2.3.4. Disease Symptoms and Disease Index

Pre-emergence damping-off was measured after 15 days from sowing, while postemergence damping-off and survival were measured after 30 days from sowing according to Mousa et al. [49]. In addition, disease symptoms were assessed, and the disease index was recorded after 45 days from sowing according to Grünwald et al. [50]. The disease index scale (0–5) based on disease progress developed by the authors was used to measure the disease severity of *Rhizoctonia* root rot, in which 0 indicated no visible symptoms; 1, a few small soft lesions on a part of the root system and hypocotyls; 2, elongated, discolored

lesions spread on the entire root system and hypocotyls; 3, deep brown necrosis grind the stem, partial root disintegration, and yellowing of leaves; 4, stem canker, root disintegration, yellowing of leaves, and stunting; and 5, collapse and death of the plants. Disease index = (i (rating no. × no. of plants in the rating)/(total no. of plants × highest rating) × 100. Shoot length, root length, fresh and dry weight, and pigments were also measured (one gram of fresh leaves was extracted by 100 mL of 80% aqueous acetone (*v*/*v*), filtrated, and then completed the volume to 100 mL using 80% acetone). The optical density of the plant extract was measured using the spectrophotometer of three wavelengths (470, 649, and 665 nm). Pigments were calculated using the equations mentioned Mg chlorophyll (a)/g tissue = 11.63(A665) − 2.39(A649), Mg chlorophyll (b)/g tissue = 20.11(A649) − 5.18(A665), Mg chlorophyll (a + b)/g tissue = 6.45 (A665) + 17.72(A649), and Carotenoids = 1000 × O.D<sup>470</sup> − 1.82 C<sup>a</sup> − 85.02 Cb/198 = mg/g fresh weight. "A" denotes the reading of optical density, phenol (one gram dry leaves was extracted with 80% cold methanol (*v*/*v*) three times at 0 ◦C. The extract was filtered; then, the volume of sample was completed to 25 mL with cold methanol. The total phenol and total soluble protein of plants were determined in the following manner: one gram of the dried leaves was added to 5 mL of 2% phenol water and 10 mL of distilled water was added; the solution was shaken and kept overnight, filtered, and completed volume to 50 mL with distilled water; then the protein content was determined according to Alhaithloul et al. [51].
