Statistical culture-based strategies to enhance chlamydospore production by Trichoderma harzianum SH2303 in liquid fermentation

Abstract

Trichoderma-based formulations are applied as commercial biocontrol agents for soil-borne plant pathogens. Chlamydospores are active propagules in Trichoderma spp., but their production is currently limited due to a lack of optimal liquid fermentation technology. In this study, we explored response surface methodologies for optimizing fermentation technology in Trichoderma SH2303. Our initial studies, using the Plackett-Burman design, identified cornmeal, glycerol, and initial pH levels as the most significant factors (P<0.05) for enhancing the production of chlamydospores. Subsequently, we applied the Box-Behnken design to study the interactions between, and optimal levels of, a number of factors in chlamydospore production. These statistically predicted results indicated that the highest number of chlamydospores (3.6×10(8) spores/ml) would be obtained under the following condition: corn flour 62.86 g/L, glycerol 7.54 ml/L, pH 4.17, and 6-d incubation in liquid fermentation. We validated these predicted values via three repeated experiments using the optimal culture and achieved maximum chlamydospores of 4.5×10(8) spores/ml, which approximately a 8-fold increase in the number of chlamydospores produced by T. harzianum SH2303 compared with that before optimization. These optimized values could help make chlamydospore production cost-efficient in the future development of novel biocontrol agents.

Keywords: Box-Behnken design; Chlamydospore; Fermentation optimization; Plackett-Burman screening; Trichoderma harzianum SH2303.

Fig. 1

Pareto chart (a) and normal plot graph (b) identifying the screening factors Hollow shapes represent the 8 factors of PB test, and among them, orange (F, H, C, G) denotes positive effects; blue (A, B, D, E) denotes negative effects; solid shapes represent 3 blank tests (Note: for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article)

Fig. 2

Contour plots of the interaction effect of two specific factors on chlamydospore production (a) Effects of corn meal (X ₁) and pH (X ₂), and their mutual interaction; (b) Effects of corn meal (X ₁) and glycerol (X ₃), and their mutual interaction; (c) Effects of glycerol (X ₃) and pH (X ₂), and their mutual interaction

Fig. 3

3D surface graph and its corresponding contour plot of prediction of chlamydospore production (b) and its desirability (a) at the optimal concentrations of corn flour (X ₁) and glycerol (X ₃)