Untersuchungen zur Übertragung von Clavibacter michiganensis ssp. sepedonicus auf gesunde Kartoffelknollen

Zusammenfassung In den Jahren 1999 bis 2003 wurde in Freiland-, Klimakammer- und Lagerungsversuchen überprüft, ob ein Risiko für die Übertragung des Erregers der Bakteriellen Ringfäule der Kartoffel (Clavibacter michiganensis ssp. sepedonicus) besteht, wenn (a) gesunde Kartoffelknollen in Kontakt mit Maschinen und Geräten kommen, die mit dem Erreger kontaminiert sind (indirekter Kontakt) und (b) gesunde Kartoffelknollen direkt in Kontakt mit infizierten Knollen kommen (direkter Kontakt). Nach indirektem Kontakt konnte nur beim nachfolgenden Anbau der kontaminierten Knollen in der Klimakammer Befall in Kraut und Knollen festgestellt werden. Im Freiland konnte der Erreger, auch bei wiederholtem Nachbau der geernteten Knollen, nicht nachgewiesen werden. Nach direktem Kontakt und nachfolgendem Anbau der kontaminierten Knollen in der Klimakammer und im Freiland, wurde der Erreger in allen Fällen in den geerntete Knollen nachgewiesen. Befall im Kraut wurde nur in dem Klimakammerversuch und in einem Freilandversuch ermittelt. Wurden durch direkten Kontakt kontaminierte Knollen eingelagert, konnte der Erreger in allen untersuchten Knollen festgestellt werden. Insgesamt besteht ein hohes Risiko, dass gesunde Knollen infiziert werden, wenn oberflächliche Kontaminationen mit dem Erreger erfolgen. Die Wahrscheinlichkeit von Infektionen steigt mit zunehmender Kontaminationsstärke.     

Überleben von Ralstonia solanacearum Biovar 2 (Rasse 3), dem Erreger der Schleimkrankheit der Kartoffel, in Boden und auf verschiedenen Materialien (Mikrokosmos)

Microcosm studies were carried out to test the survival of Ralstonia solanacearum biovar 2 (race 3) in soil at the permanent wilting point (wp) water content and at field capacity (fc) water content and on various material. Soils were placed at permanent ?5°C, 4°C, 15°C and 20°C and weekly fluctuating ?10/0/+10°C and the material at 5, 15 °C, 20°C with relative humidity (rh) uncontrolled or at constant 10% or 90%. In soil, survival was clearly dependent on temperature independent of water content. At 20°C Ralstonia solanacearum could be reisolated up to 364 days, at 15°C up to 290 days, at 4°C up to 209 days and at fluctuating temperatures (?10/0/+10°C) only up to 18 days. The lower the temperature, the more the population declined. At 15°C and 20°C appr. 10⁷ cfu/g soil were detected after 100 days, whereas at ?5°C only 10² cfu/g soil were detected after only 18 days. The pathogen was longer detectable in sandy-clay loam than in lighter sandy soil. It could be longer reisolated at wilting point and the populations did not decline as rapidly as at field capacity. Ralstonia solanacearum could best survive on material surfaces like rubber, plastic and varnished metal with maximum survival of 40 days at 5°C and 10% rh. In general there is a low risk of Ralstonia solanacearum overwintering under European climatic conditions when the fields are cleared of plant debris and the soil is frozen. Contamined material surfaces pose the risk of pathogen transmission to healthy tubers.     

Is N‐feedback involved in the regulation of nitrogenase activity in Medicago truncatula?

To determine whether senescing leaves provoke an active nitrogen (N) remobilization that results in the reduction of nitrogenase activity, 60% of Medicago truncatula lower leaves were either darkened or individually excised for two weeks. Although a considerable amount of N was remobilized, N₂ fixation activity was found to be increased to maintain the N source/sink balance, indicating an absence of the negative N‐feedback regulation of nitrogenase activity in the senescing M. truncatula.     

Expression of porcine endogenous retroviruses (PERVs) in melanomas of Munich miniature swine (MMS) Troll

Porcine endogenous retroviruses (PERVs) are integrated in the genome of all pig breeds. Since some of them are able to infect human cells, they might represent a risk for xenotransplantation using pig cells or organs. However, the expression and biological role of PERVs in healthy pigs as well as in porcine tumours is largely unknown. Since we and others have recently shown overexpression of a human endogenous retrovirus, HERV-K, in human melanomas, we studied the expression of PERVs in melanomas of selectively bred Munich miniature swine (MMS) Troll. This breeding herd of MMS Troll is characterised by a high prevalence of melanomas, which histologically resemble various types of cutaneous melanomas in humans. Several genetic factors have been defined when studying inheritance of melanomas and melanocytic nevi in MMS Troll. Here we show that the polytropic PERV-A and PERV-B as well as the ecotropic PERV-C are present in the genome of all melanoma bearing MMS Troll investigated. Most interestingly, in the spleen, but not in other organs, recombinant PERV-A/C proviruses were found. PERV expression was found elevated in melanomas when compared to normal skin and viral proteins were expressed in melanomas and pulmonary metastasis-derived melanoma cell cultures. During passaging of these cells in vitro the expression of PERV mRNA and protein increased and virus particles were released as shown by RT activity in the supernatant and by electron microscopy. Genomic RNA of PERV-A, -B and -C were found in pelleted virus particles. Although PERV expression was elevated in melanomas and pulmonary metastasis-derived cell cultures, the function of the virus in tumour development is still unclear.     

Occurrence of helminths in pig fattening units with different management systems in Northern Germany

The helminth infections on 13 pig fattening farms with different management systems (complete or partial all-in-all-out system or continuous fattening) in North-Western Germany were investigated over at least three fattening periods. Pooled faecal samples were taken from pens once before and three times after anthelmintic treatment. At the beginning of fattening 34.9% of the samples contained helminth eggs, mainly from Oesophagostomum spp. (27.5%). Ascaris suum eggs were found in 10.5% of the samples, while other parasites were only rarely found. The number of pig-supplying farms was positively correlated with the helminth infection prevalence. Immediately after deworming, all pen samples were free of helminth eggs. However, the prevalences increased again, and by the end of fattening A. suum was found in 33.0% and strongylids in 6.0% of the samples. Pens harbouring A. suum-excreting pigs at the beginning of fattening had higher infection levels at the end, and this was also the case for nodular worms. The final prevalence of Ascaris was higher in partial exchange systems than in complete all-in-all-out systems and in old pig houses compared to new ones. Transmission of both Ascaris and Oesophagostomum was highest in autumn and winter. Thus, a single anthelmintic treatment at the beginning of fattening could not prevent infection during fattening, and the state of infection at the beginning was associated with the helminth burden at slaughter. Therefore, the purchase of parasite-free pigs in combination with appropriate hygiene management may minimise the initial infection pressure and keep subsequent infection of the herd at a minimum.     

Variation in clinical and parasitological traits in Pietrain and Meishan pigs infected with Sarcocystis miescheriana

Future prophylaxis needs new concepts, including natural disease resistance of hosts against infectious agents. Genomic approaches to detect and improve disease resistance in farm animals and the molecular mechanisms involved in host-parasite interactions depend to a high degree on the trait differences between founder breeds, i.e. on the animal model. The present study evaluates differences in susceptibility/resistance against Sarcocystis miescheriana in the European Pietrain (PI) and the Chinese Meishan (ME) pig breeds, based on 25 individuals, infected orally with 5x10(4) sporocysts of S. miescheriana. Significant differences appeared in clinical, serological, haematological and parasitological findings. The major discriminating period post infection (p.i.) was between days 42 and 45. Severity of signs was negatively correlated with specific immunoglobulin titres during the first 3 weeks p.i. and positively with the load of bradyzoites in muscle tissues of the pigs. Loads of bradyzoites in muscle tissues were 20 times higher in PI than in ME. Sarcocystis-specific differences between the two breeds were in the range of 1-2 standard deviations. The study lays the foundation for further experiments to analyse chromosomal regions, candidate genes, and thus the molecular basis of Sarcocystis susceptibility/resistance as a model for host-parasite interaction in protozoan infectious disease.     

Metabolic labeling of plant cell cultures with K15NO3 as a tool for quantitative analysis of proteins and metabolites

Strategies for robust quantitative comparison between different biological samples are of high importance in experiments that address biological questions beyond the establishment of protein lists. Here, we propose the use of ¹⁵N-KNO₃ as the only nitrogen source in Arabidopsis cell cultures in order to achieve a metabolically fully labeled cell population. Proteins from such metabolically labeled culture are distinguishable from unlabeled protein populations by a characteristic mass shift that depends on the amino acid composition of the tryptic peptide analyzed. In addition, the metabolically labeled cell extracts are also suitable for comparative quantitative analysis of nitrogen-containing cellular metabolic complement. Protein extracts from unlabeled and from standardized ¹⁵N-labeled cells were combined into one sample for joined analytical processing. This has the advantage of (i) reduced experimental variability and (ii) immediate relative quantitation at the level of single extracted peptide and metabolite spectra. Together ease and accuracy of relative quantitation for profiling experiments is substantially improved. The metabolic labeling strategy has been validated by mixtures of protein extracts and metabolite extracts from the same cell cultures in known ratios of labeled to unlabeled extracts (1:1, 1:4, and 4:1). We conclude that saturating metabolic ¹⁵N-labeling provides a robust and affordable integrative strategy to answer questions in quantitative proteomics and nitrogen focused metabolomics.