Biological Control of Twospotted Spider Mite on Hops in Ohio

Home , Mesostigmata, Phytoseiidae, Spider mite, Tetranychus urticae

Biological control of twospotted spider mite on hops in Ohio

Thesis

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in

the Graduate School of The Ohio State University

Susan Gloria Ndiaye

Graduate Program in Entomology

The Ohio State University

2018

Thesis Committee

Celeste Welty, Advisor

Mary M. Gardiner

Hans Klompen

Elizabeth Y. Long

Copyrighted by

Susan Gloria Ndiaye

2018

Abstract

The twospotted spider mite, Tetranychus urticae (Acari: Tetranychidae), is a key pest on hops. Hop production is a new industry in the Midwestern USA, and little is known about management of T. urticae in this region. During 2017, we conducted an exclusion trial at four hop yards in Ohio, to determine the services provided by predators already present in hop yards, as well as the ability of the combination of predatory mites,

Neoseiulus fallacis (Acari: Phytoseiidae) and Neoseiulus californicus (Acari:

Phytoseiidae), to effectively suppress T. urticae by augmentative releases. To determine the effectiveness of predation by N. fallacis and N. californicus, three treatments were compared: a ratio of two predators per ten adult female T. urticae, a ratio of one predator per ten adult female T. urticae, and a ratio of zero predators per ten adult female T. urticae. Each treatment was established on paired leaves; one leaf was covered with a fine mesh bag and one leaf was left uncovered, in each of 50 replicates. After two weeks, the average number of spider mite motiles on the open leaves that received zero predators was significantly less than the initial ten released per leaf, suggesting that naturally occurring predators are capable of suppressing spider mite populations. The average number of spider mite motiles on the closed leaves that received two predators was also significantly less than ten per leaf, while the average number of spider mite motiles on

ii the closed leaf that received one predator was not less than ten per leaf, showing that a ratio of one predator to five spider mites is effective at reducing spider mite populations.

During 2016, we conducted a trial to determine the efficacy of augmentative biological control to suppress T. urticae populations. Treatments compared were

Neoseiulus fallacis (Acari: Phytoseiidae), and Galendromus occidentalis (Acari:

Phytoseiidae), each released at a high and a low rate, with eight blocked replicates distributed over four hop yards. T. urticae populations were monitored on the cultivar

‘Cascade’ on leaves at a height of one meter above ground and one meter from the top of each plant. When populations reached a threshold of one T. urticae per ten leaves, predatory mites from a commercial insectary were released. Treatment had a significant effect on hop yield, but when we separated it by site, the yields only differed among treatments at two of the three sites. At one site, the yields from the plots treated with the high release rate of N. fallacis and the high release rate of G. occidentalis were greater than the yield of the control treatment. At the second site, the yields from the plots treated with the high release rate of N. fallacis and both release rate of G. occidentalis were greater than the yield of the control treatment.

During 2017, a similar augmentation study was done using earlier and more intense sampling, to ensure early detection of spider mites in a system where mite density varied widely. Treatments compared N. fallacis released at a high rate and a low rate in

17 replicates. Yields did not differ significantly among treatments.

Our studies show that when spider mites are found at low to moderate densities, natural occurring predators are able to suppress their populations in Ohio hop yards.

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Although spider mite populations never reached economically damaging levels, augmentation using predatory mites did affect the yields at two of our eight sites.

Naturally occurring predators were documented to be important in the suppression of spider mite populations and we propose that future studies should focus on biological control by conservation rather than augmentation.

Dedication

This thesis is dedicated to my husband Souleye for all of his love support and to my

amazing daughter Fatime who fills my life with joy.

Acknowledgments

Farmer Collaborators: Jamie Arthur, Dell Dine, Mike Ford, Dan Hoy, Eric

Niceswanger and Dave Volkman

Field Assistants: Basrur Abhijith, Alex Brown , Ariel Fisher, Chad Kramer

Molly Dieterich Mabin and Rebecca Welsh

Statisticians: Akira Horiguchi, Chris Riley and Qian Qian

Funding: OSU’s Integrated Pest Management Program, OSU’s Paul C. and Edna H.

Warner Endowment Fund for Sustainable Agriculture, OSU’s SEEDS Program

and NCR-SARE Graduate Student Grant

Vita

2002………………………………………..…………Diploma, Piscataway High School

Piscataway , NJ

2006……………………………………………………..……...B.S. Biology and French

Juniata College

2009-2013………………………………………………………United States Peace Corps

Kaffrine, Senegal

Fields of Study

Major Field: Entomology

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Table of Contents

Abstract ...... ii Dedication ...... v Acknowledgments...... vi Vita ...... vii List of Tables ...... ix List of Figures ...... xi Chapter 1. Measuring natural enemy impact on twospotted spider mite population growth on hops in Ohio...... 1 Introduction ...... 1 Methods...... 3 Results ...... 6 Discussion ...... 10 Tables ...... 16 Figures...... 20 Chapter 2. Augmentative release of predatory mites to control twospotted spider mites on hops in Ohio ...... 30 Introduction ...... 30 Methods...... 35 Results ...... 41 Discussion ...... 30 Tables ...... 53 Figures...... 62 References ...... 78

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List of Tables

Table 1.1. Statistical results for generalized linear model ...... 16

Table 1.2. Descriptive statistics for spider mite eggs after one week...... 16

Table 1.3. Descriptive statistics for spider mite eggs after two weeks...... 17

Table 1.4. Descriptive statistics for spider mite motiles after one week ...... 17

Table 1.5. Descriptive statistics for spider mite motiles after two weeks ...... 18

Table 1.6. Descriptive statistics for predatory mites after one week ...... 18

Table 1.7. Descriptive statistics for predatory mites after two weeks ...... 19

Table 2.1. Study sites - 2016 - 2017 ...... 53

Table 2.2. Important dates in the 2016 season ...... 54

Table 2.3. Important dates in the 2017 season ...... 55

Table 2.4. Average predatory mite densities per leaf in 2016...... 56

Table 2.5. Average predatory mite densities per leaf in 2017...... 57

Table 2.6. Analysis of variance for effect of treatment on yield, 2016 ...... 58

Table 2.7. Descripitve statistics for combined yields in 2016 ...... 58

Table 2.8. Descripitve statistics for yields at Grandpop’s Hops in 2016 ...... 58

Table 2.9. Descripitve statistics for yield at Little Miami Farms in 2016...... 59

Table 2.10. Descripitve statistics for yield at Ohio Valley Hops in 2016 ...... 59

Table 2.11. Analysis of variance for effect of treatment on yield, 2017 ...... 59

Table 2.12. Descripitve statistics for combined yields in 2017...... 59

Table 2.13. Descripitve statistics for yield at Hopalong Farm in 2017...... 60

Table 2.14. Descripitve statistics for yield at Little Miami Farms in 2017...... 60

Table 2.15. Descripitve statistics for yield at Ohio Valley Hops in 2017...... 60

Table 2.16. Cost comparison - conventional, organic, and biocontrol...... 61

List of Figures

Figure 1.1. Augmentative release plot map - 2017 ...... 20

Figure 1.2 Spider mite egg density per leaf after one week ...... 21

Figure 1.3. Spider mite egg density per leaf after two weeks ...... 22

Figure 1.4. Graph of spider mite egg density after zero, one and two weeks ...... 23

Figure 1.5. Spider mite motile density per leaf after one week ...... 24

Figure 1.6. Spider mite motile density per leaf after two weeks ...... 25

Figure 1.7. Graph of spider mite motile density after zero, one and two weeks ...... 26

Figure 1.8. Predatory mite density per leaf after one week ...... 27

Figure 1.9. Predatory mite density per leaf after two weeks...... 28

Figure 1.10. Graph of predatory mite density after zero, one and two weeks...... 29

Figure 2.1. Plot map - 2016...... 62

Figure 2.2. Seasonal trends in the average number spider mite eggs per leaf - 2016 ...... 63

Figure 2.3. Seasonal trends in the average number spider mite motiles per leaf - 2016 . 64

Figure 2.4. Seasonal trends in the average number spider mite eggs per leaf - 2017 ..... 65

Figure 2.5. Seasonal trends in the average number spider mite motiles per leaf - 2017 . 66

Figure 2.6. Seasonal trends in the average number of predatory mites per leaf - 2016 ... 67

Figure 2.7. Seasonal trends in the average number of predatory mites per leaf - 2017 ... 68

Figure 2.8. Combined hop yields at all sites in 2016 ...... 69

Figure 2.9. Hop yields at Grandpop’s Hops in 2016 ...... 70

Figure 2.10. Hop yields at Little Miami Farms in 2016 ...... 71

Figure 2.11. Hop yields at Ohio Valley Hops in 2016 ...... 72

Figure 2.12. Combined hop yields at all sites in 2017 ...... 73

Figure 2.13. Hop yields at Hopalong Farm in 2017 ...... 74

Figure 2.14. Hop yields at Little Miami Farms in 2017...... 75

Figure 2.15. Hop yields at Ohio Valley Hops in 2017 ...... 76

Figure 2.16. Cost comparison - conventional, organic and biocontrol...... 77

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Chapter 1 Measuring natural enemy impact on twospotted spider mite population growth

on hops in Ohio

1. Introduction

The twospotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), is a major pest on hops Humulus lupulus L., a key ingredient in the beer brewing process.

T. urticae colonizes the hop cones causing them to become dry and brittle with a tendency to shatter, thus lowering yield quality and quantity (Cranham 1985). During a single growing season in the Pacific Northwest, the largest hop producing region in the

United States, growers sometimes use as many as nine different pesticides to control spider mite populations (Piraneo et al. 2015). Due to a plethora of detoxification genes

(Grbić et al. 2011) , a short life cycle, abundant progeny, and arrhenotokous parthenogenesis of T. urticae, populations can rapidly develop acaricide resistance (Helle and Pijacker 1985; Van Leeuwen et al. 2010). There have been 417 reported cases of acaricide resistance in T. urticae involving 93 unique active ingredients, making T. urticae the most pesticide-resistant arthropod on record (Van Leeuwen et al. 2015). As a result, growers look for alternative pest control methods, such as biological control, to manage T. urticae.

Biological control is a pest management technique that uses natural enemies to reduce pest populations. In conservation biological control, growers enhance the 1 activities of natural enemies already present in the system by providing habitat, food, or using only narrow-spectrum, pest-specific pesticides (Ehler 1998). In augmentative biological control, growers introduce natural enemies into the system. There are two types of augmentative biological control: inoculative, in which there is a single release of a few natural enemies, and inundative, in which there are multiple releases of a large number of natural enemies. Inundative releases are sometimes used to manage T. urticae, but inoculative releases are more common (Stinner 1977). Inoculative biological control was first used in 1968 to manage T. urticae populations in greenhouses and it has been increasing in popularity ever since (Van Lenteren and Woets 1988). In greenhouses, inoculative biological control has been shown to offer nearly the same economic benefit as using conventional pesticides (Opit et al. 2009). Despite its success in greenhouses, the use of inoculative biological control is much less common in outdoor crops, although

Pickett and Gilstrap (1986) found it to be a feasible approach to controlling spider mites on field corn.

Hops have made a resurgence in Ohio after almost one hundred years with no recorded production (Hop Growers of America 2016). Pesticide resistance and a greater awareness of ecologically sound pest management practices have resulted in growers’ increased interest in biological control (Attia et al. 2013). Studies done in the Pacific

Northwest show that the predatory mites Galendromus occidentalis Nesbitt (Acari:

Phytoseiidae) and Neoseiulus fallacis Garman (Acari: Phytoseiidae) have potential for use in augmentative biological control in hops (James, 2003b; Pruszynski and Cone,

1973; Strong and Croft, 1995), and growers in this area commonly release them

(DeFrancesco and Murray 2008; Walsh et al. 2015). Despite the known use of these predatory mites, recommendations for release rates and timing vary widely. Because hop production is a new industry in the Midwestern USA, there is limited information on the effectiveness of predatory mites for management of T. urticae is Midwestern hop production.

The goal of this study was to measure the impact of natural enemies on T. urticae population growth. We sought to evaluate the ability of natural enemies already present in Ohio hop yards to suppress spider mite populations. We also wanted to determine the ability of the predatory mite, Neoseiulus fallacis, a potential species for augmentation, to control spider mite populations. We hypothesized that indigenous natural enemies would reduce spider mite populations, but fail to adequately suppress them. We also hypothesized that a higher ratio of N. fallacis to T. urticae would result in better suppression of spider mite populations as compared to a lower predator-to-prey ratio.

2. Methods

2.1 Experimental Design

During June and July of 2017, we conducted an exclusion trial to: (1) determine the services provided by generalist predators already present in hop yards, and (2) evaluate the ability of the predatory mite Neoseiulus fallacis to suppress T. urticae using augmentative releases. We used a randomized complete block design with three treatments: no predator release, release of a low density of predatory mites, and release of a high density of predatory mites. There were 17 blocks containing the three treatments on the hop cultivar ‘Cascade’ distributed among four yards: Hidden Lake Farm (Mt.

Vernon, OH), Hopalong Farm (Howard, OH), Little Miami Farms (Xenia, OH), and Ohio

Valley Hops (Maineville, OH). The plots used for this experiment were the same core plots being used for an augmentative release field trial (Fig. 1.1). Each treatment of predators for the exclusion trial was placed in plots for which the same predator treatment was used in the augmentation trial, e.g. the treatment of one predator per leaf in the exclusion trial was placed in field plots being used for the low rate of predator release in the augmentation trial. Although only one or two predators were released onto each leaf used in the exclusion trial, leaves that received predators in the exclusion study were located on the same plants that also received a release of predators as part of the augmentative release study. The trial was conducted once or twice at each hop yard, for a total of 50 replicates.

The day before we deployed our exclusion trial in the field, we placed sets of ten female spider mites on a small hop leaf in a small Petri-dish and stored them in the refrigerator overnight. We ordered N. fallacis from IPM Labs (Locke, NY). We collected several specimens from each vial to confirm the species. On the same day that we aliquoted the spider mites, we received and distributed the predators with their carrying medium into small Petri-dishes (50 mm x 9 mm), placing approximately ten predators per dish. These were stored in the refrigerator overnight (~8°C) Once in the hop yards, we selected pairs of leaves at a height of one to two meters above ground.

Both leaves in each pair were located on the same plant support string. We removed all visible arthropods from each leaf, and used a small plastic clothespin to attach one small hop leaf containing 10 pre-counted female spider mites. Both leaves in any given pair

4 received the same number of predators. We used a fine paintbrush to transfer the target number of predators from a pre-prepared Petri-dish to the leaf. As soon as the spider mites and predators were placed on the leaves, one leaf in each pair was covered with a one-gallon Trimaco SupertuffTM paint strainer bag. We closed the bags using a twist tie with a strip of cotton batting wrapped around the petiole to cushion it. Each of the three plants in the core plot received the same treatment (Fig. 1.1). Due to uncertainty about the optimal length of the enclosure period, we collected one sample in each plot after one week and the remaining two samples after two weeks. Samples were placed in paper bags, and bags were stored in coolers during transport to the laboratory. We used a dissecting microscope to count the spider mite motiles, spider mite eggs, predator motiles, and predator eggs on each leaf within 6 hours of removing the leaves from the hop plants. We collected all mite predators and stored them in 75% ethanol. We then made slides of these predators and identified them to at least family.

2.2 Statistical Analysis

An initial test of the combined yield data showed a transformation was necessary to fit the ANOVA assumptions. After the data was transformed using a negative binomial, we used a generalized linear model to compare the effect of treatment (number of predators added), status (open or closed), treatment-by-status interaction, and block on the number of spider mite motiles, the number of spider mite eggs, and the number of predators (nb.glm, RStudio version 1.0.153). We used the Mann-Whitney-Wilcoxon

(MWW) test to compare the means of the open and closed samples within each treatment

(wilcox.test, RStudio version 1.0.153). To determine whether or not treatment reduced

5 the number of spider mites, we used the MWW test to compare the mean number of spider mite motiles for each treatment to ten, the initial number of spider mites released on each leaf (wilcox.test, RStudio version 1.0.153).

3. Results

Although we ordered N. fallacis from the insectary, we determined that the predators we received and released were a mixture of Neoseiulus californicus and N. fallacis. The predators collected from leaves when the trial was terminated included N. californicus, N. fallacis, and other species of phytoseiids. In predatory mite density results presented here, all phytoseiid species are combined.

The effect of treatment, status, and block on spider mite eggs, spider mite motiles and predators varied greatly. Our results concentrate on the effect of treatment and status

(Table 1.1).

All of the open and enclosed leaf samples terminated after one week had spider mite eggs on them (Table 1.2; Fig.1.2). There was a significant difference in the mean number of eggs on the open and closed leaves (Table 1.1; z-value = -3.34, P < 0.001) and the average number of eggs on the closed leaves was greater than the average number of eggs on the open leaves in the same treatment. The closed leaf that received zero predators had a significantly greater number of eggs (95.8) than the open leaf (27.5) in the same treatment (W = 148.5, P = 0.01).

Similar to the results after one week, all of the open and enclosed leaf samples terminated after two weeks also had spider mite eggs on them (Table 1.3; Fig.1.3). There was a significant difference in the average number of eggs on the open and closed leaves

(Table 1.1; z-value = -3.00, P = 0.003). There was a significant difference in the average number of eggs on the leaves that received zero predators and the leaves that received two predators (Table 1.1; z-value = -2.13, P = 0.03). In all three treatments, the average number of eggs on the closed leaves was greater than the average number of eggs on the open leaves in the same treatment. This difference was significant for the treatments that received zero predators (closed: 20.7, open: 6.5) and one predator (closed: 18.3, open:

2.8) (Table 1.3; zero predators: W = 732, P = 0.01; one predator: W = 651, P < 0.001).

In all treatments, between the leaf samples collected after one week and the leaf samples collected after two weeks, there was a decrease in the average number of eggs

(Fig. 1.4). The difference was greatest on the closed leaves that received zero predators

(95.8 to 20.7) and the closed leaves that received one predator (78.0 to 18.3) (Table 1.3 and 1.4). The smallest difference was on the closed leaves that received two predators

(23.2 to 11.5) and the open leaves that received two predators (13.2 to 2.5) (Table 1.3 and

1.4)

After one week, the average number of spider mite motiles found on the enclosed leaves was significantly greater than the average number of motiles found on the open leaves (Table 1.1; z = -2.21, P = 0.03). Each treatment had a greater average number of motiles on the closed leaves than on the open leaves of the same treatment (Table 1.4;

Fig. 1.5). This difference was significant in the treatment that received zero predators, where the closed leaves had an average of 27.0 predators and the open leaves had an average of 18.4 predators (W = 139, P = 0.03). Most leaves showed an increase in the number of spider mite motiles from the initial ten released on each leaf, but both the open

7 and the closed leaves that received two predators had an average of less than ten spider mites. This difference was only significantly less than ten on the open leaves of this treatment (closed: V = 37, P = 0.29; open: V = 15.5, P = 0.01).

After two weeks, the average number of spider mite motiles on the open leaves was significantly less than the enclosed (Table 1.1; z-value = -4.10, P < 0.001). The average number of motiles on the leaves that received two predators was significantly less than the number of motiles on the leaves that received zero predators (Table 1.1; z- value = -4.29; P < 0.001). The average number of spider mite motiles on the closed leaves of each treatment was greater than the average number of spider mite motiles on the open leaves of the same treatment (Table 1.5; Fig. 1.6). This difference was significant in both the treatments that received zero predators (26.8 to 5.9) and the treatment that received one predator (13.5 to 5.8), but was not significant in the treatment that received two predators (7.0 to 2.9) (zero predators: W = 748.5, P = 0.007; one predator: W = 678, P < 0.001; two predators: W = 460.5, P = 0.06). All of the open leaves and the closed leaves that received two predators had an average number of spider mite motiles that was significantly less than the initial ten that every leaf received (open and zero predators: V = 112.5, P < 0.001; open and one predator: V = 54, P < 0.001; open and two predators: V = 30, P < 0.001; closed and two predators: V = 86, P =

0.004).

Between week one and week two, the average number of spider mite motiles on most of the leaves decreased, except on the closed leaves that received zero predators, where the number remained almost constant (27.0 to 26.8) (Tables 1.4 and 1.5; Fig. 1.7).

The greatest decrease was seen on the open leaves that received zero and one predator

(zero: 18.6 to 5.9; one: 17.5 to 5.8). The smallest decreases were seen on the opened and enclosed leaves that received two predators (open: 6.8 to 2.9; closed: 9.0 to 7.0).

After one week, the average number of predators on the leaves that received one predator was significantly greater than the average number of predators on the leaves that received zero predators (Table 1.1; z-value = 2.75, P = 0.006). The average number of predators on the enclosed leaves that received one and two predators was greater than the average number of predators on the open leaves in the same treatment, and the average number of predators on the open leaves that received zero predators was greater than the average number of predators on the closed leaves in the same treatment (Table 1.6; Fig.

1.8). This trend continued after two weeks (Table 1.7.; Fig. 1.9); the average number of predators on the enclosed leaf that received one predator was significantly greater than on the open leaves that received one predator (W = 585, P = 0.005).

The trend in average number of predators between week one and week two varied greatly among treatments; depending on the treatment, it increased, decreased or remained almost constant (Tables 1.6 and 1.7; Fig. 1.10). The largest increase was the open leaves that received zero predators (0.6 to 2.0). The average number of predators on the enclosed leaves in that same treatment remained almost constant (0.6 to 0.5). Both the open and enclosed leaves of the treatment that received one predator showed a decrease in the average number predators between weeks one and two, while both the open and enclosed leaves of the treatment that received two predators showed an increase in the average number of predators between weeks one and two.

4. Discussion

The first goal of our study was to evaluate the ability of natural enemies already present in Ohio hop yards to suppress spider mite populations. Our exclusion study found strong evidence of natural predation. The status of the leaf samples in our study

(open or enclosed) had a significant effect on both the average number of spider mite eggs and the average number of spider mite motiles (Table 1.1) resulting in the enclosed leaves in each treatment having a greater average number of eggs and motiles than the open leaves. This difference is evidence of natural predation of spider mites. In our control treatment, this relationship was statistically significant for both eggs and motiles at both one and two weeks, showing that without the introduction of predatory mites, natural enemies present in the hop yard are able to significantly reduce the number of both spider mite eggs and spider mite motiles. After two weeks, the average number of spider mites on the open leaf that received zero predators was significantly less than ten, the initial number of spider mites on each leaf. This means that natural enemies already present in Ohio hop yards not only feed on spider mite motiles and spider mite eggs, but they are able to suppress low to moderate spider mite populations without the addition of predatory mites.

Insects from several orders (Coleoptera, Dermaptera, Diptera, Hemiptera,

Neuroptera, Thysanoptera) are known to prey on both spider mite eggs and motiles

(Chazeau 1985). In other agricultural systems, studies have documented spider mites as prey to generalist predators such as Orius spp,, Oligota kashmirica benefica (Coleoptera:

Staphylinidae) (Takahasi et al. 2001); Feltiella acarisuga (Diptera: Cecidomyiidae),

Chrysoperla carnea (Neuroptra: Chrysopidae) (Abad-Moyano et al. 2009); Conwentzia psociformis (Neuroptra: Coniopterygidae) (García- Marí and González-Zamora 1998);

Stethorus spp. (Coleoptera: Coccinellidae) and Scolothrips spp. (Thysanoptera:

Thripidae) (Abad-Moyano et al. 2009; García- Marí and González-Zamora 1998;

Takahasi et al. 2001). Takahasi et al. (2001) found that populations of spider mite specialists in Japanese pear orchards increased with spider mite populations and almost disappeared when spider mite density became very low.

During the course of this study, we observed many different predators that are known to prey on spider mites. We collected and identified numerous species of predatory mites including several species of phytoseiids other than the species released, and one species from the family Erythraeidae. When collecting our exclusion study samples, we found earwig nymphs and adults in and on the mesh bags. We discarded the data for samples that had earwigs in the bags or had chewed holes in the bags, which occurred in 9% of the bags. While counting the mites on the leaf samples, we found

Orius nymphs. While collecting samples in the field, we observed Orius adults, lacewing eggs and larvae, and lady beetle eggs, larvae and adults.

To measure biological control by predation, one must document the presence of predators and confirm that predators are eating the species of interest. In hop yards in

Washington State, James (2003a) used synthetic herbivore-induced plant volatiles to attract beneficial insects. Although he was able to determine which volatiles attracted beneficial insects, most of the insects attracted were generalists and he did not document predation of spider mites by these beneficial insects. Because most of the species that we

11 observed in Ohio hop yards are generalist predators, their presence in the hop yard does not mean that they were feeding on spider mites, and further studies would be necessary to determine which naturally occurring predators are feeding on T. urticae and suppressing their populations.

The second goal of our study was to determine the ability of predatory mites, N. fallacis and N. californicus, to control spider mite populations. We hypothesized that a higher ratio of predators to T. urticae (1:5) would result in better suppression of spider mite populations as compared to a lower predator-to-prey ratio (1:10). Because both N. fallacis and N. californicus are selective predators that prefer to feed on spider mites rather than on other arthropods or pollen (McMurtry and Croft 1997), we were able to attribute decreases in spider mite populations to their presence.

The enclosed leaves in our trial allowed us to evaluate these predator-to-prey ratios in the absence of natural predation. Although both the treatment that received one predator per ten spider mites and the treatment that received two predators per ten spider mites resulted in a lower number of spider mite eggs and motiles than the control, only the treatment that received two predators per ten spider mites was found to have significantly fewer spider mites than the control (Table 1.1). After two weeks of exposure on the hop plants, all of the treatments on the open leaves were able to suppress the spider mite populations below the original ten spider mites per leaf, but the only treatment on the enclosed leaves that successfully suppressed the spider mite population was the treatment that received two predators per ten spider mites (Fig. 1.6). With the help from naturally occurring predators, a predator-prey ratio of 1:10 was sufficient to

12 suppress spider mite populations, but because a predator-prey ratio of 0:10 was also sufficient to suppress spider mite populations when naturally occurring predators were present, the benefits of augmentation in this system are doubtful. Without the help of naturally occurring predators, a predator-to-prey ration of 1:10 was insufficient to reduce spider mite populations and we conclude that a predator-to-prey ratio of 1:5 is needed to suppress spider mite populations in the absence of naturally occurring predation.

Although we ordered only N. fallacis from a commercial insectary, we received a combination of N. fallacis and N. californicus. N. californicus is a common contaminant of N. fallacis colonies and is known to outcompete their colonies (Carol Glenister, IPM

Labs, and Brian Spencer, Applied Bio-nomics Ltd, personal correspondence). These two species of predatory mites are active across a similar range of temperature and humidity.

Although both are selective predators that feed on spider mites more than insects or pollen, N. fallacis is more of a generalist than N. fallacis (Croft et al. 1998), and its ability to overwinter is questionable (Gotoh 2005; Pratt and Croft 2000). Studies looking at biological control agents sold by commercial insectaries found that the quality of natural enemies received by consumers varied greatly among companies (O’Neil et al. 1998;

Vasquez et al. 2004), and our own verification shows that the species ordered is not always the species that is received. These issues must be factored into recommendations to growers about purchasing biological control agents from insectaries, especially if they are trying to establish long term population in their hop yard.

One confounding variable in our experiment was our inability to remove all arthropods and arthropod eggs from the leaves we used in the trial. Some arthropod eggs

13 and/or motile arthropods avoided detection, resulting in predatory mites and other arthropods on the enclosed leaves. Predatory mites in the phytoseiid family are known to hide in leaf domatia. Domatia are present on hop leaves and we found predatory mites on

30% of the enclosed leaves that received zero predators. Despite this issue, we think it had minimal impact on our results.

In our study, we collected data after both one and two weeks of exposure in hop yards, because we did not know which duration would give us more information. After one week, the average number spider mite egg was much higher than after two weeks.

Eggs have the potential of hatching into motiles that feed on leaves, but not all eggs hatch. Although the average number of eggs on the enclosed leaves that received zero predators was almost 100 per leaf after one week and only 20 per leaf after two weeks, the number of spider mite motiles in this treatment was the same after one week and two weeks. This shows that although spider mites are prolific egg producers, not all of those eggs result in feeding motiles. After one week, the spider mite population was suppressed only in the open treatment that received two predators per ten spider mites, but after two weeks, the spider mite population was suppressed in the enclosed treatment that received two predators per 10 spider mites as well as in all three of the open treatments. As a result of our observation, we conclude that in future enclosure studies, data should be collected only after two weeks to allow time for eggs to hatch and predators to control the spider mite populations.

4.1. Conclusions

Our study aimed to measure the impact of natural enemies on T. urticae population growth in Ohio hop yards. We found that without the addition of predatory mites, predators already present in the hop yards are able to successfully suppress low to moderate spider mite populations. We hypothesize that this suppression is a result of predation by both phytoseiid mites and various predatory insects.

Even though natural predation was able to suppress low to moderate spider mite populations, augmentation may be a useful tool when spider mite populations are higher.

We also found that in the absence of natural occurring predators, a predator-to-prey ratio of 1:10 was inadequate, but a predator-to-prey ration of 1:5 is needed to suppress spider mite populations.

Tables

Table 1.1. Statistical results of generalized linear model comparing the effect of treatment (number of predators added), status (open or closed), treatment-by-status interaction, and block on the number of spider mite motiles, the number of spider mite eggs, and the number of predators. [* p < 0.05; ** p < 0.01; *** p < 0.001]

Treatment Status duration compared to zero predators compared to enclosed one predator two predators open spider mite one week P = 0.63 P = 0.08 P < 0.001*** eggs z-value = 0.48 z-value = -1.77 z-value = -3.34 two weeks P = 0.80 P = 0.03* P = 0.003** z-value = 0.26 z-value = -2.13 z-value = -3.00 spider mite one week P = 0.77 P = 0.12 P = 0.03* motiles z-value = 0.29 z-value = -1.56 z-value = -2.21 two weeks P = 0.80 P < 0.001*** P < 0.001*** z-value = -0.25 z-value = -4.29 z-value = -4.10

predators one week P = 0.006** P = 0.19 P = 0.95 z-value = 2.75 z-value = 1.31 z-value = -0.06 two weeks P = 0.23 P = 0.08 P = 0.49 z-value = 1.12 z-value = 1.78 z-value = 0.69

Table 1.2. Mean, standard deviation, and standard error for spider mite eggs after one week of exposure on hop plants.

mean # treatment status N SD SE of eggs zero predators enclosed 14 95.8 113.3 30.3 zero predators open 14 27.5 71.1 19.0 one predator enclosed 11 78.0 79.4 24.0 one predator open 10 38.8 54.3 17.2 two predators enclosed 13 23.2 27.3 7.6 two predators open 14 13.2 28.0 7.5

Table 1.3. Mean, standard deviation, and standard error for spider mite eggs after two weeks of exposure in hop plants.

mean treatment status N # of SD SE eggs zero predators enclosed 32 20.7 31.0 5.5 zero predators open 35 6.5 15.0 2.5 one predator enclosed 31 18.3 42.8 7.7 one predator open 28 2.8 8.2 1.6 two predators enclosed 28 11.5 31.4 5.9 two predators open 27 2.5 4.9 0.9

Table 1.4. Mean, standard deviation, and standard error for spider mite motiles after one week of exposure in hop plants.

mean # treatment status N of SD SE motiles zero predators enclosed 14 27.0 39.6 10.6 zero predators open 14 18.4 52.9 14.1 one predator enclosed 11 19.6 21.9 6.6 one predator open 10 17.5 20.1 6.4 two predators enclosed 13 9.0 9.0 2.5 two predators open 14 6.8 16.3 4.4

Table 1.5. Mean, standard deviation, and standard error for spider mite motiles after two weeks of exposure on hop plants.

mean # of treatment status N SD SE motiles zero predators enclosed 32 26.8 41.3 7.3 zero predators open 35 5.9 15.7 2.7 one predator enclosed 31 13.5 23.4 4.2 one predator open 28 5.8 19.3 3.7 two predators enclosed 28 7.0 14.6 2.8 two predators open 27 2.9 7.3 1.4

Table 1.6. Mean, standard deviation, and standard error for predatory mites after one week of exposure on hop plants.

mean # of treatment status N SD SE motiles zero predators enclosed 14 0.6 0.9 0.2 zero predators open 13 0.6 1.3 0.4 one predator enclosed 11 2.5 3.0 0.9 one predator open 10 1.8 3.1 1.0 two predators enclosed 13 1.1 1.4 0.4 two predators open 14 0.3 0.6 0.2

Table 1.7. Mean, standard deviation, and standard error for predatory mites after two weeks of exposure on hop plants.

mean # of treatment status N SD SE motiles zero predators enclosed 32 0.5 1.3 0.2 zero predators open 35 2.0 9.3 1.6 one predator enclosed 31 1.7 2.6 0.5 one predator open 28 1.4 5.9 1.1 two predators enclosed 28 1.7 2.8 0.5 two predators open 27 0.9 2.7 0.5

Figures

X X X

Augmentative Treatments Exclusion Treatments

control zero predators

N. fallacis – low release rate one predator N. fallacis – high release rate two predators

Figure 1.1. Augmentative Release plot map – 2017. Each box represents one hop plant. The core plots of the augmentative release study are represented by the deeply shaded boxes. ‘X’ denotes predator release in the augmentation study. The exclusion study used the three plants in core plots of the augmentation study.

a b a a

Figure 1.2. Density of spider mite eggs per leaf after one week of exposure on hop plants. Error bars represent the standard error of the mean. Within each treatment, means marked by the same letter are not statistically different (MWW test).

a a

b a

Figure 1.3. Density of spider mite eggs per leaf after two weeks of exposure on hop plants. Error bars represent the standard error of the mean. Within each treatment, means marked by the same letter are not statistically different (MWW test).

Average # of Spider Mite Eggs per Leaf 120

100 zero predators enclosed 80 zero predators open 60 one predator enclosed one predator open 40 two predators enclosed 20 two predators open

0 Week 0 Week 1 Week 2

Figure 1.4. Density of spider mite eggs per leaf after zero, one, and two weeks of exposure on hop plants.

a a

* a a initial # of spider mites

Figure 1.5. Density of spider mite motiles per leaf after one week of exposure on hop plants. Error bars represent the standard error of the mean. Within each treatment, means marked by the same letter are not statistically different (MWW test). The asterisks indicate treatments with means significantly less than 10, the initial number of spider mites received by each leaf. [* p < 0.05; ** p < 0.01; *** p < 0.001]

** ** *** b b *** a

Figure 1.6. Density of spider mite motiles per leaf after two weeks of exposure on hop plants. Error bars represent the standard error of the mean. Within each treatment, means marked by the same letter are not statistically different (MWW test). The asterisks indicate treatments with means significantly less than 10, the initial number of spider mites received by each leaf. [* p < 0.05; ** p < 0.01; *** p < 0.001]

Average # of Spider Mite Motiles per Leaf 30

20 zero predators enclosed zero predators open 15 one predator enclosed initial # of one predator open 10 spider mites two predators enclosed 5 two predators open

0 Week 0 Week 1 Week 2

Figure 1.7. Density of spider mite motiles per leaf after zero, one and two weeks of exposure on hop plants.

a a

Figure 1.8. Density of predatory mites per leaf after one week of exposure on hop plants. Error bars represent the standard error of the mean. Within each treatment, means marked by the same letter are not statistically different (MWW test).

a a

Figure 1.9. Density of predatory mites per leaf after two weeks of exposure on hop plants. Error bars represent the standard error of the mean. Within each treatment, means marked by the same letter are not statistically different (MWW test).

Average # of Predators per Leaf 3

2.5

2 zero predators zero predators 1.5 one predator one predator 1 two predators two predators 0.5

0 Week 0 Week 1 Week 2

Figure 1.10. Density of predatory mites per leaf after zero, one and two weeks of exposure on hop plants.

Chapter 2. Augmentative release of predatory mites to control twospotted spider mites on hops in Ohio

1. Introduction

After almost one hundred years with no recorded production, production of hops,

Humulus lupulus L., has recently made a resurgence in the state of Ohio. In the 1800s,

Ohio was an exporter of hops, and from 1840 to 1910 Ohio was among the top ten hop producing states in the United States (Lloyd et al. 1918; Rumney 1998). A combination of factors including declining soil fertility, increasing pest and disease pressure, and rising production costs resulted in a decline in hop production throughout the eastern

United States. In 1918, when the state of Ohio enacted laws that prohibited the production and sale of alcoholic beverages, hop production, on a commercial scale, disappeared (Rumney 1998; Edwards and Howe 2012).

The cones of the hop plant are a key ingredient in the beer brewing process because they give beer its bitter taste, help balance the sweetness imparted on the beer by the sweet malts, and act as a preservative. In the past 20 years, the number of breweries in the United States has more than quadrupled from a mere 1,000 breweries in 1996 to an historic high of 6,372 breweries at the end of 2017 (Brewers Association 2018a). This trend has not left Ohio out, and the most recent count shows Ohio has at 177 breweries

(Brewers Association 2018b).

Most new breweries are small, regional craft breweries and the majority are part of a broader food movement that emphasizes buying locally produced food and beverages

(Schnell and Reese 2014). The majority of these breweries are actively looking for ways to decrease their environmental impact and/or increase their sustainability, which includes the sourcing of local and/or organic ingredients (Hoalst-Pullen et al. 2014). In a

2012 survey of regional craft breweries, Hoalst-Pullen et al. (2014) found that breweries favor locally produced ingredients over organic ingredients. This demand for locally produced hops has resulted in a large increase in hop production in states like Ohio where hop production is possible, but little to no hops were being produced.

In addition to being intensively managed, hop yards have a very high initial investment in infrastructure with costs of over $10,000 for the establishment of one acre.

Most Ohio growers begin with one acre with plans to scale up to at least five acres. In

2014, Ohio reported 30 acres of harvested hops, the first recorded commercial production in the state since prohibition. This amount had increased to 70 acres by 2017 (Hop

Growers of America 2018). Hop production in Ohio is expected to continue to increase into the foreseeable future as more and more growers are establishing hop yards throughout the state. Ohio growers’ new interest in hop production led the Ohio State

University Extension and the Ohio Agricultural Research and Development Center to establish hop yards at two of their research facilities, as well as the creation of the Ohio

Hop Growers Guild with a membership of over 70 hop growers (Bergefurd et al. 2014;

Ohio Hop Growers Guild 2018).

Growing hops in Ohio presents many challenges, including arthropod pests like the twospotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), which can cause significant damage to hop plants (Miller 2011). Spider mites overwinter in the crown of the hop plants, and begin to feed upon hop leaves in the early spring. When the cones begin to develop in July, the spider mites migrate into the cones, where they continue feeding. Hop cones are harvested green, when they reach an average of 23 percent dry matter (Lizotte 2013); in Ohio, this is usually in August and early September.

Cones with significant spider mite damage become dry and brittle, with a tendency to shatter, thus lowering yield quality and quantity (Cranham 1985).

Acaricides are commonly used to control spider mites in agricultural crops. In

2008, growers worldwide spent approximately $558 million to control Tetranychus spp. in various crops, with T. urticae being the most common species (Van Leeuwen et al.

2015). Due to a plethora of detoxification genes (Grbić et al. 2011), a short life cycle, abundant progeny, and arrhenotokous parthenogenesis of T. urticae, their populations can rapidly develop acaricide resistance (Helle and Pijacker 1985; Van Leeuwen et al. 2010).

There have been 417 reported cases of acaricide resistance in T. urticae involving 93 unique active ingredients, making T. urticae the most pesticide-resistant arthropod on record (Van Leeuwen et al. 2015).

A combination of pesticide resistance and the high cost of effective acaricides has led growers to look for alternatives to pesticides for managing spider mites. One alternative to pesticides is biological control, a pest management technique that uses natural enemies to reduce pest populations. In inundative biological control, a large

32 number of natural enemies are released and the organisms that are released provide the control. Inundative releases are sometimes used to manage T. urticae, but inoculative releases are more common (Stinner 1977). In inoculative augmentative biological control, there is a single release of a few natural enemies. These natural enemies survive and reproduce, and their progeny will act as the biological control agents. This tactic has been used to manage T. urticae populations in greenhouses since 1968 and has been increasing in popularity ever since (Van Lenteren and Woets 1988). Although inoculative biocontrol has been shown to offer nearly the same economic benefit as using conventional pesticides in greenhouses, its use in outdoor crops is much less common

(Opit et al. 2009), although studies have found that it is a feasible approach to controlling spider mites on field corn (Pickett and Gilstrap 1986) and field grown strawberries

(Rhodes et al. 2006).

In the Pacific Northwest, the largest hops producing region in the United States, studies have shown that the predatory mites Galendromus occidentalis Nesbitt (Acari:

Phytoseiidae) and Neoseiulus fallacis Garman (Acari: Phytoseiidae) are the predatory species with the greatest potential for use in inoculative biological control of T. urticae in hops (James, 2003b; Pruszynski and Cone 1973; Strong and Croft 1995), and growers in this area commonly release them (DeFrancesco and Murray 2008; Walsh et al. 2015).

Despite the known use of these predatory mites, recommendations for release rates and timing on hops have not been developed (Walsh et al. 2015). Because hop production is a new industry in the Midwestern USA, research has not yet been done on the

33 effectiveness of predatory mites on the management of T. urticae on hops production in this region.

There are five species of predatory mites commercially available for control of spider mites: Neoseiulus californicus and Phytoseiulus persimilis (BioBest; Koppert; IPM

Laboratories 2016; Rincon-Vitova 2001), Neoseiulus fallacis, Galendromous occidentalis, and Mesoseiulus longipes (IPM Laboratories 2016; Rincon-Vitova 2001).

Both M. longipes and P. persimilis are specialized predators of species in the genus

Tetranychus; they feed exclusively on spider mites (McMurtry and Croft 1997). G. occidentalis, N. californicus, and N. fallacis are all selective predators that prefer to feed on spider mites, but can also feed on pollen and other insects (McMurtry and Croft 1997).

These selective predators are good candidates for biological control on hops in Ohio, because they have the ability to survive when spider mite populations are low (Pratt and

Croft 2000). Of these selective predators, G. occidentalis and N. fallacis have the ability to overwinter, while the ability of N. californicus to overwinter is questionable (Gotoh

2005; Pratt and Croft 2000). Both G. occidentalis and N. fallacis have the potential to establish sustainable populations of predatory mites in a hop yard, and their effects could be complimentary as G. occidentalis does well in hot, dry conditions (26-37 ºC; > 50 %

RH), while N. fallacis thrives at cooler, more humid conditions (26-37 ºC; 60-90% RH)

(Kramer and Hain 1999; IPM Laboratories 2016; Rincon-Vitova 2001).

As hop production continues to increase in Ohio, growers are looking for sustainable ways to control T. urticae. Although spider mites are only one of many challenges facing Ohio hop growers, they are a key issue that has already caused

34 significant economic loss. To help growers take full advantage of the new demand for locally produced hops, an effective spider mite management program needs to be developed. The goals of our study were to: (1) document seasonal trends of spider mite populations in Ohio hop yards, (2) determine whether G. occidentalis or N. fallacis is more effective at controlling T. urticae on hops under Ohio’s growing conditions, (3) determine if a low release rate is adequate to effectively suppress spider mite populations or if a high release rate is needed, (4) determine whether predatory mites are able to spread from plants on which they were released onto adjacent hop plants, and (5) analyze the cost of augmentative biological control in comparison to conventional and organic chemical control methods.

2. Methods

2.1. Augmentative releases - 2016

In 2016, we conducted an experiment at hop yards in Ohio using the hop cultivar

‘Cascade’. Our experiment was set up in a randomized complete block with eight replicates distributed over four hop yards with a range of one to four replicates per yard

(Table 2.1). In five of the replicates, each plant was trained to climb up one string, and in the other three replicates each plant was trained to climb up onto two strings. We compared five treatments: a control, G. occidentalis at both a high and a low release rate, and N. fallacis at both a high and low release rate. The target low rate was one predator per ten spider mites and the target high rate was one predator per five spider mites. Each treatment plot consisted of three adjacent plants, hereafter referred to as the core plot.

We monitored spider mite populations in the core plots as well as three additional plants

35 on each side, hereafter referred to as the extended plot. Each extended plot of nine plants was separated from any other extended plot by a buffer of at least three plants (Fig. 2.1)

In mid-May, we began weekly sampling to monitor spider mite populations in all extended plots. We collected a sample of two leaves from the bottom of each plant at approximately one meter from the ground. Once plants reached a height of three meters, we collected an additional sample of two leaves approximately one meter from the top of the plant. When plants reached their maximum height at the top supporting wire, which was typically between 4.5 and 6 meters above ground, we sampled leaves one meter below the wire. Leaf samples were brought back to the lab in paper bags held in a cooler.

We used a mite-brushing machine (Leedom Enterprises, Mi-Wuk Village, CA) to remove mites from the leaves and deposit them onto a glass plate coated with dish detergent, which served as an adhesive. Leaves collected from the bottom zone and top zone were always brushed and counted separately. We chose a conservative action threshold of one spider mite per ten leaves based on the spray threshold of two female spider mites used in the Pacific Northwest (Walsh et al. 2015). Before we reached our action threshold of one spider mite per ten leaves, leaves from all nine plants in each extended plot were brushed onto the same plate. After spider mite populations reached our threshold, leaves from each of the nine plants per extended plot were brushed onto separate plates. After brushing, we counted the number of spider mite eggs, spider mite motiles, predator eggs, and predator motiles on half of each plate. We only counted one eighth of the plate if more than 200 spider mites were counted in one section. We also counted other arthropods including aphids, leafhoppers, Tydeidae mites, thrips, Orius, and spiders. We

36 counted and collected all the predatory mites, and stored them in 75% ethyl alcohol. We mounted these mites on glass slides and identified them to species using Phytoseiidae of

North America and Hawaii (Denmark and Evans 2011).

At any given site, when the density of spider mites in any extended plot reached our action threshold of one spider mite per ten leaves, we released predatory mites in all core plots at that site. We ordered predatory mites from IPM Labs (Locke, NY). In our first shipment, the number of G. occidentalis per vial was much lower than expected. We immediately ordered additional G. occidentalis from Rincon-Vitova (Ventura, CA), but the number G. occidentalis we received was again lower than expected, so we made all subsequent orders from IPM Labs and compensated for the reduced number of G. occidentalis by ordering additional quantities. We separated the predators with their carrying medium into small petri dishes (50 mm x 9 mm), one to nine predators per dish, carefully counting the number of predators in each dish. These dishes were stored in a refrigerator overnight (~8°C) and the predatory mites were released the next day.

Immediately before releasing predators, we used a spray bottle to lightly mist two to ten leaves on each plant with water at a height of approximately one and a half meters from the ground. We released the predators by tapping the contents of the petri dishes onto the moist leaves and using a fine paintbrush to remove any mites left in the dish.

We released predators on three plants in each core plot (Fig. 2.1). If a plant was trellised onto two strings, we released the full release rate on each of the strings. For our first release, our rate was limited by the low number of G. occidentalis we received; we used 9 predators per string for our high rate, and 4 predators per string for our low rate. We

37 waited until two weeks after the first release to sample leaves. If spider mite populations had increased in that two-week period, then we made a second release using 20 predators per string for our high release rate, and 10 predators per string for our low release rate, which were our initial target release rates. After the second release, we waited two weeks and then continued our weekly sampling to monitor spider mite populations until harvest.

Immediately before harvest, we increased the sample size to 10 leaves from both the bottom meter and top meter of the plant.

Before harvest, we measured the final height of each plant. As soon as each grower decided that the cones were ready to harvest, we measured the yields for each extended plot. When hand harvesting, we measured the yield for each individual plant.

When using a mechanical harvester, we made two measurements; one measurement was the combined yield for the plants in core plot and the other measurement was the combined yield of the remaining plants in the extended plot. We then calculated the yield as fresh weight per meter of height.

2.2. Augmentative releases - 2017

In 2017, our experimental design was the same as in 2016 with a few adjustments.

We compared only three treatments: a control and N. fallacis, released at a high and a low rate. Our release rates changed: for both our first release and second release, we used 50 predators per string for our high rate and 10 predators per string for our low rate. The target low rate was approximately equivalent to one predator per ten spider mites and the target high rate was one predator per two spider mites. There were 17 replicates distributed among four hop yards, two new sites and two the same as 2016 (Table 2.1).

In seven of the replicates, each plant was trained to climb up one string, and in seven of replicates, each plant was trained to climb up two strings. In three replicates, the plants were never trellised because they were too young . We began our sampling in early May, two weeks earlier than in 2016, and increased the number of leaves collected from the bottom of each plant, at approximately one meter from the ground, to five. We used the same action threshold of one spider mite per ten leaves. If the threshold was exceeded before plants reached a meter in height, then the predators were sprinkled throughout the clump of leaves surrounding the base of the plant. The rest of our experimental procedures remained the same as in 2016.

2.3. Cost comparison

We compared the cost of spider mite management per acre using three common conventional acaricides at the maximum rate and maximum number of applications to the cost two common organic acaricides at the maximum rate for a single application, and to the cost of one augmentative release at both a low an high rate (10 predators per plant,

50 predators per plant). The conventional acaricides were abamectin (Agri-Mek 0.15 EC;

Syngenta Crop Protection LLC), fenpyroximate (Portal XLO 0.4EC; Nichino America,

Inc.), and etoxazole (Zeal 72WP; Valent USA Corp.). The organically-allowed acaricides were potassium salts of fatty acids (Des-X, Certis USA, LLC) and clarified hydrophobic extract of neem oil (Trilogy, Certis USA, LLC).

2.4. Statistical analysis - 2016

An initial test of the yield data for all sites combined showed that a transformation was necessary to fit the ANOVA assumptions of normality and equal variance. After the

39 data was transformed using the cubed root, we used an analysis of variance to compare the effect of treatment, block, and treatment-by-block interaction on yield (aov, RStudio version 1.0.153). We then used the least significant difference to compare the cube root transformed mean yields of each treatment (LSD.test, RStudio version 1.0.153).

An initial test of the yield data at each individual site showed that a transformation was necessary to fit the ANOVA assumptions at two of the three sites.

For uniformity and ease of comparison, we wanted to use the same transformation for each individual site. After trying several different transformation, we decided to use the non-parametric Kruskal-Wallis Rank Sum Test to compare the effect of treatment on yield at each site (kruskal.test, RStudio version 1.0.153). We then used the non- parametric Wilcoxon Rank Sum Test to make pair-wise comparisons of the means for each treatment (wilcox.test, RStudio version 1.0.153).

2.5. Statistical analysis - 2017

An initial test of the yield data for all sites combined showed a transformation was necessary to fit the ANOVA assumptions of normality and equal variances. After using the log transformation, we used an analysis of variance to compare the effect of treatment, block, and treatment-by-block interaction on yield (aov, RStudio version

1.0.153). We then used the least significant difference test to compare the log transformed mean yields of each treatment (LSD.test, RStudio version 1.0.153).

An initial test of the yield data at each individual site showed a transformation was necessary to fit the ANOVA assumptions at one of the three sites. For uniformity and ease of comparison, we wanted to use the same transformation for all three sites.

After log transformation, we used an analysis of variance to compare the effect of treatment and block on yield at each site (aov, RStudio version 1.0.153). For each site, we used the least significant difference test to compare the log transformed mean yields of each treatment (LSD.test, RStudio version 1.0.153).

3. Results

3.1. T. urticae population trends - 2016

Once sampling began on 19 May at all four sites, the date of first detection of T. urticae varied by site. First detection occurred on 19 May at Grandpop’s Hops, on 1 June at Enon Artisan Hops, on 9 June at Little Miami Farms, and on 28 June at Ohio Valley

Hops (Table 2.2).

Spider mite populations began to increase rapidly in mid-June to early-July. We saw no lag time between increases in spider mite egg density and spider mite motile density (Fig. 2.2 & 2.3). The populations of eggs and motiles at the bottom and the top of the plants peaked at the same time (Table 2.2). Peak density of the spider mite motile population ranged from early July to early August. The peak populations in the bottom and top zones of the plants at each site occurred on the same day except at Grandpop’s

Hops where samples were not collected from the top of the plant on the day the population in bottom zone peaked. In the bottom zone, peak population density ranged from 16 motiles per leaf at Little Miami Farms to 45 motiles per leaf at Enon Artisan

Hops. In the top zone, peak population density ranged from 11 motiles per leaf at Ohio

Valley Hops to 37 motiles per leaf at Grandpop’s Hops. All spider mite populations decreased abruptly in late summer. The first spider mite population to crash was at

Artisan Hops in late July. The populations at the other three farms crashed just before harvest. Both the spider mite motile and spider mite egg density showed high variation between plants, and this variation increased as the density of eggs and motiles increased.

3.2. Treatment effects on T. urticae populations over time - 2016

After predator release, we expected to see a decrease in spider mite egg and motile densities in the predator release treatments and a continued increase in the control treatment. After our first release, populations of spider mite eggs and motiles continued to increase, warranting a second release. After our second release, most spider mite populations tended to decrease, however this was seen in our control treatment well as in our predator-release treatments. The treatment with the highest density of spider mites varied among sites: the low release rate of G. occidentalis at Enon Artisan Hops, the control at Grandpop’s Hops, the high release rate of N. fallacis at Little Miami Farms, and the low release rate of N. fallacis at Ohio Valley hops.

3.3. T. urticae population trends - 2017

The date of first detection varied by site (Table 2.3). First detection of spider mites at Little Miami Farms, the older planting at Hidden Lake Farm, and Hopalong

Farm, corresponded with the first sampling, 3 May at Little Miami Farms and 8 May for the other two farms. First detection at Ohio Valley Hops was not until 16 May. The first detection in the new planting at Hidden Lake Farm was on 31 May, immediately after the replacement of dead plants.

At Hopalong Farm and Hidden Lake Farm, we saw a peak in egg density two weeks before motile populations peaked (Fig. 2.4 and 2.5). Mite populations across all

42 sites peaked in late June and July. Peak motile population densities ranged between 6 motiles per leaf at Little Miami Farms and 59 motiles per leaf at Hopalong Farm. Little

Miami Farm’s motile population crashed in late June, although its peak was only 6 motiles per leaf. Hopalong Farm had the highest peak population density at 59 motiles per leaf and its population crashed in late July, about two weeks before harvest. There was no peak or crash at Ohio Valley Hops where the population density remained below two mites per leaf from early May till harvest in early August. Across all sites, mite populations were higher at the bottom of the hop plants than at top of the hop plants.

3.4. Treatment effects on T. urticae populations over time - 2017

After the predator release, we expected to see a decrease in egg and motile densities in the predator release treatments and a continued increase in the control treatment. Although the spider mite populations at Ohio Valley reached the threshold of one spider mite per ten leaves, the motile population went above two motiles per leaf.

We did a first release on 16 June and initially saw a slight decrease in density, followed by a slight increase in density. At the other three farms, after our first release, populations of eggs and motiles continued to increase, warranting a second release. After our second release, most populations tended to decrease, however this was seen in our control as well as in our predator release treatments. (Fig. 2.4 and 2.5; Table 2.2)

The grower at Hidden Lake Farm sprayed the new planting with spirodiclofen after the first release. When the spider mite population in the old planting at Hidden

Lake Farm continued to increase after two releases, the grower sprayed spirodiclofen, which resulted in a drastic decrease in spider mite populations. The grower also sprayed

43 the newer planting after the first release. No acaricides were applied in our plots at the other farms.

3.5. Predatory mites - 2016

In 2016, the shipments of G. occidentalis used for the releases on 1 July contained both G. occidentalis and N. fallacis. The other shipments of G. occidentalis were pure as were all the shipments of N. fallacis.

We found phytoseiids at three of the four sites before our initial release (Fig. 2.6).

Phytoseiid populations peaked in late July and early August, which roughly corresponded to spider mite population peaks (Fig. 2.6). We saw no trend in predator populations among treatments or between sites in relation to our predator releases (Table 2.4).

3.6. Predatory mites - 2017

In 2017, all of our shipments of N. fallacis were contaminated with N. californicus. We found phytoseiids at all sites before our initial release (Fig. 2.7).

Phytoseiid population peaks varied by site (Fig. 2.7). Predator populations peaked at

Hidden Lake Farm before the application of the acaricide. Populations at Little Miami and Hopalong occurred in late June and early July, while predator populations in the upper zone of the plants at Ohio Valley Hops peaked in early June, before the first predator release (Fig. 2.7). The highest peak was at Hopalong farm on 15 July with an average density of 1.5 predators per leaf. This peak corresponded with a peak in spider mite egg and motile populations in the same treatment. We saw no trend in predator populations among treatments or between sites in relation to our predator releases (Table

2.5).

3.7. Yields - 2016

In 2016, we did not harvest at Enon Artisan Hops due to negligible yield.

Comparing the cube root transformed combined yields for all three remaining sites, treatment, block and the treatment-by-block interaction had an effect on yield (Table 2.6.; treatment: df = 4, F = 5.03, P = 0.0001; block: df = 6, F = 25.32, P < 0.0001; treatment x block: df = 23, F = 2.59, P = 0.0007). When we compared the means of the cube root transformed data, we found no statistical difference (Table 2.7; Fig. 2.8).

When we separated the yields by site, treatment did not have a significant effect on the yield at Grandpop’s Hops, but it did have a significant effect on the yields at both

Little Miami Farm and Ohio Valley Hops (GPH: df = 4, χ2 = 7.30, P = 0.121; LMF: df =

4, χ2 = 16.16, P = 0.003; OVH: df = 3, χ2 = 8.74, P = 0.03). Even though treatment did not have a significant effect on the yields at Grandpop’s Hops, the average yields from the plots that received a both a low and high release rate of G. occidentalis were significantly greater than the average yield in the control plots. At Grandpop’s Hops, the average yield from the plots that received a high release rate of G. occidentalis also had a significantly higher average yield than the plots that received a high release rate of N. fallacis (Table 2.8; Fig. 2.9). At Little Miami Farm, the average yields from the plots that received a high release rate of N. fallacis, and the plots that received both a high and low release rate of G. occidentalis were significantly greater than the average yields in the both the control plots and the plots that received a low release rate of N. fallacis

(Table 2.9, Fig. 2.10). At Ohio Valley Hops, the average yields from the plots that received both a low and high release rate of N. fallacis, and the plot that received a high

45 release rate of G. occidentalis were significantly greater than the average yield in the control plot. At Ohio Valley Hops, the average yield from the plot that received a high release rate of G. occidentalis was also significantly greater than the average yield in the plot that received a low release rate of N. fallacis (Table 2.10, Fig. 2.11).

3.8. Yields - 2017

In 2017, we did not harvest at Hidden Lake Farm due to negligible yield.

Comparing the log transformed combined yields for all three remaining sites, only block had an effect on yield (Table 2.11; treatment: df = 2, F = 1.89, P = 0.16; block: df = 10, F

= 9.91, P < 0.0001; treatment x block: df = 20, F = 0.45, P = 0.98). When we compared the means of the cube root transformed data, we found no statistical differences (Table

2.12; Fig. 2.12).

When we separated the yields by site, treatment did not have a significant effect on the log transformed yields at any of the sites. When we compared the log transformed means within each site, there was no statistical difference at any of the sites (Tables 2.13

– 2.15; Fig. 2.13 – 2.15)

3.9. Cost comparison

The cost to treat one acre of hops with conventional acaricides is lower than treating it with organic acaricides, and both are less expensive than using augmentative releases (Table 2.16, Fig. 2.16)

4. Discussion

4.1. Seasonal trends

The first goal of our study was to document seasonal trends in spider mite populations in Ohio hop yards. In other hop growing regions, spider mites overwinter in surface layers of the soil in hop yards and become active after hop plants begin to grow in the spring (Cranham 1985). We found this is also the case in Ohio. Earlier and more intense sampling in 2017 compared to 2016 led to earlier detection of spider mites. We found that phytoseiids can also overwinter in hop yards. In 2016, we did not observe phytoseiids until a few weeks after the first detection of spider mites, but in 2017 we detected at the same time as the spider mites at all four farms.

Spider mite populations on hops are patchy in distribution. Strong et al. (1999) found that throughout the season, 69-75% of hop leaves had neither predators nor spider mites on them. We saw variation in spider mite population density among leaves of the same plant and among plants. Variation was low at the beginning of the season. As the population increased, localized spots of high density led to increased variation among plants. This was most evident in 2017 at Hopalong Farm. Spider mite populations at

Hopalong were well below a mean of five spider mites per leaf until the end of June (Fig.

2.5). On 30 June, we saw a large and sudden increase in populations on two adjacent plants in the core plot in one of our low predator release rate treatments. The bottom zone of one plant had an average of 688 mites per leaf and the adjacent plant had an average of 185 mites per leaf. The plants on either side of these had an average of 14.4 and 2.4 mites per leaf in the bottom zones of the plants. During the same week, the next

47 highest average number of spider mites per leaf was 14.4 mites per leaf. This extreme variation makes it difficult to get a representative population count.

Spider mite population densities were higher on average in 2016 than in 2017, which may have been a result due to cooler and wetter in 2017. An abundance of Eastern flower thrips, Frankliniella tritici, in 2017, could also have been a factor in lower spider mite populations. Eastern flower thrips are important prey for generalist predators such as

Orius insidious, and an abundance of thrips may have contributed to an increase the number of generalist predators, as has been observed by Ramachandran et al. (2001).

We observed variation in hop yard maintenance with respect to weed and disease control; however despite these differences, mite population trends in growth and decline were similar although densities varied widely. There was a significant effect of site on the hop cone yields in 2016, however this was likely a result of differences in spider mite population densities, because population trends were very similar.

Populations of spider mites crashed at all farms before harvest in 2016 and 2017.

The cause of the population crashes has not been determined. One possible explanation for the crash could be the onset of diapause in T. urticae. Day length plays an important role in the induction of diapause in T. urticae. The critical day length for T. urticae is about 14 hours, which does not occur in Ohio until early August (Veerman 1985).

Diapausing female T. urticae are easily identifiable, because of their orange-red coloration, but we observed very few diapausing females at the end of the season, so this explanation is unlikely.

4.2. Augmentative Releases

The second goal of our study was to determine if either G. occidentalis or N. fallacis is more effective at controlling T. urticae on hops under Ohio’s growing conditions. Although 2016 was cool and wet, which would favor N. fallacis, our results suggested that both G. occidentalis or N. fallacis were equally effective in managing spider mite populations. We also found that there is not a reliable commercial source of

G. occidentalis. We order vials of 1000 G. occidentalis, but when we carefully counted the mites from five vials from two different shipments, we found an average 184 per vial with totals ranging from 116 to 305 per vial. As a result, we have determined that although equally effective, G. occidentalis is not a feasible candidate for augmentative biological control unless a reliable source can be found.

The third goal of our study was to determine if a low release rate is adequate to effectively suppress spider mite populations or if a high release rate is needed. In 2016, we found that our augmentative releases had a treatment effect on hop yields. When we examined the results by site, the plots that received a high release rate of both G. occidentalis and N. fallacis had a significantly greater average yield than the control plots at two of the three sites. At one site, we also found that the plots that received a low release rate of N. fallacis also had a significantly greater average yield than the control plot. These three sites had drastically different management practices that may have affected the yields. In 2016, at the site in which there was no significant difference in the yields, most of the hop cones had powdery mildew damage, which may have negatively affected the yield. This same site also had on average higher spider mite populations,

49 which also may have affected the efficacy of the releases. In 2017, the treatment did not have a significant effect on yield, even though our high release rate was two and a half times what it was in 2016.

Weihrauch (2005) compared hop cone yield of untreated plants and acaricide treated plants in the Hallertau region of Germany and found that populations of 90 spider mites per leaf were tolerable at harvest time and posed little or no economic risk. Despite this finding, the currently used threshold in the Pacific Northwest is two female spider mites per leaf in June and early July, and five to ten mites per leaf after mid July (Walsh et al. 2015). Our spider mite populations did not approach an average of 90 mite per leaf at any site in 2016 or 2017, which may explain why we only saw treatment effects on the yields at two out of eight sites. If this experiment were conducted during a hot, dry year with heavy spider mite pressure, one might find different results. It would help the mid-

Western hop industry to have a formal study on the economic injury level of spider mites.

Strong and Croft (1995) found that inoculative releases of predatory mites have the potential to manage spider mites on hops in the Pacific Northwest, but more research was needed to establish a viable protocol. A recent study using augmentative biological control to manage spider mites on hops in New York State found that it failed to control spider mite populations (Tim Weigel, Cornell IPM, personal correspondence).

Successful examples of augmentative biological control in outdoor agricultural crops are rare. A meta-analysis of studies augmentative biological control studies found that only 5 of 31 studies (~15 %) were able to suppress pest populations below target densities

(Collier and Van Steenwyk 2004).

Our fourth goal was to determine if predatory mites were able to spread from plants on which they were released onto adjacent hop plants. We were unable draw any firm conclusions about predator spread, but the set-up of the typical hop yard is not conducive to predator spread. Unlike spider mites that can balloon, predators usually spread by walking. Once the hop plants reach one meter in height, ‘Casacade’ hops maintain a narrow form so that adjacent plants do not touch each other above a meter in height, which prevents movement of predatory mites from one plant to another. Other varieties take a wider form and plants do touch higher off of the ground.

4.3. Cost comparison

Our fifth goal was to analyze the cost of augmentative biological control in comparison to conventional and organic control methods. Our augmentative biological control had minimal biological impact as we found a significant treatment effect on hop cone yield at only two sites of the eight sites. At the same time, implementing augmentative biological control would have a large economic impact because the purchase and release of predatory mites is expensive, with little demonstrated impact on yields. Using acaricides is less expensive an more reliable.

4.4. Conclusions

Although augmentative biological control does have the potential to improve hop yields in Ohio, it is unreliable and the cost outweighs the benefits. During the course of our study, the average number of spider mites per leaf exceed the action threshold of two female mites per leaf, but never came close to the proposed economic threshold of 90 mites per leaf. This suggests that spider mites do not pose an economic problem to hop

51 growers in Ohio in most years. We observed many different predators that are known to prey on spider mites. We collected and identified numerous species of predatory mites including several species of phytoseiids and one species from the family Erythraeidae.

While counting the mites, we found Orius nymphs, lacewing eggs and larvae, and lady beetle eggs and larvae. While collecting samples in the field, we observed Orius adults, lacewing eggs and larvae, and lady beetle eggs, larvae and adults, and earwigs. This natural predator complex seems to be able to keep spider population under the economic threshold and future studies should concentrate on determining which of these predators are feeding on spider mites and then enhancing their activity.

Tables

Table 2.1. Study sites – 2016 and 2017.

Number of replicates Farm 2016 2017 Enon Artesian Hops 1 0 Springfield, OH Grandpop’s Hops 4 0 Marysville, OH Hidden Lake Farm of Mt. Vernon 0 6 Mt. Vernon, OH Hopalong Farm 0 5 Howard, OH Little Miami Farms 2 4 Xenia, OH Ohio Valley Hops 1 2 Maineville, OH total replicates 8 17

Table 2.2. Important dates in the 2016 season. # denotes that a sample from the top of the plant was not taken the week that the population at the bottom of the plant peaked. (GPH – Grandpop’s Hops, EAH – Enon Artisan Hops, LMF – Little Miami Farms, OVH – Ohio Valley Hops)

First Detection Peak Peak First First Second Population Site Reps of Spider (bottom of (top of Harvest Sampling Release Release Crash Mite plant) plant) Motiles GPH 4 May 19 May 19 June 26 July 22 July 22 August 3# August 22 August 22

EAH 1 May 19 June 1 June 26 July 22 July 7 n/a July 22 n/a

LMF 2 May 19 June 9 July 1 July 29 July 15 July 15 August 11 August 11

OVH 1 May 19 June 28 July 1 July 29 July 29 July 29 August 10 August 10

Table 2.3. Important dates in the 2017 season. * indicates the hop yard was sprayed with acaricide prior to the date of the crash. # indicates that mean populations were never higher than one mite per leaf. (HAF – Hopalong Farm, nHLF – new planting Hidden Lake Farm, oHLF – old planting Hidden Lake Farm, LMF – Little Miami Farms, OVH – Ohio Valley Hops)

First Detection Miticide Peak Peak First First Second Population Site Reps of Spider Spray (bottom (top of Harvest Sampling Release Release Crash Mite of plant) plant) Motiles HAF 5 May 8 May 8 June 15 Jul 12 n/a July 12 July 12 July 26 August 8 nHLF 3 May 8 May 31 June 7 July 8 June 23 June 21 n/a July 8* n/a oHLF 3 May 8 May 8 May 18 June 7 June 23 June 21 n/a July 8* n/a

LMF 2 May 3 May 3 June 9 July 7 n/a June 23 # n/a August 9

OVH 1 May 3 May 16 June 16 n/a n/a # # n/a August 10

Table 2.4. Average predatory mite densities per leaf in 2016. (GPH – Grandpop’s Hops, EAH – Enon Artisan Hops, LMF – Little Miami Farms, OVH – Ohio Valley Hops) (NF = N. fallacis; GO = G. occidentalis; low = 1:10 predator-to-prey ratio; high = 1:5 predator-to prey ratio)

Immediately Two weeks Immediately Two weeks Site Treatment before 1st after 1st before 2nd after 2nd Release release release release GPH control 0.00 0.00 - 1.08 NF low rate 0.20 0.00 - 0.75 NF high rate 0.00 0.00 - 0.83 GO low rate 0.09 0.00 - 0.25 GO high rate 0.00 0.00 - 1.00 EAH control 0.00 0.33 0.00 0.00 NF low rate 0.00 0.00 0.00 0.00 NF high rate 0.00 0.33 0.33 0.33 GO low rate 0.00 0.00 0.00 0.00 GO high rate 0.00 0.67 0.00 0.00 LMF control 0.00 0.00 0.17 0.13 ` NF low rate 0.00 0.17 0.17 0.33 NF high rate 0.00 0.17 0.00 0.07 GO low rate 0.00 0.00 0.00 0.03 GO high rate 0.00 0.00 0.33 0.07 OVH control 0.00 0.00 1.00 0.80 NF low rate 0.33 0.00 0.00 0.20 NF high rate 0.33 0.33 0.67 0.07 GO high rate 0.67 0.00 0.00 0.13

Table 2.5. Average predatory mite densities per leaf in 2017. # denotes that the acaricide spray took place between the first and second release. (HAF – Hopalong Farm, nHLF – new planting at Hidden Lake Farm, oHLF – old planting at Hidden Lake Farm, LMF – Little Miami Farms, OVH – Ohio Valley Hops) (NF = N. fallacis; low = 1:10 predator- to-prey ratio; high = 1:2 predator-to-prey ratio)

Two Two Immediately Immediately After weeks weeks Site Treatment before 1st before 2nd acaricide after 1st after 2nd Release release spray release release HAF control 0.00 0.00 0.07 0.13 n/a NF low rate 0.03 0.00 0.00 0.00 n/a NF high rate 0.02 0.00 0.00 0.00 n/a nHLF control 0.00 0.00 0.00 0.09# 0.00 NF low rate 0.00 0.00 0.09 0.00# 0.09 NF high rate 0.00 0.30 0.18 0.04# 0.18 oHLF control 0.05 0.18 0.13 0.58 0.18 NF low rate 0.00 0.13 0.30 0.22 0.31 NF high rate 0.00 0.13 0.28 0.41 0.13 LMF control 0.16 0.00 0.08 0.00 n/a NF low rate 0.00 0.00 0.00 0.00 n/a NF high rate 0.04 0.00 0.00 0.00 n/a OVH control 0.00 0.00 n/a n/a n/a NF low rate 0.27 0.00 n/a n/a n/a NF high rate 0.00 0.00 n/a n/a n/a

Table 2.6. Statistical results for analysis of variance comparing the effect of treatment, block, and treatment-by-block interaction on yields, 2016. The asterisks indicate a significant effect on the yield. [* p < 0.05; ** p < 0.01; *** p < 0.001]

df F-value p-value Treatment 4 5.03 0.001** Block 6 25.32 > 0.001*** Treatment x Block 23 2.59 > 0.001***

Table 2.7. Effect of predatory mite treatment on hop cone yield at all sites combined in 2016: mean yield, standard deviation and standard error.

Treatment N yield (g/m) sd se control 26 64.3 41.8 8.2 N. fallacis low rate 27 57.78 34.8 6.7 N. fallacis high rate 25 63.5 28.1 5.6 G. occidentalis low rate 23 62.6 32.0 6.7 G. occidentalis high rate 27 80.1 34.7 6.7

Table 2.2. Effect of predatory mite treatment on hop cone yield at Grandpop’s Hops in 2016: mean yield, standard deviation and standard error.

Treatment N yield (g/m) sd se control 12 90.8 45.6 13.2 N. fallacis low rate 12 74.4 41.5 12.0 N. fallacis high rate 10 62.3 28.1 8.9 G. occidentalis low rate 11 85.9 32.3 9.7 G. occidentalis high rate 12 102.7 35.3 10.2

Table 2.9. Effect of predatory mite treatment on hop cone yield at Little Miami Farm in 2016: mean yield, standard deviation and standard error.

Treatment N yield (g/m) sd se control 11 34.2 15.3 4.6 N. fallacis low rate 12 35.6 12.5 3.6 N. fallacis high rate 12 55.6 24.7 7.1 G. occidentalis low rate 12 41.3 7.9 2.3 G. occidentalis high rate 12 52.4 8.4 2.4

Table 2.10. Effect of predatory mite treatment on hop cone yield at Ohio Valley Hops in 2016: mean yield, standard deviation and standard error.

Treatment N yield (g/m) sd se control 3 69.1 6.5 3.7 N. fallacis low rate 3 80.0 7.0 4.0 N. fallacis high rate 3 99.0 17.2 9.91 G. occidentalis high rate 3 100.7 11.1 6.4

Table 2.3. Statistical results for analysis of variance comparing the effect of treatment, block, and treatment-by-block interaction on yields, 2017. The asterisks indicate a significant effect on the yield. [* p < 0.05; ** p < 0.01; *** p < 0.001]

df F-value p-value Treatment 2 1.89 0.16 Block 10 9.94 < 0.001*** Treatment x Block 20 0.45 0.96

Table 2.12. Effect of predatory mite treatment on hop cone yield at all sites combined in 2017: mean yield, standard deviation and standard error.

Treatment N yield (g/m) sd se control 35 48.3 38.1 6.4 N. fallacis low rate 33 42.6 37.4 6.5 N. fallacis high rate 32 48.3 30.8 5.4

Table 2.4. Effect of predatory mite treatment on hop cone yield at Hopalong Farm in 2017: mean yield, standard deviation and standard error.

Treatment N yield (g/m) sd se control 5 35.6 16.0 7.2 N. fallacis low rate 5 29.3 14.3 6.4 N. fallacis high rate 5 41.2 14.9 6.7

Table 2.5. Effect of predatory mite treatment on hop cone yield at Little Miami Farms in 2017: mean yield, standard deviation and standard error.

Treatment N yield (g/m) sd se control 24 34.0 17.7 3.6 N. fallacis low rate 24 30.4 17.0 3.5 N. fallacis high rate 23 39.1 17.9 3.7

Table 2.6. Effect of predatory mite treatment on hop cone yield at Ohio Valley Hops in 2017: mean yield, standard deviation and standard error.

Treatment N yield (g/m) sd se control 6 116.3 39.4 16.1 N. fallacis low rate 4 124.53 44.5 22.2 N. fallacis high rate 4 109.8 37.0 18.5

Table 2.7. Cost to treat one acre of hops using conventional acaricides (maximum rate, maximum applications), organically-approved acaricides (maximum rate, one application), and augmentative releases of predatory mites.

Type Treatment Cost Conventional Agri-Mek 0.15 EC $20.04 Conventional Portal XLO $42.12 Conventional Zeal $79.44 Organic Des-X $112.00 Organic Trilogy $210.00 Biological Control 10 predators/plant $238.00 Biological Control 50 predators/plant $595.00

Figures

X X X X X X

X X X

Treatments control

G. occidentalis – low release

G. occidentalis – high release rate N. fallacis – low release rate

N. fallacis – high release rate

Figure 2.1. Plot map – 2016. Each box represents one hop plant. Deeply shaded boxes are the core plots for predatory mite treatments. Lightly shaded boxes are extended plots that include plants monitored for predatory mite spread. ‘X’ denotes predatory mite release.

no high samples taken, majority of plants never reached 3 m tall

not harvested



 Figure 2.2. Seasonal trends in the average number spider mite eggs per leaf, 2016. The error bars indicate the standard error of the mean. Graphs in the upper row show the spider mite density in the top zone of plants, 1 m below the top of the plant. Graphs in the lower row show the spider mite density in the bottom zone of plants, 1 m above the ground. The black arrows indicate predatory mite release dates.

no high samples taken, majority of plants never reached 3 m tall

not harvested



 Figure 2.3. Seasonal trends in the average number spider mite motiles per leaf, 2016. The error bars indicate the standard error of the mean. Graphs in the upper row the show the spider mite density in the top zone of plants, 1 m below the top of the plant. Graphs in the lower row show the spider mite density in the bottom zone of plants, 1 m above the ground. The black arrows indicate predatory mite release dates.

Hopalong Farm

upper portion of new planting, plants decimated by never trellised Japanese beetles in mid-June not harvested not harvested



 Figure 2.4. Seasonal trends in the average number spider mite eggs per leaf. The error bars indicate the standard error of the mean. Graphs in the upper row show the spider mite density in the top zone of plants, 1 m below the top of the plant. Graphs in the lower row show the spider mite density in the bottom zone of plants, 1 m above the ground. The black arrows indicate predatory mite release dates. The red arrows indicate application of an acaricide (spirodiclofen).

Hopalong Farm

upper portion of plants decimated by new planting, Japanese beetles in never trellised mid-June

not harvested not harvested



 Figure 2.4. Seasonal trends in the average number spider mite motiles per leaf, 2017. The error bars indicate the standard error of the mean. Graphs in the upper row show the spider mite density in the top zone of plants, 1 m below the top of the plant. Graphs in the lower row show the spider mite density in the bottom zone of plants, 1 m above the ground. The black arrows indicate predatory release dates. The red arrows indicate application of an acaricide (spirodiclofen).

no high samples taken, majority of plants never reached 3 m tall

not harvested

 Figure 2.6. Seasonal trends in the average number of predatory mites per leaf, 2016. The error bars indicate the standard error of the mean. The graphs in the upper row show the predatory mite density in the top zone of plants, 1 m below the top of the plant. The graphs in the lower row show the predatory mite density in the bottom zone of plants, 1 m above the ground. The black arrows indicate predatory mite release dates.

Hopalong Farm upper portion of plants decimated by new planting, Japanese beetles in never trellised mid-June

not harvested not harvested

 Figure 2.7. Seasonal trends in the average number of predatory mites per leaf, 2017. The error bars indicate the standard error of the mean. Graphs in the upper row show the predatory mite density in the top zone of plants, 1 m below the top of the plant. Graphs in the lower row show the predatory mite density in the bottom zone of plants, 1 m above the ground. The black arrows indicate predatory mite release dates. The red arrows indicate application of an acaricide (spirodiclofen).

a a a a

Figure 2.5. Combined hop yields at all sites in 2016. Error bars represent the standard error of the mean. Means shown are actual yields, but comparisons were made using cubed root transformed data. Means with the same letter are not significantly different (LSD). (NF = N. fallacis; GO = G. occidentalis; low = 1:10 predator-to-prey ratio; high = 1:5 predator-to-prey ratio)

bc ab bc

Figure 2.9. Hop yields at Grandpop’s Hops 2016. Error bars represent the standard error of the mean. Means with the same letter are not significantly different (Wilcoxon Rank Sum Test). (NF = N. fallacis; GO = G. occidentalis; low = 1:10 predator-to-prey ratio; high = 1:5 predator-to-prey ratio)

bc c

ab a a

Figure 2.10. Hop yields at Little Miami Farms 2016. Error bars represent the standard error of the mean. Means with the same letter are not significantly different (Wilcoxon Rank Sum Test). (NF = N. fallacis; GO = G. occidentalis; low = 1:10 predator-to-prey ratio; high = 1:5 predator-to-prey ratio)

bc c

b a

Figure 2.6. Hop yields at Ohio Valley Hops 2016. Error bars represent the standard error of the mean. Means with the same letter are not significantly different (Wilcoxon Rank Sum Test). (NF = N. fallacis; GO = G. occidentalis; low = 1:10 predator-to-prey ratio; high = 1:5 predator-to-prey ratio)

a a

Figure 2.7. Combined hop yields at all sites in 2017. Error bars represent the standard error of the mean. Means shown are actual yields, but comparisons were made using log transformed data. Means with the same letter are not significantly different (LSD). (NF = N. fallacis; low = 1:10 predator-to-prey ratio; high = 1:2 predator-to-prey ratio)

Figure 2.13. Hop yields at Hopalong Farm 2017. Error bars represent the standard error of the mean. Means shown are actual yields, but comparisons were made using log transformed data. Means with the same letter are not significantly different (LSD). (NF = N. fallacis; low = 1:10 predator-to-prey ratio; high = 1:2 predator-to-prey ratio)

Figure 2.8. Hop yields at Little Miami Farms 2017. Error bars represent the standard error of the mean. Means shown are actual yields, but comparisons were made using log transformed data. Means with the same letter are not significantly different (LSD). (NF = N. fallacis; low = 1:10 predator-to-prey ratio; high = 1:2 predator-to-prey ratio)

a a a

Figure 2.15. Hop yields at Ohio Valley Hops 2017. Error bars represent the standard error of the mean. Means shown are actual yields, but comparisons were made using log transformed data. Means with the same letter are not significantly different (LSD). (NF = N. fallacis; low = 1:10 predator-to-prey ratio; high = 1:2 predator-to-prey ratio)

Conventional Acaricides Organic Acaricides Augmentation

Figure 2.9. Cost comparison of conventional acaricides, organically-allowed acaricides and augmentative biological control.

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