QUANTIFICATION OF FUNGAL HYPHAE IN LEAVES OF DECIDUOUS TREES BY AUTOMATED IMAGE-ANALYSIS

An optical method to quantify the fungal hyphae within decomposing lea ves of deciduous trees was developed. The plant matrix was partially d estroyed under hydrolytic conditions, and fungal hyphae and cellulose residues within the leaves were stained with Calcofluor M2R. Cellulose residues were subsequently depolymerized by cellulase, and fungal hyp hae were separated from the remaining plant matrix with a pressurized air-water mixture. An image analysis program to quantify the fungal hy phae was written. The program included the recognition of fungal hypha e, the elimination of stomata from the images, and the measuring of le ngths of fungal hyphae. The optical method was verified by a chemical method relying on glucosamine as an indicator of fungal biomass. The f ungal biomass in leaves of Fagus silvatica and Quercus petraea at earl y states of decomposition was 0.2 to 0.4% of the leaf weight. The biom ass reached a maximum within 2 to 4 weeks (optical method, 0.5 to 0.7% ; chemical method, 1 to 1.4% of the initial Leaf weight) and decreased thereafter.