Electron microscopy of gametangial interaction and oospore development in Phytophthora capsici

Gametangial development and oospore formation were studied, with emphasis on cell wall morphogenesis, on mated cultures (A¹xA²) of Phytophthora capsici. In this species, the oogonial and antheridial hyphae interact to produce a typical amphigynous antheridium. The following developmental steps were recognized: 1) contact between oogonial and antheridial initials; 2) penetration of the antheridial initial by the oogonial initial; 3) reemergence of the oogonial initial; 4) oogonial expansion; 5) gametangial delimitation and oogonial wall thickening; 6) penetration of the oogonium by the antheridial fertilization tube; 7) oosphere formation; 8) periplasm degeneration and outer oospore wall formation; and 9) inner oospore wall formation. Electron micrographs were obtained of steps 3–9. Steps 1 and 2 were reconstructed from subsequent events. Steps 3–6 are stages of active wall formation with clear indication of intensive dictyosome activity leading to the formation of numerous wall-destined vesicles of two different sizes and electron densities. No vesicles were seen associated with the development of the inner oospore wall; however, by this stage of development the oosphere cytoplasm exhibited an overall intense electron density that obscured fine detail. Cytoplasmic appearance changed enormously during differentiation, from a developing oogonium rich in mitochondria, ribosomes, rough endoplasmic reticulum, dictyosomes and their vesicles, through an oosphere filled with large finger-print vacuoles and lipid-like bodies, to a mature oospore with a large central vacuole (ooplast) surrounded by a cortex of numerous lipid-like bodies; other organelles are confined to the interstitial space between these storage bodies.     

Development and ultrastructure of the marine,parasitic oomycete,Lagenisma coscinodisci Drebes (Lagenidiales)

The thalli of Lagenisma coscinodisci are coenocytic. In young thalli each nucleus is associated with two procentrioles and four dictyosomes. Before nuclear division two pairs of procentrioles are found on two opposite poles each lying in a pocket of the nuclear membranes. The spindle microtubules radiate from these poles; the nuclear membrane remains completely closed in all divisional stages. The thallus transforms holocarpically into a zoosporangium. The differentiation begins after the breakdown of the host with the formation of a cell wall and with the development of a discharge tube. During these processes peripheral dilated tubules are observed which are in close, local contact with small infoldings of the plasmalemma. The discharge tube develops independent of the point of entry of the infection tube and enlarges by tip growth. There are several light and electron microscopical observations which suggest that the last nuclear divisions in zoosporangia of old cultures are meiotic.They give rise to sexually determined zoomeiospores.     

ITS region of the rDNA of Pythium longandrum, a new species; its taxonomy and its comparison with related species

Pythium longandrum (F-73.0) was isolated, from soil samples taken in Lille in northern France. Morphologically the fungus resembles closely Pythium rostratum, however its antheridial characters are unique. The oogonia of this species are provided with hypogynous and monoclinous antheridia. The antheridial cells are inflated and are probably the largest and longest for the genus. The internal transcribed spacer region of its nuclear ribosomal DNA indicates that it is entirely different from all other species of Pythium. This new species is characterized by its spherical to elongated sporangia, smooth-walled oogonia and hypogynous to monoclinous antheridia bearing long antheridial cells closely applied to the oogonia. Morphological features of this new species, together with the sequences of the ITS region of its nuclear ribosomal DNA and comparison with related species are discussed here.     

A new species of Pythium with filamentous sporangia having pectinolytic activities, isolated in the Burgundy region of France

A new species, Pythium pectinolyticum (F-83.1), isolated from soil samples taken in the Burgundy region is being described here. This species is characterised by its filamentous non-inflated type of sporangia, smooth-walled mostly catenulate oogonia and very rare antheridia. This fungus is a very slow-growing organism on most of the solid media, but it grows well in liquid media and also on those containing pectin as the sole source of carbon. Morphological features are described here together with the sequences of the internal transcribed spacer 1 region of the nuclear ribosomal DNA of the fungus, its comparison with related species, and its pectinolytic behaviour.     

Pythium contiguanum nomen novum (syn. Pythium dreschleri Paul), its antagonism to Botrytis cinerea, ITS1 region of its nuclear ribosomal DNA, and its comparison with related species

Pythium drechsleri Paul was described as a new species from soil samples taken in a salt-marsh of Arzew, Algeria [Paul, B. (1988) Une nouvelle espèce de Pythium isolée d'une saline de l'ouest Algérien. Cryptogam. Mycol. 9, 325-333]. The name of the fungus, P. drechsleri, is a nomen invalidum, as it is a later homonym of P. drechsleri Rajgopalan and Ramakrishnan [Rajagopalan, S. and Ramakrishnan, K. (1971) Phycomycetes in agricultural soils with special reference to the Pythiaceae. Madras Univ. J. Sect. B 37,38, 100-117]. A new name, Pythium contiguanum is now being given to P. drechsleri Paul. This species is characterised by its contiguous inflated type of sporangia, smooth-walled oogonia and mostly monoclinous antheridia. Although the fungus is not known to be a pathogen, it has a very well developed appressorial system comprised of both sickle shaped and coiled appressoria. Morphological features, its antagonism towards the grape-vine pathogen Botrytis cinerea, together with the sequences of the ITS1 region of its nuclear ribosomal DNA and its comparison with related species are given here.     

Fungi isolated from skins and pens of healthy animals in Nigeria

The mycoflora of 220 skin scrapings, hair, nail samples and pens’ materials of apparently healthy animals including cows, sheep, goats, rabbits, pigs and dogs were determined. Twenty eight species of fungi belonging to ten genera were recovered. Chrysosporium spp. were the most common and C. keratinophilum was recovered from all animals. Dermatophytes which are known causal agents of dermatophytosis were also isolated indifferent frequencies (Microsporum gypseum, M. canis, Trichophytonmentagrophytes, T. rubrum). This revised version was published online in June 2006 with corrections to the Cover Date.     

The effect of dung beetle activity on the discharge ofPilobolus (Fungi,mucorales) sporangia in cattle,sheep and horse faeces

The effect ofAphodius sphacelatus andA. ater activity in cattle faeces andSphaeridium bipustulatum in sheep and horse faeces on the discharge ofPilobolus sporangia was investigated under laboratory conditions. In five trials, the median number of sporangia produced was significantly less (P<0.01) in experimental chambers (with 1 or 2 beetles/g faeces) than in control chambers (without beetles). In addition, median percentage reduction of sporangia was significantly higher (P<0.01) forA. sphacelatus (80.9%) than forA. ater (56.9%) at 1 beetle/g cattle faeces.

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Résumé L'effet de l'activité d'Aphodius spacelatus et deA. ater dans les bouses de vaches et deSphaeridium bipustulatum dans les fèces de moutons et de chevaux, sur la libération de sporanges parPilobolus a été étudié en conditions de laboratoire. Sur 5 essais le nombre médian de sporanges libérés dans des cellules expérimentales (avec 1 ou 2 bousiers/g fèces) était significativement inférieur (P<0.01) à celui obtenu dans des cellules témoins (sans bousier). De plus, le pourcentage médian de réduction des sporanges est significativement plus élevé (P<0,01) pourA. sphacelatus (80,9%) que pourA. ater (56,9%) à la densité de 1 bousier/g de bouse.

     

Discharge papilla development in the phycomycete Allomyces

The process of discharge papilla (DP) formation in Allomyces macrogynus was studied by light and electron microscopy. The plug of the DP was first deposited between the plasmalemma and the wall of the zoosporangium (ZS). The wall above the plug subsequently was eroded away. Deposition of a new inner wall layer in the sporangium held the plug in place and thickening of the layer formed a collar around the plug. Further deposition of material after this stage resulted in the characteristic pulley-shape. The plug material appeared homogeneous in electron micrographs but there was evidence of an outer layer. Digestion of the plug at the time of spore release was from within.Abbreviations DP discharge papilla - ZS zoosporangium     

Three species of Lagenidiales isolated from the eggs and zoeae of the marine crabPortunus pelagicus

Three species of Lagenidiales were isolated from the eggs and zoeae of the marine crabPortunus pelagicus. One of them,Lagenidium callinectes, is reported for the first time in Japan, with fungal infection in the eggs.Haliphthoros milfordensis was discovered from a zoea of the crab.Atkinsiella okinawaensis was also isolated from a zoea of the crab and described as a new species. Growth temperature range and optimum temperature of the fungi were examined. All of the isolates grew at various concentrations of NaCl or KCl, and optimum growth was observed on PYGS agar containing seawater. Pathogenicity to zoeae of the swimming crabPortunus trituberculatus was demonstrated by using a zoospore suspension of each fungus at 25°C.     

Gametangial development in Allomyces macrogynus

The possible role of mitochondria in determining the sex of the gametangia of Allomyces macrogynus was investigated. Quantitative studies of mitochondrial distribution in vegetative hyphae confirmed previous reports of apical mitochondrial clustering. However, by the time the male and female gametangia were partitioned off, no significant difference in mitochondrial distribution between the two sexes was present. Possible mechanisms for the redistribution of mitochondria during early differentiation are discussed. In addition, cytochrome oxidase activity was demonstrated in all mitochondria of both male and female gametangia by the use of diaminobenzidine. It is concluded that neither mitochondrial distribution nor differential mitochondrial activity plays a determining role in the differentiation of the sexual cells in Euallomyces.Abbreviations %M percent area occupied by mitochondria - DAB diaminobenzidine     

Ultrastructural observations on the oogenesis of Triops cancriformis (Crustacea,Notostraca)

Summary The first stages of the oogenesis of Triops cancriformis have been studied. At the outset the oocyte is smaller than the nurse cells. Meiosis begins with typical synaptonemal complexes. The significance of these complexes and of some other peculiar structures of germ cells, i.e., pore complexes and annuli within the nucleus, and annulate lamellae within the cytoplasm are discussed. The morphofunctional uniformity of some cytoplasmic structures (annulate lamellae, concentrically arranged ER, and yolk globules) in the oocyte as well as its nurse cells is also discussed.     

Ultrastructural observations on the oogenesis of Triops cancriformis (Crustacea,Notostraca)

Summary Each ovarian follicle of Triops cancriformis is four-celled; these cells (one oocyte and three nurse cells) are interconnected by cytoplasmic bridges. In the course of differentiation, the nurse cells are early recognizable; they increase in size more than the oocyte and their nuclei contain many nucleoli. For the first time in Arthropoda, yolk globules are reported to be present in nurse cell cytoplasm; these globules arise from the smooth endoplasmic reticulum. The functional significance of the intercellular bridges and the trophic role of the nurse cells are discussed.The authors are grateful to Dr. Bruno Sabelli for his support and to Mr. Francesco Monte for his technical assistance     

Fungi associated with the Egyptian cotton leafwormSpodoptera littoralis Boisdoval

A total of 28 species representing 15 fungal genera were found to be associated with the all different stages of the Egyptian cotton leafworm,Spodoptera littoralis Boisd., its faeces and the air of the containers where it was reared on Czapek Dox agar medium at 28°C.Aspergillus was the most common genus and was frequently isolated from all substrates.A. flavus was the only fungus associated with all insect stages. At least two well known facultative pathogens were obtained:Beauveria alba andCephaliophora tropica. Nearly all fungi recovered from insect faeces or its environment were also encountered from different insect stages. Forty-two isolates were tested for their abilities for growth on a synthetic medium (nitrogen- and carbon-free) containing partially purified chitin. Most (76.2%) had moderate growth rates.     

Expression and cellular localization of the Toucan protein during Drosophila oogenesis

The toucan (toc) gene is required in the germline for somatic cell patterning during Drosophila oogenesis. To better understand the function of toc, we performed a detailed analysis of the distribution of the Toucan protein during oogenesis. Toc expression is restricted to the germline cells and shows a dynamic distribution pattern throughout follicle development. Mislocalization of the Toc protein in mutant follicles in which the microtubule network is altered indicates that microtubules play a role in Toc localization during oogenesis.     

Fungi from the roots of the common terrestrial orchid Gymnadenia conopsea

The fungal community associated with the terrestrial photosynthetic orchid Gymnadenia conopsea was characterized through PCR-amplification directly from root extracted DNA and cloning of the PCR products. Six populations in two geographically distinct regions in Germany were investigated. New ITS-primers amplifying a wide taxonomic range including Basidiomycetes and Ascomycetes revealed a high taxonomic and ecological diversity of fungal associates, including typical orchid mycorrhizas of the Tulasnellaceae and Ceratobasidiaceae as well as several ectomycorrhizal taxa of the Pezizales. The wide spectrum of potential mycorrhizal partners may contribute to this orchid's ability to colonize different habitat types with their characteristic microbial communities. The fungal community of G. conopsea showed a clear spatial structure. With 43 % shared taxa the species composition of the two regions showed only little overlap. Regardless of regions, populations were highly variable concerning taxon richness, varying between 5 and 14 taxa per population. The spatial structure and the continuous presence of mycorrhizal taxa on the one hand and the low specificity towards certain fungal taxa on the other hand suggest that the fungal community associated with G. conopsea is determined by multiple factors. In this context, germination as well as pronounced morphological and genetic differentiation within G. conopsea deserve attention as potential factors affecting the composition of the fungal community.     

Fungi associated with storage rots of cocoyams (Colocasia spp.) in Nsukka,Nigeria

Cocoyam (Colocasia spp.) corms and cormels showing spoilage symptoms were collected from many stores in Nsukka locality and examined for rot and associated fungal pathogens. Aspergillus niger, Botryodiplodia theobromae, Corticium rolfsii, Geotrichum candidum, Fusarium oxysporum, and F. solani were recovered from rotten cocoyams. The representative isolates of these species caused cocoyam rot in pathogenicity tests. The rot due to A. niger, B. theobromae and C. rolfsii was extensive resulting in complete maceration of cocoyam tissue. Potassium sorbate (0.1 mg/ml) protected cocoyams from fungal rot with the exception of C. rolfsii.     

Untersuchungen zur Dotterbildung in den Oocyten von Xiphophorus helleri (Heckel, 1848) (Teleostei,Poeciliidae)

Zusammenfassung Die Verteilung von Nucleinsäuren, Polysacchariden, Proteinen und Fetten in den verschiedenen Entwicklungsstadien der Oocyten von Xiphophorus helleri wurde histochemisch untersucht. Junge Oocyten enthalten viel RNS und Proteine, aber wenig Fett. Während der Vitellogenese steigt der Gehalt an Fetten, Glycoproteide werden eingebaut. Die dazu nötigen Stoffe werden aus dem mütterlichen Körper in niedermolekularer, löslicher Form über das Follikelepithel zur Oocyte transportiert. Diese histochemischen Befunde werden durch Ergebnisse der Elektrophorese gestützt. In elektronenmikroskopischen Untersuchungen wurden den histochemischen Fakten weitere morphologische Grundlagen zugeordnet. Während der Prävitellogenese tritt ein Dotterkern auf, der sich vor Beginn der Dotterbildung auflöst. Coated vesicles nehmen Material aus dem perioocytären Interzellularraum auf. Zellstrukturen der Oocyte beteiligen sich am Dotteraufbau. Besonders auffallend sind die Veränderungen an den Mitochondrien. In der Zusammensetzung des Dotters und in der Beteiligung der Organellen gibt es Unterschiede zu Lebistes. Injektionen von Myofer runden die Ergebnisse ab. Vom maternalen zum embryonalen Gewebe erfolgt ein Transport von Nährstoffen, nicht nur Gasaustausch. Die Definition der Ovoviviparie muß daher erweitert werden.

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Studies on yolk formation in oocytes of Xiphophorus helleri

Summary The distribution of nucleic acids, polysaccharides, proteins, and fats in different developmental stages of oocytes of Xiphophorus helleri was studied using histochemical methods. Young oocytes had high RNA and protein contents. During vitellogenesis, the fat content increased and glycoproteins were deposited. These substances were transported from the maternal body into the oocyte in a soluble, small molecular form. The histochemical results were supported by our studies using electrophoretic and electron microscopic methods. During previtellogenesis, a yolk nucleus appeared. Coated vesicles took up material from the intercellular space around the oocyte. Differences between Xiphophorus and Lebistes were observed in the composition of yolk and in the prevalent types of ultrastructural organelles present. In Xiphophorus, labelling by Myofer (intraperitoneal or intramuscular injections) verified the transport of nutritive material from the maternal body into oocytes (Stage III and IV) and embryonic tissues. This suggests that in Xiphophorus the definition of ovoviviparity has to be modified.

     

A multi-gene phylogeny of Clavicipitaceae (Ascomycota, Fungi): identification of localized incongruence using a combinational bootstrap approach

Multi-gene phylogenetic analyses were conducted to address the evolution of Clavicipitaceae (Ascomycota). Data are presented here for approximately 5900 base pairs from portions of seven loci: the nuclear ribosomal small and large subunit DNA (nrSSU and nrLSU), beta-tubulin, elongation factor 1alpha (EF-1alpha), the largest and second largest subunits of RNA polymerase II (RPB1 and RPB2), and mitochondrial ATP Synthase subunit 6 (mtATP6). These data were analyzed in a complete 66-taxon matrix and 91-taxon supermatrix that included some missing data. Separate phylogenetic analyses, with data partitioned according to genes, produced some conflicting results. The results of separate analyses from RPB1 and RPB2 are in agreement with the combined analyses that resolve a paraphyletic Clavicipitaceae comprising three well-supported clades (i.e., Clavicipitaceae clade A, B, and C), whereas the tree obtained from mtATP6 is in strong conflict with the monophyly of Clavicipitaceae clade B and the sister-group relationship of Hypocreaceae and Clavicipitaceae clade C. The distribution of relative contribution of nodal support for each gene partition was assessed using both partitioned Bremer support (PBS) values and combinational bootstrap (CB) analyses, the latter of which analyzed bootstrap proportions from all possible combinations of the seven gene partitions. These results suggest that CB analyses provide a more consistent estimate of nodal support than PBS and that combining heterogeneous gene partitions, which individually support a limited number of nodes, results in increased support for overall tree topology. Analyses of the 91-taxa supermatrix data sets revealed that some nodes were more strongly supported by increased taxon sampling. Identifying the localized incongruence of mtATP6 and analyses of complete and supermatrix data sets strengthen the evidence for rejecting the monophyly of Clavicipitaceae and much of the current subfamilial classification of the family. Although the monophyly of the grass-associated subfamily Clavicipitoideae (e.g., Claviceps, Balansia, and Epichlo?) is strongly supported, the subfamily Cordycipitoideae (e.g., Cordyceps and Torrubiella) is not monophyletic. In particular, species of the genus Cordyceps, which are pathogens of arthropods and truffles, are found in all three clavicipitaceous clades. These results imply that most characters used in the current familial classification of Clavicipitaceae are not diagnostic of monophyly.     

Fungi associated with terrestrial orchid mycorrhizas,seeds and protocorms

The identity and ecological role of fungi in the mycorrhizal roots of 25 species of mature terrestrial orchids and in 17 species of field incubated orchid seedlings were examined. Isolates of symbiotic fungi from mature orchid mycorrhizas were basidiomycetes primarily in the generaCeratorhiza, Epulorhiza andMoniliopsis; a few unidentified taxa with clamped hyphae were also recovered. More than one taxon of peloton-forming fungus was often observed in the cleared and stained mycorrhizas. AlthoughCeratorhiza andEpulorhiza strains were isolated from the developing protocorms, pelotons of clamped hyphae were often presents in the cleared protocorms of several orchid species. These basidiomycetes are difficult to isolate and may be symbionts of ectotrophic plants. The higher proportion of endophytes bearing clamp connections in developing seeds than in the mycorrhizas is attributed to differences in the nutritional requirements of the fully mycotrophic protocorms and partially autotrophic plants. Most isolates ofCeratorhiza differed enzymatically fromEpulorhiza in producing polyphenol oxidases. Dual cultures with thirteen orchid isolates and five non-orchid hosts showed that some taxa can form harmless associations with non-orchid hosts. It is suggested that most terrestrial orchid mycorrhizas are relatively non-specific and that the mycobionts can be saprophytes, parasites or mycorrhizal associates of other plants.