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Pheromone-Based Mating Disruption of Planococcus ficus(Hemiptera: Pseudococcidae) in California VineyardsAuthor(s): Vaughn M. Walton, Kent M. Daane, Walter J. Bentley, Jocelyn G.Millar, Thomas E. Larsen, and Raksha Malakar-KuenenSource: Journal of Economic Entomology, 99(4):1280-1290. 2006.Published By: Entomological Society of AmericaDOI: http://dx.doi.org/10.1603/0022-0493-99.4.1280URL: http://www.bioone.org/doi/full/10.1603/0022-0493-99.4.1280

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HORTICULTURAL ENTOMOLOGY

Pheromone-Based Mating Disruption of Planococcus ficus(Hemiptera: Pseudococcidae) in California Vineyards

VAUGHN M. WALTON,1, 2 KENT M. DAANE,1, 3 WALTER J. BENTLEY,4 JOCELYN G. MILLAR,5

THOMAS E. LARSEN,6 AND RAKSHA MALAKAR-KUENEN1

J. Econ. Entomol. 99(4): 1280Ð1290 (2006)

ABSTRACT Experiments were conducted to test a mating disruption program for the mealybugPlanococcus ficus (Signoret) (Hemiptera: Pseudococcidae) in California vineyards. The sprayable,microencapsulated formulation of the racemic sex pheromone lavandulyl senecioate was applied withan air-blast sprayer, using three and four applications in 2003 and 2004, respectively. Mating disruptionwas combined with an application of buprofezin (2004) in June. Compared with a no-pheromonecontrol, there were signiÞcantly lower season-long trap catches of adult males, season-long mealybugdensities (2003 only), and crop damage in mating disruption plots. The amount of mealybug reductionand mechanisms that resulted in lower crop damage in mating disruption plots is discussed. In samplestaken during the growing season (April to September), mealybug density was only 12.0 � 15.6 and31.1 � 11.6% lower in the mating disruption plots than in control plots in 2003 and 2004, respectively.In the mating disruption treatment, mealybug egg production was signiÞcantly lower (2003 only), aswere the proportion of ovisacs and crawlers produced. There was no treatment impact on percentageof parasitism. Mealybug density inßuenced treatment impact. In 2004, vines were categorized as havinglow, medium, or high mealybug densities during a preapplication survey. After treatment application,mealybug density was reduced by 86.3 � 6.3% on vines in the low mealybug density category, but itwas unchanged on vines in the high density category. Another factor that reduced treatment impactwas the relatively short effective lifetime of the sprayable formulation.

KEY WORDS Planococcus ficus, mating disruption, vineyard, sex pheromone, microencapsulatedpheromone

The mealybug, Planococcus ficus (Signoret) (Hemi-ptera: Pseudococcidae), has spread from its likely or-igins in the Mediterranean basin to become a primaryinsect pest of vineyards in California (Godfrey et al.2003, Daane et al. 2005), South Africa (Walton et al.2004), and Mexico (Castillo et al. 2005). When leftuncontrolled, P. ficus populations can build to suchlevels as to destroy the crop and even kill the vine(K.M.D. and V.M.W., unpublished data). Besides in-festing the grape clusters, the mealybugs excrete largequantities of honeydew that encrust the leaves, canes,and clusters, resulting in further crop damage, defo-liation, and the growth of sooty molds and bunch rots.Moreover, P. ficus is a vector of several viral diseases(Engelbrecht and Kasdorf 1990) and therefore is con-sidered an economic pest even at low infestation lev-els. In California, the invasive P. ficus is now found in

almost all grape-growing regions in the state (Daaneet al. 2006). Its rapid spread since arriving in southernCalifornia in the early 1990s was most likely facilitatedby the spread of infested nursery material (Havilandet al. 2005).

The serious consequences of P. ficus infestationshave led to tolerance levels that are relatively low,compared with those for other mealybug species. Theresult is that even low vine mealybug densities areoften treated with multiple insecticide applications.In California, the suggested insecticide treatmentsfor P. ficus include a delayed-dormant applicationand/or postharvest application of an organophosphate(chlorpyrifos), and one or more in-season applica-tions of an organophosphate (dimethoate), carbamate(methomyl), insect growth regulator (IGR) (bupro-fezin), or neonicotinoid (imidacloprid) (Bentley et al.2004). Similar programs are used in South Africa (Wal-ton 2003) and Mexico (Castillo et al. 2005), typicallywith even greater reliance on organophosphate ma-terials. Insecticides are limited in their effectiveness;however, because the P. ficus population can feed onall sections of the vine (Daane et al. 2003, Godfrey etal. 2003) and a proportion of the population remainsprotected from insecticide sprays under the bark or onthe roots. Repeated insecticide use also adversely im-

1 Division of Organisms and Environment, Center for BiologicalControl, University of California, Berkeley, CA 94720Ð3114.

2 Current address: Department of Horticulture, Oregon State Uni-versity, Corvallis, OR 97331Ð7304.

3 Corresponding author, e-mail: daane@uckac.edu.4 University of California Integrated Pest Management Program,

Kearney Agricultural Center, Parlier, CA 93648.5 Department of Entomology, University of California, Riverside,

CA 92521.6 Suterra LLC, 213 SW Columbia, Bend, OR 97702Ð1013.

0022-0493/06/1280Ð1290$04.00/0 � 2006 Entomological Society of America

pacts mealybug natural enemies (Walton and Pringle1999). For these reasons, effective, species-speciÞc,and environmentally safe control tools to work incombination with or as an alternative to insecticideprograms need to be developed (Daane et al. 2006).

Here, we present results of Þeld research directedtoward the development of a pheromone-based mat-ing disruption program for P. ficus. The mealybug sexpheromone, which is produced by the female to at-tract the winged adult male, was initially identiÞed byHinkens et al. (2001) and then commercially devel-opedandsuccessfullyused inpheromone-baitedmon-itoring traps (Millar et al. 2002, Walton et al. 2004).The effectiveness of the synthetic pheromone, and itsrelatively inexpensive means of production, presentedthe opportunity to investigate its use for mating dis-ruption. To date, successful mating disruption pro-grams have most often targeted lepidopteran (Cardeand Minks 1995, Suckling 2000) and occasionally co-leopteran(Sciarappaet al. 2005)pests, but researchersin Israel have suggested that this control techniquemay be effective for Planococcus species (Franco et al.2004). We conducted Þeld trials to evaluate the ef-fects of a microencapsulated formulation of the syn-thetic sex pheromone on P. ficus population densitiesand crop damage. SpeciÞcally, we sampled sections ofcommercial vineyards that were treated with phero-mone and sections that were not treated and measured1) season-long mealybug population dynamics, 2)mealybug egg production, 3) mealybug percentageparasitism, 4) crop damage, and 5) the effective Þeldlifetime of the microencapsulated pheromone formu-lation.

Materials and Methods

Mealybug Male Flight Activity and Mating. P. ficushas four to Þve generations per year in CaliforniaÕsSan Joaquin Valley (K.M.D. et al., unpublished data).Male ßights, as recorded by pheromone-baited traps,are not spread evenly over the growing season orrepresented by well developed peaks and valleys, apattern that may be explained by several aspects ofP. ficus biology (Daane et al. 2006). Most of the P. ficuspopulation overwinters as mated females; the Þrstmealybug ßight that is large enough to be monitoredthus consists largely of offspring from the overwin-tered generation and does not occur until May. As theseason progresses, the mealybug generations begin tooverlap and by late July all mealybug stages can befound, resulting in an overlap of male ßights. In ad-dition, toward the end of the growing season a para-sitoid, Anagyrus pseudococci Girault (Hymenoptera:Encyrtidae), increases in effectiveness (Daane et al.2004), reducing the August and September popula-tions and consequently diminishing the Þnal maleßights of the season. For these reasons, we targetedour applications of the pheromone from April (beforethe initial ßight activity) to August (the end of sig-niÞcant male ßight activity in the San Joaquin Valley).Field Sites and Treatment Application. In 2002,

experiments were conducted in three commercial

vineyards located near Del Rey, CA. In 2003 and2004, experiments were conducted in six commercialvineyards, located near Del Rey, Sanger, and Fowler,CA, and the Kearney Agricultural Center (2004 only),located near Parlier, CA. The vineyards ranged from5 to 12 ha, and each was furrow-irrigated, clean-cultivated, and planted on a Hanford sandy loam soil.The vines were mature (�10-yr-old), caned-prunedÔThompson SeedlessÕ, with canes supported by a T-trellis (two or four-wire) system. In each vineyard, weestablished a plot for the microencapsulated phero-mone and another for a no-pheromone control. Ineach vineyard, the treatment plots were separated bybuffer strips of �20Ð120 m, depending on the vine-yard size, to minimize pheromone drift into the no-pheromone control. Mating disruption and controltreatments were then randomly assigned, by using asplit-plot design. The synthetic pheromone used wasracemic lavandulyl senecioate (details on chemicalproduction in Millar et al. 2002), produced by KurarayFine Chemicals (Tokyo, Japan). The synthetic pher-omone was microencapsulated by Suterra Inc. (Bend,OR), with the formulation containing 16.30 and10.81% (AI) by weight of the pheromone, lavandulylsenecioate, in 2002Ð2003 and 2004, respectively. Themicrocapsules average �100 �m (particle size analy-sis � 152 �m) and were formed using a proprietarypolymer matrix (Suterra Inc.) that controls phero-mone release. The application rate, application dates,plot size, and insecticide treatments varied each year,as follows.

In 2002, the sprayable pheromone was Þrst availablein June, well after the mealybug male ßights and mat-ing began. Therefore, this initial trial was designed totest application methodology for use in the next sea-sonÕs Þeld trial. The microencapsulated formulationwas mixed with water (1 ml; 7.6 liter) and applied tovines using an airblast spray rig at a rate of 6.32 g(AI)/ha on 7 June (two vineyards) and 6 July (onevineyard). In all three vineyards, 0.1Ð0.2-ha matingdisruption and control treatment plots were estab-lished. No insecticides were planned for the matingdisruption or control plots during the trial period.

In 2003, the sprayable pheromone was mixed withwater (1 ml; 7.6 liter) and applied at a rate of 10.69 g(AI)/ha per application in each of Þve vineyards.Application dates were on or between 12 and 15 May,which was before any adult male mealybugs werecaught in pheromone traps, then on 19 June and2Ð4 August. In total, 32.07 g (AI)/ha per season wasapplied in the mating disruption plots. Plot size variedfrom 1.5 to 2.2 ha per treatment plot (20Ð35 vine rowsby 50Ð100 vines), with �50Ð120 m between plots ineach vineyard. In addition to the pheromone appli-cations, a delayed dormant application of an organo-phosphate (chlorpyrifos) was applied at the full labelrate (Lorsban 4E, 4.7 liter/ha in 1,400 liters of water),uniformly to all plots between 17 and 25 February2003.

In2004, sprayablepheromonewasmixedwithwater(1 ml; 7.6 liter) and applied at a rate of 10.69 g (AI)/haper application in each of Þve vineyards. Application

August 2006 WALTON ET AL.: MATING DISRUPTION FOR P. ficus 1281

dates were 20 April, 19 May, 16 June, and 19 July. Intotal, 53.45 g (AI)/ha per season was applied in themating disruption plots. Plot size varied from 0.15 to0.29 ha per treatment plot (5Ð10 vine rows by 25Ð50vines), with �20Ð30 m separating plots in each vine-yard. In addition to the pheromone applications, anin-season application of an IGR (buprofezin) was ap-plied at 50% of the full label rate, uniformly to all plotsbetween 10 and 16 June 2004. We also note that matingdisruption using dispensers loaded at 60 mg per dis-penser (Suterra Inc.) and 40 and 160 mg per dispenser(Shin Etsu Chemical Co.-Biocontrol Ltd., Mt. Crosby,Australia), dispersed at 101.2 dispensers per ha, wastested in these same vineyards, using similarly sizedplots and buffer zones as reported for the sprayableformulation.Insect Sampling. To monitor male mealybug ßight

periods and densities, three red Pherocon Delta IIIDsticky traps (Trece Inc., Adair, OK) baited with luresloaded with 100 �g of the sex pheromone (lavandulylsenecioate) (Suterra, Inc.) were placed in each treat-ment plot. Traps were hung from trellis wires such thatthey were positioned above the cordon but within thevine canopy. Traps were spaced evenly within eachtreatment plot, typically with a minimum spacing ofthree rows and 10 vines between each trap. Traps andlures were replaced every 2Ð4 wk, depending on theseasonal period and removed during each applicationof the sprayable pheromone. Trapped insects werecounted using a dissecting microscope.

Mealybug densities were determined using a timedvisual count, based on a method developed for themealybug Pseudococcus maritimus (Ehrhorn) (Geigerand Daane 2001). In each plot, two to four vines wererandomly selected in each of three to Þve rows (10vines per plot per sample date; vines and rows on theplot edges were not sampled). On each vine, a 3-minsearch was conducted, and all visible mealybugs wererecorded as the following developmental categoriesÑcrawlers and Þrst instars, second and third instars,adults, and ovisacsÑand at the following vine loca-tionsÑground (from 5 cm below the soil line to 30 cmabove on the trunk), trunk, cordon, old canes, newcanes, leaves, and grape bunches (when present).Mealybug Egg Production. To determine the effect

of the pheromone mating disruption treatment onmealybug egg production, as a measurement of fe-cundity, we collected and isolated female mealybugsto compare egg production between treatment plots.Approximately 100 female mealybugs were collectedfrom each treatment plot every 2Ð4 wk from 3 June to8 October 2003 and from 15 April to 26 July 2004.Collected mealybugs were placed individually in gel-atin capsules and held at 22 � 4�C until oviposition andegg hatch were complete, a process that was typicallycompleted in 1Ð2 mo. In 2003, we collected all mea-lybug stages, whereas in 2004 we collected only ma-ture (third instar and preovipositional adult) mealy-bugs. The numbers of unhatched eggs and crawlerswere recorded for each female. The percentage ofparasitism also was determined from the collectedmealybugs to provide some indication of the effects of

the pheromone treatment on natural enemy behaviorand abundance in the Þeld.CropDamage. In the 2003 and 2004 trials, mealybug

crop damage was rated using a 0Ð3 scale, as describedby Geiger and Daane (2001), where 0 indicates nomealybug damage; 1 indicates a grape bunch withhoneydew (an indication of mealybug presence); 2 isa bunch with honeydew and mealybugs, where part ofthe bunch is salvageable; and 3 represents a total lossof the sampled bunch. In 2003, crop damage was eval-uated for Þve clusters on 20 (two plots) or 50 (threeplots) randomly selected vines per plot (950 clustersper treatment). Sampling in 2003 indicated that mea-lybug distribution within the vineyard tended to beclumped; and to reduce sample variation resultingfrom a clumped distribution, this procedure was mod-iÞed in 2004 so that more vines were sampled. A singlecluster was sampled on each of 100 vines (500 clustersper treatment). The vines were located in the centerthree rows of each plot, with vines randomly selectedin each sampled row. Clusters located near the trunkand, when possible, touching either the trunk or cor-don were preferentially sampled because they aremore susceptible to mealybug infestation (Geiger etal. 2001).Effect of Mealybug Density. Results in 2003 indi-

cated that the mating disruption treatment had little orno effect in the vineyard with relatively high mealy-bug population density (simply deÞned here as a den-sity that caused �10% crop loss). We modiÞed themethods in 2004 to better follow pheromone treat-ment effects on different mealybug population den-sities; our goal was to sample vines that had differentinitial mealybug densities within the same vineyardthroughout the season. To select vines, we surveyedeach vineyard on 7 April, before treatments wereapplied, by using the 3-min visual search of randomlyselected vines. The vines were classiÞed as having low,medium, or high mealybug densities based on thefollowing criteria. Low density-infested vines had novisible mealybug infestation, and, additionally, thesevines were treated with an IGR (buprofezin) at thefull label rate on 12 April. Medium-infested vines hadno ant activity or discoloration of the vine resultingfrom honeydew, with �10 mealybugs found during a3-min visual search. High-infested vines had sometending ant activity [Formica aerata (Francoeur)],honeydew or sooty mold blackened trunk or cordonsections, and �10 mealybugs found during a 3-minvisual search. In each treatment plot, we selected fourvines of each category to repeatedly sample through-out the season. The 3-min sampling method is destruc-tive and can alter mealybug densities through theremoval of bark or canes (Geiger and Daane 2001).For this reason, we used a nondestructive leaf sampleto enumerate population changes in the vine canopy.For every 2 wk throughout the season, two basal leaveswere sampled on each vine and all mealybugs wererecorded.Pheromone Residue. The microencapsulated for-

mulation starts emitting sex pheromone immediatelyupon exposure to the atmosphere. The small size of

1282 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 99, no. 4

these capsules may cause rapid depletion of the pher-omone due to evaporation and degradation by expo-sure to heat and sunlight. Therefore, to optimize thepresence of a “pheromone cloud” throughout the sea-son, the effective Þeld lifetime of the microcapsuleswas determined. On 10 July 2003, the microencapsu-lated formulation was applied at a rate of 19.78 g(AI)/ha to 10 vines in a Thompson Seedless vineyardlocated at the Kearney Research and Extension Cen-ter, Parlier, CA. The vineyard had no P. ficus and noinsecticides were applied. From these pheromone-treated vines and water-treated control vines (located�50 m away), we randomly collected 10 leaves at 1, 7,14, 21, 28, 35, and 42 d after the formulation wasapplied. The leaves were placed individually onto thesticky surface of Pherocon Delta IIID pheromonetraps, which were then randomly placed at 1Ð3-mdistances from a P. ficus population inside an insectaryroom for 24 h. Adult males were counted on each trap.Statistics. Results are presented as treatment

means � SEM. For the pheromone trap counts andvisual mealybug counts, we compared season-longtreatment effects (treatment � sample date) by usingrepeated measures analysis of variance (ANOVA)(Systat 2000). Data were transformed (log[x�1]) asneeded to stabilize the variance. To compare egg pro-duction between treatments, we used a t-test for eachsample date, because collected mealybugs were inde-pendent of population density. For cluster damage, asmeasured by the rating scale, treatment effects werecompared in a 2 by 2 contingency table with treat-ments separated using SpearmanÕs rank order test(Systat 2000). To compare mealybug densities fromleaf samples on vines categorized as low, medium, orhigh densities, we Þrst plotted the season-long mea-lybug density in the control plots for each of thesecategories and used repeated measures ANOVA tovalidate the accuracy of our preseason categorizationof mealybug density. For this comparison, data weretransformed (log[x� 1]) to stabilize the variance, andpairwise comparisons were made of three possiblecategorical combinations, with � set at P � 0.0167(0.05/possible combinations). An assessment of treat-ment impact in each density category was then madeusing TukeyÕs honestly signiÞcant difference (HSD)test on the percentage change of the mealybug densityin the mating disruption treatment compared withcorresponding control plots (Systat 2000). Here, weused data collected on two sample dates (17 June and1 July 2004), when mealybug densities peaked andtreatment differences were greatest. The data on per-centage of reduction were transformed ((x� 0.5))to stabilize the variance. For each trial, statistical de-tails are listed in the corresponding Þgure captionswhere appropriate.

Results

Field Trials. In 2002, one of the three vineyardsused was treated with methomyl during the 3-wk trial,a result of the collaborating growersÕ concerns withpotentially damaging mealybug densities in the ex-

perimental plot (where we used crop destruction afterharvest) moving to uninfested portions of the vine-yard. A second vineyard was pulled out, in part be-cause of high mealybug densities and low raisin cropprices. In 2003, one of Þve vineyards was treated withchlorpyrifos during the trial period, with material ap-plied to all but three rows each of the mating disrup-tion and control plots. Although we continued to sam-ple these vineyards, separating the insecticide-treatedsections from the control sections (three rows) in2003, these data are not included in the analysis unlessotherwise noted.Mealybug Male Flight. The microencapsulated

pheromone was Þrst available for application in July2002, too late in the season to affect mealybug density.Therefore, our goal that year was to test whether themicroencapsulated application method could prop-erly deliver pheromone, which we did by measuringmale mealybug trap catches 3 wk after application. Inthe vineyard that was not disrupted by the growersÕactions, there were signiÞcantly fewer mealybugmales captured in the pheromone-treated plot (0.33 �0.33 males per trap) than the control plot (12.16 � 5.45males per trap) (t � 2.17; df � 10; P � 0.05; pairedt-test, with sample date and vineyard row used to pairtrap counts). These preliminary results suggested thatour Þeld application methods could be used in largerÞeld trials.

In 2003 and 2004, the sprayable pheromone wasapplied throughout the Þeld season. In the controlplots, male ßight activity, as recorded by the phero-mone-baited traps, was Þrst detected in May, with trapcounts rising to a peak in late July, and steadily de-clining thereafter (Fig. 1). Season-long trap catcheswere signiÞcantly lower in the mating disruption plotsthan in the control plots in 2003 (Fig. 1A) and 2004(Fig. 1B). However, male mealybugs were still caughtin the mating disruption plots and although numberswere lower overall, the seasonal ßight pattern wassimilar to that in the controls in 2004. A particularlystrong indication of the treatment impact is the num-ber of male mealybugs per trap per week from Junethrough August, during the most active period, whichwere 17.6 and 4.4 times greater in the control than themating disruption treatment in 2003 and 2004, respec-tively (2003: t� 3.86, df � 25, P� 0.001; 2004: t� 2.74,df � 29, P � 0.01).Mealybug Population Density. In 2003 and 2004,

mealybug populations were detected throughout thesampling period (April through October), reßectingthe year-round presence of the mealybug in the testedvineyards. Population density, as determined by the3-min search, increased rapidly from June to July(2003) and from April to June (2004), followed by adecrease from late July into August. In 2003, there wasno season-long treatment impact on the density ofsettled mealybugs (second instar to adult mealybugs)(Fig. 2A), whereas in 2004, there were signiÞcantlyfewer mealybugs in the mating disruption treatmentthan the control (Fig. 2B).

We compared the proportion of the six differentmealybug developmental categories present (Þrst,

August 2006 WALTON ET AL.: MATING DISRUPTION FOR P. ficus 1283

second, and third instars, and adult, adult with ovisac,and ovisac with eggs) as determined by the 3-mincounts, on sample dates when the pheromone appli-cations would have an impact on mealybug mating(18 June 2003Ð28 August 2003 and 2 June 2004 to23 August 2004). In 2003, there were signiÞcantlymore Þrst instars and fewer ovisacs in the matingdisruption than in the control treatment (Fig. 3A). In2004, there were signiÞcantly fewer Þrst instars andovisacs in the mating disruption treatment (Fig. 3B).Egg Production. In 2003, average egg production

from Þeld-collected adult mealybugs (isolated in gel-atin capsules) was 46.8 � 1.7 eggs per female acrossall treatments and sample dates. During the sampledates when the sprayable pheromone was newly ap-plied (3 June to 29 July 2003) and excluding parasit-ized mealybugs, egg production was lower in the mat-ing disruption plots (32.7 � 3.4 eggs per female, n �174) than in the control plots (55.0 � 2.6 eggs perfemale, n � 299) (t � 5.09, df � 471, P � 0.001). The

greatest impact on egg production was that 32.7% ofcollected mealybugs from the mating disruption treat-ment did not produce eggs, whereas only 9.0% ofmealybugs from the control treatment did not pro-duce eggs. Egg production on individual collectiondates provides a clear description of treatment impact.On three of four sampling dates during this period, eggproduction was signiÞcantly lower in the mating dis-ruption treatment, whereas there were no differenceson the two sample dates after the mating disruptionsprays were discontinued (Fig. 4A). In 2004, averageegg production was 23.2 � 0.8 eggs per female acrossall treatments and sample dates. During the sampledates when the sprayable pheromone was newly ap-plied (3 May to 26 July 2004), there were 22.2 � 1.3(n� 863) and 24.2 � 1.3 (n� 859) eggs per female inthe mating disruption and control treatments respec-tively. On individual sample dates, egg productionwas signiÞcantly different between treatments foronly two samples, and there egg production was bothhigher (14 May) and lower (26 July) in the matingdisruption plots (Fig. 4B).

Fig. 1. Season-longcountsofmaleP.ficus(mean�SEM)caught in pheromone-baited Delta traps. Counts were sig-niÞcantly lower in mating-disruption plots, compared withthe control, in the (A) 2003 season (F� 83.24; df � 1, 8; P�0.001) but (B) not in the 2004 season (F� 4.08; df � 1, 8; P�0.078). Data used for the repeated measures ANOVA anal-yses were from May through August collections in both 2003and 2004. Solid arrows indicate application dates for thesprayable pheromone, and the open arrow indicates theapplication date of buprofezin in 2004.

Fig. 2. Season-long density of settled (second instar toadult) P. ficus (mean � SEM), as measured by timed countson randomly sampled vines, in mating disruption and thecontrol plots in the (A) 2003 season (F� 0.19; df � 1, 6; P�0.68) and (B) 2004 season (F� 5.77; df � 1, 8; P� 0.04). Solidarrows indicate application dates for the sprayable phero-mone, and the open arrow indicates the application date ofbuprofezin in 2004.

1284 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 99, no. 4

Parasitoid Activity. In 2003, of 2,654 mealybugs iso-lated in gelatin capsules, 41.4 � 1.0% were parasitized.The mealybug developmental stage isolated inßu-enced percentage of parasitism, with the third andsecond instars more commonly parasitized (51.1 � 1.7and 56.3 � 2.1%, respectively) than the Þrst instar andadult stages (29.0 � 2.5 and 28.7 � 1.4%, respectively).Of the parasitoids reared to the adult stage (n� 593),A. pseudococci was the most common (86.3 � 1.4%),followed by Allotropa sp. (Platygastridae) (11.5 �1.3%) andLeptomastideaabnormis(Girault) (Encyrti-dae) (2.2 � 0.6%). There were no differences in levelsof parasitoid activity, as measured by either numbersof mummies counted during the 5 min search on vines(Fig. 5A), or the percentage of mummies obtainedfrom mealybugs collected and isolated in gelatin cap-sules (Fig. 5B).

In 2004, of 4,390 mature mealybugs (third instar andadults) isolated in gelatin capsules, only 2.8 � 0.3 wereparasitized, all by A. pseudococci. There was no sig-

niÞcant difference in parasitism levels between mat-ing disruption and control treatments on any sampledate (n � 9). Similarly, there was no season-longdifference in levels of parasitoid activity, as measuredby numbers of mummies counted during the 5-minsearch of vines (F � 0.17; df � 1, 8; P � 0.69).CropDamage. SigniÞcantly lower crop damage rat-

ings were recorded in mating disruption than controltreatments in 2003 and 2004 (Fig. 6). Fewer grapeclusters were rated as having “moderate” or “severe”damage in mating disruption plots (3.1 and 4.0% in2003 and 2004, respectively) compared with the con-trols (9.1 and 11.8% in 2003 and 2004, respectively).Effect ofMealybugDensity.There was a signiÞcant

difference in mealybug density on vines in the controlplots, which followed the predicted pattern of low,medium, and high mealybug densities (Fig. 7A). Wethen compared the change in mealybug densities onleaves in thematingdisruptionplots, as comparedwiththe control plots. Mealybug densities were reduced by86.3 � 6.3% on vines that were previously categorizedas having a low pretreatment mealybug density,whereas vines that were categorized as having a highpretreatment mealybug density had only a 9.0 � 35.7%reduction (Fig. 7B).

Fig. 3. Proportion of P. ficus development stages fromtimed counts of Þeld populations taken from May throughAugust collections in (A) 2003 and (B) 2004 showing non-signiÞcant treatment differences for the percentage of Þrstinstars in 2003 (t� 1.81, df � 174, P� 0.071) but signiÞcantdifferences in 2004 (t � 2.01, df � 468, P � 0.045), andsigniÞcant differences in ovisacs produced in 2003 (t� 2.37,df � 174, P � 0.018) but not 2004 (t � 1.67 df � 468, P �0.095). Code above each pair of bars denote � values at NS,not signiÞcant; *, �0.05; and **, �0.01.

Fig. 4. Numbers of eggs produced per Þeld-collectedadult female (mean � SEM), isolated individually in gelatincapsules in (A) 2003 and (B) 2004. Code above each pair ofbars denote � values at NS, not signiÞcant; *, �0.05; **, �0.01;and ***, �0.001. Numbers inside bars indicate the number offemales collected.

August 2006 WALTON ET AL.: MATING DISRUPTION FOR P. ficus 1285

Effective Field Lifetime of MicroencapsulatedPheromone. From 1 to 28 d after the microencapsu-lated pheromone was applied to leaves, signiÞcantlymore male mealybugs were caught in pheromonetraps baited with a leaf with adhering microcapsulesthan in traps baited with a water-sprayed leaf (Fig. 8).The initial trap catches (day 1) were low, which mayhave been a reßection of the colony size rather thantreatment impact. SigniÞcantly more male mealybugswere collected on sticky traps baited with pheromone-treated leaves, compared with the control, on days1Ð28, and there was no difference by day 35. Theresults indicate that the pheromone release rates de-cline after 3 wk and are no longer effective after 5 wk.

Discussion

The use of synthetic pheromones for mating dis-ruption has proven to be commercially effective for anumber of insect pests, most notably lepidopteraninsects in orchard crops, cotton, or tomatoes (Cardeand Minks 1995, Welter et al. 2005). To test the fea-

sibility of developing a commercial mating disruptionprogram for P. ficus, we applied a microencapsulatedpheromone formulation to sections of commercialvineyards, in combination with insecticide applica-tions. We observed a signiÞcant reduction in the num-ber of male mealybugs caught in traps. This result is anindication of pheromone effects, but it does not nec-essarily signify successful mating disruption; the re-duction in trap catches among treatments is not alwaysproportional to the reduction of crop damage orchanges in pest population density, as shown for ori-ental fruit moth,Grapholitamolesta (Busck) (Kovanciet al. 2004), and pink bollworm, Pectinophora gossyp-iella (Saunders) (Lykouressis et al. 2005). In fact,when we later measured mealybug population densityon the vines, we found that the level of mealybugreduction as measured by pheromone trap catches(Fig. 1) was much greater than that recorded by visualcounts of mealybugs (Fig. 2). Most important was thereduction of mealybugs and their damage in the grapeclusters, which were signiÞcantly lower in combina-tion mating disruption and insecticide treatments,compared with insecticide treatment alone.

In both 2003 and 2004, there were signiÞcantlyfewer ovisacs produced, as a proportion of the mea-lybug population, in the mating disruption treatmentthan in the controls. It is expected that mating dis-ruption would proportionally reduce the number ofovisacs and newly hatched crawlers, as a result ofunmated females. Of greater interest is the proportionof ovisacs and the number of eggs per ovisac. P. ficusegg production in South African vineyards reportedlyranges from 75 to 316 eggs per female (Walton 2003).

Fig. 5. Parasitoid activity in the 2003 season as measuredby (A) the number of mummies found during timed Þeldcounts (repeated measures ANOVA: F� 0.01; df � 1, 6; P�0.999) and (B) the number of parasitoids reared from Þeld-collected mealybugs isolated in gelatin capsules. Code aboveeach pair of bars denote � values at NS, not signiÞcant; and*, P � 0.05. Solid arrows indicate application dates for thesprayable pheromone, and the open arrow indicates theapplication date of buprofezin in 2004.

Fig. 6. Cluster damage ratings for insecticide and controltreatments in (A) 2003 and (B) 2004, where 0 is no mealybugdamage, 1 is honeydew (indicating the presence of mealy-bugs), 2 is honeydew and mealybugs but the cluster is har-vestable, and 3 is unmarketable. There was signiÞcantly lessdamage in the mating disruption treatment in 2003 (Pearson�2 � 54.81, df � 3, P� 0.001) and 2004 (Pearson �2 � 37.39,df � 3, P � 0.001).

1286 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 99, no. 4

Studies in California using the same methodologyÑisolating Þeld-collected adult mealybugs in gelatincapsulesÑshowthat themajorityof adults collected invineyards isolated from any mating disruption treat-ment produced an ovisac and an average egg produc-tion �150 eggs per female (K.M.D. and R.M.-K., un-published data). In contrast, in the 2003 trials, theoverall egg production per female was �70 eggs perfemale, across all treatments; a signiÞcant number of

mealybugs did not produce eggs, especially in themating disruption treatment. In the 2004 trials, theproportion of mealybugs producing an ovisac was�40%, with only 23 eggs per female produced acrossall treatments. Two factors may have decreased eggproduction across both treatments in 2004 comparedwith 2003. First, in-season application of IGR insecti-cide, applied only in 2004, should have retarded eggproduction. Second,differences in the sizeofplots andbuffer zones might also be an issue. The 2004 trials,compared with the 2003 trials, were conducted insmaller plots (�0.3 versus �1.5 ha) with smaller bufferzones (20Ð30 versus 50Ð120 m) between treatments,as described previously. Moreover, nearby sections ofeach vineyard were used to compare mating disrup-tion by using different pheromone loads dispersed inplastic dispensers. We speculate that pheromone driftinto the control plots may have lowered egg produc-tion. The relatively small plot size was an unavoidablecomponent of working with the sprayable formulationof the P. ficus sex pheromone, which is not yet regis-tered for Þeld use and thus necessitated crop destruc-tion in the treated plots. Plot size can impact treatmentdifferences; for example, McLaughlin et al. (1994)showed that mating disruption of the diamondbackmoth, Plutella xylostella (L.), was more successful inthe interior portions of a treated Þeld than near theperimeter.

One aspect not addressed in this study is the sexratio of the developing offspring. Some mealybugspecies have atypical reproductive systems, anadaptation known as arrhenotokous haplodiploidy,whereby males develop from unfertilized eggs (Nor-mark 2003). In practical pest management terms, this

Fig. 7. (A) Average mealybug counts per leaf � SEM onvines in control plots categorized as having low, moderate, orhigh mealybug densities during a survey on 19 April 2004.Mealybug density, as determined by repeated measuresANOVA by using sampling dates between 16 May and29 June, differed signiÞcantly among each category (F �18.70; df � 2, 9; P � 0.001), with pairwise comparisonsshowing a signiÞcant difference between low versus high(F� 61.83; df � 1, 6; P� 0.001) and medium versus high (F�27.86; df � 1, 6; P� 0.002), whereas there was no differencebetween low versus medium (F � 2.87; df � 1, 6; P � 0.14)categories. (B) Percentage reduction of mealybugs variedsigniÞcantly among mealybug density categories (F � 5.88;df � 2, 12; P� 0.016) and was greater in the low versus highdensity category (the medium- versus high-density categorywas P � 0.069).

Fig. 8. Numbers of adult male P. ficus (mean � SEM)caught in Delta traps baited with vine leaves treated with themicroencapsulated pheromone versus a water-sprayed con-trol leaf on day 1 (t � 5.02, P � 0.001), day 7 (t � 5.23, P �0.001), day 14 (t � 3.73, P � 0.001), day 21 (t � 2.99, P �0.008), and day 28 (t� 3.53,P� 0.002), after which there wasno signiÞcant difference (P � 0.05). Codes above each pairof bars denote � values at NS, not signiÞcant; *, � 0.05;**, � 0.01; and ***, � 0.001.

August 2006 WALTON ET AL.: MATING DISRUPTION FOR P. ficus 1287

suggests that the longer term effects of mating dis-ruption will be most evident in the second and sub-sequent generations after treatment. That is, the Þrstgeneration after treatment will consist primarily of areduced population with the sex ratio skewed towardmales. The effect should be even more pronounced inthe next generations, with fewer mealybugs overall,and an increasingly biased sex ratio.

Parasitism levels were not disrupted by the matingdisruption treatment, a result that was unexpected. Incontrast, earlier studies with the P. ficus pheromoneshowed that the parasitoid A. pseudococci was at-tracted to the pheromone traps (Millar et al. 2002),and we saw an increase in parasitism levels in matingdisruption trials in South Africa (V.M.W. and K.M.D.,unpublished). In the current study, two factors mayhave inßuenced parasitism, reducing the differencebetween treatments. First, in 2003 trials the vineyardshad high levels of parasitism in both treatments(Fig. 5B), a result of reduced insecticide use andinoculative release of A. pseudococci in adjacent vine-yards from 2001 to 2003. Second, in 2004 trials thevineyards received an in-season application of theIGR in June, which is a critical period for the over-wintered A. pseudococci to locate and oviposit in ex-posed hosts (Daane et al. 2004). There have beenmixed reports of natural enemy activity in matingdisruption programs. BeneÞcial arthropods were moreabundant in peach orchards under a reduced risk pestmanagement program, which included mating disrup-tion for the oriental fruit moth, compared with con-ventionally farmed orchards (Atanassov et al. 2003).Biddinger et al. (1994) compared the parasitoid com-plex of the tufted apple bud moth, Platynota idaeusalis(Walker), in orchards by using either conventionalbroad-spectrum insecticides or mating disruption, andthey reported mixed results on parasitism levels de-pending on the seasonal sampling period. In contrast,parasitism levels of the grape berry moth, EndopizavitanaClemens, were lower in plots treated with mat-ing disruption than in plots treated with conventionalinsecticides (Williamson and Johnson 2005). In thesestudies, changes in beneÞcial fauna were discussedwith respect to the elimination of broad-spectruminsecticide use, replaced by mating disruption. Wesuggest that the attraction of A. pseudococci to theP. ficus pheromone results in foraging parasitoidsspending more time host searching in mating disrup-tion plots and may even draw parasitoids in fromnearby Þelds.

For commercialization of a sprayable formulationto be adopted, the effective Þeld lifetime of the for-mulations must be improved. The efÞcacy of thesprayable formulation used in our studies clearly de-clined after only 3 wk, with the pheromone totallydepleted after 5 wk. The short Þeld lifetime of theformulation may explain, in part, the better perfor-mance of the mating disruption program in 2004,where there were four applications, compared with2003, when only three applications were made. How-ever, residual activity in the vineyard may havebeen slightly better because microcapsules in shaded

areas, bark cracks, and other protected areas may havehad a slightly longer effective lifetime (Karg et al.1994). The effective lifetime of microencapsulatedmaterials is dependent on the microcapsule porosityand coating composition (Stipanovic et al. 2004),which can be controlled, and ambient temperature,rainfall, and sunlight exposure (Bradley et al. 1995,Millar 1995, Waldstein and Gut 2004), which cannotbecontrolled.Wesuggest thatproblemswitheffectivelifetime can be overcome with better formulation ofthe microencapsulated particles. Improvement of theeffective Þeld lifetime is clearly required to develop arobust and reliable control program.

For mating disruption programs, there are severaladvantages to a microencapsulated formulation com-pared with other types of dispensers (discussed inTrimble et al. 2004). Of particular importance forcontrol of small insects such as P. ficus is that thesprayable formulation provides relatively completecoverage, with the microencapsulated pheromone innumerous point sources on each vine. The distributionof mealybugs in a vineyard is often clumped (Geigerand Daane 2001), and the fragile adult males are notstrong ßiers. Therefore, male-to-female distances areoften small, and the pheromone coverage provided bya few hundred large point sources of pheromone perhectare, such as the plastic tube dispensers commonlyused for mating disruption of Lepidoptera may not beadequate for disruption of insects separated by only afew centimeters. Additionally, the sprayable formula-tion has the advantage of being amenable to mixeswith other pesticide applications or pheromones(Carde and Minks 1995, Judd et al. 2005).

Regional temperatures also may affect program suc-cess. We should note that our studies were conductedin CaliforniaÕs San Joaquin Valley, where ambientsummer temperatures during the trial averaged 33.3 �0.3�C (high) and 18.3 � 0.4�C (low). We suggest thata mating disruption program for P. ficusmay be moresuccessful in regions with cooler climates, where theremay be a longer residual activity of the applied pher-omone and fewer mealybug generations, resulting ina seasonally shorter male ßight period.

Our research identiÞes several key factors requiringfurther improvement for a commercially successfulmating disruption program for P. ficus and suggestsfurther experimental studies to better reßect the po-tential of this program. Most evident was the effect ofmealybug density on the effectiveness of mating dis-ruption: the proportional reduction of mealybugdensity was much greater on vines with low initialmealybug densities. It is well known that the perfor-mance of mating disruption can decline with in-creased pest population density (Carde and Minks1995). For this reason, a combination of control tacticsmay prove more effective than a single tactic, as re-vealed in other pest systems: 1) mating disruption, treebanding, and sterile insect release for codling moth,Cydia pomonella (L.) (Judd and Gardiner 2005); and2) mating disruption and insecticide applications fororiental fruit moth (Trimble et al. 2001). Contrarily,application of mating disruption in combination with

1288 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 99, no. 4

fewer applications of insecticides to apple orchardsdid not lower damage of the obliquebanded leaf-roller, Choristoneura rosaceana (Harris) (Trimble andAppleby 2004). Our results are consistent with codlingand fruit moth studies and suggest that commercial-ization of this program may include some use of in-secticides or other practices to lower the initial mea-lybug density to a level at which the mating disruptionis effective.

Further studies also are needed to determine theoptimal application rates and intervals, as has beendone with mating disruption of pests such as orientalfruit moth (Kovanci et al. 2005) and obliquebandedleafroller (Judd et al. 2005). In our studies, we alsoused a formulation of �99% chemically pure lavan-dulyl senecioate, which greatly increased productioncosts. If similar levels of efÞcacy could be obtainedwith lower purity and less expensive technical gradematerial, overall costs of a mating disruption programcould be greatly reduced. Further study and manip-ulation of the formulation to enhance longevity mayshow that mating disruption is an effective, econom-ical, and sustainable tool to be implemented as part ofa mealybug management program.

Acknowledgments

We thank Suterra Inc. for the microencapsulated phero-mone, and the vineyard owners and farm managers for Þeldsupport and use of vineyards. Lee Marvin coordinated Þeldresearch and Doug Middleton, Josh Woods, Juan Sanchez,Glenn Yokota, and Rodney Yokota provided Þeld and labo-ratory help. Funding was provided by the California TableGrape Commission, the California Raisin Marketing Board,and the American Vineyard Foundation.

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Received 11 December 2005; accepted 26 February 2006.

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