Zoospore Ultrastructure Research Articles

Antimicrobial mechanisms of g-C3N4 nanosheets against the oomycetes Phytophthora capsici: Disrupting metabolism and membrane structures and inhibiting vegetative and reproductive growth

To understand the potential of urea-synthesized g-C3N4 nanosheets (0.125–1 mg/mL) as antimicrobial agents against oomycetes, an investigation of the interaction mechanism between g-C3N4 nanosheets and Phytophthora capsici was conducted. Transcription analysis showed that after being exposed to g-C3N4 nanosheets for 1 h, P. capsici triggered a sharp upregulation of antioxidant activities and structural constituents and a downregulation of metabolic pathways, including ATP generation, autophagy disruption, membrane system disorders and other complex adaptive processes. All the life stages of P. capsici, including mycelial growth, sporangium formation, zoospore numbers and zoospore germination were remarkably inhibited and even injured. A mutual mechanism is proposed in this work: ROS stress upon exposure to visible irradiation and, combined with their sharp nanosheet structure, cause perturbations of the cell membrane and induce damage to the ultrastructure of mycelial growth, sporangium and zoospores. Given that the antimicrobial action of g-C3N4 nanosheets were derived from the damage throughout the duration of treatment and was not limited to a single target, these complex mechanisms could favor the avoidance of drug resistance and benefit other oomycetes management. More importantly, in addition to restraining P. capsici infection in host plants, g-C3N4 nanosheets promoted pepper plant growth. Hence, g-C3N4 nanosheets have potential as a new non-metal antimicrobial agent to control oomycotal disease in crops.

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

ABSTRACT 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 species of aquatic chytrids from Oman

ABSTRACT Oman is a desert country in the south of the Middle East. Springs and other water sources that harbor aquatic organisms can be separated by hundreds of kilometers. In Oct 2019, we isolated four freshwater aquatic fungi (Chytridiomycota) from benthic detritus baited with pine pollen on a general nutrient medium near Salalah, Oman. Database queries of nuc 28S rRNA (28S) and internal transcribed spacer region ITS1-5.8S-ITS2 (ITS) revealed that one of these strains was Dinochytrium kinnereticum, a recently described algal pathogen from the Sea of Galilee. The other three strains had low molecular identity to available ITS sequences. These unknown strains varied in size and released endogenously swarming zoospores through papillae from mature zoosporangia. Zoospore ultrastructure was consistent with described species in the Rhizophydiales, and molecular phylogenetic results grouped these three strains into a clade in the genus Rhizophydium. We circumscribe these three strains as a sp. nov., thereby expanding the diversity within Rhizophydium described as the new species R. jobii. In doing so, we provide the first report of Chytridiomycota from Oman.

Quaeritorhiza haematococci is a new species of parasitic chytrid of the commercially grown alga, Haematococcus pluvialis

ABSTRACT Aquaculture companies grow the green alga Haematococcus pluvialis (Chlorophyta) to extract the carotenoid astaxanthin to sell, which is used as human and animal dietary supplements. We were requested to identify an unknown pathogen of H. pluvialis from an alga growing facility in the southwestern United States. To identify this zoosporic fungus and determine its phylogenetic placement among other chytrids, we isolated it into pure culture, photographed its morphology and zoospore ultrastructure, and sequenced and analyzed portions of nuc rDNA 18S and 28S genes. The organism belongs in the Chytridiomycota, but a comparison of rDNA with available representatives of the phylum did not convincingly place it in any described order. The unique zoospore ultrastructure supports its indeterminate ordinal position, and the morphology, as determined by light microscopy, did not match any described species. Consequently, we have placed this chytrid in the new genus, Quaeritorhiza, and described it as the new species Q. haematococci in the family Quaeritorhizaceae but otherwise incertae sedis in the Chytridiomycetes. This new taxon is important because it increases the known diversity of Chytridiomycota and the organism has the ability to disrupt agricultural production of an algal monoculture.

Boothiomyces angulosus and Boothiomyces elyensis: two new combinations in the Terramycetaceae (Rhizophydiales, Chytridiomycota)

The genus Rhizophydium was proposed by A. Schenk in 1858 to accommodate the inoperculate taxa previously placed in Chytridium. The morphological delineation encompassed around 235 species that have now been segregated into different genera based on molecular and zoospore ultrastructural analyses. However, some taxa have never been investigated for phylogenetic position or zoospore ultrastructural characters. The aim of this study was to use morphology, zoospore ultrastructure and molecular analyses to verify the placement of our isolates of Rhizophydium angulosum and R. elyense in the Rhizophydiales phylogeny. These isolates produced angular zoosporangia, characteristic of Terramycetaceae representatives, and grouped within the Boothiomyces clade in analyses of complete ITS and partial LSU regions of rDNA. Transmission electronic microscopy (TEM) analysis revealed that R. angulosum produces zoospores with the same ultrastructural characters described from Boothiomyces representatives. In addition, R. elyense presented sufficient characteristics that support its morphological delineation from Boothiomyces macroporosus, the type species of the genus. Based on molecular, morphological, and ultrastructural analyses, we transfer R. angulosum and R. elyense to Boothiomyces.

Taxonomic revision of the genus Zygorhizidium: Zygorhizidiales and Zygophlyctidales ord. nov. (Chytridiomycetes, Chytridiomycota).

During the last decade, the classification system of chytrids has dramatically changed based on zoospore ultrastructure and molecular phylogeny. In contrast to well-studied saprotrophic chytrids, most parasitic chytrids have thus far been only morphologically described by light microscopy, hence they hold great potential for filling some of the existing gaps in the current classification of chytrids. The genus Zygorhizidium is characterized by an operculate zoosporangium and a resting spore formed as a result of sexual reproduction in which a male thallus and female thallus fuse via a conjugation tube. All described species of Zygorhizidium are parasites of algae and their taxonomic positions remain to be resolved. Here, we examined morphology, zoospore ultrastructure, host specificity, and molecular phylogeny of seven cultures of Zygorhizidium spp. Based on thallus morphology and host specificity, one culture was identified as Z. willei parasitic on zygnematophycean green algae, whereas the others were identified as parasites of diatoms, Z. asterionellae on Asterionella, Z. melosirae on Aulacoseira, and Z. planktonicum on Ulnaria (formerly Synedra). According to phylogenetic analysis, Zygorhizidium was separated into two distinct order-level novel lineages; one lineage was composed singly of Z. willei, which is the type species of the genus, and the other included the three species of diatom parasites. Zoospore ultrastructural observation revealed that the two lineages can be distinguished from each other and both possess unique characters among the known orders within the Chytridiomycetes. Based on these results, we accommodate the three diatom parasites, Z. asterionellae, Z. melosirae, and Z. planktonicum in the distinct genus Zygophlyctis, and propose two new orders: Zygorhizidiales and Zygophlyctidales.

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 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.

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.

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.

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.

A New Parasitic Chytrid, Staurastromyces oculus (Rhizophydiales, Staurastromycetaceae fam. nov.), Infecting the Freshwater Desmid Staurastrum sp.

Chytrids are a diverse group of ubiquitous true zoosporic fungi. The recent molecular discovery of a large diversity of undescribed chytrids has raised awareness on their important, but so far understudied ecological role in aquatic ecosystems. In the pelagic zone, of both freshwater and marine ecosystems, many chytrid species have been morphologically described as parasites on almost all major groups of phytoplankton. However, the majority of these parasitic chytrids has rarely been isolated and lack DNA sequence data, resulting in a large proportion of "dark taxa" in databases. Here, we report on the isolation and in-depth morphological, molecular and host range characterization of a chytrid infecting the common freshwater desmid Staurastrum sp. We provide first insights on the metabolic activity of the different chytrid development stages by using the vital dye FUN®-1 (2-chloro-4-[2,3-dihydro-3-methyl-[benzo-1,3-thiazol-2-yl]-methylidene]-1-phenylquinolinium iodide). Cross infection experiments suggest that this chytrid is an obligate parasite and specific for the genus Staurastrum sp. Phylogenetic analysis, based on ITS1-5.8S-ITS2 and 28S rDNA sequences, placed it in the order Rhizophydiales. Based on the unique zoospore ultrastructure, combined with thallus morphology, and molecular phylogenetic placement, we describe this parasitic chytrid as a new genus and species Staurastromyces oculus, within a new family Staurastromycetaceae.

Morphological, molecular, and ultrastructural characterization of Rozella rhizoclosmatii, a new species in Cryptomycota

Rozella is a genus of unwalled endoparasites of a variety of hosts including Oomycota (Stramenopiles), Blastocladiomycota and Chytridiomycota (Fungi), and one green alga (Coleochaete, Chlorophyceae). It currently includes more than 20 formally described species, and no new species of Rozella have been described since 1987. We discovered a new Rozella species parasitizing Rhizoclosmatium globosum (Chytridiales, Chytridiomycota) and investigated its morphology, ultrastructure, and phylogenetic position. Herein named as Rozella rhizoclosmatii sp. nov., the organism induces hypertrophy of the host. Its zoospore is ultrastructurally similar to that of Rozella allomycis, although it has a unique zoospore ultrastructural feature, a lattice of perpendicular rods about the nucleus. The 18S rDNA molecular sequence of R. rhizoclosmatii is similar to that of the previously sequenced ‘Rozella ex Rhizoclosmatium’. This is the first study to inclusively characterize a new species of Rozella with morphological, ultrastructural and molecular data. As this is only the second Rozella species to be examined ultrastructurally, and because it is parasitic on a member of Chytridiomycota and not Blastocladiomycota, this research supports the conservative nature of zoospore ultrastructure to help define the genus.

Characterization of a new chytrid species parasitic on the dinoflagellate, Peridinium gatunense

Only a few chytrid fungi have been reported as parasites of dinoflagellates. Among these reports, chytrids are periodically observed growing on the dinoflagellate, Peridinium gatunense, in Lake Kinneret (Sea of Galilee), Israel. Because of the distinctive roles of parasitic chytrid fungi in decreasing phytoplankton populations and in transforming inedible algae into chytrid biomass which zooplankton grazers can eat, characterizing dinoflagellate parasites contributes to our understanding of the sustainability of this important water resource. An undescribed chytrid parasite of P. gatunense from Lake Kinneret has recently been brought into pure culture (KLL_TL-060613), facilitating exploration of its infection process. To evaluate the ability of this chytrid to affect host populations, we determined the effect of: (1) temperature and light (or dark) on prevalence of infection and (2) host growth phase and parasite:host ratio on percentage of infection. The greatest amplification in host infection occurred in cultures grown in the dark at 25 C. The percentage of host cells infected increased as the availability of host cells compared to parasite cells increased. These results demonstrate that environmental factors influence the chytrid’s potential to affect Peridinium gatunense populations. Because this chytrid had not been described taxonomically, we characterized its thallus morphology, development, zoospore ultrastructure and phylogenetic relationships. Zoospore ultrastructure was compatible with the Group II type zoospore characteristic of the family Chytridiaceae in the Chytridiales. Consistent with this observation, phylogenetic analyses of nuc 28S rDNA D1-D3 domains (28S) placed the chytrid in a clade among described taxa in the Chytridiaceae. Because thallus morphology was distinct from these other taxa, as well as other described parasites of dinoflagellates, this chytrid is described as a new genus and species, Dinochytrium kinnereticum.

A new family and four new genera in Rhizophydiales (Chytridiomycota)

Many chytrid phylogenies contain lineages representing a lone taxon or a few organisms. One such lineage in recent molecular phylogenies of Rhizophydiales contained two marine chytrids, Rhizophydium littoreum and Rhizophydium aestuarii. To better understand the relationship between these organisms, we increased sampling such that the R. littoreum/R. aestuarii lineage included 10 strains of interest. To place this lineage in Rhizophydiales, we constructed a molecular phylogeny from partial nuc 28S rDNA D1–D3 domains (28S) of these and 80 additional strains in Rhizophydiales and examined thallus morphology and zoospore ultrastructure of our strains of interest. We also analyzed sequences of the nuc rDNA region encompassing the internal transcribed spacers 1 and 2, along with the 5.8S rDNA (ITS) of our 10 strains of interest to assess sequence similarity and phylogenetic placement of strains within the lineage. The 10 strains grouped together in three well supported clades: (i) Rhizophydium littoreum + Phlyctochytrium mangrovei, (ii) three strains of Rhizophydium aestuarii and (iii) five previously unidentified strains. Light microscopic observations revealed four distinct thallus morphologies, and zoospore ultrastructural analyses revealed four distinct constellations of ultrastructural features. On the bases of morphological, ultrastructural and molecular evidence we place these strains in the new family Halomycetaceae and four new genera (Halomyces, Paludomyces, Ulkenomyces, Paranamyces) in Rhizophydiales.

Cyclopsomyces plurioperculatus: a new genus and species of Lobulomycetales (Chytridiomycota, Chytridiomycetes) from Japan

Lobulomycetales is one of the smallest orders of Chytridiomycota, containing only four genera and five species. In a survey in Japan we isolated a chytrid from a soil sample collected in a broadleaf forest, which grouped in Lobulomycetales by BLAST query. To identify this chytrid and determine its taxonomic position, thallus development and morphology were observed by light microscopy and zoospore ultrastructure was examined using a transmission electron microscopy. We conducted a phylogenetic analysis using nuc 28S rDNA sequences. Thallus morphology was characterized by a spherical zoosporangium with multiple operculate discharge papillae, which is different from that of any other species in Lobulomycetales. This chytrid is similar to Chytriomyces multioperculatus in having multiple operculate discharge papillae, but these are distinguished by characters of the discharge papillae and rhizoidal systems. Zoospores of this chytrid had electron-dense material in the kinetosome, a unique character in the order. Our 28S phylogeny placed it in a distinct clade, sister to all described species in Lobulomycetaceae. Based on these results, we propose a new genus and species of Lobulomycetales, Cyclopsomyces plurioperculatus.

Fayochytriomyces, a new genus within Chytridiales

Chytriomyces is a complex genus in Chytridiales. The morphological concept of the genus expanded as new taxa were added, and studies of zoospore ultrastructure and molecular phylogenies have revealed the genus to be polyphyletic. One problematic taxon is C. spinosus Fay, a distinctive species characterized by whorls of spines on the zoosporangium and a large accumulation of vesicle material beneath the operculum. With light-, scanning-electron and transmission-electron microscopy, we examined a culture (WJD186) isolated from a muck sample collected from a temporary forest pond. We also analyzed the D1–D2 variable domains of the nuc 28S rDNA (28S) sequences to confirm the phylogenetic placement of the species relative to the type of Chytriomyces, C. hyalinus Karling. The morphology of culture WJD186 is consistent with features Fay described for C. spinosus, and the zoospore ultrastructure is consistent with the Group I-type zoospore characters of Chytriomycetaceae (Chytridiales). In our molecular phylogeny C. spinosus does not group with the type of Chytriomyces. Consequently, we erect a new genus in Chytriomycetaceae and present the new combination Fayochytriomyces spinosus.

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.

Gromochytrium mamkaevae gen. & sp. nov. and two new orders: Gromochytriales and Mesochytriales (Chytridiomycetes).

During the last decade several new orders were established in the class Chytridiomycetes on the basis of zoospore ultrastructure and molecular phylogeny. Here we present the ultrastructure and molecular phylogeny of strain x-51 CALU - a parasite of the alga Tribonema gayanum, originally described as Rhizophydium sp. based on light microscopy. Detailed investigation revealed that the zoospore ultrastructure of this strain has unique characters not found in any order of Chytridiomycetes: posterior ribosomal core unbounded by the endoplasmic reticulum and detached from the nucleus or microbody-lipid complex, and kinetosome composed of microtubular doublets. An isolated phylogenetic position of x-51 is further confirmed by the analysis of 18S and 28S rRNA sequences, and motivates the description of a new genus and species Gromochytrium mamkaevae. The sister position of G. mamkaevae branch relative to Mesochytrium and a cluster of environmental sequences, as well as the ultrastructural differences between Gromochytrium and Mesochytrium zoospores prompted us to establish two new orders: Gromochytriales and Mesochytriales.