Diversity of basidiomycetes in Michigan agricultural soils

We analyzed the communities of soil basidiomycetes in agroecosystems that differ in tillage history at the Kellogg Biological Station Long-Term Ecological Research site near Battle Creek, Michigan. The approach combined soil DNA extraction through a bead-beating method modified to increase recovery of fungal DNA, PCR amplification with basidiomycete-specific primers, cloning and restriction fragment length polymorphism screening of mixed PCR products, and sequencing of unique clones. Much greater diversity was detected than was anticipated in this habitat on the basis of culture-based methods or surveys of fruiting bodies. With "species" defined as organisms yielding PCR products with >= 99% identity in the 5' 650 bases of the nuclear large-subunit ribosomal DNA, 241 "species" were detected among 409 unique basidiomycete sequences recovered. Almost all major clades of basidiomycetes from basidiomycetous yeasts and other heterobasidiomycetes through polypores and euagarics (gilled mushrooms and relatives) were represented, with a majority from the latter clade. Only 24 of 241 "species" had 99% or greater sequence similarity to named reference sequences in GenBank, and several clades with multiple "species" could not be identified at the genus level by phylogenetic comparisons with named sequences. The total estimated "species" richness for this 11.2-ha site was 367 "species" of basidiomycetes. Since > 99% of the study area has not been sampled, the accuracy of our diversity estimate is uncertain. Replication in time and space is required to detect additional diversity and the underlying community structure.