Screening of White-Rot Fungi Isolates for Decolorization of Pulp and Paper Mill Effluent and Assessment of Biodegradation and Biosorption Processes.

Ten white-rot fungal isolates were evaluated for the decolorization potential of pulp and paper mill effluent. Trametes elegans PP17-06, Pseudolagarobasidium sp. PP17-33, and Microporus sp.2 PP17-20 showed the highest decolorization efficiencies between 42 and 54% in 5 d. To reveal the mechanisms involved in decolorization and assess the long-term performance, PP17-06, which showed the highest decolorization efficiency, was further investigated. It could reduce the ADMI color scale by 63.6% in 10 d. However, extending the treatment period for more than 10 d did not significantly enhance the decolorization efficiencies. The maximum MnP activity of 3.27 U L-1 was observed on the 6 d during the biodegradation. In comparison, laccase activities were low with the maximum activity of 0.38 U L-1 (24 d). No significant LiP activities were monitored during the experiment. Dead fungal biomass showed an optimum decolorization efficiency of 44.18% in 8 d employing the biosorption mechanism. No significant changes in the decolorization efficiency were observed after that, suggesting the equilibrium status was reached. These results revealed that PP17-06 has the potential to decolorize pulp and paper mill effluent by employing both biodegradation and biosorption processes.

Bilateral Trichosporon asahii keratitis after ptosis correction: A case report.

RATIONALE: Fungal keratitis (FK) is a severe vision-threatening disease that can lead to corneal perforation or endophthalmitis despite proper treatment. It is important to diagnose the disease promptly due to its indolent nature and disproportionate disease symptoms. Trichosporon asahii is reported rarely as the causative organism of FK. We report a case of highly unusual bilateral T asahii keratitis following ptosis surgery. PATIENT CONCERNS: An 86-year-old female underwent bilateral levator resection surgery for ptosis. Postoperatively, the patient complained of gradually worsening bilateral ocular pain and a decrease in visual acuity associated with a chronic non-healing epithelial defect. DIAGNOSES: Both eyes of the patient were evaluated using best-corrected visual acuity, intraocular pressure, slit-lamp examination, fundus examination, and corneal culture. Multifocal deep stromal infiltrates were found in both corneas. Cultures from both corneal ulcers revealed growth of T asahii. Optical coherence tomographic examination showed bilateral macular edema. INTERVENTIONS: The patient was treated with revisional ptosis surgery, an antifungal agent for the corneal ulcer, and intravitreal injection of steroid for macular edema. OUTCOMES: Both eyes recovered well. Her best-corrected visual acuity improved from 20/200 to 20/40 in the right eye and from 20/100 to 20/40 in the left eye. LESSONS: FK can develop in the cornea when certain risk factors are present, including recent lid surgery, chronic keratitis, and steroid eye drop use. Identification and correction of risk factors can be beneficial in the treatment of FK.

Phlebopus roseus, a new edible bolete from China, is associated with insects and plants.

Phlebopus roseus is described as new based on collections from southwest China. Phylogenetic analyses of nuclear rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS) and portions of nuclear 28S rDNA (28S), translation elongation factor 1-alpha (tef1), and the largest and second largest subunits of RNA polymerase II (rpb1, rpb2) support P. roseus as a novel species in the genus Phlebopus (Boletinellaceae, Boletales). The new species resembles P. portentosus but differs from it in that mature basidiomata have a bright rose-red-colored stipe and a radiate tubular hymenophore with nested pores. Despite extensive searching, P. roseus has only been found at four sites within a 24-hectare orchard dominated by Eriobotrya japonica, which is agriculturally important given its fruit production (loquats). Therefore, this species appears to be endemic and geographically restricted. The ecology of this bolete is also unique. In line with the trophic behavior of other species in the Boletinellaceae, our observations indicate that P. roseus forms a symbiotic association with the scale insect Coccus hesperidum, identified through sequence analysis of its mitochondrial cytochrome c oxidase subunit I (COI) region, to form fungus-insect galls that develop on roots of E. japonica trees. Phlebopus roseus is an edible mushroom species and is collected from the type location by farmers and sold commercially in limited quantities at local markets alongside P. portentosus and other fungi.

Biological activities of melanin pigment extracted from Bombyx mori gut-associated yeast Cryptococcus rajasthanensis KY627764.

Melanin pigment has been produced and extracted from a wide variety of living forms ranging from microorganisms to higher organisms. Owing to the therapeutic nature of the pigment, various microbial populations have been explored for its production. Hence, we isolated a melanin producing yeast from the insect Bombyx mori gut microflora and identified it as Cryptococcus rajasthanensis based on the molecular characterization. The isolated yeast produced enhanced melanin pigment when cultured in the minimal L-tyrosine broth as compared to the Saboraud medium. The pigment was extracted and characterized as melanin based on UV-Visible spectroscopy, FTIR (Fourier-transform infrared) spectroscopy and 1H NMR (Nuclear magnetic resonance). The melanin pigment was evaluated as a potent bioactive molecule with bioactivity like antimicrobial, antioxidant, anti-inflammatory, and anticancer activity that describes the therapeutic nature of the extracted melanin pigment. Distinct from the biologically active role the melanin pigment isolated from the yeast, the Cryptococcus extract also exhibited killer toxin activity against the pathogenic yeast Candida albicans.

Characteristics of the Airway Microbiome of Cystic Fibrosis Patients.

Microbiota as an integral component of human body is actively investigated, including by massively parallel sequencing. However, microbiomes of lungs and sinuses have become the object of scientific attention only in the last decade. For patients with cystic fibrosis, monitoring the state of respiratory tract microorganisms is essential for maintaining lung function. Here, we studied the role of sinuses and polyps in the formation of respiratory tract microbiome. We identified Proteobacteria in the sinuses and samples from the lower respiratory tract (even in childhood). In some cases, they were accompanied by potentially dangerous basidiomycetes. The presence of polyps did not affect formation of the sinus microbiome. Proteobacteria are decisive in reducing the biodiversity of lung and sinus microbiomes, which correlated with the worsening of the lung function indicators. Soft mutations in the CFTR gene contribute to the formation of safer microbiome even in heterozygotes with class I mutations.

Bloodstream Infection of a Cancer Patient by Cystobasidium minutum: A Case Report and Literature Review.

Emergent fungal infections are uncommon conditions which frequently lead to death. To our knowledge, only a few cases of invasive infection by Cystobasidium minutum (previously known as Rhodotorula minuta) have been reported. Moreover, several factors are responsible for deep site infections, such as catheter-related fungemia. This report describes the first case report of Cystobasidium minutum causing fungemia in Brazil. The pathogens fungemia was demonstrated by catheter and blood culture-proven, and both yeasts were identified by sequences of D1/D2 rDNA region. After the end of antifungal therapy and catheter removal, a second blood culture was found to be negative and the clinical signs and symptoms of the patient improved.

Cryptococcus magnus Periprosthetic Shoulder Infection: A Case Report.

CASE: We present a 68-year-old diabetic man with a periprosthetic shoulder infection (PSI) by Cryptococcus magnus. The fungus was identified 4 weeks after a revision surgery for a chronic bacterial PSI. Treatment was successful with a 2-week course of intravenous liposomal amphotericin B (5 mg/kg) followed by 6 weeks of fluconazole (200 mg/d). CONCLUSION: To the best of our knowledge, only 2 periprosthetic infections due to Cryptococcus neoformans have been previously reported. This is the first report of cryptococcal involvement of a prosthetic shoulder, and the first identified osteoarticular infection ever by C. magnus.

Rhinosinusitis in an Australian mare caused by Flavodon flavus, a recently recognized invasive fungal pathogen of the horse.

We describe herein the clinical, endoscopic, computed tomography (CT), pathologic, and microbiologic features of an infection caused by an under-recognized fungal pathogen, Flavodon flavus, in a 25-y-old Australian Quarter Horse. The horse had a unilateral obstructive nasal mass, resulting in stertor and dyspnea. On endoscopy, the mass was tan, multinodular, and completely obstructed the nasal passage. CT analysis revealed a large, soft tissue-attenuating and partially mineralized mass in the right nasal passage and dorsal-conchofrontal sinus, expanding into adjacent paranasal sinuses with associated bone lysis and rhinosinusitis. Histopathology of the mass on 2 occasions revealed suppurative inflammation initially, and pyogranulomatous inflammation subsequently. The inflammatory reaction surrounded numerous spherical fungal structures (~60-80 µm diameter) that stained positively on periodic acid-Schiff and Grocott methenamine silver stains. PCR for the fungal internal transcribed spacer 1 and 2 regions followed by Sanger sequencing on a cultured isolate identified the agent as F. flavus, which has only been reported previously as pathogenic in one horse in the United States, to our knowledge. Previous reports described this fungus as a nonpathogenic, environmental commensal fungus associated with insects and plants.

Insights into fungal diversity of a shallow-water hydrothermal vent field at Kueishan Island, Taiwan by culture-based and metabarcoding analyses.

This paper reports the diversity of fungi associated with substrates collected at a shallow hydrothermal vent field at Kueishan Island, Taiwan, using both culture-based and metabarcoding methods. Culture of fungi from yellow sediment (with visible sulfur granules), black sediment (no visible sulfur granules), the vent crab Xenograpsus testudinatus, seawater and, animal egg samples resulted in a total of 94 isolates. Species identification based on the internal transcribed spacer regions of the rDNA revealed that the yellow sediment samples had the highest species richness with 25 species, followed by the black sediment (23) and the crab (13). The Ascomycota was dominant over the Basidiomycota; the dominant orders were Agaricales, Capnodiales, Eurotiales, Hypocreales, Pleosporales, Polyporales and Xylariales. Hortaea werneckii was the only common fungus isolated from the crab, seawater, yellow and black sediment samples. The metabarcoding analysis amplifying a small fragment of the rDNA (from 18S to 5.8S) recovered 7-27 species from the black sediment and 12-27 species from the yellow sediment samples and all species belonged to the Ascomycota and the Basidiomycota. In the yellow sediments, the dominant order was Pleosporales and this order was also dominant in the black sediment together with Sporidiobolales. Based on the results from both methods, 54 and 49 species were found in the black and yellow sediments, respectively. Overall, a higher proportion of Ascomycota (~70%) over Basidiomycota was recovered in the yellow sediment and the two phyla were equally abundant in the black sediment. The top five dominant fungal orders in descending order based on species richness were Pleosporales>Eurotiales>Polyporales>Hypocreales>Capnodiales in the black sediment samples, and Polyporales>Pleosporales>Eurotiales>Capnodiales>Hypocreales in the yellow sediment samples. This study is the first to observe a high diversity of fungi associated with various substrates at a marine shallow water hydrothermal vent ecosystem. While some fungi found in this study were terrestrial species and their airborne spores might have been deposited into the marine sediment, several pathogenic fungi of animals, including Acremonium spp., Aspergillus spp., Fusarium spp., Malassezia spp., Hortaea werneckii, Parengyodontium album, and Westerdykella dispersa, were recovered suggesting that these fungi may be able to cause diseases of marine animals.

New species of Bannoa described from the tropics and the first report of the genus in South America.

The genus Bannoa consists of four described species associated with dead leaves in southwestern Japan. In this study, we describe three new species, Bannoa guamensis, B. rosea, and B. tropicalis, from the South Pacific island of Guam and Guyana in South America. Isolates were obtained from surfaces of diseased and healthy leaves of plants in the Euphorbiaceae, Asteraceae, and Poaceae. DNA sequences from four gene regions, including nuc rDNA internal transcribed spacer ITS1-5.8S-ITS2 (ITS), D1-D2 domains of nuc 28S rDNA (28S), nuc 18S rDNA (18S), and a portion of tef1, which encodes translation elongation factor 1-alpha, were produced for phylogenetic analysis. Intercompatibility tests were performed, and subsequent development of clamp connections and basidia were documented for B. tropicalis. Potential life history strategies and association with diseased leaves, including rust-infected leaves, were evaluated across the genus. This is the first report of a species of Bannoa from South America.

Apiotrichum veenhuisii isolated from a pediatric patient with acute myeloid leukemia: The first case in humans.

This is the first report of the yeast Apiotrichum veenhuisii (formerly Trichosporon veenhuisii) causing disease in humans; its virulence and in vitro behavior against antifungals were also studied. The sample was isolated from biopsy fragments of disseminated lesions on the skin of a pediatric patient with acute myeloid leukemia. The studied virulence factors evidenced that the strain tested negative for secretion of the enzymes proteinase, phospholipase, and hemolysin. The isolate was characterized as low biofilm producer. Except for amphotericin B and voriconazole, the sample presented high minimum inhibitory concentration values against azole and echinocandins.

The first survey of Cystobasidiomycete yeasts in the lichen genus Cladonia; with the description of Lichenozyma pisutiana gen. nov., sp. nov.

The view of lichens as a symbiosis only between a mycobiont and a photobiont has been challenged by discoveries of diverse associated organisms. Specific basidiomycete yeasts in the cortex of a range of macrolichens were hypothesized to influence the lichens' phenotype. The present study explores the occurrence and diversity of cystobasidiomycete yeasts in the lichen genus Cladonia. We obtained seven cultures and 56 additional sequences using specific primers from 27 Cladonia species from all over Europe and performed phylogenetic analyses based on ITS, LSU and SSU rDNA loci. We revealed yeast diversity distinct from any previously reported. Representatives of Cyphobasidiales, Microsporomycetaceae and of an unknown group related to Symmetrospora have been found. We present evidence that the Microsporomycetaceae contains mainly lichen-associated yeasts. Lichenozyma pisutiana is circumscribed here as a new genus and species. We report the first known associations between cystobasidiomycete yeasts and Cladonia (both corticate and ecorticate), and find that the association is geographically widespread in various habitats. Our results also suggest that a great diversity of lichen associated yeasts remains to be discovered.

Liver abscess due to Sterigmatomyces halophilus in a boy with acute lymphoblastic leukemia.

We report the first case of liver abscess due to Sterigmatomyces halophilus. Because this pathogen grows poorly in culture medium without added salts, it was identified by sequencing analysis targeting the rRNA gene internal transcribed spacer (ITS) region. This method could be useful for pathogens that cannot be cultured using standard methods.

Proteomic fingerprinting for the fast and accurate identification of species in the Polyporoid and Hymenochaetoid fungi clades.

Basidiomycotan fungi play significant roles in the biogeochemical cycle of carbon as wood decomposers and are used in the food industry for mushroom production and in biotechnology for the production of diverse bioactive compounds and bioremediation. The correct identification of basidiomycotan isolates is crucial for understanding their biology and being able to expand their applications. Currently, the identification of these organisms is performed by analyzing morphological and genomic characteristics, primarily those based on DNA biomarkers. Despite their efficiency, such methods require considerable expertise and are both time-consuming and error-prone (multistep workflow). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged in the last decade as an accurate, fast, and powerful alternative for the identification of microorganisms. MALDI-TOF MS has been widely applied for the identification and taxonomical characterization of both bacteria and ascomycotan fungi from clinical origins. However, species of Basidiomycota have been poorly evaluated using this method. In the present study, we assessed the performance of MALDI-TOF MS using basidiomycotan isolates of two distinct taxonomical families: Polyporaceae and Hymenochaetaceae. Using a simple protocol, which eliminates the protein extraction step, we obtained high-quality mass spectra data and demonstrated that this method is efficient for the discrimination of isolates at the species level. SIGNIFICANCE: In this study, the MALDI-TOF mass spectrometry was employed to test its accuracy on the recognition of fungal species with high biotechnological and environmental interest. Using a simple and fast protocol, we obtained high-quality mass-spectra (protein fingerprinting) and proved that MALDI-TOF MS is sufficiently robust to the identification at species level and to evaluate the relationships among the isolates of the polyporoid and hymenochaetoid clades (Basidiomycota).

Metagenomic insights into effects of wheat straw compost fertiliser application on microbial community composition and function in tobacco rhizosphere soil.

The application of fertilisers incorporated with plant residues improves nutrient availability in soils, which shifts the microbial community structure and favours plant growth. To understand the impact of wheat straw compost fertiliser on soil properties and microbial community structure, tobacco planting soils were treated with four different fertilisers using varied amounts of straw compost fertiliser and a no fertiliser control (CK). Results showed that different fertilisers affected available soil nutrient contents differently. Treatment of tobacco soil with application of combined chemical fertiliser/wheat straw compost led to improved soil chemical properties, and increased soil organic matter and available phosphorus and potassium content. Treatment with FT1 200 kg/mu straw was found to be superior in improving soil fertility. Metagenomic DNA sequencing revealed that different fertiliser treatments resulted in changes in the microbial community composition. In soil treated with FT2 300 kg/mu straw for 60 days, the predominant bacterial phyla were Proteobacteria, Actinobacteria, and Verrucomicrobia, whereas Cyanobacteria, Basidiomycota, and Chlorophyta were found in high abundance in soil samples treated with FT1 200 kg/mu straw for 30 days. Functional annotation of metagenomic sequences revealed that genes involved in metabolic pathways were among the most abundant type. PCoA analysis clearly separated the samples containing straw compost fertiliser and chemical fertiliser. A significant correlation between soil properties and the dominant phyla was identified.

Cystobasidium halotolerans sp. nov., a novel basidiomycetous yeast species isolated from the Arabian Gulf.

Two strains, 1Y129 and 3Y383, which represent a single novel anamorphic yeast species, were isolated from the Arabian Gulf surrounding Qatar. The two strains have identical sequences in the D1/D2 domains of the large subunit (LSU) and the internal transcribed spacer (ITS) regions of the rRNA gene. On the basis of this sequence analysis the two strains were found to be closely related to Cystobasidium slooffiae CBS5706T with 97. 3 % nucleotide substitutions (12 nt; four gaps) in D1/D2 regions of the LSU rRNA and 97.0 % nucleotide substitutions (14 nt; two gaps) in ITS regions. In contrast to Cystobasidium halotolerans sp. nov., the related species Cystobasidium slooffiae, Cystobasidium minutum and Cystobasidium fimetarium were unable to assimilate d-galactose, soluble starch, galactitol, methanol or nitrate, and grew at 37 °C. The name proposed for this undescribed species is Cystobasidium halotolerans sp. nov. The ex-type strain is QCC/Y31/17 (=MUCL057192=CGMCC2.5570).

Ethyl-methanesulfonate mutagenesis generated diverse isolates of Puccinia striiformis f. sp. tritici, the wheat stripe rust pathogen.

Puccinia striiformis f. sp. tritici (Pst) is an obligate biotrophic fungal pathogen causing stripe rust, one of the most important diseases of wheat worldwide. Mutation is considered as one of the major mechanisms causing virulence changes in the pathogen population, but experimental evidence is limited. To study the effect of mutation on pathogen variation, we developed 33 mutant isolates by treating urediniospores of Pst race PSTv-18, avirulent to all of the 18 Yr single-gene lines used to differentiate Pst races, with ethyl methanesulfonate (EMS). These isolates were characterized as 24 races, including 19 new races, through virulence testing on the set of 18 wheat Yr single-gene differential lines; and as 21 multi-locus genotypes with 19 simple sequence repeat and 48 single-nucleotide polymorphism markers. Most of the mutant isolates had more than one avirulence gene and more than one marker locus changed compared to the wild type isolate, indicating that EMS is able to cause mutations at multiple genome sites. The results showed that mutation can cause substantial changes in both avirulence and other genomic regions. The different frequencies of virulence among the mutant isolates suggested homozygous or heterozygous avirulence loci in the parental isolate, or relative ease of mutation at some avirulence loci. The results are useful for understanding evolutionary mechanisms of the important fungal pathogen.

Anti-tumour Effect and Pharmacokinetics of an Active Ingredient Isolated from Inonotus hispidus.

Inonotus hispidus is an anti-tumour drug used in folk medicine. (4S,5S)-4-Hydroxy-3,5-dimethoxycyclohex-2-enone (HDE) is a compound isolated from Inonotus hispidus for the first time. However, the mechanisms underlying its therapeutic effects have not been elucidated. In this study, the in vitro screening, in vivo anti-tumour effects, mechanism of action, pharmacokinetics, and tissue distribution of HDE were investigated. HDE could inhibit the proliferation of HepG2 cells. Additionally, its half-maximal inhibitory concentration was 7.9 µg/mL. Increasing HDE concentrations significantly increased apoptosis rate in a dose-dependent manner. Furthermore, HDE was rapidly absorbed into mouse plasma, reaching a maximum concentration at 30 min. The area under the plasma HDE concentration-time curves for the studied organs were as follows: spleen > liver > lung > kidney > muscle > thymus > heart > brain. HDE also inhibited tumour growth up to 69%. The weights of organs harvested from HDE-treated mice were not significantly different from those harvested from control mice. Furthermore, HDE upregulated Fas expression and downregulated FasL expression in HepG2 cells. HDE significantly increased caspase-3 and caspase-8 activity. The anti-tumour effect of HDE might be realized by activating the Fas-mediated apoptotic pathway. We also found that HDE undergoes enterohepatic circulation or is quickly absorbed by the body, and the drug is released back into systemic circulation. In conclusion, HDE significantly inhibited H22 hepatocarcinoma cells (H22)tumour growth in mice without damaging organs; therefore, it may be suitable for treating liver cancer.

Mrakia hoshinonis sp. nov., a novel psychrophilic yeast isolated from a retreating glacier on Ellesmere Island in the Canadian High Arctic.

Four strains isolated from sediment sampled at the front of a retreating glacier on northern Ellesmere Island in the Canadian high Arctic, namely JCM 32575T, JCM 32576, JCM 32577 and JCM 32578, belong to a novel psychrophilic basidiomycetous yeast species in the genus Mrakia. Molecular phylogenetic analysis indicated that these strains are most closely related to the type strains of Mrakia aquatica and Mrakianic combsii, but with 8-9 and 7-12 nt substitutions in ITS and in the D1/D2 domain of the LSU rRNA gene, respectively. The strains grew at sub-zero temperatures and in vitamin-free media, with lipase and cellulase highly active even at -3 °C. These characteristics likely allow this yeast species to grow and survive in extremely cold, oligotrophic environments, such as the fronts of retreating glaciers in the high Arctic. The name Mrakia hoshinonis sp. nov. is proposed, with type strain JCM 32575T (UAMH 11969) and MycoBank number MB 825484.