Klaus Tietjen

Efficacy of plant metabolites of imidacloprid against Myzus persicae and Aphis gossypii (Homoptera: Aphididae)

The metabolism of the chloronicotinyl insecticide imidacloprid is strongly influenced by the method of application. Whilst in foliar application most of the residues on the leaf surface display unchanged parent compound, most of the imidacloprid administered to plants by soil application or seed treatment is metabolized more or less completely, depending on plant species and time. The present study revealed that certain metabolites of imidacloprid which have been described in crop plants are highly active against aphid pests in different types of bioassays. Some of these metabolites showed a high oral activity against the green peach aphid (Myzus persicae), and the cotton aphid (Aphis gossypii). The aphicidal potency of the metabolites investigated was weaker in aphid dip tests than in oral ingestion bioassays using artificial double membranes. The most active plant metabolite was the imidazoline derivative of imidacloprid. The LC50 values of this metabolite for M. persicae and A. gossypii in oral ingestion bioassays were in the lower ppb-range, i.e. 0·0044 and 0·0068 mg litre-1, respectively. Most of the other reported metabolites showed much weaker activity. Compared to imidacloprid, the imidazoline derivative showed superior affinity to housefly (Musca domestica) head nicotinic acetylcholine receptors, while all other metabolites were less specific than imidacloprid. It seems possible that, after seed treatment or soil application, a few of the biologically active metabolites arising are acting in concert with remaining levels of the parent compound imidacloprid, thus providing good control and long-lasting residual activity against plant-sucking pests in certain crops.

Aphicidal activity of imidacloprid against a tobacco feeding strain of Myzus persicae (Homoptera: Aphididae) from Japan closely related to Myzus nicotianae and highly resistant to carbamates and organophosphates

We investigated the resistance potential of a red-coloured Japanese strain (JR) of a tobacco feeding form of Myzus persicae (Sulzer) of the M. persicae species complex closely related to the tobacco aphid Myzus nicotianae Blackman. Bioassays were performed with a range of insecticides, imidacloprid, nicotine and cartap, thought to act on nicotinic acetylcholine receptors in vivo , as well as with two conventional insecticides, pirimicarb and oxydemeton-methyl, acting on acetylchol-inesterase (AChE). Compared to a susceptible strain, JR showed high resistance to pirimicarb and oxydemeton-methyl, but was far less resistant to nicotine, cartap and imidacloprid. Imidacloprid was, among the insecticides tested, the most active compound in contact and ingestion bioassays. Compared to the susceptible strain, JR showed four-to seven-fold resistance to imidacloprid depending on the type of bioassay. Resistance factors for other insecticides tested in an oral ingestion bioassay were: cartap five-fold, nicotine nine-fold, oxydemeton-methyl 107-fold and pirimicarb > 385-fold. JR showed high carboxylesterase activity. Polyacrylamide gel electrophoresis indicated esterase FE4 as the major carboxylesterase. As for most M. persicae strains and some Greek strains of M. nicotianae , JR was monomorphic for glutamate oxalacetate transaminase. Studies with pirimicarb showed a marked insensitivity of AChE to inhibition by this chemical, whilst such insensitivity could not be detected with the organophosphate insecticide oxydemeton-methyl. Receptor binding assays with [ 3 H]-imidacloprid in aphid homogenates revealed I 50 -values of 0.4 to 0.8 nM and no statistical difference between the JR and susceptible strain.

Antifeedant effect, biological efficacy and high affinity binding of imidacloprid to acetylcholine receptors in Myzus persicae and Myzus nicotianae

It is known from laboratory studies that tobacco-associated forms of Myzus persicae (Sulzer) and the closely related tobacco aphid Myzus nicotianae (Blackman) are often somewhat less susceptible to imidacloprid than non-tobacco strains of M. persicae. Choice tests (floating leaf technique) showed that tobacco aphids were also less susceptible to the antifeedant potential of imidacloprid in contact bioassays. Synergists like piperonyl butoxide or DEF did not enhance the susceptibility of tobacco-associated morphs of Myzus ssp. to imidacloprid, thus providing evidence that neither oxidative detoxication nor hydrolytic metabolization took place. However, in an attempt to study the influence of endosymbiotic bacteria on the efficacy of imidacloprid, we allowed small populations of tobacco aphids to feed on diets containing the antibiotic chlortetracycline prior to imidacloprid treatment. While the effectiveness of imidacloprid, i.e. lower LC50 values, could be improved in all strains, including the susceptible reference strain, there was no change in overall tolerance factors. In order to investigate any possible alteration of the target site, the affinity of imidacloprid and nicotine to nicotinic acetylcholine receptors in whole-aphid homogenates was measured. All strains (and clones) showed the same high-affinity binding sites and no detectable difference. Studies using the FAO dip method revealed that the lower susceptibility of M. nicotianae is not restricted to chloronicotinyls like imidacloprid or acetamiprid, because other insecticides with different modes of action such as pymetrozine and fipronil were also affected in laboratory studies. It is considered that the observed tolerance to chloronicotinyls in certain strains of Myzus ssp. is a natural variation in response, probably not coupled with any known mechanism of resistance in this species complex.

High throughput screening in agrochemical research.

The demand for new herbicides, insecticides and fungicides led to a steady increase in the number of compounds being tested to find novel market products. To keep pace with the rising workload, high throughput screening (HTS) technologies have been introduced. In agrochemical research miniaturised in vivo tests on whole real target organisms are now possible and are an integral part of the screening cascade. A complementary target based in vitro HTS has also been established in agrochemical research. Target based HTS allows a directed approach towards untouched market shares by novel modes of action. Selection of the best suited targets is the most crucial issue in this approach. Genomic methods thereby deliver many essential genes as candidate targets. Consideration of further criteria such as druggability notably narrows down the number of promising targets. Though target to hit to lead progression still is as in pharmaceutical research a complex and therefore risky process, the implementation of novel bioscience technologies has entailed the transition to an integrated innovative agrochemical research perspective.

Chloronicotinyl Insecticides: A Success of the New Chemistry

The insecticide world market, worth about 7 billion at the beginning of the 1990s, has long been dominated by old and well-established products belonging to the organophosphate, carbamate, and organochlorine classes and the newer class of pyrethroids. All these commercially important insecticides act at targets in the central nervous system and were responsible for more than 85% of worldwide insecticide sales. Inhibitors of the nicotinic acetylcholine receptor, the target of chloronicotinyl insecticides, were represented by earlier products like nicotine itself or nereistoxin analogs without any real commercial relevance at that time (Figure 1).

Ligands of the nicotinic acetylcholine receptor as insecticides

Insect nicotinic acetyl receptors (nAChR) are targets of growing importance and, since the early 1990s, the number of such highly effective insecticides as imidacloprid and spinosyn has grown. Several natural compounds, eg dihydro-β-erythroidine, methyl caconitine and paraherquamide, showing high affinity to the same receptor, were considerably less active as insecticides, most likely because of their antagonistic action. Our observations on aphids after ingestion of the antagonistic compound dihydro-β-erythroidine revealed anti-feedant-like properties. As a consequence, the symptomology of poisoning was totally different between agonists and antagonists of the nAChR. Electrophysiological (whole-cell voltage clamp) measurements in isolated housefly neurones revealed that agonism seems to be a prerequisite for insecticidal activity. Furthermore, we were able to demonstrate the existence of two different subtypes of the nAChR in isolated locust neurones with different pharmacology and ion-channel properties.

Identification of Ustilago maydis Aurora kinase as a novel antifungal target.

Infestation of crops by pathogenic fungi has continued to have a major impact by reducing yield and quality, emphasizing the need to identify new targets and develop new agents to improve methods of crop protection. Here we present Aurora kinase from the phytopathogenic fungus Ustilago maydis as a novel target for N-substituted diaminopyrimidines, a class of small-molecule kinase inhibitors. We show that Aurora kinase is essential in U. maydis and that diaminopyrimidines inhibit its activity in vitro. Furthermore, we observed an overall good correlation between in vitro inhibition of Aurora kinase and growth inhibition of diverse fungi in vivo. In vitro inhibition assays with Ustilago and human Aurora kinases indicate that some compounds of the N-substituted diaminopyrimidine class show specificity for the Ustilago enzyme, thus revealing their potential as selective fungicides.

Chapter 32. High Throughput Screening in Agrochemical Research

The demand for new herbicides, insecticides and fungicides led to a steady increase in the number of compounds being tested to find novel market products. To keep pace with the rising workload, high throughput screening (HTS) technologies have been introduced. In agrochemical research miniaturised in vivo tests on whole real target organisms are now possible and are an integral part of the screening cascade. A complementary target based in vitro HTS has also been established in agrochemical research. Target based HTS allows a directed approach towards untouched market shares by novel modes of action. Selection of the best suited targets is the most crucial issue in this approach. Genomic methods thereby deliver many essential genes as candidate targets. Consideration of further criteria such as druggability notably narrows down the number of promising targets. Though target to hit to lead progression still is as in pharmaceutical research a complex and therefore risky process, the implementation of novel bioscience technologies has entailed the transition to an integrated innovative agrochemical research perspective.

Binding of Triazines and Triazinones in the QB-Binding Niche of Photosystem II

Abstract A series of 20 triazines (derivatives of 2-alkylamino-4-benzylam ino-6-chloro-1,3,5-triazines) and 37 triazinones (derivatives of 3-alkyl-4-amino-6-phenyl-1,2,4-triazin-5-ones) is tested for inhibitory potency in photosynthetic electron flow through photosystem II of wild type Chlamydomonas reinhardtii and of five mutants with aminoacid substitutions in the D 1 protein at valine 219, alanine 251, phenylalanine 255, serine 264, and leucine 275. The data are used for computer modelling of the possible location of the compounds within a three dimensional model of the QB-binding niche of the D 1 protein.