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

Five different Brazilian soils were examined for the incidence of Bacillus nitrogen fixers. These counts were set in relation to the total spore count, and the number of facultative anaerobic spores. Twenty-four Bacillus strains were isolated using media lacking a nitrogen source, identified and their acetylene-reducing ability was determined. Eighteen of these strains were identified asB. polymyxa, and the other 6 were related to this species, but were different in some taxonomical tests. Of the 24 isolated strains, only 13 reduced acetylene; similarly, only 3 out of 9B. polymyxa reference strains were able to reduce acetylene.

Bacillus nitrogen fixers from Brazilian soils

The physiology and virulence of Ralstonia solanacearum biovar 2 strain 1609, kept in water at 4 and 20 degrees C, were studied. At 20 degrees C, total cell and plate count (colony forming units; CFU) numbers were similar, between log 5.03 and log 5.55 CFU, and log 5.03 and log 5.51 cells per ml, at days 0 and 132, respectively. However, CFU in the cultures kept at 4 degrees C dropped from log 6.78 CFU/ml at day 0 to below detection after 84 days. The presence of catalase in the agar resulted in higher CFU, and at day 84, log 1.95 CFU/ml still was detectable. No colonies were observed at day 125. The presence of viable-but-nonculturable (VBNC) cells in the 4 degrees C cultures was confirmed using SYTO9 viability staining. Viable cell numbers were log 1.77 higher than CFU on plates with catalase. At day 84 and after 125 days, log 3.70 viable cells per ml still were present. Shifts in subpopulations differing in viability were found by flow cytometric sorting of 4 degrees C-treated cells stained with SYTO9 (healthy) and propidium iodide (PI; compromised). The SYTO9-stained cell fractions dropped from 99 to 39%, and the PI-stained fractions increased from 0.7 to 33.3% between days 0 and 125. At 20 degrees C, the SYTO9-stained fraction remained stable at 99% until day 132. SYTO9-stained cells sorted from 4 degrees C cultures at day 100 were injected into tomato plants. Upon incubation for 30 days, these plants did not show wilting. However, more than log 4.19 CFU and log 8.17 cells were recovered from these plants. Cells from colonies isolated from the nonwilted plants did not regain their virulence as demonstrated by subsequent injection into several new sets of tomato plants. Cells from 4 degrees C cultures injected at day 125 were not able to cause wilting of, or proliferate in, tomato plants. The threat posed by VBNC R. solanacearum cells upon incubation at 4 degrees C was thus ephemeral because cells lost their capacity to cause disease after 125 days.

/pdf/the-low-temperature-induced-viable-but-nonculturable-state-4k5q0o9ztz.pdf

The low-temperature-induced viable-but-nonculturable state affects the virulence of Ralstonia solanacearum biovar 2

Pesticides: microbial degradation and effects on microorganisms.

Transfer of plasmid RP4 between introduced strains of Pseudomonas fluorescens was studied in 2 soils, Ede loamy sand and Guelph loam, in non-rhizosphere and rhizosphere soil using soil chambers and microcosm systems. Short-term organism survival was generally at high levels (> 106/g dry soil), in both soils, whereas long-term survival was poorer, particularly in the loamy sand. Amendment of this soil with bentonite clay improved bacterial survival. Plasmid transfer between donor and recipient strains freshly introduced into separate portions of Ede loamy sand, which were subsequently mixed, was only detected in the vicinity of growing wheat roots, suggesting roots stimulate bacterial migration and/or growth. However, no transfer was detected between resident donor and recipient cell populations (introduced 48 days previously), due to poor organism survival. Plasmid transfer was detected in the rhizosphere between established, resident donor cell populations, and newly-introduced recipients, and vice-versa, in both soils. These data suggested that plant roots enhance the frequency of bacterial matings not only between organisms present in the same niches, but also between organisms from different niches, or in different conditions of stress, probably by stimulating bacterial migration and/or growth, or by providing additional surfaces for cell-to-cell contact.

Transfer of plasmid RP4 between pseudomonads after introduction into soil; influence of spatial and temporal aspects of inoculation

The effects of protozoan inoculum density and spatial separation of (prey) organisms on the turnover and transport of specific microbial populations in sterilized soil were studied in soil microcosms, applying a combination of techniques. The fate of introduced microbial biomass was followed by monitoring 14 C-CO 2 evolution. Dynamics of and genetic transfer between genetically marked bacteria were followed by selective plating using appropriate antibiotics. Three levels of protozoan densities, i.e. no protozoa, protozoa and a 1: 10 diluted protozoan inoculum were established. Two methods of soil inoculation, i.e. joint introduction and separate introduction of protozoa with a donor population and of a 14 C-labelled recipient population, respectively, were applied. The activity of protozoa, as judged from increased CO 2 evolution compared to soils not inoculated with protozoa, was larger when 100% of the soil was inoculated with protozoa (joint introduction) compared to inoculation of protozoa into only 33% of the soil which was mixed with 66% of a soil portion not inoculated with protozoa (separate introduction). The activity of protozoa accelerated the turnover of 14 C compared to soils without protozoa. The more protozoa added, the faster 14 C was respired both in soils with joint, and in soils with separate, introduction. The transfer of genetic material between introduced donor and recipient cells was not influenced by the presence of protozoa but was reduced when separate introduction was applied compared to joint introduction. Transfer of genetic material was only found during the first 2 days of the incubation. Substantial loss of genetic material (plasmid RP4) in the donor population was detected; the presence of protozoa enhanced both donor survival and plasmid maintenance. A stimulating effect of protozoa on cellular metabolism of substrate used by the donor population is hypothesized. These results picture soil as a system in which the distribution of microorganisms in the absence of fluctuations in climatic conditions is not changing in time: protozoa hardly moved through the soil and minimal translocation of bacteria was found.

Population dynamics and activity of bacteria and protozoa in relation to their spatial distribution in soil

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