Untersuchung von Saatgutbehandlungsmitteln / Resistenzinduktoren mit Wirksamkeit gegen Gersten- und Weizenflugbrand (Ustilago nuda var. nuda u. U. nuda var. tritici)

Ziel dieses Projektes war es, alternative Saatgutbehandlungsmittel mit systemisch-fungizider oder resistenzinduzierender Wirkung gegen Ustilago nuda zu finden. Es wurden Pflanzenstärkungsmittel, Resistenzinduktoren, Pflanzenextrakte und Mikroorganismen als Samenbehandlung erprobt. Unter kontrollierten Bedingungen konnte an der Weizensorte Apogee eine Wirksamkeit von 30 bis 60 % in der Gruppe der Pflanzenextrakte gefunden werden: Bei den Pflanzenstärkungsmitteln erzielte „EnviRepel“ eine Wirksamkeit von 41,96 % und das Pseudomonas-Präparat „Pro Radix“ 69,70 %. Aus der Gruppe der mikrobiellen Antagonisten konnten neben einem Pilzisolat ausschließlich Trichoderma-Isolate erhöhte Wirksamkeiten bis zu 65 % erreichen. Bei wiederholter Testung war die Wirksamkeit allerdings nicht konsistent. In einem Feldversuch an Sommergerste ließ sich bei den Extrakten von Beinwell, Bärenklau, Stechapfel und Beifuß eine Wirksamkeit zwar tendenziell bestätigen, allerdings war der Wirkungsgrad mit 30% zu gering für die Anwendung in der Praxis. Nach den vorliegenden Erfahrungen scheint eine Beschränkung auf die Samenbehandlung nicht sinnvoll. Eine zusätzliche Behandlung zu einem späteren Entwicklungszeitpunkt (Keimlingsstadium) könnte eventuell die Wirksamkeit steigern. Eine weitere Strategie wäre es, die Infektion durch Mittelapplikation während der Blütezeit zu verhindern. Zudem wurde ein immunologischen Frühdiagnossystem über ELISA weiterentwickelt und standardisiert. Dabei konnte infiziertes Saatgut eindeutig von gesundem Saatgut unterschieden werden. Bei Untersuchungen junger Pflanzen konnte der Flugbranderreger in den Stadien EC 14 und EC 30 nicht nur im Vegetationspunkt, sondern auch in den Knoten der Haupt- und Seitentriebe nachgewiesen werden. Eine hinreichend exakte Vorhersage des späteren Ährenbefalls konnte allerdings weder durch ELISA-Messungen von bis zu 100 Einzelkörnern noch durch Untersuchungen des Sprossansatzes oder der Knoten von Jungpflanzen getroffen werden. Hier wären weitere Optimierungen der Methodik nötig

Light microscopic studies on the development of Beauveria bassiana and other putative endophytes in leaf tissues

Die vorliegende Untersuchung beinhaltete sechs Testpilze, über die in der Literatur Berichte als Endophyten vorliegen (Beauveria bassiana, Metarhizium anisopliae, Isaria fumosorosea, Trichoderma harzianum, Fusarium proliferatum, Chaetomium globosum), zwei phytopathogene Pilze (Ascochyta fabae, Plenodomus lingam) und vier Wirtspflanzen (Vicia faba, Brassica napus, Phaseolus vulgaris, Zea mays). Die Konidien oder Blastosporen bzw. Ascosporen der Testpilze wurden durch Sprühen auf die Blatt­ober­fläche oder durch Infiltration durch die Spaltöffnungen appliziert. Die lichtmikroskopische Untersuchung zeigte, dass die Sporen auf der Blattoberfläche auskeimten, aber nicht aktiv in die Blätter eindrangen. Im Blatt­inneren schienen Sporenkeimung und Hyphenwachstum auf Bereiche mit Zell- bzw. Gewebeschädigung beschränkt zu sein. Verschiedene Wirtsreaktionen wurden beobachtet, wie die Verbräunung von Epidermiszellen und die Bildung von Papillen. Eine Besiedlung des Gewebes vergleichbar der mit den Pathogenen A. fabae (bei Ackerbohne) und P. lingam (bei Raps) wurde nicht beobachtet. Erst nach Auslegen von inokuliertem Blattmaterial auf Agarmedium setzten Sporenkeimung und Hyphenwachstum im Blattinneren ein. Die Ergebnisse deuten eher auf eine saprotrophe als auf eine endophytische Lebensweise der untersuchten Pilze im Blattgewebe der untersuchten Wirtspflanzen hin.The study involved six test fungi previously recorded in the literature as being endophytes (Beauveria bassiana, Metarhizium anisopliae, Isaria fumosorosea, Trichoderma harzianum, Fusarium proliferatum, Chaetomium globosum), two plant pathogenic fungi (Ascochyta fabae, Plenodomus lingam) and four host plants (Vicia faba, Bras­sica napus, Phaseolus vulgaris, Zea mays). Aerial conidia, blastospores, or ascospores, respectively were applied to leaf surfaces by spraying or by infiltrating spore suspensions through stomata directly into the leaves. Obser­vations using light microscopy showed that the test fungi germinated on the leaf surface but did not enter actively into the leaves. Within the leaves, germination of spores and growth of hyphae appeared to depend on the presence of damaged plant tissue. Various host reactions such as browning of epidermal cells and formation of papillae were observed. Colonization of healthy leaves by the test fungi in a manner similar to the pathogens A. fabae (on Faba bean) and P. lingam (on oilseed rape) was not observed. Spore germination and hyphal growth commenced when inoculated leaves were placed on agar medium. The results indicate that the test fungi possessed a saprotrophic rather than an endo­phytic life style when associated with leaf tissue of the studied hosts

Exploring receptive and expressive language components at the age of 36 months in siblings at risk for autism spectrum disorder

Background: Language difficulties are highly prevalent in children with autism spectrum disorder (ASD) as well as in their younger siblings (high-risk (HR) sibs). Children with ASD show substantial heterogeneity in difficulties with different language components, but it remains unknown whether this variability is also present in HR-sibs. Method: Receptive (RL) and expressive language (EL) were evaluated in siblings of typically developing children (low-risk (LR) sibs, N = 33) and HR-sibs (N = 30) at 36 months, using the Mullen Scales of Early Learning (MSEL), the Dutch version of the Reynell Developmental Language Scales - 2nd edition (RDLS-2) and spontaneous language analysis (SL). Next, composite scores for receptive and expressive phonology, grammar, semantics and pragmatics were formed. Group comparisons were performed and delays in the different language components were explored. Results: HR-sibs scored significantly lower than LR-sibs on all standardized measures of RL. For EL, significantly lower scores were only found using the MSEL and not using the RDLS-2 nor using SL. HR-sibs scored significantly lower than LR-sibs for receptive and expressive semantics and receptive grammar. HR-sibs with characteristics of ASD presented with less language difficulties than HR-sibs without characteristics of ASD. The majority of HR-sibs showed a delay in one or more language components but these were not consistently detected by the different measures. Conclusions: Language delays are highly prevalent in HR-sibs and their representation resembles the language profile of children with ASD. Evaluation of language at the level of phonology, grammar, semantics and pragmatics will detect considerably more children experiencing delayed language than holistic approaches

Isolation and Characterization of fungal causal agents of black bark disease of apple using the “apple test”

In den letzten, häufig von Trockenheit geprägten Jahren hat sich der Schwarze Rindenbrand an Apfel- und Birnenbäumen in verschiedenen Teilen Deutschlands zunehmend zu einem Problem entwickelt. Ziel der vorliegenden Arbeit war es daher, methodische Grundlagen zur Isolierung der Erreger aus Apfelbäumen und ihrer Charakterisierung im Labor zu erarbeiten. Die Experimente beinhalteten die klassische Isolierung durch Auslegen von erkranktem Gewebe auf Agarmedien sowie ein in der Literatur als „Apfeltest“ beschriebenes Verfahren, bei dem befallene Rinde in Apfelfrüchte gesteckt wird. In der Folge reichern sich Pathogene im Gewebe an und können aus den entstehenden Faulstellen isoliert werden.Aus dem Stammholz eines im Wipfelbereich welkenden Baumes wurden nach Auslegen auf Agarmedium Trametes versicolor, Diaporthe eres sowie Diplodia seriata (bekannt als einer von mehreren Erregern des Schwarzen Rindenbrandes) isoliert. Diese Art wurde auch nach Auslegen befallener Rinde auf Kartoffel-Dextrose-Agar erhalten, ebenso wie Diplodia malorum. Durch Einstecken befallener Rindenstücke in Apfelfrüchte wurden weitere Isolate von D. malorum, drei Isolate von Diplodia bulgarica sowie Lambertella corni-maris, Penicillium sp. und Sclerotinia sclerotiorum gewonnen. Im Biotest mit künstlicher Inokulation mit Reinkulturen riefen die drei letztgenannten Isolate, alle Diplodia-Isolate sowie Diaporthe eres Fruchtfäulen hervor. Die Diplodia-Arten wurden darüber hinaus hinsichtlich der Geschwindigkeit des Hyphenwachstums und der Sporenmorphologie charakterisiert.During the previous years characterized by summer-drought, black canker disease of apple and pear has become a problem in different parts of Germany. The aim of the current work was therefore to develop basic techniques for isolation of the inciting pathogens from apple trees and their characterization in the laboratory. The experiments included the standard isolation of putative pathogens by placing symptomatic tissue on agar media as well as a method described in the literature as „apple test”. For the latter, symptomatic bark pieces are inserted into apple fruits and colonization of the apple tissue is visible as rotting spots. Candidate pathogens can be easily isolated from the colonized apple tissue.After placement of tissue from the trunk of the top of an apple tree expressing wilt symptoms, Trametes versicolor, Diaporthe eres and Diplodia seriata (one of several agents known to cause black canker disease) were isolated. Together with Diplodia malorum, the latter species was also obtained after placement of symptomatic bark pieces on potato dextrose agar. Insertion of diseased bark into apple fruits led to the isolation of further isolates of D. malorum, three isolates of Diplodia bulgarica, Lambertella corni-maris, Penicillium sp. and Sclerotinia sclerotiorum. After inoculation with pure cultures into apple fruits, the latter three, all Diplodia-isolates and Diaporthe eres were pathogenic and caused fruit rots. The Diplodia isolates were further characterised regarding speed of hyphal growth and spore morphology

Location of Pathogen Inoculum in the Potting Substrate Influences Damage by Globisporangium ultimum, Fusarium culmorum and Rhizoctonia solani and Effectiveness of Control Agents in Maize Seedlings

The aim of this study was to determine the impact of the location of the pathogen inoculum on damage caused by Globisporangium (syn. Pythium) ultimum, Fusarium culmorum and Rhizoctonia solani in pot tests with maize. For this purpose, pathogen inoculum was added to potting substrate, and the resulting mix was used to fill the whole pot volume, the upper half, or the lower half of pots. The remaining volume was filled with non-inoculated substrate. In a second experimental approach, maize seeds were germinated in non-inoculated potting substrate and the seedlings were transferred to inoculated substrate. The seeds were untreated, treated with the chemical thiram, or treated with a bacterial or a fungal biocontrol agent. With each of the pathogens, the damage to the developing maize seedlings was the strongest when the seeds germinated in the inoculated potting substrate. When only the roots were in contact with the inoculum, there was limited damage by R. solani and F. culmorum, and no damage by G. ultimum. This implies that in experiments with artificial inoculation, the seeds should always be in immediate contact with the inoculum if a strong pathogenic effect is desired. Conversely, seed treatments must, in the first place, be able to protect the spermosphere, while the requirement to protect the roots at a distance from the seed seems to depend on the pathogen

Modification of Bupivacaine-Induced Myotoxicity with Dantrolene and Caffeine In Vitro

BACKGROUND: Local anesthetics, especially bupivacaine, have myotoxic effects in clinically used concentrations and context. Detailed mechanisms of these effects are unknown, but an increase in intracellular calcium levels is suspected to be the most important trigger. Dantrolene and caffeine modify cellular calcium release from the sarcoplasmic reticulum. The aim of our study was to investigate the effect of dantrolene and caffeine on bupivacaine-induced myotoxicity in vitro. METHODS: A cell culture model of primary muscle cells of BALB/c AnNCrl mice was established. Cells were incubated simultaneously with increasing concentrations of bupivacaine, dantrolene, and caffeine. The fraction of dead cells was calculated after staining with propidium iodide and analysis by flow cytometry. The half-maximal inhibitory concentration of bupivacaine was calculated for each concentration. Group differences were determined by using 1-way analysis of variances with subsequent post hoc 1-way Dunnett t test. RESULTS: Both dantrolene and caffeine alone had no effect on muscle cell survival. Increasing concentrations of bupivacaine caused increasing cell death. Dantrolene dose-dependently reduced the fraction of necrotic cells, whereas caffeine dose-dependently increased the fraction of dead cells. CONCLUSIONS: Dantrolene attenuated, and caffeine enhanced, bupivacaine-induced myotoxicity, presumably by modifying sarcoplasmic calcium release. This indicates that intracellular calcium release is an important factor for local anesthetic-induced cell death

miRNAs in ancient tissue specimens of the Tyrolean Iceman

The analysis of nucleic acids in ancient samples is largely limited to DNA. Small noncoding RNAs (microRNAs) are known to be evolutionary conserved and stable. To gain knowledge on miRNAs measured from ancient samples, we profiled microRNAs in cryoconserved mummies. First, we established the approach on a World War One warrior, the “Kaiserj€ager”, which has been preserved for almost one century. Then, we profiled seven ancient tissue specimens including skeletal muscle, stomach mucosa, stomach content and two corpus organ tissues of the 5,300-year-old copper age mummy Iceman and compared these profiles to the presence of organ-specific miRNAs in modern tissues. Our analyses suggest the presence of specific miRNAs in the different Iceman’s tissues. Of 1,066 analyzed human miRNAs, 31 were discovered across all biopsies and 87 miRNAs were detected only in a single sample. To check for potential microbiological contaminations, all miRNAs detected in Iceman samples and not present in ancient samples were mapped to 14,582 bacterial and viral genomes. We detected few hits (3.9% of miRNAs compared with 3.6% of miRNAs). Interestingly, the miRNAs with higher abundance across all ancient tissues were significantly enriched for Guanine (P value of 10–13) and Cytosine (P value of 10–7). The same pattern was observed for modern tissues. Comparing miRNAs measured from ancient organs to modern tissue patterns highlighted significant similarities, e.g., formiRNAs present in themuscle. Our first comprehensive analysis of microRNAs in ancient human tissues indicates that these stable molecules can be detected in tissue specimens after 5,300 years