From field sampling to pneumatic bioreactor mycelia production of the ectomycorrhizal mushroom Laccaria trichodermophora.

In order to increase survival rates of greenhouse seedlings destined for restoration and conservation programs, successful mycorrhization of the seedlings is necessary. To reforest forest ecosystems, host trees must be inoculated with ectomycorrhizal fungi and, in order to guarantee a sufficient supply of ectomycorrhizal inoculum, it is necessary to develop technologies for the mass production of ectomycorrhizal fungi mycelia. We selected the ectomycorrhizal fungus Laccaria trichodermophora, due to its ecological traits and feasible mycelia production in asymbiotic conditions. Here, we report the field sampling of genetic resources, as well as the highly productive nutritional media and cultivation parameters in solid cultures. Furthermore, in order to achieve high mycelial production, we used strain screening and evaluated pH, carbon source concentration, and culture conditions of submerged cultures in normal and baffled shake flasks. The higher productivity culture conditions in shake flasks were selected for evaluation in a pneumatic bioreactor, using modified BAF media with a 10 g/L glucose, pH 5.5, 25 °C, and a volumetric oxygen transfer coefficient (KLa) of 36 h-1. Under those conditions less biomass (12-37 %) was produced in the pneumatic bioreactor compared with the baffled shake flasks. This approach shows that L. trichodermophora can generate a large biomass concentration and constitute the biotechnological foundation of its mycelia mass production.

Out of Asia: Biogeography of fungal populations reveals Asian origin of diversification of the Laccaria amethystina complex, and two new species of violet Laccaria.

Purple Laccaria are ectomycorrhizal basidiomycetes associated with temperate forests all over the Northern Hemisphere in at least two taxa: Laccaria amethysteo-occidentalis in North America, and L. amethystina complex in Eurasia, as shown by Vincenot et al. (2012). Here, we combine a further study of the genetic structure of L. amethystina populations from Europe to southwestern China and Japan, using neutral Single Sequence Repeat (SSR; microsatellite) markers; and a systematic description of two novel Asian species, namely Laccaria moshuijun and Laccaria japonica, based on ecological, morphological, and molecular criteria (rDNA sequences). Population genetics provides evidence of the ancient isolation of three regional groups, with strong signal for speciation, and suggests a centre of origin of modern populations closest to present-day Chinese populations. Phylogenetic analyses confirm speciation at the molecular level, reflected in morphological features: L. moshuijun samples (from Yunnan, China) display strongly variable cheilocystidia, while L. japonica samples (from Japan) present distinctive globose to subglobose spores and clavate cheilocystidia. This study of a species complex primarily described with an extremely wide ecological and geographical range sheds new light on the biodiversity and biogeography of ectomycorrhizal fungi.

Examining the efficacy of a genotyping-by-sequencing technique for population genetic analysis of the mushroom Laccaria bicolor and evaluating whether a reference genome is necessary to assess homology.

Given the diversity and ecological importance of Fungi, there is a lack of population genetic research on these organisms. The reason for this can be explained in part by their cryptic nature and difficulty in identifying genets. In addition the difficulty (relative to plants and animals) in developing molecular markers for fungal population genetics contributes to the lack of research in this area. This study examines the ability of restriction-site associated DNA (RAD) sequencing to generate SNPs in Laccaria bicolor. Eighteen samples of morphologically identified L. bicolor from the United States and Europe were selected for this project. The RAD sequencing method produced anywhere from 290 000 to more than 3 000 000 reads. Mapping these reads to the genome of L. bicolor resulted in 84 000-940 000 unique reads from individual samples. Results indicate that incorporation of non-L. bicolor taxa into the analysis resulted in a precipitous drop in shared loci among samples, suggests the potential of these methods to identify cryptic species. F-statistics were easily calculated, although an observable "noise" was detected when using the "All Loci" treatment versus filtering loci to those present in at least 50% of the individuals. The data were analyzed with tests of Hardy-Weinburg equilibrium, population genetic statistics (FIS and FST), and population structure analysis using the program Structure. The results provide encouraging feedback regarding the potential utility of these methods and their data for population genetic analysis. We were unable to draw conclusions of life history of L. bicolor populations from this dataset, given the small sample size. The results of this study indicate the potential of these methods to address population genetics and general life history questions in the Agaricales. Further research is necessary to explore the specific application of these methods in the Agaricales or other fungal groups.

Genomic and transcriptomic analysis of Laccaria bicolor CAZome reveals insights into polysaccharides remodelling during symbiosis establishment.

Ectomycorrhizal fungi, living in soil forests, are required microorganisms to sustain tree growth and productivity. The establishment of mutualistic interaction with roots to form ectomycorrhiza (ECM) is not well known at the molecular level. In particular, how fungal and plant cell walls are rearranged to establish a fully functional ectomycorrhiza is poorly understood. Nevertheless, it is likely that Carbohydrate Active enZymes (CAZyme) produced by the fungus participate in this process. Genome-wide transcriptome profiling during ECM development was used to examine how the CAZome of Laccaria bicolor is regulated during symbiosis establishment. CAZymes active on fungal cell wall were upregulated during ECM development in particular after 4weeks of contact when the hyphae are surrounding the root cells and start to colonize the apoplast. We demonstrated that one expansin-like protein, whose expression is specific to symbiotic tissues, localizes within fungal cell wall. Whereas L. bicolor genome contained a constricted repertoire of CAZymes active on cellulose and hemicellulose, these CAZymes were expressed during the first steps of root cells colonization. L. bicolor retained the ability to use homogalacturonan, a pectin-derived substrate, as carbon source. CAZymes likely involved in pectin hydrolysis were mainly expressed at the stage of a fully mature ECM. All together, our data suggest an active remodelling of fungal cell wall with a possible involvement of expansin during ECM development. By contrast, a soft remodelling of the plant cell wall likely occurs through the loosening of the cellulose microfibrils by AA9 or GH12 CAZymes and middle lamella smooth remodelling through pectin (homogalacturonan) hydrolysis likely by GH28, GH12 CAZymes.

Genet dynamics and ecological functions of the pioneer ectomycorrhizal fungi Laccaria amethystina and Laccaria laccata in a volcanic desert on Mount Fuji.

To understand the reproduction of the pioneer ectomycorrhizal fungi Laccaria amethystina and Laccaria laccata in a volcanic desert on Mount Fuji, Japan, the in situ genet dynamics of sporocarps were analysed. Sporocarps of the two Laccaria species were sampled at fine and large scales for 3 and 2 consecutive years, respectively, and were genotyped using microsatellite markers. In the fine-scale analysis, we found many small genets, the majority of which appeared and disappeared annually. The high densities and annual renewal of Laccaria genets indicate frequent turnover by sexual reproduction via spores. In the large-scale analysis, we found positive spatial autocorrelations in the shortest distance class. An allele-clustering analysis also showed that several alleles were distributed in only a small, localised region. These results indicate that Laccaria spores contributing to sexual reproduction may be dispersed only short distances from sporocarps that would have themselves been established via rare, long-distance spore dispersal. This combination of rare, long-distance and frequent, short-distance Laccaria spore dispersal is reflected in the establishment pattern of seeds of their host, Salix reinii.