In Situ Localization of Azospirillum brasilense in the Rhizosphere of Wheat with Fluorescently Labeled, rRNA-Targeted Oligonucleotide Probes and Scanning Confocal Laser Microscopy

The colonization of wheat roots by Azospirillum brasilense was used as a model system to evaluate the utility of whole-cell hybridization with fluorescently labeled, rRNA-targeted oligonucleotide probes for the in situ monitoring of rhizosphere microbial communities. Root samples of agar- or soil-grown 10- and 30-day-old wheat seedlings inoculated with different strains of A. brasilense were hybridized with a species-specific probe for A. brasilense, a probe hybridizing to alpha subclass proteobacteria, and a probe specific for the domain Bacteria to identify and localize the target bacteria. After hybridization, about 10 to 25% of the rhizosphere bacteria as visualized with 4(prm1),6-diamidino-2-phenylindole (DAPI) gave sufficient fluorescence signals to be detected with rRNA-targeted probes. Scanning confocal laser microscopy was used to overcome disturbing effects arising from autofluorescence of the object or narrow depth of focus in thick specimens. This technique also allowed high-resolution analysis of the spatial distribution of bacteria in the rhizosphere. Occurrence of cells of A. brasilense Sp7 and Wa3 was restricted to the rhizosphere soil, mainly to the root hair zone. C-forms of A. brasilense were demonstrated to be physiologically active forms in the rhizosphere. Strain Sp245 also was found repeatedly at high density in the interior of root hair cells. In general, the combination of fluorescently labeled oligonucleotide probes and scanning confocal laser microscopy provided a very suitable strategy for detailed studies of rhizosphere microbial ecology.

[1]  Y. Okon,et al.  Agronomic applications of azospirillum: An evaluation of 20 years worldwide field inoculation , 1994 .

[2]  R. Amann,et al.  In situ analysis of microbial consortia in activated sludge using fluorescently labelled, rRNA‐targeted oligonucleotide probes and confocal scanning laser microscopy , 1994, Journal of microscopy.

[3]  K. Schleifer,et al.  Identification and in situ Detection of Gram-negative Filamentous Bacteria in Activated Sludge , 1994 .

[4]  M. Van Montagu,et al.  Root colonization and systemic spreading of Azoarcus sp. strain BH72 in grasses , 1994, Journal of bacteriology.

[5]  C. Cavanaugh,et al.  Independent phylogenetic origins of methanotrophic and chemoautotrophic bacterial endosymbioses in marine bivalves , 1994, Journal of bacteriology.

[6]  S. J. Caldwell,et al.  Multicellular Organization in a Degradative Biofilm Community , 1994, Applied and environmental microbiology.

[7]  E. Delong,et al.  Design and application of rRNA-targeted oligonucleotide probes for the dissimilatory iron- and manganese-reducing bacterium Shewanella putrefaciens , 1993, Applied and environmental microbiology.

[8]  B. Jørgensen,et al.  Distribution of sulfate-reducing bacteria, O2, and H2S in photosynthetic biofilms determined by oligonucleotide probes and microelectrodes , 1993, Applied and environmental microbiology.

[9]  K. Schleifer,et al.  Dominating Role of an Unusual Magnetotactic Bacterium in the Microaerobic Zone of a Freshwater Sediment , 1993, Applied and environmental microbiology.

[10]  Anton Hartmann,et al.  Direct identification, and localization of Azospirillum in the rhizosphere of wheat using fluorescence‐labelled monoclonal antibodies and confocal scanning laser microscopy , 1993 .

[11]  Dittmar Hahn,et al.  Whole-Cell Hybridization of Frankia Strains with Fluorescence- or Digoxigenin-Labeled, 16S rRNA-Targeted Oligonucleotide Probes , 1993, Applied and environmental microbiology.

[12]  K. Schleifer,et al.  Probing activated sludge with oligonucleotides specific for proteobacteria: inadequacy of culture-dependent methods for describing microbial community structure , 1993, Applied and environmental microbiology.

[13]  E. Delong,et al.  Application of rRNA-based probes for observing marine nanoplanktonic protists , 1993, Applied and environmental microbiology.

[14]  D A Stahl,et al.  Use of rRNA fluorescence in situ hybridization for measuring the activity of single cells in young and established biofilms , 1993, Applied and environmental microbiology.

[15]  K. Schleifer,et al.  Phylogenetic Oligodeoxynucleotide Probes for the Major Subclasses of Proteobacteria: Problems and Solutions , 1992 .

[16]  J. Kloepper,et al.  A review of issues related to measuring colonization of plant roots by bacteria , 1992 .

[17]  R. Amann,et al.  Dual staining of natural bacterioplankton with 4',6-diamidino-2-phenylindole and fluorescent oligonucleotide probes targeting kingdom-level 16S rRNA sequences , 1992, Applied and environmental microbiology.

[18]  K. Schleifer,et al.  Detection of micro-organisms in soil after in situ hybridization with rRNA-targeted, fluorescently labelled oligonucleotides. , 1992, Journal of general microbiology.

[19]  R. Amann,et al.  Molecular and microscopic identification of sulfate-reducing bacteria in multispecies biofilms , 1992, Applied and environmental microbiology.

[20]  J. Costerton,et al.  Optical sectioning of microbial biofilms , 1991, Journal of bacteriology.

[21]  K. Schleifer,et al.  Identification in situ and phylogeny of uncultured bacterial endosymbionts , 1991, Nature.

[22]  Y. Bashan,et al.  Current status of Azospirillum inoculation technology: Azospirillum as a challenge for agriculture , 1990 .

[23]  R. Amann,et al.  Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations , 1990, Applied and environmental microbiology.

[24]  D. M. Ward,et al.  16S rRNA sequences reveal numerous uncultured microorganisms in a natural community , 1990, Nature.

[25]  V. Torsvik,et al.  High diversity in DNA of soil bacteria , 1990, Applied and environmental microbiology.

[26]  D A Stahl,et al.  Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology , 1990, Journal of bacteriology.

[27]  E. Delong,et al.  Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells. , 1989, Science.

[28]  C. You,et al.  Non-nodular endorhizospheric nitrogen fixation in wetland rice , 1989 .

[29]  M. Fordham,et al.  An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy , 1987, The Journal of cell biology.

[30]  T. Hurek,et al.  Cross-Reaction of Predominant Nitrogen-Fixing Bacteria with Enveloped, Round Bodies in the Root Interior of Kallar Grass , 1987, Applied and environmental microbiology.

[31]  C. Neyra,et al.  Flocculation in Azospirillum brasilense and Azospirillum lipoferum: exopolysaccharides and cyst formation , 1985, Journal of bacteriology.

[32]  J. Döbereiner,et al.  Sites and processes of association between diazotrophs and grasses , 1983 .

[33]  Mahavir Singh,et al.  Isolation and characterization of Azospirillum mutants excreting high amounts of indoleacetic acid , 1983 .

[34]  G. D. Johnson,et al.  A simple method of reducing the fading of immunofluorescence during microscopy. , 1981, Journal of immunological methods.

[35]  K. Porter,et al.  The use of DAPI for identifying and counting aquatic microflora1 , 1980 .

[36]  J. Döbereiner,et al.  Light microscopy observations of tetrazolium-reducing bacteria in the endorhizosphere of maize and other grasses in Brazil. , 1978, Canadian journal of microbiology.

[37]  O. Maaløe,et al.  Dependency on medium and temperature of cell size and chemical composition during balanced grown of Salmonella typhimurium. , 1958, Journal of general microbiology.

[38]  M. Lebuhn,et al.  Indolic and Phenolic Compounds , 1996 .

[39]  J. Baak,et al.  Confocal laser microscopy and image processing for three-dimensional microscopy: technical principles and an application to breast cancer. , 1994, Human pathology.

[40]  G. Bowen,et al.  Improving plant productivity with rhizosphere bacteria , 1994 .

[41]  D. Kluepfel.  The Behavior and Tracking of Bacteria in the Rhizosphere , 1993 .

[42]  John R. Lawrence,et al.  Confocal Laser Microscopy and Digital Image Analysis in Microbial Ecology , 1992 .

[43]  A. Hartmann,et al.  Development of gene-probes for Azospirillum based on 23S-rRNA sequences , 1992 .

[44]  J. Döbereiner.  History and new perspectives of diazotrophs in association with non-leguminous plants , 1992 .

[45]  J. Ueckert,et al.  Nitrate Reductase Activity of Azospirillum Brasilense SP7 and SP245 V — and C — Forms in Continuous Culture. , 1991 .

[46]  J. Lynch,et al.  Anatomy and community structure of the rhizosphere. , 1990 .

[47]  D. Shotton,et al.  Pe(ltu((($;: Emerging Techniques Confocal scanning" microscopy: three-dimensional biological imaging , 1989 .

[48]  J. Kloepper.  Plant Growth-Promoting Rhizobacteria and Plant Growth Under Gnotobiotic Conditions , 1981 .

[49]  E. L. Schmidt,et al.  The Immunofluorescence Approach in Microbial Ecology , 1980 .

[50]  J. Döbereiner,et al.  A taxonomic study of the Spirillum lipoferum group, with descriptions of a new genus, Azospirillum gen. nov. and two species, Azospirillum lipoferum (Beijerinck) comb. nov. and Azospirillum brasilense sp. nov. , 1978, Canadian journal of microbiology.