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INFLUENCE OF ENDOPHYTIC PERENNIAL RYEGRASS, Lolium perenne L., ON

THE POPULATION DENSITY OF TWO AND SPECIES

COMPOSITION IN MIXED TURFGRASS SWARDS

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the

Graduate School o f The Ohio State University

By

DOUGLAS SCOTT RICHMOND, B.S., M.S.

*****

The Ohio State University

1999

Dissertation Committee:

David J. Sheiiar, Adviser Approved by

Tom K. Danneberger

Ronald B. Hammond

Casey W. Hoy Advisor

Harry D. Niemczyk Department of Entomology UMI Number: 9919904

UMI Microform 9919904 Copyright 1999, by UMI Company. All rights reserved.

This microform edition is protected against unauthorized copying under Title 17, United States Code.

UMI 300 North Zeeb Road Ann Arbor, Ml 48103 Copyright by Douglas Scott Richmond 1999 ABSTRACT

The goal of this research was a practical strategy for using endophyte-mediated resistance to manage epigeal insects in turfgrass. Overseeding stands of Kentucky bluegrass, Poa pratemis L., (KB) with mixtures of endophytic and non-endophytic perennial ryegrass, Lolium perenne L., (PR+ and PR- respectively) altered turfgrass sward composition over time according to the seeding rate (kg/ha) and the cultivars used.

Bluegrass billbug, parvulus Gyllenhal, larval population density and damage were significantly reduced by overseeding, but damage varied between cultivars.

In mixtures of PR+, PR-, and KB, S. parvulus larval populations decreased as the proportion of PR+ increased. No further decrease in S. parvulus populations was observed in swards containing >38.4±3.8% PR+. Plant-systemic insecticide application had little effect on sward composition or larval S. parvulus numbers when S. parvulus populations were low. However, when S. parvulus populations were higher, a lower proportion of PR+ was found in insecticide-treated plots. In swards containing only PR- and PR+, S. parvulus larval populations also decreased as the proportion of PR+ increased. However, no further decrease in S. parvulus populations was observed in swards containing >68.0±28.7% PR+.

11 In laboratory choice tests, adult male S. parvulus showed no preference between

KB, PR- and PR+ . In non-choice assays more feeding punctures were observed on PR- and PR+ than KB. Feeding was strongly correlated with plant stem diameter. Longevity of adult male S. parvulus was reduced on PR+ compared with PR-, but was highly variable on KB. Bluegrass webworm, Parapediasia teterrella (Zincken), larvae showed no preference for KB or PR-, but both were preferred over PR+. P. teterrella survival to pupation and fresh pupal mass was the same on KB and PR-, but no larvae survived to pupation on PR+.

In pots containing mixtures of PR+, and KB, P. teterrella larval survival decreased as the proportion of PR+ increased to 40.3±19.0%. No further reductions were observed in mixtures containing higher proportions of PR+. Emigration was only a significant determinant of larval population density in monocultures of PR+.

Moderate proportions of PR+ significantly reduced pest population densities whereas higher proportions of PR+ provided no further reduction in insect populations. However, the relationship between insect population density and the proportion of PR+ is mediated by plant species composition. Plant characteristics, such as tiller diameter, provide additional sources of insect resistance for plant breeding.

Insect movement plays an important role in determining insect density in polycultures of endophytic and non-endophytic , buy does not fully account for the relationship between insect population density and the proportion of PR+. Further study of the role of natural enemies, dose acquisition, and the physiological influence of endophyte toxins in determining insect population density in mixed stands of turfgrass are warranted.

iii ACKNOWLEDGMENTS

I am indebted to my parents Stephen and Barbara Richmond for their support during my graduate education. I would like to extend my most sincere gratitude to Dr.

David J. Shetlar for his patience, guidance, and support as my mentor. The helpful advise and support of my dissertation committee members, Drs. T. Karl Danneberger, Ronald B.

Hammond, Casey W. Hoy and Harry D. Niemczyk is also profoundly appreciated. I would like to recognize the turfgrass entomology laboratory at OARDC, Mark Belcher,

Jason Holton, Eric Lingenfelter, Kevin McClure, Wade Pinkston, and Kevin Power, who’s knowledge and effort were instrumental in seeing this project to fruition. 1 also thank the entomology journal club at OARDC (Charlotte Bedet, Leann Beanland, Tim

Ebbert, Graham Head, Lisa Fiorina, John Lloyd, and Motshwari Obopile) for many helpful discussions and general esprit de corps. 1 am grateful for the counsel of several faculty including Drs. Benjamin Stinner, Daniel Herms, David Horn, Richard Hall,

William Lyon, Parwinder Grewal, and Robin Taylor. The cooperation of the

OARDC/Secrest Arboretum grounds crew, and Jim Karcher in particular were also vital to completing this project. I thank Dr. M. Siegel of the University of Kentucky and Carl

Petelle of Leisure Inc. for providing materials. Many thanks go to Dr. Kenneth

Cochran, Director of Secrest Arboretum, for providing space for field studies.

IV VITA

October 7, 1967 ...... Bom - Wooster, Ohio

December 22, 1990 ...... B. S., Biological Sciences

Bowling Green State University,

Bowling Green, OH

September 1991-March 1994 ...... Graduate Teaching Associate,

Graduate Research Associate,

The Ohio State University,

Columbus, OH

March 18, 1994 ...... M. S., Entomology

The Ohio State University,

Columbus, OH

April 1994-Present ...... Graduate Research Associate

OARDC, The Ohio State University,

Wooster, OH REFEREED JOURNAL PUBLICATIONS

Richmond, D.S. and D.J. Shetlar 1996. Eclosion Time and Spatial Distribution of Overwintering Spruce Spider Mite (Acari: Tetranychidae) Eggs on Colorado Spruce. J. Econ. Entomol. 89(2):447-452

Keeney, G., M.S. Ellis, D. Richmond and R.N. Williams 1994. A Preliminary Study of the Nitidulidae (Coleoptera) in Shawnee State Forest, Ohio. Ent. News 105(3): 149-158

Williams, R.N., J.L. Blackmer, D.S. Richmond, and M.S. Ellis 1992. Nitidulidae (Coleoptera) Diversity in Three Natural Preserves in Portage County, Ohio. Ohio J. Sci. 92(4):82-87

FIELDS OF STUDY

Major Field: Entomology

VI TABLE OF CONTENTS

PAGE

ABSTRACT ...... ii

ACKNOWLEDGMENTS ...... iv

VITA ...... V

LIST OF TABLES ...... x

LIST OF FIGURES...... xi

CHAPTERS:

I. INTRODUCTION...... I

Definition of Turfgrass Arena to be Studied ...... 2 Economically Important Insects ...... 3 Endophytic Fungi ...... 6 Role of Endophytes in Host Plant Resistance ...... 7 Chemicals Associated with Fungal Endophytes ...... 9 Influence of Insect Feeding on Plant Species Composition ...... 11 Other Factors Influencing Turfgrass Sward Com position ...... 14 Vegetation Texture ...... 17 Quantifying Turfgrass Sward Composition ...... 23 Model Description ...... 25 Study Objectives...... 28 Thesis Statement ...... 29 Experimental Subjects ...... 31

V ll OVERSEEDING ENDOPHYTIC PERENNIAL RYEGRASS. Lolium perenne L., INTO ESTABLISHED STANDS OF KENTUCKY BLUEGRASS, L., TO MANAGE BLUEGRASS BILLBUGS, Sphenophorus parvulus GYLLENHAL ...... 35

Materials and Methods ...... 37 Results ...... 41 Discussion ...... 44 C onclusion ...... 48

3. BLUEGRASS BILLBUG, Sphenophorus parvulus GYLLENHAL. LARVAL POPULATION DENSITY IN PURE STANDS OF PERENNIAL RYEGRASS, Lolium perenne L„ WITH VARYING PROPORTIONS OF ENDOPHYTIC TILLERS...... 58

Materials and Methods ...... 59 Results ...... 64 Discussion ...... 66 C onclusion ...... 69

4. RELATIONSHIP BETWEEN BLUEGRASS WEBWORM, Parapediasia teterrella (Zincken), LARVAL POPULATION DENSITY AND THE PROPORTION OF ENDOPHYTIC PERENNIAL RYEGRASS, Lolium perenne L., IN MIXED STANDS WITH KENTUCKY BLUEGRASS, Poa pratensis L...... 80

Materials and Methods ...... 82 Results ...... 87 Discussion ...... 90 Conclusion ...... 93

5. CONCLUDING REMARKS...... 101

Overseeding Rate and Sward Composition ...... 101 Seed Source and Sward Composition ...... 102 Sward Composition and Insect Population D ensity ...... 103 Sward Composition and Insect Movement ...... 105 Sward Composition and Insect Damage ...... 105 Conclusions ...... 107

via LIST OF REFERENCES...... 110

APPENDIX A. Detection of /o//7 Using TPIB ...... 123

APPENDIX B. Overseeding Data ...... 125

APPENDIX C. Billbug Populations in L perenne Monocultures Data ...... 127

APPENDIX D. Adult Male Billbug Longevity D a ta ...... 129

APPENDIX E. Adult Male Billbug Feeding Preference Data ...... 131

APPENDIX F. Bluegrass Webworm Non-Choice Data ...... 136

APPENDIX G. Bluegrass Webworm Choice Data ...... 140

APPENDIX H. Bluegrass Webworm Survival Data ...... 141

APPENDIX I. Bluegrass Webworm Emigration D ata ...... 143

IX LIST OF TABLES

TABLE PAGE

1. adversely affected by endophytes in perennial ryegrass and tall fescue...... 32

2. Parameter estimates and their standard errors resulting from segmented regression analysis of S. parvulus larval population density on the proportion of endophytic tillers in mixed stands of perennial ryegrass and Kentucky bluegrass ...... 50

3. Parameter estimates and their standard errors for segmented regression of S. parvulus larval population density on the proportion of endophytic tillers in pure stands of perennial ryegrass ...... 71

4. Survival to pupation and fresh pupal mass of P. teterrella larvae fed clippings of endophytic (PR+) and non-endophytic (PR-) perennial ryegrass, and Kentucky bluegrass (KB) in the laboratory, and survival of larvae after 30 d on PR+, PR-, and KB in pots placed outside ...... 94

5. Parameter estimates and standard errors for the segmented regression model describing the relationship between P. teterrella larval population density and the proportion of endophytic perennial ryegrass tillers in mixed stands with Kentucky bluegrass ...... 95 LIST OF FIGURES

FIGURE PAGE

1. Hypothetical model of endophjde-plant-insect interactions ...... 33

2. Components of the endophyte-plant-insect interactions model addressed in the current series of studies ...... 34

3. S. parvulus larval population densities in Kentucky bluegrass plots treated in 4 different manners (control, overseeded with endophytic perennial ryegrass at 48.8 and 97.6 kg/ha, and overseeded at 48.8 kg/ha+imidacloprid insecticide). Overseeding performed October 1995 and imidacloprid applied May 1996 and 1997. S. parvulus samples taken July 1996 and 1997 (point=mean, box=standard error, whiskers=standard deviation) ...... 51

4. Scatter plot of the relationship between the percentage of endophyte infected perennial ryegrass tillers (n=48) and S. parvulus larval population density in mixed stands of Kentucky bluegrass and perennial ryegrass. Endophyte infection calculated from 40 perennial ryegrass tillers taken randomly from each plot and multiplied by the total proportion of perennial ryegrass determined from 200 tillers taken randomly from 4-900cm" areas within each plot. Line represents result of segmented regression analysis with inflection point at arrow ...... 52

5. Visual rating of S. parvulus larval damage (10=no damage, l=complete damage) during July 1997 in plots of Kentucky bluegrass treated in 4 different manners (control, overseeded with endophytic perennial ryegrass at 48.8 and 97.6 kg/ha, and overseeded at 48.8 kg/ha+imidacloprid insecticide). Overseeding performed October 1995 using two different seed sources (Repell 11™ and Triple Play™) and imidacloprid applied May 1996 and 1997 (point=mean, box=standard error, whiskers=standard deviation) ...... 53

XI 6. Regression of S. parvulus \asual damage rating on S. parvulus larval population density (July 1997) in stands of Kentucky bluegrass overseeded (October 1995) with two sources of endophytic perennial ryegrass (Repell n™ and Triple Play™ ) ...... 54

7. The proportion of perennial ryegrass tillers evaluated each June and October (1996 and 1997) in plots of Kentucky bluegrass overseeded (October 1995) with two sources of perennial ryegrass (Repell IT™ and Triple Play™) as determined from 200 tillers taken randomly from 4- 900cm" areas within each plot (point=mean, box=standard error. whiskers=standard deviation) ...... 55

8. The proportion of perennial ryegrass tillers in plots of Kentucky bluegrass treated in 4 different manners (control, overseeded with endophytic perennial ryegrass at 48.8 and 97.6 kg/ha, and overseeded at 48.8 kg/ha+imidacloprid insecticide). Overseeding performed October 1995 and imidacloprid applied May 1996 and 1997 (point=mean, box=standard error, whiskers=standard deviation) ...... 56

9. Components of the endophyte-plant-insect interactions model addressed in Chapter 2 (See Chapter 1, Model Description for details)...... 57

10. Mean number (bar) ± SE (whiskers) of feeding punctures per adult male S. parvulus per day on endophytic perennial ryegrass (PR+), non- endophytic perennial ryegrass (PR-), and Kentucky bluegrass (KB) in non-choice feeding assays (bar=mean. whiskers=standard e rro r) ...... 72

11. Relationship between number of adult male S. parvulus feeding punctures and tiller diameter using 3 types of grass, (endophytic (PR+) and non- endophytic (PR-) perennial ryegrass, Kentucky bluegrass (KB)) ...... 73

12. Mean (point)±SE (box) and SD (whiskers) tiller diameters of endophytic (PR+) and non-endophytic (PR-) perennial ryegrass, and Kentucky bluegrass (KB) obtained from greenhouse grown fla ts ...... 74

13. Mean (point)±SE (box) and SD (whiskers) survival time of field collected adult male S. parvulus on endophytic (PR+) and non-endophytic (PR-) perennial ryegrass, Kentucky bluegrass (KB), and 3 different mixtures of PR+ and KB (40, 60, and 80% P R + ) ...... 75

14. S. parvulus larval population densities in plots of Kentucky bluegrass (KB) and perennial ryegrass (PR) containing different proportions of endophytic tillers (July 1995)...... 76

XU 15. Mean (point)±SE (box) and SD (whiskers) S. parvulus larval population density in plots of Kentucky bluegrass (KB) and perennial ryegrass (PR) with ^ 15% endophytic tillers ...... 77

16. Scatter plot of the relationship between the percentage of endophyte infected perennial ryegrass tillers and S. parvulus larval population density in pure stands of perennial ryegrass. Endophyte infection calculated from 40 perennial ryegrass tillers taken randomly from each plot. Line represents result of segmented regression analysis ...... 78

17. Components of the endophyte-plant-insect interactions model addressed in Chapter 3 (See Chapter 1, Model Description for details) ...... 79

18. Mean (point)±SE (box) and SD (whiskers) proportion of larval P. teterrella on endophytic (PR+) and non-endophytic (PR-) perennial ryegrass and Kentucky bluegrass (KB) after 24 h in choice tests ...... 96

19. Mean (point)±SE (box) and SD (whiskers) survival of larval P. teterrella on endophytic (PR+) and non-endophytic (PR-) perennial ryegrass and Kentucky bluegrass (KB) at 7, 14, 21, 28, 35, and 42 d in the lab ...... 97

20. Frequency scatter plot of the relationship between the percentage of endophyte infected perennial ryegrass plants in pots containing mixtures of Kentucky bluegrass and perennial ryegrass, and the percentage of P. teterrella larvae recovered from the pots after 30 d. Line represents result of segmented regression analysis ...... 98

21. Mean (point)±SE (whiskers) cumulative proportion of P. teterrella larvae (n=60) emigrating from pots containing endophytic perennial ryegrass (PR+), Kentucky bluegrass (KB) or a mixture of PR+ and KB in the greenhouse over the course of 40 d ...... 99

22. Components of the endophyte-plant-insect interactions model addressed in Chapter 4 (See Chapter 1, Model Description for details) ...... 100

23. Revised hypothetical model of endophyte-plant-insect interactions based on the literature and the current series of investigations (See Chapter 1. Model Description for details) ...... 109

X lll CHAPTER 1

INTRODUCTION

Certain species of ryegrass {Lolium) and fescue (Festuca) have long been known to harbor fungal endophytes (Freeman 1904; Sampson 1935; Neill 1940. 1941). The original impetus for studies on the relationship between these fungal endophytes and their grass hosts was centered around livestock toxicities (e.g., staggers) associated with cattle and sheep grazing on endophyte infected forage grasses (Bacon et al. 1977;

Fletcher and Harvey 1981; Fletcher 1982; Schmidt et al. 1982). This research focused mainly on how to detect and eliminate fungal endophytes. Only since the association of insect resistance with endophyte infected grasses (Prestidge et al. 1982) have endophytes been considered beneficial because of their possible use for insect pest management.

Although the ability of fungal endophytes to reduce insect populations and damage has been demonstrated, the relationship between insect population density and the proportion of endophytic plants in a stand of turfgrass is unknown. Likewise, it is unclear how insects may influence patterns of change in the composition of turfgrass swards containing endophytic plants. 1 developed a hypothetical model of a turfgrass system and used it as an experimental template for addressing endophyte-plant-insect interactions in turfgrass (Figure 1). Experiments were developed to address some of the key components of this model. The relationship between insect population density and the proportion of endophyte infected grass was investigated and the influence of insect damage on sward composition was evaluated.

These experiments provide information on how introductions of endophytic grasses into established stands changes insect pest populations and their damage, and how insect pests change the composition of turfgrass swards over time.

Definition of Turfgrass Arena to be Studied

Turfgrasses are among the most common components of urban ecosystems.

Recent surveys indicate turfgrass occupies more than 10 million hectares of land area in the United States alone (Gibb and Buhler 1995) and this number continues to increase with population growth (Waddington et al. 1992). Turfgrasses can provide a number of important ecological, functional and aesthetic benefits including erosion control and soil improvement, noise abatement and temperature moderation, water infiltration and recharge as well as reduced athletic injuries and improved mental health (Beard and

Green 1994). Pest management in this system has traditionally relied heavily on the use of chemical pesticides. However, concerns about environmental and human health risks, loss of pesticide registrations and problems associated with the overuse of pesticides have increased receptiveness to management alternatives and increased research activity on biological and cultural management tools (Braman 1995).

General recreation/home lawn turfgrass systems with moderate maintenance inputs represent about 90% of the total turfgrass area in the United States. Approximately $10 million is spent annually to maintain these areas (Gibb and Buhler 1995) and a substantial portion of this budget is spent on management (Danneberger 1993).

This is the experimental arena chosen for the present study because it is the most obvious scenario for use of endophytic plants. Endophytic grasses may provide a cost effective and safe cultural management alternative suitable for use in this system.

Economically Important Insects

Two economically important insects, were used in the following studies. The bluegrass billbug, Sphenophorus parvulus GyllenhaI,(Coleoptera: ), and bluegrass webworm, Parapediasia teterrella (Zincken) (: Pyraiidae), are important pests in areas of the United States where cooi-season turfgrasses are grown

(Tashiro 1987, Brandenburg and Villani 1995, Watschke et al. 1995). Because these insects feed mainly on the aerial portions of grass plants, where endophyte mycelia and metabolites are concentrated, they are generally more susceptible to endophyte-mediated resistance (Johnson-Cicalese and White 1990. Kanda et al. 1994). Because adult and larval feeding methods and reproductive strategies of these insects are very different, they can serve as models to determine similarities and differences in their reactions to populations of endophytic tillers in swards of turfgrass.

Sphenophorus parvulus

S. parvulus usually has 1 generation per year in Ohio (Niemczyk and Joyner 1982,

Watschke et al. 1995), but a partial 2nd generation has been implicated by relatively high larval population densities during mid-September in southern Ohio (Niemczyk and Frost

1978). Survival of 2nd generation larvae has not been thoroughly investigated. These insects overwinter as adults in the thatch or other protected areas such as building perimeters (Harry D. Niemczyk, pers. com.). When spring soil temperatures rise above

18.3°C, adults become active, wandering about in search of suitable food plants

(Watschke et al. 1995). After a short period of feeding, females begin to deposit eggs, singly or in groups of 2 or 3, into feeding holes in individual grass stems. Depending on temperature, eggs hatch in about 6 d and the young larvae begin to tunnel up and down the interior of the stem. When the larvae have consumed the contents of the stem or have become to large to remain inside, they exit to continue to feeding on the crown and roots of grass plants. After 35 to 55 d, larval development is completed and pupation takes place in the soil. Pupal development is completed in 8 to 10 d and the tineral adult emerges. These adults feed briefly before seeking out suitable overwintering sites.

Damage from larval S. parvulus feeding is most obvious in late June and July.

Whereas adult billbugs do little damage to turf, larval damage results mostly from feeding on the roots and crowns of plants. In high maintenance turfgrass, light infestations can result in small dead spots which resemble dollar spot disease (Sclerotinia homoeocarpa

F. T. Bennett) (Shetlar 1995). More severe infestations are capable of causing thinning or complete destruction of the turf. Parapediasia teterrella

P. teterrella normally has 2 generations per year in most of the mid-western U.S.

(Watschke et al. 1995). Larvae overwinter in the thatch or soil in silk-lined chambers.

The larvae feed in April and early May and most have pupated by mid- to late May.

Pupae take between 5 and 15 d to mature. Adults emerge at night, mate and begin laying eggs the night after mating, depositing as many as 200 eggs. Eggs are dropped into the turf canopy as the females . Eggs are not attached to any substrate. After about

5 to 6 d at 21.1 °C, the eggs hatch and the 1st instar larvae immediately seek out a suitable host plant where they begin to spin webbing along a leaf blade (Ainslie 1922). The young larvae feed on leaf tissues under the protection of this webbing imtil they have molted once or twice. At this point the larvae drop to the ground where they form a tube-like silken tunnel in the thatch. Older larvae feed at night by clipping blades and stems of grass located near the opening of their silken webbing. Larvae take approximately 40 to

45 d to mature. First generation larvae feed in June and July whereas 2nd generation larvae, which partially mature in the fall, dig deeper into the thatch or soil to overwinter.

In temperate regions of North America, P. teterrella larvae cause injury to turfgrasses in a number of settings ranging from home to golf courses (Shurtleff et al. 1987, Watschke et al. 1995). In closely mown, golf course turf, damage mainly results from birds foraging for P. teterrella larvae (Harry D. Niemczyk, pers. com.). As the larvae grow, high populations may cause irregularly shaped areas of dead or dying turf

(Watschke et al. 1995). Damage is rare in home lawns and higher cut turf areas but, high larval populations may damage turf quality (Watschke et al. 1995). Endophytic fungi

Clavicipitaceous fungi, belonging to the tribe Balansiae, systemicaily infect host plants in 3 plant families, the , Cyperaceae and Juncaceae (Clay 1989). Most of these hosts are grasses. There are 5 genera and approximately 30 species in the tribe

(Diehl 1950, Luttrell and Bacon 1977). The genera are distinguished primarily on the basis of conidial fruiting structures which may be microconidial, macroconidial, both or neither (White 1994). Fungus-host interactions may further be categorized as epibiotic or endophytic, throughout the plant (systemic) or localized, moderately to strongly pathogenic or mutualistic (Glenn et al. 1996). A progression in the reproductive cycle of these fungi can be observed from those which fruit on their hosts and produce ascospores and conidia to those which never fruit and are transmitted maternally through the plant hosts seed (Clay 1989). Reproductive variation among these fungi may represent an evolutionary trend toward reduced complexity in their life stages and a greater dependence on their host for dissemination (Clay 1988).

Fungi in the genus Neotyphodium {—Acremonium) are endophytic, systemic, seed- bom mutualists of grasses. These fungi produce no conidia and offer the greatest potential for economic use because plant quality is not reduced by infection. Two of the most important turfgrass species infected with Neotyphodium endophytes are tall fescue,

Festuca arundinacea Schreb., infected with Neotyphodium coenophialum (Morgan-Jones

& Gams) and perennial ryegrass, Lolium perenne L., infected with Neotyphodium lolii

(Latch, Christensen & Samuels). The association of insect resistance with endophyte infected grasses was 1st

reported by Prestidge et ai. (1982) after substantial differences in Argentine stem weevil.

Listronotus bonariensis (Kuschel), populations and damage levels were observed

between endophyte-free and endophyte-infected plots of perennial ryegrass. This initial

observation of protection against insect herbivory, provided to a grass by its endophyte.

aroused interest and laid the foundation for subsequent observations of this phenomenon.

As a measure of the spectrum of activity endophytic plants posses. Table I contains 40

insect species which are adversely affected by the presence of endophytic fungi in

perennial ryegrass and tall fescue.

Role of Endophytes in Host Plant Resistance

Endophyte-mediated resistance to insects encompasses all 3 major modalities of

host-plant resistance; antixenosis, antibiosis and tolerance. Antixenosis may be broadly

defined as plant factors that alter insect behavior in a way that is favorable for the plant,

whereas antibiosis describes the negative influence of a resistant plant on insect biology

(Smith 1989). Tolerance has little to do with any effect on the insect but rather describes

the ability of a plant to outgrow or recover from insect damage (Smith 1989).

Antixenosis against the Argentine stem weevil has been documented in perennial ryegrass infected with Neotyphodium lolii. Antixenosis was expressed in the form of deterred feeding (Prestidge et al. 1982) and deterred oviposition (Barker et al. 1984).

Additionally, many species o f aphids are deterred from feeding on endophytic plants of perennial ryegrass and tall fescue (Latch 1993, Breen 1992). Other examples of endophyte mediated resistance fail under the category of antibiosis. Latch et al. (1985) found that aphids feeding on tall fescue infected with N. coenophialum had much higher mortality rates than those feeding on uninfected conspecifics. Fall armyworms, Spodoptera frugiperda (J. E. Smith), reared on endophyte-infected perennial ryegrass and tall fescue showed decreased larval weights and delayed development when compared to those reared on uninfected plants (Clay et al.

1985). Johnson-Cicalese and Funk (1988) found little difference in adult S. parvulus feeding on endophyte-infected and uninfected perennial ryegrass but did observe significantly greater mortality on infected grass. Similar results were obtained by Ahmad et al. (1985) using house crickets fed on endophyte-infected perennial ryegrass. These results are consistent with the action of an antibiotic mechanism.

The 3rd form of resistance displayed by endophytic turfgrasses is tolerance. Much of the resistance in this form can be attributed to the influence endophytes have on the growth of their hosts. Latch et al. (1985) found that perennial ryegrass infected with N. lolii grew more vigorously and produced more foliage than uninfected plants under controlled conditions. Under extreme pressure from the fall armyworm, endophytic perennial ryegrass produced twice as much biomass as non-endophytic plants (Clay et al.

1993). Clay (1987) showed that endophytic perennial ryegrass and tall fescue had consistently higher germination rates than uninfected plants and that endophyte-infected tall fescue plants set significantly more seed than uninfected plants. Stands of endophytic grass will therefore persist and recover more rapidly from defoliation. Chemicals Associated with Fungal Endophytes

The chemical basis for endophyte-mediated resistance is the production of alkaloids within the plant (Porter 1994). Because the fungal mycelia are generally confined to aerial portions of the plant, most of the toxins associated with insect resistance are also located there. However, some toxins are apparently translocated to plant roots (Siegel et al. 1989). In endophyte infected perennial ryegrass, 3 classes of fungal metabolites are mainly responsible for insect resistence: the indole diterpenes

(Betina 1984), the ergot alkaloids (Rutschmann and Stadler 1978), and peramine (Rowan et al. 1986).

A) The indole diterpenes identified from endophytic perennial ryegrass include the lolitrems A-E, lolitriol and paxilline (Gallagher et al. 1984; Miles et al. 1992, 1993;

Weedon and Mantle 1987). Paxilline and lolitrems are potent, tremorgenic neurotoxins in mice and sheep (Gallagher and Hawkes 1986), and deter feeding by the Argentine stem weevil (Popay et al. 1990, Prestidge and Gallagher 1988).

B) The ergot alkaloids present in endophytic perennial ryegrass can be divided into 3 structural classes: ergopeptine alkaloids, clavine alkaloids, and lysergic acid derivatives (Rutschmann and Stadler 1978). Whereas approximately 6 ergopeptine alkaloids have been identified in perennial ryegrass, ergovaline is the most prevalent

(Porter 1994). The biological activity of erogpeptine alkaloids has been demonstrated against the fall armyworm larvae, Argentine stem weevil adults and Japanese grubs

Popillia japonica Newman (Clay and Cheplick 1989, Dymock et al. 1989, Patterson et al.

1991) and is toxic to livestock at concentrations normally encountered in the field (Rowan and Latch 1994). The clavine alkaloids, agroclavine and elymoclavine, reduced feeding and growth of the fall armyworm (Clay and Cheplick 1989). The lysergic acid derivatives are considered minor mycotoxins because they have only been reported from endophytes grown in culture (Lyons et al. 1986).

C) Peramine occurs widely in endophyte infected grasses and seems to be important in the resistance of ryegrass to the Argentine stem weevil, sod webworms

(Pyralidae:Crambinae) and the greenbug aphid, Schizaphis graminum (Rondani), (Siegel et al. 1990). Although peramine is a strong feeding deterrent to the Argentine stem weevil, it apparently has low chronic toxicity to both the weevil and to mice (Rowan et al.

1986). Peramine has been implicated as a marker enabling Argentine stem weevil to avoid other more potent endophytic toxins (Rowan and Latch 1994).

Within endophytic plants, alkaloid production may vary seasonally, and is changed by plant nutrition, temperature, and rainfall. Belesky et al. ( 1987) observed increased concentrations of ergovaline in tall fescue fertilized at a rate of 336 kg N/ha compared to tall fescue fertilized at 134 kg N/ha. They also noted a strong tendency for ergovaline concentrations to be higher in the spring and fall. However, developmental rates of insects reared on endophytic tall fescue fertilized at 150 and 300 kg/ha/y were unchanged or slightly increased in comparison to insects reared on unfertilized endophytic tall fescue (Davidson and Potter 1995). The preference of greenbug aphids for endophyte-free perennial ryegrass was greater at 14 and 21 °C than at 7 or 28°C.

These temperatures were correlated with peramine production by endophytic plants

10 (Breen 1992). The ability of endophytic plants to reduce insect damage in swards of turf may therefor depend on turfgrass management practices.

The endophyte, its host, and their interaction can determine the types and concentration of alkaloids produced. Individual endophyte isolates which produce neither peramine, lolitrems nor ergovaline have been found in perennial ryegrass (Rowan and

Latch 1994). Roylance et al.(1994) were able to extract, culture and reintroduce endophyte strains into hosts with different genomes. They demonstrated that peramine and ergovaline production were independently regulated and controlled by both plant and endophyte genotype. Johnson-Cicalese et al. (1998) reported variation in chinch bug resistance among host grasses inoculated with 3 different endophytes, demonstrating an interaction between host plant and endophyte genotype. There are also endophyte strains known to be toxic to insects but not livestock (Steven L. Clement, pers. comm.).

Variability in the types and concentrations of alkaloids produced by different endophyte- plant combinations provides an infinite number of breeding possibilities. As a result, cultivars or blends of endophytic seed may vary in their resistance to insects even when similar proportions of seed contain viable endophyte. Ultimately, it may be possible to develop site specific, prescription grasses for turf or pasture use.

Influence of Insect Feeding on Plant Species Composition

Insect outbreaks influence plant species composition. The influence of insect feeding on plant establishment, growth, and reproductive success is well documented.

Louda (1983) found that seed predation by several insects was a major influence on the

11 ability of the temperate , Haploppapus squarrosns H. and A. (Asteraceae) to establish in southern California. Evidence from annual tree ring growth indicates long term, continuous feeding by a phytophagous insect guild has severely depressed growth and productivity in sub-alpine Eucalyptus forests (Morrow and LaMarche 1978). In

Grass and cereal crops, the stem boring activity of frit , L., often causes reduced seed yields (Guile 1981). Janzen (1969) reported decreases in the reproductive success of several Acacia species do to feeding by bruchid .

By altering plant growth, survival, and reproductive success, insects may change the competitiveness of certain species over time. McBrien et al. (1983) published the 1st study documenting the effect of insect herbivory on patterns of change in species composition of terrestrial plant communities, although previous authors had acknowledged this possibility (Connell and Slatyer 1977, White 1979). McBrien et al.

(1983) demonstrated reductions in percent goldenrod cover, Solidago canadensis L.

(Asteraceae), by 3 chrysomelid beetles, Trirhabda virgata, T. canadensis, and T. borealis

Le Conte, resulting in a return to dominance by earlier stage perennial grasses. Likewise.

Bach (1994) showed that selective feeding by the willow flea beetle, Altica subplicata, influenced sand dune succession in Michigan by decreasing growth and survivorship of sand-dune willow, Salix cordata. Reid and Harmsen further (1974) suggest that sublethal levels of defoliation could lead to a switch in the dominance hierarchy within a plant community. If this is true, then insects need not kill host plants in order to change the competitive balance between plants.

12 Classic examples of biological control of weeds further serve to illustrate the ability of insect attacks to influence the composition of vegetation. Within a 10 y period

(1930-1940) the Argentine , Cactoblastis cactorum (Berg), essentially eradicated the prickly pear cactus. Opuntia stricto, from Australia where it had become a major pest

(Huffaker 1958). The chrysomelid beetle, Chrysolina quadrigemina (Suffnan), was successful in reducing St. Johnswort, Hypericum perforatum L., infestations in California to 1% of its original abundance over a period of approximately 15 y (Huffaker 1958).

The proportion of endophyte infected grass plants in a stand may also change over time. In a survey of ryegrass endophytes in the United Kingdom, Lewis and Clements

(1986) found endophyte mycelia in 14 of 61 swards, 12 of which were 15 y or older.

Over the course of 3 y, Francis and Baird (1989) observed increases in the proportion of endophytic ryegrass plants from 3% to 83% even though Argentine stem weevil numbers were low. It has been argued that endophyte-induced changes in morphology and physiology influence vigor and persistence of infected plants independently of their resistance to insects (Clay 1989). Indeed, Neotyphodium endophytes enhance plant growth and vigor (Latch et al. 1985), germination and seed set (Clay 1987), as well as drought stress tolerance (Belesky et al. 1987, Siegel et al. 1987, West et al. 1987).

However, Clay et al. ( 1994) found that endophytic perennial ryegrass produced twice the biomass of non-endophytic ryegrass in the presence of extremely high fall armyworm populations (>8,000/m‘) even though biomass of the 2 grasses was very similar when fall armyworm was absent. These later findings suggest an alteration of the competitive hierarchies among grasses due to insect feeding and fungal endophyte infection. It is

13 likely that insect attack enhances the competitive advantage to endophytic plants thereby causing change in the composition of the turfgrass plant communities. However, studies are needed to address this question on a larger scale and under normal field conditions.

Other Factors Influencing Turfgrass Sward Composition

Aside from insect feeding, a variety of biotic and abiotic factors may influence the composition of turfgrass swards. Although not all of these factors can actually be manipulated, irrigation, fertility, soil pH and mowing are common cultural management concerns capable of influencing competition between plants. Inherent differences in stress tolerance and vigor between cultivars of the same species implies that cultivar selection may also influence sward composition.

Water is essential to plant growth and survival. Water-use rate, typically measured as évapotranspiration (mm/day = total water required for growth + amount transpired from grass plant + amount evaporating from soil surface), is an important indicator of the water requirements of turfgrass (Danneberger 1993). Water-use rates vary considerably among turfgrass species and among cultivars of the same species.

Beard (1986) categorized évapotranspiration rates of 18 major turfgrass species with

Kentucky bluegrass and tall fescue ranking higher than perennial ryegrass in potential

évapotranspiration. Further, évapotranspiration rates may differ as much as 64% among cultivars of Kentucky bluegrass (Shearman 1986). However, water use rates are not necessarily a good indicator of drought tolerance. Although tall fescue has a high

évapotranspiration rate, it is considered drought tolerant due to its deep root system

14 (Danneberger 1993). As an alternative, Aronson et ai. (1987) measured drought tolerance gravimetrically. They observed decreased plant water potentials of 50-75% in Kentucky bluegrass and perennial ryegrass at soil water potentials of -80 KPa, whereas red and chewings fescue remained unaffected until a soil water potential of -400 KPa was achieved.

Because of variability in nutrient requirements among turfgrasses (Beard 1973), fertility practices, particularly nitrogen fertility, can also influence sward composition. In a 3 y study, withholding nitrogen fertilization gave creeping bentgrass a competitive advantage over annual bluegrass, resulting in an increase in the proportion of creeping bentgrass in experimental plots (Dest and Guillard 1987). Likewise, Williams (1984) reported decreases in the proportion of red fescue in fertilized grassland plots compared to unfertilized controls. Differences in nitrogen requirements have also been observed between cultivar of the same species. Nitrogen requirements between cultivars of

Kentucky bluegrass can differ by up to 50% (Beard 1973).

The influence of soil pH on turfgrass performance has been reported in many studies. Soil pH may influence plants directly through altering the availability of essential and or toxic elements, or indirectly through changing soil microbiology (Beard

1973). Fertility practices, particularly the application of NH^-based fertilizers, can also change soil pH (Turner and Hummel 1992) which can, in turn, influence sward composition. Studies indicate that tall fescue is more tolerant of acid soils than Kentucky bluegrass (Murray and Foy 1980) but more sensitive to those same soils than the fine fescues (Murray and Foy 1978). Turfgrass mixtures can also be effected by soil pH.

15 Musser (1948) observed higher proportions of Kentucky bluegrass, in mixtures with creeping bentgrass, under low soil acidity than under higher soil acidity.

Mowing can also influence sward composition because plant responses to mowing varies across species (Watchke and Schmidt 1992). Most studies have focused on differences in plant tolerance to mowing height although mowing frequency can also influence plant competition. The influence of mowing height on stand composition are generally attributed to plant growth characteristics (Beard 1973). For example, Davis

(1958) found that close mowing enhanced invasion by prostrate growing annual bluegrass into Kentucky bluegrass turf. Conversely, Madison (1962) observed that bermudagrass invaded tall fescue more slowly at lower mowing heights because the stolons of the bermudagrass plants were removed. With regard to mowing frequency, Haley et al.

(1981) found that more frequent mowing (twice a week) resulted in a higher quality

Kentucky bluegrass turf under moderate fertility.

Within a turfgrass species, different cultivars can vary considerably in their competitive abilities. As previously stated, évapotranspiration rates (Shearman 1986) and nitrogen requirements (Beard 1973) can vary considerably between cultivars. Reinert and

Busey (1983) found differences in damage were observed between Bermudagrass selections even though tropical sod webworm, Herpatogramma phaeopteralis Guenée, population densities were very similar. Differences in water-use rates, drought tolerance, fertility and pH requirements, responses to mowing height and frequency, and expression of damage among turfgrass species and cultivars means that management practices and

16 cultivar selection can determine the competitive hierarchy among plants and influence sward composition.

Vegetation Texture

The spatial relationship between host and non-host plants (e.g., adjacent neighbors, spacing, relative heights, and architecture) is referred to as vegetation texture

(Kareiva 1983). Studies have suggested that host plants surrounded by plants of another species are less attractive to specialist herbivores. Root (1973) found that "insect herbivore loads" were consistently higher in pure stands of collards than in collards planted among diverse meadow vegetation. Likewise, Horn (1981) reported lower populations of the green peach aphid, Myzus persicae (Sulzer), in weedy patches of collards than in mowed or tilled patches. Such "associational resistance," sensu

Tahvanainen and Root (1972), to insect pests has caused agricultural scientists to note the potential for controlling insect pests by manipulating cropping patterns (Cromartie 1981.

Hare 1983).

Two hypotheses have been proposed to account for the relationship between insect outbreaks and vegetation texture (Root 1973). The "enemies hypothesis" predicts predatory insects and parasitoids will be more effective in controlling herbivorous insects in more vegetationally diverse systems. A variety of mechanisms (e.g., providing harborage, alternate food sources) have been hypothesized and reviewed in this regard

(Russell 1989, Sheenan 1986). However, experimental results on the subject have shown a high degree of dependence on the specific systems and pests studied. Speight and

17 Lawton (1976) found higher numbers of carabid beetles and higher predation rates in fields where annual bluegrass, Poa annua L., was abundant than in areas where it was scarce. They postulated that P. annua provided refuge for the beetles. More recently, Hickman et al. (1995) found syrphid larvae provided more efficient biological control of aphids in cereal fields where the flowering plant, Phacelia tanacetifolia

Bentham (Hydrophyllaceae), was present as a source of pollen for adult syrphids.

Conversely, Andow and Risch (1985) found higher densities of the predacious coccinellid, Coleomegilla maculata De Geer, in monocultures than in maize-bean- squash polycultures. C maculata had a higher foraging success rate and stayed in monocultures longer than polycultures. In Russell’s (1989) review of the enemies hypothesis, 9 studies reported higher mortality from predation or parasitism in polycultures, 2 reported lower mortality rates and 2 reported no difference. But. most studies on the enemies hypothesis only show correlations between vegetation texture and the population density of natural enemies. Actual mortality rates due to natural enemies have been measured in only a few cases (Kareiva 1983).

The 2nd hypothesis, "the resource concentration hypothesis," predicts that insects with a narrow host range are more likely to find and remain in areas where their resources

(i.e.. host plants) are in denser, purer, or larger patches (Root 1973). This hypothesis is similar to a related view from island biogeography which considers the number of insects on a given plant as a balance between immigration and emigration rates and the association of these rates with factors such as host patch size and degree of isolation

(Janzen 1968). Experimental evidence supports the assertion that habitat texture

18 influences herbivore densities by altering movement and searching behavior (Kareiva

1983). Bach (1980) showed differences in cucumber beetle, Acalymma vittata (Fab.), population density, between cucumbers in polycultures and monocultures, were related to how long they stayed in a patch, and movement patterns among plots, rather than colonization, reproduction or predation. She later found similar results in a natural plant community using a closely related beetle, Acalymma innubum (Fab.), and its host.

Cayaponia americana, in open and forested patches (Bach 1984).

Diverse vegetation may simply make host plants difficult for insects to find. This may, in turn, cause an increase in mortality while searching for food. Kemp and

Simmons (1979) argued that 2nd instar spruce budworms, Choristoneura fumiferana

(Clem.), suffer high mortality during dispersal because finite energy reserves provide little time to find a suitable host. Emerging larvae must find food quickly or perish in the process of searching. However, the act of foraging itself has risks. Bergelson and

Lawton (1988) demonstrated that larvae of the geometrid moth, Apocheima pilosaria

Denis and Schiff, were more vulnerable to predation by ants when forced to move in search of food. Martson et al. (1978) showed that caterpillar movement increased spined soldier bug, Podisus maculiventris (Say), predation.

Vegetation texture may also influence insect population density by altering the suitability of individual plants for insect growth and reproduction. Dalrymple and Rogers

(1983) found western ragweed. Ambrosia psilostachya DC., root extracts reduced shoot and root growth of 16 other plant species by an average of 56.8% indicating a chemical basis for allelopathy. However, Sturz et al. (1998) showed that bacterial endophyte

19 associations in ciover-potato crop rotations actually increased shoot height and wet weight as well as root wet weight of potato plants. Bach (1980) found leaf area, growth rate and vine length of cucumber plants were reduced in polycultures compared to monocultures. She further found that for plots with equal amounts of leaf area monocultures had significantly more cucumber beetles than polycultures. These results suggest host plant quality can differ between monocultures and polycultures not only because of allelopathic activity but, also from competitive or beneficial plant interactions.

The influence of host plant quality on insect feeding and development has been investigated by many authors and has yielded mixed results. Spodoptera pectinicornis

(Hampson) had greater pupal mass when fed the floating aquatic weed, Pistia stratiotes

L. (Araceae), grown under a higher fertility regime; and, females were more likely to lay eggs singly, rather than in groups, when fed plants from a low fertility regime (Wheeler et al. 1998). Wheeler et al. (1998) speculated that the increased percentage of solitary eggs laid by females from the low quality larval diet may be an adaptive response to decrease competition among progeny. Conversely, Hacker and Bermess (1995) found high populations of the salt marsh aphid, Uroleucon ambrosiae (Thomas), on shorter, less healthy host plants, Iva frutescens L., whereas taller, healthier plants had fewer aphids.

Interestingly, this difference was explained by the fact that predacious coccinellids landed on tall plants more often than shorter plants thereby excluding aphids from tall plants and restricting them to low-quality resources.

There is obvious overlap between the "enemies hypothesis" and the "resource concentration hypothesis." Through altering movement patterns of insect herbivores,

20 diluted resources may enhance the activity of natural enemies. Indeed, several authors have acknowledged that these 2 hypotheses are not mutually exclusive (Andow 1991) and may be complimentary (Russell 1989). Insect populations are regulated by natural enemies as well as by the availability of host plants, and the relative importance of these two mechanisms varies.

Inter-planting endophytic and non-endophytic grasses has never been examined as an insect management strategy. However, based on the idea that mixtures provide a wider range of stress tolerances, inter-planting has been used as a strategy to enhance turfgrass persistence and durability. In theory, deterioration from disease and insects is less likely to occur in mixed stands than monocultures of turf (Beard 1973). Despite the lack of investigations of inter-planting endophytic and non-endophytic grasses, there is evidence implying that such a scheme may be amenable to pest management.

Studies have shown that endophytic grass plants alter insect movement and vulnerability to natural enemies and may also alter the quality of surrounding plants. For example, the argentine stem weevil is deterred from feeding on endophytic grasses by the fungal metabolite peramine and tends to seek out non-endophytic grasses (Rowan et al.

1986). Prolonged exposure to endophytic perennial ryegrass caused increased flight muscle development in adult Argentine stem weevil, possibly indicating a genetic predisposition to migrate from areas where endophytic plants are common (Barker et al.

1989). Grewal et al. (1995) demonstrated that Japanese beetle larvae. Popillia japonica

Newman, were more susceptible to infection by the entomopathogenic nematode.

Heterorhabditis bacteriophora Poinar, when feeding on endophyte infected tall fescue

21 and chewings fescue, Festuca rubra commutata Guad., than on either grass when endophyte free. Endophyte infection also enhances a plants ability to compete for available space, water and nutrients when in mixtures with other plants (Clay 1993).

Allelochemicals present in endophyte infected tall fescue seed caused reduced root hair length and density in several species of clover. Trifolium spp. (Springer 1996). Reduced root hair length and density can adversely influence a plant’s ability to compete for nutrients and water.

Because of the toxicity of alkaloids associated with endophyte infection, mixtures of endophytic and non-endophytic grasses may have additional effects on insect populations beyond the effects normally associated with polyculture. Insect that do not discriminate between resistant and susceptible plants are vulnerable to the influence of antibiosis resistance (e.g., effects on insect biology). The impact of antibiosis resistance on insect survival and fecundity has been demonstrated. For instance. Johnson-Cicalese and White (1990) did not find differences in feeding or oviposition by adult bluegrass billbugs on endophytic and non-endophytic tall fescue and perennial ryegrass, but adult mortality was much higher on both grasses when endophyte infected. Antibiotic effects of resistant tall wheatgrass, Agropyron elongatum (Host), on the Russian wheat aphid.

Diuraphis noxia (Mordvilko), included delayed reproductive maturity, shorter reproductive life-span, and reduced fecundity rates (Kindler et al. 1995). Similarly, antibiosis resistance in wild dry bean, Phaseolus vulgaris L., significantly reduced fecundity of FI female been weevils, subfasciatus (Boheman) and

Acanthoscelides obtectus (Say), raised on resistant varieties (Cardona et al. 1989).

22 Conversely, some insects can discriminate between endophyte-infected and endophyte-free plants. Breen (1992) demonstrated antixenosis (e.g., effects on insect behavior) to the green bug aphid, Schizaphis graminum (Rondani), in endophyte-infected perennial ryegrass, and Latch et al. (1985) showed the bird-cherry oat aphid,

Rhopalosiphum L.,padi avoided tall fescue infected with endophyte. In theory, insects capable of such discrimination would not suffer significant mortality due to defensive chemicals but rather, mortality would be related to increased movement and or vulnerability. However, the initiation of a behavioral responses (e.g., movement away from endophytic plants) usually requires acquisition of at least a low dose of toxic material (Hoy et al. 1998). Therefore, repeated sampling of toxic plant materials, which should be a function of the frequency of plants containing the toxin, could have a cumulative influence on herbivorous insects.

Mixtures of endophytic and non-endophytic grasses may therefore lower insect population density through increasing movement and susceptibility to attack by natural enemies or reducing the overall nutritional quality of surrounding plants. Additionally, because of the toxicity of alkaloids associated with endophyte infection, mixtures of endophytic and non-endophytic plants may lower insect population density by directly increasing mortality and or reducing fecundity.

Quantifying Turfgrass Sward Composition

A stand or sward of turfgrass may be characterized in several ways. Measures of turf biomass, density, and species composition are commonly used to describe turfgrass

23 swards and the strengths and weaknesses of different sampling techniques have been addressed by various authors (Mahdi and Stoutemyer 1953, Mitchell and Glenday 1958,

Lush ans Franz 1991). The most important attribute of a sampling method is that it provides an accurate picture in the context of the research problem being addressed.

The present series of studies were mainly concerned with the chance of insects encountering toxins produced by endophytic plants and how this probability changes over time. Because this is based on the insect’s perception of the habitat, sward composition was characterized in terms of the proportion (0-1.0) of tillers (stems, or shoots) representing a given grass species or type (Kentucky bluegrass, endophytic perennial ryegrass and non-endophytic perennial ryegrass). Stands of grass have been regarded as populations of tillers, each capable of growth and vegetative reproduction and with a finite life span (Mitchell and Glenday 1958). Not only is the tiller the vegetative unit of grasses, it serves mechanically as the feeding and or reproductive unit for several turfgrass insects. For instance, S. parvulus oviposits directly into individual tillers and larvae spend their 1st few instars feeding within these tillers (Shetlar 1995). Neonate P. teterrella larvae select individual tillers where they conceal themselves under a protective layer of silk on the stem, leaf, or leaf axial ( Ainslie 1922). Larger P. teterrella larvae often consume lower portions of plant tillers (Niemczyk pers. com.). Such tiller-centric behavior makes the tiller an obvious choice as the unit for sampling sward composition with respect to insect activity.

24 Model Description

The above literature review combined with basic ecological principles provides a foundation for a hypothetical model of how endophyte-plant-insect interactions might operate at a community level. Mixtures of endophytic and non-endophytic grasses should reduce insect herbivore populations and it is likely that insect feeding enhances the competitive advantage to endophytic plants. Therefore, this system may be thought of as a feedback system. The proportion of endophytic plants (as determined by sampling plant tillers) in a stand should dictate insect population density and the accumulation of damage. Because insect damage to non-endophytic plants is generally greater, shifts in sward composition toward more endophytic plants should occur, and this shift should occur more rapidly in areas where insect population densities are high.

Consequently, the interactions between fungal endophytes, plants, and insects may be generalized to fit a basic model (Figure 1). Five interacting variables were chosen to represent the state of the turfgrass-insect pest system investigated. These variables are called state variables because they are dynamic; their status changing according to other interacting forces in the system. State variables in the present system can be broadly grouped as those describing sward composition, and those describing insect population density and damage to the stand. The 1 st 3 state variables describe the relative proportion of the 3 main grasses present, endophytic and non-endophytic perennial ryegrass (PR+ and PR- respectively), and Kentucky bluegrass (KB). Another state variable describes insect population density and a final variable describes plant material damaged by insect feeding. Values for the grass state variables are based strictly on the proportion of tillers

25 representing each grass species and must therefore sum to one. Insect population density

is the number of insects per unit area sampled. Although damaged grass could be directly measured as biomass removed per unit area, it was estimated visually in the current study

(see Rating in the model). The status of these variables was important for testing hypotheses about the influence of various experimental treatments.

State variables are dynamic and may fluctuate according to biotic and abiotic

factors, including other state variables. Factors that cause a state variable to increase or decrease can be combined to form rate equations. These rate equations may be thought of as valves that determine the rate material (e.g., insect larvae, grass tillers) enters or leaves the system. Auxiliary variables are factors that do not directly interact with the system but can influence the rate equations (e.g., SR = seeding rate) or may calculated from a state variable (e.g.. Rating as an estimate of actual insect damage)

The most fundamental process in this model is the initial act of modifying the turfgrass stand by introducing endophytic plants (SR). In a typical management scenario, this would involve overseeding endophytic grass into an existing stand. Complete renovation from bare ground is another option but somewhat less viable because of expense and acceptability to the turf owner/manager. The resulting composition of an overseeded sward will likely depend on the amount o f seed introduced (kg/ha) (hereafter referred to in the term commonly used by turfgrass managers - seeding rate). However, the cultivar(s) used, the proportion of seed containing viable endophyte (% E+ seed), the existing species composition and cultural management (fertility, H^O. pH, and mowing) may also be important.

26 The proportion of endophytic plants should influence insect population density through I or more of the mechanisms previously discussed (emigration (Em),

immigration (Im), fecundity, and mortality). However, because management practices

(fertility and water), and the cultivar used can influence the types and concentrations of alkaloids produced, they may mediate the effects of endophytes on insect population density. Regardless of the mechanisms at work, it is likely that mixtures of endophytic and non-endophytic grasses will support fewer foliage feeding pests than pure stands of non-endophytic grass.

Endophytic plants likely suffer less damage due to insect herbivory than non- endophytic plants (See Figure I : insect damage information flow #4 to KB and PR-).

Although, endophytic plants may be more vigorous than non-endophytic plants in the absence of insect feeding, it is likely that strong, anti-herbivore defenses will provide an even greater benefit to endophytic grasses in communities where insect pressure is high.

Because endophytic turfgrasses posses anti-herbivore defenses, differential insect feeding should further alter the competitive hierarchy between endophytic and non-endophytic grasses. The rate at which endophytic grasses increase should reflect the competitive abilities of the different grasses in a sward under existing insect densities.

In the present turfgrass system, it is hypothesized the seeding rate (kg/ha) will influence sward composition only during the establishment event. However, because the seeding rate (SR), the percentage of seed containing viable endophyte (%E+ seed), management practices (fertility, H^O, pH, mowing) and the cultivar(s) used for seeding can influence plant competition, they may influence sward composition over time. Insect

27 population density will be a function of the proportion of PR+ which may influence insect

fecimdit>% mortality, emigration or immigration (rate equations B. Mort., Em, and Im respectively). Because cultivar, fertility, and water (HjO) can influence the types and concentrations of alkaloids produced, they may mediate the relationship between insect population density and the proportion of PR+ in the sward. Because different grass species may be more or less suitable to insects, the relationship between insect population density and the proportion o f PR+ may depend the species composition of the sward.

It is assumed that insect damage will be a function of insect population density (see rate equation D in Figure 1). However, damage may vary among turfgrass species and cultivars, and with fertility and water availability. Because the competitive interactions between grasses may depend on the amount of insect damage accrued, insect feeding may alter sward composition.

Study Objectives

A subset of the interactions represented in Figure 1 were investigated in the current series of studies (Figure 2). The ultimate goal of this research was a practical strategy for using endophyte-mediated resistance to manage epigeal insects in turfgrass.

Therefore, experiments were conducted to determine if overseeding endophyte infected perennial ryegrass, Lolium perenne L., into existing stands of Kentucky bluegrass. Poa pratensis L., would confer resistance to common insect pests. The influence of overseeding on sward composition was investigated to determine the relationship between insect population density and the proportion of endophyte infected tillers.

28 Insects were excluded from selected overseeded plots to determine if insect feeding could influence sward composition. The relationship between insect population density and the proportion o f endophytic tillers was also measured in pure stands of perennial ryegrass to determine if plant species composition influenced this relationship. Feeding assays were performed to provide baseline measurements of feeding preference and host suitability of the various grasses in the studies.

Results suggest how endophytic plants can be introduced into existing stands as a cultural insect management tool. Findings could also be used to create mixtures of seed that incorporate highly desirable turfgrass cultivars, which may be susceptible to insect herbivory, with resistant cultivars at ratios that effectively suppress insect pest populations while meeting certain aesthetic requirements. These studies will be useful for establishing a baseline for implementing endophyte enhanced and other insect resistant grasses into turfgrass insect management strategies.

Thesis Statement

Although the dynamics of endophyte-plant-insect interactions are likely complex, these interactions may be described by a generalized model (Figure 1). The research described in the following chapters tests hypotheses arising from the professed generalized model, contributing to the ecological understanding needed for a practical control strategy based on endophytic grasses. Overseeding with endophytic seed alters sward composition and increases the frequency of endophytic plants. Because endophytes provide grass plants with protection from herbivory, pest insect population

29 density and damage decrease as the incidence of endophyte infected tillers increases. The model predicts and my research demonstrates that greater increases in the proportion of endophytic tillers occur in areas where insect pest densities are higher. The model also predicts and my research also demonstrates that the proportion of endophytic tillers will increase to a certain level above which insect pests no longer influence the incidence of endophytic tillers.

30 Experimental Subjects

Herbivores:

Bluegrass billbug Sphenophorus parvulus (Gyllenhai) (Coleoptera:

Curculionidae)

Bluegrass webworm Parapediasia teterrella (Zincken) (Lepidoptera: Pyralidae)

Plants:

Perennial ryegrass Lolium perenne L. (Poaceae)

Kentucky bluegrass Poa pratensis L. (Poaceae)

Endophytic Fungi:

Ryegrass endophyte Neotyphodium lolii Glenn, Bacon, Price & Hanlon

31 Species Reference

ACARI Cereal Rust Mite Abacarus hystra Frost 1993

COLEOPTERA Argentine Stem Weevil Latronutus bonartensa Barker et al. 1984 Bluegrass Billbug Sphenophorus parvulus Ahmad et al. 1986 Com Flea Beetle Chaetocnema puiicarm Kirfman et. al 1986 Uneven Billbug Sphenophorus mequaiis Johnson-Cicalese and Funk 1988 Little Billbug Sphenophorus mmtrtms Johnson-Cicalese and Funk 1988 Hunting Billbug Sphenophorus venants Johnson-Cicalese and Funk 1988 Flour Beetle Tnbolium castaneum Cheplick and Clay 1988 Black Beetles arator Ball and Prestidge 1992 Crass Grub Costelytra zealandica Popay et al. 1993 Southern Masked Chafer Cyclocephala lunda Potter et al. 1992 Blackheaded Cockchafer Aphodius tasmantae Quigley et al. 1993

HEMIPTERA Milkweed Bug Onocopelnts fasctants Johnson et al. 1985 Hairy Chinch Bug Blissus leucopterus htrnts Saha et al. 1987

HOMOPTERA Aphid Rhopalosiphum padi Latch et al. 1985 Greenbug Aphid Schcaphis graminum Latch et al. 1985 Leafhopper Agallia consmcia Kirfman et al. 1986 Leafhopper Endria mimica Kirfman et al. 1986 Sharpshooters Draeculacephale antica Kirfman et al. 1986 Mealy bug Balanococcus poae Pearson 1988 Leafhopper Draeculocephala spp. M ueggeetal. 1990 Leafhopper Exttianus exiliosus M ueggeetal. 1990 Leafhopper Craminella mgnfrons Mueggeetal. 1990 Froghopper Prosapia bicmcta M ueggeetal. 1990 Aphid Diuraphis noxia Latch 1993 sugarcane Aphid Sipha Jlava Funk et al. 1993

ORTHOPTERA House Crickets Acheta domesnca Ahmad et al. 1985 Migratory Locust Locusta migratona Lewis et al. 1993 Black Field Cricket Teleogryllus commodus Quigley et al. 1993 Queensland Field Cricket Teleogryllus oceanicus Quigley ct al 1993

LEPIDOPTERA Cutworm Agroiis segelum Schmidt 1986 Cutworm Graphania muians Dymock et al. 1989 Sod Webworm Crambus spp. Funk et al. 1983 Fall Armyworm Spodoptera frugtperda Clay et al. 1985 Southern Armyworm Spodoptea ertdania Ahmad et al. 1987 Common Cutworm Agrotts injusa Quigley et al. 1993 Common Armyworm Mythimna convecta Quigley et al. 1993 Bluegrass Webworm Parapediasia teterrella Kandaetal. 1992 Soutfiem Armyworm Persectants Ewtngii Quigley et al. 1993 Pasture Tunnel Moth Philobota productella Quigley et al. 1993

Table 1. Arthropods adversely affected by endophytes in perennial ryegrass and tall fescue.

32 Legend IsaariCdmpomioiil ' I 1-^ %E* .Seed cath w ll IlFenmqrll pH jiMcmnfl

- Sovce/Siek © -AmulWy 1 r - tofomwrion Flow PR+ PR. KB R

9

% < Insect PopulatioD Density

Fenility 1 H,0 -tr' NatunI Enemies

Figure 1. Hypothetical model of endophyte-plant-insect interactions.

1 ) Sward composition is influenced by the seeding rate (SR), the percentage of seed containing viable endophyte (% E+ seed), the cuitivar(s) used, and cultural management (HiO, fertility, pH, mowing)(information flow #1). The proportions of all grasses (PR+. PR-, KB) must sum to 1. 2) Because fertility, irrigation (H^O), and cultivar may influence the types and concentrations of alkaloids produced, they may mediate the influence of PR+ on insect population density (information flow #2). Further, PR+ may influence insect population density indirectly through altering insect movement (emigration (Em) and immigration (Im)), or directly through reducing fecundity (F). or increasing mortality due to toxicity (M). Plant species composition can also influence insect population density. 3) Damage resulting from insect feeding (Insect Damage) is a frmction of insect population density, the proportion of endophytic plants, plant species composition, cultivar, fertility, and irrigation (information flow #3, rate equation D). A visual estimate (Rating) represents the amount of damaged plant material on a scale of 1- 10. 4) Insect damage will influence stand composition by reducing the proportion of PR- and KB (information flow #4).

33 I Sward Composhio^

SR Jl ^ J(Cultivar)

T _ _ x

PR+ PR- KB

“ f t d d \

/ i

Insect insect Population Damage \ æ ^ Density Em

Figure 2. Components of the endophyte-plant-insect interactions model addressed in the current series of studies.

1) Sward composition is a function of the overseeding rate, the percentage of endophyte infected seed, and the cultivar used for seeding (Chapter 2). 2) Insect population density is a function of the proportion of endophytic L. perenne (Chapters 2, 3. and 4). the cultivar(s) used for overseeding (Chapter 2), and plant species composition (Chapter 3). 3) Insect damage is a function of insect population density, the proportion of endophytic plants, plant species composition, and the cultivar(s) used for overseeding (Chapter 2). 4) Insect damage alters sward composition by reducing the proportion of non-endophytic grasses (Chapter 2).

34 CHAPTER 2

OVER-SEEDING ENDOPHYTIC PERENNIAL RYEGRASS, Lolium perenne L.. INTO

ESTABLISHED STANDS OF KENTUCKY BLUEGRASS, Poa pratensis L.. TO

MANAGE BLUEGRASS BILLBUGS, Sphenophorus parvulus Gyllenhal

Neotyphodium fungal endophytes can have profound effects on the growth and fitness of their grass mutualists. These endophytes depend on their grass host for nutrition whereas infected plants enjoy enhanced growth and vigor (Latch et al. 1985), germination and seed set (Clay 1987), drought tolerance (Belesky et al. 1987), pathogen

(Gwinn and Gavin 1992), and insect resistance (Breen 1994). The ability of

Neotyphodium endophytes to reduce insect pest population density in swards of turfgrass has been demonstrated in many studies (Breen 1994). As a result, endophytic grasses have been widely promoted for insect management. However, the relationship between insect population density and the proportion of endophytic plants in a turfgrass sward is unknown. Clarifying this relationship will provide fundamental information that may increase the usefulness of these grasses in pest management.

The composition of a turfgrass sward is usually altered in 1 of 2 ways. Complete renovation is the removal of all existing plants followed by planting of new seed consisting of the desired mixture of plant species and cultivars. This method is time consuming, expensive and is extremely disruptive to the turfgrass site (Watschke and

35 Schmidt 1992). Over-seeding into existing stands provides a quicker, less expensive and less disruptive method for altering the composition of a sward. This method can be employed to introduce endophytic plants into an existing stand. The utility of overseeding endophytic grasses to manage pest insects has not been scientifically examined. Therefore, overseeding rates needed to introduce sufficient endophyte infected plants useful for pest management are not known.

Once endophytic plants have been introduced into a sward of turfgrass, interactions between endophytic and non-endophytic plants, and between different plant species will determine the composition of the sward. Several studies found the proportion o f endophytic plants increased over time (Clay 1990; Francis and Baird 1989;

Siegel et al. 1987) or was positively correlated with population age (Lewis and Clements

1986). Brede and Duich (1986), found differences in competitiveness between Kentucky bluegrass, Poa pratensis L., and perennial ryegrass, Lolium perenne L., both above and below ground. Although many factors could influence the composition of turfgrass swards, no attempt has been made to extricate the influence of insect herbivory in the field.

This study measured the effect of overseeding perennial ryegrass, Lolium perenne

L. (PR), infected and uninfected with the fungal endophyte, Neotyphodium lolii Glenn.

Bacon, Price & Hanlon (PR+ and PR- respectively), into established stands of Kentucky bluegrass, Poa pratensis L. (KB), on Sphenophorus parvulus Gyllenhal, the bluegrass billbug, and it’s damage. Two sources of PR seed, differing in the proportion of PR+, were sown at 2 different rates into established stands containing =99 % Kentucky

36 bluegrass. S. parvulus larval population density and turfgrass sward composition were monitored for 2 y after the ryegrass introduction. S. parvulus was excluded from 1 of the treatments with a plant-systemic insecticide to determine if this species was capable of influencing short term changes in turfgrass sward composition.

I hypothesized that 5. parvulus larval population density would decrease as overseeding rate and the resulting proportion of PR+ increased. The overall proportion of

PR in the turfgrass swards was expected to increase with overseeding rate. Because

Neotyphodium endophytes enhance plant vigor, resulting proportions of PR should be higher in plots overseeded with seed containing a higher proportion of PR+ seed. Anti­ herbivore defenses provided by ftmgal endophytes to their grass mutualists should afford a greater competitive advantage to plants when insect herbivores are present. Therefore, the proportion of PR+, as well as the overall proportion of PR, should be lower in plots where S. parvulus was excluded with the insecticide.

Materials and Methods

Plot Design

Based on pre-treatment sampling, a total o f 64 plots (4.5 x 4.5 m) consisting of

99.7 ± 0.009% KB, were randomly assigned to 1 of 3 different treatments or designated as a control. Treatments 1 and 2 were over-seeded with PR at 48.8 kg/ha (1 lb/1000 ft^) but treatment 2 also received an application of imidacloprid (Merit™ 0.5 G, Bayer Corp..

Kansas City, MO), a plant systemic insecticide, in May of 1996 and 1997 (to target billbugs) at the labeled rate of 0.34 kg ai/ha. Treatment 3 was overseeded at 97.6 kg/ha

37 (2 lbs/1000 ff). Two sources of PR seed, differing in the proportion of PR-*-, were used.

Repell n™ (LofTs Seeds Inc., Willmington, OH) is a synthetic cultivar containing 95%

PR+, whereas Triple Play™ (Fine Lawn Research Inc., Lake Oswego, OR) (63% PR+ seed) is a blend of 3 synthetic cultivars: 38.64% Pebble Beach™ (29% PR+ seed),

38.64% Stallion Select™ (58% PR+ seed), and 19.73% Pennant™ (91% PR+ seed).

Control plots were vertically sliced but no seed was dispensed.

Seeding and slicing were performed the 1st week of October, 1995 using an

Olathe™ Aero-Seeder with knives set to 7.62 cm on center (Olathe Manufacturing Inc.,

Industrial Aiport, KS). The experimental design was a split-plot. repeated-measures design with 8 whole plot replicates. The whole plot factor was seed source and the sub­ plot factors were the over-seeding treatments. Plots were fertilized at a rate of 195.3 kg

N/ha/yr (4 lb N/1,000 ft^/yr) in a total of 4 applications using a 50% slow release fertilizer

(32-5-7 N-P-K in 1996 and 24-4-12 N-P-K in 1997). Broadleaf weeds were controlled with spring and fall applications of Dicamba, MCPP and 2,4 D (Three-Way™. Lesco

Inc., Rocky River, OH) at the labeled rate. Plots were mowed weekly to a height of 7.6 cm.

Insect Population Density

S. parvulus larval populations were sampled during July of 1996 and 1997 using a standard golf course cup cutter (10.795 cm dia., 91.5 cm- area). Five turf/soil cores were taken from each plot and destructively examined for larvae. The mean, untransformed number of larvae per sample was reported for analysis.

38 Aesthetic Rating

Plots were rated for aesthetic quality in July 1997 after S. parvulus larval populations were sampled. No ratings were assigned in 1996 because damage was not visible. Although insect damage could have been measured directly as the biomass of damaged plants, visual quality is of primary importance in most managed turfgrass systems. Therefore, each plot was surveyed from its southern edge and assigned a visual rating based on the estimated proportion of the turf damaged by S. parvulus. A rating scale of 1 to 10 was used with Incomplete damage and 10=no visible damage.

Sward Composition

Sward composition was sampled twice (June and October) in both 1996 and 1997.

Plots were sampled by removing all above ground shoots from 4-900 cm' quadrats. The

4 samples from each plot were combined and mixed by hand. Four groups of 50 individual shoots (200 shoots/plot) were drawn at random from the samples and identified to species.

The proportion of PR+ tillers was determined only in July, 1997 by randomly cutting 40 tillers of PR from each plot. Tillers were returned to the lab for tissue print- immunoblot (TPIB), modified from Gwinn et al. (1991) (Appendix A). The proportion of

PR+ was multiplied by the proportion of PR in the stand to arrive at the total proportion of PR+ in the stand.

39 Statistical Analysis

Split-plot ANOVA was used to evaluate the influence of seed source and over­ seeding treatment on S. parvulus larval population density whereas univariate repeated measures ANOVA was used to examine turfgrass sward composition through time. A segmented regression model was chosen to describe the relationship between S. parvulus larval population density and the proportion PR+. The model was similar to that used by

Neter et al. (1985) and is as follows:

Y=bo+b I * X+bi (X-brkpnt) * (X>brkpnt)

where;

Y=the average number of larvae per sample

X=the proportion of PR+ brkpnt=the inflection point in the relationship

bo, b, and bn=regression coefficients.

The logical expression in the model (X>brkpnt) serves as a multiplier. If the expression is true (i.e., the proportion of endophytic tillers is greater than the breakpoint), it will evaluate to 1; if it is false, it will evaluate to 0. Therefore this equation simultaneously represents two models. For turfgrass swards where X is less than or equal to the estimated breakpoint (i.e., [X>brkpnt] is false, and equal to 0):

Y=bo+b,*X

40 For swards where X is greater than the estimated breakpoint (i.e., (X>brkpnt) is true, and equal to 1):

Y=bo+b, *X+b 2 *(X-brkpnt)

For values of X greater than the estimated breakpoint, the slope is equal to bj+b, and the

intercept is equal to (bo-brkpnt*b 2 ). This model was chosen over the polynomial regression models because it was more biologically realistic (i.e., the polynomial model predicts an eventual increase in insect population density with increasing proportions of endophytic tillers), and it described a similar percentage of the total variation (polynomial

R- = 26.7 %, segmented R- = 22.3%). A Quasi-Newton estimation method was used and a starting point of 50 was specified for the inflection point. Linear regression was used to describe the relationship between S. parvulus larval population density and damage rating. All statistical analyses were performed using Statistica® 5.0 (StatSoft Inc.).

Results

Billbug Larval Population density

Mean S. parvulus larval densities were much lower in 1996 (39.5±8.I larvae/m’) than in 1997 (285.7±33.4 larvae/m-) (F=86.9; df^l, 126; P<0.00001). In 1996. no differences in S. parvulus larval densities were observed between seed sources or over­ seeding treatments (F=0.1; df=l, 7; P>0.7 and F=2.0; df=3, 42; P>0.1 respectively)

(Figure 3).

41 In 1997, no differences in S. parvulus larval densities were observed between seed

sources (F=0.02; df=l, 7; P>0.8) but, larval densities were lower in all over-seeded plots

than in the controls (F=17.8; df=3, 42; P<0.0001) (Figure 3). Larval densities were lower

in plots receiving insecticide than in plots over-seeded at 48.8 kg/ha (F=6.88; df=l, 60;

P<0.05) but only slight differences were observed between insecticide treated plots and

those over-seeded at 97.6 kg/ha (F=3.95; df=l. 60; P=0.05I). Larval densities did not

differ between the 2 seeding rates (F=0.40; df=l, 60; P>0.5).

Segmented regression indicated that S. parvulus larval population density

decreased linearly as the proportion of PR+ increased up to 38.4±3.8% (Table 2, Figure

4). However, there was no significant decrease in S. parvulus larval population density as

the proportion of PR+ increased beyond this point. There was no significant relationship

between larval densities and the proportion of PR- in the stands (F=1.7644; df=l, 46;

P=0.191).

Aesthetic Rating

Low billbug larval populations resulted in no apparent aesthetic damage in 1996.

In 1997, significant differences in aesthetic damage were observed between seed sources

and over-seeding treatments. Plots over-seeded with Repell II sustained less damage than

those over-seeded with Triple Play (F=I3.38; df=l, 7; P<0.01) but all over-seeded plots

sustained significantly less damage than the controls (F=282.46; df=l, 60; P

(Figure 5). Billbugs caused significantly more visible damage in plots over-seeded at

48.8 kg/ha than in plots receiving insecticide or those over-seeded at 97.6 kg/ha

42 (F=l4.0l; df=l, 60; P<0.01 and F=5.86; df=l, 60; P<0.05 respectively). There was no difference in visible damage between insecticide treated plots and those over-seeded at

97.6 kg/ha (F=1.74; df=l, 60; P=0.192). Damage caused by billbugs increased linearly with S. parvulus larval population density (F=7.5; df=l,62; P<0.00001) (Figure 6).

Damage decreased linearly as the proportion of PR+ increased (F=7.4; df=l,30; P<0.03).

Sward Composition

The proportion of PR+ was initially lower in plots overseeded with Repell II ™ than in plots over-seeded with Triple Play™ blend but the reverse was true by June 1997

(seed source x time interaction, F=26.68; df=3, 168; P<0.05) (Figure 7). Plots over­ seeded at the higher rate (97.6 kg/ha) resulted in greater proportions of PR in June and

October of 1996 when compared to plots over-seeded at the lower rate (48.8 kg/ha)

(F=7.15; df=l, 48; P<0.05 and F=6.28; df=l.48; P<0.05 respectively) but, no significant differences in the proportion of PR was detected between the 2 over-seeding rates in June or October of 1997 (F=l.8l; df=l. 48; P>0.1 and F=3.33, df=l, 48; P>0.05 respectively)

(Figure 8). Over-seeding rate had no influence on the proportion of L. perenne tillers infected with N. lolii (F=0.013; df=l, 14; P>0.1). A greater proportion of Repell II™ tillers were infected with N. lolii than Triple Play™ tillers (58.5 and 30.0 % respectively)

(F=52.10; df=l, 7; P<0.01). As a percentage of total tillers, PR+ comprised an average of

41.7 and 16.1% of the total stand for Repell If™ and Triple Play™ respectively in July of

1997.

43 Though an immediate trend suggested slightly lower proportions of PR in plots where billbugs were excluded (insecticide treated), differences were not significant at a=0.05 until October 1997 following a severe infestation of billbugs (Figure 8). In plots overseeded with Repell H™, PR represented 56.3% of the total tillers in insecticide treated plots compared to 68.4% in untreated plots during October 1997 (F=5.5; df=l. 28;

P<0.05). Likewise PR+ comprised 50.0% of the total PR in insecticide treated plots compared to 60.0% PR+ in untreated plots during July 1997 (F=6.2; df=l, 28; P<0.05).

In plots over-seeded with Triple Play™ , insecticide application had only a minor influence on the proportion of PR at any sampling date (F<3.2; df=l, 28; P^0.08) and no effect on the proportion of PR+ (F=0.6; df=I, 28; P>0.1).

Discussion

This study demonstrates the utility of overseeding PR+ into pre-existing stands of

KB as a cultural insect management tool. Results indicate that significant reductions in S. parvulus larval populations densities and damage can be attained through overseeding established stands of KB with PR+ at rates commonly used in the field (48.8 and 97.6 kg/ha).

Although sward composition did not differ significantly between the two overseeding rates in 1997, the higher overseeding rate (97.6 kg/ha) reduced S. parvulus larval population densities to levels comparable with the plant-systemic insecticide imidacloprid. This is likely due to differences in plant density resulting from the two overseeding rates. Because twice as much seed was placed into the same area at the

44 higher overseeding rate, plant density was likely higher in these plots. In turfgrass, increases in plant density generally result in smaller plants (Lush 1990) that are not favored by S. parvulus (Bnineau 1983).

The relationship between S. parvulus larval population density and the proportion of PR+ was best described by a segmented-linear model. This model indicated a decrease in S. parvulus larval population density as the proportion of PR+ increased to =38%. No further reductions in larval populations were observed in swards containing greater proportions of PR+. This result indicates a broad range of turfgrass situations can benefit from overseeding with PR+. Addition of even relatively low levels of PR+ will provide some benefit in terms of reduced S. parvulus populations.

Even though plots overseeded with the 2 seed sources differed in their resulting proportion of PR+ in 1997, S. parvulus larval populations were very similar. This finding is not surprising in leu of the probable variation in the types and concentrations of alkaloids produced between cultivars (Roylance et al. 1994) and the relationship between

S. parvulus larval population density and the proportion PR+ found in this study. As the proportion of PR+ increased above =38.4±3.8%, insect population density essentially leveled out. Differences, between seed sources, in the mean proportion of PR+ was probably not great enough to result in significantly different S. parvulus population densities. Differences in alkaloid production among cultivars would only further blur any distinctions between insect suppression provided by the different seed sources.

Initial sward composition (June 1996) varied with the overseeding rate but. the percentage of seed infected with N. lolii had no effect in this regard. It is somewhat

45 surprising that plots overseeded with Repell 0™ (1995) resulted in lower initial proportions of PR (1996) because Repell U™ contained a greater proportion of PR+ seed than Triple Play™. Clay (1987) found that endophyte infection increased seed germination. Therefore, higher initial proportions of PR were expected in plots overseeded with Repell H™. Inherent genetic differences between the two seed sources were apparently more important in determining initial sward composition than the proportion of PR+ seed.

There was a significant linear relationship between S. parvulus larval population density and visual damage. A significant proportion of the visible damage was accounted for by variation in S. parvulus larval population density (31.6 %), indicating that visual turf damage can be directly related to larval population density even in mixed stands of

PR+, PR- and KB. The proportion of PR+ explained only 19.8% of the variation in damage between cultivars. However, visual ratings of turf damage do not only reflect insect population density and may depend on the grass species or cultivars ability to recover from damage. Reinert and Busey (1983) observed similar numbers of adult

Herpatogramma phaeopteralis Guenée emerging from several bermudagrass selections, even though differences in damage were observed. Thorogood (1993) observed temporal differences in the proportion of dead and senescing vegetative material among breeding lines of L perenne. Even though no differences in larval population density were observed between seed sources in the current study, different levels of damage were observed. This finding indicates that differences between cultivars are likely to influence

46 the aesthetic quality of the turf regardless of insect population density but that damage

will decrease as the proportion of PR+ increases.

Although increases over time in the proportion of PR were observed in all

overseeded plots, increases were more modest in plots receiving the systemic insecticide.

Likewise, the proportion of PR+ was significantly higher in plots that did not receive

insecticide. These differences in sward composition between insecticide treated and

untreated plots were only observed when S. parvulus populations were high. Greenhouse

studies have shown that insect herbivory can influence the competitive interactions

between endophytic and non-endophytic plants over relatively short time spans (Clay et al. 1993). However, this has never been shown in the field. By excluding S. parvulus

with a systemic insecticide, the competitive advantage enjoyed by PR+, because of insect

resistance, was reduced. Thus, S. parvulus was able to influence sward composition by altering the competitive hierarchy between PR+, PR- and KB.

Because tiller density was not measured, differences in sward composition between insecticide treated and untreated plots may only reflect short term changes.

Differences in growth habit between PR and KB and issues relating to the power rule of population biology {sensu Lush 1990) indicate a need for caution in extrapolating results of the current study to more long-term shifts in turfgrass sward composition. For instance, it is unclear whether differences in sward composition were due in part to differences in tiller density or a replacement process whereby the death or morbidity of tillers allowed encroachment by neighboring tillers (i.e., self thinning). There was no obvious difference in plant canopy density between insecticide treated and un-treated

47 plots because full ground cover was preserved during all periods when sward composition was sampled. There have been no reports in the literature of the insecticide

(imidacloprid) differentially affecting 1 plant species or type over another and any influence of imidacloprid on Neotyphodium endophytes has not been described.

Nonetheless, increases in the proportion of PR in mixed the swards occurred more quickly where S. parvulus populations were high. The fact that the systemic insecticide application made no differences in the proportion of PR+ or the total proportion of PR in plots overseeded with Triple Play™ may be due to genetic differences between seed sources.

Conclusion

The results of this study provide information relevant to key aspects of the hypothesized endophyte-plant-insect model represented in Figure 9. Turfgrass swards composed of as little as 38% PR+ reduced S. parvulus population densities significantly whereas higher proportions of PR+ provided little additional benefit. Sward composition changed over the course of 2 y depending on the overseeding rate and cultivar(s) used.

Differences in damage between cultivars or blends were observed even though S. parvulus population densities were similar. These aesthetic differences between plots seeded with different seed sources were not directly related to differences in the proportion of endophyte infected plants but, were more likely related to inherent genetic differences between the 2 seed sources. By applying a systemic insecticide, S. parvulus populations and their damage were nearly eliminated and the anti-herbivore defenses

48 provided less of a competitive benefit to plants under these conditions. As a result, patterns of change in sward composition differed between insecticide treated and un­ treated plots with un-treated plots converting more quickly to a greater proportion of PR and a greater proportion of PR+ at least over the short term.

49 Variable Estimate Std. Error t P Intercept 2.34 0.20 11.52 <0.0001 Slope 1 -0.03 0.01 -2.90 0.0058 Brkpnt. 38.44 3.75 10.25 <0.0001 Slope 2 0.02 0.04 0.45 0.6571 R^O.22; Brkpnt is the estimated inflection point, slope 1 is the slope of the line above the inflection point and slope 2 is the slope of the line below the inflection point.

Table 2. Parameter estimates and their standard errors resulting from segmented regression analysis of 5. parvulus larval population density on the proportion of endophytic tillers in mixed stands of perennial ryegrass and Kentucky bluegrass

50 o 1996 • 1997

Control 48.8 kg/ha 97.6 kg/ha + innitidtDpri

Overseeding Treatment

Figure 3. S. parvulus larval population densities in Kentucky bluegrass plots treated in 4 different manners (control, overseeded with endophytic perennial ryegrass at 48.8 and 97.6 kg/ha, and overseeded at 48.8 kg/ha+imidacloprid insecticide). Overseeding performed October 1995 and imidacloprid applied May 1996 and 1997. S. parvulus samples taken July 1996 and 1997 (point=mean, box=standard error, whiskers=standard deviation).

51 ir> S. parvulus / 91.5 cm“=2J4-0.03*(%cndophyte)+0.02*(%endophyte-38.44)*(%endophyte>38.44) CII R-=22.3%

ON ON

10 20 30 40 50 70 % Endophytic Perennial RyegrassTiilers

Figure 4. Scatter plot of the relationship between the percentage of endophyte infected perennial ryegrass tillers (n=48) and S. parvulus larval population density in mixed stands of Kentucky bluegrass and perennial ryegrass. Endophyte infection calculated from 40 perennial ryegrass tillers taken randomly from each plot and multiplied by the total proportion of perennial ryegrass determined from 200 tillers taken randomly from 4-900cm' areas within each plot. Line represents result of segmented regression analysis with inflection point at arrow.

52 o Tnple Play Repell 11

Control 48.8 kg/ha 48.8 kg/ha 97.6 kg/ha + imidacloprid Overseeding Treatment

Figure 5. Visual rating of S. parvulus larval damage (10=no damage, l=complete damage) during July 1997 in plots of Kentucky bluegrass treated in 4 different manners (control, overseeded with endophytic perennial ryegrass at 48.8 and 97.6 kg/ha, and overseeded at 48.8 kg/ha+imidacloprid insecticide). Overseeding performed October 1995 using two different seed sources (Repell H™ and Triple Play™) and imidacloprid applied May 1996 and 1997 (point=mean, box=standard error, whiskers=standard deviation).

53 o ON • Repell II O Triple Play % 8 •• 3 Rating=7.54- 1.28*( S.parvulus/91.5 cm ~) W) 7 o • • • • o R “ = 33.7% « o o o o O O r I 5 • o o o Q 4 o o 1CO

o • • •

I - • • O • 0

0 1 2 3 4 5 6 SI parvulus Larvae / 91.5 cm (July 1997)

Figure 6. Regression of S. parvulus visual damage rating on S. parvulus larval population density (July 1997) in stands of Kentucky bluegrass overseeded (October 1995) with two sources of endophytic perennial ryegrass (Repell II™ and Triple Play™ ).

54 1.0 Tf (N # Repell II II 0.9 C O Triple Play 5 0.8

en 0.7 a 0.6 D 0.5 "S 0.4

Ia. 0.3 OG r 0.2 a.o o 0.1

0.0 June 1996 October 1996 June 1997 October 1997 Date

Figure 7. The proportion of perennial ryegrass tillers evaluated each June and October (1996 and 1997) in plots of Kentucky bluegrass overseeded (October 1995) with two sources of perennial ryegrass (Repell IT™ and Triple Play™) as determined from 200 tillers taken randomly from 4-900cm‘ areas within each plot (point=mean. box=standard error. whiskers=standard deviation).

55 0 1.0 II c ■ Control 0.9 A 48.8 kg/ha 1 0.8 ♦ 48.8 kg/ha + imidaclopri p • 97.6 kg/ha c/3 0.7

0>I 0.6

0.5

0.4 1eu c 0.3 o c 0.2 o a. 2 0.1 eu 0.0 June 1996 October 1996 June 1997 October 1997 Date

Figure 8. The proportion of perennial ryegrass tillers in plots of Kentucky bluegrass treated in 4 different manners (control, overseeded with endophytic perennial ryegrass at 48.8 and 97.6 kg/ha, and overseeded at 48.8 kg/ha+imidacloprid insecticide). Overseeding performed October 1995 and imidacloprid applied May 1996 and 1997 (point=mean, box=standard error, whiskers=standard deviation).

56 Sward Compositioc

SR %E+ Seed Cultivar

PR+ PR- fCB

Cultivar

Insect Insect Population Damage Density

Rating.

Figure 9. Components of the endophyte-plant-insect interactions model addressed in Chapter 2 (See Chapter 1, Model Description for details).

I ) Sward composition is a function of the overseeding rate and the cultivar(s) used but not the percentage of endophytic seed. 2) Insect population density is a function of the proportion of endophytic L. perenne but not the cuitivarfs) used for overseeding. 3) Insect damage to the sward is a function of insect population density, the proportion of endophytic plants, plant species composition and the cultivar(s) used for overseeding. 4) Insect damage results in a decrease in the proportion of non-endophytic L perenne and P. pratensis, and an increase in the proportion of endophytic L. perenne.

57 CHAPTERS

BLUEGRASS BILLBUG, Sphenophorus parvulus Gyllenhal, LARVAL POPULATION

DENSITY IN PURE STANDS OF PERENNIAL RYEGRASS, Lolium perenne L.,

WITFI VARYING PROPORTIONS OF ENDOPHYTIC TILLERS

There is a clear relationship between bluegrass billbug, Sphenophorus parvulus

Gyllenhal (Coleoptera: Curculionidae), larval population density and the proportion of endophytic perennial ryegrass, Lolium perenne L. (PR+), in the environment. However, the species composition of a stand of turfgrass may also influence insect population density. Plant species composition may influence insect population density simply due to differences in plant architecture (Bach 1981) and plant physical characteristics can influence oviposition and feeding (Pandey et al. 1990), as well as vulnerability to natural enemies (Freese 1995) in stem boring weevils like S. parvulus. Additionally, tiller or shoot size has been positively correlated with S. parvulus larval population density and damage in turfgrass (Bruneau 1983).

The main goal of this study was to investigate the relationship between S. parvulus larval population density and the proportion of PR+ in pure stands of perennial ryegrass (PR) in the field. Because the mechanism by which resistant plants avoid herbivory can influence their effectiveness in pest management (Smith 1989), the influence of grass species, the proportion of PR+, and tiller diameter on adult feeding

58 preference and longevity was investigated. The proportion of PR+ and S. parvulus larval populations were monitored in the field over the course of 3 y. Pure stands of Kentucky bluegrass Poa pratensis L. (KB), were incorporated into the experiment for comparison with PR plots containing only low levels of PR+. By examining the relationship between

S. parvulus larval population density and the proportion of PR+ in a context which allows comparison between grass species, the relative influence of each may be clarified. 1 hypothesized that plant species characteristics may influence adult feeding preference and longevity despite endophyte infection. Adult feeding preference and longevity could vary with between PR+, PR- , and KB, and may influence larval populations in the field.

Information provided may help direct research efforts aimed at breeding and using turfgrass varieties resistant to S. parvulus.

Materials and Methods

Choice Tests and Adult Longevity

Field collected, adult male S. parvulus were placed in petri dishes ( 15 cm) with moist filter paper. Four S. parvulus were placed in each dish and 5. freshly cut, whole tillers of PR-, PR+, and KB were added according to I of 6 treatments. The 1st 3 treatments consisted of 100% PR-, 100% KB and 100% PR+ tillers respectively. The remaining 3 treatments were mixtures of KB and PR+ tillers at 40%, 60% and 80% PR+ respectively. Each treatment was replicated 8 times. Petri dishes containing the billbugs were placed in an environmental chamber set at 23°C and a 14:10 (L:D) photo period.

Old tillers were removed and fresh tillers of grass were provided daily.

59 During the 1st 10 d of the study, each tiller of grass was examined microscopically. The type of tiller (PR-, PR+, KB), number of feeding puncture and the diameter of each tiller at the base o f the 1st leaf were recorded. Longevity of the adult male S. parvulus in each of the dishes was observed daily until 100% mortality.

Plot Establishment.

Two 22.7 kg bags of PR seed (> 95% PR+) (var. Repell If™ ) were acquired in

January 1993 from Loft’s Seeds Inc. (Wilmington, OH). One bag of Repell U was kept in a heated storage area for 6 months to kill most of the ftmgal endophytes in the seed while another bag was placed in a refrigerated storage area held at 5°C. In March 1994. 100 seeds from each bag were planted in greenhouse flats and allowed to germinate. After 42 d, samples of sheath material from each tiller were stained using a standard 0.5% Rose

Bengal staining solution following the protocol from Saha et al. (1988). The percentage of PR+ and germination rates were recorded and used to create blends of seed in an attempt to establish field plots with different proportions of PR+.

Fifty-4.5 X 4.5 m plots were established during June 1994 by killing the existing grass with a single application glyphosate (Round-up ™ , Monsanto Co.. St. Louis. MO).

Alleys were left unsprayed to allow a 1.5 m zone of existing grasses between plots and to provide a reservoir population of insects. Seed blends were created to establish perennial ryegrass plots representing , 15, 30, 65, and 90% PR+. Additional plots were seeded with

100% KB (var. Barron™, Lofts Seed Inc., Willmington, OH). Seeding was performed 2 w after the glyphosate application. All plots were verti-tilled and raked by hand to

6 0 remove dead plant material and a drop-seeder was used for seeding. Perennial ryegrass was seeded at a rate of 225.8 kg/ha whereas Kentucky bluegrass plots were seeded at

150.5 kg/ha. After seeding, plots were again verti-tilled to ensure seed-soil contact. Each of the 5 treatments were replicated 10 times in a randomized complete block design.

Plots were irrigated from June through September of 1994 to keep the soil moist and ensure germination and establishment

Endophyte Detection and Sward Composition.

In July of 1995, 96 and 97, plots were divided visually into 4 equal quadrants.

Ten tillers were selected randomly from within each quadrant cut at ground level, wrapped in a moist paper towel and placed in a refrigerator at 5°C until needed. In 1995, the proportion of PR+ was determined microscopically using the standard Rose Bengal staining procedure reported in Saha et al. (1988). Analysis of the resulting data indicated a high level of variability in the proportion of PR+ within the planned treatments. Based on the data from sampling in 1995, representative plots with low (<20%), medium (>20% but <80%) and high (>80%) proportions ofPR+ were selected for monitoring in 1996 and

1997. A tissue print-immunoblotting technique (TPIB), modified from Gwinn et al.

(1991) (Appendix A) was used to determine the proportion of PR+ in 1996 and 1997.

Billbug Larval Population Density

S. parvulus larval population density was monitored over the course of 3 y. In

1995, all plots were sampled whereas only selected PR plots were sampled in 1996 and

61 1997. Sampling was performed using a golf course cup cutter ( 10.795 cm diameter) which was forced into the soil a depth of = 10 cm. The soil and turf core was then extracted and destructively examined for S. parvulus larvae. Five soil cores per plot were examined and the mean number of S. parvulus larvae per sample was recorded for analysis.

Statistical Analysis

Feeding preference was determined by comparing the observed proportion of feeding on PR+ to the expected proportion, based on the null hypothesis that no differences in feeding preference existed, using a X'-goodness of fit test including only 2 of the 6 treatments (40% PR+:60% FCB and 60% PR+:40% KB). The influence of grass type (PR-, PR+ and KB) on adult feeding was determined using repeated-measures analysis of covariance (ANCOVA) where tiller diameter was used as a changing covariate (the number of feeding marks and the diameter of each tiller were measured during every day of the experiment). The influence of the 6 feeding treatments (PR-.

PR+, KB and combinations of KB and PR+) on average times to 25, 50. 75 and 100 % mortality, and the relationship between grass type and tiller diameter were determined using analysis of variance (ANOVA). Regression was used to determine the relationship between adult longevity the proportion of PR+ using the 100% KB and 100% PR- treatments combined and independently as the 0% endophyte treatment. Regression was used to determine if year-to-year changes in the proportion of PR+ were related to S. parvulus larval population densities and or sward composition the previous year.

62 Analysis of covariance (ANCOVA) was used to determine the influence of grass species

(KB or PR) on S. parvulus larval population density using the proportion PR+ as the covariate. Larval population density in Kentucky bluegrass plots and low endophyte (<15

%) perennial ryegrass plots were compared using analysis of variance (ANOVA). A segmented regression model, developed by Neter et al. (1985), was used for analysis of the relationship between S. parvulus larval population density and the proportion o f PR+ in the PR plots:

Y=bo+b, * X+b; * (X-brkpnt) * (X>brkpnt)

where;

Y=the average number of larvae per sample

X=the proportion of PR+ brkpnt=the inflection point in the relationship

bo, b| and bn=regression coefficients.

The logical expression (X>brkpnt) acts as a multiplier in the model. If the expression is true, (X>brkpnt) will evaluate to 1 ; if it is false, (X>brkpnt) will evaluate to 0. For turfgrass swards where X is less than or equal to the estimated breakpoint:

Y=bo+b,*X

For swards where X is greater than the estimated breakpoint:

Y=bo+b, *X+b,*(X-brkpnt)

63 For values of X greater than the estimated breakpoint, the slope is equal to bj+b, and the

intercept is equal to (bo-brkpnt*b 2 ). This model was chosen over a polynomial regression model because it was more biologically realistic, and it provided a better fit to the data

(polynomial: R" = 39.0 %, segmented: R* = 47.9%). A Quasi-Newton estimation method was used and a starting point of 50 was specified for the breakpoint. Because S. parvulus larval densities were extremely low in 1996, only data collected in 1995 and 1997 were used for analysis. Endophyte infection data collected in 1995 and 1997 were averaged and the means were used for analysis.

Results

Adult Feeding and Longevity Assays

Adult male S. parvulus did not discriminate between PR+ and KB (X~ = 0.26; d f=

23; P >0.05) when given a choice between the 2 grasses. However, when provided with either KB, PR+ or PR-, significantly less feeding was observed on KB than either PR+

(F=28.7; df=l. 191; P<0.0001) or PR- (F=47.9; df=l, 191; P<0.0001) and no difference was observed between PR+ and PR- (F=0.1; df= 1,191; P>0.1) (Figure 10). There was a positive linear relationship between tiller diameter and the proportion of feeding across all treatments (F>3.9 df= 9, 225 P< 0.001)(Figure 11) and KB tillers were significantly greater in diameter than either PR+ or PR- (Figure 12). Tiller diameter accounted for an average of 19.5% of the total variation in feeding over all but the 1st day of the feeding experiment when tiller diameter was not a significant covariate at a=0.05. The overall influence of the treatments on longevity of adult male billbugs was significant (F = 2.8; df

64 = 1,42; p < 0.05). There was no difference in adult longevity between KB and either PR-

or PR+ (F=3.4; df=l, 42; P>0.05 and F=2.1; df=l, 42; P>0.05 respectively) because

longevity on KB was extremely variable. However, longevity was significantly reduced

on PR+ treatment compared to PR- treatment (F = 7.53, df = 1,42 P<0.01) (Figure 13).

Regression indicated a significant linear decrease in adult longevity as the proportion of

PR+ increased from 40 to 100% (F=5.85; df=l, 30; P<0.05 ) or from 0 to 100% when the

100% PR- treatment was included (F=21.0; df=I, 38; P<0.001 ). When the 100% KB

treatment was included, adult longevity was not influenced by the proportion of PR+

(F=2.78;df^l,38; P>0.05).

Endophyte Infection

Though the proportion of PR+ in the ryegrass plots varied slightly from one year

to the next, each year was a good predictor of the proportion PR+ the following year

(F> 107.2; df=l,18; P<0.0001). The proportion of PR+ in 1997 was highly correlated with

the proportion PR+ in 1996 (R=97.1), which was well correlated with the previous years data (1995) (R=92.5). Neither 5. parvulus larval population densities nor the proportion of PR+ had any influence on changes in the proportion of PR+ from year to year (F<0.05; df=l,18; P>0.1 and F<0.20; df=l,18; P>0.1 respectively)

Larval Population density

S. parvulus larval population density was higher in KB plots when compared to all

PR plots during the 1st year of the study (F=25.62; df=l,47; ?<0.00001) but the

65 proportion of PR+ was a significant covariate with S. parvulus larval population density declining linearly as the proportion of PR+ increased (F=6.l I; df=l,47; P<0.05) (Figure

14). S. parvulus larval population density was significantly higher in KB plots than PR plots with < 15% PR+ (F=8.82; df=l,13; P<0.05) (Figure 15). Segmented regression indicated that S. parvulus larval population density decreased linearly as the proportion of

PR+ increased to 68.0% but that further reductions in S. parvulus larval population densities were not observed in swards containing higher proportions of PR+ (Table 2,

Figure 16).

Discussion

Laboratory assays show how adult male S. parvulus feeding and longevity are influenced by different host plants. Previous studies by Johnson-C icalese and White

( 1990) demonstrated that field collected adult billbugs (mixed sexes) fed equally on endophytic and non-endophytic grasses but adult longevity was significantly reduced on endophytic grass. The results herein corroborate these findings but also indicate differences in feeding between KB and PR, and differences in longevity between grass

KB, PR+ and PR-. Although KB is considered the primary host for S. parvulus (Shetlar

1995), results of the present non-choice tests showed significantly less adult male feeding on KB than either PR- or PR+. This result was somewhat unexpected primarily because average adult longevity was essentially the same on KB and PR+. PR- may be a superior host for S. parvulus, at least in terms of adult feeding and survival. However, other plant characteristics may be important in determining host suitability.

66 Differences in adult feeding were correlated with tiller diameter. Host-plant physical characteristics such as stem diameter (Freese 1995), height and crown width

(Pandey et al. 1990) have been shown to influence oviposition and feeding (Pandey et al.

1990) as well as vulnerability to natural enemies (Freese 1995) with other stem boring weevils. Indeed, S. parvulus larvae spend the 1st few larval instars completely within the stems of grass plants. When they reach a critical point in their development or when they become restricted by the size of the stem, the larvae exit and drop to the base of the plant and continue feeding within the crown. The apparent affinity of adult males for tillers of greater diameter may reflect a female behavioral mechanism selecting for larger tillers for oviposition thus optimizing larval survival. If so, then this behavior is deeply ingrained in both sexes. Evidence suggests that the ability of PR+ to influence adult feeding and survival must be considered in the context of overall turfgrass sward composition (i.e.. grasses that are mixed with PR+) and may also indicate direction for further research in developing turfgrass varieties with physical characteristics which billbugs do not prefer.

Although differences in insect survival between endophytic and non-endophytic grasses of the same species can be demonstrated, these differences do not necessarily translate directly into useful applied information.

The results of this experiment bolster the hypothesis that differences in S. parvulus larval population density may be influenced not only by the presence of PR+ but also by other plant species present. Plant characteristics, which may differ among species or cultivars, can influence insect population density (Castane and Albajes 1992) and activity (Weston et al. 1997). Adult feeding was more closely linked with tiller diameter,

67 a species characteristic in this case, than endophyte infection but, the implications for oviposition and reproductive success were not investigated. However, Bruneau (1983) found that Kentucky bluegrass tiller diameter in field plots was positively correlated with both S. parvulus larval population density and damage.

It makes sense biologically that S. parvulus larval population density could increase with the diameter of tillers in a sward. Because S. parvulus larvae feed and develop within the stems of grass plants, they are briefly somewhat protected from certain predators and pathogens. The larger the diameter of the grass stem, the further development may proceed before the must exit the stem and move to feed in the crown. Therefore, larvae which develop in stems with a larger diameter would be protected from predators and pathogens for a longer period of time. As a result, their survival may be enhanced. As the present results show, significantly lower larval densities were observed in PR plots than in KB plots, even when the proportion of PR+ was relatively low (<15%).

The fact that sward composition influences S. parvulus larval population density regardless of the PR+ was not surprising. Field trials have demonstrated differences in susceptibility to S. parvulus infestation among cultivars of a single grass species

(Shearman et al. 1982). Segmented regression using only perennial ryegrass plots illustrated a somewhat different picture of the relationship between S. parvulus larval population density and the proportion PR+ than when the same analysis was used in mixed stands of PR+, PR-, and KB (Chapter 2). The estimated inflection point in the relationship between these 2 variables in the present study was approximately 30% higher

68 than the estimate for the KB-PR mixture. The estimated inflection point for the PR monoculture was accompanied by a relatively large standard error, a product of the distribution of the data along the X axis, which was exacerbated by the lack of data between 40 and 65% PR+. However, adult male longevity was much more variable on

KB than PR-, implying that PR- is a superior host for adult S. parvulus. If adults live longer and deposit more eggs in monocultures of PR, a greater proportion of PR+ may be necessary to reduce larval population densities comparable to those found in mixed species stands (KB, PR). Conversely, PR- may be inferior to KB as a larval host, due to smaller tiller diameters. An interaction between the proportion of PR+ and plant species composition may account for the continued decrease in S. parvulus larval populations beyond those observed in mixtures of KB, PR-, and PR+. Differences in S. parvulus population density between KB monocultures and low endophyte PR monocultures imply a tendency for reduced S. parvulus population densities in PR regardless of the proportion of PR+. More extensive research will be needed to determine how the interaction between plant species composition and the proportion of PR+ influences S. parvulus larval population density.

Conclusion

The study provides information relevant to particular aspects of the hypothetical model of endophyte-plant-insect interactions presented in Chapter 1 (see Figure 17).

Results show that S. parvulus larval densities were lower in pure stands of PR than in pure stands of KB even when the proportion of PR+ was relatively low. This fact

69 may be attributed to interspecific differences in plant physical characteristics, such as tiller diameter, which alter the vulnerability of larvae to predators and pathogens.

However, if adult male feeding preference reflects female ovipositional behavior, fewer eggs would be deposited on plants with thinner tillers and resulting larval densities would be lower. S. parvulus larval population densities in monocultures of PR were significantly reduced in swards containing 68.0±28.7% PR+ but higher proportions of

PR+ had no further suppressive influence on the insects. The fact that relatively high proportions of PR+ may be needed to reduce S. parvulus population densities in monocultures of PR may be due to the fact that PR- is a superior host for adult S. parvulus. Further research is needed to clarify how the interaction between grass species and the proportion of endophytic plants influences insect population density. The present study provides compelling evidence that the physical characteristics (such as tiller diameter) of grass species are important in insect resistance. Research aimed at breeding grasses which resist S. parvulus infestation must include consideration of plant physical characteristics that make them more or less susceptible to the insect.

70 Variable Estimate Std. Error t P Intercept 0.76 0.11 6.66 <0.00001 Slope 1 -0.01 0.001 -2.04 0.048 Brkpnt. 68.00 28.66 2.37 0.023 Slope 2 0.01 0.01 0.91 0.371 R^=47.9; Brkpnt is the estimated inflection point, slope I is the slope of the line above the inflection point and slope 2 is the slope of the line below the inflection point.

Table 3. Parameter estimates and their standard errors for segmented regression of S. parvulus larval population density on the proportion of endophytic tillers in pure stands of perennial ryegrass

71 ooo h ¥ Q ¥

00

g 3 O § a.

1u u.

Figure 10. Mean number (bar) ± SE (whiskers) of feeding punctures per adult male S. parvulus per day on endophytic perennial ryegrass (PR+), non-endophytic perennial ryegrass (PR-), and Kentucky bluegrass (KB) in non-choice feeding assays (bar=mean, whiskers=standard error).

72 35

30 o • PR+ CN R=0.44 25 U p ü 15 u c eu3 10

"3u üu 5

0

Tiller Diameter (mm)

Figure 11. Relationship between number of adult male S. parvulus feeding punctures and tiller diameter using 3 types of grass, (endophytic (PR+) and non- endophytic (PR-) perennial ryegrass, Kentucky bluegrass (KB))

73 5.0

4.5

§ 4.0 I 3.5 B B u 2.5

0.5

0.0 PR- KB PR+ Grass

Figure 12. Mean (point)±SE (box) and SD (whiskers) tiller diameters of endophytic (PR+) and non-endophytic (PR-) perennial ryegrass, and Kentucky bluegrass (KB) obtained from greenhouse grown flats.

74 140

120 abc

? 100 II c

es > 'E3 C /3

100% PR- 100% KB 40% PR+ 60% PR+ 80% PR+ 100% PR+ Food Source

Figure 13. Mean (point)±SE (box) and SD (whiskers) survival time of field collected adult male S. parvulus on endophytic (PR+) and non-endophytic (PR-) perennial ryegrass, Kentucky bluegrass (KB), and 3 different mixtures of PR+ and KB (40, 60, and 80% PR+).

75 4.0 •

• KB 3.5 ■ • O PR II C • f'i 3.0 • E U ~> 5 • CN • N 2.0 ■ • > • ca • 1.5 ■ • 1 1.0 ■ Q. • o q OO cd 0.5 ■ O • ,c?o 80 5 0 0 0 oo 00 0.0 • • 0 0 20 40 60 80 100 % Endophytic Tillers

Figure 14. S. parvulus larval population densities in plots of Kentucky bluegrass (KB) and perennial ryegrass (PR) containing different proportions of endophytic tillers (July 1995).

76 CJ

o\ « 2.0 g -J

s I Co n=10

PR(E=<15) Grass

Figure 15. Mean (point)±SE (box) and SD (whiskers) S. parvulus larval population density in plots of Kentucky bluegrass (KB) and perennial ryegrass (PR) with < 15% endophytic tillers.

77 2.0 s. p a rv u lu / s91.5 cm~=0.76-0.0I*(%endophyte)+0.01*(%endophyte-68.0)*(%endophyte>68.0) • R"=47.9%

1.5 o UO Os Î 1.0 •• g • -j • • 0.5 • m • # • #

• • • ' ###

0.0 •• # # # « #

10 20 30 40 50 60 70 80 90 100 % Endophytic Perennial Ryegrass Tillers

Figure 16. Scatter plot of the relationship between the percentage of endophyte infected perennial ryegrass tillers and S. parvulus larval population density in pure stands of perennial ryegrass. Endophyte infection calculated from 40 perennial ryegrass tillers taken randomly from each plot. Line represents result of segmented regression analysis.

78 Sward Compositioi

S R II ilCnltivar)

WCî y 0>(a

PR+ PR- RB

t> ( ^

é

Culuvar

Insect Insect Population Density / Damage

Rating

Figure 17. Components of the endophyte-plant-insect interactions model addressed in Chapter 3 (See Chapter I, Model Description for details).

2) The relationship between insect population density and the proportion of endophyte infected L. perenne is a function of plant species composition.

79 CHAPTER 4

RELATIONSHIP BETWEEN BLUEGRASS WEB WORM, Parapediasia teterrella

(Zincken), LARVAL POPULATION DENSITY AND THE PROPORTION OF

ENDOPHYTIC PERENNIAL RYEGRASS, Lolium perenne L., IN MIXED STANDS

WITH KÜENTUCKY BLUEGRASS, Poa pratensis L

Mutualistic symbioses have evolved between certain endophytic fungi and grasses. These fungi provide their grass mutuaiists with an array of anti-herbivore alkaloids whereas the fungi primarily rely on the plant for nutrition (Hill 1994).

Endophyte infected grasses have been widely promoted for use in pest management programs because of their enhanced competitive abilities and resistance to insect herbivory (Murphy et al. 1993).

Perennial ryegrass, Lolium perenne L. (PR), and several species of fescue, Festuca spp., have long been known to harbor fungal endophytes (Sampson 1935, Neill 1940.

1941). These grasses have been commercially developed and are presently available for use in turfgrass. However, the relationship between insect population density and the proportion of endophytic plants in a stand of grass is presently unknown. A closer examination of this relationship will provide a target for implementing endophytic grasses in pest management.

80 Knowledge of the mechanism by which endophytic plants lower insect population density will also be important. Studies have shown that interspersion of host and non­ host plants can lower insect population density through altering movement and searching behavior of the pest insect, host plant suitability, or by enhancing the action of natural enemies (Coll and Bottrell 1994). However, the size of experimental plots is important in experiments designed to address insect responses to mixtures of host and non-host plants because movement may cause reductions in insect population density in small plots that do not hold true at a larger scale (Kareiva 1983).

The goal of this study was to quantify bluegrass webworm, Parapediasia teterrella Zincken. larval survival in relation to the proportion of endophytic perennial ryegrass plants, L. perenne L. (PR+), in stands mixed with Kentucky bluegrass. Poa pratensis L. (KB). The role of insect movement in this regard was also investigated.

Three sets of experiments were performed. The relationship between larval population density and the proportion of PR+ was determined in a series of pots, planted with different mixtures of PR+ and KB, and placed outside in the field. Initial larval density in the pots was manipulated through a controlled introduction of neonates to determine if initial insect population density had any influence on the relationship between sward composition and insect population density at a later time. Choice and non-choice feeding assays were performed in the laboratory to determine P. teterrella larval feeding preference and host suitability of the grasses, and to help separate the effects of grass species from the effects of endophyte infection. Movement assays were performed in the greenhouse to determine if emigration from the pots explained the observed relationship

81 between larval population density and the proportion of PR+. It was hypothesized that

larval population density would not vary linearly with the proportion of PR+ and that a

point may exist at which increases in the proportion of PR+ would not result in additional reductions in P. teterrella populations. If larval movement is the primary mechanism

through which PR+ reduce P. teterrella larval populations in the mixtures with KB, the

relationship between emigration from pots in the greenhouse and the proportion of PR+ should roughly oppose the relationship the percentage of larvae remaining in the pots at the end of the field study and the proportion of PR+.

Materials and Methods

Plant Material

Plant material for all experiments was procured as follows: Two 22.7 kg bags of

PR seed (> 95% PR+) (var. Repell 11 ) from the same seed lot and a 22.7 kg bag of KB

(var. Barron) were acquired in January 1995 from Loft’s Seeds Inc. (Wilmington. OH).

Half of 1 bag of the Repell II was placed in a heated storage area until October 1995 to kill most of the ftmgal endophytes in the seed. The remaining Repell II and the KB were stored at 5°C. In November, 1995, both the heat-treated and cold-stored Repell 11. along with the KB, were seeded into greenhouse flats (53 x 21 cm ) containing commercial potting soil (Promix™ BX, Premier Horticulture Inc., Red Hill, PA). Three flats of each type of grass were seeded for a total of 9 flats. Flats were maintained in the greenhouse through March 1996. Flats were fertilized monthly with 15-10-15 (N:P:K) water soluble

fertilizer and irrigated as needed. During April 1996, individual PR plants were

82 examined microscopically for endophyte presence using the Rose-Bengal staining technique described by Saha et al. (1983). Two mature tillers from each plant were examined and plants were again separated into flats according to their status (E+ or E-).

When needed, individual plants were trimmed to 5 tillers each and 10 plants were randomly placed in 15.2 cm pots. One month was allowed for plants to establish before being used in experiments. Four flats of each grass type were kept to obtain leaves for use in choice / non-choice studies. Flats were maintained in the greenhouse and pots were moved outside to a large sandbox area or placed in the greenhouse for the emigration study. All plant material was maintained at 7.6 cm by hand clipping once a week.

Choice Tests

To investigate feeding preference, a choice test arranged in a randomized block design was used. This experiment was designed to assess the ability of larval P. teterrella to distinguish between 3 plant types:PR+, PR-, and KB (without endophyte). Second- generation adult female P. teterrella were field collected and placed individually into 20 ml scintillation vials to oviposit. Eggs were removed daily, washed under suction using a

1% Clorox® solution, and immediately rinsed with distilled water. Eggs were placed in groups of =50 in petri dishes (9 cm) containing moist filter paper and kept in an environmental chamber maintained at 20°C until eclosion (egg hatch). Within 12 h of eclosion, groups of 10 neonate larvae were placed in the center of experimental arenas presenting a choice of either PR+ versus PR-, PR+ versus KB or PR- versus KB. Two

83 fresh clippings (=2 cm long) of leaf tissue were placed opposing each other, 3 cm apart, in small (4 cm) petri dishes with moist filter paper. The petri dishes containing clippings and larvae were kept in the environmental chamber, and after 24 h, the number of larvae on each type of clipping was recorded. Each treatment was replicated 3 times in each of 3 trials for a total of 9 replicates.

Non-Choice Tests

A randomized block design was also used to evaluate suitability of PR+, PR-, and

KB as host for P. teterrella. Moist filter paper (3 cm) was placed in the bottom of individual cells in a plastic rearing tray. Each tray consisted of 32-4 x 4 cm cells. Eight to 10 leaf clippings (=2 cm long) were placed in the cells so that each contained only 1 type of grass. Five neonate P. teterrella larvae were added to each cell. The tray containing the grass and larvae was placed in an environmental chamber held at 20°C.

Each treatment was replicated a total of 8 times. Fresh clippings were provided every 7 d and as needed. Observations included mortality at 7, 14,21, 28, 35, and 42 d, survival to pupation, and wet pupal mass.

Larval Population Density and Sward Composition

This experiment was performed to assess the relationship between the proportion of PR+ and P. teterrella larval population density. Two different initial larval population densities were used to determine if the proportion of PR+ had a population density dependent effect on the larvae. During June 1996, 15.2 cm (6 inch) pots planted to

84 specific mixtures of KB and PR were moved from the greenhouse to an outdoor arena

(3x3 m) filled 20 cm deep with sand. Treatments included: I) 100% PR-; 2) 100% KB;

3) 100% PR+; 4) 20% PR+, 80% KB, 5) 40% PR+, 60% KB; 6) 60% PR+. 40% KB; and

7) 80% PR+, 20% KB. After irrigating, the plants were allowed to dry and 10 or 20 neonate webworms were added to each of the pots for a 7x2 factorial design (7 plantings

X 2 larval densities) with 6 replicates. Plants were not covered and were irrigated as needed to keep grass from wilting. However, no irrigation or rain was permitted for 2 d after the larvae were introduced. Larvae were allowed to feed and develop in the pots for

30 d, after which, pots were removed to recover the larvae. The contents o f each pot were removed and placed upright in a 10 liter bucket, then drenched with = I liter of soapy water (25 ml Joy® dish washing detergent in 2 liters of water) dispensed from a sprinkling can to disclose the larvae. The pot contents were allowed to set for 10 minutes then a hose with a sprinkling nozzle was used to agitate the surface of the plug until the bucket was almost full. Pot contents were then removed and all water poured through a sieve series (5 mm, 2 mm, 0.8 mm). Contents of the sieves were carefully examined and the number of live larvae found was recorded.

Larval Emigration

Larval emigration from pots in the greenhouse was studied to determine if larval movement caused the observed changes in larval population density recorded from the pots placed in the outside arena. Because of limited numbers of P. teterrella larvae, only

3 of the 7 treatments were used for the emigration study. These included 100% KB,

85 100% PR+ and 50% KB:50% PR+. Pots were arranged on a greenhouse bench in a randomized block design with 12 replicates. Each pot was placed in the middle of a 30 cm styrofoam plate filled with soapy water to catch the insect larvae as they emigrated from the pots. Soapy water was replenished daily. In order to elevate the pots above the soapy water, each pot of plants was stacked inside 2 additional empty 15.2 cm standard pots. Five neonate webworms were placed in each pot during the 1st week of September,

1997. Daily observations were made and the number of emigrating larvae from each pot trapped in the soapy water was recorded. Observations were made for 40 d and cumulative daily emigration (proportion) was used for analysis.

Statistical Analysis

Larval feeding preference was determined using Chi-square replicated goodness of fit. The proportion of larvae on each type of grass after 24 h was reported for analysis.

Non-choice data, including percent survival at 7, 14, 21,28, 35, and 42 d. and percent survival to pupation were arcsin-square root transformed prior to analysis whereas mean pupal masses were subjected to cubic transformation. Repeated-measures ANOVA was used to elucidate differences in larval survival between the different grasses through time and MANOVA was used to determine differences in survival to pupation and fresh pupal mass. Analysis of variance and contrast analysis were also used to determine differences in larval survival between the 3 major grass types (KB, PR- and PR+) in the field pots.

Regression analysis was used to determine the influence of the proportion of PR+. initial larval population density, and their interaction, on the percentage of P. teterrella larvae

86 recovered from pots in the field. The proportion of larvae recovered from the field pots was arcsin-square root transformed to meet the assumptions of the analysis. Arcsin- square root transformed cumulative emigration data were analyzed using repeated- measures ANOVA. All statistical analyses were performed using Statistica® release 5

(StatSoft® Inc., Tulsa, OK, 1995).

Results

Choice Tests

A significantly larger proportion of P. teterrella larvae chose KB and PR- over

PR+ after 24 h, indicating a strong preference for both over PR+ (X‘ =45.2. df = 17, P <

0.01 and X' = 36.8, df = 17, P < 0.01, respectively)(Figures 18a and 18b). There were no differences in the number of larvae choosing KB and PR- (X" = 9.4, df = 17, P = 0.927)

(Figure 18c). Some evidence of feeding was observed on all plant material.

Non-Choice Tests

Larval mortality at 7 d was significantly greater on PR+ compared to both the KB and PR- (F = 277; df = 1, 21; P < 0.0001) but did not differ significantly between the KB and PR- (F = 1.56; d f=1, 21; P = 0.226) (Figure 19). Larval mortality at 14 d did not differ significantly between KB and PR- (F = 0.309 d f=1, 21, P = 0.584) but mortality on

PR+ was higher than both (F=229; df=l, 21; P<0.0001). No larvae survived to 21 d on

PR+, but no difference in survival was detected between KB and PR- at 21, 28, 35, or 42 d (F<1.01; df=l, 21; P>0.430 for all comparisons) (Fig. 19). No larvae on PR+ survived

87 to pupation and there was no significant difference in survival to pupation between PR- and KB (F = 0.24, df = 21, P = 0.629) (Table 4). Fresh pupal weights were not significantly different between PR- and KB (F = 0.990, df = 21, P = 0.331)(Table 4).

Again, evidence of leaf feeding was observed on all 3 types of grass used in the study.

Larval Population density and the Proportion of Endophytic Plants

Larval survival was significantly lower in pots containing 100% PR+ when compared to either 100% PR- or 100% KB treatment (F=49.99; df=l. 33; P<0.01 and

F=56.48; df=l, 33; P<0.01 respectively) (Table 4). No difference in larval survival was detected between pots containing 100% PR- and 100% KB (F=0.20; df=l. 33; P=0.659).

Regression indicated there was no interaction between initial larval population density and the proportion of PR+(T=0.13; df=68; P=0.897) and initial larval population density did not influence the percentage of larvae remaining in the pots after 30 d (T=0.38; df=68; P=0.705). The relationship between the proportion of PR+ and the percentage of larvae remaining in the pots after 30 days was best described by a segmented regression model (Table 5) as follows:

Y=bo+b,*X+b 2 * (X-brkpnt) * (X>brkpnt) where;

Y= % larval survival after 30 d

X= proportion PR+

Brkpnt= inflection point

bo, bj, and b,=regression coefficients

88 The logical expression (X>brkpnt) acts as a multiplier in the model. If the expression is true, (X>brkpnt) will evaluate to 1; if it is false, (X>brkpnt) will evaluate to 0. For turfgrass swards where X is less than or equal to the estimated breakpoint:

Y=bo+b,*X

For swards where X is greater than the estimated breakpoint:

Y=bo+b, *X+b 2 *(X-brkpnt)

For values of X greater than ± e estimated breakpoint, the slope is equal to bj+b^ and the intercept is equal to (bo-brkpnt^bi).

Results indicated a strong negative relationship between final larval population density and the proportion of PR+ (slope 1: T=-4.08; df=67; P<0.0001) (Table 5. Figure

20). However, only slight decreases in larval population density were observed beyond

40.3 ± 19.0% endophytic plants (slope 2: T=1.875; df=67 P=0.065).

Larval Emigration

A significant treatment x time interaction indicated that the shape of the cumulative emigration curves differed between treatments over time (F=2.7; df=78, 936;

P<0.0001) (Figure 21). Cumulative emigration from the PR+ monoculture increased sharply in the lst-2 d and remained higher than either the KB monoculture or the PR+/KB polyculture for the remainder of the experiment (F>4.5; df=l, 25; P<0.05). There were no significant differences between cumulative emigration curves for the polyculture and

KB monoculture at any point in the experiment (F<2.8; df=l, 24; P>0.10). After 40 d,

89 10.0,21.6, and 48.3% of the larvae had emigrated from the KB monoculture, the

polyculture and the PR+ monoculture respectively.

Discussion

By far the most important determinant of host suitability and preference for P. teterrella was presence of the endophyte, N. lolii, in the plant. Both PR- and KB were equally suitable and preferable hosts. In mixed stands of PR and KB, the largest plant

influence on P. teterrella populations will be the proportion of PR+. Therefore, results from the multi-species stands of grass used in the present studies should translate to monocultures of PR as well.

The choice and non-choice assays provided a background for evaluating survival of P. teterrella larvae in mixed stands of PR+ and KB. The choice assay affirmed that larval webworms avoid PR+ and seek PR- and KB. This fact, coupled with the high mortality suffered by larvae forced to feed on PR+, imply both antixenosis and antibiosis resistance accompany N. lolii infection. Indeed, both modalities have been previously documented (Johnson-Cicalese and Funk 1988, Latch 1993) with endophytic grasses.

However, this is essential mechanistic knowledge for describing insect pest population dynamics in this system. Although P. teterrella larvae actively moved away from PR+. they still bit into infected leaf tissue. Therefore, the possibility of sublethal or cumulative toxic effects must be considered. Although the dose of toxin required to elicit a behavioral response is usually much lower than that required to induce mortality, acquisition of at least some small dose is usually required to elicit a behavioral response

90 (Hoy et al. 1998). Further study is needed to determine if repeated sampling of endophytic plants has a cumulative influence on insects.

Segmented regression analysis provided a useful tool for determining the relationship between the proportion of PR+ and P. teterrella larval population density.

The analysis indicated a strong negative relationship between larval population density and the proportion of PR+ up an inflection point at 40.3%. After the inflection point, the relationship between the proportion of PR+ and larval population density did not have a significant slope at a=0.05. This indicates that increases in the proportion of PR+ beyond

40.3% may not cause continued reductions in larval population density. Instead, larval population density can be minimized at in swards containing as little as 40% PR+. These results are somewhat similar to those of Fribourg et al. (1991) who reported decreases in the performance (e.g., weight gain) of cattle in relation to the proportion of endophyte infected tillers in pastures, but found that performance was not further depressed in pastures containing more than 35% endophytic tall fescue

The fact that some larvae survived and remained in 100% PR+ treatment of the field pot study was somewhat surprising. The cultivar of PR used (Repell II) is a synthetic cultivar. This means that although all of the perermial ryegrass plants were similar, none were genetically identical. Because endophytic fungi are inherited maternally through the seed, the variation represented by the endophytes within these plants would be a direct reflection of the actual number of female parents used to synthesize the cultivar. Results of earlier work (Roylance et al.l994) imply that the

91 interaction between host plants and endophyte genotypes could produce endophyte-plant combinations that express only sublethal levels of toxins.

Alternatively, individual larvae may have varied in their susceptibility to the toxins. If such variation exists as a heritable genetic trait, insects capable of metabolizing these toxins would gain an ecological advantage in areas where PR+ is common (e.g., monostands of endophytic grass). As a result, localized populations of resistant insects could develop. The black cutworm, Agrotis ipsilon Hufhagel, is relatively unaffected by endophyte toxins, indicating that metabolism of these alkaloids by other Lepidoptera is possible (Williamson and Potter 1997). The present results imply that high proportions of PR+ could actually be detrimental to the long term sustainability of fungal endophytes as a source of insect resistance in turfgrass. The presence of réfugia, in the form of non-endophytic plants, may relax potential selection pressure thereby increasing the durability of endophyte-mediated resistance without sacrificing the potential utility of these grasses in integrated pest management. Further research will be needed to address this idea.

Larval emigration from pots in the greenhouse did not explain reductions in population density observed in the pots placed in the field. Whereas larval population density in the field pots was essentially minimized at 40% PR+, cumulative emigration in the greenhouse did not differ between the KB monocultures and pots containing 50%

PR+ (PR+/KB polyculture). Emigration from the PR+ monoculutres was significantly higher than either of the other treatments. However, larval densities in the field pots containing 100% PR+ did not decrease accordingly. The influence of natural enemies

92 and resource concentration may be complimentary mechanisms in lowering herbivore numbers in some polycultures (Russell 1989), but more detailed study is needed to clarify the influence of each in this system. The action of natural enemies or the cumulative influence of repeatedly sampling endophytic plants need also be considered in future studies.

Conclusion

The present study provides important information with regard to the relationship between insect population density and the proportion of PR+, and demonstrates how emigration plays a role in this relationship (Figure 22). These studies show that P. teterrella larval densities are generally lower in mixed stands containing PR+, but this relationship may plateau in stands containing more than 40.3±19.0 % PR+. Emigration from the pots was probably only partially responsible for these reductions even though larvae clearly avoided PR+ after contact with the toxins. It is possible that cumulative toxic effects and natural enemies may act in concert to lower P. teterrella larval densities in mixed swards of KB and PR+. Future research efforts should endeavor to clarify the interaction between the various components which influence P. teterrella populations in mixed stands of PR+ and KB. Such studies will be useful for establishing a baseline for implementing these and other resistant grasses into insect management strategies.

93 Variable PR+ PR- KB

% Survival to Pupation (lab) 0.0 52.5±7.5 55.0±9.1

Average Fresh Pupal Mass (mg) a 16.3±0.9 15.3±0.8

% Survival in Pots 1.7±1.1 24.6±4.2 27.1±4.4 a = no survival to pupation

Table 4. Survival to pupation and fresh pupal mass of P. teterrella larvae fed clippings of endophytic (PR+) and non-endophytic (PR-) perennial ryegrass, and Kentucky bluegrass (KB) in the laboratory, and survival of larvae after 30 d on PR+, PR-, and KB in pots placed outside.

94 Parameter Coefficient std. error t P

Intercept 0.483 0.071 6.845 0.000

% Endophyte below breakpoint -0.007 0.002 -4.085 0.000

Breakpoint 40.339 18.964 2.127 0.037

% Endophyte above breakpoint 0.004 0.002 1.875 0.065

R—53.3

Table 5. Parameter estimates and standard errors for the segmented regression model describing the relationship between P. teterrella larval population density and the proportion of endophytic perennial ryegrass tillers in mixed stands with Kentucky bluegrass

95 B o CN

c/j I 0 8 o * c 06 o du g

c o o 02 oCL

00 KB PR+PR. PR+ KB PR-

Choice Test

Figure 18. Mean (point)±SE (box) and SD (whiskers) proportion of larval P. teterrella on endophytic (PR+) and non-endophytic (PR-) perennial lyegrass and Kentucky bluegrass (KB) after 24 h in choice tests.

96 100 I

80 CQ > I E 3 60 cr "S cs -J 40

20

0

Days

Figure 19. Mean (point)±SE (box) and SD (whiskers) survival of larval P. teterrella on endophytic (PR+) and non-endophytic (PR-) perennial ryegrass and Kentucky bluegrass (KB) at 7, 14, 21, 28, 35, and 42 d in the lab.

97 6 0 • 1 pot • 2 pots • 3 pots 50 • 4 pots • 5 pots I 40 • 6 pots uo e 7 pots 30 10 pots * • a 20

10

20 40 60 80 100 % Endophytic Perennial Ryegrass

Figure 20. Frequency scatter plot of the relationship between the percentage of endophyte infected perennial ryegrass plants in 12 pots containing mixtures of Kentucky bluegrass and perermial ryegrass, and the percentage of P. teterrella larvae recovered from the pots after 30 d. Line represents result of segmented regression analysis.

98 0.35 100% PR+ 50% PR+ 100% KB

S 0.25 ÜD

Figure 21. Mean (point)±SE (whiskers) cumulative proportion of P. teterrella larvae (n=60) emigrating from pots containing endophytic perennial ryegrass (PR+), Kentucky bluegrass (KB) or a mixture of PR+ and KB in the greenhouse over the course of 40 d.

99 Sward Compositiot

SR %E+ Cultivar Seed

ixr%CD ^ CD Cp

PR-PR+ KB

Cultivar

Insect Insect Population Density Damage

Rating

Figure 22. Components of the endophyte-plant-insect interactions model addressed in Chapter 4 (See Chapter I, Model Description for details).

2) The proportion of endophytic L. perenne dictates insect population density mainly through increasing mortality. Emigration is likely not an important mechanism through which endophytic plants lower insect populations until the proportion of endophytic L perenne is extremely high.

100 CHAPTERS

CONCLUDING REMARKS

This dissertation provides information relevant to key aspects of the turfgrass ecosystem model proposed in Chapter I (see Figure 23). Changes in the composition of existing Kentucky bluegrass, Poa pratensis L., (KB) turf communities were measured after overseeding with perennial ryegrass, Lolium perenne L. (PR), containing both endophytic (PR+) and non-endophytic (PR-) seed, at two different rates (kg/ha) using two different sources of seed (Chapter 2). The relationship between bluegrass billbug,

Sphenophorus parvulus Gyllenhal, population density and the proportion of PR+ was measured in pure stands of PR (Chapters 3) and mixed swards of KB and PR (Chapter 2

& 4). Field studies also addressed the relationship between S. parvulus population density and damage in mixed sward of turfgrass (Chapter 2). Results suggest that insect feeding alters turfgrass sward composition, at least over the short term (Chapter 2).

Overseeding Rate and Sward Composition

Overseeding PR into established stands of KB (Chapter 2) allowed an initial establishment of PR+ and PR-. The proportion of PR then increased over the duration of the study (2 y). The higher overseeding rate (97.6 kg/ha) initially produced swards with a greater proportion of PR. However, over time the proportion of PR resulting from the

101 two different overseeding rates converged and no difference in sward composition was observed between the two overseeding rates by the second year of the study. Because there was less available space per seed in the plots overseeded at the higher rate, the proportion of PR in those plots increased at a slower rate. Swards of KB overseeded with

PR at different rates ultimately converged upon a similar sward composition because of

intraspecific competition for limited space and because management practices (e.g., mowing height, fertility and irrigation) were the same for all plots.

Seed Source and Sward Composition

The seed source was an important determinant of sward composition through time

(Chapter 2). Over time, sward composition was not a direct reflection of the proportion of PR+ seed, but was more closely related to differences between culivars. Differences in tiller density, growth habit and tillering capacity, as well as tolerance to temperature extremes and shade are known to exist between cultivars of PR (Beard 1973). However, the literature suggest that cultivars containing a greater proportion of PR+ seed should establish more quickly and compete better than cultivars with a lower percentage of PR+ seed (Clay 1987). My results indicate differences in the composition of stands overseeded with different cultivars of PR are more likely due to agronomic characteristics that vary between cultivars.

102 Sward Composition and Insect Population Density

Sward composition did influence insect population density. This part of the model was explored using S. parvulus larvae in the field in monostands of PR (Chapter 3) and mixed stands of PR+, PR-, and KB (Chapter 2). Bluegrass webworm, Parapediasia teterrella Zincken, larvae were used for the same purpose in pots planted to specific mixtures of KB and PR+ (Chapter 4). In each instance, a segmented-Iinear equation describing the relationship between insect population density and the proportion of PR+ was generated. In the case of S. parvulus, the relationship between the proportion of PR+ and larval population density differed somewhat depending on the species composition of the sward (e.g., PR monoculture or PR - KB polyculture). In mixed swards of PR and

KB. resulting from overseeding, S. parvulus larval population density steadily decreased as the proportion of PR+ increased to approximately 38% of the total stand. Further reductions in S. parvulus larval population density were not observed in swards containing higher proportions of PR+. In contrast, S. parvulus larval population density in PR monocultures continued to decrease until PR+ comprised approximately 68% of the total sward although with no data points between 42 and 65% PR+, the point where S. parvulus populations no longer decreased was more difficult to estimate acurately . The relationship between the proportion of PR+ and S. parvulus larval population density may be mediated by the species composition of the stand.

In mixed stands of KB and PR, the relationship between larval population density and the proportion of endophyte infected tillers was similar for P. teterrella and S. parvulus. In this case, P. teterrella larval population density decreased as the proportion

103 of PR+ increased to approximately 40%. Again, higher proportions of PR+ did not cause further reductions in larval population density. These studies indicate that moderate proportions of PR+ will provide significant reductions in the population density of S. parvulus and P. teterrella in polystands of PR and KB. However, if PR- is a superior host by comparison to P. pratensis (e.g., adult male S. parvulus) a higher proportion of endophytic tillers may be needed to have the same activity in monocultures of perennial ryegrass. Alternatively, if PR- is an inferior larval host due to smaller tiller diameters, an interaction between the proportion of PR+ and plant species composition may account for the continued decrease in S. parvulus larval populations beyond those observed in mixtures of KB, PR-, and PR+.

The observed relationship between insect population density and the proportion of

PR+ in mixed swards may partially explain why the proportion of endophyte infected individuals found in wild grass populations is notably low considering the profound impact endophytes can have on host fitness (Lewis et al. 1997). This pattern occurs because plants that are more abundant are generally less palatable to herbivores and their presence creates "microsites" where herbivory is reduced and more palatable plants can establish (Hay 1986). In the absence of unpalatable plants, more palatable plants may become locally extinct. Hence, the cost of being associated with another plant may be less than the cost of increased herbivory in the absence of that plant. This type of associational resistance to herbivory may actually maintain plant diversity (the number of different kinds of plants present) within a community because the most common competitor can increase the number of different kinds of plants that can establish and

104 survive (O'Dowd and Williamson 1979). Because moderate levels of endophytic plants severely reduce insect populations, non-endophytic plants may benefit. Further research will be needed to address this idea in an evolutionary context.

Sward Composition and Insect Movement

The greenhouse emigration studies provided a partial mechanistic explanation for the relationship between the proportion of PR+ and P. teterrella population density

(Chapter 4). In theory, insect movement (into, out of. or within a stand of vegetation) is the primary mechanism through which vegetation texture influences insect population density. However, studies performed in the greenhouse showed that cumulative larval emigration from the pots was significantly elevated only in pure stands of PR+. This result indicates that insect movement, on the scale measurable in these experiments, was not significantly increased through moderate alteration of turfgrass swards with PR+.

Because immigration was not permitted, the cumulative influence of repeated ingestion of toxic alkaloids and or the action of P. teterrella's natural enemies are mechanisms worthy of further investigation.

Sward Composition and Insect Damage

Introduction of PR+ into established stands of KB enhanced the visual quality of the turf through lowering insect population density (Chapter 3). The visual rating of the turf stand decreased linearly as S. parvulus larval population density increased in the field. However, laboratory assays indicated that factors other than endophyte infection

105 (e.g., tiller diameter) are important determinants of adult S. parvulus feeding behavior.

Therefore, plant species composition or the physical characteristics of the plants used, will also determine the degree to which billbugs damage a sward. This was not the case for P. teterrella. Instead, endophyte infection was more important than plant species as a determinant of P. teterrella feeding. However, because other plant characteristics (e.g.. leaf thickness, pubescence, cuticle thickness, etc...) were not measured in the P. teterrella feeding assays, their possible influence on host plant selection is unknown.

Plots treated with the systemic insecticide imidacloprid had lower S. parvulus larval populations and less damage than untreated plots overseeded at the same rate. By the end of the study, stands that received no insecticide had a significantly greater proportion of PR than stands where billbugs were excluded with insecticide.

Furthermore, PR+ comprised a greater proportion of PR in stands where insecticide was not applied. These data imply that the competitive advantage provided to PR+ by the endophyte was reduced in stands where billbugs were excluded with insecticide. S. parvulus larval feeding caused damage to KB and PR-, thereby further decreasing their competitive abilities in relation to PR+. Theory predicts a physiological tradeoff between anti-herbivore defenses and plant growth (Herms and Mattson 1992). To synthesize anti­ herbivore alkaloids, endophytes use plant resources which may otherwise be used by the plant for metabolic processes or structural purposes. However, such a physiological tradeoff does not necessarily have ecological consequences for the plant. For instance, tannins which act as barriers to insect herbivory, may also enhance drought tolerance by increasing the resilience of cell walls (Pizzi and Cameron 1986). A trait may be selected

106 for because it provides stress tolerance and because it provides defense against herbivory

(correlated selection).

Conclusions

The initial hypothesis that this system may be viewed as a feedback system (sward composition determines insect population density which determines damage to the stand which again determines sward composition) appears to be correct. The proportion of

PR+ in a stand will dictate insect population density and the expression of damage, although plant species composition must also be considered. Shifts in sward composition toward stands with more PR+ occurred more rapidly when insect population densities were high. Results of the studies presented in Chapters 3 and 4 indicate a need for more research on the mechanism through which PR+ reduces insect population density in turfgrass. Although much of the variation in the data was explained by various factors in the model, much remains unexplained. It is likely that tiller density would explain additional variation in sward composition, and insect population density and damage between overseeding treatments. The types and concentrations of alkaloids produced by particular endophyte-grass combinations may also explain variation in the relationship between insect population density and the proportion of PR+. However, results of the current studies indicate that spatial distribution of the toxins (i.e., proportion of PR+). not their concentrations within the plant, is a more important determinant of insect population density. Future research efforts should endeavor to clarify the possible action of natural enemies in turfgrass swards containing PR+. A closer inspection of the dose acquisition

107 process is also needed to clarify how the alkaloids associated with endophyte infection influence the behavioral and physiological responses of insects. Studies on the dose acquisition process should address the cumulative influence of repeatedly sampling toxic plant material in mixed stands containing PR+. Investigations of this type will provide further explanation of the relationship between insect population density and the spatial distribution of PR+ and should enhance the utility of fungal endophytes in pest management.

108 ^w vd

SR

oc% 'C>c>

PR- KB

tosect Populatioo Density Fenility 1 H p

Ratmgl

Figure 23. Revised hypothetical model of endophyte-plant-insect interactions based on the literature and the current series of investigations (See Chapter I. Model Description for details)

1 ) Sward composition is influenced by the seeding rate (SR) the cultivar(s) used, and cultural management (HjO, fertility, pH, mowing). 2) Because fertility, irrigation (HiO), and cultivar may influence the types and concentrations of alkaloids produced, they may mediate the influence of PR+ on insect population density. Further, PR+ may influence insect population density indirectly through altering insect movement (emigration (Em) and immigration (Im)), or directly through reducing fecundity (F), or increasing mortality due to toxicity (M). Aside from endophytic plants, plant species composition can also influence insect population density. 3) Damage resulting from insect feeding is a frmction of insect population density, the proportion of endophytic plants, plant species composition, cultivar, fertility, irrigation, and the overseeding rate. 4) Insect damage will influence stand composition by reducing the proportion of PR- and KB.

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122 APPENDIX A

Detection of Neotyphodium lolii Using TPIB.

Mature vegetative tillers were collected by cutting at soil level. Dead or senescent leaf material was removed and 2 cross sections ( = Imm) were taken from the cut end of the sheath area of each tiller using a razor blade or scalpel. The cross sections were placed cut side down (1 at a time) on a 0.45 micron nitrocellulose membrane (14 x 14 cm) [Trans-Blot® Transfer Medium, Bio Rad, Hurcules, CA]. Materials were layered in the following order: filter paper, nitrocellulose membrane, tissue cross section. The tissue cross sections were covered with wax paper and pressure was applied using a small pestle or similar object. After all samples were blotted, the nitrocellulose membrane was baked in a vacuum oven @ 80°C for 1 h. The baked nitrocellulose membranes were then placed in sealable plastic containers (Rubbermaid® Servin' Saver 2 cups). Blocking solution (50 ml) [(0.5% Carnation nonfat dry milk in Tris buffered saline (0.02 M Tris, pH 7.5 + 0.05 M NaCl)] was added and the containers were shaken overnight @ 4°C.

The next morning, the Blocking Buffer was poured off and replaced with antiserum diluted 1:1000 in Blocking Buffer. The nitrocellulose was shaken again with the antiserum diluted in blocking solution at room temperature for 2-4 h. The antiserum was then removed and the blots were rinsed with Blocking Buffer 5 times at 6 minutes each

(pour off old Blocking Buffer, add new Blocking Buffer and place on shaker for 6

123 minutes). After the final rinse. Protein A-alkaline Phosphatase [Anti-Rabbit IgG (whole molecule) Sigma A-8025, Sigma-Aldrich, St. Louis, MO) was added at a 1:1000 dilution in Blocking Buffer (or 0.4 pg/ml in Blocking Solution) and allowed to shake at room temperature for 2-4 h. The blots were then rinsed as before, 2 to 3 times in Blocking

Buffer.

During the last rinse, color reagents were weighed, mixed and filtered. Fast red solution was made to 1.33 mg/ml concentration using 0.2 M Tris Buffer, pH 8.2 . The other reagent was Naphthol AS-MX Phosphate made in 0.2 M Tris Buffer, pH 8.2

(0.1%). This was made in a separate container and required 0.03 g/30 ml Buffer. Both solutions were filtered separately, added to the containers and mixed (the Naphthol solution was added 1st). 30 ml of each reagent solution was used for each container.

The blots were then placed on the shaker. Best color development was observed at 15-30 minutes. The color reaction was stopped by rinsing with distilled water. Arcsin square- root transformed proportion infected shoots was reported for the analysis.

124 APPENDIX B

Overseeding Data

Cultivar Rep Tmt RyeJune96 BB96 RyeOct96 RyeJune97 BB97 RyeOct97 Endc

III 0.000 .50 0.00 .025 5.00 0.00 0.00 1 I 2 .330 .24 .36 .835 1.20 .78 .60 II 3 .175 .20 .42 .625 .40 .73 .50 I 1 4 .320 .24 .47 .860 1.20 .83 .60 I 2 4 .420 .52 .44 .915 .40 .92 .68 1 2 3 .405 .39 .35 .855 .20 .67 .46 I 2 2 .320 .62 .36 .810 .60 .74 .48 1 2 1 .005 .26 0.00 0.000 2.60 0.00 0.00 1 3 3 .315 .36 .38 .715 1.20 .49 .45 1 3 I 0.000 .75 0.00 0.000 2.80 0.00 0.00 1 3 4 .435 .64 .52 .820 1.80 .75 .43 1 3 2 .210 .34 .30 .760 1.20 .70 .70 I 4 3 .175 .08 .32 .555 .20 .53 .43 1 4 4 .360 .63 .47 .775 1.80 .69 .58 1 4 1 0.000 .75 0.00 0.000 2.40 0.00 0.00 1 4 2 .180 .33 .27 .600 1.40 .67 .79 1 5 3 .165 .32 .18 .465 1.00 .43 .40 1 5 1 0.000 .25 0.00 0.000 3.60 0.00 0.00 I 5 4 .250 .47 .47 .665 1.60 .74 .55 1 5 2 .240 .22 .37 .610 .80 .64 .52 1 6 3 .075 .17 .30 .665 .20 .62 .40 1 6 2 .260 .97 .47 .800 2.20 .78 .48 I 6 4 .385 .38 .50 .800 1.60 .81 .55 I 6 1 0.000 .13 0.00 0.000 3.00 0.00 0.00 1 7 2 .300 .61 .39 .675 .80 .73 .60 1 7 3 .225 .34 .34 .755 1.40 .61 .70 1 7 I 0.000 .50 0.00 0.000 2.40 0.00 0.00 1 7 4 .270 .10 .46 .595 1.40 .67 .60 1 8 I 0.000 0.00 0.00 0.000 3.40 0.00 0.00 1 8 3 .285 .11 .30 .570 .40 .44 .63 1 8 2 .270 .35 .39 .710 2.20 .45 .60 1 8 4 .220 .09 .35 .615 .40 .60 .63 2 1 1 .005 .38 0.00 .020 2.20 .02 0.00 2 I 2 .295 .36 .46 .480 .80 .46 .55 2 I 3 .370 .50 .31 .370 1.20 .30 .18 2 1 4 .390 .63 .28 .430 2.20 .35 .13 2 2 4 .315 .36 .44 .475 2.00 .58 .38

125 Cultivar Rep Tmt RyeJune96 BB96 RyeOct96 RyeJune97 BB97 RyeOct97 Endc

2 2 3 .125 .31 .20 .480 1.60 .41 .13 2 2 2 .255 .47 .41 .485 2.80 .43 .30 2 2 1 0.000 .25 0.00 0.000 3.20 0.00 0.00 2 3 3 .190 .33 .22 .460 1.20 .55 .33 2 3 1 .035 .15 .01 .005 2.80 .02 0.00 2 3 4 .430 27 .51 .635 220 .60 .19 2 3 2 .315 .86 .36 .525 420 .65 .13 2 4 3 250 .10 .33 .290 .80 .51 .28 2 4 4 .300 .23 .42 .455 .60 .59 .35 2 4 1 0.000 .13 .01 0.000 .80 0.00 0.00 2 4 2 .205 21 .38 .465 1.40 .56 .23 2 5 1 0.000 0.00 0.000 2.20 0.00 0.00 2 5 2 .345 .29 .540 .80 .53 .33 2 5 3 .360 .51 .475 1.20 .45 .20 2 5 4 .370 .34 .585 2.00 .59 .65 2 6 I .005 0.00 .005 .80 0.00 0.00 2 6 2 .260 .41 .600 1.60 .74 .30 2 6 3 .300 29 .600 .40 .62 .40 2 6 4 .410 .42 .600 .60 .84 .28 2 7 4 .540 .53 .775 1.00 .71 .15 2 7 3 .395 .44 .645 .60 .60 .18 2 7 2 .405 .52 .555 1.20 .72 .25 2 7 I 0.000 0.00 0.000 1.40 0.00 0.00 2 8 4 .560 .55 .670 1.60 .78 .33 2 8 I 0.000 0.00 0.000 .80 .01 0.00 2 8 3 .585 .61 .665 1.00 .56 .33 2 8 2 .550 .45 .645 2.20 .74 .28

126 APPENDIX c

Billbug Population InJL perenne Monocultres Data

Plot Year PctEndo BBn

102 1 20.0 1.000 105 1 76.0 0.000 201 1 14.0 .670 203 1 96.0 0.000 205 1 96.0 0.000 305 1 34.0 .670 401 1 76.0 0.000 402 I 94.0 .330 403 1 96.0 .170 405 1 54.0 .500 504 1 12.0 .670 601 1 68.0 .330 605 1 48.0 .500 705 1 14.0 .670 801 1 18.0 .170 803 1 92.0 0.000 903 1 94.0 .170 904 I 4.0 .500 1001 1 94.0 .330 1005 1 8.0 .170 102 2 20.0 .250 105 2 90.0 0.000 201 2 0.0 0.000 203 2 90.0 0.000 205 2 95.0 0.000 305 2 25.0 .250 401 2 75.0 0.000 402 2 100.0 0.000 403 2 75.0 0.000 405 2 55.0 0.000 504 2 15.0 .250 601 2 75.0 0.000 605 2 35.0 0.000 705 2 15.0 0.000 801 2 15.0 .125 803 2 95.0 .125 903 2 75.0 .125 904 2 5.0 .125

127 Plot Year PctEndo BBn

1001 2 85.0 0.000 1005 2 5.0 .250 102 3 30.0 .400 105 3 90.0 0.000 201 3 13.0 .800 203 3 100.0 0.000 205 3 93.0 0.000 305 3 10.0 0.000 401 3 53.0 0.000 402 3 93.0 0.000 403 3 100.0 0.000 405 3 10.0 .600 504 3 10.0 .600 601 3 83.0 .200 605 3 23.0 200 705 3 7.0 .400 801 3 7.0 .400 803 3 93.0 200 903 3 96.0 0.000 904 3 3.0 1.000 1001 3 93.0 0.000 1005 3 10.0 1.800

128 APPENDIX D

Adult Male Billbug Longevity Data

Rep Tmt Pctendo Avg Long I 1 0 110.5 1 2 0 35.8 1 3 40 116.5 1 4 60 45.5 1 5 80 40.8 1 6 100 38.8 2 1 0 91.0 2 2 0 30.5 2 3 40 40.8 2 4 60 44.5 2 5 80 60.0 2 6 100 36.8 3 I 0 90.3 3 2 0 55.5 3 3 40 58.5 3 4 60 42.5 3 5 80 71.5 3 6 100 54.8 4 1 0 113.8 4 2 0 121.5 4 3 40 117.3 4 4 60 46.5 4 5 80 28.3 4 6 100 51.0 5 1 0 78.0 5 2 0 155.3 5 3 40 55.5 5 4 60 104.5 5 5 80 105.3 5 6 100 44.3 6 I 0 107.8 6 2 0 35.0 6 3 40 87.3 6 4 60 75.3 6 5 80 55.5 6 6 100 78.8 7 1 0 86.3 7 2 0 66.3 7 3 40 108.3

129 Rep Tmt Pctendo Avg Long

7 4 60 62.5 7 5 80 38.0 7 6 100 26.5 g I 0 70.0 8 2 0 33.3 8 3 40 30.5 8 4 60 36.3 8 5 80 50.5 8 6 100 32.5

130 APPENDIX E

Adult Male Billbug Feeding Preference

Day Rep Tmt Tildia Feedmrk 1 I I 2.28 3.80 1 I 2 2.72 .80 1 1 6 2.12 3.20 1 2 1 1.84 3.00 I 2 2 3.00 3.20 1 2 6 2.04 320 1 3 1 2.12 3.00 1 3 2 2.68 .60 I 3 6 2.12 0.00 1 4 1 1.84 4.00 I 4 2 2.16 1.40 1 4 6 2.36 1.80 1 5 1 1.84 .20 1 5 2 2.68 2.20 1 5 6 2.04 5.40 1 6 1 2.16 3.80 I 6 2 2.76 1.40 I 6 6 1.60 5.40 1 7 1 2.12 3.20 I 7 2 2.60 1.20 1 7 6 1.84 3.60 1 8 1 1.76 4.40 I 8 2 2.60 1.20 1 8 6 2.20 .80 2 1 1 2.48 3.80 2 I 2 3.96 3.40 2 I 6 2.28 0.00 2 2 1 2.36 5.60 2 2 2 3.52 .60 2 2 6 2.20 1.20 2 3 1 2.60 4.60 2 3 2 3.00 2.40 2 3 6 2.72 1.40 2 4 1 2.64 4.80 2 4 2 3.12 2.60 2 4 6 1.84 2.00 2 5 1 2.36 1.60 2 5 2 3.08 1.60 2 5 6 2.24 4.60

131 Day Rep Tmt Tildia Feedmrk

2 6 I 2.04 3.80 2 6 2 3.52 3.40 2 6 6 2.40 3.40 2 7 I 2.24 2.00 2 7 2 324 120 2 7 6 2.44 3.40 2 8 1 2.76 3.20 2 8 2 3.56 1.00 2 8 6 2.04 2.40 1 I 2.40 6.20 I 2 3.44 4.00 1 6 2.28 4.80 2 1 3.00 5.80 2 2 3.96 3.80 2 6 2.68 3.00 3 1 2.68 5.40 3 2 3.32 2.00 3 6 2.36 2.80 4 1 2.48 2.00 4 2 3.20 9.20 4 6 2.00 6.00 5 1 2.56 3.80 5 2 3.36 4.40 5 6 2.48 5.80 6 1 2.72 5.40 6 2 2.88 2.40 6 6 2.40 5.00 7 1 2.56 2.40 7 2 2.92 3.80 7 6 2.36 5.40 8 t 2.32 4.80 8 2 3.00 2.80 3 8 6 2.04 2.80 4 1 1 2.48 8.40 4 1 2 3.72 6.00 4 1 6 2.72 7.80 4 2 1 2.84 5.80 4 2 2 3.00 4.60 4 2 6 3.20 4.40 4 3 1 2.76 6.80 4 3 2 3.60 7.20 4 3 6 3.16 6.80 4 4 1 2.40 6.00 4 4 2 3.64 6.00 4 4 6 2.60 10.00 4 5 1 2.52 6.20 4 5 2 2.56 5.60 4 5 6 2.44 6.80 4 6 1 2.68 11.40 4 6 2 3.08 8.40

132 Day Rep Tmt Tildia Feedmrk

4 6 6 2.52 7.60 4 7 I 2.44 4.00 4 7 2 3.36 7.00 4 7 6 2.52 7.60 4 8 1 2.76 5.40 4 8 2 3.04 520 4 8 6 2.72 8.20 5 I 1 2.68 6.20 5 I 2 2.76 420 5 1 6 2.40 8.00 5 2 1 2.20 9.40 5 2 2 3.36 3.20 5 2 6 2.36 4.00 5 3 1 2.64 720 5 3 2 2.76 6.20 5 3 6 2.64 5.00 5 4 1 2.44 820 5 4 2 3.00 1.80 5 4 6 2.56 7.40 5 5 1 2.60 3.80 5 5 2 2.84 7.60 5 5 6 2.32 4.80 5 6 1 2.72 5.20 5 6 2 2.80 5.20 5 6 6 2.60 5.00 5 7 1 2.32 420 5 7 2 2.68 4.20 5 7 6 2.88 5.80 5 8 I 2.56 6.60 5 8 2 2.60 3.00 5 8 6 2.28 4.00 6 1 1 2.20 6.80 6 1 2 3.08 6.00 6 I 6 1.92 6.00 6 2 1 2.08 5.00 6 2 2 2.84 5.20 6 2 6 2.40 1.40 6 3 1 2.44 5.80 6 3 2 2.52 6.40 6 3 6 2.16 4.60 6 4 1 1.96 6.60 6 4 2 3.00 5.00 6 4 6 2.16 5.20 6 5 1 2.48 4.60 6 5 2 3.04 5.20 6 5 6 2.16 5.80 6 6 I 2.36 6.40 6 6 2 3.28 4.20 6 6 6 2.40 5.80 6 7 1 2.24 3.80

133 Day Rep Tmt Tildia Feedmrk

6 7 2 2.76 2.80 6 7 6 228 420 6 8 1 2.60 820 6 8 2 3.24 5.80 6 8 6 2.40 520 7 1 1 2.36 6.00 7 1 2 2.52 1.40 7 1 6 2.48 2.60 7 2 I 2.08 5.20 7 2 2 2.36 2.80 7 2 6 3.08 5.20 7 3 1 2.00 5.20 7 3 2 3.08 7.40 7 3 6 2.68 7.60 7 4 I 2.16 7.80 7 4 2 2.64 6.40 7 4 6 2.04 6.60 7 5 1 2.00 4.80 7 5 2 2.52 7.00 7 5 6 2.00 7.00 7 6 1 2.28 6.80 7 6 2 3.04 4.20 7 6 6 2.20 0.00 7 7 I 2.04 2.00 7 7 2 2.36 3.00 7 7 6 1.80 2.40 7 8 I 2.08 4.00 7 8 2 2.36 4.80 7 8 6 1.84 6.20 8 1 I 1.96 4.40 8 1 2 3.12 3.00 8 1 6 2.68 3.00 8 2 I 1.88 3.00 8 2 2 3.72 5.20 8 2 6 2.64 1.40 8 3 1 1.88 3.60 8 3 2 3.60 9.20 8 3 6 2.52 5.40 8 4 1 1.96 1.40 8 4 2 2.84 4.60 8 4 6 2.48 6.40 8 5 I 2.28 0.00 8 5 2 3.28 7.40 8 5 6 1.88 4.40 8 6 1 1.96 4.00 8 6 2 3.44 5.80 8 6 6 2.64 1.00 8 7 1 1.92 2.80 8 7 2 2.84 4.00 8 7 6 2.44 2.40

134 Day Rep Tmt Tildia Feedmrk

8 8 I 1.60 5.00 8 8 2 3.16 4.60 8 8 6 2.52 4.80 9 I 1 1.96 9.20 9 1 2 2.12 2.20 9 1 6 2.12 7.40 9 2 1 1.56 4.40 9 2 2 1.88 4.80 9 2 6 2.00 3.80 9 3 1 1.56 9.40 9 3 2 2.52 8.20 9 3 6 1.84 5.40 9 4 1 1.36 5.60 9 4 2 2.04 5.60 9 4 6 1.80 6.60 9 5 1 1.80 3.20 9 5 2 2.16 8.40 9 5 6 1.76 4.80 9 6 I 1.68 4.60 9 6 2 2.00 820 9 6 6 1.88 4.60 9 7 I 1.92 2.00 9 7 2 2.24 8.80 9 7 6 2.28 1.80 9 8 I 1.56 3.80 9 8 2 2.08 6.00 9 8 6 2.00 5.40

135 APPENDIX F

Bluegrass Webworm Non-Choice Data

Rep Tmt Day % Survival Survival to Pupation Avg.

I I 7 0 0.00 1 2 7 80 .80 14.60 1 3 7 100 .60 16.50 2 1 7 0 0.00 ------2 2 7 100 .20 19.80 2 3 7 100 .60 14.10 3 1 7 0 0.00 3 2 7 100 .40 19.80 3 3 7 100 .80 18.10 4 1 7 0 0.00 ------4 2 7 100 .60 12.60 4 3 7 100 .20 18.20 5 1 7 20 0.00 5 2 7 100 .40 15.30 5 3 7 100 .40 13.60 6 1 7 20 0.00 ------6 2 7 100 .80 15.70 6 3 7 100 .40 11.40 7 1 7 20 0.00 7 2 7 80 .40 15.60 7 3 7 100 1.00 15.90 8 I 7 0 0.00 8 2 7 100 .60 16.80 8 3 7 100 .40 14.80 I I 14 0 1 2 14 80 1 3 14 100 2 I 14 0 2 2 14 100 2 3 14 80 3 1 14 0 3 2 14 100 3 3 14 100 4 I 14 0 4 2 14 100 4 3 14 80 5 I 14 20

136 Rep Tmt Day % Survival

5 2 14 80 5 3 14 100 6 1 14 0 6 2 14 100 6 3 14 100 7 1 14 0 7 2 14 80 7 3 14 100 8 1 14 0 8 2 14 100 8 3 14 100 1 1 21 0 1 2 21 80 I 3 21 100 2 1 21 0 2 2 21 100 2 3 21 80 3 1 21 0 3 2 21 100 3 3 21 100 4 1 21 0 4 2 21 100 4 3 21 80 5 1 21 0 5 2 21 80 5 3 21 100 6 1 21 0 6 2 21 100 6 3 21 100 7 1 21 0 7 2 21 80 7 3 21 100 8 1 21 0 8 2 21 100 8 3 21 100 I 1 28 0 I 2 28 80 1 3 28 100 2 1 28 0 2 2 28 100 2 3 28 60 3 I 28 0 3 2 28 100 3 3 28 100 4 1 28 0 4 2 28 100 4 3 28 80 5 1 28 0 5 2 28 80 5 3 28 100

137 Rep Tmt Day % Survival

6 I 28 0 6 2 28 100 6 3 28 100 7 I 28 0 7 2 28 80 7 3 28 100 8 I 28 0 8 2 28 100 8 3 28 100 I 1 35 0 1 2 35 80 1 3 35 80 2 1 35 0 2 2 35 60 2 3 35 60 3 1 35 0 3 2 35 100 3 3 35 100 4 1 35 0 4 2 35 100 4 3 35 80 5 1 35 0 5 2 35 60 5 3 35 100 6 I 35 0 6 2 35 100 6 3 35 100 7 1 35 0 7 2 35 40 7 3 35 100 8 I 35 0 8 2 35 100 8 3 35 100 1 I 42 0 I 2 42 80 1 3 42 80 2 1 42 0 2 2 42 60 2 3 42 60 3 1 42 0 3 2 42 80 3 3 42 100 4 1 42 0 4 2 42 100 4 3 42 80 5 1 42 0 5 2 42 60 5 3 42 100 6 1 42 0 6 2 42 100

138 Rep Tmt Day % Survival

6 3 42 100 7 1 42 0 7 2 42 40 7 3 42 100 8 1 42 0 8 2 42 100 8 3 42 60

139 APPENDIX G

Bluegrass Webworm Choice Data kb vs e+rye kb vs e-rye e-rye vs e+rye

.800 .200 .500 .500 .500 .500 1.000 0.000 .600 .400 1.000 0.000 .800 .200 .500 .500 .500 .500 .900 .100 .800 .200 .800 .200 .700 .300 .500 .500 .900 .100 .700 .300 .600 .400 .800 .200 .600 .400 .400 .600 .700 .300 .800 .200 .500 .500 .500 .500 .900 .100 .700 .300 .700 .300 1.000 0.000 .500 .500 .600 .400 1.000 0.000 .400 .600 1.000 0.000 .900 .100 .500 .500 .500 .500 .900 .100 .100 .900 .900 .100 .900 .100 .500 .500 1.000 0.000 .800 .200 .200 .800 1.000 0.000 .600 .400 .500 .500 .900 .100 .800 .200 .400 .600 .500 .500 .900 .100 .300 .700 .800 .200

140 APPENDIX H

Bluegrass Webworm Survival Data

Rep Tmt Pctendo Density Pctsnrv

1 0 0 10 50 1 1 0 10 60 1 2 20 10 30 1 3 40 10 30 I 4 60 10 10 1 5 80 10 0 1 6 100 10 0 2 0 0 20 20 2 I 0 20 40 2 2 20 20 20 2 3 40 20 0 2 4 60 20 0 2 5 80 20 5 2 6 100 20 0 3 0 0 10 10 3 1 0 10 30 3 2 20 10 10 3 3 40 10 20 3 4 60 10 10 3 5 80 10 0 3 6 100 10 10 4 0 0 20 15 4 1 0 20 30 4 2 20 20 15 4 3 40 20 20 4 4 60 20 5 4 5 80 20 5 4 6 100 20 0 5 0 0 10 40 5 1 0 10 20 5 2 20 10 10 5 3 40 10 0 5 4 60 10 0 5 5 80 10 0 5 6 100 10 0 6 0 0 20 15 6 1 0 20 20

141 s O O OOO >0 0*0>0 OOO 0>T)000‘0 ooooo ooo o CL (Ni/1—‘I/IOM — — OOOO — n — — OOOrOTf — 0000>or

X ooooooooooooooooooooooooooooooooooooooooooooooo Lj M M (N n N — — — — M n n M IN (N M — — — — — — — M m n n n n n — — — — — — — M m N m N n n

I H o o ooooo u ooooo oooooooooo ooooo ooooo ooooo ooooo uu r>iTfvooo — ooc'i'tvooo- oor'i'if'ooo- oolN'S'vooo — ooiN -^vooo- oor'iTt'ooo — oor'irfvooo — I

CL

Bluegrass Webworm Emigration Data

Tmt Pctendo Day 1 Day 10 Day 20 Day 30 Day 40

1 0 0.00 0.00 0.00 0.00 0.00 3 100 .20 .80 .80 .80 .80 2 50 0.00 0.00 .40 .40 .40 3 100 0.00 .80 .80 .80 .80 2 50 0.00 .40 .40 .60 .60 I 0 0.00 0.00 0.00 0.00 0.00 2 50 0.00 0.00 .20 .20 .20 1 0 0.00 0.00 0.00 .20 .20 3 100 0.00 .20 .20 .40 .40 2 3 100 0.00 .20 .20 .20 .40 2 2 50 0.00 0.00 0.00 .20 .20 2 1 0 0.00 0.00 0.00 0.00 0.00 2 2 50 0.00 0.00 0.00 0.00 0.00 2 1 0 0.00 0.00 0.00 0.00 0.00 2 3 100 .20 .40 .40 .40 .40 2 1 0 0.00 0.00 0.00 0.00 0.00 2 3 100 0.00 .20 .20 .20 .20 2 2 50 0.00 0.00 .40 .40 .60 3 1 0 0.00 0.00 0.00 0.00 0.00 3 3 100 0.00 .80 .80 1.00 1.00 3 2 50 0.00 0.00 0.00 0.00 0.00 3 3 100 0.00 .20 .20 .20 .20 3 2 50 0.00 0.00 0.00 0.00 0.00 3 1 0 0.00 0.00 0.00 0.00 0.00 3 2 50 0.00 0.00 0.00 0.00 0.00 3 1 0 0.00 0.00 0.00 0.00 0.00 3 3 100 0.00 0.00 0.00 0.00 0.00 4 2 50 0.00 .20 .40 .40 .40 4 3 100 .20 .40 .40 .60 .60 4 1 0 0.00 0.00 .20 .40 .60 4 I 0 0.00 0.00 0.00 .20 .20 4 2 50 0.00 0.00 0.00 0.00 0.00 4 3 100 0.00 .40 .40 .40 .60 4 3 100 0.00 .60 .60 .60 .60 4 1 0 0.00 0.00 0.00 0.00 .20 4 2 50 0.00 0.00 0.00 0.00 .20

143 IMAGE EVALUATION TEST TARGET (Q A -3 ) y

y

% V,

%

123 1 2 3 1.0 liS Ua 12.2 i

1.25 1.4 1.6

150mm

V

V /A P P L IE D A IIWJGE . Inc 1653 East Main Street Rochester. NY 14609 USA Phone: 716/482-0300 Pax: 716/288-5989

/; 0 1993. Applied Image. Inc.. All Rights Resented

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