Functional principal component data analysis: a new method for analysing microbial community fingerprints.

A common approach to molecular characterisation of microbial communities in natural environments is the amplification of small subunit (SSU) rRNA genes or genes encoding enzymes essential for a particular ecosystem function. A range of 'fingerprinting' techniques are available for the analysis of amplification products of both types of gene enabling quantitative or semi-quantitative analysis of relative abundances of different community members, and facilitating analysis of communities from large numbers of samples, including replicates. Statistical models that have been applied in this context suffer from a number of unavoidable limitations, including lack of distinction between closely adjacent bands or peaks, particularly when these differ significantly in intensity or size. Current approaches to the analysis of banding structures derived from gels are typically based on standard multivariate analysis methods such as principal component analysis (PCA) which do not consider structure of DGGE gels but treat the intensity of each band as independent from the other bands, ignoring local neighbourhood structures. This paper assesses whether a new statistical analytical technique, based on functional data analysis (FDA) methods, improves the discriminatory ability of molecular fingerprinting techniques. The approach regards band intensities as a mathematical function of the location on the gel and explicitly includes neighbourhood structure in the analysis. A simulation study clearly reveals the weaknesses of the standard PCA approach as opposed to the FDA approach, which is then used to analyse experimental DGGE data.

Plant host habitat and root exudates shape soil bacterial community structure.

The rhizosphere is active and dynamic in which newly generated carbon, derived from root exudates, and ancient carbon, in soil organic matter (SOM), are available for microbial growth. Stable isotope probing (SIP) was used to determine bacterial communities assimilating each carbon source in the rhizosphere of four plant species. Wheat, maize, rape and barrel clover (Medicago truncatula) were grown separately in the same soil under (13)CO(2) (99% of atom (13)C) and DNA extracted from rhizosphere soil was fractionated by isopycnic centrifugation. Bacteria-assimilating root exudates were characterized by denaturing gradient gel electrophoresis (DGGE) analysis of (13)C-DNA and root DNA, whereas those assimilating SOM were identified from (12)C-DNA. Plant species root exudates significantly shaped rhizosphere bacterial community structure. Bacteria related to Sphingobacteriales and Myxococcus assimilated root exudates in colonizing roots of all four plants, whwereas bacteria related to Sphingomonadales utilized both carbon sources, and were identified in light, heavy and root compartment DNA. Sphingomonadales were specific to monocotyledons, whereas bacteria related to Enterobacter and Rhizobiales colonized all compartments of all four plants, used both fresh and ancient carbon and were considered as generalists. There was also evidence for an indirect important impact of root exudates, through stimulation of SOM assimilation by a diverse bacterial community.

Studying plant-microbe interactions using stable isotope technologies.

Interactions between plants and microorganisms in the rhizosphere are complex and varied. They include the general transfer of nutrients and specific interactions mediated by the release of signalling molecules from plant roots. Until recently, understanding the nature of these interactions was limited by a reliance on traditional, cultivation-based techniques. Stable isotope probing provides the potential for cultivation-independent characterisation of organisms actively assimilating carbon derived from plant root exudate or added to the soil. Current applications have focused on interactions with relatively low-level specificity, but there is significant potential for mechanistic studies of more specific interactions, particularly if the sensitivity of the technique can be increased.

Stable isotope probing analysis of the influence of liming on root exudate utilization by soil microorganisms.

Rhizosphere microorganisms play an important role in soil carbon flow, through turnover of root exudates, but there is little information on which organisms are actively involved or on the influence of environmental conditions on active communities. In this study, a 13CO2 pulse labelling field experiment was performed in an upland grassland soil, followed by RNA-stable isotope probing (SIP) analysis, to determine the effect of liming on the structure of the rhizosphere microbial community metabolizing root exudates. The lower limit of detection for SIP was determined in soil samples inoculated with a range of concentrations of 13C-labelled Pseudomonas fluorescens and was found to lie between 10(5) and 10(6) cells per gram of soil. The technique was capable of detecting microbial communities actively assimilating root exudates derived from recent photo-assimilate in the field. Denaturing gradient gel electrophoresis (DGGE) profiles of bacteria, archaea and fungi derived from fractions obtained from caesium trifluoroacetate (CsTFA) density gradient ultracentrifugation indicated that active communities in limed soils were more complex than those in unlimed soils and were more active in utilization of recently exuded 13C compounds. In limed soils, the majority of the community detected by standard RNA-DGGE analysis appeared to be utilizing root exudates. In unlimed soils, DGGE profiles from 12C and 13C RNA fractions differed, suggesting that a proportion of the active community was utilizing other sources of organic carbon. These differences may reflect differences in the amount of root exudation under the different conditions.

Flux and turnover of fixed carbon in soil microbial biomass of limed and unlimed plots of an upland grassland ecosystem.

The influence of liming on rhizosphere microbial biomass C and incorporation of root exudates was studied in the field by in situ pulse labelling of temperate grassland vegetation with (13)CO(2) for a 3-day period. In plots that had been limed (CaCO(3) amended) annually for 3 years, incorporation into shoots and roots was, respectively, greater and lower than in unlimed plots. Analysis of chloroform-labile C demonstrated lower levels of (13)C incorporation into microbial biomass in limed soils compared to unlimed soils. The turnover of the recently assimilated (13)C compounds was faster in microbial biomass from limed than that from unlimed soils, suggesting that liming increases incorporation by microbial communities of root exudates. An exponential decay model of (13)C in total microbial biomass in limed soils indicated that the half-life of the tracer within this carbon pool was 4.7 days. Results are presented and discussed in relation to the absolute values of (13)C fixed and allocated within the plant-soil system.

Physiological and genetic characterization of fluorescent Pseudomonas associated with Cantharellus cibarius.

Fluorescent Pseudomonas spp. isolated from fruiting bodies (FB) of Cantharellus cibarius were characterized physiologically and genetically and were compared with fluorescent Pseudomonas from forest soil and with sequences from the GenBank database. Pseudomonas spp. from FB differed physiologically from isolates from soil lacking FB and had some similarities with the strains obtained from soil underneath the FB. Analyses of the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) patterns and partial sequencing analysis of the 16S-rDNA region indicated that the bacteria isolated from these environments were different. However, there was no specific Pseudomonas genotype restricted to the FB environment. Utilization of the reported fungal exudates trehalose and mannitol may explain how millions of bacteria survive in the C. cibarius FB without deteriorating the fungal mycelium. The importance of the metabolic characterization of bacteria and the possible mechanisms involved in the association with C cibarius are discussed. Our study showed that standard processes for bacterial identification, e.g., Biolog and 16S-rDNA are insufficient until databases for different ecosystems are created.

Use of different nitrogen sources by the edible ectomycorrhizal mushroom Cantharellus cibarius.

The growth of three strains of Cantharellus cibarius on liquid media containing ammonium, nitrate and bovine serum albumin (BSA) in different combinations was determined. The most readily utilisable source of N was ammonium. BSA utilisation was limited compared with media containing ammonium. Growth on nitrate was also poor, suggesting a limited capacity of C. cibarius to metabolise this nitrogen source. There was some indication of considerable intraspecific variation within C. cibarius in the utilisation of nitrogen sources. Possible links between atmospheric nitrogen deposition and the observed decrease of sporocarp formation by C. cibarius in Europe are discussed. We highlight the potential ecological significance of bacteria associated with C. cibarius which may circumvent the need for fungal extracellular enzymes to access complex nitrogen sources.

A 13C-NMR study of exudation and storage of carbohydrates and amino acids in the ectomycorrhizal edible mushroom Cantharellus cibarius.

(13)C-NMR analyses of Cantharellus cibarius growth media were performed. We found exudation of trehalose and mannitol, which may explain the phenomenon of reproducing Pseudomonas bacteria observed inside fruit bodies. Exudation varied with strain and environment. NMR analyses of stored (13)C was also performed. Trehalose, mannitol, and arginine were revealed. The mannitol pathway seems to play an important role for trehalose production in this species. This is the first study of the fate of the photosynthetically derived carbon in the highly appreciated edible ectomycorrhizal mushroom Cantharellus cibarius.