Integrating Biological Control and Chemical Control of Cabbage Caterpillar Pests

Home , Alyssum, Cabbage looper, Campopleginae, Caterpillar, Ceramica (moth), Compsilura concinnata

Integrating biological control and chemical control of cabbage caterpillar pests

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

Emily Kathryn Linkous

Graduate Program in Entomology

The Ohio State University

2013

Master's Examination Committee:

Dr. Celeste Welty (advisor)

Dr. Mark A. Bennett

Dr. Mary M. Gardiner

Dr. Luis A. Cañas

Copyrighted by

Emily Kathryn Linkous

2013

Abstract

Lepidopteran pests Plutella xylostella, Pieris rapae, and Trichoplusia ni are the major species that attack crops in the family Brassicaceae. They are typically controlled by insecticide application, but there are parasitoid species that contribute to mortality.

However, parasitoids alone are typically unable to exert sufficient control to reduce pest numbers below economic thresholds. Pest density tends to be lower in more diverse systems, including those that integrate flowering plants that parasitoids use as food resources. These resources are lacking in most agricultural landscapes. Parasitoids tend to be negatively impacted by exposure to broad-spectrum insecticides.

The first goal of this research was to investigate the integration of habitat manipulation and insecticide treatment through the planting of floral resources and use of selective microbial insecticides. The integration of sweet alyssum (Lobularia maritima) insectary strips and the insecticide Bacillus thuringiensis (B.t.) was investigated in a cabbage field trial conducted in 2011 and 2012. The results of this study are discussed in

Chapter 2. The objectives of this study were: 1) to determine the effects of insectary strips on parasitism rates and pest density; and 2) to determine the effects of insecticides on parasitism rates and pest density. The main plot treatment was the presence or absence of insectary strips and the subplot treatment was insecticide. Plots were sampled weekly.

Insectary strips were found to have no significant effect on pest density in or parasitism in 2011. In 2012, insectary strips were found to have increased pest density but also ii increased parasitism. Parasitism rates were lower in subplots treated with B.t. than in untreated subplots. Pest density in subplots treated with B.t. was not significantly different than subplots treated with cyfluthrin. These findings indicate that insectary strips can increase parasitism but may also increase pest density in some circumstances.

The use of microbial insecticide did not increase parasitism but did keep pest density low.

Because parasitism appeared to be density dependent, use of lower rates or longer intervals of B.t. may increase parasitism while maintaining low pest density.

Another goal of the research was to determine what parasitoid species were present in Ohio. The diversity and relative abundance of parasitoid species found in the survey are discussed in Chapter 3. The objectives of this study were: 1) to determine parasitoid diversity and parasitism rate on commercial farms; and 2) to determine whether differences in parasitism rates among farms are associated with differences in insecticide use as measured by the environmental impact quotient (EIQ). Ten commercial fields were used for the surveys in 2011 and 2012. Caterpillars were collected and held until the emergence of parasitoids or pests. Eleven parasitoid species were found. Species abundance varied between years. Farms with a higher EIQ rating tended to have decreased species abundance and percent parasitism, but this correlation was not significant. These findings indicate that many species of parasitoids are present in Ohio and that insecticide usage can impact parasitoid species diversity and abundance.

Diadegma insulare and Cotesia rubecula should be the primary target of conservation biocontrol tactics within integrated pest management programs for cabbage.

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Dedicated to my wonderful friends and family and the love of my life.

Acknowledgments

I would like to thank my advisor, Dr. Celeste Welty, for her support, guidance, and enthusiasm. I also thank my committee for their helpful feedback throughout my research project and Zhiguang Xu for his assistance with the statistical analysis of my data. My research project would not have been possible without the work of the farm crews at the North Central Agricultural Research Station in Fremont, Ohio, the Waterman

Agricultural and Natural Resources Laboratory in Columbus, Ohio, and the Muck Crops

Agricultural Research Station in Celeryville, Ohio. My field assistants Amanda Lord,

Jeremy Wells, Greg Holthaus, Ryan Caesar, and Mackenzie Dorner were essential for the efficient collection of data, no matter what the weather was like. I thank Stan Gahn and

Mark Koenig for their assistance in finding cabbage fields for my survey.

I also thank my colleagues, friends, and family for their unending support during my graduate school career. Thanks for being a sounding board or letting me vent. Special thanks to my loving boyfriend Justin Franzen for keeping me sane and always being there to help me through the rough times.

This research would not have been possible without my funding sources – thanks to SEEDS at OARDC and the Ohio Vegetable and Small Fruit Research and

Development Program for financial support.

Vita

May 2006 ...... Wilmington High School

May 2010 ...... B.S. Biology, Wittenberg University

2010 to present ...... Graduate Teaching Associate, Center for

Life Sciences Education, The Ohio State

University

Fields of Study

Major Field: Entomology

Table of Contents

Abstract ...... ii

Acknowledgments...... v

Vita ...... vi

List of Tables ...... x

List of Figures ...... xi

Chapter 1: A review of previous research on biological control and chemical control of cabbage pests ...... 1

1.1 Introduction ...... 1

1.2 Objectives ...... 4

1.3 Biology and ecology of Lepidopteran cabbage pests and their common

parasitoids...... 6

1.4 A review of the proposed hypotheses and the theoretical basis for the research .... 10

1.5 Previous research on floral resources, pesticides, and natural enemies, and their

integration into control programs ...... 14

1.6 Previous research on parasitoid diversity and abundance in cole crops in North

America ...... 19

vii

1.7 References ...... 22

Chapter 2: Integrating alyssum insectary strips and selective insecticides in cabbage .... 31

2.1 Introduction ...... 31

2.2 Methods ...... 34

2.2.1 Field trials at agricultural research stations ...... 34

2.2.2 Harvest evaluation ...... 38

2.2.3 Alyssum vacuum sampling ...... 38

2.2.4 Data analysis ...... 39

2.3 Results ...... 40

2.3.1 Caterpillar density...... 40

2.3.2 Parasitism...... 41

2.3.3 Harvest ...... 43

2.3.4 Alyssum vacuum sampling ...... 44

2.4 Discussion ...... 45

2.5 References ...... 47

2.6 Tables ...... 52

2.7 Figures ...... 57

Chapter 3: Caterpillar parasitoid diversity and abundance in cabbage fields in northern

Ohio...... 66

viii

3.1 Introduction ...... 66

3.2 Methods ...... 68

3.2.1 Data collection ...... 68

3.2.2 Data analysis ...... 69

3.3 Results ...... 70

3.3.1 Parasitoid diversity and contribution to percent parasitism ...... 70

3.3.2 Hill’s N1 and EIQ ...... 72

3.4 Discussion ...... 73

3.5 References ...... 74

3.6 Tables ...... 77

3.7 Figures ...... 80

Bibliography ...... 82

List of Tables

Table 1: Percent parasitism of P. xylostella, P. rapae, and T. ni by main plot factor in

Ohio cabbage field trials, 2011 and 2012...... 52

Table 2: Percent parasitism of P. xylostella, P. rapae, and T. ni by subplot factor in Ohio cabbage field trials, 2011 and 2012...... 53

Table 3: Yield and caterpillar damage rating based on Greene scale per plant by main plot and subplot factor in Ohio cabbage field trial in 2011 ...... 54

Table 4: Yield, caterpillar damage rating based on Greene scale, and thrips damage rating per plant by main plot and subplot factor in Ohio cabbage field trial in 2012...... 55

Table 5: Abundance and diversity of pests and predators collected from vacuum samples of alyssum insectary strips in four cabbage field locations in Ohio, 2012...... 56

Table 6: Overall percent parasitism and relative contribution of parasitoid species to parasitism of the diamondback moth P. xylostella, imported cabbageworm P. rapae, and cabbage looper T. ni in commercial cabbage fields in northern Ohio, 2011 and 2012. ... 77

Table 7: Diversity and abundance of parasitoid species emerging from collected P. xylostella, P. rapae, and T. ni larvae in commercial cabbage fields in northern Ohio, 2011 and 2012...... 78

Table 8: Comparison of mean environmental impact quotient (EIQ), percent parasitism, and number of abundant species (Hill's N1) values for parasitoid species emerging from

P. xylostella larvae collected from commercial cabbage fields in northern Ohio, 2011 and

2012...... 79

List of Figures

Figure 1: Layout of alyssum main plot in Ohio cabbage field trial, 2011 and 2012...... 57

Figure 2: P. xylostella and P. rapae density over time by main plot treatment in Ohio cabbage field trial in 2011...... 58

Figure 3: P. xylostella and P. rapae density over time by subplot treatment in Ohio cabbage field trials in 2011 ...... 59

Figure 4: Number of caterpillars encountered during visual inspection surveys in 2011 and 2012 ...... 60

Figure 5: P. xylostella, P. rapae, and T. ni density over time by main plot treatment in

Ohio cabbage field trial, 2012...... 61

Figure 6: P. xylostella, P. rapae, and T. ni density over time by subplot treatment in Ohio cabbage field trial in 2012...... 62

Figure 7: Percent parasitism of P. xylostella and P. rapae in subplot treatments in Ohio cabbage field trial in 2011...... 63

Figure 8: Percent parasitism of P. xylostella, P. rapae, and T. ni in subplot treatments in

Ohio cabbage field trial in 2012...... 64

Figure 9: Parasitoid species collected in alyssum vacuum samples from four cabbage field trial locations in Ohio in 2012...... 65

Figure 10: Map of commercial fields used in commercial cabbage field surveys in northern Ohio, 2011 and 2012...... 80

Figure 11: Parasitoids of P. xylostella, P. rapae, and T. ni that emerged from caterpillar collected during commercial cabbage field surveys, 2011 and 2012...... 81

xii

Chapter 1: A review of previous research on biological control and chemical control of cabbage pests

1.1 Introduction

Cabbage, Brassica oleracea var. capitata L., is grown throughout North America and the rest of the world. Ohio is the tenth largest grower of cabbage in the United States, with approximately 1,200 acres of fresh market and 1,500 acres of processing cabbage harvested each year for a total of 2700 acres (USDA ERS 2008). Cabbage is plagued by a variety of insect pests, the majority of which are Lepidopteran. Control of these pests costs U.S. cabbage growers $110-$180 per acre each year (Gianessi 2009). In Ohio, the three major Lepidopteran pests of this crop are the imported cabbageworm Pieris rapae

L. (Lep.: Pieridae), diamondback moth Plutella xylostella L. (Lep.: Plutellidae), and cabbage looper Trichoplusia ni Hübner (Lep.: Noctuidae). The larvae of these three species feed on the leaves of the cabbage plant before and after head formation, causing significant economic damage to head quality and in severe cases reducing the yield of the cabbage plant (Kirby and Slosser 1984). Left unmanaged, the portion of marketable heads in a field ranges from 0%-38% compared to 99% when insecticides are used to control insect pests (Kirby and Slosser 1984). All species can be effectively controlled with synthetic or biorational insecticides, though in some areas T. ni and P. xylostella have developed resistance to most insecticide classes including organophosphates,

1 pyrethroids, and Bacillus thuringiensis (B.t.) (Shi et al. 2004, Sarfraz and Keddie 2005,

Maxwell and Fadamiro 2006)

In addition to the three major species, there are a few occasional Lepidopteran pests of cole crops. They include the cross-striped cabbageworm Evergestis rimosalis

Guenée (Lepidoptera: Crambidae), celery leaftier Udea rubigalis Guenée (Lepidoptera:

Crambidae), and zebra caterpillar Ceramica picta Harris (Lepidotera: Noctuidae). They are rarely numerous enough to warrant additional treatment (Welty 2009). Other common non-Lepidopteran pests of cabbage include the cabbage aphid, Brevicoryne brassicae L.

(Hemiptera: Aphididae); the onion thrips, Thrips tabaci Lindeman (Thysanoptera:

Thripidae); and the harlequin stink bug, Murgantia histrionica Hahn (Hemiptera:

Pentatomidae). They are generally secondary pests that do not significantly affect yield or marketability in most areas of the country. However, these pests sometimes require additional insecticide treatment, particularly T. tabaci, which can cause damage deep within cabbage heads that significantly reduces the marketability (Stoner and Shelton

1988, Shelton et al. 2008)

On most conventional fields during the growing season, insecticides are applied based on calendar intervals or after scouting a portion of cabbage plants to determine whether caterpillar density exceeds threshold. Typically, a density of one larval unit per plant is enough to warrant insecticide treatment. A larval unit is the equivalent of one half of a large cabbage looper (Shelton et al. 1982). On large commercial cabbage farms, growers primarily use pyrethroids, neonicotinoids, and other insect nerve poisons to kill the larvae while organic growers tend to rely on spinosad or B.t. (Precheur et al. 2012,

Caldwell et. al 2005).

Suppression of these Lepidopteran pests may also be achieved through conservation of natural enemies. Wasp and fly parasitoids attack cabbage caterpillars and are able to parasitize 72-90% of caterpillars (Mitchell et al. 1997, Sourakov and Mitchell

2000). There have been augmentative releases multiple times in the past but currently there are many self-sustaining parasitoid populations in North America (Xu et al. 2001,

Wold-Burkness et al. 2005). Despite their numbers, parasitoids alone often fail to provide an adequate level of control to meet commercial growers’ standards (Biever et al. 1992,

Sarfraz et al. 2005). The parasitoids’ efficacy may be limited in part by a lack of food resources for the adult stage or by the use of broad-spectrum insecticides that can have negative effects on non-target insects through elimination of hosts or direct effects on fecundity or mortality. These are issues that an integrated pest management system may be able to address (Landis et al. 2000, Wratten et al. 2003, Sarfraz et al. 2005, Desneux et al. 2007). The use of flowering plants within the field is a form of conservation biological control that can benefit parasitoids as well as other natural enemies by providing them diverse resources such as nectar, pollen, and shelter (Landis et al. 2000, Heimpel and

Jervis 2005, Tompkins et al. 2010). These flowering plants may be integrated through allowing non-crop vegetation within a field, annual plantings of non-crop flowering plants, or establishment of a perennial flower bank adjacent to a crop field (Gurr et al.

2003). Use of more selective pesticides can also have a positive effect on parasitism rates; many broad-spectrum pesticides used on conventional farms can harm parasitoids

(Cordero et al. 2007, Bommarco et al. 2011). Knowledge of the parasitoid guild within

3 the area is necessary for determining what floral resource would be the most effective; parasitoid diversity and abundance varies by region and there are over 130 species of parasitoids that are known to attack diamondback moth alone (Furlong et al. 2004,

Jankowska and Wiech 2006, Bertolaccini et al. 2011).

The main focus of this research was to determine whether the integration of insectary strips of sweet alyssum (Lobularia maritima L.) in a cabbage field would have an effect on the number of caterpillars parasitized and thus the caterpillar density in the field. This type of habitat manipulation in cabbage fields has been studied before but to date no studies examined possible interactions between pesticide treatments and floral strips (Al-Doghairi and Cranshaw 1999, Lee and Heimpel 2005). A field trial was conducted to determine the effects of floral strips and pesticides on parasitoid activity and caterpillar density in cabbage plots. The results of this study are presented in Chapter 2.

This chapter also includes the results of vacuum sampling of the insectary strips to determine what natural enemies were present in the plots. Another goal of this research was to determine the composition of the parasitoid community in Ohio. There has never been a formal survey in Ohio of the wasp and fly parasitoids specific to cabbage pests.

Chapter 3 discusses the results of a survey of commercial cabbage farms.

1.2 Objectives

Objective 1: Determine the effects of alyssum insectary strips on pests and parasitoids in

cabbage in conjunction with microbial insecticide, conventional insecticide, or no

insecticide (Chapter 2).

Hypothesis 1: Access to nectar affects parasitoid diversity, abundance, and rate of

parasitism.

Prediction: Alyssum insectary strips will have a positive effect on parasitism and

will increase parasitoid diversity and abundance.

Hypothesis 2: Parasitoid diversity and rate of parasitism are affected by

conventional insecticides.

Prediction: Parasitism and parasitoid diversity will be higher in subplots treated

with microbial insecticides and untreated subplots than in subplots treated with

conventional insecticides.

Hypothesis 3: Parasitoids will have a density-dependent impact on caterpillars.

Prediction: Caterpillar density will be lower in areas with more parasitoid

activity.

Objective 2: Examine the effect of alyssum insectary strips on predators and parasitoids

of cabbage pests (Chapter 2).

Hypothesis: Predators and parasitoids prefer the presence of abundant nectar

resources and more complex patches over simple patches with few nectar

resources.

Prediction: Predators and parasitoids will be more abundant in fields with

alyssum insectary strips and caterpillar density will decrease as a result.

Objective 3: Determine the diversity, abundance, and distribution of caterpillar

parasitoids among commercial cabbage fields in Ohio (Chapter 3).

Hypothesis: Parasitoid diversity and abundance varies as a result of pesticide

usage.

Prediction: The parasitoid guild will be more diverse in fields treated with less

environmentally harsh insecticides as determined by the environmental impact

quotient (EIQ); parasitism will be higher in those fields than in fields treated with

more harsh insecticide with a higher EIQ.

1.3 Biology and ecology of Lepidopteran cabbage pests and their common parasitoids

Pieris rapae, which in larval form is called the imported cabbageworm and in adult form is called the cabbage white, is a cosmopolitan species that originated in

Europe and has since spread throughout the world. Its larvae feed on all species within the family Brassicaceae, though they prefer cultivated plants like cabbage over wild relatives (Richards 1940). Individuals overwinter as pupae in sheltered areas near host plants (Lasota and Kok 1989). Adults emerge from these pupae in spring. After they mate, they lay eggs singly on the underside of leaves. Adults locate their host through a combination of visual cues and olfactory detection of the glycoside sinigrin (Root 1973).

The eggs hatch within five days and the first instars immediately attack their host plant.

Development from first instar to the fifth and final instar takes 14-21 days, depending on temperature (Richards 1940). After a period of wandering, fifth instar larvae produce a silken platform to attach themselves to the plant and pupate. Unless they are entering diapause, adult P. rapae emerge 5-7 days after pupation (Richards 1940). In the northern

United States there are typically 2-3 generations per year (Lasota and Kok 1989).

Plutella xylostella, the diamondback moth, is another serious pest of cabbage and other cole crops. Larvae are small and generally increase in number over the course of the

6 growing season (Khan et al. 2005). First instar larvae feed on the mesophyll while later instars consume all of the leaf except the upper epidermis, leaving a characteristic

“windowpane” on the leaf. When disturbed, larvae wiggle vigorously to avoid the disturbance and often drop from the plant on a silken thread. Diamondback moth pupae are covered by a fine mesh cocoon made of silk. Adults emerge within five days of pupation and live an average of 17 days (Harcourt 1957). Females can lay up to 356 eggs over their lifespan (Harcourt 1957). Adults tend to remain in hiding during the day and become more active at dusk. They feed on nectar from flowers near agricultural fields, mainly from cruciferous weeds. The first generation of diamondback moth usually develops on these weeds; subsequent generations tend to feed on cultivated Brassicaceae.

There are five to six generations of diamondback moth per year in northern areas.

Diamondback moth does not readily overwinter in areas with a hard frost, so the first generation in the northern part of the United States usually migrates from the south

(Harcourt 1957, Khan et al. 2005).

The third common species of cabbage pest is the cabbage looper T. ni. This species is also multivoltine and infests the northern United States through migration from the south, like the diamondback moth (Reid and Bare 1952). Eggs must incubate for five to ten days before emergence of the first instar larva, depending on temperature. Larvae pass through five instars before pupation. Larvae spend approximately two weeks feeding on their host before forming a silken cocoon near the base of the plant and molting into a prepupa. Total time to develop from egg to adult is approximately 25 days (Shorey et al.

1962). Adults become active shortly before sunset and flight activity increases until

7 several hours afterward. Females may mate multiple times and tend to lay single eggs or small clusters on the lower parts of their host plants (Shorey et al. 1962).

There are many parasitic wasps and flies that attack the three common cabbage caterpillars. All species are obligate parasitoids that develop within the host’s midgut or integument (Sarfraz et al. 2007, Gates et al. 2012). The most common species in the northern United States are Diadegma insulare Cresson (Hymenoptera: Ichneumonidae) and Oomyzus sokolowskii Kurdjumov (Hymenoptera: Eulophidae), which are both diamondback moth parasitoids; Cotesia glomerata L. and C. rubecula Marshall

(Hymenoptera: Braconidae), which attack imported cabbageworm; and Voria ruralis

Fallen (Diptera: Tachinidae), a tachinid parasitoid of cabbage looper and many other

Lepidopteran species (Sarfraz et al. 2005, Wold-Burkness et al. 2005, Elsey and Rabb

1968, Shelton et al. 2002, Chamberlin and Kok 1986, van Driesche 2008). There are many other species present in the United States that occur with varying frequency dependant on region, weather, or presence of other, more desirable hosts (Wold-Burkness et al. 2005, Sarfraz et al. 2005).

Larval parasitoids are considered to have the greatest potential for controlling cabbage caterpillar pests due to efficient host-searching and high fecundity (Sarfraz et al.

2005, Azidah et al. 2000, Talekar and Shelton 1993). In North America, D. insulare has been shown to parasitize over 80% of larvae and is able to find hosts within 8-10 seconds of landing on a damaged leaf (Xu et al. 2001). Larvae parasitized by D. insulare consume between 35-80% less than unparasitized larvae on the same plant (Monnerat et al. 2002,

Sourakev and Mitchell 2000). Parasitization of P. rapae by C. rubecula causes a similar

8 decrease in leaf surface consumption compared to unparasitized larvae (Parker and

Pinnell 1972a) C. rubecula kills the imported cabbageworm larvae in its third instar, well before the majority of damage occurs, and was recently introduced to the United States

(Cameron and Walker 1995, McDonald and Kok 1992, Puttler et al. 1970). It is beginning to displace C. glomerata in New England, which is beneficial to growers hoping to integrate biological control; C. glomerata does not kill the parasitized larvae until its fifth instar, well after the majority of the damage is already done (Parker and

Pinnell 1972b, van Driesche 2008). Caterpillar eggs are attacked by parasitoid wasps such as Trichogramma spp., but their impact on the population is small compared to the impact of egg predators and larval parasitoids; control is only achieved through frequent mass releases of the adults (Shelton et al. 2002, Parker et al. 1971, Sarfraz et al. 2005,

Talekar and Shelton 1993). There are also a few pupal parasitoids present in North

America like Pteromalus puparum L. (Hymenoptera: Pteromalidae), a gregarious wasp that attacks P. rapae pre-pupae and pupae. However, since pupal parasitoids kill the organism after the damage to the plant has occurred, their presence affects only the number of adults in the next generation (Wold-Burkness et al. 2005).

Releases of novel parasitoids in North America for classical biological control have occurred infrequently in the last century, though many augmentative releases have occurred (Parker et al. 1971, McDonald and Kok 1992, Sarfraz et al. 2005, Parker and

Pinnell 1972). Introductions in other parts of the world have been more common but have not produced consistent control of the target pest (Talekar and Shelton 2000). Therefore, most of the species found in field surveys have been established for years with the

9 exceptions of Compsilura concinnata Say (Diptera: Tachinidae), a polyphagous fly parasitoid which only became common in the mid 1990s, and the aforementioned C. rubecula (Wold-Burkness et al. 2005, Puttler et al. 1970).

1.4 A review of the proposed hypotheses and the theoretical basis for the research

Specialist insect pests tend to be less abundant in diversified agroecosystems than in monocultures (Root 1973, Risch et al. 1983, Bjӧrkman et al. 2010, Fiedler et al. 2008).

Risch et al. (1983) reported that in 53% of the 150 studies reviewed, the herbivore species decreased in abundance in the more diverse system; in 18% abundance increased and in 20% the response of the herbivore varied. Polyphagous herbivores were less affected by the relative diversification of the agroecosystem than specialist herbivores.

This phenomenon of lower herbivore load in polycultures and the resultant decrease in yield loss is termed associational resistance (Andow 1991)

Root (1973) proposes two hypotheses that attempt to explain the decrease in herbivore abundance in diversified systems. One explanation, the enemies hypothesis, states that natural enemies are more abundant in diverse agroecosystems and exert more control on the herbivore population. In complex ecosystems, more microhabitats and alternative prey are available, enabling the population of predators and parasitoids to be relatively stable. Floral resources, if present, provide food to adult parasitoids and other nectar- or pollen-dependent natural enemies (Baggen et al. 1999, Heimpel and Jervis

2005, Jonsson et al. 2010, Hogg et al. 2011b). A stable population of natural enemies may also blunt or prevent large outbreaks of herbivores by keeping populations in check

10 before a positive feedback loop occurs (Root 1973). However, the enemies hypothesis has proven difficult to assess in a field setting (Root 1972, Bjӧrkman et al. 2010). Natural enemies have shown differing responses to the diversification of agroecosystems, and increases in natural enemy populations do not always correlate with a decrease in pest populations (Risch et al. 1983, Andow 1991, Hooks and Johnson 2003).

The other hypothesis proposed by Root is the resource concentration hypothesis.

This hypothesis predicts that specialist herbivores are more likely to discover and remain in more pure stands of their host plant than in diversified stands. Only few specialist herbivore species will be abundant in the monoculture, and as a result interspecific competition will be lower and populations will grow to damaging levels more quickly. In contrast, diverse ecosystems that incorporate non-host plants may allow more interspecific competition between generalists and specialists, causing higher mortality of pests overall. Integration of non-host plants may also disrupt oviposition behavior of herbivores, lowering the herbivore load in the stand (Root 1973, Gurr et al. 2003).

Incorporating alternative host plants may also have a similar effect on pest load if they are more attractive to herbivores and pull them away from the crop plant (Bjӧrkman et al.

2010). These two hypotheses are not mutually exclusive, but the relative contribution of the mechanisms described to the associational resistance seen in diversified agroecosystems is still unclear (Andow 1991, Bjӧrkman et al. 2010). In general, however, the resource concentration hypothesis has been found to have more support than the enemies hypothesis, though the two have rarely been investigated simultaneously

(Andow 1991, Bjӧrkman et al. 2010).

Biological control of pests via insect natural enemies is typically achieved through one of three methods: classical biocontrol, conservation biocontrol, or augmentative biocontrol (Parker et al. 1971, Sarfraz et al. 2005). In classical biocontrol, an exotic predator, parasitoid, or pathogen is imported to suppress a pest population that is also typically exotic (Caltagirone 1981, Batra 1982). Augmentative biological control involves the release of natural enemies within a field crop or a greenhouse to enhance a natural enemy population that is already extant in the area (Batra 1982). Conservation biological control involves using integrated pest management tactics like strip cropping or polyculture to conserve and enhance the efficacy of natural enemies present in the field

(Batra 1982). Integrating floral resources within an agroecosystem is typically used as a part of conservation biocontrol of parasitoids specifically; they require nectar or pollen resources in their adult form and are known to respond to the inclusion of floral resources in some crop systems (Andow 1991, Baggen et al. 1999, Wratten et al. 2003, Heimpel and Jervis 2005, Jonsson et al. 2010). Conservation biocontrol is used in conjunction with other integrated pest management techniques like crop rotation, mulching, and intercropping with other crops or floral resources; these techniques are all types of habitat manipulation (Batra 1982, Jonsson et al. 2010).

Diversification of an agroecosystem can be achieved through the incorporation of another harvestable plant such as herbs like dill, but non-crop plants are often used instead (Gurr et al. 2003). Non-crop plants may flower during some or all of the growing season of the crop plant. Floral resources included in a polyculture can be referred to as floral strips, nectary plants, cover crops, living mulch or insectary strips; the latter term

12 will be used throughout this chapter (Baggen et al. 1999, Keller and Baker 2002, Gurr et al. 2003, Hogg et al. 2011b) In general, incorporation of insectary strips is most beneficial if the target natural enemy is nectar-limited (Wratten et al. 2003). However, if both pests and parasitoids benefit from the presence of nectar resources, control by natural enemies may not be enough to circumvent an increase in herbivore numbers

(Baggen et al. 1999). Hooks and Johnson (2003) looked at the response of natural enemies of cole crops specifically to crop diversification. They found habitat manipulation had a neutral-to-positive effect on predators and parasitoids of cabbage root fly Delia radicum L. (Diptera: Anthomyiidae); inclusion of non-host plants also tended to decrease pest populations. D. radicum oviposition rates decreased in intercropped fields relative to monocultures, possibly due to disruption of olfactory or visual stimuli used by adults to locate hosts as predicted by the resource concentration hypothesis. Coccinellid, staphylinid, and other predators had a three-fold increase in abundance in polycultures with non-Brassica weeds compared to monoculture (Schellhorn and Sork 1997).

Parasitoid response to inclusion of non-crop floral resources varies widely and is dependent on floral architecture and timing of flowering (Lavandero et al. 2006).

When evaluating the enemies hypothesis or the resource concentration hypothesis, distance between treatments is an important component of any study involving wasp or fly parasitoids. Schellhorn et al. (2008) reports that Diadegma semiclausum Hellen

(Hymenoptera: Ichneumonidae) marked with a fluorescent dye quickly moved up to 100 meters from their release point. They suspected that wasps dispersed even further than that within a few days of release. Other studies have also noted the mobility of wasp

13 parasitoids, underscoring the need for widely-spaced plots to reduce the chance of spillover between experimental treatments (Schellhorn and Silberbauer 2002, Schellhorn et al. 2004, Lavandero et al. 2006).

1.5 Previous research on floral resources, pesticides, and natural enemies, and their

integration into control programs

Adult parasitoid wasps and flies rely on nectar for sustenance and on caterpillar hosts for reproduction. Other natural enemy species like syrphid flies and lacewings contribute to caterpillar control and also use floral resources for sustenance. Though caterpillar hosts are common in cabbage fields, floral nectar sources are often in short supply and may be a limiting factor in colonization of an area (Hooks and Johnson 2003,

Heimpel and Jervis 2005, Hogg et al. 2011a). Having access to floral resources is not enough; flowering plants must also be structured such that they are accessible to the wasps and in bloom for long enough to both attract and keep them within an area

(Wäckers 2004). Patt et al. (1997) established that wasps must be able to easily access nectar in flowers to get the maximum benefit to longevity and/or fecundity. Floral architecture is therefore an important factor in determining what plants species is optimal for different species of wasps which may have widely varying head widths. Some features like corolla depth and width severely limit nectar access and the flowers therefore have no effect on longevity even if they are initially attractive to the wasps due to visual cues like color (Wäckers 2004, Vattala et al. 2006). Parasitoid wasps in the cabbage caterpillar complex can have varying responses to the same floral resource. For

14 instance, Daucus carota L. (Apiales: Apiaceae) is sometimes visited by C. glomerata in the field, despite not being the most optimal nectar source for this wasp, because it is white like other common nectar sources (Wäckers 2004). In contrast, D. insulare longevity and fecundity were positively affected by access to shaded D. carota.

Longevity increased by sixteen days compared to water only and was comparable to longevity of wasps that were provided with honey and water (Idris and Grafius 1995)

Laboratory studies done on D. insulare and C. glomerata have confirmed that some nectar sources can increase the longevity and fecundity of parasitoid wasps when compared with no resources. Johanowicz and Mitchell (2000) found that provisioning D. insulare and cabbage looper parasitoid Cotesia marginiventris Cresson (Hymenoptera:

Braconidae) with sweet alyssum increased their longevity by 12.7 and 4.8 times, respectively, over individuals provided only water. Both species fed readily on the alyssum. Fecundity of D. insulare females was significantly affected by the availability of carbohydrate resources from a honey-water mixture and some wildflowers, particularly wild mustard Brassica kaber L. (Idris and Grafius 1995, Vattala et al. 2006).

Habitat manipulation to increase nectar resources in the form of insectary strips have shown some enhancement of parasitoid success in field trials that use cole crops, though results are mixed (Landis et al. 2000, Heimpel and Jervis 2005, Lee and Heimpel

2005). Insectary strips are sometimes referred to as nectaries, nectary strips or floral strips (Hogg et al. 2011a).

Pfiffner et al. (2009) planted wildflower strips at two sites in Switzerland. At one site parasitism of caterpillars was not affected, while at the other parasitism of P. rapae

15 larvae was significantly enhanced by the presence of wildflowers. Pfiffner et al. speculated that the inconsistency may be due to the mixture of flowers used; it may have been that only a few of the flower species had nectaries that were accessible or attractive to the parasitoids.

Al-Doghairi et al. (2004) examined the effectiveness of buckwheat, vetch, dill, alyssum, or a blend of native wildflowers called the ‘Good Bug Blend’ as insectary strips in cabbage plots in Colorado. Plots with different insectary strips were planted close together with sweet corn or other non-host crops as a barrier. In both years of the study, parasitism of imported cabbageworm was significantly enhanced by the presence of some insectary strips: Good Bug Blend in the first year, buckwheat and vetch in the second year. However, the insectary strips did not have a similar effect on parasitism of diamondback moth or cabbage looper. Density of all three common pest species was not consistently affected by any treatment.

Keller and Baker (2002) integrated alyssum and pak choi insectary strips in broccoli fields in Australia. Parasitism in plots with or without insectary strips was high but diamondback moth density was never affected and increased throughout the growing season. Parasitoids were observed feeding on both types of flowers but it is unknown whether they were able to access enough nectar to affect parasitism. However, localized increases in biological control may not have been apparent due to the short distance between experimental and control plots.

Although buckwheat is often used as a model floral resource in other studies, sweet alyssum was found to be superior to many other species in attraction of

Ichneumonidae and Braconidae, the two most common parasitoid families that attack cabbage caterpillars (Heimpel and Jervis 2005, Rohrig et al. 2008, Sivinski et al. 2011).

Like buckwheat, alyssum does not appear to have any direct positive effects on pest density and or longevity (Lee and Heimpel 2005, Lavandero et al. 2006). Chaney (1998) found that alyssum was the optimal plant to use as an insectary for syrphid flies in lettuce fields, and Hogg et al. (2011b) successfully controlled aphid populations in lettuce fields through enhancing predation by syrphid flies by integrating alyssum insectary strips.

Alyssum has partially hidden nectaries with cup shaped flowers that are accessible to most wasp parasitoids, including D. insulare (Johanowicz and Mitchell 2000, Vattala et al. 2006, Winkler et al. 2009). In addition to parasitoid-accessible nectar, it flowers throughout the growing season, does not grow taller than the cabbage, and is easily cultivated (Johanowicz and Mitchell 2000, Wäckers 2004, Hogg et al. 2011a). Field borders of an unsprayed Brassicaceous plant like alyssum could also be a reservoir for some caterpillars to slow the proliferation of mutations conferring pesticide resistance

(Idris and Grafius 1996).

Floral strips are only one way to enhance natural enemies’ control of caterpillar pests. Use of more selective or biorational insecticides can also increase parasitism or predation of caterpillars; many broad-spectrum, synthetic insecticides negatively affect natural enemies and may sometimes increase the damage caused by pests by reducing the number of parasitoids and predators within the field (Croft and Brown 1975, Hassan et al.

1998, Desneux et al. 2007, Bommarco et al. 2011). Many studies have looked at the effects of synthetic insecticides on parasitoid wasps specifically. Adults of D. insulare

17 and O. sokolowskii are susceptible to emamectin benzoate, acetamiprid, and spinosad, among others, but are not susceptible to B.t. toxins or azadirachtin (Idris and Grafius

1993, Xu et al. 2004, Haseeb et al. 2005, Cordero et al. 2007. Both O. sokolowskii and

Cotesia plutellae Kurdjumov, a braconid parasitoid of diamondback moth, were also negatively affected by exposure to emamectin benzoate, avermectin, and fipronil; up to

100% mortality within 24 hours was reported for the highest dose of each of these insecticides, though avermectin had no impact on C. plutellae at a lower dose (Shi et al.

2004, Haseeb et al. 2005). Parasitoid wasps can be killed very quickly after contact with some insecticides; in some cases, incapacitation occurs after only thirty minutes of exposure (Hill and Foster 2000). Ingestion of B.t. or azadirachtin-derived insecticides has no effect on mortality or rate of parasitism for D. insulare but ingestion of indoxacarb, spinosad, and lambda-cyhalothrin was highly toxic (Xu et al. 2004).

The persistence of the pesticide within the environment is typically correlated with an increase in the number of deaths of adult parasitoids. Using lower doses or more sporadic applications of less toxic synthetic insecticides like B.t. or azadirachtin is likely to have a positive effect on parasitism rates and lifespan of the wasps (Hassan et al.

1998). The environmental impact quotient (EIQ) is one technique that can be used to estimate the negative effects of insecticides on beneficial insects like parasitoids (Kovach et al. 1992). It incorporates the effects of an insecticide on applicators, the surrounding environment, and beneficial insects into one measurement that can be used to compare the effects of multiple insecticides on the environment as a whole.

1.6 Previous research on parasitoid diversity and abundance in cole crops in North

America

There are hundreds of parasitoids that attack the three main cabbage pests but the exact composition of the parasitoid community varies by region (Jankowska and Wiech

2006, Wold-Burkness et al. 2005, Kfir 1997, Shelton et al. 2002). Accurate information on the natural enemies present within a crop system is essential in conservation biological control programs (Shelton et al. 2002, Sarfraz et al. 2005). Surveys of both commercial cabbage fields and field trials at agricultural research stations are important for gathering the information needed to integrate habitat manipulation for integrated pest management systems.

Relatively few long-term, formal surveys of the caterpillar parasitoid complex in

North America have been conducted. Wold-Burkness et al. (2005) conducted field studies over eleven field seasons starting in 1991 in Minnesota at an agricultural experiment station, but historical data from as early as 1968 was also used. They found that the parasitoid complex for the three major pest species sometimes changed dramatically over the course of the study. V. ruralis only parasitized 2% of T. ni larvae in the historical data and was present in larger numbers for much of the contemporary study but disappeared for a few years. Researchers also found eleven different species of parasitoid from 1991 onward though in historical data only two species were recovered.

The P. xylostella parasitoid complex was more stable; D. insulare was the dominant parasitoid across all years. Three other species of parasitoid also contributed to diamondback moth control, though their contribution was small compared to D. insulare.

P. rapae percent parasitism was relatively stable over all years though in 1994 a new tachinid parasitoid was found and in 2002 the introduced C. rubecula was encountered at low levels. The tachinid Phryxe pecosensis Townsend (Diptera: Tachinidae), P. puparum, and C. glomerata were the dominant contributors to parasitism. The changes seen over the thirty-three year period underscore the need for frequent natural enemy surveys to ensure that integrated pest management programs are effective.

In New York, Shelton et al. (2002) collected cabbage caterpillars over seven seasons (1979-1981 and 1991-1994). Collections were made in untreated fields at an agricultural experiment station and in commercial fields treated with insecticides. P. rapae was parasitized by three larval parasitoids, the most common of which was C. glomerata, and two pupal parasitoids. P. xylostella larvae were predominately parasitized by D. insulare though three other Hymenopteran species were occasionally seen.

Parasitism of T. ni was low compared to the two other species and only one tachinid species and one Encyrtid wasp species emerged from collected larvae (Shelton et al.

2002).

An earlier study in Virginia also found a different parasitoid complex from 1981-

1982. Chamberlin and Kok (1986) surveyed untreated cabbage and commercial farms in southwestern Virginia. Relatively few P. xylostella were collected compared to T. ni and

P. rapae and no parasitoids were reported. T. ni was predominately parasitized by V. ruralis and occasionally parasitized by two ichneumonids, four tachinid species, and

Bracymeria ovata Say (Hymenoptera: Braconidae). P. rapae was also occasionally attacked by the latter, but the primary parasitoids were C. glomerata and P. puparum

(Chamberlin and Kok 1986).

There have been more surveys recently in countries outside of North America.

Most of the surveys focused on P. xylostella parasitoids due to its insecticide resistance and its status as the most destructive cole crop pest in the world (Talekar and Shelton

2000, Sarfraz et al. 2005). Kfir (1997) conducted a two-year survey of diamondback moth parasitoids on untreated cabbage in South Africa. In this area, C. plutellae was the most common parasitoid collected and the novel Diadegma species endemic to the area played a lesser role in control than in other countries. Eleven species of larval or pupal parasitoids were identified, as well as two egg parasitoids and eight hyperparasitoid species. Parasitism of diamondback moth was greater than 90% on some dates. In

Argentina, Bertolaccini et al. (2011) collected samples of P. xylostella from a commercial cabbage field and found a different parasitoid complex. D. insulare and C. plutellae were the most common parasitoids, followed by O. sokolowskii. Only one other parasitoid was recovered from the larvae.

In Australia, Furlong et al. (2004) looked at the effects of pesticide usage on the parasitoid complex of P. xylostella on commercial cabbage farms. Diamondback caterpillars reared from a laboratory colony were placed in cabbage fields on sentinel plants and were protected from any pesticide applied to the rest of the field. Five species of parasitoid were recovered from the fields, including Diadromus collaris Gravenhorst

(Hymenoptera: Ichneumonidae), a parasitoid that was uncommon or nonexistent in other areas. Background pesticide application did not have a significant effect on the species found, though overall parasitism was low throughout the study sites.

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Stoner, K. A., and A. M. Shelton. 1988. Effect of winter storage on thrips damage to cabbage. New York’s Food and Life Sciences Bulletin. 9–12.

Talekar, N. S., and A. M. Shelton. 2000. Biology, ecology, and management of the diamondback moth. Annual Review of Entomology. 24: 85–113.

Tompkins, J.-M. L., S. D. Wratten, and F. L. Wäckers. 2010. Nectar to improve parasitoid fitness in biological control: Does the sucrose:hexose ratio matter? Basic and Applied Ecology. 11: 264–271.

(USDA-ERS) U.S. Department of Agriculture Economic Research Service. Cabbage statistics, 1960-2007. USDA, Cornell, NY. van Driesche, R. G. 2008. Biological control of Pieris rapae in New England: host suppression and displacement of Cotesia glomerata by Cotesia rubecula (Hymenoptera: Braconidae). Florida Entomologist. 91: 22-25.

Vattala, H., S. Wratten, C. Phillips, and F. Wäckers. 2006. The influence of flower morphology and nectar quality on the longevity of a parasitoid biological control agent. Biological Control. 39: 179–185.

Wäckers, F. L. 2004. Assessing the suitability of flowering herbs as parasitoid food sources: flower attractiveness and nectar accessibility. Biological Control. 29: 307– 314.

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Chapter 2: Integrating alyssum insectary strips and selective insecticides in cabbage

2.1 Introduction

Nectar is an essential resource in fields where biological control is used. Adult parasitoid wasps and flies rely on nectar for sustenance and on caterpillar hosts for reproduction (Landis et al. 2000, Heimpel and Jervis 2005). Other natural enemy species such as syrphid flies and lacewings prey on herbivores and also use nectar and pollen from floral resources as their main source of food in one life stage (Hogg et al. 2011a).

Agricultural fields usually lack plant diversity and therefore lack food resources for natural enemies (Landis et al. 2000, Sarfraz et al. 2005, Fiedler et al. 2008).

Caterpillar hosts are common in cabbage fields but floral nectar sources are often in short supply and may be a limiting factor in colonization by parasitoids and other natural enemies (Hooks and Johnson 2003, Heimpel and Jervis 2005, Hogg et al. 2011a).

Having access to floral resources is not enough; flowering plants must also be structured such that nectar within the flowers is accessible to the wasps, and the flowers must be in bloom long enough to both attract and sustain them within an area (Wäckers 2004). The nectar resource used should be visually or chemically attractive to the parasitoids and should not have a positive effect on pest colonization of the field (Baggen et al. 1999,

Wäckers 2004, Fiedler and Landis 2007). Habitat manipulation in the form of insectary strips has used in previous studies to promote parasitoid success in cole crops, though this 31 has not always been successful (Landis et al. 2000, Keller and Baker 2002, Al-Doghairi and Cranshaw 2004, Heimpel and Jervis 2005, Lee and Heimpel 2005). Some studies have been conducted with little separation between experimental plots; since parasitoid wasps are highly mobile, this may have confounded the evaluation of effects of the insectary strips on parasitism and pest control (Keller and Baker 2002, Al-Doghairi and

Cranshaw 2004, Schellhorn et al. 2008). The flowers used may have been unsuitable for caterpillar parasitoids or may not have flowered consistently throughout the growing season (Lee and Heimpel 2005, Pfiffner et al. 2009).

Herbivorous pests tend to be less abundant in diverse patches, including those that incorporate flowering plants (Risch et al. 1983, Heimpel and Jervis 2005, Sarfraz et al.

2005). There are two hypotheses that attempt to explain the decrease in pests in diversified fields: 1) the enemies hypothesis: increased plant diversity provides a more attractive habitat for natural enemies; 2) the resource concentration hypothesis: host plants in diversified settings are less attractive to herbivores, or harder to find, than host plants in monocultures (Root 1973). The enemies hypothesis has been partially supported by a review by Andow (1991) in which he found that 43% of predator species and 75% of parasitoid species had higher densities in polycultures than in monocultures. However, the review and studies by Björkman et al. (2010), Adati et al. (2011), and others also provide evidence that resource concentration is the most important factor for herbivore colonization of a field in some crop systems (Andow 1991, Björkman et al. 2010, Adati et al. 2011). Integration of floral resources would diversity the field and could lead to an

32 increase in natural enemy community or a reduction in pest colonization, as predicted by these two hypotheses.

Use of selective or biorational insecticides can also increase parasitism or predation of caterpillars in contrast to the use of broad-spectrum insecticides that reduce the number of parasitoids and predators within the field (Croft and Brown 1975, Hassan et al. 1998, Cordero et al. 2007, Desneux et al. 2007, Bommarco et al. 2011). An increase in parasitism could lead to a decrease in pest density over time if parasitoids exert enough control. Studies on parasitoid wasps of cabbage pests have established that many common insecticides are highly toxic to wasps and can cause up to 100% mortality after

24 hours of exposure in lab bioassays (Idris and Grafius 1993, Shi et al. 2004, Xu et al.

2004, Haseeb et al. 2005, Cordero et al. 2007). Whether the interactions of insectary strips and insecticides can affect caterpillar pest density and parasitism in cole crops has not been reported in published studies to date.

Cabbage, Brassica oleracea L., is a crop that is well suited to biological control because it is attacked by a variety of Lepidopteran pests that are attacked by a variety of parasitoids. The three most common caterpillar pests are imported cabbageworm Pieris rapae L. (Lep.: Pieridae), diamondback moth Plutella xylostella L. (Lep.: Plutellidae), and cabbage looper Trichoplusia ni Hübner (Lep.: Noctuidae). These pests are typically controlled in commercial fields through the application of broad-spectrum insecticides as determined by action thresholds (Hines and Hutchison 2001, Precheur et al. 2012,

Caldwell et al. 2005). However, since many conventional broad-spectrum insecticides are known to negatively affect parasitoids, use of more selective biorational insecticides may

33 increase parasitoids’ contribution to caterpillar control (Cordero et al. 2007, Bommarco et al. 2011, Kawazu et al. 2011).

The objectives of this study were to determine: 1) the effects of sweet alyssum insectary strips on caterpillar density and parasitism rate in cabbage; 2) the effects of two classes of insecticides on pest density and parasitism rate; and 3) the composition of the natural enemy community found in sweet alyssum insectary strips. Sweet alyssum

(Lobularia maritima L.) was selected as the floral resource due to its parasitoid- accessible nectaries, long flowering period, and its use in other biological control studies

(Johanowicz and Mitchell 2000, Keller and Baker 2002, Al-Doghairi and Cranshaw

2004, Hogg et al. 2011b, Sivinski et al. 2011). The primary hypothesis of this study was that access to floral resources and treatment with insecticide would affect pest density and parasitism; parasitism would be higher in plots treated with biorational insecticides than those treated with broad-spectrum insecticides and pest density would be lower as a result, as predicted by the enemies hypothesis. Access to floral resources would also increase parasitism and decrease pest density. Cole crops like cabbage are ideal for testing the effectiveness of integrated pest management techniques because pests are usually present in large numbers and the parasitoid community is known to be diverse and relatively common.

2.2 Methods

2.2.1 Field trials at agricultural research stations

A field trial was conducted in 2011 and 2012 using a split plot experimental design with a randomized complete block within each main plot. The main factor in each plot

34 was the presence or absence of sweet alyssum (Lobularia maritima L.) insectary strips.

The subplot factor was the type of insecticide treatment used. One replicate consisting of two main plots of cabbage was located at each of three farms: the Ohio Agricultural

Research and Development Center Muck Crop Agricultural Research Station

(OARDC/MCARS) in Celeryville, OH; the OARDC North Central Agricultural Research

Station (OARDC/NCARS) in Fremont, OH; and the Ohio State University’s Waterman

Agricultural and Natural Resources Laboratory (WANRL) in Columbus, OH. In 2012, an additional replicate was planted at the WANRL site.

There were four replicate blocks of three subplots in each main plot. Each main plot had 18 rows of cabbage with 120 plants in each row. Strips of sweet alyssum were planted in one of the main plots at each location, selected at random. One alyssum strip was planted every six rows within the field and one strip was planted on each of the longest sides of the main plot (Figure 1). The main plot treatment without alyssum strips had bare ground in place of the alyssum. Main plots were separated by a minimum of 300 meters to reduce parasitoid dispersal which could confound treatment differences. Each replicate block was separated by a 3 meter alley at NCARS and MCARS but not at

Columbus due to space constraints. Main plot dimensions were 46 meters by 24.5 meters at NCARS and MCARS, and 30.5 meters by 24.5 meters at WANRL.

Cabbage (Brassica olaracea capitata L. cv. ‘Bravo’, Rispens Seeds, Inc. Beecher,

Illinois) and sweet alyssum (cv. ‘Carpet of Snow’, Ferry Morse Seed Company, Fulton,

Kentucky) seeds were grown in 200 cell plastic plug trays (Hummert International, Earth

City, Missouri) filled with Metro-Mix 260 (Hummert International, Earth City, Missouri)

35 in a greenhouse for five-six weeks prior to transplanting into the field. No pesticide was applied before transplanting. Cabbage and alyssum plugs were transplanted between 22-

26 May 2011 and 11-15 May 2012. Rows were spaced 0.76 meter apart with 0.3 meter spacing within rows at Fremont and Celeryville. Between row spacing was 1.07 meters and within-row spacing was 0.36 meter at Columbus due to the dimensions of the field equipment.

Each subplot consisted of two rows of cabbage flanked by two untreated guard rows on either side to reduce spray drift. There were thirty plants per row in each subplot.

There were four replicate blocks of three subplots in each main plot. Subplots were of equal size in each replicate block. B.t. and cyfluthrin were the two pesticide treatments used; each replicate block also had an untreated subplot. Beta-cyfluthrin (Baythroid XL,

Bayer CropScience, Research Triangle Park, North Carolina) was applied at the highest label rate of 233 ml per hectare. B.t. (DiPel DF and Xentari DF, Valent BioSciences,

Libertyville, Illinois) was also applied at the highest label rate of 1.12 kg per hectare for

DiPel and 1.68 kg per hectare for Xentari. Subplots were treated every two weeks with cyfluthrin or every week with B.t., alternating DiPel and Xentari, starting two weeks after transplanting and continuing until harvest. All insecticides were applied with a tractor drawn boom sprayer at Fremont and Celeryville, and with a backpack sprayer at

Columbus. The pre-emergent herbicide s-metolachlor (Dual Magnum, Syngenta Crop

Protection, Inc., Greensboro, North Carolina) was used immediately after transplanting.

At Fremont and Celeryville, fields were treated once per week with fungicide (Kocide

2000, DuPont, Wilmington, Delaware). Weeds were removed by cultivation and hoeing.

Due to drought conditions in 2012, fields at Fremont and Celeryville were irrigated throughout the growing season and fields at Columbus were occasionally irrigated while plants were young.

Fields were sampled every week to determine pest density. One of the two treated rows was scouted each week to determine the presence of eggs, larvae, and pupae of

Lepidopteran pests by visual plant inspection. Ten randomly selected plants were visually inspected in each subplot for the first three weeks, with a decrease as the plants increased in sizeto eight plants for two weeks and then six plants for the remainder of the growing season. The same row was used for scouting each week at each site. All pests encountered were classified and recorded by species, relative size, and life stage. The presence of parasitoids or predators was also noted. The second treated row was used as a collection site for caterpillars to determine the rate of parasitism for each species. Every week in 2011 or every two weeks in 2012, randomly selected plants were thoroughly surveyed and all pupae, pre-pupae, and second instar or larger larvae were collected in small plastic Petri dishes and stored in a cooler while in transit to the laboratory. The number of plants surveyed was always the same as the number of plants visually inspected. Collected larvae and pupae were transported back to the laboratory for observation within five hours of collection. Individuals were separated based on species, with a maximum of three larvae in 50 mm x 9 mm Petri dishes and six larvae in 100 mm x 15 mm Petri dishes to minimize cannibalism and transmission of disease. Individuals were provided with fresh cabbage leaves daily and reared until the emergence of either parasitoid or adult pest. Larvae and pupae were held for up to thirty days in ambient light

37 and temperature conditions. The number of dead larvae and emerged adult pests or parasitoids was recorded each day. Parasitoid adults were identified to species based on morphological descriptions and known ranges described by Azidah et al. 2000, Fitton and

Walker 1984, and McDonald and Kok 1992; voucher specimens were preserved in 80% ethanol and are available in the Museum of Biological Diversity at the Ohio State

University. Parasitoid specimens were sent to the Systematic Entomology Laboratory at

USDA-ARS in Beltsville, Maryland for identification confirmation. Sampling and surveying began two weeks after transplanting and ended the week before harvest in

August.

2.2.2 Harvest evaluation

At harvest, ten randomly selected heads from the row used for scouting were weighed and evaluated for damage using the Greene scale of marketability (Greene et al.

1969). In 2012, an additional evaluation for damage by the onion thrips (Thrips tabaci

Lindeman (Thysanoptera: Thripidae) was conducted on a subsample of three heads from each subplot. Ten layers of each cabbage head were stripped and evaluated for thrips damage individually; each layer was rated on a scale of 0-5, with 0 indicating no damage and 5 indicating severe damage, similar to Stoner and Shelton (1988).

2.2.3 Alyssum vacuum sampling

Vacuum samples were collected from sweet alyssum insectary strips in 2012 to determine what arthropods were present and whether community composition changed over time. A vacuum (Craftsman 25cc/1.5 cu. in. 2-cycle 200 mph gasoline powered blower/vac, model no. 358.794701, Sears Brands Management Corporation, Hoffman

Estate, Illinois) was adapted to hold fine-mesh collection bags. Panty hose (Hanes too

Day Sheer Knee Highs, Hanesbrands, Inc. Winston-Salem, North Carolina) or a small one-mm mesh bag was secured to the opening of the vacuum tube via a collection tube made of cardboard. Four vacuum samples were taken every two weeks from each alyssum main plot at each field site. Each sample was collected from one thirty foot strip of alyssum in each replicate, so that 25% of the alyssum present in the main plot was sampled during each collection event. Vacuum sampling of a strip was conducted by walking slowly down both sides of the strip with the vacuum tube held vertically over the plants at a height just below the majority of the flowers. Strips were randomly selected.

Samples were stored in a freezer. The contents of each sample were examined to determine the number and life stage of pests, predators, and parasitoids. Parasitoids were identified to species and predators were identified to family. Voucher specimens were preserved in 80% ethanol and deposited at the Museum of Biological Diversity at the

Ohio State University.

2.2.4 Data analysis

A repeated measures analysis of variance (ANOVA) was performed on weekly pest scouting data using PROC MIXED in Statistical Analysis Software (SAS) (SAS

Institute, Cary, NC). Main plots with or without alyssum were compared in 2011 and

2012. Subplots within each main plot were also compared. PROC NLMIXED in SAS was used to determine interaction effects. Parasitism was analyzed using logistic regression in SAS. Both total parasitism and species-specific parasitism were analyzed unless species appeared too sporadically to evaluate. Parasitism rates were pooled over

39 all sites for both years because the small sample size on some dates would have negatively influenced the power of the statistical analyses.

A one-way ANOVA with PROC MIXED in SAS was used to analyze differences in damage caused by caterpillars and by thrips. Yield was also analyzed with a one-way

ANOVA. For the alyssum vacuum samples, differences in natural enemy community composition were analyzed using PROC GLM in SAS.

2.3 Results

2.3.1 Caterpillar density

In 2011, caterpillar density ranged from 0 to 2.33 caterpillars per plant with a peak mid season (Figure 2). Density was relatively stable throughout the season and tended to remain under one caterpillar per plant even in untreated subplots. Caterpillar density may have been low due to the relatively high level of precipitation in Ohio in

2011. Subplots treated with B.t. and cyfluthrin never had more than a mean of 0.25 caterpillar per plant on any given date. Caterpillars were more abundant in untreated subplots than in either treated subplot (Figure 3). Imported cabbageworm and diamondback moth were the primary pests encountered. Cabbage looper was encountered infrequently in 2011 and its incidence was analyzed separately from other species in 2012 only.

Alyssum insectary strips did not significantly affect diamondback moth density

(F1,12 = 1.51, P = 0.24) or imported cabbageworm density (F1,12 = 0.56, P = 0.47) in 2011

(Figure 2). However, there was a consistent trend of lower density in alyssum main plots than in main plots with no alyssum for both species. Diamondback moth density was

40 higher in untreated subplots than in insecticide-treated subplots (F2,12 = 8.73, P = 0.005) but imported cabbageworm density was not. Pest density did not differ between subplots treated with cyfluthrin or B.t (Figure 3).

Caterpillar density was higher overall in 2012 than in 2011, ranging from 0 to 5.3 caterpillars per plant. The number of caterpillars encountered increased from 434 diamondback moth larvae in 2011 to 2236 in 2012. Cabbage looper was encountered in significant numbers (Figure 4). Unlike the previous year, diamondback moth density was significantly higher in alyssum main plots than in control plots (F1,18 = 9.92, P = 0.005)

(Figure 5). At one location, alyssum was not in bloom for the last three weeks of the study and those late-season data were excluded from analyses. Imported cabbageworm and cabbage looper density was higher overall in alyssum main plots, but the difference was not statistically significant for either species (Figure 5). Diamondback moth, imported cabbageworm, and cabbage looper were significantly more abundant in untreated subplots than treated subplots (Figure 6). When all caterpillar species were pooled, density was significantly higher in alyssum main plots in 2012 (F1,18 = 6.34, P =

0.02). Though untreated subplots held more caterpillars, there was again no difference between B.t. and cyfluthrin subplots.

2.3.2 Parasitism

The overall rate of parasitism was relatively high (>30%) in both years of the study. Few caterpillars were recovered from B.t. and cyfluthrin subplots and some of those collected died before parasitoid or pest emergence, leading to a small sample size relative to untreated subplots.

Mean diamondback moth parasitism in main plots was 40.6% in alyssum plots and 37.7% in control plots in 2011 (Table 1). Parasitism in 2012 was somewhat lower:

36.4% in alyssum plots and 30.3% in control plots. The presence of alyssum insectary strips had no effect on overall percent parasitism in 2011 (χ2 = 1.23, df = 1, P = 0.268) but significantly increased percent parasitism in 2012 (χ2 = 4.52, df = 1, P = 0.034).

Treatment had a significant effect on overall percent parasitism; untreated subplots had a higher rate of parasitism relative to treated subplots in 2011 (χ2 = 6.16, df = 2, P = 0.046)

(Figure 7a) and 2012 (χ2 = 10.9, df = 2, P = 0.0043) (Figure 8a). However, there was no difference between subplots treated with B.t. and subplots treated with cyfluthrin.

Diadegma insulare contributed the most to parasitism in all subplots but was more common in untreated subplots. Incidence of all parasitoid species was higher in untreated subplots than in either treated subplot (Table 2). Oomyzus sokolowskii was the only species significantly affected by the presence of alyssum insectary strips; significantly more diamondback moth larvae were parasitized by it in alyssum main plots than in control main plots in 2012 (F1,12 = 5.76, P = 0.022) (Table 1).

Imported cabbageworm parasitism was unable to be analyzed in 2011 due to small sample size for some subplots. Mean parasitism was highest in untreated subplots at 26.7% (Table 2). The small sample size from treated subplots could have caused differences between treatments to be less clear than for diamondback moth. Parasitism of both species tended to increase over time in the alyssum plot but not in the control plot in

2011. Imported cabbageworm parasitism was not significantly different between main plots in 2012 (χ2 = 3.05, df = 1, P = 0.081) but tended to be higher in plots without

42 alyssum insectary strips (Table 1). Subplot treatment also did not have a significant effect on percent parasitism (Figure 8b). Cotesia glomerata attacked more imported cabbageworm in alyssum main plots than in control plots in 2012 (F1,12 = 4.64, P =

0.048) (Table 1). Pteromalus puparum was also significantly more abundant in alyssum main plots (F1,12 = 6.3, P = 0.003).

In 2011, only six cabbage loopers were collected from the field and none were parasitized, thus no analysis was performed. Cabbage looper parasitism in 2012 tended to be higher in alyssum main plots and untreated subplots, but differences were not significant (Table 1). No species of cabbage looper parasitoid was affected by the presence or absence or alyssum insectary strips, and unlike other species parasitism tended to be higher in subplots treated with cyfluthrin, though this was not significant

(Figure 8c).

2.3.3 Harvest

Cabbage yields in 2011 and 2012 were not significantly different between main plots or subplots (Table 3, Table 4). There were no differences in caterpillar damage rating between subplots treated with B.t. and cyfluthrin in either main plot in 2011, though untreated subplots had a significantly higher mean damage rating (3.78 vs. 1.68 mean for treated subplots) (F2,66 = 66.6, P < 0.0001). Cabbage damage ratings in 2012 were also not significantly different between main plots but were again significantly higher in untreated subplots than in either treated subplot (Table 4).

In 2012, the average thrips damage rating was not different between main plots

(F1,90 = 0.00, P = 0.996) but was significantly lower in subplots treated with cyfluthrin

43 than in untreated subplots (F2,90 = 3.18, P = 0.043) (Table 4). B.t. subplots were not significantly different from either subplot. Depth of damage and number of layers rated 3 or above did not differ between subplots or main plots.

2.3.4 Alyssum vacuum sampling

Parasitoids, predators, and pests were collected in the alyssum insectary strips.

The most common predators were Hemipterans; Orius insidiosus Say (Hem.:

Anthocoridae) was the most numerous; individuals from families Geocoridae, Nabidae, and Reduviidae were also collected. Other predators included Coccinellidae,

Chrysopidae, and Syrphidae (Table 5). Diamondback moth adults and larvae were also numerous in the vacuum samples. Seven parasitoid species known to attack cabbage caterpillars were collected; P. puparum was the most common species collected (Figure

The composition of the parasitoid community recovered during vacuum sampling was different than the parasitoid community that emerged from collected caterpillars.

Only seven species were found in vacuum samples compared to eleven species that emerged from caterpillars in 2012 (Figure 9). No tachinid parasitoids were collected, though they might have been present but too mobile to collect with the vacuum.

Replicate locations were similar in diversity of natural enemies with the exception of Syrphid predators, which were significantly more abundant in Fremont than in any other site (F3,8 = 77.0, P < 0.0001). The only pests encountered in the samples were diamondback moth adults and larvae; Columbus South had the fewest pests and Fremont

44 had the highest number of pests, but this did not appear to be related to natural enemy density or abundance (Table 5).

2.4 Discussion

Parasitism rates for diamondback moth were consistent over both years, indicating that alyssum does have a positive impact on percent parasitism of this species.

However, the increase in parasitism was not correlated with a decrease in pest density.

Parasitism for imported cabbageworm and cabbage looper was more variable between the two years, but still did not affect trends in pest density.

The differences between the two study years may have been caused in part by the weather. 2011 was one of the rainiest years on record in Ohio, while 2012 was a drought year. In a drought year, the provision of alyssum flowers in irrigated fields could have attracted more adult pests to the field if wildflower nectar loads were diminished due to dry conditions, which would have contributed to the increase in pest density in those main plots.

It was surprising that parasitism in subplots treated with B.t. was more similar to parasitism in cyfluthrin subplots than to the untreated subplots. Parasitoid mortality is known to be unaffected by B.t., so the low level of parasitism seen seems to indicate that parasitism is more affected by pest density than by insecticide. If parasitism is density- dependent then usage of any insecticide, even parasitoid-safe insecticides, would decrease percent parasitism. This has implications for the use of insecticides in integrated pest managements systems; even insecticides deemed safe for natural enemies may

45 decrease the contribution of parasitoids to pest control. This could be managed by less frequent use of B.t. or use of B.t. at lower application rates.

Pest density of all three species in B.t. and cyfluthrin plots never differed significantly, though the number of pests found in B.t. subplots tended to be higher. Pest density in subplots treated with B.t. was never significantly higher than density in cyfluthrin subplots, despite having no difference in parasitism rate. This is surprising because many growers are afraid to rely solely on B.t. or other microbial insecticides due to concerns about its efficacy in reducing pest density below economic thresholds. The fact that all three major species were adequately controlled by B.t. will be of interest to growers who may be hesitant to reduce use of broad-spectrum insecticides. Since parasitism was similar between the two treatments, growers may find that using B.t. in place of cyfluthrin would give them similar yields while reducing applicator exposure to toxic chemicals. However, growers may still need to use some conventional insecticides due to other pest concerns like thrips.

The presence of predators and parasitoids in the alyssum insectary strips indicates that the plant was attractive and provided some nectar or prey resources for natural enemies. Parasitoids were seen feeding on the alyssum and were captured in significant numbers, but provision of alyssum did not influence parasitism enough that pest density was also affected. It is known that parasitoids are very mobile; the nectar provided by the alyssum could have allowed them to travel further in search of other prey (Schellhorn et al. 2008). There were also differences in parasitoid ratio between parasitoids collected in alyssum vacuum samples and parasitoids that emerged from collected caterpillars,

46 indicating that some species may be more attracted to alyssum than others. This may account for a lack of dramatic changes in parasitism and pest density. If the most effective host searchers like D. insulare are not as attracted by alyssum than less abundant species like Conura spp., this flower species may not be the optimal one to integrate into the field.

Future research should focus on insectary strip deployment, such as the ratio of crop rows to insectary rows or whether complete rows of insectary are needed. Use of insectary mixes as opposed to one type of flower may aid in attracting more parasitoid species while reducing any positive effects of nectar provision on pest species. Changes in insecticide usage, particularly B.t., may also increase parasitism rate while continuing to keep pest density below action thresholds.

2.5 References

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Al-Doghairi, M. A., and W. S. Cranshaw. 2004. The effect of interplanting of necteriferous plants on the population density and parasitism of cabbage pests. Southwestern Entomologist. 29: 61–68.

Andow, D. A. 1991. Vegetational diversity and arthropod population response. Annual Review of Entomology. 36: 561–586.

Bommarco, R., F. Miranda, H. Bylund, and C. Björkman. 2011. Insecticides suppress natural enemies and increase pest damage in cabbage. Journal of Economic Entomology. 104: 782–791.

Caldwell, B., E. B. Rosen, E. Sideman, A. M. Shelton, and C. D. Smart. 2005. Resource Guide for Organic Insect and Disease Management. http://www.nysaes.cornell.edu/pp/resourceguide/index.php.

Croft, B. A., and A. W. Brown. 1975. Responses of arthropod natural enemies to insecticides. Annual Review of Entomology. 20: 285–335.

Desneux, N., A. Decourtye, and J.-M. Delpuech. 2007. The sublethal effects of pesticides on beneficial arthropods. Annual Review of Entomology. 52: 81–106.

Fiedler, A. K., and D. A. Landis. 2007. Attractiveness of Michigan native plants to arthropod natural enemies and herbivores. Environmental Entomology. 36: 751– 765.

Fiedler, A. K., D. A. Landis, and S. D. Wratten. 2008. Maximizing ecosystem services from conservation biological control: The role of habitat management. Biological Control. 45: 254–271.

Fitton, M. and A. Walker. 1984. Hymenopterous parasitoids associated with diamondback moth: the taxonomic dilemma. In: Diamondback Moth and Other Crucifer Pests: Proceedings of the Second International Workshop. Ed. N.S. Talekar. 225-232.

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Hines, R. L. and W. D. Hutchison. 2001. Evaluation of action thresholds and spinosad for Lepidopteran pest management in Minnesota cabbage. Journal of Economic Entomology. 94: 190-196.

Hogg, B. N., R. L. Bugg, and K. M. Daane. 2011. Attractiveness of common insectary and harvestable floral resources to beneficial insects. Biological Control. 56: 76–84.

Hogg, B. N., E. H. Nelson, N. J. Mills, and K. M. Daane. 2011. Floral resources enhance aphid suppression by a hoverfly. Entomologia Experimentalis et Applicata. 141: 138–144.

Hooks, C. R. R., and M. W. Johnson. 2003. Impact of agricultural diversification on the insect community of cruciferous crops. Crop Protection. 22: 223–238.

Kawazu, K., T. Shimoda, and Y. Suzuki. 2011. Effects of insecticides on the foraging behaviour and survival of Cotesia vestalis, a larval parasitoid of the diamondback moth, Plutella xylostella. Journal of Applied Entomology. 135: 647–657.

Landis, D. A., S. D. Wratten, and G. M. Gurr. 2000. Habitat management to conserve natural enemies of arthropod pests in agriculture. Annual Review of Entomology. 45: 175–201.

Lee, J. C., and G. E. Heimpel. 2005. Impact of flowering buckwheat on Lepidopteran cabbage pests and their parasitoids at two spatial scales. Biological Control. 34: 290–301.

McDonald, R. C. and L. T. Kok. 1992. Colonization and hyperparasitism of Cotesia rubecula (Hym.: Braconidae), a newly introduced parasite of Pieris rapae, in Virginia. Entomophaga. 37: 223-228.

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Wäckers, F. L. 2004. Assessing the suitability of flowering herbs as parasitoid food sources: flower attractiveness and nectar accessibility. Biological Control. 29: 307– 314.

2.6 Tables

Table 1: Percent parasitism of P. xylostella, P. rapae, and T. ni by main plot factor in Ohio cabbage field trials, 2011 and 2012.

Percent parasitism (N)* 2011 2012 Species Alyssum No alyssum Alyssum No alyssum Plutella xylostella 40.6 (143) 37.7 (183) 36.4 (459) 30.3 (228) Diadegma insulare 32.9 (47) 31.1 (57) 21.4 (98) 24.6 (56) Oomyzus sokolowskii 7.7 (11) 6.0 (11) 13.7 (63) 4.4 (10) Cotesia plutellae - 0.5 (1) - 0.9 (2) Conura spp. - - 1.3 (6) 0.4 (1) Pieris rapae 32.4 (173) 15.4 (143) 33.9 (274) 44.7 (206) Cotesia rubecula 12.1 (21) 8.4 (12) 8.8 (24) 21.8 (45) Cotesia glomerata 3.5 (6) 0.7 (1) 5.1 (14) 3.8 (8) Pteromalus puparum 5.2 (9) 0.7 (1) 11.7 (32) 6.8 (14) Hyper. Tetrastichus galactopus 9.2 (16) 3.5 (5) 4.0 (11) 7.3 (15) Hyper. Conura spp. 2.3 (4) 2.1 (3) 1.5 (4) 0.5 (1) Trichoplusia ni 0.0 (4) 0.0 (2) 20.9 (129) 26.9 (93) Voria ruralis - - 20.2 (26) 23.7 (22) Copidosoma floridanum - - 0.8 (1) 3.2 (3) * Total number of each pest species collected or each parasitoid species emerged.

Table 2: Percent parasitism of P. xylostella, P. rapae, and T. ni by subplot factor in Ohio cabbage field trials, 2011 and 2012.

Percent parasitism (N)* 2011 2012 Species Cyfluthrin B.t. Untreated Cyfluthrin B.t. Untreated Plutella xylostella 36.0 (50) 26.3 (57) 43.1 (218) 19.6 (51) 20.9 (86) 37.7 (536) Diadegma insulare 28.0 (14) 21.1 (12) 35.8 (78) 7.8 (4) 15.1 (13) 24.4 (131) Oomyzus sokolowskii 8.0 (4) 5.3 (3) 6.9 (15) 9.8 (5) 4.7 (4) 11.9 (64) Cotesia plutellae - - 0.5 (1) - - 0.4 (2) Conura spp. - - - 2.0 (1) 1.2 (1) 0.9 (5) Pieris rapae 9.1 (11) 0.0 (20) 26.7 (285) 9.1 (11) 50.0 (10) 38.2 (458) Cotesia rubecula - - 11.2 (32) - 10.0 (1) 14.0 (64) Cotesia glomerata - - 2.7 (7) - - 4.8 (22) Pteromalus puparum 9.1 (1) - 3.2 (9) 9.1 (1) 20.0 (2) 9.4 (43) 53 Compsilura concinnata - - - - 20.0 (2) 3.3 (15)

Hyper. Tetrastichus galactopus - - 7.4 (21) - - 5.7 (26) Hyper. Conura spp. - - 2.5 (7) - - 1.1 (5) Trichoplusia ni 0.0 (1) 0.0 (3) 0.0 (2) 33.3 (21) 20.0 (55) 23.3 (146) Voria ruralis - - - 28.6 (6) 16.4 (9) 22.6 (33) Copidosoma floridanum - - - 4.8 (1) 3.6 (2) 0.7 (1) * Total number of each pest species collected or each parasitoid species emerged.

Table 3: Yield and caterpillar damage rating based on Greene scale per plant by main plot and subplot factor in Ohio cabbage field trial in 2011.

Caterpillar Caterpillar damage damage Main plot Yield (kg)* rating* Subplot Yield (kg)** rating ** Cyfluthrin 1.35a 1.64a Alyssum 1.37a 2.36a B.t. 1.29a 1.62a Untreated 1.13a 3.80b Cyfluthrin 1.65a 1.74a No alyssum 1.57a 2.42a B.t. 1.68a 1.74a Untreated 1.53a 3.78b * Within a column, means followed by the same letter are not significantly different between main plots. ** Within a column, means followed by the same letter are not significantly different between subplots within a main plot.

Table 4: Yield, caterpillar damage rating based on Greene scale, and thrips damage rating per plant by main plot and subplot factor in Ohio cabbage field trial in 2012.

Caterpillar Thrips Caterpillar Thrips Yield damage damage Yield damage damage Main plot (kg)* rating* rating* Subplot (kg)** rating** rating** Cyfluthrin 1.83a 1.80a 2.76a Alyssum 1.55a 2.78a 3.25a B.t. 1.83a 2.04a 2.86a Untreated 1.59a 4.46b 4.11b Cyfluthrin 1.97a 1.68a 2.88a No 1.57a 2.68a 3.25a B.t. 1.82a 2.01a 3.37b alyssum Untreated 1.63a 4.32b 3.50b * Within a column, means followed by the same letter are not significantly different between main plots. ** Within a column, means followed by the same letter are not significantly different between subplots within a main plot.

Table 5: Abundance and diversity of pests and predators collected from vacuum samples of alyssum insectary strips in four cabbage field locations in Ohio, 2012.

Pests Predators by family Plutellidae Plutellidae Location adult larvae Coccinellidae Chrysopidae Anthocoridae Geocoridae Nabidae Reduviidae Syrphidae Columbus 181 156 29 2 29 46 52 1 1 North

Columbus 50 23 4 4 136 6 22 5 4 South

Fremont 97 305 5 12 92 0 27 0 88

Celeryville 168 160 - 4 76 12 2 0 7

2.7 Figures

Figure 1: Layout of alyssum main plot in Ohio cabbage field trial, 2011 and 2012. The arrow indicates the direction of the rows

0.6 A) Diamondback moth Alyssum 0.5 No alyssum

0.4

0.3

0.2

No. of caterpillars/plant of No.

0.1

0.0 B) Imported cabbageworm

0.5

0.4

0.3

0.2

No. of caterpillars/plant of No.

0.1

0.0 06/08/13 06/22/13 07/06/13 07/20/13 08/03/13 08/17/13

Date

Figure 2: P. xylostella (A) and P. rapae (B) density over time by main plot treatment in Ohio cabbage field trial in 2011. Error = SEM.

1.0 A) Diamondback moth Cyfluthrin B.t. 0.8 Untreated

0.6

0.4

No. of caterpillars/plant of No.

0.2

0.0 B) Imported cabbageworm

0.8

0.6

0.4

No. of caterpillars/plant of No.

0.2

0.0 06/08/11 06/22/11 07/06/11 07/20/11 08/03/11 08/17/11

Date

Figure 3: P. xylostella (A) and P. rapae (B) density over time by subplot treatment in Ohio cabbage field trial in 2011. Error = SEM.

Figure 4: Species composition and relative abundance of caterpillars encountered during visual inspection surveys in Ohio cabbage field trial in 2011 (A) and 2012 (B).

1.6 A) Diamondback moth 1.4 1.2 Alyssum No alyssum 1.0 0.8 0.6 0.4

No.of caterpillars/plant 0.2 0.0 B) Imported cabbageworm 1.4 1.2 1.0 0.8 0.6 0.4

No. of caterpillars/plant of No. 0.2 0.0 C) Cabbage looper 1.4 1.2 1.0 0.8 0.6 0.4

No.of caterpillars/plant 0.2

0.0 05/29/12 06/12/12 06/26/12 07/10/12 07/24/12 08/07/12 Date

Figure 5: P. xylostella (A), P. rapae (B), and T. ni (C) density over time by main plot treatment in Ohio cabbage field trial, 2012. Error = SEM.

3.0 A) Diamondback moth

2.5 Cyfluthrin B.t. 2.0 Untreated

1.5

1.0

No. of caterpillars/plant of No. 0.5

0.0 B) Imported cabbageworm 2.5

2.0

1.5

1.0

No. of caterpillars/plant of No. 0.5

0.0 C) Cabbage looper 2.5

2.0

1.5

1.0

No. of caterpillars/plant of No. 0.5

0.0 05/29/12 06/12/12 06/26/12 07/10/12 07/24/12 08/07/12

Date

Figure 6: P. xylostella (A), P. rapae (B), and T. ni (C) density over time by subplot treatment in Ohio cabbage field trial in 2012. Error = SEM.

100 A) Diamondback moth 90

60 a 50 a

40 a Percent parasitism Percent 30

0 B) ImportedCyfluthrin cabbageworm B.t. Untreated 90 Subplot 80

Percent parasitism Percent 30

0 Cyfluthrin B.t. Untreated

Subplot

Figure 7: Percent parasitism of P. xylostella (A) and P. rapae (B) in subplot treatments in Ohio cabbage field trial in 2011. P. rapae parasitism rates by subplot were unable to be statistically analyzed due to small sample size. Error = SEM. Within each species graph, bars with the same letters to not differ significantly (P < 0.05).

100 A) Diamondback moth 90 80 70 60 50 a 40 b b 30

Percent parasitism Percent 20 10 0 90 B) ImportedCyfluthrin cabbagewormB.t. Untreated 80 Subplot 70 a 60 50 a a 40 30

Percent parasitism Percent 20 10 0 1 2 3 90 C) Cabbage looper 80 Subplot 70 60 50 a 40 a a 30

Percent parasitism Percent 20 10 0 Cyfluthrin B.t. Untreated

Subplot

Figure 8: Percent parasitism of P. xylostella (A), P. rapae (B), and T. ni (C) in subplot treatments in Ohio cabbage field trial in 2012. Error = SEM. Within each species graph, bars with the same letters to not differ significantly (P < 0.05). 64

Figure 9: Parasitoid species abundance and diversity based on vacuum samples of alyssum insectary strips at four Ohio cabbage field trial locations in 2012.

Chapter 3: Caterpillar parasitoid diversity and abundance in cabbage fields in northern Ohio

3.1 Introduction

Caterpillar pests cause extensive damage of cabbage and other cole crops, which causes growers to use tactics to suppress caterpillars as a critical part of cole crop management. The most economically important species in the North Central region of the

United States are Pieris rapae L. (Lepidoptera: Pieridae), Plutella xylostella L.

(Lepidoptera: Plutellidae), and Trichoplusia ni Hübner (Lepidoptera: Noctuidae) (Mahr et al. 1993, Welty 2009). On large commercial farms, these pests are primarily managed through the application of broad-spectrum insecticides based on action thresholds or calendar dates (Caldwell et al. 2005, Hines and Hutchison 2001).

In cole crops, there are currently many self-sustaining parasitoid populations in

North America, with at least fifteen species known to attack the three most common species of caterpillar pest (Chamberlin and Kok 1986, Xu et al. 2001, Shelton et al. 2002,

Wold-Burkness et al. 2005). Despite their numbers, parasitoids alone often fail to provide an adequate level of control to meet commercial growers’ standards (Biever et al. 1992,

Sarfraz et al. 2005). The composition of natural enemy communities, particularly parasitoid complexes, varies by region and year (Janowska and Wiech 2006, Wold-

Burkness et al. 2005, Kfir 1997, Shelton et al. 2002). Wasp and tachinid parasitoids are 66 the most effective biological control agents for cabbage caterpillar pests and have been released in many different locations over the course of the last century (Xu et al. 2001,

Wold-Burkness et al. 2005). Some have failed to establish or have had their efficacy reduced by hyperparasitoids, but most have successfully established either regionally or nationally (McDonald and Kok 1992).

No formal survey of cabbage pest parasitoids had been conducted in Ohio. The most recent survey in the North Central region was conducted in Minnesota in the mid

2000’s by Wold-Burkness et al. (2005), who collected data from 1991 to 2003, although historical data from as early as 1968 were also used. Larvae, pre-pupae, and pupae were collected multiple times each year to determine the species composition and overall parasitism rate (Wold-Burkness et al. 2005). Diadegma insulare Cresson (Hymenoptera:

Ichneumonidae) was the primary parasitoid of diamondback moth in this study. P. rapae was attacked by Cotesia glomerata L. (Hymenoptera: Braconidae), Compsilura concinnata Meigan (Diptera: Tachinidae), and Cotesia rubecula Marshall (Hymenoptera:

Braconidae) while T. ni was primarily parasitized by tachinid parasitoid Voria ruralis

Fallen (Diptera: Tachinidae). Shelton et al. (2002) conducted a survey over seven years in

New York and found a similar parasitoid complex and percent parasitism, though no V. ruralis or C. rubecula were collected. However, in Virginia, Chamberlin and Kok (1986) found a higher rate of parasitism for P. rapae and collected three wasp parasitoids of T. ni that were never found in the other two locations at all. They also did not report any parasitism in diamondback moth. The differences in these studies highlight the need for periodic, region-specific surveys of parasitoids.

The objectives of this study were to determine: 1) the composition of the parasitoid community in Ohio cabbage fields and 2) the effects of insecticide treatment programs on parasitism rate and parasitoid diversity. Information on the diversity and abundance of caterpillar parasitoids is useful to growers who want to incorporate conservation biological control tactics into their management system (Shelton et al. 2002,

Sarfraz et al. 2005). Information on effects of common insecticides on parasitoid in the field would also be useful to growers, particularly organic or fresh market growers that are interested in organic or integrated pest management (Cordero et al. 2007, Bommarco et al. 2011).

3.2 Methods

3.2.1 Data collection

Commercial cabbage fields managed by seven growers or companies were surveyed once per month during the summers of 2011 and 2012 to determine parasitoid diversity and relative abundance. Fields were sampled in June, July, and August in 2011 and July,

August, and September in 2012. All fields were in northern Ohio; two were located in

Ottawa County and one in Seneca County while the rest were located in Sandusky

County (Figure 10). Most fields surveyed were managed as commercial processing cabbage and treated with conventional pesticides, but only three fields were planted with fresh market cabbage and two were managed organically. In 2011, ten fields were surveyed; in 2012, eight fields were surveyed.

Caterpillars, pre-pupae, and pupae were collected from each field for forty-five person-minutes. A maximum of 50 larvae, pre-pupae, and pupae were taken from each

68 field. Not all fields contained caterpillars at the time of survey due to insecticide treatments and weather conditions. Collected individuals were placed in 50 mm x 9 mm

Petri dishes, stored in a cooler, and transported to the laboratory within 5 hours of collection. All dishes were kept at ambient light and temperature conditions. Caterpillars and pupae were held for a maximum of thirty days. Caterpillars were provided with untreated cabbage leaves that were replaced every two days until pupation or death. To reduce mortality, caterpillars were separated by species with a maximum of three individuals in 50 mm x 9 mm Petri dishes and six individuals in 100 mm x 15 mm Petri dishes. Emergence of parasitoids or adult pests and caterpillar death was recorded each day. Parasitoids were identified to species using morphological descriptions and known ranges described by Azidah et al. 2000, Fitton and Walker 1984, and McDonald and Kok

1992. Specimens were sent to USDA-ARS in Beltsville, Maryland for confirmation.

Voucher specimens were deposited in the Museum of Biological Diversity at the Ohio

State University.

3.2.2 Data analysis

The average percent parasitism of pupae and larvae was combined. All individuals that died from causes unrelated to parasitism were excluded from the analysis. The

Shannon diversity index (Equation 1) and Hill’s N1 (Equation 2) were used to determine the abundance and relative diversity of parasitoid species that emerged from each pest species. For Shannon’s index, H’ is the average uncertainty that a random individual will belong to a given species in a community made of s* species with proportional abundances pi. The result was used to determine Hill’s N1 to measure how many species

69 were abundant in a given year (Ludwig and Reynolds 1988, Wold-Burkness et al. 2005).

Values were analyzed with Fisher’s protected LSD in Statistical Analysis Software (SAS)

(SAS Institute 2008, Cary, NC).

(1)

(2)

To determine the effects of pesticide treatment, the environmental impact quotient

(EIQ) was calculated for each surveyed field the methods of Kovach et al. (1992) and the online EIQ calculator (Kovach et al. 2012). The EIQ is a measure of the impact of pesticides on the environment and integrates a number of factors including the impact on beneficial insects (Kovach et al. 1992). Differences in EIQ and percent parasitism between the two survey years were analyzed using a one-way ANOVA in Minitab. A logistic regression was used to determine the relationship between EIQ and percent parasitism and Hill’s N1 among surveyed fields within a year.

3.3 Results

3.3.1 Parasitoid diversity and contribution to percent parasitism

Parasitoid diversity and abundance varied by location and year. Caterpillars were generally more numerous in 2011 than in 2012, possibly due to a lack of consistent insecticide application in 2011 during the growing season due to excessive rain which prevented growers from entering the field with spray equipment. Average parasitism was higher in 2011 for diamondback moth and higher in 2012 for imported cabbageworm and 70 cabbage looper. Most growers used pyrethroid or spinosyn insecticides to control caterpillars except for the two organic growers who relied on B.t.

All three common pest species were found in 2011 and 2012. P. xylostella was the most numerous species found, with 290 and 71 individuals collected in 2011 and 2012, respectively (Table 6). Average percent parasitism was 45.5% in 2011 and 23.9% in

2012. D. insulare was the most common parasitoid, accounting for 80% of P. xylostella parasitism in 2011 and 88.2% in 2012. Four other species of wasp parasitoid emerged from collected larvae: Oomyzus sokolowskii Kurdjumov (Hymenoptera: Eulophidae),

Cotesia plutellae Kurdjumov (Hymenoptera: Braconidae), Microplitis plutellae

Muesebeck (Hymenoptera: Braconidae), and Conura spp. Cresson (Hymenoptera:

Chalcidae), in order of abundance (Figure 11a). All four species were seen in 2011 but only D. insulare and O. sokolowskii were collected in 2012. It was surprising to collect C. plutellae in Ohio because it has generally been released in subtropical or tropical climates

(Talekar and Shelton 2000).

P. rapae was parasitized by Cotesia rubecula exclusively in 2011, though in 6 of the 11 parasitism events the gregarious hyperparasitoid Tetrastichus galactopus

Ratzeburg (Hymenoptera: Eulophidae) or hyperparasitoid Conura spp. emerged from the pupa. In 2012 eighteen larvae were collected and there were only four parasitism events, two caused by generalist tachinid parasitoid Compsilura concinnata Meigan (Diptera:

Tachinidae) and two by pupal parasitoid Pteromalus puparum L. (Hymenoptera:

Pteromalidae) (Figure 11b). Average percent parasitism was 15.3% in 2011 and 22.2% in

2012 (Table 6). P. rapae larvae were the least numerous of the three common caterpillar

71 pests, possibly due to this species’ greater susceptibility to insecticide relative to T. ni and

P. xylostella (Maxwell and Fadamiro 2006).

T. ni larvae were present in 2011 but were not parasitized. In 2012 they were attacked by tachinid parasitoid V. ruralis as well as polyembryonic wasp Copidosoma floridanum Ashmead (Hymenoptera: Encyrtidae) (Figure 11c). Percent parasitism was

36.6% in 2012 with 15 instances of parasitism (Table 6). The number of T. ni larvae collected was similar both years (56 in 2011 vs. 41 in 2012). V. ruralis is a generalist tachinid parasitoid that may attack alternate hosts some years (Wold-Burkness et al.

2005).

3.3.2 Hill’s N1 and EIQ

The number of abundant parasitoid species for P. xylostella, as determined by

Hill’s N1 was 2.1 in 2011 and 1.4 in 2012. P. rapae parasitoid species abundance ranged from 2.2 to 1.9 species and T. ni parasitoid species abundance was 0 in 2011 and 1.3 in

2012 (Table 7). T. ni parasitoid species abundance was only higher in 2012 due to the lack of parasitism in 2011. There were no significant differences between years by species.

The average EIQ calculated in 2011 was significantly lower than the average EIQ in 2012, indicating that the insecticide programs used in 2012 had more negative impact on the environment (F1,24 = 4.61, P = 0.042) (Table 8). In 2011, mostly pyrethroids, B.t., and imidacloprid were used; in 2012, a wider variety of insecticides including carbamate and neonicotinoids were used and treatments were applied more frequently. Percent parasitism and parasitoid abundance as measured by Hill’s N1 were also significantly

72 higher in 2011 compared to 2012 for P. xylostella (F1,20 = 8.14, P = 0.01; F1,20 = 7.18, P

= 0.014) (Table 8). Due to small sample size both years, analyses for P. rapae and T. ni were not conducted. There was not a significant correlation between EIQ and percent parasitism or Hill’s N1 value, but there was a trend of decreased parasitism and species abundance as EIQ increased.

3.4 Discussion

Some of the differences in parasitoid community composition may be due to the stark difference between 2011 and 2012 in terms of weather. 2011 had record rainfall in

Ohio during the majority of the growing season. Some growers were unable to enter the field for pesticide application at the correct times due to mud and flooding. In contrast,

2012 was a drought year, so growers were able to treat their fields with insecticide as needed; they also treated more often and caterpillars were less numerous. The increased use of insecticide may account for the decrease in diversity of diamondback moth parasitoids and the decrease in percent parasitism overall.

A high EIQ was associated with low parasitism and a decrease in parasitoid species abundance. This may be due to direct toxicity to the parasitoids or an indirect effect due to the decrease in caterpillar density that makes it difficult for less common parasitoid species to find hosts and compete against more numerous species. Although specific insecticides’ effects were not analyzed, the increased use of neonicotinoids and spirotetramat may have had an effect on parasitoid mortality; their EIQ values tend to be higher than most pyrethroid and biorational insecticides (Kovach et al. 2012).

The appearance of tachinid parasitoids in 2012 may be due to changes in abundance of the tachinids’ other hosts. Both V. ruralis and C. concinnata are generalists and can attack a wide variety of Lepidopteran species (Wold-Burkness et al. 2005). Neither species appeared to be as affected by pesticide usage as parasitoid wasps, which may indicate that they are more tolerant of pesticides, as suggested in other studies (Respicio and Forgash 1984). However, it is likely that their contribution to parasitism varies widely from year to year depending on the presence of alternate or more desirable prey, so growers can not depend on them.

There did not seem to be significant variance in parasitoid wasp species between locations surveyed, though there was some variance between years. This seems to indicate that the most common species of wasp parasitoids have stable populations in

Ohio and are likely to be present every year. In Ohio, it appears that D. insulare, C. rubecula, and O. sokolowskii are the most abundant parasitoids and control programs should be based on attracting or enhancing them specifically. Because T. ni’s primary parasitoid is a tachinid fly, control methods for this pest species should focus on surveys in early summer to determine whether or not it is present and prepare to use insecticides if it is not present.

3.5 References

Biever, K. D., R. L. Chauvin, G. L. Reed, and R. C. Wilson. 1992. Seasonal occurrence and abundance of Lepidopterous pests and associated parasitoids on

collards in the northwestern United States. Journal of Entomological Science. 27: 5– 18.

Bommarco, R., F. Miranda, H. Bylund, and C. Björkman. 2011. Insecticides suppress natural enemies and increase pest damage in cabbage. Journal of Economic Entomology. 104: 782–791.

Caldwell, B., E. B. Rosen, E. Sideman, A. M. Shelton, and C. D. Smart. 2005. Resource guide for organic insect and disease management. http://www.nysaes.cornell.edu/pp/resourceguide/index.php.

Chamberlin, J. R., and L. T. Kok. 1986. Cabbage Lepidopterous pests and their parasites in southwestern Virginia. Journal of Economic Entomology. 79: 629–632.

Fitton, M. and A. Walker. 1984. Hymenopterous parasitoids associated with diamondback moth: the taxonomic dilemma. In Diamondback Moth and Other Crucifer Pests: Proceedings of the Second International Workshop. Ed. N.S. Talekar. 225-232.

Hines, R. L. and W. D. Hutchison. 2001. Evaluation of action thresholds and spinosad for Lepidopteran pest management in Minnesota cabbage. Journal of Economic Entomology. 94: 190-196.

Kfir, R. 1997. Parasitoids of Plutella xylostella (Lep. Plutellidae) in South Africa: an annotated list. Entomophaga 42: 517–523.

Kovach, J., C. Petzoldt, J. Degnil, and J. Tette. 1992. A method to measure the environmental impact of pesticides. New York’s Food and Life Sciences Bulletin. 139.

Kovach, J., C. Petzoldt, J. Degnil, and J. Tette. 2012. EIQ calculator. http://cceeiq- lamp.cit.cornell.edu/EIQCalc/input.php.

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3.6 Tables

Percent parasitism (N)* Species 2011 2012 Plutella xylostella 45.5 (290) 23.9 (71) Diadegma insulare 36.2 (105) 21.1 (15) Oomyzus sokolowskii 4.5 (13) 2.8 (2) Cotesia plutellae 3.8 (11) - Conura spp. 0.3 (1) - Microplitis plutellae 0.7 (2) - Pieris rapae 15.3 (72) 22.2 (18) Cotesia rubecula 6.9 (5) - Pteromalus puparum - 11.1 (2) Compsilura concinnata - 11.1 (2) Hyper. Tetrastichus galactopus 6.9 (5) - Hyper. Conura spp. 1.4 (1) - Trichoplusia ni 0.0 (56) 36.6 (41) Voria ruralis - 34.1 (14) Copidosoma floridanum - 2.4 (1) * Total number of each pest species collected or each parasitoid species emerged.

Table 7: Diversity and abundance of parasitoid species emerging from collected P. xylostella, P. rapae, and T. ni larvae in commercial cabbage fields in Northern Ohio, 2011 and 2012.

Plutella xylostella Pieris rapae Trichoplusia ni Number of Number of Number of abundant abundant abundant Sum of species Sum of species Sum of species Year species (Hill’s N1) species (Hill’s N1) species (Hill’s N1) 2011 5 2.1 3 2.2 - - 2012 2 1.4 2 1.9 2 1.3

Table 8: Comparison of mean environmental impact quotient (EIQ), percent parasitism, and number of abundant species (Hill's N1) values for parasitoid species emerging from P. xylostella larvae collected from commercial cabbage fields in northern Ohio, 2011 and 2012. Error = SEM. Within a column, means followed by the same letter are not significantly different.

Environmental Number of impact quotient Percent abundant species Year (EIQ) parasitism (Hill’s N1) 2011 3.83 ± 0.86 a 50.43 ± 7.75 a 1.46 ± 0.18 a 2012 10.33 ± 2.89 b 19.55 ± 6.56 b 0.74 ± 0.20 b

3.7 Figures

Figure 10: Map of commercial fields used in commercial cabbage field surveys in northern Ohio in 2011 and 2012. Red dots indicate fields used in 2011, blue dots indicated fields used in 2012.

Figure 11: Parasitoids of P. xylostella (A), P. rapae (B), and T. ni (C) that emerged from caterpillar collected during commercial cabbage field surveys in 2011 and 2012.

Bibliography