A guide to Agrobacterium-mediated transformation of the chytrid fungus Spizellomyces punctatus.

Chytrid fungi play key ecological roles in aquatic ecosystems, and some species cause a devastating skin disease in frogs and salamanders. Additionally, chytrids occupy a unique phylogenetic position- sister to the well-studied Dikarya (the group including yeasts, sac fungi, and mushrooms) and related to animals- making chytrids useful for answering important evolutionary questions. Despite their importance, little is known about the basic cell biology of chytrids. A major barrier to understanding chytrid biology has been a lack of genetic tools with which to test molecular hypotheses. Medina and colleagues recently developed a protocol for Agrobacterium -mediated transformation of Spizellomyces punctatus. In this manuscript, we describe the general procedure including planning steps and expected results. We also provide in-depth, step-by-step protocols and video guides for performing the entirety of this transformation procedure on protocols.io (dx.doi.org/10.17504/protocols.io.x54v9dd1pg3e/v1).

Mucilage protects the planktonic desmid Staurodesmus sp. against parasite attack by a chytrid fungus.

Zoosporic fungi of the phylum Chytridiomycota are ubiquitous parasites of phytoplankton in aquatic ecosystems, but little is known about phytoplankton defense strategies against parasitic chytrid attacks. Using a model chytrid-phytoplankton pathosystem, we experimentally tested the hypothesis that the mucilage envelope of a mucilage-forming desmid species provides protection against the parasitic chytrid Staurastromyces oculus. Mucilage-forming Staurodesmus cells were not accessible to the chytrid, whereas physical removal of the mucilage envelope rendered the same Staurodesmus sp. strain equally susceptible to chytrid infections as the original non-mucilage-forming host Staurastrum sp. Epidemic spread of the parasite only occurred in Staurastrum sp., whereas non-mucilage-bearing Staurodesmus sp. allowed for co-existence of host and parasite, and mucilage-bearing Staurodesmus sp. caused parasite extinction. In addition to the mucilage defense barrier, we also demonstrate the ability of both Staurastrum sp. and Staurodesmus sp. to resist infection by preventing chytrid development while still remaining viable and being able to reproduce and thus recover from an infection. This study extends our knowledge on phytoplankton defense traits and the functional role of mucilage in phytoplankton as a physical barrier against fungal parasites.

Integrative approach to species delimitation in Rhizophydiales: Novel species of Angulomyces, Gorgonomyces, and Terramyces from northern Thailand.

The Chytridiomycota is a phylum of zoosporic eufungi that inhabit terrestrial, freshwater, and oceanic habitats. Within the phylum, the Rhizophydiales contains several monotypic families theorized to hold a diverse assemblage of fungi yet to be discovered and properly described. Based on morphology alone, many species in this order are difficult or impossible to identify. In this study, we isolated three chytrids from northern Thailand. Phylogenetic analyses placed the isolates in three monotypic genera within Rhizophydiales. Intrageneric genetic distances in the internal transcribed spacer (ITS) ranged between 1.5 and 8.5%. Angulomyces solicola sp. nov. is characterized by larger sporangia, spores, and fewer discharge papilla than A.argentinensis; Gorgonomyces thailandicus sp. nov. has larger zoospores and fewer discharge papillae in culture compared to G. haynaldii; Terramyces chiangraiensis sp. nov. produces larger sporangia than T. subangulosum. We delimited species of Angulomyces, Gorgonomyces and Terramyces using a tripartite approach that employed phylogeny, ITS genetic distances and Poisson tree processes (PTP). Results of these approaches suggest more than one species in each genus. This study contributes to the knowledge of chytrids, an understudied group in Thailand and worldwide.

New nephridiophagid genera (Fungi, Chytridiomycota) in a mallow beetle and an earwig.

Nephridiophagids are unicellular fungi (Chytridiomycota) that infect the Malpighian tubules of insects. Most species have been found in cockroach hosts and belong to the genus Nephridiophaga. Three additional genera have been described from beetles and an earwig. Here, we characterise morphologically and molecular phylogenetically the nephridiophagids of the European earwig Forficulaauricularia and the mallow beetle Podagricamalvae. Their morphology and life cycle stages resemble those of other nephridiophagids, but their rRNA gene sequences support the existence of two additional genera. Whereas the earwig nephridiophagid (Nephridiochytriumforficulaegen. nov. et sp. nov.) forms a sister lineage of the Nephridiophaga cluster, the mallow beetle nephridiophagid (Malpighivincopodagricaegen. nov. et sp. nov.) represents the earliest divergent lineage within the nephridiophagids, being sister to all other species. Our results corroborate the hypothesis that different insect groups harbour distinct nephridiophagid lineages.

The new chytridiomycete Paradinomyces triforaminorum gen. et sp. nov. co-occurs with other parasitoids during a Kryptoperidinium foliaceum (Dinophyceae) bloom in the Baltic Sea.

A new chytrid genus and species was isolated and cultured from samples obtained in the Baltic Sea during a dinoflagellate bloom event. This species is characterized by having a spherical sporangium without papillae and zoospores of 2-3 µm in diameter that are released through 3 discharge pores. Molecular phylogeny based on ribosomal operon showed its sister position to the Dinomyces cluster in Rhizophydiales. Zoospores lack fenestrated cisternae but contain a paracrystalline inclusion, found in a Rhizophydiales representative for the first time. Additionally, the kinetid features are uncommon for Rhizophydiales and only observed in Dinomyces representatives so far. These morphological features and its phylogenetic relationships justify the description of the new genus and speciesParadinomyces triforaminorum gen. nov. sp. nov. belonging to the family Dinomycetaceae. The chytrid was detected during a high-biomass bloom of the dinoflagellate Kryptoperidinium foliaceum. Laboratory experiments suggest this species is highly specific and demonstrate the impact it can have on HAB development. The chytrid co-occurred with three other parasites belonging to Chytridiomycota (Fungi) and Perkinsea (Alveolata), highlighting that parasitic interactions are common during HABs in brackish and marine systems, and these multiple parasites compete for similar hosts.

Effects of algae and fungicides on the fate of a sulfonylurea herbicide in a water-sediment system.

The impact of pesticide mixtures on various soil parameters has been extensively studied, whereas research on effects in the aquatic environment is scarce. Furthermore, investigations on the consequences of chemical mixtures on the biodegradation kinetics of parent compounds remain deficient. Our research intended to evaluate potential effects by combined application of an agriculturally employed tank mixture to aquatic sediment systems under controlled laboratory conditions. The mixture contained two fungicides and one radiolabeled herbicide of which the route and rate of degradation was followed. One set of aquatic sediment vessels was incubated in the dark. A second set of vessels was controlled under identical conditions, except for being continuously irradiated to promote algal growth. In addition, the algal biomass in irradiated aquatic sediment was monitored to determine its effects and a potential role in the biodegradation of iodosulfuron-methyl-sodium. The study results showed that the herbicide, although hydro- and photolytically stable throughout the study, metabolized faster (DT50 1.1-1.2-fold and DT90 2.8-4.5-fold) when continuously irradiated in comparison to dark aquatic sediment. Both fungicides had a significant prolonging effect on the biodegradation rate of the herbicide. In the presence of fungicides, DT90 values increased 1.5-fold in the irradiated, and 2.5-fold in the dark systems. Additionally, algae may have influenced the metabolization of the herbicide in the irradiated systems, where shorter DT90 values were evaluated. Even so, the algal influence was concluded to be indirect.

Laboratory Maintenance of the Chytrid Fungus Batrachochytrium dendrobatidis.

The chytrid fungus Batrachochytrium dendrobatidis (Bd) is a causative agent of chytridiomycosis, a skin disease associated with amphibian population declines around the world. Despite the major impact Bd is having on global ecosystems, much of Bd's basic biology remains unstudied. In addition to revealing mechanisms driving the spread of chytridiomycosis, studying Bd can shed light on the evolution of key fungal traits because chytrid fungi, including Bd, diverged before the radiation of the Dikaryotic fungi (multicellular fungi and yeast). Studying Bd in the laboratory is, therefore, of growing interest to a wide range of scientists, ranging from herpetologists and disease ecologists to molecular, cell, and evolutionary biologists. This protocol describes how to maintain developmentally synchronized liquid cultures of Bd for use in the laboratory, how to grow Bd on solid media, as well as cryopreservation and revival of frozen stocks. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Reviving cryopreserved Bd cultures Basic Protocol 2: Establishing synchronized liquid cultures of Bd Basic Protocol 3: Regular maintenance of synchronous Bd in liquid culture Alternate Protocol 1: Regular maintenance of asynchronous Bd in liquid culture Basic Protocol 4: Regular maintenance of synchronous Bd on solid medium Alternate Protocol 2: Starting a culture on solid medium from a liquid culture Basic Protocol 5: Cryopreservation of Bd.

Parasitic Chytrids Upgrade and Convey Primary Produced Carbon During Inedible Algae Proliferation.

Microbial parasites have only recently been included in planktonic food web studies, but their functional role in conveying dietary energy still remains to be elucidated. Parasitic fungi (chytrids) infecting phytoplankton may constitute an alternative trophic link and promote organic matter transfer through the production of dissemination zoospores. Particularly, during proliferation of inedible or toxic algal species, such as large Cyanobacteria fostered by global warming, parasites can constitute an alternative trophic link providing essential dietary nutrients that support somatic growth and reproduction of consumers. Using phytoplankton-parasites associations grown under laboratory controlled conditions we assessed the fatty acids and biochemical composition of species with different nutritional quality and followed the metabolic pathway from the algal host and their parasites zoospores using compound-specific stable isotope analysis. This study demonstrated that chytrids are trophic upgraders able to retain essential nutrients that can be transferred to upper trophic levels both in terms of organic matter quantity and nutritional quality. Through the production of zoospores, nutritionally important long-chain polyunsaturated fatty acids that can be consequently assimilated by consumers. We conclude that parasitism at the base of aquatic food webs may represent a crucial trophic link for dietary nutrients and essential biomolecules alternative to herbivory or bacterivory, which can be particularly crucial during the proliferation of inedible or nutritionally inadequate algal species fostered by climate change.

Chytrid rhizoid morphogenesis resembles hyphal development in multicellular fungi and is adaptive to resource availability.

Key to the ecological prominence of fungi is their distinctive cell biology, our understanding of which has been principally based on dikaryan hyphal and yeast forms. The early-diverging Chytridiomycota (chytrids) are ecologically important and a significant component of fungal diversity, yet their cell biology remains poorly understood. Unlike dikaryan hyphae, chytrids typically attach to substrates and feed osmotrophically via anucleate rhizoids. The evolution of fungal hyphae appears to have occurred from rhizoid-bearing lineages and it has been hypothesized that a rhizoid-like structure was the precursor to multicellular hyphae. Here, we show in a unicellular chytrid, Rhizoclosmatium globosum, that rhizoid development exhibits striking similarities with dikaryan hyphae and is adaptive to resource availability. Rhizoid morphogenesis exhibits analogous patterns to hyphal growth and is controlled by ß-glucan-dependent cell wall synthesis and actin polymerization. Chytrid rhizoids growing from individual cells also demonstrate adaptive morphological plasticity in response to resource availability, developing a searching phenotype when carbon starved and spatial differentiation when interacting with particulate organic matter. We demonstrate that the adaptive cell biology and associated developmental plasticity considered characteristic of hyphal fungi are shared more widely across the Kingdom Fungi and therefore could be conserved from their most recent common ancestor.

New Member of Gromochytriales (Chytridiomycetes)-Apiochytrium granulosporum nov. gen. et sp.

Molecular phylogenetic analysis of 18S rRNA gene sequences of nearly any species of Chytridiomycota has typically challenged traditional classification and triggered taxonomic revision. This has often led to the establishment of new taxa which, normally, appears well supported by zoospore ultrastructure, which provides diagnostic characters. To construct a meaningful and comprehensive classification of Chytridiomycota, the combination of molecular phylogenies and morphological studies of traditionally defined chytrid species is needed. In this work, we have studied morphological and ultrastructural features based on light and transmission electron microscopy as well as molecular phylogenetic analysis of a parasite (strain X-124 CCPP ZIN RAS) morphologically similar to Rhizophydium granulosporum living on the yellow-green alga Tribonema gayanum. Phylogenetic analysis of the 18S rRNA gene sequence of this strain supports that it represents a new genus and species affiliated to the recently established order Gromochytriales. The ultrastructure of X-124 confirms its phylogenetic position sister to Gromochytrium and serves as the basis for the description of the new genus and species Apiochytrium granulosporum. The 18S rRNA gene of A. granulosporum contains a S943 group I intron that carries a homing endonuclease pseudogene.

A New Parasitic Chytrid, Staurastromyces oculus (Rhizophydiales, Staurastromycetaceae fam. nov.), Infecting the Freshwater Desmid Staurastrum sp.

Chytrids are a diverse group of ubiquitous true zoosporic fungi. The recent molecular discovery of a large diversity of undescribed chytrids has raised awareness on their important, but so far understudied ecological role in aquatic ecosystems. In the pelagic zone, of both freshwater and marine ecosystems, many chytrid species have been morphologically described as parasites on almost all major groups of phytoplankton. However, the majority of these parasitic chytrids has rarely been isolated and lack DNA sequence data, resulting in a large proportion of "dark taxa" in databases. Here, we report on the isolation and in-depth morphological, molecular and host range characterization of a chytrid infecting the common freshwater desmid Staurastrum sp. We provide first insights on the metabolic activity of the different chytrid development stages by using the vital dye FUN®-1 (2-chloro-4-[2,3-dihydro-3-methyl-[benzo-1,3-thiazol-2-yl]-methylidene]-1-phenylquinolinium iodide). Cross infection experiments suggest that this chytrid is an obligate parasite and specific for the genus Staurastrum sp. Phylogenetic analysis, based on ITS1-5.8S-ITS2 and 28S rDNA sequences, placed it in the order Rhizophydiales. Based on the unique zoospore ultrastructure, combined with thallus morphology, and molecular phylogenetic placement, we describe this parasitic chytrid as a new genus and species Staurastromyces oculus, within a new family Staurastromycetaceae.

Chytridiomycosis of Marine Diatoms-The Role of Stress Physiology and Resistance in Parasite-Host Recognition and Accumulation of Defense Molecules.

Little is known about the role of chemotaxis in the location and attachment of chytrid zoospores to potential diatom hosts. Hypothesizing that environmental stress parameters affect parasite-host recognition, four chytrid-diatom tandem cultures (Chytridium sp./Navicula sp., Rhizophydium type I/Nitzschia sp., Rhizophydium type IIa/Rhizosolenia sp., Rhizophydium type IIb/Chaetoceros sp.) were used to test the chemotaxis of chytrid zoospores and the presence of potential defense molecules in a non-contact-co-culturing approach. As potential triggers in the chemotaxis experiments, standards of eight carbohydrates, six amino acids, five fatty acids, and three compounds known as compatible solutes were used in individual and mixed solutions, respectively. In all tested cases, the whole-cell extracts of the light-stressed (continuous light exposure combined with 6 h UV radiation) hosts attracted the highest numbers of zoospores (86%), followed by the combined carbohydrate standard solution (76%), while all other compounds acted as weak triggers only. The results of the phytochemical screening, using biomass and supernatant extracts of susceptible and resistant host-diatom cultures, indicated in most of the tested extracts the presence of polyunsaturated fatty acids, phenols, and aldehydes, whereas the bioactivity screenings showed that the zoospores of the chytrid parasites were only significantly affected by the ethanolic supernatant extract of the resistant hosts.

Zoosporic parasites infecting marine diatoms - A black box that needs to be opened.

Living organisms in aquatic ecosystems are almost constantly confronted by pathogens. Nevertheless, very little is known about diseases of marine diatoms, the main primary producers of the oceans. Only a few examples of marine diatoms infected by zoosporic parasites are published, yet these studies suggest that diseases may have significant impacts on the ecology of individual diatom hosts and the composition of communities at both the producer and consumer trophic levels of food webs. Here we summarize available ecological and morphological data on chytrids, aphelids, stramenopiles (including oomycetes, labyrinthuloids, and hyphochytrids), parasitic dinoflagellates, cercozoans and phytomyxids, all of which are known zoosporic parasites of marine diatoms. Difficulties in identification of host and pathogen species and possible effects of environmental parameters on the prevalence of zoosporic parasites are discussed. Based on published data, we conclude that zoosporic parasites are much more abundant in marine ecosystems than the available literature reports, and that, at present, both the diversity and the prevalence of such pathogens are underestimated.

Molecular diversity and distribution of marine fungi across 130 European environmental samples.

Environmental DNA and culture-based analyses have suggested that fungi are present in low diversity and in low abundance in many marine environments, especially in the upper water column. Here, we use a dual approach involving high-throughput diversity tag sequencing from both DNA and RNA templates and fluorescent cell counts to evaluate the diversity and relative abundance of fungi across marine samples taken from six European near-shore sites. We removed very rare fungal operational taxonomic units (OTUs) selecting only OTUs recovered from multiple samples for a detailed analysis. This approach identified a set of 71 fungal 'OTU clusters' that account for 66% of all the sequences assigned to the Fungi. Phylogenetic analyses demonstrated that this diversity includes a significant number of chytrid-like lineages that had not been previously described, indicating that the marine environment encompasses a number of zoosporic fungi that are new to taxonomic inventories. Using the sequence datasets, we identified cases where fungal OTUs were sampled across multiple geographical sites and between different sampling depths. This was especially clear in one relatively abundant and diverse phylogroup tentatively named Novel Chytrid-Like-Clade 1 (NCLC1). For comparison, a subset of the water column samples was also investigated using fluorescent microscopy to examine the abundance of eukaryotes with chitin cell walls. Comparisons of relative abundance of RNA-derived fungal tag sequences and chitin cell-wall counts demonstrate that fungi constitute a low fraction of the eukaryotic community in these water column samples. Taken together, these results demonstrate the phylogenetic position and environmental distribution of 71 lineages, improving our understanding of the diversity and abundance of fungi in marine environments.

Copper (II) lead (II), and zinc (II) reduce growth and zoospore release in four zoosporic true fungi from soils of NSW, Australia.

This study examined the responses of a group of four zoosporic true fungi isolated from soils in NSW Australia, to concentrations of toxic metals in the laboratory that may be found in polluted soils. All isolates showed greatest sensitivity to Cu and least sensitivity to Pb. All isolates showed significant reduction in growth at 60 ppm (0.94 mmol m(-3)) for Cu, while three declined significantly at 60 ppm (0.92 mmol m(-3)) Zn. The growth of two isolates declined significantly at 100 ppm (0.48 mmol m(-3)) Pb and one at 200 ppm (0.96 mmol m(-3)) Pb. The rate of production of zoospores for all isolates was reduced when sporangia were grown in solid PYG media with 60 ppm Cu. Three isolates significantly declined in production at 60 ppm Zn and three at 100 ppm Pb. All isolates recovered growth after incubation in solid media with 60 ppm Zn or 100 ppm Pb. Two isolates did not recover growth after incubation in 60 ppm Cu. If these metals cause similar effects in the field, Cu, Pb, and Zn contamination of NSW soils is likely to reduce biomass of zoosporic true fungi. Loss of the fungi may reduce the rate of mineralisation of soil organic matter.

Cytology and molecular phylogenetics of Monoblepharidomycetes provide evidence for multiple independent origins of the hyphal habit in the Fungi.

The evolution of filamentous hyphae underlies an astounding diversity of fungal form and function. We studied the cellular structure and evolutionary origins of the filamentous form in the Monoblepharidomycetes (Chytridiomycota), an early-diverging fungal lineage that displays an exceptional range of body types, from crescent-shaped single cells to sprawling hyphae. To do so, we combined light and transmission electron microscopic analyses of hyphal cytoplasm with molecular phylogenetic reconstructions. Hyphae of Monoblepharidomycetes lack a complex aggregation of secretory vesicles at the hyphal apex (i.e. Spitzenkörper), have centrosomes as primary microtubule organizing centers and have stacked Golgi cisternae instead of tubular/fenestrated Golgi equivalents. The cytoplasmic distribution of actin in Monoblepharidomycetes is comparable to the arrangement observed previously in other filamentous fungi. To discern the origins of Monoblepharidomycetes hyphae, we inferred a phylogeny of the fungi based on 18S and 28S ribosomal DNA sequence data with maximum likelihood and Bayesian inference methods. We focused sampling on Monoblepharidomycetes to infer intergeneric relationships within the class and determined 78 new sequences. Analyses showed class Monoblepharidomycetes to be monophyletic and nested within Chytridiomycota. Hyphal Monoblepharidomycetes formed a clade sister to the genera without hyphae, Harpochytrium and Oedogoniomyces. A likelihood ancestral state reconstruction indicated that hyphae arose independently within the Monoblepharidomycetes lineage and in at least two other lineages. Cytological differences among monoblepharidalean and other fungal hyphae are consistent with these convergent origins.

Eukaryotic pathogens (Chytridiomycota and Oomycota) infecting marine microphytobenthic diatoms - a methodological comparison.

Using sediment samples from the Solthörn tidal flat (southern North Sea, Germany), collected in bi-weekly intervals from June to July 2012, a range of qualitative and quantitative screening methods for oomycete and chytrid pathogens infecting benthic diatoms were evaluated. Pre-treatment of sediment samples using short ultrasound pulses and gradient centrifugation, in combination with CalcoFluor White, showed the best results in the visualization of both pathogen groups. The highest number of infected benthic diatoms was observed in mid July (5.8% of the total benthic diatom community). Most infections were caused by chytrids and, in a few cases, oomycetes (Lagenisma Drebes (host: Coscinodiscus radiatus Ehrenberg) and Ectrogella Zopf (hosts: Dimeregramma minor in Pritchard and Gyrosigma peisonis). Among the chytrids, sporangium morphology indicated the presence of five different morphotypes, infecting mainly epipelic taxa of the orders Naviculales (e.g., Navicula digitoradiata) and Achnanthales (e.g., Achnanthes brevipes Agardh). The presence of multiple pathogens in several epipelic diatom taxa suggests a significant role for fungal parasitism in affecting microphytobenthic diatom succession.

Phytoplankton chytridiomycosis: fungal parasites of phytoplankton and their imprints on the food web dynamics.

Parasitism is one of the earlier and common ecological interactions in the nature, occurring in almost all environments. Microbial parasites typically are characterized by their small size, short generation time, and high rates of reproduction, with simple life cycle occurring generally within a single host. They are diverse and ubiquitous in aquatic ecosystems, comprising viruses, prokaryotes, and eukaryotes. Recently, environmental 18S rDNA surveys of microbial eukaryotes have unveiled major infecting agents in pelagic systems, consisting primarily of the fungal order of Chytridiales (chytrids). Chytrids are considered the earlier branch of the Eumycetes and produce motile, flagellated zoospores, characterized by a small size (2-6 µm), and a single, posterior flagellum. The existence of these dispersal propagules includes chytrids within the so-called group of zoosporic fungi, which are particularly adapted to the plankton lifestyle where they infect a wide variety of hosts, including fishes, eggs, zooplankton, algae, and other aquatic fungi but primarily freshwater phytoplankton. Related ecological implications are huge because chytrids can killed their hosts, release substrates for microbial processes, and provide nutrient-rich particles as zoospores and short fragments of filamentous inedible hosts for the grazer food chain. Furthermore, based on the observation that phytoplankton chytridiomycosis preferentially impacts the larger size species, blooms of such species (e.g., filamentous cyanobacteria) may not totally represent trophic bottlenecks. Besides, chytrid epidemics represent an important driving factor in phytoplankton seasonal successions. In this review, I summarize the knowledge on the diversity, community structure, quantitative importance, and functional roles of fungal chytrids, primarily those who are parasites of phytoplankton, and infer the ecological implications and potentials for the food web dynamics and properties. I reach the conclusion that phytoplankton chytridiomycosis represents an important but as yet overlooked ecological driving force in aquatic food web dynamics and network organization.