ASSESSMENT OF HERBIVORE INDUCED PLANT VOLATILES IN

JUVENILE HOPS BY EXPOSURE TO METHYL SALICYLATE

By

RITA M. ABDELLA

A thesis submitted in partial fulfillment of

the requirements for the degree of

MASTER OF SCIENCE IN CHEMISTRY

WASHINGTON STATE UNIVERSITY

Department of Chemistry

DECEMBER 2010

ii

To the Faculty of Washington State University:

The members of the Committee appointed to examine the thesis of RITA MARIE ABDELLA

find it satisfactory and recommend that it be accepted.

__________________________

Vincent R. Hebert, PhD., Chair

___________________________

Herbert H. Hill, PhD.

____________________________

Steven G. Metcalf, PhD.

_____________________________

William D. Samuels, PhD.

iii

ACKNOWLEDGEMENT

First, I would like to thank Dr. Vincent Hebert for acting as my advisor throughout the

completion of this thesis. Dr. Hebert‟s keen eye and attention to detail were most appreciated

during the writing of the manuscript and thesis. I would also like to thank Jane Le Page for all

the analytical work that was performed during this study. A big thank you goes out to Dr.

William Samuels, who has provided me with the encouragement to keep going and for the

instruction over the course of this degree. Many thanks also go out to the remaining members of

my committee; Dr Herbert Hill and Dr. Steven Metcalf.

Lastly, I would like to thank my grandchildren: Linden, Loghan and Makena Kastl, for

being patient with Grammy for not making a lot of their sports events and to my husband Charlie

McDonald and my daughter, Astrid Kastl for seeing me through this. And finally I want to thank

my father, Richard Abdella, for instilling the importance of education in me and in memoriam to

my mother Mary Abdella, who wasn‟t here to see me finish. Mom, you would have been proud.

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ASSESSMENT OF HERBIVORE INDUCED PLANT VOLATILES IN

JUVENILE HOPS BY EXPOSURE TO METHYL SALICYLATE

Abstract

by Rita Marie Abdella

Washington State University

December 2010

Chair: Vincent R. Hebert

The volatile secondary plant product methyl salicylate (MeSA) exhibits numerous properties,

including the attraction of beneficial insects to plants undergoing attack from herbivores. This

capability to attract beneficial species is being explored as a “green” alternative to pesticides in

hop fields and grape vineyards. While some field studies have been performed using MeSA as

an attractant for predatory species or herbivore repellant, its capability to act as a priming

mechanism to incite the production of herbivore induced plant volatiles (HIPVs) has not been

examined. To examine if MeSA can intra-specifically induce production of volatiles beneficial

to insect attraction, juvenile hops plants were placed in environmentally controlled enclosures in

close proximity to MeSA controlled release dispensers over a 72-hour exposure period.

Immediately after exposure, replicate exposed plants (and respective control plants) were placed

into individual glass chambers. The chamber atmosphere from both MeSA exposed and control

hop plants were sampled by solid phase microextraction (SPME) to chemically profile volatile

emissions after the 72-hour MeSA exposure period. SPME collections were analyzed by gas

chromatography-mass spectroscopy (GC-MS) to identify volatile compound emissions. After

v

completion of the above examinations, the control hop and the treated hop plan were combined

within in a common enclosure to assay if MeSA primed hop plants could chemically “cross-talk”

with untreated plants to warn of impending herbivory. After this combined period, plant

chamber emissions were chemically profiled by SPME-GC/MS. Spectral profiling assessment

suggests MeSA exposed hops may increase production of the sesquiterpene, (E, E) α farnesene,

an identified HIPV. These year-to-year emission observations lend support to previous field

investigations that indicate airborne MeSA emissions may elicit production of HIPVs that can

serve as beneficial insect attractants. In the second combined control-MeSA hop treated

chamber evaluation series, the control plant head space volatiles showed a relative increase in

production of the sesquiterpene, α –caryophyllene, a known HIPV. The observed trend in

increased production of α–caryophyllene suggests a volatile signal(s) between plants in close

proximity may induce the production of chemicals needed in plant defense from insect

herbivory. Although the head space emission information collected from chamber evaluations

indicate relatively greater production of certain HIPVs after MeSA hop exposure, differences in

plant rearing (rhizomes versus soft wood cuttings), other environmental conditions (greenhouse

versus growth chamber plant rearing) likely resulted in the appreciable variation in emission

profiles among the replicated MeSA exposure trials conducted from 2007 through 2009. The

emission data herein should be viewed as qualitative. Certain evident trends in HIPV production,

however, bears further investigation. The experimental procedures developed herein should serve

to better design future evaluations for understanding and characterizing chemically induced plant

defense from herbivory.

vi

Table of Contents

Page

Acknowledgements…………………………………………………………………………iii

Abstract……………………………………………………………………………………..iv

Table of Contents…………………………………………………………………………...vi

List of Tables……………………………………………………………………………….vii

List of Figures……………………………………………………………………………...viii

Acronyms and Abbreviations………………………………………………………………ix

Preface………………………………………………………………………………………1

CHAPTER 1 Literature Review………………………………………………….4

Introduction…………………………………………………………………………5

Section I: Plant Response to Damage………………………………………………5

.

Section II: Recent developments and future applications for herbivore induced plant

volatile…………………………………………………………………22

Section III: Most recent controversies and trends in HIPV research……………...24

Section IV: Conclusions……………………………………………………….…..27

CHAPTER 2 Assessment of the Production of Herbivore Induced Plant Volatiles in

Juvenile Hops Using Methyl Salicylate…………………………….….40

1. Introduction…………………………………………….………41

CHAPTER 3 Final Conclusions and Future Work……………………….………66

APPENDIX A Analytical Summary Report……………………………….………71

vii

Tables

Chapter 2 Tables

Table 1 Summary of Compounds Collected in Experiment 1from unexposed (Control) and

Methyl Salicylate (MeSA) exposed juvenile hop H. humulus by treatment year….. 57

Table 2 Summary of Compounds Collected in Experiment 2 from unexposed (Control)

and Methyl Salicylate (MeSA) exposed juvenile hop H. humulus following 48 hours

combined exposure by treatment year…………………………………………….…58

Appendix Tables

Table 1 Sample Inventory and History………………………………………….……76

Table 2 Interval Data……………………………………………………………….…83

Table 3 Treated and Control Hop Replicate Data……………………………….……93

Table 4 Greenhouse Air Sample Data…………………………………………….…..97

Table 5 Treated and Control Plant Before and After Combined Exposure………….98

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Figures

Chapter 1 Figures

Figure 1 History of research on airborne plant-plant signaling…………………….…35

Figure 2 Impact of C6 Volatiles on Herbivore-Plant Interaction……………………..36

.

Figure 3 Volatile compounds from corn seedlings (Zea mays) undamaged (A),

first hour after feeding (B) and several hours after feeding (C)……………..37

Figure 4 Factors causing and traits affected by volatile-mediated signaling

among plants………………………………………………………………...38

Figure 5 Relative Distances for HIPV Emission ………………………………….…..39

Chapter 2 Figures

Figure 1 Chromatographic profiles of volatiles from unexposed (A) and MeSA

treated hops (B)……………………………………………………………….59

Figure 2 Structural identification of ten of sixteen compounds in head space……..….60

Figure 3 Structural identification of six of sixteen compounds in head space…….…..61

Figure 4 Volatiles from control plant by growth age……………………………..….…62

Appendix Figures

Figure 1 Comparative data from 3/22/07-Treated vs. Control………………………...103

Figure 2 Treated vs. Treated after 48 hrs combination…………………………….…..104

ix

Acronyms and Abbreviations

Compounds with six carbons C6

Green Leaf Volatiles GLV

Herbivore Induced Plant Volatiles HIPV

Irrigated Agricultural Experimental Research Center IAREC

Jasmonic Acid JA

Methyl Jasmonate MeJA

Methyl Salicylate MeSA

Salicylic Acid SA

Systemic Acquired Resistance SAR

(E,E)-4,8,12- trimethyl-1,3,7,11-tridecatraene TDTT

Volatile Organic Carbon VOC

1

Preface

The field of chemical ecology has been exploring the communication between plants

using chemical signaling since the 1980‟s. Early studies indicate that interspecies

communication existed as a function of perceived threat from insect damage. It was noted that in

interspecies communication certain chemical signals were emitted either via transpiration

through leaves or via a wound area caused by insect damage. This signal provided warning to

adjacent plants, which in turn continued the process and became part of a large network of

communication. These signals were relayed over a large area, providing plants of the same

species a means to develop defense mechanisms as well as provide a warning signal.

The concept of induced chemical plant protection as well as communication with

beneficial insects was considered controversial in the late 1970‟s. Studies in the early 1980‟s

indicated, however, that various secondary chemicals were emitted by plants that served as not

only alarm signals in the general vicinity, but were used by predatory insects to locate the

herbivorous insects causing injury to the plants. The concept of this plant-herbivore-predator

interaction provided the basis for an exciting research area in the study of plant-insect

interactions. Recent research in the field of chemical ecology has yielded a great deal of

information regarding the use of specific plant generated volatile chemicals as a green alternative

to pesticides.

The primary research addressed in this thesis is “can airborne plant exposure to methyl

salicylate (MeSA),”prime” inter and intra plant signaling by the production of herbivore

2

induced plant volatiles (HIPVs) in hop plants?” In conjunction with Dr. David James of the

Irrigated Agricultural Research Center (IARC) and his staff, particularly Larry Wright, juvenile

hop plants were exposed to MeSA air emissions from a commercially produced dispenser for a

period of 72 hours to assess the capability of this volatile compound to stimulate intra-plant

production of HIPVs. To assay if MeSA could trigger secondary metabolite production, the

treated plants as well as an unexposed control plant were placed in individual glass chambers

(developed by the James group) and transported to the Food and Environmental Quality (FEQL)

at Washington State University-Tri-Cities campus for analysis by solid phase microextraction

(SPME) and gas chromatography-mass spectroscopy (GC-MS). All lab analyses were performed

by FEQL Research Analyst, Jane Le Page. My primary role in the research was the collection

and interpretation of chemical emission data and reporting.

In this body of work Chapter 1 “Herbivore Induced Plant Volatiles and Chemical

Signaling- Effects on the Plant-Prey-Predator Interaction” discusses the relevant literature

regarding the plant synthesis of volatile organic compounds such as terpenes, fatty acid

derivatives and phenolics that provide “direct” defenses against attack from herbivorous pests, as

well as bacteria and fungi. These induced volatile emissions can also serve the plant in mounting

an “indirect” defense by attracting predatory insects while under herbivore attack. In turn, plants

in the vicinity of the stressed plant can “eavesdrop” on these SOS signals and use the signal to

begin mounting their own lines of defense. Chapter 2 “Assessment of Herbivore Induced Plant

Volatiles in Juvenile Hops by Exposure to Methyl Salicylate” is in the process of being

3

developed as a manuscript submission for the Journal of Chemical Ecology. Lastly, Chapter 3

discusses my final summary remarks and possibilities for future work. Appendix A is the

Analytical Summary Report. This regulatory science document provides a detailed overview of

the initial trials as performed in 2007.

4

Chapter 1

Herbivore Induced Plant Volatiles and Chemical Signaling

Effects on Plant-Prey-Predator Interaction

5

Introduction

Natural product chemical communication and the understanding of how this

communication affects plant-prey-predator interactions is an important aspect of chemical

ecology. While the above chemical communication is receiving increasing investigative

attention, considerable debate on the mechanisms of biosynthesis and function of these plant

volatile compound emissions still exists. This chapter review covers the plant‟s myriad of

responses to pathogen-insect injury, the production and biosynthesis of the herbivore induced

plant volatiles that include the C6 green leaf volatiles, the terpenes/terpenoids as well as other

plant secondary metabolites that can repel harmful but attract beneficial insects. The importance

of herbivore induced plant volatiles is highlighted; specifically their importance on plant-prey-

predator relationships and the impact of recent studies in the field of chemical ecology. My

intent is to provide a framework from the existing literature on the primary tenet of this thesis

“the ability of the secondary metabolite, methyl salicylate to “prime” intra and inter-plant

stimulus of the production of herbivore induced volatiles.”

Section 1: Plant response to damage

Plants emit volatile organic compounds in response to feeding by herbivorous insects.

These emissions vary from plant to plant, genotype and cultivar, species and age of herbivore

and time of the duress that the plant undergoes. Although mechanistically the purpose for these

6

secondary plant metabolites are not fully understood, assays have verified that emitted chemical

compounds act as toxins or repellents to drive away insect pest or provide antibiotic properties as

a defense against pathogens, may have developed a biosynthetic pathway to release volatile

chemical blend that were attractive to parasitic or predatory insects. (Turlings et al., 1995;

Walling, 2000). Research in this area of chemical ecology is rapidly growing. As an example,

Karban and Heil (2010) illustrates in Figure 1 the recent investigative history and acceleration of

plant-to-plant signaling research in forest, agricultural crop, and native plant communities.

Section 1.1 Direct and indirect response to herbivore damage

Section 1.1.1 Direct responses to herbivore damage

Plants have developed a wide variety of mechanisms to control injury from foraging

insects. Biosynthesized chemicals that provide an immediate negative impact against herbivore

damage are classified as direct defense compounds (Arimura et al., 2005). These defenses

include the production of terpenoids, alkaloids, and other biosynthesized secondary metabolites

which are toxic or repellent to the insect initiated through the production of defense genes

(Arimura et al., 2005). The plant phytohormone, ethylene, was suggested to be the chemical

messenger for the transport of volatile signals between adjoining plant systems, providing the

means to turn on defense genes in the plant as protection from herbivores (Farmer and Ryan,

1990). These investigators suggest that the defense genes initiate proteinase inhibitors to make

the plant less palatable to herbivores, and also acted as part of the plant‟s systemic alarm system

to carry the damage information throughout the entire plant. Although no biological evidence

7

existed at that time that any other compound than ethylene could provide atmospheric signaling,

exogenous application of the plant lipid compound, methyl jasmonate (MeJA) by these

investigators did elicit proteinase response in tomatoes (Lycopersicom esculentum) at a rate

much higher than exhibited by mechanical wounding. When treated plants were housed with

control plants, the controls developed low levels of proteinase inhibitor indicating that the

volatile methyl jasmine was affecting the control plants. Tobacco and alfalfa plants that were

also exposed to MeJA treated plants by these researchers expressed proteinase inhibition.

Section 1.1.2 Indirect responses to herbivore damage

Analogous to direct response, indirect responses are also induced by herbivore attack.

However, indirect responses initiate predator attraction to the herbivore through chemical

signaling. Herbivore injured leaves emit volatile compounds of varying types and amounts. The

release of plant volatile compounds is a cyclic process occurring primarily on a diurnal cycle.

(Turlings et al., 1995). Many plants release six carbon (C6) green leaf volatiles upon the first

indication of herbivore injury (Turlings et al.,1995). Other plants, such as cotton (Gossypium

hirsutum L.), store compounds in the leaves which are released from lysegenous glands upon

injury (Rose et al., 1996). The stored compounds largely exist as the C6 volatiles and mono and

sesquiterpenes. As the injury continues, secondary metabolites are biosynthesized for up to three

days following the injury. Identified metabolites include α and β farnesene, (E) -4, 8 dimethyl-1,

3, 7 nonatriene and the terpenoid, linalool (Rose et al, 1996). Cucumber plants undergoing

attack by spider mites show an increase in the terpenes, (E)-β-ocimene and (E)-1, 4-dimethyl-1,

8

3, 7 –nonatriene. Comparatively, the green leaf volatiles, (Z)-3-hexen-1-yl, and (Z)-3-hexen-1-ol

produced by non–infested plants comprise 35 to 50% of total volatiles. These researchers

observed that chemical concentrations were reduced to approximately 1% of total volatile

emissions in spider mite infested plants. An increase in the production of terpenes peaking

several hours following the initial damage and coordinated with the photocycle, indicate the

plant‟s ability to optimize production of volatile emissions at the time when the predator is most

likely in search of prey (Turlings et al.,1995). These herbivore induced chemical signals allow

predators to seek their prey by honing in on the volatile blend being emitted by the plant and

discriminately choose their prey.

Section 1.2 Providing a warning

Plants under attack can benefit from the presence of associated plants by emitting odors

that attracted predators, repel herbivores or mask the attractive odor of a host plant (Price et al.,

1980). Baldwin and Schultz (1983) observed that mechanically damaged poplar ramets and

sugar maple seedling emitted phenolic compounds. Wounded seedlings placed in proximity to

undamaged poplar and sugar maple seedlings emitted these compounds at a higher rate than the

true controls and continued to emit phenolic compounds for approximately 75 hours after

exposure. The authors suggested that ethylene may have played a part in the transmission of

these signals, since it is a product of wounding in many species of plant and has the capability to

influence the plant‟s ability to biosynthesize these phenolic compounds. This was the first

observation that plants may be employing volatile chemicals as a mode of communication.

9

However, the concept of plant derived compounds providing signals to chemically cross-talk to

adjoining plants was largely written off to “methodological grounds” by many (Dicke et al.,

1990). This was due in part to a lack of supporting quantifiable data to illustrate that the plants

were emitting chemical compounds and poor understanding of intra-interspecies chemical

signaling. However, later investigations performed on cotton plants indicated that this transfer of

chemical-based information between plants could be assayed. Cotton plants sharing air space in

close proximity to herbivore injured cotton plants emit similar amounts of the homoterpene, (E,

E)-4, 8, 12-trimethyl-1, 3,7,11 tridecatraene (Rőse et al., 1996). This data indicates a possible

induction factor from the injured plant to the control indicating impending damage. In 2006,

Karban et al., performed a series of experiments with sagebrush (Artemisia tridentata) to

determine the effectiveness of the transfer of volatiles from wounded plants to conspecifics in the

area. They concluded that while volatiles do in fact transfer information from a single plant to

adjacent plants, airflow is necessary to carry the volatile signal both to nearby plants as well

from one section of the plant to another. These airborne signals can affect plants up to 60 cm

(approximately 2 feet) away from the source. This is an increase of their initial estimate from

their first trials where they reported that volatiles were only carried less than an inch away

(Karban et al., 2003).

Section 1.3 Attraction of predatory insects

While the stimulation of the proteinase inhibiting genes was found to be directly

correlated to the plant chemical emission (Ryan and Green, 1990) the full breadth of the effect of

10

plant volatiles on herbivores and their natural enemies has yet to be established. It was proposed

that the relationship between the releases of volatile semiochemicals (A chemical emitted by a

plant or animal that evokes a behavioral or physiological response in another organism) were the

direct result of injury by herbivores (Pare and Tumlinson 1996; Dicke, 1999). The release of

semiochemicals triggered the movement of prey movement and provided the subsequent

attractants to predators. Herbivores use the presence of these airborne natural products to aid in

the decision to stay in a specific location and feed or take off and search for food. These plant

volatile emissions are important in the movement of the predator as well. Predatory mites will

remain in an area where prey related volatiles exist until they are no longer detected, and can

distinguish between volatile semiochemicals related to different species. In a 1994 study of the

bird-cherry-oat aphid, Rhopalosiphum padi, the compound methyl salicylate was found to inhibit

the settling of R. padi aphids on barley (Hordeum sativum) (Poacaea) and spring wheat, (Tricium

aestivum), when the plants were sprayed with methyl salicylate. Petterson and co-workers

(1994), hypothesized that the compound may have acted as a repellent to R. Padi, inhibiting

colonization of the aphid, thereby preventing overfeeding. This information coincides with a

study performed by Campbell et al. (1993), where they noted that while methyl salicylate as part

of a volatile blend containing hexenal and β caryophyllene emitted from hops was attractive to

the hop aphid, Phorodon humuli, increased levels of methyl salicylate eliminated positive

response. It was noted by these investigators that these volatiles increased as a function of aphid

population, and it is thought to have the effect of repellence to prevent over-colonization of the

aphids, therefore limiting food supplies. Price and others (1980) first recognized that plants

11

often communicate with beneficial insects via chemical emissions that are attractive to these

insects. These emissions may prevent herbivory to an adjacent species by providing a repellent

or masking odor that reduce attack from insect pests, while attracting the predatory insect to

them (Dicke et al., 1990). In order to initiate this relationship, plants provide predators with a

distinct blend of chemicals that provide an odor trail to lead them to the injured plant. This

release is a distinct response to herbivore injury releasing volatiles that incite a systemic response

in the plant (Turlings, 1995). However, in order for these chemical cues to be beneficial to the

predator, Turlings suggest that signals must be clear enough to clearly seek out their prey without

interference from background odors to in a reliable manner.

Herbivore induced volatile chemical blends must be produced in sufficient amounts to

attract the predator, specific to the plant species, and genotype. Dicke (1999) found that

volatiles in the headspace volume of Cox Orange Pippin apples infested with the two spotted

spider mite (Tetranychus urticae) contained the terpenes (E)-β ocimene and (E, E) –α farnesene

representing 5% of the total blend, while the headspace analysis of Summer Red apples infested

by the same species exhibited terpenoid levels that represented 25% and 55% of the total volatile

blend respectively. Specialist parasitoid wasps can distinguish between non-host and prey

infested corn, cotton and tobacco plants by responding to herbivore induced volatile emissions

(Arimura et al., 2005). This task becomes more difficult when the herbivore feeds on several

different plant species. Pare and Tumlinson (1999) have observed that specific chemical blends

are associated with a positive interaction with the host, increasing the wasp‟s attraction to the

specific volatile blend. It has been suggested that the volatile blend emitted by plants undergoing

12

herbivore injury may not be the only method beneficial insects use to identify herbivore

presence. Environmental conditions, competition from other insect predators-enemies,dietary

needs or behavioral factors can affect a predator‟s response to plant volatiles, therefore

questioning the reliability of the herbivore induced volatile blend as a valid indicator of

herbivore presence on predator attractions (Dicke, 1999).

Section 1.4 Herbivore Induced Plant Volatiles and Plant Signaling

Plants are capable of emitting complex volatile chemical blends upon damage from

wounding. Wounding can occur from mechanical damage from ripping or tearing, or from the

chewing action of herbivorous insects. In research conducted by Turlings and Tumlinson

(1992), corn seedlings (Zea mays) were mechanically damaged in manner that mimics feeding

injury from caterpillars of the beet armyworm (Spodotera exigua) by scratching the leaves of the

seedling with a razor blade, and placing the regurgitant of the caterpillar into the wounded area.

The damaged leaves were removed and placed into a volatile collection device and the headspace

volatiles collected. Similarly, undamaged leaves from the same plant as well as control plants

were sampled and analyzed. The leaves damaged by the caterpillar regurgitant responded as if

caterpillar damage had occurred producing the terpenoid: linalool, and the terpene (E)-1, 4-

dimethyl-1, 3, 7–nonatriene and β-farnesene. Undamaged leaves from the same plants exhibited

production of (Z)-3-hexen-1-yl acetate and indole. These investigators report that the production

of the volatiles, (Z)-3-hexen-1-yl acetate and indole indicate that plant defenses are systemic,

and are induced by the damaged leaves themselves. In a 2000 study by Ozawa et al., lima beans

13

Phaseolus lunatus were infested with beet army worm Spodoptera exigua, the common army

worm, Mythinma separata, and the phytophagous mite, Tetranychus urticae to induce herbivore

volatiles. A second series of experiments using un-infested lima bean plants were conducted to

evaluate if chemical induction can be associated with insect feeding injury. Aqueous solutions of

methyl jasmonate (MeJA), a product of the jasmonic acid (JA) pathway, together with aqueous

and gaseous methyl salicylate (MeSA) concentrations (salicylic acid (SA) pathway product)

were assayed. It was earlier thought that the JA and SA pathways were mutually exclusive.

However, application of aqueous MeJA to leaves produced volatiles that mimicked damage

invoked by S. exigua and M. separata, while leaves treated with aqueous MeSA emitted volatiles

similar to those from T. urticae damaged leaves. Surprisingly, the application of gaseous MeSA

followed by aqueous MeJA and followed with a second application of gaseous MeSA, produced

volatiles mimicking those elicited by T. urticae. This information was exciting in the

identification of the biosynthesis of volatiles based on the method of predator attack. This

research indicates that chewing herbivores elicit the JA pathway, while phloem feeding insects

elicit the SA pathway.

Other investigations show that the corn leaf aphid Rhopalosiphum maidis has a distinct

preference for the odor of healthy corn seedlings over those that have been treated with the

sesquiterpene, β-farnesene when offered both odors in a y-tube olfactometer. These choices

were elicited without the aphids having any physical or visual contact with the plants. β-

farnesene is a known aphid repellent and the clean air sample was overwhelmingly chosen by

aphids regardless of gender or age (Bernasconi et al., 1998).

14

Section 1.5 Biosynthesis and induction

Plant species differ in their chemical response to herbivore damage. The chemical

composition of volatiles vary among the type of plant tissue (cotton leaves, bolls), varieties and

cultivars (de Moraes et al., 1998), genotype (Loughrin et al., 1995), attacking herbivore

(Takabayashi and Dicke 1996; Dicke, 1999) and even the developmental stage of the herbivore

(Takabayashi et al., 1995). European corn borer larvae (2nd and 3rd

instar Ostrinia nubilalis)

were allowed to feed on corn seedlings. Another set of plants experienced the same feeding with

older more experienced larvae (6th

instar). Headspace analysis of the plants fed on by the young

caterpillars revealed a volatile blend that included green leaf volatiles, while the plants fed upon

by the older caterpillars released volatiles that were not significantly different than those seen in

mechanical wounding (Takabayashi et al., 1999). This data conflicts with information by

Turlings et al., 1995; Pare et al. 1998 who demonstrated that plant response to mechanical

wounding produced a much different volatile blend than insect feeding. (de Moraes et al., 1998)

It is apparent that the species of plant determines the type and timing of herbivore

induced plant volatiles. Corn (Z. mays) releases C6 green leaf volatiles initially when under

insect attack, as do tomato, potato and tobacco and others (Tumlinson and Waecker, 2004).

Cotton plants immediately release the terpenes, α-pinene and β-caryophyllene. This is likely due

to the terpenes acting as toxins or repellents to discourage further attack by herbivores. Higher

concentrations of (Z)-3-hexen-1-ol, α-pinene, (Z)-3-hexenyl acetate and β-caryophyllene were

exhibited by cotton plants after 24 hrs following damage. (de Moraes et al., 1998).

15

1.6 Biosynthesis of herbivore induced plant volatiles

The production of herbivore induced volatiles has been shown to serve a number of

functions, including the attraction of predators, the repellence of herbivores, a means of

communication among plants and the production of antimicrobial compounds to aid the plant in

wound damage. The production of these secondary plant metabolites cannot be fully understood

until we gain a better understanding of the biosynthetic pathways and the relationship of these

pathways in plant defense. While earlier research suggested that volatile organic compounds

were carried by ethylene gas (Ryan et al., 1980), research in the mid to late 1990‟s indicates

volatile plant emissions were produced by one of the three biosynthetic pathways- the

lipoxygenase (octadecanoid) pathway-producing the green leaf volatiles (which are not

systemically released, but are produced by plants when freshly cut or damaged), the jasmonic

acid pathway (known to initiate the plant defense system), the isoprenoid pathway (which

produces terpenoids), and the shikimic acid/tryptophanic pathway which produces indole and

methyl salicylate (Pare and Tumlinson,1995).

Section 1.7 Jasmonic acid, green leaf volatiles and the octadecanoic pathway

Jasmonic acid (JA) is biosynthesized by many plants by the octadecanoid pathway. This

pathway produces the green leaf volatiles produced by corn and other plants immediately

following herbivore injury (Turlings et al., 1995; Figure 2).

16

JA has a dual role in the production of herbivore induced plant volatiles. As the end

product of this biosynthetic pathway, JA appears to be the key information pathway in plant

discrimination between mechanical wounding and herbivore injury. Exogenous application of

JA were found to elicit a volatile blend that more closely resemble that from herbivore damage

and produced defense gene initiation that is similar to observed production following herbivore

damage (de Bruxelles et al., 2001). Wild type lima bean plants treated with exogenous JA

effectively reacted to treatment that mimicked herbivore feeding, and suffered less from the

consequences of herbivore attack than control plants (Heil, 2004). JA has been shown to attract

predatory mites and prohibit embryonic development and increase egg mortality in the two

spotted mite, Tetranychus urticae on tomato plants (Ament et al., 2004) and JA increased the

attraction of the parasitic wasp Anagrus nilaparvatae to rice plants and decreased the settling

behavior of the brown leaf hopper Nilaparavata lugens. Volatile production from infestation

from N. lugens and the application of JA was shown to produce different volatile blends and it is

thought that N. lugens feeding does not trigger the JA pathway. This information indicates that

different pathways may cause the production of different volatile blends, however the resultant

volatiles are attractive to the parasitic wasp (Lou et al., 2005).

Research performed with cis-jasmone (3-methyl-(cis-2-penten-1-yl)-2-cyclopentyl-1-one)

indicates the compound is beneficial as an aphid repellent, and as an attractant for aphid

predators such as the mite eating ladybeetle, Stethorus punctum picipes. Laboratory trials and

electroanntenaography studies indicate that cis-jasmone is far better as an attractant for predators

than JA and further increases plant defense gene production over JA (Birkett et al., 2000). To

17

assess the possibility of the cis-jasmone on the predatory wasp, Cotesia marginventris, a known

parasitoid of the beet armyworm, wind tunnel tests were used to facilitate the wasps‟ choice

between control plants, mechanically damaged plants and those mechanically damaged and

treated (Turlings and Tumlinson, 1992). Overall these researchers found that the wasps showed

a distinct preference for the treated plants indicating the plant emissions distinctly attracted the

wasp. C6 Green leaf volatiles are the precursors for JA production. The C6 volatiles are normally

emitted at low levels by healthy plants and are released rapidly following herbivore or

mechanical damage. These compounds stimulate wound response gene production, reduce

reproductive rates in aphids and spider mites and reduce caterpillar feeding, and can be

attractants for the Colorado potato beetle and specialist aphids. C6 volatiles also have

antimicrobial and antifungal properties (Walling, 2000; Figure 3).

Mechanical damage from chewing insects elicits different response from that of sucking

or piercing insects. Phloem feeding insects such as aphids and whiteflies do not mechanically

damage the plant tissue in the same manner as do the chewing caterpillars and beetles. These

insects as well as those classified as cell content feeders, puncture the plant cells and rupture the

membranes. Since this method of feeding does not impact the plant in the same manner as the

chewing damage of most herbivores, the plant does not often recognize this insect attack, instead

it is perceived as a pathogen (Walling, 2000). The pathogen response initiates the JA pathway

and the SA pathways which stimulate the production of defense genes and provide secondary

metabolites such as MeSA, a known aphid repellent (Shulaev et al., 1997, Bernasconi et al.,

1998). The production of the volatiles MeJA and MeSA is the result of these types of herbivore

18

piercing injury. Corn seedlings (Z. mays) exposed to caterpillar induced green leaf volatiles, (Z)-

3- hexenal, (Z)-3-hexenol and (Z)-3-hexenyl acetate induced increased production of JA

(Engelberth et al., 2004).

The C6 green leaf volatiles can be used to determine the presence of pathogenic

bacterium or molds affecting plant species. Lima beans release the C6 volatiles 3-hexenol and

(E)-2-hexenal 15-24 hours following inoculation with Pseudnomona syringae pv.phaseolicoa

and pepper leaves release similar C6 aldehydes and alcohols following inoculation with the

bacterial pathogen Xanthanmonas campestris pv. vesicatoria (De Moraes et al., 2004). The

cassava green mite (Munonychelhs tanajoa ) feeds on the cassava plant. Cassava is an important

food crop in the tropics and losses due to the herbivore damage are costly. The mite‟s natural

enemy is an entomopathogenic fungus, Neozygites tanajoae, which is found on the plant and

attacks by the emission of spores which are fatal to the mite. A number of green leaf volatiles

affect the rapid sporulation of the fungi. These included Z-(3) –hexen-1-ol; the terpenes E-

(trans)β- ocimene and (E)-4,8 dimethyl 1,3,7 nonatriene and, the terpenoid linalool as well as

methyl salicylate (Hountondjietal et al., 2005).

Section 1.8 The isoprenoid pathway- the terpenes and the terpenoids

The majority of all volatiles systemically released by herbivore feeding are terpenes (Pare

and Tumlinson, 1996), The most commonly identified terpenes from herbivore injured plants

include (E,E)-4,8,12-trimethyl-1,3,7,11 tridecatraene (TDTT) and (E,E)-1,4-dimethyl-1,3,7 –

nonatriene. These compounds appear in the herbivore induced volatile blends of many plant

19

species including corn, lima beans, tomatoes, tobacco, cotton and others. Cotton plants

damaged by herbivores release relatively high amounts of the terpenes: (E,E) –α-farnesene, (E)-

β-ocimene, (E,E)-4,8,12-trimethyl-1,3,7,11 tridecatraene (TDTT) and (E,E)-1,4-dimethyl-1,3,7 –

nonatriene and the terpenoid, linalool. While herbivore wounding initiates the release of the

terpenoids, β-caryophyllene and α-pinene, which are stored in the leaves (Rodriguez-Saona et al.,

2002) the sesquiterpenoids such as linalool are released hours following continual insect injury.

This indicates that the compounds are biosynthesized via a pathway that allows the plant to

produce these compounds de novo to purportedly enhance the attraction of predators (Rőse et al.,

1996). Corn seedlings (Z. mays) produce a volatile blend while undergoing herbivore attack

from the pea aphid, Acyrthsiphon pisum that attracts the twelve spot lady beetle, Coleomegilla

maculata and the green lacewing, Chyrsoperla carnea (Zhu et al., 1999). The components of the

blend include the sesquiterpenes, β-farnesene and β-caryophyllene and the C6 volatile 3-hexen-

1-ol (Z). The above predator species strongly responded via electroanntenography to β-

farnesene above all others. This is likely due to the fact that this terpene is a known aphid alarm

signal, allowing the predators to seek out their prey by using the volatile as a scent trail (Zhu et

al., 1999).

Section 1.9 Methyl salicylate as a volatile semiochemical

Methyl salicylate (MeSA), the methyl ester of salicylic acid, another common plant

volatile was the subject of further research for its role in chemical signaling. In a 1997 study by

Shulaev et al., tobacco plants inoculated with the tobacco mosaic virus exposed to airborne

20

methyl salicylate displayed increased disease resistance. When these treated plants were housed

with control plants, the exposed plants transmitted signals that increased the production of the

defense mechanism gene in controls (Shulaev et al., 1997). This would indicate that plant

volatile production is a systemic response and is not simply located within the individual plant‟s

boundaries, and that they are capable of influencing like species of plant that are in the vicinity

of the threat. While both JA and SA play important roles in the defense of plants against

herbivore attack both directly and indirectly, there is a differing opinion in the literature on the

benefits of each. The JA pathway may inhibit the SA pathway which provides a separate

secondary pathway to the induction of defense genes in response to wounding (Walling, 2000).

SA levels are not affected by mechanical wounding, indicating that salicylic acid is a secondary

elicitor of defense gene production (Walling, 2000). Lima beans undergoing attack by the two

spotted spider mite (T. urticae) exhibited a different volatile response than leaves treated with the

exogenous application of JA (de Bruxelles et al., 2001). This response could not be replicated

until the application of methyl salicylate was applied or application of jasmonic acid followed by

methyl salicylate was used. This response indicates that some species such as (T.urticae) elicit a

duel response through the JA-SA pathway. Other recent research indicates that there may be an

antagonistic relationship between the JA and SA pathways. Traditionally, the SA defense

pathways are activated in defense of pathogenic attack, while JA dependent defenses are initiated

by herbivore wounding. The SA and JA pathways are considered to be mutually exclusive, each

providing its own defense mechanism. Recent evidence has shown that cross talk between the

pathways does exist and can be detrimental to the plant. Tomato plants exposed to BTH

21

(benzothiadiazol), a defense mimicking chemical, exhibited increased resistance to the pathogen

P. syringae, but increased optimum feeding conditions for the corn ear worm (Heliocoverpa zea)

(Piertse et al., 2001). In contrast, increased SA levels have been shown to lower resistance from

attack by spotted cucumber beetles while undergoing pathogen induced systemic acquired

resistance from the fungus, Colltotrichum orbiculare (Piertse et al., 2001).

Many predatory insect species including the predatory mite, Phytoseiulus persimilis, the

green lacewing, Chrysopa nigricornis and the predatory bug Anthocoris nemoralis are attracted

to MeSA (James, 2003b). MeSA baited traps were placed in juice grape vineyards and hop yards

by James and Price (2004) to assess the attractiveness of predators of two spotted mite (T.

urticae) and hop aphid (Phorodon humuli). Collection of both prey and predators were

performed using sticky bait traps, canopy shake and leaf sampling and then assessed. Four

families of insects including hover flies, wasps, dance flies and flesh flies along with a

significant number of parasitic wasps were trapped on the sticky traps in the methyl salicylate

baited vineyard blocks. The hop yard baited traps also yielded high numbers of the mite eating

ladybird beetle, S. punctum picipes and the predatory bug, Orius tristicolor. These numbers

coincided with a decrease in aphid and mite numbers. It was noted by James that predator

numbers reached the highest concentration in late July. This timing may be a function of the

higher airborne MeSA concentrations that occurred in the vineyard during spring that may have

been a deterrent to some predators (James and Price, 2004).

22

Section 2.0 Recent developments and future applications for herbivore induced plant

volatiles

Plant specific volatile emission blends have been proven to attract predatory

insects to food sources without visual or vibrational cues that may otherwise be an attractant.

But is the primary function of chemical signaling the communication between plant and

predator? Or are there multifaceted uses for chemical signaling? Is it possible that plants may

use chemical signaling as a warning to potential threat to “keep off”? Often times, the chemical

signaling serves a third and very distinct purpose as a repellent, therefore keeping potential

attackers or females seeking a place for their eggs at a safe distance. Plant volatiles may provide

parasitic wasps cues on the location of their hosts. The parasitic wasp, Trissolcus basalis, uses

plant volatiles to locate plants damaged by and containing egg masses of their prey, the southern

green stink bug, Nezara viridula. Undamaged leaves or egg masses alone were unattractive to

the wasp, as were newly hatched larvae. This information clearly links the production of plant

volatiles as a consequence of the specialized situation of herbivore damage and oviposition

(Colazza et al., 2003).

The research of the past twenty years has brought a new wave of interest. Could

biosynthetic volatiles be manufactured and combined and used to draw beneficial insects? The

need to perform field trials was understood to be the best way to assess the effect of volatiles on

predators. In a follow up field study of his 2003 and 2004 experiments, James (2005) tested

fifteen synthetic HIPV‟s in an open field and a hop yard in Washington State. The experiment

23

consisted of a number of sticky cards baited with the synthetic volatiles. James noted that eleven

different species and families of insects were attracted to thirteen of the synthetic volatiles.

It is apparent that identifying the specific attractant(s) for predatory and parasitic insects

can serve as a tool in integrated pest management. The recruitment of specialized natural

enemies can affect pest populations and reduce agricultural losses. Augmenting the biosynthesis

of plant production of specific volatiles, priming plants with volatile blends that are attractive to

predators, and the production of synthetic herbivore induced volatile blends are all being

investigated. To date, the mouse ear cress (Arabidposis thaliana) has been transgenically

modified to change the C6 green leaf volatile production following herbivore attack by cabbage

butterfly larvae (Pieris rapae), as well as the response to grey mold Botyris cinerea (Halitscke et

al., 2008). The parasitic wasp, Cotesia glomerata, is known to be attracted to the green leaf

volatiles (Z)-3-hexenal and (E)-2-hexenal. Following wounding, (Arabidposis) demonstrated a

thirty fold increase in the production of (Z)-3-hexenal following wounding (Shiojiri et al., 2006).

Two strains of the plant were genetically modified. One strain was modified to enhance C6

volatile production to twice the level of that of the control plants and the other modified to

suppress C6 volatile levels.(≈ 25% of controls). A similar study performed with genetically

volatile inhibited wild tobacco plants (Nicotiana attenuata) dramatically decreased attraction of

the generalist predator Geocoris pallens (Halitschke et al., 2008).

The use of herbivore induced plant volatiles to prime plants is attracting a great deal of

interest. The knowledge of the plants use of these signals and their uses in the application of

agricultural pest control are key areas of research for improving crop management and

24

improvement of plant defense. This concept has garnered worldwide attention and interest,

particularly in countries such as Brazil that rely heavily on agricultural products (Arab and

Bento, 2006). Manipulation of the expression of volatiles from plants as a means to attract

predatory insects is a beneficial tool in the control of insect pests, but more research is necessary.

Further research in crop plants must be preformed and the volatile chemical signals used must be

carefully selected to target only those species that effectively control the desired pest population,

as well as consideration of the effects that these volatiles may have upon neighboring species

(Turlings and Ton, 2006). In order to successfully use volatiles as a recruitment tool for pest

control and crop management, the plant must be able to identify and prioritize these signals to

their advantage (Choudary et al., 2008). For example, (Z)-3-hexenol, was found to be the

primary attractant for the parasitic wasp Opius dissitus, in the control of the pea leafminer,

Liriomyza huidobrensis, a pest that has invaded all zoogeographic regions and has a broad

variety of hosts (Wei et al., 2007).

Section 3 Recent controversies and trends in HIPV research

Section 3.1 HIPV’s act as priming agents for intra-plant signaling

Heil and Silva Bueno (2007) specifically identify the so-called green leaf volatiles as one

of the most important series of compounds for plant signaling In their estimation, the fact that

these compounds are gaseous and easily transported through the air aids in the contact of more

plant area than any of the other lesser volatile herbivore induced secondary metabolites. While

the question of benefits to the signaling plants fitness remains an important topic, it does appear

25

that within plant as well as within a community of plants, all reap some benefit from the

herbivore induced chemical communication. They hypothesize that C6 volatiles may be the

quickest method to intra-specifically prime the plant‟s defense system-even quicker than

systemic defense hormones JA and SA thereby improving the timing of response. Plant internal

signals would be much slower traveling through the xylem and phloem. In this investigation, the

authors‟ state that what has previously identified as plant to plant signaling is perhaps “plants

„eavesdropping‟ on what is within plant signaling worn on the outside.” In their 2008 study,

Frost et al., (2008) further questioned the “communication” aspect of herbivore induced

volatiles. It was noted that plants that receive the message of impending herbivory, by the

presence of herbivore induced plant volatiles begin the defense mechanisms necessary to ward

off the threat. But unlike the plant under attack, the receptor plant pays fewer costs to overall

fitness than the plant sending the message out. Furthermore, this study calls out the differences

between “priming” and “induction” as two separate functions, citing the application of C6

volatiles to tomato plants, which in turn triggered the release of a number of volatile terpenes,

but direct defenses were not measured and priming effects could not be inferred. Frost and co-

workers further clarified the difference by stating that “if induction of volatile compounds in

response to herbivory is a cry for help, the induction of volatiles in response to a volatile signal

may be more of a whisper, which appears to be correspondingly less attractive to predators”. In

a 2008 addendum to the article, Frost et al., further clarification on the specialization of HIPVs

can have on plant behavior was discussed. In particular, the C6 volatiles were previously

believed to provide the signal from plants under going herbivory as a “cry for help”. While

26

capable of stimulating both priming and defenses in a number of plant species, they do not

appear to convey “any context–dependent information” (Frost et al., 2008). The problem with

these compounds, as was pointed out by Heil and Bueno is that unlike the sesquiterpenes, the C6

volatiles are rapidly dispersed and quickly dissipated thus making them excellent messengers for

the interplant priming capabilities. Frost et al. (2008) hypothesized that the reason for this is the

fact that the C6 volatiles primary purpose is to act as an intra plant signal for priming. This

function alleviates the problem of conflicting information from nearby plant communities who

have different enemies releasing the same or similar HIPV‟s. But as the authors point out, the

HIPV‟s including the C6 volatiles are capable of being detected by parasitoids and predators, up

to tens of meters away, indicating that volatile transport distances may not be the limiting factor.

It is more likely a function of the receiving plants capability to detect and employ the signal at

some threshold concentration. Frost et. al. (2008) proposed that this threshold concentration

may vary widely due to the physiological differences in the ability of insects and plants to detect

and use these signals. This purported ability would allow the receiving plant to ignore the signals

of a distant plant–which may not be providing accurate information on impending herbivore

injury (Figure 5). This information appears to fall in line with Dicke and Baldwin (2009) who

proposed that perhaps the absence rather than the presence of a volatile signal may in fact

provide relevant information.

27

Section 3.2 Production of Herbivore Induced Plant Volatiles and Fitness

Consequences

An interesting concept that requires a great deal of further study is the question of the

fitness of the emitting plant, a topic that was recently addressed by Dicke and Baldwin (2009). .

The fitness of the HIPV emitting plant to date has never been assessed, likely due to the inability

to accurately measure the exchange of volatiles between plants in the field. While evidence

exists that both lima bean tendrils (Heil and Silva Bueno, 2007) and sagebrush (Karban et al.,

2003), exhibited increased defenses following exposure to volatiles from herbivore and

mechanically injured plants, the effect on the emitting plant still remains unanswered. More

likely, the emitting plant is more subject to herbivory than its uninjured neighbor, and improving

its neighbors fitness may in fact make it even more attractive to attack. Heil and Karban (2009)

recently posed the question “should the phenomenon therefore be termed „eavesdropping‟ rather

than „communication‟? Likely, the answer may be yes, since it is hypothesized that plant

signaling evolved from intra plant signaling and over time, plants may have developed receptors

to use the signal to benefit themselves and other within a community. Dicke and Baldwin (2010)

agree with this concept stating that until a HIPV “deaf” and “mute” plant species is developed it

will be difficult to discern the effects of HIPV‟s as a part of a community, and the overall effects

of the compounds holistic effect on the plant-rather than the individual tri-trophic interactions.

28

Section 4 Conclusion

The production of herbivore induced plant volatiles is an important area for research.

The understanding of the effects of the production of these compounds as a function of overall

plant fitness as well as the effects of the production of these compounds is an area for more

research.

The study of the production of these compounds reaches across many fields of interest;

chemistry, biology, ecology, entomology and agriculture. The increasing concerns with the

effects of agricultural chemicals on human health and the environment make this an important

topic for further research.

Recent information in the literature indicates a trend into the investigation of the

determination of the mechanisms of interplant signaling and the concepts of eavesdropping by

nearby plants. This is an area that is rapidly expanding in both basic and applied integrated pest

management research for conservation biology.

29

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35

Figure 1: History of research on airborne plant-plant signaling from Karban and Heil

36

Figure 2 Impact of C6 Volatiles on Herbivore Plant Interaction from Walling, 2000

37

Figure 3:Volatile compounds from corn seedlings (Zea mays) undamaged (A), first hour after

feeding(B) and several hours after feeding (C) Turlings and Tumlinson, 1992

38

Figure 4: Factors causing and traits affected by volatile-mediated signaling among plants

from Karban and Heil, 2010

39

Figure 5: Relative distances for HIPV emissions from Frost et al., 2008

A schematic representing differences in the relative distances over

which parasitoids and plants can respond to herbivore-induced volatile

(HIPV) emissions. The herbivore-wounded plant (far left) is wounded by

herbivores and releases HIPVs (represented by gray arrow). Based on our

recent work and work from other systems, systemic regions of the wounded

plant can respond (☑) to these HIPVs. Undamaged conspecific and heterospecific

neighboring plants close to the wounded plant may also respond (?)

to the HIPVs in what has been termed „eavesdropping‟. However, parasitoids

evidently respond to HIPVs from a greater distance than do plants. A better

understanding of the mechanisms of HIPV reception in plants is required to

understand the basis for such apparent distance limitations on plant-to-plant signaling

40

Chapter 2

Assessment of Herbivore Induced Plant Volatiles in Juvenile Hops

by Exposure to Methyl Salicylate

41

INTRODUCTION

Methyl salicylate (MeSA), the methyl ester of salicylic acid, is a secondary plant

metabolite (Hardie et al.,1994, Pare and Tumlinson 1996 and Walling 2000) MeSA has been

found to be an initiator for the production of defense genes against pathogens (Shulaev et. al,

1997) as well as an airborne plant signaling compound (Dicke et al., 1990; Bernasconi et al.,

1998, Hardie et al., 1994, Pickett et al., 2003, Karban et al., 2003, Kessler and Baldwin, 2001,

Arimura et al., 2005, Ozawa et al., 2000). Along with MeSA, a number of other volatile

compounds are released when plants are damaged by feeding from herbivores. These compounds

commonly known as herbivore induced plant volatiles (HIPV)‟s are believed to provide

communication between plants giving warnings of possible insect infestation, allowing plants to

defend themselves (Choudhary et. al. 1998, Dicke, 1998, Dicke 1999, Karban and Baldwin

1997).

MeSA has also been identified as an attractant for beneficial insects (Turlings and Ton,

2006, Turlings and Waecker, 2004; Zhu et al., 2007; James 2003, 2003a; James and Price, 2004;

Heil and Bueno, 2007) including the predatory mite, Phytoselius persimilis, the green lacewing,

Chrysopa nigricornis, and the predatory bug, Anthocoris nemoralis (James 2003a, James and

Price 2004, Hardie et. al 1994). Olfactory studies performed by Hardie et al., 1994 and

Bernasconi et al., 1998 indicate that MeSA also repels several aphid species including the black

bean aphid, Aphis fabae.

The specific use of MeSA as a signaling attractant of beneficial insects has received

attention but has not been thoroughly investigated. To examine the use of MeSA as an attractant

42

to beneficial insects, James and Price, (2004) placed MeSA dispensers in a 10 ha juice grape

(var. Concord) vineyard and a ca. 1 ha hop yard during the growing season. Analysis of

populations of predatory and herbivorous arthropods demonstrated a sharp rise in beneficial

insects in MeSA treatment compared to untreated crop areas. As a follow-up to field

examinations of James, we focused on a controlled environment-chamber study design to

address the following questions: (1) does MeSA exposure initiate intra-plant production of

herbivore induced volatiles?, and (2) can exposure to MeSA stimulate inter-plant signaling? To

address the first question, juvenile hop plants were exposed for 72 hours to dispensers emitting

MeSA then evaluated to determine if production of HIPVs could be stimulated. To address our

second question, the MeSA exposed hop plants were combined with non-exposed control hops

plants to assay if volatile emissions from the treated plants could in turn stimulate HIPV

production in control plants. HIPV emission profiles were obtained from glass chambers by

sampling the air headspace volatiles of exposed, combined, and control hop plants using space

solid phase microextraction (SPME). Mass spectral assessment of chromatographically resolved

volatile emission peaks were performed using gas chromatography–mass spectroscopy (GC-

MS).

METHODS and MATERIALS

Growing conditions for MeSA exposed and non-exposed control hop plants: Juvenile hops

(Humulus lupulus L.) Var. Chinook were grown from rhizomes under greenhouse conditions

(16:8 day:night, average temperatures 18.9-36.7 ºC) for the 2007 and the first three experiments

43

of 2008 . Hop plants were exposed to MeSA when they reached a height of approximately 10-12

cm (ca. 30 day growth). In the later experiments of 2008 and in the 2009 trials, juvenile hops

grown from root cuttings were used. These root-cutting plants were ca. 60 days old before

exposure to MeSA. Root cuttings were grown in environmentally controlled growth chambers

at 25 ◦ C and an 8:16 L: D photoperiod.

Plant Treatment: For all evaluations, Hop plants were separated into control and treated

environmental growth systems. In 2007 greenhouse evaluations, single plants were exposed to

air concentrations of methyl salicylate from impregnated sachets for approximately 72 hrs.

Following exposure, both treated and control plants (from separate greenhouses) were enclosed

in separate 2L borosilicate glass chambers (Figure 1). Two slotted 10 cm x 10 cm aluminum

base plates were aligned to minimize ambient air leakage and possible cross-contamination. The

glass chambers were then strapped down to secure them to the base plates. The chambered

plants were transported to the Washington State University –Tri Cities Food and Environmental

Quality Lab (FEQL) for SPME GC/MS spectral determination of chamber head space volatiles.

The experiment was replicated 4 times in 2007, 6 times in 2008 and three times in 2009.

44

Figure 1: Juvenile Hops in head space chambers

Volatile Collection and Analysis In all trials, the 2L plant-chamber systems were allowed to

equilibrate to room temperature ca. 2 hrs before the first series of headspace evaluations

(Experiment 1). The 2L glass chambers were outfitted with septa to allow collection of volatiles

by solid phase micro extraction (SPME; Figure 2). After 48 hrs, the control and treated plants

were placed into a common 10 L glass container and the two plants remained side-by-side for 48

hrs, at which time they were separated and placed into individual chambers (Experiment 2).

These separated chambered plants were allowed to equilibrate ca. 4 hrs before headspace SPME

sampling to examine possible inter-plant chemical signaling.

45

Figure 2: SPME sampling septa

A Carbowax-DVB film SPME fiber (Supelco, PA, USA) was inserted into the septa and

exposed to the headspace air for 5 minutes. The SPME fiber was removed then immediately

thermally desorbed at 200 C for 30 seconds into the injection port of an Agilent 6890 Gas

Chromatograph connected to a 5973 Mass Spectrometer. Analyses were duplicated at each

timed interval and performed on both treated and control plants. All analyses were performed

using the Agilent 6890 Gas Chromatograph with the 5973 Mass Spectrometer. Compounds were

desorbed into the injection port using a pulse splitless mode at 20⁰ C into an Alltech EC-WAX

column (30m x 0.32 mm I.D. x 0.25 μM film thickness). After an initial column temperature of

50 ⁰C for one minute, the temperature was raised 5

⁰ C/minute to a final temperature of 260

⁰ C for

46

5 minutes. The column was directly coupled to the ion source of the Agilent 5973 Mass

Spectrometer which was operating in EI (electron ionization) mode scanning from mass 50 to

350 amu at 2.5 scans/ sec. Spectra for the resolved compounds were searched using the National

Institute of Standards Technology (NIST) 98 library where they were compared for best match.

The percentage of total area counts associated with each individual resolved peak was noted and

compared against total area count among all combined peak areas in an attempt to chemically

profile the relative contribution of each compound spectrally identified. It is important to note

that the area counts are not used for quantification of the identified compounds, but as a means to

characterize the instrument response at each interval and to compare the response between the

treated and control plants at each sampling interval.

Results

Table 1 and 2 summarize the proportional contributions of HIVPs emanating from

unexposed control and MeSA exposed juvenile hop plants. Table 1 provides profile data for

plants exposed or not exposed to MeSA (Experiment 1), while Table 2 provides profile data for

non-exposed plants from the first experiment after combination with the MeSA-treated plants

(Experiment 2). Figure 1 chromatographically displays separation of 6 of the 16 reported HIPVs

observed during the course of this study. Figure 2 shows the differences in identified HIPV

emissions from juvenile control plants raised as rhizomes (2007-2008) and from root cuttings

(2008-2009).

47

Experiment 1: 2007 evaluations of volatiles from MeSA exposed hops

During March-May 2007, a total of four sets of juvenile hops (treatments and controls)

raised under greenhouse conditions were sampled and the headspace volatiles analyzed after

exposure to MeSA. Each area count in Table 1 represents the average response from duplicate

evaluations conducted for each interval treated and control chamber sample.

Controls: A total of ten compounds were identified in the head space analysis. The

control plant volatile blend consisted largely of the green leaf volatile 3-hexen-1-ol, (Z) acetate,

(ca. 34% of total area counts), and the alkyl aldehydes, nonanal and decanal (ca. 3% and 4%,

respectively). Low to moderate levels of the sesquiterpenes, iso-caryophyllene, α–caryophyllene

and α – farnesene were also present (ca. 2%, 6% and 2%, respectively). Best match spectra

identified germacrene B at 3%. Methyl salicylate comprised ca. 4% of total area counts.

Treated: The sesquiterpene, (E, E) α–farnesene showed a three-fold higher level (2% -

6%) than in the control. Methyl salicylate comprised ca. 17% of total area counts, > 4 times

higher than the control. Total area counts were lower in 3-hexen-1-ol, (Z) acetate (ca. 25%),

while the nonanal area counts were slightly higher at ca. 7%. Decanal counts were slightly lower

at 2%. Overall levels of α–caryophyllene were the same as the control (6%), while iso-

caryophyllene levels decreased slightly (1%).

Experiment 1: 2008 evaluations of volatiles from MeSA exposed hops

A series of six sets of control and MeSA exposed treated chamber evaluations was

conducted in 2008. In the first three trials, the juvenile hops were grown under similar

48

greenhouse conditions as in 2007. However, in the latter trials of 2008, (9/20-10/2), hop plants

were grown in individual growth chambers to exclude thrips injury to foliage. All other

handling, including sampling and analysis were performed as before.

Controls: A total of ten volatile compounds were identified. Volatiles from control plants

showed lower amounts of 3-hexen-1-ol, (Z) acetate (21 % of total area counts), α-farnesene (2%)

and α- caryophyllene (4%) compared to 2007 control plants. Several long chain fatty acids:

nonanoic acid (ca 10%), tetradecanoic acid (ca.3%) and the fatty acid alcohol, dodecanol (ca.

4%), as well as the sesquiterpene germacrene B (ca.3%), were present. A C6 volatile, 3-hexen-

1-ol, contributed trace (ca.<1% ) amounts to total counts. MeSA contributions comprised ca. 3%

of total area counts.

Treated: Nine compounds were identified in the treated plant head space analysis. The

contribution of 3-hexen-1-ol, (Z), acetate (ca. 25 to 29%) was slightly higher than in the control

plant. The sesquiterpene, germacrene D (ca. 2%), MeSA (7%) and α-farnesene (ca. 1%) were

also higher than the control. Spectral contributions from α–caryophyllene (trace), tetradecanoic

acid (ca. 1%) and nonanoic acid (trace) were lower than obtained with the control plant.

Experiment 1: 2009 evaluations of volatiles from MeSA exposed hops

Control: A total of five compounds were identified. The C6 volatiles 3-hexen-1-ol, (Z),

acetate and 3-hexen-1-ol proportionally contributed ca. 36 % and 2% to the total area counts,

respectively. The sesquiterpene, α-caryophyllene contributed ca. 3 % and β-caryophylllene, was

49

present in trace amounts. α-farnesene, contributed ca. 4% and MeSA contributed ca. 3% to the

total area counts.

Treated: A total of six compounds were identified. The C6 volatiles 3-hexen-1-ol, (Z),

acetate and 3-hexen-1-ol contributed 41 and 2% to the area counts, respectively. α-

caryophyllene occurred at trace levels while MeSA was not detected. α-farnesene was present at

ca. 5 % of total area counts. Figure 3

Experiment 2: 2007-2009 evaluations of volatiles from unexposed hops after exposure to hops

previously treated with MeSA.

To determine if MeSA exposed hops use the elevated expressions of volatile signals to

provide a conspecific plant with information regarding potential herbivore attack, we combined

the control and treated hops from the first experiment commencing in the latter trials of 2007.

In 2007, volatiles from control plants (i.e. the control plants from the earlier experiment)

following exposure to the MeSA treated plants comprised five compounds: 3-hexen-1-ol, Z

acetate: nonanal, decanal, iso-caryophyllene and α-caryophyllene. These compounds yielded 2%,

9%, 21%, 4% and 6 % respectively to the total area counts, with all but 3-hexen-1-ol, Z acetate;

and α-caryophyllene showing elevated levels compared to the levels prior to exposure to the

treated plant. α-farnesene, MeSA and the fatty acids, nonanoic and tetradecanoic acid, as well as

dodecanol were non-detectable. Analysis of volatiles from the MeSA-treated plants showed

higher levels of 3-hexen-1-ol, (z) acetate (ca.12%) and nonanal (ca..10%) than the control plants

but lower levels of decanal (ca.19%) α –caryophyllene (ca. 3%) and iso caryophyllene (ca2%).

50

In 2008 higher levels of α –caryophyllene (ca. 23%), MeSA (ca. 4%) and nonanoic acid

(ca. 10%) occurred in volatiles from the control plants after exposure to the MeSA treated plants,

compared to previous levels. Levels of the C6 volatile 3-hexen-1-ol, (Z) acetate (ca. 20%) and

3-hexen-1-ol (ca. 5%) were similar to previous levels.

In 2009 levels of α-caryophyllene (ca. 25%) and β caryophyllene (9%) were higher in

the control plants after exposure to MeSA treated plants than before exposure (ca. 4% and not

detected, respectively). In the MeSA-treated plants the presence of β-caryophyllene (ca.10%), α-

farnesene (ca. 8%) and α-caryophyllene (ca 29%) were notable.

Discussion

The data provided here suggest that young hop plants exposed to airborne MeSA are

stimulated to produce certain volatiles at elevated levels compared to non-exposed plants. Some

of these volatiles may be attractants for predators and parasitoids of hop plant herbivores. In our

experiments hop plants were not damaged by herbivores, simply exposed to one of the major

volatiles emitted by plants (including hops) when attacked by herbivores. Thus, the volatile

responses we have demonstrated may be part of „defense priming‟ rather than full defense.

Defense priming is a process in which the responses to an anticipated challenge (mediated by

volatiles from neighboring plants suffering attack) from a herbivore or a pathogen, are initiated

(Engleberth, 2006). Intra plant defense priming has been observed in lima beans (Phaseolus

lunatus) (Heil and Silva Bueno, 2007; Ozawa et al., 2000). The process of priming allows the

receiving plant to begin the production of systemic defenses without seriously affecting plant

51

fitness. This allows the plant to keep the necessary resources in reserve for full defense

production in the event of actual herbivore damage (Karban and Heil, 2009). Plants are also

capable of priming conspecifics (Karban et al., 2003; Heil and Silva Bueno, 2007), thereby

establishing the interspecific communication network to allow the receiving plant to begin the

production of systemic defenses in anticipation of herbivore attack.

Volatile production in juvenile hop plants exposed to airborne MeSA

Spectral evaluation of the head space SPME of MeSA treated hop plants identified

sixteen compounds previously associated with herbivore damage. In 2007, analyses of head

space volatiles revealed an increase in average area counts of three HIPV‟s : nonanal, (E,E) α

farnesene and MeSA. Contributions to total area counts from 3-hexen-1-ol (Z) acetate, decreased

slightly in the treated plant but increased contributions from this compound occurred in 2008 and

2009. Another volatile associated with herbivore injury, 3-hexen-1-ol was detected in 2009. All

of these compounds have been identified as part of the initial “call to arms” signal of plants

undergoing herbivore damage (Engelberth, 2006)

While the increase in the aliphatic aldehyde, nonanal, showed a greater than two fold

increase over control plants and MeSA production was > four times that of control plants, the

increased production of the sesquiterpene (E,E) α-farnesene was particularly notable. In 2007

(E,E) α-farnesene levels in MeSA treated plants were three times higher than in control plants

(ca 2% to 6%), a trend that also occurred in 2008 and 2009 (levels of 1 and 5% in treated plants,

not detected in control plants).

52

(E,E) α-farnesene was identified in head space analysis of lima beans (P. lunatus)

damaged by the two-spotted spider mite (T. urticae) (Ozawa et al., 2000), as well as in the head

space of Psylla infested pears (Pyrus communis L) (Scutareanu et al., 1997). Farnesene was

shown to be an olfactory stimulant or attractant for at least two species of predatory ladybird

beetles (Zhu et al. 1999; Francis et al., 2004), a predatory bug (Scutareanu et al., 1997) and a

predatory mite (Kong et al., 2005), and it is likely that this compound plays a role in attracting

natural enemies of pest arthropods. Farnesene is also produced in aphids as an alarm pheromone

and repels conspecifics (Pickett et a;. 1992).

While values for all of the identified compounds were inconsistent across the three trials,

the data indicate notable differences in emission profiles of non exposed control plants and

MeSA treated plants. All of the compounds we have identified in hop plant emissions have also

been found in the volatile blends from corn (Turlings and Tumlinson, 1992); lima bean (Shimoda

et al. 2000, Dicke et al., 1990); and pear (Scutareaneu et al., 1996), following herbivore injury.

The use of MeSA in these experiments was to attempt to prime hop plants to begin the

production of HIPV‟s . A review of the existing literature (Ozawa et.al, 2000) indicates that

MeSA can prime plants to begin the process of stepping up their defense mechanisms, and as a

result of the initiation of this mechanism, begin the increased production of volatiles related to

herbivore injury. We believe that the data provided here illustrates the ability of MeSA to initiate

the production of HIPV‟s in juvenile hop plants.

53

Volatile production in hop plants confined with conspecifics previously exposed to MeSA

In Experiment 2, control hop plants from the first experiment were combined for 48 hrs

with MeSA treated hops from the same experiment. Our goal was to test the hypothesis that a

„naive‟ hop plant can „eavesdrop‟ on volatiles from a conspecific previously exposed to MeSA,

and produce its own blend of volatiles. Our data suggest that a hop plant can indeed respond to

the bouquet of volatiles produced by another hop plant that has been stimulated by exposure to

MeSA. This response is characterized by production of volatiles that may prime the plant for

defense against herbivores or provide chemical signals for attracting natural enemies of

herbivores. The volatiles produced by eavesdropping hop plants at elevated levels (4-25 X) were

decanal, iso-caryophyllene, α-caryophyllene and methyl salicylate. Methyl salicylate is well

established as an attractant for a number of natural enemy species (James, 2003; James and

Price, 2004) and caryophyllene has been reported as an attractant for the green lacewing,

Chrysoperla carnea (Flint et al., 1979).

Variability in Composition of Volatiles Produced

A number of environmental issues and problems as well as the type of SPME fiber we

used may have contributed to data inconsistencies seen during the three years of this study. For

example, possible airflow contamination between greenhouses, occasional pest incidence and

differences in plant ages may have contributed to poor reproducibility. Herbivore induced plant

volatiles differ greatly in type and amount as a function of the growth age of the plant. We used

hops grown from rhizomes with a leaf age of ca. thirty days in 2007 and the first three trials in

54

2008. In the later trials of 2008 and in 2009, hops grown from soft wood cuttings, with a leaf

age of ca. sixty days were used. In studies on soybeans (Glycine max) and pears (Pyrus

communis L.) younger plants produced higher levels of fatty esters, aldehydes and alcohols as

well as sesquiterpenes such as (E,E) α-farnesene and β-caryophyllene. (Zhu et al., 2005,

Scutareanu et al., 1997). Krofta and Nesvadba (2005) noted that the variety of hop, hop age and

type of matrix sampled can affect the volatile blend. Our data on volatiles released according to

plant age (Figure 2) support this assumption and this should be considered in future research.

The use of the growth chamber in the later trials of 2008 and in 2009 may also have had

some effect on volatile production. Treatments that limited or reduced air flow between plants

prevented induced resistance in sagebrush (Artemesia tridentata) (Karban et al., 2006).

Similarly, Heil and Silva Bueno (2007) noted that intra plant communication in lima beans ( P.

lunatus L.) was also affected by the lack of air flow. They hypothesized that lianas such as the

lima bean have large areas of separation between plant areas and that air flow was necessary to

provide the access to the volatiles to begin systemic defense. While not a liana, hops do have a

similar configuration, raising the question of the effects of the importance of air flow to provide

volatile signals capable of reaching areas a significant distance from the source of herbivore

injury.

Krofta and Nesvadba (2005) reviewed an analytical procedure to assess the use of SPME

to isolate hop oils from different matrices. β-pinene, myrcene, limonene, linalool, gerianol, β-

caryophyllene, α-humulene, β-farnesene, MeSA and γ-cadinene were identified in HS-SPME

analysis of hops. Several types of SPME fibers including PDMS (polydimethylsiloxane) of

55

various sizes were used to analyze hop essential oils. In the method development of head space

SPME analysis for this study, we investigated several types of SPME fiber for optimal adsorbent

capability including 7μm and 100 μm polydimethylsiloxane (PDMS) fibers, as well as a 75 μm

Carboxen/polydimethylsiloxane and a 75μm Carbowax/divinylbenzene (CW/DVB) bonded

fiber. After some experimentation, the 75μm Carbowax/divinylbenzene (CW/DVB) (Supleco,

USA) was chosen to perform the volatile analysis. Krofta and Nesvadba (2005), suggested a

PDMS 30μm fiber (Supelco, USA) was best suited for isolation of hop oil volatiles from female

plants, while a PDMS/DVB (Divinyl Benzene) SPME filter was better suited for head space

analysis of male plants. This is due to the higher concentration of lupulin glands present in the

male plants. Since we did not sex segregate the plants used in this study, this may have played a

factor in the adsorptive capability of the fiber.

The presence of elevated levels of α-caryophyllene and β-caryophyllene was notable.

These terpenes have been identified in HIPV blends released by lima beans and kidney beans

and are used by the two-spotted spider mite predator Phytoseiulus persimilis (Acarina:

Phytoseiidae) to locate prey (Maeda and Takabayashi 2001).

Although methyl salicylate has previously been identified as a compound deserving

investigation into its signaling capabilities (Pare and Tumlinson 1999, James and Grassowitz,

2005), its capability to function as a priming compound is less certain. Engelberth (2006)

suggested that salicylic MeSA along with MeJA were among the few volatiles with potential to

serve in inter plant communication”.

56

While the data are relatively inconsistent, our results indicate trends that may illustrate

that MeSA is capable of inducing the production of herbivore induced plant volatiles in exposed

plants. Furthermore, the data also indicated trends in the production of HIPV‟s in plants exposed

to MeSA may be capable of eliciting production of herbivore induced plant volatiles in nearby

plants. However, much more research is required to fully characterize the emission of volatiles

from hop plants exposed to MeSA or conspecifics exposed to MeSA. Primed or fully alerted hop

plants mediated by deployment of synthetic MeSA in the field, may have great potential in

improving plant defense and enhancing biological control, thus reducing crop damage and

pesticide use.

6057

57

57

a % GC area expresses the proportions of each compound in the total blend of volatiles, calculated from the mean of two repetitions b N= 4 Mean of four sets of juvenile hop plants for 2007 trial c N=6 Mean of six sets of juvenile hop plants for 2008 trial d N=3 Mean of three sets of juvenile hop plants for 2009 trial

trace = <1.0% of overall area counts

ND= Not detected

2007 2008 2009

%GC area

N=4b %GC area

N=6c %GC area

N=3d

Volatile Control

C1

MeSA exposed

T1

Control

C1

MeSA

Exposed

T1

Control

C1

MeSA

Exposed

T1

3-hexen-1-ol (Z ), acetate 34 25 21 29 36 41

3-hexen-1-ol ND ND trace trace 2 2

Nonanal 3 7 ND ND ND ND

Decanal 4 2 ND ND ND ND

Iso-caryophyllene 2 1 ND ND ND trace β-caryophyllene ND ND ND ND trace trace

α-caryophyllene 6 6 4 trace 3 4

germacrene D ND trace 3 2 ND ND

α-farnesene 2 6 ND 1 ND 5

Methyl Salicylate 4 17 3 7 4 ND

germacrene B trace trace 3 ND ND ND

muurolene ND ND ND ND ND ND

γ cadinene ND ND ND ND ND ND

dodecanol 1 trace 4 ND ND ND

nonanoic acid trace ND 10 trace ND ND

Tetradecanoic acid ND ND 3 1 ND ND

Table 1: Summary of compounds collected in Experiment 1 from unexposed (control) and methyl salicylate (MeSA) exposed

juvenile hops a

6058

58

58

Table 2: Summary of compounds collected in Experiment 2 from unexposed (control) and methyl salicylate (MeSA)

exposed juvenile hop plants following combination for 48 hrs a

2007 2008 2009

%GC area %GC area %GC area

Volatile Unexposed

Controlb

C2

Control

C1

MeSA

exposed

T1

Unexposed

Control

C2

Control

C1

MeSA

Exposed

T1

Unexposed

Control

C2

Control

C1

MeSA

Exposed

T1

3-hexen-1-ol (Z ), acetate 34 2 12 20 20 27 30 14 19

3-hexen-1-ol ND ND ND 7 5 6 2 2 ND

Nonanal 3 9 10 ND ND ND ND ND ND

Decanal 4 21 19 ND ND ND ND ND ND

Iso-caryophyllene 2 4 2 2 3 2 ND ND ND

β-caryophyllene ND ND ND 2 ND ND trace 9 10

α-caryophyllene 6 6 3 4 23 9 ND 25 29

germacrene D ND ND ND ND ND 1 ND ND ND

α-farnesene 2 ND ND ND ND trace 16 9 8

Methyl Salicylate 4 ND 2 trace 4 ND 1 2 1

germacrene B trace ND ND ND ND ND ND ND ND

murrolene ND ND ND ND trace trace trace ND ND

γ cadinene ND ND ND trace 1 1. ND ND ND

dodecanol 1 ND ND ND trace ND ND ND ND

nonanoic acid trace ND ND ND 10 trace ND ND ND

Tetradecanoic acid ND ND ND ND 2 1 ND ND ND

a % GC area expresses the proportions of each compound in the total blend of volatiles

b Unexposed control plant was not used in 2007 trials. Data is original control plant data from Table 1

trace = <1.0% of overall area counts

ND= Not detected

6059

59

59

B

Figure 1: Chromatographic profiles of 6 of the 16 retention times of volatiles by unexposed control hop plants (A) and MeSA

treated plants (B). Peaks: (1) 3-hexen-1-ol, (Z) acetate,(2) 3-hexen-ol, (3). β-caryophyllene) (4) α-caryophyllene (5) (E,E) α-

farnesene (6) Methyl salicylate

A

B

6060

60

60

Figure 2: Structural identification of ten of sixteen identified compounds

6061

61

61

Figure 3: Structural identification of remaining six of sixteen compounds

62

Figure 4: Volatiles from control plant by growth age

Volatiles from root cuttings plant ca. 60 days growth

38 1

5

2 3

3-hexen-1-ol Z acetate

3-hexen-1-ol

α-caryophyllene

MeSA

α farnesene

Volatiles from rhizome

ca. 30 days growth

20

2 1 1 2 3

2

4

6 8 2

3-hexen-1-ol Z acetate Nonanal

3-hexen-1-ol

Decanal

iso-caryophyllene

alpha caryophyllene Alpha farnesene

MeSA

Dodecanol

Nonanoic acid

tetradecanoic acid

63

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66

ZHU, J., COSSE, A. L., OBRYCKI, J. J., BOO, K. S. and T. C. BAKER, 1999. Olfactory

reactions of the twelve-spotted ladybeetle, Coleomegilla maculata and the green lacewing,

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67

Chapter 3

Final Conclusions and Future Work

68

This 3-year plant chamber volatile head space emission evaluation showed trends that

imply MeSA exposed hop plants can elicit plant defense by chemically induced intra and inter

plant communication.

The chamber head space SPME emission information profiled the relative percent

contribution of sixteen herbivore induced plant volatiles to total volatile emission. Comparisons

of MeSA exposed to control plants suggest MeSA itself may elicit HIPV production aiding in

plant defense. When MeSA exposed plants were in close proximity to MeSA treated plants, a

relative rise in certain sesquiterpenes may indicate that HIPV chemical induction pathways

become operational. Previous studies with sagebrush and tobacco (Karban et al., 2004) and lima

beans (Heil and Silva Bueno, 2007) support the hypothesis that uninjured plants located near

plants undergoing herbivore injury will begin the production of volatile compounds. The

compounds emitted from the injured plant provide the uninjured plant with information to

increase its production of defense mechanisms. As the uninjured plant begins its own production

of volatiles, other nearby plants may use these volatiles to begin the process of increasing their

defense mechanisms in anticipation of possible herbivore injury.

While some field studies have been performed using methyl salicylate as an attractant for

predatory species (James, 2003, James and Price, 2004) or as a repellent for herbivory

(Pettersson et al., 2004), the use of methyl salicylate as a priming mechanism to elicit the

production of HIPVs in hops has not been performed. Although the head space emission

information collected from chamber evaluations indicate relatively greater production of certain

HIPVs after MeSA hop exposure, differences in plant rearing (rhizomes versus soft wood

69

cuttings) and other environmental conditions (greenhouse versus growth chamber plant rearing)

could account for appreciable variation in emission profiles among the replicated MeSA

exposure trials conducted from 2007 through 2009. In order to evaluate the capability of plants

to chemically communicate the need for defense, further field testing must be performed. The

data collected in these screening experiments will serve to better design future evaluations in

order to understand the function of intra-plant as well as inter-plant signaling .

70

Appendix A

Analytical Summary Report

71

ANALYTICAL SUMMARY REPORT

Characterization of Volatile Emissions from Juvenile Hop Plants after exposure to Methyl Salicylate by Gas Chromatograph/Mass

Spectroscopy

Author

Rita Abdella

Testing Facility Food and Environmental Quality Laboratory

Department of Entomology

Washington State University

2710 University Drive

Richland, WA 99354-1671

FEQL Study No.: 0507

Laboratory Research Manager

Dr. Vince Hebert

Research Conducted for:

Dr. David James

WSU-AERC, Prosser, WA

Study Timetable

Study Initiation Date: 3/2007

Experimental Termination Date: 9/2007

Report Date

7/2007

72

CERTIFICATION

The undersigned hereby declare that this study was performed under my supervision according

to the procedures described herein, and that this report provides a true and accurate record of the

results obtained.

Analytical Research Director: ___________________________ Date:

Vincent R. Hebert, Food and Environmental Quality Laboratory

Washington State University, Tri-Cities Campus, Richland WA

Analytical work performed by:

Rita Abdella, WSU-FEQL Graduate Researcher

Jane LePage, WSU-FEQL Research Chemist

73

TABLE OF CONTENTS

Page

Certification 2

Table of Contents 3

Archives (location of raw data) 4

Analytical Summary 5

I. Objective/Introduction 5

II. Sample Inventory/History 5

Table 1: Sample Inventory & History

III. Standard Preparation 7

IV. Analytical Procedure 8

A. Air Sampling 8

B. SPME Data Analyses 9

V. Results and Discussion 10

Table 2: Interval Data 11

Table 3 Treated and Control Hop Plant Replicate data 18

Table 4 Greenhouse Air Sample Data 21

Table 5 Treated and Control Hop Data before/after

Combination 26

Figure 2 28

74

ARCHIVES (LOCATION OF RAW DATA)

The original raw data, correspondence logs, and all relevant information for the study titled: “Characterization of Volatile Emissions from juvenile Hop Plants after Exposure to Methyl Salicylate by Gas Chromatography/Mass Spectroscopy” FEQL project number 0507, along with certified originals of the signed analytical summary report will be maintained by the testing facility. Exact copies of the analytical summary report and relevant information for the construction of this study will be transferred to WSU-

AERC, Prosser upon request.

Laboratory Research Director: Vincent Hebert

Testing Facility: Food and Environmental Quality Laboratory

Department of Entomology

Washington State University

Richland, WA 99354-1671

75

ANALYTICAL SUMMARY REPORT

I. Objectives/Introduction

The Food and Environmental Quality Laboratory (FEQL) in Richland, WA evaluated the volatile

emissions from juvenile hop plants following a timed exposure to methyl salicylate. Methyl

salicylate (MeSA) is a common volatile produced by plants when attacked by insect pests and can

aid natural enemies in locating their prey (Shulaev, et al., 1997; James, 2003, Kunart, et al., 2002).

Besides being a plant to insect signal, it has been suggested that MeSA elicits plant to plant

communication as well with the consequence that the hop plants themselves may start producing

their own predator-attracting volatiles (David James personal communication). To test this

hypothesis, a series of greenhouse/laboratory experiments were conducted from March 20 through

May 14th

2007 to evaluate volatile emission profiles from juvenile hop plants exposed to MeSA

when compared to control (non-exposed) plants. These studies were conducted at the WSU Prosser

Agricultural Research Center greenhouse facilities to evaluate chemical signaling following

exposure to methyl salicylate. The plants were contained in separate control and treated

greenhouses under comparable environmental conditions. The plants were of the same approximate

growth age and possessed an approximate total surface area of 30-50 cm2 when evaluated. For each

of the four trials, the treated plant was exposed to methyl salicylate from an impregnated sachet for

approximately 72 hrs. After the exposure interval, a control plant and a treated plant were enclosed

individually in 2.0 liters glass chambers that contained a sampling portal. The two chambered

plants were then transported to the Food and Environmental Quality Lab of Washington State

University –Tri-Cities. (WSU-FEQL). Upon arrival, the plants were allowed to equilibrate at room

temperature for ca. 2-hours before sampling the air surrounding each plant by Solid Phase Micro-

Extraction (SPME). The fiber of this diffusion-based sampling devise was exposed to the chamber

air for five minutes before thermal desorption into an Agilent 6890 Gas Chromatograph with a 5973

Mass Spectrometer. After 24 hrs a second set of SPME analyses were performed to evaluate

changes in chemical emission among the control and treatment chambered plants. After analyzing

the 24-hour SPME samples, the control and treatment plants were removed from their individual

containers, and placed together in a 10 liter glass chamber for 48 hours and then again separated to

their individual chambers. After allowing four hours for equilibration, a final set of SPME analyses

were drawn on the individual containers to measure the effects of exposure. The gas

chromatographic identity profiles from four replicate side-by side control-treatment evaluations are

reported herein.

76

II. Sample Inventory/History

Control and treated chambered juvenile hop plants were delivered by personal vehicle to the FEQL

testing facility on the following dates: March 20; April 10, and May 7 and May 14, 2007. Upon

arrival the plants were allowed to equilibrate for ca. 2 hours before five minutes SPME fiber

exposures conducted in the respective control and treatment chambers. Low volume air samples in

both the control and treated greenhouses were acquired with the exception of the March 20,2007

sampling date.

Table 1 provides an inventory of the samples received at FEQL, sample coding information , and

the chronological information for the handling and analyses of the air samples.

Table 1: Sample Inventory & History

Receiving sample ID Qty

received FEQL assigned ID

Specified sample

analysis

information

Date of

sample

analysis

Number

of

replicates

analyzed Control chambered hop plant

3/20/07 1 0507-032007-C

3/20/07 time

zero

3/21/07 24-

hour

2

Treated chambered hop plant

3/20/07 1 0507-032007-T

3/20/07 time

zero

3/21/07 24-

hour

2

Control and treated plants combined on

3/22/07

3/26/07 2

Control hop plant 4/10/07

1 0507-041007-C

4/10/07 time

zero

4/11/07 24

hour

2

Treated hop plant 4/10/07

1 0507-041007-TC

4/10/07 time

zero

4/11/07 24

hour

2

Control and treated

plants combined on

(note)

N/A

50/100 mg Tenax air sampling

cartridges

4

041007-AIR-C

041007-AIRCD

041007-AIR-T

4.5 l/min for 15

minutes

4.5 l/min for 15 minutes

4.5 l/min for 15

minutes

4/10/07

1

77

Receiving sample ID Qty

received FEQL assigned ID

Specified sample

analysis

information

Date of

sample

analysis

Number

of

replicates

analyzed 041007-AIR-TD 4.5 l/min for 15

minutes

Control hop plant 5/7/2007

1 0507-050707-C

5/7/07 time

zero

5/8/07 24

hour

3

Treated hop plant 5/7/2007

1 0507-050707-T

5/7/07 time

zero

5/8/07 24

hour

3

Control and treated

plants combined on

5/9/07

5/11/07

2

50/100 mg Tenax air sampling

cartridges

4

050707-AIR-C

050707-AIRCD

050707-AIR-T

050707-AIR-TD

4.25 L/min for 15

minutes

4.25L/min for 15 minutes

4.5L/min for 15

minutes

4.5L/min for 15

minutes

5/7/07

1

Control hop plant 5/14/2007

1 0507-051407-C

5/14/07

Time Zero

5/15/07 24

hour

3

Treated hop plant 5/14/2007

1 0507-051407-T

5/14/07

Time Zero

5/15/07 24

hour

3

Control and treated plants combined on

5/15/07

5/17/07 2

50/100 mg Tenax air sampling

cartridges

4

051407-AIR-C

051407-AIRCD

051407-AIR-T

051407-AIR-TD

4.25 L/min for 15

minutes

4.25 L/min for 15

minutes

4.5L/min for 15

minutes

4.5L/min for 15

minutes

5/14/07

1

78

III. Standard Preparation

Standards were prepared to bracket the range of residues in the samples. The following test

substances, standards, and standard dilutions were used throughout this study:

Test substance

Compound Reference No. Source

Methyl Salicylate 13141 Sigma-Aldrich

Stock Solution

Compounds Solution Number Solvent Concentration

Methyl salicylate 13142 Hexane 1 mg/mL

Dilutions of Stock Solutions

Compounds Solution Number Solvent Concentration

Methyl Salicylate 13142-1 Ethyl Acetate 0.5 ug/mL

Methyl Salicylate 13142-2 Ethyl Acetate 1.0 ug/mL

Methyl Salicylate 13142-3 Ethyl Acetate 2.00.5 ug/mL

Methyl Salicylate 13142-4 Ethyl Acetate 5.0 ug/mL

Methyl Salicylate 13142-5 Ethyl Acetate 10.0 ug/mL

Methyl Salicylate 13142-6 Ethyl Acetate 0.2. ug/mL

Methyl Salicylate 13142-7 Ethyl Acetate 100 mg/mL

Methyl Salicylate 13142-8 Ethyl Acetate 0.005 u/mL

Methyl Salicylate 13142-9 Ethyl Acetate 0.1 ug/mL

All standard solutions were stored in the freezer at ca.-20˚ C (freezer I.D. Comet). The dilutions

made during the course of the study are recorded in the FEQL analytical laboratory standards book.

The expiration date of the linearity standards is 10/15/2007.

79

IV. Analytical Procedure

A. Air Samples

Air sampling was performed using 50/100 mg Tenax cartridges for both control and treated

plant greenhouses to assess ambient methyl salicylate concentrations on three of the four

interval sampling dates. Two 50/100 mg Tenax cartridges were collocated on a specially

designed air sample device that was attached to a portable low volume air sample pump

(Figure 1). Air samples were collected for 15 minutes and flow rates were verified using a

Gilmont flow meter following air sampling. Flow rates are presented in Table 1.

After the air samples were collected, the Tenax tubes were eluted with 3 mL of ethyl

acetate, into a 15 mL graduated centrifuge tube and the final volume adjusted to 3mL with

ethyl acetate for analysis by gas chromatograph/mass spectroscopy (GC/MS). Extracts were

analyzed by GC/MS Selective Single Ion Monitoring (SIM) mode to quantify methyl

salicylate (see chromatographic conditions below).

B. SPME Data Analysis

Each interval set of chambered plants (control and treatment) taken on a particular interval

date were analyzed in duplicate by SPME. Upon arrival, the plants were allowed to

equilibrate at room temperature for ca. 2-hours before sampling the air surrounding each

plant by Solid Phase Micro-Extraction (SPME). A 7 µm film thickness polysiloxane fiber

was inserted into the respective control/treatment chamber for 5 or 10 minutes. Afterwards,

the exposed fiber was thermal desorbed for 30 seconds into the hot (200oC) injection port of

a Agilent 6890 Gas Chromatograph.

Methyl salicylate and other compounds were determined using an Agilent 6890N Gas

Chromatograph with 5973N Mass Selective Detector (MSD) in total ion chromatography

(TIC) mode. Typical operating conditions are described below:

GC Conditions:

Columns: Alltech 19654 EC-WAX 30mx0.32mmI.D. x0.25 μm film thickness

(Alltech Inc.)

Carrier gas: Ultrapure helium

80

Oven Program: Initial temp 50˚C for 1 minute

Ramps:

5˚C/minute to 260˚C hold for 5 minutes

Injection Volume: 2 μL

Injector: Pulsed Splitless, 200˚C

Detector: Temperatures:

MS Quad 280˚C

Scan : 50-350 m/z or Single Ion Monitoring (SIM) at 152 m/z for MeSA in Tenax

The chromatograms generated in each analysis were integrated to indicate retention time and

a library search using the Data Analysis function of the ChemStation software was

performed to identify chromatographic peaks of interest. Following peak identification, the

peaks of interest were compared against the NIST library to determine the best match. The

relative proportion (percentage of area counts) associated with individual peaks per run was

also generated to compare chromatographic results of combined control and treatment

evaluations.

V. Study Findings

On March 20, 2007 results from SPME evaluations indicated that the control plant methyl

salicylate emissions made up approximately 4% to 8% of total peak area counts. A

moderate response (approximately 25% total area count) of 3-Hexen-1-ol was observed with

lesser air emissions of formate (10%), acetic acid (10%), and 1 to3% of other organic acids

being detected. Nonanol was present in very low concentrations (approximately 2% of total

area counts). The treated plant organic emissions indicated a higher percentage of methyl

salicylate (23%-25% of total area counts) present together with α-farnasene (18%-20%).

Detected air emissions of 3-hexen-1-ol were considerably lower in the treated samples (8-

9%). After approximately 24 hours, the second set of analysis on both the control and treated

plants indicated that in the control, methyl salicylate concentrations remained stable (5% of

total area counts), while nonanal concentration increased slightly (2 to 6.9% of total area

counts). The relative proportion of methyl salicylate, nonanal and α-farnasene in the treated

plant data increased (8.6%-13%; 25%-35% and 20%-44% respectively). Other trace level

organic acids such as propanoic, and 1-dodecanol concentrations were decreased overall.

On April 10, 2007 another set of control and similarly exposed MeSA treatment juvenile

hop plants were obtained and transported to the FEQL for analysis. SPME were performed

in duplicate for each sample in an identical manner as before. Analysis of the control plant

indicates the presence of methyl salicylate (2% of total area counts). 3-hexen-1-ol, formate

and hexen-1-ol, acetate were present (22 % and 40% of total area counts), respectively. The

treated plants were sampled for 5 minutes and analyzed. Total contribution due to methyl

salicylate in the treated sample was12% to 13% of the total area counts, while hexen-1-ol,

81

acetate and 3-hexen-1-ol, formate levels contributed 38% and 40% of the total area counts;

α-farnasene contributed approximately 2.4% to total area counts. The plants were placed in

a common container and after 24 hours, removed and isolated. After a short time for

equilibration, the plants were sampled, using a five minute sample time and the data

reviewed The control plant indicated an increase in contribution to area counts due to

methyl salicylate from 2.5% to 13% of total area counts. The contribution due to α-

farnasene increased from 2.4% to 24 % after the combined exposure. The treated plant

analysis suggests that there was no change in methyl salicylate. However, a decrease in the

contribution due to hexen-1-ol acetate from 22% to 7% and a reduction in α-farnasene to

17% was observed.

On May 7, 2007 the third set of juvenile hop plants were obtained and transported to WSU-

FEQL. The control plant was sampled using a 5 minute sample time and the data was

analyzed. Butanoic acid contributed approximately 15% of the total area counts, and 3-

hexen-1-ol contributed approximately 5% of the total area counts. No indication of α-

farnasene or methyl salicylate was present. The treated plant was sampled using a five

minute sample time. The data indicated that the majority of the contribution to total area

counts came from. α-farnasene (12% of total area counts), 2% from 3-hexen-1-ol, Z formate

and 2% was due to 3-hexen-1-ol, acetate and methyl salicylate. Following the resulting

exposure of the control plant to the treated, and the allowed time for equilibration after

separation, the plants were resampled for 5 minutes. The control plant indicated no presence

of methyl salicylate or α-farnasene. Several metabolites of methyl salicylate were indicated,

with benzoic acid, 2,4 bis-tri-methyl increasing from 4% in the original sample to 29% after

exposure. Benzoic acid was also present in the sample following exposure contributing

approximately 38% of the total area counts. The treated plant indicated a two fold increase

in levels of 3-hexen-1-ol acetate (5.5% to 12.6%) and α-farnasene (11% to 22%). Methyl

salicylate increased only slightly from 3% to 4 %

On May 14, 2007 the final set of plants was obtained and transported to WSU-FEQL for

sampling and analysis. The control plant was sampled for five minutes and the data

analyzed. Of the compounds present, 3-hexen-1-ol, acetate contributed 63% of the total area

counts, while 3-hexen-1-ol, formate contributed 6%of the total area counts, with 2% of the

area counts were attributable to α-farnasene and 3% to methyl salicylate. The treated plant

sample indicated 73% of the total area counts attributable to 1,4 hexadiene, 66% of the total

area counts attributable to 3-hexen-1-ol, acetate and 19-22% attributable to methyl

salicylate. After the exposure of the control plant to the treated plant, the control plant

exhibited a twofold decrease in the area counts of 3-hexen-1-ol (63%-32%) and an increase

in decanal area counts form 1% to 32% and an increase in nonanal area counts from 7% to

11%. The treated plant exhibited a reduction in 1,4 hexadiene area counts from 73% to 12%,

and a increase in methyl salicylate area counts from 19% to 22.5%. Decanal, not present in

the original sample, contributed 33% to the total area counts. The use of total area counts is

used as an indicator of instrument response to the compounds identified in this study. It is

82

important to note that the area counts are not used for quantification of the identified

compounds, but as a means to characterize the instrument response at each interval and to

compare the response between the treated and control plants at each sampling interval.

Table 2 provides the raw data for each sampling interval.

83

Table 2

Interval Data Sample Identification Compound ID Area Counts % Total

Area

Total

Area

counts

0507-032007T Treated Hop Plant #1 3-hexen-1-ol, acetate(Z) 814065 8.605 9459860

Nonanal 326078 3.447

Decanal 458094 4.843

α- Farnesene 1521523 16.084

α- Farnesene 432908 4.576

2-(2-ethoxyethoxy)ethanol 469708 4.965

Methyl Salicylate 2397953 25.349

0507-032007T Treated Hop Plant #2 3-hexen-1-ol, acetate(Z) 768558 12.92 5947831

Nonanal 127756 2.15

Decanal 94991 1.6

α- Farnesene 1414151 23.8

α- Farnesene 397482 6.7

2-(2-ethoxyethoxy)ethanol 297726 5.006

Methyl Salicylate 2418796 40.667

0507-032007T Analyzed-3/21/07 3-hexen-1-ol, acetate(Z)

Nonanal a

Decanal a

α- Farnesene a

α- Farnesene a

2-(2-ethoxyethoxy)ethanol a

Methyl Salicylate a 0

0507-0320007-T+C Analyzed 3/26/07 3-hexen-1-ol, acetate(Z) 219837 3.8 7616994

Nonanal 431364 5.7

84

Sample Identification Compound ID Area Counts % Total

Area

Total

Area

counts

Decanal 1563544 20.57

α- Farnesene 625386 8.2

α- Farnesene 185276 2.4

2-(2-ethoxyethoxy)ethanol 288022 3.8

Methyl Salicylate 0 0

0507-032007-C Control Hop Plant 3-hexen-1-ol, acetate(Z) 2329355 30.583 5723682

Nonanal 220425 2.890

Decanal 451755 5.9

2-(2-ethoxyethoxy)ethanol 755645 9.92

α-caryophyllene 173026 2.9

α- Farnesene 89497 1.17

Methyl Salicylate 351522 4.6

0507-032007-C Control Hop Plant 3-hexen-1-ol, acetate(Z) 1300934 33.959 46278881

Nonanal 220425 2.894 46278881

Decanal 451755 5.9 46278881

2-(2-ethoxyethoxy)ethanol 755645 9.92 46278881

α- Farnesene 130565 5.6 10351201

α- Farnesene 3.84

α-caryophyllene 203753 2.27 10351201

Methyl Salicylate

0507-032007-C Control Hop Plant-Analyzed

4/12/07

3-hexen-1-ol, acetate(Z) a

Nonanal a

Decanal

2-(2-ethoxyethoxy)ethanol a

α- Farnesene

85

Sample Identification Compound ID Area Counts % Total

Area

Total

Area

counts

α-caryophyllene

Methyl Salicylate

0507-032007-T+C Analyzed 3-hexen-1-ol, acetate(Z) 422220 3.1

Nonanal 518683 1.6

Decanal 1392646 10.27

2-(2-ethoxyethoxy)ethanol ND NA

α- Farnesene 640638 4.7

α- Farnesene 231907 1.7

α-caryophyllene 916545 6.76

Methyl Salicylate ND NA

0507-041007T Treated Hop Plant #1 3-hexen-1-ol, acetate(Z) ND NA

Nonanal ND ND

Decanal 1118089 4.9

α- Farnesene ND NA

α- Farnesene ND NA

2-(2-ethoxyethoxy)ethanol ND NA

Methyl Salicylate 239808 7.2

0507-041007T Treated Hop Plant #2 3-hexen-1-ol, acetate(Z) 912455 22.4 4081824

Nonanal ND NA

Decanal ND NA

α- Farnesene 827617 20.3

2-(2-ethoxyethoxy)ethanol ND NA

Methyl Salicylate 293808 7.2

0507-041007T Analyzed 4/11/07 3-hexen-1-ol, acetate(Z) ND NA 4894139

86

Sample Identification Compound ID Area Counts % Total

Area

Total

Area

counts

1,4 Hexadiene 74077 1.5

Decanal ND NA

α-caryophyllene 193386 3.95

α- Farnesene ND NA

2-(2-ethoxyethoxy)ethanol 76157 1.6

Methyl Salicylate 259129 5.3

0507-04100007-T+C Analyzed 4/1207 3-hexen-1-ol, acetate(Z) ND NA Nonanal 208334 5.2 Decanal 1563544 α- Farnesene ND NA 2-(2-ethoxyethoxy)ethanol 58403 1.5 Methyl salicylate 703476 17.7 0507-041007-C Control Hop Plant 3-hexen-1-ol, acetate(Z) 1819153 5.2 35538598

Nonanal 2731535 7.7

Decanal 66156617 18.6

2-(2-ethoxyethoxy)ethanol 1558482 4.4

α-caryophyllene 158727 0.5

α- Farnesene 233958 .7

α- Farnesene 389787 1.1

Methyl Salicylate 316555 0.9

0507-041007-C Control Hop Plant 3-hexen-1-ol, acetate(Z) 1193144 15.9 7494237

Nonanal ND NA Decanal ND NA 2-(2-ethoxyethoxy)ethanol ND NA α- Farnesene 167961 2.2 α- Farnesene ND NA

87

Sample Identification Compound ID Area Counts % Total

Area

Total

Area

counts

α-caryophyllene ND NA Methyl Salicylate ND NA 0507-041007-C Control Hop Plant-Analyzed

4/11/07

3-hexen-1-ol, acetate(Z) 4452532 14.936 2295343

Nonanal 445232 14.936 Decanal 64963 2.18 2-(2-ethoxyethoxy)ethanol 542430 1.8 α- Farnesene 703697 23.6 α- Farnesene 386125 12.95 α-caryophyllene 047063 1.58 Methyl Salicylate ND 8.5 0507-041007-T+C Analyzed 4/12/07 3-hexen-1-ol, acetate(Z) 204035 8.9 2295343

Nonanal 161730 7.05 Decanal 335964 14.637

2-(2-ethoxyethoxy)ethanol ND

α- Farnesene 384013 16.7

α- Farnesene 103568 4.51

α-caryophyllene ND 0

Methyl Salicylate 195957 8.5 0507-050707T Treated Hop Plant #1 3-hexen-1-ol, acetate(Z) 9179031 20.064 Nonanal ND NA Decanal 129729 0.284 2-(2-ethoxyethoxy)ethanol ND NA α- Farnesene 1927476 4.2 α- Farnesene 589647 1.3 α- -caryophyllene 9716891 21.2

88

Sample Identification Compound ID Area Counts % Total

Area

Total

Area

counts

Methyl Salicylate 4669829 10.2 0507-050707T Treated Hop Plant #2 3-hexen-1-ol, acetate(Z) 6968532 16.9 Nonanal ND NA Decanal ND NA 2-(2-ethoxyethoxy)ethanol ND NA α- Farnesene 2147307 5.20 α- Farnesene 643020 1.6 α- -caryophyllene 1283423 3.11 Methyl Salicylate 4181594 10.134 0507-050707T Analyzed-5/8/07 3-hexen-1-ol, acetate(Z) 415025 5.51 Nonanal 198353 2.63 Decanal 400859 5.33 α-caryophyllene 214003 2.84 α- Farnesene 718421 9.53 α- Farnesene 183674 2.63 2-(2-ethoxyethoxy)ethanol ND NA Methyl Salicylate 198394 2.6 0507-050707-T+C Analyzed 5/11/07 3-hexen-1-ol, acetate(Z) 440886 12.6 Nonanal ND NA Decanal 80203 2.29 α-caryophyllene ND NA α- Farnesene 562628 16.11 α- Farnesene 229122 6.56 2-(2-ethoxyethoxy)ethanol ND NA Methyl Salicylate 143342 4.10 0507-0507007-C Control Hop Plant 3-hexen-1-ol, acetate(Z) 3207682 40.9 7830680

89

Sample Identification Compound ID Area Counts % Total

Area

Total

Area

counts

2,4 Hexadiene 157109 2.00 Decanal 124481 1.6 α-caryophyllene 743635 9.5 α- Farnesene ND NA Methyl Salicylate 238036 3.0 0507-050707-C Control Hop Plant 3-hexen-1-ol, acetate(Z) 1572577 45.8 3431347

2,4 Hexadiene 98949 2.9

Decanal ND NA

2-(2-ethoxyethoxy)ethanol ND NA

α- Farnesene ND NA

α-caryophyllene 507225 14.8

Methyl Salicylate 172376 5.0

0507-050707-C Control Hop Plant-Analyzed 5/8/07 3-hexen-1-ol, acetate(Z) 61513 5.3 1158917

Nonanal 172576 14.5

Decanal 314223 27.1 2-(2-ethoxyethoxy)ethanol ND NA α- Farnesene ND NA α-caryophyllene 103434 8.9 Methyl Salicylate ND NA 0507-0050707-T+C Analyzed 5/11/07 3-hexen-1-ol, acetate(Z) 92578 3.4 2723343

Nonanal ND NA Decanal ND NA 2-(2-ethoxyethoxy)ethanol ND NA

α- Farnesene ND NA

α-caryophyllene ND NA

Methyl Salicylate ND NA

90

Sample Identification Compound ID Area Counts % Total

Area

Total

Area

counts

0507-051407T Treated Hop Plant #1 3-hexen-1-ol, acetate(Z) 665224 72.99 911396

Nonanal ND NA

Decanal ND NA

2-(2-ethoxyethoxy)ethanol ND NA

α- Farnesene ND NA

α- -caryophyllene 72414 7.9

Methyl Salicylate 173758 19.1

0507-051407T Treated Hop Plant #2 3-hexen-1-ol, acetate(Z) 406461 66.02 615634

Nonanal ND NA Decanal ND NA 2-(2-ethoxyethoxy)ethanol ND NA α- -caryophyllene 69893 11.4 α- Farnesene ND NA Methyl Salicylate 139280 22.62 0507-051407T Analyzed-5/8/07 3-hexen-1-ol, acetate(Z) 96874 11.7 825568 Nonanal 98288 11.9 Decanal 270914 32.82 α-caryophyllene ND NA α- Farnesene ND NA 2-(2-ethoxyethoxy)ethanol ND NA Methyl Salicylate 186533 22.6 0507-0541407-T+C Analyzed 5/17/07 3-hexen-1-ol, acetate(Z) 210563 11.9 1771614

Nonanal 291961 16.5

Decanal 632736 35.8

α-caryophyllene ND NA α- Farnesene ND NA

91

Sample Identification Compound ID Area Counts % Total

Area

Total

Area

counts

2-(2-ethoxyethoxy)ethanol ND NA Methyl Salicylate ND NA 0507-0514007-C Control Hop Plant 3-hexen-1-ol, acetate(Z) 4172270 63 6610360

2,4 Hexadiene 444197 6.7 Decanal 66558 1.0 α-caryophyllene 392699 5.94 α- Farnesene 87774 1.33 α- Farnesene 77023 1.2 Methyl Salicylate 227442 3.44 0507-0514007-C Control Hop Plant 3-hexen-1-ol, acetate(Z) 2117851 64.99 3258699

2,4 Hexadiene 193580 5.94

Decanal 68089 2.09

α- Farnesene ND NA α-caryophyllene 225693 6.926 Methyl Salicylate 155260 4.76 0507-051407-C Control Hop Plant-Analyzed

5/15/07

3-hexen-1-ol, acetate(Z) 487363 32.7 1491983

Nonanal 244290 16.4 Decanal 404379 27.1 2,4 bis[trimethyl],benzoic acid 39791 2.7 α- Farnesene ND NA α-caryophyllene ND0 NA Methyl Salicylate ND0 NA 0507-0051407-T+C Analyzed 5/17/07 3-hexen-1-ol, acetate(Z) ND NA 2088546

Nonanal 368825 17.7

Decanal 860842 41.22

92

Sample Identification Compound ID Area Counts % Total

Area

Total

Area

counts

2,4 bis[trimethyl],benzoic acid 76764 3.7

α- Farnesene ND NA

α-caryophyllene ND NA

Methyl Salicylate ND NA

ND = None detected

NA = Not applicable a Different SPME device used for comparison

93

Table 3

Treated and Control Hop Replicate Data

Sample Identification Compound ID Area Counts % Total

Area

Total Area

counts

0507-032007T Treated Hop Plant #1 3-hexen-1-ol, acetate(Z) 814065 8.605 9459860

Nonanal 326078 3.447

Decanal 458094 4.843

α- Farnesene 1521523 16.084

α- Farnesene 432908 4.576

2-(2-ethoxyethoxy)ethanol 469708 4.965

Methyl Salicylate 2397953 25.349

0507-032007T Treated Hop Plant #2 3-hexen-1-ol, acetate(Z) 768558 12.92 5947831

Nonanal 127756 2.15

Decanal 94991 1.6

α- Farnesene 1414151 23.8

α- Farnesene 397482 6.7

2-(2-ethoxyethoxy)ethanol 297726 5.006

Methyl Salicylate 2418796 40.667

0507-032007-C Control Hop Plant 3-hexen-1-ol, acetate(Z) 2329355 30.583 5723682

Nonanal ND ND

Decanal 451755 5.9

α-caryophyllene 173026 2.9

α- Farnesene 640638 NA

α- Farnesene 231907 NA

Methyl Salicylate ND NA

0507-032007-C Control Hop Plant 3-hexen-1-ol, acetate(Z) 2280630 48.895 4664357

Nonanal 108436 2.325

94

Sample Identification Compound ID Area Counts % Total

Area

Total Area

counts

Decanal 141391 3.031

2-(2-ethoxyethoxy)ethanol 615503 13.2

α-caryophyllene 222789 4.8

α- Farnesene 88849 1.91

α- Farnesene ND NA

Methyl Salicylate 141103 3.025

0507-041007T Treated Hop Plant #1 3-hexen-1-ol, acetate(Z) ND NA 10351201 Nonanal ND ND Decanal 1118089 4.9 α- Farnesene ND NA α- Farnesene ND NA 2-(2-ethoxyethoxy)ethanol ND NA Methyl Salicylate 239808 7.2 0507-041007T Treated Hop Plant #2 3-hexen-1-ol, acetate(Z) 912455 22.4 4081824

Nonanal ND NA

Decanal ND NA

α- Farnesene 827617 20.3

α- Farnesene ND NA

2-(2-ethoxyethoxy)ethanol ND NA

Methyl Salicylate 293808 7.2

0507-041007-C Control Hop Plant 3-hexen-1-ol, acetate(Z) 1819153 5.2 35538598

Nonanal 2731535 7.7

Decanal 66156617 18.6

2-(2-ethoxyethoxy)ethanol 1558482 4.4

α-caryophyllene 158727 0.5

α- Farnesene 233958 .7

α- Farnesene 389787 1.1

Methyl Salicylate 316555 0.9

0507-041007-C Control Hop Plant 3-hexen-1-ol, acetate(Z) 1193144 15.9 7494237

95

Sample Identification Compound ID Area Counts % Total

Area

Total Area

counts

Nonanal ND NA Decanal ND NA 2-(2-ethoxyethoxy)ethanol ND NA α- Farnesene 167961 2.2 α-caryophyllene 507225 14.782 Methyl Salicylate ND NA 0507-050707T Treated Hop Plant #1 3-hexen-1-ol, acetate(Z) 9179031 20.064 Nonanal 1085129 2.37 Decanal 129729 0.284 2-(2-ethoxyethoxy)ethanol ND NA α- Farnesene 1927476 4.2 α- Farnesene 589647 1.3 α- -caryophyllene 1421623 3.10 Methyl Salicylate 4669829 21.239 0507-050707T Treated Hop Plant #2 3-hexen-1-ol, acetate(Z) 6968532 16.9 Nonanal 729633 1.78 Decanal ND NA 0 2-(2-ethoxyethoxy)ethanol 729633 1.768 α- Farnesene 2147307 5.20 α- Farnesene 643020 1.6 α- -caryophyllene 1283423 3.11 Methyl Salicylate 4181594 10.134 0507-050707-C Control Hop Plant 3-hexen-1-ol, acetate(Z) 4172270 63.1 6610360

2,4 Hexadiene 444197 6.72 Decanal 66558 1.0 α-caryophyllene 392699 5.94 α- Farnesene 87774 1.33

96

Sample Identification Compound ID Area Counts % Total

Area

Total Area

counts

α- Farnesene 77023 1.2 Methyl Salicylate 227442 3.44 0507-050707-C Control Hop Plant 3-hexen-1-ol, acetate(Z) 2117851 64.99 3258699

2,4 Hexadiene 193580 5.94

Decanal 68089 2.09

2-(2-ethoxyethoxy)ethanol ND NA α- Farnesene ND NA α-caryophyllene 225693 6.926 Methyl Salicylate 155260 4.76 0507-050707-T+C Analyzed 5/11/07 3-hexen-1-ol, acetate(Z) 440886 12.6 Nonanal ND NA Decanal 80203 2.29 α-caryophyllene 215205 6.1 α- Farnesene 562628 16.11 α- Farnesene 229122 6.56 2-(2-ethoxyethoxy)ethanol ND NA Methyl Salicylate 143342 4.10

97

Table 4

Greenhouse Air Sample Data

Receiving

sample ID

Fortification

(ug) Peak Count

Calculated

Concentration

(ug/mL)

Final

Volume

(mL)

Dilution

Factor

Total

MeSA

(ug)

Total

Air

Volume

(m3

)

MeSA

Air Conc.

(ug/m3

)

0507-050707-C1 195698 0.112 3 3 0.33 0.11 2.98

0507-050707-C2 1622658 0.094 3 3. 0.28 0.11 2.51

0507-050707-T1 277593 0.155 3 3 0.47 0.11 4.15

0507-050707-T2 521720 0.286 3 3 0.86 0.11 7.63

0507-FS-1 0.3 178157 0.102 3 3 0.31

0507-FS-2 0.3 123137 0.073 3 3 0.22

0507-051407-C1 ND 3 3 0.00 0.05 0.00

0507-051407-C2 ND 3 3 0.00 0.04 0.00

0507-051407-T1 219647 0.141 3 3 0.42 0.05 9.42

0507-051407-T2 163045 0.111 3 3 0.33 0.05 6.33

98

Table 5

Treated and Control Plant Before and After Combined Exposure

Sample Identification Compound ID

Average

Area Counts c

Average

% Total

Area

Average

Total Area

Counts

0507-032007T Treated Hop Plant 3-hexen-1-ol, acetate(Z) 791311.5 10.76 7703845.5

Nonanal 226917 2.8

Decanal 276542.5 3.27

α-caryophyllene ND NA

α- Farnesene 1467837 19.94

α- Farnesene 415195 5.6

2-(2-ethoxyethoxy)ethanol 383717 4.99

Methyl Salicylate 2408374.5 33.0

0507-0320007-Treated T+C Analyzed 3/26/07 3-hexen-1-ol, acetate(Z) 219837 3.8 7616994

Nonanal 431364 5.7

Decanal 1563544 20.57

α-caryophyllene ND NA

α- Farnesene 625386 8.2

α- Farnesene 185276 2.4

2-(2-ethoxyethoxy)ethanol 288022 3.8

Methyl Salicylate 2418796 40.667

0507-032007-C Control Hop Plant 3-hexen-1-ol, acetate(Z) 2304992.5 39.8 5194019.5

Nonanal 164430.5 2.325

Decanal 296573 2.8

2-(2-ethoxyethoxy)ethanol 685574 11.6

α-caryophyllene 97652.5 3.9

α- Farnesene 89173 1.5

99

Sample Identification Compound ID

Average

Area Counts c

Average

% Total

Area

Average

Total Area

Counts

α- Farnesene ND NA

Methyl Salicylate 246312.5 3.8

0507-032007-T+C Analyzed 3-hexen-1-ol, acetate(Z) 422220 3.1

Nonanal 518683 1.6

Decanal 1392646 10.27

2-(2-ethoxyethoxy)ethanol ND NA

α- Farnesene 640638 4.7

α- Farnesene 231907 1.7

α-caryophyllene 916545 6.76

Methyl Salicylate

0507-041007T Treated Hop Plant 3-hexen-1-ol, acetate(Z) 912455 22.4 Nonanal ND ND Decanal 1118089 4.9 α- Farnesene 827617 20.3 α- Farnesene ND NA α-caryophyllene 2-(2-ethoxyethoxy)ethanol ND NA Methyl Salicylate 249468.5 6.3 0507-041007-T+C Analyzed 4/12/07 3-hexen-1-ol, acetate(Z) 204035 8.9 2295343

Nonanal 161730 7.05 Decanal 335964 14.637

2-(2-ethoxyethoxy)ethanol ND

α- Farnesene 384013 16.7

α- Farnesene 103568 4.51

α-caryophyllene ND 0

Methyl Salicylate 195957 8.5

10

0

Sample Identification Compound ID

Average

Area Counts c

Average

% Total

Area

Average

Total Area

Counts

0507-041007-C Control Hop Plant 3-hexen-1-ol, acetate(Z) 1506148.5 10.5 21516418

Nonanal 2731535 7.7

Decanal 66156617 18.6

2-(2-ethoxyethoxy)ethanol 1558482 4.4

α-caryophyllene 158727 0.5

α- Farnesene 200960 2.9

α- Farnesene 389787 1.1

Methyl Salicylate 316555 0.9

0507-041007-T+C Analyzed 4/12/07 3-hexen-1-ol, acetate(Z) 204035 8.9 2295343

Nonanal 161730 7.05 Decanal 335964 14.637

2-(2-ethoxyethoxy)ethanol ND NA

α- Farnesene 384013 16.7

α- Farnesene 103568 4.51

α-caryophyllene ND NA

Methyl Salicylate 195957 8.5 0507-050707T Treated Hop Plant #1 3-hexen-1-ol, acetate(Z) 8073782 18.5 43506992

Nonanal ND NA Decanal 129729 0.284 2-(2-ethoxyethoxy)ethanol ND NA α- Farnesene 2037392 4.7 α- Farnesene 616334 1.5 α- -caryophyllene 5569257 12.2 Methyl Salicylate 4425712 10.2 0507-050707-T+C Analyzed 5/11/07 3-hexen-1-ol, acetate(Z) 440886 12.6

Nonanal ND NA

Decanal 80203 2.29

10

1

Sample Identification Compound ID

Average

Area Counts c

Average

% Total

Area

Average

Total Area

Counts

α-caryophyllene ND NA α- Farnesene 562628 16.11 α- Farnesene 229122 6.56 2-(2-ethoxyethoxy)ethanol ND NA Methyl Salicylate 143342 4.10 0507-050707-C Control Hop Plant 3-hexen-1-ol, acetate(Z) 1572577 45.8 3431347

2,4 Hexadiene 98949 2.9

Decanal ND NA

2-(2-ethoxyethoxy)ethanol ND NA

α- Farnesene ND NA

α-caryophyllene 507225 14.8

Methyl Salicylate 172376 5.0

0507-0050707-T+C Analyzed 5/11/07 3-hexen-1-ol, acetate(Z) 92578 3.4 2723343

Nonanal ND NA Decanal ND NA 2-(2-ethoxyethoxy)ethanol ND NA

α- Farnesene ND NA

α-caryophyllene ND NA

Methyl Salicylate ND NA 0507-051407T Treated Hop Plant 3-hexen-1-ol, acetate(Z) 535843 69.5 763515

Nonanal ND NA

Decanal ND NA

2-(2-ethoxyethoxy)ethanol ND NA

α- Farnesene ND NA

α- -caryophyllene 71154 9.7

Methyl Salicylate 156429 20.9

0507-0541407-T+C Analyzed 5/17/07 3-hexen-1-ol, acetate(Z) 210563 11.9 1771614

10

2

Sample Identification Compound ID

Average

Area Counts c

Average

% Total

Area

Average

Total Area

Counts

Nonanal 291961 16.5

Decanal 632736 35.8

α-caryophyllene ND NA α- Farnesene ND NA 2-(2-ethoxyethoxy)ethanol ND NA Methyl Salicylate ND NA 0507-0514007-C Control Hop Plant 3-hexen-1-ol, acetate(Z) 3145062 64 4934530

2,4 Hexadiene 318889 6.3 Decanal 67324 1.6 α-caryophyllene 309196 6.4 α- Farnesene 87774 1.33 α- Farnesene 77023 1.2 Methyl Salicylate 191351 4.1 0507-0051407-T+C Analyzed 5/17/07 3-hexen-1-ol, acetate(Z) ND NA 2088546

Nonanal 368825 17.7

Decanal 860842 41.22

2,4 bis[trimethyl]benzoic acid 76764 3.7

α- Farnesene ND NA

α-caryophyllene ND NA

Methyl Salicylate ND NA

10

3

Figure 1

Comparative Data from 3/22/07 Sampling Data

Treated vs. Control 3/22/07

0 5000000 10000000 15000000 20000000 25000000 30000000

3-hexen-1-ol, acetate(Z)

Nonanal

Decanal

α-caryophyllene

α-Farnesene

2-(ethoxy)ethanol

Methyl Salicylate

Area Counts TIC

3/22 Treated

3/22 Control

10

4

Figure 2 : Treated vs. Treated after 48hrs combination 3/22/07

Treated vs Treated After Combination

0 1000000 2000000 3000000 4000000 5000000 6000000

3-hexen-1-ol, acetate(Z)

Nonanal

Decanal

α-caryophyllene

α- Farnesene

α- Farnesene

2-(2-ethoxyethoxy)ethanol

Methyl Salicylate

Treated Hop 3/22/07

Treated Hop After Comb