Stress-Induced Cross-Protection and Combined Stress Responses in Extremotolerant Black Yeasts

Extremotolerant fungi inhabit environments with multiple overlapping stressors, yet most studies examine stresses individually. We tested whether preconditioning with salt, cold, or both improves survival after desiccation and freezing, and whether combined salinity and temperature effects on growth are additive or synergistic. We studied Aureobasidium pullulans, Aureobasidium subglaciale, Aureobasidium melanogenum, and Hortaea werneckii (haploid and diploid). All preconditioning treatments significantly increased long-term desiccation survival in A. pullulans, reflecting its generalist capacity to activate cross-protective responses. H. werneckii displayed smaller improvements, consistent with a specialist strategy. Freezing survival without cryoprotectants remained ~100% in both species, indicating high intrinsic tolerance. Growth analyses revealed synergistic effects of salinity and temperature in Aureobasidium spp. Species differed in salinity sensitivity (A. melanogenum > A. pullulans > A. subglaciale) and thermal preferences. A. melanogenum and A. pullulans grew faster at higher temperatures, while A. subglaciale showed the opposite trend. In H. werneckii, salinity governed growth. Haploids slowed as salinity increased, while the diploid remained unaffected. This is the first confirmation of the long-standing suggestion that hybrid diploid genomes of many H. werneckii are an adaptation to osmotic stress. These findings illustrate two pathways to extremotolerance: inducible flexibility in Aureobasidium versus constitutive halotolerance in H. werneckii.