Roles of the Aspergillus nidulans UDP-galactofuranose transporter, UgtA in hyphal morphogenesis, cell wall architecture, conidiation, and drug sensitivity

Galactofuranose (Galf) is the 5-member-ring form of galactose found in the walls of fungi including Aspergillus, but not in mammals. UDP-galactofuranose mutase (UgmA, ANID_3112.1) generates UDP-Galf from UDP-galactopyranose (6-member ring form). UgmA-GFP is cytoplasmic, so the UDP-Galf residues it produces must be transported into an endomembrane compartment prior to incorporation into cell wall components. ANID_3113.1 (which we call UgtA) was identified as being likely to encode the A. nidulans UDP-Galf transporter, based on its high amino acid sequence identity with A. fumigatus GlfB. The ugtAΔ phenotype resembled that of ugmAΔ, which had compact colonies, wide, highly branched hyphae, and reduced sporulation. Like ugmAΔ, the ugtAΔ hyphal walls were threefold thicker than wild type strains (but different in appearance in TEM), and accumulated exogenous material in liquid culture. AfglfB restored wild type growth in the ugtAΔ strain, showing that these genes have homologous function. Immunostaining with EBA2 showed that ugtAΔ hyphae and conidiophores lacked Galf, which was restored in the AfglfB-complemented strain. Unlike wild type and ugmAΔ strains, some ugtAΔ metulae produced triplets of phialides, rather than pairs. Compared to wild type strains, spore production for ugtAΔ was reduced to 1%, and spore germination was reduced to half. UgtA-GFP had a punctate distribution in hyphae, phialides, and young spores. Notably, the ugtAΔ strain was significantly more sensitive than wild type to Caspofungin, which inhibits beta-glucan synthesis, suggesting that drugs that could be developed to target UgtA function would be useful in combination antifungal therapy.     

Aspergillus nidulans UDP-glucose-4-epimerase UgeA has multiple roles in wall architecture,hyphal morphogenesis,and asexual development

Aspergillus nidulans UDP-glucose-4-epimerase UgeA interconverts UDP-glucose and UDP-galactose and participates in galactose metabolism. The sugar moiety of UDP-galactose is predominantly found as galactopyranose (Galp, the six-membered ring form), which is the substrate for UDP-galactopyranose mutase (encoded by ugmA) to generate UDP-galactofuranose (Galf, the five-membered ring form) that is found in fungal walls. In A. fumigatus, Galf residues appear to be important for virulence. The A. nidulans ugeAΔ strain is viable, and has defects including wide, slow growing, highly branched hyphae and reduced conidiation that resemble the ugmAΔ strain. As for the ugmAΔ strain, ugeAΔ colonies had substantially reduced sporulation but normal spore viability. Conidia of the ugeAΔ strain could not form colonies on galactose as a sole carbon source, however they produced short, multinucleate germlings suggesting they ceased to grow from starvation. UgeA purified from an expression plasmid had a relative molecular weight of 40.6 kDa, and showed in vitro UDP-glucose-4-epimerase activity. Transmission electron microscope cross-sections of wildtype, ugeAΔ, and ugmAΔ hyphae showed they had similar cytoplasmic contents but the walls of each strain were different in appearance and thickness. Both deletion strains showed increased substrate adhesion. Localization of UgeA-GFP and UgmA-GFP was cytoplasmic, and was similar on glucose and galactose. Neither gene product had a longitudinal polarized distribution. Localization of a UgmA-mRFP in a strain that resembled the ugmAΔ strain was cytoplasmic and lacked a longitudinal polarized distribution. The roles of UgeA in A. nidulans growth and morphogenesis are consistent with the importance of Galf, and are related but not identical to the roles of UgmA.     

Aspergillus nidulans UDP-galactopyranose mutase, encoded by ugmA plays key roles in colony growth, hyphal morphogenesis, and conidiation

Growing resistance to current anti-fungal drugs is spurring investigation of new targets, including those in fungal wall metabolism. Galactofuranose (Galf) is found in the cell walls of many fungi including Aspergillus fumigatus, which is currently the most prevalent opportunistic fungal pathogen in developed countries, and A. nidulans, a closely-related, tractable model system. UDP-galactopyranose mutase (UGM) converts UDP-galactopyranose into UDP-Galf prior to incorporation into the fungal wall. We deleted the single-copy UGM sequence (AN3112.4, which we call ugmA) from an A. nidulans nkuADelta strain, creating ugmADelta. Haploid ugmADelta strains were able to complete their asexual life cycle, showing that ugmA is not essential. However, ugmADelta strains had compact colonial growth, which was associated with substantially delayed and abnormal conidiation. Compared to a wildtype morphology strain, ugmADelta strains had aberrant hyphal morphology, producing wide, uneven, highly-branched hyphae, with thick, relatively electron-dense walls as visualized by transmission electron microscopy. These effects were partially remediated by growth on high osmolarity medium, or on medium containing 10 microg/mL Calcofluor, consistent with Galf being important in cell wall structure and/or function.     

Aspergillus nidulans Cell Wall Composition and Function Change in Response to Hosting Several Aspergillus fumigatus UDP-Galactopyranose Mutase Activity Mutants

Deletion or repression of Aspergillus nidulans ugmA (AnugmA), involved in galactofuranose biosynthesis, impairs growth and increases sensitivity to Caspofungin, a β-1,3-glucan synthesis antagonist. The A. fumigatus UgmA (AfUgmA) crystal structure has been determined. From that study, AfUgmA mutants with altered enzyme activity were transformed into AnugmA▵ to assess their effect on growth and wall composition in A. nidulans. The complemented (AnugmA::wild type AfugmA) strain had wild type phenotype, indicating these genes had functional homology. Consistent with in vitro studies, AfUgmA residues R182 and R327 were important for its function in vivo, with even conservative amino (RK) substitutions producing AnugmA? phenotype strains. Similarly, the conserved AfUgmA loop III histidine (H63) was important for Galf generation: the H63N strain had a partially rescued phenotype compared to AnugmA▵. Collectively, A. nidulans strains that hosted mutated AfUgmA constructs with low enzyme activity showed increased hyphal surface adhesion as assessed by binding fluorescent latex beads. Consistent with previous qPCR results, immunofluorescence and ELISA indicated that AnugmA▵ and AfugmA-mutated A. nidulans strains had increased α-glucan and decreased β-glucan in their cell walls compared to wild type and AfugmA-complemented strains. Like the AnugmA▵ strain, A. nidulans strains containing mutated AfugmA showed increased sensitivity to antifungal drugs, particularly Caspofungin. Reduced β-glucan content was correlated with increased Caspofungin sensitivity. Aspergillus nidulans wall Galf, α-glucan, and β-glucan content was correlated in A. nidulans hyphal walls, suggesting dynamic coordination between cell wall synthesis and cell wall integrity.     

Aspergillus nidulans galactofuranose biosynthesis affects antifungal drug sensitivity

The cell wall is essential for fungal survival in natural environments. Many fungal wall carbohydrates are absent from humans, so they are a promising source of antifungal drug targets. Galactofuranose (Galf) is a sugar that decorates certain carbohydrates and lipids. It comprises about 5% of the Aspergillus fumigatus cell wall, and may play a role in systemic aspergillosis. We are studying Aspergillus wall formation in the tractable model system, A. nidulans. Previously we showed single-gene deletions of three sequential A. nidulans Galf biosynthesis proteins each caused similar hyphal morphogenesis defects and 500-fold reduced colony growth and sporulation. Here, we generated ugeA, ugmA and ugtA strains controlled by the alcA(p) or niiA(p) regulatable promoters. For repression and expression, alcA(p)-regulated strains were grown on complete medium with glucose or threonine, whereas niiA(p)-regulated strains were grown on minimal medium with ammonium or nitrate. Expression was assessed by qPCR and colony phenotype. The alcA(p) and niiA(p) strains produced similar effects: colonies resembling wild type for gene expression, and resembling deletion strains for gene repression. Galf immunolocalization using the L10 monoclonal antibody showed that ugmA deletion and repression phenotypes correlated with loss of hyphal wall Galf. None of the gene manipulations affected itraconazole sensitivity, as expected. Deletion of any of ugmA, ugeA, ugtA, their repression by alcA(p) or niiA(p), OR, ugmA overexpression by alcA(p), increased sensitivity to Caspofungin. Strains with alcA(p)-mediated overexpression of ugeA and ugtA had lower caspofungin sensitivity. Galf appears to play an important role in A. nidulans growth and vigor.     

Aspergillus fumigatus RasA regulates asexual development and cell wall integrity

The Ras family of proteins is a large group of monomeric GTPases. Members of the fungal Ras family act as molecular switches that transduce signals from the outside of the cell to signaling cascades inside the cell. A. fumigatus RasA is 94% identical to the essential RasA gene of Aspergillus nidulans and is the Ras family member sharing the highest identity to Ras homologs studied in many other fungi. In this study, we report that rasA is not essential in A. fumigatus, but its absence is associated with slowed germination and a severe defect in radial growth. The DeltarasA hyphae were more than two times the diameter of wild-type hyphae, and they displayed repeated changes in the axis of polarity during hyphal growth. The deformed hyphae accumulated numerous nuclei within each hyphal compartment. The DeltarasA mutant conidiated poorly, but this phenotype could be ameliorated by growth on osmotically stabilized media. The DeltarasA mutant also showed increased susceptibility to cell wall stressors, stained more intensely with calcofluor white, and was refractory to lysing enzymes used to make protoplasts, suggesting an alteration of the cell wall. All phenotypes associated with deletion of rasA could be corrected by reinsertion of the wild-type gene. These data demonstrate a crucial role for RasA in both hyphal growth and asexual development in A. fumigatus and provide evidence that RasA function is linked to cell wall integrity.     

A fungus-specific ras homolog contributes to the hyphal growth and virulence of Aspergillus fumigatus

The Ras family of GTPase proteins has been shown to control morphogenesis in many organisms, including several species of pathogenic fungi. In a previous study, we identified a gene encoding a fungus-specific Ras subfamily homolog, rasB, in Aspergillus fumigatus. Here we report that deletion of A. fumigatus rasB caused decreased germination and growth rates on solid media but had no effect on total biomass accumulation after 24 h of growth in liquid culture. The DeltarasB mutant had an irregular hyphal morphology characterized by increased branching. Expression of rasBDelta113-135, a mutant transgene lacking the conserved rasB internal amino acid insertion, did not complement the deletion phenotype of delayed growth and germination rates and abnormal hyphal morphology. Virulence of the rasB deletion strain was diminished; mice infected with this strain exhibited approximately 65% survival compared to approximately 10% with wild-type and reconstituted strains. These data support the hypothesis that rasB homologs, which are highly conserved among fungi that undergo hyphal growth, control signaling modules important to the directional growth of fungal hyphae.     

dewA encodes a fungal hydrophobin component of the Aspergillus spore wall

An anonymous cDNA clone, pCAN4, was shown previously to correspond to an mRNA that accumulates preferentially during asexual sporulation of the filamentous fungus Aspergillus nidulans. The peptide encoded by pCAN4 is a fungal hydrophobin, a group of small, hydrophobic cell wall proteins. When the CAN4 gene was disrupted, conidia and conidiophores appeared to be normal, but sporulating colonies wetted more rapidly with detergent solutions than did the wild type. We renamed CAN4 dewA for the detergent wettable phenotype and mapped it to chromosome V, 24 map units from cysC. The A. nidulans rodA gene also encodes a sporulation-specific fungal hydrophobin. Spores of a dewA rodA double mutant were less hydrophobic than those of either mutant alone, showing that dewA and rodA contribute independently to spore-wall hydrophobicity, Immuno-localization of DewA by epitope tagging demonstrated that DewA is present in the spore wall, but not in the walls of germ tubes, hyphae or cells of the spore-producing conidiophore. We conclude that dewA encodes a new fungal hydrophobin component of the conidial wall.     

Reciprocal oxylipin-mediated cross-talk in the Aspergillus-seed pathosystem.

In Aspergilli, mycotoxin production and sporulation are governed, in part, by endogenous oxylipins (oxygenated, polyunsaturated fatty acids and metabolites derived therefrom). In Aspergillus nidulans, oxylipins are synthesized by the dioxygenase enzymes PpoA, PpoB and PpoC. Structurally similar oxylipins are synthesized in seeds via the action of lipoxygenase (LOX) enzymes. Previous reports have shown that exogenous application of seed oxylipins to Aspergillus cultures alters sporulation and mycotoxin production. Herein, we explored whether a plant oxylipin biosynthetic gene (ZmLOX3) could substitute functionally for A. nidulans ppo genes. We engineered ZmLOX3 into wild-type A. nidulans, and into a DeltappoAC strain that was reduced in production of oxylipins, conidia and the mycotoxin sterigmatocystin. ZmLOX3 expression increased production of conidia and sterigmatocystin in both backgrounds. We additionally explored whether A. nidulans oxylipins affect seed LOX gene expression during Aspergillus colonization. We observed that peanut seed pnlox2-3 expression was decreased when infected by A. nidulansDeltappo mutants compared with infection by wild type. This result provides genetic evidence that fungal oxylipins are involved in plant LOX gene expression changes, leading to possible alterations in the fungal/host interaction. This report provides the first genetic evidence for reciprocal oxylipin cross-talk in the Aspergillus-seed pathosystem.     

Characterization of Fusarium graminearum Mes1 reveals roles in cell-surface organization and virulence

The surfaces of fungal hyphae are mosaics of carbohydrates and cell-surface proteins. Presently, very little is known about the role of these proteins and their organization at the cell surface. Here, we characterize two Fusarium graminearum genes implicated in cell-surface organization, mes1 (FGSG_06680) and pls1 (FGSG_08695). Mes1 is a homologue of mesA, which is required for the formation of stable polarity axes in Aspergillus nidulans. Pls1 encodes a tetraspanin, which belongs to a class of proteins that have been shown to aggregate in membrane rafts along with integrins and other signaling proteins. Our results indicate that Pls1 is dispensable for saprophytic growth and wheat head infection by F. graminearum (a pathogen that does not form appressoria). However, deletion of mes1 reduces sexual and asexual reproduction, severely perturbs the shape of macroconidia and hyphae, alters the pattern of cell wall deposition and the organization of sterol-rich rafts, and attenuates virulence on wheat heads. Our results provide a basis for identifying determinants of fungal virulence that may localize to specialized domains at the cell surface.     

Male and female roles in crosses of Aspergillus nidulans as revealed by vegetatively incompatible parents

To resolve the role of male and female nuclei and mitochondria in cleistothecium formation in the model organism Aspergillus nidulans, we analysed the genetic constituents of cleistothecia from crosses between vegetatively compatible and incompatible parents. We used markers that enabled us to determine the nuclear genotype of the cleistothecial wall and the nuclear and mitochondrial genotype of the ascospores. In compatible parents, nuclear genomes and cytoplasm usually mix in the vegetative hyphae prior to the formation of the sexual stage after which any cleistothecial composition is possible. In incompatible parents, the maternal strain contributes the nuclei for the cleistothecial wall and one nucleus as well as mitochondria for the ascospore origin. The paternal strain donates one nucleus for the ascospore origin. Only in crosses between vegetatively incompatible partners, it is possible to assign a female and male role to the parental strains. Our results confirm that the vegetative heterokaryotic stage is not a prerequisite for cleistothecium formation. Using this tool, we analysed sexual sporulation mutants for male or female sterility.     

Fungal surface remodelling visualized by atomic force microscopy

Most fungal growth is localized to the tips of hyphae, however, early stages of spore germination and the growth of certain morphological mutant strains exhibit non-polarized expansion. We used atomic force microscopy (AFM) to document changes in Aspergillus nidulans wall surfaces during non-polarized growth: spore germination, and growth in a strain containing the hypA1 temperature sensitive morphogenesis defect. We compared wall surface structures of both wild-type and mutant A. nidulans following growth at 28 ° and 42 °C, the latter being the restrictive temperature for hypA1. There was no appreciable difference in surface ultrastructure between wild-type and hypA1 spores, or hyphal walls grown at 28 °C. When dry mature A. nidulans conidia were wetted they lost their hydrophobin coat, indicating an intermediate stage between dormancy and swelling. The surface structure of hypA1 germlings grown at 42 °C was less organized than wild-type hyphae grown under the same conditions, and had a larger range of subunit sizes. AFM images of hyphal wall surface changes following a shift in growth temperature from restrictive (42 °C) to permissive (28 °C), showed a gradient of sizes for wall surface features similar to the trend observed for wild-type cells at branch points. Changes associated with the hyphal wall structure for A. nidulans hypA1 offer insight into the events associated with fungal germination, and wall remodelling.     

Reciprocal oxylipin-mediated cross-talk in the Aspergillus–seed pathosystem

In Aspergilli, mycotoxin production and sporulation are governed, in part, by endogenous oxylipins (oxygenated, polyunsaturated fatty acids and metabolites derived therefrom). In Aspergillus nidulans , oxylipins are synthesized by the dioxygenase enzymes PpoA, PpoB and PpoC. Structurally similar oxylipins are synthesized in seeds via the action of lipoxygenase (LOX) enzymes. Previous reports have shown that exogenous application of seed oxylipins to Aspergillus cultures alters sporulation and mycotoxin production. Herein, we explored whether a plant oxylipin biosynthetic gene ( ZmLOX3 ) could substitute functionally for A. nidulans ppo genes. We engineered ZmLOX3 into wild-type A. nidulans , and into a Δ ppoAC strain that was reduced in production of oxylipins, conidia and the mycotoxin sterigmatocystin. ZmLOX3 expression increased production of conidia and sterigmatocystin in both backgrounds. We additionally explored whether A. nidulans oxylipins affect seed LOX gene expression during Aspergillus colonization. We observed that peanut seed pnlox2–3 expression was decreased when infected by A. nidulans Δ ppo mutants compared with infection by wild type. This result provides genetic evidence that fungal oxylipins are involved in plant LOX gene expression changes, leading to possible alterations in the fungal/host interaction. This report provides the first genetic evidence for reciprocal oxylipin cross-talk in the Aspergillus –seed pathosystem.     

Sporogenesis in Streptomyces melanochromogenes

The mode of spore differentiation in a strain of Streptomyces melanochromogenes was followed by analysis of ultrathin sections of sporulating aerial hyphae at various stages of sporogenesis. A special accent was laid on the formation of the sporulation septum and its alterations in the course of spore delimitation and separation. Distinct differences in formation and substructure have been observed between the cross walls of vegetative hyphae and the sporulation septa.Cross walls of vegetative hyphae are formed in a way typical for Gram-positive bacteria by a centripetal annular ingrowth of cytoplasmic membrane, on which wall material immediately is deposited. The development of the sporulation septa is characterized by the accumulation of amorphous material in addition to the newly synthesized wall layer inside the invaginating cytoplasmic membrane. This amorphous septal material will later be decomposed presumably by two lytic systems which cause the separation of the spores. The central region of the finished sporulation septum is perforated by microplasmodesmata. Spores are released by a break down of the surface sheath. The complete spores are enveloped by a twolayered cell wall and the spiny surface sheath.     

Heterotrimeric G-proteins of a filamentous fungus regulate cell wall composition and susceptibility to a plant PR-5 protein

Membrane permeabilizing plant defensive proteins first encounter the fungal cell wall that can harbor specific components that facilitate or prevent access to the plasma membrane. However, signal transduction pathways controlling cell wall composition in filamentous fungi are largely unknown. We report here that the deposition of cell wall constituents that block the action of osmotin (PR-5), an antifungal plant defense protein, against Aspergillus nidulans requires the activity of a heterotrimeric G-protein mediated signaling pathway. The guanidine nucleotide GDPbetaS, that locks G-proteins in a GDP-bound inactive form, inhibits osmotin-induced conidial lysis. A dominant interfering mutation in FadA, the alpha-subunit of a heterotrimeric G-protein, confers resistance to osmotin. A deletion mutation in SfaD, the beta-subunit of a heterotrimeric G-protein also increases osmotin resistance. Aspergillus nidulans strains bearing these mutations also have increased tolerance to SDS, reduced cell wall porosity and increased chitin content in the cell wall.     

Elastic properties of the cell wall of Aspergillus nidulans studied with atomic force microscopy

Currently, little is known about the mechanical properties of filamentous fungal hyphae. To study this topic, atomic force microscopy (AFM) was used to measure cell wall mechanical properties of the model fungus Aspergillus nidulans. Wild type and a mutant strain (deltacsmA), lacking one of the chitin synthase genes, were grown in shake flasks. Hyphae were immobilized on polylysine-coated coverslips and AFM force--displacement curves were collected. When grown in complete medium, wild-type hyphae had a cell wall spring constant of 0.29 +/- 0.02 N/m. When wild-type and mutant hyphae were grown in the same medium with added KCl (0.6 M), hyphae were significantly less rigid with spring constants of 0.17 +/- 0.01 and 0.18 +/- 0.02 N/m, respectively. Electron microscopy was used to measure the cell wall thickness and hyphal radius. By use of finite element analysis (FEMLAB v 3.0, Burlington, MA) to simulate AFM indentation, the elastic modulus of wild-type hyphae grown in complete medium was determined to be 110 +/- 10 MPa. This decreased to 64 +/- 4 MPa for hyphae grown in 0.6 M KCl, implying growth medium osmotic conditions have significant effects on cell wall elasticity. Mutant hyphae grown in KCl-supplemented medium were found to have an elastic modulus of 67 +/- 6 MPa. These values are comparable with other microbial systems (e.g., yeast and bacteria). It was also found that under these growth conditions axial variation in elastic modulus along fungal hyphae was small. To determine the relationship between composition and mechanical properties, cell wall composition was measured by anion-exchange liquid chromatography and pulsed electrochemical detection. Results show similar composition between wild-type and mutant strains. Together, these data imply differences in mechanical properties may be dependent on varying molecular structure of hyphal cell walls as opposed to wall composition.     

The Aspergillus nidulans putative kinase, KfsA (kinase for septation), plays a role in septation and is required for efficient asexual spore formation

In Aspergillus nidulans nuclear division and cytokinesis are coupled processes during asexual sporulation. Metulae, phialides and conidia contain a single nucleus. Here we describe the role of a putative Saccharomyces cerevisiae Kin4-related kinase, KfsA (kinase for septation) in the control of septum formation in A. nidulans. The kfsA deletion caused an increase in the number of conidiophores with septa in their stalks from 20% in wild type to 60% in the mutant strain. Interestingly, 7% of metulae contained two nuclei and the corresponding phialides remained anucleate, suggesting septum formation before proper segregation of nuclei. This points to a checkpoint control of KfsA, which prevents septum formation before nuclear separation. KfsA localized to the cortex and septa in hyphae and in conidiophores but not to the spindle-pole bodies, as it was shown for Kin4 in yeast. KfsA appeared at septa after actin disappeared, suggesting an additional role of KfsA late during septum formation.     

The push-pull mechanism of bacteriophage Ø29 DNA injection

Regulators of G-protein signalling play a crucial role in controlling the degree of heterotrimeric G-protein signalling. In addition to the previously studied flbA, we have identified three genes (rgsA, rgsB and rgsC) encoding putative RGS proteins in the genome of Aspergillus nidulans. Characterization of the rgsA gene revealed that RgsA downregulates pigment production and conidial germination, but stimulates asexual sporulation (conidiation). Deletion of rgsA (DeltargsA) resulted in reduced colony size with increased aerial hyphae, elevated accumulation of brown pigments as well as enhanced tolerance of conidia and vegetative hyphae against oxidative and thermal stress. Moreover, DeltargsA resulted in conidial germination in the absence of a carbon source. Deletion of both flbA and rgsA resulted in an additive phenotype, suggesting that the G-protein pathways controlled by FlbA and RgsA are different. Morphological and metabolic alterations caused by DeltargsA were suppressed by deletion of ganB encoding a Galpha subunit, indicating that the primary role of RgsA is to control negatively GanB-mediated signalling. Overexpression of rgsA caused inappropriate conidiation in liquid submerged culture, supporting the idea that GanB signalling represses conidiation. Our findings define a second and specific RGS-Galpha pair in A. nidulans, which may govern upstream regulation of fungal cellular responses to environmental changes.     

A new sterigmatocystin-producing Emericella variant from agricultural desert soils

An unusual, sterigmatocystin-producing taxon with characteristics of both Emericella nidulans (anamorph Aspergillus nidulans) and Emericella rugulosa (anamorph Aspergillus rugulovalvus, formerly A. rugulosus) was isolated repeatedly during a mycofloral survey of desert cotton field soils where aflatoxin is a chronic problem. Members of this taxon had ascospores with smooth convex walls like E. nidulans but grew slowly like E. rugulosa; moreover, they were similar to an industrial echinocandin B-producing strain which had been classified as "Aspergillus nidulans var. roseus." These new desert isolates were compared with "A. nidulans var. roseus" and representative wild-type isolates of E. nidulans and E. rugulosa using traditional morphological characters, secondary metabolite profiles of mycelial extracts, and Southern blot analysis of genomic DNA. The desert isolates and "A. nidulans var. roseus shared morphological, physiological and molecular characters with E. rugulosa. These isolates constitute a new non-rugulose variant of E. rugulosa.