Two new species of Chytriomycetaceae: Morphological, phylogenetic, and ultrastructural characterization.

Systematics of Chytridiales has been deeply influenced by analyses of molecular loci and zoospore ultrastructure. Even though the Chytridiales is the largest order within Chytridiomycota, Brazilian isolates of this clade have been poorly integrated. Here, we isolated seven species and documented their morphology, including zoospore ultrastructure for Siphonaria aurea, and phylogenetic positions for all based on analyses of nuc 18S and 28S rDNA. Phylogenetic results support the placement of these species in Chytriomycetaceae and Chytridiaceae, with two new species described, Rhizidium crepaturum and Siphonaria aurea, and Rodmanochytrium sphaericum recorded for the first time from Brazil.

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.

Morphology, zoospore ultrastructure, and phylogenetic position of Polyphlyctis willoughbyi, a new species in Chytridiales (Chytridiomycota).

The purpose of our research is to investigate the morphology, zoospore ultrastructure, and molecular phylogenetic placement of a chytrid from Australia. From a survey of chytrid fungi in New South Wales, Australia, we isolated strain PL AUS 026 and putatively identified it as Polyphlyctis unispina. Light microscopic evaluation determined strain PL AUS 026 to be similar to two other strains of P. unispina characterized in the literature but to have a more complex thallus than that of the type. Molecular phylogenetic analyses placed our strain as sister of or basal to Chytridiaceae, Chytridiales. Ultrastructural analysis of the zoospore of strain PL AUS 026 revealed unique features. On the basis of our analyses we designate strain PL AUS 026 as a new species, Polyphlyctis willoughbyi. This research extends our concept of Chytridiaceae systematics and ultrastructural variation in the Chytridiales zoospore.

Zopfochytrium is a new genus in the Chytridiales with distinct zoospore ultrastructure.

While surveying chytrid diversity in lakes and streams, we found on cellulosic bait a chytrid that had both monocentric and polycentric thallus forms. We brought this chytrid into axenic culture from three sites in eastern North America, studied its thallus development and zoospore ultrastructure, and compared its 28S rDNA sequence with those of other members of the Chytridiomycota. Thallus morphology matched that described for the rare chytrid, Cladochytrium polystomum Zopf. Sporangia were spherical and produced numerous long discharge tubes. After discharge, zoospores remained in spherical clusters at the tips of the inoperculate openings of discharge tubes. After 10-30 min zoospores either swam away or encysted in place. Zoospore ultrastructural features included a cell coat, flagellar plug, and paracrystalline inclusion, features typical of members of the Chytridiales. However, the flagellar apparatus structure and organellar organization differed from that of zoospores previously described. Based on its molecular phylogeny and its zoospore ultrastructural features, we classify C. polystomum as a member of the Chytridiaceae in the Chytridiales. Because its thallus development and its ribosomal DNA sequences diverged decidedly from those of Cladochytrium tenue Nowak, the type species of Cladochytrium, we erected Zopfochytrium as a new genus for this chytrid.

Collimyces mutans gen. et sp. nov. (Rhizophydiales, Collimycetaceae fam. nov.), a New Chytrid Parasite of Microglena (Volvocales, clade Monadinia).

Chytrids are early diverging lineages of true fungi that reproduce with posteriorly uniflagellate zoospores. In aquatic ecosystems, parasitic chytrids of algae have important ecological roles by influencing the population dynamics of phytoplankton and transferring nutrients and energy from inedible algae to zooplankton via zoospores. Despite their ecological importance, information on parasitic chytrids is lacking in the current systematics of chytrids. Here, we investigated a novel chytrid culture KS100 that parasitizes the green alga, Microglena coccifera (Volvocales). A cross-inoculation experiment revealed that KS100 infection was specific to the genus Microglena. Thallus morphology of KS100 is characterized by spherical or subspherical zoosporangium, which becomes slightly angular during zoospore discharge, 2-3 small and inoperculate pores from where zoospores are discharged, and rhizoids branching at the base that extends in a fan-like shape. This combination of characteristics was distinct from any other known chytrids. In molecular phylogeny, KS100 was placed in the order Rhizophydiales and was distinguished from any known families in the order. Zoospores of KS100 possessed a kinetosome-associated structure whose morphology and positioning were unique among the Rhizophydiales. Based on these results, we describe this chytrid as Collimyces mutans gen. et sp. nov. in the new family Collimycetaceae.

Irineochytrium, a new genus in Chytridiales having zoospores and aplanospores.

Many described chytrids exhibit distinct morphological features that permit positive identification by light microscopy. Chytriomyces annulatus is one such species. It has a flap-like operculum and its sporangial wall is ornamented with multiple collar-like annulations proximal to the rhizoidal axis, features that, in combination, do not occur in any other described chytrid. Recent molecular phylogenies placed C. annulatus in the Chytridiaceae (Chytridiales) lineage, which is characterized by a Group II zoospore. Here we use light microscopy and transmission electron microscopy to examine thallus morphology of an isolate (JEL 729) of C. annulatus to confirm its identity and transmission electron microscopy to examine zoospore ultrastructure to confirm its phylogenetic placement. Light microscopic examinations confirmed its identity, and transmission electron microscopy analysis revealed both motile spores (zoospores) and nonmotile spores (aplanospores). Zoospores had a unique suite of ultrastructural features characteristic of the Group II zoospore; aplanospores had similar ultrastructure minus a flagellum. Chytriomyces annulatus does not group with the Chytriomycetaceae (Chytridiales) lineage containing the type of Chytriomyces, C. hyalinus, nor does it have a zoospore typical of that lineage. These arguments support the recognition of a distinct genus in Chytridiaceae, including one species, Irineochytrium annulatum.

Hypothesized evolutionary trends in zoospore ultrastructural characters in Chytridiales (Chytridiomycota).

Chytridiales is an order of zoosporic fungi currently comprising species representing 19 genera. Although morphologically and genetically diverse, these taxa have in common a zoospore with a suite of ultrastructural characters unique among Chytridiomycota. However, multiple states have been reported for almost every character that defines the Chytridiales zoospore. Two zoospore types have been recognized, each corresponding to a family. Here we examine zoospore ultrastructure of 52 isolates in Chytridiales and assess states for six characters to hypothesize evolutionary trends, using parsimony ancestral state reconstruction for evolutionary analysis. Based on suites of character states, we describe four additional zoospore types in Chytridiales. Five of the six characters ([i] location of the nucleus, [ii] morphology of the kinetosome-associated structure, [iii] complexity of the microtubular root, [iv] microbody-lipid globule complex cisterna structure and [v] thickness of the flagellar plug) revealed ancestral and derived states. The sixth character, structure of the paracrystalline inclusion, did not resolve ancestral and derived states. In each of the lineages within Chytridiales, the evolutionary trend appears to have been from a more complex zoospore to a less complex zoospore with reduced features. As we isolate and analyze additional taxa, we discover new ultrastructural character states that assist in taxon delineation and phylogenetic interpretation.

Dendrochytridium crassum gen. et sp. nov., a taxon in Chytridiales with unique zoospore ultrastructure.

A water culture of detritus collected from an Australian tree canopy yielded multiple isolates (designated JEL 352, JEL 353, JEL 354) of an unidentified chytrid that grew on pollen bait and encysted spores of a Dictyuchus sp. oomycete. Morphological information from JEL 352 and genetic information from JEL 354 of this unidentified chytrid have been in several publications but the organism has not been named. Because isolates JEL 352 and JEL 354 are no longer viable, we sequenced partial SSU and LSU rDNA of isolate JEL 353, documented its thallus morphology with light microscopy and determined its zoospore ultrastructure via transmission electron microscopy. DNA evidence placed JEL 353 in Chytridiaceae, and its genetic composition was identical to that of JEL 354. Thallus morphology of JEL 353 was similar to that of JEL 352. Its zoospore ultrastructure is less complex compared to other members of Chytridiaceae. In pure culture, the rhizoidal system differed from other members of the family in being unevenly broad and not tapering to fine tips. Based on genetic, morphological and ultrastructural evidence, we place this chytrid in a new genus in Chytridiaceae and describe it as the new species Dendrochytridium crassum.

Three new genera in Chytridiales from aquatic habitats in Argentina.

Sampling for chytrids in a variety of habitats has resulted in pure cultures that when analyzed have yielded hypotheses of relationships based on molecular and zoospore ultrastructural markers. To extend our understanding of diversity of Chytridiales in eastern Argentina and USA, we isolated and examined the morphology, ultrastructure and 28S and ITS1-5.8S-ITS2 rDNA sequences of numerous chytrids from aquatic habitats from these two regions. Three family-level lineages (Chytridiaceae, Chytriomycetaceae, family incertae sedis) are represented in our molecular phylogeny, and three new genera (Avachytrium, Odontochytrium in Chytriomycetaceae, Delfinachytrium in family incertae sedis) are described. These findings of new genera and species emphasize the potential for discovery of additional diversity.

Pseudorhizidium is a new genus with distinct zoospore ultrastructure in the order Chytridiales.

A chytrid isolate (JEL 221) we identified as the rarely reported species, Rhizidium endosporangiatum Karling, was cultured axenically for the first time. The purposes of this study are to characterize the developmental morphology of isolate JEL 221 and to elucidate its zoospore ultrastructural features. Thallus development and morphology of isolate JEL 221 are characteristic of R. endosporangiatum as it was originally described. However, thallus morphology of R. endosporangiatum is not entirely typical of the genus Rhizidium, especially that of the type R. mycophilum. The presence of an endosporangium, a layer of material encapsulating the edges of the protoplast protruding through multiple discharge pores, makes this a distinctive species. Consistent with its published molecular-based phylogenetic placement, we found that isolate JEL 221 shared ultrastructural features with the two major zoospore types described for the Chytridiales but had distinct zoospore architecture. A new genus, Pseudorhizidium, is erected for this chytrid based on its thallus morphology, molecular phylogenetic placement and unique zoospore ultrastructure. This new genus does not fit into either of the described families (Chytridiaceae or Chytriomycetaceae) in the Chytridiales because of its unique zoospore ultrastructure, especially the two-layered nature of the electron-opaque plug in the base of the flagellum.

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

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

Germ tube mediated invasion of Batrachochytrium dendrobatidis in amphibian skin is host dependent.

Batrachochytrium dendrobatidis (Bd) is the causative agent of chytridiomycosis, a fungal skin disease in amphibians and driver of worldwide amphibian declines.We focussed on the early stages of infection by Bd in 3 amphibian species with a differential susceptibility to chytridiomycosis. Skin explants of Alytes muletensis, Litoria caerulea and Xenopus leavis were exposed to Bd in an Ussing chamber for 3 to 5 days. Early interactions of Bd with amphibian skin were observed using light microscopy and transmission electron microscopy. To validate the observations in vitro, comparison was made with skin from experimentally infected frogs. Additional in vitro experiments were performed to elucidate the process of intracellular colonization in L. caerulea. Early interactions of Bd with amphibian skin are: attachment of zoospores to host skin, zoospore germination, germ tube development, penetration into skin cells, invasive growth in the host skin, resulting in the loss of host cell cytoplasm. Inoculation of A. muletensis and L. caerulea skin was followed within 24 h by endobiotic development, with sporangia located intracellularly in the skin. Evidence is provided of how intracellular colonization is established and how colonization by Bd proceeds to deeper skin layers. Older thalli develop rhizoid-like structures that spread to deeper skin layers, form a swelling inside the host cell to finally give rise to a new thallus. In X. laevis, interaction of Bd with skin was limited to an epibiotic state, with sporangia developing upon the skin. Only the superficial epidermis was affected. Epidermal cells seemed to be used as a nutrient source without development of intracellular thalli. The in vitro data agreed with the results obtained after experimental infection of the studied frog species. These data suggest that the colonization strategy of B. dendrobatidis is host dependent, with the extent of colonization most likely determined by inherent characteristics of the host epidermis.

Thoreauomyces gen. nov., Fimicolochytrium gen. nov. and additional species in Geranomyces.

Powellomycetaceae (Spizellomycetales) contain a diverse group of exogenously developing chytrids found by baiting water preparations of soils and manure with pollen. A previous molecular phylogenetic study indicated that some lineages within this family represent undescribed genera and species. Description of genera within the Spizellomycetales traditionally has relied on ultrastructural characters of zoospores, whereas species have been based on thallus development and morphology. We analyzed Powellomycetaceae chytrids that had not yet had ultrastructural and thallus morphologies determined. Because these chytrids vary little morphologically, we used a linear discriminant function analysis of thallus characters to determine features most useful for separating species when grown in pure culture on identical media. Zoosporic ultrastructures of two groups of isolates differed from those of the two described genera in the family, and we describe the new genera Thoreauomyces with one new species and Fimicolochytrium with two new species. Also, we describe three new species within Geranomyces. Linear discriminant function analysis, although helpful for determining more stable morphological characters, was not completely accurate in assigning chytrids to the correct genus or species, thus emphasizing the importance of molecular characters for identifying these taxa.

Fluorescent microscopy of viable Batrachochytrium dendrobatidis.

Batrachochytrium dendrobatidis ( Bd ), a chytrid fungus, is a causative agent of chytridiomycosis and amphibian population declines worldwide. The sequenced genome of Bd provides information necessary for studying the fungus and its molecular biology. Fluorescent microscopy is a technique used to image targeted molecules in live or fixed organisms to understand cellular trafficking and localization, but the use of fluorescent microscopy with Bd has not yet been demonstrated. Two fluorescent stains were tested for their use in live-cell imaging of Bd , i.e., the cell wall-specific fluorophore Solophenyl Flavine 7GFE and the DNA-specific fluorophore DRAQ5. These specific staining patterns were observed in live cultures of Bd when visualized with laser-scanning confocal microscopy.

The Polychytriales ord. nov. contains chitinophilic members of the rhizophlyctoid alliance.

During the past 5 y the Rhizophydiales, Cladochytriales and Lobulomycetales have been segregated from the formerly recognized Chytridiales. Descriptions of new chytridiomycete orders are based on molecular and ultrastructural characters, which have been phylogenetically mutually supportive. The Polychytrium clade has consisted of a few chitinophilic, soil and aquatic chytrids that clustered in phylogenetic hypotheses but have not been placed in a new order. We isolated additional putative members of this clade, sequenced their nucSSU and nucLSU rDNA and examined zoospores of some of the isolates with TEM. Our isolates are in a well supported clade with previous Polychytrium clade members, but zoospore ultrastructural types vary within the clade, with characters that often are conserved within other orders (e.g. flagellar plug, rumposome) being either present or absent. Based on the isolates in culture we describe the Polychytrium clade as the Polychytriales. This order contains Polychytrium, Lacustromyces, Karlingiomyces, two new genera (Arkaya and Neokarlingia) and additional undescribed taxa.

Alogomyces tanneri gen. et sp. nov., a chytrid in Lobulomycetales from horse manure.

The order Lobulomycetales contains chytrids from soil, freshwater and marine habitats; environmental DNA sampling has indicated that representatives of this order might be found in deep ocean localities. We describe Alogomyces tanneri as the first lobulomycetalean chytrid isolated from horse manure; A. tanneri is also the first species in the order to possess a rumposome in its zoospore. This species widens the range of habitats, ultrastructural variation and thallus morphology for Lobulomycetales.

Zoospore ultrastructure and phylogenetic position of Phlyctochytrium aureliae Ajello is revealed (Chytridiaceae, Chytridiales, Chytridiomycota).

From forest soils in Scotland Phlyctochytrium aureliae was observed and brought into pure culture. Previously included in a molecular phylogenetic study of Chytridiales as Phlyctochytrium sp. KP 061, the organism groups with Phlyctochytrium planicorne, P. bullatum, Chytridium olla and C. lagenaria in the family Chytridiaceae. Thallus morphology and development as well as zoospore ultrastructure are detailed herein. The sporangium is epibiotic, spherical or subspherical, apophysate or non-apophysate, and ornamented with dentate enations. The overall zoospore ultrastructural features are consistent with the Group II type zoospore that characterizes family Chytridiaceae in the Chytridiales, although the zoospore also has two character states unique to this taxon: the MLC cisterna fenestrations are one-third to one-half the diameter of fenestrations in other Chytridiaceae zoospores and an accumulation of electron-dense material (a kinetosome-associated structure, or KAS) proximal to the kinetosome and non-flagellated centriole is extensive and unique. This study verifies that zoospore ultrastructure of P. aureliae zoospores places this species in the Chytridiales and Chytridiaceae, as indicated in a previous molecular phylogenetic study.

Olpidium bornovanus-mediated germination of ascospores of Monosporascus cannonballus: a host-specific rhizosphere interaction.

Monosporascus cannonballus, a host-specific root-infecting ascomycete, is the causal agent of a destructive disease of melon (Cucumis melo L.) known as vine decline. Ascospores germinate only in the rhizosphere of melon plants growing in field soil. However, no germination occurs in the rhizosphere of melon plants if the field soil is heated to temperatures >50°C prior to infestation with ascospores. This observation suggested that germination is mediated by one or more heat-sensitive members of the soil microflora. Although bacteria or actinomycetes were heretofore suspected as the germination-inducing microbes, our data demonstrate that Olpidium bornovanus, an obligate, host-specific, root-infecting zoosporic fungus, is responsible. In four experiments conducted in autoclaved field soil amended with various population densities of culturally produced ascospores, significant ascospore germination was recorded only in the rhizosphere of cantaloupe seedlings colonized by O. bornovanus.

Operculomyces is a new genus in the order Rhizophydiales.

In recent years molecular phylogenies based on analyses of rDNA sequences have advanced knowledge of the Chytridiomycota, but much alpha taxonomic work remains. We have discovered an operculate chytrid that molecular phylogenies placed in the Rhizophydiales, an order that currently contains a single operculate species, the rest having inoperculate zoospore release. Because descriptions of genera in the Chytridiomycota now rely on transmission electron microscopic features of the zoospore as well as molecular evidence we studied the zoospore ultrastructure of the isolate to confirm its placement in the Rhizophydiales and to evaluate it as a new genus. Its suite of ultrastructural characters confirmed its placement within the Rhizophydiales, and its J-shaped, multilayered spur and the position of the nonflagellate centriole at a slight angle to the kinetosome make it unique within the Rhizophydiales, thus supporting molecular evidence indicating that the isolate represents a new genus. Herein we describe zoospore ultrastructure and scanning electron and light microscopic level morphology of this monocentric, endogenous, operculate chytrid and describe it as a new genus and species, Operculomyces laminatus.

Molecular phylogenetic and zoospore ultrastructural analyses of Chytridium olla establish the limits of a monophyletic Chytridiales.

Chytridium olla A. Braun, the first described chytrid and an obligate algal parasite, is the type for the genus and thus the foundation of family Chytridiaceae, order Chytridiales, class Chytridiomycetes and phylum Chytridiomycota. Chytridium olla was isolated in coculture with its host, Oedogonium capilliforme. DNA was extracted from the coculture, and 18S, 28S and ITS1-5.8S-ITS2 rDNA were amplified with universal fungal primers. Free swimming zoospores and zoospores in mature sporangia were examined with electron microscopy. Molecular analyses placed C. olla in a clade in Chytridiales with isolates of Chytridium lagenaria and Phlyctochytrium planicorne. Ultrastructural analysis revealed C. olla to have a Group II-type zoospore, previously described for Chytridium lagenaria and Phlyctochytrium planicorne. On the basis of zoospore ultrastructure, family Chytridiaceae is emended to include the type of Chytridium and other species with a Group II-type zoospore, and the new family Chytriomycetaceae is delineated to include members of Chytridiales with a Group I-type zoospore.