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 mycorrhizal fungi suppress ammonia-oxidizing bacteria but not archaea across agricultural soils.

Arbuscular mycorrhizal (AM) fungi are supposedly competing with ammonia-oxidizing microorganisms (AO) for soil nitrogen in form of ammonium. Despite a few studies directly addressing AM fungal and AO interactions, mostly in artificial cultivation substrates, it is not yet clear whether AM fungi can effectively suppress AO in field soils containing complex indigenous microbiomes. To fill this knowledge gap, we conducted compartmentalized pot experiments using four pairs of cropland and grassland soils with varying physicochemical properties. To exclude the interference of roots, a fine nylon mesh was used to separate the rhizosphere and mesh bags, with the latter being filled with unsterile field soils. Inoculation of plants with AM fungus Rhizophagus irregularis LPA9 suppressed AO bacteria (AOB) but not archaea (AOA) in the soils, indicating how soil nitrification could be suppressed by AM fungal presence/activity. In addition, in rhizosphere filled with artificial substrate, AM inoculation did suppress both AOB and AOA, implying more complex interactions between roots, AO, and AM fungi. Besides, we also observed that indigenous AM fungi contained in the field soils eventually did colonize the roots of plants behind the root barrier, and that the extent of such colonization was higher if the soil has previously been taken from cropland than from grassland. Despite this, the effect of experimental AM fungal inoculation on suppression of indigenous AOB in the unsterile field soils did not vanish. It seems that studying processes at a finer temporal scale, using larger buffer zones between rhizosphere and mesh bags, and/or detailed characterization of indigenous AM fungal and AO communities would be needed to uncover further details of the biotic interactions between the AM fungi and indigenous soil AO.

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.

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.

Separation of triacylglycerols containing positional isomers of hexadecenoic acids by enantiomeric liquid chromatography-mass spectrometry.

Triacylglycerols (TAGs) containing positional isomers of hypogeic (Hy), palmitoleic (Po), and palmitvaccenic (Pv) acids from three microorganisms (top-fermenting brewer's yeast Saccharomyces cerevisiae, green alga Coccomyxa elongata, and arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis) were analyzed. Dozens of regioisomers and enantiomers of TAGs containing one, two or three hexadecenoic acids have been identified by means of reversed phase chromatography/mass spectrometry (RP-HPLC/MS). The regioisomers of TAGs containing two palmitic acids and any hexadecenoic acid were separated. Analysis of regioisomers of TAGs having one Pv residue showed that asymmetric molecular species such as PvPP or PPPv were dominant in Rhizophagus. TAGs were also analyzed on a chiral phase column and nine molecular species of TAGs containing two palmitic and any of three hexadecenoic acids were separated and identified. In the case of TAGs containing one palmitic and two hexadecenoic acids, the separation was successful only if the hexadecenoic acids were identical. Separation of TAGs containing three hexadecenoic acids was successful only if all three hexadecenoic acids were identical. Regardless of the type of TAG, it was found that TAGs in the AM fungus and containing palmitvaccenic acid bound at the sn-1 position of the glycerol backbone were dominant, suggesting similarity in the biosynthesis of the different TAGs. The covalent adduct chemical ionization method was used for identification of TAGs as adduct with (1-methyleneimino)-1-ethenyl ion, which reacted with double bond of the unsaturated fatty acid. Tandem MS thus makes it possible to identify TAGs containing various hexadecenoic acids.

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.

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.