Giacomo Pietramellara

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.

Use of humic substances as soil conditioners to increase aggregate stability

Abstract In many agricultural soils in the semi-arid and and mediterranean climates, exposure to cyclic wetting and drying (WD) can reduce aggregate stability. The extent to which soil pretreatment with coal-derived humic substances (HS) can increase aggregate stability in soils exposed to cyclic wetting and drying (WD) was evaluated in this study. The soils studied are an Acireale silty clay loam from Sicily, a Principina silt loam from Tuscany and a Bovolone loam from Venetia in Italy. On each soil eight rates of the HS (0, 0.001, 0.01, 0.05, 0.10, 0.50, 1.00 and 10.00 g/kg of soil) and four WD cycles (0, 3, 6 and 9) were used to assess any changes in stability. On the Principina and Bovolone soils dominated by illitic and smectic clay minerals, successive WD cycles reduced aggregate stability. On the Acireale soil, dominated by kaolinitic clay mineral, after initial decrease in stability following three WD cycles, the aggregates regained stability as cyclic WD continued. Amending the soils with low rates of HS (equivalent to 100–200 kg/ha) not only improved aggregate stability significantly (p = 0.05) on all the three soils, but also reduced substantially the disaggregating effects of WD cycles. This work indicates that exogenous humic substances have a potential as soil conditioners in conservation practices aimed at increasing the structural stability of soils.

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.

Effect of molecular characteristics of DNA on its adsorption and binding on homoionic montmorillonite and kaolinite

Abstract. Adsorption or binding of DNA by montmorillonite or kaolinite, homoionic to Ca2+, was not affected by base composition, blunt or cohesive ends. Fitting data to both Freundlich and Langmuir adsorption isotherms showed that the amount of lower molecular mass DNA adsorbed and bound by both clay minerals was higher than that of the higher molecular mass DNA. The relevance of phosphate groups for the adsorption of DNA by clay minerals was investigated by adding sodium metaphosphate before and after the addition of DNA to clay minerals: DNA was partially not adsorbed even at low concentrations of sodium metaphosphate. The fact that the observed DNA was partially desorbed by washing with double-distilled H2O indicated that bonds with different degrees of strength were formed between DNA molecules and clay minerals. The higher molecular mass DNA could interact with a larger number of binding sites on the external surface of clay mineral than the lower molecular mass DNA. The number of external surface binding sites was higher on kaolinite than on montmorillonite.

Microbial biomass, respiration and enzyme activities after in situ aided phytostabilization of a Pb- and Cu-contaminated soil.

We conducted a pilot-scale experiment to study the effects of an aided phytostabilisation on soil microbial and biological endpoints in an ore dust-contaminated soil. Soil was amended with alkaline fly ashes plus peat to reduce mobility of trace elements and vegetated with a proprietary grass/herb mixture. Results indicated that the proposed aided phytostabilization approach of Cu-Pb contaminaed soil significantly increased microbial biomass and respiration, reduced microbial stress and increased key soil enzyme activities. Further research is needed to unambiguously determine whether the soil biochemical endpoints that were studied responded more to decreased metal mobility or to general soil amelioration.

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

Effects of air-drying and wetting cycles on the transforming ability of DNA bound on clay minerals

Abstract Chromosomal DNA from Bacillus subtilis and plasmid pHV14 bound on montmorillonite (M) and kaolinite (K) homoionic to Ca were subjected to repeated cycles of air-drying and wetting. After each cycle the ability of the bound DNA to transform competent cells was evaluated. Chromosomal DNA bound on clay retained its transforming efficiency after three to four air-drying and wetting cycles, whereas plasmid DNA lost the transforming ability after one to two cycles. This loss was neither due to the desorption of DNA from the complexes nor to the negative effects on DNA bound on clay of the acidic pH, that develops in the water film surrounding the clay surface during the air-drying. The clay—DNA complexes required 6 h of wetting to regain their transforming ability. Free DNA retained its transforming ability for longer than bound DNA and did not require 6 h of wetting to regain its transforming ability. These results indicate that the different behaviour of DNA—clay complexes after air-drying is related to the molecular form of the DNA and to a conformational change in the DNA molecule bound on clay.

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.

Determination of extracellular neutral phosphomonoesterase activity in soil

A new approach was carried out to determine the extracellular neutral phosphomonoesterase activity of soil. Surface soils under continuous winter wheat, and characterized by different amounts of organic matter, were sampled, air-dried and stored for 2 years or kept moist and investigated immediately. Glucose and nitrate were added to each soil and phosphomonoesterase and ATP content were monitored for up to 96 h. In moist soils the enzyme activity was significantly (P < 0.05) correlated with the ATP content, an indicator of microbial biomass. At zero ATP, a positive intercept of neutral phosphomonoesterase activity on the ordinate was observed and this value was assumed to be equal to the extracellular component. The correlations were less significant in dry than fresh soil. Probably the negative intercepts observed in two air-dried soils were due to underestimation of the intracellular activity present in non-proliferating microorganisms whose number was higher than in moist samples as the result of the prolonged storage under dry conditions.