Hydrogen peroxide is suspected of being highly implicated in mushroom nutrition and in substrate bleaching during cultivation. The parameters for measuring H
2O
2 in compost samples were examined and the methodology was applied to samples from both compost colonized by cultivars and wild isolates of
Agaricus bisporus, and wheat straw or coffee pulp colonized by
Pleurotus spp. Laccase and peroxidase activities were also measured. H
2O
2 concentration measured after heating at 80 °C for inactivating laccases and peroxidases was probably both H
2O
2 pre‐existing in the compost and H
2O
2 generated from quinones and active oxygen species. This potential H
2O
2 concentration increased during the vegetative growth for all the strains, in agreement with a direct relationship between H
2O
2 concentration and active biomass of
A. bisporus or
Pleurotus spp. in their cultivation substrates. Correlations were observed between H
2O
2 concentration and manganese peroxidase activity in cultivation substrates at the stage of primordia formation. At this stage of development, H
2O
2 generation via biotic or abiotic mechanisms should be an important physiological trait of mushrooms. Copyright © 2007 Society of Chemical Industry
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