Increasing fungal blastospore desiccation tolerance with compatible solutes

In the process from fermentation, to formulation, to desiccation, and finally to storage of biocontrol agents, the low desiccation tolerance of certain active ingredients remains one of the major hurdles to overcome. A noteworthy example here are blastospores formed by the entomopathogenic fungi M. brunneum. Blastospores are easily produced in large quantities, but are vulnerable to desiccation. To achieve protection most desiccation-tolerant organisms are known to accumulate trehalose. Intracellularly, trehalose helps to maintain membrane flexibility by acting as water replacer, upholds protein hydration, and promotes vitrification to critically decelerate decay processes. The effectiveness is highly dependent on intracellular trehalose concentration and homogeneity.

Our aim is therefore to increase intracellular trehalose levels in M. brunneum blastospores through hypotonicity-induced exchange of dispensable solutes with trehalose.

Several hypotonic trehalose treatments were performed with M. brunneum blastospores and the influence on viability was monitored. Additionally, GC-MS was employed to reveal levels of trehalose and other solutes. Lastly, a fluorescence-based investigation of an osmotic drying process was conducted in a microfluidic environment on single-cell basis to identify critical areas in cells and stress phases.

We found a significant increase in viability of hypotonically treated blastospores after desiccation and rehydration, mirrored by significantly lower stress levels during microfluidic desiccation. Blastospore survival was increased from 7.6 % to 62.0 %. Treated blastospores exhibited significantly raised trehalose levels. The microfluidic observation revealed that cellular stress levels were only slightly elevated during the desiccation, but strongly increased during rehydration. Newly sporulated hyphae accumulated the greatest amount of stress marker.

A hypotonic treatment led to an increased intracellular trehalose uptake which lowered the amount of stress received during de- and rehydration and increased blastospore viability. Fluorescence based single-cell monitoring gains valuable insights into critical phases during drying.