Browsing by Subject "Root exudates"

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Characterisation of natural and synthetic nitrification inhibitors and their potential use in tomato cultivation
(2008) Souri, Mohammad Kazem; Römheld, Volker
Summary Besides commercial NIs, many chemicals could also inhibit nitrification. In our study (Chapter 3) regarding efficiency of chloride compared to 3,4-Dimethylpyrazole phosphate (DMPP), it was found that chloride at applied concentration of 30.5 mg per 100g dry soil, could effectively inhibit nitrification. Despite a lag period of 3 weeks in detectable net nitrification, inhibitory effect of chloride continued to persist even after 7 weeks of soil incubation compared to control. Nevertheless, DMPP particularly with higher concentration (2 % of N-NH4+ instead of 1%) stabilized ammonium more strongly than Cl-1. The extent of nitrification inhibition after 5 and 7 week of incubation was in order of: (2 % of N-NH4+) DMPP > (1 % of N-NH4+) DMPP> NH4Cl > KCl > control. The residue ammonium in the soil as well as the produced nitrate concentrations in samples showed a significant NI activity of chloride in both forms NH4Cl and KCl. Nitrification-induced pH decrease, however, showed a better correlation with measured nitrate than ammonium in this experiment. In a second series of experiments undertaken to identify whether the reported NI release by Brachiaria humidicola accession 26159 is an active or passive phenomenon, root exudates of plants grown under various treatments, have been collected in distilled water or in 1 mM NH4Cl. Under various pre-culture conditions such as N form (NH4+ versus NO3-), N concentrations (1, 2, 4 mM), light intensities (180, 240, 350 µmol m-2 s-1), plant age (3-weeks old versus 7-weeks old) and collecting periods (24 versus 6 h), there was no significant NI activity when root exudates were collected in distilled water. However, NI activity was detectable in root washings when the plants were exposed to extended collection times (24 h) in combination with NH4+ supply, but not after short term collection (6 h) or with NO3- in the collection solution. This observation is consistent with the results of Subbarao et al., (2006, 2007), but it also strongly suggests that the observed release of NI compounds was rather a consequence of membrane damage (passive phenomena) due to inadequate collection conditions, than mediated by controlled exudation from undamaged roots. It has been assumed that supplying only ammonium (1 mM) in distilled water as root washing medium over extended time periods (24 h) could lead to rapid ammonium uptake and medium acidification associated with the risk of Ca2+ desorption, which is an important element required for membrane stabilisation and integrity. To test the hypothesis that NI compounds are released from damaged plant cells of Brachiaria, the NI potential of fresh root and shoot homogenates was measured after soil incorporation and incubation. Surprisingly, NI potential was detected in shoot but not in root homogenates. The NI effect of soil-incorporated shoot tissues lasted for at least 8 d, while root tissue even stimulated nitrification with increasing incubation time. This NI effect was independent of the N form. However, the variability of data increased with NO3- form, higher light intensity or higher N concentrations during plant pre-culture. Independent of N forms, further extraction and characterisation of NI compounds in shoot tissue of Brachiaria plants revealed a particularly high activity in the ethanol-soluble fraction, both in plants with NH4+ and NO3- pre-culture. In a third experiment, the role of Ca2+ ions on improvement of tomato growth under ammonium nutrition was investigated. In this experiment root damage, probably by membrane damage and cytosolic sensitivity were hypothesised to be the main cause of toxicity symptoms of NH4+ on tomato plants. At application of 2 mM N as NH4+, plant biomass, number of lateral shoots, and transpiration were strongly inhibited and an increased Ca2+ application into the nutrient solution counteracted these observed negative effects. Transpiration or water consumption was found to be a good indicator of plant performance under NH4+ nutrition. Plants grown under nitrate nutrition had the highest transpiration rates, as well as the best growth characteristics. There was a positive correlation between nitrate concentrations and transpiration rates. On the other hand, plants grown in ammonium (as control, or 3 and 6 split applications of NH4+ during 4 days) showed severe toxicity symptoms including growth inhibition and leaf abscission. However, when ammonium was applied together with 10 mM Ca2+ (as CaSO4), or in a buffered solution of pH 6.6 with CaCO3 (pH or/and Ca2+ effect), transpiration and other growth factors (e.g. root and shoot dry matter, number of lateral shoots), as well as the nutrients especially N concentrations in the biomass were significantly improved. In other words, shoot and particularly root growth inhibited when NH4+ treated plants (control and split applications) did not received CaSO4 or CaCO3. Micro molar concentrations of NH4+ in 6 split applications also could not prevent ammonium toxicity symptoms.
Einfluss von erhöhtem atmosphärischen CO2 auf die N2-fixierende Symbiose von Trifolium repens L. und Rhizobium leguminosarum biovar trifolii
(2007) Stöber, Sara; von Wirén, Nicolaus
CO2 is one of the main greenhouse gases strongly influencing the climate and the terrestrial ecosystem. Up to know little is known about the impact of elevated atmospheric CO2 on symbiotic interactions in the rhizosphere, especially on the N2-fixing symbiosis between Trifolium repens and Rhizobium leguminosarum biovar trifolii. First results of a ten-year Free-Air CO2 enrichment experiment (Swiss FACE) showed that after three years of CO2 fumigation the genetic composition of the Rhizobium population in the root nodules of T. repens had changed. The first part of this thesis set out to clarify the question whether a genetic difference in the Rhizobium population of root nodules of white clover could still be detected after ten years of CO2 fumigation or if an adaptation of the nodule bacteria to elevated CO2 concentrations had occurred. Furthermore the thesis addressed the question whether elevated atmospheric CO2 leads to quantitative and qualitative changes in the root exudation of T. repens particularly with regard to exudation of signal substances during the nodulation process. In summer 2002 white clover plants were collected from plots fumigated with CO2 and control plots of the Swiss FACE. Rhizobium strains were isolated from the clover root nodules and used for rep-PCR DNA fingerprinting. Results clearly showed that after ten years of CO2 enrichment changes in the genetic composition of the R. l. bv. trifolii could no longer be observed. Thus, CO2-induced changes in the population structure of rhizobia seemed to be transient. This can be traced back to the possibility that over the experimental period a new C/N equilibrium in the grassland ecosystem has been established. At the beginning of the FACE experiment an increase in the C/N ratio of the soil was detected, which could be balanced in the course of time through enhanced symbiotic N2 fixation and consequently a higher N input into the ecosystem. The observed stabilisation of the grassland ecosystem most likely caused a reduction of the indirect CO2 impact on the microorganisms. This might explain why a change in the genetic composition of Rhizobium strains was not longer detected after ten years in the Swiss FACE. To investigate an influence of elevated atmospheric CO2 concentration on the release of signalling compounds clover plants were cultivated hydroponically in two independent climate chamber trials under axenic and non-axenic conditions at ambient and elevated CO2 concentrations (400 and 800 ppm) and different levels of N supply. Root exudates were collected over a period of seven hours and at three and four different plant ages, respectively. Phenolic compounds were extracted by solid-phase extraction and afterwards analysed with HPLC and LC-MS. Additionally, the isolated fractions were tested for their ability to induce the nodulation genes of R. l. bv. trifolii using a nod-gene induction test. The CO2 enrichment caused an increase in shoot and root growth in both experimental setups, but did not provoke a change in the C/N ratio of the roots. Besides the known signal compound 7,4?-dihydroxyflavone new phenolic substances could be detected, which have not yet been described in literature. The fractions were identified by their polarity, light absorption and molecular weight as aglyca and flavones. All of these had the ability for nod-gene induction except one fraction (fraction 2). CO2 influenced the exudation of signalling compounds quantitatively but not qualitatively. The enhanced exudation, especially of 7,4?-dihydroxyflavone, could be attributed to the higher root mass under elevated CO2 but also to a higher release rate on a root fresh weight basis. The CO2 reaction of the clover plants, for the biomass production as well as for the root exudation, was clearly dependent on the N supply and only significant under axenic conditions. In individual cases the N impact was more pronounced than the CO2 effect: with increasing N demand axenic clover plants enhanced the exudation of the nod-gene inducing fraction C. It is concluded that this fraction, identified as a hydroxyflavone, has therefore an important signal function under N limitation. Besides the CO2 concentration and N supply, root exudation by T. repens was considerably influenced by the plant age, which caused a reduction of the signal exudation in older plants and qualitative changes of the released phenols, especially under non-axenic conditions. The present study suggests that the genetic shift of R. l. bv. trifolii detected at the beginning of the Swiss FACE experiment was most likely a consequence of the enhanced exudation of phenolic signal compounds of T. repens under elevated atmospheric CO2 concentrations.
Interactions between non-symbiotic N2-fixing bacteria and plant roots in plant-microbial associations
(2009) Calvo Alegre, Olga-Cristina; von Wirén, Nicolaus
The development of biofertilizers on the basis of plant growth promoting rhizobacteria (PGPR) may be a promising approach to partially substitute costly and energy-consuming mineral fertilizers in agricultural plant production and to support agriculture in developing countries. A successful and competitive rhizosphere colonization of PGPR strains has been identified as a prerequisite for the expression of plant growth promoting effects. Apart from a wide range of external factors with an impact on the colonization process, such as soil properties, temperature, soil moisture and fertilization levels, in particular plant-microbial interactions may play an important role for the successful establishment of compatible associations. In this context, certain plant root exudates may act as signals to mediate bacterial responses with importance for root colonisation (e.g. motility and chemotaxis, production of extracellular polysaccharides). On the other hand, the induction of bacterial plant growth promotion may also depend on ability of the host plant to respond to the presence and the activity of the associated bacteria. It was therefore the aim of this thesis to investigate the contribution of putative PGPR to growth and N uptake in wheat plants and characterize the underlying mechanisms in root-bacterial associations. In the first part of this thesis, the contribution of various non-symbiotic diazotrophic rhizobacteria to plant growth promotion and N nutrition has been studied in a series of greenhouse pot inoculation experiments with wheat (Triticum aestivum L.). Different bacterial inoculants, plant genotypes, soil properties, water regimes and N fertilization levels have been varied as factors with potential impact on plant growth promotion by diazotrophs. The contribution of biological nitrogen fixation was assessed by the 15N dilution method. Plant growth and grain yield were influenced by the different N fertilization levels but no stimulation of growth or N uptake was note upon bacterial inoculation. These observations suggested a high degree of specificity or limiting factors, determining a successful plant-microbial association. The second part describes possible mechanisms that may be involved in the establishment of diazotrophs in the rhizosphere of suitable host plants. As an initial step of the colonization process, a targeted movement of the bacteria to the root surface is required and root exudates may act as attractants. Since dicarboxylic acids are known to exert chemotactic activity on diazotrophic bacteria, seed and root exudates of two graminaceous crops (Triticum aestivum L. and Zea mays L.) and for comparison also of a non-graminaceous plant species (Phaseolus vulgaris L.) were collected in hydroponic culture with and without N supply, and organic acid profiles in these root exudates were analysed. Bacterial motility assays were conducted with the major carboxylates detected in the root exudates of the selected plant species and compared to glucose and water, using Brevibacillus reuszeri as a model bacterium. Pure malate, which was found at high levels in root exudates of bean and wheat, and particularly malonate (bean) and t-aconitate (maize) stimulated the motility of Brevibacillus reuszeri as compared with glucose or water. A particularly intense promotion of bacterial motility was recorded in the presence of crude root exudates of wheat and maize plants grown under N limitation, which was not observed for root exudates of bean. However, this was not related with comparable changes of malate or t-aconitate concentrations in the root exudates. In wheat exudates, malate concentrations even decreased in response to N limitation. These findings suggest the presence of specific factors released in root exudates of N-deficient cereals, promoting the rhizosphere colonisation with B. reuszeri. For an identification of the respective factors, a more comprehensive profiling of the root exudates is necessary. In associations with diazotrophic bacteria, host plants are supplied with ammonium by the bacterial partner. This raised the question whether plant ammonium uptake systems have an impact on the efficiency of the association. To address this problem, an antisense approach was conducted with tomato, with characterised ammonium transporters (LeAMT1;1 and LeAMT1;2). The final goal was the inhibition of the ammonium transporters by production of LeAMT antisense lines to study their putative role in plant associations with diazotrophic bacteria. Northern blot analysis revealed a strong repression of LeAMT1;2 expression in three independent antisense lines associated with a lower ammonium uptake capacity under N-sufficient and N-deficient growth conditions. In contrast, LeAMT1,1 expression was only weakly repressed in antisense lines and there was no impact on N uptake. A faster decline of chlorophyll in older leaves indicates a physiological function of LeAMT1;1 and LeAMT1;2 in ammonium uptake and retrieval in shoot and root cells. The absence of consistent effects on N acquisition of the investigated antisense lines limited the suitability of this approach for studies on associations with diazotrophic bacteria.
Role of benzoic acid and lettucenin A in the defense response of lettuce against soil-borne pathogens
(2021) Windisch, Saskia; Walter, Anja; Moradtalab, Narges; Walker, Frank; Höglinger, Birgit; El-Hasan, Abbas; Ludewig, Uwe; Neumann, Günter; Grosch, Rita
Soil-borne pathogens can severely limit plant productivity. Induced defense responses are plant strategies to counteract pathogen-related damage and yield loss. In this study, we hypothesized that benzoic acid and lettucenin A are involved as defense compounds against Rhizoctonia solani and Olpidium virulentus in lettuce. To address this hypothesis, we conducted growth chamber experiments using hydroponics, peat culture substrate and soil culture in pots and minirhizotrons. Benzoic acid was identified as root exudate released from lettuce plants upon pathogen infection, with pre-accumulation of benzoic acid esters in the root tissue. The amounts were sufficient to inhibit hyphal growth of R. solani in vitro (30%), to mitigate growth retardation (51%) and damage of fine roots (130%) in lettuce plants caused by R. solani, but were not able to overcome plant growth suppression induced by Olpidium infection. Additionally, lettucenin A was identified as major phytoalexin, with local accumulation in affected plant tissues upon infection with pathogens or chemical elicitation (CuSO4) and detected in trace amounts in root exudates. The results suggest a two-stage defense mechanism with pathogen-induced benzoic acid exudation initially located in the rhizosphere followed by accumulation of lettucenin A locally restricted to affected root and leaf tissues.
Transcriptional profiling of Bacillus amyloliquefaciens FZB42 in response to seed and root exudates collected under different nutrient regimes
(2010) Carvalhais Costa, Lilia; von Wirén, Nicolaus
Plant growth-promoting rhizobacteria (PGPR) live in close association with plants and improve their growth. Bacillus amyloliquefaciens strain FZB42 is a prominent plant root-colonizing bacterium that is able to stimulate the growth of maize. To decipher the molecular cross-talk between B. amyloliquefaciens and crop plants, an exploratory analysis of the effect of seed and root exudates on the transcriptome of Bacillus amyloliquefaciens FZB42 was performed. Root exudates were collected from maize plants grown in an axenic hydroponic system under nutrient sufficiency or under deficient supply of nitrogen (N), phosphorus (P), iron (Fe) or potassium (K). An analysis of primary metabolites in the exudates was carried out, compared between treatments, and correlated with the transcriptional profiles of Bacillus amyloliquefaciens FZB42 that were gained after incubation of the bacterial culture with the root exudates. Higher exudation rates of citrate were found under Fe deficiency and greater release of ã-amino butyric acid under P deficiency. Based on a negative correlation observed between the average diffusion coefficient of N, P, K, and Fe in soils and the exudation rates of primary metabolites under conditions of N, P, K, or Fe deficiency, it was hypothesized that the exudation of sugars, amino acids and organic acids may reflect the availability and mobility of plant nutrients in soils. In the presence of seed and root exudates collected from nutrient-sufficient plants, genes involved in spore germination, transport and utilization of nutrients, biosynthesis pathways, multidrug transporters, motility and competence development were differentially expressed. In comparison to P, Fe and K, N-deficient maize root exudates caused a more distinguished change in the transcriptome of bacteria when they were in the logarithmic growth phase. During this growth phase, a number of genes coding for ribosomal proteins were down-regulated by N-deficient maize root exudates, indicating that bacterial activity was repressed. Exclusively in the presence of P-deficient maize root exudates, several genes associated to bacterial motility were induced. Moreover, a gene involved in the biosynthesis of the auxin precursor tryptophan was up-regulated by all deficiency treatments. In the transitional growth phase of Bacillus amyloliquefaciens FZB42, several genes were commonly down-regulated in different deficiency treatments. This finding is in agreement with previous studies showing that quorum-sensing and starvation-sensing are integrated to regulate cell entry into the transient phase. Taken together, this is the first study comparing the effect of different nutrient deficiencies on the composition of primary metabolites in root exudates of one plant species and evaluating systematically the transcriptional response of a Gram-positive PGPR to seed and root exudates collected from plants grown under different nutrient regimes. This analysis provides new information about the early communication between plant roots and PGPR and points to involved genes and processes that merit further investigation.