Using entomopathogenic nematodes for biological control of plum curculio, Conotrachelus nenuphar: Effects of irrigation and species in apple orchards

Biological Control 67 (2013) 123–129

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Using entomopathogenic nematodes for biological control of plum curculio, Conotrachelus nenuphar: Effects of irrigation and species in apple orchards David I. Shapiro-Ilan a,⇑, Starker E. Wright b, Arthur F. Tuttle c, Daniel R. Cooley c, Tracy C. Leskey b a b c

USDA-ARS, Southeastern Fruit and Tree Nut Research Lab, Byron, GA 31008, United States USDA-ARS, Appalachian Fruit Research Station, Kearneysville, WV 25430, United States Stockbridge School of Agriculture, University of Massachusetts, Amherst, MA 01003, United States

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 Efficacy of entomopathogenic

a r t i c l e

i n f o

Article history: Received 5 May 2013 Accepted 30 July 2013 Available online 8 August 2013 Keywords: Biological control Conotrachelus nenuphar Entomopathogenic nematode Plum curculio Steinernema

12

A

2011

# Pc emerged

10 8 6 B

4 2

C 0

Control

Sf

Sr

Treatment 4

A

2012

3

# Pc emerged

nematodes was assessed in apple orchards in WV and MA.  Steinernema riobrave caused >95% plum curculio control (average of four trials).  Steinernema feltiae caused plum curculio suppression, but at a lower level (77.5%).  Irrigation did not impact entomopathogenic nematode efficacy.  High potential for incorporation into an integrated management program is indicated.

2 B 1 B 0

Control

Sf

Sr

Treatment

a b s t r a c t The plum curculio, Conotrachelus nenuphar, is a major pest of stone and pome fruit (e.g., apples, pears, peaches, cherries, etc.). Entomopathogenic nematodes (Steinernema spp. and Heterorhabditis spp.) are virulent to ground-dwelling stages of C. nenuphar and may be incorporated into an integrated management strategy. Two significant questions that must be addressed prior to implementation are: (1) which nematode is most effective in suppressing the target pest under field conditions? and (2) what is the impact of various irrigation levels on field efficacy? We addressed these questions by comparing the efficacy of two nematodes, Steinernema riobrave and Steinernema feltiae (two nematodes that showed the highest virulence in prior lab assays and field trials) and an untreated control, at three irrigation levels 0, 1 and 6 irrigation events applied during a two-week period post-application. C. nenuphar larvae were added to mini-plots prior to treatment application. Treatment effects were assessed by comparing the number of C. nenuphar adults emerging from each plot. The experiments were conducted at two field sites (apple orchards in Kearneysville, West Virginia and Belchertown, Massachusetts) in 2011 and 2012. Relative to the untreated check, S. riobrave caused 85.0% and 97.3% control in 2011 and 2012 in Massachusetts (respectively) and 100% control in West Virginia both years, whereas S. feltiae caused 0% and 84.6% control in 2011 and 2012 (respectively) in Massachusetts, and 78.2% and 69.7% control in West Virginia. The level of C. nenuphar suppression caused by S. riobrave was higher than suppression by S. feltiae at both sites in 2011 (no differences were detected in 2012). Irrigation did not have a significant effect on C. nenuphar suppression at both sites and in both years of the experiments. The lack of irrigation effects is an unusual finding for field applications using entomopathogenic nematodes, and may have been due to

⇑ Corresponding author. Address: USDA-ARS, SE Fruit & Tree Nut Research Lab, 21 Dunbar Road, Byron, GA 31008, United States. Fax: +1 478 956 2929. E-mail address: [email protected] (D.I. Shapiro-Ilan). 1049-9644/$ - see front matter Published by Elsevier Inc. http://dx.doi.org/10.1016/j.biocontrol.2013.07.020

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the level of precipitation and water-holding capacity in the field soils tested. The results of this study have positive implications for the potential of incorporating entomopathogenic nematodes into a C. nenuphar management program. Published by Elsevier Inc.

1. Introduction The plum curculio, Conotrachelus nenuphar (Herbst), is a key pest of pome and stone fruit in North America (Racette et al., 1992; Horton and Johnson, 2005; Leskey et al., 2009). Adult weevils enter orchards from overwintering sites in the spring to feed, and oviposit in fruit. Attacked fruit aborts or is damaged rendering it non-saleable. Larvae develop in fallen fruit, exit as fourth instars, and burrow into the soil (1–8 cm) to pupate (Racette et al., 1992). After emergence, adults feed on fruit and migrate to overwintering sites in the orchard or surrounding area (Racette et al., 1992; Olthof and Hagley, 1993). In the southern United States, an additional generation may occur on prior to overwintering (Horton and Johnson, 2005). Currently, control recommendations for C. nenuphar consist solely of above-ground applications of chemical insecticides to suppress adults (Horton et al., 2013). Due to various environmental and regulatory concerns, development of alternative and sustainable control strategies is warranted. Entomopathogenic nematodes are one of the potential control options (Shapiro-Ilan et al., 2002a, 2004, 2008, 2011). Entomopathogenic nematodes (EPNs) in the genera Steinernema and Heterorhabditis are obligate parasites of insects (Poinar, 1990; Lewis and Clarke, 2012). These nematodes have a mutualistic relationship with a bacterium (Xenorhabdus spp. are associated with steinernematids and Photorhabdus spp. are associated with heterorhabditids) (Poinar, 1990). Infective juveniles nematodes (IJs), the only free-living stage, enter hosts through natural openings (mouth, anus, and spiracles), or in some cases, through the cuticle. After entering the host’s hemocoel, nematodes release their symbiotic bacteria and the nematodes molt and complete up to three generations within the host, after which IJs exit the cadaver to search out new hosts (Kaya and Gaugler, 1993). Entomopathogenic nematodes are effective at controlling a variety of economically important pests including the larvae of several weevil species (Coleoptera: Curculionidae) (Shapiro-Ilan et al., 2002b, 2005). Due to the nematode’s sensitivity to desiccation and ultraviolet radiation (Kaya, 1990; Shapiro-Ilan et al., 2006) below-ground stages of C. nenuphar are the preferred targets for nematode applications (Shapiro-Ilan et al., 2004, 2008). Our overall goal is to use EPNs as one component of an integrated management program that targets multiple stages of C. nenuphar. The concept entails using volatile lures to attract adult C. nenuphar to selected sentinel trees on the orchard perimeter; the canopies of sentinel trees will then be sprayed with adult-killing insecticides while the other trees will remain pesticide free (Leskey et al., 2008). This approach (only spraying sentinel trees) can reduce the total number of trees that receive insecticide treatment by more than 90% (Leskey et al., 2008). Given that particularly high populations of C. nenuphar can be expected in sentinel trees, some damage in these trees is expected. Once the infested fruit in the sentinel trees drops, EPNs will be applied to suppress the ground-dwelling stages of C. nenuphar and thereby protect the interior of orchard from damage, and prevent subsequent generations of C. nenuphar from developing and reproducing. Applications of EPNs to ground-dwelling stages of C. nenuphar (particularly to the larval stage) can be highly efficacious (Shapiro-Ilan et al., 2004, 2008; Pereault et al., 2009). For example, in field trials conducted in peach orchards in Georgia and Florida,

Steinernema riobrave Cabanillas, Poinar and Raulston exhibited high levels of suppression (control averaged 94% in four trials) when targeting below-ground stages of C. nenuphar (Shapiro-Ilan et al., 2004). The ability of S. riobrave to cause high levels of suppression, e.g., >90%, in soil applications was also confirmed in later studies in Georgia (Shapiro-Ilan et al., 2008) and Michigan (Pereault et al., 2009). However, before broad application can be implemented, it is necessary to determine which nematode(s) may be best suited to each region where C. nenuphar occurs. Nematode efficacy can vary in different localities due to environmental factors inherit in the target site (e.g., temperature, moisture, soil characteristics, etc.) (Shapiro-Ilan et al., 2006, 2012). For example, in Georgia Steinernema feltiae (Filipjev) was not effective in suppressing C. nenuphar (Shapiro-Ilan et al., 2004), yet in contrast Alston et al. (2005) observed moderate control when using S. feltiae in field tests conducted in Utah. In the present study, we focused on field efficacy in regions north of the southeastern US. Toward determining which nematode species or strains may be most suitable for C. nenuphar control under various conditions, we recently conducted a broad laboratory screening for virulence to C. nenuphar larvae (Shapiro-Ilan et al., 2011). The screening included 13 EPN strains (comprising 9 species), which were tested at three temperatures and two different soil types. The idea was to narrow down candidate nematodes to test further under field conditions (i.e., to obtain data to build upon in the current study). Overall, among the most virulent nematodes across the conditions tested were two species that were commercially available, S. riobrave and S. feltiae. Thus the first objective in this study was to compare the efficacy of S. riobrave to S. feltiae in two regions where C. nenuphar occurs but EPNs had not previously been tested, i.e., in appleproducing agroecosystems of the mid-Atlantic and northeastern regions of the US. Adequate soil moisture is a critical factor to achieve success in biocontrol applications with EPNs because the nematodes are highly sensitive to desiccation and require a water film to disperse (Kaya, 1990; Shapiro-Ilan et al., 2006, 2012). Thus, in addition to selection of the optimum nematode for C. nenuphar suppression, another important issue is ensuring that sufficient irrigation is applied. It is not clear how much irrigation may be necessary for EPNs to achieve C. nenuphar control in different regions or agroecosystems. Therefore, our second objective was to determine the effects of different irrigation regimes on efficacy of EPNs when applied to soil-dwelling stages of C. nenuphar.

2. Materials and methods 2.1. Nematodes, insects and field sites Nematodes, S. riobrave (355 strain) and S. feltiae (SN) strain, were reared on commercially obtained last instar Galleria mellonella (L.) at 25 °C according to procedures described in Kaya and Stock (1997) and Shapiro-Ilan et al. (2002c). Following harvest, nematodes were stored at 13 °C for less than 2 week before experimentation. C. nenuphar were reared at the USDA-ARS laboratory in Kearneysville, West Virginia at 25 °C and 14L:10D on a diet of green thinning apples based on the methods of Amis and Snow (1985). Fourth-instar C. nenuphar were collected in drop trays upon exit from fruit and separated into groups of 100. To limit self-in-

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flicted injury during holding and transport, groups of 100 larvae were placed in 200 ml plastic cups (Dixie, Georgia-Pacific Consumer Products, Atlanta, GA) filled with superfine grade vermiculite (W.R. Grace and Company, Cambridge, MA). Field sites consisted of apple orchards located at the Appalachian Fruit Research Station, Kearneysville, West Virginia and the University of Massachusetts Cold Spring Orchard site near Belchertown, Massachusetts. At the Kearneysville site, apple trees (Gale Gala and Sun Fuji varieties) were spaced 4.9 m between rows and 3.7 m or 2.4 m between trees. In Belchertown, apple trees (McIntosh variety) were spaced 3 m between trees and 4.3 m between rows. Soil at the Kearneysville site is classified as a clay loam with 25:40:35 percentage sand:silt:clay, 2% organic matter, a pH of 6.5, and gravimetric field capacity of 32%. Soil at the Belchertown site is classified as a fine sandy loam with 57:33:10 percentage sand:silt:clay, 4.8% organic matter, a pH of 6.1, and gravimetric field capacity of 22%. 2.2. Field experiments Each plot consisted of a single tree and its understory with a mini-plot cage buried approximately 1 m from the trunk. There was a buffer of at least one tree between each plot. The mini-plot cage served as a representative of the insect-infested orchard floor and was the area that C. nenuphar larvae were added to and received nematode applications. There was no vegetation or ground cover within the mini-plots. The mini-plot cages (made of polyvinyl chloride) were 11.43 cm diam and 17 cm in height (cages were buried to 15 cm deep). One day prior to treatment applications, C. nenuphar larvae were added to the soil surface of each mini-plot. The number of larvae added to each plot was 65 and 35 at the Belchertown site (in 2011 and 2012, respectively), and 65 and 22 at the Kearneysville site (in 2011 and 2012, respectively); the number of larvae added varied from year to year or between sites due to the number available from the laboratory colony. Treatments were arranged in a factorial with three nematode levels (S. feltiae, S. riobrave, and a no-nematode control) and 3 levels of irrigation. There were five replicate plots of each nematode-irrigation treatment combination resulting in 45 plots at each site (9 treatments  5 replicates); the plots were arranged in a randomized complete block design. Irrigation levels consisted of one irrigation event (i.e., application), six irrigation events, or no irrigation. The six irrigation events were hand-applied (via beaker) every second or third day over a two week period following nematode application. Treatments receiving one irrigation event were applied immediately after nematode application. The amount of irrigation applied to mini-plots during each irrigation event was 15 liters/m2; this level of irrigation was based on a range of recommended quantities for young apple orchards (Roper and Frank, 2004; ). The nematodes were shipped overnight from Byron, GA to the field locations; the nematodes were stored under refrigeration for one additional day, and then applied to field sites. In the first experiment that was conducted, which was at the Belchertown site (application made on July 8, 2011), nematodes were applied at 100 IJs/cm2 in each mini-plot. Subsequently we realized this rate, though within the range of commercial application rates (Shapiro-Ilan et al., 2002b, 2006, 2012) provided less than 200 IJs/larva within each plot, which is less than the number of IJs used in laboratory studies to achieve efficacy (Shapiro-Ilan et al., 2002a; 2011); hence subsequent trials at the Belchertown site (application on July 10, 2012) and Kearneysville site (applications on August 18, 2011 and September 11, 2012) employed a rate of 400 IJs/cm2. The nematodes were applied by pouring them from culture flasks evenly over each plot in a volume of 154 ml. After nematodes were applied and all irrigation events were completed, a trap-top

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(inverted cone shaped capturing device) was placed on each mini-plot to capture emerging C. nenuphar adults. The surviving C. nenuphar adults that emerged into the trap-top were monitored until emergence ceased (approximately 5–7 weeks post-application depending on the year and location) (Shapiro-Ilan et al. 2004, 2008). Average weekly soil temperature and precipitation was monitored during course of the experiments (from nematode application to the end of C. nenuphar emergence). 2.3. Statistical analyses The number of C. nenuphar adults that emerged from plots was compared among treatments using ANOVA (SAS, 2002). The factorial analysis focused only on the two main effects (i.e., nematode and irrigation effects) because no interaction was detected between them (see Results section) (Southwood, 1978; Steel and Torrie, 1980). If a significant treatment effect was detected in the ANOVA, then the treatment differences were further elucidated through the Student–Newman–Keuls’ test (SAS, 2002). The numbers of C. nenuphar that emerged from each plot were square-root transformed prior to analysis; non-transformed means are presented in the figures. The alpha level for all statistical tests was 0.05. 3. Results For the Belchertown site, no interactions between main effects (irrigation and nematode) were detected in 2011 or 2012 (F = 0.20; df = 4, 32; P = 0.9365 in 2011, and F = 1.30; df = 4, 32; P = 0.2923 in 2012); therefore the analyses focused on main effects.

Fig. 1. Total number of adult plum curculio (PC), Conotrachelus nenuphar, emerged from field plots following application of Steinernema feltiae (Sf), S. riobrave (Sr), or an untreated control. The experiment was conducted in an apple orchard near Belchertown, Massachusetts in 2011 and 2012. Different letters above bars indicate statistically significant (Student–Newman–Keuls’ test, a = 0.05).

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No irrigation effects were detected in either year (F = 0.21; df = 2, 32; P = 0.8127 in 2011, and F = 0.87; df = 2, 32; P = 0.4296 in 2012). Nematode effects are illustrated in Fig. 1. In 2011, the number C. nenuphar adults that emerged in plots treated with S. riobrave was significantly less than the plots treated with S. feltiae or the control (and S. feltiae treatment was not different from the control) (Fig. 1) (F = 12.48; df = 2, 32; P < 0.0001). In 2012, lower C. nenuphar emergence was observed in both nematode treatments relative to the control (F = 27.50; df = 2, 32; P < 0.0001), and there was no difference between plots treated with S. riobrave versus S. feltiae (Fig. 1). At the Kearneysville site, similar to results in at the Belchertown site, no interactions between main effects were detected in 2011 or 2012 (F = 0.22; df = 4, 32; P = 0.9252 in 2011, and F = 0.35; df = 4, 32; P = 0.8455 in 2012). Additionally, no irrigation effects were detected for either year of the study (F = 0.07; df = 2, 32; P = 0.9311 in 2011, and F = 0.32; df = 2, 32; P = 0.7285 in 2012). Nematode effects are illustrated in Fig. 2. In 2011, the numbers of C. nenuphar that emerged in nematode-treated plots were less than in control plots, but plots receiving S. riobrave exhibited lower emergence than plots treated with S. feltiae (Fig. 2) (F = 36.38; df = 2, 32; P < 0.0001). In 2012, lower C. nenuphar emergence was observed in both nematode treatments relative to the control, and there was no difference between plots treated with S. riobrave versus S. feltiae (Fig. 2) (F = 11.44; df = 2, 32; P = 0.0002). The results of average weekly soil temperatures and precipitation measurements are illustrated in Figs. 3 and 4. Maximum soil temperatures did not exceed 25 °C at either location in 2012, whereas temperatures did occasionally exceed 25 °C in 2011. Total precipitation during the experimental periods were 142.2 mm and 16.6 mm at the Belchertown site (2011 and 2012, respectively),

Fig. 2. Total number of adult plum curculio (PC), Conotrachelus nenuphar, emerged from field plots following application of Steinernema feltiae (Sf), S. riobrave (Sr), or an untreated control. The experiment was conducted in an apple orchard near Kearneysville, West Virginia in 2011 and 2012. Different letters above bars indicate statistically significant (Student–Newman–Keuls’ test, a = 0.05).

and 417.2 mm and 301.2 mm at the Kearneysville site (2011 and 2012, respectively). In both years, the majority of precipitation occurred during the first half of the experimental period.

4. Discussion Steinernema riobrave was found to be a highly efficacious nematode for suppression of ground-dwelling stages of C. nenuphar in two apple orchards found in the mid-Atlantic region and New England. In both years and at both locations of the study we observed 85% to 100% control in plots treated with S. riobrave (the average level of control from the four trials was >95%). Our results are similar to previous findings that also indicated S. riobrave to be highly efficacious in reducing C. nenuphar in soil applications. For example, 77.5% to 100% control was reported in studies conducted in peach orchards or wild plum thickets in Georgia and Florida (average of all trials was > 94% control; Shapiro-Ilan et al., 2004, 2008). Additionally, Pereault et al. (2009) observed 80% to 89% reduction of C. nenuphar in Michigan apple and cherry orchards. Thus, our research extends that of previous findings and indicates that S. riobrave is highly effective in suppressing ground-dwelling stages of C. nenuphar in a variety of cropping systems and geographic locations. To-date, it is the most effective nematode tested for C. nenuphar control. We observed that S. feltiae can suppress ground-dwelling stages of C. nenuphar, but the level of suppression was not consistent and was lower than the level provided by S. riobrave. Among the four trials conducted in our study, we observed 0% to 84.6% control (an average of all four trials was approximately 58% control). The variable and reduced impact of S. feltiae on C. nenuphar has also been observed in other studies. In Utah, Alston et al. (2005) observed limited suppression of C. nenuphar larvae in wild plum sites with mortality levels not exceeding 40%, whereas no suppression was observed when using S. feltiae in Georgia and Florida peach orchards (Shapiro-Ilan et al., 2004). One factor that may have affected the S. feltiae efficacy differentially among trials and locations is temperature; S. feltiae is known to be a relatively cold-adapted EPN (Grewal et al., 1994). In laboratory comparisons, S. feltiae exhibited high levels of virulence to C. nenuphar larvae at 12 °C, 18 °C, and 25 °C. In the studies conducted in Georgia and Florida peach orchards, maximum soil temperatures were >25 °C, and thus temperatures may have reduced S. feltiae efficacy (Shapiro-Ilan et al., 2004). Alston et al. (2005) also suggested that higher temperatures may have reduced S. feltiae efficacy in their field studies (ambient temperatures were 27– 39 °C). Soil temperatures in the present study were for the most part more moderate, particularly in 2012 (both locations), which may explain the superior levels of control observed relative to previous studies. In Belchertown 2011, no suppression was observed in the S. feltiae treated plots even though soil temperatures did not appear to be lower than at the Kearneysville site the same year. Therefore, temperature alone does not seem to be a likely explanation for the lack of efficacy at the Belchertown site in 2011. The lack of treatment effects in the S. feltiae treated plots at the Belchertown site may have been due to the reduced rate of EPN application that was used in the 2011 trial. Apparently, the rate was suitable for S. riobrave, which appears to be innately more virulent than S. feltiae, but may have been insufficient for S. feltiae; alternatively, other factors such as soil antagonists (Kaya, 2002; El-Borai et al., 2009) may have contributed to the lack of efficacy. The lack of irrigation effects in all four trials (two locations and two years) was unexpected. Entomopathogenic nematodes require adequate moisture for viability, dispersal, and infectivity (Kaya, 1990; Womersley, 1990; Koppenhöfer et al., 1995). Thus, general recommendations for EPN applications in soil consistently call for

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Fig. 3. Average weekly temperatures (degrees C) and rainfall (mm) at Belchertown, Massachusetts field sites 2011 (top) and 2012 (bottom).

irrigation to ensure sufficient moisture (Georgis and Gaugler, 1991; Kaya and Gaugler, 1993; Shapiro-Ilan et al., 2006; Lacey and Shapiro-Ilan, 2008). For example, in contrast to our results, efficacy of Heterorhabditis bacteriophora Poinar in suppressing larvae of the Japanese beetle, Popillia japonica Newman, was severely curtailed as irrigation levels were reduced (Georgis and Gaugler, 1991). Possibly, the water holding capacities of the soils in our test sites combined with adequate rains negated the need for irrigation. It is conceivable that lower levels of precipitation would have produced different results; in future research, we will control the amount of precipitation that enters plots by monitoring soil moisture over time and covering soil to prevent additional moisture penetration. Nonetheless, the potential for requiring little or no irrigation for control of C. nenuphar could be highly beneficial as many fruit growers in areas infested with C. nenuphar may not utilize irrigation in their orchards. Our findings, however, need to be confirmed in various other soils and locations; results could be disastrous if growers were to assume (based on this study) that no irrigation is needed in all cases. Definitive models are needed for each soil type based on the level of soil moisture required to achieve efficacy in C. nenuphar control. The density of C. nenuphar larvae in cages utilized in this study was high. The natural density of C. nenuphar larvae can vary greatly

depending on fruit load and size of the tree. In the approach we are pursuing, i.e., the sentinel tree approach, the number of fruit that drop due to infestation under trap trees (and thus the number of larvae in the soil) can be ten times the amount observed in nonbaited trees (unpublished data). Therefore, it was important to test the ability of nematodes to control C. nenuphar when the target pest occurs at high densities. Furthermore, a high density was used to ensure having adequate numbers for analysis. Additional research is required to determine the efficacy of nematode applications at natural larval densities. The rate of nematode application we used (e.g., (400 IJs/cm2) was also high, particularly if one were to broadcast such a rate over the entire acreage, but this was not our intention. The high rate was used to parallel the high density of larvae in the cages. Additionally, in the approach that we are advocating, only three to five sentinel trees may be treated per hectare and therefore the total amount of nematodes required would be relatively low. Indeed, we calculate that the number of nematodes required per hectare using a sentinel tree approach (even at an application rate of 400 IJs/cm2) would be less than 10% of the amount needed if one were applying nematodes to the entire acreage at the minimum recommended rate of 2.5 billion IJs per hectare (Shapiro-Ilan et al., 2002b). If nematodes are to be applied in a standard ap-

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Fig. 4. Average weekly temperatures (degrees C) and rainfall (mm) at Kearneysville, West Virginia field sites 2011 (top) and 2012 (bottom).

proach (not using sentinel tree method) and thus applying the nematodes to a large area, lower rates might also achieve suppression as observed in trials conducted in Georgia (Shapiro-Ilan et al., 2004, 2008); nonetheless, additional research on minimum rates required to suppress C. nenuphar in various regions is needed. In summary, S. riobrave exhibited high levels of efficacy in suppressing ground-dwelling stages of C. nenuphar; S. feltiae also caused mortality in C. nenuphar albeit at a lower level compared with S. riobrave. Additionally, irrigation did not affect EPN efficacy. Our findings support the use of EPNs for control of C. nenuphar in various ground applications including incorporation into the sentinel tree approach (where EPNs would be applied under sentinel trees upon fruit drop). The economic feasibility of using EPNs in the sentinel tree approach is very promising because, even if high rates of nematodes were needed, the applications would only need to be made to a small proportion of the acreage. Also in cases where irrigation may be required to bolster EPN efficacy in orchards that do not have full irrigation systems, only a few trees per ha would require irrigation, thereby reducing the burden. In future research we will test the efficacy of EPNs in the sentinel tree approach and determine potential to control C. nenuphar on a large scale with this method.

Acknowledgments We thank Terri Brearley, Kathy Halat, Torri Hancock, Leighann Fall, and Stacy Byrd for technical assistance and the USDA-NIFA-

SCRI program for funding a portion of this research (under Grant # 2009-51181-06005).

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