What Goes in Must Come out: Testing for Biases in Molecular Analysis of Arbuscular Mycorrhizal Fungal Communities

Arbuscular mycorrhizal (AM) fungi are widely distributed microbes that form obligate symbioses with the majority of terrestrial plants, altering nutrient transfers between soils and plants, thereby profoundly affecting plant growth and ecosystem properties. Molecular methods are commonly used in the study of AM fungal communities. However, the biases associated with PCR amplification of these organisms and their ability to be utilized quantitatively has never been fully tested. We used Terminal Restriction Fragment Length Polymorphism (TRFLP) analysis to characterise artificial community templates containing known quantities of defined AM fungal genotypes. This was compared to a parallel in silico analysis that predicted the results of this experiment in the absence of bias. The data suggest that when used quantitatively the TRFLP protocol tested is a powerful, repeatable method for AM fungal community analysis. However, we suggest some limitations to its use for population-level analyses. We found no evidence of PCR bias, supporting the quantitative use of other PCR-based methods for the study of AM fungi such as next generation amplicon sequencing. This finding greatly improves our confidence in methods that quantitatively examine AM fungal communities, providing a greater understanding of the ecology of these important fungi

Responses of arbuscular mycorrhizal fungi to long-term inorganic and organic nutrient addition in a lowland tropical forest

Improved understanding of the nutritional ecology of arbuscular mycorrhizal (AM) fungi is important in understanding how tropical forests maintain high productivity on low-fertility soils. Relatively little is known about how AM fungi will respond to changes in nutrient inputs in tropical forests, which hampers our ability to assess how forest productivity will be influenced by anthropogenic change. Here we assessed the influence of long-term inorganic and organic nutrient additions and nutrient depletion on AM fungi, using two adjacent experiments in a lowland tropical forest in Panama. We characterised AM fungal communities in soil and roots using 454-pyrosequencing, and quantified AM fungal abundance using microscopy and a lipid biomarker. Phosphorus and nitrogen addition reduced the abundance of AM fungi to a similar extent, but affected community composition in different ways. Nutrient depletion (removal of leaf litter) had a pronounced effect on AM fungal community composition, affecting nearly as many OTUs as phosphorus addition. The addition of nutrients in organic form (leaf litter) had little effect on any AM fungal parameter. Soil AM fungal communities responded more strongly to changes in nutrient availability than communities in roots. This suggests that the 'dual niches' of AM fungi in soil versus roots are structured to different degrees by abiotic environmental filters, and biotic filters imposed by the plant host. Our findings indicate that AM fungal communities are fine-tuned to nutrient regimes, and support future studies aiming to link AM fungal community dynamics with ecosystem function

Where less may be more: how the rare biosphere pulls ecosystems strings

Rare species are increasingly recognized as crucial, yet vulnerable components of Earth’s ecosystems. This is also true for microbial communities, which are typically composed of a high number of relatively rare species. Recent studies have demonstrated that rare species can have an over-proportional role in biogeochemical cycles and may be a hidden driver of microbiome function. In this review, we provide an ecological overview of the rare microbial biosphere, including causes of rarity and the impacts of rare species on ecosystem functioning. We discuss how rare species can have a preponderant role for local biodiversity and species turnover with rarity potentially bound to phylogenetically conserved features. Rare microbes may therefore be overlooked keystone species regulating the functioning of host-associated, terrestrial and aquatic environments. We conclude this review with recommendations to guide scientists interested in investigating this rapidly emerging research area

Metatranscriptomic reconstruction reveals RNA viruses with the potential to shape carbon cycling in soil.

Viruses impact nearly all organisms on Earth, with ripples of influence in agriculture, health, and biogeochemical processes. However, very little is known about RNA viruses in an environmental context, and even less is known about their diversity and ecology in soil, 1 of the most complex microbial systems. Here, we assembled 48 individual metatranscriptomes from 4 habitats within a planted soil sampled over a 22-d time series: Rhizosphere alone, detritosphere alone, rhizosphere with added root detritus, and unamended soil (4 time points and 3 biological replicates). We resolved the RNA viral community, uncovering a high diversity of viral sequences. We also investigated possible host organisms by analyzing metatranscriptome marker genes. Based on viral phylogeny, much of the diversity was Narnaviridae that may parasitize fungi or Leviviridae, which may infect Proteobacteria. Both host and viral communities appear to be highly dynamic, and rapidly diverged depending on experimental conditions. The viral and host communities were structured based on the presence of root litter. Clear temporal dynamics by Leviviridae and their hosts indicated that viruses were replicating. With this time-resolved analysis, we show that RNA viruses are diverse, abundant, and active in soil. When viral infection causes host cell death, it may mobilize cell carbon in a process that may represent an overlooked component of soil carbon cycling

Soil Candidate Phyla Radiation Bacteria Encode Components of Aerobic Metabolism and Co-occur with Nanoarchaea in the Rare Biosphere of Rhizosphere Grassland Communities.

Candidate Phyla Radiation (CPR) bacteria and nanoarchaea populate most ecosystems but are rarely detected in soil. We concentrated particles of less than 0.2 μm in size from grassland soil, enabling targeted metagenomic analysis of these organisms, which are almost totally unexplored in largely oxic environments such as soil. We recovered a diversity of CPR bacterial and some archaeal sequences but no sequences from other cellular organisms. The sampled sequences include Doudnabacteria (SM2F11) and Pacearchaeota, organisms rarely reported in soil, as well as Saccharibacteria, Parcubacteria, and Microgenomates. CPR and archaea of the phyla Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, and Nanohaloarchaeota (DPANN) were enriched 100- to 1,000-fold compared to that in bulk soil, in which we estimate each of these organisms comprises approximately 1 to 100 cells per gram of soil. Like most CPR and DPANN sequenced to date, we predict these microorganisms live symbiotic anaerobic lifestyles. However, Saccharibacteria, Parcubacteria, and Doudnabacteria genomes sampled here also harbor ubiquinol oxidase operons that may have been acquired from other bacteria, likely during adaptation to aerobic soil environments. We conclude that CPR bacteria and DPANN archaea are part of the rare soil biosphere and harbor unique metabolic platforms that potentially evolved to live symbiotically under relatively oxic conditions. IMPORTANCE Here, we investigated overlooked microbes in soil, Candidate Phyla Radiation (CPR) bacteria and Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, and Nanohaloarchaeota (DPANN) archaea, by size fractionating small particles from soil, an approach typically used for the recovery of viral metagenomes. Concentration of these small cells (<0.2 μm) allowed us to identify these organisms as part of the rare soil biosphere and to sample genomes that were absent from non-size-fractionated metagenomes. We found that some of these predicted symbionts, which have been largely studied in anaerobic systems, have acquired aerobic capacity via lateral transfer that may enable adaptation to oxic soil environments. We estimate that there are approximately 1 to 100 cells of each of these lineages per gram of soil, highlighting that the approach provides a window into the rare soil biosphere and its associated genetic potential

Signal beyond nutrient, fructose, exuded by an arbuscular mycorrhizal fungus triggers phytate mineralization by a phosphate solubilizing bacterium

Cooperation is a prevalent phenomenon in nature and how it originates and maintains is a fundamental question in ecology. Many efforts have been made to understand cooperation between individuals in the same species, while the mechanisms enabling cooperation between different species are less understood. Here, we investigated under strict in vitro culture conditions if the exchange of carbon and phosphorus is pivotal to the cooperation between the arbuscular mycorrhizal fungus (AMF) Rhizophagus irregularis and the phosphate solubilizing bacterium (PSB) Rahnella aquatilis. We observed that fructose exuded by the AMF stimulated the expression of phosphatase genes in the bacterium as well as the rate of phosphatase release into the growth medium by regulating its protein secretory system. The phosphatase activity was subsequently increased, promoting the mineralization of organic phosphorus (i.e., phytate) into inorganic phosphorus, stimulating simultaneously the processes involved in phosphorus uptake by the AMF. Our results demonstrated for the first time that fructose not only is a carbon source, but also plays a role as a signal molecule triggering bacteria-mediated organic phosphorus mineralization processes. These results highlighted the molecular mechanisms by which the hyphal exudates play a role in maintaining the cooperation between AMF and bacteria