Carmine Crecchio, Silvia Pascazio, and Pacifico Ruggiero
Abstract Bacteria and fungi play a key role in promoting soil organic matter (SOM) turn over and consequent nutrient availability to plants uptake. During SOM degradation, they contribute to transform highly complex biomolecules to smaller compounds, which are either immobilized by soil microflora or self-associated in humified and microbially stable superstructures. These processes rapidly occur in the rhizosphere where soil adheres to plant roots and microbial populations are more abundant and active than in bulk soil. Despite the difficulties in determining the composition of soil microbial communities, their genetic and functional diversities are fundamental to maintain soil quality and productivity, even under environmental stress or alteration. Within the national MESCOSAGR project, we provided indications on the composition and diversity of bacterial communities in soils subjected to carbon sequestration treatments. Nucleic acids were extracted from rhizosphere and bulk soils, purified and amplified by polymer-ase chain reaction (PCR), targeting a conserved region of 16S rRNA gene. Amplicons were separated by denaturing gradient gel electrophoresis (DGGE) and cluster analysis of relative electrophoretic profiles was used to evaluate the diversity of bacteria communities in soils under different soil management practices. The PCR products have also been cloned and sequenced in order to identify and characterize the microbial groups and species which populated the experimental soils. Our results, relative to field trials of the first 2 years and three experimental sites, indicate that the application of different molecular approaches contribute to reach an advanced characterization of structure and diversity of soil bacteria, as well as an appraisal of their variation, as a consequence of specific soil management practices. In particular, it appears that only the amendments with mature compost had a significant effect on the soil microbial communities, while
Dipartimento di Biologia e Chimica Agroforestale ed Ambientale, Universita di Bari Aldo Moro, Via Amendola 165/a, 70126 Bari, Italy e-mail: [email protected]
A. Piccolo (ed.), Carbon Sequestration in Agricultural Soils,
DOI 10.1007/978-3-642-23385-2_8, © Springer-Verlag Berlin Heidelberg 2012
other soil treatments such as that with the iron-porphyrin biomimetic catalyst did not have any effect.
Soil microorganisms are determinant in functioning and stability of soil ecosystem because of their influence in nutrient cycles and energy fluxes (Wertz et al. 2007). In particular, bacteria and fungi promote organic matter turn over and the consequent nutrient availability for mineral nutrition of plants (Ladd et al. 1996). In fact, in the degradation step of soil organic matter (SOM), they contribute to the decomposition of highly complex compounds to more simply compounds that, in turn, are immobilized by soil microflora or are aggregated in the humification process, giving rise to compounds recalcitrant to further microbial degradation (Smith et al. 1993).
The dynamics of the organic matter degradation depends on the nature and the quality of decomposing materials. Hodge et al. (2000) demonstrated that the amount of decomposed organic matter depends on C/N ratio of the decomposing material more than on the C and N needs of soil microorganisms. Compound with a low C/N ratio are easily degradable and rapidly incorporated by microbial biomass while compounds with a higher C/N ratio typically contain polymers such as lignin and polyphenols that are slowly degraded and only partially assimilated by soil microflora (Nicolardot et al. 2001). Furthermore, poorly degradable compounds might induce a kind of selection among microbial communities by favoring species with low metabolism (Lagomarsino et al. 2006). Repeated applications of manure for 10 years favored development of different microbial communities from those present in soils amended with chemical fertilizers (Toyota and Kuninaga 2006). Other authors demonstrated that microbial metabolites largely contributed the stable pool of C (Kiem and Kogel-Knabner 2003; Kindler et al. 2006; von Lutzow et al. 2006; Simpson et al. 2007).
An important role is attributed to the rhizosphere, that is the soil adhered to plant roots, and where microbial populations are more abundant and active than in bulk soil (Curl and Truelove 1986). Long-term effects of rhizodeposition on soil C turnover have been proposed as a means to contrast greenhouse effects (Smit et al. 1997; Paustian et al. 1998). Experiments under laboratory scale conditions (Kuikman et al. 1990a, b; Hassink et al. 1993; Foereid and Yearsley 2004) showed an increased C sequestration due to rhizosphere microorganisms in grazing grounds.
Genetic and functional diversity of soil microbial communities are fundamental to keep high levels of soil quality and productivity, even under stress conditions and in altered environments. On the other hand, it is very difficult to determine the composition of microbial communities in the very complex and dynamic soil biological system (Nannipieri et al. 2003).
For a complete view of soil environment conditions, it is important to apply analytical methods capable to investigate the diversity of microbiota, their function and distribution in soil (Hill et al. 2000). Size and composition of microbial communities can be obtained by extraction, quantification and characterization of biomarkers, which are molecules highly specific for microbial groups and species. The qualitative and quantitative determinations of biomarkers allow the investigation of the whole communities, including those species that are either poorly or not at all cultivable under laboratory conditions. Many cell components can be assumed as biomarkers. In particular, nucleic acids (DNA, RNA) give useful information on the structure and phylogenic composition of soil microorganisms (Griffiths et al. 2000).
Since bacteria can be found within soil aggregates or on their surface, the capacity to separate bacterial cells from soil particles is a key step in the study of biodiversity (Trevors 1998). There are two basic methods to extract nucleic acids from soil: a direct and an indirect method. The direct method was applied for the first time by Torsvik (1980), and consists of a preliminary desorption of bacterial cells from soil particles, followed by their lysis and further purification of the released nucleic acids. The direct method was proposed for the first time by Ogram et al. (1987). It entails the direct lysis of bacterial cells without a previous separation from soil and subsequent purification steps. This method has been successfully applied to investigate extracellular nucleic acids naturally present in soil microcosms. In both methods, cell lysis can be performed by a combination of mechanical (glass bead beater), chemical (detergents, NaOH) and enzymatic (lisozyme, protease K) methods. The efficient cell lysis and nucleic acids extraction, as well as the purity and size of extracted nucleic acids represent the major critical steps for further molecular characterizations (Ogram 2000).
Among the various molecular approaches, the more useful method for qualitative/quantitative characterization of microbial communities comprises the polymerase chain reaction (PCR) for prokaryotic genes coding the small subunit of ribosomal RNA (16SrDNA) (Ward et al. 1992). PCR is a polymerization reaction catalyzed by a thermo-stable DNA polymerase (Saiki et al. 1988) that allows the selective enrichment, of small target genome regions, without cloning into vectors/ host cells. The characterization of microbial communities by amplified genomic sequences coding for ribosomal RNA can be carried out by denaturing gradient gel electrophoresis (DGGE) (Muyzer et al. 1993), a technique that gives information at the level of microbial groups more than at that of species (Nannipieri et al. 2003). By PCR, amplicons of the same size but even slightly differing in nucleotide sequence can be separated according to their melting temperature. During electro-phoresis, each fragment remains as double strand until an exact concentration of denaturing chemicals (a linear gradient of urea and formamide at 60°C) is reached in the gel run and a denaturation occurs, thus causing conformational changes and loss of electrophoretic mobility (Heuer and Smalla 1997). The presence of a 40-45 GC stretch (GC-clamp), within the sequence of one of the primers used to amplify the target region, increases the melting point of the amplified fragments, thus avoiding a complete denaturation of amplicons and enhancing the fragments separation capacity up to 500 bp (Muyzer et al. 1993). The 16SrDNA PCR/DGGE method produces electrophoretic profiles in which the number of bands corresponds to the richness of bacterial species (at least the more representative), while the band intensities indicate the relative abundance of species. Moreover, a computational analysis of electrophoretic profiles allows the comparison among different samples and the build-up of phylogenetic trees. The amplification of DNA or RNA leads to discriminate either the whole bacterial population or the metabolically active microbiota, respectively (Prosser 2002).
A better comprehension of the relationship existing between microbial diversity and soil functions requires the use of high-resolution techniques (Nannipieri et al. 2003), being the cloning/sequencing of genomic regions undoubtedly one of them. Once again, the main target is represented by highly conserved regions of 16S rDNA that, after PCR amplification, can be easily cloned to give libraries, whose sequencing are representative of whole communities (Britschgi and Giovannoni 1991). Alternatively, PCR amplicons can be directly sequenced by the very recent pyro-sequencing approach (Acosta-Martinez et al. 2008). The matching of nucleotide sequences with databases such as NCBI GenBank or Ribosomal Database Project allows to establish a phylogenetic taxonomy of microbial species in different soil environments, as well as to compare their diversity, distribution, and abundance in soils (Maidak et al. 1997; Prosser 2002). By this approach, Curtis et al. (2002) showed that a gram of soil may contain up to 7,000 different bacteria species, that is the same amount estimated by traditional methods (Torsvik et al. 2002). Nevertheless, the definition of the number of sequences which may be adequate to represent a soil microbial diversity is still under debate.
Within the national MESCOSAGR project (see the Foreword and Chap. 3), the aim of our contribution was to provide indications about composition/diversity of bacterial communities of soils treated with carbon sequestration methods. Nucleic acids (DNA and RNA) have been extracted and purified by rhizosphere and bulk soil of three experimental fields and amplified by PCR targeting a conserved region of 16S rRNA gene. Amplicons have been separated by DGGE and cluster analysis of electrophoretic profiles has been used to evaluate the diversity of bacterial communities in soils under different soil management practices. PCR products have also been cloned and sequenced to identify microbial groups and species inhabiting the experimental soils.
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