Efficiency of the traditional practice of traps to stimulate black truffle production, and its ecological mechanisms.

The black truffle Tuber melanosporum was disseminated all over the world, propelled by the development of a wide variety of empirical practices. A widespread practice, called 'truffle trap', consists of placing pieces of truffles into excavations dug under host trees, and of collecting truffle in these traps in the next years. This research aims at (1) evaluating the effect of this practice on fruitbody production based on the analysis of 9924 truffle traps installed in 11 orchards across T. melanosporum native area in France and (2) exploring the mechanisms involved in fruitbody emergence using traps where the genotypes of introduced truffles were compared with those of fruitbodies collected in the same traps. We confirmed that truffle traps provide a major and highly variable part of truffle ground production, representing up to 89% of the collected fruitbodies. We evidenced a genetic link between introduced spores and collected fruitbodies, and then demonstrated that truffle growers provide paternal partners for mating with local maternal mycelia. We also highlighted that soil disturbance stimulate the vegetative development of established maternal mycelia. This research supports that a widely used traditional practice enhances fruitbody production by shaping favorable conditions and providing sexual partners required for fruiting.

How the truffle got its mate: insights from genetic structure in spontaneous and planted Mediterranean populations of Tuber melanosporum.

The life cycles and dispersal of edible fungi are still poorly known, thus limiting our understanding of their evolution and domestication. The prized Tuber melanosporum produces fruitbodies (fleshy organs where meiospores mature) gathered in natural, spontaneously inoculated forests or harvested in plantations of nursery-inoculated trees. Yet, how fruitbodies are formed remains unclear, thus limiting yields, and how current domestication attempts affect population genetic structure is overlooked. Fruitbodies result from mating between two haploid individuals: the maternal parent forms the flesh and the meiospores, while the paternal parent only contributes to the meiospores. We analyzed the genetic diversity of T. melanosporum comparatively in spontaneous forests vs. plantations, using SSR polymorphism of 950 samples from South-East France. All populations displayed strong genetic isolation by distance at the metric scale, possibly due to animal dispersal, meiospore persistence in soil, and/or exclusion of unrelated individuals by vegetative incompatibility. High inbreeding was consistently found, suggesting that parents often develop from meiospores produced by the same fruitbody. Unlike maternal genotypes, paternal mycelia contributed to few fruitbodies each, did not persist over years, and were undetectable on tree mycorrhizae. Thus, we postulate that germlings from the soil spore bank act as paternal partners. Paternal genetic diversity and outbreeding were higher in plantations than in spontaneous truffle-grounds, perhaps because truffle growers disperse fruitbodies to maintain inoculation in plantations. However, planted and spontaneous populations were not genetically isolated, so that T. melanosporum illustrates an early step of domestication where genetic structure remains little affected.

Isolation and characterization of new polymorphic microsatellite loci in the mixotrophic orchid Limodorum abortivum L. Swartz (Orchidaceae).

Here, we report the isolation and characterization of 11 polymorphic microsatellites in Limodorum abortivum. Allele variability has been characterized in three populations from Southern Italy and France. The number of alleles ranged from one to six per locus with an average of 3.8 alleles per locus. Observed and expected heterozygosity values ranged from 0.000 to 1.000 and from 0.492 to 0.806, respectively, with striking differences among populations. These microsatellites should be valuable tools for studying fine-scale genetic structure of scattered Limodorum abortivum populations, patterns of relationship with closely related taxa and the evolutionary ecology of its mycorrhizal interactions.

Inefficient photosynthesis in the Mediterranean orchid Limodorum abortivum is mirrored by specific association to ectomycorrhizal Russulaceae.

Among European Neottieae, Limodorum abortivum is a common Mediterranean orchid. It forms small populations with a patchy distribution in woodlands, and is characterized by much reduced leaves, suggesting a partial mycoheterotrophy. We have investigated both the photosynthetic abilities of L. abortivum adult plants and the diversity of mycorrhizal fungi in Limodorum plants growing in different environments and plant communities (coniferous and broadleaf forests) over a wide geographical and altitudinal range. Despite the presence of photosynthetic pigments, CO2 fixation was found to be insufficient to compensate for respiration in adult plants. Fungal diversity was assessed by morphological and molecular methods in L. abortivum as well as in the related rare species Limodorum trabutianum and Limodorum brulloi. Phylogenetic analyses of the fungal internal transcribed spacer (ITS) sequences, obtained from root samples of about 80 plants, revealed a tendency to associate predominantly with fungal symbionts of the genus Russula. Based on sequence similarities with known species, most root endophytes could be ascribed to the species complex encompassing Russula delica, Russula chloroides, and Russula brevipes. Few sequences clustered in separate groups nested within Russula, a genus of ectomycorrhizal fungi. The morphotypes of ectomycorrhizal root tips of surrounding trees yielded sequences similar or identical to those obtained from L. abortivum. These results demonstrate that Limodorum species with inefficient photosynthesis specifically associate with ectomycorrhizal fungi, and appear to have adopted a nutrition strategy similar to that known from achlorophyllous orchids.

Diversity and specificity of ectomycorrhizal fungi retrieved from an old-growth Mediterranean forest dominated by Quercus ilex.

We analysed the ectomycorrhizal (ECM) fungal diversity in a Mediterranean old-growth Quercus ilex forest stand from Corsica (France), where Arbutus unedo was the only other ECM host. On a 6400 m2 stand, we investigated whether oak age and host species shaped below-ground ECM diversity. Ectomycorrhizas were collected under Q. ilex individuals of various ages (1 yr seedlings; 3-10 yr saplings; old trees) and A. unedo. They were typed by ITS-RFLP analysis and identified by match to RFLP patterns of fruitbodies, or by sequencing. A diversity of 140 taxa was found among 558 ectomycorrhizas, with many rare taxa. Cenococcum geophilum dominated (35% of ECMs), as well as Russulaceae, Cortinariaceae and Thelephoraceae. Fungal species richness was comparable above and below ground, but the two levels exhibited < 20% overlap in fungal species composition. Quercus ilex age did not strongly shape ECM diversity. The two ECM hosts, A. unedo and Q. ilex, tended to share few ECM species (< 15% of the ECM diversity). Implications for oak forest dynamics are discussed.

Chlorophyllous and achlorophyllous specimens of Epipactis microphylla,(Neottieae, Orchidaceae) are associated with ectomycorrhizal septomycetes, including truffles.

Mycoheterotrophic species (i.e., achlorophyllous plants obtaining carbon from their mycorrhizal fungi) arose many times in evolution of the Neottieae, an orchid tribe growing in forests. Moreover, chlorophyllous Neottieae species show naturally occurring achlorophyllous individuals. We investigated the fungal associates of such a member of the Neottieae, Epipactis microphylla, to understand whether their mycorrhizal fungi predispose the Neottieae to mycoheterotrophy. Root symbionts were identified by sequencing the fungal ITS of 18 individuals from three orchid populations, including achlorophyllous and young, subterranean individuals. No rhizoctonias (the usual orchid symbionts) were recovered, but 78% of investigated root pieces were colonized by Tuber spp. Other Pezizales and some Basidiomycetes were also found. Using electron microscopy, we demonstrated for the first time that ascomycetes, especially truffles, form typical orchid mycorrhizae. All identified fungi (but one) belonged to taxa forming ectomycorrhizae on tree roots, and four of them were even shown to colonize surrounding trees. This is reminiscent of mycoheterotrophic orchid species that also associate with ectomycorrhizal fungi, although with higher specificity. Subterranean and achlorophyllous E. microphylla individuals thus likely rely on tree photosynthates, and a partial mycoheterotrophy in individuals plants can be predicted. We hypothesize that replacement of rhizoctonias by ectomycorrhizal symbionts in Neottieae entails a predisposition to achlorophylly.

Founder effect in a young Leccinum duriusculum (Schultzer) Singer population.

The genetic diversity of a Leccinum duriusculum population growing under <20-year-old Populus alba on former farming soil was analysed from 1998 to 2001 and compared in 2000 to the two nearest populations found under >70-year-old P. alba. Genets were recognized using RAPD amplifications with three different primers, while their conspecificity was assessed by sequencing the nuclear ITS and mitochondrial large ribosomal subunit. The young population was colonized by a large genet that persisted from 1998 to 2001 (most distal sporophores were 10.4 m apart in 2001) and a second genetically related genet appeared in 2001. Five and six genets, respectively, of smaller size were found in the two other populations, while the investigated area was slightly smaller (72.25 m2) and the three populations were strongly divergent genetically (>33%). The genetic uniformity, as well as the high speed of radial growth of the lasting genet under <20-year-old P. alba (radial growth: 1 m/year), are interpreted in the framework of a founder effect. The slow recruitment of genets is proposed to lower the intraspecific competition and to entail large, fast-growing genets. The differences from ectomycorrhizal populations due to secondary colonization, which have been investigated often, are also emphasized.

Basal hymenomycetes belonging to the Sebacinaceae are ectomycorrhizal on temperate deciduous trees.

• Heterobasidiomycetous species of the Sebacinaceae family, previously considered as saprophytes or parasites, are shown here to form ectomycorrhizas on temperate forest trees. • Ectomycorrhizas were collected under sebacinoid sporophores and near root systems of Neottia nidus-avis, an orchid symbiotic with sebacinoids. To identify the partners each ectomycorrhiza was submitted to amplification and sequencing of the plant and fungal internal transcribed spacer (ITS), and further investigated by light and electron microscopy whenever a sebacinoid ITS was found. • Molecular and microscopic analyses correlated well. Two sebacinoids of divergent rDNA sequences were demonstrated to form similar ectomycorrhizas, with a well-developed Hartig net and a hyphal mantle having thick-walled outer mantle hyphae. The ultrastructure of the septal pore (dolipore with imperforate caps) was typical for sebacinoids. In one case, intracellular colonization was seen. The ectomycorrhizal host range of these sebacinoids was not specific and included Betulaceae, Fagaceae and Tiliaceae. • Sebacinoids probably represent an overlooked ectomycorrhizal group and ectomycorrhizal symbiosis may be common among basal lineages of hymenomycetes.

Reducing the genome size of organelles favours gene transfer to the nucleus.

Endosymbiotic organelles exhibit strong genetic erosion during their evolution as a result of the loss of unnecessary genes and of gene transfer to the nucleus. The reasons for this erosion are much debated. Unidirectionality of DNA exchange between cell compartments could favour biased gene transfer, but selection might also act to favour nuclear localization of genes, for example, because organelles accumulate more mutations than do nuclei. Selection for rapid replication might be a general cause of organelle genome reduction. This selection also accounts for the compactness of organelle genomes.

Effects of detergents on P-glycoprotein atpase activity: differences in perturbations of basal and verapamil-dependent activities.

P-glycoprotein (P-gp), a plasma membrane glycoprotein associated with the multidrug resistance phenotype, is responsible for the ATP-dependent efflux of various amphiphilic drugs. Using membrane vesicles prepared from the multidrug resistant cell line DC-3F/ADX, we studied the perturbation of the basal (i.e. in the absence of drug) and verapamil-dependent P-gp ATPase activities induced by various detergents, at non-solubilizing, as well as at solubilizing, concentrations. The progressive membrane solubilization with increasing detergent concentration was monitored by light scattering and centrifugation experiments. For non-solubilizing detergent concentrations, all tested detergents except DOC induced a partial inhibition of P-gp ATPase activity, which was not correlated with the amount of the various tested detergents incorporated in the membranes. Analysis of the verapamil-induced P-gp activation reveals that P-gp ATPase activity is differently modulated by the various detergents at non-solubilizing concentrations. Thus, specific interactions between P-gp and detergents are more likely to occur rather than a global membrane perturbation. After solubilization by the various tested detergents, the basal P-gp ATPase activity was virtually completely inhibited, except in the presence of CHAPS which was able to preserve this activity at a level comparable to that measured in native membranes. However, the verapamil-induced P-gp ATPase activation was lost during P-gp solubilization by CHAPS, but recovered after dilution of CHAPS below its critical micellar concentration. These observations indicate specific interactions between P-gp and CHAPS molecules within the mixed micelles. On the whole, our data evidencing specific interactions P-gp/detergents are consistent with the location of the drug transport sites on P-gp transmembrane domains.

The land flora: a phototroph-fungus partnership?

Numerous mutualistic associations between phototrophs and fungi exist in the extant land biota. Some are widespread, such as lichens and mycorrhizae, but some are less well known or restricted to special ecological conditions, such as endophytes in plants and algae. Recent molecular data and fossils suggest that associations arose repeatedly and that some of them are ancient, and even ancestral in the case of land plants. Mutualism, that provides various adaptations to terrestrial constraints, may have played a crucial role during terrestrialization and evolution of land phototrophs.

Meiotic segregation and recombination of the intergenic spacer of the ribosomal DNA in the ectomycorrhizal basidiomycete Laccaria bicolor.

The aim of this study was to clarify the inheritance of the nuclear ribosomal DNA (rDNA) in the ectomycorrhizal basidiomycete Laccaria bicolor S238N in order to resolve inter- and within-strain relationships in forest ecosystems. PCR amplification of the intergenic spacer (IGS) was carried out in the dikaryotic mycelium and its haploid progeny. In the dikaryotic mycelium, multiple amplification products were produced for the 25s/5s (IGS1) and 5s/17s (IGS2) intergenic spacers. The 4.5- and 4.0-kb fragments of IGS2 (haplotypes alpha and beta, respectively) were observed to occur in a 1:1 ratio within the haploid progeny as a result of divergent IGS haplotypes in the two separate nuclei. Recombinant monokaryons having both types of IGS2 occurred at a low frequency (6.5%; 60 kb per centimorgan) during meiosis. Haplotypes alpha and beta of IGS1 cross-hybridized forming heteroduplexes during the PCR temperature cycle. The two IGS1 haplotypes differed only by the repeat number of a TA2C3 motif and co-segregated with the IGS2 haplotypes. Heteroduplex formation and IGS polymorphism provide information that is helpful in distinguishing between introduced exotic L. bicolor S238N and indigenous populations of Laccaria spp. in forest ecosystems.