LEAF TOTAL NITROGEN CONCENTRATION AS AN INDICATOR OF NITROGEN STATUS FOR PLANTLETS AND YOUNG PLANTS OF EUCALYPTUS CLONES

The use of leaf total nitrogen concentration as an indicator for nutritional diagnosis has some limitations. The objective of this study was to determine the reliability of total N concentration as an indicator of N status for eucalyptus clones, and to compare it with alternative indicators. A greenhouse experiment was carried out in a randomized complete block design in a 2 × 6 factorial arrangement with plantlets of two eucalyptus clones (140 days old) and six levels of N in the nutrient solution. In addition, a field experiment was carried out in a completely randomized design in a 2 × 2 × 2 × 3 factorial arrangement, consisting of two seasons, two regions, two young clones (approximately two years old), and three positions of crown leaf sampling. The field areas (regions) had contrasting soil physical and chemical properties, and their soil contents for total N, NH+4-N, and NO−3-N were determined in five soil layers, up to a depth of 1.0 m. We evaluated the following indicators of plant N status in roots and leaves: contents of total N, NH+4-N, NO−3-N, and chlorophyll; N/P ratio; and chlorophyll meter readings on the leaves. Ammonium (root) and NO−3-N (root and leaf) efficiently predicted N requirements for eucalyptus plantlets in the greenhouse. Similarly, leaf N/P, chlorophyll values, and chlorophyll meter readings provided good results in the greenhouse. However, leaf N/P did not reflect the soil N status, and the use of the chlorophyll meter could not be generalized for different genotypes. Leaf total N concentration is not an ideal indicator, but it and the chlorophyll levels best represent the soil N status for young eucalyptus clones under field conditions.

Small, stable shuttle vectors based on the minimal pVS1 replicon for use in gram-negative, plant-associated bacteria.

The minimal replicon of the Pseudomonas plasmid pVS1 was genetically defined and combined with the Escherichia coli p15A replicon, to provide a series of new, oligocopy cloning vectors (5.3 to 8.3 kb). Recombinant plasmids derived from these vectors were stable in growing and nongrowing cells of root-colonizing P. fluorescens strains incubated under different environmental conditions for more than 1 month.

Detection of live and antibiotic-killed bacteria by quantitative real-time PCR of specific fragments of rRNA.

Assessing bacterial viability by molecular markers might help accelerate the measurement of antibiotic-induced killing. This study investigated whether rRNA could be suitable for this purpose. Cultures of penicillin-susceptible and penicillin-tolerant (Tol1 mutant) Streptococcus gordonii were exposed to mechanistically different penicillin and levofloxacin. Bacterial survival was assessed by viable counts and compared to quantitative real-time PCR amplification of either the 16S rRNA genes or the 16S rRNA, following reverse transcription. Penicillin-susceptible S. gordonii lost > or =4 log(10) CFU/ml of viability over 48 h of penicillin treatment. In comparison, the Tol1 mutant lost < or =1 log(10) CFU/ml. Amplification of a 427-bp fragment of 16S rRNA genes yielded amplicons that increased proportionally to viable counts during bacterial growth but did not decrease during drug-induced killing. In contrast, the same 427-bp fragment amplified from 16S rRNA paralleled both bacterial growth and drug-induced killing. It also differentiated between penicillin-induced killing of the parent and the Tol1 mutant (> or =4 log(10) CFU/ml and < or =1 log(10) CFU/ml, respectively) and detected killing by mechanistically unrelated levofloxacin. Since large fragments of polynucleotides might be degraded faster than smaller fragments, the experiments were repeated by amplifying a 119-bp region internal to the original 427-bp fragment. The amount of 119-bp amplicons increased proportionally to viability during growth but remained stable during drug treatment. Thus, 16S rRNA was a marker of antibiotic-induced killing, but the size of the amplified fragment was critical for differentiation between live and dead bacteria.

Plants respond to pathogen infection by enhancing the antifungal gene expression of root-associated bacteria.

Plant health and fitness widely depend on interactions with soil microorganisms. Some bacteria such as pseudomonads can inhibit pathogens by producing antibiotics, and controlling these bacteria could help improve plant fitness. In the present study, we tested whether plants induce changes in the antifungal activity of root-associated bacteria as a response to root pathogens. We grew barley plants in a split-root system with one side of the root system challenged by the pathogen Pythium ultimum and the other side inoculated with the biocontrol strain Pseudomonas fluorescens CHA0. We used reporter genes to follow the expression of ribosomal RNA indicative of the metabolic state and of the gene phlA, required for production of 2,4-diacetylphloroglucinol, a key component of antifungal activity. Infection increased the expression of the antifungal gene phlA. No contact with the pathogen was required, indicating that barley influenced gene expression by the bacteria in a systemic way. This effect relied on increased exudation of diffusible molecules increasing phlA expression, suggesting that communication with rhizosphere bacteria is part of the pathogen response of plants. Tripartite interactions among plants, pathogens, and bacteria appear as a novel determinant of plant response to root pathogens.

Analysis of bioavailable arsenic in rice with whole cell living bioreporter bacteria.

A multiwell plate bioassay was developed using genetically modified bacteria (bioreporter cells) to detect inorganic arsenic extracted from rice. The bacterial cells expressed luciferase upon exposure to arsenite, the activity of which was detected by measurement of cellular bioluminescence. The bioreporter cells detected arsenic in all rice varieties tested, with averages of 0.02-0.15 microg of arsenite equivalent per gram of dry weight and a method detection limit of 6 ng of arsenite per gram of dry rice. This amounted to between approximately 20 and 90% of the total As content reported by chemical methods for the same sample and suggested that a major proportion of arsenic in rice is in the inorganic form. Calibrations of the bioassay with pure inorganic and organic arsenic forms showed that the bacterial cells react to arsenite with highest affinity, followed by arsenate (with 25% response relative to an equivalent arsenite concentration) and trimethylarsine oxide (at 10% relative response). A method for biocompatible arsenic extraction was elaborated, which most optimally consisted of (i) grinding rice to powder, (ii) mixing with an aqueous solution containing pancreatic enzymes, (iii) mechanical shearing, (iv) extraction in mild acid conditions and moderate heat, and (v) centrifugation and pH neutralization. Detection of mainly inorganic arsenic by the bacterial cells may have important advantages for toxicity assessment of rice consumption and would form a good complement to total chemical arsenic determination.

Monitoring mortality as an indicator of influenza in Catalonia, Spain.

This study aimed to investigate the behaviour of two indicators of influenza activity in the area of Barcelona and to evaluate the usefulness of modelling them to improve the detection of influenza epidemics. DESIGN: Descriptive time series study using the number of deaths due to all causes registered by funeral services and reported cases of influenza-like illness. The study concentrated on five influenza seasons, from week 45 of 1988 to week 44 of 1993. The weekly number of deaths and cases of influenza-like illness registered were processed using identification of a time series ARIMA model. SETTING: Six large towns in the Barcelona province which have more than 60,000 inhabitants and funeral services in all of them. MAIN RESULTS: For mortality, the proposed model was an autoregressive one of order 2 (ARIMA (2,0,0)) and for morbidity it was one of order 3 (ARIMA (3,0,0)). Finally, the two time series were analysed together to facilitate the detection of possible implications between them. The joint study of the two series shows that the mortality series can be modelled separately from the reported morbidity series, but the morbidity series is influenced as much by the number of previous cases of influenza reported as by the previous mortality registered. CONCLUSIONS: The model based on general mortality is useful for detecting epidemic activity of influenza. However, because there is not an absolute gold standard that allows definition of the beginning of the epidemic, the final decision of when it is considered an epidemic and control measures recommended should be taken after evaluating all the indicators included in the influenza surveillance programme.

Epidemiology of foodborne Norovirus outbreaks in Catalonia, Spain

Background: Noroviruses are one of the principal biological agents associated with the consumption of contaminated food. The objective of this study was to analyse the size and epidemiological characteristics of foodborne outbreaks of gastroenteritis in Catalonia, a region in the northeast of Spain.Methods: In all reported outbreaks of gastroenteritis associated with food consumption, faecal samples of persons affected were analysed for bacteria and viruses and selectively for parasites. Study variables included the setting, the number of people exposed, age, sex, clinical signs and hospital admissions. The study was carried out from October 2004 to October 2005.Results: Of the 181 outbreaks reported during the study period, 72 were caused by Salmonella and 30 by norovirus (NoV); the incidence rates were 14.5 and 9.9 per 100,000 person-years, respectively. In 50% of the NoV outbreaks and 27% of the bacterial outbreaks (p = 0.03) the number of persons affected was ¿10; 66.7% of NoV outbreaks occurred in restaurants; no differences in the attack rates were observed according to the etiology. Hospitalizations were more common (p = 0.03) in bacterial outbreaks (8.6%) than in NoV outbreaks (0.15%). Secondary cases accounted for 4% of cases in NoV outbreaks compared with 0.3% of cases in bacterial outbreaks (p < 0.001)Conclusion: Norovirus outbreaks were larger but less frequent than bacterial outbreaks, suggesting that underreporting is greater for NoV outbreaks. Food handlers should receive training on the transmission of infections in diverse situations. Very strict control measures on handwashing and environmental disinfection should be adopted in closed or partially-closed institutions.

Foodborne norovirus outbreak: the role of an asymptomatic food handler

Background: In July 2005 an outbreak of acute gastroenteritis occurred on a residential summer camp in the province of Barcelona (northeast of Spain). Forty-four people were affected among residents and employees. All of them had in common a meal at lunch time on 13 July (paella, round of beef and fruit). The aim of this study was to investigate a foodborne norovirus outbreak that occurred in the residential summer camp and in which the implication of a food handler was demonstrated by laboratory tests. Methods: A retrospective cohort study was designed. Personal or telephone interview was carried out to collect demographic, clinical and microbiological data of the exposed people, as well as food consumption in the suspected lunch. Food handlers of the mentioned summer camp were interviewed. Ten stool samples were requested from symptomatic exposed residents and the three food handlers that prepared the suspected food. Stools were tested for bacteries and noroviruses. Norovirus was detected using RT-PCR and sequence analysis. Attack rate, relative risks (RR) and its 95% confidence intervals (CI) were calculated to assess the association between food consumption and disease. Results: The global attack rate of the outbreak was 55%. The main symptoms were abdominal pain (90%), nausea (85%), vomiting (70%) and diarrhoea (42.5%). The disease remitted in 24-48 hours. Norovirus was detected in seven faecal samples, one of them was from an asymptomatic food handler who had not eaten the suspected food (round of beef), but cooked and served the lunch. Analysis of the two suspected foods isolated no pathogenic bacteria and detected no viruses. Molecular analysis showed that the viral strain was the same in ill patients and in the asymptomatic food handler (genotype GII.2 Melksham-like). Conclusions: In outbreaks of foodborne disease, the search for viruses in affected patients and all food handlers, even in those that are asymptomatic, is essential. Health education of food handlers with respect to hand washing should be promoted.

Phosphorus content of ditch sediments as indicator of critical source areas

Selostus : Kilpailukykyinen menetelmä pistemäisten fosforikuormitusalueiden paikantamiseen

Monitoring of aglycons of yew glycosides (3,5-dimethoxyphenol, myrtenol and 1-octen-3-ol) as first indicator of yew presence.

The toxicity of yew (Taxus spp) is well known from ancient times and is mainly due to taxins acting as inhibitors of calcium and sodium transport across the cell membrane of cardiac myocytes. The confirmation of yew taxins in body fluids can be carried out by liquid chromatography-tandem mass spectrometry (LC-MS/MS). However, before selecting this precise but expensive technique, an orientation test should be done to ascertain yew presence as toxic agent in the organism. As the 3,5-dimethoxyphenol (3,5-DMP), myrtenol and 1-octen-3-ol appear as glycosidically bound volatile compounds and are very yew specific, the detection of 3,5-DMP and the measurement of 1-octen-3-ol / myrtenol concentration ratio constitute reliable indicators of yew presence in forensic cases. The detection of these compounds is easily performed by gas chromatography-mass spectrometry (GC-MS) (SIM) after an enzymatic hydrolysis (β-glucosidase) allowing the release of volatile compounds from yew glycosides. Copyright © 2012 John Wiley & Sons, Ltd.

Use of inoculated lactic acid bacteria in fermenting sour cabbage

Selostus: Maitohappobakteerien hyödyntäminen hapankaalin fermentoinnissa

The structure of nervous tissue and of Gram-positive bacteria studied by cryo-electron microscopy of vitreous sections

Résumé La structure, ou l'architecture, des êtres vivants définit le cadre dans lequel la physique de la vie s'accomplit. La connaissance de cette structure dans ses moindres détails est un but essentiel de la biologie. Son étude est toutefois entravée par des limitations techniques. Malgré son potentiel théorique, la microscopie électronique n'atteint pas une résolution atomique lorsqu'elle est appliquée ä la matièxe biologique. Cela est dû en grande partie au fait qu'elle contient beaucoup d'eau qui ne résiste pas au vide du microscope. Elle doit donc être déshydratée avant d'être introduite dans un microscope conventionnel. Des artéfacts d'agrégation en découlent inévitablement. La cryo-microscopie électronique des sections vitreuses (CEMOVIS) a ëté développée afin de résoudre cela. Les spécimens sont vitrifiés, c.-à-d. que leur eau est immobilisée sans cristalliser par le froid. Ils sont ensuite coupés en sections ultrafines et celles-ci sont observées à basse température. Les spécimens sont donc observés sous forme hydratée et non fixée; ils sont proches de leur état natif. Durant longtemps, CEMOVIS était très difficile à exécuter mais ce n'est plus le cas. Durant cette thèse, CEMOVIS a été appliqué à différents spécimens. La synapse du système nerveux central a été étudiée. La présence dans la fente synaptique d'une forte densité de molécules organisées de manière périodique a été démontrée. Des particules luminales ont été trouvées dans Ies microtubules cérébraux. Les microtubules ont servi d'objets-test et ont permis de démontrer que des détails moléculaires de l'ordre du nm sont préservés. La compréhension de la structure de l'enveloppe cellulaire des bactéries Grampositives aété améliorée. Nos observations ont abouti à l'élaboration d'un nouveau modèle hypothétique de la synthèse de la paroi. Nous avons aussi focalisé notre attention sur le nucléoïde bactérien et cela a suscité un modèle de la fonction des différents états structuraux du nucléoïde. En conclusion, cette thèse a démontré que CEMOVIS est une excellente méthode poux étudier la structure d'échantillons biologiques à haute résolution. L'étude de la structure de divers aspects des êtres vivants a évoqué des hypothèses quant à la compréhension de leur fonctionnement. Summary The structure, or the architecture, of living beings defines the framework in which the physics of life takes place. Understanding it in its finest details is an essential goal of biology. Its study is however hampered by technical limitations. Despite its theoretical potential, electron microscopy cannot resolve individual atoms in biological matter. This is in great part due to the fact. that it contains a lot of water that cannot stand the vacuum of the microscope. It must therefore be dehydrated before being introduced in a conventional mìcroscope. Aggregation artefacts unavoidably happen. Cryo-electron microscopy of vitreous sections (CEMOVIS) has been developed to solve this problem. Specimens are vitrified, i.e. they are rapidly cooled and their water is immobilised without crystallising by the cold. They are then. sectioned in ultrathin slices, which are observed at low temperatures. Specimens are therefore observed in hydrated and unfixed form; they are close to their native state. For a long time, CEMOVIS was extremely tedious but this is not the case anymore. During this thesis, CEMOVIS was applied to different specimens. Synapse of central nervous system was studied. A high density of periodically-organised molecules was shown in the synaptic cleft. Luminal particles were found in brain microtubules. Microtubules, used as test specimen, permitted to demonstrate that molecular details of the order of nm .are preserved. The understanding of the structure of cell envelope of Gram-positive bacteria was improved. Our observations led to the elaboration of a new hypothetic model of cell wall synthesis. We also focused our attention on bacterial nucleoids and this also gave rise to a functional model of nucleoid structural states. In conclusion, this thesis demonstrated that CEMOVIS is an excellent method for studying the structure of bìologìcal specimens at high resolution. The study of the structure of various aspects of living beings evoked hypothesis for their functioning.