First report of fossil representative of Zygosporium mont. With stacked chained vesicular conidiophores from India.

In the present manuscript, we describe and illustrate a novel foliicolous fossil-species of Zygosporium Mont. (Zygosporiaceae: Xylariales: Sordariomycetes) on compressed monocot leaf recovered from the Middle Siwalik sediments (Late Miocene) of Himachal Pradesh, western Himalaya. The new fossil-species characterized by macronematous, dark brown, unbranched or occasionally branched conidiophores with a chain of up to four integrated dark brown, curved, thick-walled, swollen, hook-like, alternately or sub-oppositely arranged vesicles is described here as Zygosporium himachalensis sp. nov. This is the first fossil evidence of Zygosporium having stacked chained vesicular conidiophores and is so significant data for both paleomycologists and mycologists. The in-situ occurrence of Z. himachalensis on monocot leaf cuticles suggests a possible host-saprophyte relationship that might have existed in the ancient forest of Himachal Pradesh during the Miocene.

Three new species of Agaricus from Baiyun Mountain, Guangzhou, China.

Agaricus is a species-rich genus with more than 600 species around the world. In this work, three new species, Agaricus cacainus, A. baiyunensis, and A. praeclarefibrillosus are described from the specimens collected at Baiyun Mountain, Guangzhou, China, a subtropical area with a monsoon maritime climate, based on phylogenetic analyses and morphological examinations of internal transcribed spacer (ITS1-5.8S-ITS2 = ITS), D1/D2 domains of the large subunit of ribosomal DNA (28S), and a part of translation elongation factor 1-alpha (TEF1). Agaricus cacainus in A. sect. Amoeni is characterized by a parabolic to applanate, slightly depressed pileus covered with chocolate brown, appressed, triangular squamules against white background, a white, furfuraceous stipe, an unchanging context when cut, a fragile and evanescent annulus, usually 4- or 2-spored basidia, and mostly pyriform cheilocystidia. Agaricus baiyunensis in A. sect. Minores has a pileus with a slightly truncate top covered with light brown, downy-wooly fibrillose scales and a light yellowish stipe with membranous annulus. Agaricus praeclarefibrillosus in A. sect. Brunneopicti is characterized by a pileus surface with brownish, triangular, recurved scales and longitudinally splitting lines toward margin, a cottony stipe with white, tiny, recurved fibrils, a single annulus, and variously shaped cheilocystidia, with sparsely ornamented basidiospores. The detailed comparison of their morphological characteristics with closely related species is provided.

The Spo13/Meikin pathway confines the onset of gamete differentiation to meiosis II in yeast.

Sexual reproduction requires genome haploidization by the two divisions of meiosis and a differentiation program to generate gametes. Here, we have investigated how sporulation, the yeast equivalent of gamete differentiation, is coordinated with progression through meiosis. Spore differentiation is initiated at metaphase II when a membrane-nucleating structure, called the meiotic plaque, is assembled at the centrosome. While all components of this structure accumulate already at entry into meiosis I, they cannot assemble because centrosomes are occupied by Spc72, the receptor of the γ-tubulin complex. Spc72 is removed from centrosomes by a pathway that depends on the polo-like kinase Cdc5 and the meiosis-specific kinase Ime2, which is unleashed by the degradation of Spo13/Meikin upon activation of the anaphase-promoting complex at anaphase I. Meiotic plaques are finally assembled upon reactivation of Cdk1 at entry into metaphase II. This unblocking-activation mechanism ensures that only single-copy genomes are packaged into spores and might serve as a paradigm for the regulation of other meiosis II-specific processes.

Nuclear conditions of basidiospores and hyphal cells in the edible mushroom Oudemansiella aparlosarca.

Oudemansiella aparlosarca is an edible mushroom possessing medicinal and health benefits. Although there are studies on the cultivation of O. aparlosarca, only a few studies have focused on its genetics and life cycle. Therefore, the main objective of this study was to identify the nuclear conditions of basidiospores and homokaryotic and heterokaryotic hyphal cells and to determine the influence of different nuclear conditions on basidiospore diameter in O. aparlosarca. Two parental strains: strain-55 and strain-81 were used. Staining of basidiospores and hyphal cells in the apical region was performed. We observed the following nuclear conditions: non-nucleate, mononucleate, binucleate, and multinucleate. In both parental strains, binucleate spores were predominant, while the number of non-nucleate spores was the lowest. The diameter of non-nucleate spores was the smallest, being 11.52 µm and 12.15 µm in parental strain-81 and strain-55, respectively, while multinucleate spores had the largest diameter, being 14.78 µm in both parental strains. Both homokaryotic and heterokaryotic strains were identified in isolated single spores from parental strains. Binucleate cells were majorly present in heterokaryotic hyphal cells, and multinucleate cells were predominant in homokaryotic hyphal cells. We conclude that O. aparlosarca contains homokaryotic and heterokaryotic basidiospores, which indicates an amphithallic life cycle. The observed binucleate spores might be the result of post-meiotic mitosis.

Strongwellsea crypta (Entomophthorales: Entomophthoraceae), a new species infecting Botanophila fugax (Diptera: Anthomyiidae).

A new species from the genus Strongwellsea (Entomophthorales: Entomophthoraceae) is described: Strongwellsea crypta Eilenberg & Humber from adult Botanophila fugax (Meigen) (Diptera: Anthomyiidae). The description is based on pathobiological, phenotypical and genotypical characters. The abdominal holes in infected hosts develop rapidly and become strikingly large and edgy, almost rhomboid in shape. The new species S. crypta differs from S. castrans, the only described species infecting flies from Anthomyiidae, by: (a) naturally infecting another host species, (b) by having significantly longer primary conidia, and (c) by genotypical clustering separately from that species when sequencing ITS2.

Geometric morphometric analysis of spore shapes improves identification of fungi.

Morphology of organisms is an essential source of evidence for taxonomic decisions and understanding of ecology and evolutionary history. The geometric structure (i.e., numeric description of shape) provides richer and mathematically different information about an organism's morphology than linear measurements. A little is known on how these two sources of morphological information (shape vs. size) contribute to the identification of organisms when implied simultaneously. This study hypothesized that combining geometric information on the outline with linear measurements results in better species identification than either evidence alone can provide. As a test system for our research, we used the microscopic spores of fungi from the genus Subulicystidium (Agaricomycetes, Basidiomycota). We analyzed 2D spore shape data via elliptic Fourier and principal component analyses. Using flexible discriminant analysis, we achieved the highest species identification success rate for a combination of shape and size descriptors (64.7%). The shape descriptors alone predicted species slightly better than size descriptors (61.5% vs. 59.1%). We conclude that adding geometric information on the outline to linear measurements improves the identification of the organisms. Despite the high relevance of spore traits for the taxonomy of fungi, they were previously rarely analyzed with the tools of geometric morphometrics. Therefore, we supplement our study with an open access protocol for digitizing and summarizing fungal spores' shape and size information. We propagate a broader use of geometric morphometric analysis for microscopic propagules of fungi and other organisms.

Prey sensing and response in a nematode-trapping fungus is governed by the MAPK pheromone response pathway.

Detection of surrounding organisms in the environment plays a major role in the evolution of interspecies interactions, such as predator-prey relationships. Nematode-trapping fungi (NTF) are predators that develop specialized trap structures to capture, kill, and consume nematodes when food sources are limited. Despite the identification of various factors that induce trap morphogenesis, the mechanisms underlying the differentiation process have remained largely unclear. Here, we demonstrate that the highly conserved pheromone-response MAPK pathway is essential for sensing ascarosides, a conserved molecular signature of nemaotdes, and is required for the predatory lifestyle switch in the NTF Arthrobotrys oligospora. Gene deletion of STE7 (MAPKK) and FUS3 (MAPK) abolished nematode-induced trap morphogenesis and conidiation and impaired the growth of hyphae. The conserved transcription factor Ste12 acting downstream of the pheromone-response pathway also plays a vital role in the predation of A. oligospora. Transcriptional profiling of a ste12 mutant identified a small subset of genes with diverse functions that are Ste12 dependent and could trigger trap differentiation. Our work has revealed that A. oligospora perceives and interprets the ascarosides produced by nematodes via the conserved pheromone signaling pathway in fungi, providing molecular insights into the mechanisms of communication between a fungal predator and its nematode prey.

An international inter-laboratory study on Nosema spp. spore detection and quantification through microscopic examination of crushed honey bee abdomens.

Nosemosis is a microsporidian disease causing mortality and weakening of honey bee colonies, especially in the event of co-exposure to other sources of stress. As a result, the disease is regulated in some countries. Reliable and harmonised diagnosis is crucial to ensure the quality of surveillance and research results. For this reason, the first European Interlaboratory Comparison (ILC) was organised in 2017 in order to assess both the methods and the results obtained by National Reference Laboratories (NRLs) in counting Nosema spp. spores by microscopy. Implementing their own routine conditions of analysis, the 23 participants were asked to perform an assay on a panel of ten positive and negative samples of crushed honey bee abdomens. They were asked to report results from a qualitative and quantitative standpoint. The assessment covered specificity, sensitivity, trueness and precision. Quantitative results were analysed in compliance with international standards NF ISO 13528 (2015) and NF ISO 5725-2 (1994). Three results showed a lack of precision and five a lack of trueness. However, overall results indicated a global specificity of 98% and a global sensitivity of 100%, thus demonstrating the advanced performance of the microscopic methods applied to Nosema spores by the NRLs. Therefore, the study concluded that using microscopy to detect and quantify spores of Nosema spp. was reliable and valid.

Cross-talk between autophagy and sporulation in Saccharomyces cerevisiae.

Unicellular organisms, like yeast, have developed mechanisms to overcome environmental stress conditions like nutrient starvation. Autophagy and sporulation are two such mechanisms employed by yeast cells. Autophagy is a well-conserved, catabolic process that degrades excess and unwanted cytoplasmic materials and provides building blocks during starvation conditions. Thus, autophagy maintains cellular homeostasis at basal conditions and acts as a survival mechanism during stress conditions. Sporulation is an essential process that, like autophagy, is triggered due to stress conditions in yeast. It involves the formation of ascospores that protect the yeast cells during extreme conditions and germinate when the conditions are favorable. Studies show that autophagy is required for the sporulation process in yeast. However, the exact mechanism of action is not clear. Furthermore, several of the core autophagy gene knockouts do not sporulate and at what stage of sporulation they are involved is not clear. Besides, many overlapping proteins function in both sporulation and autophagy and it is unclear how the pathway-specific roles of these proteins are determined. All these observations suggest that the two processes cross-talk. Individually, some key features from both the processes remain to be studied with respect to the source of membrane for autophagosomes, prospore membrane (PSM) formation, and closure of the membranes. Therefore, it becomes crucial to study the cross-talk between autophagy and sporulation. In this review, the cross-talk between the two pathways, the common protein machineries have been discussed.

Atypical chlamydoconidium-producing Trichophyton tonsurans strains from Ceará State, Northeast Brazil: investigation of taxonomy by phylogenetic analysis and biofilm susceptibility.

Chlamydoconidium-producing Trichophyton tonsurans strains isolated in Northeastern Brazil have morphological features different from the classic description of this dermatophyte species. This study investigated the phylogenetic relationship of chlamydoconidium-producing T. tonsurans strains isolated in Northeastern Brazil. Also, the effect of terbinafine and farnesol on mature biofilms of T. tonsurans strains was evaluated. The mass spectra of T. tonsurans strains were investigated by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The ITS and LSU loci regions of rDNA and the partial ß-tubulin gene were sequenced and the phylogenetic tree was analysed. The effects of terbinafine and farnesol on mature T. tonsurans biofilms were evaluated through the analysis of metabolic activity, quantification of biomass and observation by scanning electron microscopy. MALDI-TOF MS spectra of the chlamydoconidium-producing T. tonsurans strains differed from the spectrum of the control strain (ATCC 28942), presenting an intense ion peak at m/z 4155 Da. Phylogenetic tree analysis showed that the chlamydoconidium-producing strains isolated in Northeastern Brazil are allocated to a single cluster, differing from strains isolated from other countries. As for mature T. tonsurans biofilms, farnesol reduced biomass and metabolic activity by 64.4 and 65.9 %, respectively, while terbinafine reduced the biomass by 66.5 % and the metabolic activity by 69 %. Atypical morphological characteristics presented by chlamydoconidium-producing T. tonsurans strains result from phenotypic plasticity, possibly for adaptation to environmental stressors. Also, farnesol had inhibitory activity against T. tonsurans biofilms, demonstrating this substance can be explored for development of promising anti-biofilm drugs against dermatophytes.

Tetra disseminated microsporidiosis: a novel disease in ornamental fish caused by Fusasporis stethaprioni n. gen. n. sp.

A novel microsporidial disease was documented in two ornamental fish species, black tetra Gymnocorymbus ternetzi Boulenger 1895 and cardinal tetra Paracheirodon axelrodi Schultz 1956. The non-xenoma-forming microsporidium occurred diffusely in most internal organs and the gill, thus referring to the condition as tetra disseminated microsporidiosis (TDM). The occurrence of TDM in black tetra was associated with chronic mortality in a domestic farmed population, while the case in cardinal tetra occurred in moribund fish while in quarantine at a public aquarium. Histology showed that coelomic visceral organs were frequently necrotic and severely disrupted by extensive infiltrates of macrophages. Infected macrophages were presumed responsible for the dissemination of spores throughout the body. Ultrastructural characteristics of the parasite developmental cycle included uninucleate meronts directly in the host cell cytoplasm. Sporonts were bi-nucleated as a result of karyokinesis and a parasite-produced sporophorous vesicle (SPV) became apparent at this stage. Cytokinesis resulted in two spores forming within each SPV. Spores were uniform in size, measuring about 3.9 ± 0.33 long by 2.0 ± 0.2 µm wide. Ultrastructure demonstrated two spore types, one with 9-12 polar filament coils and a double-layered exospore and a second type with 4-7 polar filament coils and a homogenously electron-dense exospore, with differences perhaps related to parasite transmission mechanisms. The 16S rDNA sequences showed closest identity to the genus Glugea (≈ 92%), though the developmental cycle, specifically being a non-xenoma-forming species and having two spores forming within a SPV, did not fit within the genus. Based on combined phylogenetic and ultrastructural characteristics, a new genus (Fusasporis) is proposed, with F. stethaprioni n. gen. n. sp. as the type species.

A revision of Agaricus section Arvenses with nine new species from China.

Agaricus sect. Arvenses includes numerous species that are potential candidates for cultivation, and some have high nutritional and medicinal interests. Between 2012 and 2017, 147 specimens of A. sect. Arvenses were collected in China. For this study, nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS), nuc 28S rDNA (28S), and translation elongation factor 1-alpha (tef1) sequences were used to assess species boundaries of these samples from China. Combined with morphological examination, we recognize 22 species of A. sect. Arvenses from China, of which 12 are known species, one is new record for China, and nine are proposed as new.

Open the Pores: Electroporation for the Transformation of Trichoderma reesei.

During the electroporation of T. reesei, linearized exogenous DNA is absorbed into swollen conidia by an electrical impulse. The advantage of this method is that it is less time-consuming, less expensive, and easier to perform than the classical protoplast transformation while at the same time having a comparable efficiency.

Flow Cytometry for Filamentous Fungi.

Flow cytometry is a powerful high-throughput method, which enables a fast and multi-parameter analysis of single cells and particles. A plethora of different dyes for flow cytometry are available to label different parts of a cell in addition to in vivo markers like fluorescent proteins. Trichoderma species as well as other filamentous fungi show hyphal growth, which  makes analysis in a flow cytometer difficult. Nevertheless, conidia can be readily analyzed in conventional flow cytometers. Many different applications can be envisaged. This protocol describes how conidia  can be prepared for flow cytometry and the occurrence of genetic markers such as GFP can be measured. Furthermore, a guideline how to fix and stain cells is given.

Molecular Identification of Trichoderma reesei.

Fungi comprise one of the most diverse groups of eukaryotes with many cryptic species that are difficult to identify. In this chapter, we detail a protocol for the molecular identification of the most industrially relevant species of Trichoderma-T. reesei. We first describe how a single spore culture should be isolated and used for the sequencing of the diagnostic fragment of the tef1 gene. Then, we provide two alternative methods that can be used for molecular identification and offer the diagnostic oligonucleotide hallmark of the tef1 sequence that is present in sequences of all T. reesei strains known to date and that is therefore suitable for reliable and straightforward identification.

Conidiobolus lunulus, a new entomophthoralean species isolated from leafcutter ants.

Entomophthoralean fungi with pathogenic abilities to infect social insects are rare. Here, we describe a fungus isolated from leafcutter ants. Morphologically, the fungus has spherical primary conidia and two types of microconidia: one with the same shape as the primary conidia and another with an elliptical to half-moon shape. The fungus also produces villose conidia known previously only from Conidiobolus coronatus. A multilocus phylogenetic analysis was performed with nuc rDNA sequences from three regions (28S, 18S, and internal transcribed spacer [ITS]). Our isolates are distinguished as a new species, described here as Conidiobolus lunulus, and is more closely related to C. brefeldianus than to C. coronatus, despite the greater morphological resemblance to the latter. Morphological differences, unique phylogenetic placement, and isolation from an altogether new host support this finding. This is the first record of an entomophthoralean species isolated from leafcutter ants.

Dispersal of Arbuscular Mycorrhizal Fungi: Evidence and Insights for Ecological Studies.

Dispersal is a critical ecological process that modulates gene flow and contributes to the maintenance of genetic and taxonomic diversity within ecosystems. Despite an increasing global understanding of the arbuscular mycorrhizal (AM) fungal diversity, distribution and prevalence in different biomes, we have largely ignored the main dispersal mechanisms of these organisms. To provide a geographical and scientific overview of the available data, we systematically searched for the direct evidence on the AM fungal dispersal agents (abiotic and biotic) and different propagule types (i.e. spores, extraradical hyphae or colonized root fragments). We show that the available data (37 articles) on AM fungal dispersal originates mostly from North America, from temperate ecosystems, from biotic dispersal agents (small mammals) and AM fungal spores as propagule type. Much lesser evidence exists from South American, Asian and African tropical systems and other dispersers such as large-bodied birds and mammals and non-spore propagule types. We did not find strong evidence that spore size varies across dispersal agents, but wind and large animals seem to be more efficient dispersers. However, the data is still too scarce to draw firm conclusions from this finding. We further discuss and propose critical research questions and potential approaches to advance the understanding of the ecology of AM fungi dispersal.

The secondary metabolite regulator, BbSmr1, is a central regulator of conidiation via the BrlA-AbaA-WetA pathway in Beauveria bassiana.

The filamentous fungus Beauveria bassiana, an insect fungal pathogen, is widely used for pest biocontrol. Aerial conidia are infectious propagules, and their yield and viability greatly affect the field application of this fungus; however, little is known about the molecular regulatory mechanism of the triggered conidiation. In the present study, we find that the secondary metabolite regulator BbSmr1 is involved in the regulation of asexual conidiation development and stress response in B. bassiana. A deficiency in Bbsmr1 results in a prominent fluffy-like phenotype on solid medium, decreased conidial yield, accelerated conidial germination, as well as increased tolerance to H2 O2 stress and cell wall inhibitors. The deletion of Bbsmr1 also leads to thickened conidial cell walls and changed cell epitopes. Overexpressing either BbbrlA or BbabaA in the ∆Bbsmr1 strain can rescue the phenotypes of conidial development and stress response. BbSmr1 activates BbbrlA transcription by directly binding to the A4GA3 sequence of the BbbrlA promoter. BbBrlA in turn binds to the promoter of Bbsmr1 and negatively regulates the expression of Bbsmr1. These results indicate that BbSmr1 positively regulates conidial development in B. bassiana by activating the central development pathway BrlA-AbaA-WetA and provides insights into the developmental regulatory mechanism of entomopathogenic fungi.

Extracts from Eleutherococcus senticosus (Rupr. et Maxim.) Maxim. Roots: A New Hope Against Honeybee Death Caused by Nosemosis.

Pollinators, the cornerstones of our terrestrial ecosystem, have been at the very core of our anxiety. This is because we can nowadays observe a dangerous decline in the number of insects. With the numbers of pollinators dramatically declining worldwide, the scientific community has been growing more and more concerned about the future of insects as fundamental elements of most terrestrial ecosystems. Trying to address this issue, we looked for substances that might increase bee resistance. To this end, we checked the effects of plant-based adaptogens on honeybees in laboratory tests and during field studies on 30 honeybee colonies during two seasons. In this study, we have tested extracts obtained from: Eleutherococcus senticosus, Garcinia cambogia, Panax ginseng, Ginkgo biloba, Schisandra chinensis, and Camellia sinensis. The 75% ethanol E. senticosus root extract proved to be the most effective, both as a cure and in the prophylaxis of nosemosis. Therefore, Eleutherococcus senticosus, and its active compounds, eleutherosides, are considered the most powerful adaptogens, in the pool of all extracts that were selected for screening, for supporting immunity and improving resistance of honeybees. The optimum effective concentration of 0.4 mg/mL E. senticosus extract responded to c.a. 5.76, 2.56 and 0.07 µg/mL of eleutheroside B, eleutheroside E and naringenin, respectively. The effect of E. senticosus extracts on honeybees involved a similar adaptogenic response as on other animals, including humans. In this research, we show for the first time such an adaptogenic impact on invertebrates, i.e., the effect on honeybees stressed by nosemosis. We additionally hypothesised that these adaptogenic properties were connected with eleutherosides-secondary metabolites found exclusively in the Eleutherococcus genus and undetected in other studied extracts. As was indicated in this study, eleutherosides are very stable chemically and can be found in extracts in similar amounts even after two years from extraction. Considering the role bees play in nature, we may conclude that demonstrating the adaptogenic properties which plant extracts have in insects is the most significant finding resulting from this research. This knowledge might bring to fruition numerous economic and ecological benefits.

Loss of FZO1 gene results in changes of cell dynamics in fission yeast.

Mitochondrial fission and fusion dynamics are critical cellular processes, and abnormalities in these processes are associated with severe human disorders, such as Beckwith­Wiedemann syndrome, neurodegenerative diseases, Charcot­Marie­Tooth disease type 6, multiple symmetric lipomatosis and microcephaly. Fuzzy onions protein 1 (Fzo1p) regulates mitochondrial outer membrane fusion. In the present study, Schizosaccharomyces pombe (S. pombe) was used to explore the effect of FZO1 gene deletion on cell dynamics in mitosis. The mitochondrial morphology results showed that the mitochondria appeared to be fragmented and tubular in wild­type cells; however, they were observed to accumulate in fzo1Δ cells. The FZO1 gene deletion was demonstrated to result in slow proliferation, sporogenesis defects, increased microtubule (MT) number and actin contraction defects in S. pombe. The FZO1 gene deletion also affected the rate of spindle elongation and phase time at the metaphase and anaphase, as well as spindle MT organization. Live­cell imaging was performed on mutant strains to observe three distinct kinetochore behaviors (normal, lagging and mis­segregation), as well as abnormal spindle breakage. The FZO1 gene deletion resulted in coenzyme and intermediate metabolite abnormalities as determined via metabolomics analysis. It was concluded that the loss of FZO1 gene resulted in deficiencies in mitochondrial dynamics, which may result in deficiencies in spindle maintenance, chromosome segregation, spindle breakage, actin contraction, and coenzyme and intermediate metabolite levels.