Postharvest biocontrol of Monilinia laxa,Monilinia fructicola and Monilinia fructigena on stone fruit by two Aureobasidium pullulans strains

The antagonistic effects of yeasts, L1 and L8, isolated from carposphere of ‘Redhaven’ peaches were tested for the first time in the same experiment against three Monilinia species (Monilinia laxa, Monilinia fructicola and Monilinia fructigena) in in vitro and in vivo trials. The two antagonists were selected after preliminary assays for their ability to reduce brown rot in peaches and nectarines, and both were identified by molecular and morphological tools as Aureobasidium pullulans. In in vivo trials, neither the autoclaved cells, nor the sterile culture filtrates of either antagonist showed any significant reduction of rot incidence produced by inocula of the three Monilinia species, while the washed cells of L1 and L8 completely inhibited M. laxa and M. fructicola rots and reduced M. fructigena infections by 70% and 90%, respectively. In other trials, nectarines treated with antagonist cells and inoculated with the pathogens were stored at 0 °C for 21 days, plus 7 days at 20 °C. The low temperature reduced brown rot development, since all fruit were free from disease symptoms on removal from cold storage. However after 7 d at 20 °C, untreated fruit were rotted over 45% depending on the Monilinia species but the antagonists completely inhibited M. laxa and M. fructicola, while M. fructigena infections were reduced by 89.8% and 91.2% by L1 and L8, respectively. For both strains, 10⁸ CFU ml^?1 was the most active concentration, although L1 showed good activity at a concentration of 10⁷ CFU ml^?1. Isolate L8 at the concentration of 10⁷ CFU ml^?1 was ineffective against M. fructicola and M. fructigena, showing no difference between treated fruit and the control, excepting the case of nectarines inoculated with M. laxa, where L8 at the concentration of 10⁷ CFU ml^?1 reduced the brown rot infections with respect to the control. The increase in population density of A. pullulans strains L1 and L8 in the wounds of nectarines stored at 0° or 20 °C was low but sufficient to control brown rot. In conclusion, the present preliminary study identified two antagonistic strains of A. pullulans as active ingredients for the development of biofungicides for postharvest application against three Monilinia species that are responsible for high economic losses in stone fruit crops.     

Production of volatile organic compounds by Aureobasidium pullulans as a potential mechanism of action against postharvest fruit pathogens

Two Aureobasidium pullulans strains (L1 and L8), effective against some fruit postharvest pathogens were evaluated for VOCs production as a part of their modes of action towards five pathogens (Botrytis cinerea, Colletotrichum acutatum, Penicillium expansum, Penicillium digitatum and Penicillium italicum). The VOCs were assayed with a double petri dish assay against conidia germination of target pathogens. Results obtained showed that the VOCs generated by the antagonists inhibited significantly the conidia germination of all pathogens compared to the control. In particular, the conidia germination of all Penicillium was completely inhibited by VOCs produced by L1 and L8. In in vivo tests, apples and oranges were artificially inoculated with pathogen conidia and then biofumigated with VOCs emitted by both antagonists. The antagonistic treatment controlled significantly pathogen infection, confirming the results obtained in vitro tests. The best L1 and L8 VOCs activity was observed on apple inoculated with B. cinerea where the lesion diameter reduction observed was greater than the 88%. The compounds emitted by L1 and L8 strains were identified with the solid-phase microextraction (SPME)–gas chromatographic technique. Compounds as 2-phenyl, 1-butanol-3-methyl, 1-butanol-2-methyl and 1-propanol-2-methyl belonging to the group of alcohols were mainly produced for both strains, in the first 96 h of growth. These compounds were confirmed by comparison with standards. The pure compounds of VOCs cited above were used to determine the EC₅₀ values for conidia germination of pathogens. The 1-propanol-2-methyl was the VOC least active against all tested fungi, with the EC₅₀ values over 0.8 μl ml⁻¹, while the 2-phenethyl alcohol was the most active with EC₅₀ values lower than 0.8 μl ml⁻¹, except for the C. acutatum (1.97 μl ml⁻¹). The present study demonstrated, for the first time, that the production of VOCs could play an essential role in the antagonistic activity of two A. pullulans strains against five fruit postharvest pathogens.     

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.     

Simultaneous determination of midazolam and its metabolites 1-hydroxymidazolam and 4-hydroxymidazolam in human serum using gas chromatography–mass spectrometry

A method for the quantitation of midazolam and its metabolites 1-hydroxymidazolam and 4-hydroxymidazolam from human serum capable of monitoring concentrations achieved under therapeutic conditions is presented. The substances were extracted under basic conditions with toluene and the hydroxy metabolites transformed to their tert-butyldimethylsilyl derivatives with N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide. The samples were measured by gas chromatography–mass spectrometry. The limits of detection are 0.2 ng ml⁻¹ for midazolam and 0.1 ng ml⁻¹ for 1-hydroxy- and 4-hydroxymidazolam. The coefficients of variation are 3.9% at 5 ng ml⁻¹ for midazolam, 6.7% at 2 ng ml⁻¹ for 1-hydroxymidazolam and 8.8% (22.2%) at 0.5 (0.2) ng ml⁻¹ for 4-hydroxymidazolam.     

Biocontrol of major postharvest pathogens on apple using Rhodotorula glutinis and its effects on postharvest quality parameters

The biocontrol activity of Rhodotorula glutinis on gray mold decay and blue mold decay of apple caused by Botrytis cinerea and Penicillium expansum, respectively, was investigated, as well as its effects on postharvest quality of apple fruits. The results show there was a significant negative correlation between concentrations of the yeast cells and the disease incidence of the pathogens. The higher concentration of the R. glutinis, the better effect of the biocontrol capacity. At concentrations of R. glutinis 1 × 10⁸ CFU ml^?1, the amount of gray mold decay was completely inhibited after 5 days incubation at 20 °C, after challenge with B. cinerea spores suspension of 1 × 10⁵ spores ml^?1; While the blue mold decay was completely inhibited at concentrations of 5 × 10⁸ CFU ml^?1, at challenged with P. expansum spores suspension of 5 × 10⁴ spores ml^?1_. These results demonstrated that the efficacy of R. glutinis in controlling of gray mold decay of apples was better than the efficacy of controlling blue mold. R. glutinis within inoculated wounds on apples increased in numbers at 20 °C from an initial level of 9.5 × 10⁵ CFU per wound to 2.24 × 10⁷ CFU at 20 °C after 1 day. The highest population of the yeast was recovered 4 days after inoculation, the yeast population in wounds increased by 56.9 times. After that, the population of the yeast began to decline very slowly. R. glutinis significantly reduced the incidence of natural infections on intact fruit from 75% in the control fruit to 28.3% after 5 days at 20 °C, and from 58.3 to 6.7% after 30 days at 4 °C followed by 4 days at 20 °C. R. glutinis treatment had no deleterious effect on quality parameters after 5 days at 20 °C or after 30 days at 4 °C followed by 4 days at 20 °C.     

Production of the postharvest biocontrol agent Bacillus subtilis CPA-8 using low cost commercial products and by-products

The aim of this research was to identify a low cost medium based on commercial products and by-products that provided maximum Bacillus subtilis CPA-8 growth and maintained biocontrol efficacy. Low cost media combining economical nitrogen and carbon sources such as yeast extract, peptone, soy products, sucrose, maltose and molasses were tested. Tests were carried out in 250-ml flasks containing 50 ml of each tested medium. Maximum cell growth (>3 × 10⁹ CFU ml^?1) was obtained in defatted soy flour 44% combined with sucrose or molasses media. Second, CPA-8 production was scaled up in a 5-l fermenter and CPA-8 population dynamics, pH and oxygen consumption in the optimized medium (defatted soy flour 44% – molasses) was recorded. In these tests, there was a 5-h lag phase before growth, after which exponential growth occurred and maximum production was 3 × 10⁹ CFU ml^?1 after 20 h. Fruit trials with cells and cell free supernatants from CPA-8 grown in optimized medium maintained biocontrol efficacy against Monilinia fructicola on peaches, resulting in disease reductions up to 95%. CPA-8 populations survived in wounds on inoculated peaches, regardless of the culture media used. The results show that B. subtilis CPA-8 can be produced in a low cost medium combining inexpensive nitrogen and carbon sources (40 g l^?1 defatted soy flour 44%, 5 g l^?1 molasses plus mineral trace supplements) in shake flasks and a laboratory fermenter (5 l). The results could be used to provide a reliable basis for scaling up the fermentation process to an industrial level.     

Impact of microcarrier coverage on using permittivity for on-line monitoring high adherent Vero cell densities in perfusion bioreactors

The paper presents the interest of on-line permittivity monitoring to estimate the density of Vero cells growing on microcarriers (MCs), even when high cell densities were reached in perfusion bioreactors (4.5 × 10⁶ cells ml⁻¹). Cultures were performed with various MCs concentrations in a reactor equipped with a settling tube. A linear correlation between on-line permittivity and off-line volumetric cell concentration was observed provided that MCs are not fully covered by cells. High permittivities such as 250 pF cm⁻¹ could be measured without signal saturation of the Fogale Biomass system^®. The correlation was no longer linear when cell density per carrier exceeded 100% cell confluency corresponding to 150 cells MC⁻¹ (0.15 × 10⁶ cells cm⁻²). This behaviour was attributed to the decrease of cell volume when cells saturated MCs surface. It mainly happened when low MCs concentration and continuous medium renewing were used. Therefore, permittivity sensor can be considered as a reliable tool to monitor on-line adherent cell densities not exceeding total cell confluency. Moreover, it could be useful to detect when cell confluency occurs.     

Expression and purification of recombinant feline interferon in the baculovirus-insect larvae system

Feline interferons (FeIFNs) are cytokines with antiviral, antitumor and immunomodulatory functions used as therapeutic agents in a variety of veterinary diseases. In this work, FeIFN-α7 and FeIFN-α7xArg containing eight residues of arginine were expressed in Sf9 cells and insect larvae. At 4 days post-infection (dpi), the concentrations of FeIFN-α7 and FeIFN-α7xArg in suspension culture were (1.28 ± 0.15) × 10⁶ U ml⁻¹ and (1.3 ± 0.2) × 10⁶ U ml⁻¹ respectively. The maximum expression levels of FeIFN-α7 and FeIFN-α7xArg were (3.7 ± 0.2) × 10⁶ U ml⁻¹ and (3.5 ± 0.4) × 10⁶ U ml⁻¹ at 2 dpi in Rachiplusia nu larvae and (1.1 ± 0.2) × 10⁶ U ml⁻¹ and (1.0 ± 0.15) × 10⁶ U ml⁻¹ at 5 dpi in Spodoptera frugiperda larvae respectively. R. nu was a better host for FeIFN-α7 and FeIFN-α7xArg expression. The 8xArg tag did not affect the biological activity of FeIFN-α7 and was useful to promote the FeIFN-α7xArg adsorption on ion exchange chromatography (IEC), allowing its purification in a single step from supernatant culture and R. nu larvae. FeIFN-α7xArg was purified from the larval extract with a yield of 70% and a purification factor of 25 free of viruses. We conclude that R. nu larvae are new low-cost hosts for the expression of recombinant FeIFN-α7.     

Differential effects of typhoons on ichthyotoxic Cochlodinium polykrikoides red tides in the South Sea of Korea during 2012–2014

The harmful dinoflagellate Cochlodinium polykrikoides is known to cause fish death by gill-clogging when its abundance exceeds approximately 1000 cells ml⁻¹. Thus, red tides of this dinoflagellate have caused considerable loss in the aquaculture industry worldwide. Typhoons carrying strong winds and heavy rains may alter the process of red tide events. To investigate the effects of typhoons on C. polykrikoides red tides, daily variations in the abundance of C. polykrikoides, and wind speeds in three study areas in the South Sea of Korea were analyzed during the periods of C. polykrikoides red tides and the passage of 14 typhoons during 2012–2014. The typhoons differentially affected Cochlodinium red tides during the study period, and the daily maximum wind speed generated by the typhoon was critical. Four typhoons with daily maximum wind speeds of >14 m s⁻¹ eliminated Cochlodinium red tides, while three typhoons with daily maximum wind speed of 5–14 m s⁻¹ only lowered the abundance. However, other typhoons with daily maximum wind speeds of <5 m s⁻¹ had no marked effect on the Cochlodinium abundance. Therefore, typhoons may sometimes eliminate C. polykrikoides red tide events, or reduce cell abundances to a level that is not harmful to caged fish cultivated in aquaculture industries. Thus, typhoons should be considered when compiling red tide dynamics and fish-kill models.     

Glucocorticoid receptors on and in a unicellular organism,Cryptobia salmositica

This is the first report to our knowledge that demonstrates a functional steroid hormone receptor in a protozoon. The study used Cryptobia salmositica, a pathogenic haemoflagellate found in salmonid fishes. It has been previously shown that cortisol and dexamethasone (a synthetic glucocorticoid) enhanced the multiplication of C. salmositica under in vitro conditions indicating the presence of glucocorticoid receptors on/in the parasite. Also, the glucocorticoid receptor antagonist, mifepristone (RU486), inhibited the stimulatory effect of the two glucocorticoids on parasite multiplication. In the present study, we used an antibody (produced in a rabbit against glucocorticoid receptor protein) agglutination test and confocal microscopy with immunohistofluorescence staining to demonstrate cortisol-glucocorticoid receptor-like protein receptors on the plasma membrane and in the cytoplasm of the parasite. In two in vitro studies, the addition of 50 ng ml⁻¹ of RU486 was more effective in inhibiting parasite replication in cultures with 7,000 parasites ml⁻¹ than in cultures with 14,000 parasites ml⁻¹. Also, 100 ng ml⁻¹ of RU486/ml was more effective than 50 ng ml⁻¹ in inhibiting parasite multiplication in the 14,000 parasites ml^-1 cultures. These in vitro studies indicate that the number of binding sites on/in the parasite is finite. The findings may be important in future studies especially on steroid receptor signalling pathways and dissection of ligand–receptor interactions, and for evaluating the adaptations that develop in pathogens as part of the host–parasite interaction.     

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.     

Efficacy assessment of Pichia guilliermondii strain Z1, a new biocontrol agent,against citrus blue mould in Morocco under the influence of temperature and relative humidity

The interactions of Penicillium italicum, which causes blue mould, and antagonistic yeast Pichia guilliermondii strain Z1 were examined in controlled environments, to determine the influence of relative humidity (RH) (45%, 75%, 85%, 98%, and 100%) and temperature (T) (5, 10, 15, 20, and 25 °C). All main effects and interactions were significant (P ? 0.05), with the exception of interactions RH×T and strain Z1 (BCA)×RH×T. In the pathogen control, the lesion diameter of blue mould developed under all environmental conditions but was the largest at a RH range between 98% and 100%, independent of the temperature. The efficacy of strain Z1 appeared to be independent of the environment and reduced disease incidence by more than 85% in all environmental conditions. Rapid colonization of the antagonistic yeast strain Z1 on citrus wounded sites was recorded during the first week at 5 °C. Colonization then stabilized at ±6.9 × 10⁶ CFU/ml for 30 days. This indicates that P. guilliermondii is able to adapt itself and colonize the wound sites prior to the arrival of the pathogen, displaying greater efficiency than when colonizing wounds after pathogen. The antagonist was capable of growing in low concentrations of orange juice (0.1–5%), with greatest growth at 5%. Applying strain Z1 (1 × 10⁸ CFU/ml) as a formulated product significantly reduced the incidence of infected fruits and the percentage of infected wounds relative to the pathogen control. Disease control with formulated product (45%) was slightly lower than that obtained with thiabendazole (20%) or strain Z1 culturable cells (25%). These results suggest that strain Z1 may be a useful BCA for control of blue mould under varying environmental conditions, and control may be enhanced by combining with other eco-friendly post-harvest treatments or improved formulation.     

Effect of Pichia membranaefaciens in combination with salicylic acid on postharvest blue and green mold decay in citrus fruits

The separate or combined effects of Pichia membranaefaciens and salicylic acid (SA) on the control of blue and green mold decay in citrus fruits were investigated. Results indicate that combining P. membranaefaciens (1 × 10⁸ CFU ml⁻¹) with SA (10 μg ml⁻¹) either in a point-inoculated or dipped treatment provided a more effective control of blue and green mold than separately applying yeast or SA. SA (10 μg ml⁻¹) did not significantly affect P. membranaefaciens growth in vitro but slightly increased the yeast population in fruit wounds. P. membranaefaciens plus SA effectively enhanced the phenylalanine ammonia-lyase, peroxidase, polyphenoloxidase, chitinase, and β-1,3-glucanase activities and stimulated the synthesis of phenolic compounds. The combined treatment did not impair quality parameters such as weight loss or titratable acidity, but resulted in low average natural infection incidence and increased total soluble solids and ascorbic acid contents in citrus fruits after 14 d at 20 °C.     

Scales characterising a high density thin layer of Dinophysis acuta Ehrenberg and its transport within a coastal jet

An investigation into the distribution of Dinophysis spp. in coastal waters off the south coast of Ireland was carried out in July 2007. Dinophysis acuta was present as a sub surface layer containing up to 55,000 cells L⁻¹. The population had a high percentage of viable cells (mean: 89%; median: 94%; n = 24) with a high specific division rate (∼0.55 d⁻¹). The layer, of approximately 5 m thickness, did not coincide with the fluorescence maximum and was present as a patch of horizontal dimension less than 10 km × 7 km. Both conventional and towed undulating CTD used in conjunction with high vertical resolution sampling methods showed the patch of Dinophysis to move with a similar speed and direction as the coastal flow, which ran parallel to the coast in the form of a coastal jet with speed of the order of 6.5–7 km day⁻¹. The implications of the alongshore transport of populations of harmful species in coastal jets for monitoring programmes and predictive models are discussed.     

Biological control of Botrytis bunch rot in Atlantic climate vineyards with Candida sake CPA-1 and its survival under limiting conditions of temperature and humidity

Candida sake CPA-1 is an antagonistic yeast that has previously been shown to effectively control Botrytis bunch rot in grapes. The efficacy of biological control agents is dependent on their survival, which may also depend on climatic conditions. However, few studies have evaluated the effect of abiotic factors affecting the survival of biological control agents, such as temperature (T) or relative humidity (RH). In this study, efficacy of C. sake (5 × 10⁷ CFU mL⁻¹), which was applied with the additive Fungicover (FC; 50 g L⁻¹), was tested against BBR in the laboratory and in field trials under the Atlantic climate conditions of the Bordeaux region (France). The study also evaluated the survival of C. sake under T and RH regimes simulated in climatic chambers. Two or five applications of C. sake plus FC during the growing season significantly reduced BBR severity at harvest by 48% and 82%, respectively, when compared to the control. Similar reductions were achieved after inoculation with selected virulent Botrytis cinerea strains (75% compared to control) in laboratory experiments. C. sake populations showed minimal decreases between field applications and were favored by simulated Atlantic climate conditions. The survival pattern of C. sake exposed to 40 and 45 °C combined with 30% and 100% of RH was described, demonstrating a sharp decrease during the first 24 h. Allowing 48 h for C. sake to incubate and become established on fruits prior to the exposure to 40 °C and 30% RH increased survival (P < 0.05). These results confirm the efficacy of treatment with C. sake plus FC under favorable climatic conditions for BBR development, while survival studies may help to improve the survival and efficacy of yeast BCAs, such as C. sake CPA-1.     

Estimation of the nutrient consumption by various cell sizes of the diatom Eucampia zodiacus: A representative organism causing bleaching of aquacultured nori

The diatom Eucampia zodiacus is a harmful species that indirectly causes bleaching to nori (Pyropia) cultivation through competitive utilization of nutrients during its bloom, however cellular storage and changes in physiology by asexual reproduction remains unclear. In the present study, we experimentally investigated the nitrate (N), phosphate (P) and silicic acid (Si) consumption by various cell sizes of E. zodiacus strains, the apical axis length of which ranged from 10.2 to 77.3 μm. Nutrient cell quotas of E. zodiacus ranged from 2.7 to 8.4 pM cell⁻¹ for N, 0.34–0.76 pM cell⁻¹ for P and 1.7–7.3 pM cell⁻¹ for Si, and they increased with cell size, in which there is a significant correlation between these two elements. The N and P quotas were estimated to be several times higher than the minimum cell quotas. In contrast, the Si cell quotas were approximately equal to those of the minimum values. Based on the present cell quotas, total nitrate consumption by E. zodiacus population when the blooms reached maximum cell density (=1000 cells ml⁻¹) were estimated to be 6.5 μM. Monthly mean concentrations of dissolved inorganic nitrogen (DIN) range from 3.5 to 8.2 μM during the period of late nori harvest season when E. zodiacus blooms occur, and nori bleaching is reported at the condition of DIN concentration of less than 3 μM in Harima-Nada, eastern Seto Inland Sea, Japan. Therefore, the present results suggest that E. zodiacus causes serious damage to nori cultivation due to high levels of nutrient consumption.     

Alteration of plankton communities and biogeochemical cycles by harmful Cochlodinium polykrikoides (Dinophyceae) blooms

Cochlodinium polykrikoides is a globally distributed, ichthyotoxic, bloom-forming dinoflagellate. Blooms of C. polykrikoides manifest themselves as large (many km²) and distinct patches with cell densities exceeding 10³ ml⁻¹ while water adjacent to these patches can have low cell densities (<100 cells ml⁻¹). While the effect of these blooms on fish and shellfish is well-known, their impacts on microbial communities and biogeochemical cycles are poorly understood. Here, we investigated plankton communities and the cycling of carbon, nitrogen, and B-vitamins within blooms of C. polykrikoides and compared them to areas in close proximity (<100 m) with low C. polykrikoides densities. Within blooms, C. polykrikoides represented more than 90% of microplankton (>20 μm) cells, and there were significantly more heterotrophic bacteria and picoeukaryotic phytoplankton but fewer Synechococcus. Terminal restriction fragment length polymorphism analysis of 16S and 18S rRNA genes revealed significant differences in community composition between bloom and non-bloom samples. Inside the bloom patches, concentrations of vitamin B₁₂ were significantly lower while concentrations of dissolved oxygen were significantly higher. Carbon fixation and nitrogen uptake rates were up to ten times higher within C. polykrikoides bloom patches. Ammonium was a more important source of nitrogen, relative to nitrate and urea, for microplankton within bloom patches compared to non-bloom communities. While uptake rates of vitamin B₁ were similar in bloom and non-bloom samples, vitamin B₁₂ was taken up at rates five-fold higher (>100 pmol⁻¹ L⁻¹ d⁻¹) in bloom samples, resulting in turn-over times of hours during blooms. This high vitamin demand likely led to the vitamin B₁₂ limitation of C. polykrikoides observed during nutrient amendment experiments conducted with bloom water. Collectively, this study revealed that C. polykrikoides blooms fundamentally change microbial communities and accelerate the cycling of carbon, some nutrients, and vitamin B₁₂.     

THE EFFECT OF OXYGEN DEPRIVATION ON INORGANIC NITROGEN UPTAKE IN AN ANTARCTIC MACROLICHEN

Snow meltwater containing 36 ng ml⁻¹NO₃⁻-N (raised here to between 95–101 ng ml⁻¹NO₃⁻-N) and 112 ng ml⁻¹NH₄⁺-N was sprayed onto illuminatedUsnea sphacelataat 2°C in a 2-1 capacity transparent perspex chamber force-ventilated with either air or O₂- (and CO₂-) free N₂. The NO₃-concentration in meltwater recirculated through a layer ofU. sphacelatafell toc. 8 ng ml⁻¹after 1·25 h. Although the pattern of decline was broadly comparable in both air and N₂, the initial rate of decline was lower in N₂. When undepleted meltwater was continuously sprayed onto the lichen and the effluent collected for analysis, the lichen was found to retain 55% of the wet deposited NO₃⁻in air but only 27% under N₂. Up to 90% of NH₄⁺supplied in a continuous spray of meltwater was retained by the lichen but this was affected little by O₂and CO₂deprivation.     

Quantitative analysis of sesquiterpene lactones and thymol derivatives in extracts from Telekia speciosa

A high-performance liquid chromatographic method based on a gradient elution and the application of core–shell type stationary phase was developed to estimate contents of sesquiterpene lactones and monoterpenoid thymol derivatives in the tissues of Telekia speciosa. The detection and quantification limits of the analytes were 0.05–0.29 μg ml⁻¹ and 0.15–0.89 μg ml⁻¹, respectively. Calibration curves showed good linearities (R² > 0.9996) within the test ranges. Intra-day and inter-day precisions were satisfactory with RSD < 2.6%. The recoveries of the standards tested ranged from 96 to 107%. The overall time of analysis was less than 35 min. Using the method, seven major sesquiterpene lactones and one thymol derivative were quantified in different organs of the plant and plants of different origin. Aerial parts of T. speciosa accumulated miscellaneous sesquiterpene lactones, mainly of guaiane, pseudoguaiane, xanthane and eudesmane type, whereas roots of the plant contained almost exclusively isoalantolactone – an eudesmanolide of antiproliferative and anti-inflammatory activity (up to 1.2% dry weight). Flowers of T. speciosa proved to be an excellent source of xanthanolide – 8-epi-tomentosin (0.16–0.94%). Provenience of the plant material strongly influenced its biosynthetic potential.     

Characterization and comparison of thermostability of purified β-glucosidases from a mesophilic Aureobasidium pullulans and a thermophilic Thermoascus aurantiacus

The thermophilic fungus Thermoascus aurantiacus 179-5 and the mesophilic Aureobasidium pullulans ER-16 were cultivated in corn-cob by solid state fermentation for β-glucosidase production. After fermentation both enzymes were purified. The β-glucosidases produced by the strains A. pullulans and T. aurantiacus were most active at pH 4.0–4.5 and 4.5, with apparent optimum temperatures at 80 and 75 °C, respectively. Surprisingly, the enzyme produced by the mesophilic A. pullulans was stable over a wider range of pH (4.5–9.5 against 4.5–6.5) and more thermostable (98% after 1 h at 75 °C against 98% after 1 h at 70 °C) than the enzyme from the thermophilic T. aurantiacus. The t_(1/2) at 80 °C were 90 and 30 min for A. pullulans and T. aurantiacus, respectively. β-Glucosidase thermoinactivation followed first-order kinetics and the energies of denaturation were 414 and 537 kJ mol⁻¹ for T. aurantiacus and A. pullulans, respectively. The result showed that β-glucosidase obtained from the mesophilic A. pullulans is more stable than that obtained from the thermophilic T. aurantiacus.