Acetic Acid Fumigation of Fruit Storage Bins to Control Diapausing Codling Moth Larvae

Fumigation with glacial acetic acid (AA) vapor successfully kills post-harvest pathogens on tree fruits and berries and reduces their spoilage in storage. In this study, we investigated whether a similar approach could be implemented to eradicate diapausing larvae of the codling moth, Cydia pomonella (L.), from fruit harvest bins they commonly infest. In 24-h tests conducted in 0.023-m fumigation chambers using two concentrations of vaporized AA [117,360 and 174,823 cumulative parts per million-hours (ppm-h)], mortality of diapausing larvae was 81% and 100%, respectively. A similar 24-h exposure to a 61,940 cumulative ppm-h treatment of AA caused no mortality. A 24-h fumigation of diapausing codling moth larvae placed in scaled-down plastic fruit bins treated with 55 mL of AA evaporated into a 1-m chamber caused 100% mortality. The same fumigation treatment of artificially infested, scaled-down wooden fruit bins caused no significant mortality of test larvae. Atmospheric concentrations of AA vapor in 1-m fumigation chambers containing wooden bins could not be maintained at levels necessary to cause insect death, even after multiple injections of AA. We hypothesize that either the wood or the moisture contained therein absorbs or in some other way interacts with the AA vapor. The use of AA as a fumigant targeting codling moth larvae in wooden bins is not practical or economical at this time. Fumigation of plastic fruit bins with AA would provide an economical and environmentally friendly method of killing diapausing codling moth larvae. The successful disinfestations of plastic fruit bins of codling moth would prevent these bins from being an external source of infestation, thereby decreasing overall codling moth infestation in orchards, which in turn benefits current densitydependent management practices used for the area-wide control of codling moth. chemical name: Glacial Acetic Acid (AA). Codling moth, Cydia pomonella L. (Lepidoptera: Tortricidae), is a key pest of pome fruits worldwide. This insect overwinters as a diapausing, fifth-instar larva located within a silken cocoon usually constructed in cracks or crevices of bark on host apple and pear trees. In most temperate pome-fruit production regions, codling moth larvae destined for diapause exit host fruit in late summer and early fall and wander in search of overwintering sites. While doing this, the diapause-destined larvae frequently encounter and infest fruit bins that are placed in the orchards before harvest (Higbee et al., 1999). Bins with harvested fruit are moved to packing houses where they are emptied and stored outdoors before being redistributed to orchards the next season. Bins that are inadvertently infested with diapausing larvae become unpredictable sources of adult codling moth that can significantly impact orchard pest management programs. In particular, these external sources of wild codling moth are problematic for orchardists relying on sterile insect release programs or pheromone-based mating disruption because wild moths emerging on or near bins outside an orchard can become mated before entering the orchard control area (Higbee et al., 2001; Judd and Gardiner, 2005; Newcomer, 1936; Proverbs and Newton, 1975). One solution to the issue of codling mothinfested bins would be to disinfest the empty bins before returning them to orchards. Several disinfestation methods have been tested, including use of steam washing (Newcomer, 1936), submergence in water tanks containing entomopathogenic nematodes (Cossentine et al., 2002; DeWaal et al., 2010; Lacey and Chauvin, 1999; Lacey and Unruh, 1998; Lacey et al., 2005), fumigation with carbon dioxide (Cossentine et al., 2004), and heat treatment (Hansen et al., 2006). Although many of these methods are effective, none are currently used commercially to eliminate diapausing codling moth in fruit bins, primarily as a result of the high cost of handling large quantities of bins (Higbee et al., 2001). In choosing an effective method to treat bins, it would be advantageous to use a technique that is economically viable, environmentally safe, and has the potential to control insects and microorganisms that are known to infest bins. Fumigation with glacial AA is a proven method for surface sterilization and removal of pathogens from a wide range of fruits and other plant materials (Sholberg, 2009). AA is also a relatively inexpensive and naturally occurring compound that poses little or no hazard to human health when used at the very low concentrations required to kill fungal spores. For example, Sholberg and Gaunce (1995) showed that AA concentrations of 2 to 4.7 mg L were extremely effective at killing conidia of Botrytis cinerea [Fuckeliana (de Bary) Fuckel] or Pencillium expansum Link on apples without causing any phytotoxic effects. AA vapor has also been used to protect grapes and strawberries (Moyls et al., 1996), stone fruits (Sholberg and Gaunce, 1996), and ‘d’Anjou’ pears (Sholberg et al., 2004) from fungal rots that cause spoilage in storage. Other studies have indicated that AA vapor will eliminate powdery mildew infections of apple buds, eliminate Erwinia amylovora [(Burrill) Winslow] and Pseudomonas syringae pv. syringae (van Hall) as surface contaminants of dormant scion wood (Sholberg et al., 2005), and even control common bunt of wheat (Sholberg et al., 2006). The goals of this study were 1) to determine whether AA could be used as a fumigant to kill wild diapausing codling moth larvae; and 2) to compare the efficacy of a fumigation treatment when applied to scaleddown fruit harvest bins made of plastic or wood. Materials and Methods Insects In June 2008, corrugated cardboard strips were wrapped around tree trunks in an infested apple orchard as described by Judd et al. (1997). Wild, diapause-destined codling moth larvae exited infested fruit and spun cocoons inside these cardboard strips. Cardboard was removed from trees in Oct. 2008, placed in plastic garbage bags, and held in an outdoor screenhouse through winter. In Spring 2009, a subset of these diapausing larvae was transferred to the Pacific AgriFood Research Center, Summerland, British Columbia, Canada, where they were stored in complete darkness at 2 C until needed for experiments. When needed for experiments, the cardboard backing surrounding each singular cocooned larva was cut from the larger strip with fine scissors so as to leave the cocoon attached to a small amount of cardboard and the larva undisturbed in its cocoon. All test larvae were stored like this at 2 C for up to 2 weeks before receiving fumigation treatments. Fumigation chambers Two different types of custom-made fumigation chambers were used for these experiments. The first fumigation chamber was a modification of the one described in detail by Sholberg et al. (2000). This modified chamber consisted of a 23-L (0.023 m) heavy duty aluminum cooking pot and lid (Russell Food Equipment, Vancouver, BC, Canada). The underside lip of each lid was fitted with a rubber seal. Four, hooked draw latches (Southco, Concordville, PA) were attached on the outside of the pot and evenly spaced to ensure an Received for publication 15 July 2011. Accepted for publication 4 Oct. 2011. To whom reprint requests should be addressed; e-mail [email protected]. 1634 HORTSCIENCE VOL. 46(12) DECEMBER 2011 airtight seal. An evaporator was made from an aluminum rod (25 mm diameter · 75 mm length) that had a hole drilled in one end to allow fitting of a 9.5-mm-diameter 150-W cartridge heater (CIR No. 2012/120; Omega Engineering, Stamford, CT). A 40-mm long groove large enough to hold 3 mL of water or AA was milled on the top of the rod. The evaporator was attached to the underside of the chamber lid. Liquids were placed in the evaporator using a 250-mL Hamilton glass syringe (Hamilton, Reno, NV) inserted through a hole drilled into the clear polycarbonate viewing port on the top of the lid. Placement and evaporation of all liquids and functioning of the evaporator were observed through the viewing port. During each experiment, all of the liquid boiled off in less than 2 min of operation with very little release of heat into the chamber (Sholberg et al., 2000). During evaporation of all liquids, a 75-mm tube axial fan (Papst Typ 8300N, 115 V 50 to 60 Hz 12 W; Papst Motoren Gmbn. and Co., Georgen, Germany), also attached to the inner side of the lid, was in operation throughout the fumigation. The second fumigation chamber was a 1-m wooden chamber described by Gaunce et al. (1981). This chamber was modified from its original design when the interior surfaces were covered with 0.3 mm of aluminum flashing and all seams and exposed wood were covered with aluminum foil tape (Home Hardware, Penticton, BC, Canada). The fumigation chamber had a removable front, which enabled the placement of scalesized bins inside. The edges of the door were sealed with a 0.33 · 7.6-cm piece of white rubber tape (3M brand; Home Hardware). Two Jay-Bee toggle clamps (JBT FA240; Acklands Grainger, Penticton, BC, Canada) were installed on each of the four sides of the chamber. Each of four chambers was fitted with an evaporator. Each evaporator was custom made from an aluminum rod (38 mm diameter · 76 mm length) milled to hold 62 mL of liquid. This evaporator was drilled to fit a 9.5-mm diameter 150-W cartridge heater (CIR-2021–120V; Omega Engineering). Each chamber had a small hatch (21.5 · 31.5 cm) through which water or AA was added to the evaporator. The hatch was secured using four Jay-Bee toggle clamps. A 115-mm 115-V 105 CFM tube axial fan (Model CFA11512038HS) was positioned in front of the evaporator to direct airflow over the device and circulate the air–acid mixture. Fumigation procedures Determining an effective acetic acid vapor dose to kill diapausing codling moth larvae. The objective of this experiment was to identify a dose of AA vapor that would cause 100% mortality (LD100) of diapausing codling moth larvae. Fifty test larvae were placed on the floor of each of four identical 23-L chambers. Relative humidity in each chamber was adjusted to 60% to 70% by evaporating sterile distilled water before applying fumigation treatments. The same volume of water was added to each chamber. The control chamber was fitted with a dual-sensor relative humidity– temperature probe (Model ST-616CT; Alaron Instruments, Newmarket, ON, Canada) to monitor temperature and humidity during the fumigation. This probe could not be used in treatment chambers because AA corrodes the sensor and affects its operation. The assumption is that temperature and humidity conditions in the treatment chamber were the same as the control chamber. Control and AA treatments were randomly assigned to each chamber. Heating elements in control and treatment chambers were activated simultaneously. In each treatment chamber, an initial amount (Table 1) of 99.5% glacial AA (Alphachem Ltd., Mississauga, ON, Canada) was evaporated from the small evaporator during the first minute of operation. Additional amounts of AA were added to each fumigation chamber at 20 and 200 min, because it was important to have sufficient concentration of AA vapor acting on the insect over the duration of the treatment (Bond, 1984). AA concentrations (mg L) were monitored throughout experiments by withdrawing samples of air from the chamber headspace using a 1 mL gastight syringe immediately after the AA was vaporized and at regular intervals (5 to 10 min) during the first 5 and last 3 h of fumigation. The 1-mL gas samples were injected into a Buck Model 910 gas chromatograph (GC) (Questron Technologies Corp., Mississauga, ON, Canada) fitted with a flame ionization detector and fused silica capillary column (Zebron ZB-FFAP; Phenomenex, Torrance, CA). Cumulative exposure to AA in ppm-h was calculated by multiplying the concentration (ppm) of AA · the hours of fumigation (Bond, 1984; Luvisi et al., 1992). After fumigation, all chambers were placed in a fume hood and the lid opened and vented for 15 min. All larvae were removed from each chamber, placed in separate screened 739-mL plastic containers (GladWare ; Glad Products Company, Oakland, CA), and transferred to a 20 C environmental chamber where they were held under a 16:8-h light:dark photoperiod. Starting 10 d after fumigation, all larvae were examined daily and the numbers of moths that had emerged were recorded. After 50 d, all cocoons were opened and examined for any remaining live insects. Larvae were considered to be dead if they were dry and hard. Fumigation treatment of diapausing codling moth in scale bins in 1-m chambers. The fumigation procedure in 1-m chambers was similar to that used with the 23-L chambers with the exception that it took 5 min to evaporate the AA. Air samples were collected from treatment chambers using a vacuum pump (Doerr Electric Corp., Cedarburg, WI), which drew headspace air into a 250-mL sampling bulb (Mandel Scientific, Guelph, ON, Canada). A 1-mL gas sample was taken from this bulb and injected into the GC. Fifteen diapausing larvae were placed on the bottom of each of the four scale plastic (48· 33 · 29 cm) or wooden bins (61 · 48.3 · 27.3 cm) in each chamber. The 1:21st scale plastic bins were made of high-density polyethylene (Thunderbird Plastics Ltd., Burnaby, BC, Canada). The 1:12th scale wooden bins described by Gaunce et al. (1981) were used to mimic bins used in the British Columbia fruit industry. Four bins were stacked two deep and two high in each chamber and relative humidity was adjusted to 60% to 70% by evaporating 60 mL of sterile distilled water. The control chambers containing either four wooden or four plastic bins did not receive any AA. Thirty milliliters of glacial AA was evaporated in each of the test chambers. An additional 15 and 10 mL of AA were similarly added and evaporated at 30 and 210 min after initiation in each treatment chamber, respectively. No additional glacial AA was added during the remaining 24 h. All fumigations were conducted at 26 to 28 C and ambient atmospheric pressure. Treatments were randomly assigned to each chamber and replicated four times. After fumigation, all chambers were vented for 20 min. To determine if there were any differences in mortality at different locations within each chamber, larvae from each bin within a chamber were held in separate screened 739-mL plastic containers until moth emergence. Maintenance of acetic acid levels. Our observation of low cumulative ppm-h of AA in the chambers containing scale wooden bins led to a fumigation study in which we monitored and attempted to maintain AA levels in an empty 1-m chamber, a 1-m chamber with six scale plastic bins, and a 1-m chamber with four scale wooden bins for 24 h. At the start of the fumigation, 20 mL of AA was added to each chamber. In the chamber containing scale wooden bins, an additional 10 mL of AA was added at 20 and 50 min followed by two 15-mL injections at 70 and 110 min. The chamber with wooden bins received 70 mL of AA in total. Fumigation treatments ended at 1300 min. Table 1. Volumes of glacial acetic acid (AA) and times it was injected into 23-L fumigation chambers and the cumulative exposure rates (ppm-h) for the different fumigation treatments used to determine an effective concentration required to kill diapausing codling moth larvae. Time (T) acetic acid was added The amount of AA (mL) injected at each time interval per treatment Control Treatment 1 Treatment 2 Treatment 3 T = –1 min 0 220 440 660 T = 20 min 0 110 220 330 T = 200 min 0 55 110 220 Total amount of AA added (mL) 0 385 77