Immunological detection of the fungal parasite,Pythium sp.; the causative organism of red rot disease in Porphyra yezoensis

The red rot disease of Porphyra yezoensis Ueda (Rhodophyta) is caused by a parasitic fungus, Pythium sp. To facilitate the detection of this pathogen in infected thalli of P. yezoensis, polyclonal and monoclonal antibodies were prepared. Antibodies were raised against antigen prepared from an isolate of fungal hyphae obtained from red-rot infected thallus of P. yezoensis from Aichi Prefecture. Polyclonal antibody was obtained from the antisera of immunized rabbits. Monoclonal antibody was obtained from the culture supernatant of a hybridoma which had been established by cell fusion between a myeloma cell line and spleen cells of immunized mice. Hyphae were detected by means of indirect fluorescent antibody technique. Titers of polyclonal antibodies obtained were too low to recognize fungal hyphae that had penetrated the thalli of P. yezoensis; however, monoclonal antibody was useful for the detection of fungi that had penetrated algal thalli. The monoclonal antibody was specific for the Pythium sp. from red-rot infected thalli of P. yezoensis from Saga (western Japan) and from Aichi Prefectures (central Japan), but was ineffective for infections from Miyagi Prefecture (northern Japan). It is evident, therefore, that Pythium sp. can give rise to immunologically distinct groups of red rot disease. Based on chemical and enzymatic treatments, the antigenic determinant appeared to localize on the sugar chains of glycoconjugates or the polysaccharides of the hyphal cell wall.     

Porphyra monospore system (Bangiales, Rhodophyta): A model for the developmental biology of marine plants

We have developed an experimental system of cohort monospores from clonal culture of leafy gametophytes in Porphyra yezoensis Ueda (strain TU-1). This system is quite different from traditional systems for algal protoplast experimentation, which require expensive enzymatic treatment and utilize an ineffective method of preservation. Cohort monospores were obtained by utilizing a mode of asexual reproduction in the culture strain (monospores) and artificial regulation (thallus length, temperature, light, etc.) of monospore release. When the leafy gametophytes that formed monospores were frozen at - 20°C in a cryoprotective solution composed of 5% DMSO and 5% dextran in 100% seawater, about 98% survived for 3 months. When stored at 5°C without cryoprotectants, these leafy gametophytes could be kept without monospore release for 1 week. Maximum monospore yield was about 3000 spores per 100 gametophytes, and germination rate was about 70%, This system will accelerate developmental biology studies in Porphyra.     

VARIATIONS IN THE CELL WALLS AND PHOTOSYNTHETIC PROPERTIES OF PORPHYRA YEZOENSIS (BANGIALES,RHODOPHYTA) DURING ARCHEOSPORE FORMATION1

The formation of archeospores is characteristic of Porphyra yezoensis Ueda and is important for Porphyra aquaculture. Recently, it has been regarded as a valuable seed source for propagation of thalli in mariculture. Cell wall composition changes are associated with archeospore formation in P. yezoensis. Here, we report changes of cell walls of P. yezoensis during archeospore formation. The surfaces of vegetative cells that were originally smooth became rougher and more protuberant as archeosporangia were formed. Ultimately, the cell walls of archeosporangia ruptured, and archeospores were released from the torn cell walls that were left at distal margins of thalli. With changes in cell walls, both effective quantum yield and maximal quantum yield of the same regions in thalli gradually increased during the transformation of vegetative cells to archeospores, suggesting that the photosynthetic properties of the same regions in thalli gradually increased. Meanwhile, photosynthetic parameters for different sectors of thalli were determined, which included the proximal vegetative cells, archeosporangia, and newly released archeospores. The changes in photosynthetic properties of different sectors of thalli were in accordance with that of the same regions in thalli at different stages. In addition, the photosynthetic responses of archeosporangia to light showed higher saturating irradiance levels than those of vegetative cells. All these results suggest that archeosporangial cell walls were not degraded prior to release but were ruptured via bulging of the archeospore within the sporangium, and ultimately, archeospores were discharged. The accumulation of carbohydrates during archeospore formation in P. yezoensis might be required for the release of archeospores.     

Photosynthesis-dependent Ca2+ influx and functional diversity between phospholipases in the formation of cell polarity in migrating cells of red algae

Unicellular spore cells, designated as monospores (also called archeospores), are well known as migrating plant cells, in which establishment of the anterior-posterior axis directs asymmetrical distribution of F-actin. Since the mechanisms of cell polarity formation are not yet fully elucidated in monospores, we investigated the roles of phosphoinositide signaling systems and Ca²⁺ mobilization in migration. Although we have already found the critical involvement of phosphatidylinositol 3-kinase in the establishment of cell polarity, we recently demonstrated the important roles of extracellular Ca²⁺ influx, phospholipase C (PLC) and phospholipase D (PLD). The remarkable characteristics of these factors are that Ca²⁺ influx depends on photosynthetic activity and that PLC and PLD play roles in the establishment and maintenance of cell polarity, respectively. These findings could provide new insight into the regulation of migration in eukaryotic cells.Key words: Ca²⁺ influx, cell polarity, phospholipase C, phospholipase D, photosynthesis, Porphyra yezoensisMonospores are responsible for asexual and clonal propagation of the marine multicellular red algae Porphyra and have an exceptional characteristic as migrating plant cells.^1–5 Monospores possess a round shape just after release from gametophytic blades (Fig. 1A and B), then undergo morphological change during migration. The establishment of cell polarity leads to the determination of anterior-posterior axis and asymmetrical localization of F-actin (Fig. 1C). After migration, monospores adhere to the substratum in which the apical-basal axis has been established for further development (Fig. 1D). Asymmetrical distribution of F-actin is also found in chemotaxic migration of Dictyostelium cells and leukocytes.^6,7 In these cells, reciprocal local accumulation of phosphoinositides, such as phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P₃] at the leading edge and phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P₂] at the trailing side, is critical for the establishment of cell polarity. Phosphatidylinositol 3-kinase (PI3K) and PtdIns(3,4,5)P₃-specific D-3-phosphatase PTEN have been identified as key modulators in the establishment of cell polarity, bringing asymmetrical distribution of these two phosphoinositides in plasma membranes.^6,8 Similarly, we found the involvement of PI3K activity in the establishment of cell polarity in migrating monospores,³ suggesting the evolutional conservation of the function of PI3K in migrating eukaryotic cells. On the other hand, the importance of cell wall synthesis has been found in the maintenance of the cell polarity during monospore migration⁴ as reported in Fucus zygotes.^9,10 Therefore, the establishment and maintenance of cell polarity are thought to be regulated separately in monospores of P. yezoensis. In this addendum, further evidence of differential regulation of cell polarity formation by extracellular Ca²⁺ influx and phospholipases in migrating monospores of red algae is documented according to our recent report.⁵Open in a separate windowFigure 1Establishment and maintenance of cell polarity in monospores from the red alga P. yezoensis. (A) Discharge of unicellular monospores from a multicellular gametophytic blade of P. yezoensis strain TU-1. Scale bar = 20 µm. (B–D) Asymmetrical distribution of F-actin during early development of monospores. F-actin was stained with alex Flour 488 phalloidin. (B) Discharged monospore. (C) Migrating monospore. (D) Adhering monospore. Upper and lower photos in each panel show bright-field and fluorescent images, respectively. Arrow in (C) indicates the direction of migration. Scale bars = 5 µm. (e) Schematic representation of our working hypothesis about the formation of cell polarity required for monospore migration. Photosynthesis-dependent [Ca²⁺]_cyt increase regulates PLC and PI3K for the establishment of cell polarity, while PLD is required for the maintenance of the established cell polarity. DG, diacylgycerol; IP3, inositol-1,4,5-trisphosphate; IP3r, IP3 receptor; PC, phosphatidylcholine.     

Porphyra yezoensis bei Helgoland — eine entwicklungsgeschichtliche Studie

APorphyropsis-like epiphytic specimen found in the harbour of Helgoland was grown in culture and proved to be identical with the JapanesePorphyra yezoensis. Life history studies on this economically important alga resulted in some interesting and hitherto unknown details. The variability of the adult frond is fundamentally determined by the pattern of spore germination. Settled onChaetomorpha filaments, monospores elongate within 20 minutes; the epiphytic germlings are attached to the substrate by a typical basal cell and give rise exclusively to elongated fronds provided with a cuneate base. Unattached spores, however, germinate into buds with rhizoids; they develop into elongated elliptical to oval fronds provided with round or cordate bases. Only plants with male areas were observed in the cultures, butConchocelis was abundantly produced from cells of aged thalli. Grown in mussel-shells, the filamentous phase liberated conchospores for a long time.

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Herrn Paul-Heinz Sahling zum 75. Geburtstag gewidmet, mit Anerkennung und Dank für 50jährige Zusammenarbeit.     

Visualization of Phosphoinositides via the Development of the Transient Expression System of a Cyan Fluorescent Protein in the Red Alga Porphyra yezoensis

Phosphoinositides (PIs) play important roles in signal transduction pathways and the regulation of cytoskeleton and membrane functions in eukaryotes. Subcellular localization of individual PI derivative is successfully visualized in yeast, animal, and green plant cells using PI derivative-specific pleckstrin homology (PH) domains fused with a variety of fluorescent proteins; however, expression of fluorescent proteins has not yet been reported in any red algal cells. In the present study, we developed the system to visualize these PIs using human PH domains fused with a humanized cyan fluorescent protein (AmCFP) in the red alga Porphyra yezoensis. Plasma membrane localization of AmCFP fused with the PH domain from phospholipase Cδ1 and Akt1, but not Bruton’s tyrosine kinase, was observed in cell wall-free monospores, demonstrating the presence of phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol-3,4-bisphosphate in P. yezoensis cells. This is the first report of the successful expression of fluorescent protein and the monitoring of PI derivatives in red algal cells. Our system, based on transient expression of AmCFP, could be applicable for the analysis of subcellular localization of other proteins in P. yezoensis and other red algal cells.     

TWO SPECIFIC CAUSES OF CELL MORTALITY IN FREEZE‐THAW CYCLE OF YOUNG THALLI OF PORPHYRA YEZOENSIS (BANGIALES,RHODOPHYTA)1

Porphyra yezoensis Ueda is an important marine aquaculture crop with single‐layered gametophytic thalli. In this work, the influences of thallus dehydration level, cold‐preservation (freezing) time, and thawing temperature on the photosynthetic recovery of young P. yezoensis thalli were investigated employing an imaging pulse‐amplitude‐modulation (PAM) fluorometer. The results showed that after 40 d of frozen storage when performing thallus thawing under 10°C, the water content of the thalli showed obvious effects on the photosynthetic recovery of the frozen thalli. The thalli with absolute water content (AWC) of 10%–40% manifested obvious superiority compared to the thalli with other AWCs, while the thalli thawed at 20°C showed very high survival rate (93.10%) and no obvious correlation between thallus AWCs and thallus viabilities. These results indicated that inappropriate thallus water content contributed to the cell damage during the freeze‐thaw cycle and that proper thawing temperature is very crucial. Therefore, AWC between 10% and 40% is the suitable thallus water content range for frozen storage, and the thawing process should be as short as possible. However, it is also shown that for short‐term cold storage the Porphyra thallus water content also showed no obvious effect on the photosynthetic recovery of the thalli, and the survival rate was extremely high (100%). These results indicated that freezing time is also a paramount contributor of the cell damage during the freeze‐thaw cycle. Therefore, the frozen nets should be used as soon as time permits.     

The sporophyte of Liagora farinosa lamour

Carpospores of Liagora farinosa from the western Mediterranean in culture gave rise to an Acrochaetium-like phase with filamentous prostrate and sparsely branched erect systems. This phase reproduced readily by tetraspores and monospores. The tetrasporangia were found to go through meiosis and the four haploid spores then gave rise to filamentous protonemal growths on which buds arose that developed into typical (though usually uncalcified) Liagora thalli. The monospores appeared to repeat the Acrochaetium-phase. These observations suggest that Liagora farinosa is another example of those members of the Nemalionales which show a heteromorphic life-history, with the sporophyte generation respresented by a dwarf phase.

Some aspects of the developmental physiology of the species are discussed.     

Regeneration from Laminaria japonica Areschoug (Laminariales, Phaeophyceae) protoplasts isolated with bacterial alginase

Summary The conditions for effective isolation of viable protoplasts from Laminaria japonica with an alginase produced by marine bacterium Alteromonas sp. and a commercially available cellulase were investigated. The highest yields of viable protoplasts (7.910.4x10⁶ cells g^–1 FW) were obtained with a hypertonic solution containing 50 % seawater, 25 mM MgCl₂, 5 mM HEPES buffer system, and 0.5 M mannitol. Protoplasts were not obtained from thalli of L. japonica when an abalone alginase (abalone acetone powder; AAP: Sigma) was used instead of the bacterial alginase. The isolated protoplasts were cultured in an PESI medium at 5 °C. Complete cell wall formation was observed within 7 days, and dividing cells were first observed in a 9-day-old culture. Some protoplasts regenerated into sheet-shaped thalli and rhizoid structures were also observed on some thalli after 30 to 40 days in culture. This is the first report of protoplast regeneration into plantlets of L. japonica Areschoug (Laminariales, Phaeophyceae).Abbreviations FW Flesh weight - AAP Abalone acetone powder - HEPES N-2-hydroxy-ethylpiperazine-N-2-ethanesulfonic acid - Tris Tris(hyrdoxymethyl)aminomethane - PESI Provasoli's enriched seawater with iodine     

Preliminary comparison of atmospheric CO2 enhancement to photosynthesis of Pyropia yezoensis (Bangiales,Rhodophyta) leafy thalli and filamentous thalli

Intertidal macroalgae are submerged in seawater at high tide and exposed to air at low tide. When they are exposed to the air, CO₂ is the main inorganic carbon source. In this study, the photosynthetic performances of PSI and PSII were measured in different generations of Pyropia yezoensis (leafy thalli and filamentous thalli) that had been exposed to air containing different CO₂ concentrations. Changes in photosynthesis during dehydration and salt treatment under the different CO₂ concentrations were also analyzed. The results showed that in leafy thalli, the effective photochemical quantum yield of PSII (YII) was enhanced as CO₂ increased, which suggested that CO₂ assimilation was enhanced and that they can utilize CO₂ in the air directly, even when they are subjected to moderate stress. These findings could explain why, in P. yezoensis aquaculture, moderate exposure to air does not lead to a decrease in crop yield. However, in filamentous thalli, there were no significant differences in YII at the CO₂ concentrations tested. The expression of genes involved in the Calvin cycle in leafy thalli was higher than that in filamentous thalli. CO₂ uptake and biomass of P. yezoensis leafy thalli is larger than filamentous thalli, which may be due to its different carbon utilization mechanism and the adaptation of intertidal environment in the evolutionary process.     

CRYOPRESERVATION OF THE CONCHOCELIS OF PORPHYRA (RHODOPHYTA) BY APPLYING A SIMPLE PREFREEZING SYSTEM1

The conchocelis cells of four strains of Porphyra yezoensis Udea and four other Porphyra species were cryopreserved in liquid nitrogen (LN) using a programmable freezer or a simple prefreezing system, which consisted of a styrofoam box and a deep-freezer at ?40° C. The cells differed in their freezing tolerance but survived maximally when prefrozen to ?40° C in a cryoprotective solution composed of 10% dimethylsulfoxide and 0.5 M sorbitol in 50% seawater. The cryopreservation was successfully performed by applying the simple prefreezing system as well as by a programmable freezer. Conchocelis cells thawed from the LN temperature formed colonies and retained the ability to form conchospores that grew into gametophytic thalli. This technique using a simple prefreezing system will accelerate the spread of Porphyra cryopreservation.     

Spore Dimorphism in a Microsporidan Isolate*

A microsporidan isolate currently considered to represent a mixed infection of Nosema necatrix Kramer, 1965 and Thelohania diazoma Kramer, 1965 was subjected to cultivation in hosts held at various temperatures. The ratio of the Nosema (monospore) to the Thelohania (octospore) forms at these temperatures was found to vary from 1:1 at 16 C to 1:0 at 32 C. Isolation technics using mechanical, temperature and temporal methods separated monosporous from octosporous forms for inoculation purposes. However, microscopic examination of hosts receiving these inocula revealed the presence of both monospores and octospores. Electrophoretic analysis of monospores and monospore-octospore mixtures indicated equivalent hydrophobic protein spectra. These observations suggest that this isolate has the ability to produce either single spores or spores in groups of eight. This microsporidan was not considered a member of the genus Stempellia since spores in groups of 2 or 4 were not observed. Retention of the name Nosema necatrix Kramer is suggested.     

The life history of Porphyra endiviifolium from the South Shetland Islands, Antarctica

This paper describes the reproduction and life history of an intertidal species, Porphyra endiviifolium, from Antarctica. Field specimens were examined microscopically, prepared for electron microscopy and used to establish cultures. Wild populations comprised two kinds of leafy thalli, morphologically similar but distinguished by their mode of reproduction, either sexual or asexual. Carpospores from monoecious leafy gametophytes developed into conchocelis filaments in culture, and under “winter-spring” conditions these formed conchospores that germinated to produce leafy thalli. Monospores from asexual leafy thalli developed directly into two different forms of leafy thalli. Only one of the cultured morphotypes became fertile, reproducing asexually by monospores. We conclude that the phases of the life history of P. endiviifolium show different ecological strategies, the conchocelis phase reproducing in response to short days unlike the leafy thalli in which growth and reproduction respond primarily to irradiance. Received: 2 May 1997 / Accepted: 11 October 1997     

Freezing tolerance of <Emphasis Type="Italic">Porphyra yezoensis</Emphasis> (Bangiales,Rhodophyta) gametophyte assessed by chlorophyll fluorescence

In January and February 2010, heavy sea ice formed along the coast of the Bohai Sea and the northern Yellow Sea, China. Intertidal organisms were subjected to serious freezing stress. In this study, we investigated the freezing tolerance of the upper intertidal economic seaweed Porphyra yezoensis. The maximum photochemical efficiency of PS II (F _v/F _m) in undehydrated thalli remained high after 24 h at −2°C and that in dehydrated thalli decreased in a proportion to thallial water loss. F _v/F _m dropped sharply after 24 h at −20°C, regardless of absolute cellular water content (AWC). The F _v/F _m in frozen thalli recovered rapidly at 0–20°C. A wide range of water loss in the thalli enhanced their tolerance to freezing. F _v/F _m values in undehydrated thalli dropped sharply after 3 d at −2°C or 10 d at −20°C while those in dehydrated thalli (20–53% AWCs) remained at high levels after 9 d at −2°C or 30 d at −20°C. These results indicate that P. yezoensis has high freezing tolerance by means of dehydration during the ebb tide and rapid recovery of F _v/F _m from freezing. A strategy of P. yezoensis industry to avoid heavy loss during freezing season is discussed based on these findings.     

Beispiel der Wirkung der Einsporkulturauslese als züchterische Methode beim Kulturchampignon

Zusammenfassung Drei nach einer Reihe von Vorversuchen als ertragreich selektierte Einsporkulturen wurden in sieben Ertragsprüfungen mit der Handelssorte verglichen, von der sie in der F₂ abstammen. Es konnten statistisch gesicherte Mehrerträge festgestellt werden. Die Einsporkulturauslese ist demnach eine für die züchterische Bearbeitung des Kulturchampignons geeignete Methode.

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On the use of monospores in breeding selected strains of cultivated mushrooms

Summary Three monospore cultures, selected for their high yield in earlier tests, were cultivated in seven yield tests and compared with the commercial strain from which they had been derived in F₂. Statistically significant higher yields were found. Selection by means of monospore cultures has thus been shown to be a method suitable for breeding strains of cultivated mushrooms.

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Angenommen durch W. Seyffert     

Nuclear division of the vegetative cells, conchosporangial cells and conchospores of Porphyra yezoensis (Bangiales, Rhodophyta)

To confirm the position and timing of meiosis in Porphyra yezoensis Ueda, the nuclear division of vegetative cells, conchosporangial cells and conchospores was observed. An improved staining method using modified carbol fuchsin was introduced to stain the chromosomes of Porphyra. Pit‐connections between conchosporangial cells also stained well with this method. Leptotene, zygotene, pachytene, diplotene, diakinesis, metaphase, anaphase and telophase were observed in the conchosporangial cells. During the germination of conchospores, no characteristics of meiosis I were found. No difference between the nuclear division of vegetative cells and that of conchospores was observed, and 2–3 days were needed for the first cell division both in vegetative cells and conchospores. Therefore, the cell division that occurs during conchospore germination is not meiosis I. Our results indicate that the prophase of meiosis I begins during the formation of conchosporangial branches, and metaphase I, anaphase I and telophase I take place during the maturation of conchosporangial branches. Then the three‐bivalent nucleate sporangia complete cell division to form two individual conchospores, each with one three‐univalent nucleus. The conchospores released from the sporangia are at meiotic interphase. Meiosis II occurs at the first nuclear division during conchospore germination, which is a possible explanation for the observation of mosaic thalli in mutant germlings of P. yezoensis. The mosaic thalli might also arise from gene conversion/post meiotic segregation events, comparable to those in Sordaria fimicola (Roberge ex Desm.) Ces. & De Not. and Neurospora crassa Shear & B.O. Dodge.     

Artificial Seed Production and Cultivation of the Edible Brown Alga, Sargassum Fulvellum (Turner) C. Agardh: Developing a new Species for Seaweed Cultivation in Korea

Sargassum fulvellum is a brown alga recently introduced to the seaweed cultivation industry in Korea. There is current interest in the commercial scale of aquaculture of this species. For the artificial seeding and cultivation of this alga, growth and maturation were investigated from September 2002 to August 2003. Indoor culture experiments for maturation induction were also conducted at temperatures of 5, 10, 15, 20 and 25 ^∘C and irradiances of 20, 50, 80 and 100 μmol photons m⁻² s⁻¹ under 16:8 h (L:D) photoperiod. Within a given culture test range, higher temperature and irradiance levels favoured the maturation of receptacles in S. fulvellum. Using temperature and irradiance control for thalli, artificial seed production of this species could be done one month earlier than thalli matured in nature. Under natural condition, receptacle formation of the plants began in February, and the eggs were released from March to April. For mature thalli of 200 g wet wt., artificial seeding was complete enough for attachment on seed strings of 100 m. Mean production obtained from the artificial seeding technique in situ was 3.0 kg wet wt m⁻¹ of culture rope during the cultivation period.     

Isolation of Porphyran-Degrading Marine Microorganisms from the Surface of Red Alga,Porphyra yezoensis

Marine microorganisms degrading porphyran (POR) were found on the surface of thalli of Porphyra yezoensis. Fifteen crude microorganism groups softened and liquefied the surface of agar-rich plate medium. Among these, 11 microorganism groups degraded porphyran that consisted of sulfated polysaccharide in Porphyra yezoensis. Following isolation, 7 POR-degradable microorganisms were isolated from the 11 POR-degradable microorganism groups.