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An experimental test of the effect of management strategies and rotation on plant-pathogen suppression by soil microbial communities
Postma, J. ; Pinochet, X. ; Smalla, K. ; Heuer, H. ; Lumini, E. ; Bianciotto, V. ; Schilder, M.T. ; Termorshuizen, A.J. - \ 2015
In: IPM Innovation in Europe, Poznan, Poland, 14-16 January 2015. - Poznan, Poland : Institute of Plant Protection - p. 67 - 67.
Structural and functional response of the soil bacterial community to application of manure from difloxacin-treated pigs
Jechalke, S. ; Focks, A. ; Rosendahl, I. ; Groeneweg, J. ; Siemens, J. ; Heuer, H. ; Smalla, K. - \ 2014
FEMS Microbiology Ecology 87 (2014)1. - ISSN 0168-6496 - p. 78 - 88.
mediated quinolone resistance - antibiotic-resistance - sulfadiazine - genes - plasmid - fate - rhizosphere - evolution - integrons - residues
Difloxacin (DIF) belongs to the class of fluoroquinolone antibiotics that have been intensively used for the treatment of bacterial infections in veterinary and human medicine. The aim of this field study was to compare the effect of manure from DIF-treated pigs and untreated pigs on the bacterial community structure and resistance gene abundance in bulk soil and rhizosphere of maize. A significant effect of DIF manure on the bacterial community composition in bulk soil was revealed by denaturing gradient gel electrophoresis (DGGE) of bacterial 16S rRNA gene fragments amplified from total community DNA. In few samples, quinolone resistance genes qnrB and qnrS1/qnrS2 were detected by PCR and subsequent hybridization, while qnrA was not detected. Quantitative PCR revealed an increased abundance of the integrase gene intI1 of class I integrons and sulfonamide resistance genes sul1 and sul2 in DIF manure-treated bulk soil and rhizosphere, relative to 16S rRNA genes, while traN genes specific for LowGC-type plasmids were increased only in bulk soil. Principal component analysis of DGGE profiles suggested a manure effect in soil until day 28, but samples of days 71 and 140 were found close to untreated soil, indicating resilience of soil community compositions from disturbances by manure.
Cultivation-Independent Screening Revealed Hot Spots of IncP-1, IncP-7 and IncP-9 Plasmid Occurrence in Different Environmental Habitats
Dealtry, S. ; Ding, G.C. ; Weichelt, V. ; Dunon, V. ; Schluter, A. ; Martini, M.C. ; Papa, M.F. Del; Lagares, A. ; Amos, G.C.A. ; Wellington, E.M.H. ; Gaze, W.H. ; Sipkema, D. ; Sjoling, S. ; Springael, D. ; Heuer, H. ; Elsas, J.D. ; Thomas, C. ; Smalla, K. - \ 2014
PLoS One 9 (2014)2. - ISSN 1932-6203
resistance genes - naphthalene - pseudomonas - adaptation - prevalence - transposon - diversity - sediment - biobeds
IncP-1, IncP-7 and IncP-9 plasmids often carry genes encoding enzymes involved in the degradation of man-made and natural contaminants, thus contributing to bacterial survival in polluted environments. However, the lack of suitable molecular tools often limits the detection of these plasmids in the environment. In this study, PCR followed by Southern blot hybridization detected the presence of plasmid-specific sequences in total community (TC-) DNA or fosmid DNA from samples originating from different environments and geographic regions. A novel primer system targeting IncP-9 plasmids was developed and applied along with established primers for IncP-1 and IncP-7. Screening TC- DNA from biopurification systems (BPS) which are used on farms for the purification of pesticide-contaminated water revealed high abundances of IncP-1 plasmids belonging to different subgroups as well as IncP-7 and IncP-9. The novel IncP-9 primer-system targeting the rep gene of nine IncP-9 subgroups allowed the detection of a high diversity of IncP-9 plasmid specific sequences in environments with different sources of pollution. Thus polluted sites are "hot spots'' of plasmids potentially carrying catabolic genes.
|Effect of management strategies and rotation on plant-pathogen suppression by soil microbial communities
Postma, J. ; Pinochet, X. ; Landé, Nathalie ; Smalla, K. ; Heuer, H. ; Lumini, E. ; Bianciotto, V. ; Termorshuizen, A.J. ; Schilder, M.T. ; Begg, G. - \ 2013
In: Book of Abstracts Conference Future IPM in Europe, Rive del Garda, Italy, 19-21 March 2013. - s.n. - p. 85 - 85.
Enhancement of disease suppressive properties of soils limits disease development and is, as a result, essential for sustainable agriculture. Moreover, it can be a profitable strategy for farmers to manage diseases with lowered levels or without pesticides. Agricultural soils differ in their suppressiveness towards soil-borne plant pathogens, which can be attributed to soil type, organic matter content, as well as by management practices such as crop rotation, tillage and fertilization. The objective of the current research is to devise management strategies supporting disease suppression based on the response of pathogen-suppressing soil microbial communities. A field trial is being conducted to test experimentally the effect of crop rotation and management treatments on crop health and yield, as well as the suppressive response of the soil and its physical, chemical and biological properties. An experimental site with an on-station field trial with a winter-wheat rotation in the North of France has been selected. Soil samples are taken twice in the cropping seasons of winter wheat in 2012 and 2013. Soil suppressiveness is tested in bioassays with three different soil-borne pathogens in winter wheat under controlled environmental conditions. Biological targets which are assumed to provide complementary information on soil quality and diseases suppressive capacity of soil have been selected: 1) fungal and bacterial diversity and community structure, being important quality parameters of soil life; 2) arbuscular mycorrhizal fungi (AMF), obligatory mutualistic symbionts that supply plants with inorganic nutrients and protect them against diverse abiotic and biotic stresses; 3) Lysobacter spp., a recently described antagonist that correlates with soil suppressiveness in Dutch soils; 4) community structure of nematodes, expressed as maturity index, proposed as indicator for soil quality. A preliminary consideration of the results so far shows a variable response to crop sequence and management for bacterial and fungal, AMF, and nematode communities. Some limited changes in disease suppression were detected. Lysobacter spp. is present in the selected field, but is not influenced by the soil treatments. Correlation between the treatments and the different measurements will be performed when all samples have been analysed (i.e. mid 2013
Effect of management strategies and rotation on plant-pathogen suppression by soil microbial communities
Postma, J. ; Pinochet, X. ; Landé, Nathalie ; Smalla, K. ; Heuer, H. ; Lumini, E. ; Bianciotto, V. ; Termorshuizen, A.J. ; Schilder, M.T. ; Begg, G. - \ 2013
|Soil metagenomics to identify novel mechanisms of antagonism and antifungal activity for the improved control of phytopathogens
Overbeek, L.S. van; Smalla, K. ; Bailey, M. ; Jansson, J. ; Sjöling, S. ; Vogel, T. ; Nalin, R. ; Elsas, J.D. van - \ 2008
Wageningen : Plant Research International
Objective: This project will explore & exploit the anti-phytopathogen diversity present in selected European suppressive soils representing a wide geographical spread. The untapped functional potential present in the unculturable part of the microbiota of these soils will be accessed using metagenomic cloning of soil DNA. Moreover, as a baseline, antagonistic organisms will also be isolated. Libraries constructed on the basis of the total and the active cell fractions, as well as the isolates, will be screened for anti-phytopathogen function. Selected clones will be as said with respect to the nature of the antagonistic compounds and production loci, involving sequencing. Potential producer strains will be optimised as to gene expression, growth and stability. Based on the gene sequences obtained, molecular tools will be developed and used to monitor soil suppressiveness. The impact (biosafety) of potential applications will be assessed.
Screening of bacterial isolates from various European soils for in vitro antagonistic activity towards Rhizoctonia solani and Fusarium oxysporum: Site-dependent composition and diversity revealed
Adesina, M.F. ; Lembke, A. ; Costa, R. ; Speksnijder, A.G.C.L. ; Smalla, K. - \ 2007
Soil Biology and Biochemistry 39 (2007)11. - ISSN 0038-0717 - p. 2818 - 2828.
microbial community structure - biological-control - suppressive soils - plant-pathogens - pseudomonas - wilt - microorganisms - rhizosphere - mechanisms - diseases
A cultivation-based approach was used to determine the in vitro antagonistic potential of soil bacteria towards Rhizoctonia solani AG3 and Fusarium oxysporum f. sp. lini (Foln3). Four composite soil samples were collected from four agricultural sites with previous documentation of disease suppression, located in France (FR), the Netherlands (NL), Sweden (SE) and the United Kingdom (UK). Similarly, two sites from Germany (Berlin, G-BR; and Braunschweig, G-BS) without documentation of disease suppression were sampled. Total bacterial counts were determined by plating serial dilutions from the composite soil samples onto R2A, AGS and King's B media. A total of 1,788 isolates (approximately 100 isolates per medium and site) was screened for antifungal activity, and in vitro antagonists (327 isolates) were found amongst the dominant culturable bacteria isolated from all six soils. The overall proportion of antagonists and the number of isolates with inhibitory activity against F. oxysporum were highest in three of the suppressive soils (FR, NL and SE). Characterization of antagonistic bacteria revealed a high phenotypic and genotypic diversity. Siderophore and protease activity were the most prominent phenotypic traits amongst the antagonists. The composition and diversity of antagonists in each soil was site-specific. Nevertheless, none of the antimicrobial traits of bacteria potentially contributing to soil suppressiveness analyzed in this study could be regarded as specific to a given site.
Community structure of actively growing bacterial populations in plant pathogen suppressive soil
Hjort, K. ; Lembke, A. ; Speksnijder, A.G.C.L. ; Smalla, K. ; Jansson, J.K. - \ 2007
Microbial Ecology 53 (2007)3. - ISSN 0095-3628 - p. 399 - 413.
gradient gel-electrophoresis - 16s ribosomal-rna - bromodeoxyuridine immunocapture - plasmodiophora-brassicae - molecular-cloning - microbial ecology - chitinase gene - fungi - identification - amplification
The bacterial community in soil was screened by using various molecular approaches for bacterial populations that were activated upon addition of different supplements. Plasmodiophora brassicae spores, chitin, sodium acetate, and cabbage plants were added to activate specific bacterial populations as an aid in screening for novel antagonists to plant pathogens. DNA from growing bacteria was specifically extracted from the soil by bromodeoxyuridine immunocapture. The captured DNA was fingerprinted by terminal restriction fragment length polymorphism (T-RFLP). The composition of the dominant bacterial community was also analyzed directly by T-RFLP and by denaturing gradient gel electrophoresis (DGGE). After chitin addition to the soil, some bacterial populations increased dramatically and became dominant both in the total and in the actively growing community. Some of the emerging bands on DGGE gels from chitin-amended soil were sequenced and found to be similar to known chitin-degrading genera such as Oerskovia, Kitasatospora, and Streptomyces species. Some of these sequences could be matched to specific terminal restriction fragments on the T-RFLP output. After addition of Plasmodiophora spores, an increase in specific Pseudomonads could be observed with Pseudomonas-specific primers for DGGE. These results demonstrate the utility of microbiomics, or a combination of molecular approaches, for investigating the composition of complex microbial communities in soil
|Impact of three selected biotechnological strategies for potato pathogen control on the indigenous soil microbiot
Overbeek, L.S. van; Smalla, K. ; Trigalet, A. ; Lopez, M.M. ; Jansson, M. ; Sessitsch, A. ; Elsas, J.D. van - \ 2006
Wageningen : Plant Research International
In this project, we investigate possibilities to control plant diseases caused by bacteria according to three different approaches, i.e. genetic modification of plants (two approaches) and application of avirulent competitive bacterial strains. For that purpose we selected Ralstonia solanacearum and Erwinia carotovora as model organisms for controlling diseases, caused by both agents, in genetically modified potato lines and constructed avirulent mutants of R. solanacearum for suppression of brownrot in unmodified potato lines. Genetically modified potato lines are obtained from MPB Cologne (Germany) and from the Austrian Research Centre , Seibersdorf and these lines possesses insertions of genes encoding for bacterial lytic proteins. The lines obtained from MPB Cologne, denoted DL11, DL 12 and DL13 containT4 Lysozym gene from bacterial fage T4, whereas lines mas2C4-mas1 C4 and mas2C4-mas1 Att posses genes cecropin and both cecropin and attacin, respectively, from the giant silk moth Hyalophora cecropia. Further, mutants of R. solanacearum corrupted in expression of virulence genes via transposon insertion into the hrpB gene are used to control brownrot disease in potato. Using transgenic potato lines and non-pathogenic R. solanacearum strains, greenhouse and field studies are performed to investigate the efficacy of both approaches. Next to efficacy screening, the impact of heterologous gene expression and the presence of non-pathogenic R. solanacearum mutants on the microbial phytosphere community of potato will be investigated.
PCR detection of oxytetracycline resistance genes from diverse habitats in total community DNA and in streptomycete isolates.
Nikolakopoulou, T.L. ; Egan, S. ; Overbeek, L.S. van; Guillaume, G. ; Heuer, H. ; Wellington, E.M.H. ; Elsas, J.D. van; Collard, J.M. ; Smalla, K. ; Karagouni, A.D. - \ 2005
Current Microbiology 51 (2005)4. - ISSN 0343-8651 - p. 211 - 216.
environmental bacteria - efflux protein - mycobacterium - prevalence - validation - plasmids - clusters - rimosus - primers - soil
A range of European habitats was screened by PCR for detection of the oxytetracycline resistance genes otr(A) and otr(B), found in the oxytetracycline-producing strain Streptomyces rimosus. Primers were developed to detect these otr genes in tetracycline-resistant (TcR) streptomycete isolates from environmental samples. Samples were obtained from bulk and rhizosphere soil, manure, activated sludge and seawater. The majority of TcR streptomycetes originated from bulk and rhizosphere soil. Fewer TcR streptomycetes were isolated from manure and seawater and none from sewage. By PCR, three out of 217 isolates were shown to contain the otr(A) gene and 13 out of 217 the otr(B) gene. Surprisingly, these genes were detected in taxonomic groups not known as tetracycline-producing strains. The majority of the otr gene¿carrying strains was assigned to S. exfoliatus or S. rochei and originated from all habitats from which TcR streptomycetes were obtained. Our results indicated that the occurrence of otr(A) and otr(B) genes in natural environments was limited and that otr(B), in comparison to otr(A), seemed to be more common
|Fate of Ralstonia solanacearum biovar 2 as affected by conditions and soil treatments in temperate climate zones
Elsas, J.D. van; Overbeek, L.S. van; Bailey, M.J. ; Schönfeld, J. ; Smalla, K. - \ 2005
In: Bacterial wilt disease and the Ralstonia solanacearum species complex / Allen, C., Prior, P., Hayward, A.C., St. Paul, USA : APS Press - ISBN 0890543291 - p. 39 - 49.
Specific and sensitive detection of Ralstonia solanacearum in soil on the basis of PCR amplification of fliC fragments
Schonfeld, J. ; Heuer, H. ; Elsas, J.D. van; Smalla, K. - \ 2003
Applied and Environmental Microbiology 69 (2003)12. - ISSN 0099-2240 - p. 7248 - 7256.
polymerase-chain-reaction - 16s ribosomal-rna - blood-disease bacterium - pseudomonas-solanacearum - flagellin gene - escherichia-coli - sequence - virulence - identification - diversity
Ralstonia solanacearum is the causative agent of bacterial wilt in many important crops. A specific and sensitive PCR detection method that uses primers targeting the gene coding for the flagella subunit, fliC, was established. Based on the first fliC gene sequence of R. solanacearum strain K60 available at GenBank, the Ral_fliC PCR primer system was designed; this system yielded a single 724-bp product with the DNAs of all of the R. solanacearum strains tested. However, R. pickettii and four environmental Ralstonia isolates also yielded amplicons. The Ral_fliC PCR products obtained with 12 strains (R. solanacearum, R. pickettii, and environmental isolates) were sequenced. By sequence alignment, Rsol_fliC primers specific for R. solanacearum were designed. With this primer system, a specific 400-bp PCR product was obtained from all 82 strains of R. solanacearum tested. Six strains of R. pickettii and several closely related environmental isolates yielded no PCR product; however, a product was obtained with one Pseudomonas syzygii strain. A GC-clamped 400-bp fliC product could be separated in denaturing gradient gels and allowed us to distinguish P. syzygii from R. solanacearum. The Rsol_fliC PCR system was applied to detect R. solanacearum in soil. PCR amplification, followed by Southern blot hybridization, allowed us to detect about one target DNA molecule per PCR, which is equivalent to 103 CFU g of bulk soil-1. The system was applied to survey soils from different geographic origins for the presence of R. solanacearum.
|Monitoring the identity and survival of genetically modified or non-modified plant growth promoting bacteria and their impact on soil microbial communities
Smalla, K. ; Schönwalder, A. ; Wei, L. ; Min, L. ; Elsas, J.D. van - \ 2002
In: Proceedings Biosafety Symposium, Beijing, Oct. 2002. - [S.l.] : [s.n.], 2002
plant growth promoting bacteria
The complete nucleotide sequence and environmental distribution of the cryptic, conjugative, broad-host-range plasmid pIPO2 islated from bacteria of the wheat rhizosphere
Tauch, A. ; Schneiker, S. ; Selbitschka, W. ; PÜhler, A. ; Overbeek, L.S. van; Smalla, K. ; Thomas, C.M. ; Bailey, M.J. ; Forney, L.J. ; Weightman, A. ; Ceglowski, P. ; Pembroke, T. ; Tietze, E. ; Schröder, G. ; Lanka, E. ; Elsas, J.D. van - \ 2002
Microbiology 148 (2002). - ISSN 1350-0872 - p. 1637 - 1653.
The flood of sequence data resulting from the large number of current genome projects has increased the need for a flexible, open source genome annotation system, which so far has not existed. To account for the individual needs of different projects, such a system should be modular and easily extensible. We present a genome annotation system for prokaryote genomes, which is well tested and readily adaptable to different tasks. The modular system was developed using an object-oriented approach, and it relies on a relational database backend. Using a well defined application programmers interface (API), the system can be linked easily to other systems. GenDB supports manual as well as automatic annotation strategies. The software currently is in use in more than a dozen microbial genome annotation projects. In addition to its use as a production genome annotation system, it can be employed as a flexible framework for the large-scale evaluation of different annotation strategies. The system is open source
|Occurrence and reservoirs of antibiotic resistance genes in the environment
Seveno, N. ; Kallifidas, D. ; Smalla, K. ; Elsas, J.D. van; Collard, J.M. ; Karagouni, A. ; Wellington, E.M.H. - \ 2002
Reviews in medical microbiology 13 (2002). - ISSN 0954-139X - p. 15 - 27.
antibiotic resistance genes
Antibiotic resistance genes have become highly mobile since the development of antibiotic chemotherapy. A considerable body of evidence exists proving the link between antibiotic use and the significant increase in drug-resistant human bacterial pathogens. The application of molecular detection and tracking techniques in microbial ecological studies has allowed the reservoirs of antibiotic resistance genes to be investigated. It is clear that the transfer of resistance genes has occurred on a global scale and in all natural environments. The considerable diversity of bacteria and mobile elements in soils has meant that the spread of resistance genes has occurred by all currently known mechanisms for bacterial gene transfer. Trans-kingdom transfers from plants to bacteria may occur in soil. Hot spots for gene transfer in the soil/plant environment have been described and colonized niches such as the rhizosphere and other nutrient-enriched sites, for example manured soil, have been identified as reservoirs of resistance genes. Although exposure and selection for tolerance of antibiotics is considerable in clinical environments there is increasing evidence that selection for resistant phenotypes is occurring in natural environments. Antibiotic-producing bacteria are abundant in soil and there is evidence that they are actively producing antibiotics in nutrient-enriched environments in soil. In addition there is clear evidence that the self-resistance genes found within antibiotic gene clusters of the producers have transferred to other non-producing bacteria. Perhaps most important of all is the use of antibiotics in agriculture as growth promotants and for treatment of disease in intensively reared farm animals. These treatments have resulted in gut commensal and pathogenic bacteria acquiring resistance genes under selection and then, due to the way in which farm slurries are disposed of, the spread of these genes to the soil bacterial community. Integrons with multiple resistance gene cassettes have been selected and disseminated in this way; many of these cassettes carry other genes such as those conferring heavy metal and disinfectant resistance which have been co-selected in bacteria surviving in effluents and contaminated soils, further maintaining and spreading the antibiotic resistance genes.
Gentamicin resistance genes in environmental bacteria: prevalence and transfer
Heuer, H. ; Krögerrecklenfort, E. ; Wellington, E.M.H. ; Egan, S. ; Elsas, J.D. van; Overbeek, L.S. van; Collard, J.M. ; Guillaume, G. ; Karagouni, A. ; Nikolakopoulou, D. ; Smalla, K. - \ 2002
FEMS Microbiology Ecology 42 (2002). - ISSN 0168-6496 - p. 289 - 302.
A comprehensive multiphasic survey of the prevalence and transfer of gentamicin resistance (Gmr) genes in different non-clinical environments has been performed. We were interested to find out whether Gmr genes described from clinical isolates can be detected in different environmental habitats and whether hot spots can be identified. Furthermore, this study aimed to evaluate the impact of selective pressure on the abundance and mobility of resistance genes. The study included samples from soils, rhizospheres, piggery manure, faeces from cattle, laying and broiler chickens, municipal and hospital sewage water, and coastal water. Six clusters of genes coding for Gm-modifying enzymes (aac(3)-I, aac(3)-II/VI, aac(3)-III/IV, aac(6')-II/Ib, ant(2'')-I, aph(2'')-I) were identified based on a database comparison and primer systems for each gene cluster were developed. Gm-resistant bacteria isolated from the different environments had a different taxonomic composition. In only 34 of 207 isolates, mainly originating from sewage, faeces and coastal water polluted with wastewater, were known Gmr genes corresponding to five of the six clusters detected. The strains belonged to genera in which the genes had previously been detected (Enterobacteriaceae, Pseudomonas, Acinetobacter) but also to phylogenetically distant bacteria, such as members of the CFB group, - and -Proteobacteria. Gmr genes located on mobile genetic elements (MGE) could be captured in exogenous isolations into recipients belonging to -, - and -Proteobacteria from all environments except for soil. A high proportion of the MGE, conferring Gm resistance isolated from sewage, were identified as IncP plasmids. Molecular detection of Gmr genes, and broad host range plasmid-specific sequences (IncP-1, IncN, IncW and IncQ) in environmental DNA indicated a habitat-specific dissemination. A high abundance and diversity of Gmr genes could be shown for samples from faeces (broilers, layers, cattle), from sewage, from seawater, collected close to a wastewater outflow, and from piggery manure. In the latter samples all six clusters of Gmr genes could be detected. The different kinds of selective pressure studied here seemed to enhance the abundance of MGE, while an effect on Gmr genes was not obvious.
Prevalence of streptomycin resistance genes in bacterial populations in European habitats
Overbeek, L.S. van; Wellington, E.M.H. ; Karagouni, A. ; Smalla, K. ; Collard, J.M. ; Elsas, J.D. van - \ 2002
FEMS Microbiology Ecology 42 (2002). - ISSN 0168-6496 - p. 277 - 288.
The prevalence of selected streptomycin (Sm)-resistance genes, i.e. aph (3''), aph (6)-1d, aph (6)-1c, ant (3'') and ant (6), was assessed in a range of pristine as well as polluted European habitats. These habitats included bulk and rhizosphere soils, manure from farm animals, activated sludge from wastewater treatment plants and seawater. The methods employed included assessments of the prevalence of the genes in habitat-extracted DNA by PCR, followed by hybridisation with specific probes, Sm-resistant culturable bacteria and exogenous isolation of plasmids carrying Sm-resistance determinants. The direct DNA-based analysis showed that aph (6)-1d genes were most prevalent in the habitats examined. The presence of the other four Sm-modifying genes was demonstrated in 58 f the tested habitats. A small fraction of the bacterial isolates (8€did not possess any of the selected Sm-modifying genes. These isolates were primarily obtained from activated sludge and manure. The presence of Sm-modifying genes in the isolates often coincided with the presence of IncP plasmids. Exogenous isolation demonstrated the presence of plasmids of 40–200 kb in size harbouring Sm-resistance genes from all the environments tested. Most plasmids were shown to carry the ant (3'') gene, often in combination with other Sm-resistance genes, such as aph (3'') and aph (6)-1d. The most commonly found Sm-modifying gene on mobile genetic elements was ant (3''). Multiple Sm-resistance genes on the same genetic elements appeared to be the rule rather than the exception. It is concluded that Sm-resistance genes are widespread in the environmental habitats studied and often occur on mobile genetic elements and ant (3'') was most often encountered
Effect of an Alcaligenes faecalis inoculant strain on bacterial communities in flooded soil microcosms planted with rice seedlings
Lin, M. ; Smalla, K. ; Heuer, H. ; Elsas, J.D. van - \ 2000
Applied Soil Ecology 15 (2000)2. - ISSN 0929-1393 - p. 211 - 225.
The fate and impact of Alcaligenes faecalis strain A1501R, a rifampicin-resistant derivative of a rice inoculant strain, were studied in flooded silt loam microcosms planted with rice seedlings. Selective plating revealed that strain A1501R survived at high, initially stable and later slowly declining population sizes (108-106 CFU per gram of dry soil), for 60 days. Inoculant survival in the rice rhizosphere showed a similar trend, and only at one time point (15 days), the inoculant CFU numbers were significantly higher in the rhizosphere than in corresponding bulk soil. A probe for the detection of strain A1501R in soil, based on the 16S rDNA variable region V6, was obtained via PCR amplification with specific primers. Alignment of the sequence of this V6 region with that of a range of different bacterial species indicated that it provided a tool for the detection of strain A1501R in a soil background. Dot blot hybridization of randomly isolated strains and of soil DNA confirmed the usefulness of the probe. Use of the probe in a dilution dot blot hybridization experiment with soil DNA revealed a dynamics of strain A1501R over time similar to that indicated by the CFU counts. However, the presence of background in soil did not allow the detection of inoculant numbers below an estimated 105-106 cells per gram of soil. At regular times, i.e. 3 h, 15, 30 and 40 days after introduction, bacterial community fingerprints were generated via bacterial 16S rDNA based PCR of soil DNA followed by denaturing gradient gel electrophoresis (DGGE). The introduced strain could be clearly detected by the appearance of a novel, initially strong and progressively weaker, band in the community fingerprints up to roughly 30-40 days of incubation. Indigenous organisms underlying the comigrating bands found after 30-40 days were found to react with the strain A1501R V6 probe. These isolates were identified as Pseudomonas spp. The introduction of strain A1501R did not result in substantial changes in the bacterial community fingerprints. The fingerprints obtained over time in bulk soil were >90-95% similar to each other, and there was no clear trend evidenced via cluster analysis. For the rhizosphere fingerprints, three main clusters (each of >95% similarity), representing the day-0 plus day-15, the day-30, and the day-40 bacterial communities, were found. The communities apparently changed more as a result of rice root growth than due to the presence of A1501R. Community level physiological profiling (CLPP) based on the use of Biolog GN plates was then applied using the soil microbial communities sampled over time. The potential for utilization of substrates of the Biolog system by these microbial communities remained, with a few, varying, exceptions, largely unchanged in spite of the release of strain A1501R. However, significant differences in the utilization of selected substrates were observed between the control and inoculated soils, as evidenced by the application of PCR - DGGE to the bacterial communities inhabiting selected wells of the Biolog plates. In particular, strain A1501R was found to be highly competitive in the presence of lactic acid. (C) 2000 Elsevier Science B.V.
Transformation of Acinetobacter sp. strain BD413 (pFG4deltanptII) with transgenic plant DNA in soil microcosms and effects of kanamycin on selection of transformants
Nielsen, K.M. ; Elsas, J.D. van; Smalla, K. - \ 2000
Applied and Environmental Microbiology 66 (2000). - ISSN 0099-2240 - p. 1237 - 1242.
|Safety issues in antibiotic resistance marker genes in transgenic crops
Nielsen, K.M. ; Elsas, J.D. van; Smalla, K. - \ 2000
In: Proc of the 6th International Feed Product Conference, Piacenza, 27-28/11/2000 / Piva, G., Masoero, F., - p. 146 - 162.