Drought is the most important environmental factor that significantly reduces the growth and productivity of grape (Vitis vinifera L.) plants. Calcium acts as an important signaling molecule in plants, mediating the actions of many hormones and environmental factors, including abiotic stresses. Serendipita indica, as the model endophytic root fungus, enhances plant growth under abiotic stress in various plants, but the combined effects of S. indica and calcium on grapevine drought tolerance remain under-investigated. We conducted a factorial experiment based on a completely randomized design to investigate the impact of S. indica and calcium on grapevine growth and its morphophysiological parameters under different drought levels (-0.2 as control, -0.7 and -1.2 MPa, as moderate and severe drought stress, respectively). Additionally, expression analysis of ABA-dependent and ABA-independent genes (ABF2, CIPK3, ABA2, AREB, CBL4, and CBF4) was investigated. The results indicated significant impacts of drought level, fungal inoculation, calcium treatment, and their interactions on the measured traits. With increasing stress, soluble carbohydrates (23%), malondialdehyde (2–14 times), electrolyte leakage (18%), proline (49%), phenol content (11%), and antioxidant enzyme activity increased, while cell membrane stability, relative water content (6%), soluble proteins (3%), and chlorophyll content (18%) decreased. Root inoculation with S. indica fungus, foliar application of calcium, and their interaction displayed positive effects in alleviating drought stress. The combined effects of S. indica and calcium increased the levels of total chlorophyll (4%), protein (3%), carbohydrate (17%), proline (13%), phenol (27%), and antioxidant enzymes (by 26% to 2.28 times), while decreasing malondialdehyde (18%) and electrolyte leakage (6%). Differential gene expression was observed in grape leaves after S. indica colonization and calcium treatment. S. indica, calcium, and their interaction increased the expression of all genes except AREB1 under stress condition. Notably, the interaction of calcium and fungus decreased AREB1 expression under both well-watered and water-deficit conditions. This study shows that the synergistic effects of calcium and S. indica may improve grapevine drought tolerance through physiological improvements and modulation of gene expression.
