JIA-2019-11

2539 Slaven Jurić et al. Journal of Integrative Agriculture 2019, 18(11): 2534–2548 sporulation. The best spores yield was recorded at 1.5 mol dm –3 . In comparison with the control suspension (NS=5×10 6 mL –1 ), the number of spores was smaller by almost one order of magnitude indicating the addition of calcium ions somewhat slows sporulation but did not prevent it. Microphotograph of freshly prepared microsphere taken under crossed polarizers shows T . viride spores are distributed inside the alginate matrix (Fig. 4-A). After 10 days of aging on plates at room temperature, microspheres become visibly surrounded with green mycelium (Fig. 4-B). Maximum mycelial growth and sporulation were observed between 1 and 1.5mol dm –3 ofcalciumchloride concentration. At the highest concentration, mycelium did not cover the whole substrate and sporulation was absent. Results of concentration-dependent sporulation in suspension or mycelial growth and sporulation on PDA substrate are in accordance with literature data. The presence of calcium cations was found to induce sporulation of T . viride in the submerged (Šimković et al . 2008) and solid (Kryštofova et al . 1995, 1996) media in a concentration and time-dependent manner. Section of microspheres prepared with labeled T . viride spores revealed the formation of branched hyphae inside the matrix (Fig. 4-C). Section close to the surface showed the presence of numerous spores and a few germ tubes penetrating through microsphere surface (Fig. 4-D). CLSM microphotographs show mycelium formed around microspheres were generated by germination inside the matrix and germ tubes protruding out of microspheres. 0.5 1.0 1.5 2.0 600 000 800 000 1 000 000 NS (no. mL –1 ) c (CaCl 2 ) (mol dm –3 ) Tv +CaCl 2 Fig. 3 Changes in the number of Trichoderma viride spores (NS), suspended in solutions with various calcium chloride concentrations (0.5, 1.0, 1.5, and 2.0 mol dm –3 ). c (CaCl 2 ) represents the concentration of a calcium chloride solution. 75 μm 25 μm A B C D Fig. 4 A, microphotograph of microsphere obtained under polarized light (Bar=250 µm). B, a plate with microspheres surrounded with Trichoderma viride mycelium. C and D, confocal laser scanning microscope (CLSM) microphotographs of mycelium inside matrix (C) and germ tube penetrated through microsphere surface (marked with a white arrow) in transmitted mode (D). ALG/ (Ca+ Tv ) microspheres were prepared at initial calcium chloride concentration, c i (CaCl 2 )=1 mol dm –3 , and the number of T. viride spore (NS)=1.8×10 6 mL –1 . Bars are indicated.