## 3 Results and Discussion  
### Compatible Solutes in Salt-Adapted A. sydowii Cells  
To avoid the stress induced by nutritional deprivation, we cultured the fungus with glucose as carbon source [30; 32]. In a shaken flask cultivation, most microorganisms will grow exponentially until the stationary phase, where active growth ceases due to nutrient scarcity and other biologically imposed limits. The metabolic response of the cells is expected to be different in each growth phase, and therefore the growth rate of _A. sydowii_ in different salinities was determined to establish the time needed to analyze the osmolyte content (Figure 1A). The duration of the growth phases was different between the tested conditions, reinforcing the notion that there are three different biological scenarios: the fungus growing under optimal salinity condition, or under either hyperosmotic or hypoosmotic conditions. An initial adaptation (or lag) phase was evident in the condition without NaCl, while this phase was nearly inexistent in the other three conditions. The exponential growth phase lasted until day 9 of culture under the optimal conditions and without NaCl, but only lasted until day 2 at 2.0 M NaCl. The doubling time of _A. sydowii_ on 0.5 M and 1.0 M NaCl were 10 and 11 h, respectively, while in the absence of salt and 2.0 M NaCl were 13.5 and 14.6 h, respectively. In further experiments we used 0.5 M NaCl as the optimal salinity condition. These results confirm that _A. sydowii_ is a moderate halophile with optimal growth in the salinity range 0.5 to 1.0 M NaCl (Figure 1A and Supplementary Table S2). Previous reports of marine _A. sydowii_ strains isolated from corals reinforce the notion that this fungus thrives in salinities near the seawater NaCl concentration (0.6 M NaCl) [64; 65]. In this environment the fungus infects corals causing tissue purpling and galling. Although _A. sydowii_ has a terrestrial origin, marine isolates have caused a pandemic that has reduced the coral reef population [64; 65]. Nevertheless, terrestrial isolates, as is the case of the strain BMH-0004 analyzed in this study, are not pathogenic to corals [64; 65]. Osmolyte accumulation in _A. sydowii_ was evaluated at three time points: 5-, 7-, and 11-days post-inoculation, which cover different states of growth under all conditions. Given the unique profiles of the growth curves, it was difficult to determine equivalent physiological states for the cultures. At days 5 and 7, the fungus is in exponential growth phase in the cultures without NaCl or with 0.5 M NaCl. At day 11, all the cultures had reached the stationary phase. In the cultures with 2.0 M NaCl, the exponential growth phase was not sampled because the mycelium grows in a small window of time until a stationary state where the fungus remains viable but ceases to grow (Figure 1A). This pattern is similar to the growth of fungi such as _Rhizopus microsporus_ and _A. fumigatus_ on nutritionally poor media [66]. It has been proposed that under osmotic stress, cells respond with an increased synthesis of compatible solutes, regardless of the solute, causing the lowering of water activity [67]. In _A. sydowii_ the largest accumulation of compatible solutes (mannitol, erythritol and arabitol) was observed under optimal conditions of growth (Figure 1B). This result contradicts the general notion that osmolytes are mainly accumulated under hyperosmotic conditions and cellular stress [11; 67; 68; 69; 70; 71]. However, it has been documented that other fungi exhibit higher polyol concentration when growing on their optimal NaCl and KCl concentration [72]. Our results also showed that _A. sydowii_ increased the concentration of erythroid and glycerol only when the cells were exposed to hyper-salinity (Figure 1B). In the media with  
Figure 1: Growth rate of _A. sydowii_ (**A**) and osmolyte accumulation (**B**) in optimal, hypo- and hyper osmotic conditions. Data are means \(\pm\) SD calculated from three independent experiments (\(n=3\)). Statistical analyses are detailed in Supplementary Table S3.