Mycelial Production and Mycorrhizal Establishment of Terfezia arenaria and Tirmania pinoyi on Helianthemum canariense: A Morphological and Molecular Study

Desert truffles are hypogeous edible fungi of ecological and economic importance in arid and semi-arid ecosystems, where they establish mycorrhizal associations with Helianthemum species. This study evaluated the effects of pH, culture medium, and fungal species on the apical growth and mycelial biomass production of Terfezia arenaria and Tirmania pinoyi under controlled conditions. Fungal identification was confirmed by sequencing the ITS region. Both species were cultured on Potato Dextrose Agar and Malt Extract Agar at four pH levels (4.55, 5.6, 6.0, and 6.5) at 28 °C. Mycelial biomass was further evaluated at pH 6.0 and 6.5, and the resulting mycelial suspensions (100 mL) were used to inoculate Helianthemum canariense seedlings under controlled conditions. The resulting suspension was applied directly to the root system of young seedlings at a rate of 20 mL per plant. The results revealed highly significant effects of pH and culture medium, slightly significant effects of fungal species, and highly significant interactions between all factors, including the three-way interaction medium, pH and fungal species. T. arenaria exhibited strong growth across all pH levels on PDA but showed marked inhibition on MEA at pH ≥ 6.0. In contrast, T. pinoyi responded more favorably to PDA than MEA at higher pH levels. For mycelial biomass production, no significant effect of species, pH, or medium was found when considered individually. However, a highly significant interaction was detected between species and solid-state culture for both wet- and dry-weight mycelium biomass production. T. arenaria has produced a greater biomass on MEA, while T. pinoyi performed better on PDA. In contrast, pH has shown no significant effect within the tested conditions. Inoculated H. canariense roots exhibited abundant fungal hyphae, vesicles, and endospores, together with a fungal mantle and Hartig net, confirming the establishment of a dual ectomycorrhizal–endomycorrhizal symbiosis. Overall, the results highlight strong species-specific responses and demonstrate that successful desert truffle cultivation requires optimization of culture medium and pH according to fungal species.