Diversity of ascomycete laccase gene sequences in a southeastern US salt marsh

The diversity of ascomycete laccase sequences was surveyed in a southeastern US salt marsh using a degenerate primer set designed around copper binding sites conserved in fungal laccases. This gene was targeted for diversity analysis because of its potential function in lignin degradation in the salt marsh ecosystem and because few studies have assessed functional gene diversity in natural fungal communities. Laccase sequences were amplified from genomic DNA extracted from 24 isolates (representing 10 ascomycete species) cultured from decaying blades of Spartina alterniflora, and from DNA extracted directly from the decaying blades. Among the ascomycete isolates, 21 yielded a PCR product of expected size (900 bp) that was tentatively identified as laccase based on sequence similarities to previously published laccase sequences from related organisms. Overall, 13 distinct sequence types, containing 39 distinct sequences, were identified among the isolates, with several species yielding multiple distinct laccase types. PCR amplifications from early and late decay blades of S. alterniflora yielded seven laccase types. Of these, five were composed of sequences >96% similar at the amino acid level to sequences from three cultured ascomycetes previously found to be dominant members of the fungal communities on decaying S. alterniflora blades. Two of the laccase types from the natural-decay clone library were novel and did not match any of the sequences obtained from the cultured ascomycetes. The 39 distinct sequences and 15 distinct laccase sequence types retrieved from the S. alterniflora decay system demonstrate high sequence diversity of this functional gene in a natural fungal community.