Arbuscular Mycorrhizal Fungi, Especially <i>Rhizophagus intraradices</i> as a Biostimulant, Improve Plant Growth and Root Columbin Levels in <i>Tinospora sagittata</i>

<i>Tinospora sagittata</i> is a columbin-rich medicinal plant, but its columbin levels are reduced under artificial cultivation conditions. The objective of this study was to analyze the effects of inoculations with <i>Diversispora versiformis</i> (<i>Dv</i>), <i>Funneliformis mosseae</i> (<i>Fm</i>), <i>Rhizophagus intraradices</i> (<i>Ri</i>), and mixed inoculation (<i>Dv</i> + <i>Fm</i> + <i>Ri</i>) (Mix) on growth performance, root morphology, leaf photosynthetic physiology, and root columbin levels in <i>T. sagittata</i>. These arbuscular mycorrhizal fungi (AMF) were able to colonize the roots, as evidenced by a root mycorrhizal colonization rate ranging from 17% to 48% and soil hyphal lengths ranging from 17.51 cm/g to 32.02 cm/g, with the Mix treatment being the greatest. AMF inoculations improved plant height (16–151%), leaf number (119–283%), shoot (37–211%), and root biomass (22–318%) to varying extents, with <i>Ri</i> and Mix treatments being the most prominent. AMF-treated plants presented relatively greater root total length, projected area, surface area, volume, and average diameter, especially those treated with <i>Ri</i> and Mix. AMF inoculations also significantly improved the leaf nitrogen balance index, transpiration rate, and stomatal conductance, while the photosynthesis rate and chlorophyll index varied by AMF species, along with a decrease in intercellular CO₂ levels. Root columbin levels ranged from 0.524 mg/g to 5.389 mg/g, and AMF inoculation significantly increased root columbin levels by 228–928%, with <i>Ri</i> being the most significant. Root columbin levels were significantly positively correlated with soil hyphal length, root total length, root projected area, root surface area, root volume, and root average diameter, but not root AMF colonization rate. This study demonstrates for the first time that AMF, especially <i>Ri</i>, can be employed as a biostimulant to promote growth as well as root columbin levels in <i>T. sagittata</i>, where AMF-triggered improvement in root morphology is an important reason for promoting root columbin levels.