P I Cicadomorpha Insects Associated With Bacterial Leaf ...

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Cicadomorpha Insects Associated With Bacterial Leaf Scorch InfectedOak in Central New Jersey

JIANXIN ZHANG,1,2 JAMES LASHOMB,1 ANN GOULD,3 AND GEORGE HAMILTON1

Environ. Entomol. 40(5): 1131Ð1143 (2011); DOI: http://dx.doi.org/10.1603/EN10083

ABSTRACT Potential insect vectors for transmission of oak leaf scorch caused by Xylella fastidiosaWells et al., in pin and red oaks in New Jersey were surveyed by placing yellow sticky card traps intree canopies and fogging with pyrethrin insecticide during 2002Ð2006. Thirty-seven Cicadomorphaspecies were collected from 20 genera in Membracidae, Cicadellidae, Aphrophoridae, and Clastop-teridae. Of the 12,880 potential vectors collected, 91.4% were Membracidae, 6.9% were Cicadellidae,and 1.7% were Aphrophoridae and Clastopteridae. Fogging collected more insect species and indi-viduals than sticky card collections. Sticky card sampling, done more frequently and at a larger numberof locations provided similar community structure information as fogging. Sticky card collections ofthe dominant treehopper species,Ophiderma definitaWoodruff were male biased when females weregravid.O. definita populations peaked in early June, comprised 68.2% of the total collection, and weremore abundant in pin oaks than red oaks.Graphocephala versuta (Say) peaked in mid-July, comprising6.2% of the total collection. Higher Cicadomorpha populations were observed in asymptomatic oakcanopies than in neighboring X. fastidiosa infected oaks. Individual insect specimens collected fromoaks were subjected to a X. fastidiosa DNA assay by polymerase chain reaction ampliÞcation. Theaverage X. fastidiosa positive rate was 13.89% for all specimens tested. Eleven treehopper species, sixleafhopper species, and four spittlebug species tested DNA positive for X. fastidiosa.

KEY WORDS Xylella fastidiosa, Membracidae, Cicadellidae, Aphrophoridae, Clastopteridae

Little is known about the insect vectors of bacterialleaf scorch, especially in oaks in the northeasternUnited States. Xylella fastidiosa Wells et al. is thecausal agent of oak bacterial leaf scorch (Hearon et al.1980) as well as many other plant diseases such asalmond leaf scorch (Mircetich et al. 1976), citrus var-iegated chlorosis (Lee et al. 1993), grape PierceÕs dis-ease (Hopkins and Mollenhauer 1973), maple leafscorch (Sherald et al. 1987), oleander leaf scorch(Huang et al. 2003), periwinkle wilt (McCoy et al.1978), phony peach disease (Hopkins et al. 1973),plum leaf scald (Kitajima et al. 1975), and sycamoreleaf scorch (Sherald et al. 1983). Sharpshooter leaf-hoppers and spittlebugs are known X. fastidiosa vec-tors of the almond leaf scorch (Purcell 1980a), citrusvariegated chlorosis (Brlansky et al. 1996, Krugner etal. 2000), phony peach disease (Turner and Pollard1959), periwinkle wilt (McCoy et al. 1978), PierceÕsdisease (Severin 1949, 1950, Purcell 1980b; Adlerz andHopkins 1979; Almeida and Purcell 2003), oleanderleaf scorch (Costa et al. 2000), and possibly bacterialleaf scorch of street trees. Sharpshooters and spittle-bugs are usually larger in size than phloem feeding

leafhoppers (Novotny and Wilson 1997) with the abil-ity to feed on negatively pressured xylem ßuid.

Northern red oak Quercus rubra L. and pin oak Q.palustrisMuenchh are major street trees in New Jer-sey and are susceptible to X. fastidiosa infection. Dur-ing the past 20 yr, bacterial leaf scorch disease of oakhas spread throughout the state with infections re-ported in every county (A. Gould et al. unpublisheddata). Although live bacterium and DNA of X. fasti-diosa have been detected in many Cicadomorpha (J.Zhang et al. unpublished data), their seasonal appear-ance and relationship to disease incidence is yet to bedetermined. Our main objective was to record theCicadomorpha species complex in bacterial leafscorch infected oak. We then wanted to report theproportion of each species harboring X. fastidiosa. Toaccomplish these goals we used two sampling methodsfogging (absolute) and sticky trap (relative). We com-ment on the strength of both sampling methods in theoak system.

Appropriate sampling techniques can provide ac-curate estimates of potential insect vectors contrib-uting to disease progression in the oak tree canopy.Fogging, an absolute insect sampling method that fu-migates with a fog formed by heated oil mixed withknockdown insecticide, has been used to study ar-thropods within tropical forests (Basset et al. 2003)and insect food web structures (Barbosa et al. 2000).Stork and Hammond (1997) compared various sam-

1 Department of Entomology, Rutgers University, New Brunswick,NJ 08901.

2 Corresponding author, e-mail: [email protected] Department of Plant Biology and Pathology, Rutgers University,

New Brunswick, NJ 08901.

0046-225X/11/1131Ð1143$04.00/0 � 2011 Entomological Society of America

pling techniques from tree crowns and found thatcanopy fogging provided the most accurate insectpopulation estimates in oak canopies. Sticky-traps pro-vide a relative estimator of insect populations and arewidely used in agricultural ecosystems (Southwoodand Henderson 2000), as well as forest arthropod stud-ies (Basset et al. 1997). Sticky card sampling is inex-pensive, ßexible in time and space, enables a largenumber of replicates to be taken, and is highly suitablefor the study of insect spatial distributions and strat-iÞcation. With a combined sampling of fogging andsticky cards, we determined the Cicadomorpha spe-cies complex relative to the progression ofX. fastidiosainfection in oak landscapes in central New Jersey.

Materials and Methods

Sample Sites. Four sites in central New Jersey wereselected based on the oak species present and thelandscape setting. Duke Farms is located in Hillsbor-ough with 1,100 ha of farmland and landscaped gar-dens, where mature pin oak trees are planted in dou-ble row allees lining on streets. Mercer CountyCommunity College is located in West Windsor withmid-sized pin oak lining the collegeÕs parking lots.Riverview Cemetery located on the east bank of theDelaware River, in Trenton is a 20 ha historical cem-etery founded in 1699 and shaded by various randomlyplanted northern red oaks and pin oaks. Rutgers Gar-dens, a 20 ha botanical collection located in NorthBrunswick has northern red oaks lining the roads(Table 1).

All sampled oaks were identiÞed as either symp-tomatic for bacterial leaf scorch or asymptomatic.Symptomatic oaks were identiÞed if over 25% ofbranches showed dieback in May and tested positivefor X. fastidiosa by ELISA (Agdia “DAS ELISA forXylella fastidiosa” kit) (Agdia, Inc., Elkhart, IN) inSeptember. Asymptomatic trees were classiÞed assuch if they had �5% branch dieback in May and X.fastidiosa was undetectable in September usingELISA.Sticky Trap Sampling. In 2002, 2Ð7 yellow Sticky

Strips Insect Traps (7.5 � 12.5 cm) (Olson Products,Medina, OH), depending on the tree size for an ap-proximately uniform sample volume or coverage foreach card, were placed in 25 pin oak trees at DukeFarms and 20 northern red oak trees at Rutgers Gar-dens. In 2003, eight northern red oaks and Þve pin oaksin Riverview Cemetery were added to the survey. In

2004, a new site at Mercer County Community Collegewas added, where 20 symptomatic and 20 asymptom-atic pin oaks were sampled with three sticky traps, 120degrees apart, 30 cm from the edge of each pin oakcanopy. In 2006, sticky card sampling was repeatedonly at Duke Farms and Mercer County CommunityCollege. All sticky cards were attached to a bindingclip tied to a Þshing line. A free spinning Þshing swivelÞxed on the branch was then tied to the line holdingthe sticky card. A Þshing sinker was tied to a binderclip attached to the other end of the card to stabilizethe card in the wind. When the oak canopy was toohigh to reach, the Þshing swivel was connected to aÞshing line that was run through screw eyes placed ona branch above the trap and at eye level on the trunk.A Þshing reel was used to raise or lower the cardoperated from ground level. Sticky cards were re-placed weekly from May to August.Canopy Fogging. A gas generated DYNA-FOG

Golden Eagle fogger, model 2610E, series three (Cur-tis DYNA-FOG Ltd., WestÞeld, IN) that containedone part Pyrethrin (3%) (Prentox Pyronyl oil con-centrate, Prentiss Incorporated, Floral Park, NY)mixed with two parts of a low odor base oil (RocklandCorporation, Roanoke, VA) was used to fog trees. Toobtain an absolute insect population estimate foggingwas carried out on pin oaks similar to those used forsticky card sampling and with no prior sampling his-tory at Duke Farms and Mercer County CommunityCollege. Each oak was fogged only once. Three symp-tomatic oaks and three asymptomatic oaks werefogged on 16 June, 22 July, and 23 August 2004. Fog-ging was done four times in 2006: 24 May, 21 June, 24July, and 23 August. Fog sampling was conductedbetween 6:00Ð7:00 a.m. EDT before onset of air tur-bulence. Plastic drop sheets were placed under eachoak canopy to be sampled. At Mercer County Com-munity College, where pin oaks were �14 m high,each tree was fogged from the ground until fog en-veloped each tree canopy. At Duke Farms, oak trees�35 m tall were fogged from a bucket truck. Thebucket was extended to the canopy level and the fogwas released until it enveloped the canopy. In all cases,insects began dropping from the canopy �10 min afterfogging had ceased. Collection from the drop sheetsbegan 15 min after fogging and continued for 90 min.Preliminary studies showed that few insects droppedafter 90 min.Specimen Identification and Data Analysis. All in-

sects collected from sticky cards or fogging were in-

Table 1. Cicadomorpha insect sampling sites in central New Jersey

SiteTrees

sampledTree

height (m)

Crowndiameter

(m)Year Host

Latitude Longitude

(40o north) (74o west)

Duke Farms 67 35 13 2002 2003 2004 Q. palustris 33Õ12.74” 37Õ18.79”Mercer County Community

College82 14 10 2004 2006 Q. palustris 15Õ17.80” 38Õ48.86”

Riverview Cemetery 13 26 23 2003 2004 2006 Q. rubra Q.palustris

11Õ39.75” 45Õ20.16”

Rutgers Garden 20 22 12 2002 2003 2004 Q. rubra 28Õ26.05” 25Õ13.81”

1132 ENVIRONMENTAL ENTOMOLOGY Vol. 40, no. 5

dividually placed in 0.6 or 1.5 ml centrifuge tubesaccording to the specimen size and stored at �20�C forspecies identiÞcation, sex determination, and X. fas-tidiosa DNA assay. Taxonomic terminology and phy-logenetic systems to the Cicadomorpha family levelused in this study were from Cryan (2005) and Di-etrich (2005). Species identiÞcations were based onliterature available for identifying leafhoppers: De-Long 1948, Nielson 1968, Dietrich 2005; treehoppers:Osborn 1940, Dennis 1952, 1965; Kopp and Yonke1973a, 1973b, 1973c, 1974; Deitz 1975; Dietrich et al.2001; Cryan et al. 2004; and spittlebugs: Doering 1930,1941, Hanna and Moore 1966, Hamilton 1982, Dietrich2005. Pinned voucher specimens were deposited inthe Insect Museum, Department of Entomology, Rut-gers University, New Brunswick, NJ.

Numbers of insects per sample were square roottransformed and analyzed with PROC GLM using SAS9.1.3 (SAS Institute 2006). The GLM main treatmentswere sample location (Duke Farms and MercerCounty Community College), year (2004 and 2006),month (May, June, July, August), and bacterial leafscorch infection (asymptomatic and symptomatic).Interaction of location and month was incorporatedinto the GLM model after dropping all other nonsig-niÞcant interactions. Insect community analysis wasperformed on untransformed data.Molecular Assay of X. fastidiosa. There were 1,601

insects dissected to remove eyes, legs, abdomen, andwings under Stereo Star (Reichert ScientiÞc Instru-ments) dissecting scope to prevent potential PCR in-hibition in the following procedures. Each specimenwas placed in a 2 ml screw top sterilized tube pre-loaded with two 5 mm glass beads and submerged inliquid nitrogen for 20 s immediately before beingground using a Mini-bead Beater (BioSpec Products,Inc., Bartlesville, OK) for 20 s at low speed (2,500 rpm)(Bextine 2004).X. fastidiosaDNA was extracted usinga modiÞed Qiagen DNeasy Blood and Tissue Kit.Brießy, we added 180 �l ATL buffer immediately aftergrinding and shook well 1 min after the sample hadthawed. The sample was then brießy centrifuged tospin down the solution to avoid cross contamination.DNA was eluted with 50 �l AE buffer after incubationof the membrane for 15 min. Elution was repeatedonce.Polymerase Chain Reaction. A nested polymerase

chain reaction (PCR) procedure was used to detectX.fastidiosa presence in insect samples with externalprimers 272Ð1-ext (5�-AGCGGGCCAATATTCAAT-TGC-3�) and 272Ð2-ext (5�-AGCGGGCCAAAAC-GATGCGTG-3�) followed by internal primers 272-1-int (5�-CTGCACTTACCCAATGCATCG-3�) and272Ð2-int (5�-GCCGCTTCGGAGAGCATTCCT-3�)(Pooler and Hartung 1995). The presence of X. fasti-diosa DNA was further assessed by two additionalprimer sets RST31 (5�-GCGTTAATTTTCGAAGT-GATTCGATTGC-3�)/RST33 (5�-CACCATTCGTA-TCCCGGTG-3�) (Minsavage et al. 1994) andG1 (5�-GAAGTCGTAACAAGG-3�) and L1 (5�-CAAGGCATCCACCGT-3�) (Hendson et al. 2001,Mehta and Rosato 2001). PCRs were carried out in a

Biometra UNO-Thermoblock (Biometra, Goettin-gen, Germany). PCR products were analyzed by0.9% agarose gel electrophoresis, stained with SYBRSafe DNA gel stain by adding 10 �l of 10,000Xconcentrate in DMSO (Molecular Probes, Inc., Eu-gene, OR) per 100 ml gel solution right before pour-ing. The resulting image was captured with KodakDigital Science 1D LE software, version 3.02 usinga Kodak Electrophoresis Documentation and Anal-ysis System 120 digital camera over a 312 nm UVtransilluminator FBT1 88 (Fisher Biotech, Subiaco,WA). The gel image was stored as a TIFF format Þleand processed with Adobe Photoshop CS2, version9.02 (Adobe 2005).X. fastidiosa PCR products were puriÞed with

QIAquick gel extraction kit (Qiagen Inc., Valencia,CA) and sent with their corresponding primers fordirect PCR product sequencing (GeneWiz, SouthPlainÞeld, NJ). The sequences of the PCR products,472 bp for 272-int primers, 553 bp for 16S-23S inter-vening spacer region, and 722 bp for RST31/33 prim-ers, were used as a query sequence for a GenBankdatabase searched by BLASTn (Basic local alignmentsearch tool) (Altschul et al. 1997). Fresh PCR prod-ucts of each NCBI conÞrmed X. fastidiosa isolateswere then cloned into a plasmid vector, pCR4-TOPOusing the TOPO TA Cloning Kit (Invitrogen Corpo-ration, Carlsbad, CA). Desired clones were puriÞedwith QiagenÕs QIAprep spin miniprep kit. Both strandsof the inserts were sequenced at GeneWiz. DNA se-quences were analyzed with DNASTARÕs Lasergenesequence analysis software (DNASTAR, Inc., Madi-son, WI) (Burland 2000).

Results

Cicadomorpha Species Diversity. Four families ofpotential insect X. fastidiosa vectors (Order Hemi-ptera: Suborder Auchenorrhyncha, Infraorder Cica-domorpha) were collected: Membracidae (91.4%)and Cicadellidae (6.9%) from Membracoidea super-family, Aphrophoridae (1.1%) and Clastopteridae(0.5%) from Cercopoidea superfamily for a total of12,880 potential vectors (Table 2).Membracidae. A total of 11,662 treehoppers were

collected from sticky cards and fogging during the 4 yrof sampling (Table 2). Many were known oak feeders(Wood 1983, 1993). The predominant treehopperspecies collected was Ophiderma definitaWoodruff(Woodruff 1919), comprising 75% of the collections,followed by Telamona monticola (Fitch) 8%, Archasiabelfragei(F.)5%,T. tiliaeBall4%,Glossonotusacuminatus(F.) 3%, and Cyrtolobus fenestratus (Fitch) 1%. The re-maining 19 Membracid species comprised �4% of thetotal treehopper collection (Table 2).Cicadellidae. Most of the Cicadellidae collected

were in the subfamilies Athysaninae, Neocoelidii-nae, and Typhlocybinae, which are not known xy-lem feeders and may not be important for bacterialleaf scorch disease transmission. Known X. fastid-iosa vectors are the relatively large leafhoppers fromthe Cicadellinae subfamily, which have an inßated

October 2011 ZHANG ET AL.: CICADOMORPHA ASSOCIATED WITH BACTERIAL LEAF SCORCH 1133

clypeus with enlarged pump that enables them tofeed on xylem ßuid. There were 893 Cicadellinaeleafhoppers from six species collected during the 4yr sampling period. Graphocephala versuta (Say)was the most prevalent Cicadellinae species (90.2%)collected followed by G. coccinea (Forster) (4.9%),Draeculacephala portola Ball (1.6%), Oncometopiaorbona (F.) (1.5%), Aulacizes irrorata (F.) (1.2%),and Draeculacephala anguilfera (Walker) (0.7%).Aphrophoridae and Clastperidae. The spittlebugs

belonged to two families: Aphrophoridae and Clas-topteridae. All have an inßated clypeus with enlargedfeeding pump that allows xylem feeding and are thusall are potential Xylella vectors. Despite being col-lected during the entire season, spittlebug populationswere relatively low. Of the 214 specimens collectedfrom both fogging and sticky card traps, 32.7% (70)were Clastoptera obtusa (Say), 31.9% (68) were Phi-

laenus spumarius (L.), and 24.3% (52) were P. pallidus(L.).Sticky Traps versus Canopy Fogging. Sticky cards

deployed only on fogging days collected fewer spec-imens than fogging (Table 2). Only 299 specimensfrom 13 species in 11 genera were collected from 1,004sticky trap cards placed in 105 pin oaks, while 6,121Cicadomorpha insects from 29 species of 19 generawere collected from 84 pin oaks by fogging. Eachspecies collected from sticky cards was also collectedduring fogging except forG. coccinea,which was foundonly on sticky cards. Seventeen species were presentin the fogging samples only, in addition to the 12common species present in both sampling methods,indicating that the sampling efÞciency of sticky cardsis low when compared with the fogging. However, ifcomparing sticky card collection from all samplingdays of all sites to fogging, a total of 6,759 individuals

Table 2. Cicadomorpha insect collection by sticky trap cards and fogging methods in pin and red oak canopies in central New Jerseyduring 2002–2006 and their X. fastidiosa DNA assay

SpeciesStickycards

Fogging TotalCard on fog

daysTotaltested

X. fastidiosapositive (%)

Total 6,758 6,122 12,880 299 1,601 13.89Cercopoidea: Aphrophoridae: AphrophorinaeAphrophora quadrinotata Say 19 19 15 33.33Aphrophora cribrata (Walker) 2 2 1 0Philaenus marginellus (L.) 2 1 3 3 0Philaenus pallidus (L.) 34 18 52 3 51 12.28Philaenus spumarius (L.) 49 19 68 10 68 19.23

Cercopoidea: Clastopteridae: ClastopterinaeClastoptera obtusa (Say) 13 57 70 5 42 7.14

Membracoidea: Cicadellidae: CicadellinaeDraeculacephala anguilfera (Walker) 5 1 6 5 20Draeculacephala portola Ball 14 14 9 11.11Graphocephala coccinea (Forster) 44 44 6 28 3.57Graphocephala versuta (Say) 800 5 805 97 431 20.19Aulacizes irrorata (F.) 10 1 11 2 10 10Oncometopia orbona (F.) 13 13 7 14.29

Membracoidea: Membracidae: HoplophorinaePlatycotis vittata (F.) 13 2 15 4 0

Membracoidea: Membracidae: MembracinaeEnchenopa binota (Say) 24 2 26 2 17 11.76

Membracoidea: Membracidae: SmiliinaeAcutalis tartarea (Say) 1 2 3 1Archasia belfragei Stål 341 292 633 81 120 3.33Atymna querci Fitch 4 21 25Carynota mera (Say) 1 1Cyrtolobus maculifrontis (Emmons) 1 1 1 0Cyrtolobus discoidalis (Emmons) 1 1 2 1 100Cyrtolobus fenestratus (Fitch) 106 9 115 45 4.44Cyrtolobus fuscipennis (Van Duzee) 1 1 1 0Cyrtolobus gratiosusWoodruff 11 11 5 0Cyrtolobus puritanusWoodruff 4 4 3 0Entylia carinata Forster 1 1 2Glossonotus acuminatus (F.) 234 105 339 33 114 11.4Helonica excelsa Fairmaire 6 2 8 1 1 0Ophiderma definitaWoodruff 3,944 4,838 8,782 54 226 15.49Ophiderma evelynaWoodruff 9 9Ophiderma flava Goding 8 10 18 1 0Smilia fasciata (Amyot & Serville) 11 44 55 22 4.55Telamona concava Fitch 2 2 4 2 0Telamona extrema Ball 4 4 1 100Telamona monticola (F.) 697 202 899 134 15.67Telamona tiliae Ball 348 75 423 121 9.92Telamona unicolor Fitch 25 4 29 4 22 0

Membracoidea: Membracidae: StegaspidinaeMicrocentrus perditus (Amyot & Serville) 1 110 111 90 12.22

Nymphs 254 254


of 33 species were from sticky cards, which is morethan from fogging. There were 25 common speciespresent in both fogging and sticky card samples, eightunique species present only in sticky card collections,and four unique species present only in fogging col-lections. All unique species were collected at levelsfewer than 44 adults regardless of method used(Table 2).

Sampling efÞciency also varied with insect mobility.O. definita was the most collected species from bothfogging (�80%) and sticky cards (�58%). The 22%difference is attributable to the low number of femalesattracted to sticky cards relative to fogging (Fig. 1).Fewer gravid female adults were collected on stickycards when they were carrying eggs, leading to malebiased collections that underestimate the population.Compared with treehoppers, leafhoppers are active

ßyers and were collected more on sticky cards thanfogging. G. versuta made up 32.4% (97) of the totalsticky card collection on fogging days compared with0.1% (5) of the total fogging collection. NoG. coccineawere collected using fogging. However, the earedhopper, Microcentrus perditus (Amyot and Serville),was almost exclusively collected by fogging except fora single individual collected on a sticky card. Lowsticky card collections for this species may be due alack of adult mobility. Immature treehoppers werecollected only from fogging (Table 2).Seasonal Dynamics. Although abundance varied

with location and year, the temporal pattern was sim-ilar between sites each year. Cicadomorpha werepresent from May to September, but most occurredfrom the end of May to mid July. An initial peakappeared during the Þrst 3 wk of June and was fol-lowed by a second small peak from the end of June tothe second week of the July.O. Definita. Adults were collected on sticky cards

from the end of May to the end of June with a clearpeak in the Þrst week of June (Fig. 2). O. definitapopulation abundance varied among locations, possi-bly because of the oak species composition. Low pop-ulations were observed each year at the RiverviewCemetery and Rutgers Gardens sites. Comparisons ofthe O. definita adults on sticky cards collected at Riv-erview Cemetery indicated that there were signiÞ-cantly moreO. definita adults trapped in pin oak thanin red oaks (F 1, 1644 � 19.26; P� 0.001) (Fig. 3). Adultcatches also varied between years. High populationsappeared in 2002 at Duke Farms and in 2006 at MercerCounty Community College where the only specieswas pin oaks.

Adult O. definita numbers differed between males

Fig. 1. O. definita female proportion from sticky cardtrapping and fogging and female with eggs ratio (femaleswith eggs:total females) during 2006.

Fig. 2. O. definita adult seasonal abundance (mean SE) on yellow sticky cards in pin oak canopies at Mercer CountyCommunity College (MCCC), Duke Farms (Duke), Rutgers Garden (Rutgers), and Riverview Cemetery (Trenton) during2002, 2003, 2004, and 2006.


and females in both fogging and sticky card collection.Female proportion from fogging varied from 43.4% on24 May 2006 to a high of 85.7% on 21 June 2006 (Fig.1). By the end of the season, only females were col-lected. However, much lower percentages of femaleO. definita were observed in the sticky card collec-tions. O. definita female proportion increased from2.7% on 31 May-19.8% when the population was at itspeak and then declined to 6.2% on 14 June, and 0.8%on 28 June 2006. Thirty one percent (31.1%) of thefemales collected carried eggs on 7 June during thepopulation peak. The percentage of egg carrying fe-males reached 75% around 28 June when the lowestfemale adult catch (indicating a low ßight activity)occurred on the sticky cards. Female proportionreached 100% and most of the females carried eggsafter mid July. This may indicate that females livelonger than males or males leave the canopy in lateseason.G. versuta. Adult G. versuta populations had one

major peak that extended from late June to late Julyand followed immediately afterO. definita (Fig. 4) on

sticky cards. Unlike O. definita, adult G. versuta wereactive until the end of August. Since there were fewcatches of G. versuta from fogging, it is difÞcult todetermine true female proportion; however, a higherpercentage of females appeared on the sticky cards.Female proportion from sticky cards ranged between0.60Ð0.66 during the peak period of 16 June-14 July(Fig. 5). A smaller proportion (6Ð10%) of femalescarried eggs during 16 June-28 July when comparedwith the percentage of egg-carrying O. definita.G. versuta adult population abundance varied be-

tween sites and years. The highest population oc-curred in 2004 for all sites. As with O. definita, thepopulation in 2003 was the lowest for all three sites.Although G. versuta adult populations were signiÞ-cantly higher in red oak canopies than in pin oaks inTrenton (F1, 1648 � 12.36;P� 0.0005), the highest adultcatches in 2004 and 2006 were observed at MercerCounty Community College where only pin oaks arefound. The red oaks at Rutgers Gardens had highpopulations in both 2002 and 2004, whereas, adult G.versuta populations at Duke Farms were the lowest of

Fig. 3. O. definita adult catches (mean SE) on sticky card traps in pin and red oak canopies in Riverview Cemetery,Trenton during 2004 and 2006.

Fig. 4. G. versuta adult seasonal abundance (mean SE) on sticky cards at Mercer County Community College (MCCC),Duke Farms (Duke), Rutgers Garden (Rutgers), and Riverview Cemetery (Trenton) during 2002, 2003, 2004, and 2006.


all four sites in 2004 suggesting that G. versuta mayprefer red oak to pin oak.Cicadomorpha Harboring X. fastidiosa. Fourteen

percent (13.9%) of 1,601 specimens tested were X.fastidiosaDNA positive (Table 2). Twenty one of the32 Cicadomorpha species tested were X. fastidiosaDNA positive with treehoppers making up the largestgroup of 11 species potential insect vectors. Elevenpercent (11.0%) of the tested treehoppers were pos-itive for X. fastidiosa DNA. All six Cicadellinae spe-cies tested X. fastidiosa positive with 18.8% of the490 individuals tested testing DNA positive. Fifteenpercent (15.5%) of the most dominant species, O.definita tested positive for X. fastidiosa DNA.Twenty percent (20.2%) of the tested G. versutacarried X. fastidiosa DNA while a 3.6% positive ratewas detected for a similar species, G. coccinea. Al-though spittlebug populations were low, four of sixCercopoidea species tested were X. fastidiosa pos-itive with a 15.1% DNA positive rate for the 194individuals tested.

Although the treehopperX. fastidiosaDNA positiverate was lower than for leafhoppers or spittlebugs, the

total number of positive treehoppers was substantiallyhigher. Based on the X. fastidiosa DNA positive ratefor each insect group, an estimated 1,311 treehoppers,168 leafhoppers, and 32 spittlebugs from the 12,880individuals collected might carry and potentially vec-tor the X. fastidiosa bacterium. Since X. fastidiosacarrying treehopper numbers were 655.5% largerthan the combined numbers of leafhoppers and spit-tlebugs, treehoppers may play a major role in thebacterial leaf scorch disease transmission in pin andred oak.X. fastidiosa bacteria were detected from collected

insects from May to August. Throughout the summer,X. fastidiosa occurred in treehoppers at fairly consis-tent levels: 15.7% in May, 9.1% in June, 12.1% in July,and 13.3% in August. Twenty percent (20.4%) of theleafhoppers from six Cicadellinae species collectedin July (when populations peaked) tested X. fasti-diosa DNA positive, whereas 15.3 and 12.9% testedwhen collected in June and August (when popula-tions were very low) were positive, respectively.The percentage of tested spittlebug specimens car-rying X. fastidiosawas similar to that of the Cicadel-linae (11.0, 15.9, and 18.0% for June, July, and Au-gust, respectively).Impact of Oak Bacterial Leaf Scorch Infection onCicadomorpha Abundance and Diversity. Absolutesampling using fogging showed that the total numberof Cicadomorpha was signiÞcantly higher at DukeFarms than at Mercer County Community College(F1, 74 �26.04;P�0.0001).The totalnumbercollectedin 2006 was greater than in 2004 (F1, 74 � 3.47; P �0.0665) (Fig. 6) and was because of the signiÞcantlyhigher insect density in May when compared withlater months (F3, 74 � 81.10, P � 0.0001). There wasa signiÞcant interaction between location (DukeFarms vs. Mercer County Community College) and

Fig. 5. Seasonal dynamics ofG. versuta adult female pro-portion and the ratio of females carrying eggs during thesummer in central New Jersey.

Fig. 6. Total oak feeding Cicadomorpha insect population abundance (mean SE) in pin oak canopies sampled byfogging at Duke Farms (Duke) and Mercer County Community College (MCCC) in 2004 and 2006.


time (month) of the year on the total population (F3,

74 � 9.03, P � 0.0001). Populations peaked in earlyseason and decreased in July and August in both2004 and 2006 at Mercer County Community Col-lege. The same trend occurred at Duke Farms in2006. However, the total number of insects washigher in August than in June and July at DukeFarms in 2004. The highest abundance (575 indi-viduals) was observed from a single asymptomaticpin oak tree in May at Duke Farms. The averagenumber collected from asymptomatic pin oaks was90.1 20.3 per tree and was signiÞcantly �56.1 17.1 per tree collected from symptomatic pin oaks(F

1, 74� 8.65; P � 0.0044) (Fig. 6).

The majority (96.0%) of the Cicadomorpha col-lected were membracids, of which 82.1% were O.definita, the most abundant species in pin oaks at DukeFarms and Mercer County Community College. Mem-bracidae population abundance also varied with loca-tion and time of year (F3, 74 � 8.99; P� 0.0001) (Fig.7) following the same trend described for total Cica-domorpha. Membracidae populations at Duke Farmswere highest in May 2006 when compared with allother times at Mercer County Community College orDuke Farms. Membracidae population abundance(86.9 20.3 per pin oak) in asymptomatic trees was62.6% and signiÞcantly greater than in symptomatictrees (53.4 17.0 per oak) (F1, 74 � 8.57; P� 0.0045)(Fig. 7).

SigniÞcantly more Cicadellinae leafhoppers (0.29 0.10 per tree) were found in the asymptomatic treesamples than in symptomatic trees (0.21 0.13 pertree) (F1, 74 � 8.57; P � 0.0045) (Fig. 8). In addition,more leafhoppers were found in early season at Mer-cer County Community College, while signiÞcantlymore leafhoppers were found during late season atDuke Farms (F3, 74 � 8.99; P � 0.0001).

Few spittlebugs were found in pin oak canopiesusing either sticky cards (98) or fogging (116). Al-though they can be found throughout the summerseason from June to August, signiÞcantly more spit-tlebugs were found in June than in other months(F3, 74 � 6.55; P� 0.0005) (Fig. 9). Interestingly, fewspittlebugs were found in 2006 at Duke Farms or in2004 at Mercer County Community College com-pared with those found in 2004 Duke Farms and in2006 at Mercer County Community College (Fig. 9).X. fastidiosa infection showed no signiÞcant impacton the spittlebug population density (F1, 74 � 1; P�0.3216).

Bacterial leaf scorch infection in pin and red oakscontributed to the difference in abundance of insectspecies. Adult O. definita populations collected inasymptomatic pin oaks (72.7 18.8 per tree) were71.3% signiÞcantly greater than in symptomatic tree(42.5 15.6 per tree) canopies (F1, 74 � 7.49; P �0.0078). For the remaining species, only T. tilaeshowed signiÞcant population differences based oninfection status. Eighty-Þve percent (84.6%) signif-icant more T. tiliae were collected in asymptomaticpin oaks (1.1 0.3 per tree) than in symptomatic pinoaks (0.6 0.2 per tree) (F1, 74 � 7.21; P� 0.0089).Bacterial leaf scorch infection did not affectpopulation differences for any other insect spe-cies. There were 24.5% more M. perditus (F1, 74 �2.30; P � 0.1334), 68.9% more T. monticola (F1, 74 �1.42; P � 0.2378), 80.0% more Smilia fasciata(F1, 74 � 0.45; P � 0.5064), and 121.5% more tree-hopper nymphs (F1, 74 � 1.6; P � 0.2095) found inasymptomatic canopies than that in symptomaticpin oak canopies. Conversely, 25.4% more A. bel-fragei (F1, 74 � 0.13; P � 0.7209) and 62.5% more G.acuminatus (F1, 74 � 0.56; P � 0.4557) adults were

Fig. 7. Membracidae population abundance (mean SE) in pin oak canopies sampled by fogging at Duke Farms (Duke)and Mercer County Community College (MCCC) in 2004 and 2006.


found in the symptomatic trees than in asymptom-atic trees.Impact of Bacterial Leaf Scorch Infection on Rel-ative Cicadomorpha Population Abundance. Rela-tive sampling using sticky traps did not detect anysigniÞcant insect population difference betweensymptomatic oaks and asymptomatic oaks at DukeFarms (F1, 1840 � 0.70, P � 0.4012 for total Cicado-morpha; F1, 1840 � 0.70, P � 0.4023 for Membraci-dae; F1, 1840 � 0.59, P � 0.4439 for Cicadellinae; andF1, 1840 � 0.21, P� 0.6455 for Cercopoidea). The same

trend was observed at Mercer County CommunityCollege: total Cicadomorpha (F1, 3164 � 0.32; P �0.5723) and family (F1, 3164 � 0.34, P � 0.5576 forMembracidae; F1, 3164 � 0.16, P � 0.6901 for Cicadel-linae; F1, 3164 � 2.30, P � 0.1296 for Cercopoidea).Total sticky card catches were signiÞcantly higherin 2006 than in 2004 (F1, 3164 � 626.65; P � 0.0001).This was explained by signiÞcant higher Membra-cidae catches observed in 2006 (F1, 3164 � 669.16;P � 0.0001), because of the large numbers of O.definita (F1, 3164 � 708.31; P� 0.0001). The averageO.

Fig. 8. Cicadellinae population abundance (mean SE) in pin oak canopies sampled by fogging at Duke Farms (Duke)and Mercer County Community College (MCCC) in 2004 and 2006.

Fig. 9. Spittlebug population abundance (mean SE) in pin oak canopies sampled by fogging at Duke Farms (Duke)and Mercer County Community College (MCCC) in 2004 and 2006.


definita catch per card was 0.53 in 2006 and 0.03 in 2004at Mercer County Community College. O. definitacatches comprised 96.1% of the Membracidae, or89.6% of all insects collected in 2006.

Discussion

X. fastidiosa may be transmitted in oak trees by aCicadomorpha species complex that includes treehop-pers, leafhoppers, and spittlebugs. Of the 25 speciesfound in our study, 11 treehopper species tested pos-itive for X. fastidiosa DNA. Vector studies of X. fasti-diosa have been conducted in southern agriculturesystems such as grape, peach, and citrus where leaf-hoppers are more abundant and few treehoppers arepresent. Most of the known X. fastidiosa vectors ingrape (Severin 1949, 1950; Purcell 1980; Hill and Pur-cell 1995; Costa et al. 2000; Almeida and Purcell 2003)have not been detected in oak canopies in New Jersey.However, we detected X. fastidiosa DNA in speciesknown in other plant systems (Purcell 1980) such asthe spittlebug Philaenus spumarius (Almond leafscorch), and leafhopper Oncometopia orbona (Phonypeach disease). We also detected X. fastidiosa DNAfrom leafhoppers G. coccinea, G. versuta, and Draecu-lacephala anguilfera as well as spittlebug species Phi-laenus pallidus and Clastoptera obtuse.

DNA sequencing analysis indicated that X. fastid-iosa DNA from those insects is identical to the DNAextracted from host oak petioles (J. Zhang et al. un-published data). Among the X. fastidiosa positive Ci-cadomorpha, only O. definita and G. versuta had anysizable populations each year. All other species hadlow populations and may not play a critical role indisease transmission. O. definita nymphs hatch as oakbuds break in late April/early May. X. fastidiosa pres-ent in oak stems grow into the petiole and leaf veinsas the leaf emerges and grows. Although the cell con-centration in oak petioles or other feeding sites has yetto be determined, by the time adult O. definita pop-ulations peak in early June, bacterial populationsshould be large enough for efÞcient acquisition. If thebacterial population is not high enough in early Junefor acquisition to occur, it must be high enough whenpopulations of G. versuta, the second major Cicado-morpha, peak in July. We extracted X. fastidiosaDNAfrom various Cicadomorpha in June (J. Zhang et al.unpublished data) over a month earlier than the timewhen live bacteria were isolated from treehopper O.definita in late July. This indicates the bacteria hadbeen present in the insect for some period of time ata level hard to isolate.

We compared fogging with yellow sticky card sam-pling for Cicadomorpha population determination inpin and red oaks. When the data from fogging andsticky cards are combined, 37 species of Cicadomor-pha from 21 genera were collected from oak canopiesin central New Jersey. Fogging in this study collectedmany more species and individuals than sticky cards.However, the disadvantages of sticky card samplingcan be compensated by extended sampling with morelocations, and by weekly sampling over multiple years.

At the species level, collections from fogging andsticky card sampling did not provide similar results.Treehoppers were collected in both fog and stickycard sampling, while Cicadellinae leafhoppers werecollected mostly from sticky card traps. Possible rea-sons for this difference between fogging and stickycard trapping may be variation in leafhopper distri-bution through time and space, ßight activity, or thetiming of fogging events. In this study, adultG. versutapopulation peaks detected by fogging were similar tocollections from sticky cards when sampled the sameweek as the fogging. However, these insects in oakcanopies may have been low on the days of fogging.Leafhopper populations peaked around 6 July (rang-ing from 21 June to 9 August) for both 2004 and 2006according to the sticky card trap collections. Foggingwas conducted on 16 June 2004, 22 July 2004, 21 June2006, and 24 July 2006 when leafhopper populationswere at the beginning or end of the July peak. Sec-ondly, Cicadellinae leafhoppers may have a patchyspatial distribution pattern, in which case fogging mayhave simply missed oaks harboring leafhoppers.Thirdly, sharpshooters are extremely vagile (Turnerand Pollard 1959) and are active ßyers in the oakcanopy so sticky cards may overestimate the actualleafhopper population when compared with the fog-ging. Lastly, the leafhopper catches on sticky cardsmay have been because of dispersal ßights, since stickycards catch insects throughout the day while foggingonly catches those insects present in the canopy at thetime of sampling.

Cicadomorpha species abundance varied betweenyears. O. definita populations were highest in 2002,followed by those in 2006, and very low in 2003 and2004. Meanwhile G. versuta populations were higheston 2002 and 2004, and moderately abundant in 2006.Yearly ßuctuations were also observed in Kentucky,where populations in 1994 were less than half those in1993 (Johnson and Freytag 1997). One explanationmight be annual temperature variation. The cumula-tive degree-days (�10�C) from the Þrst day of the yearto April and May were much higher in 2002 and 2006than in 2003 and 2004 (Fig. 10). Population abundanceofO. definita peaked in early June (Fig. 2), consistentwith a degree-day explanation. However, populationabundance of the midsummer species,G. versuta (Fig.4), did not show this relationship.

Fig. 10. Cumulative effective temperature (�10�C sinceÞrst day of the year) in central New Jersey between April andMay during 2003Ð2006.


We found more Cicadomorpha insects in oak can-opies where no bacterial leaf scorch symptoms wereobserved, than in infected oaks. One characteristic ofthis disease in oaks is the apparent randomness ofinfected branches within infected trees. Within a lo-cality it is common to Þnd an apparent healthy oak treenext to a severely infected one. In spring, bacterial leafscorch infected branches usually foliate later thanuninfected trees. Oak leaves are usually smaller andbranches tend to dieback after serious infection (J.Zhang et al., unpublished data). Mizell and French(1987) reported that H. vitripennis (Germar) feedspredominantly on trees which did not display X. fas-tidiosadisease symptoms when provided a choice. Thenutritional quality of xylem in bacterial leaf scorchinfected oak trees or branches may have some adverseimpact on insects or deter them from feeding or ovi-position, hence promoting insect foraging behaviorthat results in the dispersal of individuals to uninfectedoak trees in the vicinity.

Unlikewhathasbeendemonstratedby fogging,datafrom sticky cards did not show signiÞcant differencesin insect population abundance between bacterial leafscorch infected oaks and asymptomatic oaks. Thismight be because sticky card trapping is a relativesampling method that indicates insect ßight activity, inaddition to abundance. As discussed above, insect vec-tors may increase their ßight activity and move tohealthier asymptomatic oaks for feeding and oviposi-tion when bacterial infection progresses and oak hostquality declines. High mobility of these insects mayresult in high sticky card catches in the symptomaticoak trees. However, a healthy oak tree provides notonly a better food resource that will reduce the ne-cessity for dispersal ßights, but also higher leaf den-sities which can block or reduce insect mobility lead-ing to lower numbers on sticky cards in asymptomaticoak trees.

In conclusion, the fogging method measures theactual population abundance of potential vectors re-siding in each oak canopy, and represents the numer-ical consequence of the bacterial leaf scorch impact.Sticky card trapping detects insect behaviors that areinvolved with the process of bacterial leaf scorch in-fection of oaks. A high catch using sticky cards rep-resents high mobility of the insect vector, whereas alow population in bacterial leaf scorch infected oaks inthe neighborhood of asymptomatic oaks would beover-estimated using the sticky cards and would ap-pear similar to the catches from uninfected oaks. Byintegration of these two sampling methods, one cancapture the dynamics of the entire oak canopy insectpopulation when bacterial leaf scorch infectionoccurs.

Acknowledgments

Thanks to Duke Farms, Hillsborough, Somerset County;Mercer County Community College, Mercer County; andRiverview Cemetery, Trenton, Mercer County, NJ, for theirsupport in the specimen collection. Special thanks to PaulSmith, Gene Huntington, Tom Allmendinger at Duke Farms

for their generous help in providing collection equipmentand staff assistance. This study would not have been possiblewithout the excellent technical assistance from Colleen Con-over, Asa Dewan, Nadishani Dissanayaka, Dan Duran, NidhiShree Karingula, Rosemary Kim, Christine King, Sneha Shah,Tracey Switek, Steven Wong, and Chiao-Han Yu. Insectspecies identiÞcation was conÞrmed by Jason Cryan, Labo-ratory for Conservation and Evolutionary Genetics, NY StateMuseum, Albany, NY 12230. We appreciate Daniel Herms,Department of Entomology, The Ohio State University, forhis critical review of a prior draft. This is New Jersey Agri-cultural Experiment Station publication number D-08-08289-03-10.

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Received 5 April 2010; accepted 21 June 2011.


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