Centrin association with the flagellar apparatus in spores ofPhytophthora cinnamomi

Summary Antibodies raised against the calcium-binding protein centrin, were used to identify and localise centrin containing structures in the flagellar apparatus of zoospores and cysts of the oomycetePhytophthora cinnamomi. Immunoblotting of extracts from zoospores indicates that theP. cinnamomi centrin homologue is a 20 kDa protein. Immunofluorescence microscopy with anti-centrin antibodies reveals labelling in the flagella, the basal body connector and co-localisation along the microtubular R1 root (formerly called AR3) that runs from the right side of the basal body of the anterior flagellum into the anterior of the zoospore close to the ventral surface. The centrin (R1^cen) and tubulin components of the R1 root split into four loops on the right hand side of the ventral groove and rejoin along the left hand side of the groove. The R1 root continues down the left hand side of the zoospore past the basal bodies and parallel to the R4 root. We propose that at least inP. cinnamomi there is no R2 root. Immunogold labelling confirms that centrin is a component of the basal body connector complex. When the zoospores become spherical during encystment, the R1^cen pivots by approximately 90 ° with respect to the nucleus.     

Phytophthora nicotianae transformants lacking dynein light chain 1 produce non-flagellate zoospores

Biflagellate zoospores of the highly destructive plant pathogens in the genus Phytophthora are responsible for the initiation of infection of host plants. Zoospore motility is a critical component of the infection process because it allows zoospores to actively target suitable infection sites on potential hosts. Flagellar assembly and function in eukaryotes depends on a number of dynein-based molecular motors that facilitate retrograde intraflagellar transport and sliding of adjacent microtubule doublets in the flagellar axonemes. Dynein light chain 1 (DLC1) is one of a number of proteins in the dynein outer arm multiprotein complex. It is a 22 kDa leucine-rich repeat protein that binds to the catalytic motor domain of the dynein γ heavy chain. We report the cloning and characterization of DLC1 homologues in Phytophthora cinnamomi and Phytophthora nicotianae (PcDLC1 and PnDLC1). PcDLC1 and PnDLC1 are single copy genes that are more highly expressed in sporulating hyphae than in vegetative hyphae, zoospores or germinated cysts. Polyclonal antibodies raised against PnDLC1 locallized PnDLC1 along the length of the flagella of P. nicotianae zoospores. RNAi-mediated silencing of PnDLC1 expression yielded transformants that released non-flagellate, non-motile zoospores from their sporangia. Our observations indicate that zoospore motility is not required for zoospore release from P. nicotianae sporangia or for breakage of the evanescent vesicle into which zoospores are initially discharged.     

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

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

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     

Encystment of zoospores of the fungus,Phytophthora cinnamomi,is induced by specific lectin and monoclonal antibody binding to the cell surface

Summary Only two of a number of macromolecules that bind to the surface of zoospores of the dieback fungus,Phytophthora cinnamomi, induce encystment when added to a suspension of actively swimming zoospores. One, the lectin Concanavalin A (ConA), binds to the entire surface of the zoospores including the surface of both flagella. Within 10 minutes more than 70% of the cells have encysted in the presence of 5 g/ml ConA. This encystment is inhibited by preincubation of the lectin with its hapten sugar, -methyl-D-mannoside. The other effective molecule, a monoclonal antibody designated Zf-1, is one of 35 that have been raised to components on the surface of zoospores and cysts ofP. cinnamomi. The antigen for Zf-1 occurs only on the surface of the two flagella. Purified Zf-1 at 15 g/ml causes encystment of 75% of the zoospores in 13minutes. To show that the induction of encystment by these two probes is not due simply to the presence of protein either in solution or bound to the zoospore a number of other proteins were tested, including other antibodies that bind to the zoospore surface. None of these other molecules caused encystment even at concentrations greater than 200 g/ml. The results are consistent with the surface components that bind ConA and Zf-1 being involved in the critical step of triggering encystment at the surface of a potential host during infection.     

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.     

Protein storage in large peripheral vesicles in Phytophthora zoospores and its breakdown after cyst germination

The fate of large vesicles that line the periphery of Phytophthora cinnamomi zoospores was monitored by immunogold labeling during encystment and germination. Labeling was carried out using a monoclonal antibody, Lpv-1, directed against glycoprotein components of these vesicles. The results show that the vesicles are retained inside the zoospores during encystment, but their contents are degraded after germination. During initial stages of degradation, the large peripheral vesicles dilate to form small vacuoles containing fine electron-dense fibrillar material which is immunoreactive. Eventually, these small vacuoles fuse together to form large electron-lucent vacuoles which contain very little immunoreactive material. Immunoblot analysis of germinating cysts with Lpv-1 shows that the level of glycoprotein components in the vesicles declines dramatically following germination. It is proposed on the basis of this evidence that the large peripheral vesicles contain storage protein which acts as an endogenous supply of nitrogen reserves for the growing germ tube. Lpv-1 also labels peripheral vesicles in two other species of Phytophthora, P. parasitica var. nicotiana and P. nicotiana var. nicotiana. As in P. cinnamomi, these vesicles do not undergo exocytosis during encystment. The large peripheral vesicles thus appear to be analogous to protein bodies found in seeds of higher plants, and they may be a common feature of Phytophthora zoospores.     

Cell surface antigens ofPhytophthora spores: biological and taxonomic characterization

Summary The oomycetes are a class of protists that produce biflagellate asexual zoospores. Members of the oomycetes have close phylogenetic affinities with the chromophyte algae and are widely divergent from the higher fungi. This review focuses on two genera,Phytophthora andPythium, which belong to the family Pythiaceae, and the order Peronosporales. These two genera contain many species that cause serious diseases in plants. Molecules on the surface of zoospores and cysts of these organisms are likely to play crucial roles in the infection of host plants. Knowledge of the properties of the surface of these cells should thus help increase our understanding of the infection process. Recent studies ofPhytophthora cinnamomi andPythium aphanidermatum have used lectins to analyse surface carbohydrates and have generated monoclonal antibodies (MAbs) directed towards a variety of zoospore and cysts surface components. Labelling studies with these probes have detected molecular differences between the surface of the cell body and of the flagella of the zoospores. They have been used to follow changes in surface components during encystment, including the secretion of an adhesive that bonds the spores to the host surface. Binding of lectin and antibody probes to the surface of living zoospores can induce encystment, giving evidence of cell receptors involved in this process. Freeze-substitution and immunolabelling studies have greatly augmented our understanding of the synthesis and assembly of the zoospore surface during zoosporogenesis. Synthesis of a variety of zoospore components begins when sporulation is induced. Cleavage of the multinucleate sporangium is achieved through the progressive extension of partitioning membranes, and a number of surface antigens are assembled onto the zoospore surface during cleavage. Comparisons of antibody binding to many isolates and species ofPhytophthora andPythium have revealed that surface components on zoospores and cysts exhibit a range of taxonomic specificities. Surface antigens or epitopes may occur on only a few isolates of a species; they may be species-specific, genus-specific or occur on the spores of both genera. Spore surface antigens thus promise to be of significant value for studies of the taxonomy and phylogeny of these protists, as well as for disease diagnosis.Abbreviations MAbs monoclonal antibodies - ConA Concanavalin A - SBA soybean agglutinin - WGA wheat germ agglutinin - gps glycoproteins     

Thrust reversal by tubular mastigonemes: immunological evidence for a role of mastigonemes in forward motion of zoospores ofPhytophthora cinnamomi

Summary The role of tubular mastigonemes in the reversal of thrust of the anterior flagellum ofPhytophthora cinnamomi was analysed using mastigoneme-specific monoclonal antibodies and immunoflu-orescence and video microscopy. Exposure of live zoospores ofP. cinnamomi to the mastigoneme-specific Zg antibodies caused alterations in the arrangement of mastigonemes on the flagellar surface and at Zg concentrations above 0.3 /ml, mastigonemes became detached from the flagellum. As a consequence of antibody binding to the mastigonemes there were concentration-dependent perturbations in zoospore swimming behaviour and anterior flagellum beat pattern. With increasing antibody concentration zoospores swam more slowly and other parameters of their swimming pattern, such as the wavelength of the swimming helix and the frequency of rotation, were also reduced. The effects of Zg antibodies were specific at two levels: control immunoglobulins or antibodies that bound to other flagellar surface components did not have an effect on motility, and Zg antibodies did not interfere with the motility of zoospores of oomycete species to which they did not bind. The effects of antibody-induced disruption of mastigoneme arrangement strongly support previous hypotheses that tubular mastigonemes are responsible for thrust reversal by the anterior flagellum, enabling it to pull the cell through the surrounding medium.     

Forecasting infections of the red rot disease on Porphyra yezoensis Ueda (Rhodophyta) cultivation farms

Pythium porphyrae is a fungal pathogen responsible for red rot disease of the seaweed Porphyra (Rhodophyta). Infection forecasts of Porphyra by P. porphyrae were estimated from the epidemiological observations of Porphyra thalli and numbers of zoospore of P. porphyrae in laboratory and cultivation areas. Four features of forecasting infections were determined by relating zoospore concentrations to the incidence of thallus infection; infection (in more than 1000 zoospores L⁻¹), microscopic infection [less than 2 mm in diameter of lesion (in from 2000 to 3000 zoospores L⁻¹)], macroscopic infection [more than 2 mm in diameter of lesion (in from 3000 to 4000 zoospores L⁻¹), and thallus disintegration (in more than 4000 zoospores L⁻¹). High zoospore concentrations led to more infection. The tendency that zoospore concentration of P. porphyrae increased with the rate of infection of Porphyra thalli was generally observed in forecasting infections in both the laboratory and in cultivation areas. Based on the Porphyra cultivation areas, the accuracy and consistency of forecasting infections suggest that this method could be employed to manage and control red rot disease.     

Genes expressed in zoospores of<Emphasis Type="Italic"> Phytophthora nicotianae</Emphasis>

The genus Phytophthora includes many highly destructive plant pathogens. In many Phytophthora species, pathogen dispersal and initiation of plant infection are achieved by motile, biflagellate zoospores that are chemotactically attracted to suitable infection sites. In order to study gene expression in zoospores, we have constructed a cDNA library using mRNA from zoospores of Phytophthora nicotianae. The library was arrayed and screened using probes derived from mycelium or zoospore mRNA. More than 400 clones representing genes preferentially expressed in zoospores were identified and sequenced from the 5 end of the insert. The expressed sequence tags (ESTs) generated were found to represent 240 genes. The ESTs were compared to sequences in GenBank and in the Phytophthora Genome Consortium database, and classified according to putative function based on homology to known proteins. To further characterize the identified genes, a colony array was created on replicate nylon filters and screened with probes derived from four Phytophthora developmental stages including zoospores, germinating cysts, vegetative mycelium and sporulating hyphae, and from inoculated and uninoculated tobacco seedlings. Data from sequence analysis and colony array screening were compiled into a local database, and searched to identify genes that are preferentially expressed in zoospores for future functional analysis.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by C. A. M. J. J. van den Hondel     

Ultrastructure of chytridiomycete and oomycete zoospores using spray-freeze fixation

Summary The ultrastructure of zoospores of several zoosporic fungi was examined using a modified cryofixation technique. An atomizer was used to spray a zoospore suspension into the cold propane reservoir of a conventional plunge freeze-substitution apparatus. Spray-freeze fixation and freeze-substitution of zoospores porvided better fixation of vacuolar structures, membranes and the extracellular coat than that obtained with chemical fixation. The overall shape of cryofixed spores was closer to that seen in living zoospores. Two types of vacuoles were seen in cryofixed zoospores ofMonoblepharella andChytridium. One type of vacuole contained electron-opaque material within the lumen while the other type had no visible internal material in the lumen and appeared to be part of the water expulsion vacuole complex. Coated pits and coated vesicles were observed associated with both the water expulsion vacuoles and the plasma membrane inMonoblepharella andPhytophthora, suggesting that endocytosis of the plasma membrane and expulsion vacuoles is part of membrane recycling during osmoregulatory events. An extracellular coat was seen on the outer surface of cryofixed zoospores ofMonoblepharella sp.,Chytridium confervae andPhytophthora palmivora without the use of carbohydrate-specific stains. The spray-freeze method gave good and reproducible fixation of the wall-less spores in quantities greater than those obtained in previously described zoospore cryofixation studies. The technique is potentially useful for cell suspensions in that freeze damage from excess water is limited.Abbreviations ddH₂O deionized distilled water - PME Pipes/MgCl2/EGTA buffer - WEV water expulsion vacuole     

Immunological Discrimination of Phytophthora cinnamomi from other Phytophthorae Pathogenic on Chestnut

A polyclonal antiserum (A379) against water soluble proteins from Phytophthora cinnamomi mycelium was produced in rabbit. In ELISA, the 1 : 10 000 diluted antiserum revealed only Phytophthora isolates, not allowing a clear‐cut discrimination among congenerous species, in spite of a generally higher reactivity on P. cinnamomi proteins. The antiserum gave positive reactions in Western blot analyses against mycelial proteins from nine species of Phytophthora and Pythium sp. (grown on rich media), but not with Rhizoctonia solani, binucleate Rhizoctonia, Verticillium dahliae, Fusarium oxysporum and Cryphonectria parasitica. All Phytophthora species showed common epitopes on proteins of molecular masses 77, 66, 51 and 48 kDa. However, a species‐specific protein of 55 kDa was immunodecorated only in P. cinnamomi samples, thus allowing univocal identification of this species. When tested against total proteins from the same fungi grown on water, the antibody revealed diagnostic bands of 55 and 51 kDa in P. cinnamomi only. The antiserum is therefore suitable for the specific identification of P. cinnamomi emerging in distilled water from infected tissues of chestnut, blueberry and azalea.     

Ultrastructural observations of mature and encysting zoospores ofPythium proliferum de Bary

Summary The process of zoospore maturation and encystment inP. proliferum was studied by electron microscopy. General ultrastructural features of the mature, swimming zoospore were found to be similar to those previously described for other oomycetes in both the attachment and ultrastructure of the flagella as well as the type and distribution of cellular organelles. Associated with extensive areas of RER in the mature zoospores were unusual, electrondense, bar-like structures. These structures were found in the groove region of young zoospores and at the periphery of encysting zoospores. Their possible function is discussed. The five main types of vesicles observed during encystment, as seen grouped in this study, along with the vesicles described in previous studies of oomycete encystment, were in table form and individually discussed. Interesting correlations appear to exist in the types of vesicles that are present within the oomycetes studied thusfar.     

Zoospore Production Biology of Pythiaceous Plant Pathogens

Zoospores are major dispersal and infective propagules of pythiaceous species. Built upon a recently developed ‘wet‐plate’ method, the objectives of this study were to develop a better understanding about zoospore production biology. Four broth media and five incubation temperatures were evaluated with 12 isolates of Phytophthora nicotianae and 17 other pythiaceous species in this study. The ‘wet‐plate’ method worked the best for heterothallic species, especially those isolates that do not produce chlamydospores. These species included Phytophthora citrophthora, P. nicotianae, Phytophthora palmivora and Phytophthora tropicalis. They readily produced 10⁵–10⁶ zoospores/ml. Overall, most species and isolates produced more zoospores with 20% clarified V8 broth than the other three media: rye, lima bean and carrot. The optimal temperature for nutrient‐deprived culture without free‐flowing water to produce sporangia typically is 5°C cooler than that for vegetative growth. Fresh and revived cultures are more prolific than those that had been subcultured multiple times. These findings will assist oomycete researchers, adding quality, productivity and efficiency to their future zoospore‐based studies.     

The heat shock response in hydra: immunological relationship of hsp60, the major heat shock protein of Hydra vulgaris,to the ubiquitous hsp70 family

The major heat shock protein (hsp) of Hydra vulgaris has recently been found to be a 60 kDa protein. Since in all organisms studied so far, the major heat shock protein is a 70 kDa protein, we have analyzed the relationship of hydra hsp60 to the highly conserved 70 kDa heat shock protein family. Genes and proteins related to the 70 kDa class of stress proteins are present in hydra. However, antibodies known to cross-react with hsp70 proteins in several different organisms do not cross-react with hydra hsp60 suggesting that hsp60 is not related to the conserved hsp70 proteins.     

Studies on Antagonistic Effects of Trichoderma Isolates Against Phytophthora cactorum

Bioassays were used to demonstrate the antibiotic effect of Trichoderma isolates on P. cactorum. When both fungi were grown on benomyl-containing PDA medium, the mycelial growth of Trichoderma was suppressed. However, the production of antibiotics by this fungus remained active, leading to inhibition of the mycelial growth of P. cactorum. The antibiotic effect of Trichoderma on zoospores and cysts was tested on a PDA substrate precultured with Trichoderma on cellophane sheets. On the substrate of some Trichoderma isolates, lysis of zoospores, formation of extracellular vesicles, and hypertrophy of the water expulsion vesicle did occur, both resulting in the death of the zoospores. Conidial suspensions of Trichoderma isolates also induced zoospore lysis. It is presumed that membrane-active peptide antibiotics (peptaibols) are involved in zoospore lysis. The peptaibol paracelsin caused lysins of zoospores at a concentration of 2.5 × 10^?4 M. The effect on cysts depended on the Trichoderma isolate tested and the age of Trichoderma preculture. Old cultures (after beginning of sporulation) affected cysts more severely than young cultures (before sporulation) which usually were not lethal to the cysts but induced preferably microsporangium formation, inhibition of cyst germination, and retardation of germ tube growth.     

Ultrastructural evidence for the biflagellate origin of the uniflagellate fungal zoospore

Summary The morphological similarities between the kinetosome and the second centriole of the zoospores of Phlyctochytrium kniepii and P. punctatum (Chytridiomycetes) suggest that the second centriole in the chytrid zoospore is a vestigial flagellum base. It is suggested that the term vestigial kinetosome may also be used when referring to the structure which is presently termed the second centriole of the chytrid zoospore. Morphological similarities between the chytrid zoospores of P. kniepii and P. punctatum and the zoospores of Rhizidiomyces apophysatus (Hyphochytridiomycetes) are noted. The possible biflagellate origin of fungi with uniflagellate zoospores is discussed. The third fiber (C fiber) of the kinetosome triplet is shown to form as an outgrowth of the B fiber of the kinetosome doublet.     

Real-Time Polymerase Chain Reaction Assays for Rapid Detection and Quantification of <Emphasis Type="Italic">Noctiluca scintillans</Emphasis> Zoospore

Application and availability of real-time polymerase chain reaction (PCR) assay to detect and quantify the Noctiluca scintillans zoospore were investigated seasonally. Specific primer set for N. scintillans 18S rDNA was designed and applied to real-time PCR assay using the serial dilutions of N. scintillans zoospores. The real-time PCR assays with Ns63F and Ns260R primers were applied to sea water samples collected weekly in Manazuru Port of Sagami Bay, Japan from April 2005 to June 2006. We developed effective DNA preparation steps for collecting the template DNA of N. scintillans zoospore: size fraction and filter concentration of the water samples, fixation with Lugol solution, cell lysis, and purification. This method is useful for the monitoring of the zoospores of N. scintillans, and can also be used for other small and physiologically fragile planktonic cell. Variation in the density of zoospore was successfully detected in the field samples. The peak density of N. scintillans zoospore was observed to occur just before or at the same time as the peak of the vegetative cells. Moreover, zoospores were detected in seawater even when the vegetative cells were not observed. The presence of zoospore was found all year round in the present study. In this regards, this information is essential for the study of the life cycle and seasonal variation of N. scintillans in the coastal waters.