Mitochondrial DNA analysis of Exophiala jeanselmei and Exophiala dermatitidis

Mitochondrial DNA (mtDNA) analysis with Hae III, Hind. III and Msp I was performed in 45 Exophiala jeanselmei strains (30 Phialophora jeanselmei and 15 Phialophora gougerotii strains) and 31 Exophiala dermatitidis strains. The results were as follows, 1) P. jeanselmei and P. gougerotii are identical, 2) E. jeanselmei is classified into 18 types based on restriction profiles, 3) two strains of E. jeanselmei CBS 577.76 and CBS 578.76 are identified as E. dermatitidis, 4) E. dermatitidis has no intraspecific variation and is definitely distinct from E. jeanselmei, 5) E. jeanselmei is suggested to be a complex organism because of extensive mtDNA polymorphism.     

Application of flow cytometry to differentiating Exophiala dermatitidis E. moniliae and E. jeanselmei from each other

We applied a flow cytometry apparatus (FCM) to differenciating Exophiala dermatitidis, E. moniliae and E. jeanselmei from each other. The wavelength of the argon laser emitted from the FCM was 488 nm and the aperture of nozzle from which the stream of fluid containing single cells was blown out was 100 m. By irradiating the stream with laser by either the forward light scatter (FLS) or by the perpendicular light scattr (PLS), we were able to get two pieces of informations. Histograms displayed by the FLS indicate the cell size, while dot displays by the PLS reflect the cell structure. As a result, E. dermatitidis was clearly differenciated from either E. moniliae or E. jeanselmei by their histograms by FLS. In addition, dot displays by the PLS differenciated E. moniliae from E. jeanselmei.In conclusion, flow cytometry is available for differenciating E. dermatitidis, E. moniliae and E. jeanselmei from each other.     

Nutritional physiology of type isolates of currently accepted species ofExophiala andPhaeococcomyces

Nutritional physiological and tolerance tests were performed for all type strains of species currently classified in the black yeast generaExophiala andPhaeococcomyces, including some additional type strains of taxa recently reidentified asExophiala species. Most describedExophiala species can be distinguished by physiological characters.Exophiala jeanselmei with its varieties, andE. castellanii should all be retained as separate taxa. The pairs of strainsMycotorula schawii/Exophiala dermatitidis, Hormodendrum negronii/Exophiala jeanselmei var.lecaniicorni andSporotrichum gougerotii/Torula bergeri were found to be conspecific. Phenetic analyses of physiological data support the identity ofPhaeococcomyces exophialae as a yeast-like synanamorph ofExophiala spinifera. The taxonomic positions of the generaNadsoniella, Phaeoannellomyces andWangiella are discussed. The generaExophiala andPhaeococcomyces are unrelated.     

Phylogenetic analysis of ten black yeast species using nuclear small subunit rRNA gene sequences

The nuclear small subunit rRNA genes of authentic strains of the black yeastsExophiala dermatitidis, Wangiella dermatitidis, Sarcinomyces phaeomuriformis, Capronia mansonii, Nadsoniella nigra var.hesuelica, Phaeoannellomyces elegans, Phaeococcomyces exophialae, Exophiala jeanselmei var.jeanselmei andE. castellanii were amplified by PCR and directly sequenced. A putative secondary structure of the nuclear small subunit rRNA ofExophiala dermatitidis was predicted from the sequence data. Alignment with corresponding sequences fromNeurospora crassa andAureobasidium pullulans was performed and a phylogenetic tree was constructed using the neighbor-joining method. The obtained topology of the tree was confirmed by bootstrap analysis. Based upon this analysis all fungi studied formed a well-supported monophyletic group clustering as a sister group to one group of the Plectomycetes (Trichocomaceae and Onygenales). The analysis confirmed the close relationship postulated betweenExophiala dermatitidis, Wangiella dermatitidis andSarcinomyces phaeomuriformis. This monophyletic clade also contains the teleomorph speciesCapronia mansonii thus confirming the concept of a teleomorph connection of the genusExophiala to a member of the Herpotrichiellaceae. However,Exophiala castellanii did not belong to this clade. Therefore, this species is not the anamorph ofCapronia mansonii as it was postulated.     

Further studies on the phylogenesis of the genus Exophiala and Hortaea

The conidial ontogenesis of the pathogenic black yeasts is studied at an ultrastructural level and their phylogenesis is discussed. Five cultures of Exophiala dermatitidis, four of E. jeanselmei, one of E. moniliae, one of E. spinifera and six of H. werneckii were observed using a scanning electron microscope.The conidial ontogenesis of the Exophiala species is not pleomorphic but only annellidic. There are definite differences in morphology of annellated tips among the Exophiala species. The ontogenesis of Hortaea werneckii consists of a combination of sympodial and annellidic conidiogenesis. Its sympodial anamorph is unique and the annellidic anamorph is considered to be a homology of the sympodial one.     

Mitochondrial DNA analysis of Exophiala jeanselmei var. lecanii-corni and Exophiala castellanii

A Japanese clinical isolate (KU-A-0094) which was identified by de Hoog et al. as Exophiala jeanselmei var. lecanii-corni with difficulty, was compared with 5 strains including the type cultures of E. jeanselmei var. lecanii-corni, var. jeanselmei and E. castellanii using RFLP (restriction fragment length polymorphism) patterns of mtDNA (mitochondrial DNA). RFLP patterns of KUA-0094 were identical with those of E. jeanselmei var. lecanii-corni and different from those of E. castellanii with restriction enzymes of HaeIII, MspI and hindIII. Therefore, de Hoog et al.'s identification of KU-A-0094 was confirmed. Additionally, mtDNA-RFLP patterns of E. jeanselmei var. lecanii-corni and E. jeanselmei var. jeanselmei were also different from each other. Consequently E. jeanselmei var. lecanii-corni seem to be a species in its own right rather than a variant of E. jeanselmei. This revised version was published online in June 2006 with corrections to the Cover Date.     

Detection and identification of opportunistic Exophiala species using the rolling circle amplification of ribosomal internal transcribed spacers

Deep infections by melanized fungi deserve special attention because of a potentially fatal, cerebral or disseminated course of disease in otherwise healthy patients. Timely diagnostics are a major problem with these infections. Rolling circle amplification (RCA) is a sensitive, specific and reproducible isothermal DNA amplification technique for rapid molecular identification of microorganisms. RCA-based diagnostics are characterized by good reproducibility, with few amplification errors compared to PCR. The method is applied here to species of Exophiala known to cause systemic infections in humans. The ITS rDNA region of five Exophiala species (E. dermatitidis, E. oligosperma, E. spinifera, E. xenobiotica, and E. jeanselmei) was sequenced and aligned in view of designing specific padlock probes to be used for the detection of single nucleotide polymorphisms (SNPs) of the Exophiala species concerned. The assay proved to successfully amplify DNA of the target fungi at the level of species; while no cross-reactivity was observed. Amplification products were visualized on 1% agarose gels to verify the specificity of probe-template binding. Amounts of reagents were minimized to avoid the generation of false positive results. The sensitivity of RCA may help to improve early diagnostics of these difficult to diagnose infections.     

Molecular diversity of oligotrophic and neurotropic members of the black yeast genus Exophiala,with accent on E. dermatitidis

Analysis of ITS rDNA of the black yeast Exophiala dermatitidis revealed a close phylogenetic relationship to the meristematic fungus Sarcinomyces phaeomuriformis. As most strains ofS. phaeomuriformis have a yeast-like phenotype corresponding to the anamorph genus Exophiala, a new combination in Exophiala is proposed. On the basis of ITS sequence, M-13 fingerprint and SSU intron data, two main entities could be distinguished within E. dermatitidis. One of these (B) contained prevalently strains from environmental sources, while the other (A) mainly comprised strains from clinical sources. This may be due to a difference in virulence. All strains from severe brain and disseminated infections in East Asia clustered in group A. However, strains of group A caused a relatively mild fungemia in patients outside East Asia. This revised version was published online in June 2006 with corrections to the Cover Date.     

Mitochondrial DNA analysis ofExophiala moniliae

Mitochondrial DNA(mtDNA) analysis with restriction enzymes, Hae III, Hind III and Msp I was performed in 17Exophiala moniliae strains. The results were as follows: (1)E. moniliae could be classified into 10 types based on restriction patterns, (2)E. moniliae is suggested to be a complex organism because of extensive mtDNA polymorphism among strains likeE. jeanselmei and (3) two types ofE. moniliae are identical with two types ofE. jeanselmei. These results suggest thatE. moniliae is not genetically defined fromE. jeanselmei and the taxonomical status ofE. moniliae requires reevaluation     

Dematiaceous fungal pathogens isolated from nature

This study was conducted to demonstrate the presence of pathogenic dematiaceous fungi in nature. Using hamster and mouse inoculation techniques, 43 isolates of dematiaceous fungi were recovered from 39 samples of woody plant material and soil from the Virginia environment. Thirteen species were identified and included 4 Phialophora spp., 3 Cladosporium spp., 2 Exophiala spp., Sporothrix sp., Wangiella dermatitidis, Bispora betulina, and Scytalidium lignicola. Evidence is presented for the first isolations of C. trichoides from nature in the United States; these isolates proved to be pathogenic for mice in which they produced disease and death in a course similar to that seen in man. Natural isolates of Phialophora verrucosa, Phialophora repens, Exophiala jeanselmei, and Wangiella dermatitidis were identical to those species isolated from man using the following criteria: morphology, 12% gelatin reaction, and survival in laboratory animals.     

Invasion of a soft contact lens by Exophiala jeanselmei

The invasion of a soft contact lens by Exophiala jeanselmei is documented. All species in this genus are pathogenic. In humans E. jeanselmei is a recognized cause of mycetoma, phaeohyphomycosis and keratomycosis. This fungus has not been previously listed among lens invaders.     

Binding and precipitating activities ofErythrina lectins with complex type carbohydrates and synthetic cluster glycosides. A comparative study of the lectins fromE. corallodendron,E. cristagalli,E. flabelliformis,andE. indica

Erythrina lectins possess similar structural and carbohydrate binding properties. Recently, tri- and tetra-antennary complex type carbohydrates with non-reducing terminal galactose residues have been shown to be precipitated as tri- and tetravalent ligands, respectively, with certainErythrina lectins [Bhattacharyya L, Haraldsson M, Brewer CF (1988) Biochemistry 271034-41]. The present work describes a comparative study of the binding and precipitating activities of fourErythrina lectins,viz. E. corallodendron, E. cristagalli, E. flabelliformis, andE. indica, with multi-antennary complex type carbohydrates and synthetic cluster glycosides. The results show that though their binding affinities are very similar, theErythrina lectins show large differences in their precipitating activities with the carbohydrates. The results also indicate significant dependence of the precipitating activities of the lectins on the core structure of the carbohydrates. These findings provide a new dimension to the structure-activity relationship of the lectins and their interactions with asparagine-linked carbohydrates.Abbreviations EAL, ECorL, ECL, EFL, and EIL represent the lectins from the seeds ofErythrina arborescens, - E. corallodendron, E. cristagalli, E. flabelliformis, andE. indica respectively - AFOS thetri-antennary complex type oligosaccharide from asialofetuin - AFGP the tri-antennary glycopeptide from asialofetuin - MeGal methyl -d-galactopyranoside Unless stated otherwise all sugars are in thed-configuration.     

Hydroxylation of steroids by Fusarium oxysporum, Exophiala jeanselmei and Ceratocystis paradoxa

The potential of Fusarium oxysporum var. cubense UAMH 9013 to perform steroid biotransformations was reinvestigated using single phase and pulse feed conditions. The following natural steroids served as substrates: dehydroepiandrosterone (1), pregnenolone (2), testosterone (3), progesterone (4), cortisone (5), prednisone (6), estrone (7) and sarsasapogenin (8). The results showed the possible presence of C-7 and C-15 hydroxylase enzymes. This hypothesis was explored using three synthetic androstanes: androstane-3,17-dione (9), androsta-4,6-diene-3,17-dione (10) and 3α,5α-cycloandrost-6-en-17-one (11). These fermentations of non-natural steroids showed that C-7 hydroxylation was as a result of that position being allylic. The evidence also pointed towards the presence of a C-15 hydroxylase enzyme.The eleven steroids were also fed to Exophialajeanselmei var. lecanii-corni UAMH 8783. The results showed that the fungus appears to have very active 5α and 14α-hydroxylase enzymes, and is also capable of carrying out allylic oxidations.Ceratocystis paradoxa UAMH 8784 was grown in the presence of the above-mentioned steroids. The results showed that monooxygenases which effect allylic hydroxylation and Baeyer–Villiger rearrangement were active. However, redox reactions predominated.     

Degradation of monohydroxylated benzoates by strain KUFI-6N of the yeast-like fungus Exophiala jeanselmei

Strain KUFI-6N of Exophiala jeanselmei, a cyclohexanol-utilizing yeast-like fungus, was found to grow on 3 isomers of hydroxybenzoate that functioned as the sole carbon sources. Distinct and highly specific hydroxylases converted p- and m-hydroxybenzoate to protocatechuate and o-hydroxybenzoate to catechol.     

Exophiala xenobiotica sp. nov., an opportunistic black yeast inhabiting environments rich in hydrocarbons

A new black yeast species, Exophiala xenobiotica, is described, a segregant of the Exophiala jeanselmei complex. It is morphologically very similar to E. jeanselmei, though with less melanized conidiogenous cells, but deviates unambiguously on the basis of molecular phylogeny. The species is a relatively common agent of cutaneous infections in humans, whereas E. jeanselmei is associated with subcutaneous infections. Environmental strains of E. xenobiotica are frequently found in habitats rich in monoaromatic hydrocarbons and alkanes.     

The ‘A’ genome donor of Eleusine coracana (L.) Gaertn. (Gramineae)

Summary In an attempt to discover A and B genome donor(s) to finger millet, Eleusine coracana, or its progenitor species, E. africana (both allotetraploid 2n=4x=36), five diploid species, E. Indica, E. Floccifolia, E. multiflora, E. tristachya and E. intermedia, were crossed to finger millet and its progenitor taxon. Crosses were successful only with E. coracana. Three combinations of triploid hybrids E. coracana x E. indica, E. coracana x E. floccifolia, and E. coracana x E. multiflora were obtained and analysed. Meiotic behaviour was perfectly normal in parental species. The regular number of 18 bivalents in E. coracana, 9 bivalents in E. indica, E. intermedia, E. tristachya and E. floccifolia and 8 bivalents in E. multiflora were invariably noticed. In E. coracana x E. indica hybrids a mean chromosome pairing of 8.84_I+8.80_II+0.03_III+0.10_IV per cell was found. About 86.5% of the cells showed the typical 9_I+9_II configuration, suggesting that E. indica (AA) is one of the diploid genome donors to cultivated species E. coracana. A mean chromosome pairing of 11.08_I+7.63_II+0.16_III+0.04_IV per cell was found in E. coracana x E. floccifolia hybrids. Two to ten bivalents and varying numbers of univalents were seen in 55% of the cells. About 45% of the cells showed the 9_I+9_II configuration. Various evidence suggests that perennial E. floccifolia is a primitive member of the A genome group of Eleusine species, and it may not be a genome donor to E. coracana. In E. coracana x E. multiflora hybrids (2n=26) mean chromosome pairing of 21.45_I+1.97_II+0.13_III+0.04_IV per cell was found. About 91% of the cells were observed to have 20–26 univalents. Only a small percentage of the cells contained bivalents or multivalents. This pairing behaviour indicates that E. multiflora lacks genomic homology with the A or B genome of E. coracana. Genomically E. multiflora is a distinct species and a genomic symbol of C is assigned to it. Identification of the B genome donor species to cultivated millet. E. coracana remains elusive.     

The genetics of blackleg [Leptosphaeria maculans (Desm.) Ces. et De Not.] resistance in rapeseed (Brassica napus L.)

The genetic control of adult-plant blackleg [Leptosphaeria maculans (Desm.) Ces. et De Not.] resistance in rapeseed (Brassica napus L.) was studied in the F₂ and first-backcross populations of the cross Maluka (blackleg-resistant) x Niklas (highly susceptible). A L. maculans isolate possessing high levels of host specificity (MB2) was used in all inoculations. Resistance/susceptibility was evaluated using three separate measures of crown-canker size, i.e. the percentage of crown girdled (%G), external lesion length (E) and internal lesion area (%II). Disease severity scores for the F₂ and first-backcross populations based on E and %II gave discontinuous distributions, indicating major-gene control for these measures of resistance; but those for %G were continuous, indicating quantitative genetic control for this measure. Chi-square tests performed on the (poorly-defined) resistance classes, based on E, in the F₂ and first-backcross populations indicated the likelihood for resistance being governed by a single, incompletely dominant major gene. Although the distributions of the F₂ and first-backcross populations, based on%II, were clearly discontinuous, the observed segregation ratios for resistance and susceptibility did not fit any of the numerous Mendelian ratios which were considered. Differences in inheritance of resistance according to the assessment method and blackleg isolate used, were discussed.     

Microbial transformation of ginsenoside Rb1 by Rhizopus stolonifer and Curvularia lunata

Of 49 microbial strains screened for their capabilities to transform ginsenoside Rb₁, Rhizopus stolonifer and Curvularia lunata produced four key metabolites: 3-O-[-d-glucopyranosyl-(1,2)--d-glucopyranosyl]- 20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ene (1), 3-O-[-d-glucopyranosyl-(1,2)--d- glucopyranosyl]-20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ol (2), 3-O-[-d-gluco- pyranosyl-(1,2)--d-glucopyranosyl]-3, 12, 20(S)-trihydroxydammar-24-ene (3), and 3-O--d-glucopyranosyl-3, 12, 20(S)-trihydroxydammar-24-ene (4), identified by TOF-MS, ¹H- and ¹³C-NMR spectral data. Metabolites 1, 3 and 4 were from the incubation with R. stolonifer, and 1 and 2 from the incubation with C. lunata. Compound 2 was identified as a new compound.