A combined microscopy and single-cell sequencing approach reveals the ecology, morphology, and phylogeny of uncultured lineages of zoosporic fungi.

Environmental DNA analyses of fungal communities typically reveal a much larger diversity than can be ascribed to known species. Much of this hidden diversity lies within undescribed fungal lineages, especially the early diverging fungi (EDF). Although these EDF often represent new lineages even at the phylum level, they have never been cultured, making their morphology and ecology uncertain. One of the methods to characterize these uncultured fungi is a single-cell DNA sequencing approach. In this study, we established a large data set of single-cell sequences of EDF by manually isolating and photographing parasitic fungi on various hosts such as algae, protists, and micro-invertebrates, combined with subsequent long-read sequencing of the ribosomal DNA locus (rDNA). We successfully obtained rDNA sequences of 127 parasitic fungal cells, which clustered into 71 phylogenetic lineages belonging to seven phylum-level clades of EDF: Blastocladiomycota, Chytridiomycota, Aphelidiomycota, Rozellomycota, and three unknown phylum-level clades. Most of our single cells yielded novel sequences distinguished from both described taxa and existing metabarcoding data, indicating an expansive and hidden diversity of parasitic taxa of EDF. We also revealed an unexpected diversity of endobiotic Olpidium-like chytrids and hyper-parasitic lineages. Overall, by combining photographs of parasitic fungi with phylogenetic analyses, we were able to better understand the ecological function and morphology of many of the branches on the fungal tree of life known only from DNA sequences. IMPORTANCE Much of the diversity of microbes from natural habitats, such as soil and freshwater, comprise species and lineages that have never been isolated into pure culture. In part, this stems from a bias of culturing in favor of saprotrophic microbes over the myriad symbiotic ones that include parasitic and mutualistic relationships with other taxa. In the present study, we aimed to shed light on the ecological function and morphology of the many undescribed lineages of aquatic fungi by individually isolating and sequencing molecular barcodes from 127 cells of host-associated fungi using single-cell sequencing. By adding these sequences and their photographs into the fungal tree, we were able to understand the morphology of reproductive and vegetative structures of these novel fungi and to provide a hypothesized ecological function for them. These individual host-fungal cells revealed themselves to be complex environments despite their small size; numerous samples were hyper-parasitized with other zoosporic fungal lineages such as Rozellomycota.

Effects of water movement and temperature on Rhizophydium infection of Planktothrix in a shallow hypereutrophic lake.

Grand Lake St. Marys (GLSM) is a popular recreational lake located in western Ohio, United States, generating nearly $150 million in annual revenue. However, recurring algal blooms dominated by Planktothrix agardhii, which can produce harmful microcystin toxins, have raised concerns about water safety and negatively impacted the local economy. Planktothrix agardhii is host to a number of parasites and pathogens, including an obligate fungal parasite in the Chytridiomycota (chytrids). In this study, we investigated the potential of these chytrid (Rhizophydium sp.) to infect P. agardhii blooms in the environment by modifying certain environmental conditions thought to limit infection prevalence in the wild. With a focus on temperature and water mixing, mesocosms were designed to either increase or decrease water flow compared to the control (water outside the mesocosm). In the control and water circulation mesocosms, infections were found infrequently and were found on less than 0.75% of the Planktothrix population. On the other hand, by decreasing the water flow to stagnation, chytrid infections were more frequent (found in nearly 3x as many samples) and more prevalent, reaching a maximum infection rate of 4.12%. In addition, qPCR coupled with 16S-18S sequencing was utilized to confirm the genetic presence of both host and parasite, as well as to better understand the effect of water circulation on the community composition. Statistical analysis of the data confirmed that chytrid infection was dependent on water temperature, with infections predominantly occurring between 19°C and 23°C. Additionally, water turbulence can significantly reduce the infectivity of chytrids, as infections were mostly found in stagnant mesocosms. Further, decreasing the water circulation promoted the growth of the cyanobacterial population, while increasing water agitation promoted the growth of green algae (Chlorophyta). This study starts to explore the environmental factors that affect chytrid pathogenesis which can provide valuable insights into controlling measures to reduce the prevalence of harmful algal blooms and improve water quality in GLSM and similarly affected waterbodies.

Multi-year molecular quantification and 'omics analysis of Planktothrix-specific cyanophage sequences from Sandusky Bay, Lake Erie.

Introduction: Planktothrix agardhii is a microcystin-producing cyanobacterium found in Sandusky Bay, a shallow and turbid embayment of Lake Erie. Previous work in other systems has indicated that cyanophages are an important natural control factor of harmful algal blooms. Currently, there are few cyanophages that are known to infect P. agardhii, with the best-known being PaV-LD, a tail-less cyanophage isolated from Lake Donghu, China. Presented here is a molecular characterization of Planktothrix specific cyanophages in Sandusky Bay. Methods and Results: Putative Planktothrix-specific viral sequences from metagenomic data from the bay in 2013, 2018, and 2019 were identified by two approaches: homology to known phage PaV-LD, or through matching CRISPR spacer sequences with Planktothrix host genomes. Several contigs were identified as having viral signatures, either related to PaV-LD or potentially novel sequences. Transcriptomic data from 2015, 2018, and 2019 were also employed for the further identification of cyanophages, as well as gene expression of select viral sequences. Finally, viral quantification was tested using qPCR in 2015-2019 for PaV-LD like cyanophages to identify the relationship between presence and gene expression of these cyanophages. Notably, while PaV-LD like cyanophages were in high abundance over the course of multiple years (qPCR), transcriptomic analysis revealed only low levels of viral gene expression. Discussion: This work aims to provide a broader understanding of Planktothrix cyanophage diversity with the goals of teasing apart the role of cyanophages in the control and regulation of harmful algal blooms and designing monitoring methodology for potential toxin-releasing lysis events.

Interactions between chytrids cause variable infection strategies on harmful algal bloom forming species.

Cyanobacteria have a great diversity of natural enemies, such as herbivores and pathogens, including fungal pathogens within the Chytridiomycota (chytrids). While these pathogens have been previously described on a select number of cyanobacterial hosts and are suspected to play a significant ecological role, little is understood about species interactions and how competition between parasites can affect epidemic development and bloom formation. Here, three Planktothrix agardhii isolates from Sandusky Bay, Lake Erie (OH, USA) were challenged in monoculture and polyculture against infection by three isolates (C1, C2, C10) of their obligate chytrid fungal pathogen, Rhizophydiales sp. The chytrid isolates were inoculated as single isolates or a mixture of up to three different isolates. In monoculture, host isolates were characterized as highly susceptible (P. agardhii 1030), moderately susceptible (P. agardhii 1808) or mostly resistant (P. agardhii 1801). Co-infection of chytrid isolates on the highly susceptible host isolate had an additive effect on chytrid prevalence, leading to a culture crash where 2 or 3 chytrid isolates were present. Co-infection of chytrid isolates on the moderately susceptible and mostly resistant isolates had no effect on chytrid infection outcome or prevalence compared to infection with a single isolate. In polyculture, the effect on host growth was most significant in the single chytrid isolate treatment, which was attenuated with the addition of mixed chytrid treatments. Genetic analysis of the resulting population after the experimental period showed a tendency for the chytrid isolate C1 and P. agardhii 1801 to dominate in mixed population samples. Two different interspecific interactions seem to be in play; varied parasite infection strategies allow for the amplification of infection prevalence due to mixed chytrids in a susceptible monoculture, or competition allows for the dominance of a single chytrid isolate in monoculture and the reduction of infection prevalence in a host polyculture. This work thus highlights how interactions between chytrid infections can change the course of epidemic development and harmful algal bloom formation.

Genomic comparison of Planktothrix agardhii isolates from a Lake Erie embayment.

Planktothrix agardhii is a filamentous cyanobacterial species that dominates harmful algal blooms in Sandusky Bay, Lake Erie and other freshwater basins across the world. P. agardhii isolates were obtained from early (June) blooms via single filament isolation; eight have been characterized from 2016, and 12 additional isolates have been characterized from 2018 for a total of 20 new cultures. These novel isolates were processed for genomic sequencing, where reads were used to generate scaffolds and contigs which were annotated with DIAMOND BLAST hit, Pfam, and GO. Analyses include whole genome alignment to generate phylogenetic trees and comparison of genetic rearrangements between isolates. Nitrogen acquisition and metabolism was compared across isolates. Secondary metabolite production was genetically explored including microcystins, two types of aeruginosin clusters, anabaenopeptins, cyanopeptolins, microviridins, and prenylagaramides. Two common and 4 unique CRISPR-cas islands were analyzed for similar sequences across all isolates and against the known Planktothrix-specific cyanophage, PaV-LD. Overall, the uniqueness of each genome from Planktothrix blooms sampled from the same site and at similar times belies the unexplored diversity of this genus.

Environmental factors affecting chytrid (Chytridiomycota) infection rates on Planktothrix agardhii.

Planktothrix agardhii dominates the cyanobacterial harmful algal bloom biomass in Sandusky Bay, Lake Erie (USA) from May until September. This filamentous cyanobacterium known parasites including the chytrid fungal species Rhizophydium sp. C02, which was previously isolated from this region. The purpose of our work has been to establish how parasitic interactions affect Planktothrix population dynamics during a bloom event. Samples analyzed from the 2015 to 2019 bloom seasons using quantitative PCR investigate the spatial and temporal prevalence of chytrid infections. Abiotic factors examined in lab include manipulating temperature (17-31°C), conductivity (0.226-1.225 mS/cm) and turbulence. Planktothrix-specific chytrids are present throughout the bloom period and are occasionally at high enough densities to exert parasitic pressure on their hosts. Temperatures above 27.1°C in lab can inhibit chytrid infection, indicating the presence of a possible upper thermal refuge for the host. Data suggest that chytrids can survive conductivity spikes in lab at levels three-fold above Sandusky Bay waters if given sufficient time (7-12 days), whereas increased turbulence in lab severely inhibits chytrid infections, perhaps due to disruption of chemical signaling. Overall, these data provide insights into the environmental conditions that inhibit chytrid infections during Planktothrix-dominated blooms in temperate waters.

Isolation and Characterization of Rhizophydiales sp. (Chytridiomycota), Obligate Parasite of Planktothrix agardhii in a Laurentian Great Lakes Embayment.

Planktothrix agardhii dominates the cyanobacterial harmful algal bloom community in Sandusky Bay, Lake Erie (USA) from May through September. This filamentous cyanobacterium is host to a known obligate parasite; the chytrid Rhizophydium sp. During the 2018 bloom season, by utilizing dilution and single filament isolation techniques, 7 chytrid and 12 P. agardhii strains were isolated from Sandusky Bay. These 7 chytrids and a selection of P. agardhii hosts were then characterized with respect to infection rates. Infections by the isolated chytrids were specific to Planktothrix planktonic species and were not found on other filamentous cyanobacterial taxa present in the bay (Aphanizomenon sp. and Cuspidothrix sp.). Even among the potential P. agardhii host strains, individual chytrid isolates had different degrees of infectivity and showed preference for different host isolates, suggesting possible ecological partitioning even within the same sample population. Examining mechanisms of chytrid pathogenesis, the zoospores displayed a swarming pattern to attack and fracture the host filament and create new infection sites at the trichome termini. Infections by these parasitic chytrids also led to a release of intracellular microcystin toxins from the hosts. Additionally, infections were dependent on media type, highlighting the importance of media choice on experimental outcomes. Media in which chytrid swarming was observed closely matched the ionic strength of the natural environment. Understanding pathogenesis by fungal parasites will assist future efforts aimed at determining environmental factors favoring loss mechanisms for Planktothrix agardhii-dominated blooms.IMPORTANCE Whereas many studies have focused on the factors contributing to the establishment and persistence of cyanobacterial harmful algal blooms (cHABs), few studies have examined bloom pathogenesis. Chytrid fungi infect cyanobacteria and stimulate food web interactions through manipulation of previously hard to digest filaments and the release of nutrients to support heterotrophic microbes. Specifically, chytrids infective on filamentous Planktothrix agardhii exhibit a species-specific infection that fragments trichomes into shorter units that can be consumed more easily by grazers. Chytrid zoospores also serve as a high-quality food source for the lower food web. Understanding host-pathogen relationships and mechanisms of pathogenesis on cyanobacteria will be necessary to effectively model the ecology of cHABs.

Dissolved Microcystin Release Coincident with Lysis of a Bloom Dominated by Microcystis spp. in Western Lake Erie Attributed to a Novel Cyanophage.

Western Lake Erie (Laurentian Great Lakes) is prone to annual cyanobacterial harmful algal blooms (cHABs) dominated by Microcystis spp. that often yield microcystin toxin concentrations exceeding the federal EPA recreational contact advisory of 8 µg liter-1 In August 2014, microcystin levels were detected in finished drinking water above the World Health Organization 1.0 µg liter-1 threshold for consumption, leading to a 2-day disruption in the supply of drinking water for >400,000 residents of Toledo, Ohio (USA). Subsequent metatranscriptomic analysis of the 2014 bloom event provided evidence that release of toxin into the water supply was likely caused by cyanophage lysis that transformed a portion of the intracellular microcystin pool into the dissolved fraction, rendering it more difficult to eliminate during treatment. In August 2019, a similar increase in dissolved microcystins at the Toledo water intake was coincident with a viral lytic event caused by a phage consortium different in composition from what was detected following the 2014 Toledo water crisis. The most abundant viral sequence in metagenomic data sets was a scaffold from a putative member of the Siphoviridae, distinct from the Ma-LMM01-like Myoviridae that are typically documented to occur in western Lake Erie. This study provides further evidence that viral activity in western Lake Erie plays a significant role in transformation of microcystins from the particulate to the dissolved fraction and therefore requires monitoring efforts from local water treatment plants. Additionally, identification of multiple lytic cyanophages will enable the development of a quantitative PCR toolbox to assess viral activity during cHABs.IMPORTANCE Viral attack on cHABs may contribute to changes in community composition during blooms, as well as bloom decline, yet loss of bloom biomass does not eliminate the threat of cHAB toxicity. Rather, it may increase risks to the public by delivering a pool of dissolved toxin directly into water treatment utilities when the dominating Microcystis spp. are capable of producing microcystins. Detecting, characterizing, and quantifying the major cyanophages involved in lytic events will assist water treatment plant operators in making rapid decisions regarding the pool of microcystins entering the plant and the corresponding best practices to neutralize the toxin.

Physical drivers facilitating a toxigenic cyanobacterial bloom in a major Great Lakes tributary.

The Maumee River is the primary source for nutrients fueling seasonal Microcystis-dominated blooms in western Lake Erie's open waters though such blooms in the river are infrequent. The river also serves as source water for multiple public water systems and a large food services facility in northwest Ohio, USA. On 20 September 2017, an unprecedented bloom was reported in the Maumee River estuary within the Toledo metropolitan area, which triggered a recreational water advisory. Here we (1) explore physical drivers likely contributing to the bloom's occurrence, and (2) describe the toxin concentration and bacterioplankton taxonomic composition. A historical analysis using ten-years of seasonal river discharge, water level, and local wind data identified two instances when high-retention conditions occurred over ≥10 days in the Maumee River estuary: in 2016 and during the 2017 bloom. Observation by remote sensing imagery supported the advection of cyanobacterial cells into the estuary from the lake during 2017 and the lack of an estuary bloom in 2016 due to a weak cyanobacterial bloom in the lake. A rapid-response survey during the 2017 bloom determined levels of the cyanotoxins, specifically microcystins, in excess of recreational contact limits at sites within the lower 20 km of the river while amplicon sequencing found these sites were dominated by Microcystis. These results highlight the need to broaden our understanding of physical drivers of cyanobacterial blooms within the interface between riverine and lacustrine systems, particularly as such blooms are expected to become more prominent in response to a changing climate.

A Multiplex Analysis of Potentially Toxic Cyanobacteria in Lake Winnipeg during the 2013 Bloom Season.

Lake Winnipeg (Manitoba, Canada), the world's 12th largest lake by area, is host to yearly cyanobacterial harmful algal blooms (cHABs) dominated by Aphanizomenon and Dolichospermum. cHABs in Lake Winnipeg are primarily a result of eutrophication but may be exacerbated by the recent introduction of dreissenid mussels. Through multiple methods to monitor the potential for toxin production in Lake Winnipeg in conjunction with environmental measures, this study defined the baseline composition of a Lake Winnipeg cHAB to measure potential changes because of dreissenid colonization. Surface water samples were collected in 2013 from 23 sites during summer and from 18 sites in fall. Genetic data and mass spectrometry cyanotoxin profiles identified microcystins (MC) as the most abundant cyanotoxin across all stations, with MC concentrations highest in the north basin. In the fall, mcyA genes were sequenced to determine which species had the potential to produce MCs, and 12 of the 18 sites were a mix of both Planktothrix and Microcystis. Current blooms in Lake Winnipeg produce low levels of MCs, but the capacity to produce cyanotoxins is widespread across both basins. If dreissenid mussels continue to colonize Lake Winnipeg, a shift in physicochemical properties of the lake because of faster water column clearance rates may yield more toxic blooms potentially dominated by microcystin producers.

Isolation and screening of carboxydotrophs isolated from composts and their potential for butanol synthesis.

Carboxydotrophs are known for their ability to convert carbon monoxide (CO) to butanol through fermentation. Such a platform offers a promising alternative approach to biofuel production from synthesis gas feedstocks. In this study, carboxydotrophs were isolated from various manure compost. Out of 500 isolates, only 11 carboxydotrophs (7 mesophiles and 4 thermophiles) were found to utilize CO as the sole source of carbon and energy. To assess the biochemical basis for their ability to produce biofuel (butanol), the level of activities of CO dehydrogenase (CODH), hydrogenase and butanol dehydrogenase (BDH) enzymes for these isolates against the known carboxydotroph, Butyribacterium methylotrophicum was assessed. All isolates showed evidence of enzyme activities (0.16-2.20 micromol min(-1)), with the majority exhibiting higher activities compared with the known carboxydotroph, B. methylotrophicum (0.33-0.71 micromol min(-1)). The level of activities for CODH and BDH ranged from 0.163-3.59 micromolmin(-1) and 0.19-2.2 micromolmin(-1), respectively. Three isolates (M7-1, T2-22, and T3-14) demonstrated enzymatic activity three to seven times higher than B. methylotrophicum. Of these, T2-22 exhibited the highest BDH activity and shows great promise in the conversion of toxic CO into butanol more so than other carboxytotrophs known thus far. This study revealed some biochemical basis for butanol production from CO by carboxydotrophs. However, more research is needed to discover a direct biological route for butanol production from CO to strengthen their potential for synthesis gas bioprocessing. Follow-up work will focus on whole-genome sequencing of the promising isolate T2-22 to provide system-level insights into how carboxydotrophs utilize and regulate their molecular machineries for butanol production.