Aspergillus incahuasiensis sp. nov., isolated from soil in the semi-arid region of northern Chile.

During a study of the fungi from a semi-arid region of northern Chile, a novel species of Aspergillus was encountered in the soil from an area where pepper trees (Schinusmolle) were growing. Marker genes were sequenced to identify these isolates. The ß-tubulin, calmodulin and DNA-dependent RNA polymerase loci all indicated that this was a novel species in Aspergillus section Nidulantes and in the Aspergillus multicolorclade. The new species was studied morphologically and differences between it and the other members of the A. multicolor clade are described. We provide a name and description for these isolates as Aspergillus incahuasiensis sp. nov.

Aspergillus olivimuriae sp. nov., a halotolerant species isolated from olive brine.

A facultative halo-tolerant Aspergillus strain was isolated from olive brine waste, the effluent from the debittering process of table olives. Phenotypic and molecular characteristics showed clearly that the isolate represents a novel species. Based on the source of isolation, the new species has been named Aspergillus olivimuriae. It was found tolerant to high concentrations of NaCl (15 %) or sucrose (60 %) and it exhibits substantial growth under these conditions. Although the new species grew profusely at 37 °C, no growth was observed at 40 °C, conidia en masse were avellaneous on all media. The description of the new species Aspergillus olivimuriae brings the total species of Aspergillus sect. Flavipedes to 15. The type strain of A. olivimuriae sp. nov. is NRRL 66783 (CCF 6208), its whole genome has been deposited as PRJNA498048.

Aspergillus asper sp. nov. and Aspergillus collinsii sp. nov., from Aspergillus section Usti.

In sampling fungi from the built environment, two isolates that could not confidently be placed in described species were encountered. Phenotypic analysis suggested that they belonged in Aspergillus sect. Usti. In order to verify the sectional placement and to assure that they were undescribed rather than phenotypically aberrant isolates, DNA was isolated and sequenced at the beta-tubulin, calmodulin, internal transcribed spacer and RNA polymerase II loci and sequences compared with those from other species in the genus Aspergillus. At each locus, each new isolate was distant from existing species. Phylogenetic trees calculated from these data and GenBank data for species of the section Usti excluded the placement of these isolates in existing species, with statistical support. Because they were excluded from existing taxa, the distinct species Aspergillus asper (type strain NRRL 35910T) and Aspergillus collinsii (type strain NRRL 66196T) in sect. Usti are proposed to accommodate these strains.

Phylogenetic classification of Aureobasidium pullulans strains for production of feruloyl esterase.

OBJECTIVE: The objective was to phylogenetically classify diverse strains of Aureobasidium pullulans and determine their production of feruloyl esterase. RESULTS: Seventeen strains from the A. pullulans literature were phylogenetically classified. Phenotypic traits of color variation and endo-ß-1,4-xylanase overproduction were associated with phylogenetic clade 10 and particularly clade 8. Literature strains used for pullulan production all belonged to clade 7. These strains and 36 previously classified strains were tested for feruloyl esterase production, which was found to be associated with phylogenetic clades 4, 11, and particularly clade 8. Clade 8 strains NRRL 58552 and NRRL 62041 produced the highest levels of feruloyl esterase among strains tested. CONCLUSIONS: Production of both xylanase and feruloyl esterase are associated with A. pullulans strains in phylogenetic clade 8, which is thus a promising source of enzymes with potential biotechnological applications.

Revision of Aspergillus section Flavipedes: seven new species and proposal of section Jani sect. nov.

Aspergillus section Flavipedes contains species found worldwide in soils and rhizospheres, indoor and cave environments, as endophytes, food contaminants and occasionally as human pathogens. They produce many extensively studied bioactive secondary metabolites and biotechnologically relevant enzymes. The taxa were revised based on phylogenetic analysis of sequences from four loci (ß-tubulin, calmodulin, RPB2, ITS rDNA), two PCR fingerprinting methods, micro- and macromorphology and physiology. Section Flavipedes includes three known and seven new species: A. ardalensis, A. frequens, A. luppii, A. mangaliensis, A. movilensis, A. polyporicola and A. spelaeus. The name A. neoflavipes was proposed for Fennellia flavipes a distinct species from its supposed asexual state A. flavipes. Aspergillus iizukae, A. frequens and A. mangaliensis are the most common and widely distributed species, whereas A. flavipes s. str. is rare. A dichotomous key based on the combination of morphology and physiology is provided for all recognized species. Aspergillus section Jani is established to contain A. janus and A. brevijanus, species previously classified as members of sect. Versicolores, Terrei or Flavipedes. This new section is strongly supported by phylogenetic data and morphology. Section Jani species produce three types of conidiophores and conidia, and colonies have green and white sectors making them distinctive. Accessory conidia found in pathogenic A. terreus were found in all members of sects. Flavipedes and Jani. Our data indicated that A. frequens is a clinically relevant and produces accessory conidia during infection.

Genetic relatedness versus biological compatibility between Aspergillus fumigatus and related species.

Aspergillus section Fumigati contains 12 clinically relevant species. Among these Aspergillus species, A. fumigatus is the most frequent agent of invasive aspergillosis, followed by A. lentulus and A. viridinutans. Genealogical concordance and mating experiments were performed to examine the relationship between phylogenetic distance and mating success in these three heterothallic species. Analyses of 19 isolates from section Fumigati revealed the presence of three previously unrecognized species within the broadly circumscribed species A. viridinutans. A single mating type was found in the new species Aspergillus pseudofelis and Aspergillus pseudoviridinutans, but in Aspergillus parafelis, both mating types were present. Reciprocal interspecific pairings of all species in the study showed that the only successful crosses occurred with the MAT1-2 isolates of both A. parafelis and A. pseudofelis. The MAT1-2 isolate of A. parafelis was fertile when paired with the MAT1-1 isolates of A. fumigatus, A. viridinutans, A. felis, A. pseudoviridinutans, and A. wyomingensis but was not fertile with the MAT1-1 isolate of A. lentulus. The MAT1-2 isolates of A. pseudofelis were fertile when paired with the MAT1-1 isolate of A. felis but not with any of the other species. The general infertility in the interspecies crossings suggests that genetically unrelated species are also biologically incompatible, with the MAT1-2 isolates of A. parafelis and A. pseudofelis being the exception. Our findings underscore the importance of genealogical concordance analysis for species circumscription, as well as for accurate species identification, since misidentification of morphologically similar pathogens with differences in innate drug resistance may be of grave consequences for disease management.

Clinical, morphological, and molecular characterization of Penicillium canis sp. nov., isolated from a dog with osteomyelitis.

Infections caused by Penicillium species are rare in dogs, and the prognosis in these cases is poor. An unknown species of Penicillium was isolated from a bone lesion in a young dog with osteomyelitis of the right ilium. Extensive diagnostic evaluation did not reveal evidence of dissemination. Resolution of lameness and clinical stability of disease were achieved with intravenous phospholipid-complexed amphotericin B initially, followed by long-term combination therapy with terbinafine and ketoconazole. A detailed morphological and molecular characterization of the mold was undertaken. Sequence analysis of the internal transcribed spacer revealed the isolate to be closely related to Penicillium menonorum and Penicillium pimiteouiense. Additional sequence analysis of ß-tubulin, calmodulin, minichromosome maintenance factor, DNA-dependent RNA polymerase, and pre-rRNA processing protein revealed the isolate to be a novel species; the name Penicillium canis sp. nov. is proposed. Morphologically, smooth, ovoid conidia, a greenish gray colony color, slow growth on all media, and a failure to form ascomata distinguish this species from closely related Penicillium species.

Aspergillus pragensis sp. nov. discovered during molecular reidentification of clinical isolates belonging to Aspergillus section Candidi.

The identity of nine clinical isolates recovered from Czech patients and presumptively identified as Aspergillus sp. section Candidi based on colony morphology was revised using sequences of ß-tubulin, calmodulin gene sequence, and internal transcribed spacer rDNA. Six isolates were from suspected and proven onychomycosis, one from otitis externa, and two associated with probable invasive aspergillosis. The results showed that one Aspergillus candidus isolate was the cause of otitis externa, and both isolates obtained from sputa of patients with probable invasive aspergillosis were reidentified as A. carneus (sect. Terrei) and A. flavus (sect. Flavi). Three isolates from nail scrapings were identified as A. tritici, a verified agent of nondermatophyte onychomycosis. One isolate from toenail was determined to be A. candidus and the two isolates belonged to a hitherto undescribed species, Aspergillus pragensis sp. nov. This species is well supported by phylogenetic analysis based on ß-tubulin and calmodulin gene and is distinguishable from other members of sect. Candidi by red-brown reverse on malt extract agar, slow growth on Czapek-Dox agar and inability to grow at 37°C. A secondary metabolite analysis was also provided with comparison of metabolite spectrum to other species. Section Candidi now encompasses five species for which a dichotomous key based on colony characteristics is provided. All clinical isolates were tested for susceptibilities to selected antifungal agents using the Etest and disc diffusion method. Overall sect. Candidi members are highly susceptible to common antifungals.

Aureobasidium thailandense sp. nov. isolated from leaves and wooden surfaces.

Aureobasidium thailandense sp. nov. is described from cultures of material collected on leaves and wooden surfaces in Thailand and the type isolate is NRRL 58539(T). Phylogenetically it is distinct from other species of the genus Aureobasidium. Phenotypically it is distinguished by its cardinal growth temperatures, salt tolerance and production of reddish brown hyphal pigmentation in PDA cultures, but micro-morphologically it is not clearly distinguishable from Aureobasidium pullulans. Unlike A. pullulans, A. thailandense sp. nov. produces a non-pullulan extracellular polysaccharide whose characteristics are unknown. The two known isolates of A. thailandense sp. nov. possess an approx. 500 bp type I intron in the 18S rRNA gene that is present in ITS amplifications using primers ITS4 and ITS5. A. pullulans isolates uniformly lack this intron.

Aspergillus waksmanii sp. nov. and Aspergillus marvanovae sp. nov., two closely related species in section Fumigati.

Two new and phylogenetically closely related species in Aspergillus section Fumigati are described and illustrated. Homothallic Aspergillus waksmanii sp. nov. was isolated from New Jersey soil (USA) and is represented by the ex-type isolate NRRL 179(T) ( = CCF 4266(T) = Thom 4138.HS2(T) = IBT 31900(T)). Aspergillus marvanovae sp. nov. was isolated from water with high boracic acid anions content in Dukovany nuclear power station (Czech Republic). The sexual stage of this species is unknown, but the MAT1-1 locus was successfully amplified suggesting that the species is probably heterothallic and teleomorphic but is represented by only the ex-type isolate CCM 8003(T) ( = CCF 4037(T) = NRRL 62486(T) = IBT 31279(T) = IFM 60873(T)). Both species can be distinguished from all previously described species in section Fumigati based on morphology, maximum growth temperature, sequence data from five unlinked loci and unique secondary metabolites profiles.

Taxonomic revision of Eurotium and transfer of species to Aspergillus.

Aspergillus section Aspergillus contains economically important, xerophilic fungi that are widely distributed in nature and the human environment and are known for their ability to grow on substrates with low water activity. The taxa were revised based on sequence data from four loci, PCR fingerprinting, micro- and macromorphology, and physiology. The number of taxa was reduced to 17 species, all of which can be distinguished with sequence data from either the caM or RPB2 locus. The original description of A. proliferans was supplemented by a description of its teleomorph. This species seems to be relatively common and often has been confused with A. glaucus. In addition, green sporulating isolates of A. niveoglaucus isolated from food and several other substrates are indistinguishable in phenotype from A. glaucus. A dichotomous key based on ascospore size and ornamentation and the ability to grow at specific combinations of temperature and water activity is provided for identification of species. In response to recent changes in the botanical code, we transferred the Eurotium species to Aspergillus and selected one name for each species.

Sexual reproduction in Aspergillus tubingensis from section Nigri.

A sclerotium-forming member of Aspergillus section Nigri was sampled from a population in a single field in North Carolina, USA, and identified as A. tubingensis based on genealogical concordance analysis. Aspergillus tubingensis was shown to be heterothallic, with individual strains containing either a MAT1-1 or MAT1-2 mating-type gene. Strains of opposite mating type were crossed on mixed cereal agar and incubated for 5-6 months. Stromata typically formed 1-2 indehiscent ascocarps containing asci and ascospores within the pseudo-parenchymatous matrix in a manner similar to the Petromyces sexual stage from section Flavi, which is closely related to section Nigri. Ascospores of A. tubingensis differed from those of section Flavi species in the reticulate ornamentation of ascospores and the presence of two crests that form an equatorial furrow. Sexual reproduction in A. tubingensis may be useful for enhancing enzyme and organic acid production through recombination-mediated genetic engineering of industrial strains.

Aspergillus tanneri sp. nov., a new pathogen that causes invasive disease refractory to antifungal therapy.

The most common cause of invasive aspergillosis (IA) in patients with chronic granulomatous disease (CGD) is Aspergillus fumigatus followed by A. nidulans; other aspergilli rarely cause the disease. Here we review two clinical cases of fatal IA in CGD patients and describe a new etiologic agent of IA refractory to antifungal therapy. Unlike typical IA caused by A. fumigatus, the disease caused by the new species was chronic and spread from the lung to multiple adjacent organs. Mycological characteristics and the phylogenetic relationship with other aspergilli based on the sequence analysis of Mcm7, RPB2, and Tsr1 indicated that the new species, which we named as A. tanneri, belongs to Aspergillus section Circumdati. The species has a higher amphotericin B, voriconazole, and itraconazole MIC and causes more chronic infection in CGD mice than A. fumigatus. This is the first report documenting IA in CGD patients caused by a species belonging to the Aspergillus section Circumdati that is inherently resistant to azoles and amphotericin B. Unlike the results seen with many members of Aspergillus section Circumdati, ochratoxin was not detected in filtrates of cultures grown in various media. Our phenotypic and genetic characterization of the new species and the case reports will assist future diagnosis of infection caused by A. tanneri and lead to more appropriate patient management.

Aspergillus and Penicillium identification using DNA sequences: barcode or MLST?

Current methods in DNA technology can detect single nucleotide polymorphisms with measurable accuracy using several different approaches appropriate for different uses. If there are even single nucleotide differences that are invariant markers of the species, we can accomplish identification through rapid DNA-based tests. The question of whether we can reliably detect and identify species of Aspergillus and Penicillium turns mainly upon the completeness of our alpha taxonomy, our species concepts, and how well the available DNA data coincide with the taxonomic diversity in the family Trichocomaceae. No single gene is yet known that is invariant within species and variable between species as would be optimal for the barcode approach. Data are published that would make an MLST approach to isolate identification possible in the most well-studied clades of Aspergillus and Penicillium.

Poly(ß-L-malic acid) production by diverse phylogenetic clades of Aureobasidium pullulans.

Poly(ß-L-malic acid) (PMA) is a natural biopolyester that has pharmaceutical applications and other potential uses. In this study, we examined PMA production by 56 strains of the fungus Aureobasidium pullulans representing genetically diverse phylogenetic clades. Thirty-six strains were isolated from various locations in Iceland and Thailand. All strains from Iceland belonged to a newly recognized clade 13, while strains from Thailand were distributed among 8 other clades, including a novel clade 14. Thirty of these isolates, along with 26 previously described strains, were examined for PMA production in medium containing 5% glucose. Most strains produced at least 4 g PMA/L, and several strains in clades 9, 11, and 13 made 9-11 g PMA/L. Strains also produced both pullulan and heavy oil, but PMA isolated by differential precipitation in ethanol exhibited up to 72% purity with no more than 12% contamination by pullulan. The molecular weight of PMA from A. pullulans ranged from 5.1 to 7.9 kDa. Results indicate that certain genetic groups of A. pullulans are promising for the production of PMA.

Three new species of Aspergillus section Flavi isolated from almonds and maize in Portugal.

Three new aflatoxin-producing species belonging to Aspergillus section Flavi are described. They are Aspergillus mottae, A. sergii and A. transmontanensis. These species were isolated from Portuguese almonds and maize. An investigation examined morphology, extrolite production and DNA sequence data to characterize these isolates and describe the new species. Phylogenetic analysis showed that A. transmontanensis and A. sergii form a clade with A. parasiticus whereas A. mottae shares a most recent common ancestor with the combined A. flavus and A. parasiticus clade.

Applications of Machine Learning to Improve the Clinical Viability of Compton Camera Based in vivo Range Verification in Proton Radiotherapy.

We studied the application of a deep, fully connected Neural Network (NN) to process prompt gamma (PG) data measured by a Compton camera (CC) during the delivery of clinical proton radiotherapy beams. The network identifies 1) recorded "bad" PG events arising from background noise during the measurement, and 2) the correct ordering of PG interactions in the CC to help improve the fidelity of "good" data used for image reconstruction. PG emission from a tissue-equivalent target during irradiation with a 150 MeV proton beam delivered at clinical dose rates was measured with a prototype CC. Images were reconstructed from both the raw measured data and the measured data that was further processed with a neural network (NN) trained to identify "good" and "bad" PG events and predict the ordering of individual interactions within the good PG events. We determine if NN processing of the CC data could improve the reconstructed PG images to a level in which they could provide clinically useful information about the in vivo range and range shifts of the proton beams delivered at full clinical dose rates. Results showed that a deep, fully connected NN improved the achievable contrast to noise ratio (CNR) in our images by more than a factor of 8x. This allowed the path, range, and lateral width of the clinical proton beam within a tissue equivalent target to easily be identified from the PG images, even at the highest dose rates of a 150 MeV proton beam used for clinical treatments. On average, shifts in the beam range as small as 3 mm could be identified. However, when limited by the amount of PG data measured with our prototype CC during the delivery of a single proton pencil beam (~1 × 109 protons), the uncertainty in the reconstructed PG images limited the identification of range shift to ~5 mm. Substantial improvements in CC images were obtained during clinical beam delivery through NN pre-processing of the measured PG data. We believe this shows the potential of NNs to help improve and push CC-based PG imaging toward eventual clinical application for proton RT treatment delivery verification.

Measuring prompt gamma-ray emissions from elements found in tissue during passive-beam proton therapy.

Prompt gamma detection during proton radiotherapy for range verification purposes will need to operate in both active and passive treatment beam environments. This paper describes prompt gamma measurements using a high resolution 2″ × 2″ LaBr3detector for a 200 MeV clinical passive-scatter proton beam. These measurements examine the most likely discrete prompt gamma rays emitted from tissue by detecting gammas produced in water, Perspex, carbon and liquid-nitrogen targets. Measurements were carried out at several positions around the depth corresponding to the location of the Bragg peak for water and Perspex targets in order to investigate prompt gamma emission as a function of depth along the beam path. This work also focused on validating the Geant4 Monte Carlo model of the passive-scatter proton beam line and LaBr3detector by making a direct comparison between the simulated and experimental results. The initial prompt gamma measurements were overwhelmed by the high amount of scattered radiation when measuring at isocenter, shifting the target further downstream from the final collimator significantly reduced the background radiation. Prompt gamma peaks were then clearly identified for the water, Perspex and graphite targets. The developed Geant4 Monte Carlo model was able to replicate the measured prompt gamma ray energy spectra, including production for important photopeaks to within 10%, except for the 4.44 MeV peak from the water target, which had more than a 50% overestimation of the number of produced prompt gamma rays. The prompt gamma measurements at various depths correlated well with the proton dose deposition; the 4.44 and 6.13 MeV photopeak profiles peaked within 1 cm of the Bragg peak and the R50%value for the 3-7 MeV energy range predicted the proton range within 8 mm.

Aspergillus alabamensis, a new clinically relevant species in the section Terrei.

Phylogenetic analyses of sequences generated from portions of three genes coding for the proteins enolase (enoA), beta-tubulin (benA), and calmodulin (calM) of a large number of isolates within the section Terrei, genus Aspergillus, revealed the presence of a new cryptic species within this section, Aspergillus alabamensis. Most members of this new cryptic species were recovered as colonizing isolates from immunocompetent patient populations, had decreased in vitro susceptibilities to the antifungal drug amphotericin B, and were morphologically similar to but genetically distinct from Aspergillus terreus isolates.

Genus Hamigera, six new species and multilocus DNA sequence based phylogeny.

Genus Hamigera was erected for Talaro-myces species that make asci singly instead of in chains. Initially it contained two species, H. avellanea and H. striata. We describe six new species in the genus, H. fusca, H. inflata, H. insecticola, H. pallida, H. paravellanea and H. terricola. Merimbla ingelheimensis is a distinct anamorphic species in the Hamigera clade. None of our DNA sequence data (BT2, calmodulin, ITS, 1su rDNA, RPB2, Tsr1 and Mcm7) supported the placement of H. striata in the same clade as H. avellanea, thus we accepted Talaromyces striatus. In addition to Hamigera species we examined the phylogenetic disposition of Warcupiella spinulosa, Penicillium megasporum, Penicillium arenicola and Merimbla humicoloides. Despite nominal similarity of some of these species to Merimbla, none of these species are part of the Hamigera clade and M. humicoloides is placed in Penicillium to have a monophyletic genus Hamigera.