*3.3. Allelopathic Bioassay*

The allelopathic activity of the extracted EO from the above-ground parts of *C. amblyocarpa* was evaluated in vitro against the weed, *Dactyloctenium aegyptium*. The ripe seeds of the weed were collected from a cultivated field near Gamasa city, northern Mediterranean coast, Egypt (31◦26 19.3 N 31◦34 12.9 E). The uniform seeds were surface-sterilized with sodium hypochlorite (0.3%), rinsed with distilled sterilized water, and dried under sterile conditions [34]. To test the allelopathic activity, serial concentrations (10–100 μg mL−1) of the EO were prepared using 1% polysorbate 80 (Sigma-Aldrich, Darmstadt, Germany) as an emulsifier. In a Petri plate, 20 sterilized seeds of *D. aegyptium* were arranged over wetted Whatman filter paper (Sigma-Aldrich, Darmstadt, Germany), either with each concentration or polysorbate 80 (as positive control). The plates were sealed with Parafilm® tape (Sigma, St. Louis, MO, USA) and kept in a growth chamber adjusted at 25 ◦C.

The plates were checked every day, and after 7 days, the number of germinated seeds was counted, and the lengths (mm) of seedling roots and shoots were measured. The inhibition of the germination, root growth, and shoot growth of seedlings was calculated according to the following equation:

$$Inhibition\left(\%\right) = 100 \times \left(\frac{\text{Number} / \text{Length}\_{\text{control}} - \left(\text{Number} / \text{Length}\_{\text{treatment}}\right)}{\text{Number} / \text{Length}\_{\text{control}}}\right) \tag{1}$$

Additionally, the IC50, which is the concentration of the EO required for 50% inhibition of seed germination or seedling growth, was calculated by linear regression of the inhibition values versus various EO concentrations using MS-EXCEL 2016.
