Approaches to Assess the Effects and Risks of Veterinary Antibiotics Applied with Manure to Soil

In veterinary medicine, large quantities of antibiotic substances are administered each year for therapeutic and prophylactic purposes or to promote growth. As a consequence, the antibiotics and bacteria carrying transferable antibiotic resistance genes are excreted by the animals and reach the environment through run-off, leaching, and/or following manure application to agricultural fields, where they have been found to affect the structure and function of soil bacterial communities. However, we are only beginning to understand the global effects of environmental pollution with antibiotics and resistance determinants and the resulting risks for human health. For regulatory purposes, there is urgent need for criteria and methods that allow reliable and reproducible assessment of risks associated with release of realistic concentrations of antibiotics and resistance determinants into the environment following manure application. In this chapter, we will summarize recent advances, limitations, and research needed to optimize the methods to quantify and evaluate the effects and risks associated with these compounds. Approaches that are discussed focus on antibiotic resistance genes and include classical tools such as cultivation and PCR detection as well as quantitative real-time PCR and next-generation sequencing technologies used in combination with functional screening

Multitrophic interactions among Western Corn Rootworm, Glomus intraradices and microbial communities in the rhizosphere and endorhiza of maize

The complex interactions among the maize pest Western Corn Rootworm (WCR), Glomus intraradices (GI-recently renamed Rhizophagus intraradices) and the microbial communities in both rhizosphere and endorhiza of maize have been investigated in view of new pest control strategies. In a greenhouse experiment, different maize treatments were established: C (control plants), W (plants inoculated with WCR), G (plants inoculated with GI), GW (plants inoculated with GI and WCR). After 20 days of WCR root feeding, larval fitness was measured. Dominant arbuscular mycorrhizal fungi (AMF) in soil and maize endorhiza were analyzed by cloning of 18S rRNA gene fragments of AMF, restriction fragment length polymorphism and sequencing. Bacterial and fungal communities in the rhizosphere and endorhiza were investigated by denaturing gradient gel electrophoresis of 16S rRNA gene and ITS fragments, PCR amplified from total community DNA, respectively. GI reduced significantly WCR larval development and affected the naturally occurring endorhiza AMF and bacteria. WCR root feeding influenced the endorhiza bacteria as well. GI can be used in integrated pest management programs, rendering WCR larvae more susceptible to predation by natural enemies. The mechanisms behind the interaction between GI and WCR remain unknown. However, our data suggested that GI might act indirectly via plant-mediated mechanisms influencing the endorhiza microbial communities

Mikrobielle Vielfalt in der Rhizosphäre und im Boden

Bodenmikroorganismen sind von enormer Bedeutung für funktionierende Stoffkreisläufe und die Pflanzengesundheit. Die Entwicklung und Anwendung von Nukleinsäure-basierten Untersuchungstechniken hat ein neues Verständnis der Vielfalt von Mikroorganismen in der Rhizosphäre und im Boden ermöglicht. Mit Hilfe dieser kultivierungsunabhängigen Methoden konnte gezeigt werden, dass die mikrobielle Diversität durch eine Vielzahl biotischer und abiotischer Faktoren beeinflusst wird. Basierend auf der Sequenzanalyse von 16S rRNA-Genen und ITS-Fragmenten, die aus Boden-DNA amplifiziert wurden, wurde die taxonomische Zuordnung (Gattungen, Arten) der Mikroorganismen ermöglicht, deren Abundanz durch die Pflanzenart oder - sorte, den Standort oder Änderungen in der landwirtschaftlichen Praxis beeinflusst wird. Die Diversifizierung innerhalb einer Art und die Anpassung von Bakterienpopulationen an sich ändernde Umweltbedingungen erfolgen häufig durch mobile genetische Elemente. Pathogenitätsdeterminanten, Gene für die Interaktion mit der Wirtspflanze oder Antibiotika- und Schwermetallresistenzgene sind häufig auf Plasmiden lokalisiert. Dieser Artikel fasst die Forschungsarbeiten zur mikrobiellen Diversität im Boden an der BBA und im JKI in Braunschweig zusammen.Stichwörter: Mikrobielle Diversität, Bakterien, Pilze, DNA-Extraktion, Kultivierbarkeit, genetische Flexibilität, Gentransfer, mobile genetische ElementeMicrobial diversity in rhizosphere and bulk soilSummarySoil microbes are of enormous importance for functioning nutrient cycles as well as for plant health. The development and application of nucleic acid based techniques provided new insights into the diversity of bulk and rhizosphere soil microbes. The microbial diversity in soils was shown by means of cultivation-independent methods to be influenced by various biotic and abiotic factors. Sequence based analysis of 16S rRNA genes and ITS fragments amplified from total DNA extracted directly from soil allows to determine the taxonomic affiliation (phylum, class, order, genera and species) of those taxa influenced in their relative abundance by the plant species or genotype, the site, or changes in agricultural management. The diversification within a species and the adaptation of bacterial populations to changing environmental conditions is often fostered by mobile genetic elements. Pathogenicity determinants, genes responsible for the bacterial interaction with its host plant or antibiotic and heavy metal resistance genes are often localized on plasmids. This review summarizes research on microbial diversity performed over more than twenty years at the BBA and now in the JKI in Braunschweig. Keywords: Microbial diversity, bacteria, fungi, DNA extraction, culturability, genetic flexibility, gene transfer, mobile genetic element