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The symbiotic establishment of mycorrhizal fungi on plant roots is affected in various ways by the other microorganisms of the rhizosphere, and more especially by bacteria. This review discusses the case of some of these bacteria which consistently promote mycorrhizal development, leading to the concept of 'mycorrhization' helper bacteria (MHBs). Examples of MHB evidence are given from the literature, with special reference to the Douglas fir (Pseudotsuga menzeisii Mirb. Franco) Laccaria laccata Scop, ex Fr. ectomycorrhizal combination which has been more extensively studied. The fungal specificity of some MHBs and the various mechanisms underlying their effect are discussed, considering five hypotheses: effects on the receptivity of the root, effects on the root-fungus recognition, effects on the fungal growth, modification of the rhizospheric soil, and effects on the germination of the fungal propagule. MHBs are then considered for their ecological and evolutionary implications, and examples of practical applications in forest nurseries are given: when added to the fungal inoculum, MHBs can improve the success of ectomycorrhizal inoculation of planting stocks with fungi selected for their outstanding growth stimulation after outplanting. The conclusion points out a number of fundamental questions which remain unanswered about mycorrhization helper bacteria and suggests some investigation priorities in this new field of mycorrhiza research. Contents Summary 197 I. Introduction 197 II. Evidence for helper bacteria 198 III. Fungus-specificity of MHBs 200 IV. Mechanisms underlying the MHB effect 201 V. Ecological and evolutionary implications of MHBs 205 VI. Practical applications of MHBs 206 VII. Conclusions and perspectives 208 Acknowledgements 208 References 208.

The fungal symbionts forming ectomycorrhizas, as well as their associated bacteria, benefit forest trees in a number of ways although the most important is enhancing soil nutrient mobilization and uptake. This is reciprocated by the allocation of carbohydrates by the tree to the fungus through the root interface, making the relationship a mutualistic association. Many field observations suggest that ectomycorrhizal fungi contribute to a number of key ecosystem functions such as carbon cycling, nutrient mobilization from soil organic matter, nutrient mobilization from soil minerals, and linking trees through common mycorrhizal networks. Until now, it has been very difficult to study trees and their fungal associates in forest ecosystems and most of the work on ECM functioning has been done in laboratory or nursery conditions. In this review with discuss the possibility of working at another scale, in forest settings. Numerous new techniques are emerging that makes possible the in situ study of the functional diversity of ectomycorrhizal communities. This approach should help to integrate developing research on the functional ecology of ectomycorrhizas and their associated bacteria with the potential implications of such research for managing the effects of climate change on forests. (C) 2010 Elsevier Ltd. All rights reserved.

International audience; In a declining oak forest, a thinning treatment was performed in August 2004, targeting trees belonging to two decline classes. The whole ectomycorrhizal (EM) community was dominated by the fungal symbionts Clavulina cinerea, Tomentella sublilacina and Russula spp. The potential activities of eight secreted enzymes, involved in mobilizing nutrients (N, P) from soil organic matter, were measured on these three EM types in winter and spring 2006 using multiwell microplate photometric and fluorogenic methods. The enzymatic activities recorded in winter were generally significantly higher than in spring. Most of the enzyme activities studied, and particularly phosphatase and b-glucosidase, changed according to both decline class and sylvicultural treatment. In spring, each anatomotype displayed different enzymatic profile according to the decline class. These results suggest that the potential enzymatic activity of ectomycorrhizae adapts to the changes resulting from the sylvicultural treatment and reacts to the anthropic disturbance by adjusting to the new resource structure. 2007 Elsevier Ltd. All rights reserved.

Fungi represent an essential component of biodiversity, not only because of the large number of species, but also for their ecological, evolutionary and socio-economic significance. Yet, until recently, fungi received scant consideration in ecology, especially invasion ecology. Their under-representation is largely the result of a lack of scientific knowledge of fungal biodiversity and ecology. With the exception of pathogenic fungi, which cause emergent infectious diseases, the impact of fungal invasions is often difficult to quantify owing to limited baseline data on fungal communities. Here, we aim to raise awareness among mycologists and ecologists of the fungal dimension of invasions and of the need to intensify research in fungal ecology to address issues of future introductions.

The mycorrhiza helper Pseudomonas fluorescens BBc6R8 promotes the presymbiotic survival and growth of the ectomycorrhizal fungus Laccaria bicolor S238N in the soil. An in vitro fungal–bacterial confrontation bioassay mimicking the promoting effects of the bacteria on fungal growth was set up to analyse the fungal morphological and transcriptional changes induced by the helper bacteria at three successive stages of the interaction. The specificity of the P. fluorescens BBc6R8 effect was assessed in comparison with six other rhizobacterial strains possessing mycorrhiza helper or pathogen antagonistic abilities. The helper BBc6R8 strain was the only strain to induce increases in the radial growth of the colony, hyphal apex density and branching angle. These morphological modifications were coupled with pleiotropic alterations of the fungal transcriptome, which varied throughout the interaction. Early stage-responsive genes were presumably involved in recognition processes and transcription regulation, while late stage-responsive genes encoded proteins of primary metabolism. Some of the responsive genes were partly specific to the interaction with P. fluorescens BBc6R8, whereas others were mutually regulated by different rhizobacteria. The results highlight the fact that the helper BBc6R8 strain has a specific priming effect on growth, morphology and gene expression of its fungal associate L. bicolor S238N.

International audience; The year-round dynamics of laccase and acid phosphatase activities of ectomycorrhizas (EM) were monitored in an oak forest, using a microplate photometric method using individual EM root tips. Among the most frequent and abundant EM morphotypes, those of Lactarius quietus and Cortinarius anomalus showed a peak of laccase activity in spring, while those of Xerocomus chrysenteron displayed highest laccase activities in summer and autumn. In comparison, less seasononal differences were observed in root EM acid phosphatase activity. This suggests that the different EM types within the EM community perform complementary functions depending on season. q 2005 Published by Elsevier Ltd.

We tested the response of seedlings of Dicorynia guianensis, a major timber tree species of French Guiana, to mycorrhizal symbiosis and water limitation in a semi-controlled experiment under natural light conditions. Under well-watered conditions, mycorrhizal colonization resulted in an increase of net photosynthesis, growth and phosphorus uptake. When submitted to water stress, no growth reduction of mycorrhizal seedlings was observed. Mycorrhizal seedlings were more sensitive to drought than non-mycorrhizal ones in terms of carbon assimilation, but not with regard to stomatal closure. In contrast to previous studies on temperate tree seedlings, this result precludes a mycorrhizal effect on the hydraulic properties of this species. Furthermore, our results suggest that below a specific threshold of soil moisture, carbon assimilation of D. guianensis seedlings was decreased by the mycorrhizal symbiosis. This is probably related to the competition between the plant and its host fungus for carbon allocation under low light intensity, even though it did not seem to have a significant effect on mortality in our experiment.

International audience; Here we characterized the effect of the ectomycorrhizal symbiosis on the genotypic and functional diversity of soil Pseudomonas fluorescens populations and analysed its possible consequences in terms of plant nutrition, development and health. - Sixty strains of P. fluorescens were isolated from the bulk soil of a forest nursery, the ectomycorrhizosphere and the ectomycorrhizas of the Douglas fir ( Pseudostuga menziesii ) seedlings- Laccaria bicolor S238N. They were characterized in vitro with the following criteria: ARDRA, phosphate solubilization, siderophore, HCN and AIA production, genes of N 2 -fixation and antibiotic synthesis, in vitro confrontation with a range of phytopathogenic and ectomycorrhizal fungi, effect on the Douglas fir– L. bicolor symbiosis. - For most of these criteria, we demonstrated that the ectomycorrhizosphere significantly structures the P. fluorescens populations and selects strains potentially beneficial to the symbiosis and to the plant. - This prompts us to propose the ectomycorrhizal symbiosis as a true microbial complex where multitrophic interactions take place. Moreover it underlines the fact that this symbiosis has an indirect positive effect on plant growth, via its selective pressure on bacterial communities, in addition to its known direct positive effect.

Summary • This work aimed at understanding the role of mycorrhizal status in phosphorus efficiency of tree seedlings in the tropical rainforest of French Guyana. • Mycorrhizal colonization, growth, phosphorus content, net photosynthesis and root respiration were determined on three occasions during a 9-month growth period for seedlings of two co-occurring species (Dicorynia guianensis and Eperua falcata) grown at three soil phosphorus concentrations, with or without inoculation with arbuscular mycorrhizas. • Seedlings of both species were unable to absorb phosphorus in the absence of mycorrhizal association. Mycorrhizal seedlings exhibited coils that are specific of Paris-type mycorrhizae. Both species benefited from the mycorrhizal symbiosis in terms of phosphorus acquisition but the growth of E. falcata seedlings was unresponsive to this mycorrhizal improvement of phosphorus status, probably because of the combination of high seed mass and P reserves, with low growth rate. • The two species belong to two different functional groups regarding phosphorus acquisition, D. guianensis being an obligate mycotrophic species.

National audience; La croissance de semis de Chêne (Quercus petraea et Quercus robur) à racines nues, mycorhizés par Thelephora terrestris ou quatre autres champignons par inoculation en pépinière, a été comparée après plantation dans 12 parcelles forestières coupées à blanc dans le Nord-Est de la France. Certaines souches de Paxillus involutus et de Laccaria bicolor se sont révélées être de bonnes candidates pour l’inoculation en routine des pépinières de Chêne, par rapport à Thelephora terrestris qui est le principal symbionte fongique associé aux plants commerciaux de Chêne en France. Elles procurent un gain de croissance significatif, avec comme résultat une moindre compétition de la part de la végétation. Cependant, l’effet est nul si les semis sont plantés dans une prairie de graminées.

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

Forest trees are involved in root symbioses with hundreds of species of ectomycorrhizal fungi which constitute functional guilds able to improve the water and mineral nutrition of host trees. In temperate ecosystems, water shortage is a main factor limiting tree vitality. To assess how soil water conditions affected the physiological state of beech (Fagus silvatica L.) ectomycorrhizal roots, we monitored glucose respiration of two ectomycorrhizal types (Lactarius sp. and Cenococcum geophilum) during two complete growing seasons. Five stands of contrasting soil conditions were chosen in north-eastern France. The top soil horizons were equipped with micropsychrometers for measuring water potential and temperature. Glucose respiration on individual ectomycorrhizas was measured in vitro by trapping [14C]-CO2 from radiolabelled glucose. For soil water potential

Summary • The asexual ectomycorrhizal fungus Cenococcum geophilum, known for its wide host and habitat range, has been suggested to provide isolate-dependant drought protection to fine roots. However, little is known about its genetic structure at the fine scale. • Genetic diversity and population structure of C. geophilum at the regional and stand scales was surveyed in five beech (Fagus silvatica) forests in northeastern France. The stands were selected for their contrasting climatic and edaphic features to assess the effect of environmental factors on population structure. • The genetic diversity of C. geophilum was estimated using RAPD, PCR/RFLP of the rDNA internal transcribed spacer (ITS) and PCR/RFLP and sequencing of an anonymous sequence characterized amplified region (SCAR) on ectomycorrhizas and sclerotia-derived mycelial cultures. • A high degree of genetic diversity was observed between and within beech stands in C. geophilum populations. These results suggest the occurrence of a high rate of mitotic or meiotic recombination and an effect of stand features on population structure.

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

International audience; The influence of mycorrhizal infection rate and light environment on growth traits was examined for 50-week-old Dicorynia guianensis Amshoff tree seedlings. The seedlings were grown on two soil substrates (control and inoculated) in shade tunnels under three relative light levels (50% , 14% and 1% of full sunshine). For seedlings growing under 1% of full sunlight no significant differences between control and inoculated plants were observed in plant traits though a high rate of endomycorrhizal infection was recorded. In partial shaded sunlight, 14% and 50% , the rate of mycorrhizal infection was positively related to the growth performances of seedlings. The optimal growth was obtained under 14% of full sunlight, showing a greater efficiency of the mycorrhizas.; Effet des mycorhizes et de la lumière sur la croissance des semis de Dicorynia guianensis Amshoff, une césalpiniacée de la forêt tropicale humide de Guyane française. Des semis de D. guianensis ont été cultivés en pots sur un sol désinfecté, inoculé ou non avec du sol forestier, dans des serres tunnels sous trois régimes lumineux (1 % , 14 % , 50 % du plein découvert). Des paramètres de croissance des plants et la colonisation endomycorhizienne des racines ont été mesurés au bout de 50 semaines. Les semis soumis à 1 % d'éclairement et croissant sur les deux types de sol ne présentaient aucune différence significative pour aucun des caractères mesurés, bien qu'un taux élevé de mycorhization aie été noté chez les plants sur sol inoculé. En éclairement partiel, 14 et 50 % , les performances de croissance des semis étaient positivement reliées au taux d'infection mycorhizienne. L'optimum de croissance était obtenu pour l'intensité lumineuse moyenne (14 % ), montrant ainsi une meilleure efficacité des mycorhizes.

We inoculated disinfected soil at two Douglas-fir bareroot forest nurseries with three doses (8×105, 8×107 and 8×109 cfu m−2) of the rifampin-resistant mycorrhiza helper bacterium Pseudomonas fluorescens strain BBc6R8 and the ectomycorrhizal fungus Laccaria bicolor strain S238N. In one of the two nurseries, two doses of fungal inoculum (50 and 100 mg m−2 dry weight (DW) mycelium entrapped in alginate beads at the constant dose of 1 l m−2) were tested. For all bacterial treatments the density of P. fluorescens BBc6R8 in the soil, determined by dilution plating, dropped below the detection limit (10−2 cfu g−1 DW soil) 2 weeks after inoculation. Fifteen weeks after inoculation, the introduced bacterium could be detected by enrichment only in the treatments inoculated with the highest bacterial dose. Two years after inoculation, P. fluorescens BBc6R8 could not be detected in the soil of any of the bacterial treatments. Five months after inoculation and sowing, bacterial inoculation significantly increased the percentage of mycorrhizal short roots on plants inoculated with either low or high amounts of L. bicolor, in one of the two nurseries. The lowest bacterial dose increased mycorrhizal colonization from 45 to 70% in plants inoculated with the low amount of fungal inoculum, and from 64 to 77% in plants inoculated with the high amount of fungal inoculum. The lowest bacterial dose increased mycorrhizal colonization more than the highest bacterial dose. The same L. bicolor mycorrhizal index (70%) was obtained with 50 mg m−2 DW mycelium plus the bacterium than with twice this fungal dose and no bacterium (64%). Two years after inoculation, the height of the mycorrhizal Douglas-firs in the other nursery was significantly increased by the lowest bacterial dose (from 40.7 to 42.6 cm). These results indicate that co-inoculating a helper bacterium together with an ectomycorrhizal fungus can be an efficient way of optimizing controlled mycorrhization techniques for the production of high-quality Douglas-fir planting stocks. They also confirm that BBc6R8 acts at a low population density (less than 102 cfu g−1 soil); this contrasts with most PGPR effects discussed in the literature, where the minimal inoculation dose of 105 cfu g−1 soil is required to obtain the beneficial effect.

Roots of 75 tree species, belonging to 28 families, were sampled on different types of soil in the forest of French Guiana. Both seedlings and mature trees of each species were studied. The roots were first observed for ectomycorrhizas, then thinned and stained to observe and quantify endomycorrhizal associations. Ectomycorrhizas were found only on two genera: Coccoloba (Polygonaceae) and Neea (Nyctaginaceae). All the species studied had zygomycetous endomycorrhizas with hyphal coils and vesicles. It is remarkable that some families (Caesalpiniaceae, Myrtaceae, Tiliaceae, Euphorbiaceae, Lauraceae), considered as frequently ectomycorrhizal in other regions of the world, were not so in our sample. In addition, the results contradict the theory that ectomycorrhizal species are dominant in the poorest soils in tropical rainforests (tropical podzols). Key words: tropical rainforest, symbioses, mycorrhizas, French Guiana.

In deciduous oaks, spring reactivation modifies the capability of ectomycorrhizal fungi (EMf to mobilise carbon from soil organic matter. Plant carbon budget is also unbalanced during spring reactivation: carbon demand is high to build new tissues and carbon may be provided by reserves only as new photoassimilates are not yet available. Using a stable isotope approach with C-13-labelled litter, we have estimated that after a six months experiment, less than 1% of carbon is derived from litter during spring reactivation and is channelled from the soil to the thick roots of Quercus petraea via ectomycorrhiza, even when leaves are photosynthetically active. Our results are promising for future detailed quantification of different carbon sources (i.e. reserves, photosynthesis and litter derived) in EMf oak trees. They also support the hypothesis that oak trees of Q petraea in north-eastern France are partially mixotrophic plants. (C) 2012 Elsevier Ltd. All rights reserved.

A simple and rapid method was developed for the separation and determination of a large number of organic acids by using capillary zone electrophoresis, with hydrostatic injection for 30 s and UV detection at 254 nm. Electropherograms were obtained with 4-hydroxybenzoate buffer (5 m M ) adjusted to pH 4.75 at 25°C. The electroosmotic flow modifier (commercial solution) was added to the buffer at a concentration of 25 ml 1 −1 . Co-migration problems could be solved by modifying the pH of the electrophoresis buffer or by addition of Ca 2+ to the buffer.

The relatively recent evidences leading to the MHB concept (mycorrhization helper bacteria, which specificaly enhance mycorrhiza formation) ask many new questions about their degree of interdepende...

The aim of a joint effort by different research teams was to provide an improved procedure for enzyme activity profiling of field-sampled ectomycorrhizae, including recommendations on the best conditions and maximum duration for storage of ectomycorrhizal samples. A more simplified and efficient protocol compared to formerly published procedures was achieved by using manufactured 96-filter plates in combination with a vacuum manifold and by optimizing incubation times. Major improvements were achieved by performing the series of eight enzyme assays with a single series of root samples instead of two series, reducing the time needed for sample preparation, minimizing error-prone steps such as pipetting and morphotyping, and facilitating subsequent DNA analyses due to the reduced sequencing effort. The best preservation of samples proved to be storage in soil at 4?6°C in the form of undisturbed soil cores containing roots. Enzyme activities were maintained for up to 4 weeks under these conditions. Short-term storage of washed roots and ectomycorrhizal tips overnight in water did not cause substantial changes in enzyme activity profiles. No optimal means for longer-term storage by freezing at −20°C or storage in 100% ethanol were recommended. Fil: Pritsch, Karin. German Research Center for Environmental Health. Institute of Soil Ecology; Alemania Fil: Courty, Pierre Emanuel. Institut National de la Recherche Agronomique; Francia. University of Basel. Botanical Institute; Suiza Fil: Churin, Jean Louis. Institut National de la Recherche Agronomique; Francia Fil: Cloutier Hurteau, Benoit. Institut National de la Recherche Agronomique; Francia Fil: Ali, Muhammad Arif. Institut National de la Recherche Agronomique; Francia Fil: Damon, Coralie. Université Claude Bernard Lyon 1; Francia Fil: Duchemin, Myriam. Institut National de la Recherche Agronomique; Francia Fil: Egli, Simon. Swiss Federal Research Institute WSL; Suiza Fil: Ernst, Jana. German Research Center for Environmental Health. Terrestrial Ecogenetics. Institute of Soil Ecology; Alemania Fil: Fraissinet Tachet, Laurence. Université Claude Bernard Lyon 1; Francia Fil: Kuhar, José Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Micología y Botánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Micología y Botánica; Argentina. German Research Center for Environmental Health. Helmholtz Zentrum München. Institute of Soil Ecology; Alemania Fil: Legname, Elvira. Institut National de la Recherche Agronomique; Francia Fil: Marmeisse, Roland. Université Claude Bernard Lyon 1; Francia Fil: Müller, Alex. Swiss Federal Research Institute WSL; Suiza Fil: Nikolova, Petia. German Research Center for Environmental Health. Institute of Soil Ecology; Alemania Fil: Peter, Martina. Swiss Federal Research Institute WSL; Suiza Fil: Plassard, Claude. Institut National de la Recherche Agronomique; Francia Fil: Richard, Franck. Centre d’Ecologie Fonctionnelle et Evolutive; Francia Fil: Schloter, Michael. German Research Center for Environmental Health. Terrestrial Ecogenetics. Institute of Soil Ecology; Alemania Fil: Selosse, Marc André. Centre d’Ecologie Fonctionnelle et Evolutive; Francia Fil: Franc, Alain. Institut National de la Recherche Agronomique; Francia Fil: Garbaye, Jean. Institut National de la Recherche Agronomique; Francia

International audience

International audience; Introduction Important nutrients in forest soils such as nitrogen and phosphorus are mostly recycled from natural polymeric compounds contained in litter and organic debris-for example nucleic acids, proteins, or chitin. Objectives Activities of enzymes such as phosphatases, proteases, cellulases, chitinases and laccase were shown in saprotrophic but also in ectomycorrhizal fungi and there is increasing evidence that these enzymes contribute not only to the functioning of the symbiosis but also to the mobilisation of nutrients. In the present review, we describe how enzyme secretion and localisation on fungal hyphae may be connected to the potential role in soil nutrient cycling. Results Recently developed methods for enzyme activity studies of ectomycorrhizae directly assayed in or collected from the field such as enzyme activity profiling and soil imprinting are described. Their value and limitations in different examples of ecological studies is highlighted and discussed also with respect to the role of other soil microorganisms associated with ectomycorrhizae. Conclusion The conclusion from our review is that enzyme activities of ECM and their associated microorganisms provide a potentially enormous plasticity of mycorrhizosphere functionality which is an open field for further research. Enzymes secretes par les champignons ectomycorhiziens et exploitation des elements mineraux contenus dans la matiere organique du sol.

Mycorrhization helper bacteria (MHBs) isolated and selected from the Douglas fir-Laccaria laccata symbiotic system have previously been shown to be fungus-specific: they promote ectomycorrhizal establishment of Laccaria laccata but inhibit mycorrhiza formation by other fungi. In this paper, two experiments in a nursery producing two years-old bare-root Douglas-fir planting stocks confirm the specificity of MHBs under field conditions. They also show that, by selectively helping the introduced L. laccata against the resident symbionts, MHBs are an interesting alternative (safer and easier) to soil fumigation for the success of routine controlled mycorrhization of planting stocks in forest nurseries.

Norway spruce (Picea abies (L.) Karst.) seedlings were grown in a glasshouse pot experiment in soils from 11 declining and 7 healthy spruce stands from France and Germany. In soils from 9 declining stands, seedlings showed decline symptoms (needle yellowing). Soil pasteurization suppressed the symptoms, and reinoculation of the pasteurized soil with a rhizospheric extract from the corresponding stand re-induced yellowing. This suggests that a deleterious soil microflora is associated with spruce decline. The occurrence of this microflora seems to be correlated with the main chemical characteristics of the soils (low pH, low saturation of the adsorbing complex, low exchangeable Ca2+ and Mg2+, and high level of exchangeable Al). ei]R F Huettl

International audience; Liming is a forest practice used to counteract forest decline induced by soil acidification. It consists of direct Ca and Mg input in forest soil and restores tree mineral nutrition, but also causes drastic changes in nutrient availability in soil. Ectomycorrhizal (ECM) fungi significantly contribute in nutrient uptake by trees, and can recover them through organic acid secretion or through enzymatic degradation of organic matter. The symbiotic fungi use their extraradical mycelium for nutrient uptake, and then store them into the ECM mantle. In this study we measured how liming influences element contents in the mantle of Lactarius subdulcis ECMs, an abundant and particularly active in oxalate and laccase secretion in beech stands. For this purpose we used SEM observation coupled with energy- (EDX) and wavelength-dispersive-X-ray microanalyses (WDX). Results showed that ECM mantles of this species presented significantly higher Ca, Mg, Mn, K, Si, Al and Fe contents in limed plots. The nutrient amounts of L. subdulcis ECMs were significantly different between individuals for all the elements, showing a differential storage ability between individuals. The storage role of the ECM mantle can be interpreted in two different ways: i) a detoxification role for Al or heavy metals and ii) an increased potential nutrient resource by the fungus, which can benefit the tree.

International audience; The ectomycorrhizal community of an oak forest has been monitored monthly throughout fifteen months. Eight enzymatic activities secreted by the ectomycorrhizal root tips and involved in the mobilization of nutrients from soil organic matter have been measured using microplate assays, resulting in potential activity patterns of individual fungal species. Both the species structure of the community and the specific activity level of each individual species changed with the season and soil horizon. This versatility may be an adaptative response of the ectomycorrhizal fungal community to a highly variable environment. The results also suggest that some ectomycorrhizal fungi behave as occasional saprobes and contribute to the decomposition of soil organic matter and nutrient cycling together with true saprotrophic fungi.

A survey of mycorrhizae — vesicular-arbuscular endomycorrhizae (VAM) and ectomycorrhizae (ECM) — was carried out in roots and soil samples collected in young (2- to 15-month-old) Eucalyptus viminalis plantations in two forestry regions in the Santa Catarina State, southern Brazil. Percentages of root colonization and spore counts in soils were assessed in May, August and November or January. The overall trend of mycorrhizal colonization showed an early decline for VAM and a later increase for ECM as the plants aged. Vesicular-arbuscular endomycorrhizae colonization was consistently predominant in young stands (up to 7–8 months old), while ECM were predominant as the stands aged. Both types co-existed in all stands, regardless of the region and sampling period. Regional differences were found in the intensity of VAM colonization, but not in ECM colonization. Numbers of spores in soil were not, in most cases, correlated with stand age or VAM colonization. A trend towards a decrease in spore numbers in November and January was detected in both regions. The role of some ecological factors in the dynamics of the dual mycorrhizal system is discussed.

Liming is used to counteract forest decline induced by soil acidification. It consists of Ca and Mg input to forest soil and not only restores tree mineral nutrition but also modifies the availability of nutrients in soil. Ectomycorrhizal (ECM) fungi are involved in mineral nutrient uptake by trees and can recover them through dissolution of mineral surface. Oxalate and siderophore secretion are considered as the main agents of mineral weathering by ECMs. Here, we studied the effects of liming on the potential oxalate secretion and iron complexation by individual beech ECM root tips. Results show that freshly excised Lactarius subdulcis root tips from limed plots presented a high potential oxalate exudation of 177 μM tip(-1) h(-1). As this ECM species distribution is very dense, it is likely that, in the field, oxalate concentrations in the vicinity of its clusters could be very high. This points out that not only extraradical mycelium but also ECM root tips of certain species can contribute significantly to mineral weathering. Nonmetric multidimensional scaling (NMDS) separated potential oxalate production by ECM root tips in limed and untreated plots, and this activity was mainly driven by L. subdulcis ECMs, but NMDS on potential activity of iron mobilization by ECM root tips did not show a difference between limed and untreated plots. As the mean oxalate secretion did not significantly correlated with the mean iron mobilization by ECM morphotype, we conclude that iron complexation was due to either other organic acids or to siderophores.

International audience; Liming (Ca–Mg soil amendment) is a forestry practice used to correct soil acidification and restore health and productivity in declining stands. Liming is known to modify tree mineral nutrition beyond the sole Ca and Mg. We hypothesized that liming also modifies the very functioning of the tree absorbing system (that is the ectomycorrhizal fine roots) in a way that facilitates the mobilization of mineral nutrients, particularly those entrapped in soil organic matter. This hypothesis has been tested here in beech and Norway spruce stands in North-Eastern France. In autumn, we compared the ectomycorrhizal community structure and the enzymatic profiles of ectomycorrhizal root tips in limed and untreated plots by measuring the activities of eight enzymes related to the degradation of soil organic matter. The results show that the ectomycorrhizal community responds to the Ca–Mg amendment and to the resulting soil modifications by modified enzyme activity profiles and ability to mobilize nutrients from soil organic matter. The effects of liming on the belowground functioning of the tree stands result essentially from specialized ECM fungal species such as Clavulina cristata (with strong glucuronidase activity), Lactarius subdulcis (with strong laccase activity) or Xerocomus pruinatus (with strong leucine aminopeptidase activity).

International audience; The impact of past Roman occupation on the composition of ectomycorrhizal (ECM) communities was analysed in 12 Roman settlements in an oak forest in Central France. At each Roman settlement, soils and ECM roots were sampled from two plots (600 m2 each), one plot close to the remains of the buildings (

Douglas fir (Pseudotsuga menziesii) seedlings in two bare-root forest nurseries were inoculated with the ectomycorrhizal fungus Laccaria laccata, together or not with one of five mycorrhization helper bacteria isolated from L. laccata sporocarps or mycorrhizas and previously selected by in vitro and glasshouse screenings. With the most efficient MHB isolates, when compared to the control with no bacteria, the percent of mycorrhizal short roots was increased from 60 to 90 or from 80 to 100, depending on the nursery, with inoculation doses as low as 106 living cells per m2. A dual inoculum made of calcium alginate beads containing the two microorganisms appears to be a valuable technique for increasing the efficiency of ectomycorrhizal inoculation of planting stocks in forest nurseries.

Laccaria laccata (Scop ex Fr) Cke est un champignon basidiomycete ectomycorhizien tres efficace pour la mycorhization controlee du douglas (Pseudotsuga taxifolia Poir Britt). L'etude de la biologie de cette symbiose a conduit a la mise au point d'un certain nombre de techniques experimentales pour realiser sa synthese en conditions aseptiques ou non aseptiques. Cette note decrit le traitement des graines, la preparation de l'inoculum fongique, les solutions nutritives, les substrats, les systemes experimentaux aseptiques (tubes a essais et boites de Petri) et non aseptiques (experiences en pots en serre et techniques de pepiniere a racines nues) (.)

SUMMARY Two isolates of the ectomycorrhizal fungus Pisolithus tinctorius (Pers.) Coker and Couch were inoculated on to roots of Eucalyptus urophylla S. T. Blake in vitro and a comparison made of the rate of ectomycorrhizal development. Both isolates initiated ectomycorrhizal roots but the time of onset of root colonization and the rate of formation varied with the isolate. An isolate cultured from a sporocarp collected from under eucalypts in Australia rapidly colonized roots of E. urophylla and initiated mycorrhizas after 2 d incubation, whereas an isolate obtained from sporocarps collected from under pines in the United States developed ectomycorrhizas only after 7 d incubation. Dual inoculation of E. urophylla seedlings using a paper-sandwich technique again showed the rapid colonization of roots by the eucalypt isolate even when roots were inoculated 7 d previously with the pine isolate. These in vitro observations support field observations that there are variable degrees of plant species - fungal isolate compatibility, implying specificity, and this is an important factor influencing successful ectomycorrhiza formation and development.

(...) une premiere experimentation en serre a ete conduite sur des plants de chene rouge eleves pendant deux ans dans des conteneurs de grande capacite en presence ou non de molinie. Le substrat de culture est constitue d'un horizon A 1 sableux, enrichi ou non en engrais, et recevant une alimentation en eau jamais limitante. A la fin de la 2eme saison de vegetation, on a compare la biomasse des tiges et des racines dans les differentes modalites et note le statut mycorhizien sur un echantillon restrient de celles-ci. On a mis en evidence un effet depressif tres net de la molinie sur la croissance des chenes (...)

Contents Summary 22 I. Introduction 23 II. Evidence for the occurrence of MHB 23 III. Ecological and evolutionary implications of MHB 23 IV. The question of MHB specificity 27 V. Mechanisms of the MHB effect 28 VI. Role of MHB in mycorrhizal functions 31 VII. Conclusions and research priorities 32 References 33 Summary In natural conditions, mycorrhizal fungi are surrounded by complex microbial communities, which modulate the mycorrhizal symbiosis. Here, the focus is on the so-called ‘mycorrhiza helper bacteria’ (MHB). This concept is revisited, and the distinction is made between the helper bacteria, which assist mycorrhiza formation, and those that interact positively with the functioning of the symbiosis. After considering some examples of MHB from the literature, the ecological and evolutionary implications of the relationships of MHB with mycorrhizal fungi are discussed. The question of the specificity of the MHB effect is addressed, and an assessment is made of progress in understanding the mechanisms of the MHB effect, which has been made possible through the development of genomics. Finally, clear evidence is presented suggesting that some MHB promote the functioning of the mycorrhizal symbiosis. This is illustrated for three critical functions of practical significance: nutrient mobilization from soil minerals, fixation of atmospheric nitrogen, and protection of plants against root pathogens. The review concludes with discussion of future research priorities regarding the potentially very fruitful concept of MHB.

International audience

International audience; The effects of a sylvicultural treatment (thinning) on the ectomycorrhizal community structure and diversity were studied each season for 2 years in a declining Quercus robur woodland in northern Italy. Sixteen dominant oak trees were chosen as representative of two decline classes (eight per class). Fine root status, ectomycorrhizal community composition and structure were analysed before and after drastically altering tree density by felling all the neighbouring trees of four selected trees in each decline class. The ectomycorrhizal community was found to be extremely rich, with numerous infrequent species: of the 64 anatomotypes found, only 23 were present both before and after the thinning treatment, demonstrating that community structure changes rapidly after disturbance. Following an initial reduction, 10 months after removing competition and independently of health status, the biodiversity had recovered its previous level, demonstrating the high resilience of the ectomycorrhizal community to disturbance. ©2007 Elsevier B.V. All rights reserved.

Tuber magnatum, an ascomycetous fungus and obligate ectomycorrhizal symbiont, forms hypogeous fruit bodies, commonly called Italian white truffles. The diversity of bacterial communities associated with T. magnatum truffles was investigated using culture-independent and -dependent 16S rRNA gene-based approaches. Eighteen truffles were classified in three groups, representing different degrees of ascocarp maturation, based on the percentage of asci containing mature spores. The culturable bacterial fraction was (4.17 +/- 1.61) x 10(7), (2.60 +/- 1.22) x 10(7) and (1.86 +/- 1.32) x 10(6) cfu g(-1) for immature, intermediate and mature ascocarps respectively. The total of bacteria count was two orders of magnitude higher than the cfu g(-1) count. Sequencing results from the clone library showed a significant presence of alpha-Proteobacteria (634 of the 771 total clones screened, c. 82%) affiliated with Sinorhizobium, Rhizobium and Bradyrhizobium spp. The bacterial culturable fraction was generally represented by gamma-Proteobacteria (210 of the 384 total strains isolated, c. 55%), which were mostly fluorescent pseudomonads. Fluorescent in situ hybridization confirmed that alpha-Proteobacteria (85.8%) were the predominant components of truffle bacterial communities with beta-Proteobacteria (1.5%), gamma-Proteobacteria (1.9%), Bacteroidetes (2.1%), Firmicutes (2.4%) and Actinobacteria (3%) only poorly represented. Molecular approaches made it possible to identify alpha-Proteobacteria as major constituents of a bacterial component associated with T. magnatum ascoma, independently from the degree of maturation.

International audience; The species of an ectomycorrhizal (ECM) community were investigated in a temperate oak forest by morphotyping and ITS rDNA sequencing. Thirty-six ECM morphotypes were found at the site. The niche effect (as organic soil, mineral soil or dead woody debris artificially introduced in the soil) on the ECM community structure and on the potential catabolic activities of the most abundant morphotypes was studied. The morphotypes in each niche were subjected to enzymatic tests developed for hydrolytic and oxidative enzymes involved in the decomposition of organic compounds. The ECM community structure varied widely depending on the soil horizon or habitat patch. The species richness was higher in the A1 horizon than in the other niches. Different ECM species had different activity patterns for the eight enzymatic tests while co-occurring in the same niche. Catabolic activities also changed within species between niches. Dead woody debris were extensively colonized by two saprotrophic fungi (Megacollybia platyphylla and Armillaria sp.) and, in this particular niche, ECM morphotypes predominantly belonged to the genera Lactarius and Tomentella. These morphotypes showed high chitinase activities. This study suggested also that some ECM fungi could obtain nutrients via the chitin degradation of dead or live saprobes.

International audience; The ectomycorrhizas (ECM) formed by Lactarius quietus, an ECM fungus specifically associated with Quercus spp., are abundant all the year long. Root and stem growth, beginning before bud break in oak, are strong carbon sinks partially fulfilled with carbohydrate reserves. We hypothesized that L. quietus contributes to providing trees with carbon at bud break through enzymatic activities before photosynthesis begins. Activities of eight secreted enzymes (xylosidase, glucuronidase, cellobiohydrolase, β-glucosidase, N-acetyl-glucosamine, leucine aminopeptidase, acid phosphatase and laccase) relevant to carbon cycling and the release of phophorus and nitrogen from soil organic matter were measured on L. quietus ECMs before, during and after the bud break. Phenological, climatic and pedoclimatic parameters were also measured. Laccase, glucuronidase, cellobiohydrolase and β-glucosidase activities proved to be significantly related to tree reactivation and climate. All these activities can help the formation of new tissues by supplying carbon. L. quietus can behave saprotrophically, using soil organic matter as substrate. This is consistent with the hypothesis that it provides the oak trees with carbon when demand is high and photoassimilates are not yet available.

International audience; To test the hypothesis that, depending on the fungal symbiont, ectomycorrhizas are differentially affected by severe drought stress, we developed a simple method to quantify the loss of vitality of excised ectomycorrhizal tips subjected to drying under controlled conditions. The method uses 96-well microtitration plates with one single ectomycorrhizal tip per well, and is based on measuring the loss of volume and the loss of electrolytes before and after the imposed stress. This approach very significantly discriminated the two ectomycorrhizal morphotypes formed with beech (Fagus silvatica) by Lactarius subdulcis and Cenococcum geophilum, which confirmed the ability of the latter fungal species to protect roots against desiccation already suggested by previous works. The new method should contribute to the present effort in deciphering the functional diversity of complex ectomycorrhizal communities.

International audience; Mycorrhization helper bacteria (MHB), isolated from phylogenetically distinct ectomycorrhizal symbioses involving Lactarius rufus, Laccaria bicolor or Suillus luteus, were tested for fungus specificity to enhance L. rufus–Pinus sylvestris or L. bicolor–P. sylvestris mycorrhiza formation. As MHB isolated from the L. rufus and S. luteus mycorrhiza were originally characterised using a microcosm system, we assessed their ability to enhance mycorrhiza formation in a glasshouse system in order to determine the extent to which MHB are system-specific. Paenibacillus sp. EJP73, an MHB for L. rufus in the microcosm, significantly enhanced L. bicolor mycorrhiza formation in the glasshouse, demonstrating that the MHB effect of this bacterium is neither fungus-specific nor limited to the original experimental system. Although the five MHB strains studied were unable to significantly enhance L. rufus mycorrhiza formation, two of them did have a significant effect on dichotomous short root branching by L. rufus. The effect was specific to Paenibacillus sp. EJP73 and Burkholderia sp. EJP67, the two strains isolated from L. rufus mycorrhiza, and was not associated with auxin production. Altered mycorrhiza architecture rather than absolute number of mycorrhizal roots may be an important previously overlooked parameter for defining MHB effects.

Summary • Data on the diversity and distribution of enzyme activities in native ectomycorrhizal (ECM) communities are inadequate. •A microplate multiple enzymatic test was developed which makes it possible to measure eight enzyme activities on 14 individual, excised ECM root tips. Hydrolytic and oxidative enzymes are involved in the decomposition of lignocellulose, chitin and phosphorus-containing organic compounds. This test system was used to describe the functional diversity of ECM communities in two forest sites. • This set of tests proved to be accurate and sensitive enough to reveal a high diversity of activity profiles, depending on the fungal symbiont and the soil horizon. Ectomycorrhizas can be classified into specialists and generalists, and appear to complement each other in the same horizon to collectively perform all eight activities studied. • By including a higher number of different assays for more detailed analyses, ECM activity profiling will provide a valuable tool for studying the functional diversity of ECM communities.

This work was aimed at understanding how the functional diversity of ectomycorrhizas (ECM) is driven by environmental factors and how it adapts to the structure of the forest stand. Superficial fine roots were sampled 21 times during an entire year in two adjacent plots (no thinning and strong thinning) of a mature beech (Fagus silvatica) forest. Individual ectomycorrhizal root tips were morphologically characterised and the symbiotic fungi were molecularly identified. ECM were also tested for dehydrogenase and acid phosphatase activities, and soil moisture and temperature were recorded. The results provide a description of ECM community dynamics over a whole year in the two stands. The main conclusions are threefold: (1) the species structure of the ECM community and metabolic activity of each morphotype change depending on the season, temperature and soil moisture, and a number of morphotypes are more abundant and active in winter than in summer, (2) the silviculture treatment (strong thinning) modifies the ectomycorrhizal community structure, and (3) the overall function of the ECM community results from the individual time pattern and specialisation of each morphotype.

International audience; Fluorescence in situ hybridization, associated with confocal laser scanning microscopy or epifluorescence microscopy with deconvolution system, has allowed the detection of a community of intracellular bacteria in non-axenic samples of the ectomycorrhizal fungus Laccaria bicolor S238N. The endobacteria, mainly alpha -proteobacteria, were present in more than half of the samples, which consisted of ectomycorrhizae, fungal mats and fruit bodies, collected in the glasshouse or in the forest. Acridine orange staining suggests that the endobacteria inhabit both live and dead fungal cells. The role of these endobacteria remains to be clarified.

International audience; The knowledge of the survival of inoculated beneficial fungal and bacterial strains in the field and the effects of their release on the indigenous microbial communities has been of great interest since the practical use of selected natural or genetically modified microorgaanisms has been developing. The aim of this study was to monitor, 4 years after plantation into the field site, the effects of Douglas fir (Pseudotsuga menziesii) co-inoculation with the mycorrhiza helper bacterial strain Pseudomonas fluorescens BBc6R8 and/or the fungal strain Laccaria bicolor S238N on seedling growth and on the indigenous bacterial and ectomycorrhizal communities using quantitative and qualitative approaches. The field persistence of the inoculated strains was also monitored. The seedling shoot volume estimate was statistically significantly higher in the fungal inoculated plots in comparison to the non-inoculated plots but no treatment-related changes in the quantitave or qualitative microbial measurements were observed and the inoculated strains could not be detected after 4 years