François Le Tacon

Survival of inoculated Laccaria bicolor in competition with native ectomycorrhizal fungi and effects on the growth of outplanted Douglasfir seedlings

The survival, development and mycorrhizal efficiency of a selected strain of Laccaria bicolor along with naturally occurring ectomycorrhizal fungi in a young plantation of Douglas fir was examined. Symbionts were identified and their respective colonization abilities were determined. Eight species of symbiotic fungi, which may have originated in adjacent coniferous forests, were observed on the root systems. Mycorrhizal diversity differed between inoculated (5 taxa) and control (8 taxa) seedlings. Ectomycorrhizal fungi which occurred naturally in the nursery on control seedlings (Thelephora terrestris and Suillus sp.) did not survive after outplanting. Both inoculated and naturally occurring Laccaria species, as well as Cenococcum geophilum, survived on the old roots and colonized the newly formed roots, limiting the colonization by other naturally occurring fungi. Other fungi, such as Paxillus involutus, Scleroderma citrinum and Hebeloma sp. preferentially colonized the old roots near the seedlings collar. Russulaceae were found mainly in the middle section of the root system. Mycorrhizal colonization by Laccaria species on inoculated seedlings (54%) was significantly greater than on controls (13%) which were consequently dominated by the native fungi. Significant differences (up to 239%) were found in the growth of inoculated seedlings, especially in root and shoot weight, which developed mainly during the second year after outplanting. Seedling growth varied with the species of mycorrhizae and with the degree of root colonization. Competitiveness and effectiveness of the introduced strain on improving growth performances of seedlings are discussed.

Fine-scale spatial genetic structure of the black truffle (Tuber melanosporum) investigated with neutral microsatellites and functional mating type genes.

The genetic structure of ectomycorrhizal (ECM) fungal populations results from both vegetative and sexual propagation. In this study, we have analysed the spatial genetic structure of Tuber melanosporum populations, a heterothallic ascomycete that produces edible fruit bodies. Ectomycorrhizas from oaks and hazels from two orchards were mapped and genotyped using simple sequence repeat markers and the mating type locus. The distribution of the two T. melanosporum mating types was also monitored in the soil. In one orchard, the genetic profiles of the ascocarps were compared with those of the underlying mycorrhizas. A pronounced spatial genetic structure was found. The maximum genet sizes were 2.35 and 4.70 m in the two orchards, with most manifesting a size < 1 m. Few genets persisted throughout two seasons. A nonrandom distribution pattern of the T. melanosporum was observed, resulting in field patches colonized by genets that shared the same mating types. Our findings suggest that competition occurs between genets and provide basic information on T. melanosporum propagation patterns that are relevant for the management of productive truffle orchards.

Carbon Transfer from the Host to Tuber melanosporum Mycorrhizas and Ascocarps Followed Using a 13C Pulse-Labeling Technique

Truffles ascocarps need carbon to grow, but it is not known whether this carbon comes directly from the tree (heterotrophy) or from soil organic matter (saprotrophy). The objective of this work was to investigate the heterotrophic side of the ascocarp nutrition by assessing the allocation of carbon by the host to Tuber melanosporum mycorrhizas and ascocarps. In 2010, a single hazel tree selected for its high truffle (Tuber melanosporum) production and situated in the west part of the Vosges, France, was labeled with 13CO2. The transfer of 13C from the leaves to the fine roots and T. melanosporum mycorrhizas was very slow compared with the results found in the literature for herbaceous plants or other tree species. The fine roots primarily acted as a carbon conduit; they accumulated little 13C and transferred it slowly to the mycorrhizas. The mycorrhizas first formed a carbon sink and accumulated 13C prior to ascocarp development. Then, the mycorrhizas transferred 13C to the ascocarps to provide constitutive carbon (1.7 mg of 13C per day). The ascocarps accumulated host carbon until reaching complete maturity, 200 days after the first labeling and 150 days after the second labeling event. This role of the Tuber ascocarps as a carbon sink occurred several months after the end of carbon assimilation by the host and at low temperature. This finding suggests that carbon allocated to the ascocarps during winter was provided by reserve compounds stored in the wood and hydrolyzed during a period of frost. Almost all of the constitutive carbon allocated to the truffles (1% of the total carbon assimilated by the tree during the growing season) came from the host.

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

Abstract 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 α and β, 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 α and β 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.

Black truffle-associated bacterial communities during the development and maturation of Tuber melanosporum ascocarps and putative functional roles

Although truffles are cultivated since decades, their life cycle and the conditions stimulating ascocarp formation still remain mysterious. A role for bacteria in the development of several truffle species has been suggested but few is known regarding the natural bacterial communities of Périgord Black truffle. Thus, the aim of this study was to decipher the structure and the functional potential of the bacterial communities associated to the Black truffle in the course of its life cycle and along truffle maturation. A polyphasic approach combining 454-pyrosequencing of 16S rRNA gene, TTGE, in situ hybridization and functional GeoChip 3.0 revealed that Black truffle ascocarps provide a habitat to complex bacterial communities that are clearly differentiated from those of the surrounding soil and the ectomycorrhizosphere. The composition of these communities is dynamic and evolves during the maturation of the ascocarps with an enrichment of specific taxa and a differentiation of the gleba and peridium-associated bacterial communities. Genes related to nitrogen and sulphur cycling were enriched in the ascocarps. Together, these data paint a new picture of the interactions existing between truffle and bacteria and of the potential role of these bacteria in truffle maturation.

The nuclear rDNA intergenic spacer of the ectomycorrhizal basidiomycete Laccaria bicolor : structural analysis and allelic polymorphism

The nuclear rDNA intergenic spacer (IGS) of the ectomycorrhizal basidiomycete Laccaria bicolor was amplified and sequenced to identify the source of its intraspecific polymorphism. The IGS was amplified by PCR in several L. bicolor strains and shown to exhibit multiple bands and length polymorphism. The IGS loci were shown to segregate in a 1:1 ratio within haploid progenies in three dikaryotic strains, suggesting that divergent IGS haplotypes were present in the two nuclei of these strains. The two haplotypes of L. bicolor S238N were sequenced: the beta-haplotype was 4160 bp in length, whereas the size of the alpha-haplotype was estimated to be about 4700 bp. These represent the largest published fungal IGS sequences to date. These sequences can be subdivided into three main regions, IGS1, 5S rDNA and IGS2. The IGS sequences are AT-rich and contain numerous occurrences of three types of subrepeats (e.g. T2-AC3). The length polymorphism, observed between the IGS sequence of the alpha- and beta-haplotypes, results from the insertion of various numbers of a 71 bp subrepeat, called B, occurring in IGS2. This variation in subrepeat number suggests that the two haplotypes resulted from unequal cross-overs. The L. bicolor IGS was aligned with IGS sequences of two other Tricholomataceae (i.e. Tricholoma matsutake and Collybia fusipes). No sequence similarity was observed between these IGSs, but homologous subrepeats were found in L. bicolor and T. matsutake. Analysis of IGS length polymorphism is therefore an efficient tool for investigating genetic relationships between genets and within progenies in natural fungal populations.

Identification of quantitative trait loci affecting ectomycorrhizal symbiosis in an interspecific F1 poplar cross and differential expression of genes in ectomycorrhizas of the two parents: Populus deltoides and Populus trichocarpa

A Populus deltoides × Populus trichocarpa F1 pedigree was analyzed for quantitative trait loci (QTLs) affecting ectomycorrhizal development and for microarray characterization of gene networks involved in this symbiosis. A 300 genotype progeny set was evaluated for its ability to form ectomycorrhiza with the basidiomycete Laccaria bicolor. The percentage of mycorrhizal root tips was determined on the root systems of all 300 progeny and their two parents. QTL analysis identified four significant QTLs, one on the P. deltoides and three on the P. trichocarpa genetic maps. These QTLs were aligned to the P. trichocarpa genome and each contained several megabases and encompass numerous genes. NimbleGen whole-genome microarray, using cDNA from RNA extracts of ectomycorrhizal root tips from the parental genotypes P. trichocarpa and P. deltoides, was used to narrow the candidate gene list. Among the 1,543 differentially expressed genes (p value ≤ 0.05; ≥5.0-fold change in transcript level) having different transcript levels in mycorrhiza of the two parents, 41 transcripts were located in the QTL intervals: 20 in Myc_d1, 14 in Myc_t1, and seven in Myc_t2, while no significant differences among transcripts were found in Myc_t3. Among these 41 transcripts, 25 were overrepresented in P. deltoides relative to P. trichocarpa; 16 were overrepresented in P. trichocarpa. The transcript showing the highest overrepresentation in P. trichocarpa mycorrhiza libraries compared to P. deltoides mycorrhiza codes for an ethylene-sensitive EREBP-4 protein which may repress defense mechanisms in P. trichocarpa while the highest overrepresented transcripts in P. deltoides code for proteins/genes typically associated with pathogen resistance.

Characterization of Transposable Elements in the Ectomycorrhizal Fungus Laccaria bicolor

Background The publicly available Laccaria bicolor genome sequence has provided a considerable genomic resource allowing systematic identification of transposable elements (TEs) in this symbiotic ectomycorrhizal fungus. Using a TE-specific annotation pipeline we have characterized and analyzed TEs in the L. bicolor S238N-H82 genome. Methodology/Principal Findings TEs occupy 24% of the 60 Mb L. bicolor genome and represent 25,787 full-length and partial copy elements distributed within 171 families. The most abundant elements were the Copia-like. TEs are not randomly distributed across the genome, but are tightly nested or clustered. The majority of TEs exhibits signs of ancient transposition except some intact copies of terminal inverted repeats (TIRS), long terminal repeats (LTRs) and a large retrotransposon derivative (LARD) element. There were three main periods of TE expansion in L. bicolor: the first from 57 to 10 Mya, the second from 5 to 1 Mya and the most recent from 0.5 Mya ago until now. LTR retrotransposons are closely related to retrotransposons found in another basidiomycete, Coprinopsis cinerea. Conclusions This analysis 1) represents an initial characterization of TEs in the L. bicolor genome, 2) contributes to improve genome annotation and a greater understanding of the role TEs played in genome organization and evolution and 3) provides a valuable resource for future research on the genome evolution within the Laccaria genus.

Mycorhizes, pépinières et plantations forestières en France.

La première démonstration de la nécessité de l’association des arbres forestiers avec des champignons mycorhiziens a été faite il y a déjà plus d’un siècle (Frank, 1894). L’objectif de la mycorhization contrôlée des arbres forestiers est d’associer, au stade de la pépinière, les jeunes plants avec des souches fongiques mycorhiziennes performantes. Après transfert de ces plants en forêt, les souches fongiques inoculées peuvent survivre et assurer ainsi une meilleure reprise ou une meilleure croissance des arbres.

Certainties and uncertainties about the life cycle of the Périgord black truffle (Tuber melanosporum Vittad.)

Abstract• Key messageSeveral aspects of the life cycle of the Périgord black truffle have been elucidated only recently, while others remain either controversial or unstudied. In this paper, we present a revised life cycle of this fungus and highlight key aspects that have yet to be addressed or require further understanding.• ContextThe hypogeous sporophores of several Tuber species, renowned for their aromatic and gustatory qualities, are widely commercialized. One of the most valuable species is Tuber melanosporum Vittad., the Périgord black truffle also known as “the black diamond”. However, many aspects of T. melanosporum life cycle remain unsolved.• AimsIn this work, we examine past and recent findings on the life cycle of T. melanosporum, currently regarded as a model system for Tuber species, with the view of highlighting aspects of its life cycle which remain unsolved.• ResultsSeveral aspects of its life cycle have recently been elucidated (i.e. characterization of two mating type genes, heterothallism, prevalence of sexual reproduction on vegetative propagation, exclusion of one mating type by its opposite on ectomycorrhizas, dependency of ascocarps on their host for carbon allocation), while others remain unaddressed.• ConclusionNumerous additional aspects of the T. melanosporum life cycle remain unsolved, such as exclusion or competition mechanisms between ectomycorrhizal mating types, factors involved in ascocarp initiation, the nature of the connection linking ascocarps and mycorrhizas and atmospheric nitrogen fixation.