Basidiomycetous Yeast Research Articles

Soil amendments impact root‐associated fungal communities of balsam poplar on a phosphogypsum reclamation site

During reclamation organisms like fungi directly interact at the soil–plant interface and can potentially impact plant growth, health, and overall revegetation success. Therefore, a study was conducted to determine how topsoil amendments (mycorrhizae, biochar, compost, manure) influence root‐associated fungal communities and growth of fast growing Populus balsamifera trees on a phosphogypsum (PG) reclamation site in Alberta, Canada. Tree height, diameter, biomass, and diversity of Ascomycota and Basidiomycota were assessed using Illumina MiSeq sequencing targeting the fungal internal transcribed spacer region 1 (ITS1) and ITS2. Topsoil amendments and root location (topsoil vs. PG within a treatment), greatly affected the root fungal community. Basidiomycota preferentially grew in PG, and had a high treatment specific root fungal community with highest diversity of ectomycorrhizal fungi and basidiomycete yeast in biochar PG and compost PG. Populus balsamifera roots were associated with a high diversity of Ascomycota, found equally in roots in topsoil and PG, with a trend towards higher diversity and abundance in PG. The core genus found across all samples was Fusarium, a potential plant pathogen. All topsoil amendments strongly reduced abundance of Fusarium and other Ascomycota in poplar roots. Biochar and compost reduced number and abundance of potential fungal pathogens and increased number of potential plant growth promoting Basidiomycota, indicating favorable growth conditions for P. balsamifera on a phosphygypsum reclamation site by application of these amendments. This study indicates compost and biochar can be effective soil amendments to support P. balsamifera establishment and growth on a PG reclamation site.

Distinct Features Based on Partitioning of the Endophytic Fungi of Cereals and Other Grasses.

Endophytic fungi form a significant part of the plant mycobiome. Defining core members is crucial to understanding the assembly mechanism of fungal endophytic communities (FECs) and identifying functionally important community members. We conducted a meta-analysis of FECs in stems of wheat and five wild cereal species and generated a landscape of the fungal endophytic assemblages in this group of plants. The analysis revealed that several Ascomycota members and basidiomycetous yeasts formed an important compartment of the FECs in these plants. We observed a weak spatial autocorrelation at the regional scale and high intrahost variations in the FECs, suggesting a space-related heterogeneity. Accordingly, we propose that the heterogeneity among subcommunities should be a criterion to define the core endophytic members. Analysis of the subcommunities and meta-communities showed that the core and noncore members had distinct roles in various assembly processes, such as stochasticity, universal dynamics, and network characteristics, within each host. The distinct features identified between the community partitions of endophytes aid in understanding the principles that govern the assembly and function of natural communities. These findings can assist in designing synthetic microbiomes. IMPORTANCE This study proposes a novel method for diagnosing core microbiotas based on prevalence of community members in a meta-community, which could be determined and supported statistically. Using this approach, the study found stratification in community assembly processes within fungal endophyte communities (FECs) in the stems of wheat and cereal-related wild species. The core and noncore partitions of the FECs exhibited certain degrees of determinism from different aspects. Further analysis revealed abundant and consistent interactions between rare taxa, which might contribute to the determinism process in noncore partitions. Despite minor differences in FEC compositions, wheat FECs showed distinct patterns in community assembly processes compared to wild species, suggesting the effects of domestication on FECs. Overall, our study provided a new approach for identifying core microbiota and provides insights into the community assembly processes within FECs in wheat and related wild species.

Yeasts associated with mines on tree leaves in the urban areas.

Mines on tree leaves and undamaged leaves were studied to investigate yeast complexes in urban areas (Aesculus hippocastanum, miner - Cameraria ohridella; Betula verrucosa, miner - Caloptilia betulicola; Populus nigra, miner - Lithocolletis populifoliella; Quercus robur, miner - Tischeria companella; Salix caprea, miner - Trachys minuta; Syringa vulgaris, miner - Caloptilia syringella; Tilia cordata, miner - Phyllonorycter issikii; Ulmus laevis, miner - Carpatolechia fugitivella). The abundance and taxonomic structure of yeasts were studied using a surface plating method on solid media (GPY agar). Identification of yeast species was based on the ITS rDNA nucleotide sequence. The average abundance of yeasts during the first stages of mine formation in the internal tissues of leaves was 103cfu/g. After 23-25days, during the last stage of larval metamorphosis before mine destruction, the abundance of yeasts in the mines increased by two orders of magnitude to 105cfu/g. No significant differences were observed in the abundance of yeasts in mines formed by different insects on different trees. A total of twelve yeast species were observed. The fast-growing ascomycetous yeasts Hanseniaspora uvarum and H. occidentalis dominated the mines. On undamaged leaves, the basidiomycetous yeasts Papiliotrema flavescens and Rhodotorula mucilaginosa, typical in the phyllosphere, dominated. The opportunistic yeast Candida parapsilosis was detected in the yeast complexes of all mines examined and was not found on the surface of leaves. Comparison of the relative abundance of yeast species between the studied mines and undamaged leaves using principal component analysis showed that all studied yeast communities in the mines were significantly different from the epiphytic yeast complexes of the undamaged leaves. Thus, miners in urban environments provoke the formation of short-lived endophytic yeast complexes with high abundance of Hanseniaspora. For leaf miners, the yeasts serve primarily as a food source for insect larvae rich in vitamins and amino acids. The adult leaf miners, in turn, contribute to the reproduction of the yeasts and create favorable conditions for their development.

Metabolic mechanism of astaxanthin biosynthesis in Xanthophyllomyces dendrorhous in response to sodium citrate treatment

Astaxanthin is an important ketocarotenoid widely used in industries. However, its application is limited because of its low yield. Sodium citrate (Na-citrate), one of the major carbon sources for microorganisms, can promote cell growth and product accumulation. The basidiomycetous red yeast Xanthophyllomyces dendrorhous was thus used to study the effect of Na-citrate on cell growth and astaxanthin synthesis. The highest biomass and astaxanthin yield (6.0 g/L and 22.5 mg/L) were obtained in shake-flask when 3 g/L Na-citrate was added at 24 h and were 1.8 and 2.0 times higher than those of the control group, respectively. Furthermore, metabolomics and real-time reverse transcription PCR (qRT-PCR) analysis were conducted to study the metabolic pathways of X. dendrorhous in response to Na-citrate. The qRT-PCR assay revealed that Na-citrate facilitated glucose consumption, promoted the metabolic flux from glycolysis, and regulated the tricarboxylic acid (TCA) cycle, providing more energy and substrates for the synthesis of astaxanthin. The gene analysis revealed that adding Na-citrate significantly upregulated the expression of six key genes (ICL, HMGS, crtE, crtYB, crtI, and crtS) involved in pathways related to astaxanthin biosynthesis. These results suggest that exogenous Na-citrate treatment is a potentially valuable strategy to stimulate astaxanthin production in X. dendrorhous.

Co-evolution of large inverted repeats and G-quadruplex DNA in fungal mitochondria may facilitate mitogenome stability: the case of Malassezia

Mitogenomes are essential due to their contribution to cell respiration. Recently they have also been implicated in fungal pathogenicity mechanisms. Members of the basidiomycetous yeast genus Malassezia are an important fungal component of the human skin microbiome, linked to various skin diseases, bloodstream infections, and they are increasingly implicated in gut diseases and certain cancers. In this study, the comparative analysis of Malassezia mitogenomes contributed to phylogenetic tree construction for all species. The mitogenomes presented significant size and gene order diversity which correlates to their phylogeny. Most importantly, they showed the inclusion of large inverted repeats (LIRs) and G-quadruplex (G4) DNA elements, rendering Malassezia mitogenomes a valuable test case for elucidating the evolutionary mechanisms responsible for this genome diversity. Both LIRs and G4s coexist and convergently evolved to provide genome stability through recombination. This mechanism is common in chloroplasts but, hitherto, rarely found in mitogenomes.

Biodiversity of Basidiomycetous Yeasts Associated with Cladonia rei Lichen in Japan, with a Description of Microsporomyces cladoniophilus sp. nov.

For more than a century, lichens have been used as an example of dual-partner symbiosis. Recently, this has been challenged by the discovery of various basidiomycetous yeasts that coexist in multiple lichen species, among which Cladonia lichens from Europe and the United States were discovered to be highly specifically associated with the basidiomycetous yeast of the family Microsporomycetaceae. To verify this highly specific relationship, we investigated the diversity of basidiomycetous yeasts associated with Cladonia rei, a widely distributed lichen in Japan, by applying two approaches: yeast isolation from the lichen thalli and meta-barcoding analysis. We obtained 42 cultures of Cystobasidiomycetous yeast which were grouped into six lineages within the family Microsporomycetaceae. Unexpectedly, although the cystobasidiomycetes-specific primer was used, not only the cystobasidiomycetous yeasts but species from other classes were also detected via the meta-barcoding dataset; in particular, pucciniomycetous yeasts were found at a high frequency in some samples. Further, Halobasidium xiangyangense, which was detected in every sample with high abundance, is highly likely a generalist epiphytic fungus that has the ability to associate with C. rei. In the pucciniomycetous group, most of the detected species belong to the scale insect-associated yeast Septobasidium genus. In conclusion, even though Microsporomyces species are not the only yeast group associated with Cladonia lichen, our study demonstrated that the thalli of Cladonia rei lichen could be a suitable habit for them.

The first two mitochondrial genomes from Apiotrichum reveal mitochondrial evolution and different taxonomic assignment of Trichosporonales

Apiotrichum is a diverse anamorphic basidiomycetous yeast genus, and its mitogenome characterization has not been revealed. In this study, we assembled two Apiotrichum mitogenomes and compared them with mitogenomes from Agaricomycotina, Pucciniomycotina and Ustilaginomycotina. The mitogenomes of Apiotrichum gracile and A. gamsii comprised circular DNA molecules, with sizes of 34,648 bp and 38,096 bp, respectively. Intronic regions were found contributed the most to the size expansion of A. gamsii mitogenome. Comparative mitogenomic analysis revealed that 6.85–38.89% of nucleotides varied between tRNAs shared by the two Apiotrichum mitogenomes. The GC content of all core PCGs in A. gamsii was lower than that of A. gracile, with an average low value of 4.97%. The rps3 gene differentiated the most among Agaricomycotina, Pucciniomycotina and Ustilaginomycotina species, while nad4L gene was the most conserved in evolution. The Ka/Ks values for cob and rps3 genes were > 1, indicating the two genes may be subjected to positive selection in Agaricomycotina, Pucciniomycotina and Ustilaginomycotina. Frequent intron loss/gain events and potential intron transfer events have been detected in evolution of Agaricomycotina, Pucciniomycotina and Ustilaginomycotina. We further detected large-scale gene rearrangements between the 19 mitogenomes from Agaricomycotina, Pucciniomycotina and Ustilaginomycotina, and fifteen of the 17 mitochondrial genes shared by Apiotrichum varied in gene arrangements. Phylogenetic analyses based on maximum likelihood and Bayesian inference methods using a combined mitochondrial gene dataset revealed different taxonomic assignment of two Apiotrichum species, wherein A. gamsii had a more closely relationship with Trichosporon asahii. This study served as the first report on mitogenomes from the genus Apiotrichum, which promotes the understanding of evolution, genomics, and phylogeny of Apiotrichum.

Functional genome annotation and transcriptome analysis of Pseudozyma hubeiensis BOT-O, an oleaginous yeast that utilizes glucose and xylose at equal rates

Pseudozyma hubeiensis is a basidiomycete yeast that has the highly desirable traits for lignocellulose valorisation of being equally efficient at utilization of glucose and xylose, and capable of their co-utilization. The species has previously mainly been studied for its capacity to produce secreted biosurfactants in the form of mannosylerythritol lipids, but it is also an oleaginous species capable of accumulating high levels of triacylglycerol storage lipids during nutrient starvation. In this study, we aimed to further characterize the oleaginous nature of P. hubeiensis by evaluating metabolism and gene expression responses during storage lipid formation conditions with glucose or xylose as a carbon source.The genome of the recently isolated P. hubeiensis BOT-O strain was sequenced using MinION long-read sequencing and resulted in the most contiguous P. hubeiensis assembly to date with 18.95 Mb in 31 contigs. Using transcriptome data as experimental support, we generated the first mRNA-supported P. hubeiensis genome annotation and identified 6540 genes. 80% of the predicted genes were assigned functional annotations based on protein homology to other yeasts. Based on the annotation, key metabolic pathways in BOT-O were reconstructed, including pathways for storage lipids, mannosylerythritol lipids and xylose assimilation. BOT-O was confirmed to consume glucose and xylose at equal rates, but during mixed glucose-xylose cultivation glucose was found to be taken up faster. Differential expression analysis revealed that only a total of 122 genes were significantly differentially expressed at a cut-off of |log2 fold change| ≥ 2 when comparing cultivation on xylose with glucose, during exponential growth and during nitrogen-starvation. Of these 122 genes, a core-set of 24 genes was identified that were differentially expressed at all time points. Nitrogen-starvation resulted in a larger transcriptional effect, with a total of 1179 genes with significant expression changes at the designated fold change cut-off compared with exponential growth on either glucose or xylose.

Culturable Diversity of Lichen-Associated Yeasts through Enrichment Strategies

Lichens are symbiotic partnerships between a filamentous fungus and a photosymbiotic “alga”. Studies show that lichens harbor endothallic fungi, but that some taxa have been difficult to isolate from the main filamentous thallus-forming fungus and other faster growing lichenicolous/endothallic fungi. Therefore, we aimed to develop and evaluate liquid yeast-enrichment strategies to (1) isolate lichen-associated yeasts in pure culture, and (2) determine the taxonomic placement and breadth of the diversity of culturable yeasts. Eighty-two lichen samples were collected and washed with distilled water, and healthy thalli were ground up and added to seven different yeast-enrichment broths. Yeast colonies were isolated in pure culture and identified using molecular techniques. Initial isolates were identified using BLASTn analysis, and a taxonomic refinement was completed using PhyML analysis. In total, 215 isolates were obtained. The most prevalently isolated ascomycetous yeasts were within the Dothideomycetes (Aureobasidium, Plowrightia, and Dothiora), while the most frequently isolated basidiomycetous yeasts belonged to the genera Curvibasidium, Sporobolomyces, and Tremella. The generic placements could not be determined for 17 isolates, and in total 25 novel species were recovered. The results of this research indicate that (1) lichen-associated yeasts are diverse, (2) employing liquid enrichment strategies is effective for isolating many of these, and (3) lichen thalli represent a valuable untapped reservoir of diverse and novel yeast species.

Hannaella floricola sp. nov., a novel basidiomycetous yeast species isolated from a flower of Lantana camara in Portugal.

During a survey of floricolous yeasts in Portugal, a basidiomycetous yeast representing a novel species in the genus Hannaella was isolated in Portugal from the flower of Lantana camara, an ornamental exotic species native to Central and South America. A combination of phylogenetic analyses of DNA barcode sequences used in yeast molecular systematics, namely the D1/D2 domain and the complete internal transcribed spacer (ITS) region supported the recognition of a new species of Hannaella, that we designate Hannaella floricola sp. nov. (ex-type strain PYCC 9191T=CBS 18097T). Although the assignment of the new species to the genus Hannaella was evident, the detection of its closest relatives appeared more problematic. Nevertheless, our analyses suggested that H. floricola sp. nov. belongs a clade that also includes H. coprosmae, H. oryzae and H. surugaensis, together four candidate novel species. In addition we provide the molecular identification of several unidentified strains whose D1/D2 and ITS sequences are available from GenBank.

Exogenous glucosylglycerol and proline extend the chronological lifespan of Rhodosporidium toruloides.

Glucosylglycerol (GG) is an osmolyte found in a few bacteria (e.g., cyanobacteria) and plants grown in harsh environments. GG protects microbes and plants from salinity and desiccation stress. In the industry, GG is synthesized from a combination of ADP-glucose and glycerol-3-phosphate in a condensation reaction catalyzed by glucosylglycerol phosphate synthase. Proline, on the other hand, is an amino acid-based osmolyte that plays a key role in cellular reprograming. It functions as a protectant and a scavenger of reactive oxygen species. Studies on lifespan extension have focused on the use of Saccharomyces cerevisiae. Rhodosporidium toruloides, also known as Rhodotorula toruloides, is a basidiomycetous oleaginous yeast known to accumulate lipids to more than 70% of its dry cell weight. The oleaginous red yeast (R. toruloides) has not been intensely studied in the lifespan domain. We designed this work to investigate how GG and proline promote the longevity of this red yeast strain. The results obtained in our study confirmed that these molecules increased R. toruloides' viability, survival percentage, and lifespan upon supplementation. GG exerts the most promising effects at a relatively high concentration (100 mM), while proline functions best at a low level (2 mM). Elucidation of the processes underlying these favorable responses revealed that GG promotes the yeast chronological lifespan (CLS) through increased catalase activity, modulation of the culture medium pH, a rise in ATP, and an increase in reactive oxygen species (ROS) accumulation (mitohormesis). It is critical to understand the mechanisms of these geroprotector molecules, particularly GG, and the proclivity of its lifespan application; this will aid in offering clarity on its potential application in aging research.

Rapid bacterial and fungal successional dynamics in first year after chaparral wildfire.

The rise in wildfire frequency and severity across the globe has increased interest in secondary succession. However, despite the role of soil microbial communities in controlling biogeochemical cycling and their role in the regeneration of post-fire vegetation, the lack of measurements immediately post-fire and at high temporal resolution has limited understanding of microbial secondary succession. To fill this knowledge gap, we sampled soils at 17, 25, 34, 67, 95, 131, 187, 286, and 376 days after a southern California wildfire in fire-adapted chaparral shrublands. We assessed bacterial and fungal biomass with qPCR of 16S and 18S and richness and composition with Illumina MiSeq sequencing of 16S and ITS2 amplicons. Fire severely reduced bacterial biomass by 47%, bacterial richness by 46%, fungal biomass by 86%, and fungal richness by 68%. The burned bacterial and fungal communities experienced rapid succession, with 5-6 compositional turnover periods. Analogous to plants, turnover was driven by "fire-loving" pyrophilous microbes, many of which have been previously found in forests worldwide and changed markedly in abundance over time. Fungal secondary succession was initiated by the Basidiomycete yeast Geminibasidium, which traded off against the filamentous Ascomycetes Pyronema, Aspergillus, and Penicillium. For bacteria, the Proteobacteria Massilia dominated all year, but the Firmicute Bacillus and Proteobacteria Noviherbaspirillum increased in abundance over time. Our high-resolution temporal sampling allowed us to capture post-fire microbial secondary successional dynamics and suggest that putative tradeoffs in thermotolerance, colonization, and competition among dominant pyrophilous microbes control microbial succession with possible implications for ecosystem function.

Different Geographic Strains of Dinoflagellate Karlodinium veneficum Host Highly Diverse Fungal Community and Potentially Serve as Possible Niche for Colonization of Fungal Endophytes.

In numerous studies, researchers have explored the interactions between fungi and their hosting biota in terrestrial systems, while much less attention has been paid to the counterpart interactions in aquatic, and particularly marine, ecosystems. Despite the growing recognition of the potential functions of fungi in structuring phytoplankton communities, the current insights were mostly derived from phytoplankton hosts, such as diatoms, green microalgae, and cyanobacteria. Dinoflagellates are the second most abundant group of phytoplankton in coastal marine ecosystems, and they are notorious for causing harmful algal blooms (HABs). In this study, we used high-throughput amplicon sequencing to capture global snapshots of specific fungal assemblages associated with laboratory-cultured marine dinoflagellate. We investigated a total of 13 clonal cultures of the dinoflagellate Karlodinium veneficum that were previously isolated from 5 geographic origins and have been maintained in our laboratory from several months to more than 14 years. The total recovered fungal microbiome, which consisted of 349 ASVs (amplicon sequencing variants, sequences clustered at a 100% sequence identity), could be assigned to 4 phyla, 18 classes, 37 orders, 65 families, 97 genera, and 131 species. The fungal consortium displayed high diversity and was dominated by filamentous fungi and ascomycetous and basidiomycetous yeasts. A core set of three genera among all the detected fungi was constitutively present in the K. veneficum strains isolated from geographically distant regions, with the top two most abundant genera, Thyridium and Pseudeurotium, capable of using hydrocarbons as the sole or major source of carbon and energy. In addition, fungal taxa previously documented as endophytes in other hosts were also found in all tested strains of K. veneficum. Because host-endophyte interactions are highly variable and strongly case-dependent, these fungal taxa were not necessarily genuine endosymbionts of K. veneficum; instead, it raised the possibility that dinoflagellates could potentially serve as an alternative ecological niche for the colonization of fungal endophytes. Our findings lay the foundation for further investigations into the potential roles or functions of fungi in the regulation of the growth dynamics and HABs of marine dinoflagellates in the field.

Diversity of duckweed (<i>Lemnaceae</i>) associated yeasts and their plant growth promoting characteristics

<abstract> <p>The diversity of duckweed (<italic>Lemnaceae</italic>) associated yeasts was studied using a culture-dependent method. A total of 252 yeast strains were isolated from 53 duckweed samples out of the 72 samples collected from 16 provinces in Thailand. Yeast identification was conducted based on the D1/D2 region of the large subunit (LSU) rRNA gene sequence analysis. It revealed that 55.2% and 44.8% yeast species were Ascomycota and Basidiomycota duckweed associated yeasts, respectively. Among all, <italic>Papiliotrema laurentii</italic>, a basidiomycetous yeast, was found as the most prevalent species showing a relative of frequency and frequency of occurrence of 21.8% and 25%, respectively. In this study, high diversity index values were shown, indicated by the Shannon-Wiener index (<italic>H′</italic>), Shannon equitability index (<italic>E<sub>H</sub></italic>) and Simpson diversity index (<italic>1-D</italic>) values of 3.48, 0.86 and 0.96, respectively. The present results revealed that the yeast community on duckweed had increased species diversity, with evenness among species. Principal coordinate analysis (PCoA) revealed no marked differences in yeast communities among duckweed genera. The species accumulation curve showed that the observed species richness was lower than expected. Investigation of the plant growth promoting traits of the isolated yeast on duckweed revealed that 178 yeast strains produced indole-3-acetic acid (IAA) at levels ranging from 0.08–688.93 mg/L. Moreover, siderophore production and phosphate solubilization were also studied. One hundred and seventy-three yeast strains produced siderophores and exhibited siderophores that showed 0.94–2.55 activity units (AU). One hundred six yeast strains showed phosphate solubilization activity, expressed as solubilization efficiency (SE) units, in the range of 0.32–2.13 SE. This work indicates that duckweed associated yeast is a potential microbial resource that can be used for plant growth promotion.</p> </abstract>

Begerowomyces aurantius sp. nov., a novel yeast isolated from koalas' habitat in a Japanese zoological park.

Koalas are iconic mammals indigenous to Australia. These rare animals and their habitats are occasionally associated with pathogenic fungi, including species of Cryptococcus, and consequently, monitoring the mycobiota of areas inhabited by koalas is of considerable importance. In this report, we describe a novel basidiomycetous yeast isolated from a site in Kanazawa Zoo, Japan, associated with captive koalas. Swab samples were collected from koala breeding environments, from which we isolated a novel unencapsulated yeast characterized by ovoid to ellipsoidal cells (3.2-4.9 × 3.5-5 μm). These cells were observed to undergo polar budding and grow as parent bud pairs, with an optimal growth temperature of 28°C. Colonies grown on yeast extract peptone dextrose agar at 28°C have a characteristic coral pink color. On the basis of physiological, morphological, and molecular characters, the new species was placed in the genus Begerowomyces, and the name Begerowomyces aurantius JCM33898T(LSEM1333T=CBS16241T) is proposed.

<i>Leucosporidium egoroviorum</i> f.a., sp. nov., a New Yeast Species Isolated from Zucchini

Abstract—Leucosporidium egoroviorum f.a., sp. nov., a new anamorphic species of the basidiomycetous yeasts, was isolated as an endophyte from the fruits of Cucurbita pepo subsp. pepo (zucchini). According to its genetic, physiological, and morphological characteristics, the new species differed significantly from the closely related species L. fellii, L. intermedium, and L. krtinense. The holotype of the new specise, KBP Y-6804T, is preserved in a metabolically inactive state; its isotype strains are VKM Y-3065, DSM 113574, and CBS 17590. The MycoBank number is MB 842805.

JMM Profile: Trichosporon yeasts: from superficial pathogen to threat for haematological-neutropenic patients.

Trichosporon yeasts are classical agents of superficial mycoses, and they are ranked as the first to second predominant basidiomycetous yeast able to cause invasive infections. The clinical presentation of Trichosporon infections varies with the affected anatomical site, with fungaemia present in the majority of invasive trichosporonosis cases. Only a limited number of antifungal compounds can be used to treat Trichosporon infections. Azoles are the first choice due to their intrinsic resistance to echinocandins. Better laboratory methods and up-to-date databases of commercial platforms are required to improve identification, susceptibility testing and surveillance of this potentially threating infection.

RNAi machinery regulates nutrient metabolism and fluconazole resistance in the pathogenic fungus Cryptococcus deneoformans.

The RNAi machinery has been extensively studied in plant and animal cells for their crucial roles in the regulation of genome function. However, the potential roles of RNAi in controlling fungal growth and development have been poorly studied, especially in the basidiomycetous yeast Cryptococcus deneoformans. To characterize the biological functions of RNAi in the pathogenic fungus, a comparative analysis of mRNA profiles using high-throughput sequencing technology was performed for the wild type and the RNAi mutants of C. deneoformans. The results revealed a clear difference in the expression of genes associated with metabolic processes in the RNAi mutants. Besides, the growth under nutrient-limited conditions was significantly reduced in the ago2Δ mutant, suggesting the essential roles of Ago2 in nutrient metabolism. Further investigations revealed the differentially expressed transporters in the RNAi mutants, in which transporters involved in fluconazole efflux were significantly up-regulated. More importantly, on account of the upregulated transporters, RNAi mutant strains developed resistance to fluconazole. By disrupting AFR1 gene using the 'suicide' CRISPR-Cas9 system, we verified that the upregulated ABC transporter Afr1 in the RNAi mutants contributed to the fluconazole resistance. In summary, our data demonstrate that in C. deneoformans the RNAi pathway participates in nutrient metabolism and plays a role in the repression of fluconazole resistance, which provides a deep insight into RNAi mechanisms in Cryptococcus and brings great hints for the clinical treatment of cryptococcosis.

Legacy effects of fumigation on soil bacterial and fungal communities and their response to metam sodium application

BackgroundSoil microorganisms are integral to maintaining soil health and crop productivity, but fumigation used to suppress soilborne diseases may affect soil microbiota. Currently, little is known about the legacy effects of soil fumigation on soil microbial communities and their response to fumigation at the production scale. Here, 16S rRNA gene and internal transcribed spacer amplicon sequencing was used to characterize the bacterial and fungal communities in soils from intensively managed crop fields with and without previous exposure to metam sodium (MS) fumigation. The effect of fumigation history, soil series, and rotation crop diversity on microbial community variation was estimated and the response of the soil microbiome to MS application in an open microcosm system was documented.ResultsWe found that previous MS fumigation reduced soil bacterial diversity but did not affect microbial richness and fungal diversity. Fumigation history, soil series, and rotation crop diversity were the main contributors to the variation in microbial β-diversity. Between fumigated and non-fumigated soils, predominant bacterial and fungal taxa were similar; however, their relative abundance varied with fumigation history. In particular, the abundance of Basidiomycete yeasts was decreased in fumigated soils. MS fumigation also altered soil bacterial and fungal co-occurrence network structure and associations. In microcosms, application of MS reduced soil microbial richness and bacterial diversity. Soil microbial β-diversity was also affected but microbial communities of the microcosm soils were always similar to that of the field soils used to establish the microcosms. MS application also induced changes in relative abundance of several predominant bacterial and fungal genera based on a soil’s previous fumigation exposure.ConclusionsThe legacy effects of MS fumigation are more pronounced on soil bacterial diversity, β-diversity and networks. Repeated fumigant applications shift soil microbial compositions and may contribute to differential MS sensitivity among soil microorganisms. Following MS application, microbial richness and bacterial diversity decreases, but microbial β-diversity was similar to that of the field soils used to establish the microcosms in the short-term (< 6 weeks). The responses of soil microbiome to MS fumigation are context dependent and rely on abiotic, biotic, and agricultural management practices.

Opportunistic Pathogens of the Genus Cryptococcus in Louis Pasteur Days and in 200th Anniversary of his Birth

Abstract This year we are celebrating the 200th anniversary of the birth of Louis Pasteur, one of the fathers of microbiology. Interestingly, the time when Pasteur disproved the doctrine of „spontaneous generation” and announced the “germ theory of disease” coincides with the discovery of Cryptococcus neoformans and its role in cryptococcosis. Today, only in the realm of guesswork can remain the correct answer to the question „whether the observed parallelism of these events was accidental?” or „whether Pasteur’s discoveries constituted a solid foundation of the research on the etiological factors of cryptococcosis?”. Until recently, it might seem that all major virulence factors of pathogenic fungi of the Cryptococcus species complex have been thoroughly described. Meanwhile, the simultaneous publication in 2018 of three in vitro protocols for the induction of Titan cells, also known as giant cells, opened up new possibilities for research on the relatively uncharacterized virulence factor that is crucial for Cryptococcus spp. Research on the titanization process makes us realize how little we know about the virulence factors of these fungi, and how much more can be improved in the context of the treatment and prevention of cryptococcosis. The following review is not only a historical outline of research on Cryptococcus spp. and cryptococcosis, but also synthetically describes the virulence factors of these basidiomycetous yeasts, with particular emphasis on the titanization process. The phenomenon of titanization as a process of a specific morphological transformation, like Titan cells, are completely new terms in Polish literature, which will be introduced to readers here. We live in a post-antibiotic era where the lack of effective and non-toxic drugs affects patients all over the world. Specifically, the availability of only fluconazole, amphotericin B and flucytosine in therapy of cryptococcosis constitutes a significant limitation. For this reason, research on the virulence factors of Cryptococcus spp. will allow to find new effective antimycotics, including inhibitors of the titanization process.