The application of nanotechnology in agriculture has received much attention in order to improve crop yield, quality and food safety. In the present study, a Cd-tolerant endophytic fungus Colletotrichum fructicola KL19 was first ever reported to produce SeNPs, and the production conditions were optimized using the Box–Behnken design in the Response Surface Methodology (RSM-BBD), achieving a peak yield of 1.06 mM under optimal conditions of 2.62 g/20 mL biomass, 4.56 mM Na2SeO3, and pH 6.25. Following this, the properties of the biogenic SeNPs were elucidated by using TEM, DLS, and FTIR, in which the 144.8 nm spherical-shaped SeNPs were stabilized by different functional groups with a negative zeta potential of −18.3 mV. Furthermore, strain KL19 and SeNPs (0, 5, 10, 20 and 50 mg/L) were inoculated in the root zone of small-leaf spinach (Spinacia oleracea L.) seedlings grown in the soil with 33.74 mg/kg Cd under controlled conditions for seven weeks. Impressively, compared with Cd stress alone, the strain KL19 and 5 mg/L SeNPs treatments significantly (p < 0.05) exhibited a reduction in Cd contents (0.62 and 0.50 folds) within the aboveground parts of spinach plants and promoted plants’ growth by improving the leaf count (0.92 and 1.36 folds), fresh weight (2.94 and 3.46 folds), root dry weight (4.00 and 5.60 folds) and root length (0.14 and 0.51 folds), boosting total chlorophyll synthesis (0.38 and 0.45 folds), enhancing antioxidant enzymes (SOD, POD) activities, and reducing the contents of reactive oxygen species (MDA, H2O2) in small-leaf spinach under Cd stress. Overall, this study revealed that utilizing endophytic fungus C. fructicola or its derived SeNPs could mitigate reactive oxygen species generation by enhancing antioxidant enzyme activity as well as diminish the absorption and accumulation of Cd in small-leaf spinach, promoting plant growth under Cd stress.
