Mushroom production is highly affected by green mould disease, which causes severe losses worldwide. This study aimed to characterize Trichoderma spp. isolates collected from Agaricus bisporus‐ and Pleurotus ostreatus‐growing facilities based on their molecular features and evaluate their aggressiveness in mushroom production in vitro and in vivo. Fifty‐six Trichoderma isolates from mushroom farms in Aegean and Marmara regions were analysed for molecular characteristics. In a phylogenetic tree constructed based on internal transcribed spacer (ITS) and tef1 sequences, they were classified into five groups, which included Trichoderma spp. such as T. aggressivum, T. europaeum, T. pleuroticola, T. virens, T. harzianum and T. afroharzianum. Phylogenetic analyses revealed that tef1 sequences could distinguish between T. aggressivum f. aggressivum and T. aggressivum f. europaeum more specifically. Consequently, tef1 sequence variation identified 15 T. aggressivum f. europaeum isolates compared with ITS sequence variation. In vitro conditions based on the dual‐culture tests of the mycelial growth of A. bisporus showed growth inhibition between 50% and 75% depending on the isolates. Two Trichoderma isolates with different ITS and tef1 sequences (3/9 and 7/5 isolates, respectively) and more lytic and mycoparasitic activities were analysed further to determine their effects on yield and mushroom quality parameters. Therefore, T. aggressivum f. aggressivum is not the only species that affects mushroom production, and T. aggressivum f. aggressivum and T. aggressivum f. europaeum isolates significantly (p < 0.05) inhibited A. bisporus growth in vitro and in vivo.

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