Arbeitskreis
Phytopharmakologie

1. Xylemapplikation im Weinbau - Systementwicklung und Wirkstofftransport
2. Wie kommt man zum "mode of action" einer herbizidwirksamen Substanz?
3. The Development of the Food Value Chain - the Impact on Agriculture and Crop Production
4. Degradation of Metolachlor in the presence of different antibiotics in the soil.
5. Enzymes encoded by the farnesyl diphosphate synthase gene family in the Big Sagebrush Artemisia tridentata
6. Barley leaf spot complex as target for fungicide application
7. Fungizid induzierte Genexpression in Colletotrichum graminicola (Cesati) Wilson
8. 2D gel electrophoresis, mass spectrometry and video imaging are useful tools for mode of action studies
9. Influence of a recombinant human P450 system on endogenous ingredients in transgenic plants of Nicotiana tabacum L.
10. Metabolic Profiling zur Aufklärung der Wirkmechanismen von Herbiziden
11. Insecticide mode of action and mechanisms of resistance with special reference to sweet-potato whiteflies, Bemisia tabaci (Homoptera: Aleyrodidae)
12. Beeinflussung der Flavonoid-Biosynthese der Birne durch den Einsatz des Bioregulators Prohexadion-Ca
13. Einfluss des Wachstumregulators Prohexadion-Ca auf den Metabolismus phenolischer Verbindungen bei der Weinrebe Vitis vinifera L.
14. A novel in vitro technique for evaluating fungicidal activity against apple scab and powdery mildew
15. Selektionsmuster von Fungiziden bei Pflanzenpathogenen
16. Resistenzinduktion durch Pflanzenstärkungsmittel in Apfelblättern
17. Adsorption of s-Triazin derivatives on Na-bentonites - influence of humic acids and pH
18. Scintillation Proximity: On-line Measurement of Radiolabel Uptake as Precise Parameter of Fungal Life
19. Very-long-chain fatty acid elongases as herbicide targets
20. Immune-related proteomics and genomics in insects: A new challenge for plant protection?

Düker, A.; Kubiak, R.
Dienstleistungszentrum Ländlicher Raum (DLR) - Rheinpfalz, Abteilung Agrarökologie, Breitenweg 71, 67435 Neustadt

Die Xylemapplikation von Pflanzenschutzmitteln stellt eine ökologisch sinnvolle Methode des Pflanzenschutzes dar. Die applizierten Wirkstoffe gelangen dabei direkt, ohne in die Umwelt zu gelangen, über die Xylembahnen in die Zielorgane.
Einmalige Punktapplikationen in die Leitbahnen von Bäumen werden bereits seit den 70er Jahren erfolgreich angewendet. Allerdings müssen diese über längere Zeiträume ständig wiederholt werden.
Im Weinbau ließe sich die Xylemapplikation vor allem in Gewässernähe und Ortsrandlage sinnvoll etablieren. Aus ökonomischer Sicht würde hierfür ein vernetztes, einmalig zu montierendes, aber gleichwohl mehrjährig nutzbares System benötigt - welches weiterhin ungeahnte arbeitstechnische Vorteile in den Steillagen des Deutschen Weinbaus bieten könnte.
In einer vom BMBF geförderten Kooperation des Fachbereichs Ã-kologie des DLR-Rheinpfalz mit dem Fachbereich Maschinenbau der Uni-Kaiserslautern entstanden die ersten Prototypen eines vernetzten Stammapplikationssystems für den Weinbau. An Topfreben wurde weiterhin der Verbleib von xylemappliziertem 14C-markiertem BAS 500 untersucht.

What it takes to get a herbicide's mode of action.
Physionomics: a classical way in a new complexion

Klaus Grossmann
BASF Aktiengesellschaft, Agrarzentrum, D-67117 Limburgerhof

Die Auffindung von neuen Herbiziden mit neuartigen Wirkungsmechanismen ist Ziel der modernen Pflanzenschutzforschung. Für neue, herbizidwirksame Verbindungen, die im Verlauf der Gewächshaus-Prüfung identifiziert werden, stellt sich damit die Frage, auf welche Weise sich möglichst rasch und mit vertretbarem Aufwand eine Charakterisierung des Wirkungsmechanismus erreichen lässt. Zur Zeit werden intensiv molekulargenetische ("functional genomics", "transcriptomics"), biochemische ("proteomics") und stoffwechselanalytische ("metabolomics") Ansätze diskutiert. Bisher nur wenig Beachtung fand die Möglichkeit, am Ende der Prozess-Kette, auf der Ebene des komplexen Systems der Pflanze, die klassische Methode der Schadbild-Diagnose ("phenotyping") weiter zu entwickeln. Dies wird erreicht durch die Konzeption einer Palette von "funktionalen" Biotests, die eine Differenzierung des pflanzlichen Organismus nach Organisationseinheit (Pflanze, Gewebe, Zelle), Entwicklungsstadium und physiologischen und metabolischen Eigenschaften erlaubt. Das nach Prüfung aus den Ergebnissen abgeleitete Physiologie-Profil ("fingerprint") einer Substanz ist typisch für ihren physiologischen Wirkungsmechanismus, der sich direkt durch Interpretation der Ergebnisse und/oder durch Analogie-Vergleich mit Profilen von bekannten Hemmstoffen erschließen lässt. Diese als "physionomics" bezeichnete Technologie stellt für uns die Grundlage dar, nachgeschaltet aus einer Auswahl aufwendigerer molekularer, biochemischer, histochemischer und analytischer Methoden die geeignete(n) einzusetzen, um den Wirkungsmechanismus weiter einzugrenzen und den herbiziden Wirkort zu identifizieren.

Josef Haber
Consumer Care, BASF AG

Several factors are drivers of the change processes in the food value chains: saturated markets for food products, scandals like BSE, dioxin etc., concentration of food companies and food retailers, the influence of NGO's and consumer advocates. In Europe the General Food Law with its paragraph about traceability plays an important role.
Key players in the chain look for differentiation from their competitors by offering new products to the consumers. Food safety and food quality are the decisive factors. Both tangible criteria (residues of pesticides, fat content) and intangible arguments (sustainable agriculture, organic) are frequently used.
In the same time the change in the CAP exposed agriculture even more to open market conditions. The products and services coming from farms will be produced to fulfil specific market needs which require a close cooperation with downstream partners.
In the same time competition between players at the same level of the chain (e.g. mills or juice producers) will extended into competition of organised "chains" (e.g. wheat-flour-bread) where chain management is a key success factor.
Improvements in the quality of the raw materials (physical properties, nutritional value) are the contribution of farmers and input suppliers. The respective products should be based on innovative research and development in agriculture and nutrition.

Hashim, M.A.1); Accinelli, C.2); Schneider, R.J.1); Christian, T.1); Goldbach, H.E.1)
1)Institute of Plant Nutrition, University of Bonn, Karlrobert-Kreiten-Str. 13, 53115 Bonn
2) Department of Agroenvironmental Sciences and Technology, Viale Fanin 44, 40127 Bologna, Italy

Metolachlor (2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methyl-1-methylethyl) acetamide) is a chloroacetanilide herbicide which is primarily used on maize in Germany as well as worldwide. Structurally, the herbicide consists of a mixture of two enantiomers, i.e. S-enantiomer and R-enantiomer, of which the first is basically responsible for the herbicidal activity of Metolachlor. Nowadays the herbicide is being marketed as a mixture of about 88 % S-enantiomer and 12 % R-enantiomer.
A considerable share of agricultural soils receive liquid manure as an organic fertilizer. It is now known that this manure may contain veterinary antibiotics in the ppb range. Until now it is not clear, if these concentrations can interfere with the microbial biomass in soils.
As the degradation process of metolachlor is mostly biotic, and hence most probably enantioselective, it is worthy to investigate how the degradation of the herbicide R,S-metolachlor would proceed in the presence of antibiotics in the soil.
In order to do so, the degradation of metolachlor in soil (initial concentration of 5 μg/g) was followed up in the presence of different concentrations and combinations of the following antibiotics: Sulfadimidin (50 and 500 nmol/kg), Enrofloxacin, Erythromycin, Gentamycin, Oxytetracyclin, Penicillin G, Sulfadiazin, Streptomycin, Trimethoprim and Tylosin (mix of 50 nmol/kg each). It appears that degradation of metolachlor is not retarded but enhanced probably due to a co-metabolic process.

Andrea Hemmerlin1, 2*, Susan B. Rivera1, Hans K. Erickson1 and C. Dale Poulter1
1Department of Chemistry, University of Utah, Salt Lake City, Utah 84112-0850. 2Centre National de la Recherche Scientifique (UPR 2357), Institut de Biologie Moléculaire des Plantes, F-67083 Strasbourg
*corresponding author: Andrea.Hemmerlin@ibmp-ulp.u-strasbg.fr

Reference:
Hemmerlin, A., Rivera, S.B., Erickson, H.K. and Poulter, C.D. (2003) Enzymes encoded by the farnesyl diphosphate synthase gene family in the Big Sagebrush Artemisia tridentata ssp. spiciformis. J. Biol. Chem., 278(34): 32132-32140

M. Heß, H. Hausladen und Ingrid Heiser

The formation of spots on the upper, light exposed leaves followed by rapid loss of green leaf area and early ripening is a phenomenon observed frequently on winter barley. Because of the absence of significant infections by barley pathogens like mildew, rust, Rynchosporium or Drechlera teres these spots are often attributed as Physiological Leaf Spot (PLS). Apart from a physiological reaction to light exposure as a abiotic factor, Ramularia collo-cygni as a biotic factor is discussed as causal agent for leaf spots since heavy sporulation by this fungus can be found on necrotic leaf areas.
Despite the uncertainty of the causal agent, leaf spot symptoms can be reduced effectively by fungicide treatment. Field trials show, that the effect of the fungicide treatment is dependent on the fungicide used and timing of application. The effect on leaf spot formation and yield for the different treatments will be shown.
To optimise the fungicide treatment, a better understanding of the epidemiology of the leaf spot complex is needed. Results from PCR diagnosis for Ramularia collo-cygni and the finding of a photodynamic toxin, produced by Ramularia collo-cygni, offer new opportunities for epidemiological research and further investigation of the mode of action of the fungicide treatment.

Horbach, R.; Reimann, S.; Deising, H. B.
Martin-Luther-Universität Halle-Wittenberg, Institut für Pflanzenzüchtung und Pflanzenschutz, 06099 Halle (Saale)

Stefan Irmler, Christian Pillonel
Syngenta Crop Protection AG, 4002 Basel, Switzerland

Ref.:

1. Pillonel and Meyer (1997), Effect of phenylpyrroles on glycerol accumulation and protein kinase activity of Neurospora crassa; Pesticide Science 49, p. 229-236
2. Zhang et al. (2002), Osmoregulation and fungicide resistance: the Neurospora crassa os-2 gene encodes a HOG1 mitogen-activated protein kinase homologue; Applied and Environmental Micorbiology 68, p. 532-538

Joußen, N., Schuphan, I. and Schmidt, B.
Department of Biology V, Aachen University (RWTH), Worringerweg 1, 52056 Aachen

The aim of the investigation was to examine, if a human gen influences the secondary metabolism in transformed plants of Nicotiana tabacum L..
For that, leaves of N. tabacum L. were transformed with human Cyp1a2 by Agrobacterium tumefaciens. This gene encodes for an enzyme of the cytochrom P450-family (P450 or CYP) consisting of monooxygenases, that play an important role both in natural metabolism of animals and plants and in metabolism and biotransformation of xenobiotics. More than 30 regenerated plants were tested by SDS-PAGE and Western blot for level of proteins as well as by PCR for level of DNA to find out, if they were successfully transformed and expressing Cyp1a2. The ECOD assay was used in addition to prove the functional ability of the human enzyme. After that, leaf material from 29 transgenic plants was extracted. The extracts were analyzed by HPLC. In man, P450 isozyme CYP2A6 is responsible for metabolism of nicotine to cotinine or different hydroxylated derivatives. According to literature, CYP1A2 itself shows a poor turnover of nicotine. Therefore, a lower content of nicotine and the presence of metabolites of the alkaloid in the transgenic plants were suspected. Apart from nicotin, another compound was detected, but it was impossible to characterize it due to lack of a corresponding standard. According to the UV spectrum, this substance should resemble nicotine structurally.
It was ascertained that most of the examined transgenic plants contained less nicotin in comparison to nontransgenic plants or contained no nicotin at all.

Grit Laue, Klaus Kreuz, Ernst Gassmann, Tilman Brodmeier, Joachim Blanz
Syngenta CP AG, Basel

Sauter H., Lauer M., Fritsch H.: Metabolic Profiling of plants a new diagnostic technique.
In: Baker D.R., Fenyes J.G., Moberg W.K. (Eds), American Chemical Society Symposium Series No. 443, 1991, 288-299
Aranibar N., Singh B.K., Stockton G.W., Ott K.: Automated Mode-of-Action Detection by Metabolic Profiling
Biochemical and Biophysical Research Communications 286, 2001, 150-155

Ralf Nauen
Bayer CropScience AG, Research, GBI, Insect Toxicology and Resistance, 40789 Monheim, Germany

The sweet-potato whitefly, Bemisia tabaci (Homoptera: Aleyrodidae) is a serious pest in numerous cropping systems worldwide and this species occurs in different, sometimes also geographically separated biotypes such as the Q- and B-type. The most common biotype, designated type B and sometimes described as Bemisia argentifolii Bellows & Perring, is by far the most devastating one. Biotype determinations of Spanish populations of B. tabaci indicated at least two different biotypes, the B-type and a so-called non B-type (also referred to as Q-type).
Resistance to insecticides with different modes of action such as organophosphates, carbamates, pyrethroids and insect growth regulators such as buprofezin is well described for several strains collected from different geographic areas. One of the latest group of insecticides introduced to the market for whitefly control were the neonicotinoids (chloronicotinyls) which act agonistically on insect nicotinic acetylcholine receptors. Resistance to neonicotinoid insecticides has recently been shown to occur in B. tabaci Q- and B-types in some places, especially in Almeria, Spain. Studies to investigate the biochemical mechanisms of resistance were carried out on both biotypes.
Metabolic enzymes providing pesticide resistance such as esterases, glutathione S-transferases and cytochrome P450-dependent monooxygenases were studied in many species of insects and also spider mites and a few examples were given. Another important mechanism of resistance is a mutation of the binding site in target proteins affected by insecticides, so-called target site resistance.
A fluorometric microplate assay with 7-ethoxycoumarin as substrate revealed a clear correlation between neonicotinoid resistance and enhanced microsomal 7-ethoxycoumarin-O-deethylase activity. Metabolism of [14C]imidacloprid in vivo was investigated in several B. tabaci strains, too. Two metabolites were detected, 5-hydroxy-imidacloprid and the olefine compound. The olefine showed the same insecticidal acrivity and binding affinity to nAChR than imidacloprid itself, whereas 5-hydroxy-imidacloprid was more than 10 times less active. Furthermore we demonstrated that neonicotinoid-resistance is not due to an altered [3H]imidacloprid binding site of nicotinic acetylcholine receptors in Q- and B-type strains.

S. Peterek und D. Treutter
Fachgebiet für Obstbau, Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt, 85350 Freising, Alte Akademie 16

Der Bioregulator Prohexadion-Ca (Regalis©; BASF) wurde bislang vor allem zur Kontrolle des vegetativen Wachstums im Obstbau eingesetzt. In verschiedenen Studien konnten nun nach einer Prohexadion-Ca-Anwendung zusätzlich zu der Wachstumsreduktion, ein verminderter Befall von Krankheitserregern wie beispielsweise dem Feuerbranderreger (Erwinia amylovora) und dem Schorferreger (Venturia inaequalis) festgestellt werden. Aufgrund verschiedener Forschungsergebnisse, in denen phenolische Verbindungen mit einer erfolgreichen Pathogenabwehr in Zusammenhang gebracht wurden, geht man davon aus, dass die Resistenzinduktion nach einem Prohexadion-Ca-Einsatz ebenso auf Veränderungen im Flavonoidstoffwechsel beruht.
Um den Zusammenhang zwischen phenolischen Inhaltsstoffen und dem Resistenzverhalten von Birnen gegen den Feuerbranderreger zu untersuchen, wurden Birnenblättern nach Einsatz des Wachstumsregulators Prohexadion-Ca analysiert. Die Ergebnisse hieraus bestätigen eine bereits in Studien am Apfel festgestellte Beeinflussung der Flavonoid-Biosynthese. Neben Modifikationen im regulären Biosyntheseweg, konnten auch neuartige phenolische Substanzen nachgewiesen werden. So findet zum Beispiel eine Neuinduktion des 3-Deoxycatechins Luteoliflavan und des Flavanons Eriodictyol 7-glukosid statt. Zusätzlich zu diesen Akkumulationen war eine Konzentrationszunahme der monomeren Flavanole Catechin und Epicatechin sowie deren entsprechenden Procyanidine (B1, B2, B5, E-B5) zu beobachten. Im Gegensatz dazu verringerten sich die Gehalte der Flavonolglykoside.
Die Bedeutung dieser Veränderungen in der Flavonoid-Biosynthese in Birnenblätter wird im Rahmen dieser Arbeit zusätzlich in Bezug auf den Feuerbrand untersucht. Welche Rolle dabei einzelne Substanzen oder Phenolklassen auf den Infektionsverlauf spielen, wird derzeit erforscht.

Diese Arbeit wurde aus dem EU-Projekt 1999-01583: Quality of Life and Management of Living Resources (ERWINIA) finanziert

Puhl,I. 1, J.V. Hermann2, H. Hofmann2, D. Treutter1
1 Fachgebiet für Obstbau, Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt, Technische Universität München, Alte Akademie 16, 85350 Freising
2 Bayerische Landesanstalt für Weinbau und Gartenbau ,Veitshöchheim

Der Wachstumregulator Prohexadion-Ca ist strukturell dem 2-Oxoglutarat ähnlich und aufgrund dieser Eigenschaft ist es in der Lage, alle Enzyme des Phenolstoffwechsels, die 2-Oxoglutarat als Co-Substrat benötigen, zu inaktivieren (1).
Deswegen wurden nach der Behandlung mit Prohexadion-Ca in jungen Rebenblättern neuartige phenolische Verbindungen sowie Verschiebungen im Metabolismus konstitutiver phenolischer Verbindungen nachgewiesen (2). Die neuen Substanzen wurden als Flavanone und 3-Deoxyflavanoide identifiziert, deren Vorkommen in der Weinrebe bisher noch nicht beschrieben wurde (2).
Der Gehalt an konstitutiven phenolischen Verbindungen in den behandelten Blättern verändert sich unterschiedlich in Abhängigkeit von deren Struktur. Während der Gehalt an Flavonolen (Quercetin-Glycosiden) abnahm, zeigte die Konzentration der Flavanole eine deutliche Zunahme (2).
Flavanole und Deoxyflavanoide haben toxische Eigenschaften gegen Pathogene (3,4), wodurch die Wirkung von Prohexadion-Ca gegen Krankheiten der Weinrebe erklären werden kann.

• Rademacher, W. 2000: Growth Retardants: Effects on Gibberellin Biosynthesis and Other Metabolic Pathways. Plant Mol. Biol., 51: 501-531
• Gosch, C., Puhl, I., Halbwirth, H., Schlangen, K., Römmelt, S., Andreotti, C., Costa, G., Fischer, T.C., Treutter, D., Stich, K., Forkmann, G. 2003: Effect of Prohexadione-Ca on Various Fruit Crops: Flavonoid Composition and Substrate Specificity of their Dihydroflavonol 4-Reductases. Europ. J. Hort. Sci., 68: 144-151
• Scalbert, A. 1991: Antimicrobial properties of tannins. Phytochemistry, 30: 3875-3883
• Römmelt, S., Fischer, T.C., Halbwirth, H., Peterek, S., Schlangen, K., Speakman, J.-B., Treutter, D., Forkmann, G., Stich, K. 2003: Effect of Dioxygenase Inhibitors on the Resistance-related Flavonoid Metabolism of Apple and Pears: Chemical, Biochemical and Molecular Biological Aspects. Europ. J. Hort. Sci., 68: 129-136

K. Schulze, A. Rimke, L. Schreiber and J. Schönherr

Apple scab (Venturia inaequalis) and powdery mildew (Podospheara leucotricha) belong to the most important fungal pathogens in fruit production. Using isolated cuticular membranes (CM), we developed a novel in-vitro-technique for testing fungicidal activity of chemicals against these two pathogens.
With both pathogens germination of conidia and formation of appressoria initiates the infection process. V. inaequalis can easily be propagated on artificial media and conidia readily germinate on artificial surfaces such as glass, agar and teflon®. P. leucotricha is an obligate biotrophic fungus which forms conidia only on leaf tissue. In our experiments conidia of P. leucotricha did not germinate on artificial surfaces but it germinated readily on the outer surface of isolated CM. Cuticular membranes can be isolated enzymatically from host leaves and both structure and chemical composition are identical to the leaf surface which are normally colonised by scab and mildew. When applied to isolated CM conidia of both pathogens germinated with a germtube and they formed appressoria, which is the prerequisite for a successful penetration of the cuticle. This is an important advantage of this technique compared to tests on agar. On agar V. inaequalis does not produce appressoria and P. leucotricha did not even germinate. This test was used to evaluate fungicidal activity of chemicals on germination and formation of appressoria. With V. inaequalis penetration of the CM could also be investigated. Investigation of the fungus/cuticle interactions can be studied before, during and after germination.
The technique offers several advantages: (1) It is rapid and results are obtained within a few days without laborious sample preparations. (2) Fungicidal effects can be evaluated quantitatively using a vital stain and fungicidal and fungistatic effects can be distinguished. (3) The test can be conducted throughout the year and does not rely on living plants, with the exception of the production of conidia for P. leucotricha. (4) Compared to screenings on agar, the specific effects of substances on the formation of appressoria and penetration of the cuticle can be studied. Results obtained with this new technique are encouraging. It can be used to study cuticle/pathogen interactions and to establish dose-effect relations for new active ingredients or endogeneous compounds involved in resistance.

Helge Sierotzki*, Lucijana Pavic*, Ursula Hugelshofer*, Carole Stanger**, Sally Cleere**, John Windass** und Ulrich Gisi*
*Syngenta Crop Protection AG, CH 4332 Stein, Schweiz
**Syngenta, Jealotts Hill Int. Research Cen, Bracknell, Berkshire, RG42 6EY, United Kingdom

Fungizide mit neuartigem Hemmmechanismus treffen im Normalfall auf vorwiegend sensitive Individuen einer Pathogenpopulation. Ein oft sehr kleiner Teil der Population ist aber entweder resistent oder zumindest vermindert sensitiv gegenüber dem neuen Fungizid. Die Veränderung der Population unter Selektion des Fungizids hängt von quantitativen und qualitativen Eigenschaften der Resistenz ab. Man kann zwei grundsätzlich verschiedene Wege beschreiben: Disruptiv, wenn sich die sensitiven und resistenten Individuen durch einen grossen Resistenzfaktor unterscheiden (RF>100) und kontinuierlich, wenn Zwischenstufen bezüglch der Sensitvität in der Population gefunden werden und der Resistenzfaktor relativ klein ist (RF<3<50).
Als Beispiel wird die Resistenz von Mycosphaerella graminicola (anamorph Septoria tritici), ein Pathogen auf Weizen, gegenüber den QoI-Fungizden, Hemmer der Atmungskette, und den DMI-Fungiziden, Hemmer der Strolbiosynthese, besprochen.

Induction of resistance by plant activators in apple leaves

Strissel, T.1,2, Halbwirth, H.2, Hoyer, U.1,2, Zistler, C.1,2, Stich, K.2 und Treutter, D.1
1 Technische Universität München - Weihenstephan, Fachgebiet Obstbau, Alte Akademie 16, D-85350 Freising
2 Technische Universität Wien, Institut für Verfahrenstechnik, Umwelttechnik und Technische Biowissenschaften, Getreidemarkt 9, A-1060 Wien

Für Apfel konnte in früheren Untersuchungen die Bedeutung der Phenylpropanoide, insbesondere der Catechine und Procyanidine, bei der Abwehr des Apfelschorfes, verursacht durch Venturia inaequalis, aufgezeigt werden. Insbesondere eine schnelle Neusynthese dieser Substanzen nach Befall scheint dabei von wesentlicher Bedeutung zu sein. Im Rahmen dieses Projektes wurde die Beeinflussung der Flavonoid-Biosynthese durch Pflanzenstärkungsmittel an jungen Apfelblättern der Sorten 'Golden Delicious' und 'Rewena' sowohl auf enzymatischer als auch auf chemisch-analytischer Ebene untersucht. Eine Beeinflussung der Induktion durch unterschiedliche N-Ernährungszustände wurde ebenfalls berücksichtigt.
Pflanzenstärkungsmittel auf Basis von Gesteinsmehlpräparaten (z.B. Myco-Sin und SilKaBen), Pflanzenextrakten (z. B. Phyto-Vital), Huminsäuren (z. B. Biokal 01 und 02) und K-Wasserglas wurden an jungen Trieben appliziert. Die Aktivität von Phenylalaninammoniumlyase (PAL), Chalkonsynthase/-isomerase (CHS/CHI), Flavanon 3-Hydroxylase (FHT) und Dihydroflavonol 4-Reduktase (DFR) wurde zu drei Zeitpunkten nach der Behandlung bestimmt. Zeitgleich wurden Proben für chemisch-analytische Untersuchungen genommen. Dabei wurden die Gehalte an Hydroxyzimtsäuren (u.a. p-Cumarsäure), Phloretinderivaten (u.a. Phloretin und Phloridzin), acylierten Dihydrochalkonen, Flavonolen (Quercetinglukoside) und Flavanolen (Catechine und Procyanidine) ermittelt.
Es zeigte sich, dass die Behandlung mit Pflanzenstärkungsmitteln grundsätzlich zu einer Induktion der Flavonoid-Biosynthese beitragen konnte, die Reaktionsgeschwindigkeit aber von Mittel, Sorte und dem N-Ernährungszustand beeinflusst wurde. Die Behandlung mit Gesteinsmehlpräparaten führte auf enzymatischer Ebene im Vergleich zu den übrigen untersuchten Mitteln zu stärkeren Induktionen der Schlüsselenzyme PAL und CHS/CHI. Als Ursache hierfür ist eine mögliche Reaktion auf oberflächliche Verwundungen durch die Silikatbestandteile zu sehen. Die Applikation von Myco-Sin trug bei 'Golden Delicious' zu einer zeitlich aufeinanderfolgenden Induktion der Enzyme PAL, CHS/CHI, FHT und DFR bei. Im Apfelblatt konnte mit den Enzymaktivitäten korrelierend ein Anstieg von Hydroxyzimtsäuren, Phloretinderivaten, Flavonolen und Flavanolen beobachtet werden, was zu einem verbesserten Abwehrpotential gegen Venturia inaequalis im Freiland beigetragen hat. Eine erhöhte Stickstoffversorgung führte zu einem Rückgang der spezifischen Enzymaktivitäten der Schlüsselenzyme PAL und CHS/CHI. Hierin ist eine Ursache für den Rückgang an phenolischen Inhaltsstoffen zu sehen, wie er an Bäumen unter luxuriösem N-Ernährungszustand beobachtet wurde. Bei luxuriös N-versorgten Bäumen wurde in diesem Zusammenhang auch eine schlechtere Induktion der Schlüsselenzyme gemessen.

Telscher M., Schäffer A. und Schmidt B.
Institut für Biologie V, Technische Universität Aachen, Worringer Weg 1, 52056 Aachen

The fate and behaviour of pesticides in the environment is mainly by adsorption to different soil components. Therefore in this study, the sorption behaviour of two metabolites of the herbicide terbuthylazine, hydroxy-desethyl-terbuthylazine (HDT) and desethyl-terbuthylazine (DT) to Na-bentonite alone and in the presence of peat humic acids was determined. Experiments were carried out in dialysis chambers build from Teflon. A dialysis membrane with mwco (middle weight cut off) 1000 Da seperated the two half cells. Via lockable openings in each of the two half cells, easy sampling with an Eppendorf-pipette and measurement as well as variation of the pH value could be made. Measurements of pH values were made using a pH electrode with 3 mm in diameter. Humic acids were extracted from peat with sodium-hydroxide-solution and dialyzed against 1000 Da, Na-bentonite was perchased from Süd-Chemie and used without cleaning.
The adsorption to different soil components was strongly influenced by the physico-chemical properties of the analytes. Maximum adsorption was at pH equal to pKa for DT and pKa1 for HDT. The impact of humic acids on the adsorption of HDT to Na-bentonite was negligible, whereas the adsorption of DT to Na-bentonite was substantially smaller in the presence of humic acids and maximum of adsorption was shifted from pH 1.3 to pH 2.0. At pH ≥ 8 adsorption of HDT to Na-bentonite was in general smaller than adsorption of DT due to deprotonation of the hydroxyl group of HDT (pKa2). Adsorption potential of HDT in pH range of 5 to 7 was considerably higher than that of DT. So the risk of leaching is regarded greater for DT than for HDT.

H.G.Teutsch

Accumulation of nutrients is a characteristic for a fungus' aliveness. Scintillation proximity technology, in 96-well format, allows to measure the accumulation of any radiolabelled compound by fungal spores and hyphae as long as they grow at the bottom of the wells. Botrytis cinerea and Phytophthora infestans do virtually stick to plastic surfaces and grow horizontally during the first 24 hours after germination.
This makes it possible to precisely measure the influence of chemicals on fungi at very intersting growth stages:

• before and during germination
• young mycelium, where the ratio of the very active hyphal tips vs. "old", inactive hyphae is high.

1. The growth inhibition by a fungicide is nicely reflected by the course of nutrient uptake and is characteristic for the type and the concentration of a fungicide (slow / fast / with lag phase / fungistatic / fungicidal / transient effect etc.). Several Modes of Action can be classified by this method.
2. Because of the precision of the measurements, even subtle influences of additives can be observed: reversion of inhibition by a fungicide, optimisation of a growth medium etc.
3. Due to the adhesion of the mycelium to the surface, the supernatant can easily be changed (for example to wash away a fungicide, data not shown).
4. The bottom of the wells being transparent, the mycelium can also be looked microscopically and the symptoms after fungicide treatment can be related to the physiology.

Sandra Trenkamp und Klaus Tietjen
Bayer CropScience AG, Target Research, Geb. 6240, 51368 Leverkusen, Germany

In order to study the mode of action of HRAC-class K3 and N herbicides we conducted a pilot study analysing phenotype and gene expression of flufenacet- and benfuresate-treated Arabidopsis thaliana plants. Treatments with either herbicide caused phenocopies of the known Arabidopsis mutant fiddlehead, displaying fused organs and the typical fiddlehead-like inflorescence. Herbicide treatments of other plant species, including monocots, also gave rise to analogous organ fusions, indicating the presence of the target in a broad range of plants. The FIDDLEHEAD gene encodes a putative very-long-chain fatty acid elongase (VLCFAE), which corroborates earlier biochemical results pointing to the inhibition of VLCFA synthesis as mode of action of flufenacet. Gene expression profiles of herbicide-treated plants using the first 8247 gene Arabidopsis gene array of Affymetrix provided additional clues in support of inhibition of VLCFA synthesis. Of the 21 genes encoding VLCFAEs from Arabidopsis thaliana, we could express 17 heterologously in Saccharomyces cerevisiae. Six VLCFAEs, including three known elongases (FAE1, KCS1 and KCS2) and three previously uncharacterized gene products (encoded by At5g43760, At1g04220 and At1g25450) were found to be enzymatically active with endogenous yeast fatty acid substrates. The spectrum of VLCFAs accumulated in expressing yeast strains was determined by gas chromatography/mass spectrometry. The active VLCFAEs revealed highly distinct patterns of differential sensitivity to oxyacetamides, chloroacetanilides, and other compounds tested, while yeast endogenous VLCFA production, which involves its unrelated elongase (ELO) for sphingolipid synthesis, was unaffected. These findings pinpoint VLCFAEs as the target of the widely used herbicides, which have been in commercial use for 50 years, and provide important clues as to why spontaneous resistance to this class is rare.

Andreas Vilcinskas
Institute of Biochemistry and Biology, University of Potsdam, Potsdam, Germany

Insects exhibit a tremendous evolutionary success and possess powerful immune system which is based on cellular and humoral components. Insect innate immune responses are characterised by the rapid and transient synthesis of antimicrobial proteins and proteinase inhibitors that are active against pathogen-associated proteases. Investigating immune-related genomics and proteomics of the Greater wax moth, Galleria mellonella, we identified a number of antibacterial and antifungal proteins. Recently, we have cloned and expressed a recombinant antifungal protein from G. mellonella exhibiting similarity with plant defensins and Drosomycin from Drosophila. The recombinant peptide is neither active against Gram-positive and Gram-negative bacteria nor against yeast, but inhibits in vitro growth of the entomopathogenic fungus Metarhizium anisopliae. Therefore, the antifungal protein from G. mellonella has been named Gallerimycin.
Among the proteins that are induced and secreted within the hemolymph of G. mellonella after activation of innate immune responses we discovered a number of novel proteinase inhibitors which are active against proteases produced by M. anisopliae. Simultaneous synthesis of Gallerimycin and inhibitors of M. anisopliae proteases during G. mellonella innate immune response implies functional synergism in vivo.
Screening for activity of recombinant antifungal insect proteins against phytopathogenic fungi will be one part of our attempts to explore the therapeutic potential of antifungal insect proteins for the protection of crops. The potent insect innate immune system may represent a promising resource of genes for the design of transgenic plants that are resistant against phytopathogens.