Figure III.7: Effect of the soil horizon and the compartment of origin of the bacterial and fungal isolates on the discolouration diameters on CAS medium and on TCP medium. A and B: bacterial isolates, C and D: fungal isolates, A and C: test on CAS medium, B and D: test on TCP medium. Grey: upper horizon, black: lower horizon. Bars represent standard error. For the upper horizon, values with different letters (a, b or c) are significantly different according to a one-factor (compartment of origin of the isolates) ANOVA (P < 0.05) and the Bonferroni-Dunn test. For the lower horizon, values with different letters (A, B or C) are significantly different according to a one-factor (compartment of origin of the isolates) ANOVA (P < 0.05) and the Bonferroni-Dunn test. Values with asterisks are significantly different according to a one-factor (horizon of origin of the isolates) ANOVA (P < 0.05) for each compartment of origin of the isolates.

A

<figure><img src="image_32.png" /></figure>

B

<figure><img src="image_33.png" /></figure>

C

<figure><img src="image_34.png" /></figure>

D

<figure><img src="image_35.png" /></figure>
media, whereas the majority of isolates from the bulk soil was unable to mobilize either. This effect of the isolate origin compartment on iron and phosphorus mobilization potentials was confirmed by a one-factor (isolate origin compartment) ANOVA performed on the mean diameter values of the discolouration areas that appeared around the bacterial spots (P=0.001). The Bonferroni-Dunn test showed that the mean diameters of the discolouration area on the CAS or TCP media differed between the bulk soil isolates, the soil-mycorrhiza interface isolates and the mycorrhiza isolates (Figures III.7A and III.7C respectively), whatever the soil horizon from which they come. Interestingly, according to a χ2 test (P<0.001), the majority of the bacterial isolates from the soil-mycorrhiza interface and the mycorrhizae was able to mobilize both iron and phosphorus whereas the majority of the isolates from the bulk soil was unable, no matter the sampling soil horizon (Figure III.6C).

## Fungal isolates

Because the reproducibility along time of the quantitative effect of the reference isolate PML2(10) on the discolouration of the CAS and TCP media was low, only about 30 fungal isolates by horizon (the ones that were analysed in the same experiments where the effect of the isolate PML2(10) was not significantly different) were kept for statistical analyses. Because of the small number of isolates per compartment of origin, the χ2 analyses were not performed. Nevertheless, the impact of the origin compartment of the fungal isolate on iron and phosphorus mobilization was analysed by a one-factor (isolate origin compartment) ANOVA at the threshold level of P=0.05 and the Bonferroni-Dunn test. Whatever the sampling horizon, the mobilization of iron by the bulk soil isolates did not significantly differ from that of the soil-mycorrhiza interface isolates (P=0.3356 and 0.5698 for the upper and the lower horizons respectively) (Figure III.7B). Similarly, in the 0-3 cm horizon, the mobilization of phosphorus by the bulk isolates did not significantly differ (P=0.4442) from that of the soil-mycorrhiza interface. In contrast, in the case of the isolates from the 5-10 cm horizon, the mobilization of phosphorus by the soil-mycorrhiza interface isolates was significantly higher (P=0.0335) that of the bulk soil isolates (Figure III.7D).
* Effect of the origin depth of the microbial isolates

*Bacterial isolates*

In the case of the isolates from the bulk soil and the mycorrhizae, the mean diameters of the discolouration areas on the CAS medium did not significantly differ between the upper and the lower horizons (P=0.2642 for the bulk soil isolates and 0.3136 for the mycorrhiza ones). In contrast, there was a significant effect (P=0.0142) of the origin depth of the isolates in the case of the soil-mycorrhiza interface isolates (Figure III.7A). In the case of the isolates from the bulk soil, the mean diameters of the discolouration areas on the TCP medium did not significantly differ (P=0.7286) between the upper horizon and the lower horizon. In contrast, there was a significant effect (P=0.0186 and 0.0001) of the origin depth of the isolates in the case of the soil-mycorrhiza interface and the mycorrhiza isolates respectively (Figure III.7C).

*Fungal isolates*

Whatever the fungal isolate origin compartment, the mean diameters of the discolouration areas on the CAS and TCP media did not significantly differ between the upper horizon (P=0.0846 and 0.7602, respectively) and the lower horizon (P=0.1256 and 0.2445, respectively) (Figures III.7B and III.7D).

**Discussion**

The direct effect of the ectomycorrhizal symbiosis on tree nutrition is now well established: *via* their extramatrical mycelium, the ectomycorrhizal fungi colonize the soil surrounding the tree short roots and release the nutrients required for tree growth, in particular by weathering the primary minerals (Landeweert *et al.*, 2001 ; Schack-Kirchner *et al.*, 2000). In contrast, very few studies have assessed a possible indirect effect of the ectomycorrhizal symbiosis on tree nutrition, *via* its selective pressure on the microbial communities that colonize the ectomycorrhizosphere. In this study, we analysed the impact of the ectomycorrhizal symbiosis on the functional diversity of the soil bacterial and fungal communities in a forest stand, in relation to the mobilization of nutrients from soil minerals and tree nutrition. We focused on phosphorus and iron, which are central nutrients for plant growth but poorly available in natural soils (Barber, 1984).
We confirmed that the CAS and TCP *in vitro* tests are well adapted to the analysis of the functional diversity among a bacterial isolate collection, as previously shown on fluorescent *Pseudomonas* by Frey-Klett *et al.* (2005). We demonstrated that the effect of a reference bacterial isolate on the discolouration of the CAS and TCP media was reproducible along time, in independent experiments. Moreover, we observed a small variability (less than 5%) of the bacterial growth and the diameter of the discolouration area between the three replicates of the same bacterial inoculum. Because the two variables "diameters of the discolouration areas" and "efficacy of iron or phosphorus mobilization" were proved to be correlated, the CAS and TCP tests constitute an inexpensive and simple method for characterizing the functional diversity of bacterial isolates. Indeed, only visual observation of the bacterial growth and measurement of the diameter of the discolouration areas are required. In contrast, the two CAS and TCP tests must still be improved if a quantitative analysis of the functional diversity among a fungal isolate collection is to be pursued, as the growth of the fungal colonies and the diameters of the discolouration areas on the two media vary too much over time in independent experiments and between the three replicates for a same isolate in a same experiment. One hypothesis would be that the vitality of the fungal plugs used as inoculum was too variable, even if always sampled at the periphery of the fungal colonies.

It is well known that ectomycorrhizal fungi contribute to the phosphorus nutrition of their host plants (Read and Perez-Moreno, 2003), *via* their impact on minerals and organic matter (Landeweert *et al.*, 2001). Other soil bacterial and fungal microorganisms also solubilize and mineralize phosphorus from inorganic and organic pools, thus influencing the availability of phosphorus to plants (Souchie *et al.*, 2006; Vassilev *et al.*, 1996; Wakelin *et al.*, 2004). Our results revealed that in the soil of a forest stand, considering all sampling compartments together, the majority of the bacterial isolates (67%) and the totality of the fungal isolates from our microbial collections were able to mobilize phosphorus from inorganic phosphate. The phosphorus-mobilization capacity of fungi and bacteria is known to be related to the production of protons and organic acids (Cunningham and Kuiack, 1992; Illmer and Schinner, 1995). As the same mechanisms are involved in the microbial weathering of primary minerals (Welch *et al.*, 1999; Wenzel *et al.*, 1994), our results suggest that the presence of phosphate-solubilising microbial isolates in the soil of our experimental
forest stand could contribute to nutrient bioavailability and therefore to tree nutrition. This hypothesis has already been partly confirmed since we were able to demonstrate the impact of three bacterial isolates from this bacterial collection, on mineral weathering and tree seedling nutrition in controlled conditions (Calvaruso et al., 2006).

Due to the poor availability of iron in the soils and its essential role in many metabolic processes, certain plants and microorganisms have developed strategies for iron acquisition, e.g. synthetising and releasing siderophores, which are low molecular weight organic ligands with a strong affinity for iron (Hinsinger et al., 2001; Marschner and Römheld, 1994; Neilands, 1995). The CAS medium we used here was originally developed by Schwyn and Neilands (1987) to detect siderophore production by bacteria. In the studied soil, our results revealed that, when considering all sampling compartments together, 62% of the tested bacterial isolates and 96% of the fungal ones had the ability to mobilize iron from the CAS medium. Our results are consistent with those of Baakza et al. (2004) who isolated ten fungal species from both marine and terrestrial environments and observed that except a marine isolate from the *Aspergillus flavus* species, all isolates were able to mobilize iron *in vitro*. Because microbial siderophores have been demonstrated to be involved in the dissolution of common silicates (Liermann et al., 2000; Watteau and Berthelin, 1994), one could conclude from our results that many bacterial and fungal isolates, present in the forest upper and lower soil horizons we analysed, could behave as mineral weathering agents.

By comparing the intensities of iron and phosphorus mobilization by the fungal and bacterial strains isolated from ectomycorrhizae and bulk soil, we demonstrated that the oak-S. citrinum ectomycorrhizal symbiosis determines the functional structure of the culturable bacterial communities. These results obtained in 25 years-old stand confirm those obtained by Frey-Klett et al. (2005) in a nursery experiment, with nine month-old Douglas fir seedlings associated with another ectomycorrhizal fungus, *Laccaria bicolor*. These authors demonstrated that the Douglas fir-L. bicolor ectomycorrhizosphere selected *Pseudomonas fluorescens* isolates with higher iron-mobilization efficacy and phosphorus-mobilization ability in comparison to those of the bulk soil isolates. Our results therefore suggest that the so-called "mycorrhizosphere effect" observed on the functional diversity of the bacterial communities is quite general. It depends neither on the bacterial group, nor on the age and the species of the host tree, the nature of the
fungal symbiont, the environment (nursery vs forest). Interestingly, the majority of the bacterial isolates which were sampled in the soil-mycorrhiza interface and the mycorrhiza compartment were able to mobilize both iron and phosphorus. These results underline the fact that the bacterial isolates in the mycorrhizosphere possess a double competency in mobilizing nutrients and raise the question as to the mechanisms involved.

We did not observe a structuring effect of the oak-S. citrinum ectomycorrhizal symbiosis on the iron-mobilization ability of the fungal isolates, whatever the horizon. This observation could result from the small number of isolates kept for statistical analysis, taking into account the variability of the behaviour of the reference isolate we used in our experiments. The fact that the selective pressure of the ectomycorrhizal symbiosis on the fungal and bacterial communities was mainly detected in the lower horizon raises the question as to a causal relationship between the low organic matter content of this niche and the ability of the bacteria it contains to weather minerals. Such an impact of soil properties on the diversity of bacterial communities has often been reported, in as far as the bacterial genotypic and metabolic diversity is concerned (for a review see Garbeva et al., 2004). In contrast, the impact of the soil type on the functional diversity of microbial communities was so far poorly documented.

Some recent studies have demonstrated that the microbial biomass and the composition and activity of microbial communities are influenced by the soil horizons (Fierer et al., 2003; LaMontagne et al., 2003; Rosling et al., 2003). In the same experimental forest site where we sampled our fungal and bacterial collections, Lejon et al. (2005) observed the decrease of microbial biomass and the modifications of the genetic composition of fungal and bacterial communities with increasing soil depth. Similarly, Rogers and Tate (2001) and Goberna et al. (2005) showed a significant effect of soil depth on the metabolic diversity of microbial communities, as revealed by community level physiological profile (CLPP) analyses. As previously demonstrated by Krave et al. (2002), this significant stratification was related to the organic matter composition of the soil layers. In the present study, we compared the functional diversity of the culturable bacterial and fungal communities in two soil horizons differing by their organic matter content. Our results reveal for the first time a vertical stratification of the functional diversity of the culturable bacterial communities in relation to phosphorus and iron mobilization potential. Whether this stratification is linked to a change in the genetic composition of the bacterial communities related to the soil depth, as
demonstrated in other studies (Fierer *et al.*, 2003; LaMontagne *et al.*, 2003), remains to be clarified. In the same way, the mechanisms involved in this stratification, especially the impact of the mineral composition of the soil on the selection by the ectomycorrhizal symbiosis of bacterial associates with high iron and phosphorus mobilizing efficacy will require further researches.

In conclusion, our work demonstrates that the ectomycorrhizal symbiosis has a selective pressure on the functional diversity of the bacterial and non-mycorrhizal fungal communities as it selected bacteria and fungi potentially able to mobilize iron and/or phosphorus, and to consequently weather the minerals surrounding the mycorrhizal roots. Therefore we confirm once again, but this time in a natural nutrient-poor forest ecosystem, that the ectomycorrhizal symbiosis not only has a direct positive effect on plant growth *via* the nutritional competencies of its ectomycorrhizal fungal associate but also has an indirect positive effect *via* its selective pressure on ectomycorrhizosphere bacterial and fungal communities. Further researches will be needed to determine whether this mycorrhizosphere effect leads to 'physiological complementarity' between the ectomycorrhizosphere microbial communities and the ectomycorrhizal fungi, as previously stated by Koide and Kabir (2001), when studying the interactions between ectomycorrhizal and associated saprotrophic fungi. Moreover, we showed for the first time that the functional diversity of the bacterial communities varies with soil depth in the ectomycorrhizosphere only: the bacterial isolates which possess the highest efficacies for iron and phosphorus mobilization was concentrated in the ectomycorrhizosphere niche in the lower nutrient-poor horizon. This incites us to propose a new hypothesis to explain the sustainability of forest ecosystems on nutrient-poor soils: in way of their ectomycorrhizal fungal associates, tree roots would select mycorrhizosphere bacteria highly efficient for mobilizing the poorly-available nutrients required for tree growth.

## Acknowledgements

We acknowledge J. Garbaye for critical review of the manuscript and K. Bateman for review of the English language. F. Guinet, M.L. Clausse, B. Palin, P. Ray and B. Satrani for technical help, N. Amellal for her contribution to the sampling and isolation of the microbial collections. This work was supported by the French Research Ministry
(programme ACI Quantitative), by Andra (Agence nationale pour la gestion des déchets radioactifs) and the Lorraine Region.

## 3.2 Effet de la mycorhizosphère sur la diversité génotypique et métabolique des communautés bactériennes du sol en relation avec leur capacité à altérer un minéral-test *in vitro*

Le travail qui fait l'objet de cette deuxième partie avait deux objectifs : (i) comparer la capacité de souches bactériennes isolées dans le sol global et dans l'ectomycorhizosphère d'un arbre forestier à altérer un minéral-test, la biotite, et (ii) décrire et analyser la diversité génotypique et métabolique des souches bactériennes potentiellement impliquées dans le processus d'altération en forêt. Ce travail est le fruit d'une collaboration avec Stéphane Uroz, chercheur dans l'unité mixte de recherche INRA "Interactions Arbre - Microorganismes", qui a pris en charge la description génotypique et métabolique des souches bactériennes.
## Article : Mycorrhizosphere effect on the genotypic and metabolic diversity of the soil bacterial communities involved in mineral weathering in a forest soil.

Uroz, S.¹*, Calvaruso, C.², Turpault, M.P.², Pierrat, J.C.³, Mustin, C.⁴ and Frey-Klett, P.¹.

¹INRA, UMR 1136 "Interactions Arbres-Microorganismes", Centre INRA de Nancy, 54280 Champenoux.

²INRA, UR 1138 "Biogéochimie des Ecosystèmes Forestiers", Centre INRA de Nancy, 54280 Champenoux,

³INRA-ENGREF, Ecologie Forestière, 54042 Nancy.

⁴CNRS LIMOS, UHP Nancy 1, Faculté des Sciences, Domaine Victor Grignard, B.P.239, 54506 Vandoeuvre les Nancy, France.

Soumis pour publication à *Applied and Environmental Microbiology* le 02 octobre 2006.

## Résumé

Plusieurs espèces bactériennes ont déjà été décrites comme étant capables d'augmenter l'altération minérale et d'améliorer la nutrition et la croissance de la plante. Cependant, la possible relation entre le potentiel d'altération minérale, l'identité taxonomique et la capacité métabolique des souches bactériennes n'a jamais été étudiée jusqu'à présent. Ici, nous avons caractérisé une collection de 61 souches bactériennes isolées à partir de l'ectomycorhizosphère et d'ectomycorhizes de chêne - *Scleroderma citrinum* et du sol global. La capacité des souches bactériennes à altérer un minéral-test, la biotite, a été évaluée à l'aide d'un nouveau biotest en microplaques qui mesure le pH et la quantité de fer libéré en solution à partir du minéral. Nous avons révélé que les bactéries altérantes étaient significativement plus fréquentes au voisinage de *S. citrinum* que dans le sol global. De plus, l'efficacité d'altération des isolats bactériens de l'ectomycorhizosphère était significativement plus élevée que celle des isolats du sol global. Tous les isolats bactériens ont été identifiés après analyse de séquences partielles du gène *rrs*, comme appartenant aux genres *Burkholderia*, *Collimonas*, *Pseudomonas* et *Sphingomonas*, et ont été caractérisés pour leur métabolisme du carbone par la méthode BIOLOG®. Les isolats les plus efficaces dans l'altération de la biotite appartiennent aux deux genres *Burkholderia* et *Collimonas*. Des analyses multivariées ont mené à la
définition de trois groupes métaboliques. L'un d'entre eux rassemble principalement les isolats bactériens associés à *S. citrinum*, qui présentent un haut potentiel d'altération minérale. Il se compose d'isolats consommant un nombre limité de substrats carbonés, notamment le tréhalose, un carbohydrate accumulé dans le mycélium de *S. citrinum*. Nos résultats supportent donc l'hypothèse que par l'intermédiaire de son métabolisme carboné, le champignon sélectionnerait dans le réservoir sol, des communautés bactériennes à fort potentiel d'altération. Ils posent la question de l'existence d'une complémentarité fonctionnelle entre le champignon et les bactéries associées au sein du complexe ectomycorhizien, pour l'amélioration de la nutrition de l'arbre.

**Mots-clés** : Altération minérale, *Burkholderia*, *Collimonas*, effet mycorhizosphère, sol forestier.

Abstract

To date, several bacterial species have been described as capable to promote mineral weathering and to improve plant nutrition and growth. However, the possible relationships between mineral weathering potential, taxonomic identity and metabolic ability have not been investigated so far. Here we characterized a collection of 61 bacterial strains isolated from *Scleroderma citrinum* mycorrhizae, mycorrhizosphere and adjacent bulk soil in a oak forest stand. The bacterial ability to weather biotite was assessed with a new microplate bioassay that measures the pH and the quantity of iron released from the mineral. We showed that weathering bacteria were more frequent in the vicinity of *S. citrinum* than in the bulk soil. Moreover the weathering efficacy of the mycorrhizosphere bacterial isolates was significantly higher to the one of the bulk soil isolates. All the bacterial isolates were identified by partial *rrs* gene sequence analysis, as members of the *Burkholderia*, *Collimonas*, *Pseudomonas* and *Sphingomonas* genera, and characterized for their carbon metabolism by the BIOLOG® method. The most weathering efficient isolates belonged to the *Burkholderia* and *Collimonas* genera. Multivariate analysis led to the definition of three metabolic groups, one of these mainly gathered bacterial isolates associated to *S. citrinum*, which harboured high mineral weathering potential. Our results therefore support the hypothesis that by way of its carbon metabolism, the fungus select in the bulk soil reservoir a bacterial community with high weathering ability, and address the question of functional complementation
between mycorrhizal fungi and bacteria within the ectomycorrhizal complex, for the promotion of tree nutrition.

**Key-words:** *Burkholderia*, *Collimonas*, forest soil, mineral weathering, mycorrhizosphere effect.

**Introduction**

Minerals constitute a reservoir of nutrients in the soils. Their physical, chemical and biological weathering play a major role in forest ecosystems with no fertilization or amendment by humans because it releases numerous nutrients required for tree growth (phosphorus, potassium, magnesium, calcium, iron). Evidences are presently accumulating which indicate that mineral weathering by soil microorganisms affects ion cycling and plant nutrition (Calvaruso et al., 2006; Toro et al., 1997; Wallander, 2000). However to date, little is known about the taxonomic and metabolic diversity of the soil microorganisms involved in this process.

The ability to solubilize poorly soluble calcium phosphates such as hydroxyapatite or to weather silicates was described for a range of bacterial genera belonging to the α-proteobacteria (*Agrobacterium*, *Rhizobium*), the β-proteobacteria (*Achromobacter*, *Burkholderia*), the γ-proteobacteria (*Acidithiobacillus*, *Aerobacter*, *Citrobacter*, *Enterobacter*, *Erwinia*, *Pseudomonas*), and the Gram positive group (*Bacillus*, *Micrococcus*) (Puente et al., 2004) as well as for a range of mycorrhizal fungal genera (*Pisolithus*, *Paxillus* or *Suillus* ; Altomare et al., 1999, Paris et al., 1996) and non-mycorrhizal ones (*Aspergillus*, *Penicillium* ; Reddy et al., 2002; Wakelin et al., 2004). Phosphate-solubilizing bacteria were isolated from various environments such as eutrophic lake (Gen-Fu and Xue-Ping, 2005), mangrove tree roots (Vasquez et al., 2000), cactus rhizoplane (Puente et al., 2004), and tree rhizospheres (Leyval and Berthelin., 1991 ; Illmer and Schinner, 1995). However, only a limited number of studies have taken into account all together the taxonomic identity, the niche of origin and the mineral solubilization efficacy of a collection of bacterial isolates (Frey-Klett et al., 2005; Kuklinsky-Sobral et al., 2004).

When studying the genotypic and functional diversity of fluorescent pseudomonads in a forest nursery soil, Frey et al. (1997) and Frey-Klett et al. (2005) demonstrated that the Douglas fir-*Laccaria bicolor* ectomycorrhizal symbiosis genotypically and functionally structures the fluorescent pseudomonads communities. This would result from the
biochemical impact of the symbiosis on the surrounding soil, so-called mycorrhizosphere (Rambelli, 1973; Barea *et al.*, 2002; Johansson *et al.*, 2004; de Boer *et al.*, 2005). Because the mycorrhizosphere selects strains able to solubilize inorganic phosphate and very efficient for iron mobilization, in comparison to the fluorescent pseudomonads communities from the bulk soil, Frey-Klett *et al.* (2005) suggested that the symbiosis has an indirect effect on plant nutrition via its selective pressure on bacterial communities. To confirm this hypothesis, further researches are needed, notably concerning the impact of the mycorrhizosphere on the structure of the genotypic and metabolic diversity of the soil bacterial communities involved in mineral weathering, which has not been documented so far.

This is the aim of the present study. For this purpose, we selected 61 bacterial strains among 140 strains isolated from bulk soil, soil-mycorrhiza interface (mycorrhizosphere) and oak-*Scleroderma citrinum* symbiotic mantle, in a 25-year-old oak stand. We genotypically characterised them by amplifying and sequencing a portion of their 16S rDNA gene. We also determined their metabolic potential by the Biolog® method and their mineral weathering potential in a microplate assay we developed (Mustin *et al.*, in preparation). Data were then statistically analysed in order to determine if there was a relationship between the weathering ability of the bacterial isolates, their genotypic identity and/or their metabolic potential, and their compartment of origin.

## Material and methods

* Collection of bacterial strains

A collection of 140 bacterial strains was performed from oak (*Quercus petraea*)-*Scleroderma citrinum* ectomycorrhizas and bulk soil, sampled in the mineral soil horizon (5-10 cm deep) in an experimental forest site located at Breuil, in central France (Calvaruso *et al.*, 2006, Calvaruso *et al.*, submitted). The bacterial isolates were sampled in three compartments: the bulk soil (PN), the soil-mycorrhiza interface *ie* mycorrhizosphere (PML) and the symbiotic mantle (PMB). For the present study, 61 isolates were subsampled at random among the three compartments in order to be representative of the whole collection of 140 isolates: 21 PN isolates, 20 PML isolates and 20 PMB isolates. Two reference strains of *Collimonas fungivorans* named Ter6 and Ter331 (kindly provided by W. de Boer, Netherlands Institute of Ecology, Heteren), were also used during this study. All the bacterial isolates used in this study were
cryopreserved in -80°C in 20% glycerol and then cultivated on 1/10-strength tryptic soy agar (TSA) medium (3 g.L⁻¹ Tryptic Soy Broth from Difco and 15 g.L⁻¹ agar) and incubated at 25°C.

* Phenotypic and metabolic fingerprinting of the isolates.

Bacterial strains were examined for their colony morphology. Gram determination was performed using the aminopeptidase test from Sigma. Cell morphology and motility were observed with a BX41 Olympus microscope at x 1000 magnification and in phase interference contrast, on unstained living bacteria. The sole carbon utilization of the isolates was analysed with Biolog GN2 (Gram negative) microplates according to the manufacturer's instructions. The formazan accumulation in the bacterial cells was measured as the optical density at 590 nm by an automatic microplate reader (Bio-rad Model 550).

The quantitative data of carbon source utilization by each isolate were compared by multivariate analysis. Principal-component analysis was used to visualize the relationships between the isolates using the Splus software. All the variables being in the same unit, we analyzed the variance-covariance matrix instead of the correlation matrix, to better evidence distances between the variables. Classification of the isolates into homogeneous metabolic groups was achieved using the K-means method. Firstly, initial groups are formed and centroids are calculated as barycenters of the clusters. Secondly, the algorithm assigns each observation of the group with the closest centroid and new centroids are calculated. These steps are repeated until the centroids no longer move (McQueen, 1967).

* Mineral weathering potential of the isolates

Bacterial isolates were cultivated overnight in liquid LBm medium (Vaudequin-Dransart, et al., 1995). One hundred mL of each culture were washed (v/v) three times with sterile ultra-pure water. Optical density (A<sub>600nm</sub>) of the resulting suspensions was then adjusted ca. 0.8-1. Twenty µL of each suspension was transferred into a well of a sterile Multiscreen microplate (MAGVN22, 0.22 µm pore size, Millipore) containing 10 mg of biotite particles (size 200-500µM) and 180 µL of Bushnell-Hass medium (BHm: KCl, 20 mg.L⁻¹; MgSO₄·7H₂O, 150 mg.L⁻¹; NaH₂PO₄·2H₂O, 80 mg.L⁻¹; Na₂HPO₄·2H₂O, 90 mg.L⁻¹; (NH₄)₂SO₄, 65 mg.L⁻¹; KNO₃, 100 mg.L⁻¹ and CaCl₂, 20 mg.L⁻¹).
mg.L⁻¹), buffered at pH 6.5 and supplemented with glucose or mannitol (2 g.L⁻¹). The biotite was from Bancroft (Canada), a 2:1 phyllosilicate, frequently present in acid soil, which weathers relatively quickly and holds K, Mg and Fe nutrient elements. It is a pure homogeneous mineral and its composition is in g.kg⁻¹: SiO₂, 410.1 ; Al₂O₃, 109 ; Fe₂O₃, 22.1 ; FeO, 100.5 ; MnO, 2.7 ; MgO, 189 ; Na₂O, 4.1 ; K₂O, 94.6 ; TiO₂, 22.8 ; F, 44.2 and Zn, 0.8. Its structural formula is (Si₃Al₁) (Fe³⁺₀.₁₂ Fe²⁺₀.₆₁ Mg₂.₀₆ Mn₀.₀₂ Ti₀.₁₃) and K₀.₈₈ Na₀.₀₆ O₁₀ (OH₀.₉₈ F₁.₀₂). Biotite and culture media were sterilized by autoclaving (20 min. at 120°C). Glucose and mannitol were added after autoclaving. Multiscreen microplates containing the 10 mg biotite particles were sterilized by UV treatment during 30 min before filling with sterile liquid media.

For each bacterial strain, 8 wells were filled with the same inoculum (4 wells for pH determination and 4 wells for iron measurement). In each experiment, 8 wells were used as a control without bacteria: they received 20 µL of BHm medium only. Two bacterial strains named PN3(3) and PML1(4) were used as negative and positive controls, respectively, in each experiment.

The Multiscreen microplates were incubated at 25°C for 48H under constant shaking (orbital, 350 rounds per minute). After incubation, the total liquid content of each well was filtered at 0.22 µM by centrifugation using MultiScreen microplates, to discard bacterial cells and biotite particules. To assess biotite weathering, filtrates were recovered in a new microplate containing 20 µL of ferrospectral® (Merck, for iron quantification) or bromocresol (1 g.L⁻¹, Sigma, for pH determination). The pH and the amount of total iron (Fe²⁺ and Fe³⁺) released from biotite in the solution were both established by measuring the optical density at 595 nm with a Bio-rad model 550 microplate reader. Calibration curves were built to determine the relationship between the optical density and the amount of protons or of iron released, respectively for the bromocresol green and the ferrospectral dyes. Average value of the four replicates for iron quantification and for pH measurements were used to determine the weathering potential of each isolate.

In order to investigate which mechanisms could explain the weathering ability of the bacterial isolates of the collection, the same microplate weathering assay was performed with serial dilutions of citric acid (10⁻³ M, pH adjusted from 6 to 2) and hydrochloric acid (concentration adjusted to be from pH 6 to 2), in the absence of bacteria. Citric acid was used as a model of chelating molecule and hydrochloric acid as model of acidifying molecule. Experiments were performed in triplicate in the same conditions as described
above. The data obtained were used to draw two reference curves corresponding to the complexation and acidification reactions that occur during the weathering process.

* Molecular fingerprinting of the isolates.

Rrs gene amplification was performed using the universal set of primers pA (5'-AGAGTTTGATCCTGGCTCAG-3') and pH (5'-AAGGAGGTGATCCAGCCGCA-3'), which allowed the amplification of almost the entire gene (Bruce *et al.*, 1992). Polymerase chain reactions were performed in a total reaction volume of 50 µL containing 1x PCR Mastermix (Eppendorf ®), 0.1µM primers and 1µL cell extract. The following temperature cycle was used: an initial denaturation step of 5 min at 95°C followed by 30 cycles of 1 min denaturation at 95°C, 1 min annealing at 56°C and 1 min 30 s extension at 72°C, and a final extension step of 10 min at 72°C. PCR products were purified and concentrated using the Qiaquick gel extraction kit (Qiagen). Sequencing reaction was accomplished using the multicapillary BECKMAN CEQ8000 system. The sequencing primer used was 518r (5'ATTACCGCGGATGCTGG-3') (Reiter *et al.*, 2002). The sequences were compared with those in the GenBank databases (www.ncbi.nlm.nih.gov.blast), using the Blast programme (Altschul *et al.*, 1997).

* Phylogenetic analysis.

The partial 16S rRNA gene sequences from the bacterial isolates were aligned with published 16S rRNA gene sequences from α-, β- and γ-proteobacteria by use of Clustal X, version 1.8. Phylogenetic algorithms and tree design (DNA-DIST, NEIGHBOR, and SEQBOOT) were operated using the PHYLIP 3.65 package, written by J. Felsenstein (1993). Bootstrap analysis was based on 1,000 replicates.

* Statistical analysis

The effects of the compartment of origin and of the sugar source (glucose or mannitol) on the weathering ability of the bacterial isolates were determined using analysis of variance (ANOVA) at the threshold level of *P* = 0.05, and the Bonferroni-Dunn test. The Superanova software was used for all these analysis. Correlation between pH value and the amount of released iron was analysed using a linear regression analysis at the threshold level of *P* = 0.05 with the Statview software.
Figure III.8: Relationship between the mean values of iron released from the biotite (up on the graph), the mean values of pH (down on the graph) and the three compartments of origin of the bacterial isolates: Bulk soil (PN), white, mycorrhizosphere (PML), grey and mycorrhiza (PMB), black. Bars correspond to standard deviation.

<figure><img src="image_36.png" /></figure>

Figure III.9: Projection of the 61 bacterial isolates in function of their individual biotite weathering potential. Each plot represents a bacterial isolate from one of the three following compartments of origin: bulk soil (PN, grey rhombus), mycorrhizosphere (PML, white square) and mycorrhiza (PMB, black triangle). The two reference strains Collimonas fungivorans Ter6 and Ter331 are represented by white circles. Each plot is the mean value of four replicates. The weathering efficacies of PML and PMB isolates were significantly higher than the ones of PN isolates, according to a one-factor ANOVA (compartment of origin, P=0.0001) and the Bonferroni-Dunn test, performed on the iron concentrations in the culture filtrates. The two curves correspond to the weathering effect of a complexing agent (citric acid, top curve) and a strong acid (hydrochloric acid, bottom curve). The relative distribution of the bacterial spots with respect to these two curves suggests mechanisms for the weathering effect of the bacterial isolates.

<figure><img src="image_37.png" /></figure>
## Results

* Relationship between mineral weathering potential and origin of the bacterial isolates

A microplate assay was developed to quantify the weathering potential of bacterial isolates, by measuring the amount of iron and protons released in solution from the biotite. The weathering efficacies of PML and PMB isolates were significantly higher than the ones of PN isolates, according to a one-factor ANOVA (compartment of origin, *P* = 0.0001) and the Bonferroni-Dunn test, performed on the iron concentrations in the culture filtrates (Figures III.8 and III.9). The distribution of the 61 bacterial isolates according to their zone of origin and their weathering efficacy following their belonging to three weathering efficacy classes (< 0.1 ; < 0.8 and > 0.8 mg.L⁻¹ of iron released) highlights that the frequency of the most weathering efficient isolates was higher in the sampling compartments associated to mycorrhizosphere than in the bulk soil compartment (Figure III.10).

* Relationship between mineral weathering potential and weathering mechanisms

Most of the bacterial isolates able to release high amounts of iron from the biotite had also the ability to produce a high quantity of protons: (Figure III.9). This tendency was confirmed by a linear regression analysis between the amount of iron released from the biotite and the log value of the pH (Y=14.88+236.26X, R²=0.75, *P* = 0.0001). To illustrate, the majority of the isolates able to release ca. 1 mg.L⁻¹ of iron acidified the solution to pH values between 3.64 to 3.09 with the exception of the isolate PMB4(3) (pH = 5.07). Among the 61 isolates tested, PMB3(1) was the most efficient one; it released ca. 2.9 mg.L⁻¹ of iron and acidified the solution to pH 3.09. The majority of the bacterial spots, represented in figure III.9, was distributed along the reference curve of acidification.

* Relationship between taxonomy and origin of the bacterial isolates

The analysis of the nucleotide sequence of an amplified portion (ca. 400 b) of the *rrs* gene of each isolate revealed that the 61 isolates belong to the three α-, β- and γ-proteobacteria lineages only. All of the isolates analysed showed strong homology (95
Figure III.10: Distribution of the 61 bacterial isolates according to their zone of origin and their biotite weathering efficacy in three weathering classes (in mg.l-1 of iron released): < 0.1 (in white), < 0.8 (in grey) and > 0.8 (in black).

<figure><img src="image_38.png" /></figure>
<figure><img src="image_39.png" /><figcaption>Figure III.11: Phylogenetic tree constructed with the neighbor-joining method showing the relationship between the 61 bacterial isolates of the present study and reference strains from the literature, based on PCR-sequencing of a portion of the 16S rRNA gene (pA-pH primers). A bootstrap analysis was performed with 1000 repetitions, and only values above 50 are shown. According to the bootstrap analysis, six clusters named A, B, C, D, E and F were defined. The bacterial isolates from this study are named according to their compartment of origin as follow: bulk soil (PN), mycorrhizosphere (PML) and symbiotic mantle (PMB).</figcaption></figure>
to 100%) with cultivated and uncultivated environmental bacteria from the four genera *Burkholderia*, *Collimonas*, *Pseudomonas* and *Sphingomonas*.

According to the phylogenetic analysis, the bacterial isolates were clustered into six groups (Figure III.11). Eighty-four percent of the 61 bacterial isolates (cluster C, D and E) belonged to the *Burkholderia* genus. Clusters C and D gathered isolates coming from all the three sampling compartments. On the contrary, cluster E gathered only isolates coming from the mycorrhizosphere and the symbiotic mantle, showing 97 to 100% sequence homology with the *Burkholderia glathei* species. In the same way, cluster B gathered only isolates from the mycorrhizosphere and the symbiotic mantle, that belonged to the *Collimonas*. The isolates PML3(4), PML3(7), PML3(8) and PMB3(1) showed 99% sequence homology with the *rrs* gene of the *Collimonas fungivorans* species. Clusters A and F gathered only one *Sphingomonas* strain (PML4(2)) from the mycorrhizosphere and two *Pseudomonas* strains (PN1(10) and PN2(8)) from the bulk soil, respectively.

* Relationship between taxonomy, mineral weathering efficacy and origin of the bacterial isolates

Among the different bacterial genera that were identified, the *Collimonas* genus, that was isolated in association with the mycorrhizas only, gather the bacterial isolates that were significantly the most active for weathering the biotite (1.49 mg.L⁻¹ ± 0.3 of iron released in average), according to a one-factor (genus identity) ANOVA (*P* = 0.0001) and the Bonferroni-Dunn test. In contrast, the only *Sphingomonas* isolate harboured a low efficacy value (0.94 mg.L⁻¹ of iron released). Concerning the 51 *Burkholderia* isolates, the ones from the soil-mycorrhiza interface (PML ; 0.83 mg.L⁻¹ ± 0.15 of iron released) and from the symbiotic mantle (PMB ; 0.59 mg.L⁻¹ ± 0.14 of iron released) were significantly more efficient for biotite weathering than the PN isolates (0.31 mg.L⁻¹ ± 0.08 of iron released), according to a one-factor ANOVA (*P* = 0.0001) and the Bonferroni-Dunn test.

* Impact of the carbon substrate on the weathering efficacy

The potentials of mineral weathering discussed above were measured with glucose as the sole carbon substrate in the microplate assay. As sugar utilization varies among the soil bacteria and could influence directly or indirectly their weathering ability, mannitol
Figure III.12: Influence of the carbon source on the biotite weathering potential. The weathering potential of 20 bacterial isolates from bulk soil (PN, grey rhombus), 16 isolates from mycorrhizosphere (PML, white square) and 17 isolates from symbiotic mantle (PMB, black triangle), was assayed with either mannitol or glucose as sole carbon source. Each plot is a mean value of four replicates and represents the amount of iron (mg.L⁻¹) released from the biotite. For legibility reasons standard errors were not presented and a straight line corresponding to equivalent amount of iron released (ratio 1:1) from biotite in presence of glucose or mannitol was added. Whatever the origin of the isolates, the weathering efficacies of the isolates were significantly higher in presence of glucose than in the presence of mannitol and could be classified as follow: PML > PMB > PN according to a two-factor ANOVA (P = 0.0001) and the Bonferroni-Dunn test. Whatever the carbon source provided to the isolates, the mycorrhizosphere isolates (PML) were significantly more efficient than the bulk soil isolates (PN), according to a one-factor ANOVA (P = 0.0431) and the Bonferroni-Dunn test.

<figure><img src="image_40.png" /></figure>
was also tested for 20 isolates from PN, 16 from PML and 17 from PMB (Figure III.12). This sugar, metabolised by ca 94% of the tested bacterial isolates according to BIOLOG analysis, is frequently detected in fungal exudates. In the presence of mannitol as a sole carbon source, the weathering efficacy of the PN isolates was significantly lower than the ones of the PML and PMB isolates. In contrast, the weathering efficacies of the PML and PMB isolates were not significantly different, according to a one-factor ANOVA (P = 0.0431) and the Bonferroni-Dunn test. Whatever the compartment of origin of the isolates, the weathering efficacies were significantly higher when the bacteria were incubated in the presence of glucose as a sole carbon source than in the presence of mannitol, according to a two-factor (carbon source, origin of the isolates) ANOVA (P = 0.0001) and the Bonferroni-Dunn test, with the exception of isolate PMB2(9) which was more efficient in the presence of mannitol (0.265 ± 0.018 mg.L⁻¹ of iron released) than in the presence of glucose (0.058 ± 0.32 mg.L⁻¹ of iron released). Conversely, four isolates from the symbiotic mantle (PMB1(9), PMB1(11), PMB3(12) and PMB4(5)), one isolate from the mycorrhizosphere (PML1(23)) and two isolates from the bulk soil (PN2(10), PN4(7)) were able to weather the biotite in the presence of glucose only. Finally, whatever the carbon source provided to the bacteria, the weathering efficacy of the PML strains was significantly higher than the one of the PMB strains, that was also significantly higher than the one of the PN strains, according to the same two factor ANOVA (P = 0.0001).

* Multivariate analyses of the metabolic fingerprints.

As carbon metabolism may influence mineral weathering, the 61 isolates were characterized by the BIOLOG method in order to determine if some metabolic traits can be related to the weathering efficacy of the bacterial isolates. Microplates GN2 were used as the aminopeptidase test stated that all the bacterial isolates were gram negative. The quantitative data of the carbon source utilization (95 data per isolate) were subjected to multivariate analysis, to cluster the isolates sharing the same metabolic traits and to determine the characteristic traits of each cluster. Principal-component analysis was used to visualize the relationships between bacterial isolates. As several bacterial isolates harboured high sequence homology with the *Collimonas* genus, two reference strains named Ter6 and Ter331 were added to this study. According to this analysis, the projection of the 61 isolates and of the two reference strains on the plane
Figure III.13: Projection of the 61 isolates on the plane defined by the first two principal components. The classification of the isolates into three homogeneous metabolic groups named A, B and C was achieved using the K-means method. Compartments of origin of the bacterial isolates are presented as follows: bulk soil (PN, grey rhombus), mycorrhizosphere (PML, white square) and symbiotic mantle (PMB, black triangle). The two reference Collimonas strains Ter6 and Ter331 are presented in grey cross.

<figure><img src="image_41.png" /></figure>

Figure III.14: Component principal analysis of the metabolic profiles of the 61 bacterial isolates. Projection of the 95 carbon substrates from the Biolog GN microplates on the plane defined by the first two principal components. For legibility reasons, only the most characteristic substrates of groups A, B and C are identified (aminoacids (grey square), sugars (black rhombus) and carboxylic acids (grey rhombus)). The positions of the other substrates are indicated by white rhombus.

<figure><img src="image_42.png" /></figure>
defined by the first two principal components, which account for 42 and 10%, respectively, of the total variance, reveals that most of the bacterial isolates (ca. 86%) from the bulk soil (PN) gathered below the F1 axis, whereas 64% of the isolates from the mycorrhiza (PML+PMB) gathered above (Figure III.13). Using the K-means method, the data set was classified into 3 clusters named A, B and C (number fixed a priori) (Figure III.13). Isolates from the mycorrhizosphere and from the symbiotic mantle were equally distributed in the three clusters contrary to the isolates from the bulk soil that were concentrated in cluster A (ca. 33%) and C (62%) only.

The analysis of the metabolic data showed that the F1 axis was positively correlated with all the substrates except the trehalose, the L-glycyl-L-glutamic acid and the L-alanyl-glycine. This analysis allowed also to classify the most discriminating substrates for each cluster (Figure III.14). Bacterial isolates from cluster A seem to use a limited number of substrates contrary to isolates from clusters B and C. Trehalose, L-glycyl-L-glutamic acid and L-alanyl-glycine appeared as the preferentially consumed substrates of cluster A. Isolates from cluster B seemed to preferentially use carbohydrates, contrary to isolates from cluster C which seemed to prefer amino acids. The ten most characteristic substrates for cluster B, which gather isolates from the mycorrhizosphere and the symbiotic mantle only, were D-saccharic acid, galacturonic acid, D-glucosaminic acid, D-glucuronic acid, D-mannitol, D-arabitol, D-sorbitol, α-D-glucose, L-aspartic acid, bromosuccinic acid and for group C were quinic acid, β-hydroxybutyric acid, L-glutamic acid, N-acetyl-D-glucosamine, D-L-succinic acid, lactic acid, D-fructose, formic acid, L-phenylalanine, D-alanine (in decreasing order of importance).

## Discussion

Soil colonization by microorganisms depends both on their ability to have access and to metabolise the organic and inorganic nutrients present in the soil, and to interact with other microorganisms, especially root-associated ones like mycorrhizal fungi. In the rhizosphere of forest trees, ectomycorrhizal fungi, microfungi and bacteria physically interact and form multitrophic ectomycorrhizal complexes that contribute to tree nutrition (Frey-Klett *et al.*, 2005). So far, very few studies have addressed the question of the functional diversity among the different microbial components of these complexes in relation to mineral weathering, a central process for gross production and nutrient cycling. In this context, our objective was to focus on the analysis of the
diversity of the bacterial communities associated to ectomycorrhizae, in a nutrient-poor forest ecosystem. In our study, we observed that the bacterial isolates from the bulk soil were significantly less efficient for biotite weathering than the isolates from the mycorrhizosphere and the symbiotic mantle. Moreover, the frequency of isolates with the highest weathering efficacy was significantly higher in the sampling compartments associated to mycorrhizae, than in the bulk soil. Therefore, our results confirm once again that ectomycorrhizosphere structures the functional diversity of cultivable bacterial communities, as previously demonstrated by Frey *et al.* (1997) and Calvaruso *et al.* (submitted) using the two CAS and TCP *in vitro* tests which relate to iron and phosphorus mobilization. Because very little is known about the possible relationship between genotype, metabolism and weathering potential in soil bacterial communities, the 61 bacterial strains we isolated were also characterized by PCR-sequencing of a portion of the *rrs* gene and the BIOLOG® method.

Our 61 bacterial isolates belong to the *Burkholderia*, *Collimonas*, *Pseudomonas* and *Sphingomonas* genera, the *Burkholderia* genus being the most frequent one. Strains closely related to known species of the *Burkholderia* and *Pseudomonas* genera have already been described to be the dominant cultivated bacteria associated with ectomycorrhizal fungi (Duponnois *et al.*, 1991; Poole *et al.*, 2001; Bending *et al.*, 2002). *Collimonas* bacterial strains have been previously characterized as growing at the expense of living fungi (de Boer *et al.*, 2004). To our knowledge, this is the first time that bacterial strains belonging to the *Collimonas* and *Sphingomonas* genera are described for their weathering ability. In our study, the six *Collimonas* strains we isolated were among the most efficient ones, like two other *Collimonas* strains, named Ter6 and Ter331 (de Boer *et al.*, 2004), that were assayed in this work as a reference. Interestingly, these strains were only isolated from the vicinity of the *S. citrinum* mycorrhizae and according to the BIOLOG method, they proved to use very few carbon sources only, especially trehalose which is a fungus disaccaharide accumulated in *S. citrinum* mycelium (data not shown). This strongly suggests that our *Collimonas* isolates are fungus-dependant. In the *Scleroderma*-oak ectomycorrhizosphere, the *Collimonas* isolates could use trehalose exudated by the fungus or, because of their chitin-degrading ability (de Boer *et al.*, 2005) they could hydrolyse the hyphae for releasing it. De Boer *et al.* (2001) suggested that *Collimonas* strains could use their chitinolytic ability more for cohabiting with fungal hyphae than for predation. Therefore, we could here make the hypothesis of a reciprocal beneficial relationship in
which *Scleroderma* favours *Collimonas* by releasing trehalose and *Collimonas* stimulates *Scleroderma* growth by releasing inorganic nutrients from the soil minerals.

According to the phylogenetic analyse of the *rrs* gene, our 61 bacterial isolates were clustered in six groups. Among them, the *Burkholderia* isolates which represented the more frequent genus, were clustered in three groups corresponding to variable weathering efficacies. The isolates from the two first groups C and D came from all the sampling compartments and presented a mean weathering efficacy respectively of 0.17 mg.L⁻¹ and 0.39 mg.L⁻¹. On the contrary, the isolates from group E harboured a significantly higher weathering efficacy of 1.13 mg.L⁻¹. These isolates presented high sequence homology with the *B. glathei* species and came from the mycorrhizosphere and the symbiotic mantle. Interestingly, one of these strains named PML1(12) was recently described as being able to efficiently solubilize iron and inorganic phosphate *in vitro*, to significantly increase biotite weathering and pine growth (Calvaruso *et al.*, 2006). If various *Burkholderia* strains have already been described for their ability to solubilize phosphate (Lin *et al.*, 2006; Vassilev *et al.*, 2006), this is to our knowledge only the second report for the *B. glathei* species (Kim *et al.*, 2005).

Multivariate analysis of the metabolic fingerprints confirmed that bacterial isolates belonging to the same genera gathered together. The F2 axis allowed to distinguish the bulk soil isolates from the isolates of the mycorrhizosphere and the symbiotic mantle. Interestingly, the isolates from the bulk soil were present in only two (groups A and C) of the three groups that were identified with the multivariate analysis. The third group (group B) exclusively gathered strains from the mycorrhizosphere and the symbiotic mantle. Such a regrouping of isolates from a mycorrhizosphere origin, according to BIOLOG data, was also observed in the mycorrhizosphere of Douglas fir-*Laccaria bicolor* in a forest nursery soil (Frey *et al.*, 1997). The BIOLOG fingerprints were used to identify carbon-substrates preferentially metabolised by each group of isolates. As already reported by Frey *et al.* (1997), bacterial isolates from bulk soil preferentially used aminoacids and carboxylic acids. Among the 61 bacterial isolates tested, the quinic acid which was preferentially used by isolates from the mycorrhizosphere and the symbiotic mantle, was the most discriminating substrate. It is an abundant plant product. Another discriminating substrate that characterized these isolates was the N-acetyl-D-glucosamine, which was also previously described as a metabolite preferentially used by bacterial isolates from the Douglas fir-*Laccaria bicolor* mycorrhizosphere (Frey *et al.*, 1997). N-acetyl-D-glucosamine could either result from the release of plant
metabolites or from the chitin hydrolysis of the *S. citrinum* cell wall. Interestingly, bacterial isolates from group A, mainly from mycorrhizosphere and symbiotic mantle origin, were able to use a restricted number of carbon substrates, like trehalose, L-glycyl-L-glutamic acid and L-alanyl-glycine. A proportion of 23% of the 61 bacterial isolates were trehalose-users: only two isolates from the bulk soil and 13 isolates from the vicinity of *S. citrinum*. So, the high proportion of trehalose-users associated with *S. citrinum* suggests that this symbiont, which accumulates trehalose, could exert a trehalose-mediated selection on bacterial communities as previously shown by Frey *et al.* (1997) on fluorescent *Pseudomonas* communities. Concerning the question of the mechanisms of the selective pressure of the symbiosis on the bacterial communities, it was recently demonstrated that ectomycorrhizae can exert a direct effect on the genetic diversity of soil bacterial communities which is not mediated by their promoting effect of the host roots (Assigbetse *et al.*, 2005).

To address the question of a possible relationship between the carbon metabolism of the 61 bacterial strains and their mineral weathering efficacy, we calculated the mean weathering efficacy corresponding to each BIOLOG group. Groups A and B, which mainly contained isolates from the vicinity of the symbiosis, harboured the highest weathering efficacies. This result illustrates in a different manner the fact that ectomycorrhizosphere selects bacterial isolates with a high weathering potential. Because the weathering ability of the isolates was assessed *in vitro* in the presence of glucose as sole carbon source, one should wonder if the weathering efficacies we measured and the glucose utilization by the isolates were correlated. This was not the case (data not shown). Nevertheless, the carbon source we have used in our weathering bioassay could have influenced our results. Indeed it has been demonstrated that carbon source determines the activity of various phosphate-solubilizing bacteria (Nautiyal *et al.*, 2000). Some bacterial strains were proved to be more active for phosphate-solubilization when using xylose, and others when using lactose or mannitol.

As the aim of this study was to compare the weathering efficacy of the 61 isolates from different origins and to assess the impact of the ectomycorrhizosphere on the structure of the bacterial community, only two substrates were tested in the weathering bioassay: glucose and mannitol. These sugars were chosen because i) glucose utilization is a classical bacterial trait and ii) mannitol is a common sugar found in the fungal exudates (Martin *et al.*, 1984, 1985, 1998 ; Rangel-Castro *et al.*, 2000). Interestingly, whereas most of the isolates where able to use mannitol whatever their compartment of origin,
only one isolate of *Burkholderia*, sampled in the ectomycorrhizosphere, was significantly more efficient for biotite weathering in the presence of mannitol rather than in the presence of glucose. Variations of the phosphate-solubilizing ability were observed when ammonium or different sources of nitrates were used as nitrogen source (Nautiyal *et al.*, 2000). Here, to avoid this problem, the medium used for the weathering assay was amended with ammonium and nitrate in a ratio 1:1. Many other parameters were also proved to influence the phosphate-solubilizing ability of bacteria, like low levels of calcium, pH and temperature (Nautiyal *et al.*, 2000). Consequently, these results suggest a link between the bacterial metabolism, the soil conditions and the phosphate-solubilizing ability of the soil bacteria. But a fundamental question remains, as suggested by Bennet *et al*. (2001): "Does the enhanced weathering of mineral by microorganisms offer an advantage to that population, or is it simply a coincidental by-products of metabolism?". The authors showed that in low phosphate environment, even P-containing feldspars were weathered rapidly and thus suggested that silicate weathering is sometimes driven by the nutrient requirements of the microbial consortium.

Microorganisms are known to influence the formation or the dissolution of soil minerals by different mechanisms, playing a central role in soil fertility (Ehrlich, 1996; Banfield *et al.*, 1999). Two main mechanisms are suspected to be involved in mineral weathering in aerobic conditions: acidification and complexation. One hypothesis is that these mechanisms are linked to the production of geochemically reactive by-products of the bacterial metabolism. Indeed acidification is generally linked to the production of microbial organic acids and protons, and complexation to the production of microbial chelating molecules like siderophores or organic acids. In our study, the comparison of the weathering efficacies of the 61 bacterial isolates to the reference curves of acidification and complexation, suggests that both mechanisms were involved. Most of the bacterial isolates that effectively weathered the biotite harboured the ability to strongly acidify the culture media. This acidification suggested the production of organic acids from glucose metabolism. Several strains of *Burkholderia*, *Pseudomonas* and *Serratia* were described as being able to alter the dissolution of phosphate by producing gluconic acid (Rodriguez *et al.*, 2000; Krishnaraj and Goldstein, 2001; Kim *et al.*, 2005; Lin *et al.*, 2006; ). This acid results from glucose oxidation by the quinoprotein dehydrogenase (Babu-Khan *et al.*, 1995). Kim *et al*. (2005) demonstrated that a strain of *B. glathei*, able to produce this acid, was more active to solubilize
phosphate than a simple addition of gluconic acid. These authors suggested that the organic acid could chelate $Ca^{2+}$ during $PO_4^{3-}$ solubilization. However very recently, Lin *et al.* (2006) suggested that the protons associated with the gluconic acid would in fact explain the phosphate solubilisation. Interestingly Goldstein *et al.* (1999) previously demonstrated that the production of gluconic acid by a phosphate-solubilizing bacterial isolate could be induced by a root extract from a plant growing in a phosphate-limited desert environment. The weathering effect of oxalic acid and other acids was also previously described, but in studies involving fungi (Vassilev *et al.*, 1996; Gadd, 1999; Wallander, 2000). In our work, the identification of a bacterial strain more efficient for biotite weathering in the presence of mannitol than in the presence of glucose, and without any significant impact on medium acidification (pH value maintained ca. 5), suggested the involvement of another weathering mechanism, as chelation. Such a mechanism is suspected to be also involved in the case of two other bacterial strains PN3(12) and PMB4(3) that were able to release ca. 0.9 $mg.L^{-1}$ of iron with an associated pH value of respectively 5.29 and 5.07. The role of bacterial chelators or siderophores in the dissolution of mineral has already been assessed (Liermann *et al.*, 2000; Kalinowski *et al.*, 2000b). These authors presumed that the catechol derivative produced by a strain of *Streptomyces* sp. could influence hornblende dissolution. All these observations strongly suggest that mineral weathering by microorganisms is a complex equilibrium between several mechanisms such as acidification and complexation.

Mycorrhizal fungi are well known plant growth promoters (Finlay, 2004). This characteristic results from the ability of these fungi to efficiently remove organic and inorganic nutrients from soil organic matter and minerals (Blum *et al.*, 2002; Landeweert *et al.*, 2001). In nature, mycorrhizal fungi live in close association with bacterial communities, forming multitrophic complexes. Here, we demonstrated that the weathering potential of the bacterial isolates from the vicinity of *Scleroderma* was higher than the one of the isolates from the surrounding bulk soil. Therefore one should suspect that some of the functional activities like mineral weathering, that were classically attributed to ectomycorrhizal fungi, could result at least partially, from the activity of the associated exo or even endo-bacterial communities (Bertaux *et al.*, 2005), that would have been selected by the ectomycorrhizal symbiosis. In this context, one should also address the question of possible functional complementation between the ectomycorrhizal fungi and the associated bacterial communities, which would improve
the weathering efficacy of the ectomycorrhizal complexes. Indeed such cooperation between microfungi and their rhizobial associates was recently described in the context of rock phosphate solubilization (Jayasinghearachchi and Seneviratne, 2006). To confirm our hypotheses, one major bottleneck to overcome in the future will be to develop tools for assessing weathering potential at the microbial community level in addition to the strain level approach we presented here.

## Acknowledgements

This work was made possible by grants from ACI Ecologie quantitative "Quantification de l'altération biologique des minéraux des sols" and from Région Lorraine to M.P. Turpault, P. Frey-Klett and S. Uroz. All of these are gratefully acknowledged.

The authors thank Dr. J. Garbaye for critical reviewing of the manuscript, Dr. F. Martin for helpful discussions and C. Delaruelle, B. Palin, M.L. Clausse for technical assistance. We thank Dr. W. de Boer for kindly providing *Collimonas* strains.

## 3.3 Effet de différentes essences forestières sur la structuration fonctionnelle des communautés bactériennes du sol en relation avec leur capacité à altérer un minéral-test *in vitro*.

Afin de vérifier la stabilité dans le temps de la structuration fonctionnelle des communautés bactériennes telluriques et ectomycorhizosphériques, nous avons réalisé une seconde collection de souches bactériennes dans le peuplement de chêne du site de Breuil trois années après l'isolement de la première collection. A cette occasion, nous avons décidé de réaliser deux autres collections de souches bactériennes sous des essences d'arbres connues pour avoir des nutritions azotées très distinctes sur ce site, le hêtre et l'épicéa. Cette étude devait nous permettre de comparer l'effet des essences forestières sur la structuration fonctionnelle des microorganismes dans le sol.
**Article :** Forest trees select high mineral weathering bacteria to balance low nutrient availability

Calvaruso C.¹, ², Turpault M.P.¹, Uroz S.², Leclerc-Cessac E.³, Ranger J.¹, Garbaye J.², Frey-Klett P.²

¹ INRA, UR 1138 "Biogéochimie des Ecosystèmes Forestiers", Centre INRA de Nancy, 54280 Champenoux, France.

² INRA, UMR 1136 "Interactions Arbres-Microorganismes", Centre INRA de Nancy, 54280 Champenoux, France.

³ ANDRA, Direction Scientifique/Service Transferts, 92298 Châtenay-Malabry, France.
*En préparation pour letters to Nature*

## Synthèse

Les forêts tempérées sont principalement développées sur des sols non fertilisés et fréquemment sur des sols acides où la disponibilité en nutriments minéraux est le principal facteur limitant de croissance des arbres (Badeau *et al.*, 1999). Dans le contexte de développement durable des écosystèmes forestiers, une hypothèse proposée par Richards (1987) serait que les racines d'arbres sélectionneraient les communautés bactériennes du sol impliquées dans des processus biogéochimiques centraux, comme l'altération minérale (Barker and Banfield, 1998; Welch *et al.* 2002; Calvaruso *et al.*, 2006) et la minéralisation de la matière organique (Chen *et al.*, 2003; Petsch *et al.*, 2004), contribuant ainsi à la mobilisation des nutriments et à la croissance des arbres. Cependant, l'impact des essences d'arbres sur la diversité fonctionnelle des communautés bactériennes en relation avec le processus d'altération de la phase minérale est inconnu. Dans ce travail, nous montrons dans un écosystème forestier naturel soumis à des substitutions d'essences d'arbres, que le chêne (*Quercus sessiliflora* Smith) et le hêtre (*Fagus sylvatica* L.) n'utilisent pas la même stratégie que l'épicéa (*Picea abies* Karst.) pour acquérir les nutriments à partir des minéraux du sol. Dans un peuplement forestier âgé de 28 ans, nous avons montré que le chêne et le hêtre, dont les pH de la rhizosphère et du sol global sont très proches (|ΔpH (rhizosphère - sol global)| ~ 0), sélectionnent autour de leurs racines mycorhizées des souches bactériennes très efficaces dans l'altération de la phase minérale *in vitro*. Cet effet structurant n'existe pas dans la rhizosphère de l'épicéa, dont le pH rhizosphérique est
non seulement significativement plus faible comparativement à celui du chêne et du hêtre, mais aussi nettement plus faible que le pH du sol global ($|ΔpH (rhizosphère - sol global)| \sim 0.3$). Cette acidification plus marquée dans la rhizosphère de l'épicéa s'explique principalement par une libération massive de protons par les racines afin de compenser l'absorption d'ions $NH_4^+$ (Turpault *et al.*, en préparation). En effet, contrairement au chêne et au hêtre, la nitrification est très faible dans les sols sous épicéa sur le site de Breuil (Zeller *et al.*, en préparation), impliquant que la source majeure d'azote disponible pour les racines d'épicéa est la forme $NH_4^+$ (Colin Belgrand *et al.*, 2003). Parce que la production de protons est un des mécanismes majeurs responsables de l'altération minérale, nos résultats démontrent que certaines essences d'arbres ont développé des stratégies indirectes pour altérer le minéral dans des sols pauvres en nutriments, qui repose sur la sélection de communautés bactériennes possédant un fort potentiel d'altération. Dans le contexte actuel de changement climatique qui pourrait mener à un changement dans la distribution des essences d'arbres à travers l'Europe, nos résultats suggèrent que les essences d'arbres auront la capacité de s'adapter à leur nouvel environnement microbien et de sélectionner parmi les souches indigènes, celles qui amélioreront leur nutrition et par conséquent, contribueront à la durabilité des écosystèmes forestiers dans les prochaines décennies.

## Synthesis

Temperate forests are mainly developed on non fertilized and nutrient-poor soils where the mineral nutrient availability is a tree growth limiting factor (Badeau *et al.*, 1999). A hypothesis for sustainable forest development proposes that tree roots select soil bacterial communities involved in central biogeochemical processes that contribute to nutrient mobilization and tree growth (Richards, 1987), such as mineral weathering (Barker and Banfield, 1998; Welch *et al.* 2002; Calvaruso *et al.*, 2006) and organic matter mineralization (Chen *et al.*, 2003; Petsch *et al.*, 2004). However, the impact of tree species on the functional diversity of soil bacterial communities in relation to mineral weathering is unknown. Here we show in a natural forest ecosystem submitted to tree species substitution that oak (*Quercus sessiliflora* Smith) and beech (*Fagus sylvatica* L.) species do not use the same strategy than Norway spruce (*Picea abies* Karst.) species for accessing to nutrients from soil minerals. In a 28-year-old forest stand, we found that oak and beech species, which are characterized by a very little
difference between the rhizosphere and the bulk soil pH (|ΔpH (rhizosphere - bulk soil) | ~ 0) in the 5 to 10 cm soil horizon, select around their mycorrhizal roots soil bacterial strains very efficient for mineral weathering in vitro. This structuring effect does not exist in the rhizosphere of Norway spruce, which is characterized by a significantly stronger acidification of the rhizosphere in comparison with the oak and the beach, and an important difference between the rhizosphere and the bulk soil pH (|ΔpH (rhizosphere - bulk soil) | ~ 0.3) in the 5 to 10 cm soil horizon, due to a massive release of protons by the roots to balance $NH_4^+$ absorption (Turpault *et al.*, in preparation). In fact, contrary to oak and beech, the nitrification process is very weak in the soil under Norway spruce in the site of Breuil (Zeller *et al.* in preparation), implicating that the principal available nitrogen source in the soil for the Norway spruce roots is the $NH_4^+$ form (Colin Belgrand *et al.*, 2003). Because the production of proton is one of the main mechanisms responsible for mineral weathering, our results demonstrate that certain tree species have developed indirect strategies for mineral weathering in nutrient-poor soils, which lie on the selection of bacterial communities with high weathering potential. In today's context of global climate change which will give rise to a shift in the distribution of forest tree species across Europe, our result suggest that the migrating tree species will have the ability to adapt to their new microbial environment and to select among the indigenous soil microbial communities the ones that will improve their nutrition and therefore contribute to the durability of the temperate forest ecosystems in the next decades.

## Results and discussion

We analysed the structuration of soil culturable bacterial communities in relation with the mineral weathering process *in situ* at the Breuil-Chenue experimental forest site (Morvan, France) where we had at our disposal a 28-year-old experimental design that mimics a natural forest ecosystem submitted to tree species substitution (Ranger *et al.*, 2004). We isolated 627 bacterial strains in an organo-mineral soil horizon and under three different tree species, namely oak (*Quercus sessiliflora* Smith), beech (*Fagus sylvatica* L.) and Norway spruce (*Picea abies* Karst.). Bacterial strains were sampled in two soil compartments: the bulk soil which corresponds to the soil not influenced by tree root activity and the *Scleroderma citrinum* ectomycorrhizosphere (Calvaruso *et al.*, submitted). *Scleroderma citrinum* is a widely distributed ectomycorrhizal fungus in our
Figure III.15: Iron concentration (A) issue from biotite weathering and protons (B) generated by bacterial activity in function of their compartment of origin under oak, beech and Norway spruce. In white, bacterial strains isolated from the bulk soil and in grey, bacterial strains isolated from the mycorrhizosphere. Bars represent standard deviations. Each value is the mean value of 18 to 20 replicates. Treatments associated with the same letter are not significantly different according to a two-factor (compartment of the bacterial strains and tree species) ANOVA (P = 0.05) and the Bonferroni-Dunn test.

<figure><img src="image_43.png" /></figure>

Figure III.16: Projection of the bacterial isolates in function of their individual biotite weathering potential. Each plot represents a bacterial isolate from one of the two following compartments of origin: bulk soil (PN ; white), mycorrhizosphere (PML ; grey) and the three tree species: oak (circle), beech (square) and Norway spruce (triangle). The two curves correspond to the weathering effect of a complexing agent (citric acid, top curve) and a strong acid (hydrochloric acid, bottom curve).

<figure><img src="image_44.png" /></figure>
experimental forest site, which is not very efficient for mineral weathering *in vitro* (data not shown). By the way of an *in vitro* microplate assay that we developed for the screening of bacterial collections according to their mineral weathering potential (Uroz *et al.*, submitted), we evaluated the ability of a representative sub-sample of 166 bacterial isolates to decrease the pH, a mechanism potentially involved in the mineral weathering processes, and we quantified their mineral weathering efficacy by measuring the quantity of iron mobilized from a mineral-test, the biotite (Mustin *et al.*, submitted).

We observed that the majority of the tested isolates from the bacterial collections (>65%) were able to decrease pH (up to 3.5 pH units) and to mobilize iron (up to 2 mg.l⁻¹) by weathering the biotite. These results confirm in a forest environment a common knowledge: soil bacteria produce acids and complexing compounds that can weather minerals and mobilize nutrients (Ullman *et al.*, 1996; Kalinowski *et al.*, 2000a; Liermann *et al.*, 2000a et b), thus contributing to nutrient cycling and ecosystem functioning. So far, few studies have shown that plants, by the way of their root exudates, alter the structure and the activity of microbial communities and selectively favour those which are beneficial to them (Cook *et al.*, 1995; Dakora and Phillips, 2002). Here we observed that the bacterial strains isolated from the ectomycorrhizosphere of oak or beech-*S. citrinum* produce more acid compounds and mobilize more iron from the biotite than the strains from the bulk soil (Figures III.15 and III.16), suggesting that these ectomycorrhizal symbioses select bacterial strains potentially able to weather the minerals surrounding the roots and thus indirectly improve tree nutrition. This hypothesis has already been partly confirmed since we were able to demonstrate that a *Burkholderia glathei* isolate from the oak-*S. citrinum* ectomycorrhizosphere, sampled in our experimental forest site in 2001, significantly weathers biotite and improves pine seedling nutrition and growth in controlled conditions (Calvaruso *et al.*, 2006). The structuring effect of the *S. citrinum* ectomycorrhizosphere on bacterial communities is in accordance with the results from previous studies which compared the functional diversity of bacterial communities isolated from bulk soil and oak-*S. citrinum* ectomycorrhizosphere in the same experimental forest site and at the same season, but four years before (Calvaruso *et al.* submitted and Uroz *et al.* submitted). Therefore we demonstrate for the first time that the functional structure of the culturable soil bacterial community in relation to the mineral weathering process is stable along time. So far, the few studies that have
Figure III.17: Iron (A) and proton (B) concentrations in the solution, resulting from biotite weathering, and generated by bacterial activity in function of their compartment of origin and their sampling time: 2001 (void) and 2004 (hatched). Bars represent standard deviations. Each value is the mean of 20 to 27 replicates. For each soil compartment, treatments associated with the same letter are not significantly different according to a one-factor (tree species) ANOVA (P = 0.05) and the Bonferroni-Dunn test.

<figure><img src="image_45.png" /></figure>

Table III.4: Bulk and rhizosphere soil pH in the 3-10 cm horizon under oak and Norway spruce in the site of Breuil. Each value is the mean of 18 to 20 replicates. In each line, values with different letters (a or b) are significantly different according to a one-factor (tree species) ANOVA (P = 0.05) and the Bonferroni-Dunn test. In each column, values with asterisks are significantly different according to a one-factor (compartment of origin of the isolates) ANOVA (P = 0.05) and the Bonferroni-Dunn test.

<table><thead><tr><th rowspan="2">Compartment of origin</th><th colspan="3">Tree species</th></tr><tr><th>Oak</th><th>Beech</th><th>Norway spruce</th></tr></thead><tbody><tr><td>Bulk soil</td><td>4.3 <sup>a</sup></td><td>4.5 <sup>a</sup></td><td>4.3 <sup>a</sup></td></tr><tr><td>Rhizosphere</td><td>4.3 <sup>a</sup></td><td>4.5 <sup>a</sup></td><td>4.0 <sup>b*</sup></td></tr></tbody></table>
addressed the question of the year-to-year variation of the functional diversity of bacterial communities have concluded to a high temporal variation of the soil bacterial diversity (Berardesco et al., 1998; Grayston et al., 2001). One should therefore wonder why these data contrast with our results (Figure III.17). To explain the temporal stability of the structuring effect of the symbiosis on the functional diversity of bacterial communities in relation to mineral weathering we propose the hypothesis that this effect is a functional requirement, which is strongly linked to the strategy that certain tree species have developed for improving their mineral nutrition. This hypothesis is supported by previous results of Frey-Klett et al. (2005). These authors demonstrated in a different context (9 months vs 28 years old trees, Douglas fir vs oak, *Laccaria bicolor* vs *S. citrinum*, culturable *Pseudomonas fluorescens* populations vs total culturable bacterial communities, nursery vs forest stand) that the Douglas fir-*Laccaria bicolor* ectomycorrhizal symbiosis also selects *Pseudomonas fluorescens* isolates suspected to be beneficial to the nutrition of Douglas fir seedlings due to their high phosphorus and iron mobilizing abilities. Because distinct differences among tree species in the microbial community structure have been reported (Priha et al., 2001; Grayston and Prescott, 2005), we compare the functional diversity among the bacterial collections we sampled in the beech, oak and Norway spruce stands. Whatever the tree species, the abilities of the bacterial strains from the bulk soil compartment to release protons and to mobilize iron were not significantly different (Figure III.15). In contrast the quantity of protons and iron released by the bacterial strains from the oak and beech-*S. citrinum* ectomycorrhizospheres were significantly higher than the ones released by the Norway spruce ectomycorrhizosphere isolates, which were themselves not significantly different from those released by the bulk soil isolates (Figure III.15). Our result therefore demonstrate for the first time that the structuring effect of the ectomycorrhizal symbiosis on the functional diversity of bacterial communities in relation to mineral weathering depends on the tree species.

Bulk and rhizosphere soils were sampled 20 times under the three tree species to measure the pH (water). In the 5-10 cm horizon, the difference between the pH values in the rhizosphere and the pH values in the bulk soil were not significant in the oak and the beech stands (ΔpH ~ 0) (Table III.4). In contrast, this difference was significant in the Norway spruce stand (ΔpH ~ 0.3) in which the tree rhizosphere is significantly more acid than the oak and the beach rhizosphere (Table III.4). According to Turpault et al.
(in preparation) who did their investigations in the same experimental forest site as us, the lower pH in the Norway spruce rhizosphere would result from the presence of large amounts of ammonium (NH<sub>4</sub><sup>+</sup>) in the soil under this species. Indeed, when absorbing ammonium, the Norway spruce roots exudate large amounts of protons to maintain charge equilibrium (Marschner et al., 1991). In the beech and oak rhizosphere in contrast, higher pH would result from the presence of important amounts of nitrate (Zeller et al., in preparation). Our results therefore highligh the fact that tree species differently affects the physicochemical characteristics of the rhizosphere soil.

Because the mycorrhizal symbiosis alters the biological and physicochemical properties of the surrounding soil and thus modify the microbial equilibrium around the mycorrhizal roots, Linderman (1988) proposed the concept of “mycorrhizosphere effect” to qualify the structuring effect of the mycorrhizal symbiosis on microbial communities. Here we demonstrated for the first time that this mycorrhizosphere effect depends on the tree species. To explain the mycorrhizosphere effect they observed around Douglas fir and oak roots, Frey et al. (1997) and Uroz et al. (submitted) suggested that the two ectomycorrhizal fungi *L. bicolor* and *S. citrinum*, which both accumulate trehalose -a common fungal carbohydrate rarely detected in plants-, exert a trehalose-mediated selection on the functional diversity of bacterial communities. One should wonder why such a structuring effect which exists in the oak and beech ectomycorrhizosphere, does not exists in contrast in the Norway spruce ectomycorrhizosphere. We hypothesize that, because of its nitrogen nutrition based on NH<sub>4</sub><sup>+</sup> uptake, Norway spruce releases large amounts of protons in its rhizosphere which is sufficient to mobilize nutrients from soil minerals and sustain tree nutrition. This hypothesis is supported by the results obtained of Augusto *et al.* (2000) and Calvaruso *et al.* (in preparation) in the same site of Breuil, who showed that due to a reduced pH, the dissolution of a feldspar and the clay fraction, respectively, is significantly higher under Norway spruce than under oak. The availability of the nutrients from soil minerals is expected to be lower in the oak and beech stands than in the spruce one because of the soil pH. In this context, our result demonstrates that oak and beech trees use another strategy than Norway spruce trees to balance this low nutrient availability. They direct the structure of the functional bacterial communities and select bacterial strains with high mineral weathering potential. Interestingly, it was previously shown *in situ* in a beech forest that the weathering of soil minerals is significantly higher in the
rhizosphere than in the bulk soil in the poor-nutrient soil only and not in the fertilized one (Turpault *et al.*, submitted). One should thus ask if the structuring effect of the beech ectomycorrhizosphere on the functional diversity of the bacterial communities still exists in fertilized soils where the functional contribution of the bacterial communities is no more a requirement.

## Conclusion

Our work illustrates the hypothesis that tree influences on soil processes are mediated through their effects on soil microorganisms, and that conversely, soil microbial communities are influenced by tree roots (Grayston and Prescott, 2005). However it highlights the fact that this relationships strongly depends on the tree species as far as mineral weathering is concerned. In the current context of tree species migration due to global climate change, the fact that tree species are able to modify the physicochemical and biological properties of their surrounding soil in function of their nutritional needs incite us to be quite confident concerning the sustainability of temperate forest ecosystems in the next decades.

## Acknowledgements

The authors thank C. Delaruelle, B. Palin, M.L. Clausse and G. Nourrisson for technical assistance.

This work was supported by the Andra (agence nationale de gestion des déchets radioactifs), the French Research Ministry (ACI quantitative ecology project) and the region "Lorraine".

# 4. SYNTHESE ET DISCUSSION

Méthodologiquement, les tests *in vitro* en boîtes de Petri et en microplaques se sont avérés très performants pour la caractérisation de souches bactériennes cultivables en relation avec la mobilisation d'éléments minéraux. Ces tests sont fiables, reproductibles dans le temps et permettent une bonne discrimination des souches bactériennes. En
<figure><img src="image_46.png" /><figcaption>Figure III.18: Schéma de synthèse représentant la structuration des communautés bactériennes isolées du sol du site de forestier de Breuil sous les différentes essences d'arbres testées.</figcaption></figure>
revanche, ces tests sont peu adaptés à la caractérisation des communautés fongiques :
les données de croissance et de mobilisation d'éléments par un même isolat fongique
sont très variables au cours du temps, probablement du fait d'une grande variabilité de
vitalité des implants. L'utilisation d'inoculum liquide contenant une concentration de
spores calibrée pourrait être une alternative intéressante. Cependant, elle ne permettra
de caractériser que les espèces de champignons sporulantes. Contrairement aux tests en
boîtes de Petri qui n'offrent qu'une appréciation indirecte du potentiel d'altération des
isolats, basée sur la mesure du potentiel de mobilisation du fer et du phosphore à partir
de milieux synthétiques, le test en microplaques permet d'apprécier l'effet direct des
isolats sur l'altération d'un minéral-test *via* la quantité de fer mobilisée. De plus, ce test
nous permet d'aborder la question des mécanismes mis en œuvre *via* l'étude de la
relation entre la quantité de fer en solution et le pH de la solution. Toutefois, ces tests,
nécessitant l'isolement de communautés bactériennes et fongiques à partir d'un sol
forestier ne permettent de tester que les souches microbiennes cultivables qui ne
constituent qu'une faible proportion des communautés réellement présentes dans les
sols forestiers et qui n'est, par conséquent peut-être pas représentative de l'ensemble des
communautés (Torsvik *et al*., 1998 ; Anderson et Cairney, 2004). C'est pourquoi, des
tests globaux en microplaques sont en cours de mise au point par Stéphane Uroz
(INRA-Nancy, UMR 1136). Ils permettraient de tester la capacité d'altération d'un
minéral-test par l'ensemble des communautés microbiennes présentes dans un
compartiment du sol. Un des verrous techniques réside dans la préparation, à partir d'un
échantillon de sol, d'un inoculum microbien non pollué chimiquement par des
particules minérales ou des composés organiques.

Grâce à ces tests, nous avons clairement démontré, une structuration fonctionnelle des
communautés bactériennes cultivables du sol en relation avec la fonction altération de la
phase minérale : les isolats présentant le plus fort potentiel d'altération des minéraux
colonisent l'ectomycorhizosphère de *Scleroderma citrinum* sous chêne et sous hêtre
(Figure III.18). Ces souches bactériennes pourraient améliorer la nutrition des plantes et
ainsi agir comme des bactéries promotrices de la croissance des arbres forestiers en
améliorant la disponibilité des nutriments dans le sol. En revanche, aucune structuration
des communautés bactériennes n'a pu être mise en évidence sous épicéa (Figure III.18),
démontrant un effet de l'essence forestière sur la structuration des communautés
bactériennes dans le sol. Les analyses physico-chimiques des sols déjà présentées dans
le second chapitre, apportent un élément de réponse pouvant expliquer ces différences
<figure><img src="image_47.png" /><figcaption>Figure III.19: Schéma de synthèse représentant les différences de comportement des racines ectomycorhizées d'arbres forestiers dans la mobilisation des éléments par altération de la phase minérale.</figcaption></figure>
entre essences. En effet, l'épicéa acidifie plus le sol rhizosphérique (ΔpH (pH sol global – pH rhizosphère ~ 0.3) que ne le font les essences feuillues (ΔpH (pH sol global – pH rhizosphère) ~ 0) dans cet horizon de sol (5-10 cm). Cette acidification plus marquée dans la rhizosphère de l'épicéa est corrélée à une forte production de protons par ses racines afin de compenser (équilibre de charges) l'absorption massive d'azote sous forme d'ions $NH_4^+$ (cf. article intitulé "Forest trees select high mineral weathering bacteria to balance low nutrient availability"). Cette différence de structuration des communautés bactériennes observée entre deux essences feuillues (chêne et hêtre) et l'épicéa pourrait être tout simplement due aux conditions physico-chimiques différentes régnant dans le sol sous ces essences. En effet, les microorganismes du sol sont très sensibles à ces conditions et notamment au pH (Marschner et al., 2004 ; Pommerening-Röser et Koops, 2005). Une autre hypothèse serait que ce sont les racines d'arbres elles-mêmes qui contrôleraient la structuration des communautés bactériennes dans les sols forestiers (Brant et al., 2006), favorisant par ce biais la sélection et la croissance de souches bactériennes qui leur sont favorables, en étant notamment capables de compenser une faible biodisponibilité en éléments minéraux dans le sol. Ainsi, les racines de chêne et de hêtre et leurs champignons symbiotiques associés, produisant moins d'agents altérants acides que celles d'épicéa, sélectionneraient, par l'intermédiaire de leurs exsudats, des communautés bactériennes aptes à mobiliser les éléments minéraux.

Si nos hypothèses sont exactes, nous pouvons proposer deux mécanismes de mobilisation des nutriments minéraux très différents en fonction de l'essence (Figure III.19):

- pour l'épicéa : une exsudation massive de protons, en réponse à une absorption majoritaire d'azote sous forme d'ions $NH_4^+$, qui altèrent le minéral et mobilisent ainsi une importante quantité de nutriments. Cette hypothèse est en accord avec les résultats obtenus sur le site forestier de Breuil et présentés dans le chapitre précédent. Ils confirment également les résultats des travaux de Berger et al. (2006) qui ont montré que la forte mobilisation de calcium et de cations basiques par les racines d'épicéa est principalement due à une acidification résultant du fonctionnement de cette essence.

- pour le chêne et le hêtre : une exsudation de composés sélectionnant certaines souches bactériennes à fort potentiel d'altération.

Les biotests que nous avons mis au point *in vitro* en boîtes de Petri et en microplaques, même s'ils sont simples et rapides, ne permettent de caractériser uniquement le potentiel
d'altération de souches bactériennes et fongiques purifiées. L'efficacité réelle de ces souches *in situ* en présence de racines d'arbres et d'une microflore indigène reste encore à vérifier. C'est pourquoi, afin de prendre en considération les interactions rhizosphériques et pouvoir mesurer l'effet respectif de chacun des partenaires biologiques dans le processus d'altération des minéraux, nous avons mis en place des expérimentations plus complexes, en colonnes. Les résultats que nous avons obtenus font l'objet du chapitre suivant.
## CHAPITRE IV:

### QUANTIFICATION DE LA PART RESPECTIVE
DES DIFFERENTS PARTENAIRES BIOLOGIQUES
DE LA RHIZOSPHERE DANS LE PROCESSUS

### D'ALTERATION DES MINERAUX :

#### EXPERIENCES EN COLONNES
<figure><img src="image_48.png" /><figcaption>Figure IV.1: Photo du dispositif expérimental en colonnes.</figcaption></figure>
# 1. CONTEXTE ET OBJECTIFS

De nombreux travaux ont montré que les systèmes racinaires d'une part et les microorganismes du sol d'autre part, étaient capables d'altérer les minéraux silicatés (Leyval et Berthelin, 1991 ; Hinsinger et al., 1992 ; Paris et al., 1995 ; Welch et al., 1999). Cette altération peut résulter d'un fractionnement physique lié à la croissance des racines, mais elle est surtout associée à l'exsudation de métabolites aux propriétés altérantes par les racines et les microorganismes (Robert et Berthelin, 1986 ; Drever, 1994 ; Hinsinger and Gilkes, 1997 ; Jones, 1998 ; Ström, 1997). Si les mécanismes impliqués ont été déjà beaucoup étudiés, on connaît bien moins la part respective de chacun des partenaires biologiques de la rhizosphère dans les processus d'altération des minéraux et de mobilisation des éléments dans les sols car il est très difficile de découpler les effets inhérents à chacun d'entre eux. De plus, dans les écosystèmes naturels, les flux d'énergie et de matière entrants et sortants sont très variables au cours du temps et sont de ce fait difficiles à estimer (Kolka et al., 1996). Par ailleurs, malgré le rôle essentiel de la rhizosphère dans l'altération, jusqu'à présent très peu de travaux ont eu pour but de caractériser l'impact de facteurs environnementaux sur le processus d'altération des minéraux par les arbres (Berner et Berner, 2006).

C'est pourquoi, ce chapitre a pour objectifs de quantifier la part respective des différents partenaires biologiques de la rhizosphère (racines, bactéries, champignons saprophyte et ectomycorhizien) dans le processus d'altération de la phase minérale et de mesurer l'impact de composés toxiques (uranium et cadmium) sur la croissance, l'activité biologique et le processus d'altération des minéraux.

# 2. DISPOSITIF EXPERIMENTAL EN COLONNES

Après une première étape de caractérisation du potentiel d'altération de souches bactériennes et fongiques du sol via des tests simples et rapides in vitro (cf. chapitre III), nous avons mis en place trois expérimentations plus complexes, en colonnes (Figure IV.1).
<table><caption>Tableau IV.1 : Paramètres des différentes expériences d'interactions réalisées à l'aide du dispositif expérimental en colonnes.</caption><thead><tr><th>Paramètres de l'expérience</th><th>Expérience 1*</th><th>Expérience 2</th><th>Expérience 3</th></tr></thead><tbody><tr><td>Durée</td><td>21 jours</td><td>76 jours: expérience<br>"solution nutritive de base"<br>102 jours: expérience<br>"solution nutritive enrichie en éléments"</td><td>56 jours</td></tr><tr><td>Conditions de culture</td><td>18°C - 25°C,<br>humidité: 60 - 70%, 17 heures de jour, éclairage<br>fort:20000 Lux</td><td>18°C - 25°C,<br>humidité: 60 - 70%, 17 heures de jour, éclairage<br>fort:20000 Lux</td><td>18°C - 25°C,<br>humidité: 60 - 70%, 17 heures de jour, éclairage<br>fort:20000 Lux</td></tr><tr><td>Plante</td><td></td><td>Semis de pin</td><td>Semis de pin</td></tr><tr><td>Bactéries</td><td>Burkholderia glathei<br>PML1(12)<br>Burkholderia glathei<br>PMB4(3)</td><td></td><td>Pseudomonas fluorescens<br>BBc6R8</td></tr><tr><td>Champignon ectomycorrhizien</td><td></td><td></td><td>Laccaria bicolor S238N</td></tr><tr><td>Champignons</td><td>PML2(10)<br>SN2(3)</td><td></td><td></td></tr><tr><td>Minéral</td><td>Biotite</td><td>Biotite</td><td>Biotite</td></tr><tr><td>Solution nutritive</td><td>De base<br>Enrichie en glucose</td><td>De base<br>Enrichie en nutriments</td><td>De base<br>Enrichie en uranium<br>Enrichie en cadmium</td></tr></tbody></table>

* Expérience 1 = "Interactions microorganismes - minéral - solution"

Expérience 2 = "Interactions arbre forestier - bactéries - minéral - solution"

Expérience 3 = "Interactions arbre forestier - champignon ectomycorrhizien - bactéries - minéral - solution"
• Le matériel expérimental

Le tableau IV.1 résume les différentes expériences d'interactions mises en place dans les mêmes conditions de culture en chambre de croissance, et utilisant le même matériel végétal et minéral ainsi que la même solution nutritive. Ce matériel a été choisi sur deux critères : (i) être présents dans les écosystèmes forestiers et (ii) être adaptés à ce type d'expérimentation en colonnes.

- Le pin sylvestre, *Pinus sylvestris* (provenance : forêt de Haguenau) a l'avantage d'avoir une croissance relativement rapide, ce qui convient à des expériences de courte durée comme les nôtres. Les semis de pins ont été cultivés pendant six semaines en serre dans un substrat non stérile tourbe - vermiculite (1:1) dans les conditions suivantes : 60% d'humidité, des températures de 15°C la nuit et 22°C le jour, une période d'éclairage de 16h. Les racines des semis ont ensuite été minutieusement nettoyées au pinceau dans quatre bains successifs d'eau ultra-pure stérile afin de retirer toutes les particules de sol. Après avoir éliminé l'excès d'eau à l'aide d'un papier absorbant, les semis ont été pesés individuellement et repiqués dans les colonnes à raison d'un semis par colonne.

- La biotite de Bancroft (Canada) est un phyllosilicate 2:1 qui s'altère relativement rapidement et porte des éléments nutritifs (K, Mg, Fe) indispensables à la plante, ainsi que du césium. De plus, ce minéral est un mica fréquent dans les sols acides comme les sols forestiers. Il s'agit d'un minéral pur (sauf calcite), homogène et de composition 41.01 % de SiO₂, 10.9 % de Al₂O₃, 13.26 % de Fe₂O₃, 0.27 % de MnO, 18.9 % de MgO, 0.41 % de Na₂O, 9.46 % de K₂O, 2.28 % de TiO₂, 4.42 % de F, 1650 ppm de Li, 824 ppm de Zn et 56 ppm de Cs. Sa formule structurale est : (Si₃Al₁) (Fe³⁺₀.₁₂ Fe²⁺₀.₆₁ Mg₂.₀₆ Mn₀.₀₂ Ti₀.₁₃) K₀.₈₈ Na₀.₀₆ O₁₀ (OH₀.₉₈, F₁.₀₂). Après retrait des cristaux de calcite, effeuillage et élimination des bordures, les cristaux de biotite sont broyés, soumis à un traitement aux ultrasons pour retirer les particules fines puis tamisés afin de ne conserver que les particules de diamètre compris entre 0.5 et 1 mm. Ils sont stérilisés par autoclavage (20 min. à 120°C). Des cristaux purs de quartz sont concassés, traités aux ultrasons afin d'obtenir deux fractions de granulométrie 0.5 à 1 mm et 1 à 2 mm (Augusto et al., 2001) puis stérilisés par autoclavage (20 min. à 120°C). Il a été vérifié que l'autoclavage ne modifie pas la vitesse de dissolution de la biotite et du quartz. Les quantités respectives de minéraux introduits dans les colonnes sont : 2.8 g de biotite (0.5-1 mm), 10 g de quartz (0.5-1 mm) et 35 g de quartz (1-2 mm).
- La composition de la solution nutritive de base est établie de telle sorte qu'elle n'apporte aux végétaux que les éléments indispensables à la croissance de la plante mais absents de la composition de la biotite, et à des concentrations de l'ordre de celles observées dans le sol du site atelier de Breuil, soit 1.5 mg.l⁻¹ de sodium, 2 mg.l⁻¹ de phosphore (1 g NaH₂PO₄, 2H₂O), 2.3 mg.l⁻¹ de calcium, 1.9 mg.l⁻¹ de soufre (1 g CaSO₄, 2H₂O), 11.2 mg.l⁻¹ de N (3.2 g NO₃NH₄) et 1.5 ml de solution de microéléments contenant du molybdène (Mo) et cuivre (CuSO₄, 4H₂O). Le pH de cette solution nutritive est de 5.6.

En revanche, le matériel microbiologique varie suivant les expérimentations mais la préparation des inocula bactériens et fongiques est similaire quelle que soit l'expérimentation. Elle est décrite en annexe 7.

## Le calcul du bilan d'altération

Ce dispositif, grâce au contrôle des flux d'éléments entrants et sortants de la colonne, permet de faire un bilan de l'altération du minéral-test. Ainsi, le bilan d'altération (B.A.) d'un élément minéral au sein de la colonne peut être représenté par la formule générale suivante :

$$ B.A. = (S_f - S_i) + (P_f - P_i) + R \quad (2) $$

Avec :

S<sub>f</sub> est la quantité d'un élément dans la solution récoltée après percolation dans la colonne : S<sub>f</sub> correspond à la concentration de la solution drainée multipliée par le volume de cette solution,

S<sub>i</sub> est la quantité d'un élément dans la solution nutritive initiale,

P<sub>f</sub> est la quantité d'un élément dans le plant de pin en fin d'expérimentation,

P<sub>i</sub> est la quantité d'un élément dans le semis de pin en début d'expérimentation. Elle est estimée à l'aide de semis de pin témoins sacrifiés en début d'expérience,

(P<sub>f</sub> - P<sub>i</sub>) correspond donc à l'immobilisation I d'un élément par le plant au cours de l'expérience,

R est la reprécipitation d'un élément dans la colonne.
<figure><img src="image_49.png" /><figcaption>Tableau IV.2 : Résumé des analyses effectuées lors des différentes expériences d'interactions réalisées à l'aide du dispositif expérimental en colonnes.</figcaption></figure>

<table><thead><tr><th>Analyses</th><th>Expérience 1*</th><th>Expérience 2</th><th>Expérience 3</th></tr></thead><tbody><tr><td>Volume de solution percolée</td><td>OUI</td><td>OUI (expérience "solution nutritive de base")</td><td>OUI</td></tr><tr><td>pH de la solution</td><td>OUI</td><td>OUI (expérience "solution nutritive de base")</td><td>OUI</td></tr><tr><td>Concentration d'éléments dans la solution percolée</td><td>OUI</td><td>OUI (expérience "solution nutritive de base")</td><td>OUI</td></tr><tr><td>Quantité d'éléments dans les plants initiaux et finaux</td><td></td><td>OUI</td><td>OUI</td></tr><tr><td>Pesée des plants en début et en fin d'expérience</td><td></td><td>OUI</td><td>OUI</td></tr><tr><td>Quantification microbienne</td><td>OUI</td><td>OUI</td><td>OUI</td></tr><tr><td>Quantification des mycorhizes</td><td></td><td></td><td>OUI</td></tr><tr><td>Observations au DRX</td><td></td><td>OUI</td><td></td></tr><tr><td>Observations au MEB</td><td>OUI</td><td>OUI</td><td>OUI</td></tr></tbody></table>

<footnote>* Expérience 1 = "Interactions microorganismes - minéral - solution"</footnote>

<footnote>Expérience 2 = "Interactions arbre forestier - bactéries - minéral - solution"</footnote>

<footnote>Expérience 3 = "Interactions arbre forestier - champignon ectomycorrhizien - bactéries - minéral - solution".</footnote>
Par exemple, pour le potassium qui est un élément absent de la solution nutritive et qui ne reprécipite pas dans la colonne (confirmation par des analyses chimiques de la surface de particules de biotite et de quartz en fin d'expérience) :

$$ B.A. = S_f + I \quad (Figure IV.2) \qquad (3) $$

## Les analyses

Le calcul du bilan d'altération d'un minéral implique donc un nombre conséquent d'analyses sur la plante, sur la solution et également sur la phase minérale. Le tableau IV.2 résume les différents types d'analyses effectuées lors de chacune des expériences en colonnes. Ces analyses sont détaillées ci-dessous :

### Les percolats

- mesure du volume et du pH (pHmètre à électrodes SENTRON type Argus) lors de chaque prélèvement pour les quatre répétitions.

- dosage des éléments minéraux chaque semaine pour toutes les répétitions par spectrométrie d'émission à plasma ICP-AES (JY180 ULTRACE). Pour le césium (expérience d'interaction plante – microorganismes – minéral – solution), une mesure globale en fin d'expérience a été effectuée par ICP-MS au CEA de Saclay pour les quatre répétitions.

### Les plants

Afin de calculer l'augmentation de biomasse et la quantité d'éléments minéraux immobilisée par les plants de pin au cours de l'expérience, il est nécessaire de connaître la masse des parties racinaires et aériennes des plants et la quantité d'éléments minéraux dans les plants au moment de la plantation dans les colonnes. Ces analyses étant destructive, nous avons cultivé des plants de pin dans les mêmes conditions qui ont été sacrifiés au lancement de l'expérimentation.

- pesée des parties aériennes et racinaires des plants en début (plants sacrifiés) et en fin d'expérience pour les quatre répétitions,

- après une minéralisation à l'acide perchlorique des plants de pin, (protocole détaillé dans la partie "matériel et méthodes" de l'article intitulé "Root-associated bacteria
<figure><img src="image_50.png" /><figcaption>Figure IV.4: Localisation des souches bactériennes PML1(12) et PMB4(3) (cf. chapitre III) sur un diagramme fer issu de l'altération de la biotite / pH de la solution, réalisé à partir des résultats obtenus lors du test en microplaques de l'ensemble des souches bactériennes de la collection réalisée sous le peuplement de chêne en 2001. Chaque point est la moyenne de quatre répétitions. La courbe supérieure correspond à l'effet altérant d'un acide complexant (acide citrique) et la courbe inférieure correspond à l'effet altérant d'un acide chlorhydrique).</figcaption></figure>

<figure><img src="image_51.png" /></figure>
3. RESULTATS

3.1 Quantification du processus d'altération au cours de l'interaction biotite - microorganismes – solution

Notre première étude visait à quantifier l'altération d'un minéral-test, la biotite, engendrée par deux souches bactériennes et deux souches fongiques rhizosphériques en présence et en l'absence de glucose.

Matériel et méthodes

Ne sont décrits ci-dessous que les spécificités du matériel inhérent à cette
expérience qui divergent du protocole général présenté dans la partie "dispositif
expérimental en colonnes".

* Matériel microbiologique

Les souches bactériennes *Burkholderia glathei* PML1(12) et PMB4(3) ont été isolées
dans l'horizon organo-minéral 5-10 cm, respectivement au niveau de l'interface "racines
de chêne mycorhizées par *Scleroderma citrinum* - sol" et dans le manchon symbiotique
des mycorhizes de *S. citrinum* (Figure IV.3). Ces souches ont été choisies pour leurs
fortes capacités à mobiliser le fer et le phosphore *in vitro*. Les tests en microplaques ont
par ailleurs révélé que la souche PML1(12) semble plutôt altérer la biotite par
acidification alors que la souche PMB4(3) agirait plutôt par un mécanisme de
complexation (Figure IV.4).

Les souches fongiques SN2(3) et PML2(10) isolées respectivement dans le sol global de l'horizon organo-minéral 0-5 cm sous chêne et au niveau de l'interface "racines de chêne mycorhizées par S. citrinum - sol" de l'horizon organo-minéral 5-10 cm (Figure IV.3). Ces souches ont également été choisies pour leurs fortes capacités à mobiliser le fer et le phosphore *in vitro*.
<table><caption>Tableau IV.3: Les différents traitements de l'expérience "interactions microorganismes - minéral - solution". Les chiffres correspondent au nombre de répétitions.</caption><thead><tr><th rowspan="2">Souches microbiennes</th><th colspan="2">Solution nutritive</th></tr><tr><th>Avec glucose</th><th>Sans glucose</th></tr></thead><tbody><tr><td>Bactérie PML1(12)</td><td>4</td><td>4</td></tr><tr><td>Bactérie PMB4(3)</td><td>4</td><td>4</td></tr><tr><td>Champignon SN2(3)</td><td>4</td><td>4</td></tr><tr><td>Champignon PML2(10)</td><td>4</td><td>4</td></tr></tbody></table>

<figure><img src="image_52.png" /><figcaption>Figure IV.5: Densités de bactéries cultivables mesurées après 21 jours d'expérimentation dans le substrat des colonnes. Chaque histogramme correspond à la moyenne de 4 répétitions. Les barres représentent les écarts-types. Pour chaque traitement microbien, les valeurs associées à des lettres différentes sont significativement différentes selon une ANOVA (P = 0.05) à un facteur (type de solution nutritive) et le test Bonferroni-Dunn. Pour chaque type de solution nutritive, les histogrammes avec un astérisque sont significativement différents selon une ANOVA (P = 0.05) à un facteur (traitement microbien) et le test Bonferroni-Dunn.</figcaption></figure>
* Solution nutritive

La solution nutritive de base à laquelle est ajoutée, pour la moitié des traitements, une source de carbone, en l'occurrence du glucose à une concentration de 1 g.l⁻¹, pour compenser l'absence d'exsudat racinaire.

* Traitements

Le tableau IV.3 présente les différents traitements mis en place dans cette expérience.

Résultats

Les densités bactériennes sont significativement plus importantes dans les colonnes alimentées en solution nutritive avec glucose comparativement à celles des colonnes alimentées en solution nutritive sans glucose et ce, quel que soit le traitement considéré (Figure IV.5). Les densités bactériennes sont significativement plus fortes dans les traitements ayant subi l'inoculation avec les souches bactériennes PML1(12) et PMB4(3) mais uniquement lorsque la solution contient du glucose (Figure IV.5).

Quel que soit le paramètre observé (pH, quantités de magnésium et de potassium dans la solution), les résultats obtenus sur les colonnes témoins non inoculées, alimentées avec une solution nutritive contenant du glucose ne sont pas significativement différents de ceux obtenus avec les colonnes alimentées avec la même solution nutritive mais dépourvue de glucose (Tableau IV.4).

Les valeurs de pH des traitements non inoculés alimentés en solution nutritive pourvue ou non en glucose ne sont pas significativement différentes et sont stables dans le temps. A l'inverse, les valeurs de pH des traitements inoculés par les souches bactériennes ou les souches fongiques, montrent une acidification en présence de glucose qui est plus ou moins marquée et plus ou moins rapide suivant le traitement (Figure IV.6). Ainsi, la souche bactérienne PMB4(3) acidifie rapidement la solution pour atteindre des valeurs minimales de pH de 3.8 après 7 jours d'expérimentation, la souche bactérienne PML1(12) acidifie, quant à elle, plus fortement le milieu avec des valeurs de pH de l'ordre de 3.2 mais après seulement 14 jours d'expérimentation. Les souches fongiques acidifient moins la solution avec des valeurs de pH de 4.6 pour PML2(10) et 5.5 pour SN2(3) après 21 jours d'expérimentation.
<table><caption>Tableau IV.4: Quantités de magnésium et de potassium dans la solution drainée et pH de la solution drainée dans les traitements témoins non inoculés, après 21 jours d'expérimentation. Chaque valeur correspond à la moyenne de quatre répétitions. Pour les différentes variables mesurées (quantité de magnésium en solution (µg.l⁻¹), quantité de potassium en solution (µg.l⁻¹) et pH de la solution), les moyennes associées à des lettres identiques (a, A ou α) ne sont pas significativement différentes selon une ANOVA (P = 0.05) à un facteur (traitement solution nutritive), et le test de Bonferroni-Dunn.</caption><thead><tr><th>Solution nutritive</th><th>Quantité de magnésium en solution (µg.l<sup>-1</sup>)</th><th>Quantité de potassium en solution (µg.l<sup>-1</sup>)</th><th>pH de la solution</th></tr></thead><tbody><tr><td>Sans glucose</td><td>754<sup>a</sup></td><td>378<sup>A</sup></td><td>7.1<sup>a</sup></td></tr><tr><td>Avec glucose</td><td>774<sup>a</sup></td><td>446<sup>A</sup></td><td>7.0<sup>a</sup></td></tr></tbody></table>

<figure><img src="image_53.png" /><figcaption>Figure IV.6: Evolution du pH de la solution drainée au cours du temps pour les différents traitements bactériens et fongiques alimentés en solution nutritive avec glucose et sans glucose. Chaque point est la moyenne de quatre répétitions.</figcaption></figure>
Figure IV.7: Quantités totales de magnésium (A) et de potassium (B) libérées en solution au cours des 21 jours d'expérimentation. Chaque histogramme est la moyenne de quatre répétitions. Les barres représentent les écarts-types. Pour chaque type de solutions nutritives, les valeurs associées à des lettres différentes sont significativement différentes selon une ANOVA (P = 0.05) à un facteur (traitement microbien) et le test Bonferroni-Dunn.

<figure><img src="image_54.png" /></figure>
Les bilans d'altération réalisés après trois semaines d'expérimentation montrent qu'en
absence de glucose dans la solution nutritive, il n'y a aucune différence entre les
différents traitements inoculés et le traitement non inoculé concernant les quantités de
magnésium et de potassium en solution (Figures IV.7A et IV7.B). En revanche, lorsque
la solution nutritive est pourvue en glucose, les quantités de magnésium libérées en
solution sont significativement plus importantes en présence des souches bactériennes
PML1(12) et PMB4(3) (1.8 et 1.9 mg, respectivement) et à un degré moindre, en
présence des champignons (1.3 et 1.5 mg, respectivement) comparativement au
traitement non inoculé (0.8 mg) (Figure IV.7A). De même, lorsque la solution nutritive
est pourvue en glucose, les quantités de potassium libérées en solution sont
significativement plus importantes en présence des souches bactériennes PML1(12) et
PMB4(3) (0.9 et 1.0 mg, respectivement) comparativement au traitement non inoculé
(0.4 mg), ce qui n'est pas le cas des traitements inoculés avec les souches fongiques (0.5
et 0.6 mg, respectivement) (Figure IV.7B).

**Interprétation et discussion des résultats**

Quelle que soit la variable observée (pH, quantités de magnésium et de potassium dans la solution), le traitement non inoculé alimenté en solution nutritive sans glucose n'est pas différent du traitement non inoculé alimenté en solution nutritive avec glucose. Ce qui implique que le glucose, en lui-même, (i) n'interagit pas directement avec la phase minérale et (ii) n'a pas stimulé la croissance d'une microflore contaminante ayant des propriétés altérantes dans les colonnes de l'ensemble du dispositif. Ces résultats suggèrent qu'il est possible de comparer les différents traitements inoculés, avec et sans glucose.

L'acidification de la solution que nous avons uniquement observée en présence de glucose et de microorganismes inoculés suggère la production d'acides organiques résultant du métabolisme microbien du glucose. Plusieurs souches microbiennes et plus particulièrement des bactéries telles que *Burkholderia*, *Pseudomonas* et *Serratia* ont été décrites comme étant capables d'accélérer la dissolution de phosphates par la production d'acide gluconique (Rodriguez *et al.*, 2000 ; Krishnaraj & Goldstein, 2001 ; Kim *et al.*, 2005 ; Lin *et al.*, 2006). Cet acide résulte de l'oxydation du glucose par la quinoprotéine déhydrogénase (Babu-Khan *et al.*, 1995). Welch et Ullman (1999) ont suggéré que la production d'acide gluconique par les microorganismes catalyserait la
dissolution d'un feldspath. Plus récemment, Kim *et al.* (2005) ont même démontré que la production de cet acide par une souche de *B. glathei* était plus efficace pour solubiliser le phosphate qu'une simple addition d'acide gluconique. Lin *et al.* (2006) ont fait l'hypothèse que les protons associés à l'acide gluconique seraient à l'origine de la solubilisation des phosphates. En produisant une grande quantité de protons, les microorganismes vont dissoudre le phyllosilicate et mobiliser le magnésium et le potassium (Robert et Berthelin, 1986). Dans le cas des souches que nous avons étudiées, à savoir les souches bactériennes *B. glathei* PML1(12) et PMB4(3) et les souches fongiques SN2(3) et PML2(10), la production de protons issus de la dégradation du glucose lors du métabolisme microbien pourrait être le mécanisme majeur de l'altération de la biotite mais cela reste à confirmer. D'autant plus que les mesures de densités bactériennes mettent en évidence un effet stimulant du glucose sur la croissance bactérienne dans les colonnes inoculées avec les souches bactériennes PML1(12) et PMB4(3). Ceci suggère que le glucose pourrait également agir comme source d'énergie pour les microorganismes, stimulant ainsi leur activité et la production d'autres métabolites ayant des propriétés altérantes comme les acides organiques ou les enzymes (Robert et Berthelin, 1986). Le comportement des souches bactériennes PML1(12) et PMB4(3) et des souches fongiques SN2(3) et PML2(10) sur le milieu CAS (cf chapitre III) suggère que ces souches seraient également capables de synthétiser des sidérophores. En effet, le milieu CAS que nous avons utilisé, a été, à l'origine, mis au point dans le but de caractériser l'aptitude de microorganismes à produire des sidérophores (Alexander et Zuberer, 1990).

Nos résultats permettent par ailleurs de hiérarchiser l'efficacité des microorganismes à altérer de la biotite dans nos conditions expérimentales en colonnes. En effet, les souches bactériennes *B. glathei* PML1(12) et PMB4(3) augmentent la dissolution de 20 à 50% par rapport aux souches fongiques SN2(3) et PML2(10). Ceci résulterait (i) de la plus grande capacité des souches bactériennes à dégrader le glucose et à produire des composés acides comme l'indiquent les pH de la solution entre 3 et 4 pour les souches bactériennes alors qu'ils sont proches de 5 pour les champignons et (ii) de l'utilisation préférentielle de cette source de carbone par les bactéries qui engendre donc une augmentation considérable de la biomasse et de l'activité bactérienne dans les colonnes ainsi que la production d'une plus grande quantité de composés altérants comparativement aux champignons.
Pour finir, il est intéressant de noter que les valeurs de pH de la solution obtenues pour les deux souches bactériennes *B. glathei* PML1(12) et PMB4(3) sont comparables dans notre expérimentation en colonnes alors que les tests *in vitro* en microplaques indiquaient des comportements différents : la souche PML1(12) dissolvant la biotite principalement par un mécanisme d'acidification, avec un pH de la solution proche de 3, alors que la souche PMB4(3) semblait plutôt agir par un mécanisme de complexation, avec un pH de la solution voisin de 5. Ces observations mettent en évidence l'importante influence des conditions du milieu (aération, pH et composition de la solution nutritive,..) sur l'activité altérante des microorganismes, notamment en ce qui concerne les mécanismes mis en œuvre pour altérer la phase minérale et mobiliser les éléments.

## 3.2 Quantification du processus d'altération au cours de l'interaction biotite - arbre forestier -bactéries – solution

Après avoir testé l'effet des microorganismes seuls sur l'altération de la biotite, nous avons mis en place de nouveaux dispositifs en colonnes faisant interagir la plante avec quelques souches bactériennes rhizosphériques potentiellement altérantes. Cette étude devait permettre de (i) quantifier l'altération d'un minéral-test engendrée par différents acteurs biologiques (plante, bactéries), seuls ou en association, et de (ii) mesurer l'effet de l'inoculation de souches bactériennes rhizosphériques, seules ou en association, sur la croissance de la plante
**Article :** Root-associated bacteria contribute to mineral weathering and to mineral nutrition in trees: a budgeting analysis.

Calvaruso C.¹,², Turpault M.P.¹, Frey-Klett Pascale²*

UR INRA 1138 "Biogéochimie des Ecosystèmes Forestiers", Centre INRA de Nancy, 54280 Champenoux, France.

UMR INRA-UHP 1136 "Interactions Arbres-Microorganismes", Centre INRA de Nancy, 54280 Champenoux, France.

Publié dans *Applied and Environmental Microbiology* 72, 2 : 1258-1266

Résumé

La source principale de nutriments dans les écosystèmes forestiers provient de l'altération des minéraux du sol qui résulte de la circulation des eaux et de l'activité des plantes et des microorganismes. Les objectifs principaux de ce travail étaient de quantifier l'effet respectif des plantes et des bactéries associées aux racines dans l'altération des minéraux et leurs conséquences sur la croissance et la nutrition des semis d'arbres. C'est pourquoi, nous avons mis en place deux expériences en colonnes contenant un substrat quartz - biotite. Les colonnes étaient plantées ou non avec un semis de pins, et inoculées ou non avec trois souches bactériennes ectomycorhizosphériques. Ceci avait pour but de quantifier l'altération de la biotite et la croissance des plants de pin et de déterminer comment les bactéries augmentent la croissance des plants de pin.

Nous avons montré que les racines de pin augmentent l'altération de la biotite par un facteur 1.3 pour le magnésium et 1.7 pour le potassium. Nous avons démontré également que l'inoculation de la souche bactérienne *Burkholderia glathei* PML1(12) augmente significativement l'altération de la biotite par un facteur 1.4 pour le magnésium et 1.5 pour le potassium en comparaison avec les plants de pin non inoculés. De plus, nous avons observé un effet positif significatif de *B. glathei* PMB1(7) et PML1(12) sur la croissance des pins et sur la morphologie du système racinaire (nombre de racines latérales et de poils absorbants). Nous avons démontré que PML1(12) augmente la croissance des pins quand les plants sont alimentés avec une solution nutritive qui ne contient pas les nutriments présents dans la biotite. En revanche, aucune augmentation de croissance des pins liée à l'inoculation de PML1(12)
n'a été observée quand les plants étaient alimentés avec une solution nutritive contenant tous les nutriments nécessaires à la croissance des pins. Par conséquent, nous suggérons que l'effet promoteur de croissance de *B. glathei* PML1(12) résulterait principalement d'une amélioration de la nutrition de la plante *via* une augmentation de l'altération du minéral.

## Abstract

The principal nutrient source for the forest trees derives from the weathering of the 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 seedlings 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 in order 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 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.

## Introduction

In ecosystems with low inputs and without any fertilisation or soil amendment by man, the nutrients available to plants come from atmospheric inputs and weathering of soil minerals. This is mainly the case with forest ecosystems which, in addition, are
frequently located on poor and acidic soils (Badeau *et al.*, 1999). Plants developing on acidic soils are subjected to two major constraints: i) high concentrations of ions like Al³⁺ and H⁺ which inhibit root growth and ii) low mineral nutrient availability as a result of low reserves and impaired uptake due to high H⁺ concentrations (Marschner, 1991).

In temperate and boreal forest ecosystems, the vast majority of trees lives in close association with symbiotic fungi, the ectomycorrhizal fungi, which connect tree roots to the soil environment *via* a broad network of hyphae. These fungi contribute to plant nutrition by carrying far away from the roots and from very small pores, the water and the nutrients they released by weathering the primary minerals (Landeweert *et al.*, 2001; Van Breemen *et al.*, 2000). In addition, the ectomycorrhizal symbiosis modifies root exudation qualitatively and quantitatively by changing the metabolic functions of the roots (Leyval and Berthelin, 1993, Rambelli 1973; Rygiewicz and Andersen, 1994). It creates a special environment called mycorrhizosphere, where the bacterial equilibrium is different from that of the bulk soil (soil without any influence of roots) (Linderman, 1988). These modifications of the bacterial equilibrium in the mycorrhizosphere are likely to contribute to plant nutrition (Frey-Klett *et al.*, 2005). However, the mechanisms involved still remain to be clarified, especially the role of mycorrhizosphere bacteria on the weathering of soil primary minerals.

It is already known that the physico-chemical changes in the rhizospheric soil, induced by roots and their associated microflora, influence the weathering processes and the nutrients uptake by plants (Hinsinger, 1998, Jones and Darrah, 1994, Jones *et al.* 2003, Landeweert *et al.*, 2001, Marschner *et al.*, 1986). However, the respective quantitative contributions of plant and rhizosphere micro-organisms on mineral weathering processes and their consequences on plant nutrition still remain to be determined, especially in the case of nutrient-poor forest ecosystems. This study presents the first part of a project which purpose is to quantify the contribution of the different microbial components of mycorrhizosphere to the mineral weathering processes and the tree nutrition. It is focused on the bacterial component, which contribution to mineral weathering in forest soil has been completely neglected so far.

To determine the respective contribution of tree roots and mycorrhizosphere bacteria to mineral weathering, we carried out a column experiment in a growth chamber, associating pine seedlings with different combinations of three strains of *Burkholderia glathei*. The inputs and outputs of potassium and magnesium in solution, as well as the
immobilisation of these elements by pine seedlings, were measured in order to compare
the weathering budget in the different treatments. Because the bacterial strain B. glathei
PML1(12) simultaneously promoted mineral weathering and plant growth, we carried
out a complementary experiment in a greenhouse in order to confirm these effects and
to test if both effects were linked. For that purpose, we compared the effect of this
bacterial strain on the growth of pine seedlings supplied either with a deficient or a
complete nutrient solution.

**Material and methods**

* Bacterial strains

Origin

A collection of 140 bacterial strains were isolated from oak (Quercus petreae)-
*Scleroderma citrinum* ectomycorrhizas and bulk soil, sampled in the mineral soil
horizon in an experimental forest site located at Breuil, in central France. The
ecosystem is oligotrophic and very sensitive to nutrient deficiency and acidification.
The soil is an alocrisol with acid mull type humus developed in aeolian silt, over a
granite parent material containing two micas (Dejou, 1967). The bacterial isolates were
preserved at -80°C in Nutrient Broth glycerol medium (8 g.l⁻¹ Nutrient Broth from
Difco, 850 ml of demineralised water and 150 ml of glycerol).

Selection of three bacterial strains

Among our collection of 140 bacterial strains, the three strains PMB1(7), PML1(4) and PML1(12), which were isolated from oak-S. citrinum ectomycorrhizas, were chosen because they showed the highest potential for mineral weathering in three different biotests (Table IV.5). The first two biotests (CAS and TCP Petri dish tests), which measure the bacterial efficacy for phosphorus and iron mobilisation, were performed according to Frey-Klett et al. (2005). The third biotest, which allows to quantify the bacterial impact on biotite weathering, consisted in incubating each bacterial suspension (20 microliters, A<sub>600nm</sub> = 0.9) with 160 microliters of nutrient solution: 1.5 mg.l<sup>-1</sup> of sodium, 2 mg.l<sup>-1</sup> of phosphorus (10 mg.l<sup>-1</sup> of NaH<sub>2</sub>PO<sub>4</sub>, 2H<sub>2</sub>O), 2.3 mg.l<sup>-1</sup> of calcium, 1.9 mg.l<sup>-1</sup> of sulphur (10 mg.l<sup>-1</sup> of CaSO<sub>4</sub>, 2H<sub>2</sub>O), 11.2 mg.l<sup>-1</sup> of nitrogen (32 mg.l<sup>-1</sup> of NO<sub>3</sub>NH<sub>4</sub>) and 20 microliters of glucose (20 g.l<sup>-1</sup> of glucose), and 10 mg of biotite (200
Tableau IV.5: Capacity of the three bacterial strains selected for the column experiments to mobilise iron and phosphorus in three in vitro tests.

x efficacy of iron mobilisation = diameter of the discoloration zone on the CAS medium corresponding to the zone of iron solubilisation / bacterial concentration (CFU/ml).

y efficacy of phosphorus mobilisation = diameter of the discoloration zone on the TCP medium corresponding to the zone of phosphorus solubilisation / bacterial concentration (CFU/ml).

z a reference strain which presents low efficacy to mobilise nutrients in the different biotests.

¹ effect of the non-inoculated complete solution (same composition than the deficient nutrient solution used for the column experiments plus 2 g.l⁻¹ of glucose) on the mobilisation of potassium and iron in the biotest with mineral.

For each variable (efficacy of Fe and P mobilisation, solution pH, [Fe] and [K] released after biotite weathering), treatments associated with the same letter are not significantly different according to a one-factor (bacterial treatment) ANOVA (P=0.05), and the Bonferroni-Dunn test.

<table><thead><tr><th rowspan="2">Strains</th><th colspan="3">Petri dish tests</th><th colspan="2">Biotest with mineral</th></tr><tr><th>Efficacy of Fe mobilisation<sup>x</sup></th><th>Efficacy of P mobilisation<sup>y</sup></th><th>Solution pH</th><th>[Fe] released after biotite weathering</th><th>[K] released after biotite weathering</th></tr></thead><tbody><tr><td>PMB1(7)</td><td>0.23<sup>c</sup></td><td>0.70<sup>c</sup></td><td>2.93<sup>c</sup></td><td>2.26<sup>c</sup></td><td>2.59<sup>c</sup></td></tr><tr><td>PML1(4)</td><td>0.15<sup>b</sup></td><td>0.44<sup>bc</sup></td><td>2.68<sup>d</sup></td><td>3.32<sup>d</sup></td><td>3.37<sup>d</sup></td></tr><tr><td>PML1(12)</td><td>0.27<sup>c</sup></td><td>0.26<sup>b</sup></td><td>3.05<sup>c</sup></td><td>2.29<sup>c</sup></td><td>2.55<sup>c</sup></td></tr><tr><td>PN4(4)<sup>z</sup></td><td>0.00<sup>a</sup></td><td>0.00<sup>a</sup></td><td>4.05<sup>b</sup></td><td>1.28<sup>b</sup></td><td>1.90<sup>b</sup></td></tr><tr><td>Nutrient solution<sup>t</sup></td><td></td><td></td><td>5.15<sup>a</sup></td><td>0.94<sup>a</sup></td><td>1.44<sup>a</sup></td></tr></tbody></table>

Tableau IV.6: Molecular identification of the three bacterial strains.

ª top match as determined by the BLAST method. The BLAST programme was described by Altschul et al. (1997).

<table><thead><tr><th>Isolate</th><th>rrs sequence length</th><th>Top match [GenBank accession n°]<sup>a</sup></th><th>Nucleotide identity</th></tr></thead><tbody><tr><td>PMB1(7)</td><td>453</td><td>Burckholderia glathei AY154378</td><td>98%</td></tr><tr><td>PML1(4)</td><td>764</td><td>Burckholderia glathei AY154374</td><td>99%</td></tr><tr><td>PML1(12)</td><td>677</td><td>Burckholderia glathei AY154378</td><td>98%</td></tr></tbody></table>
to 500 micrometers) during 65 hours, in shaken cultures (350 rounds per minute). In
each of these three biotests, the strain PN4(4), which was isolated from bulk soil, was
used as a control because it presents a very low efficacy for P and Fe mobilisation, and
biotite weathering. The results presented in the Table IV.5 underline the great diversity
of behaviours obtained in the different biotests for the four bacterial strains.

Molecular identification

Partial sequencing of the 16S ribosomal DNA genes of the bacterial isolates was carried
out as described by Bertaux *et al.* (2003). After purification, the 16S ribosomal DNA
gene products were sequenced with the eubacterial primer 27F (Weisburg *et al.*, 1991).
The sequences were compared with those in the GenBank databases
([www.ncbi.nlm.nih.gov.blast](http://www.ncbi.nlm.nih.gov.blast), using the Blast programme (Altschul *et al.*, 1997)
(Table IV.6).

Preparation of the bacterial inocula

Bacteria were grown on 10% TSA medium (3 g.l⁻¹ Tryptic Soy Broth from Difco and 15 g.l⁻¹ agar) at 25°C for 36h. Bacteria were suspended in sterile ultra-pure water, washed twice. The inoculum concentrations were then adjusted to 4.10⁷ CFU.ml⁻¹ according to a A₆₀₀nm standard curve calibrated by plate enumeration.

* Plant material

Non mycorrhizal scots pine seedlings (*Pinus sylvestris*; provenance: Haguenau forest, France) were grown in a non-sterile peat-vermiculite substrate in a greenhouse: 60% humidity, a night temperature of 15°C and a day temperature of 22°C, and a 16 hour period of daylight, two watering of two minutes by day. After six weeks, the seedling roots were washed very carefully with a brush in sterile ultra-pure water, four times successively, to remove remaining peat and vermiculite particles. The excess of water was then removed using absorbent paper, and the seedlings were weighed individually in the case of the growth chamber experiment.
* Mineral material

The mineral chosen was a biotite from Bancroft (Canada), a 2:1 phyllosilicate, frequently present in acid soils, which weathers relatively quickly and holds K, Mg and Fe nutrient elements (which are indispensable for plants). It is a pure homogeneous mineral and its composition is: 41.01% SiO₂, 10.9% Al₂O₃, 2.21% Fe₂O₃, 10.05% FeO, 0.27% MnO, 18.9% MgO, 0.41% Na₂O, 9.46% K₂O, 2.28% TiO₂, 4.42% F and 824 ppm Zn. Its structural formula is (Si₃Al₁) (Fe³⁺₀.₁₂ Fe²⁺₀.₆₁ Mg₂₀.₀₆ Mn₀.₀₂ Ti₀.₁₃) and K₀.₈₈ Na₀.₀₆ O₁₀ (OH₀.₉₈ F₁.₀₂).

The biotite crystals were ground, washed with distilled water and treated ultrasonically (two minutes, three times at 100 volts), to remove the fine particles that electrostatically adhere to the particles with the size required, and then sieved to obtain the size fraction between 0.5 and 1 mm. Pure quartz crystals were prepared the same way and sieved to obtain two size fractions of 0.5 to 1 mm and 1 to 2 mm (Augusto *et al.*, 2001). Biotite and quartz preparations were then sterilized by autoclaving (20 min. at 120°C). A previous experiment showed that the dissolution kinetics of both minerals was not modified by autoclaving.

* Nutrient solutions

*Growth chamber experiment*

Only a deficient nutrient solution was used. The composition of this deficient nutrient solution was adjusted in order that it only contained elements that were essential for pine growth but were absent from the biotite composition, and in concentrations equivalent to those found in the soils at the experimental forest site at Breuil: 1.5 mg.l⁻¹ of sodium, 2 mg.l⁻¹ of phosphorus (10 mg.l⁻¹ NaH₂PO₄, 2H₂O), 2.3 mg.l⁻¹ of calcium, 1.9 mg.l⁻¹ of sulphur (10 mg.l⁻¹ of CaSO₄, 2H₂O), 11.2 mg.l⁻¹ of nitrogen (32 mg.l⁻¹ of NO₃NH₄) and trace-element: 0.3 mg.l⁻¹ of molybden (0.3 mg.l⁻¹ of Mo) and 0.2 mg.l⁻¹ of copper (0.6 mg.l⁻¹ of CuSO₄, 5H₂O).

*Greenhouse experiment*

Two nutrient solutions were used. The composition of the deficient nutrient solution was the same than the one used in the growth chamber experiment. In contrast, the composition of the complete solution was the same that the deficient nutrient one but
enriched by potassium, magnesium, iron, manganese and zinc in concentrations equivalent to those found in the soils at the experimental forest site at Breuil: 4.2 mg.l⁻¹ of potassium (8 mg.l⁻¹ of KCl), 1 mg.l⁻¹ of magnesium (5 mg.l⁻¹ of MgSO₄), and trace-element 0.5 mg.l⁻¹ of iron (2.5 mg.l⁻¹ FeSO₄, 7H₂O), 0.2 mg.l⁻¹ of manganese (0.6 mg.l⁻¹ of MnSO₄, H₂O), and 0.2 mg.l⁻¹ of zinc (1 mg.l⁻¹ ZnSO₄, 7H₂O).

The pH of the two nutrient solutions was about 6. Given the quantity of nutrient solutions required for the pine seedlings during the time of the experiments, the solutions were concentrated 100 times, sterilised (20 min. at 120°C) and stored at 4°C in 50 ml sterile tubes to limit all the risk of contamination, and protected from the light. Each week, the nutrient solutions were replaced by new ones, prepared by mixing the concentrated nutrient solutions with sterile ultra-pure water (20 min. at 120°C).

* Experimental devices and culture conditions

*The columns*

They were made from sterile inert polypropylene Falcon tubes, 15 cm high and 4 cm in diameter, with a hole drilled at the bottom. They contained a layer of pre-treated (by percolation with HCl solution at pH 1 for 1 night and with sterile ultra-pure water for 1 day) and sterilized (autoclaved 20 min. at 120°C) glass wool (8 mm thick), on which a mixture of 2.8 g of biotite (0.5 to 1 mm diameter), 10 g of quartz (0.5 to 1 mm) and 35 g of quartz (1 to 2 mm), were placed. A preliminary experiment with fluorescein solution showed that this mixture allows a good flow in the whole column. The columns were filled individually under a laminary flow hood. In each column, one pine seedling was planted, except for those of the control treatment without plant (WP) which simulates geochemical processes. Then 1.8 ml of bacterial inoculum with a concentration of 4.10⁷ CFU/ml was added to each column, except for the two control treatments: Non-inoculated plant (P) and without plant (WP) (see below), which received 1.8 ml of sterile ultra-pure water only.

*Growth chamber experimental device*

The deficient nutrient solution was stored in a 20 l sterile inert container, which was protected from the light by aluminium foil. Two peristaltic pumps were calibrated to supply the columns with the nutrient solution at a rate of 1 ml per hour, *via* tygon
tubing. The nutrient solution percolating through the columns was collected individually in 100 ml polyethylene tubes placed below each column. The experiment included 9 treatments (with 4 replicates): 1 control without biological material (WP), 1 control with non-inoculated plant (P) and 7 treatments with a plant and one bacterial strain (PMB1(7), PML1(4) or PML1(12)) or associations of strains (PMB1(7)+PML1(4), PMB1(7)+PML1(12), PML1(4)+PML1(12), and PMB1(7)+PML1(4)+PML1(12)). The columns were placed in a growth chamber with 60% humidity, a night temperature of 18°C and a day temperature of 25°C, and a 17 hour period of daylight.

*Greenhouse experimental device*

Fifteen ml of sterile nutrient solutions were injected manually three times per week (Monday, Wednesday and Friday) in each column with sterile syringes. The experiment included 4 treatments (with 10 replicates): two control treatments with non-inoculated plants, supplied by either a deficient nutrient solution or a complete solution, and two inoculated plant treatments with the bacterial strain PML1(12), supplied by either a complete solution or a deficient nutrient solution. The columns were placed in a greenhouse with 60% humidity, a night temperature of 18°C and a day temperature of 25°C, and a 17 hour period of daylight.

* Sampling*

*Growth chamber experiment*

Forty pine seedlings pre-grown under the same conditions as those used in the column experiment were sampled at the beginning of the experiment (before the bacterial inoculation) to quantify the initial dry weight biomass and mineral content of the seedlings. For the chemical analyses, the solutions from the four replicates of each treatment were collected throughout the experiment. Four weathering budgets for the drainage solution were thus obtained for each treatment. After 76 days, all the pine seedlings were sampled to observe root morphology and to quantify their final biomass and mineral content. Four weathering budgets for the plant immobilisation were thus obtained for each treatment.
*Greenhouse experiment*

One hundred and two days after bacterial inoculation, all the pine seedlings were sampled for analyses: shoot length, fresh and dry weight of the root and aerial part, length, diameter and volume of the roots.

* Analyses*

*Growth chamber experiment*

The volumes of all the drainage solutions were measured each week. The potassium and magnesium contents of all solutions sampled were measured by ICP emission spectrometer (Plasma torch JY180 ULTRACE). A kinetics follow-up of the potassium and magnesium released from the biotite into the drainage solution was made for the treatment without plant in order to check that the experiment was in a stationary regime (data not shown).

After having washed the pine seedlings, 0.25 g of oven-dried (65°C) aerial or root parts were ground, pre-treated with 5 ml of 30% hydrogen peroxide ($H_2O_2$) during one night. Then, 5 ml of perchloric acid ($HClO_4$) were introduced and the solutions were placed on a warming plate during one night (40°C). These two strong oxidants digest all the organic matter to let and leave behind only the mineral elements. Then the solutions were measured by ICP emission spectrometer.

To observe the weathering surfaces of the biotite, 8 particles adhering to the seedling roots after manual shaking were sampled for each treatment (two particles per replicate). The particles were air dried and glued to a glass slide with varnish. Then, the slides were coated with carbon. Images of the biotite surface and a semi-quantitative analysis of the constitutive elements of the sample were made simultaneously using a Hitachi S2500 SEM connected to a Thermonoran microanalysis system.

To observe the root morphology and the root-mineral interface, 8 root pieces (two per replicate) were sampled for each treatment. A low vacuum (high pressure) Leo 1450VP SEM, coupled to an Oxford microanalysis system was used. It differs from classical scanning electron microscope by using a controlled pressure function, thus the sample can be observed in a primary vacuum known as a Low Vacuum. The Low Vacuum mode means that one can work without a preliminary coating and thus without altering the sample. In this way root architecture and absorbent hairs in contact with the mineral
surface could be observed. To semi-quantitatively estimate the number of lateral roots
and the number of root hairs, we used a binocular microscope Leica MZ 6.

Greenhouse experiment

The shoot length (from the leaf insertion basis to the terminal bud) was measured for
each pine seedling at the end of the experiment. At sampling, the roots were washed
very carefully in sterile ultra-pure water three times successively to remove the
remaining mineral particles. The excess of water was removed using absorbent paper
and the fresh weight of the seedlings was determined. Then each root system was
photocopied, taking into account the necessity to reduce root overlapping. To measure
the different parameters relative to root architecture, the WinRhizo software was used.
It allows to quantify root length and total root surface area. Then, the plants were dried
at 65°C during five days in order to determine the dry weight of the root and aerial
parts.

* Total weathering budget

In each column of the growth chamber experiment, the total weathering budget W was
calculated as follows: W = (D-N) + I for the potassium and the magnesium, two
elements which were not re-precipitated and not exchanged in the columns as confirmed
by MEB observations.

D was the quantity of the element in the drainage solution,

N was the quantity of the element in the nutrient solution (= 0 for potassium and magnesium),

I was the quantity of the element immobilised by the pine seedling during the experiment and I = If-Ii, where Ii and If were the quantities of the element in the pine seedling at the beginning and at the end of the experiment, respectively.

* Statistical analyses of the results

The effect of the plant and the bacterial inoculations on the weathering budgets, on the
growth of the pine seedlings and their root architecture were determined using analyses
of variance (ANOVA) at the threshold level of *P* = 0.05, and the Bonferroni-Dunn test.
The Superanova software was used for all these analyses.
Figure IV.8: Mass-balance of potassium (A) and magnesium (B) released into leaching solution (grey) and taken up by plants (black). Each plot is a mean value of four replicates. Bars represent standard deviation referring to the values of the weathering budget. For each variable (sum of potassium or magnesium quantities released into leachates and taken up by plants), treatments associated with the same letter are not significantly different according to a one-factor (plant and bacterial treatment) ANOVA (P = 0.05), and the Bonferroni-Dunn test.

<figure><img src="image_55.png" /></figure>

<figure><img src="image_56.png" /></figure>
## Results

* Weathering budget

The total amounts of potassium and magnesium mobilised from biotite with non-inoculated plants or with plants inoculated by the different bacterial strains were significantly higher than those in the treatment without plant (WP) (Figure IV.8). They were also significantly higher in the treatment inoculated with strain PML1(12), relatively to the non-inoculated plant treatment (P). In this case, not only the amounts of magnesium leached into the solution but also the one taken up by the plant were significantly higher in the treatment inoculated with the strain PML1(12) in comparison to the non-inoculated plant treatment, according to a one-factor ANOVA (P = 0.05) and the Bonferroni-Dunn test (comparison to control) (data not shown). In the same way, the amounts of potassium taken up by the plant were significantly higher in the treatment inoculated with the strain PML1(12) in comparison to the non-inoculated plant treatment, according to a one-factor ANOVA (P = 0.05) and the Bonferroni-Dunn test (comparison to control) (data not shown). In contrast, when the strain PML1(12) was co-inoculated with the strains PMB1(7) or PML1(4), the quantities of potassium and magnesium mobilised from the biotite were no more significantly higher than that of the treatment with plant alone (Figure IV.8). In the special case of the triple inoculation of strains PMB1(7) + PML1(4) + PML1(12), only the amount of magnesium leached into the solution was significantly higher than the one in the treatment with the non-inoculated plant, according to a one-factor ANOVA (P = 0.05) and the Bonferroni-Dunn test (comparison to control) (data not shown).

* Analysis of the biotite weathering processes

The SEM observations clearly showed that the surface of the biotite particles from the PML1(12)-inoculated plant treatment was more weathered than the one in the treatment without plant (Figure IV.9). Bacterial cell accumulations were preferentially observed in carbon rich areas (probably root exudates). Some carbon precipitates associated with sulphur and phosphorus as well as iron precipitates were also visible (white areas). Conversely, potassium, magnesium or aluminium precipitates were not observed. The observations showed also that the majority of the biotite particles sampled along the roots was not transformed into vermiculite. This result was confirmed by plotting the total quantity of potassium released from biotite (the quantity of potassium in solution +
Figure IV.9: SEM photography of biotite surfaces without plant and bacteria (A) and with plant inoculated with the bacterial strain PML1(12) (B and C). The white and black areas presented with a black arrow correspond respectively to iron precipitates and carbon deposits. Part (C) corresponds to an enlargement of the square region in part (B). The dotted arrow present bacteria on the biotite surface.

<figure><img src="image_57.png" /></figure>
Figure IV.10: Relation between the magnesium and the potassium quantities leached into the solution and taken up by plants, i.e. mobilised in the biotite. The black linear regression curve (y = 1.29x + 59.10 and R² = 0.321) was obtained with the experimental data set. This regression is significative at P < 0.001. The dotted curve represents the Mg/K stoichiometry in the Bancroft biotite which refers to a congruent dissolution process. The area under the dotted curve corresponds to the transformation domain of the biotite into vermiculite.

<figure><img src="image_58.png" /></figure>

Figure IV.11: Increase of the root (black) and total plant (grey) biomass during the 76 days of the growth chamber experiment in the different treatments. Each plot is a mean value of four replicates. Bars represent standard deviation. For each variable (total plant growth or root growth), treatments associated with the same letter are not significantly different according to a one-factor (bacterial treatment) ANOVA (P = 0.05), and the Bonferroni-Dunn test.

<figure><img src="image_59.png" /></figure>
the quantity of potassium mobilised by the seedlings) against the total quantity of magnesium released (Figure IV.10): potassium and magnesium were released in a nearly stoichiometric way; there was no preferential release of interlayer potassium responsible for the transformation of biotite into vermiculite.

* Growth of the pine seedlings

*Growth chamber experiment*

The pine seedlings inoculated with the following bacterial strains, PMB1(7), PML1(12), PMB1(7) + PML1(12), and PMB1(7) + PML1(4) + PML1(12), produced significantly more total and root biomass than the non-inoculated plants (Figure IV.11). These data were in accordance with Low Vacuum SEM and binocular microscopy observations, which showed a higher number of lateral roots and a higher density of root hairs on PML1(12)-inoculated roots in comparison with non-inoculated plants (Figure IV.12 and Table IV.7).

In contrast to the significant positive effect of some of the bacterial associations, the co-inoculation of the strain PML1(4) with PMB1(7) significantly reduced the promoting effect of strain PMB1(7) on the growth of the pine seedlings.

*Greenhouse experiment*

When the pine seedlings were supplied by a complete nutrient solution, the inoculation of bacterial strain PML1(12) had no effect on plant growth, according to the five measured parameters: total seedling biomass, shoot length, root biomass, total length and total surface area of the roots (Figure IV.13). In contrast, as observed in the growth chamber experiment, the bacterial strain PML1(12) significantly promoted the growth of the pine seedlings which were supplied by the deficient nutrient solution: the inoculated plants had a significantly higher seedling biomass (Figure IV.13A), shoot-length (Figure IV.13B), root biomass (Figure IV.13C), total length of the roots (Figure IV.13D) and total surface area of the roots (Figure IV.13E) than the non-inoculated seedlings.
<figure><img src="image_60.png" /><figcaption>Figure IV.12: Low vacuum SEM photography of a non-inoculated root (A) and a root inoculated with the bacterial strain PML1(12), which presents many root hairs (B).</figcaption></figure>

Tableau IV.7: Semi-quantitative estimation of the number of lateral roots and the number of root hairs in the different treatments according to binocular microscopy observations.

a name of the bacterial strain inoculated.

b treatment without bacterial inoculation.

c semi-quantitative estimation of the number of the lateral roots and the number of the root hairs, with four levels (+/-, +, ++, +++).

<table><thead><tr><th>Treatment</th><th>Number of lateral roots</th><th>Number of root hairs</th></tr></thead><tbody><tr><td>P+PMB1(7) <sup>a</sup></td><td>+++ <sup>c</sup></td><td>++<sup>c</sup></td></tr><tr><td>P+PML1(4)</td><td>++</td><td>+</td></tr><tr><td>P+PML1(12)</td><td>+++</td><td>+++</td></tr><tr><td>P+PMB1(7) PML1(4)</td><td>++</td><td>++</td></tr><tr><td>P+PMB1(7) PML1(12)</td><td>++</td><td>++</td></tr><tr><td>P+PML1(4) PML1(12)</td><td>++</td><td>++</td></tr><tr><td>P+PMB1(7) PML1(4) PML1(12)</td><td>+++</td><td>++</td></tr><tr><td>P<sup>b</sup></td><td>++</td><td>+/-</td></tr></tbody></table>
Figure IV.13: Growth of the plants during the 102 days of the greenhouse experiment in the different treatments. A = total seedling biomass, B = shoot length measured above cotyledons, C = root biomass, D = total of the root length, E = total of the root surface area. Black plots correspond to plants inoculated with the bacterial strain PML1(12), white ones correspond to non-inoculated plants. Each plot is a mean value of ten replicates. Bars represent standard deviation. For each variable, treatments associated with the same letter are not significantly different according to a one-factor (bacterial treatment) ANOVA (P = 0.05), and the Bonferroni-Dunn test.

<figure><img src="image_61.png" /></figure>

<figure><img src="image_62.png" /></figure>

<figure><img src="image_63.png" /></figure>

<figure><img src="image_64.png" /></figure>
Discussion

In this work, we quantified potassium and magnesium immobilised by pine seedlings or drained out from the columns, and the biomass increase of the pine seedlings, in order i) to quantify the respective contribution of roots and root-associated bacteria to the weathering of biotite and ii) to assess the effect of the weathering process on seedling growth. To carry out this work, we chose to perform column experiments that only contain a mineral substrate (quartz and biotite) for plant growth and no organic matter, in order to mimic the conditions that exist in the natural forest soil horizons B and C, in which the weathering of minerals is the sole source of potassium and magnesium. To be in accordance with this experimental strategy, all the bacterial strains we used for this work were isolated from S. citrinum-oak ectomycorrhizas sampled in a forest soil mineral horizon.

* Effect of pine roots and bacteria on biotite weathering

Root effect

All the results obtained by the different approaches (weathering budget and SEM
observations) converge: the pine roots significantly increased biotite weathering by a
factor 1.5 in comparison to the treatment without plant. Our results are in accordance to
those of Bormann *et al.* (1998) who demonstrated in an experimental forest that red pine
(Pinus resinosa) plants improved by 2.4 and 1.8 factors the quantities of calcium and
magnesium released by weathering the primary minerals in a forest soil, respectively. In
the same way, our results are in accordance to those of Bakker *et al.* (2004) who
measured plagioclase weathering in a laboratory experiment at pH 3-4 and 25°C, and
showed that the release of Si, Mg, Ca and Al was increased by a 1.8 factor in the
presence of Douglas fir and Scots pine under these acidic conditions.

This effect of plant roots on mineral weathering may result from physical and/or
biochemical processes. Indeed, plant roots induce the formation of macropores (Gibbs
and Reid, 1988), which play a major role in the preferential flow phenomenon and thus
the weathering process (Beven and Germann, 1982). Moreover, plant roots fragment
soil minerals and thus increase the number of reactive surfaces (Berthelin, 1985 ;
Mottershead et al., 2003). In addition, through the nutrient uptake, the plants modify the
nutrient concentrations in the rhizosphere and generate zones of depletion or
accumulation of nutrients (Barber, 1995). Plant roots also modify the redox potential and the pH in the rhizosphere (Marschner et al., 1986; Marschner, 1995; Muofhe, 1997; Nye, 1981; Römheld, 1987). For example, the difference between the pH in the rhizosphere soil and in the bulk soil can be up to two pH units (Marschner and Römheld, 1983; Riley and Barber, 1971). This acidification of the rhizospheric soil results from the production of CO₂ during respiration, from the release of organic compounds in the root exudates (Drever, 1994; Hinsinger and Gilkes, 1997; Jones, 1998; Ström, 1997), and from the balance of ion charges within the roots which depends on the excretion of one H⁺ ion for every cation absorbed (Haynes, 1990). These modifications of physico-chemical conditions in the rhizosphere make the cations from the minerals more accessible to the plant (Dakora and Phillips, 2002; Grayston et al., 1996; Ochs et al., 1993). Besides, this phenomenon could also be regulated by nutrient deficiencies because plants that grow in potassium or phosphorus deficient conditions are able to modify the composition and quantity of root exudates, thus improving the mobilisation of these elements (Kraffczyk et al., 1984; Lipton et al., 1987).

## Bacterial effect

The molecular identification of the three bacterial strains we chose for this study revealed that they all belong to the *B. glathei* species, which occurrence in the soil has already been mentioned. The column experiments showed that the weathering rate of biotite varied with the different bacterial strains. These results contrast from the ones of the *in vitro* tests which demonstrated that all the three bacterial strains used had previously shown great capacities to mobilise iron, potassium and phosphorus. Our results thus underline the importance of taking into account biotic and abiotic environment, like the composition of root exudates (Marschner et al., 2001), the presence of ectomycorrhizal fungi and the soil properties (pH, aeration and physico-chemical characteristics), when characterizing the weathering effect of a given bacterial strain because these parameters may influence the expression of the weathering ability of the bacteria. Moreover, our results also suggest that the weathering ability of the bacteria, which involves the production of protons, organic acids, siderophores and organic ligands (Grayston et al., 1996; Paul and Clark, 1989; Rogers and Bennett, 2004; Welch et al., 1999) depends on the bacterial strain. Finally, the fact that multiple strain inoculation can be less efficient in mineral weathering than strains inoculated
alone, suggests for the first time the importance of microbial interactions in the mineral weathering process.

*Plant-bacterial interaction on mineral weathering*

Our results clearly demonstrate a significant positive interaction between pine roots and the bacterial strain *B. glathei* PML1(12) on mineral weathering. They are consistent with the ones of Leyval and Berthelin (1989) on a beech-*Agrobacterium radiobacter* model and the ones of Puente *et al.* (2004) on cactus inoculated or not with different rhizoplane bacteria. In our study, the positive interaction between pine roots and *B. glathei* PML1(12) could simply result from an additive effect of the weathering abilities of the two partners. Another possibility is that the weathering increase is due to a synergistic effect, which could result from three hypothetical processes: (i) the fragmentation of the mineral caused by root activity increases the direct positive effect of the bacteria on mineral weathering by increasing the reactive surfaces (Barker and Banfield, 1998); (ii) the root exudates indirectly provides substrates required for the production of weathering metabolites by the bacteria (Grayston *et al.*, 1996) or (iii) the production of growth phytohormones by the bacteria, in addition to weathering agents, stimulates root development, modify root physiology (Gamalero *et al.*, 2002 ; Patten and Glick, 1996) and root exudation which improves mineral weathering and nutrient uptake (Gahoonia *et al.*, 1997).

*Biological processes involved in biotite weathering*

The biotite can undergo two types of weathering mechanisms (i) dissolution by the destruction of the mineral structure (congruent way) or transformation into vermiculite (incongruent way) by the release of interlayer potassium (Robert *et al.*, 1979). Our weathering budgets as well as SEM observations and analyses showed that, whatever the bacterial treatments, potassium and magnesium were released in a nearly stoichiometric way: the interlayer potassium was not released faster than the magnesium from the biotite structure. Consequently, biotite had mainly been dissolved by a congruent phenomenon in our experiment. Our results contrast with those from many other studies which showed evidences of incongruent dissolution of biotite under the influence of plant root activity (Hinsinger *et al.*, 1992) and/or under the influence of rhizospheric micro-organisms (Leyval and Berthelin, 1991 ; Paris *et al.*, 1995). This
discrepancy could result from different experiment durations. In our experiment, the
exudation and the uptake by the roots could only play an important role in the
weathering process during the last 30 days of the experiment, when root development
was large enough to colonise the whole column. This obviously contrasts with the
experiments conducted by Hinsinger *et al.* (1992), who put the mineral directly in
contact with an already established root mat which readily produced large quantities of
weathering substances and was therefore able to take up abundant quantities of
potassium. In the same way, Leyval (1990) observed a partial vermiculitisation of
another phyllosilicate (phlogopite) in the rhizosphere of pine seedlings inoculated with
*Agrobacterium* sp., after one year of experimentation only.

* Effect of bacteria on the growth of pine seedlings

According to the results of the growth chamber experiment, the two mycorhizosphere
bacterial strains PMB1(7) and PML1(12) behave as plant growth promoting
rhizobacteria (PGPR) (28). The beneficial effect of these strains could result from two
non-exclusive effects: (i) an indirect effect of the bacteria on plant nutrition, *via* an
increased bacterial mobilisation of nutrients from the mineral, and/or (ii) a direct effect
of the bacteria on plant roots by means of phytohormones, like indoleacetic acid (IAA),
cytokinins and ethylene, which stimulate the formation of lateral roots and absorbent
root hairs (Patten and Glick, 1996). Indeed, an increase in the number of lateral roots
was observed in the treatments inoculated with the strains PMB1(7), PML1(12) as well
as with the association PMB1(7) + PML1(4) + PML1(12). Moreover, for the roots
inoculated with the strain PML1(12), a large number of absorbing root hairs were also
observed.

The greenhouse experiment demonstrated that the beneficial effect of the bacterial strain PML1(12) on plant growth, only occurred under conditions of nutrient deficiency. Therefore, notwithstanding a possible direct effect of the bacteria on plant growth *via* phytohormones, the bacteria mainly acted by an indirect way, *via* mineral weathering which increased the amount of nutrients available for the plant. Indeed, if the bacteria had only promoted plant growth by a direct way, we would have observed a significant increase of the growth of the plants in the nutrient-rich conditions too. Our results are in accordance with those of Toro *et al.* (1997) who demonstrated the beneficial effect of two phosphate-solubilizing bacteria (*Enterobacter* sp. and *Bacillus subtilis*) on the
growth of onion (*Allium cepa* L.) mycorrhized with *Glomus intraradices* via the release of P from rock phosphate. They are also in accordance with those of Leyval and Berthelin (1989) who performed *Pinus sylvestris* cultures in lysimetric conditions where the culture substrate contained sand, phlogopite and rock phosphate. These authors showed that *Agrobacterium radiobacter*, known to solubilize insoluble phosphates, significantly enhanced beech growth and P, Mg, Al, K and Fe concentrations in roots and stems. However, the addition of soluble potassium to compensate the potassium deficiency observed after one year of experiment prevented the authors from measuring the potassium issued from the weathering of phlogopite and consequently from establishing a total weathering budget for potassium.

## Conclusion

We confirmed the significant impact of plant roots on mineral weathering and demonstrated that a bacterial isolate, *B. glathei* PML1(12), significantly improved plant nutrition and promoted plant growth, mainly because of its effect on mineral weathering. We also showed that interactions between different bacterial strains significantly modify the mineral weathering budgets. This is the first step in understanding the mechanisms of sustainability in forest ecosystems on nutrient-poor soils: by their joined contribution to mineral weathering, root and mycorrhizosphere bacteria are efficient enough for mobilising the nutrients required to maintain tree growth. Further researches are now needed to quantify the respective contribution of the ectomycorrhizal fungi and the associated bacteria in the mineral weathering process, in relation to tree mineral nutrition.

## Acknowledgements

We acknowledge J. Garbaye for critical review of the manuscript, J. Ranger and F. Lapeyrie for helpful advices, G. Nourrisson, C. Hossann, P. Bonnaud, S. Bienaimé, D. and L. Gelhaye for technical analyses, J. L. Churin, P. Vion, B. Palin, F. Guinet, B. Simon, A. Normand, M. Cuny, D. Le Thiec, A. Kohler, A. Nys, S. Uroz, J. Jaffrain and N. Angeli for technical help.

This work was supported by the French Research Ministry (ACI quantitative ecology project) and by the Lorraine Region.
3.3 Quantification du processus d'altération au cours de l'interaction biotite -
arbre forestier -microorganismes – solution

Nous avons complexifié notre plan d'expérience en intégrant le champignon
eyectomycorhizien dans notre dispositif en colonnes. Cette partie de notre travail visait (i)
à quantifier l'altération d'un minéral-test engendrée par différents acteurs biologiques
(plante, bactérie, champignon ectomycorhizien), seuls ou en association, et (ii) à
quantifier l'effet de l'inoculation d'une souche bactérienne, de l'inoculation d'un
champignon ectomycorhizien et de la double inoculation bactérie - champignon
eyectomycorhizien, sur la croissance de la plante. En complément, l'effet de deux
composés toxiques, comme l'uranium et le cadmium sur le niveau de fonctionnement
symbiotique et de la fonction altération, a été testé. Dans une perspective d'évaluation
de la dynamique du césium dans les écosystèmes forestiers, problématique prise en
charge par l'Andra dans le cadre de ses études de sûreté, une étude préliminaire de
faisabilité du dosage de césium dans les solutions et la biomasse végétale a été
entreprise à l'aide de notre dispositif en colonnes.

Matériel et méthodes

Ne sont décrits ci-dessous que les spécificités du matériel inhérent à cette
expérience qui divergent du protocole général présenté dans la partie "dispositif
expérimental en colonnes".

* Matériel microbiologique :

Cette expérience devait initialement associer le pin sylvestre, le champignon
ectomycorhizien *Scleroderma citrinum*, souche isolée sur le site de Breuil, et la souche
bactérienne *B. glathei* PML1(12) isolée dans l'ectomycorhizosphère du couple *S.
citrinum* - chêne. Cependant plusieurs tentatives de mycorhization du pin sylvestre par
*S. citrinum* n'ont pas abouti. C'est pourquoi nous avons décidé d'utiliser le champignon
ectomycorhizien *Laccaria bicolor* S238N car ce champignon, dont la mycorhization
contrôlée est maîtrisée dans le laboratoire INRA "Interaction Arbres-Microorganismes",
est capable de former des mycorhizes avec le pin sylvestre. La souche bactérienne
*Pseudomonas fluorescens* BBc6R8, auxiliaire de la mycorhization du Douglas par le
champignon *L. bicolor* S238N, isolée par Frey-Klett *et al*. (1997), a été choisie pour ses
<table><caption>Tableau IV.8 : Les différents traitements de l'expérience "interactions arbre forestier - microorganismes - minéral - solution". Les chiffres correspondent au nombre de répétitions</caption><thead><tr><th rowspan="2">Traitements biologiques</th><th colspan="3">Traitements chimiques</th></tr><tr><th>Solution nutritive de base</th><th>Solution nutritive de base + uranium</th><th>Solution nutritive de base + cadmium</th></tr></thead><tbody><tr><td>Sans pin</td><td>4</td><td></td><td></td></tr><tr><td>Pin non inoculé</td><td>4</td><td></td><td></td></tr><tr><td>Pin + P. fluorescens BBc6R8</td><td>4</td><td></td><td></td></tr><tr><td>Pin + L. bicolor S238N</td><td>4</td><td></td><td></td></tr><tr><td>Pin + P. fluorescens BBc6R8 + L. bicolor S238N</td><td>4</td><td>4</td><td>4</td></tr></tbody></table>

<table><caption>Tableau IV.9 : Taux de mycorhization des racines courtes de plants de pin en début et après 56 jours d'expérimentation.</caption><thead><tr><th rowspan="2">Traitements biologiques</th><th colspan="2">Pourcentage de racines courtes de plants de pin mycorhizés par L. bicolor S238N</th></tr><tr><th>En début d'expérience</th><th>En fin d'expérience</th></tr></thead><tbody><tr><td>Pin non inoculé</td><td>0</td><td>0</td></tr><tr><td>Pin + P. fluorescens BBc6R8</td><td>0</td><td>0</td></tr><tr><td>Pin + L. bicolor S238N</td><td>65*</td><td>28</td></tr><tr><td>Pin + P. fluorescens BBc6R8 + L. bicolor S238N</td><td>65*</td><td>23<sup>1</sup></td></tr><tr><td>Pin + P. fluorescens BBc6R8 + L. bicolor S238N</td><td>65*</td><td>23<sup>2</sup></td></tr><tr><td>Pin + P. fluorescens BBc6R8 + L. bicolor S238N</td><td>65*</td><td>21<sup>3</sup></td></tr></tbody></table>

<footnote>* Taux de mycorhization initial moyen de 12 plants de pin cultivés dans les mêmes conditions et sacrifiés en début d'expérimentation.</footnote>

<footnote n="1">¹ Traitement solution nutritive.</footnote>

<footnote n="2">² Traitement solution nutritive + uranium.</footnote>

<footnote n="3">³ Traitement solution nutritive + cadmium.</footnote>
interactions positives avec ce champignon. De plus, cette souche présente l'avantage
d'être marquée (résistance à 100 mg.l⁻¹ de rifampicine) et elle a également montré de
bonnes potentialités à mobiliser le fer et le phosphore lors des tests *in vitro*.

* Solutions nutritives :

Pour certains traitements, 59.5 mg.l⁻¹ d'uranium sous forme d'uranyl-acétate complexé ou 0.1 mg.l⁻¹ de cadmium sous forme de chlorure de cadmium ont été ajoutés à la solution nutritive de base.

* Traitements

Le tableau IV.8 présente les différents traitements mis en place dans cette expérience.

Résultats

* Effet des partenaires biologiques de la rhizosphère :

Le taux de mycorhization des racines courtes des plants de pin par le champignon *L. bicolor* S238N avoisinait les 65% en début d'expérience pour atteindre des valeurs inférieures à 30% en fin d'expérience et ce quel que soit le traitement biologique. Ainsi, l'inoculation de la souche *P. fluorescens* BBc6R8 n'a eu aucun effet sur le taux de mycorhization. (Tableau IV.9).

L'augmentation de biomasse des plants de pins inoculés avec la souche fongique *L. bicolor* S238N seule et celle des plants ayant subi la double inoculation, souche fongique *L. bicolor* S238N - souche bactérienne *P. fluorescens* BBc6R8, sont significativement plus importantes que celle des plants non inoculés (Figure IV.14). En revanche, l'inoculation avec la souche bactérienne *P. fluorescens* BBc6R8 seule, n'a pas d'effet promoteur de croissance sur les plants de pin (Figure IV.14). L'effet bénéfique de l'inoculation fongique et de la double inoculation s'observe uniquement sur la croissance des parties aériennes des plants de pin (Tableau IV.10).

Les quantités totales de potassium libérées par altération de la biotite sont significativement plus importantes en présence de la plante, en présence de la plante mycorhizée par *L. bicolor* S238N, et en présence de la plante mycorhizée par *L. bicolor* S238N et inoculée par *P. fluorescens* BBc6R8 par rapport au traitement non inoculé (Figure IV.15). En revanche, le traitement inoculé par *P. fluorescens* BBc6R8 n'est pas
Figure IV.14: Augmentation de la biomasse sèche des plants de pin durant les 56 jours de l'expérience en colonnes. Les barres représentent les écart-types. Chaque histogramme est la moyenne de quatre répétitions. Les moyennes associeés à des lettres différentes sont significativement différentes selon une ANOVA (P = 0.05) à un facteur (traitement biologique) et le test de Bonferroni-Dunn

<figure><img src="image_65.png" /><figcaption>Tableau IV.10: Augmentation de biomasse sèche des parties aériennes et racinaires des plants de pin (mg) durant les 56 jours de l'expérimentation. Les valeurs représentent la moyenne de quatre répétitions. Dans chaque colonne, les moyennes associées à des lettres différentes sont significativement différentes selon une ANOVA (P = 0.05) à un facteur (traitement biologique), et le test de Bonferroni-Dunn.</figcaption></figure>

<table><thead><tr><th rowspan="2">Traitements biologiques</th><th colspan="2">Augmentation de biomasse sèche (mg)</th></tr><tr><th>Partie aérienne</th><th>Partie racinaire</th></tr></thead><tbody><tr><td>Pin non inoculé</td><td>128<sup>a</sup></td><td>40<sup>a</sup></td></tr><tr><td>Pin + P. fluorescens BBc6R8</td><td>145<sup>a</sup></td><td>36<sup>a</sup></td></tr><tr><td>Pin + L. bicolor S238N</td><td>222<sup>b</sup></td><td>47<sup>a</sup></td></tr><tr><td>Pin + P. fluorescens BBc6R8 + L. bicolor S238N</td><td>257<sup>b</sup></td><td>47<sup>a</sup></td></tr></tbody></table>
**Figure IV.15:** Quantité de potassium (drainée en solution et immobilisée par le plant de pin) mobilisée par altération de la biotite durant les 56 jours de l'expérience en colonnes. Les barres représentent les écart-types. Chaque histogramme est la moyenne de quatre répétitions. Les moyennes associées à des lettres différentes sont significativement différentes selon une ANOVA (P = 0.05) à un facteur (traitement biologique) et le test de Bonferroni-Dunn.

<figure><img src="image_66.png" /></figure>

**Figure IV.16:** Pourcentage de potassium immobilisé par les plants de pin (= quantité de potassium immobilisée par le plant de pin / (quantité de potassium drainée en solution + quantité de potassium immobilisée par le plant de pin)) durant les 56 jours de l'expérience en colonnes. Les barres représentent les écart-types. Chaque histogramme est la moyenne de quatre répétitions. Les moyennes associées à des lettres différentes sont significativement différentes selon une ANOVA (P = 0.05) à un facteur (traitement biologique) et le test de Bonferroni-Dunn.

<figure><img src="image_67.png" /></figure>
significativement différent du traitement témoin non inoculé (Figure IV.15). Si l'on
compare les traitements biologiques, l'altération de la biotite est significativement plus
élevée en présence de la plante seule comparativement aux traitements "plante inoculée
par *P. fluorescens* BBc6R8" et "plante inoculée par le couple *L. bicolor* S238N - *P. fluorescens* BBc6R8" (Figure IV.15). Le pourcentage de potassium prélevé par la plante
est significativement plus important lorsque le plant de pin a subi l'inoculation par *L. bicolor* ou la double inoculation (*L. bicolor* et *P. fluorescens*) comparativement au plant
de pin non inoculé (Figure IV.16).

* Effet de l'uranium et du cadmium

Seuls les plants mycorhizés par *L. bicolor* S238N et inoculés par la souche bactérienne *P. fluorescens* ont subi ces traitements chimiques.

Nous n'avons observé aucun effet de l'uranium ou du cadmium sur le taux de mycorhization des racines courtes de plants de pin par le champignon *L. bicolor* S238N (Tableau IV.9). En revanche, la croissance des plants de pin était significativement plus faible pour le traitement chimique avec uranium comparativement au traitement témoin (solution nutritive sans uranium ni cadmium) (Figure IV.17). L'effet inhibiteur de l'apport d'uranium s'observait uniquement sur la croissance des parties aériennes des plants de pin (Tableau IV.11). En ce qui concerne le traitement cadmium, nous n'observons pas observé d'effet inhibiteur sur la croissance des plants de pin (Figure IV.17). La densité de la microflore bactérienne cultivable totale est significativement plus faible pour le traitement chimique avec uranium comparativement au traitement témoin (Figure IV.18). En revanche, nous n'observons pas d'effet inhibiteur du cadmium sur cette densité bactérienne (Figure IV.18).

Les quantités totales de potassium mobilisées (drainées en solution + prélevées par la plante) par altération de la biotite sont significativement plus faibles dans le cas du traitement chimique avec uranium comparativement au traitement témoin (Figure IV.19). De même, le pourcentage de potassium immobilisé dans les plants est significativement plus faible dans le traitement ayant reçu une solution nutritive avec uranium comparativement au traitement témoin (Figure IV.20). Nous n'observons aucun effet significatif du cadmium sur la mobilisation du potassium et son immobilisation dans les plants (Figure IV.19).
Figure IV.17: Augmentation de la biomasse sèche des plants de pin durant l'expérience en colonnes. Les barres représentent les écart-types. Chaque histogramme est la moyenne de quatre répétitions. Les moyennes associées à des lettres différentes sont significativement différentes selon une ANOVA (P = 0.05) à un facteur (traitement chimique) et le test de Bonferroni-Dunn.

<figure><img src="image_68.png" /><figcaption>Tableau IV.11: Augmentation de biomasse sèche des parties aériennes et racinaires des plants de pin (mg) durant les 56 jours de l'expérimentation. Les valeurs représentent la moyenne de quatre répétitions. Les valeurs associées à des lettres différentes sont significativement différentes selon une ANOVA (P = 0.05) à un facteur (traitement chimique), et le test de Bonferroni-Dunn.</figcaption></figure>

<table><thead><tr><th rowspan="2">Traitements</th><th colspan="2">Augmentation de biomasse sèche (mg)</th></tr><tr><th>Partie aérienne</th><th>Partie racinaire</th></tr></thead><tbody><tr><td>Pin + <i>P. fluorescens</i> BBc6R8 +<br><i>L. bicolor</i> S238N</td><td>257<sup>a</sup></td><td>47<sup>a</sup></td></tr><tr><td>Pin + <i>P. fluorescens</i> BBc6R8 +<br><i>L. bicolor</i> S238N + uranium</td><td>83<sup>b</sup></td><td>41<sup>a</sup></td></tr><tr><td>Pin + <i>P. fluorescens</i> BBc6R8 +<br><i>L. bicolor</i> S238N + cadmium</td><td>198<sup>a</sup></td><td>45<sup>a</sup></td></tr></tbody></table>
Figure IV.18: Logarithme des densités de la microflore bactérienne cultivable totale après 56 jours d'expérimentation. Les barres représentent les écart-types. Chaque histogramme est la moyenne de quatre répétitions. Les moyennes associées à des lettres différentes sont significativement différentes selon une ANOVA (P = 0.05) à un facteur (traitement chimique) et le test de Bonferroni-Dunn

<figure><img src="image_69.png" /></figure>

Figure IV.19: Quantité de potassium (drainée en solution et immobilisée par le plant de pin) mobilisée par altération de la biotite durant les 56 jours de l'expérience en colonnes. Les barres représentent les écart-types. Chaque histogramme est la moyenne de quatre répétitions. Les moyennes associées à des lettres différentes sont significativement différentes selon une ANOVA (P = 0.05) à un facteur (traitement chimique) et le test de Bonferroni-Dunn.

<figure><img src="image_70.png" /></figure>
Figure IV.20 : Pourcentage de potassium immobilisé dans les plants de pin (= quantité de potassium immobilisée par le plant de pin / (quantité de potassium drainée en solution + quantité de potassium immobilisée par le plant de pin)) au cours des 56 jours de l'expérience en colonnes. Les barres représentent les écart-types. Chaque histogramme est la moyenne de quatre répétitions. Les moyennes associées à des lettres différentes sont significativement différentes selon une ANOVA (P = 0.05) à un facteur (traitement chimique) et le test de Bonferroni-Dunn

<figure><img src="image_71.png" /></figure>
Figure IV.21: Quantité de césium (drainée en solution et immobilisée par le plant de pin) mobilisée par altération de la biotite durant l'expérience en colonnes durant les 56 jours de l'expérience en colonnes. Les barres représentent les écart-types. Chaque histogramme est la moyenne de quatre répétitions. Les moyennes associées à des lettres différentes sont significativement différentes selon une ANOVA (P = 0.05) à un facteur (traitement biologique) et le test de Bonferroni-Dunn.

<figure><img src="image_72.png" /></figure>

Tableau IV.12: Pourcentage de césium immobilisé par les plants de pin (= quantité de césium immobilisée par le plant de pin / (quantité de césium drainée en solution + quantité de potassium immobilisée par le plant de pin)) durant les 56 jours de l'expérience en colonnes. Les barres représentent les écart-types. Chaque valeur est la moyenne de quatre répétitions. Pour les plants alimentés avec la solution nutritive de base, les traitements associés à des lettres identiques (a) ne sont pas significativement différents selon une ANOVA (P = 0.05) à un facteur (traitement biologique) et le test de Bonferroni-Dunn. Pour les plants ayant subi la double inoculation et alimentés en solution nutritive enrichie en uranium ou en cadmium, les moyennes associées à des lettres différentes (A ou B) sont significativement différentes selon une ANOVA (P = 0.05) à un facteur (traitement chimique) et le test de Bonferroni-Dunn.

<table><thead><tr><th>Traitements chimiques</th><th>Traitements chimiques</th><th>% de césium immobilisé par les pins</th></tr></thead><tbody><tr><td>Pin non inoculé</td><td>aucun</td><td>84<sup>a</sup></td></tr><tr><td>Pin + P. fluorescens BBc6R8</td><td>aucun</td><td>86<sup>a</sup></td></tr><tr><td>Pin + L. bicolor S238N</td><td>aucun</td><td>84<sup>a</sup></td></tr><tr><td>Pin + P. fluorescens BBc6R8 + L. bicolor S238N</td><td>aucun</td><td>91<sup>aA</sup></td></tr><tr><td>Pin + P. fluorescens BBc6R8 + L. bicolor S238N</td><td>uranium</td><td>69<sup>B</sup></td></tr><tr><td>Pin + P. fluorescens BBc6R8 + L. bicolor S238N</td><td>cadmium</td><td>87<sup>A</sup></td></tr></tbody></table>
• Essai d'un bilan d'altération sur le césium

Notre travail montre qu'il est possible de réaliser un dosage du césium en solution et
dans les parties racinaires et aériennes de la plante. Nous avons mis en évidence un effet
significatif de la plante non inoculée et de la plante associée avec les différents
partenaires biologiques de la rhizosphère sur la mobilisation du césium issu de
l'altération de la biotite (Figure IV.21), la très grande majorité du césium en solution
étant prélevée par les plants quels que soient les traitements considérés (Tableau IV.12).
Le bilan d'altération réalisé sur le césium confirme l'effet inhibiteur de l'uranium sur
l'altération de la biotite (Figure IV.22) et sur la quantité de césium prélevée par
l'association symbiotique (Tableau IV.12).

Interprétation et discussion des résultats

Cette expérimentation en colonnes avait pour but de faire interagir les différents partenaires biologiques de la rhizosphère d'un arbre forestier avec la phase minérale et la solution du sol. En ce sens, elle simulait un écosystème forestier simplifié, nous permettant de déterminer la contribution de chacun des partenaires biologiques dans l'altération minérale et d'envisager les interactions qui peuvent régner entre les différentes composantes du système.

Nous avons observé une augmentation significative de la croissance des plants de pin en
présence du champignon *L. bicolor* S238N, associé ou non à la souche bactérienne *P. fluorescens* malgré le faible pourcentage de mycorhization des semis de pin par *L. bicolor* S238N en fin d'expérience. Il est à noter que la souche *P. fluorescens* BBc6R8 n'a pas stimulé la mycorhization des semis par *L. bicolor* S238N dans cette expérience.
Ceci peut s'expliquer par le fait que cette souche a été inoculée sur des semis déjà mycorhizés et non pas au moment de l'inoculation du champignon ectomycorhizien comme cela est pratiqué couramment dans les expériences visant à quantifier l'effet auxiliaire de cette bactérie (Frey-Klett et al., 1997). En effet, Brulé et al. (2001) ont suggéré que l'effet auxiliaire de cette souche bactérienne résulterait principalement d'une stimulation par la bactérie de la survie et de la croissance du champignon en condition présymbiotique. Dans notre dispositif en colonnes, les conditions expérimentales n'auraient donc pas été propices au potentiel auxiliaire de la souche *P. fluorescens* BBc6R8. Les champignons ectomycorhiziens sont connus pour améliorer la croissance des arbres grâce à de nombreux mécanismes tels que la protection contre les
Figure IV.22: Quantité de césium (drainée en solution et immobilisée par le plant de pin) mobilisée par altération de la biotite durant les 56 jours de l'expérience en colonnes. Les barres représentent les écart-types. Chaque histogramme est la moyenne de quatre répétitions. Les moyennes associées à des lettres différentes sont significativement différentes selon une ANOVA (P = 0.05) à un facteur (traitement chimique) et le test de Bonferroni-Dunn

<figure><img src="image_73.png" /></figure>
pathogènes, la production d'hormones de croissance ou bien en favorisant l'acquisition
de nutriments via une mobilisation accrue de la réserve minérale du sol et/ou un
prélèvement supérieur par les racines (Jongmans et al., 1997 ; Van Breemen et al.,
2000 ; Landeweert et al., 2001 ; Van schöll et al., 2005). Ce dernier mécanisme semble
être à l'origine de l'effet promoteur de croissance des plants de pin observé dans notre
expérience. En effet, nos résultats montrent que les quantités de potassium mobilisées
par les plants de pin mycorhizés sont inférieures à celles mobilisées par les plants non
mycorhizés alors que les pourcentages de potassium prélevés par les plants mycorhizés
sont très nettement supérieurs à ceux prélevés par les plants non mycorhizés. Ce n'est
donc pas la production de composés altérants par les racines mycorhizées mais bien
l'augmentation des quantités de potassium accessibles aux racines de pin qui serait le
mécanisme majeur de la promotion de croissance des plants mycorhizés observés dans
notre expérience. Ces résultats s'opposent à ceux de Glowa et al. (2003) et Wallander et
al. (2005) qui ont montré que des champignons ectomycorhiziens appartenant à d'autres
espèces telles que Piloderma pouvaient fournir plus de potassium disponible à la plante
hôte en accélérant l'extraction du potassium des feuillets interfoliaires de la biotite.
Cependant, Blum et al. (2002) ont démontré que dans un sol pauvre en calcium, les
champignons ectomycorhiziens associés aux racines d'épicéa fournissaient 95% du
calcium accumulé dans le feuillage des arbres grâce à leur capacité à absorber
directement les ions minéraux issus de l'altération de l'apatite avant que les ions
calcium n'entrent dans le complexe d'échange du sol. Il semblerait donc que les
champignons ectomycorhiziens puissent contribuer à la nutrition minérale de la plante
par des mécanismes différents. Ces mécanismes sont-ils dépendants de la souche, de
l'espèce ou du genre de champignons ectomycorhiziens ? Pour le moment, la question
reste ouverte car à notre connaissance, aucune étude n'a encore été menée dans ce sens.
L'inoculation de la souche bactérienne P. fluorescens sélectionnée pour sa capacité à
mobiliser le phosphore et le fer in vitro et présentant un bon potentiel d'altération de la
biotite dans le test en microplaques, n'a pas eu d'effet stimulant sur la croissance des
plants de pin mycorhizés ou non, ni sur l'altération de la biotite. L'hypothèse la plus
plausible pour expliquer ces résultats réside dans le fait que la souche BBc6R8 qui a
apparemment totalement disparu des colonnes où elle avait été inoculée (résultats non
présentés), n'a peut-être pas trouvé dans nos conditions expérimentales la niche dont
elle avait besoin pour exprimer son potentiel d'altération. Il a été suggéré que cette
souche serait, non pas rhizosphérique, mais hyposphérique. Sa survie dépendrait en
effet de la présence du champignon *L. bicolor* S238N (Brulé, 2001). En conséquence, le faible taux de mycorhization des semis au cours de notre expérience permet peut-être d'expliquer la disparition apparente de la souche BBc6R8 dans les colonnes durant l'expérimentation. Il est important de noter que nous ne pouvons pas affirmer que la souche a réellement disparu dans la mesure où nous n'avons pas recherché la présence éventuelle de cellules bactériennes viables non cultivables. De plus, il aurait été pertinent de suivre l'évolution de la souche BBc6R8 au cours du temps de manière à apprécier la vitesse de décroissance de cette population dans nos conditions expérimentales. Malheureusement, une telle expérience qui impose obligatoirement des prélèvements destructifs n'a pu être réalisée, du fait du nombre limité de colonnes que nous pouvions alimenter en solution nutritive avec les pompes péristaltiques à notre disposition.

Malgré tout, dans notre expérience, un effet positif du pin, de l'association symbiotique pin - champignon ectomycorhizien *L. bicolor* et de l'association symbiotique pin - champignon ectomycorhizien *L. bicolor* - souche bactérienne *P. fluorescens* BBc6R8 sur l'altération de la biotite a pu être observé. La production d'agents altérants (H⁺, acide carbonique, acides organiques,...) par la plante et les microorganismes ainsi que le prélèvement et la fixation de cations de la solution sur les parois racinaires et fongiques et les membranes bactériennes, induisant un déséquilibre solution - minéral, pourraient expliquer cette stimulation du processus d'altération de la phase minérale.

La seconde partie de notre travail a consisté à évaluer la toxicité de l'uranium et du cadmium ajoutés dans la solution nutritive sur l'activité de la relation tripartite pin - champignon ectomycorhizien *L. bicolor* - souche bactérienne *P. fluorescens*, et le processus d'altération des minéraux. Notre résultat majeur est l'effet inhibiteur très marqué de l'uranium sur la croissance des plants de pin, les densités bactériennes et fongiques et l'activité biologique des plantes et des microorganismes associés, se traduisant par une nette diminution de l'altération de la biotite et un prélèvement de potassium par la symbiose ectomycorhizienne inférieur au traitement témoin. A l'inverse, le cadmium n'a pas eu d'effet dépressif. Ceci pourrait être dû à la faible concentration en cadmium utilisée dans cette expérience (0.1 mg.l⁻¹) qui s'avérait ainsi inférieure au seuil de toxicité de cet élément sur la symbiose ectomycorhizienne. Weissenhorn *et al*. (1993) ont montré que la dose de cadmium nécessaire pour provoquer la mortalité de 50% de la population d'un champignon endomycorhizien *Glomus mossae* était de 0.8 mg.l⁻¹. Une autre hypothèse non exclusive serait que le
<figure><img src="image_74.png" /><figcaption>Figure IV.23 : Schéma de synthèse résumant tous les effets observés dans les expérimentations en colonnes. La largeur des flèches représente le niveau d'altération de la biotite, plus la largeur de la flèche est importante, plus l'altération a été forte. La même symbolique a été utilisée pour caractériser la croissance des parties racinaires et des parties aériennes des plants de pin. Seuls les niveaux d'altération de la biotite associées à une même expérimentation (flèches de même couleur) sont comparables. En orange : l'expérience d'interactions minéral - microorganismes. En vert : l'expérience d'interactions minéral - pin - bactéries. En bleu, l'expérience d'interactions pin - champignon ectomycorhizien - bactéries.</figcaption></figure>
champignon ectomycorhizien L. bicolor S238N pourrait protéger la plante hôte vis-à-vis
de faibles doses en cadmium. Il a en effet déjà été montré que de nombreux
champignons ectomycorhiziens augmentent la tolérance aux métaux lourds de leurs
hôtes (Wilkins, 1991) en piégeant les métaux dans le mucilage à la surface de leurs
hyphes, dans leurs parois ou dans leurs vacuoles (Kottke, 1992).

Enfin, en ce qui concerne notre tentative de bilan d'altération sur le césium, nous avons
pu constater que le césium drainé en solution et le césium immobilisé par la biomasse
végétale sont à des concentrations mesurables. Les résultats obtenus avec le césium
confirment l'effet positif de la plante sur l'altération de la biotite et l'effet inhibiteur de
l'uranium sur le prélèvement. De plus, nous avons montré dans les traitements sans
uranium, que la majeure partie du césium mobilisé (entre 84 et 91%) est prélevée par la
plante alors que seulement 26 à 48% du potassium mobilisé est quant à lui prélevé,
supportant ainsi l'hypothèse de Broadley et Wiley (1997) selon laquelle le césium
pourrait être prélevé et transféré dans la plante à la place du potassium. Des études
complémentaires sont certes nécessaires pour valider les résultats obtenus en faisant
notamment varier les paramètres expérimentaux tels que l'âge des plants qui influe
considérablement sur le prélèvement, et la structure des minéraux argileux qui contrôle
fortement la libération du césium en solution (Smolders, 2001). Dans le cadre des
études de sûreté de l'Andra, ces résultats pourraient se révéler très utiles.

4. SYNTHESE ET DISCUSSION

La figure IV.23 résume tous les résultats obtenus dans ces expérimentations en
colonnes.

Nos expérimentations en colonnes ont permis de quantifier la part respective de
l'altération de la biotite, engendrée par la solution seule, par les plantes et diverses
souches bactériennes et fongiques, seules ou en association. Les résultats obtenus ont
démontré que les souches bactériennes *B. glathei* PML1(12) et PMB4(3) et, à un degré
moindre, les souches fongiques SN2(3) et PML2(10), isolées dans
l'ectomycorhizosphère du couple chêne - *Scleroderma citrinum* sur le site forestier de
Breuil, augmentent l'altération de la biotite par des facteurs compris entre 1.7 et 2.4
(magnésium mobilisé) comparativement au traitement abiotique mais uniquement en
présence de glucose. Ce dernier, qui n'a pas agi directement sur la phase minérale, pourrait avoir indirectement accéléré l'altération de la biotite de deux manières distinctes dans notre expérimentation :

(i) la dégradation du glucose par les communautés microbiennes de la colonne pourrait générer la production de métabolites acides capables d'attaquer la surface minérale. Des pH de 3.2 et 3.8 ont été atteints respectivement en présence des souches bactériennes B. glathei PML1(12) et PMB4(3) alors que le pH du traitement abiotique était resté proche de la neutralité tout au long de l'expérience. Il est important de préciser que dans ce type d'expérimentation en colonnes utilisant un minéral-test, la quantité de protons réellement produite par les microorganismes est considérablement sous-estimée car l'altération de la biotite est un processus consommateur de protons : la biotite joue un rôle de tampon de l'acidité. D'ailleurs toutes les expérimentations en microplaques et en colonnes, même celles utilisant un acide fort comme l'acide chlorhydrique concentré, ont montré l'existence d'un seuil du pH avoisinant des valeurs de 3 qui est probablement lié à cet effet tampon de la biotite. De même, des pH considérablement plus acides peuvent exister au niveau de certains microsites (Liermann et al., 2000).

(ii) le glucose a certainement stimulé non seulement la croissance mais aussi l'activité des communautés microbiennes comme en atteste le suivi des densités bactériennes dans les différents traitements après 21 jours d'expérimentation : la microflore bactérienne cultivable a été multipliée par des facteurs 100 pour les traitements non inoculés et les traitements inoculés avec les souches fongiques SN2(3) et PML2(10), à 1000 pour les traitements inoculés avec les souches bactériennes (PML1(12) et PMB4(3), en présence de glucose. Dans de telles conditions, l'activité microbienne accrue a pu générer un certain nombre de métabolites aux propriétés altérantes tels que les sidérophores (Schmitt et al., 2003). En considérant nos données actuelles, il ne nous est pas possible de trancher entre ces deux hypothèses. En effet, nous ne disposons d'aucune mesure des densités de souches bactériennes PML1(12) et PMB4(3) et des souches fongiques SN2(3) et PML2(10) dans les colonnes en fin d'expérimentation. De plus, nous ne connaissons pas la composition en acides organiques de la solution drainée. Des expérimentations ultérieures seront nécessaires pour acquérir ces nouvelles données.

Les résultats obtenus dans nos différentes expériences mettent tous en évidence un effet positif de la plante sur l'altération de la phase minérale comparativement au traitement sans plante ni microorganismes. Ces résultats confirment les résultats d'un grand
nombre d'études (Hinsinger et Gilkes, 1997 ; Hinsinger et al., 2001) mais pour la première fois, ils permettent également une quantification précise de l'effet de la plante sur l'altération de la biotite. L'effet positif des plants de pin sur l'altération varie entre +30% et +100%. Aucune transformation de la biotite n'a pu être mise en évidence dans nos expériences. Nous avons observé une destruction de la structure de la phase minérale par une simple dissolution congruente. Cette observation contraste avec bon nombre de données de la littérature mettant en évidence des transformations des minéraux au contact des racines (Hinsinger et al., 1992). Ces divergences peuvent s'expliquer principalement par la courte durée de nos expériences. Leyval et al. (1990) a observé, dans la rhizosphère de plants de pin, une transformation partielle en vermiculite d'un autre phyllosilicate, la phlogopite, mais après un an d'expérimentation. De plus, la plupart des expérimentations présentées dans la littérature plaçait le minéral dans un environnement racinaire très dense produisant d'importantes quantités d'agents altérants et prélevant abondamment et plus spécifiquement certains éléments comme le potassium. Dans notre expérience, du fait d'une densité racinaire moyenne dans les colonnes et étant donné le déplacement de la racine lié à sa croissance, une particule de biotite sera soumise à l'activité altérante de la racine et plus particulièrement de l'apex, durant une courte période de temps, probablement insuffisante pour transformer la biotite.

Un effet additionnel de la souche bactérienne *B. glathei* PML1(12) sur l'altération de la biotite (+40% par rapport au traitement plante seule) et sur la croissance des plants de pin (+50% par rapport au traitement plante seule) a été observé. Ce dernier effet est également observé pour la souche bactérienne *B. glathei* PMB1(7). Des études complémentaires ont montré que l'effet promoteur de croissance exercé par la souche *B. glathei* PML1(12) sur les plants de pin était principalement lié à sa capacité à mobiliser des nutriments tels que le potassium et le magnésium par altération de la biotite. Nos travaux confirment ainsi les observations faites par Jackson et Voigt (1971) pour le calcium, Boyle et Voigt (1973) pour le potassium, Kloepper et al. (1980) pour le fer, Leyval et Berthelin (1989) pour le magnésium, à savoir une amélioration de la nutrition des plantes lors de l'inoculation de certaines souches bactériennes rhizosphériques. Une co-inoculation de la souche PML1(12) avec la souche *B. glathei* PML1(4) inhibe ce potentiel d'altération et de stimulation de la croissance de la plante. Des phénomènes de compétition entre souches peuvent être invoqués (Curl et Truelove, 1986 ; Davet, 1996). Bent et al. (2001) suggèrent que la présence d'autres souches bactériennes peut inhiber