Coleoptera: Scarabaeidae - BioOne

BIOLOGICAL CONTROL-MICROBIALS

Incidence of Turf-Damaging White Grubs (Coleoptera: Scarabaeidae) and Associated Pathogens and Parasitoids on Kentucky Golf Courses CARL T. REDMOND

AND

DANIEL A. POTTER1

Department of Entomology, S-225 Agriculture Science Bldg. N., University of Kentucky, Lexington, KY 40546-0091

Environ. Entomol. 39(6): 1838Ð1847 (2010); DOI: 10.1603/EN10172

ABSTRACT Root-feeding grubs (Coleoptera: Scarabaeidae) were sampled from damaged areas of 61 irrigated roughs on 32 Kentucky golf courses to determine species composition and natural enemy incidence, the Þrst such survey in the United StatesÕ transitional turfgrass climatic zone. Masked chafers (Cyclocephala lurida Bland and C. borealis Arrow) and Japanese beetle (Popillia japonica Newman) accounted for ⬇73 and 26% of grubs found in an autumn survey, with Cyclocephala spp. predominating at most sites, although mixed infestations were common. Only a few Phyllophaga spp., and no exotic species other than P. japonica were found. Cyclocephala spp. also predominated in seasonal and statewide surveys regardless of whether a course had cool- or warm-season grass fairways. Pathogenic bacteria, Paenibacillus and Serratia spp., and the autumn-active parasitoid Tiphia pygidialis Allen were the main enemies associated with Cyclocephala spp. Predominant enemies of P. japonica were Paenibacillus, Serratia, and Metarhizium spp. in autumn, and eugregarines, Stictospora sp. (probably S. villani Hays and Clopton) and Tiphia vernalis Rohwer in spring. Entomopathogenic nematodes and the microsporidian Ovavesicula popilliae Andreadis & Hanula were nearly absent in our samples. No predictive relationships were found between soil parameters and proportionate abundance of Cyclocephala or P. japonica, or with natural enemy incidence at particular sites. Although incidence of individual enemies was generally low (⬍20%; often ⬍5%) in these point-in-time surveys, collectively and over their hostsÕ prolonged development they may take a signiÞcant toll on grub populations. KEY WORDS Popillia japonica, Cyclocephala, Paenibacillus, Stictospora, Serratia

Root-feeding white grubs (Coleoptera: Scarabaeidae) are the most destructive turfgrass insect pests in the northern two-thirds of the United States (Potter 1998, Vittum et al. 1999). In the transitional climatic zone, a wide belt extending from eastern New Mexico through southern Missouri and Kentucky to the midAtlantic states where both cool- and warm-season turfgrass species can be grown (Beard 2002), native white grubs, especially northern and southern masked chafers, Cyclocephala borealis Arrow and Cyclocephala lurida Bland, often co-occur with Japanese beetle larvae, Popillia japonica Newman (Potter et al. 1996). Where their ranges overlap, the relative extent to which Cyclocephala spp. or P. japonica damage golf course turf, and factors affecting their relative abundance at particular sites, are poorly known. From 1920 Ð1933, the former U.S. Bureau of Entomology imported numerous natural enemies of P. japonica from Asia for colonization in the eastern United States (Fleming 1968). Only three species became established, the larval parasitoids Tiphia vernalis Rohwer and Tiphia popilliavora Rohwer (Hymenoptera: Tiphiidae), and the adult parasitoid Istocheta aldrichi (Mesnil) (Diptera: Tachinidae). Although the extent 1

Corresponding author, e-mail: [email protected].

of their present geographical distributions is uncertain, T. vernalis is abundant in central Kentucky (Rogers and Potter 2004) and Connecticut (Ramoutar and Legrand 2007) but apparently is not established in Michigan (Cappaert and Smitley 2002), whereas I. aldrichi appears to be restricted to the New England states (Vittum et al. 1999). Japanese beetle and masked chafer grubs are also naturally infected by milky disease-causing bacteria, Paenibacillus spp., entomopathogenic nematodes, fungi (Metarhizium and Beauveria spp.) and other soil-inhabiting pathogens (Klein 1995, Vittum et al. 1999) although Paenibacillus strains isolated from P. japonica were the only ones intentionally introduced (Fleming 1968). Only two systematic surveys of P. japonica natural enemies have been published, both from northern states. Hanula and Andreadis (1988) found 50 Ð100% infection of P. japonica by cephaline gregarines, Stictospora sp. (Apicomplexa: Eugregarinida: Actinocephalidae), ⬇25% infection by the microsporidean Ovavesicula popilliae Andreadis and Hanula, and 3.5 and 1.2% infection by Paenibacillus spp. and Metarhizium anisopliae (Metchnikoff) Sorokin, respectively, across 49 turf sites in Connecticut. A survey of golf courses and other sites in southern Michigan (Cappaert and Smitley 2002), where P. japonica more re-

0046-225X/10/1838Ð1847$04.00/0 䉷 2010 Entomological Society of America

December 2010

REDMOND AND POTTER: WHITE GRUB NATURAL ENEMIES ON GOLF COURSES

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Table 1. Density and species of scarab larvae associated with damage to irrigated roughs of six central Kentucky golf courses, Autumn 2006, showing predominance of Cyclocephala spp. (MC) at 15 of the 18 sample sites Location University Club Midway, KY Lexington CC Lexington, KY The Bull GC Richmond, KY London CC, London, KY Connemara GC Nicholasville, KY Kearney Hill GC Lexington, KY

Site no.

Densitya

1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

19.0 12.0 22.7 9.6 10.8 12.6 8.8 7.5 9.8 11.1 12.7 11.2 12.5 10.0 36.0 21.8 13.3 8.3

Total

Grub species in samplesb MC

JB

% MC

P

149 81 160 80 77 94 89 95 104 119 63 96 58 34 5 156 84 42 1586

34 24 4 23 15 15 15 5 1 4 22 4 38 59 224 12 19 53 571

81.4 77.1 97.6 77.7 83.7 86.2 85.6 90.4 98.1 96.7 74.1 96.0 60.4 35.4 2.1 92.9 81.6 44.2 73.2

⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001 0.05 0.01 ⬍0.001 ⬍0.001 ⬍0.001 0.3 ⬍0.001

Mean no. of grubs per 0.1m2. MC, JB are masked chafer (Cyclocephala lurida and C. borealis) or Japanese beetle grubs, respectively; P value from continuity-corrected Z-test for departure from equal proportions of MC and JB at a given site. a

b

cently established, revealed 36% incidence of gregarines (probably Stictospora villani Hays and Clopton) but almost no O. popilliae or milky disease, and no Tiphia wasps or other parasitoids. Paenibacillus spp. and T. vernalis occur in Kentucky (Redmond and Potter 1995, Rogers and Potter 2004), but those and other natural enemies of P. japonica have not previously been systematically surveyed anywhere in the transitional climatic zone. Masked chafer grubs are attacked by a naturally occurring Cyclocephala-speciÞc strain of Paenibacillus (Warren and Potter 1982, Kaya et al. 1992) and by a native tiphiid, Tiphia pygidialis Allen (Rogers and Potter 2004), but their natural enemies have not previously been systematically surveyed anywhere in the United States. We report herein the results of three systematic surveys of natural enemies of scarabaeid grubs associated with turf damage on Kentucky golf courses, the Þrst such data from the transitional climatic zone. Relative occurrence of different grub species was documented and possible relationships between soil characteristics and the relative proportion of either P. japonica or Cyclocephala spp. at particular sites also were tested. Methods and Materials Transitional zone golf courses may use cool-season grasses; for example, creeping bentgrass, Agrostis palustris L. and perennial ryegrass, Lolium perenne L.; or warm season grasses; for example, Bermuda grass, Cynodon dactylon L. Pers. and zoysiagrass, Zoysia spp. on their fairways. Roughs typically are Kentucky bluegrass, Poa pratensis L., tall fescue, Lolium arundinaceum (Schreber) Darbyshire, perennial ryegrass, or mixtures of those grasses. Creeping bentgrass is commonly grown on tees and greens. Grass species no-

menclatures used herein are as deÞned by the USDAARS Germplasm Resources Information Network (2010). Autumn 2006 Survey. Six central golf courses (Table 1) were scouted and sampled for white grubs between 13Ð21 September 2006 to quantify which genera and species are most commonly associated with damage to such sites. Three different nontreated irrigated primary roughs with visible grub damage, at least 100 m apart, were sampled at each course. At each site, 6 Ð9 pits (0.1 ⫻ 0.1 m, 0.1 m deep) were excavated within about a 4 ⫻ 6 m area of turf. The grass, thatch, and soil were removed with a spade, broken apart and examined for grubs that were counted and identiÞed to genus (Cyclocephala, and a few Phyllophaga spp.) or species (P. japonica, and a few Cotinis nitida L. and Ataenius spretulus Haldeman) by their distinctive raster patterns (Vittum et al. 1999). Larvae of C. borealis and C. lurida cannot be separated by known morphological characteristics (Potter 1980, 1998). Seasonal Survey. A seasonal survey was conducted from autumn 2007 through spring 2008 to assess temporal changes in grub populations and natural enemy incidence at six central Kentucky golf courses. The courses included Cherry Blossom Golf Course (GC; Georgetown), Lexington Country Club (CC), and Idle Hour CC (Lexington), Connemara GC (Nicholasville), The Bull GC (Richmond), and University Club GC (Midway). Sampling took place during four 1-wk intervals: late August 2007, mid-September 2007, early October 2007, and early May 2008. At each golf course, three untreated roughs with grub damage, at least 100 m apart, were sampled as described in the autumn 2006 survey. Some of the same courses had been sampled in the autumn 2006 survey, but different roughs were sampled for the seasonal survey.

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ENVIRONMENTAL ENTOMOLOGY

On the Þrst sample date, the Þrst 25 grubs encountered at each site (75 grubs per course) were collected into individual 37-ml cups (Dart, Mason, MI) with moist peat moss. If 25 grubs were not found by the ninth sample, the site was rejected and another with higher densities was found. The same spots were avoided on subsequent dates because the damage to previously sampled sites remained evident. After the Þrst sample date, a maximum of nine samples was taken at each site even if 25 grubs had not been obtained. Grubs that had been damaged by sampling were counted in the site density data if a head capsule was present, but were not included among those used for natural enemy assessment. Tiphia cocoons, which may occur to depths of 12Ð16 cm (Rogers et al. 2003) were also collected and identiÞed as described below. The collected grubs were brought to the lab in coolers where they were examined for Tiphia eggs or larvae, and for macroscopic symptoms of nematode infection or disease. The grubs were then placed into individual 118 ml plastic cups (Dart) containing 50 g moist sterilized silt loam soilÐpeat moss mix (1:1 by volume) and 4 g of a turfgrass seed mixture consisting of 2:1:1 Kentucky bluegrass, annual ryegrass (Lolium multiflorum Lamarck), and creeping red fescue (Festuca rubra L). Grubs were held in the dark at 26 Ð28⬚C for 30 d to allow any pathogens to develop, and then held in the same cups at 10⬚C until they could be dissected. Ten-year (2001Ð2010) daily maximum soil temperatures (⬚C, range) measured 10 cm under grass sod in Lexington (wwwagwx.ca.uky.edu/) averaged 25.9 (23.9 Ð29.0) for July, 25.8 (23.2Ð29.0) for August, and 23.9 (21.1Ð29.4) for 1Ð15 September, and would likely be higher still in the upper 5 cm of soil where grubs typically feed, so the aforementioned holding regime was within the soil temperature range experienced by grubs and pathogens in late summer in Kentucky. Additional moisture and grass seed were added as needed, and any deceased grubs were immediately dissected. Natural enemy incidence was assessed as described below. Samples of 25 Cyclocephala grubs were collected from each golf course in May 2008 and adults were reared to determine relative species abundance. C. borealis was distinguished from C. lurida by its slightly larger size, longer pygidial pubescence, and numerous erect hairs on the elytra which are absent in C. lurida (Potter 1980). Statewide Survey. A statewide survey was conducted in autumn 2007 by mailing collection kits to superintendents of 33 golf courses across Kentucky. Each kit consisted of an insulated box (SmurÞt-Stone Container, Sioux City, IA) with 30, 37-ml plastic cups, dry peat moss, a soil sample bag, and a questionnaire asking about predominant grass species in the fairways and sampled roughs. Superintendents were asked to collect the Þrst 30 grubs encountered from a heavily infested untreated rough, place grubs in individual cups with peat, include a soil sample (top 5 cm of soil) from the collection site, and return the kits with a prepaid overnight mailing label. Sampling took place during the third week of September. Golf courses from

Vol. 39, no. 6

which kits were returned (n ⫽ 25) were grouped into one of three regions: Eastern (latitude 84.06 Ð 84.49), Central (between latitudes 84.50W and 85.49W) or Western Kentucky (latitudes 85.50 Ð 88.49W). When kits arrived, the grubs were identiÞed and examined for natural enemies as described earlier. They were placed into individual plastic cups with medium and grass seed and held for 30 d as described for the seasonal survey, and then dissected and examined for pathogens as described below. Soil samples were analyzed by the University of Kentucky Regulatory Service Laboratories for pH, and percentages of organic matter, sand, silt, and clay. Assessing Natural Enemy Incidence. Parasitoid and pathogen assessments focused on Cyclocephala spp. and P. japonica grubs because larvae of other species collectively comprised ⬍4% of the grubs at all sample sites. Diagnostic symptoms were as described by Hanula and Andreadis (1988) and Cappaert and Smitley (2002). Parasitoids. Grubs were inspected for externally attached Tiphia eggs or larvae during initial macroscopic inspection and again before dissection. T. pygidialis parasitizes second- and third-instar Cyclocephala from mid-August to early October whereas T. vernalis parasitizes postoverwintered third-instar P. japonica from mid-April to early June (Rogers and Potter 2004). Tiphia cocoons with live inhabitants found in early autumn were considered as T. vernalis and in spring as T. pygidialis because adults of those respective species would have been active during the spring and autumn sampling intervals. Cocoons found with live prepupae or pupae in late autumn could not reliably be assigned to a species, and cocoons with adult emergence holes were not counted in the survey. Paenibacillus spp. DNA-Þngerprinting has shown such a close relatedness between species and strains of milky disease bacteria infecting P. japonica and other scarabs that their taxonomic separation is unclear (Dingman 2008, 2009). Therefore, we refer to them collectively as Paenibacillus spp. Grubs were examined for macrosopic symptoms of milky disease, a characteristic clouding of hemolymph by mass bacterial sporulation during the Þnal phase of the disease cycle (Klein and Jackson 1992). Hemolymph smears then were taken from all grubs (living or deceased) before dissection and examined with a phase contrast microscope at 400⫻ for Paenibacillus spp. vegetative rods and sporangia (Redmond and Potter 1995). Entomopathogenic Nematodes. A late-instar greater wax moth larva (waxworm), Galleria mellonella (L.) (New York Worms, Long Island, NY) was added to cups with dead or moribund grubs showing symptoms of nematode infection to distinguish between pathogenic and saprophytic (nonpathogenic) species. Waxworms were exposed for 7 d, then strains of nematodes were isolated by placing cadavers on modiÞed White traps (White 1927) to collect infective juveniles. To distinguish between pathogenic and saprophytic (nonpathogenic) species, third stage infective juveniles were isolated by stereoscopic observation and placed both singly and in pairs into individual wells of

December 2010


24-well culture plates (Cellstar, Monroe NC) with 2 g of moistened sterilized sand. Each well was provisioned with a waxworm, maintained in the dark at 24⬚C, and checked daily. As a waxworm died, its color was noted and it was moved to a modiÞed White trap for emergence and collection after 10 Ð14 d (Kaya and Stock 1997). Hosts infected by Steinernema or Heterorhabditis spp. are yellowish to brown, or brick red, respectively, and limp when held with forceps (Woodring and Kaya 1988). Voucher specimens were identiÞed to species by analysis of DNA markers at the University of Arizona. UnidentiÞed mermithid nematodes were isolated in the digestive tract of a few living grubs. Because their entomopathogenicity was unproven and such infection was uncommon, these incidences are not reported in the tables. Metarhizium sp. When assessing grubs for macroscopic disease symptoms, Metarhizium sp. (probably M. anisopliae) infections were evident only as green fungal conidia on the epidermis of some cadavers. No conidia were observed on live grubs. During dissection, grubs were again inspected for external conidia or conidiophores and a hemolymph smear was examined for presence of spores or hyphal bodies (Cappaert and Smitley 2002). Ovavesicula Popilliae. Malpighian tubules of dissected grubs were observed under stereoscope for white callus-like growths caused by this microsporidian. Infection was conÞrmed by examining tubule mounts in normal saline at 400⫻ with a phase contrast microscope for presence of sporophorous vesicles (Andreadis and Hanula 1987). Stictospora sp. (Probably S. villani). Mid- and hindguts of dissected grubs were opened and examined for presence of gamonts of this gregarine (Cappaert and Smitley 2002, Hays et al. 2004). Larger gamonts were visible through the dorsal abdominal epidermis of infected grubs even before dissection. Serratia spp. During dissection, a hemolymph smear from each grub was tested for Serratia spp. using a three-step typing system based on the use of selective agars (OÕCallaghan and Jackson 1993, Jackson et al. 1993, Klein 1997). In step one, a hemolymph smear was placed on caprylate-thallous agar (CTA) media which allows Serratia spp. to grow, while repressing other microorganisms (Starr et al. 1976). If present, creamcolored (positive) isolates from the CTA agar were then transferred to DNase agar upon which Serratia spp. develop a pink halo around the whitish, growing colonies after 24 h incubation at 30⬚C. Finally, individual such colonies were spotted onto adonitol agar, upon which S. entomophila produces yellow colonies, and S. proteamaculans produces blue/green colonies (Klein 1997). In addition, the soil samples taken at each site were tested for Serratia spp. using the CTA assay. Statistical Analysis. Relative proportions of Cyclocephala spp. and P. japonica grubs in samples from the August 2006 and seasonal surveys were tested withinsite against the null hypothesis of a 1:1 ratio (twotailed Z-tests; P ⫽ 0.05). For the seasonal survey, grub

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Table 2. Density and composition of scarab grubs in independent samples from different spots in the same 18 irrigated roughs at six central Kentucky golf courses; Seasonal survey, 2007–2008 Sampling dates

Densitya

Late Aug. Mid Sept. Early Oct. Early May

20.4 ⫾ 2.0 a 9.8 ⫾ 0.7 b 6.3 ⫾ 0.8 c 2.8 ⫾ 0.8 d

Grub type (% of total)b MC

JB

Other

74.9 ⫾ 5.9 66.7 ⫾ 4.0 65.9 ⫾ 4.9 44.8 ⫾ 6.0

24.2 ⫾ 5.6 32.3 ⫾ 4.1 27.1 ⫾ 4.6 45.9 ⫾ 6.2

0.9 ⫾ 0.6 1.1 ⫾ 0.6 7.0 ⫾ 2.1 9.3 ⫾ 4.0

a Mean grubs per 0.1 m2; F ⫽ 49.7; df ⫽ 3, 15; P ⬍0.01. Means not followed by the same letter differ signiÞcantly (LSD, P ⬍0.05). b MC, masked chafers (Cyclocephala spp); JB, Japanese beetle (P. japonica); Other includes Phyllophaga spp. and a few C. nitida. See text for statistical analysis of proportions.

densities (per 0.1 m2) and percentages of grubs parasitized or infected by particular pathogens were compared between the independent sample dates by twoway analysis of variance (ANOVA). Samples from the three sites on each golf course were averaged and analyzed for seasonal differences by two-way ANOVA using courses as replicates. For the statewide survey, incidence of normal and diseased grubs was compared between samples from eastern, central, and western Kentucky golf courses by one-way ANOVA, with relative proportions that were Cyclocephala spp. or P. japonica compared between regions by a ␹2 test for heterogeneity. Spearman rank correlations were done to test for relationships between soil parameters as independent variables, and grub density, relative percentage of masked chafer or Japanese beetle grubs, and percentage infection by particular pathogens as independent variables. Percentages were arcsine square root-transformed, and all analyses were done with Statistix 9.0 (Analytical Software 2008). Data are presented as original means ⫾ SE. Results Autumn 2006 Survey. Cyclocephala spp. and P. japonica accounted for 73.2 and 26.4%, respectively (99.6% collectively), of the 2166 grubs sampled from the 18 irrigated golf course roughs on six central Kentucky golf courses. Only a few Phyllophaga spp. were found. Mean grub density across all sites was 13.9 ⫾ 1.7 per 0.1 m2 (range, 7.5Ð36.0). Cyclocephala were proportionately more abundant than P. japonica at Þve of the six courses, and at 15 of the 18 individual sample sites (Table 1). Seasonal Survey. Grub densities in irrigated roughs declined from late August though early October, and from October to May (Table 2). Cyclocephala were signiÞcantly more abundant than P. japonica throughout the autumn. The proportion of masked chafers to Japanese beetle grubs, pooled across sites, declined from October to May (␹2 ⫽ 41.0; df ⫽ 1; P ⬍ 0.001). Masked chafers were still proportionately more abundant in the early October samples from nine of the 18 roughs; with equal proportions at eight sites, and P. japonica predominating at just one site (Z-tests, P ⱕ 0.05; individual site data not shown). Phyllophaga spp.

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Table 3. Seasonal incidence of natural enemies of masked chafer (Cyclocephala spp.) and Japanese beetle (P. japonica) larvae in grub-damaged areas of 18 irrigated roughs at six central Kentucky golf courses, 2007–2008 Sample dates Cyclocephala spp. grubs Late Aug. Mid Sept. Early Oct. Early May Popillia japonica grubs Late Aug. Mid Sept. Early Oct. Early May

Percentage of grubs infected or parasitized bya,b

No. grubs examined

Pan

Ser

Meta

Stic

Ova

Hb

Tiphia

Normal

337 298 290 142

15.9 ⫾ 4.1a 19.0 ⫾ 3.0a 17.8 ⫾ 3.7a 2.6 ⫾ 1.3b

6.9 ⫾ 1.5a 5.8 ⫾ 1.3a 6.4 ⫾ 1.4a 0.0 ⫾ 0.0b

1.1 ⫾ 0.5 1.0 ⫾ 0.6 2.9 ⫾ 0.9 2.1 ⫾ 2.1

0.0 ⫾ 0.0 0.0 ⫾ 0.0 0.0 ⫾ 0.0 2.6 ⫾ 1.8

0⫾0 0⫾0 0⫾0 0⫾0

0⫾0 0⫾0 0⫾0 0⫾0

6.2 ⫾ 2.2 3.1 ⫾ 1.3 5.3 ⫾ 2.2 3.3 ⫾ 2.5

69.8 ⫾ 4.5a 71.2 ⫾ 4.0a 67.5 ⫾ 3.3a 83.1 ⫾ 7.2b

109 142 119 158

13.7 ⫾ 4.6 8.3 ⫾ 2.6 7.6 ⫾ 3.2 3.6 ⫾ 1.7

2.2 ⫾ 1.3ab 5.4 ⫾ 2.0ab 10.2 ⫾ 5.5a 0.0 ⫾ 0.0b

6.3 ⫾ 3.5 4.6 ⫾ 2.9 5.3 ⫾ 2.4 0.8 ⫾ 0.4

0.0 ⫾ 0.0a 0.0 ⫾ 0.0a 0.0 ⫾ 0.0a 20.4 ⫾ 5.5b

0.0 ⫾ 0.0 3.3 ⫾ 2.8 0.0 ⫾ 0.0 0.3 ⫾ 0.3

0.0 ⫾ 0.0 0.0 ⫾ 0.0 0.0 ⫾ 0.0 0.3 ⫾ 0.3

0.0 ⫾ 0.0a 0.0 ⫾ 0.0a 0.0 ⫾ 0.0a 19.7 ⫾ 8.8b

77.8 ⫾ 6.3a 78.5 ⫾ 5.6a 76.9 ⫾ 6.1a 49.8 ⫾ 7.9b

a Pan ⫽ Paenibacillus spp., Ser ⫽ Serratia spp., Meta ⫽ Metarhizium sp., Stic ⫽ Stictospora sp., Ova ⫽ Ovavesicula popilliae, Hb ⫽ Heterorhabditis bacteriophora, Tiphia ⫽ Tiphia pygidialis on Cyclocephala or T. vernalis on P. japonica. b Separate analyses were run for Cyclocephala or P. japonica. Within columns, means not followed by the same letter differ signiÞcantly (one-way ANOVA, LSD; P ⬍0.05; except for variables having all zeroes on some dates, for which the nonparametric Kruskal-Wallis test was used). Absence of letters after means indicates no signiÞcant differences.

and C. nitida collectively accounted for only 1% of the grubs in samples from late August and mid-September, and 7.0 ⫾ 2.1 and 9.3 ⫾ 4.0% in the early October and early May samples, respectively. The cohorts of 25 Cyclocephala spp. grubs reared from each golf course yielded similar proportions of C. lurida and C. borealis adults with both species represented in samples from each course (means 9.2 ⫾ 1.7 vs. 7.5 ⫾ 1.7, respectively; t ⫽ 0.98, df ⫽ 5; P ⫽ 0.37). Percentages of Cyclocephala spp. grubs infected by Paenibacillus or Serratia differed between sample dates (F ⫽ 4.74, 6.49, respectively; df ⫽ 3, 65; P ⬍ 0.01), with higher incidence in late summer and autumn than in the following spring (Table 3). Together those pathogens infected ⬇25% of the masked chafer grubs sampled in mid-September. Low levels (⬍3%) of Metarhizium sp. infection were present on all sample dates, whereas Stictospora sp., also at low levels, was only found in postoverwintered grubs. No entomopathogenic nematodes or Ovavesicula sp. were found in Cyclocephala grubs. In addition, ⬇6% of the Cyclocephala spp. sampled in late August had been parasitized by T. pygidialis, and cocoons containing T. pygidialis overwintering prepupae were found on later sample dates. Although Cyclocephala grub densities in the sampled areas of roughs declined ⬎90% from August to early May, the percentage of normal grubs (i.e., not infected or parasitized) was highest in the postoverwintered population (F ⫽ 16.5; df ⫽ 3, 65; P ⬍ 0.01; Table 3). Paenibacillus, Serratia, and Metarhizium spp. were the most abundant pathogens infecting P. japonica grubs in late summer and autumn (Table 3). Infection rates at the golf course level ranged from 0 to 33% for Paenibacillus, 0 Ð22% for Metarhizium, and 0 Ð 41% for Serratia spp. on one or more of those sample dates. O. popilliae was uncommon or absent on all sample dates, and out of 528 total Japanese beetle grubs autopsied, only a single larva from the May sample contained entomopathogenic nematodes (conÞrmed as H. bacteriophora). Stictospora sp., which was not detected in late summer and autumn, was the predominant patho-

gen in postoverwintered P. japonica grubs, infecting on average ⬇20% of the third instars sampled in May. In addition, T. vernalis had also parasitized nearly 20% of the grubs sampled in early May. There was a signiÞcant decline in percentage of normal P. japonica grubs from October to early May (F ⫽ 16.5; df ⫽ 3, 65; P ⬍ 0.001; Table 3). Soil characteristics varied across the 18 sites sampled for the seasonal survey. Mean ⫾ SE (range) for soil pH and percentage organic matter were 6.5 ⫾ 0.1 (5.3Ð7.1) and 7.0 ⫾ 0.4 (4.7Ð11.3), respectively; and percentages of sand, silt, and clay were 24.1 ⫾ 1.1 (17.4 Ð35.3), 62.7 ⫾ 1.4 (49.6 Ð71.2), and 13.3 ⫾ 0.9 (8.2Ð21.4), respectively. Except for soil clay content, which was negatively correlated with proportionate abundance of P. japonica in a given sample (Spearman rank correlation, r ⫽ ⫺0.51; P ⫽ 0.03), no soil parameter was correlated with predominance of either Cyclocephala spp. or P. japonica at a given site, or with percentage infection by particular pathogens (Spearman rank correlations, P ⬎ 0.05). Statewide Survey. Seven of the 10 eastern region golf courses from which samples were returned had cool season grasses in the fairways, six of the nine central Kentucky courses had warm season grass (mostly Zoysia sp.) fairways, and all six western region courses had Bermuda grass (Cynodon sp.) fairways (Table 4). Cyclocephala grubs predominated on most golf courses (Table 4). Overall, Cyclocephala spp., P. japonica, and Phyllophaga spp. accounted for 64.2, 29.4, and 6.4% of the 732 total grubs received, and overall proportions of each grub type did not differ between pooled samples from courses having cool- or warm season grass fairways (␹2 ⫽ 2.0; df ⫽ 2; P ⫽ 0.37). The percentage of grubs that were P. japonica did not differ between samples from eastern central, or western Kentucky golf courses (F ⫽ 1.34; df ⫽ 3, 22; P ⫽ 0.28). Milky disease bacteria (Paenibacillus spp.) were the most frequent pathogens in the September-collected grubs in the statewide survey (Table 5). Milky disease infection ranged from 0 to 38% for Cyclocephala spp. and from 0 to 33% for P. japonica across the 25 sample sites.

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Table 4. Grass type in fairways and relative abundance of masked chafer (MC), Japanese beetle (JB), and May beetle (MB) in 30-grub samples collected from grub-damaged irrigated roughs of 25 golf courses in the Kentucky statewide survey Course

Fairwaya

East

London Winchester CC Gibson Bay Houston Oaks Arlington AJ Jolly GrifÞn Gate Spring Valley Eagles Nest Lakeview Springs Regional totals

Central

West

Region

Type of grubsb MC

JB

MB

% MC

P

PR B CB CB Z CB/PR PR PR B PR

28 20 15 28 15 29 6 16 12 16 185

0 6 9 0 12 1 23 13 12 6 82

2 2 5 2 2 0 0 1 6 4 24

93 71 52 93 52 97 21 53 40 61 63.6

⬍0.001 0.01 0.3 ⬍0.001 0.7 ⬍0.001 0.005 0.7 1.0 0.06 0.001

Cardinal Maywood Polo Fields Oldham County Owl Creek Harmony Landing Hunting Creek Big Spring Audubon Regional totals

CB B CB Z Z PR Z Z Z

11 25 16 25 10 21 30 29 28 195

17 2 14 2 19 9 0 0 0 63

1 2 0 3 0 0 0 0 2 8

38 86 53 83 34 70 100 100 93 73.3

0.34 ⬍0.001 0.86 0.001 0.13 0.04 ⬍0.001 ⬍0.001 ⬍0.001 0.001

Elizabethtown Lindsey Rolling Hills Russellville Summit Silos Regional totals

B B B B B B

15 26 20 22 7 0 90

9 4 10 8 12 27 70

5 0 0 0 10 0 15

52 86 67 73 24 0 51.4

0.3 ⬍0.001 0.1 0.02 0.4 ⬍0.001 0.13

a

Fairway grass species as reported by superintendent: PR, perennial ryegrass; CB, creeping bentgrass; B, bermudagrass; Z, zoysiagrass. MC, JB, MB are masked chafer, Japanese beetle, or May beetle (Phyllophaga spp.), respectively. P value for departure from equal proportions of MC and JB at a given site (Z-tests). b

Despite trends for higher incidence of milky disease (F ⫽ 2.51; df ⫽ 2, 17; P ⫽ 0.11) and lower overall pathogen infection of P. japonica grubs in samples from western Kentucky (F ⫽ 2.82; df ⫽ 2, 17; P ⫽ 0.09), no pathogen was signiÞcantly more or less prevalent in samples from a particular region (Table 5). Metarhizium and Serratia spp. were present in similarly low levels in both Cyclocephala spp. and P. japonica grubs from all three regions. Ovavesicula, entomopathogenic nematodes, and Stictospora were not detected in grub samples from the statewide survey. A few of the masked chafer grubs sent in by

superintendents had attached larvae of T. pygidialis. T. vernalis attacks overwintered third-instar P. japonica in spring so its larvae would not have been present during the autumn seasonal survey. Tiphia popilliavora (Rohwer), an introduced parasitoid that attacks P. japonica grubs in late summer, was not found in any of our surveys and is not known to be established in Kentucky. Soil characteristics varied widely from site to site. Means ⫾ SE (range) for soil pH and percentage organic matter were 6.3 ⫾ 0.1 (5.2Ð7.6) and 4.8 ⫾ 0.4 (2.2Ð9.8), respectively. Mean (range) percentages of

Table 5. Natural enemy presence in 30-grub autumn samples sent in by superintendents of 22 Kentucky golf courses (8, 8, and 6 courses, respectively, in the Eastern, Central, and Western regions) in the Statewide survey, 2007 Region Cyclocephala spp. grubs Eastern Central Western Popillia japonica grubs Eastern Central Western

Percentage of grubs infected or parasitized bya

Total no. dissected

Pan

Meta

Ser

Hb

Ova

Stic

Tiphia

Normal

185 195 90

21.3 ⫾ 4.1 22.4 ⫾ 4.9 17.0 ⫾ 1.3

4.4 ⫾ 1.6 4.3 ⫾ 2.1 4.7 ⫾ 2.7

3.4 ⫾ 1.2 3.8 ⫾ 1.3 3.9 ⫾ 2.8

0⫾0 0⫾0 0⫾0

0⫾0 0⫾0 0⫾0

0⫾0 0⫾0 0⫾0

0 ⫾ 0.0 1.0 ⫾ 1.0 0.8 ⫾ 0.8

70.9 ⫾ 4.0 68.4 ⫾ 6.2 73.7 ⫾ 4.3

82 63 70

5.6 ⫾ 2.5 5.7 ⫾ 4.0 17.1 ⫾ 5.1

2.4 ⫾ 1.6 3.4 ⫾ 2.3 4.2 ⫾ 4.2

1.9 ⫾ 1.4 1.9 ⫾ 1.9 3.1 ⫾ 1.9

0⫾0 0⫾0 0⫾0

0⫾0 0⫾0 0⫾0

0⫾0 0⫾0 0⫾0

NA NA NA

90.1 ⫾ 2.8 89.1 ⫾ 4.4 75.6 ⫾ 6.3

a Pan ⫽ Paenibacillus spp., Ser ⫽ Serratia spp., Meta ⫽ Metarhizium sp., Stic ⫽ Stictospora sp., Ova ⫽ Ovavesicula popilliae, Hb ⫽ Heterorhabditis bacteriophora, Tiphia ⫽ T. pygidialis on Cyclocephala; NA indicates that T. vernalis was not active at the time of sampling. There were no signiÞcant regional differences in enemy incidence for either Cyclocephala spp. or P. japonica grubs (one-way ANOVA or Kruskal-Wallis tests, P ⬎ 0.05).

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sand, silt, and clay were 29.0 ⫾ 4.7 (8.4 Ð91.6), 60.4 ⫾ 4.3 (5.6 Ð79.5), and 10.6 ⫾ 0.8 (2.9 Ð18.5), respectively. There were no overall regional differences for any soil parameter (one-way ANOVA, P ⱖ 0.2). None of the soil parameters was signiÞcantly correlated with proportionate abundance of Cyclocephala spp., P. japonica, or Phyllophaga spp. grubs, incidence of particular pathogens, or overall percentage of infected or parasitized grubs in the samples (Spearman rank correlations, P ⱖ 0.05). Discussion This study, the Þrst of its type done in the transitional climatic zone, conÞrmed native Cyclocephala spp. as the white grubs most often associated with turf damage on Kentucky golf courses. In samples from grub-damaged areas of 61 irrigated roughs on 32 courses, masked chafers predominated at 37 sites, Japanese beetles predominated at Þve sites, and similar proportions of both were found at 19 sites. Cyclocephala spp., once thought to feed more on soil organic matter and therefore to be relatively less damaging than other scarab species (Tashiro 1987), are in fact larger than P. japonica and, grub for grub, consume more grass roots (Potter et al. 1992, CrutchÞeld and Potter 1995). Both C. borealis and C. lurida are abundant in central Kentucky, often at the same sites (Potter 1980, Haynes and Potter 1995). Phyllophaga spp. grubs, which are turf pests especially in the southern United States (Doskocil et al. 2008), were never found at high enough density to damage turf on Kentucky golf courses. Asiatic garden beetle, Maladera castanea Arrow, European chafer, Rhizotrogus majalis (Razoumowsky), and oriental beetle, Anomala orientalis (Waterhouse), important exotic turf pests in the northeast United States, were not found in this study and are not known to be established in Kentucky. Ritcher (1940), who sampled mostly in pastures and agricultural land, reported Phyllophaga spp. to be the predominant white grubs in Kentucky with only passing mention of C. lurida (formerly C. immaculata) and C. borealis as potential pests of urban sod. Since the 1940s, large expanses of farmland have been developed as managed turf which, at least in Kentucky, seems to have been accompanied by a shift in white grub species dominance from Phyllophaga with 2Ð3 yr life cycles to annual grub species. Although P. japonica was present in Kentucky as early as 1943 (Hawley and Dobbins 1945), it had not become widespread until the 1970s when it was reported in 68 of 120 counties (Cooperative Agricultural Pest Survey, unpublished data). No predictive relationships were found between soil parameters and the proportionate representation of Cyclocephala spp. or P. japonica at particular sites. Both types of grubs were found in soils ranging widely in pH, texture, and percentage organic matter. Dalthorp et al. (2000) also found no consistent relationships between soil variables and density of larval P. japonica at sites on a single intensively sampled golf course and, in pot studies, such grubs survived equally

Vol. 39, no. 6

well across the range of soil pH at which turfgrasses are grown (Vittum and Tashiro 1980). Numerous factors including soil moisture, proximity to adult food plants for P. japonica (Cyclocephala adults do not feed), propensity of adults to disperse from natal sites, and past history of insecticide usage inßuence spatial distributions of larval scarabs (e.g., Potter 1983, Villani and Wright 1990, Potter et al. 1996, Dalthorp et al. 2000). On a given golf course, grub density and species often markedly differ over a few metersÕ distance despite no obvious difference in grass cover or soil characteristics (Dalthorp et al. 2000, C.T. Redmond and D.A. Potter, personal observations). Pathogens associated with P. japonica in Kentucky are similar to those found in Connecticut (Hanula and Andreadis 1988) but more diverse than in southern Michigan (Cappaert and Smitley 2002). No new genera of pathogens were discovered. Differences in methodology between the surveys preclude any statistical comparison of pathogen loads between the respective states. In Connecticut, sample sites included town parks as well as golf courses and the same area of turf (0.56 m2) was examined at each site regardless of the number of grubs present. The southern Michigan survey compared older (⬎20 yr) populations and more recently infested areas (⬍15 yr) and like ours, focused on irrigated golf course roughs with relatively high grub densities. Hanula and Andreadis (1988) found Stictospora sp. (probably S. villani) at 42 of their 49 sample sites, with 54 Ð100% infection rates, and O. popilliae at 34 locations, with 25% overall infection, so those pathogens appear to be much more prevalent in P. japonica in Connecticut than in Kentucky. In contrast, estimated statewide infection by Paenibacillus (3.5%), Metarhizium (1.2%), and Serratia (0%) was lower in Connecticut than in Kentucky. Except for Stictospora sp., found in 36% of the sampled grubs, pathogens were scarce or absent in southern Michigan (Cappaert and Smitley 2002) where O. popilliae, Paenibacillus spp., and nematodes were absent at all but a few sites (⬍1% overall incidence), and no Serratia or Metarhizium were found. In all three surveys, pathogen incidence was highly variable from site to site. Infection of P. japonica by native pathogens (e.g., Ovavesicula, Serratia, and Stictospora) suggests those agents evolved in association with native North American grub species but have the ability to attack the invasive host. Serratia isolates have previously been recovered from scarab larvae, including P. japonica and Cyclocephala spp., in the United States (Klein and Kaya 1995). Although the ones isolated from grubs in our surveys matched the appearance of S. entomophila based on typing on selective agars (OÕCallaghan and Jackson 1993, Klein 1997), conÞrming their identity requires taxonomic and molecular characterization and is beyond the scope of this study. S. entomophila was known only from the grass grub Costelytra zealandica (White) in New Zealand pastures (Jackson et al. 1993) until recent identiÞcation of a putative Mexican strain pathogenic to several species of Phyllophaga larvae (Nun˜ ez-Valdez et al. 2008).

December 2010


When P. japonica expands its geographical range it tends to initially attain very high population densities, followed by decline and stabilization over the next 10 Ð20 yr (Fleming 1972). That pattern has been attributed to buildup of natural enemies facilitated by the initially dense host population (Fleming 1968, 1972). Cappaert and Smitley (2002) cited the paucity of enemies except for Stictospora sp. in southern Michigan compared with Connecticut, and higher incidence of Stictospora in southernmost Michigan counties where the beetle has been established longer than farther north in Michigan, as supporting FlemingÕs (1968) stabilization-by-enemies hypothesis. We found, however, some natural enemies to apparently be more prevalent in Kentucky than previously reported in Connecticut, and no differences between eastern and central Kentucky as opposed to the morerecently colonized western portion of the state. Regional climatic factors doubtless also affect the dynamics of natural enemy buildup in P. japonica after range expansions. C. borealis and C. lurida are sympatric in Kentucky, sexually cross-attractive as adults (Potter 1980, Haynes and Potter 1995), and equally vulnerable to parasitism by T. pygidialis (Rogers and Potter 2004) so they probably are susceptible to the same soil-dwelling pathogens. Naturally occurring Paenibacillus spp. (Warren and Potter 1982) were the predominant pathogen of Cyclocephala in our surveys, infecting ⬇18 Ð20% of third instars with milky disease in late summer and autumn. Serratia and Metarhizium collectively infected ⬇8% of third instars in autumn but other pathogens (Stictospora, Ovavesicula, entomopathogenic nematodes) were scarce or absent. No previously unreported pathogens of Cyclocephala spp. were discovered. Almost no information is published on natural enemies of C. lurida or C. borealis from states other than Kentucky so it is not known if our Þndings are typical across those speciesÕ wide distribution in the United States. The near-absence of entomopathogenic nematodes in samples of Kentucky white grubs is consistent with very low (0 and 0.3%) total infection rates reported in P. japonica in Connecticut and southern Michigan (Hanula and Andreadis 1988, Cappaert and Smitley 2002). None of the 2,326 total grubs autopsied in our seasonal and statewide surveys showed macroscopic symptoms of nematode infection at the time they were collected. It is unlikely that pathogenic nematodes completing their development before cadavers were autopsied were missed because few grubs (⬍3%) died during holding period and in those cases no such nematodes were found either in the still-moist cadavers or medium. Most nematodes found in association with grubs held for disease expression and autopsy were subsequently conÞrmed to be saprophytic species associated with cadavers of hosts killed by other agents. Our procedures for isolating pathogenic nematodes would not have detected nematodes that did not produce progeny (e.g., because only one male or female nematode of an amphimictic species infected, or for which the infection and ensuing development

1845

were not completed), or potentially grub-virulent strains that do not do well in wax moth larvae. Those scenarios are unlikely, however, given the absence of nematode-suspicious grubs other then the few H. bacteriophora infected ones accounted for. Although surveys of this type underestimate generational mortality because only recently infected grubs or nondegraded cadavers can be observed, nematodes seem to inßict relatively little mortality on grub populations on Kentucky golf courses. T. vernalis, which was introduced into the eastern United States in the 1920s, was found at most sample sites conÞrming that it is a well-established enemy of P. japonica in Kentucky. Parasitism rates as high as 58% have been observed at some central Kentucky sites (Rogers and Potter 2004). T. popilliavora was not found in our surveys. I. aldrichi, which parasitizes adult Japanese beetles, would not be detected in larval samples but, like T. popilliavora, we have not observed it in ⬎30 yrÕ research on Japanese beetles in Kentucky. It is established in New England (Vittum et al. 1999) but apparently not in Michigan (Cappaert and Smitley 2002). T. pygidialis, the only reported parasitoid of turfinfesting Cyclocephala spp. in the United States (Rogers and Potter 2004), was probably underrepresented in our statewide survey because it induces its host to burrow down 12Ð16 cm into the soil (Rogers et al. 2003), deeper than cooperating superintendents would have sampled. Parasitism rates of 33% or more have been documented at some sites (Rogers and Potter 2004). Nothing is published on the distribution of T. pygidialis or its impact on Cyclocephala spp. except in Kentucky, but as a native species it probably is widespread in the central United States where its hosts are abundant. Surveys of this type provide point-in-time estimates of infection or parasitism and unavoidably underestimate natural enemiesÕ collective lifetime impact on long-lived hosts such as white grubs. Many pathogens, for example, cause infected hosts to stop feeding which depletes fat bodies and reduces overwintering capability (Jackson et al. 1993, Giddens et al. 2000). In our samples, pathogen infection was highest in autumn and grub densities markedly declined from autumn to spring, suggesting many infected grubs failed to overwinter. Furthermore, multiple agents may attack the same grub, and in some cases infection by one pathogen increases the hostÕs susceptibility to others (e.g., Thurston et al. 1994). Surveys also fail to account for sub-lethal pathogen effects on host reproduction. Infection by O. popilliae, for example, can delay oviposition and reduce Japanese beetle fecundity by 50% (Hanula 1990). Although it has been suggested, based on its ubiquity in Connecticut, that S. villani is avirulent (Hanula and Andreadis 1988), we have found Stictospora-infected, postoverwintered P. japonica to be ⬇50% less likely to develop to adulthood (Redmond 2010). Sub-lethal exposure to pesticides can compromise grubsÕ defense against pathogens (Koppenho¨ fer et al. 2000). Therefore, while the impact of individual natural enemies may be relatively small at

1846


a given time, collectively over the 10 Ð11 mo that their hosts develop in the soil such agents may signiÞcantly impact white grub populations. Acknowledgments We are grateful to the Kentucky golf course superintendents who allowed us access to study sites and cooperated in the sampling, and to C. Brady and J. Condra for many hours spent dissecting grubs. We also thank P. S. Grewal (The Ohio State University) for advice about nematode isolation, and P. Stock (University of Arizona) for polymerase chain reaction (PCR) conÞrmation of their identiÞcations. This work was supported by a grant from the United States Golf Association Turfgrass and Environmental Research Program. This is paper no. 10-08-85 of the Kentucky Agricultural Experiment Station.

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Hawley, I. M., and T. N. Dobbins. 1945. The distribution and abundance of the Japanese beetle from 1935 through 1943, with a discussion of some of the known factors that inßuence its behavior. J.N.Y. Entomol. Soc. 53: 1Ð20. Haynes, K. F., and D. A. Potter. 1995. Chemically mediated sexual attraction of male Cyclocephala lurida (Coleoptera: Scarabaeidae) and other scarabaeid beetles to immature stages. Environ. Entomol. 24: 1302Ð1306. Hays, J., R. E. Clopton, D. L. Cappaert, and D. R. Smitley. 2004. Revision of the genus Stictospora and description of Stictospora villani, N. Sp. (Apicomplexa: Eugregarinida: Actinocephalidae) from the larvae of the Japanese beetle, Popillia japonica (Coleoptera: Scarabaeidae) in Michigan. J. Parasitol. 90: 1450 Ð1456. Jackson, T. A., A. M. Huger, and T. R. Glare. 1993. Pathology of amber disease in the New Zealand grass grub Costelytra zealandica (Coleoptera: Scarabaeidae). J. Invertebr. Pathol. 61: 123Ð130. Kaya, H. K., and S. P. Stock. 1997. Techniques in insect nematology, pp. 281Ð324. In L. Lacey (ed.), Manual of Techniques in Insect Pathology. Academic, New York. Kaya, H. K., M. G. Klein, T. M. Burlando, R. E. Harrison, and L. A. Lacey. 1992. Prevalence of two Bacillus popilliae Dutky morphotypes and blue disease in Cyclocephala hirta Leconte (Coleoptera: Scarabaeidae) populations in California. Pan-PaciÞc Entomol. 68: 38 Ð 45. Klein, M. G. 1995. Microbial control of turfgrass insects, pp. 95Ð100. In R. L. Brandenburg and M. G. Villani (eds.), Handbook of Turfgrass Insect Pests. Entomological Society of America, Lanham, MD. Klein, M. G. 1997. Bacteria of soil-inhabitating insects, pp. 101Ð116. In L. Lacey (ed.), Manual of Techniques in Insect Pathology. Academic, New York. Klein, M. G., and T. A. Jackson. 1992. Bacterial diseases of scarabs, pp. 43Ð 61. In T. A. Jackson and T. R. Glare (eds.), Use of Pathogens in Scarab Pest Management. Intercept, Andover, United Kingdom. Klein, M. G., and H. K. Kaya. 1995. Bacillus and Serratia species for scarab control. Mem. Inst. Oswaldo Cruz. 90: 87Ð95. Koppenho¨ fer, A. M., P. S. Grewal, and H. K. Kaya. 2000. Synergism of entomopathogenic nematodes and imidacloprid against white grubs: the mechanism. Entomol. Exp. Applic. 94: 283Ð293. Nun˜ ez-Valdez, E., M. A. Caldero´ n, E. Aranda, L. Herna´ ndez, R. M. Ramı´rez-Gama, L. Lina, Z. Rodrı´guez-Segura, M. del C. Gutie´rrez, and F. J. Villalobos. 2008. IdentiÞcation of a putative Mexican strain of Serratia entomophila pathogenic against root-damaging larvae of Scarabaeidae (Coleoptera). Appl. Environ. Microbiol. 74: 802Ð 810. O’Callaghan, M., and T. A. Jackson. 1993. Isolation and enumeration of Serratia entomophila: a bacterial pathogen of the New Zealand grass grub, Costelytra zealandica. J. Appl. Microbiol. 75: 307Ð314. Potter, D. A. 1980. Flight activity and sex attraction of northern and southern masked chafers in Kentucky turfgrass. Ann. Entomol. Soc. Am. 73: 414 Ð 417. Potter, D. A. 1983. Effect of soil moisture on oviposition, water absorption, and survival of masked chafer eggs. Environ. Entomol. 12: 1223Ð1227. Potter, D. A. 1998. Destructive turfgrass insects: biology, diagnosis, and control. Ann Arbor Press, Chelsea, MI. Potter, D. A., C. G. Patterson, and C. T. Redmond. 1992. Feeding ecology of Japanese beetle and southern masked chafer grubs (Coleoptera: Scarabaeidae): Inßuence of turfgrass species and tall fescue endophyte. J. Econ. Entomol. 85: 900 Ð909.

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Tashiro, H. 1987. Turfgrass insects of the United States and Canada. Cornell University Press, Ithaca, NY. Thurston, G. S., H. K. Kaya, and R. Gaugler. 1994. Characterizing the enhanced susceptibility of milky diseaseinfected scarabaeid grubs to entomopathogenic nematodes. Biol. Control. 4: 67Ð73. (USDA) U.S. Department of Agriculture-ARS Germplasm Resources Information Network (GRIN). 2010. Washington DC: U.S. Department of Agriculture-ARS. (www. ars-grin.gov/cgi-bin/npgs/html/tax search.pl). Villani, M. G., and R. J. Wright. 1990. Environmental inßuences on soil macroarthropod behavior in agricultural systems. Annu. Rev. Entomol. 35: 249 Ð269. Vittum, P. J., and H. Tashiro. 1980. Effect of soil pH on survival of Japanese beetle and European chafer larvae. J. Econ. Entomol. 73: 577Ð579. Vittum, P. J., M. G. Villani, and H. Tashiro. 1999. Turfgrass insects of the United States and Canada, 2nd ed. Cornell University Press, Ithaca, NY. Warren, G. W., and D. A. Potter. 1982. Pathogenicity of Bacillus popilliae (Cyclocephala strain) and other milky disease bacteria in grubs of the southern masked chafer (Coleoptera: Scarabaeidae). J. Econ. Entomol. 76: 69 Ð74. White, G. F. 1927. A method for obtaining infective nematode larvae from cultures. Science 66: 302Ð303. Woodring, J. L., and H. K. Kaya. 1988. Steinernematid and heterorhabditid nematodes: a handbook of techniques. Southern Cooperative Series Bulletin. 331. Arkansas Agricultural Experiment Station, Fayetteville, AK. Received 6 July 2010; accepted 28 September 2010.