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.

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.

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.

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.

Evaluation of Infliximab Therapy in Children with Crohn's Disease Using Trough Levels Predictors.

BACKGROUND: In adults, infliximab (IFX) levels correlate with disease activity, and antibodies to IFX (ATIs) predict treatment failure. We aimed to determine the association of IFX levels and ATIs with disease activity in a paediatric population. We prospectively collected blood, stool, and clinical data from 65 patients (age 10.5-15.1 years) with Crohn's disease (CD) before IFX administration, and measured IFX trough levels, ATIs, and faecal calprotectin levels (CPT). Samples were collected during maintenance therapy. We used multivariate analysis to identify the predictors of IFX levels. SUMMARY: Lower levels of IFX were associated with ATIs positivity (OR 0.027, 95% CI 0.009-0.077). Higher C-reactive protein (CRP) level, erythrocyte sedimentation rate, and CPT levels were found in patients with lower IFX levels. The optimal combination of sensitivity (0.5) and specificity (0.74) for disease activity was calculated for IFX levels ≥1.1 µg/mL using CRP level <5 mg/L as a marker of laboratory remission. In a model that used CPT ≤100 µg/g as the definition of remission, the optimal IFX trough level was 3.5 µg/mL. No independent association between remission and ATIs was found in our study population. However, we found an independentz association between IFX levels and serum albumin levels (OR 1.364, 95% CI 1.169-1.593), p < 0.001. Key Messages: The paediatric population was similar to adult populations in terms of the association between IFX and ATIs as well as between IFX and disease activity.

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.

Extremely Acidic Soils are Dominated by Species-Poor and Highly Specific Fungal Communities.

Highly acidic soils (pH < 3) represent an environment which might potentially offer new biotechnologically interesting fungi. Nevertheless, only little data on fungal communities in highly acidic habitats are available. Here, we focused on the diversity of cultivable filamentous microfungi in highly acidic soils (pH < 3) in the Czech Republic. Altogether, 16 soil samples were collected from four sampling sites and were processed by various approaches. In total, 54 fungal taxa were isolated and identified using classical as well as molecular markers. All dominant species were found both as living mycelia and as resistant stages. Numerous recently described or unknown taxa were isolated. The core of the fungal assemblage under study consisted of phylogenetically unrelated and often globally distributed fungi exclusively inhabiting highly acidic habitats like Acidiella bohemica, Acidomyces acidophilus, and unidentified helotialean fungus, as well as taxa known from less acidic and often extreme environments like Acidea extrema, Penicillium simplicissimum s.l., and Penicillium spinulosum. The large number of identified specialized species indicates that highly acidic environments provide suitable conditions for the evolution of specialist species. The occurrence of ubiquitous fungi in highly acidic substrates points to the principal role of competition in the colonization of such environments. The detected taxa did not require low pH to survive, because they can grow in a broad range of pH.

Tuber aestivum association with non-host roots.

Mycorrhizal fungi provide direct and functional interconnection of soil environment with their host plant roots. Colonization of non-host plants have occasionally been described, but its intensity and functional significance in complex plant communities remain generally unknown. Here, the abundance of ectomycorrhizal fungus Tuber aestivum was measured in the roots of host and non-host (non-ectomycorrhizal) plants in a naturally occurring T. aestivum colony using a quantitative PCR approach. The roots of non-host plant species found inside the brûlé area were extensively colonized by T. aestivum mycelium, although the levels were significantly lower than those found in host Carpinus betulus roots. However, fungal biomass concentration in the non-host roots was one to two orders of magnitude higher than that in the surrounding soil. This indicates existence of an important biotic interaction between T. aestivum mycelium and the non-host, mostly herbaceous plants. Roots, either host or non-host, thus probably constitute hot spots of T. aestivum activity in the soil ecosystem with as yet uncovered functional significance.

Nutrient-dependent cross-kingdom interactions in the hyphosphere of an arbuscular mycorrhizal fungus.

Introduction: The hyphosphere of arbuscular mycorrhizal (AM) fungi is teeming with microbial life. Yet, the influence of nutrient availability or nutrient forms on the hyphosphere microbiomes is still poorly understood. Methods: Here, we examined how the microbial community (prokaryotic, fungal, protistan) was affected by the presence of the AM fungus Rhizophagus irregularis in the rhizosphere and the root-free zone, and how different nitrogen (N) and phosphorus (P) supplements into the root-free compartment influenced the communities. Results: The presence of AM fungus greatly affected microbial communities both in the rhizosphere and the root-free zone, with prokaryotic communities being affected the most. Protists were the only group of microbes whose richness and diversity were significantly reduced by the presence of the AM fungus. Our results showed that the type of nutrients AM fungi encounter in localized patches modulate the structure of hyphosphere microbial communities. In contrast we did not observe any effects of the AM fungus on (non-mycorrhizal) fungal community composition. Compared to the non-mycorrhizal control, the root-free zone with the AM fungus (i.e., the AM fungal hyphosphere) was enriched with Alphaproteobacteria, some micropredatory and copiotroph bacterial taxa (e.g., Xanthomonadaceae and Bacteroidota), and the poorly characterized and not yet cultured Acidobacteriota subgroup GP17, especially when phytate was added. Ammonia-oxidizing Nitrosomonas and nitrite-oxidizing Nitrospira were significantly suppressed in the presence of the AM fungus in the root-free compartment, especially upon addition of inorganic N. Co-occurrence network analyses revealed that microbial communities in the root-free compartment were complex and interconnected with more keystone species when AM fungus was present, especially when the root-free compartment was amended with phytate. Conclusion: Our study showed that the form of nutrients is an important driver of prokaryotic and eukaryotic community assembly in the AM fungal hyphosphere, despite the assumed presence of a stable and specific AM fungal hyphoplane microbiome. Predictable responses of specific microbial taxa will open the possibility of using them as co-inoculants with AM fungi, e.g., to improve crop performance.

Organic nitrogen utilisation by an arbuscular mycorrhizal fungus is mediated by specific soil bacteria and a protist.

Arbuscular mycorrhizal (AM) fungi lack efficient exoenzymes to access organic nutrients directly. Nevertheless, the fungi often obtain and further channel to their host plants a significant share of nitrogen (N) and/or phosphorus from such resources, presumably via cooperation with other soil microorganisms. Because it is challenging to disentangle individual microbial players and processes in complex soil, we took a synthetic approach here to study 15N-labelled chitin (an organic N source) recycling via microbial loop in AM fungal hyphosphere. To this end, we employed a compartmented in vitro cultivation system and monoxenic culture of Rhizophagus irregularis associated with Cichorium intybus roots, various soil bacteria, and the protist Polysphondylium pallidum. We showed that upon presence of Paenibacillus sp. in its hyphosphere, the AM fungus (and associated plant roots) obtained several-fold larger quantities of N from the chitin than it did with any other bacteria, whether chitinolytic or not. Moreover, we demonstrated that adding P. pallidum to the hyphosphere with Paenibacillus sp. further increased by at least 65% the gain of N from the chitin by the AM fungus compared to the hyphosphere without protists. We thus directly demonstrate microbial interplay possibly involved in efficient organic N utilisation by AM fungal hyphae.

Arbuscular Mycorrhiza Mediates Efficient Recycling From Soil to Plants of Nitrogen Bound in Chitin.

Symbiosis between plants and arbuscular mycorrhizal (AM) fungi, involving great majority of extant plant species including most crops, is heavily implicated in plant mineral nutrition, abiotic and biotic stress tolerance, soil aggregate stabilization, as well as shaping soil microbiomes. The latter is particularly important for efficient recycling from soil to plants of nutrients such as phosphorus and nitrogen (N) bound in organic forms. Chitin is one of the most widespread polysaccharides on Earth, and contains substantial amounts of N (>6% by weight). Chitin is present in insect exoskeletons and cell walls of many fungi, and can be degraded by many prokaryotic as well as eukaryotic microbes normally present in soil. However, the AM fungi seem not to have the ability to directly access N bound in chitin molecules, thus relying on microbes in their hyphosphere to gain access to this nutrient-rich resource in the process referred to as organic N mineralization. Here we show, using data from two pot experiments, both including root-free compartments amended with 15N-labeled chitin, that AM fungi can channel substantial proportions (more than 20%) of N supplied as chitin into their plants hosts within as short as 5 weeks. Further, we show that overall N losses (leaching and/or volatilization), sometimes exceeding 50% of the N supplied to the soil as chitin within several weeks, were significantly lower in mycorrhizal as compared to non-mycorrhizal pots. Surprisingly, the rate of chitin mineralization and its N utilization by the AM fungi was at least as fast as that of green manure (clover biomass), based on direct 15N labeling and tracing. This efficient N recycling from soil to plant, observed in mycorrhizal pots, was not strongly affected by the composition of AM fungal communities or environmental context (glasshouse or outdoors, additional mineral N supply to the plants or not). These results indicate that AM fungi in general can be regarded as a critical and robust soil resource with respect to complex soil processes such as organic N mineralization and recycling. More specific research is warranted into the exact molecular mechanisms and microbial players behind the observed patterns.

Correlative evidence for co-regulation of phosphorus and carbon exchanges with symbiotic fungus in the arbuscular mycorrhizal Medicago truncatula.

Research efforts directed to elucidation of mechanisms behind trading of resources between the partners in the arbuscular mycorrhizal (AM) symbiosis have seen a considerable progress in the recent years. Yet, despite of the recent developments, some key questions still remain unanswered. For example, it is well established that the strictly biotrophic AM fungus releases phosphorus to- and receives carbon molecules from the plant symbiont, but the particular genes, and their products, responsible for facilitating this exchange, are still not fully described, nor are the principles and pathways of their regulation. Here, we made a de novo quest for genes involved in carbon transfer from the plant to the fungus using genome-wide gene expression array targeting whole root and whole shoot gene expression profiles of mycorrhizal and non-mycorrhizal Medicago truncatula plants grown in a glasshouse. Using physiological intervention of heavy shading (90% incoming light removed) and the correlation of expression levels of MtPT4, the mycorrhiza-inducible phosphate transporter operating at the symbiotic interface between the root cortical cells and the AM fungus, and our candidate genes, we demonstrate that several novel genes may be involved in resource tradings in the AM symbiosis established by M. truncatula. These include glucose-6-phosphate/phosphate translocator, polyol/monosaccharide transporter, DUR3-like, nucleotide-diphospho-sugar transferase or a putative membrane transporter. Besides, we also examined the expression of other M. truncatula phosphate transporters (MtPT1-3, MtPT5-6) to gain further insights in the balance between the "direct" and the "mycorrhizal" phosphate uptake pathways upon colonization of roots by the AM fungus, as affected by short-term carbon/energy deprivation. In addition, the role of the novel candidate genes in plant cell metabolism is discussed based on available literature.

Soil Matrix Determines the Outcome of Interaction Between Mycorrhizal Symbiosis and Biochar for Andropogon gerardii Growth and Nutrition.

Biochar has been heralded as a multipurpose soil amendment to sustainably increase soil fertility and crop yields, affect soil hydraulic properties, reduce nutrient losses, and sequester carbon. Some of the most spectacular results of biochar (and organic nutrient) inputs are the terra preta soils in the Amazon, dark anthropogenic soils with extremely high fertility sustained over centuries. Such soil improvements have been particularly difficult to achieve on a short run, leading to speculations that biochar may need to age (weather) in soil to show its best. Further, interaction of biochar with arbuscular mycorrhizal fungi (AMF), important root symbionts of a great majority of terrestrial plants including most agricultural crops, remains little explored. To study the effect of aged biochar on highly mycotrophic Andropogon gerardii plants and their associated AMF, we made use of softwood biochar, collected from a historic charcoal burning site. This biochar (either untreated or chemically activated, the latter serving as a proxy for freshly prepared biochar) was added into two agricultural soils (acid or alkaline), and compared to soils without biochar. These treatments were further crossed with inoculation with a synthetic AMF community to address possible interactions between biochar and the AMF. Biochar application was generally detrimental for growth and mineral nutrition of our experimental plants, but had no effect on the extent of their root colonized by the AMF, nor did it affect composition of their root-borne AMF communities. In contrast, biochar affected development of two out of five AMF (Claroideoglomus and Funneliformis) in the soil. Establishment of symbiosis with AMF largely mitigated biochar-induced suppression of plant growth and mineral nutrition, mainly by improving plant acquisition of phosphorus. Both mycorrhizal and non-mycorrhizal plants grew well in the acid soil without biochar application, whereas non-mycorrhizal plants remained stunted in the alkaline soils under all situations (with or without biochar). These different and strong effects indicate that response of plants to biochar application are largely dependent on soil matrix and also on microbes such as AMF, and call for further research to enable qualified predictions of the effects of different biochar applications on field-grown crops and soil processes.

Organic Nitrogen-Driven Stimulation of Arbuscular Mycorrhizal Fungal Hyphae Correlates with Abundance of Ammonia Oxidizers.

Large fraction of mineral nutrients in natural soil environments is recycled from complex and heterogeneously distributed organic sources. These sources are explored by both roots and associated mycorrhizal fungi. However, the mechanisms behind the responses of arbuscular mycorrhizal (AM) hyphal networks to soil organic patches of different qualities remain little understood. Therefore, we conducted a multiple-choice experiment examining hyphal responses to different soil patches within the root-free zone by two AM fungal species (Rhizophagus irregularis and Claroideoglomus claroideum) associated with Medicago truncatula, a legume forming nitrogen-fixing root nodules. Hyphal colonization of the patches was assessed microscopically and by quantitative real-time PCR (qPCR) using AM taxon-specific markers, and the prokaryotic and fungal communities in the patches (pooled per organic amendment treatment) were profiled by 454-amplicon sequencing. Specific qPCR markers were then designed and used to quantify the abundance of prokaryotic taxa showing the strongest correlation with the pattern of AM hyphal proliferation in the organic patches as per the 454-sequencing. The hyphal density of both AM fungi increased due to nitrogen (N)-containing organic amendments (i.e., chitin, DNA, albumin, and clover biomass), while no responses as compared to the non-amended soil patch were recorded for cellulose, phytate, or inorganic phosphate amendments. Abundances of several prokaryotes, including Nitrosospira sp. (an ammonium oxidizer) and an unknown prokaryote with affiliation to Acanthamoeba endosymbiont, which were frequently recorded in the 454-sequencing profiles, correlated positively with the hyphal responses of R. irregularis to the soil amendments. Strong correlation between abundance of these two prokaryotes and the hyphal responses to organic soil amendments by both AM fungi was then confirmed by qPCR analyses using all individual replicate patch samples. Further research is warranted to ascertain the causality of these correlations and particularly which direct roles (if any) do these prokaryotes play in the observed AM hyphal responses to organic N amendment, organic N utilization by the AM fungus and its (N-unlimited) host plant. Further, possible trophic dependencies between the different players in the AM hyphosphere needs to be elucidated upon decomposing the organic N sources.

Mycorrhizal hyphae as ecological niche for highly specialized hypersymbionts - or just soil free-riders?

Mycorrhizal fungi interconnect two different kinds of environments, namely the plant roots with the surrounding soil. This widespread coexistence of plants and fungi has important consequences for plant mineral nutrition, water acquisition, carbon allocation, tolerance to abiotic and biotic stresses and interplant competition. Yet some current research indicates a number of important roles to be played by hyphae-associated microbes, in addition to the hyphae themselves, in foraging for and acquisition of soil resources and in transformation of organic carbon in the soil-plant systems. We critically review the available scientific evidence for the theory that the surface of mycorrhizal hyphae in soil is colonized by highly specialized microbial communities, and that these fulfill important functions in the ecology of mycorrhizal fungal hyphae such as accessing recalcitrant forms of mineral nutrients, and production of signaling and other compounds in the vicinity of the hyphae. The validity of another hypothesis will then be addressed, namely that the specific associative microbes are rewarded with exclusive access to fungal carbon, which would qualify them as hypersymbionts (i.e., symbionts of symbiotic mycorrhizal fungi). Thereafter, we ask whether recruitment of functionally different microbial assemblages by the hyphae is required under different soil conditions (questioning what evidence is available for such an effect), and we identify knowledge gaps requiring further attention.

Terminal restriction fragment length measurement errors are affected mainly by fragment length, G+C nucleotide content and secondary structure melting point.

Several methods of molecular analysis of microbial diversity, including terminal restriction fragment length polymorphism (T-RFLP) analysis are based on measurement of the DNA fragment length. Significant variation between sequence-determined and measured length of restriction fragments (drift) has been observed, which can affect the efficiency of the identification of microorganisms in the analyzed communities. In the past, this variation has been attributed to varying fragment length and purine content. In this study, principal component analysis and multiple regression analysis were applied to find the contributions of those and several other fragment characteristics. We conclude that secondary structure melting point and G+C nucleotide content, besides the fragment length, contribute to the variation observed, whereas the contribution of purine content is less important. Incomplete denaturation of the sample at the start of electrophoretic separation of fragments has been excluded as a major cause of the variation observed. Our regression model explains the observed drift variation by approximately 56%, with standard deviation of the prediction equal to approximately 1.3 bp.