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Interspecific variation among 87 sporocarps of fungi belonging to 15 genera and 39 species were evaluated by analyzing the internal transcribed spacer (ITS) of the rDNA region using restriction fragment length polymorphism (RFLP). The ITS region was first amplified by polymerase chain reaction (PCR) with specific primers and then cleaved with different restriction enzymes. Amplification products, which ranged between 500 and 950 base pairs (bp), were obtained for all the isolates analyzed. The degree of polymorphism observed did not allow proper identification of most of the species. Cleavage of amplified fragments with the restriction enzymes Alu I, EcoR I and Hinf I revealed extensive polymorphism. The fifteen genera and most species presented specific restriction patterns. The only species not identifiable by a specific pattern belonged to the genera Russula (R. decipiens and R. straminea). These species might be considered as closely related species. The Pisolithus sporocarps had two ITS-RFLP types with one dominating. ITS sequencing confirms that the two RFLP types correspond to two distinct species of Pisolithus. Our data show the potential of ITS region PCR-RFLP for the molecular characterization of ectomycorrhizal fungi and their identification and monitoring in artificial inoculation programs.

tThe taxonomic status of eleven rhizospheric bacterial strains belonging to the genus Burkholderia andisolated from roots of Costularia (Cyperaceae), tropical herbaceous pioneer plants growing on ultramaficsoils in New Caledonia, was investigated using a polyphasic taxonomic approach. The genetic analyses(16S rRNA genes, gyrB, recA, nreB and cnr) confirmed that all strains are Burkholderia and cluster into twoseparated groups. The DNA hybridization results showed low relatedness values to the closest relativesBurkholderia species. The phenotypic analyses confirmed that the two groups of strains could be differ-entiated from each other and from other known Burkholderia species. This polyphasic study revealedthat these two groups of strains represent each a novel species of Burkholderia, for which the namesBurkholderia novacaledonica sp. nov. (type strain STM10272T= LMG28615T= CIP110887T) and B. ultra-mafica sp. nov. (type strain STM10279T= LMG28614T= CIP110886T) are proposed, respectively. Thesestrains of Burkholderia presented specific ecological traits such as the tolerance to the extreme edaphicconstraints of ultramafic soils: they grew at pH between 4 and 8 and tolerate the strong unbalancedCa/Mg ratio (1/19) and the high concentrations of heavy metals i.e. Co, Cr, Mn and Ni. Noteworthy B.ultramafica tolerated nickel until 10 mM and B. novacaledonica up to 5 mM. The presence of the nickel(nreB) and cobalt/nickel (cnr) resistance determinants encoding for protein involved in metal tolerancewas found in all strains of both groups. Moreover, most of the strains were able to produce plant growthpromoting molecules (ACC, IAA, NH3and siderophores). Such ecological traits suggest that these newspecies of Burkholderia might be environmentally adaptable plant-associated bacteria and beneficialto plants.

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International audience

Distribution and Diversity of Glomalean Endomycorrhizal Fungi of Senegal. In arid and semi-arid tropical areas characterized by plant ever-increasing damage, arbuscular endomycorrhizal fungi seem to play a major role in the conquest of new terrestrial spaces and are essential for the sur-vival of most plant communities under diverse edaph-ic and environmental conditions. However, the distribution and the diversity of indigenous fungal species in Senegal have been very little investigated to date. The goal of this study was to evaluate spore populations of Glomales from soil samples withdrawn from several sites located in the sahelian, sahelo-soudanian and soudanian areas. The spores extracted by wet-sieving and decanting method were counted and identified. Our findings highlight the presence of the three genera Glomus, Scutellospora and Gigaspora and the absence of Acaulospora, Entrophospora and Sclerocystic species in different sites in which soil spore contents were generally poor. However, diversity and relative abundance of spore populations in some sites seemed related to the increase of rainfall as well as soils and plants factors.

International audience

International audience

Nickel (Ni)-tolerant ectomycorrhizal Pisolithus albus was isolated from extreme ultramafic soils that are naturally rich in heavy metals. This study aimed to identify the specific molecular mechanisms associated with the response of P. albus to nickel. In presence of high concentration of nickel, P. albus Ni-tolerant isolate showed a low basal accumulation of nickel in its fungal tissues and was able to perform a metal efflux mechanism. Three genes putatively involved in metal efflux were identified from the P. albus transcriptome, and their overexpression was confirmed in the mycelium that was cultivated in vitro in the presence of nickel and in fungal tissues that were sampled in situ. Cloning these genes in yeast provided significant advantages in terms of nickel tolerance (+ 31% Ni EC50) and growth (+ 83% u) compared with controls. Furthermore, nickel efflux was also detected in the transformed yeast cells. Protein sequence analysis indicated that the genes encoded a P-type-ATPase, an ABC transporter and a major facilitator superfamily permease (MFS). This study sheds light on a global mechanism of metal efflux by P. albus cells that supports nickel tolerance. These specific responses to nickel might contribute to the fungal adaptation in ultramafic soil.

Metal-rich soils, especially heavy metal-polluted soils and ultramafic soils, are generally toxic to non-adapted plants and microorganisms. The role of mycorrhizal fungi in the metal tolerance of adapted plant species has become clear in the last decade. This review aims to synthesize the findings of representative studies of the effects of mycorrhizas on the alleviation of heavy metal toxicity on plants and on the absorption/accumulation of heavy metals in their roots and shoots. The adaptation to heavy metals by mycorrhizal symbionts is associated with their efficiency in metal-rich soils. More than 80 % of the studies have indicated a positive role of mycorrhizal fungi in the adaptation of plants to heavy metals in these soils, but the relationships between plant tolerance to heavy metals and the absorption of metals are complex and depend on a range of biological, physical and chemical factors.

Arbuscular mycorrhizal fungi (AMF) are obligatory biotrophs that have a symbiotic evolutionary relationship with about 80% of all terrestrial plant species. The fungus mainly supplies water and nutrients to the plant and receives photoassimilates. The AMF diversity affects both the competition among species and floristic composition of an area. Fluctuations in the population of this group of microorganisms can cause fluctuations in plant populations above ground. In this work, the AMF community profile in areas with different ages of revegetation was evaluated by morphological identification of spores and denaturing gradient gel electrophoresis (DGGE) techniques. We found 12 AMF species and dominance of the species Glomus macrocarpum determined by spore density. Since the richness level observed in each plot was low and there was predominance of one species of AMF, it can be concluded that these areas still present a high degree of disturbance. It was possible to detect complex band profiles by DGGE analyses for the two plant species studied, Visnia latifolia and Cecropia hololeuca. No relationship between AMF diversity and revegetation time was observed in these areas.

The fungus Pisolithus albus forms ectomycorrhizal (ECM) associations with plants growing on extreme ultramafic soils, which are naturally rich in heavy metals such as nickel. Both nickel-tolerant and nickel-sensitive isolates of P.similar to albus are found in ultramafic soils in New Caledonia, a biodiversity hotspot in the Southwest Pacific. The aim of this work was to monitor the expression of genes involved in the specific molecular response to nickel in a nickel-tolerant P.similar to albus isolate. We used pyrosequencing and quantitative polymerase chain reaction (qPCR) approaches to investigate and compare the transcriptomes of the nickel-tolerant isolate MD06-337 in the presence and absence of nickel. A total of 1 071 375 sequencing reads were assembled to infer expression patterns of 19 518 putative genes. Comparison of expression levels revealed that 30% of the identified genes were modulated by nickel treatment. The genes, for which expression was induced most markedly by nickel, encoded products that were putatively involved in a variety of biological functions, such as the modification of cellular components (53%), regulation of biological processes (27%) and molecular functions (20%). The 10 genes that pyrosequencing analysis indicated were induced the most by nickel were characterized further by qPCR analysis of both nickel-tolerant and nickel-sensitive P.similar to albus isolates. Five of these genes were expressed exclusively in nickel-tolerant isolates as well as in ECM samples in situ, which identified them as potential biomarkers for nickel tolerance in this species. These results clearly suggest a positive transcriptomic response of the fungus to nickel-rich environments. The presence of both nickel-tolerant and nickel-sensitive fungal phenotypes in ultramafic soils might reflect environment-dependent phenotypic responses to variations in the effective concentrations of nickel in heterogeneous ultramafic habitats.

International audience

Nous avons effectué des inoculations ponctuelles sur le système racinaire de Medicago laciniata, cultivée in vitro La bactérie symbiotique Sinorhizobium meliloti et le champignon mycorhizien arbusculaire Glomus sp. S043 ont été utilisés. Nous avons étudié la réponse chimique du tissu racinaire en présence et en l'absence de la bactérie ou du champignon par observation en microscopie visible et à épi-fluorescence. L'inoculation ponctuelle de Sinorhizobium meliloti ou Glomus sp. S043 induit une accumulation de flavonoïdes localisée autour du point d'inoculation. Les autres zones de la racine ne présentent aucune accumulation de flavonoïdes. Il n'a été observé d'accumulation de tannins ni chez le contrôle ni dans les racines inoculées. Le rôle de ces flavonoïdes est discuté.

Afin d'améliorer les reboisements, certaines espèces d'Acacia d'origine australienne comme A. holosericea ou A. mangiuni ont été introduites au Sénégal. Toutefois, il s'est avéré que ces 2 espèces étaient très sensibles aux nématodes du genre Meloidogyne et plus particulièrement à M. javanica, espèce prédominante au Sénégal. Cette sensibilité entraîne 3 inconvénients majeurs dans l'utilisation de ces 2 essences : une inhibition de la croissance de l'arbre en plantation, une inhibition de la fixation biologique de l'azote et enfin la création d'un réservoir permanent pour ces nématodes phytoparasites susceptibles ainsi d'attaquer les cultures légumières adjacentes. En conséquence, il est nécessaire d'équiper les jeunes plants de microorganismes capables d'agir contre ces pathogènes. Les champignons mycorhiziens peuvent résoudre ce problème en contrôlant le développement des nématodes. Les acacias australiens peuvent présenter 2 types de symbioses mycorhiziennes : des endomycorhizes à vésicules et à arbuscules et des ectomycorhizes. Nous avons étudié l'effet de la symbiose ectormycorhizienne sur le cycle de développement de M. javanica. Des souches de Pisolithus sp. (champignon ectornycorhizogène) ont été isolées et mises en culture pure à partir de carpophores collectés dans les régions centre et Sud du Sénégal. Ensuite, des tests de compatibilité entre ces isolats fongiques et de jeunes A. holosericea ont été réalisés en conditions axéniques. Sur 33 isolats fongiques, seulement 4 ont la capacité d'infecter A. holosericea. Ces souches ont été étudiées pour leur impact sur 2 étapes du cycle de développement de Meloidogyne : éclosion des oeufs et pénétration des juvéniles dans la racine. Les exSudats de 2 isolats (COI 019 et COI 032) induisent une toxicité vis-à-vis de la viabilité des oeufs et inhibent la pénétration des juvéniles dans la racine. L'utilisation de ces champignons ectomycorhiziens dans des programmes agroforestiers est discutée.