Combined molecular and biochemical approach identifies Aspergillus japonicus and Aspergillus aculeatus as two species

We examined nine Aspergillus japonicus isolates and 10 Aspergillus aculeatu s isolates by using molecular and biochemical markers, including DNA sequen ces of the ITS1-5.8S rRNA gene-ITS2 region, restriction fragment length pol ymorphisms (RFLP), and secondary-metabolite profiles. The DNA sequence of t he internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene coul d not be used to distinguish between A. japonicus and A. aculeatus but did show that these two taxa are more closely related to each other than to oth er species of black aspergilli. Aspergillus niger pyruvate kinase (pkiA) an d pectin lyase A (pelA) and Agaricus bisporus 28S rRNA genes, which were us ed as probes in the RFLP analysis, revealed clear polymorphism between thes e two taxa. The A. niger pkiA and pelA probes placed six strains in an A. j aponicus group and 12 isolates in an A. aculeatus group, which exhibited in traspecific variation when they were probed with the pelA gene. The seconda ry-metabolite profiles supported division of the isolates into the two spec ies and differed from those of other black aspergilli. The strains classifi ed as A. japonicus produced indole alkaloids and a polar metabolite, while the A. aculeatus isolates produced neoxaline, okaramins, paraherquamidelike compounds, and secalonic acid. A. aculeatus CBS 114.80 showed specific RFL P patterns for all loci examined. The secondary-metabolite profile of strai n CBS 114.80 also differed from those of A. japonicus and A. aculeatus. The refore, this strain probably represents a third taxon. This study provides unambiguous criteria for establishing the taxonomic positions of isolates o f black aspergilli, which are important in relation to industrial use and l egal protection of these organisms.