16S ribosomal RNA gene (rDNA) amplicon analysis remains the standard approach for the cultivation-independent investigation of microbial diversity. The accuracy of these analyses depends strongly on the choice of primers. The overall coverage and phylum spectrum of 175 primers and 512 primer pairs were evaluated in silico with respect to the SILVA 16S/18S rDNA non-redundant reference dataset (SSURef 108 NR). Based on this evaluation a selection of 'best available' primer pairs for Bacteria and Archaea for three amplicon size classes (100-400, 400-1000, ≥ 1000 bp) is provided. The most promising bacterial primer pair (S-D-Bact-0341-b-S-17/S-D-Bact-0785-a-A-21), with an amplicon size of 464 bp, was experimentally evaluated by comparing the taxonomic distribution of the 16S rDNA amplicons with 16S rDNA fragments from directly sequenced metagenomes. The results of this study may be used as a guideline for selecting primer pairs with the best overall coverage and phylum spectrum for specific applications, therefore reducing the bias in PCR-based microbial diversity studies.

Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies

https://web.science.uu.nl/pmi/publications/PDF/2012/TiPS-Berendsen-2012.pdf

The rhizosphere microbiome and plant health

Since the 1900s, the link between soil biotic activity, soil organic matter (SOM) decomposition and stabilization, and soil aggregate dynamics has been recognized and intensively been studied. By 1950, many studies had, mostly qualitatively, investigated the influence of the five major factors (i.e. soil fauna, microorganisms, roots, inorganics and physical processes) on this link. After 1950, four theoretical mile-stones related to this subject were realized. The first one was when Emerson [Nature 183 (1959) 538] proposed a model of a soil crumb consisting of domains of oriented clay and quartz particles. Next, Edwards and Bremner [J. Soil Sci. 18 (1967) 64] formulated a theory in which the solid-phase reaction between clay minerals, polyvalent cations and SOM is the main process leading to microaggregate formation. Based on this concept, Tisdall and Oades [J. Soil Sci. 62 (1982) 141] coined the aggregate hierarchy concept describing a spatial scale dependence of mechanisms involved in micro- and macroaggregate formation. Oades [Plant Soil 76 (1984) 319] suggested a small, but very important, modification to the aggregate hierarchy concept by theorizing the formation of microaggregates within macroaggregates. Recent research on aggregate formation and SOM stabilization extensively corroborate this modification and use it as the base for furthering the understanding of SOM dynamics. The major outcomes of adopting this modification are: (1) microaggregates, rather than macroaggregates protect SOM in the long term; and (2) macroaggregate turnover is a crucial process influencing the stabilization of SOM. Reviewing the progress made over the last 50 years in this area of research reveals that still very few studies are quantitative and/or consider interactive effects between the five factors. The quantification of these relationships is clearly needed to improve our ability to predict changes in soil ecosystems due to management and global change. This quantification can greatly benefit from viewing aggregates as dynamic rather than static entities and relating aggregate measurements with 2D and 3D quantitative spatial information.

A history of research on the link between (micro)aggregates, soil biota, and soil organic matter dynamics

Biofilms are complex communities of microorganisms attached to surfaces or associated with interfaces. Despite the focus of modern microbiology research on pure culture, planktonic (free-swimming) bacteria, it is now widely recognized that most bacteria found in natural, clinical, and industrial settings persist in association with surfaces. Furthermore, these microbial communities are often composed of multiple species that interact with each other and their environment. The determination of biofilm architecture, particularly the spatial arrangement of microcolonies (clusters of cells) relative to one another, has profound implications for the function of these complex communities. Numerous new experimental approaches and methodologies have been developed in order to explore metabolic interactions, phylogenetic groupings, and competition among members of the biofilm. To complement this broad view of biofilm ecology, individual organisms have been studied using molecular genetics in order to identify the genes required for biofilm development and to dissect the regulatory pathways that control the plankton-to-biofilm transition. These molecular genetic studies have led to the emergence of the concept of biofilm formation as a novel system for the study of bacterial development. The recent explosion in the field of biofilm research has led to exciting progress in the development of new technologies for studying these communities, advanced our understanding of the ecological significance of surface-attached bacteria, and provided new insights into the molecular genetic basis of biofilm development.

Microbial Biofilms: from Ecology to Molecular Genetics

A global perspective on the use, sales, exposure pathways, occurrence, fate and effects of veterinary antibiotics (VAs) in the environment

Characterization of the diversity of ecologically important microbes by rep-PCR genomic fingerprinting.

Microbiological and molecular biological methods for monitoring microbial inoculants and their effects in the soil environment

Previous work in our laboratory showed that several bacterial strains, either singly or in association with other bacteria (community migration), were capable of migrating together with the saprotrophic fungus Lyophyllum sp. strain Karsten through soil microcosms. A possible involvement of the type III secretion system (TTSS) in migration was indicated. In this study, we addressed the basis of the community migration, which might lie in a migration helper effect exerted by particular single-strain migrators on other members of the community. Different culturing (plating) as well as culture-independent (PCR-DGGE) methods were applied to assess the effects of putative bacterial helpers in the migration. We used, as a model, the migration-proficient Burkholderia terrae BS001 as the canonical helper strain. PCR-DGGE analysis of the soil system with or without added strain BS001, revealed that the latter consistently stimulated the migration of different bacterial species through the soil. This was observed both following introduction of the organism to a bacterial community from soil and on the basis of a similar organism that was naturally present. One strain, Dyella japonica BS003, was identified as an avid comigrator with B. terrae BS001, although it appeared to lag behind the latter strain in its migration speed. Further examination of the B. terrae BS001/D. japonica BS003 interaction at Lyophyllum sp. strain Karsten hyphae showed that the presence of the D. japonica strain did not negatively affect the growth and migration of B. terrae BS001 with the fungus. A biofilm of B. terrae BS001 was formed on the fungal hyphal front, and we postulate a role for this biofilm in the migration helper effect. (C) 2010 Published by Elsevier Ltd.

Hitchhikers on the fungal highway: The helper effect for bacterial migration via fungal hyphae

Vertical soil microcosms flushed with groundwater were used to study the influence of water movement on survival and transport of a genetically engineered Pseudomonas fluorescens C5t strain through a loamy sand and a loam soil. Transport of cells introduced into the top 1 cm of the vertical soil microcosms was dependent on the flow rate of water and the number of times microcosms were flushed with groundwater. The presence of wheat roots growing downward in the microcosms contributed only slightly to the movement of P. fluorescens C5t cells to lower soil regions of the loamy sand microcosms, but enhanced downward transport in the loam microcosms. Furthermore, the introduced P. fluorescens C5t cells were detected in the effluent water samples even after three flushes of groundwater and 10 days of incubation. As evidenced by a comparison of counts from immunofluorescence and selective plating, nonculturable C5t cells occurred in day 10 soil and percolated water samples, primarily of the loamy sand microcosms. Vertical soil microcosms that use water movement may be useful in studying the survival and transport of genetically engineered bacteria in soil under a variety of conditions prior to field testing.

https://aem.asm.org/content/aem/56/2/401.full.pdf

Transport of a genetically engineered Pseudomonas fluorescens strain through a soil microcosm.

Microbial diversity in soil: the need for a combined approach using molecular and cultivation techniques.

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