In situ Lokalisierung, PGPR-Effekt und Regulation des ipdC-Gens der Azospirillum brasilense Stämme Sp7 und Sp245 bei verschiedenen Weizensorten, sowie endophytische Kolonisierung durch Herbaspirillum sp. N3

The aim of this PhD thesis was to examine the endophytic
colonization behavior of Azospirillum brasilense and Herbaspirillum
sp. N3 on wheat roots. The application of the FISH method using
species specific phylogenetic oligonucleotide probes and GFP
tagging facilitated the detection of a differential colonization
behavior by the A. brasilense strains Sp7 and Sp245 on three
different wheat varieties (Triticum aestivum). For this purpose a
confocal laser scanning microscope (CLSM) was used, which enabled
three-dimesional analysis of bacterial colonization of the root.
Especially GFP tagged strains were well suited for this
application, as there was no pretreatment or sectioning of the root
sample necessary. Strain Sp7 was only located on the root surface
of all wheat cultivars, whereas strain Sp245 was also found inter-
and intracellulary in the outer root cortex layers. There was no
recognizable connection between the growth stimulating effect of
the inoculum (PGPR-effect) and the localization of the bacteria.
The most pronounced PGPR-effect could be observed with the
Brazilian wheat cultivar, which seemed to gain greatest benefit of
its partnership with A. brasilense due to a certain adaptation to
the inoculum. As the production of the auxin IAA (indole-3-acetic
acid) plays a major role in stimulating plant growth, the
expression of the key gene ipdC (indole-3-pyruvate decarboxylase)
was examined. For this, several methods were tested to generate a
fusion of the ipdC promoter with a gfp or rfp reporter gene.
Constructing a translational promoter fusion with the gfp variant
mut3 on plasmid level made expression analyses possible. With this
method the promoter region of strain Sp7 located directly upstream
of the ipdC start codon was found to differ only in a few bases
from strain Sp245. But further upstream a region of about 150 bases
was identified in strain Sp245, which was missing in strain Sp7.
For Sp245 two different fusions were constructed, which contained
the Sp245 promoter region homologous to strain Sp7 and the whole
promoter region of Sp245, respectively. With these constructs the
importance of the promoter region only present in strain Sp245 for
control and intensity of ipdC expression in A. brasilense Sp245
could be demonstrated. Additionally an induction of the
corresponding ipdC promoter fusions of Sp7 and Sp245 was achieved
when adding phenylalanine or tyrosine. Total promoter activity was
higher in strain Sp7 than in strain Sp245, and ipdC expression
appeared to be subject to a stricter control in strain Sp245. These
results were confirmed, when the strains containing the promoter
fusions were used as reporters for ipdC expression on wheat roots.
A demonstration of the induction of the ipdC promoter by root
exudates in situ was possible. Finally, isolate N3 from surface
sterilized wheat roots was characterized in detail. According to
the 16S rDNA sequence data the isolate was phylogenetically
allocated to the genus Herbaspirillum. But a subsequent DNS-DNS
hybridization ruled out, that the strain belonged to any of the
known Herbaspirillum species. Thus, the isolate, which might
represent a new species, was named Herbaspirillum sp. N3. A
specific, 16S rRNA targeted probe was constructed, which
facilitated the examination of wheat roots colonization by this
bacteria using FISH. Additionally the strain was GFP tagged to
enable the detection in uncut root material. By this, an
unequivocal demonstration of the endophytic colonization by
Herbaspirillum sp. N3 mainly within the intercellular spaces was
possible.