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.     

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.     

Ultrastructure of chytridiomycete and oomycete zoospores using spray-freeze fixation

Summary The ultrastructure of zoospores of several zoosporic fungi was examined using a modified cryofixation technique. An atomizer was used to spray a zoospore suspension into the cold propane reservoir of a conventional plunge freeze-substitution apparatus. Spray-freeze fixation and freeze-substitution of zoospores porvided better fixation of vacuolar structures, membranes and the extracellular coat than that obtained with chemical fixation. The overall shape of cryofixed spores was closer to that seen in living zoospores. Two types of vacuoles were seen in cryofixed zoospores ofMonoblepharella andChytridium. One type of vacuole contained electron-opaque material within the lumen while the other type had no visible internal material in the lumen and appeared to be part of the water expulsion vacuole complex. Coated pits and coated vesicles were observed associated with both the water expulsion vacuoles and the plasma membrane inMonoblepharella andPhytophthora, suggesting that endocytosis of the plasma membrane and expulsion vacuoles is part of membrane recycling during osmoregulatory events. An extracellular coat was seen on the outer surface of cryofixed zoospores ofMonoblepharella sp.,Chytridium confervae andPhytophthora palmivora without the use of carbohydrate-specific stains. The spray-freeze method gave good and reproducible fixation of the wall-less spores in quantities greater than those obtained in previously described zoospore cryofixation studies. The technique is potentially useful for cell suspensions in that freeze damage from excess water is limited.Abbreviations ddH₂O deionized distilled water - PME Pipes/MgCl2/EGTA buffer - WEV water expulsion vacuole     

Studies on British chytrids XXVIII. Rhizophydium nobile sp. nov., parasitic on the resting spore of Ceratium hirundinella O.F.Müll. from the plankton

The life history of a new species of the Chytridiales Rhizophydium nobile is described. It occurs in the autumn on resting spores of the alga Ceratium hirundinella O.F.M. in Blelham Tarn, Windermere and Esthwaite Water, lakes in the English Lake District. The sporangium develops from the zoospore and possesses a branched rhizoidal system. The zoospores are fully formed in the sporangium. On dehiscence part of the sporangium content flows out surrounded by a vesicle which eventually bursts and liberates the zoospores. Only a few resting spores were seen associated with the sporangial stage. They were small spherical thick-walled bodies containing several globules. Further observations need to be made upon this stage.     

STUDIES OF THE MOTILE CELLS OF CHYTRIDS. V. FLAGELLAR RETRACTION IN POSTERIORLY UNIFLAGELLATE FUNGI

Presented are observations of 17 species in the following 14 genera. Blastocladiales : Allomyces and Blastocladiella. Chytridiales : Asterophlyctis, Catenochytridium, Chytridium, Chytriomyces, Cladochytrium, Entophlyctis, Phlyctochytrium, Rhizophlyctis, Rozella, Septochytrium, and two undescribed cladochytriaceous genera. Encystment without flagellar retraction is briefly considered. Recognized and described are four basic developmental patterns or methods of flagellar retraction: lash-around, body-twist, straight-in, and vesicular. Variations in and combinations of the four basic methods are described. Observations, most of which are illustrated with brightfield, darkfield, phase-contrast, and electron microscopy, along with what is known from the literature, are discussed and serve as the basis for generalized, summary diagrams or two-dimensional models of patterns of flagellar retraction in posteriorly uniflagellate fungi. An endeavor is made to put the findings reported on in this paper in historical, morphogenetic, and phylogenetic perspective.     

The flagellar apparatus and striated rhizoplast of the zoospore ofOlpidium brassicae

Summary The flagellar apparatus and its associated structures of the zoospore ofOlpidium brassicae are described and compared with observations of other zoospores of the uniflagellatePhycomycetes. The zoospore ofO. brassicae is shown to have an extensive cone-shaped rhizoplast fused to both the functional and the vestigial kinetosomes. Three-dimensional reconstructions were made of the kinetosomal region. The vestigial kinetosome differs from the functional, as it only has triplet bundles of microtubules and it lacks a system of props. The proximal termination of the central pair of flagellar microtubules occurs within the axoneme. No terminal plate is observed. The occurrence of dictyosomes in theChytridiales, Monoblepharidales, andHyphochytriales is discussed and it is concluded that a dictyosome may be present in the encysting zoospore and the maturing zoosporangium ofO. brassicae but only vestiges of a dictyosome are to be found in the free-swimming 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.     

CATENOPHLYCTIS,A NEW GENUS OF THE CATENARIACEAE

Catenophlyctis gen. nov. is established in the family Catenariaceae to include the fungus previously known as Phlyctorhiza variabilis Karling. This ubiquitous saprophyte was formerly included in the genus Phlyctorhiza Hanson of the Chytridiales on the grounds that its eucarpic thallus is predominantly monocentric and because it develops usually from an enlargement of the germ tube. Additional studies on this species in India have confirmed the previous observations that the thallus of some strains frequently becomes extensively polycentric and Catenaria-like. Also, its zoospores have been found to be similar in structure to those of species of the Catenariaceae. Accordingly, this species cannot be retained in Phlyctorhiza as Hanson defined the genus, and it is transferred from the Chytridiales to a new genus of the family Catenariaceae in the order Blastocladiales.     

Ultrastructural observations of mature and encysting zoospores ofPythium proliferum de Bary

Summary The process of zoospore maturation and encystment inP. proliferum was studied by electron microscopy. General ultrastructural features of the mature, swimming zoospore were found to be similar to those previously described for other oomycetes in both the attachment and ultrastructure of the flagella as well as the type and distribution of cellular organelles. Associated with extensive areas of RER in the mature zoospores were unusual, electrondense, bar-like structures. These structures were found in the groove region of young zoospores and at the periphery of encysting zoospores. Their possible function is discussed. The five main types of vesicles observed during encystment, as seen grouped in this study, along with the vesicles described in previous studies of oomycete encystment, were in table form and individually discussed. Interesting correlations appear to exist in the types of vesicles that are present within the oomycetes studied thusfar.     

Ultrastructural description of a new chytrid genus of caecum anaerobe, Caecomyces equi gen. nov., sp. nov., assigned to the Neocallimasticaceae

Vegetative and reproductive stages of Caecomyces equi gen. nov., sp. nov. isolated from the horse caecum were examined by light and electron microscopy. This organism, which is similar to isolates known as Sphaeromonas communis, produces uniflagellate, uninucleate zoospores whose perikinetosomal structures, i.e. circumflagellar ring, spur, struts and scoop, are similar in many respects to those described in species of Neocallimastix. Microtubular roots extend basally from the spur and associate with hydrogenosomes and the nucleus. Another group of microtubules radiates laterally in a fan-shaped array close to the plasmalemma. Zoospores encyst, shedding their flagella with basal bodies, and germinate to diglobular thalli. Either coralloid or bulbous rhizoids form in plant material, but only the latter in axenic culture. Incipient zoospores are produced from a multinucleate eucarpic thallus and devlop within cleavage vacuoles containing flagella. An isolate from the cow rumen was found to be similar to C. equi in morphology and zoospore ultrastructure. On the basis of zoospore ultrastructure, we assign the new genus to the Neocallimasticaceae of the order Spizellomycetales. Organisms previously described as Sphaeromonas communis and Piromonas communis are renamed Caecomyces communis and Piromyces communis and assigned to the same family.     

Zoosporic fungi of Oceania. IV. Additional monocentric chytrids

Summary Several monocentric chytrid species of the generaPhlyctochytrium,Rhizidium,Chytridium,Chytriomyces andKarlingia were isolated on various substrata from soils of Oceania. Among these are two new species,Phlyctochytrium megastomum andRhizidium endosporangiatum. The former species is characterized chiefly by sporangia with an usually large apical, subapical, or a lateral exit orifice, the persistence of remnants of the pailla wall at the edge of the exit orifice, and by small zoospores. Rhizidium endosporangiatum is characterized at first primarily by predominantly spherical sporangia which develop 1 to 8 broad papillae. At maturity these papillae give them an angular shape. The exit papillae deliquesce at the tip, and through them protrude prominent arms of an endosporangium which expands and in which the zoospores usually swarm briefly. Willoughby's Chytridium parasiticum occurred abundantly as a parasite ofNowakowskiella sp.,Pythium sp., andPhytophthora sp. and developed epibiotic resting spores like those ofChytriomyces. Accordingly, this parasite is transferred to this genus and renamedChytriomyces willoughbyii because the nameparasiticum is preempted by the author's previously describedChytriomyces parasiticus.This study has been supported by a grant from the National Science Foundation, Washington, D.C.     

The zoospore and meiospore of the aquatic phycomyceteCatenaria anguillulae

Summary The zoospore and meiospore of the aquatic phycomyceteCatenaria anguillulae (Phycomycetes, Blastocladiales, Catenariaceae) have a nuclear cap enclosing the cellular ribosomes within a double membrane, and a side body complex which is very similar to that observed in zoospores ofBlastocladiella andCoelomomyces and is structurally related to the side body complex observed in spores ofAllomyces. The structural organization of the side body complex and striated rootlet is analyzed from serial sections.The meiospore also contains an array of flattened cisternae which are in direct contact with, and appear to be derived from, the outer nuclear membrane and the backing membrane of the side body complex.The structural organization of the zoospore and meiospore ofC. anguillulae is compared to and contrasted with the structural organization observed in spores of members of theChytridiales, Blastocladiales, Monoblepharidales, andHarpochytriales. It is concluded that the structural organization of the spores of theBlastocladiales, Monoblepharidales, andHarpochytriales is similar, and affinities in spore organization can be found in some members of theChytridiales.     

RELATIONSHIP OF TEMPERATURE TO ZOOSPORE PRODUCTION IN TETRASPORA GELATINOSA

A strain of Tetraspora gelatinosa was isolated from a field collection and a technique for measuring quantitatively the production of zoospores was devised. The method employed compound colonies grown on agar from zoospore suspensions. The effect of 3 temperatures on zoospore production was tested: no zoospores were produced by the colonies at 3 C; zoospores were produced at 11 and 23 C and the final yield of zoospores at the 2 temperatures was not significantly different. However, at 11 C the maximum density of zoospores produced, measured by cell count, occurred after IS hr, while the maximum density at 23 C occurred after 21 hr.     

A simple in-vitro 'wet-plate' method for mass production of Phytophthora nicotianae zoospores and factors influencing zoospore production

A simple in-vitro ‘wet-plate’ method for mass-producing Phytophthora nicotianae zoospores at ≥ 1.0 × 10⁶ zoospores/ml is described. Temperature critically affected zoospore production; 22 °C was optimum, while 36 °C was completely inhibitory. Zoospores being the most important propagule of P. nicotianae, temperature of recycled irrigation water may be manipulated to reduce diseases in irrigated nursery crops.     

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.     

禾谷多粘菌游动孢子超微结构观察

本文描述了寄生在大麦根部的禾谷多粘菌Polymyxa graminis Led.的次生游动孢子的超微结构,包括核、内质网、高尔基体、线粒体、脂质粒、排泄泡、小囊、具膜小囊、核糖体以及鞭毛基体(Kinetosome)和鞭毛杆等细胞器。游动孢子中未见微体。同时也在电镜下观察了游动孢子接触和穿透根细胞时所形成的管腔(Rohr)和棘杆(Stachel)以及游动孢子穿透细胞壁的详细过程。     

The effect of the weight of the seedling-derived tuber on subsequent clonal generations in a potato breeding programme

Cultures of Polymyxa graminis were maintained in roots of barley plants grown in sand at different temperatures using Wisconsin soil temperature tanks. At 17 – 20°C, the minimum time from inoculation with cystosori to the production of zoospores from the inoculated roots was 2 – 3 wk. At 11 – 20°C many zoospores were produced but the incubation period was longer at the lower temperatures. Above 20°C little fungal development occurred. The duration of motility of zoospores ranged from c. 1 h to > 24 h. Bovine serum albumen (BSA) prolonged motility but glycine and glucose had no effect or, at higher concentrations, were toxic. Zoospores were rapidly immobilised by zinc ions in solution at or above 10μg/ml. In some experiments BSA added to the zoospore suspension greatly increased transmission of barley yellow mosaic virus (BaYMV) while glucose, glycine and ovalbumen decreased it. When seedlings were incubated with zoospore suspensions for 24 h at different temperatures, BaYMV transmission was high (> 60%) at 10, 15 and 20°C but there was little at 5 or 25°C. In experiments to determine the time taken for zoospore penetration, seedlings were incubated in suspension for different periods of time and then rinsed in zinc sulphate solution to kill free zoospores. Between 3 and 3·5 h was needed for zoospores to establish infection. Transmission occurred equally to plants of various ages between 3 days and 7·5 wk.