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Biological Control 44 (2008) 207–215 www.elsevier.com/locate/ybcon

Control of plum curculio, nenuphar, with entomopathogenic nematodes: Effects of application timing, alternate host plant, and nematode strain

David I. Shapiro-Ilan a,*, Russell F. Mizell III b, Ted E. Cottrell a, Dan L. Horton c

a USDA-ARS, SE Fruit and Tree Nut Research Laboratory, 21 Dunbar Road, Byron, GA 31008, USA b Department of Entomology, University of Florida, Quincy, FL, USA c Department of Entomology, University of Georgia, Athens, GA, USA

Received 30 May 2007; accepted 28 July 2007 Available online 11 August 2007

Abstract

In prior research, we established that soil applications of Steinernema riobrave in orchards that target plum curculio, Conotra- chelus nenuphar, larvae can result in high levels of control (78–100%). In this study, we investigated the potential of using entomopath- ogenic nematodes to control C. nenuphar further by evaluating (1) field efficacy of late-term nematode applications, i.e., that target C. nenuphar after the onset of adult emergence, (2) control C. nenuphar larvae in an alternate host, i.e., wild plum, Prunus angustifolia, and (3) laboratory virulence of different Steinernema carpocapsae and S. riobrave strains. In 2004 and 2005, late-term applications of S. carpocapsae (All strain) and S. riobrave (355 strain) in a peach orchard in Quincy, Florida failed to cause any measurable control. In field tests targeting C. nenuphar larvae in a wild plum thicket (Byron, Georgia), S. riobrave (355 strain) caused 100% control (both years), and S. riobrave (3-8b strain) caused 98.6% and 87.9% control in 2005 and 2006, respectively. Applications in alternate hosts could reduce C. nenuphar immigration into the orchard. Laboratory experiments detected differential virulence to C. nenuphar larvae in S. carpocapsae (All strain) and six strains of S. riobrave; among the most virulent were the 3-2, 3-3, and 355 strains of S. riobrave. Path- ogenicity to adult C. nenuphar in the laboratory was not detected in five S. riobrave strains, but was detected in four S. carpocapsae strains (All, Agriotos, Italian and Sal) but not in two others (Kapow and Mexican). We conclude that soil applications with entomo- pathogenic nematodes can cause high levels of C. nenuphar control in peach or alternate hosts, but timing of application and the choice of nematode species or strain can be important. Published by Elsevier Inc.

Keywords: Biological control; Conotrachelus nenuphar; Entomopathogenic nematode; Peach; Plum; Steinernema

1. Introduction fallen fruit, exit as fourth instars, and burrow into the soil (1–8 cm) to pupate (Racette et al., 1992). After emergence, The plum curculio, Conotrachelus nenuphar (Herbst), is adults feed on fruit and migrate to litter in or surrounding a major pest of stone and pome fruit in North America the orchard to overwinter (Racette et al., 1992; Olthof and (Racette et al., 1992). Adults weevils enter trees in the orch- Hagley, 1993). In the southern United States, an additional ard from overwintering sites in the spring, feed, and ovi- generation may occur on many Prunus species prior to posit in fruit. Attacked fruit aborts or is deformed overwintering (Horton et al., 2006). rendering it non-saleable. Larvae continue to develop in Current control recommendations for C. nenuphar con- sist solely of above-ground applications of chemical insec- ticides to suppress adults (Be`lair et al., 1998; Olthof and * Corresponding author. Fax: +1 478 956 2929. Hagley, 1993; Horton et al., 2006). Due to environmental E-mail address: [email protected] (D.I. Shapiro-Ilan). and regulatory concerns, research on developing alterna-

1049-9644/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.biocontrol.2007.07.011 208 D.I. Shapiro-Ilan et al. / Biological Control 44 (2008) 207–215 tive control strategies is warranted. Entomopathogenic nematodes are warranted. In this study we explore several nematodes are one potential control option (Shapiro-Ilan factors that could affect development of a C. nenuphar et al., 2002a, 2004). management tactic, i.e., effects of application time, host Entomopathogenic nematodes in the genera Steinernema plant, and nematode strain. In prior research, nematode and Heterorhabditis are obligate parasites of (Poin- field applications were made shortly after or during larval ar, 1990; Grewal et al., 2005). These nematodes have a emergence (the application directly targeted the larval mutualistic symbiosis with a bacterium (Xenorhabdus spp. stage) (Shapiro-Ilan et al., 2004; Alston et al., 2005). A and Photorhabdus spp. for steinernematids and heterorhab- question that may be raised is whether nematode applica- ditids, respectively) (Poinar, 1990). Infective juveniles (IJs), tions might also be effective if targeted closer to or after the only free-living stage, enter hosts through natural open- the onset of adult emergence. One potential hindrance to ings (mouth, anus, and spiracles), or in some cases, through controlling C. nenuphar with soil applications in the orch- the cuticle. After entering the host’s hemocoel, nematodes ard (regardless of what stage is targeted) is the presence release their bacterial symbionts, which are primarily of nearby alternate plant hosts. In some areas such as responsible for killing the host within 24–48 h, defending Georgia, USA, immigration of second generation adult against secondary invaders, and providing the nematodes C. nenuphar from alternate hosts outside of the orchard with nutrition (Dowds and Peters, 2002). The nematodes can be substantial, e.g., the most predominant source in molt and complete up to three generations within the host Georgia is the wild plum, Prunus angustifolia Marshall after which IJs exit the cadaver to search for new hosts (Jenkins et al., 2006a). These immigrant populations cause (Kaya and Gaugler, 1993). damage as the insects enter the orchard to oviposit and Entomopathogenic nematodes can effectively control a feed. Possibly, the problem could be ameliorated if nema- variety of economically important pests including a todes are applied for suppression of larvae under alternate number of weevil species (Klein, 1990; Shapiro-Ilan et al., host plants. Entomopathogenic nematode efficacy when 2002b; Grewal et al., 2005). Due to the nematode’s sensitiv- targeting the same insect can vary among host plant spp. ity to desiccation and ultraviolet (UV) radiation, applica- (Jaworska and Ropek, 1994; Barbercheck et al., 1995; tions to soil or cryptic habitats tend to be most Head et al., 2004; Jagdale et al., 2004); thus, we hypothe- efficacious (Kaya and Gaugler, 1993). Therefore, fourth sized that efficacy versus C. nenuphar larvae in an alternate instar, pupae, and adult C. nenuphar that occur in or on host may not be the same as observed previously in a peach the soil (Racette et al., 1992) are potential targets. orchard (Shapiro-Ilan et al., 2004). In experiments con- Research on use of entomopathogenic nematodes to ducted thus far, S. carpocapsae (All strain) or S. riobrave suppress soil-dwelling stages of C. nenuphar has shown (355) have shown the most promise for adult C. nenuphar promise. Initial studies indicated two species, Steinernema control, and S. riobrave (355) has been most successful in feltiae (Filipjev), and Steinernema carpocapsae (Weiser), controlling the larval stage (Shapiro-Ilan et al., 2002a, to be pathogenic to C. nenuphar larvae in the laboratory 2004). Different nematode strains within a species may dif- (Tedders et al., 1982; Olthof and Hagley, 1993). Also in fer in various traits important to biocontrol (Shapiro-Ilan laboratory studies, Shapiro-Ilan et al. (2002a) compared et al., 2003; Grewal et al., 2004); thus, it may be beneficial the virulence of six steinernematid and heterorhabditid spe- to determine the potential of other S. carpocapsae or S. rio- cies to C. nenuphar larvae and adults and concluded that brave strains to suppress C. nenuphar. Hence, our objec- Steinernema riobrave Cabanillas Poinar and Raulston and tives were to (1) evaluate the field efficacy of late-term S. carpocapsae showed the greatest virulence to adult nematode applications for C. nenuphar suppression (after C. nenuphar, whereas S. feltiae and S. riobrave were most the onset of adult emergence), (2) determine if nematodes virulent to the larval stage. In one field study, S. carpocap- can control C. nenuphar larvae in wild plum, and (3) com- sae, applied to suppress adult C. nenuphar, failed to provide pare different strains of S. carpocapsae and S. riobrave for acceptable levels of fruit protection (Be`lair et al., 1998); this virulence to C. nenuphar larvae and adults in the failing, however, was likely due to exposure of the nema- laboratory. todes to UV radiation and desiccating conditions because the applications were made above-ground without any pro- 2. Materials and methods tective formulations (Be`lair et al., 1998). Significantly more potential has been shown in field tests targeting the larval 2.1. Nematodes and insects stage. Alston et al. (2005) reported up to 39% larval mor- tality in a northern population of C. nenuphar following For each experiment, nematodes (S. carpocapsae and or applications of S. feltiae. Shapiro-Ilan et al. (2004) S. riobrave) were reared in parallel on last instar Galleria observed substantially higher mortality (78–100%) in a mellonella (L.) at 25 C according to procedures described southern population of C. nenuphar when applying in Kaya and Stock (1997). Galleria mellonella larvae were S. riobrave (355 strain) in peach, Prunus persica (L.), orch- obtained from Webster’s Waxieworms (Webster, WI). Fol- ards (S. feltiae was not effective in those tests). lowing harvest, nematodes were aerated and stored at Based on the results described above, further investiga- 13 C for less than 2 weeks before experimentation. Prior tions on control of C. nenuphar with entomopathogenic to the experiments described herein, the number of pas- D.I. Shapiro-Ilan et al. / Biological Control 44 (2008) 207–215 209 sages that the nematodes had been cultured through The experiment was repeated in the same plots in 2005. G. mellonella since their original isolation did not exceed Experimental procedures were identical except that, rather seven. than placing infested on plots, 50 C. nenuphar lar- vae obtained from naturally infested peaches or plums 2.2. Late-term nematode applications for C. nenuphar (Shapiro-Ilan et al., 2002a), were placed approximately control 5 cm below the soil surface within the plots’ perimeters. The larvae were placed in the center of each plot. Based Field studies were conducted in 2004 and 2005 to deter- on C. nenuphar behavior (Quaintance and Jenne, 1912; mine the efficacy of late-term nematode application for Snapp, 1930) movement of larvae across plots was extre- control of soil-dwelling C. nenuphar. Late-term applica- mely unlikely. To mimic natural conditions, and to avoid tions were defined by targeting the insect after the onset ant predation (Jenkins et al., 2006b), larvae were then cov- of adult emergence. The experiments were conducted in ered with soil (from the same location). Larvae were buried Quincy, Florida, at the University of Florida, North Flor- on May 4, 2005 and nematodes applied on May 31, 2005; ida Research and Education Center. Plots (0.39 m2) were methods of application and monitoring were as described located in the rows equidistant between peach trees for the 2004 experiment. (9-year-old University of Florida test variety M2-6 trees with 4.5 · 6 m spacing in a fine sandy loam soil); there 2.3. Nematode applications in wild plum was no ground cover within the plots. Methods of evalua- tion were based on those described by Shapiro-Ilan et al. Nematode efficacy in suppression of C. nenuphar larvae (2004) except, rather than directly targeting C. nenuphar was evaluated in 2005 and 2006 in a wild plum (P. angust- larvae, nematodes were applied after the onset of adult ifolia) thicket adjacent to a peach orchard at the USDA- emergence. The goal was to wait until adult emergence ARS research farm in Byron, Georgia (soil was a loamy was first initiated and then apply nematodes shortly sand). Methods were based on previous field evaluations thereafter. of nematode efficacy versus C. nenuphar larvae in peach On April 16, 2004, 100 peaches that had fallen from orchards (Shapiro-Ilan et al., 2004). The plum thicket trees in the experimental site and were infested with was cleared of underbrush leaving only the plum trees C. nenuphar (indicated by the distinctive crescent shaped standing in the area to be used. The experiment was orga- scars on the fruit) (Racette et al., 1992) were placed on bare nized in a randomized block design with five replicates per ground within each plot and larvae were allowed to emerge treatment. Circular plots (0.66 m2) were arranged with a naturally. In order to estimate the number of larvae emerg- minimum distance between plots of 1.3 m. Cone traps ing in each plot, 100 infested peaches were also placed in made of aluminum screening (hole size 0.03 cm diameter, each of six Berlese funnels and larval exit was recorded and dimensions of 109 cm bottom diameter tapering to approximately every 2–3 days until no larvae were recorded 5 cm diameter, height 91.4 cm) fitted with boll weevil traps on two consecutive sample dates. The Berlese funnels were on top were placed over each plot and secured around the located in the orchard under peach trees adjacent to the edges with soil from the surrounding area. plots and were about 25 cm above the ground. To monitor In 2005, 3 days before nematode application, 100 adult emergence in plots, cone traps made of aluminum C. nenuphar larvae obtained from naturally infested pea- screening (hole size 0.03 cm diameter, and dimensions of ches or plums (Shapiro-Ilan et al., 2002a), were placed 70.5 cm bottom diameter fitted with boll weevil traps on approximately 5 cm below the soil surface within the plots’ top (Boethel et al., 1976; Duncan et al., 2001) were placed perimeters. Larvae were then covered with soil (from the over each plot and secured around the edges with soil from same location). Two strains of S. riobrave (355 and 3-8b) the surrounding area. were applied on June 6, 2005 at a rate of 100 IJs/cm2 in Treatments (five replicates of each in a completely ran- each plot. Similar to the experiments conducted at Quincy, domized design) were applied on May 27, 2004. Treatments Florida, each treatment was applied in 200 ml of water via included S. riobrave (355 strain) and S. carpocapsae (All) at a watering can. A non-treated control received 200 ml a rate of 100 IJs/cm2 in each plot; control plots received water only. To offer additional protection a light layer water only. Each treatment was applied in 200 ml of water (ca. 1 cm) of soil from the site was distributed to cover via a watering can. A non-treated control received 200 ml the application site. Following application, each plot water only. To offer additional protection a light layer received an additional 100 ml of water. Adults were col- (ca. 1 cm) of soil from the site was distributed to cover lected daily until no adults were collected for three consec- the application site. Following application, each plot utive days. Plots were watered every 2–3 days for 2 weeks, received an additional 100 ml of water. Plots were watered unless rain amounts were sufficient (e.g., >10 mm); approx- every 2–3 days for 2 weeks, unless rain amounts were suf- imately 500 ml of water was added per plot on each irriga- ficient (e.g., >10 mm). Adults were collected daily until no tion event. In 2006, 64 larvae were placed in the soil 3 days adults were collected for 3 consecutive days. Only weevils prior to nematode application and nematodes (S. riobrave emerging after the treatment date were included in the [355 and 3-8b]) were applied on June 29, 2006 at a rate analysis. of 61 IJs/cm2 (a lower rate was used due to a shortage in 210 D.I. Shapiro-Ilan et al. / Biological Control 44 (2008) 207–215 our nematode yield). Otherwise, treatment applications, was significant mean separation was elucidated through irrigation, and assessment of adult emergence were as Student–Newman–Keuls’ multiple range test (SAS, 2001). described for 2005. In the laboratory experiments, data from identical experi- ments repeated in time were combined, and variation 2.4. Virulence comparison among nematode strains among trials was accounted for as a block effect. Prior to analysis the number of adults emerged in field experiments Separate experiments were conducted to compare was square root transformed, and percentage C. nenuphar S. carpocapsae and S. riobrave strains for virulence to survival in the laboratory was arcsine transformed (South- C. nenuphar adults in the laboratory. Procedures were wood, 1978; Steel and Torrie, 1980). based on those described by Shapiro et al. (1999, 2002a). Additionally, in field experiments when treatment effects Experimental units consisted of plastic cups (Bioserv Inc., were detected, percentage control (relative to the number Frenchtown, NJ) (3–4 cm i.d., 3.5 cm deep) that were filled of weevils emerged in the non-treated check) was calculated with 27 g (oven-dried) autoclaved soil from the USDA- using Abbott’s formula (Abbott, 1925). To display the pat- ARS research farm (Byron, GA), and contained one larva tern of natural C. nenuphar emergence in the field relative or adult insect each. After autoclaving, the soil (a loamy to nematode application date, cumulative percentage emer- sand with 84:10:6 percentage sand:silt:clay, pH 6.1, and gence (relative to total weevils captured) was calculated for organic matter = 2.8% by weight) was kept at 25 C for the sampling periods. Average daily soil temperatures (at at least 2 weeks before use (Kaya and Stock, 1997). Mois- 10 cm depth) were recorded for all field experiments from ture levels in each cup were brought to 14% (approximate the time nematodes were applied until the last date of field capacity) prior to addition of insects or nematodes. C. nenuphar emergence. One experiment included the following S. carpocapsae strains: All, Italian, Sal, Kapow, Mexican, and Agriotos. The experiment contained four replicates of 10 cups per 3. Results treatment. The second experiment addressing virulence of adult C. nenuphar included the following strains of 3.1. Late-term nematode applications for C. nenuphar S. riobrave: 3-2, 3-3, 3-8b, 355, 7-8a, and 7-12. This exper- control iment had four replicates of seven cups per treatment. For purposes of qualitative comparison across experiments, Late-term applications of S. carpocapsae and S. riobrave S. riobrave (355) was included in the first experiment and did not cause C. nenuphar suppression; no differences in S. carpocapsae (All) was included in the second. In both adult emergence were detected in the 2004 and 2005 exper- experiments, following addition of insects, 500 IJs were iments (F = 0.5; df = 2, 12; P = 0.618, and F = 0.76; applied to each cup by pipette in 0.5 ml tap water, and a water-only control was included. Cups were stored at 30 25 C and insect survival was evaluated 5 days post-treat- 25 A ment. The experiments were repeated once (2 trials). 20 An experiment was also conducted to evaluate nema- A 15 tode strains for virulence to C. nenuphar larvae. Based on A 10 previously observed efficacy in controlling C. nenuphar 5 larvae (Shapiro-Ilan et al., 2002a, 2004), this experiment # weevils emerged 0 focused primarily on comparison of different S. riobrave Control Sc Sr strains. The following nematode treatments were included: Treatment S. riobrave (3-2, 3-3, 3-8b, 355, 7-12, and TP strains) and S. carpocapsae (All). Although S. carpocapsae was not 14 A expected to exhibit superior virulence (Shapiro-Ilan et al., 12 A A 2002a), the nematode was included as an additional ‘‘stan- 10 8 dard’’ and to ensure that a treatment difference could be 6 detected in the test. The experiment was conducted as 4 described above for the adult C. nenuphar evaluation. 2 # weevils emerged There were four replicates of 10 cups per treatment, and 0 Control Sc Sr the experiment was repeated once. Treatment

2.5. Data analysis Fig. 1. Nematodes versus late-stage C. nenuphar in the field. Mean (SEM) emergence of adult Conotrachelus nenuphar from larval infested peaches The average number of weevils emerged per plot in field following application of entomopathogenic nematodes (100 infective juveniles/cm2) in a peach orchard in Quincy, Florida 2004 (a) and 2005 experiments, and the average percentage C. nenuphar sur- (b). Control = water only, Sf = Steinernema feltiae (SN strain), Sr = S. vival in laboratory experiments were analyzed for treat- riobrave (355 strain). Different letters above bars indicate statistically ment effects through analysis of variance; if the F-test significant differences in emergence (SNK test, = 0.05). D.I. Shapiro-Ilan et al. / Biological Control 44 (2008) 207–215 211 df = 2, 12; P = 0.416, for 2004 and 2005, respectively) a % Control # weevils emerged (Fig. 1a and b). Berlese funnel captures indicated that an 100 A 20 average (±SD) of 116.5 ± 13.3 larvae emerged per 100 infested peaches in 2004. Adult weevil emergence patterns 80 16 indicated that at least 10% of the total weevils that emerged 60 12 were captured by the date of nematode application, and 40 8 % Control 100% were captured 40 and 13 days post-application in 20 4

B B # weevils emerged 2004 and 2005, respectively (Fig. 2a). The more succinct 0 0 emergence pattern observed in 2005 relative to 2004 was Control Sr3-8b Sr355 likely due to a more uniform age structure in the buried lar- Treatment vae (2005) relative to the insects that emerged over time % Control # weevils emerged from naturally infested peaches (2004). Average daily soil b temperatures (±SD) during the experimental periods were 100 16 27.1 ± 0.9 C and 26.8 ± 0.9 C in 2004 and 2005, 80 A 12 respectively. 60 8 40 % Control 3.2. Nematode applications in wild plum 4 20 # weevils emerged B B Applications of S. riobrave 3-8b and 355 strains caused 0 0 Control Sr3-8b Sr355 suppression of C. nenuphar emergence in wild plum thickets. Treatment In 2005 and 2006, the number of adult weevils that emerged in control plots was greater than the number that emerged in Fig. 3. Nematodes versus larvae in plum thickets. Mean (SEM) emergence of adult Conotrachelus nenuphar from larvae buried under soil, and treated plots (F = 19.39; df = 2, 12; P = 0.0002 and percentage control (according to Abbott’s formula), after exposure to F = 4.26; df = 2, 12; P = 0.04, for 2005 and 2006, respec- entomopathogenic nematodes (100 infective juveniles/cm2) in a wild plum tively), and no difference in emergence was detected between thicket in Byron, Georgia 2005 (a) and 2006 (b). Control = water only, the two nematodes treatments (Fig. 3a and b). Based on Sr = S. riobrave (355 or 3-8b strain). Different letters above bars indicate Abbott’s formula the 355 strain caused 100% control during statistically significant differences in emergence (SNK test, = 0.05).

2004 2005 100 100 90 90 80 80 70 70 60 60 50 50 40 40 30 30 20 20

% Cumulative Emergence 10 10 % Cumulative Emergence 0 0

0 7 3 5 0 0 2 6 8 -6 1 20 2 32 4 -8 10 15 Days Post Application Days Post Application

2005 2006 100 100 90 90 80 80 70 70 60 60 50 50 40 40 30 30 20 20 10 10 % Cumulative Emergence 0 % Cumulative Emergence 0

8 0 2 6 8 4 8 0 2 4 6 8 0 2 4 1 2 2 24 2 2 30 32 3 1 2 2 2 2 2 3 3 3 Days Post Application Days Post Application

Fig. 2. Cumulative emergence. Cumulative percentage emergence of Conotrachelus nenuphar adults from larvae buried or from larval infested fruit in Quincy, Florida (a) or Byron, Georgia (b). A total of 109, 55, 69, and 33 weevils emerged (from five replicate plots) in Quincy 2004, Quincy 2005, Byron 2005, and Byron 2006, respectively. 212 D.I. Shapiro-Ilan et al. / Biological Control 44 (2008) 207–215 both years of the experiment; no weevils emerged from the 100 A plots treated with S. riobrave (355 strain). The 3-8b caused B 98.6% and 87.9% control in 2005 and 2006, respectively 80 B B BC (Fig. 3a and b). Adult weevil emergence patterns indicated 60 CD CD that 10% of the total weevils that emerged were captured D by 21 and 19 days post-application, and 100% were captured 40 % Survival 31 and 30 days post-application in 2005 and 2006, respec- 20 tively (Fig. 2b). Average daily soil temperatures (±SD) dur- 0 ing the experimental periods were 26.9 ± 0.9 C and l ol -2 -3 tr cAl 3 3 29.1 ± 1.1 C in 2005 and 2006, respectively. n SrTp o S Sr Sr Sr355 C Sr3-8b Sr7-12 Treatment 3.3. Virulence comparison among nematode strains Fig. 5. Nematodes versus C. nenuphar larvae in the lab. Survival of In laboratory experiments, although most S. carpocap- Conotrachelus nenuphar larvae after exposure to entomopathogenic sae strains caused reduced survival in C. nenuphar adults nematodes at a rate of 500 infective juveniles per insect. Control = water only (no nematodes), Sc = Steinernema carpocapsae,Sr=S. riobrave. relative to the non-treated control, no differences in viru- Nematode treatments included S. carpocapsae (All strain) and the lence were detected among the strains. Specifically, C. nenu- following S. riobrave strains: 3-2, 3-3, 3-8b, 355, 7-12, and TP. Different phar adult survival was suppressed relative to the control in letters above bars indicate statistical significance (SNK test, = 0.05). all S. carpocapsae treatments except the Kapow and Mex- ican strains (F = 2.66; df = 7, 55; P = 0.019); additionally survival in the S. riobrave (355) treatment was not different virulence to C. nenuphar adults among S. riobrave strains, from the control (Fig. 4a). In the comparisons focusing on weevil survival in all S. riobrave treatments as well as S. carpocapsae (All strain) was not different from the control (F = 2.07; df = 6, 48; P = 0.075) (Fig. 4b). a 100 Steinernema riobrave strains exhibited differential viru- 80 A lence to C. nenuphar larvae in our laboratory study

60 (F = 10.46; df = 7, 55; P < 0.0001) (Fig. 5). All nematode AB treatments caused lower larval survival than the control 40 % Live B AB B AB B (Fig. 5). However, larval survival in the S. riobrave (3-2 B 20 strain) treatment was lower than survival in the 3-8b, 7- 12, and TP treatments, but not different from the 3-3 or 0 355 strains. Lower survival was observed in the 3-3 and 355 treatments compared with 3-8b and TP. Larval sur- ScAgr ScAll ScItal ScSal Sr355 Control ScMex vival following application of S. carpocapsae (All) was ScKapow Treatment greater than survival following applications of S. riobrave strains 3-2, 3-3, and 355, and similar to the other strains. b 100 4. Discussion 80 A 60 A A Our results from the peach orchard (in Quincy, FL) A A A 40 indicate that applying entomopathogenic nematodes for % Live A control of soil-dwelling C. nenuphar after the onset of adult 20 emergence is not an effective strategy. These results diverge 0 from the high levels of efficacy observed in peach orchards by Shapiro-Ilan et al. (2004) when earlier applications were ScAll Sr3-2 Sr3-3 Sr355 Sr7-12 Control Sr3-8b made. The differences in timing of applications and result- Treatment ing difference in insect phenology between the two studies are evident. Shapiro-Ilan et al. (2004) applied nematodes Fig. 4. Nematodes versus C. nenuphar adults in the lab. Survival of 10 or less days after infested fruit was placed or 3 days after Conotrachelus nenuphar adults after exposure to entomopathogenic nematodes at a rate of 500 infective juveniles per insect. Control = water larvae were buried in the plots, whereas in the present only (no nematodes), Sc = Steinernema carpocapsae,Sr=S. riobrave. One study, applications were made more than 5 weeks after experiment (a) included S. riobrave (355 strain) and the following S. fruit was put down (2004) or 27 days after larvae were bur- carpocapsae strains: Agriotos (Agr), All, Italian (Ital), Kapow, Mexican ied (2005). Furthermore, in the present study at least 10% (Mex) and Sal. Another experiment (b) included S. carpocapsae (All emergence was observed by the application date, whereas strain) and the following S. riobrave strains: 3-2, 3-3, 3-8b, 355, and 7-12. Different letters above bars indicate statistical significance (SNK test, Shapiro-Ilan et al. (2004) did not observed 10% emergence = 0.05). until more than 2 weeks post-application. D.I. Shapiro-Ilan et al. / Biological Control 44 (2008) 207–215 213

Possibly the lack of biocontrol efficacy observed in our in 2005, yet no significant suppression was observed. These late-application in the Quincy, Florida experiments relative results emphasize the need to focus on earlier applications to the results of Shapiro-Ilan et al. (2004) was due to differ- to achieve control. ential susceptibility among different ages or stages of Structural or species diversity among host plants can C. nenuphar. Based on the timing of applications, Shap- affect insect nutrition and fitness (e.g., due to secondary iro-Ilan et al. (2004) clearly targeted the larval stage, i.e., plant compounds) and create different environmental con- initial exposure of nematodes was to larval stage and pri- ditions that may in turn affect entomopathogenic nematode marily larvae that had only recently emerged from fruit efficacy. Several studies have indicated host plant effects on (e.g., within 7 days). In the present study, due to variation entomopathogenic nematode activity when testing plant in insect development, we do not know what the exact species of different genera (Jaworska and Ropek, 1994; stages of the insects in the soil were at the time of applica- Barbercheck et al., 1995; Head et al., 2004). In our study, tion, yet it is apparent they were substantially further devel- one might have expected little difference between the results oped compared with those in Shapiro-Ilan et al. (2004), i.e., we observed in wild plum relative to results observed in constituting older larvae, pupae or adults. We know from peach by Shapiro-Ilan et al. (2004) because the two host prior studies that entomopathogenic nematodes are patho- plants are in same genus (Prunus), and otherwise the exper- genic to both adult and larval stage C. nenuphar (Shapiro- imental protocols were essentially the same. On the other Ilan et al., 2002a), but the two were never compared hand, the structural diversity (such as due to distance directly for susceptibility, and pupae have yet to be tested. between plants) between the two hosts used in this study Nonetheless, it is well established in other insects that both could conceivably have altered soil conditions in a manner insect stage as well as age within stage can affect suscepti- that would affect nematode efficacy (Kaya, 1990). Yet this bility to nematodes (Fuxa et al., 1988; LeBeck et al., was apparently not the case. Despite not having directly 1993; Shapiro et al., 1999; Shapiro-Ilan, 2001), and this compared applications in the two host plants, our results may be the case for C. nenuphar as well. in wild plum are clearly in agreement with the prior study Environmental conditions were not likely to have been conducted in peach (Shapiro-Ilan et al., 2004). The nema- crucial in limiting efficacy of the late-term applications. tode applications targeting C. nenuphar larvae in wild plum The average temperatures recorded in the late-term appli- caused high levels of control; complete or nearly complete cation experiments (Quincy, FL) were more narrow and insect suppression was observed with both nematode within range of those recorded in the Byron, Georgia strains in both years of the study. Therefore, our results experiments (where high levels of efficacy were observed), confirm that S. riobrave is highly effective at controlling and furthermore the temperatures might be considered C. nenuphar larvae in the soil, and suggests that this is close to ideal for nematode activity (Grewal et al., 1994). the case regardless of host plant (at least among those Soil moisture was not likely an issue given that the plots tested thus far). were irrigated regularly. Additionally, other soil parame- Our results from the Byron, Georgia experiments indi- ters (e.g., texture) in the Quincy, Florida experiments in cate that growers may be able to reduce levels of C. nenu- 2004 and 2005 were not likely to have been detrimental phar immigration into the orchard by treating alternate to nematode activity because high levels of C. nenuphar lar- host plants in the surrounding area shortly after fruit drop. val mortality had been previously been observed in the Such an approach would have to be weighed against eco- same orchard (Shapiro-Ilan et al., 2004). nomic constraints. Conceivably, growers could also use A reduced term of exposure may have contributed to the alternate hosts such as wild plum as a trap crop surround- lack of efficacy in the late-term application Quincy, Florida ing the orchard, and apply nematodes to protect the inte- experiments. The period that nematodes and insects over- rior. Additional studies are required to determine if such lapped in the ground in this study was considerably shorter alternate host treatments, combined with management than the period reported in Shapiro-Ilan et al. (2004). within the orchard would indeed yield economic benefits. Therefore, the relatively shorter duration of exposure Using a variety of strains that were included in this may have contributed to reduced efficacy in the Quincy, study, Shapiro-Ilan et al. (2003) observed differential viru- Florida experiments; longer exposure can increase the lence of S carpocapsae strains to pecan weevil (Curculio chance of infection. Furthermore the earlier applications caryae [Horn]) larvae and adults, and Stuart et al. (2004) made in Shapiro-Ilan et al. (2004) would increase the observed differential virulence of S. riobrave strains to lar- chances for boosting efficacy through nematode recycling. vae of the Diaprepes root weevil, Diaprepes abbreviatus However, despite the shorter duration, it is still plausible (L.). Therefore, we hypothesized that differences among that late-term applications would significantly suppress these strains would also exist in virulence to C. nenuphar C. nenuphar. Given that the insects are only 1–8 cm below larvae and adults. However, within species differences (and thus host-seeking was not a substantial barrier), nem- among the nematodes were not detected when assaying atode suppression could conceivably have commenced by C. nenuphar adults. Furthermore, based on the results of 2–4 days post-application. In the Quincy, Florida experi- Shapiro-Ilan et al. (2002a), virulence to adults in this study ments weevils continued to emerge after nematode applica- was lower than expected. Possibly, the discrepancy in tion for more than 5 weeks in 2004 and close to two weeks results was due to the use of different insect populations 214 D.I. Shapiro-Ilan et al. / Biological Control 44 (2008) 207–215 in the two studies; in addition to being collected in different Duncan, L.W., McCoy, C.W., Stansly, P.A., Graham, J.H., Mizell, R.F., years, the insects used in the prior study were obtained 2001. Estimating the relative abundance of adult citrus root weevils from Monticello, Florida, and those used in this study were (Coleoptera: ) with modified Tedders traps. Environ- mental Entomology 30, 939–946. obtained from Quincy, Florida and Byron, Georgia. Differ- Fuxa, J.R., Agudelo-Silva, F., Richter, A.R., 1988. Effect of host age and ences in nematode susceptibility among C. nenuphar popu- nematode strain on susceptibility of Spodoptera frugiperda to Steiner- lations have been observed previously (Alston et al., 2005), nema feltiae. Journal of Nematology 20, 91–95. albeit with populations that were more geographically sep- Grewal, P.S., Power, K.T., Grewal, S.K., Suggars, A., Haupricht, S., 2004. arated than those used in this study. Another hypothesis Enhanced consistence in biological control of white grubs (Coleoptera: Scarabaeidae) with new strains of entomopathogenic nematodes. may be that the nematodes attenuated over time due to Biological Control 30, 73–82. repeated culturing (Shapiro et al., 1996; Wang and Grewal, Grewal, P.S., Ehlers, R.-U., Shapiro-Ilan, D.I. (Eds.), 2005. Nematodes as 2002; Bilgrami et al., 2006). Attenuation, however, seems Biocontrol Agents. CABI, New York, NY. unlikely to have been a major factor because high levels Grewal, P.S., Selvan, S., Gaugler, R., 1994. Thermal adaptation of of virulence were observed in the laboratory and field entomopathogenic nematodes—niche breadth for infection, establish- ment and reproduction. Journal of Thermal Biology 19, 245–253. experiments targeting larvae. Conceivably, C. nenuphar Head, J., Lawrence, A.J., Walters, K.F.A., 2004. Efficacy of the adults are not as susceptible to entomopathogenic nema- entomopathogenic nematode, Steinernema feltiae, against Bemisia todes as previously thought; a lower than expected suscep- tabaci in relation to plant species. Journal of Applied Entomology 128, tibility would also be consistent with the lack of efficacy 543–547. observed in our late-term field applications. Horton, D., Brannen, P., Bellinger, B., Ritchie, D., 2006. Southeastern peach, nectarine, and plum pest management and culture guide, Within species variation among nematode strains was Bulletin 1171. University of Georgia, Athens, GA. detected for virulence to C. nenuphar larvae. The high levels Jagdale, G.B., Casey, M.L., Grewal, P.S., Lindquist, R.K., 2004. of virulence observed in some previously untested S. rio- Application rate and timing, potting medium, and host plant effects brave strains (e.g., 3-2 and 3-3) though not superior to on the efficacy of Steinernema feltiae against the fungus gnat, Bradysia the already field-proven and commercially available 355 coprophila in floriculture. Biological Control 29, 296–305. Jaworska, M., Ropek, D., 1994. Influence of host-plant on the suscep- strain, may warrant further study. These strains exhibiting tibility of Sitona lineatus L. (Col., Curculionidae) to Steinernema high virulence may possess other attributes that are supe- carpocapsae Weiser. 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