Analysis of mycelial growth and development of the desert truffle "Terfezia claveryi" Chatin and microorganisms associated to desert truffle mycorrhizal plants

Resumen

Terfezia claveryi Chatin is an edible hypogeous fungus belonging to the group known as "desert truffles", mainly found in arid and semi-arid ecosystems of the Mediterranean basin. In its natural habitat, T. claveryi establishes ectendomycorrhizal symbiosis with several species of the genus Helianthemum and its fruiting is in spring. Moreover, it is the first species of desert truffle to be cultivated and has become an alternative agricultural crop in semi-arid areas of the Iberian Peninsula, which has been increasing in recent years. The main objective of this thesis is to study the mycelial behaviour of the desert truffle T. claveryi, both in the laboratory and in the field, as well as the microorganisms associated with the rhizosphere of host plants of the genus Helianthemum. To this end, five partial objectives were developed, which led to the following results and conclusions. First, the mycelial growth of T. claveryi was evaluated under in vitro conditions by testing the MMN culture medium (solid and liquid) with different concentrations of macronutrients, micronutrients and vitamins, and under different conditions of pH, initial inoculum size and C/N ratio. The results yielded an optimised MMN culture medium that improved the mycelial biomass produced. In addition, the mycelium produced in liquid culture in bioreactor was used for inoculation and production of desert truffle mycorrhizal plants. Secondly, the seasonal dynamics of T. claveryi s.l. mycelium was studied in soil during 4 years in different natural areas and in plantations in the Region of Murcia. For this purpose, specific primers were designed for DNA quantification using real-time PCR in each of the seasons. SYBR-Green based qPCR protocol was developed with the primer pair Tc452F/TerclaR as a suitable candidate to detect the specific target. Mycelial distribution in different soils was independent of the geographical features of the experimental site. The analysis revealed that year as the only factor that significantly separated the mycelial data into two groups (years 1-4 and 2-3), where significant differences were only found for the winter and spring seasons. Moreover, winter mycelium was strongly correlated with agroclimatic variables in autumn season, in which precipitation, aridity index and relative humidity were positive and maximum temperature, vapor pressure deficit and evapotranspiration were negative correlated. Thirdly, bacteria were isolated from the rhizosphere of T. claveryi x H. almeriense across seasons, or from the different stages of host plant phenology. Phenotypic, biochemical, molecular and PGPR activities of the bacterial colonies (phosphorus solubilisation, auxin release, siderophore production and ACC-deaminase activity) were characterized. Statistical analyses revealed that there was a significant enrichment in phosphorus solubilising bacteria (organic acid releasing) and bacteria with ACC-deaminase activity during the fruiting season of the fungus (spring). Moreover, it was also confirmed that a change in the phenological state of the plant determined a change in the bacterial community linked to its PGPR traits. Finally, to understand the fungal community associated with ascocarp productive plants versus non-productive plants, large-scale sequencing tools for DNA were used to perform a metagenomics assay, both in soil and root. Different patterns in fungal species composition were found by productivity. In addition, some trophic modes of the fungal species obtained were identified as relevant for having a positive or negative effect on ascocarp productivity in plantation. Furthermore, a group of significant OTUs associated with productive areas were found, which could be used as markers for the localization of desert truffles.