The contribution of 1,8-dihydroxy naphthalene (DHN) melanin to cadmium (Cd) tolerance in two dark septate endophytes (DSE) of the genus Cadophora with different melanin content was investigated in vitro. The DSE isolate Cad#148 with higher melanin content showed higher tolerance to Cd than the less melanised Cad#149. Melanin synthesis was significantly reduced by Cd in both isolates with uninhibited melanin synthesis, in a dose-dependent manner. Inhibition of melanin synthesis by tricyclazole reduced the relative growth of Cad#148 exposed to Cd and did not affect Cad#149. Cd accumulation was not altered by tricyclazole in the two isolates, but it increased catalase and reduced glutathione reductase activity in more melanised Cad#148, indicating higher stress levels. In contrast, in Cad#149 the enzyme activity was less affected by tricyclazole, indicating a more pronounced role of melanin-independent Cd tolerance mechanisms. Cd ligand environment in fungal mycelia was analysed by extended EXAFS (X-ray absorption fine structure). It revealed that Cd was mainly bound to O- and S-ligands, including hydroxyl, carboxyl, phosphate and thiol groups. A similar proportion of S- and O- ligands (~35% and ~65%) were found in both isolates with uninhibited melanin synthesis. Among O-ligands two types with Cd-O-C- and Cd-O-P- coordination were identified. Tricyclazole altered Cd-O- ligand environment in both fungal isolates by reducing the proportion of Cd-O-C- and increasing the proportion of Cd-O-P coordination. DHN-melanin, among other tolerance mechanisms, significantly contributes to Cd tolerance in more melanised DSE fungi by immobilising Cd to hydroxyl groups and maintaining the integrity of the fungal cell wall.
Keywords: Antioxidant enzymes; Cd tolerance; DSE; EXAFS; Inhibitor tricyclazole; Melanin.
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