Subterranean Chemical Ecology of Tritrophic Interactions

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Subterranean Chemical Ecology of Tritrophic Interactions Citrus Roots, Root Weevils and Entomopathogenic Nematodes
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english
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Ali,Jared Gregory
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Doctorate ( Ph.D.)
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University of Florida
Degree Disciplines:
Entomology and Nematology
Committee Chair:
Stelinski, Lukasz L.
Committee Members:
Duncan, Larry W
Liburd, Oscar E
Syvertsen, James P
Alborn, Hans

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Subjects / Keywords:
belowground -- chemical -- citrus -- diaprepes -- entomopathogenic -- pregeijerene -- root
Entomology and Nematology -- Dissertations, Academic -- UF
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Entomology and Nematology thesis, Ph.D.
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theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
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Abstract:
In response to herbivore feeding, plants release odors that benefit them by attracting natural enemies of herbivorous insects. Such interactions have been thoroughly examined aboveground. It has become increasingly evident that similar interactions occur belowground. The root-weevil (Diaprepes abbreviatus) is a serious citrus pest. Entomopathogenic nematodes (EPNs) have varying, and unpredictable, efficacy in controlling the weevil. Interactions between the plant, insect and nematode are poorly understood. In root-zone bioassays, root-weevil infested rootstock (Swingle) recruited significantly more EPNs than non-infested or mechanically damaged roots, or larvae alone. GC-MS analysis detected unique volatiles released from roots in response to weevil feeding. We compared attraction to volatiles of infested and non-infested roots from the hybrid, Swingle rootstock, and a parent line of the hybrid, P. trifoliata (Pt). Volatiles from Swingle infested by weevils were more attractive to both EPNs and plant parasitic nematodes than non-infested roots irrespective of foraging strategy. Pt, attracted EPN species irrespective of insect herbivory. Analysis of root volatiles revealed that Pt released the attractive cue constitutively, regardless of weevil feeding. A different non-hybrid species (C. aurantium) released the attractive cue only in response to larval feeding. Pregeijerene (1, 5-dimethylcyclodeca-1, 5, 7-triene) was identified as the major constituent of EPN attraction released from weevil-damaged roots. The release of pregeijerene by citrus roots peaked 9-12 hr after initiation of larval root feeding. Volatile collections from above/belowground portions of citrus plants revealed that aboveground adult feeding does not induce production of pregeijerene analogous to that induced by root damage nor does damage by larvae belowground induce a similar cue aboveground. Through the development of novel in-situ volatile sampling methods, pregeijerene release was detected from roots of mature trees in the field. In field experiments, lab-collected citrus volatiles from infested roots and isolated pregeijerene increased mortality of beetle larvae compared with controls. Using species-specific probes designed to identify EPN species, we determined by quantitative real-time PCR that field application of pregeijerene increased pest mortality by attracting four species of EPNs native to Florida. This and similar chemicals may have broad application for controlling agriculturally significant root pests.
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In the series University of Florida Digital Collections.
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Includes vita.
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by Jared Gregory Ali.
Thesis:
Thesis (Ph.D.)--University of Florida, 2011.
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Adviser: Stelinski, Lukasz L.
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RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-08-31

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1 SUBTERRANEAN CHEMICAL ECOLOGY OF TRITROPHIC INTERACTIONS: CITRUS ROOTS, ROOTS WEEVILS AND ENTOMOPATHOGENIC NEMATODES By JARED GREGORY ALI A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PART IAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011

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2 2011 Jared Gregory Ali

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3 To the Monkey, the Tiger, the Rabbit, the Cock, and the Cub

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4 ACKNOWLEDGMENTS I thank my family for all of the lo ve and strength they have provided. for the opportuniti es Lukasz L. Stelinski has given me, along with his time, patience and guidance throughout this work. I thank Hans T. Alborn, I walked away from every talk we had with an idea in my mind a nd a smile on my face. I thank Larry Duncan for bringing to light the small world that can only be studied with a shovel and an eyelash. I addition to my committee there were a number of people that helped to mak e this experience a special one: Wendy Meyer become a part of her family. Ian Jack son, the hardest workingman in L.A. always ready to hand out a smile. Finally you, thank you for everything and for what i s to c ome in the future Oh yes, the Beast

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ .......... 9 ABSTRACT ................................ ................................ ................................ ................... 11 CHAPTER 1 I NTRODUCTION ................................ ................................ ................................ .... 13 Plants and Insect Herbivores ................................ ................................ .................. 13 Herbivore Induced Plant Volatiles ................................ ................................ ........... 14 The M odel System ................................ ................................ ................................ .. 17 Biology and N atural H istory of Diaprepes abbreviatus ................................ ............ 20 Entomopathogenic N ematodes of D. abbreviatus ................................ ................... 22 Objectives ................................ ................................ ................................ ............... 24 Research Questions ................................ ................................ ............................... 24 2 SUBTERRANEAN HERBIVORE INDUCED VOLA TILES RELEASED BY CITRUS ROOTS UPON FEEDING BY DIAPREPES ABBREVIATUS RECRUIT ENTOMOPATHOGENIC NEMATODES ................................ ................................ 30 Materials and Methods ................................ ................................ ............................ 33 Insects ................................ ................................ ................................ .............. 33 Nematodes ................................ ................................ ................................ ....... 33 Plants ................................ ................................ ................................ ............... 33 Olfactometer ................................ ................................ ................................ ..... 34 Volatile C ollections ................................ ................................ ........................... 35 GC MS Analysis ................................ ................................ ............................... 36 EPN R esponse to R oot E xtracts ................................ ................................ ...... 36 Statistical Analysis ................................ ................................ ............................ 38 Results ................................ ................................ ................................ .................... 38 Olfactometer Bioassays ................................ ................................ .................... 38 GC MS Analysis ................................ ................................ ............................... 38 EPN R esponse to R oots E x tracts ................................ ................................ ..... 39 Discussion ................................ ................................ ................................ .............. 39 3 CONSTITUTIVE AND INDUCED SUBTERRANEAN PLANT VOLATILES ATTRACT BOTH ENTOMOPATHOGENIC AND PLANT PARASITIC NEMATODES ................................ ................................ ................................ ......... 48

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6 Materials and Methods ................................ ................................ ............................ 53 Insects ................................ ................................ ................................ .............. 53 Nematodes ................................ ................................ ................................ ....... 53 Plants ................................ ................................ ................................ ............... 54 Nematode B ehavior ................................ ................................ .......................... 54 Above versus B elow ground V olatile C ollections ................................ ............ 55 Volati le C ollection from I nfested versus N on infested P lants ........................... 56 GC MS A nalysis ................................ ................................ ............................... 57 Statistical A nalysis ................................ ................................ ............................ 58 Results ................................ ................................ ................................ .................... 58 Nematode B ehavior ................................ ................................ .......................... 58 Effect of Below versus A bove ground H erbivory on R elease of N ematode A ttractants ................................ ................................ ................................ ..... 59 Subteranean R elease of V olatiles by V arious P lant S pecies ............................ 59 Discussion ................................ ................................ ................................ .............. 59 4 MANIPULATING NATIVE POPULATIONS O F ENTOMOPATHOGENIC NEMATODES WITH HERBIVORE INDUCED PLANT VOLATILES TO ENHANCE PEST CONTROL ................................ ................................ ................. 74 Materials and Methods ................................ ................................ ............................ 76 Insect larvae ................................ ................................ ................................ ..... 76 Plants ................................ ................................ ................................ ............... 77 Nematodes u sed for L aboratory B ioassay s and qPCR ................................ .... 77 In situ Volatile Collection from Infested Roots ................................ .................. 78 In situ V olatile C ollection from I nfested R oots in the F ield ................................ 78 GC MS Analysis ................................ ................................ ............................... 79 Isolation and P urification of P regeijerene ................................ ......................... 79 Two choice B ioassay to D etermine O ptimal D osage to A ttract EPNs .............. 80 Application of HIPVs in the Field ................................ ................................ ...... 81 Detection, I dentificati on and Q uantification of E ntomopathogenic N ematodes using R eal T ime qPCR ................................ .............................. 83 NMR A nalysis of Pregeijerene ................................ ................................ .......... 85 Results ................................ ................................ ................................ .................... 85 In situ V olatile C ollection from I nfested R oots in the F ield ................................ 85 Release and P urification of 1, 5 D imethylcyclodeca 1, 5, 7 T riene .................. 86 Identification of P regeijerene ................................ ................................ ............ 86 Optimum P regeijerene C oncentration ................................ .............................. 87 Field V erification of I ncreased B eetle M ortality by B elowground HIPVs ........... 87 Real time qPCR D etermination of EPN D iversity, and A ttraction to HIPVs ...... 88 NMR A nalysis of Pregeijerene ................................ ................................ .......... 89 Discussion ................................ ................................ ................................ .............. 90 5 CONCLUSIONS ................................ ................................ ................................ ... 106 LIST OF REFERENCES ................................ ................................ ............................. 113

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7 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 132

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8 LIST OF TABLES Table page 2 1 GC MS identification of volatiles from Swingle citrumelo rootstock (Citrus paradise Poncirus trifoliate ................................ ................................ .............. 43 3 1 Trophic level, foraging strategy and ecological status of nemat odes tested ....... 68 3 2 GC MS identification of volatiles from various citrus rootstocks ......................... 69 4 1 Species of entomopathogenic nematod es identified and quantified in response to HIPV deployment in the field. ................................ .......................... 94 4 2 1 H (600 MHz), 13 C (151 MHz), HMBC and NOESY NMR spectroscopic data for pregeijerene in C 6 D 6 .. ................................ ................................ .................... 95 4 3 1 H (600 MHz), 13 C (151 MHz), HMBC and NOESY NMR spectroscopic data for geijerene in C 6 D 6 ................................ ................................ ........................ 96

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9 LIST OF FIGURES Figure page 1 1 Diaprepes abbreviatus resting on citrus leaf. ................................ ...................... 26 1 2 Diaprepes abbreviatus adults damage to citrus leaves (notching) ..................... 27 1 3 Young (left) and older larvae of the Diaprepes root weevil on cakes of an artificial diet developed by ARS. ................................ ................................ ......... 28 1 4 A generalized depiction entomopath ogenic nematode life cycle. ...................... 29 2 1 Schematic diagram of sand column assay unit.. ................................ ................. 44 2 2 Mean number of S. diaprepesi attrac ted to chambers. ................................ ....... 45 2 3 Example chromatograms showing volatile profiles of D. abbreviatus infested plants, non infested plants and larvae alone. ................................ ..................... 46 2 4 Mean number of nematodes attracted to volatiles from D. abbreviatus infested roots compared with volatiles from undamaged roots. .......................... 47 3 1 Schematic diagram of simul taneous above and below ground volatile collection apparatus (ARS, Gainesville, FL, USA).. ................................ ............ 70 3 2 Responses of Tylenchulus semipenetrans, Steinernema carpocapse, S. riobrave, S. diaprepesi, and Heterorhabditis indica. ................................ ........... 71 3 3 Example chromatograms depicting volatile profiles from simultaneous collections of root and shoot volatiles of Swingle ( Citrus paradisi Poncirus trifoli ata ). ................................ ................................ ................................ ............. 72 3 4 Example chromatogram showing volatile profiles from roots .............................. 73 4 1 Representation of soil probe design used to sa mple volatiles belowground ...... 97 4 2 Conversion of Pregeijerene to Geijerene. ................................ ........................... 98 4 3 Chromatograms showing the initial crude ex tract prior to purification and final purified Pregeijerene. ................................ ................................ ......................... 99 4 4 Schematic diagram of the deployment and sampling procedure for field experiments ................................ ................................ ................................ ...... 100 4 5 Chromatograms of volatiles taken from intact citrus roots in the field ............... 101 4 6 Time course of pregeijerene (1, 5 dimethylcyclodeca 1, 5, 7 triene) release fo llowing initiation of root weevil (D. abbreviatus) feeding on citrus roots. ........ 102

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10 4 7 Optimal dosage of pregeijerene ( 1, 5 dimethylcyclodeca 1, 5, 7 triene ) for attracting entomopathogenic nem atodes ( S. riobrave and H. indica ). .............. 103 4 8 Mean percentage of larval mortality for treatments with or without D. abbreviatus fed upon root volatiles. ................................ ................................ .. 104 4 9 Effect of pregeijerene on weevil mortality and associated attraction of EPN species. ................................ ................................ ................................ ............ 105

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11 Abstract o f Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy SUBTERRANEAN CHEMICAL ECOLOGY OF TRITROPHIC INTERACTIONS: CITRUS ROOTS, ROOTS WEEVILS AND ENTOMOPATHOGENIC NEMATODES By Jared Gregory Ali August 2011 Chair: Luk asz L. Stelinski Major: Entomology and Nematology In response to herbivore feeding plants release odors that benefit them by attracting natural enemies of herbivorous insects. Such interactions have been thoroughly examined aboveground. It has become in creasingly evident that similar interactions occur belowground. The root weevil ( Diaprepes abbreviatus ) is a serious citrus pest Entomopathogenic nematodes (EPNs) have varying, and unpredictable, eff icacy in controlling the weevil. I nteractions between th e plant, insect and nematode are poorly understood. In root zone bioassay s root weevil infested rootstock ( Swingle ) recruited significantly more EPNs than non infested or mechanically damaged roots, or larvae alone. GC MS analysis detected unique v olatile s released from roots in response to weevil feeding. We compared attraction to volatiles of infested and non infested roots from the hybrid, Swingle rootstock, and a parent line of the hybrid, P. trifoliata (Pt). Volatiles from Swingle infested by weevils were more attractive to both EPNs and plant parasitic nematodes than non infested roots irrespective of foraging strategy. Pt, attracted EPN species irrespective of insect herbivory. Analysis of root volatiles revealed that Pt released the attractive cue c onstitutively, regardless of weevil feeding. A different non hybrid species ( C. aurantium ) released the attractive cue only in

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12 response to larval feeding. P regeijerene ( 1, 5 dimethylcyclodeca 1, 5, 7 triene) was identified as the major constituent of EPN a ttraction released from weevil damaged roots. The release of p regeijerene by citrus roots peaked 9 12 hr after initiation of larval root feeding. Volatile collections from above/belowground portions of citrus plants revealed that aboveground adult feeding does not induce production of pregeijerene analogous to that induced by root damage nor does damage by larvae belowground induce a similar cue aboveground. Through the development of novel in situ volatile sampling methods, pregeijerene release was detecte d from root s of mature trees in the f ield. In field experiments, lab collected citrus volatiles from infested root s and isolated pregeijerene increased mortality of beetle larvae compared with controls. Using species specific probes designed to identify EP N species, we determined by quantitative real time PCR that field application of pregeijerene increased pest mortality by attracting four species of EPNs native to Florida. This and similar chemicals may have broad application for controlling agriculturall y significant root pests.

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13 CHAPTER 1 INTRODUCTION Plants and Insect Herbivores Autotrophic green p lants provide virtually all of the total energy available to terrestrial organisms P lants have been in engaged in an arms race with herbivores over millio ns of years of evolution and have developed defenses that protect them from herbivory This coevolutionary process has led to the development of tremendous biodiversity which is highly evident in insects Concurrent selection pressures have simultaneously pushed the evolution of resistance traits in plants and traits in insect herbivores to overcome plant defenses. Plants have a variety of defensive strategies against insects. Chemical, p hysical, and biotic defenses can reduce herbivory and increase plant fitness. Physical features on the tissues of plants can drastically influence herbivore acceptance of host plants. The presence of trichomes and wax crystal structures on the plant surface, leaf thickness and toughness, sclerotization and high silica conte nt may cause avoidance behavior. Plants may also store toxic or repellent compounds in their leaf tiss ues. These are a ll form s of constitutive defense (Karban & Baldwin 1997) Plants also produce toxic or repellent compounds only in response to insect dam age, and this process is termed induce d defense (Karban & Baldwin 1997) Most plants display multiple defenses, which vary in intensity and effectiveness, and can operate over different temporal and spatial scales against different attackers. These defense s can be classified as direct, when exerting a negative im pact on herbivore s, or indirect, when manipulati n g of organisms in higher trophic levels to negative ly impact the herbivore

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14 Direct defenses may prevent herbivores from feeding due to physical (spin es, thorns tric homes, and waxes), or chemical defenses (secondary plant metabolites, phenylpropanoids, terpinoids, alkaloids, and proteinase inhibitors) (Karban & Baldwin 199 7 ) Indirect defenses are adaptations that result in the recruitment and /or preser vation of organisms that protect plants against herbivores (Karban & Baldwin 199 7 ) These can range from constitutive for mation of domatia, which serve as domatia for beneficial organisms such as ants, mites, and even bacteria to the production of foliar n ectaries and nutritive structures that can also be used by natural enemies of herbivores (Boethel & E ikenbary 1986; Whitman 198 8 ). Plant indirect defenses can also be induced. During the last two decades, it has been revealed that plants respond to herbivo re feeding by producing and releasing odors ( herbivore induced plant volatiles or HIPVs) that are exploit ed by natural enemies that use these cues t o locate their prey and hosts ( T urlings & W ckers 2004; Dicke & Vet 1999; Dicke et al. 2003). Herbivore Indu ced Plant Volatiles HIPVs are known to play various important roles in plant arthropod interactions, in addition to natural enemy recruitment. For example t hey are known to deter ovipositon by Lepidoptera (Landolt 1993). There is also mounting evidence th at HIPVs are involved in plant plant communication (Engelberth et al. 2004; Arimura et al 2000; Kessler & Baldwin 2001; Baldwin et al 2002). The composition of HIPVs is kn own for many plant herb i vore systems (Pare & Tumlinson 1999). Some HIPVs are taxon specific, such as glucosinolate breakdown products in Brassica species (Mattiacci et al 1995), whereas other s appear to be common to many different plant families ( Boom et al 2004) These compound s include six carbon (C6) generally released by plant leaves

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15 immediately after wounding. These include isomers of hexanol, hexanal, and hexenyl actetate (Hatanaka 1993). In general green leaf volatiles are present directly after wounding with (Z) 3 hexenyl acetate as an excep tion (Matsui et al 2000), and they may be involved in trigger ing terpenoid production (Farag & Pare 2002), causing the accumulation of jasmonic acid (JA) as well as the expression of defense genes (Bate & Rot hestein 1998; Engelberth et al 2004). It has a lso bee n suggested that C6 volatiles play a direct role in plant defense in addition to a possible antimicrobial function. For e xample C6 aldehydes and alcohols reduce tobacco ap hid fecundity (Hildebrand et al 1993). In addition s ome C6 compounds may f unction as indirect defenses (Kessler & A lessandro & T urlings 2005) or play a role in signaling within or between plants that result s in up re gulation of genes associated with defense (Arimura et al 2001). In contrast to C6 aldehydes and alcohols, the emission of (Z) 3 hexenyl acetate can be observ ed a few hours after feeding or mechanical damage suggesting a similar signaling pathway as other herbivore induced terpinoids (Turlings et al 1995; Arimura et al 200 1 ). Herbivore induced l eaf volatiles also include terpe noids, encompassing monoterpenes (C10), sesquiterpenes (C15) and homot erpenes (C11 or C16). All terpenoi ds are synthesized through the condensation of isopentyl diphosphate and its allylic isomer dimethylallyl diphosphate in ei th er the cytosol or the plastids (Pare & T umlinson 1999; Arimura et al. 2005). Indole is a common and dominating nitrogenous compound found in HIPVs derived from the Shikemate acid pathway (Frey et al. 2000) C ontinuous mechanical damage of plant tissues can result in the emission of volatile blends resembling those occurring after herbivore damage ( M ithofer et al.

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16 2005), but commonly the emission o f these volatiles can be enhanced and prolonged by eliciting facto rs from a feeding insect. These factors als o elicit odor emission when they are taken up via the stem of the plant or even via the petiole of a leaf; the response to their elicitors has been shown to be systemic (Dicke et al. 1990, T urlings et al. 1993). Plant defense responses have been ascribed to a wide variety of chemical elicito rs that activate specific down stream si gnal transduction pathways (Par e et al. 2005). Two major classes of insect derived eli citors are the Beta glucosidase discovered in regurgitant of Pieris brassicae larvae, which faci litates the emission of glucosinolate breakdown products (Mattiacci et al 1995); and the fatty acid derivative volicitin and related compounds that induce the release of the full blend of volatiles normally induced by caterpillar feeding (Alborn et al. 199 7). T he wide variety of elicitors is often the result of slight changes to chemical precursors which can have strong e ffects on the volatile blend emitted from the p l ant (e.g. De Moraes et al. 2001, Kessler & Baldwin 2001, van P oecke & Dicke 2004). Moreov er, biosynthesis and release of HIPVs can be affected by biotic f actors such as plant hormones (F armer 2001; Thaler et al. 2002), microorganisms ( Piel et al 1997; Cardoza et al. 2002), and abiotic factors such as temp erature, light (Takabayashi et al 199 4, Gouingguene & T urlings 2002), or O 3 and CO 2 (Vuorinen et al. 2004). Although the series of specific defense responses that are activated depend s on the precise plant herbivore interaction, several common global responses have emerged. Herbivore feeding usually triggers defense responses mediated by ethylene and jasmonic acid tha t act synergistically (Kahl et al 2000; Schmelz et al. 2003), whereas pathogen attack typically elevates salicylic acid levels in a plant (Vranova et al.

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17 2002). On the other han d it seems that plant response signals can be highly variable depending on plant genotype (Takabayashi et al 1991; Loughrin et al. 1995; Gouinguene et al. 2001), p lant parts ( T urlings et al. 1993), or growth stages of a plant (Gouinguene et al 2001). P l ant s are additionally capable of respond ing differentially to specific herbivores (De Moraes et al. 1998; Turlings et al 1998), and to different life stages of the same herbivore (Takabayashi et al. 1995). With respect to research on HIPVs and their intera ction s with herbivores, substantial focus has been given to the aboveground parts of plants and only recently have interactions investigated belowground ( von T ol et al. 2001; R asmann et al. 2005; this dissertation ). Van Tol et al (2001) showed that plants recruit e ntomopahtogenic nematodes to thei r herbivore damaged roots Furthermore, maize roots infested with larvae of the Western corn root worm ( Diabrotica virgifera ) production ( E ) caryophyllene, which attracts entomopathogenic nematodes (Rasmann et al 2005). Spiking soil with synthetic ( E ) caryophyllene decreases the emergence of adult corn root worms from maize by half compared with untreated control plots due to enhanced nema tode attraction (Rasmann et al 2005). More recently research has acknowledged that plants mediate interactions between two communities e.i. those found above or below ground (van Dam & Heil 2011, Erb et al. 2011) These interactions are highly diverse, a nd be coming an important aspect of investigati n g plant defense. The Model System Diaprepes abbreviatus (Linnaeus) (Coleoptera: Curculionidae), (Figure 1 1) was first introduced into Florida in 1964 (Beavers & Selhime 1975). Over the past 40 plus years i t has significantly contributed to the spread of disease and damage to citrus,

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18 ornamental plants, and other crops. D. abbreviatus is a native economic pest of the Caribbe an where at least 19 additional species are known within the genus (Wolco tt 1936 ). Diapr epes abbreviatus has spread over a large area of central and southern Florida where it caus es approximately $70 million in damage annually (Weissling et al 2002; Lapointe 2000). The initial area of infestation was an estimated 6,500 acres in Apopka, FL an d ha s now increased to an estimated 164,000 acres over 20 counties in central and southern Florida (Weissling et al 200 2). Diaprepes abbreviatus has a wide host range, attacking ap proximately 293 different plant species including citrus, sugarcane, veget ables, potatoes, strawberries, woody fieldgrown ornamentals, sweet potatoes, papaya, guava, mahogany, containerized ornamentals, and non cultivated wild plant s (Simpson et al. 1996, 2000). D iaprepes abbreviatus damage to the vegetative portion of plants is most often seen as notching on the margins of young leaves (Fennah 1940) (Fig ure 1 2 ). This is a key trait characterizing D. abbreviatus infestation. Adults continue to feed on fol iage and lay eggs between older leaves (Schroeder 1992; Fennah 1940). Howeve r, t he greatest damage is caused by larvae feeding below ground. U pon hatching, t he larvae fall to the soil and make their way to the roots of plants where later instars feed and develop (Schroeder 1992). This feeding can girdle the taproot causing damage that disables the plant from taking up water and nutrients resulting in plant dea th (Schroeder 1992). This type of damage also facilitates secondary infections by Phythophora oomycete species (Graham et al 1996). Young hosts can be killed by a single larv a whi le several larvae can result in serious decline of older, established hosts (Weissling et al 2002). Since

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19 larvae develop below ground, it is difficult to detect them before decline of above ground vegetation of the host is observed. Current chemical control of D. abbreviatus includes foliar insecticides (Bullock et al. 1988), ovi cides and oil sprays (Schroeder 1996) to red uce adult feeding, oviposition, and viable egg production. Soil applied insecticides l ike Brigade WSB and Capture 2EC are used as a soil barrier to decrease lar val entry (Knapp 1999). Foliar chem ical spray applications such as Danitol 2.4EC, Imidan 70WP Kryocide 96 WP, and Micromite 80WGS are most effective during peak seasonal D. abbreviatus abundance. C hemical controls are less ef fective than earlier available treatments comprised of the now banned organochlorine soil pesticides (Dun can et al. 1999; McCoy 1999). The most effective method for controlling the more damaging mid to late instars found on roots appears to be entomopathog enic nemat odes, which are roundworms from the genera Heterorhabditis or Steinernema They are obligate parasites that kill their host with the aid of a symbiotic bacterium (Poina r 1990). Native and introduced entomopathogenic nematodes are infectious to al l la rval stages and possibly adults (Adair 1994 ; Schr oeder 1 990). Releases of mass produced entomopathogenic nematodes (EPNs) have been used by citrus growers for over 20 years (Duncan et al. 1999). It has also been shown tha t use of EPNs can reduce larval populations of D. abbreviatus (Shroeder 1990; Downing et al. 1991; Schroeder 1992 ; Duncan et al. 199 9 ; Bullock et al. 1999) and thus resulting adult populations (Bullock et al. 1999, Duncan et al. 2007) However, improvement of the efficacy of EPN treatment is still desired. Presently D. abbreviates control using EPNs has been inconsistent and depende nt on nematode species and soil composition (Adair 1994 ; Duncan et al. 199 9 ). One

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20 approach to enhance the effectiveness of EPNs and control of D. abbreviatus m ay be l defenses. Exploiting h erbivore induced plant volatile emission s may represent a ne w approaches in integrated pest management (IPM). Plants benefit by releasing HI PV s wh en they recruit natural enemies of subterranean herbivores (van Tol et al. 2001; Neveu et al. 2002; Aratchige et al. 2004). For example, entomopathogenic nematodes are attracted to exudates of Thuja plants ( Thuja occidentalis ) infested with larvae of the vine weevil (van Tol et al. 2001). F urthermore, maize roots infested with larvae of the Western corn root worm ( Diabrotica virgifera ) release ( E ) caryophyllene, which attracts entomopathogenic nematodes (Rasmann et al 2005). Spiking soil with synthetic ( E ) caryophyllene decreases the emergence of adult corn root worms from maize by half compared with untreated control plots (Rasmann et al 2005 ). Identification of the signal s that mediate the interactions between D. abbreviatus infested plants and entomopathogenic nematodes could advanc e understand of this relationship Determining whether citrus release s specific chemicals that recruit entomop athogen ic nematodes upon weevil damage may improve the efficacy of these biological control agents. Following identificat ion, application of such chemicals to the soil may attract n aturally occurring nematodes as well as improve the host finding capability of exogenously applied nematodes leading to substantial impro vement in the efficacy of this biological control tactic. Biology and natural history of Diaprepes abbreviatus The root weevil, Diaprepes abbreviatus ranges from 1936). It has various color morphs that differ in hues of yellow, gray, orange and black (Lapointe USDA 2000). The larvae are white legless and grow to about 1 inch in length

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21 (Figure 1 3) It is native to the Caribbean region. Diaprepes abbreviatus beca me a sig nificant pest in the early 1900 crops su ch as sugar cane (Lapointe USDA 2000). Increased incidence of D. abbreviatus damage may be correlated with the introduction of the mongoose as a biologic al control agent for rats. The mongoose failed to control the rats but successfully killed off populations of many bird and lizard species that preyed on D. abbreviatus (Watson 1903). D. abbreviatus was considered a significant pest of sugar cane in Barbad os by 1921 (Bourne 1921). In 1964 it wa s introduced into Florida in an ornamental ship ment from Puerto Rico (Woodruff 1968). It has since spread throughout Florida and may still threaten other states. D. abbreviatus became established in citrus groves in the Rio Grande Valley of Texas as of 2000 (Skaria & French 2001). Since 1974, D. abbreviatus infestation ha d threatened California, which is a major producer of citrus and other host plants of this pol yphagous pest and has since been found in agricultural areas of California (Grafton Cardwell et al. 2004). Although adults may emerge year round, there are two peak emergence periods. The first occurs during the spring from May to June. The second peak emergence is in the fall from August to September (Duncan et al 2001). Mating and egg laying occur throughout both of these periods. A single female can lay up to 5,000 eggs during her 3 4 month life span (Wolcott 1936) The eggs are laid between leaves and typically hatch within 7 10 days. The larvae will fall onto the ground and make their way into the soil to the fibrous roots of host plants where they feed until pupation begins. The period of larval to adult emergence varies from several mont hs to more than a year (Wolcott 1936).

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22 There are two main features o f life cycle that have made it difficult to control as a pest of cultivated crops. First, its life stages are active in the field throughout the majority of the annual season. Second, adults and larvae occupy separate habitats (above and be low ground); therefore, each life stage must be targeted separately (Georgis et al. 2005). Given that adults continuously emerge from soil to produce offspring, which in turn return to the soil, control methods that target only adults or larvae will only s poradically reduce the pest population density. Because persistent insecticides (e.g., dieldrin and chlordane) are no longer available, a combination of non persistent tactics timed to kill both life phases of the population is a strategy often used by gro wers (Georgis et al. 2005). Growers have widely adopted the use of commercially formulated entomopathogenic nematodes since they became available in 1990 to manage the soil stages of the weevil (B u llock et al. 1999 ; Schroeder 1992). Entomopathogenic nemato des of D. abbreviatus Two families of nematodes are commonly us ed as biological control agents: Steinermenatidae and Het erorhabditidae. These families vector a symbiotic bacterium into the body cavitie s of insects. The life cycle of entomopathogenic nemato des consists of these major steps: 1) penetration into the body cavity of the potential host, 2) release of bacteria, 3) development of mature adults, 4) mating and reproduction of injective juveniles, and 5) emergence of infective juveniles in search of a new host (Figure 1 4). The infective juv enile is a third stage juvenile and is morphologically and physiologically adapted to remain for extended periods without ingesting food (Poinar 1990). Infection with e ntomopathogenic nematodes can result in death o f their insect host within 48 hr. Entomopathogenic nematodes have been investigated and implemented for management of D. abbreviatus larvae in Florida citrus for almost two decades. Early

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23 invesitgations focused on Steinernema glaseri S. carpocapsae and H bacteriophora for control of the weevil (Bullock & Miller 1994; Downing et al. 1991; Schroeder 1992). Current formulations containing S. riobrave have become adopted commercially for D. abbreviatus management. Of the several species evaluated in laborator y bioassays and greenhouse trails, S. riobrave and a Florida isolate of H. indica were the most effective against the Diaprepes root weevil, and reproduction by H. indica in the weevil exceeded that of other species (Shapiro Ilan & McCoy 2000a; Shapiro Ila n & McCoy 2000b). S. riobrave is currently the only nematode species commercially marketed for the Florida citrus industry. H. indica (no longer available) wa s formulated as a paste and S. riobrave can be obtained in water dispersible granular formulations In 1999, approximately 20% of the hectares infested with this weevil were treated with nematodes (Shapiro Ilan et al. 2002). Given that reported efficacy of entomopathogenic nematodes ranges fro m 0% to >90% suppression (Adair 1994; Bullock et al. 1999; D uncan et al. 199 9 ; McCoy et al. 2000) improved efficacy of this tactic is desired. One potential means by which to improve the efficacy of EPN s is by gaining a better understanding of their foraging strategies in order to more effectively exploit nematode behavior. Often, nematode species can be categorized according to their foraging behavior. Ambush (sit and wait) and cruise (wide search) strategies, are generally considered as the di poles of a continuum of sal ta to ry search strategies (Lewis et al 1992,1 993; Campbell & Gaugler 1997; Grewal et al 1996). Cruisers allocate more of their time to scanning for resource associated cues as they move through the environment, and exhibit only brief pauses, and are therefore more effective at finding sedentary and cryptic host s Ambush foragers scan during long pauses and allocate

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24 less time to moving through their environment. They wait for resources to come to them, making ambushers effective at finding resources with high mobility. It is important to consider thes e alternative foraging strategies using a comparative approach when investigating the use of HIPVs to enhance biological control. Objectives Assess behavioral responses of entomopathogenic nematodes to Diaprepes infested plants : Quantify EPN response to weevil damaged, mechanically damaged versus undamaged plants, or weevils alone. Identify plant released chemicals that recruit entomopathogenic nematodes to Diaprepes infested plants : Determine attractiveness of HIPVs to various EPN species. Evaluate the r elative efficacy of recruitment chemical (s) for improving biological control of D. abbreviatus in the field : Test whether HIPVs to recruit EPN to caged D. abbreviatus in a citrus grove Research Questions The present dissertation addresses the following qu estions: Do citrus roots that are attacked by larvae of the citrus root weevil produce induced volatiles tha t attract entomopathogenic nematodes? It has been demonstrated that aboveground plant produced organic volatile compounds induced by the feeding of folivores can cause the attraction of their natural enemies such as parasitoids (Turlings & W ckers 2004). Recently the focus has gone belowground (van Tol et al 2001 ; Rasmann et al 2005). The aim of the study presented in C hapter 2 was to asse s s if Dia prepes abbreviatus infested roots produced compounds that could at tract the entomopathogenic nematode, Steinernema diaprepesi The chapter also

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25 introduces a novel method for in situ volatile collection from roots and the bioassay of EPN s to these cues Doe s release of HIPV nematode attractant from citrus roots vary depending on citrus variety? Does response of nematodes vary depending on species foraging strategy and trophic level ? Although recent work has shown that EPN s can respond to cues emitted from r oots of plants while fed upon by their roots herbivores (Rasmann et al. 2005 ; Ali et al. 2010), little is known about the variation in release amongst citrus roots and variation in between response of various nematode s pecies to these cues. Chapter 3 prese nts a study that evaluated various rootstock cues and responses of various nematode species both entomopathogenic and plant parasitic, to these cues. The chapter demonstrates broad attraction of HIPVs to both plant parasit ic and entomopathogenic nematodes as well as demonstrating variation in responses to these cues based on nematode foraging strategy. Can this cue be used to manipulate entomopathogenic nematode s in the field to increase larval mortality? Although many plants have been shown to release vo latiles that attract natural enemies of their herbivores ( T urling & W ckers 2004), f ew studies have been able to translate t hese basic findings into practical field application ( De Moraes et al 1998;Thaler 1999; Johnson 2004). Only one study has evaluated this interaction belowground ( R asmann et al 2005). In C hapter 4 isolate d and purifie d pregeijerene was evaluated in a field trial s to determine if this HIPV could increase larval mortality by attracting various species of EPNs. Moreover this study presen ts a novel approach to the quantification of naturally occurring EPN species that were attracted by deployment of HIPVs

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26 Figure 1 1. Diaprepes abbreviatus resting on citrus leaf. Photograph by Peggy Greb USDA ARS 2010.

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27 Figure 1 2 Diaprepes abbrev iatus adults damage to citrus leaves (notching) Photograph by Jared Gregory Ali 2008

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28 Figure 1 3. Young (right ) and older larvae (left) of the Diaprepes root weevil on cakes of an artificial diet developed by ARS. Photograph by Peggy Greb USDA ARS.

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29 Figure 1 4 A generalized depi ction entomopathogenic nematode life cycle. Diagram by David I. Shaprio IIan, USDA ARS, SEFTNRL, Byron,GA and Randy Gaugler, Department of Entomology, Rutgers University, New Brunswick, NJ. ( From: Nematodes (Rhabditida: St einernemat idae &Heterorhabditidae. http://www.biocontrol.entomology.cornell.edu/pathogens/nematodes.html )

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30 CHAPTER 2 S UBTERRANEAN HERBIVOR E INDUCED VOLATILES RE LEASED BY CITRUS ROOTS UPON FEEDING B Y DIAPREPES ABBREVIA TUS RECRUIT ENTOMOPATHOGENIC NEM ATODES Plants produce an array of signals with diverse roles, providing them with responses necessary to survive in their dynamic environment. Examples of plants luring organism s to facilitate their reproductive requirements are ubiquitous and often taken for granted (Pichersky & Gershenzon 2002). Less acknowledged is the ability of a plant to manipulate the behavior of organisms to serve defensive roles (Turlings & Wckers 2004) However, examples of such tritrophic interactions, between plants, herbivores, and natural enemies are quite common (Agrawal & Rutter 1998; Agrawal & Karban 1999; Baldwin & Preston 1999; Dicke et al 2003). Herbivore feeding on plants results in release of volatile compounds, which may attract arthropod predators and/or parasitoids. For instance, lima bean plants ( Phaseolus lunatus ), release volatiles when infested with spider mites ( Tetranychus urticae ), which attract the predatory mite Phytoseiulus pers imilis (Takabayashi & Dicke 1996). Oviposition can also stimulate plant exudates that are attractive to egg parasitoids; the legume, Vicia faba emits volatiles which attract the egg parasitoid, Trissolcus basalis after oviposition by the Pentatomid, Nezara viridula ( Colazza et al 2004). Specific compounds from both the plant and salivary elicitors from the herbivore have been shown to mediate these interactions (Alborn et al 1997). For example, the plant volatile methyl salicylate attracts herbivore preda tors (e.g. De Boer & Dicke 2004). Volicitin, found in oral secretions of caterpillars ( Spodoptera exigua ), has been well characterized and shown to induce volatile production in maize (Alborn et al 2000; Turlings et al 2000). As the details of above grou nd tritrophic interactions have become substantially

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31 resolved (Vet et al 1991; Vet & Dicke 1992), recent attention has focused on analogous communication systems in the subterranean environment. Volatile signaling by plant roots can contribute to belowgr ound defense by acting as antimicrobial or antiherbivore substances (Bais et al 2006; Tumlinson et al 1992, 1999; Neveu et al 2002). Plants can also benefit by releasing herbivore induced volatile emissions that recruit natural enemies of subterranean h erbivores, as recently shown by van Tol et al (2001), Aratchige et al (2004), and Rasmann et al (2005). The pressure from belowground pests of plants is significant and likely imparts selection pressure for evolution of induced plant responses. Diaprepe s abbreviatus (L.) is a significant belowground pest of plant roots on more than 290 plant species including citrus, sugarcane, vegetables, potatoes, strawberries, woody field grown ornamentals, sweet potatoes, papaya, guava, mahogany, containerized orname ntals, and non cultivated wild plants (Simpson et al 2000). D. abbreviatus was first introduced into Florida in 1964 (Beavers & Selhime 1975). Over the past 40 years it has significantly contributed to the spread of disease and damage to citrus, ornamenta l plants, and other crops causing approximately $70 million in damage annually (Weissling et al 2002). D. abbreviatus damage the vegetative portion of plants by notching young leaves (Fennah 1940). Mature adults lay eggs between older leaves and emerging first instar larvae drop to the soil where they develop and feed on roots causing the most severe damage to plants (Schroeder 1992; Fennah 1940). Currently, the most effective method for controlling the larval stage is with entomopathogenic nematodes (EPN) from the genera Heterorhabditis or Steinernema (Downing et al 1991; Schroeder 1992).

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32 EPNs are obligate parasites that kill their host with the aid of a symbiotic bacterium (Poinar 1990). Mass produced EPNs have been used for control of D. abbreviatus b y citrus growers for over 20 years (Duncan et al 1999). Mass release of EPNs can effectively reduce larval populations of D. abbreviatus (Downing et al 1991; Schroeder 1992; Bullock et al 1999). However, the reported efficacy of EPNs against D. abbrevia tus ranges from 0% to >90% suppression (Adair 1994; Bullock et al 1999; McCoy et al 2000) and thus improved consistency of this tactic is desired. One approach to enhance the effectiveness of EPNs against D. abbreviatus may be to y produced chemical defenses. Recent work has shown EPNs ( Heterorhabditis megidis ) are attracted to exudates of Thuja plants ( Thuja occidentalis ) infested with larvae of the vine weevil ( Otiorhynchus sulcatus ) (van Tol et al 2001). Furthermore, maize root s infested with larvae of the western corn rootworm ( Diabrotica virgifera ) release terpenoids, typically ( E ) caryophyllene, which attracts EPNs ( Heterorhabditis megidis ) (Rasmann et al 2005). In this investigation, we quantified the behavior of the entomopathogenic nematode, Steinernema diaprepesi Nguyen & Duncan, in response to citrus plants damaged by larva l D. abbreviatus We show that EPNs are attracted to weevil damaged roots, but not so to mechanically damaged roots, undamaged roots or larvae alone. We also identified volatile compounds induced by weevil feeding and show that EPN response is specifically mediated by solvent extracts of infested roots. Identification of the signals that mediate interactions between D. abbreviatus infested plants and the associated EPNs could advance biological control of D. abbreviatus by selectively increasing the functio nal and/or numerical response of its natural enemies.

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33 Materials and Methods Insects D. abbreviatus Research and Education Center (CREC) in Lake Alfred, FL. This culture was periodically supplemented from a large culture maintained at the Division of Plant Industry Sterile Fly Facility in Gainesville, FL. Larvae are reared on an artificial diet developed by Beavers (1982) using procedures described by Lapointe and Shapiro (1999). Larvae us ed in experiments were 3 rd to 6 th instars. Nematodes S. diaprepesi were isolated from D. abbreviatus larvae buried in a commercial citrus orchard in Florida. The nematodes were then reared in last instar greater wax moth larvae, Galleria mellonella (L.) (L epidoptera: Pyralidae), at approximately 25C according to procedures described in Kaya and Stock (1997). Infective juveniles (IJs) that emerged from insect cadavers into White traps (White 1927) were stored in shallow water in transfer flasks at 15C for up to 2 weeks prior to use. Plants Citrus paradisi Macf. Poncirus trifoliata L. Raf.) rootstock is very prominent in commercial citrus production. The prevalence of this genotype is due to its tolerance to blight, citrus tristeza vir us, plant parasitic nematodes and Phytophthora spp as well as cold tolerance (Stover & Castle 2002). The extensive use of this rootstock in commercial citrus production justified its use in this investigation. All plants were grown and maintained at the CREC in Lake Alfred, FL, USA in a greenhouse at 26 C, and 60 80% RH.

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34 Olfactometer EPN response to D. abbreviatus infested roots was tested with a root zone olfactometer (Analytical Research Systems, Gainesville, FL, USA) according to the design described in Rasmann et al (2005). The olfactometer consists of a central glass chamber (8 cm in diameter and 11 cm deep) attached by 6 side arms to 6 glass pots (5 cm in diameter and 11 cm deep) in which various plants/treatments were tested. The side arms are jo ined to the 6 treatments pots with Teflon connectors fitted with a fine mesh filter impervious to nematodes (2300 mesh, Smallparts, Inc., Miramar, FL). For all tests the olfactometer was filled with sand that had been autoclaved for 1 hr at 250C and then adjusted to 10% moisture (dry wt. sand:water volume; W/V). In tests involving plants, seedlings were given three days to adjust to their sand filled olfactometer for each experiment. In the first experiment, we tested nematode response to weevil infested plants versus non infested controls. Infested plants were subjected to three days of feeding by 3 rd 6 th instar weevil larvae. Non infested plants were not exposed to weevils. Three of the arms of the olfactometer were randomly assigned to a weevil infeste d plant while the remaining three received the non infested control. IJ nematodes (2500) were released into the central olfactometer chamber. Twenty four hours after nematode release, the olfactometer was disassembled and nematodes from each connecting arm were recovered from soil using Baermann extractors; extracted nematodes were collected and counted with a dissection scope. The tests were replicated with ten nematode releases for each treatment. In a second experiment, we compared the response of EPNs t o weevil infested plants with larvae alone in sand. The bioassay consisted of three chambers with plants

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35 infested with six larvae each (as above) and three chambers containing six larvae in sand only. The experimental protocol and sampling procedures were otherwise identical to E xperiment 1. In a third experiment, EPN response was assayed to weevil infested plants (as above) versus mechanically damaged roots. The treatments compared consisted of two mechanically damaged plants, two infested plants, and two sand only control arms. Treatments were randomly assigned to chambers. Plant roots were mechanically damaged by stabbing roots five times daily with a metal corkborer for 3 days prior nematode release (7 mm in diameter). This damage procedure was used bec ause it visually resembled the type of damage inflicted by feeding D. abbreviatus larvae after 72 hr All other experimental and sampling procedures were identical to those described for E xperiment 1 Volatile C ollections The objective of this experiment w as to identify volatiles emitted by citrus roots damaged by weevil larvae. Volatiles were collected from 1) sand alone (negative control), 2) larvae alone in sand, 3) non infested plant roots, and 4) weevil infested roots. Each treatment was prepared withi n a chamber and connecting arm of the 6 chambered olfactometer and filled with the same 10% moistened sand as in the bioassays. Larvae, non infested plants, and infested plants were maintained for three days before sampling. All plants were maintained in t he olfactometer chambers for three days prior to weevil infestation. Thereafter, each chamber of the olfactometer containing a treatment was connected to a vacuum pump (ARS, Gainesville, FL, USA) for 24 hr with a suction flow of 0.8 ml/min. Compounds emitt ed from chambers were collected on adsorbent traps filled with 50 mg Super Q,800 1000 mesh (Alltech Deerfield, IL, USA)

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36 held in glass fittings between the chamber and vacuum pump. Thereafter, Super Q traps were rinsed with 150 L of dichloromethane into in dividual 2.0 m L clear glass vials (Varian, Palo Alto, CA, USA, part number: 392611549 equipped with 500 L glass inserts). GC MS Analysis A 1 L aliquot of each dichloromethane extract was injected onto a GC MS gas chromatograph (HP 6890) equipped with 30 m 0.25 mm ID, 0.25 m film thickness DB 5 capillary column (Quadrex, New Haven, CT, USA), interfaced to a 5973 Mass Selective Detector (Agilent, Palo Alto, CA, USA), in both electron impact and chemical ionization modes. The column was held at 40C for 1 min after inje ction and then programmed at 10 C/min to 260C. The carrier gas used was helium at a flow average velocity of 30 cm/sec. Isobutane was used as the reagent gas for chemical ionization, and the ion source temperature was set at 250C in CI and 220C in EI. EI Spectra library search was performed using a floral scent database compiled at the Department of Chemical Ecology, Gteborg Sweden, the Adams2 terpenoid/natural product library (Allured Corporation, Adams 1995) and the NIST05 library. When available, mass spectra and retention times were compared to that of authentic standards. EPN R esponse to R oot E xtracts The objective of this experiment was to compare EPN response to solvent extracts of citrus roots before and after weevil feeding. Citr us plants were placed individually into chambers of the 6 arm olfactometer for three days as previously described. Thereafter, volatiles were collected from chambers for 24 hr as described above in the volatile collections procedure. Six larvae were then p laced into each

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37 chamber containing a plant and allowed to feed for 3 d ays Thereafter, volatiles were collected a second time from the intact feeding system for 24 hr The adsorbent Super Q traps from both treatments (before and after feeding) were extract ed by rinsing with 150 L of dichloromethane directly after their 24 hr collections as described above. To quantify EPN response to the root extracts collected, a two choice sand filled olfactometer was used ( Figure 2 1 ). The olfactometer consists of three detachable sections: two opposing glass jars ( Figure 2 1A) (16 m L BTL, sample type 111, CLR, SNAPC, Wheaton, Millville, NJ), which contained treatments and a central connecting tube 3cm in length (Blue Max tm 50 m L polypropylene conical tube 30x115 mm, Bec ton Dickinson Labware, Becton Dickinson Company, Franklin Lakes, NJ, USA), with an apical hole into which nematodes were applied ( Figure 2 1B). Extracts were placed on filter paper, which was allowed to dry 30 s for solvent evaporation. Thereafter, filter papers were placed on the bottom of each glass jar ( Figure 2 1C) which were subsequently filled with 10% saturated, sterilized sand as described above. The central chamber connecting the two jars (arms of the olfactometer) was also filled with sterilized a nd moistened sand. The entire olfactometer was 8 cm in length when assembled with two possible extract treatments at opposite ends of the nematode release point. Nematodes (200 IJs) were applied into the central orifice of the connecting tube and given 8 h r to respond. Thereafter, the column was disassembled and the contents of the two collection pots were sampled using Baermann extractors; extracted nematodes were collected and counted. The exper iment was replicated ten times.

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38 Statistical Analysis Paired t tests were used to compare nematode response in experiments testing root extracts in the two choice olfactometers ( df =9). Data from experiments using the six arm olfactometer were analyzed with a log linear model. Given that these data did not conform to simple variance assumptions implied in using the multinomial distribution, quasi likelihood functions were used to compensate for the over dispersion of nematodes within the olfactometer (Turlings et al 2004). The model was fitted by maximum quasi likelih ood estimation in the software package R (R Development Core Team 2004). Results Olfactometer Bioassays Significantly more EPNs were found attracted to D. abbreviatus infested roots than non infested control roots ( F =12.76, df =1, 58, P <0.001) ( Figure 2 2A ). Infested roots attracted significantly more EPNs per arm than those containing larvae alone ( F =13.78, df =1, 58, P <0.001) ( Figure 2 2B). Significantly more EPNs were attracted to D. abbreviatus infested roots than to either mechanically damaged roots or the sand control ( F =12.34, df =2, 57, P <0.001) ( Figure 2 2C). There was no significant attraction to mechanically damaged roots as compared with the sand control ( P =0.34) ( Figure 2 2C). GC MS Analysis pinene were identified in non infe sted and infested plant roots by GC MS (Table 2 1). D. abbreviatus infested roots released four additional unique compounds that were not present in non infested roots (Table 2 1). Two sesquiterpenes were the most abundant and were consistently present in infested roots.

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39 These were geijerene and its precursor pregeijerene ( Figure 2 3). On column GC/MS analyses showed significantly less geijerene and a comparable increase of pregeijerene strongly suggesting a thermal degradation of geijerene to pregeijerene during GC analyses with splitless injection. It is therefore an open question how much geijerene might actually be released by the infested roots. The above six compounds were absent from pots containing larvae alone (Table 2 1). EPN R esponse to R oots E x tr acts Significantly more EPNs were found in arms containing solvent extracts of D. abbreviatus infested roots than non infested roots ( P =0.03) ( Figure 2 4). Discussion Interactions between EPNs and their host insects, competitors and natural enemies are w ell documented, but the degree to which herbivore induced plant signals alter EPN orientation is largely unknown (Duncan et al 2007; Jaffee & Strong 2005). Carbon dioxide has long been known to attract nematodes to plant roots (Prot & Van Gundy 1981; Gaug ler et al 1980). However, functioning alone, such an ambiguous signal might not allow efficient host location by EPNs. Van Tol et al (2001) postulated that plants produce induced compounds that attract EPNs; this hypothesis has been confirmed in two syst ems (Boff et al 2002; Rasmann et al 2005). Furthermore, ( E ) caryophyllene has been identified as the specific EPN recruitment signal emitted by maize roots damaged by corn rootworms (Rasmann et al 2005). The current results indicate that Swingle citru melo rootstock releases herbivore induced response. These sesquiterpenes have not been described for citrus previously; however, they are known for insecticidal, antifeeda nt and oviposition deterrent effects in

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40 leaves of other rutaceous plant species (Kiran et al 2006; Kiran & Devi 2007). Geijerenes have also been described in hairy root cultures of Pimpinella anisum (Santos et al 1998). Although these compounds were cons istently present in infested root samples and are presumed candidate attractants for S. diaprepesi we have yet to confirm the behavioral activity of the individual compounds. Solvent extracts of infested roots attracted S. diaprepesi suggesting that one o r a blend of these compounds may be active. Fractionation studies of the induced compounds via preparative gas chromatography in concert with two choice bioassays of the partitioned profile may enable us to resolve the role of individual compounds on EPN b ehavior. Recent identification of an EPN recruitment chemical is in the initial stages of application for crop protection and has been promising (Turlings & Ton 2006; Degenhardt et al 2003, 2009). Direct application of ( E ) caryophyllene to soil has been shown to reduce rootworm damage through enhanced action of their EPNs (Rasmann et al 2005). Furthermore, recent advances in biochemistry/molecular genetics have made it possible to engineer cultivated maize to release ( E ) caryophyllene to recruit EPNs and protect roots from herbivore damage (Degenhardt et al 2003, 2009; Hiltpold et al 2010). The currently investigated citrus rootstock system is very different from the annual maize cropping system for which EPN recruitment is already being developed fo r corn rootworm management. Perennial systems characterized by fewer disturbances are believed to support more effective biological control than annually disturbed crops (Southwood & Comins 1976). Thus, augmenting the impact of S. diaprepesi in a perennial tree fruit system by application of recruitment chemicals may prove even more effective than in annual crops.

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41 It will also be informative to investigate the parent lines of the Swingle rootstock, Citrus paradisi and Poncirus trifoliata to determine if ei ther or both lines exhibit the herbivore induced EPN recruitment seen in the hybrid. Furthermore, we plan to investigate if other non citrus hosts of D. abbreviatus release induced recruitment signals. Given the wide host range of D. abbreviatus, it will b e important to determine the breath of this EPN recruitment response among its diverse host plants. Several nematode species attack D. abbreviatus. Steinernema glaseri S. carpocapsae and Heterorhabditis bacteriophora were initially investigated as possib le control agents (Downing et al 1991; Schroeder 1992). Of the species evaluated in laboratory bioassays and greenhouse trails, S. riobrave and a Florida isolate of H. indica were the most effective (Shapiro Ilan & McCoy 2000). Currently, S. riobrave and H. indica are formulated for commercial application against D. abbreviatus in Florida citrus. These two EPN species, in addition to S. diaprepesi will be evaluated and compared in similar future studies to determine whether the tentatively identified EPN recruitment signals are specific to the natively occurring EPN associated with the weevil or whether these signals function more broadly for other EPN species. We also report here for the first time an in situ method for sampling subterranean herbivore in duced volatiles during real time insect feeding. Previously used methods involve freeze drying and crushing root samples (Rasmann et al 2005), which will affect and badly represent volatile production from intact roots. The currently described method allo ws identification of belowground volatiles as they are released over time without disturbance to the system.

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42 The current results indicate that a commercially used citrus rootstock emits induced volatile chemicals in response to herbivore feeding that attra ct beneficial nematodes. Identification of the specific active compounds may lead to the development of an augmentive EPN recruitment tactic that improves biological control of D. abbreviatus Also, such identification would be the first step towards devel opment of genetically engineered citrus rootstocks for enhanced recruitment of EPNs. Alternatively, it is possible that engineering plants for increased release of terpenes in general may prove effective (Schnee et al 2006).

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43 Table 2 1. GC MS identificati on of volatiles from Swingle citrumelo rootstock (Citrus paradise Poncirus trifoliate 1 Synthetic standard comparison. 2 Indentification was based on comparisons of retention times with stand ard and spectral data from Adams, EPA, and Nist05 Libraries. Infested root Non infested root Larvae only Peak # RT Name CAS# Presence 1 7.50 pinene 1,2 000080 56 8 + + 2 8.08 pinene 1,2 000127 91 3 + + 3 10.81 Gei jerene 2 006902 73 4 + 4 12.93 Pregeijerene 2 020082 17 1 + 5 14.75 Santalene 2 000512 61 8 + 6 14.93 Z Bergamotene 2 018252 46 5 +

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44 Figure 2 1 Schematic diagram of sand column assay unit. Glass jar (17 ml) with samples at base (A), connecting tube (3 cm) with hole for nematode application (B), extracts placed on filter pa per (C), arena was filled with heat sterilized sand at 10% moisture for all assays.

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45 Figure 2 2 Mean number of S. diaprepesi attracted to chambers containing weevil infested plants versus non infested control plants (A), weevil infested plants versus l arvae alone (B), weevil infested plants, mechanically damaged plants or sand control (C). Each panel represents a separate experiment (n=10) conducted in a 6 arm olfactometer.

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46 Figure 2 3 Example chromatograms showing volatile profiles of D. abbrevia tus infested plants, non infested plants and larvae alone. Volatile profile of infested Citrus paradise Poncirus trifoliate rootstock (A) Volatile profile of non infested Citrus paradise Poncirus trifoliate rootstock (B) Volatile profile of D. abbrevia tus alone in sand (C). All samples were collected for a 24 hr Geijerene (3), Pregeijerene (4), Z Bergamotene (6). (Compound numbers correspond to Table 2 1).

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47 Fig ure 2 4 Mean number of nematodes attracted to volatiles from D. abbre viatus infested roots compared with volatiles from undamaged roots

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48 CHAPTER 3 CONSTITUTIVE AND IND UCED SUBTERRANEAN PL ANT VOLATILES ATTRAC T BOTH ENTOMOPATHOGENI C AND PLANT PARASITI C NEMATODES Plant insect predator (parasite) interactions are often describ ed in the above ground terrestrial environment. However, analogous below ground plant herbivore interactions should also be considered (van Dam 2009). General understanding of plant communication has greatly improved since early insights into plant insect mutualisms, which arise as plants meet their reproductive requirements (Erlich & Raven 1964). Our understanding of the impact that herbivore induced plant volatiles (HIPVs) have on the tertiary trophic level continues to increase and is expanding beyond th e general understanding that HIPVs attract predators (Turlings et al 1990; Heil 2008; Dicke & Baldwin 2010). Above ground plant defense by HIPV signalling is now considered a common and broadly understood phenomenon (Agrawal & Rutter 1998; Agrawal & Karban 1999; Baldwin & Preston 1999; Dicke et al 2003; Turlings & Wckers 2004). Herbivore induced plant volatiles are often only released after herbivore feeding. For instance, lima bean plants ( Phaseolus lunatus ) release volatiles when infested with spider mi tes ( Tetranychus urticae ), which attract the predatory mite Phytoseiulus persimilis (Dicke & Sabelis 1988). It is known that compounds associated with the feeding insect can mediate such plant response (Alborn et al 1997). Most known are volicitin and oth er fatty acid amides, found in oral secretions of herbivores, which induce volatile production in plants (Alborn et al 2000; Turlings et al 2000). Over the past decade the role of subterranean release of HIPVs and their indirect impact on plant defense has become increasingly evident (van Tol et al 2001; Aratchige et al 2004; Rasmann et al 2005; Ali et al 2010). Below ground herbivory likely imparts significant selection pressure for evolution of induced plant responses

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49 (Blossey & Hunt Joshi 2003). In plant herbivore systems in which the lifecycle of the herbivore is partitioned between above and below ground plant zones, a unique opportunity exists for investigating plant defense s in both above ground and subterranean environments in response to da mage from a single herbivore species. Furthermore, investigations of HIPV release with perennial, cultivated plant species allow insights into the evolution of responses in both naturally occurring and artificially selected genotypes (Kllner et al 2008; Degenhardt et al 2009). Larvae of the weevil Diaprepes abbreviatus feed on the roots of more than 290 plant species including citrus, sugarcane, potatoes, strawberries, woody field grown ornamentals, sweet potatoes, papaya, guava, mahogany, ornamentals, a nd non cultivated wild plants (Simpson et al 1996). Diaprepes abbreviatus was first introduced to Florida in 1964 (Woodruff 1964). Over the past 40 years, it has contributed significantly to the spread of disease and damage (Weissling et al 2002). Above ground, D. abbreviatus damages the vegetative portion of plants by notching young leaves (Fennah 1940). Mature adults lay eggs between older leaves and emerging first instars drop to the soil where they develop and feed on roots causing the most severe dam age to plants (Fennah 1940; Schroeder 1992). Entomopathogenic nematodes (EPNs) from the genera Heterorhabditis or Steinernema (Downing et al. 1991; Schroeder 1992) are known to infect this insect (McCoy et al 2000). Entomopathogenic nematodes are parasito ids that kill their host with the aid of a symbiotic bacterium (Poinar 1990). Citrus paradisi Poncirus trifoliata ) fed upon by D. abbreviatus attract entomopathogenic

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50 nematodes ( S. dia prepesi ) (Ali et al 2010). We found that weevil infested roots release volatile compounds not found in undamaged roots and suggested this to be an indirect defense associated with attraction of beneficial nematodes. Of the four main compounds released by damaged roots, the C 12 terpenes pregeijerene and its breakdown product, geijerene, were the main two volatiles potentially associated with attraction of beneficial nematodes, and preliminary research supports the hypothesis that in this system the geijere nes are the major nematode attractants (unpublished). commonly used due to its resistance to diseases, plant parasitic nematodes and adverse environmental conditions (St over & Castle 2002). The question therefore arose how broadly release of nematode attracting cues occurs among various citrus varieties. Diaprepes abbreviatus is the main root weevil species affecting citrus and thus the major interest of our present rese arch. However, a complex of related insect species also attack citrus roots (Duncan et al 1999), thus nematode attraction may have broad significance for citrus defense Therefore, i n addition to determining the extent of nematode attraction among various citrus varieties, we also investigated the breadth of responsiveness among several ent omopathogenic nematode species. Entomopathogenic nematodes can be categorized according to their foraging and wait) versus ide search radius) strategies are generally considered as dipoles of a continuum of salutatory search tactics (Lewis et al. 1992, 1993; Grewal et al 1994). Cruisers allocate more of their time scanning for resource associated cues as they move through the ir environment, exhibiting only brief pauses, and are therefore more effective at finding sedentary and cryptic hosts (Lewis

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51 et al 1995; Lewis et al 2006). In contrast, ambush foragers scan during long pauses and allocate less time to active movement thr ough their environment (Campbell & Gaugler 1993). They are thought to wait for resources to come to them, increasing effectiveness of finding highly mobile prey. Steinernema carpocapsae (nictating species) is a representative ambush type EPN, while H. indi ca (non nictating) is a typical cruise type EPN (Lewis 2002). Steinernema diaprepesi is a recently discovered D. abbreviatus and is considered intermediate on the spectrum betw een ambushers and cruisers (Nguyen & Duncan 2002). Finally, Steinernema riobrave was discovered in Texas and it is also considered intermediate with respect to foraging strategy (Cabanillas et al. 1994). In addition to investigating the above EPN species, we also included a plant parasitic species as a trophic level outgroup. The citrus nematode, Tylenchulus semipenetrans is one of the most significant parasites of plants reducing citrus yield by 6 12% worldwide In Flo rida, it is estimated to affect 25 % of described citrus species ( Esser et al. 1991 ). The life cycle of T. semipenetrans consists of an egg and four larval stages followed by a sexually reproducing adult stage. Second stage larvae are the infective juveniles that infest citrus roots. This lar val stage penetrates deeply into feeder root cortical tissues, where they become immobile, establishing permanent, specialized feeding sites within the root (Munn & Munn 2002). Second stage larvae moult three times, increasing in size with each moult to fo rm large, posteriorly swollen females capable of depositing ca. 75 500 eggs per female (Munn & Munn 2002).

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52 Above ground plant stress elicits defensive responses in both above and below ground tissues (Kaplan et al 2008a, b; Erb et al 2008; van Dam 2009 ; van Dam & Heil this issue). Additionally, many studies have found an increase in the levels of shoot defense s following root herbivory (Bezemer et al 2004; van Dam, Raaijmakers & van der Putten 2005; Soler et al 2005). Analogously, levels of root defen se s can be affected by shoot herbivory (Soler et al 2007; Tiwari et al 2010; Erb et al 2011 ). Above ground below ground cascades of plant defense can be reciprocally beneficial or detrimental between plant shoots and roots (van Dam & Heil 2011 ). However it was unclear in our system whether above ground stress induced an associated below ground response to root feeding or vice versa Therefore, we investigated if release of nematode attracting cues is a localized root response or whether it is also media ted by shoot herbivory. Our on going investigations of herbivore induced nematode attraction using citrus as a study system have addressed the breadth of this response among various citrus species as well as the breadth of responsiveness to the plant prod uced cues by a diversity of nematode species. Additionally, the current investigation explored whether releasing a plant volatile that could potentially attract beneficial parasitoids of insect herbivores was associated with ecological cost of attracting p lant pathogens. Our findings suggest that a species and hybrid line more vulnerable to phytopathogenic nematodes can reduce the associated costs by emitting nematode attracting volatiles only when it is necessary, that is, when roots are attacked by herbiv ores. In contrast, a species that is not susceptible to root parasites produces these cues constantly, investing more into constitutive defense

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53 Materials and Methods Insects Diaprepes abbreviatus larvae were obtained from a culture maintained at Universit FL, USA. This culture was periodically supplemented from a larger culture maintained at the Division of Plant Industry Sterile Fly Facility in Gainesville, FL, USA. Larvae were rear ed on a commercially prepared diet (Bio Serv, Inc., Frenchtown, NJ) as described in Beavers (1982) using procedures described by Lapointe & Shapiro (1999). Larvae used in experiments were from third to sixth instars. Female adults were used two weeks after emergence. Nematodes Nematode foraging strategy and trophic level status are summarized in Table 3 1. The entomopathogenic nematodes, S. diaprepesi S. riobrave S. carpocapsae and H. indica were isolated from D. abbreviatus larvae buried in commercial ci trus orchards in Florida. Steinernema riobrave and S. carpocapsae isolates were descendants of commercial formulations intended for field application to manage D. abbreviatus All EPN species were cultured in last instar larvae of the greater wax moth, Gal leria mellonella at approximately 25 C according to procedures described in Kaya & Stock (1997). Infective juveniles (IJs) that emerged from insect cadavers into White traps (White 1927) were stored in shallow water in transfer flasks at 15 C for up to 2 weeks prior to use. Tylenchulus semipenetrans were obtained from infected field grown citrus. Infected roots and surrounding soil were soaked and IJ nematodes were subsequently extracted via sieving and centrifugation flotation (Southey 1986).

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54 Plants All plants were grown and maintained at the CREC in Lake Alfred, FL, USA, in a greenhouse at 26 C, and 60 80% relative humidity. Poncirus trifoliata is a common rootstock for commercial production of oranges, grapefruit, most mandarins and lemons. Its prevalen ce is based on advantages such as resistance to Phytophthora fungi, T. semipenetrans citrus tristeza virus, as well as cold tolerance and high fruit quality (Stover & Castle 2002). A major drawback is its slow growth (Stover & Castle 2002). It is typicall y hybridized to blend its desirable qualities with the faster growth of other varieties (Gardner & Horanic 1967). Swingle citrumelo, C. paradisi P. trifoliata rootstock is one of these hybrids and is very prominent in commercial citrus production (Hutch inson 1974; Stover & Castle 2002). Sour orange, Citrus aurantium, is one of the oldest and most common rootstocks used for commercially grown citrus (Stover & Castle 2002). However, its susceptibility to tristeza virus and T. semipenetrans has decreased it s prevalence in the past decade (Stover & Castle 2002) These three rootstocks were chosen in an effort to determine the breadth of nematode attraction among diverse citrus varieties with and without hybridization. Nematode B ehavior The behavioural respons es of nematodes to collected root samples were quantified in a two choice sand filled olfactometer described thoroughly by Ali et al (2010). Briefly, the olfactometer consists of three detachable sections: two opposing 16 mL glass jars which contained tr eatments and a central connecting tube 3 cm in length with an apical hole into which nematodes were applied (Ali et al 2010). For each plant species, root volatiles were collected and extracted from the collection filters according to the methods describe d by Ali et al (2010). An adsorbent trap was connected to the

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55 bottom opening of the glass root zone chamber; treatments were non destructively sampled with a vacuum connected to the adsorbent trap that pulled air from the chamber. Trap extracts from infes ted and non infested roots were placed on filter paper, which was allowed to dry 30 s for solvent evaporation. Thereafter, filter papers were placed on the bottom of each glass jar, which were subsequently filled with 10% saturated (dry wt. sand: water vol ume; W/V), sterilized sand (Ali et al 2010). The central chamber connecting the two arms of the olfactometer was also filled with sterilized and moistened sand. Nematodes ( c. 200 IJs) were applied into the central orifice of the connecting tube and given 8 hr to respond. Following the incubation period, the column was disassembled and the nematodes from the 2 collection jars were extracted using Baermann Funnels. The experiment was replicated ten times for each nematode species and plant rootstock combinat ion. The control treatment for each nematode species consisted of solvent blanks placed in each arm of the olfactometer. This double blank treatment produced identical results for each nematode species (no response), and thus a mean for all nematode specie s examined is reported for this treatment. Above versus B elow ground V olatile C ollections By simultaneous collection of root and shoot volatiles using a headspace guillotine chamber coupled with a root zone collection chamber ( Figure 3 1) we examined whet her adult feeding on Swingle shoots induce a nematode attracting plant root response analogous to that observed in response to root damage by larvae. Similarly, we investigated if typical induced root volatiles were released above ground in response to roo t damage by larvae. Plants were initially placed in glass root zone chambers (ARS, Gainesville, FL, USA) filled with sand that had been autoclaved for 1 hr at 250 C and then adjusted to 10% moisture as described in Ali et al (2010). The chambers and

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56 plant s were placed below a platform on which a Teflon guillotine was attached ( Figure 3 1). The shoots of the plant passed through the guillotine opening and Teflon slides were positioned at the base to seal off the upper portions of the plant from the root zon e. A glass chamber (ARS, Gainesville, FL, USA) was then placed on the Teflon platform containing all upper portions of the exposed plant. Charcoal purified and humidified air was drawn over plants and pulled out at a rate of 300 mL min 1 through a trap con taining 50 mg of Super Q adsorbent (Alltech Assoc., Deerfield, Illinois, USA). Volatiles were collected for 24 hr after which Super dichloromethane into individual 2.0 mL clear glass vials as described above. Volatiles fr om both roots and shoots of plants were initially sampled three days after preparation to determine baseline volatile production. On day four, plants were infested with either six larvae at the root zone or six female adults were placed on leaves above gro und. The below and above ground chambers of each infestation type were simultaneously sampled for three subsequent days after infestation. Beetle feeding was easily noticeable in damaged leaves above ground and was visually confirmed on roots after the fe eding interval (Ali et al 2010). Each infestation treatment was replicated 5 times. Volatile C ollection from I nfested versus N on infested P lants The objective of this experiment was to compare volatile release by roots of P. trifoliata and Sour orange ( C. aurantium ) that were damaged by D. abbreviatus feeding or left undamaged. Plants were potted in sand filled glass root zone chambers as previously described. Seedlings were given 3 days to adjust to their sand filled chambers. Infested plants were subject ed to an additional 3 days of feeding by weevil larvae. Non infested plants were not exposed to weevils during this period. Thereafter,

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57 each root zone chamber was connected to a vacuum pump (ARS, Gainesville, FL, USA) for 24 hr with a suction flow of 80 mL min 1 (Ali et al 2010). Compounds emitted from chambers were collected on adsorbent traps filled with 50 mg Super Q, (800 1000 mesh, Alltech Deerfield, IL, USA) held in glass fittings between the chamber and vacuum pump (Ali, Alborn & Stelinski 2010). Th ereafter, Super Q traps were rinsed with mL clear glass vials (Varian, Palo Alto, CA, USA, part number: 392611549 equipped with 500 et al 2010). GC MS A nalysis All samples were injected as 1 chromatograph (HP 6890) equipped with 30 m length 0.25 mm internal diameter, 0.25 1 capillary column (Quadrex, New Haven, CT, USA), interfaced to a 5973 Mass Selective Detector (Agilent, Palo Alto, CA, USA), in both electron impact and chemical ionization modes. Samples were introduced eithe r by splitless injection at 220 C or by cold on column injection. In the second case, a 1 m fused silica deactivated retention gap was added between injector and analytical column and the injector was programmed to follow the oven tempera ture. The column was held at 40 C for 1 min after injection and then programmed f or a temperature increase of 10 C min 1 to 260 C. The carrier gas used was hel ium at an average flow velocity of 30 cm s 1 Isobutane was used as the reagent gas for chemical ionization, and the ion so urce temperature was set at 250 C in chemical ionization (CI) and 220C in electric ionization (EI). Electric ionization spectra libr ary search was performed using a floral scent database compiled at the Department of Chemical Ecology, Gteborg Sweden, the Adams2 terpenoid/natural product library (Allured Corporation, Adams

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58 1995) and the NIST05 library. When available, mass spectra and retention times were compared to those of authentic standards. Statistical A nalysis Nematode response investigated in the two choice bioassay chambers was analysed with a two factor analysis of variance (ANOVA) with root extract treatment and nematode spec ies comprising the two factors. Where ANOVA showed significant in the software package R (R Development Core Team 2004). Given that a lack of response to the double blank control occurred consistently for each nematode species tested, the responses of each species were pooled for this treatment. Results Nematode B ehavior Entomopathogenic nematodes of all species responded similarly either arms of the blank negative control (F = 3.0, df = 2, 72, P = 0.087) ( Figure 3 2A). However, when D. abbreviatus infested and uninfested P. trifoliata roots were tested, most nematode species preferred either root treatment over the blank control (F=35.66, df = 2,129, P <0.001). The only exc eption was the ambush forager type S. carpocapsae ( P = 0.134) ( Figure 3 2A). All tested nematode species preferred Swingle plants infested with D. abbreviatus larvae over the paired uninfested controls ( P < 0.001) ( Figure 3 2B). In addition, movement of S. diaprepesi in response to D. abbreviatus infested Swingle rootstocks was significantly greater than that observed for the other nematode species tested ( P < 0.001) ( Figure 3 2B).

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59 Effect of B elow versus A bove ground H erbivory on R elease of N ematode A ttrac tants Feeding by D. abbreviatus larvae on citrus roots induced production of pregeijerene in the subterranean root zone; however, no pregeijerene or related compounds were found in the volatile collections of above ground shoots in response to larval feedi ng ( Figure 3 3A). Conversion of pregeijerene to geijerene was found to be an artefact of heat exposure in a splitless GC injector and thus the total production of pregeijerene in response to herbivory turned out to be the combination of the observed pregei jerene and geijerene peaks ( Figure 3 3A). These C 12 terpenes are thought to elicit nematode attraction (Ali et al 2010). Adult beetle feeding on above ground shoots did not induce production of pregeijerene or other volatiles typically released in respons e to root damage ( Figure 3 3B); however, release of limonene from above ground shoots was increased ( Figure 3 3B). Subteranean R elease of V olatiles by V arious P lant S pecies Pregeijerene was released constitutively by P. trifoliata roots and the release was not affected by larval D. abbreviatus feeding ( Figure 3 4A). In contrast, pregeijerene was released by Swingle roots (Table 3 2 and Ali et al 2010) and Sour Orange rootstocks ( Figure 3 4B) only in response to D. abbreviatus larval feeding ( Figure 3 4B, Tab le 3 2). Discussion The rhizosphere within which nematodes forage to find resources has been the subject of investigation for several decades. Nematode host searching behaviour is typically mediated by cues from host(s) or their immediate environment (Lewi s et al 2006) that can be either volatile and diffuse through soil or dissolved in and moving through the

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60 water film surrounding soil particles. Cues emanating from plant roots, a necessary habitat for many insect hosts, can also influence the behaviour o f EPN nematodes (Bird & Bird 1986; Choo et al 1989; Lei et al. 1992; van Tol et al 2001; Boff, van Tol & Smits 2002; Neveu et al 2002). In addition to organic compounds, environmental factors such as temperature, substrate vibrations, electric potential carbon dioxide and various inorganic compounds can mediate the behaviour of nematodes as they search for hosts (Jansson & Nordbringhertz 1979; Torr et al 2004). Until recently, little was known about EPN chemotaxis in response to herbivore induced cues (Rasmann et al 2005; Hiltpolt et al 2010; Ali et al 2010). However, herbivore feeding triggers production of EPN attracting volatiles in annual grasses (Rasmann et al 2005) and recently, we showed EPNs ( S. diaprepesi ) in response to herbivory by larval D. abbreviatus root weevils and that the attraction was due to an induced release of subterranean volatiles (Ali et al 2010). In both cases, the nematode attractants appear to be terpenoids. We dete rmined that in response to herbivory, the Swingle hybrid, as well as another common non hybridized species, sour orange ( C. aurantium ), produced pregeijerene, the proposed nematode attractant. Surprisingly, we found that one of the parents of the Swingle h ybrid, P. trifoliata attracted nematodes independent of herbivory and that this could be explained by constant release of pregeijerene. Thus, our observations show pregeijerene can be produced constitutively as well as in response to damage among diverse citrus varieties. I t is possible that plant breeding to develop the cultivable Swingle hybrid may have created an herbivore induced response similar to that observed with the non hybridized sour orange ( C. aurantium ) species by

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61 loss of the trait responsibl e for constant signalling observed in one of its parents A similar genetic consequence was observed in maize, where a below ground cue found in wild relatives and European lines was lost during the breeding of North American maize lines (Kllner et al 20 08). W e intend to utilize microarray analysis to resolve gene regulation in response to herbivory among these different citrus varieties. Our results indicate that all EPN species tested exhibited attraction to herbivore induced volatiles irrespective of their foraging strategy (Fig ure 3 3 ). Specifically, the S. carpacapse H. indica (Lewis 2002), as well as the two species thought to exhibit an intermediate behavioural foraging strategy (Lewis et al 1992; Lewis 2002) were all attracted to D. abbreviatus damaged roots of the Swingle rootstock. Analogously, the Swingle parent line, P. trifoliata also attracted nematodes of all species (except for S. carpocapse, ambusher) independent of damage ( Figure 3 2A). Thus, these results support the hypothesis that pregeijerene likely explains this attraction. Of the EPN species investigated, S. diaprepesi exhibited the greatest behavioural response even though this species is thought to be intermediate on the spectrum between pur versus S. diaprepesi is a n endemic species and may have considerable advantages in attacking D. abbreviatus weevils (Nguyen & Duncan 2002) and thus it appears that specialization rather than foraging strategy may better exp Steinernema carpocapsae (ambusher) is a less effective entomopathogen of D. abbreviatus (Schroeder 1994; Duncan et al. 1996; Bullock et al 1999) than S. riobrave (intermediate between ambusher and cruiser) (L ewis 2002). It is thought that active and

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62 explain this difference (Grewal et al 1994; Lewis et al 1995). Nematode attraction to damaged citrus root chemicals in the current in vestigation appeared to differ based on foraging strategy. Our results are congruent with the proposed foraging strategy behaviours of the nematode species tested, similarly to that observed for other EPN ambushing species investigated ( S. carpocapsae) did not move in the olfactometer when pregeijerene was ubiquitous and coming from each possible direction of movement ( Figure 3 2 A ); however, it did respond when the cue was present in only one of the two arm s ( Figure 3 2B ). In contrast, the cruising and intermediate foraging strategy species always responded to these volatiles, whether they were in one or both arms of the two choice test chamber (Figs. 3 2, 3 3). To date, investigations of nematode response t o below ground volatiles have focused on entomopathogens (Lewis et al 1993; Lewis, Grewal & Gaugler 1995; Rasmann et al 2005; Hiltpolt et al 2010; Ali et al 2010). Entomopathogenic nematode host finding is mediated by both long range cues that facilita te finding of the root zone as well as shorter range cues that facilitate host location within the root zone (Choo & Kaya 1991; Kanagy & Kaya, 1996; Hui & Webster 2000; van Tol et al 2001; Rasmann et al 2005). The attraction of plant parasitic nematodes to below ground HIPVs was hitherto unknown. It is generally accepted that plant roots release various attractants that mediate response by the infective stages of plant parasitic nematodes (Prot 1980). A variety of physio chemical gradients exist around ph ysiologically active roots including amino acids, ions, pH, carbon dioxide and sugars (Perry & Aummann 1998). However, little is understood regarding the specific cues that mediate attraction of plant parasitic

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63 nematodes to preferred feeding sites. Our res ults suggest that plant parasitic nematodes are attracted to specific roots volatiles, whose production is in some cases enhanced by herbivore damage. These root specific volatiles may facilitate host finding among opportunistic plant parasitic nematodes t hat likely use a multitude of cues to locate feeding sites. It is puzzling that the parental P. trifoliata line of the commercial Swingle rootstock constantly produced and released attractants for beneficial nematodes that also were utilized by plant para sitic nematodes. Selection for an herbivore induced signalling response should be strongest in the direction toward channelling resources caries a high physiological c ost (Zangerl & Rutledge 1996; Agrawal & Karban 1999; Karban et al 1999; Strauss et al 2002; Heil 2002; van Dam 2009). However, constant release of volatiles that attracted EPN species appeared to carry the ecological cost of also attracting a plant patho genic species, Therefore, it is less surprising that the faster growing Swingle commercial hybrid only released this cue upon herbivory. However, the apparent correlation between defense and growth rate needs to be carefully tested. The current laboratory based investigation did not resolve the many potential competitive interactions between beneficial and parasitic nematodes and with their natural enemies that might occur in the field (Jansson & Nordbringhertz 1979). Costs for P. trifoliata resistance to T semipenetrans infection require further evaluation. Exploitation of plant defense constitutive or induced. Citrus aurantium is highly susceptible to T. semipenetrans infection. Ther efore an induced response may have been selected for in this species

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64 given the associated ecological costs of attracting potential parasites. Costs of defense s are well known above ground (Puustinen et al 2004; Adler & Irwin 2005). Our results are consist ent with the notion that defense s against diverse enemies may evolve independently but not without associated direct ecological costs in terms of reduced vigor and/or increased susceptibility to different threats and situations (Heil 2002; van Dam & Heil 2 011 ). Our results suggest that these terpenoid volatiles cannot be easily categorized as synomones (mutually beneficial) as was previously thought (Ali et al 2010). It appears that in citrus, they might function as both kairomones (disadvantageous to its emitter, beneficial to its receiver) and synomones, depending on the trophic context. Resolution of their total impact on plant defense may serve a number of additional functions. Potential antibiotic effects and plant microbe signalling were not investigated here. Depending on the nematode fauna in a particular location, the beneficial effect of attracting entomophathogens may be negated by concurrent attraction of plant parasites. This complex interaction oc curring within the citrus system will need to be investigated in a field setting and also deserves further investigation in other below ground systems which attempt to categorize plant volatiles. As observed previously compounds that are characterized for defensive roles can also render plants more attractive to specialist herbivores (Dicke & van Loon 2000; Heil 2008) Although distinct, the shoots and roots of plants act synergistically using primary resources from both above and below ground plant organs to produce organic matter. These ecologically valuable plant products are constantly threatened by primary

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65 consumers. Plants have thus developed numerous strategies to withstand the impacts of herbivores, pathogens and parasites. For several decades there has been an emphasis on the above ground mechanisms of plant defense (Zangerl & Bazzaz 1992; Howe & Jander 2008). However, the synergy between below and above ground organs associated with plant growth is likely paralleled by interactions that contribute to plant defense (Masters & Brown 1992; Bezemer et al 2004; Bezemer & van Dam 2005; Erb et al 2009). Roots synthesize a number of secondary metabolites that are known leaf defense s, including furocoumarins, alkaloids, terpenoids, aldehydes and nicotine (Erb et al 2009). Until recently, pregeijerene had only been detected in herbivore damaged roots of Swingle citrus (Ali et al 2010). In the current investigation, we simultaneously sampled volatiles from the above and below ground appendages of plants w hile they were actively damaged at the root or shoot zone by different stages of the same holometabolous insect herbivore. Pregeijerene was only released by roots in response to below ground herbivory by D. abbreviatus larvae ( Figure 3 3A). Neither roots n or foliage released this putative nematode attractant upon above ground herbivory by adult beetles ( Figure 3 3B). Although our results indicate that the major constituent of nematode attraction is unique to the below ground portions of the plant, it remain s possible that correlations exist between above ground and below ground herbivory in this system. In the current investigation, we did not address attraction of above ground natural enemies of D. abbreviatus adults in response to below ground or above gro und herbivory. However, our results suggest an above ground HIPV release in response to adult beetle feeding (i.e. increased production o f limonene from leaves ( Figure 3 3B ), which deserves further investigation.

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66 With respect to the influence of above gro und herbivory on below ground plant defense we hypothesized that adult beetle feeding may induce production of an EPN instar larvae drop and burrow into the soil, we pos tulated that it would be advantageous for the plant to attract a community of entomopathogens as herbivore larvae are dropping to the soil and before they have established active feeding sites on roots. Our results provide no evidence in support of such a priming hypothesis based on induction of nematode attracting cues as the attractants were only induced by below ground herbivory. Yet, it is established that other responses in roots could be primed during above ground herbivory which could facilitate defe nse (Rasmann & Turlings 2007; Erb et al 2008; van Dam 2009; Erb et al 2011 ). It may be possible that defense is augmented via above ground feeding, either directly by a build up of defensive compounds in the roots or indirectly by an increased release ra te of defensive cues, both of which require further investigation. We provide evidence that nematode attracting cues are released by a diversity of citrus species. These cues can be released constantly or only in response to herbivore damage. A diversity o f nematode species were attracted to these cues including entomopathogens and plant parasites. It seems that these nematode attractants have host specialization appeared to play a more imp ortant role than foraging strategy in terms of efficiency of chemotaxis in response to these cues. The surprisingly similar response of a plant parasitic species to that of several entomopathogens suggests that these cues cannot be easily categorized as ei ther kairomones or synomones. It seems the citrus spp. more

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67 vulnerable to phytopathogentic nematodes reduce related costs by emitting nematode attracting volatiles only when it is crucial, that is, when herbivores are feeding. In contrast, non susceptible species invest more in constitutive defense given the lack of cost associated with attracting pathogens. This hypothesis warrants further investigation, in a context that measures the associated cost of producing this attracting cue.

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68 Table 3 1 Trophic l evel, foraging strategy and ecological status of nematodes tested Nematode spp. Trophic Level Foraging Strategy Ecological Status Steinernema diaprepesi Entomopathogen Intermediate Indigenous to Florida S. carpocapsae Entomopathogen Ambush Comm ercially introduced S. riobrave Entomopathogen Intermediate Commercially introduced Heterorhabditis indica Entomopathogen Cruiser Commercially applied; indigenous to Florida Tylenchulus semipenetrans Plant parasite Sedentary root endoparasite Ag ricultural pest; citrus parasite

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69 Table 3 2 GC MS identification of volatiles from various citrus rootstocks Swingle (Citrus paradisi Poncirus trifoliata) Poncirus (Poncirus trifoliata) Sour Orange (Citrus aurantium) RT Names CAS# Infe sted Non infested Infested Non infested Infested Non infested 7.25 pinene a,b 000080 56 8 + + + + 7.90 pinene a,b 000127 91 3 + + + + 8.69 Limonene a,b 000138 86 3 12.94 Geijerene b 006902 73 4 + + + + 10.81 Pregeijerene b 020082 17 1 + + + + a Synthetic standard comparison. b Identification was based on comparisons of retention times (RT)with standard and spectral data from Adams, EPA and Nist05 Libraries

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70 Figure 3 1 Schematic diagram of simultaneous above and below ground volatile collection apparatus (ARS, Gainesville, FL, USA). The guillotine volatile collection chambers used for above ground collections received a constant flow of charcoal purified and humidified air, which was suctioned at a rate of 300 mL min 1 through a trap containing 50 mg of Su per Q adsorbent (Alltech Assoc., Deerfield, Illinois, USA). Root zone collection chambers used to collect below ground volatiles were filled with heat sterilized sand standardized at 10 % saturation.

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71 Figure 3 2 Responses of Tylenchulus semipenetrans, Steinernema carpocapse, S. riobrave, S. diaprepesi, and Heterorhabditis indica when presented with: A) V olatiles from roots of Poncirus trifoliata infested with Diaprepes abbreviatus lavare vs. volatiles from undamaged P. trifoliata roots or B) V olatiles f rom roots of Citrus paradisi P. trifoliata (Swingle hybrid) infested with D. abbreviatus larvae vs volatiles from undamaged C. paradisi P. trifoliata roots in two choice olfactometer.

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72 Figure 3 3 Example chromatograms depicting volatile profiles fr om simultaneous collections of root and shoot volatiles of Swingle ( Citrus paradisi Poncirus trifoliata ) in response to A) B elow ground and B) A bove ground herbivory by Diaprepes abbrevatus larvae and adults, respectively. All samples were collected for 24 h r

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73 Figure 3 4 Example chromatogram showing volatile profiles from roots of A) Poncirus trifoliata or B) Sour orange ( Citrus aurantium ) in response to Diaprepes abbrevatus herbivory upon roots or undamaged controls. All samples were collected for 2 4 h r

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74 CHAPTER 4 MANIPULATING NATIVE POPULATIONS OF ENTOM OPATHOGENIC NEMATODE S WITH HERBIVORE INDUC ED PLANT VOLATILES T O ENHANCE PEST CONTR OL Natural enemies of herbivores use flexible foraging strategies that often incorporate environmental cues from the signals that mediate interactions between herbivore damaged plants and species within the third trophic level are well documented (e.g. Turlings et al 1990; Takabayashi & Dicke 1996; Tumlinson et al 1993; Turl ings & Wackers 2004 ). However, it has recently been shown that subterranean defenses also mediate HIPVs and reduce herbivore performance and population densities ( De Moraes et al 1998; Thaler 1999; Kessler & Baldwin 2001, 2004 ) by attracting natural enemi es of the herbivore. Thus below ground induced defense might be as complex and important as above ground induced plant defense ( van Tol et al. 2001 ; Rasmann et al 2005 ; Hiltpold et al 2010; Ali et al 2010 ; 2011 ). Furthermore, it could be utilized in agroecosystems to enhance the effectiveness of natural enemies ( Pickett & Poppy 2001; Degenhardt et al 2003; Aharoni et al 2005; Turlings & Ton 2006 ) as has been demonstrated for maize ( Rasmann et al 2005 ). The disparity in the number of aboveground in vestigations versus analogous belowground research on indirect defense is largely due to technical limitations rather than lack of interest or relevance ( Hunter 2001 ; Rasmann & Agrawal 2008 ). Larvae of the weevil Diaprepes abbreviatus (L), that was first f ound in Florida in 1964 ( Beaver & Selhime 1978 ), feed on the roots of more than 290 plant species including citrus, sugarcane, potatoes, strawberries, woody field grown ornamentals, sweet potatoes, papaya, guava, mahogany, containerized ornamentals, and no n cultivated wild plants ( Simpson et al 1996 ). Over the past 40 years, it has contributed

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75 significantly to the spread of disease and agricultural damage ( Graham et al 2002 ). Pesticide applications are expensive, environmentally hazardous and often ineffe ctive ( Bullock et al 1999; Duncan et al 1999 ). Currently, the most effective method for controlling D. abbreviatus is with application of commercially formulated entomopathogenic nematodes (EPNs) from the genera Heterorhabditis and Steinernema ( Downing e t al 1991; Schroeder 1994 ). EPNs are obligate parasites that kill their host with the aid of a symbiotic bacterium ( Poinar 1990 ). Over 20 years of use, the mass release of EPNs as a biopesticide for D. abbreviatus has been reported as varying and unpredic table with an efficacy ranging anywhere between 0% to >90% ( Downing et al 1991 ; Schroeder 1994 ; Graham et al 2002 ; Duncan et al 1999 ). In addition many orchards in Florida, especially on the central ridge harbor rich communities of naturally occurring EP N species capable of suppressing weevil populations below economic thresholds ( Stuart et al 2008 ). Promoting plant attractiveness to natural enemies is a novel agrochemical alternative to traditional broad spectrum pesticides that indiscriminately kill p redators and parasitoids leading to subsequent pest resurgence and secondary pests ( Bruce 2010 ). The use of natural products to enhance biocontrol is typically compatible with integrated pest management; deploying HIPVs above ground by controlled release dispensers has been shown to increase plant recruitment and retention of beneficial parasites or predators ( Thaler 1996 ; James & Grasswitz 2005 ). In an analogous belowground investigation EPN infection of Diabrotica virgifera virgifera larvae was increase d by spiking soil surrounding maize roots with the HIPV, ( E ) caryophyllene ( Rasmann et al 2005 ). We have also recently shown that some citrus root stock s

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76 ( Citrus paradisi Macf. Poncirus trifoliata L. Raf. and Citrus aurantium ) release HIPVs in response to feeding by the weevil, D. abbreviatus, that attract EPN species endemic to Florida ( Ali et al 2010 2011 ). In this investigation, we identify the specific HIPV attractant as 1, 5 dimethylcyclodeca 1, 5, 7 triene (pregeijerene) and show its real time release in response to herbivory. We also demonstrate that field application of this volatile increases mortality of belowground root feeding weevils by attracting their natural enemies. Furthermore, we demonstrate the presence of this compound in the root zone of fully grown trees in root weevil infested o rchards Recently developed qPCR primers and probes were used to detect and enumerate cryptic species of EPNs allowing for species specific quantification of nematode response to attractants belowground. The use of plant produced signals, such as damage induced r elease of pregeijerene along with conservation biological control strategies could extend the usefulness of EPNs in citrus and other crops damaged by belowground herbivores. Given the broad effect of pregeijerene on a plurality of EPN species, it is possib le that this chemical could b e widely used for enhancing EPN based biological control of subterranean insect pests of agricultural and urban plants. Materials and Methods Insect L arvae Diaprepes abbreviatus larvae were obtained from a culture maintained at FL, U.S.A. This culture was periodically supplemented from a larger culture maintained at the Division of Plant Industry Sterile Fly Facility in Gainesville, FL, U.S.A. La rvae were reared on a commercially prepared diet (Bio Serv, Inc., Frenchtown, NJ) as

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77 described in Beavers (1982) using procedures described by Lapointe and Shapiro (1999). Larvae used in experiments were from third to sixth instars. Plants Citrus paradisi Macf. Poncirus trifoliata L. Raf.) rootstock is very prominent in commercial citrus production (Castle & Stover 2001). The extensive use of this rootstock in commercial citrus production justified its use in this investigation. All pla nts were grown and maintained at the CREC in Lake Alfred, FL, U.S.A. in a greenhouse at 26 2 C, and 60 80% RH. Ruta graveolens L. was purchased as full y grown plants 18 plants were immediately bare rooted and rinsed removing as much soil material as possible, and placed in vials containing Dichloromethane for further extractions and purification. The remaining plant material was discarded. Nematodes used for L aboratory B ioassays and qPCR The entomopathogenic nematodes, Steinernema di aprepesi S. riobrave S. glaseri(x) and H eterorhabditis indica were isolated from D. abbreviatus larvae buried in commercial citrus orchards in Florida. S. riobrave isolates were descendants of commercial formulations intended for field application to ma nage D. abbreviatus All EPN species were cultured in last instar larvae of the greater wax moth, Galleria mellonella at approximately 25C according to procedures described in Kaya and Stock (1997). Infective juveniles (IJs) that emerged from insect cada vers into White traps (White 1927) were stored in shallow water in transfer flasks at 15C for up to 2 wk prior to use.

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78 In situ Volatile Collection from Infested Roots Citrus plants (Swingle citrumelo, Citrus paradisi Poncirus trifoliata ) were grown and maintained at the CREC in Lake Alfred, FL, U.S.A. in a greenhouse at 26C, and 60 80% RH. Six plants were initially placed in glass root zone chambers (ARS, Gainesville, FL, U.S.A.) filled with sand that had been autoclaved for 1 hr at 250C and then adju sted to 10% moisture as described in Ali et al ( 2010 ). All seedlings were given three days to adjust to their sand filled chambers. Three of the plants were subjected to three days of feeding by weevil larvae. During this period each of the six root zone chamber were connected to a vacuum pump (ARS, Gainesville, FL, U.S.A.) with a suction flow of 80 ml / min ( Ali et al 2010 ). Compounds emitted from chambers were collected on adsorbent traps filled with 50 mg Super Q, (800 1000 mesh, Alltech Deerfield, IL, U.S.A.) held in glass fittings between the chamber and vacuum pump ( 2010 ). Super Q traps were changed every 3h for a 72h period to track the time course of volatile release. The removed Super dichloromethane into individual 2.0 m L clear glass vials (Varian, Palo Alto, CA, U.S.A., part number: 392611549 equipp L glass inserts) (Ali et al 2010 ). The undamaged plants served as a control. In situ V olatile C ollection from I nfested R oots in the F ield V olatiles were collected from the soil beds surrounding citrus trees in a non managed, privately owned field site. A soil probe ( Figure 4 1 ) was used to sample at a depth of 20 cm and at distance s of 1 m or 10 m from the trunks of citrus trees A vacuum pum p was used to pull air at a rate of 200 m L /min for a total of 30min. Compounds were collected on adsorbent traps filled with 50 mg Super Q, (800 1000 mesh, Alltech Deerfield, IL, U.S.A.) attached to the top of the soil probe ( Figure 4 1)

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79 The Super Q traps L of dichloromethane into individual 2.0 m L clear glass vials (Varian, Palo Alto, CA, U.S.A., part number: L glass inserts). GC MS Analysis L aliquots of dich loromethane extracts onto a gas chromatograph (HP 6890) equipped with 30 m0.25 mm 1 or DB35 capillary column (Agilent, Palo Alto, CA, U.S.A.), interfaced to a 5973 or 5975 Mass Selective Detector (Agilent, Palo Alto, CA, U.S. A.), in both electron impact and chemical ionization modes. Samples were introduced using either splitless injection at 220C or by cold on column injection. In the second case, a 1m fused silica deactivated retention gap was added between injector and ana lytical column and the injector was programmed to follow the oven temperature. The column was held at 35C for 1 min after injection and then programmed at 10C/min to 260C. The carrier gas used was helium at an average flow velocity of 30 cm/s. Isobutane was used as the reagent gas for chemical ionization, and the ion source temperature was set at 250C in CI and 220C in EI. EI Spectra library search was performed using a floral scent database compiled at the Department of Chemical Ecology, Gteborg Swed en, the Adams2 terpenoid/natural product library (Allured Corporation, Adams 1995) and the NIST05 library. When available, mass spectra and retention times were compared to those of authentic standards in addition to internal standard (nonyl acetate (4 g/ L ) ). Isolation and P urification of P regeijerene Although pregeijerene (1, 5 dimethylcyclodeca 1, 5, 7 triene) was collected from citrus roots damaged by D. abbreviatus it was necessary to find an alternative and abundant source of the pure compound for la boratory bioassay and field testing.

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80 Hydrodistilled common rue ( Ruta graveolens ) essential oil contains geijerene as a major constiuent (67% of the total volatile compounds) ( Kuzovkina et al. 2008 ). However, at temperatures exceeding 120C ( Kubeczka 1974 Figure 4 2 ) the macrocyclic pregeijerene will rearrange to geijerene thus by on column analyses of common rue root extracts we, as anticipated, found large quantities of pregeijerene rather than geijerene. For isolation of pregeijerene, rue roots were crus hed in dichloromethane. GC/MS analyses showed pregeijerene to constitute about 95% of the terpene content in addition to large quantities of more polar compounds, mostly furanocoumarins. To remove the furanocoumarins the dichloromethane extract was first e liminated by gently evaporating the sample to a small volume (0.5m L ) and was re suspending it in 4m L of pentane. After centrifugation, the supernatant was again gently concentrated and re suspended in 4ml of pentane and again centrifuged to remove solids. An attempt to use a silica column resulted in a partial conversion of pregeijerene to co geijerene. The yellow solution was therefore slowly passed through a diol column, successfully removing the cyanocoumarins while maintaining intact pregeijerene ( Fig ure 4 3). The two remaining impurities were removed by first repeatedly partitioning the hexane extract with methanol followed by a slow filtering through a quartenary amin ion exchange column ( Figure 4 3 ) The final hexane solution was analyzed by GC MS f or purity and by GC/FID with nonyl acetate as an internal standard for quantification. Serial dilutions were made from this extract providing five concentrations (30 L aliquots ) of pregeijerene (8.0g/ L ; 0.80g/ L ; 0.08g/ L ; 0.008g/ L ; 0.0008g/ L ). Two choice B ioassay to D etermine O ptimal D osage to A ttract EPNs The behavioral responses of nematodes to collected pregeijerene were quantified in a two choice sand filled olfactometer described thoroughly by Ali et al ( 2010 ). Briefly,

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81 the olfactometer consi sts of three detachable sections: two opposing 16 m L glass jars which contained treatments and a central connecting tube 3 cm in length with an apical hole into which nematodes were applied. Dilutions from the purified R. gravleolens root extract were plac ed on filter paper, which was allowed to dry 30 s for solvent evaporation. Thereafter, filter papers were placed on the bottoms of each glass jar, which were subsequently filled with 10% saturated (dry wt. sand: water volume; W/V), sterilized sand (Ali et al 2010 ). The central chamber connecting the two arms of the olfactometer was also filled with sterilized and moistened sand. Nematodes ( ca. 200 IJs) were applied into the central orifice of the connecting tube and given 8 hr to respond. Following the inc ubation period, the column was disassembled and the nematodes from the 2 collection jars were extracted using Baermann Funnels. The experiment was replicated five times for each dilution for two species of EPN, S. riobrave and H. indica t tes t was used to compare nematode response in the two choice olfactometer. Since responses of both species to pregeijerene versus the solvent controls were identical, data for both species were combined prior to analysis ( df = 18). The dosage at which we det ected a significant proportion of EPN s attracted to the treatment arm was selected for our field trial. Application of HIPVs in the Field The experiment was conducted in a sandy soil citrus grove at the Citrus Research and Education Center, in Lake Alfred (28 07 26.84 N, 81 42 55.31 W; 97:2:1, sand:silt:clay; pH 7.1; 0.1% OM). The experiment was placed within a section of mature orange trees spaced (without beds) 4.5 m within and 8.1 m between rows that were irrigated with microsprinklers. A randomized desi gn was used to place treatments

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82 between trees in eight adjacent rows. Cylindrical wire mesh cages containing autoclaved sandy soil (10% moisture) and a single larva of D. abbreviatus (reared on h the tree canopies. Cages were made of 225 mesh stainless steel cylinders (7 3 cm diam.) secured at each end with polypropylene snap on caps ( McCoy et al 2000 ). Six cages were placed equidistant from one another in a circle pattern (48cm diam.) for eac h treatment (n=10) ( Figure 4 4). The cages contained one of two treatments ( 1 ) Soil with a single D. abbreviatus larva and infested roots volatiles, or ( 2 ) Soil with a single D. abbreviatus larva and blank solvent control. Treatments were applied as 30 L aliquots to 3 cm diameter filter paper discs (Whatman, Maidstone, U.K.). Solvent was allowed to evaporate for 30 s, prior to insertion of filter papers at the base of each cage. The cages were left buried for 72 hr. Eight soil core samples (2.5 cm dia. 3 0 cm deep) were taken from soil surrounding the treatment arena before the cages were removed. Recovered larvae were rinsed and placed on moistened filter paper in individual P etri dishes for observation. Mortality of the larvae was recorded from 0 to 72 h r after removal from soil. We also investigated the effect of isolated pregeijerene on weevil mortality. The methods for this experiment were similar to that described above, except that the soil remaining within the six cages from each replication was pl aced in a container and homogenized for later nematode DNA extraction (n=10). Soil cores taken from the surrounding treatment arena were also homogenized and stored for nematode DNA extraction (n=10) ( Figure 4 4) DNA extraction took place after sucrose ce ntrifugation extraction of all nematodes (see further materials and methods). DNA extracted from

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83 both soil samples were analyzed using species specific primers for five EPN species hed standard curve ( Campos Herrera et al 2011 ). In the second experiment cages were left buried t test s were used to determine the effect of treatment on mean weevil mortalities ( df = 18). Dete ction, I dentification and Q uantification of E ntomopathogenic N ematodes using R eal T ime qPCR Real time qPCR was used to identify and quantify attraction of naturally occurring EPN species to volatiles applied in the field and to identify nematodes to specie s. This technique targeted six EPN species ( Steinernema diaprepesi S. riobrave S. scapterisci, Heterorhabditis indica H. zealandica and an undescribed species in the S. glaseri group(x)) ( Campos Herrera et al 2011a, b ). Briefly, species specific prime rs and TaqMan probes were designed from the ITS rDNA region using sequences of the target steinernematid and heterorhabditid species as well as closely related species recovered from the NCBI database or generated by the authors in that study Multiple al ignments of the corresponding sequences were performed ( Larkin et al 2007 ) to select areas of variability in the ITS region. The designed primers and probes (Primer Blast, Rozen & Skaletsky 2000 ) provided no non specific amplification when they were teste d using other EPN species. Standard curve points were obtained from DNA dilution. Four independent DNA extractions were performed from Eppendorf tubes containing 300 IJs in 100 L (Ultra Clean Soil TM DNA kit, MO BIO) and mixed to avoid the differences betw een DNA elution in the final step of extraction ( Torr et al 2007 Campos Herrera et al 2011 ). Dilutions corresponding with 100, 30, 10, 3 and 1 were prepared using serial dilution of the appropriate DNA.

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84 Nematodes from soil samples were extracted by suc rose centrifugation ( Jenkins 1964 ) from a liquots of 500 cm 3 from the mixed composite sample Each nematode community was concentrated in an 1.5 m L Eppendorf tube. DNA was processed using the UltraClean TM soil DNA extraction Kit and quantification was perfo rmed for each DNA extraction using the nanodrop system with the control program ND 1000 v3.3.0. All DNA samples were adjusted to a 0.2 ng/ L that is required for nematode quantification ( Campos Herrera et al 2011 ). Real time PCR was performed in optical 9 6 well reaction plates (U.S.A. Scientific, Orlando, FL, U.S.A.) on an ABI Prism 7000 (Applied Biosystem). All reactions were performed in a final volume of 20 L with 10 L of TaqMan Universal PCR Master Mix (AB, manufactured by Roche, Branchburg, NJ), us ing the appropiate primer and probe concentration for each species previously described ( Campos Herrera et al 2011 ). In all tests a negative control was included by adding sterile de ionized water instead of template DNA, and the positive control was the corresponding standard curve Thermal cycling was performed as described in Campos Herrera et al ( 2011 ), using 59C as annealing temperature and 35 cycles. All the samples/controls were run in duplicate. Data from the standard curves were log (x) transfo rmed and a linear regression was performed of log (number of nematodes) and threshold cycle value (Ct) was performed to estimate the efficieny and accuracy of the system ( SPSS 18.0 software for Windows XP SPSS Inc., Chicago, IL, U.S.A.) Then, a correct ion factor was applied to transform the qPCR data to the real value according with each dilution. The resulting real values were analyzed with an ANOVA for the EPN species recovered ( F = 41, df = 5, 204). s HSD test

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85 was conducted to discriminate among means in the software R (R Development Core Team 2004). NMR A nalysis of Pregeijerene P regeijerene wa s purifed for NMR using prepGC as a mixture of pregeijere and geirene 70:30 ratio. The p regeijere ne and geijerene mixture (~60 ug) in ~150 L of C 6 D 6 (Cambridge Isotope Laboratories Inc.) was placed in a 2.5 mm NMR tube (Norell). One dimentional 1 H and nuclear overhauser enhancement (NOE) difference experiments and two dimensional NMR spectroscopy, including gradient correlation spectroscopy, heteronuclear single quantum coherence, heteronuclear multiple bond correlation and NOE spectroscopy were used to characterize pregeijerene. All 2D NMR spectra were acquired at 24C and an additional 1D NOE dif ference experiment was conducted at 10C using a 5 mm TXI CryoProbe and a Bruker Avance II 600 console (600 MHz for 1 H, 151 MHz for 13 C). Residual C 6 D 6 was used to referen ce c hemical 6 H 6 ) = 7.16 ppm for 1 6 H 6 ) = 128.2 ppm for 13 C (Fulmer et al. 2010). NMR spectra were processed using Bruker Topspin 2.1 and MestreLabs MestReNova software packages. Numbering is based on Jones and Southerland (1968). The H and 13 C NM R data in C 6 D 6 are presented for pregeijerene and geijerene in Tables 4 2 and 4 3 because the original NMR data was obtained in carbon tetrachloride solution. Results In situ V olatile C ollection from I nfested R oots in the F ield Volatiles collected from 1m and 10m distance s detected both pregeijerene and geijeriene ( Figure 4 5). Thus, demonstrating the presence of this cue under natural field conditions.

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86 Release and P urification of 1, 5 D imethylcyclodeca 1, 5, 7 T riene Volatiles were non destructively samp led every 3 hours from seedlings in sandy soil using root zone chambers (ARS, Gainesville, FL, U.S.A.) as previously described ( Ali et al. 2010, 2011 ). Three hours after the introduction of D. abbreviatus larvae to citrus roots, 1, 5 dimethylcyclodeca 1, 5, 7 triene (pregeijerene) was identified as a dominating root volatile, reaching a maximum release between 9 and 12 hr after initiation of larval feeding ( Figure 4 6 ). There was no appreciable increase of any additional volatiles. After the initial spike, the release of pregeijerene decreased progressively over time ( Figure 4 6 ). Initially it was a challenge to find sufficient amounts of pregeijerene for bioassays and field testing. However, it was previously established ( Kuzovkina et al 2008 ) that a hy drodestillate of common rue ( Ruta graveolens ) roots contained the related terpene geijerene as a major constituent (67% of the total volatile compounds). It is known that pr egeijerene easily converts to g eijerene, for example at temperatures exceeding 120 C ( Kubeczka 1974 ) ( Figure 4 2 ) thus on column GC/MS analyses confirmed pregeijerene as the naturally occurring main terpene in roots of common rue that easily could be extracted and purified from crushed roots using a series of solid phase extractions ( Figure 4 3 ). Identification of P regeijerene Pregeijerene isolated from common rue and in the citrus root volatiles was found to be identical by EI and CI GC/MS analyses on DB1, DB5 and DB35 GC columns. Although the EI mass spectra matched pregeijerene in t he Adams 2 library the lack of a standard made it necessary to confirm the structure by NMR analyses (see NMR results)

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87 Optimum P regeijerene C oncentration Serial 10 fold dilutions were made in dichloromethane from purified pregeijerene providing five conce ntrations. The behavioral responses of EPNs to pregeijerene were quantified in two choice sand filled olfactometers ( Ali et al 2010, 2011 ) identifying 8ng/ L (in 30 L aliquots) as an optimally attractive dosage to EPNs ( S. riobrave and H. indica ) ( Figure 4 7 ). Field V erification of I ncreased B eetle M ortality by B elowground HIPVs Field tests were conducted to determine whether application of infested root volatiles affects EPN inflicted mortality of sentinel D. abbreviatus larvae deployed in a citrus orchar d by increasing response of naturally occurring and introduced EPN species. Cylindrical mesh cages containing autoclaved sandy soil ( McCoy et al 2000 ) contained one of two treatments ( 1 ) Soil with a single D. abbreviatus larva and a standardized collectio n of infested roots volatiles, or ( 2 ) Soil with a single D. abbreviatus larva and blank solvent control. Cages were buried 20 cm below ground between citrus tree canopies ( Figure 4 4 ). Mortality of larvae placed in cages with the infested root volatiles wa s significantly higher than that for larvae placed in cages with solvent alone ( Figure 4 8 ). A second experiment was conducted to test whether also pregeijerene alone would increase mortality of larvae by attracting naturally occurring EPN species. Two tr eatments were deployed within the root zone of mature citrus as described above. EPNs were quantified from soil samples taken within cages and from the surrounding soil of each treatment arena using real time qPCR. Nematodes were extracted from collected s oil and analyzed using species specific primers for EPN species known to be

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88 ( Campos Herrera et al 2011a, b ). Average mortality of larvae buried with purified pregiejerene wa s significantly higher than that of larvae buried with the solvent control ( Figure 4 9 A ). The number of EPNs recovered from cages containing the purified compound was significantly higher than that from cages with the solvent control ( Figure 4 9 B ). There w ere also significantly more EPNs found in the soil samples surrounding cages containing pregeijerene than in solvent control cages ( Figure 4 9 C ). Real time qPCR D etermination of EPN D iversity, and A ttraction to HIPVs Real time qPCR was employed to quantif y the attraction of naturally occurring entomopathogenic nematodes (EPN) in the field and identify them to species. In this study, we employed a technique for identification of six EPN species known to either naturally occur in Florida ( Steinernema diaprep esi, Heterorhabditis indica H. zealandica as well as an undescribed species in the S. glaseri group (x)) or which were commercially applied in citrus groves in Florida ( S. riobrave S. scapterisci ) ( Campos Herrera et al 2011 ) (Table 4 1). Species speci fic primers and TaqMan probes were designed from the ITS rDNA region using sequences of the target steinernematid and heterorhabditid species as well as closely related species recovered from the NCBI database ( http://www.ncbi.nlm.nih.gov/Genbank/ ) or generated by the authors in that study. Comparisons of EPN species were based on standard curve points obtained from DNA dilutions ( Holeva et al 2006 Leal et al 2007 Torr et al 2007 Campos Herrera et al 2011 ). Steinernema glaseri(x), S. diaprepesi H. indica and H. zealandica were detected in soil samples in which mortality of D. abbreviatus was increased by the presence of HIPVs (Table 1). Tukey HSD test indicated H. indica and H. zealandica were si gnificantly more abundant than the S. glaseri(x) and S. diaprepesi EPN species

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89 (P < 0.0001 i n all comparisons). Species that could have been exogenously applied biopesticides, S. riobrave and S. scapterisci were not detected in any of the samples (Table 4 1). NMR A nalysis of Pregeijerene The 1 H NMR data (Table 4 2) for pregeijerene with reported proton chemical shifts and J couplings for pregeijerene A (Jones & Southerland 1968) are consistent, but not with pregeijerene B (Cool & Adams 2003). Jones and S outherland (1968) did not report 13 C NMR data, thus we compared the 13 C NMR data with Germacrene C containing a cyclodecadiene ring like pregeijerene with the exception of an isopropyl substitution at C8 position. Both 1 H and 13 C NMR data agreed with germ acrene C (Colby et al 1998) except for carbons adjacent to C8 as expected. The two dimentional NOESY experiment at room temperature (24C) resulted in two very weak NOE. The flexi ble cyclodecadiene ring wa s found to exist in three different conformation al isomers for germecrine A at or lower than 25C (Faraldos et al 2007). Therefore, NOE difference experiments were done on the two methyl groups at C1 and C5 at 10C, above freezing temperature, and 30C in C 6 D 6 Overall NOEs were small, but signal inte nsity was better at 10C for NOE difference experiments. The protons of methyl group at C5 had NOEs to proton 6.52 of C7, 2.08 of C4 and 1.94 of C3/1.97 of C9. The protons of the methyl group at C1 had NOEs to 1.73 of C10 and 1.97 of C9/.94 of C3. The NO E results agree with pregeijerene and flexible cyclodecadiene ring structure s (Jones & Southerland 1968; Colby et al 1998; Faraldos et al 2007). In Addition we found that chemical shifts of protons at C2, C7 and C8 are sensitive to temperature changes.

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90 Discussion The present research identifies pregeijerene as an HIPV associated with the indirect defense of citrus plant roots. Field application of this compound increased mortality of root weevils by its corresponding attraction of EPN In addition to citrus roots, other rutaceus plant species are known to produce pregeijerene ( Santos et al. 1998, Kuzovkina et al. 2008 ). ( E ) caryophyllene is the only other identified volatile terpenoid known to attract EPNs ( Rasmann et al 2005 ). While the sesquiterpene ( E ) caryophyllene is known to play an ecological role for numerous arthropod and nematode species ( Turlings et al 1998 Kigathi et al. 2009 ), this is the first description of an ecological role for the C 12 terpene pregeijerene. In this investigation we combined both recent and novel techniques of in situ detection of belowground cues and enumeration of cryptic EPN species using real time qPCR to describe this subterranean interaction in detail. Two major obstacles account for difficulties in evaluating applied volatiles for the attraction of belowground natural enemies in the field. First, quantifying mortality of the targ et pest is usually measured by emerging adults ( Degenhardt et al 2009 Rasmann et al 2011 ). In field experiments there is a high potential for low recovery of applied herbivores. This technique also gives no confirmation for the specific cause of mortali ty. Second, it can be difficult to quantify populations of naturally occurring EPNs. The number of EPNs in soil is usually estimated indirectly by baiting soil with sentinel insects ( Koppenhofer et al. 1998 ; Mracek et al 2005 ). Such estimates are imprecise because infection rates are species specific and are dependent on environmental conditions such as soil moisture, temperature and porosity ( Stuart et al 2006 ). Low r ecovery of EPNs after application can make accurate quantitative comparisons of EPN attra ction to treatments difficult in addition to the time

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91 consuming and intensive task of identifying the recovered nematodes to species which very few have the expertise to do accurately Quantitative real time PCR is an efficient method for quantifying cry ptic organisms such as bacteria, fungi, and nematodes from soil samples ( Klob et al 2003 Atkins et al 2005 Zhang et al 2006 ) and has been recently employed to investigate EPN diversity in natural habitats ( MacMillan et al 2006 Torr et al 2007 Camp os Herrera et al 2011 a, b ). This technique allowed quantification of increased mortality of weevil larvae in response to field application of the HIPV, pregeijerene. Furthermore, we proved that weevil mortality was caused by EPN species naturally occurrin g in Florida rather than those which could have been previously applied in the form of biopesticides. These results are not due to a lack of behavioral response by the commercially formulated species entirely, as shown in the laboratory experiment ( Figure 4 6 ). Non native EPM species have been introduced into Florida citrus in the form of biopesticides. In our field tests, only native species of nematodes responded to field applications of the HIPV Therefore manipulation of naturally occurring EPNs with p regeijerene without the need for exogenous application of non native EPNs appears to be a viable tactic. Furthermore in orchards with established EPN populations, large scale introduction of non native species may displace native populations due to troph ic cascades and limited resources or may cause an increase in populations of nematophagous fungi that eliminate EPN populations ( Duncan et al 1996 Koppenhofer et al 1998 McCoy et al 2000 Stuart et al 2008 ). Although it is known that the artificiall y reared and commercially formulated EPN can persist, it is possible that natives have advantages associated with habitat acclimation and response to HIPVs

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92 ( Hiltpolt et al 2010 ) thus further investigation of enhancing conservation biological control of be lowground pests in concert with behavioral modification via HIPVs is warranted. The obstacles of investigating above belowground chemically mediated interactions between plants and animals are being overcome and refocused ( van Dam 2009 Johnson 2008 van Dam & Heil 2011 ). One of the most important areas of focus in both aboveground and belowground systems remains understanding induced plant responses to herbivory that can indirectly reduce preference or performance of herbivores. Although it was originally postulated as a potential novel approach to pest management in agricultural systems ( Green & Ryan 1972 ) and insect herbivore population regulation ( Haukioja & Hakala 1975 ), few studies ( Khan et al 1997 De moraes et al 1998 Birkett et al 2000 Kessler & Baldwin 2001 Ockroy et al 2001 ) of induced responses (particularly volatile) have addressed this practical application beyond fundamental concepts in ecology and evolutionary biology ( Hunter 2002 van Dam & Heil 2011 ). There are even fewer attempts to investigate these dynamics belowground. At least half of all plant biomass is attacked by underground herbivores and pathogens, living amongst a complex ecological foodweb ( De Deyn & van der Putten 2005 ) HIPVs are likely important mediators of tritrophic interactions that afford indirect plant defense within the root zone. Direct field sampling of root volatiles is a promising method for evaluating these belowground interactions in real time. We also for the first time detected not only direct increases in larval insect mortality associated with use of a belowground HIPV attractant, but demonstrated a corresponding quantitative increase in subterranean natural enemies Our previous

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93 research suggests that volatile production in response to herbivore feedin g differs between citrus species ( Ali et al 2010 ). Our findings could have broad impact on rootstock selection in commercial agriculture, by screening and recommending or developing rootstocks that release attractants which promote accumulation of EPN com munities. Further investigation is needed to evaluate genetic expression of this response, which could optimize development of transgenic rootstocks to attract beneficial nematodes in response to pest damage. Pregeijerene may have extensive application for enhancing native biological control of root feeding insects given its broad attractiveness to a plurality of nematode species, including those which attack a wide range of belowground herbivores ( Choo 2002 ).

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94 Table 4 1. Species of entomopathogenic nematod es identified and quantified in response to HIPV deployment in the field. EPN Species Ecological Status Detected Representation (%) Steinernema diaprepesi Native + 1 S. glaseri(x) Native + 1 S. scapterisci Commercial product 0 S. riobrave Commercial product 0 Heterorhabditis indica Native + 54 H. zealandica Native + 44

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95 Table 4 2. 1 H (600 MHz), 13 C (151 MHz), HMBC and NOESY NMR spectroscopic data for pregeijerene in C 6 D 6 13 C was also detected directly (126 MHz) using a 5 mm Cryoprobe. Chemica l shifts referenced to residual proton signal in C 6 D 6 1 H) = 7.16 ppm for 1 6 D 6 H) = 128.2 ppm for 13 C. Position 13 C [ppm] 1 H [ppm] J coupling constants [Hz] HMBC correlations (C.No) NOE peaks 1 140.6 2 125.2 1H 4.83 # ddt J = 11.5, 4.9, 1.4 2.45* 3 27.6 2H 2.06, 1.94 2.06, 1H, m 1.94, 1H, m 1.94 1.19* 4 39.8 2H 2.08, 1.67 2.08, 1H, dt J = 11.5, 3.4 1.67, 1H, dt J = 4.4, 12.0 1.67 C6, C3 (weak), CH3 of C5 5 $ 6 128.9 1H 5.39 br d J = 9.7 C4, C8 *2.28, *1.67 7 130.0 1H 6.52 # t J = 10 1.49* 8 127.5 1H 5. 53 # ~dt J = 10.0, 8 9 29.5 2H 2.28, 1.97 2.28, 1H, m 1.97, 1H, m 1.97 1.19* 10 39.1 2H 1.73, 2.45 1.73, 1H, dt J = 4.6, 12.8 2.45, 1H, ~ddd J = 12.8, 6.0, 1.9 1.73 1.19* CH3 C1 20.6 3H 1.19 d J = 1.1 C1, C2, C10 **1.73, **1.96/1.97 CH3 C5 16. 2 3H 1.49 s C6, C4 **6.52, **2.08, **1.94/1.97 weak NOEs observed with 2D NOESY experiment at 24C, **observed from 1D NOE difference experiments at 10C. # Chemical shifts are temperature sensitive. $ We think carbon chemical shifts of C5 and C6 overlap. C arbons numbered based on Jones and Sutherland (1968).

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96 Table 4 3. 1 H (600 MHz), 13 C (151 MHz), HMBC and NOESY NMR spectroscopic data for geijerene in C 6 D 6 13 C was also detected directly (126 MHz) using a 5 mm Cryoprobe. Chemical shifts referenced to r esidual proton signal in C 6 D 6 1 H) = 7.16 ppm for 1 6 D 6 H) = 128.2 ppm for 13 C. For convenience, the pregeijerene numbering is retained after cope rearrangement to geijerene. Position 13 C [ppm] 1 H [ppm] J coupling constants [Hz] HMBC correlations (C.No) Unique NOESY peaks 1 38.0 2 149.0 1H 5.86 dd J = 17.5, 10.8 3 110.6 2H 4.99, 4.94 4.99, 1H, dd J=17.5, 1.3 4.94, 1H, dd J = 10.8, 1.3 4.99 C1 4.95 C1 4.99 0.96 4 114.2 2H 4.82, 4.97 4.82, 1H, br s 4.97, 1H, m 5 146.7 6 51.5 1H 2.7 quintet J = 2.7 7 126.2 1H 5.66 dddd J = 10.1, 2.2, 3.5, 3.5 8 129.9 1H 5.59 dddd J = 10.1, 3.2, 2.1, 2.1 9 22.6 2H 1.91 m 0.96 10 33.4 2H 1.43 m CH3 C1 20.9 3H 0.96 s C1, C2, C6, C10 1.72 CH3 C5 24.3 3H 1.72 br s C5, C4, C6 0.96

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97 Figure 4 1. Representation of soil probe design used to sample volatiles belowground. Probe is inserted into soil and connected to a vacuum pump.

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98 Figure 4 2. Conversion of Pregeijerene(A) to Geijerene(B).

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99 Figure 4 3. Chro matograms showing the initial crude extract prior to purification and final purified Pregeijerene.

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100 Figure 4 4 Schematic diagram of the deployment and sampling procedure for field experiments in which sentinel traps with root weevils were deployed wit h or without HIPVs. One treatment replicate is depicted.

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101 Figure 4 5. Chromatograms of volatiles taken from intact citrus roots in the field at 1m and 10m distances from the trunk of the tree.

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102 Figure 4 6 Time course of pregeijerene (1, 5 dimethylc yclodeca 1, 5, 7 triene) release following initiation of root weevil (D. abbreviatus) feeding on citrus roots. fed controls.

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103 Figure 4 7 Optimal dosage of pregeijerene ( 1, 5 dimethy lcyclodeca 1, 5, 7 triene ) for attracting entomopathogenic nematodes ( S. riobrave and H. indica ) based on the log scale dilution of purified compound. Picture in upper right displays sand filled two choice olfactometers used for nematode bioassays. = P v alue < 0.05.

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104 Figure 4 8 Mean percentage of larval mortality for treatments with or without D. abbreviatus fed upon root volatiles. ** = P value < 0.01. (N = 10, t = 3.25, P = 0.005)

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105 Figure 4 9 Effect of pregeijerene on weevil mortality and assoc iated attraction of EPN species A) Average mortality of larvae buried with purified pregeijerene compared with the solvent control ( N = 10, t = 4.01, P = 0.0008 ) B) Mean number of EPNs recovered from cages containing the purified pregeijerene compared wit h cages containing the solvent control ( N = 10, t = 5.33, P = 0.00005 ) C) Mean number of EPNs recovered from soil samples surrounding cages containing pregeijerene compared with the solvent control ( N = 10, t = 5.67, P = 0.00003 )

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106 CHAPTER 5 CONCLUSIONS P lants are under constant pressure from higher trophic levels which attempt to exploit their autotrophic resources. Herbivorous i nsects (primary consumers) are the most successful herbivores harvesting primary resources, as evident by their overwhelming num bers and diversity. However, the success and abundance of primary consumers causes selection pressure for evolution of plant defense This has resulted in the evolution of complex interactions between plants, insect herbivores, and their natural enemies As stated by Price et al. ( 1980 ) all terrestrial communities based on living plants are composed of at least three interacting trophic levels: plants, herbivores, and natural enemies. This seminal paper introduced an important relationship which has led to over 3 0 years of investigations by ecologist s and e ntomologists focusing on predators and parasitoids of herbivores There is increasing evidence of plants using natural enemies as bodyguards. Mechanisms of this relationship are found in plants' product ion of food and shelter for these natural enemies or by producing herbivores induced volatiles that expose the presence of herbivores. The chapters outlined in this dissertation provide evidence for the use of herbivore induced volatiles by entomopathogeni c nematodes to locate their hosts. This is only the second stud y to identify a belowground cue in a tritrophic interaction. However, to fully define this relationship as a plant defense additional terms must be met. Understanding the evolution of this pote ntial defensive interaction will require a connection to the plant trait which affects the entomopathogen, and the direct/indirect effects the entomopathogen has on the fitness of the plant. Observations made in the

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107 studies of this work can be ev aluated on their implications for both agricultural biological control strategies and the evolutionary consequence s of these relationships. Chapter 2 demonstrated in a root zone bioassay, that root weevil infested rootstock (Swingle citrumelo) recruited significant ly more EPN ( Steinernema diaprepesi ) than non infested or mechanically damaged roots, or larvae alone. By dynamic in situ collection and GC MS analysis of volatiles from soil, in combination with a two choice sand column bioassay it was found that Swingle citrus roots release induced volatiles in response to herbivore feeding and that some of these induced volatiles function as attractants for EPN s This study was the first step in drawing a connection to a trait which affects the entomopathogen. Although w e introduce an in situ method for detecting belowground signals and correlate these specific infested root volatiles to the attraction of EPN s further work was necessary to reveal the breadth of this interaction C hapter 3 examined the extent to which bel owground recruitment signals modify behavior of nematode species representing various foraging strategies, and trophic levels. We compared attraction to extracts of infested roots and non infested roots from hybrid, Swingle citrus rootstock, and a parent l ine of the hybrid, P. trifoliata (Pt). Swingle roots infested by weevils attracted more nematodes than non infested roots irrespective of nematode foraging strategy and trophic status. The parental line of the swingle rootstock, Pt, attracted all nematode species irrespective of insect herbivory. Dynamic in situ collection and GC MS analysis of soil volatiles revealed that Pt roots released recruitment signals constitutively, regardless of weevil feeding. A different non hybrid citrus species (Sour orange, C. aurantium ) released nematode recruitment signals only in response to larval feeding. Volatile collections from above/belowground portions of

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108 citrus plants revealed that aboveground feeding does not induce production of nematode recruitment signals analo gous to that induced by root damage nor does damage by larvae belowground induce a similar signal abov eground. This study demonstrated that roots can have induced or constitutive release of nematode cues. It rely associated with the attraction of beneficial nematodes. The plants susceptible to the plant parasites seemed to have a strategy which circumvents the ecological cost of attracting its parasites by only releasing these cues when fed upon by the root we evils. The roots which constitutively release these cues were not susceptible to the phytopathogens. The cue thus seems to be heavily context dependent and should be evaluated for use only in systems which are not threatened by citrus specific plant parasi tes. It became evident that evaluations of the abilit y to increase mortality of root weevil s would be necessary to further define this cue as an indirect defense. In the fourth chapter the main constituent released by damaged citrus roots was identified as pregeijerene ( 1, 5 dimethylcyclodeca 1, 5, 7 triene) and field assays of lab collected citrus root HIPVs proved attraction of native EPN and associated increased mortality of beetle larvae compared with controls was possible We determined b y quantitative real time PCR that field application of pregeijerene increased pest mortality by attracting four species of EPNs native to Florida, U.S.A. This was a first step in evaluating the potential to reduce herbivore populations. Alth ough it holds promising implications for biological control, we must still evaluate this interaction from the pre spective of the plant s fitness/yield. This study identified a specific belowground cry for help and then showed how this cue could reduce herb ivore numbers but we are

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109 yet to show protection of the plant via reduced damage and thus increased yield. Future studies must show this protection relationship for the ecological requirements of defining this response as a defense and for the implementati on of these responses in biological control strategies. The se studies have made a number of contributions to the field of chemical ecology and biological control. By developing methods for the underground detection of responses to herbivory in real time we have made the first direct quantifications of belowground HIPV release Combining both recent and novel techniques of in situ detection of belowground cues and enumeration of cryptic EPN species using real time qPCR it bec a me possible to describe this s ubterranean interaction in detail. ( E ) caryophyllene is the only other identified volatile terpenoid known to attract EPNs (Rasmann et al 2005). While the sesquiterpene ( E ) caryophyllene is known to play an ecological role for numerous arthropod and nematode species (Turlings et al 1998 Kigathi et al. 2009), this is the first description of an ecological role for the C 12 terpene pregeijerene. The next steps of this investigation must test the potential for this compound to protect citrus roots from damage This research has two very dif ferent aspects and implications First, the observations in th is dissertation have implications in the field s of natural defense ecology and evolutionary biology. Second, we are examining the phenomena within an agroecological system with domesticated crop s. These two points bring about a paradox in their respective methodological applications. Can we impose observations of domesticated plant s in an agricultural context on evolutionary theory of natural plant

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110 defense s ? We must first take into consideration the properties associated with agricultural systems. T raditional farming practices thrived on large genetic crop diversity (Marshall 1977) Although, outbreaks of insect pest s often devastate d yield s the high level of resistance maintained allowed for low er, but consistent and sustainable levels of production. Decades of p lant breeding to meet the demands of growers, processors, packers and consumers has produced dramatic increases in yield and qualities of crop plants (Marshall 1977) This consequentially led to an increasing level of uniformity in viable crop products. Locally adapted genotypes have been substituted with widely adapted cultivars that are characterized by insufficient defense against attack by specialized insects. Feeny (1976) developed an extension of coevolution defense theory, based on apparency. Apparent plant they are predicted to be well defended and not readily susceptible to counter adaptation. Unapparent plant s on the other hand are not predictably distributed and defenses are susceptible to counter adaptation. The plight of modern agriculture has been to domesticate unapparent plants and make them apparent. Managed ecosystems are characterized by low plant diversity and low genetic v ariability compared with natural ecosystems It is well known that reduced heterogeneity can enhance the colonization of plants by insect pests (Andow 1990), and it can also influence the performance of natural enemies (Price et al. 1980, Price 1986). Howe ver, biological control studies are often viewed as "ecological experiments on a grand scale, and illustrate both the 'escape' of pest species relieved of natural enemies and their demise when enemies are restored to the system" (Strong et al. 1984), and

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111 m any early biological control practitioners (e.g., DeBach and Rosen 1991, Waage 1992) consider ed there to be no fundamental ecological difference between successful classical biological control and the action of native natural enemies ('naturalcontrol' sens u DeBach [1964]). A distinguishing feature instantly appears as we consider the indirect defense interactions in agroecosystems. The objective of biological control in agriculture is to maximize the effectiveness of a natural enemy complex in suppressing p ests and ultimately in enhancing crop yield (Debach & Ros e n, 1991; Norris et al. 2003; Denno et al. 2008). In ecological contexts, trophic cascades are predator prey interactions that indirectly alter the abundance, biomass or productivity of a community a cross more than one trophic link in a food web (Hawkins et al. 1999; Pace et al. 1999). In this way the extent to which herbivore populations are constrained by natural enemies in agroecosystems is si gnificantly different and their broad ecological implica tions may be limited. Although the findings of this largely agricultural study may have limited ecological relevance, there are too few investigations of belowground community inte potential grand scale implications on plant defense strategy. The majority of terrestrial studies, investigating enemy propagated trophic cascades have focused on arthropods or vertebrates as predators in aboveground food webs, whether in natural or managed ecosystems (Rosenheim et al. 1995; Hawkins et al. 1999; Snyder et al. 2005; Rasmann & Agrawal 2008). Soil dwelling organisms comprising belowground food webs have been virtually ignored (Hunter 2001; Rasmann & Agrawal 2008) Nematodes, despite their prevalence in

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112 natural and agricultural h abitats, are highly under represented in studies of population and food web dynamics and in particular in those investigating trophic cascades (Stuart et al. 2006). Thus based on the knowledge obtained from the study of aboveground arthropod food webs, an alogous information is critical for understanding the broader ecological and ev olutionary mechanisms involved in EPN herbivore plant indirect defense. Thus the broader evolutionary implications of research presented in this work are open to debate. Howeve r there is a common focal point for the future directions of this study which would be relevant to both evolutionary relationships and agricultural applications. Next it will be most important to study how such interactions enhance or reduce plant biomass and yield. As stated earlier trait must be made between the natural enemy and the fitness of the plant to develop evolutionary predications o n the adaptive role of this relationship Additionally, investigating t his EPN h erbivore p lant interaction (and the manipulation of cues within it) to potentially influence yield of citrus and other crops is of paramount importance to agriculture and biological control.

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113 LIST OF REFERENCES A dair R. C. 1994. A four year field trial of entomopathogenic nematodes for control of Diaprepes abbreviatus in Flatwoods citrus grove. Proceedings Fl orid a S tate Horticultural Soc iety 107 63 68. A dams R. P. 1995. Identification of Essential Oil Components by Gas Chromatography/Mass Spectr ometry. Allured, Carol Stream, IL. Adler, L.S. & Irwin, R.E. 2005 Ecological costs and benefits of defenses in nectar. Ecology 86 2968 2978. Agrawal, A. A., & Karban, R. 1999. Why induced defenses may be favored over constitutive strategies in plant s. In The Ecology and Evolution of Inducible Defenses eds. R. Tollrian and C. D. Harvell, pp. 45 61. Princeton: Princeton Uni versity Press. A grawa L, A. A., & R utter M. T. 1998. Dynamic anti herbivore defense in ant plants: the role of induced responses. O ikos 83 227 236. Aharoni A Jongsma M A & Bouwmeester H J 2005 Volatile science? Metabolic engineering of terpenoids in plants. Trends in Plant Science 10 594 602. Alborn, H.T., Jones, T.H., Stenhagen, G.S., & Tumlinson, J.H. 2000. Identifica tion and synthesis of volicitin and related components from beet armyworm oral secretions. Journal Chemical Ecology, 26 203 220 A lborn, H. T ., T urlings T.C. J., J ones, T. H., S tenhagen G., L oughrin J. H., & T umlinson J. H. 1997. An elicitor of plant vo latiles from beet armyworm oral secretion Science 276 945 949. Ali, J.G., Alborn, H.T. & Stelinski, L.L. 2010. Subterranean herbivore induce volatiles released by citrus roots attract entomopathogenic nemtaodes. Journal of Chemical Ecology 36 361 36 8. Ali J.G., Alborn, H.T., Stelinski, L.L. 2011. Constitutive and Induced Subterranean Plant Volatiles Attract Both Entomopathogenic and Plant Parasitic Nematodes. Journal of Ecology 99 26 35. Andow, D. A. 1990 Vegetational Diversity and arthropod respo nse. Annual Review of Entomology, 36 561 586. A ratchige N. S., L esna I., & S abelis M. W. 2004. Below ground plant parts emit herbivore induced volatiles: olfactory responses of a predatory mite to tulip bulbs infested by rust mites. Experimental and Applied Acaro logy, 33 21 30.

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115 Birkett, M.A., Campbell, C.A.M., Chamberlain, K., G uerrieri, E., Alastair, J.H., Martin, J.L., Matthes, M., Napier, J.A., Petterson, J., Pickett J.A., Poppy, G.M., Pow, E.M., Pye, B.J., Smart, L.E., Wadhams, G.H., & Woodcock, C.M. 2000. New roles for cis jasmone as an insect semiochemical and inplant defense Proceedings of the National Academy of Sciences U.S.A ., 97 9329 9334. Blossey, B. & Hunt Joshi, T. R. 2003. Belowground herbivory by insects: influence on plants and aboveground herbivores. Annual Review of Entomology 48 521 547 B oethel D.J. & E ikenbary R.D 1986. Interactions of plant resistance and parasitoids and predators of insects. Chichester, Ellis Horwood limited. B off M. I. C., van Tol, R. H. W. M., & S mits P. H. 2002. Behavioural response of Heterorhabditis megidis towards plant roots and insect larvae. Biocontrol, 47 67 83. Bourne, B. A. 1921. Insect att acks reported or observed. Annual Report of the Department of Agriculture : Barbados 1919 1920, pp. 12 13 Bullock, R.C., McCoy, C. W., & Fojtik. J. 1988. Foliar sprays to control adults of thecitrus ro ot weevil complex in Florida. Pr oceedings of Florida State Horticultural Society 101 1 5. Bullock R.C. & Miller, R.W 1994. Suppression of Pachnaeus litus and Diaprepes abbreviatus (Coleoptera: Curculionidae) adult emergence with Steinernema carpocapsae (Rhabditida: Steinernematidae) soil drenches in field evaluations, Proceedings of the Florida State Horticultural Society, 107 pp. 90 92. B ullock, R. C., P elosi, R.R., & K iller. E. E. 1999. Management of citrus root weevils (Coleoptera: Curculionidae) on Florida citrus with soil applied entomopathogenic nematodes (Nematoda: Rhabditida). Florida Entomol ogist, 82 1 7. Bruce T J A 2010. in Plant Communication from Ecological Perspective, eds Ninkovic Verlag, Heidelberg), pp 215 227 Cabanillas, H.E., Poinar, G.O. & Raulston, J.R. 1994. Steinernema riobravis n. sp. (Rhabditida: Steinernematidae) from Texas. Fundamental & Applied Nematology 17 123 131. Campbell, J.F. & Gaugler R. 199 7 Nictation behavior and its ecological implica tions in the host search strategies of entomopathogenic nematodes (Heterorhabditidae and Steinernematidae). Behaviour 126 3 4. Campos Herrera R, Johnson E G El Borai F E Stuart R J Graham J H Duncan L W 2011. Long term stability of entomop athogenic nematode spatial

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132 BIOGRAPHICAL SKETCH Jare d Gregory Ali was born in Philadelphia, Pennsylvania. Jared was educated in Quaker school s for elementary, middle, and high school H ere he developed a longing to explore an alternative path of knowledge Jared left high school his junior year and traveled through the United States, Canada and Mexico. Reading many classic works of inspired Jared to approach his education through the study of biological interactions and evol ution. After completing his GED and writing a n expressive letter to the University of Delaware, he was offered admission to the College of Arts and Sciences. He majored in biological sciences as an undergraduate and continued on to complete his M.Sc. in En tomolog y & Wildlife Ecology under the advisement of Dr. Douglas W. Tallamy studying sexual selection and chemical communication He moved to Florida to pursue a Ph.D. under Dr. Lukasz L. Stelinski where his research encompassed belowground multitrophic i nteractions and chemical ecology. Upon the completion of his Ph.D. at the University of Florida Jared accept ed an opportunity to study plant defense a nd mu lt itrophic interactions in the department of Ecology & Evolutionary Biology at Cornell University u nder the supervision of Dr. Anurag Agrawal.