First application of loop-mediated isothermal amplification (LAMP) assays for rapid identification of mating type in the heterothallic fungus Aspergillus fumigatus

Background Loop-mediated isothermal amplification (LAMP) assays, which operate at a single temperature and require no post-reaction processing, have been described for rapid species-specific detection of numerous fungi. The technology has much less commonly been applied to identification of other key genetic traits such as fungicide resistance, and has not yet been applied to mating-type determination in any fungus. Objectives To develop first LAMP assays for mating-type identification in a fungus, in this instance with the saprophytic mould and human opportunistic pathogen Aspergillus fumigatus, a heterothallic ascomycete requiring isolates of opposite mating type (MAT1-1, MAT1-2) for sexual reproduction. Methods New LAMP primer sets, targeted to MAT gene sequences, were screened against 34 A. fumigatus isolates (of known mating type) from diverse clinical, environmental and geographic sources to establish if they could distinguish MAT1-1 or MAT1-2 genotypes. Results and conclusions The new assays, operating at a single temperature of 65°C, correctly identified the mating-type of A. fumigatus isolates in <20 minutes, and thus have numerous research and practical applications. Similar MAT LAMP assays could now be developed for other fungi of agricultural, environmental, industrial and/or medical importance

Taxonomic novelties in Aspergillus section Fumigati: A. tasmanicus sp. nov., induction of sexual state in A. turcosus and overview of related species

The phylogenetic position of two Aspergillus strains isolated from Australian soil and phenotypically resembling A. unilateralis was investigated by using multigene phylogeny based on β-tubulin (benA), calmodulin (CaM), actin (act), and RNA polymerase II second largest subunit (RPB2) genes. The analysis supported their placement into a separate lineage within a well-supported clade containing 10 other members of section Fumigati (“A. unilateralis clade”). Comparisons of extrolite profiles, taxonomically informative morphological and physiological characters were made, and it was discovered that the two strains can be differentiated from all relatives by their low maximum growth temperature, short stipes, and ornamentation of conidia. The data justified the proposal of a new species, A. tasmanius sp. nov. Amplification of mating-type genes showed that the A. unilateralis clade contains five heterothallic species. Only the MAT1-1-1 idiomorph was detected among isolates of A. unilateralis, A. tasmanicus, and A. marvanovae, while isolates having both opposite mating types were detected in A. turcosus and A. nishimurae. The sexual state of A. turcosus was induced by mating experiments and is described in this study. Ascospores of this species were unique by their smooth to finely verrucose convex surface and two well-visible equatorial crests. Some exometabolites detected in A. marvanovae and A. tasmanicus are also indicative of a perfect state, thus supporting the hypothesis that these species have cryptic sexual cycles. The epitype and ex-epitype culture is designated for A. nishimurae to facilitate further taxonomic work with this species