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The objective of this study was to characterize the microbial communities attached to clay (< 2 μm), fine silt (2-20 μm), coarse silt (20-63 μm) and sand-sized fractions [> 63 μm; including particulate organic matter (POM)] of an arable soil and analyse their response to more than 100 years of two different fertilization regimes. Mild ultrasonic dispersal, wet-sieving and centrifugation allowed the separation of soil particles with the majority of bacterial cells and DNA still attached. Fertilizations increased soil organic carbon (SOC), total DNA and the abundance of bacterial, archaeal and fungal rRNA genes more strongly in the larger-sized fractions than in fine silt, and no effect was seen with clay, the latter representing above 70% of the total microbial populations. A highly positive correlation was found between microbial rRNA genes and the surface area provided by the particles, while the correlation with SOC was lower, indicating a particle-size-specific heterogeneous effect of SOC. The prokaryotic diversity responded more strongly to fertilization in the larger particles but not with clay. Overall, these results demonstrate that microbial responsiveness to long-term fertilization declined with smaller particle sizes and that especially clay fractions exhibit a high buffering capacity protecting microbial cells against changes even after 100 years under different agricultural management.

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Cultivation-independent analyses based on genetic profiling of partial bacterial 16S rRNA genes by PCRsingle-strand conformation polymorphism (PCR-SSCP), reverse transcriptase (RT)-PCR-SSCP of the 16S rRNA itself, and stable isotope probing (SIP), followed by RT-PCR-SSCP, were applied to characterize the diversity of metabolically active bacteria in the larval gut of Manduca sexta bred on tobacco leaves under greenhouse conditions. For SIP, hatching larvae were fed with leaves from tobacco plants grown in a 13 CO 2 -enriched atmosphere. Dominant SSCP bands were sequenced and phylogenetically analyzed. Only one major gut colonizer, an Enterococcus relative, was detected; it occurred in the heavy RNA fraction, demonstrating its metabolic activity, and it originated from eggs, where its metabolic activity was also indicated by rRNA-based SSCP profiles. In contrast, a Citrobacter sedlakii relative was detected on eggs by DNA-SSCP, but rRNA-SSCP and SIP-rRNA-SSCP were negative, suggesting that these bacterial cells were inactive. A Burkholderia relative was dominant and metabolically active on the tobacco leaves but inactive inside the gut, where it was also quantitatively reduced, as suggested by lower band intensities in the DNA-based SSCP profiles. SIP-RNA-SSCP detected another metabolically active gut bacterium (Enterobacter sp.) and more bacteria in the light RNA fraction, indicating low or no metabolic activity of the latter inside the gut. We conclude that the larval gut supported only a low diversity of metabolically active bacteria.Insects represent one of the largest reservoirs of bacterial diversity on Earth, and more than 250 million years of coevolution have resulted in manifold interactions between insects and bacteria, ranging from pathogenicity to highly sophisticated symbiotic relationships (13, 56). One of the most striking interactions is that bacteria have extended the nutritional range of insects by supplying nutrients as endosymbionts (14) and by accessing otherwise indigestible substrates, such as lingo-cellulose-derived organic matter from soils, with the help of gut bacteria (7).Manduca sexta (Sphingidae) represents a lepidopteran species adapted to extreme habitat conditions because their larvae can feed exclusively on the leaves of tobacco plants (43). Tobacco plants typically contain high amounts of alkaloids which are highly toxic to other insects and vertebrates but not to M. sexta (21,49,57). Once hatched from eggs deposited on tobacco leaves, the M. sexta larvae consume large amounts of such leaves and pass through five larval stages (instars) within 2 to 3 weeks, increasing their individual body weight from approximately 1 mg to 10 g (22, 43). While this simple feeding strategy is a large problem for tobacco growers (23), it is, under controlled conditions, an excellent system to generate larvae for biological research, which explains the broad use of M. sexta in laboratories worldwide (for examples, see references 26, 29, and 52).Despite their wide laboratory dissemination and their ...