No abstract

The efficacy of all major insecticide classes continues to be eroded by the development of resistance mediated, in part, by selection of alleles encoding insecticide insensitive target proteins. The discovery of new insecticide classes acting at novel protein binding sites is therefore important for the continued protection of the food supply from insect predators, and of human and animal health from insect borne disease. Here we describe a novel class of insecticides (Spiroindolines) encompassing molecules that combine excellent activity against major agricultural pest species with low mammalian toxicity. We confidently assign the vesicular acetylcholine transporter as the molecular target of Spiroindolines through the combination of molecular genetics in model organisms with a pharmacological approach in insect tissues. The vesicular acetylcholine transporter can now be added to the list of validated insecticide targets in the acetylcholine signalling pathway and we anticipate that this will lead to the discovery of novel molecules useful in sustaining agriculture. In addition to their potential as insecticides and nematocides, Spiroindolines represent the only other class of chemical ligands for the vesicular acetylcholine transporter since those based on the discovery of vesamicol over 40 years ago, and as such, have potential to provide more selective tools for PET imaging in the diagnosis of neurodegenerative disease. They also provide novel biochemical tools for studies of the function of this protein family.

From 1997 onward, the strobilurin fungicide azoxystrobin was widely used in the main banana-production zone in Costa Rica against Mycosphaerella fijiensis var. difformis causing black Sigatoka of banana. By 2000, isolates of M. fijiensis with resistance to the quinolene oxidase inhibitor fungicides were common on some farms in the area. The cause was a single point mutation from glycine to alanine in the fungal target protein, cytochrome b gene. An amplification refractory mutation system Scorpion quantitative polymerase chain reaction assay was developed and used to determine the frequency of G143A allele in samples of M. fijiensis. Two hierarchical surveys of spatial variability, in 2001 and 2002, found no significant variation in frequency on spatial scales <10 m. This allowed the frequency of G143A alleles on a farm to be estimated efficiently by averaging single samples taken at two fixed locations. The frequency of G143A allele in bulk samples from 11 farms throughout Costa Rica was determined at 2-month intervals. There was no direct relationship between the number of spray applications and the frequency of G143A on individual farms. Instead, the frequency converged toward regional averages, presumably due to the large-scale mixing of ascospores dispersed by wind. Using trap plants in an area remote from the main producing area, immigration of resistant ascospores was detected as far as 6 km away both with and against the prevailing wind.

Azoxystrobin is a common fungicide used by farmers of Solanaceous crops against Alternaria solani, but there was growing concern about decreased sensitivity with repeated applications. In 2002 and 2003, monitoring of A. solani from commercial potato fields in Wisconsin indicated increased frequency and a statewide distribution of isolates with decreased in vitro sensitivity to azoxystrobin. Mean effective concentration in inhibiting spore germination by 50% values gathered in 2002 and 2003 were approximately 20-fold higher than baseline isolates of A. solani collected in 1998 from fields that had never been treated with azoxystrobin. This sensitivity decrease was correlated with site-specific mutations in the cytochrome b detected by quantitative real-time polymerase chain reaction. The F129L and the G143A substitution have been shown to cause a reduction in sensitivity or resistance, respectively, to quinone outside inhibitors. All of the recovered A. solani isolates collected in 2002 and 2003 were wild type at position 143. However, all three mutations responsible for the F129L substitution (TTA, CTC, and TTG) were detected in our samples. In addition, the frequency of this amino acid substitution in A. solani isolates was statistically different across sampling sites and years, indicating that sensitivity changes depended on specific disease management practices.

The I1781L amino acid substitution in the target ACCase enzyme causes broad resistance to ACCase inhibitor herbicides in several monocotyledenous weeds of agronomic importance. This mutation results from a substitution of an adenine (A) residue by either a thymine (T) or cytosine (C) at position 5341 in Alopecurus myosuroides Huds and at an equivalent position in Lolium species, Avena fatua L. and Setaria viridis (L.) Beauv. Two different procedures, the PCR‐based allele‐specific assay (ASA) and the derived cleaved amplified polymorphic sequence (dCAPS) method, have previously been described for detecting this mutation. These methods are, however, only amenable to low sample throughput and are used in the analysis of single plants. Here, an alternative high‐throughput ARMS/Scorpion real‐time quantitative PCR (Q‐PCR) method for measuring levels of the I1781L mutation in pools of leaf and seed samples of Lolium populations is presented. The limit of detection for C and T mutant alleles in a background of wild‐type A is 0.02 and 0.0003% respectively. In this study, DNA from batches of 24 leaf segments measuring 0.5 cm from different plants or 1000 seeds could be conveniently extracted and accurately analysed. As part of assay validation, the comparative analysis of five geographically distinct Lolium populations with dCAPS and Q‐PCR procedures demonstrated the accuracy of the latter method, and the three possible II1781, IL1781 and LL1781 ACCase genotypes being distributed as predicted by the Hardy–Weinberg principle. Given the dominance of the L1781 over the I1781 allele at recommended field rates for most ACCase inhibitors, the frequency of herbicide survivors in the field owing only to the presence of the I1781L mutation is thus predicted to be 2pq + q2, where p and q are the frequencies of the I1781 and L1781 alleles as determined by Q‐PCR. The Q‐PCR assay established allows detection of very low levels of the L1781 ACCase mutation before resistance would normally be discernible in the field. Therefore, it offers the opportunity to tackle resistance at its very onset, potentially avoiding implementation of complicated and often costly weed management practices. Copyright © 2006 Society of Chemical Industry