Patching-up of major influencing factors of Rhizophagus irregularis cultivation for industrial production of consistent spore density through cascade statistical optimization

Arbuscular mycorrhizal fungi (AMF) play a crucial role as a climate-change solution, aiding nutrient uptake, interplant communication, and stress mitigation. However, its industrial-scale production remains limited. This study aimed to optimize production of Rhizophagus irregularis (Apex-MYK-01) in carrot root organ cultures using a cascading approach to achieve consistent spore yields. The first phase identified ideal conditions: 2 × 2 cm inoculum size, 100 mL media, and using 90-day-old inoculum in box-shaped vessels. The second phase revealed four critical factors (p < 0.05) influencing output. Final optimization of those four parameters via central composite design pinpointed optimal values: 80-day incubation, 1.25 cm media height, 0.875 % sugar, and 2.75 mg KH₂PO₄. When tested at industrial scale, these conditions boosted spore production by 16.8 % (2213 ± 11 vs. lab-scale 1895 ± 15 spores/g) while maintaining high root colonization rates (82 ± 11 %). This cascading strategy demonstrates a viable path to standardized, large-scale AMF production.