Analysis of internal transcribed spacer (ITS) regions of rRNA genes in fungal communities in a southeastern U.S. salt marsh.

Abstract

The ascomycete community colonizing decaying Spartina alterniflora blades in a southeastern U.S. salt marsh was characterized by analysis of internal transcribed spacer (ITS) regions of fungal rRNA genes. ITS sequences were amplified with ascomycete-specific primers from DNA extracted from S. alterniflora blades at two stages of decay (early and late) and were identified based on sequence analysis of a companion ascomycete culture collection. The S. alterniflora ITS libraries were dominated by clones from three species of ascomycetes: Mycosphaerella sp. 2, Phaeosphaeria spartinicola, and Phaeosphaeria halima. ITS sequences from five other less abundant ascomycete species were also found in the clone libraries, only two of which could be identified based on the culture collection, Hydropisphaera erubescens and a new species nicknamed '4clt'. Ascospore expulsion assays indicated dominance by the same three species as the ITS analysis, although this non-molecular approach differed from the molecular method in relative ranking of the dominant species and in characterization of minor species. Analysis of ITS amplicons from three replicate plots by terminal restriction fragment length polymorphism (T-RFLP) analysis showed significant spatial homogeneity in ascomycete community composition for both early- and late-stage decay. ITS sequence analysis identified morphologically cryptic subgroups for two of the three dominant salt marsh ascomycetes.