JIA-2019-11

2538 Slaven Jurić et al. Journal of Integrative Agriculture 2019, 18(11): 2534–2548 surface hydrophobicity (ranged from 12 to 52%), i.e., less hydrophobic cell surfaces were less negatively charged. With the increase in calcium chloride concentration T . viride spores become less negatively charged due to electrostatic binding of calcium cations (Fig. 2-A). Trichoderma species belong to the group of microorganisms, which can survive in high concentrations of different metals and have the potential to bind them. Calcium ions bind mainly to fungal cell wall structure (López Errasquı́n and Vázquez 2003) which is composed of polysaccharides and glycoproteins, as well as lipids and other minor components (pigments and inorganic salts). Melanin, which in other fungi is associated with chitin, was shown to replace this polymer in the spore wall of Trichoderma species (Fogarty and Tobin 1996). Melanin contains carboxyl, phenolic, hydroxyl, and amine functional groups which serve as potential binding sites for metal ions. A reversal of charge around 0.7 mol dm –3 of calcium ions suggested that besides electrostatic interactions some other mechanisms are responsible for calcium binding; probably ion exchange and/or some other physicochemical interactions between cations and functional groups on the fungal cell wall. Effect of Ca 2+ addition on the size of particles in suspensions is presented in Fig. 2-B. Calcium ions did not significantly change the particle size at a lower concentration. The increase of calcium ions concentration from 0.005 to 2 mol dm –3 resulted in less negatively charged spore surface due to electrostatic interactions between Ca 2+ and T . viride spores. Diminishing of electrostatic repulsions between spores led to their aggregation. Microscopic observation of spores suspended at a higher concentration of calcium ions confirmed significant aggregation (Fig. 1-C). Effect of calcium ions on the sporulation and growth of T. viride Fig. 3 presents changes in the number of T . viride spores suspended in calcium chloride solutions. The number of spores increased with increasing calcium concentration indicating positive calcium impact on A B C 50 μm 50 μm Fig. 1 Scanning electron microscope (SEM) image of dry Trichoderma viride spores (A), optical microscopy images of T. viride spores suspended in calcium chloride solution at 0.005 mol dm –3 (B) and 1.5 mol dm –3 (C), respectively. The number of T. viride spores (NS) is 1.8×10 6 mL –1 . Bars are indicated. Fig. 2 Changes in the average zeta potential ( ζ , A), and the mean hydrodynamic diameter (d, B) of Trichoderma viride spores with different calcium chloride ( Tv +CaCl 2 ) concentrations at a constant number of T. viride spores in suspension (NS) is 1.8×10 6 mL –1 . c (CaCl 2 ) represents the concentration of CaCl 2 solution. The error bars indicate the standard deviation of the means. 0 0.5 1.0 1.5 2.0 –12 –8 –4 0 4 8 ζ (mV) Tv +CaCl 2 A B 0 0.5 1.0 1.5 2.0 0 1 000 2 000 3 000 4 000 5 000 d (nm) c (CaCl 2 ) (mol dm –3 ) c (CaCl 2 ) (mol dm –3 ) Tv +CaCl 2