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Evaluating the performance of Orius insidiosus as a predator of thrips in greenhouse-grown geraniums

Christopher Moseley Epes

Major Project/Report submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Online Master of Agricultural and Life Sciences In: Environmental Science

Alejandro Del-Pozo-Valdivia, Ph.D., Assistant Professor & Extension Specialist, Virginia Polytechnic Institute and State University, Department of Entomology, Hampton Roads Agricultural Research and Extension Center Laurie Fox, Ph.D. Peter B. Schultz, Ph.D. Joyce Latimer, Ph.D.

Date of Submission: 07/15/2021

Keywords: Biocontrol, Greenhouse, Geraniums, Orius, Thrips

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Evaluating the performance of Orius insidiosus as a predator of thrips in greenhouse-grown geraniums

Christopher Moseley Epes

ABSTRACT

Thrips are among the most common and economically damaging greenhouse pests in the world. Due to the demanding nature of chemical control programs in greenhouses for thrips, biocontrol strategies are growing in popularity in greenhouse pest control programs. Geraniums are common in spring annual greenhouse production programs, and like many other crops demand thrips management strategies. This project lays the groundwork for exploring the performance of minute pirate bug, Orius insidiosus, to reduce thrips densities in greenhouse geraniums. In a laboratory, Orius and thrips were released into controlled, no-choice predation arenas using both geranium leaves and flowers, and thrips mortality was assessed. Orius and thrips were then released onto finished potted geranium plants under no-choice conditions in an insect proof cage under greenhouse conditions, after which thrips mortality was assessed. The cumulative results show that Orius prey upon thrips on geranium leaves and florets, and could be used as a tool to help reduce thrips populations in greenhouse-grown geraniums.

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Table of Contents ABSTRACT ...... 2 Chapter 1: Literature Review ...... 5 1.1 Rationale ...... 5 1.2 Research Project Background ...... 7 1.3 Insect Background Information ...... 8 1.4 Environmental condition requirements for insects of interest ...... 11 Chapter 2 - Assessing the predation potential of Orius and Amblyseius on thrips infesting geranium under controlled and no-choice conditions ...... 12 2.1 Introduction ...... 12 2.2 Materials and Methods ...... 14 2.21 Test Plants ...... 14 2.22 Predation Arenas ...... 15 2.23 Insects/Mites ...... 16 2.24 Experimental Design ...... 18 2.25 Procedure ...... 21 2.3 Data Analysis ...... 22 2.4 Results ...... 23 2.41 Predation rates in the leaf arenas ...... 23 2.42 Predation in Floret Arenas ...... 24 2.5 Discussion ...... 26 2.51 Predation in Leaf Arenas ...... 26 2.52 Predation in Floret Arenas ...... 29 Chapter 3 – Assessing the predation potential of Orius on thrips infesting geranium under greenhouse conditions in no-choice insect cages ...... 30 3.1 Introduction ...... 30 3.2 Materials and Methods ...... 31 3.21 Test Plants ...... 31 3.22 Arenas ...... 31 3.23 Insects ...... 32 3.24 Experimental Design ...... 33 3.25 Procedure ...... 36 3.26 Data Analysis ...... 40 3.3 Results ...... 40 4

3.31 Fall 2020 ...... 40 3.32 Spring 2021 ...... 41 3.4 Discussion ...... 43 Conclusion ...... 44 References ...... 46 Appendix ...... 49

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Chapter 1: Literature Review

1.1 Rationale

From 2013 to 2017 I worked as a professional greenhouse production manager of a wide variety of florist-quality annuals and perennials, including an array of different types of geraniums. Among the cornerstones of our spring production and sales were the zonal and interspecific red geranium varieties. Typically, geranium crops began as plugs transplanted to

4.5”, 6” and 12” containers in successions throughout February and March, ready for market throughout April and up to Mother’s Day. Until the last frost date had passed (forecasted for late

March/early April in Virginia Beach, the region of discussion) insecticides were not necessary.

The only potential later winter/early spring pest we had to contend with were fungus gnats, which could easily be kept well below economic thresholds with patient water management and good sanitation.

The problems arose after the last frost when western flower thrips (Thysanoptera:

Thripidae) populations began to build up. During fall, we used the house to produce poinsettias, to which we applied to each pot a dose of granular imidacloprid (Mode of Action 4A, neonicotinoid insecticide) sufficient to provide season-long systemic control of whitefly for each plant. This control measure was categorically successful every year. We did not have any other long-term systemic control options available to us that weren’t neonicotinoids, so we chose not to use this chemical class on any other crops produced in this greenhouse, because we might reduce their efficacy on the poinsettias.

As temperatures began to warm each spring, we would begin a rotating insecticide application program on the geraniums consisting of various pyrethroids, carbamates, 6 organophosphates, and azadirachtin, which would last until Mother’s Day when the house would be emptied. We found these control measures to be moderately effective up until mid-April, but virtually ineffectual on our resident western flower thrips population between that time and

Mother’s Day. The beginning of our loss of control appeared to coincide annually with the point in time when night-time low temperatures consistently climbed to 60°F. This annual 3-4 week interval spent aggressively battling the buildup of western flower thrips populations on the back end of our spring geranium crop, constituted what was undoubtedly our single most arduous and resource depleting pest control task each year, and was the catalyst for my wondering if there was a better way to manage thrips.

In late 2017, after I had left my greenhouse position and moved onto extension work, I came across an article in GrowerTalks Magazine entitled ‘Orius insidiosus: A Natural Thrips

Killer.’ Immediately bringing back memories of my annual western flower thrips difficulties, my attention was captured. By the time I had left greenhouse production I had developed a curiosity for biocontrol programs, but had not had the opportunity to implement anything in my operation.

I was familiar with predatory mite and parasitic wasp programs, but not this particular predator, otherwise known as minute pirate bug. The article was written by a head grower for regional wholesale plug production operation, and he described how, by using rearing techniques and insectary plants throughout winter, Orius insidiosus (Hemiptera: Anthocoridae) had become a fixture in his highly successful spring and summer thrips control program in the greenhouse. The grower deduced that a single minute pirate bug would consume any crawling stage of a thrips, at up to 80 thrips per day. Moreover, he found ample evidence that the predator was also proliferating outdoors in his production fields during the warm season, providing a significant degree of control there as well. After reading this article, I immediately endeavored to discover 7 the provenance of Orius insidiosus, and found that its native range is the Americas, and it is known to be most abundant in the eastern United States. Subsequently, minute pirate bug has been a fixture in the homeowner-level horticultural IPM education I do as an Extension Agent.

When the time came to choose a research topic for my Master Program, Orius insidiosus predation of plant pests, specifically western flower thrips, seemed like a natural fit. Using greenhouse geraniums as the growing medium for this project was the logical choice.

1.2 Research Project Background

Last year, a colleague who works for Plant Products, Inc., a large seller of biocontrol insects, brought to my attention that growers in the Hampton Roads region who have used Orius insidiosus in biocontrol programs were successfully using a new variety of ever-blooming sunflower called ‘Sunfinity,’ which was released by Syngenta in 2017, as an insectary plant for in-situ rearing of the pirate bugs. Insectary plants attract, shelter, and provide alternative food to various species of insects that parasitize or prey upon other smaller insects (Becker et al, 2018). I presumed, given how recently ‘Sunfinity’ was introduced to the market, that research into its efficacy as an insectary plant had probably not taken place. After I confirmed this via a literature search, we began designing our research project.

It quickly became apparent however that due to the complicated nature of this research question, and the fundamentally complicated field of biocontrol research, we would have to begin by answering the basic question, does Orius insidiosus display predation of any thrips species, including the western flower thrips on geraniums? There is ample research that supports this in a variety of contexts, but the goal was to be able to definitively state that in the context of our research, at our facility, under our influence, that yes, Orius insidiosus will prey upon thrips. 8

After we could safely answer this question, we would be able to build off that using a more complicated research framework. We chose to divide the project into subsequent chapters.

First, we would, upon the suggestion of my committee chair, Alejandro Del-Pozo-

Valdivia, create non-choice bioassay predation arenas using geranium leaf discs placed in agar- filled petri dishes, and release Orius insidiosus and thrips into each arena to observe what transpires. This method is based upon Aphid Susceptibility Test Method 019, established by the

Insecticide Resistance Action Committee’s (IRAC’s) Insecticide Resistance Task Force

(https://irac-online.org/methods/aphids-adultnymphs/). In addition to gauging mortality of thrips released with Orius insidiosus in these arenas, we also decided to incorporate another predator for comparative purposes and create bioassay predator arenas in the same fashion for them as well. The predator we chose for comparison is commonly used in biocontrol programs for thrips suppression: the predatory mite Amblyseius cucumeris. My colleague from Plant Products, Inc.,

Andrew Eye, volunteered to supply us with the Orius and Amblyseius needed for the project.

Second, if predation was clearly observed in the previous set of studies, we would build thrips-proof insect cages large enough to house potted, flowering geranium plants. We would then release thrips onto the flowers, followed by a release of Orius insidiosus and observe the degree of thrips mortality.

1.3 Insect Background Information

Orius insidiosus, otherwise known as insidious flower bug, is a type of minute pirate bug in the order Hemiptera and family Anthocoridae (Sprague, Funderburk, 2016). Orius insidiosus is known to naturally prey on a number of soft-bodied insects considered to be economically important agricultural pests, including thrips such as the invasive western flower thrips

(Frankliniella occidentalis) (Xu et al 2006). Insidious flower bug occurs most commonly in the 9 eastern United States, but is widely distributed throughout southern United States, southward into Mexico, Central and South America, as well numerous islands throughout the Caribbean Sea

(Herring, 1966).

Adult Orius insidiosus, approximately 3 mm in length, are elliptical in shape with white and black-patched wings that extend just beyond the length of the abdomen (Sprague,

Funderburk, 2016). The abdomen, thorax and head of adults are black, their eyes are red, and a they have a long, piercing-sucking proboscis which they use to kill and consume prey. The climate zone will determine how long Orius insidiosus is active throughout the year. In USDA

Hardiness Zone 8A, where our research is located, insidious flower bug is likely to be active from late-March/early-April through late-October/early-November (Funderburk, 2016). Orius insidiosus is known to thrive on variety of crops and wild plant species, especially peppers, okra and cotton, where they are able to persist even when prey populations are low, by subsisting off of plant pollen and plant juices, which rarely results in damage to the plant (Funderburk, 2016).

Prey for adult and nymphal stages of Orius insidiosus consists of a wide array of smaller, soft- bodied economically important pests such as aphids, mites, and whiteflies, as well as the eggs of these and other pest species; however, it demonstrates a clear preference for flower thrips, specifically western flower thrips (Frankliniella occidentalis), on whom it preys on both adult and, more robustly, larval forms (Funderburk et al 2000. Baez at al 2004).

Frankliniella occidentalis, otherwise known as western flower thrips, is arguably the most destructive greenhouse pest in the world. Native to the western United States, over the last fifty years this pest has become economically problematic in floriculture greenhouses worldwide, seemingly indiscriminately of crop (Frank and Baker, 2009). The host ubiquity of western flower thrips both indoor and outdoor, coupled with their tiny size, reproductive capacity, flight ability, 10 developmental characteristics and propensity to spread disease have come to make them supremely difficult to control in modern floriculture (Frank and Baker, 2009).

Western flower thrips, about 1-2 mm long at full size, will feed on soft tissue throughout the plant including in buds, terminal shoots, and flowers by scraping and puncturing plant tissue with their mouthparts and feeding on the sap in the plant cells causing them to collapse (Frank and Baker 2009). Leaves that have been damaged can display pale spots and become distorted and twisted at full emergence, and flowers, to which western flower thrips is most attracted display pale, sometimes necrotic splotches and patches that contrast unattractively with the flower color and can cause the petals to prematurely fall (Frank and Baker 2009). There are six developmental stages of this pest: egg, two larval stages, a prepupal stage, pupal stage, and adult,

(Frank and Baker 2009). The eggs of western flower thrips are oviposited directly into plant tissue by the adult females, who are capable of laying between 150-300 eggs over their lifetimes

(Frank and Baker 2009). The eggs hatch and first instar larvae emerge as pale to translucent white. While feeding on plant tissue they grow into the second instar larvae which are the same color, but are similar in size to an adult (Frank and Baker 2009). At the end of the of the second larval stage the larvae will drop from the plant to the soil below, where it will pupate without feeding into an adult through a prepupal and pupal stage (Frank and Baker 2009). At the end of the pupal stage in the soil, the adult will emerge, females being slender, yellow to light brown in color with fringed wings, and males being slightly smaller than the female and light yellow in color (Frank and Baker 2009). The whole developmental process from egg to adult can take about 13 days, and adults can live for about 28 days (Frank and Baker, 2009). 11

1.4 Environmental condition requirements for insects of interest

Understanding the environmental conditions necessary for the insects to thrive was a critical component for project success. Information gleaned from environmental conditions from previous research was used to establish environmental parameters for both the bioassays and the greenhouse component. Orius insidiosus and western flower thrips would necessitate constant temperatures of approximately 24°C-28°C (75°F-82°F). We would be able to replicate this in the bioassays via environmental control growth chambers (Percival Model I-30BLL). The greenhouse system hardware was not sufficient to allow that degree of control over daytime high temperatures and overnight low temperatures. Additionally, Orius insidiosus would necessitate a constant 60-75% relative humidity range, which like the temperature range would be attainable in the bioassays via the environmental control chambers, but would be subject to potential fluctuation outside of that range in the greenhouse environment. Lastly, the necessary photoperiod for Orius insidiosus activity would need to be at least L14:D10 to prevent them from going into a resting state, which would be possible in both the bioassays and the greenhouse component via supplemental lighting (Ruberson et al, 2000).

Predatory mites (Amblyseius cucumeris) would require constant temperatures between

23°C-26°C (73°F-79°F), and the preferred range of relative humidity would need to be 70-75%, feasible in the bioassays but not the greenhouse. The predatory mite would, as with Orius and thrips, necessitate a photoperiod of L14:D10, which would be attainable in the bioassays and the greenhouse. 12

Chapter 2 - Assessing the predation potential of Orius and Amblyseius on thrips infesting geranium under controlled and no-choice conditions

2.1 Introduction

Flower thrips are known to be pests of a variety of economically important floriculture crops (Frank and Baker, 2009). Biological control measures are commonly sold throughout the industry to help control flower thrips, including reared and packaged Orius for thrips predation, as well as the predatory mite Amblyseius cucumeris. The literature available on Orius and

Amblyseius predation of thrips is limited to only a handful of ornamental plant species relative to the whole of ornamental floriculture production.

Flower thrips are known to infest species of geranium, Pelargonium sp., a fixture in the floriculture industry (USDA, 2019). Literature on thrips predation by Orius in geraniums is limited. To begin exploring efficacy of Orius on thrips in geraniums, it was first be critical to prove that Orius would prey upon thrips under controlled conditions by using geranium plant tissue as a medium in predation arenas. Amblyseius cucumeris was evaluated in addition to Orius in this fashion.

Review of literature focusing on thrips predation by Orius and Amblyseius on other plant species in controlled arenas revealed common threads in protocol that may be used for zonal geraniums. Controlled arenas for testing largely consisted of a small plastic container or petri dishes in which either a plant part or seedling was placed, after which thrips were released first, then the predator was released within 24 hours, and thrips mortality was evaluated (Baez et al,

2004, Chow et al, 2008, Chow et al, 2010, Deligeorgidis, 2002, Enkegaard and Xu, 2009, 13

Meiracker and Sabelis, 1999, Xu et al, 2005, Xu and Enkegaard, 2010). The materials used for the arenas and the way the arenas were engineered varied, but they typically fell into three categories of protocol:

1. A seedling was grown in a small pot, over which a clear plastic dome or cover was placed

when the seedling was ready; the dome or cover had one or more holes through which

insects could be introduced into the arena. The holes were sealed with thrips-proof insect

screening following release of the insects (Enkegaard and Xu, 2009, Sprague and

Funderburk, 2016).

2. A flower or leaf, still attached to the plant, was inserted through a hole or gap in clear

plastic container large enough to hold the plant part, and that hole or gap was sealed

around the flower stem or petiole; insects were introduced into the arena via holes in the

plastic container which were subsequently sealed with thrips-proof insect screening

(Chow et al, 2008, Chow et al, 2010).

3. A whole flower, whole leaf or leaf disc was removed from the plant and placed into a

petri dish. The insects were placed into the arena and the arena was sealed either with the

petri dish lid or with thrips-proof insect screening (Baez et al, 2004, Deligeorgidis, 2002,

Meiracker and Sabelis, 1999, Xu and Enkegaard, 2010).

Looking at the predator-to-prey ratios in literature helped provide a context for Orius-to- thrips density ratios to use in each arena. The majority of the literature used included ratios of

1:5, 1:10, 1:20, and 1:30 in controlled predation arenas, each of which illustrated successful predation and control of thrips (Baez et al, 2004, Chow et al, 2008, Chow et al, 2010,

Deligeorgidis, 2002, Enkegaard and Xu, 2009). Xu et. al used Orius-to-thrips ratios of 1:50,

1:80, 1:100, and 1:160. In addition to observing success predation, they concluded that as thrips 14 density increases, Orius predation increases in kind (Xu et al, 2005). For Amblyseius assessment a predator-to-prey density of 3:1 was chosen to by us in an attempt to assure some degree of observable predation.

2.2 Materials and Methods

2.21 Test Plants

Interspecific hybrid geranium variety ‘Calliope Dark Red’ was used as the test plant for this experiment. The Calliope series is a zonal/interspecific hybrid which is known for its heat tolerance and is widely grown for retail in the America southeast. Two sets of bioassays were done to complete this chapter, the first taking place in fall of 2020, and the second taking place in the spring of 2021.

For both sets of bioassays, geraniums were ordered as plugs (rooted cuttings) from Lucas

Greenhouses in Monroeville, New Jersey. The fall plugs arrived on ship-week 39 (September 21) in 2020, and the spring plugs arrived on ship-week 11 (March 17) in 2021. All 200 fall plugs were transplanted the week of their arrival into 4.5” (one pint) round ITML pots that each contained PRO-MIX BX potting soil (75-85% spaghnum peat moss, 10-14% perlite, vermiculite, limestone, wetting agent, mycorrhizae). A teaspoon of 15-9-12 Osmocote controlled-release fertilizer was added to each pot. In spring, 150 plugs were transplanted into 4.5” pots as in fall, but the remaining 50 were transplanted into 6” (one quart) ITML pots also containing PRO-MIX

BX potting soil but with two teaspoons of 15-9-12 Osmocote instead of one. In both spring and fall, the pots were then placed in a greenhouse under a conventional spritzer/spaghetti tube irrigation system. They were fertigated at every watering via Dosatron injecting Nature’s Source

10-4-3 liquid Professional Plant Food at a rate of 150 ppm. The plants were grown for five weeks. The nighttime low temperature heating threshold was 60°F and daytime high temperature 15 cooling threshold was 80°F. After five weeks under these conditions in both spring and fall, the geraniums had produced leaves and flowers sufficiently to harvest and use in predation arenas.

No pesticides were used during the production process, and no pests were detected.

2.22 Predation Arenas

The predation arenas were created using the Insecticide Resistance Action Committee

Aphid Susceptibility Test Method 019 as a template (https://irac-online.org/methods/aphids- adultnymphs/). Two types of arenas were created for evaluation, leaf arenas and floret arenas, which constituted the two repetitions of the fall bioassay experiment.

One hundred mm diameter x 15mm deep petri dishes were used for the leaf arenas in fall of 2020. The petri dishes were filled halfway with prepared, hot, liquid agar, which was allowed to cool long enough to cause no damage to the geranium leaf tissue but not long enough for the agar to solidify. In spring of 2021, 2 oz. clear plastic souffle (condiment cups) with airtight lids were used for the leaf arenas. In both fall and spring, 5 cm diameter geranium leaf discs were cut from randomly harvested whole geranium leaves and then placed onto the agar surface (Figures

1, 2, 3). The bottom of the leaf disc sealed to the agar surface, and a seal was created along the entire circumference of the leaf disc and the inner edge of the petri dish.

The souffle cups were used for the floret arenas in both fall 2020 and spring 2021. This was done to create an arena tall enough to allow setting a geranium floret pedicel-down into agar. The souffle cups were filled halfway with prepared, hot, liquid agar, as with the leaf arenas.

A single floret was then randomly cut from the geranium flower head halfway down the pedicel, and placed pedicel-down into the agar. The undersides of the floret petals sealed to the agar surface, and the outer edges of the petals contacted and sealed throughout the circumference of the inside of the souffle cups, effectively creating a sealed predation arena similar to the leaf 16 arenas. This left the upward facing parts of the floret, including the entire pistil and stamen available as a viable arena. The cups were sealed with the airtight lids.

2.23 Insects/Mites

Tobacco thrips (F. fusca), western flower thrips (WFT, F. occidentalis) and eastern flower thrips (EFT, F. tritici) are all active in Southeast Virginia and can be found during the growing season at the Virginia Tech Hampton Roads Agriculture Research and Extension Center

(Virginia Tech HRAREC) in Virginia Beach. Tobacco thrips are readily distinguishable from

WFT and EFT by the dark brown/black color and larger size of adults, and bright orange color of immatures. WFT and EFT adults are easy to distinguish from tobacco thrips, but not readily distinguishable from one another with the naked eye. Due to our inability to locate a WFT colony from which we could obtain them exclusively for the project, and due to the time necessary to separate large numbers of WFT from the EFT, we opted not to separate WFT and

EFT after collection from the field. As such, the random combination of the two is what was used throughout this project, designated “WFT/EFT.”

2.231 Fall 2020 Bioassays

In the fall of 2020 when the thrips were collected to begin building a colony, tobacco thrips were present in the landscape in much higher numbers than WFT/EFT. Thrips were collected from Gardenias (Gardenia jasminoides) and Camellias (Camellia japonica) at the

Virginia Tech HRAREC demonstration gardens. The tobacco thrips and WFT/EFT that were collected were separated into two separate colonies.

The thrips rearing method used for the colony is an unpublished variation of the Steiner and Goodwin method, using green beans as a medium (Steiner and Goodwin, 1998). While using this protocol, the WFT/EFT colony dwindled and ultimately perished, presumably due to the 17 natural population decline during fall. The tobacco thrips colony remained steady however, and we chose to use tobacco thrips for the experiments.

The predators in question for the fall data set, minute pirate bug, Orius insidiosus, and the predatory mite Amblyseius cucumeris, were purchased from Plant Products, Inc., and arrived two days before being introduced into the arenas.

2.232 Spring 2021 Bioassays

When preparing for the spring 2021 portion of the predation experiment, we again began collecting thrips from the Virginia Tech HRAREC demonstration gardens, from Viburnum

(Viburnum alnifolium), Fothergilla (Fothergilla gardenii) and Hawthorn (Crataegus sp.). Each of these spring-blooming shrubs was host to tobacco thrips as well as WFT/EFT. More attempts were made to create colonies for both species, but as in fall, these colonies collapsed within two to three days. We decided to collect thrips and release them into the predation arenas immediately following collection. Contrasting with fall, the amount of WFT/EFT was high, so we elected to use WFT/EFT in addition to tobacco thrips.

Orius insidiosus alone was used for the spring data set, as the fall data on Amblyseius cucumeris were determined to be inconclusive. The Orius was purchased from Plant Products,

Inc. Two shipments of Orius were used for the spring data set, one for the first and second replication, and a separate shipment for the third replication. This was done to assure that treatment affects could not be attributed to specific shipments. 18

2.24 Experimental Design

2.241 Fall 2020 bioassays

The fall 2020 portion of this experiment contained two repetitions utilizing a randomized block design. The first repetition was designed to assess mortality of tobacco thrips (F. fusca) in the presence of Orius and Amblyseius on geranium leaf disc bioassays, and the second repetition was designed to assess mortality of tobacco thrips in the presence of Orius and Amblyseius on geranium floret bioassays. Each of these repetitions contained four replications. Each replication consisted of three treatment arenas that contained Orius and thrips, three treatment arenas that contained Amblyseius and thrips, and a single control treatment arena that contained only thrips.

This amounted to a total of seven treatments per replication, 28 total treatments per repetition, and 56 experimental units in this experiment.

For both Orius vs. thrips and Amblyseius vs. thrips, we tested the predators against eight adult thrips, eight immature thrips, and a combination of four adult thrips + four immature thrips.

For the Orius treatments, four unsexed adult Orius were used in each treatment (representing a

1:2 predator-to-prey ratio), and for the Amblyseius treatments, 24 unsexed Amblyseius were used in each treatment (3:1 predator-to-prey ratio) in random combinations of adults and immatures

(Table 1, Appendix).

2.242 Spring 2021 bioassays

The spring portion of this experiment utilized six total repetitions, three leaf repetitions and three flower repetitions. The first leaf repetition and first flower repetition were done together as Repetition Group 1. The second leaf repetition and second flower repetition were done together as Repetition Group 2, and the third leaf and third flower repetitions as Repetition

Group 3. Amblyseius was omitted entirely from the spring 2021 bioassays. Additionally, we 19 decided to focus entirely on adult thrips and omit the immature thrips in spring of 2021. This allowed for a simplified experimental design. Subsequently, each repetition contained two treatments, a control treatment in which thrips alone were released into the arena, and an

Orius+Adult thrips treatment. There were six replications of each of those treatments, amounting to 12 total replications per repetition.

In Repetition Group 1, we used tobacco thrips as prey in order to supplement the results of the fall 2020 experiment. The predation arenas for Repetition Group 1 contained four unsexed

Orius and eight unsexed, adult tobacco thrips. Repetition Groups 1 and 2 however contained four unsexed Orius and eight WFT/EFT. The design is illustrated in Table 2, found in the Appendix.

Figure 1. Cutting leaf discs for arenas.

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Figure 2. Creating leaf disc arenas.

Figure 3. Completed leaf and floret arenas. 21

2.25 Procedure

2.251 Fall 2020 bioassays

One day before initiation of the experiment, via aspirator the Orius, Amblyseius and the thrips were each aliquoted into small, sealed plastic condiment cups per the quantities needed for the treatments for staging (24 Orius cups, each containing four Orius, 24 Amblyseius cups, each containing 24 Amblyseius, and 56 thrips cups, 24 with eight adult thrips, 16 with eight immatures, and 16 with four adult + four immature thrips). The intent was for the cups to contain nothing but the insects, allowing a starvation period of 24 hours prior to release. After the insects were aliquoted into the cups, the cups were then placed into the environmental control chambers.

The day of the experiment, geraniums were randomly chosen from the greenhouse crop.

After inspection to assure no pest or disease activity, 12 plants were transported into the laboratory to use for the leaf disc and floret bioassays described above. Twenty-eight leaf disc arenas and 28 floret arenas were made and labeled. The agar in the arenas was allowed to set and cool, after which the cups containing the Orius, Amblyseius and thrips were emptied into the arenas by hand. After the insects were emptied into the arenas, the arenas were sealed and placed into the environmental chambers for 24 hours. The arenas were then removed and assessed for predation by counting the dead adult thrips, living adult thrips, dead immature thrips, and living immature thrips.

2.252 Spring 2021 bioassays

On the setup day of each spring repetition, flowers were collected from the landscape at the Virginia Tech HRARC in plastic, gallon-sized resealable bags, and the bags were placed upright in the lab, allowing the thrips within the bag to begin migrating upward toward the seal.

Immediately following flower collection, flowering geraniums were randomly chosen from the 22 greenhouse. The leaf disc and flower arenas were created using the same procedure as in fall; however, we suspected that static electricity was linked to thrips mortality in the plastic containers used in the fall experiment, so all plastic containers used in spring were treated immediately prior to use with an ionizing gun to remove static electricity.

As soon as the agar cooled and settled, approximately one hour after the arenas were created, the thrips collection bags were opened and adult thrips were aspirated directly from the collection bags into small plastic vials at a count of eight adult thrips per vial. All plastic vials were also treated prior to use with an ionizing gun to remove static electricity. After 24 thrips- vials had been created, unsexed Orius adults were aspirated directly from their shipping cannister into plastic vials at a count of four Orius per vial. After 12 Orius vials had been created, all of the thrips and Orius vials were opened and dumped one-by-one into the arenas, after which the arenas were immediately sealed and placed into the environmental control chambers for 24 hours.

2.3 Data Analysis

Data sets from both fall 2020 and spring 2021 were subjected to an ANOVA using a generalized mixed linear model (PROC MIXED, SAS 2011) to determine any treatment effects.

Individual analyses were run on the leaf tissue and the flower assays. The Tukey-Kramer method was used to separate treatment means post ANOVA, looking at thrips counted dead at the end of the experiment as the response variable. When needed, the data were log-transformed to comply with normality. Back-transformed means and standard errors are represented in the bar graphs. 23

2.4 Results

2.41 Predation rates in the leaf arenas

2.411 Fall 2020 bioassays

The results of thrips mortality in the leaf disc arenas varied (Table 3, Appendix) and there were statistical differences (DF=6,18; F=38.33; P<0.0001). On average, in the Orius+Adults treatments, 7.5 adult thrips out of eight were found dead, representing a 96% mortality rate

(Figure 4). This treatment context represented statistically the highest mortality of the leaf treatments. The second highest statistical mortality of the treatments occurred in the

Amblyseius+Adults treatments, where on average seven adult thrips out of eight were found dead. In the control treatments, 3.3 adults were found dead on average.

Figure 4. Leaf tissue bioassay results Fall 2020, average number dead thrips in Orius+adult treatments. 2.412 Spring 2021

Analysis was run on the number of dead thrips per Orius arena relative to the Orius-free control arenas throughout the repetitions (Table 4, Appendix). A significant difference was found between the control and the Orius arenas. The Orius leaf arenas illustrated an average of 6.4 24 thrips dead per arena, representing an 80% mortality rate across all the repetitions, whereas the control leaf arenas illustrated an average of less than one thrips dead per control arena across all repetitions, representing a 8% mortality (Figure 5).

Figure 5. Leaf tissue bioassay results Spring 2021, average number of dead adult thrips in all leaf repetitions. Leaf repetition 1, which used tobacco thrips as prey, illustrated an average of 5.5 thrips dead per Orius arena, representing a 69% mortality rate, while the control arenas illustrated an average of zero thrips dead per arena. Leaf repetitions 2 and 3, which used WFT/EFT as prey, illustrated a statistical average of 7.1 thrips dead per Orius arena, representing a mortality rate of

89%, while the control arenas illustrated an average of 0.5 thrips dead per arena, representing a

6% mortality rate.

2.42 Predation in Floret Arenas

2.421 Fall 2020

The results of the fall 2020 floret arenas showed significant differences between treatments (Table 5, Appendix). Analysis was run on dead adult thrips found in the floret arenas 25

(DF=6,18; F=30.73; P<0.0001). On average, in the Orius+Adults treatments, 7.5 adult thrips out of eight were found dead (Figure 6), representing a 94% mortality rate. In the Amblyseius+Adults treatments, on average one adult was found dead, representing a 13% mortality rate. In the control treatments, on average 0.5 adults were found dead, representing a 6% mortality rate.

Figure 6. Floret tissue bioassay Fall 2020 results, average number dead thrips in Orius+Adult treatments. 2.422 Spring 2021

Analysis was run on the average number of dead adult thrips per Orius arena relative to the number of dead thrips per control arena across the flower repetitions (Table 6, Appendix). A significant difference was found between the control arenas and Orius arenas (DF=1,66;

F=409.39; P<0.0001). The Orius arenas resulted in an average of 6.2 thrips dead per Orius arena across all flower repetitions, representing a 77% mortality rate, whereas the control arenas across all flower repetitions resulted in an average of only 0.9 thrips dead per arena, representing an

11% mortality rate (Figure 7). 26

Figure 7. Floret tissue bioassay Spring 2021 results, average number dead adult thrips in all floret repetitions. Flower repetition 1, which used tobacco thrips as prey, illustrated an average of 6.2 thrips dead per Orius arena, representing a 77% mortality rate, whereas the control arenas illustrated an average of just 0.2 thrips dead per arenas, representing a 2% mortality rate. Flower repetitions 2 and 3, which used WFT/EFT as prey, likewise illustrated an average of 6.2 thrips dead per Orius arena (77% mortality rate), and 1.3 thrips dead per control arena (16% mortality rate) respectively.

2.5 Discussion

2.51 Predation in Leaf Arenas

The Orius treatments across all repetitions showed a predation correlation between Orius and thrips, both adult and immatures. Unfortunately, in the fall 2020 repetitions, problems were encountered in the control treatments that compromised the data. The average statistical mortality across the control treatments was 45.6%. With this high of a number appearing in the 27 control, it makes it very difficult to draw definitive conclusions about whether or not the dead thrips in the Orius arenas had succumbed to predation or had died by other means.

There were a few reasons why mortality occurred at such high rates in the control arenas.

First, it was obvious when depositing the Orius, Amblyseius and thrips into the arenas from their staging cups, that the staging cups contained enough static electricity to make the process difficult. We hypothesized that not only could the static electricity been responsible for killing thrips, so could have the cumbersome process of getting them from the staging cups into the arenas due to the static electricity. Second, the leaf disc arenas built up a noticeable amount of condensation within the arenas. It is possible that this was responsible for some thrips mortality.

We also found that staging Orius for an extended period of time was problematic, as the Orius in the staging cups would prey upon one another, and at the end of the staging period many of the cups contained at least one dead Orius which had to be replaced with a living one that had not been starved for 24 hours.

Unlike the Orius arenas, the Amblyseius arenas did not show a clear predation correlation, even absent the control. The living and dead thrips counts in the Amblyseius arenas tended to fluctuate such that it did not appear as though predation was a factor. Other factos such as the static electricity and condensation could have impacted the experiment. Additionally, there were factors that may have contributed to the Amblyseius uncertainty. First, the mite shipment contained almost entirely immature mites. Immature mites are smaller and slower than adult thrips, so we can assume predation would be minimal or non-existent. There was still a possibility that the immature Amblyseius would prey upon the immature thrips, but mortality rates in the Amblyseius+Immatures treatment arenas did not clearly reflect this. 28

Secondly, it is possible that the Amblyseius may not have been adequately starved of food after being aliquoted into their 24-hour staging cups prior to release into the arenas. The

Amblyseius arrived from Plant Products, Inc. in a container that, in addition to the mites, held a fine mixture of vermiculite and a substance that was later confirmed by Plant Products to be a supplemental food source meant to sustain the mites during shipping. When the mites were aspirated from the container into the staging cups, it was difficult to transfer them without picking up vermiculite and the food source in the process. As such, this debris ended up in both the staging cups and the treatment arenas.

Subsequently we took a more straightforward route with the spring 2021 bioassays. We opted to simplify the experiment by using only Orius and adult thrips. Additionally, we utilized an ionizing gun to remove static electricity from the plastic components before every use. Lastly, we chose to use the souffle cups for the spring 2021 leaf bioassays, the rationale being, with a larger, deeper arena, the potential impact of static electricity and condensation would be reduced.

The results of our spring 2021 leaf bioassays demonstrate a robust predation response of thrips mixed with Orius on geranium leaves under controlled conditions. This is promising because geranium leaves contain trichomes (leaf hairs), that may interfere with a predator’s ability to hunt. The Orius+adult leaf arenas from both fall 2020 and spring 2021 however collectively resulted in 84% thrips mortality, in stark contrast to the control leaf arenas, which averaged 11% thrips mortality across both fall and spring. This infers that leaf texture is likely not an impediment to Orius movement and predation of thrips. Geraniums have not previously been the subject of Orius predation research of thrips. These data unequivocally supports the continued use of geraniums as a medium for Orius predation research of thrips, and may ultimately support the use of Orius in greenhouse geranium thrips biocontrol programs. 29

Further, when compared to tobacco thrips, the results for the WFT/EFT indicate a possible predation preference for WFT/EFT by Orius. The total average tobacco thrips mortality in the Orius leaf arenas of spring 2021 repetition 1 was 69%, whereas the total average

WFT/EFT mortality in leaf repetitions 2 and 3 was 89%, a 20% difference between species.

2.52 Predation in Floret Arenas

For the same reason the Amblyseius data were inconclusive for the leaf disc bioassays in the fall of 2020, it was difficult to draw any definitive conclusions about predation in the floret bioassays in the fall of 2020 as well. It was thus decided to abandon the Amblyseius treatments in spring and focus on the relationship between Orius and adult thrips. Both the fall and spring floret Orius treatments were as strong as the leaf disc treatments.

In the Orius+Adults floret arenas for fall 2020, the thrips mortality was 97%. In the subsequent spring 2021 floret arenas (all Orius+Adult treatments), the thrips mortality was 77%.

The control arena results were significantly different from the Orius arenas in both fall and spring: 9% mortality across the fall control floret arenas, and 11% mortality across the spring control floret arenas. This demonstrates a strong predation response by Orius of thrips on geranium florets under controlled conditions. The floret results effectively build on the leaf arena results to support the idea that Orius predation of thrips is not hindered by the basic anatomical structures of geranium florets (petals, stamen, pistil). These flower structures are much more complicated than a flat leaf surface and provide more places for thrips to hide. The Orius in our experiment demonstrated an ability to search for and prey upon the thrips. These results effectively supplement the fall 2020 leaf arena results to support the continued use of geraniums as a research medium for Orius predation of thrips, and potentially the use of Orius for thrips biocontrol programs in greenhouse geranium production systems. 30

Chapter 3 – Assessing the predation potential of Orius on thrips infesting geranium under greenhouse conditions in no-choice insect cages

3.1 Introduction

Flower thrips are among the most economically important greenhouse floriculture pests in the world. Their size, reproductive capacity, insecticide resistance development, and flight ability make for very intensive control programs even in sophisticated production systems.

Moreover, flower thrips have an indiscriminate taste for a range of flower species, making them all the more ubiquitous across different indoor production systems.

Flower thrips chemical control programs call for diverse chemical class rotations in order to prevent pesticide resistance development. Classes of labeled pesticides for thrips control in greenhouses include but are not limited to carbamates, pyrethroids, organophosphates, and neonicotinoids. Bio-insecticides derived from plants and bacteria are also available for thrips control in the greenhouse, and beneficial insects are commercially available for thrips control

(Enkegaard and Xu, 2009). Typically, predatory mites, specifically Amblyseius cucumeris and

Amblyseius swirskii, are commonly used for control of first and second instar larvae of thrips before below-ground pupation. Alternatively, Orius insidiosus is also sold as a means of biological control of flower thrips in larval and adult form, although this is less commonly used on a large scale because Orius is more expensive, often more than ten times the cost of predatory mites on a per-predator basis (Table 7, Appendix). Research discussed in this manuscript demonstrate a potentially valuable predation response from Orius on flower thrips on at least a few commercially grown greenhouse crops (Baez et al, 2004, Chow et al, 2010, Fransen and

Tolsma, 1992, Funderburk et al, 2000, Shipp and Wang, 2003) 31

As evidenced by the predation arena bioassays in Chapter 2 of this manuscript, in controlled environments there is a direct predation correlation between the minute pirate bug

Orius insidiousus and at least three species of flower thrips that occur in this region, tobacco thrips, Frankliniella fusca, western flower thrips, Frankliniella occidentalis, and eastern flower thrips, Frankliniella tritici. Establishing a usable arena context in the greenhouse was the goal of the next research phase.

3.2 Materials and Methods

3.21 Test Plants

The details of plant selection and cultivation prior to initiation of the experiment are identical to that which was summarized in Chapter 2. A treatment type was included to compare thrips mortality in insecticide-treated geraniums with thrips mortality in the control groups and the Orius groups. A repetition was done in fall of 2020, and a second repetition was done in spring 2021.

The day the experiment was initiated in both the fall 2020 and spring 2021 repetitions, geraniums were chosen randomly from the geranium crop for the insecticide treatment cages.

This group of geraniums was given a drench application of a commercially available pesticide labeled for thrips, Flagship 25WG insecticide (active ingredient thiamethoxam, IRAC Group 4A, neonicotinoid). The application rate used for the drench was 8.5 oz of product per 100 gallons water, as directed by the product label.

3.22 Arenas

The arenas were designed and built in-house with the goal of being functionally similar to commercially available insect cages, which are typically comprised of a lightweight frame 32 covered by insect proof netting of varying grades. The cages for this experiment were rectangular, sized to 24” X 18” X 15” so that they would be able to house three potted, flowering geraniums. The frames of the cages were constructed with ½” PVC piping, after which the four

24” sides of the cages were covered with fine-grade, thrips-proof insect netting (BioQuip

Products, No-Thrips insect screen, 150 µm X 150 µm). The netting was attached to the PVC frame with screws, then hot glue was used to seal the entirety of the insect netting seam to the

PVC frame (Figure 8).

The two 18” X 15” “ends” of the cages were left open initially so that an irrigation line could be run through the cage. Pots were connected within the cages for watering during the experiment (Figure 9). After connecting the pots to the irrigation line, polyethylene covers that fit the “ends” of the cages were cut to size and hot-glued to the bottom of the cage frames around the irrigation lines (Figure 10). Three 4.5” potted geraniums were used per cage in the fall 2020 portion, and a single 6” potted geranium was used per cage in the spring 2021 portion. After the potted geraniums were placed into the cages and connected to irrigation, the polyethylene covers were hot-glued to the cage frame, sealing all of the edges. Twelve cages were created, one of which contained a remote temperature sensor to monitor heat buildup within the cages.

3.23 Insects

3.231 Fall 2020

WFT/EFT were collected from the various plants in the landscape at the Virginia Tech

HRAREC, and were used for the fall 2020 greenhouse repetition.

Due to the difficulties associated with assessing Amblyseius predation of thrips in the fall

2020 bioassay portion of this research, predatory mites were omitted from Chapter 3 entirely. 33

Orius alone was used as a predator in Chapter 3. They were purchased from Plant Products, Inc. and arrived the day prior to initiation of the fall repetition.

Twenty four adult WFT/EFT were released per cage. 12 Orius were released per Orius cage to achieve a 1:2 predator:prey ratio as in Chapter 2.

3.232 Spring 2021

WFT/EFT were widely available throughout the Virginia Tech HRAREC landscape for collection in spring 2021. As was done in the lab bioassays, flowers were collected into collection bags, and WFT/EFT were aspirated from the bags into holding containers in preparation for release into the cages.

The Orius used in this experiment were ordered from Plant Products, Inc., and arrived the same day that the spring repetition was initiated.

Thrips numbers were increased from 24 adults per cage in the fall 2020, to 300 adult

WFT/EFT released per cage in spring 2021. Orius numbers were also increased to 130 per Orius cage. It was estimated that roughly 10-15% of the thrips would die of causes unrelated to predation by Orius, so releasing 130 Orius would allow for an approximate 1:2 predator:prey ratio.

3.24 Experimental Design

3.241 Fall 2020

The greenhouse contained four long tables running parallel with one another. Each table corresponded to a replication (block) (Figure 11). Each of the four replications (blocks) within this repetition contained three treatments (cages), all of which contained thrips: An Orius+thrips cage, a thrips-only control cage, and an insecticide-treated thrips cage. Each cage contained three 34 potted geraniums, and each pot contained four designated treatment sub-units in which thrips mortality would be assessed following the treatment period: Flower #1, Flower #2, Remaining

Flower Tissue, and All Vegetative Tissue. In total, each cage contained 12 measurements, for a total of 36 across each replication (Table 8, Appendix). The cage layout of each replication was randomized to account for unforeseen environmental factors.

3.242 Spring 2021

Replication and treatment design were the same for spring 2021. However, in an effort to simplify the experiment at the pot-level, only one 6” potted, flowering geranium was used per cage. Each pot was designated four treatment sub-units, in which thrips mortality was assessed: flower tissue, vegetative tissue, the geranium pot, and the container that was used to release the thrips into the cage (this container was left in the cage after release). In total, each cage contained four measurable sub-units, and each replication contained 12 measurable sub-units (Table 9,

Appendix).

Figure 8. Thrips-proof netting attached to cage frames with screws and hot glue. 35

Figure 9. Cages left open on both ends to allow irrigation line to run through cage and connect to pots.

Figure 10. Polyethylene end-covers for the cages were sealed around the irrigation pipes with hot glue. 36

Chapter 3 Greenhouse Experimental Layout

(North) Greenhouse entry/exit

TABLE/BLOCK 1 TABLE/BLOCK 2 TABLE/BLOCK 3 TABLE/BLOCK 4 Air intakeAir vent intake Air vent

OriusOrius Insecticide Orius  Treatment Treatment Treatment Treatment 101 201 301 401

Control Insecticide Orius Control  Treatment Treatment 102 202 302 402 Exhaust Fan Exhaust

Insecticide Control Control Insecticide  Treatment Treatment 103 203 303 403

Greenhouse entry/exit (South) Figure 11. Experiment layout within greenhouse, same for Fall 2020 and Spring 2021 repetitions. 3.25 Procedure

3.251 Fall 2020

One day before initiation of the fall repetition, via aspirator the Orius and the adult

WFT/EFT were aliquoted into small, sealed plastic condiment cups for staging, per the quantities needed for the cages (four Orius cups, each containing 12 Orius, 12 thrips cups, each containing

24 adult WFT/EFT). The thrips were collected from landscape plants at the Virginia Tech

HRAREC the day prior and stored in environmental control chambers until initiation of the experiment. The Orius were aspirated directly out of the container in which they had been 37 shipped. After the insects were aliquoted into the plastic cups, the cups were then placed in the environmental control chambers.

The day the experiment was initiated, geraniums were randomly chosen from the crop for the cages. Twelve of the geraniums were given a drench application of a commercially available pesticide labeled for thrips, Flagship 25WG insecticide (active ingredient thiamethoxam, IRAC

Group 4A, neonicotinoid). The application rate used for the drench was 8.5 oz product per 100 gallons water, as directed by the product label. After the drench was applied and the drenched pots had ceased draining (30 minutes), all of the geraniums were placed into their respective cages and hooked up to the spaghetti-tube irrigation spritzers.

Following the insecticide drench, one of the two polyethylene faces on each cage was sealed completely, and the other was sealed on two sides, leaving a corner flap on each cage hanging open. The thrips cups were removed from the environmental control chambers, the tops of the cups were opened, and the cup was placed upside down on top of the geranium flower closest to the opening, and the cup was left there for the duration of the experiment. The flower on which the cup was placed was marked with a white piece of tape around the flower stem to designate that flower as the release point for each cage. The release flower was designated as

Flower 1. Designating a release point would give insight to the degree of insect movement throughout the plants into the other designated sub-units (Flower 2, Vegetative Tissue, etc.).

After the thrips cup was placed onto Flower 1, the polyethylene flap was closed and sealed with hot glue to the cage frame.

Approximately three hours after the thrips were sealed into the cages, the Orius staging cups were removed from the environmental control chambers and transported to the greenhouse.

Because the cages had already been sealed, the polyethylene had to be cut open to release the 38

Orius. Using a razor blade, an “L” was cut, approximately 6” X 6”, into the polyethylene directly adjacent to Flower 1. This created an opening suitable to insert the Orius cup, tip the cup upside down, dump the 12 Orius in the cup onto Flower 1, and remove the cup. The “L” shaped slice in the polyethylene was then immediately sealed with two pieces of greenhouse repair tape. This type of tape is made of polyethylene with adhesive on one side, and is commonly used to repair rips and cuts in greenhouse plastic. The tape created a reliable seal in the plastic that prevented insects from escaping.

After seven days, the cages were opened one by one, starting with the control cages. Each cage had 12 collection buckets assigned to it: four buckets for each plant, one bucket for each measurable sub-unit of the plant (Flower 1, Flower 2, Vegetative Tissue, etc.). The flowers and vegetative tissues were cut and placed into their respective buckets, and the buckets were taken to the lab to be analyzed for the presence of dead or living thrips and Orius.

3.252 Spring 2021

The day before initiation of the spring repetition, flowers were collected from the landscape at the Virginia Tech HRAREC in plastic, gallon-sized resealable bags, and the bags were placed upright in the lab, allowing the thrips within the bag to begin migrating upward toward the seal. Because of the warming weather, the thrips populations in the landscape was flourishing, and the bags contained ample amounts of WFT/EFT. After the WFT/EFT had begun migrating toward the top of the collection bags, the bags were opened one by one, and WFT/EFT were aspirated directly out of the bags into pint-sized buckets that had been retrofitted to an aspirator. These holding buckets were treated with an ionizing gun prior to use to eliminate static electricity. Three hundred adult WFT/EFT were aspirated into each of 12 buckets, one for each cage. The buckets were then placed into the environmental control chambers. 39

The day the experiment was initiated, geraniums were randomly chosen from the 6” geranium crop. Four of these geraniums were given a drench application of a commercially available pesticide labeled for thrips, Flagship 25WG insecticide (see details above).

As in fall 2020, one of the two polyethylene faces on each cage was then sealed completely, and the other was sealed on one side, leaving a corner flap on each cage hanging open. The pint-sized thrips holding buckets were removed from the environmental control chambers, tipped upside down above the plant and lightly tapped to dislodge thrips. The bucket was then placed adjacent next to the geranium pots so the two were touching, and the plant canopy was hanging into the buckets, allowing the remaining thrips in the bucket unfettered access into the plant canopy. The bucket was left there for the duration of the repetition. The polyethylene was then sealed with hot glue.

During the three-hour interim period between the thrips release and Orius release, the

Orius were aspirated directly from their shipping container into four pint-sized holding buckets, at 130 Orius per bucket. This was done immediately before release to mitigate the likelihood of cannibalism occurring within the buckets and reducing Orius numbers before release. The buckets were treated with an ionizing gun to eliminate static electricity prior to use. At three hours after thrips-release, the Orius were released into the cages in the same fashion as in fall

2020. An “L” was cut into one of the polyethylene faces on each cage, the bucket was inserted into the cage and tipped upside down, and the Orius were deposited onto the geranium. The bucket was then removed and the “L” sealed with greenhouse repair tape.

After seven days, the cages were opened one by one, starting with the control cages. Each cage had four collection buckets assigned to it: one bucket for flowers, one bucket for leaf tissue, one bucket for the geranium pot, and one bucket into which the thrips release bucket would be 40 placed. The flowers and vegetative tissues were cut and placed into their respective buckets, the pot and release buckets were collected, and the buckets were taken to the lab to be analyzed for the presence of dead or living thrips and Orius.

3.26 Data Analysis

Data sets were subjected to an ANOVA using a generalized mixed linear model (PROC

MIXED, SAS 2011) to determine any effects of the treatments. The Tukey-Kramer method was also used to separate treatment means post ANOVA, using the number thrips counted alive at the end of the experiment as the response variable. When needed, data were also log-transformed to comply with normality. Back-transformed means and standard errors are presented in the bar graphs.

3.3 Results

3.31 Fall 2020

The control cage buckets were assessed first for the presence of living adult thrips. On average, 7.5 adult WFT/EFT were found per cage, 3.5 living and four dead. Second, the Orius cage buckets were assessed, in which on average 6.3 adult WFT/EFT were found per cage. Of the 6.3, on average 2.8 were living and 3.5 were dead. Due to the extraordinary amount of time and labor necessary to assess the buckets from each cage, coupled with the low results of the control and Orius cages, the decision was made not to assess the insecticide treatment cages, and to regroup in order to improve the procedure for the spring 2021 repetition. Data analysis was inconclusive for the fall 2020 repetition and as such not included here. 41

3.32 Spring 2021

The average number of living adult WFT/EFT across the control cages was 10.8 out of

300 released, and they were predominantly found in the flower tissue (Table 10, Appendix). The average number of dead adult WFT/EFT in the control cages was 94.8; however, the thrips- release buckets contained a large proportion of the dead thrips in the control cages. In the Block

1 control, 69% of dead adults were found still in the release bucket. In the Block 2 control, 33% of dead adult thrips were in the release bucket. In Block 3, 47% of the dead adult thrips were in the release bucket, and in Block 4, 49% of dead adult thrips were in the release bucket. The majority of remaining dead thrips were found in the vegetative tissue in each control cage.

The average number of living WFT/EFT nymphs found across the control cages was

30.5. In the Block 1 control, 90% of those nymphs were found in the flower tissue and the remaining nymphs were found in the leaf tissue. In the Block 2 control, 74% of those nymphs were found in the flower tissue and the remaining nymphs found in the leaf tissue. In the Block 3 control, only 7% of those nymphs were found in the flower tissue and the remaining were found in the leaf tissue. In the Block 4 control, 24% of the living nymphs were found in the flower tissue, with the remaining found in the leaf tissue.

The average number of living adult WFT/EFT across the Orius cages was 3.5 out of 300 released. The average number of dead adult WFT/EFT in the Orius cages was 93.3. As in the control cages, a significant proportion of those dead adult thrips were found in the thrips-release buckets. In fact, on average 47% of the dead thrips found per cage were found in the release the bucket. The majority of the remaining dead thrips were found predominantly in the vegetative tissue. 42

Only one out of the four Orius cages in the spring 2021 greenhouse experiment contained any living WFT/EFT nymphs: Block 3, in which only 4 WFT/EFT nymphs were found in the flower tissue.

There being a noticeable different between the number of living thrips

(adults+immatures) in the control cages versus the Orius cages, statistical analysis was run on total living thrips under those two criteria (DF=1,3; F=12.61; P<0.0380). The control cages averaged 42.3 total living thrips per cage, while the Orius cages averaged 4.5 total living thrips per cage, with a standard error value of 6.1 (Figure 12).

Figure 12. Average number of all living thrips (adults+immatures) in Orius+adults and Control.

The numbers of living and dead adult Orius were quite low across all blocks. On average, only 8.3 living Orius out of the 300 released were counted per cage. However, in three of the four cages, Orius nymphs were identified: six in Block 1, six in Block 2, and 18 in Block 3. 43

Going into the spring 2021 repetition, the planned counting criteria was adult thrips. Given the low counts of living adult thrips in the control cages (11.8 on average), the extraordinary amount of time and labor needed to assess the cage buckets, and the similarly low results from the fall

2020 repetition, the decision was made after assessing the control cage buckets and Orius cage buckets not to assess the insecticide cage buckets.

3.4 Discussion

Although the fall 2020 greenhouse repetition did not provide viable data for analysis, it did provide information that was helpful in framing the spring 2021 repetition procedure, which in turn did provide useful data. The repetitions collectively contribute to a successfully developing protocol, and demonstrate the potential for using Orius in future biocontrol research.

The temperature and humidity levels monitored within the cages (data not presented) did not become elevated relative to the conditions outside the cages, demonstrating that the cage design did not negatively impact the growing environment.

Far too little thrips were utilized in the fall 2020 repetition. There was no access to a viable colony, and the outdoor populations from which to collect were far below what was necessary for the experiment. In spring, thrips populations were sufficient in the landscape, and many more could be collected for the experiment. Though 300 adult thrips were used per cage in the spring repetition, we recommend increasing that number as much as is practical in future research projects.

In the second repetition we hypothesized that approximately 10-15% of the thrips population would die during the transfer process from causes unrelated to predation and likely related to being aspirated multiple times and transported. This was inferred based on our lack of 44 success rearing thrips in the laboratory colonies between fall of 2020 and spring 2021. Thus, in an effort to achieve a 1:2 predator-to-prey ratio, we introduced 130 Orius into the cages rather than 150. On average, out of 300 thrips released per Orius cage, 42.9 were found dead in the release bucket. This equates to 14.3% release bucket mortality per cage. Out of the 300 thrips released per control cage, an average of 47.3 thrips were found dead in the release buckets, equating to an average of 16% bucket mortality per cage. This appears to confirm our approximate estimate of 10-15% thrips mortality for reasons unrelated to predation.

The results of the second repetition show a significant difference in the amount of total living thrips nymphs and adults between the control cages and the Orius cages. It is likely that

Orius preyed upon any thrips life -stage present, lowering thrips densities in the cages with this predator present. This experiment showcased the potential of using Orius to reduce thrips populations in geranium crops under greenhouse conditions.

Moreover, Orius nymphs were also identified in the Orius cages, which points to the possibility that Orius were feeding on thrips sufficiently to begin oviposition. This may potentially indicate that Orius not only suppresses thrips, but also proliferates in their presence.

Conclusion

Thrips are a major, global, economically damaging pest in greenhouse production. While intensive pesticide rotation programs can be effective in management of thrips, biocontrol research shows promise in the use of beneficial insect predators to manage greenhouse pests including thrips, without the danger of insecticide resistance buildup. There is however still much work to be done. The existing body of research only applies to a miniscule fraction of 45 existing insect species, commercially produced plant species and environmental contexts. This makes new biocontrol research on economically important plants critical.

Greenhouse geraniums are a staple in spring annual greenhouse production virtually everywhere, and well known as targets for thrips infestation. Our research is the first effort to assess Orius predation of thrips on greenhouse geraniums. The results of our bioassay experiments illustrate that Orius can prey upon multiple species of thrips on geraniums leaves and florets. Further, the results of our greenhouse cage work show that Orius may not only inhibit thrips development on geraniums, but also oviposit and develop new Orius generations on geraniums in the presence of abundant prey. While further research is necessary to shore up our greenhouse cage experiments, ultimately, our results show that Orius could be harnessed to prey upon thrips in geraniums. Ultimately, this research effort could be considered as a foundational piece for further investigation on biocontrol strategies for thrips in greenhouse-produced geraniums.

46

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4. Chow A, Chau A, Heinz KM (2010). Compatibility of Amblyseius (Typhlodromips) swirskii (Athias-Henriot) (Acari: Phytoseiidae) and Orius insidiosus (Hemiptera: Anthocoridae) for biological control of Frankliniella occidentalis (Thysanoptera: Thripidae) on roses, Biological Control, Volume 53, Issue 2, 2010, Pages 188-196, ISSN 1049-9644, https://doi.org/10.1016/j.biocontrol.2009.12.008. (https://www.sciencedirect.com/science/article/pii/S1049964409003211)

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6. Enkegaard A, Xu X (2009). Prey preference of Orius sauteri between western flower thrips and spider mites. Entomologia experimentalis et applicata. 2009;132(1):93-98. doi:10.1111/j.1570-7458.2009.00867.x

7. Frank S., Baker J. (2009). Entomology Insect Notes: Western Flower Thrips. North Carolina State Cooperative Extension Publication, July 1, 2009; Revised November 13, 2020.

8. Fransen JJ, Tolsma J (1992). Releases of the minute pirate bug, Orius insidiosus (Say) (Hemiptera: Anthocoridae), against western flower thrips, Frankliniella occidentalis 47

(Pergande), on chrysanthemum. Mededelingen- faculteit landbouwwetenschappen rijksuniversiteit gent. 1992;57(2):479-479.

9. Funderburk J, Stavisky J, Olson S (2000). Predation of Frankliniella occidentalis (Thysanoptera: Thripidae) in field peppers by Orius insidiosus (Hemiptera: Anthocoridae). Environmental entomology. 2000;29(2):376-382. doi:10.1603/0046- 225X(2000)029[0376:POFOTT]2.0.CO;2

10. Herring, JL (1966). The genus Orius of the Western Hemisphere (Hemiptera: Anthocoridae). Annals of the Entomological Society of America. 59: 1093-1109.

11. Meiracker RAF, Sabelis MW (1999). Do functional responses of predatory arthropods reach a plateau? A case study of Orius insidiosus with western flower thrips as prey. Entomologia experimentalis et applicata. 1999;90(3):323-329. doi:10.1046/j.1570- 7458.1999.00452.x

12. Ruberson JR, Shen YJ, Kring TJ (2000). Photoperiod sensitivity and diapause in the predator Orius insidiosus (Heteroptera: Anthocoridae). Annals of the Entomological Society of America: 93: 1123-1130.

13. Shipp JL, Wang K (2003). Evaluation of Amblyseius cucumeris (Acari: Phytoseiidae) and Orius insidiosus (Hemiptera: Anthocoridae) for control of Frankliniella occidentalis (Thysanoptera: Thripidae) on greenhouse tomatoes. Biological control. 2003;28(3):271- 281. doi:10.1016/S1049-9644(03)00091-4

14. Sprague D., Funderburk J. (2016). Featured Creatures: Insidious Flower Bug. University of Florida, North Florida Research and Education Center. Publication#: EENY-665, September 2016.

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16. Xu X, Borgemeister C, Poehling H (2005). Interactions in the biological control of western flower thrips Frankliniella occidentalis (Pergande) and two-spotted spider mite Tetranychus urticae Koch by the predatory bug Orius insidiosus Say on beans. Biological control. 2006;36(1):57-64. doi:10.1016/j.biocontrol.2005.07.019

17. Xu X, Enkegaard A (2010). Prey preference of the predatory mite, Amblyseius swirskii between first instar western flower thrips Frankliniella occidentalis and nymphs of the twospotted spider mite Tetranychus urticae. Journal of insect science. 2010;10(149):1- 11. doi:10.1673/031.010.14109 48

18. United States Department of Agriculture National Agriculture Statistics Service (2019). Floriculture Crops, 2018 Summary. ISSN: 1949-0917

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Appendix

REPETITION 1 - LEAF - Fall 2020 REPETITION 1 - FLOWER - Fall 2020 Orius Amblyseius Frankliniella Orius Amblyseius Frankliniella Rep. # Treatment (unsexed) (unsexed) Adult Immature Rep. # Treatment (unsexed) (unsexed) Adult Immature 101 1 4 0 8 101 1 4 0 8 102 2 40 8 102 2 40 8 103 3 4 0 44 103 3 4 0 44 104 4 0 24 8 104 4 0 24 8 105 5 0 24 8 105 5 0 24 8 106 6 0 24 4 4 106 6 0 24 4 4

BLOCK 1 BLOCK 107 7 (Control) 0 0 8 1 BLOCK 107 7 (Control) 0 0 8

Orius Amblyseius Frankliniella Orius Amblyseius Frankliniella Rep. # Treatment (unsexed) (unsexed) Adult Immature Rep. # Treatment (unsexed) (unsexed) Adult Immature 201 1 4 0 8 201 1 4 0 8 202 2 40 8 202 2 40 8 203 3 4 0 44 203 3 4 0 44 204 4 0 24 8 204 4 0 24 8 205 5 0 24 8 205 5 0 24 8 206 6 0 24 4 4 206 6 0 24 4 4

BLOCK 2 BLOCK 207 7 (Control) 0 0 8 2 BLOCK 207 7 (Control) 0 0 8

Orius Amblyseius Frankliniella Orius Amblyseius Frankliniella Rep. # Treatment (unsexed) (unsexed) Adult Immature Rep. # Treatment (unsexed) (unsexed) Adult Immature 301 1 4 0 8 301 1 4 0 8 302 2 40 8 302 2 40 8 303 3 4 0 44 303 3 4 0 44 304 4 0 24 8 304 4 0 24 8 305 5 0 24 8 305 5 0 24 8 306 6 0 24 4 4 306 6 0 24 4 4

BLOCK 3 BLOCK 307 7 (Control) 0 0 8 3 BLOCK 307 7 (Control) 0 0 8

Orius Amblyseius Frankliniella Orius Amblyseius Frankliniella Rep. # Treatment (unsexed) (unsexed) Adult Immature Rep. # Treatment (unsexed) (unsexed) Adult Immature 401 1 4 0 8 401 1 4 0 8 402 2 40 8 402 2 40 8 403 3 4 0 44 403 3 4 0 44 404 4 0 24 8 404 4 0 24 8 405 5 0 24 8 405 5 0 24 8 406 6 0 24 4 4 406 6 0 24 4 4

BLOCK 4 BLOCK 407 7 (Control) 0 0 8 4 BLOCK 407 7 (Control) 0 0 8 Table 1. Experiment plan Fall 2020. 50

Predation Baseline Bioassays (Tobacco Thrips) Predation Baseline Bioassays (Tobacco Thrips)

Date: 4/28/21 Date: 4/28/21

Repetition 1 - Leaf Repetition 1 - Flower

Orius Thrips Orius Thrips Rep. # Treatment(unsexed) (Adults) Rep. # Treatment(unsexed) (Adults) 101 1 4 8 101 1 4 8 102 2 0 8 102 2 0 8 103 3 4 8 103 3 4 8 104 4 0 8 104 4 0 8 105 5 4 8 105 5 4 8 106 6 0 8 106 6 0 8 107 7 4 8 107 7 4 8 108 8 0 8 108 8 0 8 109 9 4 8 109 9 4 8 110 10 0 8 110 10 0 8 111 11 4 8 111 11 4 8 112 12 0 8 112 12 0 8

Predation Baseline Bioassays (Eastern/Western) Predation Baseline Bioassays (Eastern/Western)

Date: 4/29/21 Date: 4/29/21

Repetition 2 - Leaf Repetition 2 - Flower

Orius Thrips Orius Thrips Rep. # Treatment(unsexed)(Adults) Rep. # Treatment(unsexed) (Adults) 101 1 4 8 101 1 4 8 102 2 0 8 102 2 0 8 103 3 4 8 103 3 4 8 104 4 0 8 104 4 0 8 105 5 4 8 105 5 4 8 106 6 0 8 106 6 0 8 107 7 4 8 107 7 4 8 108 8 0 8 108 8 0 8 109 9 4 8 109 9 4 8 110 10 0 8 110 10 0 8 111 11 4 8 111 11 4 8 112 12 0 8 112 12 0 8

Predation Baseline Bioassays (Eastern/Western) Predation Baseline Bioassays (Eastern/Western)

Date: 5/12/21 date: 5/12/21

Repetition 3 - Leaf Repetition 3 - Flower

Orius Thrips Orius Thrips Rep. # Treatment(unsexed)(Adults) Rep. # Treatment(unsexed) (Adults) 101 1 4 8 101 1 4 8 102 2 0 8 102 2 0 8 103 3 4 8 103 3 4 8 104 4 0 8 104 4 0 8 105 5 4 8 105 5 4 8 106 6 0 8 106 6 0 8 107 7 4 8 107 7 4 8 108 8 0 8 108 8 0 8 109 9 4 8 109 9 4 8 110 10 0 8 110 10 0 8 111 11 4 8 111 11 4 8 112 12 0 8 112 12 0 8 Table 2. Experiment Plan Spring 2021. 51

REPETITION 1 - LEAF Collected by Chris Epes on 11/5/20 Orius Amblyseius Frankliniella Remaining Frankliniella Missing Adult Immatures Rep. # Treatment (unsexed) (unsexed) Living Dead Living Dead Adult Immatures 101 1 4 00800 0 0 102 2 4 00008 0 0 103 3 4 00404 0 0 104 4 0 24 160 0 1 0 105 5 0 24 005 1 0 2 106 6 0 24 503 0 0 1

BLOCK 1 BLOCK 107 7 (Control) 0 06200 0 0

Collected by Julie Brindley on 11/5/20 Orius Amblyseius Frankliniella Remaining Frankliniella Missing Adult Immatures Rep. # Treatment (unsexed) (unsexed) Living Dead Living Dead Adult Immatures 201 1 4 00600 2 0 202 2 4 00008 0 0 203 3 4 00404 0 0 204 4 0 24 260 0 0 0 205 5 0 24 005 3 0 0 206 6 0 24 314 0 0 0

BLOCK 2 BLOCK 207 7 (Control) 0 05200 1 0

Collected by Julie Brindley on 11/5/20 Orius Amblyseius Frankliniella Remaining Frankliniella Missing Adult Immatures Rep. # Treatment (unsexed) (unsexed) Living Dead Living Dead Adult Immatures 301 1 4 00800 0 0 302 2 4 00006 0 2 303 3 4 00404 0 0 304 4 0 24 170 0 0 0 305 5 0 24 006 2 0 0 306 6 0 24 042 1 0 1

BLOCK 3 BLOCK 307 7 (Control) 0 03500 0 0

Collected by Chris Epes on 11/5/20 Orius Amblyseius Frankliniella Remaining Frankliniella Missing Adult Immatures Rep. # Treatment (unsexed) (unsexed) Living Dead Living Dead Adult Immatures 401 1 4 00800 0 0 402 2 4 00007 0 1 403 3 4 01304 0 0 404 4 0 24 190 1 0 0 405 5 0 24 004 3 0 1 406 6 0 24 134 0 0 0

BLOCK 4 BLOCK 407 7 (Control) 0 02400 2 0 Table 3. Results of fall 2020 leaf bioassay repetition. 52

Predation Baseline Bioassays - Tobacco Thrips

Date: 4/28/21

Repetition 1 - Leaf - Tobacco Thrips Results - 4/29/21 Orius Thrips Thrips Thrips Thrips Rep. # Treatment (unsexed) (Adults) Living Dead Missing 101 1 4 8 5 3 0 102 2 0 8 7 0 1 103 3 4 8 4 4 0 104 4 0 8 8 0 0 105 5 4 8 1 7 0 106 6 0 8 8 0 0 107 7 4 8 4 4 0 108 8 0 8 8 0 0 109 9 4 8 1 7 0 110 10 0 8 8 0 0 111 11 4 8 0 8 0 112 12 0 8 8 0 0

Predation Baseline Bioassays (Eastern/Western) date: 4/29/21

Repetition 2 - Leaf - Eastern/Western Results - 4/30/21 Orius Thrips Thrips Thrips Thrips Rep. # Treatment (unsexed) (Adults) Living Dead Missing 101 1 4 8 0 8 0 102 2 0 8 7 0 1 103 3 4 8 0 7 1 104 4 0 8 8 0 0 105 5 4 8 0 8 0 106 6 0 8 7 1 0 107 7 4 8 2 6 0 108 8 0 8 8 0 0 109 9 4 8 1 7 0 110 10 0 8 7 1 0 111 11 4 8 1 7 0 112 12 0 8 6 2 0

Predation Baseline Bioassays (Eastern/Western)

Date: 5/12/21

Repetition 3 - Leaf Results - 5/13/21 Orius Thrips Thrips Thrips Thrips Rep. # Treatment (unsexed) (Adults) Living Dead Missing 101 1 4 8 2 6 0 102 2 0 8 8 0 0 103 3 4 8 1 7 0 104 4 0 8 7 1 0 105 5 4 8 1 7 0 106 6 0 8 9 0 0 107 7 4 8 1 8 0 108 8 0 8 8 0 0 109 9 4 8 1 8 0 110 10 0 8 8 0 0 111 11 4 8 3 6 0 112 12 0 8 6 1 1 Table 4. Results of spring 2021 leaf bioassay repetitions 1, 2 and 3. 53

REPETITION 1 - FLOWER Collected by Shannon Bradley on 11/5/20 Orius Amblyseius Frankliniella Remaining Frankliniella Missing Adults Immatures Rep. # Treatment (unsexed) (unsexed) Living Dead Living Dead Adult Immatures 101 1 4 008000 0 102 2 4 000040 4 103 3 4 013010 3 104 4 0 24 71 0 0 0 0 105 5 0 24 00 6 2 0 0 106 6 0 24 40 4 0 0 0

BLOCK 1 BLOCK 107 7 (Control) 0 062000 0

Collected by Chris Epes on 11/5/20 Orius Amblyseius Frankliniella Remaining Frankliniella Missing Adults Immatures Rep. # Treatment (unsexed) (unsexed) Living Dead Living Dead Adult Immatures 201 1 4 017000 0 202 2 4 000000 8 203 3 4 004030 1 204 4 0 24 53 0 0 0 0 205 5 0 24 00 4 1 0 3 206 6 0 24 31 1 2 0 1

BLOCK 2 BLOCK 207 7 (Control) 0 080000 0

Collected by Julie Brindley on 11/5/20 Orius Amblyseius Frankliniella Remaining Frankliniella Missing Adults Immatures Rep. # Treatment (unsexed) (unsexed) Living Dead Living Dead Adult Immatures 301 1 4 017000 0 302 2 4 000010 7 303 3 4 040200 2 304 4 0 24 80 0 0 0 0 305 5 0 24 00 2 1 0 5 306 6 0 24 41 1 1 0 2

BLOCK 3 BLOCK 307 7 (Control) 0 070001 0

Collected by Chris Epes on 11/5/20 Orius Amblyseius Frankliniella Remaining Frankliniella Missing Adults Immatures Rep. # Treatment (unsexed) (unsexed) Living Dead Living Dead Adult Immatures 401 1 4 008000 0 402 2 4 001120 5 403 3 4 013120 1 404 4 0 24 80 0 0 0 0 405 5 0 24 10 4 3 0 1 406 6 0 24 40 1 3 0 0

BLOCK 4 BLOCK 407 7 (Control) 0 080000 0 Table 5. Results of fall 2020 floret bioassay repetition.

54

Predation Baseline Bioassays - Tobacco Thrips

Date: 4/28/21

Repetition 1 - Flower - Tobacco Thrips Results - 4/29/21 Orius Thrips Thrips Thrips Thrips Rep. # Treatment (unsexed) (Adults) Living Dead Missing 101 1 4 8 1 5 2 102 2 0 8 8 0 0 103 3 4 8 0 6 2 104 4 0 8 7 0 1 105 5 4 8 0 8 0 106 6 0 8 8 0 0 107 7 4 8 2 6 0 108 8 0 8 5 0 3 109 9 4 8 4 4 0 110 10 0 8 6 1 1 111 11 4 8 0 8 0 112 12 0 8 8 0 0

Predation Baseline Bioassays (Eastern/Western) date: 4/29/21

Repetition 2 - Flower - Eastern/Western Results - 4/30/21 Orius Thrips Thrips Thrips Thrips Rep. # Treatment (unsexed) (Adults) Living Dead Missing 101 1 4 8 1 7 0 102 2 0 8 7 1 0 103 3 4 8 0 7 1 104 4 0 8 7 1 0 105 5 4 8 0 6 2 106 6 0 8 8 0 0 107 7 4 8 1 7 0 108 8 0 8 8 0 0 109 9 4 8 1 4 3 110 10 0 8 8 0 0 111 11 4 8 1 7 0 112 12 0 8 8 0 0

Predation Baseline Bioassays (Eastern/Western) date: 5/12/21

Repetition 3 - Flower Results - 5/13/21 Orius Thrips Thrips Thrips Thrips Rep. # Treatment (unsexed) (Adults) Living Dead Missing 101 1 4 8 3 7 0 102 2 0 8 8 0 0 103 3 4 8 4 4 0 104 4 0 8 6 3 0 105 5 4 8 3 4 1 106 6 0 8 6 3 0 107 7 4 8 2 6 0 108 8 0 8 7 2 0 109 9 4 8 1 8 0 110 10 0 8 6 4 0 111 11 4 8 0 7 1 112 12 0 8 4 1 3 Table 6. Results of spring 2021 floret bioassay repetitions 1, 2, and 3. 55

Part Number Description Retail Price $

15018 Amblyseius Cucumeris Vermiculite 250,000 (5 L) $87.53 15045 Amblyseius Andersoni 25,000 (1 L) $145.36 15100 BI, Cucumeris Mini Sachet (1000/case)(Hooks) $69.35 15101 BI, Cucumeris Mini Sachet (500/case)(Sticks) $46.79 15102 BI, Cucumeris Mini Sachet (1000/case)(Sticks) $86.85 15103 BI, Swirskii 25,000 (1 L Bottle) $45.90 15104 BI, Swirskii Sachets (500 / case) (Hooks) $163.39 15119 BI, Persimilis 25,000 (16 Oz Bottle) $139.57 15120 BI, Swirskii 125,000 (5 L Bag) $192.21 15121 BI, Swirskii Sachet (500/case)(Sticks) $202.32 15122 BI, Cucumeris Sachet (200/Case)(Hooks) $46.15 15201 BI, Cucumeris 250,000 (5 L) Vermiculite $69.63 16914 BI, Cucumeris 250,000 Sprinkler Bag (5 L) $77.56 16915 Amblyseius Cucumeris Sprinkler 20,000 (1 L) Tube $7.67 16916 Amblyseius Cucumeris Breeder Slow Release Tube $6.91 16917 Amblyseius Cucumeris 20,000 Vermiculite $11.89 17006 Atheta 500 (1 L) $37.32 17007 Amblyseius Cucumeris 250,000 w/Bran (5 L Bucket) $77.56 17014 ABS Mini Cucumeris Sachets (1000/Case)(Stakes) $86.85 17015 Amblyseius Cucumeris 50,000 Sprinkler (1 L) $18.40 17020 Amblyseius Degenerans 500 (30 ml) $46.00 17022 ABS Mini Cucumeris Sachets (500/Case)(Stakes) $46.43 17110 Orius 500 (250 ml) $29.38 17112 Orius 1,000 (250 ml) $58.43 17114 Orius 2,000 (500 ml) $116.29 17115 BI, Orius 1,000 (8 Oz Bottle) $64.15 17134 ABS Mini Cucumeris Sachets (500/Case)(No Hooks) $34.08 17136 ABS Mini Cucumeris Sachets (500/Case)(Hooks) $38.50 17137 ABS Mini Cucumeris Sachets (1000/Case)(Hooks) $70.77 17141 Persimilis SD (sawdust carrier) 10,000 (1 L) $67.94 17142 Phytoseiulus Persimilis 2,000 (250 ml) $18.06 17144 Phytoseiulus Persimilis 25,000 (1 L) $145.22 17148 Swirskii 25,000 (500 ml) $45.90 17200 Swirskii Sachets 500/Case $163.39 17219 Swirskii Breeding System 100 Sachet Bags $50.75 17220 A. Cucumeris 50,000 Vermiculite System (1 L) $26.04 17221 ABS Mini Cucumeris Sachets (1000/Case)(No Hooks) $62.39 17233 Phytoseiulus Persimilis 10,000 $67.94 17235 Steinernema System 600 Million (12 X 50 Million) $225.40 17236 Aphi-Mix System 750 (100 ml) $59.05 17237 Swirskii Long Life System 500 Sachets $220.80 Table 7. 2021 Commercial biocontrol pricelist, Plant Products, Inc., the partnering company for the project (Orius ≈ 6 cents/insect; Amblyseius ≈ 6/100 of a cent per mite).

56

Table 8. Experiment Plan for greenhouse fall 2020. 57

Table 9. Experimental plan for greenhouse spring 2021. 58

Table 10. Results from spring 2021 greenhouse repetition.