| Summary: | The diversity of orchid mycorrhizal fungi (OMF) and other beneficial root-associated fungi in temperate forests has scarcely been examined. This study aimed to analyze the diversity of mycorrhizal and rhizosphere-associated fungal communities in the terrestrial orchids <i>Gavilea lutea</i> and <i>Chloraea collicensis</i> growing in high-orchid-population-density areas in the piedmont of the Andes Cordillera with native forest (<i>Nothofagus-Araucaria</i>) and Coastal Cordillera with an exotic plantation (<i>Pinus-Eucalyptus</i>) in south-central Chile. We focused on rhizosphere-inhabiting and peloton-associated OMF in a native forest (Andes Cordillera) and a mixed forest (Coastal Cordillera). The native terrestrial orchids <i>G. lutea</i> and <i>C. collicensis</i> were localized, mycorrhizal root segments were taken to isolate peloton-associated OMF, and rhizosphere soil was taken to perform the metabarcoding approach. The results revealed that Basidiomycota and Ascomycota were the main rhizosphere-inhabiting fungal phyla, showing significant differences in the composition of fungal communities in both sites. <i>Sebacina</i> was the most-abundant OMF genera in the rhizosphere of <i>G. lutea</i> growing in the native forest soil. In contrast, <i>Thanatephorus</i> was the most abundant mycorrhizal taxa growing in the rhizosphere of orchids from the Coastal Cordillera. Besides, other OMF genera such as <i>Inocybe</i>, <i>Tomentella</i>, and <i>Mycena</i> were detected. The diversity of OMF in pelotons differed, being mainly related to <i>Ceratobasidium</i> sp. and <i>Tulasnella</i> sp. These results provide evidence of differences in OMF from pelotons and the rhizosphere soil in <i>G. lutea</i> growing in the Andes Cordillera and a selection of microbial communities in the rhizosphere of <i>C. collicensis</i> in the Coastal Cordillera. This raises questions about the efficiency of propagation strategies based only on mycorrhizal fungi obtained by culture-dependent methods, especially in orchids that depend on non-culturable taxa for seed germination and plantlet development.
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