Long-term preservation of Arbuscular mycorrhizal fungi

Field: Agricultural and Biological Sciences (Life Sciences)

Arbuscular mycorrhizal fungi (AMF) are obligate root symbionts, forming associations with most existing terrestrial plants. The plants obtain inorganic nutrients (e.g. N, P) via their fungal partners in exchange of which they provide the fungi with carbon compounds. AMF improve plant growth, health and productivity and as such, represent key organisms in agro-ecosystems. Currently, AMF diversity is maintained via continuous culture; in vivo on trap plants under greenhouse facilities, and in vitro in association with excised, transformed or nontransformed roots. However, these methods are time and work-consuming and the risks of contaminations and loss of genetic stability are not excluded. The objective of this thesis was to develop a long-term preservation method adapted to different AMF species and genera that guarantee their viability and stability over unlimited time of storage. In a first step, we tested different long-term preservation protocol (e.g. lyophilization, vitrification, cryopreservation) on Rhizophagus sp. MUCL 43204 produced in vitro. Cryopreservation appeared the most reliable method and was used in a second step on a large number of AMF isolates cultured in vitro. After 6 months storage at -130 °C, the viability of AMF isolates and their ability to colonize plant roots and reproduce the life cycle were tested. In a third step, we evaluated the morphology, physiological activity and genetic stability of 6 months-cryopreserved AMF isolates belonging to the genus Rhizophagus. Finally we applied the cryopreservation protocol developed on a large array of AMF isolates cultured either in vitro or in vivo. Our results demonstrated that the cryopreservation by encapsulation-drying and storage at -130°C of AMF propagules was effective, at least for 6 months, for different Rhizophagus isolates cultured in vitro. The method comprised five steps (1) the encapsulation in alginate beads of AMF propagules (i.e. spores and mycorrhizal root pieces) isolated from 5 months old cultures, (2) the incubation overnight in trehalose (0.5M), (3) the drying at 27°C for 2 days (i.e. at 8.1 ± 4.6% of beads water content), (4) the cryopreservation at -130 C in a freezer following a 2 steps decrease in temperature: a fast decrease (~12°C min-1) from room temperature (+20°C) to -110°C followed by a slow decrease in temperature (~1°C min-1) from -110°C to -130°C and (5) the direct thawing in a water bath set at +35°C. The morphology, physiological activity and genetic stability evaluated after 6 months storage at -130°C were similar to the non-cryopreserved controls. This method was also successfully applied to isolates belonging to Glomus, Claroideoglomus, Septoglomus, Paraglomus and Gigaspora cultured either in vitro or in vivo. Ours findings highlights the possibility to use the encapsulation-drying method for the cryopreservation and storage at -130°C of AMF isolates produced either in vitro or in vivo. These results further open the door to isolates that remain recalcitrant to different forms of long-term preservation at ultra-low temperature.