Evidence for the importance of enzymatic digestion of epidermal walls during subepidermal sporulation and pustule opening in white blister rusts (Albuginaceae)

Albugo candida, A. ipomoeae-panduratae, Pustula tragopogonis, Wilsoniana bliti and W. portulacae are widespread obligate biotrophic plant pathogens causing white blister diseases on a variety of flowering plants. Their subepidermal mode of sporulation is unique amongst Oomycetes and leads to blister-like structures on their hosts similar to those produced by true rusts (Uredinales). Unlike in true rusts, sporangia are colourless and produced in chains; the first formed, primary sporangium, differing in size and morphology from subsequent secondary sporangia. According to current interpretations of pustule development the rising pressure of the growing chains of sporangia tear off the epidermal layer from the mesophyll and, in the end, ruptures the epidermis to release the sporangia. This is not convincing considering the rigidity of the epidermal layer and the fact that thin-walled mesophyll cells show no signs of pressure endurance. Our detailed light-, scanning electron-, and transmission electron microscopic observations provide evidence that pustule development and opening are regulated and delicate processes that involve directed enzymatic dissection of host tissue cell walls. The process starts when intercellular hyphae separate the epidermal layer from the parenchyma, forming a cavity in which sporulation takes place. Then thick-walled sporogenous hyphae with club-shaped but thin-walled tips develop and produce sporangia in basipetal succession from the apices of the sporogenous hyphae. The short-living primary sporangia attach tightly to the inner cell walls of the epidermal layer and undergo dramatic cytological changes during pustule maturation, including vacuolisation and development of numerous electron-dense vesicles that might deliver cell wall degrading enzymes. In ripe pustules, the disintegration of areas of epidermal cells leads to the opening of the pustules and to the release of the secondary sporangia. Also the comparison of samples prepared for scanning electron microscopy with fresh pustules, as well as the comparison of the inner epidermal layers detached by the pathogens and detached by force supports our conclusion that delicate enzymatic activity and not force are involved in pustule development and opening by these highly sophisticated pathogens.     

Differentiation of Phytophthora infestans Sporangia from Other Airborne Biological Particles by Flow Cytometry

The ability of two different flow cytometers, the Microcyte (Optoflow) and the PAS-III (Partec), to differentiate sporangia of the late-blight pathogen Phytophthora infestans from other potential airborne particles was compared. With the PAS-III, light scatter and intrinsic fluorescence parameters could be used to differentiate sporangia from conidia of Alternaria or Botrytis spp., rust urediniospores, and pollen of grasses and plantain. Differentiation between P. infestans sporangia and powdery mildew conidia was not possible by these two methods but, when combined with analytical rules evolved by genetic programming methods, could be achieved after staining with the fluorescent brightener Calcofluor white M2R. The potential application of these techniques to the prediction of late-blight epiphytotics in the field is discussed.     

Observations on Nothia aphylla Lyon ex Høeg

Naked branched axes associated with sporangia, originally described by Kidston and Lang (1920) as the probable fertile region of Asteroxylon mackiei, were later shown by Lyon (1964) to belong to an unknown plant which he named Nothia aphylla. The name was validated by Høeg in 1967. Nothia aphylla is described here in greater detail from new material collected from the Rhynie locality. The sporangia are lateral in terminal branched spikes. Their arrangement ranges from spiral to semiverticillate. The fertile axes terminate in clusters of 2–5 sporangia. The stalked sporangia are rather reniform with a distal transversely-extended dehiscence slit. Systematically Nothia has similarities to both Zosterophyllophytina and Rhyniophytina.     

Aulacotheca collicola n.sp. (medullosaceae) from the early Pennsylvanian of the Illinois basin

Compressed synangia referable to the medullosan pollen organ genus Aulacotheca Halle have been discovered in Early Pennsylvanian sediments from Rock Island County, Illinois, U.S.A. Specimens were recovered from localized shales containing floral elements suggesting an upland environment. Based on synangial size, morphology, sporangial number, and prepollen type, two distinct forms, A. collicola n.sp. and Aulacotheca sp., are recognized. Synangia of A. collicola are 11–17 × 2.5 – 4.0 mm and have four to six sporangia. Free sporangial tips are acutely pointed and dehiscence is through a longitudinal slit to the inside of the synangium. Prepollen averages 181 × 129 μm and has a monolete suture with median deflection. Sexine on proximal and distal surfaces shows narrow, closely spaced, anastomosing inpockets; distal and lateral walls are separated by a deep, encircling fold. Synangia of Aulacotheca sp. are 21–33 × 3.8 – 5.5 mm and have 4–6 sporangia. Distally, free sporangial tips have a narrow extension, giving a mucronate appearance. Features of these species suggest that greater morphological diversity in synangial and pollen structure occurs in Aulacotheca and the medullosan pteridosperms than previously recognized.     

Two new species,Pythium agreste and P. wuhanense,based on morphological characteristics and DNA sequence data

In an investigation of Pythium species in China, two new species, P. agreste and P. wuhanense, were identified based on morphological characteristics and DNA sequence data. Pythium agreste has slightly inflated sporangia, oogonia encompassed by antheridia and antheridial stalks forming a very complicated knot, and plerotic oospores. It differs from the morphologically similar P. volutum which has inflated sporangia, bigger oogonia, and aplerotic oospores; from P. kashmirense which has contiguous inflated sporangia, catenulate oogonia and coiled or bent oogonial stalks; and from P. pectinolyticum which has catenulate oogonia and bigger oogonia and oospores. Pythium wuhanense can be differentiated from morphologically similar P. emineosum and P. paroecandrum by its possession of intercalary cylindrical to elongated sporangia and intercalary oogonia catenulate with sporangia and antheridia. Phylogenetic analyses showed that these two new species were clearly separated from morphologically similar Pythium species, based on the internal transcribed spacer (ITS) region and cytochrome C oxidase subunit I (COI) gene sequences using maximum parsimony and Bayesian methods. The two new species are described and illustrated in detail.     

Neoleptonema yongpilii E.-Y. Lee & I.K. Lee,gen. et sp. nov. (Phaeophyceae), based on morphological characters and RuBisCO spacer sequences

An elachistacean epiphyte, Neoleptonema yongpilii E.-Y. Lee & I.K. Lee, gen. et sp. nov., is reported from Korean coasts. The plants are distinguished by having unbranched assimilatory filaments with intercalary plurilocular sporangia as well as lateral plurilocular sporangia from the cortex. The new genus differs from the genera Leptonematella P. Silva and Halothrix Reinke by having pod-shaped plurilocular sporangia on the medulla, and from Elachista Duby and Proselachista Y.P. Lee & Garbary by having intercalary plurilocular sporangia and a poorly developed medulla. The phylogenetic relationships of Neoleptonema yongpilii were inferred from the spacer sequences between the genes coding for the large and small subunits of the RuBisCO gene. The new genus is a member of a poorly resolved clade consisting of several genera within the Elachistaceae, within which it is more closely related to Halothrix and Elachista nipponica than to Leptonematella.     

Infection Efficiency of Four Phytophthora infestans Clonal Lineages and DNA-Based Quantification of Sporangia

The presence and abundance of pathogen inoculum is with host resistance and environmental conditions a key factor in epidemic development. Therefore, several spore-sampling devices have been proposed to monitor pathogen inoculum above fields. However, to make spore sampling more reliable as a management tool and to facilitate its adoption, information on infection efficiency and molecular tools for estimating airborne sporangia concentration are needed. Experiments were thus undertaken in a growth chamber to study the infection efficiency of four clonal lineages of P. infestans (US-8, US-11, US-23, and US-24) by measuring the airborne sporangia concentration and resulting disease intensity. The relationship between the airborne sporangia concentration and the number of lesions per leaf was exponential. For the same concentration, the sporangia of US-23 caused significantly more lesions than the sporangia of the other clonal lineages did. Under optimal conditions, an airborne sporangia concentration of 10 sporangia m⁻³ for US-23 was sufficient to cause one lesion per leaf, whereas for the other clonal lineages, it took 15 to 25 sporangia m⁻³ to reach the same disease intensity. However, in terms of diseased leaf area, there was no difference between clonal lineages US-8, US-23 and US-24. Also, a sensitive quantitative real-time polymerase chain reaction (qPCR) tool was developed to quantify P. infestans airborne sporangia with detection sensitivity of one sporangium. The specificity of the qPCR assay was rigorously tested for airborne inoculum and was either similar to, or an improvement on, other published PCR assays. This assay allows rapid and reliable detection and quantification of P. infestans airborne sporangia and thereby, facilitates the implementation of spores-sampling network.     

A reconsideration of Dimeripteris cornuta SCHWEITZER and CAI, a diminutive fossil plant from the Middle Devonian of Yunnan, China

Small plant fragments previously attributed to Dimeripteris cornuta SCHWEITZER and CAI and new specimens are described from the Middle Devonian (Givetian) Xichong Formation of Wuding, Yunnan Province, southwest China. A predominantly trifurcate branching pattern is recognized in lower orders of branching. Erect fusiform sporangia are borne in pairs terminally on a short pedicel on up to three times dichotomously-branching fertile unit. The beaked tips of the paired sporangia point outwards. This plant is distinguished from known plants by the trifurcate branching pattern and sporangia morphology, but is of problematic affinity. The plant is named Tauritheca cornuta (SCHWEITZER and CAI) WANG and BERRY nov. gen. and nov. comb.     

SPORE MORPHOLOGY IN THE CYATHEACEAE. I. THE PERINE AND SPORANGIAL CAPACITY: GENERAL CONSIDERATIONS

The literature on cyatheaceous spore morphology relative to the presence of a perine layer is reviewed, and evidence based on a sodium-hydroxide assay is presented indicating that the outer scultpine layer in certain cyatheaceous spores is perine. Perine so defined characterizes Metaxya, paleotropical and certain neotropical species of Sphaeropteris, nearly all species of Alsophila, all species of Nephelea, and certain species of Trichipteris and Cyathea. It is lacking in Lophosoria, many species of Trichipteris and Cyathea, and all species of Cnemidaria. Two major patterns of spore number per sporangium in the family are reported. Lophosoria, Sphaeropteris, Trichipteris, Cyathea, Cnemidaria, and probably Metaxya are characterized by 64-spored sporangia, whereas most species of Alsophila and all species of Nephelea are characterized by 16-spored sporangia. The congruence of this generic distribution of sporangial-capacity types with Tryon's phyletic arrangement of cyatheaceous genera supports the naturalness of his system. The intrasporangial germination of spores retained in dehisced and dispersed sporangia supports the suggestion that decreased spore number per sporangium in Alsophila and Nephelea may relate to the role of the sporangia as dispersal units. The decreased number of spores per sporangium is associated with a trend toward increase in the number of sporangia per sores, with the highest known count approaching 1000 sporangia per sorus. The Alsophila-Nephelea evolutionary line has probably not been ancestral in the phylogeny of the more advanced groups of ferns.     

A Strategy for the Proliferation of Ulva prolifera,Main Causative Species of Green Tides,with Formation of Sporangia by Fragmentation

Ulva prolifera, a common green seaweed, is one of the causative species of green tides that occurred frequently along the shores of Qingdao in 2008 and had detrimental effects on the preparations for the 2008 Beijing Olympic Games sailing competition, since more than 30 percent of the area of the games was invaded. In view of the rapid accumulation of the vast biomass of floating U. prolifera in green tides, we investigated the formation of sporangia in disks of different diameters excised from U. prolifera, changes of the photosynthetic properties of cells during sporangia formation, and development of spores. The results suggested that disks less than 1.00 mm in diameter were optimal for the formation of sporangia, but there was a small amount of spore release in these. The highest percentage of area of spore release occurred in disks that were 2.50 mm in diameter. In contrast, sporangia were formed only at the cut edges of larger disks (3.00 mm, 3.50 mm, and 4.00 mm in diameter). Additionally, the majority of spores liberated from the disks appeared vigorous and developed successfully into new individuals. These results implied that fragments of the appropriate size from the U. prolifera thalli broken by a variety of factors via producing spores gave rise to the rapid proliferation of the seaweed under field conditions, which may be one of the most important factors to the rapid accumulation of the vast biomass of U. prolifera in the green tide that occurred in Qingdao, 2008.     

AN UNUSUAL BOTRYOPTERID SPORANGIAL AGGREGATION FROM THE MIDDLE PENNSYLVANIAN OF NORTH AMERICA

A new fertile species of Botryopteris (Botryopteridaceae: Filicales) is described from four incomplete Middle Pennsylvanian specimens. Fertile pinnae of B. cratis sp. n. consist of branched frond members bearing numerous globose sporangia. Surrounding the sporangial aggregations are larger sterile frond members (0.5-1.5 mm diam). Fertile pinnae are oval in transverse section and possess an eccentrically developed cortex composed chiefly of fibers. Some frond members show the typical botryopterid xylem configuration with three protoxylem strands. Spherical sporangia are loosely aggregated on the smallest pinnae by short, broad stalks. The annulus is band-like, two cells high, and extends transversely across the lower half of the sporangium for approximately half the circumference. Spores are oval, trilete, verrucate, and covered by a thin separable layer. Sporangium morphology is like that of Botryopteris antiqua, but the spores closely resemble those of B. globosa. The new species is unlike previously described fructifications of Botryopteris in exhibiting a small pinna system which surrounds smaller pinnae bearing sporangia in an aggregation. The new form is considered to be less specialized than previously described globosoid forms because the sporangia are much less crowded. Isolated frond members, believed to belong to the new species, have a large central arm in the pinna xylem trace that resembles the Stephanian taxon B. renaultii. Small stems attached to the adaxial surface of frond members are radial, protostelic, centrarch, and have a three-zoned cortex. The inner cortical zone contains large elongate cells with distinctive layered deposits. Stems are covered with uniseriate multicellular hairs on multicellular bases. Stems compare closely with B. mucilaginosa in histological features.     

Two novel species representing a new clade and cluster of Phytophthora

Phytophthora stricta sp. nov. and Phytophthora macilentosa sp. nov. are described based on morphological, physiological and molecular characters in this study. Phytophthora stricta represents a previously unknown clade in the rRNA internal transcribed spacer (ITS)-based phylogeny. Phytophthora macilentosa, along with nine other species, consistently forms a high temperature-tolerant cluster within ITS clade 9. These observations are supported by the sequence analysis of the mitochondrial cytochrome c oxidase 1 gene. Both species are heterothallic and all examined isolates are A1 mating type. Phytophthora stricta produces nonpapillate and slightly caducous sporangia. This species is named after its characteristic constrictions on sporangiophores. Phytophthora macilentosa produces nonpapillate and noncaducous sporangia, which are mostly elongated obpyriform with a high length to breadth ratio. Both species were recovered from irrigation water of an ornamental plant nursery in Mississippi, USA and P. stricta was also recovered from stream water in Virginia, USA.     

IDANOTHEKION GEN. N., A SYNANGIATE POLLEN ORGAN WITH SACCATE POLLEN FROM THE MIDDLE PENNSYLVANIAN OF ILLINOIS

Idanothekion glandulosum gen. et sp. n. is a synangiate pollen organ represented by approximately 30 specimens contained in coal balls from the middle Pennsylvanian of Illinois. Each synangium is composed of seven to nine elongate sporangia that are fused laterally for approximately four-fifths of their length, and are radially arranged about, and fused to, a short central column; the central column is restricted to the proximal one-third of the synangium. Distal to the column the sporangia surround a hollow central area. Dehiscence occurred by means of a longitudinal slit along the mid-line of the inner face of each sporangium. The outer walls of the sporangia have a complex histology involving an external epidermis, a middle presumably glandular layer containing scattered enlarged cells, and an inner layer made up of thin-walled parenchyma. Vascular tissue is present in the central column and outer walls of the sporangia. Each sporangium has a prominent, attenuate, multicellular tip. Large numbers of saccate pollen grains similar to those found in numerous fossil and extant coniferophytes as well as some Mesozoic pter-idosperms were produced in each sporangium. Idanolhekion resembles some synangia assignable to Paleozoic members of the Marattiales; however, the new genus compares most closely with pollen organs believed to have been produced by members of the Pteridospermales. It seems most likely that Idanothekion represents the pollen organ of some member of the Lyginopteridaceae that produced pollen of a type which up to now has not been known from Paleozoic seed ferns.     

Coelomomyces psorophorae vartasmaniensis Couch+Laird (1988) (Coelomomycetaeceae: Blastocladiales), a fungal pathogen of the mosquitoAedes australis

The germination of sporangia inCoelomomyces psorophorae vartasmaniensis (C. p. tas.) is uncoordinated and thus there is a mixture of developmental stages in any given population. Continuous urografin gradients separated out the critical stages of germinating sporangia giving four bands, each band representing a consecutive stage of germination. These stages were investigated for changes in the sporangial wall using Transmission Electron Microscopy (TEM). The sporangia have a typical two-layered wall, an electron dense outer layer which can be divided into three distinct sub-layers D1, D2, and D3 and an inner electron transparent secondary wall. Stage 3 sporangia have an intact D1 layer on their outer wall. In the subsequent stages (4 & 4b) there is a progressive breakdown of this layer.     

P protein in the phloem of Cucurbita. I. The development of P-protein bodies

Light and electron microscopical observations of the cells of the phloem of Cucurbita maxima have shown that two distinct types of P-protein bodies are formed: a larger type which arises as fine fibrils and a smaller type which apparently arises as groups of tubules. The tubules of the smaller type of body measure 242 ± 3.6 (SE) A (n = 48) and appear morphologically identical with the P1-protein tubules of Nicotiana tabacum L. In some of these P1-protein bodies the tubules are arranged in a regular manner with a center-to-center distance of 295 A. The P protein of the larger type of P-protein body is first apparent in the cytoplasm as small aggregates of fine fibrils. This P-protein component has been designated P3 protein. As the P3 protein accumulates it is organized into large bodies. Some of these bodies contain only P3 protein, others a tubular form of protein, and still others a combination of P3 protein and a tubular form. This variability indicates that there is a developmental sequence of the formation of tubules from the P3-protein fibrils. These tubules measure 179 ± 8.2 (SE) A (n = 31) and have been designated P4 protein.     

Germination of the resting sporangia ofPhysoderma maydis,the causal agent ofPhysoderma disease of maize

Summary The structural and developmental characteristics of the resting sporangium in uniflagellate phycomycetes, together with the type of zoospore, are of high taxonomic value. Among these fungi, however, only a few electron microscopic investigations have been published on this topic, mainly due to technical problems. In the present study ofPhysoderma maydis (Blastocladiales) these problems were overcome as the resting sporangia in this species are formed synchronously, in large numbers, the germination is readily induced and the impermeability of the resting sporangium wall can be circumvented by shaking the prefixed sporangia with glass beads.The germination of the resting sporangia ofP. maydis is described by correlative light and electron microscopic studies and discussed in relation to related investigations on sporogenesis: The germination process starts by a breakdown of large electron-dense accretions found in the resting stage. Simultaneously, the peripheral location of the lipid bodies is lost. The large operculum is pushed open by a protrusion of the inner sporangial wall; an additional wall layer is formed during this process. Synaptonemal complexes are found in the nuclei at this stage, as are nuclear division figures which suggests anEuallomyces type of life cycle for this fungus. Cleavage vesicles, formed from dictyosomes or endoplasmic reticulum, ultimately separate the sporangial content into meiospores. The sequential assembly of organelles into the side body complex is described. Sequestering of the ribosomes into a nuclear cap is interpreted as taking place immediately prior to zoospore discharge.     

Fertile organs and in situ spores of a new dipteridaceous fern Hausmannia sinensis from the Jurassic of northern China

As a representative fossil member of the dipteridaceous fern, genus Hausmannia was reported worldwide from the Mesozoic strata; however, little is known about the fertile structures, including sporangia and in situ spores, of this genus. In this study, a new species Hausmannia sinensis was identified from the Middle Jurassic of Nei Mongol (Inner Mongolia), northern China. The specimens are compressions and are well preserved with details of sporangia and in situ spores. The leaf laminae are broadly fan-shaped, with an almost entire margin. Primary and lateral veins dichotomously branch to form square or polygonal meshes. Each ultimate mesh bears one to two circular sori of 0.4 mm in diameter. Sori are exindusiate; each sorus contains three to six round to ovoid sporangia. The annulus is developed and oblique, with stomial region present in proximal position. Spores are trilete, circular to oval in shape. Both proximal and distal surfaces are covered with baculate to subverrucate sculptures. Spores range from 20 to 30 μm in diameter (average 28 μm), and are comparable to the dispersed genera Baculatisporites Thomas and Pflug and Apiculatisporis Potonié and Kremp. Hausmannia sinensis represents the first compression species of genus Hausmannia form Eurasia, which shows the combination of well-preserved sori, sporangia, annuli and in situ spore characters, and is therefore helpful for further understanding the diversity and evolution of the Dipteridaceae fern lineage through time.     

Plasmopara invertifolia sp. nov. causing downy mildew on Helichrysum bracteatum (Asteraceae)

Plasmopara invertifolia sp. nov. causes severe leaf distortion and necrosis on Helichrysum bracteatum, a beautiful and important ornamental plant for trade in Brazil. This oomycete pathogen is distinguished from other species of Plasmopara on Asteraceae by its smaller sporangia and larger sporangiophores, which justifies the proposition of a new taxon in the genus Plasmopara to accommodate it. The phylogenetic analysis of cox2 gene sequence data supports such placement and also shows that P. invertifolia is close to the P. halstedii complex. Plasmopara invertifolia is then described, illustrated and discussed.