RESISTANCE AND ALTERNATIVE MANAGEMENT TO

LINEOLARIS (PALISOT DE BEAUVOIS), , IN

STRAWBERRY GENOTYPES, FRAGARIA XANANASSA, AND RASPBERRY,

RUBUS IDEA USh.

A Thesis

Presented to

The Faculty of Graduate Studies

of

The University of Guelph

by

CYNTHIA ANN ROUGOOR

In partial fulfillment of requirements

for the degree of

Master of Science

November, 2008

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PLANT RESISTANCE AND ALTERNATIVE MANAGEMENT TO LYGUS LINEOLARIS (PALISOT DE BEAUVOIS), TARNISHED PLANT BUG, IN STRAWBERRY GENOTYPES, FRAGARIA XANANASSA, AND RASPBERRY, RUBUSIDEAUSL.

Cynthia A. Rougoor Advisors: University of Guelph, 2008 Professor A. Dale Professor R. Hallett

Tarnished plant bug feeds on developing and fruit tissue of strawberries.

The wild species Fragaria virginiana displays resistance and has been crossed with commercial day-neutral strawberries, producing resistant hybrids. The purpose of this study was to elucidate possible mechanisms of resistance and rank the resistance of hybrids. The effect of pollen load was also examined. Field trials compared four commercial day-neutral cultivars on three farms in southern on a weekly basis.

Cultivar Fort Laramie consistently had lower damage compared to other cultivars at all sites. Laboratory experiments verified resistance and assessed the relationship of pollen to fruit damage. Caged choice and no-choice bioassays determined families 703, 704 and

708 and Fort Laramie consistently showed resistant qualities and reduced damage.

Susceptible with pollen had higher damage and resistant plants did not. This study

shows resistant genotypes were consistent and that pollen load plays a role in

susceptibility. &r^

~I would like to dedicate this thesis to my grandmother, Helen Grimwood, for without her I wouldn't be the person that I am today, nor have such an appreciation of plants and . We miss you.~

1 Acknowledgments:

There are a number of people I would like to thank for their help and support during the completion of this project, I couldn't have done this without them.

Many thanks to my committee and supervisors for help with production of this manuscript. I would like to thank the cooperators who facilitated the field component,

John Cooper, Strawberry Tyme Farms Inc., Simcoe, Dan Tigchelaar, Tigchelaar Berry

Farm Inc., Vineland, Paul Watson, Watson Farms Ltd., Bowmanville.

I would like to thank Pam Fisher and Al Sullivan for their support, encouragement and help with promoting my research. Thanks to Michelle Edwards and Peter MaCaskell for their help with statistical analysis and the SAS programming. Thanks to Bruce

Broadbent and Jay Whistlecraft for their help and expertise with establishing my tarnished plant bug colony. Also, thanks to Ray Hutchisen for helping produce the cages for my bioassays. I would like to thank Wally Andres and Larry Frost for technical assistance at the Simcoe Elesearch Station.

A sincere thanks to Sarah Stephenson, my partner in crime, for all the support through the whole learning process and being the best office mate. A big thank you to

Gary Pigeau for all his help and support, when things became overwhelming you were always there to reassure me. I don't know what I would do without you.

Also, I would like to thank my family, especially my Dad, Mom and Cathy, for all their help, guidance support and understanding, thanks for being there for me.

u Chapter 1

1 Literature Review 1

1.1 Introduction 1 1.2 Strawberry Production 2 1.2.1 2 1.2.2 Botanical Description 3 1.2.3 Economics 4 1.3 Raspberry Production 4 1.3.1 Taxonomy 4 1.3.2 Botanical Description 5 1.3.3 Economics 5 1.4 Biology of the Tarnished Plant Bug 6 1.4.1 Taxonomy 6 1.4.2 Life History 6 1.4.3 Description 7 1.4.4 Distribution 9 1.4.5 Damage 11 1.5 Management of Tarnished Plant Bug 14 1.5.1 Monitoring 14 1.5.2 Cultural Control 16 1.5.3 Biological Control 18 1.5.4 Chemical Control 19 1.5.4.1 Strawberry 19 1.5.4.2 Raspberry 19 1.5.5 Host Plant Resistance 20 1.6 Mechanisms of Resistance 21 1.6.1 Antixenosis 21 1.6.2 Antibiosis 23 1.6.3 Tolerance 23 1.6.4 Damage Variation in Strawberries 24 1.7 Conclusions 26

Chapter 2

2 Comparison of the susceptibility of four commercial strawberry cultivars to tarnished plant bug 28

2.1 Abstract 28 2.2 Introduction 28 2.3 Purpose 31 2.4 Materials and Methods 31 2.4.1 Experimental Design 32 2.4.2 Field Sites 32 2.4.3 Sampling and Damage Evaluation 33

m 2.4.4 Statistical Analysis 34 2.5 Results 37 2.5.1 Field Data Results in 2006 37 2.5.2 Incidence and Temporal Distribution of Tarnished Plant Bug in 2006 42 2.5.3 Field Data Results in 2007 44 2.5.4 Incidence and Temporal Distribution of Tarnished Plant Bug in 2007 44 2.6 Discussion and Conclusions 47

Chapter 3

3 Investigating Tarnished Plant Bug Resistance in Strawberries 53

3.1 Abstract 53 3.2 Introduction 54 3.3 Objectives 60 3.4 Materials and Methods 61 3.4.1 Rearing and Maintenance 61 3.4.2 Plant Maintenance 61 3.4.3 Laboratory Trials 62 3.4.4 Experimental Design 64 3.4.4.1 Determination of Optimal Nymph Numbers 64 3.4.4.2 Determination of Susceptible Stage of Strawberry Development 65 3.4.4.3 Determination of Strawberry Fruit Susceptibility to Adult Plant Bug 65 3.4.4.4 Choice Bioassays: Resistant and Susceptible Strawberry Genotype Pairs 65 3.4.4.5 No-choice Bioassay: Resistant versus Susceptible Genotypes 66 3.4.4.6 Influence of Pollen Load on Damage Levels 66 3.4.5 Statistical Analyses 66 3.4.5.1 Determination of Optimal Nymph Numbers, Susceptible Stage of Strawberry Development and Susceptibility to Adult Plant Bug 66 3.4.5.2 Choice and No-choice Bioassays: Resistant and Susceptible Strawberry Genotypes 67 3.4.5.3 Influence of Pollen Load on Damage Levels 68 3.5 Results 68 3.5.1 Determination of Optimal Nymph Numbers 68 3.5.2 Determination of Susceptible Stage of Strawberry Development 71 3.5.3 Determination of Strawberry Fruit Susceptibility to Adult Plant Bug 71 3.5.4 Choice Bioassays: Resistant and Susceptible

IV Strawberry Genotype Pairs 71 3.5.5 No-choice Bioassay: Resistant versus Susceptible Genotypes 73 3.5.6 Choice versus No-choice Exposure 76 3.5.7 Influence of Pollen Load on Damage Levels 76 3.6 Discussion and Conclusions 76

Chapter 4

4 Determination of Raspberry Fruit Susceptibility to Tarnished Plant Bug 86 4.1 Abstract 86 4.2 Introduction 86 4.3 Objective 87 4.4 Materials and Methods 87 4.4.1 Insect Rearing 87 4.4.2 Bioassay 88 4.4.3 Statistical Analysis 89 4.5 Results 90 4.6 Discussion and Conclusions 90

Chapter 5

5 Discussion and Conclusions 95

5.1 Discussion 95 5.2 Conclusion 106 5.3 Literature Cited 107

v List of Tables

Table 2.1 Combined field analysis of fruit damage for 2006. 38 Table 2.2 Combined density analysis for 2006. 38 Table 2.3 Individual field analysis of fruit damage for 2006. 38 Table 2.4 Individual field severity index analysis for 2006. 39 Table 2.5 Individual field density analysis for 2006. 39 Table 2.6 Mean percent fruit damage, severity index of damage and mean number of tarnished plant bug nymphs and adults for (2006). 41 Table 2.7 Damage and severity index variation on mulch treatments for 2006. 46 Table 2.8 Nymph density variation amoung replications for 2006. 46 Table 2.9 Combined field analysis of fruit damage and severity index for 2007. 46 Table 3.1 Combined comparison of hybrids to nymph number on the proportion and severity index of damage. 69 Table 3.2 Comparison of the effect of nymph number on the proportion and severity index of damage for individual hybrid strawberry genotypes Seascape and 703-43. 69 Table 3.3 Choice Bioassay: Comparison within families of resistant and susceptible hybrids. 72 Table 3.4 No-choice Bioassay: Comparison of resistant to susceptible hybrids exposed to different nymph numbers. 74 Table 3.5 Comparison of damage between resistant and susceptible hybrids. 75 Table 3.6 Choice versus No-choice experiments. 77 Table 3.7 Affect of pollen load on damage proportion. 77 Table 4.1 Analysis of unfilled and aborted raspberry drupelets. 91

VI List of Figures

Figure 1.1 Life history of tarnished plant bug. 8 Figure 1.2 Fruit deformation. 13 Figure 2.1 Severity index of damage rating scale for tarnished plant bug damage. 35 Figure 2.2 Effect of replication on the proportion of damage. 43 Figure 2.3 Frequency histograms for 2006. 45 Figure 3.1. Example of pair selection for laboratory bioassays. 63 Figure 3.2 Mean proportion of damaged strawberry fruit. 70 Figure 4.1 Mean proportion of damaged raspberry drupelets. 92

vn Glossary

Cultivar - a taxonomic subdivision of a species consisting of a naturally occurring or selectively bred populations or individuals that differ from the remainder of the species in certain minor characteristics

Families - a group of genetically different individuals raised from the same parents

Genotype - an individual with a unique genetic makeup; a set of individuals from the same genotype are often called a clone

vin Chapter 1

1 Literature Review

1.1 Introduction

Tarnished plant bug, Lygus lineolaris (Palisot de Beauvois) is a ubiquitous native

North American insect. It is widely distributed and feeds on more than fifty economically valuable crops.

In Ontario, tarnished plant bug is a serious pest on strawberry fruit but little is known about its status on raspberry fruit. Adults and nymphs feed on immature berries

and interfere with the translocation of growth hormones in strawberries. Plant bug

feeding results in abnormal fruit development and reduces the marketable crop yield. In untreated fields the level of damage can increase drastically and can reduce yields

(Schaefers, 1981; Wold and Hutchison, 2003).

Plant bug populations are typically controlled by and most of the

chemicals used have a seven day pre-harvest interval, (PHI) required between spray

application and harvest to allow the pesticide to breakdown. In day-neutral production,

limited options are available since plants produce flowers and fruit

simultaneously which does not allow enough time for the required pre-harvest interval.

Some available insecticides have a three day pre-harvest interval such as Malathion but

will no longer be available for control of plant bug in strawberry crops. New methods are required for effective plant bug control on day-neutral cultivars. One possibility is to use host-plant resistance.

Strawberry breeders have incorporated wild strawberry genotypes that exhibit resistance to tarnished plant bug damage into commercial cultivars to select for resistant

1 traits. Since little is known about the mechanisms of resistance within the wild strawberry

plants, bioassays need to be conducted to determine the properties which make these

plants less preferred by tarnished plant bug.

1.2 Strawberry Production

1.2.1 Taxonomy

The cultivated strawberry, Fragaria X ananassa L. is a member of the Rosaceae

family (Eriksson et al., 2003). Wild strawberry species are widely distributed, and

seventeen species exist around the globe. In temperate climates there are two types of

fruiting habits (Hancock et al., 1996). The first to be discovered was June-bearing or

single crop habit. In Ontario, fruit develops in May and is usually harvested in June over

a two to three week period. Flowers are initialized when the photoperiod is less then 14

hours and/or temperatures are less than 15.5° C (Hancock, 1999).

The second is everbearing, which can be divided into two subgroups on the

basis of sensitivity to their photoperiod: long day and day-neutral (Hancock, 1999). Long

day cultivars fruit two or more times per growing season and long photoperiods govern

flower production; while day-neutral cultivars produce flowers and fruit continuously

from early spring until late fall and flowering is not dependent on length of photoperiods

(Hancock et al., 1996).

June-bearing cultivars have a lower fruit yield compared to day-neutral cultivars,

since day-neutrals produce fruit continuously throughout the growing season with a peak

in fruit harvest every six weeks after June (Darrow, 1966). Plant breeders developed day-

neutral cultivars to extend fruit production and produce plants with higher yield and

2 better quality berries. Runner production is reduced in day-neutrals since most metabolic

resources are allocated to flowers and immature fruit.

1.2.2 Botanical Description

Strawberries are herbaceous low growing perennials which spread via runners.

Leaves are trifoliate arising from the crown, a reduced stem, and are produced every eight

to twelve days. Strawberry grow in a branched style called cymes, which

have five levels where the top floret opens first and the rest bloom sequentially along the

peduncle (Dale et al, 2000).

Three types of flowers are produced: pistillate (or female) flowers that do not

have anthers, staminate (or male) flowers that have anthers but non-functional pistils, and

hermaphrodite (or perfect) flowers that have both functional anthers and pistils (Hancock

et al., 1996). Sex is regulated by three alleles which have different levels of dominance;

female is dominant to hermaphrodite which is dominant to male (Hancock, 1999).

Strawberry flowers are composed of 50-100 pistils with an accompanying style and

stigma for . Once fertilized, the ovule grows into an achene which is the true

fruit of a strawberry plant found on the surface of the receptacle (Dale et al., 2000). In

young strawberry fruit, the achenes are clustered together and separate as the receptacle

expands. The hormone, indole acetic acid, is synthesized in the achenes and translocated

to the receptacle, which stimulates cell enlargement and receptacle expansion (Handley

and Pollard, 1993; Veluthambi and Poovaiah, 1984). The induced cells of the receptacle

enlarge producing the swollen red berry within thirty days of pollination (Dale et al.,

2000).

3 1.2.3 Economics

In Canada, strawberry fruit had a farm gate value of $52 million in 2002 (Makki,

2002). and Ontario are the two major producers, contributing approximately $30 million to the overall Canadian value (Mailvaganam, 2007). In Ontario, the annual farm

gate value of strawberries was $18 million in 2002. In 2002, there were 4,100 acres of fruit-bearing strawberry plants and 800 acres of non-bearing plants (Mailvaganam, 2007).

Approximately 40-45% of Ontario's strawberry production is pick-your-own, where the

farmer allows customers to enter the field and pick berries for themselves (Dale et al.,

2000). Fresh market retail and wholesale production both supply fresh picked berries to .

markets which are sold directly to the public. Less than 10% of strawberry production is

used by confectionaries to create jams, ice creams or yogurts (Dale et al., 2000).

1.3 Raspberry Production

1.3.1 Taxonomy

Raspberries are a diverse group which belong to the genus Rubus, family

Rosaceae and are closely related to strawberries in the Rosoideae subfamily (Crandall,

1995). Species of Rubus are commonly referred to as brambles, which denotes their

thorny canes (Goulart et al., 1991). There are four species of horticultural importance, blackberry (Rubus ursinus subgenus Rubus), red raspberry (Rubus ideaus L., Rubus

strigosus) L. and black raspberry (Rubus occidentalis L).

Selective breeding has led to the introduction of new cultivars with different

fruiting habits, fruit colour and thornlessness. There are two types of fruiting habits

within raspberry cultivars: summer bearing and fall bearing. Summer bearing cultivars

4 produce fruit in their second year, while fall bearing cultivars are able to produce fruit in the fall of their first year and again the next summer (Crandall, 1995).

1.3.2 Botanical Description

Raspberry canes are erect thorny shrubs, which have perennial roots and crowns, and biennial shoots. The shoots, or canes, emerge as root suckers and grow vegetatively in their first growing season and become dormant in the winter. The next spring they produce lateral branches which will flower and produce fruit. Vegetative canes in their first growing season are primocanes, while reproductive canes in their second growing season are floricanes (Goulart et al., 1991).

Lateral branches aire produced from the top of the plant to the base; with lateral branches at the top blossoming before those closer to the base. Fruiting lateral branches are composed of a terminal bud and axillary buds. Terminal flowers blossom first while axillary flowers bloom successively enabling an extended harvest (Louws, 1996).

Raspberry flowers are composed of five , five and approximately 150 pistils. Each pistil enlarges to form an ovary which is covered with flesh and skin to form a drupe (Crandall, 1995). Raspberry fruit are aggregate since one berry is composed of tiny drupelets that are held together on the central receptacle. Each drupelet is an entire fruit, where 80-100 drupelets are required to produce a normally developed berry.

Flowers are self-fruiting and can be pollinated by flowers from the same plant. It takes thirty to thirty five days to produce a mature fruit after pollination (Crandall, 1995).

1.3.3 Economics

Red raspberries are a minor crop in Ontario with an estimated fresh market value of $2.5 million, which represents 3% of the total farm gate value of all Ontario fruit crops

5 (Mailvaganam, 2007). The Canadian area of production devoted to raspberry production

is approximately 9,370 acres valued at $28 million (Makki, 2002). Raspberry production

has declined because of continuous disease, pressure and high costs of production

(Louws, 1996). The first economic return is three years after establishment. Raspberries

are sold in pick-your-own and fresh market operations. They are also used in processed jams and juices.

1.4 Biology of the Tarnished Plant Bug

1.4.1 Taxonomy

Tarnished plant bug belongs to the order , suborder Heteroptera and the

family ( or plant bugs). Hemiptera are "true bugs" with piercing-sucking

mouthparts that originate at the tip of the head and extend posteriorly underneath the

body (Elzinga, 2000). Plant bug species are distributed throughout and

also . Lygus rugulipennis (Poppius) is found in Europe, while Lygus hesperus

(Knight) is found in western North America and L. lineolaris in eastern North America.

Plant bug adults appear flattened because the wings are held flat over the thorax

and abdomen. The forewings are differentiated with a thickened base and a membranous

tip (Elzinga, 2000).

1.4.2 Life History

Tarnished plant bug undergoes incomplete metamorphosis, where nymphs

resemble the adult stage and there is no pupal stage. Adults emerge in the spring when

the temperature reaches 8°C, they feed on strawberry buds and shoots. Oviposition on

plant material begins once the temperature is 20°C (Cermak and Walker, 1992;

Bostanian, 1994). Females oviposit for 10-31 days, laying approximately five eggs per

6 day which are inserted into stems, leaf mid ribs, petioles, buds or florets (Cermak and

Walker, 1992). Eggs are 1 mm long, creamy white and have a flask shaped appearance

(Figure 1.1). Nymphs hatch within 7-10 days emerging in mid-May during strawberry bloom and feed on new shoots and flowers. The nymph population typically peaks in

August and early September (Schwartz and Foottit, 1992). There are five nymphal ins tars, which occur over twelve to thirty-four days. The fifth instar undergoes metamorphosis to the adult stage in late June and July (Cermak and Walker, 1992).

Tarnished plant bug are multivoltine with up to five overlapping generations per year in

Ontario (Anon. 1999). Nymphs mature into adults by the fall and adults over-winter in diapause among dead , leaf litter and under tree bark.

1.4.3 Description

Most Lygus species have a stink gland that secretes allomones, which is a defense chemical with a foul smell that is released to repel potential predators when the insect is disturbed (Gueldner and Parrott, 1978; Wardle et al., 2003). The volatiles secreted by plant bug are comprised of a few constituents, whereby the major component in females is hexanyl butyrate and in males hexyl butyrate (Gueldner and Parrott, 1978; Wardle et al., 2003). It was thought that the volatiles could be used as plant protectants but it was found that the compounds do not have a long-term effect on the behaviour of conspecifics and would not be useful as a repellent (Wardle et al., 2003).

Adult tarnished plant bugs are named for their tarnished colour, which ranges from black to dull brown. Adults are typically 6-7mm long, 2.5 mm wide and oval in shape (Figure 1.1) (Schwartz and Foottit, 1992; Cermak and Walker, 1992). Two sets of wings allow adults to fly. The back half of the forewings are membranous and form a "V

7 Figure 1.1. Life history of tarnished plant bug. View of tarnished plant bug instars developmental progression from first instar to adult. A, Tarnished plant bug egg (http://www.gaipm.org/top50/images/2722028.jpg), B, first instar; C, second instar; D third instar with single black spot on the thorax; E fourth instar with the five black spot pattern and initiated wing pads; F, fifth instar with elongated wing pads, and G, adult which shows the key characteristic of Lygus lineolaris adults, the membranous tip of the forewing indicated by M and yellow triangle on the scutellum indicated by T.

8 shape and are bent on a downward angle (Anon. 2005). A distinct characteristic is the yellow triangular mark that is present on the scutellum or dorsal side of the thorax

(Figure 1.1).

Nymphs are small, ranging from 1-5 mm long, and are bright green in colour when they are young and darken as they mature (Schwartz and Foottit, 1992). Five nymphal instars which can be identified by a thoracic mark. The first instar is typically faint green in colour and l-2mm in length (Figure 1.1), while the second instar is 2-3mm

in length and has a stronger green hue (Figure 1.1). The third instar has a single black

spot develops on the thorax and wing pads have initiated which lengthen as the nymph

matures to the fifth instar (Figure 1.1) (Schwartz and Foottit, 1992). Fourth instars have

five black spots on the thorax while the fifth is significantly darker in colour with longer

wing pads (Figure 1.1).

1.4.4 Distribution

Lygus lineolaris is the most widely distributed species throughout North America

and is found at low and high elevations from Alaska to southern Mexico (Bostanian,

1994). They are very adaptable and feed on over three hundred and fifty plant species and

fifty commercial crops including: alfalfa, {Medicago sativa L.), apples, (Malus domestica

Borkh.), celery, (Apium graveolens L.), , ( hirsutum L.), plums, (Prunus

domestica), and tomatoes, (Solatium lycopersicum L.) (Bostanian, 1994). Typically they

move to different host plants throughout the seasons and feed on plants which have new

growth or flowers. In the spring, early emerging weeds, such as common mullein,

( thapsus L.), sorrel, (Rumex acetosa L.), and fleabane, (Erigeron annuuss L.

Pers), provide food for adults which come out of diapause (Bostanian, 1994). In the

summer, oxe-eye daisy, (Chrysanthemum leucanthemum L.), shepherd's purse, (Capsella

9 bursa-pastoris L.), pigweed, (Amaranthus retroflexus L.), lamb's quarters,

(Chenopodium album L.), and knapweed, (Centaurea jacea L.), are available. In the

autumn weed hosts such as goldenrod, {Ambrosia artemisiifolia L.), asters, (Aster puniceus L.), thistle, (Cirsium arvense L.), and fleabane are available until snowfall

(Bostanian, 1994; Schwartz and Foottit, 1992). Although plant bug is native to North

America, many of the plants they feed on are not and may not be recognized by native

parasites. As such, native parasitoids are unable to adequately control the plant bug

(Anon 1998) and are unavailable for use as a natural biocontrol. Tarnished plant bugs are

highly attracted to flowers and reproductive tissues (Loeb et al., 2006). Since these are

normally the marketable structures of crop plants, it should be determined which plants

and weeds are more attractive to tarnished plant bug in order to reduce plant bug damage

on commercial crops.

Plants vary in their attractiveness to tarnished plant bug. Mustard, (Brassica juncea (L.) Czern.), is a preferred host over alfalfa and cotton, which includes both

nectarless and standard cultivars (Hatfield et al., 1983). Adults preferred to spend most of

their time on mustard while alfalfa was least preferred, which indicates mustard is highly

attractive to plant bug. Wold and Hutchison (2003) completed a further assessment of

hosts and found in the early growing season (March-June) alfalfa had the highest density

of tarnished plant bug compared to a strawberry field, wooded area and a fence row.

However, the plant bug population moved to the strawberry field in mid-June once bloom

occurred.

Loeb et al. (2006) found that tarnished plant bugs were more abundant in habitats

with flowers. Nymph populations peaked shortly after the onset of flower production

10 within weedy plots, which indicates that flower production is a determinant of colonization (Loeb et al., 2006).

1.4.5 Damage

Plant bug species feed on the reproductive organs or apical meristems of plants causing the fruit to be malformed or aborted (Bostanian, 1994). Plant bug feeding causes fruiting structures to abscise and damage to the seeds (Schwartz and Foottit, 1992). They repeatedly probe and puncture the tissue of a host plant with their mandibular stylet to

determine whether the plant is palatable, subsequently causing mechanical damage to plant tissue (Schwartz and Foottit, 1992).

Plant bugs usually feed when the strawberry flower is between anthesis and

fall (Riggs, 1990; Handley and Pollard, 1993). Strawberry fruit at the green fruit stage are

the most susceptible to plant bug damage. Primarily plant bugs feed on the achenes of the fruit but as the receptacle begins to enlarge the preferred feeding site changes from the

achenes to the receptacle tissue (Handley and Pollard, 1993).

Fruit with symptomatic plant bug damage are associated with a high percentage of hollow achenes (Handley and Pollard, 1993). As the plant bug probes the achene, it

injects digestive enzymes (pectinases and polygalacturonase), into the plant tissue

(Handley and Pollard, 1993; Cohen and Wheeler, 1998; Backus et al., 2007). The pectinases break down the interior cell wall of the endosperm of the achene, creating a

solubilized slurry which is consumed by the bug, leaving the achene hollow (Rodriguez-

Saona et al., 2002). Pectinases remove one galacturonic acid molecule at a time from

pectin molecules, which destroys the cell wall (Cohen and Wheeler, 1998). The

destruction of the endosperm in early fruit development restricts indole acetic acid

11 synthesis and its translocation to the receptacle. Hence the receptacle cells are not

stimulated to grow and seedy pockets are formed (Handley and Pollard, 1993; Laurema and Varis, 1991). This condition is called apical seediness or cat facing (Figure 1.2A). As the fruit develops, the achenes enlarge and become lignified, which makes it difficult for the plant bug stylet to penetrate, thus reducing the susceptibility of the fruit (Handley and

Riggs, 1990; Pollard, 1993). If only a few achenes are damaged, there is little effect on the fruit since each achene is able to stimulate receptacle growth for a distance of several

millimeters (Allen and Gaede, 1963).

Fruit malformation from plant bug damage can be confused with poor pollination

and heat damage. The type of malformation can be distinguished by the size of the

achenes in the damaged area. Symptoms of tarnished plant bug damage are identified by the presence of achenes that are of equal size, whereas poor pollination results in the presence of achenes that vary in size in the malformed area (Figure 1.2) (Cermak and

Walker, 1992).

The consequences of tarnished plant bug damage are dependent on the

developmental stage of strawberry flowers when feeding occurs and its duration. Feeding that occurs prior to petal fall results in blossom death, while feeding from petal fall until

achene separation causes apical seediness on the fruit (Handley, 1991; Easterbrook, 2000;

Wold and Hutchison, 2003). Handley (1991) assessed the impact of feeding duration on

strawberry fruit and found 8-24 hours of exposure resulted in slight deformities, at 48 hours the highest proportion of apical seediness occurs, while greater than 48 hour exposure caused blossom death.

12 Figure 1.2. Fruit deformation. A) Full view of tarnished plant bug damage on strawberry fruit, B) Close up of apical seediness caused by plant bugs feeding on the achene, C) Full view of poor pollination, D) Close up of fruit malformation caused by poor pollination.

13 1.5 Pest Management of Tarnished Plant Bug

Integrated pest management (IPM) is a comprehensive approach that enables multiple methods to be used that are effective, economically sound and environmentally friendly to reduce pest damage. Methods that can be used in the IPM approach include biological, genetic, cultural and chemical controls (Pedigo, 2002). Pest populations can be monitored in an IPM program to maximize control of the pest, while reducing unnecessary spray applications, and minimizing risk to the environment.

1.5.1 Monitoring

Many Ontario growers use scouts to monitor and estimate population densities of tarnished plant bug in order to optimize timing of insecticide applications, rather than use a calendar-based insecticide spray program. Scouts monitor fields with a sequential sampling program that determines insect pest density and minimizes the number of samples needed (Mailloux and Bostanian, 1989). By sampling the proportion of infested units, i.e. flower clusters, the mean pest density is estimated to determine whether the economic threshold has been reached (Mailloux and Bostanian, 1989). In strawberry production, plants are monitored every 3-4 days beginning at first bloom in the spring, to determine the number of early nymphal instars, that hatch on the flowers (Mailloux and

Bostanian, 1990; Dale et al., 2000). Flower clusters are tapped over a shallow dish and examined with a hand lens, the number of nymphs counted and recorded, and the dish emptied after each tap. The recommended economic threshold for strawberries is 2.25 nymphs per 15 clusters or 0.15 per cluster (Cermak and Walker, 1992). At this threshold,

2% of the fruit will suffer severe damage. If insecticides are not applied until 3-5 weeks after threshold has been reached, 5% of the fruit will be damaged (Cermak and Walker,

14 1992). Second year strawberry plants have a higher number of nymphs earlier in the growing season than first year plants (Jay et al., 2004). Adults become established in the field during the first growing season and overwinter there, allowing them to colonize the field sooner the next spring. Since adults can be present earlier scouting needs to be initiated prior to July (Jay et al., 2004). Optimal times to sample are in the morning or at dusk because insects move off the plant for shelter at high temperatures. Wet and windy conditions cause nymphs to become inactive, thus scouting should be delayed because populations may be underestimated (Rancourt et al., 2000).

The role of plant phenology in strawberry production has been the focus of research to determine if flower abundance and yield affect the incidence of tarnished plant bug oviposition and damage in strawberries. The number of nymphs found can vary per cultivar and per plant structure (Rhainds and English-Loeb, 2003b). Ovipositing females and nymphs are found on the receptacle of the fruit, which indicates nymphs prefer to feed and females prefer to lay eggs in stems and around the flower

(Rhainds and English-Loeb, 2003b).

Nymph density increases with increased strawberry plot sizes but does not translate into increased incidence of damage. Rhainds and English-Loeb (2003 a) used 0.4 m and 1.0 m plot sizes and found that nymph densities increased per flower with increased plot size of June-bearing and day-neutral strawberries. Although plant bug densities increased, there was not an increase in the incidence of damage. Smaller patches of strawberry flowers had a lower density of nymphs and higher incidence of damage, than did large patches, which had a higher density of nymphs but lower incidence of damage. These results suggest a dilution effect where if the number of flowers is

15 increased there is less fruit damage, because some flowers are not fed upon (Rhainds and

English-Loeb, 2003a).

1.5.2 Cultural Control

A broad spectrum of crop management techniques can be used to alter the production system to reduce pest populations or avoid pest injury. Growers can select

optimal sites to plant and use resistant cultivars to reduce pest populations, that migrate

into their crop. To reduce tarnished plant bug migration, strawberries should be planted

away from adult overwintering sites (i.e. hedgerows), and flowering crops (i.e. alfalfa), that are highly attractive to them. Trap crops can be used to lure the plant bug away from

valuable crops and reduce crop damage (Norris et al., 2003), and also act as reservoirs for predators and parasitoids of pests (Easterbrook and Tooley, 1999). Alfalfa is an effective trap crop as long as the crop has staggered flower production to prevent tarnished plant bug from migrating into the strawberries (Norris et al., 2003).

German chamomile (Matricaria recutita L.) has been recommended as a trap crop

around strawberries to control L. rugulipennis. Plots which had M. recutita planted

around the perimeter had delayed plant bug populations by two and a half weeks where

(Easterbrook and Tooley 1999). However, the plant bug population was not reduced all

season long. It is essential to maintain the quality of the trap crop or nymphs will migrate

into the main crop, resulting in a higher than normal population. Easterbrook and Tooley

(1999) suggest that an insecticide should be applied to the trap crop before migration

occurs to minimize damage in the adjacent strawberry crop.

Plastic mulches can be used as a method of control to reduce initial insect

infestation levels by repelling migrating insects. The colour of the plastic reflects

16 different amounts of UV light which affects the insect's behaviour and ability to find a host (Kring and Schuster 1992). Mulch with high UV reflectance directly repels insects

as a group, while long-wavelength light is more attractive (Kring 1972; Summers et al.,

1995; Stavisky et al, 2002; Doring et al., 2004). Silver plastic mulch has been found to

consistently increase yield in strawberry plants and suppress the density of plant bug

nymphs in June-bearing cultivars throughout the growing season (Rhainds and English-

Loeb, 2001). The mulch increased the number of fruit per plant. This provides further

support to their dilution theory where increased numbers of flowers and fruit results in a

lower incidence of plant bug damage. Rhainds and English-Loeb (2001) found the

reflective mulch reduced plant bug damage as much as one application of Malathion.

Colonization patterns and visual cues can be used to improve monitoring

techniques and develop improved trap crop systems. Plant phenology plays a key role in

determining tarnished plant bug colonization; in particular, plants with a high number of

flowers are more attractive (Loeb et al., 2006). Typically the nymph population peaked

shortly after the onset of flower production, which indicated colonization was not

random, but coincident with blossom development of different plant species as the season

progresses (Loeb et al., 2006). Colour and smell can affect how attractive a host is to

tarnished plant bug, since adults prefer white cards over red, specifically ones scented

with Erigeron canadensis L., fleabane (Loeb et al., 2006).

Insecticides and can be used to reduce the initial migration of adults

and to prevent high nymph populations later on in the crop (Hardman et al., 2004).

Insecticides were applied to an apple orchard before bloom to target migrating adults.

The lowest rate of fruit injury was in plots where full rates of the pyrethroids cyhalothrin-

17 lambda or cypermethrin were applied before bloom (Hardman et al., 2004). To reduce

insecticide use and adult migration, herbicides were applied to reduce weed hosts that would attract plant bug adults. Early applications of herbicides before the period of fruit

susceptibility, reduced fruit damage (Hardman et al., 2004; Snodgrass et al., 2005).

1.5.3 Biological Control

Biological control uses beneficial insects such as natural predators and parasitoids to reduce pest populations (Pedigo, 2002). An effective biological control agent could

save more than $1 billion annually for many crops and reduce the insecticide load in the

environment (Williams et al., 2003).

Peristenus diagoneutis Loan (Hymenoptera: Braconidae) is an effective parasitoid

wasp of tarnished plant bug nymphs in Europe and as a result it is not considered a pest

of strawberries on that continent (Tilmon and Hoffman, 2003). Peristenus diagoneutis

was successfully released in northern New Jersey in the 1980's and has dispersed northwards to New York (Tilmon and Hoffman, 2003). Leiophron argentinensis Shaw

(Hymenoptera: Braconidae) is another parasitoid wasp that parasitizes mirids in

Argentina and Paraguay (Williams et al., 2003).

Female wasps of P. diagoneutis and L. argentinensis lay a single egg within an

early instar of a plant bug nymph, and after hatching the larva feeds on the nymph's

internal tissues (Williams et al., 2003). The mature larva emerges from the dead nymph,

and pupates in the soil. Typically there are two generations per year, with pupae of the

second generation overwintering in the soil and adults emerging in the spring (Williams

et al., 2003).

Entomopathogenic fungi can also be used for biological control of plant bug. Liu

18 et al. (2003), found Beauveria bassiana can reduce plant bug populations by 81%. Fungal

spores attach to the external body surface of the insect and germinate under the right environmental conditions. The hyphae then colonize the insect and break down the

internal tissues causing death (Liu et al., 2003).

1.5.4 Chemical Control

1.5.4.1 Strawberry

The most common method to control tarnished plant bug is insecticides, which

are applied when populations reach the economic threshold. The active ingredients in

insecticide formulations endosulfan, dimethoate and cypermethrin are used to control tarnished plant bug. These have at least a 7 day preharvest interval (Cermak and Walker,

1992). Insecticides can be effectively applied to June-bearing cultivars to control tarnished plant bug since fruit is harvested only once, and does not conflict with the days to harvest requirements.

Insecticides are restricted for day-neutral production because fruit is harvested

every 3-4 days which does not allow enough time for the specified preharvest interval.

Insecticides used for plant bug also may have a non-target effect on pollinating insects

since flowers and fruit occur simultaneously (Easterbrook and Tooley, 1999).

1.5.4.2 Raspberry

There are limited chemical options available for control of insects on raspberries.

Since there are no economic threshold levels for raspberry fruit the grower must decide

when plants should be treated to control insects. Plant bugs have been observed on the

fruit and flowers of raspberry canes but it is unknown whether nymphs and adults

19 actually cause damage to the fruit (Crandall, 1995). As a result, there are no products registered for control of tarnished plant bug on raspberries in Canada.

1.5.5 Host Plant Resistance

In nature, plants and insects have been a part of an evolutionary arms race and have placed selective forces upon each other. Plants have evolved and produced

numerous ways to reduce the amount of damage sustained by the insects which feed on

them. Plant breeders have crossed plants with resistant traits to produce new cultivars for

crop production which sustain low levels of pest damage. Growers can incorporate

insect-resistant cultivars as one of the most cost effective components of an IPM

program, especially since they are compatible with other control strategies. There are two

major sources of genes for resistance: native genes which occur within the same or

closely related species, and genes that are genetically engineered or moved from another

species.

There are two types of resistance that can be expressed in plants, vertical and

horizontal resistance (Pedigo, 2002). Vertical resistance refers to plant species that are

resistant to one, or a very few, insect species and is controlled by a single gene. Some

resistant genes are so effective at controlling damage it is known as complete resistance.

In contrast, horizontal resistance refers to plant species that are resistant to a broad range

of insect species and is controlled by many genes. Since a large number of genes are

involved this resistance is known as in complete because there may be less damage

caused but damage still occurs. In the case of pathogens horizontal resistance may slow

the infection process so that the pathogen may not grow as well or spread to another

plant. There are two types of resistance traits: oligogenic and polygenic (Pedigo, 2002).

20 Oligogenic resistance or major gene resistance is when one gene expresses resistance, typically to one species of pest. Polygenic resistance or minor gene resistance is when resistance to one or more pests is controlled by a number of genes.

Resistant plants can be crossed with susceptible plants to amplify the expression

of resistance in susceptible cultivars. Progeny must continually be bred with resistant plants to increase expression of the resistant trait effectively. The main disadvantage of this type of resistance is that it places a selective pressure on the insect population, and

may result in development of individuals that are able to overcome the plant resistance

(Pedigo, 2002).

1.6 Mechanisms of Resistance

Many plant species have evolved unique defense traits which can affect pest behaviour or physiology. These traits can range from morphological to in

nature. There are three mechanisms of resistance that may be expressed by plants in order

to reduce insect damage: antixenosis, antibiosis and tolerance (Painter, 1951; Kogan and

Ortman, 1978).

1.6.1 Antixenosis

Plants are able to interfere with insect host plant selection behaviours through

either structural or chemical traits. Antixenosis are plant characteristics that drive insects

away from a host. This type of resistance mechanism reduces the ability of an insect from being able to settle, colonize or penetrate plant surfaces and thus results in reduced damage to the plant (Kogan and Ortman, 1978). Antixenosis is also known as 'non- preference', which refers to the response of insects to plants with antixenotic traits

21 (Painter, 1951). Resistant plants may have a deterrent to reduce insect feeding or may be lacking the attractant which is found in susceptible plants.

Morphological traits such as , waxes or a thick epidermis can physically prevent the insect from landing and feeding on the plant. Plants can also produce and emit volatile compounds to prevent or deter insect predation and oviposition. The defensive chemicals produced by plants, known as allomones, are found in the plant tissue. These chemicals are offensive and unpalatable and prevent insect feeding (Pedigo,

2002). As insects select a host for feeding, a sample bite is taken to determine if the host is suitable. If unpalatable allomones are present in the tissue the insect will not continue feeding and reject the plant as a host, thus reducing damage to the plant. Expression of these traits is genetically based and can be manipulated to increase the expression of resistance within the plant.

White mustard (Sinapis alba L.) uses two methods of defense that reduce the rate that Lygus lineolaris feed upon seedpods: high concentrations of p-hydroxybenzyl glucosinolate (sinalbin) and many long trichomes (Bodnaryk, 1996). When trichomes were removed from the pods and were compared to pods with intact trichomes, plant bugs fed twice as much on shaved seedpods than on unshaved ones. The trichomes appear to be a physical barrier which prevents the plant bugs from reaching the pod to feed.

In cotton, Gossypium hirsutum L., and maize, Zea mays L., several volatile compounds are emitted following feeding by plant bugs (Rodriguez-Saona et al., 2002).

Some of the volatiles emitted were: (Z)-3-hexenyl acetate, (£)-|3-farnesene, (E, E)-a- farnesene. Feeding by nymphs and adults induced volatile emissions on maize plants,

22 while only adult feeding induced chemical release from cotton. There may be an elicitor

in the plant bug saliva that induces production and release of these volatiles, which may

attract beneficial predators of the pest (Rodriguez-Saona et al., 2002).

1.6.2 Antibiosis

The production of which have adverse lethal or sublethal effects

on an insect is known as antibiosis (Painter, 1951). The chemicals produced are called

allelochemicals and impair the pest's physiology and metabolic processes when

consumed (Painter, 1951). The effects induced on the insects are due to long term

exposure, since the insects feed on the plant and consume a large quantity. Allomones

can have a variety of effects and can cause mortality, sterility, reduce fecundity, delay

maturity, or induce physical or behavioral abnormalities. Plants with high levels of

allomone expression can be hybridized to produce progeny with elevated levels of the

allomone.

1.6.3 Tolerance

Tolerance occurs when the plant is able to produce a satisfactory yield in spite of

supporting a pest population equal to that which would damage a susceptible plant

(Painter, 1951). Plants with this type of resistance are typically able to repair or replace

damaged parts. Some plants defend themselves by denaturing the digestive enzymes

released in the saliva of insects with piercing sucking mouthparts (Laurema and Varis,

1991). Quinones and are a biochemical defense used by plants to prevent the

breakdown of the cellular walls by insect feeding which reduces damage to the plant.

Tolerance differs from antixenosis and antibiosis in the predominant part played

by the plant (Painter, 1951). Antixenosis and antibiosis are based on insect plant-

23 relationships and require am active response by the insect. The main disadvantage of

antixenosis and antibiosis is that it places a selective pressure on the insect population,

and may result in development of individuals that are able to overcome the plant

resistance (Pedigo, 2002).

Tolerance does not exert amy selective pressure on the insects to develop resistance which

prevents the selection of resistant insects in the population.

Among several everbearing strawberry cultivars examined, the cultivar Bolero

appeared to tolerate Lygus rugulipennis damage (Easterbrook and Simpson, 2000).

Bolero had less damage but had the same number of nymphs as the other cultivars. The

level of tolerance was also passed onto progeny of Bolero. Although, they showed

tolerance to damage it was only seen in the first year and dissipated in subsequent years.

The mechanism in Bolero could have been antibiosis since nymphal weights were

reduced in choice tests. However, Easterbrook and Simpson (2000) concluded the

resistance mechanism exhibited was tolerance because tarnished plant bug nymph

numbers did not differ between cultivars in field experiments. Therefore, Bolero and its

progeny do not exhibit antibiosis characteristics but rather a form of tolerance to

tarnished plant bug feeding.

1.6.4 Damage Variation in Strawberries

There is a wide range of variability in tarnished plant bug damage sustained by

different strawberry cultivars. The June-bearing cultivars Honeoye, Conaga, Veestar and

Sparkle display minimal tarnished plant bug damage, while in Kent and Micmac

experienced higher amounts of damage (Handley et al., 1991). Handley et al. (1991)

speculated that plant genetics play a role in the amount of damage sustained. The cv.

24 Honeoye and Canoga both have the resistant cv. Holiday as a parent. Also, cv. Veestar had a similar amount of damage to its parent, Sparkle (Anon 1997). Kent and MicMac

were highly susceptible and both share Tioga as a parent. Handley et al. (1993) found

some of the unsprayed June-bearing cultivars had a lower amount of plant bug injury than

cultivars that were sprayed three times with insecticides. Susceptible cultivars benefited

from spray applications and resistant cultivars did not. Kent and MicMac continued to

show the highest incidence of damage while Honeoye had reduced plant bug injury even

when unsprayed with insecticide.

Since certain cultivars were found to resist tarnished plant bug, efforts have been

made to identify the mechanisms of resistance. Cultivars with suspected resistance have

been studied to determine unique characteristics which may make them unsuitable as

plant bug hosts. High pollen levels were found to be significantly correlated with plant

bug damage in 28 cultivars, while flower size, flower number, length, peduncle

length and petiole length were uncorrelated (Handley and Dill, 2003). Cultivars with high

amounts of pollen could be highly attractive to tarnished plant bug. However, tarnished

plant bug pressure was particularly low during this study, so these results may not

represent this situation at peak pest pressure.

Grunfeld et al. (1989) compared the composition of pollen and nectar from 8

strawberry cultivars. The free amino acids and proteins in the pollen and nectar varied in

strawberry cultivars, but they were unable to relate the differences to insect preference.

In cotton, damage was highest on flowers where plant bugs specifically fed on

anthers and pollen grains (Williams and Tugwell, 2000). Damaged pollen and anthers

were characterized as being irregularly shrunken and dark. The study by Williams and

25 Tugwell (2000) concurs with Handley and Dill's (2003) study, supporting high pollen levels as an attractant for plant bug and damage.

The wild species, Fragaria virginiana Duch., exhibits resistance to tarnished plant bug damage (Dale et al., 2008). Commercial cultivars were crossed with F. virginiana Duch. in attempt to select for plants with the resistant traits from the wild species and agronomic traits from the commercial cultivars. The genotypes produced had an intermediate level of resistance and exhibited less damage than commercial cultivars

(Dale et al., 2008). Resistant traits were shown to be recessive (Dale et al., 2007).

Although resistance was found within the hybrids, the mechanisms behind the reduced amount of damage have not yet been determined.

1.7 Conclusions

Tarnished plant bug is a very opportunistic insect pest, with a wide range of hosts which makes population control very difficult. Plant bug populations that feed on day- neutral strawberries cannot be adequately controlled with currently registered insecticides. The wild strawberry species, F. virginiana, expresses resistance to tarnished plant bug damage and yields fruit with reduced amounts of damage. Genotypes from F. virginiana crosses show variation in the amount of damage observed.

Several mechanisms have been proposed to clarify the variation observed in the amount of tarnished plant bug damage between strawberry cultivars. The purpose of the research was to: 1.) investigate differences between several day-neutral strawberry cultivars in terms of tarnished plant bug populations and damage level; 2.) investigate the mechanisms of resistance observed in advanced day-neutral selections in a controlled

26 greenhouse environment; and 3.) determine the effect of tarnished plant bug feeding on raspberries.

Field trials were used to assess relative susceptibility of several day-neutral cultivars to tarnished plant bug and determine if geographic differences exist among tarnished plant bug populations preferences. Laboratory bioassays were performed to validate previous field studies (Dale et al., 2008) and examine possible mechanisms of resistance found in these plants. Elucidating the mechanism(s) of resistance may aid in expanding production of day-neutral cultivars with tarnished plant bug resistance and could potentially increase the farm gate value of the Ontario strawberry crop.

27 Chapter 2

2 Comparison of the susceptibility of four commercial strawberry cultivars to tarnished plant bug

2.1 Abstract

Tarnished plant bug (Lygus lineolaris (Palisot de Beauvois)) is a major pest in commercial strawberry production in North America. Adults and nymphs feed on the soft developing seeds and fruit tissue, which causes fruit deformation and results in yield reduction and economic loss. A field study was conducted to determine whether tarnished plant bug consistently damaged 4 commercial day-neutral strawberry cultivars 1.) Fort

Laramie; 2.) Seascape; 3.) Tristar; and 4.) Tribute at 3 field sites in southern Ontario.

Plants were monitored weekly to assess and compare the number of tarnished plant bug and their damage on fruit. The cultivars ranked consistently for the proportion of damaged fruit at the different field sites. Fort Laramie consistently had fewer fruits damaged, while Tribute had the highest with 12 times the amount of damage than Fort

Laramie. The position of the plant within the plot did not affect the proportion and severity index of damage. Plants on black plastic mulch had more damage than those on white plastic mulch. At the Vineland site, nymphs did not migrate far into the strawberry field and the highest proportion of damage was located near the edge of the field. This study showed that tarnished plant bug damage to cultivars was consistent across southern

Ontario. Cultivar Fort Laramie exhibited resistant characteristics which could be enhanced through breeding programs.

2.2 Introduction

Tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), is the most common pest in day-neutral strawberry production in Ontario. Nymphs and adults feed on

28 developing achenes, preventing receptacles from developing normally and reducing the marketable yield for growers (Handley and Pollard, 1993; Laurema and Varis, 1991).

Tarnished plant bug is considered the most important deterrent to the expansion of day- neutral strawberry production in North America (Dale et al., 2000).

The most common method to control plant bug populations in strawberries is with insecticides. In June-bearing cultivars, fruit are produced only in the spring and early blooming cultivars flower before plant bug is present in the field. On day-neutral strawberries, plant bug populations cannot be controlled with currently registered insecticides because their days-to-harvest requirements are incompatible with continual fruit harvest characteristic of this type of strawberry (Dale et al, 2000). Several control strategies can be combined to potentially control plant bug, including, reflective mulch

(Rhainds and English-Loeb, 2001), biological control (Tilmon and Hoffman, 2003) and resistant cultivars (Handley et al., 1991; 1993).

Strawberry cultivars vary considerably in the amount of damage that results from tarnished plant bug feeding. Handley et al. (1991) found significant and consistent variability in the amount of plant bug injury between twenty June-bearing cultivars in the field. The results showed some cultivars were less susceptible than others and there may be a genetic role in the range of susceptibility (Handley et al., 1991). The cultivars

Honeoye and Canoga had the lowest amount of injury and both have Holiday as a parent, which also had the lowest amount of injury in another cultivar study (Handley and Dill unpublished data). Kent and MicMac were highly susceptible and both share Tioga as a parent, which suggests susceptibility to plant bug damage is genetic.

29 Handley et al. (1993) found that some unsprayed June-bearing cultivars had a lower amount of plant bug injury than cultivars that were sprayed 3 times with insecticides. Kent and MicMac had the highest incidence of damage while Honeoye had reduced plant bug injury even when no insecticide was applied thus exhibiting a form of resistance. The results of the study were consistent with their previous study and Handley et al. (1993) stated that further resistance screening of strawberry germplasm should be undertaken.

Resistance to tarnished plant bug damage in genotypes of everbearing strawberries was first studied by Easterbrook and Simpson (2000). The cultivar Bolero appeared to tolerate L. rugulipennis damage as compared to several everbearing strawberry cultivars (Easterbrook and Simpson, 2000). No difference was found in the proportion of eggs hatched on the plants but there was a difference in the amount of damage observed. Although the nymph numbers were the same, those on Bolero weighed less suggesting mechanisms of antibiosis. However, it was concluded Bolero exhibited tolerance because nymph numbers between cultivars did not differ in field experiments.

When comparing day-neutral cultivars, cv. Fort Laramie was the most resistant to fruit damaged by plant bug, while cv. Seascape was the most susceptible (Dale et al.,

2008). Dale et al. (2008) completed a field study which exposed several commercial, wild and hybrid strawberry cultivars to indengenous populations of tarnished plant bug.

Significant differences were observed in the amount of damage incurred on each cultivar.

Cultivars Tribute and Tristar showed low levels of damage but varaitions from year to year occur (Dale et al., 2008). The wild strawberry, F. virginiana, had the lowest level of plant bug damage, even lower than cv. Fort Laramie (Dale et al., 2008). Crosses have

30 been completed between F. virginiana and several commercial cultivars, (cv Fort

Laramie, Fern, Evangeline, 21K74 and 127A71) in an attempt to produce hybrids which have elevated levels of resistant to plant bug damage. In the field study conducted by

Dale et al. (2008) some of the hybrid crosses showed low levels of fruit damage and indicate levels of resistance, however the mechanism(s) of resistance is unknown.

2.3 Purpose

Field experiments were conducted to:

1. detect how uniform the response of tarnished plant bug damage was on several day-neutral cultivars at 3 field sites in southern Ontario,

2. determine the range of day-neutral cultivar susceptibility with respect to proportion and severity index index of damage caused by tarnished plant bug among several day-neutral cultivars,

3. establish whether tarnished plant bug damage increases on resistant cultivars when they are planted next to highly susceptible cultivars; and,

4. determine whether different coloured plastic mulch can be used to reduce the proportion and severity index of damage caused by tarnished plant bug.

2.4 Materials and Methods

A field study was conducted at 3 commercial field sites in southern Ontario in

2006 and 2007. The field sites were: Strawberry Tyme Farms Inc., Simcoe (Latitude

42.8359, Longitude - 80.3052, N), Tigchelaar Berry Farm Inc., Vineland (Latitude

43.1599, Longitude - 79.3944, N) and Watson Farms Ltd., Bowmanville (Latitude

43.9138, Longitude - 78.6884, N). In May 2006, 4 commercial day-neutral strawberry cultivars - Fort Laramie, Tribute, Tristar, Seascape, with different levels of resistance to tarnished plant bug were selected for the field trial (Dale et al., 2008).

At all sites, dormant crowns were planted on beds covered with plastic mulch.

They were trickle irrigated and fertilized according to the fruit production

31 recommendations (Dale et al., 2000). Indigenous populations of tarnished plant bug were

allowed to colonize the field plots and no insecticides were applied.

2.4.1 Experimental Design

At each site, a randomized complete block design was used in which the 4

cultivars were replicated 8 times. Each plot consisted of 12 plants. Between each plot, 4

cv. Seascape plants were planted to act as a buffer between cultivars and to attract

tarnished plant bugs to the site.

2.4.2 Field Sites

Vineland soil beds were 30cm (H) x 61cm (W) and with a row spacing of 91cm.

Beds were covered with black plastic mulch with straw bedding between rows to

suppress weed growth. Plants were hand planted in two-staggered rows per bed with

28cm within and 13cm between row spacing. The research plot was 4 beds wide and

15.5m long. A 3m buffer at each end of the rows served to protect the research plot from

pesticide drift from adjacent commercial strawberry beds. Two buffer rows were planted

on each side of the 18.5m long research plot. The outskirt rows of the Vineland field plot

were beside a lane of grass, a peach orchard and a romaine field which are highly

attractive to tarnished plant bug.

Bowmanville soil beds were 30cm (H) x 122cm (W) and with a row spacing

76cm. Plot replicates 1-4 were covered with black plastic and plot replicates 5-8 were

covered with white plastic mulch to suppress weed growth. Plants were hand planted in

four-staggered rows per bed with 30cm within and 25cm between row spacing. The research plot was 4 beds wide and 10m long. Two meters of buffer plants were planted at

32 each end of the rows, while 2 buffer rows were planted on each side of the 12m long research plot.

Simcoe soil beds were 30cm (H) x 122cm (W) and with a row spacing 76cm. The beds were covered with white plastic mulch to suppress weed growth. Plants were hand planted in four-staggered rows per bed with 30cm within and 25cm between row spacing.

The research plot was 4 beds wide and 10m long. Twenty-four meters of buffer plants were planted at each end of the rows, while 2 buffer rows were planted on each side of the 34m long research plot.

2.4.3 Sampling and Damage Evaluation

Two plants were sampled in the center of each plot to evaluate each cultivar, and two plants were sampled at the edge of each plot to determine whether or not proximity to the cv. Seascape buffer plants affected tarnished plant bug damage levels. Plants were monitored and fruit was harvested weekly from June 6th to October 26th, 2006 and May

9th to July 5th, 2007.

The number of tarnished plant bug were counted on sample plants using a flower tapping technique described by Dale et al. (2000). Monitoring was initiated once the terminal blossom on the sample plant was in bloom and was only done on calm sunny days, when nymphs are more active (Rancourt et al., 2000). All flower clusters on a plant were tapped 3 times onto a white paper plate and the number of nymphs and adults were recorded per plant. One hundred and twenty eight plants were sampled every week per site.

All ripe fruit was picked, counted and the severity index of damage rated for tarnished plant bug damage on a 0 to 4 scale, where: 0= no damage, 1=

33 damaged, 2= 1/4 to 1/3 of the berry damaged, 3= 1/3 to 2/3 of the berry damaged, and 4=

>2/3 of the berry damaged (Figure 2.1). After plants were monitored and rated, all remaining ripe fruit on the unsampled plants was removed. Monitoring, sampling and damage evaluations were conducted in both 2006 and 2007.

2.4.5 Statistical Analysis

The proportion of damaged fruit was calculated as the number of damaged fruit divided by the total number of fruit harvested from the plots. A severity index for damaged fruit was calculated as the sum of [(lx score rating 1) + (2 x score rating 2) + (3 x score rating 3) + (4 x score rating 4)] divided by the total number of fruit multiplied by

4 (Dale et al., 2008). Fruit given a severity score of 0 was used in the class of normally developed fruit.

Data from all field sites were tested to meet statistical assumptions and data normality. A univariate procedure was performed to determine if data were normally distributed using the Shapiro Wilk test. The heterogeneity of variance was tested to determine whether data was homogeneous using the Brown and Forsythe homogeneity of variance test. When necessary, heterogeneity of variance was reduced by subjecting data to an arcsin transformation.

Data from all the field sites in 2006 and 2007 were initially pooled together to compare varietal differences, however the univariate normalization procedure and Brown and Forsythe homogeneity of variance test (SAS Institute, version 9.1, 2002, Cary, NC) of the damage proportion data indicated the data from each year could not be pooled together. Univariate normalization procedure and Brown and Forsythe homogeneity of variance test were used to compare data from all field sites in the 2006 field season. Data

34 Figure 2.1. Severity index of damage rating scale for tarnished plant bug damage. Damage was rated on a scale of 0 to 4, where: 0= no damage, 1= 2/3 of the berry damaged. Damage caused by plant bug was mostly concentrated on the tip of all the fruit.

35 from the Simcoe site could not be pooled with the other field sites because very few fruit

were damaged due to very low tarnished plant bug populations in 2006. When the data

from Simcoe was removed from the pooled analysis, the assumptions of the variance

analysis were met.

The number of nymphs, adults, proportion of damaged fruit and severity index by

plant bug was compared for different cultivars from all sites by an analysis of variance,

using PROC GLM (SAS Institute, version 9.1, 2002, Cary, NC). Tables are reported

using least significant difference for planned paired comparisons.

For Vineland and Bowmanville, data was analyzed utilizing a split-plot design,

with replicates within sites treated as main plots, strawberry cultivar as subplots and edge

effect as sub-subplots for proportion and severity index. When comparing the number of

nymphs and adults at each site, replicates were used as main plots and cultivars were

subplots. The mean number of nymphs and adults per sample and the proportion of fruit

damaged by tarnished plant bug for different sites were compared. To calculate the F-

ratio, replicate*site mean square was used as the error mean square for replicates and

sites. The replicate*site*cultivar mean square for cultivar and site by cultivar interaction

and the residual mean square was used for the edge effect and its interactions. Also, since

the site by cultivar interaction varied significantly, the two sites were analyzed separately.

A regression was completed to determine the effect of distance from the edge of

the field and the proportion of damaged fruit and nymph number, where the means and

the results of the equation were graphed. The regression model used where the response

(Y) was Y= bix + b2X + e, where (x) is the number of nymphs, bi and \>i are the linear

and quadratic polynomial regression coefficients and e is the random error term. The

36 intercept was constrained to zero since no tarnished plant bug damage could occur in the absence of nymphs. Linear and quadratic effects were tested for the independent variable and the regression equations calculated for the significant linear effects.

For the 2007 field data the Bowmanville field site was prematurely ploughed under by the grower so no data was collected from the site in 2007. Monitoring at the

Vineland and Simcoe field site was stopped in mid June since the strawberry harvest had ended and the plot was ploughed under.

2.5 Results

2.5.1 Field Data Results in 2006

In 2006, tarnished plant bugs were present throughout the strawberry growing season. For the pooled analyses of the Bowmanville and Vineland field sites both site and site x cultivar varied significantly for all characteristics measured which required each site to be analyzed separately.

An analysis of each site indicated that the cultivars differed significantly at the

Vineland and Bowmanville sites for the proportion of damage and severity index caused by tarnished plant bug and number of plant bug nymphs (Table 2.3-2.5). The number of adults did not vary significantly. Replicates did not differ significantly at the Simcoe and

Bowmanville sites which indicated that the data collected from the plots were uniform.

However at Vineland, replicates were found to be a significant source of variation for the severity index and nymph and adult numbers. At Simcoe, cultivars did not vary significantly for any of the characteristics measured because there was a low population level of tarnished plant bug.

The amount of tarnished plant bug damage for the different cultivars ranged from

37 Table 2.1. Combined field analysis of fruit damage for 2006. Combined analysis of variance of the proportion of fruit damaged and the severity index of damage tarnished plant bug damage in 2006 for four cultivars at Bowmanville and Vineland, ON. Bold numbers indicate significant differences, P=0.05. Proportion of Fruit Severity Index of Damaged Damage Source df F ratio P F ratio P Site 2 16.2 0.0002 22.5 0.0001 Replicate 7 3.2 0.0073 1.4 0.22 Error Mean Square 7 1.21 0.30 1.75 0.10 Cultivar 3 44.6 0.0001 50.4 0.0001 Site*cultivar 3 4.5 0.0069 7.2 0.0004 Error Mean Square 49 0.016 0.50 0.033 0.50 Edge 1 33.0 0.091 2.8 0.10 Site*edge 1 1.8 0.18 1.1 0.31 Site*cultivar*edge 6 0.86 0.53 0.70 0.65 Error Mean Square 128 0.012 0.025

Table 2.2. Combined density analysis for 2006. Combined analysis of variance of the number of nymphs and adults in 2006 for four cultivars at Bowmanville and Vineland, ON. Bold numbers indicate significant differences. Nymphs Adults Source df F ratio P F ratio P Site 2 205.8 0.0001 79.7 0.0001 Replicate 7 1.06 0.40 1.42 0.2114 Error Mean Square 14 3.81 0.0001 1.71 0.06 Cultivar 3 11.32 0.0001 2.76 0.048 Site*cultivar 6 4.97 0.0002 2.90 0.013 Error Mean Square 77 1.00 0.50 1.00 0.50

Table 2.3. Individual field analysis of fruit damage for 2006. Individual analyses of variance of the proportion of damage caused by tarnished plant bug in 2006 for four cultivars at Vineland, Bowmanville and Simcoe. Bold numbers indicate significant differences. Vineland Bowmanville Simcoe Source df F ratio P F ratio P F ratio P Replicate 7 1.9 0.11 2.2 0.081 0.50 0.83 Cultivar 3 10.9 0.0002 63.0 0.0001 2.5 0.084 Error Mean Square 21 0.024 0.074 0.0083 0.48 0.0051 0.56 Edge 1 3.8 0.055 0.10 0.76 0.02 0.88 Cultivar* edge 3 0.70 0.56 1.2 0.31 2.4 0.073 Error Mean Square 92 0.016 0.0084 0.0055

38 Table 2.4. Individual field severity index analysis for 2006. Individual analyses of variance of the severity index caused by tarnished plant bug in 2006 for four cultivars at Vineland, Bowmanville and Simcoe. Bold numbers indicate significant differences. Vineland Bowmanville Simcoe Source df F ratio P F ratio P F ratio P Replicate H 3.06 0.0062 0.84 0.50 0.74 0.65 Cultivar J 13.0 0.0001 45.0 0.0001 2.5 0.089 Error Mean Square 21 0.041 0.18 0.030 0.20 0.0068 0.59 Edge 3.2 0.077 0.25 0.62 0.38 0.54 Cultivar* edge J 0.78 0.51 0.68 0.57 3.1 0.032 Error Mean Square 92 0.031 0.021 0.0076

Table 2.5. Individual field density analysis for 2006. Individual analyses of variance of the number of nymphs and adults found in 2006 for four cultivars at Vineland, Bowmanville and Simcoe. Bold numbers indicate significant differences. Vineland Bowmanville Simcoe F F F Source df ratio P ratio P ratio P Nymphs Replicate 7 3.29 0.0037 3.64 0.06 0.42 0.88 Cultivar 3 4.72 0.011 10.6 0.0026 1.14 0.36 Error Mean Square 127 3.72 15.8 0.11 Adults Replicate 7 0.55 0.79 0.42 0.75 0.91 0.52 Cultivar 3 2.03 0.14 2.02 0.18 0.80 0.51 Error Mean Square 127 0.78 2.70 0.21

39 0.24% to 15% in 2006 (Table 2.6). The cv. Fort Laramie had significantly less damaged fruit than cv. Tribute, Tristar and Seascape and also the lowest severity index of damage rating, while Tribute had the highest at both sites (Table 2.6).

Fort Laramie also supported significantly fewer nymphs and adults than the other cultivars (Table 2.6). At the Vineland field site, cv. Seascape had the most nymphs and adults; while at Bowmanville, cv. Tribute that had the most but not significantly more than Tristar (Table 2.6).

At the Simcoe field site an analysis of variance indicated there was no significant source of variation among the cultivars (Table 2.6). However, the general trend at the site reiterates the findings from the other two field sites.

The proportion of damage and severity index did not differ significantly between planting position within a plot at all three sites indicating an absence of an edge effect

(Table 2.1, 2.3, 2.4). A significant cultivar by edge interaction was found for severity index at the Simcoe site (P=0.032).

At the Bowmanville field site, two different colours of plastic mulch were used on the raised beds, black and white. There was a significantly higher percent of damaged fruit on the plants grown on the black plastic mulch (7.78%) compared to the white plastic mulch (5.50%), but no difference in the severity index.

An analysis of the proportion of damage per replicate indicated that Vineland had unique results compared to the other two sites. Regression analyses showed that there was a significant linear relationship between the plot replication and number of tarnished plant bug nymphs present and proportion of damage (Table 2.3). The quadratic effect was not significant. Both characteristics decreased as distance from the edge of the field

40 Table 2.6: Mean percent fruit damage, severity index of damage and mean number of tarnished plant bug nymphs and adults for (2006). The mean percent damage, severity index and the mean number of nymphs and adults for each cultivar for Vineland, Bowmanville and Simcoe field sites in 2006. Mean Percent Fruit Mean Severity Number of Number of Damage Index of Damage Tarnished Plant Tarnished Plant Strawberry Untransformed Untransformed Bug Bug Site Cultivar (Transformed) (Transformed) Nymphs ± SE Adults ± SE Vineland Tribute 9.7 (0.32) a 0.17 (0.42) a 2.9 ± 2.5 b 0.53 ± 0.88 b Seascape 3.3 (0.18) b 0.05 (0.23) b 3.5 ±2.4 a 1.0 ±1.3 a Tristar 3.5 (0.19) b 0.056 (0.24) b 2.2 ±1.7 be 0.50 ± 0.76 b Fort Laramie 0.92 (0.097) c 0.012 (0.11) c 1.4 ±1.7 c 0.41 ± 0.67 b LSD (0.056) (0.079) 0.91 0.47 Bowmanville Tribute 14.8 (0.40) a 0.29 (0.57) a 10.5 ±4.0 a 2.8 ±1.6 a Seascape 10.8 (0.34) b 0.22 (0.48) b 7.7 ± 4.8 b 2.3 ± 2.10 ab Tristar 3.6 (0.19) c 0.051 (0.23) c 10.2 ±4.8 a 2.2±1.8ab Fort Laramie 1.4 (0.12) d 0.02 (0.14) d 5.0 ± 2.8 c 1.3 ± 1.1b LSD (0.041) (0.064) 1.8 0.82 Simcoe Tribute 0.82 (0.091) a 0.0083 (0.092) ab 0.13 ±0.34 a 0.19 ±0.40 a Seascape 0.82 (0.091) a 0.011 (0.11) a 0.22 ± 0.42 a 0.28 ±0.68 a Tristar 0.79 (0.089) a 0.0081 (0.091) ab 0.063 ± 0.25 a 0.094 ± 0.30 a Fort Laramie 0.24 (0.050) a 0.0026 (0.052) b 0.094 ± 0.30 a 0.19 ±0.40 a LSD (0.042) (0.050) 0.21 0.30

41 increased (Figure 2.2).

2.5.2 Incidence and Temporal Distribution of Tarnished Plant Bug in 2006

Although the cultivars were damaged similarly at the three sites, the number of plant bugs varied with time between sites. During the 2006 growing season, Bowmanville had the highest population of nymphs and adults on all cultivars. The Simcoe site had the lowest population of nymphs and showed very irregular patterns (Figure 2.3). The nymph population increased in July at all field sites and peaked in August. Nymphs were consistently found at Vineland and Bowmanville over the growing season, but not at

Simcoe.

When the pattern of nymph and adult abundance was compared with the amount of damage observed, the nymphs appeared to cause more damage. Since the fruit takes about 4 weeks to ripen, there is a 4 week lag between when they feed and when the damage is seen on the ripe fruit. At the Bowmanville field nymph numbers increased in

July corresponding to increased fruit damage in August.

Adults were consistently found over the growing season at the Vineland and

Bowmanville sites but not at Simcoe. The adult population increased in the spring and remained high in August and September at the Vineland and Bowmanville field sites, and remained high in October at Bowmanville (Figure 2.3). At the Simcoe site, adults were found in the spring but not in the fall season.

Damaged fruit was first seen in July and was consistently found throughout the growing season at all field sites (Figure 2.3). At Vineland, the amount of damage declined over the growing season on most cultivars except Tribute which peaked in damage in August. At Bowmanville the amount of damage peaked in August and then

42 0.07 - r 60

0.06- i^^N^ " - 50 ^X^V. Damage § 0.05 ^ts/^X. y = -0.0701 x + 66 40 *. O R2 = 8603 CD y = -9E-05X + 0.076 ^^^vT^^X. °- .Q o 0.04 2 N SS>S E CL R = 0.8727 ^" NT V. 3 a> Nymph Number X^ ^X. - 30 2 «D) 0.03 - .Qc. E CO a • X >» CzO g 0.02 x^ - 20 4-1 a> o 5 - 10 0.01 -

0- I I I I I I 0 (3 100 200 300 400 500 600 700 Distance from the Field Edge (cm)

Figure 2.2. Effect of replication on the proportion of damage and total nymphs number. As the replication number increases so does the distance from the edge of the weedy field. Comparison of mean proportion of fruit damaged by tarnished plant bug (•) and total sum of plant bug nymphs (•) for each replication within the Vineland field site.

43 declined. Seascape and Tribute had the highest amount of damage in August and

September at Bowmanville. Minimal damage was found at the Simcoe site during the

2006 growing season. Damage increased in the spring at the Simcoe site and peaked in

August, while in the fall the amount of damage drastically declined (Figure 2.3).

2.5.3 Field Data Results in 2007

In 2007 the plant bug population was exceptionally low at both the Vineland and

Simcoe sites and the threshold was not reached in the buffer zone or within the plots. An analysis of the proportion of damage and severity index indicated no significant sources of variation for the effect of cultivar, replication and edge (Table 2.9).

2.5.4 Incidence and Temporal Distribution of Tarnished Plant Bug in 2007

The population of nymphs at the Vineland field site was very low in 2007 and started to increase in at the end of May. No adults were seen in the samples at this field site over the growing season. Even though there was a lack of nymphs and adults recorded over the growing season, some plant bug damage was seen on the fruit. Damage was recorded at the end of May, with a peak in mid June, which started to increase in early June.

Nymphs were found in the Simcoe field plots at the end of May 2007 and peaked at the end of June. Adults were first seen at the end of May on two cultivars, Seascape and Fort Laramie, and were steady through the two-month growing season. Some damage was observed in the plots and was initially found at the end of May. The amount of damage began to increase in early July but the field was ploughed. The proportion of fruit damaged in 2007 was much lower than in 2006.

44 Vineland Bowmanville Simcoe 7 6- 5- 4- E 3- 2 2- 1 *J rlMi ill Jne JJy Ajg Sept C« June JJy Aig Sept Cd June JJy Ajg Sept Cct

1 Q8 0.6 Q4 Q2 A 0 \L jLtie JJy Aug Sept Oct Jine JJy Aug Sept Oct June JuV Aug Sept Oct

„ 80-. E0 80-1

BO 60-

43 40 CD C Q.• Q 3D-J 3D 20

0 ill II l I, 0 *n rll Jl 0 June JJy Ajg Sept Cfct June JJy Ajg Sept Cct Jine JLty Ajg S^t Qt

Figure 2.3. Frequency histograms for 2006. Tarnished plant bug populations and damage to the commercial strawberry cultivars • Fort Laramie,B Seascape,B Tribute and H Tristar at Vineland, Bowmanville and Simcoe field sites in 2006. Comparison of the mean number of nymphs, adults, and percent of fruit damaged by tarnished plant bug.

45 Table 2.7. Damage and severity index variation on mulch treatments for 2006. Analysis of variance of the proportion of damage and severity index caused by tarnished plant bug on white and black mulch in 2006 for four cultivars at Bowmanville. Bold numbers indicate significant differences. Proportion Damage Severity Index Source df F ratio P F ratio P Plastic 1 14.0 0.034 0.95 0.40 Replicate 3 3.1 0.19 1.06 0.48 Error Mean Square 3 0.0048 0.64 0.024 0.34 Cultivar 3 58.8 0.0001 60.9 0.0001 Error Mean Square 3 0.54 0.66 0.53 0.67 Edge 1 0.10 0.76 0.25 0.62 Cultivar* edge 3 1.2 0.31 0.68 0.57 Plastic*edge 1 0.000014 0.97 0.04 0.85 Error Mean Square 88 0.0085 0.021

Table 2.8. Nymph density variation amoung replications for 2006. Analysis of variance of the number of nymphs found per replication at the Vineland field site 2006. Bold numbers indicate significant differences. Source Df Mean Square F value P Replication 7 0.047 3.06 0.0062 Linear 1 0.019 3.99 0.048 Quadratic 1 0.0061 1.30 0.26 Error Mean Square 127 0.016

Table 2.9. Combined field analysis of fruit damage and severity index for 2007. Analysis of variance of the proportion of damage and severity index caused by tarnished plant bug in 2007 for four cultivars at Vineland and Simcoe. Bold numbers indicate significant differences. Vineland Simcoe Proportion of Severity Proportion of Severity Damaged Fruit Index Damaged Fruit Index F F F F Source df ratio P ratio P ratio P ratio P Replicate 7 1.5 0.21 1.8 0.14 1.1 0.41 1.1 0.39 Cultivar 3 0.78 0.52 0.57 0.64 0.31 0.82 0.33 0.81 Error Mean 21 1.1 0.32 1.0 0.45 1.3 0.17 1.3 0.19 Square Edge 1 1.3 0.26 1.5 0.23 0.81 0.37 1.2 0.27 Edge*cultivar 3 0.62 0.61 0.63 0.60 0.57 0.63 0.54 0.66 Error Mean 92 0.0011 0.0014 0.0026 0.0037 Square

46 2.6 Discussion and Conclusions

In my study, tarnished plant bug damage on strawberry cultivars at different field sites was uniform and rank consistent. These results indicate that cultivars can be selected for southern Ontario as a whole; there is no need to breed specific cultivars for specific regions.

Differences were found in my study in the proportion of plant bug damage and severity index of damage among the 4 commercial cultivars tested. These results are similar to the variability found in the percent damage of cultivated June-bearing strawberry cultivars previously reported by Handley et al. (1991; 1993). My study is the third to report on differences in commercial everbearing and day-neutral cultivars.

Easterbrook (2000) also found variation in damage among commercial cultivars and Dale et al. (2008) reported differences in damage among commercial cultivars and experimental genotypes. The study by Dale et al. (2008) was the first to report variation in the amount of tarnished plant bug damage found on F. virginiana hybrids and my results indicate Fort Laramie sustains the lowest amount of damage.

Tarnished plant bug nymphs had a greater impact than adults on the amount of damaged fruit in the field. The amount of damage observed was proportional to the population size of tarnished plant bug within the field. Tarnished plant bug nymphs and adults were consistently found at the Vineland and Bowmanville field. At the

Bowmanville site, nymphs may have had a greater contribution to the percent of fruit damaged. As the number of nymphs increased so did the amount of damage. As the growing season progressed the nymph population and the amount of damage began to decrease even though there was still a high number of adults present in the field. A

47 similar trend was seen at the Vineland field site but since there was a lower population, the trend was not as significant.

Fort Laramie may exhibit antixenosis because it consistently had the lowest number of plant bug nymphs, which resulted in the lowest proportion and severity index of tarnished plant bug damage at all field sites. These results confirm the work by Dale et al. (2008) who also found Fort Laramie had reduced fruit damage than other day-neutral cultivars but did not state a possible explanation of resistance. Highly susceptible cultivars supported high numbers of plant bug nymphs, which resulted in a high amount of fruit injury. My study shows that the amount of injury observed is related to the number of nymphs present. These results are consistent with the findings of Handley et al. (1993) where resistant cultivars harbored fewer nymphs than the more susceptible cultivars. Easterbrook (2000) also showed that lower numbers of nymphs were correlated with a lower incidence of damage. Dale et al. (2008) found that F. virginiana genotypes supported fewer nymphs and also had a lower percentage of damaged fruit.

Plant bug nymphs appear to travel short distances into crops. The results of this study suggests that tarnished plant bugs remain on susceptible cultivars and do not readily move to more resistant ones, which means that several cultivars can be grown within proximity of each other with minimal consequences. Nymphs did not move between plots of different cultivars providing no evidence of edge effects. At the

Vineland field plot, strawberry plants at the perimeter of the field were found to have a higher incidence of damage versus plants at the center. These results are consistent with

Dale et al. (2007) who found the further away the row is from the weedy perimeter of the field, the lower the percentage of misshapen fruit. In the current study, eggs may have

48 been laid in the grass near the edge of the strawberry field allowing nymphs to migrate into the strawberry crop. Since nymphs are unable to walk long distances, higher numbers of them were found on the outer perimeter rows of the strawberry field and this would increase the incidence of damage observed. Stinner et al. (1983) found that adult leafhoppers emerging from overwintering sites colonized the outer perimeter of an apple orchard resulting in increased damage. To reduce tarnished plant bug from migrating into the perimeter of the strawberry crop, weeds could be controlled with herbicides to reduce attractive hosts that could lead plant bug into the strawberries. Early applications of herbicides in apple orchards before the period of fruit susceptibility, significantly reduced fruit damage caused by plant bug (Hardman et al., 2004; Snodgrass et al., 2005).

Plastic mulch with high UV reflectance appears to directly repel tarnished plant bugs. In my study, strawberry plants grown on white plastic mulch had reduced plant bug injury compared to plants grown on black plastic. This finding support Rhainds et al.

(2001) work where silver reflective mulch consistently reduced the density of tarnished plant bug nymphs on strawberry flower clusters. Since the mulch increased yield as well, it may have caused a dilution effect on the number of damaged fruit, thus reducing the amount of damage observed in the field. They also found that reflective mulch reduced plant bug damage as much as can be achieved with one application of Malathion.

The behavioural responses of insects to light and colour are related to the portion of the compound eye that is illuminated, where different amounts of reflected light can either be attractive or repellent (Kring 1972; Kring and Schuster 1992; Summers et al.,

1995; Rhainds and English-Loeb, 2001; Stavisky et al., 2002; Reitz et al, 2003; Momol and Stavisky, 2004; Demirel and Cranshaw, 2006). Kring and Schuster (1992) speculate

49 the lowered insect populations are due to the level of UV light reflected since the percentage of UV reflectance of aluminum plastic is four times greater than black mulch.

Mulch with high UV reflectance, i.e. silver, does not eliminate insect pests but reduces the initial infestation levels by repelling migrating insects (Kring and Schuster 1992).

Thus the reflective nature of the white plastic at visible wavelengths in this study may have affected the plant bugs' sensory ability to search for a host food source and thus reduced the amount of damage observed.

New integrated pest management programs that combine resistant cultivars with barrier technologies could be used to reduce damage on susceptible cultivars. Fort

Laramie has been found to have reduced damage but is not grown due to poor fruit quality; while Seascape is the most common cultivar used in production despite its known susceptibility to plant bug. My study has shown Fort Laramie is highly resistant to plant bug damage, and has also shown nymphs do not migrate past the edge of the field.

Seascape may be a susceptible cultivar however consumers prefer it's fruit quality over other day-neutral fruit rendering Seascape as the most popular day-neutral cultivar grown in commercial production. Although Fort Laramie is resistant, the cultivar lacks good fruit quality. Seascape has large fruit, with firm flesh and bright colour, while Fort

Laramie has smaller berries, with soft flesh that is easily damaged and is dull in colour. A new field design could be developed where the highly susceptible, yet commercially successful, cv. Seascape could be grown in a block surrounded by a perimeter of resistant

Fort Laramie plants.

Since Fort Laramie is less preferred by plant bug nymphs, migration of nymphs into the field from weedy lanes should be reduced, providing protection to the susceptible

50 Seascape plants in the center. The width of the Fort Laramie perimeter will also need to be tested to determine how many rows could reduce plant bug migration. To strengthen the program, all the plants should be grown on white plastic mulch, which will further reduce the population of plant bug. As well, weeds at the edge of the field should also be controlled in attempt to reduce attractants to the strawberry field. This new field plan would enable growers to produce the susceptible Seascape fruit to satisfy consumers and allow higher quality fruit.

Field experiments should be performed to determine if migrating nymphs or migrating adults are most responsible for fruit injury. Seascape plants should be planted in a center square of the field with several rows of Fort Laramie plants surrounding. The number of nymphs and amount of damage should be sampled from the buffer rows of

Fort Laramie and in the center planting of Seascape. If a high number of nymphs and injury were found in the center Seascape plants and low numbers in the Fort Laramie plants, this would indicate that adult migration into the field is the main cause of fruit injury. Conversely, if the number of nymphs and injury found in the center Seascape plants is low, this would indicate that migrating nymphs from the field edges cause the most injury and were repelled by Fort Laramie plants, thus reducing damage to Seascape fruit.

The emphasis of my study was to identify the response of several day-neutral cultivars to tarnished plant bug feeding at different sites in Ontario, as insect populations can evolve biotypes at different locations. At the sites examined in this study, the cultivars were consistently ranked in the same order of most to least susceptible, indicating no geographical differences in plant bug preferences. A range of damage was

51 found between cultivars in the amount of damage and this indicates that some cultivars are more resistant to plant bug damage than others, with Fort Laramie being the most resistant. Plants grown on white plastic mulch show reduced plant bug damage to the fruit and indicates this colour of mulch can be used as a cultural control. The variability found in the amount of damage between cultivars indicates a potential mode of resistance within the plants. The mechanism of resistance was not elucidated in my study and further examination is required to determine what mode of resistance could be present and whether it could be used as an effective method of control for tarnished plant bug.

Laboratory bioassays could be used to assess the variability observed and provide a starting point in determining the potential mechanisms of resistance.

The variability in the amount of damage between cultivars could play a crucial role in breeding programs and my study indicates these programs could be applicable in several areas of southern Ontario. Consequently resistant plants and white mulch could be combined thus reducing the proportion of fruit damaged by tarnished plant bug and benefiting commercial producers.

52 Chapter 3

3 Investigating Tarnished Plant Bug Resistance in Strawberries

3.1 Abstract

Resistance to tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), has been used to explain the variation in damage found between different strawberry cultivars

{Fragaria X ananassa L.) Breeding for tarnished plant bug resistance has produced strawberry hybrids which show less injury, but these have not been ranked by level of resistance. My study established protocols to determine: 1) the most susceptible stage of fruit development; 2) the number of nymphs required to induce an adequate amount of fruit damage to elucidate differences between genotypes; and, 3) a severity index to characterize damage caused by plant bug adults feeding on strawberry fruit. Strawberry hybrids and the commercial day-neutral cultivars - Fort Laramie and Seascape, were tested in choice and no-choice bioassays in order to rank the genotypes and determine which consistently had the lowest proportion of damage. The amount of pollen on the flowers was also manipulated to determine if pollen load affects the severity of damage.

Fruit at the green stage showed the highest proportion of damage and flowers at petal fall became aborted, while exposed flowers developed normal fruit. The more nymphs there were, the greater the damage and severity index. There was no significant difference in the proportion of damage and severity index caused by adults and nymphs, which indicates that control of adults is just as important as control of nymphs. In no-choice experiments, the 704-43 Fragaria virginiana hybrid was damaged less than the susceptible cv. Seascape, which indicates that the genotype may exhibit elevated resistant qualities. Choice experiments confirmed resistant strawberry hybrids of families 703, 704

53 and 708, and cv. Fort Laramie showed less damage than their susceptible comparators.

No difference was detected in the proportion of damage found between choice and no- choice experiments, which indicates the differences between genotypes were consistent.

Flowers of the susceptible genotype with pollen present had a higher proportion of damage than with pollen absent. The resistant genotype had a lower proportion of damage than the susceptible genotype and the occurrence of damage was not related to the presence or absence of anthers. Genotype, 708-58 was identified to consistently have reduced damage and exhibit resistant qualities.

3.2 Introduction

The biology and relationship of host and pest interactions need to be understood in order to make recommendations for improved integrated crop management. When investigating the basis for differences in resistance and susceptibility to insects among cultivars and/or genotypes, we must determine the most susceptible phenological stage of the crop and understand how the life stage of the insect affects the amount and severity of damage incurred. The type of damage caused by tarnished plant bug to strawberry fruit is dependent on the developmental stage of the crop, since some specific stages are more susceptible than others. In previous experiments, flowers and fruit at various stages of development (i.e. closed bud, open flower, achenes enlarging and achenes just separated) were caged and exposed to tarnished plant bug 3rd instar nymphs and adults. Damage to strawberry flowers from petal fall to achene separation resulted in apical seediness, while fruit with pink receptacles were not damaged (Allen and Gaede, 1963; Handley, 1991;

Easterbrook, 2000). However, one study has shown fruit at the green stage exhibits the highest amount of damage while exposed flowers in bloom exhibit the second most

54 damage (Allen and Gaede, 1963) and another showed that open flowers became aborted

(Handley, 1991; Easterbrook, 2000).

Discrepancies have been identified and Handley's (1991) results were challenged by Wold and Hutchison (2003) who state Handley's sample size was too small to detect significant differences in the susceptibility of the different fruit stages. Wold and

Hutchison (2003) stated that Allen and Gaede (1963) did not show that damage differed among the growth stages and concluded that all growth stages were susceptible to plant bug. In Wold and Hutchison's (2003) study, high amounts of damage were required to determine differences among hybrid genotypes. Further investigation is required to verify which developmental stage is the most susceptible to damage.

The duration of fruit exposure to plant bugs also contributes to the type of damage observed. Low exposure durations result in a minimal amount of damage while longer exposures can cause blossom death (Handley, 1991; Easterbrook, 2000). A high tarnished plant bug population can also affect the amount of damage; as the number of nymphs increases the amount of damage also increases (Easterbrook, 2000). Ultimately, exposing fruit to a high number of plant bug nymphs or adults for 48 hours when fruit is between the petal fall and achene separation stage will result in the highest amount of apical seediness (Handley, 1991; Easterbrook, 2000). This information is important in developing a methodology focused on finding differences in resistance between strawberry genotypes. It is still unknown whether 4th or 5th instar nymphs cause more damage than adults, but it has been shown that 1st and 2nd instar nymphs cause the least amount of injury (Allen and Gaede, 1963). However the 3rd instar nymphs were not ranked at all.

55 The effect of each life stage on developing plants needs to be understood to determine which stage has the most impact on which plant structure as well to determine which stage should be targeted in integrated pest management programs. Adult plant bugs have been found in high numbers on the reproductive structures of day-neutral cultivars (Rancourt et al., 2000). The mobility and distribution of plant bug suggests that the exclusion of adults from population estimates may be inappropriate for everbearing cultivars, since fruit is produced for several months until fall. However, it has not been verified that adult tarnished plant bugs cause more or less damage to immature strawberry fruit than nymphs. Cotton plants, Gossypium hirsutum L. swept with nets, had more tarnished plant bug adults on the vegetative structures, while nymphs were found on fruiting structures (Zink and Rosenheim, 2004). Although nymphs may be found on fruiting structures, it is not known whether nymphs actually cause more damage to developing fruit.

Greenhouse studies have shown that the presence of L. hesperus adults significantly increased strawberry fruit deformity and would constitute a problem for growers of day-neutral species (Riggs, 1990). In that study, secondary flowers of the day- neutral strawberry cultivar Tristar were exposed to adult L. hesperus in caged experiments for 48 hours. Adults caused damage to developing fruit but it was not compared to that caused by nymphs (Riggs, 1990).

Damage by tarnished plant bug in June-bearing cultivars is thought to be minimal since there is slow buildup of population numbers in the spring, but this has been determined only for the New York region and may not be true for other regions

(Schaefers, 1981).

56 The proportion of fruit damaged by plant bug varies between commercial day- neutral cultivars and in wild species. Wild strawberries, F. virginiana, are damaged less by tarnished plant bug than cultivated strawberry and have been used in crossing experiments (Dale et al., 2008). A range of day-neutral genotypes have been selected to develop families, which vary in day-neutrality and susceptibility to tarnished plant bug damage. Previous field trials have showed consistent variability in the proportion of damage at several field sites in Ontario (Chapter 2).

For the purpose of my study, experimental protocols need to be developed to identify differences among hybrid genotypes. Many studies have shown differences in plant bug damage in the field, but few have performed such research on strawberries in the laboratory. The methodology used in previous bioassays was modified for my study to quantify differences in damage and to elucidate potential reasons for differences.

Cultivars have shown differences in the amount of injury sustained but the underlying mechanism is unknown. These bioassay procedures are designed to determine the mechanisms of resistance exhibited in plants: antibiosis, antixenosis and/or tolerance

(Painter, 1951; Kogan and Ortman, 1978). Choice bioassays determine antixenosis (non- preference) qualities, where insects will select the better host; while no-choice bioassays identify antibiotic qualities which determine host suitability (Diaz-Montano et al., 2006).

Plants in no-choice bioassays will be exposed to the same population of nymphs which would allow a damage comparison between resistant and susceptible plants and could identify tolerance as a mechanism of resistance.

Antixenotic qualities can be identified when a resistant and susceptible genotype are exposed simultaneously to an insect herbivore. Choice studies can evaluate insect

57 preference for particular plant genotypes by exposing the pest to several host options

(Painter, 1951). Individual genotypes can be exposed to insects in no-choice experiments to determine how the plant affects the metabolic processes of the insect. If the resistant plants exhibit antibiotic characteristics, then the insect may not mature or reproduce normally (Painter, 1951).

No-choice bioassays can be used to effectively show resistance by means of antibiosis. Of the few strawberry studies that have been performed, no-choice bioassays on the strawberry cultivars Bolero, Everest and Evita showed significant differences in nymph weights among cultivars, indicating resistance by means of antibiosis for Bolero

(Easterbrook and Simpson, 2000). Plant bug nymphs and ovipositing females were also found to exhibit preferences for plants with more flowers and larger fruit (Rhainds and

English-Loeb, 2003).

Antibiosis and antixenosis traits were found in resistant soybean genotypes,

Glycine max (L.), to soybean aphid, Aphis glycines, in choice and no-choice experiments

(Diaz-Montano et al., 2006). In choice experiments a lower number of adults were found on the resistant genotypes compared to susceptible, indicating a strong antixenotic effect.

While in no-choice experiments, fewer nymphs hatched on the resistant genotypes, indicating a strong antibiotic effect was also present (Diaz-Montano et al., 2006).

Resistant wheat genotypes of Triticum aestivum can affect the host choice behaviour of the greenbug, Schizaphis graminum, and also affect the development and fecundity of nymphs which are reared on the plants. Choice and no-choice tests were used to categorize wheat genotypes as antixenotic and antibiotic to greenbug (Zhu et al.,

2005). Resistant wheat genotypes had a reduced number of adults in choice experiments,

58 which indicated antixenotic qualities (Zhu et al., 2005). Late instar nymphs exposed to resistant wheat genotypes in no-choice experiments were found to have delayed maturity and lower fecundity rates than nymphs on susceptible genotypes. The abnormal biological development of the nymphs strongly indicates antibiosis as the resistance mechanism (Zhu et al., 2005).

The level of expression of resistant properties can vary in resistant genotypes, having strong effects on specific pests. Resistant sweet potato germplasm, (Ipomoea batatas L.) Lam. (Convolvulaceae) has shown strong antibiotic properties against banded cucumber beetle (Diabrotica balteata LeConte) in bioassays. Beetles that fed on resistant genotypes had reduced longevity by 111 days (Jackson and Bohac, 2007)._Beetles in no- choice bioassays never initiated feeding on resistant genotypes and most beetles died before they fed, indicating antixenotic affects (Jackson and Bohac, 2007).

The morphology of the strawberry plants could influence plant bug behaviour and affect the amount of damage observed. Specific physical features of strawberry plants have yet to be studied to determine how plant morphology affects the behaviour of plant bug and amount of damage observed. Resistant strawberry plants possibly exhibit antixenotic morphological qualities, which may deter tarnished plant bug. Pollen is highly attractive for flower pollinators and could also attract insects that damage the plant as well. The amount of pollen may influence the preference of plant bug and could affect the incidence of damage.

Investigation of floral characteristics of strawberry cultivars has shown high amounts of pollen are significantly correlated with damage (Handley and Dill, 2003).

However, constituents such as sugar, amino acid and protein content within nectar and

59 pollen were unrelated to the attractiveness of strawberry cultivars to tarnished plant bug

(Grunfeld et al., 1989). Adult tarnished plant bugs generally restrict feeding to male

reproductive structures on cotton flowers, G.hirsutum L., in caged experiments (Williams

and Tugwell, 2000). Furthermore, plant bug damage on cotton plants was concentrated

on the anthers and pollen grains, which were characterized as being irregularly shrunken

and dark (Pack, 1973 reviewed in Williams and Tugwell, 2000). Most of the research on

pollen load has been performed on cotton and research pertaining to strawberry

production is lacking. Further research is required to investigate if the amount of pollen

affects the preferences of tarnished plant bug to strawberries cultivars.

3.3 Objectives

Laboratory bioassays were conducted to develop techniques and to elucidate possible mechanisms of resistance. The bioassays were conducted:

1. to verify which strawberry growth stage is the most susceptible to tarnished plant bug,

2. to determine the optimal number of nymphs to use in experiments to identify differences between genotypes,

3. to determine the extent and severity index of damage to immature fruit caused by tarnished plant adults,

4. to confirm results of the Dale et al. (2008) field experiment in a controlled environment, where day-neutral hybrids and commercial cultivar Fort Laramie had less plant bug damage,

5. to use differences in the proportion and severity index of damage for tarnished plant bug to compare genotypes and identify the presence of resistance and associate mechanisms, and,

6. to determine if the presence or absence of pollen in strawberry flowers affects the incidence of damage caused by tarnished plant bug nymphs.

60 3.4 Materials and Methods

3.4.1 Insect Rearing and Maintenance

Adult tarnished plant bug were collected from an alfalfa field near Guelph,

Ontario, Canada in September and October 2005. They were collected with a sweep net and aspirated into vials for transportation to adult colony cages. The adults were split equally between two cages which were made with a wooden frame (43cmx 23cmx 41cm) with five sides covered in mesh and one Plexiglas side with a nylon access port.

Nymphs were raised in 4L white Plastipak plastic buckets with mesh lids for aeration.

Both adults and nymphs were given green beans, snow peas and romaine lettuce as food sources. Potato sprouts were provided to the adults as an oviposition substrate. Adults were provided water using a 500mL mason jar placed upside down on a petri dish and held in placed with elastics. A toothpick was inserted to maintain water flow. Food and oviposition substrate were replaced every 2 to 3 days and placed in 4L buckets to allow eggs to hatch. Buckets of nymphs were inspected every two to three days and any adults were removed and placed into the adult cages for . Colonies were maintained at the University of Guelph growth room until May 2006 at 25 ±1°C, with 16:8 hour lightdark regime and 50% RH. The colony was moved from Guelph to the Simcoe

Research Station growth cabinets in May 2006; both adult and nymphal colonies were kept in a growth chamber set at 25 ± 1°C, with 16:8 hour lightdark regime and 30% RH.

3.4.2 Plant Maintenance

Strawberry genotypes were selected from a previous work which studied the inheritance of tarnished plant bug resistance (Dale et al., 2008). Two individual genotypes from five crosses with the least and greatest tarnished plant bug damage were

61 selected as matched pairs to be used in my study, Figure 3.1. The hermaphrodite genotypes used were: 703-48 and 43 progeny of Fern (susceptible) and 21K74 (resistant);

704-12 and 63 progeny of Fern and Evangeline (resistant); 705-69 and 49 progeny of

Fern and 127A71 (susceptible); 708-60 and 58 progeny of Fort Laramie (resistant) and

21K74 (resistant); 709-24 and 36 progeny of Fort Laramie and Evangeline (resistant); along with Seascape and Fort Laramie.

The matched plant pairs were transplanted into 15 cm diameter pots and maintained in a glasshouse with daily drip irrigation of a 20% nutrient solution (Plant

Prod® 20-20-20 water soluble fertilizer, mixing rate of 300g/100L) for 120 seconds per day. Flowers were removed to promote runner production and thus increase in the number of plants available for experimentation. Once the runners had leaflets they were cut and planted into thirty-six cell packs to promote root development. Plants were potted into a 1:1:1:1 soil mixture of vermiculite (Therm-o-Rock® Vermiculite and Perlite, New

Eagle, PA), sand, peat moss (Premier Pro Mix®, Quakertown, PA) and turface (Turface

Athletics MVP®, Buffalo Grove, IL), which was premixed before use. Plants were maintained in a glasshouse with temperatures between 20-25°C. The plants were exposed to natural light during the spring and summer, and supplementary lighting was provided during the winter, 5:00pm to 6:00am from October to May. The light was generated by

400W high pressure sodium lamps suspended 2m above the bench and each lamp illuminated approximately 2m of bench space.

3.4.3 Laboratory Trials

Plants used for experimentation were selected based on the growth stage of the . Plants were used in experiments when the primary flower was between

62 Fort Laramie x Evangeline = 709 genotypes

Fl Fl Fl Fl Fl Fl Fl 36 26 29 47 30 41 24

Susceptible < —• Resistant

Figure 3.1. Example of pair selection for laboratory bioassays. Fort Laramie and Evangeline were crossed, producing the new family 709 genotypes. Progeny within the family were ranked in order of damage where the plant with the most and least damage were used a matched pair in the bioassay experiments.

63 petal fall and the separation stage and the secondary flower was in blossom.

Flowers were hand pollinated twice at anthesis, before and after plant bug

exposure, to ensure consistent pollination. Anthers were removed from other strawberry

plants and placed into a Petri dish to dehisce. Pollen was then transferred to the

experimental flowers using a camelhair brush. Plants were placed into a wooden test cage

(43cmx 23cmx 41cm) which had five sides covered in mesh and a Plexiglas lid with a

nylon access port.

In no-choice single plant bioassays, nymphs were released on the plant; while in

choice two plant bioassays, nymphs were dropped out of vials between the pots. After

two days the nymphs were removed and counted, and the developmental stages of all

flowers and fruit were recorded. Fruit was harvested when the receptacle tissue was fully

red. Damage was rated on a 0 to 4 point scale, where 0= no damage, 1=

damaged, 2= 1/4 to 1/3 of the berry damaged, 3= 1/3 to 2/3 of the berry damaged, and 4=

>2/3 of the berry damaged.

3.4.4 Experimental Design

3.4.4.1 Determination of Optimal Nymph Numbers

Experiments to determine the optimal nymph number to distinguish among

resistant cultivars were conducted between September 6th and November 27th, 2007. Two

day-neutral genotypes (Seascape and 703-43) were individually exposed to 0, 1, 3 or 6

tarnished plant bug nymphs. Ten replicates were conducted where each replicate

consisted of a single plant per cage. Plants were exposed to nymphs for 72 hours. Once

fruit developed and ripened, berries were harvested and damage was rated.

64 3.4.4.2 Determination of Susceptible Stage of Strawberry Development

Experiments to determine the most susceptible fruit stage were conducted between October 22nd and November 30th, 2007. Single day-neutral plants each with a range of different developmental stages of fruit were exposed to 6 tarnished plant bug nymphs for 72 hours. Ten replicates were conducted where each replicate consisted of a single caged plant. Once fruit developed and ripened, berries were harvested and damage was rated.

3.4.4.3 Determination of Strawberry Fruit Susceptibility to Adult Plant Bug

To determine susceptibility of strawberry fruit to adult plant bug experimentation was conducted November 12l to 30 , 2007. Single day-neutral plants were exposed to either 6 nymphs or 6 adult tarnished plant bug for 72 hours. Ten replicates were tested each of which consisted of a single plant per cage. Once fruit developed and ripened, berries were harvested and damage was rated.

3.4.4.4 Choice Bioassays: Resistant and Susceptible Strawberry Genotype Pairs

Experiments were conducted from November 23rd, 2007 to January 7th, 2008. Six matched pairs of day-neutral strawberry genotypes (703-48 and 43, 704-12 and 63, 705-

49 and 69, 708-58 and 60, 709-24 and 36, Fort Laramie and Seascape) were used to confirm the results of Dale et al. (2008) for resistance to tarnished plant bug. Each replicate consisted of 2 plants per cage, i.e. one susceptible and one resistant plant of the same genetic background, which allowed the nymphs a choice of 2 hosts to feed on. Ten to 14 replicates were recorded for each genotype pair. Six tarnished plant bug nymphs were placed per cage and retrieved after 72 hours. Once fruit ripened, they were harvested and damage was rated.

65 3.4.4.5 No-choice Bioassay: Resistant versus Susceptible Genotypes

All the experimental genotypes, except for family 705, were evaluated. Family

705 was excluded because the plants of 705-69 were not flowering at the time.

Ten genotypes were individually exposed to tarnished plant bug nymphs. Three or

6 tarnished plant bug nymphs were placed in each cage to determine how much damage was induced by a given number of nymphs. Ten to 14 replicates of each genotype were conducted. Once fruit developed and ripened, berries were harvested and damage was rated.

3.4.4.6 Influence of Pollen Load on Damage Levels

Experiments were conducted between November lx , 2007 and January 7l , 2008.

Ten, 2 plant replicates of the day-neutral genotypes, 704-63 and 704-12, were conducted.

In each replicate one plant had the anthers removed from all the flowers and on the other the anthers were left in place. The plants were placed into a wooden test cage and exposed to three tarnished plant bug nymphs for 72 hours. Once fruit developed and ripened, the berries were harvested and damage was rated.

3.4.5 Statistical Analyses

3.4.5.1 Determination of Optimal Nymph Numbers, Susceptible Stage of Strawberry Development and Susceptibility to Adult Plant Bug

The proportion of damaged fruit was calculated as the number of damaged fruit divided by the number of normal and damaged fruit. The severity index of damaged fruit was calculated as the sum of [(lx score rating 1) + (2 x score rating 2) + (3 x score rating

3) + (4 x score rating 4)] divided by the total number of fruit multiplied by 4 (Dale et al.,

2008). Fruit given a severity score of 0 was used in the class of normally developed fruit.

66 Data from all treatments were tested to meet statistical assumptions and data normality. A univariate normalization procedure was performed to determine if data were normally distributed, while Brown and Forsythe homogeneity of variance test determined whether the data was homogeneous (SAS Institute, version 9.1, 2002, Cary, NC). When the data were not normally distributed the data were transformed with the arcs in square root transformation.

The proportion of damaged fruit and severity index was compared for different genotypes with an ANOVA using PROC GLM, with a=0.05 (SAS Institute, version 9.1,

2002, Cary, NC).

For optimal nymph number, the proportion of damaged fruit and severity index were regressed against the number of nymphs with PROC GLM (SAS Institute, version

9.1, 2002, Cary, NC) with the number of tarnished plant bug nymphs as the continuous variable. The regression model used was Y= bxi + bx2g + b3gx , where Y is the response, x is the number of nymphs exposed, b is the main effect of treatment and g is genotype.

The intercept was constrained to zero since no tarnished plant bug damage could occur in the absence of nymphs. When the quadratic coefficients were not significantly different from zero, the model was refitted with these terms set to zero.

3.4.5.2 Choice and No-choice Bioassays: Resistant and Susceptible Strawberry Genotypes

The arcsin square root transformation of the proportion of damaged fruit and severity index from both experiments were analyzed by ANOVA, using PROC GLM

(SAS Institute, version 9.1, 2002, Cary, NC) with genotype as fixed effects. In the no- choice experiments, mean comparisons with contrast statements compared the proportion of damage means of the related genotypes. Data collected from no-choice experiments

67 exposing plants to 3 nymphs or 6 nymphs were compared to each other, mean comparisons with contrast statements compared the proportion of damage means of the related genotypes.

3.4.5.3 Influence of Pollen Load on Damage Levels

The proportion of damaged fruit and severity index from all treatments were analyzed by ANOVA, using PROC GLM (SAS Institute, version 9.1, 2002, Cary, NC) with pollen and replication as independent variables. Means separations were conducted using Tukey's Honestly Significantly Different test, with a=0.05.

3.5 Results

3.5.1 Determination of Optimal Nymph Numbers

The combined regression analysis of both genotypes showed that there was a significant linear relationship between tarnished plant bug number and both the proportion of damaged fruit and severity index (Table 3.1). Genotype did not vary significantly, however both genotype by linear coefficient interactions and the genotype by quadratic coefficient interaction for the proportion of damaged fruit varied significantly.

Individual genotype regression analyses showed that for proportion of damaged fruit and severity index there was a significant linear relationship for genotype 703-43 and a significant quadratic relationship for cv. Seascape (Table 3.2). For both genotypes, more damage and more severe damage occurred when the flowers were exposed to higher numbers of tarnished plant bug nymphs (Figure 3.2). Although damage increased with increasing nymph number for both genotypes, the proportion of damage for 703-43 was lower than that for cv. Seascape when exposed to high numbers of nymphs. This is

68 Table 3.1. Combined comparison of hybrids to nymph number on the proportion and severity index of damage. Combined analysis of variance of the proportion of fruit damaged and severity index by varied number of tarnished plant bug nymphs in 2007 for the strawberry genotype 703-43 and commercial cultivar Seascape. Bold numbers indicate significant differences. Proportion of Fruit Damaged Severity Index of Damage Source df F ratio P F ratio P Replicate 9 5.13 0.027 8.18 0.0055 Genotype 0.0 0.98 0.12 0.73 Linear 77.0 0.0001 48.0 0.0001 Quadratic 0.04 0.85 0.01 0.93 Geno*Linear 8.8 0.0041 7.4 0.0082 Geno* Quadratic 5.01 0.028 3.4 0.068 Error Mean Square 79 0.10 0.016

Table 3.2. Comparison of the effect of nymph number on the proportion and severity index of damage for individual hybrid strawberry genotypes Seascape and 703-43. Separate analysis of variance of the proportion and severity index of fruit damaged by tarnished plant bug nymphs in 2007 for the strawberry genotype 703-43 and commercial cultivar Seascape. Bold numbers indicate significant differences. Seascape 703-43 Proportion of Severity Proportion of Severity Fruit Index of Fruit Damaged Index Damaged Damage F F F F Source df ratio P ratio P ratio P ratio P Replicate 9 1.3 0.26 1.26 0.29 1.48 0.20 1.5 0.20 Linear 1 0.72 0.41 0.17 0.69 16.70 0.0006 11.4 0.0032 Quadratic 1 6.58 0.017 7.29 0.013 2.95 0.10 2.0 0.17 Error Mean Square 39 0.013 0.13 0.024 0.15

69 A.

0.45 n

0 12 3 4 5 6 7 Number of Nymphs

0 12 3 4 5 6 7 Number of Nymphs

Figure 3.2. Mean proportion of damaged strawberry fruit. Mean proportion of damage caused by plant bug nymphs to strawberry inflorescences (A) and severity index (B) of Seascape (•) and 703-43(>) in caged experiments.

70 the first study to find that as the number of tarnished plant bug nymphs increases so does the proportion of damage incurred on strawberry fruit.

3.5.2 Determination of Susceptible Stage of Strawberry Development

All flowers that were exposed to tarnished plant bug nymphs at bloom developed into undamaged fruit. When flowers were exposed at the petal fall stage, 75% of the flowers were aborted, 6% were damaged and 19% were undamaged. When green fruit was exposed, 67% of the fruit were damaged and 33% were undamaged.

3.5.3 Determination of Strawberry Fruit Susceptibility to Adult Plant Bug

Plants exposed to tarnished plant bug adults and nymphs had 91% damaged fruit.

The proportion of damaged fruit and severity index caused by adults and nymphs did not differ significantly. Adults damaged 33% of the fruit and nymphs damaged 38% (df=9

F= 0.32, P= 0.60), and the severity index was 0.62 and 0.79, for adults and nymphs, respectively (df=9 F= 0.34, P= 0.60).

3.5.4 Choice Bioassays: Resistant and Susceptible Strawberry Genotype Pairs

An analysis of the proportion of damage caused by tarnished plant bug in choice experiments revealed that genotype varied significantly for only two of the matched pairs

(Table 3.3): the 708 family genotypes and cv. Fort Laramie/Seascape comparisons. Fort

Laramie and Seascape also differed significantly in the severity index, whereas family

708 genotypes was marginally insignificant (Table 3.3). The mean proportion of damage in Fort Laramie (0.072) was 1/4 of Seascape (0.26), and the mean severity index in Fort

Laramie (0.072) was 1/5 than in Seascape (0.38). The genotype 708-58 had a 1/9 lower proportion of damage (0.048) than 708-60 (0.43). Replicate was not a significant source of variation for any of the matched pair comparisons.

71 Table 3.3. Choice Bioassay: Comparison within families of resistant and susceptible hybrids. Analysis of variance of the proportion of fruit damaged by tarnished plant bug and the severity index in choice experiments between two genotypes of the hybrid families 703, 704, 705, 708, 709, and commercial cultivars Fort Laramie and Seascape. Bold numbers indicate significant differences. Proportion of Fruit Severity Index of Damaged Damage Source df F ratio F ratio 703-48 vs 703-43 1 0.67 0.43 0.12 0.74 Replicate 11 0.98 0.51 1.14 0.42 Error Mean Square 23 0.065 0.44

704-12 vs 704-63 1 0.93 0.35 1.2 0.29 Replicate 13 0.64 0.79 0.57 0.84 Error Mean Square 27 0.025 0.098

705-69 vs 705-49 1 0.42 0.53 0.00 0.95 Replicate 9 1.3 0.34 2.01 0.16 Error Mean Square 19 0.026 0.11

708-60 vs 708-58 1 5.3 0.04 4.8 0.057 Replicate 11 0.98 0.52 0.68 0.73 Error Mean Square 23 0.17 0.14

709-24 vs 709-36 1 0.67 0.43 0.48 0.51 Replicate 9 0.66 0.73 0.64 0.74 Error Mean Square 19 0.0099 0.075

Fort Laramie vs Seascape 1 9.25 0.011 8.7 0.013 Replicate 11 0.97 0.52 1.14 0.41 Error Mean Square 23 0.024 0.064

72 The number of nymphs retrieved from each of the matched plants was not significantly different between resistant and susceptible genotypes (data not shown).

3.5.5 No-choice Bioassay: Resistant versus Susceptible Genotypes

Combined analyses for the proportion of damage and severity index in no-choice experiments revealed that genotypes and, genotype by nymphs varied significantly but number of nymphs used did not (Table 3.4).

Within genotypes, the matched pairs in families 704, 708 and Fort Laramie and

Seascape differed significantly from each other in proportion of damage and the severity index. The matched pairs in the family 703 only differed significantly from each other in the proportion of damaged fruit (Table 3.4).

When the matched pair contrasts were examined individually the nymph number by Fort Laramie/Seascape interaction was significant for the severity index only.

Significant residual interactions were found for both the proportion of damage and the severity index (Table 3.4). The mean proportion of damage in Fort Laramie was 1/3 less and mean severity index was 1/5 less than in Seascape (Table 3.5). The genotype 708-58 had 1/4 lower mean proportion of fruit damaged and 1/5 lower mean severity index than did 708-60 (Table 3.5). The mean proportion of damage in 704-63 was 1/4 less than in

704-12 and the mean severity index was 1/3 less in 704-63 than in 704-12. The genotype

703-43 had 1/3 lower mean proportion of damage and mean severity index than did 703-

48 (Table 3.5). The number of nymphs retrieved from no-choice experiments with the matched families did not significantly differ between resistant and susceptible genotypes.

73 Table 3.4. No-choice Bio assay: Comparison of resistant to susceptible hybrids exposed to different nymph numbers. Combined analysis of variance of the proportion of damage and severity index caused by tarnished plant bug in comparisons of resistant and susceptible genotypes and commercial cultivars when exposed to three or six nymphs in no-choice experiments. An analysis of variance also compared the genotype results of three nymphs vs six nymphs. Bold numbers indicate significant differences. Proportion of Severity Index of Fruit Damaged Damage Source df F ratio F ratio Replication 2.13 0.022 1.5 0.15 Nymph Number 0.46 0.5 1.1 0.30 Genotype 9 4.6 0.0001 3.4 0.0008 703-43 vs 703-48 1 4.1 0.046 3.0 0.087 704-12 vs 704-63 1 15.9 0.0001 7.3 0.0079 708-58 vs 708-60 1 10.5 0.0015 5.7 0.018 709-24 vs 709-36 1 0.05 0.83 0.1 0.75 Fort Laramie vs Seascape 1 6.6 0.011 12.02 0.0007 Residual 4 2.08 0.087 0.71 0.59 Nymphs * Genotype 9 2.06 0.037 2.28 0.021 Nymph vs (703-43 vs 703--48) 1 0.28 0.59 0.84 0.36 Nymph vs (704-12 vs 704--63) 1 1.5 0.22 1.2 0.29 Nymph vs (708-58 vs 708-60) 1 0.05 0.82 0.36 0.55 Nymph vs (709-24 vs 709-36) 1 0.47 0.49 1.1 0.29 Nymph vs (Fort vs Seascape) 1 1.88 0.17 4.3 0.041 Residual 4 3.59 0.0081 3.2 0.015 Error Mean Square 135 0.029 0.11 *Nymph means number of nymphs

74 Table 3.5. Comparison of damage between resistant and susceptible hybrids. Mean damage proportions and severity index for each nymph treatment on 8 strawberry genotypes and 2 commercial cultivars. LSD compared all genotype means within the same number of nymphs. Proportion of Damage Severity Index of Damage 3 6 3 6 Genotype Nymphs Nymphs Mean Nymphs Nymphs Mean 703-43 0.069 0.083 0.076 0.078 0.20 0.14 703-48 0.24 0.17 0.21 0.41 0.30 0.36

704-63 0.11 0.043 0.077 0.18 0.083 0.13 704-12 0.38 0.21 0.30 0.57 0.28 0.43

708-58 0.031 0.094 0.063 0.030 0.093 0.062 708-60 0.21 0.30 0.26 0.24 0.45 0.33

709-36 0.16 0.094 0.13 0.35 0.19 0.27 709-24 0.14 0.15 0.15 0.19 0.28 0.24

Fort Laramie 0.065 0.16 0.11 0.066 0.16 0.11 Seascape 0.14 0.39 0.27 0.25 0.83 0.54 LSD 0.19 0.16 0.37 0.31

75 3.5.6 Choice versus No-choice Exposure

Choice and no-choice experiments were compared between the family pairs.

None of the choice and no-choice main effects nor their interactions varied significantly

(Table 3.6). Whether nymphs were given a choice or not, there was no significant difference in the amount of damage observed. In both bioassays, only the differences between susceptible and resistant plants varied significantly. This indicates both bioassays detect real differences in fruit damage and these are based solely on susceptibility.

3.5.7 Influence of Pollen Load on Damage Levels

When the nymphs were removed from the cages, 60% of the nymphs on genotype

704-63 and 67% on genotype 704-12 were retrieved from plants which had anthers present.

Plants with intact anthers had significantly more damaged fruit and higher severity indices than those with the anthers removed from the flowers for the genotype

704-12 (susceptible) but not for 704-63 (resistant) (Table 3.7). The presence of pollen elicited seven times the damage compared to the absence of pollen for genotype 704-12.

Replication was not a significant source of variation which indicates that the conditions for each replication were similar.

3.6 Discussion and Conclusion

High amounts of frait damage resulted when developing strawberry fruit were exposed to high numbers of tarnished plant bug. The results from these experiments confirm those of Allen and Gaede (1963), Riggs (1990), Handley (1991) and Easterbrook

(2000), which showed that fruit exposed to nymphs at the green stage resulted in the

76 Table 3.6. Choice versus No-choice experiments. Analysis of variance of the proportion and severity index of tarnished plant bug damage in 2007 comparing all hybrid genotypes and commercial cultivars exposed to six nymphs in choice versus no- choice experiments. The term susceptibility defines the comparison of all hybrid genotypes, which are deemed susceptible and resistant plants, while the term choice defines the experiment type comparing the results from the different choice and no- choice bioassays. Bold numbers indicate significant differences Proportion of Fruit Severity Index of Damaged Damage Source df F ratio F ratio Family 4 2.2 0.015 1.6 0.17 Susceptibility 1 28.0 0.0001 20.0 0.0001 Suscep*Fam 4 2.0 0.0966 2.4 0.054 Choice 1 0.23 0.62 0.03 0.87 Choice*Fam 4 1.9 0.11 2.0 0.093 Choice*Suscep 1 0.11 0.74 0.15 0.70 Choice*Sus*Fam 4 0.33 0.86 0.65 0.63

Table 3.7. Affect of pollen load on damage proportion. Analysis of variance and means of the proportion of damage and severity index caused by tarnished plant bug with the presence and absence of anthers for two strawberry genotypes 704-12 (susceptible) and 704-63 (resistant). Bold numbers indicate significant differences. 704-12 704-63 Severity Severity Proportion of Index of Proportion of Index of Fruit Damage Damage Fruit Damage Damage F F F F Source df ratio P ratio P ratio P ratio P Replicate 9 0.89 0.57 0.67 0.72 0.38 0.92 0.51 0.84 Pollen 1 16.5 0.0028 12.6 0.0063 0.02 0.91 0.44 0.52 Error Mean Square 19 0.022 0.67 0.036 1.8 Means Anthers Present 0.31 1.0 0.12 1.0 Anthers Absent 0.045 0.20 0.13 0.60

77 highest incidence of plant bug damage. In my experiments, flowers exposed to nymphs at petal fall were aborted by harvest. This is consistent with Handley (1991) and

Easterbrook (2000). Unlike previous studies, the current research quantified the damage caused by a given number of nymphs and found that as the number of nymphs increased, so did the proportion of damage incurred.

In this experiment, blossoms exposed to tarnished plant bug feeding developed normal fruit, while Easterbrook (2000) and Handley (1991) found that exposed flowers were aborted. The reason for this may be the different test conditions between studies. In my study, plants were exposed when the primary berry was at the green fruit stage and secondary flowers were in bloom, whereas, the previous studies exposed single flowers.

Nymph behaviour and hence damage would differ if nymphs preferred the green berry over the flower. If the study was repeated and green berries were absent, the flowers may become aborted as they did in the studies of Easterbrook (2000) and Handley (1991).

Adult tarnished plant bugs cause damage to flowers and developing fruit. My research demonstrated adults do cause damage, but nymphs and adults cause similar amounts damage. This is in contrast with the results of Easterbrook (2000) and Handley

(1991) who reported that adults could cause significant amounts of damage to strawberry fruit but comparisons were not made between damage caused by the two stages. In contrast, Zink and Rosenheim (2004) stated that nymphs caused more damage on immature fruit than adults. Based on my results, both nymphs and adults should be controlled equally in the field, as both can cause significant damage to strawberry crops.

These results are consistent with the conclusions of Rancourt et al. (2000) who state that excluding adults from population estimates may be inappropriate for everbearing

78 cultivars, since fruit is produced throughout the summer and is therefore highly susceptible. An insecticide can be applied in early spring to day-neutral cultivars to control adults that emerge from overwintering sites. If adults are controlled early in the growing season it will reduce the amount of eggs that are laid and thus reduce initial population growth (Cooley et al., 2003).

The hybrid genotype 703-43, consistently showed reduced damage compared to

Seascape plants at the same insect population number and potentially shows resistance.

Exposed Seascape plants showed a drastic increase in the amount of damage with increased nymph numbers, while only a minimal increase was seen for the genotype 703-

43. By definition, the difference in damage when plants were exposed to the same number of nymphs indicates the genotype 703-43 exhibits a form of tolerance to tarnished plant bug injury (Painter, 1951). As the number of nymphs was increased, the difference in damage between the two plant cultivars further increased, emphasizing the differences in level of resistance.

The greatest difference in damage between the two cultivars was observed with exposure to 6 nymphs. In further experiments to distinguish resistance levels between genotypes, fruit at the immature green fruit stage should be exposed to 6, 2nd and 3rd instar tarnished plant bug nymphs, since resistance was evident when genotypes were challenged with 6 nymphs but not 3. It is possible that as the plants are exposed to a higher number of nymphs the resistance or tolerance levels will become more pronounced and easier to identify when compared to highly susceptible plants.

Variation between hybrid genotypes in plant bug damage was consistent in both choice and no-choice bioassays. The proportion of damage observed was not dependent

79 on the number of nymphs but was specific to the genotype. Some had low levels of damage regardless of the number of nymphs they were exposed to, which indicated that these genotypes exhibited resistance. The severity index of damage was also dependent on the specific genotype; some genotypes had more severe damage as the number of nymphs increased, indicating a high level of susceptibility. Genotypes that had low proportions of damaged fruit also had a low severity index.

Fort Laramie had consistently lower damage and, thus, is more resistant than

Seascape. In both the choice and the no-choice experiments, Fort Laramie had 1/3 to 1/4 the damage compared to Seascape. The genotype 708-58 had the least amount of damage of all genotypes tested and showed the strongest resistance to plant bug injury.

In a field trial, Fort Laramie was found to be more resistant than other strawberry genotypes, consistent with Dale et al. (2008) and in the field trial of this thesis, had 11- times less damage than the other cultivars (Chapter 2). In the field, tarnished plant bugs are able to detect differences between strawberry cultivars since fewer nymphs were found on Fort Laramie (Chapter 2). However in choice and no-choice bioassays, Fort

Laramie exhibited a form of tolerance, since nymphs numbers were the same on both

Fort Laramie and Seascape, but resulted in different proportions of damage.

Variability in the proportion of damage was found between optimal nymph number and no-choice experiments for genotype 703-43. The optimal nymph number and no-choice experiments had similar methodology and should have yielded similar results.

While this was true for Seascape, 703-43 differed in the proportion of damage between the experiments to determine optimal nymph number and the no-choice experiments regardless of nymph number. The reason for this difference is unclear. However, since

80 the optimal nymph experiment for 703-43 was one of the first experiments to be completed, there may have been some additional variability during the development of the bioassay methodology. Another potential source of variability could have been the limited number of plants available for no-choice experiments on 703-43. As experiments progressed, the number of these plants decreased. These plants continued to meet the morphological criteria for experimentation, but may have not been in optimum health.

Fruit from these experiments may have developed abnormally and would not have been rated as damaged, since achenes varied in size indicating poor pollination, thus decreasing the proportion of damage in the no-choice experiments.

The resistant plants show strong traits of tolerance in choice experiments.

Resistant plants of the matched genotypes exhibited a reduced proportion of damage compared to their matched susceptible genotype. Some of the resistant genotypes continued to have a lower proportion of damage even in no-choice experiments, indicating that even in the absence of alternate food, the nymphs found the resistant plants to be unsuitable hosts. The reduced amount of damage on these strawberry genotypes could suggest antixenotic properties. However, nymphs retrieved after experimentation were collected off the fruit of the plants and not off the cage walls. There were no differences in the number of nymphs retrieved from the resistant and susceptible genotypes. Since no difference in nymph number on the plants was observed it suggests that the resistant genotypes do not influence the feeding behavior of plant bug and point to a tolerance mechanism.

The plants did not show any immediate antibiotic effect on the nymphs. At the end of the exposure time there was no difference in the number of nymphs retrieved,

81 regardless of the genotype. Therefore I assume there are no acutely toxic biochemicals present within the plant. However the occurrence of sublethal effects was not examined, so antibiosis cannot be entirely excluded as a potential resistance mechanism.

In order to determine if an antibiotic mechanism is present within the genotypes, experiments should be conducted to examine long-term survival, maturation and reproduction of exposed nymphs. Bioassays could expose plant bug adults to resistant and susceptible genotypes to determine if long-term antibiotic effects exist. Adults which feed and reproduce on the plants may have reduced fecundity and nymphs may have affected developmental rates. Bioassay studies by Diaz-Montano et al. (2006) on soybean aphid, Aphis glycines, showed that adults which fed on resistant genotypes produced fewer nymphs, indicating a strong antibiotic effect. Strong antibiotic affects have been found in everbearing strawberry fruit, where significant differences were found between the weights of tarnished plant bug nymphs reared on different cultivars (Easterbrook and

Simpson 2000).

Further bioassays could examine the performance of the resistant genotypes when paired in choice bioassays with another resistant genotype of a different family. Such experiments would further help to rank the resistant genotypes and identify which has the overall lowest proportion of damage. Most importantly, the mechanism of resistance within the resistant genotypes should be determined. Further experimentation to determine how the plants affect the biology and metabolic processes of the nymphs could narrow down the source of the resistance traits. If bioassays indicate that the biology of the nymphs is affected, this would warrant chemical analysis to determine if there is a

82 difference in the phytochemistry of the resistant and susceptible genotypes and may identify an underlying mechanism of resistance.

Tarnished plant bug may be attracted to flowers with pollen. My study demonstrated the presence of pollen is associated with increased tarnished plant bug damage on susceptible genotypes, but this relationship was not observed for the resistant genotypes. These results are consistent with that of Handley and Dill (2003) who found tarnished plant bug injury was highly correlated with the amount of pollen present within the flower. Adults may prefer to lay eggs on plants with high pollen levels, as this may be an indicator of host fitness (Handley and Dill, 2003).

More nymphs were retrieved from flowers with intact anthers for both the resistant and susceptible genotypes; which indicates that flowers with little pollen are less preferred. However, relative abundance of pollen does not explain resistance observed in genotype 704-63. The genotype 704-63 exhibits a form of resistance which outweighs the contribution of pollen, which either makes it more tolerant to tarnished plant bug damage or which deters nymphs from feeding. Thus, susceptible plants with no pollen should have a reduced amount of plant bug injury, while resistant plants may not show a pollen- dependent difference in plant bug injury, due to the larger contribution of their resistant traits.

Further research should be conducted with genotypes 704-12 and 63 and other matched pairs to quantify pollen abundance, and to compare resistant and susceptible genotypes with anthers removed in choice studies. This research would help to confirm the relative roles of pollen and resistance factors in strawberry genotypes.

83 To determine if susceptible genotypes have more anthers present to produce more pollen, the experiment should be repeated with other strawberry cultivars to support the results found in my study. A field study would also be of interest and could include hermaphrodite and dioecious plants to determine which have a higher proportion of damage.

Protocols were established in this study to elucidate possible mechanisms of resistance in strawberry plants. Strawberry fruit at the green growth stage was identified as the most susceptible. The amount of damage and severity index of damage was found to be dependent on the number of nymphs exposed. Adults and nymphs were characterized as causing the same amount of damage, therefore control of both life stages at the green fruit stage is equally important.

A number of strawberry genotypes with fruit at the immature green fruit stage were exposed to tarnished plant bug nymphs. Some genotypes maintained a low amount of damage despite the number of nymphs exposed, thus exhibiting resistance. Fort

Laramie and genotype 708-58 had the least amount of damage and showed the most resistance to tarnished plant bug in choice and no-choice experiments. The reduced amount of damage incurred in both bioassays indicates true resistance through a mechanism of tolerance. High pollen load was associated with increased tarnished plant bug damage on susceptible but not on resistant genotypes and this may to due to the overriding contribution of resistant traits.

The information obtained from these bioassays show promise for advancing the development of new strawberry cultivars with elevated resistance to tarnished plant bug.

84 These new cultivars could lead to the production of day-neutral cultivars which would effectively reduce tarnished plant bug damage and aid commercial strawberry production.

85 Chapter 4

4 Determination of Raspberry Fruit Susceptibility to Tarnished Plant Bug

4.1 Abstract

Tarnished plant bug (Lygus lineolaris) is a well documented pest on strawberry fruit, but no scientific evidence has been reported on how it affects raspberry fruit. In cage experiments fruiting lateral branches of the raspberry {Rubus idaeus L.) cultivar

Nova were exposed to four numbers of tarnished plant bug nymphs 0, 1, 5 and 10 per lateral branch. Once fruit matured, the number of unfilled, filled, and aborted drupelets was counted to determine the damage caused by the nymphs. Results showed that tarnished plant bug nymphs do cause damage to raspberry fruit. As the number of nymphs per lateral branch increased, the number of unfilled drupelets increased and aborted drupelets decreased. When the lateral branches were exposed to five or more nymphs, more drupelets were unfilled, which caused unsightly blemishes to the fruit, rendering them unmarketable for fresh market sale. These results indicate that five nymphs per lateral branch may be a suitable economic or action threshold.

4.2 Introduction

Raspberries, Rubus ideaus L., are an aggregate fruit, which is in high demand in

Canada but only 9.4 thousand acres are devoted to its production in Canada (Goulart,

1991; Makki, 2002). Growers face many problems with insect pests because there are few insecticides available and economic thresholds have been determined for few of the pests. This is the case with tarnished plant bug, where little is known about its effect on raspberry fruit and no thresholds have been established. Tarnished plant bug is not mentioned as a problem pest of raspberries in the Ontario Ministry of Agriculture Food

86 and Rural Affairs production guide nor is it listed in the spray calendar (OMAFRA,

2006).

Damage caused by plant bug has not been extensively studied and in the United

Kingdom is considered unimportant (Schaefers et al., 1991). A single North American extension publication indicates that feeding by tarnished plant bug on immature fruit results in failed drupelets and malformed fruit and this causes significant yield reductions

(Schaefers et al., 1981). Very little research has subsequently been performed to determine whether plant bug actually affects the fruit and what the damage looks like.

Tarnished plant bugs have been found to live on raspberry plants and fruit (Kieffer et al.,

1983), but no scientific literature has documented their impact on the fruit. Further research is needed to determine the type and severity of damage caused by tarnished plant bug to raspberry fruit.

4.3 Objective

Laboratory experiments were performed in order to:

1. determine whether tarnished plant bug nymphs feed on and cause malformation of raspberry fruit; and,

2. determine the impact of nymph density on the proportion and severity of damaged fruit.

4.4 Materials and Methods

4.4.1 Insect Rearing

Adult plant bugs were collected from an alfalfa field near Guelph, Ontario,

Canada in September and October 2005. Plant bugs were collected with a sweep net and aspirated into vials for transportation. The adults were split equally between two cages which were made with a wooden frame (43cmx 23cmx 41cm) with 5 sides covered in

87 mesh and one Plexiglas side with a nylon access port. Nymphs were raised in 4L white

Plastipak® plastic buckets with mesh lids for aeration. Both adults and nymphs were given green beans, snow peas and lettuce as food sources. Potato sprouts were provided to the adults as an oviposition substrate. Adults were given water using a 500mL mason jar placed upside down on a petri dish and held with elastics. A toothpick was then inserted to allow for water flow. Food and oviposition substrate were replaced every 2-3 days and potato sprouts were placed in 4L buckets to allow eggs to hatch. Buckets with nymphs were inspected every two to three days and any adults were removed and placed into the adult cages for reproduction. Colonies were maintained at the University of

Guelph until May 2006 at 25 ±1°C, with 16:8 hour light:dark regime and 50% RH. The colony was moved from Guelph to the Simcoe Research Station in May 2006; both adult and nymph colonies were kept in a growth chamber set at 25 ± 1°C, with 16:8 hour lightdark regime and 30% RH.

4.4.2 Bioassay

Overwintered raspberry plants, cv. Nova, were obtained from Strawberry Tyme

Farms Inc., Simcoe, Ontario in February 2006 and grown in a greenhouse with ambient light at 16-24°C at the Simcoe Research Station, Simcoe, Ontario.

When the plants started to flower in May 2006, five plants were selected which had a progression of blossom development on the lateral branches (i.e. flower trusses).

On each plant, 4 lateral branches with only the terminal flower open were selected and randomly assigned to each treatment. Second to 3rd instar nymphs were placed on the lateral branches (0, 1,5 or 10 per lateral branch) which were then covered with a muslin bag. Cotton batting was wrapped around the base of the branch and the bag was tied shut.

88 Muslin bags were removed after 72 hours and the number of retrieved nymphs was counted. Any open flowers were hand-pollinated before and after the lateral branches were placed in the muslin bags. Flowers were observed every other day and any open blossoms were pollinated until all flowers on the lateral branch were open. Each plant was considered a replicate. For the first replicate, the treatment of 5 nymphs was absent since the lateral branch was accidentally broken off.

Twenty-six days after the start of the experiment, all plants were moved from the

Simcoe Research Station to the University of Guelph main campus for further observation. Plants were placed into an outdoor, temporary greenhouse with screen mesh and placed in a shaded area to allow fruit to ripen. Once fruit developed and turned pink in colour, the ripe fruit were cut off and dissected to count the number of filled, unfilled and aborted drupelets. Filled drupelets were identified as normally developed fleshy red fruit with a round appearance and contained a single seed. Unfilled drupelets were fruit with the seed surrounded by dehydrated flesh, while aborted fruit were brown undeveloped seeds with no flesh and the style attached.

4.4.3 Statistical Analysis

The proportion of unfilled drupelets was calculated as the number of unfilled drupelets divided by the number of unfilled, filled and aborted drupelets. The proportion of aborted drupelets was calculated as the number of aborted drupelets divided by the number of unfilled, filled and aborted drupelets. Fruit with only aborted drupelets were omitted from the analysis.

Data were subject to testing by PROC UNIVARIATE (SAS Institute, version 9.1,

2002, Cary, NC) to satisfy the assumptions of normal distribution and homogeneity of

89 variance prior to statistical analyses. When necessary, heterogeneity of variance was reduced by subjecting data to arcsin square root transformation. The proportion of unfilled drupelets and aborted drupelets was compared for different nymph treatments by regression analysis using PROC GLM (SAS Institute, version 9.1, 2002, Cary, NC) with number of nymphs as the independent variable.

The regression model used where the response (Y) was Y= bix + b2X + e, where

(x) is the number of nymphs exposed, bi and b2 are the linear and quadratic polynomial regression coefficients and e is the random error term. The intercept was constrained to zero since no tarnished plant bug damage could happen when there were no plant bugs in the treatment of zero nymphs. When the quadratic coefficients were not significantly different from zero, the model was refitted with these terms omitted. Linear and quadratic effects were tested for the independent variable and the regression equations calculated for the significant linear effects.

4.5 Results

A significant linear relationship was found between tarnished plant bug number and both the proportion of unfilled and aborted drupelets (Table 4.2). The quadratic effect was not significant. More unfilled drupelets occurred when the flowers were exposed to increasing numbers of tarnished plant bug nymphs (Figure 4.1 A), whereas the proportion of aborted drupelets decreased when the flowers were exposed to increasing nymph numbers (Figure 4. IB).

4.6 Discussion and Conclusions

My study found that tarnished plant bug nymphs damage raspberry fruit and that the proportion of unfilled drupelets increased with increased nymph number.

90 Table 4.1. Analysis of unfilled and aborted raspberry drupelets. Analysis of variance of the proportion of unfilled raspberry drupelets caused by tarnished plant bug and number of aborted drupelets found. Variable df Mean Square F value P Unfilled Replication 4 0.020 0.49 0.74 Linear 1 1.61 50.1 <0.0001 Quadratic 1 0.025 0.76 0.38 Error Mean Square 112 0.042 Aborted Replication 4 0.19 0.51 0.73 Linear 1 3860.0 12.4 0.0006 Quadratic 1 60.8 0.20 0.66 Error Mean Square 112 0.37

91 • (o 0.4 "3 0.35 3 D 0.3 - 0) I 0.25 i ^s^+ y = 0.0316X + 0.0886 = 0.2 -i 2 o R = 0.9396 = 0.15 - .2 < o 0.1 Q. £ 0.05 -

0 - i I I I 4 6 8 10 12 Number of Nymphs

B.

«2 0.4 J • 0> y = -0.0115x +0.3211 §. 0.35- 2 3 R = 0.2184 O 0.3- •

J?3 0.25- < 0.2- o | 0.15- • 2 0.1 - O. O £ 0.05 -

0- I I • • ...... , 4 6 8 10 12 Number of Nymphs

Figure 4.1. Mean proportion of damaged raspberry drupelets. Mean proportion of unfilled and aborted raspberry drupelets exposed to nymphs. Regression analyses of A. mean proportion of unfilled drupelets, and B. mean proportion of aborted drupelets in 'Nova' raspberry fruit in relation to exposure to tarnished plant bug nymphs (0,1,5, 10 per lateral branch).

92 Similar results were seen in experiments to determine the optimal number of nymphs for trials on strawberries (Chapter 3). The increase in the proportion of unfilled drupelets with increasing nymph number indicates that nymphs fed upon the seed, thus preventing flesh development and causing malformation of fruit. My study confirms the work of Schaefers et al. (1991) since immature fruit exposed to plant bug nymphs resulted in failed drupelets and malformed fruit.

There was a negative relationship between nymph number and proportion of aborted drupelets. The proportion of aborted drupelets decreased with higher nymph number. Aborted drupelets may be due to poor pollination and could have been caused by poor hand pollination technique. The low occurrence of aborted drupelets in the 5 and

10 nymph treatments indicates that movement of plant bugs on the flowers may have helped to improve pollination. This possibility requires further investigation. Large amounts of pollen should be collected in the future to ensure that the fruit is hand pollinated effectively, so that the fruit develop successfully and that the numbers of aborted drupelets are reduced.

There are no recommendations available as to the number of aborted or unfilled drupelets that lead to a raspberry fruit being considered unmarketable in the fresh market sector. Consumers may have zero tolerance for unfilled drupelets and want to purchase berries which are perfectly filled out. There may be a higher tolerance for fruit with aborted drupelets than fruit with unfilled drupelets since aborted drupelets are small and unseen while unfilled drupelets are more pronounced and unsightly, blemishing the whole berry. If 1/4 - 1/2 of the berry has aborted or unfilled drupelets, then the consumer would likely reject the product; whereas consumers may accept damage to 10% of the

93 berry. Fruit exposed to ten nymphs had 16% damage which would likely render them unmarketable, while fruits exposed to five nymphs had 5% damage which may be more tolerable to the consumer.

Further research is needed to determine action thresholds for insecticide application in raspberries. Sequential sampling methods are used in strawberry production to determine whether economic thresholds have been reached and if pesticides should be used to control pests, i.e. tarnished plant bug and clipper weevil (Dale et al.,

2000). A model for sequential sampling should be established to demonstrate how many raspberry lateral branches should be sampled to determine whether an action threshold has been reached and whether insecticides should be applied to the raspberry field.

Thresholds would benefit growers since it would help determine the most effective time to spray pesticides, increasing marketable yield, and potentially reducing production costs and environmental effects.

94 Chapter 5

5 Discussion and Conclusion

5.1 Discussion

Tarnished plant bug is a major deterrent to the commercial expansion of day- neutral strawberry production in Ontario. Resistant cultivars are a potential approach to reduce the effects of tarnished plant bug feeding damage in this crop. Breeding has produced strawberry genotypes which exhibit resistance to tarnished plant bug damage, but the levels of resistance have not been quantified or ranked and the mechanisms underlying this trait have not been identified.

Plants are considered resistant if they are able to produce a larger, good quality crop compared to others with the same insect population pressure, continually exhibit this character under different conditions and consistently sustain a low level of damage

(Painter, 1951). Considering these criteria, cv. Fort Laramie can be defined as a resistant.

The hybrid genotype 708-58 is the progeny of a Fort Laramie cross with 21K74 and also sustains low plant bug damage. This result may be of interest to breeders because it shows that resistance to tarnished plant bug damage is a heritable trait which can be transferred to progeny. New genotypes could be developed which have the fruit aesthetics and quality desired by the consumer as well as insect resistance traits to help increase production for strawberry growers.

A trait may be present in resistant strawberry plants which deters tarnished plant bug feeding. Cultivar Fort Laramie and other select day-neutral hybrids (708-58 and 703-

43) consistently exhibited reduced tarnished plant bug damage compared to other commercial cultivars and hybrid genotypes throughout my studies.

95 Differences in the number of nymphs observed feeding on different cultivars in my study and in Dale et al. (2008) indicates antixenosis resistance mechanism. In the field, resistance in the form of antixenosis was observed because cv. Fort Laramie had the lowest number of plant bug nymphs and proportion of damage.

The resistant strawberry cultivars examined in this study exhibited resistance by means of tolerance since the number of nymphs retrieved after the bioassay did not differ but the degree of injury did. When nymphs were retrieved from choice experiments, the same number of nymphs were present on both resistant and susceptible plants, thus providing similar plant bug pressure. Even with the same plant bug pressure the resistant plants exhibited less damage and thus show resistance by means of tolerance.

Fruit exposed to nymphs at the green stage sustained more damage than fruit exposed at other developmental stages, which is consistent with previous studies (Allen and Gaede, 1963; Riggs, 1990; Handley, 1991; Easterbrook, 2000). However, in this study, the damage caused by a given number of nymphs was quantified. On susceptible genotypes, the proportion of damaged fruit was positively correlated with increased number of nymphs. In contrast, on resistant genotypes of the families 703, 704 and 708, the number of nymphs did not affect the amount of damage incurred. Similar results led to the designation of the cv. Bolero as a tarnished plant bug tolerant cultivar (Easterbrook and Simpson, 2000).

The hybrid genotypes in my study did not show resistance by an acute antibiotic effect since mortality of nymphs did not differ among genotypes. However, this does not rule out a sublethal antibiotic mechanism of resistance, where the allelochemicals responsible could affect the metabolic processes of plant bugs as they mature and

96 reproduce. To test this hypothesis, nymphal growth, length of the nymphal stage and reproduction should be compared over several generations on resistant and susceptible genotypes.

Analysis of plant defense compounds could determine if the phytochemistry of resistant and susceptible genotypes differs and may identify an underlying chemical trait conveying resistance. Previous work has shown that defensive compounds are present in strawberries and that they affect tarnished plant bug development, but these compounds have not been identified (Easterbrook and Simpson, 2000). Research in other crops has shown that defensive compounds that are consumed by herbivorous insects can affect maturity and fecundity (Zhu et al., 2005; Diaz-Montano et al., 2006). Trichomes of wild tomato leaves produce and secrete volatile compounds that are toxic to certain insects

(Hamilton-Kemp et al., 1988). Naturally occurring oils in wild strawberry leaves (F. chiloensis and F. viridis) are responsible for resistance to the two-spotted ,

Tetranychus urticae Koch. (Khanizadeh and Belanger, 1997). Thirty-four essential oils were either present or absent in the wild species when compared to the commercial cultivars, but it has not yet been determined which may contribute to resistance or susceptibility. The authors speculated that specific combinations of oils may be cultivar- specific and that their abundance may be affected by environmental factors and cultural practices (Khanizadeh and Belanger, 1997).

Induced resistance may occur in strawberry plants in response to fruit harvest. It has been shown that plants that were susceptible to two spotted spider mites during fruit production were resistant to damage 2 weeks after harvest was complete (Hamilton-

Kemp et al., 1988). When strawberry fruit are removed from a plant, the production of

97 methyl salicylate increases 10-fold (Hamilton-Kemp et al., 1988) and high concentrations of this chemical repel mites (Rodriguez et al., 1976). It is possible induced resistance should be considered/examined in plants exposed to tarnished plant bug nymphs.

Induced resistance could be examined in resistant strawberry hybrids with respect to subsequent exposure to nymphs. Resistant strawberry plants could be exposed to tarnished plant bug damage and exposed a second time to see whether the plants show an increased level of resistance. By re-exposing plants to nymphs it would be possible to determine if exposure to nymphs results in induced resistance in strawberries. Further breeding could be performed to produce cultivars which are extremely resistant.

Damage was observed on all genotypes in all bioassays, but resistant genotypes

708-58, 704-63 and 703-43 had a significant reduction in tarnished plant bug damage.

Plants that show a level of resistance equal or greater than that of cv. Fort Laramie, represent only one generation of crosses, so there is considerable potential to increase resistance levels within these genotypes. If resistance is conveyed by a phytochemical, it should be possible to breed plants that produce that compound in amounts sufficient to render the plant completely unpalatable to tarnished plant bug. Until then, resistance cannot exclusively control this pest, but cultivars with lower levels of resistance could be used in combination with other control methods to reduce both the amount of injury sustained and insecticides used. The hybrid genotypes identified in my study could play a key role in breeding programs to produce cultivars which are less susceptible to tarnished plant bug damage. Morphological traits could also contribute to resistant levels and can also be selected by breeders to enhance resistance.

98 Tarnished plant bug appears to prefer plants with high pollen levels, but this seems secondary to plant resistance. Susceptible plants with pollen removed were less attractive to tarnished plant bug nymphs and showed reduced damage as compared to those with a full pollen load. However, resistant genotypes had reduced damage whether pollen was present or not. Adults have been found to prefer to oviposit on plants with high pollen levels as this may be an indicator of host fitness (Handley and Dill, 2003).

Dioecious Fragaria virginiana clones are generally more resistant than hermaphroditic F. x ananassa genotypes (Dale et al., 2008). In a dioecious population many of the plants are female, and anthers and pollen are absent, possibly contributing to less plant bug damage. The finding that damage levels on resistant genotypes is unrelated to the presence or absence of pollen is consistent with findings from choice and no-choice bioassays and indicates the likely presence of an antixenotic resistance mechanism. To examine this hypothesis, the number of anthers should be counted on resistant and susceptible genotypes to determine if resistant plants have naturally reduced amounts of pollen. Controlled laboratory experiments between cultivars with natural differences in pollen load would confirm if tarnished plant bug prefers hosts with higher pollen loads. A similar field study could determine the agricultural relevance of this relationship and could also include comparisons with hermaphroditic and dioecious plants which sustain higher tarnished plant bug damage.

The presence of pollen affects the behaviour of pollinators and could also impact the host selection behaviour of plant bugs looking for food. Female flowers receive half as many visits from pollinators, since their floral scent composition differs from that of the males (Ashman et al., 2005). If plant bugs respond to olfactory cues from strawberry

99 flowers and preferentially respond to plants with more pollen, then female plants should experience less plant bug damage. Plant bugs do not feed on flower pollen but the ovaries of developing fruit and may only be attracted to pollen as an indicator of an ovary food source. Fruit on hermaphroditic plants may be more susceptible to damage than dioecious females since both pollen cue and ovaries are present. Plant bug may visit male dioecious flowers because of the pollen cue but are unable to find the ovary food source.

If male plants are found to be more attractive than hermaphrodites, then male plants could be planted around the perimeter of susceptible plants as a trap row. The trap row could be sprayed to reduce the plant bug population and thus increase the number of marketable fruit in the center of the field.

Experimental conditions may affect the outcome of feeding studies with tarnished plant bug. In my study, flowers exposed to plant bugs at bloom developed normal fruit, while Handley (1991) and Easterbrook (2000) found that flowers exposed to 1 plant bug nymph for 48 hours were aborted. In my study, experimental plants had the primary berry at the green fruit stage and secondary flowers in bloom at the time of plant bug exposure, whereas the previous studies (Handley 1991; Easterbrook 2000) exposed flowers alone.

Preference of nymphs for green berries over flowers resulted in no damage or abortion of flowers which may have occurred if green berries were absent.

The laboratory bioassays should be confirmed in the field to determine if the rankings of reduced damage are consistent. Choice and no-choice bioassays have indicated that Fort Laramie and 708-58 sustain the least amount of plant bug damage compared to matched pairs from each family. This result suggests that the differences in resistance to tarnished plant bug are real, reproducible and consistent with previous

100 studies of Dale et al. (2008). Field tests should be conducted on 708-58 to confirm the resistance under pressure from natural populations of tarnished plant bug in a commercial production setting.

There is no evidence for differences in feeding preference among plant bug populations in southern Ontario, as strawberry cultivars ranked consistently at all field sites. My study is the first of its kind to identify that plant bug feeding behaviour on strawberry cultivars does not vary at different geographical locations. These results are agronomically significant since cultivars can now be bred for southern Ontario as a whole. However, it may be prudent to evaluate adults and nymphs from other strawberry production regions in order to confirm that there is only a single plant bug biotype in

Ontario.

Tarnished plant bugs detect differences between and exhibit preferences for certain strawberry cultivars, as has been demonstrated in this and other studies (Handley et al., 1991, 1993; Easterbrook and Simpson, 2000; Dale et al., 2008). In addition, neighbouring plants appear not to affect plant bug preferences as damage to resistant cultivars was the same whether or not they were grown directly beside the highly susceptible cv. Seascape plants. Susceptible plants did not experience less damage when beside resistant plants.

Nymph migration into the field from a weedy perimeter affects the spatial distribution of damaged fruit observed in the strawberry field. At Vineland ON, tarnished plant bug damage was found to be highest in rows at the perimeter of the field plots and decreased further into the field. Previous studies show nymphs migrate into the field from the weedy perimeter and remain in high numbers since they are unable to walk long

101 distances (Stinner et al., 1983; Dale et al., 2007). The higher population results in more fruit damage at the perimeter and results in less damage incurred the further away.

Insecticide applications should be targeted at perimeter rows, a practice which may reduce the amount of insecticide used, particularly if reduction in perimeter plant bug populations results in little or no damage further into the field. Cultivar preferences of nymphs and adults could also be exploited as a cultural control tactic. Resistant cultivars could be planted around the perimeter of the field to reduce nymph migration. This new strategy would enable growers to plant susceptible, but agronomically superior, cultivars as the main crop, but utilize the traits of resistant cultivars to reduce fruit injury.

Control of both the adult and nymphal plant bug stages is required in day-neutral production since both life stages are present and cause significant amounts of damage to strawberry crops. Handley (1991) and Easterbrook (2000) showed that adults caused damage to developing fruit but these studies did not examine damage caused by nymphs.

Allen and Gaede (1963) and Zink and Rosenheim (2004) stated that nymphs cause more damage on developing fruit than adults, but the damage was not quantified. Rancourt et al. (2000) found that adults and nymphs caused equal damage to the flower and fruits, which is confirmed by my results.

Plant bug behaviour can also be affected by the reflectivity of the surface that plants are grown on. Less plant bug injury occurred on strawberry plants grown on white plastic than on black plastic. Since the white plastic reflects a higher amount of UV light

(Kring and Schuster 1992), it acts as a repellant by affecting the behaviour of the insects

(Kring 1972; Stavisky et al., 2002; Doring et al., 2004; Demirel and Cranshaw 2006).

The Simcoe research site experienced less damage than others and was the only site to

102 have all plants grown on white plastic mulch. The low level of damage could have been due to high levels of UV reflectance in this field, which repelled plant bug adults, deterred egg-laying and thus reduced both the plant bug population and damage at the

Simcoe site. Consistent with my study, decreased tarnished plant bug damage was observed on strawberries grown with silver reflective mulch, which was considered to be as effective as one malathion application (Rhainds et al., 2001).

Tarnished plant bugs damage developing raspberry fruit and damage increases with plant bug number. This is the first report of controlled studies demonstrating that tarnished plant bugs damage individual drupelets. An earlier report showed that plant bugs damaged raspberries based on field observations (Schaefers, 1991). An unexpected result of my study was the reduction in aborted drupelets with increasing nymph numbers. Raspberry flowers are self-fertilized but require insects for adequate pollination

(Louws, 1996). Since the raspberry flowers were enclosed and not exposed to natural means of pollination, the mechanical pollination may not have sufficed. It appears that the movement of nymphs about the plant resulted in pollination of more drupelets when more nymphs were present. Tarnished plant bug nymphs have not been previously reported to be pollinators and this result is unique to my study.

In susceptible strawberries and raspberries the amount of fruit damaged depended on the number of tarnished plant bug nymphs present and the developmental stage of the fruit. The more nymphs exposed to fruit, the more damage is incurred on the fruit. Mature fruit sustains less damage than young fruit since as the fruit ripens, the achenes or seeds enlarge and become lignified, making it difficult for the plant bug stylet to penetrate the tissue (Handley and Pollard, 1993; Riggs, 1990).

103 A successful integrated pest management program for tarnished plant bug in strawberries should be made up of a number of components, including resistant cultivars, border plantings and/or bairrier crops, perimeter application of insecticides, and highly reflective plastic mulches. Growers could either use resistant plants as their main crop or plant them as a border around susceptible plants in order to reduce movement of plant bug populations into susceptible plantings.

Further research needs to be conducted to better understand how different management practices could control tarnished plant bug in strawberry production. The use of resistant cultivars as a crop barrier has not been studied in strawberry production since only a limited number of cultivars are resistant to plant bug. Typically, June- bearing cultivars have been identified as resistant but have not been used to produce new crop management techniques. The impact of a resistant cultivar barrier on adult and nymph migration needs to be assessed in order to optimize day-neutral planting patterns.

For example, a field experiment is needed to compare plant bug abundance and damage to Seascape plants in a solid planting versus those in a planting with Seascape in a center square of the field surrounded by several rows of cv. Fort Laramie plants. If a high number of nymphs and damage occur in the center Seascape plants and low numbers in the cv. Fort Laramie plants, this would indicate that adult migration past the resistant border into the field is the main factor influencing fruit injury. Conversely, if the number of nymphs and damage in the center Seascape plants is low, this would indicate that Fort

Laramie plants effectively prevent migration of adult tarnished plant bug into the field.

An experiment may also be needed with different border to centre cultivar ratios in order to determine the optimal planting pattern for both plant bug control and profitability. A

104 split-plot design could be used to evaluate the effectiveness of combining a resistant barrier with white mulch.

Coloured plastic mulch has been effective in controlling insect pests in other horticultural crops but has not been extensively studied in strawberry production and its effect on tarnished plant bug damage. The previously discussed field of Seascape plants surrounded with a barrier of resistant cv. Fort Laramie could be divided with each half planted with or without white plastic mulch. The amount of damage could be compared to determine which management is the most effective.

This is the first report that the pollen load of strawberry flowers affects the proportion of plant bug damage in strawberries. Further investigation should compare the use of dioecious and hermaphrodite flowers in the field to determine which will have the least amount of damage. A field plot which consists of a resistant border, and the center divided with each half planted with or without white plastic mulch could be further divided in to quarter sections. The quarter sections could be planted with either hermaphrodite plants or with dioecious plants with a higher percentage of female plants to ensure a lower pollen load. This experimental field plot design would assess which cultural combinations are the most effective to reduce damaged fruit. New management practices would be an asset for strawberry growers in order to obtain control of tarnished plant bug populations and also have quality fruit production.

Continued research is needed to better understand how tarnished plant bug impacts fruit damage in the field and to assess potentially resistant plants. This new information will help Ontario strawberry farmers produce higher crop yields and enable them to compete with farmers in other countries.

105 5.2 Conclusion

Tarnished plant bug is one of the major pests in strawberry production in Ontario and is a major constraint to the expansion of day-neutral strawberry production. My study has found that: resistance is a consistent trait, plastic mulch reduces tarnished plant bug feeding, high pollen levels increase damage on susceptible cultivars and nymphs do not move far from the perimeter of the field.

The cv. Fort Laramie consistently showed low damage and some of the new genotypes showed even lower levels. This result is consistent with previous work (Dale et al., 2008) and could be beneficial in future breeding programs.

The source of reduced injury of resistant genotypes remains unknown, but possible mechanisms have been revealed. Antixenosis and tolerance are found to be the mechanisms of resistance by field experiments and choice/no-choice experiments, respectively. Therefore, strawberry resistance to tarnished plant bug feeding appears to be a combination of at least two separate mechanisms.

Insect resistant host plants are not a stand-alone solution to pest problems. Host plant resistance is merely a tool, which should be used and integrated with other pest management tactics. The combined approaches of resistant cultivars with lower pollen production, grown on white mulch, with pesticides sprays targeted at a perimeter planting of resistant plants around a susceptible cultivar, should effectively reduce the proportion of strawberry fruit damaged by tarnished plant bug and benefit commercial producers.

106 5.3 Literature Cited

Anonymous (1998) Day, W.H. Alternatives to Insecticides for Managing Vegetable Insects Proceedings of a Farmer/Scientist Conference: Current Status of Biological Control of the Tarnished Plant Bug in Northeast Alfalfa and Research on Extension of This Method to Other Crops. Site accessed September 9, 2006 http://vvv.state.ct.us/caes/AlternativestoInsecticides/alternatives.htm

Anonymous (1999) Howitt, A.H. State University. Fruit IPM Factsheet: Tarnished Plant Bug. Site accessed June 9, 2005 http ://www. canr. msu. edu/vanburen/tarnish.htm

Anonymous (2005) Perm State Entomological Notes: Tarnished Plant Bug Site accessed June 1, 2005 http://www.ento.psu.edu/extension/factsheets/tarnished_plant_bug.htm

Allen, W.W. and Gaede, S.E. (1963). The relationship of Lygus bugs and thrips to fruit deformity in strawberries. Journal of Economic Entomology. 56: 823-825

Ashman, T.L., Bradburn, M., Cole, D.H., Blaney, B.H., and Raguso, R.A. (2005) The scent of a male: the role of floral volatiles in pollination of a gender dimorphic plant. Ecology. 86(8): 2099-2105

Backus, E.A., Cline, A.R., Ellerseick, M.R. and Serrano, M.S. (2007) Lygus hesperus (Hemiptera: Miridae) feeding on cotton: new methods and parameters for analysis of nonsequential electrical penetration graph data. Annals of the Entomological Society of America. 100(2): 296-310

Bodnaryk, R.P. (1996) Physical and chemical defences of pods and seeds of white mustard (Sinapis alba L.) against tarnished plant bugs, Lygus lineolaris (Palisot de Beauvois) (Heteroptera: Miridae). Canadian Journal of Plant Science. 76: 33-36

Bostanian, N.J. (1994) The tarnished plant bug and strawberry production. Research Branch Agriculture and Agri-Food Canada, Quebec

Cermak, P. and Walker, G.M. (1992) Tarnished plant bug: a major pest of strawberry. Ontario Ministry of Agriculture and Food Factsheet

Cohen, A.C. and Wheeler Jr, A.G. (1998) Role of saliva in the highly destructive fourlined plant bug (Hemiptera: Miridae: Mirinae). Annals of the Entomology Society of America. 91(1): 94-100

Cooley, D.R., Schloemann, S.G., and Tuttle, A.F. (1993) Development and implementation of integrated pest management for strawberries in Massachusetts. Advances in Strawberry Research. 12: 1-10

107 Crandall, P.C. (1995) Growth and development: morphology, physiology. In Bramble production: the management and marketing of raspberries and blackberries. Gough, R.E. (ed.) pp 17-31. New York, United States of America: The Haworth Press, Inc.

Dale, A., Galic, D., and Hallett, R. (2008) Fragaria virginiana tolerates tarnished plant bug. Entomologia Experimentalis et Applicata. 126: 203-210

Dale, A., Hughes, B.R., Hallett, R., Galic, D. and Rougoor, C. 2007. Progress toward reduced tarnished plant bug injury in strawberry through cultivar resistance and modified cultural practices. In: Takeda, F., D.T. Handley, and E. B. Poling (eds.) Proc. 2007. N. American Strawberry Symposium. North American Strawberry Growers Association, Kemptville, ON, Canada. CD and paper versions pp. 77-80.

Dale, A., Walker, G., and Fisher, P. (2000) Commercial strawberry production: economics, The strawberry plant: crown development, flower and fruit development. In Growing strawberries in Ontario, Publication 513. pp 2-8. Toronto, Canada: Queen's Printer for Ontario

Darrow, G.M. (1966) History, breeding and physiology. In The strawberry, pp. 20-28. New York, United States of America: Holt, Rinehart and Winston

Demirel, N. and Cranshaw, W. (2006). Relative effect of colour mulches to potato/tomato psyllid, Paratrioza cockerelli (Sulc) (Homoptera:Psyllidae), on garden tomato plants. Journal of Entomology. 3(2): 189-193

Diaz-Montano, J., Reese, J.C., Schapaugh, W.T. and Campbell, L.R. (2006) Characterization of antibiosis and antixenosis to the soybean aphid (Hemiptera: Aphididae) in several soybean genotypes. Entomological Society of America. 99(5): 1884-1889.

Doring, T.F., Kirchner, S.M., Kuhne, S., and Saucke, H. (2004). Response of alate aphids to green targets on coloured backgrounds. Entomologia et Applicata. 113: 53-61

Easterbrook, M.A. (2000) Relationships between the occurrence of misshapen fruit on late-season strawberry in the United Kingdom and infestation by insects, particularly the European tarnished plant bug, Lygus rugulipennis. Entomologia Experimentalis et Applicata. 96: 59-67

Easterbrook, M.A. and Tooley, J.A. (1999) Assessment of trap plants to regulate numbers of the European tarnished plant bug, Lygus rugulipennis, on late-season strawberries. Entomologia Experimentalis et Applicata. 92: 119-125

Easterbrook, M.A. and Simpson, D.W. (2000) Susceptibility of everbearing cultivars of strawberry to the European tarnished plant bug, Lygus rugulipennis. Journal of Horticultural Science and Biotechnology. 75(4): 405-408

108 Elzinga, R.J. (2000) Classification: hemiptera. In Fundamentals of entomology 5 Edition. Harvey, L. Carnis, M. (ed.) pp 382-387. New Jersey, United States of America: Prentice Hall Inc.

Eriksson, T., Hibbs, M. S., Yoder, A. D. Delwiche, C. F. & Donoghue, M. J. (2003) The phylogeny of Rosoideae. International Journal Plant Science. 164(2): 197-211

Goulart, B.L. (1991) Introduction: the genus Rubus. In Compendium of raspberry and blackberry diseased and insects. Ellis, M.A., Converse, R.H., Williams, R.N. Willamson, B. (ed.) pp 1-2. Minnesota, United States of America: The American Phytopathological Society

Grunfeld, E., Vincent, C. and Bagnara, D. (1989) High-performance liquid chromatography analysis of nectar and pollen of strawberry flowers. Journal of Agricultural Food Chemistry. 37: 290-294

Gueldner, R.C. and Parrott, W.L. (1977) Volatile constituents of the tarnished plant bug. Insect Biochemistry. 8: 389-391

Hamilton-Kemp, T.R., Anderson, R.A., Rodriguez, J.G., Loughrin, J.H., Patterson, C.G. (1988) Strawberry foliage headspace vapor components at periods of susceptibility and reistance to Tetranychus urticae Koch. Journal of Chemical Ecology. 14(3): 789-796

Hancock, J.F., Scott D.H. and Lawrence F.J. (1996) Strawberries. In Fruit Breeding Vol II. Vine and Small Fruits. Janick, J and Moore, N. pp 419-470. New York, New York: John Wiley & Sons Inc.

Hancock, J.F. (1999) Strawberries. New York, New York: CAB I Publishing

Handley, D.T. (1991) Strawberry fruit development and the effects of feeding by the tarnished plant bug, Lygus lineolaris. In American Strawberry Conference. Dale, A. and Luby, J.J. (ed.) pp 209-216. Portland, Oregon: Timberland Press

Handley, D.T., Dill, J.F. and Pollard, J.E. (1991) Field susceptibility of twenty strawberry cultivars to tarnished plant bug injury. Fruit Varieties Journal. 45(3): 166-169

Handley, D.T., Dill, J.F. and Pollard, J.E. (1993) Tarnished plant bug injury on six strawberry cultivars treated with differing numbers of insecticide sprays. Fruit Varieties Journal. 47(3): 133-137

Handley, D.T. and Pollard, J.E. (1993) Microscopic examination of tarnished plant bug (Heteroptera: Miridae) feeding damage to strawberry. Horticultural Entomology. 86(2): 505-510

109 Handley, D.T. and Dill, J.F. (2003) Vegetative and floral characteristics of six strawberry cultivars associated with fruit size, yield and susceptibility to tarnished plant bug injury. International Society for Horticultural Science. 626: 161-167

Hardman, J.M., Jensen, K.I.N., Moreau, D.L., Franklin, J.L. and Bent, E.D. (2004) Effects of ground cover treatments and insecticide use on population density and damage caused by Lygus lineolaris (Heteroptera: Miridae) in apple orchards. Journal of Economic Entomology. 97(3): 993-1002

Hatfield, L.D., Ferreira, J. and Frazier, J.L. (1983) Host selection and feeding behaviour by the tarnished plant bug, Lygus lineolaris (Hemiptera: Miridae). Annals of the Entomological Society of America. 76(4): 688-691

Jackson, D.M and Bohac, J.R. (2007) Resistance of sweetpotato genotypes to adult Diabrotica beetles. Journal of Economic Entomology. 100(2): 566-572

Jay, C.N., Cross, J.V., and Burgess, C. (2004) The relationship between populations of European tarnished plant bug (Lygus rugulipennis) and crop losses due to fruit malformation in everbearer strawberries. Crop Protection. 24: 825-834

Khanizadeh, S. and Belanger, A. (1997) Classification of 92 strawberry genotypes based on their leaf composition. Acta Horticulturae. 439: 205-210

Kieffer, J.N., Shanks, C.H. and Turner, W.J. 1983. Populations and control of insects and spiders contaminating mechanically harvested red raspberries in Washington and Oregon. Journal of Economic Entomology. 76 (3): 649-653.

Kogan, M. and Ortman, E.E., (1978) Antixenosis — a new term proposed to replace Painter's "Nonpreference" modality of resistance. ESA Bull. 24

Kring, J.B (1972). Flight behaviour of aphids. Annual Review of Entomology. 17: 461- 483

Kring, J.B and Schuster, D.J. (1992). Management of insect on pepper and tomato with UV-reflectance mulches. Florida Entomologist. 75(1): 119-129

Laurema, S. and Varis, A.L. (1991) Salivary amino acids in Lygus species (Heteroptera: Miridae). Insect Biochemistry. 21(7): 759-765

Liu, H, Skinner, M., and Parker, B.L. (2003) Bioassay method for assessing the virulence of Beauveria bassiana against tarnished plant bug, Lygus lineolaris (Hem., Miridae). Journal of Applied Entomology. 127: 299-304

Loeb, G., Carroll, J. and Cha, D. (2006). Understanding habitat colonization by tarnished plant bug as basis for developing an attraction-based management system for berry crops.

110 Progress report to North American Strawberry Growers Association. Cornell University, New York State. Site accessed December 10, 2006 http://www.nysipm.cornell.edu/grantspgm/projects/proj06/fruit/loeb.asp

Louws, F.J. (1996) Red raspberries: planning and planting. In Growing red raspberries in Ontario, Publication 105. Dale, A., Clarke, T., Huffmon, L., Hoffman, D., Watson, T., Sullivan, A., Whittamore, M. (ed.) pp 3-5. Toronto, Ontario: Queen's Printer.

Mailloux, G. & Bostanian, N.J. (1989). Presence-absence sequential decision plans for management of Lygus lineolaris (Hemiptera:Miridae) on strawberry. Environmental Entomology. 18(5): 829-834

Mailloux, G. & Bostanian, NJ. (1990) The phenological development of strawberry plants and its relation to tarnished plant bug seasonal abundance. Advances in Strawberry Production. 10: 30-36

Mailvaganam, S. (2003) Strawberries: area, production, and farm value by county and district, Ontario, 2003. Site accessed December 10, 2005 http ://www. gov. on. ca/OM AFR A/english/stats/hort/ctystraw02 .htm

Mailvaganam, S. (2007) Area, production and farm value of specified commercial fruit crops, Ontario, 2005. Site accessed July 5, 2007 http: //www. go v. on. ca/O M AFR A/engl ish/stats/hort/cty s tr a w02 .htm

Makki, F. (2002) 2002/2003 Canadian fruit situation and trends including apples, tender fruits, grapes (Vinifera) and berries. Agriculture and Agri-Food Canada. Ottawa, Ontario, pp 0-23

Momol, M.T. and Stavisky, J. (2004). Integrated management of tomato spotted wilt on field-grown tomatoes. Plant Disease. 88: 882-890

Norris, R.F., Caswell-Chen, E.P. and Kogan, M. (2003) Cultural management of pests: trap crops. In Concepts in integrated pest management, pp 435-436. New Jersey, United States of America: Prentice Hall Inc.

Ontario Ministry of Agriculture, Food and Rural Affairs. 2006. Fruit production recommendations 2006-2007 Publication 360. pp 175-181. Toronto, Canada: Queen's Printer for Ontario.

Painter, R.H. (1951) The mechanisms of resistance: preference and non-preference, antibiosis, tolerance. In Insect resistance in crop plants, pp 24-83. New York, United States of America: The Macmillan Company

Pedigo, L.P. (2002) Managing insects with resistant plants: mechanisms of resistance. In Entomology and pest management 4th Edition, pp 441- 476. New Jersey, United States of America: Prentice Hall Inc.

Ill Rancourt, B., Vincent, C. and Oliveira. D.D. (2000) Circadian activity of Lygus lineolaris (Hemiptera: Miridae) and effectiveness of sampling techniques in strawberry fields. Journal of Economic Entomology. 93(4): 1160-1166

Reitz, S.R., Yearby, E.L., Funderburk, J.E., Stavisky, J., Momol, M.T. and Olson, S.M. (2003). Integrated management tactics for Frankiniella thrips (Thysanoptera: Thripidae) in field-grown pepper. Journal of Economic Entomology. 96(4): 1201-1214

Rhainds, M. Kovach, J., Dosa, E.L., and English-Loeb, G. (2001) Impact of reflective mulch on yield of strawberry plants and incidence of damage by tarnished plant bug (Heteroptera: Miridae). Journal of Economic Entomology. 94(6): 1477-1484

Rhainds, M. and English-Loeb, G. (2003a) Testing the resource concentration hypothesis with tarnished plant bug on strawberry: density of hosts and patch size influence the interaction between abundance of nymphs and incidence of damage. Ecological Entomology. 28: 348-358

Rhainds, M. and English-Loeb, G. (2003b). Variation in abundance and feeding impact of tarnished plant bug (Hemiptera: Miridae) for different cultivars of strawberry: role of flowering phenology and yield attributes. Journal of Economic Entomology. 96(2): 432- 440

Riggs, D.I.M. (1990) Greenhouse studies on the effect of Lygus bug feeding on "Tristar" strawberry. Advances in Strawberry Production. 9: 40-43

Rodriguez, J.G., Kemp, T.R. and Dabrowski, Z.T. (1976) Behaviour of Tetranychus urticae toward essential oil mixtures from strawberry foliage. Journal of Chemical Ecology. 2: 221-230

Rodriguez-Saona, C, Crafts-Brandner, S.J., Williams III, L., and Pare, P.W. (2002) Lygus hesperus feeding and salivary gland extracts induce volatile emissions in plants. Journal of Chemical Ecology. 28(9): 1733-1747

Schaefers, G.A. (1981). Pest management systems for strawberry insects. In CRC handbook of pest management in agriculture. Pimental, D. (ed.) pp 377-393. CRC, Boca, Raton, Florida

Schaefers, G.A. (1991). Insects that damage fruit: Lygus bugs. Ellis, M.A., Converse, R.H., Williams, R.N. Willamson, B. Compendium of raspberry and blackberry diseases and insects, (pp 66-67). Minnesota United States of America: The American Phytopathological Society.

Schwartz, M.D. and Foottit, R.G. (1992) Lygus bugs on the prairies, biology, systematics and distribution. Research Branch Agriculture Canada, Biological Resources Division, Ottawa

112 Snodgrass, G.L., Scott, W.P., Abel, C.A. Robbins, J.T., Gore, J., and Hardee, D.D. (2005) Tarnished plant bug (Heteroptera: Miridae) populations near fields after early season treatment. Pest Management. 34(3): 705-711

Stavisky, J., Funderburk, J., Brodbeck, B.V., Olson, S.M. and Anderson, P.C. (2002). Population dynamics of Frankiniella spp. and tomato spotted wild incidence as influenced by cultural management tactics in tomato. Journal of Economic Entomology. 95(6): 1216-1221

Stinner R.E., Barfield, C.S., Simac, J.L. and Dohse, L. (1983). Dispersal and movement of insect pests. Annual Review Entomology. 28: 319-335

Summers, C.G. and Stapleton, J.J. (2002). Management of corn leafhopper (Homoptera: Cicadellidae) and corn stunt disease in sweet corn using reflective mulch. Journal of Economic Entomology. 95(2): 325-330

Summers, C.G., Stapleton, J.J, Newton, A.S., Duncan, R.A. and Hart, D. (1995). Comparison of sprayable and film mulches in delaying the onset of aphid-transmitted virus diseases in zucchini squash. Plant Disease. 79(11): 1126-1131

Tilmon, K.J. and Hoffman, M.P. (2003) Biological control of Lygus lineolaris by Peristenus spp. in strawberry. Biological Control. 26: 287-292

Velutherambi, K. and Poovaiah, B.W. (1984) Auxin-regulated polypeptide changes at different stages of strawberry fruit development. Plant Physiology. 75: 349-353

Wardle, A.R., Borden, J.H., Pierce, H.D. and Gries, R. (2003) Volatile compounds released by disturbed and calm adults of the tarnished plant bug. Journal of Chemical Ecology. 29(4): 931-944

Williams, L. and TugwelL, N.P. (2000) Histological description of tarnished plant bug (Heteroptera: Miridae) feeding on small cotton floral buds. Journal of Entomological Science. 35(2): 187-195.

Williams, L, Logarzo, G.A., Shaw, S.R., Price, L.D. and Manrique, V. (2003) Leiophron argentinensis Shaw (Hymenoptera: Braconidae): a new species of parasitoid from and Paraguay-information on life history and potential for controlling Lygus bugs (Hemiptera: Miridae). Annals of the Entomological Society of America. 96(6): 834- 846

Wold, S.J. and Hutchison, W.D. (2003) Phenology of Lygus lineolaris (Hemiptera: Miridae) in Minnesota June-bearing strawberries: comparison of sampling methods and habitats. Journal of Economic Entomology. 96(6): 1814-1820

113 Zink, A.G. and Rosenheim, J.A. (2004). State-dependent sampling bias in insects: implications for monitoring western tarnished plant bugs. Entomologia Experimentalis et Applicata. 113: 117-123.

Zhu, L.C., Smith, CM. and Reese, J.C. (2005) Categories of resistance to greenbug (Homoptera: Aphididae) biotype K in wheat lines containing Aegilops tauschii genes. Annals of Entomological Society of America. 98(6): 2260-2265.

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