Use of Insect Pathogen Bacillus Thuringiensis for Control of Grubs (Coleoptera: Scarabaeidae) in Turf Tracy Ellis Michaels Iowa State University

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2000 Use of insect pathogen Bacillus thuringiensis for control of grubs (Coleoptera: Scarabaeidae) in turf Tracy Ellis Michaels Iowa State University

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Use of insect pathogen Bacillus thuringiensis for control of grubs (Coleoptera: Scarabaeidae)

in turf

Tracy Ellis Michaels

A dissertation submitted to the graduate faculty

in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

Major: Entomology

Major Professors: Joel R. Coats and Leslie C. Lewis

Iowa State University

Ames, Iowa

2000 UMI Number 9962834

I® UMI

UMI Microfomi9962834 Copyright 2000 by Bell & Howell Infomnation and Leaming Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code.

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This is to certify that the doctoral dissertation of

Tracy Ellis Michaels

has met the dissertation requirements of Iowa State University

Signature was redacted for privacy.

Co-major Professor

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Co-major Professor

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For the Major Program

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For the Graauate College iii

This representation of my efforts in education is dedicated to the very many people and other god's creatures in my life who have guided my thoughts and been a source of

inspiration and strength. I only hope that my deep gratitude has been shown and shared with

you during life, and whether you are in heaven or on earth, you know who you are. iv

TABLE OF CONTENTS

LIST OF TABLES vi

CHAPTER I. GENERAL INTRODUCTION 1 Indigenous and Introduced U.S. Scarab Grub Pests in Turf I Damage, Life Cycle, and Behavior 3 Chemical Control 4 Challenges to Safe and Effective Insecticide Application 6 Irrigation as a Tool for use in Grub Control 7 Biological Control 8 Objectives for Dissertation Studies 10 Dissertation Organization 10

CHAPTER 2. SUSCEPTIBILITY OF U.S. TURFGRASS GRUBS TO BACILLUS THURJNGIENSIS SUBSPECIES TOLWORTHI, KUMAMOTOENSIS, P^mJAPONENSIS. 12 Abstract 12 Introduction 13 Methods 18 Bacillus thuringiensis preparations 18 Bioassays 20 Anomala cuprea 20 Cotinis sp 21 Cyclocephala boreal is 22 Cyclocephala lurida 22 Cyclocephala pasademe 22 Popillia japonica 23 Phyllophaga sp. 24 Bacillus popilliae preparation 24 Midgut pH measurements 25 Results 25 Anomala cuprea 25 Atenius sp. 25 Cotinis sp 25 Cyclocephala borealis 26 Cyclocephala lurida 26 Cyclocephala pasadenae 26 Popillia japonica 27 Phyllophaga sp. 28 V Bacillus popilliae against Cydocephala sp. 28 pH 28 Discussion 28 References 34

CHAPTER 3: USE OF LEPTINOTARSA RUBIGINOSA (COLEOPTERA: CHRYSOMELIDAE) TO ASSAY PLACEMENT OF BACILLUS THURINGIENSIS TOLWORTHIUNDER TWO APPLICATION RATES IN CLAY AND SANDY TURF SOIL 41 Abstract 41 Introduction 41 Materials and Methods 44 Application 44 Sampling 44 Bioassay 45 Results 45 Discussion 48 References 49

CHAPTER 4: BACILLUS THURINGIENSIS JAPONENSIS PLACEMENT IN TURF AFTER STANDARD APPLICATION AND IRRIGATION REGIMES 53 Abstract 53 Introduction 53 Materials and Methods 55 Results 57 Weather 57 Soil Moisture 57 Untreated plots 60 Log transformation 60 Block^Day interaction 63 Measurements over time 64 Effect of application rate 64 Effect of post-treatment irrigation volumes 69 Recovery of Bacillus thuringiensis japonensis one day after application 69 Discussion 73 References 76

GENERAL CONCLUSIONS 81 GENERAL REFERENCES 86 ACKNOWLEDGEMENTS 96 vi

LIST OF TABLES

Table I. Description of Bacillus thuringiensis subspecies in this study 19

Table 2. Host range spectra of four Bacillus thuringiensis subspecies against U.S. grub pests 28

Table 3. Digestive system pH of five fall-collected Cyclocephala pasadenae grubs 31

Table I. Percentage mortality of Leptinotarsa rubiginosa (Coleoptera: Chrysomelidae) in bioassay of turf soil samples from four depths after treatment with Bacillus thuringiensis tolworthi to clay-containing turf soil at Lawrence Welk Golf Course, Escondido CA (1,870 and 9,350 i/ha volume of «800 ug/ml application followed by 0.75-cm post-treatment irrigation) 46

Table 2. Percentage mortality of Leptinotarsa rubiginosa (Coleoptera: Chrysomelidae) in bioassay of turf soil samples from four depths after treatment with Bacillus thuringiensis tolworthi to sandy turf soil at Torrey Pines Golf Course, San Diego, CA (1,870 and 9,350 1/ha volume of w800 ug/ml application followed by 0.75-cm post-treatment irrigation) 47

Table 1. Weather conditions on sampling days at ISU Horticulture Station 58

Table 2. Percentage moisture of samples in untreated and treated plots on Day 1 59

Table 3. Colony-forming units (CFU)/g sample in untreated and treated plots 61

Table 4. F-Vaiues from Type III Analysis of Variance (ANOVA) model for tests of hypotheses using corrected error terms on logio CFU/g sample from six turf sampling depths for all sampling dates 62

Table 5. Treated plots logio CFU/g sample by block 65

Table 6. Treated plots logio CFU/g sample by each sampling day 66

Table 7. Gain (+) or Loss (-) in logio CFU/g sample between sampling time points 67

Table 8. Application-volume effect on placement of logio CFU/g sample 68 vii

Table 9. Irrigation (post-application) effect on placement logio CFU/g sample 70

Table 10. Summary of Day I log lo CFU/g sample and moisture. Percentage logio CFU/g soil (% of recovery) and moisture (% of moisture) expressed as a fraction of the three-depth sum for application (1/ha) and irrigation (cm) treatments. Observations taken at 0-1-cm grass and thatch, 1-3-cm grass root zone and 3-5-cm mineral soil depths 71 1

CHAPTER 1. GENERAL INTRODUCTION

Indigenous and Introduced U.S. Scarab Grub Pests in Turf

An estimated 1,395 species and 135 genera of insects belonging to the family

Scarabaeidae are believed to exist in the U.S. and Canada (Ritcher 1966; Amett 1985). The characteristic C-shaped "scarabaeidaeiform" larvae of this family, often referred to as white grubs, herein will be referred to as grubs. Less than twenty genera, some of which are indigenous and some introduced, are noted to be regular pests of turfgrass (Tashiro 1987).

May and June beetles {Phyllophaga sp. subfamily Melolonthinae) have several hundred species that are indigenous to the U.S. and Canada (Amett 1985). Life cycles of

Phyllophaga sp., also called "true white grub", are from 1 to 4 years. Phyllophaga sp. can cause com stand reduction in Iowa when the population exceeds one grab per 0.09 m" (one sq. ft) in the com field before planting (McLeod et al. 1999). In turfgrass however, estimated populations of Phyllophaga should reach at least 3-4 grubs per 0.09 m^ before insecticide application is considered (Crocker et al. 1995). The smaller indigenous black turfgrass

Ataenius, Ataenius spretulus (Haldeman) (subfamily Aphodinae) is a widely distributed, sporadic pest, especially in southern states where two generations may occur each season.

The damage threshold greatly exceeds that of Phyllophaga and treatment is recommended for

Ataenius sp. when numbers exceed 30-50 gmbs per 0.09 m^ (Vittum 1995). Indigenous green June beetle Cotinis nitida (L.) and Cotinis mutablis Gory & Perchcron (subfamily

Cetoniinae) found in the southern U.S. is unique due to causing damage symptoms by burrowing, tunneling, and excavation (Tashiro 1987). Less than five Cotinis sp. gmbs per

0.09 m' is the estimated treatment threshold for perennial ryegrass/bentgrass fairways

(Hellman 1995). The indigenous masked chafers (subfamily Dynastinae), including the 2 northern masked chafer Cyclocephala borealis Arrow and the southern masked chafer C. lurida Bland, are distributed across the U.S. The northern and southern masked chafers are the most economically important annual white grubs in Iowa and other mid-westem turfgrass

(Brandenburg and Villani 1995; Potter 1995). A complex of Cyclocephala sp., including C. pasadenae Casey are a sporadic problem in California (Ali 1989; Kayaet al. 1992).

Treatment threshold estimates for this common pest vary, depending on turf type and condition, and range from 8-20 per 0.09 m^ (Potter 1995). Several introduced turfgrass pests with economic importance are found in the northeast and the mid-Atlantic states in mixed populations (Aim et al. 1995). The European chafer Rhizotrogus (Amphimallon) majalis

(Razoumowsky) (subfamily Melolonthinae) introduced from Europe, is a large grub with damage thresholds 5-10 grubs per 0.09 m for low maintenance turf and 15-20 per 0.09 m' for irrigated turf (Smitley 1995). The Oriental beetle Anomala orientalis (Waterhouse)

(subfamily Rutelinae) introduced from Asia, is also a damaging pest (Aim et al. 1995). The annual Asiatic garden beetle Maladera castanea (Arrow) (subfamily Melolonthinae) feeds more deeply in soil than most grubs and has a treatment threshold of 18-20 grubs per 0.09 m^

(Heller 1995). The Japanese beetle Popillia japonica Newman (subfamily Rutelinae) is a serious pest with a treatment threshold of six to ten grubs per 0.09 m^ (Vittum 1995).

Japanese beetles occur in the eastern states and as far west as Illinois (Brandenburg and

Villani 1995). The population threatens to spread westward, and the species is quarantined in many states, including Iowa (Potter 1991). Initially in 1975, small areas of infestation were noted in Iowa, but populations died out, and only small groups or individuals were found until 1994. Since 1994, Japanese beetles have been recovered from 11 different Iowa counties. Recoveries by citizens and the Iowa Department of Agriculture and Land 3

Stewardship have been steady with the prevalent captures at several locations over several years in Scott, Linn, and Dubuque counties, although recoveries have also been made in other eastern counties i.e. Lee, Muscatine, Clinton, Clayton, Johnson, Black Hawk, Floyd, and Page. The isolated nature of the infestations suggests that introductions have been from infested nursery stock while small and new recoveries can often be explained as newly arrived hitch hikers (D.R. Lewis, personal communication).

Damage, Life Cycle, and Behavior

Grubs with one-year life cycles that prune grass roots are considered to be the most important pest of cool season turf (Tashiro 1987). It is typically either fall or early spring when damage to turf is apparent. Damage is recognized as browning patches, where the turf is loosely bound to the soil. Additional damage occurs when skunks, moles and birds forage for grubs. Life cycle parameters pose a challenge to the entomologist when attempting to control grub pests. Eggs are deposited in soil by adult female beetles during May/June/July, depending on the species and the weather. Once the eggs hatch the grubs migrate laterally only a short distance from the original location of oviposition. Damage from grub feeding is typically visible from August through September when the grubs are in the third and final instar (Tashiro 1987). By the time damage is apparent, grubs are usually nearing completion of the larval stage. The fully grown larvae from summer and fall feeding migrate below the frost line to over-winter. There are variable numbers of grubs that survive over-wintering depending on weather conditions. Damage may also be visible in the spring when some species such as the Japanese beetle migrate upwards from their over-wintering site to feed in the root zone and replenish fat reserves. After feeding is complete in May/June/July the grubs build an earthen cell to pupate. Within two weeks following pupation, the beetles 4 emerge (Tashiro 1987). Some grub species e.g. Japanese beetles have feeding adult stages, while others such as the masked chafers, do not. Adult beetles are often found during

May/June/July at porch lights or clinging to window screens. Beetles use pheromones to locate each other to mate. Once fertilized, females choose a suitable location for egg deposition. Conditions considered attractive by the females for egg deposition have not been suitably characterized to be predictive of susceptible turf (Allsopp et al. 1992). Soil characteristics such as type, moisture, and temperature have been observed as important factors in oviposition location, egg and larval survival, adult emergence and vertical and horizontal movement of grubs (Cowles and Villani 1994; Villani and Nyrop 1991; Villani and Wright 1988; Potter and Braman 1991). Monitoring of the species and the timing of adult emergence allows timing of grub population surveys, which is important for implementation of an integrated pest management program in turf (Peacock and Smart 1995).

Pheromone traps are commercially available for Japanese beetles, while other beetles may be sampled with light traps. Adults are identified not only by size and color but also by the shape of the terminal segment of the antenna, the abdominal segments, adult internal genitalia, and the distribution of setae or spine patterns near the larval anal slit and raster

(Tashiro 1987; Brandenburg and Villani 1995).

Chemical Control

The optimal time to apply an insecticide is when grubs are first- and second-instars.

However, infestations often escape detection until after visible damage is apparent because of the difficulty of estimating a population of underground, widely scattered, small first- or second-instars. The technique to monitor for grubs is to turn over sod and take multiple samples in a defined area (0.09 m^). An insecticide application from July to early August 5

will prevent further damage from occurring in the current season. If damage occurs in the

fall, an application of insecticide does not necessarily protect an area from damage the

following summer. When damage is apparent insecticide application will kill only the late

larval stages after most damage has occuned. Likewise, applications in the spring do not

protect an area from reinfestation by the subsequent generation. An application of an

insecticide in the fall may, however, prevent spring losses in rare cases where larvae feed at the root zone just before pupating (Tashiro 1987).

The insecticides used to reduce grub populations have historically been organophosphates such as fonofos (Crusade®, Mainstay®), ethoprop (MoCap®), isazofos

(Triumph®), isofenphos (Oftanol®), diazinon (Diazinon®), chlorpyrifos (Dursban®), trichlorfon (Dylox®, Proxol®) or carbamates such as bendiocarb (Turcam®), carbaryl

(Sevin®). A synthetic pyrethroid, permethrin (Astro® Torpedo®), and a chloronicotinyl imidacloprid (Merit®) have also been used on turf (Gaussoin et al. 1994; Mullins 1993;

Ware 1994).

In Iowa, the Iowa State University Extension Service survey of 150 golf courses in

1990 indicated that grubs ranked second as the most commonly chemically treated insect pest in greens and tees, while being the most commonly treated insect pest in golf course fairways

(Agnew and Lewis 1993). Although insecticide usage to control turfgrass insect pests

(including grubs) accounted for less than 20% of total chemical applications to turfgrass, the amount of insecticide active ingredient applied for all golf courses included in the survey was approximately 2,200 kg total statewide (Agnew and Lewis 1993). The most conmionly used insecticides in Iowa turf were carbaryl, isofenphos and bendiocarb (Agnew and Lewis 1993). 6

Challenges to Safe and Effective Insecticide Application

The principles governing movement of chemical pesticides in agricultural soil systems also apply to turfgrass environments (Arnold and Briggs 1990; Branham et al. 1993).

Additional concerns affecting safe and effective use on turf are the increased likelihood of human proximity, presence of grass, presence of thatch, as well as the common use of irrigation (Odanaka et al. 1993; Kenna 1995; Branham et al. 1995). Chemical properties of the insecticide, the physical and chemical properties of the soil, and other environmental variables including turfgrass variety and maturity, thickness of the thatch layer, microbial populations, temperature, and soil moisture, either by irrigation or rainfall, all affect the fate of insecticides in turf (Branham et al. 1995; Kerma 1995; Branham et al. 1993; Odanaka et al.

1993). Insecticides with a moderate to high adsorption coefficient greater than 100, bind to organic matter in the thatch layer and soil, and reduce the risk for groundwater contamination

(Kenna 1995). Several insecticides used in turf rapidly bind to the organic matter in the thatch layer, while the remainder may volatilize, degrade by chemical or microbial processes, or leach into soil possibly through macropores (Branham et al. 1993; Starrett et al. 1996).

Golf course mineral soil beneath the thatch layer is constructed for aeration and gaseous diffusion for oxygen supply to the roots. The majority soil of golf course is sand particles which offers the desired porosity for optimal root respiration. Sandy soils have little organic matter to act as adsorptive surfaces for pesticide used in turf (Tate 1995). Increased leaching of pesticides has been observed in less mature turfgrass and types of grass with less aggressive growth, presumably because of reduced volume of thatch than in mature stands

(Spieszalski et al. 1994; Branham et al. 1993). It has been suggested that turfgrass systems are more favorable than agricultural systems for reducing runoff of pesticides due to the 7 increased adsorptive properties of the grass and thatch layers (Starrett et al. 1996; Branham et al. 1993).

Low to moderate persistence (less than 120 days), as measured by time it takes for an insecticide to degrade to one half its initial concentration (DT50), would reduce likelihood of non-target effects and groundwater contamination (Kenna 1995; Branham et al. 1995).

Insecticides with low volatility, a tendency for photolysis, hydrolysis, or microbial utilization, each balanced with the need for insecticide effectiveness, present reduced risks to the environment in contrast to insecticides with greater stability and persistence (Kenna

1995; Odanaka et al. 1993). Irrigation is commonly recommended after pesticide application to rinse the pesticide off the surface of the turf and grass blades. The practice is thought to increase safety to humans as well as pest control effectiveness by decreasing likelihood of unintentional exposures, degradation by mowing, volatilization, or photolysis (Kenna 1995;

Branham et al. 1993),

Irrigation as a Tool for use in Grub Control

Although the practice of post-application irrigation was probably initiated for safety and effectiveness of pesticides in general, it has also been shown to have an important role for use in grub control, and it has also been used for improvement of effectiveness of pathogen-based insecticides. Studies showed that isazofos and isofenphos were mainly recovered from the thatch layer and showed very little leaching of the insecticides to the first

2.5-cm root zone or deeper in mineral soil (Niemczyk 1987; Niemczyk and Krueger 1987;

Vittimi 1985). These observations did not explain how the grubs, which presumably fed in the root zone, received the treatment, except to suggest that grubs were feeding on the thatch, or that a lethal breakdown product leached into the root zone. Later studies showed that 8 irrigation did not simply move the insecticides downward in the soil profile, but that moisture affects movement patterns of grubs (Cowles and Villani 1994; Villani and Wright 1988).

Grubs were observed to respond to moisture provided by irrigation by moving upwards in the soil profile, to the thatch-soil interface, where they receive a lethal dosage.

Biological Control

There have been two pathogen-based conmiercial insecticides for control of grubs in the U.S. (Klein 1992, 1995; Tanada and Kaya 1993). The very first ever commercial pathogen-based insecticide was targeted against Japanese beetle (Dutky 1941). Strains of

Bacillus popilliae Dutky were cultured in vivo to sell for inundative and inoculative releases

(Doom® by Fairfax Biological Labs, Clinton Comers NY). Bacillus popilliae, which causes milky spore disease, can be isolated from persistent low level epizootics in populations of

Japanese beetle and Cyclocephala hirta (Bulla et al. 1978; Klein 1997; Tanada and Kaya

1993; Kaya 1992). B. popilliae spores can persist in the soil environment, but they can vary in effectiveness of host population suppression. They may be recycled by the host grub for many years (Klein 1992,1995; Tanada and Kaya 1993). Steimrmma glaseri (Steiner), an entomopathogenic nematode, was the second pathogen marketed against Japanese beetle

(Klein 1995). Under certain favorable conditions, other nematodes, e.g. Steinernema carpocapsae (Weiser), Heterorhabditis heliothidis (Khan, Brooke, and Hirshman),

Heterorhabditis bacteriophora (Steiner) Steinernema glaseri (Steiner), may also persist over multiple generations in the field providing varying levels of biological control (Klein 1995;

Tanada and Kaya 1993; Kaya and Stock 1997). Infective juvenile stages of die nematodes

Steinernema sp. and Heterorhabditis sp. carry intestinal bacterial symbionts Xenorhabdus sp. and Photorhabdus sp. (Enterobacteriaceae). The bacteria are highly virulent to the insect 9 after being carried into the host by the nematode and reaching in the midgut or the hemocoel.

(Forst et ai. 1997). Entomopathenogenic nematodes used to control grubs have been shown to have enhanced survival and infection rates when pre-treatment and post-treatment irrigation was used (Downing 1994; Shedar et al. 1988), although effectiveness varied depending on nematode genera and soil types (Zimmerman and Cranshaw 1991; Cowles and

Villani 1994).

Other pathogens, parasites and predators are known to perform as biological control agents on populations of grubs in the field (Fleming 1968) but have not been cultured for sale in the U.S. (Klein 1995). Serratia entomophilia, a bacterium and causative agent of amber disease, is registered for use in New Zealand (Jackson et al. 1992). Research on the combined efficacy of biological controls such as bacteria and nematodes has been conducted and shown to cause enhanced efficacy (Koppenhofer et al. 1999; Koppenhofer and Kaya

1997). Bacteria and fungi cultured from grubs include Rickettsiella popilliae Dutky, causative agent of blue disease (Klein 1988), Metarhizium anisopliae (Melchnikof), a green muscardine fungus and Beaiiveria bassiana (Bals.) a white-muscardine fungus.

Microsporidia such as Ovavesicula popilliae (Hanula 1990) have been isolated from grubs

(Klein 1995; Tanada and Kaya 1993). Parasitoids and predators of grubs include tachinids

(Diptera: Tachinidae), tiphia (Hymenoptera: Tiphiidae), digger wasps (Hymenoptera:

Sphecidae), ground beetles (Coleoptera: Carabidae), and various ants (Hymenoptera:

Formicidae) (Fleming 1968). Vertebrate predators include skunk, raccoon, moles, opossums, armadillos, and crows, whose efforts to hunt grubs underground can account for significant secondary damage (Potter 1991; Brandenburg and Villani 1995; Tashiro 1987). 10

Objectives for Dissertation Studies

The objectives of this dissertation are 1) to broaden and more greatly define the host range of Bacillus thuringiensis (Bt) subspecies against U.S. turfgrass grub pests 2) investigate a novel technique for use of Bt, specifically its use in turf to control white grubs

(Coleoptera: Scarabaeidae) and 3) evaluate parameters for the successful safe and effective application of Bt in turfgrass for control of white grubs.

Dissertation Organization

This dissertation is composed of an introduction, three journal papers, and a conclusion. The General Introduction (Chapter 1) gives an overview of grub (Coleoptera:

Scarabaeidae) pests in U.S. and both chemical and biological control measures used to control these insects in turf. The first paper (Chapter 2) addresses the host range of three Bt subspecies, the biological activity of which two against U.S. turfgrass grub pests have not formerly been described except in patent literature. Chapter 2 which describes the host range of three Bt subspecies kumamotoensis, tolworthi and japonensis against U.S. pests will be submitted to the Journal of Invertebrate Pathology. The second paper (Chapter 3) discusses small plot studies evaluating Bt after spraying to turf using insect bioassay as an analytical tool to show recovery of Bt tolworthi from the soil. Due to the unexpected results of obtaining Bt toxin from depths of 2-cm depths firom two turf study sites, this paper will also be submitted to the Journal of Invertebrate Pathology to demonstrate the feasibility of using

Bt in a novel way for subterranean pest control. The third paper (Chapter 4) discusses the influence of commonly employed application and irrigation practices on the placement of Bt subspecies japonensis in turf soil. The study shows that post-treatment irrigation practices, 11 similar to those used for chemical pesticide application, may also positively benefit Bt biological pesticide effectiveness. This paper will be submitted for horticultural and turf interest to the Journal of Economic Entomology. A General Conclusion is followed by a

General Reference section at the end of the thesis. The General Reference section includes citations used in both the General Introduction and the General Conclusion. An

Acknowledgment section follows the reference section. 12

CHAPTER 2. SUSCEPTIBILITY OF U.S. TURFGRASS GRUBS TO BACILLUS

SUBSPECIES TOLWORTHI, KUMAMOTOENSIS, AND

JAPONENSIS.

A paper to be submitted to Journal Invertebrate Pathology Tracy Ellis Michaels''^, Gregory A. Bradfisch', Leslie C. Lewis^"^, and Joel R. Coats^

Abstract

An evaluation was performed for host range of Bacillus thuringiensis tolworthi, kumamotoensis, and japonensis against the following scarab turfgrass pests (Coleoptera:

Scarabaeidae) Anomala cuprea, Atenius sp., Cotinis sp., Cyclocephala borealis,

Cyclocephala lurida, Cyclocephala pasadenae, Popillia japonica, Phyllophaga sp. Results showed Bacillus thuringiensis tolworthi and Bacillus thuringiensis japonensis to have activity against U.S. economic grub pests Cyclocephala borealis, Cyclocephala lurida, and

Popillia japonica while Bacillus thuringiensis kumamotoensis was active against Cotinis sp.

Other U.S. turfgrass pests tested, Atenius sp. and Phyllophaga sp. had no detectable susceptibility to any of the three isolates. Bacillus popilliae was inactive against

Cyclocephala pasadenae.

' Dow AgroSciences, 5501 Oberlin Drive, San Diego CA 92121 ^ USDA-ARS Com Insects and Crop Genetics Research Unit, Genetics Laboratory, ISU, Ames, lA 50011 ^ Department of Entomology, Iowa State University, Ames, lA SOOl 1 13

Introduction

Bacillus thuringiensis (Bt) Berliner (Bacillaceae) is known as a source of insecticidal proteins, which are produced as cytoplasmic protein inclusions called crystals, or parasporal bodies (Burges 1973). The insecticidal proteins and the genes that code for theui are the basis for the majority of commercial pathogen-based insecticides worldwide (Andrews et al.

1987; van Frankenhuyzen 1993). The spores of many subspecies of Bt have been isolated from thousands of different environments (Martin and Travers 1989; Meadows et al. 1992) and cultured for mass production (McGuire et al. 1997). Bt is believed to be a soil bacterium yet researchers have noted that sporulated Bt strains are poorly adapted for survival in the natural soil environment (Burges 1973). Monitoring studies on the deterioration of Bt proteins and spores in soil have rarely detected germination and reproduction of the bacterium (West and Burges 1985; West et al. 1985a; West et al. 1985b). Only when competitor microorganisms were removed and nutrients, moisture and the pH adjusted

(pH>5.5) was Bt shown to germinate when inoculated to a soil (West et al. 1985a).

Consequently Bt is believed to need an insect host for reproduction (Ellar 1990). Infected insect cadavers are thought to inoculate the soil with spores (Meadows 1993). Under various conditions, epidemics have been noted in beehives, silkworm rearing, dry food stores and insect rearing situations (Tanada and Kaya 1993; van Frankenhuyzen 1993). Rarely has Bt been shown to cause wide spread epizootics in the outdoors; nor has it been shown to be a natural, low-profile pathogen in scarab grub populations in the soil (Klein 1995).

The incentive to develop products based on Bt insecticidal protein has spurred an intense international effort to define the gene sequences and further understand the mode of action of Bt insect-selective proteins, which are known to cause a trans-membrane pore of 14

-1.0 nanometers in the insect midgut epithelium (Li et al. 1991; Menestrina and Semjen

1999; Ellar 2000). Some details on mode of action, such as the receptor molecule, may differ

with each insect and toxin combination, possibly accounting for Bt toxin host-selectivity

(Knowles and Ellar 1987; Knowles et al. 1991). The activity of the CrylAc protein in

Manduca sexta (Lepidoptera:Sphingidae) is known to begin with solubili2ation of the

inclusion or crystal in the alkaline pH of the larval midgut (Gill 1992). The protein is cleaved by midgut proteinases from a length of 135 kDa to 66 kDa, to form the truncated or activated toxin (Tanada and Kaya 1993; Gill 1992). The solubilized and activated protein is

what is often chosen for expression in transgenic plants (Ely 1993). A section of the activated toxin binds to a receptor such as aminopeptidase-N on the epithelial cell membrane,

while other sections of the molecule orient and group to form a pore in the membrane

(Carroll et al. 1997; Ellar 2000). The pore disrupts the exchange of nutrients and ions occurring between the digestive tract and the hemolymph (Knowles and Ellar 1987; Knowles et al. 1991). Also, the midgut pore allows gut contents to pass to the hemocoel, causing the observed septicemia and insect death.

Many unique Bt insecticidal proteins, sometimes called delta-endotoxins, have been discovered in the past twenty years. The current system classifies crystal (cry) toxins based on insecticidal protein amino acid sequence identity (Crickmore et al. 1998; Crickmore et al.

1999; van Frankenhuyzen and Nystrom 1998). The Bt nomenclature system was established

(Hofte and Whitley 1989) and is evolving. The gene for the insecticidal crystal from Bacillus

popilliae, the causative agent of milky spore disease, was cloned in 1997 (Zhang et al. 1997).

This is in contrast to the vast knowledge on the molecular biology of at least 100 proteins

produced by Bt subspecies and strains, a small percentage of which have activity against 15 beetles (van Frankenhuyzen and Nystrom 1998; Crickmore et al. 1999). Despite lack of information on Bt infecting grubs in natural habitats, screening efforts around the globe have demonstrated that several strains of Bacillus sp. have selective activity against some species of grubs.

One of the first records of insecticidal activity of Bt against grubs was that by Sharpe

(1976). Japanese beetle grubs were noted to be susceptible to Btgalleriae (Sharpe 1976).

The susceptibility of the grubs to Bt galleriae was observed to be related to the physiological state of the insect. Sharpe noticed a difference in grub susceptibility to Bt between spring- collected and fall-collected populations. The fall-collected grubs showed a lower susceptibility to Bt galleriae than did the spring-collected grubs (Sharpe and Detroy 1979).

Japanese beetle grubs were previously measured to have a steady midgut pH of 9.5 (Swingle

1931). Upon measuring gut pH and making observations, Sharpe and Detroy (1979) observed that actively feeding grubs with normal amounts of food in the digestive system tended to have higher pH values. Midgut pHs of fall-collected Japanese beetle larvae varied from 6.8 to 10.2, but tended to have pH values below 9.0, compared with consistently higher pH«10 of the spring-collected grubs. Sharpe and Detroy (1979) speculated that decreased susceptibility of fall-collected grubs was caused by a decrease in gut pH as the larvae emptied their guts and prepared for winter diapause, in contrast to spring-collected grubs which had resumed active feeding before pupation. The spring-collected grubs, with the alkaline midguts, were presumably in a physiological condition to solubilize Bt inclusions, the first step in activating Bt protein. The physiological state of the grub, possibly affecting the midgut likelihood firom solubilizing or digesting the protoxin to the active form, may be an important factor when considering grub response to Bt toxins. This factor is especially 16 important to consider for Bt research with grubs, because grubs are often field-collected in the fall and held in storage for a few months at cool temperatures for use in laboratory experiments.

Since the research by Sharpe and Detroy (1979), there have been no reports that have further characterized activity of Btgalleriae against grubs (Glare et al. 1992) however, there has been extensive resources devoted to finding other Bt subspecies with protein toxins active against Coleoptera. Insecticidal screening investigations regularly used laboratory- reared Colorado potato beetle Leptimtarsa decemlineata (Say) (Coleoptera: Chrysomelidae) for discovery of initial Bt activity (Robert et al. 1994). In one investigation Bt tenebrionis containing Cry3 Aa (formerly CrylllA) was tested against several agronomically important coleopteran pests, including Colorado potato beetle and Japanese beetle. Cry3Aa toxin had high levels of activity on Colorado potato beetle but no detectable activity on Japanese beetle

(Macintosh et al. 1990). In another observation of Bt activity against grubs, a New Zealand scarab pest Costelytra zealandica was reported to be susceptible to an undescribed Bt subspecies, which caused growth inhibition at dosages of 1 mg/ml in artificial diet (Wigley and Chilcott 1990). In another case, coleopteran-active toxins other than Bt tenebrionis

Cry3 A toxin identified by bioassays on Leptinotarsa rubiginosa, a relative of the Colorado potato beetle, were discovered to have shared activity on white grubs (Michaels 1993;

Michaels et al. 1993; Michaels et al. 1996). These were Bt tolworthi and Bt kumamotoensis

(Sick et al. 1990; Grossman 1992; Donovan et al. 1993; Narva and Fu 1993; Michaels 1993;

Michaels et al. 1993; Michaels et al. 1996; Robert et al. 1994). Bt tolworthi was determined to produce proteins Cry3Bai, Cry3Bb2 (formerly CryUIB, CryIIIB3,) and Bt kumamotoensis identified to produce CrySBai (formerly CrylllE). Laboratory and turf plug tests by Ecogen 17

Inc., Langhom, PA and Chemiawn Research and Development, Delaware, OH demonstrated

a Cry3Bb2 toxin to cause 81% mortality of third-instar Japanese beetle. These tests showed

northern masked chafer, Cyclocephata borealis to also be susceptible to CrySBb? (Grossman

1992). Bt tenebrionis Cry3Aa was active against Colorado potato beetle but not Japanese

beetle, while Cry3Bai and Cry3Bb2 toxins produced by Bt tolworthi strains were active against both a relative of the Colorado potato beetle and the Japanese beetle (Grossman 1992;

Sick et al. 1990; Donovan et al. 1993; Michaels 1993; Michaels et al. 1993; Michaels et al.

1996). The host spectrum specificity of Bt toxins against Coleoptera varied further, as Bt kimamotoensis, producer of CrySA protein, was discovered to be toxic to green June beetle grub Cotinis sp. (Narva and Fu 1993; Michaels 1993; Michaels et al. 1993; Michaels 1996).

In 1992 a Japanese laboratory discovered a toxin CrySC (formerly CrylllF), named from a

previously undescribed Bt subspecies. The subspecies was named japonensis (Btj) after the country where it was isolated, and was nicknamed "buibui" after the susceptible insect, a white grub Cupreus chafer/(nowa/a cuprea Hope (lizuka et al. 1998; Ohba et al. 1992).

The isolates discovered to be active on various white grubs in the 1990s were all quite different from one another in protein profiles, as measured by polyacrylamide gel electrophoresis. The delta-endotoxins or alkali-soluble proteins considered protoxins from Bt tolworthi are 75,68, and 61 kDa; Bt kumamotoemis is a doublet 130 kDa; and Bt japonensis is a 131 kDa length protein (Sugimura et al. 1997). Not only are the protein profiles defined, but the gene sequence data are available for all scarab-active delta-endotoxins described to date (lizuka et al 1998; Sato et al. 1994; Donovan et al. 1993; Michaels et al. 1993; Michaels et al. 1996; Crickmore et al. 1998; Grossman 1995; Crickmore et al. 1999; van

Frankenhuyzen and Nystrom 1998). 18

The host range of Btj buibui has been well defined for several insect pests of economic importance in Japan (Suzuki et al. 1992). Cupreus chafer, Japanese beetle, and several Japanese pest species in the subfamily Rutelinae including Anomala albopilosa Hope,

Anomala rufocuprea Motschulsky, Anomala daimiana Harold, Anomala shonfeldti Ohaus are susceptible to Btj buibui in laboratory tests (Suzuki et al. 1992). Anomala orientalis, the

Oriental beetle, an imported U.S. pest, limited in its range to the northeast, is also susceptible to Btj buibui (Aim et al. 1997; Suzuki et al. 1992).

The objectives of this research were to further explore the host range of toxins from

Bt tolworthi, Bt kumamotoenis, and Bt japonensis against grubs which are important pests In the U.S. and to show the relative toxicity of the toxins.

Methods

Bacillus thuringiensis preparations

Bt treatments (Table I) consisted of Bt sporulated cultures which were stored as lyophilized powders and mixed with water to form a suspension for the bioassay. The lyophilized powders were provided by Dow AgroSciences, San Diego, CA. The total protein of the powders was estimated by comparison to a bovine serum albumin standard Sigma-

Aldrich Chem. Co., St. Louis, MO using sodium dodecyl sulfate polyacrylamide gel electrophoresis Bio-Rad Laboratories, Hercules, CA and densitometry Molecular Dynamics,

Sunnyvale, CA. Suspensions were mixed to contain I to 2.5 mg of protein per ml of water.

This suspension was added to the rearing medium or insect diet to give a known amount of protein per unit weight or volume. 19

Table 1. Description of Bacillus thuringiensis subspecies in this study.

Associated Inclusion/Crystal Characteristics

Example Protein Bt subspecies Strain Shape Classification® Size (kDa)

kumamotoensis PS50C' ellipse CrySA 130 tolworthi PS86B1^ sphere Cry3Bb2 74 japonemis buibui^ sphere Cry8C 130 tenebrionis B.t.s.d.'' square CrySA 72 kurstaki HD-73^ bipjnramid CrylA 130

' Narva et al 1993; Michaels 1993; Michaels et al. 1993; Michaels et al. 1996; Crickmore 1999 " Sick et al 1990; Michaels 1993; Michaels et al. 1993; Michaels et al. 1996; Crickmore 1999 ' Ohba 1992; Sato etal. 1994; lizukaetai. 1998; Crickmore 1999 * Sick et al 1990; van Frankenhuyzen and Nystrom 1998 ^ Hofle and Whiteley 1989; Crickmore 1999 ® Crickmore et al. 1999; van Frankenhuyzen and Nystrom 1998 20

Bioassays

Bioassays were conducted at several different locations where grubs were available either by cooperative researchers or by senior author. The same general protocol was used in all bioassays with modifications due to grub condition and availability. Protein suspensions were mixed to contain approximately I mg protein/ml of water as quantified by SDS-PAGE unless otherwise noted. To prepare the diet for the grub bioassay, annual ryegrass (Lolium multiflonim) was sprouted in flats with 1:5 peat:sand soil mixture. The grass was 7.5-cm tall when removed from the 60 x 30 x 5-cm flat and the roots rinsed free of most soil particles.

The roots were allowed to dry at room temperature on the lab bench for -20 hours before they were cut with scissors into sections less than 1-cm long. Roots were mixed with the materials being tested at the rate of 3 g dried roots with 6 ml toxin suspension and distributed among ten Plastic SOLO™ 29.5 ml (loz.) cups before infestation with one grub per cup.

Plastic SOLO™ perforated lids were used to cap the cups. The larval bioassay cups were held at 26±2°C and 50-80% RH until mortality and/or weight data were taken, generally on day 7 unless otherwise noted. Mortality in comparison to the untreated control was used as the indicator of susceptibility to the Bt toxins. Where adequate numbers of insects were available to conduct concentration-response bioassays, Probit analyses were performed on the mortality data (LeOra Sofhvare 1987). The following sections describe slight deviations from the main protocol that occurred for each insect tested.

Anomala cuprea

First instar y4«oma/a cuprea Cupreus chafer were obtained fi*om a laboratory colony at Kubota Corporation, Osaka, Japan where bioassay was performed. One gram of compost was weighed, placed in each cup, and mixed with 1 ml of the Bt suspension with a spatula 21 before a grub was added to each cup. Bt tolworthi was bioassayed at a final estimated concentration of 500 ug protein/ g peat-mix against ten grubs in each treatment.

Atenius sp.

Second and third instar black turfgrass Atenius grubs were collected at T.awrence

Welk Golf Course, Escondido, CA. The larvae were returned to the laboratory at Dow

AgroSciences, San Diego, CA in collection site soil and bioassayed that day. One ml of 100 mg powder/ml treatment suspension was mixed with 4 g soil at in each cup before 5-10 grubs were added per cup. Unventilated lids were used. Mortality observations were taken on

Day 7. Preparations of Bt tolworthi, Bt kumamotoensis, and Bt japonensis tested at a final estimated concentration of 750-1,000 ug protein/ g soil against 41, 21, and 26 grubs respectively.

Cotinis sp.

Grubs were field-collected from compost piles at 1110 Fern Street, Escondido, CA or

Balboa Park, San Diego, CA and held at 10°C in peat mulch from the collection site until the bioassay. Bioassay was performed at Dow AgroSciences, San Diego, CA. Each protein suspension was mixed with Canadian sphagnum peat moss by stirring with a spatula in a 0.25

L plastic cup, approximately 2.5 g placed per 29.5 ml cup, to give a 1:1 ratio by weight of suspension:peat-moss. Preparations of Bt tolworthi, Bt kumamotoensis, Bt tenebrionis and Bt kurstaki were bioassayed at a final estimated concentration of 750 ug protein/ g peat-mix against nine grubs in each treatment for the first test. For the test on different instars, grubs were separated into groups based on size; first instar, early-second instar, and late-second instar, which were each approximately 1.25 cm, 2 cm, or 3 cm in length respectively and were bioassayed at a final estimated concentration of750 ug protein/ g peat-mix against ten 22 first instar, ten second instar and four third in each Bt kumamotoemis and Bt kurstaki treatment.

Cyclocephala borealis

The one-dosage bioassay was performed at Chemlawn Research and Development

Center, Columbus, OH, where grubs had been field collected and held in cold storage for several months before the bioassay. To perform the bioassay, the top 4 cm of roots and thatch fi-om a Kentucky bluegrass turf field were dried overnight on the laboratory bench then cut into approximately 1 gram pieces. Each turf piece was individually dipped into the test solution then placed in a cup before being infested with a third-instar northern masked chafer. Cups were held at 26°C for 7 days until mortality observations were taken.

Preparations of Bt tolworthi and Bt kumamotoemis were bioassayed against twenty-five grubs at a final estimated concentration of 750 ug protein/ g root-mix.

Cyclocephala lurida

Bioassays were performed in late August at the University of Nebraska, Lincoln, NE.

Grubs and turf roots were gathered from a grub collection site at a golf course near Lincoln,

NE, the roots rinsed with tap water and dried on the bench ovemight, while the grubs were stored for less than a week in soil at 10-15°C. Roots were mixed with the materials being tested before infestation with two second-instar grubs. Sixty and ninety grubs were used for the first and second assay respectively. Estimated dosages ranged from 400 - 1,300 ug protein /g diet for Bt tolworthi and 200- 600 ug protein/g diet for Bt japonensis preparations.

Cyclocephala pasadenae

Third instar Cyclocephala pasadenae were collected at Fairbanks Ranch Golf Course,

Rancho Santa Fe, CA or Balboa Park, San Diego, CA and stored in a wash basin layered with 23 old newspaper and a 3-cni layer collection site soil at 10-15°C until bioassay. Bioassays were performed at Dow AgroSciences, San Diego, CA. The first C. pasadenae bioassay included Bt tolworthi, Bt kumamotoensis, Bt tenebrionis, and Bt kurstaki tested at approximately 600 ug protein/g diet with ten grubs each. The second bioassay included Bt tolworthi tested at an estimated 300, 600, and 900 ug protein/g soil and Bt kimamotoensis tested at an estimated 2,400 ug protein/g soil with ten grubs each. Two concentration- response bioassays of Bt tolworthi at protein concentrations ranging from 53 to 850 ug protein/g diet against six C. pasadenae grubs/treatment with Bt kurstaki and water control as negative controls were performed. In addition, the resuhs of three tests with protein concentrations ranging from 15 -500 ug protein/g diet of Bt tolworthi and 1-60 ug protein/g diet of Bt Japonensis with 50-60 grubs per test was analyzed to compare the activity of the two materials.

Popiliia japonica

Popillia japonica was tested at two locations, University of Rhode Island and

University of Kentucky. At the University of Rhode Island, Kingston, RI, the grubs were field collected in the spring and held for one month in storage in the laboratory at 10±2°C until the bioassay. One third-instar was introduced into each cup, which was then enclosed with a ventilated snap cap lid. The bioassay was held at 27±l°C and 50±20% RH. Each day the cups were checked daily for moisture level. If condensation was observed, larger holes were made in the lid. If the cups were drying, the trays were placed in a large loosely closed plastic bag. The two bioassays conducted in Rhode Island included Bt tolworthi and Bt japonensis tested at estimated dosages of 150,75, and 35 ug protein/ g root-mix with ten grubs per dosage. 24

Bioassays were performed in early September at the University of Kentucky,

Lexington, KY. Grubs and turf roots were gathered from grub collection site at a sod farm near Lexington KY, the roots were rinsed with tap water and dried on the bench overnight, and grubs were stored for less than a week in soil at 10-15°C. Roots were mixed with the materials being tested at the rate of 6 g dried roots with 6 ml toxin suspension before infestation with one second instar. Two bioassays with five larvae per dosage where Bt tolworthi was tested a concentrations ranging from 60 - 1000 ug protein/g diet and Bt japonensis from 30 - 500 ug protein/g diet.

Phyllophaga sp.

Phyllophaga sp., true white grubs were collected in December in Monahans, Texas where Phyllophaga submucida usually predominates, and held at 15°C for less than one week before the bioassay. Bioassay was performed at the Texas Agricultural Extension

Service, Fort Stockton, Texas. One third-instar was placed in each cup then closed with ventilated snap cap lid. The bioassay was held at 25±3®C and 60±20% RH. Each day they were checked daily to maintain moisture level. If condensation was observed, larger holes were made in the lid. If the cups were drying, the trays were placed in a large loosely closed plastic bag. Preparations of Bt tolworthi, Bt kumamotoensis and Bt japonensis were tested at

750 ug protein/g diet against ten grubs in each treatment.

Bacillus popiiiiae preparation

Milky spore powder manufactured by Rueter Laboratories, 2405 James Madison

Highway, Haymarket VA 22069 (703-754-4167) contains not less than 100 million viable B. popiiiiae spores per gram of powder, 0.016% inert ingredients, 0.400% parts of P.japonica, and 99% diluents. EPA Registration No. 36488-1. Milky spore powder was weighed. 25 suspended in water at a rates of 100, 50, and 25 mg/mi and added to dried grass roots.

Midgut pH measurements

The grubs were collected in early November and held at 10-15°C until the pH evaluation was performed. A micro manipulator was used to place the microelectrodes into the digestive system to take measurements of the foregut, anterior midgut, posterior midgut, hindgut and rectal sac. Measurements were taken from five dissected grubs which were kept moistened with buffered pH 7.5 saline solution.

Results

Anomaia cuprea

A preparation of Bt tolworthi tested at a final estimated concentration of 500 ug protein/ g soil against ten grubs showed 0% mortality. The A. cuprea bioassay had zero water-control mortality.

Atenius sp.

Preparations of Bt tolworthi, Bt kumamotoensis, and Bt japonensis tested at a final estimated concentration of 750 ug protein/ g soil against 41,21, and 26 grubs were observed to cause 22%, 71%, and 26% mortality, respectively. There was 70% mortality in the untreated check.

Cotinis sp.

Preparations of Bt tolworthi, Bt kumamotoensis, Bt tenebrionis and Bt kiirstaki were bioassayed at a final estimated concentration of 750 ug protein/ g peat-mix against nine grubs each. Mortality of second and third instars, except in the Bt kumamotoensis treatment where

89% mortality was observed, was below 11%, including the water-control with no mortality.

Observations were confinned by a test of Bt kumamotoensis in comparison to Bt kurstaki at 26 the same rates against first, second and third instar (10, 10, and 4 grubs each) which showed

100% mortality of first and second instar and 75% mortality of third instar in the Bt kumamotoensis treatment in comparison to 0% mortality in all Bt kurstaki treatments. A third bioassay of 5-10 grubs per treatment evaluated Bt kumamotoensis at three reduced rates of an estimated 500,150, and 40 ug protein/g peat-mix and resulted in a 80,60, and 20% mortality respectively of Cotinis. There was the no activity of Bt tolworthi against Cotinis sp. However, an initial test of Bt japonensis at an estimated 300 ug protein/g peat-mix caused 100% mortality.

Cyclocephala borealis

Preparations of Bt tolworthi and Bt kumamotoensis were bioassayed against this pest at a final estimated concentration of 750 ug protein/ g root-mix. The twenty-five grubs exposed to each subspecies had 79% and 12% mortality, respectively. There was 4% mortality in the untreated check.

Cyclocephala lurlda

Results from pooled data from two concentration-response tests (n=150) estimated the LC50 for Bt tolworthi and Bt japonensis to be 50 ug/g (95% confidence intervals range 7-

85 ug/g), and 5 ug protein/ g root-mix respectively.

Cyclocephala pasadenae

The first C. pasadenae bioassay included Bt tolworthi, Bt kumamotoensis, Bt tenebrionis, and Bt kurstaki tested at approximately 600 ug protein/g diet with ten grubs each. All treatments had zero mortality with the exception of the Bt tolworthi treatment in which 85% of the grubs died. A second bioassay evaluated rates of Bt tolworthi at 900,600, and 300 ug protein/g root-mix with a flat concentration-response of 80%, 60% and 60% 27 mortality, respectively. The non-activity of Bt kumamotoensis confirmed with zero mortality, the same as the water control, at estimated rates as high as 2.4 mg protein/g root- mix. A second concentration-response bioassay of Bt tolworthi against six C pasadenae grubs/treatment continued to show minimal rate response between 25% and 74% mortality caused by concentrations between 53 and 850 ug protein/ g root-mix, and mortality by Bt kurstaki and the water control remained under 10%. Results from pooled data from three concentration-response tests with five dosages each (n=184) estimated the LC50 for Bt tolworthi and Bt japonensis to be 241 and 45 ug protein/ g root-mix (no confidence intervals calculated by the analysis due to variation in grub response), respectively.

Popiiiia japonica

The Popiiiia japonica bioassays conducted in Rhode Island included Bt tolworthi and

Bt japonensis tested at estimated dosages of 150, 75, and 35 ug protein/ g root-mix with ten grubs per dosage. There was 20% and 0% mortality of insects in the first and second test water control treatment respectively. The average mortality of the grubs at the top rate of

150 ug/g for both preparations was 90%; this was followed by a similar concentration- response at the 75 ug/g and 35 ug/g rates of 75% and 20% in the Bt tolworthi treatment and

80% and 10% in the Bt japonensis treatment.

The bioassays performed in Kentucky against Popiiiia japonica had similar results to the Rhode Island tests, where Bt tolworthi and Bt japonensis were estimated to have an LC50 of 69 and 61 ug protein/ g root-mix (no confidence intervals calculated by the analysis due to variation in grub response) in two tests with 29 and 30 grubs. 28

Phyllophaga sp.

Preparations of Bt tolworthi, Bt kumamotoensis and Bt japonemis caused 10%, 30%, and 30% mortality of Phyllophaga sp. in the single-concentration test of approximately 750 ug protein/g diet against ten grubs in each treatment. There was 20% mortality in the untreated check.

Bacillus popilliae against Cyclocephala sp.

The grubs exposed to the milky spore showed no visible signs of disease within the time frame of the study. The top rate (66 mg/g roots) of milky spore caused mortality of 30%

Bt kurstaki mortality of 16%, and water mortality of 0% by Day 7. By Day 18 the mortality in the top rate of milky spore increased to 50%; however mortality in the Bt kurstaki treatment (50%); and water treatment (16%) were higher as well.

Average gut pH (not including measurements from rectal sac) of digestive system was pH»9.0, whereas the rectal sac average was slightly reduced at pHw8.5 (Table 3). These measurements were not correlated with susceptibility of the larvae to Bt.

Discussion

Signifrcant improvements to the methodology of grub bioassay are necessary to obtain reliable estimates on response to Bt toxins. The field collected insects were variable in condition and few in number, leading to the variability of the results and difficulty in calculation of confidence intervals in the Probit analysis. However, this research was important to learning the host range for a group of Bt toxins against several U.S. grub pests

(Table 2). 29

Table 2. Host range spectra of four Bacillus thuringiensis subspecies against U.S. grub pests.

Indication of Susceptibility of U.S. Grub Pests'

Anomala .Uenius Cotinis Cyclocephala Phyllophaga Popillia

Bt subspecies sp. sp sp. sp. sp. japonica

X __2 __2 _3 tenebrionis X X _2 ^4,7 __2 _2 _2 kumamotoemis X _2 __2 _2 _|_4,5 _2 ^4,5 tolworthi _2 _2 Japonensis +'

(+) indicates susceptibility, (-) indicates no observed effect, (x) not reported " Michaels et al. reported herein ' Macintosh et al. 1990 ^Michaels 1993; Michaels et al. 1993; Michaels et al. 1996 'Grossman 1992 ® Suzuki etai. 1992 'Robert etal. 1994 30

An LC50 estimation for Bt tolworthi and japonensis against the Japanese beetle,

Popillia Japonica, Southern masked chafer Cyclocephala lurida, and Pasadena masked chafer Cyclocephala pasademe had similarly variable results due to low numbers of grubs in the test and the variable conditions of the field collected grubs. However, these tests confirm both Bt tolworthi and Bt Japonensis do have activity against Japanese beetle and masked chafer grubs, which are the most significant economically important grub pests in the U.S.

Results of these tests suggest similar relative activity of Bt japonensis and Bt tolworthi against the Japanese beetle. Published results of Bt japonensis against first instar

Japanese beetle show this grub to be highly susceptible i.e. 25 ug protein/ g compost of 130 kDa Bt japonensis caused 100% mortality (Suzuki et al. 1992). This differed from results of this study where at both locations Nebraska and Kentucky, second instars were susceptible to

65 ug protein/ g compost of both Bt japonensis and Bt tolworthi. These results may mean that Japanese beetle is much less susceptible than cupreus chafer to Bt japonensis, which has been reported to have an estimated LC50 (ug insecticidal protein/g diet) of 0.09, 0.13 and 11 ug/g for first, second, and third instar cupreus chafer grubs respectively (Suzuki et al. 1993).

Results from these studies with Cyclocephala species pasadenae and lurida suggest a higher level of effectiveness of Bt japonensis than of Bt tolworthi, i.e. an estimated LC50 value of 45 and 241 ug protein/g root-mix for third instar pasadenae and 5 and 50 ug protein/root-mix for second instar C. lurida. Similarly, the results from this study show Bt japonensis to be more efficacious than Bt kumamotoenis against Cotinis sp.

Field collected grubs cannot be synchronized to their physiological age as can laboratory-reared insects. The condition of the grubs can be quite variable even when 31

Table 3. Digestive system pH of Ave fall-collected Cyclocephala pasadenae grubs.

Point of Measurement

Anterior Posterior Rectal Grub # Foregut Midgut Midgut Hindgut Sac

1 9.5 - 9.0 8.8 8.4 2 9.2 9.7 8.5 8.3 8.3 3 - 9.5 9.4 8.8 8.6 4 - 8.8 9.9 8.5 8.3 5 8.0 9.1 9.3 9.3 8.9

Average 8^9 9J 9^2 8J ^ 32 collected at the same time from the same location. In some cases, the results of insecticide tests may not have this variability, however, due to the mode of action of Bt and the requirement for solubilization of the protein in the gut, the physiological state of the insect may affect its susceptibility. It may be possible that the results from this study were made more variable due to changes in midgut pH as a result of physiological age as was observed by Sharpe (1979).

Behavioral differences were noted for Cyclocephala pasadenae used in the two treatments. None of the surviving grubs in the Bt tolworthi treatment had reduced the feed to waste, leaving roots intact in the cup, whereas in the Bl kumamotoensis treatment, there were

10 cups of the 14 total cups in which all of the food including roots had been consumed by

Day 17. Also, grubs that were actively feeding in the cups were curled in a feeding position with the food positioned between abdomen and mouth. Surviving grubs in the Bt tolworthi treatment were tightly wrapped in a C-shaped position with no food being held. Evidence of active feeding by amount of food consumption and feeding position may be a useful indicator in the bioassay evaluation process for determining the health and activity of the grub.

Exploitation of the state of the food material during data collection may assist in making a quick assessment of mortality.

Although several of the stains, Bt tenebrionis, Bt tolworthi and Bt kumamotoensis used in this study are similarly active on the chrysomelids Leptinotarsa sp., (Crickmore et al.

1999 Sick et al. 1990; Grossman 1992; Donovan et al. 1993; Narva and Fu 1993; Robert et al. 1994) such as the Colorado potato beetle, these same toxins show individual differences in their effects against scarab grubs (Grossman 1992; Donovan et al. 1993; Narva and Fu 1993;

Michaels 1993; Michaels et al. 1993; Michaels et al. 1996; Robert et al. 1994). Toxins from 33 kumamotoensis and tolworthi, active against Colorado potato beetle, are each active against a different scarab pest (Grossman 1992; Michaels 1993; Michaels et al. 1993; Michaels et al.

1996; Robert et al. 1994). This study addressed the questions on the host spectrum of these toxins to determine if grubs of greater economic importance were also susceptible. Results indicated that the northern masked chafer Cyclocephala borealis, and southern masked chafer Cyclocephala liirida were susceptible, as their responses to Bt toxins were very similar to those of Pasadena masked chafer Cyclocephala pasadenae. This suggests that grub relatedness, especially on the genera level or subfamily level, may be an important predictor of grub response to Bt subspecies. Supporting the similarity of same genera response are the observations on susceptibility of several grubs in the geneta Anomala to Bt Japonensis

(Suzuki et al 1992). It is possible that other species in the subfamily Cetoniinae, as observed by Robert (1994), are susceptible to toxins produced by Bt kumamotoensis. Thus far, Bt toxins have not been reported or described which cause lethality to grubs such as

Phyllophaga sp. as reported in this study, in the subfamily Melolonthinae, which include many grubs of major economic importance, especially in crops grown in regions other than the U.S. Also there have been no physiological explanations offered as to why different genera and subfamilies sometimes share susceptibility to Bt toxins, as exemplified by

Japanese beetle (subfamily Rutelinae) and masked chafer (subfamily Dynastinae in their susceptibility to Bt tolworthi and Bt japonensis.

Bacteria in the family Bacillaceae, especially Bacillus thuringiensis, have been observed as insect pathogens for at least a century (Tanada and Kaya 1993). Knowledge on the host spectrum of Bt subspecies has expanded from the discovery by Pasteur and Ishiwata in the 1860s and early 1900s of toxicity against Lepidoptera to the discovery of toxicity to 34 aquatic Diptera in the 1970s and Coleoptera in the 1980s. Infomiation on the host range spectrum of Bt toxins continues to expand, from those earliest observations differentiating Bt toxin susceptibilities by insect Order, to gaining finer resolution of Bt host range to insect

Family, or even Subfamily. It is evident from the literature is that host range of a Bt toxin may not be easily predicted. The host spectrum of Bt toxins has become so broad that current reforms to the nomenclature on Bt toxins minimizes emphasis on host range, which was once thought to have a pattern, instead relying on amino acid sequence identities for detailed study of the gene products from Bt. Discovery efforts in the 1990s on grubs, such as presented in this paper, may add to the potential conmiercial usefulness of previously discovered Bt coleopteran toxins.

References

Andrews, R. E., Jr., R. M. Faust, H. Wabiko, K. C. Raymond, L. A. Bulla Jr. 1987. The

biotechnology of Bacillus thuringiensis. CRC Critical Reviews in Biotechnology. 6:163-

232.

Surges, H.D. 1973. Enzootic disease of insects. Annals of the New York Academy of

Sciences. 217:31-49

Carroll, J., M.G. Wolfersberger, and D.J. Ellar. 1997. The Bacillus thuringiensis Cry 1 Ac

toxin-induced permeability change in Manduca sexta midgut brush border membrane

vesicles proceeds by more than one mechanism. J. Cell Sci.. 110:3099-3104.

Crickmore, N., D.R. Zeigler, J.F. Feitelson, E. Schnepf, J. Van Rie, D. Lereclus, J. Baum and

D.H. Dean. 1998. Revision of the nomenclature for the Bacillus thuringiensis pesticidal

crystal proteins. Microbiol. MolecuL Biol, Rev. 62:807-813. 35

Crickmore, N., D.R. Zeigler, E. Schnepf, J. Van Rie, D. Lereclus, J. Baum, A. Bravo, and

D.H. Dean, 1999. Bacillus thuringiensis toxin nomenclature.

fhttpV/www.biols.susx.ac.uk/Home/Neil Crickmore/^//index.html).

Donovan, W.P., M.J. Rupar, and A.C. Slaney. 1993. Bacillus thuringiensis crylllC(b) toxin

gene and protein toxic to coleopteran insects. United States Patent, 5,264,364.

Hilar, D.J. 1990. Pathogenic determinants of entomopathogenic bacteria, pp. 298-302. In

Proceedings, 5"' International Colloquium on Invertebrate Pathology and Microbial

Control, 1990 Adelaide, Incorporating the 23Annual Meeting of the Society for

Invertebrate Pathology, Proceeding as and Abstracts, August 20-24, Adelaide, Australia.

Hilar, D.J. 2000. Summary of Research Programmes.

(http://www.bio.cam.ac.uk/deDt^iochem/UTQs/Hllar.htmn.

Hly, S. 1993. Chapter 5 the engineering of plants to express Bacillus thuringiensis delta-

endotoxins pp. 105-124. In P. F. Entwistle, J. S. Corey, M. J. Bailey, S. Higgs [eds],

Bacillus thuringiensis an environmental biopesticide: theory and practice, John Wiley

and Sonb' Ltd., West Sussex, England

Gill, S. S., E.A. Cowles, and P.V. Pietrantonio. 1992. The mode of action of Bacillus

thuringiensis QT^doXoKxns. Ann. Rev. Entomol. 37:615-636.

Glare, T.R., N.M. Upadhyaya, and H. K. Mahanty. 1992. Genetic manipulation towards

strain improvement of scarab pathogens, pp. 153-165. In T.A. Jackson and T.R. Glare

[eds], Use of pathogens in scarab pest management. Intercept Ltd, Andover, Hampshire,

England.

Grossman, J. 1992. Conference notes: Entomological Society of America's 1991 annual

meeting-part one. IPM Pract. 14 (2):15-18 36

Grossman, J. 1995. Conference notes: Entomological Society of America's 1994 annual

meeting-part one. IPM Pract. 17(3):14-15.

Hofte, H., and H.R. Whiteley. 1989. Insecticidal crystal proteins of Bacillus thuringiensis.

Microbiol. Rev. 53:242-255. lizuka, T., M. Tagawa, S. Arai, M. Niizeki, and T. Miyake. 1998. Bacillus strain and harmful

organism controlling agents. United States Patent, 5,736,514.

Klein, M.G. 1995. Microbial control of turfgrass insects pp. 72-75. 1995. In R. L.

Brandenburg and M.G. Villani [eds.], Handbook of Turfgrass Insect Pests. The

Entomological Society of America, Lanham, MD.

Knowles, B.H., P.J. Knight, and D.J. Ellar. 1991. N-acetyl galactosamine is part of the

receptor in insect gut epithelia that recognizes an insecticidal protein from Bacillus

thuringiensis. Proc. R. Soc. Lond. B. 245:31-35.

Knowles B., and D.J. Ellar. 1987. Colloid-osmotic lysis is a general feature of the

mechanisms of action of Bacillus thuringiensis delta-endotoxins with different insect

specificity. Biochim. Biophys. Acta. 924:509-518.

LeOra Software. 1987. POLO-PC: User's Guide to Probit or Logit Analysis. LeOra

Software, Berkeley, CA.

Li, J., J. Carroll, and D.J. Ellar. 1991. Crystal structure of insecticidal delta-endotoxin from

Bacillus thuringiensis at 2.5 angstrom resolution. Nature. 353: 815-21.

Macintosh, S.C., T.B. Stone, S.R. Sims, P.L. Hunst, J.T. Greenplate, P.G. Marrone, F.J.

Perlak, D.A. FischhofF, and R.L. Fuchs. 1990. Specificity and efficacy of purified

Bacillus thuringiensis proteins against agronomically important insects. J. Invert. Path.

56:258-266. 37

Martin P.A.W. and R.S. Travers. 1989. WorldAvide abundance and distribution of Bacillus

thuringiensis isolates. Appl. Environ. Microbiol. 55:2437-2442.

McGuire, M.R., L.J. Galan-Wang, and P. Tamez Guerra. 1997. Chapter III-3: Bacteria:

bioassay of Bacillus thuringiensis against lepidopteran larvae, pp. 91-99. In L.A. Lacey

[ed], manual of techniques in insect pathology. Academic Press, Inc., San Diego, CA.

Meadows, M.P., D.J. Ellis, J. Butt, P. Jarrett and H.D. Burges. 1992. Distribution, frequency,

and diversity of Bacillus thuringiensis in an animal feed mill. Appl. Environ. Microbiol..

58:1344-1350.

Meadows, M. P. 1993. Chapter 9 Bacillus thuringiensis in the environment, pp. 193-220. In

P. F. Entwistle, J. S. Corey, M. J. Bailey, S. Higgs [eds], Bacillus thuringiensis an

environmental biopesticide: theory and practice, John Wiley and Sons Ltd., West Sussex,

England.

Menestrina, G., and B.V. Semjen. 1999. Chapter 14: Biophysical methods and model

membranes for the study of bacterial pore-forming toxins, pp. 287-309. In The

comprehensive sourcebook of bacterial protein toxins, 2"'' Edition. Academic Press, Inc.,

San Diego, CA.

Michaels, T.E. 1993. Process for controlling scarab pests with Bacillus thuringiensis isolates.

United States Patent, 5,185,148.

Michaels, T.E., K.E. Narva, and L. Foncerrada. 1993. Bacillus thuringiensis toxins active

against scarab pests. World Intellectual Property Organization patent, WO 93/15206.

Michaels, T.E., K,E. Narva, and L. Foncerrada. 1996. Bacillus thuringiensis toxins active

against scarab pests. United States Patent, 5,554,534. 38

Molecular Dynamics. 1994. User's Guide for ImageQuaNT 5.1® for Microsoft

Windows®NT. Sunnyvale, CA.

Narva, K.E., Fu, J. 1993. Novel coleopteran-active toxins from Bt, isolate 50Ca serovar

kumamotoensis. (unpublished data submitted to EMBL/GenBank DDBJ databases Dec.

1993).

Ohba, M., H. Iwahana, S. Asano, N. Suzuki, R. Sato, and H. Hori. 1992. A unique isolate of

Bacillus thuringiensis serovar japonensis with a high larvicidal activity specific for

scarabaeid beetles. Letters Appl. Microbiol. 14: 54-57.

Robert, P., J. Chaufaux, M. Marchal. 1994. Sensitivity of larval Oxythyrea Junesta

(Coleoptera: Scarabaeidae, Cetoniinae) to three strains of Bacillus thuringiensis (Subsp.

Tenebrionis). J. Invert. Path. 63:99-100.

Sato, R., K. Takeuchi, K. Ogiwara, M. Minami, Y. Kaji, N. Suzuki, H. Hori, S. Asano, M.

Ohba, and H. Iwahana. 1994. Cloning, heterologous expression, and localization of a

novel crystal protein gene from Bacillus thuringiensis serovar japonensis strain buibui

toxic to scarabaeid insects. Curr. Microbiol. 28:15-19.

Sharpe, E. S. 1976. Toxicity of the parasporal crystal of Bacillus thuringiensis to Japanese

beetle larvae. J. Invert. Path. 27:421-422.

Sharpe, E. S., R. W. Detroy. 1979. Susceptibility of Japanese beetle larvae to Bacillus

thuringiensis: associated effects of diapause, midgut pH, and milky disease. J. Invert.

Path. 34:90-91 (1979).

Sick, A.J., F. Gaertner, A. Wong. 1990. Nucleotide sequence of a coleopteran-active toxin

gene from a new isolate of Bacillus thuringiensis subspecies tolworthi. Nucl. Acids Res..

18:1305. 39

Sugimura, M., R. Sato, and H. Iwahana. 1997. Unusual proteolytic processing of a delta-

endotoxin from Bacillus thuringiensis strain buibui by larval midgut-juice of Anomala

cuprea Hope (Coleoptera: Scarabaeidae). Appl. Entomol. Zool. 32(4):533-540.

Suzuki, N., H. Hori, K. Ogiwara, S. Asano, R. Sato, M. Ohba and H. Iwahana. 1992.

Insecticidal spectrum of a novel isolate of Bacillus thuringiensis serovar japonensis.

Biol. Control. 2: 138-142.

Suzuki N., H. Hori, S. Asano. 1993. Sensitivity of cupreus chafer, Anomala cuprea

(Coleoptera: Scarabaeidae) in different larval stages to Bacillus thuringiensis serovar

japonensis strain buibui. Appl. Entomol. Zool. 28:403-405.

Suzuki, N. H. Hori, M. Tachibana and S. Asano. 1994. Bacillus thuringiensis strain buibui

for control of cupreous chafer, Anomala cuprea (Coleoptera: Scarabaeidae), in turfgrass

and sweet potato. Biol. Control. 4:361-365.

Swingle, M.C. 1931. Midgut pH of Japanese beetle. Ann. Entomol. Soc. Amer. 20:489-495.

Tanada. Y. and H.K. Kaya. 1993. Insect Pathology. Academic Press, Inc., San Diego, CA. van Frankenhuyzen, K. and C. Nystrom. 1998. The Bacillus thuringiensis toxin specificity

database. fhttp://www.glfc.forestrv.ca/english/res/B/ HomePaee/netintro.htm van Frankenhuyzen, K.V. 1993. The challenge of Bacillus thuringiensis pp. 1-35. In P. F.

Entwistle, J. S. Corey, M. J. Bailey, S. Higgs [eds]. Bacillus thuringiensis an

environmental biopesticide: theory and practice, John Wiley and Sons Ltd., West Sussex,

England.

West, A.W and H.D. Burges. 1985. Persistence of Bacillus thuringiensis and Bacillus cereus

in soil supplemented with grass or manure. Plant Soil. 83:389-398. 40

West, A.W., H.D. Burges, T.J. Dixon, and C.H. Wybom. 1985a. Survival of Bacillus

thuringiensis and Bacillus cereus spore inocula in soil: effects of pH, moisture, nutrient

availability and indigenous microorganisms. Soil Biol. Biochem. 17:657-665.

West, A.W., H.D. Burges, T.J. Dixon and C.H. Wybom. 1985b. Effect of incubation in non-

sterilized and autoclaved arable soil on survival of Bacillus thuringiensis and Bacillus

cereus spore inocula. New Zealand Jour, of Ag. Res. 28:559-566.

Wigley, P. and C. Chilcott. 1990. Bacillus thuringiensis isolates active against the New

Zealand pasture pest, Costelytra zealandica (Coleoptera: Scarabaeidae), pp. 344. In

Proceedings and Abstracts. Fifth Intemational Colloquium on Invertebrate Pathology and

Microbial Control, Adelaide, Australia.

Zhang, J., T.C. Hodgman, L. Krietger, W. Schnetter, and H.U. Schairer. 1997. Cloning and

analysis of the first cry gene from Bacillus popilliae. J. Bacteriol. 179:4336-4341. 41

CHAPTER 3: USE OF LEPTINOTARSA RUBIGJNOSA (COLEOPTERA:

CHRYSOMELIDAE) TO ASSAY PLACEMENT OF BACILLUS THURINGIENSIS

TOLWORTHIUNDER TWO APPLICATION RATES IN CLAY AND SANDY TURF

SOIL

A paper to be submitted to Journal Invertebrate Pathology Tracy Ellis Michaels''^, Gregory A. Bradfisch', Leslie C. Lewis^'^, and Joel R. Coats^ Abstract

Leptinotarsa rubiginosa (Coleoptera: Chrysomelidae) which is susceptible to Bacillus thuringiensis (Bt) tolworthi was used to indicate placement of the bacterium after application to turf. The bioassay indicated that Bt tolworthi was recovered the same day from both sites up to depths of 2 cm after a suspension of 800 ug/ml was applied at rates of 1,870 and 9,350

1/ha (200 and 1,000 gallons per acre) followed by 0.75 cm irrigation.

Introduction

Bacillus thuringiensis (Bt) has generally been dismissed as a control agent for use against subterranean insects, and before the era of pesticidal transgenic plants, has largely been considered for commercialization only as a spray-on bioinsecticide for foliar feeding pests, such as Lepidoptera larvae or the Colorado potato beetle, Leptinotarsa decemlineata

(Coleoptera: Chrysomelidae). The paradigm of the 1980's on the use of Bt to control soil

' Dow AgroSciences, SSOl Oberlin Drive, San Diego CA 92121 ^ USDA-ARS Com Insects and Crop Genetics Research Unit, Genetics Laboratory, ISU, Ames, lA SOOI 1 ^ Department of Entomology, Iowa State University, Ames, lA SOOI 1 42 insects was summed up by A. W. West on the fate of Bt in soil, "from the practical consideration of insect pest control, the rapid loss of crystal insecticidal activity upon exposure to the soil greatly reduces the potential of Bt to control soil pests," (West 1984b).

The loss of protein insecticidal activity is a serious concern; another preoccupying concern was whether delivery of the bioinsecticide to depths in the turf soil where subterranean insects inhabited was even possible. Due to this reasoning, many investigators did not consider applying Bt for use in controlling soil pests until the 1990's when activity of Bt against white grubs was discovered in the laboratory (Grossman 1992; Michaels 1993;

Michaels et al. 1993; Michaels et al. 1996; Robert et al. 1994; Ohba 1992)). Once white grub susceptibility to Bt was established, the first step for assessing the feasibility of developing a

Bt product for control of grubs, was to establish whether the material could be applied to the grub habitat and be efficacious.

Insect bioassay has been used as an analytical tool for indicating the presence and estimating persistence of Bt strains in the soil environment. Studies employing bioassay as a tool have generally addressed environmental concerns, rather than using soil bioassay to optimize insecticidal application parameters. Studies using Galleria mellonella

(Lepidoptera: Pyralidae) as a bioassay subject implicated microorganisms to have an important role in degradation of the protein toxin from Bt aizawai by showing decreased insecticidal activity in natural soil compared to autoclaved soil (West et al. 1984a; West et al.

1984b). Manduca sexta (Lepidoptera: Sphingidae) has been used to show Bt kurstaki to have insecticidal activity whether sorbed or desorbed to clays and humic acids prior to being utilized by soil microorganisms (Tapp and Stotzky 1995b; Tapp and Stotzky 1998; Crecchio and Stotzky 1998; Koskella and Stotzky 1997). Insect bioassay of soil samples has also been 43 used as a comparison in the validation of other monitoring tools such as dot-blot inununoassay for use in estimating Bt presence and persistence in soil (Tapp and Stotzky

1995a). The use of insect bioassay of Bt protein from soil is a method of detection that has also been used to follow the time frame of degradation of Bt transgenic crops after harvest

(Sims and Holden 1996).

Most white grubs which are pests in turf have only one generation per year, such as

Japanese beede Popillia japonica and masked chafers Cyclocephala sp. (Coleoptera:

Scarabaeidae) and they not readily cultured in the laboratory. Usually grubs must be field- collected for laboratory studies, which means subjects for study are In short supply and are variable (Cowles and Villani 1994; Villani and Nyrop 1991; Villani and Wright 1988; Potter and Braman 1991). These characteristics make studies to define insecticide placement characteristics using the intended grub target reserved for the most important questions. A meaningful alternative to using field-collected grubs for laboratory studies on insecticide placement would be helpful for answering preliminary questions on soil placement.

Similarly studies have employed insect bioassay as a means to measure Bt persistence in agricultural soils (Tapp and Stotzky 1995b; Sims and Holden 1996). The study reported herein employed the use of insect bioassay to determine if Bt tolworthi lethal to Japanese beetle and masked chafers, could be detected after application to turf soil by using a surrogate bioassay insect. L rubiginosa (Coleoptera: Chrysomelidae) is used as a surrogate because it shares similar susceptibility to Bt tolworthi as do the key U.S. white grub pests e.g.

Japanese beede and northern and southern masked chafers C. borealis and C. lurida, respectively. The bioassay of the grub-active toxin Bt tolworthi against a shared host L. rubiginosa is tool for making distinctions on features of insecticide application when white 44 grubs are not available. The objective of this study was to determine if Bt tolworthi could be recovered following application to soil using L. rubiginosa as an assay insect.

Materials and Methods

Application

The Bt tolworthi aqueous suspension provided by Dow AgroSciences was estimated by SDS-PAGE to contain 0.8 mg protein/ml was applied to the turf with a CO2 pressurized backpack sprayer with an 8001 EVS flat-fan nozzle (Spraying Systems Co., Wheaton, IL).

The spray was applied to an area defined by perforations on a cardboard sheet placed over the turf of 15 X 30-cm (0.5 sq. ft) and was timed to deliver an equivalent of 1,870 and 9,350

1/ha at spraying times of 2 and 10 seconds (200 and 1,000 gallons per acre, total applied approximately 7 and 35 mg protein toxin respectively to treatment areas). The application was followed by approximately 0.75-cm of irrigation, applied with the same equipment and flow rate as the treatments. The sites used for the study were in the organic sandy clay loam in the rough at lower elevations of the Lawrence Welk Golf Course, Escondido, CA characterized by (10-35% clay, 20-50% sand and native silt) and the Torrey Sandstone soil at

Torrey Pines Golf Course, San Diego, CA.

Sampling

The same day of application, two soil cores were removed from each treatment plot and one from the untreated check with a lO-cm diameter golf-cup cutter. The cores were placed in plastic bags and kept upright during transport. In the laboratory each soil core was placed horizontally and cross-sections were cut through the core using a hacksaw. The four cross-sections of the core consisted of (a) the grass and thatch, (b) mineral soil with roots to an approximate depth of 0.6 cm, (c) soil from an approximate depth 0.6-1.2 cm, and (d) soil 45 from an approximate depth 1.3-2.0 cm. Scissors were used to remove lO-g sections from the interior of the circular cross-sections.

Bioassay

A ten-gram sample taken from each cross-section of a soil core was placed in 20 ml water and mixed for 30 seconds with a Vortex Genie 2™ (Scientific Industries, Bohemia,

NY) before 3-5 tomatillo leaves were added to the suspension and wetted by inverting the tube five times. The 3-5 tomatillo leaves per soil sample were surface dried before being placed into petri dishes with five first-instar L rubiginosa. Samples from cach cross section were tested with 3-4 replications. Two days later the larvae were observed for mortality. A

Chi-Square statistical test was performed on the L rubiginosa bioassay data to determine if the Bt tolworthi treatments (at either application rate) were different from the untreated soil samples (Zar 1984). The Chi-Square test between treatments was performed on data from each depth and each soil type (SPSS Inc. 1996).

Results

Recovery of Bt tolworthi as indicated by L rubiginosa mortality was evident at all depths sampled for both Lawrence Welk golf course (Table 1) or the Torrey Pines golf course (Table 2) turf soils. The bioassay demonstrated the percolation of Bt tolworthi to the lowest depths sampled, i.e., to a depth of ~2.0-cm in both soil types. There was a clear difference of effect on L rubiginosa between the Bt tolworthi-tieated and the untreated check at all depths from both turf soils sampled (Chi-Square Test, p=0.05). Percentage mortality for L rubiginosa in the Bt tolworthi-txeaisd samples averaged across both application volumes from the Lawrence Welk site were 86%, 92%, 77%, 43% for (a) the grass and thatch, (b) mineral soil with roots to an approximate depth of 0.6-cm, (c) soil from an 46

Table 1. Percentage mortality of Leptinotarsa rubiginosa (Coleoptera: Chrysomelidae) in bioassay of turf soil samples from four depths after treatment with Bacillus thuringiensis tolworthi to clay-containing turf soil at Lawrence Welk Golf Course, Escondido CA (1,870 and 9,350 I/ha volume of «800 ug/ml application followed by 0.75-cm post-treatment irrigation).

% Mortality of L. rubiginosa

Insecticide Application Volume

Soil Samples 1,8701/ha 9,3501/ha 0 p-value'

Grass/Thatch 75 97 5 0.001

Roots/mineral soil

~0-0.6 cm 90 95 5 0.001

Roots/mineral soil

-0.6-1.3 cm 75 80 10 0.001

Roots/mineral soil

-1.3-2.0 cm 42 45 6 0.003

'/7-value from Chi-Square Test is given. 47

Table 2. Percentage mortality of Leptinotarsa rubiginosa (Coleoptera: Chrysomelidae) in bioassay of turf soil samples from four depths after treatment with Bacillus thuringiensis tolworthi to sandy turf soil at Torrey Pines Golf Course, San Diego, CA (1,870 and 9350 I/ha volume of »800 ug/ml application followed by 0.75-cm post-treatment irrigation).

% Mortality of L. rubiginosa

Insecticide Application Volume

Soil Samples 1,8701/ha 9,3501/ha 0 p-value'

Grass/Thatch 93 100 6 0.001

Roots/mineral soil

~0-0.6 cm 96 100 0 0.001

Roots/mineral soil

-0.6-1.3 cm 63 100 7 0.001

Roots/mineral soil

-1.3-2.0 cm 70 90 0 0.001

p-value from Chi-Square Test is given. 48 approximate depth 0.6-l.3-cm, and (d) soil from an approximate depth 1.3-2.0-cm mineral soil. Whereas percentage mortality for L. rubiginosa in the untreated check from the

Lawrence Welk site were 5%, 5%, 10%, 6% for (a) the grass and thatch, (b) mineral soil with roots to an approximate depth of 0.6-cm, (c) soil from an approximate depth 0.6-1.3-cm, and

(d) soil from an approximate depth 1.3-2.0-cm mineral soil.

Similar results were obtained for the Torrey Pines site. Percentage mortality for L. rubiginosa in the Bt tolworthi-txtdXtd samples averaged across both application volumes from the Torrey Pines site were 96%, 98%, 81%, 80% for (a) the grass and thatch, (b) mineral soil with roots to an approximate depth of 0.6-cm, (c) soil from an approximate depth 0.6-1.3-cm, and (d) soil from an approximate depth 1.3-2.0-cm mineral soil.

Percentage mortality for L. rubiginosa in the untreated check from the Torrey Pines site were

6%, 0%, 7%, 0% for (a) grass/thatch, (b) 0-0.6-cm, (c) 0.6-1.3-cm, and (d) 1.3-2.0-cm mineral soil.

Discussion

This study indicates Bt tolworthi is recoverable to a depth of «2 cm when applied either at «7 mg protein toxin per ha or «35 mg protein toxin per ha (1,870 and 9,350 1/ha) followed by 0.75 cm irrigation. The turf soil bioassayed against L rubiginosa from these test conditions had lethal concentrations at depths below the thatch, as mortality observed was greater than 70% in 13 of the 16 soil observations. This means that the concenfration of the

Bt toxin on the tomatillo leaf surface was generally greater than the LCso concentration estimated we have observed for the strain Bt tolworthi on this insect (1.2 ug/ml data not shown). There was an inverse relationship between mortality and depth of the soil sample in a bioassay. 49

Use of Bt for pest control in soil was considered an impossibility until studies like this one demonstrated that Bt could be applied and recovered from turf soil. Although insects

have been used to characterize insecticidal activity to address environmental concerns after

application to soil, insect bioassay such as demonstrated in this paper has not yet been extensively employed to evaluate insecticide application parameters for use against insects in

the soil. This study used a novel method for addressing the question of Bt placement in turf soil. Since this initial study was completed, researchers further considering the development of Bt in turf have demonstrated the efficacy of Bt against grubs in the field (Aim et al 1997;

Koppenhofer et al. 1999; Koppenhofer and Kaya 1997; Suzuki et al. 1994). Furthermore, a surrogate insect as an analytical tool to study Bt placement could serve to measure other

parameters of importance to insecticide efficacy when the target insect is not available, as is common in studies of white grubs.

References

Aim, S.R., M.G. Villani, T. Yeh, and R. Shutter. 1997. Bacillus thuringiensis serovar

Japonensis strain buibui for control of Japanese and Oriental beetle larvae (Coleoptera:

Scarabaeidae). Appl. Entomol. Zool. 32:477-484.

Cowles, R.S. and M.G. Villani. 1994. Soil interactions with chemical insecticides and

nematodes used for control of Japanese beetle (Coleoptera: Scarabaeidae) larvae. J. Econ.

Entomol. 87(4) 1014-1021.

Crecchio, C. and G. Stotzky. 1998. Insecticidal activity and biodegradation of the toxin from

Bacillus thuringiensis subsp. kurstaki bound to humic acids from soil. Soil Biol.

Biochem. 30: 463-470. 50

Grossman, J. 1992. Conference notes: Entomological Society of America's 1991 annual

meeting-part one. IPM Pract. 14 (2): 15-18

Koppenhofer, A.M., H.Y. Choo, H.K. Kaya, D.W. Lee and W.D. Gelemter. 1999. Increased

field and greenhouse efficacy against scarab grubs with a combination of an

entomopathogenic nematode and Bacillus thuringiensis. Biol. Cont. 14:37-44.

Koppenhofer, A.M. and H.K. Kaya. 1997. Additive and synergistic interaction between

entomopathogenic nematodes and Bacillus thuringiensis for scarab grub control. Biol.

Cont. 8:131-137.

Koskella, J. and G. Stotzky. 1997. Microbial utilization of free and clay-bound insecticidal

toxins from Bacillus thuringiensis and their retention of insecticidal activity after

incubation with microbes. Appl. Environ. Microbiol. 63: 3561-3568.

Michaels, T.E. 1993. Process for conU:olling scarab pests with Bacillus thuringiensis isolates.

United States Patent, 5,185,148.

Michaels, T.E., K.E. Narva, and L. Foncerrada. 1996. Bacillus thuringiensis toxins active

against scarab pests. United States Patent, 5,554,534.

Ohba, M., H. Iwahana, S. Asano, N. Suzuki, R. Sato, and H. Hori. 1992. A unique isolate of

Bacillus thuringiensis serovar Japonensis with a high larvicidal activity specific for

scarabaeid beetles. Letters Appl. Microbiol. 14: 54-57.

Potter, D.A. and S.K. Braman. 1991. Ecology and management of turfgrass insects. Annu.

Rev. Entomol. 36:383-406. 51

Robert, P., J. Chaufaux, M. Marchal. 1994. Sensitivity of larval Oxythyreajunesta

(Coleoptera: Scarabaeidae, Cetoniinae) to three strains of Bacillus thuringiensis (Subsp.

Tenebrionis). J. Invert. Path. 63:99-100.

Sims, S.R. and L.R. Holden. 1996. Insect bioassay for determining soil degradation of

Bacillus thuringiensis subsp. kurstaki CrylA(b) protein in com tissue. Environ. Entomol.

25:659-664.

SPSS Inc. 1996. SYSTAT® 6.0 for Windows®. SPSS Inc. Chicago, IL.

Suzuki, N., H. Hori, M. Tachibana, L.S. Indrasith and A. Fujiie. 1994. Residual effects of

the toxin from Bacillus thuringiensis serovar japonensis strain buibui in the soil. Appl.

Entomol. Zool. 29: 610-613.

Tapp, H. and G. Stotsky. 1995a. Dot blot enzyme-linked immunosorbent assay for

monitoring the fate of insecticidal toxins from Bacillus thuringiensis in soil. Appl.

Environ. Microbiol. 61:602-609.

Tapp, H. and G. Stotzky. 1995b. Insecticidal activity of the toxins from Bacillus

thuringiensis subspecies kurstaki and tenebrionis adsorbed and bound on pure and soil

clays. Appl. Environ. Microbiol. 61:1786-1790.

Tapp, H. and G. Stotzky. 1998. Persistence of the insecticidal toxin from Bacillus

thuringiensis subspecies kurstaki in soil. Soil. Biol. Biochem. 30:471-476.

Villani, M. G., and R. J. Wright. 1988. Use of radiography in behavioral studies of turfgrass

infesting scarab grub species (Coleoptera: Scarabaeidae) grubs in turf. J. Econ. Entomol.

78: 172-180. 52

Villani, M.G. and J.P. Nyrop. 1991. Age-dependent movement patterns of Japanese beetle

and European chafer (Coleoptera: Scarabaeidae) grubs in soil-turfgrass microcosms.

Environ. Entomol. 20(1) 241-251.

West, A.W., H.D. Burges, and C.H. Wybom. 1984a. Effect of incubation in natural and

autoclaved soil upon potency and viability of Bacillus thuringiensis. J. Invert. Path.

44:121-127.

West, A.W., H.D. Burges, R.J. White and C.H. Wybom. 1984b. Persistence of Bacillus

thuringiensis parasporal crystal insecticidal activity in soil. J. Invert. Path 44: 128-133.

Zar, J. H. 1984. Biostatistical Analysis, 2"*^ Edition. Prentice-Hall, Inc., Englewood Cliffs,

NJ. 53

CHAPTER 4: BACILLUS THURINGIENSIS JAPONENSIS PLACEMENT IN TURF

AFTER STANDARD APPLICATION AND IRRIGATION REGIMES

A paper to be submitted to Journal of Economic Entomology Tracy Ellis Michaels'"'*, Paul Bystrak^, Leslie C. Lewis^''*, and Joel R. Coats'^ Abstract

A sampling regime of rifamycin-resistant Bacillus thuringiensis japonensis was used to study placement of this bacterium in U.S. Golf Association creeping bentgrass green at

Iowa State University Horticulture Station. Thirty ml of the Bt suspension containing ~5.7 x lO" colony forming units was sprayed in an application volume equivalent of 467,935 and

1870 1/ha proceeded by 0, 1 or 2 cm of irrigation equivalent to 1.2 x 0.9-m turf plots. Turf soil was sampled to a depth of 11 cm on Days 0, 1,6 and 60. Higher volumes of irrigation resulted in greater recovery of colony-forming units beneath the grass and thatch layer and were successful in placing the Bt where grubs would presumably be feeding. Soil moisture levels were high throughout the duration of the study possibly diminishing the strength of the evidence for the importance of post-treatment irrigation on the placement of the bacterium.

Introduction

There are several methods to characterize and detect the status and fate of Bacillus thuringiensis {Bt) in soil which do not employ the use of insects. Methods exist to detect Bt

DNA in soil, Bt proteins in soil, and Bt spores in soil (Chapman and Carlton 1985). Bt

' Dow AgroSciences, SSOl Oberlin Drive, San Diego CA 92121 ^ Mycogen Seeds, 301 Campus Drive, PO Box 280, Huxley, lA 50124 ^ USDA-ARS Com Insects and Crop Genetics Research Unit, Genetics Laboratory, ISU, Ames, LA SOOl 1 * Department of Entomology, Iowa State University, Ames, LA SOOl 1 54 strains have been used as models for the study of DNA plasmid transfer between bacteria in the soil envirorunent. To study the frequency of plasmid transfer in soil, a marker or reporter gene which confers resistance to an antibiotic or some combination of antibiotics is used on the plasmid of interest (Thomas 2000). In this case, soil samples can be plated onto antibiotic-containing media to estimate the transfer frequency of DNA plasmid encoding for antibiotic-resistance. Direct DNA detection is a second potential way to measure DNA exchange between microorganisms in soil as well as a tool for understanding fate of microorganisms used for insect pest control (van Elsas et al. 1998). However detection and quantification of DNA from soil currently has complications due to the interference by soil factors on the chemical reactions used to hybridize and amplify DNA (Romanowski et al.

1993; Vettori et al. 1996). Immunological techniques may also be employed to monitor the protein delta-endotoxin from Bt in the environment (Cheung et al. 1988). The protein must be recovered from the environmental samples and be able to react with an antibody, which is labeled with a reporter molecule such as a dye, and the strength of the reported signal quantified by image analysis. Another method, also the one used in this study, to measure the fate of Bt in soil after intentional application to the environment is to measure the recovery of Bt spores or colony-forming units from the sample (Smith and Barry 1998). This method can be used to make quantitative estimates on the levels of Bt populations and the persistence of the spores in the soil (Smith and Barry 1998; Wantabe and Hayano 1995; West et al. 1985; Yara et al. 1997). Field experiments have employed the use of antibiotic- resistant strains to measure characteristics of Bt after application (Eskils and Lovgren 1997;

Young et al. 1995, Pedersen et al. 1995). Antibiotic-resistant strains may be recovered on selective media from envirorunental samples with little background interference from the 55 sample. This method tends to be less costly and time-consimiing as well. Each method for measuring the fate of Bt after application to the environment has its benefits, depending on the types of questions being asked. The soil environment, in all its complexity of characteristics, poses further technical challenges to monitoring fate of Bt (Jepson et al.

1994). Use of Bt in turf represents a unique environment for investigating Bt placement and fate for purposes of insect control. This study employed a rifamycin-resistant subspecies of

Bacillus thuringiemis Japonensis (Btj) to investigate parameters for optimal placement of Bt for grub pest control in turf.

Materials and IVIethods

The field study was performed at Iowa State University (ISU) Horticulture Station,

Ames, lA, and soil samples were processed at USDA-ARS Com Insects and Crop Genetics

Research Unit, Genetics Laboratory, ISU. The 2,000 m^ creeping bentgrass green, constructed to meet U.S. Golf Association (USGA) standards for greens and tees, was divided into 40 plots with alleyways between plots in a randomized complete block design with nine treatments and one untreated check plot within each of the four blocks. The 3x3 factorial array of treatments consisted of three application volumes of 467,935 and 1870 l/ha or 50, 100 and 200 gallons per acre (GPA) and three post-application irrigation treatments of

0, I, and 2 cm of water. The turf had 1-cm uncompressed thatch and mineral sandy loam soil composition was 80:10:10 sand:peat:loam.

The rifamycin-B-resistant liquid bioinsecticide of BtJ provided by Dow AgroSciences

(formerly Mycogen Corporation, San Diego CA) contained spores at the concentration of

~1.9xl0'° spores/ml. The manually powered, CO2 pressurized track sprayer consisted of three flat-fan nozzles on an aluminum boom held over the plot by a 0.9 x 1.5 x 0.2-m copper 56

pipe frame (Spraying Systems Co., Wheaton, IL). The sprayer was used to distribute 30 ml

of the Btj suspension containing -5.7 x lO" colony forming units in an application volume

equivalent of467, 935 and 18701/ha water (pH=7.7). The sprayer was then used to apply

water (pH=6.7) for the post-application irrigation treatments over the 1.2 x 0.9-m plots. On

each sampling day, two cores were randomly sampled without replacement from two

locations in each plot with a 2.54-cm diameter soil probe. The 10-cm" surface area sampled

for each plot had a theoretical total of 5.1 x 10* spores. Each soil probe was washed free

from soil particles with a sponge in a 20 liter bucket of soapy water containing 0.25 ml/L

Ultra Palmolive Original concentrated dishwashing soap (Colgate-Palmolive Company, New

York, NY) before it was thoroughly rinsed with a squirt bottle of 70% ethanol and allowed to

drip dry before taking the next sample. The soil cores were sectioned into six depths (O-1, 1-

3,3-5,5-7, 7-9 and 9-11 cm) with alternating knives held in 70% ethanol and the section

from each depth pooled for each plot into a sterile Whirlpac® bag. The turf sections were

frozen at 0°C until spore recovery measurements were taken. The wet and dry weights were

recorded before serial dilutions were suspended in phosphate buffered saline and plated on

two plates per dilution of nutrient agar containing rifamycin B (15 |ag/ml), Nystatin (50

^g/nll) and cyclohexamide (100 ^ig/ml) (Sigma-Aldrich Chemical Company, St. Louis MO).

The average from the plated dilution with 30-300 colony-forming units (CPU) was used to

calculate the colony-forming units per gram soil used in the analysis. Statistical Analysis

Software (SAS Institute 1990), and Systat 6.0 (SPSS Inc. 1996) were used for statistical

analysis and compilation. 57

Results

Weather

Weather was rainy with an estimated 1.25 cm of rainfall during the 24 hr prior to bioinsecticide application and post-application irrigation. Precipitation on Day 0 was a light mist with a 5-10 mph east breeze while the plots were being sprayed, yet had stopped during sampling of Blocks 1-3. But a fast-moving lightning storm interrupted the completion of

Block 4 sampling on Day 0. Maximum/minimum soil temperature for the ISU horticulture station at 20/50-cm depths were 24/19°C and 21/21, 22/17 and 17/17, 15/10 and I7/13°C for monthly periods ending 9-1-94, 10-1-94 and 11-1-94 respectively. Weather conditions were recorded at the plots each sampling day (Table I).

Soil Moisture

Percentage moisture content for the main treatments (dry weight/wet weight) was examined. No appreciable differences were discerned in the moisture content at each depth from samples taken at different blocks, days, application rate, or irrigation volume. The average moisture content observations "Mean (95% C.I.)" of the treated plots demonstrated greatest moisture content within mineral soil samples, depth 3-5-cm depth at 62% (60-65), whereas the 1-3-cm root zone had 58% (56-60) or the 0-1-cm grass and thatch 31% (29-33) moisture. The moisture content observations "Mean (95% C.I.)" of the untreated soil were similar to the treated plots over the course of the study with depth 3-5-cm in mineral soil at

63% (56-69), 1-3-cm root zone at 60 % (53-67), and 0-1-cm grass and thatch at 33% (28-37),

The data on Day 1 moisture content of the untreated plots was similar to the treated plots

(Table 2). Also, the percentage of moisture was found to be similar at each depth sampled across ail nine treatments for Day 1. The exception was the 0-1-cm grass and thatch layer in Table 1. Weather conditions on sampling days at ISU Horticulture Station.

Meterological Data

Sampling Day Condition 0 16 60

Date 9-24-94 9-25-94 9-30-94 11-20-94

Daily MaxTMin Air Temperature (°C) 15/5 21/7 29/12 5/2

Air Temperature at Plots during Sampling (°C) 13 16 23 2

Plot Soil Temperature (°C) 2-cm depth 17 24 24 14

Plot Soil Temperature (°C) 7-cm depth 15 19 21 14

Wind Velocity (mph) and Direction 10, east gusting to 25 10-15, north 5, northwest 5-10, northeast

Relative Humidity (%) 80 80 60 60

Cloud Cover (%) 100 100 80 100

Precipitation 24 hr Pre-Sampling (cm) 1.3 0.8 0 0

Precipitation 24 hr Post-Sampling (cm) 0.8 0 0 2.0 Table 2. Percentage moisture of samples in untreated and treated plots on Day 1.

MeantSEM % Moisture (wt/wt)

Untreated Plots Treated Plots Turf Sample N % N %

Grass & Thatch (0-1 cm) 4 28.2±2.7 36 25.911.0

Root Zone (1-3 cm) 4 47.4±0.9 36 46.310.5

Mineral Soil (3-5 cm) 4 51.0±0.6 36 50.010.4

Mineral Soil (5-7 cm) 4 51.3±0.4 36 52.110.2

Mineral Soil (7-9 cm) 4 53.210.6 36 52.9dt0.1

Mineral Soil (9-11 cm) 4 54.1+0.6 36 53.6+0.2 60

the 1870 1/ha application rate followed by 2 cm of irrigation where the moisture content was elevated (24%) in comparison to the mean of the other treatments (21%). Significant correlation between sample moisture and logioCFU/g was not uncovered for any treatment factors. Otherwise a relationship between sample moisture content and treatment effect was not evident. The turf soil samples consistently contained high moisiure levels. Moisture observations suggest the turf soil may have been near maximum water holding capacity when the treatments were applied on Day 0 and suggest the plots remained moist during the study.

Untreated plots

The plating method to detect 5//-rifamycin-resistant spores detected background counts of fl(/-rifamycin-resistant-like organisms in the untreated check. The untreated plots were not included in the analysis of variance which examined application and irrigation treatment effects. Averaging across all treatments and days (Table 2) CFU/g soil means were

102-103 CFU/ g soil greater in the treated plots (Table 3). Although the background readings from the untreated plots were not exactly homogeneous, the counts in the treated plots were examined for trends due to treatment effects.

Log transformation

Normal probability graphs of counts of colony-forming units per gram soil demonstrated die data for each depth to be positively skewed. Consequently, the CPU/ g soil data was logarithmically transformed using the equation X'= logio(X+l). This transformation improved normality and avoided problems with zero counts. The transformation was performed before treatment effects were analyzed for each depth. Table 3. Colony-forming units (CFU)/g sample in untreated and treated plots.

CFU/g Sample

Untreated Plots Treated Plots Turf Sample N CFU/g soil N CFU/g soil

Grass & Thatch (0-1 cm) 15 2.6 X 10^ 135 1.4 X 10'

Root Zone (1-3 cm) 15 2.2 X 10' 135 3.2 X 10^

Mineral Soil (3-5 cm) 15 1.9 X 10^ 135 2.1 X 10'

Mineral Soil (5-7 cm) 15 2.5 X lO' 135 7.9 X lO'

Mineral Soil (7-9 cm) 15 1.3 X 10' 135 4.3 X 10®

Mineral Soil (9-11 cm) 15 4.6 X 10' 135 2.0 X 10® Table 4. F-Values from Type ill Analysis of Variance (ANOVA) model for tests of hypotheses using corrected error terms on logio CFU/g sample from six turf sampling depths for all sampling dates.

F-Values from ANOVA (* indicates significance p<0.05)

Depth Grass & Term Thatch Root Zone Mineral Soil Mineral Soil Mineral Soil Mineral Soil (0-1 cm) (1-3 cm) (3-5 cm) (5-7 cm) (7-9 cm) (9-11 cm)

BLOCK (BL) 38.9» 10.4* 25.2* 4.5* 0.3 0.5

APPLICATION 2.0 5.2* 3.9* 1.2 0.6 0.1 (L/HA)

IRRIGATION (IRR) 7.4» 39.6* 53.1* 35.2* 35.1• 33.3*

L/HA*IRR 4.1• 7.5* 4.2* 4.8* 3.4* 5.2*

BL*L/HA*iRR 1.3 0.9 1.4 2.3 2.4 1.5

DAY 6.2* 10.3* 19.7* 7.S* 28.8* 11.8*

L/HA* DAY 0.4 l.S 0.5 0.6 1.2 1.3

IRR*DAY 0.2 3.2* 4.1• 2.8* 4.5* 2.6*

BL*DAY 14.7* 7.5* 7.0^ 2.3* 0.7 0.5

L/HA*IRR*DAY 0.6 0.8 1.3 0.7 1.0 1.2

N 135 135 135 135 135 135 R^ 0.84 0.82 0.87 0.84 0.87 0.80 63

Block*Day interaction

The initial analysis of variance model (ANOVA) fitted to the transformed data

demonstrated a significant interaction effect fi*om BLOCK*DAY. The final ANOVA model

accounted for 70 of 134 degrees of freedom by employing BLOCK (blocks 1-4), 1/ha (liters

per hectare 467, 935,1870), and IRR (post-treatment irrigation 0, I & 2 cm) as whole-plot

treatments. In addition the model used DAY (samples taken Day 0, I, 6 & 60) as sub-plot

treatments. The BLOCK*DAY interaction term remained in the sub-plot analysis as an

identifiable source of non-random error. F-test values (Table 4) and Tukey's Studentized

Range Test for whole-plot treatments were calculated using an error mean square error computed by pooling sums of squares corresponding to interactions between blocks and the

whole plot factors. (Whole plot mean squares for depths 0-1, 1-3, 3-5, 5-7, 7-9, & 9-11 cm,

respectively, equaled 0.13,0.18,0.23,0.34, 0.34, & 0.30). F-test values (Table 4) and

Tukey's Studentized Range Test for analyses related to DAY effects, and their interactions

with whole plot factors were calculated using an error mean square computed by pooling sums of squares for all remaining interactions with blocks except for the BLOCK*DAY

interaction (subplot error mean squares at depths 0-1, 1-3, 3-5, 5-7, 7-9, & 9-11 cm, respectively, equaled 0.09, 0.19, 0.15, 0.15, 0.13, & 0.20).

Differences in counts between Block 1 and the other blocks may explain why a significant BLOCK*DAY interaction was evident (Table 4). Block 1 showed slightly elevated logioCFU/g soil counts (Table 5) especially noticeable in the grass and thatch layer

(0-1 cm). 64

Measurements over time

DAY effects were highly significant, both individually and as part of interaction terms (Table 4). The combined treatment means from each DAY were compared using

Tukey's Pairwise Mean Comparison Test (Table 6). From Day 0 to 60 slight changes in logioCFU/g were detected (Table 7). LogioCFU/g soil quantities decreased in the 0-1-cm grass and thatch layer, while detection logioCFU/g soil increased in the lower mineral soil layers of 3-5 cm, 5-7 cm, 7-9 cm and 9-11 cm (Table 7). The slight trend showed a decrease in detectable spores in the top layer of grass and thatch. This suggests possible gravitational settling of logioCFU/g soil to lower depths.

Effect of application rate

The L/HA application rate treatment had the lowest significant F-values from

ANOVA and was significant when it was part of interaction terms with irrigation (Table 4).

Few differences were detected for application rate at each depth using Tukey's Pairwise

Mean Comparison Test (Table 8). Rate increases within the application range tested did not significantly change the number of detectable spores captured by the 0-1-cm grass and thatch layer (Table 8). The application rates chosen for the study were within parameters for turf insecticide application, but seemed to have a minor influence on rinsing Btj spores through the grass and thatch. However, at the l-3-cm and 3-5-cm depths, the higher application rate treatments of 935 and 1870 1/ha led to higher levels of LogioCFU/g soil detected at those depths. This suggests a minor influence of application rates on the placement of spores to the

1-3- and 3-5-cm potential grub habitat. Table 5. Treated plots logio CFU/g sample by block.

Mean±SEM Logio CFU/g Sample'

Block Turf Sample N 1 2 3 4'

Grass & Thatch (0-1 cm) 4 6.8±0.1. 6.2+0.1 b 6.210.1 b 6.110.1 b

Root Zone (1-3 cm) 4 6.3+0.1. 5.7±0.1b 6.010.1, 6.1+0.1,

Mineral Soil (3-5 cm) 4 5.5±0.1 , 6.2±0.1b 6.010.1b 5.710.1,

Mineral Soil (5-7 cm) 4 5.3±0.1 , 5.6±0.1b 5.610. lb 5.510.1b

Mineral Soil (7-9 cm) 4 5.110.1 a 5.1±0.1, 5.110.1, 5.310.1,

Mineral Soil (9-11 cm) 4 4.7±0.1 a 4.8±0.1, 4.710.1. 4.810.1,

* At each depth means followed by the same letter are not statistically different (Tukey's p=O.OSO). ^ Block 4 was sampled three days instead of four (N=3). Table 6. Treated plots logio CFU/g sample by each sampling day.

Mean±SEM Logio CFU/g Sample '

Day Turf Sample N 0^ 1 6 60

Grass & Thatch (0-1 cm) 36 6.6±0.1 a 6.4±0.1 ab 6.210.1 be 6.210.1 c

Root Zone (1-3 cm) 36 6.2±0.1 . 5.7±0.1 b 6.010.1 a 6.210.1 a

Mineral Soil (3-5 cm) 36 5.4+0.2 . 5.5±0.1 b 6.110.1 b 5.910.1 b

Mineral Soil (5-7 cm) 36 5.2±0.1 , 5.5±0.1 b 5.710.1 b 5.510.1 b

Mineral Soil (7-9 cm) 36 4.6±0.1 a 5.210.1 b 5.510.1 c 5.210.1 b

Mineral Soil (9-11 cm) 36 4.610.1 a 4.710.1 a 5.110.1 b 4.510.1 a

' At each depth means followed by the same letter are not statistically different (Tukey's p=0.050). ^ Day 0 is average of three blocks instead of four (N=27). Table 7. Gain (+) or Loss (-) in logio CFU/g sample between sampling time points.

+/- Change in Logio CFU/g Sample *

Time Points (Sampling Day)

Turf Sample Otol 0 to 6 0to60 lto6 lto60 6 to 60

Grass & Thatch (0-1 cm) -0.1 -0.3* -0.4* -0.1 -0.2* -0.1

Root Zone (1-3 cm) -0.4* -0.1 +0.1 +0.3* +0.5* +0.2

Mineral Soil (3-5 cm) +0.5* +0.7* +0.5* +0.1 -0.1 -0.2

Mineral Soil (5-7 cm) +0.3* +0.5* +0.4» +0.1 0.0 -0.1

Mineral Soil (7-9 cm) +0.6* +0.9* +0.6* +0.3* 0.0 -0.3*

Mineral Soil (9-11 cm) +0.2 +0.5* -0.3 +0.4* -0.2 -0.6*

' (*)Indicates a statistically significant change between time points (Tukey*s p=O.OSO) Table 8. Application-volume effect on placement of logio CFU/g sample.

Mean±SEM Logio CFU/g Sample *

Application Rate (1/ha) Turf Sample N MSD 467 935 1870

Grass & Thatch (0-1 cm) 45 0.1 6.3±0.1 , 6.3±0.1 a 6.410.1 a

Root Zone (1-3 cm) 45 0.2 5.9±0.1, 6.1±0.1 b 6.110.1 b

Mineral Soil (3-5 cm) 45 0.2 5.7±0.1 a 5.8±0.1 ab 6.010.1 b

Mineral Soil (5-7 cm) 45 0.3 5.4±0.1, 5.4±0.1ab 5.610.1 b

Mineral Soil (7-9 cm) 45 0.3 5.110.1, 5.0±0.1 a 5.210.1 a

Mineral Soil (9-11 cm) 45 0.2 4.7±0.1, 4.8±0.1 a 4.710.1 a

' At each depth means followed by the same letter are not statistically different (Tukey's p=0.050) 69

Effect of post-treatment irrigation volumes

With the exception of BLOCK at the 0-1-cm grass and thatch layer, F-test values were consistently largest for terms containing irrigation IRR (Table 4). The F-values indicating significance of the post-application irrigation factor were especially noticeable for the 3-5-cm, 5-7-cm 7-9-cm and 9-11-cm depths containing mineral soil, as well as the 1-3- cm root zone. At these depths significance was evident when irrigation was a main and sub plot interaction effect. Tukey's Pairwise Mean Comparison Test was used to compare means at the depths sampled for the main treatment effect of post-application irrigation volume

(Table 9). The 2-cm irrigation treatment significantly reduced logioCFU/g soil from the 0-1- cm grass and thatch layer in comparison to the lesser irrigation volumes (6.2±0.1 vs 6.4±0.1

& 6.5±0.1)(Table 9). Interestingly, logioCFU/g soil detected in the 2-cm irrigation treatment had a corresponding increase in the 1-3-cm root zone layer (6.3±0.1 Vs 5.6±0.1) just below the grass and thatch, compared to no irrigation. The trend of logioCFU/g soil influence by the irrigation treatments suggested greater penetration of the bioinsecticide past the grass and thatch layer to the root zone and mineral soil with Increasing volumes of irrigation (Table 9).

Recovery of Bacillus thuringiensis japonensis one day after application

Day 1 logioCFU/g soil recovery patterns and the moisture levels from the nine treatments of application rate and irrigation volume were considered (Table 10). The data from Day 1 was examined because it represented a complete data set closest in time to when the application was made. The data from the top three sample depths of 0-1 cm, 1-3 cm and

3-5 cm were summarized because those are depths where grubs would potentially be located.

The sum of logioCFU/g soil recovered in the top three depths (Total logioCFU/g) was used as the basis for percentage (% of recovery) at each depth shown (Table 10). The sum of % Table 9. Irrigation (post-application) effect on placement logio CFU/g sample.

Mean±SEM Logio CFU/g Sample *

Irrigation Volume (cm) Turf Sample N MSD 0 1 2

Grass & Thatch (0-1 cm) 45 0.1 6.5±0.1 a 6.4±0.1 a 6.2±0.1 b

Root Zone (1-3 cm) 45 0.2 5.6±0.1 a 6.2±0.1 b 6.3±0.1 b

Mineral Soil (3-5 cm) 45 0.2 5.210.1, 6.1±0.1 b 6.2±0.1 b

Mineral Soil (5-7 cm) 45 0.3 4.9±0.1 a 5.6±0.1 b 6.0±0.1 c

Mineral Soil (7-9 cm) 45 0.3 4.6±0.1. 5.2±0.1 b 5.6±0.1 c

Mineral Soil (9-11 cm) 45 0.2 4.2±0.1. 4.7±0.1 b 5.2±0.1 e

' At each depth means followed by the same letter are not statistically different (Tukey's p^O.050) Table 10. Summary of Day 1 logio CFU/g sample and moisture. Percentage logio CFU/g soil (% of recovery) and moisture (% of moisture) expressed as a fraction of the three- depth sum for application (1/ha) and irrigation (cm) treatments. Observations taken at 0-1-cm grass and thatch, 1-3-cm grass root zone and 3-5-cm mineral soil depths.

Mean ± SEM Logio CFU/g Sample

% of Recovery of Logio CFU/g Soil from Total of Top Three Depths

% of Moisture in Each Depth from Total % Moisture from Top Three Depths

Post-Application Irrigation Volume (cm) 0 1 2

Application Rate (1/ha) Application Rate (1/ha) Application Rate (1/ha)

Depth N 467 935 1870 467 935 1870 467 935 1870

0-1 cm 4 6.0±03 6.9±0.7 6.7Hb0.4 6.4±0.5 6.3±0.5 6.7±0.3 6.4±0.4 6.1 ±0.2 6.2±0.5

% of recovery 39 37 37 34 35 35 34 34 34

% of moisture 21 19 22 21 20 21 22 21 24

1-3 cm 4 4.8±0.2 6.0±0.4 5.7±0.4 6.0±0.3 5.8±0.3 6.0±0.5 6.0±0.3 5.8±0.4 5.5±0.5

% of recovery 32 33 32 31 32 32 33 32 30

% of moisture 38 39 38 38 39 38 38 38 36 Table 10. (continued)

Mean ± SEM Logio CFU/g Sample

% of Recovery of Logip CFl)/g soil from Total of Top Three Depths

% of Moisture in Each Depth from Total % Moisture from Top Three Depths

Post-Application Irrigation Volume (cm) 0 1 2

Application Rate.(l/ha) Application Rate (1/ha) Application Rate (1/ha) Depth N 467 935 1870 467 935 1870 467 935 1870

3-5 cm 4 4.4±0.2 5.5±0.2 S.7±0.2 6.7±0.2 6.1±0.3 6.3±0.1 6.0±0.3 6.1±0.3 6.4±0.1

'/•of recovery 29 30 31 35 33 33 33 34 36

% of moisture 41 42 40 41 41 41 40 41 40

Total Log,0 15.2 18.4 18.1 19.1 18.2 19.0 18.4 18.0 18.1 CFU/g

Total Moisture 118.1 119.0 122.3 121.6 121.3 123.0 124.1 121.5 128.9 % 73

moisture content (Total Moisture %) in each of the top three depths was used as the basis for percentage (% of moisture) at each depth (Table 10).

The-%-of-recovery summary for Day I (Table 10) confirms the ANOVA and

Tukey's Pairwise Mean Comparison Test observations. Increased irrigation volumes reduced the level of recoverable logioCFU/g sample in the 0-1-cm grass and thatch layer.

The inverse was true for the 3-5-cm mineral soil layer where recovered logioCFU/g sample was increased by increased irrigation volume. The % recovered logioCFU/g sample in the 1-

3-cm-root zone was observed to be no different in this study among the nine treatments.

Discussion

This study employed the use of an antibiotic-resistant strain of Btj. Although environmental concerns may exist regarding the use of antibiotic-resistant strains of 5/ for study (Haack et al 1996), in one study a streptomycin-resistant Bt israelensis was shown to have very slow growth and few lasting effects on the indigenous bacterial population (Eskils et al. 1997). Spore measurements from soils showed no proliferation and a two-year persistence following aerial forest application of Bt (Smith 1998).

The study presented herein may have represented an extreme in soil moisture conditions of near-saturation upon application. Although the data showed no clear relationship between soil moisture and treatment effect, the conditions of turf and soil sample moisture may have been a substantial underlying factor affecting the results. This suggests effects of BtJ application rate and post-application irrigation may have differed in a study

(unlike this one) where samples showed low soil moisture. High levels of soil moisture may be responsible for masking the slightly evident trends in this study, where irrigation was 74 found to cause increased Btj penetration from the grass and thatch layer into the root zone and mineral soil in turf. Furthermore the pre-existing high soil moisture content may have been important to allowing penetration of rifamycin-resistant Btj spores to the observed depths. One study in mulberry plantations showed no translocation after Bt application followed by irrigation even though irrigation was found more than 10 cm below the surface, but the soil characteristics were not reported and may not have been similar (Akiba 1991).

However in the mulberry plantation study, Bt was recovered to a depth of up 2-6 cm.

Furthermore in laboratory irrigated soil column tests, applied Bt followed by imgation was detected in the flow-through from sandy columns but not columns with volcanic ash soil

(Akiba 1991). This suggests Bt may percolate in sandy soil that is irrigated.

The principles governing movement of chemical pesticides may also apply to understanding of Bt performance in turfgrass environments (Arnold and Briggs 1990;

Branham et al. 1993). The same concerns which apply to chemical insecticides, such as the physical and chemical properties of the soil, other environmental variables including turfgrass variety and maturity, thickness of the thatch layer, microbial populations, temperature, and soil moisture, may similarly apply to Bt outcome in turf (Branham et al.

1995; Kenna 1995; Branham et al. 1995; Odanaka et al. 1993).

The increased adsorptive properties of the grass and thatch layers of turfgrass, by binding chemical insecticides, may reduce unintended runoff into waterways (Starrett et al.

1996; Branham et al. 1993). Insecticides with a moderate to high adsorption coefficient greater than 100, bind to organic matter in the thatch layer and soil, and reduce the risk tor groundwater contamination (Kenna 1995). Studies showed that isazofos and isofenphos were mainly recovered from the thatch layer and showed very little leaching of the 75 insecticides to the first 2.5-cm root zone or deeper mineral soil (Niemczyk, 1987; Niemczyk and Krueger 1987; Vittum 1985). Bt deita-endotoxins studied have shown Bt proteins to have high adsorption coefficient values and that Bt proteins bind strongly to organic matter

(Sundaram 1996). In one sense, Bt may behave similarly to several other insecticides used in turf, where binding to the organic matter in the thatch layer occurs (Branham et al. 1993;

Starrett et al. 1996).

Post-application irrigation of insecticides in turf has been initiated for safety and effectiveness of pesticides in general. It is used to reduce the risk of exposure to insecticides on the surface of the soil, but also may be important to transporting the insecticide off of the grass and thatch layer while it is still in a mobile form, to the depths where grubs are feeding

(Niemczyk, 1987; Niemczyk and Krueger 1987; Vittum 1985). Irrigation is important to promoting chemical insecticide movement downward in the soil profile, but to promote movement of grubs upward (Cowles and Villani 1994; Villani and Wright 1988). Grubs were observed to respond to moisture provided by irrigation by moving upwards in the soil profile, to the thatch-soil interface, where presumably grubs receive the lethal dosage.

Likewise, irrigation of Bt after application to turfgrass may be important to effectiveness.

The results of this study suggested the practice of irrigation to have a role in movement of Bt spores ofT of the grass and thatch layer into the root zone, especially apparent in the higher volumes of irrigation where fewer quantities of spores were recovered in the grass and thatch.

The depth of spore transport in this study can be explained by the near saturation conditions of the turf during the study as mentioned above. Additionally, as similar to observed with chemical insecticides, leaching to depths in the soil has been observed to occur 76 through macropores (Branham et al. 1993; Starrett et al. 1996) and may explain some of the observations in this study. The characteristics of the turf mineral soil beneath the thatch layer at the study site may also be an important contributing factor to the depths of CFU recovery in the soil, as it was constructed for good drainage, aeration and gaseous diffusion for oxygen supply to the roots. Whereas the organic matter in the grass and thatch layer may bind Bt, the sandy characteristics of the study site had low organic matter for adsorptive surface, and may be a contributing factor to spores recovery at the depths observed.

Chemical insecticides must withstand degradation to some extent in order to provide insecticide effectiveness, and this may also be a similar for Bt to be an effective insect control in turf grass soil (Kenna 1995; Odanaka et al. 1993). Hopefully, Bt as a soil insecticide presents reduced risks to the environment in contrast to insecticides with greater stability and persistence. This study demonstrated that under well-drained turf field conditions, treatment of turf with BtJ under average application parameters, should deliver the bioinsecticide to where the grubs reside.

References

Akiba, Y. 1991. Assessment of rainwater-mediated dispersion of field sprayed Bacillus

thuringiensis in the soil. Appl. Entomol. Zool. 26:477-483.

Arnold, D.J. and G.G. Briggs. 1990. Chapter Five, Fate of pesticides in soil: predictive and

practical aspects, pp. 101-122. In D.H. Hutson and T.R. Roberts [eds.]. Environmental

Fate of Pesticides. John Wiley & Sons Ltd, West Sussex, England.

Branham, B., E. Miltner, and P. Rieke. 1995. Potential groundwater contamination fi-om

pesticides and fertilizers used on golf courses. USGA Green Section Record. Jan/Feb;34-

37. 77

Branham, B.E., Smitley, D.R. and E.D. Miltner. 1993. Chapter 14, Pesticide fate in turf:

studies using model ecosystems, pp. 156-167. In K.D. Racke and A.R. Leslie [eds.].

Pesticides in Urban Environments: Fate and Significance. Division of Agrochemicals,

203^'' National American Chemical Society Meeting, San Francisco, CA April 5-10,

1992. ACS Symposium Series, American Chemical Society, Washington D.C.

Chapman, J.S., and B.C. Carlton. 1985. Conjugal transfer in Bacillus thuringiensis, pp. 453-

467. In D.R. Helinski, N. Cohen, D.B. Clewell, D.A. Jackson, and A. Hollaender [eds],

Plamids in Bacteria. Plenum Press, New York, NY.

Cheung, P.Y.K., and B.D. Hammock. 1988. Monitoring Bacillus thuringiensis in the

environment with enzyme-linked immunosorbent assay pp. 359-372. In P.A. Hedin, J.J.

Menn, R. M. Hollingworth [eds.], Biotechnology for Crop Protection, ACS Symposium

Series no. 379. American Chemical Society, Washington, D.C.

Cowles, R.S. and M.G. Villani. 1994. Soil interactions with chemical insecticides and

nematodes used for control of Japanese beetle (Coleoptera: Scarabaeidae) larvae. J. Econ.

Entomol. 87(4) 1014-1021.

Eskils, K., and A. Lovgren. 1997. Release of Bacillus thuringiensis subsp. israelensis in

Swedish soil. FEMS Microbiol. Ecol.. 23:229-237.

Haack, B.J., R.E. Andrews, and T.E. Loynachan. 1996. Tn916-mediated genetic exchange in

soil. Soil Biol. Biochem. 28:765-771,

Jepson, P.C., B. C. Croft, and G.E. Pratt. 1994. Test systems to determine the ecological

risks posed by toxin release from Bacillus thuringiensis genes in crop plants. Molec.

Ecol. 3:81-89. 78

Kenna, M.P. 1995. What happens to pesticides applied to golf courses? USGA Green Section

Record. Jan/Feb: 1-9.

Niemczyk, H.D. 1987. The influence of application timing and post-treatment irrigation on

the fate and effectiveness of isofenphos for control of Japanese beetle (Coleoptera:

Scarabaeidae) larvae in turfgrass. J. Econ. Entomol. 80:465-470.

Niemczyk, H.D. and H.R. Krueger. 1987. Persistence and mobility of isazofos in turfgrass

thatch and soil. J. Econ. Entomol. 80:950-952.

Odanaka, Y., T. Taniguchi, Y. Shimamura, K. lijima, Y. Koma, T. Takechi, and O. Matano.

1994. Runoff and leaching of pesticides in golf courses. J. Pestic. Sci. 19:1-10.

Pedersen J.C., P.H. Damgaard, J. Eilenberg, and B.M. Hansen. 1995. Dispersal of Bacillus

thuringiensis vac. kurstaki in an experimental cabbage field. Can. J. Microbiol. 41: 118-

125.

Romanowski, G., M.G. Lorenz, and W. Wackemagel. 1993. Use of polymerase chain

reaction and electroporation of Escherichia-coli to monitor the persistence of

extracellular plasmid DNA introduced into natural soils. Appl. Environ. Microbiol. 59:

3438-3446.

SAS Institute. 1990. SAS® User's Guide: Statistics. SAS Institute, Cary, NC.

Smith, R.A, and J.W. Barry. 1998. Environmental persistence of Bacillus thuringiensis

spores following aerial application. J. Invert. Path. 71:263-267.

SPSS Inc. 1996. SYSTAT® 6.0 for Windows®. SPSS Inc. Chicago, IL.

Starrett, S.K., N.E. Christians, and T.A. Austin. 1996. Soil processes and chemical transport:

movement of pesticides under two irrigation regimes applied to turfgrass. J. Environ.

Qual. 25:566-571. 79

Sundaram, K.M.S. 1996. Sorptive interactions and binding of deita-endotoxin protein from

Bacillus thuringiensis subsp kurstaki in forest soils. J. of Environ. Sci. Health Part B.

31:1321-1340.

Thomas, J. A. W. Morgan, J.M. Whipps, and J.R. Saunders. 2000. Plasmid transfer

between the Bacillus thuringiensis subspecies kurstaki and tenebrionis in laboratory

culture and soil and in lepidopteran and coleopteran larvae. Appl. Environ.Microbiol.

66:118-124.

Van Elsas, J.D., G.F. Duarte, A.S. Rosado, and K. Smalla. 1998. Microbiological and

molecular biological methods for monitoring microbial inoculants and their effects in the

soil environment. J. Microbiol. Method 32:133-154.

Vettori, C., D. Paffetti, G. Pietramellara, G. Stotsky, and E. Gallori. 1996. Amplification of

bacterial-DNA bound on clay-minerals by the random amplified polymorphic DNA

(RAPD) technique. FEMS Microbiol. Ecol. 20: 251-260.

Villani, M.G. and R.J. Wright. 1990. Environmental influences on soil macroarthropod

behavior in agricultural systems. Annu. Rev. Entomol. 35:249-269.

Vittum, P.J. 1985. Effect of timing of application on effectiveness of isofenphos, isazophos

and diazinon on Japanese beetle (Coleoptera: Scarabaeidae) grubs in turf. J. Econ.

Entomol. 78: 172-180.

Wantabe, K., and K. Hayano. 1995. Seasonal variation of soil protease activities and their

relation to proteolytic bacteria and Bacillus spp. in paddy field soil. Soil Biol. Biochem.

27:197-203. 80

West, A.W., H.D. Surges, T.J. Dixon, and C.H. Wybom. 1985. Survival of Bacillus

thuringiensis spore inocula in soil: effects of pH, moisture, nutrient availability and

indigenous microorganisms. Soil Biol. Biochem. 17:657-665.

Yara, K., Y Kunimi and H. Iwahana. 1997. Comparative studies of growth characteristic and

competitive ability in Bacillus thuringiensis and Bacillus cereus in soil. Appl. Entomol.

Zool. 32: 625-634.

Young, C.S., G. Lethbridge, L.J. Shaw, and R.G. Bums. 1995. Survival of inoculated

Bacillus cereus spores and vegetative cells in non-planted and rhizosphere soil. Soil

Biol. Biochem. 27:1017-1026. 81

GENERAL CONCLUSIONS

This research was to explore the use of Bacilliis thuringiensis (JBi) toxins against turfgrass grub pests (Coleoptera: Scarabaeidae). Part of this dissertation research demonstrated newly described relationships between toxins produced by Bt and grub susceptibility, A survey of several U.S. grub pests was performed to understand the host range of Bacillus thuringiensis tolworthi, kumamotoensis, and Japonensis. The following scarab turfgrass pests (Coleoptera: Scarabaeidae) were evaluated: Anomala cuprea, Atenius sp., Cotinis sp., Cyclocephala borealis, Cyclocephala lurida, Cyclocephala pasadenae,

Popillia j'aponica, Phyllophaga sp. Results showed Bacillus thuringiensis tolworthi and

Bacillus thuringiensis japonensis to have activity against U.S. economic grub pests

Cyclocephala borealis, Cyclocephala lurida, and Popillia japonica while Bacillus thuringiensis kumamotoensis was active against Cotinis sp. Recently Bt toxins have now been shown to be active against white grubs, and the unique aspect of this research was the discovery of Bt tolworthi and Bt japonensis to be active against the main U.S. pests of economic significance in turf: masked chafers and Japanese beetles, as well as the activity of

Bt kumamotoensis against green firuit beetle.

Supported by the research in tiiis dissertation is that the insecticidal activity of the Bi toxins are specific against a nanow spectrum of grubs. This suggests that grub relatedness, especially on the genera level or subfamily level, may be an important predictor of grub response to Bt subspecies. Results from this dissertation research indicate the northern masked chafer Cyclocephala borealis, southern masked chafer Cyclocephala lurida Pasadena masked chafer Cyclocephala pasadenae had similar susceptibility profiles in their responses 82 to Bt toxins. This result was similar to susceptibility of several grubs in the gensra. Anomala to Bt japonensis (Suzuki et al 1992). It is possible that other species in the subfamily

Cetoniinae, as observed in one report (Robert et al 1994), are susceptible to toxins produced by Bt kumamotoensis. Also this dissertation confirmed the observations that grubs must be actively feeding to orally consume and be susceptible to the Bt protein as the insects must be in a receptive physiological state for susceptibility to Bt. Also, the activity of performing laboratory studies in this research confirmed previous worker's comments (Cowles and

Villani 1994; Villani and Nyrop 1991; Villani and Wright 1988; Potter and Braman 1991) on the challenges to studying soil insects such as grubs. The subterranean habits and length of life cycle are also factors in contributing to the variability of response of the grubs to the Bt toxins.

But soil such as turf soil presents a unique environment, and it challenges the optimizing of insecticide application. Prior knowledge on the use of as an effective spray- on insecticide has thus far been limited to foliar and aquatic environments (Tanada and Kaya

1993). While it was acceptable to dismiss the use of Bt toxins for use against soil pests for decades (West et al. 1984), this dissertation explored soil applications of Bt as a biological insecticide with applications to the field. In this dissertation, studies demonstrated the recovery of Bt from the soil, suggesting the feasibility for a novel use as a commercial pathogen for subterranean insects. In this dissertation a bioassay insect Leptinotarsa rubiginosa (Coleoptera: Chrysomelidae), also susceptible to Bt tolworthi, was used to indicate placement of the bacterium after application to turf. The bioassay indicated that Bt tolworthi was recovered the same day from both sites up to depths of 2 cm after a suspension of 800 ug/ml was applied at rates of 1,870 and 9,3501/ha (200 and 1,000 gallons per acre) 83 followed by 0.75 cm irrigation. These initial results confirm other studies which had positive results for use of Bt in controlling soil-inhabiting grubs in turfgrass (Aim 1997; Koppenhofer and Kaya 1997; Koppenhoffer et al. 1999; Suzuki et al. 1994).

Fortunately for the purposes of evaluating risks posed to the environment by the possibility of using Bt for grub control, Bt strains have been used as models for the study of

DNA plasmid transfer between bacteria in the soil envirormient, and methods are established to detect Bt DNA in soil, Bt proteins in soil, and Bt spores in soil (Chapman and Carlton

1985). A conunon method employed to measure Bt spores in soil was employed to determine what aspects of conventional insecticide application may apply to successful Bt use. A sampling regime of rifamycin-resistant Bt japonensis (Btj) was used to study placement of this bacterium in U.S. Golf Association creeping bentgrass green using standard insecticide application volume equivalent of467,935 and 18701/ha proceeded by 0, 1 or 2 cm of irrigation to 1.2 x 0.9-m turf plots. Sampling of the turf soil to a depth of 11 cm on

Days 0, 1, 6 and 60 showed irrigation to be an important factor in placement of Btj spores.

Higher volumes of irrigation resulted in greater recovery of colony-forming units beneath the grass and thatch layer and were successful in placing the Bt where grubs would presumably be feeding. Factors such as indigenous soil moisture may have diminished the on importance of post-treatment irrigation, however this practice has a strong influence on the placement of the bacterium. More importantly the research suggests that Btj fits within program of a turfgrass manager when applying pesticides to turf by being compatible with routine chemical application using standard equipment and practices.

In hindsight Bt efficacy against grubs in soil might not be surprising, since environmental studies on Bt protein have indicated that Bt may have some physical and 84 chemical properties that are consistent with desirable characteristics for the use as a soil bioinsecticide (Sundaram 1996; Tapp and Stotzky 1995; Tapp and Stotzky 1998; Crecchio and Stotzky 1998; Koskella and Stotzky 1997). Bt delta-endotoxins studied have been shown to have high adsorption coefficient values and bind strongly to organic matter (Sundaram

1996). This binding to soil organic matter, in addition to preventing leaching, may assist in retaining insecticidal activity. Yet concern of undesirable environmental persistence of Bt in most modem manufactured golf courses may be mediated by the use of sandy soil in greens and tees to provide aeration for grass roots. Under these circumstances, Bt proteins may have lower adsorption to golf course soil than agricultural soil and may be more exposed to degradation by factors of the environment. Btj spores however do persist up to 60 days in turfgrass soil. This persistence of spores may also be important to the retention of activity of native Bt active protein toxin against grubs due the recycling of bacteria in dead and dying insects in the soil, as has been thought to occur in Bacillus popilliae epizootics (Thomas et al.

2000; Klein 1992, 1995; Tanada and Kaya 1993) and long-term control observed for grub control by Btj in sweet potato crops in Japan (Suzuki et al. 1994). Although the environmental concerns should be further addressed (Jepson et al. 1994), Bt for use in the soil may be viewed similarly as Bt for other applications, as an important tool for integrated pest management.

Bt is a reduced-risk insecticide, and with the combined knowledge of how Bt performs for grub control, as well as its known environmental characteristics, all information from this dissertation points to a safe and effective new use for this well-established biopesticide for use against turfgrass pests. This dissertation demonstrates that Bt host range extends to coleopterans previously not known to be susceptible, suggests the use of Bt for a 85 new and novel purpose of turfgrass grub control, and offers insight that the use bioinsecticide application to turf fits with conventional turfgrass management. 86

GENERAL REFERENCES

Agnew, M. and D.R. Lewis. 1993. A survey of pesticides used on Iowa golf courses in 1990:

FG-460. Iowa State University Extension Service, Ames, lA.

Ali, A.D. 1989. Insect and Related Pests of Turfgrass, pp. 49-68. In A.D Ali and C.L.

Elmore. Turfgrass Pests: University of California Cooperative Extension Sei-vice

Publication No. 4053. Division of Agriculture and Natural Resources, University of

California, Oakland CA.

Allsopp, P.G., M.G. Klein, and E.L. McCoy. 1992. Effect of soil moisture and soil texture on

oviposition by Japanese beetle and rose chafer (Coleoptera: Scarabaeidae). J. Econ.

Entomol. 85(6): 2194-2200.

Aim, S.R., M.G. Villani, T. Yeh, and R. Shutter. 1997. Bacillus thuringiensis serovar

japonensis strain buibui for control of Japanese and Oriental beetle larvae (Coleoptera:

Scarabaeidae). Appl. Entomol. Zool. 32:477-484.

Aim, S.R., M.G. Villani and M.G. Klein. 1995. Oriental Beetle, pp. 81-83. In R. L.

Brandenburg and M.G. Villani [eds.], Handbook of Turfgrass Insect Pests. The

Entomological Society of America, Lanham, MD.

Amett, R.H., Jr. 1985. American insects: a handbook of the insects of America north of

Mexico. Van Nostrand Reinhold Company Inc., New York, NY.

Arnold, D.J. and G.G. Briggs. 1990. Chapter Five, Fate of pesticides in soil: predictive and

practical aspects, pp. 101-122. In D.H. Hutson and T.R. Roberts [eds.]. Environmental

Fate of Pesticides. John Wiley & Sons Ltd, West Sussex, England. 87

Aronson, A.I., and W. Beckman. 1987. Transfer of chromosomal genes and plamids in

Bacillus thuringiensis. Appl. Environ. Microbiol. 53:1525-1530.

Brandenburg R. L. and M.G. Villani [eds,]. 1995. Handbook of Turfgrass Insect Pests. The

Entomological Society of America, Lanham, MD.

Branham, B., E. Miltner, and P. Rieke. 1995. Potential groundwater contamination from

pesticides and fertilizers used on golf courses. USGA Green Section Record. Jan/Feb;34-

37.

Branham, B.E. and D.J. Wehner. 1985. The fate of diazinon applied to thatched turf Agron.

Jour. 77:101-104.

Branham, B.E., Smitley, D.R. and E.D. Miltner. 1993. Chapter 14, Pesticide fate in turf:

studies using model ecosystems, pp. 156-167. In K.D. Racke and A.R. Leslie [eds.],

Pesticides in Urban Environments: Fate and Significance. Division of Agrochemicals,

203"* National American Chemical Society Meeting, San Francisco, CA April 5-10,

1992. ACS Symposium Series, American Chemical Society, Washington D.C.

Bulla. L.A., Jr., R.N. Costilow, and E.S. Sharpe. 1978. Biology of Bacillus popilliae. Adv.

Appl. Microbiol. 23: 1-18.

Chapman, J.S., and B.C. Carlton. 1985. Conjugal transfer in Bacillus thuringiensis, pp. 453-

467. In D.R. Helinski, N. Cohen, D.B. Clewell, D.A. Jackson, and A. Hollaender [eds],

Plamids in Bacteria. Plenum Press, New York, NY.

Cowles, R.S. and M.G. Villani. 1994. Soil interactions with chemical insecticides and

nematodes used for control of Japanese beetle (Coleoptera: Scarabaeidae) larvae. J. Econ.

Entomol. 87(4) 1014-1021. 88

Crecchio, C., and G. Stotsky. 1998. Binding of DNA on humic acids: effect on

transformation of Bacillus subtilis and resistance to DNAse. Soil Biol. Biochem.

30:1061-1067.

Crocker, R.L., W.T. Naiion, Jr., and J.A. Reinert. 1995. May and June Beetles, pp. 72-75. In

R. L. Brandenburg and M.G. Villani [eds.]. Handbook of Turfgrass Insect Pests. The

Entomological Society of America, Lanham, MD.

Downing, A.S. 1994. Effect of irrigation and spray volume on efficacy of entomopadiogenic

nematodes (Rhabditida: Heterorhabditidae) against white grubs (Coleoptera:

Scarabaeidae). J. Econ. Entomol. 87:643-646.

Dutky, S.R. 1941. Testing the possible value of milky diseases for control of soil-inhabiting

larvae. J. Econ. Entomol. 34:(2):217.

Fleming, W.E. 1968. Biological control of die Japanese beede. Technical Bulletin 1383

Agricultural Research Service. United States Department of Agriculture, Washington

D.C.

Forst, S., B. Dowds, N. Boemare, and E. Stackebandt. 1997. Xenorhabdus and Photorhabdus

spp.: bugs that kill bugs. Annu. Rev. Microbiol. 51:47-72.

Gaugler, R. 1988. Ecological considerations in the biological control of soil-inhabiting

insects with entomopathogenic nematodes. Agric. Ecosys. Environ. 24:351-360.

Gaussoin, R., Baxendale, F., J. Watkins, and S. Hygnstrom. 1994. Pesticide selection guide

for Nebraska professional turfgrass managers EC 94-1558-A. Nebraska Cooperative

Extension Service, Lincoln, NE. 89

Hanula, J.L. 1990. Epizootiological investigations of the microspcridium Ovavesicula

popilliae and bacterium Bacillus popilliae in field populations of the Japanese beetle

(Coleoptera: Scarabaeidae). Environ. Entomol. 19:1552-1557.

Heller, P.R. 1995. Asiatic garden beetle, pp. 26-28. In R. L. Brandenburg and M.G. Villani

[eds.], Handbook of Turfgrass Insect Pests. The Entomological Society of America,

Lanham, MD.

Hellman, L. 1995. Green June beetle, pp. 57-59. In R. L. Brandenburg and M.G. Villani

[eds.], Handbook of Turfgrass Insect Pests. The Entomological Society of America,

Lanham, MD.

Jackson, T.A., J.R. Pearson, M.O. O'Callaghan, H.K.Mahanty, and M.J. Willocks. 1992.

Pathogen to product development of Serratia entomophilia (Enterobacteriaceae) as a

commercial biological control agent for the New Zealand grass grub (Costelytra

zealandica), pp. 191-198. In T.A. Jackson and T.R. Glare [eds], Use of pathogens in

Scarab pest management. Intercept Ltd, Andover, Hampshire, England.

Jepson, P.C., B. C. Croft, and G.E. Pratt. 1994. Test systems to determine die ecological

risks posed by toxin release from Bacillus thuringiensis genes in crop plants. Molec.

EcoL 3:81-89.

Kaya, H.K. and S.P. Stock. 1997. Chapter VI: Techniques in insect pathology, pp. 281-322.

In L.A. Lacey [ed]. Manual of Techniques in Insect Pathology. Academic Press, Inc., San

Diego, CA. 90

Kaya, H.K., M.G. Klein, T.M. Burlando, R.E. Harrison, and L.A. Lacey. 1992. Prevalence of

two Bacillus popilliae Dutky morphotypes and blue disease in Cyclocephala hirta

LeConte (Coleoptera: Scarabaeidae) populations in California. Pan-Pacific Entomol..

68(1): 38-45.

Kenna, M.P. 1995. What happens to pesticides applied to golf courses? USGA Green Section

Record.Jan/Feb: 1-9.

Klein, M.G. 1992. Use of Bacillus popilliae in Japanese beetle control, pp. 179-189. In T.A.

Jackson and T.R. Glare [eds], Use of pathogens in Scarab pest management. Intercept

Ltd, Andover, Hampshire, England.

Klein, M.G. 1995. Microbial control ofturfgrass insects pp. 72-75. 1995. In R. L.

Brandenburg and M.G. Villani [eds.]. Handbook of Turfgrass Insect Pests. The

Entomological Society of America, Lanham, MD.

Klein, M.G. 1997. Chapter III-4: Bacteria of soil inhabiting pests, pp. lOl-l 13. In L.A. Lacey

[ed], Manual of Techniques in Insect Pathology. Academic Press, Inc., San Diego, CA.

Koppenhofer, A.M. and H.K. Kaya. 1997. Additive and synergistic interaction between

entomopathogenic nematodes and Bacillus thuringiensis for scarab grub control. Biol.

Cont. 8:131-137.

Koppenhofer, A.M., H.Y. Choo, H.K. Kaya, D.W. Lee and W.D. Gelemter. 1999. Increased

field and greenhouse efficacy against scarab grubs with a combination of an

entomopathogenic nematode and Bacillus thuringiensis. Biol. Cont. 14:37-44.

Koskella, J. and G. Stotzky. 1997. Microbial utilization of free and clay-bound insecticidal

toxins from Bacillus thuringiensis and their retention of insecticidal activity after

incubation with microbes. Appl. Environ. Microbiol. 63:3561-3568. 91

Lewis, D.R. 1997. Insect pests of turfgrass, pp. 36-37. In D.R. Lewis, M. Gleason, R.

Hartzler, J. lies, B. Whipker, and J. Homstein [eds], Iowa Commercial Pesticide

Applicator Manual Category 3: Ornamental, Turf, and Greenhouse Pest Management CS-

15. Iowa State University Extension Service, Ames, lA.

Lewis, D.R. 2000. Personal Communication. Department of Entomology and Extension

Entomologist, Iowa State University, Ames, lA.

McLeod, M., M. Weiss, and M.E. Rice. 1999. White Grubs, pp. 115-117. In K.L. Steffey,

M.E. Rice, J. All, D.A. Andow, M.E. Gray, and J.W. Van Duyn [eds] Handbook of Com

Insects. Entomological Society of America, Lanham, MD.

Mullins, J.W. 1993. Chapter 13 Imidacloprid: a new nitroguanidine insecticide. In S.O.

Duke, J.J. Menn and J.R. Plimmer [eds.], Pest Control with Enhanced Environmental

Safety: American Chemical Society Symposium Series No. 524. American Chemical

Society, Washington, D.C.

Niemczyk, H.D. 1987. The influence of application timing and post-treatment irrigation on

the fate and effectiveness of isofenphos for control of Japanese beetle (Coleoptera:

Scarabaeidae) larvae in turfgrass. J. Econ. Entomol. 80:465-470.

Niemczyk, H.D. and H.R, Krueger. 1987. Persistence and mobility of isazofos in turfgrass

thatch and soil. J. Econ. Entomol. 80:950-952.

Odanaka, Y., T. Taniguchi, Y. Shimamura, K. lijima, Y. Koma, T. Takechi, and O. Matano.

1993. Rimoff and leaching of pesticides in golf courses. J. Pestic. Sci. 19:1-10.

Peacock, C.H. and M.M. Smart. 1995. IPM, monitoring, and management plans - a mandate

for the future. USGA Green Section Record. May/June 1995:10-14. 92

Potter, D.A. 1995. Masked Chafers, pp. 70-72. In R. L. Brandenburg and M.G. Villani

[eds.]. Handbook of Turfgrass Insect Pests. The Entomological Society of America,

Lanham, MD.

Potter, D.A. and S.K. Braman. 1991. Ecology and management of turfgrass insects. Annu.

Rev. Entomol. 36:383-406.

Ritcher, P.O. 1966. White Grubs and Their Allies: A Study of North American Scarabaeoid

Larvae. Oregon State University Press, Corvallis, OR.

Robert, P., J. Chaufaux, M. Marchal. 1994. Sensitivity of larval Oxythyrea fiinesta

(Coleoptera: Scarabaeidae, Cetoniinae) to three strains of Bacillus thuringiensis (Subsp.

Tenebrionis). J. Invert. Path. 63:99-100.

Sharpe, E. S., R. W. Detroy. 1979. Susceptibility of Japanese beetle larvae to Bacillus

thuringiensis: associated effects of diapause, midgut pH, and milky disease. J. Invert.

Path. 34:90-91 (1979).

Shetlar, D.J., P.E. Suleman, and R. Georgis. 1988. Irrigation and use of entomogenous

nematodes, Neoaplectana spp. and Heterorhabditis heliothidis (Rhabditida:

Stememematidae and Heterorhabditidae), for control of Japanese beetle (Coleoptera:

Scarabaeidae) grubs in turfgrass. J. Econ. Entomol. 81: 1318-1322.

Smitley, D.R. 1995. European chafer, pp. 50-52. In R. L. Brandenburg and M.G. Villani

[eds.], Handbook of Turfgrass Insect Pests. The Entomological Society of America,

Lanham, MD.

Spieszalski, W.W., H.D. Niemczyk, and D.J. Shetlar. 1994. Sorption of chlorpyrifos and

fonofos on four soils and turfgrass thatch using membrane filters. J. Environ. Sci. Health.

B29(6):I117-1136. 93

Stanett, S.K., N.E. Christians, and T.A. Austin. 1996. Soil processes and chemical transport:

movement of pesticides under two irrigation regimes applied to turfgrass. J. Environ.

Qual. 25:566-571.

Sundaram, K.M.S. 1996. Sorptive interactions and binding of delta-endotoxin protein from

Bacillus thuringiensis subsp kurstaki in forest soils. J. of Environ. Sci. Health Part B.

31:1321-1340.

Suzuki, N. H. Hori, M. Tachibana and S. Asano. 1992. Bacillus thuringiensis strain buibui

for control of cupreous chafer, Anomala cuprea (Coleoptera: Scarabaeidae), in turfgrass

and sweet potato. Biol. Control. 4:361-365.

Suzuki, N. H. Hori, M. Tachibana and S. Asano. 1994. Bacillus thuringiensis strain buibui

for control of cupreous chafer. Anomala cuprea (Coleoptera: Scarabaeidae), in turfgrass

and sweet potato. Biol. Control. 4:361-365.

Tanada. Y. and H.K. Kaya. 1993. Insect Pathology. Academic Press, Inc., San Diego, CA.

Tapp, H., and G. Stotsky. 1995. Dot blot enzyme-linked immunosorbent assay for monitoring

the fate of insecticidal toxins from Bacillus thuringiensis in soil. Appl. Environ.

Microbiol. 61:602-609.

Tapp, H. and G. Stotzky. 1998. Persistence of the insecticidal toxin from Bacillus

thuringiensis subspecies kurstaki in soil. Soil. Biol. Biochem. 30:471-476.

Tashiro, H. 1987. Turfgrass Insects of the United States and Canada. Cornell University

Press, Ithaca, NY.

Tate, R.L. 1995. Soil Microbiology. John Wiley & Sons. New York, NY. 94

Thomas, J. A. W. Morgan, J.M. Whipps, and J.R. Saunders. 2000. Plasmid transfer

between the Bacillus thiiringiensis subspecies Jcurstaki and tenebrionis in laboratory

culture and soil and in lepidopteran and coleopteran larvae. Appl. Environ.Microbiol.

66:118-124.

Villani, M. G., and R. J. Wright. 1988. Use of radiography in behavioral studies of turfgrass

infesting scarab grub species (Coleoptera: Scarabaeidae) grubs in turf J. Econ. Entomol.

78: 172-180.

Villani, M.G. and J.P. Nyrop. 1991. Age-dependent movement patterns of Japanese beetle

and European chafer (Coleoptera: Scarabaeidae) grubs in soil-turfgrass microcosms.

Environ. Entomoi. 20(1) 241-251.

Villani, M.G. and R.J. Wright. 1990. Environmental influences on soil macroarthropod

behavior in agricultural systems. Annu. Rev. Entomol. 35:249-269.

Vittum, P.J. 1985. Effect of timing of application on effectiveness of isofenphos, isazophos

and diazinon on Japanese beetle (Coleoptera: Scarabaeidae) grubs in turf. J. Econ.

Entomol. 78: 172-180.

Vittum, P.J. 1995. Black turfgrass ataenius, pp. 35-37. In R. L. Brandenburg and M.G.

Villani [eds.], Handbook of Turfgrass Insect Pests. The Entomological Society of

America, Lanham, MD.

Vittum, P.J. 1995. Japanese Beetle, pp. 66-69. In R. L. Brandenburg and M.G. Villani [eds.].

Handbook of Turfgrass Insect Pests. The Entomological Society of America, Lanham,

MD.

Ware, G. W. 1994. The Pesticide Book 4*'' Ed. Thomson Publications, Fresno, CA. 95

West, A.W. 1984. Effect of incubation in natural and autoclaved soil upon potency and

viability of Bacillus thuringiensis. J. of Invert. Path. 44:121-127.

Zimmerman, R.J. and W.S. Cranshaw. 1991. Short-term movement of Neoaplectana spp.

(Rhabditida: Steinemematidae) and Heterorhabditis "HP-88" strain (Rhabditida:

Heterorhabditidae) through turfgrass thatch. J. Econ. Entomol. 84:875-878. 96

ACKNOWLEDGEMENTS

This research was supported by Dow AgroSciences, San Diego CA (formerly

Mycogen Corporation) by providing grants-in-aid for laboratory supplies to Department of

Entomology, Iowa State University, by providing research materials, technical expertise, laboratory facilities, and flex-time to me as a (currently 12-year) employee. Numerous co workers, supervisors, managers, current or former associates at DAS/Mycogen and Kubota

Corporation cooperatively gave their knowledge and guidance in many ways. Specifically I would like to thank L. Hickle, J. Payne, P.Bystrak, R. Shutter, E. Schnepf. B. Sturgis, M.

Walz, J. Bing, G. Schwab, S. Kerbs, C. Conlan, M. Farrow, J. Petell, G. Bradfisch, S.

Stanley, S. Glasnapp, L. Nygaard, K. Ogiwara, Mr. Takahashi, S. Evans, Dr. S. Asano, Dr.

Suzuki, Dr. Hori, K. Narva, R. Reynolds, J. Cesarek, G. Soares, B. Stockhoff, K. Wanner, P.

Zomer and W. Gelemter.

My major professors. Dr. Joel Coats and Dr. Leslie Lewis, are surrounded by fabulous helpful people in the Insecticide Toxicology Group of the Department of

Entomology as well as USDA-ARS Com Insects Research Group. Committee Members Dr.

T. Loynachan and Dr. R. Andrews and graduate student Dr. B. Haack also provided invaluable technical guidance. The people 1 met while enrolled at ISU over the past seven years were a pleasure to know, and I would like to thank Dr. R. Cao, Dr. T. Anderson, Dr. J.

Cink, Dr. Pamela Rice, Dr. S. Lee, Dr. E. Arthur, Dr. Patricia Rice, Dr. F. Lam, Dr. R.

Pingle, C. Peterson, J. Anhalt, Dr. J. Tollefson, Kelly Kyle, J. Dyer, R. Gunnerson, N.

Gallagher, J. Robbins, and Dr. J. Van Dyke. A special thanks for the laboratory assistance of

Auli Rainio and Kristine Dunbar. 97

The turfgrass insect research community with special thanks to Dr. F. Baxendale, Dr.

M. Klein, Dr. S. Aim, Dr. R. Cowles, Dr. H. Niemczyk, Dr. T. Jackson, Dr. H. Kaya, Dr. D.

Shetlar, Dr. C.Allen, Dr. D. Potter, Dr. P. Vittum, Dr. A. Suggars, Dr. M. Villani, Dr. R.

Brandenburg, and Dr. D. Lewis who generously shared bountiful knowledge and experience.

Several golf courses and the ISU Horticultural Station were also very generous by allowing me to utilize (and damage) turf for research purposes. The assistance of the ISU Statistics

Department especially Dr. Kenneth Koehler was greatly appreciated.