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.     

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.     

Ingestion of the dinoflagellate, Pfiesteria piscicida, by the calanoid copepod, Acartia tonsa

The dinoflagellate, Pfiesteria piscicida, can form harmful algal blooms in estuarine environments. The dominant copepod species usually found in these waters is Acartia tonsa. We tested the ability of A. tonsa to graze the non-toxic zoospore stage of P. piscicida and thus serve as a potential biological control of blooms of this algal species. A. tonsa grazed the non-toxic zoospore stages of both a non-inducible P. piscicida strain (FDEPMDR23) and a potentially toxic strain (Tox-B101156) at approximately equal rates. Ingestion of P. piscicida increased with cell concentration and exhibited a saturated feeding response. Both the maximum number of cells ingested (I_max) and the slope of the ingestion curve (α) of A. tonsa feeding on P. piscicida were comparable to these ingestion parameters for A. tonsa fed similar-sized phytoplankton and protozoan species. When these laboratory ingestion rates were combined with abundance estimates of A. tonsa from the Pocomoke Estuary and Chesapeake Bay, we found that significant grazing control of the non-toxic zoospore stage of P. piscicida by A. tonsa would only occur at high copepod abundances (>10 copepods L⁻¹). We conclude that under most in situ conditions the potential biological control of blooms of P. piscicida is exerted by microzooplankton grazers. However, in the less saline portions of estuaries where maximum concentrations of copepods often occur with low abundances of microzooplankton, copepod grazing coefficients can be similar to the growth rates of P. piscicida.     

Actin filaments predominate in morphogenic cell stages, whereas plaques predominate in non-morphogenic cell stages in Peronosporomycetes

We investigated the structural distribution of both types of actin arrays, filaments and plaques, in a soil-borne phytopathogenic peronosporomycete (oomycete), Aphanomyces cochlioides, under standardized host-free bioassays. The phenomenon was monitored during progression through all the asexual developmental processes of the organism. It was noted that the filamentous-form of actin was predominant during the morphogenic (morphologically active) stages of development. Conversely, during non-morphogenic (morphologically quiescent) stages, plaques dominated. From these analyses, we proposed a criterion that predominance of an actin form relates to, and precedes the morphological behaviour of a cellular stage in Peronosporomycetes. A decrease in the quantity of plaques in the encysted zoospore (non-morphogenic stage) during its developmental progression into morphogenic stages, both in germination and regeneration processes, asserted the notion that plaques function as the organization centres and are related to the reorganization of cell structure and the transition of the cell into a new stage. Furthermore, polymerization of filamentous-form during emergence stages in zoospore regeneration process revealed that filaments render motility to a developing zoospore. This unprecedented function of filaments in the developing zoospores was demonstrated using nicotinamide (0.8 × 10⁻⁶ m), which did not cause actin disruption, but could induce zoospore encystment, and its further replacement with water triggered the zoospore emergence process. Additionally, by using latrunculin B, an actin polymerization inhibitor, we also demonstrated the functional necessity of actin during various developmental processes in Aphanomyces.     

Differences in the virulence of Heterorhabditis bacteriophora and Steinernema scarabaei to three white grub species: The relative contribution of the nematodes and their symbiotic bacteria

Because susceptibility of white grub species to entomopathogenic nematodes differs, we compared the virulence of Photorhabdus temperata and Xenorhabdus koppenhoeferi, the symbiotic bacteria of the nematodes Heterorhabditis bacteriophora and Steinernema scarabaei, respectively, to the three white grub species, Popillia japonica, Rhizotrogus majalis, and Cyclocephala borealis. Both bacteria were pathogenic to all three grub species even at 2 cells/grub. However, the median lethal dose at 48 h post injection and median lethal time at 20 cells/grub showed that P. temperata was more virulent than X. koppenhoeferi to C. borealis. Although H. bacteriophora is less pathogenic than S. scarabaei to R. majalis and P. japonica, their symbiotic bacteria did not differ in virulence against these two grub species, and they also showed similar growth patterns both in vitro and inside R. majalis larvae at 20 °C. We then tested the pathogenicity of oral- and intrahemocoel-introduced H. bacteriophora to R. majalis to determine whether nematodes are able to successfully vector the bacteria into the hemolymph. Hemocoel injected H. bacteriophora was pathogenic to R. majalis indicating successful bacterial release, but orally introduced H. bacteriophora were not. Dissection of grubs confirmed that the orally introduced H. bacteriophora were unable to penetrate into the hemolymph through the gut wall. We conclude that the low susceptibility of R. majalis to H. bacteriophora is not due to the symbiotic bacteria but rather to the nematode’s poor ability to penetrate through the gut wall and the cuticle to vector the bacteria into the hemolymph.     

中国产石韦属植物孢子形态特征及分类学意义

利用光镜和扫描电镜,对石韦属(Pyrrosia)19种植物的孢子纹饰进行了研究。结果表明:19种石韦属孢子都为黄色,形态均为肾形,两侧对称,单裂缝,说明该属植物是一个自然类群。表面纹饰类型有3种,即瘤状、瘤状—疣状和瘤状—网脊状。孢子表面纹饰特征性状稳定,在种间存在较大差异。该研究结果可以为形态近似种的分类提供参考依据,同时也为石韦属属下分类系统的建立提供了重要证据。     

Characterization of zoospore and cyst surface structure in saprophytic and fish pathogenicSaprolegnia species (oomycete fungal protists)

Summary The importance of the surface structure and chemistry in zoospores and cysts of oomycetes is briefly reviewed and the organelle systems associated with encystment described. The surface structure and chemistry of primary and secondary zoospores and cysts ofSaprolegnia diclina (a representative saprophytic species) andS. parasitica (a representative salmonid fish pathogen) were explored using the lectins concanavilin A (Con A) and wheat germ agglutinin (WGA) and monoclonal antibodies (MAbs) raised against a mixed zoospore and cyst suspension ofS. parasitica. The binding of lectins and antibodies to spores was determined using immunofluorescence microscopy with fluorescein isothiocyanate-labelled probes and with electron microscopy with gold-conjugated probes applied to spore suspensions post-fixation. In both species Con A, which is specific for glucose and mannose sugars, bound to both the surface of primary and secondary zoospores (the surface glycocalyx) and their cyst coats and readily induced zoospore encystment. The binding to the cysts appeared to be mainly associated with the matrix material released from the primary and secondary encystment vesicles and which appeared to diminish with time. No binding to germ tube walls was observed with this lectin. The MAb labelling showed a generally similar binding pattern to the primary and secondary cysts to that observed with Con A, although the binding to zoospores was more variable. Primary zoospores bound the antibodies but secondary zoospores appeared less reactive. It is suggested that the MAbs share a common epitope with one or more of the Con A-binding components. In both species WGA, which is specific for amongst other things the sugar N-acetyl glucosamine, bound to localised apical patches on the primary zoospores. This lectin also binds to the ventral groove region of secondary zoospores ofS. diclina, which were induced to encyst by this lectin. In contrast secondary zoospores ofS. parasitica were not induced to encyst by the addition of WGA and showed a patchy dorsal binding with this lectin. WGA also binds to both the inner wall of discharged primary cysts and the young germ tube walls of both species. These observations are discussed both in relation to other oomycete spores and to their possible functional and ecological significance.Abbreviations BSA bovine serum albumin - Con A Concanavalin A - DBA Dolichos biflorus agglutinin - ELISA enzyme-linked immunosorbent assay - EM electron microscope - EV encystment vesicles - FCS foetal calf serum - FITC Fluorescein isothiocyanate - FV peripheral fibrillar vesicles - G+F 0.2% glutaraldehyde and 2.0% formaldehyde primary fixative solution - 2G 2% glutaraldehyde primary fixative - LM light microscopy - MAbs monoclonal antibodies - LPV large peripheral vesicles - PBS phosphate buffered saline - PCV flattened peripheral cisternae - PEV primary encystment vesicle - PIPES piperazine-N,N1-bis(2-ethane sulfonic acid) - PNA Ricinus communis agglutinin - RAM-FITC/Au_10–20 Fluorescein isothiocyanate/gold (10 or 20 nm) labelled rabbit anti-mouse immunoglobulin - RCA Ricinus communis agglutinin - SEM scanning electron micrograph - SBA soybean agglutinin - SEV secondary encystment vesicles - TEM transmission electron micrograph - UEA I Ulex europaeus agglutinin - WGA wheat germ agglutinin     

A newHalophytophthora species,H. porrigovesica, from subtropical and tropical mangroves

A new oomycete was found from intertidal fallen leaves of mangroves in Japan and Thailand and is described here asHalophytophthora porrigovesica. This species is characterized by having an epapillate, ovate zoosporangium with a lens-shaped dehiscence plug-like material at the apex, and by forming an expanding long cylindrical vesicle prior to zoospore release. A key to 14 species and 2 varieties ofHalophytophthora including the new species is proposed. The subtropical (Iriomote is., Japan) strains and tropical (Thailand) strains were different in physiological properties and especially in the asexual reproduction. The subtropical strains showed a lower optimal temperature and wider range of suitable temperature and salinity for zoosporangium formation, whereas the tropical strains showed a higher optimal temperature and narrower range of temperature and salinity. These differences are explained as adaptations of the strains to the environmental conditions of their respective habitats. From the subtropical mangroves, six strains of the new species have been isolated only from submerged leaves ofSonneratia alba, while several strains have been isolated from tropical mangroves from the leaves of three species of mangrove trees,S. alba, Bruguiera gymnorrhiza andAvicennia alba. This indicates a change of taxon selectivity (host specificity) with the geographical distribution.     

Biological control of Tipula paludosa (Diptera: Nematocera) using entomopathogenic nematodes (Steinernema spp.) and Bacillus thuringiensis subsp. israelensis

Tipula paludosa (Diptera: Nematocera) is the major insect pest in grassland in Northwest Europe and has been accidentally introduced to North America. Oviposition occurs during late August and first instars hatch from September until mid-October. Laboratory and field trials were conducted to assess the control potential of entomopathogenic nematodes (EPN) (Steinernema carpocapsae and S. feltiae) and Bacillus thuringiensis subsp. israelensis (Bti) against T. paludosa and to investigate whether synergistic effects can be exploited by simultaneous application of nematodes and Bti. Results indicate that the early instars of the insect are most susceptible to nematodes and Bti. In the field the neonates prevail when temperatures tend to drop below 10 °C. S. carpocapsae, reaching >80% control, is more effective against young stages of T. paludosa than S. feltiae (<50%), but the potential of S. carpocapsae might be limited by temperatures below 12 °C. Mortality of T. paludosa caused by Bti was not affected by temperature even at 4 °C but the lethal time increased with decreasing temperatures. Synergistic effects of Bti and EPN against T. paludosa were observed in 3 out of 10 combinations in laboratory assays but not in a field trial. The potential of S. carpocapsae was demonstrated in field trials against early instars in October reaching an efficacy of >80% with 0.5 million nematodes m⁻² at soil temperatures ranging between 3 and 18 °C. Results with Bti were strongly influenced by the larval stage and concentration. Against early instars in autumn between 74 and 83% control was achieved with 13 kg ha⁻¹ Bti of 5,700 International Toxic Units (ITUs) and 20 kg ha⁻¹ of 3,000 ITUs. Applications in spring against third and fourth instars achieved between 0 and 32% reduction. The results indicate that application of Bti and nematodes will only be successful and economically feasible during the early instars and that the success of S. carpocapsae is dependent on temperatures >12 °C. Synergistic effects between S. carpocapsae and Bti require more detailed investigations in the field to determine maximal effect.     

下延叉蕨和芽胞叉蕨配子体发育的研究

利用光学显微镜详细观察了叉蕨属(Tectaria)下延叉蕨(Tectaria decurrens)和芽胞叉蕨(T.fauriei)的配子体发育过程,记录了配子体各发育阶段的特征。结果表明:(1)下延叉蕨和芽胞叉蕨的孢子均为单裂缝,具周壁,由周壁形成纹饰,孢子极面观椭圆形,赤道面观豆形或肾形。(2)孢子萌发方式为向心型。(3)原叶体发育方式为三叉蕨型。(4)成熟原叶体心脏形,两翼向斜上方扩展。(5)均具单细胞和多细胞毛状体,在丝状体或片状体阶段出现。研究认为,从配子体发育角度看,叉蕨属是较进化的陆生真蕨类;毛状体的类型、位置和出现时间等特征在叉蕨属种间存在差异,可作为该属种间分类的特征。     

Stromata, sporangiomata and chlamydosori of Phytophthora ramorum on inoculated Mediterranean woody plants

Three types of multihyphal structures, stromata, sporangiomata and chlamydosori, are described for the plant pathogen Phytophthora ramorum. Their morphology, morphogenesis and position on the host organ were observed by dissecting, compound and scanning electron microscopy. Stromata were consistently formed one to two weeks after zoospore inoculation of detached leaves and fruits of an assortment of Mediterranean sclerophyll shrubs. Stroma initials appeared subcuticularly or subepidermally and developed as small hyphal aggregates by repeated branching, budding, swelling and interweaving, eventually forming a prosenchyma. They always emerged through the adaxial side of the leaf by rupture of the overlying host tissue. Occasionally sporangia and chlamydosori (packed clusters of chlamydospores) were formed on the stromata. Sporangiomata bore short sporangiophores and clusters of 20–100 sporangia and resembled sporodochia of the mitosporic fungi. The biological significance of these multihyphal structures is discussed. Some epidemiological aspects were also studied: several understorey species of the holm oak (Quercus ilex) woodland were susceptible to in vitro infection with three isolates of P. ramorum originally collected from different ornamental hosts. The risk of spread to this ecosystem is evaluated.     

目标树经营初期对柏木人工林土壤线虫群落的影响

为研究目标树经营初期对柏木人工林土壤线虫群落的影响,对遂宁市安居区处于竞争生长阶段的柏木林进行3种不同密度目标树经营,共计样地12个(目标树密度为6株/667 m~2、9株/667 m~2、12株/667 m~2以及对照各3个)。在目标树经营1年后,采集春、夏、秋、冬4个季节样品共252个,采用改进的Baermann湿漏斗法分离土壤线虫,共鉴定出土壤线虫59科143属。其中Prionchulus为优势属,食细菌性线虫38属,食真菌性线虫12属,植食性线虫36属,杂食/捕食性线虫57属。土壤线虫生活史策略以c-p 4类群为主,占总数的44.33%,其中目标树密度为9株/667 m~2整个样地(9N)的c-p 4占比最大。目标树经营增加了土壤线虫的数量,目标树密度为9株/667 m~2的目标树单株(9C)线虫密度达到最大;目标树经营处理中均以杂食/捕食性线虫数量最多,其中9C杂食/捕食性线虫数量均高于其他处理;目标树经营提高了多样性指数(H′)、均匀度指数(J)、线虫营养多样性指数(TD)、线虫成熟度指数(MI),目标树经营改善了土壤线虫群落结构,其中目标树密度为9株/667 m~2样地的土壤线虫群落结构最优。研究结果表明,采用目标树经营有利于提升土壤线虫群落多样性,使柏木人工林生态系统向更稳定的方向发展,其中目标树密度为9株/667 m~2的效果最为显著。     

膜叶铁角蕨属的形态系统学研究

膜叶铁角蕨属隶属于铁角蕨科,全世界约有30种,我国分布有18种,是该属植物的分布中心之一。到目前为止,膜叶铁角蕨属的物种数目和物种分类还存在很大争议,一些物种的界限和定义还模糊不清,为了得到一个自然的膜叶铁角蕨属分类系统,还需要对膜叶铁角蕨属的物种分类做进一步研究。该文在前人研究的基础上,对膜叶铁角蕨属10种植物的形态特征,包括孢子形态特征、叶柄和根状茎上的鳞片形态特征、叶片形态、羽片形状以及叶脉特征等进行详细观察分析,探讨了各个形态特征间的关系以及膜叶铁角蕨属植物的物种分类。结果表明:膜叶铁角蕨属植物的叶片及羽片等形态特征存在很大区别,叶柄和根状茎上的鳞片以及孢子形态的种间差异虽然不大,但其在大小、形状、颜色等方面的细微差别仍可作为部分种类的鉴定依据。该研究结果为膜叶铁角蕨属植物的物种分类及进一步研究提供了重要依据。     

小麦赤霉病拮抗菌JB7的拮抗活性及鉴定

由禾谷镰刀菌(Fusarium graminearum, Fg)引起的赤霉病是限制小麦生产的主要病害之一。生物防治是一种高效且可持续的防治方法。【目的】从小麦种子内筛选具有抑制禾谷镰刀菌的菌株并对其生防潜力进行评估,为小麦赤霉病生防制剂的开发与利用提供菌种资源及理论支撑。【方法】采用平板对峙、孢子萌发法和无菌上清液抑菌试验筛选小麦种子内对禾谷镰刀菌具有拮抗活性的内生菌株;利用扫描电镜(scanning electron microscope, SEM)和共聚焦扫描电镜(confocal laser scanning microscope, CLSM)观察并分析无菌上清液对Fg的分生孢子形态、膜完整性以及胞内活性氧的影响;通过盆栽试验验证内生菌对小麦赤霉病的生防效果;应用二代Illumina HiSeq测序平台进行全基因组测序。【结果】从小麦种子中分离出一株高效抑制Fg生长的内生菌株JB7,其衰亡期无菌上清液对Fg孢子萌发抑制率高达85.23%。菌株JB7的无菌上清液使Fg孢子表面凹陷,破坏其细胞膜,造成核酸和蛋白质的渗漏,诱导Fg菌丝活性氧的累积,引起Fg菌丝可溶性蛋白和丙二醛含量的显著升高。该菌株具有分泌蛋白酶、纤维素酶、葡聚糖酶和产铁载体的能力。盆栽试验表明菌株JB7能显著降低小麦赤霉病的病情指数(P<0.05)。经全基因组学鉴定为甲基营养型芽孢杆菌(Bacillus methylotrophicus) JB7,该菌株基因组中含有12个抑菌功能的次级代谢产物合成基因簇。【结论】菌株JB7能抑制禾谷镰刀菌的生长,对小麦赤霉病有较强的防效,可作为生物防治小麦赤霉病的候选菌株。     

Detection and quantification of Entomophaga maimaiga resting spores in forest soil using real-time PCR

Environmental sampling to monitor entomopathogen titre in forest soil, a known reservoir of insect pathogens such as fungi and viruses, is important in the evaluation of conditions that could trigger epizootics and in the development of strategies for insect pest management. Molecular or PCR-based analysis of environmental samples provides a sensitive method for strain- or species-based detection, and real-time PCR, in particular, allows quantification of the organism of interest. In this study we developed a DNA extraction method and a real-time PCR assay for detection and quantification of Entomophaga maimaiga (Zygomycetes: Entomophthorales), a fungal pathogen of the gypsy moth, in the organic layer of forest soil. DNA from fungal resting spores (azygospores) in soil was extracted using a detergent and bead mill homogenization treatment followed by purification of the crude DNA extract using Sephadex–polyvinylpolypyrrolidone microcolumns. The purification step eliminated most of the environmental contaminants commonly co-extracted with genomic DNA from soil samples but detection assays still required the addition of bovine serum albumin to relieve PCR inhibition. The real-time PCR assay used primers and probe based on sequence analysis of the nuclear ribosomal ITS region of several E. maimaiga and two E. aulicae strains. Comparison of threshold cycle values from different soil samples spiked with E. maimaiga DNA showed that soil background DNA and remaining co-extracted contaminants are critical factors determining detection sensitivity. Based on our results from comparisons of resting spore titres among different forest soils, estimates were best for organic soils with comparatively high densities of resting spores.     

The explosive diversification of the diatom genus Chaetoceros across the Eocene/Oligocene and Oligocene/Miocene boundaries in the Norwegian Sea

Eocene to middle Miocene stratigraphic changes in species richness, abundance and valve size of Chaetoceros resting spores in the Norwegian Sea (DSDP Site 338) were investigated in order to understand past productivity and paleoenvironmental changes in upwelling regions. As a result, drastic resting spore events were recognized in a 6 myr interval across the Eocene/Oligocene boundary (EO Event), the Oligocene/Miocene boundary (OM Event) and in the early middle Miocene (emM Event). The EO Event was characterized by explosive diversification at both the morpho-generic and specific levels, an increase in abundance, and a decrease in valve size from the upper Eocene through the lowest Oligocene. The OM Event was defined by a two-fold increase in species richness. During the emM Event spore abundance decreased rapidly, and species richness and valve size decreased gradually. These changes may indicate changes in the nutrient supply, especially in upwelling regions. The increased species richness suggests a change from a stable water column with a constant nutrient supply in the Eocene to an unstable one with a sporadic nutrient supply by increased vertical mixing in the Oligocene, based on evaluation of the ecologic differences between dinoflagellate cysts and Chaetoceros resting spores. The role of main primary producer might have switched from dinoflagellates and/or nannoplankton in the Eocene to diatoms, especially Chaetoceros, in the Oligocene in the Norwegian Sea. Increased resting spore species richness during the OM Event may show that environmental changes such as global cooling and nutrient mixing led to a diversification of the spore producing genus Chaetoceros. The emM Event might have been affected by changes in paleoceanographic conditions, perhaps a decrease in nutrient supply. This study presents the first paleoceanographic analysis using not only the total resting spore abundance but also the abundances of individual species, and establishes the value of spore taxonomy and diatom analysis including spores.     

The ultrastructure of cell wall formation and of gamma particles during encystment of Allomyces macrogynus zoospores

Structural changes during cell wall formation by populations of semisynchronously germinating zoospores were studied in the water mold Allomyces macrogynus. Fluorescence microscopy using Calcofluor white ST (which binds to -1,4-linked glycans) demonstrated that Calcofluor-specific material was deposited around most cells between 2–10 min after the induction of encystment (beginning when a wall-less zoospore retracts its flagellum and rounds up). During the first 15 min of encystment there was a progressive increase in fluorescence intensity. Ultrastructural analysis of encysting cells showed that within 2–10 min after the induction of encystment small vesicles 35–70 nm diameter were present near the spore surface, and some were in the process of fusing with the plasma membrane. The fusion of vesicles with the zoospore membrane was concomitant with the appearance of electron-opaque fibrillar material outside the plasma membrane. Vesicles similar to those near the spore surface were found within the gamma () particles of encysting cells. These particles had a crystalline inclusion within the electron-opaque matrix. During the period of initial cyst cell wall formation numerous vesicles appeared to arise at the crystal-matrix interface. Approximately 15–20 min was required for the cell wall to be formed. We suggest that the initial response of the zoospore to induction of encystment is the formation of a cell wall mediated by the fusion of cytoplasmic vesicles with the plasma membrane.Non-Standard Abbreviations GlcNac N-Acetylglucosamine - DS sterile dilute salts solution - PYG peptone-yeast extract-glucose broth     

Prosthecium species with Stegonsporium anamorphs on Acer

Data from microscopic morphology, single-spore cultures, and DNA analyses of teleomorphs and anamorphs support the recognition of five species of Prosthecium with Stegonsporium anamorphs on Acer: P. acerinum sp. nov., the teleomorph of S. acerinum; P. acerophilum comb. nov., formerly known as Dictyoporthe acerophila; P. galeatum comb. nov., originally described as Massaria galeata; P. opalus sp. nov.; and P. pyriforme sp. nov., the teleomorph of S. pyriforme s. str. The morphology of both type specimens and freshly collected material was investigated. The teleomorphs have brown ellipsoidal ascospores with five distosepta and often a longitudinal distoseptum. The anamorphs of all species described here belong to Stegonsporium; their connection to the Prosthecium teleomorphs was demonstrated by morphology and DNA sequences of single spore cultures derived from both ascospores and conidia. The anamorphs and teleomorphs of all five Prosthecium species are described and illustrated by LM images, and a key to these species is provided. As perceived from this work, S. pyriforme is restricted to Europe and does not occur in North America, whereas S. acerinum is restricted to North America, not found in Europe. The host associations given in the literature are revised and evidence is provided that only A. opalus, A. pseudoplatanus, and A. saccharum are confirmed hosts of Prosthecium with Stegonsporium anamorphs. Molecular phylogenetic analyses of tef1, ITS rDNA, and partial nuLSU rDNA sequences confirm that the species with Stegonsporium anamorphs are closely related to P. ellipsosporum, the generic type species. Stilbospora macrosperma is confirmed as the anamorph of P. ellipsosporum by DNA data of single spore isolates obtained from both ascospores and conidia.