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.

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.

Arbuscular common mycorrhizal networks mediate intra- and interspecific interactions of two prairie grasses.

Arbuscular mycorrhizal fungi form extensive common mycorrhizal networks (CMNs) that may interconnect neighboring root systems of the same or different plant species, thereby potentially influencing the distribution of limiting mineral nutrients among plants. We examined how CMNs affected intra- and interspecific interactions within and between populations of Andropogon gerardii, a highly mycorrhiza dependent, dominant prairie grass and Elymus canadensis, a moderately dependent, subordinate prairie species. We grew A. gerardii and E. canadensis alone and intermixed in microcosms, with individual root systems isolated, but either interconnected by CMNs or with CMNs severed weekly. CMNs, which provided access to a large soil volume, improved survival of both A. gerardii and E. canadensis, but intensified intraspecific competition for A. gerardii. When mixed with E. canadensis, A. gerardii overyielded aboveground biomass in the presence of intact CMNs but not when CMNs were severed, suggesting that A. gerardii with intact CMNs most benefitted from weaker interspecific than intraspecific interactions across CMNs. CMNs improved manganese uptake by both species, with the largest plants receiving the most manganese. Enhanced growth in consequence of improved mineral nutrition led to large E. canadensis in intact CMNs experiencing water-stress, as indicated by 13C isotope abundance. Our findings suggest that in prairie plant communities, CMNs may influence mineral nutrient distribution, water relations, within-species size hierarchies, and between-species interactions.

Plant Community Richness Mediates Inhibitory Interactions and Resource Competition between Streptomyces and Fusarium Populations in the Rhizosphere.

Plant community characteristics impact rhizosphere Streptomyces nutrient competition and antagonistic capacities. However, the effects of Streptomyces on, and their responses to, coexisting microorganisms as a function of plant host or plant species richness have received little attention. In this work, we characterized antagonistic activities and nutrient use among Streptomyces and Fusarium from the rhizosphere of Andropogon gerardii (Ag) and Lespedeza capitata (Lc) plants growing in communities of 1 (monoculture) or 16 (polyculture) plant species. Streptomyces from monoculture were more antagonistic against Fusarium than those from polyculture. In contrast, Fusarium isolates from polyculture had greater inhibitory capacities against Streptomyces than isolates from monoculture. Although Fusarium isolates had on average greater niche widths, the collection of Streptomyces isolates in total used a greater diversity of nutrients for growth. Plant richness, but not plant host, influenced the potential for resource competition between the two taxa. Fusarium isolates had greater niche overlap with Streptomyces in monoculture than polyculture, suggesting greater potential for Fusarium to competitively challenge Streptomyces in monoculture plant communities. In contrast, Streptomyces had greater niche overlap with Fusarium in polyculture than monoculture, suggesting that Fusarium experiences greater resource competition with Streptomyces in polyculture than monoculture. These patterns of competitive and inhibitory phenotypes among Streptomyces and Fusarium populations are consistent with selection for Fusarium-antagonistic Streptomyces populations in the presence of strong Fusarium resource competition in plant monocultures. Similarly, these results suggest selection for Streptomyces-inhibitory Fusarium populations in the presence of strong Streptomyces resource competition in more diverse plant communities. Thus, landscape-scale variation in plant species richness may be critical to mediating the coevolutionary dynamics and selective trajectories for inhibitory and nutrient use phenotypes among Streptomyces and Fusarium populations in soil, with significant implications for microbial community functional characteristics.

Common mycorrhizal networks amplify competition by preferential mineral nutrient allocation to large host plants.

Arbuscular mycorrhizal (AM) fungi interconnect plants in common mycorrhizal networks (CMNs) which can amplify competition among neighbors. Amplified competition might result from the fungi supplying mineral nutrients preferentially to hosts that abundantly provide fixed carbon, as suggested by research with organ-cultured roots. We examined whether CMNs supplied (15) N preferentially to large, nonshaded, whole plants. We conducted an intraspecific target-neighbor pot experiment with Andropogon gerardii and several AM fungi in intact, severed or prevented CMNs. Neighbors were supplied (15) N, and half of the target plants were shaded. Intact CMNs increased target dry weight (DW), intensified competition and increased size inequality. Shading decreased target weight, but shaded plants in intact CMNs had mycorrhizal colonization similar to that of sunlit plants. AM fungi in intact CMNs acquired (15) N from the substrate of neighbors and preferentially allocated it to sunlit, large, target plants. Sunlit, intact CMN, target plants acquired as much as 27% of their nitrogen from the vicinity of their neighbors, but shaded targets did not. These results suggest that AM fungi in CMNs preferentially provide mineral nutrients to those conspecific host individuals best able to provide them with fixed carbon or representing the strongest sinks, thereby potentially amplifying asymmetric competition below ground.

Mycorrhizal phenotypes and the Law of the Minimum.

Mycorrhizal phenotypes arise from interactions among plant and fungal genotypes and the environment. Differences in the stoichiometry and uptake capacity of fungi and plants make arbuscular mycorrhizal (AM) fungi inherently more nitrogen (N) limited and less phosphorus (P) limited than their host plants. Mutualistic phenotypes are most likely in P-limited systems and commensal or parasitic phenotypes in N-limited systems. Carbon (C) limitation is expected to cause phenotypes to shift from mutualism to commensalism and even parasitism. Two experiments compared the influence of fertilizer and shade on mycorrhizas in Andropogon gerardii across three naturally N-limited or P-limited grasslands. A third experiment examined the interactive effects of N and P enrichment and shade on A. gerardii mycorrhizas. Our experiments generated the full spectrum of mycorrhizal phenotypes. These findings support the hypothesis that mutualism is likely in P-limited systems and commensalism or parasitism is likely in N-limited systems. Furthermore, shade decreased C-assimilation and generated less mutualistic mycorrhizal phenotypes with reduced plant and fungal biomass. Soil fertility is a key controller of mycorrhizal costs and benefits and the Law of the Minimum is a useful predictor of mycorrhizal phenotype. In our experimental grasslands arbuscular mycorrhizas can ameliorate P-limitation but not N-limitation.

Rhizosphere bacterial communities associated with long-lived perennial prairie plants vary in diversity, composition, and structure.

The goal of this research was to investigate the variation in rhizosphere microbial community composition, diversity, and structure among individual Andropogon gerardii Vitman (big bluestem) and Lespedeza capitata Michx. (bush clover). Bacterial communities from the rhizosphere of 10 plants of each species (n = 20 plants total) were explored using a culture-independent pipeline. Microbial communities associated with both host plants had high bacterial diversity within individual plant rhizosphere and taxa unique to individual rhizospheres. Bacterial communities associated with the rhizosphere of A. gerardii were consistently more diverse than those associated with L. capitata, and there were significant differences between plant species in rhizosphere bacterial community composition. Differences included microbial taxa with no known functional relationship with their preferred host species, including sulfide-methylating obligate anaerobes (Holophaga), complete denitrifiers (Rhodoplanes), sludge inhabitants (Ktedonobacter), and nitrate oxidizers (Nitrospira). These results suggest the potential for plant species to have significant impacts on a broad array of ecosystem functions (e.g., cycling of carbon, nitrogen sulfurs, metals, and trace elements) via their selective impacts on soil microbes. However, sequence-based community analysis and the corresponding lack of intact microbial cultures limits understanding of the potential influences of enriched microbial taxa on plant hosts and their roles in ecosystem functioning.

Resource availability and imbalance affect plant-mycorrhizal interactions: a field test of three hypotheses.

Ecological stoichiometry can explain major trends in how interactions among species change across fertility gradients, but important questions remain. For example, stoichiometry predicts that fertilization should cause plants to reduce carbon allocation to arbuscular mycorrhizal fungi and, consequently, reduce fungal abundance, but responses in the field are highly variable. In a field experiment, we tested three hypotheses that could drive this variation: (1) fungi are nitrogen limited in very nitrogen-poor soils, so fertilization increases their abundance; (2) the N:P ratio of fertilization affects plant carbon allocation to fungi; (3) plant species differences affect fungal response. Our results support all three hypotheses: stoichiometry and species idiosyncrasies jointly determined fungal response to fertilization. We provide field evidence in support of the hypothesis that nitrogen can limit fungal abundance in temperate grasslands. We also show that fungal abundance in soil (hyphal length) differed beneath two dominant plant species: big bluestem (Andropogon gerardii) and smooth brome (Bromus inermis). These grass species also differed in the degree to which they reduced root colonization with fertilization, but these differences in allocation did not lead to differential responses to fertilization in terms of fungal abundance in the soil. This study shows that, while ecological stoichiometry is a useful framework for understanding the effects of eutrophication on this important and widespread species interaction, including these subtleties can increase the predictive power of the theory.

Common mycorrhizal networks amplify size inequality in Andropogon gerardii monocultures.

Arbuscular mycorrhizal fungi can interconnect plant root systems through hyphal common mycorrhizal networks, which may influence the distribution of limiting mineral nutrients among interconnected individuals, potentially affecting competition and consequent size inequality. Using a microcosm model system, we investigated whether the members of Andropogon gerardii monocultures compete via common mycorrhizal networks. We grew A. gerardii seedlings with isolated root systems in individual, adjacent containers while preventing, disrupting or allowing common mycorrhizal networks among them. Fertile soil was placed within the containers, which were embedded within infertile sand. We assessed mycorrhizas, leaf tissue mineral nutrient concentrations, size hierarchies and the growth of nearest neighbors. Plants interconnected by common mycorrhizal networks had 8% greater colonized root length, 12% higher phosphorus and 35% higher manganese concentrations than plants severed from common mycorrhizal networks. Interconnected plants were, on average, 15% larger and had 32% greater size inequality, as reflected by Gini coefficients, than those with severed connections. Only with intact common mycorrhizal networks were whole-plant dry weights negatively associated with those of their neighbors. In the absence of root system overlap, common mycorrhizal networks likely promote asymmetric competition below ground, thereby exaggerating size inequality within A. gerardii populations.

Role of mycorrhizal colonization in plant establishment on an alkaline gold mine tailing.

The potential role of arbuscular mycorrhizal fungi (AMF) in the revegetation of an alkaline gold mine tailing was studied in Barberton, South Africa. The tailing, characterized by a slow spontaneous plant succession, is colonized by the shrub Dodonaea viscosa and the grasses, Andropogon eucomus and Imperata cylindrica, all colonized by AMF. The effectiveness of mycorrhizal colonization in grasses was tested under laboratory conditions using fungal isolates of various origins. Both grasses were highly mycorrhiza dependent, and the presence of mycorrhizal colonization significantly increased their biomass and survival rates. The fungi originating from the gold tailing were better adapted to the special conditions of the tailing than the control isolate. Although the total colonization rate found for native fungi was lower than for fungi from non-polluted sites, they were more vital and more effective in promoting plant growth. The results obtained might serve as a practical approach to the phytostabilization of alkaline gold tailings.

Analysis of ribosomal RNA indicates seasonal fungal community dynamics in Andropogon gerardii roots.

Use of the reverse-transcribed small subunit of the ribosomal RNA (rRNA) was tested for exploring seasonal dynamics of fungal communities associated with the roots of the dominant tallgrass prairie grass, Andropogon gerardii. Ribosomal RNA was extracted, reverse-transcribed, and PCR-amplified in four sampling events in May, July, September, and November. Analyses of cloned PCR amplicons indicated that the A. gerardii rhizospheres host phylogenetically diverse fungal communities and that these communities are seasonally dynamic. Operational taxonomic units with Basic Local Alignment Search Tool affinities within the order Helotiales were dominant in the rhizosphere in May. These putative saprobes were largely replaced by arbuscular mycorrhizal fungi with likely affinities within Glomerales suggesting that the fungal communities are not only compositionally but also functionally dynamic. These data suggest replacement of functional guilds comprised of saprobic fungi by mutualistic fungi in the course of a growing season.

Isolation and characterization of pyrene metabolizing microbial consortia from the plant rhizoplane.

Most research on the ecology of PAH degrading bacteria in the rhizosphere has focused on individual strains that grow on specific PAHs. Thus, there are fundamental questions as to importance of microbial consortia for PAH degradation in the plant rhizosphere. The study reported here characterized cultivable pyrene degrading rhizoplane microbial communities from two different plant species using a root printing technique on agar plates. Colonies were revealed by formation of clearing zones on medium containing a thin film of pyrene on the surface of a mineral nutrient agar. Prints of the rhizoplane colonies were obtained from roots of Melilotus officinalis (sweet yellow clover) and Andropogon gerardii (big bluestem) plants. Phylogenetic characterizations of selected pyrene degrading colonies were assessed by PCR-DGGE and DNA sequencing. Results showed that different populations of cultivable pyrene degraders were obtained from representative consortia that were examined. Many of the PAH degrading consortia consisted of mixtures of bacterial species that were unable to degrade pyrene by themselves. While this study focused on culturable PAH degraders, the results suggest that pyrene degradation in the rhizosphere commonly involves the activity of bacterial consortia in which various species of bacteria interact to achieve PAH degradation.

Ontogeny and phylogeny of a Scutellospora heterogama mutant, with implications for morphological recognition of species in Glomeromycota.

A putative mutant of Scutellospora heterogama has been maintained for 19 pot culture generations over 15y. The mutant differed phenotypically from the wild-type parental lineage in characteristics of the spore wall: albino versus pigmented red-brown color, high plasticity in acidic mutants versus rigid and finely laminate, deep red-purple reaction versus no reaction in Melzer's reagent, respectively. This variation was equivalent to or greater than that between any two species in Scutellospora or any other genus in Glomeromycota. Comparison of spore ontogenesis revealed that the terminal (mature) state of the albino mutant was similar to a transient intermediate stage in the wild-type. The albino phenotype, therefore, did not result from emergence of a unique morphological innovation. Rather, it arose from a mutation that led to premature termination of spore ontogenesis so that a unique transient juvenile stage became permanent in mature spores. Because this mutation was homogeneous in all progeny populations, it is hypothesized to be a recessive trait expressed only after the allele was distributed in all nuclei of the fungal thallus. Sampling of the genomes of the putative mutant and wild-type isolates by microsatellite-primed PCR suggested a local mutation. The profile of the mutant was identical to that of the wild-type parent and was 60-97 % similar to those of four other S. heterogama isolates. Phylogenetic analysis of the D1-D2 domains of the 25S rRNA gene and a ß-tubulin gene with and without three variable introns placed the albino mutant solidly within the S. heterogama clade. These results suggest that stability of morphological traits is not a suitable criterion by itself to recognize species. The albino phenotype was a discrete and heritable mutation that became fixed in a population and was stable through time and space. In the absence of negative selection, this mutation could persist, disperse and then be misinterpreted as a new species in nature. Genetic markers expose this mutation as a population-level variant and therefore of no macroevolutionary significance. Assessment of genetic divergence amongst multiple isolates is important in ascertaining the contribution of morphological characters toward recognition of species in glomeromycotan clades.

Variation in aluminum resistance among arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal (AM) fungi mediate interactions between plants and soils, and are important where nutrient or metal concentrations limit plant growth. Variation in fungal response to edaphic conditions may influence the effectiveness of the plant-mycorrhizal association in some soil environments. Andropogon virginicus (broomsedge) colonizes disturbed sites in the eastern United States, including acidic mine soils where aluminum (Al) is phytotoxic, and Al resistance in broomsedge has been associated with colonization by the AM fungus Glomus clarum. In the present study, inter- and intra-specific variation to confer Al resistance to broomsedge was assessed among selected species of AM fungi. Broomsedge seeds were grown in sand culture inoculated with one of five isolates of three species of fungi (G. clarum, Acaulospora morrowiae, and Scutellospora heterogama). Plants were exposed to 0 or 400 microM Al in nutrient solution and harvested after 4 or 9 weeks of growth. Mean infection percentage, plant biomass, and plant tissue Al and phosphorus (P) concentrations were measured. G. clarum conferred the greatest Al resistance to broomsedge, with the lowest variability among isolates for colonization and growth inhibition by Al [tolerance indices (TI) between 22.4 and 92.7%]. Broomsedge plants colonized by A. morrowiae were consistently the most sensitive to Al, with little variation among isolates (TI between 1.6 and 12.1%). Al resistance by S. heterogama isolates was intermediate and wide-ranging (TI between 3.9 and 40.0%). Across all AM fungal isolates, resistance was associated with high rates of colonization and low tissue Al concentrations of broomsedge plants. The functional diversity in Al resistance displayed by these AM fungi reflect variation in acclimation mechanisms operating in the mycorrhizal symbiosis under environmental stress.

Defoliation effects on the community structure of arbuscular mycorrhizal fungi based on 18S rDNA sequences.

The effects of defoliation on arbuscular mycorrhizal (AM) associations in the field were investigated in terms of the community structure of AM fungi colonizing roots of grassland plants; the carbohydrate balance of the host plants was also determined. We focused on two plant species dominating Japanese native grasslands: the grazing-intolerant species Miscanthus sinensis and the grazing-tolerant species Zoysia japonica. Community structures of AM fungi were determined from 18S rRNA gene sequences. The dominant fungal group in both plant species was the Glomus clade, which was classified into several phylogenetic groups based on genetic distances and topology. In Miscanthus roots, the Glomus-Ab, Glomus-Ac, and Glomus-Ad groups were detected almost equally. In Zoysia roots, the Glomus-Ab group was dominant. Defoliation effects on the community structure of AM fungi differed between the plant species. In Miscanthus roots, the percentage of root length colonized (%RLC) by the Glomus-Ac and Glomus-Ad groups was significantly reduced by defoliation treatment. On the other hand, AM fungal group composition in Zoysia roots was unaffected by defoliation except on the last sampling date. Decreased %RLC by Glomus-Ac and Glomus-Ad coincided with decreased non-structural carbohydrate (NSC) levels in host plants; also, significant positive correlations were found between the %RLC and some NSC levels. On the other hand, the %RLC by Glomus-Ab in both plant species was unaffected by the NSC level. These results suggest that AM fungal groups have different carbohydrate requirements from host plants.

Arbuscular mycorrhizal fungi enhance aluminium resistance of broomsedge (Andropogon virginicus L.).

In the eastern United States, broomsedge (Andropogon virginicus L.) is found growing on abandoned coal-mined lands that have extremely acidic soils with high residual aluminium (Al) concentrations. Broomsedge may be inherently metal-resistant and nutrient-efficient or may rely on the arbuscular mycorrhizal (AM) fungal association to overcome limitations on such sites. Broomsedge plants were grown with and without an acidic ecotype AM fungal consortium and exposed to controlled levels of Al in two experiments. The AM fungal consortium conferred Al resistance to broomsedge. Arbuscular mycorrhizal fungi reduced Al uptake and translocation in host plants, potentially reflecting measured reductions in inorganic Al availability in the rhizosphere of mycorrhizal plants. Mycorrhizal plants exhibited lower shoot P concentrations, higher phosphorus use efficiency, and lower root acid phosphatase rates than non-mycorrhizal plants. Aluminium significantly reduced calcium (Ca) and magnesium (Mg) tissue concentrations in both mycorrhizal and non-mycorrhizal plants. However, plant response to any change in nutrient acquisition was substantially less pronounced in mycorrhizal plants. The exclusion of Al and greater stability of tissue biomass accretion-tissue nutrient relationships in mycorrhizal broomsedge plants exposed to Al may be important mechanisms that allow broomsedge to grow on unfavourable acidic soils.