The Effects of Strawberry Cropping Practices on the Strawberry Tortricid (: ), Its Natural Enemies, and the Presence of Nematodes Author(s): Lene Sigsgaard, Cyril Naulin, Solveig Haukeland, Kristian Kristensen, Annie Enkegaard, Nauja Lisa Jensen, Jørgen Eilenberg Source: Journal of Science, 14(122):1-18. Published By: Entomological Society of America URL: http://www.bioone.org/doi/full/10.1673/031.014.122

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Journal of Insect Science: Vol. 14 | Article 122 Sigsgaard et al.

The effects of strawberry cropping practices on the strawberry tortricid (Lepidoptera: Tortricidae), its natural enemies, and the presence of nematodes

Lene Sigsgaard1a, Cyril Naulin1, Solveig Haukeland2, Kristian Kristensen3, Annie Enkegaard4, Nauja Lisa Jensen5, Jørgen Eilenberg1

1University of Copenhagen, Department of Plant and Environmental Sciences, Section of Organismal Biology, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark 2Bioforsk, Norwegian Institute for Agricultural and Environmental Research, Ås, Norway 3Aarhus University, Faculty of Science and Technology, Department of Agroecology, Research Centre Foulum, Blichers Allé 20, Postboks 50, DK-8830 Tjele, Denmark 4Aarhus University, Faculty of Science and Technology, Department of Agroecology, Research Centre Flakkebjerg, DK-4200 Slagelse, Denmark 5HortiAdvice Scandinavia A/S, Hvidkærvej 29, DK-5250 Odense SV, Denmark

Abstract Cropping practice can affect pests and natural enemies. A three-year study of the strawberry tor- tricid, Acleris comariana (Lienig and Zeller) (Lepidoptera: Tortricidae), its Copidosoma aretas Walker (: ), and its entomopathogenic fungi was conducted in seven pairs of organic and conventional farms to test the hypothesis that farming practice (organic versus conventional) will affect the level of pest infestation and will affect the natural enemies. In addition, the number of years with strawberries on the farm, field age, and other factors that may affect pests and their natural enemies were considered. Farms were charac- terized by their cropping practices, cropping history, and other parameters. Field-collected larvae were laboratory reared to assess mortality from and entomopathogenic fungi. In 2010, a survey of nematodes was made to assess the response of an unrelated taxonomic group to crop- ping practice. 2,743 larvae were collected. Of those, 2,584 were identified as A. comariana. 579 A. comariana were parasitized by C. aretas and 64 A. comariana were parasitized by other para- sitoid species. Finally, 28% of the larvae and pupae of A. comariana died from unknown causes. Only two of the field-collected A. comariana larvae were infected by entomopathogenic fungi; one was infected by Isaria sp. and the other by Beauvaria sp. The density of A. comariana was on average four times lower in organic farms, which was significantly lower than in conventional farms. A. comariana was more dominant on conventional farms than on organic farms. The effect of crop age (One, two, or three years) on A. comariana infestation was significant, with higher infestations in older fields. Crop age had no effect on A. comariana infestation in a comparison of first- and second-year fields in 2010. Cropping practice did not lead to significant differences in the level of total parasitism or in C. aretas parasitism; however, C. aretas contributed to a higher proportion of the parasitized larvae on conventional farms than on organic farms. Mortality from unknown causes of A. comariana was higher in organic farms than conventional farms, and un- known mortality was two to seven times higher in second-generation A. comariana than in first

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Journal of Insect Science: Vol. 14 | Article 122 Sigsgaard et al. generation. Entomopathogenic nematodes were found on three organic farms and one conven- tional farm. Plant parasitic nematodes were found in more samples from conventional farms than from organic farms. The low density of A. comariana in organic farms exposes the specialist C. aretas to a higher risk of local extinction. In organic farms, where the density of A. comariana is low, other parasitoids may play an important role in controlling A. comariana by supplementing C. aretas. Other tortricid species may serve as alternative hosts for these other parasitoids, con- tributing to conserving them in the habitat. The higher unknown mortality of larvae from organic fields may be the result of non-consumptive parasitoid or predator effects. This study reports an example of the effects of cropping practice on an insect pest, with similar effects on nematodes. An understanding of the responsible factors could be used to develop more sustainable cropping systems.

Keywords: Acleris comariana, Copidosoma aretas, parasitoid, organic farming Correspondence: a [email protected] Editor: Robert Jetton was editor of this paper. Received: 2 September 2012 Accepted: 2 May 2013 Published: 1 September 2014 Copyright: This is an open access paper. We use the Creative Commons Attribution 3.0 license that permits unrestricted use, provided that the paper is properly attributed. ISSN: 1536-2442 | Vol. 14, Number 122

Cite this paper as: Sigsgaard L, Naulin C, Haukeland S, Kristensen K, Enkegaard A, Jensen NL, Eilenberg J. 2014. The effects of strawberry cropping practices on the strawberry tortricid (Lepidoptera: Tortricidae), its natural enemies, and the presence of nematodes. Journal of Insect Science 14(122). Available online: http://www.insectscience.org/14.122

Introduction soma aretas Walker (Hymenoptera: Encyr- tidae) and entomopathogenic fungi that infect Cropping practice can affect pests and natural A. comariana larvae. We also recorded the enemies with effects on crop health and yield. presence of plant‒parasitic and entomopatho- As a result of reduced use of agrochemicals genic nematodes in soils of the same 14 farms and more semi-natural vegetation, defined as a to assess the response of an unrelated taxo- habitat within or outside a crop containing a nomic group of organisms to cropping community of non-crop species (J. Holland, practice. Game and Wildlife Conservation Trust, UK, personal communication), on organic farms, A complex of six species of tortricids (Lepi- organic cropping practice is believed to lead doptera: Tortricidea) can attack strawberry to increased species diversity (Bengtsson et al. crops. These include the strawberry tortricid 2005) and to increased natural enemy pest Acleris comariana, which is the most common control (Crowder et al. 2010). species, Celypha lacunana (Denis and Schiff), Clepsis spectrana (Treits), Cacoecimorpha A three-year study at 14 farms in Denmark pronubana (Hübn.), Cnephasia asseclana was conducted to assess the effects of crop- (Denis and Schiffermüller), and Lozotaenia ping practice on the strawberry tortricid, forsterana (Bovien and Thomsen 1950, Acleris comariana (Lienig and Zeller) (Lepi- Stenseth 1982, Alford 1984, Cross et al. doptera: Tortricidae), and its natural enemies 2001). Although A. comariana is one of the with a focus on the major parasitoid Copido- main pests of strawberry, additional

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Journal of Insect Science: Vol. 14 | Article 122 Sigsgaard et al. major pests include the strawberry weevil An- pared the presence of entomopathogenic nem- thonomus rubi Herbst, spider mites and atodes in different cropping systems. tarsonemid mites (Lindhard et al. 2003). Tor- tricid larvae feed on the leaves and flowers of In this study, we aimed to analyse the effect of strawberries. Using pesticides to control moth organic or conventional cropping practices on larvae is harmful to many natural enemies and A. comariana and its natural enemies and how may contribute to the propagation of other these cropping practices affect the interaction pests, such as mites; therefore, there is a need between A. comariana, its parasitoids, and to look at alternative methods for moth control entomopathogenic fungi. Nematodes were (Sigsgaard and Petersen 2008). An under- sampled in the same fields to test whether standing of the effects of cultivation practice cropping systems affect a taxonomic group on A. comariana numbers and how mortality unrelated to and fungi in a similar way. factors can contribute to the control of A. co- mariana is fundamental to developing Materials and Methods conservation biological control strategies. Farms and fields The main parasitoid of A. comariana is the The project was designed to have six pairs of egg‒larval parasitoid Copidosoma aretas farms, which matched as closely as possible in (Sigsgaard et al. 2013). This parasitoid is the terms of geography, area, and number of years dominant parasitoid of A. comariana in the with strawberries. Because there are few or- United Kingdom (Turner 1968; Alford 1975, ganic strawberry farms, the organic farms 1976), yet the effect of cropping practice on were selected before the conventional farms. this parasitoid is not known. Insect pathogenic Two more farms were subsequently included fungi have not been recorded for A. comaria- in the study: in 2009 a large conventional na, but they are known from other strawberry production (conventional farm 7), Lepidopterans (Guzmán-Franco et al. 2008). and in 2010 an organic farm (organic farm 7). Insect pathogenic fungi can be an important The inclusion of organic farm 7 overlapped in mortality factor and subsequently are of rele- time with farm 3, which it later replaced when vance for conservation biological control. organic farm 3 phased out strawberry produc- tion in 2010. These two latter farms, Entomopathogenic nematodes occur naturally conventional 7 and organic 7, constituted the in the soil (as infective non-feeding juvenile last pair of farms. The farms were selected to stages) and parasitize the soil inhabiting stag- represent a range of sizes and cropping histo- es of their insect hosts. Entomopathogenic ries (e.g., either a long history or on new sites) nematodes are common in cultivated and un- (see Table 1). cultivated land, and many species occur more frequently in sandy soils (Hominick 2002, One field in each farm was studied. The fields Griffin et al. 2005, Haukeland et al. 2006). selected in 2009 were one-year strawberry, Studies on the natural populations of ento- indicating that they had been planted the pre- mopathogenic nematodes have been vious year, and this was the first year of concerned mostly with their prevalence in cer- harvest. In 2010, samplings were continued in tain habitats or altitudes and the discovery of the same fields, which were then two-year new species. Few studies, if any, have com- strawberry. The exception was that in farm 7, the field in 2009 was a three-year crop, but it

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Journal of Insect Science: Vol. 14 | Article 122 Sigsgaard et al. Table 1. Farms sampled. Cropping practice, number, locality, geographical UTM coordinates (Zealand includes zones 32 and 33, we used the modified map where all of Zealand is in zone 32), area with strawberry, total area belonging to farm, years with strawberry (2009), distance to other farms with strawberry production, use of insecticide (I), stems/m row ± SE (2011).

was replaced by a two-year crop in 2010. An two collections were made per generation, assessment in 2010 of tortricid numbers in when the larvae were second to third instar new one-year crops compared with the two- and again when they reached fourth to fifth year crops was made to provide a separate da- instar. Subsequent samplings included least 30 ta set to analyse the effect of crop age on samples, and this was increased up to a tortricid infestation. The new one-year crops maximum of 60 samples when larval density replaced the old fields for samplings of the was low. If necessary, more samples were summer generation in 2010 in those cases obtained by using an additional diagonal where the two-year crop had been removed. In transect walk, which created a sampling path spring 2011, conventional farms 2, 3, and 7 in the form of an X. Infestation was recorded and organic farm 7 had three-year-old fields by registering the number of larvae per 1 m of and the remaining fields were two-years old. row, equivalent to 3–3.5 Honeoye plants.

Field assessments 2009‒2011 The summer larvae of 2009 were sampled Farms were characterized according to twice, with a 10-day interval, in late July and cropping practice, cropping history, area early August. Sampling continued in spring dedicated to strawberry cultivation, and and summer of 2010 and spring of 2011. In proximity to other strawberry farms. Tortricid 2010, a one-year field was also sampled at infestation was recorded from April 2009 to each farm to allow a direct comparison of the June 2011, covering five generations of A. effect of field age on infestation. comariana. In 2009, spring larvae were sampled only once in early May in one-year Parasitism of A. comariana fields when larvae were about third to fifth To assess mortality caused by parasitoids and instar. One field was sampled in each farm entomopathogenic fungi, larvae were reared (Table 1). When possible, fields with the individually in 35 mL containers at 20 ± 1°C. widely used early strawberry, Fragaria × The bottom of each container was covered ananassa Duch. variety ‘Honeoye,’ were with 1% water agar. A strawberry leaf was selected to reduce variation. Thirty random inserted in the agar for the larva to feed on. samples were made during a diagonal transect Larvae were transferred to a new container walk of the field. In the following samplings, with a fresh leaf twice a week. The larvae

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Journal of Insect Science: Vol. 14 | Article 122 Sigsgaard et al. were examined every second day, and any 1997). The containers were held at room tem- mortality was recorded. perature, and the larvae were examined for mortality and entomopathogenic nematode Emerged parasitoids were preserved for later infection after one and two weeks. Dead lar- determination. Copidosoma aretas was identi- vae were placed in humid Petri dish chambers fied to species (Guerreri and Noyes 2005); to allow for nematode development. Nema- other parasitoids were determined to the fami- todes were identified to genus by ly level only. Dead larvae without signs of morphological examination of the infective parasitism were incubated to screen for insect juvenile stages (Adams and Nguyen 2002). pathogenic fungi. Field-collected larvae that Free-living plant parasitic nematodes were emerged as adults were likewise identified to extracted from a 250 mL sub-sample from species. Finally, pupae that did not emerge each soil sample using the elutriation tech- were dissected. nique (Seinhorst 1962). Plant parasitic nematodes were identified to genus by mor- Prevalence of insect pathogenic fungi. phological examination of the adult stages. Dead larvae without signs of parasitism were placed in humid chambers to enhance fungal Yield growth. Spore preparations were made on a Strawberry yield was assessed at all farms in microscope slide for identification (Lacey 2011 in the same fields as used in the study. In 1997). Normally infestation is clearly visible late June, the number of flowers, fruit, and after two weeks, but to ensure that no patho- empty stems were counted in eight to 10 ran- gens were missed, all samples were retained domly selected 1 m rows, equivalent to 3–3.5 for three weeks; others, for which there was plants. The sum of flowers, fruit, and empty some doubt, were retained for up to five stems represented the production potential of weeks. the crop. This measure replaced a yield esti- mate because frost damage affected fruit Influence of cropping practice on nema- formation of several fields in 2011. todes In mid-September 2010, nematodes were Data analysis sampled in the same fields from which the Prevalence of A. comariana in the field. The insect samples were collected, providing sev- number of observed tortricids was summa- en organic and seven conventional samples. rized for each field. Data (sum of larvae per Samples were taken using a soil corer. Each field per sampling date) were then analysed in sample comprised 20 pooled cores of 2.5 cm a generalised linear mixed model, where the diam taken to a depth of ≈15 cm. Cores were number of larvae, based on analyses for best taken as evenly as possible along a “W” pat- fit, was found to be Poisson distributed with tern within each field and mixed to give one an over dispersion within each year and pair. sample of ≈1 kg/ field. The mean depended on cropping practice, year, crop age, and generation, as well as in- To recover entomopathogenic nematodes, a teraction between these (as systematic subsample from each soil sample was placed effects). In addition, we attempted to include in a 500 mL plastic container and baited with characteristics for the individual farms/fields three late larval instars of Galleria mellonella as explanatory variables, with the possibility L. (Lepidoptera: ) (Kaya and Stock that the effect depended on the cropping prac-

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Journal of Insect Science: Vol. 14 | Article 122 Sigsgaard et al. tice. The farms were ordered in pairs, and we the presence of either Pratylenchus or Lon- attempted to include one organic and one con- gidorus; and 2 meant that the plant parasitic ventional farm in each pair. These pairs and nematode genera co-occurred. For the ento- year, field, and generation within field were mopathogenic nematodes belonging to the included as a random effects. The over- genus Steinernema, samples were by the na- dispersion parameter and random effects were ture of the baiting method either 0 in cases included to take into account variations not where the beneficial was not found or 1 when caused by random sampling, for example, var- it was present. Data for both groups of nema- iation caused by soil and environmental todes were analyzed using the Wilcoxon effects within and between fields. Finally, the signed rank test. Data for plant parasitic over-dispersion effect was estimated and tak- nematodes Pratylenchus and Longidorus were en into account. To accommodate the slightly also analysed in a generalised linear mixed varying numbers of samples per field, the log- model; the number of nematodes, based on arithm to the number of samples was included analyses for best fit, was found to be Poisson as an offset-variable. The offset-variable also distributed with a mean that depended on included the correction needed to account for cropping practice. We attempted to include sampled tortricids that were not A. comariana. additional characteristics for the individual fields as explanatory variables, with the possi- Mortality factors. The proportion of A. co- bility that the effect depended on the cropping mariana larvae parasitized by C. aretas and practice. The farms were ordered in pairs with by other parasitoids as well as the proportion one organic and one conventional farm in of deaths and adult emergence were analysed each pair. These pairs were included as a ran- using a generalized linear mixed model as- dom effect. suming proportion parasitized, proportion dead, and proportion of adult emergence to Yield. Yield was analysed using a linear follow a binominal distribution with an over- mixed model, with cultivation method as fixed dispersion within each year and pair. In the effects and pairs together with farms as a ran- model, the logit of the binomial probability dom factor. depended on the fixed effects of cultivation method, generation, year, the two-way inter- Results actions among these, and the random effects of pair and pair within year. Finally the over- Effect of cropping practice and other man- dispersion effect was estimated and taken into agement practices on tortricid infestation of account. The over-dispersion parameter and strawberry random effects were included for the same In this study, 2,743 tortricid larvae were col- reason as mentioned in section above; howev- lected. Of these 1,198 adult A. comariana er, non-significant interaction effects of fixed emerged. 159 other tortricids were found, effects were removed from the final model. some of which completed development to adults: these were Celypha lacunana Denis Nematodes. To analyze the effect of cropping and Schiffermüller (n = 40 adults, 22 from practice on soil parasitic nematodes, soil sam- conventional fields), Cnephasia asseclana ples were ranked according to presence of Denis and Schiffermüller (n = 49 adults, 20 plant parasitic nematodes: 0 indicated no plant from conventional fields), and Clepsis spec- parasitic nematodes were present; 1 indicated trana Treitschke (n = 2, both from

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Journal of Insect Science: Vol. 14 | Article 122 Sigsgaard et al. conventional fields). The larvae of these spe- A. comariana was the dominant tortricid in cies are normally darker than A. comariana organic and conventional farms, but less so in larvae and can be distinguished from A. co- organic farms. In organic farms, A. comariana mariana in the laboratory. The larvae of the made up 85.2% of the individuals, and in con- three other tortricid species feed on a variety ventional fields it made up 96.9% of the of herbaceous plants. The densities of A. co- individuals (F1,48 = 31.7, P < 0.0001). The in- mariana per meter row, equivalent to 3–3.5 teraction effect of the time of sampling plants for the study period, are presented in (following the two generations of A. comaria- Fig. 1, which is corrected for the presence of na) and the year on proportion of other other tortricids. species was also significant (F1,48 = 4.3, P = 0.04). Thus, in 2009, other tortricid species The densities of A. comariana, corrected for made up 2.7% of the individuals collected the presence of other tortricids, depended during sampling for the first generation, strongly on cropping practice (organic or con- whereas other tortricids made up only 0.4% of ventional) (Fig. 1), with higher densities under the individuals sampled for the second genera- conventional practice (F1,24.6 = 14.75 , P = tion, equivalent to seven times less. For 2010, 0.001). No significant main effect was found the corresponding percentages were 6.2% and for the year of study or generation (spring, 3.3%, equivalent to half as many other tortri- summer). There was a significant effect of cids found while sampling for the second crop age (one, two, or three year) (F2,45.8 = generation. In 2011, only the first generation 3.43, P = 0.041), with a higher infestation in was sampled, and 11.0% of the individuals in older fields. Interaction effects were non- this sampling belonged to other tortricid spe- significant. One explanatory variable was cies. found to be significant and was included in the analysis: Universal Transverse Mercator Effect of crop age (UTM) North (F1,8.7 = 11.1, P = 0.008), the In 2010, we sampled new first-year strawberry log(x) estimate being (‒0.00007 ± 0.00022), fields at the same time as the second-year indicating that the numbers of A. comariana fields, once for both generations (the second decreased by 0.07 times per km north. One sampling), to test whether crop age influenced other explanatory variable, the number of infestation. An analysis of the density of A. years a farm had produced strawberries, was comariana as an effect of cropping practice, near significant (F1,14.7 = 3.48, P = 0.082). generation (first or second), crop age, and all interaction effects showed that the average Insecticide use was not a significant explana- number of A. comariana per m row found in tory variable for densities of A. comariana. the first-year fields (conventional 0.63 ± 0.14, Conventional farms 5, 6, and 7 used insecti- organic 0.14 ± 0.05) was not significantly dif- cides in strawberries. Pyrethroid was applied ferent from that in the second-year fields in spring or early summer to control A. comar- (conventional 0.81 ± 0.14, organic 0.11 ± iana or A. rubi, or both. The remaining 0.05) (F2,36 = 0.55, P = 0.584), whereas there conventional farms used only herbicides and was a highly significant effect of cropping fungicides, except for conventional farm 3, practice (F1,21.1 = 11.13, P = 0.003), with a which did not use herbicides. All of the con- higher density of A. comariana in convention- ventional farms used fertilizer. al fields (lsmeans conventional: 11.9 ± 1.9, organic 2.8 ± 1.9) and a significant effect of

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Journal of Insect Science: Vol. 14 | Article 122 Sigsgaard et al. generation (lsmeans first generation 9.1 ± 1.7, second generation; in organic farms, it was second generation 5.7 ± 1.7) (F1,23.5 = 4.71, P 26.2% in the first generation and 39.7% in the = 0.040). There were no significant interaction second generation (F1,44 = 5.08, P = 0.029). effects. There was also a significant interaction effect of year and generation on mortality. In 2009, Natural parasitism of A. comarian it was 6.2% in the first generation and 33.3% The dominant parasitoid was C. aretas. Of the in the second generation. In 2010, it was A. comariana larvae collected, 579 were para- 19.7% in the first generation, and 30.4% in the sitized by C. aretas. Other parasitoids were second. In 2011, when only the first genera- recovered from 64 A. comariana larvae or pu- tion was sampled, mortality was 26.4% (F1,44 pae. They were primarily Hymenopterans = 17.53, P < 0.001). (Ichneumonidae [31] and Braconidae [15]) but also included a few Dipterans (Tachinidae A near-significant higher proportion of larvae [6]). Dissection of un-emerged pupae showed were parasitized by C. aretas in conventional that 12 were parasitized by hymenopterans, farms (24.9%) than in organic farms (15.8%) which had died before emergence. Finally, (F1,46 = 3.9, P = 0.054). There was also a near- 664 larvae and 122 pupae died from unknown significant difference among the three year factors, i.e., neither parasitism nor fungal crops —30% individuals were parasitized by pathogens could be detected. The number of C. aretas in 2009, 18% in 2010, and 21% in larval deaths from unknown factors was 2011 (F1,46 = 3.9, P = 0.054). equivalent to an unknown mortality of 28% (Fig. 2). There was no difference in total parasitism of A. comariana between conventional farms An analysis of the proportion of A comariana (26.8%) and organic farms (21.0%) (F1,46 = adults emerging from collected larvae as a 0.04, P = 0.839). The main effects of year and function of cropping practice (organic or con- generation were also not significant. However, ventional), generation (first or second), and the proportion of total parasitism of A. comar- year (2009, 2010, 2011) (Proc Glimmix, SAS iana by C. aretas was higher in conventional Institute, www.sas.com) showed a significant farms (93.1%) than in organic farms (74.3%) main effect of generation, reflecting that the (F1,34 = 15.0, P < 0.001). successful emergence in first generations were higher (51.7%) than in second generations Natural occurrence of insect pathogenic (42.8%) (F1,32.3 = 5.83, P = 0.022), and no in- fungi (prevalence) teraction effects were significant. Only two of the incubated larvae were natural- ly infected with insect pathogenic fungi. One The proportion of larvae that died of unknown larva, collected in 2011 from conventional causes was analysed as a function of cropping farm 6, was infected by a species of Beau- practice (organic or conventional), generation veria. The second larva, also collected in 2011 (first or second), and year (2009, 2010, 2011) but from conventional farm 7, was infected by (Proc Glimmix). There was a significant main a species of Isaria. Both isolates are main- effect of cropping practice on unknown mor- tained in the fungal culture collection at the tality, with a higher mortality in the second Department of Plant and Environmental Sci- generation. In conventional farms, it was ences at the University of Copenhagen. 20.9% in the first generation and 30.7% in the

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Journal of Insect Science: Vol. 14 | Article 122 Sigsgaard et al. Table 2. Presence of entomopathogenic nematodes and plant parasitic nematodes (number of samples (fields) with nematodes) and density of plant parasitic nematodes (mean number of nematodes per 250 mL soil) in seven conventionally and seven organi- cally grown strawberry fields.

Effect of cropping type on nematode popu- because frost damage made it impossible to lation compare harvested yield. Yield was not signif- Entomopathogenic nematodes belonging to icantly different in conventional and organic the genus Stenernema were recovered from fields (F1,11 = 1.2, P = 0.30). A model includ- one conventionally grown field and from three ing A. comariana density as an explanatory organically grown fields. Plant parasitic variable was not significant. nematodes were also present. The dominant plant parasitic nematode genera were Discussion Pratylenchus and Longidorus. Pratylenchus was present in seven conventionally grown Across the three years of the study, there was fields and six organically grown fields; Lon- a highly significant lower infestation of A. gidorus was present in four conventionally comariana in organic strawberry fields. Also, grown fields and one organically grown field there was a significant effect of crop age. No (Table 2). The presence of entomopathogenic other factors were significant, and there were nematodes was not significantly different be- no interaction effects. A single covariable, tween organic and conventionally grown UTM-north, was significant, with infestation fields (Wilcoxon S = ‒2.5, P = 0.62). Also the of A. comariana decreasing from south to presence of plant parasitic nematodes (ranked) north by 0.07 times per km. It is normally as- was not significantly higher in conventional sumed that problems in strawberry fields build fields than in organic (Wilcoxon S = 3, P = up over time, but multiple years with straw- 0.25). A mixed linear model of total number berry production on a farm was only a near- of plant parasitic nematodes as a function of significant covariable and of much less signif- cropping practice with farm pair as a random icance than cropping practice. Comparisons variable and with years with strawberry and between organic and conventional cropping soil textural class as covariables showed no practice at the farm scale are more heteroge- significant interaction effects, a near- neous than studies at plot scale, as found in a significant effect of soil textural class (F1, 6.3 = meta-analysis (Bengtsson et al. 2005). Such 5.18, P = 0.061), and no significant effect of heterogeneity can mask the effect of covaria- cropping practice (F1,8.1 = 0.92, P = 0.366). bles but does not exclude an influence by them. Yield The yield of the 14 fields in 2011, excluding Although we found an effect of crop age dur- organic field 3, which was no longer cultivat- ing the three-year study, when we compared ed in 2011, is presented (Table 1) as the sum A. comariana infestation in first- and second- of flowers, fruit, and empty stems per m row year fields within individual farms, no differ- by late June. This number, which represents ence in infestation level was found. This may potential rather than actual yield, was used be because field distances between old and

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Journal of Insect Science: Vol. 14 | Article 122 Sigsgaard et al. young strawberry crops within farms were small. In fact, it is still a common practice to The proportion of parasitism by C. aretas was plant old and young strawberry crops next to highest in conventional farms, with a higher each other. Young and old crops may thus be contribution of parasitism by other parasitoids separated by just a few meters, especially on in the organic farms. A strong correlation be- farms where strawberry is a minor product, tween A. comariana density and parasitism by whereas distances of up to 100 m between C. aretas underlines the potential of this para- young and old strawberry crops existed on the sitoid to regulate A. comariana (Sigsgaard et large farms. Short distances facilitate the mi- al. 2013); it can also help to explain the low gration of insects between old and young density of this parasitoid in organic farms. crops. If the effective mobility of A. comaria- Where the density of A. comariana is low, the na is restricted to a few hundred meters, as specialist C. aretas is exposed to a higher risk noted by Turner (1968) and as indicated for of local extinction. For those systems in which tortricids in general (Jeanneret and Charmillot extinction probabilities have been quantified, 1995), a farm layout with larger temporal and the parasitoid is more prone to extinction than spatial distances between strawberry crops its host, supporting the theoretical expectation could, in all likelihood, reduce this tortricid that higher trophic levels are at greater risk of pest. extinction than lower trophic levels (Cronin and Reeve 2005). A. comariana was most dominant in conven- tional farms. Although the actual number of Results indicate that at low densities of A. co- other tortricid species was comparable be- mariana, as in organic farms, other parasitoids tween organic and conventional farms, the provide supplementary control to that of the proportion of these other tortricid species in specialist C. aretas. Other tortricid species organic strawberry fields was significantly may serve as alternative hosts for these other higher. These other tortricid species were gen- parasitoids, which in turn may serve to main- eralists with a wide host-plant range and it is tain these other parasitoids in the habitat, e.g., likely that they originated in the surrounding the parasitoid Colpoclypeus florus (Walker) of landscape or other crops. The higher nutrient leafrollers in apple (Pfannenstiel et al. 2010). levels normally found in conventional fields are predictably correlated with higher densi- The higher unknown mortality of A. comaria- ties of generalists (Staley et al. 2010); na larvae collected in organic farms calls for however, our findings do not agree with this. further study. Identical rearing conditions for all collected larvae exclude the possibility that Although total parasitism of A. comariana this was an effect of rearing. A possible un- was not different between the two cropping known mortality factor could be non- practices, the proportion of larvae parasitized consumptive predator effects (McCauley et al. by C. aretas was almost significantly higher 2011) or from parasitoid probing. The higher in conventional farms than in organic farms. unknown mortality in the second generation The percentage of parasitism by C. aretas in of A. comariana, during a period of the sum- this study (organic 16%, conventional 25%) is mer when there are more natural enemies, higher than the <10% reported by Turner could support this hypothesis. (1968) and Vernon (1971) but lower than the 76% found by Alford (1976).

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Journal of Insect Science: Vol. 14 | Article 122 Sigsgaard et al. The lower dominance of A. comariana and C. in the use of agrochemicals on organic farms. aretas in organic fields is in accordance with However, only three of the conventional farms the findings of a meta-study on the effect of in this study used insecticides, and insecticide organic farming practice on diversity and fur- was not a significant covariable. All of the ther demonstrates that there is a higher conventional farms used fertilizers, herbicides diversity associated with organic farming (except one), and fungicides. Herbicides and practices (Bengtsson et al. 2005). Likewise, in fungicides may either affect direct- a comparison of parasitoid diversity between ly or have indirect effects through changes in 10 organic and 10 conventional farms, floral composition. Noxious effects of herbi- Macfadyen et al. (2011) found significantly cides on the eggs of parasitoids have been more parasitoid species on organic farms, with reported for the encyrtid Trichogramma preti- more species in each functional group. osum Riley (Bueno et al. 2008). Although parasitism was not higher on the organic The higher diversity of tortricids and parasi- farms, the higher unknown mortality of larvae toids in organic fields found in this study may sampled from these farms points to a higher be the result of higher floral diversity than in activity of natural enemies as one explanation conventional fields. For A. comariana and C. for the much lower infestation by A. comaria- aretas, it is known that flowering plants may na under organic cropping practice. increase the longevity of both species (Sigsgaard et al. 2013), which in turn may Very low prevalence of entomopathogenic lead to higher densities. The abundance of fungi on A. comariana flowering plant species and species richness The very low natural prevalence of ento- within strawberry fields and hedgerows of mopathogenic fungal infestation of A. conventional farms 1, 2, 3, 5, and 7 and organ- comariana larvae indicates that natural regula- ic farms 1, 2, 4, and 7 according to a study by tion by this group of natural enemies is not E. J. Ahrenfeldt et al. (University of Copen- likely. This may be attributed to the fact that hagen, Denmark) is comparable. Thus the A. comariana larvae are not in contact with floral species richness in conventional fields soil, although entomopathogenic fungi can was 12, 12, 7, 5, and 7 plant species, respec- also be found on the phylloplane (Meyling tively; and in the four studied organic fields, and Eilenberg 2007). No previous studies plant species richness was 13, 10, 3, and 10 have assessed the prevalence of entomopatho- species, respectively. Richness in convention- genic fungal infection of A. comariana; al hedgerows adjacent to the conventional however, Entomophthora was reported from fields was 6, 13, 7, 5, and 6 plant species, re- the congener A. minuta (Robinson) (MacLeod spectively, and in organic hedgerows was 9, 9, and Müller-Kögler 1973), and according to 6, and 6 species respectively. Differences in Zimmermann and Weizer (1991), fungi are plant or crop diversity at larger scales, such as often found on tortricid larvae and pupae. between fields, cannot be excluded. Effect of cropping practice on nematodes Organic cropping practice is thought to lead to Data may indicate a higher prevalence of en- increased species diversity (Bengtsson et al. tomopathogenic nematodes in organic 2005) and to increased natural enemy pest strawberry fields, whereas plant parasitic control (Crowder et al. 2010) as a result of nematode levels appear slightly higher in con- more semi-natural vegetation and a reduction ventional farms, although not significantly so.

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Journal of Insect Science: Vol. 14 | Article 122 Sigsgaard et al. A higher presence of entomopathogenic practices and conservation biological control nematodes in organically grown fields may be strategies. The findings also suggest ento- linked to greater insect host availability in mopathogenic nematodes may benefit from such fields, compared with conventionally organic cropping practice with a trend of low- grown fields, if chemical measures are im- er infestation with entomopathogenic plemented in the latter. A parallel Norwegian nematodes; however, this remains to be stud- study found a similar response by ento- ied further. Previous studies at the farm scale, mopathogenic nematodes to cropping practice, where organic and conventional farms were with a lower infestation by plant parasitic matched according to landscape structure, also nematodes in organic fields (N. Trandem, show significant differences in terms of diver- Bioforsk, Norwegian Institute of Agricultural sity and natural enemies, but they also reflect and Environmental Research, Ås, Norway, highly heterogeneous results (Bengtsson et al. personal communication). Further studies to 2005). For further studies aimed at improving elucidate which factors favor the apparent cropping practices to control A. comariana, higher occurrence of entomopathogenic nema- plot experiments may be needed to obtain todes in organically grown fields are worth clearer results, although large plots are needed pursuing. Nematode data from the present to study this mobile insect. The low preva- study and from a study in Norway (N. Tran- lence of fungi probably excludes the use of dem, personal communication) suggest that fungi in conservation biological control. En- cropping practices may have comparable ef- tomopathogenic fungi may still have potential fects on this unrelated system. Likewise, for inundation or inoculation biological con- Gliessmann et al. (1996), in a comparison be- trol; however, this still remains to be tested. tween conventional and organic strawberry production during a three-year conversion, Acknowledgements found higher levels of beneficial nematodes in organic fields. This project was supported by Danish Minis- try of Environment, Environmental Protection Estimated yield was not different between Agency (EPA), as part of the project “Biolog- cropping practices, pointing to the possibility ical control of tortricids and aphids in of reduced A. comariana infestation and re- strawberry” 2009–2012. Special thanks to the duced pesticide and agrochemical input farmers who hosted the study. Also, thanks to without a compromise in yield. In contrast to Cathrine Betzer, Maja Rohr Hansen, Shannon this study, yields found in fields under con- E. Myers, and Catherine Hepp for their assis- version to organic production, studied by tance with field sampling and laboratory work Gliessmann et al. (1996), were less, possibly during the project. Thanks to two anonymous because of a conversion decline. referees and to Anja Amtoft Wynns for their comments on this paper. Findings that A. comariana numbers are con- sistently three to four times lower in organic References practices than in conventional cropping prac- tices encourages further study into the Adams, B. J., and K. B. Nguyen. 2002. responsible factors. Understanding such fac- and systematics, pp. 1–33. In R. tors may provide information that can aid in Gaugler (ed.). Entomopathogenic the development of more sustainable cropping Nematology. CABI, Wallingford, UK.

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Journal of Insect Science: Vol. 14 | Article 122 Sigsgaard et al. Alford, D. V. 1975. Specific feeding agriculture promotes evenness and natural preferences of tortricid larvae on flowering pest control. Nature 466: 109–123. strawberry plants. Plant Pathol. 24: 54–58. Gliessman, S., M. Werner, S. Swezey, E. Alford, D. V. 1976. Observation on Caswell, J. Cochran, and F. Rosado-May. Litomastix aretas, an encyrtid parasite of the 1996. Conversion to organic strawberry strawberry tortrix moth. Ann. Appl. Biol. 84: management changes ecological processes. 1–5. Calif. Agric. 50: 24–31.

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Journal of Insect Science: Vol. 14 | Article 122 Sigsgaard et al. Turner, J. R. G. 1968. The ecological genetics of Acleris comariana (Zeller) (Lepidoptera: Tortricidae), a pest of strawberry. J. Anim. Ecol. 37: 489–520.

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Figure 1. Field densities of Acleris comariana in organic and conventional strawberry fields from 2009–11. Columns to the left are the conventional fields, columns to the right the organic fields, (a) spring 2009, (b) summer 2009, (c) spring 2010, (d) sum- mer 2010, (e) spring 2011. High quality figures are available online.

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Figure 2. Mortality factors of A. comariana for organic fields number 1 to 7 and conventional fields number 1 to 7. The height of stacked columns shows the total number of larvae collected from each field. From below they are: the number emerging as adult A. comariana (50% grey), number dead (hatched lower left to upper right), parasitized by C. aretas (hatched, bold line, up- per left to lower right), Ichneumonidae (80% black), Braconidae (chequered), Tachinidae (zigzag lines). (a) 2009 first generation, which was sampled only once; all subsequent generations were sampled twice; (b) 2009, second generation; (c) 2010 first gen- eration; (d) 2010 second generation; (e) 2011, first generation. High quality figures are available online.

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Figure 3. Production potential of strawberry in seven conventional and seven organic farms in 2011, represented by averaged total number of flowers, fruit and empty stems, i.e. stems per 1 m row, counted in 8–10 randomly selected 1 m rows, equivalent to 3–3.5 plants in late June. This measure replaced a yield estimate because frost damage affected fruit formation of several fields. High quality figures are available online.

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