Presence and distribution of heavy metal tolerant fungi in surface soils of a temperate pine forest

Natural soils represent a rich reservoir of novel and diverse microbial communities. Heavy metal tolerant fungi have been described in contaminated soils and water, but it is unknown how abundant and diverse they are in natural systems. We isolated and identified heavy metal tolerant fungi from a temperate pine forest (Duke Forest, NC, USA) and determined their soil abundance and distribution using amplicon sequencing. Soil serial dilutions were inoculated on malt extract agar with metal concentrations between 100 and 1000 ppm of FeSO4, ZnSO4, CuSO4, Al-2(SO4)(3) , Pb(NO3)(2) , NiCl2, CdCl2 , and K2Cr2O7 , depending on the metal used, at 25 degrees C. A total of 425 fungal isolates were obtained, from which the majority were isolated in Pb enriched-medium and the least from Cr and Al enriched media. A total of 62 unique Operational Taxonomic Units (OTUs) were identified at 97% sequence similarity using the ITS nuclear ribosomal RNA. The most common and diverse genera isolated were Penicillium and Trichoderma, with 126 and 92 isolates, representing 10 and 6 unique OTUs, respectively. Umbelopsis, Pochonia, Pseudogymnoascus, Trichocladium, and Ilyonectria are reported for the first time as tolerant taxa to multiple metals. The use of culture-based methods also revealed the presence of taxa that could not be detected using soil amplicon sequencing; these non-detectable OTUs corresponded to cultures with low isolation rates. The most commonly isolated taxa were detected using amplicon sequencing in 90-100% of the soil samples and soil depths using direct sequencing techniques, showing consistent results with the cultured-based methods. Penicillium, Trichoderma, Umbelopsis, and Saitozyma sequences were detected in 100% of the soil samples (156 samples) at all soil depths. Our study shows that this natural environment contained abundant and diverse communities of heavy metal tolerant fungi similar to those reported in metal contaminated sites. These commonly recovered taxa showed wide levels of adaptation to different metals and toxicity levels. The combination of culture-based and direct sequencing approaches were useful to describe the abundance and distribution of heavy metal tolerant fungi in natural soils.