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Li, Dan (2011): Phenotypic variation and molecular signaling in the interaction of the rhizosphere bacteria Acidovorax sp. N35 and Rhizobium radiobacter F4 with roots. Dissertation, LMU München: Faculty of Biology
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Abstract

The aim of this doctoral thesis was to investigate the factors relevant in plant interaction of two plant growth promoting rhizobacteria (PGPR). For this, the strain Acidovorax sp. N35 isolated from surface sterilized wheat roots and the two strains F4 and F7 of Rhizobium radiobacter, a bacterium associated with the plant growth promoting fungus Piriformospora indica, were chosen. First of all, the isolate N35 was characterized using phylogenetic and taxonomic methods. The 16S rRNA gene sequence analysis showed that strain N35 has the closest sequence similarities (98.2, 98.5 and 99.0 %) to the environmental Acidovorax species A. delafieldii, A. facilis and A. defluvii. The DNA-DNA hybridization values clearly separated the isolate from these three species. Additionally, phenotypic properties, such as substrate metabolization profiles as determined by a Biolog GN2 assay and cell wall fatty acid profiles concerning the fatty acids C16:0, C16:1ω7cis/trans, C17:0cyclo and C18:0cyclo and C19:0cyclo, facilitated the differentiation of the newly isolated strain N35 from its closest relatives. Thus, the strain N35 was classified as representative of a new species within the genus Acidovorax, and the name Acidovorax radicis sp. nov. is suggested. “Cand. A. radicis” N35 undergoes an irreversible phenotypic variation, resulting in different colony shapes on agar plate. In soil system, both phenotypes showed a plant growth promoting effect both on barley roots and shoots. The wild type N35 (rough colony type) had a better plant growth promoting effect on barley in comparison with phenotype variant N35v (smooth colony type). Wild type and phenotype variant cells of “cand. A. radicis” N35 were labeled with GFP and/or YFP and their separate and co-colonization behavior was investigated in a monoxenic system and a soil system using a CLSM for detection. Both types of N35 could endophytically colonize barley roots after 12 weeks inoculation in the soil system. Competitive root colonization behavior was observed after co-inoculation with differentially labeled wild type N35 and phenotype variant N35v bacteria, where the wild type showed dominant colonization of barley roots compared to the phenotype variant. Moreover, the variant N35v lost its motility due to missing flagella and swarming ability. The differences of both types at genetic level were investigated using whole genome sequence data obtained from 454 pyrosequencing (Roche) using the GS FLX Titanium chemistry. As only difference in the genome sequence, a 16 nucleotides deletion was identified in the mutL gene, which encodes for the mismatch repair protein MutL. In phenotype N35v, the frame shift caused by this deletion leads to the formation of a stop codon in the coding gene, resulting in a truncated MutL protein with a missing functional MutL C-terminal domain. This mutation occurred in exactly the same way in all investigated phenotype variants. These results suggest that MutL might be directly or indirectly responsible for the phenotypic variation in “cand. A. radicis” N35. Quorum sensing signaling molecules produced by “cand. A. radicis” N35 were identified using biosensors as well as Fourier transform ion cyclotron resonance - mass spectrometry (FT-ICR-MS) and ultra performance liquid chromatography (UPLC). Both types of “cand. A. radicis” N35 possess the same AraI/AraR quorum sensing system, which belongs to the LuxI/LuxR type. The two N35 phenotypes produced nearly the same amount of 3-OH-C10-HSL in the exponential growth phase. A co-inoculation experiment of AHL producing wild type N35 and a constructed AHL negative mutant N35 ΔaraI showed that wild type N35 had a dominant colonization behavior compared to the AHL negative mutant on barley roots in a monoxenic system. These data indicate that quorum sensing is involved in regulation of root colonization by “cand. A. radicis” N35. The second examined PGPR, R. radiobacter, which occurs naturally as endofungal bacterium in the plant growth promoting fungus P. indica, was demonstrated to colonize the surface of barley roots with fluorescence in situ hybridization (FISH) in a monoxenic system. The interaction of P. indica harboring R. radiobacter with other rhizobacteria was investigated using plate confrontation assays. Antibiotics and lipopeptides produced and excreted by the plant growth enhancing rhizobacterium Bacillus amyloliquefaciens FZB42 and the biocontrol rhizobacterium Pseudomonas fluorescens SS101 were shown to be responsible for the observed inhibition of P. indica by these bacteria. R. radiobacter F4 and F7 were able to synthesize a variety of oxo- and hydroxyl-C8- to C12-HSL compounds. In addition, both strains also produced coumaroyl-HSL when coumaric acid was supplied in the medium. The lactonase expressing transformants F4 NM13 and F7 NM13, which are the AHL negative phenotypes, abolished the lipase and siderophore activity. Considering this, quorum sensing influences the production of metabolites including lipase and siderophores in R. radiobacter F4 and F7. Further work should be directed to the question whether quorum sensing also plays a role in the interaction of the bacterium with fungus and/or plant.