RAPID COMMUNICATION Susceptibility of the Strawberry Crown (: ) to Entomopathogenic Nematodes

1 DENNY J. BRUCK, DAVID L. EDWARDS, AND KELLY M. DONAHUE

USDAÐARS Horticultural Crops Research Laboratory, 3420 NW Orchard Avenue, Corvallis, OR 97330

J. Econ. Entomol. 101(2): 251Ð255 (2008) ABSTRACT The objective of this study was to determine the susceptibility of the strawberry crown moth, bibionipennis (Boisduval) (Lepidoptera: Sesiidae) larvae to two species of ento- mopathogenic nematodes. The entomopathogenic nematodes Steinernema carpocapsae (Weiser) strain Agriotos and Heterorhabditis bacteriophora (Steiner) strain Oswego were evaluated in labora- tory soil bioassays and the Þeld. Both nematode species were highly infective in the laboratory bioassays. Last instars were extremely susceptible to nematode infection in the laboratory, even in the protected environment inside the strawberry (Fragaria ϫ ananassa Duch.) crown. Infectivity in the laboratory was 96 and 94% for S. carpocapsae and H. bacteriophora, respectively. Field applications in late fall (October) were less effective with S. carpocapsae and H. bacteriophora, resulting in 51 and 33% infection, respectively. Larval mortality in the Þeld from both nematode treatments was signiÞcantly greater than the control, but treatments were substantially less efÞcacious than in the laboratory. Soil temperature after nematode applications in the Þeld (11ЊC mean daily temperature) was below minimum establishment temperatures for both nematode species for a majority of the post-application period. It is clear from laboratory data that strawberry crown moth larvae are extremely susceptible to nematode infection. Improved control in the Þeld is likely if nematode applications are made in late summer to early fall when larvae are present in the soil and soil temperatures are more favorable for nematode infection.

KEY WORDS strawberry, microbial control, biological control, entomopathogenic nematodes

Strawberries (Fragaria ϫ ananassa Duch.) are an im- in length when mature. Larvae of the strawberry portant small fruit crop, particularly along the west crown moth feed primarily on strawberry crowns, coast of the United States. Strawberries were grown on resulting in stunting of plants and severe stand thin- 16,000 ha in California, Oregon, and Washington in ning under heavy infestations. Heavily infested Þelds 2006, with a value in excess of $1 billion (NASS 2007). can be completely destroyed in a single growing sea- Currently, the key pests associated with straw- son. Last instars spend the winter in the crowns of berries in the PaciÞc Northwest production region strawberry plants. Overwintering larvae feed for a (Oregon and Washington) include strawberry crown short time in early spring (AprilÐMay) before pupat- moth, Synanthedon bibionipennis (Boisduval) (Lepi- ing. Adults emerge in late June and July, mate, and doptera: Sesiidae), a complex of root weevil species females lay eggs on leaves around the base of straw- (Coleoptera: Curculionidae), and cyclamen mites berry plants or directly on the crowns. Eggs hatch Stenotarsonemus pallidus (Banks) (Acari: Tarsonemi- within 2 wk, and neonates initially feed on small roots dae), among others. before tunneling into the crowns (Berry 1978). Cur- The strawberry crown moth is a univoltine pest that rent strawberry crown moth management depends on occurs throughout the western United States and Brit- the use of a pheromone (Nielsen et al. 1978) to help ish Columbia, Canada, wherever strawberries are time insecticide applications. If the conventional man- grown. Adults are a clearwing moth with a wing span agement program is not successful, larvae are very of nearly 20 mm. The adult is brightly colored, with a difÞcult to control with conventional insecticides black abdomen banded with yellow on the second, once they enter the crowns in late summer to early fall. Þfth, and sixth abdominal segments. Larvae, which are In addition to strawberries, this moth also attacks rasp- white with a dark brown head capsule, reach 20 mm berries, black raspberries, blackberries, and boysen- berries throughout the PaciÞc Northwest (J. Todd, Mention of trade names or commercial products in this publication personal communication). is solely for the purpose of providing speciÞc information and does not Entomopathogenic nematodes are attractive for use imply recommendation or endorsement by the U.S. Department of Agriculture. in biological control programs, because numerous spe- 1 Corresponding author, e-mail: [email protected]. cies are commercially available and they have been 252 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 101, no. 2 used for control of a variety of insect pests (Georgis et placed into plastic cups (5 cm in height by 7.5 cm in al. 2006). Because of their sensitivity to UV light and width) with an uninfested strawberry crown along desiccation, nematodes are most effective against with Ϸ20 g of Þeld soil and held at 21ЊC in complete pests in soil or other protected environments (Kaya darkness for 2 wk. and Gaugler 1993). Effective control relies on the Laboratory bioassays were performed using Þeld successful matching of the most effective nematode soil (Canderly Sandy Loam; 66.3:22.5:11.2, sandÐsiltÐ with the target pest (Georgis and Gaugler 1991). clay) autoclaved (1.1 kg/cm2; 121ЊC) for 2 h, left Before these studies, no data were available on the overnight, and autoclaved for an additional hour. The infectivity of nematodes against the strawberry crown soil was then placed in a drying oven at 70ЊC for 24 h moth. However, other Synanthedon spp. have been and stored (4ЊC) in zip lock bags until use. Fifty grams shown to be highly susceptible to entomopathogenic of oven-dried soil was placed into plastic cups (5 cm nematodes in the laboratory and Þeld (Bedding and in height by 7.5 cm in width). Sterile distilled water (7 Miller 1981, Miller and Bedding 1982, Deseo¨ and Miller ml) was added to each container and mixed with a 1985, Cossentine et al. 1990, Shapiro-Ilan and Cottrell sterile spatula until homogenous. Strawberry crowns 2006, McKern et al. 2007). Laboratory testing and infested with an individual last instar were placed into small-scale Þeld studies are important Þrst steps to each container, and half were covered with soil to identify nematodes with potential for managing pests simulate conditions in the Þeld. Nematodes (500 in- of unknown susceptibility. This is particularly impor- fective juveniles (IJs) per cup; 12 IJs per cm2) were tant for a pest such as the strawberry crown moth that released onto the surface of each cup in 1 ml of water is difÞcult to manage once established in the straw- so that the Þnal moisture content was standardized at berry crown. Entomopathogenic nematodes are ide- Þeld capacity (15% moisture). Containers were ally suited to attack larvae overwintering in such an capped and placed into large zip-lock bags containing environment. Nematodes move within Synanthedon several pieces of moistened paper towel, and then tipuliformis (Clerk) (Lepidoptera: Sesiidae) tunnels containers were incubated at 22ЊC in complete dark- in black currants, Ribes nigrum L. (Miller and Bedding ness for 8 d. Subsequently, each container was thor- 1982). The cryptic overwintering site inside pista- oughly searched, and the numbers of live and nema- chios, Pistacia vera L., proved to be an ideal environ- tode-infected larvae (conÞrmed by dissection) were ment to target navel orangeworm, Amyelois transitella determined. Each experiment contained an untreated (Walker) (Lepidoptera: Pyralidae), with ento- control (water only) and was arranged in a random- mopathogenic nematodes (Siegel et al. 2006). Al- ized complete block design with four replications and though Þlbertworm, Cydia latiferreana (Walsingham) Þve larvae per replicate. This experiment was re- (Lepidoptera: Tortricidae) and the Þlbert weevil Cur- peated (two complete trials) using both nematodes culio occidentalis (Casey) (Coleoptera: Curculion- species. Because of a shortage of larvae, the control idae) generally do not overwinter inside the nut, they treatment in the second run of the experiment only do emerge from the nut once it falls on the ground and contained three replications. overwinter in hibernacula in the soil under infested An experiment was also performed to determine the trees (Dohanian 1944). These overwintering larvae efÞcacy of S. carpocapsae and H. bacteriophora against were also ideally situated for targeting by ento- strawberry crown moth larval infestations in the Þeld. mopathogenic nematodes (Bruck and Walton 2007). Six replicates, consisting of 1.5 m of row (Ϸ10Ð15 Therefore, the objective of this study was to deter- plants) arranged in a randomized complete block de- mine the susceptibility of strawberry crown moth lar- sign, were marked out in an infested Þeld of ÔTotemÕ vae to two species of entomopathogenic nematodes. strawberries located in Benton County, OR. On 11 Additionally, a replicated Þeld trial was performed to October 2007, each length of row was treated with determine the efÞcacy of these nematodes against this nematodes (100 IJs per cm2) with a volume of water insect in the Þeld. equivalent to 1,900 liters water/ha by using a backpack CO2 sprayer Þtted with a fan nozzle. The experiment also contained an untreated control (water only). Soil Materials and Methods temperatures were recorded at a depth of 5 cm Two species of entomopathogenic nematodes, (HOBO U12, Onset Computer Corporation, Cape Steinernema carpocapsae (Weiser) Agriotos strain and Cod, MA). Twenty-one days after nematode applica- Heterorhabditis bacteriophora Poinar Oswego strain, tion, seven soil cores (10.8 cm in diameter; 5Ð7 cm in were used in laboratory soil bioassays to determine depth) were randomly dug from each length of row, their virulence against strawberry crown moth larvae. and the cores were returned to the laboratory. Each Nematodes were produced in vivo in last instar sample was thoroughly searched, and the numbers of Tenebrio molitor (L.) (Coleoptera: Tenebrionidae) live and nematode-infected larvae (conÞrmed by dis- (Kaya and Stock 1997). After collection, infective ju- section) were determined. veniles (IJs) were stored at 4ЊC for Ͻ1 wk before use. An arcsine transformation of the percentage of lar- Strawberry crowns infested with last instar strawberry val infection in the laboratory bioassay was performed crown moth were collected from an infested straw- to stabilize variance (Snedecor and Cochran 1989). A berry Þeld (28 September 2007) in Benton County test of homogeneity of variance was performed to Oregon. Crowns were returned to the laboratory, and detect variation between the two laboratory bioassays the larvae were removed. Larvae were individually with each insectÐnematode combination (Little and April 2008 BRUCK ET AL.: MICROBIAL CONTROL OF STRAWBERRY CROWN MOTH 253

Table 1. Mean percentage (؎ SD) of strawberry crown moth Conditions in the laboratory bioassays were optimal larvae infected with each species of entomopathogenic nematode for nematode infection, and they may not be an ac- from laboratory bioassays and field trials curate reßection of what occurs in the Þeld. There- Treatment fore, we determined the efÞcacy of S. carpocapsae and Control S. carpocapsae H. bacteriophora H. bacteriophora against strawberry crown moth lar- vae in the Þeld. Although the Þeld experiment per- Laboratory 96 (5.9)b 94 (5.25)b 0 (0)a Field 51 (19)b 33 (18)b 0 (0)a formed was limited in scale, the results indicated that even under far less than ideal environmental condi- Means with different letters in the same row are signiÞcantly dif- tions, both nematodes were infective in the Þeld (Ta- ferent (P Յ 0.05) (SAS Institute 1999). ble 1). There was a signiÞcant difference in the num- ber of dead larvae (all due to nematode infection) between the control and applications of S. carpocapsae Hills 1978). Variability was not signiÞcant between (␹2 ϭ 10.72; df ϭ 1; P ϭ 0.001) and H. bacteriophora bioassays, and data were combined for analysis. Lab- (␹2 ϭ 23.00; df ϭ 1; P ϭ 0.0001). There was no signiÞcant oratory data were analyzed using the General Linear difference in larval infection between S. carpocapsae Models procedure, with TukeyÕs multiple range test and H. bacteriophora applications (␹2 ϭ 2.82; df ϭ 1; used to separate means (SAS Institute 1999). Chi- P ϭ 0.09) (Table 1). Soil temperature postnematode square analysis was used to compare nematode efÞ- applications (Fig. 1) in the Þeld steadily declined, and cacy in Þeld applications (SAS Institute 1999). The they were below the minimum establishment temper- reference probability used throughout was P Յ 0.05. atures for both nematode species (12 and 15ЊC for S. carpocapsae and H. bacteriophora, respectively) for a Results and Discussion majority of the postapplication period (Grewal et al. Both nematode species were highly infective in 1994). Soil temperature, 2 wk before nematode appli- laboratory bioassays (F ϭ 66.87; df ϭ 2, 20; P Ͻ 0.0001) cation (i.e., immediately after larval collection), was (Table 1). All larvae in laboratory assays were inside more moderate, with warmer low temperatures and a strawberry crown at the beginning of the assay and less variation between the high and low temperature. all were found inside the crown at the conclusion of Had we been able to apply nematodes earlier in the the assay. This behavior seemed to be typical, because fall, they are likely to have been more efÞcacious. larvae were rarely (Ͻ10%) located outside of the However, we did not have the quantity of each species crown when collected from the Þeld (28 September of nematode available when larvae were initially col- 2007). Their presence in the crown in the laboratory lected in September to perform Þeld trials, and we assays did not afford them any protection from nem- were forced to wait until a fresh batch of nematodes atode infection. could be produced in vivo.

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0 1-Oct 3-Oct 5-Oct 7-Oct 9-Oct 2-Nov 4-Nov 11-Oct 13-Oct 15-Oct 17-Oct 19-Oct 21-Oct 23-Oct 25-Oct 27-Oct 29-Oct 31-Oct Date Fig. 1. Soil temperatures (ЊC) in Þeld experiments performed in 2007 to determine the efÞcacy of entomopathogenic nematodes against strawberry crown moth larvae. Downward arrow indicates when nematodes were applied. 254 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 101, no. 2

S. carpocapsae and H. bacteriophora have been as- trol in the Þeld may be improved if nematode appli- sayed against and are infective toward a wide range of cations are made before larvae enter the crowns. insect pests (Kaya and Gaugler 1993). However, there Earlier nematode application will not only target po- are no other published reports on the infectivity of tentially exposed larvae but also increased soil tem- entomopathogenic nematodes against strawberry peratures at that time, late summer to early fall, would crown moth larvae with which to compare our results. likely lead to increased levels of nematode infection. Entomopathogenic nematodes are highly infective Future work will focus on larger scale Þeld studies against a number of other Lepidoptera (Williams et al. with nematodes applied at various times to determine 2002, Cottrell and Shapiro-Ilan 2006, Shapiro-Ilan and the efÞcacy, feasibility, and economics of managing Cottrell 2006, McKern et al. 2007). Shapiro-Ilan and strawberry crown moth larvae with entomopatho- Cottrell (2006) found that steinernematids were more genic nematodes on a commercial scale. Based on virulent toward the lesser tree borer, Synanthe- these results and other published reports of nematode don pictipes (Grote & Robinson) (Lepidoptera: Sesi- efÞcacy against Sesiidae (Shapiro-Ilan and Cottrell idae), than the heterorhabditids evaluated in the lab- 2006, McKern et al. 2007), applications of steinerne- oratory. Applications of S. carpocapsae and H. matids are likely to be most efÞcacious in the Þeld. bacteriophora in the Þeld reduced the number of rasp- berry crown borer, marginata (Harris) (Lepidoptera: Sesiidae), another pest of small fruit Acknowledgments also commonly located in the crown, 53 and 33%, We thank W. Louis Tedders of Southeastern Insectaries, respectively (McKern et al. 2007). The results of McK- Inc., for providing the nematodes used in these studies. This ern et al. (2007) mirror our Þeld data; however, the work was supported solely by the USDAÐARS, PaciÞc West nematode rate used in their trial was signiÞcantly Area, Horticultural Crops Research Laboratory, Corvallis, lower (Ϸ5.4 IJs per cm2). Applications targeting P. OR, CRIS 5358-22000-032-00D. marginata in Arkansas were made in April when soil temperatures were reported to range between 8 and References Cited 16ЊC, but the duration of time at those temperatures was not reported. In addition, P. marginata trials were Bedding, R. A., and A. Miller. 1981. Disinfecting blackcur- not evaluated for a full 2 mo after nematode applica- rant cuttings of , using the insect tion in which time soil temperatures would be increas- parasitic nematode, Neoaplectana bibionis. Environ. En- ing in addition to ample time for nematode recycling tomol. 10: 449Ð453. Berry, R. E. 1978. and mites of economic importance to occur. Our trials were performed in late fall when in the Northwest. 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