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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.

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.

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.

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.

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.

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; 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; 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.

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.

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

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.

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.

International audience; Pinus pinaster seedlings were grown in a sandy dune soil either inoculated with Hebeloma cylindrosporum or let to natural colonisation. Six months later, half of the seedlings of both treatments were subjected to a 3-week moderate drought. Root colonisation analysis showed that root tips were colonised to almost 100% independent of the inoculation. DNA determination of the ectomycorrhizal morphotypes showed that inoculated seedlings were extensively mycorrhized by H. cylindrosporum (more than 75%) whereas non-inoculated seedlings were mycorrhized by the exotic species Thelephora terrestris (50%) and Laccaria bicolor (30%) and to a lesser extent by H. cylindrosporum (20%). Drought did not affect these frequencies. Total plant biomass was not affected by the mycorrhizal status or by drought but the root/shoot biomass ratio as well as the root/leaf surface area ratio were much lower in seedlings extensively colonised by H. cylindrosporum. Root hydraulic conductivity was higher in plants mainly mycorrhized by H. cylindrosporum, showing that this fungus improved the water uptake capacity of the root system as compared to T. terrestris and/or L. bicolor. This positive effect was also found under drought but to a lesser extent. H. cylindrosporumalso increased the amount of root-adhering soil as compared to the other fungal symbionts, illustrating the performance of this association in aggregating sandy soil particles and developing the rhizosheath. The origin of the reduced root hydraulic resistance by H. cylindrosporum mycorrhization is discussed for the whole path including soil, soil-root interface and root cortex.

Bacterial proliferations have recurrently been observed for the past 15 years in fermentor cultures of the ectomycorrhizal fungus Laccaria bicolor S238N, suggesting the presence of cryptic bacteria in the collection culture of this fungus. In this study, intracellular bacteria were detected by fluorescence in situ hybridization in combination with confocal laser scanning microscopy in several collection subcultures of L. bicolor S238N. They were small (0.5 μm in diameter), rare, and heterogeneously distributed in the mycelium and were identified as Paenibacillus spp. by using a 16S rRNA-directed oligonucleotide probe initially designed for bacteria isolated from a fermentor culture of L. bicolor S238N.

En Guyane française où les sols sont généralement acides et pauvres en éléments minéraux, la forêt est caractérisée par sa plurispécificité. Une approche de type écologique a montré, dans les racines des principales espèces forestières, la dominance d'endomycorhizes à structure intracellulaire particulière : des hyphes de type Paris, spiralé. Leur rôle dans l'acquisition du phosphore a été étudié expérimentalement chez deux Césalpiniacées. Les semis d'Angélique, Dicorynia guianensis Amshoff, sont très dépendants, pour leur croissance et leur teneur en phosphore, des mycorhizes à mycélium spiralé ; ce type de mycélium représente plus de 90 % de la colonisation fongique. Par contre, les semis de Wapa (Eperua falcata Aublet) forment également des mycorhizes mais ces dernières améliorent le statut phosphaté et sont sans effet sur la croissance de cette espèce. Ces résultats contribuent à la compréhension du rôle des symbioses endomycorhiziennes dans le processus de régénération naturelle et ouvrent des perspectives de recherche quant à leur intervention éventuelle dans le maintien de la diversité forestière.

Les arbres d'une plantation de Chene pedoncule (Quercus robur L.) de 16 ans dans le Nord-Est de la France, realisee avec des plants mycorhizes ou non par le champignon Paxillus involutus, ont ete etudies du point de vue de la croissance, de l'elagage naturel et des gourmands. Les resultats montrent que la stimulation de croissance initiale se traduit encore par un effet significatif sur la hauteur totale, mais que le traitement fongique n'a plus d'effet sur la hauteur ou le diametre de la premiere branche vivante. Les arbres initialement mycorhizes par Paxillus involutus tendent a avoir plus de gourmands, avec une frequence plus elevee de gourmands groupes. Cependant, la plupart de ces gourmands sont petits et de duree de vie tres limitee, et la conclusion generale est que le gain de croissance procure par la mycorhization controlee n'est en rien compromis par d'eventuels effets secondaires pouvant deprecier la qualite des tiges.

Pedunculate oak seedlings (Quercus robur) inoculated with the ectomycorrhizal fungus Laccaria lacata were grown for 1 year on fertilized sphagnum peat in two nurseries. Three factors affecting microbial populations in the substrate were studied, fungicide treatment of the seeds, peat disinfection before sowing (methyl bromide or steam pasteurization), and inoculation with mycorrhization helper bacteria. Treatment of acorns with Iprodione had no depressive effect on mycorrhiza formation. Both disinfection techniques were equivalent, stimulating or depressing mycorrhiza formation depending on the initial microflora in the peat. The introduction of two previously selected mycorrhization helper bacteria (one Pseudomonas fluorescens and one unidentified fluorescent pseudomonad), isolated from L. laccata sporocarps associated with Douglas fir—L. laccata ectomycorrhizas in other nurseries, significantly increased the mycorrhizal rate from 30 to 53% of the short roots. The implications of these results for the controlled mycorrhization of planting stocks and the specificity of mycorrhization helper bacteria are discussed.

The natural 15N abundance method for estimating symbiotic biological N2-fixation was tested on legume trees from two rain forests on contrasting soils (oxisols and spodosols) in French Guiana. When possible, the significance of N2-fixing species in the plant community was evaluated in terms of density, biomass and contribution of N2-fixation to the building up of the total nitrogen mass in the leaves. Of the two sites, the rain forest on spodosols was the less favourable for application of the [delta]15N method: the available soil nitrogen was isotopically similar to fixed-N2. Hence, the results showed that a reliable estimate of N2-fixation could not be obtained. A substantial contribution of fixed-N2 to the nitrogen nutrition of legumes was found on oxisols, with an average value of 54 % Ndfa (Nitrogen derived from the atmosphere). The contribution of the N2-fixing legumes to the biomass of the stand was estimated to be 2 t ha[minus sign]1 for the leaf biomass and 136 t ha[minus sign]1 for the total above-ground plant biomass. With 7.5 % of trees in the stand able to fix N2 (462 out of 6156), N2-fixation was estimated to be 7 kg ha[minus sign]1 y[minus sign]1. These results are the first use of the [delta]15N method to estimate nitrogen input by N2-fixing legumes to a natural rain forest. The inter-site variability observed in the [delta]15N of the non-fixing plants suggested different nitrogen-cycling processes in the two soils. The [delta]15N of the non-N2-fixing plants could be related to the soil nitrogen availability and be used as an indicator of efficient or non-efficient nitrogen-cycling rain forests. The spatial variability of the [delta]15N in the plant-available soil nitrogen pool and the nitrogen balance in tropical rain forests are discussed.

Functional aspects of biodiversity were investigated in a lowland tropical rainforest in French Guyana (5°2'N, annual precipitation 2200 mm). We assessed leaf '15N as a presumptive indicator of symbiotic N2 fixation, and leaf and wood cellulose '13C as an indicator of leaf intrinsic water-use efficiency (CO2 assimilation rate/leaf conductance for water vapour) in dominant trees of 21 species selected for their representativeness in the forest cover, their ecological strategy (pioneers or late successional stage species, shade tolerance) or their potential ability for N2 fixation. Similar measurements were made in trees of native species growing in a nearby plantation after severe perturbation (clear cutting, mechanical soil disturbance). Bulk soil '15N was spatially quite uniform in the forest (range 3-5‰), whereas average leaf '15N ranged from m0.3‰ to 3.5‰ in the different species. Three species only, Diplotropis purpurea, Recordoxylon speciosum (Fabaceae), and Sclerolobium melinonii (Caesalpiniaceae), had root bacterial nodules, which was also associated with leaf N concentrations higher than 20 mg gm1. Although nodulated trees displayed significantly lower leaf '15N values than non-nodulated trees, leaf '15N did not prove a straightforward indicator of symbiotic fixation, since there was a clear overlap of '15N values for nodulated and non-nodulated species at the lower end of the '15N range. Perturbation did not markedly affect the difference '15Nsoil m '15Nleaf, and thus the isotopic data provide no evidence of an alteration in the different N acquisition patterns. Extremely large interspecific differences in sunlit leaf '13C were observed in the forest (average values from m31.4 to m26.7‰), corresponding to intrinsic water-use efficiencies (ratio CO2 assimilation rate/leaf conductance for water vapour) varying over a threefold range. Wood cellulose '13C was positively related to total leaf '13C, the former values being 2-3‰ higher than the latter ones. Leaf '13C was not related to leaf '15N at either intraspecific or interspecific levels. '13C of sunlit leaves was highest in shade hemitolerant emergent species and was lower in heliophilic, but also in shade-tolerant species. For a given species, leaf '13C did not differ between the pristine forest and the disturbed plantation conditions. Our results are not in accord with the concept of existence of functional types of species characterized by common suites of traits underlying niche differentiation; rather, they support the hypothesis that each trait leads to a separate grouping of species.

Des semis de D guianensis ont ete cultives en pots sur un sol desinfecte inocule ou non avec un sol forestier, des racines mycorhizees de D guianensis ou des nematodes. Des parametres de croissance des plantes et la colonisation endomycorhizienne des racines ont ete mesures au bout de 200 j. Les resultats ne sont pas concluants en ce qui concerne le role des nematodes, mais montrent clairement que D guianensis est dependant de la symbiose endomycorhizienne pour son developpement : les poids de matiere seche des parties aeriennes des semis inocules avec sol ou racines (aux racines endomycorhizees a 87-84 %) sont respectivement 77 et 54 % superieurs a celui des semis temoins non inocules (non mycorhizes). En relation avec des observations anterieures en foret, ces resultats confortent l'hypothese que les endomycorhizes jouent un role important dans la regeneration de D guianensis.

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Dans la foret tropicale humide de Guyane francaise, une grande diversite de morphologie racinaire et d'associations symbiotiques a ete observee sur 21 especes d'arbres. Certaines structures inhabituelles sont decrites, comme des racines courtes noduleuses contenant des hyphes en pelotons. Les endomycorhizes constituent l'association symbiotique dominante dans ce type de foret. Des galles a nematodes (Meloidogyne sp) frequentes sur les racines des semis de Dicorynia guianensis et de Peltogyne venosa jouent probablement un role dans la regeneration de ces especes

Dans de nombreux cas de deperissement, des insuffisances d'alimentation minerale interviennentmais leurs relations avec le deperissement peuvent etre de differentes natures.RELATIONS ENTRE CARENCES MINERALES ET DEPERISSEMENTLes carences, intervenant comme cause principale ou directe du deperissement, dependent du solet peuvent provenir :— d'une insuffisance de fertilite chimique due a l'evolution du sol (vieux sols fortement alteres)ou a une pauvrete initiale de la roche-mere . Le deperissement du Pin maritime dans les Landes en1963-65 (carence en P) ou le jaunissement du Sapin et de l'Epicea dans les montagnes acides en1983-93 (carence en Mg et Ca) nous en fournissent des exemples ;— d'un manque de profondeur du sol, aggrave eventuellement par la presence de calcaire,meme en faible quantite. La carence en potassium, phosphore, azote, parfois manganese, observeesur des peuplements jaunissants de Sapin ou d'Epicea dans certaines parties du Jura en 1984-87releve de ce type de situation.Des facteurs climatiques ou de pollution peuvent interferer avec ces insuffisances d'alimentationminerale. La carence magnesienne s'est developpee dans les Vosges a la suite de sequencesclimatiques particulieres en 1983 et 1984 : printemps tres pluvieux suivis d'etes chauds et secs.Elle regresse a la faveur d'annees normalement arrosees pour s'aggraver a nouveau en cas desecheresse (Dambrine et al., 1993 ; Bonneau, 1993). Reciproquement, une carence minerale peutaccentuer les effets d'une crise climatique (exemple du deperissement du Pin maritime dans lesLandes ou l'effet du froid a ete accentue par une trop faible alimentation minerale) . La photosyn-these de jeunes epiceas eleves sur un sol pauvre en Ca et Mg diminue lorsqu'on les expose a unepollution par l'ozone, alors qu'elle n'est pas modifiee sur un sol fertilise (Selinger et al., 1986).ELIMINATION OU ATTENUATION DE CERTAINS DEPERISSEMENTS PAR FERTILISATIONLa preuve que des carences minerales interviennent, soit comme causes principales, soit commefacteurs aggravants dans certains deperissements, est fournie par l'effet favorable de la fertilisationsur le retablissement d'un etat de sante normale. Beaucoup d'exemples sont connus :— de nombreux essais dans les Landes ont montre l'effet spectaculaire d'une fertilisationphosphatee sur la restauration d'un comportement normal des jeunes peuplements (Bonneau et al.,1968)

Estivalet (1989), Estivalet et al. (1990), Devevre (1990) et Devevre et al. (1993 a, 1994) ont montre que des microorganismes deleteres, de nature fongique, etaient associes au jaunissement de l'Epicea commun en France et en Allemagne . Il s'agit d'une des formes recensees de deperissement de l'Epicea (Type 1 : spruce decline ; Roberts et al ., 1989) caracterisee par le jaunissement et la chute des aiguilles (il faut cependant rappeler que, dans de nombreux cas, la defoliation chez l'Epicea n'est pas liee a un jaunissement) . Le jaunissement est du a une carence en magnesium dans les aiguilles qui resulte d'une trop faible absorption de cet element par les racines, dans des sols acides pauvres en magnesium echangeable . C'est la reduction de la photosynthese et, par lameme, la diminution de la translocation des carbohydrates, qui entrainent une baisse de la croissance des arbres . Les champignons deleteres sont presents dans le sol des forets affectees par le jaunissement et absents dans le sol des forets saines.

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Summary — A range of bacterial isolates from Laccaria laccata mycorrhizas and sporocarps were tested for their effect on ectomycorrhizal development of Douglas fir with L laccata. The experiments were carried out in aseptic conditions and in the glasshouse under summer and winter conditions. Fourteen isolates increased mycorrhizal development after 16 wk of growth in the summer experiment. Seven bacterial isolates displayed a significant stimulating effect in the winter experiment. All bacterial isolates tested under aseptic conditions displayed a significant stimulating effect. In the winter experiment, the treatments without L laccata inoculation were contaminated by Thelephora terrestris (ectomycorrhizal basidiomycete), a natural contaminant in the glasshouse. Six bacterial isolates displayed a significant inhibiting effect towards ectomycorrhizal infection by T terrestris. Three isolates enhanced the ectomycorrhizal development of Douglas fir with L laccata in all experiments. It is confirmed that the inoculation techniques in forest nurseries could be improved by such mycorrhization helper bacteria (MHB). The results with T terrestris suggest that the mechanisms involved in interactions between bacteria and mycorrhizal establishment are partly fungus-specific. The results of the experiments in aseptic conditons suggest that the MHB act directly on the plant or/and on the fungus. Their stimulating effect is not the result of the suppression of root pathogens or other inhibitors of mycorrhizal infection. MHB could be used both for enhancing the infection by an introduced fungus and for reducing unwanted infection by inefficient symbionts such as T terrestris. Thus, the need for soil disinfection before inoculating might be reduced.

11 ref.; National audience

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A fumigated nursery bed on a sandy loam was inoculated with the ectomycorrhizal fungus Laccaria laccata and seeded with Douglas fir. Two types of inoculum were compared : mycelium grown in a vermiculite/peat mixture, and mycelium grown in liquid medium and entrapped in a calcium alginate gel with different quantities of mycelium. At the end of the first growing season, the alginate inoculum at the dose of 5 g mycelium (dry weight) per m2 proved to be the most efficient. The top dry weight of the seedlings in this treatment was 2.3 fold that of the noninoculated fumigated controls. This inoculation treatment also ensured nearly total mycorrhizal infection by L. laccata and reduced root necrosis caused by fungal pathogens., Des semis de Douglas (Pseudotsuga menziesii) ont été cultivés en pépinière sur un sol sablo-limoneux désinfecté au bromure de méthyle. L’inoculation par le champignon ectomycorhizien Laccaria laccata a été réalisée à l’aide de mycélium ayant poussé dans un mélange de vermiculite et de tourbe ou de mycélium produit en milieu liquide et inclus dans un gel d’alginate de calcium, avec trois doses différentes. Pendant toute la saison de végétation, des observations ont porté sur la croissance des semis, l’infection ectomycorhizienne et le développement des nécroses racinaires dues à Fusarium oxysporum. En fin d’année, les meilleurs résultats (par comparaison avec un témoin non inoculé) ont été obtenus avec l’inoculum inclus dans l’alginate à la dose de 5 g de mycélium (poids de matière sèche par m2), qui procure une infection presque totale par L. laccata, ramène l’intensité des nécroses racinaires à un niveau tolérable, et double la biomasse des semis. L’analyse de l’évolution de l’infection au cours de la saison de végétation montre que la supériorité de l’inoculum alginate est essentiellement due à une meilleure survie du champignon et à une infection plus étalée dans le temps que dans le cas de l’inoculum classique vermiculite/tourbe. Ces résultats sont d’un grand intérêt pratique, car on sait par ailleurs que L. laccata, dans ce type de pépinière, permet de produire des plants plus sains et de taille commerciale en 2 ans au lieu de 3 ans, et qu’il assure une meilleure reprise et une meilleure croissance initiale après transplantation en forêt.

10 ref.; National audience

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La necessite d'elargir la gamme des clones adaptes aux conditions ecologiques du nord-est de la France, a conduit la Federation de la vulgarisation forestiere de l'Est a installer en 1968, un premier reseau regional d'observations sur le comportement, en pepiniere et en plantation, de onze clones parmi lesquels certains ont fait recemment l'objet d'introductions dans la region (voir liste en annexe) . Ce premier reseau est complete actuellement par un dispositif regional interessant de nouveaux clones isoles dans divers instituts de recherches ; ce dispositif s'insere dans le programme experimental national, recemment cite par Monsieur Viart . Au niveau regional, l'ensemble de ces efforts est entrepris par les organismes charges du developpement de la foret privee en Lorraine, Alsace et Franche-Comte, avec le concours de la Commission nationale du Peuplier, du Centre technique du genie rural des eaux et des forets et du Centre national de recherches forestieres (1).