Lichtheimia species are the second most important cause of mucormycosis in Europe. To provide broader insights into the molecular basis of the pathogenicity-associated traits of the basal Mucorales, we report the full genome sequence of L. corymbifera and compared it to the genome of Rhizopus oryzae, the most common cause of mucormycosis worldwide. The genome assembly encompasses 33.6 MB and 12,379 protein-coding genes. This study reveals four major differences of the L. corymbifera genome to R. oryzae: (i) the presence of an highly elevated number of gene duplications which are unlike R. oryzae not due to whole genome duplication (WGD), (ii) despite the relatively high incidence of introns, alternative splicing (AS) is not frequently observed for the generation of paralogs and in response to stress, (iii) the content of repetitive elements is strikingly low (<5%), (iv) L. corymbifera is typically haploid. Novel virulence factors were identified which may be involved in the regulation of the adaptation to iron-limitation, e.g. LCor01340.1 encoding a putative siderophore transporter and LCor00410.1 involved in the siderophore metabolism. Genes encoding the transcription factors LCor08192.1 and LCor01236.1, which are similar to GATA type regulators and to calcineurin regulated CRZ1, respectively, indicating an involvement of the calcineurin pathway in the adaption to iron limitation. Genes encoding MADS-box transcription factors are elevated up to 11 copies compared to the 1–4 copies usually found in other fungi. More findings are: (i) lower content of tRNAs, but unique codons in L. corymbifera, (ii) Over 25% of the proteins are apparently specific for L. corymbifera. (iii) L. corymbifera contains only 2/3 of the proteases (known to be essential virulence factors) in comparision to R. oryzae. On the other hand, the number of secreted proteases, however, is roughly twice as high as in R. oryzae.

Mucor species are common soil fungi but also known as agents of human infections (mucormycosis) and used in food production and biotechnology. Mucor circinelloides is the Mucor species that is most frequently isolated from clinical sources. The taxonomy of Mucor circinelloides and its close relatives (Mucor circinelloides complex – MCC) is still based on morphology and mating behaviour. The aim of the present study was a revised taxonomy of the MCC using a polyphasic approach. Using a set of 100 strains molecular phylogenetic analysis of five markers (ITS, rpb1, tsr1, mcm7, and cfs, introduced here) were performed, combined with phenotypic studies, mating tests and the determination of the maximum growth temperatures. The multi-locus analyses revealed 16 phylogenetic species of which 14 showed distinct phenotypical traits and were recognised as discrete species. Five of these species are introduced as novel taxa: M. amethystinus sp. nov., M. atramentarius sp. nov., M. variicolumellatus sp. nov., M. pseudocircinelloides sp. nov., and M. pseudolusitanicus sp. nov. The former formae of M. circinelloides represent one or two separate species. In the MCC, the simple presence of well-shaped zygospores only indicates a close relation of both strains, but not necessarily conspecificity. Seven species of the MCC have been implemented in human infection: M. circinelloides, M. griseocyanus, M. janssenii, M. lusitanicus, M. ramosissimus, M. variicolumellatus, and M. velutinosus.

Although the number of mucormycosis cases has increased during the last decades, little is known about the pathogenic potential of most mucoralean fungi. Lichtheimia species represent the second and third most common cause of mucormycosis in Europe and worldwide, respectively. To date only three of the five species of the genus have been found to be involved in mucormycosis, namely L. corymbifera , L. ramosa and L. ornata. However, it is not clear whether the clinical situation reflects differences in virulence between the species of Lichtheimia or whether other factors are responsible. In this study the virulence of 46 strains of all five species of Lichtheimia was investigated in chicken embryos. Additionally, strains of the closest-related genus Dichotomocladium were tested. Full virulence was restricted to the clinically relevant species while all strains of L. hyalospora, L. sphaerocystis and Dichotomocladium species were attenuated. Although virulence differences were present in the clinically relevant species, no connection between origin (environmental vs clinical) or phylogenetic position within the species was observed. Physiological studies revealed no clear connection of stress resistance and carbon source utilization with the virulence of the strains. Slower growth at 37°C might explain low virulence of L. hyalospora , L. spaherocystis and Dichotomocladium ; however, similarly slow growing strains of L. ornata were fully virulent. Thus, additional factors or a complex interplay of factors determines the virulence of strains. Our data suggest that the clinical situation in fact reflects different virulence potentials in the Lichtheimiaceae.

Zygomycetes of the order Mucorales can cause life-threatening infections in humans. These mucormycoses are emerging and associated with a rapid tissue destruction and high mortality. The resistance of Mucorales to antimycotic substances varies between and within clinically important genera such as Mucor, Rhizopus, and Lichtheimia. Thus, an accurate diagnosis before onset of antimycotic therapy is recommended. Matrix-assisted laser desorption ionization (MALDI)-time of flight (TOF) mass spectrometry (MS) is a potentially powerful tool to rapidly identify infectious agents on the species level. We investigated the potential of MALDI-TOF MS to differentiate Lichtheimia species, one of the most important agents of mucormycoses. Using the Bruker Daltonics FlexAnalysis (version 3.0) software package, a spectral database library with m/z ratios of 2,000 to 20,000 Da was created for 19 type and reference strains of clinically relevant Zygomycetes of the order Mucorales (12 species in 7 genera). The database was tested for accuracy by use of 34 clinical and environmental isolates of Lichtheimia comprising a total of five species. Our data demonstrate that MALDI-TOF MS can be used to clearly discriminate Lichtheimia species from other pathogenic species of the Mucorales. Furthermore, the method is suitable to discriminate species within the genus. The reliability and robustness of the MALDI-TOF-based identification are evidenced by high score values (above 2.3) for the designation to a certain species and by moderate score values (below 2.0) for the discrimination between clinically relevant (Lichtheimia corymbifera, L. ramosa, and L. ornata) and irrelevant (L. hyalospora and L. sphaerocystis) species. In total, all 34 strains were unequivocally identified by MALDI-TOF MS with score values of >1.8 down to the generic level, 32 out of 34 of the Lichtheimia isolates (except CNM-CM 5399 and FSU 10566) were identified accurately with score values of >2 (probable species identification), and 25 of 34 isolates were identified to the species level with score values of >2.3 (highly probable species identification). The MALDI-TOF MS-based method reported here was found to be reproducible and accurate, with low consumable costs and minimal preparation time.