First detection of etoxazole resistance in Australian two-spotted mite Tetranychus urticae Koch (Acarina: Tetranychidae) via bioassay and DNA methods Grant A Herron,* Lauren K Woolley, Kate L Langfield and Yizhou Chen New South Wales Department of Primary Industries, Elizabeth Macarthur Agricultural Institute, Private Bag 4008, Narellan, 2567, Australia. Abstract Etoxazole is used in Australia to control mites on a range of crops including pome fruit and cotton. Although resistance has been widely reported elsewhere, it has not previously been detected in Australia. Here, we report the first detection of etoxazole resistance (>12 500 fold) in Australian two-spotted mite Tetranychus urticae from pome fruit via both bioassay and DNA methods. It is envisaged that the DNA-based method developed here can be used to monitor etoxazole resistance in T. urticae. Key words bioassay, PCR-restriction fragment length polymorphism (RFLP), resistance detection, resistance management. INTRODUCTION Etoxazole was first registered for use in Australia in 2004 by Sumitomo Chemical Australia Pty Limited for spider mite control (mainly Tetranychus spp.) on a range of crops including horticulture and broad acre (APVMA 2016). Etoxazole belongs to the Insecticide Resistance Action Committee (IRAC) mode of action (MOA) group 10B known as mite insect growth inhibitors of unknown or uncharacterised biological activity (IRAC 2016). Etoxazole does not affect adults directly but causes them to produce unviable eggs (Kim & Yoo 2002; Dekeyser 2005; Nauen & Smagghe 2006). Furthermore, etoxazole disrupts the moulting process of mite juvenile stages (Yoo & Kim 2000). Etoxazole resistance in Tetranychus urticae has been reported in Japan (Kobayashi et al. 2001; Uesugi et al. 2002; Asahara et al. 2008) and South Korea (Lee et al. 2004), but not in Australia. Resistance is thought under the control of a single (Kobayashi et al. 2001) autosomal recessive (van Leeuwen et al. 2012) gene. The I1017F mutation found within the last transmembrane helix of the predicted C-terminal 5 transmembrane domain of chitin synthase (CHS1) was linked to etoxazole resistance in T. urticae (van Leeuwen et al. 2012). Recently, a genome-editing approach whereby the I1017F mutation was introduced into the Drosophila melanogaster CHS1 gene (kkv) provided functional evidence for a target-site mutation in etoxazole resistance (Douris et al. 2016). Herron (2005) established bioassay methodology for etoxazole against T. urticae and a discriminating dose for use in Australian cotton, but the product was not heavily used, and resistance monitoring was not conducted. However, recent data from Australian cotton suggest that resistance frequencies against key chemical controls may compromise efficacy and resistance to abamectin is increasing (Herron & Wilson 2016). For that reason, etoxazole use in Australian cotton may increase, and monitoring would again be a priority. Woolley et al. (2015) recently discovered the etoxazole resistance causing mutation I1017F in an Australian isolate of T. urticae, but the result was not considered unequivocal as no bioassay data was available for confirmation. Here, we corroborate the result of Woolley et al. (2015) against established bioassay methodology to unequivocally confirm etoxazole resistance in Australian T. urticae for the first time and provide a verified DNA-based method for resistance monitoring. MATERIALS AND METHODS Acaricide Supplied by Sumitomo Chemical Australia Pty Limited as Paramite® or Zeal® Selective Miticide (110 g/L etoxazole suspension concentrate) that is field sprayed (pome and stone fruit) at 35 mL product/100 L. Strains tested The reference susceptible strain was collected from an unsprayed ornamental crop in the Sydney region in 1988; it has been isolated since collection and is known to be susceptible (Herron et al. 2004). The field isolate ‘Nashee f ’ was collected in 2015 from a commercial fruit crop at Banbartha, New South Wales with unknown spray history. Cultures were maintained under insecticide-free conditions in separate, purpose-built cages on potted French bean (Phaseolus vulgaris L.) at 25°C (± 4°C) under natural light and transferred onto a fresh plant weekly (Herron et al. 2004). A sub strain of ‘Nashee f ’ known as ‘Nashee p ’ was sprayed with a 0.001 g/L etoxazole discriminating dose (Herron 2005) on an ad hoc basis to maintain resistance. *grant.herron@dpi.nsw.gov.au © 2017 State of New South Wales doi: 10.1111/aen.12290 Austral Entomology (2017) ••, ••–•• bs_bs_banner