Judith Ascher

Microbial diversity and soil functions

Summary Soil is a complex and dynamic biological system, and still in 2003 it is difficult to determine the composition of microbial communities in soil. We are also limited in the determination of microbially mediated reactions because present assays for determining the overall rate of entire metabolic processes (such as respiration) or specific enzyme activities (such as urease, protease and phosphomonoesterase activity) do not allow any identification of the microbial species directly involved in the measured processes. The central problem posed by the link between microbial diversity and soil function is to understand the relations between genetic diversity and community structure and between community structure and function. A better understanding of the relations between microbial diversity and soil functions requires not only the use of more accurate assays for taxonomically and functionally characterizing DNA and RNA extracted from soil, but also high-resolution techniques with which to detect inactive and active microbial cells in the soil matrix. Soil seems to be characterized by a redundancy of functions; for example, no relationship has been shown to exist between microbial diversity and decomposition of organic matter. Generally, a reduction in any group of species has little effect on overall processes in soil because other microorganisms can take on its function. The determination of the composition of microbial communities in soil is not necessary for a better quantification of nutrient transformations. The holistic approach, based on the division of the systems in pools and the measurement of fluxes linking these pools, is the most efficient. The determination of microbial C, N, P and S contents by fumigation techniques has allowed a better quantification of nutrient dynamics in soil. However, further advances require determining new pools, such as active microbial biomass, also with molecular techniques. Recently investigators have separated 13 C- and 12 C-DNA, both extracted from soil treated with a 13 C source, by density-gradient centrifugation. This technique should allow us to calculate the active microbial C pool by multiplying the ratio between labelled and total DNA by the microbial biomass C content of soil. In addition, the taxonomic and functional characterization of 13 C-DNA allows us to understand more precisely the changes in the composition of microbial communities affected by the C-substrate added to soil.

Extracellular DNA in soil and sediment: fate and ecological relevance.

The review discusses origin, state and function of extracellular DNA in soils and sediments. Extracellular DNA can be released from prokaryotic and eukaryotic cells and can be protected against nuclease degradation by its adsorption on soil colloids and sand particles. Laboratory experiments have shown that DNA adsorbed by colloids and sand particles can be taken up by prokaryotic competent cells and be involved in natural transformation. Most of these experiments have been carried out under artificial conditions with pure DNA molecules and pure adsorbing matrices, but in soils and sediments, pure surface-reactive colloids are not present and DNA is present with other cellular components (wall debris, proteins, lipids, RNA, etc.) especially if released after cell lysis. The presence of inorganic compounds and organic molecules on both soil particles and DNA molecules can influence the DNA adsorption, degradation and transformation of competent cells. Extracellular DNA can be used as C, N and P sources by heterotrophic microorganisms and plays a significant role in bacterial biofilm formation. The nucleotides and nucleosides originated from the degradation of extracellular DNA can be re-assimilated by soil microorganisms. Extracellular DNA in soil can be leached and moved by water through the soil profile by capillarity. In this way, the extracellular DNA secreted by a cell can reach a competent bacterial cell far from the donor cell. Finally, the characterisation of extracellular DNA can integrate information on the composition of the microbial community of soil and sediments obtained by analysing intracellular DNA.

Effects of Root Exudates in Microbial Diversity and Activity in Rhizosphere Soils

The rhizosphere is the soil volume at the root-soil interface that is under the influence of the plant roots and the term was introduced by Hiltner in 1904 (Brimecombe et al. 2001). Microbial population in the rhizosphere has continuous access to a flow of low and high molecular weight organic substrates derived from roots. This continuous flow of organic compounds may affect together with specific physiochemical and biological conditions microbial activity and community structure of the rhizosphere soil (Sorensen 1997; Brimecombe et al. 2001). Current techniques still lack the adequate sensitivity and resolution for data collection at the micro-scale, and the question ‘How important are various soil processes acting at different scales for ecological function?’ is therefore challenging to answer. The nano-scale secondary ion mass spectrometer (NanoSIMS) represents the latest generation of ion microprobes, which link high-resolution microscopy with isotopic analysis. Recently Herrmann et al. (2007) have described the principles of NanoSIMS and discusses the potential of this tool to contribute to the field of biogeochemistry and soil ecology. Both microbial activity and microbial diversity of the rhizosphere have been extensively studied as testimonies by numerous chapters and books (Keister and Creagen 1991; Lynch 1990a; Pinton et al. 2001, 2007; Waisel et al. 1991). This interest depends on the important effects that microorganisms inhabiting the rhizosphere have on plant activity. Both beneficial and detrimental interactions occur between microorganisms and plants (Lynch 1990b); among the former symbiotic dinitrogen fixation, association with mycorrhizae, biocontrol against pathogens and production of plant growth promoting compounds by beneficial rhizobacteria have

Are humus forms, mesofauna and microflora in subalpine forest soils sensitive to thermal conditions?

This study focuses on the biological and morphological development of humus profiles in forested Italian Alpine soils as a function of climate. Humus form description, systematic investigation of microannelid communities and polyphasic biochemical fingerprinting of soil microbial communities (denaturing gradient gel electrophoresis (DGGE) and phospholipid fatty acid analysis (PLFA)) were performed to compare sites differing in mean annual temperature due to different altitude and exposure. Although the soil biota showed complex responses, several differences in soil biological properties seem to be due to thermal differences. Although soil acidity also determines biological properties, it is not a state factor but rather influenced by them. The thickness of the organic layer and the acidification of the subjacent mineral horizon increased under cooler conditions (north-exposure; higher altitude), whereas the thickness of the A horizon inversely decreased. Species richness of microannelid assemblages was higher under warmer conditions (south-exposure; lower altitude) and the vertical distribution of microannelids shifted along the gradient to lower temperatures from predominant occurrence in the mineral soil to exclusive occurrence in the organic layer. Microbial biomass (total PLFA) was higher at the cooler sites; the prevalence of Gram-negative bacteria could be ascribed to their better adaptation to lower temperature, pH and nutrient contents. The δ13C signatures of the PLFA markers suggested a lower decomposition rate at the cooler sites, resulting in a lower respiratory loss and an accumulation of weakly decomposed organic material. DGGE data supported the PLFA results. Both parameters reflected the expected thermal sequence. This multidisciplinary case study provided indications of an association of climate, mesofauna and microbiota using the humus form as an overall link. More data are however needed and further investigations are encouraged.

Maize lines with different nitrogen use efficiency select bacterial communities with different β-glucosidase-encoding genes and glucosidase activity in the rhizosphere

We studied the molecular diversity of β-glucosidase-encoding genes, microbial biomass, cellulase, N-acetyl-glucosaminidase, β-glucosidase, and β-galactosidase activities in the rhizosphere and bulk soil of two maize lines differing in nitrogen use efficiency (NUE). The maize lines had significant differences in diversity of β-glucosidase-encoding genes in their rhizosphere, and Actinobacteria and Proteobacteria were the dominating phyla in all samples, but representatives of Bacteroidetes, Chloroflexi, Deinococcus-Thermus, Firmicutes, and Cyanobacteria were also detected. Among the Proteobacteria, β-glucosidase genes from α-, β-, and γ-Proteobacteria were dominant in the rhizosphere of the high NUE maize line, whereas δ-Proteobacteria β-glucosidase genes were dominant in the rhizosphere of the low NUE maize line. The high NUE maize line also showed higher glucosidase activities in the rhizosphere than the low NUE maize line. We concluded that plants with high NUE select bacterial communities in the rhizosphere differing in the diversity of β-glucosidase-encoding genes which likely result in higher C-hydrolyzing enzyme activities. These effects on the diversity of β-glucosidase-encoding genes may influence the C dynamics in the agro-ecosystems.

Purification and isotopic signatures (δ13C, δ15N, Δ14C) of soil extracellular DNA

The aim of this work was to obtain pure extracellular DNA molecules so as to estimate their longevity in soil by an isotope-based approach. Extracellular DNA molecules were extracted from all horizons of a forest soil and purified by the procedure of Davis (Purification and precipitation of genomic DNA with phenol–chloroform and ethanol. In: Davis LG, Dibner MD, Battey JF (eds) Basic methods in molecular biology. Appleton & Lange, Norwalk, 16–22, 1986) without (DNA1) or with (DNA2) a successive treatment with binding resins followed by elution. The two differently purified DNA samples were compared for their A260/A280 ratio, polymerase chain reaction (PCR) amplification and natural abundance of stable (13C and 15N) and radioactive (14C) isotopes. The purity index and the PCR amplification did not differentiate the efficiency of the two purification procedures. The isotopic signature of DNA was more sensitive and was strongly affected by the purification procedures. The isotopic measurements showed that the major contaminant of extracellular DNA1 was the soil organic matter (SOM), even if it is not possible to exclude that the similar δ13C, δ15N and Δ14C values of DNA and SOM could be due to the use of SOM-deriving C and N atoms for the microbial synthesis of DNA. For extracellular DNA2, extremely low values of Δ14C were obtained, and this was ascribed to the presence of fossil fuel-derived substances used during the purification, although in amounts not revealed by gas chromatography-mass spectrometry analysis. The fact that it is not possible to obtain contaminant-free DNA molecules and the potential use of soil native organic compounds during the microbial synthesis of DNA make it not achievable to estimate the age of soil extracellular DNA by radiocarbon dating.

Experimental discrimination and molecular characterization of the extracellular soil DNA fraction

We experimentally discriminated and quali-quantitatively characterized the extracellular fraction of a forest soil DNA pool. We sequentially extracted and classified the components of extracellular DNA by its strength of interaction with soil colloids as: (1) extractable in water, free in the extracellular soil environment or adsorbed on soil colloids; and as (2) extractable in alkaline buffer after previous extraction in water, bound on soil colloids. The comparative molecular analysis (fluorometer, gel electrophoresis, genetic fingerprinting) of directly and sequentially extracted extracellular DNA revealed quantitative and qualitative differences, also in terms of genetic information about microbial communities. The sequential extraction of extracellular DNA revealed differences in molecular weight, indicating a relationship between DNA fragment length and strength of interaction with soil colloids. The sequential extraction was also suitable to assess the presence of tightly bound DNA, providing information about the DNA-colloid interactions naturally occurring in the soil environment.

Recent Advances in Functional Genomics and Proteomics of Plant Associated Microbes

Research in soil microbiology has concerned the determination of the presence of gene sequences so as to assess microbial diversity rather than the determination of gene expression. Generally these molecular techniques are based on the specific amplification of the target nucleic acid by polymerase chain reaction (PCR) with either restriction analysis or separation by denaturing or conformational properties of the resulting amplicons (Lynch et al. 2004). On the other hand microbial activities in soil have been measured by classical techniques such as those for determining soil respiration, enzyme activities, N mineralization, adenylate energy charge, leucine and thymidine incorporation, etc., with no idea of gene expression. The rhizosphere effects on microbial diversity and activity are discussed in Chap. 14 of this book. Rapid progress in genomics has led to the availability of full genome sequences of hundreds of microorganisms, mostly bacteria (DeLong 2002). Combinations of new molecular methodology and genomics have been used successfully to link microbial phylogeny with function in several ecological studies and the same approach could provide significant insights into plant–microbe interactions in the rhizosphere. The functional genomics is based on a holistic or systemic approach with studying information flow within a cell and this requires the application of high throughput methods using automated technologies, which allow functional analysis of genome, proteome and metabolome of an organism (Wren 2000). In this way it is possible to get an insight on interplay of a large number of gene products and the relative consequences of this communication to the physiology of a cell. In contrast, molecular biologists have followed the reductionistic approach by studying single genes, and the individual actions of genes with a step-by-step characterization of metabolic pathways. Thus, an efficient DNA extraction with

Land use and seasonal effects on a Mediterranean soil bacterial community

To evaluate the effects of management practices and seasons on a soil bacterial community and the composition of ammonia-oxidizing bacteria (AOB), molecular screenings were compared among Mediterranean (Sardinia) soils with different plant covers and different agricultural practices, namely cork oak forest, tilled/non-tilled vineyard, hay crop and pasture. We compared the fingerprints from both independent replicates and pooled samples to ascertain the best approach for studying the environmental effects on bacterial composition. The soil microbial biomass, which was estimated from the amounts of extracted soil dsDNA, was 2 to 3 folds higher in the spring than in the autumn; in the spring, it was negatively correlated with the intensity of land use. A 16S rDNA DGGE experiment confirmed that both the land use and season markedly affect the composition of the soil bacterial community. Tilled vineyard soil exhibited the lowest similarities in community structures, suggesting that tillage induced the most marked disturbance among the tested land management methods. Distinct AOB populations were found for each type of land use; among these types, the cork oak forest proved to be a protective habitat for AOB against environmental changes. Our results suggest that the comparative community level and group-specific fingerprinting enabled an accurate evaluation of multiple factors in soil bacterial structures when performed with both independent and pooled replicates.