bioRxiv preprint doi: https://doi.org/10.1101/092452; this version posted December 11, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. El-Sayed and Suckling: Use of phenylacetonitrile plus acetic acid to monitor Pandemis leafroller 2016 components. Further studies are warranted Use of phenylacetonitrile to develop phenylacetonitrile and possibly other aromatic plant volatiles as bisexual plus acetic acid to lures for the range of tortricid pests monitor Pandemis attacking horticultural crops. pyrusana (Lepidoptera: KEY WORDS: codling moth, leafroller, host- plant volatile, phenylacetonitrile Tortricidae) in apple INTRODUCTION Ashraf M. El-Sayed1*, David M. Suckling1,2 Pandemis spp. leafrollers are an important group of tortricid pests attacking pome 1The New Zealand Institute for Plant & Food fruits (apple, Malus domestica Borkhausen, Research Limited, and pear, Pyrus communis L.), in North Gerald Street, 7608, America (Chapman 1973, Dombroskie and Lincoln, New Zealand Sperling 2012) and in Europe and Asia (Dickler 1991). These species are often 2School of Biological Sciences, University of Auckland bivoltine and overwintering larvae in the fall- Tamaki Campus, Building 733, spring and summer generations feed on the Auckland, New Zealand skin of developed fruits adjacent to leaves creating cull fruits (Brunner 1996). *Author for correspondence: Insecticides are most often used to manage [email protected] these pests; although sex pheromone mating disruption has been developed and ABSTRACT A recent discovery has implemented on a relatively small acreage in demonstrated that herbivore induced plant North America (Judd and Gardiner 2008). volatile compounds from apple trees infested Sex pheromone-baited traps are used to with larvae were highly attractive to con- monitor adult populations to assess both specific adult male and female leafrollers. density and phenology, but pest managers However, this work (conducted in New feel they are insensitive to local population Zealand and Canada) tested only low doses densities within the orchards (Brunner of kairomone. Our US study here was 1984). Alternative lures, including acetic conducted to assess the attractiveness of acid, have been evaluated to monitor both higher doses of the six apple volatiles sexes of Pandemis pyrusana (Kearfott) putatively identified from apple trees (Knight, 2001); however, acetic acid alone infested by tortricid larvae to the leafroller, was considered to be a weak lure and was Pandemis pyrusana Kearfott. These volatiles not adopted by Washington State growers to included: β-caryophyllene, germacrene D, monitor P. pyrusana (Alway 2003). benzyl alcohol, phenylacetonitrile, (E)- nerolidol, and indole. No volatiles were Apple seedlings uniquely release several attractive to P. pyrusana when used alone. compounds, including acetic acid, acetic However, traps baited with anhydride, benzyl alcohol, phenylacetonitrile plus acetic acid caught phenylacetonitrile, indole, 2-phenylethanol, both sexes of P. pyrusana. Traps baited with and (E)-nerolidol only when infested by the other volatiles plus acetic acid caught larvae of light brown apple moth (LBAM), zero to only incidental numbers of moths (< Epiphyas postvittana (Walker), the 1.0). Interestingly, traps baited with obliquebanded leafroller (OBLR), C. phenylacetonitrile plus acetic acid caught rosaceana (Harris), and the eye-spotted bud significantly more P. pyrusana than traps moth (ESBM), Spilonota ocellana (Denis & baited with a commercial sex pheromone Schiffermüller) (Suckling et al. 2012; El- lure. The evaporation rate of the acetic acid Sayed et al. 2016). Recently El-Sayed et al. co-lure was an important factor affecting (2016) found that a binary blend of the two catches of P. pyrusana with herbivore-induced plant volatile compounds phenylacetonitrile, and studies are needed (HIPVCs), phenylacetonitrile + acetic acid to optimize the emission rates of both lure and 2-phenylethanol + acetic acid, attracted 1 bioRxiv preprint doi: https://doi.org/10.1101/092452; this version posted December 11, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. El-Sayed and Suckling: Use of phenylacetonitrile plus acetic acid to monitor Pandemis leafroller 2016 a significant number of conspecific male and Sept. 2013. In addition, the proprietary female adult LBAM in New Zealand. Further lures: Pherocon CM-DA lure loaded with investigation with other leafrollers pear ester, the Pherocon CM-DA combo lure (Tortricidae) in Canada, including the ESBM loaded with codling moth sex pheromone and OBLR, revealed similar systems. The and pear ester, and the Pherocon PLR lure study reported by El-Sayed et al. (2016) (#3147) loaded with the P. pyrusana sex targeted mainly ESBM and OBLR using a pheromone were obtained from Trécé Inc. lower doses of the new kairomone. In this study we have investigated the response of Field experiment protocols. Studies evaluated P. pyrusana to the compounds reported by single, binary, and ternary blends of El-Sayed et al. (2016) using higher doses of selected host-plant volatiles and acetic acid the HIPVCs in Washington State during co-lures using a standardized protocol in 2012–2014. Also, studies were conducted to 2012 and 2013. Studies were conducted in evaluate possible interactions with any of two apple orchards, the USDA research farm the attractive volatiles when combined with situated east of Moxee, WA (46o30’N, pear ester and acetic acid in binary and 120o10’W), and in a commercial orchard ternary blends for moth catches of both C. located southwest of Naches, WA (46o43’N, pomonella and P. pyrusana in the same trap. 120o42’W). Orange delta traps with sticky inserts (Pherocon VI, Trécé Inc.) were used MATERIALS AND METHODS in all studies. Five replicates of each lure Chemicals and Lures. Chemical purity of the treatment were randomized and spaced 20– volatile apple chemicals used in our trials 30 m apart. Traps were placed at a 2 m were as follows: benzyl alcohol (99%), (E)- height in the canopy. All lures were placed nerolidol (85%), phenylacetonitrile (99%), on the liner. Acetic acid vials with 3.2 mm indole (99%), β-caryophyllene (97%), and holes were used in all studies unless germacrene D (96%) (Sigma Aldrich, St. specified otherwise. Traps baited with a Louis, MO). These chemicals were loaded blank sachet lure were used as a negative (100 mg) into plastic sachet. Sachet lures control in each study. Studies were repeated consisted of a heat-sealed, semi-permeable a variable number of times and typically polyethylene bag (45 mm x 50 mm, 150 µm each test ran for 5–7 d. When studies were wall thickness), with a piece of cellulose repeated on different dates all traps were acetate filter (15 mm × 45 mm, Moss collected, new lures were transferred to new Packaging Co. Ltd., Wellington, New liners, and traps were re-randomized in the Zealand) inserted as the carrier substrate. field. Moths were sexed in all studies. Two acetic acid (glacial acetic acid (99.7%), Sigma Aldrich) co-lures were made by Comparison of induced volatiles. Plant drilling either 1.0 or 3.2 mm holes in the cap volatiles, reportedly induced by tortricid of 8 ml Nalgene vials (Nalg-Nunc larval feeding on apple and attractive to International, Rochester, NY) and loading adults (El-Sayed et al., 2016), were each vial with two small cotton balls and 5 evaluated in two consecutive studies in the ml of glacial acetic acid. A third acetic acid Moxee orchard during 2013. The first study lure, a proprietary round (3.4 cm diameter) compared moth catches from 12–19 Aug. in plastic membrane cup (Pherocon AA) was traps baited with phenylacetonitrile, benzyl provided by Trécé Inc. (Adair, OK). The mean alcohol, indole, (E)-nerolidol, β- weekly weight loss (evaporation rate) of caryophyllene, or germacrene D; and the acetic acid from the three lures was combination of phenylacetonitrile with an recorded from 30 Aug. to 26 Sept. 2013. acetic acid co-lure. The second study Individual lures (N = 10) were placed in red compared these same six volatiles all in Pherocon VI delta traps (Trécé Inc.) without combination with acetic acid co-lures sticky liners that were hung in the canopy of against acetic acid alone from 19–28 Aug. five linden trees, Tilia cordata Miller, at the Yakima Agricultural Research Laboratory, Combination lures with phenylacetonitrile. Wapato, WA. Similarly, the weight loss of Studies conducted in both 2012 and 2013 sachet lures loaded with phenylacetonitrile evaluated phenylacetonitrile alone, and in (N = 5) was recorded from 28 Aug. to 19 combination with either pear ester or acetic 2 bioRxiv preprint doi: https://doi.org/10.1101/092452; this version posted December 11, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. El-Sayed and Suckling: Use of phenylacetonitrile plus acetic acid to monitor Pandemis leafroller 2016 acid, pear ester with acetic acid, and plant volatiles during Test 1 in the Moxee phenylacetonitrile plus pear ester and acetic orchard (Table 1). In comparison, small acid in order to determine whether numbers of moths were caught when acetic interactions would affect catches of either or acid was added as a co-lure to traps baited both target species. Traps in 2012 were with phenylacetonitrile (Table 1). During thehe initially placed in the Moxee orchard on 1 second test comparing these six volatiles in Aug. and traps were checked and re- combination with acetic acid, no moths werere randomized on 8 Aug.