PLANT FUNGAL ENDOSYMBIONTS ALTER HOST-PARASITE RELATIONSHIPS
BETWEEN GENERALIST HERBIVORES (LEPIDOPTERA: NOCTUIDAE) AND AN
ENTOMOPATHOGENIC NEMATODE
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy
in the Graduate School of The Ohio State University
By
Brian Albert Kunkel, B.S., M.S.
*****
The Ohio State University
2003
Dissertation Committee: Approved by:
Parwinder S. Grewal, Advisor
A. Raymond Miller ______
Dave J. Horn Advisor
Martin F. Quigley Department of Entomology
Douglas J. Doohan
ABSTRACT
Perennial ryegrass (Lolium perenne L.) and tall fescue (Festuca arundinacea
Schreb.) contain symbiotic fungi (Neotyphodium spp.) that provide several ecological
advantages to host plants: enhanced resistance to drought, disease, and insect herbivory.
The resistance to insect herbivory is the result of fungus-produced alkaloids that are toxic
to most herbivores. However, black cutworm, Agrotis ipsilon, is a generalist herbivore
that is able to feed and develop on endophytic perennial ryegrass. As some insects can
use plant secondary compounds to defend themselves against predators, I hypothesized
that the cutworms fed on endophytic grasses would exhibit greater defense against a
lethal endoparasitic nematode, Steinernema carpocapsae. To test this hypothesis, I
developed a method to remove the fungal endophytes from the grass seed through a heat
treatment. Laboratory experiments involving 4-5th instars support the hypothesis that A.
ipsilon feeding on plants with high (>90%) incidence of endophyte are less susceptible to
entomopathogenic nematodes than those feeding on plants with no or low incidence of
endophyte. Field studies show decreased susceptibility to S. carpocapsae when larvae
were confined to areas of endophytic grass (>75% infected). Early (2-3rd) instars were equally susceptible to nematode attack regardless of host plant.
ii
Endophytic grass fed to cutworm larvae did not influence nematode attachment
behavior, or their ability to penetrate and successfully develop into adults. I examined
the effects of ergot alkaloids that are produced by N. lolii such as, ergotamine,
ergonovine, ergocryptine, ergocristine, and seed extract on nematode viability and
infectivity. Ergonovine malate increased and ergocristine decreased the rates of
nematode infectivity, whereas other treatments had no significant effect. I investigated
the effects of ergocristine on Xenorhabdus nematophila, the symbiotic bacterium of S. carpocapsae. Bacterial growth and pathogenicity were significantly reduced when bacteria were grown in nutrient broth containing 200 µg/ml concentration of ergocristine.
Further research demonstrated that several fungal alkaloids, including ergocristine, persist in or are sequestered by black cutworm and another noctuid, the fall armyworm. I conclude that A. ipsilon developing on endophytic grasses may acquire some level of resistance against entomopathogenic nematodes. This resistance is mediated through the effects of alkaloids; thus our results underscore the ability of N. lolii to affect trophic interactions through the production of alkaloids.
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I dedicate this work to my wife and parents.
iv
ACKNOWLEDGMENTS
I would like to express my sincere gratitude to my major advisor Parwinder
Grewal for his guidance, support, and patience, as being my mentor. I am grateful for the
advice I received from my dissertation committee members, Drs. Ray Miller, Dave Horn,
Martin Quigley, and Doug Doohan. I thank everyone in our laboratory, Kevin Power,
Kevin McClure, Corrie Yoder, and Drs. Sukhbir Grewal, Gunpati Jagdale, Doug
Richmond, Seppo Salminen, Somu Nethi, and Tim Miklasiewicz, for assistance and discussion about my various research projects. The use of Dr. Dan Herms’s freeze-dryer
and Dr. Saskia Hogenhout’s capillary tube puller was appreciated.
I would like to thank the department of entomology and Parwinder Grewal for
providing financial support throughout my studies here at The Ohio State Unviversity.
Additionally, I thank the The Ohio State University – OARDC competitive grants
committee for awarding me a competitive grant.
I am thankful for the support provided by my parents throughout my graduate
education. I would like to thank my wife, Patti Kunkel, for all her assistance and support
that she has provided throughout my graduate education.
v
VITA
January 26, 1971………………………………….. Born – Covington, KY
December 19, 1994……………………………….. B.S., Biological Sciences
Northern Kentucky University
Highland Heights, KY
June 1995 - June 1998…………………………… Graduate Research Assistant
University of Kentucky
Lexington, KY
August 1998……………………………………….. M.S., Entomology
University of Kentucky
Lexington, KY
July 1998 – present ……………………………….. Graduate Research Associate
The Ohio State University
Wooster, OH
PUBLICATIONS
Refereed Journal Publications
1. Kunkel, B.A., D.A. Potter & D.W. Held. 2001. Lethal and sublethal effects of bendiocarb, halofenozide, and imidacloprid on Harpalus pennsylvanicus (Coleoptera: Carabidae) following different modes of exposure in turfgrass. J. Econ. Entomol. 94:60-67.
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2. Kunkel, B.A., D.A. Potter, & D.W. Held. 1999. Impact of halofenozide, imidacloprid, and bendiocarb on beneficial invertebrates and predatory activity in turfgrass. J. Econ. Entomol. 92:922-930.
3. Kunkel, B.A. & J.M. Hastings. 1996. Intersexual difference in feeding frequency and prey size in the robber fly Promachus albifacies (Diptera:Asilidae): possible influence of male mating behavior. Trans. Kentucky Acad. Sci. 57:1-5.
4. Luken, J.O., T.C. Tholemeier, L.M. Kuddes, & B.A. Kunkel. 1995. Performance, plasticity, and acclimation of the nonindigenous shrub Lonicera maackii (Caprifoliaceae) in contrasting light environments. Can. J. Bot. 73:1953-1961.
5. Luken, J.O., T.C. Tholemeier, B.A. Kunkel, & L.M. Kuddes. 1995. Branch architecture plasticity of Amur honeyscukle (Lonicera maackii (Rupr.) Herder): initial response in extreme light environments. Bull. Tor. Bot. Club 122(3):190-195.
FIELDS OF STUDY
Major Field: Entomology
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TABLE OF CONTENTS
PAGE
ABSTRACT……………………………………………………………. ii DEDICATION…………………………………………………………. iv ACKNOWLEDGEMENTS……………………………………………. v VITA……………………………………………………………………. vi LIST OF TABLES……………………………………………………… x LIST OF FIGURES…………………………………………………….. xi
CHAPTERS:
Chapter 1
Introduction………………………………………………………. 1
Chapter 2 A simple method to remove Neotyphodium endophytes from perennial ryegrass and tall fescue seed
2.1 Abstract……………………………..……………………….……… 12 2.2 Introduction……………………………………………….………… 13 2.3 Materials and Methods…………………………………………..….. 14 2.4 Results………………………………………………………………. 18 2.5 Discussion……………………………………………………….….. 20 2.6 Acknowledgments……………………………………………….….. 23
Chapter 3 Endophyte infection in perennial ryegrass reduces the susceptibility of black cutworm to an entomopathogenic nematode
3.1 Abstract……………………………………………………………… 37 3.2 Introduction……………………………………………………. …… 38 3.3 Materials and Methods………………………………………… …… 40 3.4 Results…………………………………………………………. …… 47
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3.5 Discussion…………………………………………………………… 50 3.6 Acknowledgments…………………………………………………… 54
Chapter 4 Endophyte infection in perennial ryegrass reduces the susceptibility of black cutworm to an entomopathogenic nematode: the mechanism
4.1 Abstract…………………………………………………………….. 63 4.2 Introduction………………………………………………………… 64 4.3 Materials and Methods…………………………………………….. 66 4.4 Results…………………………………………………………. ….. 73 4.5 Discussion…………………………………………………………. 76 4.6 Acknowledgments…………………………………………………. 80
Chapter 5 Fate of Neotyphodium lolii - produced alkaloids in generalist herbivores, Agrotis ipsilon and Spodoptera frugiperda and possible ecological consequences
5.1 Abstract…………………………………………………………….. 92 5.2 Introduction…………………………………………………….….. 92 5.3 Materials and Methods……………………………………………... 95 5.4 Results……………………………………………………………… 100 5.5 Discussion………………………………………………………….. 101 5.6 Acknowledgments…………………………………………………. 106
Bibliography………………………………………………………………… 111
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LIST OF TABLES
Table Page
5.1 The amount of endophyte produced alkaloids (:g/g dry weight) and perloline methyl ether (:g/g dry weight) found in pupae or hemolymph S. frugiperda larvae fed on endophyte infected (HE) or endophyte free (NE) grass clippings…. 110
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LIST OF FIGURES
Figure Page
2.1 Effect of seed treatments on ‘Goalkeeper’ perennial ryegrass infected with Neotyphodium endophyte. Percentage (± SEM) of plants infected with endophyte grown in 1999 from seeds stored in woven polypropylene bags during treatment, • = 4°C seed treatment and ∆ = 37°C seed treatment. Weeks refers to the number of weeks seeds were exposed to treatments…………..……………………. 24
2.2 Effect of seed treatments on ‘Palmer III’ perennial ryegrass infected with Neotyphodium endophyte. Percentage (± SEM) of plants infected with endophyte grown in 1999 from seeds stored in woven polypropylene bags during treatment, • = 4°C seed treatment and ∆ = 37°C seed treatment. Weeks refers to the number of weeks seeds were exposed to treatments……………………………….. 25
2.3 Effect of seed treatments on ‘Alamo’ tall fescue infected with Neotyphodium endophyte. Percentage (± SEM) of plants infected with endophyte grown in 1999 from seeds stored in woven polypropylene bags during treatment, • = 4°C seed treatment and ∆ = 37°C seed treatment. Weeks refers to the number of weeks seeds were exposed to treatments..………………………………………… 26
2.4 Effect of seed treatments on ‘Goalkeeper’ perennial ryegrass germination when infected with the Neotyphodium endophyte. Percent seeds (± SEM) germinated in 1999 that were stored in woven polypropylene bags during treatment, • = 4°C seed treatment and ∆ = 37°C seed treatment. Weeks refers to the number of weeks seeds were exposed to treatments.………………………..………… 27
2.5 Effect of seed treatments on ‘Palmer III’ perennial ryegrass germination when infected with the Neotyphodium endophyte. Percent seeds (± SEM) germinated in 1999 that were stored in woven polypropylene bags during treatment, • = 4°C seed treatment and ∆ = 37°C seed treatment. Weeks refers to the number of weeks seeds were exposed to treatments.……………..…………………… 28
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2.6 Effect of seed treatments on ‘Alamo’ tall fescue germination when infected with the Neotyphodium endophyte. Percent seeds (± SEM) germinated in 1999 that were stored in woven polypropylene bags during treatment, • = 4°C seed treatment and ∆ = 37°C seed treatment. Weeks refers to the number of weeks seeds were exposed to treatments.…………………………………..……… 29
2.7 Effect of seed treatments on ‘Alamo’ tall fescue infected with the Neotyphodium endophyte. Percentage of plants (± SEM) infected with endophyte grown in 2000 from seeds stored in paper bags during treatment, • = 4°C seed treatment and ∆ = 37°C seed treatment. Weeks refers to the number of weeks seeds were exposed to treatments…………………..………………………………………………. 30
2.8 Effect of seed treatments on ‘Goalkeeper’ perennial ryegrass infected with the Neotyphodium endophyte. Percentage of plants (± SEM) infected with endophyte grown in 2000 from seeds stored in paper bags during treatment, • = 4°C seed treatment and ∆ = 37°C seed treatment. Weeks refers to the number of weeks seeds were exposed to treatments..………………………………………… 31
2.9 Effect of seed treatments on ‘Palmer III’ perennial ryegrass infected with the Neotyphodium endophyte. Percentage of plants (± SEM) infected with endophyte grown in 2000 from seeds stored in paper bags during treatment, • = 4°C seed treatment and ∆ = 37°C seed treatment. Weeks refers to the number of weeks seeds were exposed to treatments.…………………………………………. 32
2.10 Effect of seed treatments on ‘Goalkeeper’ perennial ryegrass germination when infected with the Neotyphodium endophyte. Percent seeds (± SEM) germinated in 2000 that were stored in paper bags during treatment, • = 4°C seed treatment and ∆ = 37°C seed treatment. Weeks refers to the number of weeks seeds were exposed to treatments.……………………………………………………… 33
2.11 Effect of seed treatments on ‘Palmer III’ perennial ryegrass germination when infected with the Neotyphodium endophyte. Percent seeds (± SEM) germinated in 2000 that were stored in paper bags during treatment, • = 4°C seed treatment and ∆ = 37°C seed treatment. Weeks refers to the number of weeks seeds were exposed to treatments..……………………………………………… 34
2.12 Effect of seed treatments on ‘Alamo’ tall fescue germination when infected with the Neotyphodium endophyte. Percent seeds (± SEM) germinated in 2000 that were stored in paper bags during treatment, • = 4°C seed treatment and ∆ = 37°C seed treatment. Weeks refers to the number of weeks seeds were exposed to treatments.……………………………………………………… 35
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2.13 Percentage of plants infected with Neotyphodium endophytes in field plots when planted with either seed stored at 37° or 4°C respectively. ………….. 36
3.1 Weight gain (+SEM) by neonate Agrotis ipsilon fed on endophyte infected (▼) or endophyte free (●) perennial ryegrass to pupation. ………………… 55
3.2 Average mortality (+ SEM) of 4th instar Agrotis ipsilon due to Steinernema carpocapsae over time. Larvae were fed for 14 d on perennial ryegrass, Lolium perenne, plants containing high (>90% plants infected), medium (40-60% plants infected), and low (<20% plants infected) prevalence of Neotyphodium lolii endophytes. Treatments were as follows: high endophyte and nematodes (▼), medium endophyte and nematodes (■), low endophyte and nematodes (●), high endophyte and no nematodes (∇), medium endophyte and no nematodes (□), and low endophyte and no nematodes (○). Nematode concentration used in this experiment was 60 infective juveniles/insect……………………….. 56
3.3 Average cumulative mortality over time (+ SEM) of Agrotis ipsilon larvae. 2-3rd instars (open bars) and 4-5th instars (solid bars) were exposed to 0, 10, 20, or 40 Steinernema carpocapsae infective juveniles per insect. Bars with different letters are significantly different at α = 0.05. ………………………… 57
3.4 Average cumulative mortality Mortality (± SEM) of 2-3rd (○) and 4-5th (●) instar Agrotis ipsilon exposed to Steinernema carpocapsae over time (Time*Instar interaction). ……………………………………………………………… 58
3.5 Average mortality (+SEM) of 4-5th instars reared on greenhouse grown plants containing either >95% infected with endophyte (E+) or without endophyte infection (E-). Nematode concentration used in this experiment was about 35 infective juveniles/insect. Treatments were as follows: fed E+ and exposed to nematodes (●), fed E+ and not exposed to nematodes (○), fed E – and exposed to nematodes (▲), and fed E – and not exposed to nematodes (∆)……… 59
3.6 Average mortality (+ SEM) due to Steinernema carpocapsae of 2nd —3rd instar Agrotis ipsilon fed on endophyte infected >75% (E +) or endophyte free (E -) perennial ryegrass. Nematode concentration used in both experiments was 20 infective juveniles/insect. Treatments were as follows: fed E+ and exposed to nematodes (●), fed E+ and not exposed to nematodes (○), fed E – and exposed to nematodes (▲), and fed E – and not exposed to nematodes (∆)……….. 60
3.7 Average mortality (+ SEM) due to Steinernema carpocapsae of 2nd —3rd instar Agrotis ipsilon fed on endophyte infected >95% (E+) or endophyte free (E -) perennial ryegrass. Nematode concentration used in both experiments was 20 infective juveniles/insect. Treatments were as follows: fed E+ and exposed to nematodes (●), fed E+ and not exposed to nematodes (○), fed E – and exposed to nematodes (▲), and fed E – and not exposed to nematodes (∆)……… 61
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3.8 Field experiment. Mortality of 4th instar Agrotis ipsilon exposed to Steinernema carpocapsae for 36- 38 h in field plots of endophyte-free and endophyte-infected ‘Goalkeeper’ perennial ryegrass (Lolium perenne). Larvae were confined using 21 cm diameter PVC cylinders. Nematodes used in the field experiment were applied at 0.5 billion/acre rate, which is one-half the recommended rate….. 62
4.1. Mean number (+ SEM) of Steinernema carpocapsae infective juveniles found attached to Agrotis ipsilon 4-5th instar hosts. Larvae were exposed to about 80 infective juveniles for 1.5, 3, 6, 12, and 20 h before being washed and removed from treated arenas. Larvae fed on endophytic grass (full bars) and larvae fed on endophyte free grass (empty bars) are presented…………………………. 81
4.2 Mean number (+ SEM) of Steinernema carpocapsae adults found within Agrotis ipsilon larvae. Larvae were dissected 72 h after the start of the experiment to count the number of nematodes penetrated and developed to adults. Larvae fed on endophytic grass (full bars) and larvae fed on endophyte free grass (empty bars) are presented………………………………………………………………. 82
4.3 Average mortality (+ SEM) for Agrotis ipsilon larvae removed after 1.5, 3, 6, 12, 20 h of exposure to nematodes. Larvae were removed from the challenge arena after exposure period and maintained in petri dishes containing no nematodes. Larva mortality was observed at 36, 48 and 72 h after initial exposure to nematodes. Larvae fed on endophytic grass (full bars) and larvae fed on endophyte free grass (empty bars) are presented. Bars with different lower case letters are significantly different at α= 0.05………………………………… 83
4.4 Mean % mortality (± SEM) of Galleria mellonella when exposed to Steinernema carpocapsae treated with ergocristine solutions for 3 d. Alkaloid concentrations tested were 200 and 100 µg/mL. Dimethyl sulfoxide (DMSO) treatments and water served as controls. All treatments had a G. mellonella larva exposed to one infective juvenile that had been stored in one of the following treatments for 3 d: 200 µg/mL concentration of alkaloid (●), 100 µg/mL concentration of alkaloid (○), water (□), 5% dimethyl sulfoxide (DMSO; only with ergocristine and ergocryptine; ▲), or a water control (no nematodes; ■)………………….… 84
4.5 Mean % mortality (± SEM) of Galleria mellonella when exposed to Steinernema carpocapsae treated with ergocryptine solutions for 3 d. Alkaloid concentrations tested were 200 and 100 µg/mL. Dimethyl sulfoxide (DMSO) treatments and water served as controls. All treatments had a G. mellonella larva exposed to one infective juvenile that had been stored in one of the following treatments for 3 d: 200 µg/mL concentration of alkaloid (●), 100 µg/mL concentration of alkaloid (○), water (□), 5% dimethyl sulfoxide (DMSO; only with ergocristine and ergocryptine; ▲), or a water control (no nematodes; ■)…………………. 85
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4.6 Mean % mortality (± SEM) of Galleria mellonella when exposed to Steinernema carpocapsae treated with ergonovine solutions for 3 d. Alkaloid concentrations tested were 200 and 100 µg/mL. Water served as controls. All treatments had a G. mellonella larva exposed to one infective juvenile that had been stored in one of the following treatments for 3 d: 200 µg/mL concentration of alkaloid (●), 100 µg/mL concentration of alkaloid (○), water (□), and a water control (no nematodes; ■)…………………………………………………………….. 86
4.7 Mean % mortality (± SEM) of Galleria mellonella when exposed to Steinernema carpocapsae treated with ergotamine solutions for 3 d. Alkaloid concentrations tested were 200 and 100 µg/mL. Water served as controls. All treatments had a G. mellonella larva exposed to one infective juvenile that had been stored in one of the following treatments for 3 d: 200 µg/mL concentration of alkaloid (●), 100 µg/mL concentration of alkaloid (○), water (□), 5% dimethyl sulfoxide (DMSO; only with ergocristine and ergocryptine; ▲), or a water control (no nematodes; ■). ………………………………………………………………………….… 87
4.8 Mean % mortality (± SEM) of Galleria mellonella when exposed to Steinernema carpocapsae treated with different concentrations of seed extract/methanol, methanol, or water for 3 d. Our treatments were as follows: 10% seed extract (●), 10% methanol (○), 5% seed extract (!), 5% methanol (∇), 2.5% seed extract (■), 2.5% methanol (□), water controls (!). …………………………… 88
4.9 Mean number of colony forming units (cfus) (±SEM) exposed to 0.5% DMSO (full bars) or 200 µg/mL of ergocristine (empty bars) in nutrient broth for 18 h. Results for three trials are presented……………………………………….. 89
4.10 Mean % mortality (± SEM) of Galleria mellonella over time when injected with 1 µl suspension of X. nematophila, the bacterial symbiont of Steinernema carpocapsae. Number of X. nematophila colony forming units (cfus) injected in first experiment (A) was about 6 and 4 for 0.5% DMSO control (□) and 200 µg/mL ergocristine (!), or 0 for nutrient broth and no bacteria (") respectively. ………………………………………………………………………………. 90
4.11 Mean % mortality (± SEM) of Galleria mellonella over time when injected with 1 µl suspension of X. nematophila, the bacterial symbiont of Steinernema carpocapsae. The number of cfus injected was about 10 and 6 for 0.5% DMSO control (□), 200 µg/mL ergocristine plus bacteria (!) respectively, and 0 for 200 µg/mL ergocristine with no bacteria (") or nutrient broth with no bacteria ("; controls). …………………………………………………………………… 91
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5.1 Concentrations of various alkaloids (+SEM) found in endophyte infected perennial ryegrass (HE), Lolium perenne L. PerMet stands for perloline methyl ether, a plant produced alkaloid; whereas, the remaining alkaloids presented are produced by the fungus Neotyphodium lolii…………………………………. 107
5.2 Concentration of various alkaloids found in black cutworm, Agrotis ipsilon, fed on Neotyphodium lolii infected perennial ryegrass (high proportion of plants infected, HE) until larvae were 5-6th instar. No N. lolii produced alkaloids were detected in S. frugiperda larvae fed on N. lolii free perennial ryegrass. ECI = ergocristine, ECY = ergocryptine, EGN = ergonovine, EVAL = ergovaline……………………………………………..……………………. 108
5.3 Concentration of various alkaloids found in fall armyworm, Spodoptera frugiperda, fed on Neotyphodium lolii infected perennial ryegrass (high proportion of plants infected, HE) until larvae were 5-6th instar. No N. lolii produced alkaloids were detected in S. frugiperda larvae fed on N. lolii free perennial ryegrass. ECI = ergocristine, ECY = ergocryptine, EGN = ergonovine, EVAL = ergovaline, PMET = perloline methyl ether ……………………………………………………………………………… 109
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CHAPTER 1
INTRODUCTION
As plants evolve new defenses to escape their insect herbivores, a fitness benefit
is thought to spur a burst of radiation. Insects that overcome new plant defenses may
colonize the under exploited resource free of competition and eventually may evolve into
a specialist on that particular plant. Insects that specialize on chemically defended plants
may use those chemicals as stimulants for host plant recognition. Additionally, those
insects may sequester the plant-produced compounds for their own defense (Dobler,
2001). However, the consequences of this co-evolutionary race between plants and
insect herbivores on the third trophic level have rarely been documented. Perennial
ryegrass, Lolium perenne L., forms a symbiotic relationship with the fungus
Neotyphodium lolii (Latch, Samuels & Christensen) Glenn, Bacon & Hill that grows intercellularly throughout the foliage (van Heeswijck and McDonald 1992; Saikkonen et al., 1998). This symbiotic association with the fungus provides increased drought tolerance and resistance to disease and herbivory, which enables endophytic plants to dominate plant communities over time (Clay 1996). The enhanced resistance against herbivory is due to the production of different alkaloids by the fungus. The classes of alkaloids produced by Neotyphodium spp. are: ergot alkaloids, indole alkaloids (lolitrem
B), pyrrolizidine (lolines, tall fesuce only), pyrollopyrazine (peramine) and a few others such as ergosterol
1
(Porter 1994). Lolitrem B and peramine can be found in leaf sheaths, stems, older leaves, regrowth, reproductive stems, and crowns (Ball et al., 1997a, Ball et al., 1997b). These alkaloids provide protection from both vertebrate and invertebrate herbivores; however, like many plants that produce secondary metabolites, the concentrations can vary between and within individual plants. The alkaloids and their concentrations can vary due to interactions between plant host and endophyte (Roylance et al., 1994), and these interactions lead to varied experimental results (Latch, 1997). Abiotic factors such as temperature, water and fertilization can influence production of both lolitrem B and peramine (van Heeswijck and McDonald, 1992).
To evaluate the role of grass-endophyte interactions in grassland communities, a method of removing the endophyte from large amounts of seed is needed. Endophytes are usually removed from infected plants by fungicide (Siegel et al., 1984; Williams et al., 1984; De Battista et al., 1990) or removed from seed by heat treatments (Siegel et al., 1984; Williams et al., 1984; Welty et al., 1987), however, these are usually applicable to small amounts of seed (Latch and Christensen, 1982; Welty et al., 1987;
Bishop et al., 1997). The fungicides used to remove the endophyte can be phytotoxic, may delay seedling emergence, distort leaves, and is not cost effective for large areas of land (Latch and Christensen, 1982; Latch, 1983; Siegel et al., 1984).
The indole alkaloids include lolitrem A-E, paxilline, lolitriol, and indoleacetic acid (Miles et al., 1992; Porter, 1994). Lolitrem alkaloids are known to be lipophilic, complex chemicals that act as tremorgenic neurotoxins in mammals. Lolitrem B is thought to be responsible for ryegrass staggers in livestock (van Heeswijck and
McDonald, 1992). Ergot alkaloids can be divided into the following five groups,
2
ergopeptine, simple lysergic acid amides, ergopeptam, clavine, and lysergic acid. The
ergopeptine group is further subdivided into the ergotamine, ergoxine, and ergotoxine
sub-groups (Porter, 1994).
Many insects are deterred from feeding on endophytic grasses, but the insect still
consumes some of the plants and experience toxins within the plants. An insect that
detects a toxin within its host plant and receives a sub-lethal dose of the compound may
respond behaviorally and move to find alternate hosts (Hoy et al., 1998). This is one way
to avoid severe physiological effects of the toxin. Richmond and Shetlar (2000) found that chinch bug nymphs are more likely to emigrate from stands containing high proportions of endophytic grasses. Additionally, sod webworms and black cutworms are
likely to emigrate more quickly from stands of grass containing high proportions of
endophytic grass, than are those in a mixed stand of grasses (Richmond and Shetlar 1999,
Richmond and Shetlar 2001). Some insects suffer increased predation due to slower
development rates or movement (Bergelson and Lawton, 1988; Haggstrom and Larsson,
1995). Over 30 insect species are either deterred or suffer toxic consequences from
feeding on endophytic perennial ryegrass (Breen, 1994; Prestidge and Ball, 1997).
Endophyte-infected grasses should not directly affect the density of generalist
predators. Davidson and Potter (1995) found that endophytic tall fescue did not have
direct adverse affects on predator density. Some of the more abundant generalist
predators found in turfgrass are ants, carabids, spiders, and staphylinids (Kunkel et al.
1999). Predation pressure should be the same in areas planted with either endophytic or
non-endophytic grasses. Most insect herbivores that consume endophytic grasses suffer
the toxic effects of reduced growth, deterrence, or death (Breen, 1994). van Heeswijck
3
and McDonald (1992) suggest that consumption of endophytic grass may increase the exposure of susceptible life stages of an insect to extrinsic mortality factors such as natural enemies and entomopathogens. In fact, Grewal et al. (1995) found that Popillia japonica Newman larvae fed on roots of endophytic grasses or pure alkaloids suffered greater mortality rates from the entomopathogenic nematode, Heterorhabditis bacteriophora. These white grubs lost weight and were more prone to nematode infection. Thus, biological control of insect pests may be enhanced in areas planted with endophytic grasses.
However, it also appears as if the effects of endophytic grasses may be detrimental to biological control in some cases. Barker and Addison (1996) found that the parasitoid, Microctonus hyperodae, of the weevil, Listronotus bonariensis, attacked insects fed on grass with or without endophyte with equal frequency, but parasitoid development in weevils fed on endophytic grass was retarded because the host was nutritionally inadequate. Bultman et al. (1997) found that two Euplectrus parasitoids suffered detrimental effects when they parasitized Spodoptera frugiperda (fall armyworm) fed on endophytic tall fescue. Omacini et al. (2001) demonstrated that parasitism rates of aphids, fed on the endophytic grass Lolium multiflorum (Italian ryegrass), were lower than those fed non-endophytic grass. Primary parasitoids were less successful on the aphids fed on non-endophytic grass because they suffered greater attack by secondary parasitoids. These studies underscore the possibility that the endophytic grasses, tall fescue and perennial ryegrass, may have either beneficial or adverse effects on biological control agents; this has implications on a community wide scale. The impact of partial
4
plant resistance on biological control needs to be evaluated to obtain a better
understanding of the interactions among plants, pests, and natural enemies (Verkerk et
al., 1998).
Many insect orders contain representatives that sequester alkaloids, increasing the
herbivores' defenses against natural enemies (Blum 1981). Brown (1984) found that
some butterflies were protected from spiders when they consumed pyrrolizidine alkaloids
as larvae. Predators that use visual cues for locating prey learn to avoid aposematically
colored insects (Bowdish and Bultman, 1993). The moth Utetheisa ornatrix receives a
nuptial gift containing sequestered pyrrolizidine alkaloids from males during courtship
that protects her from spiders (Rossini et al, 2001) and her eggs from generalist predators such as green lacewings (Eisner et al., 2000)
A group of natural enemies referred to as entomopathogenic nematodes in the families Steinernematidae and Heterorhabditidae have received significant attention due to their commercial availability and potential of controlling a broad spectrum of insect pests (Grewal and Georgis 1998). Nematodes in both families possess a symbiotic relationship with bacteria that cause rapid insect mortality. Steinernema carpocapsae
Weiser (Rhabditida: Steinernematidae) has been one of the most widely studied species of entomopathogenic nematodes (Kaya and Gaugler, 1993). S. carpocapsae can infect
over 200 different insect species in laboratory tests; thus it has become an excellent
candidate as a biological control agent (Grewal and Georgis, 1998). This nematode
forms a symbiotic relationship with the bacterium, Xenorhabdus nematophila Thomas and Poinar. The third stage infective juveniles of S. carpocapsae attack suitable insects
5
in or on the soil (Campbell and Gaugler, 1993; Grewal et al., 1997), invade through
natural body openings, and release the symbiotic bacterium into the hemocoel. The
bacterial infection usually kills the host within 2—3 days (Kaya & Gaugler, 1993). The
infective juveniles feed on multiplying bacteria and insect body contents and reproduce.
After 1-3 generations, the next generation of infective juveniles leaves the cadaver
seeking new hosts.
Grewal et al. (1994) found that S. carpocapsae does not exhibit a directional
response to host cues, and does not move great distances in search of hosts; therefore
they concluded that it ambushes potential hosts. Campbell and Gaugler (1993) found that
the nictation behavior (body waving) of S. carpocapsae allows it to raise >95% of its body off of the substrate, thus facilitating attachment to potential hosts as they pass by.
S. carpocapsae is capable of jumping distances that are on average nine times its own body length and a jump height of seven times the body length (Campbell and Kaya,
1999). The ambusher nematodes are most effective against potential prey that are mobile, and have been demonstrated to provide substantial control of lepidopteran pests
(Kaya and Hara, 1981; Grewal et al., 1994).
Shannag et al. (1994) demonstrate that S. carpocapsae (Mexican) strain was quite
effective at protecting squash plants from damage caused by the pyralid, Dianphania
nitidalis (Stoll), a notorious pest of cucurbits in the southeast. Many examples exist
where S. carpocapsae (All) or (Mexican) strains were used to study invasion efficiency,
host exposure time, instar susceptibility, or pest control (Fuxa et al., 1988; Epsky and
Capinera, 1993; Epsky and Capinera, 1994a; Shannag and Capinera, 1995; Medeiros et
6
al., 2000). Shannag and Capinera (2000) recently discovered that S. carpocapsae
(Mexican strain) interferes with the parasitoid, Cardiochiles diaphaniae, which could
affect control of the melonworm.
The symbiotic bacteria, X. nematophila, are Gram negative facultative anaerobic
bacteria, that are members of the family Enterobacteriaceae and have phase I and II
variants (Boemare, 2002). Phase I variants are held in S. carpocapsae, and phase II variants form after phase I bacteria have reached the stationary phase in culture or in the cadavers. Phase II is less virulent and reduces nematode development compared to phase
I X. nematophila (Dowds and Peters, 2002).
Lamberty et al. (1999) report that lepidopteran insects contain inducible antibacterial peptides called cecropins that are capable of killing both Gram negative and positive bacteria. During metamorphosis, some lepidopterans release antibacterial peptides into the midgut that is effective against E. coli (Russell and Dunn, 1996). The most commonly used insect for the propagation of entomopathogenic nematodes in the laboratory is the lepidopteran, Galleria mellonella. This lepidopteran is easily infected because there is no non-self recognition (Dunphy and Webster, 1988). Additionally, S. carpocapsae and its symbiotic bacteria, may be insensitive to the bactericidal proteins, or it might inhibit the induction or function of bactericidal polypeptides produced by the insect (Forst et al., 1997). X. nematophila produces two cytolytic activities against different cell targets in Spodoptera littoralis (Brillard et al., 2001). Park and Kim (2000) found that X. nematophila targets eicosanoid production in S. exigua which disrupts the immune response and results in immunodepressive hemolymph septicemia. Both phases of the bacteria exhibited antifungal activities, but phase II bacteria were less virulent
7
(Chen et al., 1994). Dunphy and Webster (1988) state that release of lipopolysaccharides
from X. nematophila and subsequent hemocyte damage is part of the mechanism of the bacterial virulence. S. carpocapsae overcomes the insect immune systems in three ways: tolerance, evasion, and suppression (Dowds and Peters, 2002).
As endophyte infected perennial ryegrass influences interactions between invertebrate herbivores and their natural enemies, I wanted to investigate the effect of the endophytes on the entomopathogenic nematodes. I chose to work with the generalist herbivore, the black cutworm (Agrotis ipsilon Hufnagel; Lepidoptera: Noctuidae) because it is a voracious pest of grass and other crops such as, wheat, soybeans, corn, velvetleaf, morning glory, and many vegetables (Rings et al., 1975; Bushing and Turpin
1977). Bushing and Turpin (1976) found that Rumex crispus L., curled dock; Barbarea vulgaris R. Br., yellow rocket; and alfalfa, Medicago sativa L. (Vernal) had the highest percentage of eggs and generally oviposition was on the stem or underside of the leaves.
Harris et al. (1962) found that A. ipsilon reared in the laboratory had six to seven instars with a life cycle of egg to adult lasting about 45 days. Rings et al. (1975) state that vegetables are more likely to be damaged when planted in areas formerly maintained as grasslands.
A. ipsilon feeds on many types of grasses including creeping bentgrass, perennial ryegrass, and tall fescue, thus is an important pest of turfgrasses (Williamson and Potter
1997a). A. ipsilon can develop on Festuca and Lolium spp. grasses that form symbiotic relationships with endophytic fungi, Neotyphodium spp. which confer protection to the grass against many herbivorous insects. Williamson and Shetlar (1995) found that females oviposit on the distal 25% of creeping bentgrass leaf blades; therefore, increasing
8
the likelihood of removal from the plants when the grass is cut. They also propose that
future putting green infestations may originate from peripheral areas. Black cutworms
prefer to feed on creeping bentgrass, but it does consume endophytic perennial ryegrass
and tall fescue; however these grasses do not necessarily provide resistance (Williamson
and Potter, 1997a). They also found that black cutworms fed on endophytic perennial
ryegrass show slight decreases in weight gain. Most early A. ipsilon instars (3-4th) encountered were feeding on bentgrass on the surface; whereas, older larvae (4-5th) were found feeding in burrows (Williamson and Potter, 1997b).
Fall armyworm, Spodoptera frugiperda (J.E. Smith), is another voracious generalist that feeds on cotton, tobacco, legumes, vegetables, and warm and cool season grasses (i.e., perennial ryegrass). I included this species in some experiments because it is easier to maintain in the laboratory. These insects usually feed around dusk or dawn, but they can be active at other times. Neonates scrape the undersides of leaves initially, and as the larvae develop, the larvae consume all foliage down to the crown of the plant
(Potter, 1998). Breen (1993a) found that S. frugiperda suffered decreased weight gain and delayed development when fed on endophytic perennial ryegrass, but results varied between the different genotypes. Molina-Ochoa et al. (1999) found that resistant corn silks enhanced the susceptibility of S. frugiperda to S. carpocapsae. However,
Richmond et al. (2003) found that S. frugiperda fed on endophytic perennial ryegrass had increased resistance to S. carpocapsae. S. frugiperda increases detoxification enzymes when it consumes the allelochemical, xanthotoxin (Yu, 1999).
Interest in biological control of black cutworm and fall armyworm has increased due to the recent bans on the use of chemical insecticides. The nematode S. carpocapsae
9
has been shown to be an effective biological control agent for both pest species (Buhler
and Gibb, 1994); however the effect of endophytes on A. ipsilon susceptibility to S.
carpocapsae has never been investigated. I hypothesized that those A. ipsilon larvae fed
on endophytic perennial ryegrass would show decreased susceptibility to the nematode.
Before I could evaluate this interaction, I needed endophytic and non-endophytic plants
of the same cultivar of perennial ryegrass. I developed a rapid method that used heat to
remove most of the endophyte from grass seed within six weeks. Afterwards, the treated
seed was planted and the plants were used in experiments to test the effects of endophyte
on A. ipsilon susceptibility to S. carpocapsae.
The biology of A. ipsilon allowed me to investigate the effect of endophytic perennial ryegrass on the third trophic level. Many insects that feed on endophytic grasses are unable to complete development, but A. ipsilon can complete development and only suffers slower development. Since A. ipsilon is very mobile, it has a greater probability of encountering an ambushing natural enemy such as S. carpocapsae.
Perennial ryegrass is frequently planted in ecosystems where A. ipsilon can be found, thus it is possible that A. ipsilon larvae might consume endophytic grass and later encounter S. carpocapsae. Therefore, I made the following predictions:
Prediction 1: Exposure of large quantities of endophyte infected seed to 37°C would kill
Neotyphodium endophytes without compromising seed viability.
Prediction 2: A. ipsilon larvae fed on endophytic perennial ryegrass would be less
susceptible to S. carpocapsae than those feeding on endophyte free grass.
Prediction 3: The fungus N. lolii was responsible for the observed resistance to S.
10
carpocapsae.
Prediction 4: A. ipsilon and S. frugiperda store N. lolii produced alkaloids within their
body as they feed on infected perennial ryegrass.
From the above predictions, I constructed hypotheses that I have further developed and tested in the following chapters.
11
CHAPTER 2
A simple method to remove Neotyphodium endophytes from perennial ryegrass and tall
fescue seed
2.1 ABSTRACT
Perennial ryegrass (Lolium perenne L.) and tall fescue (Festuca arundinacea
Schreb.) contain symbiotic fungi (Neotyphodium spp.). These endophytic fungi provide several ecological advantages to host plants: enhanced resistance to drought, disease, and insect herbivory. The endophytic fungi are only maternally transmitted through the seed.
To evaluate the ecological significance of this symbiosis a method is needed to remove the endophyte from the seed to permit comparisons between infected and non-infected plants of the same cultivar in the field. I evaluated the effect of temperature on the survival of the endophyte in the seed of three cultivars. I found that ‘Alamo’ tall fescue and ‘Goalkeeper’ perennial ryegrass seed stored at 37°C lost most of the viable endophyte within six weeks when kept in woven polypropylene bags with a 7-14% loss in seed germination. The loss of viable endophyte was not as rapid for ‘Palmer III’ perennial ryegrass. When seed was stored in brown paper bags only ‘Alamo’ lost most of its viable endophyte. Field plots established using the heat-treated seed confirmed the removal of
12
the endophyte. This method may be used for the removal of endophyte from large
quantities of seed to facilitate field studies examining grass-endophyte interactions within
the same cultivar.
2.2 INTRODUCTION
Tall fescue (Festuca arundinacea Schreb.) and perennial ryegrass (Lolium
perenne L.) are common pasture and turf grasses that may be infected with symbiotic fungi. Tall fescue alone accounts for millions of hectares of pastures in the United
States. Tall fescue and perennial ryegrass are infected with maternally transmitted fungal endophytes (Neotyphodium spp.) (Schardl and Phillips, 1997). These asexual fungi live
intercellularly in above ground plant tissues producing a variety of toxic alkaloids. These
alkaloids improve plant fitness by providing enhanced drought tolerance, protection from
herbivory, and disease resistance. However, when present in forage grasses, they cause
fescue toxicosis or ryegrass staggers in cattle and sheep (Breen, 1994).
There has been difficulty in the establishment of endophyte free swards of grass
due to the greater competitive ability of endophytic grasses already planted in vast acres
(Schardl and Phillips, 1997). Recent ecological research suggests that endophytes could
possibly alter plant communities through the following mechanisms: invasiveness (Clay
and Holah, 1999), fungal-plant interactions, including possible nutrient acquisitions
(Arechavaleta et al., 1989; Vitousek and Walker, 1989; Hartnett and Wilson, 1999), root
exudates (Callaway and Aschehoug, 2000) and insect herbivory (Carrière et al., 1998).
To evaluate the role of grass-endophyte interactions in grassland communities, a method
of removing the endophyte from large amounts of seed is needed.
13
Latch (1997) states that grass and endophyte genotypes can interact, resulting in
varied experimental results. Endophytes are usually removed from infected plants by
fungicide (Siegel et al., 1984; Williams et al., 1984; De Battista et al., 1990) or removed
from seed by heat treatments (Siegel et al., 1984; Williams et al., 1984; Welty et al.,
1987). However, these treatments typically reduce germination (Latch and Christensen,
1982; Welty et al., 1987) and are applicable only to small quantities of seed (Latch and
Christensen, 1982; Welty et al., 1987; Bishop et al., 1997). Furthermore, fungicides used to remove the endophyte can be phytotoxic, may delay seedling emergence, or distort leaves (Latch and Christensen, 1982; Latch, 1983). Fungicide applications to large areas of established pastures at the concentrations necessary to remove the endophyte from the plants are not economically feasible or registered for that use (Latch, 1983; Siegel et al.,
1984). The best way to ensure having a field without endophyte is to obtain seed from
parent plants that are endophyte free (Latch, 1983; Shardl and Phillips, 1997). Therefore,
I evaluated the effect of temperature on removal of viable endophyte from large
quantities of seed of two cultivars of perennial ryegrass and one of tall fescue. I
hypothesized that seed kept at 37°C for 5-6 wks would be endophyte free without a
significant loss in germination and seed kept at 4°C would maintain a viable endophyte.
2.3 MATERIAL AND METHODS
All experimental seed was harvested in 1998 by Lofts Seed Inc. (Winston Salem,
NC) and acquired in 1999. The seed was stored at 4°C, and had 15% moisture by weight
until used in the experiments (about 7 months). Two turf type cultivars of perennial
ryegrass and one cultivar of tall fescue were used in all experiments unless otherwise
14
noted. The perennial ryegrass cultivars were ‘Goalkeeper’ and ‘Palmer III’, and the tall fescue cultivar was ‘Alamo’. The perennial ryegrass cultivars were infected with
Neotyphodium lolii (Latch, Sammuels, & Christensen) Glenn, Bacon, & Hanlin and the tall fescue cultivar was infected with Neotyphodium coenophialum (Morgan-Jones &
Gams) Glenn, Bacon, & Hanlin.
Endophyte removal experiment: 1999
The purpose of this experiment was to see if short-term storage in a hot environmental chamber would be sufficient to kill the endophytic fungi found in the seeds without a reduction in germination. The two seed treatments for this experiment were as follows: storage in a walk-in cold room at 4°C or storage in a walk-in environmental chamber (Environmental Growth Chambers, Chagrin Falls, OH) at 37°C with 60-80 % RH. About 7.9 kg seed from each cultivar was stored in woven polypropylene bags for 6 weeks (wks) in the respective treatments. The first seed sample to determine effects of temperature was taken at 2 wks and continued every week thereafter. Samples of seed were removed from the bag and planted in 35 x 17 x 11.5 cm flats (Landmark, Akron, OH) in Pro-Mix BX (Premier Horticulture Inc., Red Hill, PA) that had been moistened the day before to facilitate water holding potential. Twenty-four seeds were placed into three rows of eight seeds in each of four flats. The labeled flats were placed into a different walk-in environmental chamber (CONVIRON BDW80,
Winnipeg, Canada) and kept there for 2 wk in a 8:16 h L:D photoperiod, 20:25°C cycle at a constant 50% relative humidity. After 2 wks, percent germination data were recorded for each of the flats, and the flats were transferred to a greenhouse (20-25°C, 70-90%
15
RH) and irrigated every day. The grass was maintained at a height of about 5 cm.
Grasses were sampled for endophyte 16 wks post-treatment. Plants in each flat were uniformly numbered one to twenty-four. Plant numbers one, two, eleven, twelve, twenty- one and twenty-two were used to determine the presence of the endophyte. Sampled plants had a mature tiller removed from the base of the plant with a small scissors and the tiller was used in the tissue blot immuno-assay technique described by Gwinn et al.
(1991). Percentage of plants with endophyte was recorded for all flats.
Endophyte removal experiment: 2000.
This experiment was performed to replicate the proceeding procedure, but with smaller samples of seed stored in paper bags. About 150 g of seed were removed from the seed stored at 4°C in the preceding year. Latch and Christensen (1982) reported viable endophyte was maintained in seed when stored at 0-5°C for 7 years. Therefore, seed stored at 4°C in between experiments should not have lost any appreciable level of viable endophyte. The samples were placed in brown paper bags (12#, S&G Packaging
LLC, Plainfield, IL) and placed in the same treatments as the preceding year. Every week for 6 wks a sub sample of the seeds was removed from the paper bags and planted in flats as above. The flats were moved to a greenhouse (21°C, 43% RH) after seed germination and maintained at a height of about 5 cm. Germination and percentage of plants with endophyte were recorded as described above.
Field establishment experiment.
The seed from the ‘Endophyte removal experiment: 1999’ was used to establish turf grass plots in the autumn of 1999. The seeding rates were adjusted based upon the
16
germination results that were obtained in the above experiment. Cultivars used in this experiment were ‘Goalkeeper’ and ‘Alamo’. Plots (6.1 x 6.1 m) were seeded with seed from the cold or hot treatment on 24 September 1999. This allowed us to seed plots with either endophyte infected grass or endophyte free grass of the same cultivar. On 15
August and 13 September 2000, four sampling circles made of 21 cm diameter PVC pipe were placed around areas comprised mostly of ‘Goalkeeper’ or ‘Alamo’, respectively.
Five tillers were removed from the area encompassed by a cylinder, brought into the lab, and evaluated for the presence of endophyte as above.
Statistical Analyses.
Percent germination and viable endophyte data were arcsine square root transformed and analyzed using Repeated Measures Analysis of Variance in Statistica 6.0
(Statsoft, 1999). Field data were arcsine square root transformed and analyzed using a two-way analysis of variance (ANOVA). All interactions between variables were considered significant at α = 0.1, and simple effects were at α = 0.05. Data presented in the figures are the non-transformed values.
17
2.4 RESULTS
Endophyte removal experiment: 1999
All three grasses had significant reduction of viable endophyte in the 37°C seed treatment. Endophyte was removed from 90% of the seeds of the two perennial ryegrass cultivars, ‘Goalkeeper’ and ‘Palmer III’, by 4 and 6 wks respectively (F = 13.9 and 16.8, df = 1,6, P = 0.001 and = 0.006, respectively; Fig.2.3 & 2.7). The longer ‘Goalkeeper’ and ‘Palmer III’ seed was in the 37°C treatment the greater was the reduction in percent seed containing viable endophytes (F = 3.4, 9.1, df = 4,24, P = 0.024, P< 0.001, respectively). Although the 37°C treatment significantly reduced viable endophyte in seeds of the tall fescue cultivar ‘Alamo’ (F = 153.8, df= 1,6, P<0.001; Fig. 2.11), time interactions were not significant (P>0.2).
The 37°C treatment significantly reduced germination of ‘Goalkeeper’ by 7-14% as compared to the 4°C treatment (F = 15.8, df = 1,6, P <0.01; Fig. 2.1) but not of
‘Palmer III’ (P >0.05; Fig. 2.5). The 37°C treatment also significantly reduced tall fescue seed germination over time by 7-10% (F= 6.6, df = 4,24, P<0.001; Fig. 2.9).
Endophyte removal experiment: 2000
Only one of the grasses sustained a significant reduction in viable endophyte in the 37°C treatment in this experiment. ‘Alamo’ showed a significant reduction of seed with viable endophyte as time increased (F = 4.5, df = 5,30, P=0.003; Fig. 2.12). About
90% of the viable endophyte was removed from ‘Alamo’ seed by 3 wks and completely
18
eliminated by 4 wks (F = 17.7 and 66.0, df = 1,6, P = 0.006 and < 0.001, respectively).
However, both ‘Goalkeeper’ and ‘Palmer III’ did not show a significant reduction in the numbers of seed with viable endophyte (P>0.05; Fig. 2.4 & 2.8 respectively).
Germination was not significantly reduced for any of the grasses in any treatment
(P>0.05; Fig. 2.10—2.12) in this experiment.
I combined the data from both years to see if there was a significant effect of seed storage on germination and endophyte viability. ‘Goalkeeper’ seed in the brown paper bags (2000 experiment) had more viable endophyte in the 37°C treatment than did the woven polypropylene bags (1999 experiment)(F = 37.5, df = 1,12, P< 0.001). ‘Palmer
III’ seed stored at 37°C in the woven polypropylene bags had significantly less viable endophyte than that stored in the cold (F =16.8, df= 1,6, P = 0.006). However, this was not the case with seed stored in the brown paper bags. Regardless of seed treatment,
‘Palmer III’ seed stored in woven polypropylene bags showed a noticeable decline in the percent viable endophyte by the end of the experiment whereas the seed in brown paper bag did not (F = 2.4, df = 4,48, P = 0.07). Regardless of storage method, tall fescue seeds with viable endophyte decreased over time in the 37°C treatment (F = 3.6, df = 4,48, P =
0.01). Woven polypropylene bags reduced ‘Goalkeeper’ germination in the 37°C treatment; the brown paper bag did not (F = 16.2, df = 1,12). ‘Palmer III’ germination was not adversely affected by storage methods, seed treatment, or time in seed treatments
(P>0.05). ‘Alamo’ seed stored at 37°C in woven polypropylene bags had fewer seeds germinate than seeds in brown paper bags (F = 8.1, df = 4,48, P<0.001).
19
Field establishment experiment
The plots containing the perennial ryegrass (‘Goalkeeper’) seeds stored in the 4°C treatment had significantly higher percentages of grass tillers infected with endophyte than those plots with seed from 37°C treatment (F = 216.5, df = 1,24, P<0.001; Fig.
2.13). About 74% of these perennial ryegrass plants were infected with the endophyte when stored at 4°C; those stored at 37°C had none of the sampled plants infected. Plots seeded with ‘Alamo’ seed from the 37°C treatment had a significantly lower percentage of grass with viable endophytes than seed stored at 4°C (F = 284.0, df = 1,24, P<0.001;
Fig. 2.13). About 48% of the tall fescue plants were infected when stored at 4°C; those stored at 37°C had about 1% infected.
2.5 DISCUSSION
My results illustrate that a majority of the endophyte in tall fescue and perennial ryegrass cultivars can be removed within six weeks by storing the seed at 37°C in woven polypropylene bags. Previous work demonstrates that the viable endophyte can be removed when seed is stored at ambient temperature, but it usually takes seven to twelve months (Siegel et al., 1984; Williams et al., 1984; Rolston et al., 1986). Although
Burpee and Bouton (1993) removed viable endophyte from tall fescue seed by storing at
47°C for only a week, the amount of seed used was small. Other heat treatment methods, such as hot water baths or hot moist air injections, can be complicated and are capable of removing endophyte from seeds in as little as 2 h, but are applicable only to small quantities of seed (Latch and Christensen, 1982; Siegel et al., 1984; Welty et al., 1987;
Bishop et al., 1997). Siegel et al. (1984) stored 30.0 kg of tall fescue seed at about 20°C
20
for nine months then removed all viable endophyte by heat treatment for 1 wk at 49°C.
Additionally, they state that endophytes may be removed in seven to eleven months of
storage at 21°C; consequently, it is possible that the remaining amount of endophyte
removed by the 49°C treatment was minimal. This storage method is simple, fast, and
applicable to large quantities of seed.
Seed germination tends to be adversely affected by prolonged storage and heat
treatments (Latch and Christensen, 1982; Williams et al., 1984; Welty et al., 1987).
Latch and Christensen (1982) submersed seed in hot water baths for as little as 25
minutes at 55°C to remove endophyte, but had about 30% reduction in germination.
Welty et al. (1987) conclude that temperature, seed moisture, and time interact to
influence seed and endophyte viability. However, Bishop et al. (1997) were able to
remove endophyte from small quantities of seed within 2 h with a 50°C heat treatment
and a 50°C rinse without reducing germination. Although Burpee and Bouton (1993)
eliminated endophyte from tall fescue seed in one week at 47°C, they do not report the
effect of their treatment on seed germination. Siegel et al. (1984) treated seed with heat
(49°C) that resulted in a 9% loss of seed viability. Rolston et al. (1991) concluded that endophytes can be removed from ryegrass seed without affecting seed germination if the seed was stored at high humidity and 37°C for 3 weeks. Nott and Latch (1993) removed endophyte from a small amount of perennial ryegrass seed within 2 weeks when seed was stored under high humidity and 37°C. They had a decline in germination of about 13% by the time all viable endophyte was removed. My treatments eliminated most of the endophyte from ‘Goalkeeper’ perennial ryegrass and ‘Alamo’ tall fescue and had 7-14% reduction in germination.
21
Endophytes remain viable when stored at low temperatures (Latch and
Christensen, 1982), but viability may decrease depending on seed moisture (Rolston et al.
1986). The cold seed treatment had similar endophyte levels for the end of 1999 and the
beginning of 2000. Therefore, I concluded that the amount of viable endophyte lost due
to cold storage was minimal.
Differences in the effect of temperature on endophyte viability in the seed in the
two types of bags may be explained by relative seed moisture associated with storage.
Rolston et al. (1986) found some storage bags maintain endophyte viability better than
others, and endophytes have greater survival at lower temperatures and low relative
humidity levels (<60%). Welty et al. (1987) concluded that temperature and seed
moisture interact to influence seed germination and endophyte viability. As seed
moisture and temperature increase, viable endophyte and germination decrease.
I found ‘Palmer III’ perennial ryegrass to be more resistant to the elimination of
its endophyte compared to ‘Goalkeeper’ and ‘Alamo’, a tall fescue cultivar, although the
seed from both species were similarly treated. Host plants and the endophyte both can
influence the concentrations of various alkaloids produced by the symbiotic fungus
(Agee and Hill, 1994; Yue et al., 2000). Breen (1993b) concluded that host plant
genotypes may have greater affect on endophyte infection level within a plant than
temperature. Latch (1997) states that endophyte-host plant interactions may differ based
upon the grass species, cultivar, and endophyte. Therefore, it appears that the difference
between the cultivars and species is due to the interaction between host plant and
endophyte genotypes. Greenhouse and field experiments demonstrated the effectiveness of the 37°C seed treatment in reducing the incidence of viable endophyte from large
22
quantities of seed. Over 90% of viable endophyte was removed from cultivars of both perennial ryegrass and tall fescue when stored in woven polypropylene bags. I conclude that the 37°C seed treatment for 6 wks is sufficient to remove viable endophyte when stored in woven polypropylene bags, which are typically used to ship and store large quantities of seed. Although, the methods used by Nott and Latch (1993) removed endophyte from perennial ryegrass seed quickly, the amount of seed I used was greater and the seed was maintained at lower humidity. My results offer a simple method for quickly removing endophyte in large quantities of seed from both perennial ryegrass and tall fescue with little reduction in seed germination. This method can be used to provide grass plants of the same cultivar with and without endophyte, for large scale field studies without fungicide associated disturbances of the environment.
2.6 ACKNOWLEDGMENTS
I acknowledge Patti Kunkel, Angela Barker, Celia Boone, Somasekhar Nethi, and
Kevin McClure for their technical assistance. I thank Lofts Seed Inc. for supplying the seed used in these experiments. I also thank Dr. J. Scheerens for the use of his vacuum oven, and Dr. D. Richmond for review of the previous version of the manuscript. Ohio
Agriculture Research and Development Center provided funding through an interdisciplinary team research award to Parwinder S. Grewal.
23
1999 100 80 60
% Infection 40 20 0 0123456
Weeks
Figure 2.1 Effect of seed treatments on ‘Goalkeeper’ perennial ryegrass infected with
Neotyphodium endophyte. Percentage (± SEM) of plants infected with
endophyte grown in 1999 from seeds stored in woven polypropylene bags
during treatment, • = 4°C seed treatment and ∆ = 37°C seed treatment.
Weeks refers to the number of weeks seeds were exposed to treatments.
24
1999 100 80 60
% Infection 40 20 0 0123456
Weeks
Figure 2.2 Effect of seed treatments on ‘Palmer III’ perennial ryegrass infected with
Neotyphodium endophyte. Percentage (± SEM) of plants infected with
endophyte grown in 1999 from seeds stored in woven polypropylene bags
during treatment, • = 4°C seed treatment and ∆ = 37°C seed treatment.
Weeks refers to the number of weeks seeds were exposed to treatments.
25
1999 100 80 60
% Infected 40 20 0 0123456
Weeks
Figure 2.3 Effect of seed treatments on ‘Alamo’ tall fescue infected with
Neotyphodium endophyte. Percentage (± SEM) of plants infected with endophyte grown in 1999 from seeds stored in woven polypropylene bags during treatment, • = 4°C seed
treatment and ∆ = 37°C seed treatment. Weeks refers to the number of weeks seeds
were exposed to treatments.
26
1999 100 80 60 40 % Germination 20 0 0123456
Weeks
Figure 2.4 Effect of seed treatments on ‘Goalkeeper’ perennial ryegrass germination
when infected with the Neotyphodium endophyte. Percent seeds (± SEM)
germinated in 1999 that were stored in woven polypropylene bags during
treatment, • = 4°C seed treatment and ∆ = 37°C seed treatment. Weeks
refers to the number of weeks seeds were exposed to treatments.
27
1999 100 80 60 40 % Germination 20 0 0123456
Weeks
Figure 2.5 Effect of seed treatments on ‘Palmer III’ perennial ryegrass germination
when infected with the Neotyphodium endophyte. Percent seeds (± SEM)
germinated in 1999 that were stored in woven polypropylene bags during
treatment, • = 4°C seed treatment and ∆ = 37°C seed treatment. Weeks
refers to the number of weeks seeds were exposed to treatments.
28
1999 100 80 60 40 % Germination 20 0 0123456
Weeks
Figure 2.6 Effect of seed treatments on ‘Alamo’ tall fescue germination when
infected with the Neotyphodium endophyte. Percent seeds (± SEM)
germinated in 1999 that were stored in woven polypropylene bags during
treatment, • = 4°C seed treatment and ∆ = 37°C seed treatment. Weeks
refers to the number of weeks seeds were exposed to treatments.
29
100 2000 80 60
% Infection 40 20 0 0123456
Weeks
Figure 2.7 Effect of seed treatments on ‘Alamo’ tall fescue infected with the
Neotyphodium endophyte. Percentage of plants (± SEM) infected with
endophyte grown in 2000 from seeds stored in paper bags during
treatment, • = 4°C seed treatment and ∆ = 37°C seed treatment. Weeks
refers to the number of weeks seeds were exposed to treatments.
30
2000 100 80 60
% Infected 40 20 0 0123456
Weeks
Figure 2.8 Effect of seed treatments on ‘Goalkeeper’ perennial ryegrass infected with
the Neotyphodium endophyte. Percentage of plants (± SEM) infected with
endophyte grown in 2000 from seeds stored in paper bags during
treatment, • = 4°C seed treatment and ∆ = 37°C seed treatment. Weeks
refers to the number of weeks seeds were exposed to treatments.
31
100 2000 80 60
% Infection 40 20 0 0123456
Weeks
Figure 2.9 Effect of seed treatments on ‘Palmer III’ perennial ryegrass infected with
the Neotyphodium endophyte. Percentage of plants (± SEM) infected with
endophyte grown in 2000 from seeds stored in paper bags during
treatment, • = 4°C seed treatment and ∆ = 37°C seed treatment. Weeks
refers to the number of weeks seeds were exposed to treatments.
32
2000 100 80 60 40 % Germination 20 0 0123456
Weeks
Figure 2.10 Effect of seed treatments on ‘Goalkeeper’ perennial ryegrass germination
when infected with the Neotyphodium endophyte. Percent seeds (± SEM)
germinated in 2000 that were stored in paper bags during treatment, • =
4°C seed treatment and ∆ = 37°C seed treatment. Weeks refers to the
number of weeks seeds were exposed to treatments.
33
2000 100 80 60 40 % Germination 20 0 0123456
Weeks
Figure 2.11 Effect of seed treatments on ‘Palmer III’ perennial ryegrass germination
when infected with the Neotyphodium endophyte. Percent seeds (± SEM)
germinated in 2000 that were stored in paper bags during treatment, • =
4°C seed treatment and ∆ = 37°C seed treatment. Weeks refers to the
number of weeks seeds were exposed to treatments.
34
2000 100 80 60 40 % Germination 20 0 0123456
Weeks
Figure 2.12 Effect of seed treatments on ‘Alamo’ tall fescue germination when
infected with the Neotyphodium endophyte. Percent seeds (± SEM)
germinated in 2000 that were stored in paper bags during treatment, • =
4°C seed treatment and ∆ = 37°C seed treatment. Weeks refers to the
number of weeks seeds were exposed to treatments.
35
100 80 60 40 % Infection 20 0 4oC37oC4oC37oC Goalkeeper Alamo
Figure 2.13 Percentage of plants infected with Neotyphodium endophytes in field plots
when planted with either seed stored at 37° or 4°C respectively.
36
CHAPTER 3
Endophyte infection in perennial ryegrass reduces the susceptibility of black cutworm to
an entomopathogenic nematode
3.1 ABSTRACT
Perennial ryegrass, Lolium perenne L., forms a symbiotic relationship with
Neotyphodium lolii, a fungus that produces alkaloids. This relationship provides a competitive advantage to the host plant in grassland communities by increasing drought tolerance, and disease and herbivore resistance. Black cutworm, Agrotis ipsilon, is among the few insect species that are able to feed and develop on endophytic perennial ryegrass. As some insects can use plant secondary compounds to defend themselves against predators, I hypothesized that the cutworms fed on endophytic grasses would exhibit greater defense against a lethal endoparasitic nematode, Steinernema carpocapsae. Laboratory experiments involving 4-5th instars support the hypothesis that
A. ipsilon feeding on grass clippings from field plots with high (>90%) incidence of
endophyte infected perennial ryegrass are less susceptible to entomopathogenic
nematodes than larvae fed grass clippings from plants with little or no incidence of
endophyte. Laboratory studies resulted in similar overall mortality after 48 h. However,
field studies show decreased
37
susceptibility to S. carpocapsae when larvae were confined to areas of endophytic grass
(>75% infected). Early instars (2-3rd) fed on endophyte free grass suffered greater overall mortality at all nematode concentrations than 4-5th instars fed similarly. Early (2-
3rd) instars were equally susceptible to nematode attack regardless of food source. These results indicate that the fungal endosymbionts of grasses can influence the biology of natural enemies of an herbivorous insect.
3.2 INTRODUCTION
As plants evolve new defenses to escape their insect herbivores, a fitness benefit is thought to lead to greater speciation (Dobler, 2001). Some grasses in the family
Poaceae may form symbioses with the endophytic fungi, Neotyphodium spp. (Siegel et al., 1987). The benefit of harboring the endophytic fungi primarily stems from the acquisition of fungus-produced defensive alkaloids that provide resistance against herbivory (Breen, 1994; Porter, 1994). Grasses harboring the fungal endophytes eventually dominate a plant community (Clay, 1997).
Insects that overcome plant chemical defenses may exploit the plants as a resource free of competition and may evolve into insects that specialize on those plants.
Insect specialists may use noxious compounds produced by plants as feeding stimulants or incorporate them into their own defense; regardless these insects have evolved mechanisms to deal with these secondary compounds (Dobler, 2001). Ehrlich & Raven
(1964) referred to this evolutionary race as coevolution between plants and herbivorous insects. Black cutworm, Agrotis ipsilon Hufnagel, can feed and complete development on the endophyte-infected perennial ryegrass (Williamson & Potter, 1997a) that is
38
otherwise highly toxic to diverse insect species (Breen, 1994; Clay, 1997). The ability of this polyphagous species to feed and develop on endophytic perennial ryegrass indicates the ability to tolerate or detoxify the plant alkaloids. However, Bultman et al. (1997) found that parasitoids are affected by exposure of their host insects to endophytes. Thus, there is evidence that insects that can develop on endophytic grass gain some benefit against their natural enemies.
Omacini et al. (2001) reported that endophytic plants have the potential to alter food-web dynamics. They found that Neotyphodium lolii reduces survival of aphid parasitoids and their associated hyperparasitoids. Barker & Addison (1996) demonstrated that confinement of Listronotus bonariensis (Kuschel) weevils on endophyte infected ryegrass and switchgrass leaf segments resulted in retarded development of the parasitoid Microctonus hyperodae Loan. These multitrophic effects are expected when the development and survival of primary herbivores is affected by the poor quality diet and toxicity of the secondary metabolites resulting from the plant-fungal symbiosis. However, some insect herbivores that consume plants with toxic or deterrent secondary compounds have incorporated these chemicals into their own defenses
(Brower, 1969; Brown, 1984). Therefore, I hypothesized that A. ipsilon larvae fed on endophytic perennial ryegrass would exhibit greater defense against their natural enemies.
In this investigation, I demonstrate the acquisition of enhanced tolerance/resistance by A. ipsilon larvae fed on endophyte infected perennial ryegrass against infection by an entomopathogenic nematode with an extremely broad host range.
I selected Steinernema carpocapsae Weiser (Rhabditida: Steinernematidae) for this
39
investigation because it is a lethal endoparasite of A. ipsilon larvae and is used in its biological control (Grewal & Georgis, 1998). The third stage infective juveniles of S. carpocapsae attack suitable insects in or on the soil (Campbell & Gaugler, 1993; Grewal et al., 1997), invade through natural body openings, and release a symbiotic bacterium,
Xenorhabdus nematophila Thomas and Poinar, into the hemocoel. The bacterial infection usually kills the host within 2—3 days (Kaya & Gaugler, 1993). The infective juveniles feed on multiplying bacteria and insect body contents and reproduce. After 1-3 generations, the next generation of infective juveniles is produced and leaves the cadaver to seek new hosts.
First, I measured the effects of endophytic perennial ryegrass on weight gain and time to pupation of A. ipsilon. Next, I determined the differences in susceptibility of 2nd
and 4th instar A. ipsilon to S. carpocapsae after feeding the larvae on perennial ryegrass
containing different levels of endophyte. I also evaluated the effects of nematode dose on
both 2-3rd and 4-5th instars fed endophyte free perennial ryegrass. The susceptibility of the 4th instar A. ipsilon fed on endophytic or non-endophytic perennial ryegrass was also evaluated in the field.
3.3 MATERIAL AND METHODS
Grasses, insects, and nematodes.
Perennial ryegrass (Lolium perenne L.) was used to study the effect of the
endophyte, Neotyphodium lolii (Latch, Samuels, & Christensen) Glenn, Bacon & Hanlin,
on black cutworm susceptibility to entomopathogenic nematodes. Field plots planted
with blends of ‘Repel II’ perennial ryegrass (Loft’s Seed Inc., Wilmington, OH)
40
contained one of three levels of endophyte infected grass plants; high (>90% tillers
infected), medium (40-60% tillers infected), and low (<20% tillers infected). Endophyte
levels of all grasses were previously determined either through staining plant material or
by a modified tissue print immunoblott assay (Saha et al., 1988; Gwinn et al., 1991).
Clippings were gathered during July – September 1999 for use in assays with neonate
cutworms. Endophyte free ryegrass was clonally propagated from plants treated with
repeated applications of the fungicide, propiconazole the previous year. Endophyte
infected ‘Repel II’ ryegrass grown in the greenhouse had an infection rate of about 75%
or >95%. Grass clipping samples were taken from the ryegrass grown in the greenhouse
while larvae were being reared for use in the experiments. These samples were processed
with an HPLC-based method (Salminen & Grewal, 2001) to verify that fungal produced
alkaloids were present at the time larvae were consuming the plants. These samples
confirmed results from staining or immuno-assays that the fungus was present throughout
the experiments. The nematodes, S. carpocapsae, were reared from the last instar of G.
mellonella L. (Kaya & Stock, 1997), and were stored at 24ûC. The A. ipsilon eggs were obtained from adults reared in laboratory colonies maintained at USDA-ARS in Ames,
Iowa. For all experiments, A. ipsilon larvae were fed on grass clippings from plants containing the appropriate levels of endophyte infection for the respective experiments and were kept in an incubator at 25°C, L14:D10 cycle. In laboratory experiments, A.
ipsilon larvae were considered dead if they did not show any response to prodding with a
paintbrush. I defined larval susceptibility to S. carpocapsae as the likelihood that an
insect encountering the nematodes will become infected and die. All laboratory
experiments were conducted in an incubator at 25°C, L14:D10 cycle.
41
Effect of endophyte on A. ipsilon development.
Our methods used to rear A. ipsilon were similar to those of Williamson & Potter
(1997a) in which 10 neonates were placed in a 9 cm petri dish and fed either endophyte infected or endophyte free grass for 1 week. Larvae were weighed at 1 week and placed singly in 5 x 8 cm plastic cups to prevent cannibalism. Larvae were weighed at 8, 15, 21, and 28 days of development and total number of days required for pupation was recorded up to 40 days. Food was provided ad libitum daily, or as necessary.
Effect of endophyte infected field grown grass on 4th instar A. ipsilon susceptibility to nematodes.
This experiment was conducted to determine if A. ipsilon feeding on endophytic grass results in decreased mortality due to entomopathogenic nematodes. Larvae were divided into groups and randomly assigned a level (high, medium or low) of endophyte perennial ryegrass from the field plots. Larvae in each treatment were fed on grass clippings having the appropriate level of endophyte infection for 2 wk before being used in the experiment. Prior to exposure to the nematodes, larvae were weighed and grouped by size to determine if size influenced susceptibility. Five 4th instars were exposed to
300 nematodes per petri dish (9.0 cm diameter) with four replicates. Controls for this experiment consisted of larvae fed on all levels of endophyte perennial ryegrass (high, medium, low), but without nematodes. Mortality was recorded at 20, 24, 28, and 42 h post-exposure to the nematodes.
42
Dose experiments.
These experiments were conducted to determine the effect of nematode dose on
2-3rd and 4-5th instars. I hypothesized that 2-3rd instars would die more quickly and
would have greater overall mortality at the specified doses compared to 4-5th instars.
Larvae were fed endophyte free grass clippings from greenhouse plants until they were
either 2-3rd instar or 4-5th instar. Nematode doses used for the experiment were 0, 10, 20, and 40 infective juveniles per insect per petri dish. Each dose was applied to either two
(2-3rd instars) or three (4-5th instars) petri dishes per replicate. The susceptibility of 4-5th
instars to nematode doses was replicated four times; whereas the response of 2-3rd instars was replicated three times. The experiment was conducted a couple of weeks earlier for
2-3rd instars than 4-5th instars, and the insects came from different populations. The no
nematode dose counted as our control. Five larvae were added to each petri dish about
an hour after nematode application. Mortality readings were recorded at 16 h and every 4
h thereafter until 44 h exposure.
Effects of greenhouse grown grass on 4 - 5th instar A. ipsilon susceptibility to
nematodes.
The experiment tested the susceptibility of 4-5th instar A. ipsilon to S.
carpocapsae when the larvae had consumed grass clippings from one of the two
endophyte infection levels; no endophyte and >95% plants infected. Individual neonates
were placed in a 5 cm petri dish and fed either endophyte infected or endophyte free
grass clippings for 3 wk. A. ipsilon larvae were weighed individually and placed into
43
size classes. The 4th instar larvae of similar weight were chosen from both diets for use
in the experiment. A nematode concentration of 35 infective juveniles per larva (175
nematodes/dish) was applied to filter paper in a 9.0 cm plastic Petri dish 2 h prior to
introduction of larvae. Five larvae were placed into each dish with grass clippings. I
used two Petri dishes for each treatment within a replicate, and I replicated the
experiment three times (n = 30 larvae/treatment). The treatments were larvae fed on
endophytic grass (no nematodes), larvae fed on endophyte free grass (no nematodes), larvae fed on endophytic grass and exposed to nematodes, and larvae fed on endophyte free grass and exposed to nematodes. Mortality was recorded at 16 h and every 4 h thereafter until >80% mortality was observed. Larvae that died during this experiment were placed at 5°C after 72 h at 25°C to slow nematode development. Dead larvae from all treatments were dissected to count the number of adult nematodes. Adult nematodes are presented as number of nematodes penetrated/larva.
Effect of greenhouse grown grass on 2nd—3rd instar A. ipsilon susceptibility to
nematodes.
This experiment examined the effect of endophyte-infected grass on early instar
susceptibility to S. carpocapsae. Two trials of early instar exposure to S. carpocapsae
were conducted to account for low numbers of replicates within each separate trial.
Neonates were fed either endophytic (75% infected) or non-endophytic grass (0%
infected) from the greenhouse and kept in an incubator at 25°C, L14:D10 cycle. Controls
consisted of four larvae provided either endophyte or endophyte free perennial ryegrass
in petri dishes and not exposed to nematodes. Treatments were larvae fed either
44
endophyte or endophyte free perennial ryegrass and exposed to nematodes, or the respective control. An experimental unit consisted of 9.0 cm plastic petri dish that contained four 2nd – 3rd instar A. ipsilon. This experiment had three experimental units per treatment for each replicate and the experiment was replicated four times (n = 48 larvae per treatment). The nematodes were applied at 80 infective juveniles per dish.
Mortality in each experimental unit was recorded at 16 h and every 4 h thereafter until 48 h after exposure.
I conducted this experiment again with the same nematode concentration to verify the lack of effects of endophytic grass on early instar susceptibility to S. carpocapsae, but this time I also included larval weight as a covariate. Larvae were fed endophytic (>95% infected) or non-endophytic grass as before and mortality was recorded at identical time intervals after exposure. Data from this experiment were not combined with the previous experiment because the endophyte infection level was greater, and I wanted to determine if weight was a significant covariate to larval susceptibility. Larvae fed either endophyte or endophyte free grass clippings were weighed individually and assigned to weight classes. There were five 2nd-3rd instars in each 9.0 cm dish and 100 infective juveniles were applied per dish. Larvae of similar weight were used per replicate. There were two dishes per replicate and four replicates for the experiment (n = 40 larvae per treatment).
Controls were the larvae fed on the respective grass, but not exposed to nematodes.
Field experiment.
Turfgrass plots, containing endophyte infected (about 75%) or endophyte free
(0%) grass, were used to determine if susceptibility of A. ipsilon to nematodes was
45
similarly affected by the endophyte in the field. In this experiment, larvae were obtained
from a laboratory colony (Dow AgroSciences, Indianapolis, IN) where they had been fed
on artificial diet until 2-3rd instar. Cylinders (21 cm dia) confined 2nd – 3rd instar A. ipsilon to areas of endophyte infected or endophyte free perennial ryegrass. Ten larvae were placed in each cylinder, and a 2.5 cm band of Vaseline® was placed around the lip of each cylinder to prevent their escape. A fine mesh was placed over each cylinder to prevent predation by birds. Larvae fed within the cylinders for 10 d prior to nematode application in order to reach 4-6th instar. The nematodes were applied at 0.5 billion
infective juveniles/acre. At 36-38 h post-application, the remaining grass was clipped
close to the ground, and clippings were examined for larvae before being discarded. The
ground was drenched with a Joy® (Proctor and Gamble, Cincinnati, OH) detergent rinse
(30 mL/7.6 L) to cause the larvae to surface (Niemczyk & Shetlar, 2000). The larvae that
did not surface were considered dead. Control checks consisted of the same size
cylinders, number of larvae, endophyte or endophyte free grass and the length of time in
the field, but without nematode application.
Data analysis.
Data were analyzed using Statistica(Statsoft, 1999) software package. Data for
the larval feeding trial and effect of endophyte on A. ipsilon susceptibility were analyzed
using Multivariate Repeated Measures Analysis of Variance (ANOVA). Data were
arcsine square root transformed before analysis as a Repeated Measures ANOVA. The
remainder of the laboratory experiments was analyzed using a Univariate Repeated
Measures ANOVA and Greenhouse-Geisser adjusted degrees of freedom and P-values
46
were used where the data failed the assumptions of sphericity. The field experiment was
analyzed as an ANOVA and mortality was corrected with Abbott’s formula (Abbott,
1925). Interactions were evaluated for significance at α= 0.1 in order to minimize risks
of Type II errors. Significant interactions were evaluated using linear contrasts, and main
effects were examined using a least significant difference (LSD) means separation
procedure. The untransformed data are presented.
3.4 RESULTS
Effect of endophyte on A. ipsilon development.
A. ipsilon larvae fed on non-endophytic grass obtained greater mass quicker than larvae feeding on endophytic grass over the course of the experiment (F=2.95,df = 4, 42;
P=0.03; Fig.3.1). There was no significant difference for time to pupation or emergence
(P>0.05) between larvae fed different diets (data not presented). The average number of days to pupation for larvae fed non-endophytic grasses was 37 and those fed on endophytic grass was 38.
Effect of endophyte infected field grown grass on 4th instar A. ipsilon susceptibility
to nematodes.
Overall, the type of grass had a significant effect on 4th instar mortality induced
by S. carpocapsae (F= 4.2,df =2, 24; P= 0.03). Those larvae fed on grass from plots
containing high endophyte levels had significantly lower average cumulative mortality
than those fed on grass from plots with medium or low percentages of plants infected with endophytes. There was an interaction between endophyte level of grass and length
47
of time to mortality (P=0.06; Fig. 3.2). Early in the experiment, larvae fed on grass from
high endophyte plots had less mortality at 20 and 28 h post-exposure to nematodes than
did larvae fed grass from the low level plots (t >2.1 df =1,24, P≤0.05). I found that
almost all larvae died by 42 h post-exposure (Fig. 3.2). The weight of larvae did not
differ when fed on clippings from plants in field plots with high, medium, or low
proportions of endophytic grasses (data not shown; P>0.05).
Dose experiment.
My results support the hypothesis that the 2-3rd instars had greater rates of
mortality than 4-5th instars at each time interval (F = 2.6, df = 3,190, P = 0.05; Fig. 3.4).
Additionally, 2-3rd instars suffered significantly greater average cumulative mortality
than 4-5th instars at each nematode dose excluding controls (F = 3.6, df = 3,64, P = 0.02;
Fig 3.3). Twenty nematodes per larvae resulted in a total cumulative mortality of 87.4%
(± 8.5%) for the 2-3rd instars and in 74.2% (± 5.4%) total cumulative mortality for 4-5th instars. However, 40 nematodes per insect resulted in a similar average cumulative mortality (49.4 ± 4.1%) for 4-5th instars as the 20 nematodes per insect caused for 2-3rd
instars (55.9 ± 6.1%; Fig. 3.3). The dose of 40 nematode/insect caused a 67.2 (± 5.6%)
average cumulative mortality in 2-3rd instars. Finally, it took about 24 – 28 h for 2-3rd instars exposed to 20 nematodes/insect to cause greater than 50% mortality, whereas, for
4-5th instar it took 32 h. Those 4-5th instars exposed to 40 nematodes/insect averaged
greater than 50% mortality by 28 h. Therefore, I chose 20 nematodes per insect as the
dose for 2-3rd instars for future experiments and 40 nematodes per insect for 4-5th instars.
48
Effect of greenhouse grown grass on 4 – 5th instar A. ipsilon susceptibility to
nematodes.
In this trial I found that there was an interaction between length of time to death
and presence of endophyte (F=27.3, df = 21, 77; P<0.01). Larvae fed on non-endophytic
grass clippings had higher rates of mortality than those fed on endophytic grass clippings
at 24 h (F= 7.2, df =1,11, P=0.02; Fig. 3.5). Larvae fed on non-endophytic grass suffered
about 60% (± 11.8%) mortality by 28 h, whereas, those fed on endophytic grass suffered
only 47.5% (± 7.9%). There were no significant differences after 32 h (Fig. 3.5). Unlike
the first trial, there were no overall differences in average total cumulative larval
mortality due to endophyte. Weight of larvae was not a significant covariate for this trial.
Similar numbers of adult nematodes were found in dead larvae fed on endophyte grass
(4.9 ± 0.6 nematodes penetrated/larva) or fed on endophyte free grass (5.0 ± 0.6
nematodes/larva; P>0.05).
Effect of endophyte on 2nd—3rd instar A. ipsilon susceptibility to nematodes.
I found that there were no significant effects (P>0.05) of endophyte infection on
susceptibility of early instar larvae to entomopathogenic nematodes over time, or on
average cumulative mortality overall (Fig. 3.6). I found that larvae fed on endophyte
infected grass experienced about 50% mortality within 24-28 h of exposure to nematodes,
whereas those larvae fed on endophyte free grass suffered 50% or greater mortality
within 28-32 h of exposure (Fig. 3.6). However, this observation was not significantly
different. In the second experiment, larvae fed on endophyte-infected grass sustained
49
about 82% mortality overall; whereas, those fed on endophyte free grass clippings had
about 78% (Fig. 3.7). The second experiment also showed no significant effect of endophyte on susceptibility of A. ipsilon to S. carpocapsae (P>0.05; Fig. 3.7).
Additionally, larvae fed on either endophytic or endophyte free grass and exposed to nematodes averaged about 50% mortality around 32-36 h (51.1 ± 10.5% and 52.5 ±
11.8%, respectively; Fig. 3.7). Additionally, there was no significant correlation of weight of larvae to mortality (P >0.05).
Field experiment.
The results of this experiment were similar to the laboratory experiments using 4th instar larvae. Putative nematode-induced mortality at 36 h in the field was lower for A. ipsilon feeding on endophyte-infected ryegrass (16 ± 8.3%) than for non-endophytic ryegrass (51 ± 15.1%; F=14.3, df=1,4; P=0.02; Fig. 3.8).
3.5 DISCUSSION
The 4th instar A. ipsilon larvae fed on perennial ryegrass with high prevalence of
endophyte had lower susceptibility to S. carpocapsae than those fed on plants with lower
or no incidence of endophyte for at least some of the exposure period. Barbercheck
(1993) found that southern corn rootworm larvae (Diabrotica undecimpunctata howardi
Barber) exposed to S. carpocapsae had higher rates of mortality when fed on squash
(cucurbitacins) compared to those fed on peanuts. However, those insects fed on squash
had lower nematode progeny produced per host, thus suggesting the possibility of a
terpenoid effect. In fact, Barbercheck & Wang (1996) found that cucurbitacin D could
50
adversely affect the growth of Xenorhabdus and Photorhabdus spp, both symbiotic bacteria of entomopathogenic nematodes. Additionally, Epsky & Capinera (1994b) found that A. ipsilon fed collards containing glucosinolates were poor hosts for S. carpocapsae reproduction because of lipid content differences. Endophytic grasses can negatively affect phytopathogenic nematodes (Elmi et al., 2000). The alkaloids produced by endophytic perennial ryegrass and tall fescue can also reduce the survival and infectivity of the entomopathogenic nematode, Heterorhabditis bacteriophora Poinar
(Grewal et al., 2003). Other entomopathogens can also be adversely affected by plant secondary metabolites (Krischik et al., 1988; Costa & Gaugler, 1989). Therefore, the decrease in the 4th instar A. ipsilon susceptibility to nematodes may be due to the direct toxic effects of alkaloids on S. carpocapsae or on nematodes’ symbiotic bacterium, X. nematophila. However, there is also a possibility of the altered physiology of A. ipsilon that leads to enhanced defense against the nematodes.
White grubs (Coleoptera: Scarabaeidae) that feed on roots of endophytic grasses only suffer from sub-lethal effects including reduced weight gain and slower development (Potter et al., 1992; Crutchfield & Potter, 1995). Grewal et al. (1995) discovered that P. japonica grubs feeding on endophytic tall fescue and ryegrass were more susceptible to attack by H. bacteriophora. However, the greatest concentrations of alkaloids from endophytic grasses are typically found in aerial portions of the plants
(Carroll, 1988). Therefore, the alkaloid concentrations normally encountered by scarabs would be substantially less than the alkaloid concentrations encountered by foliage feeding pests. The enhanced susceptibility of white grubs fed on the roots of endophyte infected tall fescue to entomopathogenic nematodes may be due to the synergism
51
between the low concentrations of endophyte alkaloids and the entomopathogenic
nematodes. The decreased susceptibility of A. ipsilon to S. carpocapsae may be related to the consumption of high concentrations of alkaloids found in aerial parts of the endophytic perennial ryegrass.
Another reason A. ipsilon has a reduction in susceptibility to entomopathogenic nematodes could be the acquisition of a novel defense by the larvae during feeding on endophytic grass. Parasitoids can be affected by plant alkaloids consumed by their larval hosts (Campbell & Duffey, 1979; Barbosa et al., 1991). Alkaloids may render hosts deterrent to the natural enemies due to sequestration and inclusion of alkaloids into insect cuticle or hemolymph (Montllor et al., 1991; Schaffner et al., 1994). Current research demonstrates that early instar lepidopterans are more susceptible to entomopathogens than later instars (Huang et al., 1999; Escribano et al., 1999). Likewise, our experiments demonstrate that 2-3rd instars exposed to 10, 20, and 40 infective juveniles per insect died quicker than 4-5th instars. Apparently, age is an important factor in the susceptibility of
A. ipsilon to the nematodes. However, as nematode dose increased, differences in
mortality decreased. Early instars that consumed endophytic grass were as susceptible to
S. carpocapsae as those feeding on non-endophytic ryegrass in all experiments.
However, I found that 4-5th instars fed on endophytic perennial ryegrass were less
susceptible to the nematodes than larvae fed on endophyte free grass. Therefore, the
results suggest that developing A. ipsilon larvae receive a benefit from feeding on
endophyte-infected perennial ryegrass.
My results confirm results by Williamson & Potter (1997a) that A. ipsilon feeding
on endophytic perennial ryegrass show a decrease in weight gain, but not a significant
52
increase in developmental time. The larvae fed on endophytic grass had greater mass at
35 d in our experiments because some of the larvae fed on non-endophytic grass had
entered a non-feeding prepupae stage, and thus weighed less. I show evidence that A.
ipsilon is able to feed and develop on endophytic perennial ryegrass although other
lepidopterans are negatively affected (Richmond & Shetlar 1999).
A. ipsilon is capable of completing development when fed on exclusively endophytic perennial ryegrass; however, it does result in decreased weight gain. This suggests that endophytic perennial ryegrass is not the best host plant for the larvae to develop on especially since there is a long time to pupation. However, Grewal et al.
(1995) suggest that lack of adequate food may enhance susceptibility of white grubs to nematodes. Therefore, A. ipsilon should be easier to control with S. carpocapsae because of the poor host plant, but this does not appear to be the case. Early instars are more susceptible to S. carpocapsae, and as the larva ages it becomes more resistant to nematode infection. Larvae fed on endophytic grass and exposed to nematodes under realistic field conditions (i.e., no confinement) could result in more drastic reductions of susceptibility to S. carpocapsae compared to larvae fed on endophyte free grasses and exposed to nematodes. A possible reason for this could be that S. carpocapsae is patchily distributed in turfgrass (Campbell et al., 1996; Campbell et al., 1998) and A. ipsilon larvae move considerable distances at night (Williamson and Potter, 1997b).
Larvae confined to experimental arenas where contact with nematodes was assured may have confounded our results because larvae are not permitted to escape predation pressure. In my field experiment, confined insects fed on either endophytic or endophyte free grasses were not restricted to just grass clippings. Greater recovery rates of larvae
53
from field plots containing endophytic grass suggest that these larvae were less susceptible to the nematode. In laboratory studies, the larvae were fed exclusively on grass clippings, but in the field the entire plant may be consumed. Since the greatest concentration of the fungus is in leaf sheaths and the crown, larvae in laboratory studies may not have encountered the same concentrations of fungal hyphae as in field situations.
Although effects appear to be transitory in laboratory studies, field situations may result in an increase in longevity that permits a larva to possibly inflict economic damage in highly valued turf areas where damage thresholds are extremely low. Future research needs to determine the mechanisms of this decrease of susceptibility of A. ipsilon to the entomopathogenic nematode S. carpocapsae.
3.6 ACKNOWLEDGMENTS
I thank Patti Kunkel, Dr. John Lloyd, Dr. Elizabeth DeNardo, and Dr. Xiaodong
Wang for their technical assistance. I thank Dow AgroSciences and J. Dyer at USDA-
ARS for supplying A. ipsilon larvae used in these experiments. This work was funded by the USDA-CSREES project No. 00-35316-2949 to P.S. Grewal.
54
800 640 480 320 Mass (mg) 160 0 8 15212835 Days
Figure 3.1 Weight gain (+SEM) by neonate Agrotis ipsilon fed on endophyte infected
(▼) or endophyte free (●) perennial ryegrass to pupation.
55
100 80 60
% mortality 40 20 0 20 24 28 42 Hours exposed
Figure. 3.2 Average mortality (+ SEM) of 4th instar Agrotis ipsilon due to
Steinernema carpocapsae over time. Larvae were fed for 14 d on
perennial ryegrass, Lolium perenne, plants containing high (>90% plants
infected), medium (40-60% plants infected), and low (<20% plants
infected) prevalence of Neotyphodium lolii endophytes. Treatments were
as follows: high endophyte plus nematodes (▼), medium endophyte plus
nematodes (■), low endophyte plus nematodes (○), high endophyte and no
nematodes (∇), medium endophyte and no nematodes (□), and low
endophyte and no nematodes (●). Nematode concentration used in this
experiment was 60 infective juveniles/insect.
56
100
80 b b 60 b a a
% mortality 40 a 20
0 0102040 Dose (infective juveniles/insect)
Figure 3.3 Average cumulative mortality over time (+ SEM) of Agrotis ipsilon
larvae. 2-3rd instars (open bars) and 4-5th instars (solid bars) were exposed
to 0, 10, 20, or 40 Steinernema carpocapsae infective juveniles per insect.
Bars with different letters are significantly different at α = 0.05.
57
100
80
60
40 % mortality
20
0 16 20 24 28 32 36 44 Hours exposed
Figure 3.4 Average cumulative mortality Mortality (± SEM) of 2-3rd (○) and 4-5th (●)
instar Agrotis ipsilon exposed to Steinernema carpocapsae over time.
58
100
75
50 % mortality 25
0 16 20 24 28 32 36 40 48 Hours exposed
Figure 3.5 Average mortality (+SEM) of 4-5th instars reared on greenhouse grown
plants containing either >95% infected with endophyte (E+) or without
endophyte infection (E-). Nematode concentration used in this experiment
was about 35 infective juveniles/insect. Treatments were as follows: fed
E+ and exposed to nematodes (●), fed E+ and not exposed to nematodes
(○), fed E – and exposed to nematodes (▲), and fed E – and not exposed
to nematodes (∆).
59
100
75
50 % Mortality 25
0 16 20 24 28 32 36 40 44 48 Hours exposed
Figure 3.6 Average mortality (+ SEM) due to Steinernema carpocapsae of 2nd —3rd
instar Agrotis ipsilon fed on endophyte infected >75% (E +) or endophyte
free (E -) perennial ryegrass. Nematode concentration used in both
experiments was 20 infective juveniles/insect. Treatments were as
follows: fed E+ and exposed to nematodes (●), fed E+ and not exposed to
nematodes (○), fed E – and exposed to nematodes (▲), and fed E – and
not exposed to nematodes (∆).
60
100
75
50
% Mortality 25
0 16 20 24 28 32 36 40 44 48 Hours exposed
Figure 3.7 Average mortality (+ SEM) due to Steinernema carpocapsae of 2nd —3rd
instar Agrotis ipsilon fed on endophyte infected >95% (E+) or endophyte
free (E -) perennial ryegrass. Nematode concentration used in both
experiments was 20 infective juveniles/insect. Treatments were as
follows: fed E+ and exposed to nematodes (●), fed E+ and not exposed to
nematodes (○), fed E – and exposed to nematodes (▲), and fed E – and
not exposed to nematodes (∆).
61
100
80 b
60
40 a % Mortality
20
0 Endophyte Endophyte Infected Free
Figure 3.8 Field experiment. Mortality of 4th instar Agrotis ipsilon exposed to
Steinernema carpocapsae for 36- 38 h in field plots of endophyte-free and
endophyte-infected ‘Goalkeeper’ perennial ryegrass (Lolium perenne).
Larvae were confined using 21 cm diameter PVC cylinders. Nematodes
used in the field experiment were applied at 0.5 billion/acre rate, which is
one-half the recommended rate.
62
CHAPTER 4
Endophyte infection in perennial ryegrass reduces the susceptibility of black cutworm to
an entomopathogenic nematode: the mechanism
4.1 ABSTRACT
Perennial ryegrass forms a symbiotic relationship with the fungus Neotyphodium
lolii, which provides many benefits including resistance to herbivory through the
production of alkaloids. The impact of endophytic grass on the third trophic level has
received little attention. The black cutworm, Agrotis ipsilon, acquires reduced susceptibility to the entomopathogenic nematode, Steinernema carpocapsae when it consumes the endophytic grass. I examined the potential mechanisms of the resistance exhibited by A. ipsilon against S. carpocapsae. Although A. ipsilon larvae fed on endophytic grass had similar numbers of nematodes attach and successfully develop into adults, they did suffer significantly lower mortality than larvae fed on endophyte free grass when exposed to nematodes for only 1.5 h. I examined the effects of ergot alkaloids that are produced by N. lolii such as, ergotamine tartrate, ergonovine maleate, ergocryptine, ergocristine, and seed extract on nematode viability and infectivity.
Ergonovine malate increased and ergocristine decreased the rates of nematode infectivity, whereas other treatments had no significant effect. I investigated the effects of ergocristine on Xenorhabdus nematophila, the symbiotic bacterium of S. carpocapsae.
63
Bacterial growth and pathogenicity were significantly reduced when bacteria were grown
in nutrient broth containing 200 µg/ml concentration of ergocristine. I conclude that
herbivores capable of developing on endophytic grasses may acquire some level of
resistance against entomopathogenic nematodes due to reductions in infectivity. This
resistance is mediated through the effects of alkaloids; thus our results underscore the
ability of N. lolii to affect trophic interactions through the production of alkaloids.
4.2 INTRODUCTION
Perennial ryegrass, Lolium perenne L., forms a symbiotic relationship with the
fungus, Neotyphodium lolii (Latch, Samuels & Christensen) Glenn, Bacon & Hill, which produces alkaloids that provide resistance against herbivory (Siegel et al., 1987; Breen,
1994). The fungus colonizes the leaves, stems, crown, and reproductive structures of the plant, but is asymptomatic (Carroll, 1988). There is paucity of information on the effects of this symbiotic relationship on the third trophic level. Although Davidson and Potter
(1995) found that the densities of various arthropod predators did not differ between plots of endophytic and non-endophytic tall fescue, the fungus can adversely affect the community dynamics and growth of parasitoids (Barker and Addison, 1996; Omacini et al., 2001). Kunkel and Grewal (2003) found that the late instar black cutworms, Agrotis
ipsilon Hufnagel (Lepidoptera: Noctuidae), fed on endophyte infected perennial ryegrass
were less susceptible to the enotmopathogenic nematode, Steinernema carpocapsae
Weiser (Nematoda: Steinernematidae) than those fed on endophyte-free grass.
Agrotis ipsilon is a generalist insect herbivore that can feed and develop on endophyte-infected perennial ryegrass (Busching and Turpin, 1976, 1977; Williamson
64
and Potter, 1997a). The entomopathogenic nematode, S. carpocapsae is typically used to manage A. ipsilon (Buhler and Gibb, 1994; Baur et al., 1997). The ambush foraging strategy of S. carpocapsae allows infective juveniles to attach to mobile, surface dwelling hosts that pass nearby (Kaya and Gaugler, 1993). Campbell and Gaugler (1993) found that S. carpocapsae nictates (body waving) which aids in attachment to hosts by an increase in the proportion of the nematode in contact with a host and reduction in surface tensions that hold the nematode to the substrate. Following attachment, the nematode penetrates either through the mouth, anus, or spiracles of the insect host. After the infective juveniles reach the hemocoel, they release their symbiotic bacteria,
Xenorhabdus nematophila Thomas & Poinar, which multiply rapidly, killing the insect within 2-3 days due to septicemia (Kaya and Gaugler, 1993).
Plant secondary compounds or Neotyphodium spp. produced alkaloids that can increase susceptibility of herbivorous insects to entomopathogenic nematodes
(Barbercheck, 1993; Grewal et al., 1995). However, endophytic fungal secondary metabolites are able to inactivate and kill the plant parasitic nematode Meloidogyne incognita (Hallmann and Sikora, 1996). Previous research demonstrates that populations of some phytopathogenic nematodes are reduced in the rhizosphere of endophyte- infected tall fescue (Eerens et al., 1997; Elmi et al., 2000). Porter (1994) describes the wide array of alkaloids associated with endophyte infected perennial ryegrass and tall fescue, some of which include peramine, ergovaline, lolitrem B, lolines, ergotamine, and other alkaloids. I propose that S. carpocapsae may interact with the fungal produced alkaloids through contact with a host that has consumed infected plants. Grewal et al.
(2003) found that the alkaloids ergocryptine, ergonovine, and ergotamine significantly
65
reduce survival and infectivity of the entomopathogenic nematode, Heterorhabditis
bacteriophora Poinar. Barbercheck and Wang (1996) found that the plant secondary compound, cucurbitacin D, could reduce the growth of the symbiotic bacterium X.
nematophila. Therefore, I hypothesized that the endophyte-associated alkaloids may
reduce the infectivity of S. carpocapsae to A. ipsilon by adversely affecting the growth
and activity of X. nematophila. First, S. carpocapsae attachment and penetration into A.
ipsilon larvae fed on either endophyte-infected or endophyte free perennial ryegrass was
evaluated. Next, I examined the effects of specific alkaloids found within the endophyte-
infected plants on nematode survival and infectivity. Finally, the effects of ergocristine
on the growth and pathogenicity of X. nematophila was determined.
4.3 MATERIALS AND METHODS
Nematodes, insects, and plant material.
Entomopathogenic nematodes were obtained from laboratory cultures maintained
in the Department of Entomology of the Ohio State University in Wooster, OH.
Nematodes were reared in the last instar Galleria mellonella L. following the methods
described by Kaya and Stock (1997) and were used within 2 wks of emergence from the
host.
The black cutworm, A. ipsilon, eggs were obtained from a laboratory colony
maintained at the USDA-ARS facility at Iowa State University. Neonates were fed either
endophyte-infected or endophyte free grass clippings for 16 d. Ten neonates were placed
in every petri dish with 2 filter papers until 7 d. Thereafter, the larvae were kept
individually to prevent cannibalism. Larvae were fed every other day and dishes were
66
kept in an incubator at 25ûC and a 14:10 L:D cycle. Once the larvae reached 4-5th instar, individual larvae were weighed and used in the experiments.
Flats of endophyte-infected and endophyte-free perennial ryegrass plants were maintained in a greenhouse for the duration of the experiments. ‘Repel II’ perennial ryegrass was fertilized once a week and grown with a 14:10 L:D growing period at 18-
25ûC. Endophyte was removed from plants through repeated applications of propiconazole 3 years prior to use in experiments. Endophyte infection was confirmed through microscopic examination of stained leaf sheaths (Saha, 1988) or immuno-blot assays (Gwinn et al., 1991). Plants were divided into two groups, endophyte-infected
(>95% plants infected) and endophyte free (<10% plants infected). Clippings from either endophyte-infected or endophyte-free plants were taken with scissors and fed immediately after removal from the plant to larvae.
Nematode attachment and penetration.
I wanted to determine if larvae that consumed endophytic perennial ryegrass were deterrent to nematodes and if the endophyte had an effect on nematode development in the host. The 2x5 factorial experiment consisted of the two grasses, endophyte infected
(E+) and endophyte free (E-), and five time intervals, 1.5, 3, 6, 12, 20 h after exposure to the nematodes. Five insects, fed either E+ or E- grass, were tested individually at each time interval. An individual larva was exposed to the LC50 concentration of S.
carpocapsae (Epsky and Capinera, 1994a), or about 85 infective juveniles. This dose of
nematodes was chosen because it provided a better chance of larvae encountering a
nematode even at the short exposure times. At each time interval, the larva was removed
67
from the petri dish and washed with 0.5 mL of water using a paintbrush; afterwards the
larva was placed into a petri dish containing filter paper, grass clippings, and no
nematodes. The paintbrush was rinsed with 0.5 mL of water and combined with the
water used to clean the larva. This wash-water was examined for the number of infective
juveniles removed from each larva. Larval mortality was recorded at 36, 48, and 72 h
after exposure to S. carpocapsae. After 72 h, the petri dishes were stored at 4ºC until dead larvae were dissected. The adult nematodes found within each larva were considered to be the number of nematodes that penetrated and developed to adult stage.
Effects of pure alkaloids on nematode viability and infectivity.
This experiment was designed to examine the effects of ergotamine, ergonovine, ergocristine, and ergocryptine alkaloids on S. carpocapsae. These alkaloids were chosen because they are commercially available (Sigma, St. Louis, MO). Nematode concentrations were determined before being added to the solutions of alkaloids. About
500 infective juveniles (IJs) contained in about 0.4 mL of water were added to each well of a 24-well plate prior to the addition of alkaloid solutions. Ergotamine tartrate
(hereafter referred to as ergotamine) was mixed in water until a uniform milky suspension was obtained. Ergonovine malate (ergonovine) dissolved in water and both ergocristine and ergocryptine were dissolved in dimethyl sulfoxide (DMSO). Alkaloid concentrations were prepared as 400 µg/mL and diluted to the concentrations examined, which were 200 and 100 µg/mL. Our treatments were alkaloid concentrations of 200 or
100 µg/mL, water, and if the alkaloids were dissolved in DMSO then DMSO alone was also included as a control. The final concentration of DMSO was 5% for the alkaloid
68
treatments and control. There were 4 replicates and each replicate had 4 wells of each
treatment, and the total volume within each well was 1.0 mL (n = 16). Each well was
counted for viability every 24 h for 72 h. After 72 h, infectivity of the nematodes to G. mellonella was determined using the 1:1 sand well bioassays (Grewal et al., 1999), and larval mortality was recorded at 24, 36, 48, and 72 h. Those larvae not moving in response to prodding with a paintbrush were considered dead.
Preparation of seed extract.
Seeds from endophyte infected perennial ryegrass var. ‘Goalkeeper’ were ground into a powder using an industrial blender. Ten grams of powdered seed were weighed and placed in a screw cap vial containing 50 mL of methanol. The vial was placed on a shaker table for 2 h before filtering sample into a 250 mL beaker. Filter paper and sample were rinsed with 10 mL of methanol. An additional 100 mL of methanol was added to the seeds and shaken for 2 more hours. The methanol sample was filtered into the same beaker as before and vacuum filtered through a glass fiber filter paper (VWR,
West Chester, PA). The methanol sample was placed in a round bottom flask and evaporated to about 4 ml using a rotary evaporator. The remaining methanol was pipetted into a screw cap vial and stored at 4ûC until used in the experiments.
Effects of seed extract on nematode viability and infectivity.
The objective of this experiment was to test the hypothesis that S. carpocapsae exposed to seed extract containing a mixture of endophyte associated alkaloids would show decreased viability and infectivity. The final concentrations of seed extract used
69
were 20%, 10%, 5%, and 2.5%, and there were four replicates (n = 4). I used methanol treatments at the same concentrations as controls. Each treatment consisted of a total volume of 1.0 ml placed in a well of a 24-well plate and each concentration was maintained in separate plates to avoid methanol volatiles affecting other treatments.
Nematodes in distilled water were used as controls to compare the methanol and seed extracts. About 400 S. carpocapsae infective juveniles were exposed to each treatment.
Nematode viability was recorded at 24-28 h, 47-52 h, and 74-77 h post exposure to the treatments. After 77 h, nematode infectivity was determined using the 1:1 sand well bioassay against the last instar G. mellonella. Larval mortality was recorded every 24 h for 72 h.
Effect of ergocristine on X. nematophila growth.
I wanted to determine if bacteria grown in an alkaloid-laden medium had reduced growth rates compared to those in broth with no alkaloids. The symbiotic bacteria, X. nematophila, of S. carpocapsae was grown on nutrient agar plates for 3 d at 28°C prior to use in the experiment. A single phase I colony was removed from the plate using a flame sterilized loop and inoculated into 100 mL of nutrient broth (Difco Laboratories, Detroit,
MI) and incubated at 28°C for 18 h prior to exposure to alkaloids. Nutrient broth contained broth, bromothymol blue (0.025 g/L) and triphenyltetrazolium chloride (0.04 g/L). Our treatments were in 250 ml Erlenmyer flasks and consisted of either the nutrient broth with a final concentration of 200 µg/ml of ergocristine and 0.5% DMSO, or nutrient broth with 0.5% DMSO only. All flasks were placed on a shaker at 150 rpm and maintained at 28°C. I removed 0.5 ml of the 18 h bacteria suspension and inoculated
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each of our treatments. After 18 h of exposure to treatments, samples were serially
diluted and 100 µL of the 10-4, 10-5, 10-6, and the 10-7 dilutions were plated onto nutrient
broth triphenyltetrazolium agar (NBTA). The NBTA plates were petri dishes with
nutrient agar, bromothymol blue (0.025 g/L) and triphenyltetrazolium chloride (0.04 g/L;
Kaya and Stock, 1997). The dishes were wrapped with parafilm and placed in an
incubator at 28°C for 3 d before the number of bacterial colonies (cfus) were counted.
There were three replicates in the first experiment (n = 3). Due to the ease in counting
the distinct colonies, the 10-7 plates were used to count the number of colonies and are presented as number of colonies for experiment 1. This experiment was conducted two more times as described above with three (experiment 2) or four (experiment 3) replicates and only the 10-6 through 10-8 dilutions plated on NBTA plates.
Effect of ergocristine on X. nematophila pathogenicity.
I conducted two experiments to test if X. nematophila decreased pathogenicity to G.
mellonella when cultured in nutrient broth containing the alkaloid, ergocristine. The bacteria suspensions used in the first experiment came from experiment 2 described above and for the second from experiment 3 described above. In the first experiment, my treatments were as follows: 200 µg/mL ergocristine and 0.5% DMSO plus bacteria, 0.5%
DMSO plus bacteria, or 0.5% DMSO and no bacteria as a control. The no bacteria treatment was used to estimate mortality due to injections with our micro-injectors. A
0.5 ml sample was removed from each treatment and serially diluted to 10-8. The 10-6 through 10-8 dilutions had 100 µL removed and plated to provide an estimate of how
many cfus were injected. Micropipette injectors (micro-injector) were made using a
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micropipette puller (Industrial Science Associates Inc., Ridgewood, NY). For each
treatment, 1.0 µL of bacterial suspension was removed from the 10-6 dilution with a
micropipette and placed onto a sterile parafilm strip. Immediately afterwards, a droplet
of bacterial suspension was removed with a sterile micro-injector from the sterile
parafilm by capillary action. The bacteria suspension was injected into a proleg or
intersegmental membrane of the G. mellonella host, and larval mortality was observed at
every 6 h for 36 h. Micro-injectors were sterilized by immersing them for 24 h in 95%
ethanol and drying under UV light for at least 1 h prior to use. Thereafter injectors were
stored in containers that had been sterilized similarly. Parafilm was sterilized with 95%
ethanol. The experiment was replicated three times and each treatment had 6 insects per
replicate. The sand (1.0 g) in each well of the 24-well plate was moistened with 90 µL of
water prior to the addition of an injected insect to prevent desiccation. Each 24-well
plate was wrapped in parafilm to hold lid in place, prevent larval escape, and to minimize
desiccation.
In the second experiment the treatments were, 200 µg/mL of ergocristine, 0.5%
DMSO plus bacteria, 200 µg/mL of ergocristine and no bacteria, 0.5% DMSO and no
bacteria, and all treatments were in nutrient broth. All treatments were replicated three
times. Each treatment was serially diluted to 10-8, and 100 µL of 10-6 through 10-8 were
plated on NBTA agar plates as above. Injections were performed as above, but 12 G.
mellonella were used per treatment and observations on insect mortality began at 12 h post-injection.
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Statistical analysis.
All mortality data were arcsine square root transformed to normalize data.
Transformed data were analyzed as a Repeated Measures Analysis of Variance
(ANOVA) using Statistica software (Statsoft 1999). The main effects of the experiments
were tested at α = 0.05 and interactions were examined with α = 0.10. Significant
interactions were further evaluated with contrasts with α = 0.05. A. ipsilon mortality was
analyzed and the Geiser-Greenhouse used to determine if time interactions were
significant. In experiments with pure alkaloids, the alkaloid concentrations were
contrasted to determine if significantly different from each other, and if they were not,
they were averaged when contrasted versus their respective control treatments. In the
experiment that evaluated the effects of ergocristine on bacterial growth, the 10-7
dilutions were analyzed and Fisher’s LSD was used for means separation. Data are
presented as non-transformed means ± standard error.
4.4 RESULTS
Nematode attachment and penetration.
There was no significant effect of endophyte-infected grass on rates of nematode
attachment or on their ability to infect and develop into adults (P >0.05; Fig. 4.1, 4.2).
This was true for all time periods examined. Larvae examined at 1.5 h had about 9-12
infective juveniles attached and those larvae exposed for 20 h had significantly fewer (F
= 4.4, df = 4,130, P = 0.002), about 4-6 infective juveniles, but there were no differences between larvae fed on endophytic or endophyte free grass treatments (P >0.05; Fig. 4.1).
73
Additionally, the larvae examined at 12 and 20 h had more nematodes penetrate and
develop into adults (F = 5.9, df = 4,130, P < 0.001) compared to those at earlier time
intervals, however there were no differences between larvae fed on endophytic or
endophyte free grass (P >0.05; Fig. 4.2). Significantly fewer larvae fed on endophytic grass died when they were removed from nematode exposure at 1.5 h (F= 2.0, df= 6,31;
P = 0.08; Fig. 4.3). Those larvae fed on endophytic grass and exposed to nematodes for
1.5 h suffered 44.5 ± 17.8% mortality by 72 h whereas those larvae fed on endophyte free
grass suffered 85.7 ± 14.3% mortality by 72 h.
Effects of pure alkaloids on nematode viability and infectivity.
Effects of alkaloids on S. carpocapsae depended on the alkaloid (Fig. 4.4-4.7).
After 72 h of continuous exposure to pure alkaloids, the viability of S. carpocapsae
exceeded 98% for all alkaloids at both concentrations. There was also insufficient
evidence to suggest that ergocryptine or ergotamine adversely affected infectivity
(P>0.05; Fig. 4.5, 4.7 respectively). Ergonovine did significantly increase infectivity of
S. carpocapsae over time (F=2.1, df = 6,68, P= 0.06). Both concentrations of ergonovine had greater larval mortality (i.e., increased nematode infectivity) than the water control from 36 h until 72 h (F = 9.0, 5.9, 3.9; df =1,36, P = 0.004, 0.02, 0.06 for 36, 48, 72 h, respectively; Fig. 4.6). Ergocristine treatments significantly reduced infectivity over time compared to DMSO controls (F = 2.0; df = 9,112, P = 0.04). Reductions in infectivity became apparent at 48 and 72 h (F = 4.5, 5.1; df =1, 48, P = 0.04 and 0.03 for 48 and 72 h, respectively; Fig. 4.4).
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Effects of seed extract on nematode viability and infectivity.
Alkaloids present in the seed did not adversely affect S. carpocapsae. Both 20% seed extract and 20% methanol alone treatments caused about 98% mortality of S. carpocapsae (F = 2.2; df =16,62, P<0.05). Nematode survival exceeded 95% for all other seed extract and methanol treatments (data not shown). The results of nematode infectivity from the other seed extract concentrations were not significantly different than corresponding methanol concentrations (P>0.05). The rates of infectivity were around
55% to 78% depending upon treatment, but the 5% methanol treatment had significantly greater larval mortality compared to the water control (P= 0.01; Fig. 4.8). The 5% methanol alone treatment also had greater mortality than the 5% seed extract treatment but it was not significant (P = 0.07; Fig. 4.8).
Effect of ergocristine on X. nematophila growth.
The 0.5% DMSO treatment (i.e., control) had significantly more colony-forming units (cfus) than the 200 µg/mL ergocristine treatment for all three experiments (F= 15.3,
14.8, 17.4, df = 1,3, 1,3 and 1,6; P ≤ 0.03 for experiments 1-3 respectively; Fig. 4.9).
Experiment 3 had significantly more cfus than either experiment 1 or 2 (F = 37.1, df
=2,12, P <0.001). For example, there were about 30.67 ± 2.73 and 41 ± 3.61 cfus/100 µL of 10-7 dilution from the 200 µg/mL ergocristine treatment in experiment 1 and 2 respectively; whereas, experiment 3 had 64.25 ± 6.92 cfus/100 µL (Fig. 4.9).
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Effect of ergocristine on X. nematophila pathogenicity.
Ergocristine caused a significant decrease in the ability of X. nematophila to kill G. mellonella over time (F = 7.8, df = 8,6, P = 0.02) in the first experiment (Fig. 4.10). I found that 200 µg/mL of ergocristine caused about 60% reduction in mortality caused by
X. nematophila. In our second experiment, there was no significant difference in total cumulative mortality between the 0.5% DMSO treatment (91.6 ± 4.8%) and the 200
µg/mL ergocristine treatment (80.6 ± 2.8%)(Fig. 4.11). I noticed that 200 µg/mL of ergocristine with no bacteria resulted in 15% mortality.
4.5 DISCUSSION
The infection status of perennial ryegrass fed on by A. ipsilon did not appear to affect the ability of S. carpocapsae to attach or penetrate and subsequently develop into adults. Montllor and Bernays (1991) and Schaffner et al. (1994) found that ants and spiders are capable of detecting alkaloids within the integument of potential prey. Our data suggest that S. carpocapsae does not discriminate between A. ipsilon larvae fed on endophyte-infected or endophyte free perennial ryegrass. Therefore, the possible mechanisms of resistance exhibited by A. ipsilon when it feeds on endophyte-infected grass probably occur once the nematode enters the insect. Additionally, as expected those larvae fed on endophytic grass and removed from nematode exposure at 1.5 h had significantly lower mortality at 72 h than larvae fed on endophyte free grass and removed after 1.5 h. Williamson and Potter (1997b) demonstrated that A. ipsilon is capable of moving considerable distances at night. The deterrent nature of endophytic perennial
76
ryegrass leads to increased herbivore movement including A. ipsilon (Richmond and
Shetlar, 1999; Richmond and Shetlar, 2001). Although this should increase possible exposure to nictating nematodes such as S. carpocapsae, A. ipsilon appears to have a defense against the nematode if it has consumed sufficient quantities of endophytic perennial ryegrass.
I observed that S. carpocapsae exposed to ergonovine had greater rates of infectivity compared to nematodes maintained in water controls. This enhanced infectivity could possibly enhance biological control of some insect pests. Grewal et al.
(1995) found that H. bacteriophora infectivity was increased when Japanese beetle grubs were fed on ergotamine tartrate. Those Diabrotica undecimpunctata howardii Barber that consumed plants containing cucurbitacins were more susceptible to entomopathogenic nematodes (Eben and Barbercheck, 1997). Ergocryptine, ergotamine, and the seed extract did not have an effect on viability or infectivity of S. carpocapsae in our experiments. The 20% seed extract prepared in 20% methanol killed virtually all of
S. carpocapsae exposed to this treatment; however, the 20% methanol treatment also killed virtually all the nematodes. Therefore, the death of nematodes in 20% seed extract treatment was due to the high concentration of methanol and not due to the alkaloids found in the seed. Grewal et al. (2003) found that 3 d exposure to peramine and ergonovine did not have adverse effects on infectivity on H. bacteriophora. Furthermore, they found that perennial ryegrass seed extract resulted in no significant reduction in H. bacteriophora infectivity until 14 d of exposure. Additionally, nematodes exposed to tall fescue seed extract likewise did not reduce nematode infectivity. Contrary to our results however, Grewal et al. (2002) found significant reduction of viability of H. bacteriphora
77
when exposed to pure alkaloids or seed extracts for 30 d. Therefore, the nematode H.
bacteriophora is either more sensitive to exposure to the alkaloids, or our experiments
may have had similar results if they had continued for longer periods of time.
The alkaloid ergocristine significantly reduced the ability of S. carpocapsae to
infect the susceptible host G. mellonella when exposed in the one-on-one sand-well
bioassays. Some Drosophila acquires protection from parasitic nematodes through
development on mushrooms containing α-amanitin (Jaenike, 1985). Grewal et al. (2003)
found that 200 µg/mL ergocryptine and ergotamine significantly reduce H. bacteriophora infectivity after 6 d of continuous exposure. Additionally, endophytic grasses have negative effects on plant parasitic nematodes (West et al., 1988; Elmi et al., 2000).
Therefore, I concluded that endophyte-infected perennial ryegrass can affect the ability of
S. carpocapsae to infect potential hosts.
I found that ergocristine reduces X. nematophila growth in nutrient broth.
Ergocristine treatments had lower colony counts than the control containing the same solvent, DMSO. Additionally, I observed reduced pathogenicity of X. nematophila grown in ergocristine when injected into G. mellonella. Therefore, I conclude that this is at least one mechanism by which A. ipsilon acquires resistance against S. carpocapsae.
Barbercheck and Wang (1996) found that the plant secondary compound cucurbitacin D is capable of reducing X. nematophila growth. Epsky and Capinera (1994b) have found that A. ipsilon fed on collards containing glucosinolate allelochemicals produce fewer S. carpocapsae due to a reduction of lipid content available for the nematodes.
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Furthermore, both Beauveria bassiana and Bacillus thuringiensis var. kurstaki Berliner, have reduced colony growth when they encounter plant secondary compounds (Costa and
Gaugler, 1989; Krischik et al., 1988).
The alkaloid ergocristine was shown to adversely affect the infectivity of S. carpocapsae, and reduce the growth and pathogenicity of X. nematophila, the symbiotic bacteria carried by the nematodes. The concentrations of alkaloids can vary between plants and within plant parts. Siegel et al. (1987) found that lolines in tall fescue can be as high as 8,000 µg/g dry weight. In extreme instances ergovaline levels have been found to be as high as 175 µg/g dry weight (Lane et al., 1997). The interactions between fungus and host plant genotype can be variable, and therefore care is needed to extrapolate to different cultivars or species (Breen, 1993c). Roylance et al. (1994) found that alkaloid concentrations within a plant may vary due to plant and fungus genotype interactions.
Therefore, it is possible that the concentrations I examined of the various alkaloids occur in the plants. The predominant alkaloids found within endophyte-infected perennial ryegrass: peramine, ergovaline, and lolitrem B are commercially unavailable for testing.
Those alkaloids may provide additional protection to A. ipsilon against S. carpocapsae.
Finally, insects have been shown to incorporate plant secondary compounds into their defenses, reproduction, or to be harmful to parasitoid development (Campbell and
Duffey, 1979; Schulz et al., 1988; Nishida and Fukami. 1990) and insect herbivores that consume noxious plant material may have resistance to some predators or parasitoids even if it is just the presence of the plant material itself within the insect (Price et al.,
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1980). Therefore, our data suggest that those A. ipsilon fed on endophyte-infected
perennial ryegrass have an increased resistance to S. carpocapsae because of the
presence of the alkaloids, notably ergocristine, produced by the fungus.
4.6 ACKNOWLEDGMENTS
I thank Dr. Sukhbir K. Grewal, Dr. Douglas S. Richmond, Dr. Elizabeth
DeNardo, Dr. Seppo Salminen, and Patti Kunkel for technical expertise. Additionally, I
thank Dr. Saskia Hogenhaut for use of some of her equipment and supplies, and Dr. Jean
Dyer at the USDA-ARS, Iowa for supplying A. ipsilon eggs. The research was supported by a USDA-NRI grant no. 00-35316-9249 to P.S. Grewal and an OARDC Graduate
Competitive grant to B.A. Kunkel.
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12
9
6 No. nematodes attached/larva
3
0 1.5 3 6 12 20
Hours exposed
Figure 4.1. Mean number (+ SEM) of Steinernema carpocapsae infective juveniles
found attached to Agrotis ipsilon 4-5th instar hosts. Larvae were exposed
to about 80 infective juveniles for 1.5, 3, 6, 12, and 20 h before being
washed and removed from treated arenas. Larvae fed on endophytic grass
(full bars) and larvae fed on endophyte free grass (empty bars) are
presented.
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15
12
9
6 No. of nematodes penetrated/larva 3
0 1.5 3 6 12 20
Hours exposed
Figure 4.2 Mean number (+ SEM) of Steinernema carpocapsae adults found within
Agrotis ipsilon larvae. Larvae were dissected 72 h after the start of the
experiment to count the number of nematodes penetrated and developed to
adults. Larvae fed on endophytic grass (full bars) and larvae fed on
endophyte free grass (empty bars) are presented
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b 100
80 a 60
40
% Cutworm mortality 20
0 1.5 3 6 12 20 Hours exposed
Figure 4.3 Average mortality (+ SEM) for Agrotis ipsilon larvae removed after 1.5, 3,
6, 12, 20 h of exposure to nematodes. Larvae were removed from the
challenge arena after exposure period and maintained in petri dishes
containing no nematodes. Larva mortality was observed at 36, 48 and 72
h after initial exposure to nematodes. Larvae fed on endophytic grass (full
bars) and larvae fed on endophyte free grass (empty bars) are presented.
Bars with different lower case letters are significantly different at α= 0.05.
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100
75
50 % Mortality 25
0 24 36 48 72 Hours exposed
Figure 4.4 Mean % mortality (± SEM) of Galleria mellonella when exposed to
Steinernema carpocapsae treated with ergocristine solutions for 3 d.
Alkaloid concentrations tested were 200 and 100 µg/mL. Dimethyl
sulfoxide (DMSO) treatments and water served as controls. All treatments
had a G. mellonella larva exposed to one infective juvenile that had been
stored in one of the following treatments for 3 d: 200 µg/mL concentration
of alkaloid (●), 100 µg/mL concentration of alkaloid (○), water (□), 5%
dimethyl sulfoxide (DMSO; only with ergocristine and ergocryptine; ▲),
or a water control (no nematodes; ■).
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100
75
50 % Mortality 25
0 24 36 48 72 Hours exposed
Figure 4.5 Mean % mortality (± SEM) of Galleria mellonella when exposed to
Steinernema carpocapsae treated with ergocryptine solutions for 3 d.
Alkaloid concentrations tested were 200 and 100 µg/mL. Dimethyl
sulfoxide (DMSO) treatments and water served as controls. All treatments
had a G. mellonella larva exposed to one infective juvenile that had been
stored in one of the following treatments for 3 d: 200 µg/mL concentration
of alkaloid (●), 100 µg/mL concentration of alkaloid (○), water (□), 5%
dimethyl sulfoxide (DMSO; only with ergocristine and ergocryptine; ▲),
or a water control (no nematodes; ■).
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100
75
50 % Mortality 25
0 24 36 48 72 Hours exposed
Figure 4.6 Mean % mortality (± SEM) of Galleria mellonella when exposed to
Steinernema carpocapsae treated with ergonovine solutions for 3 d.
Alkaloid concentrations tested were 200 and 100 µg/mL. Water served as
controls. All treatments had a G. mellonella larva exposed to one
infective juvenile that had been stored in one of the following treatments
for 3 d: 200 µg/mL concentration of alkaloid (●), 100 µg/mL
concentration of alkaloid (○), water (□), and a water control (no
nematodes; ■).
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100
75
50 % Mortality 25
0 24 36 48 72 Hours exposed
Figure 4.7 Mean % mortality (± SEM) of Galleria mellonella when exposed to
Steinernema carpocapsae treated with ergotamine solutions for 3 d.
Alkaloid concentrations tested were 200 and 100 µg/mL. Water served as
controls. All treatments had a G. mellonella larva exposed to one
infective juvenile that had been stored in one of the following treatments
for 3 d: 200 µg/mL concentration of alkaloid (●), 100 µg/mL
concentration of alkaloid (○), water (□), 5% dimethyl sulfoxide (DMSO;
only with ergocristine and ergocryptine; ▲), or a water control (no
nematodes; ■).
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100
80
60 % Mortality 40
20
0 24 36 48 72 Hours exposed
Figure 4.8 Mean % mortality (± SEM) of Galleria mellonella when exposed to
Steinernema carpocapsae treated with different concentrations of seed
extract/methanol, methanol, or water for 3 d. Our treatments were as
follows: 10% seed extract (●), 10% methanol (○), 5% seed extract (!),
5% methanol (∇), 2.5% seed extract (■), 2.5% methanol (□), water
controls (!).
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a 100 b 80 a a 60 b b 40
No. cfus/petri dish 20
0 123 Experiment
Figure 4.9 Mean number of colony forming units (cfus) (±SEM) exposed to 0.5%
DMSO (full bars) or 200 µg/mL of ergocristine (empty bars) in nutrient
broth for 18 h. Results for three trials are presented.
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100 80 60 40 % Mortality 20 0 6 121824303672 Hours exposed
Figure 4.10 Mean % mortality (± SEM) of Galleria mellonella over time when
injected with 1 µl suspension of X. nematophila, the bacterial symbiont of
Steinernema carpocapsae. Number of X. nematophila colony forming
units (cfus) injected in first experiment (A) was about 6 and 4 for 0.5%
DMSO control (□) and 200 µg/mL ergocristine (!), or 0 for nutrient
broth and no bacteria (").
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100 80 60 40 % Mortality 20 0 12 18 24 30 42 Hours exposed
Figure 4.11 Mean % mortality (± SEM) of Galleria mellonella over time when
injected with 1 µl suspension of X. nematophila, the bacterial symbiont of
Steinernema carpocapsae. The number of cfus injected was about 10 and
6 for 0.5% DMSO control (□), 200 µg/mL ergocristine plus bacteria (!)
respectively, and 0 for 200 µg/mL ergocristine with no bacteria (") or
nutrient broth with no bacteria ("; controls).
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CHAPTER 5
Fate of Neotyphodium lolii - produced alkaloids in generalist herbivores, Agrotis ipsilon
and Spodoptera frugiperda and possible ecological consequences
5.1 ABSTRACT
Several insect species sequester plant alkaloids for either incorporation into sex pheromones, or defenses against natural enemies, I hypothesized that endophyte- produced alkaloids are sequestered by the black cutworm, A. ipsilon and fall armyworm,
S. frugiperda. I found that larvae with grass filled guts, guts cleared of grass of both species, and pupae of fall armyworm contained detectable amounts of ergocristine, ergocryptine, ergonovine, and ergovaline. Therefore, I conclude that endophyte- produced alkaloids do persist in these generalist herbivores and may be sequestered. The levels of alkaloids detected within the insects may be sufficient to provide partial resistance for both insect species against entomopathogenic nematodes.
5.2 INTRODUCTION
The area of plant-insect interactions has been investigated extensively over the past century (Vershaffelt, 1911; Dethier, 1941; Frankel, 1959; Feeny, 1976; Bernays and
Chapman, 1994). Ehrlich and Raven (1964) proposed that plants have evolved to avoid
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insect herbivory, whereas, the insects have evolved to deal with plant secondary
compounds. Insects have many physiological and behavioral mechanisms to avoid plant
secondary compounds. Dussourd and Eisner (1987) describe insect behavior, vein
cutting, to avoid the latex defenses of their host plant. Brattsten (1992) describes the
many different types of metabolism possessed by various herbivores that circumvent
plant allelochemicals, and among them are polysubstrate monoxygenases (cytochrome
P450 enzyme complex), oxidative or reductive metabolism, and transfer enzymes.
Duffey (1980) describes the physiological process where insects consume noxious plant
compounds and sequester them for their own defense. Some lepidopteran insects have
demonstrated the ability to consume pyrrolizidine alkaloids and incorporate them into
part of the sex pheromones used in courtship (Trigo et al., 1994). Price et al. (1980) proposed that future investigations of plant-insect interactions incorporate the influence of the third trophic level.
The impact of partial plant resistance on biological control needs to be evaluated for various cropping systems so that the interactions among plants, pests, and natural enemies can be maximized for optimal control (Verkerk et al., 1998). Price et al. (1980) suggested that enhanced control of herbivores could result from prolonged development due to nutritional deficiencies and may lead to increased susceptibility to attack from either other insects or entomopathogens. However, several insect orders contain representatives that sequester alkaloids, increasing the herbivores' defenses against natural enemies (Blum 1981). Brown (1984) found that some butterflies were protected from spiders when they consumed pyrrolizidine alkaloids as larvae. Predators that use visual cues to locate prey learn to avoid aposematically colored insects (Bowdish and
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Bultman, 1993). The female moth Utetheisa ornatrix receives a nuptial gift containing
pyrrolizidines from males that protect her from spiders (Rossini et al, 2001) and her eggs
from generalist predators (Eisner et al., 2000). Boppré (1984) redefines
pharmocophagous insects as herbivores that search for and consume plants containing
secondary compounds for benefit other than nutrition. Some insect herbivores may also
passively sequester secondary plant compounds (Rowell-Rahier and Pasteels, 1992).
Perennial ryegrass forms a symbiotic relationship with Neotyphodium lolii, an endophytic fungus that provides increased resistance to herbivory through the production of a variety of alkaloids (Porter, 1994). The few studies that examined the effects of this relationship on the third trophic level demonstrate that parasitoids are adversely affected when they parasitize host insects that fed on endophytic perennial ryegrass (Barker and
Addison, 1996; Bultman et al., 1997; Omacini, 2001). Grewal et al. (2003) found that the
entomopathogenic nematode, Heterorhabdus bacteriophora, suffers decreased infectivity and longevity when exposed to N. lolii-produced alkaloids. Agrotis ipsilon Hufnagel and
Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) larvae show decreased susceptibility to the entomopathogenic nematode, Steinernema carpocapsae, when they have consumed endophytic perennial ryegrass (Kunkel and Grewal, 2003; Richmond et al., 2003) as compared to the non-endophytic grass. Kunkel et al. (2003) found that the infectivity of S. carpocapsae is reduced when exposed to the N. lolii-produced alkaloid ergocristine. They also reported that ergocristine reduces growth and pathogenicity of
Xenorhabdus nematophila, the symbiotic bacteria of S. carpocapsae (Kunkel et al.,
2003). A. ipsilon and S. frugiperda are two cryptic generalist herbivores capable of completing development on endophyte infected grasses (Breen, 1993a; Williamson and
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Potter, 1997), whereas most herbivores that consume these grasses suffer the toxic effects
of various alkaloids (Siegel et al., 1987). However, the fate of these fungal produced
alkaloids has not been examined in insect herbivores. Since A. ipsilon and S. frugiperda fed on endophyte-infected ryegrass were less susceptible to the entomopathogenic nematodes (Kunkel and Grewal, 2003; Kunkel et al., 2003; Richmond et al., 2003), I hypothesized that these generalist herbivores are sequestering endophyte-produced alkaloids.
5.3 MATERIALS AND METHODS
Grass.
The grass used in these experiments was perennial ryegrass, Lolium perenne L., variety ‘Repell II’ infected with the fungal endophyte Neotypodium lolii. Half of our grass plants had endophyte removed with repeated applications of the systemic fungicide propiconazole three years prior to these experiments. This provided flats of grass that had either high proportions of plants infected (>95 infected; high endophyte, HE) or plants that contained no endophyte (0% infected; NE). The level of endophyte infection for each treatment was determined by immuno-assays (Gwinn et al., 1991). Grasses were maintained at 18-24ûC, 14:10 L:D cycle, and fertilized once a week throughout the growing season. The level of ergot alkaloids in the endophyte-infected and endophyte free perennial ryegrass plants was determined prior to feeding them to the insects. The amount of perloline methyl ether, a methylated plant alkaloid, present in the grass was also determined when S. frugiperda were being fed on HE and NE grass clippings, thus there is no data for A. ipsilon larvae.
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Insects.
Black cutworm, Agrotis ipsilon, eggs were obtained from a laboratory colony at
USDA-ARS in Ames, Iowa. Fall armyworm, Spodoptera frugiperda (corn strain), eggs
were obtained from AgriPest (Zebulon, NC) laboratory colonies. Neonates were
separated into groups fed on either HE or NE grass clippings. Clippings were from the
upper one-third of foliage of the plants. Ten neonates were placed in a 9.0 cm Petri dish
with two pieces of filter paper for 1 wk; thereafter insects were maintained individually
in 5.5 cm petri dishes with two pieces of moistened filter paper. Larvae were fed grass
clippings every other day, or as they needed food until they reached at least 5th instar
(about 21 d). After each feeding, the Petri dishes were wrapped with parafilm to
minimize desiccation.
Ergot alkaloid analysis.
Samples were processed and analyzed following the procedures of Salminen and
Grewal (2001). Insects and frass were frozen, freeze-dried and ground into fine powder
using a mortar and pestle. Two hundred milligrams of the powdered sample was
weighed and processed for analysis. All samples had 3 µg of ergotamine added as an
internal standard. Samples were shaken in scintillation vials containing the internal
standard and a 9:1 chloroform: 0.01 N sodium hydroxide solution for 0.5 h. Afterwards,
samples were filtered and then vacuum filtered through a SPE column. A 75:25
acetone:chloroform solution was filtered through the column, and samples were dried
with 1.0 ml of diethyl ether. Finally, the samples were eluted from the column with 2.0
mL methanol in sample vials, and evaporated off using nitrogen gas.
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Sequestration of endophyte alkaloids by A. ipsilon.
To determine if ergot alkaloids are sequestered by the insects that feed on endophytic perennial ryegrass I followed the method described by Montllor et al. (1990) in which they starved the insects for 24 h to empty their gut prior to analysis. The insects were frozen for 24 h at -20°C before being freeze-dried for 1 wk at -20°C. Fifteen insects were pooled to make up a sample and processed as described for ergot alkaloid analysis above.
Concurrently, I examined the frass of A. ipsilon fed HE or NE grass clippings for the presence of the ergot alkaloids. The frass was collected from the same insects, placed in scintillation vials, and freeze-dried at -20°C for 1 wk. The sample was pooled from all
15 insects as above and processed for ergot alkaloid detection as described above.
Since I found that the freeze-dried insects appeared to have some grass clippings remaining within the gut even after 24 h of starvation, I transferred the insects to an artificial diet for 24 h as an attempt to flush all sources of endophyte alkaloids out of their gut. Insects fed on artificial diet until there were no grass clippings visible in the frass
(about 24 h). Larvae were then placed in a 9.0 cm petri dish and placed in a freezer (-
20°C) for 24 h. The frozen insects were wrapped in cheese-cloth and freeze-dried at -
20°C for 1 wk. Freeze-dried samples were then placed in a desiccator that was stored at -
20°C. A total of 15 insects made up the pooled sample of freeze-dried insects.
Sequestration of endophyte alkaloids by S. frugiperda.
I explored if ergot alkaloids could also be detected in S. frugiperda larvae fed on
HE and NE grass clippings. Our treatments were insects fed on either HE or NE grass
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clippings until the insects reached about 4-5th instar. In order to have similar data
compared to A. ipsilon, I starved S. frugiperda for 24 h prior to processing for ergot and
lolitrem analysis. The insects were divided into three replicates of ten insects each and
were frozen for 24 h at -20°C. The frozen insects were wrapped in cheese-cloth and
freeze-dried for 1 wk at -20°C.
I repeated our procedure, as with A. ipsilon above, to determine if ergot alkaloids
could be found in the frass of S. frugiperda. Frass samples were collected from the
previously mentioned S. frugiperda larvae after the insect reached 4th instar stage. There
were three replicates of frass samples and ten insects per replicate. Frass was collected in
scintillation vials over 3 d and between collections, samples were stored in a freezer at -
20°C. After 3 d, the samples were freeze-dried for 1 wk at -20°C. The samples were
processed for ergot alkaloid analysis as described above.
Another group of S. frugiperda larvae was fed either HE or NE grass until they
reached 5-6th instar. The larvae were then switched to artificial diet to ensure that all sources of alkaloids were removed from the gut. These larvae were fed artificial diet until they defecated diet instead of grass clippings, and were then processed for ergot alkaloid analysis as described above. Each of the three replicates had 7 insects allocated to each treatment for this experiment.
A third group of S. frugiperda larvae were fed either HE or NE grass clippings
until they reached 5-6th instar to determine the presence of ergot alkaloids in the hemolymph. Afterwards, larvae in each treatment were divided into three replicates that consisted of 15 insects each. Insects were placed on an ice bath for about 20 minutes before hemolymph samples were taken. Micropipette injectors (micro-injector) were
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made using a micropipette puller (Industrial Science Associates Inc., Ridgewood, NY).
A micro-injector was used to puncture the proleg of a chilled insect that had been folded and held so that hemolymph would collect into a droplet. A 10 µl micropipette was used to collect and estimate the amount of hemolymph placed into an Eppendorf tube. The 1.5 ml Eppendorf tube had 200 µl of an ergotamine internal standard and 1.0 ml of methanol.
The Eppendorf tube was kept on ice while the hemolymph was being collected from each insect. About 300 µl of hemolymph was collected from the insects fed on NE grass; whereas, about 400 µl of hemolymph was collected from those fed on HE grass. The
Eppendorf tube was vortexed for 1 minute before being centrifuged for 17 minutes at
14,000 rpm. The supernatant was removed from the Eppendorf tube and filtered through an aminopropyl solid phase extraction (SPE) column (Burdick & Jackson, Muskegon,
MI). Filtered supernatant was evaporated under nitrogen gas, and then the sample was stored at -20ûC until analyzed with an HPLC.
Finally, S. frugiperda larvae were reared to pupation on either HE or NE grass clippings. Insect pupae were permitted to completely sclerotize and after about 24 h they were frozen at -20°C. The five pupae from each diet were pooled into one sample and freeze-dried at -20°C for 10 d. Afterwards the samples were ground and processed for ergot alkaloid analysis as described above.
Data Analysis.
Data were analyzed to determine if there were differences in the location of detected alkaloids. The data analyzed were from S. frugiperda fed HE or NE grass clippings because I only had one pooled sample value for A. ipsilon. Data were analyzed
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as main effects Analysis of Variance (ANOVA) using Statistica 6.0 (Statsoft 1999). All
alkaloid concentrations (µg/g dry weight) are relative to our ergotamine standard used
during HPLC analysis.
5.4 RESULTS
Sequestration of endophyte alkaloids by A. ipsilon.
Ergocristine, ergovaline, and ergocryptine were detectable in A. ipsilon larvae fed on HE grass (Fig. 5.1). There were no detectable alkaloids found in the frass of A.
ipsilon. Ergocristine was detected in the highest concentration in A. ipsilon larvae starved for 24 h compared to the other ergot alkaloids detected. The larvae fed on HE grass and subsequently had the gut cleared with artificial diet still had considerable amounts of ergot alkaloids (Fig. 5.1). Ergovaline (89.5 µg/ g dry weight) was the predominant ergot alkaloid found in larvae fed on HE grass after their gut had been cleared with diet. Both samples of insects, ‘starved’ and ‘gut-cleared’, had greater concentrations of ergocristine than larval frass. The A. ipsilon larvae fed on NE grass had no ergot alkaloids and perloline methyl ether concentration was not documented.
Sequestration of endophyte alkaloids by S. frugiperda.
The S. frugiperda larvae fed on HE grass had significantly greater concentrations of ergocristine, ergocryptine, ergonovine, and ergovaline than the respective frass samples for the insects (F> 1.8, df=3,7, P<0.05 for all alkaloids; Fig. 5.3). I found that
ergonovine was in highest concentration in larvae fed on HE grass, however, it was the
predominant alkaloid found in S. frugiperda frass as well (Fig. 5.3). The starved insects
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had significantly greater concentrations of ergocristine than that found in the frass (F=
79.2, df=2,6, P<0.05). Finally, the pupae that had consumed HE grass clippings also had greater concentrations of ergocristine and ergocryptine (27.8 and 46.7 :g/g dry weight, respectively) than the frass samples (2.2 and 23 :g/g dry weight, respectively; F>30.0, df= 3,7, P<0.05 for both alkaloids). Perloline methyl ether was the most abundant alkaloid detected in the hemolymph of larvae fed on endophytic or endophyte free grass clippings (Table 5.1). All ergot alkaloids that had been detected in larvae were detected in very small quantities in the hemolymph (Table 5.1).
5.5 DISCUSSION
I found that A. ipsilon and S. frugiperda larvae have detectable amounts of ergot alkaloids when they consume grass infected with the endophyte N. lolii. The alkaloids detected were ergocristine, ergocryptine, ergonovine, and ergovaline that are normally found in perennial ryegrass infected with N. lolii (Salminen and Grewal, 2001). The alkaloids, ergocristine, ergovaline, and ergocryptine, were detected even after the insect gut had been cleared with artificial diet that contained no alkaloids. The insects fed on endophyte-free grass had no detectable amounts of ergot alkaloids. Although there were detectable levels of alkaloids in frass it appears as if some of these alkaloids are maintained or persist in the insect (Fig. 5.2 – 5.3). Montllor et al. (1990) found that the moth, Uresiphita reversalis, excretes most of the alkaloids consumed, but retains some in the cuticle, which acts as a deterrent to ants and wasps. Boros et al. (1991) found that the noctuid, Lepipolys sp., sequesters iridoid glycosides and stores them through the larva
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stage until the pupa stage. The nymphalid butterfly, Junonia coenia, has a partially cryptic larval stage that sequesters iridoid glycosides through the larva stage, but the adults do not have these alkaloids (Pereyra and Bowers, 1988).
There are representatives of insects from many of the insect orders that sequester plant secondary compounds from their host plant (Blum, 1981). Klitzke and Trigo
(2000) report a hemipteran and a coleopteran that sequester pyrrolizidine alkaloids from their host plant, Senecio brasiliensis. There are many documented cases where lepidopterans sequester plant compounds. Typically, insects that sequester alkaloids are aposematic in coloration, which suggests that these alkaloids are used by the insects for their own defense (Stamp and Wilkens, 1992). The brightly colored cinnabar moth,
Tyria jacobaeae, has pyrrolizidine alkaloids in all of its life stages (van Zoelen and van der Meijden (1991). Male Utetheisa ornatrix moths bestow nuptial gifts to females that can protect the female and eggs from predation (Eisner et al., 2000). Yasui (2001) found that a geometrid, Milionia basalis, acquires protection from potential stink bug predators through sequestration of plant derived compounds. Both A. ipsilon and S. frugiperda larvae that are at least 4th instar are more resistant to the entomopathogenic nematode S. carpocapsae if they develop exclusively on endophytic perennial ryegrass (Kunkel and
Grewal, 2003; Richmond et al., 2003). Furthermore, Kunkel et al. (2003) found that the
N. lolii produced alkaloid ergocristine reduces nematode infectivity and the growth and pathogenicity of Xenorhabdus nematophila, the symbiotic bacteria of S. carpocapsae.
I propose that these insects acquire a defense against S. carpocapsae when they have consumed endophytic perennial ryegrass. Both of these insects have cryptic coloration and are nocturnal. Birds and invertebrate predators rely on vision as a means
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of locating prey, therefore these predators play a vital role in the development of
aposematic coloration in the insects that sequester noxious compounds (Stamp and
Wilkens, 1992; Nishida, 2002). Therefore I suggest that the ergot alkaloids may not
provide protection against birds or predators that rely on visual cues. Stamp and
Wilkens (1992) reported that parasitoids rely heavily on smell to locate potential hosts
and secondarily on other senses such as touch and vision. These larvae may then be
better protected versus potential parasitoids. Bultman et al. (1997) found that parasitoids
that had parasitized S. frugiperda that had consumed endophyte infected grass performed poorly. Schaffner et al. (1994) found that spiders and ants were repelled by
Rhadinoceraea nodicornis, a specialist sawfly that sequestered alkaloids. They found
that the one of the sawflies sequestered ceveratrum alkaloids in its hemolymph. I suggest
that A. ipsilon and S. frugiperda may receive increased defense against parasitoids,
entomopathogenic nematodes, and possibly other invertebrate predators.
Campbell and Duffy (1979) reported that there is a potential for plant antibiosis
and biological control to be incompatible. They found reduced pupal eclosion, decreased
size and longevity of a parasitoid when it developed on a host that had consumed
tomatine. Barbosa et al. (1991) found that levels of nicotine that had no effect on
Manduca sexta caused significant mortality of the parasitoid Cotesia congregata.
Hyposoter annulipes, a parasitoid of S. frugiperda, will less frequently parasitize larvae,
fewer parasitoids form cocoons, and adults are smaller when the host insect has
consumed nicotine in its diet (Barbosa et al., 1986). Although the concentrations of
alkaloids found in insect hemolymph can be quite low they are still able to influence
parasitoid development and survival (Barbosa et al., 1991). Jaenike (1985) suggested
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that the mushroom toxin α-amanitin protects mycophagous drosophilids from parasitic
nematodes. Additionally, plant secondary compounds can affect colony growth of
Beauveria bassiana (Costa and Gaugler, 1989) and Xenorhabdus nematophila
(Barbercheck and Wang, 1996). Grewal et al. (2003) found that Neotyphodium coencophilum produced alkaloids reduced viability and infectivity of the entomopathogenic nematode Heterorhabditis bacteriophora.
My results suggest that some ergot alkaloids remain in both A. ipsilon and S. frugiperda larvae even after they switch to a different food source. I did not prolong the insect feeding on the alternative diet for more than 24 h because I was concerned about possible prepupa or pupa formation of our insects. However, I did find that S. frugiperda
larvae that are reared exclusively on endophytic perennial ryegrass through pupation still
had detectable concentrations of ergocristine and ergocryptine within the pupae (Table
1). Nishida (2002) described various ways that noxious toxins are sequestered by insect
herbivores. For example, defensive compounds may be sequestered through simple
diffusion if they are nonpolar, whereas hydrophyllic compounds cannot diffuse across the
gut membrane. Therefore, gut permeability is a major obstacle to sequestration, and
many insects get around it via a carrier-mediated mechanism. Some insects that
sequester pyrrolizidine alkaloids reduce the N-oxide to the tertiary alkaloid where it is
passively absorbed into the hemolymph and efficiently re-N-oxidized (Nishida, 2002).
This aspect needs to be investigated further for our insects because I did not account for
the total amount of alkaloid consumed. Schaffner et al. (1994) studied a specialist
sawfly, Rhadinoceraea nodicornis, and found alkaloids in the hemolymph and excrement
whereas a polyphagous sawfly, Aglaostigma sp., appeared to degrade all ingested
104
alkaloids. Some insects that feed on pyrrolizidine alkaloids are able to alter the chemical
structures into novel compounds or precursors to insect pheromones (Trigo et al., 1994).
Lindigkeit et al. (1997) found that S. littoralis efficiently excretes pyrrolizidine alkaloids
in the tertiary or reduced state. They suggested that the alkaloid N-oxide is easily
reduced, passes into the hemolymph and is excreted.
A. ipsilon and S. frugiperda sequestered detectable concentrations of the ergot alkaloids including ergocristine, ergocryptine, ergonovine, and ergovaline. Kunkel et al.
(2003) suggest that the alkaloid ergocristine may provide resistance against S. carpocapsae to these larvae. The results reported in this paper suggest that this is a possible mechanism because the alkaloid is maintained in the larvae. However, the exact location of the alkaloids in the body of the insect has not yet been determined. Insect cuticle, gut, various glands, hemolymph, or scales on the wings of the adult are all possible locations where alkaloids may be sequestered by insects (Nishida, 2002). For maximum protection of the insect against attack by the entomopathogenic nematodes the alkaloids should be found in the hemolymph. Alkaloid concentrations in the hemolymph were extremely low compared to these found in the whole insect. The methodology of detection of alkaloids in the hemolymph was not very precise and may have confounded the results. Additionally, Forst et al., (1997) suggest that insects may actually die before the symbiotic bacteria of the nematodes reaches high titers in the hemolymph. They propose that the bacteria may be nodulated and shuttled out of circulation to a fat body for a time. I do not know if the fat bodies in either A. ipsilon or S. frugiperda contain any of these alkaloids, but this does warrant investigation for future studies. My results however, do provide a possible explanation why some parasitoids or entomopathogens
105
may be less effective in areas containing endophytic perennial ryegrass. Although, total
concentrations of alkaloids consumed by A. ipsilon and S. frugiperda could not be quantified, I propose that some of the alkaloids persist within the insects at least to the pupa stage if the insect only consumes infected grass.
5.6 ACKNOWLEDGMENTS
I thank Patti Kunkel, Bryant Chambers, Ashley Roberts, Stephanie Greinert for their technical assistance, Dr. Dan Herms for the use of his freeze drier and a dessicator, and Jean Dyer at USDA-ARS, Iowa for supplying the black cutworms used in these experiments. This research was supported by the USDA-NRI grant No. P.S. Grewal and an OARDC graduate research grant to B.A. Kunkel.
106
PerMet
Ergocristine 150 Ergocryptine 125 Ergonovine 100 Ergovaline g/g dry weight
µ 75
50
25
0
Figure 5.1 Concentrations of various alkaloids (+SEM) found in endophyte infected perennial ryegrass (HE), Lolium perenne L. PerMet stands for perloline methyl ether, a plant produced alkaloid; whereas, the remaining alkaloids presented are produced by the fungus Neotyphodium lolii.
107
100
Larvae 90
80 Frass
Clear 30 gut g/g dry weight µ 20
10
ECI ECY EGN EVAL
Figure 5.2 Concentration of various alkaloids found in black cutworm, Agrotis ipsilon, fed on Neotyphodium lolii infected perennial ryegrass (high proportion of plants infected, HE) until larvae were 5-6th instar. No N. lolii produced alkaloids were detected in A. ipsilon larvae fed on N. lolii free perennial ryegrass. ECI = ergocristine, ECY = ergocryptine, EGN = ergonovine, EVAL = ergovaline.
108
200 Larva
175 Frass 150 Clear 125 gut 100 g/g dry weight µ 75 50 25 0 ECI ECYEGN EVAL PMET
Figure 5.3 Concentration of various alkaloids found in fall armyworm, Spodoptera frugiperda, fed on Neotyphodium lolii infected perennial ryegrass (high proportion of plants infected, HE) until larvae were 5-6th instar. No N. lolii produced alkaloids were detected in S. frugiperda larvae fed on N. lolii free perennial ryegrass. ECI = ergocristine, ECY = ergocryptine, EGN = ergonovine, EVAL = ergovaline, PMET = perloline methyl ether.
109
Source Perloline Ergocristine Ergocryptine Ergonovine Methyl Ether NE fed larvae hemolymph 15.91 ± 2.83 Not detected Not detected Not detected HE fed larvae hemolymph 15.91 ± 11.62 0.01 ± 0.004 0.01 ± 0.003 0.09 ± 0.3 HE Pupae 9.0 27.8 46.7 Not detected
Table 5.1 The amount of endophyte produced alkaloids (:g/g dry weight) and perloline methyl ether (:g/ml hemolymph) found in pupae or hemolymph S. frugiperda larvae fed on endophyte infected (HE) or endophyte free (NE) grass clippings.
110
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