Development of an optimized culture medium using meso-inositol and mannitol to maximize chlamydospore production of Duddingtonia flagrans

Biological control using nematophagous fungi such as Duddingtonia flagrans offers a sustainable strategy to reduce gastrointestinal nematode populations in grazing animals. However, large-scale application requires efficient chlamydospore production without compromising fungal viability and efficacy. This study aimed to optimize a solid culture medium enriched with meso-inositol and mannitol to enhance chlamydospore production and assess its impact on nematode predatory capacity for their subsequent use as biological control agent. Different formulations were tested by supplementing Glucose Sabouraud Agar with various concentrations of meso-inositol (1.1–2 %) and mannitol (2–5 %). Cultures were incubated for up to 35 days, and chlamydospores were extracted and quantified under optical microscope at 3, 4, and 5 weeks of culturing. The highest yield – 6.95 × 10⁷ chlamydospores/plate - was obtained using 2 % meso-inositol and 5 % mannitol after 35 days of incubation at 27 ± 0.5 °C and 70 ± 5 % RH. In vitro fungal predatory activity against gastrointestinal nematode larvae from naturally parasitized sheep was maintained across all formulation treatments, with parasite larval reduction exceeding 70 % (p < 0.0001), indicating that the optimized medium did not impair nematophagous efficacy. These findings contribute to the development of scale-up fungal culture strategies for producing commercially-based D. flagrans suitable for its incorporation into integrated control programs of livestock parasites.