Discovery of highly reactive self-splicing group II introns within the mitochondrial genomes of human pathogenic fungi.

Fungal pathogens represent an expanding global health threat for which treatment options are limited. Self-splicing group II introns have emerged as promising drug targets, but their development has been limited by a lack of information on their distribution and architecture in pathogenic fungi. To meet this challenge, we developed a bioinformatic workflow for scanning sequence data to identify unique RNA structural signatures within group II introns. Using this approach, we discovered a set of ubiquitous introns within thermally dimorphic fungi (genera of Blastomyces, Coccidioides and Histoplasma). These introns are the most biochemically reactive group II introns ever reported, and they self-splice rapidly under near-physiological conditions without protein cofactors. Moreover, we demonstrated the small molecule targetability of these introns by showing that they can be inhibited by the FDA-approved drug mitoxantrone in vitro. Taken together, our results highlight the utility of structure-based informatic searches for identifying riboregulatory elements in pathogens, revealing a striking diversity of reactive self-splicing introns with great promise as antifungal drug targets.

miRNA Mediated Regulation and Interaction between Plants and Pathogens.

Plants have evolved diverse molecular mechanisms that enable them to respond to a wide range of pathogens. It has become clear that microRNAs, a class of short single-stranded RNA molecules that regulate gene expression at the transcriptional or post-translational level, play a crucial role in coordinating plant-pathogen interactions. Specifically, miRNAs have been shown to be involved in the regulation of phytohormone signals, reactive oxygen species, and NBS-LRR gene expression, thereby modulating the arms race between hosts and pathogens. Adding another level of complexity, it has recently been shown that specific lncRNAs (ceRNAs) can act as decoys that interact with and modulate the activity of miRNAs. Here we review recent findings regarding the roles of miRNA in plant defense, with a focus on the regulatory modes of miRNAs and their possible applications in breeding pathogen-resistance plants including crops and trees. Special emphasis is placed on discussing the role of miRNA in the arms race between hosts and pathogens, and the interaction between disease-related miRNAs and lncRNAs.

Chromoblastomycosis in the Amazon region, Brazil, caused by Fonsecaea pedrosoi, Fonsecaea nubica, and Rhinocladiella similis: Clinicopathology, susceptibility, and molecular identification.

Chromoblastomycosis is a chronic subcutaneous disease caused by human contact with melanized fungi occurring mainly in tropical and subtropical zones worldwide. This study assessed 12 patients with chromoblastomycosis from Rondônia, Brazil, Amazon region. In sum, 83.3% were men, 41.6% were from Monte Negro city, median age was 52.9 years, and median time to disease progression was 12.2 years. Lesions were located on the lower limbs (75%), and verruciform was prevalent form (66.6%). After 3 years of treatment with itraconazole, two patients were considered cured. The etiological agents were identified by the molecular sequence of the ribosomal internal transcribed spacer ITS1, 5.8S, and ITS2 region and ß-tubulin genes. Eight strains were identified as Fonsecaea pedrosoi, two were F. nubica, and two were Rhinocladiella similis. The antifungal activity of five drugs was evaluated, and the most active drug was terbinafine (range minimal inhibitory concentration [MIC] 0.015-0.12 µg/ml), itraconazole (range MIC 0.03-0.5 µg/ml) and voriconazole (range MIC 0.06-0.5 µg/ml). The highest MIC was 5-fluorocytosine (range MIC 2-32 µg/ml), and amphotericin B (range MIC 0.25-2 µg/ml). In conclusion, the present study expanded the epidemiological disease database and described for the first time F. nubica and R. similis as chromoblastomycosis agents in the Brazilian Amazon region. Our results confirmed the importance of using molecular methods to identify the melanized fungi and stimulate the recognition of the disease in other places where no cases have been reported.

Molecular identification and antibacterial properties of an ericoid associated mycorrhizal fungus.

BACKGROUND: The quest for novel sources of antibacterial compounds have necessitated the inclusion of ericoid mycorrhizal fungi (ERM) commonly found within the root of ericaceous plants. Agar-well diffusion method was used to detect antibacterial activity and was followed by the microbroth diffusion method [minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)]. RESULTS: The results of the phytochemical screening indicated that only alkaloids, flavonoids, phenols, saponins, cardiac glycosides and terpenoids were present, while steroids and tannins were absent. The MIC of the extracts ranged between 2 and 16 mg/mL, and the lowest MIC was obtained with Staphylococcus aureus. Also, the result of the MBC study indicated that the fungal extract was most active at concentrations of 2 and 4 mg/mL against Bacillus subtilis and S. aureus, respectively. CONCLUSIONS: This bioassay showed, for the first time, antibacterial activity of L. incrustata against some bacterial species. Subsequently, ERM fungi should be given attention when searching for antimicrobial agents because they could provide a solution to solve problems associated with conventional disease treatments (i.e. pathogenic microorganisms resistance).

Screening of bacterial endophytes as potential biocontrol agents against soybean diseases.

AIMS: This research was aimed at identifying and characterizing endophytic micro-organisms associated with soybean that have antimicrobial activity towards soybean pathogens. METHODS AND RESULTS: Soybean plants were collected from field trials in four locations of southern Brazil that were cultivated with conventional (C) and transgenic glyphosate-resistant (GR) soybeans. Endophytic bacteria isolated from roots, stems and leaves of soybeans were evaluated for their capacity to inhibit fungal and bacterial plant pathogens and 13 micro-organisms were identified with antagonistic activity. Approximately 230 bacteria were isolated and identified based on the 16S rRNA and rpoN gene sequences. Bacteria isolated from conventional and transgenic soybeans were significantly different not only in population diversity but also in their antagonistic capacity. Thirteen isolates showed in vitro antagonism against Sclerotinia sclerotiorum, Phomopsis sojae and Rhizoctonia solani. Bacillus sp. and Burkholderia sp. were the most effective isolates in controlling bacterial and fungal pathogens in vitro. Extracts and precipitates from culture supernatants of isolates showed different patterns of inhibitory activity on growth of fungal and bacterial pathogens. CONCLUSIONS: Bacillus sp. and Burkholderia sp. were the most effective isolates in controlling fungal pathogens in vitro, and the activity is mainly due to peptides. However, most of the studied bacteria showed the presence of antimicrobial compounds in the culture supernatant, either peptides, bacteriocins or secondary metabolites. SIGNIFICANCE AND IMPACT OF THE STUDY: These results could be significant to develop tools for the biological control of soybean diseases. The work brought to the identification of micro-organisms such as Bacillus sp. and Burkholderia sp. that have the potential to protect crops in order to enhance a sustainable management system of crops. Furthermore, the study provides the first evidences of the influence of management as well as the genetics of glyphosate-resistant soybean on the diversity of bacterial endophytes of soybean phytobiome.

Modeling dermatophytosis in reconstructed human epidermis: A new tool to study infection mechanisms and to test antifungal agents.

Dermatophytosis is a superficial fungal infection of keratinized structures that exhibits an increasing prevalence in humans and is thus requesting novel prophylactic strategies and therapies. However, precise mechanisms used by dermatophytes to adhere at the surface of the human epidermis and invade its stratum corneum are still incompletely identified, as well as the responses provided by the underlying living keratinocytes during the infection. We hereby report development of an in vitro model of human dermatophytosis through infection of reconstructed human epidermis (RHE) by arthroconidia of the anthropophilic Trichophyton rubrum species or of the zoophilic Microsporum canis and Arthroderma benhamiae species. By modulating density of arthroconidia in the inoculum and duration of exposure to such pathogens, fungal infection limited to the stratum corneum was obtained, mimicking severe but typical in vivo situation. Fungal elements in infected RHE were monitored over time by histochemical analysis using periodic-acid Schiff-staining or quantified by qPCR-detection of fungal genes inside RHE lysates. This model brings improvements to available ones, dedicated to better understand how dermatophytes and epidermis interact, as well as to evaluate preventive and therapeutic agents. Indeed, miconazole topically added to RHE was demonstrated to inhibit fungal infection in this model.

Nomenclatural notes on Nadsoniella and the human opportunist black yeast genus Exophiala.

The opportunistic black yeast are particularly known through the genus Exophiala, characterised by annellidic budding cells. However, this phenotype is polyphyletic within the order Chaetothyriales. Seventeen generic names are available in the family Herpotrichiellaceae, one of which is Exophiala. Future taxonomy will be based on molecular phylogeny; each multi-species clade may qualify for one of these names. This paper focuses on the genus Nadsoniella, which is the oldest valid name in the Herpotrichiellaceae. Despite its exophiala-like phenotype, the type species of Nadsoniella clusters in the jeanselmei-clade, competing with the sympodial genus Rhinocladiella. In contrast, Exophiala competes with morphologically pronounced genera Thysanorea and Veronaea. Replacing the current phenotypic system for phylogenetic nomenclature requires highly stable phylogenies, which currently are not available.

[Advances in the regulation of cephalosporin C biosynthesis - A review].

The beta-lactam antibiotic cephalosporin C is produced industrially by Acremonium chrysogenum. Its derivative 7-aminocephalosporanic acid (7-ACA) is the intermediate of most chemical modification cephalosporins that are the most frequently used antibiotics for the therapy of infectious diseases. Due to its importance, the biosynthetic pathway of cephalosporin C has been elucidated in Acremonium chrysogenum. To improve the yield of cephalosporin C and reduce the cost of production, recent studies have been focused on the sophisticated regulation of cephalosporin C biosynthesis. In this review, recent advances in cephalosporin C biosynthesis and regulation are summarized.

Seasonal and spatial variation of Deuteromycetes population in polluted cost of Kiaochow Bay.

Objective: To reveal the relationship between Deuteromycetes community and the environmental in Kiaochow Bay of the Yellow Sea. Methods: Using recorded pollution survey, we used molecular methods to study seasonal and spatial variation of Deuteromycetes community diversity in different polluted waters of Kiaochow Bay of the Yellow Sea, China. Results: Denaturing gradient gel electrophoresis fingerprints varied obviously among different sites of similar level of pollution. Moreover, sequence analysis of recovered dominant bands exhibited the existence of plenty of uncultivable fungi, among which Penicillium was the dominant genus. Furthermore, in heavily polluted estuary, there were abundant animal pathogens such as amoeba and Pythium as well as Deuteromycetes. These discoveries demonstrate that the Deuteromycetes community structure is closely related to marine environment, and are indicative of different level of marine contamination. Conclusion: The relationship between Deuteromycetes community and different level of pollution and seasons varied were closely related.

Molecular basis of the regulation of iron homeostasis in fission and filamentous yeasts.

When iron load exceeds that needed by fission and filamentous yeasts, iron-regulatory GATA-type transcription factors repress genes encoding iron acquisition systems. In contrast, under iron starvation, optimization of cellular iron utilization is coordinated by a specialized regulatory subunit of the CCAAT-binding factor that fosters repression of genes encoding iron-using proteins. Despite these findings, there is still limited knowledge concerning the mechanisms by which these iron-responsive regulators respond to high- or low-iron availability. To provide a framework for understanding common and distinct properties of iron-dependent transcriptional regulators, a repertoire of their functional domains in different fungal species is presented here. In addition, discovery of interacting partners of these iron-responsive factors contributes to provide additional insight into their properties.

A century later: rediscovery, culturing and phylogenetic analysis of Diploöspora rosea, a rare onygenalean hyphomycete.

Nearly 100 years after its first discovery, Diploöspora rosea was detected on biologically damaged parchment paper in Rome, Italy and isolated from house dust collected in Micronesia. The isolation of this culture permitted morphological study of colony characters, conidium and conidiophore development, and phylogenetic investigations using sequences of nuc 18S rDNA, internal transcribed spacers, and 28S rDNA. The results indicate that D. rosea is an onygenalean fungus, of uncertain taxonomic position, basal or sister to the Gymnoascaceae. Based on observations of the parchments using SEM-Energy Dispersive Spectroscopy, we speculate that the fungus occurs in archival and domestic environments subject to periodic wetting. Its ability to grow on all low water activity media used in the study, including malt extract agar amended with 60% sucrose, confirms its xerophilic nature.

A case of cerebral phaeohyphomycosis caused by Fonsecaea monophora, a neurotropic dematiaceous fungus, and a review of the literature.

The Fonsecaea species, which are the leading causes of chromoblastomycosis, are not considered neurotropic fungal agents. Fonsecaea pedrosoi is the primary species in the genus and is usually isolated from chromoblastomycosis cases. However, the recently distinguished species F. monophora has been reported in a few cerebral phaeohyphomycosis cases. Here, a case of cerebral phaeohyphomycosis caused by Fonsecaea monophora is presented in a 71-year-old female subject with chronic diabetes mellitus and hypertension. The identification of F. monophora was made through mycological and molecular analysis, and an isolate was differentiated from the closely related F. pedrosoi by sequence data on key bases on the ribosomal internal transcribed spacer region. The case was successfully treated with surgical and medical approaches, and the patient has remained healthy and stable after a ten-month follow up. Given the increasing incidence of this type of infection of the central nervous system (CNS), this case provides further support for the consideration that F. monophora might represent a neurotropic agent.

Phenotypic and molecular identification of Fonsecaea pedrosoi strains isolated from chromoblastomycosis patients in Mexico and Venezuela.

Chromoblastomycosis is a chronic granulomatous disease caused frequently by fungi of the Fonsecaea genus. The objective of this study was the phenotypic and molecular identification of F. pedrosoi strains isolated from chromoblastomycosis patients in Mexico and Venezuela. Ten strains were included in this study. For phenotypic identification, we used macroscopic and microscopic morphologies, carbohydrate assimilation test, urea hydrolysis, cixcloheximide tolerance, proteolitic activity and the thermotolerance test. The antifungal activity of five drugs was evaluated against the isolates. Molecular identification was performed by sequencing the internal transcribed spacer (ITS) ribosomal DNA regions of the isolated strains. The physiological analysis and morphological features were variable and the precise identification was not possible. All isolates were susceptible to itraconazole, terbinafine, voriconazole and posaconazole. Amphotericin B was the least effective drug. The alignment of the 559-nucleotide ITS sequences from our strains compared with sequences of GenBank revealed high homology with F. pedrosoi (EU285266.1). In this study, all patients were from rural areas, six from Mexico and four from Venezuela. Ten isolates were identified by phenotypic and molecular analysis, using ITS sequence and demonstrated that nine isolates from Mexico and Venezuela were 100% homologous and one isolate showed a small genetic distance.

Antifungal susceptibility patterns of opportunistic fungi in the genera Verruconis and Ochroconis.

Species of Verruconis and species of Ochroconis are dematiaceous fungi generally found in the environment but having the ability to infect humans, dogs, cats, poultry, and fish. This study presents the antifungal susceptibility patterns of these fungi at the species level. Forty strains originating from clinical and environmental sources were phylogenetically identified at the species level by using sequences of the ribosomal DNA internal transcribed spacer (rDNA ITS). In vitro antifungal susceptibility testing was performed against eight antifungals, using the Clinical and Laboratory Standards Institute (CLSI) broth microdilution method. The geometric mean MICs for amphotericin B (AMB), flucytosine (5FC), fluconazole (FLC), itraconazole (ITC), voriconazole (VRC), and posaconazole (POS) and minimum effective concentrations (MECs) for caspofungin (CAS) and anidulafungin (AFG) across the Ochroconis and Verruconis species were as follows, in increasing order. For Verruconis species, the values (µg/ml) were as follows: AFG, 0.04; POS, 0.25; ITC, 0.37; AMB, 0.50; CAS, 0.65; VRC, 0.96; 5FC, 10.45; and FLC, 47.25. For Ochroconis species, the values (µg/ml) were as follows: AFG, 0.06; POS, 0.11; CAS, 0.67; VRC, 2.76; ITC, 3.94; AMB, 5.68; 5FC, 34.48; and FLC, 61.33. Antifungal susceptibility of Ochroconis and Verruconis was linked with phylogenetic distance and thermotolerance. Echinocandins and POS showed the greatest in vitro activity, providing possible treatment options for Ochroconis and Verruconis infections.

Assessment of fungal contamination in waste sorting and incineration-case study in Portugal.

Organic waste is a rich substrate for microbial growth, and because of that, workers from waste industry are at higher risk of exposure to bioaerosols. This study aimed to assess fungal contamination in two plants handling solid waste management. Air samples from the two plants were collected through an impaction method. Surface samples were also collected by swabbing surfaces of the same indoor sites. All collected samples were incubated at 27°C for 5 to 7 d. After lab processing and incubation of collected samples, quantitative and qualitative results were obtained with identification of the isolated fungal species. Air samples were also subjected to molecular methods by real-time polymerase chain reaction (RT PCR) using an impinger method to measure DNA of Aspergillus flavus complex and Stachybotrys chartarum. Assessment of particulate matter (PM) was also conducted with portable direct-reading equipment. Particles concentration measurement was performed at five different sizes (PM0.5; PM1; PM2.5; PM5; PM10). With respect to the waste sorting plant, three species more frequently isolated in air and surfaces were A. niger (73.9%; 66.1%), A. fumigatus (16%; 13.8%), and A. flavus (8.7%; 14.2%). In the incineration plant, the most prevalent species detected in air samples were Penicillium sp. (62.9%), A. fumigatus (18%), and A. flavus (6%), while the most frequently isolated in surface samples were Penicillium sp. (57.5%), A. fumigatus (22.3%) and A. niger (12.8%). Stachybotrys chartarum and other toxinogenic strains from A. flavus complex were not detected. The most common PM sizes obtained were the PM10 and PM5 (inhalable fraction). Since waste is the main internal fungal source in the analyzed settings, preventive and protective measures need to be maintained to avoid worker exposure to fungi and their metabolites.

Fungal contamination in two Portuguese wastewater treatment plants.

The presence of filamentous fungi was detected in wastewater and air collected at wastewater treatment plants (WWTP) from several European countries. The aim of the present study was to assess fungal contamination in two WWTP operating in Lisbon. In addition, particulate matter (PM) contamination data was analyzed. To apply conventional methods, air samples from the two plants were collected through impaction using an air sampler with a velocity air rate of 140 L/min. Surfaces samples were collected by swabbing the surfaces of the same indoor sites. All collected samples were incubated at 27°C for 5 to 7 d. After lab processing and incubation of collected samples, quantitative and qualitative results were obtained with identification of the isolated fungal species. For molecular methods, air samples of 250 L were also collected using the impinger method at 300 L/min airflow rate. Samples were collected into 10 ml sterile phosphate-buffered saline with 0.05% Triton X-100, and the collection liquid was subsequently used for DNA extraction. Molecular identification of Aspergillus fumigatus and Stachybotrys chartarum was achieved by real-time polymerase chain reaction (RT-PCR) using the Rotor-Gene 6000 qPCR Detection System (Corbett). Assessment of PM was also conducted with portable direct-reading equipment (Lighthouse, model 3016 IAQ). Particles concentration measurement was performed at five different sizes: PM0.5, PM1, PM2.5, PM5, and PM10. Sixteen different fungal species were detected in indoor air in a total of 5400 isolates in both plants. Penicillium sp. was the most frequently isolated fungal genus (58.9%), followed by Aspergillus sp. (21.2%) and Acremonium sp. (8.2%), in the total underground area. In a partially underground plant, Penicillium sp. (39.5%) was also the most frequently isolated, also followed by Aspergillus sp. (38.7%) and Acremonium sp. (9.7%). Using RT-PCR, only A. fumigatus was detected in air samples collected, and only from partial underground plant. Stachybotrys chartarum was not detected in any of the samples analyzed. The distribution of particle sizes showed the same tendency in both plants; however, the partially underground plant presented higher levels of contamination, except for PM2.5. Fungal contamination assessment is crucial to evaluating the potential health risks to exposed workers in these settings. In order to achieve an evaluation of potential health risks to exposed workers, it is essential to combine conventional and molecular methods for fungal detection. Protective measures to minimize worker exposure to fungi need to be adopted since wastewater is the predominant internal fungal source in this setting.

Pathogenicity of conidia-based preparations of entomopathogenic fungi against the greenhouse pest aphids Myzus persicae, Aphis gossypii, and Aulacorthum solani (Hemiptera: Aphididae).

Seeking new isolates of entomopathogenic fungi with greater virulence against greenhouse aphid pests than those currently registered in North America for control of these insects, single-dose screening assays of 44 selected fungal isolates and 4 commercially available strains were conducted against first-instar nymphs of Myzus persicae and Aphis gossypii. The assays identified a number of Beauveria and Metarhizium isolates with virulence equal to or greater than that of the commercial strains against the nymphal aphids, but none exhibited exceptionally high virulence. Virulence of Isaria isolates was unexpectedly low (<31% mortality at doses>1000conidia/mm(2)). In dose-response assays, Beauveria ARSEF 5493 proved most virulent against M. persicae and A. gossypii; however, LC50s of this isolate did not differ significantly from those of B. bassiana commercial strain JW-1. Dose-response assays were also conducted with Aulacorthum solani, the first reported evaluations of Beauveria and Metarhizium against this pest. The novel isolate Metarhizium 5471 showed virulence⩾that of Beauveria 5493 in terms of LC25 and LC50, but 5493 produced a steeper dose response (slope). Additional tests showed that adult aphids are more susceptible than nymphs to fungal infection but confirmed that infection has a limited pre-mortem effect on aphid reproduction. Effects of assay techniques and the potential of fungal pathogens as aphid-control agents are discussed.

Filamentous fungi from the Atlantic marine sponge Dragmacidon reticulatum.

Dragmacidon reticulatum is a marine sponge of wide occurrence in the Eastern and Western Atlantic. Little is known about D. reticulatum fungal diversity. Filamentous fungi recovered from D. reticulatum were assessed in the present study using a polyphasic taxonomic approach, including classical morphology, molecular biology and MALDI-TOF ICMS. Ninety-eight fungal strains were isolated from two D. reticulatum samples by using six different culture media, which were identified up to the genus level. Sixty-four distinct fungal ribotypes were obtained, distributed among twenty-four different genera belonging to the Ascomycota and Zygomycota. Representatives of Penicillium and Trichoderma were the most diverse and abundant fungi isolated. Amongst Penicillium spp. three isolates belonged to the same ribotype can be considered as a putative new species. Data derived from the present study highlight the importance of using a polyphasic approach to get an accurate identification in order to structure a reliable culture collection.

Fungal endophyte diversity in soybean.

AIM: To determine the identity and diversity of endophytes in soybean plants using culture-dependent (CD) and culture-independent (CI) methods. METHODS AND RESULTS: Stem samples were collected from three field-grown soybean cultivars grown to a reproductive stage in Minnesota, USA. Samples were surface disinfested, and CD and CI methods were used to assess the endophytes. For the CD method, fungi were isolated and grouped based on colony morphology, and the rDNA ITS region was sequenced to identify the cultures. The most frequently isolated genera were Cladosporium (36%), Alternaria (13%), Diaporthe (9%) and Epicoccum (9%). For the CI method, DNA was extracted from the stems, and the ITS region was amplified, cloned and sequenced for identification. The most prevalent genus detected using CI method was Cladosporium (85%). CONCLUSIONS: Soybean contains a diverse array of endophytic fungi that were identified in this study. The CD method detected greater endophyte diversity (H' = 2·12) than the CI method (H' = 0·66). SIGNIFICANCE AND IMPACT OF THE STUDY: The results improve our understanding of the identity and diversity of endophytic fungi that likely have different kinds of interactions with soybean plants. The results suggest that CD and CI methods should be used to study endophytes in soybean and perhaps other annual crop plants.

Paleomycology of the Princeton Chert I. Fossil hyphomycetes associated with the early Eocene aquatic angiosperm, Eorhiza arnoldii.

The Eocene (~ 48.7 Ma, Ypresian-Lutetian) Princeton Chert of British Columbia, Canada, has long been recognized as a significant paleobotanical locality, and a diverse assemblage of anatomically preserved fossil plants has been extensively documented. Co-occurring fossil fungi also have been observed, but the full scope of their diversity has yet to be comprehensively assessed. Here, we present the first of a series of investigations of fossilized fungi associated with the silicified plants of the Princeton Chert. This report focuses on saprotrophic, facultative-aquatic hyphomycetes observed in cortical aerenchyma tissue of an enigmatic angiosperm, Eorhiza arnoldii. Our use of paleontological thin sections provides the opportunity to observe and infer developmental features, making it possible to more accurately attribute two hyphomycetes that were observed in previous studies. These comprise multiseptate, holothallic, chlamydospore-like phragmoconidia most similar to extant Xylomyces giganteus and basipetal phragmospore-like chains of amerospores like those of extant Thielaviopsis basicola. We also describe a third hyphomycete that previously has not been recognized from this locality; biseptate, chlamydosporic phragmoconidia are distinguished by darkly melanized, inflated apical cells and are morphologically similar to Brachysporiella rhizoidea or Culcitalna achraspora.