Intentional Coverage Gaps Reduce Cost of Mating Disruption for Phyllocnistis citrella (Lepidoptera: Gracillariidae) in Citrus

Transcription

1 HORTICULTURAL ENTOMOLOGY Intentional Coverage Gaps Reduce Cost of Mating Disruption for Phyllocnistis citrella (Lepidoptera: Gracillariidae) in Citrus S. L. LAPOINTE, 1,2 L. L. STELINSKI, 3 C. P. KEATHLEY, 4 AND A. MAFRA-NETO 5 J. Econ. Entomol. 107(2): 718Ð726 (2014); DOI: ABSTRACT The leafminer, Phyllocnistis citrella Stainton (Lepidoptera: Gracillariidae), is a global pest of citrus and contributes to the incidence and severity of citrus bacterial canker. SPLAT CLM (ISCA Technologies, Riverside, CA) is an emulsiþed wax that provides sustained release of (Z,Z,E)- 7,11,13-hexadecatrienal, the major component of P. citrella sex pheromone. Trials in commercial orchards demonstrated that SPLAT CLM applied to plots of varying width resulted in disruption of trap catch of male P. citrella within treated rows and across untreated rows adjacent to treated rows. SPLAT CLM applied to plots of constant width (10 rows) disrupted trap catch across an untreated gap as the square of the width of the gap. Similarly, the ability of the pheromone source in treated rows to disrupt trap catch across untreated gaps of constant size declined as the square of the width of adjacent treated areas. A coverage pattern of 4 rows skipped for every 10 treated rows resulted in a 4% reduction of trap shutdown, and reduced the product and application costs by 29%. Mining incidence by P. citrella in treated rows was reduced by 53% compared with untreated areas. Intentional coverage gaps can signiþcantly reduce the cost of mating disruption. Commercial lures for P. citrella used in this study were highly potent with respect to attracting males. Each lure was 10 3 times as attractive as an individual P. citrella female. Disruption of trap catch using commercial lures may underestimate actual mating disruption achieved in the Þeld. KEY WORDS sex pheromone, (Z,Z,E)-7,11,13-hexadecatrienal, citrus leafminer, SPLAT, Lepidoptera 1 USDAÐARS, United States Horticultural Research Laboratory, 2001 South Rock Rd., Fort Pierce, FL Corresponding author, stephen.lapointe@ars.usda.gov. 3 Entomology and Nematology Department, Citrus Research and Education Center, University of Florida, 700 Experiment Station Rd., Lake Alfred, FL USDAÐARS, 2001 South Rock Rd., Fort Pierce, FL ISCA Technologies, Inc., 1230 Spring St., Riverside, CA Recent progress in Þeld trials (Lapointe et al. 2009, Stelinski et al. 2010) has propelled commercialization of SPLAT CLM (ISCA Technologies, Riverside, CA), a mating disruptant product for the leafminer Phyllocnistis citrella Stainton (Lepidoptera: Gracillariidae), a pest of citrus crops throughout the world (Heppner 1993). Larval feeding by this species reduces photosynthetic capacity of leaves and increases the susceptibility of leaves to citrus bacterial canker, Xanthomonas axonopodis pv. citri (Gottwald et al. 2007, Hall et al. 2010). Successful mating disruption of P. citrella in commercial citrus was demonstrated (Stelinski et al. 2008) using SPLAT CLM, an emulsiþed wax formulation providing sustained release of an offratio blend of P. citrella sex pheromone over several weeks (Lapointe and Stelinski 2011). This is the Þrst use of mating disruption in Florida citrus; adoption by growers will depend on product and application costs as well as on efþcacy. Synthesis of the major component of the P. citrella sex pheromone, (Z,Z,E)-7,11,13-hexadecatrienal, involves a highly pyrophoric reagent (Leal et al. 2006, Moreira et al. 2006) that limits the scale-up potential and increases the cost above what may be acceptable to citrus growers. We previously conducted a limited Þeld experiment that demonstrated the possibility of reducing the amount of SPLAT CLM applied by incorporating intentional coverage gaps (Lapointe and Stelinski 2011) similar to what has been suggested for mating disruption of the gypsy moth, Lymanria dispar (L.) (Tcheslavskaia et al. 2005). Here, we report a more complete set of experiments and analyses that describe the effect on trap catch disruption of differently sized coverage gaps and treatment blocks in commercial orchards typical of citrus operations in Florida. Trap catch disruption (trap shutdown) is often used as a proxy measurement for mating disruption because of the difþculty of assessing the mating status of female insects in the Þeld. Typically, the number of male insects caught in traps baited with an attractive pheromone blend in areas treated with pheromone to disrupt mating is compared with trap catch in untreated areas under the assumption that the ability of pheromone lures to attract males is equivalent to that of unmated females. Disruption of trap catch is, therefore, indirect evidence of mating disruption. In practice, pheromone lures are loaded with many times the amount of pheromone released by individual females (Witzgall et al. 2008). Synthetic lures that are far more

2 April 2014 LAPOINTE ET AL.: MATING DISRUPTION OF P. citrella 719 potent than authentic females may underestimate the impact of a pheromone treatment on mating disruption. Measures of pheromone-mediated disruption may include: disruption of male moth catch in traps baited with synthetic pheromone or unmated females (Stelinski et al. 2005), quantiþcation of feral moth mating (Knight and Light 2013), direct observation of moth response to pheromone sources in the Þeld (Witzgall et al. 1999, Stelinski et al. 2004), and most importantly, assessment of crop damage (Gut et al. 2004, Knight et al. 2012). Of these, trap shutdown has been widely used as a benchmark for estimating mating disruption because of the ease of using this technique (Gut et al. 2004). In addition to assessing trap shutdown as an estimate of mating disruption, we assessed damage caused by P. citrella by evaluating the number of mines per shoot from plots treated with SPLAT CLM. We assume that the cost savings achieved by not treating a proportion of a crop is a nearly linear function of the gap size. However, it has been suggested (Lapointe and Stelinski 2011) and we hypothesize here that the loss of disruption resulting from incorporation of a repeated treatment gap pattern into a grove is related to the square of the width of the treatment gap. As a result, it should be possible to incorporate treatment gaps with signiþcant savings and only minor reductions in the effectiveness of mating disruption. Such an approach is of particular interest in the case of P. citrella because the high cost of synthesis of the pheromone is the major limitation to adoption of a mating disruption strategy for this species. To prove this point, we conducted Þeld trials that varied the width of treatment gaps, and in a separate experiment, we varied the width of treated blocks to identify appropriate parameters for an economically advantageous coverage pattern for pheromone deployment that maintains effective disruption as estimated by trap shutdown. Determining the relative attractiveness of monitoring lures versus authentic females is a required milestone to fully understand trap catch disruption data as they relate to mating disruption. We also conducted an experiment to determine the relative attraction of male P. citrella to synthetic pheromone lures of various dosages as compared with unmated females. Materials and Methods SPLAT CLM Application. SPLAT containing 0.15% (Z,Z,E)-7,11,13-hexadecatrienal (SPLAT CLM, ISCA Technologies, Riverside, CA) was applied using an applicator designed and fabricated in collaboration with International Fly Masters (Fort Pierce, FL) as previously described (Lapointe and Stelinski 2011). The applicator consisted of a pair of computer-controlled variable speed peristaltic pumps that drew the SPLAT compound from reservoirs suspended above the pumps and delivered it to nozzles located above blowers on either side of a frame mounted to a tractor by a three-point hitch. Dollops of SPLAT CLM fell by their own weight into the airßow from the blowers, and air velocity was only sufþcient to loft the dollops into the tree canopy while avoiding disruption of the dollops. The product was applied as 1 g dollops at a nominal rate of 500 g/ha with four replicates. Here, the nominal rate refers to the number of grams of SPLAT CLM applied to treated areas, while the effective rate is the amount of SPLAT CLM averaged over treated and untreated areas within a given treatment. Field trials were conducted during 2011 at TRB Groves (27 01 N, W) in northern Charlotte County, FL, and Emerald Grove (27 28 N, W), courtesy of The Packers of Indian River, located in northwestern St. Lucie County, FL. Effect of Varying Width of Treatment Gaps. At TRB Groves, SPLAT CLM was applied to Duncan grapefruit in 10-row blocks. Tree spacing was 7.3 by 3.7 m between and within rows, respectively; rows were 185 m in length. The area of each individual plot was 1.5 ha. Each row in all plots contained 54 trees. Plots were replicated four times. SPLAT CLM was applied at a nominal rate of 500 g/ha. Treated blocks were separated by gaps of varying width, either 4, 8, or 10 rows so that each untreated gap was ßanked by treated blocks of 10 rows each (10Ð4Ð10, 10Ð8Ð10, and 10Ð 10Ð10 treatedðuntreatedðtreated rows, respectively; see schematic design in Fig. 2). To assess trap catch disruption, two Pherocon VI traps (Trécé, Adair, OK), each baited with a rubber septum loaded with 0.1 mg of (Z,Z,E)-7,11,13-hexadecatrienal and mg of (Z,Z)-7,11-hexadecadienal (IT203, ISCAlure-Citrella, ISCA Technologies, Riverside, CA), were placed in the center rows of treated and untreated plots on the 18th and 36th trees in rows of 54 trees. Sticky trap liners were replaced and counted weekly for a period of 10Ð11 wk after application of SPLAT CLM. Mean weekly trap catch disruption was calculated as a percentage of the trap catch in baited traps placed in untreated rows 115 m from treated rows. Data were compared by analysis of variance (ANOVA) and Þtted to a response surface using Design-Expert software (Stat-Ease Inc., Minneapolis, MN). The experiment was repeated three times during 2011 by reapplying SPLAT CLM to the same plots on 22 April, 13 July, and 27 September. To estimate the effect of treatment gaps on trap catch disruption in a hypothetically large grove treated with the corresponding pattern (e.g., 10Ð4Ð 10), weighted means of treated and untreated areas were combined to produce an expected trap catch disruption calculated as the sum of the experimentally determined trap catch disruption in treated plots multiplied by the proportion of treated rows (number of treated rows by the total number of rows) and the experimentally determined trap catch disruption in untreated plots multiplied by the proportion of untreated rows (number of untreated rows by the total number of rows). For this exercise, trap shutdown over the entire untreated gap was assumed to be equivalent to the mean experimental trap shutdown observed in the central row of the untreated plots.

3 720 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 107, no. 2 Fig. 1. Seasonal mean SEM trap catch disruption within grapefruit plots treated with SPLAT CLM as depicted in the schematic diagram. Columns represent the trap catch disruption as measured by pheromone-baited traps located within the four untreated exterior rows (U, Ext, n 288), the four treated exterior rows (T, Ext, n 288), the six untreated interior rows (U, Int, n 432), and the eight treated interior rows (T, Int, n 576). Means were calculated as a percentage of the trap catch disruption of the grand mean of all traps. Charlotte County, FL, Effect of Alternating Treated and Untreated Double-Row Beds. An additional coverage design (designated zebra ) was tested during the same period on a production block of red grapefruit on Swingle rootstock (U.S. Department of AgricultureÐAgricultural Research Service [USDAÐARS]) at the TRB location described above using the same SPLAT CLM formulation and batch. The coverage pattern consisted of alternating double-row beds. Tree spacing was 7.3 by 3.7 m between and within rows, respectively; rows were 360 m in length. The area of each individual plot was 0.84 ha. Each row in all plots contained 100 trees. Plots were replicated three times. SPLAT CLM was applied at a nominal rate of 500 g/ha to beds (two rows) separated by untreated beds. Replicated plots consisted of six treated beds each (see schematic design in Fig. 1). Two traps baited with attractant lures were located within each treated and untreated bed for a total of 22 traps in each plot. The experiment was repeated three times during 2011 by reapplying SPLAT CLM to the same plots on 22 April, 13 July, and 27 September. Trap catch disruption was calculated as described above based on catch of traps located in adjacent untreated red grapefruit. Effect of Varying Width of Treated Area. Rows of trees within four production blocks ( 183 by 1550 m or 28 ha each) of actively managed, mature Flame grapefruit on Swingle rootstock located at Emerald Grove were divided into treatment plots, controls, and buffer areas to avoid interactions between treatments. Trees were hedged and topped at between 4 and 5 m in double-row beds spaced 3.8 m between trees within rows (48 trees per row) and 7.6 m between rows. Treatments were arranged in a randomized block design with four blocks and one replication of each treatment per block. Treatments consisted of varying the number of rows treated with SPLAT CLM from 2 to 10, while maintaining a constant number of untreated rows (10) ßanked by treated areas of equal width (see schematic design in Fig. 5). Treatments were designated 2Ð10Ð2, 4Ð10Ð4, 8Ð10Ð8, and 10Ð 10Ð10 indicating the number of treatedðuntreatedð treated rows. Each treatment was replicated four times; six blocks of 10 rows each were included as positive (treated with SPLAT CLM) and 5 blocks of 10 rows each were included as negative (untreated) controls. All plots were separated by 10 untreated rows (buffer rows). To assess disruption of the malesõ ability to orient to an attractive pheromone blend, traps (Pherocon VI, Trécé, Adair, OK) baited with a lure (ISCAlure-Citrella) were deployed in the center rows of treated and untreated plots. Two traps were deployed in the center rows of each of the two SPLAT CLM-treated areas within each treatment on approximately the 16th and 32nd trees with the 48-tree rows. Three traps were deployed at equal intervals along the center row of each of the untreated 10-row areas (gaps) within treatments on approximately the 12th, 24th, and 36th trees within the 48-tree rows. Trap liners were replaced approximately weekly and lures were replaced every 6 wk. Trap catch was expressed as the number of male P. citrella per trap per day. The experiment was repeated three times during the 2011 growing season with SPLAT CLM applications occurring on 14 April, 10 June, and 21 September. Leafminer infestation was evaluated in untreated plots and in treated plots that were 2, 4, 8, and 10 rows wide. Evaluations were conducted in rows within untreated plots that were 100m from any tree treated with SLAT CLM. In the untreated plots and within the treated and untreated areas of each of the experimental plots, we collected 10 new shoots from the middle two rows, along the center three-þfths of each row. The number of active mines on each of 10 shoots (ßush) was counted and the mean number of mines per shoot was calculated. Data were analyzed by one-way ANOVA followed by orthogonal contrasts (StudentÕs t-test) to compare the average number of mines per shoot in the untreated versus treated plots and then in treated rows versus untreated rows within treated plots. Data were square-root (x 1) transformed before ANOVA to improve homogeneity of variance. The number of mines per ßush was reported as untransformed means ( SEM). Relative Potency of Pheromone Lures. Natural rubber septa loaded with varying amounts of the attractive 3:1 blend of (Z,Z,E)-7,11,13-hexadecatrienal: (Z,Z)-7,11-hexadecadienal were prepared by ISCA Technologies (Riverside, CA) and deployed in Pherocon VI traps with adhesive liners in citrus groves at two locations (St. Lucie and Polk counties, FL) along with traps baited with virgin female P. citrella. The loading rate of pheromone was varied in Þve log steps from to 1.3 mg per lure. Commercially sold ISCAlure-Citrella lures were loaded with mg

4 April 2014 LAPOINTE ET AL.: MATING DISRUPTION OF P. citrella 721 pheromone per lure. Lures were placed in traps and deployed in a completely randomized design over 18 d in JulyÐAugust 2011 in blocks of commercial citrus in St. Lucie County, FL. Unmated female P. citrella were reared by collecting citrus leaves with mines from Þeld locations and allowing the adults to emerge in glass petri dishes provided with moist Þlter paper in an environmental chamber maintained at a photoperiod of 12:12 (L:D) h and 30 C during photophase and 26 C during scotophase. Adult females were transported to the Þeld and placed in cages within 18 h of emergence. Cages consisted of screened plastic enclosures containing sections of dental wick moistened with a dilute sucrose solution. The adhesive liners of traps baited with lures and females were replaced daily. Traps were numbered and assigned randomly to locations within the citrus orchard; locations were rerandomized and traps were relocated daily to minimize variance because of variation in P. citrella population density between trap sites. All traps were separated by 60 m. Four replicates of seven treatments were deployed: rubber septa loaded at Þve rates with a 3:1 blend of (Z,Z,E)-7,11,13-hexadecatrienal:(Z,Z)-7,11- hexadecadienal, a control trap (baited with a blank rubber septum), and caged female(s). Availability of females ßuctuated during the trial, so traps were baited with 1Ð6 individuals per trap; data were expressed as the number of males caught per female and compared with a regression of trap catch on pheromone loading rate. Statistical Analysis. The application experiments were laid out in a randomized block design with four replications. Weekly counts of trap liners from plots treated with SPLAT CLM were expressed as a percentage of the catch from traps in untreated plots (trap catch disruption). When appropriate, the angular (arsine) transformation of the percentage trap catch disruption was applied before analysis by ANOVA (Kasuya 2004). Untransformed means are reported. A three-dimensional response surface to visually demonstrate attenuation of trap catch disruption over 11 wk after application of SPLAT CLM in each of the treatments (gap width) was generated in Design-Expert (v7.0.3; State-Ease Inc., Minneapolis, MN). Post hoc partitioning of the sum of squares to generate orthogonal comparisons of seasonal means of treatments was carried out using the least square means contrast option in JMP (v. 10.0, SAS Institute Inc., Cary, NC). The Bonferroni procedure was used to adjust for determination of signiþcant differences when conducting multiple post hoc comparisons. Attenuation of trap catch disruption was plotted as the mean trap catch disruption as a function of increasing treated area width or increasing gap width and Þtted to a quadratic curve (Lapointe and Stelinski 2011). Questions regarding the relative strength of trap catch disruption as affected by location within the plot were addressed in the zebra experiment of alternating treated and untreated rows. Mean trap catch disruption was compared by ANOVA using a 2 by 2 factorial design to compare the main effects of location (interior or exterior rows) and treatment (treated or untreated with SPLAT CLM). To estimate the relative attraction of unmated females, the log of the number of male P. citrella captured in traps baited with synthetic pheromone were regressed against the log pheromone-loading rate of the lures. The number of males or female in traps baited with unmated females was Þt to the regression line to estimate the relative pheromone released by individual females and as a rough estimate of the relative strength of the commercial lures. Results Effect of Varying Width of Treatment Gaps. Mean trap shutdown was calculated as the number of male P. citrella captured in traps placed in the center row of untreated plots of varying widths ßanked by 10 rows treated with SPLAT CLM, divided by the number of males captured in traps located in untreated control plots (n 5 plots). The correlation between trap shutdown and gap width was signiþcant ( 0.006) on each of the eight dates when trap liners were collected through 9 wk after the April and June applications of SPLAT CLM, at which point trap shutdown in the treated control plots fell below 50% (Fig. 2). There was a strong and anticipated effect of time after application on trap catch disruption (P ) but there was no interaction between time and gap width (P 0.39). There was a signiþcant effect of gap width on the mean difference (loss) of trap catch disruption compared with the positive control (10 treated rows) for each of the three application dates (Þrst application: F 12.59; df 2, 21; P ; second application: F 15.70; df 2, 24; P ; third application: F 11.64; df 2, 24; P 0.001). Data for the Þrst application are presented in Table 1. To illustrate, trap catch disruption at 7 d after the Þrst application was Þtted to a quadratic expression (Fig. 3), resulting in a strong correlation between increasing gap widths and declining trap shutdown. Seasonal means ( SEM, n 8 dates) were also compared by setting trap shutdown in the positive controls to 100% and plotting the weighted mean trap shutdown of the three gap treatments as the loss of shutdown compared with positive controls for the three applications (Fig. 4). The reduction of trap shutdown compared with the positive controls in plots that included 4 skipped rows (30.5 m) compared with the trap shutdown in the center rows of adjacent plots of 10 treated rows was 4% (y 0.64x x , where y trap shutdown and x percentage of skipped area). A gap of 4 rows for every 10 treated rows corresponds to a 29% reduction in SPLAT CLM required to treat a hypothetically large area treated with a repeating pattern. The quadratic Þt of loss of trap catch disruption with increasing gap width (Figs. 3 and 4) suggests that the ability of pheromone applications in plots of constant width (10 rows) to disrupt trap catch across an untreated gap declined with the square of the width of the gap.

5 722 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 107, no. 2 Fig. 2. A three-dimensional response surface plot of trap catch disruption within treated plots with varying coverage gaps over 11 wk after the application of SPLAT CLM at a nominal rate of 500 g/ha. Red points are response values above the predicted value and pink points are response values below the predicted value. Effect of Alternating Treated and Untreated Double-Row Beds. Overall, the treatment consisting of alternating treated and untreated two-row beds performed less well compared with the other treatments (Table 1). However, the nature of the design and placement of traps differed from the other treatments because of the absence of central locations for untreated and treated areas. Mean trap catch disruption varied according to the location of traps within the plot of six treated beds alternating with untreated beds. There was no interaction between location and treatment (F 2.15; df 1, 1583; P 0.14), so main effects were examined. Treated rows had signiþcantly greater (F 9.83; d.f. 1, 1583; P ) trap catch disruption expressed as a proportion of the total trap catch disruption in the treatment ( %) compared with the untreated rows ( %). Trap catch disruption as a percentage of total trap disruption in the treatment in interior rows ( %) was signiþcantly greater (F ; df 1, 1583; P ) compared with that in the exterior rows ( %). Effect of Varying Width of Treated Area. Inspection (Fig. 5) of the seasonal means ( SEM, n 26 dates over three applications when trap catch disruption exceeded 50%) suggested the orthogonal contrasts presented in Table 2. There were signiþcant differences (P , ANOVA) in the number of male P. citrella captured in baited traps for all three applications and the mean of the three applications (Table 3). The Þrst contrast (C1) demonstrated that the number of male P. citrella captured in the positive control plots (treated) did not differ from the number captured in the treated rows of the plots that consisted of 4, 8, or 10 treated rows on any of the three dates (Table 3). The second contrast (C2) demonstrated a signiþcant difference between the number of males captured in the treated rows of plots that consisted of two treated rows and the number of males captured in the treated rows of the remaining treatments for all three applications (Table 3). The third contrast (C3) demonstrated that the trap catch in untreated gap rows in the plots that consisted of 2 treated rows and 10 untreated rows (2Ð10Ð2) was signiþcantly different from the Table 1. Trap catch disruption (percentage of trap catch in untreated plots) after application of SPLAT CLM calculated as the weighted means of treated and untreated rows in citrus plots with varying width of untreated gaps Width (m) Days after application a Grand mean b Mean decrease c a ab bc c a Entries for each date are calculated as weighted means of the mean trap catch (n 16) in untreated gaps and in treated plots (n 24) divided by the trap catch in untreated control plots (n 64). b Grand mean of dates when mean trap catch disruption in control exceeded 50%, n 8. c Means followed by the same letter do not differ by TukeyÕs honest signiþcant difference (ANOVA: F 15.11; df 3, 31; P ). Gap widths consisted of 0 (positive control), 4, 8, or 10 gap rows ßanked by 10 treated rows (gaps of 0, 30.5, 61.0, or 76.2 m, respectively) or alternating two-row beds of treated and untreated rows (gap of 15.25m). The mean decrease was calculated as mean (n 8) difference in trap catch disruption between gap treatments and treated control plots

6 April 2014 LAPOINTE ET AL.: MATING DISRUPTION OF P. citrella 723 Fig. 3. Effect of increasing width of untreated gap on mean trap catch disruption ( SEM, n 8 traps) expressed as the weighted mean of treated and untreated rows 7 d after application of SPLAT CLM. Width of gap was varied between blocks of 10 treated rows (76 by 183 m) as shown in the schematic design (not to scale and not representative of plot arrangement). Traps were placed in the center rows of treated blocks (0 gap) and untreated blocks. Charlotte County, FL, trap catch in the untreated rows of the remaining treatments for the second application (no. 2), marginally so after the Þrst application, and not signiþcant Fig. 5. Seasonal mean SEM trap catch disruption over 26 sampling dates from 14 April through 23 November, 2011, in grapefruit plots that received SPLAT CLM at a nominal rate of 500 g/ha. Plots consisted of a constant number (10) of untreated rows (gap) ßanked by a varying number (0, 2, 4, 8, and 10) of treated rows (7.6 by 183 m) as shown in the schematic design (not to scale and not representative of plot arrangement). Traps were placed in the center rows of treated and untreated blocks. St. Lucie County, FL, after the third application and for the mean of three applications (Table 3). The mean ( SEM, n 4) number of P. citrella mines per shoot differed across treatments (F 8, ; P 0.001) ranging from a high of in untreated plots to , , , and for untreated rows surrounded by 4, 8, 16, or 20 treated rows, respectively (Fig. 6). The mean ( Fig. 4. Effect of increasing gap width (percentage of total area) on mean ( SEM, n 8 approximately weekly sampling dates) trap catch disruption (weighted mean of treated untreated rows) over 62 d after application of SPLAT CLM, replicated on three dates. Gap widths were 0, 30.5, 61, and 76 m. Gaps were ßanked by 1.4 ha blocks of 10 treated rows (76 by 183m) on either side as shown in the schematic design in Fig. 2. Traps were placed in the center rows of treated blocks (0 gap) and untreated blocks. Charlotte County, FL, 2011.

7 724 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 107, no. 2 Table 2. Orthogonal contrasts applied to treatments in an experiment to compare the width of citrus plots treated with SPLAT CLM on P. citrella trap shutdown within treated and flanking untreated rows Treatment a TÐCÐS b C1 c C2 d C3 e control T Ð10Ð2 S Ð10Ð2 T Ð10Ð4 S Ð10Ð4 T Ð10Ð8 S Ð10Ð8 T Ð10Ð10 S Ð10Ð10 T a Treatments (number of treatedðuntreatedðtreated rows). b T, treated; C, untreated control; S, untreated (skipped). c Contrast 1: trap shutdown in the positive control (treated) versus that in treated rows of plots consisting of more than two rows treated. d Contrast 2: trap shutdown in the treated rows of plots consisting of two rows treated versus that in treated rows of plots consisting of more than two rows treated. e Contrast 3: trap shutdown in skipped rows of plots with two treated rows versus that in skipped rows of plots consisting of more than two rows treated. SEM, n 4) number of mines in rows treated with SPLAT CLM ranged from , , , and (n 8) for 2, 4, 8, and 10 treated rows, respectively. There were 53% fewer mines per shoot in areas that were treated or surrounded by treated areas ( , n 36) compared with the untreated control (contrast, t 4.8; P ). There were 24% fewer mines per shoot (t 2.1; P 0.05) in treated rows ( ; n 20) compared with adjacent untreated rows ( ; n 16). Relative Potency of Pheromone Lures. The number of male P. citrella caught in traps baited with variably loaded lures or unmated females was expressed as the number of males per trap per day. The data for the variably loaded lures were plotted as the log of Fig. 6. Mean ( SEM) number of mines per shoot in untreated control plots, within treated areas of varying width (number of treated rows), or untreated areas of 10 rows ßanked by treated areas of corresponding width. St. Lucie County, FL, the daily catch and Þt to an exponential model (Fig. 7). The capture of males in traps baited with a single unmated female was equivalent to the rate of capture predicted by the regression in a trap baited with 145 ng of synthetic pheromone, approximately three orders of magnitude less than the standard pheromone lure. Discussion Trap shutdown is a commonly used proxy for measurement of mating disruption (Gut et al. 2004). Trap shutdown measures disruption of attraction to a synthetic pheromone blend released at rates attractive to male moths caused by the deployment of one or more synthetic pheromone components within a crop. Field Table 3. Results of the orthogonal contrasts presented in Table 2 comparing trap catch of male P. citrella in SPLAT CLM-treated and untreated citrus plots with varying number of treated rows Statistic C1 C2 C3 Application no. 1 (April) a T value P (two-tailed) * 0.03 P (one-tailed) * * Application no. 2 (June) b T value P (two-tailed) * * P (one-tailed) * * Application no. 3 (Sept.) c T value P (two-tailed) * 0.96 P (one-tailed) * 0.48 Mean of 3 applications d T value P (two-tailed) * 0.12 P (one-tailed) * 0.06 * SigniÞcant at (Bonferroni correction). a Application no. 1: F 9, ; P b Application no. 2: F 9, ; P c Application no. 3: F 9, ; P Mean of three applications: F 9, ; P Fig. 7. Effect of amount of the attractive 3:1 triene:diene pheromone blend loaded onto rubber septa on mean daily trap catch of male P. citrella. The black circle represents the mean number of male P. citrella caught in traps baited with IT203 lures and the black square (not included in regression) represents the mean number of males per female caught in traps baited with live virgin females and Þt to the regression line to estimate relative pheromone emission of female P. citrella. St. Lucie County, FL, 2011.

8 April 2014 LAPOINTE ET AL.: MATING DISRUPTION OF P. citrella 725 trials in which the effect of proportionality of a twocomponent pheromone blend on trap shutdown was investigated for P. citrella in citrus demonstrated that trap shutdown increased with the proportion of the triene (Z,Z,E)-7,11,13-hexadecatrienal, one of two principal pheromone components of P. citrella (Lapointe et al. 2009). As a result, the triene has been chosen as the sole active ingredient for use in SPLAT CLM commercial formulation for mating disruption of P. citrella. The use of a single synthetic compound also simpliþes manufacturing. However, synthesis of the triene is costly and involves use of a highly pyrophoric reagent, tert-butyllithium. The relatively high cost of synthesis of the triene may limit adoption of mating disruption as a cost-effective tool for citrus leafminer control unless ways to reduce cost are identiþed (Lapointe et al. 2011). The purpose of the studies presented here was to explore the potential for cost savings by varying the coverage of SPLAT CLM in citrus groves by leaving a proportion of gap rows untreated. The savings to be realized by this approach include the cost of pheromone and dispenser matrix (SPLAT) as well as labor, fuel, and other costs associated with the applicator machinery. Lapointe and Stelinski (2011) reported that coverage gaps of 20 or 33% of tree rows (every Þfth row or every Þfth and sixth rows, respectively) did not signiþcantly reduce the trap shutdown obtained over the combined area with machine-applied SPLAT CLM in grapefruit. To design a coverage pattern for citrus, decisions must be made regarding the number of consecutive rows to treat and the number to leave untreated. Lapointe et al. (2011) showed that trap shutdown was greatest when pheromone dispensers were located in each tree within a grove compared with levels of aggregation of dispensers at the same rate of pheromone per unit area. Therefore, we decided to conduct two experiments: one where the number of untreated rows was varied between blocks of 10 rows of citrus trees treated with SPLAT CLM at the nominal rate of 500 g/ha (1 g triene per hectare), and a second trial where the number of untreated rows was constant and the number of rows (also treated at the same nominal rate) varied. In the trial of varying gap width, there was a good quadratic Þt of trap shutdown, indicating an exponential decline in disruption with increasing gap width, similar to that reported and predicted by Lapointe and Stelinski (2011). A gap of 4 rows between blocks of 10 treated rows (equal to a 28.6% reduction in amount of SPLAT CLM) resulted in a mean loss of trap shutdown of 4% at 7 d after application (Fig. 3) and 5% for a season-long average (Fig. 4) compared with trap shutdown in the treated control block. It should be noted that this reduction was the trap shutdown observed in the center row of the untreated area, and therefore represents the disruption that occurred at the farthest point from a pheromone source. The estimate of 4% reduction in disruption therefore overestimates the disruption that likely occurred over the entire untreated area. Regardless, the trade-off of a maximum estimated loss of efþcacy of 4% in return for a 28.6% savings in product and associated costs make a strong economic argument for this pattern of coverage. The results of varying the width of treated blocks suggest that a minimum of four treated rows are necessary to achieve trap shutdown in both the treated and untreated rows (Fig. 5). Treated widths of 4, 8, and 10 rows resulted in similar levels of trap catch disruption in both treated and untreated areas. This result may explain the poorer-than-expected performance of the 2/2 treatment consisting of 2 treated and 2 untreated rows compared with the 10/10 treatment consisting of 10 treated and 10 untreated rows that results in the same application rate per unit area (Table 1). All of the coverage patterns tested may be expected to perform better when applied to larger areas. This may be particularly true in the case of the zebra design in comparison with the other designs tested. Additional trials may be necessary to identify an optimum combination of gap and treated widths. Multiple solutions may exist or be appropriate for speciþc situations. However, based on these results, a conservative recommendation can be made to reduce SPLAT CLM application by 30% with minimal loss in trap shutdown (and presumably mating disruption) by skipping 4 rows for every 10 rows treated at the nominal rate of 500 g/ha equal to an effective rate of 350 g/ha, thereby reducing the cost of mating disruption for P. citrella. Previous experience with ISCAlure-Citrella pheromone lures (IT203) demonstrated that the lures were highly potent with respect to attracting male P. citrella (Lapointe and Leal 2007). Our results demonstrated that the IT203 lure is 10 3 times as attractive as an individual P. citrella female. This suggests that we may be underestimating the disruption achieved by using disruption of such powerful attractant lures as an estimate of actual mating disruption. Stelinski et al. (2010) were able to document reduced incidence of mines of P. citrella for several weeks after the application of SPLAT CLM to small (0.25 ha) plots. Here, we have demonstrated reductions in the number of mines at a larger scale within both treated rows and untreated gap rows ßanked by treated rows. It seems reasonable to assume that measures of trap catch disruption are an accurate gauge of mining damage for this species. Our results demonstrate that coverage patterns that incorporate intentional gaps can significantly reduce the cost of pheromone deployment while maintaining effective trap catch disruption and reducing the incidence of mining by P. citrella. Acknowledgments Larry Markle, Julia Morris, Matt Chellemi, Paul Robbins and Rocco Alessandro (USDAÐARS, Ft. Pierce, FL), Wendy Meyer, Siddharth Tiwari, and Ian Jackson (University of Florida, Lake Alfred, FL) provided technical assistance in the Þeld. Dave Robinson (International Fly Masters, Ft. Pierce, FL) provided prototype machinery and made the Þeld applications. SigniÞcant funding was provided by the Specialty Crops Block Grant, USDA and the Florida Department of Agriculture and Consumer Services. We are particularly grateful to Tom Stopyra (The Packers of Indian River, Fort Pierce, FL) and David Kemeny (TRB Groves, Punta Gorda,

9 726 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 107, no. 2 FL) for arranging access to those properties. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the USDA and does not imply its approval to the exclusion of other products that may also be suitable. References Cited Gottwald, T. R., R. B. Bassanezi, L. Amorim, and A. Bergamin-Filho Spatial pattern analysis of citrus canker-infected plantings in São Paulo, Brazil, and augmentation of infection elicited by the Asian leafminer. Phytopathology 97: 674Ð683. Gut, L. J., L. L. Stelinski, D. R. Thomson, and J. R. Miller Behavior modifying chemicals: prospects and constraints in IPM, pp. 73Ð121. In O. Koul, G. S. Dhaliwal, and G. Caperus (eds.), Integrated pest managementñpotential, constraints, and challenges. CABI, Wallingford, United Kingdom. Hall, D. G., T. R. Gottwald, and C. H. Bock Exacerbation of citrus canker by citrus leafminer Phyllocnistis citrella in Florida. Fla. Entomol. 93: 558Ð566. Heppner, J. B Citrus leafminer, Phyllocnistis citrella, in Florida. Trop. Lepidoptera 4: 49Ð64. Kasuya, E Angular transformationðanother effect of different sample sizes. Ecol. Res. 19: 165Ð167. Knight, A., and D. Light Monitoring codling moth (Lepidoptera: Tortricidae) in orchards treated with pear ester and sex pheromone combo dispensers. J. Appl. Entomol. 137: 214Ð224. Knight, A. L., L. L. Stelinski, V. Hebert, L. Gut, D. Light, and J. Brunner Evaluation of novel semiochemical dispensers simultaneously releasing pear ester and sex pheromone for mating disruption of codling moth (Lepidoptera: Tortricidae). J. Appl. Entomol. 136: 79Ð86. Lapointe, S. L., and W. S. Leal Describing seasonal phenology of the leafminer Phyllocnistis citrella (Lepidoptera: Gracillariidae) with pheromone lures: controlling for lure degradation. Fla. Entomol. 90: 710Ð714. Lapointe, S. L., and L. L. Stelinski An applicator for high viscosity semiochemical products and intentional treatment gaps for mating disruption of Phyllocnistis citrella. Entomol. Exp. et Appl. 141: 145Ð153. Lapointe, S. L., L. L. Stelinksi, T. J. Evens, R. P. Niedz, D. G. Hall, and A. Mafra-Neto Sensory imbalance as mechanism of mating disruption in the leafminer Phyllocnistis citrella: elucidation by multivariate geometric designs and response surface models. J. Chem. Ecol. 35: 896Ð903. Lapointe, S. L., L. L. Stelinksi, and R. D. Robinson A novel pheromone dispenser for mating disruption of the leafminer Phyllocnistis citrella (Lepidoptera: Gracillariidae). J. Econ. Entomol. 104: 540Ð547. Leal, W. S., A. L. Parra-Pedrazzoli, A. A. Cossé, Y. Murata, J.M.S. Bento, and E. F. Vilela IdentiÞcation, synthesis, and Þeld evaluation of the sex pheromone from the citrus leafminer, Phyllocnistis citrella. J. Chem. Ecol. 32: 155Ð168. Moreira, J. A., S. McElfresh, and J. G. Millar Identi- Þcation, synthesis, and Þeld testing of the sex pheromone of the citrus leafminer, Phyllocnistis citrella. J. Chem. Ecol. 32: 169Ð194. Stelinski, L. L., L. J. Gut, A. V. Pierzchala, and J. R. Miller Field observations quantifying attraction of four tortricid moths to high-dosage pheromone dispensers in untreated and pheromone-treated orchards. Entomol. Exp. et Appl. 113: 187Ð196. Stelinski, L. L., L. J. Gut, R. E. Mallinger, D. Epstein, T. P. Reed, and J. R. Miller Small plot trials documenting effective mating disruption of Oriental fruit moth by using high densities of wax-drop pheromone dispensers. J. Econ. Entomol. 98: 1267Ð1274. Stelinski, L. L., J. R. Miller, and M. E. Rogers Mating disruption of citrus leafminer mediated by a non-competitive mechanism at a remarkably low pheromone release rate. J. Chem. Ecol. 34: 1107Ð1113. Stelinski, L. L., S. L. Lapointe, and W. L. Meyer Season-long mating disruption of citrus leafminer, Phyllocnistis citrella Stainton, with an emulsiþed wax formulation of pheromone. J. Appl. Entomol. 134: 512Ð520. Tcheslavskaia, K., C. Brewster, K. Thorpe, A. Sharov, D. Leonard, and A. Roberts Effects of intentional gaps in spray coverage on the efþcacy of gypsy moth mating disruption. J. Appl. Entomol. 129: 475Ð480. Witzgall, P., A.-C. Bäckman, M. Svensson, U. Koch, F. Rama, A. El-Sayed, J. Brauchli, H. Arn, M. Bengtsson, and J. Löfqvist Behavioral observations of codling moth, Cydia pomonella, in orchards permeated with synthetic pheromone. Biocontrol 44: 211Ð237. Witzgall, P., L. Stelinski, L. Gut, and D. Thomson Codling moth management and chemical ecology. Annu. Rev. Entomol. 53: 503Ð522. Received 6 September 2013; accepted 17 February 2014.