Attachment, penetration and early host defense mechanisms during the infection of filamentous brown algae by Eurychasma dicksonii.

Eurychasma dicksonii is one of the most common and widespread marine pathogens and attacks a broad spectrum of more than 45 brown algal species. The present study focuses on the mechanism used by the pathogen to attach on the host cell wall and force its way into algal cells. Ultrastructural examination revealed a needle-like structure which develops within the attached spore and extends along its main axis. Particular cell wall modifications are present at the basal part of the spore (adhesorium pad) and guide the needle-like tool to penetrate perpendicularly the host cell wall. The unique injection mechanism is shared with Haptoglossa species which suggests that this is an important characteristic of early diverging oomycetes. Furthermore, the encystment and adhesion mechanism of E. dicksonii shows significant similarities with other oomycetes, some of which are plant pathogens. Staining and immunolabelling techniques showed the deposition of ß-1,3-glucans on the host cell wall at the pathogen penetration site, a strategy similar to physical responses previously described only in infected plant cells. It is assumed that the host defense in terms of callose-like deposition is an ancient response to infection.

A new oomycete species parasitic in nematodes, Chlamydomyzium dictyuchoides sp. nov.: developmental biology and phylogenetic studies.

The genus Chlamydomyzium is a little studied holocarpic oomycete parasite of nematodes of uncertain phylogenetic and taxonomic position. A new holocarpic species, Chlamydomyzium dictyuchoides, is described which has usually refractile cytoplasm and a dictyuchoid pattern of spore release. This new species infects bacteriotrophic rhabditid nematodes and was isolated from diverse geographical locations. Infection was initiated by zoospore encystment on the host surface and direct penetration of the cuticle. A sparsely branched, constricted, refractile thallus was formed which eventually occupied almost the entire host body cavity, often accompanied by complete dissolution of the host cuticle. Walled primary cysts formed throughout the thallus and each cyst released a single zoospore via an individual exit papillum, leaving a characteristic dictyuchoid wall net behind. At later stages of infection some thalli formed thick-walled stellate resting spores in uniseriate rows. Resting spore formation appeared to be parthenogenetic and was not accompanied by the formation of antheridial compartments. These spores had ooplast-like vacuoles and thick multi-layered walls, both of which suggest they were oospores. The maximum likelihood tree of sequences of the small ribosomal subunit (SSU) gene placed this new isolate in a clade before the main saprolegnialean and peronosporalean lines diverge. A second undescribed Chlamydomyzium sp., which has direct spore release forms a paraphyletic clade, close to C. dictyuchoides and Sapromyces. The fine structure of other documented Chlamydomyzium species was compared, including an undescribed (but sequenced) isolate, SL02, from Japan, Chlamydomyzium anomalum and Chlamydomyzium oviparasiticum. Chlamydomyzium as currently constituted is a paraphyletic genus that is part of a group of phylogenetically problematic early diverging clades that lie close to both the Leptomitales and Rhipidiales.

Aquastella gen. nov.: a new genus of saprolegniaceous oomycete rotifer parasites related to Aphanomyces, with unique sporangial outgrowths.

The oomycete genus Aquastella is described to accommodate two new species of parasites of rotifers observed in Brooktrout Lake, New York State, USA. Three rotifer species--Keratella taurocephala, Polyarthra vulgaris, and Ploesoma truncatum--were infected, and this is the first report of oomycete infection in these species. Aquastella attenuata was specific to K. taurocephala and Aquastella acicularis was specific to P. vulgaris and P. truncatum. The occurrence of infections correlated with peak host population densities and rotifers were infected in the upper layers of the water column. Sequencing of 18S rRNA and phylogenetic analysis of both species placed them within the order Saprolegniales, in a clade closely related to Aphanomyces. The Aquastella species were morphologically distinct from other rotifer parasites as the developing sporangia penetrated out through the host body following its death to produce unique tapered outgrowths. Aquastella attenuata produced long, narrow, tapering, finger-like outgrowths, whilst A. acicularis produced shorter, spike-like outgrowths. We hypothesize that the outgrowths serve to deter predation and slow descent in the water column. Spore cleavage was intrasporangial with spore release through exit tubes. Aquastella attenuata produced primary zoospores, whereas A. acicularis released spherical primary aplanospores, more typical of other genera in the Aphanomyces clade.

A family of small tyrosine rich proteins is essential for oogonial and oospore cell wall development of the mycoparasitic oomycete Pythium oligandrum.

The mycoparasitic oomycete Pythium oligandrum is homothallic, producing an abundance of thick-walled spiny oospores in culture. After mining a cDNA sequence dataset, we identified a family of genes that code for small tyrosine rich (Pythium oligandrumsmall tyrosine rich (PoStr)) proteins. Sequence analysis identified similarity between the PoStr proteins and putative glycine-rich cell wall proteins from the related plant pathogenic oomycete Pythium ultimum, and mating-induced genes from the oomycete Phytophthora infestans. Expression analysis showed that PoStr transcripts accumulate during oospore production in culture and immunolocalisation indicates the presence of these proteins in oogonial and oospore cell walls. PoStr protein abundance correlated positively with production of oogonia as determined by antibiotic-mediated oogonia suppression. To further characterise the role of PoStr proteins in P. oligandrum oospore production, we silenced this gene family using homology-dependent gene silencing. This represents the first characterisation of genes using gene silencing in a Pythium species. Oospores from silenced strains displayed major ultrastructural changes and were sensitive to degradative enzyme treatment. Oogonia of silenced strains either appeared to be arrested at the mature oosphere stage of development or in around 40 % of the structures, showed a complete suppression of oospore formation. Suppressed oogonia were highly vacuolated and the oogonium wall was thickened by a new inner wall layer. Our data suggest PoStr proteins are probably integral structural components of the normal oospore cell wall and play a key role in oospore formation.

The evolutionary phylogeny of the oomycete "fungi".

Molecular sequencing has helped resolve the phylogenetic relationships amongst the diverse groups of algal, fungal-like and protist organisms that constitute the Chromalveolate "superkingdom" clade. It is thought that the whole clade evolved from a photosynthetic ancestor and that there have been at least three independent plastid losses during their evolutionary history. The fungal-like oomycetes and hyphochytrids, together with the marine flagellates Pirsonia and Developayella, form part of the clade defined by Cavalier-Smith and Chao (2006) as the phylum "Pseudofungi", which is a sister to the photosynthetic chromistan algae (phylum Ochrophyta). Within the oomycetes, a number of predominantly marine holocarpic genera appear to diverge before the main "saprolegnian" and "peronosporalean" lines, into which all oomycetes had been traditionally placed. It is now clear that oomycetes have their evolutionary roots in the sea. The earliest diverging oomycete genera so far documented, Eurychasma and Haptoglossa, are both obligate parasites that show a high degree of complexity and sophistication in their host parasite interactions and infection structures. Key morphological and cytological features of the oomycetes will be reviewed in the context of our revised understanding of their likely phylogeny. Recent genomic studies have revealed a number of intriguing similarities in host-pathogen interactions between the oomycetes with their distant apicocomplexan cousins. Therefore, the earlier view that oomycetes evolved from the largely saprotrophic "saprolegnian line" is not supported and current evidence shows these organisms evolved from simple holocarpic marine parasites. Both the hyphal-like pattern of growth and the acquisition of oogamous sexual reproduction probably developed largely after the migration of these organisms from the sea to land.

Genome sequence of the necrotrophic plant pathogen Pythium ultimum reveals original pathogenicity mechanisms and effector repertoire.

BACKGROUND: Pythium ultimum is a ubiquitous oomycete plant pathogen responsible for a variety of diseases on a broad range of crop and ornamental species. RESULTS: The P. ultimum genome (42.8 Mb) encodes 15,290 genes and has extensive sequence similarity and synteny with related Phytophthora species, including the potato blight pathogen Phytophthora infestans. Whole transcriptome sequencing revealed expression of 86% of genes, with detectable differential expression of suites of genes under abiotic stress and in the presence of a host. The predicted proteome includes a large repertoire of proteins involved in plant pathogen interactions, although, surprisingly, the P. ultimum genome does not encode any classical RXLR effectors and relatively few Crinkler genes in comparison to related phytopathogenic oomycetes. A lower number of enzymes involved in carbohydrate metabolism were present compared to Phytophthora species, with the notable absence of cutinases, suggesting a significant difference in virulence mechanisms between P. ultimum and more host-specific oomycete species. Although we observed a high degree of orthology with Phytophthora genomes, there were novel features of the P. ultimum proteome, including an expansion of genes involved in proteolysis and genes unique to Pythium. We identified a small gene family of cadherins, proteins involved in cell adhesion, the first report of these in a genome outside the metazoans. CONCLUSIONS: Access to the P. ultimum genome has revealed not only core pathogenic mechanisms within the oomycetes but also lineage-specific genes associated with the alternative virulence and lifestyles found within the pythiaceous lineages compared to the Peronosporaceae.

The development, ultrastructural cytology, and molecular phylogeny of the basal oomycete Eurychasma dicksonii, infecting the filamentous phaeophyte algae Ectocarpus siliculosus and Pylaiella littoralis.

The morphological development, ultrastructural cytology, and molecular phylogeny of Eurychasma dicksonii, a holocarpic oomycete endoparasite of phaeophyte algae, were investigated in laboratory cultures. Infection of the host algae by E. dicksonii is initiated by an adhesorium-like infection apparatus. First non-walled, the parasite cell developed a cell wall and numerous large vacuoles once it had almost completely filled the infected host cell (foamy stage). Large-scale cytoplasmic changes led to the differentiation of a sporangium with peripheral primary cysts. Secondary zoospores appeared to be liberated from the primary cysts in the internal space left after the peripheral spores differentiated. These zoospores contained two phases of peripheral vesicles, most likely homologous to the dorsal encystment vesicles and K-bodies observed in other oomycetes. Following zoospore liberation the walls of the empty cyst were left behind, forming the so-called net sporangium, a distinctive morphological feature of this genus. The morphological and ultrastructural features of Eurychasma were discussed in relation to similarities with other oomycetes. Both SSU rRNA and COII trees pointed to a basal position of Eurychasma among the Oomycetes. The cox2 sequences also revealed that the UGA codon encoded tryptophan, constituting the first report of stop codon reassignment in an oomycete mitochondrion.

An ultrastructural study of development and reproduction in the nematode parasite Myzocytiopsis vermicola.

An isolate of Myzocytiopsis vermicola, a holocarpic parasite of Rhabditis nematodes, was studied with transmission electron microscopy (TEM) to follow development during infection, asexual and sexual reproduction. Nematodes became infected after attachment of apical cystospore buds to the nematode cuticle. Apical buds were packed with vesicles with dense fibrillar contents, which were absent from the thallus. Some thalli developed into sporangia while others became paired gametangial cells. Zoospore cleavage was often intrasporangial, although during the early stages of an epidemic partially differentiated zoospores usually were released via an exit tube into a fine vesicle. Packets of tripartite tubular hairs (TTH) were not observed in the cytoplasm of either developing or mature sporangia. TEM of sectioned material and whole mounts of zoospores revealed biflagellate zoospores, some without hairs and others with a proximal row of very short hairs on the anterior flagellum. Gametangial contact was via a short, walled fertilization tube and surplus antheridial and oogonial nuclei remained in their respective gametangial cells until disintegration of the periplasm. The mature oospores had a scalloped, electron opaque, epispore wall layer. These observations will be discussed in relation to the likely phylogenetic position of the Myzocytiopsidales within the oomycetes.

Relative fibrolytic activities of anaerobic rumen fungi on untreated and sodium hydroxide treated barley straw in in vitro culture.

The fibrolytic activities of rumen fungi were studied in terms of dry matter loss, plant cell wall degradation and enzyme (cellulase and xylanase) activities, when grown in vitro on either untreated or sodium hydroxide treated stems of barley straw over a 12 day period. Changes in fungal growth, development and overall biomass were followed using chitin assay and scanning electron microscopy. Treatment with sodium hydroxide resulted in a decrease in the NDF content together with the disruption of cuticle and the loosening and separation of the plant cells within the straw fragments. The enzyme activities of the anaerobic fungi have a high positive correlation (R(2)=0.99) with their biomass concentration assessed by chitin assay indicating that chitin is a valuable index for the estimation of the fungal biomass in vitro. The anaerobic fungi produced very extensive rhizoidal systems in these in vitro cultures. After incubation with rumen fungi, dry matter losses were, respectively, 35% and 38% for the untreated and treated straw samples and the overall fungal biomass, determined by chitin assay, was significantly higher in the treated samples. In vitro degradation of cellulose and hemicellulose was also higher in the treated than that of untreated cultures. Although, comparatively, xylanase activity was higher than that of cellulase, the cellulose fraction of the straw was degraded more than hemicellulose in both treated and untreated straw.

Methods for the isolation, culture and assessment of the status of anaerobic rumen chytrids in both in vitro and in vivo systems.

Anaerobic fungi were isolated from both the rumen and faeces of nine sheep and a cow. A reliable and simple method for the isolation of anaerobic fungi using 24 h rumen incubated milled straw as the inoculum source was developed. We also evaluate the use of chitin measurements as an assay of rumen fungal biomass. Chitin levels were determined from various sample sources (milled barley straw used as the fungal culture substrate in vitro; plant particulate digests from the rumen (PLP) and centrifuged strained rumen fluid (CSRF) using both HPLC and colorimetric methods. Both methods were highly correlated and consequently the simpler colorimetric method was adopted for subsequent studies. There was also a high degree of correlation between anaerobic fungal cellulase activities with the assayed chitin content of milled barley straw cultures over 12 d of an in vitro experiment. The colorimetric chitin assay protocol was then used to assess the diurnal variation and abundance of rumen fungi in in vivo assays. We assessed the distribution of chitin (mg g(-1) dry matter) in various fractions of the strained rumen fluid (SRF) and PLP samples from the rumen of sheep. Chitin was detected in all fractions of strained rumen fluid but the main source of chitin in the samples may be attributed to the fungal biomass. We did not detect any significant differences in chitin levels over a 24 h sampling period. Finally, an SEM study on subsamples of milled straw and plant particulate matter used in the chitin assays, revealed that the pattern of the fungal development on substrate material differs from the culture medium to the rumen.

Distribution and estimation of anaerobic zoosporic fungi along the digestive tracts of sheep.

The status of anaerobic zoosporic (Chytridiomycota) fungi along the entire digestive tract of sheep was assessed both analytically and microscopically. Digest samples were taken from different segments of the digestive tracts of three newly killed sheep that previously had been used in experimental dietary studies. These digest samples were tested for the presence of rumen fungi by assessing the recovery of live fungi from the samples, direct observation of digested plant remains under the scanning electron microscope (SEM), and using a chitin assay as an estimation of fungal biomass. Live anaerobic fungi were recovered from the abomasum, small and large intestine, caecum and faeces of sheep, but not from the digest samples of rumen and omasum. However, SEM examination of the samples confirmed the presence of fungal structures from all of these organs. In the large intestine and caecum samples the observed sporangial structures were rounded and showed conspicuous surface pitting. Results of the chitin assay indicated that the anaerobic fungi might account for up to 20% of the total microbial biomass in the rumen of sheep. The results of this study support the view that anaerobic fungi may be present as a resistant stage in the lower reaches of the digestive tract.

Ultrastructural morphogenesis of dimorphic arcuate infection (gun) cells of Haptoglossa erumpens an obligate parasite of Bunonema nematodes.

Haptoglossa is a genus of biflagellate organisms that has been placed in the oomycetes and is characterised by producing unique infective gun cells, which usually infect by physically rupturing the nematode cuticle. Haptoglossa erumpens is a parasite of Bunonema nematodes that produces arcuate infection cells and aplanospores that are discharged following the swelling and rupture of the thallus wall and distended host cuticle. Recent isolations of H. erumpens have revealed that the germinating aplanospores develop into two similar-sized but morphologically distinct infection cells. The uni-nucleate, convexly arcuate, gun cells were observed to fire in response to host nematodes, producing a cylindrical sporidium inside the host body. These gun cells had an apical missile chamber containing a needle with a unique arrangement of investing cones. Unlike previously described gun cells, the tube tail did not wind around the nucleus but continued into the basal vacuole where it terminated. The second type of infection cell was a concavely arcuate, bi-nucleate, cell that had an unusually large and elongate annulus component in the missile chamber. These modified bi-nucleate gun cells were never observed to fire in response to contact with Bunomena nematodes. The patterns of morphological and structural variations in these infection structures in this genus are reviewed in the light of these findings.