Pascale Frey-Klett

Mineral weathering by bacteria: ecology, actors and mechanisms

Soil microbes play an essential role in the environment by contributing to the release of key nutrients from primary minerals that are required not only for their own nutrition but also for that of plants. Although the role of fungi in mineral weathering is beginning to be elucidated, the relative impact of bacteria in this process and the molecular mechanisms involved remain poorly understood. Here, we discuss the ecological relevance of bacterial weathering, mainly in the soil and especially in acidic forest ecosystems, which strongly depend on mineral weathering for their sustainability. We also present highlights from recent studies showing molecular mechanisms and genetic determinants involved in the dissolution of complex minerals under aerobic conditions. Finally, we consider the potential applications of genomic resources to the study of bacterial weathering.

Effect of the mycorrhizosphere on the genotypic and metabolic diversity of the bacterial communities involved in mineral weathering in a forest soil.

ABSTRACT To date, several bacterial species have been described as mineral-weathering agents which improve plant nutrition and growth. However, the possible relationships between mineral-weathering potential, taxonomic identity, and metabolic ability have not been investigated thus far. In this study, we characterized a collection of 61 bacterial strains isolated from Scleroderma citrinum mycorrhizae, the mycorrhizosphere, and the adjacent bulk soil in an oak forest. The ability of bacteria to weather biotite was assessed with a new microplate bioassay that measures the pH and the quantity of iron released from this mineral. We showed that weathering bacteria occurred more frequently in the vicinity of S. citrinum than in the bulk soil. Moreover, the weathering efficacy of the mycorrhizosphere bacterial isolates was significantly greater than that of the bulk soil isolates. All the bacterial isolates were identified by partial 16S rRNA gene sequence analysis as members of the genera Burkholderia, Collimonas, Pseudomonas, and Sphingomonas, and their carbon metabolism was characterized by the BIOLOG method. The most efficient isolates belonged to the genera Burkholderia and Collimonas. Multivariate analysis resulted in identification of three metabolic groups, one of which contained mainly bacterial isolates associated with S. citrinum and exhibiting high mineral-weathering potential. Therefore, our results support the hypothesis that by its carbon metabolism this fungus selects in the bulk soil reservoir a bacterial community with high weathering potential, and they also address the question of functional complementation between mycorrhizal fungi and bacteria in the ectomycorrhizal complex for the promotion of tree nutrition.

Root-Associated Bacteria Contribute to Mineral Weathering and to Mineral Nutrition in Trees: a Budgeting Analysis

ABSTRACT The principal nutrient source for forest trees derives from the weathering of soil minerals which results from water circulation and from plant and microbial activity. The main objectives of this work were to quantify the respective effects of plant- and root-associated bacteria on mineral weathering and their consequences on tree seedling growth and nutrition. That is why we carried out two column experiments with a quartz-biotite substrate. The columns were planted with or without pine seedlings and inoculated or not with three ectomycorrhizosphere bacterial strains to quantify biotite weathering and pine growth and to determine how bacteria improve pine growth. We showed that the pine roots significantly increased biotite weathering by a factor of 1.3 for magnesium and 1.7 for potassium. We also demonstrated that the inoculation of Burkholderia glathei PML1(12) significantly increased biotite weathering by a factor of 1.4 for magnesium and 1.5 for potassium in comparison with the pine alone. In addition, we observed a significant positive effect of B. glathei PMB1(7) and PML1(12) on pine growth and on root morphology (number of lateral roots and root hairs). We demonstrated that PML1(12) improved pine growth when the seedlings were supplied with a nutrient solution which did not contain the nutrients present in the biotite. No improvement of pine growth was observed when the seedlings were supplied with all the nutrients necessary for pine growth. We therefore propose that the growth-promoting effect of B. glathei PML1(12) mainly resulted from the improved plant nutrition via increased mineral weathering.

Microbial Biodiversity: Approaches to Experimental Design and Hypothesis Testing in Primary Scientific Literature from 1975 to 1999

SUMMARY Research interest in microbial biodiversity over the past 25 years has increased markedly as microbiologists have become interested in the significance of biodiversity for ecological processes and as the industrial, medical, and agricultural applications of this diversity have evolved. One major challenge for studies of microbial habitats is how to account for the diversity of extremely large and heterogeneous populations with samples that represent only a very small fraction of these populations. This review presents an analysis of the way in which the field of microbial biodiversity has exploited sampling, experimental design, and the process of hypothesis testing to meet this challenge. This review is based on a systematic analysis of 753 publications randomly sampled from the primary scientific literature from 1975 to 1999 concerning the microbial biodiversity of eight habitats related to water, soil, plants, and food. These publications illustrate a dominant and growing interest in questions concerning the effect of specific environmental factors on microbial biodiversity, the spatial and temporal heterogeneity of this biodiversity, and quantitative measures of population structure for most of the habitats covered here. Nevertheless, our analysis reveals that descriptions of sampling strategies or other information concerning the representativeness of the sample are often missing from publications, that there is very limited use of statistical tests of hypotheses, and that only a very few publications report the results of multiple independent tests of hypotheses. Examples are cited of different approaches and constraints to experimental design and hypothesis testing in studies of microbial biodiversity. To prompt a more rigorous approach to unambiguous evaluation of the impact of microbial biodiversity on ecological processes, we present guidelines for reporting information about experimental design, sampling strategies, and analyses of results in publications concerning microbial biodiversity.

Inter-kingdom encounters: recent advances in molecular bacterium-fungus interactions.

Interactions between bacteria and fungi are well known, but it is often underestimated how intimate and decisive such associations can be with respect to behaviour and survival of each participating organism. In this article we review recent advances in molecular bacterium–fungus interactions, combining the data of different model systems. Emphasis is given to the positive or negative consequences these interactions have on the microbe accommodating plants and animals. Intricate mechanisms of antagonism and tolerance have emerged, being as important for the biological control of plants against fungal diseases as for the human body against fungal infections. Bacterial growth promoters of fungal mycelium have been characterized, and these may as well assist plant-fungus mutualism as disease development in animals. Some of the toxins that have been previously associated with fungi are actually produced by endobacteria, and the mechanisms that lie behind the maintenance of such exquisite endosymbioses are fascinating. Bacteria do cause diseases in fungi, and a synergistic action between bacterial toxins and extracellular enzymes is the hallmark of such diseases. The molecular study of bacterium–fungus associations has expanded our view on microbial communication, and this promising field shows now great potentials in medicinal, agricultural and biotechnological applications.

Survival in the soil of the ectomycorrhizal fungus Laccaria bicolor and the effects of a mycorrhiza helper Pseudomonas fluorescens

In disinfected forest nursery soils, inoculating Douglas fir (Pseudotsuga menziesii) seedlings with the ectomycorrhizal fungal strain Laccaria bicolor S238N significantly increases tree growth after outplantating. However, the success of the inoculation depends on survival of the fungal inoculum in the soil during the pre-symbiotic life of the fungus. We followed the survival of L. bicolor S238N in autoclaved nursery soil in the glasshouse, and under gnotobiotic conditions in autoclaved or γ-irradiated nursery soil. We also studied the effect of the mycorrhiza helper bacterium Pseudomonas fluorescens BBc6R8, which promotes the Douglas fir-L. bicolor S238N symbiosis, on fungal viability. In the glasshouse, fungal viability was assessed by trapping with Douglas fir seedlings. We showed that the fungus retained its viability in a pre-symbiotic state in the soil at least for 23 weeks, which is much longer than that reported in the literature for other ectomycorrhizal fungi. The bacterium did not significantly modify the survival of the fungus. In the gnotobiotic experiments, ergosterol, a specific fungal membrane component, was used to quantify fungal biomass. Fungal behaviour differed with the disinfection technique used, which modified the chemical characteristics of the initial soil. There was no fungal growth in the autoclaved soil but there was a rapid increase of fungal biomass in the irradiated soil. The effect of the bacterium on fungal biomass also varied with a significant stimulation in the autoclaved soil vs. a significant inhibition in the irradiated soil. Our results show that the beneficial effect of the bacterium on the fungus depends on the condition of the fungus, i.e. the greatest benefit occurs when the fungus is growing under unfavourable conditions.

Black truffle-associated bacterial communities during the development and maturation of Tuber melanosporum ascocarps and putative functional roles

Although truffles are cultivated since decades, their life cycle and the conditions stimulating ascocarp formation still remain mysterious. A role for bacteria in the development of several truffle species has been suggested but few is known regarding the natural bacterial communities of Périgord Black truffle. Thus, the aim of this study was to decipher the structure and the functional potential of the bacterial communities associated to the Black truffle in the course of its life cycle and along truffle maturation. A polyphasic approach combining 454-pyrosequencing of 16S rRNA gene, TTGE, in situ hybridization and functional GeoChip 3.0 revealed that Black truffle ascocarps provide a habitat to complex bacterial communities that are clearly differentiated from those of the surrounding soil and the ectomycorrhizosphere. The composition of these communities is dynamic and evolves during the maturation of the ascocarps with an enrichment of specific taxa and a differentiation of the gleba and peridium-associated bacterial communities. Genes related to nitrogen and sulphur cycling were enriched in the ascocarps. Together, these data paint a new picture of the interactions existing between truffle and bacteria and of the potential role of these bacteria in truffle maturation.

Influence of Forest Trees on the Distribution of Mineral Weathering-Associated Bacterial Communities of the Scleroderma citrinum Mycorrhizosphere

ABSTRACT In acidic forest soils, availability of inorganic nutrients is a tree-growth-limiting factor. A hypothesis to explain sustainable forest development proposes that tree roots select soil microbes involved in central biogeochemical processes, such as mineral weathering, that may contribute to nutrient mobilization and tree nutrition. Here we showed, by combining soil analyses with cultivation-dependent analyses of the culturable bacterial communities associated with the widespread mycorrhizal fungus Scleroderma citrinum, a significant enrichment of bacterial isolates with efficient mineral weathering potentials around the oak and beech mycorrhizal roots compared to bulk soil. Such a difference did not exist in the rhizosphere of Norway spruce. The mineral weathering ability of the bacterial isolates was assessed using a microplaque assay that measures the pH and the amount of iron released from biotite. Using this microplate assay, we demonstrated that the bacterial isolates harboring the most efficient mineral weathering potential belonged to the Burkholderia genus. Notably, previous work revealed that oak and beech harbored very similar pHs in the 5- to 10-cm horizon in both rhizosphere and bulk soil environments. In the spruce rhizosphere, in contrast, the pH was significantly lower than that in bulk soil. Because the production of protons is one of the main mechanisms responsible for mineral weathering, our results suggest that certain tree species have developed indirect strategies for mineral weathering in nutrient-poor soils, which lie in the selection of bacterial communities with efficient mineral weathering potentials.

Characterization of functional traits of two fluorescent pseudomonads isolated from basidiomes of ectomycorrhizal fungi

Abstract Some functional traits of Pseudomonas fluorescens 92 and BBc6, two strains isolated, respectively, from the basidiome of the ectomycorrhizal fungi Suillus grevillei and Laccaria laccata , were evaluated. A rifampicin-resistant mutant of P. fluorescens 92 ( P. fluorescens 92R1) showed a significant in vivo plant growth promotion effect on cucumber plants. Quantitative analysis of enzymatic and physiological activities on different substrates showed that P. fluorescens 92 produced about a three times higher level of avicelase than BBc6, while equivalent amounts of β-glucosidase were produced by both strains. Satisfactory levels of neutral phosphomonoesterase and medium levels of acid phosphomonoesterase were produced by both. Only P. fluorescens BBc6 produced a very low amount of phosphodiesterase. Both strains produced high amounts of IAA and siderophores. Both strains showed on an iron deficient medium a very high antagonistic activity against the phytopathogenic fungus Heterobasidion annosum . Purification of fluorescent siderophores by copper-chelate chromatography showed that P. fluorescens 92 produced one pyoverdin (Pf92) and BBc6 two pyoverdins (PfBI and PfBII). A good inhibitory activity against mycelial growth of H. annosum was also observed when using the pyoverdines purified by affinity chromatography. Further purification by reverse phase high pressure liquid chromatography produced multiple fractionation of the three pyoverdins. Analysis of the reverse-phase purified pyoverdines by electronspray ionization mass spectrometry gave for pyoverdin Pf92 the mass value of 1213.8 and for both PfBI and PfBII the mass value of 1305.7. The presence of iron-chelating forms and sodium adducts were also evidenced.

Bacteria associated with truffle-fruiting bodies contribute to truffle aroma.

Truffles, symbiotic fungi renown for the captivating aroma of their fruiting bodies, are colonized by a complex bacterial community of unknown function. We characterized the bacterial community of the white truffle Tuber borchii and tested the involvement of its microbiome in the production of sulphur-containing volatiles. We found that sulphur-containing volatiles such as thiophene derivatives, characteristic of T. borchii fruiting bodies, resulted from the biotransformation of non-volatile precursor(s) into volatile compounds by bacteria. The bacterial community of T. borchii was dominated by α- and β-Proteobacteria. Interestingly, all bacteria phyla/classes tested in this study were able to produce thiophene volatiles from T. borchii fruiting body extract, irrespective of their isolation source (truffle or other sources). This indicates that the ability to produce thiophene volatiles might be widespread among bacteria and possibly linked to primary metabolism. Treatment of fruiting bodies with antibacterial agents fully suppressed the production of thiophene volatiles while fungicides had no inhibitory effect. This suggests that during the sexual stage of truffles, thiophene volatiles are exclusively synthesized by bacteria and not by the truffle. At this stage, the origin of thiophenes precursor in T. borchii remains elusive and the involvement of yeasts or other bacteria cannot be excluded.