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 <i>Aureobasidium pullulans</i>, <i>Aureobasidium subglaciale</i>, <i>Aureobasidium melanogenum</i>, and <i>Hortaea werneckii</i> (haploid and diploid). All preconditioning treatments significantly increased long-term desiccation survival in <i>A. pullulans</i>, reflecting its generalist capacity to activate cross-protective responses. <i>H. werneckii</i> 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 <i>Aureobasidium</i> spp. Species differed in salinity sensitivity (<i>A. melanogenum</i> > <i>A. pullulans</i> > <i>A. subglaciale</i>) and thermal preferences. <i>A. melanogenum</i> and <i>A. pullulans</i> grew faster at higher temperatures, while <i>A. subglaciale</i> showed the opposite trend. In <i>H. werneckii</i>, 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 <i>H. werneckii</i> are an adaptation to osmotic stress. These findings illustrate two pathways to extremotolerance: inducible flexibility in <i>Aureobasidium</i> versus constitutive halotolerance in <i>H. werneckii</i>.