Detection of potential fungal pathogens Fusarium falciforme and F. keratoplasticum in unhatched loggerhead turtle eggs using a molecular approach

The recognition of the Fusarium solani species complex (FSSC) as a potential global threat to sea turtle eggs represents yet another instance of an emerging fungal infection impacting wildlife. Traditionally, culturing has been used to identify fungi associated with sea turtle eggs. Since culturing recovers only a subset of environmental microbes, a molecular approach was adopted instead to survey the fungal composition inside unhatched eggs. DNA was directly extracted from the embryonic fluid and biofilms in 73 fully incubated unhatched loggerhead sea turtle eggs collected from different regions of Jekyll Island, GA, USA, in 2010 and 2012. The fungal internal transcribed spacer region of the nuclear ribosomal DNA (ITS nrDNA) was amplified from the DNA samples, and ITS products were cloned, sequenced and subjected to phylogenetic analysis. Sequences corresponded to previously cultivated fungi and were dominated by the FSSC members Fusarium falciforme and F. keratoplasticum. These fungi were consistently detected in unhatched eggs throughout Jekyll Island during each nesting year examined. Fusarium falciforme and F. keratoplasticum ITS sequences were nearly identical to isolates implicated in causing disease in sea turtle embryos from Ascension Island, Australia, Central America and Cape Verde. This represents the first survey establishing these fungal groups in North American loggerhead eggs and provides confirmation of the widespread distribution of F. falciforme and F. keratoplasticum using a novel approach. As we begin to meet conservation goals of species recovery, density-dependent management issues such as emerging fungal infections become a growing concern. Assessing the long-term impacts of Fusarium may be a rising priority.