A stressing method for producing high-density Trichoderma spores in a dual-layer by utilizing a starch-based medium in a reconditioning approach

Abstract

A stressing method was adopted to produce Trichoderma harzianum TI21 spores in a dual-layer with an outer-layer spore free of media-residue by reconditioning the starch-based rice agar medium. The essential parameters were explored and optimized using statistical methods. The single factorial test's findings demonstrated that lower-pH and starch-based media encouraged sporulation while suppressing the growth of contaminants. The Box-Behnken design identified the ideal levels as pH 4, 30 °C, and 75 %. In the reconditioning approach, the first harvest produced 4.91 (3.64 × 1011 CFU/g) of spores (g/L) after 5 days of cultivation, while the second and third harvests produced 4.19 and 3.48 (respectively) after 4 days of cultivation, leaving 755 (g/L) (2.25 × 1010 CFU/g) media-mixed spores. As compared to media-mixed spores, media residue-free spores had significantly longer viability (18 months) with no clogging hazards. According to the findings, the method can be used industrially to produce stable, high-density spores for widespread agricultural applications.