Comparison of barley succession and take-all disease as environmental factors shaping the rhizobacterial community during take-all decline.

The root disease take-all, caused by Gaeumannomyces graminis var. tritici, can be managed by monoculture-induced take-all decline (TAD). This natural biocontrol mechanism typically occurs after a take-all outbreak and is believed to arise from an enrichment of antagonistic populations in the rhizosphere. However, it is not known whether these changes are induced by the monoculture or by ecological rhizosphere conditions due to a disease outbreak and subsequent attenuation. This question was addressed by comparing the rhizosphere microflora of barley, either inoculated with the pathogen or noninoculated, in a microcosm experiment in five consecutive vegetation cycles. TAD occurred in soil inoculated with the pathogen but not in noninoculated soil. Bacterial community analysis using terminal restriction fragment length polymorphism of 16S rRNA showed pronounced population shifts in the successive vegetation cycles, but pathogen inoculation had little effect. To elucidate rhizobacterial dynamics during TAD development, a 16S rRNA-based taxonomic microarray was used. Actinobacteria were the prevailing indicators in the first vegetation cycle, whereas the third cycle-affected most severely by take-all-was characterized by Proteobacteria, Bacteroidetes, Chloroflexi, Planctomycetes, and Acidobacteria. Indicator taxa for the last cycle (TAD) belonged exclusively to Proteobacteria, including several genera with known biocontrol traits. Our results suggest that TAD involves monoculture-induced enrichment of plant-beneficial taxa.

Abundance and diversity of CO2-fixing bacteria in grassland soils close to natural carbon dioxide springs.

Gaseous conditions at natural CO2 springs (mofettes) affect many processes in these unique ecosystems. While the response of plants to extreme and fluctuating CO2 concentrations ([CO2]) is relatively well documented, little is known on microbial life in mofette soil. Therefore, it was the aim of this study to investigate the abundance and diversity of CO2-fixing bacteria in grassland soils in different distances to a natural carbon dioxide spring. Samples of the same soil type were collected from the Stavesinci mofette, a natural CO2 spring which is known for very pure CO2 emissions, at different distances from the CO2 releasing vents, at locations that clearly differed in soil CO2 efflux (from 12.5 to over 200 micromol CO2 m(-2) s(-1) yearly average). Bulk and rhizospheric soil samples were included into analyses. The microbial response was followed by a molecular analysis of cbbL genes, encoding for the large subunit of RubisCO, a carboxylase which is of crucial importance for C assimilation in chemolitoautotrophic microbes. In all samples analyzed, the "red-like" type of cbbL genes could be detected. In contrast, the "green-like" type of cbbL could not be measured by the applied technique. Surprisingly, a reduction of "red-like" cbbL genes copies was observed in bulk soil and rhizosphere samples from the sites with the highest CO2 concentrations. Furthermore, the diversity pattern of "red-like" cbbL genes changed depending on the CO(2) regime. This indicates that only a part of the autotrophic CO2-fixing microbes could adapt to the very high CO2 concentrations and adverse life conditions that are governed by mofette gaseous regime.

In vitro antagonism of an actinobacterial Kitasatospora isolate against the plant pathogen Phytophthora citricola as elucidated with ultrahigh resolution mass spectrometry.

Many soil microorganisms antagonistic to soil borne plant pathogens are well known for their ability to control diseases in situ. A variety of substances, like lytic enzymes, siderophores and antibiotics, produced by these organisms have the potential to protect roots against pathogens. Understanding the ecology and a functional assessment of antagonistic microbial communities in soil requires in-depth knowledge of the mechanisms involved in these interactions, a challenging task in complex systems if low-resolution methods are applied. We propose an information-rich strategy of general relevance, composed of adequate preconcentration in conjunction with ultrahigh resolution ion cyclotron resonance Fourier transform mass spectrometry (ICR-FT/MS) and nuclear magnetic resonance (NMR) spectroscopy to identify any bioactive substances in complex systems. This approach is demonstrated on the specific example of substance identification considered responsible for in vitro antagonism of an actinobacterial antagonist isolated from European beech (Fagus sylvatica) rhizosphere soil against the oomycetous root rot pathogen Phytophthora citricola. The isolate belonging to the genus Kitasatospora exhibited strong antibiosis against the oomycete in vitro. The bioactive substance was observed to exhibit a molar mass of 281.1699 g/mol in positive electrospray ionization mass spectra, and the high mass accuracy of the ICR-FT/MS measurements allowed a precise assignment of a molecular formula that was found identical to the macrolide polyketide cycloheximide C(15)H(23)NO(4)+H(+); its identity was then unequivocally confirmed by the information-rich atomic signature of proton NMR spectroscopy. In conclusion, the combination of the near orthogonal methods (pre)fractionation, ultrahigh-resolution ICR-FT mass spectrometry (yielding molecular and MS(n) fragment signatures) and nuclear magnetic resonance spectroscopy (providing atomic signatures) has been found capable of identifying a biocontrol active compound of Kitasatospora active against Phytophthora citricola expediently, quickly, and accurately. This straightforward approach is of general applicability to elucidate biocontrol mechanisms in any complex system with improved efficiency.

A new cultivation independent approach to detect and monitor common Trichoderma species in soils.

A set of primers was developed for the detection, identification and quantification of common Trichoderma species in soil samples. Based on a broad range master alignment primers were derived to amplify an approximate 540 bp fragment comprising the internal transcribed spacer region 1 (ITS 1), 5.8S rDNA and internal transcribed spacer region 2 (ITS 2) from all taxonomic Clades of the genus Trichoderma. The primer set was applied to test strains as well as community DNA isolated from arable and forest soil. For all tested isolates the corresponding internal transcribed spacer regions of Trichoderma spp. strains were amplified, but none of non-Trichoderma origin. PCR with community DNA from soil yielded products of the expected size. Analysis of a clone library established for an arable site showed that all amplified sequences originated exclusively from Trichoderma species mainly being representatives of the Clades Hamatum, Harzianum and Pachybasioides and comprising most of the species known for biocontrol ability. In a realtime PCR approach the primer set uTf/uTr also proved to be a suitable system to quantify DNA of Trichoderma spp. in soils.

Impact of elevated atmospheric O3 on the actinobacterial community structure and function in the rhizosphere of European beech (Fagus sylvatica L.).

Many bacteria belonging to the phylum of Actinobacteria are known as antagonists against phytpathogenic microbes. This study aimed to analyze the effect of ozone on the actinobacterial community of the rhizosphere of four years old European beech (Fagus sylvatica L.) trees during different time points of the vegetation period. Effects of ozone on the total community structure of Actinobacteria were studied based on the analysis of 16S rRNA gene amplicons. In addition effects of the ozone treatment on the diversity of potential biocontrol active Actionobacteria being able to produce antibiotics were characterized by using the type II polyketide synthases (PKS) genes as marker. Season as well as ozone treatments had a significant effect on parts of the actinobacterial rhizosphere community of European beech. However on the basis of the performed analysis, the diversity of Actinobacteria possessing type II PKS genes is neither affected by seasonal changes nor by the ozone treatments, indicating no influence of the investigated treatments on the biocontrol active part of the actinobacterial community.

Single application of sewage sludge--impact on the quality of an alluvial agricultural soil.

The effects of sewage sludge on soil quality with regard to its nutrient and heavy metal content, microbial community structure and ability to maintain specific soil function (degradation of herbicide glyphosate) were investigated in a three months study using an alluvial soil (Eutric Fluvisol). Dehydrated sewage sludge significantly increased soil organic matter (up to 20.6% of initial content), total and available forms of N (up to 33% and 220% of initial amount, respectively), as well as total and plant available forms of P (up to 11% and 170% of initial amount, respectively) and K (up to 70% and 47% of initial amount, respectively) in the upper 2 cm soil layer. The increase of organic matter was most prominent 3d after the application of sewage sludge, after 3 months it was no longer significant. Contents of nutrients kept to be significantly higher in the sewage sludge treated soil till the end of experiment. Contents of some heavy metals (Zn, Cu, Pb) increased as well. The highest increase was found for Zn (up to 53% of initial amount), however it was strongly bound to soil particles and its total content was kept below the maximum permissible limit for agricultural soil. Based on molecular fingerprinting of bacterial 16S rRNA gene and fungal ITS fragment on 3rd day and 3rd month after sewage sludge amendment, significant short term effects on bacterial and fungal communities were shown due to the sewage sludge. The effects were more pronounced and more long-term for bacterial than fungal communities. The mineralization of (14)C-glyphosate in the sewage sludge soil was 55.6% higher than in the control which can be linked to (i) a higher glyphosate bioavailability in sewage sludge soil, which was triggered by the pre-sorption of phosphate originating from the sewage sludge and/or (ii) beneficial alterations of the sewage sludge to the physical-chemical characteristics of the soil.

Changes in fungal community composition in response to vegetational succession during the natural regeneration of cutover peatlands.

Despite the importance of peatlands as a major store of sequestered carbon and the role of fungi in releasing sequestered C, we know little about the community structure of fungi in peatlands. We investigated these across a gradient of naturally regenerating peatland vegetation using denaturing gradient gel electrophoresis (DGGE) and clone libraries of fragments of the fungal rRNA internal transcribed spacer (ITS) region. Significant changes in the fungal community structure of peat samples at different stages of regeneration were observed, which relate to the composition of the vegetation recolonizing these sites. Cloning and sequence analysis also demonstrated a potential shift in the relative abundance of the main fungal phyla. Some of the clones identified to genus level were highly related to fungi known to play a role in the degradation of plant litter or wood in similar ecosystems and/or form mycorrhizal associations. In addition, several fungal isolates highly related to peat clones were obtained, and their enzymic capacity to degrade structural plant tissues was assessed. Together, these results suggest that the fungal community composition of peat may be an important indicator of the status of regeneration and potential carbon sequestration of cutover peatlands.

Decolorization of synthetic dyes and production of manganese-dependent peroxidase by new fungal isolates.

Two yeasts, Debaryomyces polymorphus, Candida tropicalis, and two filamentous fungi, Umbelopsis isabellina, Penicillium geastrivorus, could completely decolorize 100 mg Reactive Black 5 (RB 5) l-1 within 16-48 h. Manganese-dependent peroxidase (MnP) activities between 60 and 424 U l-1 were detected in culture supernatants of three of these organisms indicating the color removal by enzymatic biodegradation but with P. geastrivorus there was no ligninolytic enzyme activity in its culture and the decolorization was mainly due to biosorption to mycelium. Extensive decolorization by D. polymorphus (69-94%) and C. tropicalis (30-97%) was obtained with five other azo dyes and one anthraquinone dye. Except for Reactive Brilliant Blue KNR and Reactive Yellow M-3R, the four azo dyes, Reactive Red M-3BE, Procion Scharlach H-E3G, Procion Marine H-EXL and Reactive Brilliant Red K-2BP, induced D. polymorphus to produce MnP (105-587 U l-1). However, MnP activities of 198-329 U l-1 were only detected in the culture of C. tropicalis containing Reactive Red M-3BE and Reactive Brilliant Red K-2BP, respectively.