Sensitive and rapid detection of Vero toxin-producing Escherichia coli using loop-mediated isothermal amplification.
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A loop-mediated isothermal amplification (LAMP) assay was developed to detect Vero toxin (VT)-producing Escherichia coli rapidly (within 60 min). The 24 strains of VT-producing E. coli were successfully amplified, but 6 strains of non-VT-producing E. coli and 46 bacterial species other than E. coli were not. The sensitivity of the LAMP assay was found to be >0.7 c.f.u. per test using serogroups O157, O26 and O111 of VT-producing E. coli; this sensitivity is greater than that obtained by PCR assay. Furthermore, the LAMP assay was examined for its ability to detect VT-producing E. coli in food because of the difficulty of detection in food samples. The recovery of VT-producing E. coli by LAMP assay from beef and radish sprouts inoculated with the pathogen was high, similar to that obtained using culture methods with direct plating and/or plating after immunomagnetic separation. Although PCR assay was unable to recover VT-producing E. coli from half of the radish samples, LAMP assay was successful in most samples. In addition, VT-producing E. coli was successfully detected in cultures of the beef samples by LAMP assay, but not by the culture method. The LAMP products in naturally contaminated beef samples were analysed to confirm the specific amplification of the VT-encoding gene, and were found to show a specific ladder band pattern on agarose gel after electrophoresis. Additionally the sequences of the LAMP products coincided well with the expected sequences of the VT-encoding gene. These results indicate that the proposed LAMP assay is a rapid, specific and sensitive method of detecting the VT-producing E. coli. (+info)
A novel locus involved in extracellular polysaccharide production and virulence of Xanthomonas campestris pathovar campestris.
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Xanthomonas campestris pathovar campestris (Xcc) is the causal agent of black rot disease in cruciferous plants. The extracellular polysaccharide (EPS) produced by Xcc is an important pathogenicity factor and also has a range of industrial uses. In preliminary work a number of transposon-mediated insertion mutants in Xcc with defects in EPS production were identified. Here, one of these mutated loci was investigated in detail. Six ORFs within the locus (ORFs XC3811-3816) were disrupted by plasmid integration. Mutation of XC3813, XC3814 or XC3815 resulted in significantly reduced EPS production and significantly reduced virulence on the host plant Chinese radish (Raphanus sativus). The EPS production and virulence of XC3813, XC3814 and XC3815 mutants could be restored by intact XC3813, XC3814 and XC3815 genes, respectively, when provided in trans. Although bioinformatic analysis suggested a role for XC3814 and XC3815 in lipopolysaccharide biosynthesis, the lipopolysaccharides produced by the mutants were indistinguishable from those of the wild-type, as judged by electrophoretic mobility in SDS-polyacrylamide gels. These results reveal that XC3813, XC3814 and XC3815 comprise a novel gene cluster involved in EPS production and virulence of Xcc. (+info)
Effects of dietary administration of plant-derived anthocyanin-rich colors to spontaneously hypertensive rats.
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Anthocyanins have beneficial effects such as free radical scavenging activity. We investigated the effects of continuous administration of colors from purple corn (PCC), purple sweet potato (PSC) and red radish (RRC) to spontaneously hypertensive rats (SHR). These are rich in anthocyanins. Animals were fed with diets containing PCC, PSC or RRC (1 mass% of diets) for 15 wk. While the body weight and the daily food intake of administered rats were not different from those of the non-administered control rats through the experimental period, the blood pressure and the heart rate of SHR administered each color decreased as compared to the control group from the early stage of administration. These results suggest that plant-derived colors containing anthocyanins have anti-hypertensive effects on hypertensive animals. (+info)
Genes encoding pentatricopeptide repeat (PPR) proteins are not conserved in location in plant genomes and may be subject to diversifying selection.
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BACKGROUND: The pentatricopeptide repeat (PPR) is a degenerate 35 amino acid motif that occurs in multiple tandem copies in members of a recently recognized eukaryotic gene family. Most analyzed eukaryotic genomes contain only a small number of PPR genes, but in plants the family is greatly expanded. The factors that underlie the expansion of this gene family in plants are not as yet understood. RESULTS: We show that the location of PPR genes is highly variable in comparisons between orthologous, closely related, and otherwise co-linear chromosomal regions of the Brassica rapa or radish and Arabidopsis thaliana. This observation also pertains to paralogous duplicated segments of the genomes of Arabidopsis thaliana and Brassica rapa. In addition, we show that PPR genes that seem closely linearly aligned in these comparisons are not generally found to be closely related to one another at the nucleotide and amino acid sequence level. We observe a relatively high level of non-synonomous vs synonomous changes among a group tandemly repeated radish PPR genes, suggesting that these, and possibly other PPR genes, are subject to diversifying selection. We also show that a duplicated region of the Arabidopsis genome possesses a relatively high density of PPR genes showing high similarity to restorers of fertility of cytoplasmic male sterile (CMS) systems of petunia, radish and rice. The PPR genes in these regions, together with the restorer genes, are more highly similar to one another, in sequence as well as in structure, than to other PPR genes, even within the same sub-family. CONCLUSION: Our results suggest are consistent with a model in which at least some PPR genes undergo a "birth and death" process that involves transposition to unrelated chromosomal sites. PPR genes hold certain features in common with disease resistance genes (R genes), and their "nomadic" character suggests that their evolutionary expansion in plants may have involved novel molecular processes and selective pressures. (+info)
Streptomyces turgidiscabies secretes a novel virulence protein, Nec1, which facilitates infection.
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Emergence of new, economically important plant-pathogenic species in the mostly saprophytic genus Streptomyces involves acquisition of a large, mobile pathogenicity island (PAI). Biosynthetic genes for a phytotoxin, thaxtomin A, are contained on this PAI. The Nec1 protein has necrogenic activity on excised potato tuber tissue, and the encoding gene is highly conserved in plant-pathogenic Streptomyces spp. The G+C content of nec1 indicates lateral transfer from an unrelated taxon; however, the nucleic acid and protein databases have not yielded homologs. Data presented in this article demonstrate that the Nec1 protein is necrogenic when expressed in Escherichia coli and that an active 16-kDa form of Nec1 is secreted from the plant pathogen Streptomyces turgidiscabies. Deletion analysis of nec1 demonstrated that the 151-amino-acid C-terminal region of the Nec1 protein is sufficient to confer necrogenic activity. Analysis of nec1 transcriptional start sites indicates that two mRNA species are produced and that the site of transcription initiation is influenced by glucose. S. turgidiscabies containing a nec1 deletion was greatly compromised in virulence on Arabidopsis thaliana, Nicotiana tabacum, and Raphanus sativus seedlings. The wild-type strain, S. turgidiscabies Car8, aggressively colonized and infected the root meristem of radish, whereas the deltanec1 mutant Car811 did not. Taken together, these data suggest that Nec1 is a secreted virulence protein with a conserved plant cell target that acts early in plant infection. (+info)
Parameter estimation and prediction for the course of a single epidemic outbreak of a plant disease.
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Many epidemics of plant diseases are characterized by large variability among individual outbreaks. However, individual epidemics often follow a well-defined trajectory which is much more predictable in the short term than the ensemble (collection) of potential epidemics. In this paper, we introduce a modelling framework that allows us to deal with individual replicated outbreaks, based upon a Bayesian hierarchical analysis. Information about 'similar' replicate epidemics can be incorporated into a hierarchical model, allowing both ensemble and individual parameters to be estimated. The model is used to analyse the data from a replicated experiment involving spread of Rhizoctonia solani on radish in the presence or absence of a biocontrol agent, Trichoderma viride. The rate of primary (soil-to-plant) infection is found to be the most variable factor determining the final size of epidemics. Breakdown of biological control in some replicates results in high levels of primary infection and increased variability. The model can be used to predict new outbreaks of disease based upon knowledge from a 'library' of previous epidemics and partial information about the current outbreak. We show that forecasting improves significantly with knowledge about the history of a particular epidemic, whereas the precision of hindcasting to identify the past course of the epidemic is largely independent of detailed knowledge of the epidemic trajectory. The results have important consequences for parameter estimation, inference and prediction for emerging epidemic outbreaks. (+info)
Evaluation of nutritional availability and anti-tumor activity of selenium contained in selenium-enriched Kaiware radish sprouts.
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We estimated the nutritional availability of selenium (Se) in Se-enriched Kaiware radish sprouts (SeRS) by the tissue Se deposition and glutathione peroxidase (GPX) activity of rats administered the sprouts, and examined the effect of SeRS on the formation of aberrant crypt foci (ACF) in the colon of mice administered 1,2-dimethylhydrazine (DMH) to evaluate anti-tumor activity. Male weanling Wistar rats were divided into seven groups and fed a Se-deficient basal diet or the basal diet supplemented with 0.05, 0.10, or 0.15 microg/g of Se as sodium selenite or SeRS for 28 d. Supplementation with Se dose-dependently increased serum and liver Se concentrations and GPX activities, and the selenite-supplemented groups showed a higher increase than the SeRS-supplemented groups. The nutritional availability of Se in SeRS was estimated to be 33 or 64% by slope ratio analysis. Male 4-week-old A/J mice were divided into seven groups and fed a low Se basal diet or the basal diet supplemented with selenite, SeRS, or selenite + non-Se-enriched radish sprouts (NonSeRS) at a level of 0.1 or 2.0 microg Se/g for 9 weeks. After 1 week of feeding, all mice were given six subcutaneous injections of DMH (20 mg/kg) at 1-week intervals. The average number of ACF formed in the colon of mice fed the basal diet was 4.3. At a supplementation level of 0.1 mug Se/g, only SeRS significantly inhibited ACF formation. At a supplementation level of 2.0 microg Se/g, both selenite and SeRS significantly inhibited ACF formation. The addition of NonSeRS to the selenite-supplemented diets tended to inhibit ACF formation, but this was not statistically significant. These results indicate that SeRS shows lower nutritional availability but higher anti-tumor activity than selenite. (+info)
Differential expression of three catalase genes in the small radish (Rhaphanus sativus L. var. sativus).
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Three catalase cDNA clones were isolated from the small radish (Raphanus sativus L.). Their nucleotide and deduced amino acid sequences showed the greatest homology to those of Arabidopsis. Genomic Southern blot analysis, using RsCat1 cDNA as a probe, showed that catalases are encoded by small multigene family in the small radish. Nondenaturing polyacrylamide gels revealed the presence of several catalase isozymes, the levels of which varied among the organs examined. The isozyme activities were assigned the individual catalase genes by Northern analysis using total RNA from different organs. The three catalase genes were differentially expressed in response to treatments such as white light, xenobiotics, osmoticum, and UV. Their expression in seedlings was controlled by the circadian clock under a light/dark cycle and/or in constant light. Interestingly, RsCat1 transcripts peaked in the morning, while those of RsCat2 and RsCat3 peaked in the early evening. Our results suggest that the RsCat enzymes are involved in defense against the oxidative stress induced by environmental changes. (+info)