An ascomycete H4 variant with an unknown function.

Histone variants leading to altered nucleosome structure, dynamics and DNA accessibility occur frequently, albeit rarely for H4. We carried out a comprehensive in silico scrutiny of fungal genomes, which revealed the presence of a novel H4 variant (H4E) in the ascomycetes, throughout the Pezizomycotina, in basal species of the Taphrinomycotina and also in the Glomeromycota. The coding cognate genes show a specific intron/exon organization, different from H4 canonical genes. H4Es diverge from canonical H4s mainly in the N- and C-terminal extensions, showing marked differences in the distribution and number of Lys and Arg residues, which may result in novel post-translational modifications. In Aspergillus nidulans (Pezizomycotina, Eurotiomycetes) the H4E variant protein level is low in mycelia. However, the encoding gene is well expressed at 37°C under nitrogen starvation. H4E localizes to the nucleus and interacts with H3, but its absence or overexpression does not result in any detectable phenotype. Deletion of only one of the of the two canonical H4 genes results in a strikingly impaired growth phenotype, which indicates that H4E cannot replace this canonical histone. Thus, an H4 variant is present throughout a whole subphylum of the ascomycetes, but with hitherto no experimentally detectable function.

Vertical and horizontal gene transfer shaped plant colonization and biomass degradation in the fungal genus Armillaria.

The fungal genus Armillaria contains necrotrophic pathogens and some of the largest terrestrial organisms that cause tremendous losses in diverse ecosystems, yet how they evolved pathogenicity in a clade of dominantly non-pathogenic wood degraders remains elusive. Here we show that Armillaria species, in addition to gene duplications and de novo gene origins, acquired at least 1,025 genes via 124 horizontal gene transfer events, primarily from Ascomycota. Horizontal gene transfer might have affected plant biomass degrading and virulence abilities of Armillaria, and provides an explanation for their unusual, soft rot-like wood decay strategy. Combined multi-species expression data revealed extensive regulation of horizontally acquired and wood-decay related genes, putative virulence factors and two novel conserved pathogenicity-induced small secreted proteins, which induced necrosis in planta. Overall, this study details how evolution knitted together horizontally and vertically inherited genes in complex adaptive traits of plant biomass degradation and pathogenicity in important fungal pathogens.

Development of a Multicomponent Microbiological Soil Inoculant and Its Performance in Sweet Potato Cultivation.

The cultivation and consumption of sweet potato (Ipomoea batatas) are increasing globally. As the usage of chemical fertilizers and pest control agents during its cultivation may lead to soil, water and air pollution, there is an emerging need for environment-friendly, biological solutions enabling increased amounts of healthy crop and efficient disease management. Microbiological agents for agricultural purposes gained increasing importance in the past few decades. Our goal was to develop an agricultural soil inoculant from multiple microorganisms and test its application potential in sweet potato cultivation. Two Trichoderma strains were selected: Trichoderma ghanense strain SZMC 25217 based on its extracellular enzyme activities for the biodegradation of plant residues, and Trichoderma afroharzianum strain SZMC 25231 for biocontrol purposes against fungal plant pathogens. The Bacillus velezensis strain SZMC 24986 proved to be the best growth inhibitor of most of the nine tested strains of fungal species known as plant pathogens, therefore it was also selected for biocontrol purposes against fungal plant pathogens. Arthrobacter globiformis strain SZMC 25081, showing the fastest growth on nitrogen-free medium, was selected as a component with possible nitrogen-fixing potential. A Pseudomonas resinovorans strain, SZMC 25872, was selected for its ability to produce indole-3-acetic acid, which is among the important traits of potential plant growth-promoting rhizobacteria (PGPR). A series of experiments were performed to test the selected strains for their tolerance to abiotic stress factors such as pH, temperature, water activity and fungicides, influencing the survivability in agricultural environments. The selected strains were used to treat sweet potato in two separate field experiments. Yield increase was observed for the plants treated with the selected microbial consortium (synthetic community) in comparison with the control group in both cases. Our results suggest that the developed microbial inoculant has the potential to be used in sweet potato plantations. To the best of our knowledge, this is the first report about the successful application of a fungal-bacterial consortium in sweet potato cultivation.

cotH Genes Are Necessary for Normal Spore Formation and Virulence in Mucor lusitanicus.

Mucormycosis is an invasive fungal infection caused by certain members of the fungal order of Mucorales. The species most frequently identified as the etiological agents of mucormycosis belong to the genera Rhizopus, Lichtheimia, and Mucor. The frequency of systemic mucormycosis has been increasing, mainly because of increasing numbers of susceptible patients. Furthermore, Mucorales display intrinsic resistance to the majority of routinely used antifungal agents (e.g., echinocandins and short-tailed azoles), which limits the number of possible therapeutic options. All the above-mentioned issues urge the improvement of molecular identification methods and the discovery of new antifungal targets and strategies. Spore coat proteins (CotH) constitute a kinase family present in many pathogenic bacteria and fungi and participate in the spore formation in these organisms. Moreover, some of them can act as virulence factors being receptors of the human GRP78 protein during Rhizopus delemar-induced mucormycosis. We identified 17 cotH-like genes in the Mucor lusitanicus genome database. Successful disruption of five cotH genes in Mucor was performed using the CRISPR-Cas9 system. The CotH3 and CotH4 proteins play a role in adaptation to different temperatures as well as in developing the cell wall structure. We also show CotH4 protein is involved in spore wall formation by affecting the total chitin content and, thus, the composition of the spore wall. The role of CotH3 and CotH4 proteins in virulence was confirmed in two invertebrate models and a diabetic ketoacidosis (DKA) mouse model. IMPORTANCE Current treatment options for mucormycosis are inadequate, resulting in high mortality rates, especially among immunosuppressed patients. The development of novel therapies for mucormycosis has been hampered by lack of understanding of the pathogenetic mechanisms. The importance of the cell surface CotH proteins in the pathogenesis of Rhizopus-mediated mucormycosis has been recently described. However, the contribution of this family of proteins to the virulence of other mucoralean fungi and their functionality in vital processes remain undefined. Through the use of the CRISPR-Case9 gene disruption system, we demonstrate the importance of several of the CotH proteins to the virulence of Mucor lusitanicus by using three infection models. We also report on the importance of one of these proteins, CotH4, to spore wall formation by affecting chitin content. Therefore, our studies extend the importance of CotH proteins to Mucor and identify the mechanism by which one of the CotH proteins contributes to the development of a normal fungal cell wall, thereby indicating that this family of proteins can be targeted for future development of novel therapeutic strategies of mucormycosis.

Characterization of the antagonistic potential of the glyphosate-tolerant Pseudomonas resinovorans SZMC 25872 strain against the plant pathogenic bacterium Agrobacterium tumefaciens.

The utilization of microorganisms with biocontrol activity against fungal and bacterial pathogens of plants is recognized as a promising, effective, and environment-friendly strategy to protect agricultural crops. We report the glyphosate-tolerant Pseudomonas resinovorans SZMC 25872 isolate as a novel strain with antagonistic potential towards the plant pathogenic bacterium Agrobacterium tumefaciens. In our studies, the growth of the P. resinovorans SZMC 25872 and A. tumefaciens SZMC 14557 isolates in the presence of 74 different carbon sources, and the effect of 11 carbon sources utilized by both strains on the biocontrol efficacy was examined. Seven variations of media with different carbon sources were selected for the assays to observe the biocontrol potential of the P. resinovorans strain. Also, 50% concentrations of the cell-free culture filtrates (CCF) obtained from medium amended with L-alanine or succinic acid as sole carbon source were found to be effective for the growth suppression of A. tumefaciens by 83.03 and 56.80%, respectively. The effect of 7 media on siderophore amount and the activity of extracellular trypsin- and chymotrypsin-like proteases, as well as esterases were also evaluated. Significant positive correlation was found between the siderophore amount and the percentage of inhibition, and the inhibitory effect of the CCFs obtained from medium amended with succinic acid was eliminated in the presence of an additional iron source, suggesting that siderophores produced by P. resinovorans play an important role in its antagonistic potential. The metabolic profile analysis of the P. resinovorans SZMC 25872 strain, performed by high performance liquid chromatography - high resolution mass spectrometry (HPLC-HRMS), has identified several previously not reported metabolites that might play role in the antagonistic effect against A. tumefaciens. Based on our findings we suggest that the possible inhibition modes of A. tumefaciens SZMC 14557 by P. resinovorans SZMC 25872 include siderophore-mediated suppression, extracellular enzyme activities and novel bioactive metabolites.

Molecular Characterization of Novel Mycoviruses in Seven Umbelopsis Strains.

The presence of viruses is less explored in Mucoromycota as compared to other fungal groups such as Ascomycota and Basidiomycota. Recently, more and more mycoviruses are identified from the early-diverging lineages of fungi. We have determined the genome of 11 novel dsRNA viruses in seven different Umbelopsis strains with next-generation sequencing (NGS). The identified viruses were named Umbelopsis ramanniana virus 5 (UrV5), 6a (UrV6a); 6b (UrV6b); 7 (UrV7); 8a (UrV8a); 8b (UrV8b); Umbelopsis gibberispora virus 1 (UgV1); 2 (UgV2) and Umbelopsis dimorpha virus 1a (UdV1a), 1b (UdV1b) and 2 (UdV2). All the newly identified viruses contain two open reading frames (ORFs), which putatively encode the coat protein (CP) and the RNA-dependent RNA polymerase (RdRp), respectively. Based on the phylogeny inferred from the RdRp sequences, eight viruses (UrV7, UrV8a, UrV8b, UgV1, UgV2, UdV1a, UdV1b and UdV2) belong to the genus Totivirus, while UrV5, UrV6a and UrV6b are placed into a yet unclassified but well-defined Totiviridae-related group. In UrV5, UgV1, UgV2, UrV8b, UdV1a, UdV2 and UdV1b, ORF2 is predicted to be translated as a fusion protein via a rare +1 (or -2) ribosomal frameshift, which is not characteristic to most members of the Totivirus genus. Virus particles 31 to 32 nm in diameter could be detected in the examined fungal strains by transmission electron microscopy. Through the identification and characterization of new viruses of Mucoromycota fungi, we can gain insight into the diversity of mycoviruses, as well as into their phylogeny and genome organization.

Aspergillus Was the Dominant Genus Found during Diversity Tracking of Potentially Pathogenic Indoor Fungal Isolates.

Viable airborne pathogenic fungi represent a potential health hazard when exposing vulnerable persons in quantities exceeding their resilience. In this study, 284 indoor fungal isolates from a strain collection of indoor fungi were screened for pathogenic potential through the ability to grow in neutral pH at 37 °C and 30 °C. The isolates were collected from 20 locations including 14 problematic and 6 non-problematic ordinary buildings. Out of the screened isolates, 170 isolates were unable to grow at 37 °C, whereas 67 isolates growing at pH 7.2 at 37 °C were considered as potential opportunistic pathogens. Forty-seven isolates growing at 30 °C but not at 37 °C were considered as less likely pathogens. Out of these categories, 33 and 33 strains, respectively, were identified to the species level. The problematic buildings included known opportunistic pathogens: Aspergillus calidoustus, Trichoderma longibrachiatum, Rhizopus arrhizus and Paecilomyces variotii, as well as less likely pathogens: Aspergillus versicolor, Chaetomium cochliodes, Chaetomium globosum and Chaetomium rectangulare. Opportunistic pathogens such as Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger and Aspergillus tubingensis and less likely pathogens such as Aspergillus westerdijkiae, Chaetomium globosum and Dichotomopilus finlandicus were isolated both from ordinary and from problematic buildings. Aspergillus was the dominant, most diverse genus found during screening for potentially pathogenic isolates in the indoor strain collection. Studies on Aspergillus niger and Aspergillus calidodoustus revealed that tolerance to cleaning chemicals may contribute to the adaptation of Aspergillus species to indoor environments.

Survival and growth of microscopic fungi derived from tropical regions under future heat waves in the Pannonian Biogeographical Region.

Warming and heat waves are predicted by different climate models in the near future in the Pannonian Biogeographical Region (PBR). These climatic effects may have impact on the prevalence and distribution of certain fungal species of this area. In this study the effects of predicted climate scenarios were tested on fungi being endemic or unintentionally introduced by global trade from regions of warm temperate climate. Common fungal species were selected for the study and exposed to heat waves during 7 days according to two climate scenarios: one moderately (RCP 4.5, Tavg = 27 °C, Tmax = 35 °C, RH: 100%) and one strongly pessimistic (RCP 8.5, Tavg = 30 °C, Tmax = 40 °C, RH: 100%) that include predictions for the Central Hungarian Region for July 2050. According to our results, Aspergillus flavus, Aspergillus niger, Aspergillus tubingensis and Fusarium strains introduced from tropical regions tolerated heat waves, unlike Penicillium and Talaromyces spp. and endemic Cladosporium spp. which were unable to grow under the RCP 8.5 treatment. The effects of climate change on fungi raise new issues not only from economic and health perspectives, but also in relation with plant protection and environment. Our results suggest that heat waves driven by climate change promote the colonization and growth of the tested strains of non-native fungi more likely than that of the native ones.

Characterization of Four Novel dsRNA Viruses Isolated from Mucor hiemalis Strains.

We previously screened the total nucleic acid extracts of 123 Mucor strains for the presence of dsRNA molecules without further molecular analyses. Here, we characterized five novel dsRNA genomes isolated from four different Mucor hiemalis strains with next-generation sequencing (NGS), namely Mucor hiemalis virus 1a (MhV1a) from WRL CN(M) 122; Mucor hiemalis virus 1b (MhV1b) from NRRL 3624; Mucor hiemalis virus 2 (MhV2) from NRRL 3616; and Mucor hiemalis virus 3 (MhV3) and Mucor hiemalis virus (MhV4) from NRRL 3617 strains. Genomes contain two open reading frames (ORF), which encode the coat protein (CP) and the RNA dependent RNA polymerase (RdRp), respectively. In MhV1a and MhV1b, it is predicted to be translated as a fusion protein via -1 ribosomal frameshift, while in MhV4 via a rare +1 (or-2) ribosomal frameshift. In MhV2 and MhV3, the presence of specific UAAUG pentanucleotide motif points to the fact for coupled translation termination and reinitialization. MhV1a, MhV2, and MhV3 are part of the clade representing the genus Victorivirus, while MhV4 is seated in Totivirus genus clade. The detected VLPs in Mucor strains were from 33 to 36 nm in diameter. Hybridization analysis revealed that the dsRNA molecules of MhV1a-MhV4 hybridized to the corresponding molecules.

Chaetomium and Chaetomium-like Species from European Indoor Environments Include Dichotomopilus finlandicus sp. nov.

The genus Chaetomium is a frequently occurring fungal taxon world-wide. Chaetomium and Chaetomium-like species occur in indoor environments, where they can degrade cellulose-based building materials, thereby causing structural damage. Furthermore, several species of this genus may also cause adverse effects on human health. The aims of this research were to identify Chaetomium and Chaetomium-like strains isolated from indoor environments in Hungary and Finland, two geographically distant regions of Europe with drier and wetter continental climates, respectively, and to study their morphological and physiological properties, as well as their extracellular enzyme activities, thereby comparing the Chaetomium and Chaetomium-like species isolated from these two different regions of Europe and their properties. Chaetomium and Chaetomium-like strains were isolated from flats and offices in Hungary, as well as from schools, flats, and offices in Finland. Fragments of the translation elongation factor 1α (tef1α), the second largest subunit of RNA polymerase II (rpb2) and ß-tubulin (tub2) genes, as well as the internal transcribed spacer (ITS) region of the ribosomal RNA gene cluster were sequenced, and phylogenetic analysis of the sequences performed. Morphological examinations were performed by stereomicroscopy and scanning electron microscopy. Thirty-one Chaetomium sp. strains (15 from Hungary and 16 from Finland) were examined during the study. The most abundant species was Ch. globosum in both countries. In Hungary, 13 strains were identified as Ch. globosum, 1 as Ch. cochliodes, and 1 as Ch. interruptum. In Finland, 10 strains were Ch. globosum, 2 strains were Ch. cochliodes, 2 were Ch. rectangulare, and 2 isolates (SZMC 26527, SZMC 26529) proved to be representatives of a yet undescribed phylogenetic species from the closely related genus Dichotomopilus, which we formally describe here as the new species Dichotomopilus finlandicus. Growth of the isolates was examined at different temperatures (4, 15, 20, 25, 30, 37, 35, 40, and 45 °C), while their extracellular enzyme production was determined spectrophotometrically.

Genome organization and evolution of a eukaryotic nicotinate co-inducible pathway.

In Aspergillus nidulans a regulon including 11 hxn genes (hxnS, T, R, P, Y, Z, X, W, V, M and N) is inducible by a nicotinate metabolic derivative, repressible by ammonium and under stringent control of the nitrogen-state-sensitive GATA factor AreA and the specific transcription factor HxnR. This is the first report in a eukaryote of the genomic organization of a possibly complete pathway of nicotinate utilization. In A. nidulans the regulon is organized in three distinct clusters, this organization is variable in the Ascomycota. In some Pezizomycotina species all 11 genes map in a single cluster; in others they map in two clusters. This variable organization sheds light on cluster evolution. Instances of gene duplication followed by or simultaneous with integration in the cluster, partial or total cluster loss, and horizontal gene transfer of several genes (including an example of whole cluster re-acquisition in Aspergillus of section Flavi) were detected, together with the incorporation in some clusters of genes not found in the A. nidulans co-regulated regulon, which underlie both the plasticity and the reticulate character of metabolic cluster evolution. This study provides a comprehensive phylogeny of six members of the cluster across representatives of all Ascomycota classes.

Characterization of Three Pleiotropic Drug Resistance Transporter Genes and Their Participation in the Azole Resistance of Mucor circinelloides.

Mucormycosis is a life-threatening opportunistic infection caused by certain members of the fungal order Mucorales. This infection is associated with high mortality rate, which can reach nearly 100% depending on the underlying condition of the patient. Treatment of mucormycosis is challenging because these fungi are intrinsically resistant to most of the routinely used antifungal agents, such as most of the azoles. One possible mechanism of azole resistance is the drug efflux catalyzed by members of the ATP binding cassette (ABC) transporter superfamily. The pleiotropic drug resistance (PDR) transporter subfamily of ABC transporters is the most closely associated to drug resistance. The genome of Mucor circinelloides encodes eight putative PDR-type transporters. In this study, transcription of the eight pdr genes has been analyzed after azole treatment. Only the pdr1 showed increased transcript level in response to all tested azoles. Deletion of this gene caused increased susceptibility to posaconazole, ravuconazole and isavuconazole and altered growth ability of the mutant. In the pdr1 deletion mutant, transcript level of pdr2 and pdr6 significantly increased. Deletion of pdr2 and pdr6 was also done to create single and double knock out mutants for the three genes. After deletion of pdr2 and pdr6, growth ability of the mutant strains decreased, while deletion of pdr2 resulted in increased sensitivity against posaconazole, ravuconazole and isavuconazole. Our result suggests that the regulation of the eight pdr genes is interconnected and pdr1 and pdr2 participates in the resistance of the fungus to posaconazole, ravuconazole and isavuconazole.

Fungi and their secondary metabolites in water-damaged indoors after a major flood event in eastern Croatia.

In winter and summer of 2016 and 2017, airborne fungi and house dust were collected in indoors of the village Gunja, which had been flooded, and the control village Gornji Stupnik (Croatia) in order to explore variations of fungal indoor levels, particularly Aspergilli section Nidulantes series Versicolores, as well as fungal metabolites in dust. Levels of airborne Aspergilli (Versicolores) were three times as high in winter and summer in Gunja than in the control village, while dustborne isolates were equally present in both locations. Sequencing of the calmodulin gene region revealed that among Aspergilli (Versicolores), A. jensenii and A. creber were dominant and together with A. puulaauensis, A. tennesseensis and A. venenatus produced sterigmatocystin and 5-methoxysterigmatocystin (HPLC coupled with mass spectrometry); A. amoenus, A. fructus, A. griseoaurantiacus, A. pepii, and A. protuberus produced sterigmatocystin but not 5-methoxysterigmatocystin; A. sydowii did not produce any of these toxins. A total of 75 metabolites related to Penicillium (29), Aspergillus (22), Fusarium (10), Alternaria (5), Stachybotrys (2), and other fungi (7) were detected in dust by liquid chromatography-tandem mass spectrometry. The majority of metabolites including sterigmatocystin and 5-methoxysterigmatocystin exhibited a higher prevalence in winter in Gunja.

Post-Flood Impacts on Occurrence and Distribution of Mycotoxin-Producing Aspergilli from the Sections Circumdati, Flavi, and Nigri in Indoor Environment.

Mycotoxin-producing Aspergilli (Circumdati, Flavi, and Nigri), usually associated with contaminated food, may also cause respiratory disorders and are insufficiently studied in water-damaged indoor environments. Airborne (N = 71) and dust borne (N = 76) Aspergilli collected at post-flood and control locations in Croatia resulted in eleven different species based on their calmodulin marker: A. ochraceus, A. ostianus, A. pallidofulvus, A. sclerotiorum, and A. westerdijkiae (Circumdati); A. flavus (Flavi); and A. tubingensis, A. welwitschiae, A. niger, A. piperis, and A. uvarum (Nigri). Most of the airborne (73%) and dust borne (54%) isolates were found at post-flood locations, and the highest concentrations measured in indoor air (5720 colony-forming units (CFU)/m3) and dust (2.5 × 105 CFU/g) were up to twenty times higher than in the control locations. A. flavus dominated among airborne isolates (25%) at the unrepaired locations, while 56% of the dust borne Aspergilli were identified as A. tubingensis and A. welwitschiae. The ability of identified isolates to produce mycotoxins aflatoxin B1 (AFB1), fumonisin B2 (FB2), and ochratoxin A were assessed by LC-MS analysis. All ochratoxin A (OTA)-producing Circumdati belonged to A. westerdijkiae (13.7 ± 15.81 µg/mL); in the section, FlaviA. flavus produced AFB1 (2.51 ± 5.31 µg/mL), while A. welwitschiae and A. niger (section Nigri) produced FB2 (6.76 ± 13.51 µg/mL and 11.24 ± 18.30 µg/mL, respectively). Water damage dominantly supported the occurrence of aflatoxigenic A. flavus in indoor environments. Yet unresolved, the causal relationship of exposure to indoor Aspergilli and adverse health effects may support the significance of this research.

Characterization of the Plant Growth-Promoting Activities of Endophytic Fungi Isolated from Sophora flavescens.

Endophytic fungi in symbiotic association with their host plant are well known to improve plant growth and reduce the adverse effects of both biotic and abiotic stresses. Therefore, fungal endophytes are beginning to receive increased attention in an effort to find growth-promoting strains that could be applied to enhance crop yield and quality. In our study, the plant growth-promoting activities of endophytic fungi isolated from various parts of Sophora flavescens (a medicinally important plant in Mongolia and China) have been revealed and investigated. Fungal isolates were identified using molecular taxonomical methods, while their plant growth-promoting abilities were evaluated in plate assays. Altogether, 15 strains were isolated, representing the genera Alternaria, Didymella, Fusarium and Xylogone. Five of the isolates possessed phosphate solubilization activities and twelve secreted siderophores, while all of them were able to produce indoleacetic acid (IAA) in the presence or absence of tryptophan. The endogenous and exogenous accumulation of IAA were also monitored in liquid cultures using the HPLC-MS/MS technique to refine the plate assay results. Furthermore, for the highest IAA producer fungi, the effects of their extracts were also examined in plant bioassays. In these tests, the primary root lengths of the model Arabidopsis thaliana were increased in several cases, while the biomasses were significantly lower than the control IAA treatment. Significant alterations have also been detected in the photosynthetic pigment (chlorophyll-a, -b and carotenoids) content due to the fungal extract treatments, but these changes did not show any specific trends.

Agricultural systems as potential sources of emerging human mycoses caused by Trichoderma: a successful, common phylotype of Trichoderma longibrachiatum in the frontline.

Trichoderma species are abundant in different agricultural habitats, but some representatives of this genus, mainly clade Longibrachiatum members are also emerging as causative agents of various human diseases with even fatal outcome. Strains of these species frequently show resistance to commonly used azole antifungals. Based on previous data it is hypothesized that Trichoderma isolates identified in human infections derive from environmental-including agricultural-origins. We examined Trichoderma longibrachiatum Rifai and Trichoderma bissettii Sandoval-Denis & Guarro strains recovered from four novel cases of human mycoses, along with isolates from previous case reports and different agricultural habitats, using multilocus phylogenetic analysis, BIOLOG Phenotype Microarrays and Etest. Strains attributed to T. bissettii were more abundant in both clinical and agricultural specimens compared to T. longibrachiatum. The majority of the isolates of both taxa could tolerate >256, >32 and >32 µg/ml fluconazole, itraconazole and posaconazole, respectively. None of the obtained results revealed characteristic differences between strains of clinical and agricultural origin, nor between the two taxa, supporting that agricultural environments may be significant sources of infections caused by these emerging human fungal pathogens. Furthermore, based on our findings we propose the re-classification of T. bissettii as T. longibrachiatum f. sp. bissettii.

New Species of the Genus Curvularia: C. tamilnaduensis and C. coimbatorensis from Fungal Keratitis Cases in South India.

Members of the genus Curvularia are melanin-producing dematiaceous fungi of increasing clinical importance as causal agents of both local and invasive infections. This study contributes to the taxonomical and clinical knowledge of this genus by describing two new Curvularia species based on isolates from corneal scrapings of South Indian fungal keratitis patients. The phylogeny of the genus was updated based on three phylogenetic markers: the internal transcribed spacer (ITS) region of the ribosomal RNA gene cluster as well as fragments of the glyceraldehyde-3-phosphate dehydrogenase (gpdh) and translation elongation factor 1-α (tef1α) genes. The maximum likelihood phylogenetic tree constructed from the alignment of the three concatenated loci revealed that the examined isolates are representing two new, yet undescribed, Curvularia species. Examination of colony and microscopic morphology revealed differences between the two species as well as between the new species and their close relatives. The new species were formally described as Curvularia tamilnaduensis N. Kiss & S. Kocsubé sp. nov. and Curvularia coimbatorensis N. Kiss & S. Kocsubé sp. nov. Antifungal susceptibility testing by the broth microdilution method of CLSI (Clinical & Laboratory Standards Institute) revealed that the type strain of C. coimbatorensis is less susceptible to a series of antifungals than the C. tamilnaduensis strains.

Characterization of Aspergillus tamarii Strains From Human Keratomycoses: Molecular Identification, Antifungal Susceptibility Patterns and Cyclopiazonic Acid Producing Abilities.

Aspergillus tamarii appears to be an emerging aetiological agent of human keratomycoses in South India. The investigated strains were isolated from six suspected fungal keratitis patients attending a tertiary care eye hospital in Coimbatore (Tamil Nadu, India), and were initially identified by the microscopic examinations of the scrapings and the cultures. Our data suggest that A. tamarii could be easily overlooked when identification is carried out based on morphological characteristics alone, while the sequence analysis of the calmodulin gene can be used successfully to recognize this species accurately. According to the collected clinical data, ocular trauma is a common risk factor for the infection that gradually developed from mild to severe ulcers and could be healed with an appropriate combined antifungal therapy. Antifungal susceptibility testing revealed that A. tamarii strains are susceptible to the most commonly used topical or systemic antifungal agents (i.e., econazole, itraconazole and ketoconazole) except for natamycin. Moreover, natamycin proved to be similarly less effective than the azoles against A. tamarii in our drug interaction tests, as the predominance of indifferent interactions was revealed between natamycin and econazole and between natamycin and itraconazole as well. Four and five isolates of A. tamarii were confirmed to produce cyclopiazonic acid (CPA) in RPMI-1640 - which is designed to mimic the composition of human extracellular fluids - and in yeast extract sucrose (YES) medium, respectively, which is a widely used culture medium for testing mycotoxin production. Although a ten times lower mycelial biomass was recorded in RPMI-1640 than in YES medium, the toxin contents of the samples were of the same order of magnitude in both types of media. There might be a relationship between the outcome of infections and the toxigenic properties of the infecting fungal strains. However, this remains to be investigated in the future.

Comparative genomics reveals the origin of fungal hyphae and multicellularity.

Hyphae represent a hallmark structure of multicellular fungi. The evolutionary origins of hyphae and of the underlying genes are, however, hardly known. By systematically analyzing 72 complete genomes, we here show that hyphae evolved early in fungal evolution probably via diverse genetic changes, including co-option and exaptation of ancient eukaryotic (e.g. phagocytosis-related) genes, the origin of new gene families, gene duplications and alterations of gene structure, among others. Contrary to most multicellular lineages, the origin of filamentous fungi did not correlate with expansions of kinases, receptors or adhesive proteins. Co-option was probably the dominant mechanism for recruiting genes for hypha morphogenesis, while gene duplication was apparently less prevalent, except in transcriptional regulators and cell wall - related genes. We identified 414 novel gene families that show correlated evolution with hyphae and that may have contributed to its evolution. Our results suggest that hyphae represent a unique multicellular organization that evolved by limited fungal-specific innovations and gene duplication but pervasive co-option and modification of ancient eukaryotic functions.

Detection and Molecular Characterization of Novel dsRNA Viruses Related to the Totiviridae Family in Umbelopsis ramanniana.

Umbelopsis ramanniana is an oleaginous fungus belonging to the Mucoromycotina subphylum. Our group had previously detected four double-stranded RNA (dsRNA) bands in the U. ramanniana NRRL 1296 strain by gel electrophoresis. Here we describe the molecular characterization of its dsRNA elements as well as the discovery of four novel dsRNA viruses: Umbelopsis ramanniana virus 1 (UrV1), Umbelopsis ramanniana virus 2 (UrV2), Umbelopsis ramanniana virus 3 (UrV3), and Umbelopsis ramanniana virus 4 (UrV4). Full genomes of UrV1, UrV3, and UrV4 were determined using the full-length amplification of cDNAs (FLAC) technique; they contain two open reading frames (ORF), which putatively encode the coat protein (CP) and the RNA dependent RNA polymerase (RdRp), respectively. In case of UrV2, a partial ORF encoding a partial RdRp gene could be determined. Based on the phylogeny inferred from the RdRp sequences, UrV1 and UrV4 belong to the genus Totivirus, while UrV2 may belong to the genus Victorivirus. UrV3 nested to a novel, unclassified group of Totiviridae, which is related to the genus Totivirus. Hybridization analysis revealed that the dsRNA molecules of UrV1 and UrV4 correspond to the same 5.0-kbp electrophoretic band, whilst the probe for the UrV3 hybridized to the largest, 5.3-kbp and the 3.0-kbp bands of the dsRNA pattern of U. ramanniana. Interestingly, the probe for the UrV2 sequence did not hybridized to any dsRNA bands, but it could be amplified from the isolated 3.0-kbp fragment. By transmission electron microscopy, two different isometric virus particles with about 50 and 35 nm in diameter were detected in U. ramanniana NRRL 1296 indicating that this strain harbor multiple viruses. Beside U. ramanniana, dsRNA elements were also detected in other Umbelopsis isolates with different patterns consisting of 2 to 4 discrete and different sized (0.7-5.3-kbp) dsRNA molecules. Based on a hybridization analysis with UrV1 CP and RdRp probes, the bands with the size of around 5.0-kbp, which were present in all tested Umbelopsis strains, are presumed as possible full mycovirus genomes.