Resumen de Traceability of the mycorrhizal symbiosis in the controlled production of edible mushrooms = Traçabilitat de la simbiosi micorízica en la producció controlada de fongs comestibles
The availability of most edible ectomycorrhizal mushrooms depends on their natural fructification. Therefore, mycorrhizal plant production could be an alternative for obtaining ectomycorrhizal edible fungi. The first step to establish a plantation for a certain species of mushrooms is the production of plants, inoculated with the fungus, in order to be outplanted to field. The mycorrhization of plants with species of the Boletus edulis complex has been achieved under pure culture synthesis conditions. The production of B. edulis mycorrhizal plants under nursery conditions could be achieved by acclimation of in vitro-produced mycorrhizal plants, but needs still more research to determine an appropriate procedure. Sporocarp formation of these fungi is linked to habitat characteristics and climate conditions, but these data alone do not explain all the trends of fungal fruiting and dynamics. It could be hypothesized that the amount of soil mycelia could also be related to the production of carpophores. Progress of new technologies in molecular biology and the availability of fungal genomes have led to the development, implementation and use of techniques for the traceability of edible ectomycorrhizal fungi. Through the thesis it has been demonstrated that the real- time PCR technique, with the design of specific oligonucleotides for the detection and quantification of extraradical mycelium in soil, allows us to determine the concentration of soil mycelium in forests and plantations, increasing detection limits over conventional PCR. Specific primers and probes have been designed for B. edulis and Tuber melanosporum for real-time PCR amplification, allowing the traceability of these fungal species in the controlled production of edible mycorrhizal mushrooms. The annual belowground dynamics of extraradical soil mycelium, ectomycorrhizal root tips and sporocarp production of two ectomycorrhizal fungi, B. edulis and Lactarius deliciosus, have been studied in two different pine forests (Pinar Grande and Pinares Llanos, respectively) in Soria in permanent plots. Quantification of extraradical mycelium of Rhizopogon roseolus x Pinus pinea plantations (two in Cabils) and of T. melanosporum x Quercus ilex plantation (one in Cerc), in Catalunya has been carried out from its establishment until 24 or 48 months. T. melanosporum extraradical mycelium has also been quantified by real-time PCR in a natural truffle ground and in seven truffle orchards (around 20 years old) established in Tierra Estella and Valdorba sites (Navarra). Finally, the spatial genetic structure of T. melanosporum population was analyzed in a productive orchard (France). The distribution of the two T. melanosporum mating types was monitored in the soil. Ectomycorrhizas and ascocarps were mapped and genotyped using simple sequence repeat markers and the mating type locus and their genetic profiles were compared. No direct relationships were found between soil mycelium amounts and sporocarp production for any of the studied fungal species, but it was possible to obtain positive correlations between vegetative structures (ectomycorrhiza and extraradical mycelium) for B. edulis, R. roseolus and T. melanosporum. The results obtained open the possibility of using quantification of soil mycelium by real-time PCR as a good indicator for root colonization in field conditions (in natural areas or in manmade orchards), especially when a nondestructive sampling or less time consuming analysis were required. The extraradical mycelial amounts of B. edulis, L. deliciosus and R. roseolus and T. melanosporum mycorrhizas were correlated with climate parameters as temperature, rainfall, solar radiation, relative humidity and evapotranspiration. Moreover, it has been possible to map the distribution of different T. melanosporum genotypes trough the traceability of ascocarps, ectomycorrhizas and soil samples. A pronounced spatial genetic structure was found. A nonrandom distribution pattern of the T. melanosporum was observed, resulting in field patches colonized by genets that shared the same mating types.
