Genetic characterization of the interaction between Italian ryegrass (Lolium multiflorum Lam.) and Xanthomonas translucens pv. graminis

Italian ryegrass (Lolium multiflorum Lam.) is a widely used for hay and silage production. Xanthomonas translucens pv. graminis (Xtg) is a plant pathogen and causes bacterial wilt on a number of forage grass species including L. multiflorum. The pathogen spreads through wounds caused when mowing with contaminated equipment. Resistance mechanisms of L. multiflorum and virulence mechanisms of Xtg are largely unknown. Therefore, the main objective of this thesis was to understand the interaction of Xtg with L. multiflorum. The first aim was to elucidate the existence of race-specificity in the L. multiflorum-Xtg interaction and to identify genomic regions that contribute to Xtg resistance. No race-specific interactions were found in 62 L. multiflorum genotypes infected with six Xtg isolates. Molecular marker-resistance associations revealed one simple sequence repeat marker on linkage group 5 to be significantly associated with bacterial wilt resistance. The second aim was to identify candidate genes for bacterial wilt resistance using a cDNA microarray of perennial ryegrass (L. perenne). Transcriptome analyses revealed candidate genes for bacterial wilt resistance such as the low silicon transporter and the germin-like protein 6. Virulence factors of Xtg were investigated by screening for genes encoding the type III secretion system. This revealed a sequence similar to the hrpG gene. A mutant deficient of the hrpG gene was obtained and infection with the ΔhrpG mutant lead to significantly reduced symptoms. In planta analyses revealed that this mutant was still able to multiply inside its host. The data presented here provides comprehensive knowledge of this plant-pathogen interaction and represents a valuable basis for the development of tools for marker-assisted selection. Zusammenfassung Italienisches Raigras (Lolium multiflorum) wird häufig als Futtergras angebaut. Bakterienwelke ist eine Krankheit, die durch Xanthomonas translucens pv. graminis (Xtg), hervorgerufen wird. Die Züchtung von resistenten Sorten ist die effizienteste Kontrollmassnahme gegen Bakterienwelke. Die Quelle der Krankheitsresistenz und Virulenzmechanismen der Bakterien sind noch immer unbekannt. Deshalb war das Hauptziel dieser Arbeit, die Interaktion von L. multiflorum und Xtg besser zu charakterisieren. Das erste Ziel stellte die Aufklärung der Existenz von Rassenspezifität in der L. multiflorum-Xtg Interaktion dar. Zudem wurden molekulare Marker untersucht, die mit Bakterienwelke Resistenz assoziiert sind. Es konnte keine Rassenspezifität gefunden werden in den 62 L. multiflorum Genotypen, die mit sechs verschiedenen Xtg Isolaten infiziert wurden. Die Analyse mit molekularen Markern zeigte, dass ein Mikrosatelliten Marker auf der Kopplungsgruppe 5 signifikant mit Bakterienwelke- Resistenz assoziiert war. Das zweite Ziel war, Kandidatengene für Xtg Resistenz in L. multiflorum zu identifizieren mittels einer Genexpressionsanalyse. Dazu wurde ein cDNA Microrarray verwendet, der mit Sequenzen von Englischem Raigras (L. perenne) entwickelt worden war. Die Genexpressionsstudie führte zur Identifizierung von interessanten Kandidatengenen, die mit Bakterienwelke Resistenz assoziiert sein könnten. Um auch Gene von Xtg zu kennen, die für die Interaktion mit L. multiflorum von grosser Wichtigkeit sein könnten, wurde das Xtg Genom nach Virulenzgenen des Typ III Sekretionssystems untersucht. So wurde das hrpG Gen gefunden und eine Mutante generiert, die das hrpG Gen nicht mehr hat. Diese Mutante war signifikant weniger virulent bei der Infektion von L. multiflorum Pflanzen. In planta Analysen zeigten jedoch, dass die ΔhrpG Mutanten sich noch immer vermehren konnten. Die Erkenntnisse aus dieser Studie stellen einen elementaren Baustein dar, diese Interaktion in grossem Umfang zu verstehen und das erarbeitete Wissen stellt eine essentielle Grundlage dar für die Entwicklung von Marker gestützter Züchtung

Diverse antibiotic resistance genes in dairy cow manure

Application of manure from antibiotic-treated animals to crops facilitates the dissemination of antibiotic resistance determinants into the environment. However, our knowledge of the identity, diversity, and patterns of distribution of these antibiotic resistance determinants remains limited. We used a new combination of methods to examine the resistome of dairy cow manure, a common soil amendment. Metagenomic libraries constructed with DNA extracted from manure were screened for resistance to beta-lactams, phenicols, aminoglycosides, and tetracyclines. Functional screening of fosmid and small-insert libraries identified 80 different antibiotic resistance genes whose deduced protein sequences were on average 50 to 60% identical to sequences deposited in GenBank. The resistance genes were frequently found in clusters and originated from a taxonomically diverse set of species, suggesting that some microorganisms in manure harbor multiple resistance genes. Furthermore, amid the great genetic diversity in manure, we discovered a novel clade of chloramphenicol acetyltransferases. Our study combined functional metagenomics with third-generation sequencing to significantly extend the roster of functional antibiotic resistance genes found in animal gut bacteria, providing a particularly broad resource for understanding the origins and dispersal of antibiotic resistance genes in agriculture and clinical settings

Particulate matter exposure during pregnancy is associated with birth weight, but not gestational age, 1962-1992: a cohort study

<p>Abstract</p> <p>Background</p> <p>Exposure to air pollutants is suggested to adversely affect fetal growth, but the evidence remains inconsistent in relation to specific outcomes and exposure windows.</p> <p>Methods</p> <p>Using birth records from the two major maternity hospitals in Newcastle upon Tyne in northern England between 1961 and 1992, we constructed a database of all births to mothers resident within the city. Weekly black smoke exposure levels from routine data recorded at 20 air pollution monitoring stations were obtained and individual exposures were estimated via a two-stage modeling strategy, incorporating temporally and spatially varying covariates. Regression analyses, including 88,679 births, assessed potential associations between exposure to black smoke and birth weight, gestational age and birth weight standardized for gestational age and sex.</p> <p>Results</p> <p>Significant associations were seen between black smoke and both standardized and unstandardized birth weight, but not for gestational age when adjusted for potential confounders. Not all associations were linear. For an increase in whole pregnancy black smoke exposure, from the 1^st(7.4 μg/m³) to the 25^th(17.2 μg/m³), 50^th(33.8 μg/m³), 75^th(108.3 μg/m³), and 90^th(180.8 μg/m³) percentiles, the adjusted estimated decreases in birth weight were 33 g (SE 1.05), 62 g (1.63), 98 g (2.26) and 109 g (2.44) respectively. A significant interaction was observed between socio-economic deprivation and black smoke on both standardized and unstandardized birth weight with increasing effects of black smoke in reducing birth weight seen with increasing socio-economic disadvantage.</p> <p>Conclusions</p> <p>The findings of this study progress the hypothesis that the association between black smoke and birth weight may be mediated through intrauterine growth restriction. The associations between black smoke and birth weight were of the same order of magnitude as those reported for passive smoking. These findings add to the growing evidence of the harmful effects of air pollution on birth outcomes.</p

Bacterial wilt - a puzzling disease of forage grasses

Kölliker R, Wichmann F, Vorhölter F-J, et al. Bacterial wilt - a puzzling disease of forage grasses. Agrarforschung Schweiz. 2013;40(1):32-39.Bacterial wilt - a puzzling disease of forage grasses Bacterial wilt of forage grasses is caused by the bacterium Xanthomonas translucens pv. graminis (Xtg) and leads to major damage in meadows and pastures. To support the breeding of resistant cultivars, we are studying the genetic bases of the interaction between bacteria and plants. In doing so, we have learnt that this interaction exhibits a number of unique features when compared with other diseases of rice, tomatoes or lemons caused by Xanthomonas species. For one thing, no indications were found of a strain-specific interaction between individual bacterial isolates and plant genotypes, as is characteristic for typical resistance genes. The sequencing of the Xtg genome has shown that this pathogen possesses an unusually high number of insertion sequences. In addition, the organisation of an important virulence factor differs noticeably from other Xanthomonas species. Although knock-out mutation of this virulence factor led to a significant reduction of virulence, the bacteria were still able to reproduce in the plant. These findings provide valuable bases for the further development of efficient breeding methods

Functional Repertoire of Antibiotic Resistance Genes in Antibiotic Manufacturing Effluents and Receiving Freshwater Sediments

Environments polluted by direct discharges of effluents from antibiotic manufacturing are important reservoirs for antibiotic resistance genes (ARGs), which could potentially be transferred to human pathogens. However, our knowledge about the identity and diversity of ARGs in such polluted environments remains limited. We applied functional metagenomics to explore the resistome of two Croatian antibiotic manufacturing effluents and sediments collected upstream of and at the effluent discharge sites. Metagenomic libraries built from an azithromycin-production site were screened for resistance to macrolide antibiotics, whereas the libraries from a site producing veterinary antibiotics were screened for resistance to sulfonamides, tetracyclines, trimethoprim, and beta-lactams. Functional analysis of eight libraries identified a total of 82 unique, often clinically relevant ARGs, which were frequently found in clusters and flanked by mobile genetic elements. The majority of macrolide resistance genes identified from matrices exposed to high levels of macrolides were similar to known genes encoding ribosomal protection proteins, macrolide phosphotransferases, and transporters. Potentially novel macrolide resistance genes included one most similar to a 23S rRNA methyltransferase from Clostridium and another, derived from upstream unpolluted sediment, to a GTPase HflX from Emergencia. In libraries deriving from sediments exposed to lower levels of veterinary antibiotics, we found 8 potentially novel ARGs, including dihydrofolate reductases and beta-lactamases from classes A, B, and D. In addition, we detected 7 potentially novel ARGs in upstream sediment, including thymidylate synthases, dihydrofolate reductases, and class D beta-lactamase. Taken together, in addition to finding known gene types, we report the discovery of novel and diverse ARGs in antibiotic-polluted industrial effluents and sediments, providing a qualitative basis for monitoring the dispersal of ARGs from environmental hotspots such as discharge sites of pharmaceutical effluents

The "Eyelet Sign" as an MRI Clue for Inflammatory Brown Syndrome

BACKGROUND Brown syndrome is characterized by a restrictive elevation deficit of the affected eye in adduction. Besides the well-known congenital form, different acquired etiologies including inflammation, trauma, and surgery may prevent the superior oblique (SO) tendon from gliding freely through the trochlea on attempted upgaze. We present MRI findings in pediatric and adult patients with inflammatory acquired Brown syndrome. METHODS Retrospective review of clinical and MRI findings of 6 patients (4 children: median age 8.4 years [range 6.1-8.7]; 2 adults: age 46.4 and 51.1 years). Median follow-up was 23 months (range 1-52). RESULTS In all 6 patients, orbital MRI demonstrated inflammatory changes of the SO tendon-trochlea complex. A striking feature was circumferential contrast enhancement of the trochlea with central sparing where the tendon passes, reminiscent of an eyelet. In all cases, the motility restriction improved either spontaneously or with systemic anti-inflammatory treatment. Although both adult patients had a history of known seronegative spondyloarthritis, there was no associated systemic condition in the children in our series. CONCLUSIONS Both in children and in adults, MRI can provide evidence of inflammatory changes located at the trochlea-tendon complex in acquired Brown syndrome here referred to as the "eyelet sign," which may be helpful in confirming the clinical diagnosis and guide appropriate treatment

KP4 to control Ustilago tritici in wheat: Enhanced greenhouse resistance to loose smut and changes in transcript abundance of pathogen related genes in infected KP4 plants

Ustilago tritici causes loose smut, which is a seed-borne fungal disease of wheat, and responsible for yield losses up to 40%. Loose smut is a threat to seed production in developing countries where small scale farmers use their own harvest as seed material. The killer protein 4 (KP4) is a virally encoded toxin from Ustilago maydis and inhibits growth of susceptible races of fungi from the Ustilaginales. Enhanced resistance in KP4 wheat to stinking smut, which is caused by Tilletia caries, had been reported earlier. We show that KP4 in genetically engineered wheat increased resistance to loose smut up to 60% compared to the non-KP4 control under greenhouse conditions. This enhanced resistance is dose and race dependent. The overexpression of the transgene kp4 and its effect on fungal growth have indirect effects on the expression of endogenous pathogen defense genes

The noncanonical type III secretion system of Xanthomonas translucens pv. graminis is essential for forage grass infection

Wichmann F, Vorhölter F-J, Hersemann L, et al. The noncanonical type III secretion system of Xanthomonas translucens pv. graminis is essential for forage grass infection. Molecular plant pathology. 2013;14(6):576-588