Silke Ruppel

Settlement of the diazotrophic, phytoeffective bacterial strain Pantoea agglomerans on and within winter wheat : an investigation using ELISA and transmission electron microscopy

The aim of this study was to investigate the ability of Pantoea agglomerans, a plant growth-promoting bacterium, to colonize various regions and tissues of the wheat plant (Triticum aestivum L.) by using different inoculation methods and inoculum concentrations. In addition, the enzyme-linked immunosorbent assay (ELISA) and transmission electron microscopy (TEM) were used to determine: (a) the ability of the bacterial cells to grow and survive both on the surface and within internal tissue of the plant and (b) the response of the plant to bacterial infection. After inoculation, cells of the diazotrophic bacterial strain P. agglomerans were found to be located in roots, stems and leaves. Colony development of bacterial cells was only detected within intercellular spaces of the root and on the root surface. However, single bacterial cells were observed in leaves and stems on the surface of the epidermis, in the vicinity to stomatal cells, within intercellular spaces of the mesophyll and within xylem vessels. Inoculated bacterial cells were found to be able to enter host tissues, to multiply in the plant and to maintain a delicate relationship between endophyte and host. The density of bacterial settlement in the plant in all experiments was about 106 to 107 cells per mL root or shoot sap. Establishment was confirmed by a low coefficient of variation of ELISA means at these concentrations.

Progress in cultivation-independent phyllosphere microbiology.

Most microorganisms of the phyllosphere are nonculturable in commonly used media and culture conditions, as are those in other natural environments. This review queries the reasons for their ‘noncultivability’ and assesses developments in phyllospere microbiology that have been achieved cultivation independently over the last 4 years. Analyses of total microbial communities have revealed a comprehensive microbial diversity. 16S rRNA gene amplicon sequencing and metagenomic sequencing were applied to investigate plant species, location and season as variables affecting the composition of these communities. In continuation to culture-based enzymatic and metabolic studies with individual isolates, metaproteogenomic approaches reveal a great potential to study the physiology of microbial communities in situ. Culture-independent microbiological technologies as well advances in plant genetics and biochemistry provide methodological preconditions for exploring the interactions between plants and their microbiome in the phyllosphere. Improving and combining cultivation and culture-independent techniques can contribute to a better understanding of the phyllosphere ecology. This is essential, for example, to avoid human–pathogenic bacteria in plant food.

Colonization behaviour of two enterobacterial strains on cereals

Abstract Two diazotrophic enterobacterial strains, Pantoea agglomerans D5/23 and Klebsiella pneumoniae CC12/12, were observed in sterile and non-sterile hydroponic and soil experiments in order to determine, by means of an immunological detection method (double antibody sandwich enzyme linked immunosorbent assay), their colonization sites, their migration within individuals of different plant species, and their ability to compete with indigenous organisms. To investigate the interaction between bacteria and plants, root and shoot samples were analysed using electron microscopy. Field experiments were performed to determine the growth-promoting abilities of the bacterial strains. In field experiments, inoculation with P. agglomerans led to an increase in the grain yield of different wheat (Triticum aestivum) cultivars. The same strain was also able to colonize the rhizosphere and the phyllosphere of different cereals due to its ability to migrate within the plant. Roots and media were colonized 10–100 times more intensively than shoots, with about 106 cells g–1 root and 104 cells g–1 shoot. We found that P. agglomerans colonized the root and plant-growth medium of wheat to a greater extent than those of rye (Secale cereale) and barley (Hordeum vulgare), whereas the colonization of shoots was higher in rye and barley compared to wheat. Furthermore, while cell numbers of K. pneumoniae in media and roots were 10 times higher than cell numbers of P. agglomerans, only the latter markedly increased root growth. We were able to detect significant differences in colonization numbers between treatments even if the data were not normally or log-normally distributed or the variances were not homogenous.

Genome Sequence of Enterobacter radicincitans DSM16656T, a Plant Growth-Promoting Endophyte

Enterobacter radicincitans sp. nov. DSM16656(T) represents a new species of the genus Enterobacter which is a biological nitrogen-fixing endophytic bacterium with growth-promoting effects on a variety of crop and model plant species. The presence of genes for nitrogen fixation, phosphorous mobilization, and phytohormone production reflects this microbes potential plant growth-promoting activity.

Composition of the Phyllospheric Microbial Populations on Vegetable Plants with Different Glucosinolate and Carotenoid Compositions

The plant phyllosphere is intensely colonized by a complex and highly diverse microbial population and shows pronounced plant-species-specific differences. The mechanisms and influencing factors determining whether and in which density microorganisms colonize plant phyllosphere tissues are not yet fully understood. One of the key influencing factors is thought to be phytochemical concentration and composition. Therefore, correlations between various concentrations of individual glucosinolates and carotenoids in four different plant species—Brassica juncea, Brassica campestris, Cichorium endivia, and Spinacea oleracea—and the phyllospheric bacterial population size associated with the aerial parts of the same plants were analyzed. The concentration of various individual glucosinolates and carotenoids were measured using high-performance liquid chromatography. The phyllospheric bacterial population size including both nonculturable and culturable organisms was assessed using quantitative real-time polymerase chain reaction, and the physiological profile of the culturable microbial community was analyzed using the Biolog system. Results show significant differences between plant species in both concentration and composition of secondary metabolites, bacterial population size, and microbial community composition in three consecutively performed experiments. An interesting and underlying trend was that bacterial density was positively correlated to concentrations of β-carotene in the plant phyllosphere of the four plant species examined. Likewise, the alkenyl glucosinolates, 2-propenyl, 3-butenyl, and 4-pentenyl, concentrations were positively correlated to the bacterial population density, whereas the aromatic glucosinolate 2-phenylethyl showed a negative correlation to the phyllospheric bacterial population size. Thus, we report for the first time the relationship between individual glucosinolate and carotenoid concentrations and the phyllospheric bacterial population size of nonculturable and culturable organisms and the phyllospheric microbial physiological profiles.

Impact of the PGPB Enterobacter radicincitans DSM 16656 on Growth, Glucosinolate Profile, and Immune Responses of Arabidopsis thaliana

Plant growth-promoting bacteria (PGPB) affect plant cellular processes in various ways. The endophytic bacterial strain Enterobacter radicincitans DSM 16656 has been shown to improve plant growth and yield in various agricultural and vegetable crops. Besides its ability to fix atmospheric nitrogen, produce phytohormones, and solubilize phosphate compounds, the strain is highly competitive against native endophytic organisms and colonizes the endorhizosphere in high numbers. Here, we show that E. radicincitans inoculation of the noncrop plant Arabidopsis thaliana promotes plant growth. Furthermore, high performance liquid chromatography (HPLC) analysis revealed that bacterial inoculation slightly decreased amounts of aliphatic glucosinolates in plant leaves in a fast-growing stage but increased these compounds in an older phase where growth is mostly completed. This effect seems to correlate with developmental stage and depends on the nitrogen requirement. Additionally, nitrogen deficiency studies with seedlings grown on medium containing different nitrogen concentrations suggest that plant nitrogen demand can influence the intensity of plant growth enhancement by E. radicincitans. This endophyte seems not to activate stress-inducible mitogen-activated protein kinases (MAPKs). Analyzing transcription of the defense-related genes PR1, PR2, PR5, and PDF1.2 by quantitative real time polymerase chain reaction (qPCR) revealed that E. radicincitans DSM 16656 is able to induce priming via salicylic acid (SA) or jasmonate (JA)/ethylene (ET) signaling pathways to protect plants against potential pathogen attack.

Properties of the halophyte microbiome and their implications for plant salt tolerance

Saline habitats cover a wide area of our planet and halophytes (plants growing naturally in saline soils) are increasingly used for human benefits. Beside their genetic and physiological adaptations to salt, complex ecological processes affect the salinity tolerance of halophytes. Hence, prokaryotes and fungi inhabiting roots and leaves can contribute significantly to plant performance. Members of the two prokaryotic domains Bacteria and Archaea, as well as of the fungal kingdom are known to be able to adapt to a range of changes in external osmolarity. Shifts in the microbial community composition with increasing soil salinity have been suggested and research in functional interactions between plants and micro-organisms contributing to salt stress tolerance is gaining interest. Among others, microbial biosynthesis of polymers, exopolysaccharides, phytohormones and phytohormones-degrading enzymes could be involved.

Verticillium Suppression Is Associated with the Glucosinolate Composition of Arabidopsis thaliana Leaves

The soil-borne fungal pathogen Verticillium longisporum is able to penetrate the root of a number of plant species and spread systemically via the xylem. Fumigation of Verticillium contaminated soil with Brassica green manure is used as an environmentally friendly method for crop protection. Here we present a study focused on the potential role of glucosinolates and their breakdown products of the model plant Arabidopsis thaliana in suppressing growth of V. longisporum. For this purpose we analysed the glucosinolate composition of the leaves and roots of a set of 19 key accessions of A. thaliana. The effect of volatile glucosinolate hydrolysis products on the in vitro growth of the pathogen was tested by exposing the fungus to hydrated lyophilized plant tissue. Volatiles released from leaf tissue were more effective than from root tissue in suppressing mycelial growth of V. longisporum. The accessions varied in their efficacy, with the most effective suppressing mycelial growth by 90%. An analysis of glucosinolate profiles and their enzymatic degradation products revealed a correlation between fungal growth inhibition and the concentration of alkenyl glucosinolates, particularly 2-propenyl (2Prop) glucosinolate, respectively its hydrolysis products. Exposure of the fungus to purified 2Prop glucosinolate revealed that its suppressive activity was correlated with its concentration. Spiking of 2Prop glucosinolate to leaf material of one of the least effective A. thaliana accessions led to fungal growth suppression. It is suggested that much of the inhibitory effect observed for the tested accessions can be explained by the accumulation of 2Prop glucosinolate.

Effect of ammonium and nitrate on 15N2-fixation of Azospirillum spp. and Pantoea agglomerans in association with wheat plants

Abstract The dinitrogen fixing ability of two diazotrophic bacterial strains Pantoea agglomerons and Azospirillum spp. which are proved to express N 2 -fixing activity in presence of additional inorganic nitrogen sources was tested in association with wheat plants in hydroponic experiments using 15 N 2 incubation. The effect of 100 ppm nitrogen added as NH 4 C1 or KNO 3 to wheat plants on dinitrogen fixing activity of native as well as inoculated bacteria was determined. Enrichment of 15 N, that means fixed dinitrogen, was detected in plant growth media, in roots and shoots of wheat plants grown 26 days in 15 N 2 enriched atmosphere. Highest 15 N amounts were found in wheat shoots. As well as the form of nitrogen applied and the bacterial strain inoculated effected plant growth, nitrogen uptake and the amount of biologically fixed dinitrogen. Ammonia or nitrate supply to plants did not repress 15 N 2 fixation. Distribution of 15 N within the plant and media was mainly influenced by the inoculated bacterial strain. The detected dinitrogen fixing ability in presence of inorganic nitrogen of both bacterial strains in pure culture was confirmed even in association with wheat plants. That finding offers the possibility to select diazotrophic bacterial strains in pure culture which are able to fix dinitrogen in association with plants when additional inorganic nitrogen was fertilized.

Interaction between plants and bacteria: glucosinolates and phyllospheric colonization of cruciferous vegetables by Enterobacter radicincitans DSM 16656.

For determining interactive plant-bacterial effects between glucosinolates and phyllospheric colonization by a plant growth-promoting strain, Enterobacter radicincitans DSM 16656, in cruciferous vegetables, the extent of bacterial colonization was assessed in 5 cruciferous vegetables (Brassica juncea, Brassica campestris, Brassica oleracea var. capitata, Brassica rapa var. alboglabra, Nasturtium officinale) using a species-specific TaqMan™ probe and quantitative real-time PCR. Colonization ability of inoculated E. radicincitans in the phyllosphere of these species varied from inability to colonize B. rapa up to a very good colonization rate of B. campestris. In addition to morphological factors and other plant compounds, the colonization rate was affected by different individual aromatic and aliphatic glucosinolates and their concentration, revealing that both plant pathogens and plant growth-promoting bacteria were affected by glucosinolates in their colonization behavior. In contrast, after E. radicincitans inoculation neither the total nor the individual glucosinolate concentrations in the phyllosphere of the 5 cruciferous species were affected, indicating that the nonpathogenic E. radicincitans might cause only poor cell damage by metabolizing plant cell components and does not induce a plant defense response and thus subsequently an increased glucosinolate concentration in the phyllosphere. Moreover, E. radicincitans induced no stimulation of indole glucosinolate biosynthesis by additional bacterial auxin supply.