Browsing by Subject "Polymerase-Kettenreaktion"

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Community Structure and Activity of Nitrate-Reducing Microorganisms in Soils under Global Climate Change
(2006) Deiglmayr, Kathrin; Kandeler, Ellen
Since the beginning of the Industrial Revolution, atmospheric carbon dioxide concentrations have been steadily increasing and, thus, contributed to a warming of the climate and altered biogeochemical cycles. To study the response of soil microorganisms to altered environmental conditions under global climate change, the nitrate-reducing community was regarded as a model community in the present thesis. This functional group, which performs the first step in the denitrification pathway, was selected because it is phylogenetically very diverse. In particular rising levels of atmospheric carbon dioxide as the most important catalyst of temperature rise and the retreat of glaciers in the Alps as one of the most evident consequences of climate change were investigated. The behaviour of nitrate reducers was investigated in a biphasic approach: (i) at the level of its enzyme activity of the nitrate reductase and (ii) at the level of community structure, which was characterised by RFLP (Restriction Fragment Length Polymorphism)-fingerprints using the functional gene narG. The effect of elevated atmospheric carbon dioxide concentrations on nitrate-reducing micro-organisms was studied in the Swiss FACE (Free Air Carbon dioxide Enrichment) experiment including the rhizosphere of two functional plant types (Lolium perenne and Trifolium repens), two N fertilisation levels and two sampling dates (June and October 2002). Whereas in June no significant treatment effect was observed, the nitrate reductase activity proved to be significantly reduced under elevated atmospheric carbon dioxide at the autumn sampling date. Simultaneously, elevated enzyme activities were recorded under Trifolium repens and high N fertilisation pointing to a control of nitrate reductase activity by nitrate availability at the time of sampling. The community structure of nitrate reducers, however, showed a different response pattern with sampling date and the strongly varying pH of the different experimental plots constituting the main driving factors. With respect to the three experimental factors atmospheric carbon dioxide, plant type and N fertilisation the composition of the nitrate reducers revealed a high stability. The microbial succession of nitrate-reducing microorganisms was studied in the rhizosphere of Poa alpina across the glacier foreland of the Rotmoosferner/Oetz valley. Sampling was performed in August and at the end of the short period of vegetation in September. The nitrate reductase activity increased significantly with progressing successional age, whereas organic carbon together with nitrate concentrations in the soils explained the major part of this effect. The microbial community of nitrate reducers revealed a significant shift across the glacier foreland, with pH and organic carbon representing the most important environmental factors inducing this shift. A detailed analysis of the clone libraries that were constructed for the youngest and the oldest site in the glacier foreland pointed to the tendency of lower diversity in the late succession compared to the young succession. Possibly an increasing selective pressure due to higher densities of microorganisms and, hence, a higher competition for limited resources contributed to the decline in diversity. In conclusion, the functional group of nitrate reducers responded to changing environmental conditions under global climate change particularly through altered enzyme activities. The amount and the direction of this response depended strongly on the nitrate availability and the organic carbon content in soils. The community structure of nitrate-reducing microorganisms, however, proved to be resilient towards short-term substrate fluctuations. This indicates that the genetic pool of this group of soil microorganisms possesses a high functional stability characterized by a relatively persistent composition and an independent modulation of enzyme activity.
Diagnostics and genome analysis of phloem-limited phytopathogenic bacteria
(2022) Zübert, Christina; Hölzle, Ludwig
This thesis contributes to improving the epidemiological understanding of ‘Candidatus Arsenophonus phytopathogenicus’ and bacteria of the provisional taxon ‘Candidatus Phytoplasma’ by applying genetic markers for identification, differentiation, and phylogenetic reconstruction of this economically relevant plant pathogens.
Entwicklung von spezifischen PCR-ELISA Nachweissystemen für Coxiella burnetii, Francisella tularensis und Orthopockenviren
(2001) Kohlhaußen, Simone; Böhm, Reinhard
In the present work specific systems on the basis of the PCR were developed for the detection of Coxiella burnetii, Francisella tularensis and Orthopoxviruses. The detection of the pathogens is possible without additional cultivation. In order to exclude false-negative PCR results the amplification reaction was established as a competitive PCR in which an internal control DNA is included as an amplification check. With regard to a possible automation the detection-systems were constituted in form of a 'PCR ELISA'. The colorimetric detection of the amplicons takes place thereby in streptavidin coated microtiter plates. For each system biotinylated capture probes for both the internal control DNA (ST) and the pathogen specific DNA (WT) have been developed. For the use of these proof systems in the clinical laboratory, the routine application of the tested decontamination procedure by means of UNG during the execution of the PCR ELISA is recommended. For all three systems a detection of the pathogens is possible within one working day. For the species specific detection of F. tularensis and the genus specific detection of Orthopoxviruses the PCR ELISA systems established here represent the first developments of this type with integrated amplification control and potential for quantification.
Molecular systematics of selected Diadegma species (Hymenoptera: Ichneumonidae: Campoplegine) important in biological control
(2006) Wagener, Barbara; Zebitz, Claus P. W.
The genus Diadegma (Hymenoptera: Ichneumonidae: Campopleginae) represents a large group of parasitoids with 201 species worldwide. Adult Diadegma females parasitise larvae of various lepidopteran species and some species, in particular Diadegma insulare (Cresson) and D. semiclausum (Hellén), have gained economic importance as biological control agents of Plutella xylostella (Linnaeus). A low parasitism rate of <15 % of the parasitoid complex (Diadegma sp., Oomyces sokolowskii (Kurdjumov) and Diaplazon laetatorius (Fabricius)) in unsprayed cabbage and kale fields infested with P. xylostella in eastern and southern Africa was the starting point for the development of a biological control project for P. xylostella which was implemented by the International Centre of Insect Physiology and Ecology (ICIPE), Kenya. One of the objectives of the biocontrol project was to examine the taxonomic status of Diadegma species associated with P. xylostella in eastern and southern Africa and the exotic parasitoid D. semiclausum imported to Kenya from Taiwan (Asian Vegetable Research and Development Centre, AVRDC) by cross breeding experiments and molecular methods. Thus, two different molecular regions, a fragment of the mitochondrial cytochrome c oxidase subunit (COI) and the second internal transcribed spacer (ITS2) of ribosomal DNA were amplified utilising polymerase chain reaction (PCR) and digested afterwards with several restriction enzymes (PCR-Restriction Fragment Length Polymorphism-RFLP). In the due course of the study examinations of several Diadegma species attacking P. xylostella were undertaken with the PCR-RFLP method developed previously for the African Diadegma. This molecular method could solve some taxonomic difficulties of the genus Diadegma. Sequence analyses were used to investigate the phylogenetic relationship of nine Diadegma species (D. blackburni (Cameron), D. insulare, D. leontiniae (Brèthes), D. chrysostictos (Gmelin), D. armillata (Gravenhorst), D. fenestrale (Holmgren), D. mollipla (Holmgren), D. semiclausum, D. rapi (Cameron)) and the phylogenetic relationship of the genus Diadegma within the superfamily Ichneumonoidea. Cross breeding experiments were carried out between two populations of D. mollipla from eastern and southern Africa. No significant differences in the total number of progeny per female and the number of male offspring were obtained, whereas the female progeny showed significant differences. Hybrid females resulting from both reciprocal crosses were reproductively compatible with males of both parental lines, which indicated that no genetic incompatibility was apparent between the two D. mollipla populations. In contrast, crosses between D. mollipla and D. semiclausum resulted only in the occurrence of male offspring, which is typical for unfertilised progeny in Diadegma. The laboratory cultures of D. mollipla and D. semiclausum were highly male biased. Inbreeding, where homozygosity is much higher, is leading to a higher diploid male production. Diploid males can easily be detected by isoenzyme variations as a genetic marker. Heterozygote females/males of D. semiclausum and D. mollipla were identified by phosphoglucomutase (PGM) electrophoretic banding patterns. Crosses between a mother (heterozygote, diploid) and her son (homozygote, haploid) resulted in one diploid male in D. mollipla and none in D. semiclausum. Information about diploid males in D. semiclausum detected with PGM has already been published and different methodologies might be the reason why in D. semiclausum no diploid male was detected. Therefore the present analyses with PGM as molecular marker should be seen as a preliminary study.
Nachweis von luftgetragenen Viren an Standorten der Abfall- und Abwasserentsorgung
(2002) Mayr, Constanze F.; Boehm, Reinhard
In this study, aerosols workers at waste and wastewater treatment plants were exposed to, were examined with regard to the presence of airborne human pathogenic viruses. Besides the detection of hepatitis B virus and hantaviruses the main issue was to detect hepatitis A virus, rotavirus, human enteroviruses and poliovirus. Airborne viruses were sampled in parallel with different bioaerosol samplers, using various physical collection methods. At selected locations, measurements with two special-impingers, the MD 8-sampler and two high-volume-samplers were performed. Personal exposition of waste treatment workers to aerosols was measured using the PGP-GSP-system. Before viruses could be detected, the air samples had to be prepared. Because of the different sampling methods, several protocols had to be established. For the purification and concentration of the collected virus particles the following methods were used: prefiltration, ultrafiltration (100 and 500 KD), density gradient ultrafiltration using a sucrose gradient and the digestion of gelatine with trypsin. After these sampling processing procedures the virus detection and quantification of the samples of the special-impingers, the MD 8-sampler and the high-volume-samplers were assayed for human enteroviruses and rotavirus using four different tissue cultures (VERO, BGM, A549 and MA104) and molecular biological methods (RT-nested-PCR). For all other viruses only molecular biological methods (PCR) were applied. Regarding to the low flow-rate of the PGP-GSP-System only PCR was taken for virus detection. For the detection of viruses using the PCR assay, four different commercially available extraction-kits for DNA and RNA were compared. For all viruses besides hantavirus, several nested-PCR-systems for the qualitative and quantitative (using externe standards) determination with the LightCycler technique were established. To get informations about the attitude of a possible inhibition of the PCR reaction, the prepared samples were seeded with defined amounts of equine rhinovirus and amplified with an external standard on the LightCycler. The established PCR assays for hantavirus, hepatitis A virus, human enteroviruses and poliovirus showed a higher sensitivity (to be more sensitive) than the used cell culture. For detecting rotavirus, the sensitivity of both assays were equal, the detection limit for the nested-PCR of hepatitis B virus was one gencopy per reaction. In none of the 144 aerosol samples (45 PGP-GSP-samples and 99 from the other samplers) rotavirus, hepatitis A virus, hepatitis B virus and hantaviruses could be detected with the PCR technology. Also no rotavirus was isolated with the tissue culture method. In 21 cases the PCR showed positive results for human enterovirus RNA, the calculated concentration ranged from Äq. 100,47 KID50/ml to Äq. 103,74 KID50/ml. Viral infectivity could be demonstrated in 33 out of 99 examined specimens after inoculation on several cell cultures. The virus concentration ranged from 100,7 KID50/ml to more than 103,7 KID50/ml. After plaque purification some of the isolates were sequenced and identified as poliovirus type 1. In conclusion, the results of these studies indicate that at the investigated locations high measurable levels of hepatitis A virus, hantavirus, rotavirus and hepatitis B virus can not be expected. Nevertheless, in some special cases contamination might be possible. The detection of human enterovirus in 21 % of the location-based samples shows that especially bioaerosols at the waste water treatment plants might possibly be contaminated with viruses. Therefore the risk of infections can not be excluded.