Selenium induced growth modulation and toxicity in Pleurotus florida to establish baseline parameters for substrate level biofortification.

Pleurotus florida (P. florida) mushrooms are widely valued for their nutritional, medicinal and bioactive properties. Selenium (Se) biofortification of edible mushrooms offers a sustainable strategy to mitigate global micronutrient deficiencies, however the narrow margin between the nutritional benefits and toxicity of Se necessitates precise physiological optimization. This study presents a comprehensive in vitro evaluation of Se-induced growth modulation, oxidative stress, ultrastructural responses and uptake dynamics in P. florida cultivated under sodium selenite concentrations. Mycelial growth on PDA exhibited a highly reproducible biphasic response across three independent experiments. Low Se concentrations (10-15 mg L⁻¹) significantly enhanced radial growth and biomass accumulation. Elevated concentrations (≥40 mg L⁻¹) caused sharp declines in growth and biomass, accompanied by abnormal colony morphology and reduced mycelial density. Lipid peroxidation analysis revealed a strong dose-dependent increase in oxidative membrane damage, with Se concentration explaining nearly 90% of the observed variation, indicating a shift from antioxidant support at low doses to pro-oxidant toxicity at higher levels. Scanning electron microscopy (SEM) confirmed enhanced hyphal branching and structural organization at optimal Se concentrations, while severe ultrastructural damage including hyphal collapse and filament breakage was evident under high Se stress. Scanning Electron Microscopy coupled with energy dispersive X-ray spectroscopy (SEM-EDS) analysis showed Se-induced alterations in mycelial surface composition and inductively coupled plasma mass spectrometry (ICP-MS) based mass-balance analysis demonstrated high Se removal efficiency (>80%), with excessive biomass-normalized accumulation at high concentrations reflecting stress rather than efficient biofortification. Collectively, this study defines a narrow Se tolerance window in P. florida and identifies 15 mg L⁻¹ as the optimal concentration for safe and effective Se biofortification.