Chytridiomycete Fungus Research Articles

Features of the rumen microbiome in reindeer with necrobacteriosis

Reindeer are a unique animal species that have adapted to the harsh Arctic conditions. Adaptive features of the structure of the gastrointestinal tract of reindeer allows you to eat actively digest and assimilate lichens, foliage, shrubs, snowy greens. Along with beneficial microflora, pathogenic and conditionally pathogenic microorganisms live in the reindeer rumen. The purpose of our research is to study and compare the features of the rumen microbiome in healthy and sick reindeer with necrobacteriosis. The subject of the study was samples of cicatricial contents taken from 38 reindeer (Rangifer tarandus) of the Nenets breed of different age groups. Microbiome studies were performed by NGS sequencing on the MiSeq next generation sequencing platform (Illumina, USA) using primers for the V3-V4 region of 16S rRNA.Based on the studies, it was possible to establish that the microbiome of the reindeer rumen in summer and winter is characterized by certain differences. During the summer grazing period, the presence of a greater number of pathogenic bacteria such as Fusobacterium sp., Parvimonas sp. and Porphyromonas endodontalis, while in the spectrum of pathogenic microorganisms, bacteria of the genus Fusobacterium, which are considered the main infectious agent in necrobacteriosis, were most often isolated. In the same period, the presence of bacteria of the genus Sharpea, belonging to the phylum Erysipelotrichaceae, which ferment sugars with the formation of lactic acid and can cause the development of lactic acidosis, was established in deer rumen samples. In calves, during the disease, the number of chytridiomycete fungi, which are involved in the fermentation of polysaccharides, decreases.

Cryptic but direct costs of an epidemic caused by Batrachochytrium dendrobatidis in the endangered Sardinian newt Euproctus platycephalus (Amphibia, Caudata)

Abstract Parasites can directly affect host survival by causing cryptic costs that may occur in species when mortality does not take place or is less evident. Costs can be present in differently life history stages or age cohorts or cause age-dependent effects. Here we investigate the effects of chytridiomycete fungus Batrachochytrium dendrobatidis, (Bd), that causes one of the main infectious diseases implicated in numerous mass mortalities and population declines in the class of Amphibia, including in one of the rarest and most threatened salamander in Europe, the endemic Sardinian brook newt Euproctus platycephalus. This species is known to be infected by Bd since 2006, without evident direct cost or mass mortality events being observed. During 5 years of sampling (2007-2011), we captured 273 newts in the main infected area of the Limbara Mountain in the North of Sardinia, and, through epidemiological and age analyzes, we assess that Bd directly affected juvenile recruitment, and it is related to difficulties in the development of the species, with an impact on the females growth.

Occurrence and partial genetic characterisation of Lettuce big‐vein associated virus and Mirafiori lettuce big‐vein virus infecting lettuce in Jordan

AbstractLettuce big‐vein disease (LBVD) is an economically damaging disease that occurs in most lettuce production areas worldwide. Several studies have associated LBVD with a complex of two viruses, Lettuce big‐vein associated virus (LBVaV) and Mirafiori lettuce big‐vein virus (MiLBVV). Both viruses are transmitted through soil by the zoospores of the chytridiomycete fungus Olpidium virulentus. During surveys carried out in two growing seasons from 2015 to 2016 in the main vegetable growing areas in Jordan (Jordan Valley, Jerash, and Madaba), 179 lettuce plants, 46 lettuce seedlings, and 56 weed plants were collected. RNA extraction was conducted from leaves of all samples. Associated viruses were identified using reverse transcription‐polymerase chain reaction (RT‐PCR) with specific primer pairs targeting part of the coat protein (CP) genes of LBVaV and MiLBVV. RT‐PCR results revealed that, out of 225 lettuce samples tested, 32 (14%) and 44 (20%) were singly infected with LBVaV and MiLBVV, respectively, and 73 (32%) samples were dually infected with LBVaV and MiLBVV. Overall, MiLBVV was detected in more lettuce samples (117) compared with LBVaV which was detected in 105 samples. Moreover, one sample of each weed species of Chenopodium murale and Sonchus oleraceus tested positive for MiLBVV and LBVaV, respectively. The partial nucleotide sequence of the CP genes of LBVaV and MiLBVV Jordanian isolates showed similarities to other isolates available in the NCBI database ranging between 93.9–100% and 88.3–100%, respectively. Nucleotide sequence comparison of the Jordanian isolates and inference from phylogenetic trees with strong support revealed well‐defined sub‐groups with little or no correlation with planting area or geographic origin. The occurrence of both viruses in lettuce plants was associated with the presence of resting spores and zoospores of Olpidium spp. in lettuce plants grown under field and growth chamber conditions. Lettuce plants inoculated with Olpidium spp. zoospores isolated from roots of symptomatic lettuce plants, developed LBVD‐symptoms. At 60 days post‐transplantation, both viruses were detected by RT‐PCR in most of the plants analysed. Biological and molecular assays together with the light microscopy observations of resting spores and zoospores of Olpidium spp., revealed the presence of LBVaV, MiLBVV, and O. virulentus in big‐vein diseased lettuce plants.

A New Chytridiomycete Fungus Intermixed with Crustacean Resting Eggs in a 407-Million-Year-Old Continental Freshwater Environment.

The 407-million-year-old Rhynie Chert (Scotland) contains the most intact fossilised remains of an early land-based ecosystem including plants, arthropods, fungi and other microorganisms. Although most studies have focused on the terrestrial component, fossilised freshwater environments provide critical insights into fungal-algal interactions and the earliest continental branchiopod crustaceans. Here we report interactions between an enigmatic organism and an exquisitely preserved fungus. The fungal reproductive structures are intermixed with exceptionally well-preserved globular spiny structures interpreted as branchiopod resting eggs. Confocal laser scanning microscopy enabled us to reconstruct the fungus and its possible mode of nutrition, the affinity of the resting eggs, and their spatial associations. The new fungus (Cultoraquaticus trewini gen. et sp. nov) is attributed to Chytridiomycota based on its size, consistent formation of papillae, and the presence of an internal rhizoidal system. It is the most pristine fossil Chytridiomycota known, especially in terms of rhizoidal development and closely resembles living species in the Rhizophydiales. The spiny resting eggs are attributed to the crustacean Lepidocaris rhyniensis, dating branchiopod adaptation to life in ephemeral pools to the Early Devonian. The new fungal interaction suggests that, as in modern freshwater environments, chytrids were important to the mobilisation of nutrients in early aquatic foodwebs.

Mitigating amphibian chytridiomycoses in nature.

Amphibians across the planet face the threat of population decline and extirpation caused by the disease chytridiomycosis. Despite consensus that the fungal pathogens responsible for the disease are conservation issues, strategies to mitigate their impacts in the natural world are, at best, nascent. Reducing risk associated with the movement of amphibians, non-amphibian vectors and other sources of infection remains the first line of defence and a primary objective when mitigating the threat of disease in wildlife. Amphibian-associated chytridiomycete fungi and chytridiomycosis are already widespread, though, and we therefore focus on discussing options for mitigating the threats once disease emergence has occurred in wild amphibian populations. All strategies have shortcomings that need to be overcome before implementation, including stronger efforts towards understanding and addressing ethical and legal considerations. Even if these issues can be dealt with, all currently available approaches, or those under discussion, are unlikely to yield the desired conservation outcome of disease mitigation. The decision process for establishing mitigation strategies requires integrated thinking that assesses disease mitigation options critically and embeds them within more comprehensive strategies for the conservation of amphibian populations, communities and ecosystems.This article is part of the themed issue 'Tackling emerging fungal threats to animal health, food security and ecosystem resilience'.

Amphibian survey and current absence of Batrachochytrium dendrobatidis (Bd) in Ivoloina Park, Toamasina (eastern Madagascar)

Amphibians are threatened globally by the chytridiomycete fungus Batrachochytrium dendrobatidis (Bd), which is still expanding in range. Madagascar, rich in amphibian diversity, remains one of the few places that the fungus has not invaded. Herein, we present results from a pilot survey for Bd in conjunction with a rapid amphibian survey conducted at Ivoloina Park, a forestry station near Toamasina, eastern Madagascar. This park is located on the south-western side of the Ivoloina River in a former lowland rainforest now predominantly covered by plantations of exotic trees. Our amphibian survey confirmed the presence of 12 anuran species identified by both morphology and molecular barcoding and revealed the presence of four candidate species. Real-time polymerase chain reaction screening for the presence of Bd showed that all 59 samples tested negative, thus confirming the absence of the pathogen in this area. Our survey of Ivoloina provides the first species list for a suburban park in Madagascar complemented with chytridiomycosis screening.

Assessing Risk and Guidance on Monitoring of Batrachochytrium dendrobatidis in Europe through Identification of Taxonomic Selectivity of Infection

Amphibians are globally threatened, but not all species are affected equally by different threatening processes. This is true for the threat posed by the chytridiomycete fungus (Batrachochytrium dendrobatidis). We compiled a European data set for B. dendrobatidis to analyze the trends of infection in European amphibians. The risk of infection was not randomly distributed geographically or taxonomically across Europe. Within countries with different prevalence, infection was nonrandom in certain amphibian taxa. Brown frogs of the genus Rana were unlikely to be infected, whereas frogs in the families Alytidae and Bombinatoridae were significantly more likely to be infected than predicted by chance. Frogs in the 2 families susceptible to B. dendrobatidis should form the core of attempts to develop spatial surveillance studies of chytridiomycosis in Europe. Ideally, surveys for B. dendrobatidis should be augmented by sampling the widespread genus Pelophylax because this taxon exhibits geographically inconsistent overinfection with B. dendrobatidis and surveillance of it may facilitate recognition of factors causing spatial variability of infection intensity. Several European amphibian taxa were not represented in our data set; however, surveillance of unsampled species should also occur when warranted.

Host invasion by Batrachochytrium dendrobatidis: fungal and epidermal ultrastructure in model anurans

The chytridiomycete fungus Batrachochytrium dendrobatidis (Bd) colonizes mouthparts of amphibian larvae and superficial epidermis of post-metamorphic amphibians, causing the disease chytridiomycosis. Fungal growth within host cells has been documented by light and transmission electron microscopy; however, entry of the fungus into host cells has not. Our objective was to document how Bd enters host cells in the wood frog Lithobates sylvaticus, a species at high mortality risk for chytridiomycosis, and the bullfrog L. catesbeianus, a species at low mortality risk for chytridiomycosis. We inoculated frogs and documented infection with transmission electron microscopy. Zoospores encysted on the skin surface and produced morphologically similar germination tubes in both host species that penetrated host cell membranes and enabled transfer of zoospore contents into host cells. Documenting fungal and epidermal ultrastructure during host invasion furthers our understanding of Bd development and the pathogenesis of chytridiomycosis.

Comparative study of cellulolytic activity of three rumen fungi on different substrates

Anaerobic chytridiomycete fungi are found in the gastrointestinal tracts of many domesticated ruminant and nonruminant herbivores and of a wide variety of wild herbivorous mammals. They produce high levels of cellulases and hemicellulases; these enzymes are regulated by substrate (especially soluble sugars) available to the organisms. The aim of this paper was to do a comparative study of cellulolytic activity of three rumen fungi on carboxymethyl cellulose and Avicel. The capacity of enzymes was determined by monitoring the growth on carboxymethyl cellulose (CMC) and Avicel. Enzyme activity was detected extracellularly in culture supernatants after vegetative growth. All of the isolates degraded CMC and avicel, and exhibited cellulolytic activities (carboxymethyl cellulose-(CMC-ase) and avicelase).

Structure, expression and function of Allomyces arbuscula CDP II (metacaspase) gene

Allomyces arbuscula, a primitive chytridiomycete fungus, has two Ca 2+-dependent cysteine proteases, the CDP I and CDP II. We have cloned and analyzed the nucleotide sequence of CDP II gene and domain structure of the protein. Blast analysis of the sequence has shown that the protein belongs to a newly described member of caspase superfamily protein, the metacaspase, a CD clan of C14 family cysteine protease, we hence-forth name it as AMca 2 ( Allomyces metacaspase 2). Southern hybridization studies have shown that the gene exists in a single copy per genome. The transcriptional analysis by Northern hybridization has confirmed our previous results that the protein is developmentally regulated, i.e. present in active growth phase but disappears during nutritional stress which also induces reproductive differentiation, indicating that the protein promotes cell growth, not death. The recombinant gene product expressed in E scherichia coli has all the catalytic properties of native enzyme, i.e. sensitivity to protease inhibitors and substrate specificity. There is an absolute requirement of Ca 2+ for the activation of catalytic activity and the presence of R residue at the cleavage site (P1 position) in the substrate. The presence of a second basic residue, either R or K, in the P2 position strongly inhibits the catalytic activity which is stimulated by the presence of P and to a lesser extent G at this site. Peptide substrates with D at the cleavage site are not recognised and therefore not cleaved. The enzyme activity is inhibited by EDTA–EGTA, cysteine protease inhibitors and a specific peptide inhibitor Ac GVRCHCL TFA, but not by E64, although a potent inhibitor of cysteine proteases.

Amphibian Chytridiomycosis in the Oregon Spotted Frog (RANA PRETIOSA) IN WASHINGTON STATE, USA

In recent years Batrachochytrium dendrobatidis (Bd), a chytridiomycete fungus pathogenic to amphibians (Longcore and others 1999), has been implicated as the proximate cause of amphibian declines around the world (Berger and others 1998; Daszak and others 2003; Muths and others 2003; Pounds and others 2006). Despite the insidious nature of Bd (Green and others 2002), few published data exist addressing its occurrence in the Pacific Northwest (PNW). When Pearl and others (2007) opportunistically examined 7 PNW amphibian species, they found Bd most often (57% of 21 individuals from 14 sampled populations) in the highly aquatic Oregon Spotted Frog (Rana pretiosa), an Endangered Species in Washington State (WDFW 2009). However, all R. pretiosa they sampled were from Oregon, and Bd was not detected in the Rana cascadae (Cascades Frog) and Anaxyrus boreas (Western Toad) specimens they sampled from Washington. Here, we report the detection of Bd in R. pretiosa from Washington. Since 1997 and 1998, respectively, we have been monitoring R. pretiosa populations at the Trout Lake Natural Area Preserve (TLNAP: UTM Zone 10, 610857-612950E, 5095880-5097574N, WGS84; elev. 594 to 599 m) and Conboy Lake National Wildlife Refuge (CLNWR: UTM Zone 10, 625223-635180E, 5086652-5095491N, WGS84; elev. 552 to 576 m), in Klickitat County, Washington. These sites represent 2 of only 3 areas where R. pretiosa is known to occur in Washington (McAllister and Leonard 1997). At both sites, monitoring included egg mass surveys during the annual breeding season following snowmelt (late February to mid-March at CLNWR; midMarch to early April at TLNAP). Surveys involved area-specific counts of individual egg masses and egg mass groups, each of which was marked with flags and geo-referenced using a Geographic Positioning System (GPS) to avoid double-counting. Sampling at CLNWR addressed 4 hydrologically distinct units, which were surveyed repeatedly until no new egg masses were found; at TLNAP, 3 units consistently used for breeding were sampled. We inferred a 1:1 correspondence between egg mass numbers and number of breeding females based on a combination of our direct observations of oviposition (n 5 13), the recapture of non-gravid females known to have laid eggs (n 5 84), and the relatively short interval over which the laying of new egg masses occurs (about 3 wk). Collectively, these data indicate that females lay only 1 clutch annually, and that egg mass numbers reflect the effective population numbers of adult females. Decline in egg mass numbers in 3 of the 4 surveyed units at CLNWR from 2004 to 2005 (Fig. 1) and 2 of the 3 units at TLNAP over the same period (Fig. 2), coupled with the increasing recognition of Bd as a cause of amphibian declines, motivated us to collect dead frogs found during 2006 and test them for Bd. Five dead adult R. pretiosa were tested for Bd; 1 from CLNWR and 4 from TLNAP. The CLNWR specimen collected on 13 March 2006 displayed feeble vital signs, minimal response to touch, and righting response was lacking. The frog died within 20 min of discovery, was preserved in 10% formalin, and then stored in 70% ethanol before histological examination using a standard wet-mount preparation of its epidermal tissue (Berger and others 1999), which was sloughing extensively as multiple epidermal layers. A dead adult female R. GENERAL NOTES

Batrachochytrium dendrobatidis Occurrence in Eurycea cirrigera

Several pathogens affect amphibians, but a chytridiomycete fungus, Batrachochytrium dendrobatidis, is of particular interest because this pathogen is linked to localized amphibian population declines and extinction of species. Species-specific infections and pathogen distribution are poorly understood, particularly in members of Caudata. We found B. dendrobatidis in adult Eurycea cirrigera (Southern Two-lined Salamander), a species not previously reported as susceptible to this fungal pathogen, and this report is the first record of B. dendrobatidis occurrence in Alabama.

Detection of Chytridiomycosis Caused by Batrachochytrium dendrobatidis in the Endangered Sardinian Newt (Euproctus platycephalus) in Southern Sardinia, Italy

The chytridiomycete fungus Batrachochytrium dendrobatidis is known to be focally distributed across Europe, but has only been linked to "chytridiomycosis at a few locations in Spain. Here we report the second occurrence of chytridiomycosis in European amphibians. We found a population of endangered Sardinian newts (Euproctus platycephalus) exhibiting clinical signs of disease including loss of digits and patchy, discolored skin. Molecular examination of skin samples tested positive for B. dendrobatidis. The population of E. platycephalus has been in decline on a timescale consistent with the global emergence of chytridiomycosis, and the ecology of this salamander suggests that the disease in this species warrants concern.

Climate change and outbreaks of amphibian chytridiomycosis in a montane area of Central Spain; is there a link?

Amphibian species are declining at an alarming rate on a global scale in large part owing to an infectious disease caused by the chytridiomycete fungus, Batrachochytrium dendrobatidis. This disease of amphibians has recently emerged within Europe, but knowledge of its effects on amphibian assemblages remains poor. Importantly, little is known about the environmental envelope that is associated with chytridiomycosis in Europe and the potential for climate change to drive future disease dynamics. Here, we use long-term observations on amphibian population dynamics in the Peñalara Natural Park, Spain, to investigate the link between climate change and chytridiomycosis. Our analysis shows a significant association between change in local climatic variables and the occurrence of chytridiomycosis within this region. Specifically, we show that rising temperature is linked to the occurrence of chytrid-related disease, consistent with the chytrid-thermal-optimum hypothesis. We show that these local variables are driven by general circulation patterns, principally the North Atlantic Oscillation. Given that B. dendrobatidis is known to be broadly distributed across Europe, there is now an urgent need to assess the generality of our finding and determine whether climate-driven epidemics may be expected to impact on amphibian species across the wider region.

The emerging amphibian pathogen Batrachochytrium dendrobatidis globally infects introduced populations of the North American bullfrog, Rana catesbeiana

Batrachochytrium dendrobatidis is the chytridiomycete fungus which has been implicated in global amphibian declines and numerous species extinctions. Here, we show that introduced North American bullfrogs (Rana catesbeiana) consistently carry this emerging pathogenic fungus. We detected infections by this fungus on introduced bullfrogs from seven of eight countries using both PCR and microscopic techniques. Only native bullfrogs from eastern Canada and introduced bullfrogs from Japan showed no sign of infection. The bullfrog is the most commonly farmed amphibian, and escapes and subsequent establishment of feral populations regularly occur. These factors taken together with our study suggest that the global threat of B. dendrobatidis disease transmission posed by bullfrogs is significant.

Chytrid Fungus in Europe

Chytrid Fungus in Europe

An anaerobic mitochondrion that produces hydrogen

Hydrogenosomes are organelles that produce ATP and hydrogen, and are found in various unrelated eukaryotes, such as anaerobic flagellates, chytridiomycete fungi and ciliates. Although all of these organelles generate hydrogen, the hydrogenosomes from these organisms are structurally and metabolically quite different, just like mitochondria where large differences also exist. These differences have led to a continuing debate about the evolutionary origin of hydrogenosomes. Here we show that the hydrogenosomes of the anaerobic ciliate Nyctotherus ovalis, which thrives in the hindgut of cockroaches, have retained a rudimentary genome encoding components of a mitochondrial electron transport chain. Phylogenetic analyses reveal that those proteins cluster with their homologues from aerobic ciliates. In addition, several nucleus-encoded components of the mitochondrial proteome, such as pyruvate dehydrogenase and complex II, were identified. The N. ovalis hydrogenosome is sensitive to inhibitors of mitochondrial complex I and produces succinate as a major metabolic end product--biochemical traits typical of anaerobic mitochondria. The production of hydrogen, together with the presence of a genome encoding respiratory chain components, and biochemical features characteristic of anaerobic mitochondria, identify the N. ovalis organelle as a missing link between mitochondria and hydrogenosomes.

Investigation of Batrachochytrium Dendrobatidis in Four Boreal Toad (Bufo Boreas Boreas) Populations in Wyoming

Batrachochytrium dendrobatidis (Figure 1) is a newly described chytridiomycete fungus that infects amphibian keratin (Berger et al. 1998). It is reported to cause disease and mortality in metamorphosed amphibians around the world (Lips 1998: Young et al. 2001; Bradley et al. 2002; Muths et al. 2003). In several cases, this pathogen is believed to be the primary cause of extinction for amphibian species (Daszak et al. 1999).

In Vitro Characterization of a tRNA Editing Activity in the Mitochondria of Spizellomyces punctatus, a Chytridiomycete Fungus

In the chytridiomycete fungus, Spizellomyces punctatus, all eight of the mitochondrially encoded tRNAs are predicted to have one or more base pair mismatches at the first three positions of their aminoacyl acceptor stems. These tRNAs are edited post-transcriptionally by replacement of the 5'-nucleotide in each mismatched pair with a nucleotide that can form a standard Watson-Crick base pair with its counterpart in the 3'-half of the stem. The type of mitochondrial tRNA editing found in S. punctatus also occurs in Acanthamoeba castellanii, a distantly related amoeboid protist. Using an S. punctatus mitochondrial extract, we have developed an in vitro assay of tRNA editing in which nucleotides are incorporated into various tRNA substrates. Experiments employing synthetic transcripts revealed that the S. punctatus tRNA editing activity incorporates nucleotides on the 5'-side of substrate tRNAs, uses the 3'-sequence as a template for incorporation, and adds nucleotides in a 3'-to-5' direction. This activity can add nucleotides to a triphosphorylated 5'-end in the absence of ATP but requires ATP to add nucleotides to a monophosphorylated 5'-end; moreover, it functions independently of the state of tRNA 3' processing. These data parallel results obtained in a previous in vitro study of A. castellanii tRNA editing, suggesting that remarkably similar activities function in the mitochondria of these two organisms. The evolutionary origins of these activities are discussed.

Origin, evolution, and mechanism of 5' tRNA editing in chytridiomycete fungi.

5' tRNA editing has been demonstrated to occur in the mitochondria of the distantly related rhizopod amoeba Acanthamoeba castellanii and the chytridiomycete fungus Spizellomyces punctatus. In these organisms, canonical tRNA structures are restored by removing mismatched nucleotides at the first three 5' positions and replacing them with nucleotides capable of forming Watson-Crick base pairs with their 3' counterparts. This form of editing seems likely to occur in members of Amoebozoa other than A. castellanii, as well as in members of Heterolobosea. Evidence for 5' tRNA editing has not been found to date, however, in any other fungus including the deeply branching chytridiomycete Allomyces macrogynus. We predicted that a similar form of tRNA editing would occur in members of the chytridiomycete order Monoblepharidales based on the analysis of complete mitochondrial tRNA complements. This prediction was confirmed by analysis of tRNA sequences using a tRNA circularization/RT-PCR-based approach. The presence of partially and completely unedited tRNAs in members of the Monoblepharidales suggests the involvement of a 5'-to-3' exonuclease rather than an endonuclease in removing the three 5' nucleotides from a tRNA substrate. Surprisingly, analysis of the mtDNA of the chytridiomycete Rhizophydium brooksianum, which branches as a sister group to S. punctatus in molecular phylogenies, did not suggest the presence of editing. This prediction was also confirmed experimentally. The absence of tRNA editing in R. brooksianum raises the possibility that 5' tRNA editing may have evolved twice independently within Chytridiomycota, once in the lineage leading to S. punctatus and once in the lineage leading to the Monoblepharidales.