1 Towards the Development of An

TOWARDS THE DEVELOPMENT OF AN INTEGRATED PEST MANAGEMENT PROGRAM FOR STRAWBERRIES: INVESTIGATION OF CULTIVAR SUSCEPTIBILITY TO TWOSPOTTED SPIDER MITES (TETRANYCHUS URTICAE KOCH) AND STING NEMATODES (BELONOLAIMUS LONGICAUDATUS RAU) AND INTEGRATION OF THE PREDATORY MITE (NEOSEIULUS CALIFORNICUS MCGREGOR) AND CHEMICAL TACTICS

OMOTOLA G. OLANIYI

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2018

To God and my boys: Ademola, Jedidiah, and Zuriel

ACKNOWLEDGMENTS

I am sincerely thankful to my advisor, Dr. Oscar Liburd for his guidance, advice, and support throughout the course of my study. I appreciate the effort and time he invested in getting this dissertation finished, with useful comments and edits. I would like to thank my committee members, Dr. Carlene Chase, Dr. Dakshina Seal, and Dr. Tesfamariam Mengistu for their constant support, patience, and adequate help. I am grateful to Dr. Chase for her promptness in responding to my emails, providing feedback, meeting with me to clarify issues about my field work, and caring about my personal and family life. I am thankful to all my committee members for the cordial relationships they share among themselves, which provided me with a conducive atmosphere to express myself and conduct the experiments.

I thank Dr. Phil Koehler for his constant encouragement, guidance, and mentorship at the crucial time that I needed it most. I appreciate Dr. Dan Hahn for the constant words of wisdom, advice, and encouragement that kept me going even in difficult times. I am grateful to Dr.

Jennifer Gillett-Kaufman for helping me to be a better writer and the approving smiles that motivated me. I am grateful to Dr. Richard Mankin for his genuine interest in my success, and

Dr. Alex Habteweld for his help with processing nematode samples and identifying some nematodes. I appreciate Dr. Salvador Gezan, Dr. Lazarus Mramba, Dr. Mohammed Sallam, Dr.

Edzard van Santen, and Dr. James Colee of IFAS Statistical consulting for their tremendous help with data analysis and answering all my questions. I thank Dr. Henry Fadamiro for his occasional check-ins with my progress.

I am grateful to my parents, Mr. & Mrs. Dosumu and my siblings for their constant love, support, and prayers to complete my study successfully. I am blessed to be surrounded by wonderful friends such as Mrs. Dorothy Mbuche, Dr. Tamika Garrick, Dr. Teresia Nyoike, Dr.

Eutychus Kariuki, Dr. Jerrine Foster, and Ms. Adeola Oyelade for their help, encouragement, advice, and support at various stages of my Ph.D journey. I thank the students and post-doctoral associates at the Small Fruit and Vegetable IPM Laboratory and the staff at the Plant Science and

Research Education Unit in Citra for their help with planting, harvesting, and protecting the strawberries during freeze periods, especially Mr. Buck Nelson for always picking my calls and providing me with adequate help when needed.

I am grateful to my pastor, Dr. John Cowart of Abiding Faith Christian Church (AFCC) for his prayers, advice, encouragement and mental support. I thank the following church members for their encouragement, prayers, and support; Dr. Marcia Buresch, Dr. Hellena Scott-

Okafor, Ms. Theresa Glaeser, Rev. Lydia Knighton, Ms. Patricia Stubbs, Mrs. Bridget Stokes,

Mrs. Larose Manker, and Mrs. Constance Taylor.

I thank my husband, Mr. Ademola Olaniyi Dada for his help with field work, constant encouragement, patience, and support throughout my program. I acknowledge my son, Jedidiah

Olaniyi for fueling the willpower within me to finish strong. As you would say “I did it”, mummy did it and she is proud that she achieved this milestone with you in her life. Thank you for being the bright spot in my life. Above all, I am eternally grateful to God for the strength and grace that made me complete my PhD program, and beyond even as I go further in my career path.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 9

LIST OF FIGURES ...... 10

ABSTRACT ...... 12

CHAPTER

1 INTRODUCTION ...... 14

Strawberry Production in Florida ...... 15 Justification of the Study ...... 18 Specific Objectives ...... 20

2 LITERATURE REVIEW ...... 22

Biology of the Twospotted Spider Mite, Tetranychus urticae Koch...... 22 Webbing ...... 23 Dispersal ...... 24 Diapause ...... 24 Symptoms of Injury and Management of TSSM ...... 25 Cultural control ...... 26 Chemical control ...... 27 Biological control ...... 28 Predatory Mite, Neoseiulus californicus McGregor ...... 28 Host Plant Resistance ...... 29

3 SUSCEPTIBILITY OF SELECTED COMMERCIAL STRAWBERRY CULTIVARS TO TWOSPOTTED SPIDER MITE, TETRANYCHUS URTICAE KOCH IN ORGANIC PRODUCTION ...... 32

Introduction ...... 32 Materials and Methods ...... 34 Laboratory and Greenhouse Experiments ...... 34 Twospotted spider mite colony ...... 34 Population density of twospotted spider mites ...... 35 Field Trials ...... 35 Study site ...... 35 Plot layout and experimental design ...... 36 Planting ...... 37 Sampling ...... 38 Marketable yield ...... 38

Data analysis ...... 39 Results...... 40 Population Density of Twospotted Spider Mite (TSSM) ...... 40 Greenhouse study ...... 40 Field trials ...... 41 Marketable Yield ...... 43 Discussion ...... 45 Greenhouse Trial ...... 45 Field Experiments ...... 45 Marketable Weight ...... 47

4 DISPERSAL AND MOVEMENT OF NEOSEIULUS CALIFORNICUS MCGREGOR ...... 58

Introduction ...... 58 Materials and Methods ...... 60 Laboratory Experiments ...... 60 Field Trials ...... 61 Data Analysis ...... 64 Results...... 65 Preference of Predatory Mites ...... 65 Field Trials ...... 65 Population density of the predatory mite, Neoseiulus californicus ...... 65 Dispersal of Neoseiulus californicus ...... 67 Discussion ...... 67 Preference of Predatory Mites ...... 67 Population Density of Neoseiulus californicus ...... 68 Dispersal of Neoseiulus californicus ...... 69

5 SUSCEPTIBILITY OF STRAWBERRY CULTIVARS TO STING NEMATODE, BELONOLAIMUS LONGICAUDATUS RAU ...... 78

Introduction ...... 78 Materials and Methods ...... 81 Plant Materials ...... 81 Study Site ...... 82 Experimental Study ...... 82 Sampling ...... 83 Nematode Extraction ...... 83 Marketable Yield ...... 84 Data Analysis ...... 84 Results...... 85 Population Density of Sting Nematode, Belonolaimus longicaudatus ...... 85 Marketable Yield ...... 86 Discussion ...... 87 Population Density of Sting Nematode, Belonolaimus longicaudatus ...... 87 Marketable Weight ...... 88

6 EFFICACY OF SELECTED MITICIDES AND MICROBIAL FUNGICIDES FOR MANAGEMENT OF TWOSPOTTED SPIDER MITES ...... 97

Introduction ...... 97 Materials and Methods ...... 99 Laboratory Experiments ...... 99 Field Trials ...... 101 Data Analysis ...... 102 Results...... 102 Laboratory Experiments ...... 102 Field Trials ...... 104 Discussion ...... 105 Laboratory Experiments ...... 105 Field Trials ...... 107

7 CONCLUSION...... 119

LIST OF REFERENCES ...... 122

BIOGRAPHICAL SKETCH ...... 138

LIST OF TABLES

Table page

3-1 Proportion of culls with mean marketable fruit weight for all strawberry cultivars produced at Plant Science Research and Education Unit (PSREU), Citra and Plant City, FL...... 57

4-1 Behavioral response of Neoseiulus californicus to strawberry cultivars manually infested with twospotted spider mites (TSSM)...... 71

4-2 Behavioral response of Phytoseiulus persimilis to strawberry cultivars manually infested with twospotted spider mites (TSSM)...... 72

4-3 Total number of Neoseiulus californicus recovered on strawberry cultivars in response to distances from the point of release...... 77

5-1 Total populations of A) sting and B) ring nematodes on strawberry cultivars planted using different production techniques...... 94

5-2 Total marketable weights (in kg) of strawberry on selected commercial cultivars for each production technique...... 95

6-1 Population density of the predatory mite, Neoseiulus californicus on treated and untreated strawberry leaflets at intervals after pesticide treatments in the laboratory study...... 113

6-2 Population density of the predatory mite, Neoseiulus californicus on strawberry plots before and after pesticide applications in the field study...... 118

LIST OF FIGURES

Figure page

3-1 Mean population density of twospotted spider mite (TSSM) on strawberry cultivars grown in the greenhouse on different sampling dates...... 49

3-2 Mean population density of twospotted spider mite eggs on strawberry cultivars in the greenhouse...... 50

3-3 Mean population of TSSM motiles and eggs on selected commercial cultivars established at Plant Science Research and Education Unit (PSREU) Citra, FL during the 2013-14 strawberry season...... 51

3-4 Mean population of TSSM motiles and eggs on selected commercial cultivars established at Plant City, FL during the 2013-14 strawberry season...... 52

3-5 Mean population of TSSM motiles and TSSM eggs in high tunnel system on selected strawberry cultivars under the high tunnel management system for the entire season...... 53

3-6 Mean population of TSSM motiles and eggs on selected commercial cultivars established at Plant Science Research and Education Unit (PSREU) Citra, FL during the 2014-15 strawberry season...... 54

3-7 Mean weight of culls and marketable fruit weight harvested on selected strawberry cultivars at the Plant Science Research and Education Unit (PSREU) Citra, FL during the 2013-14 growing season...... 55

3-8 Mean weight of culls and marketable fruit weight harvested on selected strawberry cultivars at a grower’s commercial farm in Plant City, FL during the 2013-14 growing season...... 56

4-1 Mean population of Neoseiulus californicus motiles and eggs on commercial cultivars established at Plant Science Research and Education Unit (PSREU), Citra during the 2013-2014 growing season...... 73

4-2 Mean population of Neoseiulus californicus motiles and eggs on commercial cultivars established at a grower’s organic farm in Plant City, during the 2013-2014 growing season...... 74

4-3 Mean population of Neoseiulus californicus motiles and eggs on commercial cultivars established at Plant Science Research and Education Unit (PSREU), Citra during the 2014-2015 growing season...... 75

4-4 Mean population of N. californicus motiles in open field strawberry in Citra, FL during the 2014-2015 strawberry growing season...... 76

5-1 Plot layout of the field experiment conducted in 2016-2017 growing season at Plant Science Research and Education Unit (PSREU), Citra...... 84

5-2 Mean population of sting nematodes on selected cultivars on different sampling dates...... 91

5-3 Mean population of sting nematode, Belonolaimus longicaudatus on selected strawberry cultivars throughout the entire strawberry season 2016-17...... 92

5-4 Mean marketable weights (in kg) of strawberry harvested for each cultivar...... 93

5-5 Relationship between strawberry yield and populations of A) sting nematode B) ring nematode...... 96

6-1 Population density of twospotted spider mites (TSSM) on treated and untreated strawberry leaflets at intervals after pesticide treatments in the laboratory...... 109

6-2 Population density of twospotted spider mite (TSSM) eggs on treated and untreated strawberry leaflets at intervals after pesticide treatments in the laboratory...... 110

6-3 Population density of twospotted spider mites (TSSM) on treated and untreated strawberry leaflets at intervals after microbial pesticide treatments in the laboratory study...... 111

6-4 Population density of twospotted spider mite (TSSM) eggs on treated and untreated strawberry leaflets at intervals after pesticide treatments in the laboratory study...... 112

6-5 Population density of twospotted spider mites (TSSM) on strawberry plants before (12/21/2015 and 3/27/2016, enclosed in a rectangle) and after chemical treatments in the field study...... 114

6-6 Population density of twospotted spider mite (TSSM) eggs on strawberry plants before (12/21/2015 and 3/27/2016, enclosed in a rectangle) and after chemical treatments in the field study...... 115

6-7 Population density of twospotted spider mite (TSSM) observed on strawberry plants before and after chemical treatments each week in the field study...... 116

6-8 Population density of twospotted spider mite (TSSM) eggs observed on strawberry plants before and after chemical treatments each week in the field study...... 117

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

Omotola G. Olaniyi

December 2018

Chair: Oscar E. Liburd Major: Entomology and Nematology

Florida is a major producer of strawberries within the United States second after

California. Strawberries are usually produced using conventional methods of production that rely mostly on chemical control tactics to manage the twospotted spider mite (TSSM) Tetranychus urticae, the key pest of strawberry in Florida. Field trials and laboratory experiments were conducted during the strawberry production seasons from 2013 to 2017 to evaluate integrated strategies for managing TSSM and potential host plant resistance to sting nematode,

Belonolaimus longicaudatus in strawberry.

The goals of this research were to evaluate the effects of selected summer cover crops on twospotted spider mite populations and the susceptibility of selected commercial strawberry cultivars to TSSM in two production systems (Chapter 3), investigate the dispersal of a mite predator, Neoseiulus californicus among selected strawberry cultivars and identify strawberry cultivars that would support high populations of N. californicus (Chapter 4), examine the susceptibility of selected strawberry cultivars to sting nematode using bare root and plug

12 transplants as production techniques (Chapter 5), and to evaluate the effectiveness of selected

OMRI-approved miticides and conventional microbial pesticides on TSSM populations and their effects on natural enemy populations (Chapter 6).

Results showed that the selected commercial cultivars have varying susceptibility to

TSSM and that production systems influence the populations of TSSM on cultivars. Neoseiulus californicus, although an efficient predator of TSSM did not disperse quickly in search of its prey, rather populations increased gradually during the season and showed a preference for

Winterstar™ and Sensation cultivars in laboratory experiments.

The strawberry production techniques of using bare roots versus plugs did not have a significant effect on the population of sting nematodes. However, cultivars like Radiance,

Sensation, and Winterstar™ tolerated lower populations of sting nematodes than Albion and

Benecia throughout the entire season. Organic miticides had varying levels of efficacy in reducing mite populations, but Aramite and Cosavet DF micronized sulfur performed best.

Microbial pesticides performed poorly, but the efficacy may improve if applied before spider mite populations become established. None of the pesticides evaluated appear to have any negative effects on the beneficial arthropods.

CHAPTER 1 INTRODUCTION

The most common cultivated strawberry species in the world is Fragaria × ananassa

Duchesne (Hancock et al. 2008). Fragaria × ananassa is a major small fruit crop commercially produced within the United States, with California as the highest producing state (USDA-NASS

2017). Florida is a major producer of strawberries for fresh market during the winter months

(Mossler and Nesheim 2007), valued at approximately $337 million in 2017 (USDA-NASS

2018). The production of strawberry is important within the US as a source of income through exports and as a diet food, rich in antioxidants (Wang and Zheng 2001), vitamins, and other nutrients that are important in boosting and maintaining human health.

Strawberries are usually produced using conventional methods, where the growers depend mainly on the use of synthetic pesticides to minimize pest damage and losses. However, consumer demand for pesticide-free fruits and vegetables have made some growers consider organic production of strawberry as a solution. In 2016, 6,249 ha of strawberry were certified organic by the United States Department of Agriculture (USDA) according to National Organic

Production (NOP) standards, of which 93 ha were harvested in Florida, with production estimated at 744,406 kg (USDA-NASS 2017). This represented two percent of total harvested area in Florida in 2016 and an increased harvested area of 19 ha from 2015.

Organic production involves the cultivation of crops and raising of livestock using techniques such as cultural control, biological control, and only approved non-synthetic chemicals to control weeds, diseases, and arthropod pests. Organic production is one approach to achieving sustainability of pesticide use in a high value crop such as strawberry. Sustainable agriculture is the production of food using techniques to meet increasing world population demands, while preserving the environment, public health, and human activities (Lal 2008). Best

14 management practices are required to increase and expand organic strawberry production in

Florida and other Southeastern states. The transition period from conventional agriculture to organic production usually lasts three years. During this period, yield is generally lower for organic production compared with conventional production (Russo and Taylor 2006). In addition, marketable strawberry yields in both conventional and organic production systems are usually reduced due to arthropod pests, weeds, and diseases (Walsh et al. 1998, Nyoike and

Liburd 2013).

Strawberry Production in Florida

In Florida, strawberries are grown as an annual crop usually in open fields, using the raised bed plasticulture system (Chandler et al. 1993, Nyoike and Liburd 2014). Commercial strawberry production involves laying drip irrigation lines within raised beds covered with black polyethylene mulch. The number of irrigation lines depends on the width of the bed. Raised beds are advantageous because they make hand harvesting easier and create an efficient system for applying soil fumigants and fertilizers (Chandler et al. 1993), whereas the polyethylene mulch helps in weed management. Planting holes are usually created at pre-determined spacing, and strawberry plants are usually planted in alternate double rows at 0.3 – 0.45 m apart.

Methyl bromide was used in strawberry production for decades to fumigate the soil to manage nematodes, pathogens, and weeds. However, because methyl bromide depletes the stratospheric ozone layer (Ducom 2006), its production and importation were gradually reduced from 2005 and was subsequently only available in response to critical use exemption applications (Gareau 2010). It was completely phased out in 2014 under the Montreal Protocol

(Gareau 2015). Due to the phase out of methyl bromide, alternative fumigants including

15 chloropicrin, 1.3-dichlopropene, metam sodium (Gilreath et al. 2008), dimethyl disulfide (Fritsch

2005), and methyl iodide (Dube 2015) have been evaluated.

Common pathogenic diseases in field-grown and greenhouse strawberry include botrytis

(Legard et al. 2001), anthracnose, leather rot, phytophthora crown rot, and powdery mildew.

These are fungal diseases and commercial strawberry production in Florida makes use of routine fungicide applications including Abound® (Azoxystrobin), Cabrio® (Pyraclostrobin), Switch®

(Cyprodinil + Fludioxonil), Flint® (Trifloxystrobin), and Bumper® (Propiconazole) to manage these diseases (Whitaker et al. 2016).

Strawberry plants produce hermaphroditic flowers. Pollination of these flowers is essential to produce well-formed fruits with optimum size (Chagnon et al. 1993). Pollinated strawberry flowers produced heavier, better quality, and more marketable fruits compared with those that were not pollinated (Malagodi-Braga and Kleinert 2004, Roselino et al. 2009).

Strawberries are mainly pollinated by wind and insects. Some common pollinators of strawberries include bees, flies, wasps, moths, butterflies, and stink bugs (Nye and Anderson

1974). Nye and Anderson (1974) reported that the most efficient pollinators were honey bees

(Apis mellifera L.), sweat bees (Halictus ligatus Say), and drone flies (Eristalis spp.).

Among the arthropods, the honey bee is an effective pollinator of strawberries and is used commercially in open field, greenhouse, and high tunnel strawberry production systems

(Goodman and Oldroyd 1988, Svensson 1991). In greenhouse production of strawberry, the use of honey bees as pollinators is limited due to possible crash of the colony. This prompted the research for alternative strawberry pollinators, with greater or equal efficiency as the honey bees.

Studies conducted showed that stingless bees such as Trigona minangkabau (Kakutani et al.

1993), Tetragonisca angustula Latreille (Malagodi-Braga and Kleinert 2004), Scaptotrigona aff.

16 depilis, Nannotrigona testaceicornis (Roselino et al. 2009) were effective strawberry pollinators, although they were less effective compared with the honey bees in terms of flower fertilization and foraging efficiency. However, pollinated strawberry flowers produced heavier, better quality, and more marketable fruits compared with those that were not pollinated (Malagodi-Braga and

Kleinert 2004, Roselino et al. 2009), thus significantly increasing fruit production.

The key arthropod pest that attacks Florida strawberry plants is the twospotted spider mite (TSSM), Tetranychus urticae Koch (Mossler and Nesheim 2003, Rhodes and Liburd 2006,

Nyoike and Liburd 2013). Other arthropod pests of strawberry are broad mites

(Polyphagotarsonemus latus Banks), armyworms (Spodoptera frugiperda Smith), aphids, whiteflies, thrips, and seed bugs (Neopamera bilobata Say). Animal pests of strawberry include rodents and birds. Some beneficial organisms found in strawberry fields include predatory mites, sixspotted thrips (Scolothrips sexmaculatus (Perg.)), big-eyed bugs (Geocoris spp.), minute pirate bugs (Orius spp.), lady bugs, and lacewings (Chrysoperla spp.).

Plant-pathogenic nematodes are common pests in Florida soils, attacking a wide variety of crop plants. They are ubiquitous, multicellular organisms, yet they are under-studied (T. M.

Mengistu, personal communication). Several nematodes have been found attacking strawberry in most production areas. These include the sting nematode, Belonolaimus longicaudatus Rau

(Abu-Gharbieh and Perry 1970, Noling et al. 2010), the root knot nematode, Meloidogyne incognita Chitwood, M. hapla Chitwood (Nyoike et al. 2012), the strawberry crimp nematode,

Aphelenchoides fragariae Ritzema-Bos, the lesion nematode, Pratylenchus spp. (Szczgiel and

Hasior 1972), the needle nematode, Longidorus elongatus (Seinhorst 1966), and Ditylenchus dipsaci Filipjev (Goodey 1951).

Nematodes are important vectors of plant pathogens. The attack of nematodes on a host plant usually weakens the host, increases its susceptibility to fungal pathogens, and disease occurrence. The disease development varies with the species of nematode and its relationship with the pathogen, as well as the pathogen itself; therefore, influencing the method of control

(Khan 1993). For example, A. fragariae vectors Rhodococcus fascians, a bacterial pathogen, which causes cauliflower disease in strawberry (Khan and Pathak 1993).

The best way to manage a pathogen-nematode complex is prevention, which can be achieved through planting disease-free planting stock and clean seeds. Qiu et al. (1993) demonstrated the effect of hot water baths on mortality of A. fragariae at different time exposures and different temperatures. He found that as water temperature increases, less exposure time is required to achieve 100% mortality of A. fragariae on infested crowns of selected strawberry cultivars. However, the exposure period of A. fragariae to hot water bath above a certain temperature may prevent or impair plant survival, plant growth, and fruiting potential. This preventive method can be used to achieve mortality of nematodes on plants prior to planting strawberry in the field.

Justification of the Study

Strawberry is a high-value crop in the United States that is attacked by several arthropod pests and nematodes. As the organic strawberry market grows, there is a need to develop compatible methods to manage pests and the consequent damage to strawberry. Integrated pest management (IPM) is the combination and application of different control strategies (chemical, biological, cultural, and host plant resistance) in a compatible manner to effectively reduce pest populations and prevent them from reaching economic injury levels (EIL), with minimum

18 negative impact on humans, non-target organisms and the environment (Flint and Bosch 1981,

Pedigo 1989).

Cultivars of a particular plant species may vary in their susceptibility to pests and diseases; therefore, host plant resistance is an important component of an IPM program. Host plant resistance as a pest management strategy is advantageous, as its effect is persistent and cumulative. With the implementation of resistant plants into the cropping system, there is no problem of toxic residue on the fruit and no need for re-application of chemicals, as is often the case with chemical control tactics. However, development of a resistant plant cultivar can take several years of research, and pests can develop biotypes that will overcome plant resistance after some time.

Neoseiulus californicus (McGregor) is a predator of TSSM and was recommended as an effective biological control agent of TSSM in Florida, because it can reduce and maintain TSSM populations below economic threshold (ET) throughout the entire strawberry season (Rhodes and

Liburd 2006). However, there is need to understand their dispersal and preference for some life activities such as mite feeding and oviposition, as influenced by leaf characteristics or varietal differences.

The development of effective management strategies to control plant-pathogenic nematodes is critical due to the significant crop losses that has been attributed to them. For example, losses in corn yield is up to 20% in some states (Koenning et al. 1999). Methyl bromide was traditionally the most effective soil fumigant used against nematodes (Zasada et al. 2010); however, its phase out process resulted in research for and adoption of alternative soil fumigants being adopted (Locascio et al. 1997, LaMondia 1999, Gilreath et al. 2008, Lopez-Aranda et al.

2009). Cultural techniques such as crop rotation (Todd 1991), intercropping (LaMondia et al.

2002), cover cropping, fallow (Rhoades 1983), soil solarization (Saleh et al. 1988), and biofumigation (Bello et al. 2002, Zasada et al. 2010) have been evaluated as alternatives to methyl bromide with varying levels of success.

There are few pesticides available for managing TSSM and other pests in organic strawberry production. Since the Organic Materials Review Institute (OMRI) evaluates pesticides only for compliance of the ingredients with the National Organic Products (NOP), not for their effectiveness in reducing pest populations, it is important to evaluate allowed pesticides to evaluate efficacy in an integrated pest management program and to ensure low or no impact on beneficial organisms.

Currently, more than 70% of strawberry growers in Florida are conventional growers (O.

E. Liburd, personal communication), and as a result, there has been less emphasis on pest management strategies for organic growers. Therefore, to address this deficiency, the goal of this study is to develop pest management techniques that can be used to manage key pests in organic strawberry production.

Specific Objectives

1a) Evaluate some commercially available strawberry cultivars for susceptibility to twospotted spider mites (TSSM) in an organic production system.

Hypothesis: Strawberry cultivars will show differential susceptibility to TSSM populations and some cultivars will be more adapted to organic production systems. b) Evaluate whether summer cover crops have any influence on TSSM population and the diversity and abundance of natural enemies.

Hypothesis: Cover crops will influence the level of pest infestation and abundance of natural enemies.

2a) Study the dispersal of N. californicus in field grown strawberries.

Hypothesis: Natural populations of N. californicus are attracted to mite-infested leaves and their dispersal is guided by the availability of TSSM. b) Identify strawberry cultivar(s) that would support high populations of the predatory mite, Neoseiulus californicus, as a biological control agent of TSSM.

Hypothesis: Strawberry cultivars vary in their physical attributes and this may influence the preference and population of N. californicus on some cultivars.

3) Evaluate the susceptibility of different strawberry cultivars to the sting nematode, Belonolaimus longicaudatus in field-grown strawberry using different production techniques.

Hypothesis: Some strawberry cultivars will be susceptible to the sting nematode and the bare root transplants will be easily accessed by sting nematodes.

4a) Identify key organic miticides that can be used in the management of TSSM in organic strawberry production.

Hypothesis: Organic miticides vary in their effectiveness to control TSSM populations; there will be differences in TSSM population between pesticide-treated and untreated plants. b) Evaluate the efficacy of selected microbial pesticides in the management of TSSM and their effect on natural enemy populations.

Hypothesis: Microbial pesticides will be effective against TSSM and specific in their action without a negative impact on predatory mites and beneficial insects.

CHAPTER 2 LITERATURE REVIEW

Biology of the Twospotted Spider Mite, Tetranychus urticae Koch

The twospotted spider mite (TSSM) is a polyphagous pest with a wide host range that feeds on the foliage of fruit, vegetable, and field crops (Ayyapath et al. 1996, Alatawi et al.

2005). It belongs to the family Tetranychidae, one of the important families in the class

Arachnida and is probably one of the most destructive mite species. Pest mites have become a major agricultural problem in many countries, as a result of the increased use of synthetic pesticides and fertilizers (Huffaker et al. 1969, Boykin et al. 1984, James and Price 2002).

Twospotted spider mites are light yellow or green in color with two dark spots, one on either side of the body. The two dark spots form as body waste accumulates, therefore, they may be absent in newly molted mites (Fasulo and Denmark 2000). Twospotted spider mites may appear red or orange during winter. The lower daily temperature and decreased photoperiod induce the female

TSSM to go into diapause (Boudreaux 1963).

The life cycle of TSSM consists of egg, larva, protonymph, deutonymph, and adult.

Twospotted spider mites feed on host plants at all life stages except at the egg stage. The eggs are round, and translucent. Mating is not required for oviposition, especially in virgin females.

The unfertilized eggs develop into exclusively male offspring (arrhenotoky) or only female offspring (thelytoky), while the fertilized eggs hatch to become both male and female offspring

(Boudreaux 1963). An adult female can mate with many males. At the larval stage, TSSM have three pairs of legs. Each immature stage is followed by a quiescent stage called chrysalis. The name of each quiescent stage is specific to the nymphal stage; therefore, each spider mite must go through the protochrysalis, deutochrysalis and teliochrysalis respectively to reach adulthood

(Boudreaux 1963).

The development rate of TSSM is mainly dependent on temperature (van de Vrie et al.

1972, White and Liburd 2005). Other environmental factors that affect development include relative humidity (Shibuya et al. 2016), wind, rainfall, light, the availability, and health of the host plants (Huffaker et al. 1969). These same environmental factors also affect oviposition, movement, and survival of TSSM. Saba (1973) reported that laboratory-reared Tetranychus males emerged 24 h before females, thus resulting in the potential mating of all females as they mature and emerge. The TSSM adults are dimorphic. The females are usually bigger with a rounded posterior end, while the males are smaller than the females, with narrow bodies, that taper at the posterior end. Adult male TSSM live for an average of 14 days and adult females live for approximately 19 days (Shih et al. 1976).

Webbing

Tetranychus urticae motiles produce silken webs that function in locating mates, as a means for dispersal (Gerson 1985), and for protecting themselves and their eggs against predation and adverse weather conditions. Twospotted spider mites spin web as they walk; a direct relationship was found between TSSM movement and the density of webs spun (Saito

1977). Web spinning is usually simultaneous with the feeding activity of TSSM (Gerson 1985).

All motile stages of TSSM produce silk threads. Webbing may be used as a communication means between sexes. A female deutonymph, before her final molt, spins a web that serves as a pheromone substrate which guides a male to her (Cone et al. 1971). The male displays territorial behavior by spinning a web over the female deutonymph to prevent other males from mating with such female upon reaching adulthood (Penman and Cone 1974). Factors that influence the amounts of silk produced include temperature, humidity, leaf texture, and species of the host plant (Gerson 1985).

Dispersal

Twospotted spider mites usually move within and among plants by walking (Bradenburg and Kennedy 1982). Long distance movement is wind-assisted (Margolies and Kennedy 1985), or by human interference such as through mechanical tools. Twospotted spider mites may disperse to new hosts by hitchhiking on insects and birds. Twospotted spider mites can disperse within a ball of silk thread to another plant.

The dispersal of TSSM is usually influenced by colonization, food quality of the host plant (Hussey and Parr 1963), and effect of pesticides. Penman and Chapman (1983) reported that the application of fenvalerate to bean plants (Phaseolus vulgaris L.) resulted in a spindown of TSSM, thus enhancing dispersal by wind (Gerson 1985). Spindown is a term used to describe the hanging down of TSSM from a single thread below a leaf surface. Spindown can occur in the absence of pesticides, but it is usually linked to repellent properties of some miticides (Gemrich et al. 1976). Aerial dispersal of TSSM is more common in adult females, less common in nymphs, but rare in males (Bradenburg and Kennedy 1982).

Diapause

Diapause is a state of dormancy or arrested development. It is genetically determined, but environmental factors may be responsible for its expression (Tauber and Tauber 1976).

Arthropods go into diapause at different life stages, depending on the stage at which they are adapted to withstand the adverse climatic conditions (Andrewartha 1952). Twospotted spider mites overwinter as adults and have a different color from non-diapausing females. During winter, TSSM motiles and their eggs appear red or orange. Diapause also accounts for differences in physiology, morphology, and behavior of spider mites (Veerman 1985). Several studies have showed that diapausing TSSM females live longer and can tolerate freezing

24 temperatures compared to non-diapausing females (van de Bund and Helle 1960, Stenseth 1965).

In sub-tropical regions, TSSM is present all year round; they prefer areas with hot and dry conditions (White and Liburd 2005). In temperate regions, the female overwinters in ground litter, tree barks, and shrubs (Takafuji and Kamibayashi 1984, Fasulo and Denmark 2000).

Hussey and Parr (1963) observed that non-diapausing TSSM moves towards source of light, while diapausing forms move away from light.

Symptoms of Injury and Management of TSSM

In Florida, TSSM is the key arthropod pest of strawberries, whether grown in the field, high tunnel, or greenhouse (Osborne et al. 1999, Rhodes and Liburd 2006, Nyoike and Liburd

2013). It is a potential pest in most strawberry producing areas (Hepworth and MacFarlane 1992,

Walsh et al. 1998). Twospotted spider mites are usually found on the lower surface of the leaves, and may go unnoticed for some time, until the leaves begin to show symptoms of injury (Opit et al. 2009), such as webbing, stunting, and discoloration.

Twospotted spider mites cause injury to strawberry plants by using their stylet-like chelicerae to pierce the leaf, and suck out sap, resulting in the destruction of the mesophyll layer.

Continuous feeding lowers chlorophyll content (Sances et al. 1979), consequently reducing the net photosynthetic rate (Hall and Ferree 1975, Park and Lee 2002), transpiration (DeAngelis et al. 1983), and ultimately impairing plant health, growth, and vigor. Bronzing and webbing are late infestation symptoms observed as the mite feeding continues. At high infestations of TSSM, the growth rate of strawberry plants and production of fruits are significantly reduced (Helle and

Sabelis 1985, Rhodes and Liburd 2006, Nyoike and Liburd 2013), the strawberry plants lose their leaves, and may die due to TSSM injury.

Management of TSSM is quite challenging, because they have a high reproductive potential, short generation times, multiple generations within a year (Huffaker et al. 1969, Tuan et al. 2016), and potential to develop resistance to miticides (Croft and van de Baan 1988).

Current management methods for TSSM include chemical control (Liburd et al. 2007), biological control (Fraulo and Liburd 2007), and cultural control (Smithley and Peterson 1991,

White and Liburd 2005).

Cultural control

Studies show that TSSM abundance is affected by irrigation rate (Smithley and Peterson

1991), irrigation method (White and Liburd 2005), relative humidity, temperature, field sanitation, and host plant quality (Huffaker et al. 1969, Wilson 1994). The rate of development is usually influenced by the prevailing temperature and soil moisture conditions. Twospotted spider mites thrive best under hot, dry conditions and on water-stressed plants (Smithley and Peterson

1991, White and Liburd 2005). A study by White and Liburd (2005) showed that the drip plus overhead irrigation method supports higher TSSM population, compared with drip irrigation alone. This finding contradicted the assumption that overhead irrigation will wash mites off the leaves (Cloyd et al. 2009) or increase the relative humidity above levels that will constrain the reproduction and survival of TSSM.

Under field conditions, the presence of weeds has been reported to increase the abundance of TSSM (Takafuji and Kamibayashi 1984, Nyoike and Liburd 2014), because they can survive on a wide range of plant species. Therefore, the removal of weeds and old plant residues, and planting of trap crops can be effective management measures to reduce mite populations. Increased rate of fertilization has also been found to increase spider mite populations (Wilson 1994, Chen et al. 2007).

Chemical control

Twospotted spider mites have been traditionally managed in strawberries using several miticides (acaricides) registered for use in Florida. Chemicals that have been used within the past decade include sulphur, bifenazate (Acramite®), Carbaryl, Etoxazole (Zeal®), Fenbutatin-oxide

(Vendex®), Hexythiazox (Savey®), Fenpyroximate (Portal®), Malathion, Abamectin (Avid®),

Naled (Dibrom®), Chlorpyrifos (Lorsban®), Bifenthrin (Capture®), Fenpropathrin (Danitol®), and paraffinic oil (JMS stylet oil) (Rhodes and Liburd 2006, Liburd et al. 2007). With the exception of JMS stylet oils and sulphur (some formulations), all of the other acaricides are not labelled for organic use and cannot be integrated into an organic pest management program for strawberries. Additional tools (miticides) need to be identified that can be used in an integrated organic management program.

The continuous reliance on conventional pesticides for mite management can lead to a host of problems including mite resistance to several classes of chemical miticides (Croft and van de Baan 1988, Kwon et al. 2010, van Leeuwen et al. 2010), harm to beneficial organisms, environmental pollution and residual effects (Prischmann et al. 2005, Hardman et al. 2006), especially when products are used incorrectly. Twospotted spider mites can rapidly develop resistance due to its ability to perform metabolic detoxification of pesticides (van Pottelberge et al. 2008, Kwon et al. 2010, van Leeuwen et al. 2010), and ability to inherit resistance from the female parent (Croft and van de Baan 1988, van Leeuwen et al. 2006). Resistance to pesticides usually develops at the population level, and it is enhanced by multiple applications of the same pesticide.

The effectiveness of miticides on TSSM is often reduced, due to their evolutionary behavior of hiding on the underside of leaves and their ability to produce webs, which may

27 protect them from chemical droplets. Therefore, the use of non-chemical approaches such as predacious mites through augmentative releases offers potential for management of TSSM.

Biological control

There are no known parasitoids of TSSM. This could be attributed to their small size and cryptic behavior, living on the underside of leaves. Natural enemies of TSSM include predatory mites namely Phytoseiulus spp., Neoseiulus spp., Metaseiulus spp., big-eyed bugs (Geocoris spp.), minute pirate bug (Orius spp.), lacewings (Chrysopa spp.), and sixspotted thrips,

(Scolothrips sexmaculatus).

Predatory mites, such as Phytoseiulus persimilis Athias-Henriot, Neoseiulus californicus

McGregor (Rhodes and Liburd 2006), and Metaseiulus occidentalis (Oatman et al. 1977a) have been evaluated for effective control of TSSM. Phytoseiulus persimilis is more aggressive in its searching ability and prey consumption than the N. californicus but does not persist in the ecosystem in the absence of TSSM, as it is a specialist predator of TSSM. In contrast, N. californicus becomes established in introduced areas and persists in the system in the absence of

TSSM because it also feeds on pollen and other mites (McMurtry and Croft 1997).

The timing and rate of release of predatory mites are important in achieving effective management of TSSM (Oatman et al. 1977b). A study by Fraulo and Liburd (2007) revealed that in Florida, one-time early release of N. californicus was effective in suppressing TSSM populations below economic threshold, throughout the strawberry growing season.

Predatory Mite, Neoseiulus californicus McGregor

The predatory mite, N. californicus is a type II selective predator (McMurtry and Croft

1997) of TSSM. A type II predator has a wider range of prey that it feeds on, although some diets may be preferred over others (McMurtry and Croft 1997). For example, N. californicus has

28 a higher oviposition rate when it feeds on Tetranychus cinnabarius than on Eutetranychus orientalis (McMurtry and Croft 1997), a higher reproductive rate, a higher survival rate, and shorter developmental period when it feeds on TSSM compared to other tetranychids like T. evansi (Escudero and Ferragut 2005). Neoseiulus californicus is an effective biological control agent of spider mites (Rhodes and Liburd 2006, Rhodes et al. 2006) and tarsonemid mites like cyclamen mites, Steneotarsonemus pallidus Banks (Easterbrook et al. 2001) on various crop plants, including strawberry without altering the arthropod diversity (Fraulo et al. 2008). It is mass produced and available commercially in many parts of the world.

Gotoh et al. (2004b) investigated the effect of temperature on the population development and fecundity of female N. californicus. The study revealed the optimum temperature range for

N. californicus was 15–35°C. At 37.5°C, the rate of oviposition declined significantly, and hatchability dropped to 0%. At 40°C, oviposition stopped. The intrinsic rate of natural increase was 0.274 at 25°C, this was lower than the intrinsic rate of 0.336 reported for TSSM by Shih et al. (1976), suggesting that N. californicus is reproducing at a lower rate, and that emerging N. californicus populations are sustained by the constant availability of TSSM. Neoseiulus californicus shows a type II functional response to consumption of TSSM eggs (Gotoh et al.

2004a, Ahn et al. 2010). In contrast, the generation time is shorter for P. persimilis and the intrinsic rate of increase is higher than N. californicus and TSSM (Escudero and Ferragut 2005).

This may explain why P. persimilis is more efficient than N. californicus at suppressing TSSM populations.

Host Plant Resistance

Host plant resistance is an important component of an IPM program. Snelling (1941) defined resistance as plant characteristics which enhance such plants to repel pests or tolerate the

29 attack of insects under similar conditions that would have caused a greater injury to other plant cultivars of the same species. Resistance can be manifested from a low level to a high level

(Pedigo and Rice 2006). At the upper extremity is immunity, this is a rare form of resistance in which a specific arthropod pest is not known to cause injury to the plant cultivar considered under any condition. At the lower extremity, a plant cultivar is considered to be susceptible if the plant cultivar showed greater than average damage by a particular arthropod pest (Kogan 1994).

Host plant resistance is conditioned by a variety of factors which pertain to the inter- relationship between the host plant and pest, representing a dynamic system. In this system, some series of requirements by the pest must be met by the plant for it to be a suitable host. If there are breaches to meeting these requirements, such plant becomes unavailable to pests, and this leads to resistance. Resistant cultivars may display certain characteristics to avoid herbivory.

Such plant characteristics include morphological factors (abundance of trichomes, leaf thickness, and leaf surface) and chemical factors (chemical compound composition) (Kogan 1994).

Resistance is genetically controlled, although its expression may be influenced by environmental factors (Painter 1954, Kogan 1994). The mechanism of host plant resistance is categorized into three kinds: antixenosis (non-preference), antibiosis, and tolerance (Painter 1951).

a) Antixenosis: Host plants possess characteristics against a guest (pest) for oviposition, feeding, and shelter (Kogan and Ortman 1978, Kogan 1994). Leaf pubescens is a morphological attribute that confers non-preference for feeding. For example, Turnipseed (1977) reported the effect of leaf pubescens on development of some insects on selected soybean genotypes. In an evaluation of five isogenic lines of the Lee variety, he found that the population of Empoasca fabae varied with the abundance of trichomes on the genotypes. The population of E. fabae was

30 lowest on PI 229358, which has long erect trichomes and the lowest hair density among all the isogenic lines that were evaluated. The highest population of insect pest was on glabrous Lee.

b) Antibiosis: Host plant possesses characteristics which may affect the biology of the pest and its development in an adverse manner. This may lead to retardation in growth, prolonged developmental period, reduction in population, and death in some cases. Plant defense include the secretion of toxins (Kogan 1994), which affects the development of the pest or the plant variety may lack essential nutrients, thereby creating an imbalance in the available nutrients for optimal growth of pest species. Secondary plant substances present in some plants can serve as toxins, therefore, leading to antibiotic resistance. Lale and Makoshi (2000) investigated chemical antibiosis of two cowpea varieties on Callosobruchus maculatus. They reported that both varieties were more susceptible to C. maculatus when their seed coats were removed, however their oviposition preference varied on the cowpea seeds in both varieties. Their results showed that the chemical content in seed coats and the concentration are important factors that determined the feeding and oviposition preferences of C. maculatus.

c) Tolerance: This represents the first phase of the damage course, in which the host plant can recover from the attack of the insect. The plant has some ability to withstand infestation, and or repair the injury it suffered without obvious damage. A susceptible plant cultivar will produce lower yield compared with a tolerant cultivar of the same plant, at the same or lower pest population (Kogan 1994).

CHAPTER 3 SUSCEPTIBILITY OF SELECTED COMMERCIAL STRAWBERRY CULTIVARS TO TWOSPOTTED SPIDER MITE, TETRANYCHUS URTICAE KOCH IN ORGANIC PRODUCTION

Introduction

Strawberry (Fragaria × ananassa Duch.) production is threatened by many arthropod pests and diseases. In Florida, the twospotted spider mite (TSSM), Tetranychus urticae (Koch) is the primary pest that reduces strawberry yield. Twospotted spider mites have not been reported to vector any plant diseases. However, they feed on strawberry leaves by sucking the cell cytoplasm, consequently reducing chlorophyll content, transpiration rate (DeAngelis et al. 1983,

Reddall et al. 2004), plant vigor, and yield (Wilson 1993).

In organic strawberry production, cultural practices are usually the first line of defense in an integrated pest management (IPM) program against pests and diseases. This involves cultural tactics such as choosing the plant material including cultivars. In assessing crop cultivars, host plant resistance is a major strategy in the IPM tool box. The cultivar’s relative tolerance or susceptibility to arthropod pests is important in organic strawberry production, because chemical control is limited to only a few pesticides of botanical or biological origin or synthetics on the

National List of Allowed and Prohibited Substances.

Generally, varietal selection for a particular crop depends on factors such as flavor, shelf life, fruit size, and quality. However, leaf surface and architecture are important factors that could serve as a characteristic or diagnostic feature among cultivars of the same plant species.

For instance, leaf architecture can determine the abundance of mites, and can influence predator- prey interactions (Walter 1996).

Plant cultivars vary in their susceptibility to insect pests (Raina et al. 1984), mite pests

(Gilbert et al. 1966, Regev and Cone 1975), diseases (Hershman et al. 1970), and weeds (Zhao et

32 al. 2006, Mahajan and Chauhan 2011). Cultivars differ genotypically, which gives rise to many characteristics including leaf texture, volatile composition, and yield. This may account for differential levels of arthropod pest establishment. Cultivar differences may also account for variation in plant vigor and rate of transpiration as influenced by the leaf thickness. Strawberry leaves of different cultivars may vary in their abundance of trichomes or leaf hairs, which could serve as habitat for predatory mites or protect TSSM from predation by insects.

Apart from the inherent traits of strawberry cultivars, the choice of cultural practices is an important factor that may influence susceptibility of crops to pests, weeds, and diseases (White and Liburd 2005). Cropping practices can influence the level of pest infestation and abundance of natural enemies. For example, Sigsgaard et al. (2014) reported lower infestation level of

Acleris comariana (Lepidoptera: Tortricidae) on organically produced strawberry compared with those produced by conventional means. In a study conducted by Iwassaki et al. (2015), TSSM population was significantly lower on strawberry fields managed with integrated (biological and chemical) control strategies compared with those managed using conventional (chemical) management methods alone. Nyoike and Liburd (2014) found that re-using plastic mulch with or without thatch had no significant effect on plant size, pest populations, and beneficial insects.

However, re-using the mulch with thatch suppressed weed growth and increased the incidence of fungal diseases in field-grown strawberries.

The use of cover crops has become a common practice in sustainable agriculture, especially because of the added fertility inputs, enhanced crop performance, and increased biodiversity (Ingels et al. 1994). Cover crops have been reported to suppress insect pest populations (McNeill et al. 2012), suppress weed growth (Teasdale 1996, Collins et al. 2008), increase soil nutrients thus, soil fertility (Ingels et al. 2005), reduce nematodes (McSorley 1998,

1999), reduce soil-borne pathogens (Hansen et al. 1990), and reduce environmental problems such as soil erosion.

Only a few studies have examined the influence of host plant cultivar differences on the fecundity of TSSM (McFarlane and Hepworth 1994, Costa et al. 2017). Cultivars bred for conventional production system may perform differently in organic production systems. This study was conducted to evaluate the susceptibility of selected commercially available strawberry cultivars to TSSM in organic systems and to identify the summer cover crops that were used. We hypothesized that different cover crops would result in different mite and predator populations and that strawberry cultivars would differ in their susceptibility to TSSM. Potential mechanism is that cover crops will improve soil structure and fertility, resulting in more robust strawberry growth and improve plant defense against pests. Another potential mechanism is that cover crops would influence off-season pests and beneficial arthropods in a manner that suppresses TSSM.

Materials and Methods

Laboratory and Greenhouse Experiments

Twospotted spider mite colony

Field-collected TSSM on strawberry plants were introduced onto established pinto bean

(Phaseolus vulgaris L.) plants in a greenhouse at the Entomology and Nematology Department,

University of Florida, Gainesville, FL. The bean seeds were purchased from a grocery store in

Gainesville, FL and plantings were staggered every five days to ensure sufficient food for TSSM and maintain their growing population. The bean seeds were planted in trays 0.5 m X 0.3 m X

0.06 m deep filled with potting soil (Jungle Growth Potting Soil with Organic Fertilizer, Home

Depot, Gainesville, FL). The bean plants were kept in the greenhouse at 21 ± 6 °C, natural photoperiod and watered regularly as required.

Population density of twospotted spider mites

Greenhouse: Strawberry bare root transplants were purchased from C. O. Keddy

Nursery Inc., Lakeville, Nova Scotia for all cultivars except the proprietary cultivars that were sourced from Driscoll’s, Watsonville, California, USA. Bare root transplants of commercially available strawberry cultivars: Albion, Camarosa, Strawberry Festival, Florida Radiance,

Treasure, Winterstar ™, Proprietary 1 and 2, were transplanted into pots in a greenhouse at the

Entomology and Nematology Department, University of Florida, Gainesville, FL on October 24,

2013. The one-gallon plastic pots were filled with commercially available Jungle Growth organic potting mix (Home Depot, Gainesville, FL). Initially, plants were watered daily until they were fully established and then watered frequently (every 2 days) as required. Strawberry cultivars were replicated five times, each replicate consisted of six individual plants of each cultivar, therefore making 30 plants of each of the eight cultivars. All bare root plants were housed in a greenhouse with weekly average temperature of 27 ± 5 °C at the Entomology and Nematology

Department, University of Florida, Gainesville, FL. Natural infestation of TSSM occurred after some weeks and increased on the strawberry cultivars over time.

Sampling was done at weekly intervals by collecting two leaflets from each cultivar per replicate. The samples were processed by identifying, counting, and recording the abundance of arthropods present on each leaflet with the aid of a stereo microscope (Leica M80) manufactured by Leica Microsystems Inc., Buffalo Grove, IL. Sampling was done for a period of four weeks.

Field Trials

Study site

Field trials were conducted twice from October 2013–March 2014 and from October

2014–April 2015. During the first growing season, the experiments were conducted at two

35 locations within Florida from October 2013 through March 2014. The first location was at the

University of Florida Plant Science Research and Education Unit (PSREU) in Citra, Marion

County, FL, with a calendar soil type and the second location was at a commercial organic strawberry farm in Plant City, Hillsborough County, FL. We chose to conduct the experiment at

Plant City because it is the major hub for strawberry production in Florida. In the second growing season, the experiment was conducted only at PSREU from October 2014 through April

2015 for easy accessibility and frequent monitoring. Prior to planting strawberries in both years, soil samples were collected and analyzed for nutrient content, sampled for presence of nematodes, and level of nematode infestation, if any.

An Organic Materials Review Institute (OMRI) approved fertilizer, Nature Safe® 10-2-8 all season fertilizer was applied at the rate of 1681 kg/ha before transplanting strawberries. After transplanting, the organic fertilizer (3-0-6; N-P-K, Howard Fertilizers, Orlando FL) was applied at 8 kg/ha N and 16 kg/ha K weekly through drip irrigation. Organic fungicides namely Double

Nickel® (Certis USA®, Columbia MD) applied at 3.36 kg/ha, Regalia® (Marrone® Bio

Innovations, Davis CA), at the rate of 2.34 L/ha and Actinovate® (Novozymes® BioAg Inc.,

Brookfield WI) at the recommended rate of 0.56 kg/ha were applied in rotations every two weeks through drip irrigation.

Plot layout and experimental design

The experimental design was a split plot design with the whole plots arranged in a randomized complete block with 4 replications. The whole plot factor consisted of cover crops and the sub-plot factor was strawberry cultivars. Eight strawberry cultivars were randomized to each whole plot following the termination of the cover crop treatments. There were 5 cover crop treatments including: 1) Indigofera hirsuta L. (hairy indigo), 2) Crotalaria juncea L. (sunn

36 hemp), 3) Crotalaria breviflora DC (short flower rattlebox), 4) Aeschynomene americana L.

(American jointvetch), and 5) a no cover crop weedy control. Individual whole plots were 6.1 m x 1.5 m spaced at 1.8 m apart, with 7.6 m alley (buffer zone) between blocks. During the 2014–

2015 growing season, only three cover crops treatments were evaluated. These treatments included: 1) I. hirsuta, 2) C. juncea, and 3) weedy control.

Planting

The cover crops were planted in July during the summer season preceding the strawberry season in both years. The cover crops were terminated and incorporated into the soil in

September of both years, in readiness for planting of strawberries.

Bare root transplants of eight commercial strawberry cultivars: Albion, Camarosa,

Strawberry Festival, Florida Radiance, Treasure, Winterstar™, Proprietary 1 and 2, were transplanted in October 2013. All the strawberry transplants except for Proprietary 1 and 2 were purchased from C. O. Keddy Nursery Inc., Lakeville, Nova Scotia. Strawberry cultivars were planted on raised beds covered with plastic mulch (Guardian®) in alternate double rows, according to standard strawberry planting techniques in Florida (Whitaker et al. 2016). The transplants were spaced at 0.4 m along and between rows. Transplants were watered regularly using the overhead sprinkler system for 24 h for the first five days and for five hours per day for the next five days to allow for plant establishment. The drip irrigation system was used afterwards until the end of the season. The drip irrigation was programmed to run for three times each day for 45 min each time, at a flow rate of 1.89 L/min/30.5 m of drip tape (Chapin®).

A similar experiment that utilized only a single cover crop, sunn hemp was conducted under the high tunnel system using a randomized complete block design with three additional

37 strawberry cultivars to the ones used in the open field system. The additional three cultivars were

Florida 127 (Sensation), Aroma 1 and 2.

During the second growing season, October 2014 through April 2015, three strawberry cultivars from the previous growing season that harbored the lowest TSSM population and high fruit yield were transplanted in the open field. Plug transplants of Winterstar™, Sensation, and

Strawberry Festival were transplanted (using a split plot design, similar to the one described in

2013). Plug transplants were used instead of bare root transplants because they require less water to establish in the field. Cropping practices such as irrigation, removing runners, tilling, and frost protection were routinely performed as necessary.

Sampling

Sampling began at four weeks after transplanting when transplants reached the five- trifoliate leaf stage. Plants were sampled weekly by randomly collecting three to five of the older and lower trifoliates of each strawberry variety from every treatment plot. The collected leaf samples were held in labelled Ziploc® bags (Racine, WI) in a cooler and transported back to the

Small Fruits and Vegetable IPM (SFVIPM) laboratory. Each trifoliate was examined under a stereo microscope (Leica M80, Buffalo Grove, IL) in the SFVIPM laboratory in the Entomology and Nematology Department, at the University of Florida, Gainesville, FL. All arthropods, at different stages of development found on the leaves were identified, counted and recorded according to the strawberry cultivar. The sampling period was approximately 19 weeks for each year.

Marketable yield

Harvesting was done once a week in the early season (November–December) and twice or three times per week during mid and late season (January–March) from all treatment plots. A

38 fruit was considered marketable if it showed no evidence of physical damage such as peck marks, holes, removed achenes (seeds), cat-faced injuries from insect, bird, or animal feeding, rots and mold from disease pathogens, and cracks from frost injury. In addition, small fruits (<

10 g) and deformed fruits were not considered marketable, because the defects could be due to nutrient deficiency and inefficient pollination, respectively. Culls (unmarketable fruits) were removed simultaneously from the field to prevent disease incidence. Marketable fruits and culls from each treatment plot were put in separate clamp shells and weighed with the aid of a scale

(TL 12001, Denver Instrument Company®, Arvada CO.).

Data analysis

Open field data in both locations were log transformed and analyzed using PROC

MIXED (SAS Institute 2010) to run a two-way analysis of variance (ANOVA) with cover crops and cultivars as the treatment effects. The interaction effect between cover crops and cultivars was also evaluated. Weekly data were pooled and one-way ANOVA was used to test for difference in the abundance of TSSM and TSSM eggs among cultivars in both locations.

Location was used as a factor to test for differences in the population of TSSM motiles on each strawberry cultivar.

In the greenhouse experiment, data were analyzed using repeated measures generalized linear model with a negative binomial distribution for TSSM motiles and square root transformation for TSSM eggs, with an unstructured correlation via PROC GLIMMIX in SAS

9.4. For the high tunnel experiment, repeated measures analysis was done using a generalized linear model with a square root transformation and an autoregressive correlation structure type 1

(ARH1) via PROC GLIMMIX in SAS 9.4.

Marketable yield and culls (unmarketable yield) for the open field production system in

2013–2014 were analyzed using PROC MIXED (SAS Institute 2012). The proportion of culls (P

Culls) for each cultivar was also analyzed using the formula 100 x total culls weight/total yield, where total yield is the sum of culls weight and marketable weight.

i.e. P Culls (%) = Culls weight x 100

Total fruit weight produced

Tukey’s multiple comparison procedure was used to perform the mean separation tests where significant difference was observed (P < 0.05).

Results

Population Density of Twospotted Spider Mite (TSSM)

Greenhouse study

In the greenhouse experiment (2013-14), the population of TSSM motiles on most cultivars fluctuated throughout the entire sampling period. The interaction effects between dates and cultivars (F = 4.49, df = 21, 43.1; P < 0.0001; Fig. 3-1) were significant. There were significant differences in TSSM population density among cultivars for motiles (F = 5.75; df = 7,

28.02; P = 0.0003) on different sampling dates (F = 10.04, df = 3, 18.67; P = 0.0004). Pooled data indicated that the highest number of TSSM motiles was recorded on Camarosa, and that the population of motiles kept increasing overtime until the last sampling date, when TSSM population declined on most cultivars.

For TSSM eggs, the interaction effects between sampling dates and cultivars were not significant (F = 1.56, df = 21, 52.34; P = 0.0991). There were significant differences among cultivars (F = 6.88, df = 7, 32; P < 0.0001), but sampling dates were not significant (F = 1.18, df

= 3, 30; P = 0.3330). Similarly, to the motiles, the highest population of TSSM eggs was on

Camarosa and this was significantly higher compared to cultivars like Proprietary 1, Proprietary

2, Treasure, and Winterstar™. The number of TSSM eggs that were oviposited on Albion,

Florida Radiance, and Festival were not significantly different from the other strawberry cultivars (Fig. 3-2).

Field trials

In 2013–2014 growing season, data were initially analyzed using repeated measures but there were no significant differences among the treatments over time. Therefore, weekly data were pooled together to examine treatment effects over the entire season. At the PSREU location, there was no significant interaction between cover crops and cultivars (F = 1.10, df =

28, 116; P = 0.3550) for TSSM motiles, but the interaction was significant for TSSM eggs (F =

1.18; df = 28, 104; P = 0.0197). There was no significant difference among the cover crop treatments for TSSM motile population (F = 0.93; df = 4, 116; P = 0.4490) and TSSM eggs (F =

0.73; df = 4, 11.7; P = 0.5897), although some cover crops numerically had lower TSSM populations. However, there were significant differences among the cultivars in the population density of TSSM motiles (F = 2.60, df = 7, 116; P = 0.0158) and eggs (F = 2.98; df = 7, 104; P =

0.0068). Cultivars like Florida Radiance, Winterstar™, and Festival had lower TSSM adult population than the other cultivars evaluated (Fig. 3-3). Subsequently these cultivars were used during the following growing season (2014-2015). At PSREU, Citra, the populations of TSSM motiles were significantly lower than Plant City. More eggs were present on cultivars that supported relatively high populations of TSSM motiles, and these cultivars (Albion and

Proprietary 2) harbored significantly more eggs than Festival, Winterstar™, and Florida

Radiance.

At the Plant City location, the result was similar to that obtained at Citra. There was no significant interaction between cover crop and cultivars for TSSM motiles (F = 0.99, df = 28,

105; P = 0.4853) and TSSM eggs (F = 0.84, df = 28, 105; P = 0.7005). However, the populations of TSSM motiles on the strawberry cultivars were significant (F = 10.57, df = 7, 105; P <

0.0001) with Festival, Winterstar™, and Florida Radiance harboring the lowest populations, but no significant difference among cover crops (F = 0.30, df = 4, 12; P = 0.8722). The population of

TSSM eggs on the cultivars were significantly different (F = 6.38, df = 7, 105; P < 0.0001), but not significantly different for cover crops (F = 1.07, df = 4, 12; P = 0.4141). The population of

TSSM eggs was lowest on the same cultivars that harbored the lowest TSSM motile populations

(Fig. 3-4).

We compared the population of TSSM in both locations and found that there were interaction effects between location and cultivar for TSSM motiles (F = 6.60, df = 7, 198; P <

0.0001). Location had a significant effect on the population of TSSM (F = 210.25, df = 1, 21.5;

P < 0.0001). In both locations, the cultivars performed differently and this was significant for

TSSM motile (F = 8.75, df = 7, 198; P < 0.0001) and egg populations (F = 2.98; df = 7, 104; P =

0.0068).

In the high tunnel system, the population of TSSM motiles was generally high compared to the open field. The interaction effects between date and the cultivars (F = 1.32; df = 198,

318.9; P = 0.0141) were significant. Also, significant differences were observed among the cultivars (F = 2.58, df = 11, 107.1; P = 0.0060) on different sampling dates (F = 30.50; df = 18,

184.3; P < 0.0001). Festival and Aroma 2 harbored more TSSM and this was significantly different from Sensation. Unlike the open field production system, Festival and Florida Radiance

42 harbored high mite populations like some susceptible cultivars, but Winterstar™ was consistent in harboring low mite populations in both open field and high tunnel systems.

The interaction effects between date and the cultivars (F = 1.50; df = 198, 353.9; P =

0.0005) were significant for TSSM eggs. The population of TSSM eggs were significant on the cultivars (F = 3.06; df = 11, 121.7; P = 0.0012) on different sampling dates (F = 31.44; df = 18,

199.8; P < 0.0001). In the high tunnel production system, Sensation had a lower TSSM population compared with Festival and Florida Radiance (Fig. 3-5) and for this, it was re- evaluated in the open field during the second year.

In 2014–2015, the interaction effects between cultivars and cover crops were significant

(F = 3.57; df = 4, 18; P = 0.0260; Fig. 3-6) in relation to the population of TSSM motiles, although there were no significant differences in TSSM motile populations on strawberry cultivars (F = 0.86; df = 2, 18; P = 0.4382) and cover crops (F = 0.75; df = 2, 6; P = 0.5100).

Among the three cultivars evaluated, Sensation had the lowest TSSM motile population during the sampling period. The populations of TSSM motiles were highest in areas where hairy indigo was mowed into the soil. Generally, we observed that TSSM population was highest for all cultivars during the midseason (weeks 7-12). There were no significant interaction effects between cover crops and cultivars (F = 1.59; df = 4, 18; P = 0.2208) in relation to the abundance of eggs oviposited on the cultivars. Also, no significant differences were observed for cover crops (F = 0.71; df = 2, 6; P = 0.5298) and cultivars (F = 1.69; df = 2, 18; P = 0.2133).

Marketable Yield

In the open field production system at Citra for the growing season 2013–2014, the interaction effects between cover crops and cultivars for marketable yield were not significantly different (F = 0.78; df = 28, 105; P = 0.7755). However, the total marketable yield was

43 significantly different among cultivars (F = 11.91; df = 7, 105; P < 0.0001; Fig. 3-7), but cover crops had no significant effect on marketable yield (F = 0.43; df = 4, 12; P = 0.7819).

The weight of culls obtained from treatment plots showed significant differences among cultivars (F = 20.67; df = 7, 105; P < 0.0001), but not for cover crops (F = 2.06; df = 4, 12; P =

0.1500). As expected, the proportion of culls varied significantly among the cultivars (F = 58.74; df = 7, 105; P < 0.0001). However, we observed that the proportion of culls depended on the total fruit weight produced, with an indirect relationship between proportion of culls and marketable fruit weight. This was true for all the cultivars except Albion that had a slightly larger proportion of culls compared to Proprietary 1 that had a lower total market weight (Table 3-1).

At Plant City, during the same growing season (2013–2014), there was no significant interaction between cultivars and cover crops (F = 0.70; df = 28, 105; P = 0.8563). Significant differences were observed among cultivars (F = 23.13; df = 7, 105; P < 0.0001) for the total marketable weight, but not for cover crops (F = 3.04; df = 4, 12; P = 0.0605). The weight of culls was significantly different among cultivars (F = 2.89; df = 7, 117; P = 0.0080) and cover crops

(F = 2.64; df = 4, 117; P = 0.0371). The proportion of culls was significantly different among cultivars only (F = 13.44; df = 7, 105; P < 0.0001), but unlike the result obtained at Citra, where

Festival had the highest marketable weight and the lowest cull weight, the lowest proportion of culls was recorded on Radiance and it had the highest marketable weight (Fig. 3-8). The lower yielding cultivars tended to have lower weight of culls; however, Proprietary 2, Albion, and

Treasure also had the highest proportion of culls.

Discussion

Greenhouse Trial

In this experiment, Proprietary 1, Winterstar™, and Proprietary 2 harbored the lowest

TSSM population. From these results, different cultivars had the highest or lowest mite population on weekly basis. We do not find these results conclusive and it requires further research under greenhouse conditions. Our results from the field studies were more consistent, which suggests that factors such as temperature, humidity, and solar radiation in greenhouse settings may influence how cultivars respond to TSSM pressure. For instance, the conditions in the greenhouse were different to the field with weekly averages in the greenhouse temperature of

27.0 ± 5.1 °C and relative humidity ~ 65% compared with the field with wide fluctuations ranging from 2.0 ± 1.0 °C to 27.0 ± 3.8 °C and relative humidity >80%. Therefore, cultivars that performed well under field conditions may fail when exposed to greenhouse settings. Another explanation is that it is possible that some spider mites moved from susceptible cultivars to other with the aid of air blowing from a cooling fan in the greenhouse and/ or mechanical tools like hose or watering can during routine watering.

Field Experiments

We saw no direct effects of the cover crops with respect to mite abundance on the different cultivars. The hypothesis is that cover crops will add to soil nutrients, which will cause some cultivars to become more robust and thus increase their defenses against TSSM infestation.

A different site location was used in the second growing season, hence there were no cumulative effects of the cover crops. This theory may be valid if cover crops are allowed to add to the soil nutrients over time (as a result of decomposition).

The population of TSSM was higher at the commercial grower’s farm in Plant City compared to the research station at Citra. We attributed the higher TSSM population to the overwintering sites and proximity of the strawberry field to other cultivated plants. Also, the planting locations used at Citra in both years were virgin lands (for strawberry production), hence the slow buildup and low populations of TSSM. However, the overall population of TSSM was higher in the second growing season (2014-15) probably because of weather conditions and overwintering adults from the previous season.

In our study, Festival, Winterstar™, and Florida Radiance had the lowest TSSM population in the open field production system. Radiance was not used in the following year because its supply was not available with our vendor, therefore Sensation was evaluated in the open field system because of its good performance in the high tunnel. Our findings are similar to previous studies that reported cultivars of a particular plant species can vary in their ability to tolerate pests (McFarlane and Hepworth 1994, Romeih et al. 2013). For example, in a similar study conducted by Rhainds et al. (2002), ‘Honeoye’ was recommended as the most suitable strawberry cultivar for organic production in the Northeastern region because of its tolerance to tarnished plant bug and high yield.

In the first growing season (2013-14) at PSREU, Citra, the overall population of TSSM on the strawberry cultivars was higher under the high tunnel system. This could be due to adaptation differences under different production systems. Costa et al. (2017) evaluated three management systems (open field, high tunnel, and low tunnel) and they found that the population of TSSM was highest under the low tunnel management system and lowest in the open field.

In the high tunnel system, we found high populations of TSSM on Florida Radiance and

Festival. These results are similar to a study conducted by Costa et al. (2017) that showed the

46 population of TSSM on several strawberry cultivars was influenced by the production system.

For example, Costa et al. (2017) found that cultivars like ‘Albion’, ‘Camino Real’, and ‘Ventana’ had higher TSSM populations in the open field system compared to the high tunnel. The low tunnel production system supported higher TSSM populations on other cultivars. Also, TSSM populations on Festival and Florida Radiance were high probably because the plants were closely planted to each other. There may be a possibility that the proximity to susceptible cultivars like

Aroma 1, Aroma 2, and Proprietary 1 made it easier for TSSM to colonize cultivars like Festival and Radiance that were not susceptible. We believe that Proprietary 1, Aroma 1, and Aroma 2 are more susceptible because they harbored more mites by the first day of sampling, which was at four weeks after transplanting. Overall, Proprietary 1 harbored the highest TSSM population in the open field production system at Citra.

Generally, the high tunnel strawberry production system is not recommended in Central and Southern Florida because of the warm temperatures that may build up within the high tunnel, thus making TSSM management difficult. The warm temperatures would trigger high reproduction rate of TSSM as the intrinsic rate of TSSM increases with higher temperatures

(Kaur and Zalom 2018) within an optimal range. This production system may work in Northern

Florida or other states with cooler climatic conditions and thus contribute to increase in strawberry production.

Marketable Weight

The marketable yield varied among cultivars in both locations. Cultivars differ genotypically and this may affect fruit characteristics such as shelf life, size, skin thickness/ penetration force (by insects) that affect marketability of fruit. Festival and Florida Radiance had the highest marketable weights in Citra and Plant City respectively. In Plant City, Festival

47 produced slightly lower yield than Florida Radiance. The marketable yield produced by Florida

Radiance was quite low in Citra, but it produced the highest yield in Plant City. We think that differences in fertility programs, weather conditions (such as less rain and frost), adaptability differences, and management tactics (fencing to prevent animals like rodents and scarecrows to prevent birds) may be responsible for the higher yield obtained at the grower’s farm in Plant

City. Also, the overall higher yield reported of cultivars in Plant City may vary due to the implementation of a different fertility program compared to Citra. Some of the factors previously discussed regarding the differences between Citra and Plant City may also account for the differences in yield obtained.

With the exception of Festival and Camarosa in Citra, other cultivars in Citra had higher cull weight compared to the marketable fruit yield. At both locations, fruits were harvested solely for fresh market with more stringent requirements at the grower’s organic farm in Plant City because the harvested fruits were marketed to a bigger strawberry firm.

We did not run a regression analysis to measure the relationship between TSSM population and marketable fruit weight, however we observe that the cultivars with the lowest

TSSM population (Festival, Winterstar, and Radiance) had the highest marketable fruit weight in

Plant City, this is not exactly true for strawberry production at Citra.

Albion F = 4.49; df = 21, 43.1; P < 0.0001 Camarosa 140 Proprietary1 Proprietary2 120 Festival Radiance Treasure 100 Winterstar

20 Mean population of TSSM motiles per per leaflet of motiles TSSMMean population 0 03/26/14 04/03/14 04/10/14 04/17/14 Sampling dates

Figure 3-1. Mean population density of twospotted spider mite (TSSM) on strawberry cultivars grown in the greenhouse on different sampling dates. Vertical error bars represent the standard error of the mean.

200 a F = 6.88; df = 7, 32; P < 0.0001 180

160

140

120 ab eggs per leaflet eggs abc 100 abc 80

60 bc 40 bc

20 c c Mean of TSSMMean population 0

Strawberry cultivars

Figure 3-2. Mean population density of twospotted spider mite eggs on strawberry cultivars in the greenhouse. Means followed by the same letters are not significantly different, Tukey’s test. P < 0.05. (SAS 9.4, 2012).

A B

C D

Figure 3-3. Mean population of TSSM motiles and eggs on selected commercial cultivars established at Plant Science Research and Education Unit (PSREU) Citra, FL during the 2013-14 strawberry season. Means followed by the same uppercase letters (for TSSM motiles) and lowercase letters (for TSSM eggs) are not significantly different, Tukey’s test. P < 0.05. Graphs without letters are not significantly different.

A B

C D

Figure 3-4. Mean population of TSSM motiles and eggs on selected commercial cultivars established at Plant City, FL during the 2013-14 strawberry season. Means followed by the same uppercase letters (for TSSM motiles) and lowercase letters (for TSSM eggs) are not significantly different, Tukey’s test. P < 0.05 (SAS 9.4, 2012). Graphs without letters are not significantly different.

TSSM 350 TSSM EGGS a a 300

250

and and eggs/trifoliate/week ab 200 ab ab ab ab 150 ab ab A AB AB 100 AB AB ab ab AB AB AB AB 50 AB AB b B

0 Mean of motiles TSSMMean population

Strawberry cultivars

Figure 3-5. Mean population of TSSM motiles and TSSM eggs in high tunnel system on selected strawberry cultivars under the high tunnel management system for the entire season. Means followed by the same lowercase letters (for orange line) and uppercase letters in common (for blue line) are not significantly different at (Tukey’s test; P < 0.05) (SAS 9.4, 2012).

Figure 3-6. Mean population of TSSM motiles and eggs on selected commercial cultivars established at Plant Science Research and Education Unit (PSREU) Citra, FL during the 2014-15 strawberry season. Means not followed by the same letters are significantly different, Tukey’s test, P < 0.05 (SAS 9.4, 2012). Graphs without letters are not significantly different.

4.5 Marketable weight 4 Culls weight a

3.5 a 3

weights for the entire season entirethe for weights 2.5 A bc b A bc bc bc

fruit AB

(x 1000 kg) 1000 (x 2 c ABC 1.5 BCD CD D D 1

0.5

0 Mean culls and marketable and cullsMean

Strawberry cultivars

Figure 3-7. Mean weight of culls and marketable fruit weight harvested on selected strawberry cultivars at the Plant Science Research and Education Unit (PSREU) Citra, FL during the 2013-14 growing season. Means for the same color bar with no letter in common are significantly different (Tukey’s test; P < 0.05) (SAS 9.4, 2012). Lower case letters (for orange bars, represent weight of culls) and uppercase letters (for blue bars, represent the marketable yield of strawberry on each cultivar).

9 Marketable weight A Culls weight 8 AB ABC 7 BC

6 CD

5 a DE ab b 000 kg) 000 ab ab E E 4 b

x 1 x b b ( 3

Mean culls and marketable fruit weights for the entire season entirethe for weights fruit marketable and cullsMean 0

Strawberry cultivars

Figure 3-8. Mean weight of culls and marketable fruit weight harvested on selected strawberry cultivars at a grower’s commercial farm in Plant City, FL during the 2013-14 growing season. Means for the same color bar with no letter in common are significantly different (Tukey’s test; P < 0.05) (SAS 9.4, 2012). Lower case letters (for orange bars, represent weight of culls) and uppercase letters (for blue bars, represent the marketable yield of strawberry on each cultivar).

Table 3-1. Proportion of culls with mean marketable fruit weight for all strawberry cultivars produced at Plant Science Research and Education Unit (PSREU), Citra and Plant City, FL. CULTIVAR Citra P Culls (%) Plant City P Culls (%)

PROPRIETARY1 64.26 bc 39.32 bc

PROPRIETARY2 78.57 a 46.35 a

ALBION 67.66 b 47.87 a

CAMAROSA 49.32 de 41.56 ab

FESTIVAL 43.82 e 34.06 c

RADIANCE 60.98 c 33.44 c

TREASURE 65.56 bc 46.36 a

WINTERSTAR 52.79 d 36.40 bc

Means followed by the same letters within columns are not significantly different ((Tukey’s test; P < 0.05) (SAS 9.4, 2012).

P Culls (%) = Culls weight x 100 Total fruit weight produced where total fruit weight produced = marketable yield + culls weight

CHAPTER 4 DISPERSAL AND MOVEMENT OF NEOSEIULUS CALIFORNICUS MCGREGOR

Introduction

The twospotted spider mite (TSSM), Tetranychus urticae Koch, feeds on a wide variety of plants. It has been reported as a pest on more than 900 plant species (Kavousi et al. 2009) such as strawberry, tomato, and bean. Twospotted spider mites usually move within and among plants by crawling. Long distance movement is wind-assisted (Margolies and Kennedy 1985) or by human interference (mechanical tools and clothing). However, predatory mites search for their prey (TSSM) by walking and can penetrate the web of TSSM for feeding purposes. Although predators learn associations between plant odors in response to herbivory (Drukker et al. 2000,

Amin et al. 2009) and in response to the presence of competitors for prey or conspecific predators on plants (Janssen et al. 1997, Zahedi-Golpayegani et al. 2007), predatory mites are often met with obstacles such as deviating routes (Sabelis and van der Weel 1993), weather conditions, and higher order predators.

Predatory mites such as Phytoseiulus persimilis Athias-Henriott and Neoseiulus californicus McGregor are effective natural enemies of TSSM (Oatman et al. 1977, Rhodes and

Liburd 2006, Fraulo and Liburd 2007). These predatory mites can locate their prey using volatiles released by plants after being fed on by TSSM (Sabelis and van der Weel 1993). The response of predators to plant volatiles depends on the composition and concentration of the emitted volatiles (Bruce and Pickett 2011). In addition to predatory mites being attracted to plant volatiles, TSSM also release volatiles termed kairomones that attract predators to them (Hoy and

Smilanick 1981). For example, Schmidt (1976) observed P. persimilis wandering around the web spun by TSSM. Predators also release volatiles as an inevitable by-product of physiological activity, which serves to deter other competitors (Janssen et al. 1997).

Neoseiulus californicus can persist in agricultural systems as a type II predator feeding on pollen and other prey in the absence of TSSM (McMurtry and Croft 1997), although their intrinsic rate of increase is higher when TSSM is the primary food source compared to other alternate food sources (Khanamani et al. 2017). To our knowledge, no research study has been conducted that examined the dispersal pattern of N. californicus in relation to the presence and abundance of TSSM. To determine how far N. californicus can move in search of TSSM, we designed an experiment using mark-release-recapture (MRR) technique.

Mark-release-recapture is a technique used to study the population and dispersal of an organism within an ecosystem (Forschler and Townsend 1996, Marini et al. 2010). This technique involves marking insects with a material (Hagler and Jackson 2001), releasing them into the field, and employing appropriate sampling techniques to recapture the marked insects after a given time interval (Hagler 1997). The choice of a marker to be used depends on the insect being marked. Factors affecting the choice of a marker include durability of the marker, cost effectiveness of the marker, the application method of the marker, size and life stage of the arthropod, and safety to the organism (Hagler 1997). Paints, dyes, and dusts (Naranjo 1990), radioactive markers (Hight et al. 2005), and protein markers (Hagler 1997, Boina et al. 2009) have all been used with this technique.

Our experiment was conducted to estimate the distance that overwintering N. californicus will move in search of its food. The specific objectives were to: 1) identify the strawberry cultivar(s) that would attract and support high population of N. californicus 2) estimate the dispersal distance of N. californicus from surrounding or adjacent vegetation into infested strawberry fields, 3) study some life history traits of N. californicus on selected commercial cultivars, and 4) study the behavioral response of N. californicus to TSSM on selected strawberry

59 cultivars. We hypothesized that natural populations of N. californicus are attracted to mite- infested leaves, and their dispersal is guided by the availability of TSSM.

Materials and Methods

Colony: Neoseiulus californicus was initially purchased from Koppert® Biological

Systems (Howell, Michigan) and were used as the parent stock to start a colony of predatory mites. A black coarse card stock was used as the habitat for raising generations of N. californicus. The coarseness was achieved by pouring liquid candle wax on the card and wire mesh was placed on the card before the wax changed to the solid state. The coarse card stock was placed on moist cotton in a 140 mm plastic square weigh boat (Morganville Scientific®,

Morganville, NJ) and N. californicus were introduced onto the coarse card. The perimeter of the plastic container is filled with 10% bleach solution to minimize the movement of mites away from the card stock. These predatory mites were reared in the Small Fruit and Vegetable

Integrated Pest Management (SFVIPM) laboratory under ambient room temperature ~ 23oC with relative humidity (RH) 65%. Predatory mites were fed on a diet of TSSM that were brushed from bean leaves with the aid of a mite brushing machine (Leedom® Enterprises, Mi Wuk Village,

CA). The predatory mites were occasionally provided with honey on a cotton wick. The TSSM that were used as diet for the predatory mites were obtained from the colony maintained on pinto bean (Phaseolus vulgaris L.) plants raised in a greenhouse (as described in chapter 3). At 21 d intervals, five adult N. californicus females were sub-cultured from the original colony to start a new batch of colonies.

Laboratory Experiments

These experiments were conducted in January 2018 on three cultivars of strawberry:

Winterstar™, FL 127 (Sensation), and Strawberry Festival. The plug transplants of these

60 cultivars were purchased from Luc Lareault Inc., Lavaltrie, QC, Canada. Strawberry plants of each cultivar were initially grown using standard commercial organic production techniques in individual one-gallon pots in a greenhouse at 27 ± 5 °C, 52 ± 20% RH, and under natural photoperiod 12:12 L:D (Florida’s winter months regime). No miticides or insecticides were applied to the pots before the release of N. californicus. Neoseiulus californicus that was used in these experiments were F1 and F2 generations obtained from the laboratory colony.

a) Preference of predatory mites: The behavioral response of predatory mites, N. californicus and P. persimilis to different strawberry cultivars was observed using the three cultivars earlier mentioned. Three leaflets of each cultivar were placed in a 10 cm diam. Petri dish equidistant from each other on a moistened Whatman® filter paper (purchased from Thermo

Fisher Scientific, Gainesville, FL) lined with cotton wool. One predatory mite of either species

(N. californicus and P. persimilis) was chosen at random and released in the center of the Petri dish and the direction of its movement was observed for 10 min or until a choice was made. Each predatory mite was used once and replaced after a choice was made or after 10 min without any choice.

Field Trials

Predatory mite introduction: Neoseiulus californicus was purchased from Koppert®

Biological Systems (Howell, Michigan). Predatory mites were kept in the laboratory for one to three days prior to use, depending on the prevailing weather conditions. A viability test was done prior to release. This involved observing mites in a 5 cm diam. Petri dish to make sure they are active before releasing them in the field. Neoseiulus californicus were released by gently shaking containers over TSSM infested strawberry plants (with the goal of achieving a 10: 1 TSSM: N. californicus ratio, respectively). Neoseiulus californicus were put in 500 ml bottles, containing ~

25,000 N. californicus at different motile stages mixed with inert materials such as vermiculite.

These predatory mites were released onto strawberry plants nine weeks after transplanting. At this time, TSSM population had established in the field.

a) Population density of Neoseiulus californicus: Strawberry plants were grown in the field using the protocol described in objective 1 (Chapter 3). As indicated during 2013, eight commercial strawberry cultivars including Albion, Camarosa, Strawberry Festival, Florida

Radiance, Treasure, Winterstar™, Proprietary 1 and 2 were transplanted at the Plant Science

Research Education Unit (PSREU) at Citra, FL and at a commercial organic farm in Plant City,

FL.

Sampling was conducted weekly at the research station in Citra, but once in two weeks at the grower’s farm in Plant City because of the distance from the university. Sampling was done by collecting three to five lower trifoliate leaves from every treatment plot. The samples were placed in labelled Ziploc® (Racine, WI) bags and taken to the Small Fruit and Vegetable

Integrated Pest Management (SVIPM) laboratory for further processing as earlier discussed in

Chapter 3. In 2014-2015 growing season, only three cultivars; Winterstar™, Festival, and

Sensation were planted and evaluated for their differential ability to support population of N. californicus.

b) Dispersal of Neoseiulus californicus: The field component of the dispersal study was conducted at PSREU in Citra, FL on three strawberry cultivars, Festival, Sensation, and

Winterstar™. Each cultivar was planted on plastic mulch beds measuring 12 m by 1.5 m. These plants were grown using standard production techniques as described in Chapter 3 without the application of miticides or insecticides. Pre-counts of TSSM and N. californicus were conducted on each plot to assess population densities prior to releasing N. californicus.

The protein marker used is the commercially available chicken egg albumin (All Whites,

100% liquid egg whites by Crystal Farms, Lake Mills, WI), hereafter referred to as egg white. It was purchased from Publix Supermarket, a grocery store located in Gainesville, FL. The egg white was diluted to 10% in water, and few drops of Tween 20 were added to the solution to reduce its surface tension. The egg white solution was applied to the strawberry leaves immediately after N. californicus release using an atomizer to spray a fine mist of the solution on the predatory mites. The leaves were sprayed until complete coverage on the plants was achieved.

Neoseiulus californicus, purchased from Koppert® Biological Systems

(www.koppertonline.com), were released on healthy strawberry leaves by shaking the motiles along with the medium (usually vermiculite or sawdust) from a 500 ml or 250 ml bottle containing ~25,000 or ~5000 N. californicus, respectively with perforated circular holes. The introduction of the protein-marked predatory mites was restricted to a 3 m area for each treatment bed. Five to 20 handpicked TSSM were placed on the remaining strawberry plants.

Sampling was done at 2 and 5 days after predatory mite release, and at one-week intervals afterwards for a period of four weeks. The undersides of leaves were examined in-situ for N. californicus at distances of 1.5 m, 3 m, 6 m, and 9 m from the point of release on each bed to track the predator movement in relation to its prey location. The experiment was repeated three times on each cultivar. To distinguish between marked predatory mites and naturally occurring ones, the presence of protein on the predatory mites will be tested using the enzyme-linked immunosorbent assay (ELISA) technique, as developed by Jones et al. (2006), and modified by

Boina et al. (2009).

Data Analysis

In the choice experiment to study the behavioral response of the two predatory mites to the different strawberry cultivars, the GENMOD procedure was used to analyze the number of times a cultivar was chosen as a proportion of the total number of the predatory mites used. A mixed logistic regression was used to test if there were significant differences among the cultivars.

Open field data in both locations were log transformed and analyzed using PROC

MIXED (SAS Institute 2012) to run a two-way ANOVA with cover crop as whole plot factor and cultivar as the subplot factor. We found no significant effect of the cover crops and although repeated measures were initially used, but there was no significant effect over time, because the populations of N. californicus motiles and eggs were too low and not found on some treatment plots. In the high tunnel experiment, repeated measures analysis was done using a generalized linear model with a square root transformation and an autoregressive correlation structure type 1

(ARH1) via PROC GLIMMIX in SAS 9.4. Tukey’s multiple comparison procedure was used to perform the mean separation tests where significant difference was observed (P < 0.05).

Results

Preference of Predatory Mites

Of the 90 N. californicus evaluated, significant differences were observed among the cultivars (χ2 = 40.07; df = 3; P < 0.0001; Table 4-1). Six of the N. californicus did not make any choice among the cultivars within the time frame allotted for observation. Twenty-seven P. persimilis were used in this experiment and we observed significant differences among the cultivars (χ2 = 23.27; df = 3; P < 0.0001; Table 4-2). Both predatory mites preferred

Winterstar™ and this was significantly different from Festival. Neoseiulus californicus showed equal preference to Winterstar™ and Sensation and this was significantly different from Festival.

Only few N. californicus moved towards infested Festival leaflets and this was not significantly different from the N. californicus that did not make any choice among the cultivars within the allotted time of observation for each predatory mite.

Field Trials

Population density of the predatory mite, Neoseiulus californicus

At the PSREU location, there were no significant interaction effects between cover crops and cultivars for N. californicus motiles (F = 0.85, df = 28, 103; P = 0.6852) and on the abundance of N. californicus eggs (F = 1.39, df = 28, 116; P = 0.1163) oviposited on the strawberry cultivars. No significant differences were observed among the cover crops in relation to the population density of N. californicus motiles that established on the strawberry cultivars (F

= 1.45, df = 4, 11.3; P = 0.2811).

The population of N. californicus motiles was significantly different among the strawberry cultivars (F = 5.76; df = 7, 103; P < 0.0001; Fig. 4-1). However, the number of eggs oviposited by predatory mites on the strawberry cultivars was generally low, and unlike the motiles, there was no significant difference among cultivars (F = 1.71, df = 7, 116; P = 0.1128),

65 but cover crops had a significant effect on the number of eggs oviposited (F = 3.18, df = 4, 116;

P = 0.0161).

At the organic commercial farm in Plant City, the interaction effects between cover crops and cultivars in relation to the population of N. californicus motiles were not significant (F =

1.55, df = 28, 105; P = 0.0594). The population of N. californicus was significantly different among cultivars (F = 9.01, df = 7, 105; P < 0.0001). The population of N. californicus was lowest on Florida Radiance and Winterstar™.

Similar results were obtained for N. californicus eggs. There were no significant interaction effects between cover crops and cultivars for N. californicus eggs (F = 0.52, df = 28,

105; P = 0.9744). However, the number of N. californicus eggs oviposited on the strawberry cultivars varied significantly (F = 4.23, df = 7, 105; P = 0.0004; Fig 4-2).

In the 2014-2015 growing season, the experiment was only conducted at PSREU, Citra.

Three cover crops and three cultivars were evaluated. We did not find any significant interactions between cover crops and cultivars in relation to populations of N. californicus motiles (F = 0.60; df = 4, 18; P = 0.6687). There were no significant differences in the populations of N. californicus motiles among the cultivars (F = 0.75; df = 2, 18; P = 0.4875; Fig

4-3). Similarly, the populations of N. californicus eggs on all three cultivars were not significantly different (F = 0.11; df = 2, 18; P = 0.9007). Because cover crops had no significant effect on the population of N. californicus, data were pooled together to observe time effects on the population of N. californicus on each cultivar. We observed that N. californicus populations increased towards the end of the strawberry season (Fig. 4-4).

Other beneficial arthropods encountered during routine sampling included ladybugs

(Coccinellidae), sixspotted thrips (Thripidae), spiders, and lacewings (Chrysopidae and

Hemerobiidae). We did not present data on the abundance of these beneficial insects.

Dispersal of Neoseiulus californicus

The total number of N. californicus recaptured was too low compared to the number of marked predatory mites that were released. Therefore, we could not justify performing ELISA to ascertain if all the predatory mites recovered were the ones we marked and released in the strawberry plots. A total number of 32 N. californicus motiles and 3 eggs were recovered during sampling. Most of the motiles were found on the Sensation cultivar (Table 4-3). This is consistent with the result obtained in the choice experiment where N. californicus motiles responded equally to Winterstar and Sensation cultivars.

Discussion

Preference of Predatory Mites

For all three cultivars evaluated, Winterstar™ was the most preferred cultivar to both predatory mites, N. californicus and P. persimilis. Although, the number of times N. californicus selected Winterstar™ was almost the same for Sensation, however more eggs were laid on

Winterstar compared to Sensation and no eggs were laid on Festival. Festival was the least preferred cultivar, this finding is similar to results obtained in field experiments in previous years

(Chapter 3) where the populations of N. californicus were higher on cultivars like Winterstar™ and Sensation even though Festival harbored more TSSM. A number of reasons could account for this.

Predatory mites search for their prey in response to plant volatiles emitted as a signal of herbivory, but we are not certain if the concentration of the volatiles increases with infestation

67 and prey abundance. The reproduction rate of predatory mites is expected to increase with prey

(TSSM) availability, however, it is possible that female predatory mites prefer Winterstar™ and

Sensation as oviposition substrates and thus move back to cultivars like Festival that may have higher TSSM abundance or it could be that egg hatchability of N. californicus is low on Festival.

Microclimate may be more suitable for egg hatchability and survival on Winterstar™ and

Sensation due to leaf morphology and characteristics, such as leaf thickness and trichomes.

We observed that the response time to make a choice among the strawberry cultivars was shorter for P. persimilis. They have higher searching capability because they are specialist predators of tetranychids (McMurtry and Croft 1997).

Population Density of Neoseiulus californicus

Cultivars like Albion, Proprietary 1, and Camarosa had the highest mean population of N. californicus. These cultivars supported high TSSM population (Chapter 3). It appears as if the populations of N. californicus on the cultivars may have been guided by the abundance of TSSM on the leaves and not by leaf morphology and characteristics, such as pubescence, waxiness, and trichomes as reported by previous studies for other predatory mites on other crops (Sabelis et al.

1999, Duso et al. 2003, Loughner et al. 2008). However, both of these factors could have influenced the population of N. californicus.

At the PSREU location in 2013-2014, the mean populations of N. californicus eggs were almost zero, except in areas that were cover cropped with sunn hemp and hairy indigo. However, in Plant City, the populations of N. californicus motiles and eggs were significantly higher compared with Citra. The difference in population density could be due to the higher prey

(TSSM) density at Plant City or it could also be due to the diverse vegetation that could serve as an alternate food source for N. californicus.

Neoseiulus californicus builds its population gradually over time, as they feed on TSSM.

This explains why the population of N. californicus increases towards the end of the season, as the population of TSSM declines. In a high tunnel (discussed under chapter 3), cultivars like

Winterstar™ and Sensation supported high populations of N. californicus despite the lower

TSSM harbored compared to Florida Radiance and Festival. This confirms that Winterstar™ and

Sensation may have chemical or physical properties that attract and support increase in population of N. californicus.

Dispersal of Neoseiulus californicus

Neoseiulus californicus does not move at long distances within a short time period unlike

TSSM, which can be dispersed by wind. Predatory mites move in search of their prey by walking, therefore leaf connectedness for crawlers like N. californicus is very important to achieve their fast colonization on infested strawberry plants. Sensation and Winterstar™ favored dispersal of N. californicus along farther distances compared to Festival. Festival usually puts out a lot of foliage compared to other cultivars, this may account for the slow movement of N. californicus as they would rather stay and feed on the TSSM available on the foliage until there are none. The absence of prey will force the predatory mite to move in search of food.

We could not conduct the ELISA test for protein on N. californicus due to the low recapture rates. However, we presumed that all sampled motiles were recaptures of our previous release. This is because previous monitoring of the field before the experiment showed that the plots were free from N. californicus. We had introduced TSSM to some areas to encourage the movement of N. californicus from the point of release towards their prey (TSSM). All the recaptured N. californicus were caught within the first week of sampling (post release of N.

69 californicus), we believe it would take longer than a week for N. californicus in the wild to walk from adjacent vegetation areas to TSSM infested plots.

Table 4-1. Behavioral response of Neoseiulus californicus to strawberry cultivars manually infested with twospotted spider mites (TSSM). Cultivar z Value Mean ± SEM Pr > |z| Festival 0.2828 0.1667 ± 0.04 b < 0.0001 Sensation 0.2174 0.3778 ± 0.05 a 0.0217 Winterstar 0.2162 0.3889 ± 0.05 a 0.0366 None 0.4226 0.0667 ± 0.03 b < 0.0001 Means followed by the same letters within the same column are not significantly different (Tukey’s test; P < 0.05) (SAS 9.4, 2012). None accounts for the number of times N. californicus did not make any choice among the strawberry cultivars within the allotted observation period of 10 min.

Table 4-2. Behavioral response of Phytoseiulus persimilis to strawberry cultivars manually infested with twospotted spider mites (TSSM). Cultivar z Value Mean ± SEM Pr > |z| Festival -3.40 0.1111 ± 0.06 b 0.0007 Sensation -2.71 0.2222 ± 0.08 b 0.0068 Winterstar 0.96 0.5926 ± 0.09 a 0.3387 None -0.73 0.0741 ± 0.05 b 0.0006 Means followed by the same letters within the same column are not significantly different (Tukey’s test; P < 0.05) (SAS 9.4, 2012). None accounts for the number of times P. persimilis did not make any choice among the strawberry cultivars within the allotted observation period of 10 min.

A B

C D

Figure 4-1. Mean population of Neoseiulus californicus motiles and eggs on commercial cultivars established at Plant Science Research and Education Unit (PSREU), Citra during the 2013-2014 growing season. Means followed by the same uppercase letters (for N. californicus motiles) and lowercase letters (for N. californicus eggs) are not significantly different, Tukey’s test. P < 0.05 (SAS 9.4, 2012). Graphs without letters are not significantly different.

A B

C D

Figure 4-2. Mean population of Neoseiulus californicus motiles and eggs on commercial cultivars established at a grower’s organic farm in Plant City, during the 2013-2014 growing season. Means followed by the same uppercase letters (for N. californicus motiles) and lowercase letters (for N. californicus eggs) are not significantly different, Tukey’s test. P < 0.05 (SAS 9.4, 2012). Graphs without letters are not significantly different.

Figure 4-3. Mean population of Neoseiulus californicus motiles and eggs on commercial cultivars established at Plant Science Research and Education Unit (PSREU), Citra during the 2014-2015 growing season.

1.6

1.4 Festival 1.2 Sensation

Winterstar

per trifoliate leaf trifoliate per

1.0

0.8 N.californicus 0.6

0.4

0.2 Mean population of of population Mean 0.0 1 3 5 7 9 11 13 15 17 19 Sampling period (weeks)

Figure 4-4. Mean population of N. californicus motiles in open field strawberry in Citra, FL during the 2014-2015 strawberry growing season.

Table 4-3. Total number of Neoseiulus californicus recovered on strawberry cultivars in response to distances from the point of release. Cultivar Distance (m) N. californicus motiles N. californicus eggs

0.3 1 0 Festival 0.6 1 0

0.9 1 0

0.3 4 0 Sensation 0.9 10 0

1.5 6 0

0.3 2 0 Winterstar 0.9 4 0

1.5 3 3

CHAPTER 5 SUSCEPTIBILITY OF STRAWBERRY CULTIVARS TO STING NEMATODE, BELONOLAIMUS LONGICAUDATUS RAU

Introduction

Plant parasitic nematodes are one of the most important biotic factors that reduce quality and quantity of strawberry yield. Sting nematode (Belonolaimus longicaudatus Rau), northern root-knot nematodes (Meloidogyne hapla Chitwood), root-lesion (Pratylenchus penetrans Cobb), and foliar nematodes (Aphelenchoides spp.) have been reported in strawberries in Florida

(Nyoike et al. 2012, Noling 2016, Desaeger and Noling 2017). The sting nematode is a common pest of many wild and commercially cultivated flowering plants, vegetables, and fruit crops

(Crow 2015), including strawberry (Abu-Gharbieh and Perry 1970). It is the primary nematode pest attacking strawberry plants in Florida (Abu-Gharbieh and Perry 1970, Noling 2016), if measured in yield reduction. It is an ectoparasite found in the soil, with a long slender body and long stylet.

Sting nematodes are usually found in sandy soils, where their reproduction rate is higher compared with other soil types (Miller 1972, Robbins and Barker 1974, Norton 1979). This nematode’s preference for sandy soils is a major factor that accounts for their abundance and distribution in Florida soils, as well as the warm climate throughout the state (Boyd and Dickson

1972). The sting nematode became a noted pest in the southeastern United States in the 1950s, and the increase in its abundance is likely due to a change of the cover crop used to suppress the nematode population (Noling 2016). For example, Noling (2016) mentioned that many growers stopped using velvet bean as a cover crop and adopted the use of sesbania as a summer cover crop prior to planting strawberry in the fall. This change was thought to be a factor in the increased population of sting nematode on strawberry.

Sting nematode injury on roots cause abnormalities at the root tip, which result in little or no new root growth, lack of fine feeder roots in plants, and the development of short stubby branches. In addition, necrotic lesions may also be produced laterally along the sides of roots

(Noling 2016). These root injuries negatively affect water and nutrient translocation, which in turn cause stunting and decline in strawberry growth. Other symptoms of the sting nematode on strawberry include gradual browning from leaf edges that spread to the midrib and entirely on the leaves, chlorosis, and eventual death (Boyd and Perry 1970).

The sting nematode exists in many physiological races, based on morphological differences and host ranges between geographic populations (Noling 2016). Abu-Gharbieh and

Perry (1970) identified three of the physiological races of B. longicaudatus in Florida. The physiological races are Fuller’s Crossing population, Gainesville population, and Sanford population. Each population performed differentially on the tested host plants: rough lemon, peanut, strawberry, and tomato. The Gainesville population is the only physiological race that caused damage to strawberry plants.

The management of sting nematode would require an integrated approach, which includes cultural, chemical, biological, and host plant resistance strategies. In the past, growers have focused the management of sting nematodes on the use of nematicides. These are chemicals used solely to reduce nematode populations. There are many commercially available nematicides, and their effectiveness varies, depending on the nematode genera. Plant-parasitic nematodes in strawberry plantings have been managed using soil fumigants, of which methyl bromide was historically one of the most effective fumigants used for their management. Methyl bromide is now banned in the production of fruits and vegetables in the US. Nevertheless, several chemical alternatives have been evaluated for nematode management in fruit crops.

These include chloropicrin, 1, 3- dichlopropene, dimethyldisulfide, metam sodium, calcium cyanamide, and propylene oxide (Gilreath et al. 2008, Zasada et al. 2010).

In organic production of crop plants, where synthetic chemicals could not be used, several tactics including the use of cover crops, crop rotation, flooding, vermicomposts, and soil sterilization have been used to manage nematode population. Cover crops have been evaluated to determine their effectiveness in suppressing nematodes (Halbrendt 1996, Abawi and Widmer

2000), as well as in reducing weed pressure on the crop of interest. Cover crops that have been used in the past include velvet bean, Mucuna deeringiana (Bort.) Merr.; joint vetch,

Aeschynomene americana L.; hairy indigo, Indigofera hirsuta L.; showy crotalaria, Crotalaria spectabilis Roth.; marigold (Reddy et al. 1986); sunn hemp, Crotalaria juncea L.; Sesbania spp.; and sudan grass, Sorghum sudanese (Piper) Stapf. (Noling 2016). Nematode injury on strawberry varies, with the cover crop planted prior to the establishment of strawberry fields (Noling

2016).

Cultural control methods that have been effective in managing nematode problems include flooding, crop rotation, and vermicomposts. Flooding helps to manage nematodes by depleting oxygen levels (Guerena 2006). Vermicomposts are organic soil amendments, with physical and chemical properties and they have been reported to increase soil nutrients and reduce plant pathogens. Arancon et al. (2002) evaluated three types of vermicomposts on strawberry, grapes, tomato, and pepper plants. They found that the nematode population was reduced significantly in vermicompost treated soils, compared with soils treated with inorganic fertilizer.

Nematodes are vectors of plant pathogens. However, the use of sterile soils limits the growth of pathogens. Soil sterilization can be used in greenhouse plantings and achieved by

80 heating the soil to a temperature of about 120°C, which is lethal to the survival of microorganisms. Biological control can be used in nematode management. Natural enemies of nematodes such as bacteria and fungi have been identified, isolated, extracted, and formulated as biopesticides. These formulated biopesticides have been applied to plants or to the soil to kill nematodes in crops. However, the effectiveness can be improved through more careful research, as this control method is promising.

One of the best control strategies for nematode management is the use of resistant crop varieties. Dale and Potter (1998) reported the differential susceptibility of some strawberry cultivars to the northern lesion nematode, Pratylenchus penetrans. However, Noling (2016) and

Zasada et al. (2010) mentioned that the sting nematode shows no differential damage to cultivars of the same plant species. Crop varieties differ in how efficiently they utilize soil nutrients, resources, and tolerating the attack of pests, diseases and weeds. Therefore, our objective is to determine if selected strawberry cultivars show differential susceptibility to the establishment of sting nematode infestation using two production techniques that included bare root and plug transplants. We hypothesized that the sting nematode as an ectoparasite, will easily locate and feed on the bare root transplants, compared to the plug transplants. Our findings will either confirm or refute the previous research reported by Noling (2016) and Zasada et al. (2010) that there are no strawberry cultivars that are tolerant to sting nematode abundance and injury.

Materials and Methods

Plant Materials

Plug and bare root transplants of six cultivars of strawberry namely Festival, Radiance,

Winterstar™, FL 127 (Sensation), Albion, and Benecia were purchased from Luc Lareault,

Lavaltrie, Canada. Vydate ®, a commercially available nematicide manufactured by

DowDuPont™ Inc. (Midland, Michigan) was ordered and purchased online

(www.dupont.com/products-and-services/crop-protection).

Study Site

The experiment was conducted at the Plant Science Research and Education Unit

(PSREU) Citra, in Marion County FL. Strawberry plants were cultivated on a plot measuring 0.2 ha, with a history of different types of nematodes such as root knot, Meloidogyne spp.; sting,

Belonolaimus longicaudatus; ring, Criconemoides spp; lance, Hoplolaimus galeatus Thorne; and lesion, Pratylenchus spp. The soil type at this location is Sparr fine sand.

Experimental Study

This experiment was conducted between October 2016 and April 2017. The experimental design was a split, split plot with whole plots arranged in a randomized complete block design.

Whole plots were with and without Vydate®, each category split with bare roots and plugs. Sub- plots were six strawberry cultivars randomized within whole plots. Sub-sub plots contained either bare root or plug transplants (Fig. 5-1). Vydate® (nematicide) was applied at the recommended rate of 2.34 L/ha through drip irrigation to the raised beds before covering with plastic mulch. This nematicide was re-applied at 15 days after planting. Each strawberry cultivar was cultivated on a polyethylene mulch bed (Brentwood Plastics Inc. St. Louis, MO) that measured 6.1 m long and 1.5 m wide. On each bed, strawberry plants were cultivated in double alternate rows according to standard strawberry growing practice (Whitaker et al. 2016). The strawberry plants were spaced at 0.4 m along and between rows. A 2 m buffer zone was maintained within blocks and 4.5 m between blocks.

Sampling

Soil samples were collected three times during the planting season. They were collected at planting in early season (on October 25, 2016), during the midseason (on February 8, 2017), and at the end of the season (on April 24, 2017). Six core samples were randomly collected from each plot at a depth of 8-12 inches using a soil auger. The soil sample from each plot was put in a labeled Ziploc® bag (Racine, WI). The bags were put in a cooler and transported to the

Nematology Assay Laboratory, University of Florida, Gainesville, FL for further processing.

Nematode Extraction

We used the sugar centrifugation protocol as developed by Jenkins (1964). Essentially, the soil in each Ziploc® bag was thoroughly mixed, to have a uniform soil population of the collected soil. A 100-cc beaker filled with a tightly packed soil sample from each treatment was emptied into a kitchen strainer nested on a two-liter stainless steel pitcher. The soil was rinsed with tap water at high flow velocity through the strainer, to agitate the soil so that it was suspended in the water. The kitchen strainer was removed, and the content in the pitcher was allowed to stand for several seconds, for the sand to settle. The suspension was decanted using a

400-mesh sieve, avoiding the sand that settled at the bottom of the pitcher from collecting on the sieve.

The isolate on the sieve was rinsed over a plastic funnel into a 100 ml centrifuge tube and placed into a centrifuge at 3500 rpm for three minutes. The supernatant was decanted without disturbing the soil plug at the bottom and the tube was filled with a 45% sucrose solution. The tubes were placed back into the centrifuge for an additional three minutes at the same speed. This process suspended the nematodes in the sugar solution. The suspension was poured off onto a

500-mesh sieve, and the nematodes were washed through a funnel into a tube, to about 20 ml by

83 volume. This process was repeated for each soil sample. The contents in each tube was poured into a Petri dish, nematodes were identified and counted under a compound microscope and recorded for each sample.

Marketable Yield

Harvesting was done once a week beginning in January from all treatment plots. At this time, all the cultivars were bearing fruits. Marketable fruits from each treatment plot were weighed and recorded. A fruit is considered marketable if it showed no evidence of physical damage such as peck marks, holes, removed achenes (seeds), or cat-faced injuries from insect, bird, or animal feeding, rots and mold from disease pathogens, and cracks from frost injury. In addition, small fruits and deformed fruits were not considered marketable, because the defects could be due to nutrient deficiency and inefficient pollination respectively. Culls (unmarketable fruits) were removed simultaneously from the field to prevent disease incidence. Marketable yield was weighed insitu using a digital portion control scale (Torrey L-EQ, Torrey®, www.torrey-electronics.com).

Data Analysis

A two-factor linear mixed model was used to analyze the data in SAS 9.4. Data were square root transformed to meet the assumptions of normality and analyzed with transplant method (plug or bare root transplant) with or without Vydate® and cultivars as the treatment effects. The experimental unit was measured multiple times; therefore, an autoregressive correlation structure type 1 with variances that were allowed to vary with time (ARH1) was used.

Tukey’s multiple comparison procedure was used to perform the mean separation tests where significant difference was observed (when P < 0.05).

A Pearson’s correlation was used to test for correlation between the population of the sting nematode and total market weight of strawberries. To do this, we summed up the weekly marketable weights for each treatment until the nematode sampling date, hence no marketable yield was recorded for the samples collected on October 25, 2016 due to the absence of fruits.

Results

Population Density of Sting Nematode, Belonolaimus longicaudatus

There were significant interaction effects between date and production technique (F =

4.83; df = 6, 204; P = 0.0001) and between date and cultivar (F = 3.16; df = 10, 204; P = 0.0009;

Fig. 5-2) in relation to the population of sting nematode. The population of sting nematodes varied on all the cultivars for the three sampling dates and this was significant (F = 294.66; df =

2, 204; P < 0.0001), with the lowest number recorded on the first sampling date. All the four production techniques (plug + Vydate®, bare roots + Vydate®, bare roots - Vydate®, and plug -

Vydate®) evaluated showed no significant difference (F = 0.67; df = 3, 9; P = 0.5895). There was a significant difference in the abundance of sting nematodes among the strawberry cultivars for the growing season (F = 7.94; df = 5, 204; P < 0.0001), with the mean sting nematode population on Radiance significantly lower than Albion and Benecia (Fig. 5-3).

Other parasitic nematodes found in the soil included ring, Criconemoides spp; root knot,

Meloidogyne spp.; lance, Hoplolaimus galeatus; and lesion, Pratylenchus spp. (data not shown).

Of these nematodes, ring nematode was the most abundant. We analyzed the population of ring nematodes and found that the interaction effects between date and production techniques (F =

2.39; df = 6, 144; P = 0.0313) had significant differences on the population of ring nematodes found in the soil. The sampling dates were significantly different (F = 13.35; df = 2, 144; P <

0.0001) with the lowest population at planting. As expected, the population of ring nematodes in

85 the soil increased over time because of the establishment of strawberries in the field (data not shown). However, their populations in the soil were not significant for cultivars (F = 1.84; df =

5, 60; P = 0.1188) cultivated on the treatment plots with the production techniques (F = 1.27; df

= 3, 9; P = 0.3435). Overall, the numerically highest populations of sting and ring nematodes were found on the bare roots (Table 5-1).

Marketable Yield

The interaction effects between date and production technique (F = 3.44; df = 3, 126; P =

0.0190), date and cultivar (F = 12.64; df = 5, 126; P < 0.0001), and production technique and cultivar (F = 2.47; df = 15, 126; P = 0.0033) had significant differences on the marketable yield.

The marketable weight of strawberry harvested on all treatment plots were significant for date (F = 214.59; df = 1, 126; P < 0.0001), for production technique (F = 11.86; df = 3, 126; P <

0.0001), and among cultivars (F = 34.08; df = 5, 15; P < 0.0001; Fig 5-4). Overall, Festival produced the heaviest fruits (175.01 kg), followed by Benecia (150.98 kg) and Winterstar

(100.67 kg).

There was a positive correlation between sting population and marketable weight (r =

0.37; P < 0.0001). However, it is a weak relationship, as the coefficient of determination, R² is

0.13 (Fig. 5-5A). A negative correlation was obtained between the ring nematode population and total market weight, but it was not significant (r = -0.08; R² = 0.006; P = 0.28; Fig. 5-5B). For most of the cultivars, treated and untreated bareroots produced more marketable fruits (Table 5-

2).

Discussion

Population Density of Sting Nematode, Belonolaimus longicaudatus

With the establishment of strawberry in the field during the fall season, the populations of sting nematodes, B. longicaudatus and other nematodes, including free-living nematodes increased in the strawberry plots and sting nematode population was highest at the end of the season. Initially, the populations of sting and ring nematodes were low but increased over time.

One possibility for the low numbers of nematodes seen initially was that the land was left fallow during the summer and there were no nutrients to nourish and support the development of the nematode populations.

The production techniques evaluated (bare roots and plugs with and without Vydate®) performed similarly and had no effect on the population of sting nematodes. A number of reasons could possibly explain for our results. Firstly, sting nematodes are ectoparasites feeding near roots; therefore, it is easier for sting nematodes to feed near exposed roots (bare roots). At the time of planting, we observed that the population of sting nematode was generally low and it was difficult to observe significant differences among production techniques when population is so low. Secondly, as time passed, the plugs disintegrated and the roots became exposed (personal observation by uprooting few plants) allowing plugs to act similarly as bare roots. This situation will facilitate equal accessibility of sting nematodes to both plugs and bare roots, accounting for the absence of significant differences. The disintegration of the plugs may be influenced by edaphic factors such as soil pH, soil organisms, and soil texture.

Vydate® has no fumigant properties, thus its effectiveness depends largely on its correct application to soil. Vydate® is not registered for sting nematode in strawberry, but it is a recommended nematicide for use in potato (Noling 2009). Also, Vydate® was applied early

87 during the season as recommended for all nematicides (Jardine and Todd 1990). Noling (2005) suggested that nematode management should be carried out before planting or at the end of the season, because post-plant corrective measures are not usually effective. Jardine and Todd

(1990) reported that nematicides often offer temporary reduction of nematodes but nematode populations return to pre-treatment levels before the season ends. This explains why the highest numerical population of nematodes was observed on treated plugs and treated bare roots during the midseason and end of season sampling. Ring nematode is not considered a major nematode pest in strawberry; however, the abundance of ring nematodes may interfere with yield and vigor of the strawberry plant.

The population of sting nematode was lowest on the Radiance cultivar. However, we cannot conclude that this cultivar is resistant to sting nematode. Resistant genes are not present in all plant cultivars with desirable agronomic traits (Roberts 1992). For example, the Mi gene has been identified in tomato (Gilbert and McGuire 1956) as the resistance gene for root knot nematodes like Meloidogyne incognita, M. arenaria, and M. javanica (Barham and Winstead

1957). To date, we are not aware of any research study that has identified resistance genes in strawberry for plant-parasitic nematodes.

Marketable Weight

We believe that bare roots produced more and/or heavier fruits because their roots are usually extensive and able to access more nutrients in the soil. Although the lowest population of sting nematode was found on Florida Radiance, yet cultivars like Festival, Benecia, and

Winterstar™ produced more and heavier fruits. This confirmed that market weight is not always dependent on the population of nematode pests, especially for pests like sting nematode that acts below ground, causing indirect injury to strawberry plants. The injury results in stunting, which

88 consequently reduces marketable yield. Noling (1999) reviewed past research studies that evaluated non-fumigant nematicides like Vydate® and concluded that most of such studies were inconsistent in managing nematodes and /or maximizing profits to the grower.

Although sting nematode cause above-ground symptoms like stunting, cultivars like

Festival and Winterstar™ usually produce much foliage (personal observation) in readiness for pollination and hence more fruits. The lowest marketable weight was produced by Albion, coincidentally this is the cultivar that harbored the highest population of sting nematodes. Sting nematode occurs in patches and this is usually evident by irregular distribution of stunted plants in the field (Noling 2005), it is possible that the patchy distribution of sting nematode accounts for the inconclusive results obtained.

N BLOCK 1 BLOCK 2 BLOCK 3 BLOCK 4 1.5 m 13.7 m

S F R F R S S F R R S F e W A e a B e B a W e A e B A W e a A a B e e W n n . l st d e s e d . l n e l . s d l d e n s . s S b i n n i S s b s n b t i b i n s S i t S t 6 m a t i a e e a t a i a e i t i a i a e a t v i i ti a o n c c n a t o t c o a v n o n c t a a v o r n c i v i c r i n i i n r a c n c i i r n l e a a a e o o a l e e a o a l n n n l 2.1 m 2.1 m Bare roots

F S R S F R R F S F S R Plug B e e a A W e A e B a W A a e B e W e e a W A B e s n d l . n l s e d . l d s e n . st n d . l e n s i S s b s s S Vydate plug t b t n i S b i t n S i i b n e a a i t a i e a t i e a t a t i i i a i v a e c t n o a t o c n a o n c t a ti n a o c v v v 4.6 m a Vydate bare roots i i c n r i n i c r n c i i r o c r n i l a a o e o a a e e a a o n e a l n n l l n 13.7 m 30.8 m R S F R F S F R S R F S a e B W e A B W A a e e W e B A a e a B e A W e d n e . s l e . l d st n . s e l d n d e s l . n s n S b n S b i s n b s b s i t i S t i i n t S a a e t i e t i a a t e i a a a e i t a i 6 m v 4.6 m i i n t c a o c a o n t a c o n t n c o a t c i i r v n i r n c a i r v i n c i c i v n r i e o a a a e l o a a e o e a a o n l n l n l n

F S R R F S S R F F S R A B e e W a a e e W A B B e W a A e W e e a B A n l e s n . d d s n . l e e . d l s . s n d e l b n s S i s S b n n s S i b S s n b t i t t t i i e a t a 4.6 m a a t i e e a t a i t a a e i i i i i o c t a n n t a o c c t a n o a ti n c o v v n i v i r c c i r n i i i r c n r v o c i n a a o e e a o a a o e a a n e a l n l n l l 68.6 m

In eac h block, the rows of cultivars are 0.9 m apart. Each plant is 0.4 m apart

Figure 5-1. Plot layout of the field experiment conducted in 2016-2017 growing season at Plant Science Research and Education Unit (PSREU), Citra. The colors represented the production techniques (whole plot treatments) and the cultivars were the subplot factors replicated four times within each block.

50 Albion Benecia 45 Festival a 40 Winterstar Sensation 35 Radiance ab 30

25 bc bc 20 c 15 a a 10 ab Mean nematodes of sting Mean population ab 5 ab b 0 10/25/2016 2/8/2017 4/24/2017 Sampling dates

Figure 5-2. Mean population of sting nematodes on selected cultivars on different sampling dates. Means followed by the same letters on the same sampling date are not significantly different. Tukey’s test; P < 0.05 (SAS 9.4, 2012).

70 F = 7.94; df = 5, 2014; P < 0.0001

60 a a 50

ab 40

30 bc bc c 20

Mean population of sting nematodes for the entire season season the nematodes of for entire sting Mean population 0 Albion Benecia Festival Radiance Sensation Winterstar Strawberry cultivars

Figure 5-3. Mean population of sting nematode, Belonolaimus longicaudatus on selected strawberry cultivars throughout the entire strawberry season 2016-17. Means followed by the same letters are not significantly different. Tukey’s test; P < 0.05 (SAS 9.4, 2012).

2/8/2017 9 4/24/2017 A 8

7 B 6

5 C

4 CD CD 3 a a (kg) of strawberry harvested per cultivar per harvested strawberry of (kg) D a ab 2 ab

1 b

Mean weight Mean 0 Albion Benecia Festival Radiance Sensation Winterstar

Strawberry cultivars

Figure 5-4. Mean marketable weights (in kg) of strawberry harvested for each cultivar. Means not connected by the same lowercase letters (for blue bars) and not connected by uppercase letters for orange bars are significantly different. Tukey’s test; P < 0.05 (SAS 9.4, 2012).

Table 5-1. Total populations of A) sting and B) ring nematodes on strawberry cultivars planted using different production techniques. A Cultivars Bare roots Bare roots + Vydate® Plug + Plug Vydate® Albion 248 406 175 204

Benecia 238 255 207 325

Festival 242 149 169 225

Radiance 122 137 60 104

Sensation 142 159 64 129

Winterstar 124 113 172 133

TOTAL 1116 1219 847 1120

B Cultivars Bare roots Bare roots + Vydate® Plug + Vydate® Plug

Albion 107 108 78 79

Benecia 75 75 97 74

Festival 101 104 53 67

Radiance 134 52 43 87

Sensation 139 80 73 126

Winterstar 61 69 42 50

TOTAL 617 488 386 483

Table 5-2. Total marketable weights (in kg) of strawberry on selected commercial cultivars for each production technique. Cultivars Bare roots Bare roots + Vydate® Plug Plug + Vydate®

2/8/17 4/24/17 2/8/17 4/24/17 2/8/17 4/24/17 2/8/17 4/24/17

Albion 3.48 11.85 5.71 18.15 1.44 4.27 1.37 4.09

Benecia 8.62 28.72 9.53 30.65 12.99 25.56 13.06 21.85

Festival 10.88 31.16 14.67 44.20 11.17 37.84 5.83 19.26

Radiance 11.49 16.57 11.06 17.73 5.65 12.61 6.56 12.42

Sensation 6.04 11.69 9.85 16.34 7.62 12.97 6.27 10.55

Winterstar 4.16 16.01 7.66 19.77 7.68 18.84 7.49 19.06

14 y = 0.0385x + 2.5537 R² = 0.1333 12

2 marketable marketable yield (kg) strawberry of on all treatment plots 0 Total 0 50 100 150 200 Population of sting nematode on each treatment plot A

14 y = -0.0298x + 3.6338 R² = 0.0061 12

Total marketable Total marketable yield (kg) strawberry of on plots 0 0 5 10 15 20 25 30 35 40 Population of ring nematode on each treatment plot B Figure 5-5. Relationship between strawberry yield and populations of A) sting nematode B) ring nematode.

CHAPTER 6 EFFICACY OF SELECTED MITICIDES AND MICROBIAL FUNGICIDES FOR MANAGEMENT OF TWOSPOTTED SPIDER MITES

Introduction

Spider mites are common pests of many crop plants and ornamentals. Over the years, spider mites have become a major problem, as a result of frequent insecticide/acaricide applications due to resistance and destruction of natural enemies that regulate mite population

(Ayyapath et al. 1996, Barati and Hejazi 2015). Twospotted spider mites (TSSM), Tetranychus urticae Koch are found everywhere, on crops grown in the field, greenhouses, and high tunnels.

The use of biological control as the sole management strategy is not an effective approach for managing spider mites. This is because the natural enemy (predatory mites) may not reproduce at an adequate rate to effectively reduce the mite population. Some conventional chemicals have been successful in controlling key arthropod pests, but the lack of host specificity has impacted the natural enemies, and other non-target organisms in the system (Jeppson 1965).

Minute pirate bugs (Orius spp.) and big-eyed bugs (Geocoris spp.) are beneficial insects found to be effective in reducing the populations of many pest species (Hoy 1994, Eubanks et al. 2002).

Past surveys of strawberry fields showed that these predators are abundant in the system.

The minute pirate bug, big-eyed bug, and sixspotted thrips, Scolothrips sexmaculatus (Perg.) are generalist predators of pests such as mites, aphids, thrips, and whiteflies (Fraulo et al. 2008).

These arthropod pests are usually found on strawberry leaves during routine sampling operations.

Pest resurgence is a common phenomenon, in which pest populations spike after a chemical control because the natural enemy populations fail to recover at the same rate as the pest population (Reissig et al. 1982, Hardin et al. 1995). This creates imbalance in the

97 agroecosystem, and often a bigger problem than before the application of pesticides. This necessitates the importance of applying only narrow-spectrum chemicals with known specificities for one pest or pest group, which are not disruptive to natural enemies. Furthermore, pesticide resistance is a common problem in the management of pest insects and mites (Helle

1962). Repeated applications of the same miticide over time may result in the displacement of the susceptible strain, selecting for the resistant strain in the population. Other factors that contribute to miticide resistance in mites include rapid reproductive ability, dispersal rate, and arrhenotoky in mite population (van Leeuwen et al. 2010).

Microbial pesticides are considered environmentally safe and specific in action against the target pest. A good example is Bacillus thuringiensis (Bt) used against lepidopteran pests at the larval stages. There are few naturally occurring pathogens of TSSM, but fungi in the genus

Neozygites have been reported as effective natural enemies of TSSM. For example, Neozygites floridana (Weiser and Muma) Remaudiére and S. Keller is a specialist natural enemy of TSSM

(Keller 1997, Wekesa et al. 2011, Westrum et al. 2014). The effectiveness of N. floridana on

TSSM has been studied on several host plants, including strawberry (Klingen and Westrum

2007). The study conducted by Klingen and Westrum (2007) showed that the effect of N. floridana is negatively impacted by the application of some fungicides. Neozygites floridana develops hyphal bodies within TSSM, killing its host and forming new spores that develop into conidia that are the infective stage of this fungus (Wekesa et al. 2011).

Some fungal pathogens that have been studied as biocontrol agents against acarine pests include Beauveria bassiana, Metarhizium anisopliae, Paecilomyces farinosus, P. fumosoroseus, and Verticillium lecanii (Chandler et al. 2000). Although Beauveria bassiana is a potential biocontrol agent of TSSM (Chandler et al. 2005) and commercially available, however, it has not

98 been fully explored in the management of TSSM because of limited knowledge and awareness of its potency.

In Florida, many growers use the conventional system in strawberry production, which relies on pesticide application irrespective of economic injury levels. The negative public perception against pesticides and genetically modified foods (GMOs) have prompted some growers to consider organic strawberry production. In organic production of crops, products that are used must be in compliance with the USDA National Organic Products (NOP) standards.

Typically, a list of these products is provided by the Organic Materials Review Institute (OMRI).

These organic pesticides are often approved for safety, but their effectiveness may not have been evaluated in the field. However, organic pesticides should be used as the last resort if preventative, cultural, and biological strategies did not affect control. There is need for periodic evaluation of commercially available miticides that are environmentally safe and effective against pests with minimal or no negative impact on beneficial organisms. This study was carried out to evaluate the efficacy of pesticides permitted in organic systemsand microbial pesticides in managing TSSM and their effect on beneficial arthropods. We hypothesized that different pesticides will vary in their ability to control TSSM and there will be differences in mite population between pesticide-treated and untreated plants. Furthermore, the pesticides will be specific in their action and will not have negative effects on the predatory mites and beneficial insects.

Materials and Methods

Laboratory Experiments

Two laboratory experiments were conducted in the Small Fruit and Vegetable IPM

(SVIPM) Laboratory to evaluate the effectiveness of selected OMRI approved chemicals and

99 microbial pesticides on TSSM. These experiments were conducted in the laboratory on a single cultivar of strawberry, Festival. Fresh strawberry leaves were collected from plants grown in the greenhouse. Each leaflet was placed on moist filter paper layered with moist cotton wool in a 5 cm diam. Petri dish. The experimental design was a completely randomized design (CRD) with four replicates.

Organic Miticides: In the first experiment, the treatments evaluated included 1) Cosavet

DF Micronized Sulfur® (Belchim Crop Protection USA, LLC Wilmington, DE), 2) Grandevo®

(Marrone Bio Innovations, Davis CA), 3) Aramite® (ExcelAg. Corp. USA, Miami, FL), and 4) tap water (control). Ten TSSM from the laboratory colony (as discussed in chapter 3) were manually (handpicked) introduced onto each strawberry leaflet maintained on a moist filter paper in a Petri dish. Each chemical treatment was applied at the manufacturer’s recommended rate using a Potter Spray Tower (Burkard Scientific, Uxbridge, UK) to create a fine mist of the chemical and achieve uniform coverage on the leaflets. Counts were done under a stereo microscope (Leica M80; Leica Microsystems, Buffalo Grove, IL) under the magnification of

10X at regular intervals of 6, 24, 48, and 72 h to assess TSSM mortality and the abundance of

TSSM eggs produced.

Conventional Microbial Pesticides: In the second experiment, the treatments evaluated included two formulations (in powdery and liquid forms) of Beauveria bassiana strain GHA at low (1 lb/acre or 1 pint/acre) and high (2 lbs/acre or 2 pints/acre) rates (Certis USA, Columbia

MD) 1a) CX-10285 (low), b) CX-10285 (high), 2a) CX-10282 (low), b) CX-10282 (high)

[totaling 4 microbial treatments], 5) Abamectin (Avid®), and 6) tap water (control). All pesticide treatments were applied at the recommended manufacturers’ rates. A single field infested leaflet was placed on moist filter paper lined with cotton wool in Petri dish. Arthropods on each leaf

100 were identified and their abundance was recorded. Each treatment was applied using the Potter

Spray Tower and the leaflets were examined under a stereo microscope (Leica M80; Leica

Microsystems, Buffalo Grove, IL) at 3 and 7 d post treatment. The treatments CX-10285 (low) and CX-10285 (high) were re-applied at day 7 and counts of arthropod that survived were done on day 10 (3 d after the second treatment application).

Field Trials

Organic Miticides: This experiment was conducted at the Plant Science Research

Education Unit (PSREU), Citra FL on Festival cultivar from October 2015–April 2016.

Individual sampling bed size was approximately 6 m x 1.5 m and strawberry plants were established on double rows (per bed) at a spacing of 0.3 along and between rows. A pre-count of

TSSM in individual plots was conducted prior to treatment application. The same four treatments used in the laboratory experiments were evaluated in the field trials. Each treatment was replicated four times in a completely randomized design (CRD). Each treatment was applied with a knapsack sprayer at the manufacturer’s recommended rate. Leaf samples were collected at

3, 7, and 14 d post treatment. The population of TSSM was assessed by randomly collecting three trifoliate leaves from plants per plot/rep and inspecting leaves under a stereo microscope

(Leica M80; Leica Microsystems, Buffalo Grove, IL) under the magnification of 10X. The number of motiles and eggs were recorded. Each treatment was applied twice (December 21,

2015 and March 27, 2016) during the entire strawberry growing season.

Conventional Microbial Pesticides: This experiment was also conducted at PSREU, Citra

FL in Marion County, FL on strawberry cultivar, FL 127 (Sensation). The treatments were the same as those used in the laboratory assay. Treatments were applied using a backpack sprayer, at the manufacturers’ recommended rates. The experimental design was CRD and replicated three

101 times. Each bed measured 6.1 m x 0.8 m. The treatments were applied three times (February 1, 8, and 15, 2018). Pre and post sampling were done weekly by randomly collecting four trifoliate leaves from each treatment plot just before applying treatments. These data provided efficacy information on previous week’s pesticide application. The samples were taken to SFVIPM laboratory for further processing. Leaf samples were examined under a stereo microscope, Leica

M80 (Leica Microsystems, Buffalo Grove, IL) and counts of all arthropods encountered were recorded.

Data Analysis

Data were analyzed with repeated measures analysis using generalized linear model with a negative binomial distribution and an unstructured correlation via PROC GLIMMIX in SAS

9.4. Tukey’s multiple comparison procedure was used to perform the mean separation tests where significant difference was observed (when P < 0.05).

Results

Laboratory Experiments

Organic Miticides: The interaction effects between time and treatments were not significant (F = 2.41; df = 9, 12.85; P = 0.0736) in relation to the population of TSSM motiles, but the interaction effects between time and treatments were significant for TSSM eggs (F =

5.38; df = 9, 11.57; P = 0.0048). Leaflets treated with Aramite® had numerically the lowest population of TSSM motiles during the sampling period, except at 48 h when the numeric lowest population was recorded on leaflets treated with micronized sulfur. Twospotted spider mite population was significantly lower on the treated leaflets compared with the control (F = 14.77; df = 3, 12; P = 0.0002) except at the 6 h and 48 h interval, when Grandevo® was not

102 significantly different from the control (Fig. 6-1). Overall, the time effect was significant (F =

5.38; df = 3, 10; P = 0.0183).

Also, the population of TSSM eggs increased significantly over time (F = 237.35; df = 3,

10.85; P < 0.0001) across all treatments and after 72 h, the population of TSSM eggs was numerically highest on leaflets treated with Grandevo® (Fig.6-2). There was no significant difference among the treatments in the number of eggs oviposited on the treated leaflets and control (F = 0.38; df = 3, 11.53; P = 0.7687).

Conventional Microbial Pesticides: Immediately after the application of microbial pesticides, the population of TSSM declined (observed at three days after treatment application), except for the control treatment that had increased mite population. The interaction effect between time and treatments was significant (F = 12.46; df = 15, 23.76; P < 0.0001). Also, there was a significant difference among the dates of treatment and observation (F = 104.72; df = 3,

16; P < 0.001) and among the treatments (F = 6.93; df = 5, 18; P = 0.0009).

The population of TSSM on leaflets treated with CX-10282 (high) was significantly lower compared with leaflets treated with CX-10285 and the control at three days and one week after treatment application (Fig. 6-3). Infact, TSSM population was eliminated on leaflets treated with CX-10282 (high) by one week after treatment application. At three days after treatment application, the populations of TSSM on leaflets treated with CX-10285 reduced numerically, but still had higher TSSM population compared with the control. At seven days after treatment application, the populations of TSSM on leaflets treated with CX-10282 and Abamectin® had declined and there was no economic justification for re-application of the chemicals, unlike leaflets that were treated with CX-10285. At the end of the experiment (day 10), TSSM population was numerically highest on the untreated leaflets (control); however, this was not

103 significantly different from the leaflets that were treated with a higher concentration of CX-

10285.

A similar result was obtained for TSSM eggs, where the interaction effects between dates and treatment (F = 4.30; df = 15, 23.76; P = 0.0008) were significant. This showed that less eggs were oviposited, many of which did not hatch due to the mortality of TSSM after being exposed to pesticides. CX-10282 and Abamectin® achieved effective control and reduced TSSM egg populations significantly compared to the CX-10285 and control (F = 3.23; df = 5, 18; P =

0.0297; Fig. 6-4). Overall, the sampling dates had significant effect (F = 24.13; df = 3, 16; P <

0.0001) on the population of TSSM eggs oviposited on the strawberry leaflets.

Other arthropods recorded on the leaflets included thrips, Frankliniella spp., predatory mite, N. californicus, and sixspotted thrips, S. sexmaculatus. The microbial pesticides performed significantly different from each other (F = 6.05; df = 5, 18; P = 0.0019) but did not have negative impact on the population of N. californicus, as the population increased on leaves treated with CX-10282 (Table 6-1).

Field Trials

Organic miticides: The interaction effects between the treatments and time period were significant for both TSSM (F = 6.56; df = 21, 84; P < 0.001) and TSSM eggs (F = 3.94; df = 21,

84; P < 0.001). The populations of TSSM motiles declined over time, thus it influenced the number of eggs oviposited on the strawberry plants. There were significant differences in TSSM population among the treatments (F = 6.96; df = 3, 12; P = 0.006), all the chemical treatments performed similarly, but significantly better than the control (Fig. 6-5). A similar result was obtained for TSSM eggs (F = 12.19; df = 3, 12; P = 0.001; Fig 6-6). Also, the time effect was

104 significant for TSSM (F = 48.26; df = 7, 84; P < 0.001) and TSSM eggs (F = 34.28; df = 7, 84; P

< 0.001).

Conventional Microbial Pesticides: The population of TSSM reduced significantly on plots treated with Abamectin® compared with other treatments (F = 10.88; df = 5, 40; P <

0.0001; Fig. 6-7). Overall, the microbial fungicides did not perform well, as the mite population fluctuated during the sampling period, except on the last sampling date that mite populations declined in all the plots, including the control plots. The treatments showed significant difference on reducing the population of TSSM eggs (F = 2.95; df = 5, 48; P = 0.0211; Fig. 6-8), and time had significant effect on the population of TSSM eggs (F = 112.51; df = 3, 48; P < 0.0001).

A similar result was obtained when the effects of these pesticides on N. californicus were evaluated. The populations of N. californicus increased on all the treatment plots but the lowest populations were recorded on plots treated with Abamectin (Table 6-2).

Discussion

Laboratory Experiments

Organic miticides: The populations of TSSM were reduced immediately following the application of the organic miticides except for Grandevo® that had only a slight decrease in

TSSM population and was similar to the control. Overall, Aramite® was the most effective treatment. At 48 h there was a slight increase in TSSM population in the Aramite® treatment.

This was probably due to the increase in the number of eggs oviposited at 24 h. Overall, the number of eggs laid by TSSM after miticide treatments was initially low but increased over time, with the highest number of eggs on Grandevo-treated leaves. Grandevo® is a microbial pesticide that contains the Chromobacterium subtsugae strain PRAA4-1. This pesticide usually targets sucking insects (Shannag and Capinera 2018) interfering with feeding (Martin et al. 2007),

105 reproduction, and development (Shannag and Capinera 2018). Although, it is labelled for mites, in our study it had little to no effects on TSSM population. The reason for this is unclear but it needs further investigation. With regard to the other miticide treatments used in our study, it appears that TSSM motiles that escaped being killed by direct contact of the miticides were able to carry on life processes such as oviposition, even at a higher rate compared to the control.

Similar findings were reported by Szczepaniec and Raupp (2013) that fecundity increased in female mites, Eurytetranychus buxi (Tetranychidae) exposed to imidacloprid compared with females on untreated boxwoods.

The decline in TSSM population towards the end of the sampling period could be attributed to the reduced leaf quality (Watson 1964, Specht 1965, Storms 1969). In the laboratory, the leaflets were not exposed to direct sunlight, therefore they could not carry out photosynthesis. As a result, the leaflets although are maintained in moist conditions could not provide enough nutrients to support the growing population of TSSM, resulting in death.

Conventional Microbial Pesticides: The microbial pesticide, Beauveria bassiana strain

GHA with formulation CX-10282 was more effective than CX-10285, and this was evident immediately after the application of the treatments. The dosage rates (low or high) did not make a statistical difference in reducing TSSM population. Therefore, a lower concentration of each microbial pesticide would be recommended for economic reasons. The differential effectiveness of the microbial pesticides is likely due to the formulation. It is possible that CX-10285 was not immediately effective because a longer time is required for the fungi to sporulate and grow conidia. Therefore, this microbial pesticide may be more effective as a preventative treatment or just as TSSM population is starting to build up. Abamectin® proved to be more effective than

CX-10285 in reducing TSSM population but did not support increased population of the

106 predatory mite, N. californicus. This was quite noticeable as lower population of N. californicus was observed in the Abamectin treatment. As expected, TSSM population increased on the untreated (control) leaflets until the leaf quality declined, and consequently resulted in a rapid decrease in TSSM population on the last sampling day.

Microbial pesticides did not seem to have negative effects on the predatory mite, N. californicus as the population did not decline in those treatments. In addition, N. californicus rate of oviposition did not appear to be significantly affected. Towards the end of the study N. californicus population was reduced. This reduction was probably due to the increase competition for TSSM (that were reduced in abundance), hence some died of starvation.

We observed the spores of Cladosporium spp. on some of the leaflets towards the end of the experiment. This may have contributed to the mortality of TSSM, as the leaf quality declined over time.

Field Trials

Organic Miticides: The findings in the field studies were similar to those obtained in the laboratory. All miticides reduced TSSM population one week after application. Since mite population in the field was decimated, the plots were left fallow for TSSM buildup, then a second application was done at 13 weeks after the initial application. We observed that TSSM population was highest on plots initially treated with Aramite®. Our findings are similar to previous work by James and Price (2002) that TSSM population increased following miticide treatments. Carey (1982) reported that hormoligotic effects of an insecticide or miticide may result in multiple fold increase of TSSM compared with an untreated population. This was also observed in a study conducted by Wang et al. (2016), where two different dosage rates of spinetoram resulted in an increase in TSSM population. A number of reasons may account for

107 the increased TSSM population, including stimulating effects of some chemicals, reduction in natural enemies, environmental factors, and resistance factors (Bartlett 1968).

We observed that Grandevo® performed differently in the laboratory experiment compared with the field trials. In the laboratory, Grandevo® was the least effective among all the miticides, but it performed best on the field after the second application. These results are unclear but interesting. It is possible that environmental factors including moisture or humidity and possible temperature interacted to allow Grandevo® to perform better in the field.

Conventional Microbial Pesticides: All the microbial pesticides performed similarly in the field experiment and were not effective in reducing TSSM populations. It appears that the microbial pesticides used in our study may perform better as a preventative tool since these microbial pesticides require time to grow spores that would become conidia for effective control of TSSM. At the time of application of these pesticides, TSSM population was already established.

Similar to our laboratory study, the lowest population of N. californicus was observed on plots treated with Abamectin® (Table 6-2). Our findings were supported by Iwassaki et al.

(2015) where the population of N. californicus was lower on conventionally managed strawberry plots treated with Abamectin compared with plots managed using integrated management (N. californicus and selective miticide).

The production system may have also contributed to the high population of mites.

Strawberry plots were cultivated and managed using low irrigation scheme; which has been shown to increase TSSM population as reported by White and Liburd (2005).

108

Control 12 Grandevo Sulfur 10 Aramite

8 TSSM motiles after treatment after motiles TSSM 6

ofsurviving 4

population Mean 0 6 h 24 h 48 h 72 h Time (in hours) after treatment application

Figure 6-1. Population density of twospotted spider mites (TSSM) on treated and untreated strawberry leaflets at intervals after pesticide treatments in the laboratory. Vertical bars represent standard error of the mean.

109

60 Control Grandevo

50 Sulfur

application Aramite

10 Mean population of TSSM eggs chemical after laid of eggs TSSMMean population 0 6 h 24 h 48 h 72 h

Figure 6-2. Population density of twospotted spider mite (TSSM) eggs on treated and untreated strawberry leaflets at intervals after pesticide treatments in the laboratory. Vertical bars represent standard error of the mean.

110

250 CX-10285 (low) CX-10285 (high) CX-10282 (low) 200 CX-10282 (high) ABAMEC CONTROL

150

100

0 DAY 0 DAY 3 DAY 7 DAY 10 Sampling Days

Figure 6-3. Population density of twospotted spider mites (TSSM) on treated and untreated strawberry leaflets at intervals after microbial pesticide treatments in the laboratory study. Day 0 represents the pre-treatment sample means. Vertical bars represent standard error of the mean.

111

250 CX-10285 (low) CX-10285 (high) CX-10282 (low) 200 CX-10282 (high) ABAMEC CONTROL

150

100

50 Mean population of TSSM eggs per leaflet of eggs TSSMMean population

0 DAY 0 DAY 3 DAY 7 DAY 10 Sampling Days

Figure 6-4. Population density of twospotted spider mite (TSSM) eggs on treated and untreated strawberry leaflets at intervals after pesticide treatments in the laboratory study. Day 0 represents the pre-treatment sample means. Vertical bars represent standard error of the mean.

112

Table 6-1. Population density of the predatory mite, Neoseiulus californicus on treated and untreated strawberry leaflets at intervals after pesticide treatments in the laboratory study. TREATMENTS DAY 0 DAY 3 DAY 7 DAY 10 CX-10285 (low) 1.50 ± 0.87 0.00 ± 0.00 b 1.25 ± 0.75 ab 1.75 ± 1.11 CX-10285 (high) 1.00 ± 0.41 1.00 ± 0.58 b 0.75 ± 0.48 ab 0.50 ± 0.50 CX-10282 (low) 2.75 ± 1.89 5.00 ± 1.47 a 4.25 ± 0.85 ab 2.50 ± 0.50 CX-10282 (high) 1.50 ± 0.65 7.50 ± 0.50 a 4.75 ± 1.84 a 2.50 ± 1.19 ABAMECTIN 0.75 ± 0.48 0.50 ± 0.29 b 0.25 ± 0.25 b 0.25 ± 0.25 CONTROL 0.75 ± 0.48 0.75 ± 0.48 b 0.75 ± 0.48 ab 1.25 ± 0.95 Means followed by the same letters in the same column are not significantly different, Tukey’s test. P < 0.05. (SAS 9.4, 2012).

113

1200

Control 1000 Sulfur Grandevo Aramite 800 Pre- treatment

600

400

200 Mean population of TSSM per treatment per trifoliateper treatment per TSSM of populationMean

0 12/21/2015 12/24/2015 12/28/2015 1/5/2016 3/27/2016 3/30/2016 4/3/2016 4/10/2016 Sampling dates

Figure 6-5. Population density of twospotted spider mites (TSSM) on strawberry plants before (12/21/2015 and 3/27/2016, enclosed in a rectangle) and after chemical treatments in the field study. Vertical bars represent standard error of the mean.

114

1200 Pre- treatment Control Pre- 1000 treatment Sulfur Grandevo Aramite 800

600

400

200 Mean population of TSSM eggs trifoliate per treatment of per eggs TSSMMean population

0 12/21/2015 12/24/2015 12/28/2015 1/5/2016 3/27/2016 3/30/2016 4/3/2016 4/10/2016 Sampling dates

Figure 6-6. Population density of twospotted spider mite (TSSM) eggs on strawberry plants before (12/21/2015 and 3/27/2016, enclosed in a rectangle) and after chemical treatments in the field study. Vertical bars represent standard error of the mean.

115

1000 CX-10285 (low) CX-10285 (high) 900 CX-10282 (low) CX-10282 (high) 800 ABAMEC CONTROL 700

600

500

400

300

200 Mean population of TSSM per of per trifoliate TSSMMean population

100

0 2/1/2018 2/8/2018 2/15/2018 2/22/2018

Treatment Application and Sampling Dates

Figure 6-7. Population density of twospotted spider mite (TSSM) observed on strawberry plants before and after chemical treatments each week in the field study. Vertical bars represent standard error of the mean.

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2000 CX-10285 (low) 1800 CX-10285 (high) CX-10282 (low) 1600 CX-10282 (high) ABAMEC

1400 CONTROL strawberry trifoliate strawberry

1200

1000

800

600

400

200

Mean population of TSSM eggs per treated of eggs TSSMMean population 0 2/1/2018 2/8/2018 2/15/2018 2/22/2018

Treatment Application and Sampling Dates

Figure 6-8. Population density of twospotted spider mite (TSSM) eggs observed on strawberry plants before and after chemical treatments each week in the field study. Vertical bars represent standard error of the mean.

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Table 6-2. Population density of the predatory mite, Neoseiulus californicus on strawberry plots before and after pesticide applications in the field study. Treatments 2/1/2018 2/8/2018 2/15/2018 2/22/2018 CX-10285 (low) 0.83 ± 0.66 1.89 ± 1.21 7.03 ± 3.72 ab 5.25 ± 2.85 CX-10285 (high) 0.08 ± 0.17 2.92 ± 1.72 16.25 ± 8.18 a 3.67 ± 2.08 CX-10282 (low) 2.25 ± 1.39 2.25 ± 1.39 6.17 ± 3.30 ab 8.83 ± 4.60 CX-10282 (high) 1.33 ± 0.93 3.33 ± 1.92 19.01 ± 9.50 a 14.48 ± 7.32 Abamectin 0.33 ± 0.37 1.17 ± 0.84 0.67 ± 0.57 b 2.75 ± 1.64 Control 0.67 ± 0.57 1.67 ± 1.10 6.08 ± 3.26 ab 7.50 ± 3.95

Means followed by the same letters in the same column are not significantly different, Tukey’s test. P < 0.05. (SAS 9.4, 2012).

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CHAPTER 7 CONCLUSION

Strawberry cultivars performed differentially under different production systems and location. Cultivars influence the population density of twospotted spider mites (TSSM). The cultivars (Strawberry Festival, Florida Radiance, Winterstar™, and FL 127 “Sensation”) with the lowest mite population will be more suitable for organic production, as less management inputs will be required, thus reducing production costs and increasing profits. This study forms the initial step that can potentially help plant breeders to isolate genes that are involved in cultivar resistance or tolerance to TSSM. These genes can be combined with other desired traits to develop cultivars that can tolerate or are more resistant to TSSM and other related plant feeding arthropods while still able to achieve other desired characteristics such as good flavor, shelf life, and high yielding.

The cover crops (sunn hemp, hairy indigo, shortflower rattlebox, and joint vetch) evaluated did not have direct effects on the strawberry cultivars with TSSM abundance.

Marketable yield was influenced by the population of TSSM populations. Cultivars with higher

TSSM populations produced less fruits compared with cultivars that harbored less TSSM populations.

The abundance of Neoseiulus californicus is mainly dependent on the availability of prey

(TSSM). However, laboratory studies and observation showed that some N. californicus preferred some cultivars (Winterstar™ and Sensation) over others. The reasons for this preference are unclear but requires further investigation of the chemical compounds and composition of cultivars like Winterstar™ and Sensation, as well as the measurement of the leaf physical characteristics that may influence the choice of N. californicus and Phytoseiulus persimilis to Winterstar™ and mobility of N. californicus on these cultivars. We recommend the

119 need to develop efficient sampling techniques to monitor for and study the dispersal of N. californicus population.

This study was the first of its kind in testing the differential susceptibility of strawberry cultivars to sting nematodes under Florida’s production condition. Tolerance of cultivars like

Radiance, Sensation, and Winterstar™ to sting nematode compared with Albion and Benecia deserves further research to confirm these results and determine other commercially available cultivars that are less susceptible to sting nematodes with high marketable yield. Bare root and plug transplants performed similarly with no effect on the population of sting nematodes.

Therefore, production technique is not a major factor to consider in the management of sting populations in strawberry fields. Other management methods would need to be explored.

Vydate® is not registered for use in strawberry, however the shrinking options of nematicides makes it imperative to explore other nematicides that may be effective in reducing sting nematode problems in strawberry. However, the use of crop rotation, other cultural management methods, and biological control (such as the use of entomopathogenic pathogens like Pasteuria spp.) may be incorporated into an integrated management of sting nematodes.

Marketable weight varied with cultivar and not with the level of sting nematode populations present in the soil. However, it is possible that at higher nematode infestations, the market weight would be severely reduced.

It is advisable that the field distribution of nematodes be studied for future studies, so that spot treatments of nematicides can be done, rather than the whole field to maximize economic returns. Also, future studies would include the evaluation of some cover crops that can be included in the integrated management of sting nematodes.

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Chemical control methods, although fast acting should remain the last resort. The results obtained in this study showed that some pesticides are better applied as a preventative management strategy rather than as a curative tactic. The chemicals did not cause total mortality, therefore the surviving TSSM could potentially start a new generation that may result in resistant populations. All the organic miticides were effective and can be incorporated into the strawberry cropping system on a rotational basis to prevent resistance development.

Future studies would involve testing each chemical at different motile stages (larva, protonymph, and deutonymph) to determine if TSSM is more susceptible at any stage. Also, we would recommend that manufacturers produce safe miticides that would be effective at preventing TSSM eggs from hatching into larvae.

Overall, based on these experiments, the cultivars suitable for organic strawberry production in Florida include Sensation, Winterstar™, Florida Radiance, and Festival. We recommend these cultivars because of their tolerance to TSSM and sting nematode and high fruiting capacity.

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BIOGRAPHICAL SKETCH

Omotola Olaniyi was born in Nigeria, where she received her bachelor’s in agriculture

(plant science) at the Department of Crop Production and Protection, Obafemi Awolowo

University (OAU) Ile-Ife in 2008. She did the mandatory one-year national youth service corps

(NYSC) at the Department of Botany, Lagos State University. She began her master’s degree in

2011 at the Florida A&M University, Tallahassee, FL, and graduated in 2013 as a top student.

During her master’s degree, she worked in the area of invasive species under the supervision of

Dr. Muhammad Haseeb and Dr. Richard Mankin. She started her doctorate degree immediately at the University of Florida in the Small Fruits and Vegetable Integrated Pest Management

Laboratory under the supervision of Dr. Oscar Liburd. Her doctoral research was based on evaluating several strategies for enhancing management of arthropod pests in organic strawberry production. She plans to continue her career with mitigating pests, especially at the border points and thus prevent their establishment.

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