| Summary: | This study aims to evaluate whether a selected arbuscular mycorrhizal fungus, <i>Diversispora spurca</i>, improves growth in drought-stressed walnut (<i>Juglans regia</i> L. cv. Qingxiang) plants and whether this improvement is associated with changes in osmolyte (fructose, glucose, sucrose, soluble protein, proline, and betaine) levels. After 60 days of soil drought treatment (50% of maximum field water-holding capacity), root <i>D. spurca</i> colonization rate and soil mycelium length decreased by 13.57% and 64.03%, respectively. Soil drought also inhibited the growth performance of aboveground (stem diameter, leaf number, leaf biomass, and stem biomass) and underground (root projected area, surface area, and average diameter) parts, with uninoculated plants showing a stronger inhibition than <i>D</i>. <i>spurca</i>-inoculated plants. <i>D. spurca</i> significantly increased these growth variables, along with aboveground part variables and root areas being more prominent under drought stress versus non-stress conditions. Although drought treatment suppressed the chlorophyll index and nitrogen balance index in leaves, mycorrhizal inoculation significantly increased these indices. Walnut plants were able to actively increase leaf fructose, glucose, sucrose, betaine, and proline levels under such drought stress. Inoculation of <i>D. spurca</i> also significantly increased leaf fructose, glucose, sucrose, betaine, proline, and soluble protein levels under drought stress and non-stress, with the increasing trend in betaine and soluble protein being higher under drought stress versus non-stress. Drought stress dramatically raised leaf hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) levels in both inoculated and uninoculated plants, while mycorrhizal plants presented significantly lower H<sub>2</sub>O<sub>2</sub> levels, with the decreasing trend higher under drought stress versus non-stress. In conclusion, <i>D. spurca</i> symbiosis can increase the growth of drought-stressed walnut plants, associated with increased osmolyte levels and decreased H<sub>2</sub>O<sub>2</sub> levels.
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