Constraining activity and growth substrate of fungal decomposers via assimilation patterns of inorganic carbon and water into lipid biomarkers.

Fungi are among the few organisms on the planet that can metabolize recalcitrant carbon (C) but are also known to access recently produced plant photosynthate. Therefore, improved quantification of growth and substrate utilization by different fungal ecotypes will help to define the rates and controls of fungal production, the cycling of soil organic matter, and thus the C storage and CO2 buffering capacity in soil ecosystems. This pure-culture study of fungal isolates combined a dual stable isotope probing (SIP) approach, together with rapid analysis by tandem pyrolysis-gas chromatography-isotope ratio mass spectrometry to determine the patterns of water-derived hydrogen (H) and inorganic C assimilated into lipid biomarkers of heterotrophic fungi as a function of C substrate. The water H assimilation factor (αW) and the inorganic C assimilation into C18:2 fatty acid isolated from five fungal species growing on glucose was lower (0.62% ± 0.01% and 4.7% ± 1.6%, respectively) than for species grown on glutamic acid (0.90% ± 0.02% and 7.4% ± 3.7%, respectively). Furthermore, the assimilation ratio (RIC/αW) for growth on glucose and glutamic acid can distinguish between these two metabolic modes. This dual-SIP assay thus delivers estimates of fungal activity and may help to delineate the predominant substrates that are respired among a matrix of compounds found in natural environments.IMPORTANCEFungal decomposers play important roles in food webs and nutrient cycling because they can feed on both labile and more recalcitrant forms of carbon. This study developed and applied a dual stable isotope assay (13C-dissolved inorganic carbon/2H) to improve the investigation of fungal activity in the environment. By determining the incorporation patterns of hydrogen and carbon into fungal lipids, this assay delivers estimates of fungal activity and the different metabolic pathways that they employ in ecological and environmental systems.

Carbon and phosphorus exchange rates in arbuscular mycorrhizas depend on environmental context and differ among co-occurring plants.

Phosphorus (P) for carbon (C) exchange is the pivotal function of arbuscular mycorrhiza (AM), but how this exchange varies with soil P availability and among co-occurring plants in complex communities is still largely unknown. We collected intact plant communities in two regions differing c. 10-fold in labile inorganic P. After a 2-month glasshouse incubation, we measured 32P transfer from AM fungi (AMF) to shoots and 13C transfer from shoots to AMF using an AMF-specific fatty acid. AMF communities were assessed using molecular methods. AMF delivered a larger proportion of total shoot P in communities from high-P soils despite similar 13C allocation to AMF in roots and soil. Within communities, 13C concentration in AMF was consistently higher in grass than in blanketflower (Gaillardia aristata Pursh) roots, that is P appeared more costly for grasses. This coincided with differences in AMF taxa composition and a trend of more vesicles (storage structures) but fewer arbuscules (exchange structures) in grass roots. Additionally, 32P-for-13C exchange ratios increased with soil P for blanketflower but not grasses. Contrary to predictions, AMF transferred proportionally more P to plants in communities from high-P soils. However, the 32P-for-13C exchange differed among co-occurring plants, suggesting differential regulation of the AM symbiosis.

Mycorrhizal effects on crop yield and soil ecosystem functions in a long-term tillage and fertilization experiment.

It is well understood that agricultural management influences arbuscular mycorrhizal (AM) fungi, but there is controversy about whether farmers should manage for AM symbiosis. We assessed AM fungal communities colonizing wheat roots for three consecutive years in a long-term (> 14 yr) tillage and fertilization experiment. Relationships among mycorrhizas, crop performance, and soil ecosystem functions were quantified. Tillage, fertilizers and continuous monoculture all reduced AM fungal richness and shifted community composition toward dominance of a few ruderal taxa. Rhizophagus and Dominikia were depressed by tillage and/or fertilization, and their abundances as well as AM fungal richness correlated positively with soil aggregate stability and nutrient cycling functions across all or no-tilled samples. In the field, wheat yield was unrelated to AM fungal abundance and correlated negatively with AM fungal richness. In a complementary glasshouse study, wheat biomass was enhanced by soil inoculum from unfertilized, no-till plots while neutral to depressed growth was observed in wheat inoculated with soils from fertilized and conventionally tilled plots. This study demonstrates contrasting impacts of low-input and conventional agricultural practices on AM symbiosis and highlights the importance of considering both crop yield and soil ecosystem functions when managing mycorrhizas for more sustainable agroecosystems.

Unraveling the diversity of hyphal explorative traits among Rhizophagus irregularis genotypes.

Differences in functioning among various genotypes of arbuscular mycorrhizal (AM) fungi can determine their fitness under specific environmental conditions, although knowledge of the underlying mechanisms still is very fragmented. Here we compared seven homokaryotic isolates (genotypes) of Rhizophagus irregularis, aiming to characterize the range of intraspecific variability with respect to hyphal exploration of organic nitrogen (N) resources, and N supply to plants. To this end we established two experiments (one in vitro and one in open pots) and used 15N-chitin as the isotopically labeled organic N source. In Experiment 1 (in vitro), mycelium of all AM fungal genotypes transferred a higher amount of 15N to the plants than the passive transfer of 15N measured in the non-mycorrhizal (NM) controls. Noticeably, certain genotypes (e.g., LPA9) showed higher extraradical mycelium biomass production but not necessarily greater 15N acquisition than the others. Experiment 2 (in pots) highlighted that some of the AM fungal genotypes (e.g., MA2, STSI) exhibited higher rates of targeted hyphal exploration of chitin-enriched zones, indicative of distinct N exploration patterns from the other genotypes. Importantly, there was a high congruence of hyphal exploration patterns between the two experiments (isolate STSI always showing highest efficiency of hyphal exploration and isolate L23/1 being consistently the lowest), despite very different (micro) environmental conditions in the two experiments. This study suggests possible strategies that AM fungal genotypes employ for efficient N acquisition, and how to measure them. Implications of such traits for local mycorrhizal community assembly still need to be understood.

Root cap is an important determinant of rhizosphere microbiome assembly.

Plants impact the development of their rhizosphere microbial communities. It is yet unclear to what extent the root cap and specific root zones contribute to microbial community assembly. To test the roles of root caps and root hairs in the establishment of microbiomes along maize roots (Zea mays), we compared the composition of prokaryote (archaea and bacteria) and protist (Cercozoa and Endomyxa) microbiomes of intact or decapped primary roots of maize inbred line B73 with its isogenic root hairless (rth3) mutant. In addition, we tracked gene expression along the root axis to identify molecular control points for an active microbiome assembly by roots. Absence of root caps had stronger effects on microbiome composition than the absence of root hairs and affected microbial community composition also at older root zones and at higher trophic levels (protists). Specific bacterial and cercozoan taxa correlated with root genes involved in immune response. Our results indicate a central role of root caps in microbiome assembly with ripple-on effects affecting higher trophic levels and microbiome composition on older root zones.

The role of arbuscular mycorrhizal symbiosis in improving plant water status under drought.

Arbuscular mycorrhizal fungi (AMF) have been presumed to ameliorate crop tolerance to drought. Here, we review the role of AMF in maintaining water supply to plants from drying soils and the underlying biophysical mechanisms. We used a soil-plant hydraulic model to illustrate the impact of several AMF mechanisms on plant responses to edaphic drought. The AMF enhance the soil's capability to transport water and extend the effective root length, thereby attenuating the drop in matric potential at the root surface during soil drying. The synthesized evidence and the corresponding simulations demonstrate that symbiosis with AMF postpones the stress onset limit, which is defined as the disproportionality between transpiration rates and leaf water potentials, during soil drying. The symbiosis can thus help crops survive extended intervals of limited water availability. We also provide our perspective on future research needs and call for reconciling the dynamic changes in soil and root hydraulics in order to better understand the role of AMF in plant water relations in the face of climate changes.

Can cardiolipins be used as a biomarker for arbuscular mycorrhizal fungi?

Specific biomarker molecules are increasingly being used for detection and quantification in plant and soil samples of arbuscular mycorrhizal (AM) fungi, an important and widespread microbial guild heavily implicated in transfers of nutrients and carbon between plants and soils and in the maintenance of soil physico-chemical properties. Yet, concerns have previously been raised as to the validity of a range of previously used approaches (e.g., microscopy, AM-specific fatty acids, sterols, glomalin-like molecules, ribosomal DNA sequences), justifying further research into novel biomarkers for AM fungal abundance and/or functioning. Here, we focused on complex polar lipids contained in pure biomass of Rhizophagus irregularis and in nonmycorrhizal and mycorrhizal roots of chicory (Cichorium intybus), leek (Allium porrum), and big bluestem (Andropogon gerardii). The lipids were analyzed by shotgun lipidomics using a high-resolution hybrid mass spectrometer. Size range between 1350 and 1550 Da was chosen for the detection of potential biomarkers among cardiolipins (1,3-bis(sn-3'-phosphatidyl)-sn-glycerols), a specific class of phospholipids. The analysis revealed a variety of molecular species, including cardiolipins containing one or two polyunsaturated fatty acids with 20 carbon atoms each, i.e., arachidonic and/or eicosapentaenoic acids, some of them apparently specific for the mycorrhizal samples. Although further verification using a greater variety of AM fungal species and samples from various soils/ecosystems/environmental conditions is needed, current results suggest the possibility to identify novel biochemical signatures specific for AM fungi within mycorrhizal roots. Whether they could be used for quantification of both root and soil colonization by the AM fungi merits further scrutiny.

Arbuscular Mycorrhiza and Nitrification: Disentangling Processes and Players by Using Synthetic Nitrification Inhibitors.

Both plants and their associated arbuscular mycorrhizal (AM) fungi require nitrogen (N) for their metabolism and growth. This can result in both positive and negative effects of AM symbiosis on plant N nutrition. Either way, the demand for and efficiency of uptake of mineral N from the soil by mycorrhizal plants are often higher than those of nonmycorrhizal plants. In consequence, the symbiosis of plants with AM fungi exerts important feedbacks on soil processes in general and N cycling in particular. Here, we investigated the role of the AM symbiosis in N uptake by Andropogon gerardii from an organic source (15N-labeled plant litter) that was provided beyond the direct reach of roots. In addition, we tested if pathways of 15N uptake from litter by mycorrhizal hyphae were affected by amendment with different synthetic nitrification inhibitors (dicyandiamide [DCD], nitrapyrin, or 3,4-dimethylpyrazole phosphate [DMPP]). We observed efficient acquisition of 15N by mycorrhizal plants through the mycorrhizal pathway, independent of nitrification inhibitors. These results were in stark contrast to 15N uptake by nonmycorrhizal plants, which generally took up much less 15N, and the uptake was further suppressed by nitrapyrin or DMPP amendments. Quantitative real-time PCR analyses showed that bacteria involved in the rate-limiting step of nitrification, ammonia oxidation, were suppressed similarly by the presence of AM fungi and by nitrapyrin or DMPP (but not DCD) amendments. On the other hand, abundances of ammonia-oxidizing archaea were not strongly affected by either the AM fungi or the nitrification inhibitors. IMPORTANCE Nitrogen is one of the most important elements for all life on Earth. In soil, N is present in various chemical forms and is fiercely competed for by various microorganisms as well as plants. Here, we address competition for reduced N (ammonia) between ammonia-oxidizing prokaryotes and arbuscular mycorrhizal fungi. These two functionally important groups of soil microorganisms, participating in nitrification and plant mineral nutrient acquisition, respectively, have often been studied in separation in the past. Here, we showed, using various biochemical and molecular approaches, that the fungi systematically suppress ammonia-oxidizing bacteria to an extent similar to that of some widely used synthetic nitrification inhibitors, whereas they have only a limited impact on abundance of ammonia-oxidizing archaea. Competition for free ammonium is a plausible explanation here, but it is also possible that the fungi produce some compounds acting as so-called biological nitrification inhibitors.

Community response of arbuscular mycorrhizal fungi to extreme drought in a cold-temperate grassland.

Climate extremes pose enormous threats to natural ecosystems. Arbuscular mycorrhizal (AM) fungi are key plant symbionts that can affect plant community dynamics and ecosystem stability. However, knowledge about how AM fungal communities respond to climate extremes in natural ecosystems remains elusive. Based on a grassland extreme drought experiment in Inner Mongolia, we investigated the response of AM fungal communities to extreme drought in association with plant communities. The experiment simulated two types of extreme drought (chronic/intense) of once-in-20-year occurrence. AM fungal richness and community composition exhibited high sensitivity to extreme drought and were more sensitive to intense drought than chronic drought. This community sensitivity (i.e. decline in richness and shifts in community composition) of AM fungi can be jointly explained by soil moisture, plant richness, and aboveground productivity. Notably, the robustness of the plant-AM fungal community co-response increased with drought intensity. Our results indicate that AM fungal communities are sensitive to climate extremes, and we propose that the plant community mediates AM fungal community responses. Given the ubiquitous nature of AM associations, their climate sensitivity may have profound consequences on plant communities and ecosystem stability under climate change.

Facilitation of plant water uptake by an arbuscular mycorrhizal fungus: a Gordian knot of roots and hyphae.

Arbuscular mycorrhizal (AM) fungi play a positive role in plant water relations, and the AM symbiosis is often cited as beneficial for overcoming drought stress of host plants. Nevertheless, water uptake via mycorrhizal hyphal networks has been little addressed experimentally, especially so through isotope tracing. In a greenhouse study conducted in two-compartment rhizoboxes, Medicago truncatula was planted in the primary compartment (PC), either inoculated with Rhizophagus irregularis or left uninoculated. Plant roots were either allowed to enter the secondary compartment (SC) or were restricted to the PC by root-excluding mesh. Substrate moisture was manipulated in the PC such that the plants were grown either in high moisture (15% of gravimetric water content, GWC) or low moisture (8% GWC). Meanwhile, the SC was maintained at 15% GWC throughout and served as a water source accessible (or not) by roots and/or hyphae. Water in the SC was labeled with deuterium (D) to quantify water uptake by the plants from the SC. Significantly, increased D incorporation into plants indicated higher water uptake by mycorrhizal plants when roots had access to the D source, but this was mainly explained by generally larger mycorrhizal root systems in proximity to the D source. On the other hand, AM fungal hyphae with access to the D source increased D incorporation into plants more than twofold compared to non-mycorrhizal plants. Despite this strong effect, water transport via AM fungal hyphae was low compared to the transpiration demand of the plants.

Dead Rhizophagus irregularis biomass mysteriously stimulates plant growth.

Arbuscular mycorrhizal (AM) fungi establish symbiotic associations with many plant species, transferring significant amounts of soil nutrients such as phosphorus to plants and receiving photosynthetically fixed carbon in return. Functioning of AM symbiosis is thus based on interaction between two living partners. The importance of dead AM fungal biomass (necromass) in ecosystem processes remains unclear. Here, we applied either living biomass or necromass (0.0004 potting substrate weight percent) of monoxenically produced AM fungus (Rhizophagus irregularis) into previously sterilized potting substrate planted with Andropogon gerardii. Plant biomass production significantly improved in both treatments as compared to non-amended controls. Living AM fungus, in contrast to the necromass, specifically improved plant acquisition of nutrients normally supplied to the plants by AM fungal networks, such as phosphorus and zinc. There was, however, no difference between the two amendment treatments with respect to plant uptake of other nutrients such as nitrogen and/or magnesium, indicating that the effect on plants of the AM fungal necromass was not primarily nutritional. Plant growth stimulation by the necromass could thus be either due to AM fungal metabolites directly affecting the plants, indirectly due to changes in soil/root microbiomes or due to physicochemical modifications of the potting substrate. In the necromass, we identified several potentially bioactive molecules. We also provide experimental evidence for significant differences in underground microbiomes depending on the amendment with living or dead AM fungal biomass. This research thus provides the first glimpse into possible mechanisms responsible for observed plant growth stimulation by the AM fungal necromass.

Abiotic contexts consistently influence mycorrhiza functioning independently of the composition of synthetic arbuscular mycorrhizal fungal communities.

The relationship between mycorrhiza functioning and composition of arbuscular mycorrhizal (AM) fungal communities is an important but experimentally still rather little explored topic. The main aim of this study was thus to link magnitude of plant benefits from AM symbiosis in different abiotic contexts with quantitative changes in AM fungal community composition. A synthetic AM fungal community inoculated to the model host plant Medicago truncatula was exposed to four different abiotic contexts, namely drought, elevated phosphorus availability, and shading, as compared to standard cultivation conditions, for two cultivation cycles. Growth and phosphorus uptake of the host plants was evaluated along with the quantitative composition of the synthetic AM fungal community. Abiotic context consistently influenced mycorrhiza functioning in terms of plant benefits, and the effects were clearly linked to the P requirement of non-inoculated control plants. In contrast, the abiotic context only had a small and transient effect on the quantitative AM fungal community composition. Our findings suggest no relationship between the degree of mutualism in AM symbiosis and the relative abundances of AM fungal species in communities in our simplified model system. The observed progressive dominance of one AM fungal species indicates an important role of different growth rates of AM fungal species for the establishment of AM fungal communities in simplified systems such as agroecosystems.

Geography and habitat predominate over climate influences on arbuscular mycorrhizal fungal communities of mid-European meadows.

Despite the crucial importance of arbuscular mycorrhizal fungi (AMF) for numerous processes within terrestrial ecosystems, knowledge of the determinants of AMF community structure still is limited, mainly because of the limited scope of the available individual case studies which often only include a few environmental variables. Here, we describe the AMF diversity of mid-European meadows (mown or regularly cut grasslands, or recently abandoned lands where grasslands established spontaneously) within a considerably heterogeneous landscape over a scale of several hundred kilometers with regard to macroclimatic, microclimatic, and soil parameters. We include data describing the habitat (including vegetation type), geography, and climate, and test their contribution to the structure of the AMF communities at a regional scale. We amplified and sequenced the ITS 2 region of the ribosomal DNA operon of the AMF from soil samples using nested PCR and Illumina pair-end amplicon sequencing. Habitat (especially soil pH) and geographical parameters (spatial distance, altitude, and longitude) were the main determinants of the structure of the AMF communities in the meadows at a regional scale, with the abundance of genera Septoglomus, Paraglomus, Archaeospora, Funneliformis, and Dominikia driving the main response. The effects of climate and vegetation type were not significant and were mainly encompassed within the geography and/or soil pH effects. This study illustrates how important it is to have a large set of environmental metadata to compare the importance of different factors influencing the AMF community structure at large spatial scales.

Allelic Variants for Candidate Nitrogen Fixation Genes Revealed by Sequencing in Red Clover (Trifolium pratense L.).

Plant-rhizobia symbiosis can activate key genes involved in regulating nodulation associated with biological nitrogen fixation (BNF). Although the general molecular basis of the BNF process is frequently studied, little is known about its intraspecific variability and the characteristics of its allelic variants. This study's main goals were to describe phenotypic and genotypic variation in the context of nitrogen fixation in red clover (Trifolium pretense L.) and identify variants in BNF candidate genes associated with BNF efficiency. Acetylene reduction assay validation was the criterion for selecting individual plants with particular BNF rates. Sequences in 86 key candidate genes were obtained by hybridization-based sequence capture target enrichment of plants with alternative phenotypes for nitrogen fixation. Two genes associated with BNF were identified: ethylene response factor required for nodule differentiation (EFD) and molybdate transporter 1 (MOT1). In addition, whole-genome population genotyping by double-digest restriction-site-associated sequencing (ddRADseq) was performed, and BNF was evaluated by the natural 15N abundance method. Polymorphisms associated with BNF and reflecting phenotype variability were identified. The genetic structure of plant accessions was not linked to BNF rate of measured plants. Knowledge of the genetic variation within BNF candidate genes and the characteristics of genetic variants will be beneficial in molecular diagnostics and breeding of red clover.

Validation of dose distribution computation on sCT images generated from MRI scans by Philips MRCAT.

AIM: To evaluate calculation of treatment plans based on synthetic-CT (sCT) images generated from MRI. BACKGROUND: Because of better soft tissue contrast, MR images are used in addition to CT images for radiotherapy planning. However, registration of CT and MR images or repositioning between scanning sessions introduce systematic errors, hence suggestions for MRI-only therapy. The lack of information on electron density necessary for dose calculation leads to sCT (synthetic CT) generation. This work presents a comparison of dose distribution calculated on standard CT and sCT. MATERIALS AND METHODS: 10 prostate patients were included in this study. CT and MR images were collected for each patient and then water equivalent (WE) and MRCAT images were generated. The radiation plans were optimized on CT and then recalculated on MRCAT and WE data. 2D gamma analysis was also performed. RESULTS: The mean differences in the majority of investigated DVH points were in order of 1% up to 10%, including both MRCAT and WE dose distributions. Mean gamma pass for acceptance criteria 1%/1 mm were greater than 82.5%. Prescribed doses for target volumes and acceptable doses for organs at risk were met in almost all cases. CONCLUSIONS: The dose calculation accuracy on MRCAT was not significantly compromised in the majority of clinical relevant DVH points. The introduction of MRCAT into practise would eliminate systematic errors, increase patients' comfort and reduce treatment expenses. Institutions interested in MRCAT commissioning must, however, consider changes to established workflow.

The direction of carbon and nitrogen fluxes between ramets in Agrostis stolonifera changes during ontogeny under simulated competition for light.

Resource sharing is universal among connected ramets of clonal plants and is driven both by the developmental status of the ramets and the resource gradients. Above-ground competition forms spatial light gradients, but the role of resource sharing in such competition is unclear. We examined translocation of resources between mother and daughter ramets of Agrostis stolonifera under light heterogeneity throughout ramet ontogeny. We labelled ramets with 13C and 15N to estimate the bidirectional translocation of resources at three developmental stages of the daughters. In addition, we compared the final biomass of integrated and severed ramets in order to estimate the effect of integration on growth. Young developing daughters were supported by carbon, whereas nitrogen was only translocated towards daughters at the beginning of rooting, regardless of the light conditions. Shading of mothers was a major determinant of resource translocation between developed ramets, with carbon being preferentially moved to daughters from shaded mothers while nitrogen translocation was limited from daughters to shaded mothers. Surprisingly, the absolute amounts of translocated resources did not decline during development. Growth of daughters was enhanced by integration regardless of the shading. Overall, A. stolonifera maximizes the resource translocation pattern in order to enable it to spread from unfavourable habitats, rather than compensating for light heterogeneity among ramets.

Appropriate nonmycorrhizal controls in arbuscular mycorrhiza research: a microbiome perspective.

Establishment of nonmycorrhizal controls is a "classic and recurrent theme" in mycorrhizal research. For decades, authors reported mycorrhizal plant growth/nutrition as compared to various nonmycorrhizal controls. In such studies, uncertainties remain about which nonmycorrhizal controls are most appropriate and, in particular, what effects the control inoculations have on substrate and root microbiomes. Here, different types of control and mycorrhizal inoculations were compared with respect to plant growth and nutrition, as well as the structure of root and substrate microbiomes, assessed by next-generation sequencing. We compared uninoculated ("absolute") control to inoculation with blank pot culture lacking arbuscular mycorrhizal fungi, filtrate of that blank inoculum, and filtrate of complex pot-produced mycorrhizal inoculum. Those treatments were compared to a standard mycorrhizal treatment, where the previously sterilized substrate was inoculated with complex pot-produced inoculum containing Rhizophagus irregularis SYM5. Besides this, monoxenically produced inoculum of the same fungus was applied either alone or in combination with blank inoculum. The results indicate that the presence of mycorrhizal fungus always resulted in stimulation of Andropogon gerardii plant biomass as well as in elevated phosphorus content of the plants. The microbial (bacterial and fungal) communities developing in the differently inoculated treatments, however, differed substantially from each other and no control could be obtained comparable with the treatment inoculated with complex mycorrhizal inoculum. Soil microorganisms with significant biological competences that could potentially contribute to the effects of the various inoculants on the plants were detected in roots and in plant cultivation substrate in some of the treatments.

Utilization of organic nitrogen by arbuscular mycorrhizal fungi-is there a specific role for protists and ammonia oxidizers?

Arbuscular mycorrhizal (AM) fungi can significantly contribute to plant nitrogen (N) uptake from complex organic sources, most likely in concert with activity of soil saprotrophs and other microbes releasing and transforming the N bound in organic forms. Here, we tested whether AM fungus (Rhizophagus irregularis) extraradical hyphal networks showed any preferences towards certain forms of organic N (chitin of fungal or crustacean origin, DNA, clover biomass, or albumin) administered in spatially discrete patches, and how the presence of AM fungal hyphae affected other microbes. By direct 15N labeling, we also quantified the flux of N to the plants (Andropogon gerardii) through the AM fungal hyphae from fungal chitin and from clover biomass. The AM fungal hyphae colonized patches supplemented with organic N sources significantly more than those receiving only mineral nutrients, organic carbon in form of cellulose, or nothing. Mycorrhizal plants grew 6.4-fold larger and accumulated, on average, 20.3-fold more 15N originating from the labeled organic sources than their nonmycorrhizal counterparts. Whereas the abundance of microbes (bacteria, fungi, or Acanthamoeba sp.) in the different patches was primarily driven by patch quality, we noted a consistent suppression of the microbial abundances by the presence of AM fungal hyphae. This suppression was particularly strong for ammonia oxidizing bacteria. Our results indicate that AM fungi successfully competed with the other microbes for free ammonium ions and suggest an important role for the notoriously understudied soil protists to play in recycling organic N from soil to plants via AM fungal hyphae.

Utilization of organic nitrogen by arbuscular mycorrhizal fungi-is there a specific role for protists and ammonia oxidizers?

Arbuscular mycorrhizal (AM) fungi can significantly contribute to plant nitrogen (N) uptake from complex organic sources, most likely in concert with activity of soil saprotrophs and other microbes releasing and transforming the N bound in organic forms. Here, we tested whether AM fungus (Rhizophagus irregularis) extraradical hyphal networks showed any preferences towards certain forms of organic N (chitin of fungal or crustacean origin, DNA, clover biomass, or albumin) administered in spatially discrete patches, and how the presence of AM fungal hyphae affected other microbes. By direct 15N labeling, we also quantified the flux of N to the plants (Andropogon gerardii) through the AM fungal hyphae from fungal chitin and from clover biomass. The AM fungal hyphae colonized patches supplemented with organic N sources significantly more than those receiving only mineral nutrients, organic carbon in form of cellulose, or nothing. Mycorrhizal plants grew 6.4-fold larger and accumulated, on average, 20.3-fold more 15N originating from the labeled organic sources than their nonmycorrhizal counterparts. Whereas the abundance of microbes (bacteria, fungi, or Acanthamoeba sp.) in the different patches was primarily driven by patch quality, we noted a consistent suppression of the microbial abundances by the presence of AM fungal hyphae. This suppression was particularly strong for ammonia oxidizing bacteria. Our results indicate that AM fungi successfully competed with the other microbes for free ammonium ions and suggest an important role for the notoriously understudied soil protists to play in recycling organic N from soil to plants via AM fungal hyphae.

Real-time PCR quantification of arbuscular mycorrhizal fungi: does the use of nuclear or mitochondrial markers make a difference?

Root colonization by arbuscular mycorrhizal fungi (AMF) can be quantified by different approaches. We compared two approaches that enable discrimination of specific AMF taxa and are therefore emerging as alternative to most commonly performed microscopic quantification of AMF in roots: quantitative real-time PCR (qPCR) using markers in nuclear ribosomal DNA (nrDNA) and mitochondrial ribosomal DNA (mtDNA). In a greenhouse experiment, Medicago truncatula was inoculated with four isolates belonging to different AMF species (Rhizophagus irregularis, Claroideoglomus claroideum, Gigaspora margarita and Funneliformis mosseae). The AMF were quantified in the root samples by qPCR targeted to both markers, microscopy and contents of AMF-specific phospholipid fatty acids (PLFA). Copy numbers of nrDNA and mtDNA were closely related within all isolates; however, the slopes and intercepts of the linear relationships significantly differed among the isolates. Across all isolates, a large proportion of variance in nrDNA copy numbers was explained by root colonization intensity or contents of AMF-specific PLFA, while variance in mtDNA copy numbers was mainly explained by differences among AMF isolates. We propose that the encountered inter-isolate differences in the ratios of mtDNA and nrDNA copy numbers reflect different physiological states of the isolates. Our results suggest that nrDNA is a more suitable marker region than mtDNA for the quantification of multiple AMF taxa as its copy numbers are better related to fungal biomass across taxa than are copy numbers of mtDNA.