Prozessregulation in der Rhizosphäre

In order to study the events occurring before mycorrhization between oaks (Quercus robur L.) and the ectomycorrhizal fungus Piloderma croceum, oak seedling morphogenesis was highlighted as basis to characterize the developmental behaviour of micro-propagated oaks in a semi-sterile Petri-dish mycorrhization system. As well as in seedlings, shoots of the micro-cuttings remained characterized be an endogen rhythmic growth under controlled culture conditions (25°C and 16 h illumination). Lateral roots of the micro-propagated oaks also displayed a rhythmic growth, whereby the root and shoot flushes were out of phase, with concomitance of apical bud swelling and maximal growth rate of the roots. In micro-cuttings inoculated with P. croceum, this concomitance was maintained, in spite of a strongly enhanced root and shoot growth induced by the fungus in its long premycorrhizal stage. The specificity of morphological modifications (for example enhanced leaf area and lateral root growth) during the prolonged pre-mycorrhizal stage was demonstrated using activated charcoal and auxine (IAA). Both compounds simultaneously advanced mycorrhizal formation and stopped the enhanced development of first order mother roots. In order to elucidate molecular mechanisms involved during this prolonged pre-mycorrhizal stage, a subtractive cDNA bank was established between roots sampled before mycorrhization and control roots mixed with fungal mycelium. After southern blot analysis 60 differentially expressed genes were sequenced and homology was determined for about 30 of them. Northern blot analysis revealed an up regulation during the pre-mycorrhizal stage of two genes encoding for metallothionein and formate-dydrogenase, respectively. The model will serve for further analyses of gen regulation at different stages of mycorrhiza formation.

Quantitative studies of root development as it depends on climate, soil and plant properties are one prerequisite to derive relations that allow for a more general, possibly predictive approach to root growth and, consequently, root nutrient uptake. Since net maize root length, e.g. amount of roots that can be measured or observed in a soil sample, increases until flowering, detailed information is needed until this stage is entered. The process of dispersion of the total amount of roots formed in soil, i.e. root penetration into soil and root proliferation in soil layers, has to be quantified.

Five field experiments, conducted in three years substantially differing in climate are used to describe root and shoot development on a silt loam soil at one location (West Lafayette, Indiana, USA). Both, total root length development and shoot growth of maize were related to the product of accumulated thermal time and solar radiation for all 5 experiments. Root growth in the topsoil layer was positively related to soil water content as calculated with a mechanistic soil water balance model.

Regression analysis of a long term experiment showed that on a silt loam soil there was a positive relation between root length at flowering in the topsoil layer and the amount of rainfall during the last weeks prior to flowering, e.g. the time of fastest root growth. Root length accumulation at the bottom of the soil profile was positively related to air temperature during the weeks following planting.

Experiments conducted in thin-layer cuvettes filled with a loamy sand soil allowed for a non-destructive and repeated observation of plant root development. The development of a software program enabled quantification of root development on the transparent front of the cuvettes. After penetration of a soil layer root growth was exponential followed by a linear growth phase. Secondary roots were formed at seminal roots approx. 2 days old, and secondary root elongation continued for 6 to 8 days. Only few tertiary roots were observed on scanner images. With decreasing depth the maximum absolute growth rate of roots decreased. Water content of soil layers (SWC) influenced penetration rate of roots into soil as well root proliferation within soil layers. Decreasing SWC increased number of secondary roots and reduced length of secondary roots, the overall effect being a reduction in absolute root growth rate. Increased soil bulk density (SBD) reduced total root length produced in cuvettes. The main reason was reduction of number of secondary roots formed. Seminal root development was not strongly influenced by soil bulk density. With decreasing depth the effect of SBD on the formation of lateral roots increased strongly.

It has been shown that during vegetative growth of the corn plant shoot growth and root abundance are interdependent, and a maximum root-shoot ratio (RSR) can be derived. Root penetration into soil and root proliferation during the exponential and linear growth phase in soil layers (root dispersion) can be described sufficiently exact by regression equations that take into account air temperature and solar radiation. A maximum absolute penetration rate (APR) and growth rate (AGR) can be derived. AGR and APR are influenced by distance from soil surface, soil water content and soil bulk density; these relations can be expressed mathematically.

On basis of the results obtained a one-dimensional mathematical model is developed for a layered soil using STELLA software. Input data are daily values for minimum and maximum air temperature, and solar radiation. The soil profile is subdivided in up to 10 layers with a minimum depth of 5 cm, and each layer is characterized by one soil bulk density. Daily values of soil water contents of soil layers are calculated using an external model of soil water balance. From these data the model calculates shoot biomass accumulation, root abundance from shoot growth, root penetration into soil, and root proliferation in soil layers. Maximum absolute root growth rate and maximum penetration rate are altered with soil depth, with changes of soil water content for soil layers over time, and as soil bulk density differs for soil layers.

The following rules were implemented:

As could be expected for the data set used for model development, calculations agreed well with data of shoot growth and root abundance. Root growth in soil layers was best reflected for the 0–15 and 15–30 cm layer, and deviations between measured and predicted values were highest for the deepest soil layers.

It is concluded that the root growth model reflects the major processes that deter­mine root abundance and dispersion for the weather and soil conditions that are characteristic for the location. Inclusion of other soil types and climates may extend the applicability of the model to other environmental conditions in the future.

The model presented here is simplistic in a number of aspects. The following list gives some keywords and is not final:

The list shows that model calculations cannot be expected to agree well with observations if one or more of these factors are dominating over the processes that are at present incorporated into the model. However, the degree of agreement may be used to decide whether or not additional processes have to be included. The presented, simplistic model can be used as a starting point.

Im Rahmen der Chemotaxonomie werden Naturstoffe häufiig dazu benutzt, um Verwandtschaftsbeziehungen und gemeinsame evolutionäre Ursprünge von pflanzlichen Taxa zu untersuchen. Das Vorkommen gleicher oder ähnlicher pflanzlicher Naturstoffe in verschiedenen Taxa wird als Indiz für eine Verwandtschaft zwischen diesen Taxa gewertet (Hegnauer 1992). Es gibt jedoch auch Naturstoffe, die so weit in der Natur verbreitet sind, dass das Vorkommen identischer oder ähnlicher Naturstoffe nicht als ein Hinweis für eine Verwandtschaft zwischen zwei Taxa gewertet werden kann. Solche Naturstoffe sind die maytansinoiden Verbindungen (Fig. 1) (wie zum Beispiel Maytansin, Maytanbutin, Maytanprin, Maytansinol oder Ansamitocin), die in Höheren Pflanzen (Celastraceen, Euphorbiaceen und Rhamnaceen), Moosen (Lembophyllaceen, Thuidiaceen und Neckeraceen) sowie in Bakterien (Actinosynnema pretiosum) vorkommen (Reider und Roland 1984, Lancini 1986).

Die Rhizosphäre ist von großer Bedeutung für die Pflanzengesundheit, wobei die funktionelle Gruppe der antagonistischen Mikroorganismen eine wichtige Komponente bildet. Der bodenbürtige Pilz Verticillium dahliae Kleb. — der Erreger der Verticillium-Welke und gleichzeitig Modellpathogen — ist weltweit für hohe Ertragsausfälle verantwortlich. Die mikrobielle Diversität antagonistischer Bakterien von Verticillium-Wirtspflanzen (Kartoffel, Raps und Erdbeere) wurde in Freilandversuchen analysiert. Eine Vielzahl von Bakterien (15642) wurde überprüft. Der Anteil antagonistischer Mikroorganismen schwankte von 3–47 % und wurde durch die Herkunft der Isolate (Pflanzenspezies/Entwicklungsstadium/Mikrohabitat) sowie von Bodenparametern beeinflusst. Nach einer ldentifizierung von 750 Isolaten konnten über 80 Bakterienspezies identifiziert werden. Eine entwickelte Screeningstrategie mit verschiedenen Schritten von Labor- zu Felduntersuchungen ermöglichte eine Überprüfung der antagonistischen Bakterien hinsichtlich ihres Potenzials für die biologische Kontrolle. Hierbei wurden fünf Isolate als Biological Control Agents ausgewählt und zu 3 Produkten entwickelt.

Gram-negative Bakterien verfügen über ein eigenes Regulationssystem, das sie zur Interaktion befähigt (Eberl 1999). Diese Art der Zell-Zell-Kommunikation erfolgt über Signalmoleküle vom Typ N-Acyl-L-Homoserin-Lacton (AHL), welche erst ab Erreichen eines bestimmten Schwellenwertes eine zellspezifische Reaktion auslösen (Quorum sensing) z.B. die Produktion von Antibiotika oder Enzymen (Swift et al. 1996). AHL-produzierende Rhizosphärenbakterien sind damit u.a. im Hinblick auf mögliche Abwehrmechanismen gegen Phytopathogene bei Pflanzen von besonderem Interesse.

Microscopy of ectomycorrhizal roots directly sampled from the field has revealed the presence of bacteria in the hyphal sheath, in the Hartig net region and on soilcolonising extramatrical hyphae. Some of these rhizosphere bacteria, named mycorrhiza helper bacteria, have been shown to improve hyphal growth and mycorrhiza formation. The mechanisms behind these processes are currently unknown. We have focused on the effect of two streptomycete strains, that induce hyphal growth and mycorrhization, on the gene and protein expression of Amanita muscaria (fly agaric). Total RNA isolated from A. muscaria mycelium, co-cultured with the streptomycete strain 505, has been used for cDNA synthesis and for the construction of a subtracted cDNA bank. Differential expression of the subtracted cDNAs has been analysed by macroarrays and partially verified by Northern hybridisation. Changes in protein patterns between streptomycete-induced A. muscaria mycelium and of aseptic fungal cultures have been analysed by twodimensional gel electrophoresis and peptide mass spectrometry. Most of the differentially expressed fungal genes show homology to members of signal transduction pathways. Others are homologous to genes involved in metabolism, cell proliferation and cell structure. This implies that the fungus-streptomycete interaction is connected with changes in signalling pathways that lead to an altered fungal physiology.

Nowadays the development of alternative clean-up techniques for heavy metal contaminated soils becomes more and more important. An ecological and economical way to remove heavy metals could be via phytoremediation with heavy metal accumulating plants. Previous research focussed on a decrease of the total contents of heavy metals in soil, even though the significance of total contents is very limited from an(eco-)toxicological point of view.

In a pot experiment with sunflowers we analysed the mobility and binding forms of heavy metals over time after adding various mobilizing agents to polluted soils in order to enhance the soil-plant transfer of the metals.

Ecological agriculture expands in industrial countries. Therefore biological nitrogen fixation receives more attention. Especially grass species in mixed stands with legumes like alfalfa or clover play an important role for fodder production, because the dry mass and N yield of grass increase in comparison with grass pure stands. The N transfer between alfalfa and ryegrass under normal and enriched CO₂ concentrations was studied through isotope dilution at a very low level of N fertilization (5,5 mg N/pot) in a pot experiment using quartz sand as substrate. The growth of grass and non nitrogen fixing alfalfa plants were limited and the N-transfer between alfalfa and ryegrass was only 0,06 kg N ha ^-1 at 360 ppm CO₂ and 1,1 kg N ha^-1 at 720 ppm CO₂ although the nitrogen fixation was nearly constant. Root/ shoot ratio and root mass of ryegrass increased at 720 ppm CO₂. We concluded that the improved root growth of grass plants promoted the uptake of fixed N from alfalfa plants. The duration of growth at 720 ppm CO₂ until harvest was only two weeks and therefore mainly root exudates from alfalfa plants should be the source of increased fixed N in grass plants.

Higher plants affect processes in the rhizosphere by root exudation. The exuded compounds may directly influence nutrient availability or may serve as a substrate for various nutrient-solubilizing soil microorganisms. Furthermore, plant growth promoting effects of rhizosphere microbes include the production of a vast range of metabolites (biological active substances) that may affect plant growth directly after being taken up by the plant, or indirectly by modifying the soil environment. Phytohormones like auxines (indole-3-acetic acid, IAA), gibberellins (gibberellic acid, Ga₃) or cytokinins (trans-zeatin, t-Z) belong to this groups of substances. It is well documented that cultures of several plant growth promoting microorganisms produce these phytohormones in considerable amounts. But there are only few data available about long-term effect of these coumpounds on rhizosphere processes of intact plants.

In the present investigation, four cultivars of maize plants (Zea mays L.) were grown in a greenhouse pot experiment with coarse sand as substrate. Control plants were supplied with nutrients by daily watering with a nutrient solution (40 mg P/1) and P-deficient plants were cultivated in a substrate containing 324 mg of sparingly soluble Ca₃ (PO₄)₂ (65 mg P). Additionaly, the concentration of IAA, GA₃ or t-Z in the rhizosphere of maize plants was maintained at 0.1 mM (IAA, GA₃) or 0.01 mM (t-Z) by daily addition of a freshly mixed hormone-containing nutrient solution to one half of each P treatment. Three factorial analysis of variance showed significant effects of hormone treatment, P availability, and genotype on growth of maize plants indicating that the presented method is suitable for induction and investigation of long-term hormone effects on rhizosphere processes of higher plants.

Root derived organic carbon compounds and their microbial metabolites are of ecological importance for the nutrient supply of higher plants. An extraction method similar to the double-lactate (DL) method (2 g soil, 100 ml solution with 5 g l^-1 substance, pH 3.7, 90 min) was used to measure the P-solubilizing ability of sugars and carboxylic acids in 10 different soils. Neutral extraction solutions were used to detect specific anion effects. Additionally a 24 h extraction at 28 °C was used to permit chemical and microbial changes of the substances in the soil.

Sugars and sugar alcohols can contribute to P mobilization in insterile soils with a pH below 7. They have no effect under alkaline conditions. The acidic extraction with gluconic acid was as efficient as DL, although the buffer capacity of the solution is relatively low. The P release increased during 24 h. However, a neutral gluconate extraction was only comparable with sugar extraction. Succinic acid was only effective under acidic conditions.

Citrate and oxalate, even in small concentrations, mobilized large P amounts under different soil conditions. Mainly the effectiveness of citrate increased with a longer incubation time. Specific anion effects are detectable with neutral extractions.

It can be concluded, that the qualitative composition of root deposits has a higher influence on their P-mobilizing efficiency than their total amount.

An artificial soil was produced by open air composting of a mixture of sewage sludge, wood shavings, green compost, and a sandy loam soil, and used to investigate the effect of plant growth (wheat) and organic matter application on turnover of organic C und N. A greenhouse experiment was carried out with and without plants (wheat) to test the turnover of N in artificial soil and a mixture of artificial soil with sand in the ratio of 1:1. The results showed that the cultivation of wheat increased the net N mineralization by about 68% to 71% in comparison to treatments without plants. A laboratory experiment was carried out to investigate the effect of incubation of straw or animal manure with artificial soil at 35 °C on C mineralization and net N mineralization. The addition of straw and animal manure caused an increase in mineralization of C. The net N mineralization was higher after addition of animal manure and without substrate application (control) than after addition of straw.

Root structural residues are one of the main sources for organic C input into the soil. However, little is known about the actual input of root cell wall residues and relevant fractions (cellulose, hemicellulose, lignin) into soil, especially during the ontogenesis of plants. In order to solve this problem we combined a repeated pulse labelling of shoots by ¹⁴CO₂ for quantification rhizosphere ¹⁴C fluxes with procedures to characterize root residues and selected fractions from root residues (lignin, carbohydrates) at the example of maize. The C flux via root decay during ontogenesis amounted to approx. 116 kg C per ha, approx. 20% of the total C input into the soil as a result of net root growth. In these root residues were 30 kg C as cellulose, 13 kg C as hemicellulose, and 24 kg C as lignin. While the root fractions of old plants showed a uniform ¹⁴C distribution, there were strong inhomogeneity with young plants in this regard.