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.     

Short-term storage and cryopreservation of milt from Atlantic salmon and sea trout

Experiments on short-term preservation of sperm were performed with Atlantic salmon (Salmo salar). Fertility was maintained for up to 10 days when 2 mm thick samples were stored at 0° C under an oxygen atmosphere in the presence of antibiotics (125 IU penicillin and 125 μg streptomycin per ml sperm). Fertility was completely lost after 24 days. Sperm stored without antibiotics fertilized 100% of eggs after 6 days.

Cryopreservation was carried out with milt from Atlantic salmon and sea trout (Salmo trutta). Semen mixed with extender was frozen on dry ice (pellets) with subsequent storage in liquid nitrogen. Sperm pellets were thawed in a 0.12-M NaHCO₃-solution (10° C) before insemination. The suitability of an extender as previously described by Stoss and Holtz and of a 0.3-M glucose solution with the addition of 10% DMSO, was tested on two different batches of sperm and eggs in Atlantic salmon and sea trout. In addition, the extender earlier reported by Mounib and an aqueous solution of 10% DMSO were only used in Atlantic salmon with one batch of gametes.

Insemination with cryopreserved Atlantic salmon sperm resulted in 36 to 91.3% eyed eggs (control = 100). The differences were caused by the type of extender and the batch of gametes employed. The very simple extender consisting of 0.3 M glucose and 10% DMSO only was the most successful. Results with cryopreserved sea trout sperm ranged between 38.6–54.8% eyed eggs, showing no difference between treatments.     

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.