Assessment of the effect of time and temperature on the availability of residual phosphate in a glasshouse study of four soils using the Olsen method

The Olsen method is an indicator of plant-available phosphorus (P). The effect of time and temperature on residual phosphate in soils was measured using the Olsen method in a pot experiment. Four soils were investigated: two from Pakistan and one each from England (calcareous) and Colombia (acidic). Two levels of residual phosphate were developed in each soil after addition of phosphate by incubation at either 10degreesC or 45degreesC. The amount of phosphate added was based on the P maximum of each soil, calculated using the Langmuir equation. Rvegrass was used as the test crop. The pooled data for the four soils incubated at 10degreesC showed good correlation between Olsen P and dry matter yield or P uptake (r(2) = 0.85 and 0.77, respectively), whereas at 45 degreesC, each soil had its own relationship and pooled data did not show correlation of Olsen P with dry matter yield or P uptake. When the data at both temperatures were pooled, Olsen P was a good indicator of yield and uptake for the English soil. For the Pakistani soils, Olsen P after 45 degreesC treatment was an underestimate relative to the 10 degreesC data and for the Colombian soil it was an overestimate. The reasons for these differences need to be explored further before high temperature incubation can be used to simulate long-term changes in the field.

Effects of host plant drought stress on the performance of the bird cherry-oat aphid, Rhopalosiphum padi (L.): a mechanistic analysis

1. The growth (increase in height and leaf number) of four grass species was reduced by a -0.5 MPa drought stress, but the performance of an associated herbivore, Rhopalosiphum padi (L.), was not affected consistently. The intrinsic rate of increase of R. padi was reduced by drought stress on three grass species, including Dactylis glomerata (L.), but was unaffected on Arrhenatherum elatius (L.). Therefore, there is no general relationship in the effect of plant drought on an insect herbivore, even among closely related host plant species. 2. Drought stress increased the quality of plant phloem sap, as indicated by increased sieve element osmotic pressure and essential amino acid concentrations. Thus, diet quality could not account for the reduced performance of R. padi under drought stress. The concentration of essential amino acids in the phloem of well-watered A. elatius was, however, lower than that of well-watered D. glomerata, correlating with the decreased performance of aphids on well-watered A. elatius. 3. There were no differences in aphid feeding duration between watering treatments or plant species but sap ingestion rates were reduced significantly under drought stress. 4. Using the measure of dietary amino acid concentrations and the estimate of sap ingestion, the essential amino acid flux through aphids was calculated. Compared with the flux through aphids feeding on well-watered D. glomerata, there was a reduction in aphids feeding on drought-stressed D. glomerata and drought-stressed A. elatius due to lower sap ingestion rates. The flux through aphids on well-watered A. elatius was also reduced due to low phloem essential amino acid concentrations. Thus, the performance of an aphid is correlated with the availability and accessibility of essential amino acids.

Detection of transgenic DNA and protein, in rumen fluid, duodenal digesta, milk, blood and faeces of lactating dairy cows

The objective was to determine the presence or absence of transgenic and endogenous plant DNA in ruminal fluid, duodenal digesta, milk, blood, and feces, and if found, to determine fragment size. Six multiparous lactating Holstein cows fitted with ruminal and duodenal cannulas received a total mixed ration. There were two treatments (T). In T1, the concentrate contained genetically modified (GM) soybean meal (cp4epsps gene) and GM corn grain (cry1a[b] gene), whereas T2 contained the near isogenic non-GM counterparts. Polymerase chain reaction analysis was used to determine the presence or absence of DNA sequences. Primers were selected to amplify small fragments from single-copy genes (soy lectin and corn high-mobility protein and cp4epsps and cry1a[b] genes from the GM crops) and multicopy genes (bovine mitochondrial cytochrome b and rubisco). Single-copy genes were only detected in the solid phase of rumen and duodenal digesta. In contrast, fragments of the rubisco gene were detected in the majority of samples analyzed in both the liquid and solid phases of ruminal and duodenal digesta, milk, and feces, but rarely in blood. The size of the rubisco gene fragments detected decreased from 1176 bp in ruminal and duodenal digesta to 351 bp in fecal samples.

Detection of unusual mutation within the VP1 region of different re-isolates of poliovirus Sabin vaccine

In the present study, a genomic analysis of full VP1 sequence region of 15 clinical re-isolates (14 healthy vaccinees and one bone marrow tumor patient) was conducted, aiming to the identification of mutations and to the assessment of their impact on virus fitness, providing also insights relevant with the natural evolution of Sabin strains. Clinical re-isolates were analyzed by RT-PCR, sequencing and computational analysis. Some re-isolates were characterized by an unusual mutational pattern in which non-synonymous mutations outnumbered the synonymous ones. Furthermore, the majority of amino-acid substitutions were located in the capsid exterior, specifically in N-Ags, near N-Ags and in the north rim of the canyon. Also mutations, which are well-known determinants of attenuation, were identified. The results of this study propose that some re-isolates are characterized by an evolutionary pattern in which non-synonymous mutations with a direct phenotypic impact on viral fitness are fixed in viral genomes, in spite of synonymous ones with no phenotypic impact on viral fitness. Results of the present retrospective characterization of Sabin clinical re-isolates, based on the full VP1 sequence, suggest that vaccine-derived viruses may make their way through narrow breaches and may evolve into transmissible pathogens even in adequately immunized populations. For this reason increased poliovirus laboratory surveillance should be permanent and full VP1 sequence analysis should be conducted even in isolates originating from healthy vaccinees.

Mechanisms of partial plant resistance to diamondback moth (Plutella xylostella) in brassicas

Artificial diet studies were used to differentiate among physical and chemical mechanisms affecting the suitability to diamondback moth (Plutella xylostella L.), of 16 food substrates obtained by growing four different brassicas in the glasshouse or field and measuring the pest's performance on either leaf discs or a diet incorporating leaf powders. Leaves of Chinese cabbage and the cabbage cultivar 'Minicole' were, respectively, the most and least suitable leaves for the insect, but this ranking was reversed on artificial diet. Leaves of glasshouse-grown plants were more suitable than those of plants grown in the fields. Differences in the suitability of leaves to diamondback moth appeared to be largely determined by leaf toughness and surface wax load. Concentrations of individual glucosinolates in the brassicas probably acted as phagostimulants, so increasing their intrinsic susceptibility to diamondback moth, but the effect of the physical factors appeared more important.

Detection of Salmonella enteritidis in eggs by the polymerase chain reaction

A polymerase chain reaction (PCR) for the specific detection of the gene sequence, sefA, encoded by all isolates of Salmonella enteritidis, was developed. The PCR could detect as few as four S enteritidis washed bacterial cells but egg contents inhibited the PCR. Eggs spiked with 50 S enteritidis bacterial cells were homogenised, inoculated into buffered peptone water and grown at 37 degrees C for 16 hours, when the PCR was successful. A positive internal control was developed to differentiate between true and false negative PCR results for the detection of S enteritidis. In a limited trial of the egg handling procedures and the PCR, one of 250 chickens' eggs from retail outlets was found to be contaminated with S enteritidis.

Detection of gyrA mutations in quinolone-resistant Salmonella enterica by denaturing high-performance liquid chromatography

Denaturing high-performance liquid chromatography (DHPLC) was evaluated as a rapid screening and identification method for DNA sequence variation detection in the quinolone resistance-determining region of gyrA from Salmonella serovars. A total of 203 isolates of Salmonella were screened using this method. DHPLC analysis of 14 isolates representing each type of novel or multiple mutations and the wild type were compared with LightCycler-based PCR-gyrA hybridization mutation assay (GAMA) and single-strand conformational polymorphism (SSCP) analyses. The 14 isolates gave seven different SSCP patterns, and LightCycler detected four different mutations. DHPLC detected 11 DNA sequence variants at eight different codons, including those detected by LightCycler or SSCP. One of these mutations was silent. Five isolates contained multiple mutations, and four of these could be distinguished from the composite sequence variants by their DHPLC profile. Seven novel mutations were identified at five different loci not previously described in quinolone-resistant salmonella. DHPLC analysis proved advantageous for the detection of novel and multiple mutations. DHPLC also provides a rapid, high-throughput alternative to LightCycler and SSCP for screening frequently occurring mutations.

Detection of Oxidation Products of 5-Methyl-2′-Deoxycytidine in Arabidopsis DNA

Epigenetic regulations play important roles in plant development and adaptation to environmental stress. Recent studies from mammalian systems have demonstrated the involvement of ten-eleven translocation (Tet) family of dioxygenases in the generation of a series of oxidized derivatives of 5-methylcytosine (5-mC) in mammalian DNA. In addition, these oxidized 5-mC nucleobases have important roles in epigenetic remodeling and aberrant levels of 5-hydroxymethyl-29-deoxycytidine (5-HmdC) were found to be associated with different types of human cancers. However, there is a lack of evidence supporting the presence of these modified bases in plant DNA. Here we reported the use of a reversed-phase HPLC coupled with tandem mass spectrometry method and stable isotope-labeled standards for assessing the levels of the oxidized 5-mC nucleosides along with two other oxidatively induced DNA modifications in genomic DNA of Arabidopsis. These included 5- HmdC, 5-formyl-29-deoxycytidine (5-FodC), 5-carboxyl-29-deoxycytidine (5-CadC), 5-hydroxymethyl-29-deoxyuridine (5- HmdU), and the (59S) diastereomer of 8,59-cyclo-29-deoxyguanosine (S-cdG). We found that, in Arabidopsis DNA, the levels of 5-HmdC, 5-FodC, and 5-CadC are approximately 0.8 modifications per 106 nucleosides, with the frequency of 5-HmdC (per 5-mdC) being comparable to that of 5-HmdU (per thymidine). The relatively low levels of the 5-mdC oxidation products suggest that they arise likely from reactive oxygen species present in cells, which is in line with the lack of homologous Tetfamily dioxygenase enzymes in Arabidopsis.

Detection of recent changes in climate using meteorological data from south-eastern Bangladesh

Analysis of meteorological records from four stations (Chittagong, Cox’s Bazar, Rangamati, Sitakunda) in south-eastern Bangladesh show coherent changes in climate over the past three decades. Mean maximum daily temperatures have increased between 1980 and 2013 by ca. 0.4 to 0.6°C per decade, with changes of comparable magnitude in individual seasons. The increase in mean maximum daily temperature is associated with decreased cloud cover and wind speed, particularly in the pre- and post-monsoon seasons. During these two seasons, the correlation between changes in maximum temperature and clouds is between -0.5 and -0.7; the correlation with wind speed is weaker although similar values are obtained in some seasons. Changes in mean daily minimum (and hence mean) temperature differ between the northern and southern part of the basin: northern stations show a decrease in mean daily minimum temperature during the post-monsoon season of between 0.2 and 0.5°C per decade while southern stations show an increase of ca. 0.1 to 0.4°C per decade during the pre-monsoon and monsoon seasons. In contrast to the significant changes in temperature, there is no trend in mean or total precipitation at any station. However, there is a significant increase in the number of rain days at the northern sites during the monsoon season, with an increase per decade of 3 days in Sitakunda and 7 days at Rangamati. These climate changes could have a significant impact on the hydrology of the Halda Basin, which supplies water to Chittagong and is the major pisciculture centre in Bangladesh.

Rapid, automated detection of stem canker symptoms in woody perennials using artificial neural network analysis

Background Pseudomonas syringae can cause stem necrosis and canker in a wide range of woody species including cherry, plum, peach, horse chestnut and ash. The detection and quantification of lesion progression over time in woody tissues is a key trait for breeders to select upon for resistance. Results In this study a general, rapid and reliable approach to lesion quantification using image recognition and an artificial neural network model was developed. This was applied to screen both the virulence of a range of P. syringae pathovars and the resistance of a set of cherry and plum accessions to bacterial canker. The method developed was more objective than scoring by eye and allowed the detection of putatively resistant plant material for further study. Conclusions Automated image analysis will facilitate rapid screening of material for resistance to bacterial and other phytopathogens, allowing more efficient selection and quantification of resistance responses.

Use of the checkerboard DNA-DNA hybridisation technique for in vivo detection of cariogenic microorganisms on metallic brackets, with or without use of an antimicrobial agent

Objective: Using checkerboard DNA-DNA hybridisation (CDDH) assay, this randomised clinical study evaluated the contamination of metallic brackets by four cariogenic bacterial strains (Streptococcus mutans, Streptococcus sobrinus, Lactobacillus casei and Lactobacillus acidophilus) and the efficacy of 0.12% chlorhexidine gluconate (CHX) mouthwashes in reducing bacterial contamination. Methods: Thirty-nine 11-33-year-old patients under treatment with fixed orthodontic appliances were enrolled in the study and had 2 new metallic brackets bonded to premolars. Nineteen patients used a 0.12% CHX mouthwash (Periogard (R)) and 20 patients used a placebo mouthwash (control) twice a week. After 30 days, the brackets were removed and samples were obtained for analysis by CDDH. Data were analysed statistically by the Kruskal-Wallis test (alpha = 0.05) using the SAS software. Results: S. mutans, S. sobrinus, L. casei and L. acidophilus were detected in 100% of the samples from both groups. However, brackets of the control group were more heavily contaminated by S. mutans and S. sobrinus (P < 0.01). In the experimental group, although all counts decreased after rinsing with the chlorhexidine solution, there was significant difference only for S. mutans (P = 0.03). Conclusions: The use of 0.12% chlorhexidine gluconate mouthwashes can be useful in clinical practice to reduce the levels of cariogenic microorganisms in patients under treatment with fixed orthodontic appliances. (C) 2011 Elsevier Ltd. All rights reserved.

Detection of urease in the cell wall and membranes from leaf tissues of bromeliad species

Urea is an important nitrogen source for some bromeliad species, and in nature it is derived from the excretion of amphibians, which visit or live inside the tank water. Its assimilation is dependent on the hydrolysis by urease (EC: 3.5.1.5), and although this enzyme has been extensively studied to date, little information is available about its cellular location. In higher plants, this enzyme is considered to be present in the cytoplasm. However, there is evidence that urease is secreted by the bromeliad Vriesea gigantea, implying that this enzyme is at least temporarily located in the plasmatic membrane and cell wall. In this article, urease activity was measured in different cell fractions using leaf tissues of two bromeliad species: the tank bromeliad V. gigantea and the terrestrial bromeliad Ananas comosus (L.) Merr. In both species, urease was present in the cell wall and membrane fractions, besides the cytoplasm. Moreover, a considerable difference was observed between the species: while V. gigantea had 40% of the urease activity detected in the membranes and cell wall fractions, less than 20% were found in the same fractions in A. comosus. The high proportion of urease found in cell wall and membranes in V. gigantea was also investigated by cytochemical detection and immunoreaction assay. Both approaches confirmed the enzymatic assay. We suggest this physiological characteristic allows tank bromeliads to survive in a nitrogen-limited environment, utilizing urea rapidly and efficiently and competing successfully for this nitrogen source against microorganisms that live in the tank water.

Expression pattern of four storage xyloglucan mobilization-related genes during seedling development of the rain forest tree Hymenaea courbaril L.

During seedling establishment, cotyledons of the rain forest tree Hymenaea courbaril mobilize storage cell wall xyloglucan to sustain growth. The polysaccharide is degraded and its products are transported to growing sink tissues. Auxin from the shoot controls the level of xyloglucan hydrolytic enzymes. It is not yet known how important the expression of these genes is for the control of storage xyloglucan degradation. In this work, partial cDNAs of the genes xyloglucan transglycosylase hydrolase (HcXTH1) and beta-galactosidase (HcBGAL1), both related to xyloglucan degradation, and two other genes related to sucrose metabolism [alkaline invertase (HcAlkIN1) and sucrose synthase (HcSUS1)], were isolated. The partial sequences were characterized by comparison with sequences available in the literature, and phylogenetic trees were assembled. Gene expression was evaluated at intervals of 6 h during 24 h in cotyledons, hypocotyl, roots, and leaves, using 45-d-old plantlets. HcXTH1 and HcBGAL1 were correlated to xyloglucan degradation and responded to auxin and light, being down-regulated when transport of auxin was prevented by N-1-naphthylphthalamic acid (NPA) and stimulated by constant light. Genes related to sucrose metabolism, HcAlkIN1 and HcSUS1, responded to inhibition of auxin transport in consonance with storage mobilization in the cotyledons. A model is proposed suggesting that auxin and light are involved in the control of the expression of genes related to storage xyloglucan mobilization in seedlings of H. courbaril. It is concluded that gene expression plays a role in the control of the intercommunication system of the source-sink relationship during seeding growth, favouring its establishment in the shaded environment of the rain forest understorey.

Mobilization of seed reserves during germination of four tropical species of the Amazon Rainforest

Seed reserve mobilization during germination of the Amazonian species Myciaria dubia (camu-camu), Eugenia stipitata(araca-boi), Dipteryx odorata (cumaru) and Hymenaea courbaril (jatoba) was evaluated. Seeds were placed in germination chambers at 30 degrees C with it 12 h photoperiod. Analysis of primary metabolites (carbohydrates, lipids and proteins) and fatty acid composition were carried out in quiescent seeds and at four germination stages after radicle protrusion. Germination was high in all species but there were statistically significant differences between species. Differences were also observed with regard to the duration of the germination period. The seeds showed variation in the content and composition of the analyzed compounds. indicating that the mobilization rates of these compounds may affect germination velocity.

Quantitative detection of periodontopathic bacteria in atherosclerotic plaques from coronary arteries

Oral pathogens, including periodontopathic bacteria, are thought to be aetiological factors in the development of cardiovascular disease. In this study, the presence of Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum-periodonticum-simiae group, Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens and Tannerella forsythia in atheromatous plaques from coronary arteries was determined by real-time PCR. Forty-four patients displaying cardiovascular disease were submitted to periodontal examination and endarterectomy of coronary arteries. Approximately 60-100 mg atherosclerotic tissue was removed surgically and DNA was obtained. Quantitative detection of periodontopathic bacteria was performed using universal and species-specific TaqMan probe/primer sets. Total bacterial and periodontopathic bacterial DNA were found in 94.9 and 92.3 %, respectively, of the atheromatous plaques from periodontitis patients, and in 80.0 and 20.0%, respectively, of atherosclerotic tissues from periodontally healthy subjects. All periodontal bacteria except for the F. nucleatum-periodonticum-simiae group were detected, and their DNA represented 47.3 % of the total bacterial DNA obtained from periodontitis; patients. Porphyromonas gingivalis, A. actinomycetemcomitans and Prevotella intermedia were detected most often. The presence of two or more periodontal species could be observed in 64.1 % of the samples. In addition, even in samples in which a single periodontal species was detected, additional unidentified microbial DNA could be observed. The significant number of periodontopathic bacterial DNA species in atherosclerotic tissue samples from patients with periodontitis suggests that the presence of these micro-organisms in coronary lesions is not coincidental and that they may in fact contribute to the development of vascular diseases.