Foliar application of humic acid and seaweed extract modulates proline accumulation and chlorophyll content in custard apple under drought conditions

Abstract Drought stress triggers osmotic adjustment through proline accumulation and compromises photosynthetic potential through chlorophyll degradation in fruit crops. Biostimulants offer sustainable approaches to enhance stress tolerance through coordinated metabolic regulation, though underlying mechanisms remain poorly characterized in underutilized species like custard apple ( Annona squamosa L.). This study provides the first field assessment of proline content and chlorophyll concentration responses to foliar-applied humic acid (HA) and seaweed extract (SWE) under drought conditions in custard apple. Sixteen treatment combinations comprising HA (1000–3000 ppm) and SWE (1000–3000 ppm), applied individually and in combination, were evaluated across two contrasting drought seasons (2022–2023) using the custard apple cv. Balanagar. Proline content and total chlorophyll were quantified before (June) and after (November–December) biostimulant applications under natural drought stress. Combined HA (3000 ppm) + SWE (2000 ppm) achieved optimal metabolic modulation, reducing proline accumulation by 13.2% (from 9.54 to 8.28 µmol g −1 FW) and enhancing chlorophyll retention by 6.9% (from 2.32 to 2.48 mg g −1 FW) relative to untreated controls. Individual high-concentration treatments (HA 3000 ppm; SWE 2000 ppm) provided significant but comparatively lower benefits. Principal component analysis revealed distinct treatment-dependent temporal trajectories, with PC1 and PC2 explaining 83% of variance in proline dynamics and coordinated metabolic responses. Year-to-year consistency despite 34% rainfall reduction (1095 vs. 718 mm) demonstrated robust stress mitigation across variable environmental conditions. Observed physiological patterns are consistent with complementary mechanisms involving HA-mediated nutrient and water relations improvements, and SWE-associated hormonal and antioxidant effects, as reported in the literature for other crops; however, these mechanisms were not directly measured in the present study. Strong inverse correlation between proline content and chlorophyll retention (r = − 0.67 to − 0.73, P < 0.001) indicates coordinated regulation of osmotic adjustment and photosynthetic pigment maintenance. These findings support the use of proline and chlorophyll as practical physiological indicators for monitoring stress responses and evaluating biostimulant effects in custard apple production systems under water-limited conditions.