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.

A Transcriptomic Atlas of the Ectomycorrhizal Fungus Laccaria bicolor.

Trees are able to colonize, establish and survive in a wide range of soils through associations with ectomycorrhizal (EcM) fungi. Proper functioning of EcM fungi implies the differentiation of structures within the fungal colony. A symbiotic structure is dedicated to nutrient exchange and the extramatricular mycelium explores soil for nutrients. Eventually, basidiocarps develop to assure last stages of sexual reproduction. The aim of this study is to understand how an EcM fungus uses its gene set to support functional differentiation and development of specialized morphological structures. We examined the transcriptomes of Laccaria bicolor under a series of experimental setups, including the growth with Populus tremula x alba at different developmental stages, basidiocarps and free-living mycelium, under various conditions of N, P and C supply. In particular, N supply induced global transcriptional changes, whereas responses to P supply seemed to be independent from it. Symbiosis development with poplar is characterized by transcriptional waves. Basidiocarp development shares transcriptional signatures with other basidiomycetes. Overlaps in transcriptional responses of L. bicolor hyphae to a host plant and N/C supply next to co-regulation of genes in basidiocarps and mature mycorrhiza were detected. Few genes are induced in a single condition only, but functional and morphological differentiation rather involves fine tuning of larger gene sets. Overall, this transcriptomic atlas builds a reference to study the function and stability of EcM symbiosis in distinct conditions using L. bicolor as a model and indicates both similarities and differences with other ectomycorrhizal fungi, allowing researchers to distinguish conserved processes such as basidiocarp development from nutrient homeostasis.

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.