3.2: Aquatic Fungi (Chytrids) - Biology

3.2: Aquatic Fungi (Chytrids) - Biology

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Like many of the earlier fungal lineages, this group has been divided into several distinct lineages. Once all classified as Chytridiomycota, these early, aquatic fungi are now grouped into Blastocladiomycota, Chytridiomycota, and Neocallimastigomycota. Though likely not directly related, these fungi share a few characteristics:

  • Primarily aquatic, though some are parasites of terrestrial plants
  • Swimming spores (zoospores) with a single whiplash flagellum

Saprotrophic Chytrids

Figure (PageIndex{1}): Chytrids in the genus Allomyces are aquatic decomposers. The top image shows sesame seeds floating in pond water with fungus growing off of them. The second image (bottom left) shows a microcopic image of a zoosporangium produced at the end of a hyphal filament (asexual reproduction). The final image (bottom right) shows two resting sporangia, the result of sexual reproduction. Photos by Tom Bruns, some rights reserved (CC-BY-NC).

Parasitic Chytrids

Figure (PageIndex{2}): This image shows Micromyces zygogonia (Chytridiomycota), a parasite with spiky structures (prosori), inside the cells of the alga Spirogyra. Photo Credit Fahrenheit_66, CC-BY-NC.

Figure (PageIndex{3}): In these images, the part of the chytrids you can see is the large, globose struture attached to the outside of the diatom. This is the part of the thallus where spores are made. "Chytrid parasites of marine diatoms. (A) Chytrid sporangia on Pleurosigma sp. The white arrow indicates the operculate discharge pore. (B) Rhizoids (white arrow) extending into diatom host. (C) Chlorophyll aggregates localized to infection sites (white arrows). (D and E) Single hosts bearing multiple zoosporangia at different stages of development. The white arrow in panel E highlights branching rhizoids. (F) Endobiotic chytrid-like sporangia within diatom frustule. Bars = 10 μm. For more details see: Hassett BT, Gradinger R (2016) "Chytrids dominate arctic marine fungal communities". Environ Microbiol, 18(6):2001–2009." Unknown author, CC BY 4.0, via Wikimedia Commons.

Figure (PageIndex{4}): A parasite on Geranium, this Synchytrium papillatum (Chytridiomycota) is forming pink growths on the calyx and peduncle of these flowers. Photo Credit: James Bailey some rights reserved (CC BY-NC).

Figure (PageIndex{5}): Likely the most infamous chytrid, Batrachochytrium dendrobatidis (Chytridiomycota) causes a lethal skin infection in many amphibians. This now widespread invasive pathogen is contributing to global amphibian declines and extinctions. Photo by Jonathan (JC) Carpenter, some rights reserved (CC BY-NC).

11.2 – Classifications of Fungi

By the end of this section, you will be able to do the following:

  • Identify fungi and place them into the five major phyla according to current classification
  • Describe each phylum in terms of major representative species and patterns of reproduction

The kingdom Fungi contains five major phyla that were established according to their mode of sexual reproduction or using molecular data. Polyphyletic, unrelated fungi that reproduce without a sexual cycle, were once placed for convenience in a sixth group, the Deuteromycota, called a “form phylum,” because superficially they appeared to be similar. However, most mycologists have discontinued this practice. Rapid advances in molecular biology and the sequencing of 18S rRNA (ribosomal RNA) continue to show new and different relationships among the various categories of fungi.

The five true phyla of fungi are the Chytridiomycota (Chytrids), the Zygomycota (conjugated fungi), the Ascomycota (sac fungi), the Basidiomycota (club fungi) and the recently described Phylum Glomeromycota ((Figure)).

Ecological Significance of Parasites

Parasites are important components of ecological communities (Thomas et al., 2005 Hatcher and Dunn, 2011). They have the potential to regulate host populations, mediate interspecific competition between hosts and other species, maintain genetic polymorphism and biodiversity, and affect community structure. Nevertheless, the effects of parasites and diseases on food webs and ecosystem dynamics have been neglected until recently (Polis and Strong, 1996 Marcogliese and Cone, 1997). New research suggests that parasites have the potential to alter food-web topology, stability, interaction strength and energy flow (Lafferty, 2006 Kuris et al., 2008 Lafferty et al., 2008).

Parasites commonly function as prey within ecosystems (Johnson et al., 2010 Thieltges et al., 2013). There are two main ways in which parasites become prey. Predators can either consume the infected hosts of parasites (concomitant predation) or their free-swimming life stage (Johnson et al., 2010). Many aquatic parasites including viruses, chytrids, trematodes, and nematodes, have a free-swimming stage that may be subject to predation (Gonzalez and Suttle, 1993 Kagami et al., 2004 Kuris et al., 2008 Johnson et al., 2010). The Chytridiomycota (chytrids) are one of the dominant groups of parasites in aquatic ecosystems. The free-living zoosporic stage of chytrids actively searches for and infects host cells, extracting nutrients and developing into mature sporangia that release new zoospores (Canter, 1967 Figure 1). There are more than 700 species of chytrids known to infect phytoplankton, zooplankton, fungi, plants, and invertebrate animals (Sparrow, 1960 Gleason et al., 2008). Here in this review, we mainly focus on parasitic chytrids that infect phytoplankton, and explain their roles in aquatic food webs as prey for zooplankton through the “mycoloop” pathway (Kagami et al., 2007a).

Figure 1. Diagram of “mycoloop.” Parasitic chytrids can transfer material from large inedible phytoplankton to zooplankton. Chytrids zoospores are excellent food for zooplankton in terms of size (2𠄵 μm in diameter), shape, nutritional quality (rich in PUFAs and cholesterols). Large colonies of host phytoplankton may also be fragmented by chytrid infections and become edible to zooplankton. On the other hand, infected host colonies may remain inedible to Daphnia, or even become less edible due to the aggregate formation of cells. Those aggregations may sink faster, and affect material cycling in lakes.

Zygomycota: The Conjugated Fungi

The zygomycetes are a relatively small group of fungi belonging to the Phylum Zygomycota. They include the familiar bread mold, Rhizopus stolonifer, which rapidly propagates on the surfaces of breads, fruits, and vegetables. Most species are saprobes, living off decaying organic material a few are parasites, particularly of insects. Zygomycetes play a considerable commercial role. The metabolic products of other species of Rhizopus are intermediates in the synthesis of semi-synthetic steroid hormones.

Zygomycetes have a thallus of coenocytic hyphae in which the nuclei are haploid when the organism is in the vegetative stage. The fungi usually reproduce asexually by producing sporangiospores ( Figure 2 ). The black tips of bread mold are the swollen sporangia packed with black spores ( Figure 3 ). When spores land on a suitable substrate, they germinate and produce a new mycelium. Sexual reproduction starts when conditions become unfavorable. Two opposing mating strains (type + and type –) must be in close proximity for gametangia from the hyphae to be produced and fuse, leading to karyogamy. The developing diploid zygospores have thick coats that protect them from desiccation and other hazards. They may remain dormant until environmental conditions are favorable. When the zygospore germinates, it undergoes meiosis and produces haploid spores, which will, in turn, grow into a new organism. This form of sexual reproduction in fungi is called conjugation (although it differs markedly from conjugation in bacteria and protists), giving rise to the name “conjugated fungi.”

Figure 2. Zygomycetes have asexual and asexual life cycles. In the sexual life cycle, plus and minus mating types conjugate to form a zygosporangium.

Figure 3. Sporangia grow at the end of stalks, which appear as (a) white fuzz seen on this bread mold, Rhizopus stolonifer. The (b) tips of bread mold are the spore-containing sporangia. (credit b: modification of work by “polandeze”/Flickr)

3. Process Engineering and Genetic Engineering

A combination of process engineering and genetic (molecular) engineering approaches can aid the successful transposition of anaerobic fungi from their natural habitat in herbivores to effective exploitation in industrial biofuel production. The discipline of process engineering will be necessary to synthetically create a suitable habitat and environment in which an unnaturally large population of anaerobic fungi can resiliently prosper as a monoculture or co-culture in the absence of the host animal. The application of process engineering should include attention to design aspects such as the structure of fermentation vessels, solid and liquid amounts and retention times, suitable inoculation and plant biomass feeding regimes (batch or continuous). Genetic engineering provides the opportunity to manipulate anaerobic fungal cells and exploit their genetic potential for the purposes of higher product yields, increased environmental resilience and faster hydrolysis of lignocellulose material. Therefore, this section will discuss the current progress, challenges and future goals relating to achieving and optimising the industrial use of anaerobic fungi via both approaches.

3.1. Process Engineering

According to Dal Pont [55], process engineering can be summarised as the understanding and application of the fundamental principles and laws of nature that allow us to transform raw material and energy into products that are useful to society, at an industrial level. As yet, understanding of the anaerobic fungi falls far short of them being able to transform lignocellulosic substrates into biofuel energy products that are useful to society at an industrial scale. Moreover, it is important to underscore the significance of the anaerobic fungal niche in the mammalian digestive tract when considering opportunities and challenges for their biotechnological exploitation. On the one hand, an alternation of generations between reproductive motile zoospores and benthic, vegetative fungal thalli represent limitations (certainly for monocentric fungi) that must be accommodated if they are to be grown successfully in industrial processes. On the other hand, much can be learnt and potentially exploited (particularly from an engineering perspective) from a detailed understanding of the way in which the anaerobic fungi thrive and deconstruct lignocellulosic substrates in their natural habitat.

3.1.1. Anaerobic Fungi in Anaerobic Digestion (AD)

Anaerobic fungi reside in and are easily isolated in culturable form from the digestive tract of large herbivorous mammals. In these environments, lignocellulosic substrates are abundant and oxygen is absent. Many other anaerobic environments contain an abundance of lignocellulose and might also support anaerobic fungi. For example, anoxic zones in landfill sites, anoxic muds and marshlands and purpose-built anaerobic digesters. Multiple studies have demonstrated that DNA extracted from these sites map unequivocally to the Neocallimastigomycota, suggesting that anaerobic fungi may be present and are not exclusively gut inhabitants [20,21,56,57,58,59,60,61]. However, the prevalence of large numbers of stress-tolerant survival structures in the faeces of mammalian herbivores means that their nucleic acid motifs will be abundant and widespread in nature. Therefore, it is to be expected that molecular signatures of anaerobic fungi will be found in a broad range of habitats outside of the gastrointestinal tract, wherever faeces are deposited. Detection of fragments of nucleic acid belonging to the anaerobic fungi in these locations should not be taken as evidence of their ability to undergo vegetative growth and reproduction. Where anaerobic fungi have been sought in landfill sites using culture methodologies, they have not been found [62].

In recent research, several studies have investigated the use of anaerobic fungi for bioaugmentation in industrial anaerobic digestion (AD) plants [57,58,63,64,65]. The rationale for inferring a role for anaerobic fungi in AD implies analogy with the digestive tract ecosystem. In both environments, complex molecules of plant origin are converted into simple organic molecules. The rationale is also cognisant of the fact that anaerobic fungi in their natural habitat form stable, syntrophic co-cultures with methanogenic archaea [5,66,67]. If anaerobic fungi could be successfully utilised in an AD plant, they could allow lignocellulose to become a major feedstock, representing an important step-change in the bioremediation process. Genetic motifs of anaerobic fungi have been found in industrial AD plants. In one study, 10 commercial plants in Germany were surveyed for transcriptional activity [57]. Anaerobic fungal 18S DNA was found, but only in plants that received cattle manure and of those, only two were found to contain GH5 endonuclease transcripts, suggesting metabolic activity. Others have also found genetic motifs of anaerobic fungi in manure-fed digesters, in landfill sites and in pond and stream muds adjacent to land grazed by livestock [58,59,60].

Anaerobic fungi are known to produce a survival stage that can exist for many months in dried livestock faeces [20,21,68]. They can also be readily isolated in culturable form from livestock manure and slurries [59,61]. Most isolates of anaerobic fungi studied in the laboratory have been obtained from livestock faeces. Thus, it seems inevitable that genetic motifs of anaerobic fungi will be detected in bioreactors, landfill sites or aqueous ecosystems where livestock manures are deliberately or accidentally introduced. It is therefore necessary to conduct this type of research in accordance with Koch’s postulates, to isolate, re-introduce and re-isolate viable cultures, before ascribing a role for anaerobic fungi in the AD environment, or indeed in any bioaugmentation study.

3.1.2. Bioreactor Design and Habitat Engineering

Many of the techniques used to culture anaerobic fungi in the laboratory are based on methods developed by Hungate [69], Bryant [70], Hungate and Macey [71], and Miller and Wolin [72]. With relatively few exceptions these methods, together with the enumeration and growth determining procedures of Joblin [41] and Theodorou et al. [43,73], are used to routinely culture and maintain anaerobic fungi at bench-scale in the laboratory. This subject area was reviewed recently by Haitjema et al. [37]. In general, anaerobic fungi are grown at 39 ଌ without agitation in small batch cultures (of 10� mL culture volume) in thick-walled glass tubes or bottles sealed with gas-tight stoppers. In order to retain culture viability, anaerobic fungi must be maintained in sequential batch culture, with a transfer interval of between 2 and 7 days [37]. While some anaerobic fungi have been grown on defined media [74], better growth is obtained on complex media where sterile rumen fluid (10�%) is an essential component of all such media. Problems associated with culture viability and the requirement for rumen fluid in culture media are noteworthy as barriers to growing anaerobic fungi in larger-scale bioreactors. The need for rumen fluid is a particular constraint to scale-up and research is required to elucidate those factors in rumen fluid that are necessary to stimulate fungal growth.

The first attempt to grow anaerobic fungi on a plant biomass concentration that was higher than that typically used in batch cultures was performed by Zhu et al. [75,76]. In their research, by continuously eluting growing cultures with fresh culture medium, they succeeded in growing an anaerobic fungus on increasing concentrations, up to 80 g dry matter (DM) L 𢄡 of wheat straw. By using a multichannel peristaltic pump to deliver fresh culture medium to several culture bottles as spent medium was removed, these authors were able to monitor replicated cultures and make treatment comparisons. When compared with results obtained from conventional batch cultures, where the fungus is grown on just 10 g DM L 𢄡 of wheat straw, their continuous-flow cultures produced up to 20 times more cell wall-degrading enzymes (CMCase and β-glucosidase) [75]. In comparisons involving anaerobic fungi grown on 80 g DM L 𢄡 of wheat straw in batch or continuous-flow cultures, up to 30 times more cell wall-degrading enzymes were produced [76]. While just 5𠄹% of the wheat straw DM was lost in batch cultures grown on 80 g DM L 𢄡 , during the same incubation period, 52�% was lost in comparable continuous-flow cultures [76]. The continuous-flow cultures described by Zhu et al. [75,76], although not representative of conventional continuous-culture systems where substrate as well as culture medium is removed, provided a simple and effective means of growing anaerobic fungi on high concentrations of plant biomass approximating those found in the rumen. The authors concluded that by using media flushing to remove the build-up of toxic fermentation end-products, the fungus was able to degrade considerably more wheat straw, produce significantly larger quantities of plant biomass degrading enzymes and survive for significantly longer periods of time in continuous-flow as opposed to batch cultures. In their 1997 publication, Zhu et al. [76] concluded that anaerobic fungi and continuous-flow cultures may have industrial potential. The effect of including in continuous-flow cultures, methanogenic and/or other non-methanogenic bacteria alongside anaerobic fungi offers intriguing possibilities and awaits further research. Important components of rumen fermentation, such as high DM concentrations, anaerobic conditions, selective retention of particulate matter, removal of toxic end-products and pulsed addition of substrate will need to be considered when developing suitable fermentation systems for the anaerobic fungi.

3.1.3. Solid Substrate Fermentation

Solid substrate fermentation is a process in which microorganisms ferment a substrate in the absence of free water or with a very low free water content [77,78]. Unlike bacteria, filamentous aerobic fungi are able to grow on a substrate in the absence of free water by utilising the bound water in the substrate [79,80]. Aerobic fungi which grow on lignocellulosic substrates tend to grow in a linear rather than exponential manner [81]. Industrial applications for microorganisms such as Trichoderma and Aspergillus involve submerged culture bioreactors, but these fungi have been highly adapted and genetically modified for this purpose [82,83]. In their natural habitat, these fungi grow on solid substrates and are not submerged in culture media. Under these circumstances, and in this particular niche, the fungi require different enzymes, cellular structures and metabolites to those grown in submerged culture [84,85,86]. In recent years, there has been much interest in harnessing aerobic fungi for the purpose of solid substrate fermentation [78] and some of the adopted approaches may be applicable to the anaerobic fungi. Figure 2 presents, in schematic format, bioreactor designs that may be suitable for industrial-scale use of anaerobic fungi. While noting that the zoospores of anaerobic fungi exist in a liquid environment, their vegetative thalli grow directly on insoluble substrates and it may therefore be possible to adapt existing solid substrate fermentation methodologies to grow anaerobic fungi at industrial scale. The culture systems commonly used for solid substrate fermentation in industry are static bioreactors (fixed bed and perforated trays), agitated bioreactors (horizontal drum, continuously/intermittently pulsed) and mixing bioreactors (rotating drum) [78].

Schematic bioreactor and anaerobic digester designs for industrial-scale use of anaerobic fungi. A lignocellulose (raft) layer forms due to biomass floating as anaerobic fungi ferment their substrate: (a) up-flow anaerobic digester where anaerobic fungi are grown ± methanogens to produce CH4, H2 and CO2 (b) plug flow anaerobic digester (c) continuous-flow bioreactor with intermittent substrate feeding and (d) high dry matter (solid-state) bioreactor where anaerobic fungi ± methanogens grow directly on moist substrate. The bioreactor is flushed with CO2 humidified with culture medium. Substrate is batch fed and residual lignocellulose can be used downstream in biotechnological processes.

In comparison to submerged culture, solid substrate fermentations are less susceptible to bacterial contamination as most bacteria require a liquid environment in order to grow and/or form a biofilm on the surface of a substrate [78,87]. Hydrolytic enzymes in solid state fermentation systems are also less prone to substrate inhibition [78,87]. If secondary metabolites, enzymes or free sugars are the desired end-product in a solid-state fermentation, then the highly concentrated effluent produced serves to eliminate the need for costly additional downstream concentration steps [78,87]. By contrast, submerged fermentation has the advantages of easier control of parameters such as pH, temperature and separation of substrate from end products [88]. As many of the existing designs of solid-substrate fermenters are unsealed to the atmosphere, maintaining a strictly anaerobic environment will be a key challenge associated with adapting solid substrate fermentation for use with anaerobic fungi. Additionally, the absence of a liquid medium presents further challenges as the buffering capacity of the growth medium and the absence of reducing agents present the risk of oxygen toxicity killing the fungus. Nevertheless, it might be feasible to develop a continuous culture system based on a plug flow digester with a very high solids content, suspended in a highly concentrated growth media, similar to the bench-scale continuous-flow systems investigated by Zhu et al. [75,76].

3.2. Genetic Engineering

Anaerobic fungi have large genomes (

100� Mb) adapted for utilisation of plant matter and survival in the gastrointestinal tract of herbivorous mammals [37,89]. Solomon et al. [4] found that fungi obtained from horse, sheep and goat contained more genes encoding carbohydrate active enzymes (CAZymes) than any other microorganism. Many of these CAZymes are found in large multiprotein cellulosomes that allow the fungus to break down lignocellulosic biomass in a synergistic manner [37]. Despite historical and recent progress in this field, the composition of these extracellular enzyme�llulosome complexes is not well described, and it is unclear whether fungal cellulosomes are predominantly secreted or bound to rhizoidal or bulbous structures [37,90,91]. With fungal genetic engineering to manipulate product selectivity and yields, anaerobic fungi show great potential for one-step processing of crude biomass. Realisation of this goal will require the development of robust genetic tools for anaerobic fungi ( Figure 3 ). In parallel, the unique and diverse arsenal of enzymes used by these organisms [4] has spurred efforts to express native fungal genes in other hosts (heterologous expression).

Anaerobic fungi show great potential for new genetic tool development and heterologous expression for biofuels production. Blue dots represent inserted or modified proteins, e.g., a fluorescent reporter attached to a knock-in cellulase. Fungi or heterologous hosts can be evolved for improved phenotypes, such as H2 production.

As a framework for investigation, Wilken et al. developed a genome-scale metabolic model for N. lanati, an anaerobic gut fungus isolated from sheep faeces [74]. This model was validated by 13 C metabolic flux analysis that identified the fluxes of carbon through glycolysis, tricarboxylic acid cycle, and in the hydrogenosome. For improved H2 production, future genetic engineering efforts may focus on directing flux through the hydrogenosome. This organelle and the pathways within it are not well characterised, with pyruvate ferredoxin oxidoreductase and/or pyruvate formate lyase potentially playing important roles in H2 production [74]. Future development of enzyme knockout strains can validate critical pathways and enable strain screening and evaluation to yield more productive enzyme and organism variants. Other potential biofuel targets include ethanol and butanol, produced by engineering strains with modified alcohol dehydrogenase and aldehyde dehydrogenase activities. Increased production of volatile fatty acids may also be beneficial when paired with other microorganisms to produce biofuels.

3.2.1. Transformation

The obligately anaerobic nature and complex life cycle [92,93,94] are challenges towards genetic engineering of these fungi. There have been no reports of stable genetic transformation of anaerobic fungi so far. Transformation requires foreign DNA entry into the organism and either integration into host genomic DNA or maintenance through replicating structures such as plasmids or artificial chromosomes.

Given the expected low transformation efficiency of the techniques described here, it will be important to use a robust selection marker. The first report of transformation on an anaerobic fungus described transient expression of the β-glucuronidase gene under control of a putative enolase promoter using a biolistic device (gene gun) approach [95]. However, these experiments were conducted without any selection pressure for the delivered gene, and the blue pigment generated upon treatment with substrate post-transformation failed to appear 7 days after transformation. Anaerobic fungi have been reported to be sensitive to hygromycin B [96], and transformation with a resistance marker can potentially be a useful selection scheme. A scheme using the hph gene encoding a hygromycin B phosphotransferase is the most common selection method used in filamentous (aerobic) fungi [97]. Investigation into anaerobic fungal autotrophs can also be fruitful, as it would enable complementation strategies. The wild type strains of yeasts such as Saccharomyces pombe, S. cerevisiae and Candida albicans are sensitive to 5-Fluoroorotic Acid (5-FOA) due to native expression of orotidine-5-monophosphate decarboxylase (OMP decarboxylase, encoded by the URA3 gene). In yeasts, URA3 is involved in uracil biosynthesis, and URA3-deficient strains are dependent on uracil supplementation for growth. Anaerobic fungi can be grown in defined media without uracil supplementation [98] and published genomes contain a putative OMP decarboxylase [4,37,89], and are therefore likely to be sensitive to 5-FOA. A strategy involving knockout of the URA3 homologue and selection with 5-FOA merits further investigation for selection.

Due to the fact that the vegetative thallus in monocentric anaerobic fungi is devoid of DNA and because fungal zoospores are reported to have relatively thin, non-chitinaceous, flexible cell walls [99], zoospores have been targeted as the most amenable life cycle stage for nucleic acid delivery and strain engineering. Calkins et al. [100] described a protocol to harvest zoospores from Pecoramyces ruminantium and later showed RNA interference-mediated knockdown of lactate dehydrogenase [101]. RNA interference (RNAi) has been observed naturally and is used in many organisms to decrease mRNA transcript number (and thus protein number) of targets [102]. Calkins and co-workers [101] identified genes required for RNAi in the genome of P. ruminantium and synthesised doubled-stranded RNA encoding a 21 base pair stretch in the lactate dehydrogenase transcript. They incubated this RNA with harvested zoospores and observed significant decreases in target gene expression (25% of untreated) and lactate production (14% of untreated) in propagated fungal mass. This work represents a promising proof-of-concept of metabolic engineering in anaerobic fungi and opens several interesting avenues of exploration. However, accompanying lactate dehydrogenase downregulation was an unwanted and non-specific downregulation of an additional 29 transcripts. Additional mechanistic investigation of siRNA targeting is needed, including understanding the duration of effect and generalisability to other genes and pathways.

A recent report by Swafford et al. [103] details the electroporation of the closely related blastoclades Batrachochytrium dendrobatidis and B. salamandrivorans. Electroporation is a widely used method of genetic transformation, in which target cells are exposed to a high electric field (typically 250� V/cm) in the presence of DNA. The electric field is thought to cause temporary holes in the cell membrane and subsequent entry of DNA [103]. In the study by Swafford and colleagues, electroporation parameters (pulse shape, voltage and timing) were optimised for dextran entry and viability, resulting in 95% of zoospores taking up payload and a 41�% survival rate, quantified by flow cytometry and motility, respectively. The authors observed that even without electroporation, some zoospores exhibited pericellular fluorescence due to dextran cell wall interactions, and analysis of electroporated cells showed intracellular signal, confirming uptake. Additionally, the authors note that the electroporation efficiency was highly dextran source-dependent, which has implications for extension to DNA transformation. Assembly of nucleic acids into polyplexes, as applied in the gene therapeutics field [104], may be necessary for high efficiency transformation of anaerobic fungi. Furthermore, uptake and persistence of DNA is dependent on long-term survival and division, and it is possible that electroporated zoospores may survive initially but fail to encyst and propagate. Careful quantification of zoospore propagation, through thallus forming unit (TFU) determinations [39], or gas pressure measurements [73], will be important for protocol validation especially when generating large and diverse gene libraries.

Other methods for nucleic acid delivery into non-model organisms are worth further exploration in their application to anaerobic fungi. Agrobacterium tumefaciens is a natural plant-targeting bacterium that has been used to integrate DNA into filamentous fungi, like Aspergillus. This system has been used to insert DNA into specific regions in the host DNA via CRISPR/Cas9. However, Agrobacterium-mediated transformation requires extended (㸶 h) co-incubation at temperatures below 30 ଌ, whereas Neocallimastix grows best at 39 ଌ, and is capable of growth only between 33 ଌ and 41 ଌ [105]. The reconciliation of growth conditions is a necessary first step for developing a general Agrobacterium-mediated transformation protocol for anaerobic gut fungi.

3.2.2. Heterologous Expression

While there is a growing effort to directly genetically manipulate anaerobic fungi, the challenges posed by these non-model organisms make the expression of genes of interest in model systems, like Escherichia coli and S. cerevisiae, appealing. A comprehensive list of reports of heterologous expression of anaerobic fungal proteins is available in Flad et al. [3]. Jones et al. [18] reported the expression and structural characterisation of anaerobic fungal glycoside hydrolases in E. coli, finding that arabinose-containing disaccharides were released by enzymatic digestion of plant-derived arabinan and arabinoxylan. In 2011, Jin reported the heterologous expression of endo-β-1,4-glucanase (EG) from an Orpinomyces strain in T. reesei [106]. Importantly, this required codon optimisation of the natively AT-rich anaerobic fungal gene. Wilken et al. describe a codon optimisation table, as well as amino acid and nucleotide-level abundance profiling of several fungal genomes, which would warrant consideration for construct design and engineering strategy development [107]. Seppälä et al. [108] expressed fluoride exporter proteins from several Neocallimastix strains in S. cerevisiae and found a higher activity variant than the wild type S. cerevisiae exporter, contributing to a higher fluoride tolerance.

Despite the rapid pace of progress in the heterologous expression of fungal enzymes, the complex, yet biotechnologically valuable anaerobic fungal cellulosome has yet to be expressed in a model organism, although en route to synthetic fungal cellulosome construction, dockerin-fused fungal enzymes have been expressed in yeast and E. coli [90]. Many heterologous proteins sourced from anaerobic fungi struggle to achieve soluble expression in model microbes, even after careful codon optimisation. This may be due to the inability of native-like post-translational modifications in the heterologous host, activation of stress responses in the host, or both [109]. Insertion of anaerobic fungal genes in currently more genetically tractable organisms can enable the use of typical protein engineering techniques such as directed evolution and structure-aided design. The creation of large protein libraries in S. cerevisiae and E. coli, in some cases exceeding 10 8 variants, makes the high throughput screening of variants possible. Once an optimal variant is identified, it can be further refined and potentially retro-inserted into the original host, completing the development cycle. This methodology may be particularly relevant for anaerobic fungi, which are exceptional lignocellulosic degraders, but not highly genetically tractable, towards efficient production of biofuels.


Fungi play a dominant role in terrestrial environments where they thrive in symbiotic associations with plants and animals and are integral to nutrient cycling in diverse ecosystems. Everywhere that moisture and a carbon source coexist in the terrestrial biosphere, fungi are expected to occur. We know that fungi can be devastating to agricultural crops, both in the field and during their storage, and cause mortality in immunocompromised patients in numbers that rival the deaths from malaria. Yet fungi can also be harnessed as sources of food, chemicals and biofuels when humans exploit fungal metabolism. Despite their central role in the health and disease of the terrestrial biosphere, much less is known about the function and potential of marine fungi. Are fungi ubiquitous in marine environments as they are on land? Do they play the same or similar roles in these ecosystems? Here we describe the state of knowledge about the abundance and functions of fungi in the marine environment with a goal to stimulate new inquiry in this very open area.

Supplementary Information

Fungi that are well adapted and constantly active in aquatic habitats.

Fungi that are less adapted to and only periodically active in aquatic habitats.

Fungi that are little adapted to and only sporadically active in aquatic habitats.

A mechanism whereby atmospheric carbon is sequestered by vertical transfer to deep waters and sediments.

Pulsed event-based disturbances referring to strong single events such as storms and droughts.

Long-term anthropogenic disturbances

Gradually increasing press disturbances such as global climate change or urbanization, both leading to species loss and shifts in community composition.

A fungus in symbiosis with a vascular plant via the root in the rhizosphere.

Fungi parasitizing on other fungi.

Parasites of a host that is also a parasite.

The precursor ribosomal RNA (rRNA) is a prespliced, full-length transcribed ribosomal operon including all functional and spacer regions.


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Diversity and Hidden Host Specificity of Chytrids Infecting Colonial Volvocacean Algae

Chytrids are zoosporic fungi that play an important, but yet understudied, ecological role in aquatic ecosystems. Many chytrid species have been morphologically described as parasites on phytoplankton. However, the majority of them have rarely been isolated and lack DNA sequence data. In this study we isolated and cultivated three parasitic chytrids, infecting a common volvocacean host species, Yamagishiella unicocca. To identify the chytrids, we characterized morphology and life cycle, and analyzed phylogenetic relationships based on 18S and 28S rDNA genes. Host range and specificity of the chytrids was determined by cross-infection assays with host strains, characterized by rbcL and ITS markers. We were able to confirm the identity of two chytrid strains as Endocoenobium eudorinae Ingold and Dangeardia mamillata Schröder and described the third chytrid strain as Algomyces stechlinensis gen. et sp. nov. The three chytrids were assigned to novel and phylogenetically distant clades within the phylum Chytridiomycota, each exhibiting different host specificities. By integrating morphological and molecular data of both the parasitic chytrids and their respective host species, we unveiled cryptic host-parasite associations. This study highlights that a high prevalence of (pseudo)cryptic diversity requires molecular characterization of both phytoplankton host and parasitic chytrid to accurately identify and compare host range and specificity, and to study phytoplankton-chytrid interactions in general.

Figure S1. Phenotypic variability in the algal culture strains PAN4 (Yamagishiella unicocca) and PAN1 (Eudorina elegans).

Figure S2. (A) Infected green algal colony “moC1, that was picked up manually with a micropipette, from a water sample from Lake Stechlin (September 2016). (B) A similar looking infected colony, from the same water sample, with multiple chytrid infections.

Table S1. GenBank accession numbers for chytrid and host strains.

Appendix S1. Material and methods.

Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Watch the video: The Devastating Chytrid Fungus (May 2022).


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