Astrid Wetzel

Legume root metabolites and VA-mycorrhiza development

Summary The communication by phenylpropane-metabolites between symbiotic microorganisms and their legume host plants was further studied by the elucidation of root segment-specific exudation of aromatic compounds. The excreted flavonoids were collected by blotting seedling roots directly onto cellulose acetate filters. The flavonoids were eluted from filter segments and subsequently analysed by capillary electrophoresis. Effects of various flavonoids on VA-mycorrhiza (VAM) development were studieti. Glomus mosseae and Glomus intraradix (Schenck and Perez, 1990) were used in these studies. Daidzein (2 to 5 µM) increased germination of spores in a period between 15 to 30 days. Myricetin increased hyphal growth of germinated spores at a concentration of 2 µM between 20 and 50 days. Quercetin had a similar effect in a concentration range of 0.8 to 2.0 µM, whereas 5 µM slightly inhibited the growth of hyphae from the germinating spores. Phytoalexin production in VA-mycorrhiza infected legume roots was studied with Vicia faba by wyerone concentration to quantify the reaction of a non-aromatic secondary root metabolite. VAM infected roots increased wyerone concentration by a factor of 3 to 5 compared with the control, however, at a very low absolute level. A phenylpropane-communication concept for Rhizobium infected legumes is discussed in relation to a still incomplete similar scheme for VAM.

Identification and characterization of flavonoids in the root exudate of Robinia pseudoacacia

Abstract Eight compounds exuded from young roots of black locust (Robinia pseudoacacia) were separated by two-dimensional HPTLC, by HPLC and GC, and were identified by spectroscopic methods (ultraviolet/visible spectroscopy and mass spectrometry) as 4′,7-dihydroxyflavone, apigenin, naringenin, chrysoeriol and isoliquiritigenin. Structural assignments were confirmed by comparison with authentic standards. The capacity to induce β-galactosidase activity in Rhizobium sp. NGR234 containing a nod box::lacZ fusion on plasmid pA27 identified these flavonoids and the chalcone as nod gene inducers. This indicates the important role of these compounds in nodulation of this legume tree.

Toxicity testing of heavy metals with theRhizobium-legume symbiosis: High sensitivity to cadmium and arsenic compounds

Legume root nodules are the site of biological nitrogen fixation in theRhizobium-legame symbiosis. Nodules are structures unique to this symbiosis and they are morphologically as well as physiologically distinct from other plant organs. Organic substances affecting the macro- or microsymbionts vitality, such as PAHs (Wetzelet al., 1991), reduce nodulation even before visible damage to the plant can be detected. We present data that the formation of nodules (nodulation) may also serve for ecotoxicological evaluation of heavy metals in different binding states.Tests were performed in petri dishes with alfalfa (lucerne) seedlings inoculated withRhizobium meliloti. Cultivation took place in growth cabinets with carefully standardized and documented growth conditions. Data from stressed plants was recorded after 14 days of cultivation on contaminated substrate. A dose responsive decrease in nodulation was found after application of cadmium acetate, cadmium iodide, cadmium chloride, sodium salts of arsenate and arsenite, arsenic pentoxide, and lead nitrate, whereas lead acetate showed no effect up to a concentration of 3 μM. The dose response curves were used to calculate EC10, EC50 and EC90 values. EC50 values for cadmium compounds range from 1.5 to9.5 pM. Testing different arsenic compounds results in EC50 from 2.6 to 20.1 μM. EC50 of lead nitrate is 2.2 μM. The sensitivity, reproducibility and reliability of this test system is discussed compared to established biotests.

Biotoxizitätstests mit PAK

ZusammenfassungIm Zusammenhang mit der Umweltchemikalienprüfung und der Untersuchung und Kontrolle kontaminierter Böden besteht großer Bedarf an Testverfahren mit höheren Pflanzen, die schnell, einfach und preisgünstig durchzuführen sind und bereits bestehende Testverfahren nach OECD- und BBA-Richtlinien sinnvoll ergänzen können. Dieser Beitrag stellt einen Biotoxizitätstest vor, der die Nodulation in der Leguminosen-Knöllchen-Symbiose als sensiblen Wirkungsparameter für die Belastung mit polyzyklischen aromatischen Kohlenwasserstoffen (PAK) benutzt. Fremdstoff, Pflanze und Mikroorganismus werden dabei auf einer festen Matrix in Kontakt miteinander gebracht; das axenische System erlaubt die Untersuchung der Fremdstoffwirkung sowohl auf den Gesamtorganismus Pflanze, als auch auf die Interaktion von Pflanze und Mikroorganismus (Knöllchenbildung). In Dose-Response-Versuchen zeigte sich der Wirkungsparameter Nodulation, gemessen als Anzahl Wurzelknöllchen pro Pflanze, sensibler als andere pflanzliche Wachstumsparameter.AbstractThere is great need for toxicity tests with higher plants for the investigation and monitoring of chemicals in the environment — especially for tests which are quick, easy, and inexpensive. Such tests should add to and improve current guidelines, e.g., OECD- and BBA-Guidelines. This article describes the nodulation of leguminous plants withRhizobium bacteria used in a bioassay with polycyclic aromatic hydrocarbons (PAH). Plants growing on a solid medium were exposed simultaneously to bacteria and PAH. This axenic culture system allows to observe the impact of PAH on plant growth and the interaction between plant and bacteria (nodulation). Dose-response-experiments indicated that nodulation measured as number of nodules per plant, was more sensitive to the inhibition by PAH than other plant growth parameters.

Bioassays for Soil Contamination Using the Legume Root Nodule Symbiosis

A new biotest system for ecotoxicological evaluation of soil pollutants was developed, using the symbiosis of Rhizobium meliloti strains with Medicago sativa cultivars, with the number of nodules formed as effect parameter. The development of the test system is described. Soil contaminating substances such as heavy metals, nitroaromatic compounds and polycyclic aromatic hydrocarbons (PAHs), reduced nodulation without damage to the plants and bacteria as separate units at distinct concentration levels. A dose-responsive decrease in nodulation was especially found after application of cadmium ions, sodium salts of arsenate and arsenite, arsenic pentoxide, and lead nitrate. The dose response curves were used to calculate EC values. EC50 values (50% inhibition) for cadmium compounds range from 1.5 to 9.5.tM. Arsenic compounds result in EC50 from 2.6 to 20.1 μM. Sensitivity, reproducibility, and reliability of this test system is discussed and compared to other established biotests such as the LumisTox-test with the example of nitroaromatic compounds such as TNT, 2,4-DNT, 2,6-DNT, and 4-ADNT.

Robinia pseudoacacia , a model tree legume

Robinia pseudoacacia has a number of attributes as a model tree legume, due to one of the highest net photosynthetic rates amoung woody plants (up to 36 µM CO2 x m-2 x s -1), resistance to a number of stresses, early flowering, production of abundant seeds already after three years and a significant genetic variation (Hanover, 1990). With these characters it has a number of advantages compared to the list of 50 nitrogen fixing trees proposed by the Nitrogen Fixing Tree Association considered for their economical or ecological importance (Brewbaker, 1990). All these specific characters can be studied in relation to nodulation and nitrogen fixation by Rhizobium loti (Werner et al., 1996), to VA-mycorrhiza infection (Werner, 1992) and also to other ecological important aspects such as growth on degraded soils due to the very plastic and efficient root system.

Flavonoide im Wurzelraum von Robinia Pseudoacacia — Isolierung, Identifikation, Interaktion

Robinia pseudoacacia als Vertreter der Baum-Leguminosen zahlt zu den wenigen Baumarten, die bei verhaltnismasig hohen jahrlichen Zuwachsraten (bis zu 2,5 m) gleichzeitg relativ dichtes und widerstandsfahiges Holz aufweisen.