Optimizacija biosinteze antagonista skladišnih patogena jabuke primenom Streptomyces hygroscopicus / Optimization of antagonists of storage apple pathogens biosynthesis by Streptomyces hygroscopicus

Apple fruits are available on the market all year round and their safety is of great importance for the consumers. Unfortunately, many phytopathogenic fungi can cause apple diseases but fungi from the genera Alternaria and Fusarium are listed among important storage pathogens of apple fruits. It is considered that health-safe apple fruits are primarily healthy fruits, without the presence of phytopathogenic fungi and without the remains of fungicides. Excessive use of pesticides in modern agriculture, including those used for control of fungal phytopathogens, has led to several problems related to pollution, environmental degradation and emergence of resistant strains. Biological control of plant pathogens by means of microorganisms is considered as an attractive alternative to chemical-based treatments, with minimal impact on the environment. The main lack of biotechnology production of the antifungal agents are the high costs, so the optimization of the biosynthesis conditions is the most important step towards its overcoming. Therefore, the aim of this research is to optimize the medium composition and process conditions (agitation and aeration rate) of the antagonists of storage apple pathogens biosinthesis by Streptomyces hygroscopicus in laboratory-scale bioreactor. Based on the obtained results, it can be concluded that the S. hygroscopicus cultivation medium containing glycerol as a carbon source, with no additional nitrogen source, proved to be the most suitable for the production of the A. alternate and F. avenaceum antagonistic agents. The results of the optimization of medium for biosynthesis of the antagonists of storage apple pathogens using S. hygroscopicus indicated the following optimal values of the examined factors: the content of glycerol of 20 g/l, (NH4)2SO4 of 0.25 g/l and K2HPO4 of 1.41 g/l. By applying the defined optimal values and using the appropriate mathematical models, the following responses are predicted: residual glycerol of 5.3418 g/l; residual nitrogen of 0.193 g/l; residual phosphates of 0.8601 g/l; inhibition zone diameters of A. alternata KA10 of 28.22 mm; inhibition zone diameters of A. alternata T1Jg3 of 36.86 mm; inhibition zone diameters of F. avenaceum KA12 of 25.54 mm and inhibition zone diameters of F. avenaceum KA13 of 26.78 mm. Examining the effect of different agitation and aeration rate on the biosynthesis of antagonists of storage apple pathogens using S. hygroscopicus in a laboratory-scale bioreactor with total volume of 3 l, the following optimal values of the investigated factors are obtained: the agitation rate of 100 rpm, the aeration of 1.2 l/l/min and 96-hour as optimal time of cultivation. Bringing together the appropriate mathematical models and defined optimal values, the following responses are predicted: residual glycerol of 8.2645 g/l; residual nitrogen of 0.1723 g/l; residual phosphates of 0.5167 g/l; biomass content of 1.5023 g/l; inhibition zone diameters of A. alternata KA10 of 62.62 mm; inhibition zone diameters of A. alternata T1Jg3 of 61.09 mm; inhibition zone diameters of F. avenaceum KA12 of 45.55 mm and inhibition zone diameters of F. avenaceum KA13 of 43.02 mm. The validity of the results was confirmed by performing the biosynthesis of the antagonists of storage apple pathogens in the medium with optimal composition and the optimal process parameters in a laboratory-scale bioreactor with the total volume of 7l. The efficacy of produced antagonistic agents effective against A. alternata (KA10 and T1Jg3) and F. avenaceum (KA12 and KA13) isolates was confirmed in vitro and in planta. The results obtained within this research represents the basis for the further enlargement of the scale, which is a key step towards the potential industrialization of the proposed production process.