Monitoring resistance allele frequency at the early stage of resistance development is important for the successful acaricide resistance management. Etoxazole is a mite growth inhibitor to which resistance is conferred by an amino acid substitution in the chitin synthase 1 (CHS1; I1017F) in T. urticae. If the susceptible allele can be specifically digested by restriction endonuclease, the ΔΔCt method using real-time PCR for genomic DNA (RED-ΔΔCt method) may be available for monitoring the resistance allele frequency. We tested whether the etoxazole resistance allele frequency in a pooled sample was accurately measured by the RED-ΔΔCt method and validated whether the resistance variant frequency was correlated with etoxazole resistance phenotype in a bioassay. Finally, we performed a pilot test using field populations. Strong linearity of the measures by the RED-ΔΔCt method with practical resistance allele frequencies; resistance allele frequency in the range between 0.5% to at least 0.75% was strictly represented. The strong linear relationship between hatchability of haploid male eggs after the etoxazole treatments (phenotype) and resistance allele frequencies in their mothers provided direct evidence that I1017F is a primary resistance factor to etoxazole in the strains used for experiments. The pilot test revealed a significant correlation between egg hatchability (including both diploid female eggs and haploid male eggs) and estimators in field populations. Consequently, we concluded that the RED-ΔΔCt method is a powerful tool for monitoring a resistance allele in a pooled sample.
Due to the development of acaricide resistance in Tetranychus urticae, a rapid and accurate monitoring method for acaricide resistance gene frequency is required for resistance management.In this study, we developed a new diagnostic gene frequency prediction method, using quantitative real-time PCR with allele-specific primer sets, for mutation of I1017F in chitin synthase I, G126S in mitochondrial cytochrome b, and H110R in the PSST subunit of mitochondrial electron transport complex I, which are involved in etoxazole, bifenazate, and pyridaben resistance in T. urticae, respectively. Resistance allele frequencies computed using the 2 −ΔΔCq method, in mixtures of resistance and wild-type (susceptible) alleles, were strongly correlated with the actual frequencies of the resistance alleles in DNA samples for all three acaricides (regression slopes of 0.945-0.996; R 2 > 0.99). This result strongly indicates that resistance allele frequency in a population can be accurately predicted using a diagnostic quantitative real-time PCR method with a resistance allele-specific primer set. Finally, we applied this method to nine field populations and successfully determined resistance allele frequencies for the three acaricides. Overall, this diagnostic prediction method may contribute to the development of acaricide resistance management strategies, via monitoring resistance allele frequencies for a wide range of acaricides.
The seasonal prevalence of the citrus red mite and its predators on Satsuma mandarin trees was surveyed by nakedeye observation and the beating method in three commercial groves in Shizuoka Prefecture in 2003 and 2004. Of the predators, only phytoseiid mites were caught mainly by beating, and their population dynamics were synchronized with those of the citrus red mite from June to September. Most adult females of phytoseiid mites caught by beating belonged to the species Neoseiulus californicus. Furthermore, one population of N. californicus caught from a surveyed grove showed development and oviposition on the eggs of the citrus red mite as well as the Tetranychus urticae eggs under experimental conditions. These results indicate that an indigenous population of N. californicus in conventionally controlled citrus groves in central Japan has the ability to suppress the population of P. citri on Satsuma mandarin.
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