mutants of Arabidopsis provides evidence that a non-mycorrhizal plant acquires organic nitrogen from agricultural soil

Although organic nitrogen (N) compounds are ubiquitous in soil solutions, their potential role in plant N nutrition has been questioned. We performed a range of experiments on Arabidopsis thaliana genetically modified to enhance or reduce root uptake of amino acids. Plants lacking expression of the Lysine Histidine Transporter 1 (LHT1) displayed significantly lower contents of 13 C and 15 N label and of U-13 C5 ,15 N2 L-glutamine, as determined by liquid chromatography-mass spectrometry when growing in pots and supplied with dually labelled L-glutamine compared to wild type plants and LHT1-overexpressing plants. Slopes of regressions between accumulation of 13 C-labelled carbon and 15 N-labelled N were higher for LHT1-overexpressing plants than wild type plants, while plants lacking expression of LHT1 did not display a significant regression between the two isotopes. Uptake of labelled organic N from soil tallied with that of labelled ammonium for wild type plants and LHT1-overexpressing plants but was significantly lower for plants lacking expression of LHT1. When grown on agricultural soil plants lacking expression of LHT1 had the lowest, and plants overexpressing LHT1 the highest C/N ratios and natural δ15 N abundance suggesting their dependence on different N pools. Our data show that LHT1 expression is crucial for plant uptake of organic N from soil.