JIA-2019-11

2536 Slaven Jurić et al. Journal of Integrative Agriculture 2019, 18(11): 2534–2548 2.2. Microsphere preparation Two kinds of microspheres were prepared, without (ALG/ Ca) and with T . viride spores (ALG/(Ca+ Tv )). They were prepared by dripping 100 mL of sodium alginate or a mixture of sodium alginate and T . viride spores into calcium chloride solution. The concentration of sodium alginate was constant (1.5%, w/v), whereas initial calcium cations concentrations varied from 0.5 to 2.0 mol dm –3 . The flow rate of sodium alginate solution was 5.5 to 7 mL min –1 (determined by using encapsulator nozzle size of 300 µm at the vibration frequency of 800 Hz and 120 mbar pressures (Encapsulator Büchi-B390, BÜCHI Labortechnik AG, Switzerland)). Microspheres were formed in the cross-linking solution under mechanical stirring, then washed several times with sterilized water and filtered through Büchner funnel. They were stored in deionized water at 277 K until further studies. A number of the microspheres were allowed to air-dry at room temperature to reach their equilibrium moisture content. 2.3. Effect of calcium concentration on T. viride growth and sporulation To verify vegetative growth capability and sporulation in the presence of calcium cations, two sets of experiments were performed. In the first set, suspension of T . viride spores was left in solution with various concentration of calcium chloride as well as inoculated on PDA substrate. In the second set of experiments, the number of spores (NS) loaded in microspheres was determined at different time intervals. Effect of calcium concentration on T. viride growth and sporulation An indigenous isolate of T . viride spores was maintained on the PDA Petri plates (diameter: 10 cm) and flooded with 15 mL of calcium chloride (from 0.5 to 2 mol dm –3 ). Suspensions were poured into plastic tubes (15 mL) and sealed. Aliquots of each suspension (0.3 mL) were inoculated on PDA substrate (Petri plate’s diameter: 10 cm), sealed and cultivated in a humid chamber at room temperature for 10 days. The number of spores in suspensions was determined with a hemocytometer, using Neubauer Counting Chambers (Hirschmann EM Techcolor, Germany). The appearance of T . viride mycelium around microspheres on Petri dishes was observed visually. Control samples were prepared with distilled water. Effect of calcium cations on T. viride spores loaded in microspheres Sporulation ability of loaded T . viride was investigated by measuring changes in the spore number per 1 g of microspheres (NS g –1 ) on freshly prepared microspheres after 1, 10 and 20 days of loading. Samples for measurements were prepared by dissolving 10 g of microspheres in 100 mL of NaHCO 3 (0.2 mol dm –3 ) and Na 2 C 6 H 5 O 7 · 2H 2 O (0.06 mol dm –3 ) mixture and mixed for 30 min at room temperature. Aseptic conditions were provided throughout the assay. A total of 5 mL of the sample was filtered through the sterilized muslin cloth (Trinci 1974). The number of spores was determined spectrophotometrically (Waghunde et al . 2010). Absorbance was measured at 550 nm using UV-VIS spectrophotometer (Shimadzu, UV-1700). The stock solution used for the calibration curve was diluted with sterilized distilled water. The number of spores in stock solution was determined with a hemocytometer, using a Neubauer Counting Chamber (Hirschmann EM Techcolor, Germany). The electrostatic charge and size of T. viride spores suspended in water The electrostatic charge and zeta potential (ζ), of T . viride spores suspended in water and calcium chloride solutions, were determined by Zetasizer Nano ZS (Malvern, UK). The zeta potential was estimated from electrophoretic mobility measurements. The hydrodynamic diameter (d) was estimated by the Einstein- Stokes equation assuming a spherical aggregate. Results are presented as the mean value of at least three to six measurements. 2.4. Methods Fourier-transform infrared spectroscopy analysis The Fourier-transform infrared spectroscopy spectra were recorded with the FTIR Instrument-Cary 660 FTIR (MIR system) spectrometer (Agilent Technologies, USA). Samples were mixed with potassium bromide (250 mg) to get pellets. Spectral scanning was done in the range of 500–4 000 cm –1 . Microscopic observations Microphotographs were taken with a light (LM), fluorescence (FM), scanning electron (SEM) and confocal laser scanning microscope (CLSM). The average diameter and overall shape of microspheres were observed by a Leica MZ16a Stereo-Microscope (Leica Microsystems Ltd., Switzerland) using Olympus Soft Imaging Solutions GmbH, version E_LCmicro_09Okt2009 (Germany). The dried T . viride spores and microspheres for scanning electron microscopy observations (FE-SEM, model JSM- 7000F, Jeol Ltd., Japan) were put on the high-conductive graphite tape. The Image J Software was used for the determination the size of T . viride spores and pores on a microsphere surface. T . viride spore suspensions and microspheres were investigated by a Confocal Laser Scanning Microscope (TCP SP2, Leica Lasertechnik, Germany). The microscope was operated in transmitted and fluorescent mode at an acceleration voltage of 80 kV. All sample preparations were