Hyphal branching in A. mellea fungi was evaluated in vitro by the paper disk diffusion method74. Primary hyphae were cultured in PDA medium containing 20 g L−1 glucose, 4 g L−1 potato powder and 14 g L−1 agar. The dishes were cultured in the dark for 5–7 days at 23 °C. Secondary hyphae emerge from primary hyphae and grow upward in a negative geotropic manner in the gel; the growth of secondary hyphae was used for the assay. Test samples were first dissolved in acetone then diluted with 70% ethanol in water. The concentration of test sample solutions of natural 5-deoxy-strigol was adjusted with reference to the calibration of synthetic (±)-5-deoxy-strigol in an HPLC analysis. Paper disks (1 cm in length, 8 mm in width) loaded with 15 μL of test sample solution were placed in front of the tips of the secondary hyphae. The control was on the opposite direction of the paper without 5-deoxy-strigol. Hyphal branch patterns were analyzed at 24 h and 48 h after treatment. The sample was scored as positive for hyphal branching if new hyphal branches formed from the treated secondary hyphae. The assay was repeated at least twice, using between three and five dishes for each concentration.