Biochemical Determination of the Viability of Fungal Spores and Hyphae

Abstract

Germination is commonly used to monitor spore viability, but many fungal spores do not germinate readily. Rapid viability tests are needed. Methods were developed to monitor viability, based on enzyme activity and ATP content of fungal spores and hyphae. ATP content, measured by the luciferin-luciferase system, was much higher in living spores than in dead spores. Hydration was an additional aid in differentiating the ATP content of viable spores compared with dead spores. Approximately 40 μg of spores were required to measure the ATP level. Preparation from 25 μg of living spores or 100–150 μg fresh hyphae contained sufficient phosphatase or glucosidase to give a positive fluorescence test for hydrolysis of methylumbelliferyl-phosphate or methylumbelliferyl-glucoside at 37 C in 2 h. Dead spores and hyphae did not fluoresce. These methods were used to test the viability of: Tilletia controversa, T. caries. Ustilago scitaminea, Puccinia striiformis, Pisolithus tinctorius, Rhizopogon colossus, and Elaphomyces granulatus spores, and hyphae of T. caries, Rhizoctonia solani, Hymenochaete tabacina, Phellinus weirii, Haematostereum sanguinolentum and Gaeumannomyces graminis var. tritici. These techniques should be useful in determining quickly the effects of fumigants and fungicides on fungal spores and hyphae.