*4.7. Phenolic Compounds*

The hydromethanolic phenolic extracts obtained according to Hagen et al.'s [59] method were then purified using the protocol described in previous paper [41]. Total phenolic determination was performed following the Folin–Ciocalteu method [60].

#### *4.8. Extraction and Spectrophotometric Assay of Lignin Content*

The lignin was extracted using the Bruce and West [61] protocol, with some modifications, in 0.5 mL of ethanol 90% (*v*/*v*), were grounded 500 mg of treated roots. Pellet obtained after centrifugation at 10,000× *g* for 20 min and at 4 ◦C was dried for 12 h at 35 ◦C. Samples of 25 mg of the dried residue were treated with 0.5 mL of thioglycolic acid and 1.25 mL of HCl solution at 2 M. The mixture was heated during 8 h at 100 ◦C. After cooling and centrifugation, 2.5 mL of NaOH was added to the pellet. The supernatant recovered after stirring for 18 h at 25 ◦C, was centrifuged (10,000× *g*, 20 min, 4 ◦C) treated with 0.5 mL of pure HCl, to precipitate lignin thioglycolic acid after incubation over 4 h at 4 ◦C. The absorbance at 280 nm was measured in 500 μL of NaOH. The lignin content was expressed in μg of lignin thioglycolic acid/g Dry Matter (DM).
