George A. Kowalchuk

Reciprocal Rewards Stabilize Cooperation in the Mycorrhizal Symbiosis

Plants and their associated fungi reward partners that offer the best resources to sustain mutualism in complex systems. Plants and their arbuscular mycorrhizal fungal symbionts interact in complex underground networks involving multiple partners. This increases the potential for exploitation and defection by individuals, raising the question of how partners maintain a fair, two-way transfer of resources. We manipulated cooperation in plants and fungal partners to show that plants can detect, discriminate, and reward the best fungal partners with more carbohydrates. In turn, their fungal partners enforce cooperation by increasing nutrient transfer only to those roots providing more carbohydrates. On the basis of these observations we conclude that, unlike many other mutualisms, the symbiont cannot be “enslaved.” Rather, the mutualism is evolutionarily stable because control is bidirectional, and partners offering the best rate of exchange are rewarded.

Nitrification in acid soils: micro-organisms and mechanisms

Abstract Nitrification in acid soils was first reported in the beginning of the 20th century. Although this finding has been well substantiated by countless studies since then, it has until recently remained unclear which micro-organisms were responsible for nitrate production at low pH. Substantial evidence now supports the role of chemolitho–autotrophic bacteria as the main nitrifying agents in most acid soils. Heterotrophs may make some contribution to nitrification in acid soils, but this is difficult to demonstrate conclusively. Current insights in the phylogenetic position of autotrophic nitrifying bacteria in acid soils and the mechanisms that may enable them to be active at low pH are presented. In addition, the spatial variability of their activity and their contribution to the flux of the greenhouse gas N2O is discussed.

Effects of above-ground plant species composition and diversity on the diversity of soil-borne microorganisms

A coupling of above-ground plant diversity and below-ground microbial diversity has been implied in studies dedicated to assessing the role of macrophyte diversity on the stability, resilience, and functioning of ecosystems. Indeed, above-ground plant communities have long been assumed to drive below-ground microbial diversity, but to date very little is known as to how plant species composition and diversity influence the community composition of micro-organisms in the soil. We examined this relationship in fields subjected to different above-ground biodiversity treatments and in field experiments designed to examine the influence of plant species on soil-borne microbial communities. Culture-independent strategies were applied to examine the role of wild or native plant species composition on bacterial diversity and community structure in bulk soil and in the rhizosphere. In comparing the influence of Cynoglossum officinale (hounds tongue) and Cirsium vulgare (spear thistle) on soil-borne bacterial communities, detectable differences in microbial community structure were confined to the rhizosphere. The colonisation of the rhizosphere of both plants was highly reproducible, and maintained throughout the growing season. In a separate experiment, effects of plant diversity on bacterial community profiles were also only observed for the rhizosphere. Rhizosphere soil from experimental plots with lower macrophyte diversity showed lower diversity, and bacterial diversity was generally lower in the rhizosphere than in bulk soil. These results demonstrate that the level of coupling between above-ground macrophyte communities and below-ground microbial communities is related to the tightness of the interactions involved. Although plant species composition and community structure appear to have little discernible effect on microbial communities inhabiting bulk soil, clear and reproducible changes in microbial community structure and diversity are observed in the rhizosphere.

Analysis of Bacterial Communities in the Rhizosphere of Chrysanthemum via Denaturing Gradient Gel Electrophoresis of PCR-Amplified 16S rRNA as Well as DNA Fragments Coding for 16S rRNA

ABSTRACT The effect of developing chrysanthemum roots on the presence and activity of bacterial populations in the rhizosphere was examined by using culture-independent methods. Nucleic acids were extracted from rhizosphere soil samples associated with the bases of roots or root tips of plants harvested at different stages of development. PCR and reverse transcriptase (RT) PCR were used to amplify 16S ribosomal DNA (rDNA) and 16S rRNA, respectively, and the products were subjected to denaturing gradient gel electrophoresis (DGGE). Prominent DGGE bands were excised and sequenced to gain insight into the identities of predominantly present (PCR) and predominantly active (RT-PCR) bacterial populations. The majority of DGGE band sequences were related to bacterial genera previously associated with the rhizosphere, such asPseudomonas, Comamonas, Variovorax, and Acetobacter, or typical of root-free soil environments, such as Bacillus and Arthrobacter. The PCR-DGGE patterns observed for bulk soil were somewhat more complex than those obtained from rhizosphere samples, and the latter contained a subset of the bands present in bulk soil. DGGE analysis of RT-PCR products detected a subset of bands visible in the rDNA-based analysis, indicating that some dominantly detected bacterial populations did not have high levels of metabolic activity. The sequences detected by the RT-PCR approach were, however, derived from a wide taxonomic range, suggesting that activity in the rhizosphere was not determined at broad taxonomic levels but rather was a strain- or species-specific phenomenon. Comparative analysis of DGGE profiles grouped all DNA-derived root tip samples together in a cluster, and within this cluster the root tip samples from young plants formed a separate subcluster. Comparison of rRNA-derived bacterial profiles showed no grouping of root tip samples versus root base samples. Rather, all profiles derived from 2-week-old plant rhizosphere soils grouped together regardless of location along the root.

Microvariation artifacts introduced by PCR and cloning of closely related 16S rRNA gene sequences

ABSTRACT A defined template mixture of seven closely related 16S-rDNA clones was used in a PCR-cloning experiment to assess and track sources of artifactual sequence variation in 16S rDNA clone libraries. At least 14% of the recovered clones contained aberrations. Artifact sources were polymerase errors, a mutational hot spot, and cloning of heteroduplexes and chimeras. These data may partially explain the high degree of microheterogeneity typical of sequence clusters detected in environmental clone libraries.

Shifting carbon flow from roots into associated microbial communities in response to elevated atmospheric CO2

Rising atmospheric CO2 levels are predicted to have major consequences on carbon cycling and the functioning of terrestrial ecosystems. Increased photosynthetic activity is expected, especially for C-3 plants, thereby influencing vegetation dynamics; however, little is known about the path of fixed carbon into soil-borne communities and resulting feedbacks on ecosystem function. Here, we examine how arbuscular mycorrhizal fungi (AMF) act as a major conduit in the transfer of carbon between plants and soil and how elevated atmospheric CO2 modulates the belowground translocation pathway of plant-fixed carbon. Shifts in active AMF species under elevated atmospheric CO2 conditions are coupled to changes within active rhizosphere bacterial and fungal communities. Thus, as opposed to simply increasing the activity of soil-borne microbes through enhanced rhizodeposition, elevated atmospheric CO2 clearly evokes the emergence of distinct opportunistic plant-associated microbial communities. Analyses involving RNA-based stable isotope probing, neutral/phosphate lipid fatty acids stable isotope probing, community fingerprinting, and real-time PCR allowed us to trace plant-fixed carbon to the affected soil-borne microorganisms. Based on our data, we present a conceptual model in which plant-assimilated carbon is rapidly transferred to AMF, followed by a slower release from AMF to the bacterial and fungal populations well-adapted to the prevailing (myco-)rhizosphere conditions. This model provides a general framework for reappraising carbon-flow paths in soils, facilitating predictions of future interactions between rising atmospheric CO2 concentrations and terrestrial ecosystems.

Positive effects of organic farming on below‐ground mutualists: large‐scale comparison of mycorrhizal fungal communities in agricultural soils

*The impact of various agricultural practices on soil biodiversity and, in particular, on arbuscular mycorrhizal fungi (AMF), is still poorly understood, although AMF can provide benefit to plants and ecosystems. Here, we tested whether organic farming enhances AMF diversity and whether AMF communities from organically managed fields are more similar to those of species-rich grasslands or conventionally managed fields. *To address this issue, the AMF community composition was assessed in 26 arable fields (13 pairs of organically and conventionally managed fields) and five semi-natural grasslands, all on sandy soil. Terminal restriction fragment length polymorphism community fingerprinting was used to characterize AMF community composition. *The average number of AMF taxa was highest in grasslands (8.8), intermediate in organically managed fields (6.4) and significantly lower in conventionally managed fields (3.9). Moreover, AMF richness increased significantly with the time since conversion to organic agriculture. AMF communities of organically managed fields were also more similar to those of natural grasslands when compared with those under conventional management, and were less uniform than their conventional counterparts, as expressed by higher beta-diversity (between-site diversity). *We suggest that organic management in agro-ecosystems contributes to the restoration and maintenance of these important below-ground mutualists.

Community analysis of arbuscular mycorrhizal fungi associated with Ammophila arenaria in Dutch coastal sand dunes.

A polymerase chain reaction‐denaturing gradient gel electrophoresis (PCR‐DGGE) approach for the detection and characterization of arbuscular mycorrhizal fungi (AMF) 18S ribosomal DNA (rDNA) was developed and applied to the study of AMF communities associated with the main sand‐stabilizing plant species of the Dutch sand dunes, marram grass (Ammophila arenaria, L.). DNA was extracted directly from plant roots, soil or isolated AMF spores, and prominent bands resulting from AMF‐specific DGGE profiles were excised for sequence analysis. This strategy provided a robust means of detecting and identifying AMF‐like species without the use of trap plant cultivation methods. A number of Glomus‐like and Scutellospora‐like sequences was detected, including a putatively novel Glomus species, and differences were observed in the dominant AMF‐like populations detected in healthy vs. degenerating stands of A. arenaria and in bulk sand dune soil. It has previously been suggested that plant pathogens, such as fungi and nematodes, may contribute to the decline of A. arenaria. Although no causal relationship can be drawn between the observed differences in the dominantly detected AMF‐like populations and the vitality of plant growth, these results indicate that mutualistic interactions between this plant and AMF should not be overlooked when examining the role of soil‐borne microorganisms in vegetation dynamics. In addition, there were discrepancies observed between the AMF‐like groups detected in spore populations vs. direct 18S rDNA analysis of root material, corroborating previous suggestions that spore inspection alone may poorly represent actual AMF population structure.

Soil characteristics more strongly influence soil bacterial communities than land-use type

To gain insight into the factors driving the structure of bacterial communities in soil, we applied real-time PCR, PCR-denaturing gradient gel electrophoreses, and phylogenetic microarray approaches targeting the 16S rRNA gene across a range of different land usages in the Netherlands. We observed that the main differences in the bacterial communities were not related to land-use type, but rather to soil factors. An exception was the bacterial community of pine forest soils (PFS), which was clearly different from all other sites. PFS had lowest bacterial abundance, lowest numbers of operational taxonomic units (OTUs), lowest soil pH, and highest C : N ratios. C : N ratio strongly influenced bacterial community structure and was the main factor separating PFS from other fields. For the sites other than PFS, phosphate was the most important factor explaining the differences in bacterial communities across fields. Firmicutes were the most dominant group in almost all fields, except in PFS and deciduous forest soils (DFS). In PFS, Alphaproteobacteria was most represented, while in DFS, Firmicutes and Gammaproteobacteria were both highly represented. Interestingly, Bacillii and Clostridium OTUs correlated with pH and phosphate, which might explain their high abundance across many of the Dutch soils. Numerous bacterial groups were highly correlated with specific soil factors, suggesting that they might be useful as indicators of soil status.

Unlocking the potential of metagenomics through replicated experimental design

Metagenomics holds enormous promise for discovering novel enzymes and organisms that are biomarkers or drivers of processes relevant to disease, industry and the environment. In the past two years, we have seen a paradigm shift in metagenomics to the application of cross-sectional and longitudinal studies enabled by advances in DNA sequencing and high-performance computing. These technologies now make it possible to broadly assess microbial diversity and function, allowing systematic investigation of the largely unexplored frontier of microbial life. To achieve this aim, the global scientific community must collaborate and agree upon common objectives and data standards to enable comparative research across the Earths microbiome. Improvements in comparability of data will facilitate the study of biotechnologically relevant processes, such as bioprospecting for new glycoside hydrolases or identifying novel energy sources.