Solute excretion improves trehalose uptake and desiccation tolerance of Metarhizium brunneum blastospores

In this study, a novel approach was developed to increase the intracellular trehalose concentration in M. brunneum blastospores by uptake from the medium, improving their desiccation tolerance. Briefly, placing the blastospores in a hypotonic environment induced solute excretion, allowing significantly increased trehalose uptake during subsequent trehalose treatment. Conductivity changes and GC–MS analysis revealed that glycerol and lactate excretion is triggered in a hypotonic NaCl solution. After hypotonic + trehalose treatment, intracellular glycerol, lactate, and glucose levels increased, while other desiccation-protective solutes remained unaffected. A fluorescence-based single-cell analysis of membrane stress and death during osmotic desiccation and rehydration showed that membrane stress and cell death were strongly amplified upon rehydration. Trehalose treatment reduced this amplification, while hypotonic + trehalose treatment minimised cell death and prevented a stress response during rehydration, though it increased membrane stress during desiccation. Newly formed germ tubes were identified as critical points of membrane failure during desiccation. Ultimately, the drying survival of blastospores was significantly improved by the hypotonic + trehalose treatment compared to untreated or trehalose-only-treated blastospores. In summary, this technique successfully enhanced blastospore desiccation tolerance and could be adapted to load cells with other solutes.