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1.  Effects of sample handling and cultivation bias on the specificity of bacterial communities in keratose marine sponges 
Complex and distinct bacterial communities inhabit marine sponges and are believed to be essential to host survival, but our present-day inability to domesticate sponge symbionts in the laboratory hinders our access to the full metabolic breadth of these microbial consortia. We address bacterial cultivation bias in marine sponges using a procedure that enables direct comparison between cultivated and uncultivated symbiont community structures. Bacterial community profiling of the sympatric keratose species Sarcotragus spinosulus and Ircinia variabilis (Dictyoceratida, Irciniidae) was performed by polymerase chain reaction-denaturing gradient gel electrophoresis and 454-pyrosequecing of 16S rRNA gene fragments. Whereas cultivation-independent methods revealed species-specific bacterial community structures in these hosts, cultivation-dependent methods resulted in equivalent community assemblages from both species. Between 15 and 18 bacterial phyla were found in S. spinosulus and I. variabilis using cultivation-independent methods. However, Alphaproteobacteria and Gammaproteobacteria dominated the cultivation-dependent bacterial community. While cultivation-independent methods revealed about 200 and 220 operational taxonomic units (OTUs, 97% gene similarity) in S. spinosulus and I. variabilis, respectively, only 33 and 39 OTUs were found in these species via culturing. Nevertheless, around 50% of all cultured OTUs escaped detection by cultivation-independent methods, indicating that standard cultivation makes otherwise host-specific bacterial communities similar by selectively enriching for rarer and generalist symbionts. This study sheds new light on the diversity spectrum encompassed by cultivated and uncultivated sponge-associated bacteria. Moreover, it highlights the need to develop alternative culturing technologies to capture the dominant sponge symbiont fraction that currently remains recalcitrant to laboratory manipulation.
doi:10.3389/fmicb.2014.00611
PMCID: PMC4235377  PMID: 25477868
holobiont; microbial cultivation; microbial diversity; pyrosequencing; symbiosis
2.  The impact of the pathogen Rhizoctonia solani and its beneficial counterpart Bacillus amyloliquefaciens on the indigenous lettuce microbiome 
Lettuce belongs to the most commonly raw eaten food worldwide and its microbiome plays an important role for both human and plant health. Yet, little is known about the impact of potentially occurring pathogens and beneficial inoculants of the indigenous microorganisms associated with lettuce. To address this question we studied the impact of the phytopathogenic fungus Rhizoctonia solani and the biological control agent Bacillus amyloliquefaciens FZB42 on the indigenous rhizosphere and phyllosphere community of greenhouse-grown lettuce at two plant stages. The rhizosphere and phyllosphere gammaproteobacterial microbiomes of lettuce plants showed clear differences in their overall and core microbiome composition as well as in corresponding diversity indices. The rhizosphere was dominated by Xanthomonadaceae (48%) and Pseudomonadaceae (37%) with Rhodanobacter, Pseudoxanthomonas, Dokdonella, Luteimonas, Steroidobacter, Thermomonas as core inhabitants, while the dominating taxa associated to phyllosphere were Pseudomonadaceae (54%), Moraxellaceae (16%) and Enterobacteriaceae (25%) with Alkanindiges, Pantoea and a group of Enterobacteriaceae unclassified at genus level. The preferential occurrence of enterics in the phyllosphere was the most significant difference between both habitats. Additional enhancement of enterics on the phyllosphere was observed in bottom rot diseased lettuce plants, while Acinetobacter and Alkanindiges were identified as indicators of healthy plants. Interestingly, the microbial diversity was enhanced by treatment with both the pathogen, and the co-inoculated biological control agent. The highest impact and bacterial diversity was found by Rhizoctonia inoculation, but FZB42 lowered the impact of Rhizoctonia on the microbiome. This study shows that the indigenous microbiome shifts as a consequence to pathogen attack but FZB42 can compensate these effects, which supports their role as biocontrol agent and suggests a novel mode of action.
doi:10.3389/fmicb.2014.00175
PMCID: PMC4001036  PMID: 24795707
lettuce microbiome; Gammaproteobacteria; soil-borne pathogens; 16S rRNA gene pyrosequencing; phyllosphere; rhizosphere; Lactuca sativa
3.  Scanning a microhabitat: plant-microbe interactions revealed by confocal laser microscopy 
No plant or cryptogam exists in nature without microorganisms associated with its tissues. Plants as microbial hosts are puzzles of different microhabitats, each of them colonized by specifically adapted microbiomes. The interactions with such microorganisms have drastic effects on the host fitness. Since the last 20 years, the combination of microscopic tools and molecular approaches contributed to new insights into microbe-host interactions. Particularly, confocal laser scanning microscopy (CLSM) facilitated the exploration of microbial habitats and allowed the observation of host-associated microorganisms in situ with an unprecedented accuracy. Here I present an overview of the progresses made in the study of the interactions between microorganisms and plants or plant-like organisms, focusing on the role of CLSM for the understanding of their significance. I critically discuss risks of misinterpretation when procedures of CLSM are not properly optimized. I also review approaches for quantitative and statistical analyses of CLSM images, the combination with other molecular and microscopic methods, and suggest the re-evaluation of natural autofluorescence. In this review, technical aspects were coupled with scientific outcomes, to facilitate the readers in identifying possible CLSM applications in their research or to expand their existing potential. The scope of this review is to highlight the importance of confocal microscopy in the study of plant-microbe interactions and also to be an inspiration for integrating microscopy with molecular techniques in future researches of microbial ecology.
doi:10.3389/fmicb.2014.00094
PMCID: PMC3945399  PMID: 24639675
plant-microbe interactions; cryptogams; confocal laser scanning microscopy (CLSM); endophyte; pathogen; GFP; DsRed; fluorescence in situ hybridization (FISH)
4.  Vertical transmission explains the specific Burkholderia pattern in Sphagnum mosses at multi-geographic scale 
The betaproteobacterial genus Burkholderia is known for its versatile interactions with its hosts that can range from beneficial to pathogenic. A plant-beneficial-environmental (PBE) Burkholderia cluster was recently separated from the pathogen cluster, yet still little is known about burkholderial diversity, distribution, colonization, and transmission patterns on plants. In our study, we applied a combination of high-throughput molecular and microscopic methods to examine the aforementioned factors for Burkholderia communities associated with Sphagnum mosses – model plants for long-term associations – in Austrian and Russian bogs. Analysis of 16S rRNA gene amplicons libraries revealed that most of the Burkholderia are part of the PBE group, but a minor fraction was closely related to B. glathei and B. andropogonis from the pathogen cluster. Notably, Burkholderia showed highly similar composition patterns for each moss species independent of the geographic region, and Burkholderia-specific fluorescent in situ hybridization of Sphagnum gametophytes exhibited similar colonization patterns in different Sphagnum species at multi-geographic scales. To explain these patterns, we compared the compositions of the surrounding water, gametophyte-, and sporophyte-associated microbiome at genus level and discovered that Burkholderia were present in the Sphagnum sporophyte and gametophyte, but were absent in the flark water. Therefore, Burkholderia is a part of the core microbiome transmitted from the moss sporophyte to the gametophyte. This suggests a vertical transmission of Burkholderia strains, and thus underlines their importance for the plants themselves.
doi:10.3389/fmicb.2013.00394
PMCID: PMC3866706  PMID: 24391630
Sphagnum fallax; Sphagnum magellanicum; Burkholderia communities; amplicon pyrosequencing; FISH-CLSM
5.  The DSF Quorum Sensing System Controls the Positive Influence of Stenotrophomonas maltophilia on Plants 
PLoS ONE  2013;8(7):e67103.
The interaction of the Gram-negative bacterium Stenotrophomonas maltophilia with eukaryotes can improve overall plant growth and health, but can also cause opportunistic infections in humans. While the quorum sensing molecule DSF (diffusible signal factor) is responsible for the regulation of phenotypes in pathogenic Stenotrophomonas, up until now, no beneficial effects were reported to be controlled by it. Our objective was to study the function of DSF in the plant growth promoting model strain S. maltophilia R551-3 using functional and transcriptomic analyses. For this purpose, we compared the wild-type strain with a mutant deficient in the rpfF (regulation of pathogenicity factors) gene that is essential for the synthesis of DSF. Oilseed rape seeds treated with the wild-type strain showed a statistically significant increase in germination rate compared with those treated with the rpfF mutant. Similarly, the wild-type strain exhibited better plant growth promotion and a greater efficiency in colonizing oilseed rape compared to the mutant strain. Moreover, only the wild-type was capable of forming structured cell aggregates both in vitro and in the rhizosphere, a characteristic mediated by DSF. Gene transcription analyses showed that numerous genes known to play a role in plant colonization (e.g. chemotaxis, cell motility, biofilm formation, multidrug efflux pumps) are controlled by the rpf/DSF system in S. maltophilia. In addition, we detected new potential functions of spermidine, primarily for both growth promotion and stress protection. Overall, our results showed a correspondence between the regulation of DSF and the positive interaction effect with the plant host.
doi:10.1371/journal.pone.0067103
PMCID: PMC3715506  PMID: 23874407
6.  Multi-Annual Fluctuations in Reconstructed Historical Time-Series of a European Lobster (Homarus gammarus) Population Disappear at Increased Exploitation Levels 
PLoS ONE  2013;8(4):e58160.
Through the history of ecology, fluctuations of populations have been a dominating topic, and endogenous causes of fluctuations and oscillations have been recognized and studied for more than 80 years. Here we analyzed an historical dataset, covering more than 130 years, of European lobster (Homarus gammarus) catches. The data shows periodic fluctuations, which are first dampened and then disappear over time. The disappearance of the periodicity coincided with a substantial increase in fishing effort and the oscillations have not reappeared in the time series. The shifting baseline syndrome has changed our perception of not only the status of the stock, but also the regulating pressures. We describe the transition of a naturally regulated lobster population into a heavily exploited fisheries controlled stock. This is shown by the incorporation of environmental and endogenous processes in generalized additive models, autocorrelation functions and periodicity analyses of time-series.
doi:10.1371/journal.pone.0058160
PMCID: PMC3616055  PMID: 23573187
7.  Sphagnum mosses harbour highly specific bacterial diversity during their whole lifecycle 
The ISME Journal  2011;6(4):802-813.
Knowledge about Sphagnum-associated microbial communities, their structure and their origin is important to understand and maintain climate-relevant Sphagnum-dominated bog ecosystems. We studied bacterial communities of two cosmopolitan Sphagnum species, which are well adapted to different abiotic parameters (Sphagnum magellanicum, which are strongly acidic and ombrotrophic, and Sphagnum fallax, which are weakly acidic and mesotrophic), in three Alpine bogs in Austria by a multifaceted approach. Great differences between bacterial fingerprints of both Sphagna were found independently from the site. This remarkable specificity was confirmed by a cloning and a deep sequencing approach. Besides the common Alphaproteobacteria, we found a discriminative spectrum of bacteria; although Gammaproteobacteria dominated S. magellanicum, S. fallax was mainly colonised by Verrucomicrobia and Planctomycetes. Using this information for fluorescent in situ hybridisation analyses, corresponding colonisation patterns for Alphaproteobacteria and Planctomycetes were detected. Bacterial colonies were found in high abundances inside the dead big hyalocytes, but they were always connected with the living chlorocytes. Using multivariate statistical analysis, the abiotic factors nutrient richness and pH were identified to modulate the composition of Sphagnum-specific bacterial communities. Interestingly, we found that the immense bacterial diversity was transferred via the sporophyte to the gametophyte, which can explain the high specificity of Sphagnum-associated bacteria over long distances. In contrast to higher plants, which acquire their bacteria mainly from the environment, mosses as the phylogenetically oldest land plants maintain their bacterial diversity within the whole lifecycle.
doi:10.1038/ismej.2011.151
PMCID: PMC3309359  PMID: 22094342
abiotic factors; bog ecosystem; FISH–CLSM; deep-sequencing; microbe+plant communities; S. magellanicum/fallax
8.  Stopover decision during migration: physiological conditions predict nocturnal restlessness in wild passerines 
Biology Letters  2009;5(3):302-305.
During migration, a number of bird species rely on stopover sites for resting and feeding before and after crossing ecological barriers such as deserts or seas. The duration of a stopover depends on the combined effects of environmental factors, endogenous programmes and physiological conditions. Previous studies indicated that lean birds prolong their refuelling stopover compared with fat birds; however, the quantitative relationship between physiological conditions and stopover behaviour has not been studied yet. Here, we tested in a large sample of free-living birds of three European passerines (whinchats, Saxicola rubetra, garden warblers, Sylvia borin and whitethroats, Sylvia communis) whether the amount of migratory restlessness (Zugunruhe) shown at a stopover site depends on physiological conditions. An integrated measure of condition based on body mass, amount of subcutaneous fat and thickness of pectoral muscles strongly predicted the intensity of Zugunruhe shown in recording cages in the night following capture. These results provide novel and robust quantitative evidence in support of the hypothesis that the amount of energy reserves plays a major role in determining the stopover duration in migratory birds.
doi:10.1098/rsbl.2008.0755
PMCID: PMC2679912  PMID: 19324648
bird; migration; stopover; migratory restlessness; Zugunruhe
9.  Multi-level trophic cascades in a heavily exploited open marine ecosystem 
Anthropogenic disturbances intertwined with climatic changes can have a large impact on the upper trophic levels of marine ecosystems, which may cascade down the food web. So far it has been difficult to demonstrate multi-level trophic cascades in pelagic marine environments. Using field data collected during a 33-year period, we show for the first time a four-level community-wide trophic cascade in the open Baltic Sea. The dramatic reduction of the cod (Gadus morhua) population directly affected its main prey, the zooplanktivorous sprat (Sprattus sprattus), and indirectly the summer biomass of zooplankton and phytoplankton (top-down processes). Bottom-up processes and climate–hydrological forces had a weaker influence on sprat and zooplankton, whereas phytoplankton variation was explained solely by top-down mechanisms. Our results suggest that in order to dampen the occasionally harmful algal blooms of the Baltic, effort should be addressed not only to control anthropogenic nutrient inputs but also to preserve structure and functioning of higher trophic levels.
doi:10.1098/rspb.2007.1752
PMCID: PMC2587786  PMID: 18460432
Baltic Sea; pelagic marine ecosystem; food web; bottom-up versus top-down control; climate; eutrophication
10.  Comparison of Different Primer Sets for Use in Automated Ribosomal Intergenic Spacer Analysis of Complex Bacterial Communities 
Applied and Environmental Microbiology  2004;70(10):6147-6156.
ITSF and ITSReub, constituting a new primer set designed for the amplification of the 16S-23S rRNA intergenic transcribed spacers, have been compared with primer sets consisting of 1406F and 23Sr (M. M. Fisher and E. W. Triplett, Appl. Environ. Microbiol. 65:4630-4636, 1999) and S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 (L. Ranjard et al., Appl. Environ. Microbiol. 67:4479-4487, 2001), previously proposed for automated ribosomal intergenic spacer analysis (ARISA) of complex bacterial communities. An agricultural soil and a polluted soil, maize silage, goat milk, a small marble sample from the façade of the Certosa of Pavia (Pavia, Italy), and brine from a deep hypersaline anoxic basin in the Mediterranean Sea were analyzed with the three primer sets. The number of peaks in the ARISA profiles, the range of peak size (width of the profile), and the reproducibility of results were used as indices to evaluate the efficiency of the three primer sets. The overall data showed that ITSF and ITSReub generated the most informative (in term of peak number) and reproducible profiles and yielded a wider range of spacer sizes (134 to 1,387) than the other primer sets, which were limited in detecting long fragments. The minimum amount of DNA template and sensitivity in detection of minor DNA populations were evaluated with artificial mixtures of defined bacterial species. ITSF and ITSReub amplified all the bacteria at DNA template concentrations from 280 to 0.14 ng μl−1, while the other primer sets failed to detect the spacers of one or more bacterial strains. Although the primer set consisting of ITSF and ITSReub and that of S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 showed similar sensitivities for the DNA of Allorhizobium undicula mixed with the DNA of other species, the S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 primer set failed to detect the DNA of Pseudomonas stutzeri.
doi:10.1128/AEM.70.10.6147-6156.2004
PMCID: PMC522057  PMID: 15466561

Results 1-10 (10)