Survival and death of Chara internodal cells in electrolyte solutions and calcium release from the cell wall

Abstract. Survival and death of Chara internodal cells were investigated in one of the alkali metal salts KCl, some of the alkali earth metal salts CaCl₂, Ca(NO₃)₂, MgCl₂, Mg(NO₃)₂, SrCl₂, Sr(NO₃)₂, BaCl₂ and Ba(NO₃)₂, potassium phosphate pH buffer solution (pH 7.0), Tris-maleate pH buffer solution (pH 7.0), HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulphonic acid)-KOH (pH 7.0) pH buffer solution, calcium buffer solutions, and deionized water. Most of the internodal cells died within a day or a few days in KCl, MgCl₂, Mg(NO₃)₂, BaCl₂ and Ba(NO₃)₂ solutions of higher concentrations, calcium buffer solutions of pCa 6.0, 10.0 mol m^-3 potassium phosphate pH buffer solution and 10.0 mol m^-3 Trismaleate pH buffer solution. However, all of the internodal cells survived more than 10 d in deionized water, 80.0 mol m^-3 CaCl₂, 80.0 mol m^-3 Ca(NO₃)₂, 80.0 mol m^-3 SrCl₂, 80.0 mol m^-3 Sr(NO₃)₂ calcium buffer solutions of pCa 4.0 and pCa 5.0, and 10.0 mol m^-3 HEPES-KOH (pH 7.0) pH buffer solution. Addition of Ca²⁺ or Sr²⁺ to K⁺, Mg²⁺ and Ba²⁺ salt solutions increased the survival rates of the internodal cells. Calcium release from the internodal cell wall was measured in deionized water, KCl, NaCl, MgCl₂, CaCl₂, SrCl₂ and BaCl₂ solutions. Except in deionized water and CaCl₂ solution, most of the calcium binding to the cell wall was released within one or a few hours in respective electrolyte solutions. Thus, survival and death of the internodal cells in the electrolyte solutions tested were interpreted in terms of the calcium release from the cell wall and the cell membrane, and intrinsic ability of Sr²⁺ to maintain the cell membrane normal.     

Synthesis and Antifungal Activities of Trichodermin Derivatives as Fungicides on Rice

Twenty new trichodermin derivatives, 2a – 5 , containing alkoxy, acyloxy, and Br groups in 4‐, 8‐, 9‐, 10‐ and 16‐positions were synthesized and characterized. The antifungal activities of the new compounds against rice false smut (Ustilaginoidea virens), rice sheath blight (Rhizoctonia solani), and rice blast (Magnaporthe grisea) were evaluated. The results of bioassays indicated that the antifungal activities were particularly susceptible to changes at 4‐, 8‐, and 16‐positions, but low to changes at 9‐ and 10‐positions. Most of these target compounds exhibited good antifungal activities at the concentration of 50 mg l^?1. Compound 4 (9‐formyltrichodermin; EC₅₀ 0.80 mg l^?1) with an CHO group at 9‐position displayed nearly the same level of antifungal activity against Ustilaginoidea virens as the commercial fungicide prochloraz (EC₅₀ 0.82 mg l^?1), while compound 3f ((8R)‐8‐{[(E)‐3‐phenylprop‐2‐enoyl]oxy}trichodermin; EC₅₀ 3.58 and 0.74 mg l^?1) with a cinnamyloxy group at C(8) exhibited much higher antifungal activities against Rhizoctonia solani and Magnaporthe grisea than the commercial fungicides prochloraz (EC₅₀ 0.96 mg l^?1) and propiconazole (EC₅₀ 5.92 mg l^?1), respectively. These data reveal that compounds 3f and 4 possess high antifungal activities and may serve as lead compounds for the development of fungicides in the future.     

Annuario della Società Botanica Italiana 1932

A sandy culture experiment was conducted to investigate the effects of exogenous CaCl₂ on the indole alkaloid accumulation in Catharanthus roseus under salt stress. One-month seedlings of C. roseus were treated with the different concentrations of NaCl (0, 50, and 100 mmol l^? 1) and 7.5 mmol l^? 1 CaCl₂. The plant samples were analyzed after 7 days of the treatments. The NaCl-stressed plants showed decrease of fresh and dry weight and increase of malondialdehyde (MDA) content compared to control. Tryptophan decarboxylase (TDC) activity increased significantly under 50 mmol l^? 1 NaCl without CaCl₂ addition, 50 mmol l^? 1 NaCl with 7.5 mmol l^? 1 CaCl₂, and CaCl₂ treatment without NaCl addition. There was a significant increase in peroxidase activity under NaCl stress compared to control. The vindoline, catharanthine, vincristine, and vinblastine contents increased under salt stress (especially with 50 mmol l^? 1 NaCl treatment with or without CaCl₂). Addition of CaCl₂ to NaCl-stressed plants increased biomass, TDC activity, vindoline, and catharanthine contents and lowered MDA and vincirstine contents compared to the plants without CaCl₂. The plants treated with CaCl₂ alone showed higher TDC activity, vindoline, catharanthine, and vinblastine content when compared to control. The results showed that exogenous CaCl₂ could promote the indole alkaloid metabolism under salt stress.     

Antiviral activity of Distictella elongata (Vahl) Urb. (Bignoniaceae), a potentially useful source of anti-dengue drugs from the state of Minas Gerais, Brazil

Aims: To investigate the in vitro antiviral activity of Distictella elongata (Vahl) Urb. ethanol extracts from leaves (LEE), fruits (FEE), stems and their main components. Methods and Results: The antiviral activity was evaluated against human herpesvirus type 1 (HSV‐1), murine encephalomyocarditis virus (EMCV), vaccinia virus Western Reserve (VACV‐WR) and dengue virus 2 (DENV‐2) by the 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) colorimetric assay. LEE presented anti‐HSV‐1 [EC₅₀ 142·8 ± 5·3 μg ml^?1; selectivity index (SI) 2·0] and anti‐DENV‐2 activity (EC₅₀ 9·8 ± 1·3 μg ml^?1; SI 1·5). The pectolinarin ( 1 ) isolated from LEE was less active against HSV‐1 and DENV‐2. A mixture of the triterpenoids ursolic, pomolic and oleanolic acids was also obtained. Ursolic and oleanolic acids have shown antiviral activity against HSV‐1. A mixture of pectolinarin ( 1 ) and acacetin‐7‐O‐rutinoside ( 2 ) was isolated from FEE and has presented anti‐DENV‐2 activity (EC₅₀ 11·1 ± 1·6 μg ml^?1; SI > 45). Besides the antiviral activity, D. elongata has disclosed antioxidant effect. Conclusions: These data shows that D. elongata has antiviral activity mainly against HSV‐1 and DENV‐2, besides antioxidant activity. These effects might be principally attributed to flavonoids isolated. Significance and Impact of the Study: Distictella elongata might be considered a promising source of anti‐dengue fever phytochemicals.     

Comparison of supplements* to enhance recovery of heat‐injured Salmonella from egg albumen

Aims: The purpose of this study was to determine the proficiency of supplements to enhance the recovery of Salmonella from heat‐treated liquid egg albumen on solid agar media. Methods and Results: Salmonella‐inoculated albumen, heated at 53·3°C for 4 min, was plated on 39 combinations of solid media with or without the addition of 12 supplements. Greater numbers of Salmonella (P < 0·05) recovered with the addition of 1·0 g l^?1 ferrous sulfate (FeSO₄) than with any other supplements, except for 0·5 or 1·0 g l^?1 3′3′‐thiodipropionic acid (TDP), which recovered equivalent populations. Addition of 1·0 g l^?1 sodium pyruvate or 6·0 g l^?1 yeast extract plus 1·0 g l^?1 sodium pyruvate supported greater resuscitation than unsupplemented tryptic soy agar (TSA) or supplementing with 0·01 or 0·1 g l^?1 N‐propyl gallate, 10 g l^?1 activated charcoal, 0·1 g l^?1 KMnO₄ or 50 mg l^?1 ethoxyquin. The remaining supplements supported recovery of equivalent numbers of Salmonella, which were fewer cells than recovered with 1·0 g l^?1 FeSO₄, yet greater populations than recovered with 50 mg l^?1 ethoxyquin. Conclusion: Supplementation of plating media with FeSO₄, TDP or sodium pyruvate enhanced recovery of sublethally injured Salmonella from albumen. Significance and Impact of the Study: Pasteurizing albumen impedes recovery of pathogens. These results suggest that the addition of supplements to plating media may assist resuscitation and colony development of heat‐injured salmonellae.     

Response surface methodology for optimization of medium components in submerged culture of Aspergillus flavus for enhanced heparinase production

Aims: Aim of the study was to develop a medium for optimal heparinase production with a strain of Aspergillus flavus (MTCC‐8654) by using a multidimensional statistical approach. Methods and Results: Statistical optimization of intracellular heparinase production by A. flavus, a new isolate, was investigated. Plackett–Burman design was used to evaluate the affect of medium constituents on heparinase yield. The experimental results showed that the production of heparinase was dependent upon heparin, the inducer; chitin, structurally similar to heparin and NH₄NO_3, the nitrogen source. A central composite design was applied to derive a statistical model for optimizing the composition of the fermentation medium for the production of heparinase enzyme. The optimum fermentation medium consisted of (g l^?1) Mannitol, 8·0; NH₄NO₃, 2·5; K₂HPO₄, 2·5; Na₂HPO₄, 2·5; MgSO₄.7H₂O, 0·5; Chitin, 17·1; Heparin, 0·6; trace salt solution (NaMoO₄.2H₂O, CoCl₂.6H₂O, CuSO₄.5H₂O, FeSO₄.7H₂O, CaCl₂), 10^?4 mol l^?1. Conclusions: A 2·37‐fold increase in heparinase production was achieved in economic and effective manner by the application of statistical designs in medium optimization. Significance and Impact of the Study: Heparinase production was doubled by statistical optimization in a cost‐effective manner. This heparinase can find application in pharmaceutical industry and for the generation of low‐molecular‐weight heparins, active as antithrombotic and antitumour agents.     

Characterizing the escape response of juvenile summer flounder Paralichthys dentatus to diel‐cycling hypoxia

Swimming speed, angular correlation and expected displacement were measured in juvenile summer flounder Paralichthys dentatus acclimated to either oxygen saturation (c. 7·8 mg O₂ l^?1; saturation‐acclimated fish) or diel‐cycling hypoxia (cycling between 11·0 and 2·0 mg O₂ l^?1) for 10 days and subsequently exposed to more severe diel‐cycling hypoxia (cycling between 7·0 and 0·4 mg O₂ l^?1). Saturation‐acclimated P. dentatus exhibited an active response to declining dissolved oxygen (DO) by increasing swimming speed, angular correlation and expected displacement to peak levels at 1·4 mg O₂ l^?1 that were 3·5, 5·5 and 4·2 fold, respectively, greater than those at DO saturation. Diel‐cycling hypoxia‐acclimated P. dentatus also exhibited an active response to declining DO, although it was relatively less pronounced. Diel‐cycling hypoxia‐acclimated P. dentatus swimming speed, however, still doubled as DO decreased from 7·0 to 2·8 mg O₂ l^?1. Diel‐cycling hypoxia‐acclimated P. dentatus did not recover as well from low DO exposure as did saturation‐acclimated fish. This was reflected in their relatively more random swimming (low angular correlation between successive moves) and poor maintenance of rank order between individuals during the recovery phase. Even saturation‐acclimated P. dentatus did not resume swimming at speeds observed at saturation until DO was 4·2 mg O₂ l^?1. Paralichthys dentatus were very sensitive to decreasing DO, even at DO levels that were not lethal or growth limiting. This sensitivity and their poor recovery may preclude juvenile P. dentatus from using highly productive nursery habitats affected by diel‐cycling hypoxia.     

Ventilatory and behavioural responses of the marbled sole Pseudopleuronectes yokohamae to progressive hypoxia

This study identified ventilatory and behavioural responses in the marbled sole Pseudopleuronectes yokohamae under experimentally induced progressive decreases in dissolved oxygen (DO) levels. Ventilation frequency showed an increase with decreasing DO levels from normoxia to 2·75 mg O₂ l^?1, followed by a decrease in ventilation frequency at decreased DO levels from 2·00 to 0·75 mg O₂ l^?1. At DO levels below 2·00 mg l^?1, behaviours at the bottom were suppressed, whereas avoidance behaviours increased. A decrease in avoidance behaviours was observed from 1·00 to 0·75 mg O₂ l^?1. Upside‐down reversal and incapacitation at DO levels of 1·00–0·75 mg O₂ l^?1 suggested that sublethal effects on P. yokohamae were induced. The responses observed before the sublethal DO level could be interpreted as an effort to maintain oxygen uptake, reduce routine activities and facilitate avoidance. The observed DO level thresholds that induce behavioural responses, in addition to sublethal effects, indicate hypoxia‐tolerance that is important for understanding the effects of hypoxia on coastal ecosystems.     

Inactivation of Bacillus cereus by Na‐chlorophyllin‐based photosensitization on the surface of packaging

Aims: This study was focused on the possibility to inactivate food‐borne pathogen Bacillus cereus by Na‐chlorophyllin (Na‐Chl)‐based photosensitization in vitro and after attachment to the surface of packaging material. Methods and Results: Bacillus cereus in vitro or attached to the packaging was incubated with Na‐Chl (7·5 × 10^?8 to 7·5 × 10^?5 mol l^?1) for 2–60 min in phosphate buffer saline. Photosensitization was performed by illuminating cells under a light with a λ of 400 nm and an energy density of 20 mW cm^?2. The illumination time varied 0–5 min and subsequently the total energy dose was 0–6 J cm^?2. The results show that B. cereus vegetative cells in vitro or attached to the surface of packaging after incubation with 7·5 × 10^?7 mol l^?1 Na‐Chl and following illumination were inactivated by 7 log. The photoinactivation of B. cereus spores in vitro by 4 log required higher (7·5 × 10^?6 mol l^?1) Na‐Chl concentration. Decontamination of packaging material from attached spores by photosensitization reached 5 log at 7·5 × 10^?5 mol l^?1 Na‐Chl concentration. Comparative analysis of different packaging decontamination treatments indicates that washing with water can diminish pathogen population on the surface by <1 log, 100 ppm Na‐hypochlorite reduces the pathogens about 1·7 log and 200 ppm Na‐hypochlorite by 2·2 log. Meanwhile, Na‐Chl‐based photosensitization reduces bacteria on the surface by 4·2 orders of magnitude. Conclusions: Food‐borne pathogen B. cereus could be effectively inactivated (7 log) by Na‐Chl‐based photosensitization in vitro and on the surface of packaging material. Spores are more resistant than vegetative cells to photosensitization‐based inactivation. Comparison of different surface decontamination treatments indicates that Na‐Chl‐based photosensitization is much more effective antibacterial tool than washing with water or 200 ppm Na‐hypochlorite. Significance and Impact of the Study: Our data support the idea that Na‐Chl‐based photosensitization has great potential for future application as an environment‐friendly, nonthermal surface decontamination technique.     

Identification of novel halotolerant bacillopeptidase F-like proteinases from a moderately halophilic bacterium, Virgibacillus sp. SK37

Aims: Virgibacillus sp. SK37 isolated from Thai fish sauce produced numerous NaCl‐activated subtilisin‐like proteinases. Our objectives were to purify, characterize and identify these extracellular proteinases. Methods and Results: Three major subtilisin‐like enzymes including 19, 34 and 44 kDa were partially purified and showed maximum activity at pH 8, 55–60°C, 25–30% NaCl and 70–100 mmol l^?1 CaCl₂. Enzymes showed stability at 0–30% NaCl and <20 mmol l^?1 CaCl₂ and were completely inhibited by phenylmethanesulphonyl fluoride but not by ethylenediaminetetraacetic acid. The isoelectric points of 19‐, 34‐ and 44‐kDa proteinases were at 3·6, 5·2 and 3·8, respectively, based on 2D electrophoresis. Peptide mass fingerprint and de novo peptide homology analysis of tryptic peptides using MALDI‐TOF and LC–MS/MS, respectively, suggested that all three enzymes were novel and homologous to bacillopeptidase F. Conclusions: The three major proteinases are a member of bacillopeptidase F‐like enzymes exhibiting thermophilic and halotolerant characteristics with high stability at 30% NaCl. Significance and Impact of the Study: This is the first report on bacillopeptidase F‐like proteinases in genus Virgibacillus with a distinct halotolerant feature. They showed potential to be a processing aid for food and biotechnological applications, particularly in high salt condition.     

Isolation and characterization of actinomycetes from healthy goat faeces

Aims: To isolate and characterize actinomycetes with probiotic activities from healthy goat faeces. Methods and Results: Faecal actinomycetes were isolated by dilution methods and identified by 16S rRNA gene sequence analysis. The hydrolytic enzyme activities were analysed by clear zone formation. The antimicrobial activities and resistance to heavy metals were tested by growth inhibition methods. The isolates belong to a small group of actinobacterial genera, including Streptomyces, Nocardiopsis and Oerskovia. The Oerskovia was the most widely distributed genus among the cultures. The proportion of streptomycete‐like strains producing amylase or protease is significantly higher than those of other actinomycetes (P < 0·05). Compared with streptomycete‐like strains, a higher proportion of (α‐ or β‐) galactase‐producing other actinomycetes was found in goat faeces. More than 50% of streptomycete‐like strains showed activities against test fungi. Streptomycetes could tolerate 0·25 mmol l^?1 Cr₂O₇^2?, 2 mmol l^?1 Ni²⁺; however, other actinomycetes are liable to 40 mmol l^?1 Fe³⁺ and 0·25 mmol l^?1 Cr₂O₇^2? and resistant to 5 mmol l^?1 Ni²⁺ and 2 mmol l^?1 Cu²⁺. Conclusions: The different physiological characteristics of the actinomycetes suggested that the cooperation in the actinomycetes might be involved in their association with goat. Significance and Impact of the Study: Probiotic mixtures based on faecal actinomycetes showed potentials in animal production.     

Increased synthesis of α‐tocopherol,paramylon and tyrosine by Euglena gracilis under conditions of high biomass production

Aims: To analyse the production of different metabolites by dark‐grown Euglena gracilis under conditions found to render high cell growth. Methods and Results: The combination of glutamate (5 g l^?1), malate (2 g l^?1) and ethanol (10 ml l^?1) (GM + EtOH); glutamate (7·15 g l^?1) and ethanol (10 ml l^?1); or malate (8·16 g l^?1), glucose (10·6 g l^?1) and NH₄Cl (1·8 g l^?1) as carbon and nitrogen sources, promoted an increase of 5·6, 3·7 and 2·6‐fold, respectively, in biomass concentration in comparison with glutamate and malate (GM). In turn, the production of α‐tocopherol after 120 h identified by LC‐MS was 3·7 ± 0·2, 2·4 ± 0·1 and 2 ± 0·1 mg [g dry weight (DW)]^?1, respectively, while in the control medium (GM) it was 0·72 ± 0·1 mg (g DW)^?1. For paramylon synthesis, the addition of EtOH or glucose induced a higher production. Amino acids were assayed by RP‐HPLC; Tyr a tocopherol precursor and Ala an amino acid with antioxidant activity were the amino acids synthesized at higher concentration. Conclusions: Dark‐grown E. gracilis Z is a suitable source for the generation of the biotechnologically relevant metabolites tyrosine, α‐tocopherol and paramylon. Significance and Impact of the Study: By combining different carbon and nitrogen sources and inducing a tolerable stress to the cell by adding ethanol, it was possible to increase the production of biomass, paramylon, α‐tocopherol and some amino acids. The concentrations of α‐tocopherol achieved in this study are higher than others reported previously for Euglena, plant and algal systems. This work helps to understand the effect of different carbon sources on the synthesis of bio‐molecules by E. gracilis and can be used as a basis for future works to improve the production of different metabolites of biotechnological importance by this organism.     

Antiviral activity of Bignoniaceae species occurring in the State of Minas Gerais (Brazil): part 1

Aims: To evaluate the antiviral activity of Bignoniaceae species occurring in the state of Minas Gerais, Brazil. Methods and Results: Ethanol extracts of different anatomical parts of bignoniaceous plant species have been evaluated in vitro against human herpesvirus type 1 (HSV‐1), vaccinia virus (VACV) and murine encephalomyocarditis virus (EMCV) by the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. A total of 34 extracts from 18 plant species selected according to ethnopharmacological and taxonomic criteria were screened. Fifteen of the 34 extracts (44·1%) have disclosed antiviral activity against one or more of the viruses assayed with EC₅₀ values in the range of 23·2 ± 2·5–422·7 ± 10·9 μg ml^?1. Conclusions: Twelve of the 34 extracts (35·3%) might be considered promising sources of antiviral natural products, as they have shown EC₅₀ ≤ 100 μg ml^?1. The present screening discloses the high potential of the Bignoniaceae family as source of antiviral agents. Significance and Impact of the Study: Active extracts were identified and deserve bioguided studies for the isolation of antiviral compounds and studies on mechanism of action.     

Effect of NaOH (caustic wash) on the viability, surface characteristics and adhesion of spores of a Geobacillus sp. isolated from a milk powder production line

Aim: To investigate the viability, surface characteristics and ability of spores of a Geobacillus sp. isolated from a milk powder production line to adhere to stainless steel surfaces before and after a caustic (NaOH) wash used in clean‐in‐place regimes. Methods and Results: Exposing sessile spores to 1% NaOH at 65°C for 30 min decreased spore viability by two orders of magnitude. The zeta potential of the caustic treated spores decreased from ?20 to ?32 mV and they became more hydrophobic. Transmission electron microscopy revealed that caustic treated spores contained breaks in their spore coat. Under flow conditions, caustic treated spores suspended in 0·1 mol l^?1 KCl were shown to attach to stainless steel in significantly greater numbers (4·6 log₁₀ CFU cm^?2) than untreated spores (3·6 log₁₀ CFU cm^?2). Conclusions: This research suggests that spores surviving a caustic wash will have a greater propensity to attach to stainless steel surfaces. Significance of Study: The practice of recycling caustic wash solutions may increase the risk of contaminating dairy processing surfaces with spores.     

Use chemometric techniques in the optimization of a specific bioassay for betalactams in milk

Aims: A microbiological bioassay using Geoacillus stearothermophilus was optimized to detect betalactams at concentrations near to the Maximum Residue Limits (MRLs), with low cross‐specificity for tetracycline. Methods and Results: A factorial design (3 × 4) was used to evaluate the effects of concentration of spores (2·0 × 10⁶, 4·0 × 10⁶ and 8·0 × 10⁶ spores ml^?1) and incubation time (3·0, 3·5, 4·0 and 4·5 h) on the response of the bioassay. Then, desirability function to raise the detection capabilities (CC_β) of tetracyclines and increase sensitivity to betalactams was implemented. Significant effects of Log[S] and incubation time [It] on the CC_β of betalactams and tetracyclines were observed. Finally, high value of global desirability (D = 0·853), adequate betalactams CC_β (3·8 μg l^?1 of penicillin ‘G’, 27 μg l^?1 of oxacillin, 8·1 μg l^?1 of ampicillin, 48 μg l^?1 of cloxacillin) and high tetracyclines CC_β (5260 μg l^?1 chlortetracycline, 1550 μg l^?1 of oxytetracycline, 1070 μg l^?1 of tetracycline) were calculated. Conclusions: The application of chemometric tools allows the optimization of a bioassay that detects betalactam residues in milk. The more robust conditions have been achieved in Log[S] = 6·30 and [It] = 4·20 h. Significance and Impact of the Study: The logistic regression model and the desirability function are adequate chemometric techniques to improve the properties of the methods, because it is possible to increase sensitivity and decrease cross‐specificity simultaneously.     

Statistical optimization of simple culture conditions to produce biomass of an ochratoxigenic mould biocontrol yeast strain

Aim: To maximize biomass production of an ochratoxigenic mould–controlling strain of Lachancea thermotolerans employing response surface methodology (RSM). Methods and Results: Using Plackett–Burman screening designs (PBSD) and central composite designs (CCD), an optimized culture medium containing (g l^?1): fermentable sugars (FS), 139·2, provided by sugar cane molasses (CMz), (NH₄)₂HPO₄ (DAP), 9·0, and yeast extract (YE), 2·5, was formulated. Maximal cell concentration obtained after 24 h at 28°C was 24·2 g l^?1cell dry weight (CDW). The mathematical model obtained was validated in experiments performed in shaken‐flask cultures and also in aerated bioreactors. Maximum yield and productivity values achieved were, respectively, of 0·23 g CDW/g FS in a medium containing (g l^?1): FS, 87·0; DAP, 7·0; YE, 1·0; and of 0·96 g CDW l^?1 h^?1 in a medium containing (g l^?1): FS, 150·8 plus DAP, 6·9. Conclusions: Optimized culture conditions for maximizing yeast biomass production determined in flask cultures were applicable at a larger scale. The highest yield values were attained in media containing relatively low‐CMz concentrations supplemented with DAP and YE. Yeast extract would not be necessary if higher productivity is the aim. Significance and Impact of the Study: Cells of L. thermotolerans produced aerobically could be sustainably produced in a medium just containing cheap carbon, nitrogen and phosphorus sources. Response surface methodology allowed the fine‐tuning of cultural conditions.     

Efficiency of osmotic and chemical treatments to improve the permeation of the cryoprotectant dimethyl sulfoxide to Japanese whiting (Sillago japonica) embryos

Insufficient cryoprotectant permeation is one of the major obstacles for successful fish embryo cryopreservation. The purpose of this study was to test the effectiveness of osmotic and chemical treatments to enhance cryoprotectant uptake by fish embryos. Japanese whiting Sillago japonica embryos at the somites and tail elongation stages were treated with hyperosmotic sugar solutions (1 M trehalose and sucrose) for 2-6 min, or a permeating agent (2-6 mg/mL pronase) for 30-120 min, and then impregnated with 10-15% DMSO in artificial sea water or aqueous solutions containing inorganic salts (0.125-0.25 M MgCl₂ and CaCl₂). The viability of the embryos after the treatments was estimated from hatching rates and the internal DMSO concentration was measured by HPLC. Treatment with trehalose for 3 min prior to impregnation with DMSO enhanced the uptake of the cryoprotectant by 45% without significantly affecting embryo viability, whereas pronase had no noticeable effect on cryoprotectant permeation. Incorporation of DMSO into the embryos was enhanced by 143-170% in the presence of 0.25 M MgCl₂ and 0.125 M CaCl₂ compared to sea water. A combination of treatments with trehalose and MgCl₂ was even more effective in promoting DMSO permeation (191% compared to untreated embryos). Tail elongation embryos were less tolerant of the treatments, but had higher DMSO impregnation. In conclusion, the use of trehalose (as dehydrating agent) and MgCl₂/CaCl₂ (as a vehicle during impregnation) greatly promoted cryoprotectant uptake and may be a promising aid for the successful cryopreservation of fish embryos.     

The mixture of kresoxim‐methyl and boscalid,an excellent alternative controlling grey mould caused by Botrytis cinerea

Grey mould, caused by the fungus Botrytis cinerea, is one of the most destructive diseases in greenhouses for which serious fungicide resistance has developed. Between 2003 and 2005, 213 isolates of B. cinerea from two geographical regions were characterised for baseline sensitivity to kresoxim‐methyl. In the absence of salicylhydroxamic acid (SHAM), the mean 50% effective concentration (EC₅₀) values were 6.67 ± 0.61 (mean ± SD) and 0.37 ± 0.10 mg L^?1 during growth and germination, respectively. In the presence of 100 mg L^?1 SHAM, baseline sensitivities were distributed as unimodal curves with mean EC₅₀ values of 2.38 ± 0.21 and 0.28 ± 0.09 mg L^?1 for inhibiting growth and inhibiting germination, respectively. The mixture of kresoxim‐methyl and boscalid showed good control efficacy against strawberry grey mould disease. After the mixture was extensively used on strawberry for 2 years, 50 isolates were collected and determined for their sensitivity to kresoxim‐methyl and boscalid, respectively. The mean EC₅₀ of germination inhibition by boscalid was 0.39 ± 0.08 mg L^?1. The mean EC₅₀ of germination inhibition by kresoxim‐methyl was 0.26 ± 0.07 mg L^?1 in the presence of 100 mg L^?1 SHAM. Sensitivities of B. cinerea to both kresoxim‐methyl and boscalid did not show any significant decrease. These results suggest that their mixture is a satisfactory alternative candidate for management of grey mould disease in greenhouses.     

PRIMARY PRODUCTION OF CRUSTOSE CORALLINE RED ALGAE IN A HIGH ARCTIC FJORD1

Crustose coralline algae occupied ～1%–2% (occasionally up to 7%) of the sea floor within their depth range of 15–50 m, and they were the dominant encrusting organisms and macroalgae beyond 20 m depth in Young Sound, NE Greenland. In the laboratory, oxygen microelectrodes were used to measure net photosynthesis (P) versus downwelling irradiance (E_d) and season for the two dominant corallines [Phymatolithon foecundum (Kjellman) Düwel et Wegeberg 1996 and Phymatolithon tenue (Rosenvinge) Düwel et Wegeberg 1996] representing> 90% of coralline cover. Differences in P‐E_d curves between the two species, the ice‐covered and open‐water seasons, or between specimens from 17 and 36 m depth were insignificant. The corallines were low light adapted, with compensation irradiances (E_c) averaging 0.7–1.8 μmol photons·m ^{? 2}·s ^{? 1} and light adaptation (E_k) indices averaging 7–17 μmol photons·m ^{? 2}·s ^{? 1}. Slight photoinhibition was evident in most plants at irradiances up to 160 μmol photons·m ^{? 2}·s ^{? 1}. Photosynthetic capacity (P_m) was low, averaging 43–67 mmol O₂·m ^{? 2} thallus·d ^{? 1} (～250–400 g C·m ^{? 2} thallus·yr ^{? 1}). Dark respiration rates averaged ～5 mmol O₂·m ^{? 2} thallus·d ^{? 1}. In ice covered periods, E_d at 20 m depth averaged ～1 μmol photons·m ^{? 2}·s ^{? 1}, with daily maxima of 2–3 μmol photons·m ^{? 2}·s ^{? 1}. During the open water season, E_d at 20 m depth averaged ～7 μmol photons·m ^{? 2}·s ^{? 1} with daily maxima of ～30 μmol photons·m ^{? 2}·s ^{? 1}. Significant net primary production of corallines was apparently limited to the 2–3 months with open water, and the small contribution of corallines to primary production seems due to low P_m values, low in situ irradiance, and their relatively low abundance in Young Sound.     

The influence of light on copper‐limited growth of an oceanic diatom,Thalassiosira oceanica (Coscinodiscophyceae)

Thalassiosira oceanica (CCMP 1005) was grown over a range of copper concentrations at saturating and subsaturating irradiance to test the hypothesis that Cu and light were interacting essential resources. Growth was a hyperbolic function of irradiance in Cu‐replete medium (263 fmol Cu′ · L^?1) with maximum rates achieved at 200 μmol photons · m^?2 · s^?1. Lowering the Cu concentration at this irradiance to 30.8 fmol Cu′ · L^?1 decreased cellular Cu quota by 7‐fold and reduced growth rate by 50%. Copper‐deficient cells had significantly slower (P < 0.0001) rates of maximum, relative photosynthetic electron transport (rETR_max) than Cu‐sufficient cells, consistent with the role of Cu in photosynthesis in this diatom. In low‐Cu medium (30.8 fmol Cu′ · L^?1), growth rate was best described as a positive, linear function of irradiance and reached the maximum value measured in Cu‐replete cells when irradiance increased to 400 μmol photons · m^?2 · s^?1. Thus, at high light, low‐Cu concentration was no longer limiting to growth: Cu concentration and light interacted strongly to affect growth rate of T. oceanica (P < 0.0001). Relative ETR_max and Cu quota of cells grown at low Cu also increased at 400 μmol photons · m^?2 · s^?1 to levels measured in Cu‐replete cells. Steady‐state uptake rates of Cu‐deficient and sufficient cells were light‐dependent, suggesting that faster growth of T. oceanica under high light and low Cu was a result of light‐stimulated Cu uptake.