Arsenic Speciation and Distribution in Three Hebeloma Species: Insights into Arsenic Handling and Transformation in Mycorrhizal Fungi.

Arsenic accumulation and biotransformation in fungi remain poorly understood, particularly in mushrooms. This study investigated arsenic distribution, speciation, and detoxification mechanisms in three mushroom species of the genus Hebeloma: H. bulbiferum, H. sinapizans, and H. mesophaeum. Wild collected fruit bodies and laboratory mycelial cultures were analysed using size-exclusion chromatography (SEC) and ion-pair reversed-phase ICP-MS to determine total arsenic concentrations and speciation patterns. H. bulbiferum exhibited the highest arsenic accumulation in fruit bodies (up to 563 mg kg⁻¹ dry mass), predominantly as dimethylarsenate, whereas H. sinapizans accumulated less total As (up to 41.2 mg kg⁻¹ dry mass), comprising mainly arsenobetaine and dimethylarsenate, and H. mesophaeum (up to 4.2 mg kg⁻¹ dry mass) was dominated by inorganic arsenic and arsenocholine. SEC revealed that arsenic was present as low-molecular-weight fractions, although a minor protein-associated peak was observed in H. sinapizans, suggesting a possible presence of an arsenic-binding protein. Mycelial cultures demonstrated species-specific tolerance to arsenate and the ability to transform inorganic arsenic into organic arsenicals, with varying degrees of arsenate reduction, arsenite efflux, and methylation. In particular, H. sinapizans and H. mesophaeum exhibited a higher degree of As(III) efflux than H. bulbiferum, indicating a more efficient As reduction and export. Notably, some organoarsenicals, dimethylarsenate and trimethylarsine oxide, were actively excreted into the growth medium, indicating a role for mushroom mycelia in environmental arsenic cycling. These findings highlight distinct arsenic detoxification strategies in Hebeloma species, reveal fungal de novo arsenobetaine synthesis, and provide insights into arsenic transformation and sequestration in ectomycorrhizal mushrooms.