Development of a rapid and highly sensitive biochemical method for the measurement of fungal spore viability. An alternative to the CFU method

A biochemical method, based on dehydrogenase activity (DHA) measurement, has been developed as an alternative to colony forming unit (CFU) enumeration, for assessing the viability of fungal spores. In viable cells, a tetrazolium salt (MTT) is reduced to a coloured formazan (MTTf) by cellular dehydrogenase enzymes. From the colorimetric assay developed by Mosmann for mammalian cells, the procedure has been adapted and optimised using P. digitatum spores as a model. Propan-2-ol has been selected as the best solvent to extract the MTTf from the spores. The sensitivity of the method has been considerably increased by determining the optimal conditions of incubation for the MTT reduction by spores: temperature at 50°C and pH 8. Using the assay, a correlation has been established between DHA and the number of viable spores as measured by the CFU method (105 CFU = 1.14 DHA -26; r2 = 0.94). This standardised procedure allows the viability of P. digitatum spores to be estimated with accuracy and reliability. Moreover, the extension of this assay to other strains has been demonstrated (Aspergillus niger and Metarhizium flavoviride).