USE OF MOLECULAR METHODS FOR IDENTIFICATION ANDIN SITU STUDIES OF DIAZOTROPHIC PLANT COLONIZING BACTERIA

G. Kirchhoe, B. Eckerrl, V.M. Reis2, V.L.D. Baldani2, B. Aßmus1, A. Hartmann1

1GSF-National Research Center for Environment and Health, Institute of Soil Ecology, D-85758 Neuherberg, Germany. 2Centro Nacional de Pesquisa de Agrobiologfa (EMBRAPA-CNPAB), Km 47, Rio de Janeiro, Brazil.

1 . Introduction

An increasing number of examples describing systernic colonization of grarnineous crops by diazotrophic Azoarcus, Acetobacter, Herbaspirillum, Burkholderia and Azospirillum are reported (Hurek et al, 1994, Reiset al, 1994, Olivareset al, 1996, Kirchhof et al, 1997a). To identify and characterize new isolates and to study their synergistic interaction with its host plants, molecular tools are advantageous. Furthermore, for the exarnination of plant colonizing competence of isolates accurate methods for the re-identification of inoculated bacteria are necessary.

2. Identification

rRNA sequence analysis and probing with rRNA directed oligonucleotides has become a common method for the identification of bacteria at all taxonornic levels (Stackebrandt, Rainey, 1995; Stahl, Amann, 1991). Overmore, fluorescently labeled oligonucleotides are weil established tools for rnicrobial ecology studies (Amann et al, 1995). Therefore, a set of rDNA-sequence derived oligonucleotides specific for diazotrophic bacteria were developed (Kirchhof et al, 1997a) and applied. The phylogenetic position of recently obtained strains isolated from rice, sugarcane or the energy crops Miscanthus sp. and Penniserum purpureum were deterrnined by total 16S rRNA gene sequence analysis. The sugarcane and rice isolates could be classified as two new Burkholderia species. Some isolates of Miscanrhus and Penniserum could be described as a new duster belonging to the genus Herbaspirillum (Kirchhof et al, 1997a, 1997b)

The occurrence of a specific target bacterium could be indicated directly in plant extracts by amplifying a 409 bp stretch of the 23S rDNA with the species-specific oligonucleotide and a more universal primer as flanking startpoints. With this approach Herbaspirillum seropedicae could be found in the aerial plant tissue of sugarcane, maize and Pennisetum grown in the field, while Acetobacter diazotrophicus could be detected in roots and leaves of three Pennisetum cultivars, stem and leaves of sugarcane and in the roots and leaves of sugarcane plants cultivated in the greenhouse (Kirchhof et al, subrnitted).

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3 . Genetic Diversity

A rapid procedure to get inforrnation about genetic diversity ofbacterial isolates is the PCR fingerprinting display, using primers targeting repetitive sequences (Rademak:er, Th Bruijn, 1997). Oligonucleotides directed to sequences derived from eucaryotic LINEs (Long INterspersed Elements) supposedly conserved in all cells were used (Srnida et al, 1996). Comparison of the PCR products obtained from 11 isolates of rice, 4 of sugarcane, 20 of Pennisetum and 26 of Miscanthus, demonstrated sirnilar patterns for the isolates recovered from one plant type. Sirnilar clusters could also be found when comparing patterns of carbon substrate utilization (BIOLOG®). These support the hypothesis, that each plant harbours a diazotrophic bacterial population with restricted variance.

4 Microscopic Localization

In situ localization of the bacteria within the plant tissue was perforrned using confocal Iaser scanning rnicroscopy (SCLM) now provides an ideal tool for investigations of multiple labeled rnicroorganisms in unsectioned specimen (Aßmus et al, 1995). The inspection of the sample is lirnited by the intensity of the ernitting fluorescence light and endophytic bacteria in deep root celllayers could not be detected. To solve this problem, an

amplification of the signal with TSA ®(tyrarnin signal amplification)-labeling (Bauwens et al, 1997) was tested. For this purpose, digoxygenin labeled oligonucleotide probes were

applied and horseradish peroxidase (HRP)-coupled a-DIG antibodies attached. Following, a Tyrarnine-linked fluorochrome was added and this very reactive substrate was oxidized by the HRP. This reaction results in the covalent binding of the fluorochrome to the surrounding arninogroups of the ribosome and intense fluorescence could be achieved.

5 References

Amann RIet al (1995) Microbiol. Rev. 59, 143-169. Aßmus B et al (1995) Appl. Env. Microbiol. 61, 1013-1019. Bauwens S et al (1997) Plant Mol. Biol. Rep., in press. Hurek T et al .(1994) J. Bac. 176, 1913-1923. Kirchhof G et al (1997a) Soil Biol. Biochem. 29, 853-862. Kirchhof G et al (1997b) Plant and Soil, in press. OlivaresFLet al (1996) Biol. Fert. Soils 21, 197-200. Rademak:er JLW, De Bruijn FJ (1997) In G Caetano-Anolles, PM Gresshoff, DNA markers: Protocols, Applications and Overviews, J.Wiley & Sons, Inc., in press. Reis VM et al (1994) World J. Microbiol. Biotech. 10, 401-405. Srnida J et al (1996) Gen. Anal. (Biomolecular Engineering) 13, 95-98. Stackbrandt E, Rainey FA (1995) In Akkerrnans, A.D.L., V an Elsas, J.D. and De Bruijn, F.J., Molecular Microbial Ecology Manual, Kluwer Acadernic Publischers, Dordrecht, Stahl DA, Amann RI (1991) In Stackebrandt E, Goodfellow M: Nucleic acid techniques in bacterial systematics, John Wiley & Sons Ltd., Chichester, England. 205-248.