Seasonal dynamics and spatial distribution of epiphytic dinoflagellates in Peter the Great Bay (Sea of Japan) with special emphasis on Ostreopsis species

Studies of epiphytic dinoflagellates in Peter the Great Bay, Sea of Japan in 2008–2011 revealed the presence of 13 species. Five of the species are known as potentially toxic: Amphidinium carterae, A. operculatum, Ostreopsis cf. ovata, O. cf. siamensis and Prorocentrum lima. The maximum species richness and abundance of epiphytic dinoflagellates were observed in autumn (from September to October). Ostreopsis spp. were most widely distributed and predominated, amounting to 99% of the total density of dinoflagellates. Multi-year seasonal dynamics of Ostreopsis spp. in Peter the Great Bay showed that these cells appear as epiphyton in August after maximum warming of surface waters (22–24 °С) and disappear in early November, when the water temperature decreases below 7 °С. Ostreopsis spp. proliferation occurred in September, when the water temperature was 17.2–21.0 °C. The highest densities of Ostreopsis spp. were recorded on September 9, 2010 on the rhodophyte Neorhodomela aculeata – 230 × 10³ cells g⁻¹ DW or 52 × 10³ cells g⁻¹ FW. The spatial distribution of epiphytic dinoflagellates was investigated in the near-shore areas of Peter the Great Bay during the second half of September 2010 to evaluate the role of hydrodynamic conditions. Epiphytic dinoflagellates were not found in sheltered sites having weak mixing hydrodynamics. However, the abundances of Ostreopsis spp. were significantly higher at sites having moderate turbulence compared to biotopes experiencing strong wave action. Densities of Ostreopsis spp. were not significantly different on macrophytes with branched thallus of all taxonomic divisions. However, the average cell densities of Ostreopsis spp. on green algae with branched thallus were significantly higher than on green algae having laminar thallus.     

Seasonality of Dinophysis spp. and Prorocentrum lima in Black Sea phytoplankton and associated shellfish toxicity

Plankton surveys, between 2001 and 2005 along the Russian Caucasian Black Sea Coast, revealed Dinophysis rotundata, D. caudata and Prorocentrum lima as the most ubiquitous of the known dinoflagellates associated with diarrhetic shellfish poisoning (DSP). Dinophysis spp. were first observed during the spring phytoplankton succession and persist throughout the late summer phytoplankton peak. The highest total concentration, 3000 cells/L, of D. rotundata and D. caudata was observed in April 2001. Unlike Dinophysis, P. lima was rarely observed in plankton samples but closely followed storm events with maximum cell counts of P. lima occurred in July 2002.The presence of Dinophysis in mussel (Mytilus galloprovincialis) hepatopancreas correlated with concentration with Dinophysis observed in the plankton samples. Conversely, P. lima could be found in most hepatopancreas samples collected during the May to October period. Therefore, planktonic concentration of P. lima does not reflect its availability for and consumption by shellfish.Samples of mussel hepatopancreas, from August 2002, with a corresponding Dinophysis concentration of 250 cells/L and no observable P. lima, were found to contain 0.03 ng OAE/g. This sample analyses by LC-MS/MS displayed okadaic acid (OA) and related congeners (DTX1) along with the pectinotoxins (PTX2 and PTX2sa). Highest observed levels of P. lima-induced DSP-toxicity in hepatopancreas was 0.41 g OA-equivalents/g corresponded to the highest observed planktonic cell counts of P. lima, 300 cell/L in August 2001. Cultures isolated from this sample were found to produce OA, DTX1 and their related diol esters.These data reveal a threat, represented by DSP-toxic species, at Black Sea coasts, and provide grounds for the introduction of phycotoxin control measures in the region.     

A red tide off the Myanmar coast: Morphological and genetic identification of the dinoflagellate composition

A red tide composed mainly of two Prorocentrum species was found near Kadan Island, southern Myanmar coast, on March 14, 2012. This is the first record of a red tide in the Myanmar coastal area. The red tide included three different harmful dinoflagellates: Prorocentrum rhathymum, Prorocentrum shikokuense and Alexandrium affine. Strains of these species were successfully cultured and examined with detailed morphological observation and DNA (28S rRNA gene) analyses. The strains were subjected to growth experiments under different temperatures to understand their growth capabilities. The experiments were carried out at four different temperature regimes (15, 20, 25 and 30 °C). A. affine exhibited low tolerance for the low temperature regime (15 °C), despite records of its presence in northern temperate regions, indicating this strain is adapted to the tropical environment in Myanmar. P. rhathymum and P. shikokuense exhibited broad tolerance to all given temperature ranges and showed high division rates, providing the physiological basis to form red tides.     

Effects of different levels of N- and P-deficiency on cell yield,okadaic acid,DTX-1, protein and carbohydrate dynamics in the benthic dinoflagellate Prorocentrum lima

Prorocentrum lima (Ehrenberg) Dodge is a cosmopolitan epiphytic dinoflagellate that produces biotoxins which are causative of diarrhetic shellfish poisoning (DPS). Here we report on effects of several nitrogen (N) and phosphorous (P) limited conditions on cell yield, okadaic acid (OA) and dinophysistoxin-1 (DTX-1) contents synoptically with cell carbohydrate, exopolysaccharide (EPS) and cell protein concentrations in a P. lima strain isolated from the Sacca di Goro lagoon (Northern Adriatic Sea). Batch culture experiments were set to assess changes induced by four nitrogen-limited levels (1/3-N, 1/10-N, 1/20-N, and 1/50-N) and four phosphorus-limited levels (1/3-P, 1/10-P, 1/20-P, and 1/50-P) with respect to control nutrient conditions (f/2 medium; NO₃⁻ and PO₄³⁻ concentrations: 883 and 36.3 μM, respectively; N/P ratio: 24). Low nutrients availability determined lower cell yields starting from 1/10-N and 1/3-P levels and the pattern observed was dependent on nutrient dynamics, as shown by N and P analyses performed in culture media during growth. Final cell yield decreased significantly up to 4.7- and 5.6-fold under 1/50-N and 1/50-P-limited levels with respect to control values, while cell volume increased with respect to control (up to 30% and 35% for N- and P-experiment, respectively). On overall, OA concentration ranged from 6.69 to 15.80 pg cell⁻¹, while DTX-1 ranged from 0.12 and 0.39 pg cell⁻¹ resulting in unusual high OA/DTX-1 ratios. The study indicates that protein, carbohydrate, EPS, and toxin concentrations displayed remarkable different patterns under the two kinds of nutrient deficiencies. The main differences can be summarised as: (i) significant decrease of cell protein concentration (up to 2-fold) under N-limitation, conversely no significant changes in protein concentration under P-limitation; (ii) significant increase of cell carbohydrate (up to 2.8-fold and 3.4-fold for N- and P-limitation, respectively) and cell OA amount (up to 1.9-fold and 2.3-fold, N- and P-limitation, respectively) under both N- and P-limitations, however different level-deficiency dependent patterns were displayed under the two nutrient conditions; (iii) significant increase of EPS concentration (up to 6.50-fold) under P-limitation, conversely no significant changes in EPS concentration under N-limitation. Data presented here indicate that P. lima adopts different eco-physiological strategies to face N-limitation or P-limitation. This study provides the first evidence for an increase in EPS production by benthic dinoflagellates under P-limited conditions; the ecological significance of this increase is discussed.     

Morphology and phylogeny of Prorocentrum caipirignum sp. nov. (Dinophyceae), a new tropical toxic benthic dinoflagellate

A new species of toxic benthic dinoflagellate is described based on laboratory cultures isolated from two locations from Brazil, Rio de Janeiro and Bahia. The morphology was studied with SEM and LM. Cells are elliptical in right thecal view and flat. They are 37–44 μm long and 29–36 μm wide. The right thecal plate has a V shaped indentation where six platelets can be identified. The thecal surface of both thecal plates is smooth and has round or kidney shaped and uniformly distributed pores except in the central area of the cell, and a line of marginal pores. Some cells present an elongated depression on the central area of the apical part of the right thecal plate. Prorocentrum caipirignum is similar to Prorocentrum lima in its morphology, but can be differentiated by the general cell shape, being elliptical while P. lima is ovoid. In the phylogenetic trees based on ITS and LSU rDNA sequences, the P. caipirignum clade appears close to the clades of P. lima and Prorocentrum hoffmannianum. The Brazilian strains of P. caipirignum formed a clade with strains from Cuba, Hainan Island and Malaysia and it is therefore likely that this new species has a broad tropical distribution. Prorocentrum caipirignum is a toxic species that produces okadaic acid and the fast acting toxin prorocentrolide.     

Characterization of the toxicity of Cochlodinium polykrikoides isolates from Northeast US estuaries to finfish and shellfish

Harmful algal blooms caused by Cochlodinium polykrikoides are annual occurrences in coastal systems around the world. In New York (NY), USA, estuaries, bloom densities range from 10³ to 10⁵ mL^?1 with higher densities (≥10⁴ cells mL^?1) being acutely toxic to multiple fish and shellfish species. Here, we report on the toxicity of C. polykrikoides strains recently isolated from New York and Massachusetts (USA) estuaries to juvenile fish (Cyprinodon variegates) and bay scallops (Argopecten irradians), as well as on potential mechanisms of toxicity. Cultures of C. polykrikoides exhibited dramatically more potent ichthyotoxicity than raw bloom water with 100% fish mortality occurring within ～1 h at densities as low as 3.3 × 10² cells mL^?1. More potent toxicity in culture was also observed in bioassays using juvenile bay scallops, which experienced 100% mortality during 3 days exposure to cultures at cell densities an order of magnitude lower than raw bloom water (～3 × 10³ cells mL^?1). The toxic activity per C. polykrikoides cell was dependent on the growth stages of cultures with early exponential growth cultures being more potent than cultures in late-exponential or stationary phases. The ichthyotoxicity of cultures was also dependent on both cell density and fish size, as a hyperbolic relationship between the death time of fish and the ratio of algal cell density to length of fish was found (～10³ cells mL^?1 cm^?1 yielded 100% fish mortality in 24 h). Simultaneous exposure of fish to C. polykrikoides and a second algal species (Rhodomonas salina or Prorocentrum minimum) increased survival time of fish, and decreased the fish mortality suggesting additional cellular biomass mitigated the ichthyotoxicity. Frozen and thawed-, sonicated-, or heat-killed-, C. polykrikoides cultures did not cause fish mortality. In contrast, cell-free culture medium connected to an active culture through a 5 μm nylon membrane caused complete mortality in fish, although the time required to kill fish was significantly longer than direct exposure to the whole culture. These results indicate that ichthyotoxicity of C. polykrikoides isolates is dependent on viability of cells and that direct physical contact between fish and cells is not required to cause mortality. The ability of the enzymes peroxidase and catalase to significantly reduce the toxicity of live cultures and the inability of hydrogen peroxide to mimic the ichthyotoxicity of C. polykrikoides isolates suggests that the toxicity could be caused by non-hydrogen peroxide, highly reactive, labile toxins such as ROS-like chemicals.     

Lethal effects of ichthyotoxic raphidophytes,Chattonella marina,C. antiqua,and Heterosigma akashiwo,on post-embryonic stages of the Japanese pearl oyster,Pinctada fucata martensii

The inimical effects of the ichthyotoxic harmful algal bloom (HAB)-forming raphidophytes Heterosigma akashiwo, Chattonella marina, and Chattonella antiqua on the early-life stages of the Japanese pearl oyster Pinctada fucata martensii were studied. Fertilized eggs and developing embryos were not affected following exposure to the harmful raphidophytes; however, all three algal species severely affected trochophores and D-larvae, early-stage D-larvae, and late-stage pre-settling larvae. Exposure to C. marina (5 × 10² cells ml⁻¹), C. antiqua (10³ cells ml⁻¹), and H. akashiwo (5 × 10³ cells ml⁻¹) resulted in decreased success of metamorphosis to the trochophore stage. A complete inhibition of trochophore metamorphosis was observed following exposure to C. antiqua at 5 × 10³ cells ml⁻¹ and C. marina at 8 × 10³ cells ml⁻¹. In all experiments, more than 80% of newly formed trochophores were anomalous, and in the case of exposure to H. akashiwo at 10⁵ cells ml⁻¹ more than 70% of D-larvae were anomalous. The activity rates of D-larvae (1-day-old) were significantly reduced following exposure to C. antiqua (8 × 10³ cells ml⁻¹, 24 h), C. marina (8 × 10³ cells ml⁻¹, 24 h), and H. akashiwo (10⁴ cells ml⁻¹, 24 h). The activity rates of pre-settling larvae (21-day-old) were also significantly reduced following exposure to C. antiqua (10³ cells ml⁻¹, 24 h), C. marina (8 × 10³ cells ml⁻¹, 24 h), and H. akashiwo (5 × 10⁴ cells ml⁻¹, 24 h). Significant mortalities of both larval stages were induced by all three raphidophytes, with higher mortality rates registered for pre-settling larvae than D-larvae, especially following exposure to C. marina (5 × 10²–8 × 10³ cells ml⁻¹, 48–86 h) and C. antiqua (10³–8 × 10³ cells ml⁻¹, 72–86 h). Contact between raphidophyte cells and newly metamorphosed trochophores and D-larvae, 1-day-old D-larvae, and 21-day-old larvae resulted in microscopic changes in the raphidophytes, and then, in the motile early-life stages of pearl oysters. Upon contact and physical disturbance of their cells by larval cilia, H. akashiwo, C. marina and C. antiqua became immotile and shed their glycocalyx. The trochophores and larvae were observed trapped in a conglomerate of glycocalyx and mucus, most probably a mixture of larval mucous and raphidophyte tricosyts and mucocytes. All motile stages of pearl oyster larvae showed a typical escape behavior translating into increased swimming in an effort to release themselves from the sticky mucous traps. The larvae subsequently became exhausted, entrapped in more heavy mucous, lost their larval cilia, sank, become immotile, and died. Although other toxic mediators could have been involved, the results of the present study indicate that all three raphidophytes were harmful only for motile stages of pearl oysters, and that the physical disturbance of their cells upon contact with the ciliary structures of pearl oyster larvae initiated the harmful mechanism. The present study is the first report of lethal effects of harmful Chattonella spp. towards larvae of a bivalve mollusc. Blooms of H. akashiwo, C. antiqua and C. marina occur in all major cultivation areas of P. fucata martensii during the developmental period of their larvae. Therefore, exposure of the motile early-life stages of Japanese pearl oysters could adversely affect their population recruitment. In addition, the present study shows that further research with early-life development of pearl oysters and other bivalves could contribute to improving the understanding of the controversial harmful mechanisms of raphidophytes in marine organisms.     

Killing potential protist predators as a survival strategy of the newly described dinoflagellate Alexandrium pohangense

Blooms caused by some species belonging to the dinoflagellate genus Alexandrium are known to cause large-scale mortality of fish. Thus, the dynamics of these species is important and of concern to scientists, officials, and people in the aquaculture industry. To understand the dynamics of such species, their growth and mortality due to predation need to be assessed. The newly described dinoflagellate Alexandrium pohangense is known to grow slowly, with a maximum autotrophic growth rate of 0.1 d⁻¹. Thus, it may not form bloom patches if its mortality due to predation is high. Therefore, to explore the mortality of A. pohangense due to predation, feeding on this species by the common heterotrophic dinoflagellates Gyrodinium dominans, Gyrodinium moestrupii, Luciella masanensis, Noctiluca scintillans, Oxyrrhis marina, Oblea rotunda, Polykrikos kofoidii, and Pfiesteria piscicida, as well as by the ciliate Tiarina fusus, was examined. None of these potential predators was able to feed on A. pohangense. In contrast, these potential predators were killed and their bodies were dissolved when incubated with A. pohangense cells or cell-free culture filtrates. The survival of G. moestrupii, O. marina, P. kofoidii, and T. fusus on incubation with 10 cells ml⁻¹ of A. pohangense was 20–60%, while that at the equivalent culture filtrates was 20–70%. With increasing A. pohangense cell-concentration (up to 1000 cells ml⁻¹ or equivalent culture filtrates), the survival rate of G. moestrupii, O. marina, P. kofoidii, and T. fusus rapidly decreased. The lethal concentration (LC₅₀) for G. moestrupii, O. marina, P. kofoidii, and T. fusus at the elapsed time of 24 h with A. pohangense cells (cultures of 11.4, 13.3, 1.6, and 3.3 cells ml⁻¹, respectively) was lower than that with A. pohangense filtrates (culture filtrates of 35.5, 30.6, 5.5, and 5.0 cells ml⁻¹, respectively). Furthermore, most of the ciliates and heterotrophic dinoflagellates in the water collected from the coast of Tongyoung, Korea, were killed when incubated with cultures of 1000 A. pohangense cells ml⁻¹ and equivalent culture filtrates. The relatively slow growing A. pohangense may form blooms by reducing mortality due to predation through killing potential protist predators.     

Seasonal shifts in seagrass bed primary producers in a cold-temperate estuary: Dynamics of eelgrass Zostera marina and associated epiphytic algae

The seasonal dynamics of seagrass and epiphytic algal primary production were measured in an eelgrass (Zostera marina) bed in the Akkeshi-ko estuary, Hokkaido, Japan (43°02′N, 144°52′E). During spring and early summer, eelgrass biomass increased, with a high production (maximum: 2.89 g C m⁻² day⁻¹), but the production and biomass of epiphytic algae remained low. In contrast, epiphytic algae bloomed in August, with a high production (5.21 g C m⁻² day⁻¹), but eelgrass production ceased and its biomass subsequently decreased. Therefore, the major primary producers in this eelgrass bed switched seasonally from eelgrass in spring and early summer to epiphytic algae in late summer and autumn. Epiphytic algae maintained similar productivity because of the change of photosynthetic kinetics and the dominant epiphytic diatom changed from highly adhesive species to less adhesive or filamentous small species during the bloom. This suggests that the change of epiphyte density and biomass was due to change of its loss rate, possibly due to herbivorous grazing rate. Moreover, competition between epiphytic algae and eelgrass for nutrients and light may also affect the dramatic seasonal changes in the major primary producers.     

Waterfowl grazing effects on submerged macrophytes in a shallow mediterranean lake

Waterfowl exclusion cages were set up in Sentiz Lake, an eutrophic shallow lake in León (NW of Spain) in order to determine the role of waterfowl herbivory on macrophyte biomass and species composition. Total macrophyte biomass was high during the study (250 g DW m⁻² in summer). The macrophyte community was mainly formed by Myriophyllum alterniflorum (95% cover), Ceratophyllum demersum (5%) and Potamogeton gramineus (<0.5%). High densities of co-occurring coots (Fulica atra; 24 ind/ha) and ducks (Anas penelope, A. strepera and A. platyrhynchos; 18 ind/ha) did not have a significant effect on macrophyte biomass in the lake. There were no statistical differences between total biomass inside and outside the exclosures, although plant biomass reached a higher value inside the cages than in the lake. Biomass species composition was significantly different inside and outside exclosures; C. demersum was more abundant in the cages than in the lake. P. gramineus, almost absent in the lake, became co-dominant with M. alterniflorum in some exclosures. The detailed study of M. alterniflorum flower buds in summer showed significant herbivory by coots. Flower bud abundance was lower in the lake (35% lower in June; 85% lower in July) than under waterfowl exclusion. The effect of waterfowl on macrophyte biomass in Mediterranean wetlands seems to be negligible as compared to effects identified in northern European lakes. Apart from an important role in dispersal, waterfowl in Mediterranean areas have a strong qualitative effect on the structure of plant communities by selecting most palatable species or their reproductive structures.     

Mixotrophy in the newly described dinoflagellate Yihiella yeosuensis: A small,fast dinoflagellate predator that grows mixotrophically,but not autotrophically

To investigate tropical roles of the newly described Yihiella yeosuensis (ca. 8 μm in cell size), one of the smallest phototrophic dinoflagellates in marine ecosystems, its trophic mode and the types of prey species that Y. yeosuensis can feed upon were explored. Growth and ingestion rates of Y. yeosuensis on its optimal prey, Pyramimonas sp. (Prasinophyceae), as a function of prey concentration were measured. Additionally, growth and ingestion rates of Y. yeosuensis on the other edible prey, Teleaulax sp. (Cryptophyceae), were also determined for a single prey concentration at which both these rates of Y. yeosuensis on Pyramimonas sp. were saturated. Among bacteria and diverse algal prey tested, Y. yeosuensis fed only on small Pyramimonas sp. and Teleaulax sp. (both cell sizes = 5.6 μm). With increasing mean prey concentrations, both specific growth and ingestion rates of Y. yeosuensis increased rapidly before saturating at a mean Pyramimonas concentration of 109 ng C mL⁻¹ (2725 cells mL⁻¹). The maximum growth rate (mixotrophic growth) of Y. yeosuensis fed with Pyramimonas sp. at 20 °C under a 14:10-h light-dark cycle of 20 μE m⁻² s⁻¹ was 1.32 d⁻¹, whereas the growth rate of Y. yeosuensis without added prey was 0.026 d⁻¹. The maximum ingestion rate of Y. yeosuensis fed with Pyramimonas sp. was 0.37 ng C predator⁻¹ d⁻¹ (9.3 cells predator⁻¹ d⁻¹). At a Teleaulax concentration of 1130 ng C mL⁻¹ (66,240 cells mL⁻¹), growth and ingestion rates of Y. yeosuensis fed with Teleaulax sp. were 1.285 d⁻¹ and 0.38 ng C predator⁻¹ d⁻¹ (22.4 cells predator⁻¹ d⁻¹), respectively. Thus, Y. yeosuensis rarely grows without mixotrophy, and mixotrophy supports high growth rates in Y. yeosuensis. Y. yeosuensis has the highest maximum mixotrophic growth rate with the exception of Ansanella graniferaamong engulfment feeding mixotrophic dinoflagellates. However, the high swimming speed of Y. yeosuensis (1572 μm s⁻¹), almost the highest among phototrophic dinoflagellates, may prevent autotrophic growth. This evidence suggests that Y. yeosuensis may be an effective mixotrophic dinoflagellate predator on Pyramimonas and Teleaulax, and occurs abundantly during or after blooms of these two prey species.     

Field and culture studies on the ecophysiology of the toxic dinoflagellate Alexandrium minutum (Halim) present in Greek coastal waters

In Greek coastal waters, the toxic dinoflagellate Alexandrium minutum (strain AJ879163) was detected for the first time in spring 2002. This species proliferated during spring–summer of 2002 and 2003 over a wide geographic range along the north-south Aegean Sea coastline, mostly at low concentrations (average: 10²–10³ cells L⁻¹) with one exception of higher abundance (average: 10⁵ cells L⁻¹). This study presents data on environmental (temperature, salinity, chl α, nutrients) and ecological (phytoplankton species composition, diversity, taxa dominance, community dissimilarities) parameters in the areas of A. minutum occurrence. A. minutum was isolated and grown in batch cultures used in a series of bioassay experiments for determination of its pigment composition by HPLC, half saturation constants (K_s) for nitrogen and phosphorus, and its response to different nitrogen to phosphorus (N:P) ratios.     

Morphology and molecular phylogeny of Nitzschia bizertensis sp. nov.—A new domoic acid-producer

A new toxin-producing marine diatom, Nitzschia bizertensis sp. nov., isolated from the Bizerte Lagoon (Tunisia, Southwest Mediterranean Sea) is, based on studies on eight different strains, characterized morphologically by light microscopy, transmission and scanning electron microscopy, and phylogenetically using the nuclear rDNA regions: SSU, ITS1, 5.8S, ITS2 and D1–D3 of the LSU. The species belongs to the sections Lanceolatae or Lineares as defined by Cleve and Grunow (1880). These sections are characterized by species having linear-lanceolate valves with an eccentric raphe where the fibulae does not extend into the valve, and are otherwise famous for the lack of characters useful for delineation of species. Nitzschia bizertensis differs from most other species in these sections by having a high density of interstriae. The morphological and phylogenetic studies and comparisons with previously described Nitzschia species showed Nitzschia bizertensis sp. nov. to be a new species. Batch culture experiments were conducted for estimations of maximum growth rate and production of domoic acid (DA). Maximum cellular DA content of the examined strains ranged from 2 × 10⁻⁴ to 3.6 × 10⁻² pg cells⁻¹. The total DA concentration (pg mL⁻¹) was high already in exponential growth phase maybe due to reinoculation of “old” stationary phase cells, and increased into stationary growth phase where it reached a stationary level varying among the strains from ca. 4500 to 9500 pg mL⁻¹. Nitzschia bizertensis represents a new domoic acid-producing diatom and is the second toxin producing Nitzschia species. The resolution of Nitzschia bizertensis and Nitzschia navis-varingica in different parts of the LSU phylogenetic tree, and the recovery of the Pseudo-nitzschia species phylogenetically distant from those two species suggests that the ability to produce DA either evolved multiple times independently or was lost multiple times.     

Efficacy of the antagonist Aureobasidium pullulans PL5 against postharvest pathogens of peach,apple and plum and its modes of action

The efficacy of Aureobasidium pullulans PL5 against different postharvest pathogens of fruits (Monilinia laxa on plums and peaches, Botrytis cinerea and Penicillium expansum on apples) were evaluated under storage conditions when applied at 10⁸ cells ml⁻¹ and their interactions were studied in vitro and in vivo to discover the possible modes of action. Under 1.2 °C and 95% relative humidity (RH) for 28 days, the efficacy of PL5 against M. laxa on plums was 45%, reducing disease incidence from 78% to 43%. Under 1 °C and 95% RH for 21 days, the efficacy against M. laxa on peaches was 63%, reducing disease incidence from 79% to 29%. Under 4 °C and 95% RH for 45 days, the efficacy against B. cinerea and P. expansum on apples was 56% and 46%, respectively. In Lilly–Barnett minimal salt medium with the fungal cell walls of pathogens as sole carbon source, PL5 produced β-1,3-glucanase, exo-chitinase and endo-chitinase. Nutrient concentrations had significant effect on pathogen growth reduction by PL5. No attachment was observed in antagonist–pathogen interactions in vitro or in vivo. PL5 completely inhibited pathogen spore germination in PDB at 10⁸ cells ml⁻¹, whereas at 10⁶ cells ml⁻¹ the efficacy was significantly decreased. However, inactivated cells and culture filtrate of PL5 had no effect on pathogen spore germination and germ tube elongation. Our results showed that A. pullulans PL5 could be introduced in commercial formulations to control postharvest pathogens on fruits and its activity was based on secretion of lytic enzymes and competition for nutrients.     

Proliferation of the toxic dinoflagellate Ostreopsis cf. ovata in relation to depth,biotic substrate and environmental factors in the North West Mediterranean Sea

In recent decades, the North West Mediterranean Sea has been seriously affected by the development of the toxic benthic dinoflagellate Ostreopsis cf. ovata, which is associated with harmful effects on human health and the environment. The present work aims to provide a large overview of the multiple environmental factors assumed to regulate or influence the growth of Ostreopsis. An intensive sampling campaign over a full annual cycle was performed along the French and Italian coasts (in six sites from Cassis to Genoa), to determine patterns of temporal and spatial distributions of both O. cf. ovata epiphytic and planktonic cells. Results highlighted substantial seasonal variations in the abundance of Ostreopsis. These variations correlated to seawater temperature, with an optimum growth temperature ranging from 23 °C to 27.5 °C. Phosphate concentration, rather than nitrogen or silicate, was also positively associated with Ostreopsis. Decreases in oxygen and increases in chlorophyll a concentrations were recorded during the summer blooming period. The maximal Ostreopsis epiphytic abundance was generally higher on Dictyota spp. than on the other two sampled macroalgae (up to 8.54 × 10⁶ cells g⁻¹ FW), even though statistical analysis did not support a clear substrate preference. Epiphytic abundances were significantly higher at a very shallow depth (0.5 m), than at 1 and/or 3 m depths. High anthropogenic pressure (related to population density) seems to have promoted the occurrence of blooms in urbanized areas, which could partly explain the strong demarcation in Ostreopsis development between Western and Eastern sampling sites. The ecological niche of Ostreopsis cf. ovata needs precise definition, which will require further in situ and in vitro experimental studies, to determine the relative importance of distinct environmental parameters.     

Occurrence of toxic Prymnesium parvum blooms with high protease activity is related to fish mortality in Hungarian ponds

The unicellular alga Prymnesium parvum has been responsible for toxic incidents with severe ecological impacts in many parts of the world, and causes massive fish kills worldwide. Recently the haptophyte microalgae have caused water-bloom (4.3 × 10⁴ cells ml⁻¹) in 6 fish ponds with high conductivity in Hungary, and caused fish mortality with typical symptoms. Toxicity of P. parvum from water samples was quantified by the assay of the influence of its cell-free filtrates on haemolysis (346 ± 42.2) and in fish and daphnia toxicity tests. High amount of proteases in P. parvum containing waterbloom samples were detected with the help of activity gel electrophoresis. The proteases of investigated P. parvum samples (125–18 kDa) showed high gelatinolytic activity and some of them showed sensitivity to EDTA (inhibitors of metalloproteases) and to PMSF (inhibitors of serine proteases).     

Feeding by the newly described heterotrophic dinoflagellate Stoeckeria changwonensis: A comparison with other species in the family Pfiesteriaceae

The feeding ecology of the newly described heterotrophic dinoflagellate Stoeckeria changwonensis was explored. The feeding behavior of S. changwonensis, and the kinds of prey species that it feeds on were investigated with several different types of microscopes and high-resolution video-microscopy. Additionally, the growth and ingestion rates of S. changwonensis as a function of prey concentration for perch (Lateolabrax japonicus) blood cells, the raphidophyte Heterosigma akashiwo, the cryptophytes Rhodomonas salina and Teleaulax sp., and the phototrophic dinoflagellate Amphidinium carterae prey were measured. S. changwonensis feeds on prey through a peduncle, after anchoring the prey by using a tow filament. This type of feeding behavior is similar to that of Stoeckeria algicida, Pfiesteria piscicida, and Luciella masanensis in the family Pfiesteriaceae; however, S. changwonensis feeds on various kinds of prey species different from those of the other heterotrophic dinoflagellates. S. changwonensis ingested perch blood cells and diverse algal species, in particular, the large thecate dinoflagellate Lingulodinium polyedrum which are not eaten by the other peduncle feeders. H. akashiwo and the perch blood cells supported positive growth of S. changwonensis, but R. salina, Teleaulax sp., and A. carterae which support positive growth of P. piscicida and L. masanensis did not support positive growth of S. changwonensis. With increasing mean prey concentration the growth rates for S. changwonensis on H. akashiwo and the perch blood cells increased rapidly and then slowly or became saturated. The maximum growth rates of S. changwonensis on H. akashiwo and the perch blood cells were 0.376 and 0.354 d⁻¹, respectively. Further, the maximum ingestion rates of S. changwonensis on H. akashiwo and the perch blood cells were 0.35 ng C predator⁻¹ d⁻¹ (3.5 cells predator⁻¹ d⁻¹) and 0.27 ng C predator⁻¹ d⁻¹ (29 cells predator⁻¹ d⁻¹), respectively. These maximum growth and ingestion rates of S. changwonensis on H. akashiwo, the perch blood cells, R. salina, Teleaulax sp., and A. carterae differed considerably from those of S. algicida, P. piscicida, and L. masanensis on the same prey species. Thus, the feeding behavior of S. changwonensis may differ from that of other species in the family Pfiesteriaceae.     

Effects of the herbicide 2,4-D on the growth of nine aquatic macrophytes

A study was conducted to determine the effect of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) on nine submersed macrophyte species. The first objective of the study was to investigate the sensitivity of various endpoints in macrophyte toxicity tests. A second objective was to investigate the implications of hormesis in the risk assessment of 2,4-D. 2,4-D was applied in concentrations ranging from 10 to 3000 μg L⁻¹. Endpoints determined 4 weeks after the start of the treatment were based on shoot and root growth in water. The EC₅₀s were calculated using models excluding and including a parameter describing hormesis. Results indicated that the total length of the roots can be regarded as a sensitive endpoint for the response of a macrophyte to 2,4-D. For the tested rooted macrophyte species, the EC₅₀ values for the length and number of the roots ranged from 92 to 997 and from 112 to 1807 μg L⁻¹, respectively. At low concentrations (10 and 30 μg L⁻¹), stimulation of some of the endpoints (hormesis) was found for several of the species. Although hormesis may have ecological implications, its importance for the ecological risk assessment of 2,4-D in this study was limited.     

Nucleic acid changes during photodynamic inactivation of bacteria by cationic porphyrins

Light activation of photosensitizing dyes in presence of molecular oxygen generates highly cytotoxic reactive oxygen species leading to cell inactivation. Nucleic acids are molecular targets of this photodynamic action but not considered the main cause of cell death. The in vivo effect of the photodynamic process on the intracellular nucleic acid content of Escherichia coli and Staphylococcus warneri was evaluated herein.Two cationic porphyrins (Tetra-Py⁺-Me and Tri-Py⁺-Me-PF) were used to photoinactivate E. coli (5.0 μM; 10⁸ cells mL^?1) and S. warneri (0.5 μM; 10⁸ cells mL^?1) upon white light irradiation at 4.0 mW cm^?2 for 270 min and 40 min, respectively. Total nucleic acids were extracted from photosensitized bacteria after different times of irradiation and analyzed by agarose gel electrophoresis. The double-stranded DNA was quantified by fluorimetry and the porphyrin binding to bacteria was determined by spectrofluorimetry.E. coli was completely photoinactivated with both porphyrins (5.0 μM), whereas S. warneri was only completely inactivated by Tri-Py⁺-Me-PF (0.5 μM). The hierarchy of nucleic acid changes in E. coli was in the order: 23S rRNA > 16S rRNA > genomic DNA. The nucleic acids of S. warneri were extensively reduced after 5 min with Tri-Py⁺-Me-PF but almost unchanged with Tetra-Py⁺-Me after 40 min of irradiation. The amount of Tri-Py⁺-Me-PF bound to E. coli after washing the cells is higher than Tetra-Py⁺-Me and the opposite was observed for S. warneri. The binding capacity of the photosensitizers is not directly related to the PDI efficiency or nucleic acid reduction and this reduction occurs in parallel with the decrease of surviving cells.