Maria Teresa Ceccherini

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.

Degradation and Transformability of DNA from Transgenic Leaves

ABSTRACT The fate of transplastomic (chloroplast genome contains the transgene) tobacco plant DNA in planta was studied when the plant leaves were subjected to decay conditions simulating those encountered naturally, including grinding, incubation with cellulase or enzymes produced by Erwinia chrysanthemi, and attack by the plant pathogen Ralstonia solanacearum. Direct visualization of DNA on agarose gels, gene extraction yield (the number of amplifiable aadA sequences in extracted plant DNA), and the frequency that recipient bacteria can be transformed by plant DNA were used to evaluate the quality and quantity of plant DNA and the transgene. These measurements were used to monitor the physical and biological degradation of DNA inside decaying plant tissues. Our results indicate that while most of the DNA will be degraded inside plant cells, sufficient DNA persists to be released into the soil.

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.

Effects of swine manure fertilization on autotrophic ammonia oxidizing bacteria in soil

Abstract Molecular and biochemical investigation methods have been exploited to evaluate the effects of pig slurry, used for three years as a fertilizer in intensive agriculture, on the presence and activity of chemolithotrophic ammonia oxidizing bacteria, the biocatalysts of the first step in the nitrification process. The evaluation was carried out, on bulk soil, comparing data from the first and the third year of fertilization. Oligonucleotide sequences selected from the 16S rRNA genes of autotrophic ammonia oxidizers have been used as specific PCR amplification primers and probes. The potential nitrification activity (PNA) has been used to determine the effects of swine manure fertilization on the ammonia oxidizing activity. Members of the genera Nitrosomonas and Nitrosospira were always detectable, but the genus Nitrosospira was more represented than Nitrosomonas . The differences between the hybridization signals were correlated with the doses of pig slurry and the number of applications. The mineral (urea) and organic fertilizations showed similar effects on the ammonia oxidisers investigated by molecular and biochemical methods. It was found that additions of swine manure to soil plots increased both potential ammonia-oxidise activity and ammonia oxidiser sequences. Stimulation of the soil ammonia-oxidise activity was due mainly to its effects on the indigenous bacterial population.

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.

Microbial dynamics in Mediterranean Moder humus

There is a growing interest in the links between humus forms and soil biota, and little is known about these links in Mediterranean ecosystems. Culture-independent techniques, such as DNA extraction followed by DGGE and enzyme activities, allowed us to compare microbial communities in two horizons of a forest soil in different seasonal conditions. Direct in situ lysis was applied for extraction of DNA from soil; intracellular DNA was separated from extracellular and used to represent the composition of microflora. The aims were to describe how biochemical and microbiological parameters correlate with topsoil properties in typical Mediterranean Moder humus. Changes in bacterial and fungal community composition were evident from DGGE profiles. Degrees of similarity and clustering correlation coefficients showed that the seasonal conditions may affect the composition and activity of bacterial and fungal communities in the OH horizon, while in the E horizon the two communities were hardly modified. In the same season, OH and E horizons showed a different composition of bacterial and fungal communities and different enzyme activities, suggesting similar behaviour of eubacteria and fungi relatively to all the variables analysed. Evidently, different organic carbon content in soil horizons influenced microflora composition and microbial activities involved in the P and N cycles.

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.

Enzyme activity and microbial community structure in the rhizosphere of two maize lines differing in N use efficiency

AimsStudy of the changes in soil microbial biomass, enzyme activity and the microbial community structure in the rhizosphere of two contrasting maize lines differing in the nitrogen use efficiency (NUE).MethodsThe Lo5 and T250 inbred maize characterized by high and low NUE, respectively, were grown in rhizoboxes allowing precise sampling of rhizosphere and bulk soil and solution. We also determined microbial biomass, enzyme activities involved in the C, N, P and S cycles, and the microbial community structure using a phylogenetic group specific PCR-DGGE approach in the rhizosphere and bulk soil of both Lo5 and T250 maize lines.ResultsHigh NUE Lo5 maize induced faster inorganic N depletion in the rhizosphere and larger changes in microbial biomass and enzyme activities than the low NUE T250 maize line. The two maize lines induced differences in the studied microbial groups in the rhizosphere, with the larger modifications induced by the high NUE Lo5 maize line.ConclusionsThe Lo5 maize line with higher NUE induced larger changes in soil chemical properties and in the enzyme activity, soil microbial biomass and community structure than the low NUE T250 maize line, probably due to differences in the root exudates of the two maize lines.