BACKGROUND
knowledge on the relationship between plant communities and soil microbial communities is still lacking in large parts, although recent ecological research focuses on potentially beneficial effects of biodiversity on ecosystem processes, including the response of ecosystems to environmental changes such as increasing atmospheric carbon dioxide levels  <cit> .
soil microorganisms are the driving force behind soil organic matter transformations such as mineralization of organic compounds.
these transformations are the basis of plant decomposition, soil aggregation, nutrient availability, soil fertility and, therefore in general, soil ecosystem functioning.
however, these transformations might be significantly influenced by different levels of plant diversity which can affect soil microbial communities regarding e.g.
population sizes, activities and taxonomic composition  of soil microbes  possibly mediated via altered growth of the plant communities  <cit> .
on both local and global scales, the wealth of soil microbial diversity is poorly appreciated and, therefore, the importance of soil organisms has been largely neglected  <cit> .
a profound understanding of soil biodiversity and its relation to ecosystem functions is vital for long-term sustainable soils  <cit> .
however, detailed knowledge on the control of ecosystem processes and functioning by this diversity is still lacking  <cit> .
soil microbial communities are characterized by two levels of diversity, namely genetic diversity and functional diversity: a high level of genetic diversity is found in many different types of soil .
recent detailed investigations based on molecular methods such as dna-dna-hybridization, 16s rrna sequencing, pcr-based methods with primers derived from rrna sequencing, fluorescence in situ hybridization , or immunological techniques revealed that soil microbial communities are composed of a vast variety of microorganisms resulting in complex microbial interactions and nutrient flows  <cit> .
the composition of these communities is usually subject to seasonal fluctuations and may vary between different locations.
in addition, organisms are not homogeneously distributed over the whole environment  <cit> .
regarding soil, it has been hypothesized that significant reductions in microbial diversity due to environmental changes are unlikely and that the genetic diversity does not represent a major factor that limits ecosystem functioning  <cit> .
elevated atmospheric co <dig> can have indirect effects on soil microbial communities via altered plant inputs .
as result, soil microbial communities and their activities are stimulated: increased carbon flow might affect the portion of culturable soil bacteria and might favor fast growing organisms  <cit> .
in turn, also the nitrogen flow  in soil ecosystems can be influenced by elevated co <dig>  <cit> , although nitrogen concentration in plant litter is not affected  <cit> .
therefore, microbial community composition and functional diversity are subject to changes under changing environmental conditions and the populations will adapt to the new conditions.
we examined soil samples from experimental grassland plots where plant communities of different species richness had been exposed for  <dig> years to ambient or elevated levels of carbon dioxide and assessed soil microbial community structure.
the two main questions were:  what is the effect of elevated co <dig> and plant diversity on bacterial richness?
to which extent are treatment-induced effects reflected in changes in the structural composition of the soil bacterial community?
an innovative feature of this study is the combination of an experimental-ecological approach and a microbiological approach to characterize the microbial populations involved.
we determined the total number of soil microbial operational taxonomic units  as a measure for "bacterial richness" as well as the dissimilitude of these otus as a measure for bacterial composition applying analyses of terminal restriction fragment length polymorphism .
RESULTS
dominant soil microbial populations  varied only very little between different soil samples .
only a few bands were observed.
the band patterns of the total community 16s rdna showed that bacterial communities consisted of five to six bacterial groups that were present in all of the differently treated soils examined.
these populations seemed to be ubiquitous and occurred in all samples, independently of experimental treatments or sampling location.
only very rarely additional otus were detected .
as a consequence, we found that soil bacterial community structure was only poorly resolved when analyzing bacterial populations by ttge.
in our case, the resolution of ttge was too low for the monitoring of soil microbial diversity.
to improve analysis on a high resolution level, soil microbial populations were assessed by t-rflp  <cit> .
different trf'-types were applied, namely  3'-bstu,  5'-bstu,  3'-mnl, and  5'-mnl .
combination ,  and  yielded  <dig>   <dig> and  <dig> operational taxonomic units , respectively, whereas  <dig> otus were obtained from combination .
every of the four different trf'-types applied may constitute on its own a possibility to determine soil microbial diversity.
a combined analysis, however, allows more powerful statistical analysis and to monitor soil microbial diversity more comprehensively regarding the different otus present.
however, combining the information obtained from all assays  showed that elevated carbon dioxide levels does not significantly influence the number of soil microbial community otus  .
moreover, also different plant diversity levels showed no significant effect on this bacterial richness measure .
no interaction was detected between carbon-dioxide and diversity treatments.
to analyze bacterial composition, all band patterns of the different enzyme/label combinations were analyzed in combination by canonical correspondence analysis.
occurrence of otus within the four replicates of each treatment combination was analyzed separately, but additionally also the frequency of occurrence of otus within the four replicates was assessed.
as shown in fig.
3b, plant diversity had a significant  effect on bacterial composition.
three distinct separate clusters can be observed, each related to one of the three different plant diversity levels.
in contrast, elevated carbon dioxide did not affect bacterial composition .
although a clustered pattern was observed, a significant difference was not detectable.
all trfs were compared with the tap-database.
only bacterial strains simultaneously matching maximally two types of enzyme/label combinations were considered .
no strains simultaneously matching three or four enzyme/label combinations were detected.
in several cases, results yielded more than one bacterial species for the same enzyme/label combination.
the frequency of occurrence of the different combinations of trfs and bacteria belonging to them was variable.
several bacterial strains were found only in specific plots and were obviously related to elevated carbon dioxide or varying plant diversity levels: escherichia coli and ferrobacterium limneticum were detected only in three samples, all showing a high plant diversity level.
the patterns referring to the unidentified strain from lake gossenkoellesee was found only in samples with medium plant diversity .
clostridium perfringens, sulfobacillus disulfidooxidans, kitasatospora paracochleata, kitasatospora melanogena and kitasatospora kifuense were found only in plots with elevated carbon dioxide levels.
furthermore, analyses of the different trf patterns by logistic regression showed that the probability of occurrence of salmonella typhimurium in plots treated by elevated co <dig> was significantly increased .
discussion
several studies found a positive relationship between elevated co <dig> and bacterial richness  <cit> , whereas others found a negative effect  <cit> .
therefore, results show a certain inconsistency  <cit> .
in our case, considering the negligible effect of elevated carbon dioxide and of different plant diversity levels on the number of otus detected, it can be concluded that both experimental treatments had no effect on bacterial richness.
furthermore, elevated carbon dioxide concentrations did not affect soil microbial composition as also reported by griffiths et al.
<cit>  or zak et al  <cit> .
in contrast, aboveground plant diversity significantly affected belowground bacterial composition.
these findings suggest that the soil microbial composition is mainly related to plant diversity  rather than altered soil carbon fluxes induced by elevated atmospheric co <dig> and subsequently increased photosynthetic activities.
our analyses showed that increased levels of carbon dioxide had no influence on soil microbial community composition .
canonical correspondence analysis  showed no statistical difference between plots at ambient co <dig> level compared with plots with elevated co <dig> level.
differences in rhizosphere carbon allocation  have been postulated and observed when plants were exposed to increased co <dig> levels in other studies, e.g.
changed quality of litter, increase in root exudates and stimulation of rhizodeposition  <cit> .
consequently, effects on soil microbial community composition might occur  <cit> .
however, in our case the aboveground exposure of plants to elevated carbon dioxide was not reflected in a belowground change of bacterial composition.
in contrast to the co <dig> treatment, plant diversity had a significant effect on belowground microbial community composition .
that is, on a genetic level, bacterial community structure in soil can be differentiated in relation to different plant diversity levels.
this is in agreement with studies reported earlier on the functional microbial diversity in soil samples exposed to different plant diversity levels  <cit> .
the differentiation between the three different plant diversity levels on the basis of the soil bacterial composition suggests that all three plant diversities exhibit their own bacterial environment.
the land where our experimental plots have been set up, has a long-time record as pasture for cows.
therefore, the occurrence of escherichia coli and salmonella sp.
is not surprising since these organisms easily originate from cowpats as shown by muirhead et al.,  <cit>  and johannessen et al.
<cit> .
anoxic micro-habitats might occur in soil allowing the existence and survival of anaerobes such as clostridia.
in addition, clostridium is an spore-forming organism able to form spores which can easily survive in soil for prolonged time periods.
since ralstonia solanacearum is commonly found in soil as plant pathogen but also free-living when host plants are absent  <cit> , the occurrence is not unusual.
therefore, we conclude that our findings reflect the true state of the soil investigated.
as stated by other authors  <cit> , soil type might be the key determinant for soil microbial communities.
since the soil type was the same throughout all experimental plots, we believe that the differences we have observed reflect the true state of the soil depending on the different treatments e.g.
elevated co <dig> or plant diversity.
CONCLUSIONS
in summary, plant diversity levels are affecting bacterial composition .
elevated carbon dioxide does not lead to quantitative alteration , whereas plant diversity is responsible for qualitative changes .

 knowledge on the relationship between plant communities and soil microbial communities is still lacking in large parts, although recent ecological research focuses on potentially beneficial effects of biodiversity on ecosystem processes, including the response of ecosystems to environmental changes such as increasing atmospheric carbon dioxide levels. soil microorganisms are the driving force behind soil organic matter transformations such as mineralization of organic compounds. these transformations are the basis of plant decomposition, soil aggregation, nutrient availability, soil fertility and, therefore in general, soil ecosystem functioning. however, these transformations might be significantly influenced by different levels of plant diversity which can affect soil microbial communities regarding e.g. population sizes, activities and taxonomic composition of soil microbes possibly mediated via altered growth of the plant communities. on both local and global scales, the wealth of soil microbial diversity is poorly appreciated and, therefore, the importance of soil organisms has been largely neglected. a profound understanding of soil biodiversity and its relation to ecosystem functions is vital for long-term sustainable soils. however, detailed knowledge on the control of ecosystem processes and functioning by this diversity is still lacking. soil microbial communities are characterized by two levels of diversity, namely genetic diversity and functional diversity: a high level of genetic diversity is found in many different types of soil. recent detailed investigations based on molecular methods such as dna-dna-hybridization, 16s rrna sequencing, pcr-based methods with primers derived from rrna sequencing, fluorescence in situ hybridization, or immunological techniques revealed that soil microbial communities are composed of a vast variety of microorganisms resulting in complex microbial interactions and nutrient flows. the composition of these communities is usually subject to seasonal fluctuations and may vary between different locations. in addition, organisms are not homogeneously distributed over the whole environment. regarding soil, it has been hypothesized that significant reductions in microbial diversity due to environmental changes are unlikely and that the genetic diversity does not represent a major factor that limits ecosystem functioning. elevated atmospheric co can have indirect effects on soil microbial communities via altered plant inputs. as result, soil microbial communities and their activities are stimulated: increased carbon flow might affect the portion of culturable soil bacteria and might favor fast growing organisms. in turn, also the nitrogen flow in soil ecosystems can be influenced by elevated co, although nitrogen concentration in plant litter is not affected. therefore, microbial community composition and functional diversity are subject to changes under changing environmental conditions and the populations will adapt to the new conditions. we examined soil samples from experimental grassland plots where plant communities of different species richness had been exposed for years to ambient or elevated levels of carbon dioxide and assessed soil microbial community structure. the two main questions were: what is the effect of elevated co and plant diversity on bacterial richness? to which extent are treatment-induced effects reflected in changes in the structural composition of the soil bacterial community? an innovative feature of this study is the combination of an experimental-ecological approach and a microbiological approach to characterize the microbial populations involved. we determined the total number of soil microbial operational taxonomic units as a measure for "bacterial richness" as well as the dissimilitude of these otus as a measure for bacterial composition applying analyses of terminal restriction fragment length polymorphism.  dominant soil microbial populations varied only very little between different soil samples. only a few bands were observed. the band patterns of the total community 16s rdna showed that bacterial communities consisted of five to six bacterial groups that were present in all of the differently treated soils examined. these populations seemed to be ubiquitous and occurred in all samples, independently of experimental treatments or sampling location. only very rarely additional otus were detected. as a consequence, we found that soil bacterial community structure was only poorly resolved when analyzing bacterial populations by ttge. in our case, the resolution of ttge was too low for the monitoring of soil microbial diversity. to improve analysis on a high resolution level, soil microbial populations were assessed by t-rflp. different trf'-types were applied, namely 3'-bstu, 5'-bstu, 3'-mnl, and 5'-mnl. combination, and yielded and operational taxonomic units, respectively, whereas otus were obtained from combination. every of the four different trf'-types applied may constitute on its own a possibility to determine soil microbial diversity. a combined analysis, however, allows more powerful statistical analysis and to monitor soil microbial diversity more comprehensively regarding the different otus present. however, combining the information obtained from all assays showed that elevated carbon dioxide levels does not significantly influence the number of soil microbial community otus. moreover, also different plant diversity levels showed no significant effect on this bacterial richness measure. no interaction was detected between carbon-dioxide and diversity treatments. to analyze bacterial composition, all band patterns of the different enzyme/label combinations were analyzed in combination by canonical correspondence analysis. occurrence of otus within the four replicates of each treatment combination was analyzed separately, but additionally also the frequency of occurrence of otus within the four replicates was assessed. as shown in fig. 3b, plant diversity had a significant effect on bacterial composition. three distinct separate clusters can be observed, each related to one of the three different plant diversity levels. in contrast, elevated carbon dioxide did not affect bacterial composition. although a clustered pattern was observed, a significant difference was not detectable. all trfs were compared with the tap-database. only bacterial strains simultaneously matching maximally two types of enzyme/label combinations were considered. no strains simultaneously matching three or four enzyme/label combinations were detected. in several cases, results yielded more than one bacterial species for the same enzyme/label combination. the frequency of occurrence of the different combinations of trfs and bacteria belonging to them was variable. several bacterial strains were found only in specific plots and were obviously related to elevated carbon dioxide or varying plant diversity levels: escherichia coli and ferrobacterium limneticum were detected only in three samples, all showing a high plant diversity level. the patterns referring to the unidentified strain from lake gossenkoellesee was found only in samples with medium plant diversity. clostridium perfringens, sulfobacillus disulfidooxidans, kitasatospora paracochleata, kitasatospora melanogena and kitasatospora kifuense were found only in plots with elevated carbon dioxide levels. furthermore, analyses of the different trf patterns by logistic regression showed that the probability of occurrence of salmonella typhimurium in plots treated by elevated co was significantly increased. discussion several studies found a positive relationship between elevated co and bacterial richness, whereas others found a negative effect. therefore, results show a certain inconsistency. in our case, considering the negligible effect of elevated carbon dioxide and of different plant diversity levels on the number of otus detected, it can be concluded that both experimental treatments had no effect on bacterial richness. furthermore, elevated carbon dioxide concentrations did not affect soil microbial composition as also reported by griffiths et al. or zak et al. in contrast, aboveground plant diversity significantly affected belowground bacterial composition. these findings suggest that the soil microbial composition is mainly related to plant diversity rather than altered soil carbon fluxes induced by elevated atmospheric co and subsequently increased photosynthetic activities. our analyses showed that increased levels of carbon dioxide had no influence on soil microbial community composition. canonical correspondence analysis showed no statistical difference between plots at ambient co level compared with plots with elevated co level. differences in rhizosphere carbon allocation have been postulated and observed when plants were exposed to increased co levels in other studies, e.g. changed quality of litter, increase in root exudates and stimulation of rhizodeposition. consequently, effects on soil microbial community composition might occur. however, in our case the aboveground exposure of plants to elevated carbon dioxide was not reflected in a belowground change of bacterial composition. in contrast to the co treatment, plant diversity had a significant effect on belowground microbial community composition. that is, on a genetic level, bacterial community structure in soil can be differentiated in relation to different plant diversity levels. this is in agreement with studies reported earlier on the functional microbial diversity in soil samples exposed to different plant diversity levels. the differentiation between the three different plant diversity levels on the basis of the soil bacterial composition suggests that all three plant diversities exhibit their own bacterial environment. the land where our experimental plots have been set up, has a long-time record as pasture for cows. therefore, the occurrence of escherichia coli and salmonella sp. is not surprising since these organisms easily originate from cowpats as shown by muirhead et al., and johannessen et al.. anoxic micro-habitats might occur in soil allowing the existence and survival of anaerobes such as clostridia. in addition, clostridium is an spore-forming organism able to form spores which can easily survive in soil for prolonged time periods. since ralstonia solanacearum is commonly found in soil as plant pathogen but also free-living when host plants are absent, the occurrence is not unusual. therefore, we conclude that our findings reflect the true state of the soil investigated. as stated by other authors, soil type might be the key determinant for soil microbial communities. since the soil type was the same throughout all experimental plots, we believe that the differences we have observed reflect the true state of the soil depending on the different treatments e.g. elevated co or plant diversity.  in summary, plant diversity levels are affecting bacterial composition. elevated carbon dioxide does not lead to quantitative alteration, whereas plant diversity is responsible for qualitative changes. 
in contrast, differences in plant diversity levels had a significant effect on bacterial composition but no influence on bacterial richness. plant diversity levels are affecting bacterial composition. we examined soil samples taken from a biodiversity × co grassland experiment where replicate plots harboring or different plant species had been exposed to ambient or elevated levels of carbon dioxide for years. changes in aboveground plant species diversity as well as variations of environmental conditions such as exposure of ecosystems to elevated concentrations of atmospheric carbon dioxide may lead to changes in metabolic activity, composition and diversity of belowground microbial communities, both bacterial and fungal. likewise, screening of samples on a high-resolution level by terminal restriction fragment length polymorphism showed that increased levels of carbon dioxide had no significant influence on both soil bacterial community composition and on bacterial richness. regarding species level, several bacterial species were found only in specific plots and were related to elevated carbon dioxide or varying plant diversity levels. elevated carbon dioxide does not lead to quantitative alteration, whereas plant diversity is responsible for qualitative changes. for example, analysis of t-rflp showed that the occurrence of salmonella typhimurium was significantly increased in plots exposed to elevated co.
 changes in aboveground plant species diversity as well as variations of environmental conditions such as exposure of ecosystems to elevated concentrations of atmospheric carbon dioxide may lead to changes in metabolic activity, composition and diversity of belowground microbial communities, both bacterial and fungal.  we examined soil samples taken from a biodiversity × co grassland experiment where replicate plots harboring or different plant species had been exposed to ambient or elevated levels of carbon dioxide for years. analysis of soil bacterial communities in these plots by temporal temperature gradient gel electrophoresis showed that dominant soil bacterial populations varied only very little between different experimental treatments. these populations seem to be ubiquitous. likewise, screening of samples on a high-resolution level by terminal restriction fragment length polymorphism showed that increased levels of carbon dioxide had no significant influence on both soil bacterial community composition and on bacterial richness. in contrast, differences in plant diversity levels had a significant effect on bacterial composition but no influence on bacterial richness. regarding species level, several bacterial species were found only in specific plots and were related to elevated carbon dioxide or varying plant diversity levels. for example, analysis of t-rflp showed that the occurrence of salmonella typhimurium was significantly increased in plots exposed to elevated co.  plant diversity levels are affecting bacterial composition. elevated carbon dioxide does not lead to quantitative alteration, whereas plant diversity is responsible for qualitative changes. 