Electron microscopy of the rodlet layer of Neurospora crassa conidia.

Neurospora crassa macroconidia possess a regularly arranged layer of small fibers (rodlets) near the spore surface. The structure and location of this layer were studied by making surface replicas, by negative staining, by freeze-fracturing and deep-etching, and by thin sectioning. When conidia were shaken vigorously in water, the layer fragmented and became separated from the surface in sheets. Negative staining of such sheets showed that the individual rodlets have a hollow central core. When conidia were shaken gently in water or fixative, large fragments of the rodlet layer often remained on the conidial surface. The fragments tended to fold back on each other such that multiple layers were sometimes seen in thin sections. It is concluded that in dry conidia the rodlets are located on the extreme outside of the spore where they form a monolayer with only occasional regions of overlap.     

Characteristics of Streptomyces coelicolor A3(2) aerial spore rodlet mosaic

Cytochemical analysis of Streptomyces coelicolor (A3(2) indicated that the aerial growth rodlet mosaic is a polysaccharide. Statistical analysis of frequency distributions of individual rodlet lengths from control and ether-reoriented spore mosaics indicated that the rodlet fibrillar image is the result of individual particulates, rather than evaginations in a continuous sheet of material. A model of the mature sport envelope was developed from freeze-etch-replicated, thin-sectioned, and critical point dried S. coelicolor A3(2) mature spores. The rodlet mosaic was situated between the outer spore wall and an external granuloma matrix. Mixture spore envelope layers from the inner surface to the external surface are plasma membrane, inner spore wall, outer spore wall, rodlet mosaic, an undefined granular matrix, and the sheath. The granular matrix had an uneven thickness and much of the matrix was frequently absent from the interspore spaces of mature spore chains. Streptomyces coelicolor A3(2) mosaic rodlets were isolated by acetic acid refluxing, then ethanol precipitation. Complete acid hydrolysis of rodlets released on sugar which cochromatographed with D-glucosamine-HCl and released acetic acid at 139% of the expected level. Cell associated rodlet mosaics and isolated mosaic rodlets were hydrolyzed with chitinase. Infrared spectra of isolated rodlets were similar to crab chitin spectra.     

ULTRASTRUCTURE OF THE PSEUDOCAPILLITIUM AND SPORES OF THE MYXOMYCETE LYCOGALA EPIDENDRUM. LICEALES

The pseudocapillitium and spores of L. epidendrum were studied by transmission (TEM) and scanning electron microscopy (SEM). The SEM reveals that the pseudocapillitial surface is covered by bands of “wartlike” processes that alternate with non-ornamented regions. Otherwise, the pseudocapillitium is a hollow structure composed of three regions. The outer region is thin, electron dense and continuous with many irregular processes. Internal to this area is an amorphous region containing scattered electron dense material. The innermost region of the pseudocapillitium is thin, inconspicuous and usually electron dense. L. epidendrum possesses spores that are covered by a surface reticulum consisting of polygonal areas which are continuous with the outermost spore layer. The outer spore layer is thin and electron dense. The inner spore layer is an electron transparent region that contains granular or fibrillar components. Sections of spores showed a dense cytoplasm possessing most of the usual organelles along with microtubules and microbodies.     

Spore ornamentation of Haplosporidium nelsoni and Haplosporidium costale (Haplosporidia), and incongruence of molecular phylogeny and spore ornamentation in the Haplosporidia

Spore ornamentation of Haplosporidium nelsoni and Haplosporidium costale was determined by scanning electron microscopy. For H. nelsoni, the spore surface was covered with individual ribbons that were tightly bound together and occurred as a single sheet. In some spores, this layer was overlaid with a network of branching fibers, about 0.05 microm in diameter, which often was dislodged from the spore at the aboral pole. For H. costale, ornamentation consisted of a sparse network of branching fibers on the spore surface. Molecular phylogenetic analysis of the phylum Haplosporidia revealed that Urosporidium, Bonamia, and Minchinia were monophyletic but that Haplosporidium was paraphyletic. All species of Minchinia have ornamentation composed of epispore cytoplasm, supporting the monophyly of this genus. The presence of spores with a hinged operculum and spore wall-derived ornamentation in Bonamia perspora confounds the distinction between Bonamia and Haplosporidium. Species with ornamentation composed of outer spore wall material and attached to the spore wall do not form a monophyletic group in the molecular phylogenetic analysis. These results suggest that the widely accepted practice of assigning all species with spore wall-derived ornamentation to Haplospordium cannot be supported and that additional genera are needed in which to place some species presently assigned to Haplosporidium.     

Direct Probing of the Surface Ultrastructure and Molecular Interactions of Dormant and Germinating Spores of Phanerochaete chrysosporium

Atomic force microscopy (AFM) has been used to probe, under physiological conditions, the surface ultrastructure and molecular interactions of spores of the filamentous fungus Phanerochaete chrysosporium. High-resolution images revealed that the surface of dormant spores was uniformly covered with rodlets having a periodicity of 10 +/- 1 nm, which is in agreement with earlier freeze-etching measurements. In contrast, germinating spores had a very smooth surface partially covered with rough granular structures. Force-distance curve measurements demonstrated that the changes in spore surface ultrastructure during germination are correlated with profound modifications of molecular interactions: while dormant spores showed no adhesion with the AFM probe, germinating spores exhibited strong adhesion forces, of 9 +/- 2 nN magnitude. These forces are attributed to polysaccharide binding and suggested to be responsible for spore aggregation. This study represents the first direct characterization of the surface ultrastructure and molecular interactions of living fungal spores at the nanometer scale and offers new prospects for mapping microbial cell surface properties under native conditions.     

The fission yeast spore is coated by a proteinaceous surface layer comprising mainly Isp3

The spore is a dormant cell that is resistant to various environmental stresses. As compared with the vegetative cell wall, the spore wall has a more extensive structure that confers resistance on spores. In the fission yeast Schizosaccharomyces pombe, the polysaccharides glucan and chitosan are major components of the spore wall; however, the structure of the spore surface remains unknown. We identify the spore coat protein Isp3/Meu4. The isp3 disruptant is viable and executes meiotic nuclear divisions as efficiently as the wild type, but isp3∆ spores show decreased tolerance to heat, digestive enzymes, and ethanol. Electron microscopy shows that an electron-dense layer is formed at the outermost region of the wild-type spore wall. This layer is not observed in isp3∆ spores. Furthermore, Isp3 is abundantly detected in this layer by immunoelectron microscopy. Thus Isp3 constitutes the spore coat, thereby conferring resistance to various environmental stresses.     

狗脊孢壁发育超微结构的研究

鳞毛蕨型孢子类型众多,初步研究表明形态相似的孢子类型其孢壁发育特征存在差异,因此有必要对各代表类群的孢壁发育进行深入地研究。该文利用透射电镜对乌毛蕨科(Blechnaceae)狗脊(Woodwardia japonica)孢壁结构和发育的超微结构进行研究。结果表明:(1)狗脊孢子囊的结构由外向内分别为孢子囊壁细胞、两层绒毡层细胞和孢子母细胞;(2)狗脊孢子具乌毛蕨型(Blechnoid type)外壁,表面光滑,由两层构成,裂缝区域具辐射状的槽;(3)周壁属于空心型(cavity type),由四层构成,从内向外分别为P1、P2、P3和P4层,前三层叠合在一起,层间有不同程度的空隙,P4层与前三层之间具有明显而连续的空腔,并隆起形成片状褶皱纹饰;(4)有小球体和小杆共同参与孢子周壁的形成,周壁部分或全部来源于孢子囊壁细胞。综上所述,狗脊孢子与同属于鳞毛蕨型的贯众(Cyrtomium fortunei)和朝鲜介蕨(Dryoathyrium coreanum)孢壁的发育在周壁结构、周壁各层的发育顺序、周壁来源和参与成壁的特征物质等方面存在差异。该研究有利于进一步理解蕨类植物孢壁所蕴含的分类和演化上的科学意义和价值。     

Surface Rodlets of Tilletia indica Teliospores

Tilletia indica teliospores were studied by use of thin sections and freeze-etch replicas. Surfaces of these spores have rodlet patterns which differ from those previously reported for spores of other fungi. The rodlets on T. indica teliospores average 240 nm in length and are not grouped into fascicles.     

A Screen for Spore Wall Permeability Mutants Identifies a Secreted Protease Required for Proper Spore Wall Assembly

The ascospores of Saccharomyces cerevisiae are surrounded by a complex wall that protects the spores from environmental stresses. The outermost layer of the spore wall is composed of a polymer that contains the cross-linked amino acid dityrosine. This dityrosine layer is important for stress resistance of the spore. This work reports that the dityrosine layer acts as a barrier blocking the diffusion of soluble proteins out of the spore wall into the cytoplasm of the ascus. Diffusion of a fluorescent protein out of the spore wall was used as an assay to screen for mutants affecting spore wall permeability. One of the genes identified in this screen, OSW3 (RRT12/YCR045c), encodes a subtilisin-family protease localized to the spore wall. Mutation of the active site serine of Osw3 results in spores with permeable walls, indicating that the catalytic activity of Osw3 is necessary for proper construction of the dityrosine layer. These results indicate that dityrosine promotes stress resistance by acting as a protective shell around the spore. OSW3 and other OSW genes identified in this screen are strong candidates to encode enzymes involved in assembly of this protective dityrosine coat.     

Cytochemical study of the yeast and mycelial cell walls ofParacoccidioides brasiliensis

Cell wall structure and macromolecular organization of the various growth forms ofParacoccidioides brasiliensis were investigated using chemical, enzymatic, and cytochemical methods. The wall of yeast-like cells was not sensitive to periodic acid and β(1 → 3) glucanase treatments, but bound calcofluor suggesting that it is composed of an α(1 → 3) glucan and of chitin. The fibrillar outer layer of bud cell initials and of abscision areas, were characterized by the presence of a strongly periodic acid- and concanavalin A-reactive substance that was sensitive to the lytic action of protease, presumably composed of mannan and proteins. The outermost layer of the mycelium was sensitive to protease, and periodic acid and peanut agglutinin positive indicating that it was composed of galactomannan and protein. The inner layer is mainly composed of a β(1 → 3), (1 → 6) glucan (sensitive to snail enzyme but not to an exo β(1 → 3) glucanase), and chitin. Septa were brightly fluorescent with calcofluor. Our results are compared with models previously proposed by other authors.     

ULTRASTRUCTURE OF DISPERSED AND IN SITU SPECIMENS OF THE DEVONIAN SPORE RHABDOSPORITES LANGII: EVIDENCE FOR THE EVOLUTIONARY RELATIONSHIPS OF PROGYMNOSPERMS

Abstract: The spore Rhabdosporites (Triletes) langii (Eisenack) Richardson, 1960 is abundant and well preserved in Middle Devonian (Eifelian) ‘Middle Old Red Sandstone’ deposits from the Orcadian Basin, Scotland. Here it occurs as dispersed individual spores and in situ in isolated sporangia. This paper reports on a detailed light microscope (LM), scanning electron microscope (SEM) and transmission electron microscope (TEM) analysis of both dispersed and in situ spores. The dispersed spores are pseudosaccate with a thick walled inner body enclosed within an outer layer that was originally attached only over the proximal face. The inner body has lamellate/laminate ultrastructure consisting of fine lamellae that are continuous around the spore and parallel stacked. Towards the outer part of the inner body these group to form thicker laminate structures that are also continuous and parallel stacked. The outer layer has spongy ultrastructure. In situ spores preserved in the isolated sporangia are identical to the dispersed forms in terms of morphology, gross structure and wall ultrastructure. The sporangium wall is two‐layered. A thick coalified outer layer is cellular and represents the main sporangium wall. This layer is readily lost if oxidation is applied during processing. A thin inner layer is interpreted as a peritapetal membrane. This layer survives oxidation as a tightly adherent membranous covering of the spore mass. Ultrastructurally it consists of three layers, with the innermost layer composed of material similar to that comprising the outer layer of the spores. Based on the new LM, SEM and TEM information, consideration is given to spore wall formation. The inner body of the spores is interpreted as developing by centripetal accumulation of lamellae at the plasma membrane. The outer layer is interpreted as forming by accretion of sporopollenin units derived from a tapetum. The inner layer of the sporangium wall is considered to represent a peritapetal membrane formed from the remnants of this tapetum. The spore R. langii derives from aneurophytalean progymnosperms. In light of the new evidence on spore/sporangium characters, and hypotheses of spore wall development based on interpretation of these, the evolutionary relationships of the progymnosperms are considered in terms of their origins and relationship to the seed plants. It is concluded that there is a smooth evolutionary transition between Apiculiretusispora‐type spores of certain basal euphyllophytes, Rhabdosporites‐type spores of aneurophytalean progymnosperms and Geminospora‐/Contagisporites‐type spores of heterosporous archaeopteridalean progymnosperms. Prepollen of basal seed plants (hydrasperman, medullosan and callistophytalean pteridosperms) are easily derived from the spores of either homosporous or heterosporous progymnosperms. The proposed evolutionary transition was sequential with increasing complexity of the spore/pollen wall probably reflecting increasing sophistication of reproductive strategy. The pollen wall of crown group seed plants appears to incorporate a completely new developmental mechanism: tectum and infratectum initiation within a glycocalyx‐like Microspore Surface Coat. It is unclear when this feature evolved, but it appears likely that it was not present in the most basal stem group seed plants.     

Two hydrophobins are involved in fungal spore coat rodlet layer assembly and each play distinct roles in surface interactions, development and pathogenesis in the entomopathogenic fungus, Beauveria bassiana

The entomogenous filamentous fungus, Beauveria bassiana expresses two hydrophobin genes, hyd1 and hyd2, hypothesized to be involved in cell surface hydrophobicity, adhesion, virulence, and to constitute the protective spore coat structure known as the rodlet layer. Targeted gene inactivation of hyd1 resulted in seemingly 'bald' conidia that contained significantly altered surface fascicles or bundles. These cells displayed decreased spore hydrophobicity, loss of water mediated dispersal, changes in surface carbohydrate epitopes and β-1,3-glucan distribution, lowered virulence in insect bioassays, but no effect on adhesion. In contrast, Δhyd2 mutants retained distorted surface bundles, but truncated/incomplete rodlets could be seen within the bundles. Δhyd2 conidia displayed both decreased cell surface hydrophobicity and adhesion, but the mutant was unaffected in virulence. The double Δhyd1Δhyd2 mutant was distinct from the single mutants, lacking both bundles and rodlets, and displaying additively decreased cell surface hydrophobicity, reduced cell attachment and lowered virulence than the Δhyd1 mutant. Epitope tagged constructs of the proteins were used to examine the expression and distribution of the proteins and to demonstrate the continued presence of Hyd2 in the Δhyd1 strain and vice versa. The implications of our results with respect to fascicle and rodlet assembly on the spore surface are discussed.     

Characterization of Laurencia arbuscula spore mucilage and cell walls with stains and FITC-labelled lectins

In most red algae, spores are liberated without a cell wall, within a sheath of mucilage that is responsible for its primary attachment. Utilizing fluorescent-labelled lectins, we identified carbohydrate residues and their location in the mucilage and cell walls of spores of Laurencia arbuscula. Cell wall formation and mucilage composition were studied with Calcofluor, Toluidine Blue (AT-O), Alcian Blue (AB) and periodic acid-Schiff (PAS). In the mucilage, we identified α-d-mannose, α-d-glucose, N-acetyl-glucosamine, N-acetyl-galactosamine and β-d-galactose. All sugar residues were found in the cell wall, in the spore body rather than in the rhizoid, which suggests that the residues may be related to initial substrate adhesion. A cell wall is produced soon after the spore's attachment, beginning with a deposition of cellulose around the spore, as indicated by Calcofluor. A polarization of the cell wall triggers the process of germination. The cell-wall matrix was positive to AB and metachromatic to AT-O, indicating acidic polysaccharides, while neutral polysaccharides were positive to PAS.     

Spore morphology and ultrastructure of Cyathea (Cyatheaceae,Pteridophyta) species from southern South America

Spore sculpture and wall structure of eight Cyathea (Cyatheaceae) species from southern South America were studied using light microscopy (LM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM) techniques. Two layers, i.e. an inner and an outer layer, were observed in the perispore. The inner layer has two strata: the inner stratum is attached to the exospore and composed of rodlets tangentially oriented to the spore surface and randomly intermixed; the outer stratum consists of a three-dimensional network of rodlets with either free or fused distal edges forming spinules. The outer layer is thin, darkly contrasted and covers the rodlets. In most cases, the exospore has two layers and a pitted surface. In Cyathea atrovirens, the exospore surface is smooth, while in C. delgadii and C. myriotricha it is verrucate. The homogeneity of perispore features within the genus Cyathea is evident, while exospore features are heterogeneous. The exospore has different kinds of surface-structures that are of potential interest for assessing evolutionary trends within the group.     

Colony development in Streptomyces carpinensis: a streptomycete with substrate mycelium spores

In this paper we present an ultrastructural study of spore formation in aerial vs. substrate mycelia of Streptomyces carpinensis. Both mycelia initiated spore formation at nearly the same time of colony development but exhibited different patterns of spatial localization of sporulation: spore formation took place throughout the aerial mycelium whereas in the substrate mycelium was confined to a narrow zone at the bottom of the colony. The ultrastructural changes leading to spore formation, however, were quite similar in both mycelia, differing only with respect to the outer components of the sporal wall. Spores formed in the aerial mycelium were covered by a thin sheath whereas the spores formed in the substrate mycelium were covered by an amorphous electron-dense material.     

A mechanism of propagule release from unilocular reproductive structures in brown algae

Summary The structure of unilocular sporangia inP. littoralis was investigated along with several other species of brown algae in order to study the mechanism by which propagules are released from unilocular reproductive structures. Unilocular sporangia inP. littoralis are composed of a spherical cell wall of two distinct layers and contain a number of zoospores. The mass of spores is surrounded and permeated by mucilaginous carbohydrates. It is suggested that the production of these carbohydrates generates the necessary pressure to weaken the sporangial wall. In addition, ultrastructural observations indicate that further weakening seems to occur due to digestion of the inner wall layer. Walls of sporangia were mechanically broken just prior to normal spore release in order to investigate whether internal pressure exists, and if it can effect spore discharge. Results show that an internal pressure does exist prior to normal spore discharge and that this pressure is not generated by turgor pressure of the spores themselves or by a semi-permeable wall osmoticum system. The discharge of spores seems to occur when the carbohydrate around the spores swells. The adsorption of water when plants are immersed by the incoming tide thus seems a likely mechanism of spore discharge. The similarities of unilocular reproductive structures and spore release in several brown algal species suggests common mechanisms of propagule discharge for members of thePhaeophyta.     

Spore Fine Structure in Clostridium cochlearium

The fine structure of Clostridium cochlearium was examined by use of thin sections, negative stains, and carbon replicas. Particular attention was given to details of the sporulation process and to fine structure of the spores. Spore coat formation was well advanced before the first evidence of cortex formation was noted. Three distinct spore coats were detected, the outermost of which was composed of seven layers. In addition, the spores possessed tubular appendages of variable length attached to one end of the spore. These differed in a number of respects from those described for other clostridia.     

SPORE WALL ULTRASTRUCTURE OF PROTOSALVINIA

Protosalvinia is an enigmatic fossil which has been historically assigned to several major taxonomic groups. Stratigraphically, the fossil occurs in a narrow range of Upper Devonian sediments. Tetrads of spores are associated with shallow depressions on the surface of approximately 5% of the specimens collected from the Ohio Shale in Columbus, OH. Spores are approximately 250 μm in diameter and have a spore wall which is composed of at least two distinct layers. The outer layer is coarsely laminated in regions where adjacent spores are in contact. Individual laminar units are thinnest toward the inside and gradually thicker toward the surface of the spore. In non-contact regions, the outer layer is composed of globular units. The inner layer of the wall has little discernable structure except for the presence of a distinct suture beneath the proximal trilete mark. This firmly establishes the meiotic nature of these structures. Comparison with eggs and tetraspores of several extant phaeophycean algae shows little similarity.     

Microscopic observations of germination and septum formation in pycnidiospores of Botryodiplodia theobromae

A population of aseptate pycnidiospores of the fungus Botryodiplodia theobromae can be induced to germinate or to form septa delimiting two cells; this developmental process is dependent upon nutritional and environmental factors. Transmission electron microscope investigations indicate that during germination of the aseptate spore, a new inner wall layer is synthesized de novo at the site of germ tube emergence. Formation of the septum also involves the de novo synthesis of an inner wall layer which comprises the majority of the septum and completely surrounds the spore. The wall of the germ tube emerging from the septate spore is a direct extension of this inner layer deposited during the formation of the septum. Although the early stages of spore germination may involve localized enzymatic degradation of the internal layers of the spore wall, transmission and scanning electron micrographs of germinating spores show that the outer wall layers are physically fractured by the emerging germ tube. It is suggested that spore germination and septum formation are initially similar processes regarding cell wall genesis but that some mechanism responsive to environmental and nutritional conditions determines the course of development.     

基于孢子粘附功能的大型海藻生态修复技术研究

为实现大型海藻人工移植高效、节约和可持续性,研究基于大型海藻孢子的粘附特性,于2020年4月,初选了几种食品添加剂作为粘附剂,与铜藻(Sargassum horneri)孢子混合后,通过室内外涂覆实验观察孢子附着、萌发和生长情况,筛选出最优的粘附剂,并通过铜藻孢子低温保存实验研究解决延长海藻人工移植周期问题.结果表明:...