JIA-2019-11

2535 Slaven Jurić et al. Journal of Integrative Agriculture 2019, 18(11): 2534–2548 plant pathogen fungi. The positive effect on plant growth and productivity is the independent ability and equally remarkable and significant as their antifungal ability. Numerous studies showed that Trichoderma species may induce changes in the microbiota composition of roots, enhance nutrient uptake, stabilize soil nutrients, promote root development, and increase root hair formation (Harman 2006; Waghunde et al . 2010; Stewart and Hill 2014). Promoting plant growth effect has been suggested to involve solubilization of otherwise unavailable mineral nutrients. The growth and sporulation of Trichoderma species depend on the interplay between various environmental factors (exposure to light, endogenous rhythms, C:N status, the ambient pH of the medium, environmental cations, physical injury to the mycelium and the presence of fungal-derived volatile organic compounds) among which environmental calcium plays an important role (Jayaswal et al . 2003; Steyaert et al . 2010). It was demonstrated that the simultaneous addition of calcium chloride with biocontrol agent can improve the activity of biocontrol agents (El-Mougy et al . 2012). The synergism of calcium and biocontrol agents can be a result of several different interactions taking place between calcium ions and biocontrol agents (Droby et al . 1997). Calcium is an essential plant macronutrient and an integral part of plant cell wall structure. It has many roles: regulating the transport of other nutrients, promoting proper plant cell elongation, helping in protecting the plant against diseases, being involved in the activation of certain plant enzymes, having a regulating effect in the cells, contributing to the stability of the plant, etc. (Jones 2012; Barker and Pilbeam 2015). Since calcium is required in relatively large concentrations for normal cell growth, it is important that a sufficient amount of calcium is always available in the root environment. The main problem in the commercial use of a biocontrol agent is to select an appropriate formulation which ensures the viability during storage and application. Encapsulation is an advanced technology by which, solids, liquids or gases may be loaded into carrier matrix (usually microspheres or microcapsules within micro- or nano-scale), protects biocontrol agents from the environment, improves their viability and allows controlled release at the right time and in the right place (Cumagun 2014). The suitable formulation should provide optimal delivery of an active agent to the site of action in order to meet adequate shelf life. Despite the array of encapsulation methodologies for encapsulated chemical agents, there are only a few data in the literature about the simultaneous delivery of biological and chemical agents (Vinceković et al . 2016, 2017). The requirement for the choice of a carrier for simultaneous delivery is to choose a material which can incorporate both, chemical and biological agents, in the same compartment without diminishing their activities. Loading in biopolymer matrices has been recognized as an effective formulation for controlled release of bioactive agents used for plant protection (Rathore et al . 2013). We have shown in our previous papers (Vinceković et al . 2016, 2017) that the presence of Trichoderma viride spores and copper cations in the same compartment do not inhibit their activity. Results obtained opened up perspectives for the future use of other gelling cations, which act at the same time as macro- or micronutrients for simultaneous loading with T . viride spores in alginate microparticles for plant nutrition and protection. Due to benefits for crop protection and nutrition as well as their compatibility, T . viride spores and calcium ions were taken as a suitable couple for loading in alginate microspheres. Providing the plant with additional calcium and T . viride could balance nutrition and protection as essential components in terms of achieving better yields and quality of crops. Having in mind that T . viride is a living organism and both amounts of alginate and concentration of gelling cations affect markedly physicochemical properties of alginate gels (Selimoglu and Elibol 2010), we have investigated the impact of calcium ions concentration on the vegetative growth and sporulation of T . viride as well as on the essential microsphere parameters at an adjusted constant concentration of sodium alginate (ALG). The main aim of the present study was to investigate the influence of calcium ions concentration on the physicochemical properties of calcium alginate microspheres prepared without and with T . viride spores to ensure the flow of bioactive agents to the rhizosphere. It was shown how to adjust physico-chemical properties of microspheres with a concentration of calcium ions. Proposed microspheres have a great potential to be used in agriculture especially on acidic soils. 2. Materials and methods 2.1. Materials Low viscosity sodium alginate (CAS Number: 9005-38-3; molecular weight: 80–120 kDa) was purchased from Sigma Aldrich (USA), calcium chloride from Kemika (Croatia) and eosin from Fluka (Buchs, Switzerland). All other chemicals were of analytical grade and used as received without further purification. An indigenous isolate of T . viride (STP) originated from parasitized sclerotia of Sclerotinia sclerotiorum was used in all experiments (Topolovec-Pintarić et al . 2013). STP was grown for 10 days in potato dextrose broth (PDB, Biolife, Italy) under the constant aeration at 295 K. Details of STP and spore suspension preparation were previously described (Vinceković et al . 2016).