Genotypic analysis of Mucor from the platypus in Australia

Mucor amphibiorum is the only disease agent known to cause significant morbidity and mortality in the free-living platypus (Ornithorhynchus anatinus) in Tasmania. Infection has also been reported in free-ranging cane toads and green tree frogs from mainland Australia, but not confirmed in platypuses from the mainland. To date, there has been little genotyping specifically conducted on M. amphibiorum. A collection of twenty-one Mucor isolates representing isolates from platypus, frogs and toads, and environmental samples were obtained for genotypic analysis. Internal transcribed spacer (ITS) region sequencing and GenBank comparison confirmed the identity of the majority of isolates. Representative isolates from infected platypus formed a clade containing the reference isolates of M. amphibiorum from the CBS repository. The M. amphibiorum isolates showed a close sequence identity with M. indicus, and consisted of 2 haplotypes, differentiated by single nucleotide polymorphisms within the ITS-1 and ITS-2 regions. With the exception of 96-4049, all isolates from platypus were in one haplotype. Multi-locus fingerprinting via the use of inter-simple sequence repeats polymerase chain reaction (ISSR-PCR) identified 19 phenotypes. Two major clusters: (1)M. amphibiorum and M. racemosus, (2)M. circinelloides, M. ramosissimus and M. fragilis were evident. Seven M. amphibiorum isolates from platypus were present in two sub-clusters, with isolate 96-4053 appearing genetically distinct from all other isolates. Isolates classified as M. circinelloides by sequence analysis formed a separate sub-cluster, distinct from other Mucor spp. The combination of sequencing and multi-locus fingerprinting has the potential to provide the tools for rapid identification of M. amphibiorum. Data presented on the diversity of the pathogen and further work in linking genetic diversity to functionaldiversity will provide critical information for its management in Tasmanian river systems.