Arbuscular Mycorrhizal Fungi and Exogenous Calcium Synergistically Alleviate Arsenic Stress in Cotton Seedlings

Cotton (Gossypium hirsutum L.) is a promising candidate for an Arsenic (As)-tolerant plant due to its low As accumulation in fibers. The individual arbuscular mycorrhizal fungi (AMF) inoculation or exogenous calcium (Ca²⁺) application is known to enhance heavy metal tolerance in higher plants; however, their synergistic mechanisms in alleviating As stress in cotton remain poorly understood. A three-factor pot experiment was conducted, including two levels of AMF (Funneliformis mosseae C.Walker & A.Schüßler) inoculation (non-inoculated/inoculated), As stress (0/100 mgAs⁵⁺·kg^-1soil), and exogenous Ca²⁺ (CaCl₂) application (0/20 mmol·L^-1 CaCl₂). AMF inoculation and Ca²⁺ application were investigated for their effects on cotton growth, root morphology, photosynthetic characteristics, osmotic regulators, antioxidant enzyme activities, and ion homeostasis under As stress. Results showed As stress significantly disrupted cotton growth (decreased plant height, shoot and root dry weight) and root morphology (reduced total root length, root area, and root fork number), photosynthetic capacity (reduced Pn, Ci, Fv/Fm, and ΦPSII), osmotic adjustment (decreased proline, soluble sugar and protein), antioxidant defense (inhibited SOD, POD, CAT activities), and K⁺/Ca²⁺ homeostasis (reduced concentration of K⁺ and Ca²⁺, and K⁺/Ca²⁺ ratio). Both AMF inoculation and Ca²⁺ application independently alleviated these adverse effects of As stress. At the same time, AMF symbiosis combined with exogenous Ca²⁺ was better than AMF inoculation or Ca²⁺ application alone in optimizing root architecture, improving stomatal function and photosynthetic efficiency, enhancing osmotic regulator accumulation and antioxidant enzyme activities, and restoring ion balance under As stress. Three-way ANOVA confirmed significant As×AMF×Ca²⁺ interactions on key parameters such as Pn and ΦPSII. In summary, both AMF inoculation and Ca²⁺ application synergistically enhanced cotton As tolerance through regulating growth, root morphology, photosynthetic characteristics, osmotic regulators, antioxidant enzyme activities, and ion homeostasis, demonstrating its potential for sustainable cotton cultivation in As-contaminated soils.