JIA-2019-11

2540 Slaven Jurić et al. Journal of Integrative Agriculture 2019, 18(11): 2534–2548 Fig. 5 presents the change in the number of spores loaded in microspheres with time. There was no significant change in NS between freshly loaded and 1 day stored in microspheres. During storage for 10 days, the number of T . viride spores increased and after 20 days somewhat decreased, but remained still higher than the number of spores in freshly prepared microspheres. Results undoubtedly showed the intermolecular interactions between calcium cations and negatively charged T . viride spores stimulated sporulation during storage. The best biomass yield in microspheres prepared at 1 mol dm –3 of initial calcium chloride concentration indicates the benefits of microspheres prepared at this concentration. Somewhat lower biomass yield at higher calcium chloride concentration might be explained with a lesser amount of water in microsphere structure. This means microspheres provide a supportive environment for germination. Intermolecular interactions between calcium cations and negatively charged T . viride spores are primarily of electrostatic origin, however, at higher calcium concentrations some other physicochemical interactions between cations and fungal cell wall should be also taken into account. Identification of molecular interactions between calcium ions, T. viride spores and sodium alginate Information on molecular interactions between calcium and T . viride spores as well as between T . viride spores and sodium alginate was obtained by comparing FTIR spectra of single components and their mixtures (Fig. 6). The spectrum of calcium chloride shows stretching vibrations at 3487, 3396, 3224 and 1629 cm –1 which relate to the vibration of –OH functional group, and its characteristic peak at 657 cm –1 (Ca–O stretching) (Cho et al . 2014). Characteristics of recorded T . viride spectrum are: a broad band around 3 321 cm –1 (overlapping of O–H and N–H stretching vibrations), frequencies at 2 921 and 2 854 cm –1 (indicative of >CH 2 and -OH groups), the small peak at 2 367 cm –1 (asymmetric stretching of –N=C=O group), the pronounced peak at 1608 cm –1 and several small peaks between 1500 and 1200 cm –1 (stretching vibration of C=O of carboxyl groups conjugated to -NH deformation), the small peak at 1 545 cm –1 (amine group stretching vibration resulting from –NH deformation mode to C=N deformation mode) and the strong peak at 1 040 cm –1 (C–F and C–Br vibrations) (Vinceković et al . 2016). The spectrum of T . viride and calcium chloride mixture shows somewhat more intense but less broad –OH and –NH stretching vibrations band around 3 385 cm –1 and a significant reduction in intensity of T . viride bands at 2 921 and 2854 cm –1 . The disappearance of small peaks between 1 500 and 1 200 cm –1 , the appearance of a peak at 1 311 cm –1 , and shifting of T . viride bands at 3321, 1608, 1207 and 663 cm –1 to 3394, 1628, 1307 and 647 cm –1 suggested that carboxylate and at least hydroxyl groups could be involved in interactions as was similarly observed for interactions between T . viride and copper ions (Vinceković et al . 2016). Characteristic FTIR bands of sodium alginate are in accordance with literature data (Sankalia et al . 2007). The strong and broad absorption band in the range 3 000– 3600 cm –1 belongs to the hydroxyl (O-H) group, stretching vibration at 2 920 cm –1 corresponds to the aliphatic C-H group, the bands at 1 595 and 1 405 cm –1 correspond to asymmetric and symmetric stretching peaks of carboxylate (–COO – ) groups, weak broad stretching vibration at 1 295 0 2 000 4 000 6 000 8 000 0.5 1.0 1.5 2.0 NS (no. g –1 ) Time (d) c i (CaCl 2 ) (mol dm –3 ) 1 10 20 Fig. 5 Changes in the number of Trichoderma viride spores (NS) in ALG/(Ca+ Tv ) (calcium alginate microspheres containing T. viride spores) microspheres prepared at a various initial calcium chloride concentration, c i (CaCl 2 ), after 1, 10 and 20 days of loading. The error bars indicate the standard deviation of the means. Fig. 6 FTIR spectra of Trichoderma viride spores ( Tv , red line), calcium chloride (CaCl 2 , blue line) and their mixture ( Tv +CaCl 2 , green line), alginate (ALG, black dash dot line) and the mixture of T. viride spores and alginate (( Tv +ALG), black dashed line). 4 000 3 500 3 000 2 500 2 000 1 500 1 000 500 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Tv CaCl 2 Tv +CaCl 2 ALG ------ Tv +ALG Transmittance Wavenumber (cm –1 )