Cicadomorpha Insects Associated with Bacterial Leaf Scorch Infected Oak in Central New Jersey

Home , Aphrophora cribrata, Cicadomorpha, Clastoptera, Clastoptera obtusa, Entylia carinata, Graphocephala coccinea

PLANT-INSECT INTERACTIONS Cicadomorpha Insects Associated With Bacterial Leaf Scorch Infected Oak 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 fastidiosa Wells et al., in pin and red oaks in New Jersey were surveyed by placing yellow sticky card traps in tree canopies and fogging with pyrethrin insecticide during 2002Ð2006. Thirty-seven Cicadomorpha species 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 individuals than sticky card collections. Sticky card sampling, done more frequently and at a larger number of locations provided similar community structure information as fogging. Sticky card collections of the dominant treehopper species, Ophiderma deﬁnita Woodruff were male biased when females were gravid. O. deﬁnita populations peaked in early June, comprised 68.2% of the total collection, and were more abundant in pin oaks than red oaks. Graphocephala versuta (Say) peaked in mid-July, comprising 6.2% of the total collection. Higher Cicadomorpha populations were observed in asymptomatic oak canopies than in neighboring X. fastidiosa infected oaks. Individual insect specimens collected from oaks were subjected to a X. fastidiosa DNA assay by polymerase chain reaction ampliÞcation. The average X. fastidiosa positive rate was 13.89% for all specimens tested. Eleven treehopper species, six leafhopper 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 bacterial leafhoppers (Novotny and Wilson 1997) with the abil- leaf scorch, especially in oaks in the northeastern ity to feed on negatively pressured xylem ßuid. United States. Xylella fastidiosa Wells et al. is the Northern red oak Quercus rubra L. and pin oak Q. causal agent of oak bacterial leaf scorch (Hearon et al. palustris Muenchh are major street trees in New Jer- 1980) as well as many other plant diseases such as sey and are susceptible to X. fastidiosa infection. Dur- almond leaf scorch (Mircetich et al. 1976), citrus var- ing the past 20 yr, bacterial leaf scorch disease of oak iegated chlorosis (Lee et al. 1993), grape PierceÕs dis- has spread throughout the state with infections re- ease (Hopkins and Mollenhauer 1973), maple leaf ported in every county (A. Gould et al. unpublished scorch (Sherald et al. 1987), oleander leaf scorch data). Although live bacterium and DNA of X. fasti- (Huang et al. 2003), periwinkle wilt (McCoy et al. diosa have been detected in many Cicadomorpha (J. 1978), phony peach disease (Hopkins et al. 1973), Zhang et al. unpublished data), their seasonal appear- plum leaf scald (Kitajima et al. 1975), and sycamore ance and relationship to disease incidence is yet to be leaf scorch (Sherald et al. 1983). Sharpshooter leaf- determined. Our main objective was to record the hoppers and spittlebugs are known X. fastidiosa vec- Cicadomorpha species complex in bacterial leaf tors of the almond leaf scorch (Purcell 1980a), citrus scorch infected oak. We then wanted to report the variegated chlorosis (Brlansky et al. 1996, Krugner et proportion of each species harboring X. fastidiosa. To al. 2000), phony peach disease (Turner and Pollard accomplish these goals we used two sampling methods 1959), periwinkle wilt (McCoy et al. 1978), PierceÕs fogging (absolute) and sticky trap (relative). We com- disease (Severin 1949, 1950, Purcell 1980b; Adlerz and ment on the strength of both sampling methods in the Hopkins 1979; Almeida and Purcell 2003), oleander oak system. leaf scorch (Costa et al. 2000), and possibly bacterial Appropriate sampling techniques can provide ac- leaf scorch of street trees. Sharpshooters and spittle- curate estimates of potential insect vectors contrib- bugs are usually larger in size than phloem feeding 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 with 1 Department of Entomology, Rutgers University, New Brunswick, knockdown insecticide, has been used to study ar- NJ 08901. thropods within tropical forests (Basset et al. 2003) 2 Corresponding author, e-mail: [email protected]. 3 Department of Plant Biology and Pathology, Rutgers University, and insect food web structures (Barbosa et al. 2000). New Brunswick, NJ 08901. Stork and Hammond (1997) compared various sam-

0046-225X/11/1131Ð1143$04.00/0 ᭧ 2011 Entomological Society of America 1132 ENVIRONMENTAL ENTOMOLOGY Vol. 40, no. 5

Table 1. Cicadomorpha insect sampling sites in central New Jersey

Crown Trees Tree Latitude Longitude Site diameter Year Host sampled height (m) o o (m) (40 north) (74 west) Duke Farms 67 35 13 2002 2003 2004 Q. palustris 33Õ12.74” 37Õ18.79” Mercer County Community 82 14 10 2004 2006 Q. palustris 15Õ17.80” 38Õ48.86” College Riverview Cemetery 13 26 23 2003 2004 2006 Q. rubra Q. 11Õ39.75” 45Õ20.16” palustris Rutgers Garden 20 22 12 2002 2003 2004 Q. rubra 28Õ26.05” 25Õ13.81” pling techniques from tree crowns and found that 2004, a new site at Mercer County Community College canopy fogging provided the most accurate insect was added, where 20 symptomatic and 20 asymptom- population estimates in oak canopies. Sticky-traps pro- atic pin oaks were sampled with three sticky traps, 120 vide a relative estimator of insect populations and are degrees apart, 30 cm from the edge of each pin oak widely used in agricultural ecosystems (Southwood canopy. In 2006, sticky card sampling was repeated and Henderson 2000), as well as forest arthropod stud- only at Duke Farms and Mercer County Community ies (Basset et al. 1997). Sticky card sampling is inex- College. All sticky cards were attached to a binding pensive, ßexible in time and space, enables a large clip tied to a Þshing line. A free spinning Þshing swivel number of replicates to be taken, and is highly suitable Þxed on the branch was then tied to the line holding for the study of insect spatial distributions and strat- the sticky card. A Þshing sinker was tied to a binder iÞcation. With a combined sampling of fogging and clip attached to the other end of the card to stabilize sticky cards, we determined the Cicadomorpha spe- the card in the wind. When the oak canopy was too cies complex relative to the progression of X. fastidiosa high to reach, the Þshing swivel was connected to a infection in oak landscapes in central New Jersey. Þshing line that was run through screw eyes placed on a branch above the trap and at eye level on the trunk. A Þshing reel was used to raise or lower the card Materials and Methods operated from ground level. Sticky cards were re- Sample Sites. Four sites in central New Jersey were placed weekly from May to August. selected based on the oak species present and the Canopy Fogging. A gas generated DYNA-FOG landscape setting. Duke Farms is located in Hillsbor- Golden Eagle fogger, model 2610E, series three (Cur- ough with 1,100 ha of farmland and landscaped gar- tis DYNA-FOG Ltd., WestÞeld, IN) that contained dens, where mature pin oak trees are planted in dou- one part Pyrethrin (3%) (Prentox Pyronyl oil con- ble row allees lining on streets. Mercer County centrate, Prentiss Incorporated, Floral Park, NY) Community College is located in West Windsor with mixed with two parts of a low odor base oil (Rockland mid-sized pin oak lining the collegeÕs parking lots. Corporation, Roanoke, VA) was used to fog trees. To Riverview Cemetery located on the east bank of the obtain an absolute insect population estimate fogging Delaware River, in Trenton is a 20 ha historical cem- was carried out on pin oaks similar to those used for etery founded in 1699 and shaded by various randomly sticky card sampling and with no prior sampling his- planted northern red oaks and pin oaks. Rutgers Gar- tory at Duke Farms and Mercer County Community dens, a 20 ha botanical collection located in North College. Each oak was fogged only once. Three symp- Brunswick has northern red oaks lining the roads tomatic oaks and three asymptomatic oaks were (Table 1). fogged on 16 June, 22 July, and 23 August 2004. Fog- All sampled oaks were identiÞed as either symp- ging was done four times in 2006: 24 May, 21 June, 24 tomatic for bacterial leaf scorch or asymptomatic. July, and 23 August. Fog sampling was conducted Symptomatic oaks were identiÞed if over 25% of between 6:00Ð7:00 a.m. EDT before onset of air tur- branches showed dieback in May and tested positive bulence. Plastic drop sheets were placed under each for X. fastidiosa by ELISA (Agdia “DAS ELISA for oak canopy to be sampled. At Mercer County Com- Xylella fastidiosa” kit) (Agdia, Inc., Elkhart, IN) in munity College, where pin oaks were Ͻ14 m high, September. Asymptomatic trees were classiÞed as each tree was fogged from the ground until fog en- such if they had Ͻ5% branch dieback in May and X. veloped each tree canopy. At Duke Farms, oak trees fastidiosa was undetectable in September using Ϸ35 m tall were fogged from a bucket truck. The ELISA. bucket was extended to the canopy level and the fog Sticky Trap Sampling. In 2002, 2Ð7 yellow Sticky was released until it enveloped the canopy. In all cases, Strips Insect Traps (7.5 ϫ 12.5 cm) (Olson Products, insects began dropping from the canopy Ϸ10 min after Medina, OH), depending on the tree size for an ap- fogging had ceased. Collection from the drop sheets proximately uniform sample volume or coverage for began 15 min after fogging and continued for 90 min. each card, were placed in 25 pin oak trees at Duke Preliminary studies showed that few insects dropped Farms and 20 northern red oak trees at Rutgers Gar- after 90 min. dens. In 2003, eight northern red oaks and Þve pin oaks Specimen Identiﬁcation and Data Analysis. All in- in Riverview Cemetery were added to the survey. In sects collected from sticky cards or fogging were in- October 2011 ZHANG ET AL.: CICADOMORPHA ASSOCIATED WITH BACTERIAL LEAF SCORCH 1133 dividually placed in 0.6 or 1.5 ml centrifuge tubes Biometra UNO-Thermoblock (Biometra, Goettin- according to the specimen size and stored at Ϫ20ЊC for gen, Germany). PCR products were analyzed by species identiÞcation, sex determination, and X. fas- 0.9% agarose gel electrophoresis, stained with SYBR tidiosa DNA assay. Taxonomic terminology and phy- Safe DNA gel stain by adding 10 ␮l of 10,000X logenetic systems to the Cicadomorpha family level concentrate in DMSO (Molecular Probes, Inc., Eu- used in this study were from Cryan (2005) and Di- gene, OR) per 100 ml gel solution right before pour- etrich (2005). Species identiÞcations were based on ing. The resulting image was captured with Kodak literature available for identifying leafhoppers: De- Digital Science 1D LE software, version 3.02 using Long 1948, Nielson 1968, Dietrich 2005; treehoppers: a Kodak Electrophoresis Documentation and Anal- Osborn 1940, Dennis 1952, 1965; Kopp and Yonke ysis System 120 digital camera over a 312 nm UV 1973a, 1973b, 1973c, 1974; Deitz 1975; Dietrich et al. transilluminator FBT1 88 (Fisher Biotech, Subiaco, 2001; Cryan et al. 2004; and spittlebugs: Doering 1930, WA). The gel image was stored as a TIFF format Þle 1941, Hanna and Moore 1966, Hamilton 1982, Dietrich and processed with Adobe Photoshop CS2, version 2005. Pinned voucher specimens were deposited in 9.02 (Adobe 2005). the Insect Museum, Department of Entomology, Rut- X. fastidiosa PCR products were puriÞed with gers University, New Brunswick, NJ. QIAquick gel extraction kit (Qiagen Inc., Valencia, Numbers of insects per sample were square root CA) and sent with their corresponding primers for transformed and analyzed with PROC GLM using SAS direct PCR product sequencing (GeneWiz, South 9.1.3 (SAS Institute 2006). The GLM main treatments PlainÞeld, NJ). The sequences of the PCR products, were sample location (Duke Farms and Mercer 472 bp for 272-int primers, 553 bp for 16S-23S inter- County Community College), year (2004 and 2006), vening spacer region, and 722 bp for RST31/33 prim- month (May, June, July, August), and bacterial leaf ers, were used as a query sequence for a GenBank scorch infection (asymptomatic and symptomatic). database searched by BLASTn (Basic local alignment Interaction of location and month was incorporated search tool) (Altschul et al. 1997). Fresh PCR prod- into the GLM model after dropping all other nonsig- ucts of each NCBI conÞrmed X. fastidiosa isolates niÞcant interactions. Insect community analysis was were then cloned into a plasmid vector, pCR4-TOPO performed on untransformed data. using the TOPO TA Cloning Kit (Invitrogen Corpo- Molecular Assay of X. fastidiosa. There were 1,601 ration, Carlsbad, CA). Desired clones were puriÞed insects dissected to remove eyes, legs, abdomen, and with QiagenÕs QIAprep spin miniprep kit. Both strands wings under Stereo Star (Reichert ScientiÞc Instru- of the inserts were sequenced at GeneWiz. DNA se- ments) dissecting scope to prevent potential PCR in- quences were analyzed with DNASTARÕs Lasergene hibition in the following procedures. Each specimen sequence analysis software (DNASTAR, Inc., Madi- was placed ina2mlscrew top sterilized tube pre- son, WI) (Burland 2000). loaded with two 5 mm glass beads and submerged in liquid nitrogen for 20 s immediately before being Results ground using a Mini-bead Beater (BioSpec Products, Inc., Bartlesville, OK) for 20 s at low speed (2,500 rpm) Cicadomorpha Species Diversity. Four families of (Bextine 2004). X. fastidiosa DNA was extracted using potential insect X. fastidiosa vectors (Order Hemi- a modiÞed Qiagen DNeasy Blood and Tissue Kit. ptera: Suborder Auchenorrhyncha, Infraorder Cica- Brießy, we added 180 ␮l ATL buffer immediately after domorpha) were collected: Membracidae (91.4%) grinding and shook well 1 min after the sample had and Cicadellidae (6.9%) from Membracoidea super- thawed. The sample was then brießy centrifuged to family, Aphrophoridae (1.1%) and Clastopteridae spin down the solution to avoid cross contamination. (0.5%) from Cercopoidea superfamily for a total of DNA was eluted with 50 ␮l AE buffer after incubation 12,880 potential vectors (Table 2). of the membrane for 15 min. Elution was repeated Membracidae. A total of 11,662 treehoppers were once. collected from sticky cards and fogging during the 4 yr Polymerase Chain Reaction. A nested polymerase of sampling (Table 2). Many were known oak feeders chain reaction (PCR) procedure was used to detect X. (Wood 1983, 1993). The predominant treehopper fastidiosa presence in insect samples with external species collected was Ophiderma deﬁnita Woodruff primers 272Ð1-ext (5Ј-AGCGGGCCAATATTCAAT- (Woodruff 1919), comprising 75% of the collections, TGC-3Ј) and 272Ð2-ext (5Ј-AGCGGGCCAAAAC- followed by Telamona monticola (Fitch) 8%, Archasia GATGCGTG-3Ј) followed by internal primers 272- belfragei (F.) 5%, T. tiliae Ball 4%, Glossonotus acuminatus 1-int (5Ј-CTGCACTTACCCAATGCATCG-3Ј) and (F.) 3%, and Cyrtolobus fenestratus (Fitch) 1%. The re- 272Ð2-int (5Ј-GCCGCTTCGGAGAGCATTCCT-3Ј) maining 19 Membracid species comprised Ͻ4% of the (Pooler and Hartung 1995). The presence of X. fasti- total treehopper collection (Table 2). diosa DNA was further assessed by two additional Cicadellidae. Most of the Cicadellidae collected primer sets RST31 (5Ј-GCGTTAATTTTCGAAGT- were in the subfamilies Athysaninae, Neocoelidii- GATTCGATTGC-3Ј)/RST33 (5Ј-CACCATTCGTA- nae, and Typhlocybinae, which are not known xy- TCCCGGTG-3Ј) (Minsavage et al. 1994) and lem feeders and may not be important for bacterial G1 (5Ј-GAAGTCGTAACAAGG-3Ј) and L1 (5Ј- leaf scorch disease transmission. Known X. fastid- CAAGGCATCCACCGT-3Ј) (Hendson et al. 2001, iosa vectors are the relatively large leafhoppers from Mehta and Rosato 2001). PCRs were carried out in a the Cicadellinae subfamily, which have an inßated 1134 ENVIRONMENTAL ENTOMOLOGY Vol. 40, no. 5

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

Sticky Card on fog Total X. fastidiosa Species Fogging Total cards days tested positive (%) Total 6,758 6,122 12,880 299 1,601 13.89 Cercopoidea: Aphrophoridae: Aphrophorinae Aphrophora quadrinotata Say 19 19 15 33.33 Aphrophora cribrata (Walker) 2 2 1 0 Philaenus marginellus (L.) 2 1 3 3 0 Philaenus pallidus (L.) 34 18 52 3 51 12.28 Philaenus spumarius (L.) 49 19 68 10 68 19.23 Cercopoidea: Clastopteridae: Clastopterinae Clastoptera obtusa (Say) 13 57 70 5 42 7.14 Membracoidea: Cicadellidae: Cicadellinae Draeculacephala anguilfera (Walker) 5 1 6 5 20 Draeculacephala portola Ball 14 14 9 11.11 Graphocephala coccinea (Forster) 44 44 6 28 3.57 Graphocephala versuta (Say) 800 5 805 97 431 20.19 Aulacizes irrorata (F.) 10 1 11 2 10 10 Oncometopia orbona (F.) 13 13 7 14.29 Membracoidea: Membracidae: Hoplophorinae Platycotis vittata (F.) 13 2 15 4 0 Membracoidea: Membracidae: Membracinae Enchenopa binota (Say) 24 2 26 2 17 11.76 Membracoidea: Membracidae: Smiliinae Acutalis tartarea (Say) 1 2 3 1 Archasia belfragei Stål 341 292 633 81 120 3.33 Atymna querci Fitch 4 21 25 Carynota mera (Say) 1 1 Cyrtolobus maculifrontis (Emmons) 1 1 1 0 Cyrtolobus discoidalis (Emmons) 1 1 2 1 100 Cyrtolobus fenestratus (Fitch) 106 9 115 45 4.44 Cyrtolobus fuscipennis (Van Duzee) 1 1 1 0 Cyrtolobus gratiosus Woodruff 11 11 5 0 Cyrtolobus puritanus Woodruff 4 4 3 0 Entylia carinata Forster 1 1 2 Glossonotus acuminatus (F.) 234 105 339 33 114 11.4 Helonica excelsa Fairmaire 6 2 8 1 1 0 Ophiderma deﬁnita Woodruff 3,944 4,838 8,782 54 226 15.49 Ophiderma evelyna Woodruff 9 9 Ophiderma ﬂava Goding 8 10 18 1 0 Smilia fasciata (Amyot & Serville) 11 44 55 22 4.55 Telamona concava Fitch 2 2 4 2 0 Telamona extrema Ball 4 4 1 100 Telamona monticola (F.) 697 202 899 134 15.67 Telamona tiliae Ball 348 75 423 121 9.92 Telamona unicolor Fitch 25 4 29 4 22 0 Membracoidea: Membracidae: Stegaspidinae Microcentrus perditus (Amyot & Serville) 1 110 111 90 12.22 Nymphs 254 254 clypeus with enlarged pump that enables them to laenus spumarius (L.), and 24.3% (52) were P. pallidus feed on xylem ßuid. There were 893 Cicadellinae (L.). leafhoppers from six species collected during the 4 Sticky Traps versus Canopy Fogging. Sticky cards yr sampling period. Graphocephala versuta (Say) deployed only on fogging days collected fewer spec- was the most prevalent Cicadellinae species (90.2%) imens than fogging (Table 2). Only 299 specimens collected followed by G. coccinea (Forster) (4.9%), from 13 species in 11 genera were collected from 1,004 Draeculacephala portola Ball (1.6%), Oncometopia sticky trap cards placed in 105 pin oaks, while 6,121 orbona (F.) (1.5%), Aulacizes irrorata (F.) (1.2%), Cicadomorpha insects from 29 species of 19 genera and Draeculacephala anguilfera (Walker) (0.7%). were collected from 84 pin oaks by fogging. Each Aphrophoridae and Clastperidae. The spittlebugs species collected from sticky cards was also collected belonged to two families: Aphrophoridae and Clas- during fogging except for G. coccinea, which was found topteridae. All have an inßated clypeus with enlarged only on sticky cards. Seventeen species were present feeding pump that allows xylem feeding and are thus in the fogging samples only, in addition to the 12 all are potential Xylella vectors. Despite being col- common species present in both sampling methods, lected during the entire season, spittlebug populations indicating that the sampling efÞciency of sticky cards were relatively low. Of the 214 specimens collected is low when compared with the fogging. However, if from both fogging and sticky card traps, 32.7% (70) comparing sticky card collection from all sampling were Clastoptera obtusa (Say), 31.9% (68) were Phi- days of all sites to fogging, a total of 6,759 individuals October 2011 ZHANG ET AL.: CICADOMORPHA ASSOCIATED WITH BACTERIAL LEAF SCORCH 1135

ßyers and were collected more on sticky cards than fogging. G. versuta made up 32.4% (97) of the total sticky card collection on fogging days compared with 0.1% (5) of the total fogging collection. No G. coccinea were collected using fogging. However, the eared hopper, Microcentrus perditus (Amyot and Serville), was almost exclusively collected by fogging except for a single individual collected on a sticky card. Low sticky card collections for this species may be due a lack of adult mobility. Immature treehoppers were collected only from fogging (Table 2). Seasonal Dynamics. Although abundance varied with location and year, the temporal pattern was similar between sites each year. Cicadomorpha were Fig. 1. O. definita female proportion from sticky card present from May to September, but most occurred trapping and fogging and female with eggs ratio (females from the end of May to mid July. An initial peak with eggs:total females) during 2006. appeared during the Þrst 3 wk of June and was followed by a second small peak from the end of June to of 33 species were from sticky cards, which is more the second week of the July. than from fogging. There were 25 common species O. Definita. Adults were collected on sticky cards present in both fogging and sticky card samples, eight from the end of May to the end of June with a clear unique species present only in sticky card collections, peak in the Þrst week of June (Fig. 2). O. definita and four unique species present only in fogging col- population abundance varied among locations, possi- lections. All unique species were collected at levels bly because of the oak species composition. Low pop- fewer than 44 adults regardless of method used ulations were observed each year at the Riverview (Table 2). Cemetery and Rutgers Gardens sites. Comparisons of Sampling efÞciency also varied with insect mobility. the O. definita adults on sticky cards collected at Riv- O. definita was the most collected species from both erview Cemetery indicated that there were signiÞ- fogging (Ͼ80%) and sticky cards (Ͼ58%). The 22% cantly more O. definita adults trapped in pin oak than ϭ Ͻ difference is attributable to the low number of females in red oaks (F 1, 1644 19.26; P 0.001) (Fig. 3). Adult attracted to sticky cards relative to fogging (Fig. 1). catches also varied between years. High populations Fewer gravid female adults were collected on sticky appeared in 2002 at Duke Farms and in 2006 at Mercer cards when they were carrying eggs, leading to male County Community College where the only species biased collections that underestimate the population. was pin oaks. Compared with treehoppers, leafhoppers are active Adult O. definita numbers differed between males

Fig. 2. O. deﬁnita adult seasonal abundance (mean Ϯ SE) on yellow sticky cards in pin oak canopies at Mercer County Community College (MCCC), Duke Farms (Duke), Rutgers Garden (Rutgers), and Riverview Cemetery (Trenton) during 2002, 2003, 2004, and 2006. 1136 ENVIRONMENTAL ENTOMOLOGY Vol. 40, no. 5

Fig. 3. O. definita adult catches (mean Ϯ SE) on sticky card traps in pin and red oak canopies in Riverview Cemetery, Trenton during 2004 and 2006. and females in both fogging and sticky card collection. sticky cards. Unlike O. definita, adult G. versuta were Female proportion from fogging varied from 43.4% on active until the end of August. Since there were few 24 May 2006 to a high of 85.7% on 21 June 2006 (Fig. catches of G. versuta from fogging, it is difÞcult to 1). By the end of the season, only females were col- determine true female proportion; however, a higher lected. However, much lower percentages of female percentage of females appeared on the sticky cards. O. definita were observed in the sticky card collec- Female proportion from sticky cards ranged between tions. O. definita female proportion increased from 0.60Ð0.66 during the peak period of 16 June-14 July 2.7% on 31 May-19.8% when the population was at its (Fig. 5). A smaller proportion (6Ð10%) of females peak and then declined to 6.2% on 14 June, and 0.8% carried eggs during 16 June-28 July when compared on 28 June 2006. Thirty one percent (31.1%) of the with the percentage of egg-carrying O. definita. females collected carried eggs on 7 June during the G. versuta adult population abundance varied be- population peak. The percentage of egg carrying fe- tween sites and years. The highest population oc- males reached 75% around 28 June when the lowest curred in 2004 for all sites. As with O. definita, the female adult catch (indicating a low ßight activity) population in 2003 was the lowest for all three sites. occurred on the sticky cards. Female proportion Although G. versuta adult populations were signiÞ- reached 100% and most of the females carried eggs cantly higher in red oak canopies than in pin oaks in ϭ ϭ after mid July. This may indicate that females live Trenton (F1, 1648 12.36; P 0.0005), the highest adult longer than males or males leave the canopy in late catches in 2004 and 2006 were observed at Mercer season. County Community College where only pin oaks are G. versuta. Adult G. versuta populations had one found. The red oaks at Rutgers Gardens had high major peak that extended from late June to late July populations in both 2002 and 2004, whereas, adult G. and followed immediately after O. definita (Fig. 4) on versuta populations at Duke Farms were the lowest of

Fig. 4. G. versuta adult seasonal abundance (mean Ϯ 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. October 2011 ZHANG ET AL.: CICADOMORPHA ASSOCIATED WITH BACTERIAL LEAF SCORCH 1137

total number of positive treehoppers was substantially higher. Based on the X. fastidiosa DNA positive rate for each insect group, an estimated 1,311 treehoppers, 168 leafhoppers, and 32 spittlebugs from the 12,880 individuals collected might carry and potentially vector the X. fastidiosa bacterium. Since X. fastidiosa carrying treehopper numbers were 655.5% larger than the combined numbers of leafhoppers and spittlebugs, treehoppers may play a major role in the bacterial leaf scorch disease transmission in pin and Fig. 5. Seasonal dynamics of G. versuta adult female pro- red oak. portion and the ratio of females carrying eggs during the X. fastidiosa bacteria were detected from collected summer in central New Jersey. insects from May to August. Throughout the summer, X. fastidiosa occurred in treehoppers at fairly consis- all four sites in 2004 suggesting that G. versuta may tent levels: 15.7% in May, 9.1% in June, 12.1% in July, prefer red oak to pin oak. and 13.3% in August. Twenty percent (20.4%) of the Cicadomorpha Harboring X. fastidiosa. Fourteen leafhoppers from six Cicadellinae species collected percent (13.9%) of 1,601 specimens tested were X. in July (when populations peaked) tested X. fasti- fastidiosa DNA positive (Table 2). Twenty one of the diosa DNA positive, whereas 15.3 and 12.9% tested 32 Cicadomorpha species tested were X. fastidiosa when collected in June and August (when popula- DNA positive with treehoppers making up the largest tions were very low) were positive, respectively. group of 11 species potential insect vectors. Eleven The percentage of tested spittlebug specimens car- percent (11.0%) of the tested treehoppers were pos- rying X. fastidiosa was similar to that of the Cicadel- itive for X. fastidiosa DNA. All six Cicadellinae spe- linae (11.0, 15.9, and 18.0% for June, July, and Au- cies tested X. fastidiosa positive with 18.8% of the gust, respectively). 490 individuals tested testing DNA positive. Fifteen Impact of Oak Bacterial Leaf Scorch Infection on percent (15.5%) of the most dominant species, O. Cicadomorpha Abundance and Diversity. Absolute deﬁnita tested positive for X. fastidiosa DNA. sampling using fogging showed that the total number Twenty percent (20.2%) of the tested G. versuta of Cicadomorpha was signiÞcantly higher at Duke carried X. fastidiosa DNA while a 3.6% positive rate Farms than at Mercer County Community College ϭ Ͻ was detected for a similar species, G. coccinea. Al- (F1, 74 26.04; P 0.0001). The total number collected ϭ ϭ though spittlebug populations were low, four of six in 2006 was greater than in 2004 (F1, 74 3.47; P Cercopoidea species tested were X. fastidiosa pos- 0.0665) (Fig. 6) and was because of the signiÞcantly itive with a 15.1% DNA positive rate for the 194 higher insect density in May when compared with ϭ Ͻ individuals tested. later months (F3, 74 81.10, P 0.0001). There was Although the treehopper X. fastidiosa DNA positive a signiÞcant interaction between location (Duke rate was lower than for leafhoppers or spittlebugs, the Farms vs. Mercer County Community College) and

Fig. 6. Total oak feeding Cicadomorpha insect population abundance (mean Ϯ SE) in pin oak canopies sampled by fogging at Duke Farms (Duke) and Mercer County Community College (MCCC) in 2004 and 2006. 1138 ENVIRONMENTAL ENTOMOLOGY Vol. 40, no. 5

Fig. 7. Membracidae population abundance (mean Ϯ SE) in pin oak canopies sampled by fogging at Duke Farms (Duke) and Mercer County Community College (MCCC) in 2004 and 2006. time (month) of the year on the total population (F Few spittlebugs were found in pin oak canopies 3, 74 ϭ 9.03, P Ͻ 0.0001). Populations peaked in early using either sticky cards (98) or fogging (116). Al- season and decreased in July and August in both though they can be found throughout the summer 2004 and 2006 at Mercer County Community Col- season from June to August, signiÞcantly more spit- lege. The same trend occurred at Duke Farms in tlebugs were found in June than in other months ϭ ϭ 2006. However, the total number of insects was (F3, 74 6.55; P 0.0005) (Fig. 9). Interestingly, few higher in August than in June and July at Duke spittlebugs were found in 2006 at Duke Farms or in Farms in 2004. The highest abundance (575 indi- 2004 at Mercer County Community College com- viduals) was observed from a single asymptomatic pared with those found in 2004 Duke Farms and in pin oak tree in May at Duke Farms. The average 2006 at Mercer County Community College (Fig. 9). number collected from asymptomatic pin oaks was X. fastidiosa infection showed no signiÞcant impact 90.1 Ϯ 20.3 per tree and was signiÞcantly Ͼ56.1 Ϯ ϭ ϭ on the spittlebug population density (F1, 74 1; P 17.1 per tree collected from symptomatic pin oaks 0.3216). (F ϭ 8.65; P ϭ 0.0044) (Fig. 6). 1, 74 Bacterial leaf scorch infection in pin and red oaks The majority (96.0%) of the Cicadomorpha col- contributed to the difference in abundance of insect lected were membracids, of which 82.1% were O. species. Adult O. deﬁnita populations collected in deﬁnita, the most abundant species in pin oaks at Duke asymptomatic pin oaks (72.7 Ϯ 18.8 per tree) were Farms and Mercer County Community College. Mem- 71.3% signiÞcantly greater than in symptomatic tree bracidae population abundance also varied with loca- (42.5 Ϯ 15.6 per tree) canopies (F ϭ 7.49; P ϭ tion and time of year (F ϭ 8.99; P Ͻ 0.0001) (Fig. 1, 74 3, 74 0.0078). For the remaining species, only T. tilae 7) following the same trend described for total Cica- domorpha. Membracidae populations at Duke Farms showed signiÞcant population differences based on were highest in May 2006 when compared with all infection status. Eighty-Þve percent (84.6%) signif- other times at Mercer County Community College or icant more T. tiliae were collected in asymptomatic Ϯ Duke Farms. Membracidae population abundance pin oaks (1.1 0.3 per tree) than in symptomatic pin Ϯ ϭ ϭ (86.9 Ϯ 20.3 per pin oak) in asymptomatic trees was oaks (0.6 0.2 per tree) (F1, 74 7.21; P 0.0089). 62.6% and signiÞcantly greater than in symptomatic Bacterial leaf scorch infection did not affect trees (53.4 Ϯ 17.0 per oak) (F ϭ 8.57; P ϭ 0.0045) population differences for any other insect spe- 1, 74 ϭ (Fig. 7). cies. There were 24.5% more M. perditus (F1, 74 ϭ ϭ SigniÞcantly more Cicadellinae leafhoppers (0.29 Ϯ 2.30; P 0.1334), 68.9% more T. monticola (F1, 74 0.10 per tree) were found in the asymptomatic tree 1.42; P ϭ 0.2378), 80.0% more Smilia fasciata ϭ ϭ samples than in symptomatic trees (0.21 Ϯ 0.13 per (F1, 74 0.45; P 0.5064), and 121.5% more tree- ϭ ϭ ϭ ϭ tree) (F1, 74 8.57; P 0.0045) (Fig. 8). In addition, hopper nymphs (F1, 74 1.6; P 0.2095) found in more leafhoppers were found in early season at Mer- asymptomatic canopies than that in symptomatic cer County Community College, while signiÞcantly pin oak canopies. Conversely, 25.4% more A. bel- ϭ ϭ more leafhoppers were found during late season at fragei (F1, 74 0.13; P 0.7209) and 62.5% more G. ϭ Ͻ ϭ ϭ Duke Farms (F3, 74 8.99; P 0.0001). acuminatus (F1, 74 0.56; P 0.4557) adults were October 2011 ZHANG ET AL.: CICADOMORPHA ASSOCIATED WITH BACTERIAL LEAF SCORCH 1139

Fig. 8. Cicadellinae population abundance (mean Ϯ 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- trend was observed at Mercer County Community ϭ ϭ atic trees. College: total Cicadomorpha (F1, 3164 0.32; P ϭ ϭ Impact of Bacterial Leaf Scorch Infection on Rel- 0.5723) and family (F1, 3164 0.34, P 0.5576 for ϭ ϭ ative Cicadomorpha Population Abundance. Rela- Membracidae; F1, 3164 0.16, P 0.6901 for Cicadel- ϭ ϭ tive sampling using sticky traps did not detect any linae; F1, 3164 2.30, P 0.1296 for Cercopoidea). signiÞcant insect population difference between Total sticky card catches were signiÞcantly higher ϭ Ͻ symptomatic oaks and asymptomatic oaks at Duke in 2006 than in 2004 (F1, 3164 626.65; P 0.0001). ϭ ϭ Farms (F1, 1840 0.70, P 0.4012 for total Cicado- This was explained by signiÞcant higher Membra- ϭ ϭ ϭ morpha; F1, 1840 0.70, P 0.4023 for Membraci- cidae catches observed in 2006 (F1, 3164 669.16; ϭ ϭ Ͻ dae; F1, 1840 0.59, P 0.4439 for Cicadellinae; and P 0.0001), because of the large numbers of O. ϭ ϭ ϭ Ͻ F1, 1840 0.21, P 0.6455 for Cercopoidea). The same deﬁnita (F1, 3164 708.31; P 0.0001). The average O.

Fig. 9. Spittlebug population abundance (mean Ϯ SE) in pin oak canopies sampled by fogging at Duke Farms (Duke) and Mercer County Community College (MCCC) in 2004 and 2006. 1140 ENVIRONMENTAL ENTOMOLOGY Vol. 40, no. 5 deﬁnita catch per card was 0.53 in 2006 and 0.03 in 2004 at Mercer County Community College. O. deﬁnita catches comprised 96.1% of the Membracidae, or 89.6% of all insects collected in 2006.

Discussion X. fastidiosa may be transmitted in oak trees by a Cicadomorpha species complex that includes treehoppers, leafhoppers, and spittlebugs. Of the 25 species found in our study, 11 treehopper species tested positive for X. fastidiosa DNA. Vector studies of X. fasti- Fig. 10. Cumulative effective temperature (Ͼ10ЊC since diosa have been conducted in southern agriculture Þrst day of the year) in central New Jersey between April and systems such as grape, peach, and citrus where leaf- May during 2003Ð2006. hoppers are more abundant and few treehoppers are present. Most of the known X. fastidiosa vectors in At the species level, collections from fogging and grape (Severin 1949, 1950; Purcell 1980; Hill and Pur- sticky card sampling did not provide similar results. cell 1995; Costa et al. 2000; Almeida and Purcell 2003) Treehoppers were collected in both fog and sticky have not been detected in oak canopies in New Jersey. card sampling, while Cicadellinae leafhoppers were However, we detected X. fastidiosa DNA in species collected mostly from sticky card traps. Possible rea- known in other plant systems (Purcell 1980) such as sons for this difference between fogging and sticky the spittlebug Philaenus spumarius (Almond leaf card trapping may be variation in leafhopper distri- scorch), and leafhopper Oncometopia orbona (Phony bution through time and space, ßight activity, or the peach disease). We also detected X. fastidiosa DNA timing of fogging events. In this study, adult G. versuta from leafhoppers G. coccinea, G. versuta, and Draecu- population peaks detected by fogging were similar to lacephala anguilfera as well as spittlebug species Phi- collections from sticky cards when sampled the same laenus pallidus and Clastoptera obtuse. week as the fogging. However, these insects in oak DNA sequencing analysis indicated that X. fastid- canopies may have been low on the days of fogging. iosa DNA from those insects is identical to the DNA Leafhopper populations peaked around 6 July (rang- extracted from host oak petioles (J. Zhang et al. un- ing from 21 June to 9 August) for both 2004 and 2006 published data). Among the X. fastidiosa positive Ci- according to the sticky card trap collections. Fogging cadomorpha, only O. definita and G. versuta had any was conducted on 16 June 2004, 22 July 2004, 21 June sizable populations each year. All other species had 2006, and 24 July 2006 when leafhopper populations low populations and may not play a critical role in were at the beginning or end of the July peak. Sec- disease transmission. O. definita nymphs hatch as oak ondly, Cicadellinae leafhoppers may have a patchy buds break in late April/early May. X. fastidiosa pres- spatial distribution pattern, in which case fogging may ent in oak stems grow into the petiole and leaf veins have simply missed oaks harboring leafhoppers. as the leaf emerges and grows. Although the cell con- Thirdly, sharpshooters are extremely vagile (Turner centration in oak petioles or other feeding sites has yet and Pollard 1959) and are active ßyers in the oak to be determined, by the time adult O. definita pop- canopy so sticky cards may overestimate the actual ulations peak in early June, bacterial populations leafhopper population when compared with the fog- should be large enough for efÞcient acquisition. If the ging. Lastly, the leafhopper catches on sticky cards bacterial population is not high enough in early June may have been because of dispersal ßights, since sticky for acquisition to occur, it must be high enough when cards catch insects throughout the day while fogging populations of G. versuta, the second major Cicado- only catches those insects present in the canopy at the morpha, peak in July. We extracted X. fastidiosa DNA time of sampling. from various Cicadomorpha in June (J. Zhang et al. Cicadomorpha species abundance varied between unpublished data) over a month earlier than the time years. O. definita populations were highest in 2002, when live bacteria were isolated from treehopper O. followed by those in 2006, and very low in 2003 and definita in late July. This indicates the bacteria had 2004. Meanwhile G. versuta populations were highest been present in the insect for some period of time at on 2002 and 2004, and moderately abundant in 2006. a level hard to isolate. Yearly ßuctuations were also observed in Kentucky, We compared fogging with yellow sticky card sam- where populations in 1994 were less than half those in pling for Cicadomorpha population determination in 1993 (Johnson and Freytag 1997). One explanation pin and red oaks. When the data from fogging and might be annual temperature variation. The cumula- sticky cards are combined, 37 species of Cicadomor- tive degree-days (Ͼ10ЊC) from the Þrst day of the year pha from 21 genera were collected from oak canopies to April and May were much higher in 2002 and 2006 in central New Jersey. Fogging in this study collected than in 2003 and 2004 (Fig. 10). Population abundance many more species and individuals than sticky cards. of O. definita peaked in early June (Fig. 2), consistent However, the disadvantages of sticky card sampling with a degree-day explanation. However, population can be compensated by extended sampling with more abundance of the midsummer species, G. versuta (Fig. locations, and by weekly sampling over multiple years. 4), did not show this relationship. October 2011 ZHANG ET AL.: CICADOMORPHA ASSOCIATED WITH BACTERIAL LEAF SCORCH 1141

We found more Cicadomorpha insects in oak can- for their generous help in providing collection equipment opies where no bacterial leaf scorch symptoms were and staff assistance. This study would not have been possible observed, than in infected oaks. One characteristic of without the excellent technical assistance from Colleen Con- this disease in oaks is the apparent randomness of over, Asa Dewan, Nadishani Dissanayaka, Dan Duran, Nidhi infected branches within infected trees. Within a lo- Shree Karingula, Rosemary Kim, Christine King, Sneha Shah, Tracey Switek, Steven Wong, and Chiao-Han Yu. Insect cality it is common to Þnd an apparent healthy oak tree species identiÞcation was conÞrmed by Jason Cryan, Labo- next to a severely infected one. In spring, bacterial leaf ratory for Conservation and Evolutionary Genetics, NY State scorch infected branches usually foliate later than Museum, Albany, NY 12230. We appreciate Daniel Herms, uninfected trees. Oak leaves are usually smaller and Department of Entomology, The Ohio State University, for branches tend to dieback after serious infection (J. his critical review of a prior draft. This is New Jersey Agri- Zhang et al., unpublished data). Mizell and French cultural Experiment Station publication number D-08-08289- (1987) reported that H. vitripennis (Germar) feeds 03-10. predominantly on trees which did not display X. fastidiosa disease symptoms when provided a choice. The nutritional quality of xylem in bacterial leaf scorch References Cited infected oak trees or branches may have some adverse Adlerz, W. C., and D. L. Hopkins. 1979. Natural infectivity impact on insects or deter them from feeding or ovi- of two sharpshooter vectors of PierceÕs disease of grape position, hence promoting insect foraging behavior in Florida. J. Econ. Entomol. 72: 916Ð919. that results in the dispersal of individuals to uninfected Almeida, R., A. H. Purcell, and H. Alexander. 2003. Trans- oak trees in the vicinity. mission of Xylella fastidiosa to grapevines by Homalodisca Unlike what has been demonstrated by fogging, data coagulata (Hemiptera: Cicadellidae). J. Econ. Entomol. 96: 264Ð271. from sticky cards did not show signiÞcant differences Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. in insect population abundance between bacterial leaf Zhang, W. Miller, and D. J. Lipman. 1997. Gapped scorch infected oaks and asymptomatic oaks. This BLAST and PSI-BLAST: a new generation of protein might be because sticky card trapping is a relative database search programs. Nucl. Acids Res. 25: 3389Ð sampling method that indicates insect ßight activity, in 3402. addition to abundance. As discussed above, insect vec- Barbosa, P., A. Segarra, and P. Gross. 2000. Structure of two tors may increase their ßight activity and move to macrolepidopteran assemblages on Salix nigra (Marsh) healthier asymptomatic oaks for feeding and oviposi- and Acer negundo L.: abundance, diversity, richness, and tion when bacterial infection progresses and oak host persistence of scare species. Ecol. Entomol. 25: 374Ð379. Basset, Y., N. D. Springate, H. P. Aberlenc, and G. Delvare. quality declines. High mobility of these insects may 1997. A review of methods for sampling arthropods in result in high sticky card catches in the symptomatic tree canopies, pp. 27Ð52. In. N. E. Stork, J. Adis, and R. K. oak trees. However, a healthy oak tree provides not Didham (eds.), Canopy Arthropods. Chapman & Hall, only a better food resource that will reduce the ne- London, United Kingdom. cessity for dispersal ßights, but also higher leaf den- Basset, Y., V. Novotny, S. E. Miller, and R. L. Kitching. 2003. sities which can block or reduce insect mobility lead- Methodological advances and limitations in canopy en- ing to lower numbers on sticky cards in asymptomatic tomology, pp. 7Ð16. In Y. Basset, V. Novotny, S. E. Miller, oak trees. and R. L. Kitching (eds.), Arthropods of tropical forests: In conclusion, the fogging method measures the spatio-temporal dynamics and resource use in the canopy. Cambridge University Press, Cambridge, United actual population abundance of potential vectors re- Kingdom. siding in each oak canopy, and represents the numer- Bextine, B., S. J. Tuan, H. Shaikh, M. Blua, and T. A. Miller. ical consequence of the bacterial leaf scorch impact. 2004. Evaluation of methods for extracting Xylella fasti- Sticky card trapping detects insect behaviors that are diosa DNA from the glassy-winged sharpshooter. J. Econ. involved with the process of bacterial leaf scorch in- Entomol. 97: 757Ð763. fection of oaks. A high catch using sticky cards rep- Brlansky, R. H., V. D. Damsteegt, D. S. Howd, and J. S. resents high mobility of the insect vector, whereas a Hartung. 1996. Transmission of the causal agent of citrus low population in bacterial leaf scorch infected oaks in variegated chlorosis, Xylella fastidiosa, with a sharp- the neighborhood of asymptomatic oaks would be shooter leafhopper vector from Florida. Phytopathology 86(11 suppl.): S74. over-estimated using the sticky cards and would ap- Burland, T. G. 2000. DNASTARÕs lasergene sequence anal- pear similar to the catches from uninfected oaks. By ysis software. Methods Mol. Biol. 132: 71Ð91. integration of these two sampling methods, one can Costa, H. S., M. S. Blua, J. A. Bethke, and R. A. Redak. 2000. capture the dynamics of the entire oak canopy insect Transmission of Xylella fastidiosa to oleander by the population when bacterial leaf scorch infection glassywinged sharpshooter, Homalodisca coagulate. Hort- occurs. science 35: 1265Ð1267. Cryan, J. R. 2005. Molecular phylogeny of Cicadomorpha (Insecta: Hemiptera: Cicadoidea, Cercopoidea and Mem- Acknowledgments bracoidea): adding evidence to the controversy. Syst. Entomol. 30: 563Ð574. Thanks to Duke Farms, Hillsborough, Somerset County; Cryan, J. R., B. M. Wiegmann, L. L. Deitz, C. H. Dietrich, and Mercer County Community College, Mercer County; and M. F. Whiting. 2004. Treehopper trees: phylogeny of Riverview Cemetery, Trenton, Mercer County, NJ, for their Membracidae (Hemiptera: Cicadomorpha: Membra- support in the specimen collection. Special thanks to Paul coidea) based on molecules and morphology. Syst. En- Smith, Gene Huntington, Tom Allmendinger at Duke Farms tomol. 29: 441Ð454. 1142 ENVIRONMENTAL ENTOMOLOGY Vol. 40, no. 5

Deitz, L. L. 1975. ClassiÞcation of the higher categories of Kopp, D. D., and T. R. Yonke. 1974. The treehoppers of the New World treehoppers (Homoptera: Membraci- Missouri: Part 4. Subfamily Smiliinae; Tribes Telamonini dae). University of North Carolina Press, Raleigh, NC. (Homoptera: Membracidae). J. Kans. Entomol. Soc. 47: DeLong, D. M. 1948. The leafhoppers, or Cicadellidae, of 80Ð129. Illinois (Eurymelinae-Balcluthinae). Bull. Illinois Natur. Krugner, R., C. Lopes, and J. S. Santos. 2000. Transmission Hist. Surv. 24: 97Ð376. efÞciency of Xylella fastidiosa to citrus by sharpshoot- Dennis, C. 1952. The Membracidae of Wisconsin. Wis. ers and identiÞcation of two new vector species, pp. Acad. Sci. Arts Lett. 41: 129Ð152. 423. In R. H. Yokomi, J. V. Da Graca, and R. F. Lee Dennis, C. 1965. Oklahoma treehoppers (Homoptera, (eds.), Proceedings of The 14th Conference of The Membracidae), pp. 50Ð64. In Proceedings, 45th of The International Organization of Citrus Virologists, 13Ð18 Oklahoma Academy of Sciences, 1964, Edmond, OK. The September 1998, Campinas, Sao Paulo State, Brazil. Oklahoma Academy of Sciences, Edmond, OK. International Organization of Citrus Virologists, De- Dietrich, C. H. 2005. Keys to the families of Cicadomorpha partment of Plant Pathology, University of California, and subfamilies and tribes of Cicadellidae (Hemiptera: Riverside, CA. Auchenorrhyncha). Fl. Entomol. 88: 502Ð517. Lee, R. F., M.J.G. Beretta, J. H. Hartung, M. E. Hooker, and Dietrich, C. H., S. H. Mckamey, and L. L. Deitz. 2001. Mor- K. S. Derrick. 1993. Citrus variegated chlorosis: conÞr- phology-based phylogeny of the treehopper family Mem- mation of Xylella fastidiosa as the causal agent. Summ. bracidae (Hemiptera: Cicadomorpha: Membracoidea). Phytopathol. 19: 123Ð125. Syst. Entomol. 26: 213Ð239. McCoy, R. E., D. L. Thomas, J. H. Tsai, and W. J. French. Doering, K. C. 1930. Synoposis of family Cercopidae (Ho- 1978. Periwinkle wilt, a new disease associated with xy- moptera) in North America. J. Kans. Entomol. Soc. 3: lem delimited rickettsia-like bacteria transmitted by a 53Ð108. sharpshooter. Plant Dis. Rep. 1022Ð1026. Doering, K. C. 1941. Synoposis A revision of two genera of Mehta, A., and Y. B. Rosato. 2001. Phylogenetic relation- North American Cercopidae (Homoptera). J. Kans. En- ships of Xylella fastidiosa strains from different hosts, tomol. Soc. 14: 102Ð134. based on 16S rDNA and 16SÐ23S intergenic spacer se- Hamilton, K.G.A. 1982. The insects and arachnids of Can- quences. Int. J. Syst. Evol. Microbiol. 51: 311Ð318. ada: part 10. The spittlebugs of Canada. Homoptera: Cer- Minsavage, G. V., C. M. Thompson, D. L. Hopkins, R. M. copidae. Canadian Government Publishing Centre, Ot- Leite, and R. E. Stall. 1994. Development of a polymer- tawa, Canada. Agriculture Canada Publication 1740. ase chain reaction protocol for detection of Xylella fas- Hanna, M., and T. Moore. 1966. The spittlebugs of Michigan tidiosa in plant tissue. Phytopathology 84: 456Ð461. (Homoptera: Cercopidae). Pap. Mich. Acad. Sci. Arts Mircetich, S. M., S. K. Lowe, W. J. Moller, and G. Nyland. Lett. 51: 39Ð73. 1976. Etiology of almond leaf scorch disease and trans- Hearon, S. S., J. L. Sherald, and S. J. Kostka. 1980. Associa- mission of the causal agent. Phytopathology 66: 17Ð24. tion of xylem-limited bacteria with elm, sycamore, and Mizell III, R. F., and W. J. French. 1987. Leafhopper oak leaf scorch. Can. J. Bot. 58: 1986Ð1993. vectors of phony peach disease: feeding site preference Hendson, M., A. H. Purcell, D. Chen, C. Smart, M. Guilhab- and survival on infected and uninfected peach, and ert, and B. Kirkpatrick. 2001. Genetic diversity of seasonal response to selected host plants. J. Entomol. PierceÕs disease strains and other pathotypes of Xylella Sci. 22: 11Ð22. fastidiosa. Appl. Environ. Microbiol. 67: 895Ð903. Nielsen, M. W. 1968. The leafhopper vectors of phytopatho- Hill, B. L., and A. H. Purcell. 1995. Acquisition and reten- genic viruses (Homoptera: Cicadellidae) taxonomy, bi- tion of Xylella fastidiosa by an efÞcient vector. Phytopa- ology, and virus transmission. USDA, ARS Tech. Bull. thology 85: 209Ð212. 1382: 384. Hopkins, D. L., and H. H. Mollenhauer. 1973. Rickettsia- Novotny, V., and M. R. Wilson. 1997. Why are there no small like bacterium associated with PierceÕs disease of grapes. species among xylem sucking insects? Evol. Ecol. 11: Science 179: 298Ð300. 419Ð437. Hopkins, D. L., H. H. Mollenhauer, and W. J. French. 1973. Osborn, H. 1940. The Membracidae of Ohio. OH Biol. Surv. Occurrence of a rickettsia like bacterium in the xylem of Bull. No. 37, VII: 51Ð101. peach with phony disease. Phytopathology 63: 1422Ð1423. Pooler, M. R., and J. S. Hartung. 1995. SpeciÞc PCR detec- Huang, Q., W. Li, and J. Hartung. 2003. Association of Xy- tion and identiÞcation of Xylella fastidiosa causing citrus lella fastidiosa with leaf scorch in Japanese beech bonsai. variegated chlorosis. Curr. Microbiol. 31: 377Ð381. Can. J. Plant Pathol. 25: 401Ð405. Purcell, A. H. 1980a. Almond leaf scorch: leafhopper and Johnson, M. P., and P. H. Freytag. 1997. Treehoppers (Ho- spittlebug vectors. J. Econ. Entomol. 73: 834Ð838. moptera: Membracidae) on pin oak in Kentucky. J. Kans. Purcell, A. H. 1980b. Environmental therapy for PierceÕs Entomol. Soc. 70: 21Ð30. disease of grapevines. Plant Dis. 64: 388Ð390. Kitajima, E. W., M. Bakarcic, and M. V. Fernandez-Valiela. SAS Institute. 2006. SAS 9.1.3 service pack 4. SAS Institute, 1975. Association of rickettsia-like bacteria with plum Cary, NC. leaf scald disease. Phytopathology 65: 476Ð479. Severin, H.H.P. 1949. Transmission of the virus of PierceÕs Kopp, D. D., and T. R. Yonke. 1973a. The treehoppers of disease by leafhoppers. Hilgardia 19: 190Ð202. Missouri: part 1. Subfamilies Centrotlnae, Hoplophorio- Severin, H.H.P. 1950. Spittle-insect vectors of PierceÕs dis- ninae, and Membracinae (Homoptera: Membracidae). J. ease virus II. Life history and virus transmission. Hilgardia Kans. Entomol. Soc. 46: 42Ð64. 19: 357Ð382. Kopp, D. D., and T. R. Yonke. 1973b. The treehoppers of Sherald, J. L., S. S. Hearon, S. J. Kostka, and D. L. Morgan. Missouri: part 2. Subfamily Smiliinae; Tribes Acutalini, 1983. Sycamore leaf scorch: culture and pathogenicity of Ceresini, and Polyglyptini (Homoptera: Membracidae). fastidious xylem-limited bacteria from scorch-affected J. Kans. Entomol. Soc. 46: 233Ð275. trees. Plant Dis. 67: 849Ð852. Kopp, D. D., and T. R. Yonke. 1973c. The treehoppers of Sherald, J. L., J. M. Wells, S. S. Hurtt, and S. J. Kostka. 1987. Missouri: part 3. Subfamily Smiliinae; Tribe Smiliini. J. Association of fastidious, xylem-inhabiting bacteria with Kans. Entomol. Soc. 46: 375Ð421. leaf scorch in red maple. Plant Dis. 71: 930Ð933. October 2011 ZHANG ET AL.: CICADOMORPHA ASSOCIATED WITH BACTERIAL LEAF SCORCH 1143

Southwood, T.R.E., and P. A. Henderson. 2000. Ecological Wood, T. K. 1983. Life history patterns of tropical mem- methods, 3rd ed. Blackwell, Malden, MA. bracids (Homoptera: Membracidae) [Treehoppers]. So- Stork, N. E., and P. M. Hammond. 1997. Sampling arthro- ciobiology 8: 299Ð344. pods from tree-crowns by fogging with knockdown in- Wood, T. K. 1993. Diversity in the New World Membraci- secticides: lessons from studies of oak tree beetle assem- dae. Annu. Rev. Entomol. 38: 409Ð435. blages in Richmond Park (UK), pp. 3Ð26. In N. E. Stork, Woodruff, L. B. 1919. A review of our local species of the J. Adis, and R. K. Didham (eds.), Canopy Arthropods. Membracid genus Ophiderma Fairmarie. J. New York Chapman & Hall, London, United Kingdom. Entomol. Soc. 27: 249Ð260. Turner, W. F. and H. N. Pollard. 1959. Insect transmission of phony peach disease. U.S. Department of Agriculture, Technical Bulletin 1193. Received 5 April 2010; accepted 21 June 2011.