Development of a Trichoderma atroviride LU132 variant active at lower temperatures for control of Botrytis cinerea on grapes using protoplast technology

Abstract

Biocontrol agents are living organisms that require stabilisation, usually through product formulation, to attain an acceptable shelf life of 1-2 years. Trehalose (a storage carbohydrate in a wide variety of organisms) is an osmo-protectant that is stable under hot and acidic conditions and is able to protect fungal spores against freezing or excess heat. Trehalose has also been shown to increase spore viability in fungi. In the current research, the ability of trehalose to prevent loss of Trichoderma atroviride conidial viability during storage under conditions of accelerated aging to simulate long-term storage was evaluated. T. atroviride conidia were produced by solid substrate fermentation, with the initial broth amended with varying amounts of trehalose (ranging from 0-24.4 mM). Conidia were harvested in sterile water and allowed to air-dry before storage at either 4 or 30°C with uniform water activity. Condial viability was assessed at monthly intervals by counting percentage germination. At harvest, trehalose amendment of the growth media resulted in a significant increase in trehalose content of T. atroviride conidia of between 18 and 42%, when compared with the untreated controls. The 0.8 mM trehalose treatment significantly increased viability of conidia, as measured by the area under the conidia viability progress curve, in both the 4 and 30°C storage treatments by 5 and 12%, respectively. However, at this concentration, trehalose did not significantly extend shelf-life of T. atroviride. The addition of higher concentrations of trehalose significantly reduced spore viability.