Quantification of the dark fungal taxon Cryptomycota using qPCR.

Fungi are present in a wide variety of natural environments, and in the last years, various studies have shown that they are quite abundant in aquatic ecosystems. In addition, a whole new highly diverse phylum, the Cryptomycota, was discovered. Nevertheless, research on aquatic fungi and a detailed evaluation of their functions and distribution are still sparse. One of the main reasons is a limitation in reliable identification and quantification methods. To bridge part of the research gap, this study aims to implement a quantitative PCR method to detect and quantify the newly discovered phylum. We developed and validated a Cryptomycota-specific qPCR primer pair targeting the 5.8S region that detects the majority of Cryptomycota, but Microsporidia. The resulting amplicon is 102 bp long. We used different environmental samples to evaluate the primer pair, various fungal sequences as negative control and positive control sequences. Obtained amplicons were sequenced using Illumina, and the obtained ASVs were all classified as Cryptomycota. The qPCR method works reliably and specifically for the quantification of Cryptomycota in environmental samples.

The Effects of Nitrogen and Phosphorus on Colony Growth and Zoospore Characteristics of Soil Chytridiomycota.

The Chytridiomycota phylum contributes to nutrient cycling and the flow of energy between trophic levels in terrestrial and aquatic ecosystems yet remains poorly described or absent from publications discussing fungal communities in these environments. This study contributes to the understanding of three species of soil chytrids in vitro-Gaertneriomyces semiglobifer, Spizellomyces sp. and Rhizophlyctis rosea-in the presence of elevated concentrations of nitrogen and phosphorus and with different sources of nitrogen. Colony growth was measured after 4 weeks as dry weight and total protein. To determine the impacts on zoospore reproduction, motility, lipid content, and attachment to organic substrates, 4- and 8-week incubation times were investigated. Whilst all isolates were able to assimilate ammonium as a sole source of nitrogen, nitrate was less preferred or even unsuitable as a nutrient source for G. semiglobifer and R. rosea, respectively. Increasing phosphate concentrations led to diverse responses between isolates. Zoospore production was also variable between isolates, and the parameters for zoospore motility appeared only to be influenced by the phosphate concentration for Spizellomyces sp. and R. rosea. Attachment rates increased for G. semiglobifer in the absence of an inorganic nitrogen source. These findings highlight variability between the adaptive responses utilised by chytrids to persist in a range of environments and provide new techniques to study soil chytrid biomass and zoospore motility by total protein quantification and fluorescent imaging respectively.

Newly emerging diseases of marine turtles, especially sea turtle egg fusariosis (SEFT), caused by species in the Fusarium solani complex (FSSC).

Sea turtles are presently considered severely endangered species that are historically threatened by many environmental factors. Recently, additional threats to sea turtles from two pathogenic species of fungi in the Fusarium solani species complex (F. falciforme and F. keratoplasticum) have been identified. These species infect marine turtle eggs, causing sea turtle egg fusariosis, and kill their embryos, with recent reports of hatch-failure in seven globally distributed species of endangered sea turtles (Caretta caretta, Chelonia mydas, Dermochelys coriaceae, Eretmochelys imbricata, Lepidochelys olivacea, Lepidochelys kempi and Natator depressus). Mycelia and spores of pathogenic species of Fusarium are produced in disturbed terrestrial soils and are transported to the ocean in coastal run off. We propose that these fungi grow on floating particles of plant tissues (leaves and wood), animal tissues, silt and plastics, which are carried by wind and currents and the turtles themselves to the beaches where the turtles lay their eggs.

Pathogenic Labyrinthula associated with Australian seagrasses: Considerations for seagrass wasting disease in the southern hemisphere.

Marine disease ecology is a growing field of research, particularly for host organisms negatively impacted by a changing climate and anthropogenic activities. A decrease in health and increase in susceptibility to disease has been hypothesised as the mechanism behind wide-spread seagrass die-offs related to wasting disease in the past. However, seagrass wasting disease and the causative pathogen, Labyrinthula, have been vastly understudied in the southern hemisphere. Our aim was to build on the current knowledge of Australian Labyrinthula descriptions and phylogeny, while also providing a first look at wasting disease ecology in Australia. Five seagrass species along a 750km stretch of coastline in southeastern Australia were sampled. The resulting 38 Labyrinthula isolates represented a diversity of morphotypes and five haplotypes of varying phylogenetic clade positions and virulence. The haplotypes clustered with previously-described phylogenetic clades containing isolates from Asia, USA and Europe. Pathogenicity tests confirmed, for the first time, the presence of at least two pathogenic haplotypes in Australia. While historically there have been no reports of wasting disease-related seagrass habitat loss, the presence of pathogenic Labyrinthula highlights the need for disease monitoring and research to understand seagrass wasting disease ecology in Australia.

Key Ecological Roles for Zoosporic True Fungi in Aquatic Habitats.

The diversity and abundance of zoosporic true fungi have been analyzed recently using fungal sequence libraries and advances in molecular methods, such as high-throughput sequencing. This review focuses on four evolutionary primitive true fungal phyla: the Aphelidea, Chytridiomycota, Neocallimastigomycota, and Rosellida (Cryptomycota), most species of which are not polycentric or mycelial (filamentous), rather they tend to be primarily monocentric (unicellular). Zoosporic fungi appear to be both abundant and diverse in many aquatic habitats around the world, with abundance often exceeding other fungal phyla in these habitats, and numerous novel genetic sequences identified. Zoosporic fungi are able to survive extreme conditions, such as high and extremely low pH; however, more work remains to be done. They appear to have important ecological roles as saprobes in decomposition of particulate organic substrates, pollen, plant litter, and dead animals; as parasites of zooplankton and algae; as parasites of vertebrate animals (such as frogs); and as symbionts in the digestive tracts of mammals. Some chytrids cause economically important diseases of plants and animals. They regulate sizes of phytoplankton populations. Further metagenomics surveys of aquatic ecosystems are expected to enlarge our knowledge of the diversity of true zoosporic fungi. Coupled with studies on their functional ecology, we are moving closer to unraveling the role of zoosporic fungi in carbon cycling and the impact of climate change on zoosporic fungal populations.

The First Isolation and Characterisation of the Protist Labyrinthula sp. in Southeastern Australia.

As a result of anthropogenic influences and global climate change, emerging infectious marine diseases are thought to be increasingly more common and more severe than in the past. The aim of our investigation was to confirm the presence of Labyrinthula, the aetiological agent of the seagrass wasting disease, in Southeastern Australia and provide the first isolation and characterisation of this protist, in Australia. Colonies and individual cells were positively identified as Labyrinthula using published descriptions, diagrams, and photographs. Their identity was then confirmed using DNA barcoding of a region of the 18S rRNA gene. Species level identification of isolates was not possible as the taxonomy of the Labyrinthula is still poorly resolved. Still, a diversity of Labyrinthula was isolated from small sections of the southeast coast of Australia. The isolates were grouped into three haplotypes that are biogeographically restricted. These haplotypes are closely related to previously identified saprotrophic clades. The study highlights the need for further investigation into the global distribution of Labyrinthula, including phylogenetic pathogenicity and analysis of host-parasite interactions in response to stressors. Given the results of our analyses, it is prudent to continue research into disease and epidemic agents to better prepare researchers for potential future outbreaks.

Visualization of the structural changes in plywood and gypsum board during the growth of Chaetomium globosum and Stachybotrys chartarum.

Fungal growth in indoor environments is associated with many negative health effects. Many studies focus on brown- and white-rot fungi and their effect on wood, but there is none that reveals the influence of soft-rot fungi, such as Stachybotrys spp. and Chaetomium spp., on the structure of building materials such as plywood and gypsum wallboard. This study focuses on using micro-computed tomography (microCT) to investigate changes of the structure of plywood and gypsum wallboard during fungal degradation by S. chartarum and C. globosum. Changes in the materials as a result of dampness and fungal growth were determined by measuring porosity and pore shape via microCT. The results show that the composition of the building material influenced the level of penetration by fungi as shown by scanning electron microscopy (SEM). Plywood appeared to be the most affected, with the penetration of moisture and fungi throughout the whole thickness of the sample. Conversely, fungi grew only on the top cardboard in the gypsum wallboard and they did not have significant influence on the gypsum wallboard structure. The majority of the observed changes in gypsum wallboard occurred due to moisture. This paper suggests that the mycelium distribution within building materials and the structural changes, caused by dampness and fungal growth, depend on the type of the material.

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.

Ecological functions of zoosporic hyperparasites.

Zoosporic parasites have received increased attention during the last years, but it is still largely unnoted that these parasites can themselves be infected by hyperparasites. Some members of the Chytridiomycota, Blastocladiomycota, Cryptomycota, Hyphochytriomycota, Labyrinthulomycota, Oomycota, and Phytomyxea are hyperparasites of zoosporic hosts. Because of sometimes complex tripartite interactions between hyperparasite, their parasite-host, and the primary host, hyperparasites can be difficult to detect and monitor. Some of these hyperparasites use similar mechanisms as their parasite-hosts to find and infect their target and to access food resources. The life cycle of zoosporic hyperparasites is usually shorter than the life cycle of their hosts, so hyperparasites may accelerate the turnaround times of nutrients within the ecosystem. Hyperparasites may increase the complexity of food webs and play significant roles in regulating population sizes and population dynamics of their hosts. We suggest that hyperparasites lengthen food chains but can also play a role in conducting or suppressing diseases of animals, plants, or algae. Hyperparasites can significantly impact ecosystems in various ways, therefore it is important to increase our understanding about these cryptic and diverse organisms.

Morphology, phylogeny, and ecology of the aphelids (Aphelidea, Opisthokonta) and proposal for the new superphylum Opisthosporidia.

The aphelids are a small group of intracellular parasitoids of common species of eukaryotic phytoplankton with three known genera Aphelidium, Amoeboaphelidium, and Pseudaphelidium, and 10 valid species, which form along with related environmental sequences a very diversified group. The phyla Microsporidia and Cryptomycota, and the class Aphelidea have recently been considered to be a deep branch of the Holomycota lineage forming the so called the ARM-clade which is sister to the fungi. In this review we reorganize the taxonomy of ARM-clade, and establish a new superphylum the Opisthosporidia with three phyla: Aphelida phyl. nov., Cryptomycota and Microsporidia. We discuss here all aspects of aphelid investigations: history of our knowledge, life cycle peculiarities, the morphology (including the ultrastructure), molecular phylogeny, ecology, and provide a taxonomic revision of the phylum supplied with a list of species. We compare the aphelids with their nearest relatives, the species of Rozella, and improve the diagnosis of the phylum Cryptomycota.

Current ecological understanding of fungal-like pathogens of fish: what lies beneath?

Despite increasingly sophisticated microbiological techniques, and long after the first discovery of microbes, basic knowledge is still lacking to fully appreciate the ecological importance of microbial parasites in fish. This is likely due to the nature of their habitats as many species of fish suffer from living beneath turbid water away from easy recording. However, fishes represent key ecosystem services for millions of people around the world and the absence of a functional ecological understanding of viruses, prokaryotes, and small eukaryotes in the maintenance of fish populations and of their diversity represents an inherent barrier to aquatic conservation and food security. Among recent emerging infectious diseases responsible for severe population declines in plant and animal taxa, fungal and fungal-like microbes have emerged as significant contributors. Here, we review the current knowledge gaps of fungal and fungal-like parasites and pathogens in fish and put them into an ecological perspective with direct implications for the monitoring of fungal fish pathogens in the wild, their phylogeography as well as their associated ecological impact on fish populations. With increasing fish movement around the world for farming, releases into the wild for sport fishing and human-driven habitat changes, it is expected, along with improved environmental monitoring of fungal and fungal-like infections, that the full extent of the impact of these pathogens on wild fish populations will soon emerge as a major threat to freshwater biodiversity.

Three dimensional quantification of biological samples using micro-computer aided tomography (microCT).

MicroCT is increasingly being used to observe soft animal and plant tissues. Conventional electron and light microscope staining protocols used to enhance the contrast of soft tissues have the potential to be adapted for use in microCT. This would increase the versatility of the microCT beyond improving qualitative observations to facilitating quantitative analysis of soft tissues. This paper describes the development of a culture system and staining protocol which has successfully been used to obtain three dimensional (3-D) quantitative data of filamentous and zoosporic soil fungi. The fungi were grown in an artificial matrix that was developed to simulate the particulate nature of soil. The combination of high contrast staining protocol and use of an X-ray translucent matrix allowed for 3-D qualitative and quantitative analysis of fungal growth. A salient point raised by this study is that the effectiveness of a protocol is reliant on the tissue or cell culture system which includes the composition of the sample, the sampling vessel, the depth of a sample and the combination of stains used. The potential use of this method extends to other fields where distribution and growth patterns in 3-D need to be quantified.

Quantitative methods for the analysis of zoosporic fungi.

Quantitative estimations of zoosporic fungi in the environment have historically received little attention, primarily due to methodological challenges and their complex life cycles. Conventional methods for quantitative analysis of zoosporic fungi to date have mainly relied on direct observation and baiting techniques, with subsequent fungal identification in the laboratory using morphological characteristics. Although these methods are still fundamentally useful, there has been an increasing preference for quantitative microscopic methods based on staining with fluorescent dyes, as well as the use of hybridization probes. More recently however PCR based methods for profiling and quantification (semi- and absolute) have proven to be rapid and accurate diagnostic tools for assessing zoosporic fungal assemblages in environmental samples. Further application of next generation sequencing technologies will however not only advance our quantitative understanding of zoosporic fungal ecology, but also their function through the analysis of their genomes and gene expression as resources and databases expand in the future. Nevertheless, it is still necessary to complement these molecular-based approaches with cultivation-based methods in order to gain a fuller quantitative understanding of the ecological and physiological roles of zoosporic fungi.

Resource seeking strategies of zoosporic true fungi in heterogeneous soil habitats at the microscale level.

Zoosporic true fungi have frequently been identified in samples from soil and freshwater ecosystems using baiting and molecular techniques. In fact some species can be components of the dominant groups of microorganisms in particular soil habitats. Yet these microorganisms have not yet been directly observed growing in soil ecosystems. Significant physical characteristics and features of the three-dimensional structures of soils which impact microorganisms at the microscale level are discussed. A thorough knowledge of soil structures is important for studying the distribution of assemblages of these fungi and understanding their ecological roles along spatial and temporal gradients. A number of specific adaptations and resource seeking strategies possibly give these fungi advantages over other groups of microorganisms in soil ecosystems. These include chemotactic zoospores, mechanisms for adhesion to substrates, rhizoids which can penetrate substrates in small spaces, structures which are resistant to environmental extremes, rapid growth rates and simple nutritional requirements. These adaptations are discussed in the context of the characteristics of soils ecosystems. Recent advances in instrumentation have led to the development of new and more precise methods for studying microorganisms in three-dimensional space. New molecular techniques have made identification of microbes possible in environmental samples.