*3.3. Allelopathic Bioassay*

The extracted EO from the aerial parts of *P. lapathifolia* was tested in vitro for its allelopathic activity against the germination and seedling growth of the weed *E. colona*. The ripened seeds of *E. colona* were collected from a cultivated field near the city of Manzala, Al-Dakahlya Governorate, Egypt (31.1691466N 31.896379E). The homogenized seeds in size and color were sterilized with 0.3% NaClO and dried in a sterilized condition. The viability of seeds was preliminarily tested and found to be 94.56% ± 3.25. For bioassay, different concentrations (0, 25, 50, 75, and 100 mg L<sup>−</sup>1) of the extracted EO were prepared in 1% Tween® 80 (Sigma-Aldrich, Darmstadt, Germany) as an emulsifier. Twenty sterilized seeds were arranged over a filter paper (Whatman No. 1) in Petri dishes. About four mL of each concentration and Tween® 80 (as control) were poured over the filter paper, and the dishes were sealed with Parafilm® tape (Sigma, St. Louis, MO, USA) to avoid the loss of EO [41]. For each concentration, five dishes were tested, and the experiment was repeated three times. A total of 75 dishes (5 treatments (4 concentrations + control) × 5 dishes (replications) × 3 times) were prepared and incubated at 27 ◦C in a growth chamber with adjusted light conditions of 16 h light and 8 h dark. After ten days of incubation, the number of germinated *E. colona* seeds was counted, and the lengths of the seedling root and shoot of the weed were measured. The inhibition of germination and growth was calculated with respect to control according to the following equation:

$$\text{Inhibition } (\%) = 100 \times \frac{\left(\frac{N\_{\text{control}}}{L}\_{\text{control}} - \frac{N\_{\text{treatment}}}{L}\_{\text{treatment}}\right)}{\frac{N\_{\text{treatment}}}{L}\_{\text{control}}}$$

where *N* is the number of germinated seeds and *L* is the length of the seedling root or shoot.
