The Impact of Beauveria Bassiana

Home , Beauveria bassiana, Cereal leaf beetle, Eriborus, European corn borer, Trichogramma

THE IMPACT OF BEAUVERIA BASSIANA, TRICHOGRAMMA, Bt SPRAYS AND SPINOSAD ON THE LEPIDOPTERAN (CRAMBIDAE) CEREAL STALK BORER, THE EUROPEAN CORN BORER (OSTRINIA NUBILALIS).

Rostern N. Tembo

A DISSERTATION

Submitted to the graduate college of Bowling Green State University in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

May 2009

Committee:

Dan Pavuk, Advisor

Alexander Tarnovsky (graduate college Rep)

Gabriela Bidart Bouzat

Ronald Hammond

Moira Van-Staaden

Rostern Tembo

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Abstract

Dr. Daniel Pavuk, Advisor

The research examined the effects of microbial and novel insecticidal control strategies

and also Trichogramma pretiosum on the European corn borer (ECB), Ostrinia nubilalis

(Hubner) (Lepidoptera: Crambidae). The treatments included Spinosad (an insecticide from

bacteria (Saccharopolyspora spinosa), entomopathogenic fungus Beauveria bassiana, the

parasitoid Trichogramma pretiosum and foliar applied Bt spray and two combinations Beauveria

bassiana plus Bt spray and Trichogramma pretiosum and Spinosad.

The infestation of corn by European corn borer larvae was severe in all control treatments

for both 2006 and 2007. The microbial treatments caused various levels of European corn borer

mortality. In all the parameters there was a significant difference between the control and other treatments with a P value of <0.001. In this research, treatment 3 (Spinosad) emerged as the most

effective biological agent in the control of the European corn borer.

The treatments had no effect on the relative abundance and composition of non-target

arthropods (P >0.05).

The possible desirable outcome of the research would be an increased awareness of the

alternative and potentially useful control strategies available for Ostrinia nubilalis. The research

provides support for underutilized control strategies and increased stakeholder adoption of

integrated pest management practices and thereby reducing the use of conventional insecticides

especially, for organic farmers.

To my God who has allowed me to earn this highest degree (doctorate) on the land.

To my parents who have both departed this life leaving a legacy of love, hope and

perseverance.

To my family members who have made many sacrifices for me in order to achieve this level in education.

Acknowledgement

I would like to thank the Lord for having given me the opportunities and abilities that helped me pursue this program and for many blessings I have received throughout my life. It was from His grace alone that this accomplishment was born.

I would like to thank Dr. Daniel Pavuk - my advisor for all the knowledge he has passed onto me over the past four years, his insight into what I was doing, his patience with me, his help and his encouragements when the work was hard, all this played a role in making me finish this research.

I would like to acknowledge the invaluable support from my doctoral committee members: Drs.

Ronald Hammond (from Ohio State University), Stan L. Smith, Moira Van Staaden and

Alexander Tarnovsky (the graduate college representative), for their help during Oral exams.

Thank you to Matt Davis - the manager and his staff of Northwestern Branch of the Ohio

Agriculture Research and Development Center (OARDC) of Ohio State University (where I did my research) for their help throughout the three years of doing this research. Thank you to my lab mates; Laura, Mary and Melanie for your assistance and friendship rendered to me in the lab.

I have learned over the years that there are many people that have a hand in our everyday lives.

Some are more noticed than others.

Special appreciation goes to my dearest wife Eddie, for her patience and countless hours devoted to supporting me in this adventure. Your help at the research center and at home, your understanding of what I was doing and your encouragement will always be remembered. To my two wonderful sons, Emmanuel and Nathan for your help also at the research center and for helping me realize that I could not remain in graduate school for ever. All of you three have tremendously helped me earn this higher degree with love, patience and in ways I will never know. Thank you!! Thank you!! Thank you!! This research was sponsored by, Katzner and vi

University Bookstore Fund For Graduate Student Research and Professional Development fund and North Central Sustainable Agriculture Research and Education (SARE).

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TABLE OF CONTENTS

CHAPTER I. GENERAL INTRODUCTION……………...……………………………… 12

Life Cycle of the European Corn Borer (Ostrinia nubilalis)…………………………. 13

Biological Control of the European Corn Borer……………………………………… 16

Bacillus thuringiensis…………………………………………………………………. 20

Control of the European Corn Borer………………………………………………….. 23

Review of Other Research Done on European Corn Borers………………………….. 24

CHAPTER II. EFFECTS OF TREATMENTS ON INFECTION

LEVELS AND DAMAGE BY EUROPEAN CORN BORER

LARVAE (OSTRINIA NUBILALIS)… ………………………………………………. 29

Objectives For This Research………………………………………………………… 29

Materials and Methods……………………………………………………………...... 29

Data Acquisition…………………………………………………………………...... 32

Results…………………………………………………………………………...... 33

Mean Number of Infested Stalks…………………………………………………….. 33

Mean Number of Larvae per Stalk………………………………………………...... 34

Mean Number of Tunnels per Stalk………………………………………………….. 34

Mean Length of Tunnels………………………………………………..……………. 35

Mean Number of Infected Cobs…..…………………………………………………. 36

Mean Yield per Plot………………………………………………………………….. 36

CHAPTER III. THE IMPACT OF THE TREATMENTS ON NON-TARGET

ARTHROPODS……...... 38

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Materials and Methods……………………………………………………………...... 38

Results of Insect Sampling………………………………………………………...... 40

Diabrotica virgifera virgifera………………………………………………………… 40

Popillia japonica…………………………………………………………………...... 40

Coccinella septempunctata…………………………………………………………… 41

Euschistus variolarius……………………………………………………………...... 41

Ostrinia nubilalis……………………………………………………………………... 41

Melanoplus femurrubrum……………………………………………………………. 42

Neoconocephalus ensiger…………………………………………………………… 42

Andrena imitatrix cresson…………………………………………………………… 42

CHAPTER IV. DISCUSSION AND CONCLUSION………………………………...... 44

REFERENCES……………………………………………………………………… 47

APPENDIX A. FIGURES…………………………………………………………. 56

APPENDIX B. TABLES…………………………………………………………… 82

LIST OF FIGURES

Figures Page

1 The Effect of European Corn Borer Larvae on Corn Leaves…………………… 56

2 The Effect of European Corn Borer Larvae on Ear Shank……………………… 57

3 Two Different Generations of the European Corn Borer……………………...... 58

4 The Effect of European Corn Borer Larvae on the Stalks………………………. 59

4b The Effect of European Corn Borers on the Stalks…………………………...... 60

5 Lydella thompsoni Adult Fly……………………………………………………. 61

6 A Macrocentrus cingulum Reinhard……………………………………………. 62

7 Eriborus terebrans Female………………………………………………………. 63

8 Grasshopper and Maggot Killed by Beauveria bassiana……………………...... 64

9 Trichogramma pretiosum Male………………………………………………….. 65

10 Bacteria Saccharopolyspora spinosa…………………………………………….. 66

11 Bt Spores………………………………………………………………………… 67

12 The Mean of Infested Stalks for 2006 and 2007………………………………… 68

13 The Mean of Larvae in Different Treatments Sampled in 2006 and 2007……… 69

14 The Mean of Tunnels in Different Treatments for 2006 and 2007……………… 70

15 The Mean of Length of Tunnels in Different Treatments for 2006 and 2007...... 71

16 The Mean of Infested Cobs for 2006 and 2007.…………………………………. 72

17 The Mean Yield of Corn for 2006 and 2007…………………………………….. 73

18 The Mean For Diabrotica virgifera virgifera for 2006 and 2007……………….. 74

19 The Mean For Popillia japonica for 2006 and 2007……………………………. 75 x

20 The Mean For Coccinella septempunctata Sampled for 2006 and 2007………... 76

21 The Mean For Euschistus variolarius for 2006 and 2007……………………….. 77

22 The Mean For Ostrinia nubilalis for 2006 and 2007……………………………. 78

23 The Mean For Melanoplus femurrubrum for 2006 and 2007…………………… 79

24 The Mean For Neoconocephalus ensiger for 2006 and 2007…………………… 80

25 The Mean For Andrena imitatrix cresson for 2006 and 2007………………...... 81

26 The Pheromones and Plots Showing Buffers…………………………………… 82

27 Time of Sampling Arthropods………………………………………………...... 83

LIST OF TABLES

Tables

1 Seven Different Treatments……………………………………………………. 84

2 Different Plots of Treatments………………………………………………...... 85

3 Mean Results of Different Treatments (2006)…………………………………. 86

3b Mean Results of Different Treatments (2007)………………………………….. 87

4 Mean Results of Different Arthropods Sampled in 2006………………………. 88

4b Mean Results of Different Arthropods Sampled in 2007………………………… 89

CHAPTER 1

GENERAL INTRODUCTION

Maize (Zea mays), or corn, together with wheat and rice, are the major cereal crops grown around the world (at least in 53 countries) (FAO Stat.2001). It ranks third in production following wheat and rice. Maize is the world’s most widely grown crop in almost all tropical areas of the world, including the tropical highlands over 3000 m in altitude and as far North as the 65th latitude in temperate areas. Because of different ecological conditions that exist between

the temperate areas and the tropics, the insect vectors and their disease agents also are different

under these different conditions (Tsai and Falk 1999). Corn is an extremely important crop

grown in the United States, Europe and Africa for both human consumption and livestock. Of the

various pests that attack maize/corn in Africa, the Lepidopteran stem /stalk borers are by far the

most injurious, particularly Chilo partellus (Kfir. 2002, Youdeowi 1989). In the United States

(Ohio, Minnesota, Iowa and other Corn Belt states), there are a number of pests that attack corn,

the European corn borer - Ostrinia nubilalis (Lepidoptera: Crambidae) is one of the most

destructive pests, significantly affecting growth and production of corn.

The European corn borer is not a native North American pest. It came to North America

during the early 1900s, possibly in broom corn imported from central Europe (Hungary and

Italy). It was found in the North-Central States in 1921. It spread slowly from Southern Michigan

and Northern Ohio. The European corn borer is the most damaging insect pest of corn

throughout the United States and Canada. Losses resulting from European corn borer damage

and control costs exceed $1 billion each year (Mason et al. 1996, University of Minnesota 2002).

During a 1995 outbreak, losses in Minnesota alone exceeded $285 million. A recent four-year

study in Iowa indicated average losses of nearly 13 bushels per acre (826.3 kg/ha) in both first

13 and second generations of European corn borer, for total losses of about 25 bushels per acre

(1589 kg/ha) (University of Minnesota 2002).

Damage to corn from the European corn borers has increased in Europe in the last several decades. This increase may be due to environmental changes, the significant increase in monoculture corn acreage, the introduction of more susceptible hybrids, and the increased use of pesticides, which could be reducing predator/parasitoid populations (Cordero, et al. 1998).

Life Cycle of the European Corn Borer (Ostrinia nubilalis)

In Ohio the European corn borer, normally goes through two generations each year (Fig 3).

There are four stages in each generation: egg, larva, pupa and adult, commonly known as the moth. The larva feeds a lot and grows more and more while shedding its exoskeleton (molting) as it grows. The larva goes through five instars, or larval stages of development. During the fifth instar, all larvae either prepare to pupate and become adults or enter diapauses (Iowa State

University, 2006). The larva overwinters as a full grown 5th instar larva in the corn stalks and cobs. The overwintered larvae pupate in the spring and emerge as moths ten to fourteen days later during May and June. Moths later gather in grassy areas of the field margins to mate and

drink dew on plants (Iowa State University, 2006). On warm evenings in June, the female moths

fly from these grassy areas into corn to lay masses of eggs (normally 15 to 25 per egg mass) on the leaves. Eggs hatch out within five to seven days depending on temperature. The newly hatched larvae establish themselves in the whorl, where they start feeding on new leaves. Early- instar larvae initially feed on foliage, causing window-pane injury, and on the tender central whorl, which subsequently leads to shot-hole injury in the emerging foliage. As the larvae reach

the third- and fourth-instar stages (about 1/2-in. long), they tunnel into the mid-ribs and stalks.

There they complete their larval development as fifth-instar larvae and transform into pupae

14 from which adult moths will emerge (Ohio State University Extension Factsheet, 2001). When the leaves grow and unroll from the whorl, the small round holes scattered in the leaves can be seen. After two weeks, the larvae leave the whorl and bore into stems, excavating tunnels in which they develop completely into pupae within the plant. In late July and August, moths emerge from the pupae and move to grassy vegetation near or inside the cornfields to mate and drink water. Summer moths may lay 85% of their eggs on the undersides of the ear leaf. After hatching the second-generation, larvae feed on leaves for a few days (Fig.1). The larvae bore into stems and ear shank (Fig.2). These larvae usually overwinter and do not pupate until the following spring.

The overwintering larvae break their diapause in the spring of the following year, with

the onset of warm weather, longer photoperiods and spring rains, the larvae pupate, and the

adults enclose and mate (Fig.3). Fertilized Ostrinia nubilalis females lay their eggs on maize; these first generation larvae damage plants by feeding on the leaves, including the midrib and tunneling in the maize stalks. The second generation larvae tunnel in stalks and ears and feed on leaf sheaths and collars (Mason et al., 1996). The tunnels that have been created by the larvae

reduce the strength of the stalks and ear shanks, thereby predisposing the corn plants to stalk breakage and ear drop and enhancement of stalk rot due to fungal infection. The feeding activities of Ostrinia nubilalis larvae result in both physiological (reduction in ear development) and physical (dropped ears and broken stalks) damage (Clark et al., 2000). The feeding of these

European corn borer larvae results in reduced plant growth, malformed ears, and reduced kernel size and harvest losses due to broken plants (Melchinger et al. 1998). Research has shown that in

Europe, corn yields are reduced 6% for one European corn borer larvae per plant and European

15 corn borer infested maize is characterized by the presence of tunnels throughout the stem (Bohn et al. 1999) (Fig.4).

In most cases, the probability of heavy infestation of corn by both generations of the

European corn borer is low (Hudon et al. 1989). This is so because the first generation moths prefer the most developed fields in an area while the second generation moths often target the least developed fields (Hudon et al., 1989). This means, therefore, that many fields will escape significant damage by one generation or the other. Corn plants heavily infested by first generation borer larvae are unattractive to egg-laying moths of the second generation (Hudon et al. 1989). Infested corn plants produce an odor that is repellent to the moths. Also the excrement and frass of the first generation larvae repel the second generation moths (Hudon et al. 1989).

Secondary infestation of corn stalks by fungi and bacteria is another form of damage associated with European corn borer larvae feeding. European corn borer larvae carry spores of fungal pathogens in the genus Fusarium moniliforme, thereby increasing the incidence of stalk, ear and root rots. Fusarium appears white (moldy corn) to salmon colored, though it may not be visible on the corn kernel (Bakan et al., 2002). Fusarium moniliforme is a common pathogen of corn, that it is found wherever corn is grown. The feeding holes left on the corn kernels by

European corn borer larvae serve as a preferential site for penetration of fungi. Close associations have been noted between susceptibility of corn hybrids to European corn borer larvae and the appearance of stalk rot (by Fusarium graminearum). Fusarium species (Fusarium culmorum (F. roseum) and F. equiseti (F. roseum) that infest corn produce toxins in the plant

tissue called Fumonisins (Bakan et al., 2002). Fumonisins are a group of mycotoxins produced

by fungi in the genus Fusarium. They are the most common mycotoxins because they are acutely

toxic to animals (pigs and horses). They have been implicated as possible cause of human

16 esophageal cancer, equine leukoencephalomalacia (ELEM), a serious disease in horses and porcine edema, a disease in swine. These animals contract such diseases from the feeding on corn containing the fungi (Bakan et al., 2002).

Biological Control of the European Corn Borer

Control measures against the European corn borer have traditionally involved the use of insecticides, resistant cultivars, and biological control organisms. Because Ostrinia nubilalis is an invasive organism, a classical biological control program was initiated against it soon after it was introduced. Between 1920 and 1938, twenty four parasitoids were released in the United

States from Europe and the orient (Baker et al. 1949). Only three species were and are subsequently recovered from Ostrinia nubilalis larvae: Lydella thompsoni (Herting) (Diptera:

Tachinidae), Macrocentrus cingulum (Goidanich), (Hymenoptera: Braconidae), and Eriborus terebrans (Gravenhorst). Lydella thompsoni is a solitary endoparasitoid of European corn borer larvae. This tachinid fly was one of twenty four species of parasitoids introduced from Europe and the Orient (Asia) as part of a USDA importation program (Baker et al., 1949). For many years after its introduction L. thompsoni was the most important parasitoid of European corn borer in many areas of the United States. Parasitization of up to 75% of the second borer generation was recorded in the early years, and it was considered a major controlling factor of borer populations. But there was an abrupt, unexplained decline in populations around 1960 and the fly disappeared from many places (Baker et al. 1949). There have been subsequent reintroductions in several locations and L. thompsoni appears to be established once more, particularly from Connecticut west to central Ohio and into South Carolina (fig.5). Macrocentrus cingulum, a braconid parasitoid of European corn borer is native to Europe and Asia. It was one of three exotic parasitoids that became established in North America from 24 species introduced

17 in 1926. Macrocentrus cingulum is most abundant in a band from northeastern Pennsylvania to eastern Virginia. It may parasitize up to 50% of corn borer larvae in these areas, but a much smaller proportion in the Midwest. Macrocentrus cingulum became established in the Midwest during the late 1940's, and was soon considered an important mortality factor of the corn borer.

Populations of Macrocentrus cingulum declined, however, in the early 1960's, about the same time the microsporidian pathogen Nosema pyrausta, which affects both the parasitoid and the

European corn borer, became established here. This wasp was detected in only five north central states in a 1987-1990 USDA survey of European corn borer parasitoids (Pavuk & Stinner 1992).

Very high levels of parasitism at a few locations in Wisconsin in the early 1990’s suggest that

Macrocentrus cingulum may be locally abundant, even though average populations are quite low

(Fig.6). The ichneumonid wasp, Eriborus terebrans, was also introduced to the United States as part of the classical biological control project to control the European corn borer. Approximately

140,000 wasps collected from both Asia and Europe were released from 1927 through 1940 in 13 states from Vermont to Virginia and as far west as Indiana and Michigan (Pavuk & Stinner

1992). Eriborus terebrans is an endoparasitoid of European corn borer larvae. It became established in the North Central Region of the U.S and is currently one of the most widely distributed parasitoids of the corn borer in the Corn Belt. This parasitoid overwinters as a larva inside overwintering corn borers, and resumes development in the spring. Adult wasps complete development in the prepupal stage of the corn borer. Emergence of first generation wasps coincides with the first larval generation of European corn borers. Females mate soon after emergence and can lay eggs within a day after emergence (Winnie & Chiang. 1992) (Fig.7).

Studies in Ohio have determined that all three of these parasitoid species occur in the state,

though Lydella thompsoni has largely disappeared, being only occasionally reared from Ostrinia

18 nubilalis larvae (e.g. Pavuk & Stinner 1992, Mason et. 1994). Predatory insects have also been shown to inflict significant mortality on the European corn borer. In particular, the ladybird beetles (Coleoptera: Coccinellidae), predatory bugs, mainly Orius insidious (Say) (Heteroptera:

Anthocoridae) and Nabis spp. (Heteroptera: Nabidae), syrphid larvae (Diptera: Syrphidae) and lacewing larvae and adult (Neuroptera: Chrysopidae) have all been observed to feed on the eggs and larvae of Ostrinia nubilalis (Phoofolo et al., 2001, Musser & Shelton, 2003). Spiders

(Araneae) are also potentially effective predators of Ostrinia nubilalis life stages. Ground beetles

(Coleoptera: carabidae) can also feed on larvae that may become dislodged from plants, although these predators are not likely to be major regulators of corn borer populations (Phoofolo et al.

2001, Musser & Shelton, 2003). Certain entomopathogens cause significant mortality in Ostrinia nubilalis populations. The microsporidian, Nosema pyrausta, can exert substantial mortality on

European corn borer populations (Zimmack & Brindley, 1957, Lewis & Lynch, 1978, Pierce et al., 2001). It is an effective and wide spread pathogen of Ostrinia nubilalis. Nosema pyrausta is an obligate parasite of Ostrinia nubilalis (Sajap and Lewis, 1988), slowing larval development and increasing mortality. This protozoan-like microbe reduces egg laying, kills some larvae, and increases over-wintering mortality. Mortality caused by this disease increases when European corn borer larvae are stressed by other factors, such as harsh weather. An unclassified microsporidium in the genus Nosema is not as common as Nosema pyrausta, but its prevalence is increasing in Iowa and Illinois. It is more virulent, thus causing greater mortality than N. pyrausta. Ostrinia nubilalis adults infected with Nosema pyrausta have reduced fecundity and longevity (Windels et al., 1976).

The entomopathogenic fungus, Beauveria bassiana, can cause mortality in Ostrinia nubilalis populations (York, 1958, Lewis et al., 2002). Beauveria bassiana is a fungus, which

19 causes a disease known as the white muscadine in insects. This fungus is insect specific and common soil borne fungus that occurs worldwide. It attacks a wide range of both immature and adult insects. Besides silkworm, the extensive list of hosts includes such important pests as whiteflies, aphids, grasshoppers, termites, Colorado potato beetle, Mexican bean beetle, Japanese beetle, boll weevil, cereal leaf beetle, bark beetles, lygus bugs, chinch bug, fire ants, European corn borer, codling moth, and Douglas fir tussock moth. Natural enemies, such as lady beetles, are susceptible too, and it has even been found infecting the lungs of wild rodents, and the nasal passages of humans. There are many different strains of the fungus that exhibit considerable variation in virulence, pathogenicity and host range. It occurs in the soil as a saprophyte. It invades the insect body by contact. The fungal conidia become attached to the insect cuticle and after germination, the hyphae penetrate the cuticle and proliferate in the insect’s body producing toxins and draining the insect of nutrients, eventually killing it (Groden 1999). High humidity is essential for conidia germination and infection establishes between 24 and 48 hours. The infected insect may live for three to five days and then dies (Fig.8).

Trichogramma pretiosum are extremely tiny wasps in the family of Trichogrammatidae.

They range in size between 0.2 and 1.5 mm. These parasitoids occur naturally in almost every terrestrial habitat and some aquatic habitats as well (Knutson, 2005). They parasitize insect eggs, especially eggs of moths and butterflies. Some of the most important caterpillar pests of field crops, forests and fruit and nut trees are attacked by Trichogramma wasps (Knutson, 2005).

Thousands of adult Trichogramma released in a corn field infested with European corn borer eggs would seek out and parasitize the eggs. The result would be, a biological “insecticide” that strikes at the target pest with no risk to other natural enemies, humans’ health or the environment

(Knutson, 2005). Nevertheless, the use of Trichogramma for control of the European corn borer

20 can be difficult because there are factors to consider before using them, such as (a) the species of

Trichogramma to use, (b) method chosen to release them in the field, (c) the numbers released and the timing of the release, (d) the quality and fitness of the parasites and (e) the crop and environmental conditions. The Trichogramma must be released prior to egg hatching of the host.

To do this one would have to accurately predict moths’ occurrence and egg density to plan parasitoid release (King et al 1985). The Trichogramma are typically shipped and released as pupae inside host eggs. The parasitized pupae are usually on a strip of paper which can be stapled to the underside of a corn leaf. Adults emerge out within four hours, once the pupae inside host eggs are exposed to light and at least to 27 ºC (Morrison, Stinner and Ridgeway

1976., Smith, 1994) (Fig.9).

Bacillus thuringiensis

Bacillus thuringiensis is a Gram-positive, soil dwelling bacterium. Spores and crystalline insecticidal proteins produced by B. thuringiensis are used as specific insecticides under trade names such as Dipel and Thuricide. Because of their specificity, these pesticides are regarded as environmentally friendly, with little or no effect on humans, wildlife, pollinators, and most other beneficial insects. B. thurigiensis-based insecticides are often applied as liquid sprays on crop plants, where the insecticide must be ingested to be effective. It is thought that the solubilized toxins form pores in the midgut epithelium of susceptible larvae. Recent research has suggested that the midgut bacteria of susceptible larvae are required for B. thuringiensis insecticidal activity (Nichole, et al. 2006). Bacillus thuringiensis, has been used extensively to control a wide range of lepidopteran pests (Gill, 1992), including Ostrinia nubilalis (Bartels & Hutchison, 1995,

Huang et al., 1999).

The insecticidal proteins produced by Bacillus thuringiensis (Bt) have provided a uniquely specific, safe, and effective tool for the control of a wide variety of insect pests. Bt has been used in spray formulations for over 40 years, where it is considered remarkably safe- mainly because specific formulations harm only a narrow range of insect species. Bt insecticidal protein genes have been incorporated into several major crops, where they provide a model for genetic engineering in agriculture. Viable bacterial spores of Bacillus thuringiensis constitute the active ingredient in Bt spray formulations. Crystal (Cry) and Cytolytic (Cyt) Insecticidal Crystal

Protein (ICPs) are the primary cause of insect’s death. However, other components in sprayed Bt pesticides can contribute, especially in insects that are not very sensitive to cry proteins. For example, the Bt spore is important to lethality to both gypsy moth larvae and to such pests as the beet army worm and the cotton boll worm. The Bt spore germinates after the gut is damaged and then begins vegetative growth, producing other insecticidal toxins and synergists. These include vegetative insecticidal proteins (VIPs), β-exotoxin, zwittermicin A, chitinases, and phospholipids. Bt sprays are used sporadically and typically over small areas. In tropical and subtropical areas populations of diamondback moth have become resistant to Bt sprays following their intensive use. Crops sprayed with traditional Bt formulations include various vegetables, tree fruits, artichokes and berries. There are advantages and disadvantages to the use of Bt in spray form. Like chemical pesticides, the timing, dosage and formulation of the application can be controlled in any growing season to meet specific pest pressure. The drawbacks are that spray can drift during application, cannot be applied uniformly to all parts of the plant, and cannot be delivered to pests that are inside the plant tissue. Furthermore, insecticidal crystals and spores of

Bt exposed on plant surfaces are highly sensitive to degradation by UV light and removed by water runoff (Schnepf, et al., 1998). The incorporation of the endotoxin gene from Bacillus

22 thuringiensis (Bt) into maize plants to produce transgenic Bt maize has revolutionized the control of Lepidopterous pests of maize (Koziel et al., 1993, Jansens et al., 1997, Graeber et al., 1999,

Hyde et al., 1999, Clark et al., 2000, Baute et al., 2002, Catangui, 2003). However, concern has been expressed concerning the development of Ostrinia nubilalis resistance to Bt corn (Van

Emden, 1999), and the use of non-transgenic refuges to manage resistance has been urged

(Onstad & Gould 1998, Caprio et al., 1999.

Spinosad is derived from the metabolites of the naturally occurring soil actinomycete,

Saccharopolyspora spinosa. Spinosad may be used to control pests in both agricultural environments and also in green houses, golf courses, gardens and around homes. Spinosad is a mixture of Spinosyn A, (C41H65NO10) and Spinosyn D, (C42H67NO10 that are tetracyclic- macrolide compounds produced by an Actinomycete, Saccharopolyspora spinosa, originally isolated from a Caribbean soil sample (Sparks et al., 1998). It is primarily a stomach poison with some contact activity and is particularly active against Lepidoptera, Diptera, some coleopter, termites, ants, and thrips (Bret et al., 1997). It is a neurotoxin with a novel mode of action targeting the nicotinic acetylcholine receptor. Exposure results in cessation of feeding followed later by paralysis. It basically causes excitation of the insect nervous system, leading to involuntary muscle contractions, prostration with tremors, and finally paralysis and death.

Spinosad is manufactured at Dow AgroScience’s facility in Harbor Beach, Michigan, using a

fermentation process in which Saccharopolyspora spinosa colonies are grown using natural

products such as soybean and cottonseed meal (Dow AgrowSciences 2003). It has been

developed to provide rapid control of Lepidoptera and other pests with minimum disruption of beneficial insects and other non target organisms. Spinosad provides users with a unique package of desirable features, such as: Effective control of many pest species, low application rates

23 resulting in low environmental load, safer for use with most beneficial insects, and low mammalian, low avian, and fish toxicity. Spinosad has been embraced by Integrated Pest

Management (IPM) practioners as one of the new generation of biorational pesticides. This has been due to its efficacy as an insecticide together with the selective toxicity characteristics of the product and favorable environmental profile.

Control of the European Corn Borer

To avoid yield losses of corn, insecticides are often used by farmers to control the

European corn borer. The use of insecticides such as Lorsban 4E (Chloropyrifos), Intrepid 2F

(Methoxyfenozide), permethrin (liquid Ambush or granular Pounce), lambda cyhalothrin, furadan (carbofuran) and Methyl Parathion has been very common, especially before the larvae tunneled into the corn stalks. However scouting for European corn borer larvae, especially second generation larvae is not easy and timing an insecticide application for maximum efficacy is difficult. Many producers did very little to manage European corn borers in Illinois and some

Midwest States (Rice & Ostlie, 1997), but overuse of insecticides is not economically sound and is environmentally hazardous. For example Chlorpyrifos (Lorsban) has a high toxicity on birds;

Carbofuran (Furandan) has a high toxicity on birds, mammals and fish, while permethrin has an extremely toxicity to fish, high on bees and low on birds (Palmer & Bromley, 1992). In recent years, elevated awareness of the impacts of pesticide use on the environment and human health has resulted in efforts to reduce reliance on chemical control. Recent experiments with the systemic insecticide fipronil indicate that it provides a high level of control of first generation corn borers (76% reduction) (John et al, 1998). Agricultural products include Chipco Choice (a product of fipronil) for use against pests of field corn, golf courses and commercial turf. Applied to the soil, fipronil is taken up in the plant tissue, where it remains active for up to 10 weeks

(John et al. 1998). Fipronil is a broad-spectrum insecticide that disrupts the insect's central nervous system (John et al, 1998).

Many countries have instituted more stringent regulations on pesticide manufacture, registration and use, thereby increasing the cost and decreasing the availability of these tools.

The need for alternatives to pesticides use is very clear. A recent report by the U.S congress,

Office of Technology Assessment (U.S congress, OTA 1995) indicates that biologically based technologies such as biological control could be more widely used to solve pressing needs in pest management.

Review of Other Research Done on European Corn Borer

Kfir, (1992) in Pretoria, South Africa speculated that parasitoids play an important role in curtailing stalk borer populations and that without their activities, annual yield losses would be much higher. Kfir, (1993) demonstrated that a partial removal of natural enemies from a cereal crop could bring about a substantial increase in stem borer numbers. This indicates that natural enemies have the ability to suppress stem borer populations and reduce pest numbers (Kfir

2002).

Biotechnology has greatly helped to bring on the market hybrids that are resistant to the

European corn borer. Plant geneticists create Bt corn by inserting selected exotic DNA into the corn plant’s own DNA. Bt corn hybrids produce an insecticidal protein derived from the bacterium Bacillus thuringiensis, commonly called Bt. The Bt corn has a gene from Bacillus thuringiensis. Because these hybrids contain an exotic gene, they are commonly called transgenic plants. The Bt gene in these plants’ produces a protein that kills European corn borer larvae. Most larvae die after taking only a few bites. The unique feature of this bacterium is its production of crystal-like proteins that selectively kills specific groups of insects. These crystal

25 proteins (cry protein) are insect stomach poisons that must be eaten to kill the insect (Gianessi et al, 2003). Once eaten, an insect’s own digestive enzymes activate the toxic form of the protein.

The cry proteins bind to specific “receptors” on the intestinal lining and rupture the cells. Insects stop feeding within two hours of a first bite and if enough toxin is eaten, die within two or three days (Gianessi et al, 2003). Consequently, Bt corn provides high levels of yield protection even during heavy oviposition pressure by the European corn borer.

The Ohio State University has done research on the corn borer injury on both Bt corn and non-Bt corn. The results indicated more injury on the non-Bt corn than on the Bt corn, and the yield of Bt corn was larger than the yield of non-Bt corn (Agricultural Extension Bulletin, 2002).

An evaluation of corn yield in Iowa in 1998 demonstrated that the yield of Bt corn hybrids was

2.9 bushels per acre (184.3 kg/ha) higher than non Bt hybrids, (Farnham & Clint, 1998). Out of

84 comparisons (61%), the Bt hybrids outperformed their non-Bt counterparts. The data showed that even without significant corn borer pressure, Bt hybrids are capable of yielding as well as, if their non-Bt counterparts (Agricultural Extension Bulletin, 2002).

As with any other technology, the use of Bt corn has raised several questions regarding potential risks. There are concerns of populations of European corn borers developing resistance to Bt corn. The only documented case of resistance occurring in the field was a result of heavy use of Bt spray against the diamondback moth (Sayyed et al, 2000). To prevent or delay the emergence of insect resistance to Bt crops, the Biotechnology Industry, the United States

Environmental Protection Agency (EPA) research entomologists and farmers have worked together to develop insect resistance management (IRM) programs. The first component of these programs is insect resistance management via a high dose/refuge strategy (Biotech Summary,

2002). This strategy dictates that the dose of the insecticides should be sufficient to kill all

26 susceptible insect pests. Bt crops provide an advantage in this regard because the Bt dosage is both high and consistently expressed in the plant. In comparison, Bt sprays are deposited on plants in varying concentrations and at various times, subjecting insects to both high and low doses. The second component of the IRM strategy is the use of refuges of non Bt corns. The success of this strategy depends on the fact that resistance to Bt has been found to be a recessive trait. This means that Bt will still be effective against an insect that carries both Bt resistant and susceptible alleles because the susceptible allele is dominant. Refuges allow Bt-susceptible insects to proliferate without selection pressure from Bt toxins. The susceptible insects are then available to mate with resistant insects that may emerge from the Bt field (EPA, 2001, Biotech

Summary 2002). This slows the spread of the recessive gene, and lowers the chance that succeeding offspring carrying two Bt resistant genes will proliferate. Strategies also have been developed to detect resistance before it develops into problem. Monitoring the early emergence of resistance in the field is difficult, especially when the resistance trait is recessive. But new research has helped to solve this problem. A change at a single gene can confer a substantial resistance in certain nematodes and lepidopterans. As these genetic changes can be detected, monitoring can provide sufficient warning to adjust insect control strategies and avoid a large population of resistant pests from emerging. Gene stacking is another way insecticides resistance can be prevented. Bt crops should incorporate two or more Bt genes (Huang et al., 1999). Insects then would have to develop resistance to two or more insecticides to survive. Wheat varieties containing multiple genes for insect resistance have been used for decades to control stem rust without the emergence of uncontrollable pests.

The other concerns associated with the use of Bt are: The potential for harm to non target organisms. The possible ecological consequences of gene flow from engineered crops to non-

27 engineered crops and wild relatives. These concerns merit continued attention on a case by case basis in order to ensure that Bt technologies have the maximum positive impact with a minimum risk to agriculture. Prudent use of Bt technologies will also be key in maintaining their usefulness for a long period of time.

The conventional agriculture community is not aware of the risk. However, the potential effects of Bt corn on predators, parasitoids and pathogens that affect the populations of European corn borer have not been studied thoroughly, especially in field research. Pilcher et al. (1997) fed

Bt corn pollen to three predatory species in the laboratory. They reported that direct consumption of Bt corn pollen by Coleomeilla maculate (DeGeer), Orius insidious (say), and Chrysoperla carnea Stephens had no detrimental effects. Results from laboratory studies in Switzerland

(Hilbeck et al., 1998) revealed increasing mortality of C. carnea larvae that preyed on European corn borers or other Lepidoptera that fed on Bt corn. Interestingly, Zwahlen et al.,(2000) reported no effects on the predator Orius majusculus (Reuter) that were fed the thrips Anophothrips obscurus (Muller) that had fed on Bt corn.

There are two economically important cereal stalk borers that were introduced to

Southern Africa, Chilo partellus and Chilo sacchariphagus. Chilo partellus was brought to

Africa from Asia in 1930. Because of the disastrous effects Chilo partellus has on corn and of the displacement of native stalk borers, in 1993, endoparasitoids Cotesia flavipes from Pakistan was introduced to Kenya in Africa, to control the Chilo partellus populations. The impact of

Cotesia flavipes on stalk borer population in Kenya was recently investigated (Zhou, 2001). A ratio dependent, host-parasitoid model was used to estimate the stalk borer density with and without the parasitoid. A reduction of stalk borer infestation from 1.1 – 1.6 stalk borers per plant was observed (Kfir, 2002). In Madagascar where Cotesia flavipes was released against Chilo

28 sacchariphagus in sugar cane, maximum levels of parasitism (60%) were not reached until six years after the releases (Btbeder-Matibet & Malinge, 1967).

CHAPTER II

Effects of Treatments on Infestation Levels and Damage by the European Corn Borer

Objectives For This Research:

The proposed research had the following objectives:

1. To compare the efficacy of Bt spray, Beauveria bassiana, Trichogramma pretiosum,

and Spinosad for the economic control of Ostrinia nubilalis.

2. To assess the impact of these treatments on the abundance and composition of non-

target arthropods.

Materials and Methods

The research involved setting up a completely random design at the Northwestern

Branch of the Ohio Agriculture Research and Development Center (OARDC), one of the fields’ research facilities owned by Ohio State University and located in wood county, Ohio. The experimental design for this research consisted of 35 plots and 7 treatments. Each plot was 12.2 meters by 12.2 meters width (Table.2). Each plot was separated from the next plot by 12.2 meters by 6.1 meters buffer that was cultivated every week to remove weedy vegetation and reduce the movement of arthropods between plots. Beck 5222 variety corn was planted on 76 cm rows using reduced tillage practices and standard fertilization procedures. Each plot had 16 rows of corn. A rough estimate on plants per row was 65-70 stalks. Each plot had approximately 900 stalks. Pheromones traps obtained from Trece Corporation in California was set to help us determine the exact time when the Ostrinia nubilalis moths would be flying around and when to start the treatments (Fig. 26). Maize or corn was planted during the second week of May as was a common agriculture practice by producers in the Northwestern Ohio area. Preemergence (Lexar,

Roundup and Ester) and post emergence (Accent, crop oil concentrate and Basagram) herbicide were used to control weed populations within plantings.

Ostrinia nubilalis egg masses were obtained from Benzon Research Inc., 7 Kuhn Drive

Carlisle, PA 17013. From fifteen to twenty egg masses were stapled to the undersides of corn leaves in each of the plots to correspond to the approximate flight period of the first generation, which is usually mid to late May through early June. These egg masses were attached to the center 5 rows of each plot, 5 meters from each end of the set of corn rows, to avoid possible edge effects. A total of fourteen thousand eggs of European corn borers were artificially applied in all the plots during the first and also during the second generation. Egg masses were examined every day until hatching; any eggs showing evidence of disease were removed and immediately replaced. The egg masses were also checked daily for any evidence of predation by ladybird beetles or other predators such as Orius insidiosus. The same procedure was used to place the egg masses in the plots to correspond to the flight of the second generation moths in late July or early August. Treatment 1 was the control treatment and had no microbial agent applied.

Treatment 2 had the Trichogramma pretiosum parasitized eggs which were attached to the leaves of corn by way of stapling the strips containing the parasitized eggs to the leaves on the same day the attachment of the egg masses of European corn borer was done. The Trichogramma pretiosum takes one or two days to hatch out. In this case they hatched out before the eggs of the

European corn borer did, and were able to parasitize the European corn borer eggs. That was good as they were difficult to keep within one plot.

In 2006, all the sprayings were done by using the portable back pack sprayer.

Spinosad: 20 mL of spinosad per 3.78 liters of water (1 gallon of water). The sprayer used was a

3 gallons (11.34 Liters) sprayer. So for 11.34 Liters (3 gallons) of water, 60 mL of Spinosad was

31 used. This was enough to spray two plots of Spinosad treatment. Another similar mixture and a half was made for the remaining three plots of spinosad.

Beauveria bassiana: 25 mL of Beauveria bassiana per 3.78 liters (1 gallon) of water. The sprayer was a 11.34 liters, so 75 mL of Beauveria bassiana was used to make a solution. This was enough for two plots only, and another mixture and a half was made for the other three plots.

Bt spray: 38g of Bt granules per 3.78 liters of water (1 gallon). For 11.34 Liters (3 gallons sprayer), 114g of Bt granules were dissolved in a sprayer of water. This was enough for two plots of Bt treatment, and again another similar mixture and a half was made for the other plots.

In 2007 a tractor was used for all sprayings for both 1st and 2nd generations of the

European corn borer. Treatment 3 was sprayed with Spinosad as soon as the European corn borer

larvae became visible. Treatment 4 was sprayed with the Beauveria bassiana. Treatment 5 had

been sprayed with Bacillus thuringiensis sprays. Treatment 6 had a combination of Beauveria

bassiana and Bt Spray (same concentration as in treatment 4 and 5), and treatment 7 consisted of

both Trichogramma pretiosum and Spinosad treatments (Table 1) (same concentration as in

treatment 2 and 3).

The spraying methodology was as follows:

Spinosad: 20 mL of spinosad per 3.78 L (1gallon) of water. Beauveria bassiana: 25 mL of

Beauveria bassiana per 3.78 L of water. Bt spray: 38g of Bt granules per 3.78 L of water.

Since spraying was done by the tractor, changes had to be made on the volume of water used

(conforming to the tank size of the tractor) and the volume of the microbial agents as well. We used 60.48 L (16 gallons) of water per each trip of spraying. Therefore, 320 mL of Spinosad was mixed with 60.48 L of water, 400 mL of Beauveria bassiana was mixed with 60.48 L (16 gallons) of water and 602.3 g of Bt spray was mixed with 60.48 L of water. Each plot was 12.2

32 meters x 12.2 meters = 148.8m2. There were 5 replications of each of the 7 different treatments.

Five plots multiplied by 148.8 m² per plot = 744 m² per treatment. 744 m2 / 4046.8 m2 per acre =

0.1838 acres per treatment. We used water as a carrier at a rate of 75.6 L/acre. 75.6 L/acre x

0.183 acres per treatment = 13.8 L of water (carrier) needed per treatment. We rounded this up to

15.12 L. We needed at least 15.12 L to keep the machine pumping at a constant rate therefore to

allow for priming the system and making sure the previous product was purged from the lines we

mixed 30.24 L of solution for each treatment. This may seem excessive but it was necessary to

have this much guarantee, we had an adequate amount of solution to complete all the plots. We

mixed microbial agent with 60.48 L of water so that we could complete 2 treatments with each

load.

After the spraying was over, the experimental plots were observed twice each week for

evidence of feeding damage by Ostrinia nubilalis larvae on the maize plants. Windowpane

feeding and midrib and stalk tunneling were visibly seen whenever one would walk in the plots.

Data Acquisition

Parameters were established to determine the level and extent of damage to the stalks and corn

by European corn borer larvae. These parameters were: number of infected stalks per plot, the

number of larvae found per stalk, number of tunnels per stalk, length of tunnels per stalk, number

of infected cobs per stalk, and the yield per plot. During mid August, 20 stalks of maize/corn

were randomly (to void being biased) selected from each plot and were visually sampled for

damaged stalks, Ostrinia nubilalis larvae per stalk, number of tunnels per plant, tunnel length per

plant, and infected cobs recorded. Every plot was imaginary divided into four parts, and from

each part five stalks were cut down making twenty stalks from every plot. Each treatment had

five replications and the mean for each treatment was recorded (Tables 3a and b). Random

33 sampling ensured that each member of the population had an equal and independent chance of being chosen as a member of the sample, but the selection of any member of the population must in no way influence the selection of any other member. During harvesting time, the total yield per plot was also noted in kilogram per hectare. The calculated data were statistically analyzed by first doing Normality Test and then later statistical analysis using Minitab 14, one-way

ANOVA and differences between parameters were obtained by using Tukey post-hoc comparison analysis. The analysis helped identify which parameters were similar and which ones were different.

Results

Mean Number of Infested Stalks

There was a significant difference in the infestation of the stalks between the control treatments and the other treatments for this parameter. There was more significant damage or infestation by the European corn borer larvae in the stalks of corn in the control treatment than in the other treatments. The control treatment had a mean of 17 infested stalks per plot, while the other treatments had much less than that (see Table 3a). There was a significant difference between the control treatment and the other treatments (One way ANOVA; DF = 6, 28, F =14.73 and P =

0.001). There was very low infestation of stalks of corn in Spinosad treatment and treatment of

Spinosad and Trichogramma pretiosum, with a mean of 3.4 (±0.98 SE) and 4.8 (±1.2 SE), respectively, of the sampled stalks (Fig.12).

For the 2007 results are somehow similar to the results obtained in 2006. There is no noticeable difference between the control treatment and treatment with Trichogramma pretiosum. There was a significant difference between the control treatments and the other treatments (One way

ANOVA; DF = 6, 28, F = 9.83 and P = 0.001). The infestation of the stalks was greater in the

34 control treatment and in the treatment with Trichogramma pretiosum with a mean of 20 (± 0.0

SE) and 19.6 (± 0.4 SE), respectively. The trend seems to be very similar with that of 2006

(Fig.12.b).

Mean Number of Larvae per Stalk

The highest number of larvae per stalk was found in the control treatment followed by the treatment with Trichogramma pretiosum. The control treatment had a mean of 34.8, while the other treatments had fewer larvae per stalk (Table 3a). There was a significant difference between treatments with Spinosad, treatment with Spinosad plus Trichogramma pretiosum with the other treatments (One way ANOVA; DF =6, 28, F = 14.37 and P < 0.001). Treatment with

Spinosad (a mean of 9.8 (± 0.58 SE) and treatment of Trichogramma pretiosum plus Spinosad

(mean of 8.6 (± 1.86 SE) had lower mean larvae per stalk than the other treatments (Table 3a).

The 2007 results were similar to the 2006 results. The highest number of larvae was found in the control treatment and also in the treatment with Trichogramma pretiosum. (One way ANOVA;

DF =6, 28, F = 7.03 and P < 0.001). There were fewer larvae per stalk in treatment with

Spinosad (a mean of 6.0 (± 1.48 SE) and also in the treatment with Trichogramma pretiosum and

Spinosad with mean of 10.8 (± 1.16 SE) than in the control treatment which had a mean of 25.8

(± 3.38 SE) (Fig.13b).

Mean Number of Tunnels per Stalk

There were more tunnels in the stalks of corn from control treatment and plots with

Trichogramma pretiosum treatment with a mean of 24.2 (± 3.8 SE) and 25.4 (± 2.44 SE), respectively than in other treatments. There was a significant difference between the control treatment and treatments with Spinosad and Trichogramma pretiosum plus Spinosad (One way

ANOVA; DF = 6, 28, F = 9.09, P = < 0.001). Treatment with Spinosad and the treatment of

Trichogramma pretiosum plus Spinosad) had low numbers of tunnels (mean of 6.6 (± 0.4 SE) and 7.6 (± 7.6 SE) respectively, per plant compared to the rest of the treatments (Fig.14).

In 2007, there were more tunnels in the control treatment with a mean of 44.8 (± 3.06 SE) and also in treatment with Trichogramma pretiosum, with a mean of 36.6 (± 3.82 SE) than in any other treatments. There were fewer tunnels in treatment with Spinosad (a mean of 11.8 (±1.28

SE) than in any other treatments. There was a significant difference between the control treatment and treatment with Spinosad (One way ANOVA; DF = 6, 28, F = 14.18, P < 0.001

(Fig.14b).

Mean Length of Tunnels

There was no significant difference between control treatment and the treatment with

Trichogramma pretiosum. Both treatments had long tunnel means of 32.6 cm (± 2.3 SE) and 32 cm (± 5.4 SE) respectively. However, control treatment and treatment with Trichogramma pretiosum differed significantly from Treatments with Spinosad, Beauveria bassiana and Bt spray and Trichogramma pretiosum and Spinosad (One way ANOVA; DF = 6, 28, F = 6.96, P =

< 0.001). The shortest length of tunnels was recorded in treatment with Trichogramma pretiosum and Spinosad with a mean of 6.8 cm (± 1.7 SE) and also in treatment with Spinosad with a mean of 14.0 cm (± 3.3 SE) (Fig.15).

In 2007, the longest tunnels were also in control treatments and treatment with Trichogramma pretiosum with a mean of 46.0 cm (± 1.9 SE) and 39.0 cm (± 3.86 SE) respectively. The shortest lengths of tunnels were in Treatment with Spinosad with a mean of 12.4 cm (± 1.5 SE). There was a significant difference between the mean length of tunnels in control treatment and the mean lengths of other treatments (One way ANOVA; DF = 6, 28, F = 15.88, P <0.001)

(Fig.15b).

Mean Number of Infested Cobs

The highest infestation of cobs was recorded in control treatment with a mean of 17.4 (±2.0 SE) and treatment with Trichogramma pretiosum which had a mean of 13.4 (± 1.3 SE). There was no significant difference between the control treatment and treatment with Trichogramma pretiosum. The lowest infestation of the cobs was in treatment with Spinosad. There was a significant difference between control treatment and treatments with Spinosad, Beauveria bassiana, Beauveria bassiana plus Bt sprays and Trichogramma pretiosum plus Spinosad (One way ANOVA; DF = 6, 28, F = 7.08, P < 0.001). There was also low infestation of cobs in treatments with Bt sprays and Trichogramma pretiosum and Spinosad) (Fig.16).

In 2007, the highest infestation of cobs was in the control treatment with a mean value of

14.2 (± 0.73 SE) followed by the plots in treatment with Trichogrammma pretiosum with a mean of 11 (± 1.14 SE). There was no significant difference between the control treatment and treatment with Trichogramma pretiosum. There was a significant difference between the control treatment and other treatments (One way ANOVA; DF = 6, 28, F = 20.19, P < 0.001). The lowest infestation of cobs was recorded in treatment with Spinosad (Fig.16b).

Mean Yield

The yield was quite lower in the control treatment with a mean of 7847.11 (±1196 SE) kilograms per hectare, than the rest of the treatments, followed by treatment with Trichogramma pretiosum with a mean yield of 9942.04 (± 484.88 SE) kilograms per hectare. There was a significant difference between the control treatment and the treatments with Spinosad, Beauveria bassiana and Trichogramma pretiosum plus Spinosad. One way ANOVA; DF = 6, 28, F = 2.49 and P <

0.046. The highest yield was in treatment with Trichogramma pretiosum plus Spinosad with a

37 mean yield of 10797.57 (± 664.03 SE) kilograms per hectare, followed by treatment with

Spinosad with a mean yield of 10756.89 (± 376.22 SE) kilograms per hectare (Fig.17).

In 2007, the yield was slightly lower in the control treatment than in the other treatments.

In control treatment the mean yield was 10360.28 (±293.8 SE) kilograms per hectare while in the other treatments the yield was higher than that (Figure 17b). The highest yield was in Spinosad treatment, with 11515.8 (±110.2 SE) kilograms per hectare followed by treatment with

Trichogramma pretiosum plus Spinosad, with a mean yield of 11335.28 (± 153.2 SE) kilograms per hectare. There was no significant difference between the yield in the control treatment and the yield in other treatments (One way ANOVA; DF = 6, 28, F = 1.44, P = 0.237) (Fig.17b).

Yield losses are primarily physiological losses due to reduced plant growth. Stalk tunneling by the European corn borer results in shorter plants with fewer and smaller leaves.

Movement of water and nutrients can be restricted over the entire kernel- filling period. During the period of kernel growth, there is between 5 and 6% loss in grain yield for each European corn borer larva per plant. During corn development stage, the loss per plant is about 2 to 4% (Mason et. al. 1996). Most yield losses can be attributed to the impaired ability of the corn plants to produce normal amounts of grain due to physiological effect of larvae feeding on the leaf and conductive tissues (Mason et al. 1996).

CHAPTER III

THE IMPACT OF THE TREATMENTS ON THE ABUNDANCE AND COMPOSITION OF

ARTHROPODS.

Another research was also conducted to find out the arthropod predatory fauna and to assess the potential impacts that these treatments could have had on the abundance and composition of arthropods in corn plantings.

Material and Method

The random sampling process of insects started three weeks after the application of treatments. Every plot was imaginary divided into four parts, and movement through each part of plot was a walk and the beat – stick method of sampling was used. On each of the four parts of the plot the stalks of corn were beaten to scare the insects and those insects that tried to fly away were trapped by the net. Every plot had four places which were sampled for insects. The beat stick method equipment consisted of a cut broomstick approximately ½ m in length and a home tray (where arthropods fell after gently beating the stalk of corn with a stick), a jar (1000cm3 in volume) with less than 100cm3 of ethyl acetate solution in it, and a net for trapping the arthropods. There were also a number of plastic bags. After opening and closing of the jar, the smell of the ethyl acetate could diminish and insects could take long before they died. During that time some of the arthropods were put in plastic bags until the jar was refilled with new Ethyl acetate. This method of collection concentrated on free moving or flying arthropods. Any insect arthropod that was seen on the corn leaves, stalk or tassels was trapped by a net and after catching it was put in a jar containing ethyl acetate solution (which kills arthropods within few minutes). Some arthropods could fly away after falling on the tray and it was at that time that the net proved very essential because they could be trapped by the net and finally caught.

During both years 2006 and 2007 identification of the arthropods was done in the lab when the whole process of sampling was over. Sampling arthropods was always done on days that were clear and dry and with a minimum temperature of at least 21.0 degrees Celsius. During

2006 and 2007 sampling of insects was done in all the 35 plots. Most arthropods sampled from different plots with various treatments were in the order of Coleoptera (lady beetles Japanese beetles and Western corn root worm), Hemiptera (Sink bugs), Hymenoptera (bees), Lepidoptera,

(Moth) and Orthoptera (Katydids and grasshoppers). The reason for having sampled few orders of arthropod could be that the changes in corn plants growth levels could have potentially affected the arthropod orientation and interaction with the plants, and not the microbial agents.

For arthropods that feed on pollen (at that stage pollen had fallen off) the changed plants could for example offer less pollen as food source. A decrease in arthropod species availability would eventually affect other arthropod populations that fed on those arthropods from the decreased population. Bees need both suitable nesting sites and pollen plants (Gathmann et al. 1994).

Parasitoids depend on spatially and temporally co-occurrence of hosts and nectar (Russell 1989).

Some monophagous insect herbivores may spend their whole life on one host plant, feeding copulating and ovipositing (Zwolfer & Harris 1971; Tscharntke 1999), and may be these could be the ones that were sampled. Most organisms do not live completely independent of other organisms, but depend on more or less intimate interactions with other species (Redfearn &

Pimm 1987).

The following were the average insects that were sampled in all the plots. Only the Means and

Standard Errors (SE) will be reported. The data were statistically analyzed by using Minitab 14, one-way ANOVA. The analysis showed no significant overall differences between the insects sampled from control treatments and other treatments (Table 4a and 4b).

Results of Insect Sampling

The Diabrotica virgifera virgifera (Western corn rootworm)

Although in 2006, there was more Diabrotica virgifera virgifera in control treatment with mean

13.0 (± 1.6 SE) than in the rest of the other treatments, there was no significant difference between the control treatments and the other treatments (One way ANOVA; DF = 6, 28, F = 1.37 and P= 0.261) (Fig.18a).

In 2007 there were more western corn root worms caught in treatment with Trichogramma pretiosum, mean 3.4 (±1.1 SE) than in the other treatments and again there was no significant difference between the control treatments and the other treatments (One way ANOVA; DF = 6,

28 F = 1.94 and P = 0. 109) (Fig.18b).

The Popillia japonica (Japanese beetles)

The Popillia japonica was present in low in numbers in all the plots in 2006. Looking at the graph (Fig.19a) one would think that treatments of Spinosad (mean of 0.00 (± 0.0 SE)) and

Beauveria bassiana (mean 0.0 (± 0.0 SE)) had lower numbers than the other plots. There was no significant difference between the control treatments and the other treatments (one way ANOVA

– DF =6, 28, F = 0.50 and P = 0.803). In 2007, in the entire entire treatments Popillia japonica was present in low numbers. Although at a glance it appears as though were more Popillia japonica in the treatments with Beauveria bassiana plus Bt spray (mean 0.6 (± 0.4 SE) and treatment with Trichogramma pretiosum plus Spinosad (mean 0.6 (± 0.4 SE) than Popillia japonica in the control treatment and the others, there was no significant difference between the control and the other treatments (Fig.19b), (One way ANOVA; DF = 6, 28, F = 0.17 and P = 0.

984).

The Coccinella septempunctata (Lady beetles)

The Control treatment seems to have had a big mean 2.0 of Coccinella septempunctata in 2006 than other plots, while treatments with Bt spray, and Trichogramma pretiosum and Spinosad appear to have had the lowest means 0.4 (± 0.2 SE) and 0.8 (± 0.2 SE) respectively, (One way

ANOVA; DF = 6, 28 F = 1.54 and P =0.200). There was no significant difference between the control treatments and other treatments (Fig.20a).

In 2007 all the Treatments had the same mean 0.2 and same Standard of Error of Mean (SE) ±

0.2, there was no significant difference between the control treatments and other treatments

(Fig.20b), (One way ANOVA; DF = 6, 28 F = 0.00 and P = 1.000).

The Euschistus variolarius (Stink bugs)

Treatment with Trichogramma pretiosum with Spinosad had the larger mean 1.8 (± 0.8 SE) for

Euschistus variolarius than any other treatment while treatment with Beauveria bassiana had the lowest mean 0.00 (± 0 SE). Despite this large difference in means, there was no significant difference between the control treatments and other treatments (Fig. 21a) in 2006 (One way

ANOVA; DF = 6, 28 F = 1.92 and the P =0.112).

In 2007 Treatment with Spinosad had the largest mean 1.0 (± 0.77 SE) the Euschistus variolarius while treatment with Trichogramma pretiosum had the lowest mean 0.4 (± 0.2 SE). There was no significant difference between the control treatments and other treatments (Fig. 21b). (One way

ANOVA; DF = 6, 28 F = 0.25 and P = 0.953).

The Ostrinia nubilalis (Moths)

There was no significant difference between the control treatment and the other treatments in

2006 (One way ANOVA; DF = 6, 28 F = 01.67 P =0.166) (Fig. 22a).

In 2007 there was also no significant difference between the control and other treatments (One way ANOVA; DF = 6, 28 F = 0.50 and P = 0.803) (Fig 22b).

Melanoplus femurrubrum (grasshoppers)

The mean in all the treatments was less than 1.0. There was no significant difference between the control treatment and the other treatments in 2006 (One way ANOVA; DF = 6, 28 F = 0.92 and

P =0.496) (Fig.23a).

In 2007, the highest mean 1.2 (± 0.7 SE) was found in treatment with Spinosad while the lowest mean 0.0 (± 0 SE) was found in treatment with Trichogramma pretiosum. There was no significant difference between the control treatment and other treatments (One way ANOVA; DF

= 6, 28 F = 1.05 and the P = 0.94 (Fig.23b).

The Neoconocephalus ensiger (Katydids)

In all the treatments the mean was less than 1.0. There was no significant difference between the control treatments and the other treatments in 2006 (Fig.24a) (One way ANOVA; DF = 6, 28 F =

1.88 and P =0.121).

In 2007 the largest mean 2.8 (± 0.5 SE) was found in treatment with Trichogramma pretiosum

plus Spinosad while the lowest mean 1.0 (± 0.3 SE) was in treatment 3. There was no significant

difference between the control treatments and the other treatments (Fig.24b) (One way ANOVA;

DF = 6, 28 F = 1.78 and P = 0. 139).

Andrena imitatrix cresson (Bees)

In 2006 all the treatments had a mean of less than 1.0. There was no significant difference between the control treatments and the other treatments (Fig.25) (One way ANOVA; DF = 6, 28

F = 0.83 and P = 0. 554.

In 2007, the mean number of Andrena imitatrix in all the plots was low, less than 1.0. There was no significant difference between the control treatments and the other treatments (Fig. 25b) (One way ANOVA; DF = 6, 28 F = 0.26 and P = 0.951).

So far the statistical analysis done regarding the abundance and composition of the arthropods in all the plots for both years, showed no significant difference. The results clearly show that the different treatments applied to various plots had no effect on the distribution and abundance of these arthropods.

CHAPTER IV

DISCUSSION AND CONCLUSION

The results of this study indicated intensive infestation of stalks, lots of European corn borer larvae in the stalks, lots of tunnels, much infestation of cobs and a yield loss of corn in the control treatments. There was also a considerable infestation of the stalks by the European corn borer larvae, a good amount of larvae and a number of tunnels, and infested cobs which consequently affected the yield in the treatment with Trichogramma pretiosum. The explanation for this is that in the control treatments there was simply the lack of control of the European corn borer larvae in those plots. Whenever there is infestation of pests and control measures are not implemented, the results will be destruction of the crops resulting in decreased yield of the same.

In the treatment with Trichogramma pretiosum, it could be that, a) it is always difficult to contain the Trichogramma in the same plots. They are flying insects and are prone to wander around or fly away to distant places. b) There is a major limitation to the use of Trichogramma due to reduced efficacy under conditions of heavy rainfall, sunshine and high temperatures (J.

Chihrane and G. Lauge, 1996). There was a week when the temperatures were over 90 0F during

this research. c) In addition to reducing the efficacy of parasitism by Trichogramma, high

temperatures cause male sterility and reductions in the rate of wasp emergence from the

capsules. The other treatments (with Spinosad, Beauveria bassiana, Bt spray, Beauveria

bassiana plus Bt spray and Trichogramma pretiosum plus Spinosad) provided a considerable

amount of control of the European corn borer in almost all parameters for both years.

Throughout this research Spinosad has emerged as the most effective biological agent in

the control of the European corn borer. Treatments with Spinosad in all parameters have shown

the efficacy of Spinosad in controlling the European corn borer larvae. The general trend so far

45 has been that these biological agents have impacted the European corn borers. The infestations on corn have been very severe in all the control treatments while the different treatments have imposed various degrees of restraints on the European corn borer population. In all the parameters there has been a significant difference between the control and other treatments with

P<0.001.

While the tradition control methods of using insecticides are sometimes environmentally hazardous, and fail to control the European corn borer larvae when the larvae are in the tunnels, these novel (underutilized) biological control methods if extensively used would provide good control measures in an integrated pest management. They would provide farmers an economically effective and environmentally sound approach to the management of the European corn borers. This is so because one of the Biological agents, Beauveria bassiana by the help of its conidia would grow into the tunnel of the stalk develop into hyphae which proliferate and kill the larvae inside the stalk. Beauveria bassiana also has no preference as to its host’s stage in life; it will attack larvae and adults. A very unique characteristic is that it affects its host upon contact, unlike many other pathogens that need to be consumed to cause infection. Upon contact the pathogen kills the host from the inside out. It produces spores, known as conidia (asexual form), that directly infect through the outside of the insect’s skin; it then proceeds to germinate. From the spores it secretes enzymes that attack and dissolve the cuticle. It also produces Beauvericin, a toxin that weakens the host’s immune system.

This research finding is relevant in boosting underutilized control strategies and increasing stakeholder adoption of integrated pest management practices and thereby reducing the use of conventional insecticide. The results are good and relevant for increasing farmers’ adoption of

Integrated Pest Management practices, reducing the use of conventional, broad-spectrum

46 chemicals for Ostrinia nubilalis control and employing less environmentally harmful insecticides. By adopting less broadly toxic chemicals in pest management, control by natural enemies of European corn borer, such as Parasitoids such as Trichogramma pretiosum and microbial pathogens may be enhanced and this would in turn reduce the need for chemical controls and make row crop farming more profitable for the farmers.

The results of the research done on the abundance and composition of the arthropods in the treatment plots clearly show that the different treatments applied to various plots had no effect on the distribution and abundance of these arthropods. All the P values obtained by one way ANOVA were bigger than 0.05.

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APPENDIX A: FIGURES

Figure 1: The effect of European corn borers on the leaves.

Figure 2 : Effect of the European corn borer larvae on ear shank

Figure 3. Two different generations of the European corn borer per year in Ohio.

Figure 4. The effect of European corn borers on the stalks.

Figure 4b. The effect of European corn borers on the stalks

Fig.5. Lydella thompsoni adult fly, which parasitizes European corn borer

Fig.6. A Macrocentrus cingulum Reinhard female preparing to insert an egg into a European corn borer larva.

Fig.7. Eriborus terebrans female, a parasite of European corn borer larvae in the north central region of the

United States.

Fig.8.Grasshoppers killed by B. bassiana Beauveria bassiana on a maggot

Fig.9. T. pretiosum male

Fig. 10. Bacteria Saccharopolyspora spinosa from where Spinosad comes from.

Fig. 11. Bt spores

Infested Stalks

18 a 16

14 a

10 b 8 c Mean (±SEM) 6 c c 4 d

0 C T S B Bt B&Bt T&S Treatment

Fig.12.The mean number of infested stalks in different treatments sampled in 2006. Treatments that share the same Letters (lower case) are not significantly different. Different letters are significantly different (One way ANOVA, P<0.001). C =Control, T =Trichogramma pretiosum, S = Spinosad, B =Beauveria bassiana, Bt =Bt spray.

Infested Stalks

20 a a

b 15 b c c

10 d Mean (±SEM)

0 C T S B Bt B&Bt T&S Treatment

Fig.12b.The mean number of infested stalks in different treatments sampled in 2007. Treatments that share the same Letters (lower case) are not significantly different. Different letters are significantly different (One way ANOVA, P<0.001). C =Control, T =Trichogramma pretiosum, S = Spinosad, B =Beauveria bassiana, Bt =Bt spray.

Larvae per Treatment 40

35 a

30 a 25 a

20 b 15 c Mean (±SEM) d 10 d

0 C T S B Bt B&Bt T&S Treatment

Fig.13.The mean number of larvae in different treatments sampled in 2006. Treatments that share the same letters (lower case) are not significantly different. Different letters are Significantly different (One way ANOVA, P<0.001). C =Control, T =Trichogramma pretiosum, S = Spinosad, B =Beauveria bassiana, Bt =Bt spray.

Larvae per Treatment 40

30 a a 25

15 b

Mean (±SEM) c c c 10 d 5

0 C T S B Bt B&Bt T&S Treatment

Fig. 13b.The mean number of larvae in different treatments sampled in 2007. Treatments that share the same letters (lower case) are not significantly different. Different letters are Significantly different (One way ANOVA, P<0.001). C =Control, T =Trichogramma pretiosum, S = Spinosad, B =Beauveria bassiana, Bt =Bt spray.

Number of Tunnels

a 25 a a

20 b

15 c

Mean (±SEM) Mean 10 d d 5

0 C T S B Bt B&Bt T&S Treatment

Fig.14.The mean number of tunnels in different treatments sampled 2006. Treatments that share the same letters (lower case) are not significantly different. Different letters are significantly different (One way ANOVA, P<0.001). C =Control, T =Trichogramma pretiosum, S = Spinosad, B =Beauveria bassiana, Bt =Bt spray.

Number of Tunnels

50 a

40 a

30 b c 20 c Mean (±SEM) c

dc 10

0 C T S B Bt B&Bt T&S Treatment

Fig.14b.The mean number of tunnels in different treatments sampled in 2007. Treatments that share the same letters (lower case) are not significantly different. Different letters are significantly different (One way ANOVA, P<0.001). C =Control, T =Trichogramma pretiosum, S = Spinosad, B =Beauveria bassiana, Bt =Bt spray.

Length of Tunnels (cm) 40

35 a a 30 a 25 b 20

15 c c Mean (±SEM)

10 dc 5

0 C T S B Bt B&Bt T&S Treatment

Fig.15.The mean of length of tunnels in different treatments sampled in 2006. Treatments that share the same letters (lower case) are not significantly different. Different letters are significantly different (One way ANOVA, P<0.001). C =Control, T =Trichogramma pretiosum, S = Spinosad, B =Beauveria bassiana, Bt =Bt spray.

Length of Tunnels (cm)

50 a

40 a

30 b c c 20 c Mean (±SEM)

d c 10

0 C T S B Bt B&Bt T&S Treatment

Fig. 15b.The mean of length of tunnels in different treatments sampled in 2007. Treatments that share the same letters (lower case) are not significantly different. Different letters are significantly different (One way ANOVA, P<0.001). C =Control, T =Trichogramma pretiosum, S = Spinosad, B =Beauveria bassiana, Bt =Bt spray.

Number of infested cobs

18 a 16

14 a a

12 b 10 c c 8

Mean (±SEM) dc 6

0 C T S B Bt B&Bt T&S Treatment

Fig.16.The mean number of infested cobs in different treatments sampled in 2006. Treatments that share the same Letters (lower case) are not significantly different. Different letters are significantly different (One way ANOVA, P<0.001). C =Control, T =Trichogramma pretiosum, S = Spinosad, B =Beauveria bassiana, Bt =Bt spray.

Infested cobs 16 a 14

12 a

Mean (±SEM) bb 4 b b b 2

0 C T S B Bt B & Bt T & S Treatment

Fig.16b.The mean number of infested cobs in different treatments sampled in 2007. Treatments that share the same Letters (lower case) are not significantly different. Different letters are significantly different (One way ANOVA, P<0.001). C =Control, T =Trichogramma pretiosum, S = Spinosad, B =Beauveria bassiana, Bt =Bt spray.

Yield in Kg/ha

12000

a a a a a 10000 a

8000 b

6000

Mean (±SEM) 4000

2000

0 C T S B Bt B&Bt T&S Treatment

Fig.17.The mean yield of different treatments sampled in 2006. Treatments that share the same letters (lower case) are not significantly different. Different letters are significantly different (One way ANOVA, P<0.046). C =Control, T =Trichogramma pretiosum, S = Spinosad, B =Beauveria bassiana, Bt =Bt spray.

Yield in kg/ha per treatment

12000 a a a a a a a 10000

8000

6000

Mean (±SEM) 4000

2000

0 C T S B Bt B & Bt T & S Treatment

Fig.17b.The mean yield of different treatments sampled in 2007. Treatments that share the same letters (lower case) are not significantly different (One way ANOVA, P<0.237). C =Control, T =Trichogramma pretiosum, S = Spinosad, B =Beauveria bassiana, Bt =Bt spray.

Diabrotica virgifera virgifera 16 (Western corn root worm)

14 a a a 12 a 10 a

8 aa 6 Mean (±SEM)

0 C T S B Bt B & Bt T & S Treatments

Fig.18a.The mean number for diabrotica virgifera virgifera sampled in 2006. There was no significant difference between the control and other treatments (One way ANOVA, P = 0.261). a = means all were same. C =Control, T –Trichogramma pretiosum, S =Spinosad, B =Beauveria bassiana, Bt =Bt spray

Diabrotica virgifera virgifera 5.0 (Western corn root worm)

4.5

4.0 a 3.5

3.0

2.5 a 2.0 a a a

Mean (±SEM) a 1.5 a 1.0

0.5

0.0 C T S B Bt B & Bt T & S Treatment

Fig.18b.The mean number for diabrotica virgifera virgifera sampled in 2007. There was no significant difference between the control and other treatments (One way ANOVA, P = 0.109). a = means all were the same. C =control, T =Trichogramma pretiosum, S =Spinosad, B =Beauveria bassiana, Bt =Bt spray.

Popillia japonica 0.45 (Japanese beetle)

0.40

0.35

0.30

0.25 a a a a a 0.20

0.15

Mean (±SEM) 0.10

0.05 a a 0.00

-0.05 C T S B Bt B & Bt T & S Treatments

Fig.19.The mean number for Popillia japonica sampled in 2006. There was no significant difference between the control and other treatments. One way ANOVA, P = 0.803. a = means all were the same. C =control, T =Trichogramma pretiosum, S =Spinosad, B =Beauveria bassiana, Bt =Bt spray.

Popillia japonica 1.1 (Japanese beetle) 1.0

0.9

0.8

0.7 a a 0.6

0.5 a a a a 0.4 Mean (±SEM) 0.3 a 0.2

0.1

0.0 C T S B Bt B & Bt T & S Treatments

Fig.19b.The Mean number for Popillia japonica sampled in 2007. There was also no significant difference between the control and other treatments. One way ANOVA, P = 0.984. a = means all were the same. C =control, T =Trichogramma pretiosum, S =Spinosad, B =Beauveria bassiana, Bt =Bt spray.

Coccinella septempunctata 2.5 (Lady beetles) a 2.0

1.5 a a

1.0 a Mean (±SEM) a

0.5 a a

0.0 C T S B Bt B & Bt T & S Treatments

Fig.20.The mean number for Coccinella septempunctata sampled in 2006. There was no significant difference between the control and other treatments. One way ANOVA, P = 0.200. a = means all were the same. C =control, T =Trichogramma pretiosum, S =Spinosad, B =Beauveria bassiana, Bt =Bt spray.

Coccinella septempunctata (lady beetles) 0.40

0.35

0.30

0.25 a a a a a a a 0.20

0.15 Mean (±SEM)

0.10

0.05

0.00 C T S B Bt B & Bt T & S Treatment

Fig.20b.The mean number for Coccinella septempunctata sampled in 2007. There was no significant difference between the control treatments and other treatments. One way ANOVA, P = 1.00. a = means all were the same. C =control, T =Trichogramma pretiosum, S =Spinosad, B =Beauveria bassiana, Bt =Bt spray.

2.8 Euschistus variolarius 2.6 (Stink bugs) 2.4 2.2 2.0 a 1.8 1.6 1.4 1.2 1.0 a 0.8 Mean (±SEM) a a a 0.6 0.4 a 0.2 a 0.0 -0.2

C T S B Bt B & Bt T & S Treatment

Fig.21.The mean number for Euschistus variolarius sampled in 2006. There was no significant difference between the control and other treatments. One way ANOVA, P = 0.112. a = means all were the same. C =control, T =Trichogramma pretiosum, S =Spinosad, B =Beauveria bassiana, Bt =Bt spray.

Euschistus variolarius (Stink bugs) 2.0

1.8

1.6

1.4

1.2 a 1.0 aa 0.8 a Mean (±SEM) a a 0.6 a 0.4

0.2

0.0 C T S B Bt B & Bt T & S Treatment

Fig.21b.The mean number for Euschistus variolarius sampled in 2007. There was no significant difference between the control and the other treatments. One way ANOVA, P = 0.953. a = means all were the same. C =control, T =Trichogramma pretiosum, S =Spinosad, B =Beauveria bassiana, Bt =Bt spray.

Ostrinia nubilalis (Moths) 1.4 1.3 1.2 1.1 1.0 0.9 a 0.8 0.7 0.6 0.5 a a a 0.4 Mean (±SEM) 0.3 0.2 0.1 aa a 0.0 -0.1

C T S B Bt B & Bt T & S Treatment

Fig.22.The mean number for Ostrinia nubilalis sampled in 2006. There was no significant difference between the control and other treatments. One way ANOVA, P = 0.166. a = means all were the same. C =control, T =Trichogramma pretiosum, S =Spinosad, B =Beauveria bassiana, Bt =Bt spray.

Ostrinia nubilalis 0.45 (Moths)

0.40

0.35

0.30

0.25 a a a a 0.20

0.15

Mean (±SEM) Mean 0.10

0.05 a a a 0.00

-0.05 C T S B Bt B & Bt T & S Treatment

Fig.22b.The mean number for Ostrinia nubilalis sampled in 2007. There was no significant difference between the control and other treatments. One way ANOVA, P = 0.803. a = means all were the same. C =control, T =Trichogramma pretiosum, S =Spinosad, B =Beauveria bassiana, Bt =Bt spray.

Melanoplus fermurrubrum 1.4 (Grasshoppers) 1.3 1.2 1.1 1.0 0.9 0.8 a 0.7 a a 0.6 0.5 a 0.4 Mean (±SEM) 0.3 a a 0.2 0.1 a 0.0 -0.1

C T S B Bt B & Bt T & S Treatment

Fig.23.The mean number for Melanoplus femurrubrum sampled in 2006. There was no significant difference between the control and other treatments. One way ANOVA, P =0.496. a = means all were the same. C =control, T =Trichogramma pretiosum, S =Spinosad, B =Beauveria bassiana, Bt =Bt spray.

Melanoplus femurrubrum (Grass hoppers) 2.0

1.8

1.6

1.4 a 1.2 a 1.0 a 0.8 a 0.6 Mean (±SEM) a 0.4 a 0.2 a 0.0

-0.2 C T S B Bt B & Bt T & S Treatment

Fig.23b.The mean number for Melanoplus femurrubrum sampled in 2007. There was no significant difference between the control and other treatments. One way ANOVA, P = 0.940. a = means all were the same. C =control, T =Trichogramma pretiosum, S =Spinosad, B =Beauveria bassiana, Bt =Bt spray.

1.4 Neoconocephalus ensiger 1.3 (Katydids) 1.2 1.1 1.0 0.9 a 0.8 0.7 0.6 0.5 0.4 Mean (±SEM) 0.3 a a a 0.2 0.1 a aa 0.0 -0.1

C T S B Bt B & Bt T & S Treatment

Fig.24.The mean number for Neoconocephalus ensiger sampled in 2006. There was no significant difference between the control and other treatments. One way ANOVA, P = 0.121. a = means all were the same. C =control, T =Trichogramma pretiosum, S =Spinosad, B =Beauveria bassiana, Bt =Bt spray.

(Neoconocephalus ensiger) (Katydids) 3.5

3.0 a

2.5 a a 2.0 a a a 1.5

Mean (±SEM) a 1.0

0.5

0.0 C T S B Bt B & Bt T & S Treatment

Fig.24b.The mean number for Neoconocephalus ensiger sampled in 2007. There was no significant difference between the control and other treatments. One way ANOVA, P = 0.139. a = means all were the same. C =control, T =Trichogramma pretiosum, S =Spinosad, B =Beauveria bassiana, Bt =Bt spray.

Andrena imitatrix 0.45 (Bees)

0.40

0.35

0.30

0.25 a a 0.20

0.15

Mean (±SEM) 0.10

0.05 a aaaa 0.00

-0.05 C T S B Bt B & Bt T & S Treatment

Fig.25.The mean number for Andrena imitatrix cresson sampled in 2006. There was no significant difference between the control and other treatments. One way ANOVA, P = 0.554. a = means all were the same. C =control, T =Trichogramma pretiosum, S =Spinosad, B =Beauveria bassiana, Bt =Bt spray.

Andrena imitatrix 0.85 (Bees) 0.80 0.75 0.70 0.65 0.60 0.55 0.50 0.45 a 0.40 0.35 0.30 0.25 Mean (±SEM) a a a a a 0.20 0.15 0.10 0.05 a 0.00 -0.05

C T S B Bt B & Bt T & S Treatment

Fig.25b.The mean number for Andrena imitatrix cresson sampled in 2007. There was no significant difference between the control and other treatments. One way ANOVA, P = 0.951. a = means all were the same. C =control, T =Trichogramma pretiosum, S =Spinosad, B =Beauveria bassiana, Bt =Bt spray.

Fig. 26. The pheromone traps and plots showing buffers between the plots.

Fig. 27. This was during the time of sampling the arthropods.

APPENDIX B: TABLES

Table 1. Seven different treatments that were used during the whole research

PLOT ENTOMOPATHOGEN / CROP

TREATMENT

1 Control Corn

2 Trichogramma pretiosum Corn

3 Spinosad Corn

4 Beauveria bassiana Corn

5 Bt Sprays Corn

6 Beauveria Bassiana & Bt

spray Corn

7 Trichogramma & Spinosad

Corn

Table 2. Different plots treatments

5 4 1

7 6 3 2

6 7 2 3

5 1 1 4

4 2 7 5

Results 3 3 6 6

2 4 5 7

1 5 4 1

7 6 3 2

Table 3a. Mean results of different treatments (2006).

Infected Larvae Tunnels Length of Infected Yield in Treatment stalks out per 20 (± per 20 (± tunnels cobs per kilograms of 20 (± SEM) SEM) per 20 (± 20 (± /hectares SEM) stalks stalks SEM) SEM) plot stalks stalks 1. Control 17 34.8 24.2 32.6 17.4 3847.11

2. Trichogramma 13.2 27 25.4 32 13.4 9942.04 Pretiosum

3. Spinosad 3.4 9.8 6.6 14 6.4 10756.89

4. Beauveria 9.2 25.4 23 26.8 14 10603.06 bassiana

5. Bt Spray 6.6 14.4 13.6 22.2 9.4 10697.14

6. Beauveria bassiana & Bt 5.4 16.8 17.8 14.4 11.4 10317.03 spray 7. Trichogramma pretiosum & 4.8 8.6 7.6 6.8 9.0 10797.57 Spinosad

Table 3b. Mean results of different treatments (2007).

Infected Larvae Tunnels Length of Infected Yield in Treatment stalks per 20 (± per 20 (± tunnels cobs per kilograms out of SEM) SEM) per 20 (± 20 (± per 20 (± stalks stalks SEM) SEM) hectare SEM) stalks stalks

1. Control 19.8 29.4 44.8 46 14.2 10360.28

2. Trichogramma pretiosum 19 25.8 36.6 39 11 10825.6

3. Spinosad 9.8 6 11.8 12.4 2.6 11515.8

4 Beauveria 14.6 15.2 25.2 27.6 3.4 10831.89 bassiana

5. Bt Spray 12.8 12 18 19.8 4.8 11203.01

6. Beauveria 16 11.8 20.8 23.2 4.8 9942.88 bassiana & Bt Spray

7. Trichogramma 14.4 10.8 17.4 21.2 3.8 11335.28 pretiosum & Spinosad

Table 4a: Mean results of different arthropods sampled in 2006.

Plots Diabrotic Popillia Coccinella Euschistus Ostrinia Melanoplus Neocono- Andrena a japonica septempunct variolariu nubilalis femurrubru cephalus imitatrix virgifera- ata s m ensiger Cresson virgifera

1 13 0.2 2 0.2 0.4 0.8 0.8 0.2

2 12.2 0.2 1.4 0.8 0.8 0.4 0 0

3 9.8 0 1.4 0.6 0 0.2 0 0.2

4 10.2 0 0.8 0 0 0.2 0 0

5 6.6 0.2 0.4 0.6 0.4 0.6 1 0

6 8.6 0 1 0.6 0 0.4 0 0

7 12.4 0.2 0.8 1.8 0 0 0 0

Table 4b: Mean results of different arthropods sampled in 2007.

Plots Diabrotica Popillia Coccinella Euschistus Ostrinia Melanoplus Neocono- Andrena virgifera- japonica septempunctata variolarius nubilalis femurrubrum cephalus imitatrix virgifera ensiger Cresson

1 1.8 0.2 0.2 0.6 0.2 0.4 1.6 0.2

2 3.4 0.4 0.2 0.4 0 0 1.6 0.2

3 1.8 0.4 0.2 1 0.2 1.2 1 0.2

4 1.8 0.4 0.2 0.6 0 0.6 2 0.4

5 1.4 0.4 0.2 0.8 0.2 0.8 2.2 0

6 1.8 0.6 0.2 0.8 0 1 1.8 0.2

7 1.2 0.6 0.2 0.6 0.2 0.2 2.8 0.2