Chemical composition and antimicrobial activity of Citrus essences on honeybee bacterial pathogen <Emphasis Type="Italic">Paenibacillus larvae</Emphasis>, the causal agent of American foulbrood

Antimicrobial properties and chemical composition of four citrus fruit essential oils to control Paenibacillus larvae, the causal agent of American foulbrood disease (AFB) were determined. This honeybee larvae disease occurs throughout the world and is found in many beekeeping areas of Argentina. Citrus fruit essential oils tested were those from grapefruit (Citrus paradisi), sweet orange (Citrus sinensis), mandarin (Citrus nobilis) and lemon (Citrus limon). The components of the essential oils were identified by SPME-GC/MS analysis. The antimicrobial activity of the oils against P. larvae were determined by the broth microdilution method. Two way ANOVA tests for minimum inhibitory concentrations (MICs) data and minimal bactericide concentrations (MBCs) data, indicated significant differences between the strains and the oils tested. The antimicrobial assays showed that the oil of C. paradisi inhibited the bacterial strains at the lowest concentrations tested, MICs and MBCs averages of 385.0 mg/l and 770.0 mg/l, respectively. This property could be attributed to the kind and percentage of the volatile components of the oil, like limonene (69.9%) and myrcene (9.6%). The use of essential oils or their specific volatile components individually against pests related to food provision may represent an alternative scope for the control of this serious disease because it does not leave toxic chemical residues in honey nor in its by products.     

Analysis of volatile compounds released from embryogenic cultures and somatic embryos of sweet oranges by head space SPME

Volatile compounds released from callus and nucellar embryo tissues of ‘Valencia late’ and ‘Washington Navel’ sweet oranges (Citrus sinensis, L. Osbeck) were collected/concentrated by head space solid phase micro extraction and analysed by gas chromatography-mass spectrometry. Friable, white embryogenic cultures released a number of volatile compounds, including some essential oils. Different samples of the same embryogenic culture showed variability, possibly related to the presence of tissues undergoing differentiation. Analyses of the somatic embryos permitted the identification of several components, including limonene and methyl anthranilate. Considering the simplicity and the very small sample required (0.3 g of fresh tissue) head space solid phase micro extraction is suitable for studies and comparisons of volatile metabolites released from in vitro Citrus tissue cultures suggesting its potential in Citrus biochemical, genetic and breeding research. This revised version was published online in June 2006 with corrections to the Cover Date.     

Chemical Composition and Antifungal Activity of the Essential Oils of Lavandula pedunculata (Miller) Cav.

The chemical composition and antifungal activity of the essential oils of Lavandula pedunculata (Miller ) Cav. , harvested in North and Central Portugal, were investigated. The essential oils were isolated by hydrodistillation and analyzed by GC and GC/MS. The minimal‐inhibitory concentration (MIC) and the minimal‐lethal concentration (MLC) of the essential oils and of their major constituents were used to evaluate the antifungal activity against different strains of fungi involved in candidosis, dematophytosis, and aspergillosis. The oils were characterized by a high percentage of oxygenated monoterpenes, the main compounds being 1,8‐cineole (2.4–55.5%), fenchone (1.3–59.7%), and camphor (3.6–48.0%). Statistical analysis differentiated the essential oils into two main types, one characterized by the predominance of fenchone and the other one by the predominance of 1,8‐cineole. Within the 1,8‐cineole chemotype, two subgroups were well‐defined taking into account the percentages of camphor. A significant antifungal activity of the oils was found against dermatophyte strains. The essential oil with the highest content of camphor was the most active with MIC and MLC values ranging from 0.32–0.64 μl/ml.     

Production of essential oils by flowers of Hyacinthus orientalis L. regenerated in vitro

Essential oils were produced by flowers of Hyacinthus orientalis L. that had been regenerated in vitro. The production of these oils was affected by the concentration of gibberellic acid and sucrose in the medium and by temperature. The highest concentration of essential oils was obtained when regenerated flowers were cultured in vitro for 3 weeks at 20 °C on Murashige and Skoog's medium that contained 30 g/l sucrose plus 1 mg/l gibberellic acid, whereas the highest amount of essential oils was obtained after a culture period of 3 weeks at 25 °C. The composition of essential oils from flowers that had been regenerated in vitro was compared with that from flowers grown in the field. Essential oils detected by gas-liquid chromatography included nine components in the case of the regenerated flowers and six and ten components in the case of stage 3 and stage 4 flowers grown in the field, respectively. There were four common components, namely, 1-hepten-3-ol, benzyl alcohol, phenethyl alcohol and cinnamyl alcohol. In the regenerated flowers, a single component, phenethyl alcohol, was a major constituent (75%), whereas two compounds, phenethyl alcohol (stage 3, 55%; stage 4, 48%) and cinnamyl alcohol (stage 3, 23%; stage 4, 29%) were the major constituents in the case of flowers grown in the field. Four and five other components were specific to flowers regenerated in vitro and field-grown flowers, respectively.Abbreviations BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - ABA abscisic acid - GA₃ gibberellic acid - ACC 1-aminocyclopropane-1-carboxylic acid - ethephon (2-chloroethyl)phosphonic acid - MS medium Murashige and Skoog's medium - GLC gas-liquid chromatography - GC-MS gas chromatography-mass spectrometry - FW fresh weight     

Antimicrobial Activity of Syzygium aromaticum and Zanthoxylum xanthoxyloides Essential Oils Against Phytophthora megakarya

The aim of this study was to assess the chemical composition and the antimicrobial activity of essential oils of dried fruits and buds of Zanthoxylum xanthoxyloides (Z. xanthoxyloides) and Syzygium aromaticum (S. aromaticum or clove), respectively, against Phytophthora megakarya (P. megakarya). Essential oils were extracted by hydrodistillation, and their composition was determined by gas chromatography and by gas chromatography coupled with Mass Spectrometry. The minimal inhibitory concentration (MIC) and minimal lethal concentration (MLC) of the essential oils against P. megakarya were assessed by the Agar dilution method. The in vivo efficacy study consisted of spraying the essential oil emulsions on cocoa pod husk pieces (CPHP), followed by the inoculation with P. megakarya zoospores. The hydrodistillation yielded 10.54 and 1.89% of essential oils for S. aromaticum and Z. xanthoxyloides, respectively. Both oils were mainly made up of oxygenated monoterpenes (89.58 and 88.2%, respectively). Eugenol (83.02%) and eugenyl acetate (9.15%) were the main components of clove oil while α‐citronelol (25.83%) and trans‐geraniol (16.49%) were mostly found in the Z. xanthoxyloides oil. Clove oil exhibited stronger antimicrobial activity with a MIC of 250 μl/l than Z. xanthoxyloides with MIC of 350 μl/l. The symptoms were totally suppressed on pod husk treated with clove oil at 2000 μl/l. The decrease in the growth rate of the necrosis (GRN) and the sporulation of P. megakarya (PS) on cocoa husk after the successful infection was significant after the treatment with essential oils. These results are promising and indicate that the studied essential oils might be further investigated as natural alternatives to synthetic fungicides for the control of cocoa black pod diseases.     

Effects of organic compounds on the degradation ofp-nitrophenol in lake and industrial wastewater by inoculated bacteria

Many microorganisms fail to degrade pollutants when introduced in different natural environments. This is a problem in selecting inocula for bioremediation of polluted sites. Thus, a study was conducted to determine the success of four inoculants to degradep-nitrophenol (PNP) in lake and industrial wastewater and the effects of organic compounds on the degradation of high and low concentrations of PNP in these environments.Corynebacterium strain Z4 when inoculated into the lake and wastewater samples containing 20 µg/ml of PNP degraded 90% of PNP in one day. Addition of 100 µg/ml of glucose as a second substrate did not enhance the degradation of PNP and the bacterium utilized the two substrates simultaneously. Glucose used at the same concentration (100 µg/ml), inhibited degradation of 20 µg of PNP in wastewater byPseudomonas strain MS. However, glucose increased the extent of degradation of PNP byPseudomonas strain GR. Phenol also enhanced the degradation of PNP in wastewater byPseudomonas strain GR, but had no effect on the degradation of PNP byCorynebacterium strain Z4.Addition of 100 µg/ml of glucose as a second substrate into the lake water samples containing low concentration of PNP (26 ng/ml) enhanced the degradation of PNP and the growth ofCorynebacterium strain Z4. In the presence of glucose, it grew from 2×10⁴ to 4×10⁴ cells/ml in 3 days and degraded 70% of PNP as compared to samples without glucose in which the bacterium declined in cell number from 2×10⁴ to 8×10³ cells/ml and degraded only 30% PNP. The results suggest that in inoculation to enhance biodegradation, depending on the inoculant, second organic substrate many play an important role in controlling the rate and extent of biodegradation of organic compounds.Abbreviations PNP p-nitrophenol     

Stimulation of adventitious root formation in non-woody stem cuttings by uridine

Uridine strongly stimulated adventitious root formation in stem cuttings of sunflower (Helianthus annuus L.), mung bean (Vigna radiata L.) and common bean (Phaseolus vulgaris L.). A dose response curve of uridine induced rooting showed that the optimum concentration of uridine was 0.1 µM. At all concentrations employed, uridine had no significant effect on root elongation. The rooting response of stem cuttings to the optimal concentration of indole-3-butyric acid (10 µM) in combination with 0.1 µM uridine did not significantly differ from their response to either of these compounds when applied alone. However, the rooting response of the cuttings to sub-optimal IBA (0.01 µM) was significantly stimulated by uridine. These findings suggested that uridine may have stimulated rooting by increasing the sensitivity of the rooting tissue to auxin.     

Degradation of phenol and phenolic compounds by a defined denitrifying bacterial culture

Phenol, a major pollutant in several industrial waste waters is often used as a model compound for studies on biodegradation. This study investigated the anoxic degradation of phenol and other phenolic compounds by a defined mixed culture of Alcaligenes faecalis and Enterobacter species. The culture was capable of degrading high concentrations of phenol (up to 600 mg/l) under anoxic conditions in a simple minimal mineral medium at an initial cell mass of 8 mg/l. However, the lag phase in growth and phenol removal increased with increase in phenol concentration. Dissolved CO₂ was an absolute requirement for phenol degradation. In addition to nitrate, nitrite and oxygen could be used as electron acceptors. The kinetic constants, maximum specific growth rate _max; inhibition constant, K _i and saturation constant, K _s were determined to be 0.206 h^–1, 113 and 15 mg phenol/l respectively. p-Hydroxybenzoic acid was identified as an intermediate during phenol degradation. Apart from phenol, the culture utilized few other monocyclic aromatic compounds as growth substrates. The defined culture has remained stable with consistent phenol-degrading ability for more than 3 years and thus shows promise for its application in anoxic treatment of industrial waste waters containing phenolic compounds.     

Biodegradation of diesel oil and production of fatty acid esters by a newly isolated Pseudomonas citronellolis KHA

The ability of a newly isolated Pseudomonas citronellolis KHA to degrade diesel oil and to synthesize fatty acid esters has been screened in aerobic batch cultures. The microorganism was able to grow with diesel oil at initial concentrations up to 126 g/l, with optimal growth at 25 g/l. Strain KHA has produced compounds showing strong emulsifying properties (E₂₄ = 75% at the end of the exponential growth phase). The crude extract reduces the surface tension of water from 72 mN m⁻¹ down to 35 mN m⁻¹ with a corresponding minimal concentration value of 60 mg/l. GC and GC–MS analysis of crude product show that the major components are those of hexadecanoic acid propyl ester and octadecanoic acid propyl ester, which have potential for applications in cosmetics, pharmaceutical and foods industries. In addition, strain KHA represents a valuable source of compounds with surface-active properties and potential for the application in clean up of the sites contaminated with hydrocarbons.     

Aliesterase- (Ali-E) activity in Daphnia magna straus as a parameter for exposure to parathion

In vivo exposure of Daphnia magna Straus to parathion in concentrations from 0.05 to 5 µg/l resulted in a concentration-dependent decrease in aliesterase activity of the homogenate of the animals. Decrease in this activity after 48 hours of exposure was maximal for concentrations > 1.5 µg/l and amounted to about 90% of the activity of the blank.     

Adaptive respiratory significance of organophosphates (ATP & GTP) in air-breathing fishes

The ATP and GTP contents of erythrocytes of Protopterus annectens, Polypterus senegalus and Clarias lazera were assessed in relation to maturation and hypoxia. The steady state normal concentrations of ATP were: 446, 316 and 475 µM/100 ml and those of GTP were 286, 93 and 85 µM/100 ml in Protopterus, Polypterus and Clarias, respectively. The level of ATP decreased and that of GTP increased with increments in body growth, especially during development of the pulmonary organs. The concentration of GTP also increased progressively with hypoxia (O₂: 7.4 to 3.6 mg/l); the level ATP remained almost unaltered. The ratio of GTP increased markedly with increased hypoxia particularly in the adaptive respiratory range of aquatic oxygen concentrations (6.5–4.8 mg/l) when the fish were forced to depart from branchial respiration and became reliant on air-breathing.     

Anisakis simplex s.l.: Larvicidal activity of various monoterpenic derivatives of natural origin against L3 larvae in vitro and in vivo

This paper describes the activity against Anisakis simplex s.l. L₃ larvae of six monoterpenic derivatives obtained from different essential oils, (α-pinene, β-pinene, ocimene, myrcene, geranyl acetate, and cineole). In in vitro assays, α-pinene, ocimene and cineole showed high activity at a concentration of 125 μg/mL (48 h) but only α-pinene and ocimene were active at 62.5 μg/mL. In in vivo assays, L₃ larvae and study compounds were simultaneously administered per os to Wistar rats. The most active compound was α-pinene, finding lesions in only 20% of treated rats versus 98% of controls. Further in vivo studies are required to investigate whether addition of these compounds to food could have a prophylactic effect, reducing the pathogenicity of A. simplex s.l. L₃ in humans, and to explore any possible synergy among compounds.     

Enhancing of erythromycin production by <Emphasis Type="Italic">Saccharopolyspora erythraea</Emphasis> with common and uncommon oils

The enhancing effect of various concentrations of 18 oils and a silicon antifoam agent on erythromycin production by Saccharopolyspora erythraea was evaluated in a complex medium containing soybean flour and dextrin as the main substrates. The oils used consisted of sunflower, pistachio, cottonseed, melon seed, water melon seed, lard, corn, olive, soybean, hazelnut, rapeseed, sesame, shark, safflower, coconut, walnut, black cherry kernel and grape seed oils. The biomass, erythromycin, dextrin and oil concentrations and the pH value were measured. Also, the kinds and frequencies of fatty acids in the oils were determined. The productivity of erythromycin in the oil-containing media was higher than that of the control medium. However, oil was not suitable as a main carbon source for erythromycin production by S. erythraea. The highest titer of erythromycin was produced in medium containing 55 g/l black cherry kernel oil (4.5 g/l). The titers of erythromycin in the other media were also recorded, with this result: black cherry kernel > water melon seed > melon seed > walnut > rapeseed > soybean > (corn = sesame) > (olive = pistachio = lard = sunflower) > (hazelnut = cotton seed) > grape seed > (shark = safflower = coconut). In media containing various oils, the hyphae of S. erythraea were longer and remained in a vegetative form after 8 days, while in the control medium, spores were formed and hyphae were lysed.     

Composition and biological activities of essential oils from Limnophila geoffrayi Bonati

Essential oils from the dried aerial parts of Limnophila geoffrayi Bonati were obtained by water-distillation. d-Pulegone (27.14%), perillaldehyde (19.13%) and limonene (9.00%) were characterized as the major constituents using gas chromatography-mass spectrometry analysis. The antimicrobial activities of the essential oils and their major components were evaluated against microorganisms encountered normally in contaminated cosmetic products, using the agar- and broth-dilution methods. Their insecticidal activities against the Oriental fruit fly Bactrocera dorsalis (Hendel) were tested using a bioassay with impregnated filter paper. The results showed that the essential oils possessed high antimicrobial activity, with minimum inhibitory concentrations ranging from 0.03 to 0.2% per unit volume (v/v). Strong insecticidal activity as a fumigant was also observed at an oil dose of 5 μl/disc, with a 94% mortality. Perillaldehyde was the most active compound among the main components of these essential oils.     

Metabolic engineering of Pichia pastoris X-33 for lycopene production

As an alternative carotenoid producer, non-carotenogenic Pichia pastoris was chosen for a reddish carotenoid lycopene production because it can grow to high cell density without accumulation of ethanol and utilize various classes of organic materials such as methanol as carbon sources. Two synthetic lycopene-pathway plasmids, pGAPZB-EBI* and pGAPZB-EpBpI*p, were designed and constructed. The pGAPZB-EpBpI*p plasmid encoded three carotenogenic enzymes that were engineered to be targeted into peroxisomes of P. pastoris whereas the pGAPZB-EBI* plasmid encoded non-targeted enzymes. After both plasmids were transformed into P. pastoris, the lycopene-producing clone containing the pGAPZB-EpBpI*p plasmid, referred to as Ω, was selected and used for further optimization study. Of the carbon sources tested, glucose resulted in the highest level of lycopene production in complex and minimal media. Batch fermentation of the Ω clone resulted in the production of 4.6 mg-lycopene/g-DCW, with a concentration of 73.9 mg/l of lycopene in minimal medium. For the first time non-carotenogenic yeast P. pastoris was metabolically engineered by heterologously expressing lycopene-pathway enzymes and the lycopene concentration of 73.9 mg/l was obtained. This serves as a basis for the development of biological process for carotenoids using P. pastoris at a commercial production level.     

Efficient transformation and regeneration of carnation cultivars usingAgrobacterium

We have developed an efficient method for transformation and regeneration of plants from carnation,Dianthus caryophyllus L. Whole leaves fromin vitro shoot cultures were mixed withAgrobacterium, cocultivated for 5 days and then plated on 2 µg/l chlorsulfuron (CS). Regenerated shoots and shoot clusters were divided into smaller sections and plated on 3 µg/l CS for selection to produce fully transformed shoots. Geneticin (G418) and kanamycin used were not as effective selective agents as CS. All regenerated shoots were vitrified. These were normalized, rooted and transferred to the greenhouse. 100% of regenerated plants were transformed based on rooting assay, GUS assay, PCR and Southern analysis.     

Composition and Biological Activity of Picea pungens and Picea orientalis Seed and Cone Essential Oils

The increasing consumption of natural products lead us to discover and study new plant materials, such as conifer seeds and cones, which could be easily available from the forest industry as a waste material, for their potential uses. The chemical composition of the essential oils of Picea pungens and Picea orientalis was fully characterized by GC and GC/MS methods. Seed and cone oils of both tree species were composed mainly of monoterpene hydrocarbons, among which limonene, α‐ and β‐pinene were the major, but in different proportions in the examined conifer essential oils. The levorotary form of chiral monoterpene molecules was predominant over the dextrorotary form. The composition of oils from P. pungens seeds and cones was similar, while the hydrodistilled oils of P. orientalis seeds and cones differed from each other, mainly by a higher amount of oxygenated derivatives of monoterpenes and by other higher molar mass terpenes in seed oil. The essential oils showed mild antimicrobial action, however P. orientalis cone oil exhibited stronger antimicrobial properties against tested bacterial species than those of P. pungens. Effects of the tested cone essential oils on human skin fibroblasts and microvascular endothelial cells (HMEC‐1) were similar: in a concentration of 0 – 0.075 μl/ml the oils were rather safe for human skin fibroblasts and 0 – 0.005 μl/ml for HMEC‐1 cells. IC₅₀ value of Picea pungens oils was 0.115 μl/ml, while that of Picea orientalis was 0.105 μl/ml. The value of IC₅₀ of both oils were 0.035 μl/ml for HMEC‐1 cells. The strongest effect on cell viability had the oil from Picea orientalis cones, while on DNA synthesis the oil from Picea pungens cones.     

In situ nutrient assays of periphyton growth in a lowland Costa Rican stream

Nutrient limitation of primary production was experimentally assessed using an in situ bioassay technique in the Quebrada Salto, a third-order tropical stream draining the northern foothills of the Cordillera Central in Costa Rica. Bioassays employed artificial substrata enriched with nutrients that slowly diffuse through an agar-sand matrix (Pringle & Bowers, 1984). Multiple comparisons of regression coefficients, describing chlorophyll-a accrual through time for different nutrient treatments, revealed positive micronutrient effect(s). Micronutrient treatment combinations (Fe, B, Mn, Zn, Co, Mo, EDTA), supplemented with and without nitrate and phosphate, exhibited significantly greater chlorophyll-a accrual over all other treatments (P < 0.05), supporting over three times that of the control after 14-d of substratum colonization. Neither of the major nutrients (N or P) produced a significant stimulation, although the N treatment displayed 50% more chlorophyll-a than the control after 14-d. Similarly, Si, EDTA, and Si + N + P treatments did not exhibit chlorophyll-a response curves that were significantly different from the control. During the experiment, mean NH₄-N and (NO₂ + NO₃)-N concentrations in the Salto were 2.0 µM (28.6 µg · l^–1) and 7.2 µM (100.2 µg · l ^–1), respectively. High concentrations of PO₄-P ( = 2.0 µM; 60.9 µg · l^–1) and TP ( = 3.0 µM; 94.0 µg · l^–1) were also found, and consequently low molar N:P ratios = 4.7). Despite the potential for N limitation in the system, both N and P appear to be at growth saturating levels. This may be due to micronutrient limitation and/or light limitation of periphyton growth in densely shaded upstream portions of the stream.     

Promoting effect of wood vinegar compounds on fruit-body formation ofPleurotus ostreatus

The promoting effect of wood vinegar compounds on the fruiting ofPleurotus ostreatus (Japanese name, Hiratake) was investigated. Not only crude wood vinegar but its components, 3,5-dimethylphenol, 2-methoxyphenol, butanoic acid and 1-pentanol, had the ability to promote fruit-body formation on liquid medium. For use of these promoters industrially, a test for practical cultivation was carried out using a commercial sawdust medium. The addition of 100 µg/ml butanoic acid and 100 µg/ml 2-methoxyphenol into the sawdust medium after removal of the surface mycelial layer (kinkaki in Japanese) produced 29 and 23% higher yields of fruit-bodies than the control cultures (137.2 g/bottle), respectively. The addition of the crude wood vinegar as a medium component into sawdust substrates in the concentration range of 0.1–6% increased yields of fruit-bodies by 21–42% over the control.     

Production,partial purification and bioassay of toxic metabolites of three plant pathogenic species ofColletotrichum in Nigeria

Colletotrichum lindemuthianum, C. truncatum andC. graminicola, produced metabolites in culture which induced necrotic lesions on susceptible hosts. The highest production was obtained from Richard's medium under shake incubation. The toxic metabolites of these pathogens fluoresce at 254 nm and 366 nm under ultraviolet light. The crude metabolites of theseColletotrichum species inhibited seed germination at the concentration of 100 µg/ml while the potency of the metabolites decreases with increase in dilution to 0.1 µg/ml. Similarly, the metabolites also inhibited growth of seedlings of hosts to the pathogen at 100 µg/ml and the potency again reduced with increasing dilution to 0.10 µg/ml.