2.6. Phytotoxicity assays
2.6.1. Garlic germination study
A seed germination study was carried out to identify EOs with phytotoxic effects. Garlic cloves with a diameter of 1.5-cm were purchased in a local market and kept at 20 ◦C in paper bags. A stock solution of liquid culture medium was prepared according to Díaz Báez et al. (2004). The medium was diluted 10 times and the pH was adjusted to 7.0. Test tubes (10 × 1.5 cm) were filled with culture medium (9 mL) and EOs were added to achieve their MFCs. The epidermis of the seeds was removed before the test. Garlic cloves were put on top of the tube with the root zone submerged in the culture medium. Tubes were incubated at 20 ◦C for 72 h in darkness. A solution of Cu2+ was used as a positive control. The neg-
ative control was growth medium without EO. After incubation, the lengths of the roots and leaves were measured. The results were expressed as roots growth inhibition (%) and leaves growth inhibition (%), using the equation [(C − T)/C] × 100, where C is the mean value of the positive control and T is the mean value of the treatment. Non-phytotoxic essential oils (NPEOs) were identified in this study.
2.6.2. Garlic yield
Previously identified NPEOs were tested in a field assay in order to determine their effect on garlic production. MFC of NPEOs and MFC plus an extra 50% (MFC50+) of this dose (to supplement the losses by volatilization and light degradation) of NPEOs were tested. Bins (16 liters) were filled with soil collected from the Experimental Station of “Facultad de Ciencias Agropecuarias” where garlic had never been cultivated, and buried at the same place. Twenty eight garlic seeds were planted in four rows of seven seeds each. After 30, 50 and 70 days of seeding, broths with the EOs were applied to the soil. After the growing season (120 days), the dry weight of garlic cloves was obtained and the yield was expressed as kg/ha.