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.     

Morphology,zoospore ultrastructure,and molecular position of taxa in the Asterophlyctis lineage (Chytridiales, Chytridiomycota)

The purpose of our research is to investigate morphology, zoospore ultrastructure, and molecular placement of six strains in the Asterophlyctis (Chytridiales) lineage. In previous molecular analyses strain JEL 186, putatively Asterophlyctis sarcoptoides, placed as basal in family Chytriomycetaceae. Recent sampling for chytrids resulted in isolation of five strains (WJD 209, MP 058, JEL 524, JEL 857, and JEL 885) molecularly related to strain JEL 186. Our morphological evaluations reveal that strains JEL 186 and WJD 209 are members of Asterophlyctis. Strain WJD 209 is considered representative of the type, A. sarcoptoides, and strain JEL 186 a new species, Asterophlyctis michiganensis. The four strains MP 058, JEL 524, JEL 857, and JEL 885 are distinct from Asterophlyctis, and we consider them as members of a new genus, Wheelerophlyctis, composed of two species, Wheelerophlyctis interior and Wheelerophlyctis interiexterior. Asterophlyctis and Wheelerophlyctis are sister taxa and we demarcate that lineage as Asterophlyctaceae. The two genera also have similar zoospore ultrastructure, which is unique among strains in Chytridiales. In consideration of their molecular position and zoospore ultrastructure, we hypothesize that Asterophlyctis and Wheelerophlyctis represent a bridge between Chytriomycetaceae and Chytridiaceae. This research expands our concepts of systematics and zoospore ultrastructural variation in Chytridiales.     

Cladochytriales—a new order in Chytridiomycota

Recently, molecular and ultrastructural analyses have resulted in revised phylogenetic hypotheses in the phylum Chytridiomycota. The order Chytridiales, once considered monophyletic, has been subdivided into several new orders. However, the most recent analyses indicate that the emended Chytridiales is also polyphyletic. One monophyletic lineage in Chytridiales includes Cladochytrium, Nowakowskiella, and five other genera. Many of the chytrids in this clade have often been observed growing on decaying plant tissue and other cellulosic substrates from aquatic habitats and moist soils. In this study we analysed combined nu-rRNA gene sequences (partial SSU and LSU) of 30 isolates from North American aquatic and soil samples. Based on molecular monophyly and zoospore ultrastructure, we designate this clade as a new order, Cladochytriales, which includes four families: Cladochytriaceae, Nowakowskiellaceae, Septochytriaceae fam. nov., and Endochytriaceae.     

Rediscovery of an unusual chytridiaceous fungus new to the order Rhizophydiales

The newest order in Chytridiomycota, Rhizophydiales, shows remarkable genetic divergence and zoospore ultrastructural diversity. From submersed mud from a marsh near an oligotrophic lake in the Patagonia region of Argentina, we isolated a previously undescribed chytrid in Rhizophydiales with unique 28S rRNA and ITS1-5.8S-ITS2 sequences and a distinctive zoospore and thallus. In a combined LSU + 5.8S phylogeny of representative taxa in the order, the nearest relative to this new species is Rhizophlyctis harderi, and their zoospores share several similar character states. A new genus is erected for this chytrid, the first to be brought into pure culture from this region.     

Pendulichytrium sphaericum gen. et sp. nov. (Chytridiales,Chytriomycetaceae), a new chytrid parasitic on the diatom,Aulacoseira granulata

During the last decade, our concept of chytrid systematics has dramatically changed based on molecular phylogeny and zoospore ultrastructure. In contrast with well-studied saprotrophic chytrids, only a few obligate parasitic chytrids have been investigated with modern methods. Here, we investigate the novel chytrid culture KS93 that is parasitic on the diatom Aulacoseira granulata. Thallus morphology of KS93 was characterized by a spherical, stalked zoosporangium with a single, apical inoperculate discharge pore and zoospore discharge as a mass in a vesicle. A cross-inoculation experiment revealed that the infection of KS93 was specific to A. granulata. Zoospores of KS93 possessed the characters of the Group I type zoospore of the Chytriomycetaceae in the Chytridiales, but does not appear to have a paracrystalline inclusion. Additionally, KS93 also possessed a globule-type KAS (kinetosome-associated structure), first reported here for members of Chytriomycetaceae. In our molecular phylogeny, KS93 was placed in the basal position of the Chytriomycetaceae and was distinguished from any known species in the family. Morphological features of KS93 were distinct from those of any other taxa in the Chytriomycetaceae and from any described chytrids. Based on these results, we describe this chytrid as Pendulichytrium sphaericum gen. et sp. nov. in the family Chytriomycetaceae.     

Production and modifications of extracellular structures during development of chytridiomycetes

Summary In development of the primitive fungi, chytridiomycetes, unwalled zoospores bearing single, posterior flagella are transformed into walled, round-cells which elaborate the thallus. Production, structural modification, or release of extracellular material are involved with each transition of developmental stage. This article reviews the variety and developmental changes of extracellular materials found at the cell surface of chytridiomycetes. A cell coat, produced from Golgi-derived vesicles during zoosporogenesis, is visible around free swimming zoospores of some chytridiomycetes. How the zoospore surface receives and transduces signals is not widely explored, but it is known that fenestrated cisternae and simple cisternae, which are integrated into the microbody-lipid globule complex, are spatially and structurally associated with the plasma membrane and flagellar apparatus. This spatial association, as well as the cytochemical localization of calcium in fenestrated cisternae, suggest a mechanism for signal transduction and for regulation of zoospore motility. Zoospores become encased in a new layer of extracellular material as the zoospore encysts. Among some chytrids the source of this material is preexisting vesicles which fuse with the plasma membrane. Among other zoospores, a readily identifiable population of encystment vesicles is not apparent, demonstrating that there is no single pattern or mechanism for zoospore encystment in chytridiomycetes. Encysted zoospores developing into thalli, typically produce cell walls with a microfibrillar substructure. Ultrastructural analysis of walls reveals distinctive architecture and remarkable sculpturing which have been used in systematics of some members of chytridiomycetes. Nothing is known as to underlying controls of cytoskeletal elements and plasma membrane enzyme complexes in wall biogenesis. Many changes in cell surface structures accompany thallus maturation. Septa, many traversed with plasmodesmata, are produced in most chytrid thallus types. As sporangia and resting spores prepare for the production and release of zoospores, additional extracellular layers of material are frequently produced. Polarized deposits of extracellular material become discharge plugs, discharge vesicles, or endoopercula. Interstitial material is also released into cleavage furrows. Circumscissile or localized digestion of walls produce operculate or inoperculate exit ports for zoospore release. Cryofixation preserves more extensive extracellular material than does conventional chemical fixation, and broader application of cryofixation may radically alter our current view of cell surface structure. Thus chytridiomycetes exhibit a range in patterns for the occurrence and subsequent modifications of extracellular materials, even for members within the same order. The most universally recognized role for these extracellular materials is protection. Although there is a reasonable view of the types of extracellular material involved in chytridiomycete development, we have only limited understandings of their biogenesis or roles in regulation and communication, areas awaiting more investigations.Abbreviations DIC Nomarski-differential contrast optics - TEM transmission electron microscopy     

Ultrastructural and molecular phylogenetic delineation of a new order,the Rhizophydiales (Chytridiomycota)

In the order Chytridiales, Rhizophydium is a morphologically defined genus based upon the production of a monocentric, inoperculate, epibiotic sporangium, an endobiotic rhizoidal axis which branches, and an epibiotic resting spore. Despite its simple morphology, over 220 species of Rhizophydium have been described. Recent phylogenetic analyses using nuLSU rRNA (28 S rRNA) gene sequences of a geographically diverse sampling of Rhizophydium cultures revealed that the classical genus Rhizophydium is genetically more variable than previously understood and actually represents multiple genera. In the present study, we use zoospore ultrastructural characters and 28 S rRNA and 5.8 S ribosomal gene sequences of 96 isolates in culture to circumscribe the monophyletic Rhizophydium clade as a new order, Rhizophydiales. Correspondingly, zoospores of members of the Rhizophydiales exhibit a unique suite of ultrastructural character states that further define the order and distinguish it from the order Chytridiales. Molecular analyses reveal several strongly supported clades within the Rhizophydiales. Three of those clades encompass a broad range of isolates and are defined as new families Rhizophydiaceae, Terramycetaceae, and Kappamycetaceae. To resolve close relationships within Terramycetaceae, combined 28 S rRNA and ITS1–5.8 S–ITS2 sequences were analysed and details of zoospore ultrastructural character states determined, with two new genera, Terramyces and Boothiomyces, described. Two species formerly classified in Rhizophydium are transferred to the new genera. This work provides a framework for additional taxonomic revisions within the new order Rhizophydiales and compares genetic variation useful in defining genera, species, and populations within this lineage of chytrids. A broader sampling of representatives is needed before taxonomic decisions can be made for remaining clades within the Rhizophydiales.     

Taxonomy, molecular phylogeny, and ultrastructural morphology of Olpidiopsis porphyrae sp. nov. (Oomycetes, straminipiles), a unicellular obligate endoparasite of Bangia and Porphyra spp. (Bangiales, Rhodophyta)

Olpidiopsis porphyrae sp. nov., a marine oomycete endoparasite that infects the commercially cultivated red alga Porphyra yezoensis, is described and its phylogenetic position based on molecular data and ultrastructural morphology is discussed. O. porphyrae infects the host Porphyra by means of encysted zoospores. Spherical-shaped holocarpic thalli develop within the cytoplasm of its algal host, which produce monoplanetic, subapically biflagellate zoospores. The characteristic features of this isolate are the ellipsoidal, unicellular thallus and simple holocarpic zoosporangial development, which show morphological similarity with the genus Olpidiopsis. Laboratory infection experiments with a wide range of green, brown, and red algae revealed that O. porphyrae infects several stages of the bangialean red algae (the genera Bangia and Porphyra). Molecular phylogenetic analyses inferred from both SSU rRNA and cox2 genes showed O. porphyrae branched before the main saprolegnian and peronosporalean lineages within the monophyletic oomycete clade, indicating its phylogenetic separation from them. A single or double K-body-like organelle, which contains tubular inclusions, is found located to one side of the zoospore nucleus and shows similarities to homologous organelles previously described in O. saprolegniae. The ultrastructural morphology of O. porphyrae with zoospore initials containing K-bodies and tubular mitochondrial cristae is characteristic of oomycetes. Group I intron-like multiple insertions were found in the SSU rRNA gene of O. porphyrae. This is the first report of SSU group I introns in the class Oomycetes.     

Ratio-dependent functional response of two common Cladocera present in farmland ponds to Batrachochytrium dendrobatidis

Batrachochytrium dendrobatidis is responsible for amphibian declines worldwide. Decreasing the aquatic density of this chytrid through consumption of its infectious zoospores by Cladocera (water fleas) may mitigate the impact of chytridiomycosis. Understanding this predator-prey relationship requires insights in the zoospore ingestion rate of an average water flea, but such data are almost non-existent. We investigated the functional response of Simocephalus vetulus and Chydorus sphaericus feeding on B. dendrobatidis zoospores. These Cladocera commonly occur in farmland ponds, which may represent a major habitat for disease control. Both water fleas actively ingested zoospores and their per capita ingestion rate was best modelled in function of zoospore-to-Cladocera ratio, implying mutual interference among water fleas during zoospore feeding. The larger S. vetulus substantially consumed more zoospores, characterised by a maximum ingestion rate of 2.5 × 10⁵ zoospores.Cladocera⁻¹.h⁻¹.mL⁻¹, which is about 12 times higher than for C. sphaericus. These findings are useful to support model-based management of chytridiomycosis.     

Ultrastructural evidence for the biflagellate origin of the uniflagellate fungal zoospore

Summary The morphological similarities between the kinetosome and the second centriole of the zoospores of Phlyctochytrium kniepii and P. punctatum (Chytridiomycetes) suggest that the second centriole in the chytrid zoospore is a vestigial flagellum base. It is suggested that the term vestigial kinetosome may also be used when referring to the structure which is presently termed the second centriole of the chytrid zoospore. Morphological similarities between the chytrid zoospores of P. kniepii and P. punctatum and the zoospores of Rhizidiomyces apophysatus (Hyphochytridiomycetes) are noted. The possible biflagellate origin of fungi with uniflagellate zoospores is discussed. The third fiber (C fiber) of the kinetosome triplet is shown to form as an outgrowth of the B fiber of the kinetosome doublet.     

ZOOSPORE STRUCTURE OF THE MYCOPARASITIC CHYTRID CAULOCHYTRIUM PROTOSTELIOIDES OLIVE

The subcellular organization of zoospores released from sessile, parasitic sporangia of Caulochytrium protostelioides was studied with light and electron microscopy. A single flagellum is posteriorly directed but laterally inserted into the cylindrical motile zoospore. A striated rhizoplast attaches the proximal end of the kinetosome to a specialized region of the nuclear envelope. A system of rough endoplasmic reticulum, smooth endoplasmic reticulum, dictyosomes and bristle-coated vesicles are associated with the one to several pulsating vacuoles typically located near the flagellar apparatus. The microbody-lipid globule complex (MLC) comprises one to many lipid globules. An extensive microbody branches around each lipid globule and encloses a portion of the rhizoplast. A reticulum of smooth surfaced cisternae interdigitates among the branches of the complex microbody, and cisternae are opposed to the surface of lipid globules opposite the microbodies. Mitochondria with predominantly circular profiles are scattered throughout the zoospore body, but several are always adjacent to the microbody, and hence, are also part of the MLC. Ribosomes are uniformly distributed throughout the zoospore, and one to several cisternae of rough endoplasmic reticulum are adjacent to the nuclear envelope. Zoospores of C. protostelioides are similar to several other chytrid zoospores, which also have the same type of microbody-lipid globule complex, but yet are structurally distinct from any other chytrid zoospore.     

THE DEVELOPMENT AND POSSIBLE RELATIONSHIPS OF A NEW ATKINSIELLA PARASITIC IN INSECT EGGS

Atkinsiella entomophaga is a holocarpic parasite in eggs of various midges and caddis flies. Primary zoospores escape through long discharge tubes and assume an abbreviated period of motility before encysting. Laterally biflagellate secondary zoospores subsequently emerge from the cysts. Coincident with discharge tube formation, the thallus undergoes strong vacuolization giving the protoplast a reticulate aspect with nuclei situated between the vacuoles and connected to one another by protoplasmic threads. Stages in zoosporogenesis resemble those of members of the Lagenidiales. It is proposed that Atkinsiella be included in the Eurychasmaceae along with Eurychasma and Eurychasmidium and that the family be transferred to the Lagenidiales. All members of this family have diplanetic zoospores.     

PSEUDULVELLA AMERICANA BELONGS TO THE ORDER CHAETOPELTIDALES (CLASS CHLOROPHYCEAE), EVIDENCE FROM ULTRASTRUCTURE AND SSU RDNA SEQUENCE DATA1

The genus Pseudulvella Wille 1909 includes epiphytic, freshwater, or marine disk‐shaped green microalgae that form quadriflagellate zoospores. No ultrastructural or molecular studies have been conducted on the genus, and its evolutionary relationships remain unclear. The purpose of the present study is to describe the life history, ultrastructural features, and phylogenetic affiliations of Pseudulvella americana (Snow) Wille, the type species of the genus. Thalli of this microalga were prostrate and composed of radiating branched filaments that coalesced to form a disk. Vegetative cells had a pyrenoid encircled by starch plates and traversed by one or two convoluted cytoplasmic channels. They had well‐defined cell walls without plasmodesmata. Asexual reproduction was by means of tetraflagellate zoospores formed in numbers of two to eight from central cells of the thallus. The flagellar apparatus of zoospores was cruciate, with four basal bodies and four microtubular roots. The paired basal bodies lay directly opposite (DO) one another. The microtubular root system had a 5‐2‐5‐2 alternation pattern, where the “s” roots contained five microtubules in a four‐over‐one configuration. A tetralobate nonstriated distal fiber connected all four basal bodies. A wedge‐shaped proximal sheath subtended each of the basal bodies. The ultrastructural features of the zoospores were those of members of the order Chaetopeltidales. Phylogenetic analyses based on SSU rDNA placed P. americana sister to Chaetopeltis orbicularis in a well‐supported Chaetopeltidales clade. Such a combination of features confirmed that this alga is a member of the order Chaetopeltidales.     

Forecasting infections of the red rot disease on Porphyra yezoensis Ueda (Rhodophyta) cultivation farms

Pythium porphyrae is a fungal pathogen responsible for red rot disease of the seaweed Porphyra (Rhodophyta). Infection forecasts of Porphyra by P. porphyrae were estimated from the epidemiological observations of Porphyra thalli and numbers of zoospore of P. porphyrae in laboratory and cultivation areas. Four features of forecasting infections were determined by relating zoospore concentrations to the incidence of thallus infection; infection (in more than 1000 zoospores L⁻¹), microscopic infection [less than 2 mm in diameter of lesion (in from 2000 to 3000 zoospores L⁻¹)], macroscopic infection [more than 2 mm in diameter of lesion (in from 3000 to 4000 zoospores L⁻¹), and thallus disintegration (in more than 4000 zoospores L⁻¹). High zoospore concentrations led to more infection. The tendency that zoospore concentration of P. porphyrae increased with the rate of infection of Porphyra thalli was generally observed in forecasting infections in both the laboratory and in cultivation areas. Based on the Porphyra cultivation areas, the accuracy and consistency of forecasting infections suggest that this method could be employed to manage and control red rot disease.     

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.     

Flow cytometric determination of zoospore DNA content and population DNA distribution in cultured Pfiesteria spp. (Pyrrhophyta)

The relative cellular DNA content from 23 different clonal cultures of Pfiesteria spp. zoospores was determined using a DNA fluorochrome and flow cytometry. Significant differences between Pfiesteria piscicida and P. shumwayae were detected, both in mean zoospore DNA content and population cell cycle DNA distribution. Intraspecific differences in DNA content were found between clonal zoospore cultures established from different geographical regions. Long-term cultures (years) of P. piscicida were available for testing, and a negative correlation was observed between zoospore DNA content and time in culture. Zoospore cell cycle-related DNA distributions were also markedly different between the two species in these clonal cultures. In most cultures tested, P. piscicida zoospores exhibited bimodal DNA flow histograms with G1-S-G2+M distributions, typical of eukaryotic asynchronously cycling cells. In contrast, cultures of P. shumwayae zoospores exhibited one DNA peak distribution, indicative of synchronized cells. The data are consistent with the hypothesis that P. shumwayae zoospores are interphasic cells, and mitosis in zoospore cultures of this species predominantly occurs as benthic or adherent non-motile division cysts. Light microscopy observations of the nuclear condition of electrostatically sorted zoospores of each Pfiesteria species also support this hypothesis. If highly conserved, this disparity in modes of vegetative reproduction would ramify the population dynamics of the two Pfiesteria species.     

Ultrastructure of early stages of Rozella allomycis (Cryptomycota) infection of its host, Allomyces macrogynus (Blastocladiomycota)

This study reconstructs early stages of Rozella allomycis endoparasitic infection of its host, Allomyces macrogynus. Young thalli of A. macrogynus were inoculated with suspensions of R. allomycis zoospores and allowed to develop for 120 h. Infected thalli at intervals were fixed for electron microscopy and observed. Zoospores were attracted to host thalli, encysted on their surfaces, and penetrated their walls with an infection tube. The parasite cyst discharged its protoplast through an infection tube, which invaginated the host plasma membrane. The host plasma membrane then surrounded the parasite protoplast and formed a compartment confining it inside host cytoplasm. The earliest host-parasite interface within host cytoplasm consisted of two membranes, the outer layer the host plasma membrane and the inner layer the parasite plasma membrane. At first a wide space separated the two membranes and no material was observed within this space. Later, as the endoparasite thallus expanded within the compartment, the two membranes became closely appressed. As the endoparasite thallus continued to enlarge, the interface developed into three membrane layers. Thus, host plasma membrane surrounded the parasite protoplast initially without the parasite having to pierce the host plasma membrane for entry. Significantly, host-derived membrane was at the interface throughout development.     

Evidence that Blastocladiella emersonii zoospore chitin synthetase is located at the plasma membrane

Blastocladiella emersonii zoospores are not encased by a cell wall and do not detectably synthesize or contain chitin; accompanying de novo cell wall formation during zoospore encystment, chitin rapidly accumulates and is incorporated into the cell wall. Essential for understanding this abrupt change in chitin synthesis is the location of zoospore chitin synthetase. The enzyme has previously been reported to the sequestered with distinctive cytoplasmic organelles (gamma particles) characteristic for the zoospore cell type. Using similar differential and equilibrium density centrifugation procedures to those reported previously, we have observed the vast majority of zoospore homogenate chitin synthetase activity in fractions distinct from the gamma particle-enriched fractions. Over 90% of the homogenate enzyme activity could be recovered in a sucrose buoyant density region (1.14–1.18 g/ml) containing membranous elements and well separated from the region enriched for gamma particles (1.30–1.34 g/ml). When zoospores were surface-labelled with [³H]concanavalin A prior to homogenization, the buoyant density regions of radioactivity and of chitin synthetase activity exhibited nearly complete coincidence. At least the bulk of zoospore chitin synthetase appears to be located at the plasma membrane, rather than in gamma particles.     

Binding of exogenous DNA to marine sediments and the effect of DNA/sediment binding on natural transformation of Pseudomonas stutzeri strain ZoBell in sediment columns

A new species of strictly anaerobic chytridiomycete was isolated from dried faeces of the Saharian ass that had been stored for up to 150 days. Because of its monocentric thallus and uniflagellate zoospores it belongs to the genus Piromyces. It exhibits a high affinity for P. mae and P. dumbonica but differs from them in its morphological and ultrastructural characteristics. Its flagellar apparatus is similar to that of all previously reported fungi.