Cx 41 1

Dual cultures of F. culmorum KF846 with Trichoderma or Clonostachys strains were used for microscopic observations. They were grown on a sterile strip of 20 mm cellophane membrane (50-μm thick) placed before fungal inoculation on a PDA medium in the middle of 8.5 cm Petri dish. Fungi were inoculated on opposite sides of Petri dishes at a distance of 5 mm from the edge. After 7 and 14 days of incubation at 25 ± 2 °C through 12 h/12 h of darkness/light, the mycelium overgrown cellophane membrane was cut with a razor blade in sterile conditions, placed on a microscope slide in a drop of distilled water and examined. Observations were carried out using a light microscope (Olympus CX-41-1 with UC-30 camera, Olympus Corporation, Tokyo, Japan). The samples were screened for loops of the Trichoderma or Clonostachys around F. culmorum hyphae and the anatomical damage of the pathogen.

The staining procedure of wheat roots treated with Trichoderma was carried out according to Chacón et al. [41 (link)]. The sections of roots were treated with 0.5% toluidine blue solution (Kolchem, Lodz, Poland) dye for 3 min, for detecting lignin, pectin, and hyphae of Trichoderma inside the wheat roots. The observations were captured using a light microscope (Olympus CX-41-1 with UC-30 camera, Olympus, Japan). Pectins were dyed a red–purple color, lignins a blue color.

The fungi method proposed by Phillips and Hayman [82 (link)] was used with slight modifications for the detection of colonized roots. Plant roots were collected and washed with deionized water. Ten fragments (1 cm long) were taken from the thinnest roots of each plant. Fragments were incubated in 5% KOH (Avantor Performance Materials Poland S.A.) for 24 h. Subsequently, KOH was removed and the material were washed three times with deionized water and incubated in 50% lactic acid (Avantor Performance Materials Poland S.A., Gliwice, Poland) for 1 h. The detection process was carried out with 0.1% trypan blue (Merck KGaA, Darmstadt, Germany, formerly Sigma, St. Louis, MO, USA) in lactoglicerol solution (1:1:1) of glycerol (Merck KGaA, Darmstadt, Germany, formerly Sigma, St. Louis, MO, USA): lactic acid: deionized water] for 2 h. All incubations were performed at room temperature. The observations were carried out using a light microscope (Olympus CX-41-1 with UC-30 camera, Olympus, Tokyo, Japan). For the estimation of colonized wheat roots, each 1 cm long fragment was observed under microscope to study mycelia, vesicles and arbuscules. Calculation of the percentage of root colonization (5) was carried out with the following formula: $\%ofrootcolonization=\frac{numberofcolonizedrootsfragment}{totalnumberofexaminatedfragments}\times 100$