Optimized assay and storage conditions for enzyme activity profiling of ectomycorrhizae.

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.

Influence of forest trees on the distribution of mineral weathering-associated bacterial communities of the Scleroderma citrinum mycorrhizosphere.

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

Effects of liming on potential oxalate secretion and iron chelation of beech ectomycorrhizal root tips.

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.

Does forest liming impact the enzymatic profiles of ectomycorrhizal communities through specialized fungal symbionts?

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).

Temporal changes in the ectomycorrhizal community in two soil horizons of a temperate oak forest.

The species structure of an ectomycorrhizal (ECM) community was assessed monthly for 15 months in the two horizons (A1 and A2) of an oak temperate forest in northeastern France. Ectomycorrhizal species were identified each month by internal transcribed spacer sequencing. Seventy-five fungal symbionts were identified. The community was dominated by Tomentellaceae, Russulaceae, Cortinariaceae, and Boletales. Four species are abundant in the study site: Lactarius quietus, Tomentella sublilacina, Cenococcum geophilum, and Russula sp1. The relative abundance of each species varied depending on the soil horizon and over time. Some species, such as L. quietus, were present in the A1 and A2 horizons. C. geophilum was located particularly in the A2 horizon, whereas T. sublilacina was more abundant in A1. Some species, such as Clavulina sp., were detected in winter, while T. sublilacina and L. quietus were present all year long. Our results support the hypothesis that a rapid turnover of species composition of the ECM community occurs over the course of a month. The spatial and temporal unequal distribution of ECM species could be explained by their ecological preferences, driven by such factors as root longevity, competition for resources, and resistance to environmental variability.

Cenococcum geophilum populations show a high degree of genetic diversity in beech forests.

• 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.

Contrasting responses to mycorrhizal inoculation and phosphorus availability in seedlings of two tropical rainforest tree species.

• 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.

Liming in a beech forest results in more mineral elements stored in the mantle of Lactarius subdulcis ectomycorrhizas.

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.

Differential ability of ectomycorrhizas to survive drying.

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.

The fungal dimension of biological invasions.

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.

Occurrence and diversity of bacterial communities in Tuber magnatum during truffle maturation.

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.

Mycorrhization helper bacteria: a case of specificity for altering ectomycorrhiza architecture but not ectomycorrhiza formation.

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.

Year-round monitoring of diversity and potential metabolic activity of the ectomycorrhizal community in a beech (Fagus silvatica) forest subjected to two thinning regimes.

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.

Activity profiling of ectomycorrhiza communities in two forest soils using multiple enzymatic tests.

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.

Ectomycorrhizal symbiosis affects functional diversity of rhizosphere fluorescent pseudomonads.

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 N2-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.