Kornelia Smalla

Bulk and Rhizosphere Soil Bacterial Communities Studied by Denaturing Gradient Gel Electrophoresis: Plant-Dependent Enrichment and Seasonal Shifts Revealed

ABSTRACT The bacterial rhizosphere communities of three host plants of the pathogenic fungus Verticillium dahliae, field-grown strawberry (Fragaria ananassa Duch.), oilseed rape (Brassica napus L.), and potato (Solanum tuberosum L.), were analyzed. We aimed to determine the degree to which the rhizosphere effect is plant dependent and whether this effect would be increased by growing the same crops in two consecutive years. Rhizosphere or soil samples were taken five times over the vegetation periods. To allow a cultivation-independent analysis, total community DNA was extracted from the microbial pellet recovered from root or soil samples. 16S rDNA fragments amplified by PCR from soil or rhizosphere bacterium DNA were analyzed by denaturing gradient gel electrophoresis (DGGE). The DGGE fingerprints showed plant-dependent shifts in the relative abundance of bacterial populations in the rhizosphere which became more pronounced in the second year. DGGE patterns of oilseed rape and potato rhizosphere communities were more similar to each other than to the strawberry patterns. In both years seasonal shifts in the abundance and composition of the bacterial rhizosphere populations were observed. Independent of the plant species, the patterns of the first sampling times for both years were characterized by the absence of some of the bands which became dominant at the following sampling times.Bacillus megaterium and Arthrobacter sp. were found as predominant populations in bulk soils. Sequencing of dominant bands excised from the rhizosphere patterns revealed that 6 out of 10 bands resembled gram-positive bacteria. Nocardiapopulations were identified as strawberry-specific bands.

Plant species and soil type cooperatively shape the structure and function of microbial communities in the rhizosphere.

The rhizosphere is of central importance not only for plant nutrition, health and quality but also for microorganism-driven carbon sequestration, ecosystem functioning and nutrient cycling in terrestrial ecosystems. A multitude of biotic and abiotic factors are assumed to influence the structural and functional diversity of microbial communities in the rhizosphere. In this review, recent studies on the influence of the two factors, plant species and soil type, on rhizosphere-associated microbial communities are discussed. Root exudates and the response of microorganisms to the latter as well as to root morphology were shown to shape rhizosphere microbial communities. All studies revealed that soil is the main reservoir for rhizosphere microorganisms. Many secrets of microbial life in the rhizosphere were recently uncovered due to the enormous progress in molecular and microscopic tools. Physiological and molecular data on the factors that drive selection processes in the rhizosphere are presented here. Furthermore, implications for agriculture, nature conservation and biotechnology will also be discussed.

Antibiotic resistance gene spread due to manure application on agricultural fields.

The usage of antibiotics in animal husbandry has promoted the development and abundance of antibiotic resistance in farm environments. Manure has become a reservoir of resistant bacteria and antibiotic compounds, and its application to agricultural soils is assumed to significantly increase antibiotic resistance genes and selection of resistant bacterial populations in soil. The genome location of resistance genes is likely to shift towards mobile genetic elements such as broad-host-range plasmids, integrons, and transposable elements. Horizontal transfer of these elements to bacteria adapted to soil or other habitats supports their environmental transmission independent of the original host. The human exposure to soil-borne resistance has yet to be determined, but is likely to be severely underestimated.

Bacterial diversity of the rhizosphere of maize (Zea mays) grown in tropical soil studied by temperature gradient gel electrophoresis

The bacterial diversity and population dynamics in the rhizosphere of two maize cultivars (Nitroflint and Nitrodent) grown in tropical soils was studied, by traditional cultivation techniques and 16S rRNA gene-based molecular analysis of DNA directly extracted from soil and rhizosphere samples. Rhizosphere and soil samples were taken at three different plant growth stages. Total aerobic bacterial counts were determined. Fingerprints of the most dominant bacterial population were generated by TGGE separation of 16S rRNA gene fragments amplified from total community DNA using eubacterial specific primers. To reduce the complexity of TGGE fingerprints or to analyse less abundant populations, primers specific for different phylogenetic groups have been used. A comparison of the cfu obtained for rhizosphere of both cultivars indicated significant differences only for rhizosphere and soil samples taken 40 days after sowing. However, a comparison of TGGE patterns indicated that the composition of the bacterial community analysed at different plant growth stages for both cultivars was similar. A comparison of α-, β-proteobacterial and actinomycete TGGE patterns of both cultivars confirmed this observation. The eubacterial TGGE profiles reflected strong seasonal population shifts in the bacterial rhizosphere community of both maize cultivars which could be also observed in the TGGE patterns of α- and β-proteobacteria and to a lesser extent for actinomycetes. The rhizosphere effect was much more pronounced for young roots compared to samples taken from mature maize plants. The rhizosphere fingerprints showed a reduced complexity for young plants with up to five dominating bands while for mature plants patterns similar to those of soil were observed. Sequencing of dominant clones indicated that the dominant population found at all plant growth stages can be assigned to Arthrobacter populations.

Plant-Dependent Genotypic and Phenotypic Diversity of Antagonistic Rhizobacteria Isolated from Different Verticillium Host Plants

ABSTRACT To study the effect of plant species on the abundance and diversity of bacterial antagonists, the abundance, the phenotypic diversity, and the genotypic diversity of rhizobacteria isolated from potato, oilseed rape, and strawberry and from bulk soil which showed antagonistic activity towards the soilborne pathogen Verticillium dahliae Kleb. were analyzed. Rhizosphere and soil samples were taken five times over two growing seasons in 1998 and 1999 from a randomized field trial. Bacterial isolates were obtained after plating on R2A (Difco, Detroit, Mich.) or enrichment in microtiter plates containing high-molecular-weight substrates followed by plating on R2A. A total of 5,854 bacteria isolated from the rhizosphere of strawberry, potato, or oilseed rape or bulk soil from fallow were screened by dual testing for in vitro antagonism towards Verticillium. The proportion of isolates with antagonistic activity was highest for strawberry rhizosphere (9.5%), followed by oilseed rape (6.3%), potato (3.7%), and soil (3.3%). The 331 Verticillium antagonists were identified by their fatty acid methyl ester profiles. They were characterized by testing their in vitro antagonism against other pathogenic fungi; their glucanolytic, chitinolytic, and proteolytic activities; and their BOX-PCR fingerprints. The abundance and composition of Verticillium antagonists was plant species dependent. A rather high proportion of antagonists from the strawberry rhizosphere was identified as Pseudomonas putida B (69%), while antagonists belonging to the Enterobacteriaceae (Serratia spp., Pantoea agglomerans) were mainly isolated from the rhizosphere of oilseed rape. For P. putida A and B plant-specific genotypes were observed, suggesting that these bacteria were specifically enriched in each rhizosphere.

Dynamics of Fungal Communities in Bulk and Maize Rhizosphere Soil in the Tropics

ABSTRACT The fungal population dynamics in soil and in the rhizospheres of two maize cultivars grown in tropical soils were studied by a cultivation-independent analysis of directly extracted DNA to provide baseline data. Soil and rhizosphere samples were taken from six plots 20, 40, and 90 days after planting in two consecutive years. A 1.65-kb fragment of the 18S ribosomal DNA (rDNA) amplified from the total community DNA was analyzed by denaturing gradient gel electrophoresis (DGGE) and by cloning and sequencing. A rhizosphere effect was observed for fungal populations at all stages of plant development. In addition, pronounced changes in the composition of fungal communities during plant growth development were found by DGGE. Similar types of fingerprints were observed in two consecutive growth periods. No major differences were detected in the fungal patterns of the two cultivars. Direct cloning of 18S rDNA fragments amplified from soil or rhizosphere DNA resulted in 75 clones matching 12 dominant DGGE bands. The clones were characterized by their HinfI restriction patterns, and 39 different clones representing each group of restriction patterns were sequenced. The cloning and sequencing approach provided information on the phylogeny of dominant amplifiable fungal populations and allowed us to determine a number of fungal phylotypes that contribute to each of the dominant DGGE bands. Based on the sequence similarity of the 18S rDNA fragment with existing fungal isolates in the database, it was shown that the rhizospheres of young maize plants seemed to select the Ascomycetes order Pleosporales, while different members of the Ascomycetes and basidiomycetic yeast were detected in the rhizospheres of senescent maize plants.

Dynamics and functional relevance of ammonia-oxidizing archaea in two agricultural soils.

Crucial steps in geochemical cycles are in many cases performed by more than one group of microorganisms, but the significance of this functional redundancy with respect to ecosystem functioning is poorly understood. Ammonia-oxidizing archaea (AOA) and their bacterial counterparts (AOB) are a perfect system to address this question: although performing the same transformation step, they belong to well-separated phylogenetic groups. Using pig manure amended with different concentrations of sulfadiazine (SDZ), an antibiotic that is frequently used in veterinary medicine, it was possible to affect AOB and AOA to different degrees. Addition of manure stimulated growth of AOB in both soils and, interestingly, also growth of AOA was considerably stimulated in one of the soils. The antibiotic treatments decreased the manure effect notably on AOB, whereas AOA were affected to a lower extent. Model calculations concerning the respective proportions of AOA and AOB in ammonia oxidation indicate a substantial contribution of AOA in one of the soils that further increased under the influence of SDZ, hence indicating functional redundancy between AOA and AOB.

Using the class 1 integron-integrase gene as a proxy for anthropogenic pollution

Around all human activity, there are zones of pollution with pesticides, heavy metals, pharmaceuticals, personal care products and the microorganisms associated with human waste streams and agriculture. This diversity of pollutants, whose concentration varies spatially and temporally, is a major challenge for monitoring. Here, we suggest that the relative abundance of the clinical class 1 integron-integrase gene, intI1, is a good proxy for pollution because: (1) intI1 is linked to genes conferring resistance to antibiotics, disinfectants and heavy metals; (2) it is found in a wide variety of pathogenic and nonpathogenic bacteria; (3) its abundance can change rapidly because its host cells can have rapid generation times and it can move between bacteria by horizontal gene transfer; and (4) a single DNA sequence variant of intI1 is now found on a wide diversity of xenogenetic elements, these being complex mosaic DNA elements fixed through the agency of human selection. Here we review the literature examining the relationship between anthropogenic impacts and the abundance of intI1, and outline an approach by which intI1 could serve as a proxy for anthropogenic pollution.

Effects of T4 Lysozyme Release from Transgenic Potato Roots on Bacterial Rhizosphere Communities Are Negligible Relative to Natural Factors

ABSTRACT Rhizosphere bacterial communities of two transgenic potato lines which produce T4 lysozyme for protection against bacterial infections were analyzed in comparison to communities of wild-type plants and transgenic controls not harboring the lysozyme gene. Rhizosphere samples were taken from young, flowering, and senescent plants at two field sites in three consecutive years. The communities were characterized in a polyphasic approach. Cultivation-dependent methods included heterotrophic plate counts, determination of species composition and diversity based on fatty acid analysis of isolates, and community level catabolic profiling. Cultivation-independent analyses were based on denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments amplified from rhizosphere DNA using primers specific for Bacteria, Actinomycetales, or α- or β-Proteobacteria. Several bands of the DGGE patterns were further characterized by sequence analysis. All methods revealed that environmental factors related to season, field site, or year but not to the T4 lysozyme expression of the transgenic plants influenced the rhizosphere communities. For one of the T4 lysozyme-producing cultivars, no deviation in the rhizosphere communities compared to the control lines was observed. For the other, differences were detected at some of the samplings between the rhizosphere community structure and those of one or all other cultivars which were not attributable to T4 lysozyme production but most likely to differences observed in the growth characteristics of this cultivar.

Microbiological and molecular biological methods for monitoring microbial inoculants and their effects in the soil environment

As the use of biotechnology products, such as genetically modified microorganisms (GMMs), in the environment might bring about undesirable ecological effects, it is important that the environmental fate of inoculant organisms, as well as any effects of their release, are assessed. Ideally, pilot studies in microcosms or small mesocosms are thus performed prior to a larger-scale (commercial) application, and the results of these studies serve to guide further environmental use of the GMM. In these pilot studies, the methods employed to assess the environmental fate and effects of the GMM will have to be fine-tuned and optimized so as to assess these phenomena in an optimal way. This review examines the methods that are currently available for the assessment of the environmental fate of genetically modified and unmodified microorganisms, as well as the impact following their release. The emphasis will be on monitoring of these phenomena in soil, as a paradigm of assessments in complex environmental matrices. Detection methods based on cultivation, which generally rely on the use of added or intrinsic markers, serve to assess the fate of the culturable fractions of released microorganisms. The specificity of this detection can be enhanced by using a combination of cultivation-based and immunology- and/or DNA-based assessments. Furthermore, specific immunofluorescence or in situ hybridization techniques are suitable to quantify populations of GMMs at the level of microscopy-detectable cells, even though fluorescent in situ hybridization is still plagued by sensitivity problems in oligotrophic environments. Detection methods based on nucleic acids (DNA or RNA) extracted from the environment offer the possibility to monitor the fate of the heterologous genes released, including that following a horizontal gene transfer. Moreover, these methods can also provide a picture of the dynamics of the total numbers of microbial cells released. For a sound assessment of the biosafety of environmental releases, a polyphasic approach to environmental monitoring is recommended, as in most analyses information will be needed on the fate of culturable and nonculturable cells, as well as on that of the specific gene sequences released. Finally, the use of impact analysis via an array of different methods is briefly reviewed, and the merits of novel approaches to assessments of community structure via molecular means (PCR/DGGE, ARDRA or T-RFLP) and metabolic profiling via Biolog are discussed as methods specifically aimed at the detection of shifts in community structure and function (metabolic complement analysis).