FUNGI FROM THE LOWER DEVONIAN RHYNIE CHERT: CHYTRIDIOMYCETES

Several different chytridiomycetes are described from the Lower Devonian (Siegenian) Rhynie chert. Included are both eucarpic and apparently holocarpic forms that occur in Palaeonitella, Aglaophyton, Lyonophyton, Horneophyton, and clusters of algal cells, as well as in the surrounding chert matrix. Holocarpic types consist of endobiotic sporangia, each characterized by one discharge tube. Sporangia can be traced from the thallus stage to the discharge of zoospores. Monocentric and polycentric eucarpic chytrids are associated with the miospores of Aglaophyton and various thick-walled fungal spores. In these forms the sporangia are variable in size and shape ranging up to 30 μm. Most appear to be inoperculate and there is evidence that the sporangium ruptured on the distal surface. Some contain zoospores with flagella. One operculate eucarpic form had parasitized the cellular gametophyte emerging from the proximal surface of an Aglaophyton spore. Several of the Rhynie chert chytrids are comparable with a number of extant forms (e.g., Olpidiaceae and Spizellomycetaceae), while others possess features that encompass several groups. These fossil fungi are discussed in the context of their interactions with other organisms in this Lower Devonian freshwater paleoecosystem.     

SPORANGIAL STRUCTURE AND ZOOSPOROGENESIS IN CHORDA TOMENTOSA (LAMINARIALES)1,2,3

Members of the genus Chorda represent the simplest form of sporophyte in the order Laminariales. The present study deals with reproduction in Chorda tomentosa, involving the initiation, growth, and structure of the sporangium and the process of zoosporogenesis. The simple tube-like sporophyte of Chorda tomentosa grows in diameter by means of repeated anticlinal divisions in a superficial meristematic layer known as the meristoderm. The onset of reproduction is marked by the conversion of the meristoderm from contributing cells to the vegetative plant body to producing 2 new cell types: paraphyses and sporangial mother cells. At the time of initiation, sporangial mother cells are crescent shaped and possess a densely staining cytoplasm. Sporangial mother cells increase in size, become ellipsoid, decrease in staining density, and undergo meiosis. After meiosis, sporangia increase in size while their nuclei undergo successive cycles of synchronous mitotic divisions. Sporangia increase to a maximum length of 120 μ;m at which time they possess the characteristic “cap” found in all members of the order studied thus far. At this stage the protoplast of the sporangium is organized into a peripheral layer of nucleus-chloroplast pairs and a central region of vacuoles. Cleavage furrows begin to form at the cell membrane and are met by furrows developing in the interior of the cytoplasm resulting in the division of the entire protoplast into separate units. Each unit is an individual zoospore. The biflagellate zoospores contain a nucleus, one chloroplast with eyespot, perinuclear Golgi, and several bodies of presumed storage carbohydrate. The occurrence of a small population of early developing sporangia is described. In essential details, the origin, development, and structure of sporangia in Chorda tomentosa are identical to all earlier observations in the Laminariales.     

MORPHOLOGY AND LIFE CYCLE OF A NEW CHYTRID WITH AERIAL SPORANGIA

An investigation of the life cycle of Caulochytrium gloeosporii Voos and Olive, a parasite of Gloeosporium, has confirmed the existence of an alternation of generations. Zoospores from sessile sporangia may develop vegetatively into similar sporangia or they may function as isogametes, fusing in pairs to produce cysts (zygotes) that give rise to slender-stalked aerial sporangia. Meiosis is believed to occur in the aerial sporangium, which at germination liberates eight asexual zoospores. The species is homothallic.     

Antibacterial Activity and Mechanisms of Ethanol Extracts from <i>Scutellaria baicalensis</i> Georgi and <i>Magnolia officinalis</i> against <i>Phytophthora nicotianae</i>

Phytophthora nicotianae causes substantial economic losses in most countries where tobacco is produced. At present, the control of P. nicotianae mainly depends on chemical methods, with considerable environmental and health issues. We investigated the effects of ethanol extracts from Scutellaria baicalensis Georgi (SBG) and Magnolia officinalis (MO). On mycelial growth, sporangium formation, and zoospore release of P. nicotianae. Both extracts inhibited the growth of P. nicotianae, with mycelial growth inhibition rates of 88.92% and 93.92%, respectively, at 40 mg/mL, and EC50 values of 5.39 and 5.74 mg/mL, respectively. The underlying mechanisms were the inhibition of sporangium formation, the reduction of zoospore number, and the destruction of the mycelium structure. At an SBG extract concentration of 16.17 mg/mL, the inhibition rates for sporangia and zoospores were 98.66% and 99.39%, respectively. At an MO extract concentration of 2.87 mg/mL, the production of sporangia and zoospores was completely inhibited. The hyphae treated with the two plant extracts showed different degrees of deformation and damage. Hyphae treated with SBG extract showed adhesion and local swelling, whereas treatment with MO extract resulted in broken hyphae. Mixture of the extracts resulted in a good synergistic effect.     

Cell-wall abnormalities of a Chlamydomonas reinhardtii mutant strain under suboptimal growth conditions

Sporangia were accumulated in autotrophically and mixotrophically growing cultures of the Chlamydomonas reinhardtii mutant strain ls entering the stationary phase. Such an accumulation of sporangia was never observed in stationary-phase cultures of wildtype strains. Sporangia harvested from stationary-phase cultures of the mutant strain ls released their zoospores after being resuspended in fresh culture medium. Liberation of zoospores was also observed during fixation of these sporangia with glutaraldehyde and OsO₄. Release of zoospores during fixation was prevented by pretreatment with 3 mol·l^–1 LiCl. Ultrastructural analyses of these LiCl-pretreated sporangia revealed that they contained abnormal sporangial walls: sporangia containing sporangia and sporangia surrounded by additional multilayered cell walls have been observed. Similar abnormal cell-wall structures were found in sporangia accumulated at the end of the dark period, when the mutant strain ls was grown photoautotrophically under a 12 h light-12 h dark regime with suboptimal aeration. When grown under optimal conditions, this particular mutant did not show any abnormal wall structures.This work has been supported by a grant from the Deutsche Forschungsgemeinschaft. The authors thank Mrs. C. Adami for the photographic work.     

FOUR NEW SPECIES OF THRAUSTOCHYTRIUM FROM ANTARCTIC REGIONS,WITH NOTES ON THE DISTRIBUTION OF ZOOSPORIC FUNGI IN THE ANTARCTIC MARINE ECOSYSTEMS

New species of the obligately marine Thraustochytriaceae Sparrow were discovered in subantarctic and antarctic waters of the southeastern Indian Ocean, the southwestern Pacific Ocean, and the antarctic Ross Sea during two cruises of the research vessel USNS ELTANIN. The life cycles of four species of Thraustochytrium in seawater-pollen and/or seawater-brine shrimp larvae cultures are described. Thraustochytrium antarcticum sp. nov. develops sporangia that may proliferate from a single basal rudiment. Flagellated zoospores are liberated from the sporangium upon complete disintegration of the sporangial wall at maturity. Thraustochytrium rossii sp. nov. and T. kerguelensis sp. nov. are both similar in that they develop sporangia that may proliferate from more than one basal rudiment. The latter species releases flagellated zoospores upon complete disintegration of the sporangial wall, but the former species liberates a mass of individually immobile zoospores from the sporangium. These remain quiescent for several hours before they swim away one after another. The protoplast of Thraustochytrium amoeboidum sp. nov. leaves the sporangium through a pore as an amoeboid body which then gives rise to nonflagellated amoebospores by successive bipartitioning. Laterally biflagellate thraustochytrioid zoospores were also observed, but the way in which they are formed remains to be determined. Zoosporic and aplanosporic phycomycetes were recovered from water samples collected in the Subtropical, Subantarctic, and Antarctic Zones of the Southern Ocean. Highest numbers of phycomycete propagules were found in antarctic waters near the Antarctic Convergence during ELTANIN's Cruise 51. In the Subtropical and Subantarctic (but not in the Antarctic) Zones fungal population densities increased with proximity to continents or islands. At each station where phycomycetes were recovered, highest numbers of propagules were generally found in the surface layers (25–250 m) of the ocean below the photic zone (lower limit 30–60 m). This peculiar distribution may indicate that phycomycetes are engaged in decomposing substances derived from the photic zone.     

New Insights in the Life Cycle and Epidemics of Phytophthora porri on Leek

White tip, caused by Phytophthora porri, is a devastating disease in the autumn and winter production of leek (Allium porrum) in Europe. This study investigated the disease cycle of P. porri in laboratory and field conditions. Oospores readily germinated in the presence of non‐sterile soil extract at any temperature between 4 and 22°C, with the formation of sporangia which released zoospores. The zoospores survived at least 7 weeks in water at a temperature range of 0 till 24°C. Microscopic examinations revealed that zoospores encysted and germinated on the leek leaf surface and hyphae entered the leaf directly through stomata or by penetrating via appressoria. Oospores were formed in the leaves within 6 days, while sporangia were not produced. By monitoring disease progress in fields with a different cropping history of leek, it could be deduced that P. porri survives in soil for up to 4 years. Disease progress during three consecutive years was correlated with average daily rainfall in the infection period. Disease incidence on leek was reduced when rain splash was excluded by growing the plants in an open hoop greenhouse. Based on these findings, we propose a disease cycle for P. porri in which oospores germinate in puddles, and zoospores reach the leaves by rain splash and survive in water in the leaf axils, from where they infect the plant by direct penetration or via stomata. When conditions become unfavourable, oospores are produced in the leaves which again reach the soil when leaves decay. Secondary spread of the disease by sporangia does not seem to be important.     

The Effect of Exposure to Decreasing Relative Humidity on the Viability of Phytophthora ramorum sporangia

Sporangia of three isolates of Phytophthora ramorum representing three different clonal lineages were subjected to relative humidity (RH) levels between 80 and 100% for exposure periods ranging from 1 to 24 h at 20°C in darkness. Plastic containers (21.5 × 14.5 × 5 cm) were used as humidity chambers with 130 ml of glycerine solution added to each container. Glycerine concentrations corresponded to 100, 95, 90, 85 and 80% RH based on refractive index measurements. Sporangia suspensions were pipeted onto nitrile mesh squares (1.5 × 1.5 cm, 15 micron pore size) which were placed in the humidity chambers and incubated at 20°C in darkness. Following exposure periods of 1, 2, 4, 8, 12 and 24 h, mesh squares were inverted onto Petri dishes of selective medium and sporangia germination assessed after 24 and 48 h. At 100% RH, we observed a mean value of 88% germination after 1 h exposure declining to 18% germination following 24 h incubation. At 95% RH, a steeper decline in germination was noted, with means ranging from 79% at 1 h to less than 1% at 24 h exposure. At 90% RH, no germination was noted after 8 or more h exposure, and values were 57%, 22% and 3% germination for the 1, 2 and 4 h exposures, respectively. Germination was only observed at 1 h exposure for both the 85% RH treatment (52% germination) and the 80% RH treatment (38% germination). The three isolates responded similarly over the range of RH values tested. The germination response of P. ramorum sporangia to RH values between 80% and 100% was comparable to that reported for other Phytophthora species. Knowledge of conditions that affect P. ramorum sporangia germination can shed light on pathogenesis and epidemic potential and lead to improved control recommendations.     

Electron microscopy of primary zoosporogenesis inPlasmodiophora brassicae

Primary zoosporogenesis in resting sporangia ofPlasmodiophora brassicae that had been incubated for 14 d in culture solution containing turnip seedlings was examined by transmission electron microscopy. A single zoospore differentiated within each sporangium, the differentiation being initiated by the emergence, of two flagella in the tight space formed by invagination of the plasma membrane within the sporangium. The differentiazing zoospore was similar in intracellular aspects to sporangia within clubroot galls. Then a deep groove formed on the zoospore cell body by further invagination of the plasma membrane. Two flagella appeared to coil around the zoospore cell body in parallel along this groove. Thereafter, the cell body lost the groove and became rounded following the protoplasmic condensation (contraction of cell body) during late development, and assumed an irregular shape at the stage of maturation. Intracellular features in, developing and mature zoospores were complicated, being characterized by electron-dense nuclei and mitochondria, microbodies, cored vesicles and various unidentified cytoplasmic vesicles and granules. A nucleolus-like region was observed only in the nucleus of the mature zoospore. A partially opened germ, pore was also seem in the sporangium containing the mature zoospore.     

Microassay for biological and chemical control of infection of tobacco byPhytophthora parasitica var.nicotianae

We developed a simple, rapid, small-scale assay for infection of tobacco seedlings byPhytophthora parasitica var.nicotianae. One 7-day-old tobacco seedling was placed in each well of a 96-well microtiter plate and inoculated with 500 zoospores ofP. parasitica var.nicotianae. After 72 h all of the inoculated seedlings of the susceptible cultivar, KY14, were infected, and the pathogen had produced sporangia that were visible on the surfaces of the seedlings. Sporangia did not develop on seedlings that were inoculated simultaneously with zoospores and either 1 µg/mL of the chemical fungicide metalaxyl or 5 µL of filtrate of a sporulated culture of the biocontrol agent,Bacillus cereus UW85. Seedlings of tobacco cultivar KY17 were infected byP. parasitica var.nicotianae, although mature plants of this variety are resistant to the pathogen. This microassay may facilitate the rapid screening of potential biological and chemical control agents and may be useful for studying mechanisms of infection and control ofPhytophthora spp. under hydroponic conditions.     

A PERSISTENT VIRUS INFECTION IN FELDMANNIA (PHAEOPHYCEAE)1

A virus infection is described within the unilocular sporangia of Feldmannia sp., a filamentous brown alga (Phaeophyceae). The alga is easily maintained in culture and vegetative growth is vigorous, but formation of icosahedral virions 150 nm in diameter completely displaces production of zoospores. The viruses, estimated at 1–5 × 10⁶ per sporangium, are eventually released by rupture of the sporangial wall. Deoxyribonucleic acid (DNA) isolated from the viruses can be readily digested with restriction endonucleases and consists of ca. 170 kbp of double-stranded DNA.     

A method to obtain rapid zoosporogenesis of Pythium insidiosum

Nine strains of Pythium insidiosum the etiologic agent of pythiosis, were inoculated on 2% water agar plus grass blades and then incubated one day at 25°C, 35°C and 37°C. Sporangium and secondary biflagellate-type zoosporas from the parasitized grass blades were noticed in induction medium after one hour of incubation at 35 °C and 37 °C. The number of sporangia and zoospores were lower at 25 °C, than 35 °C and 37 °C. Increasing the days of incubation of the parasitized grass blades resulted in the increase in the time of incubation in the induction medium. Corn meal agar, Schmitthenner medium and Sabouraud dextrose agar were also tested but the sporangium and zoosporas were always observed after five hours of incubation in induction medium.     

Sporangium Exposure and Spore Release in the Peruvian Maidenhair Fern (Adiantum peruvianum,Pteridaceae)

We investigated the different processes involved in spore liberation in the polypod fern Adiantum peruvianum (Pteridaceae). Sporangia are being produced on the undersides of so-called false indusia, which are situated at the abaxial surface of the pinnule margins, and become exposed by a desiccation-induced movement of these pinnule flaps. The complex folding kinematics and functional morphology of false indusia are being described, and we discuss scenarios of movement initiation and passive hydraulic actuation of these structures. High-speed cinematography allowed for analyses of fast sporangium motion and for tracking ejected spores. Separation and liberation of spores from the sporangia are induced by relaxation of the annulus (the ‘throwing arm’ of the sporangium catapult) and conservation of momentum generated during this process, which leads to sporangium bouncing. The ultra-lightweight spores travel through air with a maximum velocity of ~5 m s^-1, and a launch acceleration of ~6300g is measured. In some cases, the whole sporangium, or parts of it, together with contained spores break away from the false indusium and are shed as a whole. Also, spores can stick together and form spore clumps. Both findings are discussed in the context of wind dispersal.     

The biology of Peronospora viciae on pea: laboratory experiments on the effects of temperature, relative humidity and light on the production, germination and infectivity of sporangia

Germination of Peronospora viciae sporangia washed off infected leaves varied from 20% to 60%. Sporangia shaken off in the dry state gave 11–19% germination. Most sporangia lost viability within 3 days after being shed, though a few survived at least 5 days. Infected leaves could produce sporangia up to 6 weeks after infection, and sporulating lesions carried viable sporangia for 3 weeks. Sporangia germinated over the range 1–24 °C, with an optimum between 4 and 8 °C. Light and no effct. The temperature limits for infection were the same as for germination, but with an optimum between 12 and 20 °C. A minimum leaf-wetness period of 4h was required, and was independent of temperature over the range 4–24 °C. Maximum infectivity occurred after 6h leaf wetness at temperatures between 8 and 20 °C. Infection occurred equally in continuous light or in darkness. After an incubation period of 6–10 days sporangia were produced on infected leaves at temperatures between 4 and 24 °C, with an optimum of 12–20 °C. Exposure to temperatures of 20–24 °C for 10 days reduced subsequent sporulation. Sporangia produced at suboptimal temperatures were larger, and at 20 °C. smaller, than those produce at 12–16 °C. Viability was also reduced. No sporangia were produced in continuous light, or at relative humidities below 91%. For maximum sporulaiton an r.h. of 100% was required, following a lower r.h. during incubation. Oospores wre commonly formed in sporulating lesions, and also where conditons limited or prevented sporulation. The results are discussed briefly in relaiton to disease development under field conditions.     

中华水韭孢子囊发育研究

采用半薄切片法,连续观察了极度濒危级(CR)植物中华水韭大小孢子囊的发育过程,以期从无性生殖的角度,为探讨其濒危原因提供直观可靠的理论根据。结果显示:(1)中华水韭的大小孢子叶相间排列,无混生孢子囊。(2)隔丝为孢子供给营养,其体积直接影响孢子的大小、产量和育性。(3)大小孢子囊都近半数败育,小孢子囊为整齐发育,大孢子囊为不整齐发育。(4)大小孢子囊均无柄,且都不存在开裂结构,只有孢子囊壁腐烂后才能散播孢子。研究认为,中华水韭的濒危与孢子囊的发育特征密切相关,孢子囊的高频率败育、没有开裂结构以及对环境的依赖,是造成中华水韭濒危的重要因素之一;通过与近缘类群孢子囊的比较,发现仅水韭孢子的散播借助外力,对生境要求较高,即验证了水韭古老的系统学地位,同时说明水韭更具有监测生境地区环境指标的能力。     

The Ultrastructure of Sanchytrium tribonematis (Sanchytriaceae,Fungi incertae sedis) Confirms its Close Relationship to Amoeboradix

Fungi encompass, in addition to classically well‐studied lineages, an ever‐expanding diversity of poorly known lineages that include, among others, zoosporic chytrid‐like parasites. According to recent phylogenetic analysis based on 18S + 28S rRNA concatenated genes two unusual chytrid‐like fungi Amoeboradix gromovi and Sanchytrium tribonematis form a monophyletic group, the family Sanchytriaceae, which represents a new divergent taxon that remains incertae sedis within Fungi. Zoospores of Amoeboradix gromovi contain one of the longest kinetosomes known in eukaryotic cells, which are composed of microtubular singlets or doublets. However, the ultrastructure of S. tribonematis, the type species of the genus had not been yet studied. Here, we provide the results of TEM investigations of zoospores and sporangia from two strains of S. tribonematis. The two strains are endowed with unusual features. Like in A. gromovi, amoeboid zoospores of S. tribonematis contain a long kinetosome composed of microtubular singlets, and the two orthogonal centrioles in their sporangia have nine microtubular singlets with an internal ring. The morphological and ultrastructural features of S. tribonematis are now included in the improved taxonomic diagnosis for this species.     

Characterisation of the water expulsion vacuole inPhytophthora nicotianae zoospores

Summary The water expulsion vacuole (WEV) in zoospores ofPhytophthora nicotianae and other members of the Oomycetes is believed to function in cell osmoregulation. We have used videomicroscopy to analyse the behaviour of the WEV during zoospore development, motility and encystment inP. nicotianae. After cleavage of multinucleate sporangia, the WEV begins to pulse slowly but soon attains a rate similar to that seen in motile zoospores. In zoospores, the WEV has a mean cycle time of 5.7 ± 0.71 s. The WEV continues to pulse at this rate until approximately 4 min after the onset of encystment. At this stage, pulsing slows progressively until it becomes undetectable. The commencement of WEV operation in sporangia coincides with the reduction of zoospore volume prior to release from the sporangium. Disappearance of the WEV during encystment occurs as formation of a cell wall allows the generation of turgor pressure in the cyst. As in other organisms, the WEV inP. nicotianae zoospores consists of a central bladder surrounded by a vesicular and tubular spongiome. Immunolabelling with a monoclonal antibody directed towards vacuolar H⁺-ATPase reveals that this enzyme is confined to membranes of the spongiome and is absent from the bladder membrane or zoospore plasma membrane. An antibody directed towards plasma membrane H⁺-ATPase shows the presence of this ATPase in both the bladder membrane and the plasma membrane over the cell body but not the flagella. Analysis of ATPase activity in microsomal fractions fromP. nicotianae zoospores has provided information on the biochemical properties of the ATPases in these cells and has shown that they are similar to those in true fungi. Inhibition of the vacuolar H⁺-ATPase by potassium nitrate causes a reduction in the pulse rate of the WEV in zoospores and leads to premature encystment. These results give support to the idea that the vacuolar H⁺-ATPase plays an important role in water accumulation by the spongiome in oomycete zoospores, as it does in other protists.Abbreviations BMM butyl methylmethacrylate - F fix 4% formaldehyde fixation - GF fix 4% formaldehyde and 0.2% glutaraldehyde fixation - V-ATPase vacuolar H⁺-ATPase - WEV water expulsion vacuole     

Secondary Sporangium Formation in Phytophthora Palmivora (Butl.) Butl

Very few sporangia of Phytophthora palmivora germinated directlyand produced secondary sporangia in distilled water and in solutionsof amino acids and carbohydrates at 30 °C. Although 1.0per cent (w / v) peptone and yeast-extract stimulated a highpercentage of germination by formation of germ tubes, less than1.0 per cent of the germinated sporangia produced secondarysporangia. Secondary sporangium formation was induced by transferringgerminated primary sporangia from a nutrient medium of sufficientlyhigh concentration to either distilled water or dilute solutionsof organic and inorganic compounds immediately after emergenceof the germ tubes. The percentage of germinated sporangia formingsecondary sporangia was influenced by both the nature and concentrationof the medium into which they were transferred. The secondarysporangia were significantly smaller than the primary sporangia. Phytophthora palmivora, germination, sporangium, Theobroma cacao L., cocoa     

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.