Anton Hartmann

Soil amoebae rapidly change bacterial community composition in the rhizosphere of Arabidopsis thaliana

We constructed an experimental model system to study the effects of grazing by a common soil amoeba, Acanthamoeba castellanii, on the composition of bacterial communities in the rhizosphere of Arabidopsis thaliana. Amoebae showed distinct grazing preferences for specific bacterial taxa, which were rapidly replaced by grazing tolerant taxa in a highly reproducible way. The relative proportion of active bacteria increased although bacterial abundance was strongly decreased by amoebae. Specific bacterial taxa had disappeared already two days after inoculation of amoebae. The decrease in numbers was most pronounced in Betaproteobacteria and Firmicutes. In contrast, Actinobacteria, Nitrospira, Verrucomicrobia and Planctomycetes increased. Although other groups, such as betaproteobacterial ammonia oxidizers and Gammaproteobacteria did not change in abundance, denaturing gradient gel electrophoresis with specific primers for pseudomonads (Gammaproteobacteria) revealed both specific changes in community composition as well as shifts in functional genes (gacA) involved in bacterial defence responses. The resulting positive feedback on plant growth in the amoeba treatment confirms that bacterial grazers play a dominant role in structuring bacteria–plant interactions. This is the first detailed study documenting how rapidly protozoan grazers induce shifts in rhizosphere bacterial community composition.

Diazotrophic Bacterial Endophytes in Gramineae and Other Plants

Almost every plant has endophytic bacteria, which do not cause any harm to the plant, but may exert supportive effects on plant development and health. Diazotrophic bacteria were found to reside in roots, stems, and leaves of Gramineae (e.g., sugarcane, rice, maize, wheat, Miscanthus, and Pennisetum) and other plants (e.g., coffee, sweet potato, pineapple, banana). Gluconacetobacter diazotrophicus is a typical endophytic diazotroph in sugarcane roots, stems, and leaves, where its plant growth promotion is supposedly based on a combination of nitrogen fixation and phytohormonal effect. Several Rhizobia were shown to colonize gramineous plants (rice, wheat, maize) endophytically which are grown in rotation or as mixed cultivation with legumes. They exert plant growth promoting effects in their non-leguminous hosts. Some Azospirillum strains are able to colonize the root cortex of Gramineae and other plants and act as plant growth promoting agents mostly via phytohormonal stimulation of root development and activity. Herbaspirillum seropedicae is residing inside the roots and stems of sugarcane, rice, sweet potato, and other plants. It has been shown to exert plant growth promotion and nitrogen fixation in planta. Azoarcus sp. BH72 and other species are endophytes of Kallar grass (Leptochloa fusca), a halophyte in Pakistan, and also of different rice species. They colonize the aerenchyma of roots, are able to fix nitrogen in planta, and may turn to a non-culturable state inside the host plant. Several diazotrophic Burkholderia species, B. tropica, B. unamae, and B. brasilensis were described as endophytes of sugarcane, rice, maize, and teosinte plants. Most interestingly, three quite closely related Burkholderia species (B. phymatum, B. tuberum and B. mimosum) are able to form nodules in legumes (e.g., Mimosa spp.) and fix nitrogen in a symbiotic state like Rhizobia. Among the gamma-proteobacteria, Klebsiella pneumoniae 342 and Pseudomonas stutzeri A1501 were identified as effective and systemic endophytes of maize and rice, respectively.

Localization and Visualization of Microbial Community Structure and Activity in Soil Microhabitats

In recent years, the localization and microvisualization of bacterial cells and their in situ activities in environmental samples have made tremendous progress. A future goal for methodological developments must be the application of these in situ techniques to the detection of fungal communities in the environment. On the one hand, the miniaturization and improvement of the sensitivity of enzymatic measurements and molecular biological techniques enabled new achievements in the synecological understanding of microbes on very small sample scales.On the other hand, the demonstration of in situ activities and the link to phylogenetically defined microbes using the FISH—MAR or TOF/SIMS techniques tshow promise in filling the gap between micrometer scale microbial ecology and the ecological understanding of processes on larger scales. These techniques provide rather robust and relatively rapid tools for the identification of the population structure and function of microbes in diverse environmental samples. However, for application to complex heterogenic samples, the spatial organization needs to be preserved in a refined way. The conservation of the three-dimensional structure in the micrometer range by the improvement of embedding techniques which still allow the fixation and hybridization procedure to occur at reliable precision would be very useful for further in depth studies in microbial ecology. In addition, the development of digital-image analysis tools to extract the three-dimensional spatial data from the investigated specimen would further support the in situ studies of organismic interactions.

Application of Halotolerant Bacteria to Restore Plant Growth Under Salt Stress

High salinity abolishes several stages of plant life ranging from the seed germination step to maturity. Many processes are inhibited, such as phytohormone synthesis and regulation, normal root and shoot development, nutrient uptake, photosynthesis, and DNA replication. Plant growth promoting bacteria (PGPB) are naturally colonizing plants and occur in the rhizosphere or non rhizosphere soil and benefit plant growth by numerous processes. The importance of halotolerant PGPB resides in their ability to adapt to increased salinity by efficient osmoregulatory mechanism to be able to continue regular cell functions. Thus, halotolerant PGPB are able to provide plants with their activities to challenge osmotic stress by supporting them in the restoration of essential activities, e.g., in their hormonal balance. Halotolerant PGPB stimulate plant growth under high salinity by using similar mechanisms like halosensitive PGPB, such as synthesis of indole acetic acid (IAA), gibberellins (GA), cytokinins (CK), abscisic acid (ABA), solubilization of insoluble phosphate, synthesis and excretion of siderophores, and production of ACC-deaminase to reduce high growth inhibitory levels of ethylene occurring in plants at salt stress conditions. Furthermore, some halotolerant PGPB are even able to colonize plants endophytically, produce various antimicrobial metabolites against pathogenic fungi and bacteria, support plant health by improving systemic resistance and contribute to soil fertility and remediation.

Detection of the Bacterial Quorum-Sensing Signaling Molecules N -Acyl-Homoserine Lactones (HSL) and N -Acyl-Homoserine (HS) with an Enzyme-Linked Immunosorbent Assay (ELISA) and via Ultrahigh-Performance Liquid Chromatography Coupled to Mass Spectrometry (UHPLC-MS)

Quick and reliable quantitative methods requiring low amounts of sample volume are needed for the detection of N-acyl-homoserine lactones (HSL) and their degradation products N-acyl-homoserines (HS) in order to elucidate the occurrence and dynamics of these prevalent quorum-sensing molecules of Gram-negative bacteria in natural samples and laboratory model experiments. A combination of ELISA and UHPLC-MS is presented here which has proven to meet these requirements. Both methods can not only precisely detect and quantify HSLs but also their degradation products HS and thereby enable studying signaling dynamics in quorum sensing, which have been identified to play an essential role in bacterial communication.

Detection and Characterization of Endobacteria in the Fungal Endophyte Piriformospora indica

The nonpathogenic Alphaproteobacterium Rhizobium radiobacter (syn. Agrobacterium tumefaciens) (RrF4) is a subculture of the endobacterium R. radiobacter, which is intricately associated with its host, the beneficial plant-colonizing fungal basidiomycete Piriformospora indica. RrF4 is genetically very similar to the well-studied plant pathogenic R. radiobacter biovar I strain C58 (genomovar G8). Highly similar genetic content of RrF4 and C58 denotes the high potential for RrF4 to directly interact with plants. The failure to cure P. indica from its endobacterium still hampers a conclusive prediction of the bacterium’s extended role in the interaction of the fungus with a broad spectrum of host plants. However, beneficial activities shown in cereals and the Brassicaceae Arabidopsis thaliana were hardly distinguishable when induced either by RrF4 or P. indica. We discuss here the various strategies employed to detect, characterize, and eventually elucidate the endobacterium’s role in the tripartite symbiosis with its fungal host and a plant.

Role of Quorum Sensing Signals of Rhizobacteria for Plant Growth Promotion

Signaling events between rhizosphere microbes and plants substantially contribute to establish different qualities of microbe-plant interactions from beneficial cooperativity to pathogenicity. In addition to the pathogen-associated molecular patterns (PAMPs), like exo- and lipopolysaccharides or flagellins, which are effectively recognized by the plants’ innate immune system, various secondary metabolites, such as antibiotics or the so-called autoinducers involved in the quorum sensing response of bacteria, are additional modulators of plants’ perception of associated microbes. In Gram-negative bacteria, N-acyl homoserine lactones (AHLs) are the major quorum sensing autoinducing molecules, which have a central role in the differentiation of specific phenotypes of sessile cells, living in root-attached microcolonies or biofilm consortia. AHLs turned out to have profound effects on plant development and/or defense priming and development of systemic resistance against pathogens. AHLs have different structural modifications (e.g., short or long hydrocarbon chain residues). While the hydrophilic ones can be taken up by plants, the lipophilic stay in the roots. Different modes of plant growth promotion by these AHL types in various plants are summarized in this chapter. We hypothesize, that in the absence of pathogenic patterns, AHLs support a beneficial to symbiotic interaction with plants. In cases when plant pathogens use AHLs for virulence development, AHLs reinforce plant’s defense. Alternatively, AHL degradation activities of certain rhizosphere bacteria can be used to suppress the pathogenic attack. To foster beneficial interactions of rhizotrophs with plants, consortia of bacteria using the same autoinducers could be developed.

In Situ Localization and Strain-Specific Quantification of Azospirillum and Other Diazotrophic Plant Growth-Promoting Rhizobacteria Using Antibodies and Molecular Probes

A central issue in the understanding of the interaction and symbiotic function of diazotrophic bacteria with non-leguminous crop plants is detailed knowledge about the localization of the associated diazotrophic bacteria within the plant, their in situ activities in the plant-associated niches, and strain-specific quantification of inoculated bacteria. In addition to the colonization of rhizosphere soil and the rhizoplane, it has become apparent that an endophytic location of a diazotroph would provide it with a higher potential to interact more closely with the plant, particularly with respect to increasing the availability of carbon and energy nutrients derived from the plant, as well as the possibility, in return, of improving the transfer of bacterial-derived metabolites to the plant. Detailed localization of bacteria was successfully performed using fluorescence labeled ribosome-directed oligonucleotide probes in the fluorescence in situ hybridization (FISH) approach coupled to the use of confocal laser scanning microscopy (CLSM), and via immunolocalization with specific antibodies using transmission electron microscopy (TEM). Furthermore, the fate of inoculated bacteria could be traced by using specifically marked strains by applying the genes for the green or red fluorescent protein (GFP, RFP) and β-glucuronidase (GUS). Strain-specific quantification approaches for inoculants based on quantitative PCR using sequence characterized amplified regions (SCARs) and other genomic marker sequences have been developed and successfully applied. In this chapter major achievements and existing obstacles using these high resolution approaches to analyze bacteria in situ are presented together with some basic protocols.