Abstract Climate change and water scarcity have made drought stress a major challenge for agriculture and food security. This study investigates the effects of PEG-induced water deficit stress and the culture filtrate of Aspergillus niger strain ZRS14 on the physiological and biochemical traits of Rasheh grape (Vitis vinifera L. cv. Rasheh) under in vitro conditions. A factorial experiment in a completely randomized design was conducted. Water deficit was simulated using polyethylene glycol (PEG) 6000 at 0%, 2%, 4%, and 6% (w/v), while fungal culture filtrate was applied at 48-, 72-, and 120-h post-inoculation, as well as a combined 48 + 120-h treatment. An uninoculated PDB medium (ZRS14 Ctrl) served as the fungal control. Increasing PEG concentrations significantly reduced photosynthetic pigments (chlorophyll a, total chlorophyll, and carotenoids) and elevated oxidative stress markers including hydrogen peroxide (H₂O₂) and malondialdehyde (MDA). In response, antioxidant enzyme activities (CAT, POD, SOD, APX) and total phenolic content (TPC) increased, while total flavonoid content (TFC) showed minimal change. Application of A. niger culture filtrate improved drought tolerance by enhancing pigment levels and antioxidant responses, and by reducing oxidative markers. Under severe drought, TPC levels rose significantly, while TFC remained stable or slightly decreased. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) confirmed the positive role of the fungal filtrate in enhancing biochemical resilience. This is the first study to evaluate the biostimulant potential of A. niger strain ZRS14 for mitigating PEG-induced drought stress in grapevine, offering a promising strategy to improve plant performance under controlled drought conditions.
