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Biochemical Mode of Resistance to Multiple Insect Pests in a Romaine Lettuce Cultivar

Permanent Link: http://ufdc.ufl.edu/UFE0021705/00001

Material Information

Title: Biochemical Mode of Resistance to Multiple Insect Pests in a Romaine Lettuce Cultivar
Physical Description: 1 online resource (207 p.)
Language: english
Creator: Sethi, Amit
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: antifeedant, armyworm, banded, beet, beetle, bioassays, cabbage, cucumber, damage, defense, enzymes, extraction, insect, interactions, lactuca, laticifers, lepidoptera, lettuce, looper, metabolites, oxidative, plant, sativa, secondary, solvent
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Lettuce (Lactuca sativa L.) quality and yield can be reduced by feeding of several insect pests. Host plant resistance to these insects is an environmentally sound adjunct to conventional chemical control. In this study I compared the survival, development and feeding behavior of cabbage looper, Trichoplusia ni (H?bner) and beet armyworm Spodoptera exigua (H?bner) on two romaine lettuce cultivars, resistant ?Valmaine? and susceptible ?Tall Guzmaine?. The survival and development of both species was significantly less on resistant Valmaine than on susceptible Tall Guzmaine. The two insect species showed different feeding preference for leaves of different age groups on Valmaine and Tall Guzmaine. Latex from Valmaine strongly inhibited feeding of banded cucumber beetle, Diabrotica balteata LeConte compared to Tall Guzmaine when applied to the surface of artificial diet in both choice and no-choice tests. In a choice test involving diet disks treated with Valmaine latex from young leaves versus mature leaves, the beetles consumed significantly less diet treated with latex from young than mature leaves. No significance difference in feeding was found between diet disks treated with latex from young and mature Tall Guzmaine leaves in choice tests. Three solvents of differing polarity (water, methanol and methylene chloride) were tested to extract deterrent compounds from latex; Valmaine latex extracted with water:methanol (20:80) strongly inhibited beetle feeding when applied to the surface of artificial diet. These studies suggest that moderately polar chemicals within latex may account for resistance in Valmaine to D. balteata. Further fractionation of methanolic crude extract of Valmaine latex was done using reverse phase and cation exchange solid-phase extraction to isolate the deterrent compounds. Retention of deterrent compounds on cation exchange resin suggests the presence of compounds with amine group in Valmaine latex. Further bioassay directed fractionation of cation exchange extract using LC/MS indicates the presence of ten compounds in the active fraction between 3 and 4 min. The successful isolation of potent feeding deterrents against D. balteata adults provides convincing evidence of a chemical basis for host plant resistance mediated through latex in this cultivar. Latex from damaged plants of Valmaine was much more deterrent to D. balteata adults than latex from undamaged plants when applied on the artificial diet under choice conditions and no such difference was found in Tall Guzmaine choice tests. The activities of three enzymes (phenylalanine ammonia lyase, polyphenol oxidase and peroxidase) significantly increased in Valmaine latex from damaged plants over time (i.e., 1, 3 and 6 d) after feeding initiation, but they remained the same in Tall Guzmaine latex. The constitutive level of phenylalanine ammonia lyase and polyphenol oxidase was also significantly higher in the Valmaine latex than in Tall Guzmaine latex. These studies suggest that latex chemistry may change after damage due to increased activity of inducible enzymes and that inducible resistance appears to act synergistically with constitutive resistance in Valmaine latex.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Amit Sethi.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: McAuslane, Heather J.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021705:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021705/00001

Material Information

Title: Biochemical Mode of Resistance to Multiple Insect Pests in a Romaine Lettuce Cultivar
Physical Description: 1 online resource (207 p.)
Language: english
Creator: Sethi, Amit
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: antifeedant, armyworm, banded, beet, beetle, bioassays, cabbage, cucumber, damage, defense, enzymes, extraction, insect, interactions, lactuca, laticifers, lepidoptera, lettuce, looper, metabolites, oxidative, plant, sativa, secondary, solvent
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Lettuce (Lactuca sativa L.) quality and yield can be reduced by feeding of several insect pests. Host plant resistance to these insects is an environmentally sound adjunct to conventional chemical control. In this study I compared the survival, development and feeding behavior of cabbage looper, Trichoplusia ni (H?bner) and beet armyworm Spodoptera exigua (H?bner) on two romaine lettuce cultivars, resistant ?Valmaine? and susceptible ?Tall Guzmaine?. The survival and development of both species was significantly less on resistant Valmaine than on susceptible Tall Guzmaine. The two insect species showed different feeding preference for leaves of different age groups on Valmaine and Tall Guzmaine. Latex from Valmaine strongly inhibited feeding of banded cucumber beetle, Diabrotica balteata LeConte compared to Tall Guzmaine when applied to the surface of artificial diet in both choice and no-choice tests. In a choice test involving diet disks treated with Valmaine latex from young leaves versus mature leaves, the beetles consumed significantly less diet treated with latex from young than mature leaves. No significance difference in feeding was found between diet disks treated with latex from young and mature Tall Guzmaine leaves in choice tests. Three solvents of differing polarity (water, methanol and methylene chloride) were tested to extract deterrent compounds from latex; Valmaine latex extracted with water:methanol (20:80) strongly inhibited beetle feeding when applied to the surface of artificial diet. These studies suggest that moderately polar chemicals within latex may account for resistance in Valmaine to D. balteata. Further fractionation of methanolic crude extract of Valmaine latex was done using reverse phase and cation exchange solid-phase extraction to isolate the deterrent compounds. Retention of deterrent compounds on cation exchange resin suggests the presence of compounds with amine group in Valmaine latex. Further bioassay directed fractionation of cation exchange extract using LC/MS indicates the presence of ten compounds in the active fraction between 3 and 4 min. The successful isolation of potent feeding deterrents against D. balteata adults provides convincing evidence of a chemical basis for host plant resistance mediated through latex in this cultivar. Latex from damaged plants of Valmaine was much more deterrent to D. balteata adults than latex from undamaged plants when applied on the artificial diet under choice conditions and no such difference was found in Tall Guzmaine choice tests. The activities of three enzymes (phenylalanine ammonia lyase, polyphenol oxidase and peroxidase) significantly increased in Valmaine latex from damaged plants over time (i.e., 1, 3 and 6 d) after feeding initiation, but they remained the same in Tall Guzmaine latex. The constitutive level of phenylalanine ammonia lyase and polyphenol oxidase was also significantly higher in the Valmaine latex than in Tall Guzmaine latex. These studies suggest that latex chemistry may change after damage due to increased activity of inducible enzymes and that inducible resistance appears to act synergistically with constitutive resistance in Valmaine latex.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Amit Sethi.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: McAuslane, Heather J.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021705:00001


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BIOCHEMICAL MODE OF RESISTANCE TO MULTIPLE INSECT PESTS IN A ROMAINE
LETTUCE CULTIVAR




















By

AMIT SETHI


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2007
































2007 Amit Sethi



































To my beloved father, Amar L. Sethi who has been my role-model for hard work, persistence
and personal sacrifices, and who instilled in me the inspiration to set high goals and the
confidence to achieve them, and his words of encouragement and push for tenacity ring in my
ears.









ACKNOWLEDGMENTS

It gives me immense pleasure to record my thanks and sense of profound gratitude to my

major advisor, Dr. Heather J. McAuslane for her kind inspiration, constant supervision,

constructive criticism and encouragement throughout the period of my Ph.D, especially time

spent in informal discussions and training that have all been a valuable part of my learning

experience.

Expressing sense of gratitude and admiration for the kind help extended by Dr. Hans T.

Alborn (CMAVE, USDA) and Dr. Bala Rathinasabapathi (Departemnt of Horticultural Sciences)

is not mere obedience of convention, but a real appreciation. I am also highly obliged to Dr.

Gregg S. Nuessly and Dr. Russell T. Nagata (Everglades Research and Education Center), the

members of my committee for their guidance and valuable suggestions for the improvement of

this dissertation project.

I owe my sincere thanks to Jennifer Hogsette, Jennifer Meyer, and Debbie Boyd for their

timely help in the insect colony maintenance when I was away for the conferences. I am thankful

to Dr. Peter Teal (CMAVE, USDA) for providing greenhouse space for growing lettuce plants

and also Julia Meredith (CMAVE, USDA) for taking care of plants when I was away for

scientific conferences. I am also thankful to Dr. Marty Marshall (Departement of Food Science

and Food Nutrition) for use of his spectrophotometer.

Words fail me to convey the depth of my feelings and gratitude to my lab mates Jennifer

Meyer, Karla Addesso, Jennifer Hogsette and Murugesan Rangasamy for their encouragement,

generosity and memorable association.

I seize the opportunity to express my moral obligations to my brothers and their families,

and in-laws for their encouragement and moral support. My father deserves my heartiest thanks









for his magnanimity, inspiration and encouragement at times of despair that helped me in

innumerable ways in making this effort a success.

No appropriate words could be traced in the presently available lexicon to acknowledge

the sacrifices, selfless devotion, love and unflinching support extended by my beloved wife Dr.

Ramandeep Kaur to complete this study.

Putting it last, but feeling it first, I owe God who has given me courage, patience and

motivation from time to time in completing my degree successfully.









TABLE OF CONTENTS

page

A CK N O W LED G M EN T S ................................................................. ........... ............. .....

L IST O F T A B L E S ...................................................................................................... . 9

LIST OF FIGURES .................................. .. .... ..... ................. 10

ABSTRAC T ................................................. ............... 14

CHAPTER

1 R EV IEW O F LITER A TU R E ......................................................................... .......... .......... 16

Introdu action ............................................ ........... .................................. 16
O rigin and H history of L ettuce........................................................................................... 17
T ypes of L ettu ce ................................................................... ............18
Insect Pests and Lettuce ....................................................... .......... ........ ...... 18
Host Plant Resistance ....................... ...................................21
Biochem ical Basis of H ost Plant R resistance ................................... ....................................24
H ost Plant Resistance Due To Proteins................................................ ...... ......... 24
P protease inhibitors ................................................................ .. ....... ......... 2 5
Cysteine protease................................................... 25
O xidativ e enzym es .............................. .......................... .... ........ .... ..... ...... 26
Proteins of the cell w all .................. ......................... .. .. .... .. ........ .... 28
Secondary m etabolism pathw ays ........................................ ......... ............... 29
Enzymes involved in secondary metabolism ......................................................29
Host Plant Resistance Due To Secondary Plant Compounds............... ...................30
P h e n o lic s ................................................................ 3 1
F lav o n o id s .................................................................3 3
T erpenoids .................................... .. ...................................35
H ost Plant R resistance in Lettuce to Insect Pests ....................................................................37
A p h id s............... .. ..........................................................3 7
C cabbage L ooper .............................................................................................. ........39
B handed C ucum ber B eetle ..................................................................... ..... ................40
L eafm in er ................................................................4 1
H elicoverp a species........... ...... .............................................. .............. .......... ....... 42
Spodoptera species .................... .......................................42
Bemisia species or strains ............. ... ... .... ... ........... ............ ......... 43
T hrips....... ...............................................................43
R e se a rc h G o a ls ................................................................................................................. 4 4







6










2 HOST PLANT RESISTANCE IN ROMAINE LETTUCE AFFECTS LARVAL
FEEDING BEHAVIOR AND BIOLOGY OF TRICHOPLUSIA NI AND
SPODOPTERA EXIGUA (LEPIDOPTERA: NOCTUIDAE) ...........................................46

In tro du ctio n ................... .......................................................... ................ 4 6
M materials and M methods ...................................... .. .......... ....... ...... 48
P la n ts ........................................................................................... 4 8
In sects .................. ................ ................................. ......................... 4 8
Neonate Survival and Development to Third Instar............................................ 49
Survival and Development from Neonate to Adult Emergence................................50
Fecundity and Longevity of Subsequent Generation ............................................... 51
Results.................. .... .......... ....... .......... ............. 51
Neonate Survival and Development to Third Instar............................................ 51
L arval F feeding B behavior .............. ........................................................... ............... 52
Survival and Development from Neonate to Adult Emergence................ .......... 53
Fecundity and Longevity of Subsequent Generation ............................................... 54
D iscu ssion ................. .... .................. .................................................54

3 ROMAINE LETTUCE LATEX DETERS FEEDING OF BANDED CUCUMBER
BEETLE (COLEOPTERA: CHRYSOMELIDAE) ........................... ............... 68

Introdu action ................... .......................................................... ................. 6 8
M materials and M methods ...................................... .. .......... ....... ...... 71
P plants an d In sects ................................................................7 1
A artificial D iet P reparation ...................................................................... ...................73
Latex Collection and Solvent Extraction..................................................................... 74
B ioassay Conditions ..................................................................... ..........75
Choice Tests and No-choice Tests with Fresh Latex ............................................... 76
Choice Tests Using Latex from Young and Mature Leaves .......................................76
N o-Choice Tests U sing Latex Extracts ........................................ ....................... 77
Beetle Behavior in Response to Contacting Latex........................................................77
Statistical A n aly sis ................................................................7 8
R e su lts .............. .............. .... ............................................................8 0
Latex Choice and N o-Choice Tests .............................................. ........................... 80
Choice Tests Using Latex from Young and Mature Leaves .......................................81
N o-Choice Tests U sing Latex Extracts ........................................ ....................... 82
Beetle Behavior in Response to Contacting Latex........................................................83
D isc u ssio n ................... ........................................................... ................ 8 4

4 BANDED CUCUMBER BEETLE (COLEOPTERA: CHRYSOMELIDAE)
RESISTANCE IN ROMAINE LETTUCE: UNDERSTANDING LATEX
C H E M IST R Y ...................................... ................................................... 104

Introduction ................... ........................................................ ................. 104
M materials an d M eth od s .............................................................................. ..................... 10 5
P plants an d In sects ..................................................................... 10 5









Assay for Feeding Deterrence .......................................................... .............. 106
Latex Collection and Crude Extract Preparation .............................. .................... 107
Fractionation of Crude Extract Using Reversed-Phase (C-18) Cartridge ................ 107
Fractionation of Crude Extract Using C-18, SAX and SCX Cartridges Connected in
S series ..................................10 9
LC/MS Separation of SCX Fraction............................ ......... ..... .......... 110
Statistical Analysis .................................................................................... .......................111
Results ............... ........... ..... ...... ......... ......... .............. ......... 112
Fractionation of Crude Extract Using C-18 Cartridge ............................. ...............12
Fractionation of Crude Extract Using C-18, SAX and SCX Cartridges Connected in
S e r ie s .................................................................................................................... 1 1 3
Fractionation of SCX Fraction Using LC/MS....................... .................114
D iscu ssion ......... ........ ......... ............................................114

5 INVESTIGATING ENZYME INDUCTION AS A POSSIBLE REASON FOR
LATEX-MEDIATED INSECT RESISTANCE IN ROMAINE LETTUCE .....................136

Introduction ................ ...... ........................ ................... 136
M materials and M methods .................................... ... .. .......... ....... .... 138
P la n ts .........................................................................1 3 8
In se c ts ............. ..... ............ ................. .......................................................1 3 8
Artificial Diet.................... ..................................................139
Bioassay Conditions for Feeding Damage ....................................... ......... 139
Choice-tests Using Latex from Damaged and Undamaged Plants............................140
E nzym e A activity A ssay s................................................................ .......................... 140
Phenylalanine ammonia-lyase (PAL)......................................................... 141
Polyphenol oxidase (PPO ). ............................................. ........................... 141
Peroxidase (POX)............... ... ................. ..... ..........142
Statistical A naly sis ........................ .. .... ........................ .. .... .. .. ....... .... .... 142
R e su lts ...................1...................4...................3..........
Latex Characteristics from Damaged and Undamaged Plants .....................................143
Choice-tests Using Latex from Damaged and Undamaged Plants.............................143
Total Protein C content ............................................ .. .... .... ......... .. .... .. 145
Phenylalanine Ammonia Lyase ........... ..... ......... ............................ 145
P olyphenol O xidase ........... ............................................................................. .. ...... .. 146
Peroxidase ................ ... .....................................146
Relationship between Female Weight Gain and Enzyme Activity .............................147
D iscu ssion ......... ............ ......................... ............................147

6 SU M M A R Y .......................................................................... ..........................164

LIST OF REFEREN CES ......... ...................................... ......... ...... ................... 173

B IO G R A PH IC A L SK E T C H ............................................................................. ....................206






8









LIST OF TABLES


Table page

2-1 Performance of cabbage looper and beet armyworm released as neonates onto
V alm aine and Tall G uzm aine lettuce.................................................................... ...... 59

2-2 Fecundity and longevity of subsequent generation of cabbage looper and beet
armyworm reared on Valmaine and Tall Guzmaine lettuce...................... ...............60

3-1 Dry weight consumption of diet disks treated with Valmaine (Val) or Tall Guzmaine
(TG) latex under choice and no-choice tests by six D. balteata adults in 16 h .............101

3-2 Feeding deterrent activity of latex against D. balteata adults when artificial diet disks
were treated with latex from either resistant Valmaine (Val) or susceptible Tall
Guzmaine (TG) in choice and no-choice tests........................................... 102

3-3 Dry weight of diet consumed by six D. balteata adults in 16 h when given a choice
between diet disks treated with latex from either young or mature leaves of resistant
Valmaine or susceptible Tall Guzmaine lettuce cultivars................... ... .............103

5-1 Total diet consumption by six D. balteata adults on two diet disks treated with latex
from same lettuce cultivar, Valmaine or Tall Guzmaine after 24 h of their release........163









LIST OF FIGURES


Figure page

2-1 Experimental setup to study cabbage looper and beet armyworm neonate survival
and develop ent to third instar......... ...................................................... ............... 61

2-2 Larval mortality of cabbage looper and beet armyworm after 1 wk of feeding on
resistant Valmaine and susceptible Tall Guzmaine lettuce.....................................62

2-3 Instars of cabbage looper (CL) and beet armyworm (BAW) surviving for 1 wk on
resistant Valmaine and susceptible Tall Guzmaine lettuce.....................................63

2-4 Feeding of tw o lepidopterans on lettuce ................................................. ....... ........ 64

2-5 Feeding preference of cabbage looper (CL) and beet armyworm (BAW) larvae
among lettuce leaves of different ages on resistant Valmaine and susceptible Tall
Guzmaine................. ... .... ............................... 65

2-6 Feeding behavior of beet armyworm ........................................ .......................... 66

2-7 Relationships between adult weight and fecundity of cabbage looper (CL) and beet
armyworm (BAW) that developed from larvae reared on resistant Valmaine (VAL)
or susceptible Tall Guzm aine (TG) lettuce ............................................. ............... 67

3-1 Wounding of lettuce releases a milky fluid called latex ..................................................88

3-2 Colony rearing ofD. balteata. See text for description of each stage of colony
m maintenance. ..............................................................................89

3-3 Collection of latex from romaine lettuce, application on artificial diet disk and
b ioassay setu p .......................................................... ................. 90

3-4 Schem e of latex solvent extraction. .............................................................................91

3-5 Latex dissolution in different solvents...................................................... ............. 92

3-6 Feeding bioassays using fresh latex...................................................................... 93

3-7 Mean number ofD. balteata adults feeding on artificial diet disks treated with latex
from resistant Valmaine (Val), disks treated with latex from susceptible Tall
Guzmaine (TG), and control diet disks in choice tests.. ....................................... ....... 94

3-8 Mean number ofD. balteata adults feeding on two artificial diet disks treated with
latex from resistant Valmaine (Val), disks treated with latex from susceptible Tall
Guzmaine (TG), and control diet disks in no-choice tests.......................................95

3-9 Choice tests using D. balteata adults on two artificial diet disks treated with latex
from young and mature leaves of the same cultivar. ............. .................................. ....... 96









3-10 Number ofD. balteata adults feeding on artificial diet disks treated with latex from
young or mature leaves of resistant Valmaine (Val) and susceptible Tall Guzmaine
(T G ) in choice tests........................................................................... 97

3-11 No-choice tests using D. balteata adults when both the disks were smeared with
either Valmaine latex extract or Tall Guzmaine latex extract ................. ..................98

3-12 Mean number ofD. balteata adults feeding on two artificial diet disks treated with
latex extracts from resistant Valmaine (Val) and susceptible Tall Guzmaine (TG),
and controls in no-choice test.. .................... ..... ............................... ...............99

3-13 Dry weight of diet consumed by six D. balteata adults in 16 h when both diet disks
were treated with Valmaine (Val) or Tall Guzmaine (TG) latex extracts under no-
ch oice situ action s.................................................... ................ 10 0

4-1 Scheme for solid-phase extraction and fractionation of crude extract after passing
through reversed-phase (C-18) cartridge. .................................... .......... ................... 118

4-2 Scheme for solid-phase extraction and fractionation of crude extract after passing
through reversed-phase (C-18), anion (SAX) and cation (SCX) exchange cartridges
connected in series .................................................................... ..........119

4-3 Fractions obtained after HPLC analysis of cation exchange (SCX) fraction ................120

4-4 Color characteristics of fractions obtained after passing crude extract through
reversed phase C -18 cartridge......... ..................................................... ............... 121

4-5 Bioassays of C-18 fractions applied on artificial diet disks using D. balteata adults
under no-choice conditions. ...................................................................... .................. 122

4-6 Mean number ofD. balteata adults feeding after 90 min on two artificial diet disks
treated with fractions obtained after passing crude extract at three pH levels through
C -18 cartridge. ......... ......... ..................................................................... 123

4-7 Dry weight of diet consumed by D. balteata adults when disks were treated with
fractions obtained after passing crude extract with different pH levels through C-18
cartridge e ................... ............................ ......................... ................ 12 4

4-8 Color characteristics of fractions obtained after passing C-18 unbound fraction
through anion (SAX) and cation (SCX) exchange cartridges connected in series..........125

4-9 Bioassays of ion-exchange fractions applied on artificial diet disks using D. balteata
adults under no-choice conditions. ............................................................................126

4-10 Mean number ofD. balteata adults feeding after 90 min on diet disks treated with
ion-exchange fractions obtained by passing C-18 unbound fraction (original pH 6.5)
through anion (SAX) and cation (SAX) exchange cartridges connected in series..........127









4-11 Dry weight of diet consumed by D. balteata adults when disks were treated with ion-
exchange fractions obtained after passing C-18 unbound fraction (original pH 6.5)
through anion (SAX) and cation (SAX) exchange cartridges connected in series..........128

4-12 Mean number of insects feeding after 90 min on diet disks treated with fractions
obtained after LC/MS analysis of cation exchange fraction (SCX) at pH 9.0 of the
m ob ile ph ase.. ................................................................................. 12 9

4-13 Dry weight of diet consumed by D. balteata adults when disks were treated with
fractions obtained after LC/MS analysis of cation exchange fraction (SCX) at pH 9.0
of the mobile phase. .............. ............................ ..... ......... .. 130

4-14 Mean number of insects feeding after 90 min on diet disks treated with fractions
obtained after LC/MS analysis of cation exchange fraction (SCX) at pH 10.0 of the
m obile phase. ............................................................................13 1

4-15 Dry weight of diet consumed by D. balteata adults when disks were treated with
fractions obtained after LC/MS analysis of cation exchange fraction (SCX) at pH
10 .0 of the m obile phase. .............. .. .................................................. .. ..................... 132

4-16 Electrospray LC/MS total negative ion trace of active fraction between 3 and 4 min....133

4-17 Structure of sesquiterpene lactones characterized in lettuce......................................... 134

4-18 Chemical structures of flavonoids found in lettuce ............................... ............... .135

5-1 Feeding damage caused by D. balteata adults on two lettuce cultivars, Valmaine
(VAL) and Tall Guzmaine (TG). ................................... ......... ................... 152

5-2 Adults ofD. balteata feeding on diet disks treated with latex from damaged and
undamaged plants of two lettuce cultivars, Valmaine and Tall Guzmaine...................153

5-3 Number ofD. balteata adults feeding on artificial diet disks in a choice between
latex from damaged and undamaged plants of Valmaine after 1, 2, 3 and 4 h of their
release ......................................................... ..................................154

5-4 Number ofD. balteata adults feeding in a choice test using two artificial diet disks
treated with damaged and undamaged plants of lettuce cultivar, Tall Guzmaine after
1, 2, 3 and 4 h of their release................................................ .............................. 155

5-5 Artificial diet consumption after 24 h by D. balteata adults in choice test using two
diet disks treated with latex from damaged and undamaged plants of two lettuce
cultivars, Valmaine (VAL) and Tall Guzmaine (TG).............................. ... ..................156

5-6 Total protein content in two lettuce cultivars, Valmaine (VAL) and Tall Guzmaine at
1, 3 and 6 d after initiation of feeding damage by adults of D. balteata ........................157









5-7 Activity of phenylalanine ammonia lyase (PAL) in two lettuce cultivars, Valmaine
(VAL) and Tall Guzmaine at 1, 3 and 6 d after initiation of feeding damage by adults
of D balteata.. ..................................... .................. ...... ... ........ ....... 158

5-8 Activity of polyphenol oxidase (PPO) in two lettuce cultivars, Valmaine (VAL) and
Tall Guzmaine at 1, 3 and 6 d after initiation of feeding damage by adults of D.
baltea ta ............................................................ ...... ........ ................ 159

5-9 Activity of peroxidase (POX) in two lettuce cultivars, Valmaine (VAL) and Tall
Guzmaine at 1, 3 and 6 d after initiation of feeding damage by adults of D. balteata....160

5-10 Gain in fresh weight of D. balteata females over a 6-d period of feeding on two
romaine lettuce cultivars, Valmaine (VAL) and Tall Guzmaine (TG)...............................161

5-11 Relationship between fresh weight gained by D. balteata females feeding on two
lettuce cultivars, Valmaine (VAL) and Tall Guzmaine (TG) and activity of PAL,
PPO and POX enzymes after 1, 3 and 6 d of feeding damage............... ... ...............162









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

BIOCHEMICAL MODE OF RESISTANCE TO MULTIPLE INSECT PESTS IN A ROMAINE
LETTUCE CULTIVAR

By

Amit Sethi

December 2007

Chair: Heather J. McAuslane
Major: Entomology and Nematology

Lettuce (Lactuca sativa L.) quality and yield can be reduced by feeding of several insect

pests. Host plant resistance to these insects is an environmentally sound adjunct to conventional

chemical control. In this study I compared the survival, development and feeding behavior of

cabbage looper, Trichoplusia ni (Hubner) and beet armyworm Spodoptera exigua (Hubner) on

two romaine lettuce cultivars, resistant 'Valmaine' and susceptible 'Tall Guzmaine'. The

survival and development of both species was significantly less on resistant Valmaine than on

susceptible Tall Guzmaine. The two insect species showed different feeding preference for

leaves of different age groups on Valmaine and Tall Guzmaine.

Latex from Valmaine strongly inhibited feeding of banded cucumber beetle, Diabrotica

balteata LeConte compared to Tall Guzmaine when applied to the surface of artificial diet in

both choice and no-choice tests. In a choice test involving diet disks treated with Valmaine latex

from young leaves versus mature leaves, the beetles consumed significantly less diet treated with

latex from young than mature leaves. No significance difference in feeding was found between

diet disks treated with latex from young and mature Tall Guzmaine leaves in choice tests. Three

solvents of differing polarity (water, methanol and methylene chloride) were tested to extract

deterrent compounds from latex; Valmaine latex extracted with water:methanol (20:80) strongly









inhibited beetle feeding when applied to the surface of artificial diet. These studies suggest that

moderately polar chemicals within latex may account for resistance in Valmaine to D. balteata.

Further fractionation of methanolic crude extract of Valmaine latex was done using

reverse phase and cation exchange solid-phase extraction to isolate the deterrent compounds.

Retention of deterrent compounds on cation exchange resin suggests the presence of compounds

with amine group in Valmaine latex. Further bioassay directed fractionation of cation exchange

extract using LC/MS indicates the presence of ten compounds in the active fraction between 3

and 4 min. The successful isolation of potent feeding deterrents against D. balteata adults

provides convincing evidence of a chemical basis for host plant resistance mediated through

latex in this cultivar.

Latex from damaged plants of Valmaine was much more deterrent to D. balteata adults

than latex from undamaged plants when applied on the artificial diet under choice conditions and

no such difference was found in Tall Guzmaine choice tests. The activities of three enzymes

phenylalaninee ammonia lyase, polyphenol oxidase and peroxidase) significantly increased in

Valmaine latex from damaged plants over time (i.e., 1, 3 and 6 d) after feeding initiation, but

they remained the same in Tall Guzmaine latex. The constitutive level of phenylalanine ammonia

lyase and polyphenol oxidase was also significantly higher in the Valmaine latex than in Tall

Guzmaine latex. These studies suggest that latex chemistry may change after damage due to

increased activity of inducible enzymes and that inducible resistance appears to act

synergistically with constitutive resistance in Valmaine latex.









CHAPTER 1
REVIEW OF LITERATURE

Introduction

Lettuce, Lactuca sativa L., a member of the Compositae (Asteraceae), is a rosette plant that

is grown commercially for its leaves. The family Compositae includes a wide range of

herbaceous plants and accounts for one tenth of known angiosperm species. Lettuce is one of the

most important vegetable crops grown in the United States, in terms of quality and quantity as

well as its acreage (Ryder 1998). Demand for lettuce grows yearly, probably due to its use as a

healthy, low caloric, salad component of meals. It requires minimal processing, and its long

storage life, good quality and reputation as healthy food contribute to its increase in salad bars

and fast foods (Ferreres et al. 1997).

During 2006, the United States produced 2,935 thousand metric tons of head lettuce, 857.8

thousand metric tons of leaf lettuce, and 990.3 thousand metric tons of romaine lettuce harvested

over areas of 71,508, 29,056, and 24,929 hectares, respectively (Agricultural Statistics 2007).

Crisphead (iceberg) varieties predominate in the Unites States markets, particularly for extended

transport. However, romaine (Cos), butterhead, and leaf type lettuces are also produced in

considerable amounts. A number of other varieties that show variation in color from light green

and yellow to deep green are also becoming more accepted. Romaine lettuce is the most common

leaf lettuce grown throughout the United States.

California is the major producer of lettuce (77% of total production) in the United States

(Lauritzen 1999), followed by Arizona, Florida and New Jersey (Kerns et al. 1999, USDA 2002).

Lettuce production from the Everglades Agricultural areas in southern Florida contributes 90%

of the total state production (Hochmuth et al. 1994).









Origin and History of Lettuce

Lettuce originated in the Mediterranean region and its cultivation may have started in

Egypt as early as 4500 years BC (Lindquist 1960). Lettuces were supposedly grown by Persians

500 years BC, and were introduced into China between the years 600 and 900 AD. Lettuces were

mentioned in England in the fourteenth century and reached America with Columbus (Davis et

al. 1997). In 1494, Columbus introduced a non-heading type of lettuce to the New World. This

type quickly formed a seed stalk and in fact did not become a stable food crop. Head lettuce in

the United States was first reported in 1543 (Helm 1954). Salad lettuce was popular with the

ancient Greeks and Romans and it arrived in the United States during colonial days (Davis et al.

1997). Sturtevant (1886) studied the history of lettuce and observed that 83 distinct varieties of

lettuce were grown under nearly 200 names at the New York Agricultural Experiment Station.

These varieties were present in three distinct form-species, the lanceolate-leaved, the Cos and the

cabbage.

The lanceolate-leaved form was represented by one variety, 'the deer's tongue', and had a

chicory-like appearance in some stages of its growth, as mentioned and illustrated by Bauhin

(1671). This type of lettuce was submitted under the names Romaine asperge, Lactuca

angustana Hort., and L. cracoviensis Hort by Vilmorin (1883).

The Cos lettuce had upright growth of elongated, spatulate leaves. The Cos form was less

commonly grown in northern Europe as compared to the south and was seldom cultivated in

France and Germany in the sixteenth century.

Cabbage lettuce was characterized by rounded and spatulate leaves, growing less upright

than the Cos lettuce. The commentators of the sixteenth and seventeenth centuries deemed this

form-species to have been known to ancient Greeks and Romans. Pliny (23-79 AD) and

Columella (42 AD) referred to it as a variety, 'Laconicon', and 'Tartesian' or 'Baetica',









respectively. The cabbage lettuce was more wrinkled or blistered than the Cos (Sturtevant 1886).

Pinaeus (1561) identified a heading lettuce that closely resembled "the stone tennis ball" variety

of lettuce. Botanists were agreed in considering the cultivated lettuce as a modification of the

wild species L. scariola (de Candolle 1885). In conclusion, these three form-species had different

origins from different wild forms that had been cultivated in different regions of the world

(Sturtevant 1886).

Types of Lettuce

There are five modern types of lettuce based on morphological features: crisp-head, leaf,

butterhead, cos or romaine, and stem (Davis et al. 1997, Ryder 1998). The crisp-head varieties

with dense, firm heads and crisp leaves are the most significant commercial types and take about

75 130 d from planting to mature. Leaf lettuce varieties have frilled, glossy red or bright green

leaves and mature in 45 d from planting. Leaf lettuce is a good type of lettuce for home gardens,

as it matures quickly and is easy to grow. Butterhead lettuce generates an unfastened and soft

head, and inner leaves have an oily or buttery feel. Butterhead varieties produce high quality

lettuce for commercial purposes. They mature slightly earlier than crisp-head varieties. The cos

or romaine type of lettuce develops an elongated head of stiff, upright leaves about 80 d from

planting. Cos lettuce is an important lettuce type in Europe and is also gaining popularity in the

United States. Stem lettuce often is listed in catalogs under the name of Celtuce (CELery -

letTUCE). It is grown for its fleshy, elongated stem rather than its leaves.

Insect Pests and Lettuce

Lettuce is vulnerable to attack by several insect pests from seedling to reproductive stages.

The estimated average yield loss is 17 and 13% for fall and spring lettuce, respectively, due to

attack of various insect pests (Anonymous 2003). Seedling pests are bulb mites (Rhizoglyphus

spp., Tyrophagus spp.), black cutworm (Agrotis ipsilon Hufnagel), variegated cutworm









(Peridroma saucia (Hibner)), granulate cutworm (Feltia subterranean (Fabricius)), darkling

beetles tenebrionidss), field cricket (Gryllus spp.), garden symphylans (Scutigerella immaculate

(Newport)), pea leafminer (Liriomyza huidobrensis (Blanchard)), serpentine leafminer (L. trifolii

(Burgess)), vegetable leafminer (L. sativae Blanchard), and springtails.

Lepidopterous pests are responsible for major economic yield losses in lettuce, with losses

reaching 100% if control measures are not followed (Inglis and Vestey 2001). Important

lepidopterous pests include: armyworm (Pseudaletia unipuncta Haworth), beet armyworm

(Spodoptera exigua (Hibner)), corn earworm (Helicoverpa zea Boddie), tobacco budworm

(Heliothis virescens (Fabricius)), cabbage looper (Trichoplusia ni (Hibner)), alfalfa looper

(Autographa californica Speyer), and saltmarsh caterpillar (Estigmene acrea [Drury]) (Parenzan

1984, Toscano et al. 1990, McDougall et al. 2002, Anonymous 2003). In Florida, beet

armyworm (S. exigua), southern armyworm (S. eridania), cabbage looper (T. ni), corn earworm

(H. zea), black cutworm (A. ipsilon), variegated cutworm (P. saucia), and granulate cutworm (F.

subterranea) are the major lepidopterous pests (Nuessly and Webb 2003).

The coleopterous pests of lettuce include western spotted cucumber beetle, Diabrotica

undecimpunctata howardi Barber and banded cucumber beetle, D. balteata LeConte (Nuessly

and Webb 2003). In Florida, cucumber beetles are found throughout the state. The banded

species is more common in central and southern Florida whereas, the spotted species is more

prevalent in northern Florida. Beetles may cause potential losses of 100%, if not managed. Yield

loss with proper management strategies is generally less than 2.5%. Cucumber beetles became a

problem on lettuce in Washington, when peas and cucumbers were grown in lettuce growing

areas (Inglis and Vestey 2001).









The homopterous pests are foxglove aphid (Aulacorthum solani (Kaltenbach)), green

peach aphid (Myzuspersicae (Sulzer)), potato aphid (Macrosiphum euphorbiae (Thomas)),

lettuce aphid (Nasonovia ribisnigri (Mosley)), lettuce root aphid (Pemphigus bursarius (L.)), and

silverleaf whitefly (Bemisia argentifolii Bellows & Perring). In Florida, Uroleucon

pseudoambrosiae (Olive) is important (Mossler and Dunn 2005). Aphids appear annually in

lettuce production fields and cause yield losses generally less than 2% under normal

management with insecticides. Losses in Washington can range from 75 to 100% without the

timely use of chemical control measures (Inglis and Vestey 2001). Tarnished bug (Lygus

lineolaris (Palisot) and L. hesperus Knight) is also a pest of lettuce. It causes qualitative damage

due to discharge of a toxin during feeding that can be sufficiently severe to make the heads

unmarketable. This pest arises irregularly every few years, often later in the spring and early

summer. Potentially 100% of the acreage can be affected without appropriate management

(Kurtz 2001, McDougall et al. 2002, Anonymous 2003).

In the United States, about 93% of the lettuce area is highly dependent upon chemical

control for management of economic pests (Agricultural Statistics 2001). Florida, in particular,

ranks first among lettuce growing states in the usage of insecticides to manage insect pests.

Florida growers applied insecticides on 98 to 100% of the states lettuce acreage with a total

annual usage ranging from 1,900 to 4,900 pounds of active ingredient (Mossler and Dunn 2005).

High dependence on chemicals poses a potential threat to farmers, the environment, and natural

enemies of these insect pests. Dependence on chemicals is also costly. Therefore, there is a need

to look for alterative strategies for management of economic insect pests of lettuce. Host plant

resistance should be one of the major components of an integrated pest management (IPM)

program and can sustain or improve production efficiency in ways that will maintain or enhance









natural resources and the environment (Sharma and Ortiz 2002, Sadasivan and Thayumanavan

2003). Despite noticeable benefits of host plant resistance mediated through chemicals, it may

reduce the competitive ability of plants, leading to a trade-off between growth and resistance

(Herms and Mattson 1992). The production and maintenance of these chemicals require

resources that are then not available for the growth and reproduction of plants. Therefore,

metabolic costs are thought to be involved in resistance (Agrawal 1999) and resistance is always

affected by metabolic turnover of compounds (Fagerstrom 1989, Skogsmyr and Fagerstrom

1992, Gershenzon 1994).

Management of insects based on host plant resistance is more advantageous economically,

ecologically and environmentally than management based on chemical measures (Sharma and

Ortiz 2002, Sadasivan and Thayumanavan 2003). It is a very targeted and long-lasting approach

to manage economic insect pests. Dependence on fewer chemical sprays and increased yields

could provide economic benefits. Plant resistance increases ecosystem stability due to conserving

species diversity and maintains natural food webs by not disturbing natural enemies of insect

pests.

Host Plant Resistance

Plants live in a world that is inhabited by numerous adversaries (biotic and abiotic), the

major proportion of which belongs to plant-eating animals, including insects, called herbivores.

In spite of the great variety of herbivores, only parts of plants are defoliated, and the majority of

plant foliage and reproductive structures survives due to an innate capacity to tolerate herbivory

by compensating for resource losses (Constabel 1999, Strauss and Agrawal 1999), or to defend

themselves and thus to reduce the amount of damage (Constabel 1999). The ability of the plant

to defend itself against herbivores using different strategies is known as host plant resistance.

Host plant resistance is considered to be one of the most effective components of an integrated









pest management program and has been exploited to reduce the dependence on chemical

insecticides (Panda and Kush 1995). Host plant resistance is usually compatible with other

control measures like biological control and cultural controls, and maintains the food web by

conserving the natural enemies. Plants possess a natural defensive system incorporating

mechanical and chemical factors produced via transcriptional activation of corresponding genes.

These defenses operate either constitutively or after damage due to enemy attack (induced

resistance) (Vet 1999).

Many studies have investigated host plant preferences of herbivorous insects.

Morphological structures like hair and waxes (Lucas et al. 2000), hooks, spikes and trichomes

(Gilbert 1971), leaf hardness (Patanakamjorn and Pathak 1967), and physical factors, such as

water content (Scriber 1977), and nutrient content (Morrow and Fox 1980) are identified as

important factors leading to rejection of or preference for certain plant tissues by an insect. Low

nutritional quality of the plants may impede the development of insect herbivores (Scriber and

Slansky 1981). Plants are also known to be full of an array of secondary compounds, which may

be toxic, lower the nutritional quality of the foliage, or act as antifeedants (Fraenkel 1959,

Bernays and Chapman 1977, Rhoades 1979, Scriber and Slansky 1981, Constabel 1999).

Secondary chemicals are not evenly distributed in plant tissues. They are usually

concentrated in specialized structures, like vacuoles, idioblasts, glandular trichomes, cavities and

canals (Esau 1965, Fahn 1979). Plants sequester secretions within a diversity of canal systems

that include laticifers, resin ducts and phloem (Fahn 1979, Metcalfe and Chalk 1983). The canals

usually form a complex network and are effectively distributed throughout the plant. Secretions

in these canals are characteristically stored under pressure. Damage by insects causes an

immediate release of fluids down a force gradient to the place of injury (Buttery and Boatman









1976). Insects may get entrapped due to adhesiveness of some exudates (Farrell et al. 1991). The

squirt gun defense mechanism in the forest plant, Bursera trimera Bullock is a good example; a

fine spray of resins that is released just after attack by the chrysomelids Blepharida spp. causes

larval mortality (Becerra et al. 2001).

It has been shown that some insects on canal-bearing plants defuse the canalicular reaction

before feeding. The cabbage looper, T. ni, ruptures Lactuca laticifers by making a superficial

trench before actual feeding. The trench drains the latex from the distal tip and isolates that

particular section from the main canal system (Dussourd and Denno 1991, 1994). Dussourd

(1993) compared the survivorship of each instar of T. ni and yellow-striped armyworm,

Spodoptera ornithogalli (Guenee), an insect that does not trench on canal-bearing plants, to the

following instar on intact vs. detached leaves ofL. serriola. The survivorship was high for each

instar of T. ni on both leaf categories. In contrast, S. ornithogalli larvae survived only on

detached leaves. Larvae of S. ornithogalli in the first and second instar often died with their

mandibles glued together with latex. Older larvae tried to feed over and over again but invariably

starved to death. Detaching leaves, particularly of plant species with exudates, often modifies

their palatability (Bernays and Lewis 1986, Huang et al. 2003c).

Constitutive defense is common to all healthy plants and provides general protection

against invasion by herbivores. Constitutive defense has also been referred to as natural or innate

defense. On the other hand, induced defense is the mechanism that must be induced or turned on

by plant exposure to an herbivore. Unlike constitutive defense, it is not immediately ready to

come into play until after the plant is appropriately exposed to herbivore. Constitutive defense is

not specific, and is directed toward general strategic defense. Phenolic compounds were

previously regarded as quantitative defenses that are always present at high levels in plant tissues









(Feeny 1976). Recently, it has been shown that certain phenolics may increase after insect attack

or mechanical wounding (Pullin 1987, Clausen et al. 1989, Ke and Saltveit 1989, Hartley and

Lawton 1991, Brignolas et al. 1995, Constabel 1999). Thus, it seems that induced defense may

be more cost-effective for plants than constitutive defense under certain conditions. In induced

defense, secondary compounds are manufactured as reactions to insect attack or wounding, and

there is no need to maintain the compounds at a steady and effective concentration as in the case

of constitutive defense (Herms and Mattson 1992, Baldwin 1994, Gershenzon 1994). Induced

defense contributes to plant resistance by enhancing the action of natural enemies of insects

(Thaler 1999).

Biochemical Basis of Host Plant Resistance

Both proteins and secondary plant compounds contribute to defense in plants. Secondary

plant compounds are organic molecules that are not required for normal physiological processes

in growth and development. These biochemicals are also called allelochemicals, because they

influence the behavior and/or physiology of species other than their own. Generally, secondary

plant compounds have been more extensively studied than proteins, possibly due to their

interesting structural variety and advanced biological activities (Duffey and Stout 1996).

Host Plant Resistance Due To Proteins

Molecular biology has proven to be a useful tool in host plant resistance research because

plant defense responses can be studied at the level of gene expression rather than simply with

assays of the encoded proteins. Each protein in a plant is encoded by a single gene, which can be

isolated and employed for developing genetically engineered crops with improved pest

resistance. Regulation of gene expression is a principal way that defense proteins are generated

in plants and has been confirmed by the induction of mRNA after herbivory (Constabel et al.

2000).









Protease inhibitors

Protease inhibitors (PIs) are proteins that strongly bind proteolytic enzymes and thereby

hinder their activity (Ryan 1990, Richardson 1991). PIs are classified as inhibitors of serine,

cysteine, aspartic, or metallo-proteases (Ryan 1990). These inhibitors effectively block the active

site of proteases by binding to it and forming a complex with a low dissociation constant (Terra

et al. 1996, Walker et al. 1998). PIs accumulate in tomato leaves in response to insect attack

within hours of damage (Green and Ryan 1972). Low molecular weight protease inhibitors, such

as leupeptin, calpin inhibitor I, and calpeptin are strong antifeedants for adult western corn

rootworm, Diabrotica virgifera virgifera LeConte (Kim and Mullin 2003). All PIs possess a di-

or tripeptidyl aldehyde moiety, which binds covalently with sulfhydral (SH) group on the taste

chemoreceptors of insects (Kim and Mullin 2003). PIs cause hyper-production of insect digestive

enzymes, which triggers the loss of sulfur amino acids, and also reduces the quantity of proteins.

As a result, insects become weak, exhibit stunted growth and ultimately die (Shulke and

Murdock 1983).

Cysteine protease

Cysteine protease is a 33-kDa defense protein, which accumulates in resistant lines of

maize (Zea mays L.) in response to larval feeding of fall armyworm, S. frugiperda (Pechan et al.

2000). It accumulates in the mid whorl of the maize plant within 1 h of infestation and continues

to build up for as long as 7 d. This protein hinders larval growth and is responsible for 60 to 80%

weight loss (compared to control insects feeding on susceptible lines of maize) (Pechan et al.

2000), which is due to destruction of the peritrophic matrix of the gut and subsequent disruption

of the normal digestive mechanism (Pechan et al. 2002).









Oxidative enzymes

Oxidative enzymes include phenol oxidases, peroxidases, and lipoxygenases. These are

stress-associated enzymes synthesized in plants (Butt 1980). The oxidative enzymes are involved

in anti-nutritive defense in plants against various insect pests (Felton et al. 1989, Duffey and

Felton 1991, Duffey and Stout 1996). Systemin, jasmonates, and the octadecanoid defense

signaling pathway induce polyphenol oxidase and lipoxygenase in tomato and cotton, and thus

support the role of oxidative enzymes in plant defense (Constabel et al. 1995, Thaler et al. 1996,

Bi et al. 1997a, Heitz et al. 1997). Bestwick et al. (2001) characterized pro- and antioxidant

enzyme activities during the hypersensitive reaction (HR) in lettuce, and reported a prolonged

oxidative stress in lettuce cells experiencing HR. This stress is chiefly through a boost in pro-

oxidant activities primarily taking place in the absence of enhanced antioxidants.

Polyphenol oxidase. Polyphenol oxidase (PPO) uses molecular oxygen to catalyze the

oxidation of monophenolic and ortho-diphenolic compounds, and is a key factor for darkening of

many fruits and vegetables (Sherman et al. 1991, Steffens et al. 1994, Constabel and Ryan 1998).

The expression of PPO is generally high in diseased, insect-damaged and wounded tissues

(Mayer and Harel 1979, Stout et al. 1994, Constabel et al. 1995, Thaler et al. 1996). In crops,

such as potato, tomato, apple and hybrid poplar, wound-induction at the level of PPO mRNA has

been confirmed due to accessibility of PPO cDNA probes (Constabel et al. 1996). PPO contacts

its chemical substrates during insect feeding. PPO produces reactive ortho-quinones which

readily form alkylated amino acids, which ultimately results in protein modification, cross-

linking, and precipitation. This protein modification significantly impacts insect pests by

preventing efficient digestion and assimilation of nitrogen (Felton et al. 1992, Duffey and Stout

1996).









Wounding induces expression of PPO genes in damaged as well as undamaged

systemicallyy wounded) leaves (Robison and Raffa 1997, Havill and Raffa 1999). Constabel et

al. (2000) observed through southern blot analysis that hybrid poplar presumably possesses two

PPO genes, with polymorphic alleles at each locus. Similarly, tomato and potato also have seven

and six member PPO genes families, respectively (Hunt et al. 1993, Thygesen et al. 1995). Out

of seven PPO genes in tomato, only one gene is wound inducible, while the others are regulated

by development (Thipyapong and Steffens 1997, Thipyapong et al. 1997). Therefore, the wound-

induced increase in PPO activity is through transcriptional activation of PPO genes and de novo

enzyme synthesis, rather than enzyme activation (Bergey et al. 1996, Constabel et al. 2000).

Various plant PPOs require chemical activation to become active, as they are present in latent

form in the plant (Jimenez and Garcia-Carmona 1996). The younger leaves show higher PPO

activity than older leaves due to buildup of higher levels of PPO mRNA in response to restricted

damage of old leaves (Constabel et al. 2000).

Peroxidase. Peroxidase is a heme-containing enzyme that oxidizes a wide range of

biological compounds, such as phenolics, indole acetic acid, and ascorbate by utilizing hydrogen

peroxide (Butt 1980). Peroxidase plays a key role in lignification of plant tissue. The cell wall

peroxidases produce phenoxy radicals from hydroxycinnamyl alcohols that ultimately form

lignin by non-enzymatic polymerization (Douglas 1996). These enzymes also perform an

important role in suberization of tissues (Kolattukudy 1981). In addition, they are also involved

in the construction of cross-links between carbohydrates and proteins in cell walls (Fry 1986,

Cassab and Varner 1988). In various plants, like tomato, rice, peanut and bean, peroxidase level

is increased after wounding of tissues (Breda et al. 1993, Felton et al. 1994a, Ito et al. 1994,

Smith et al. 1994). Peroxidase is also involved in defense by means of cell wall reinforcement









due to its role in lignification and cross-linking of other cell wall components. Ultimately,

amplified peroxidase level affects insect performance due to increased leaf toughness (Coley

1983). Bi et al. (1997a) observed induced resistance in cotton to H. zea due to increased

peroxidase activity in previously damaged cotton foliage or squares. Similarly, Dowd and

Lagrimini (1997) found that peroxidase-overproducing transgenic tobaccos, Nicotiana sylvestris

(Spegazzini and Comes) and N. tabacum L., experienced significantly less damage by H. zea

than did wild plants, suggesting the contribution of peroxidase activity in leaf resistance to

chewing insects. Aphid infestation in barley results in ethylene production and subsequent

increase in hydrogen peroxide and total peroxidase activity. This highlights the role of ethylene

in the oxidative response of infested barley plants (Argandona et al. 2001).

Lipoxygenase. Lipoxygenase employs molecular oxygen to oxygenate unsaturated fatty

acids, like linoleic and linolenic acid, and produces fatty acid hydroperoxides (Galliard and Chan

1980, Siedow 1991). Lipoxygenase has a number of important roles in plant defense against

insect pests. Lipoxygenase produces a direct antinutritive effect on insects. This adverse effect is

due to destruction of polyunsaturated fatty acids, which are key nutrients for most insects

(Duffey and Stout 1996). Fatty acid hydroperoxides (plus extra free radicals) generated by

lipoxygenase react with essential amino acids and modify proteins. Therefore, lipoxygenase

plays an antinutritive role in plant defense similar to PPO and peroxidase (Duffey and Felton

1991).

Proteins of the cell wall

Stresses, including insect pest and pathogen attack, modify cell wall contents of plants,

such as carbohydrates, proteins, and phenolics (Bowles 1990, Carpita and Gibeaut 1993). Cell

wall proteins, such as proline-rich proteins (PRPs), hydroxyproline-rich glycoproteins (HRGPs),









arabinogalactan proteins, and glycine-rich proteins (GRPs), are induced during wounding of

leaves or stems (Showalter 1993).

Secondary metabolism pathways

Phenolics and phenylpropanoids are major classes of phytochemicals responsible for

defense reactions in plants. These chemicals are synthesized and accumulated upon insect and

pathogen attack, and mechanical wounding. The phenylpropanoids are mainly derivatives of

phenylalanine (an aromatic amino acid). Plants possess hundreds of phenylpropanoids, with

flavonoids and their derivatives constituting the major group (Heller and Forkman 1993).

Phenylpropanoid synthesis is always initiated through a common phenylpropanoid pathway.

Phenylalanine is converted through a number of steps to hydroxycinnamoyl coenzyme A (CoA)

esters, which is a branching point in phenylpropanoid biosynthesis. Lignin precursors, cell wall-

bound hydroxycinnamoyl esters, and soluble glucosides are possible end products of different

branches and ultimately form lignin and various flavonoids.

Enzymes involved in secondary metabolism

Herbivory and wounding induces various phenylpropanoid enzymes. Phenylalanine

ammonia lyase (PAL) is the first enzyme of the phenylpropanoid pathway and catalyzes

deamination of phenylalanine to cinnamic acid (Hahlbrock and Scheel 1989). PAL is inducible

by insect and pathogen attack, mechanical wounding, exposure to ethylene and abiotic stresses,

such as UV light (Hyodo et al. 1978, Jones 1984, Hahlbrock and Scheel 1989, Ke and Saltveit

1989). PAL induction is well documented in certain plant parts, like lettuce leaves (Ke and

Saltveit 1986), bean hypocotyls (Cramer et al. 1989), alfalfa (Dixon and Harrison 1990), tobacco

leaves (Pellegrini et al. 1994, Fukasawa-Akada et al. 1996), potato tubers and leaves (Rumeau et

al. 1990, Joos and Hahlbrock 1992), and parsley (Lois and Hahlbrock 1992). Induction of PAL

takes place through transcription of a single or many genes. Endogenous phenylpropanoid









pathway intermediates regulate the level of PAL transcripts by way of a feedback mechanism

(Braun and Tevini 1993, Orr et al. 1993). Various other important phenylpropanoid enzymes, in

addition to PAL, such as 4-cinnamic acid hydroxylase (4-CH) in Arabidopsis thaliana (L.) and

pea (Pisum sutivum L.) (Frank et al. 1996, Mizutani et al. 1997) and caffeic acid O-

methyltransferase in corn (Capellades et al. 1996), are also induced by insect and pathogen attack

and/or wounding. Similarly, 4-coumarate CoA ligase in tobacco, Arabidopsis, and bean

(Phaseolus vulgaris L.) is also wound inducible (Smith et al. 1994a, Ellard-Ivey and Douglas

1996, Lee and Douglas 1996). In addition, numerous shikimic acid pathway enzymes responsible

for phenylalanine biosynthesis, like 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP)

synthase in potato tubers (Solanum tuberosum L.) (Dyer et al. 1989) and shikimate

dehydrogenase from bell peppers (Diaz and Merino 1998) are induced by wounding. Peiser et al.

(1998) reported that PAL inhibitors control browning of cut lettuce.

Host Plant Resistance Due To Secondary Plant Compounds

There are wide range of secondary plant compounds found in the plant kingdom (Luckner

1990, Dey and Harborne 1997). This wide diversity of compounds is hypothesized to be the

outcome of co-evolution of plants with insects and pathogens (Harborne 1993). Secondary plant

compounds are traditionally categorized into three major groups: carbon-based phenolics and

terpenes, and nitrogen-containing compounds such as alkaloids (Taiz and Zeiger 1991). In

general, carbon-based compounds have been considered cheaper defense tools than nitrogen-

containing compounds, as nitrogen is vital and frequently in limited supply for the growth of

plants (Bryant et al. 1983). Gonzalez (1977) reported the presence of terpenes, sterols, flavonoids

and other phenolics, and alkaloids in lettuce.









Phenolics

Phenolics constitute a diverse group of chemicals, ranging from small phenolic acids to

complex polymers such as tannins and lignin (Dey and Harborne 1997). Most phenolic

compounds are derivatives of the shikimic acid and phenylpropanoid pathways and bear

aromatic rings having one or more hydroxyl groups. Phenolics can be divided into simple

phenols and polyphenols based on the number of hydroxyl group attached. Simple phenols

include the hydrobenzoic acids (e.g., vanillic acid), the hydroxycinnamic acids (e.g., caffeic acid)

and the coumarins (e.g., umbelliferone). Polyphenols are a diverse group of plant phenolics, such

as flavonoids (e.g., quercetin) and tannins (e.g., esters of gallic acid) (Schoonhoven et al. 2005).

Additional functional groups such as ester, methyl, acetyl or sugar moieties are also found in

some complicated phenolics. Stresses such as excessive light, UV, cold, nutrient deficiencies,

and attacks by insects and pathogens are most commonly responsible for the induction of

phenolics in plants (Dixon and Paiva 1995, Somssich et al. 1996). In intact plants, phenolics are

stored in vacuoles in their less toxic glycoside forms as water-soluble compounds (Hosel 1981).

The wounding of cells (e.g., by insect attack) causes release of the glycosides from their storage

site (Hosel 1981) and ultimately formation of compounds with toxic, deterrent or nucleophilic,

and nutritive-value-lowering properties after coming in contact with specific degradative

enzymes. For example, toxic hydrogen cyanide is released due to hydrolysis of harmless

cyanogenic glycosides by P-glucosidase activity (Wink 1997).

o-Substituted phenolic compounds (e.g., chlorogenic acid) produce o-quinones with the

help of oxidative enzymes which alkylate amino acids by binding to their nucleophilic groups

(Felton et al. 1989, Constabel 1999). This binding hinders the assimilation of essential amino

acids and lowers the quality of plant foliage for insects (Felton et al. 1989). As Bi et al. (1997a)

observed, wounding of cotton (Gossypium hirsutum L.) foliage induced increased activity of









oxidative enzymes that was associated with decreased levels of the nutritional antioxidant

ascorbate and increased levels of phenolic prooxidants (i.e., chlorogenic acid) and lipid

peroxides. This significant decline in host nutritional quality (due to accumulation of secondary

compounds) is responsible for induced resistance in cotton foliage and squares to herbivory by

H. zea, indicated by a decrease in larval growth when larvae fed on previously damaged foliage

or squares compared to the controls. Chemicals like phenolics also play an important role in the

inhibition of oviposition on the host plant in addition to reducing larval growth and survival

(Dethier 1970, Todd et al. 1971, Chapman 1974, Elliger et al. 1980, Corcuera 1993, Stotz et al.

1999).

Simple phenols such as ferulic, caffeic andp-coumaric acids are precursors of lignin. Upon

wounding of plant tissue, a subsequent host response occurs involving an intensive accumulation

of lignin-like polyphenolics in wounded or ruptured cells. This is followed by a rapid

hypersensitive death of the cell, giving rise to a single cell brownish necrosis (Moerschbacher et

al. 1990, Nicholson and Hammerschmidt 1992, Wei et al. 1994, Zeyen et al. 1995). Wounding of

potato tubers (Hahlbrock and Scheel 1989) and lettuce (Loaiza-Velarde et al. 1997, Loaiza-

Velarde and Saltveit 2001) induces an accumulation of phenolic conjugates, such as chlorogenic

acid (a caffeic acid conjugate). Hydrogen peroxide is required for the polymerization step in the

formation of the poly (phenolic) domain of suberized potato tubers (Razem and Bernards 2002).

Ke and Saltveit (1989) also observed an increase in phenolic compounds (e.g., chlorogenic and

isochlorogenic acids) and brown stain in lettuce tissue affected by russet spotting.

Phenolics are known to play important role in the host plant resistance (Dethier 1970, Todd

et al. 1971, Chapman 1974, Elliger et al. 1980, Corcuera 1993, Stotz et al. 1999). Ikonen et al.

(2001) reported feeding deterrence in the willow (Salix pentandra L.), to the leaf beetle









(Lochmaea capreae L.) due to high levels of chlorogenic acid in the leaves. Cole (1984)

correlated resistance to lettuce root aphid with the presence of high amounts of isochlorogenic

acid and the enzyme PAL in resistant lettuce cultivars. However, the increased concentration of

phenolics in transgenic tobacco showing differential expression of PAL does not substantiate

their role in plant resistance against the generalist tobacco hornworm (Manduca sexta L.), and

the specialist tobacco budworm (H. virescens (Fabricius)) (Bi et al. 1997b). Similarly,

Eichenseer et al. (1998) also did not find any preference in larvae ofM. sexta fed transgenic

tobacco plants that either under- or over-expressed PAL and consequently with either lower or

higher levels of phenolics than normal.

Tannins are complex polyphenols and are more prevalent in woody perennials than in

herbaceous plants (Swain 1979). They are often considered as general feeding deterrents in

plant-insect interactions, and therefore, play an important role in chemical ecology and defense

against insects (Swain 1979, Hagerman and Butler 1991). Based on their structure, they are

categorized as condensed tannins, or proanthocyanidins, and hydrolysable tannins, which are

gallic acid or ellagic acid esters of various sugars.

Caffeic acid derivatives (Ke and Saltveit 1988) and flavonoids (Hermann 1976) are the two

main classes of simple phenols and complex polyphenols, respectively, which have been

identified in lettuce. In particular, simple phenolics like monocaeffeoyl tartaric acid, chicoric

acid, 5-caffeolyquinic acid and 3,5-di-O-caffeoylquinic acid are present in lettuce (Winter and

Hermann 1996, Ferreres et al. 1997).

Flavonoids

Plants flavonoids are a large group of phenolic compounds produced by the shikimic acid

pathway. Flavonoids are grouped under major classes, such as the flavanones, flavones,

flavonols, and isoflavonoids (Harborne 1994). In the biosynthesis of flavonoids, chalcone









synthase (CHS) is the first committed enzyme that catalyzes the formation of chalcone

intermediate by condensing three malonyl-CoA and one hydroxycinnamoyl-CoA molecules.

CHS is known to be involved in the response to many forms of stress in many plants, including

to insects and pathogens (Dangl et al. 1989). Chalcone is then catalyzed to flavanone with the

help of the enzyme chalcone isomerase. In the next step, flavonoid biosynthesis splits into

different branches. In the first branch, flavones are formed from flavanones due to the action of

flavone synthase (Britsch 1990). Secondly, dihydroflavonols, which are precursors of flavonols

and anthocyanins, can be synthesized from flavanones by the enzyme flavanone-3-hydroxylase.

In the third branch, flavanones can be converted into isoflavanones in a reaction catalyzed by

isoflavanone synthase (Dixon et al. 1995).

Flavonoids are found in high concentrations in many plant species under normal conditions

as sugar conjugates (Frost et al. 1977, Feng and McDonald 1989, Jahne et al. 1993, Stapleton

and Walbot 1994), and over 50 different glycosides have been identified among the more

common-occurring flavonoids (Hermann 1976, 1988). Flavonoid accumulation in leaves is very

much increased in response to illumination with the UV-B spectrum of visible light (Koes et al.

1994, Strid et al. 1994). Flavonoids play a role in the protection of plants from the damaging

effects of UV-light, as they have good light absorbing properties in the UV spectrum (Markham

1989). Red-pigmented lettuce, such as 'Lollo Rosso', contains high concentrations of

anthocyanin with antioxidant and free-radical scavenging properties (Gil et al. 1998). Flavonols,

such as kaempferol, quercetin and myricetin, and the analogous flavones, apigenin and luteolin

are found in vegetables, fruits, and beverages (Hertog et al. 1992, 1993), and are also known to

possess antioxidant and free radical scavenging activity in foods (Shahidi and Wanasundara

1992). Flavonols, such as quercetin 3-O-glucuronide, quercetin 3-O-glucoside and quercetin 3-









O-(6-O-malonylglucoside) are also present in lettuce (Winter and Hermann 1996, Ferreres et al.

1997). The lettuce varieties 'Lollo Rosso' and 'Round' contain high amounts of quercetin,

varying from 11-911 /g g-1 of fresh weight in the outer leaves to 450 mg g-1 in the inner leaves

(Crozier et al. 1997). The polyphenol compounds (caffeic acid derivatives, quercetin and

kaempferol glycosides) are present in higher amounts in lettuce grown in the field than in a

greenhouse (Romani et al. 2002).

The behavior, development, and growth of insects are influenced by plant flavonoids

(Hedin and Waage 1986). Plant flavonoids act as feeding stimulants for the boll weevil

(Anthonomus grandis Boheman) in cotton (Hedin et al. 1988), or oviposition stimulants to the

citrus-feeding swallowtail butterfly, Papilio xuthus L. (Nishida et al. 1987). Flavonoids may be

antibiotic substances effective against phytophagous insects (Todd et al. 1971, Chan and Waiss

1978, Chan et al. 1978, Joerdens-Roettger 1979, Elliger et al. 1980, Hanny 1980, Hedin et al.

1983, Peng and Miles 1988, Ridsdill-Smith et al. 1995). Rutherford (1998) observed the

involvement of chlorogenates and flavonoids in the resistance of sugarcane to the stalk borer

(Eldana saccharina Walker). Two extreme types of flavonoid profile were found using near-

infrared spectroscopy (NIR), one coupled with susceptibility and other with resistance. Stalk

borer larvae could be induced to feed by inclusion of the susceptible-type flavonoid profile into a

defined synthetic diet. Subsequent survival of first instar larvae was greater on this diet than on

diets containing the resistant-type flavonoid profile.

Terpenoids

Terpenoids are a diverse group of chemicals which all originate from iospentenoid

precursors. Based on the number ofisoprene (five-carbon) units, terpenoids are classified as

mono-, sesqui-, di, tri- or tetra-terpenoids. They are known to have various secondary functions,









like defense against pathogens and insects as well as primary functions, such as membrane

components, pigmentation, free radical scavenging, and growth regulators (Harborne 1993).

Antibiotic, cytotoxic, and allergenic properties are also associated with terpenoids (Burnett et al.

1978).

Many sesquiterpene lactones accumulate in canals (laticifers) closely associated with the

vascular tissues of composite plants (Esau 1965). Damaged laticifers release latex containing

sesquiterpene lactones which may have analgesic, antitussive and sedative properties (Gromek et

al. 1992). Sesquiterpene lactones are extremely varied in their structure, properties and functions

(Rees and Harborne 1985). The main bitter constitutive principles of Lactuca species are

lactucin, lactucopicrin, 8-deoxylactucin and their derivatives, such as 11,13-dihydro-analogues

(Barton and Narayanan 1958, van Beek et al. 1990). Two triterpenes, the quaianolides lactucin,

and lactucopicrin have been isolated from dry latex of L. virosa. The presence of lactucin, 8-

deoxylactucin, and lactucopicrin in lettuce and chicory make them intensely bitter (Price et al.

1990). Wounding of leaves or stems ofLactuca species releases a milky latex consisting of 15-

oxalyl and 8-sulphate conjugates of lactucopicrin, which ultimately revert to the parent lactone

due to hydrolysis of unstable oxalates. The induced quaianolide sesquiterpene lactone

phytoalexin, lettucenin A, is also present in Lactuca species, but not in chicory (Sessa et al.

2000). Lettucenin A was initially characterized by Takasugi et al. (1985). It is one of the most

toxic phytoalexins ever discovered and provides resistance to lettuce downy mildew in certain

lettuce cultivars due to its strong antimicrobial properties (Bennett et al. 1994). Bestwick et al.

(1995) isolated lettucenin A from lettuce seedlings with the red spot physiological disorder. A

15-glycososyl conjugate of 11,13-dihyrolactucopicrin is found in L. tartarica roots (Kisiel et al.

1997). Likewise, the related quaianolide sesquiterpene lactone glycosides, such as picriside A









(lactucin-15-glycoside) and crepiaside A (8-deoxylactucin-15-glycoside), are found in other

members of the Lactuceae tribe (Seto et al. 1988).

Host Plant Resistance in Lettuce to Insect Pests

Aphids

Many species of aphids are known to colonize lettuce, but few are responsible for

transmission of viruses (Kennedy et al. 1962). Aphids are the most serious pests of lettuce in

North America (Alleyne and Morrison 1977, Forbes and Mackenzie 1982, Toscano et al. 1990),

Spain (Nebreda et al. 2004), and other areas of Europe (Ester et al. 1993, Ellis et al. 1996, Martin

et al. 1996, Monnet and Ricateau 1997, Parker et al. 2002). Reduction in yield of lettuce is due to

direct damage caused by aphid feeding and indirect damage by aphid-transmitted virus

infections. In addition, marketability of harvested heads is greatly reduced by the physical

presence of aphids (Dunn 1959, Rufingier et al. 1997).

The lettuce root aphid, P. bursarius, is one of the most important pests of lettuce in the

United States (Swift and Lange 1980, Blackman and Eastop 2000), Western Europe, and Canada

(Ellis 1991, Reinink and Dieleman 1993). It feeds on the youngest leaves and rapidly colonizes

the 'heart' of the lettuce (Forbes and Mackenzie 1982). The lettuce aphid, N. ribisnigri, is a

major pest in the United States, Czechoslovakia, UK, France, Germany, Netherlands and

Switzerland (Reinink and Dieleman 1993, Mosler and Dunn 2005). Uroleucon ambrosiae is a

pest of hydroponically-grown lettuce in Brazil (Auad and Moraes 2003, Miller et al. 2003) and

Turkey (Zeren 1985). Green peach aphid, M. persicae (Capinera 2004), and potato aphid, M.

euphorbiae (Reinink and Dieleman 1989), are active vectors of lettuce yellow virus.

A variety of chemicals are sprayed to control aphids in lettuce. Therefore, to reduce lettuce

growers' dependence on insecticides for aphid control, a number of alternative measures must be

used as a part of IPM program together with the use of varieties resistant to aphids (Tatchell et









al. 1998). Plant resistance as one of the components of IPM has been extensively studied to

manage aphids on lettuce. Successful transfer of resistance from wild to cultivated lettuce has

proven useful in controlling N. ribisnigri (Eenink et al. 1982). However these varieties afford

only slight to no defense against M. persicae and M euphorbiae (Reinink and Dieleman 1989,

van Helden et al. 1993). Modern varieties of lettuce resistant to P. bursarius, such as 'Avoncrisp'

and 'Lakeland', possess the dominant Lra gene (Dunn 1974, Ellis et al. 1994). The Lra gene is

also linked to the downy mildew (Bremia lactucae) resistance gene, Dm6 (Harrewijn and

Dieleman 1984, Ellis et al. 1994, Ellis et al. 2002). However, the lettuce variety 'Grand Rapid',

reported to be resistant to P. bursarius (Dunn and Kempton 1980), does not possess Dm6 (Crute

and Dunn 1980). In addition, several factors whose genetic basis have not been identified, such

as deficient nutritive value of the phloem sap, phytochemicals (toxic or deterrents), and

unacceptability of the plant surface for feeding, provide resistance to aphids (Harrewijn and

Dieleman 1984).

Wild lettuce species L. virosa L., L. saligna L., and L. perennis L. are found to be resistant

to M. persicae, causing aphid mortality and lower nymph production (Eenink and Dieleman

1982). This resistance (governed by additive genes) was transferred to cultivated lettuce by

making a series of inter-specific crosses (Eenink et al. 1982). Clones ofM. persicae exhibit

different intensities of aggressiveness on lettuce. The lettuce genotypes selected for partial

resistance to the aggressive clone WMpl possess complete or almost complete resistance to less

aggressive clones (Reinink et al. 1989). Lactuca virosa is almost completely resistant to N.

ribisnigri, causing low feeding rate, adult and nymphal mortality, and reduced reproduction

(Eenink and Dieleman 1982). Complete resistance to N. ribisnigri is governed by the presence of









the Nr gene in the plant, whereas the Nr gene provides only partial resistance to M. persicae, and

no resistance to M euphorbiae (Reinink and Dieleman 1989).

Iceberg lettuce shows resistance towards three main aphids, N. ribisnigri, M. euphorbiae

and P. bursarius (Dunn and Kempton 1980). Ester (1998) observed 100% resistance against N.

ribisnigri and M. euphorbiae in aphid-resistant butterhead lettuce cultivars. In Europe, the lettuce

butterhead cultivar 'Dynamite' shows high resistance against N. ribisnigri and P. bursarius,

some resistance to M. euphorbiae and U. sonchi, but no resistance to the glasshouse-potato

aphid, Aulacorthum solani (Kaltenbach) (van der Arend et al. 1999, van Melckebeke et al. 1999).

Butterhead cultivars are moderately to highly resistant to M. euphorbiae and U. sonchi, whereas

crisphead cultivars possess little or no resistance to either aphid species (Reinink and Dieleman

1989). The lettuce cultivar 'Charan' shows partial resistance to M euphorbiae and U. sonchi

(Reinink et al. 1995). Montllor and Tjallingii (1989) electronically monitored the probing

behavior ofM. persicae and N. ribisnigri on susceptible and resistant lettuce lines using a DC

amplifier. They proposed the possible involvement of both mesophyll and phloem factors in

conferring resistance. van Helden and Tjallingii (1993) also discussed the role of phloem vessels

in resistant lettuce. van Helden et al. (1995) compared the phloem sap of both resistant and

susceptible cultivars and found no relationship between phloem sap composition and resistance

to N. ribisnigri. However, later work by van Helden and van der Wal (1996) suggests the

presence of a resistance factor against N. ribisnigri in lettuce phloem sap. The roots of lettuce

cultivars showing resistance to P. bursarius have greater concentrations of isochlorogenic acid

and PAL as compared to susceptible cultivars (Cole 1984).

Cabbage Looper

Cabbage looper, T. ni, is a serious problem in all lettuce growing areas in the United States

(Kerns and Palumbo 1996, Kerns et al. 1999, Agnew 2000, Kurtz 2001). It is the predominant









pest of lettuce during autumn in California (Kishaba et al. 1976, Vail et al. 1989). The larvae of

T. ni often transect leaves with a narrow trench before eating to reduce exposure to exudates,

such as latex, during feeding (Dussourd 2003). Cabbage looper larvae develop faster on excised

than on attached leaves of prickly lettuce, L. serriola, signifying the suitability of these plants

when canals are inactivated (Tune and Dussourd 2000). Lactucin from lettuce latex seems to act

as a trenching stimulant, but other chemicals, such as phenylpropanoids, monoterpenes, and

furanocoumarins, show slight or no activity for inducing trenching (Dussourd 2003). The F2

plants derived from a cross between L. sativa lines and resistant lines of L. saligna were resistant

(Whitaker et al. 1974) and showed antixenosis toward T. ni (Kishaba et al. 1980).

Banded Cucumber Beetle

The banded cucumber beetle, D. balteata, is a generalist feeder that feeds upon many plant

species. In the early 1900s, this pest was mostly found in Central and South America and Mexico

(Saba 1970, Krysan 1986, Bellows and Diver 2002). Later on, it spread into the United States

and is now established in Alabama, Arizona, Arkansas, California, Florida, Georgia, Louisiana,

Mississippi, New Mexico, North Carolina, South Carolina, and Texas (CABI 2006). It is also

found throughout Florida but most commonly in the Lake Okeechobee area (Capinera 1999). It

is an economic concern for lettuce cultivation in southern Florida (Nuessly and Nagata 1993).

Diabrotica balteata has a high reproductive capacity (Pitre and Kantack 1962), and many

generations occur throughout the year (Schalk 1986).

Romaine lettuce cultivars 'Valmaine' and 'Tall Guzmaine' were analyzed to assess the

level of resistance to D. balteata (Huang et al. 2002). Valmaine was highly resistant whereas Tall

Guzmaine was susceptible to D. balteata. The mechanism of resistance was determined to be

antixenosis and such little feeding occurred on Valmaine that the reproductive structures were

not fully developed in adult females (Huang et al. 2002). However, latex from both Valmaine









and Tall Guzmaine showed antifeedant activities when applied to the surface of a preferred food,

such as lima bean (Phaseolus vulgaris L.) leaves. Valmaine plants that had been previously fed

upon showed higher resistance to D. balteata than did Tall Guzmaine after previous feeding,

suggesting involvement of physical factors and an induced mechanism of resistance in Valmaine

against the beetle (Huang et al. 2003b).

Leafminer

Plants in over 47 genera belonging to 10 families have been recorded as hosts of

leafminers. The principal leafminer species affecting lettuce include L. sativae, L. trifolii, L.

huidobrensis and L. langei Frick. Both L. trifolii and L. sativae are native to America

(Waterhouse and Norris 1987). In the United States, these two species are found commonly in

the southern United States from Florida to California and Hawaii (Capinera 1999). In Arizona, L.

sativae is predominant during the period of August to January, whereas during February L.

trifolii prevails (Kerns and Palumbo 1996, Kerns et al. 1999, Agnew 2000). In recent years,

populations of leafminers have increased in coastal areas in California (Kurtz 2001). In central

Florida, populations of leafminer are high between May and October, when minimum average

temperatures are 25C, whereas higher temperature in southern Florida favors leafminer

populations throughout the year (Anonymous 1999). Leafminer larvae cause damage by mining

the leaves, which may result in reduced photosynthetic activity. Younger plants are more

vulnerable to leafminer attack and severe damage can kill the plants (Nuessly and Webb 2003).

The romaine lettuce cultivar Valmaine was the most resistant to L. trifolii in tests involving

three additional lettuce cultivars, 'Floricos 83', 'Parris Island Cos', and Tall Guzmaine. Adults

on Valmaine had significantly reduced levels of feeding, longevity, and fecundity (Nuessly and

Nagata 1994). Liriomyza trifolii preferred to feed on the middle leaves of Valmaine plants in

contrast to Tall Guzmaine where they preferred to feed on the older and younger leaves. When









honey was supplied as a supplement to the diet of Valmaine, female survivorship and

reproductive rates increased to levels more similar to Tall Guzmaine suggesting a deficiency in a

critical diet component in Valmaine (Nagata et al. 1998).

Mou and Ryder (2003) screened 48 varieties of cultivated lettuce, L. sativa and the wild

species, L. serriola, L. saligna, and L. virosa for resistance to L. langei. Wild species had fewer

leafminer stipples per unit area than cultivated lettuce. Iceberg experienced the most stippling

damage among the genotypes tested. The progenies of crosses between the resistant genotypes

were selected to raise the level of resistance (Mou et al. 2004, Mou and Ryder 2003).

Helicoverpa species

Heliothinae are very destructive pests of many crops and frequently shift to lettuce from

surrounding crops, like cotton and corn (Kerns and Palumbo 1996, Kerns et al. 1999, Agnew

2000, Kurtz 2001). Corn earworm, H. zea, is found throughout the United States (Capinera

1999). It is found on all Florida vegetable crops (Martin et al. 1976). In Australia, H. armigera

(Htibner) and H. punctigera Wallengren are serious pests of lettuce and can cause extensive

damage (Ridland et al. 2002, Dimsey and Vujovic 2003). In India, H. armigera is found

throughout the year in lettuce fields, but is most active during March and April (Parihar and

Singh 1992).

Spodoptera species

Beet armyworm, S. exigua, is a polyphagous and widely distributed insect (CABI 1972). It

is the key pest of lettuce in the western United States (Metcalf and Flint 1962, Kerns and

Palumbo 1996, Kerns et al. 1999, Agnew 2000, Kurtz 2001). In the early stage of crop

development (between thinning and cupping stage), it does not cause any economic damage, but

feeding during the heading stage makes the lettuce unmarketable (Kerns and Palumbo 1996,

Kerns et al. 1999, Agnew 2000). Ghaffar et al. (2002) found the pupal period of S. exigua to be









the shortest (5.8 d) on lettuce as compared to eggplant (Solanum melongena L.) and field

bindweed (Convolvulus arvensis L.) (7.6 d).

Bemisia species or strains

The B strain of the cotton whitefly, Bemisia tabaci (or B. argentifolii, the silverleaf

whitefly), is one of the primary pests of fall lettuce in California and Arizona. It causes complete

destruction of early fall planted lettuce due to the extraction of large amounts of phloem sap from

seedlings (Kerns and Palumbo 1996, Kems et al. 1999, Agnew 2000). It also causes yellowing

and distortion of the leaves and can reduce dry mass accumulation by up to 41%, depending

upon population level (Costa et al. 1993). In lettuce, whitefly stylets penetrate epidermal cells

and intercellular junctions while feeding. Arrangement of vascular bundles in lettuce affects the

feeding behavior of whitefly. The length of the vascular bundle (2.8 mm per 1.0 mm2 leaf area)

is tolerably acceptable to whitefly (Cohen et al. 1996). However, fewer minor veins (fewer

vascular bundles) accounts for low success of whitefly on lettuce compared to preferred crops,

such as cantaloupe and other cucurbits (Cohen et al. 1998).

Thrips

Western flower thrips, Frankliniella occidentalis (Pergande), and onion thrips, Thrips

tabaci (Lindeman), are prevalent pests of lettuce in Arizona (Kerns and Palumbo 1996, Kerns et

al. 1999, Agnew 2000, Kurtz 2001). Western flower thrips is a native of North America, and has

a broad host range of more than 500 species representing 50 plant families (Beshear 1983, Yudin

et al. 1986). It is most commonly found in California (Bryan and Smith 1956, Rob 1989) and

Arizona (Bibby 1958) on lettuce. Thrips adults and larvae puncture and feed from epidermal

cells (Nuessly and Webb 2003), and affect quality of lettuce, as they cause leaf stippling and rib

discoloration (Kurtz 2001). Romaine lettuce is especially susceptible to thrips in Arizona (Kerns

and Palumbo 1996, Kerns et al. 1999, Agnew 2000). In Florida, Frankliniella spp. are important









carriers of tomato spotted wilt and escarole necrosis viruses (Nuessly and Webb 2003). Mollema

and Cole (1996) found a positive correlation between amino acid concentration in lettuce leaves

and western flower thrips damage suggesting that higher concentrations are important for

successful thrips development.

Research Goals

Lettuce is an important leafy vegetable grown all over the world. In the United States,

romaine lettuce is the most commonly grown leaf lettuce. It is vulnerable to attack by several

insect pests during field production. Chemical control measures are the main tools for

management of insect pests on lettuce and about 93% of lettuce grown in the United States is

under chemical management of noxious insects (Agricultural Statistics 2001). In southern

Florida, vegetable farming involves high intensity pesticide usage (>20 pounds of active

ingredient pesticide per acre/crop), and often there is more than one crop per year, which further

increases the amount of pesticides used (Agricultural Statistics Board 2001). In Florida, lettuce

production is more concentrated in the southern part of the state, which is an ecologically

sensitive area due to its proximity to the Everglades National Park and heavy precipitation and

run-off (Miles and Pfeuffer 1997). High dependence on chemicals can pose a threat to growers

and natural enemies of insect pests as well as involves a heavy cost (Sharma and Ortiz 2002,

Sadasivan and Thayumanavan 2003). Hence, there is a need to look for alterative tactics for

management of economic insect pests.

Host plant resistance is an important component of integrated pest management.

Management of insects based on host plant resistance can reduce the sole dependence on

chemical usage (Sharma and Ortiz 2002, Sadasivan and Thayumanavan 2003). Thus, it is

essential to develop resistant varieties in lettuce to reduce these economic and environmental

problems. The romaine lettuce cultivar Valmaine is known to possess a high level of resistance









against D. balteata (Huang et al. 2002) and L. trifolii (Nuessly and Nagata 1994) as compared to

three other cultivars, 'Parris White', 'Short Guzmaine', and Tall Guzmaine. Resistance was

highest in Valmaine and lowest in Parris White in confirmation with pedigree analysis (Guzman

1986). Short Guzmaine is the product of Valmaine and 'FL 1142', whereas Tall Guzmaine was

selected from progeny of a cross between Short Guzmaine and Parris White. Guzman designed

Tall Guzmaine for improvement of certain horticultural characters over Valmaine, such as

thermodormancy, premature bolting, and resistance to lettuce mosaic virus and corky root rot.

Breeders did not evaluate insect resistance when developing Tall Guzmaine. Further, previously

wounded Valmaine plants showed higher resistance to D. balteata as compared to Tall

Guzmaine suggesting the involvement of an induced mechanism of resistance in Valmaine

(Huang et al. 2003b). Thus, it would be helpful to know the biochemical mechanism of

resistance in Valmaine to different insects to aid plant-breeding programs in development of new

lettuce cultivars with both desirable horticultural characters and insect resistance.

The objectives of this study were the following:

1. To compare survival, development and feeding behavior of cabbage looper and beet

armyworm on Valmaine and Tall Guzmaine

2. To determine the potential of latex produced by Valmaine as a defense mechanism

against banded cucumber beetle using choice and no-choice tests and isolation of

deterrent compounds from the latex using solvent extraction

3. To further isolate deterrent compounds from Valmaine latex using bioassay-directed

fractionation of Valmaine latex crude extract

4. To investigate enzyme induction as a possible reason for latex-mediated insect resistance

in Valmaine









CHAPTER 2
HOST PLANT RESISTANCE IN ROMAINE LETTUCE AFFECTS LARVAL
FEEDING BEHAVIOR AND BIOLOGY OF TRICHOPLUSIA NI AND
SPODOPTERA EXIGUA (LEPIDOPTERA: NOCTUIDAE)

Introduction

Over the past 15 yr, romaine lettuce, Lactuca sativa L., has been the fastest

growing vegetable in terms of production, consumption, and exports in the United States.

During the period 2002 to 2004, romaine lettuce accounted for 22% of all lettuce

produced in the United States and per capital use of romaine lettuce has tripled (3.7 kg)

since 1992-94 (USDA 2005a). Lettuce is vulnerable to attack by several insects including

lepidopterans that can be responsible for yield losses of 100% if populations are not

managed (Inglis and Vestey 2001). In Florida, the cabbage looper, Trichoplusia ni

(Hubner), and the beet armyworm, Spodoptera exigua (Hubner) (Lepidoptera:

Noctuidae), are serious pests of lettuce (Nuessly and Webb 2003).

Economic pests are managed chemically on about 89% and 85% of head and other

lettuce acreage, respectively, in the United States (USDA 2005b). Florida ranks first

among lettuce growing states in the usage of insecticides and growers apply insecticides

on 98 to 100% of the state's lettuce acreage to manage these insect pests (Mossler and

Dunn 2005). For instance, restricted insecticides such as lambda-cyhalothrin (34% and

32% of head and other lettuce acreage, respectively) and methomyl (32% and 30% of

head and other lettuce acreage, respectively) are extensively applied on lettuce (USDA

2005c). Rapid development of insecticide resistance has been reported for Liriomyza spp.

(Diptera: Agromyzidae) against chlorinated hydrocarbons, organophosphates and the

pyrethroid permethrin (Genung 1957, Leibee 1981, Parrella and Keil 1984). The high

dependence on chemicals poses a threat to agricultural workers and natural enemies of









these insect pests and increases production cost. Therefore, the implementation of

alternative strategies, such as host plant resistance, for the management of economic

insect pests should be explored.

'Valmaine' romaine lettuce (Leeper et al. 1963) was the major cultivar grown in

Florida before the adoption of 'Tall Guzmaine'. Tall Guzmaine was selected from a cross

between 'Short Guzmaine' and 'Parris White' (Guzman 1986). Short Guzmaine was a

selection from a cross between Valmaine and 'Florida 1142'. Tall Guzmaine was selected

for resistance to thermodormancy, premature bolting, lettuce mosaic virus and corky root

rot; however, Guzman did not include insect resistance in his selection criteria (Guzman

1986). Tall Guzmaine was found to be susceptible to the leafminer, Liriomyza trifolii

(Burgess) (Nuessly and Nagata 1994) and the banded cucumber beetle, Diabrotica

balteata LeConte (Coleoptera: Chrysomelidae) (Huang et al. 2002) compared to

Valmaine. Therefore, I selected the same two cultivars to determine whether resistance in

Valmaine extends to a third order containing economically important insect pests of

lettuce, the Lepidoptera.

In this study, I tested the performance of two noctuid defoliators important to

Florida lettuce production, cabbage looper and beet armyworm on Valmaine and Tall

Guzmaine. I chose these two insect species because I was interested in how ecologically

similar but behaviorally different defoliators of lettuce would respond to the selected

lettuce cultivars. Cabbage loopers trench leaves of latex-bearing plants (Dussourd and

Denno 1994), whereas beet armyworms do not. In particular, cabbage looper has been

shown to deactivate the canalicular defenses in wild lettuce, Lactuca serriola L., by

making shallow trenches before actual feeding (Dussourd 1997). The objectives of the









study were to compare the survival, development and feeding behavior of cabbage looper

and beet armyworm on resistant Valmaine and susceptible Tall Guzmaine lettuce.

Materials and Methods

Plants

Seeds of two romaine lettuce cultivars, Valmaine and Tall Guzmaine, were

provided by R. T. Nagata (Everglades Research and Education Center, University of

Florida, FL). Seeds were germinated by placing them overnight in a Petri dish lined with

wet filter paper in the laboratory. Germinated seeds were planted in a transplant tray

filled with commercial soil mix (MetroMix 220, Grace Sierra, Milpitas, CA) in a

greenhouse with natural light at a mean temperature of 270C (32 to 240C) and 68% mean

RH (44 to 94%). After 2 wk, seedlings were transplanted to plastic pots (15 cm diameter)

filled with MetroMix 220. Plants were irrigated daily and fertilized once per week with

10 ml of a 10 g/L solution of soluble fertilizer (Peters 20-20-20, N-P-K, W.R. Grace,

Fogelsville, PA) from transplanting of seedlings to the end of the experiment. Four-week-

old plants with six to seven true leaves were used in all experiments that were conducted

in the greenhouse under ambient light.

Insects

Cabbage looper eggs were supplied by G. L. Leibee (Mid-Florida Research and

Education Centre, University of Florida, FL) from a 1-yr-old colony, which was raised on

mustard leaves. Eggs were sterilized with 500 ml 0.008 % sodium hypochlorite solution

(Clorox, Oakland, CA) for 1 min in a cylindrical container (18 cm diameter by 7.5 cm

high). Sterilized eggs were rinsed twice with distilled water and were drained into a nylon

strainer. Eggs were inverted into a 177-ml cup under running water until the cup was half

filled with water. The remaining half cup was filled with neutralizer (10% sodium









thiosulphate solution) and eggs were soaked in the cup for 2 min. Neutralizer and eggs

were drained into a nylon strainer and eggs were rinsed twice with distilled water. After

rinsing, eggs were placed on a paper towel in a cylindrical container with plastic screen

lid and placed in an incubator at 27 20C, 70 10% RH, and a photoperiod of 14:10

(L:D) h. Neonates were used for bioassays.

Egg masses of beet armyworm were collected from pepper plants in Citra, FL and

the subsequent generations (F3 through F8) were used for bioassays. Eggs were sterilized

in the same way as for cabbage looper. Newly emerged larvae were transferred onto pinto

bean diet (Guy et al. 1985) in a rectangular container (25 x 25 x 11 cm) with plastic

screen lid in an incubator at the same conditions as for cabbage looper. Pupae were

placed into paper cups and placed in the incubator. Beet armyworm adults were held in a

screen cage (30.5 x 30.5 x 30.5 cm) in the incubator. Two cotton plants with three to four

true leaves were used for oviposition and were replaced with fresh ones every other day.

Adults were fed a 20% sucrose solution dispensed on a cotton wick. Neonates were used

for bioassays.

Neonate Survival and Development to Third Instar

Thirty replicates of each cultivar were set up along greenhouse benches in a

randomized complete block design. Experiments on beet armyworm and cabbage looper

were done separately under similar greenhouse conditions. Ten neonates were placed in

the central whorl of each plant and the plant was covered with a cylindrical screen cage

(18.5 cm diameter x 61.0 cm height) to confine the insects for feeding (Fig. 2-1). Plants

were dissected 1 wk after infestation to locate the surviving larvae. Larval mortality,

weight, instar and feeding behavior were observed and recorded. Instars were determined

by measuring head capsule widths (Capinera 2005, 2006). Observations were also made









on the preferred site of feeding on a leaf and within a plant. Larval mortality and weight

for each species were analyzed using PROC GLM with cultivar as a fixed effect and

replications as random effect (SAS Institute 1999). Tukey's honestly significant

difference (HSD) test with a significance level of a = 0.05 (SAS Institute 1999) was used

for posthoc means separation. Log-likelihood ratio (G2-test) (Zar 1984) was used to

analyze the frequency of surviving instars using JMP release 5.1.2 (JMP Software, SAS

Institute Inc., Cary, NC). Differences in the preferred site of feeding within a plant were

analyzed by x2 goodness of fit tests (Freund and Wilson 1997).

Survival and Development from Neonate to Adult Emergence

Time of development from neonates to adults was investigated on both lettuce

cultivars. Thirty replicates of each cultivar were set up along greenhouse benches in a

randomized complete block design. Experiments on beet armyworm and cabbage looper

were done separately under similar greenhouse conditions. Ten neonates were placed in

the central whorl of each plant and the plant was covered with a cylindrical screen cage

(Fig. 2-1). Days required to develop from neonate to pupa and from pupa to adult

emergence were recorded. Beet armyworm larvae were provided MetroMix 220 in a 5-

cm-diameter Petri dish at the base of the plant as a pupation site. Cabbage looper pupated

on the plant and on the walls of the container so were not supplied with MetroMix. Pupae

were removed from the greenhouse, weighed and put in individual cups in the incubator

at 27 20C, 70 10% RH, and a photoperiod of 14:10 (L:D) h. Emerged adults were

sexed, killed and then dried in an oven at 50 50C, for 3 d. Larval period, pupal fresh

weight, pupal period, and dry weight of emerged adults of each insect species were

analyzed using PROC GLM with cultivar as a fixed effect and replications as random

effect (SAS Institute1999). Tukey's honestly significant difference (HSD) test with a









significance level of a = 0.05 (SAS Institute 1999) was used for posthoc means

separation. Percent successful pupation and adult emergence were analyzed by two

sample t-test using PROC TTEST (SAS Institute1999). A binominal test (using the

normal approximation with test statistic Z) was used to determine whether the numbers of

males versus females deviated from a 1:1 ratio on each cultivar. A Fisher's Exact test of

independence was used to test whether the adult sex-ratio differed between the cultivars

(Sokal and Rohlf 1995) using JMP release 5.1.2.

Fecundity and Longevity of Subsequent Generation

Fecundity and longevity were measured for nine pairs of newly emerged adults of

each species that had been reared on either Tall Guzmaine or Valmaine as larvae. Each

pair of adults was confined on a Tall Guzmaine plant using a cylindrical screen cage

(18.5 x 61.0 cm) in the greenhouse. Adults were supplied with 20% sucrose solution.

Every other day, the lettuce plant was replaced with a fresh plant. Eggs were counted on

each plant and totaled over the life of each female. Fecundity and adult longevity of each

insect species were analyzed using PROC GLM with cultivar as a main effect (SAS

Institute 1999). A simple linear regression analysis was done to study the relationship

between adult weight and fecundity using PROC REG (SAS Institute 1999).

Results

Neonate Survival and Development to Third Instar

Larval mortality of cabbage looper and beet armyworm after 1 wk of feeding was

significantly higher on Valmaine than on Tall Guzmaine (Fig. 2-2). Cabbage looper

mortality was 24 times higher on Valmaine than on Tall Guzmaine (F = 242.82; df= 1,

29; P = 0.0001) whereas beet armyworm mortality was four times higher on Valmaine

than on Tall Guzmaine (F= 187.54; df = 1, 29; P = 0.0001). Average weight of cabbage









looper feeding for 1 wk on Valmaine (75.4 + 3.7 mg, mean SEM) was significantly

lower than that of larvae feeding on Tall Guzmaine (151.2 + 3.3 mg) (F = 249.27; df = 1,

29; P = 0.0001). Beet armyworm weight was also significantly lower (1.5 + 0.1 mg) on

Valmaine than on Tall Guzmaine (8.3 0.8 mg) (F = 68.71; df= 1, 29; P = 0.0001).

The instar of the larvae surviving to plant dissection (1 wk after inoculation as

neonates) differed significantly on the two lettuce cultivars for both species (Fig. 2-3).

Cabbage looper and beet armyworm developed more slowly on Valmaine than on Tall

Guzmaine. More of the surviving neonates of both insect species were in the early instars

on Valmaine than on Tall Guzmaine. About 80% of cabbage looper surviving on

Valmaine were in either the first or second instar, whereas on Tall Guzmaine about 80%

of surviving larvae were in either the third or fourth instar (Fig. 2-3). Of the beet

armyworm surviving for 1 wk on Valmaine, 57.7% were in the first instar, whereas

78.8% were in the third instar on Tall Guzmaine (Fig. 2-3).

Larval Feeding Behavior

The insect species behaved differently on the lettuce cultivars. Cabbage looper cut

narrow trenches across veins on the leaves and then fed on the area distal to the trench

(Fig. 2-4A). This behavior released exudate from the laticifers of the leaves. Beet

armyworm did not trench; neonates made shallow scratches between the veins by feeding

on parenchymatous tissue and second instars made holes through the leaf (Fig. 2-4B).

The preferred site of feeding of cabbage looper (x = 55.42, df = 2; P = 0.0001) and beet

armyworm (2 = 35.13, df = 2; P = 0.0001) differed between the two cultivars (Fig. 2-5).

Cabbage looper preferred to feed on the lowermost fully mature leaves of Valmaine

plants and on young and middle-aged leaves of Tall Guzmaine plants (rarely feeding on

fully-matured leaves) (Fig. 2-6). Beet armyworm preferred to feed on the lowermost fully









mature leaves of Valmaine plants and on middle-aged leaves of Tall Guzmaine plants.

Both insect species preferred to feed on the distal end of leaves. Early instars of cabbage

looper preferred to feed on the underside of the leaves, whereas early instars of beet

armyworm fed on the upper side of the leaves.

Survival and Development from Neonate to Adult Emergence

Both cabbage looper (F = 82.55; df = 1, 29; P = 0.0001) and beet armyworm (F=

581.58; df = 1, 29; P = 0.0001) took significantly longer time to develop from neonate to

pupation on Valmaine than on Tall Guzmaine (Table 2-1). Larval period of cabbage

looper and beet armyworm was increased by 2.6 d and 5.9 d, respectively on Valmaine.

Feeding on Valmaine resulted in a significant reduction in successful pupation of cabbage

looper (t = 9.75; df = 58; P <0.0001) and beet armyworm (t = 13.46; df = 58; P <0.0001)

(Table 2-1). Pupae of cabbage looper (F = 41.53; df = 1, 29; P = 0.0001) and beet

armyworm (F = 63.84; df = 1, 29; P = 0.0001) weighed significantly less when reared on

Valmaine compared to Tall Guzmaine (Table 2-1). The duration of the pupal period of

cabbage looper (F = 44.53; df = 1, 29; P = 0.0001) and beet armyworm (F = 30.79; df=

1, 29; P = 0.0001) was significantly increased on Valmaine (Table 2-1), thus delaying

adult emergence. Successful emergence of adults from pupae surviving on Valmaine was

significantly reduced for cabbage looper (t = 2.40; df = 58; P = 0.0196) but not for beet

armyworm (t= 1.40; df = 58; P = 0.1649) (Table 2-1). Adults of cabbage looper (F=

83.02; df = 1, 29; P = 0.0001) and beet armyworm (F = 196.34; df = 1, 29; P = 0.0001)

surviving on Valmaine weighed significantly less than those surviving on Tall Guzmaine

(Table 2-1). The mean adult sex-ratio of cabbage looper (Z = 0.91, P = 0.3652) and beet

armyworm (Z = 0.59, P = 0.5529) did not deviate from a 1:1 ratio on Valmaine. The

mean adult sex-ratio of cabbage looper (Z = 1.30, P = 0.1950) and beet armyworm (Z=









1.33, P = 0.1845) also did not deviate from a 1:1 ratio on Tall Guzmaine. In addition, the

sex-ratios of adult cabbage looper (Fisher's Exact test of independence, P = 0.1417) and

beet armyworm (Fisher's Exact test of independence, P = 0.2077) on the two cultivars did

not differ statistically (Table 2-1).

Fecundity and Longevity of Subsequent Generation

Fecundity of cabbage looper (F = 109.36; df = 1, 8; P = 0.0001) and beet

armyworm (F = 149.14; df = 1, 8; P = 0.0001) on Valmaine was reduced by 62.8 and

67.9%, respectively, compared to that on Tall Guzmaine (Table 2-2). Significant positive

linear relationships were found between adult weight and fecundity of both insect species

on the two lettuce cultivars (Fig. 2-7). However, neither male nor female longevity of

cabbage looper (male: F= 0.47; df = 1, 8; P = 0.5121; female: F= 0.47; df= 1, 8; P=

0.5121) nor beet armyworm (male: F = 0.31; df = 1, 8; P = 0.5943; female: F = 1.33; df=

1, 8; P = 0.2815) differed on Valmaine or Tall Guzmaine (Table 2-2).

Discussion

Performance of cabbage looper and beet armyworm was greatly reduced on

resistant Valmaine compared to Tall Guzmaine. Insects surviving on poor quality hosts

are expected to have reduced survival to adult emergence and reduced fecundity (Zalucki

et al. 2001), as was shown in my study. Larval survival and development can be reduced

on poor quality hosts due to nutritional composition and/or secondary plant metabolites

(Scriber and Slansky 1981, Herms and Mattson 1992, Slansky 1992).

Nutritional composition and secondary plant metabolites vary among plants, plant

parts and developmental stages (Nelson et al. 1981, Brower et al. 1982). Cabbage looper

and beet armyworm larvae preferred to feed on mature leaves of Valmaine (Fig. 2-5). In

lettuce, mature leaves are less nutritious than young and middle-aged leaves. Young and









middle-aged lettuce leaves are more metabolically active than mature leaves, and

therefore, contain higher amounts of dry matter, ascorbic acid and soluble solids, such as

fructose, sucrose, glucose, fructans and other saccharides (McCabe et al. 2001, Siomos et

al. 2002). Moreover, mature lettuce leaves always have higher amounts of anti-nutritional

constituents, such as nitrates (Siomos et al. 2002). Leaf maturation is accompanied by a

decline in the concentrations of proteins and other nutrients (Bernays and Chapman

1994). Therefore, feeding on less nutritious mature leaves of Valmaine may have affected

the fitness of cabbage looper and beet armyworm.

Larval avoidance of young and middle-aged leaves of Valmaine may have been due

to the presence of high amounts of latex and/or the chemical constituents of latex. Latex

from young and middle-aged leaves was pure white and viscous whereas latex from

mature leaves was a watery translucent fluid (A. Sethi, pers. obs.). Young leaves of the

poinsettia, Euphorbiapulcherrima Wilenow contained higher amounts of latex and

laticifer starch than mature leaves (Spilatro and Mahlberg 1986). The proportionally

higher latex amount may have a specific purpose related to plant defense. The defensive

role of latex has been attributed to its sticky nature, which would enable the plant to

capture small insects and immobilize the mouthparts of larger insects (Farrell et al. 1991,

Dussourd 1993, Dussourd and Denno 1991, 1994). Antiherbivore function of latex has

been suggested in many plant systems (Shukla and Krishna-Murti 1971, Fahn 1979,

Konno et al. 2004, 2006). The presence of high amounts of latex with its chemical

components in young leaves (Kinghom and Evans 1975, Swain 1977, Rees and Harborne

1985) may provide for their defense compared to mature leaves. In the chicory plant,

Cichorium intybus L., sesquiterpene lactones were present in the highest amounts in the









most actively growing regions of the plant and possessed antifeedant properties against

Schistocerca gregaria (Orthoptera: Acrididae) (Rees and Harborne 1985). Various

organic compounds, like phenolics and terpenoids have been reported in latex of Lactuca

spp. (Crosby 1963, Gonzales 1977, Cole 1984, Sessa et al. 2000) and their defensive role

as phytoalexins has been reported against plant diseases (Bennett et al. 1994, Bestwick et

al. 1995). In lettuce, the density of latex is successively decreased from the base to the

apex of the leaf (Small 1916). This may account for the preference of neonate caterpillars

in my study to feed on the distal end (apex) of leaves.

Certain plant enzymes, such as phenylalanine ammonia lyase, polyphenol oxidase

and peroxidase are known for their defensive role against insects. In lettuce, Campos-

Vergas and Saltveit (2002) reported enhanced activity of phenylalanine ammonia lyase

upon mechanical wounding in young leaves compared to mature leaves. Phenylalanine

ammonia lyase is also more active in aphid-resistant cultivars ofL. sativa than in

susceptible cultivars (Cole 1984).

Outer (i.e., older) leaves of head lettuce exhibit high concentrations of flavonoids,

such as quercetin (Hohl et al. 2001). Quercetin and its derivatives are known to act as

phagostimulants to many lepidopterans (Simmonds 2003). Therefore, the feeding

location of beet armyworm and cabbage looper may have been the end result of both

antifeedant properties (either physical or chemical or both) of young and middle-aged

leaves and phagostimulant properties of mature leaves.

Cabbage looper exhibited greater fitness than beet armyworm on Valmaine. Larval

mortality of cabbage looper was less compared to beet armyworm on Valmaine and other

parameters, such as larval weight, pupal weight, percent pupation and adult weight of









cabbage looper were less affected compared to beet armyworm on Valmaine (Table 2-1).

Moreover, larval development of cabbage looper was faster than beet armyworm, as

cabbage looper larvae entered the fourth larval after 1 wk on Valmaine while beet

armyworm larvae were still in the third instar stage (Fig. 2-2). Survival and development

of yellow-striped armyworm was also affected greatly on L. serriola compared to

cabbage looper (Dussourd 1993). This superior performance of cabbage looper may be

attributed to their feeding behavior (i.e., trenching on laticiferous plants). Trenching

blocks latex flow to intended feeding sites and may act as a counter-adaptation to the

plant's defensive secretions (Dussourd and Denno 1994). In spite of their behavioral

counter-adaptation, cabbage looper performance was worse on Valmaine than on Tall

Guzmaine.

Lettuce possesses little tolerance for cosmetic damage and foliar feeding by

lepidopterous pests greatly affects its marketable production. The consumer, especially in

developed countries, will not accept produce unless it is free of all insects and blemishes

at harvest. Further, lettuce is a short-season crop and insufficient time may be present

between treatment of chemical and harvest for pesticide residues to decline to acceptable

levels (Norris et al. 2003). This limits the use of chemicals in lettuce production that do

not break down rapidly. Therefore, host plant resistance is an economically, ecologically

and environmentally advantageous method of insect management. The results of my

study have confirmed that Valmaine expresses considerable resistance to lepidopterous

pests in spite of their counter-strategies against plant resistance. In general, multiple-

insect resistance is thought to be more desirable than single-insect resistance (Smith

1989). Feeding on Valmaine resulted in reduced vigor of both insect species, which









ultimately could make them more susceptible to other biotic and abiotic factors.

However, additional research is required to determine the biochemical basis of multiple-

insect resistance in lettuce. Understanding the mechanism of resistance will certainly aid

in the development of lettuce cultivars with improved pest resistance and may result in

reduced pesticide usage.











Table 2-1. Performance of cabbage looper and beet armyworm released as neonates onto Valmaine and Tall Guzmaine lettuce.
Species Cultivar Larval % Pupal Pupal % Adult Adult Sex-ratio
period Pupation weight period emergence weight (male:
(days) (mg) (days) (mg) female)
Cabbage Valmaine 11.7 0.2a 49.3+ 172.2 4.5b 8.7 0.2a 82.4 2.5a 23.4 0.6b 1.18 : la


Tall
Guzmaine
Valmaine


2.0b
9.1 + 0.3b 79.0 +


19.3 + 0.3a


2.3a
27.3
2.6b


206.5 +2.8a 7.8 0.1b

51.4 1.4b 7.5 0.1a


90.7 1.3a

86.6 + 2.7a


29.7 + 0.6a

9.1 0.3 b


1 : 1.20a

1.20 : la


Tall 13.4 + 0.1b 65.3 68.9 1.3a 6.7 + 0.1b 93.9 + 2.0a 14.9 + 0.3a 1 : 1.22a
Guzmaine 3.8a
Means SEM followed by different letters for each parameter within insect species differed significantly (P < 0.05) using ANOVA and Tukey's HSD test for
larval period, pupal weight, pupal period and adult weight, two sample t-test for % pupation and % adult emergence, and Fisher's Exact test of independence for
sex-ratio


looper


Beet
armyworm











Table 2-2. Fecundity and longevity of subsequent generation of cabbage looper and beet armyworm reared on Valmaine and Tall
Guzmaine lettuce.
Species Cultivar Fecundity Male longevity (d) Female longevity (d)

Cabbage looper Valmaine 146.4 + 8.4a 11.8 + 0.2a 10.1 + 0.2a

Tall Guzmaine 393.3 + 18.1b 12.0 + 0.3a 10.3 0.3a

Beet armyworm Valmaine 123.2 + 10.3a 6.4 0.2a 8.1 + 0.2a

Tall Guzmaine 383.6 + 17.7b 6.3 0.2a 8.4 0.2a

Means + SEM followed by different letters for each parameter within insect species differed significantly (P < 0.05) using ANOVA and Tukey's HSD test.








'I


Site of release


Figure 2-1. Experimental setup to study cabbage looper and beet armyworm neonate
survival and development to third instar.


qIf&












60

Valmaine
o50 I Tall Guzmaine


40


30
0

20 -


10 -


0
Cabbage looper Beet armyworm

Figure 2-2. Larval mortality of cabbage looper and beet armyworm after 1 wk of feeding
on resistant Valmaine and susceptible Tall Guzmaine lettuce. Error bars
indicate 1 SEM











100
CL Valmaine

80 Tall Guzmaine


60
s.-

40-
CU
^20
(D
s 40
c



100
C



O

C:
0 20
210
I-









1 st 2nd 3rd 4th
Instar

Figure 2-3. Instars of cabbage looper (CL) and beet armyworm (BAW) surviving for 1
wk on resistant Valmaine and susceptible Tall Guzmaine lettuce. G2 tests
indicated that the instar distribution on Valmaine differed significantly from
that on Tall Guzmaine (P < 0.05).





















B




Figure 2-4. Feeding of two lepidopterans on lettuce. A) Cabbage looper cutting narrow
trenches on romaine lettuce, B) Beet armyworm damage on romaine lettuce:
(a) & (b) shallow scratches, (c) holes.














25 -

20 -

15 -

10 -

5

0

30

25 -
25

20

15

10

5

0


-I _


BAW




Valmaine
I Tall Guzmaine


Young


Middle


Mature
Mature


Leaf age
Figure 2-5. Feeding preference of cabbage looper (CL) and beet armyworm (BAW)
larvae among lettuce leaves of different ages on resistant Valmaine and
susceptible Tall Guzmaine. The y-axis depicts the total number of plants (out
of 30) on which at least some feeding occurred on leaves of the specified age
group.


I




















Figure 2-6. Feeding behavior of beet armyworm on (a) Tall Guzmaine and (b) Valmaine, and of cabbage
looper on (c) Tall Guzmaine and (d) Valmaine.













250


VAL CL


7 x- 95.3


0.82
0007


.-

o
L.


TG-CL
y = 15.9 x 166.1
R2 = 0.95
p<0.0001 .


0 20 22 24 26 28 30
Adult weight (mg)


0 28 30 32 34 36 38 40 42
Adult weight (mg)


250


200 y = 20.
R2= 0
150 p<0.0


VAL BAW
7x- 101.5
.94
1001


*o


100


600


TG BAW


> 500


o 400
U-


y = 37.3 x- 329.7
R2 = 0.86
p= 0.0003


300


10 12 14


0 16


18 20


Adult weight (mg) Adult weight (mg)
Figure 2-7. Relationships between adult weight and fecundity of cabbage looper (CL) and beet armyworm (BAW) that developed
from larvae reared on resistant Valmaine (VAL) or susceptible Tall Guzmaine (TG) lettuce.


y = 9.
R2 =
p = 0.


200


0 150
100
100


600

500

400

300


0 8


*









CHAPTER 3
ROMAINE LETTUCE LATEX DETERS FEEDING OF BANDED CUCUMBER
BEETLE (COLEOPTERA: CHRYSOMELIDAE)

Introduction

Latex is the common term used to describe a frequently milky plant exudate which

is typically stored under positive pressure within specialized vessels called laticifers (Fig.

3-1). These laticifers accompany the vascular bundles and ramify into the mesophyll to

reach the epidermis (Hayward 1938, Esau 1965, Metcalfe 1967, Olson et al. 1969,

Metcalfe and Chalk 1983, Fahn 1990, Kekwick 2001). About 12,500 to 20,000 plant

species, belonging to >900 genera from about 40 families, most of which are

dicotyledons, are known to exude latex (Esau 1965, Metcalf 1967, Lewinsohn 1991,

Kekwick 2001, Evert 2006). Latex contributes to plant defense in two different ways;

physical properties (stickiness) and chemical properties (toxic constituents). Stickiness

can result in the entrapment or gumming up of the mouthparts of herbivorous insects

(Dillon et al. 1983, Dussourd 1993, 1995, Zalucki and Malcolm 1999). Latex contains

toxic constituents including alkaloids (Roberts 1987, Valle et al. 1987, Konno et al.

2006), cardiac glycosides (Zalucki and Brower 1992, Zalucki and Malcolm 1999), and

terpenoids (Evans and Schmidt 1976, Rees and Harborne 1985, Spilatro and Mahlberg

1986). Some insects circumvent the mechanical stickiness and toxic effects of latex by

severing latex-bearing veins or by cutting trenches prior to consuming the distal tissue

(Dussourd 1993, Zalucki and Malcolm 1999, Sethi et al. 2006).

Lettuce, Lactuca sativa L., is one of the most important vegetable crops grown

throughout the world and its production grows annually (USDA 2005a). As a cultivated

crop, lettuce is vulnerable to attack by various insect pests including the banded

cucumber beetle, Diabrotica balteata LeConte (Nuessly and Nagata 1993). This insect









has a host range of >50 plant species in 23 families (Saba 1970) and a high reproductive

potential of >800 eggs per female with a 2 to 8 wk oviposition period (Pitre and Kantack

1962). It can be found throughout the year in the southern United States (Schalk 1986). In

southern Florida, foliar feeding by D. balteata adults leads to economic damage in lettuce

due to reduction in stand and marketability, decreased photosynthetic area, frass

contamination of the heads, and increased vulnerability to diseases. Chemical control of

soil-borne eggs, larvae and pupae of this insect has been ineffective (Schalk et al. 1986)

and control of the adult is the sole promising option (Schalk et al. 1990). As a result,

growers currently are dependent on pesticides (Nuessly and Nagata 1993) which can pose

a threat to the environment, farm workers and natural enemies of insect pests, and

increase production costs.

Host plant resistance was explored as an alternative strategy for the management of

this economic insect pest in a cos or romaine lettuce cultivar, 'Valmaine' (Nuessly and

Nagata 1994, Huang et al. 2002, Sethi et al. 2006). A high level of resistance was

reported in Valmaine, compared to the closely related susceptible cultivar 'Tall

Guzmaine' against serpentine leafminer, Liriomyza trifolii (Burgess) (Nuessly and

Nagata 1994), banded cucumber beetle (Huang et al. 2002) and two lepidopterans,

Trichoplusia ni (Hubner) and Spodoptera exigua (Hubner) (Sethi et al. 2006). These

studies suggested that Valmaine lacks feeding stimulants or contains feeding deterrents,

either in the leaf cuticle or the leaf interior. Huang et al. (2003a) reported that leaf surface

chemicals were not responsible for resistance in Valmaine and suggested chemicals

inside the leaf may play a role. However, incorporation of freeze-dried leaves of

Valmaine into an artificial diet did not deter feeding by D. balteata adults and neither did









application of Valmaine latex on the leaf surface of a favorite food, lima bean (Huang et

al. 2003b). It is possible that the activity of physical and/or chemical defenses in latex or

leaf tissue may have been reduced or eliminated when whole leaves were dried and

powdered. Furthermore, the physical and chemical properties of latex may have changed

when applied on lima bean leaves due to drying of the latex and/or oxidation of chemical

constituents.

In free-choice situations L. trifolii (Nuessly and Nagata 1994), D. balteata (Huang

et al. 2002), T. ni and S. exigua (Sethi et al. 2006) preferred to feed on mature leaves of

Valmaine over young or middle-aged leaves. The avoidance of young and middle-aged

leaves of Valmaine may have been due to the presence of high amounts of latex and/or

the chemical constituents of latex. The latex from young and middle-aged leaves is pure

white and viscous, whereas latex from mature leaves is watery and translucent (Sethi et

al. 2006).

In this study, I report on the possible deterrent role of latex against beetle feeding

on artificial diet treated with freshly extracted latex from either Valmaine or Tall

Guzmaine in choice and no-choice conditions. Additional tests were conducted using

latex extracted from young versus mature leaves of these two cultivars to study the role

of leaf age in the expression of latex deterrence. Lastly, samples of supernatant material

collected following dissolution of latex from both cultivars in water/methanol

combinations or methylene chloride and centrifugation were applied to diet disks under

no-choice situations to determine whether differences in latex chemistry between

Valmaine and Tall Guzmaine contribute to the multiple insect resistance observed on

Valmaine.









Materials and Methods


Plants and Insects

Valmaine and Tall Guzmaine seeds were germinated by placing them overnight in

a Petri dish lined with a wet filter paper in the laboratory. Germinated seeds were planted

in a transplant tray filled with commercial soil mix (MetroMix 220, Grace Sierra,

Milpitas, CA) in a greenhouse with natural light at a mean temperature of 27C (32 to 24

C) and 68% mean R.H. (44 to 94%). After 2 wk, seedlings were transplanted into 15-cm-

diameter plastic pots filled with MetroMix 220. Plants were irrigated daily and fertilized

once a week with 10 ml of a 10 g/1 solution of soluble fertilizer (Peters 20-20-20, N-P-K,

W.R. Grace, Fogelsville, PA).

Bush lima bean (Phaseolus lunatus L.) cultivar Fordhook 242 (Illinois Foundation

Seeds, Champagne, IL) was grown as an adult food source for the colony. Seeds were

planted in a transplant tray filled with MetroMix 200. Lima bean plants were irrigated

daily and fertilized once a week after the first true-leaf stage with the same solution used

for lettuce plants.

Adults ofD. balteata were used because previously the same insect species was

used by Huang et al. (2003b) as explained above. In addition, R. T. Nagata (Everglades

Research and Education Center, University of Florida, FL) used the same insect species

to track resistance in lettuce breeding lines. Further, adults ofD. balteata were easy to

rear and handle during bioassays. A colony ofD. balteata was established in 2003 from a

wild population of adults collected from spiny amaranth, Amaranthus spinosus L. and

primrose willow, Ludwigiaperuviana L. in Belle Glade, FL. The colony was

supplemented with wild individuals to increase genetic diversity in 2005 and 2006.

Adults ofD. balteata were fed on lima bean leaves and sweet potato tubers, and larvae









were reared on corn seedling roots (H93 x FB37, Illinois Foundation Seeds Inc., IL) as

per Huang et al. (2002) (Fig. 3-2).

Adults of the D. balteata colony were confined in a ventilated plexiglas cage (30.5

x 30.5 x 30.5 cm) in an incubator at 27 2 C and R.H. 70 10% with a photoperiod of

14:10 (L:D) h (Fig. 3-2A). Oviposition was facilitated by providing two domed-shaped

plastic containers (8.4 cm diameter x 7 cm high) with mesh-covered lids (0.1 x 0.2 cm).

These plastic containers were covered with inverted strawberry baskets upon which lima

bean leaves were placed (Fig. 3-2B). The plastic containers were filled with upright small

glass vials (20 ml) to hold tightly one moist layer of cotton balls. Two circular pads, each

containing four layers of premium paper towels (Kimberly-Clark Co., Roswell, GA), and

four layers of cheesecloth were placed between the layer of cotton balls and the meshed

lid.

The cheescloth and paper towel pads with eggs were collected every 2 d and kept in

a Petri dish with a screened lid in the same incubator. Three-day-old eggs were dipped in

sodium hypochloride solution (15 ml Clorox in 485 ml water, The Clorox Co., CA) for 1

min and then rinsed thrice with deionized water to affect surface sterilization. The

sterilized eggs were replaced in the incubator and covered with a wet paper towel within

a cylindrical container (18 cm diameter x 7.5 cm high) with a screened lid (Fig. 3-2C).

On the following day, 9 to 10 germinated corn seeds were put in the container as food for

the emerging larvae.

Larval D. balteata were raised on germinated corn seeds in containers designed to

maintain sufficient moisture for seed growth without drowing the larvae or covering them

with soil. A preassembled germination paper with a wick stapled on each end was placed









at the botton of a rectangular plastic container (32.5 x 17.2 x 10 cm) and covered with a

single layer of pregerminated corn seeds. The seeds were covered with wet paper towel

and the container was placed on the top of a water-filled tray in such a way that the two

wicks were suspended in the water. Pregerminated corn seeds were prepared by soaking

dried seeds overnight in a Clorox solution (16 ml/L of water). They were rinsed with

deionized water the following morning and stored in a refrigerator until needed. These

larval rearing containers were covered with a screened lid. One-day-old emerged larvae

on germinated corn seeds were next transferred to the rectangular rearing containers

stocked with 3-d-old germinated corn seedlings and kept at 27 + 2 C with a photoperiod

of 14:10 (L:D) h in a rearing room (Fig. 3-2D, E). After 1 wk of larval rearing in these

containers, the larvae were transferred to a second container with germinated corn to

complete their larval development (Fig. 3-2F, G, H).

Two days after putting larvae in the second container, third instar larvae were

collected into a container (18 cm x 7.5 cm high) filled with moistened and autoclaved

MetroMix 220 (Fig. 3-21) to allow for pupation and adult emergence. The container was

covered with a dampened towel to retain moisture. After 10 d, the emerged adults were

transferred into the screen cage mentioned above (Fig. 3-2J).

Artificial Diet Preparation

Dry mix for artificial diet is commercially available and has been shown to support

the adult stage ofD. balteata (Creighton and Cuthbert 1968). All materials required for

preparing and dispensing the diet were thoroughly sanitized with sodium hypochlorite

solution (Clorox, Oakland, CA) diluted 1:5 with deionized water. A 100-ml quantity of

southern corn rootworm artificial diet (Bio-Serv, Frenchtown, NJ) (Creighton and

Cuthbert 1968) was prepared as follows. Sterile deionized water (100 ml) and agar (1.74









g) were heated on a hot plate to boiling. Once the agar had cooled to approximately 40

C, KOH solution (1 ml) and diet dry mix (14.91 g) were added to it and thoroughly

mixed to avoid the formation of lumps. The liquid diet was dispensed into two glass Petri

dishes (9 cm diameter). The diet was allowed to cool before the Petri dishes were covered

with glass lids. The Petri dishes were wrapped completely in plastic wrap and aluminum

foil, and stored in a refrigerator (4-6 C) for up to 3 h.

Latex Collection and Solvent Extraction

Latex (70 il) was collected from the bases (where leaf lamina joins the stem) of

young and middle-aged leaves of individual plants, sites where there was a rapid

exudation of latex upon cutting (Fig. 3-3). The cuts were made using a disposable scalpel

blade (Feather, Osaka, Japan). The latex was collected using a silanized 100-tl glass

capillary tube inserted into a microdispenser (Drummond Scientific Company, Broomall,

PA) 60 s after the leaf base was cut.

In a pilot study, I incorporated fresh latex into the artificial diet for D. balteata

adults at two concentrations (0.1 and 0.2%) and recorded diet consumption by D. balteata

adults to investigate the potential of Valmaine latex as a mechanism of multiple insect

resistance. Latex did not deter feeding ofD. balteata adults when presented in this

manner. Therefore, in this study, I applied freshly extracted latex from either Valmaine or

Tall Guzmaine to artificial diet and confined D. balteata adults under choice and no-

choice conditions to investigate the possible deterrent role of latex against beetle feeding.

A 1.5-cm-diameter cork borer was used to punch out disks (1 cm thick) from cooled

artificial diet. Latex (70 il) from an individual plant was applied, immediately after

collection, onto the top surface and sides of a diet disk using a microdispenser.









I chose artificial diet as a substrate for application of the latex because it kept latex

moist for a longer time by providing more moisture compared to lima bean leaves. In

addition, latex treated diet disks facilitated the direct exposure ofD. balteata adults to

latex. As the diet disks were totally covered with latex on all sides, it somewhat simulated

the natural situation where an insect gnawing a lettuce plant is directly exposed to latex.

Four different solvent combinations, i.e., water, water:methanol (20:80),

water:methanol (50:50), and methylene chloride were used to extract chemical

constituents of latex (Fig. 3-5A). Latex (70 [l) was collected from an individual plant in

the same way as explained above and immediately dissolved in 10 times volume of the

solvent (Fig. 3-4). After dissolution, samples were centrifuged at 1200 xg for 20 min and

supernatant was collected (Fig. 3-5B, C). The supernatant was reduced down to 1/10

volume by evaporating with nitrogen gas. An amount of extract, equivalent to 70 [l latex,

was applied to each diet disk for use in the following bioassays (Fig. 3-4).

Bioassay Conditions

For all experiments described below, an experimental unit consisted of two diet

disks and three pairs of unfed D. balteata adults within a plastic ventilated container (10

x 10 x 8 cm). Unfed adults that had emerged within 48 h of the start of the experiment

were used in all tests. The diet disks were placed on the bottom of the container and

beetles were allowed to feed on the diet for 16 h. Each experimental unit was replicated

15 times. The experiments were carried out at 25 + 1C in a laboratory under a

photoperiod of 14:10 (L:D) h. In all tests, the number of adults feeding on each diet disk

was recorded 15, 30, 60 and 90 min after their release into the bioassay chambers.









Choice Tests and No-choice Tests with Fresh Latex

Choice tests were conducted to determine whether D. balteata adults showed a

preference between diet disks treated with latex from Valmaine or Tall Guzmaine. Three

treatment combinations were studied: latex from Valmaine versus latex from Tall

Guzmaine, latex from Valmaine versus control (untreated diet without latex), and latex

from Tall Guzmaine versus control (Fig. 3-6A). Three treatments (latex from Valmaine,

latex from Tall Guzmaine, and control) also were studied in a no-choice situation, with

each experimental unit containing two disks of the same treatment (Fig. 3-6B).

Dry weight of diet consumed in a 16-h period was calculated for comparison

among the treatments. To determine dry weight, an additional 10 diet disks from each

treatment (Valmaine latex-treated, Tall Guzmaine latex-treated and control) were

weighed individually (disk fresh weight) before they were put into an oven at 50 + 50C.

After 3 d, these diet disks were reweighed individually (disk dry weight). A dry/fresh

weight ratio was calculated for each diet disk and averaged over the 10 disks. The diet

fresh weight was determined for each disk for each treatment prior to the start of each

experiment. After 16 h of exposure to beetle feeding, the diet disk was dried in the oven

for 3 d as above, reweighed and then multiplied by the corresponding average dry/fresh

weight ratio. The dry weight of diet consumed was calculated as the difference between

initial and final dry weights.

Choice Tests Using Latex from Young and Mature Leaves

Choice tests were conducted to determine whether D. balteata adults exhibit any

preference between diet disks treated with latex from young or mature leaves of either

Valmaine and Tall Guzmaine. Two treatment combinations were studied: latex from

young leaves versus latex from mature leaves of Valmaine and latex from young leaves









versus latex from mature leaves of Tall Guzmaine. The dry weight consumption of young

and mature latex-treated diets of each cultivar in 16 h was recorded as described above.

Total diet consumed per three pairs of adults for 16 h was calculated by adding

consumption of the two diet disks in each container in each treatment.

No-Choice Tests Using Latex Extracts

Fifteen treatments were studied: five for Valmaine latex dissolved in water,

water:methanol (20:80, % v/v), water:methanol (50:50), methylene chloride, and fresh

latex without solvent; five for Tall Guzmaine latex dissolved in water, water:methanol

(20:80), water:methanol (50:50), methylene chloride, and fresh latex without solvent; and

five for control the four solvent combinations without latex and untreated diet. Each

experimental unit contained two disks of the same treatment. The dry weights of

Valmaine- and Tall Guzmaine-extract treated and control diets disks consumed in 16 h

were calculated as above.

Beetle Behavior in Response to Contacting Latex

Observations were made on beetle behavior in response to contacting latex and

latex extracts on diet disks in the choice and no-choice tests described above. In addition,

freshly collected latex (1 il) from both Valmaine and Tall Guzmaine plants was applied

to mouthparts of beetles (10 for each) using a microdispenser. Using a microscope,

salivation by treated beetles was observed immediately after latex application and

mobility of mouthparts was observed after 24 h to distinguish if toxic constituents or

stickiness contributed to the feeding deterrence of Valmaine lettuce. Individual beetles

also were confined on young leaves of either Valmaine or Tall Guzmaine (10 plants for

each cultivar) and observed for 90 min using a microscope to closely observe their

feeding behavior in response to contacting latex during test bites.









Statistical Analysis

For all choice and no-choice tests using latex, the number of adults feeding on diet

15, 30, 60 and 90 min after release was analyzed as a repeated measures design using

Proc GLIMMIX (SAS Institute 2003). In each choice test (Valmaine versus Tall

Guzmaine, Valmaine versus control, and Tall Guzmaine versus control), data on number

of adults feeding were analyzed as a 2 x 4 factorial design separately, in which latex was

treated as one factor with two levels, and time interval after beetle release was treated as

the other factor with four levels (15, 30, 60 and 90 min). In no-choice tests, data on

number of adults feeding were analyzed as a 3 x 4 factorial design, in which latex was

treated as on factor with three levels (Valmaine, Tall Guzmaine and control), and time

interval after beetle release was treated as the other factor with four levels. Both variables

(latex and time interval) were fixed. Fifteen groups of six beetles (i.e., replications) were

randomly assigned to each level of latex, meaning that beetles were nested within latex

levels. Each group of six beetles was tested four times (levels of time interval). The

model was number of beetles feeding = [latex] [replications(latex)] [time interval]

[latex*time interval]. The error degree of freedom for latex effect was calculated as levels

of latex(replications 1). The error degree of freedom for time interval effect and

interaction was calculated as levels of latex(levels of time interval 1) replicationss 1).

In no-choice tests using latex extracts, the data on number of beetles feeding was

analyzed using Proc GLM (SAS Institute 2003) separately at each time interval with latex

extract as a fixed effect and replications as a random effect. The error degree of freedom

for latex extract effect was calculated as (levels of latex extract -1)(replications -1).

The dry weights of Valmaine, Tall Guzmaine and control diets consumed in 16 h

were analyzed by paired t-tests using Proc MEANS (SAS Institute 2003) for all choice









tests and by ANOVA using Proc GLM with latex as a fixed effect and replications as a

random effect (SAS Institute 2003) for all no-choice tests. The total dry weight consumed

by adding consumption of the two diet disks in each chocie test using latex from young

and mature leaves including control disks was also analyzed by ANOVA using Proc

GLM with latex as a fixed effect and replications as a random effect (SAS Institute

2003). The error degree of freedom for latex/latex extract effect was calculated as (levels

of latex/ latex extract -1)(replications -1). Tukey's honestly significant difference (HSD)

test with a significance level of a = 0.05 (SAS Institute 2003) was used for post hoc

means separation.

Deterrence coefficients (relative and absolute) were calculated (Nawrot et al. 1986)

based on the amount of diet consumed. All the data from both choice and no-choice tests

were pooled and used to determine coefficients using the following equations:

Relative (R) = [(C T) / (C + T)] x 100 (Choice Test)

Absolute (A) = [(CC TT) / (CC + TT)] x 100 (No-choice Test)

where C and CC are the consumption of control diet (without latex) in choice and

no-choice tests, respectively; and T and TT are the consumption of latex-treated diet

(Valmaine or Tall Guzmaine) in choice and no-choice tests, respectively. The deterrent

activity of the latex-treated diets was expressed by the total coefficient of deterrence (D =

A + R). The deterrence coefficients were analyzed by two-sample t-tests using PROC

TTEST (SAS Institute 2003).









Results


Latex Choice and No-Choice Tests

Treatment of latex had significant effect on the number of insects feeding in all

three choice tests, Valmaine (Val) versus Tall Guzmaine (TG) (F = 64.83; df = 1, 28; P=

0.0001), Valmaine versus control (F = 99.27; df = 1, 28; P = 0.0001), and Tall Guzmaine

versus control (F = 5.68; df = 1, 28; P = 0.0241). Beetles avoided feeding on diet treated

with Valmaine latex (Fig. 3-6A; 3-7A, B). The number of insects feeding on diet treated

with Valmaine latex was negligible compared to the number feeding on diet treated with

Tall Guzmaine latex (Fig. 3-7A) and control diet (Fig. 3-7B). The number of insects

feeding increased over time (i.e., 15, 30, 60 and 90 min) in all choice tests (Val vs. TG: F

= 7.28; df = 3, 84: P = 0.0002, TG vs. control F= 9.83; df = 3, 84: P = 0.0001, Val vs..

control: F = 24.87; df = 3, 84: P = 0.0002) (Fig. 3-7A, C). Significant interactions were

found between latex treatment and time interval in the choice tests involving Valmaine

and Tall Guzmaine (F = 8.56; df = 3, 84; P = 0.0001) and Valmaine and the control diet

(F = 28.47; df = 3, 84; P = 0.0001). In contrast, there was no significant interaction found

in the choice test between Tall Guzmaine latex-treated diet disks and control disks (F =

1.44; df = 3, 84; P = 0.2374) (Fig. 3-7C). Beetles consumed significantly less diet treated

with Valmaine latex (Table 3-1). Beetles ate 2.9 times more on Tall Guzmaine latex

treated diet than diet treated with Valmaine latex in a choice between Valmaine and Tall

Guzmaine. Beetles also consumed 4.5 times more control diet than diet treated with

Valmaine latex in a choice between Valmaine and control. Beetles also consumed 1.5

times less diet treated with Tall Guzmaine than control diet.

In no-choice tests, latex also had significant on the number of insects feeding on

diets (F = 109.46; df = 2, 42; P = 0.0001). Significantly fewer insects fed on diet treated









with Valmaine latex than on Tall Guzmaine latex-treated and control diets (Figs. 3-6B, 3-

8). No significant interaction was found between latex treatment and time interval (F =

1.74; df = 3, 126; P = 0.1179). Beetles consumed 4.7 and 6.3 times more Tall Guzmaine

latex-treated and control diets, respectively, than Valmaine latex-treated disks (F=

168.31; df = 2, 42; P = 0.0001) (Table 3-1).

Valmaine latex exhibited strong deterrence against beetles in both choice and no-

choice bioassays (Table 3-2). Both relative and absolute coefficients of deterrence for

Valmaine latex-treated diets were significantly higher than those for Tall Guzmaine

latex-treated diets. The total coefficient of deterrence of Valmaine latex was 3.9 times

higher than that of Tall Guzmaine latex.

Choice Tests Using Latex from Young and Mature Leaves

In Valmaine choice test, latex significantly affected the number of insects feeding

on diet (F = 61.87; df = 1, 28; P = 0.0001), but it was not significantly affected by latex

treatment in Tall Guzmaine choice tests (F= 1.84; df= 1, 28; P = 0.812). Significantly

fewer insects fed on diet treated with latex from young leaves than on diet treated with

latex from mature leaves of Valmaine (Figs. 3-9, 3-10A). Adult preference for diet

treated with latex from mature leaves of Valmaine increased significantly with time (F=

30.95; df = 3, 84; P = 0.0001) (Fig. 3-10A). In the Tall Guzmaine latex choice test, the

number of beetles feeding on diet disks treated with latex from young leaves did not

differ significantly from that on disks treated with latex from mature leaves (Figs. 3-9, 3-

10B). The number of beetles feeding on both Tall Guzmaine diets increased significantly

with time (F= 39.44; df = 3, 84; P= 0.0001) (Fig. 3-10B).

Beetles consumed 7.2 times more diet treated with latex from mature Valmaine

leaves than treated with latex from young Valmaine leaves (Table 3-3). Diet consumption









did not differ significantly between diet disks treated with latex from young and mature

leaves of Tall Guzmaine. The total diet consumed in the Valmaine latex choice test (sum

of the consumption on the two disks) did not differ significantly from the amount eaten in

the Tall Guzmaine latex choice test but was significantly less than the amount consumed

in the control diet test.

No-Choice Tests Using Latex Extracts

Water extracts of both Valmaine and Tall Guzmaine were yellow in color, but the

color of the Valmaine extract was more intense than that of the Tall Guzmaine extract

(Fig. 3-5C). Water:methanol (20:80) extracts of both cultivars were colorless. The

water:methanol (50:50) extract of Tall Guzmaine was colorless, but it was yellow in the

case of Valmaine. Methylene chloride extracts of both cultivars were white in color and

sticky.

Treatment of latex extracts had significant on the number of insect feeding on diet

after 15 min (F= 11.97; df = 14, 196; P = 0.0001); 30 min (F= 12.60; df = 14, 196; P=

0.0001); 60 min (F= 24.42; df = 14, 196; P = 0.0001); and 90 min of release (F= 31.93;

df= 14, 196; P = 0.0001). Significantly fewer insects fed on diet disks treated with a

water:methanol (20:80) extract of Valmaine latex than on diets treated with all other

Valmaine and Tall Guzmaine latex extracts, as well as all the control diets (Figs. 3-11, 3-

12). In addition, diet consumption was also significantly affected by the latex extract

treatment (F = 95.01; df= 14, 196; P = 0.0001). Beetles consumed significantly less diet

treated with water:methanol (20:80) extract of Valmaine latex than diet treated with any

other latex extract or control diet (Fig. 3-13). The number of insects feeding on disks

(Fig. 3-12) and amount consumed (Fig. 3-13) on diet disks treated with the









water:methanol (20:80) extract of Valmaine latex were equivalent to those values for diet

treated with fresh Valmaine latex.

Beetle Behavior in Response to Contacting Latex

In latex choice tests, beetles flew immediately to the roof and walls of the

container whenever they approached the Valmaine latex-treated diet disk, whereas the

beetles started feeding on the Tall Guzmaine latex-treated diet disk whenever they

approached it. In latex no-choice tests, beetles generally returned to the roof and walls of

the container after approaching several times the Valmaine latex-treated diet disks. The

behavior of the beetles on diet treated with water:methanol (20:80) extracts of Valmaine

latex was similar to that for diet treated with pure Valmaine latex. Before biting a latex-

treated diet disk, beetles inspected it at a close range, antennating and palpating it. In

cases where the beetles landed directly on a disk, they appeared to sense the deterrent

with their tarsi, even before antennating and palpating the disk, and flew back to the

container walls immediately. Beetles performed frequent and more vigorous grooming of

antennae and tarsi by passing them through mouthparts after contact with Valmaine latex

compared to Tall Guzmaine latex. Further tarsal grooming was also done by scraping the

legs on the elytra.

Beetles salivated more when Valmaine latex was applied to their mouthparts with

a microdispenser compared to Tall Guzmaine latex. Mandibles and maxillae were not

gummed up and were moving freely 24 h after application of either Valmaine or Tall

Guzmaine latex, but there were some traces of dried latex on the labium and tarsi. During

test bites on a lettuce leaf and contact with the exuded latex, the beetles moved away

from the feeding site very quickly and started test bites somewhere else. The response

was very vigorous on Valmaine. On Tall Guzmaine, beetles resumed test bites in close









proximity to the previous bites, but on Valmaine tests bites were much farther away from

the previous bites.

Discussion

Evidence presented here indicates that resistance found in Valmaine romaine

lettuce against D. balteata is due to latex. Adult D. balteata were found more frequently

on diets treated with latex from Tall Guzmaine than on diets treated with Valmaine latex

in both choice and no-choice tests. The alighting behavior of the beetles observed in my

choice and no-choice tests suggests that contact chemosensilla are present on their

antennae, palps and tarsi (Chapman 2003). These types of chemosensilla have been

reported in other chrysomelids, such as on the antennae of the cabbage stem flea beetle,

Psylliodes chrysocephala L. (Isidoro et al. 1998), maxillary appendages of the western

corn rootworm, D. virgifera virgifera LeConte (Chyb et al. 1995, Eichenseer and Mullin

1996), and tarsomeres of the Klamath beetle, Chrysolina brunsvicensis Gravenhorst

(Rees 1969). Such chemsensilla have been found to discriminate between

phagostimulants and phagodeterrents. Antennal and tarsal grooming, similar to that

observed by us with D. balteata, has been reported in the crucifer flea beetle, Phyllotreta

cruciferae Goeze as an important part of the prefeeding behavior for recognizing host and

non-host crucifers (Henderson et al. 2004).

Adult D. balteata consumed significantly less Valmaine latex-treated diet

compared to Tall Guzmaine latex-treated diet in both choice and no-choice tests. Huang

et al. (2003b) reported that latex from both Valmaine and Tall Guzmaine was very

deterrent to beetle feeding when applied on lima bean leaves. I believe that Tall

Guzmaine latex in the studies of Huang et al. (2003b) showed very high deterrence due to

changes in its chemical properties (possibly oxidation) after drying on the lima bean leaf









surface. In my studies, the precisely defined quantities of latex (70 .il) applied to diet

disks did not dry significantly within the 16-h exposure period to beetles due to moisture

from the artificial diet, which perhaps prevented changes in the chemical properties of the

latex. Both cultivars produce latex upon wounding but the much higher coefficient of

deterrence for Valmaine latex compared to Tall Guzmaine latex observed in my study

argues that Valmaine latex is more deterrent than Tall Guzmaine due to its physical

or/and chemical properties. These properties may be due to the original chemicals

produced by the plants or new chemicals produced by the action of certain plant

enzymes, such as phenylalanine ammonia lyase, polyphenol oxidase and peroxidase.

Valmaine also partially or totally lost its resistance in feeding bioassays using detached

leaves or leaf disks, where latex exudation was greatly reduced (Huang et al. 2003c). This

further provided evidence about the defensive role of latex in resistant Valmaine.

The strong deterrent activity of Valmaine latex extracted with water:methanol

(20:80) provides compelling evidence for the chemical basis of resistance in Valmaine

against D. balteata. The ability of water:methanol (20:80) to extract deterrent

constituents from Valmaine latex suggests that moderately polar compounds in Valmaine

latex account for its feeding deterrence. The defensive role of latex due to it chemical

constituents against insects has been reported in many plant systems, such as milkweed

(Dussourd and Hoyle 2000), mulberry (Konno et al. 2006), papaya (Konno et al. 2004),

and chicory (Rees and Harborne 1985). Various organic compounds, such as phenolics

and terpenoids have been reported in latex ofLactuca spp. (Crosby 1963, Gonzalez 1977,

Cole 1984, Sessa et al. 2000), and their defensive role as phytoalexins has been reported

against plant diseases (Bennett et al. 1994, Bestwick et al. 1995).









Latex from young leaves of Valmaine strongly deterred the feeding of D. balteata

adults in a choice between diets treated with latex from young and mature leaves. Sethi et

al. (2006) found that latex from young and mature leaves differed in terms of milkiness

and viscosity. Such differences in milkiness arise due to differences in the refractive

indices of the dispersing particles (mainly terpenoids) and the dispersing medium (Esau

1965, Fahn 1990). Thus, latex from young leaves may be richer in dispersing particles,

and the relatively higher amount of dispersing particles may have a specific purpose

related to plant defense. Young leaves are typically better defended than mature leaves

due to the presence of higher quantities of latex and its associated chemical components

(Swain 1977, Spilatro and Mahlberg 1986). In the chicory plant, Cichorium intybus L.,

sesquiterpene lactones were present in the highest amounts in the most actively growing

regions of the plant and possessed antifeedant properties against Schistocerca gregaria

(Orthoptera: Acrididae) (Rees and Harborne 1985). Young vines of sweetpotato, Ipomoea

batatas (L.) Lam., possessed more latex and exhibited less damage due to the sweetpotato

weevil, Cylasformicarius (F.) (Coleoptera: Curculionidae) than mature vines (Data et al.

1996). Latex exudation is higher in growing regions than in mature regions of great

bindweed, Calystegia silvatica (Kit.) Griesb (Condon and Fineran 1989).

Anatomy of laticifers changes during the course of their ontogeny (Olson et al.

1969). The number of laticifers and their contents decrease with increasing proximity to

roots (Condon and Fineran 1989, Monacelli et al. 2005). In mature leaves, the protoplast

of laticifers degenerates near senescence indicating a low level of metabolism (Fineran

1982, 1983). Fusion of latex particles has also been seen in mature leaves when much of

the latex has already vanished (Dickenson 1963, Heinrich 1967, Fineran 1982). Plug-like









masses of callose have been reported at the bases of mature leaves and no or much

reduced amounts of latex exude when such leaves are severed, completely or partially,

from the plant (Spencer 1939). Young leaves have discrete files oflaticifers separated by

end walls, while laticifers differentiate by breakdown of end walls in mature leaves

(Condon and Fineran 1989). Thus, laticifers of young leaves may have more turgor

pressure resulting in profuse latex exudation compared to mature leaves, making it more

likely that insect mandibles will be exposed to latex during test bites on intact leaves.

My data support a hypothesis that latex has a definite role in the expression of

resistance in Valmaine lettuce to D. balteata, and differences in latex chemistry between

the two cultivars may account for this. Future research on the isolation of latex

constituents and their biological activity is required to better understand the mechanism

of resistance in Valmaine lettuce. This knowledge may contribute to the development of

new cultivars expressing insect resistance along with superior horticultural traits through

conventional and transgenic approaches.


















Figure 3-1. Wounding of lettuce releases a milky fluid called latex.


Orw



















-,. /


J


4-


4-


Figure 3-2. Colony rearing of D. balteata. See text for description of each stage of colony maintenance.


r































Figure 3-3. Collection of latex from romaine lettuce, application on artificial diet disk and
bioassay setup.









Lettuce plant






Applied on
diet disk
(70 pl)
F 1


SLatex
(70 pl)


Supernatant
reduced =
with N2


4


Dissolved in
solvent (1:10)1


Centrifuged
@ 1200 g
for 20 min

I
Supernatant
collected


L .U


Figure 3-4. Scheme of latex solvent extraction.


=a













Water: Methanol Water: Methanol
Water (50:50) (20:80)


Val TG al TG Val TG


Water: Methanol
Water (50:501


Methylene
chloride


Water


Water: Methanol Water: Methanol
50:5s0) r(2:8o0


Methylene
chloride


Val TO Val TO al TG al TG Ial TG


Water: Methanol
r20:801


Figure 3-5. Latex dissolution in different solvents. A) Latex dissolved in different
solvents, B) pellet settled down after centrifugation, and C) supernatant
collected after centrifugation.


1


W1m









Choice tests


B No-choice test


Figure 3-6. Feeding bioassays using fresh latex. A) Choice tests: Valmaine (Val) versus
Tall Guzmaine (TG), Valmaine versus control, Tall Guzmaine versus control.
B) No-choice tests: Valmaine (Val), Tall Guzmaine (TG), control.












- Val
TG


A Val
Control


a
bb
b


15 30 60 90


b

c c c


15 30 60 90


B TG
Control
a
ab ab ab


ilf


15 30 60


Time after release (min)


Figure 3-7. Mean number ofD. balteata adults feeding on artificial diet disks treated with latex from resistant Valmaine (Val), disks
treated with latex from susceptible Tall Guzmaine (TG), and control diet disks in choice tests. Error bars indicate SEM.
Bars topped with different letters within same panel differ significantly at the 0.05 level (Tukey's HSD test).










6 Val

_z I TG
S5 Control a a
Sab a
4 ab ab
b- b
0b
15 30 60 903
C'
c- 2


E
z C C C C
0-
15 30 60 90

Time after release (min)


Figure 3-8. Mean number ofD. balteata adults feeding on two artificial diet disks treated
with latex from resistant Valmaine (Val), disks treated with latex from
susceptible Tall Guzmaine (TG), and control diet disks in no-choice tests.
Error bars indicate SEM. Bars topped with different letters differ significantly
at the 0.05 level (Tukey's HSD test).








Valmaine


Tall Guzmaine


Control


Young Mature Young Mature

Figure 3-9. Choice tests using D. balteata adults on two artificial diet disks treated with
latex from young and mature leaves of the same cultivar.












-6
m; Val-Young
.- 5
SVal-Mature


A TG-Young
TG-Mature


a

b
C
c T"'

d cd d
d d d


15 30 60 90


ab a
bc abc abc

cd


de


15 30 60 90


Time after release (min)

Figure 3-10. Number ofD. balteata adults feeding on artificial diet disks treated with
latex from young or mature leaves of resistant Valmaine (Val) (A) and
susceptible Tall Guzmaine (TG) (B) in choice tests. Error bars indicate SEM.
Bars topped with different letters within same panel differ significantly at the
0.05 level (Tukey's HSD test).


Cn
t3
U,


L1
Cn
.0
EO
z


















Val Latex Val W


TG -W:M (50:50)


TG -W:M (20:80)


A."j

T I II-


TG -MeCI


Figure 3-11. No-choice tests using D. balteata adults when both the disks were smeared with either Valmaine latex extract or Tall
Guzmaine latex extract. W Water, M Methanol, MeCl Methylene chloride. Red circle indicates the most deterrent
extract comparable to fresh latex.


TG W










A B

MeCI ab ab
W:M (20:80) ab ab
W:M (50:50) ab ab
Water a a
Control ab a

TG-MeCI ab ab
TG-W:M (20:80) ab ab
TG-W:M (50:50) b ab
TG-Water ab ab
TG-latex ab ab
Val-MeCI ab ab






C D

MeCI ab abc
W:M (20:80) ab abc
W:M (50:50) ab abc
Water a a
Control ab ab

TG-MeCI ab abc
TG-W:M (20:80) ab abc
TG-W:M (50:50) ab
TG-Water ab abc
TG-late x ab abc

Val-MeCI b bc
VaI-W:M (20:80) C d
VaI-W:M (50:50) 0 ab bc
Val-Water Hab abc
Val-latex t c F d

0 1 2 3 4 5 6 0 1 2 3 4 5 6
Number of insects feeding / 2 disks

Figure 3-12. Mean number ofD. balteata adults feeding on two artificial diet disks
treated with latex extracts from resistant Valmaine (Val) and susceptible
Tall Guzmaine (TG), and controls in no-choice test. Error bars indicate
SEM. Bars topped with different letters within same panel (A, B, C, D)
differ significantly at the 0.05 level (Tukey's HSD test). A) 15 min, B) 30
min, C) 60 min, and D) 90 min.












MeCI
W:M (20:80)
W:M (50:50)
Water
Control


TG-MeCI
TG-W:M (20:80)
TG-W:M (50:50)
TG-water
TG-latex


a
a
a
ab
a


c
cd

cd
cd


Val-MeCI cd
Val-W:M (20:80) f
Val-W:M (50:50) cd
Val-water de
Val-latex ef

0 10 20 30 40 50 60
Diet consumed (mg)


Figure 3-13. Dry weight of diet consumed by six D. balteata adults in 16 h when both
diet disks were treated with Valmaine (Val) or Tall Guzmaine (TG) latex
extracts under no-choice situations. Error bars indicate SEM. Bars topped
with different letters differ significantly at the 0.05 level (Tukey's HSD
test).


Ii


I


1









Table 3-1. Dry weight consumption of diet disks treated with Valmaine (Val) or Tall
Guzmaine (TG) latex under choice and no-choice tests by six D. balteata
adults in 16 h.
Mean diet consumed SEM (mg)

Tests Treatment P value

Val latex TG latex Control

Choice*
Val vs. TG 5.4 + 0.5 15.5 + 0.7 0.0001

Val vs. Control 5.5 + 0.5 -24.7 0.5 0.0001

TG vs. Control 14.4+ 0.5 21.9 0.6 0.0001

No-Choicet 7.3 + 0.4c 34.6 1. lb 46.2 2.4a 0.0001

* P value from paired t-test. TMeans SEM followed by different letters in no-choice test differed
significantly (P < 0.05) using ANOVA (F = 168.31; df = 2, 42; P = 0.0001) and Tukey's HSD test.









Table 3-2. Feeding deterrent activity of latex against D. balteata adults when artificial
diet disks were treated with latex from either resistant Valmaine (Val) or
susceptible Tall Guzmaine (TG) in choice and no-choice tests.


Relative

63.6

20.7

0.0001


Deterrence coefficients
Absolute


72.7

14.4


0.0001


Total

136.3

35.0

0.0001


P value from two sample t-test.


Latex


P value









Table 3-3. Dry weight of diet consumed by six D. balteata adults in 16 h when given a
choice between diet disks treated with latex from either young or mature
leaves of resistant Valmaine or susceptible Tall Guzmaine lettuce cultivars.
Total diet
Cultivar Choice Diet consumed (mg)A consu d (et
consumed (mg)t


Valmaine


Tall Guzmaine


Young latex-treated diet vs.

mature latex-treated diet

Young latex-treated diet vs.

mature latex-treated diet


3.7 + 0.6b

26.1 + 1.9a

18.1 + 2.0a

20.5 + 2.2a


29.8 2.2 b


38.7 3.9ab


Control* 50.1 + 5.2a

*Both disks were untreated in control diet. A Means SEM followed by different letters within cultivar
differed significantly using paired t-test. TMeans SEM followed by different letters within column
differed significantly (P < 0.05) using ANOVA (F = 6.69; df = 2, 42; P = 0.0030) and Tukey's HSD test.









CHAPTER 4
BANDED CUCUMBER BEETLE (COLEOPTERA: CHRYSOMELIDAE)
RESISTANCE IN ROMAINE LETTUCE: UNDERSTANDING LATEX CHEMISTRY

Introduction

Host plant resistance is an important potential component of any integrated pest

management program for a crop pest. Many plants produce compounds that mediate host

plant suitability to insect herbivores (Rosenthal and Berenbaum 1991). These biologically

active compounds are frequently present in viscous secretions, such as latex or resin,

within specialized canal systems separate from the vascular system (Fahn 1979, Metcalf

and Chalk 1983, Farrell et al. 1991). Thus, insect mouthparts get exposed to these

compounds during test bites due to copious flow of latex at the damage site (Farrell et al.

1991). The common components of latex are polyisoprene, proteins, amino acids, fatty

acids, tetracyclic triterpenoids, glycerides, waxes, starch, flavonoids, alkaloids, water,

organic and inorganic salts and many unidentified compounds (Nielson et al. 1977,

Spilatro and Mahlberg 1986, Gazeley et al. 1988). Examples of some compounds found

in latex with activity against different insect pests include diterpenes (Evans and Schmidt

1976, Noack et al. 1980) and nonprotein amino acids in Euphorbia (Haupt 1976),

cardenolides in milkweed (Seiber et al. 1982, Nishio et al. 1983), alkaloids in poppy

(Roberts 1987, Matile 1976) and mulberry (Konno et al. 2006), sesquiterpene lactones in

chicory (Rees and Harborne 1985), and cysteine proteases in papaya and fig (Konno et al.

2004). However, latex of most of the laticiferous species within the Apocynaceae,

Compositae, Euphorbiaceae, Musaceae, Papaveraceae, and Urticaceae has not been

chemically characterized.

A cultivar of romaine lettuce (Lactuca sativa L.), 'Valmaine', possesses insect

resistance against leafminer, Liriomyza trifolii (Burgess) (Nuessly and Nagata 1994),









banded cucumber beetle, Diabrotica balteata LeConte (Huang et al. 2002) and two

lepidopterans, Trichoplusia ni (Hubner) and Spodoptera exigua (Hubner) (Chapter 2,

Sethi et al. 2006). Latex from Valmaine applied to artificial diet deters feeding of D.

balteata. Further, a crude extract prepared by dissolving Valmaine latex in a

water:methanol mixture (20:80, % v/v) also strongly inhibits beetle feeding when applied

to the surface of artificial diet (Chapter 3, Sethi et al. 2007). This suggests that Valmaine

latex contains deterrent compounds which are responsible for its resistance against

multiple species of mandibulate insects.

Here, I describe the isolation and characterization of deterrent compounds from

Valmaine latex against D. balteata adults using bioassay-directed fractionation.

Materials and Methods

Plants and Insects

Seeds of the romaine lettuce cultivar, Valmaine were germinated by putting them

on moist filter paper in a Petri dish. Germinated seeds were planted in soil-less media

(MetroMix 220 potting mixture, Grace Sierra, Milpitas, CA) and healthy seedlings were

transplanted into 15-cm-diameter plastic pots after 2 wk in a greenhouse with natural

light at a mean temperature of 27 C (22 to 300C) and 68% mean R.H. (48 to 93%).

Plants were fertilized with 10 ml of a 10 g/1 solution of Peters 20-20-20 (N-P-K) (W.R.

Grace, Fogelsville, PA) once a week. Bush lima bean (Phaseolus lunatus L.) plants of the

'Fordhook 242' cultivar (Illinois Foundation Seeds, Champagne, IL) were grown in

transplant trays and fertilized with the same solution used for lettuce plants.

A wild population of D. balteata adults was collected from spiny amaranth,

Amaranthus spinosus L. and primrose willow, Ludwigiaperuviana L. in Belle Glade, FL

in 2003. A colony was established by raising adults on lima bean leaves and slices of









sweet potato tubers, and larvae were fed on corn seedling roots as per the methods of

Huang et al. (2002) (Chapter 3). Wild individuals were added to the colony in 2005 and

2006 to increase genetic diversity. Unfed adults that had emerged within 48 h of the start

of the experiment were used in all bioassays.

Assay for Feeding Deterrence

Extracts/fractions from latex obtained as described below were bioassayed on

artificial diet for feeding deterrence towards D. balteata adults under no-choice

conditions. The southern corn rootworm artificial diet (Bio-Serv, Frenchtown, NJ), and

disks of diet for use in the assays, were prepared as described in Chapter 3 (Sethi et al.

2007). An experimental setup consisted of two diet disks placed on the bottom of a

plastic container (10 x 10 x 8 cm) with screen lid and three male-female pairs of beetles.

Both diet disks in each container were treated with the same kind of extract/fraction. The

beetles were allowed to feed on the diet for 16 h. The experiments were carried out at 25

+ 1C in a laboratory under a photoperiod of 14:10 (L:D).

In all bioassays, the number of adults feeding on two diet disks was recorded 90

min after their release into the container. The dry weights of diet consumed in 16 h were

also recorded. To compensate for the weight associated with moisture loss during the

feeding tests, individual fresh weights of 10 diet disks were recorded before they were

dried in an oven at 50 50C. Individual dry weights of these disks were recorded after 3

d and an average dry/fresh weight ratio was calculated. The diet disks for bioassays were

weighed prior to the bioassay setup. At the end of the experiment, the remaining portions

of the disks were reweighed after drying for 3 d in the oven. The amount of dry weight of

diet consumed was calculated as the difference between initial and final dry weights. Dry









weights of diet disks consumed by the beetles were computed by multiplying fresh

weight by the average dry/fresh weight ratio.

Latex Collection and Crude Extract Preparation

Cuts were made near the leaf-bases of young and middle-aged leaves of lettuce

plants (9-10 true-leaf stage) using a disposable scalpel blade (Feather, Osaka, Japan).

Fresh latex (70 [il) was collected from each plant using a 100-tl silanized glass capillary

tube inserted into a microdispenser (Drummond Scientific Company, Broomall, PA) and

immediately dissolved in 10x volume of water:methanol (20:80) mixture. After

dissolution, samples were centrifuged at 1200 xg for 20 min and then the supernatant was

collected. The supernatant (hereafter termed crude extract) was concentrated to 0.1 x

volume (the original latex volume) by evaporation under a gentle stream of nitrogen

(Chapter 3, Sethi et al. 2007).

Fractionation of Crude Extract Using Reversed-Phase (C-18) Cartridge

Reversed phase separations involve a polar or moderately polar sample matrix

(mobile phase) and a nonpolar stationary phase. The analyte of interest is moderately- to

non-polar. Alkyl bonded silica (C-18) is the most commonly used stationary phase in

solid-phase extraction (SPE) (Hennion 1999). The crude extract was first fractionated

using a C-18 cartridge (300 mg, Alltech Associates, Inc., IL) (Fig. 4-1). Prior to

application of the crude extract, the C-18 cartridge was pre-conditioned with 10 ml

methanol and then with 10 ml water. The crude extract was percolated through the C-18

cartridge at a rate of approximately 1 drop per 1.5 s using positive pressure, and the

unbound fraction was collected. After percolation of the crude extract, the cartridge with

retained compounds was washed with 10x volumes of a stepwise gradient of water-









methanol mixtures [water, water:methanol (80:20, % v/v), water:methanol (40:60), and

water:methanol (5:95)] to elute retained compounds. After percolation of each water-

methanol mixture, subsequent fractions were collected and each fraction concentrated

back to 0.1 x volume under a nitrogen stream.

In reversed-phase SPE procedures using C-18 packing, trapping of the analyte is

optimized by adjusting the pH of the conditioning solution or aqueous sample, or by

adding a small percentage of organic solvent to the sample before percolation (Hennion

1999, Simpson 2000). Adjustment of the sample pH greatly enhances retention of

ionizable compounds under their neutral form on C-18 packing by making them

sufficiently hydrophobic (Pichon 2000). The sample pH can also be adjusted for sample

clean-up so that unwanted compounds in the sample are retained on the SPE packing

(Hennion 1999, Le6n-Gonazalez and Perez-Arribas 2000, Simpson 2000). Therefore, the

above extractions were repeated separately using crude extract acidified or basified to

three different pH, i.e., at original (6.5), acidic (3.0) and alkaline (9.0) pH. Acidification

and basification of crude extract was achieved by adding 1 N phosphoric acid and 1 N

ammonium hydroxide, respectively.

The crude extract, unbound fraction, four eluted fractions [water, water:methanol

(80:20), water:methanol (40:60) and water:methanol (5:95)] and the combination of all

four eluted fractions were applied to artificial diet disks for deterrence bioassays under

no-choice conditions. An amount of each extract/fraction, equivalent to 70-[l latex, was

applied to each diet disk. For controls, five water-methanol mixture combinations without

latex extract (including water:methanol (20:80)) and untreated diet disks were used. Each









experimental unit was replicated nine times for extracts at the original pH and six times

each for extracts acidified to pH 3.0 or basified to pH 9.0.

Fractionation of Crude Extract Using C-18, SAX and SCX Cartridges Connected in
Series

Ion-exchange SPE is also a commonly used method for the extraction of charged

compounds. Negatively (anionic) and positively cationicc) charged compounds can be

isolated on anion exchange (SAX) and cation exchange (SCX) stationary phases,

respectively. Subsequently, these charged compounds can be eluted using a solution of

high ionic strength that displaces the absorbed compounds (Hennion 1999). The crude

extract at original pH was next fractionated using C-18, SAX (functional group:

quaternary ammonium, counter ion: acetate) and SCX (functional group: sulphonic acid,

counter ion: hydrogen) cartridges (Alltech Associates, Inc., IL) connected in series (Fig.

4-2). Prior to crude extract application, C-18 cartridges were pre-conditioned with 10 ml

methanol and then with 10 ml water; SAX and SCX cartridges were pre-conditioned with

10 ml water. The samples were passed by positive pressure through the cartridges at a

flow rate of approximately 1 drop per 1.5 s. The crude extract at original pH (6.5) was

percolated through a C-18 cartridge and the unbound fraction was collected. Then, the C-

18 unbound fraction was percolated through SAX and SCX cartridges connected in series

and the unbound fraction was collected. After percolation of the C-18 unbound fraction,

SAX and SCX cartridges with retained compounds were washed separately with 10x

volumes of a stepwise gradient of NaCl solutions (0.1, 0.5 and 1 M) to elute retained

compounds. After percolation of each NaCl solution, subsequent fractions were collected

and concentrated back to 0.1 x volume under a nitrogen stream.









An amount of each extract/fraction, equivalent to 70 [l latex, was applied to each

diet disk for use in the bioassays. Nine treatments were studied: crude extract; C-18

unbound fraction; SAX and SCX unbound fraction; and 0.1, 0.5, 1M NaCl fractions from

each SAX and SCX cartridge. Controls consisted of untreated diet disks, disks treated

with water:methanol (20:80) mixture, and disks treated with 0.1, 0.5 or 1 M NaCl

solutions. Each experimental unit was replicated nine times. The 0.5 M-NaCl SCX

fraction exhibited the strongest deterrent activity and was termed "SCX fraction" in the

following LC/MS separations.

LC/MS Separation of SCX Fraction

LC/MS helps in the fractionation of a sample with simultaneous characterization of

chemical compounds. Fractionating increases the sample simplicity and ultimately makes

the characterization of the compounds much easier. The SCX fraction was further

fractionated by LC/MS. A Thermo Finnigan LCQ Deca XP Max was used employing

electrospray ionization (ESI) (sheath gas, 25 arbitrary units; sweep gas, 10 arbitrary units;

spray voltage, 5.00 kV; capillary temperature 2850C; and capillary voltage, 3.0 V) with

the Thermo Separations spectra HPLC system (quaternary pump P4000, autosampler AS

3000, and diode array detector UV6000). Separations were performed on a PLRP-S

column (100 A, 3 |tm, 150 x 4.6 mm, Polymer Laboratories. Ltd., UK) with solvent A

(water with 10 mM ammonium format) and solvent B (90 acetonitrile: 10 water with

10mM ammonium format, v:v) as mobile phases for 40 min. Elution was performed

using two solvent gradient systems with column temperature maintained at 600C and a

flow rate of 1.0 ml/min. The first gradient elution system employing solvent A at pH 9.0

began with 95:5 percent (A and B) and reached 45:55 at 25 min, followed by gradient to









0:100 in 5 min. The solvent was then kept at the final composition for 5 min.. The second

gradient, with solvent A at pH 10, began with 100:0 percent (A and B) and reached 0:100

at 25 min. It was then kept at that composition for 10 min. UV absorption was monitored

at 190 800 nm, and a low-volume micro needle valve splitter P450 (Upchurch

Scientific, Oak Harbor, WA) was used to split the solvent flow between the UV detector

and MS electrospray interface up to 90:10, making it possible to collect 90% of the eluted

material in one minute fractions for bioassay while simultaneously obtain LC/MS spectra.

In the first gradient elution system at pH 9.0, fractions collected each minute were

recombined into six major fractions (Fig. 4-3) and concentrated to a volume equivalent to

70 [l of latex to treat one diet disk. Then, these six fractions (#0-3, #4-7, #8-11, #12-15,

#16-20, and #21-40) and the combination of eluted fractions were applied on the surface

of artificial diet disks. Each experimental unit was replicated six times and each unit had

two diet disks treated with same kind of fraction under no choice conditions. Untreated

diet and diets treated with crude extract and SCX fraction were used for the controls.

In the second gradient elution system at pH 10.0, fractions were collected each

minute but only eleven fractions were used for bioassays under no-choice conditions (#2,

#3, #4-6, #20, #21, #22, #23, #24, #25, #26, and #27). Controls consisted of untreated

diet disks and disks treated with crude extract and SCX fraction. Each experimental unit

was replicated three times.

Statistical Analysis

In all no-choice tests, number of adults feeding on two diet disks 90 min after

beetle release and the dry weights of diets consumed in 16 h were analyzed using Proc

GLM (SAS Institute 2003) with latex fraction as a fixed effect and replications as a

random effect. The error degree of freedom for latex fraction effect was calculated as









(levels of latex fraction -1)(replications -1). Tukey's honestly significant difference

(HSD) test with a significance level of a = 0.05 (SAS Institute 2003) was used for post

hoc means separation.

Results

Fractionation of Crude Extract Using C-18 Cartridge

Water and water:methanol (40:60) fractions were light yellow and milky white,

respectively; the 80:20 and 5:95 water:methanol fractions were colorless (Fig. 4-4).

Fractionation at original pH. Fractionation of the crude extract at its original pH

and subsequent bioassays indicated that the unbound fraction had activity equivalent to

that of the crude extract (Fig. 4-5). Latex fraction had significant effect on the number of

insects feeding on diet disks (F = 12.05; df = 12, 96; P = 0.0001). Fewer insects were

counted 90 min after their release on diet disks treated with the unbound fraction than on

disks treated with any other C-18 water-methanol mixture fraction or on control diet

disks (Fig. 4-6A). Latex fraction also significantly affected diet consumption by beetles

(F = 39.40; df = 12, 96; P = 0.0001). Beetles consumed significantly less diet treated with

the unbound fraction than diet treated with any other water-methanol mixture fraction or

control diet (Fig. 4-7A).

Fractionation of crude extract at pH 3.0. Fractionation of the crude extract

acidified to pH 3.0 on the C-18 cartridge and subsequent bioassays revealed that some of

the deterrent compounds were retained on the C-18 resin. Latex fraction significantly

affected the number of beetles feeding (F = 5.03; df = 12, 60; P = 0.0001). Significantly

more beetles were counted 90 min after their release on diets treated with the water-

methanol mixture extracts compared to diet treated with the unbound fraction (Fig. 4-6B).

Latex fraction also had significant effect on diet consumption (F= 11.49; df = 12, 60; P =









0.0001). The unbound fraction was still deterrent to beetle feeding as diet consumption

was significantly less on it, similar to that on the crude extract. But, in addition, the water

fraction also had some deterrent activity (Fig. 4-7B).

Fractionation of crude extract at pH 9.0. Bioassays of fractions obtained by

passing the crude extract basified to pH 9.0 over C-18 cartridge identified deterrent

activity again in the unbound fraction with some deterrent activity in the water fraction.

Latex fraction had significant effect on the number of insects feeding on diet (F = 4.08;

df = 12, 60; P = 0.0001). After 90 min, the number of insects feeding on diet treated with

the unbound fraction did not differ significantly from the number feeding on diet treated

with the crude extract (Fig. 4-6C). Latex fraction also affected diet consumption by

beetles (F = 4.57; df = 12, 60; P = 0.0001). Beetles consumed similar amounts of diet

treated with the unbound fraction and the crude extract (Fig. 4-7C).

Fractionation of Crude Extract Using C-18, SAX and SCX Cartridges Connected in
Series

The 0.1M NaCl fraction eluted from the SAX cartridge was colorless, but the other

two fractions (0.5 and 1M NaC1) were yellow (Fig. 4-8). All three fractions eluted from

the SCX cartridge were colorless.

The deterrent activity of the 0.5 M NaCl fraction obtained from the SCX cartridge

was similar to that of the crude extract (Fig. 4-9). Latex fraction had significant effect on

the number of insects feeding on diet (F= 31.75; df = 13, 104; P = 0.0001). Significantly

fewer insects were counted on the diet disks treated with the 0.5 M NaCl fraction from

either the SAX or SCX cartridge 90 min after their release (Fig. 4-10) compared to all

other fractions. Application of latex fraction also significantly affected diet consumption

by beetles (F = 54.67; df= 13, 104; P = 0.0001). Beetles consumed significantly less diet









treated with the 0.5 M NaCl fraction eluted from the SCX cartridge than diet treated with

any other fraction from the SAX or SCX cartridges (Fig. 4-11). Beetles also consumed

significantly less diet treated with the 0.5 M NaCl fraction from the SAX cartridge but

not as little as they did on disks treated with the crude extract.

Fractionation of SCX Fraction Using LC/MS

At pH 9.0 of solvent A. Application of latex fraction significantly affected both the

number of beetles counted on diet disks and the amount that they consumed (number of

beetles on disks: F = 18.78; df = 9, 45; P = 0.0001; consumption: F = 88.34; df = 9, 45; P

= 0.0001). Fewer beetles were counted on and consumed less of the diet disks treated

with the crude extract, the SCX fraction, LC/MS fractions #0-3, fractions #21-40 as well

as the combination of all LC/MS fractions (Figs. 4-12, 4-13). Some weak feeding

deterrent activity was also found in the #4-7 fraction.

At pH 10.0 of solvent A. Latex fraction treatment has significant effect on the

number of insects feeding on diet (F= 11.92; df = 13, 26; P = 0.0001). Diets treated with

fraction #3 were as deterrent to feeding as were disks treated with either the crude extract

or the SCX fraction (Fig. 4-14). Diet consumption by beetles was also significantly

affected due the treatment of latex fractions (F = 26.74; df = 13, 26; P = 0.0001).

Consumption was the lowest on the diet disks treated with the crude extract, the SCX

fraction and the #3 fraction (Fig. 4-15). This fraction was estimated to contain about 10

peaks based on UV absorption (190 450 nm) and M+1 ions produced when analyzed

using positive ion electrospray LC/MS (Fig. 4-16).

Discussion

The deterrent activity of the unbound fraction of the reversed-phase extraction at

the original pH indicates that the deterrents compounds were not retained on C-18 resin.









Reversed phase extractions using C-18 involve a polar or moderately polar sample matrix

(mobile phase) and a nonpolar stationary phase. The analytes of interest retained on the

cartridge are moderately- to non-polar. So, this indicates that the deterrent compounds in

the crude extract are highly polar. Many biologically active compounds are known to

exist in their glycosidic form. By binding to sugars, the toxicity of these compounds is

reduced and their solubility is increased which facilitates their storage in large amounts.

These compounds become more active after coming in contact with specific degradation

enzymes (Harborne 1979, Schoonhoven et al. 2005). In both lettuce and chicory

(Chicorium intybus L.), most of the sesquiterpenes are found in glycosidic form and the

bitterness of the plants is associated with them (Price et al. 1990). Tamaki et al. (1995)

also reported that 44, 34 and 56% of sesquiterpene lactones were in their bound form in

the wild lettuce species, L. saligna and L. virosa, and cultivated lettuce, respectively.

These sesquiterpenes exhibited low retention on C-18 cartridges (Schenck 1966, Tamaki

et al. 1995). Phenolic glycosides found in white grub-infested sugarcane (Nutt et al.

2004) and in white lupin (Lupinus albus L.) (Stobiecki et al 1997) also exhibited low

retention due to their high polarity and solubility in water.

The crude extract was fractionated at two extreme pH levels with the intention of

better retaining deterrent compounds with either acidic or alkaline characteristic. In my

study, diet consumption data indicate that some of the deterrent compounds in the crude

extract were retained on the C-18 packing both at acidic and basic pH. Some of the

compounds with deterrent activity were eluted by water at both pH levels, but also by

water:methanol (40:60) at basic pH.









Ion exchange solid-phase extraction is commonly used for the extraction of

compounds that are charged when in an aqueous solution. In my study, the deterrent

compounds were retained on the SCX packing after percolation of the unbound fraction

from the C-18 cartridge, and were eluted with 0.5 M NaCl solution. Retention of

deterrent compounds on SCX suggests a basic nature for the compounds.

During the fractionation of the SCX fraction using LC/MS with a mobile phase at

pH 9.0, the deterrent activity was found in the very early fractions, between 0 and 3 min,

indicating that this pH was not high enough to fully deprotonate a basic column, or that

the early elution could be due to additional polar constituents of the molecule, for

example sugars. Some deterrent activity was also found in the later fraction eluting

between 21 and 40 min which might indicate the aglycon form of an earlier eluting

glycosidic compound. When the pH of the mobile phase was raised to 10.0 and the

gradient elution system slightly changed to accommodate very polar compounds the

deterrent activity was retained on the column and only found in the fraction eluting

between 3 and 4 min and not in the later fractions. The change in pH appears to have

neutralized very basic compounds, and ultimately resulting in their retention on the

column. However, the loss of activity in the later elutin fraction can for the moment not

be easily explained.

Based on UV absorption and MS data, there are more than ten compounds present

in the fraction between 3 and 4 min, some of these compounds having substituted

aromatic group characteristics. Substituted aromatic compounds previously were reported

in lettuce, such as sesquiterpene lactones (lactucin, molecular weight 276; and

lactucopicrin, molecularweight 410) (Sessa et al. 2000) (Fig. 4-17) and flavonoids









(flavonol glycosides, flavone glycoside and anthocyanidin glycosides) (Dupont et al

2000) (Fig. 4-18). But their biological activity against insects has not been reported in

lettuce. However, sesquiterpene lactones provide resistance against lettuce downy

mildew and the red spot physiological disorder in certain lettuce cultivars due to its

strong antimicrobial properties (Bennett et al. 1994, Bestwick et al. 1995). Sesquiterpene

lactones play an antifeedant role in the closely related plant species chicory against

Schistocerca gregaria (Forsk.) (Rees and Harborne 1985).

The successful isolation of potent feeding deterrents for banded cucumber beetle

from a crude extract of romaine lettuce latex provides convincing evidence of a chemical

basis for host plant resistance in this variety. Deterrent compounds can be extracted using

reversed-phase and cation exchange cartridges (SCX) linked in series, and their retention

on cation exchange indicates that they are basic. In addition, LC/MS analysis indicates

the presence of substituted aromatic compounds. The chemical composition of the

fraction between 3 to 4 min is being investigated. Understanding the defensive role of

latex and its deterrent constituents (apart from physical defense due to stickiness) will

help to better comprehend the mechanisms of insect-plant interactions. Furthermore,

qualitative and quantitative knowledge of these biologically active compounds may help

plant breeders select for genotypes with an inherently high level of resistance using these

compounds as markers. Insect-susceptible but otherwise horticulturally superior cultivars

could also be made more resistant through genetic engineering.









Crude Extract

C-18





Water 100%


Water
fraction


I Water.Methanol (80.20)


I
Water Methanol (80 20)
fraction


Water.Methanol (40.60)
fraction



I


Water Methanol (40 60)


I Water Methanol (5 95)


Water Methanol (5 95)
fraction


Bioassay for activity


Figure 4-1. Scheme for solid-phase extraction and fractionation of crude extract after
passing through reversed-phase (C-18) cartridge.


Unbound
fraction









Crude Extract

C-18




C-18 Unbound
fraction



U SAX


SCX

SAX S CX

SAX and SCX
0.1 M NaCI unbound fraction 0.1 M NaCI


01M 0.1 M
NaCI NaCI
fraction fraction

0.5 M NaCI 0.5 M NaCI


05M 0.5M
NaCI NaCI
fraction fraction
1 M NaCI 1 M NaCI
1M 1M
NaCI NaCI
fraction fraction


Bioassay for activity


Figure 4-2. Scheme for solid-phase extraction and fractionation of crude extract after
passing through reversed-phase (C-18), anion (SAX) and cation (SCX)
exchange cartridges connected in series.











RT 0 00 -39 98 SM 5B


#0-3 #4-7















7 30

5 91
1 5 52 1
4 6-7
92


#E


.9 4








7 56


1 0 1 6



S-11 (#12-15









332





1 32 8
12 63

112 65


#16-20





















591 18,12


0 5 10
.


35


16 20(
Tim e (m In)


Figure 4-3. Fractions obtained after HPLC analysis of cation exchange (SCX) fraction.











































120


#21-40


23 38 27 09


31 11


36 42


I





















8O. ib


1.4. I*


fr.&I


OPIIV T r
rR CTw


-& ...-- -% W -

i 0l.aXtSract Unbound Water Water:Methanol Water:Methanol WaterMethanol Combined
^.;: 80:20 40:60 5:95 (All 4)


Figure 4-4. Color characteristics of fractions obtained after passing crude extract through reversed phase C-18 cartridge.


QNB .,rp
, P T.AaI..


Lc: -. -
















I r'L- de e...tract








,,*"N. L I : D O )


Figure 4-5. Bioassays of C-18 fractions applied on artificial diet disks using D. balteata adults under no-choice conditions.


I
'..,." Le

-v









._,_r, .l- ,_i .J A ll 41 )


-If
"A' ri ci*Tij











B) C)


Untreated
W:M (20:80)
W
W:M (80:20)
W:M (40:60)
W:M (5:95)


Crude extract f b


Unbound fraction
W
W:M (80:20)
W:M (40:60)
W:M (5:95)
Combined (All 4)


a

a
a
01 a

0 1 2 3 4 5 6


a
a
Sa
a
a

a




a
a


0 1 2 3 4 5 6


Sab
ab
ab
a
ab
a

bc


c abc
ab
ab
ab
ab

012345
0 1 2 3 4 5 6


Number of insects feeding / 2 disks

Figure 4-6. Mean number ofD. balteata adults feeding after 90 min on two artificial diet disks treated with fractions obtained after
passing crude extract at three pH levels through C-18 cartridge: A) original (pH 6.5), B) acidic (pH 3.0), and C) basic (pH
9.0). Error bars indicate SEM. Bars topped with different letters within same panel (A, B or C) differ significantly at the
0.05 level (Tukey's HSD test). (W Water, M Methanol).











B) C)


Untreated a
W:M (20:80) a
W a
W:M (80:20) a
W:M (40:60) a
W:M (5:95) a

Crude extract b


Sab
ab


a a
ab
C


d


Unbound fraction b b
W a b %c abc
W:M (80:20) // / a ab a
W:M (40:60) a a ab
W:M (5:95) a ab a
Combined (All 4) a ab abc

0 1020 3040 5060 0 1020 3040 5060 0 1020 3040 5060


Diet consumed (mg)

Figure 4-7. Dry weight of diet consumed by D. balteata adults when disks were treated with fractions obtained after passing crude
extract with different pH levels through C-18 cartridge: A) original (pH 6.5), B) acidic (pH 3.0), and C) basic (pH 9.0).
Error bars indicate SEM. Bars topped with different letters within same panel (A, B or C) differ significantly at the 0.05
level (Tukey's HSD test). (W Water, M Methanol).


.,
0o


c
tO
1" --







































Figure 4-8. Color characteristics of fractions obtained after passing C-18 unbound fraction through anion (SAX) and cation (SCX)
exchange cartridges connected in series.


WRPIUPr

:*'' : tV~ ^




















Anion Exchange (SAX) Fractions


Cation Exchange (SCX) Fractions

Figure 4-9. Bioassays of ion-exchange fractions applied on artificial diet disks using D. balteata adults under no-choice conditions.












Untreated a
S W:M (20:80) a
S 0.1M NaCI a
0
0.5M NaCI a
1 M NaCI a

Crude extract b
C18 unbound fraction b

0.1M NaCI a
0.5M NaCI b
1 M NaC a

0.1M NaCI a
X 0.5M NaCI b
c 1 M NaCI
Unbound fraction a

0 1 2 3 4 5 6

Number of insects feeding / 2 disks

Figure 4-10. Mean number ofD. balteata adults feeding after 90 min on diet disks treated
with ion-exchange fractions obtained by passing C-18 unbound fraction
(original pH 6.5) through anion (SAX) and cation (SAX) exchange
cartridges connected in series. Error bars indicate SEM. Bars topped with
different letters differ significantly at the 0.05 level (Tukey's HSD test).












Untreated a
W:M (20:80) a
S 0.1 M NaCI a
0
o 0.5M NaCI a
1M NaCI a

Crude extract c
C18 unbound fraction c

0.1 M NaCI a
S 0.5M NaCI
0.1M NaCI a

0.1M NaCI a
X 0.5M NaC c
c |1M NaCI a
SUnbound fraction a

0 10 20 30 40 50 60

Diet consumed (mg)

Figure 4-11. Dry weight of diet consumed by D. balteata adults when disks were treated
with ion-exchange fractions obtained after passing C-18 unbound fraction
(original pH 6.5) through anion (SAX) and cation (SAX) exchange
cartridges connected in series. Error bars indicate SEM. Bars topped with
different letters differ significantly at the 0.05 level (Tukey's HSD test).












Untreated


Crude extract c
SCX fraction c


# 0-3 bc
# 4-7 ab
#8-11 a
#12-15 a
# 16-20 \ a
# 21-40 c
Combined (all 6) c

0 1 2 3 4 5 6

Number of insects feeding / 2 disks

Figure 4-12. Mean number of insects feeding after 90 min on diet disks treated with
fractions obtained after LC/MS analysis of cation exchange fraction (SCX)
at pH 9.0 of the mobile phase. Error bars indicate SEM. Bars topped with
different letters differ significantly at the 0.05 level (Tukey's HSD test).












Untreated


Crude extract
SCX fraction


# 0-3
# 4-7
#8-11
# 12-15
# 16-20
#21-40
Combined (all 6)


de
e


de
c
ab

b


0 10 20 30 40 50 60
Diet consumed (mg)

Figure 4-13. Dry weight of diet consumed by D. balteata adults when disks were treated
with fractions obtained after LC/MS analysis of cation exchange fraction
(SCX) at pH 9.0 of the mobile phase. Error bars indicate SEM. Bars topped
with different letters differ significantly at the 0.05 level (Tukey's HSD
test).












Untreated a
Crude extract b
SCX fraction b


# 2 a
#3 b
# 4-6 a
# 20 a
#21 a
# 22 a
#23 a
# 24 a
#25 a
# 26 a
# 27 a

0 1 2 3 4 5 6
Number of insects feeding / 2 disks

Figure 4-14. Mean number of insects feeding after 90 min on diet disks treated with
fractions obtained after LC/MS analysis of cation exchange fraction (SCX)
at pH 10.0 of the mobile phase. Error bars indicate SEM. Bars topped with
different letters differ significantly at the 0.05 level (Tukey's HSD test).












Untreated
Crude extract
SCX fraction


#2
#3
# 4-6
# 20
#21
# 22
# 23
# 24
#25
#26
# 27


ab
Sab
ab


Sab
ab
a
ab


0 10 20 30 40 50 60
Diet consumed (mg)

Figure 4-15. Dry weight of diet consumed by D. balteata adults when disks were treated
with fractions obtained after LC/MS analysis of cation exchange fraction
(SCX) at pH 10.0 of the mobile phase. Error bars indicate SEM. Bars topped
with different letters differ significantly at the 0.05 level (Tukey's HSD
test).




















3.95



2.88 3.38 4.22
2.59 A J --4.08

1.7 198 /v 3--.27.204 8.0
1 -76


V I I I i i i I i i
1 2


I 1 P
3


I I I I I I I I i I I I I P I I I | I 1 |
4 5 68 7 8 9
Time



...." 209.93


192.I0 I ja.a
i7.1a l paL iL, ll l Ii. .11 iL IlkL.1l1.. .l l.ii h


0 0mz0 20
m/z


SSO9~


Figure 4-16. Electrospray LC/MS total negative ion trace of active fraction between 3 and 4 min.


100-



50-
50-


" --;--


'- 'L . . . . ..''


,I


~" ''~'~' "' '~'""~~'~"~~ ~1"'-1'"~1~~""~"1~'~--~--~-~--~


eoooooos



rrwoooo
sooooooo-
2aoooooo--
2000~000-
.~4000m-
roooo~xlrr
r

o-













iistiupR2


15
O


lactucin


R1 = OH; R2 =OH


lactucopicrin


R1= OH;

R= OCOCH2-C OH
R12


Figure 4-17. Structure of sesquiterpene lactones characterized in lettuce (Sessa et al.
2000).














HO 0 RO0..



OH O OH O
OH
OH OH

HO.


OR
OH

Figure 4-18. Chemical structures of flavonoids found in lettuce A) flavonol glycosides;
B) flavone glycoside; and C) anthocyanidin glycosides (kaempferol if R = H;
quercetin if R = OH) (R = glycoside) (Dupont et al. 2000).









CHAPTER 5
INVESTIGATING ENZYME INDUCTION AS A POSSIBLE REASON FOR LATEX-
MEDIATED INSECT RESISTANCE IN ROMAINE LETTUCE

Introduction

Lettuce (Lactuca sativa L.) is one of the most important vegetable crops grown

throughout the world (Ryder 1998). Lettuce growers suffer huge economic losses due to

various insect pest infestations because of the very high cosmetic standards demanded by

consumers (Palumbo et al. 2006). The romaine lettuce cultivar, 'Valmaine' exhibits a

high level of resistance against various insects, including the leafminer, Liriomyza trifolii

(Burgess) (Nuessly and Nagata 1994), banded cucumber beetle, Diabrotica balteata

LeConte (Huang et al. 2002) (Fig. 5-1), and two lepidopterans, Trichoplusia ni (Hubner)

and Spodoptera exigua (Hibner) (Chapter 2, Sethi et al. 2006). Valmaine's resistance

would be useful in an integrated pest management program however this cultivar is not

popular among growers because of its susceptibility to thermodormancy, premature

bolting, lettuce mosaic virus and corky root rot (Guzman 1986). Plant breeders have

attempted to improve the horticultural characteristics of Valmaine through breeding, but

unfortunately the horticulturally improved and currently used cultivar, 'Tall Guzmaine'

lost resistance to insects during the process (Chapter 2, Sethi et al. 2006).

My previous research revealed that Valmaine latex placed on artificial diet deterred

D. balteata feeding, whereas latex from Tall Guzmaine did not (Chapter 3, Sethi et al.

2007). I hypothesize that feeding deterrence due to constitutive levels of compounds in

latex may explain the mechanism of multiple insect resistance in Valmaine. Furthermore,

previously wounded Valmaine plants showed an increased localized resistance to feeding

by D. balteata compared to unwounded plants, suggesting the involvement of an









inducible mechanism of resistance (Huang et al. 2003b). Tall Guzmaine showed no such

inducible resistance.

Latex is an aqueous suspension or solution of complex mixtures of molecules

found in specialized secretary cells of plants known as laticifers (Evert 2006). Laticifers

possess high metabolic activity. In addition to synthesizing numerous molecules (lipids,

sugars and proteins) required to achieve their basic physiological functions, laticifers are

also known to synthesize and store diverse secondary metabolites in appreciable amounts

in latex (Moussaoui et al. 2001). Many defensive compounds with demonstrated negative

impact on insect fitness are stored in latex (Evans and Schmidt 1976, Haupt 1976, Matile

1976, Noack et al. 1980, Seiber et al. 1982, Nishio et al. 1983, Rees and Harborne 1985,

Roberts 1987, Konno et al. 2004, 2006; Ramos et al. 2007). Activity of phenylalanine

ammonia lyase, polyphenol oxidase and many other defense-related enzymes is much

higher in the laticifers than in the leaves of rubber tree (Hevea brasiliensis H.B.K.)

(Broekaert et al. 1990, Kush et al. 1990, Martin 1991, Gidrol et al. 1994, Pujade-Renaud

et al. 1994, Wititsuwannakul et al. 2002). Wounding of laticifers is also known to induce

other defense-related enzymes in latex of papayas (Azarkana et al. 2004, Kydt et al.

2007), fig tree (Ficus carica L.) (Kim et al. 2003, Taira et al 2005), rooster tree

(Calotropisprocera Ait.) (Freitas et al. 2007), and Albanian spurge (Euphorbia

characias L.) (Mura et al. 2005, 2007; Fiorillo et al. 2007). Thus, plant latex acts as a

chemical defense due to alteration in its constituents upon insect damage.

The purpose of this study was to investigate the role of inducible enzymes in the

latex-mediated multiple insect resistance in Valmaine. I asked the questions of whether

enzyme activities changed after insect feeding damage, how quickly this change









occurred, how long the elevated levels lasted, and whether elevated enzyme activity was

correlated with increased feeding deterrent activity in latex. Hence, choice experiments

were conducted with D. balteata adults between diets treated with latex from damaged

and undamaged plants within the same cultivar (Valmaine and Tall Guzmaine) to look

for changes in latex chemistry after beetle feeding. The induction of defense-related

enzymes, in particular phenylalanine ammonia lyase, polyphenol oxidase and peroxidase

in latices of resistant Valmaine and susceptible Tall Guzmaine was also compared with

and without D. balteata feeding damage.

Materials and Methods

Plants

The seeds of romaine lettuce cultivars Valmaine (resistant) and Tall Guzmaine

(susceptible) were germinated overnight on moistened filter paper. The germinated seeds

were planted in transplant trays filled with Metro Mix 200 (Grace Sierra, Milpitas, CA)

and healthy seedlings were transplanted 2 wk later into 15-cm-diameter plastic pots. The

plants were watered daily and fertilized with 15 ml of Peters 20-20-20 solution (W.R

Grace, Fogelsville, PA) every week. Six-week-old lettuce plants (9-10 true-leaf stage)

were used for the experiments. Bush lima bean seeds (Phaseolus lunatus L.) of the

cultivar Fordhook 242 (Illinois Foundation Seeds, Champagne, IL) were planted in

transplant trays filled with Metro Mix 200 and fertilized with the same solution used for

lettuce plants.

Insects

The colony ofD. balteata was started from adults collected from weeds (spiny

amaranth, Amaranthus spinosus L. and primrose willow, Ludwigiaperuviana L.) in Belle

Glade, Florida in 2003. New adults were added to the established colony in 2005 and









2006 to increase genetic diversity. Larval stages were reared on the roots of corn

seedlings and adults were fed on lima bean leaves and sweet potato tubers (Chapter 3,

Huang et al. 2002). Unfed adults, within 48 h of emergence, were used for the

experiments.

Artificial Diet

Freshly-made southern corn rootworm artificial diet (Bio-Serv, Frenchtown, NJ)

was used in all experiments. The diet was prepared according to methods previously

described (Chapter 3, Sethi et al. 2007). One-cm-thick disks were punched out from

cooled artificial diet using a 1.5-cm-diameter cork borer.

Bioassay Conditions for Feeding Damage

One hundred and eighty plants of each cultivar were placed individually in

cylindrical screen cages (18.5 cm diameter x 61.0 cm height) for use in collecting latex

from plants after timed, continuous intervals ofD. balteata feeding. Two male-female

beetle pairs were placed into half (90 plants) of the cages of each cultivar, while the other

90 plants of each cultivar were used as undamaged checks. Beetles were allowed to feed

on the plants for 6 d. Females were weighed individually before releasing them on the

plants, and again at either 1, 3 or 6 d after they were released into the cages, to determine

weight change. Latex was collected from plants 1, 3 and 6 d after they were released into

the cages. Out of these 180 plants of each cultivar, latex was collected from 60 plants (30

damaged and 30 undamaged checks) at each time interval (1, 3, and 6 d) after initiation of

feeding damage. Out of each batch of 30 plants, latex from 15 plants was used for diet

disk choice tests and latex from the other 15 was used to assess enzyme activity, as

explained below. Each group of 15 plants was further divided into 5 groups (replicates)

of 3 plants for the collection of latex. An aloquot of 70 [tl of latex was collected from









each group of three plants for use in the assays described below. Latex was collected

using a silanized microdispenser (Drummond Scientific Company, Broomall, PA) from

the leaf base (site of leaf lamina attachment to the stem, and of rapid latex exudation

upon cutting) of young and middle-aged leaves of individual plants 60 s after cutting the

tissue with a disposable scalpel blade (Feather, Osaka, Japan). The experiments were

carried out at 25 1IC in a laboratory under a photoperiod of 14:10 (L:D) h.

Choice-tests Using Latex from Damaged and Undamaged Plants

Latex (70 il) collected from plants as described above was applied onto the top and

side surfaces of a diet disk, immediately after collection. The experimental unit for the

choice-test bioassay consisted of two diet disks, one treated with latex from beetle-

damaged plants and the other one with latex from undamaged checks within each

cultivar. In the control experimental units, two untreated diet disks were used. The diet

disks were placed on the bottom of a plastic ventilated container (10 x 10 x 8 cm) and

three male-female pairs of beetles were allowed to feed on the disks for 24 h at 25 1C

in a laboratory. The number of adults feeding on each diet disk was recorded 1, 2, 3 and 4

h after their release into the bioassay units. Dry weight of the diet consumed in 24 h was

calculated as previously described in Chapter 3 and in Sethi et al. 2007. Total diet

consumed per three pairs of adults in 24 h was calculated by adding the consumption of

the two diet disks in each repplicate of each treatment.

Enzyme Activity Assays

Activity of the enzymes phenylalanine ammonia lyase, polyphenol oxidase and

peroxidase was assayed in the latices of Valmaine and Tall Guzmaine 1, 3 and 6 d after









initiation of beetle damage. Collected latex was dispensed into a -200C chilled, 1.5-ml

micro-centrifuge tube, on ice and immediately stored at -800C until analyses.

Frozen latex (70 pl) was dissolved in 5 ml of 50 mM potassium phosphate buffer

(pH 6.2) and centrifuged at 48,500 xg for 45 min at 40C (Model J2-HS, Beckman

Instruments, Fullerton, CA). The supernatant was collected and stored at -800C until

analyses. Total protein and enzyme activities were determined using a spectrophotometer

(Model DU 640, Beckman Instruments, Fullerton, CA). Total protein was estimated

according to the modified Lowry's method (Peterson 1977) using the Folin-Ciocalteau

phenol reagent (Pierce Chemical, Rockford, IL) and bovine serum albumin as a standard.

Phenylalanine ammonia-lyase (PAL). PAL activity in latex was measured as

described by Ke and Saltveit (1986) and Campos-Vergas and Saltveit (2002) with slight

modifications. The supernatant was analyzed for PAL activity after addition of 200 [il of

supernatant to 400 Cl of 50 mM L-phenylalanine (dissolved in 20 mM potassium

phosphate buffer, pH 8.8) and 400 pl of 50 mM potassium phosphate buffer pH (8.8) and

incubated at 400C for 30 min. The absorbance was measured at 290 nm before and after

incubation. PAL activity was expressed as the amount of PAL ([[mol mg-1 h-1) that

produces 1 |tmol of cinnamic acid in 1 h. Cinnamic acid (0 400 [tmol at an increment of

15 itmol) was used as a reference for quantification of PAL activity.

Polyphenol oxidase (PPO). PPO activity was assayed following the methods of

Sirinphanic and Kader (1985) and Loiaza-Velarde et al. (1997) with slight modifications.

PPO activity was assessed by incubating 10 il of supernatant with 500 pl of 1.6%

catechol (Sigma, St. Louis, MO), 100 pl of 50 mM potassium phosphate buffer (pH 6.2)

and 390 pl distilled water. Absorbance of the mixture was read at 480 nm over a period









of 2 min. One unit of PPO activity was defined as the amount of enzyme that produced an

increase in absorbance of 0.1 per min at 480 nm. The linear portion of the curve was used

to estimate the reaction rate.

Peroxidase (POX). The activity of POX was determined using the methods of

Loiaza-Velarde et al. (1997) with slight modifications. The POX activity was determined

by combining 10 ptl of H202 (30%, v/v) in 50 ptl of supernatant, 300 ptl of 18 mM

guaiacol, 100 ptl of 50 mM potassium phosphate buffer (pH 6.2) and 540 [tl of distilled

water. Absorbance of the resulting mixture was examined at 420 nm over a period of 2

min. The POX activity ([tmol mg protein-1 min-1) was determined using guaiacol molar

absorptivity (E = 26.6 M-1 cm-1) at 420 nm. The reaction rate was calculated using the

linear portion of the curve.

Statistical Analysis

Data on number of insects feeding on diet disks treated with latex collected from

plants with and without prior beetle exposure were analyzed as a repeated measures

design using Proc GLIMMIX (SAS Institute 2003). Separate analyses were run for disks

from each cultivar at each prior beetle exposure interval (1, 3 and 6 d). The variables

latex (from damaged or undamaged plants) and time interval after beetle release (1, 2, 3

and 4 h) were fixed. Fifteen groups of six beetles (i.e., replications) were randomly

assigned to each level of latex and tested four times (1, 2, 3 and 4 h). Data on dry weight

of diet consumed under choice tests were analyzed using PROC GLM (SAS Institute

2003) with latex and time interval after beetle release as fixed effects. Replications were

treated as a random effect for each cultivar.

Data on enzyme activities were analyzed using PROC GLM (SAS Institute 2003)

with cultivar, latex treatment (damaged or undamaged), and time interval after feeding









initiation on plants as fixed effects. Replications were again treated as a random effect.

Data on beetle fresh weight gain were analyzed using PROC GLM (SAS Institute 2003)

with cultivar and time interval after feeding initiation as fixed effects, and replications as

a random effect. Tukey's honestly significant difference (HSD) test with a significance

level of a = 0.05 (SAS Institute 2003) was used for post hoc means separation. Simple

regression analysis was done to study the relationship between beetle fresh weight gain

and enzymatic activities using PROC REG (SAS Institute 2003).

Results

Oberservations of Latex Characteristics from Damaged and Undamaged Plants

The latex from Valmaine plants damaged for 3 or 6 d browned faster and to a

deeper hue than did latex collected after 1 d of feeding damage. However, no such

differences were noted in the latex of Tall Guzmaine. The quantity of latex exuded by

Tall Guzmaine plants decreased with the duration of feeding damage. Tall Guzmaine

latex collected after 3 and 6 d of feeding damage was also less viscous, and more watery

and translucent, while latex quality in Valmaine did not differ visually.

Choice-tests Using Latex from Damaged and Undamaged Plants

In case of Valmaine choice tests, type of latex 1 d after feeding initiation did not

have significant effect on the number of insects feeding on the diet disks (F = 2.0851; df

= 1, 8; P= 0.1585), but latex after 3 (F= 18.96; df = 1, 8; P = 0.0001) and 6 d (F=

14.43; df= 1, 8; P = 0.0005) after feeding initiation had significant effects. The number

ofD. balteata adults feeding on disks treated with latex from Valmaine plants that had

been fed on for 1 d was not significantly different from the number feeding on disks

treated with latex from undamaged Valmaine plants (Fig. 5-2 and 5-3). However, there

were significant differences between disks treated with Valmaine latex from plants with









and without feeding after 3 and 6 d. In Tall Guzmaine choice tests, latex anytime after

feeding initiation did not have any significant effect on the number of beetle feeding on

diet disks (1 d: F= 0.0753; df = 1, 8; P = 0.7855; 3 d: F= 0.800; df = 1, 8; P = 0.7791, 6

d: F = 0.0468; df = 1, 8; P = 0.8301). The number of beetles feeding on diet disks treated

with latex from damaged Tall Guzmaine plants or with latex from undamaged plants did

not differ significantly at any time after initiation of feeding damage (Fig. 5-2 and 5-4).

In the Valmaine choice test, latex (damaged or undamaged) had significant effect

on diet consumption by the beetles (F = 72.02; df = 1, 24; P = 0.0001). Adults of D.

balteata consumed significantly less diet treated with latex from damaged plants than diet

treated with latex from undamaged plants (Fig. 5-5). Time interval (1, 3, and 6 d) after

feeding initiation on plants did not have significant effect on diet consumption by the

beetles (F = 1.08; df = 2, 24; P = 0.3548). But there was significant interaction between

latex and time interval (F = 3.67; df = 2, 24; P = 0.0406). The amount of diet eaten from

disks treated with latex from damaged plants decreased with increasing duration of beetle

feeding on plants, whereas the amount of diet eaten from disks treated with latex from

undamaged plants was constant across the three time intervals after initiation of feeding

(Fig. 5-5). In the Tall Guzmaine choice test, latex did not have any significant effect on

diet consumption by beetles (F = 0.2160; df = 1, 24; P = 0.6463). Diet consumption by

D. balteata adults on diet treated with latex from damaged plants did not differ

significantly than on diet treated with latex from undamaged plants (Fig. 5-5). Neither

significant effect of time interval (F = 0.60; df = 2, 24; P= 0.5592), nor significant

interaction between latex and time interval (F = 2.04; df= 2, 24; P = 0.1521) on diet

consumption was found.









Treatment of latex significantly affected the total diet consumption in choice tests

(F= 235.08; df = 2, 33; P = 0.0005). Total diet consumed by six D. balteata was

significantly less on Valmaine latex treated diet compared to Tall Guzmaine latex treated

and control diets (Table 5-1). Diet consumption did not change significantly when disks

were treated with latex collected from damaged plants at different time intervals (F =

1.11; df = 2, 33; P = 0.3412). No significant interaction was found between type of latex

and time interval after feeding initiation (F = 0.6330; df = 4, 33; P = 0.6425).

Total Protein Content

Type of cultivar had significant effect on the total protein content (F = 91.77; df=

1, 47; P = 0.0001). Total protein content was significantly higher (1.3 fold) in Valmaine

latex than in Tall Guzmaine latex (Fig. 5-6). No significant effect of treatment (damaged

or undamaged) was found on total protein content (F = 1.49; df = 1, 47; P = 0.2281). But

significant effect of time interval after feeding damage (1, 3 and 6 d) was found (F =

5.29; df = 2, 47; P = 0.0084). Significant interactions were found between cultivar and

treatment (damaged or undamaged) (F = 16.70; df= 1, 47; P = 0.0002), and between

cultivar and time interval after feeding damage (F= 7.61; df= 2, 47; P = 0.0013). Total

protein content in Valmaine after 6 d of feeding damage was 1.36 fold higher than after 1

d. There was no increase protein content of Tall Guzmaine latex through time.

Phenylalanine Ammonia Lyase

The effect of cultivar was significant on PAL activity (F = 289.82; df = 1, 47; P =

0.0001). The activity of PAL was significantly higher (3.44 fold) in Valmaine latex than

in Tall Guzmaine latex (Fig. 5-7). Both treatment (F = 98.45; df = 1, 47; P = 0.0001) and

time interval after feeding initiation (F = 7.96; df = 2, 47; P = 0.0010) had significant

effect on PAL activity. Significant interactions were found between cultivar and









treatment (F = 20.96; df = 1, 47; P = 0.0001), and between cultivar and time interval after

initiation of feeding damage (F= 7.36; df = 2, 47; P = 0.0016). PAL activity in Valmaine

latex was significantly increased after 3 d (1.81 fold) and 6 d (1.54 fold) of feeding

damage, relative to 1 d after initiation of feeding. No increase was seen in PAL activity in

the latex of Tall Guzmaine through time.

Polyphenol Oxidase

Type of cultivar had significant effect on PPO activity (F = 358.32; df = 1, 47; P=

0.0001). The activity of PPO was significantly higher (4.37 fold) in Valmaine latex than

in Tall Guzmaine latex (Fig. 5-8). Both treatment (F = 80.31; df = 1, 47; P = 0.0001) and

time interval after feeding initiation (F= 8.25; df = 2, 47; P = 0.0008) had significant

effect on PPO activity. Significant interactions were found between cultivar and

treatment (F = 74.86; df = 1, 47; P = 0.0001), and between cultivar and time interval after

feeding damage (F = 11.65; df= 2, 47; P = 0.0016). PPO activity was significantly

increased 3 d (1.74 fold) and 6 d (1.78 fold) after feeding damage in Valmaine latex, but

not in Tall Guzmaine latex.

Peroxidase

The POX activity was significantly affected by the type of cultivar (F= 35.49; df=

1, 47; P = 0.0001). The activity of POX was significantly higher (2.1 fold) in Valmaine

latex than in Tall Guzmaine latex (Fig. 5-9). Both treatment (F = 39.29; df = 1, 47; P =

0.0001) and time interval after feeding initiation (F = 4.92; df = 2, 47; P = 0.0113) had

significant effect on POX activity. Significant interactions were found between cultivar

and treatment (F = 35.45; df = 1, 47; P = 0.0001), and between cultivar and time interval

after feeding damage (F = 5.16; df = 2, 47; P = 0.0094). POX activity was significantly









increased 3 d (1.56 fold) and 6 d (2.1 fold) after feeding damage in Valmaine latex but

not in Tall Guzmaine latex.

Relationship between Female Weight Gain and Enzyme Activity

Cultivar had significant effect on gain in female fresh weight (F = 1269.92; df = 1,

23; P = 0.0001). Female beetles weighed significantly less when fed on Valmaine than

Tall Guzmaine (Fig. 5-10). Both time interval after feeding initiation on plants (F=

30.42; df = 2, 23; P = 0.0001) and interaction between cultivar and time interval (F=

161.35; df = 2, 23; P = 0.0001) had significant effect on female fresh weight gain.

Females feeding on Tall Guzmaine weighed 2.2, 12.1, and 50.8 times more than the

females on Valmaine after 1, 3 and 6 d of feeding on the plants, respectively. Beetles lost

weight over time on Valmaine whereas they gained weight on Tall Guzmaine (Fig. 5-10).

Furthermore, a significant negative relationship was found between female fresh weight

gain and activities of each enzyme (PAL, PPO and POX) in latex from damaged plants of

Valmaine (Fig. 5-11). No significant relationship was found between female fresh weight

gain and any of the enzyme activities of latex from Tall Guzmaine.

Discussion

Valmaine latex from damaged plants was more deterrent compared to latex from

undamaged plants. This may be due to the change in the concentration of its constituents.

Upon wounding, latex turns brown after sometime due to the production of quinones that

are catalyzed by PPO. The browning potential of the latex from damaged Valmaine

plants was observed to increase with time after feeding damage. The browning is much

darker in color in a disease-resistant clone of rubber tree than in a susceptible clone

(Wititsuwannakul et al. 2002). Increased intensity of browning may be due to the higher

activity of PPO. The intensity of browning was observed to remain the same in Tall









Guzmaine latex after beetle damage. In fact, the intensity of browning was less in latex

from undamaged Tall Guzmaine plants than in latex from undamaged Valmaine plants.

Tall Guzmaine damaged plants produced less latex which was also less viscous, and more

watery and translucent, while the amount of latex production and its viscosity and color

(miliky white) remained the same in Valmaine latex even after beetle damage. Such

differences in milkiness arise due to differences in the refractive indices of the dispersing

particles (mainly terpenoids) and the dispersing medium (Esau 1965, Fahn1990). Thus,

the production of these dispersing particles in Tall Guzmaine may have been reduced

after feeding damage or the loss of large amounts of latex during beetle feeding may have

reduced the concentration of these compounds. The amount of total protein increased in

latex from Valmaine after beetle damage while it did not change in Tall Guzmaine. Ni et

al. (2001) also found a significant increase in the total protein content in wheat cultivars

after damage by the Russian wheat aphid.

The activities of all three enzymes, PAL, PPO and POX were increased

significantly in Valmaine latex after 3 d ofD. balteata feeding damage while they were

same in Tall Guzmaine latex. Even the constitutive level of PAL and PPO in undamaged

plants was significantly higher in Valmaine latex than in Tall Guzmaine latex. Alteration

in the levels of these enzymes due to insect feeding has been observed by many other

workers (Green and Ryan 1972, Cole 1984, Hildebrand et al. 1986, Felton 1989, Felton et

al. 1994a, b; Miller et al. 1994, Rafi et al. 1996, Jerez 1998, Stout et al. 1999, Constabel

et al. 2000, Chaman et al. 2001, Ni et al. 2001, Heng-Moss et al. 2004). The rate of

secondary metabolism via the phenylpropanoid pathway, leading to production and

accumulation of soluble phenolic compounds, is greatly increased after wounding of









lettuce tissue (Tomas-Barberan et al. 1997, Saltveit et al. 2005). The production of

phenylpropanoid compounds plays an important role in plant defense (Hahlbrock and

Scheel 1989). Phenylalanine ammonia lyase is the first committed enzyme in the

phenylpropanoid pathway (Dixon and Paiva 1995). Its de novo synthesis and increased

activity is an initial response to wounding (Lopez-Galvez et al. 1997, Thomas-Barberan

et al. 1997, Campos-Vergas and Saltveit 2002) that ultimately results in increased

concentrations of phenolic compounds (Loaiza-Velarde et al. 1997). The

phenylpropanoid pathway starts with the deamination of phenylalanine to cinnamic acid

due to the action of PAL. The enhanced activity of PAL results in an increased

production and accumulation of several phenolic compounds that are sequestered in the

vacuole. These compounds can be oxidized to strong electrophillic quinones (brown

substances) by PPO when membranes become disrupted. In addition, wounding also

results in an increased expression of POX and lignin formation (Luh and Phithakpol

1972, Ribereau Gayon 1972, Robinson 1972, Hanson and Havir 1979, Rhodes et al.

1981).

Higher activity of PAL was found in resistant cultivars of lettuce infested with

lettuce root aphid, Pemphigus bursarius (L.) (Cole 1984) and barley infested with

greenbug, Schizaphis graminum (Rondani) (Chaman et al. 2003). The activity of PAL

was also increased in strawberry leaves as a result of infestation by twospotted spider

mite, Tetranychus urticae (Inoue et al. 1985).

Insect resistance in many plant species (soybean, tomato, potato, cotton, rubber

tree, poplar and barley) has been associated with higher activity of PPO (Gregory and

Tingey 1981, Hedin et al. 1983, Felton et al. 1989, Duffey and Felton 1991, Steffens and









Walter 1991, Bi et al. 1993, Felton et al. 1994a, Constabel et al. 1996, Wititsuwannakul

et al. 2002, Wang and Constabel 2004, Chaman et al. 2001). Peroxidase activity is also

known to increase in tomato and barley after infestation with corn earworm, Helicoverpa

zea Boddie (Stout et al. 1999) and greenbug (Chaman et al. 2001), respectively.

Earlier tests by Huang et al. 2003 found only localized induced resistance in

Valmaine after 2 d ofD. balteata damage. It is possible the 2 d feeding duration was not

long enough to induce increased resistance (Schoonhoven et al. 2006). In our study,

significant increases in the levels of all the three enzymes (PAL, POX and PPO) were

only found at 3 and 6 d after feeding damage, but not after 1 d of feeding on Valmaine.

Female beetles confined for 1 d on Valmaine plants had gained weight, lending support

to the hypothesis that increased resistance is only induced after at least 2 d of feeding.

Beetles were observed tunneling, and presumably feeding, in the midrib tissue near the

proximal end of the leaf. However, after 3 d, beetles did not feed much and lost weight

over the remaining 3 d of the experiment. So, beetles may have stopped feeding due to

induced resistance. Under these conditions, plants may have reached an equilibrium of

defensive compounds concentrations and enzyme activities, and stopped further

increment in the activities of these enzymes to save energy for development and growth. I

also found a strong relationship between female weight gain and activities of all the three

enzymes (PAL, PPO and POX), indicating a possible correlation between increased

enzymes activities and decreased beetle fitness.

Based on my results, I hypothesize that increased levels of PAL, PPO and POX in

Valmaine after D. balteata damage result in increased production of secondary

metabolites and other unknown defensive compounds. Consequently, induced resistance









in Valmaine acts synergistically with the constitutive resistance of latex and ultimately

enhances its resistance against D. balteata. Further research is required to characterize

these damage-inducible enzymes at the molecular level to support breeding programs for

the development of resistant cultivars with superior horticultural traits using either

conventional or transgenic approaches.

































Figure 5-1. Feeding damage caused by D. balteata adults on two lettuce cultivars,
Valmaine (VAL) and Tall Guzmaine (TG).

















*/- ] ^ a
r' T^!'' ""IBF" IB "


i.TIall Guzmaine ..-" .







Darmaged lnda~
Diinragied {ndams~~Zhedl


Figure 5-2. Adults ofD. balteata feeding on diet disks treated with latex from damaged
and undamaged plants of two lettuce cultivars, Valmaine and Tall Guzmaine.












S Damaged
Undamaged


V6

:5

4
4--
t3
U)
U)
2
4--
0
L 1
U)
E O
z


1 2 3 4


1 2 3 4


Time (h)


Figure 5-3. Number ofD. balteata adults feeding on artificial diet disks in a choice between latex from damaged and undamaged
plants of Valmaine after 1, 2, 3 and 4 h of their release. Error bars indicate SEM. Bars topped with different letters within
panel (day 1, 3 or 6) differ significantly at the 0.05 level (Tukey's HSD test).


1 2 3 4


Day 1 Day 3 Day 6




ns a a

a a a a
bb
.. b b b b b b b












Damaged
Undamaged

6
Day 1 Day 3 Day 6
5 5
nsns ns
S4-



3 4-



(A z 1 2 3 4 1 2 3 4 1 2 3 4
Time (h)


Figure 5-4. Number ofD. balteata adults feeding in a choice test using two artificial diet disks treated with damaged and undamaged
plants of lettuce cultivar, Tall Guzmaine after 1, 2, 3 and 4 h of their release. Error bars indicate SEM. Bars topped with
different letters within panel (day 1, 3 or 6) differ significantly at the 0.05 level (Tukey's HSD test).










Damaged
Undamaged

25
2C VAL ns TG
E20
C
0
15 15
E a a
S10 b
c b
O0
bc
0
1 3 6 1 3 6
Days after beetle damage


Figure 5-5. Artificial diet consumption after 24 h by D. balteata adults in choice test
using two diet disks treated with latex from damaged and undamaged plants of
two lettuce cultivars, Valmaine (VAL) and Tall Guzmaine (TG). Error bars
indicate SEM. Bars topped with different letters with panel (VAL or TG)
differ significantly at the 0.05 level (Tukey's HSD test).









-- VAL Damaged
-o- VAL Undamaged
- TG Damaged
-- TG Undamaged


Days after beetle damage


Figure 5-6. Total protein content in two lettuce cultivars, Valmaine (VAL) and Tall
Guzmaine at 1, 3 and 6 d after initiation of feeding damage by adults of D.
balteata. Error bars indicate SEM. Points topped with different letters differ
significantly at the 0.05 level (Tukey's HSD test).


30


25


20


15 /


def










-*-
-0-


10



8



6



4


2-



0


VAL Damaged
VAL Undamaged
TG Damaged
TG Undamaged


Days after beetle damage


Figure 5-7. Activity of phenylalanine ammonia lyase (PAL) in two lettuce cultivars,
Valmaine (VAL) and Tall Guzmaine at 1, 3 and 6 d after initiation of feeding
damage by adults ofD. balteata. Error bars indicate SEM. Points topped with
different letters differ significantly at the 0.05 level (Tukey's HSD test).


a





b

bcd bc
bcd

de cde cde


e -ee
e










-0--

-o-


VAL Damaged
VAL- Undamaged
TG Damaged
TG Undamaged


Days after beetle damage


Figure 5-8. Activity of polyphenol oxidase (PPO) in two lettuce cultivars, Valmaine
(VAL) and Tall Guzmaine at 1, 3 and 6 d after initiation of feeding damage by
adults ofD. balteata. Error bars indicate SEM. Points topped with different
letters differ significantly at the 0.05 level (Tukey's HSD test).













159


a a






b b

bc



cd d d









-- VAL Damaged
-o- VAL Undamaged
T TG Damaged
-- TG Undamaged


0.08
a


0.06 ab



0.04 bc



0.02 T


0.00


1 3 6


Days after beetle damage


Figure 5-9. Activity of peroxidase (POX) in two lettuce cultivars, Valmaine (VAL) and
Tall Guzmaine at 1, 3 and 6 d after initiation of feeding damage by adults of
D. balteata. Error bars indicate SEM. Points topped with different letters
differ significantly at the 0.05 level (Tukey's HSD test).












2.0
a
-*- VAL
1.5 -
a) b


4 1.0 c

t-1-

0.5
0))
.c
d e
_e I e
S0.0
L.

-0.5
1 3 6

Days after beetle damage


Figure 5-10. Gain in fresh weight of D. balteata females over a 6-d period of feeding on
two romaine lettuce cultivars, Valmaine (VAL) and Tall Guzmaine (TG).
Error bars indicate SEM. Points topped with different letters differ
significantly at the 0.05 level (Tukey's HSD test).














VAL

*y = 7.15 7.34x
R sq = 0.57
P= 0.0012


0
0.0 0.5 1.0 1.5 2.0 0
0')
E
o
c0
d


10

8

6
0)
E 4
S2
o



75

E
- 10

: 8

6
Q- A


10

S8

0 6
-4
a_ 4


2 _
O
0
0.0 0.5 1.0 1.5 2.0 2.5
Fresh weight gain / female (mg)


VAL

y = 6.06 5.95x


0.10

0.08

0.06


\ sq = u.54
S P= 0.0019 0.04
c 0.02

0 0.00
0.0 0.5 1.0 1.5 2.0
E

E

0.10
y =1.04 0.11x TG 5:
0.08
R sq = 0.04 8
P= 0.4701 > 0.06

0.04


2
0-----'-";------
0
0.0 0.5 1.0 1.5 2.0 2.5
Fresh weight gain /female (mg)


0.0 0.5 1.0 1.5 2.0


y = 0.02 0.002x
R sq = 0.15
P = 0.6646


0.02

0.00
0.0 0.5 1.0 1.5 2.0 2.5
Fresh weight gain / female (mg)


Figure 5-11. Relationship between fresh weight gained by D. balteata females feeding on two lettuce cultivars, Valmaine (VAL) and
Tall Guzmaine (TG) and activity of A) PAL, B) PPO and C) POX enzymes after 1, 3 and 6 d of feeding damage.


y = 1.55- 0.25x
R sq = 0.10
P= 0.2621


1


1









Table 5-1. Total diet consumption by six D. balteata adults on two diet disks treated with
latex from same lettuce cultivar, Valmaine or Tall Guzmaine after 24 h of
their release.


Days after Damage

1

3

6


Tall Guzmaine


Total Diet Consumption (mg)

15.21.8c

13.51.2c

12.41.2c

36.71.9b


35.40.9b

32.1+3.8b

45.81.2a

47.32.6a

46.81.5a


Means SEM followed by different letters within column differed significantly (P < 0.05) using ANOVA
and Tukey's HSD test.


Cultivar

Valmaine


Control









CHAPTER 6
SUMMARY

Lettuce, Lactuca sativa L., is one of the most important vegetable crops grown

throughout the world, especially in the United States. California is the major producer of

lettuce in the United States (77 % of all lettuce harvested) followed by Arizona, Florida

and New Jersey. In Florida, lettuce production from the Everglades Agricultural areas in

south Florida contributes 90% of the total state production. Lettuce suffers economic

losses due to several insect pests, such as cabbage lopper, Trichoplusia ni (Hubner); beet

armyworm, Spodoptera exigua (Hubner); banded cucumber beetle, Diabrotica balteata

Leconte; and leafminer, Liriomyza trifolii (Burgess). For the management of these pests,

growers are dependent on pesticides. Approximately 93% of the lettuce acreage in the

United States is treated with the insecticides. Florida ranks first among lettuce growing

states in the usage of insecticides to manage these insect pests. Therefore, there is a need

to look for alterative strategies for management of economic insect pests. Management of

insects with host plant resistance is an important component of integrated pest

management strategies.

The romaine lettuce cultivar, 'Valmaine' is known to possess a high level of

resistance to D. balteata and the leafminer. Diabrotica balteata feeding is increased after

removal of leaf surface chemicals in Valmaine with methylene chloride, but these surface

chemicals did not show any deterrence when applied to leaf surfaces of palatable lima

bean at different concentrations. Therefore, it seems that internal factors are involved

rather than external chemical factors in imparting resistance against D. balteata in

Valmaine. Further, previously wounded Valmaine plants showed an increased localized









resistance to D. balteata compared to unwounded plants suggesting the involvement of

an inducible mechanism of resistance.

The purpose of this research was to investigate the extent of resistance in the lettuce

cultivar Valmaine against another order of economically important lettuce pests, the

Lepidoptera. The second objective was to identify the mechanism of this multiple insect

resistance.

To address the first objective, I compared the survival, development and feeding

behavior of cabbage looper and beet armyworm on two romaine lettuce cultivars,

resistant Valmaine and the closely-related susceptible 'Tall Guzmaine'. Larval mortality

of both species was significantly higher on Valmaine than on susceptible Tall Guzmaine.

Significant difference between the cultivars was also observed in development. Larvae

weighed six times (beet armyworm) and two times (cabbage looper) more after feeding

for 1 wk on Tall Guzmaine than on Valmaine. Larval period was 5.9 (beet armyworm)

and 2.6 d (cabbage looper) longer on Valmaine than on Tall Guzmaine. Pupal duration of

both insect species was also increased by almost 1 d by feeding on Valmaine compared to

Tall Guzmaine. Weights of the pupa and adult of both insect species were reduced on

Valmaine compared to Tall Guzmaine. The sex ratio of progeny did not deviate from 1:1

when larvae were reared on resistant Valmaine. The fecundity of cabbage looper and beet

armyworm moths that developed from larvae reared on Valmaine was about one third

that of moths from Tall Guzmaine, but adult longevity did not significantly differ on the

two lettuce cultivars.

Feeding behavior of these moth species was also significantly affected by lettuce

cultivar. The two insect species showed different feeding preference for leaves of









different age groups on Valmaine and Tall Guzmaine. Cabbage looper preferred to feed

on the lowermost fully mature leaves of Valmaine plants and on young and middle-aged

leaves of Tall Guzmaine plants (rarely feeding on fully-matured leaves). Beet armyworm

preferred to feed on the lowermost fully mature leaves of Valmaine plants and on middle-

aged leaves of Tall Guzmaine plants. Both insect species preferred to feed on the distal

end of leaves. Early instars of cabbage looper preferred to feed on the underside of the

leaf, whereas early instars of beet armyworm fed on the upper side of the leaf. Cabbage

loopers also cut narrow trenches on the leaf before actual feeding to block the flow of

latex to the intended site of feeding. In contrast, beet armyworms did not trench; neonates

made shallow scratches between the veins by feeding on parenchymatous tissue and

second instars made holes through the leaf. The different feeding behavior of the two

species on Valmaine may explain the superior performance of cabbage looper compared

to beet armyworm.

Lettuce is a laticiferous plant, meaning that it produces a white milky fluid after

tissue damage. Latex is stored under pressure in the laticifers. Plant latex is a known

defense in certain plants through its physical and chemical properties against several

insects. Therefore to address my second objective, i.e. identification of mechanism of

resistance in Valmaine romaine lettuce, I hypothesized that latex also plays a defensive

role in lettuce. I again used two romaine lettuce cultivars, Valmaine (resistant) and Tall

Guzmaine (susceptible) to study the potential of latex as a defense mechanism against D.

balteata.

Latex from Valmaine strongly inhibited D. balteata feeding compared to Tall

Guzmaine when applied to the surface of artificial diet. The amount of diet consumed









from Valmaine latex treated disks was significantly less than that consumed from diet

treated with Tall Guzmaine latex, in both choice and no-choice tests. The number of

adults feeding on diet treated with Valmaine latex was less compared to Tall Guzmaine

latex treated diet after 15, 30, 60 and 90 minutes of their release. These studies suggest

that latex may account for resistance in Valmaine to D. balteata.

All four species that have been tested on Valmaine and Tall Guzmaine (D. balteata,

leafminer, cabbage looper and beet armyworm) prefer to feed on the lowermost fully

matured leaves of resistant cultivar Valmaine. Therefore I decided to test whether this

kind of behavior is mediated through any differences in the properties of latex from

young and mature leaves. Latex from the young leaves is more viscous and solid white,

whereas it is more watery and translucent in the mature leaves. Hence, I conducted choice

tests using two artificial diet disks, one treated with latex from young leaves and the

second one treated with latex from mature leaves.

There was a significant interaction between leaf age and variety on diet

consumption by the beetles. In Valmaine latex treated choice tests, the beetles consumed

significantly less diet treated with latex from young leaves than that consumed from diet

treated with latex from mature leaves. No significance difference in diet consumption

was found between diets treated with latex from young and mature leaves in Tall

Guzmaine latex treated choice tests. So, this may explain insect avoidance of young and

middle-aged leaves of Valmaine.

After these studies, I was confident that the multiple insect resistance observed in

Valmaine was mediated through latex. So in order to further investigate whether this

resistance was due to physical or chemical properties of latex, I prepared a crude extract









by dissolving latex in different solvents. Three solvents of differing polarity (water,

methanol and methylene chloride) were tested to extract and compare deterrent

compounds from Valmaine and Tall Guzmaine latex.

Solvents and the interaction of solvent with lettuce cultivar had significant deterrent

affects on beetle feeding. Valmaine latex extracted with water:methanol (20:80) strongly

inhibited beetle feeding when applied to the surface of artificial diet. The percentage of

beetles feeding on diet treated with Valmaine water:methanol (20:80) extract was less

compared to Tall Guzmaine water:methanol (20:80) extract treated diet at intervals of 15,

30, 60 and 90 min after their release. The amount of diet consumed in no-choice tests

from disks treated with Valmaine water:methanol (20:80) extract was significantly less

than that consumed from diet disks treated with Tall Guzmaine methanol:water (80:20)

extract.

To study the role of physical properties of latex in Valmaine resistance, I conducted

a small study by applying fresh latex on the mandibles ofD. balteata adults. Beetles

salivated more when Valmaine latex was applied to their mouthparts compared to Tall

Guzmaine latex but mandibles and maxillae were not gummed up and were moving

freely 24 h after application of either Valmaine or Tall Guzmaine latex (although there

were traces of dried latex on the labium and tarsi). These studies strongly indicated a

biochemical rather than physical basis of resistance in Valmaine to D. balteata. The

ability to extract deterrent compounds in water:methanol (20:80) suggested that

moderately polar chemicals within latex may account for the observed resistance.

The next series of steps were conducted to isolate deterrent chemicals from the

crude Valmaine latex extract (water:methanol, 20:80). The crude extract was first passed









through C-18 cartridges at three different pH levels (natural, acidic and alkaline) to

evaluate its relative polarity. No significant deterrent activity was found in the fraction

eluted from the cartridge using a step gradient of water:methanol mixtures. The activity

was only found in the unbound fraction eluting from the C-18 cartridge, indicating that

the deterrent compounds were highly polar. Next, the C-18 unbound fraction was passed

through anion exchange and cation exchange cartridges connected in series. The retained

compounds on both ion exchange cartridges were tested for feeding deterrence after they

were eluted using a NaCl salt gradient. The 0.5M fraction obtained from the cation

exchange cartridge possessed the highest deterrent activity. Retention of the deterrent

compounds in Valmaine latex on the cation exchange column indicates its basic nature. A

fraction eluting between 3 and 4 min exhibited the strongest deterrent activity during

further fractionation of cation exchange extract using HPLC-MS. UV absorption and MS

data indicated the presence of ten compounds in this active fraction and some of these

compounds have substituted aromatic structure. Hence, these results strongly support my

hypothesis that unacceptability of Valmaine to D. balteata is primarily due to chemical

constituents of latex.

Previous research showed that there was a localized induced resistance in Valmaine

plants after feeding by D. balteata. In general, induced resistance involves changes in

plant defensive chemistry due to alteration in the levels of various enzymes, such

phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO) and peroxidase (POX).

Therefore, my next steps were to evaluate the potential activity of these three enzymes in

Valmaine and Tall Guzmaine lettuce. The questions I tried to answer were if, and how

quickly such enzymes could be activated after beetle damage. If such enzymes were









present and inducible by beetle feeding then for how long were levels increased, and did

their higher activity correlate with feeding deterrent activity in the latex. To answers

these questions, I first tested for inducible enzymatic activity by giving D. balteata

adults a choice between diets treated with latex from either damaged or undamaged

plants. Separate tests were run with extracts from Valmaine and Tall Guzmaine. I

investigated the expression of inducible enzymes phenylalanine ammonia lyase,

polyphenol oxidase and peroxidase in the latex of both damaged and undamaged plants

of Valmaine and Tall Guzmaine. Diet consumption was significantly reduced when disks

were treated with latex collected from beetle-damaged Valmaine plants 3 and 6 d after

feeding initiation. No significant difference was found in the diet consumption when

disks were treated with latex from beetle-damaged Tall Guzmaine plants. Activities of all

the three enzymes were significantly enhanced in Valmaine latex after 3 and 6 d of

damage, whereas activity remained low in latex from damaged Tall Guzmaine plants.

The constitutive levels of PAL and PPO were also significantly higher in latex from

undamaged Valmaine compared to Tall Guzmaine plants. So, it seems that Valmaine is

better defended in terms of higher expression of these enzymes both at constitutive and

induced levels. On Valmaine, beetles gained weight after 1 d of feeding, but then lost

weight after being confined on the plants for 3 and 6 d. Fresh weight gain of female D.

balteata fed Tall Guzmaine plants increased in a linear fashion over the 6 d exposure

period. However, a significant negative relationship was found between weight gain and

activities of PAL, PPO and POX in Valmaine latex. These studies suggest that latex

chemistry may change after beetle feeding damage due to increased activity of inducible









enzymes, and that inducible resistance appears to act synergistically with constitutive

resistance in Valmaine latex.

Based on my findings, it appears that Valmaine possesses both constitutive and

induced resistance mechanisms, and both are mediated through latex. Solvent extraction

studies of the deterrent compounds suggest the presence of biologically active

nitrogenous compounds in Valmaine latex, while enzyme induction studies after insect

damage indicate an increase in the phenolic compounds. Hence, constitutive and induced

defenses in Valmaine may involve different biochemicals. In a situation where there is no

constant insect pressure, Valmaine exhibits a constitutive defense and is a non-preferred

host. However, in situations where there is prolonged insect pressure, and those insects

either have no choice but to feed on Valmaine or are not significantly deterred by the

constituitive defenses, inducible enzymatic activity in Valmaine may turn on the second

line of defense to protect itself from further damage. Therefore, both types of defenses

might be acting synergistically in Valmaine.

Further, Valmaine exhibited resistance only against insects having chewing

mouthparts (D. balteata adults, leafminer maggots and beet armyworm and cabbage

looper caterpillars) and not against insects having sucking mouthparts, such as whitefly

(unpublished, Heather McAuslane), aphids (unpublished, Gregg Nuessly) and thrips

(unpublished, Amit Sethi). This dichotomy may be an outcome of the mechanism of

resistance in Valmaine. Because latex is found in laticifers which run parallel to the

vascular system in the plant, chewing insects accidentally rupture the laticifers when

attempting to feed on lettuce, resulting in their exposure to latex-borne feeding deterrents.

On the other hand, most of the successful sucking insects are known to feed









intercellularly and in this way avoid or reduce the frequency of rupturing laticifers. This

may explain why Valmaine only possesses resistance against chewing insects and not

against sucking insects.

Based on studies done so far, I propose a biochemical basis for host plant resistance

in Valmaine. Further research is required to identify the deterrent compounds both at

constitutive and induced levels and also to characterize these inducible enzymes at the

molecular level so that both can be used as selection markers during breeding programs

to improve lettuce varieties.









LIST OF REFERENCES


Agnew, K. 2000. Crop profile for lettuce in Arizona. Pesticide Information and Training
Office. University of Arizona, Arizona.

Agrawal, A. A. 1999. Induced plant defense in plants: the ecology and evolution of
restrained defense, pp. 137- 166. In A. A. Agrawal, S. Tuzun, and E. Bent [eds.],
Induced plant defenses against pathogens and herbivores. Biochemistry, Ecology
and Agriculture. APS Press, St. Paul.

Agricultural Statistics. 2001. Statistical highlights of U.S. agriculture 2000-2001. USDA,
NASS. http://www.nass.usda.gov/index.asp.

Agricultural Statistics. 2003. Statistical highlights of U.S. agriculture 2002-2003. USDA,
NASS. http://www.nass.usda.gov/index.asp.

Agricultural Statistics. 2007. Vegetables 2006 Summary. January 2007. USDA, NASS.
http://www.nass.usda.gov/index. asp.

Alleyne, E. H., and F. O. Morrison. 1977. The lettuce root aphid, Pemphigus bursarius
(L.) (Homoptera: Aphidoidea) in Quebec, Canada. Ann. Entomol. Soc. Quebec 22:
171-180.

Anonymous. 1999. Crop Profile for Celery in Florida. The National Science Foundation
Center for Integrated Pest Management, North Carolina State University, Raleigh,
NC. http://cipm.ncsu.edu/cropprofiles/docs/FLCelery.html.

Anonymous. 2003. Integrated Pest Management for Cole Crops and Lettuce, pp. 112.
Agriculture and Natural Resources, University of California, Davis.
http://www.ipm.ucdavis.edu/PMG/selectnewpest.lettuce.html.

Argandona, V. H., M. Chaman, L. Cardemil, O. Munoz, G. E. Zuniga, and L. J.
Corcuera. 2001. Ethylene production and peroxidase activity in aphid-infested
barley. J. Chem. Ecol. 27: 53-68.

Auad, A. M., and J. C. Moraes. 2003. Biological aspects and life table of Uroleucon
ambrosiae as a function of temperature. Sci. Agricola 60: 657-662.

Azarkana, M., R. Wintjensb, Y. Loozeb, and D. Baeyens-Volant. 2004. Detection of
three wound-induced proteins in papaya latex. Phytochemistry 65: 525-534.

Baldwin, I. T. 1994. Chemical changes rapidly induced by folivory, pp. 1-23. In E. A.
Bernays [ed.], Insect-plant interactions, vol 5. CRC Press Incorporated, Boca
Raton.

Barton, D. H. R., and C. R. Narayanan. 1958. Sesquiterpenoids. Part X. The constituents
of lactucin. J. Chem. Soc. 1: 963-971.









Bauhin. 1671. Cited from E. L. Sturtevant. 1886. A study of garden lettuce. Am. Nat. 20:
230-233.

Becerra, J. X., D. L. Venable, P. H. Evans, and W. S. Bowers. 2001. Interaction between
chemical and mechanical defenses in the plant genus Bursera and their implications
for herbivore. Am. Zool. 41: 865-876.

Bellows, B. C., and S. Diver. 2002. Cucumber beetles: organic and biorational IPM.
ATTRA National Sustainable Agriculture Information Service, Fayetteville, AR.

Bennett, M. H., M. Gallagher, C. Bestwich, J. Rossiter, and J. Mansfield. 1994. The
phytoalexin response of lettuce to challenge by Botrytis cinerea, Bremia lactucae
and Pseudomonas syringae pv. phaseolicola. Physiol. Mol. Plant Pathol. 44: 321-
333.

Bergey, D., G. Howe, and C. A. Ryan. 1996. Polypeptide signaling for plant defensive
genes exhibits analogies to defense signaling in animals. Proc. Natl. Acad. Sci.
USA 93: 12053-12058.

Bemays, E. A., and R. F. Chapman. 1977. Deterrent chemicals as a basis of oligophagy
in Locusta gregaria L. Ecol. Entomol. 2: 1-18.

Bemeys, E. A., and R. F. Chapman. 1994. Host plant selection by phytophagous insects.
Chapman & Hall, New York.

Bemays, E. A., and A. C. Lewis. 1986. The effect of wilting on palatability of plants to
Schistocerca gregaria, the desert locust. Oecologia 70: 132-135.

Beshear, R. J. 1983. New records ofthrips in Georgia. J. Georgia Entomol. Soc. 18: 342-
344.

Bestwick, L., A. L. Adam, N. Puri, and J. W. Mansfield. 2001. Characterization of and
changes to pro- and anti-oxidant enzyme activities during the hypersensitive
reaction in lettuce (Lactuca sativa L.). Plant Sci. 161: 497-506.

Bestwick, L., M. H. Bennett, J. W. Mansfield, and J. T. Rossiter. 1995. Accumulation of
the phytoalexin lettucenin A and changes in 3-hydroxy-3-methylglutaryl coenzyme
A reductase activity in lettuce seedlings with the red spot disorder. Phytochemistry
39: 775-777.

Bi, J. L., G. W. Felton, and A. J. Mueller. 1993. Induced resistance in soybean to
Helicoverpa zea: Role of plant protein quality. J. Chem. Ecol. 20: 183-198.

Bi, J. L., G. W. Felton, J. B. Murphy, P. A. Howles, R. A. Dixon, and C. J. Lamb. 1997a.
Do plant phenolics confer resistance to specialist and generalist insect herbivores?
J. Agric. Food Chem. 45: 4500-4504.









Bi, J. L., J. B. Murphy, and G. W. Felton. 1997b. Antinutritive and oxidative components
as mechanisms of induced resistance in cotton to Helicoverpa zea. J. Chem. Ecol.
23: 97-117.

Bibby, F. F. 1958. Notes on thrips of Arizona. J. Econ. Entomol. 51: 450-452.

Blackman, R. L., and V. F. Eastop. 2000. Aphids on the world's crops. John Wiley &
Sons, Chichester, UK.

Bowles, D. J. 1990. Defense-related proteins in higher plants. Annu. Rev. Biochem. 59:
873-907.

Braun, J., and M. Tevini. 1993. Regulation of UV-protective pigment synthesis in the
epidermal layer of rye seedlings (Secale cereale L. cv. kustro). Photochem.
Photobiol. 57: 318-323.

Breda, C., D. Buffard, R. B. van Huystee, and R. Esnault. 1993. Differential expression
of two peanut peroxidase cDNA clones in peanut plants and cells in suspension
culture in response to stress. Plant Cell Rep. 12: 268-272.

Brignolas, F., B. Lacroix, F. Lieutier, D. Sauvard, A. Drouet, A. C. Claudot, A. Yart, A.
A. Berryman, and E. Christiansen. 1995. Induced response in phenolic metabolism
in two Norway spruce clones after wounding and inoculation with Ophiostoma
polonicum, a bark beetle-associated fungus. Plant Physiol. 109: 821-827.

Britsch, L. 1990. Purification and characterization offlavone synthase 1, a 2-
oxoglutarate-dependent desaturase. Arch. Biochem. Biophys. 282: 152-160.

Broekaert, W., H.-I. Lee, A. Kush, N-H Chua, and N. Raikhel. 1990. Wound induced
accumulation of mRNA containing a hevein sequence in lacticifers of rubber tree
(Hevea brasiliensis). Proc. Natl. Acad. .Sci. 87: 7633-7637.

Brower, L. P., J. N. Seiber, C. J. Nelson, P. Tuskes, and S. P. Lynch. 1982. Plant
determined variation in the cardenolide content, thin layer chromatography profiles,
and emetic potency of monarch butterflies, Danausplexippus reared on milkweed,
Asclepias eriocarpa in California. J. Chem. Ecol. 8: 579-633.

Bryan, D. E., and R. F. Smith 1956. The Frankliniella occidentalis (Pergande) complex
in California. Univ. Calif. Public Entomol. 10: 359-410.

Bryant, J. B., F. S. Chapin III, and D. R. Klein. 1983. Carbon/nutrient balance of boreal
plants in relation to vertebrate herbivory. Oikos 40: 357-368.

Burnett, W. C., S. B. Jones, and T. J. Mabry. 1978. The role of sesquiterpene lactones in
plant and animal coevolution, pp. 233-257. In J. B. Harborne [ed.], Biochemical
aspects of plant animal coevolution. Academic Press, London.









Butt, V. S. 1980. Direct oxidases and related enzymes, pp. 81-123. In E. E. Conn and
P.K. Stumpf [eds.], The biochemistry of plants, vol.2. Academic Press, New York.

Buttery, B. R., and S. G. Boatman. 1976. Water deficits and flow of latex, pp. 233-289.
In T.T. Kozlowski [eds.], Water deficits and plant growth, vol. IV. Academic Press,
New York, USA.

CABI. 1972. Distribution maps of pests. Spodoptera exigua (Hibner). Commonwealth
Agricultural Bureau, London. Series A, Map 302.

CABI, 2006. Distribution Maps of Plant Pests. Diabrotica balteata. Commonwealth
Agricultural Bureau, London. Map 681.

Campos-Vargas, R., and M. E. Saltveit. 2002. Involvement of putative chemical wound
signals in the induction of phenolic metabolism in wounded lettuce. Physiol. Plant.
114:73

Capellades, M., M. A. Torres, I. Bastisch, V. Stiefel, F. Vignols, W. R. Bruce, D.
Peterson, P. Puigdomenech, and J. Rigau. 1996. The maize caffeic acid O-
methyltransferase gene promoter is active in transgenic tobacco and maize plant
tissues. Plant Mol. Biol. 31: 307-322.

Capinera, J. L. 1999. Banded cucumber beetle. Featured Creatures [Online]. Publication
Number: EENY-105. University of Florida, Department of Entomology and
Nematology. http://creatures.ifas.ufl.edu/veg/bean/bandedcucumberbeetle.htm.

Capinera, J. L. 2004. Green peach aphid. Featured Creatures [Online]. Publication
Number: EENY-222. University of Florida, Department of Entomology and
Nematology. http://creatures.ifas.ufl.edu/veg/aphid/green_peach_aphid.htm.

Capinera, J. L. 2005. Cabbage looper. Featured Creatures [Online]. Publication Number:
EENY-116. University of Florida, Department of Entomology and Nematology.
http://creatures.ifas.ufl.edu/veg/leaf/cabbagelooper.htm.

Capinera, J. L. 2006. Beet armyworm. Featured Creatures [Online]. Publication Number:
EENY-105. University of Florida, Department of Entomology and Nematology.
http://creatures.ifas.ufl.edu/veg/leaf/beetarmyworm.htm.

Carpita, N. C., and D. M. Gibeaut. 1993. Structural models of primary cell walls in
flowering plants: consistency of molecular structure with the physical properties of
the walls during growth. Plant J. 3: 1-30.

Cassab, G. I., and J. E. Varner. 1988. Cell wall proteins. Annu. Rev. Plant Physiol. Plant
Mol. Biol. 39: 321-353.

Chaman, M. E., S. V. Copaja, and V. H. Argandon. 2003. Relationships between salicylic
acid content, phenylalanine ammonia-lyase (PAL) activity, and resistance of barley
to aphid infestation. J. Agric. Food Chem. 51: 2227-2231.









Chaman M. E., L. J. Corcuera, G. E. Zuniga, L. Cardemil, V. H. Argandona. 2001.
Induction of soluble and cell wall peroxidases by aphid infestation in barley. J.
Agric. Food Sci. 49: 2249-2253.

Chan, B. G., and A. C. Waiss. 1978. Condensed tannin, an antibiotic chemical from
Gossypium hirsutum. J. Insect Physiol. 24: 113-118.

Chan, B. G., A. C. Waiss, R. G. Binder, and C. A. Elliger. 1978. Inhibition of
lepidopterous larval growth by cotton constituents. Entomol. Exp. Appl. 24: 94-
100.

Chapman, R. F. 1974. The chemical inhibition of feeding by phytophagous insects: a
review. Bull. Entomol. Res. 64: 339-363.

Chapman, R. F. 2003. Contact chemoreception in feeding by phytophagous insects.
Annu. Rev. Entomol. 48: 455-484.

Chyb, S., H. Eichenseer, B. Hollister, C. A. Mullin, and J. L. Frazier. 1995. Identification
of sensilla involved in taste mediation in adult western corn rootworm (Diabrotica
virgifera virgifera LeConte). J. Chem. Ecol. 21: 313-329.

Clausen, T. P., P. B. Reichardt, J. P. Bryant, R. A. Werner, K. Post, and K. Frisby. 1989.
Chemical model for short-term induction in quaking aspen (Populus tremuloides)
foliage against herbivores. J. Chem. Ecol. 15: 2335-2346.

Cohen, A. C., C. C. Chu, T. J. Henneberry, T. Freeman, D. Nelson, J. Buckner, D.
Margosan, P. Vail, and L. H. Aung. 1998. Feeding biology of the silverleaf
whitefly (Homoptera: Aleyrodidae). Chin. J. Entomol. 18: 65-82.

Cohen, A. C., T. J. Henneberry, and C. C. Chu. 1996. Geometric relationships between
whitefly behavior and vascular bundle arrangements. Entomol. Exp. Appl. 78: 135-
142.

Cole R. A. 1984. Phenolic acids associated with the resistance of lettuce cultivars to the
lettuce root aphid. Ann. Appl. Biol. 105: 129-145.

Coley, P. D. 1983. Herbivory and defensive characteristics of tree species in a lowland
tropical forest. Ecol. Monographs 53: 209-233.

Condon, J. M., and B. A. Fineran. 1989. Distribution and organization of articulated
laticifers in Calystegia silvatica (Convolvulaceae). Bot. Gaz. 150: 289-302.

Constabel, C. P. 1999. A survey of herbivore-inducible defense proteins and
phytochemicals, pp. 137-166. In A. A. Agrawal, S. Tuzun and E. Bent [eds.],
Induced plant defenses against pathogens and herbivores: biochemistry, ecology
and agriculture. APS Press, St. Paul.









Constabel, C. P., D. R. Bergey, and C. A. Ryan. 1995. Systemin activates synthesis of
wound inducible tomato leaf polyphenol oxidase via the octadecanoid defense
signaling pathway. Proc. Natl. Acad. Sci. USA 92: 407-411.

Constabel, C. P., D. R. Bergey, and C. A. Ryan. 1996. Polyphenol oxidase as a
component of the inducible defense response in tomato against herbivores, pp. 231-
252. In J. T. Romeo, J. A. Saunders, and P. Barbosa [eds.], Phytochemical diversity
and redundancy in ecological interactions. Plenum Press, New York.

Constabel, C. P., Y. Peter, P. Lynn, J. Joseph, Christopher, M. E. 2000. Polyphenol
oxidase from hybrid poplar: cloning and expression in response to wounding and
herbivory. Plant Physiol. 124: 285-296.

Constabel, C. P., and C. A. Ryan. 1998. A survey of wound and methyl jasmonate-
induced leaf polyphenol oxidase in crop plants. Phytochemistry 47: 507-511.

Corcuera, L. J. 1993. Biochemical basis for the resistance of barley to aphids.
Phytochemistry 33: 741-747.

Costa, H. S., D. E. Ullman, M. W. Johnson, and B. E. Tabashnik. 1993. Association
between Bemisia tabaci density and reduced growth, yellowing, and stem
blanching of lettuce and kai choy. Plant Dis. 77: 969-972.

Cramer, C., K. Edwards, M. Dron, X. Liang, S. L. Dildine, G. P. Bolwell, R. A. Dixon,
C. J. Lamb, and W. Schuch. 1989. Phenylalanine ammonia-lyase gene organization
and structure. Plant Mol. Biol. 12: 367-383.

Creighton, C. S., and E. R. Cuthbert, Jr. 1968. A semisynthetic diet for adult banded
cucumber beetles. J. Econ. Entomol. 61: 337-338.

Crosby, D. G. 1963. The organic constituents of food. 1. Lettuce. J. Food Sci. 28: 347-
355.

Crozier, A., M. E. J. Lean, M. S. McDonald, and C. Black. 1997. Quantitative analysis of
the flavonoid content of commercial tomatoes, onions, lettuce, and celery. J. Agric.
Food Chem. 45: 590-595.

Crute, L. R., and J. A. Dunn. 1980. An association between the resistance to root aphid
(Pemphigus bursarius) and downy mildew (Bremia lactuca Regel) in lettuce.
Euphytica 29: 483-488.

Dangl, J. L., K. Harlbrock, and J. Schell. 1989. Regulation and structure of chalcone
synthase genes, pp. 155-173. In J. K. Vasil and J. Schell [eds.], Cell culture and
somatic cell genetics of plants, Academic Press, New York.

Data, E. S., S. F. Nottingham, and S. J. Kays. 1996. Effect of sweetpotato latex on
sweetpotato weevil (Coleoptera: Curculionidae) feeding and oviposition. J. Econ.
Entomol. 89: 544-549.









Davis, R. M., K. B. Subbarao, and E. A. Kurtz. 1997. Compendium of lettuce diseases.
APS Press, St. Paul, MN.

de Candolle. 1885. Origin of cultivated plants, p. 95. Cited from E. L. Sturtevant. 1886. A
study of garden lettuce. Am. Nat. 20: 230-233.

Dethier, V. G. 1970. Chemical interactions between plants and insects, pp. 83-102. In E.
Sondheimer, and J. B. Simeone [eds.], Chemical ecology. Academic Press, New
York.

Dey, P. M., and J. B. Harborne. 1997. Plant biochemistry. Academic Press, London.

Diaz, J., and F. Merino. 1998. Wound-induced shikimate dehydrogenase and peroxidase
related to lignification in pepper (Capsicum annuum) leaves. J. Plant Physiol. 152:
51-57.

Dickenson, P. B. 1963. Structure composition and biochemistry of Hevea latex, pp. 43-
51. In L. Bateman [ed.], The chemistry and physics of rubber-like substances.
Maclaren and Sons, London.

Dillon, P. M., S. Lowrie, and D. McKey. 1983. Disarming the "Evil woman": petiole
constriction by a sphingid larva circumvents mechanical defenses of its host plant,
Cnidoscolus urens (Euphorbiaceae). Biotropica 15: 112-116.

Dimsey, R., and S. Vujovic. 2003. Lettuce growing. Agriculture notes (AG1119).
Department of Primary Industries, Victoria, Australia.

Dixon, R. A., and M. J. Harrison. 1990. Activation, structure, and organization of genes
involved in microbial defense in plants. Adv. Genet. 28: 166-217.

Dixon, R. A., M. J. Harrison, and N. L. Paiva. 1995. The isoflavonoid phytoalexin
pathway: from enzymes to genes to transcription factors. Physiol. Plant. 93: 385-
392.

Dixon, R. A., and N. L. Paiva. 1995. Stress induced phenylpropanoid metabolism. Plant
Cell 7: 1085-1097.

Douglas, C. J. 1996. Phenylpropanoid metabolism and lignin biosynthesis: from weeds to
trees. Trends Plant Sci. 1:171-178.

Dowd, P. F., and L. M. Lagrimini. 1997. Examination of different tobacco (Nicotiana
spp.) type under and overproducing tobacco anionic peroxidase for their leaf
resistance to Helicoverpa zea. J. Chem. Ecol. 23: 2357-2370.

Duffey, S. S., and G. W. Felton. 1991. Enzymatic antinutritive defenses of the tomato
plant against insects, pp. 167-197. In Hedin PA [ed.], Naturally occurring pest
bioregulators. ACS Press, Washington, DC.









Duffey, S. S., and M. J. Stout. 1996. Antinutritive and toxic components of plant defense
against insects. Arch. Insect Biochem. Physiol. 32: 3-37.

Dunn, J. A. 1959. The biology of the lettuce root aphid. Ann. Appl. Biol. 47: 475-491.

Dunn, J. A. 1974. Study on inheritance of resistance to root aphid Pemphigus bursarius
in lettuce. Ann. Appl. Biol. 76: 9-18.

Dunn, J. A., and D. P. H. Kempton. 1980. Susceptibilities to attack by top aphids in
varieties of lettuce. Ann. Appl. Biol. 94: 58-59.

Dupont, M. S., Z. Mondin, G. Williamson, and K.R. Price. 2000. Effect of variety,
processing and storage on the flavonoid glycoside content and composition of
lettuce and endive. J. Agric. Food Chem. 48: 3957-3964.

Dussourd, D. E. 1993. Foraging with finesse: caterpillar adaptations for circumventing
plant defenses, pp. 92-131. In N. E. Stamp and T. Casey [eds], Ecological and
evolutionary constraints on caterpillars. Chapman and Hall, New York, USA.

Dussourd, D. E. 1995. Entrapment of aphids and whiteflies in lettuce latex. Ann.
Entomol. Soc. Am. 88: 163-172.

Dussourd, D. E. 1997. Plant exudates trigger leaf-trenching by cabbage loppers,
Trichoplusia ni (Noctuidae). Oecologia 112: 362-369.

Dussourd, D. E. 2003. Chemical stimulants of leaf-trenching by cabbage loopers: natural
products, neurotransmitters, insecticides, and drugs. J. Chem. Ecol. 29: 2023-2047.

Dussourd, D. E., and R. F. Denno. 1991. Deactivation of plant defense: correspondence
between insect behaviour and secretary canal architecture. Ecology 72: 1383-1396.

Dussourd, D. E., and R. F. Denno. 1994. Host range of generalist caterpillars: trenching
permits feeding on plants with secretary canals. Ecology 75: 69-78.

Dussourd, D. E., and A. M. Hoyle. 2000. Poisoned plusiines: toxicity of milkweed latex
and cardenolides to some generalist caterpillars. Chemoecology 10: 11-16.

Dyer, W. E., J. M. Henstrand, A. K. Handa, and K. M. Herrmann. 1989. Wounding
induces the first enzyme of the shikimate pathway in Solanaceae. Proc. Natl. Acad.
Sci., USA 86: 7370-7373.

Eenink, A. H., and F. L. Dieleman. 1982. Resistance of lettuce to leaf aphids: research on
components of resistance, on differential interactions between plant genotypes,
aphid genotypes and the environment and on the resistance level in the field after
natural infestation. Med. Fac. Landbouww. Rijksuniv. Gent 47: 607-615.









Eenink, A. H., F. L. Dieleman, J. H. Visser, and A. K. Minks. 1982. Resistance of
Lactuca accessions to leaf aphids: components of resistance and exploitation of
wild Lactuca species as sources of resistance, pp. 349-355. Proc. 5th Intl. Symp.
Insect Plant Relationships, Wageningen, The Netherlands.

Eichenseer, H., J. L. Bi, and G. W. Felton. 1998. Indiscrimination ofManduca sexta
larvae to overexpressed and underexpressed levels of phenylalanine ammonia-lyase
in tobacco leaves. Entomol. Exp. Appl. 87: 73-78.

Eichenseer, H., and C. A. Mullin. 1996. Maxillary appendages used by western corn
rootworms, Diabrotica virgifera virgifera, to discriminate between a
phagostimulant and -?deterrent. Entomol. Exp. Appl. 78: 237-242.

Ellard-Ivey, M., and C. Douglas. 1996. Role ofjasmonates in the elicitor- and wound-
inducible expression of defense genes in parsley and transgenic tobacco. Plant
Physiol. 112: 183-192.

Elliger, C. A., B. C. Chan, and A. C. Waiss. 1980. Flavonoids as larval growth inhibitors.
Naturwissenschaften 67: 358-359.

Ellis, P. R. 1991. The root of the problem. Grower 116: 11-13.

Ellis, P. R., S. J. McClement, P. L. Saw, K. Phelps, W. E. Vice, N. B. Kift, D. Astley, and
D. A. C. Pink. 2002. Identification of source of resistance in lettuce to the lettuce
root aphid Pemphigus bursarius. Euphytica 125: 305-315.

Ellis, P. R., D. A. C. Pink, and A. D. Ramsey. 1994. Inheritance of resistance to lettuce
root aphid in the lettuce cultivars 'Avoncrisp' and 'Lakeland'. Ann. Appl. Biol. 124:
141-151.

Ellis, P. R., G. M. Tatchell, R. H. Collier, and W. E. Parker. 1996. Assessment of several
components that could be used in an integrated programme for controlling aphids
on field crops of lettuce, pp. 91-97. Integrated control of field vegetable pests, vol.
19. IOBC Bull.

Esau, K. 1965. Plant anatomy. John Wiley & Sons, New York, USA.

Ester, A. 1998. Aphid resistance of butterhead lettuce tested in practice. PAV Bull.
Vollegrondsgroenteteelt No. February, 6-8.

Ester, A., J. Gut, A. M. van Oosten, and H. C. H. Pijnenburg. 1993. Controlling aphids in
iceberg lettuce by alarm pheromone in combination with an insecticide. J. Appl.
Ent. 115: 432-440.

Evans, F. J., and R. J. Schmidt. 1976. Two new toxins from the latex ofEuphorbia
poissonii. Phytochemistry 15: 333-335.









Evert, R. F. 2006. Esau's plant anatomy, meristems, cells, and tissues of the plant body:
their structure, function, and development. John Wiley & Sons, Inc. New Jersey.

Fagerstrom, T. 1989. Anti-herbivory chemical defense in plants: A note of the concept of
cost. Am. Nat. 133: 281-287.

Fahn, A. 1979. Secretory tissues in plants. Academic Press, New York, USA.

Fahn, A. 1990. Plant anatomy. Pergamon Press, New York.

Farrell, B. D., D. E. Dussourd, and C. Mitter. 1991. Escalation of plant disease: do
latex/resin canals spur plant diversification? Am. Nat. 138: 891-900.

Feeny, P. P. 1976. Plant apparency and chemical defense, pp. 1-40. In J. W. Wallace, and
R. L. Mansel [eds.], Biochemical interaction between plants and insects: recent
advances in phytochemistry. Plenum Press, New York.

Felton, G., J. Bi, C. B. Summers, A. J. Mueller, and S. Duffey. 1994b. Potential role of
lipoxygenases in defense against insect herbivory. J. Chem. Ecol. 20: 651-666.

Felton, G. W., K. K. Donato, R. M. Broadway, and S. S. Duffey. 1992. Impact of
oxidized plant phenolics on the nutritional quality of dietary protein to a noctuid
herbivore, Spodoptera exigua. J. Insect Physiol. 38: 277-285.

Felton, G. W., K. Donato, R. J. Del Vecchio, and S. S. Duffey. 1989. Activation of plant
foliar oxidases by insect feeding reduces nutritive quality of foliage for noctuid
herbivores. J. Chem. Ecol. 15: 2667-2694.

Felton, G. W., C. B. Summers, and A. J. Mueller. 1994a. Oxidative responses in soybean
foliage to herbivory by bean leaf beetle and three-cornered alfalfa hopper. J. Chem.
Ecol. 20: 639-650.

Feng, Y., and C. E. McDonald. 1989. Comparison of flavonoids in bran of four classes of
wheat. Cereal Chem. 66: 516-518.

Ferreres, F., M. I. Gil, M. Castaner, and F. A. Tomas-Barberan. 1997. Phenolic
metabolites in red pigmented lettuce: changes with minimal processing and cold
storage. J. Agric. Food Chem. 45: 4249-4254.

Fineran, B. A. 1982. Distribution and organization of non-articulated laticifers in mature
tissues of poinsettia (Euphorbiapulcherrima Willd.) Ann. Bot. 50:207-220.

Fineran, B. A. 1983. Differentiation of non-articulated laticifers in poinsettia (Euphorbia
pulcherrima Willd.). Ann. Bot. 52: 279-293.

Fiorillo, F., C. Palocci, S. Soro, and G. Pasqua. 2007. Latex lipase of Euphorbia
characias L.: An specific acylhydrolase with several isoforms. Plant Sci. 172:
722-727.









Forbes, A. R., and J. R. Mackenzie. 1982. The lettuce aphid, Nasonovia ribisnigri
(Homoptera: Aphididae) damaging lettuce crops in British Columbia. J. Entomol.
Soc. British Columbia 79: 28-31.

Fraenkel, G. S. 1959. The raison d'etre of secondary plant substances. Science 129: 1466-
1470.

Frank, M. R., J. M. Deyneka, and M. A. Schuler. 1996. Cloning of wound-induced
cytochrome P450 monooxygenases expressed in pea. Plant Physiol. 110: 1035-
1046.

Freitas, C. D. T., J. S. Oliveira, M. R. A. Miranda, N. M. R. Macedo, M. P. Sales, L. A.
Villas-Boas, and M. V. Ramos. 2007. Enzymatic activities and protein profile of
latex from Calotropisprocera. Plant Physiol. Biochem. 45: 781-789.

Frost, S., J. B. Harborne, and L. King. 1977. Identification of the flavonoids in five
chemical races of cultivated barley. Hereditas 85: 163-167.

Freund, R. J., and W. J. Wilson. 1997. Statistical methods. Academic Press, Inc, San
Diego.

Fry, S. C. 1986. Cross-linking of matrix polymers in the growing cell walls of
angiosperms. Annu. Rev. Plant Physiol. Plant Mol. Biol. 37: 165-186.

Fukasawa-Akada, T., S. Kung, and J. C. Watson. 1996. Phenylalanine ammonia-lyase
gene structure, expression, and evolution in Nicotiana. Plant Mol. Biol. 30: 711-
722.

Galliard, T., and H. W. S. Chan. 1980. Lipoxygenases, pp. 132-161. In E. E. Conn, and P.
K. Stumpf [eds.], The biochemistry of plants, vol. 4. Academic Press, New York.

Gazeley, K. F., A. D. T. Gorton, and T. D. Pendle. 1988. Latex concentrates; properties
and composition, pp-63-98. In A. D. Roberts [ed.], Natural rubber science and
technology, Oxford University Press, New York.

Genung, W. G. 1957. Some possible cases of insect resistance to insecticides in Florida.
Proc. Fla. State Hortic. Soc. 70: 148-152.

Gershenzon, J. 1994. The cost of plant chemical defense against herbivory: a biochemical
perspective, pp. 105-173. In E. A. Bernays [ed.], Insect-plant interactions, 5. CRC
Press, Inc., Boca Raton.

Ghaffar, A., M. R. Attique, and M. R. Naveed. 2002. Effect of different hosts on the
development and survival of Spodoptera exigua (Hubner) (Noctuidae:
Lepidoptera). Pakistan J. Zool. 34: 229-231









Gidrol, X., H. Chrestin, H. L. Tan, and A. Kush. 1994. Hevein, a lectin like protein from
Hevea brasiliensis (Rubber tree) is involved in coagulation of latex. J.Biol. Chem.
269:9278-9283

Gil, M. I., M. Castaner, F. Ferreres, F. Artes, and F. A. B. Thomas. 1998. Modified
atmosphere packaging of minimally processed "Lollo Rosso" (Lactuca sativa)
phenolic metabolites and quality changes. Z. Lebensm. Untere. Forsch. 206: 350-
354.

Gilbert, L. E. 1971. Butterfly plant coevolution: has Passiflora adenopoda won the
selection race with heliconiine butterflies? Science 172: 582-586.

Gonzalez, A. G. 1977. Lactuceae chemical review, pp.1081-1095. In V. H. Heywood
and J. B. Harborne [eds.], The biology and chemistry of the Compositae. Academic
Press, New York.

Green, T. R., and C. A. Ryan. 1972. Wound-induced proteinase inhibitor in plant leaves:
a possible defense mechanism against insects. Science 175: 776-777.

Gregory, P., W. M. Tingey. 1981. Chemical mechanisms of potato resistance to the
leafhopper. pp 95-99. In Breeding for Resistance to Insects and Mites. Canterbury,
England: Proc. 2nd Eucarpia/IOBC Meeting of the Working Group Breeding for
Resistance to Insects and Mites.

Gromek, D. W. Kisiel, A. Klodzinska, and E. Chojnacka-Wojcik. 1992. Biologically
active preparations from Lactuca virosa L. Phytother. Res. 6: 285-287.

Guy R. H., N. C. Leppla, J. R. Rye, C. W. Green, S. L. Barrette, and K. A. Hollien.
1985. Trichoplusia ni, pp. 487-494. In Pritam Singh and R. F. Moore [eds.],
Handbook of insect rearing, vol. 2. Elsevier Science Publishers, Amsterdam.

Guzman, V. L. 1986. Short Guzmaine, Tall Guzmaine and Florigade, three cos lettuce
cultivars resistant to lettuce mosaic virus. IFAS Univ. Fla. Agric. Exp. Stn. Circ. S-
326.

Hagerman, A. E., and L. G. Butler. 1991. Tannins and lignins, pp. 355-388. In G. A.
Rosenthal, and M. R. Berenbaum [eds.], Herbivores: their interaction with
secondary plant metabolites. Academic Press, San Diego.

Hahlbrock, K., and D. Scheel. 1989. Physiology and molecular biology of
phenylpropanoid metabolism. Annu. Rev. Plant Physiol. Plant Mol. Biol. 40: 347-
369.

Hanny, B. W. 1980. Gossypol, flavonoid and condensed tannin content of cream and
yellow anthers of five cotton (Gossypium hirsutum L.) cultivars. J. Agric. Food
Chem. 28: 504-506.









Hanson, K. R. and E. A. Havir. 1979. An introduction to the enzymology of
phenylpropanoid biosynthesis, p-91-138. In T. Swain, J. B. Harborne and C. F. Van
Sumere [eds]. Biochemistry of plant phenolics. Plenum Press, New York.

Harborne, J. B. 1979. Flavonoid pigments, pp. 619-655. In G. A. Rosenthal and D. H.
Janzen, [eds]. Herbivores: their interaction with secondary plant metabolites.
Academic Press, New York.

Harborne, J. B. 1993. Introduction to ecological biochemistry. Academic Press, London.

Harborne, J. B. 1994. The flavonoids, advances in research since 1986. Chapman and
Hall, London.

Harrewijn, F., and F. L. Dieleman. 1984. The importance of mineral nutrition of the host
plant in resistance breeding to aphids, pp.235-243. In Proc. Sixth Intl. Cong.
Soilless Cult. Lunteren, The Netherlands.

Hartley, S. E., and J. H. Lawton. 1991. Biochemical aspects and significance of the
rapidly induced accumulation of phenolics in birch foliage, pp. 105-132. In D. W.
Tallamy, and M. J. Raupp [eds.], Phytochemical induction by herbivores. John
Wiley & Sons Inc., New York.

Haupt, I. 1976. Separation of the sites of synthesis and accumulation of 3, 4-
dihydroxyphenylalanine in Euphorbia lathyris L. Nova Acta Leopoldina
Supplementum 7: 129-132.

Havill, N. P., and K. F. Raffa. 1999. Effects of elicitation treatment and genotypic
variation on induced resistance in Populus: impacts on gypsy moth (Lepidoptera:
Lymantriidae) development and feeding behavior. Oecologia 120: 295-303

Hayward, H. E. 1938. The structure of economic plants. Macmillan and Company, New
York.

Hedin, P. A., J. N. Jenkins, D. H. Collum, W. H. White, W. L. Parrot, and M. W.
MacGown. 1983. Cyanidin-3-glucoside, a newly recognized basis for resistance in
cotton to the tobacco budworm Heliothis virescens (Fab.) (Lepidoptera:
Noctuidae). Experientia 39: 799-801.

Hedin, P. A., J. N. Jenkins, A. C. Thompson, J. C. McCarty, D. H. Smith, W. L. Parrot,
and R. L. Shepherd. 1988. Effect of bioregulators on flavonoids, insect resistance
and yield of seed cotton. J. Agric. Food Chem. 36: 1055-1061.

Hedin, P. A., and S. K. Waage. 1986. Roles of flavonoids in plant resistance to insects,
pp?. In V. Cody, E. Middleton, and J. B. Harborne [eds.], Plant flavonoids in
biology and medicine: biochemical, pharmacological, and structure-activity
relationships. Liss, New York.









Heinrich, G. 1967. Licht- und elektronenmikroskopische Unterssuchungen der
Milchrohren von Taraxacum bicorne. Flora (Jena) Abt. A. 158: 413-420.

Heitz, T., D. R. Bergey, and C. A. Ryan. 1997. A gene encoding a chloroplast-targeted
lipoxygenase in tomato leaves is transiently induced by wounding, systemin, and
methyl jasmonate. Plant Physiol. 114: 1085-1093.

Heller, W., and G. Forkman. 1993. Biosynthesis of flavonoids. pp. 499-535. In J. B.
Harborne [ed.], The flavonoids, advances in research since 1986. Chapman and
Hall, London.

Helm, J. 1954. Lactuca sativa in morphologisch-systematischer Sicht. Kulturpflanze 2:
72-129.

Henderson, A. E., R. H. Hallett, and J. J. Soroka. 2004. Prefeeding behavior of the
crucifer flea beetle, Phyllotreta cruciferae, on host and nonhost crucifers. J. Insect
Behav. 17: 17-39.

Heng-Moss, T. M., G. Sarath, F. Baxendale, D. Novak, S. Bose, X. Ni, and S.
Quisenberry. 2004. Characterization of oxidative enzyme changes in buffalograsses
challenged by Blissus occiduus. J. Econ. Entomol. 97: 1086-1095.

Hennion, M.-C. 1999. Solid-phase extraction: method development, sorbents, and
coupling with liquid chromatography. J. Chromatogr. A, 856: 3-54.

Hermann, K. 1976. Flavonols and flavones in food plants: a review. J. Food Technol. 11:
433-448.

Hermann, K. 1988. On the occurrence of flavonol and flavone glycosides in vegetables.
Z. Lebensen Unters. Forsch. 186: 1.

Herms, D. A., and W. J. Mattson. 1992. The dilemma of plants: to grow or defend. Quart.
Rev. Biol. 67: 283-335.

Hertog, M. G. L., E. J. M. Fesens, P. C. H. Hollman, M. B. Katan, and D. Kromhout.
1993. Dietary antioxidant flavonoids and the risk of coronary heart disease: the
zutphen elderly study. Lancet 342: 1007.

Hertog, M. G. L., P. C. H. Hollman, and D. P. Venema. 1992. Optimization of
quantitative HPLC determination of potentially anticarcinogenic flavonoids in fruit
and vegetables. J. Agric. Food. Chem. 40: 1591.

Hildebrand, D. F., J. G. Rodriguez, G. C. Brown, K. T. Luu, C. S. Volden. 1986.
Peroxidative responses of leaves in two soybean genotypes injured by two spotted
spider mites (Acari: Tetranychidae). J. Econ. Entomol. 79: 1459-1465.









Hochmuth, G., E. Hanlon, R. Nagata, G. Snyder, and T. Schueneman. 1994. Crisphead
lettuce: fertilization recommendations for crisphead lettuce grown on organic soils
in Florida. Gainesville, FL. Florida Coop. Extn. Serv. Bull. SP-153.

Hohl, U., B. Neubert, and H. Pforte, I. Schonhof, and H. Bohm. 2001. Flavonoid
concentrations in the inner leaves of head lettuce genotypes. Eur. Food Res.
Technol. 213: 205-211.

Hosel, W. 1981. Glycosylation and glycosides, pp. 725-755. In P. K. Stumpf, and E. E.
Conn [eds.], The biochemistry of plants, vol. 7. Academic Press Inc., New York.

Huang, J., H. J. McAuslane, and G. S. Nuessly. 2003a. Effect of leaf surface extraction
on palatability of romaine lettuce to Diabrotica balteata. Entomol. Exp. Appl. 106:
227-234.

Huang, J., H. J. McAuslane, and G. S. Nuessly. 2003b. Resistance in lettuce to
Diabrotica balteata (Coleoptera: Chrysomelidae): the roles of latex and inducible
defense. Environ. Entomol. 32: 9-16.

Huang, J., G. S. Nuessly, H. J. McAuslane, and R. Nagata. 2003c. Effect of screening
methods on expression of romaine lettuce resistance to adult banded cucumber
beetle, Diabrotica balteata (Coleoptera: Chrysomelidae). Florida Entomol. 86:
194-198.

Huang, J., G. S. Nuessly, H. J. McAuslane, and F. Slansky. 2002. Resistance to adult
banded cucumber beetle, Diabrotica balteata (Coleoptera: Chrysomelidae), in
romaine lettuce. J. Econ. Entomol. 95: 849-855.

Hunt, M., N. Eannetta, H. Yu, S. Newman, and J. Steffens. 1993. cDNA cloning and
expression of potato polyphenol oxidase. Plant Mol. Biol. 21: 59-68.

Hyodo, H., H. Kuroda, and S. F. Yang. 1978. Induction of phenylalanine ammonia lyase
and increase in phenolics in lettuce leaves in relation to the development of russet
spotting caused by ethylene. Plant Physiol. 2: 31-35.

Ikonen, A., J. Tahvanainen, and H. Roininen. 2001. Chlorogenic acid as an antiherbivore
defense of willows against leaf beetles. Entomol. Exp. Appl. 99: 47-54.

Inglis, D. A. and E. Vestey. 2001. Crop profile for lettuce in Washington.
http://mtvernon.wsu.edu/plant_pathology/plant_path.htm.

Inoue, M., S. Sezaki, T. Sorin, and T. Sugiura. 1985. Change ofphenylalanine ammonia-
lyase activity in strawberry leaves infested with the two-spotted spider mite,
Tetranychus urticae Koch (Acarina : Tetranychidae). Appl. Entomol. Zool. 20:
348-349.









Isidoro, N. B., J. Ziesmann, and I. H. Williams. 1998. Antennal contact chemosensilla in
Psylliodes chrysocephala responding to cruciferous allelochemicals. Physiol.
Entomol. 23: 131-138.

Ito, H., F. Kimizuka, A. Ohbayashi, H. Matsui, M. Honma, A. Shinmyo, Y. Ohashi, A. B.
Caplan, and R. L. Rodriguez. 1994. Molecular cloning and characterization of two
complementary DNAs encoding putative peroxidases from rice (Oryza sativa L.)
shoots. Plant Cell Rep. 13: 361-366.

Jahne, A., C. Fritzen, and G. Weissenbock. 1993. Chalcone synthase and flavonoid
products in primary-leaf tissues of rye and maize. Planta 189: 39-46.

Jerez, M. I. 1998. Response of two maize inbred lines to chinch bug feeding. M.S. thesis.
Mississippi State University Mississippi.

Jimenez, M., and F. Garcia-Carmona. 1996. The effect of sodium dodecyl sulfate on
polyphenol oxidase. Phytochemistry 42: 1503-1509.

Joerdens-Roettger, D. 1979. The role of phenolic substances for host selection behaviour
of the black bean aphid, Aphisfabae. Entomol. Exp. Appl. 26: 49-54.

Jones, D. H. 1984. Phenylalanine ammonia lyase: regulation of its induction and its role
in plant development. Phytochemistry 23: 1349-1359.

Joos, H. J., and K. Hahlbrock. 1992. Phenylalanine ammonia lyase in potato (Solanum
tuberosum L.). Genomic complexity, structural comparison of two selected genes
and modes of expression. Euro. J. Biochem. 204: 621-629.

Ke, D., and M. E. Saltveit. 1986. Effects of calcium and auxin on russet spotting and
phenylalanine ammonia-lyase activity in iceberg lettuce. HortScience 21: 1169-
1171.

Ke, D., and M. E. Saltveit. 1988. Plant hormone interaction and phenolic metabolism in
the regulation of russet spotting in iceberg lettuce. Plant Physiol. 88: 1136-1140.

Ke, D., and M. E. Saltveit. 1989. Developmental control of russet spotting, phenolics
enzymes, and IAA oxidase in cultivars of iceberg lettuce. J. Am. Soc. Hortic. Sci.
114: 472-477.

Kekwick, R. G. 0. 2001. Latex and laticifers, pp. 1-6. In Encyclopedia of Life Sciences.
John Wiley & Sons, Ltd: Chichester (http://www.els.net/).

Kennedy, J. S., M. F. Day, and V. F. Eastop. 1962. A conspectus of aphids as vectors of
plant viruses, pp. 1-114. Commonwealth Institute of Entomology, London.

Kerns, D. L., M. E. Methron, J. C. Palumbo, C. A. Sanchez, D. W. Still, B. R. Tickes, K.
Umeda, and M. A. Wilcox. 1999. Guidelines for head lettuce production in
Arizona. Univ. Ariz. Coop. Extn. IPM, Ser. 12.









Kerns, D. L., and J. C. Palumbo. 1996. Lettuce IPM: southwestern USA. Yuma Valley
Agricultural Center, University of Arizona, Yuma, Arizona.

Kim, J. H., and C. A. Mullin. 2003. Antifeedant effects of proteinase inhibitors on
feeding behaviors of adult western corn rootworm (Diabrotica virgifera virgifera).
J. Chem. Ecol. 29: 795-810.

Kinghorn, A., and F. Evans. 1975. A biological screen of selected species of the genus
Euphorbia for skin irritant effects. Planta 28: 325-335.

Kishaba, A. N., J. D. McCreight, D. L. Coudriet, T. W. Whitaker, and G. R. Pesho. 1980.
Studies of ovipositional preference of cabbage looper on progenies from a cross
between cultivated lettuce and prickly lettuce. J. Am. Soc. Hortic. Sci. 105: 890-
892.

Kishaba, A. N., T. W. Whitaker, W. Berry, and H. H. Toba. 1976. Cabbage looper
oviposition and survival of progeny on leafy vegetables. HortScience. 11: 216-217.

Kisiel, W., B. Barszcz, and E. Szneler. 1997. Sesquiterpene lactones from Lactuca
tatarica. Phytochemistry 45: 365-368.

Koes, R. E., F. Quattrocchio, and J. N. M. Mol. 1994. The flavonoid biosynthetic
pathway in plants: function and evolution. Bioessays 16: 123-132.

Kolattukudy, P. E. 1981. Structure, biosynthesis, and biodegradation of cutin and suberin.
Annu. Rev. Plant Physiol. Plant Mol. Biol. 32: 539-567.

Konno, K., C. Hirayama, M. Nakamura, K. Tateishi, Y. Tamura, M. Hattori, and K.
Kohno. 2004. Papain protects papaya trees from herbivorous insects: role of
cysteine proteases in latex. Plant J. 37: 370-378.

Konno, K., H. Ono, M. Nakamura, K. Tateishi, C. Hirayama, Y. Tamura, M. Hattori, A.
Koyama, and K. Kohno. 2006. Mulberry latex rich in antidiabetic sugar-mimic
alkaloids forces dieting on caterpillars. Proc. Natl. Acad. Sci. USA 103: 1337-1341.

Krysan, J. L. 1986. Introduction: biology, distribution, and identification of pest
Diabrotica, pp. 1-23. In J.L. Krysan and T.A. Miller [eds.], Methods for the study of
pest Diabrotica. Springer-Verlag, New York.

Kurtz, E. A. 2001. Crop profile for iceberg lettuce in California. California Lettuce
Research Board, Salinas, California.

Kush, A., E. Goyvarets, M. L. Chye, and N. H. Chua. 1990. Lacticifers-specific gene
expression in Hevea brasiliensis (Rubber tree). Proc, Natl. Acad. Sci. 87: 1787-
1790.









Kyndt, T., E. J. M. V. Damme, J. V. Beeumen, and G. Gheysen. 2007. Purification and
characterization of the cysteine proteinases in the latex of Vasconcellea spp. FEBS
J. 274: 451-462.

Lauritzen, E. 1999. Monterey county agricultural commissioner's crop report. p. 32.
Monterey County, Salinas, CA: Monterey Agricultural Commissioner.

Lee, D., and C. J. Douglas. 1996. Two divergent members of a tobacco 4-coumarate:
coenzyme A ligase (4CL) gene family. cDNA structure, gene inheritance and
expression, and properties of recombinant proteins. Plant Physiol. 112: 193-205.

Leeper, P. W., T. W. Whitaker, and G. W. Bohan. 1963. Valmaine a new cos-type
lettuce. Am. Veg. Grower, September, p.716.

Leibee, G. I. 1981. Insecticidal control ofLiriomyza spp. on vegetables, pp. 216-220. In
D. J. Schuster [ed.], Proceedings IFAS industry conference on biology and control
of Liriomyza leafminers, November 3-4, 198. Lake Buena Vista, Florida.

Le6n-Gonazalez, M. E., and L. V. Perez-Arribas. 2000. Chemically modified polymeric
sorbents for sample preconcentration. J. Chromatogr. A 902: 3-16.

Lewinsohn, T. M. 1991. The geographical distribution of plant latex. Chemoecology. 2:
64-68.

Lindqvist, K. 1960. On the origin of cultivated lettuce. Hereditas 46: 319-350.

Loaiza-Velarde, J. G., and M. E. Saltveit. 2001. Heat shocks applied either before or after
wounding reduce browning of lettuce leaf tissue. J. Amer. Soc. Hortic. Sci. 126:
227-234.

Loaiza-Velarde, J. G., F. A. Tomas-Barberan, and M. E. Saltveit. 1997. Effect of
intensity and duration of heat-shock treatments on wound-induced phenolic
metabolism in iceberg lettuce. J. Amer. Soc. Hortic. Sci. 122: 873-877.

Lois, R., and K. Hahlbrock. 1992. Differential wound activation of members of the
phenylalanine ammonia-lyase and 4-coumarate:CoA ligase gene families in various
organs of parsley plants. Z. Naturforsch. 47: 90-94.

Lopez-Galvez, G. M. E. Saltveit, and M. Cantwell. 1997. Wound induced phenylalanine
ammonia lyase aivity: Factors affecting it inducton and correlation with the quality
of minimally processed lettuce. Postharvest Biol. Technol. 9: 223-233.

Lucas, P. W., I. M. Turner, N. J. Dominy, and N. Yamashita. 2000. Mechanical defenses
to herbivory. Ann. Bot. 86: 913-920.

Luckner, M. 1990. Secondary metabolism in microorganisms, plants, and animals.
Gustav Fischer Verlag, Jena.









Luh, B. S., and B. Phithakpol. 1972. Characteristics of polyphenyl oxidase related to
browning in cling peaches. J. Food. Sci. 37: 264-268.

Markham, K. R. 1989. Flavones, flavonols and their glucosides. Methods Plant Biochem.
1: 197-232.

Martin, C., L. Schoen, C. Rufingier, and N. Pasteur. 1996. A contribution to the
integrated pest management of the aphid Nasonovia ribisnigri in salad crops.
Integrated Control of Field Vegetable pests. IOBC Bull. 19: 98-101.

Martin, M. N. 1991. The latex ofHevea brasiliensis contains high levels of both
chitinases and chitinases/lysozymes. Plant Physiol. 95: 465-476.

Martin, P. B., P. D. Lingren, and G. L. Greene. 1976. Relative abundance and host
preferences of cabbage looper, soybean looper, tobacco budworm, and corn ear
worm on crops grown in northern Florida. Environ. Entomol. 5: 878-882.

Matile, P. 1976. Localizations of alkaloids and mechanism of their accumulation in
vacuoles of Chelidonium majus laticifers. Nova Acta Leopoldina Supplementum 7:
65-73.

Mayer, A. M., and E. Harel. 1979. Polyphenol oxidases in plants. Phytochemistry 18:
193-215.

McCabe, M. S., Garratt, L. C., F. Schepers,, W. J. R. M. Jordi, G. M. Stoopen, E.
Davelaar, J. H. van Rhijn, J. B. Powers, and M. R. Davey. 2001. Effects of
PSAG12-IPT gene expression on development and sequence in transgenic lettuce.
Plant Physiol. 127: 505-516.

McDougall, S., T. Napier, J. Valenzisi, A. Watson, J. Duff, G. Geitz, and T. Franklin.
2002. Integrated pest management in lettuce: information guide, pp. 154. NSW
Agriculture, Orange, Australia.

Metcalf, C. L., and W. P. Flint. 1962. Destructive and useful insects, their habits and
control, 4th edition, McGraw-Hill, San Francisco.

Metcalfe, C. R. 1967. Distribution of latex in the plant kingdom. Econ. Bot. 21:115-127.

Metcalfe, C. R., and L. Chalk. 1983. Anatomy of the dicotyledons, vol. II. Clarendon,
Oxford, England.

Miles, C. J., and R. J. Pfeuffer. 1997. Pesticides in canals of South Florida. Arch.
Environ. Contam. Toxicol. 32: 337-345.

Miller, H., Porter, D. R., Burd, J. D., Mornhinweg D. W., Burton, R. L. 1994.
Physiological effects of Russian wheat aphid (Homoptera: Aphididae) on resistant
and susceptible barley. J. Econ. Entomol. 87: 493-499.









Miller, N. J., A. J. Birley, A. D. J. Overall, and G. M. Tatchell. 2003. Population genetic
structure of the lettuce root aphid, Pemphigus bursarius (L.), in relation to
geographic distance, gene flow and host plant usage. Heredity 91: 217-223.

Mizutani, M., D. Ohta, and R. Sato. 1997. Isolation of a cDNA and a genomic clone
encoding cinnamate 4-hydroxylase from Arabidopsis and its expression manner in
plant. Plant Physiol. 113:755-763.

Moerschbacher, B. M., U. Noll, L. Gorrichon, and H. J. Reisener. 1990. Specific
inhibition of lignification breaks hypersensitive resistance of wheat to stem rust.
Plant Physiol. 93: 465-470.

Mollema, C., and R. A. Cole. 1996. Low aromatic amino acid concentrations in leaf
proteins determine resistance to Frankliniella occidentalis in four vegetable crops.
Entomol. Exp. Appl. 78: 325-333.

Monacelli, B., A. Valletta, N. Rascio, I. Moro, and G. Pasqua. 2005. Laticifers in
Camptotheca acuminata Decne: distribution and structure. Protoplasma 226: 155-
161.

Monnet, Y., and J. F. Ricateau. 1997. La lutte aphicide raisonee en cultures de laitues de
plein champs: bilan de trois annees de pratique. Quatrieme Conference
International sur les Ravageurs en Agriculture. Montpellier, France, 6-8 January.
2: 497-504.

Montllor, C. B., and W. F. Tjallingii. 1989. Stylet penetration by two aphid species on
susceptible and resistant lettuce. Entomol. Exp. Appl. 52: 103-111.

Morrow, P. A., and L. R. Fox. 1980. Effect of variation in eucalyptus essential oil on
insect growth and grazing damage. Oecologia 45: 209-219.

Mossler, M. A., and E. Dunn. 2005. Florida crop/pest management profile: lettuce. Univ.
Fla. IFAS Extn. http://edis.ifas.ufl.edu/PI070.

Mou, B., and Y. B. Liu. 2003. Leafminer resistance in lettuce. HortScience 38: 570-572.

Mou, B., and E. J. Ryder. 2003. Screening and breeding for resistance to leafminer
(Liriomyza langei) in lettuce and spinach, pp. 43-47. In Proc. Eucarpia Meeting
leafy vegetables Gen. Breeding, the Netherlands, 19-21 March, 2003. Centre for
Genetic Resources, The Netherlands.

Mou, B., E. J. Ryder, J. Tanaka, Y. B. Liu, and W. E. Chaney. 2004. Breeding for
resistance to leafminer in lettuce. Acta Hort. 637: 57-62.

Moussaoui, A. El, M. Nijs, C. Paul, R. Wintjens, J. Vincentelli, M. Azarkan and Yvan
Looze. 2001. Revisiting the enzymes stored in the laticifers of Caricapapaya in the
context of their possible participation in the plant defense mechanism. Cell Mol.
Life Sci. 58: 556-570.









Mura A., R. Medda, S. Longu, G. Floris, A. C. Rinaldi, and A. Padiglia. 2005. A
Ca2+/calmodulin-binding peroxidase from Euphorbia latex: novel aspects of
calcium-hydrogen peroxide cross-talk in the regulation of plant defenses.
Biochemistry 44: 14120-14130.

Mura, A., F. Pintus, R. Medda, G. Floris, A. C. Rinaldi, and A. Padiglia. 2007. Catalase
and Antiquitin from Euphorbia characias: two proteins involved in plant defense?
Biochemistry 72: 501-508.

Nagata, R. T., L. M, Wilkinson, and G. S. Nuessly. 1998. Longevity, fecundity, and leaf
stippling ofLiriomyza trifolii (Diptera: Agromyzidae) as affected by lettuce
cultivar and supplemental feeding. J. Econ. Entomol. 91: 999-1004.

Nawrot, J., E. Bloszyk, J. Harmatha, L. Novotny, and B. Drozdz. 1986. Action of
antifeedants of plant origin on beetles infesting stored products. Acta Entomol.
Bohemoslov. 83:327-335.

Nebreda, M., A. Moreno, N. Perez, I. Palacios, V. Seco-Fernandez, and A. Fereres. 2004.
Activity of aphids associated with lettuce and broccoli in Spain and their efficiency
as vectors of Lettuce mosaic virus. Virus Res. 100: 83-88.

Nelson, C. J., J. N. Seiber, and L.P. Brower. 1981. Seasonal and intraplant variation of
cardinolide content in California milkweed, Asclepias eriocarpa, and implications
for plant defense. J. Chem. Ecol. 7: 981-1010.

Ni, X, S. S. Quisenberry, T. Heng-Moss, J. Markwell, G. Sarath, R. Klucas, and F.
Baxendale 2001. Oxidative responses of resistant and susceptible cereal leaves to
symptomatic and nonsymptomatic cereal aphid (Hemiptera: Aphididae) feeding.
J.Econ. Entomol. 94: 743-751.

Nicholson, R. L., and R. Hammerschmidt. 1992. Phenolic compounds and their role in
disease resistance. Annu. Rev. Phytopath. 30: 369-389.

Nielson, P. E., H. Nishimura, J. W. Otvos, and M. Calvin. 1977. Plant crops as a source
of fuel and hydrocarbon like materials. Science 198: 942-944.

Nishida, R., T. Ohsugi, S. Kokubo, and H. Fukami. 1987. Oviposition stimulants of a
citrus-feeding swallowtail butterfly, Papilio xuthus L. Experientia 43: 342-344.

Nishio, S., M. S. Blum, and S. Takahashi. 1983. Intraplant distribution of cardenolides in
Asclepias humistrata (Asclepiadaceae) with additional notes on their fates in
Tetraopes melanurus (Coleoptera: Cerambycidae) and Rhyssomatus lineaticollis
(Coleoptera: Curculionidae). Mem. Coll. Agric. Kyoto Univ. 122: 43-52.

Noack, E. A., A. E. G. Cera, and G. Falsone. 1980. Inhibition of mitochondrial oxidative
phosphorylation by 4-deoxyphorbol trimester, a poisonous constituent of the latex
sap of Euphorbia iglandulosa Desf. Toxicon 18: 165-174.









Norris, R. F., E. P. Caswell-Chen, and M. Kogan. 2003. Concepts in integrated pest
management. Prentice-Hall, New Jersey.

Nuessly, G. S., and R. T. Nagata. 1993. Evaluation of damage by serpentine leafminer
and banded cucumber beetle to cos lettuce. Everglades Res. Ed. Center Res. Rpt.,
EV-1993. 2: 76-77.

Nuessly, G. S., and R. T. Nagata. 1994. Differential probing response of serpentine
leafminer, Liriomyza trifolii (Burgess), on cos lettuce. J. Entomol. Sci. 29: 330-338.

Nuessly, G. S., and S. E. Webb. 2003. Insect management for leafy vegetables (lettuce,
endive and escarole). Univ. Fla. IFAS Extn. http://edis.ifas.ufl.edu/IG161.

Nutt, K. A., M. G. O'Shea, and P. G. Allsopp. 2004. Feeding by sugarcane whitegrubs
induces changes in the types and amounts of phenolics in the roots of sugarcane.
Environ. Exp. Bot. 51: 155-165.

Olson, K. C., T. W. Tibbitts, and B. E. Struckmeyer. 1969. Leaf histogenesis in Lactuca
sativa with emphasis upon laticifer ontogeny. Amer. J. Bot. 56: 1212-1216.

Orr, J. D., R. Edwards, and R. A. Dixon. 1993. Stress responses in alfalfa (Medicago
sativa L.) XIV. Changes in the levels of phenylpropanoid pathway intermediates in
relation to regulation of 1-phenylalanine ammonia-lyase in elicitor-treated cell-
suspension cultures. Plant Physiol. 101: 847-856.

Palumbo, J., A. Fournier, P. Ellsworth, K. Nolte, and P. Clay. 2006. Insect crop losses
and insecticide usage for head lettuce in Arizona: 2004 2006. University of
Arizona College of Agriculture 2006 Vegetable Report.
http://cals.arizona.edu/pubs/crops/az1419/.

Panda, N., and G. S. Kush. 1995. Host plant resistance to insects. CAB International,
Wallingford, Oxon, U.K.

Parenzan, P. 1984. Noctuidae (Lepidoptera, Heterocera) of southern Italy (addenda).
Entomologica 19: 97-134.

Parihar, S. B. S., and 0. P. Singh. 1992. Role of host plants in development and survival
ofHeliothis armigera (Hubner). Bull. Entomol. New Delhi. 33: 74-78.

Parker, W. E., R. H. Collier, P. R. Ellis, A. Mead, D. Chandler, J. A. Blood Smyth, and
G. M. Tatchell. 2002. Matching control options to a pest complex: the integrated
pest management of aphids in sequentially-planted crops of outdoor lettuce. Crop
Prot. 21: 235-248.

Parrella, M. P., and B. C. Keil. 1984. Insect pest management: the lesson of Liriomyza.
Bull. Entomol. Soc. Am. 30: 22-25.









Patanakamjorn, J. and M. D. Pathak. 1967. Varietal resistance of rice to the Asiatic rice
borer, Chilo suppressalis (Lepidoptera: Crambidae), and its association with
various plant characters. Ann. Entomol. Soc. Am. 60: 287-292.

Pechan, T., A. Cohen, W. P. Williams, and D. S. Luthe. 2002. Insect feeding mobilizes a
unique plant defense protease that disrupts the peritrophic matrix of caterpillars.
Proc. Natl. Acad. Sci. USA 99: 13319-13323.

Pechan, T., L. Ye, Y. Chang, A. Mitra, L. Lin, F. M. Davis, W. P. Williams, and D. S.
Luthe. 2000. A unique 33-kDa cysteine proteinase accumulates in response to
larval feeding in maize genotypes resistant to fall armyworm and other
Lepidoptera. Plant Cell 12: 1031-1040.

Peiser, G., G. Lopez-Galvez, M. Cantwell, and M. E. Saltveit. 1998. Phenylalanine
ammonia lyase inhibitor controls browning of cut lettuce. Postharvest Biol.
Technol. 14: 171-177.

Pellegrini, L., O. Rohfritsch, B. Fritig, and M. Legrand. 1994. Phenylalanine ammonia-
lyase in tobacco. Molecular cloning and gene expression during the hypersensitive
reaction to tobacco mosaic virus and the response to a fungal elicitor. Plant Physiol.
106: 877-886.

Peng, S. Z., and P. W. Miles. 1988. Acceptability of catechin and its oxidative
condensation products to the rose aphid, Macrosiphum rosae. Entomol. Exp. Appl.
47: 225-265.

Peterson, G. L. 1977. A simplification of the protein assay method of Lowry et al. which
is more generally applicable. Anal. Biochem. 83: 346-356.

Pichon, V. 2000. Solid-phase extraction for multiresidue analysis of organic
contaminants in water. J. Chromatogr. A 885: 195-215.

Pinaeus, A. 1561. Hist. Plants. Cited from E. L. Sturtevant. 1886. A study of garden
lettuce. Am. Nat. 20: 230-233.

Pitre, N. H., Jr., and E. J. Kantack. 1962. Biology of the banded cucumber beetle,
Diabrotica balteata, in Louisiana. J. Econ. Entomol. 55: 904-906.

Price, K. R., M. S. Dupont, R. Shepherd, H. W. S. Chan, and G. R. Fenwick. 1990.
Relationship between chemical and sensory properties of exotic salad crops:
colored lettuce (Lactuca sativa) and chicory (Cichorium intybus). J. Sci. Food
Agric. 53: 185-192.

Pujade-Renaud, V., A. Clement, C. Perrot-Rechenmann, J.-C.Prev6t, H. Chrestin,J.-L.
Jacob, and J. Cuern. 1994. Ethylene-induced increase in glutamine synthetase
activity and mRNA levels in Hevea brasiliensis latex cells. Plant Physiol.105: 127-
132.









Pullin, A. S. 1987. Changes in leaf quality following clipping and regrowth of Urtica
dioica and consequences for a specialist herbivore, Aglais urticae. Oikos 49: 39-45.

Rafi, M. M., R. S. Zemetra, and S. S. Quisenberry. 1996. Interaction between Russian
wheat aphid (Homoptera: Aphididae) and resistant and susceptible genotypes of
wheat. J. Econ. Entomol. 89: 239-246.

Ramos, M. V., C. D. T. Freitas, F. Staniscuaski, L. L. P. Macedo, M. P. Sales, D. P.
Sousa, and C. R. Carlini. 2007. Performance of distinct crop pests reared on diets
enriched with latex proteins from Calotropisprocera: Role of laticifer proteins in
plant defense. Plant Sci. 173: 349-357.

Razem, F. A., and M. A. Bernards. 2002. Hydrogen peroxide is required for
poly(phenolic) domain formation during wound induced suberization. J. Agric.
Food Chem. 50: 1009-1015.

Rees, C. J. C. 1969. Chemoreceptor specificity associated with choice of feeding site by
the beetle, Chrysolina brunsvicensis, on its food plant, Hypericum hirsutum.
Entomol. Exp. Appl. 12: 565-583.

Rees, S. B., and J. B. Harborne. 1985. The role of sesquiterpene lactones and phenolics in
the chemical defense of the chicory plant. Phytochemistry 24: 2225-2231.

Reinink, K., and F. L. Dieleman. 1989. Resistance in lettuce to the leaf aphids
Macrosiphum euphorbiae and Uroleucon sonchi. Ann. Appl. Biol. 115: 489-498.

Reinink, K., and F. L. Dieleman. 1993. Survey of aphid species on lettuce. Bull. OILB
SROP 16: 56-68.

Reinink, K., F. L. Dieleman, and R. Groenwold. 1995. Inheritance of partial resistance to
the leaf aphids Macrosiphum euphorbiae and Uroleucon sonchi in lettuce. Ann.
App. Biol. 127: 413-424.

Reinink, K., F. L. Dieleman, J. Jansen, and A. M. Montenarie. 1989. Interactions between
plant and aphid genotypes in resistance of lettuce to Myzuspersicae and
Macrosiphum euphorbiae. Euphytica 43: 215-222.

Rhoades, D. F. 1979. Evolution of plant chemicals defense against herbivores, pp. 3-54.
In G. A. Rosenthal, and D. H. Janzen [eds.], Herbivores: their interaction with
secondary plant metabolites. Academic Press, New York.

Rhodes, M. J. C., L. S. C. Wooltorton, and A. C. Hill. 1981. Changes in phenolic
metabolism in fruit and vegetable tissue under stress. pp. 193-220. In J. Friend and
M. J. C. Rhodes [eds.] Recent advances in the biochemistry of fruits and
vegetables. Academic Press, London.

Ribereau-Gayon, P. 1972. Plant phenolics. Oliver and Boyd, Edinburgh.









Richardson, M. 1991. Seed storage proteins: the enzyme inhibitors. Methods Plant
Biochem. 5: 259-305.

Ridland, P., S. Vujovic, C. Murdoch, P. Williams, F. Goubran, R. Dimsey, and L.
Zirnsak. 2002. Improving lettuce insect pest management-Victoria. Department of
Natural Resources and Environment. Victoria, Australia.

Ridsdill-Smith, T. J., Y. Jiang, and E. L. Ghisalberti. 1995. A method to test compounds
for feeding deterrence towards red-legged earth mite (Acarina: Penthaleidae). Ann.
Appl. Biol. 127: 593-600.

Rob, K. L. 1989. Analysis of Frankliniella occidentalis (Pergande) as a pest of
floricultural crops in California greenhouses. Ph.D. Dissertation, University of
California, Riverside.

Roberts, M. F. 1987. Papaver latex and alkaloid storage vacuoles, pp. 513-528. In B.
Marin [ed.], Plant vacuoles: their importance in solute compartmentation in cells
and their applications in plant biotechnology. Plenum, New York.

Robinson, T. 1972. The organic constituent of higher plants. Cordus Press. North
Amherst, Mass.

Robison, D. J., and K. F. Raffa. 1997. Effect of constitutive and inducible traits of hybrid
poplars on forest tent caterpillar feeding and population ecology. For. Sci. 40: 686-
714.

Romani, A., P. Pinelli, C. Galadi, G. Sani, A. Cimato, and D. Heimler. 2002. Polyphenols
in green house and open air grown lettuce. Food Chem. 79: 337-342.

Rosenthal, G. A., and M. R. Berenbaum. 1991. Herbivores: their interaction with
secondary plant metabolites. 2nd ed. Vol. 1. The chemical participants. Academic
Press, New York.

Rufingier, C., L. Schoen, C. Martin, and N. Pasteur. 1997. Resistance ofNasonovia
ribisnigri (Homoptera: Aphididae) to five insecticides. J. Econ. Entomol. 90: 1445-
1449.

Rumeau, D., E. A. Maher, A. Kelman, and A. M. Showalter. 1990. Extension and
phenylalanine ammonia-lyase gene expression altered in potato tubers in response
to wounding, hypoxia, and Erwinia carotovora infection. Plant Physiol. 93: 1134-
1139.

Rutherford, R. S. 1998. Prediction of resistance in sugarcane to stalk borer Eldana
saccharina by near-infrared spectroscopy on crude budscale extracts: involvement
of chlorogenates and flavonoids. J. Chem. Ecol. 24: 1447-1463.

Ryan, C. A. 1990. Protease inhibitors in plants: genes for improving defenses against
insects and pathogens. Annu. Rev. Phytopath. 28: 425-449.









Ryder, E. J. 1998. Lettuce, endive and chicory. CABI Publishing Cambridge, UK.

Saba, F. 1970. Host plant spectrum and temperature limitations of Diabrotica balteata.
Can. Entomol. 102: 684-691.

Sadasivan S., and B. Thayumanavan. 2003. Molecular host plant resistance to pests.
Marcel Drekker, Inc. Basel.

Saltveit M. E., Y.-J Choi, and F. A. Thomas-Barberan. 2005. Involvement of components
of the phospholipids-signaling pathway in wound phenlproponoid metabolism in
lettuce (Lactuca sativa) leaf tissue. Physiol. Plant. 125: 345-355.

SAS Institute. 1999. Guide for personal computers, version 6. SAS Institute, Cary, NC.

SAS Institute. 2003. Guide for personal computers, version 9.1.3. SAS Institute, Cary,
NC.

Schalk, J. M. 1986. Rearing and handling of Diabrotica balteata, pp. 49-56. In J. L.
Krysan and T. A. Miller [eds.], Methods for the study of pest Diabrotica. Springer,
NewYork.

Schalk, J. M., J. R. McLaughlin, and J. H. Tumlinson. 1990. Field response of feral male
banded cucumber beetles to the sex pheromone 6,12-dimethylpentadecan-2-one.
Fla. Entomol. 73: 292-297.

Schalk, J. M., A. Jones, and P. D. Dukes. 1986. Factors associated with resistance in
recently developed sweet potato cultivars and germplasm to the banded cucumber
beetle, Diabrotica balteata LeConte. J. Agric. Entomol. 3: 329-334.

Schenck, P. 1966. Szintigraphische Darstellung des parastemalen Lymphsystems.
Strahlentherapie 130: 504-508.

Schoonhoven, L. M., J. J. A. Loon, and M. Dicke. 2005. Insect plant biology. Oxford
Press, New York.

Scriber, J. M. 1977. Limiting effects of low leaf water content on nitrogen utilization,
energy budget and larval growth ofHyalophora cecropia (Lepidoptera:
Saturniidae). Oecologia 28: 269-287.

Scriber, J. M., and F. Slansky Jr. 1981. The nutritional ecology of immature insects.
Annu. Rev. Entomol. 26: 183-211.

Seiber, J. N., C. J. Nelson, and S. M. Lee. 1982. Cardenolides in the latex and leaves of
seven Ascelpes species and Calotropisprocera. Phytochemistry 21: 2343-2348.

Sessa, R. A., M. H. Bennett, M. J. Lewis, J. W. Mansfield, and M. H. Beale. 2000.
Metabolite profiling of sesquiterpene lactones from Lactuca species. J. Biol. Chem.
275: 26877-26884.









Sethi, A., H. J. McAuslane, R.T. Nagata, and G. S. Nuessly. 2006. Host plant resistance
in romaine lettuce affects feeding behavior and biology of Trichoplusia ni and
Spodoptera exigua (Lepidoptera: Noctuidae). J. Econ. Entomol. 99: 2156-2163.

Sethi, A., H. J. McAuslane, R.T. Nagata, and G. S. Nuessly. 2007. Romaine lettuce latex
deters banded cucumber beetle (Coleoptera: Chrysomelidae) feeding. Entomol.
Exp. Appl. (In press).

Seto, M., T. Miyase, K. Umehara, A. Uneno, Y. Hirano, and N. Otani. 1988.
Sesquiterpenes lactones from Cichorium endivia L. and C. intybus L. and cytotoxic
activity. Chem. Pharm. Bull. 36: 2423-2428.

Shahidi, F., and P. K. Wanasundara. 1992. Phenolic antioxidants. Crit. Rev. Food Sci.
Nutr. 32: 67.

Sharma, H. C., and R. Ortiz. 2002. Host plant resistance to insects: An eco-friendly
approach for pest management and environment conservation. J. Environ. Biol. 23:
111-135.

Sherman, T. D., K. C. Vaughn, and S. O. Duke. 1991. A limited survey of the
phylogenetic distribution of polyphenol oxidase. Phytochemistry 30: 2499-2506.

Showalter, A. M. 1993. Structure and function of plant cell wall proteins. Plant Cell 5: 9-
23.

Shukla, O. P., and C. R. Krishna-Murti. 1971. The biochemistry of plant latex. J. Sci.
Indus. Res. 12: 640-662.

Shulke, R. H., and L. L. Murdock. 1983. Lipoxygenase, trypsin inhibitor and lectin from
soybeans: effects on larval growth ofManduca sexta (Lepidoptera: Sphingidae).
Environ. Entomol. 12: 787-791.

Siedow, J. 1991. Plant lipoxygenase: structure and function. Annu. Rev. Plant Physiol.
Plant Mol. Biol. 42: 145-188.

Simmonds, M. S. J. 2003. Flavonoid-insect interactions: recent advances in our
knowledge. Phytochemistry 64: 21-30.

Simpson, N. J. K. 2000. Solid phase extraction principles, strategies and applications
Marcel Dekker, New York.

Siomos, A. S., P. P. Papadopoulou, C. C. Dogras, E. Vasiliadis, A. Dosas and N.
Georgiou. 2002. Lettuce composition as affected by genotype and leaf position.
Acta Hort. 579: 635-639.

Sirinphanic, J. and A. A. Kader. 1985. Effects of total C02 on total phenolics,
phenylalanine ammonia lyase and polyphenol oxidase in lettuce tissue. J. Amer.
Soc. Hort. Sci. 110: 249-253.









Skogsmyr, I., and T. Fagerstrom. 1992. The cost of anti-herbivory defense: an evaluation
of some ecological and physiological factors. Oikos 64: 451-457.

Slansky, Jr. F. 1992. Allelochemical-nutritient interactions in herbivore nutritional
ecology, pp. 135-174. In G. A. Rosenthal and M. R. Berenbaum (eds.), Herbivores:
their interaction with secondary plant metabolites, 2nd ed. vol. 1: The chemical
participants. Academic Press, New York.

Small, J. 1916. The translocation of latex and the multiple razor. New Phytol. 15: 194-
199.

Smith, C. G., M. W. Rodgers, A. Zimmerlin, D. Ferdinando, and G. P. Bolwell. 1994.
Tissue and subcellular immunolocalisation of enzymes of lignin synthesis in
differentiating and wounded hypocotyl tissue of french bean (Phaseolus vulgaris
L.). Planta 192: 155-164.

Smith, C. M. 1989. Plant resistance to insects: a fundamental approach. John Wiley &
Sons, Inc., New York.

Sokal, R. R., and F. J. Rohlf. 1995. Biometry. W. H. Freeman & Co., New York.

Somssich, I. E., P. Wernert, S. Kiedrowski, and K. Hahlbrock. 1996. Arabidopsis
thaliana defense related protein EL13 is an aromatic alcohol: NADP+
oxidoreductase. Proc. Natl. Acad. Sci. USA 93: 14199-14203.

Spencer, H. J. 1939. On the nature of the blocking of the lactiferous system at the leaf
base of Hevea brasiliensis. Ann. Bot. 3: 231-235.

Spilatro, S. R., and P. G. Mahlberg 1986. Latex and lacticifer starch content of
developing leaves of Euphorbiapulcherrima. Amer. J. Bot. 73: 1312-1318.

Stapleton, A. E., and V. Walbot. 1994. Flavonoids can protect maize DNA from the
induction of ultraviolet radiation damage. Plant Physiol. 105: 881-889.

Steffens, J. C., and D. S. Walters. 1991. Biochemical aspects of glandular trichome-
mediated insect resistance in the Solanaceae, pp 136-149. In P. A. Hedin [ed]:
Naturally occurring pest bioregulators. ACS Symp. Ser. 489. Washington, DC:
American Chemical Society.

Steffens, J., E. Harrel, and M. Hunt. 1994. Polyphenol oxidase, pp. 276-304. In B. E.
Ellis, G. W. Kuroki, and H. A. Stafford [eds.], Genetic engineering of plant
secondary metabolism. Plenum Press, New York.

Stobiecki, M., P. Wojtaszek, and K. Gulewicz. 1997. Application of solid phase
extraction for profiling quinolizidine alkaloids and phenolic compounds in Lupinus
albus. Phytochem.Anal. 8: 153-158.









Stotz, H. U., J. Kroymann, and T. Mitchell-Olds. 1999. Plant-insect interactions. Curr.
Opin. Plant Biol. 2: 268-272.

Stout, M. J., A. L. Fidantsef, S. S. Duffey, and R. M. Bostock. 1999. Signal interactions
in pathogen and insect attack: systemic plant-mediated interactions between
pathogens and herbivores of the tomato, Lycopersicon esculentum. Physiol. Mol.
Plant Pathol. 54: 115-130.

Stout, M. J., J. Workman, and S. S. Duffey. 1994. Differential induction of tomato foliar
proteins by arthropod herbivores. J. Chem. Ecol. 20: 2575-2594.

Strauss, S. Y., and A. A. Agrawal. 1999. The ecology and evolution of plant tolerance to
herbivory. Trends Ecol. Evol. 14: 179-185.

Strid, A., W. S. Chow, and J. M. Anderson. 1994. UV-B damage and protection at the
molecular level in plants. Photosynthesis Res. 39: 475-489.

Sturtevant, E. L. 1886. A study of garden lettuce. Am. Nat. 20: 230-233.

Swain, R. 1977. Secondary compounds as protective agents. Annu. Rev. Plant. Physiol.
28: 279-501.

Swain, T. 1979. Tannins and lignins, pp. 657-682. In G. A. Rosenthal and D. H. Janzen
[eds.], Herbivores: their interaction with secondary plant metabolites. Academic
Press, San Diego.

Swift, J. E., and W. H. Lange. 1980. Lettuce root aphid, p. 2. Leaflet No. 2668,
University of California.

Taira, T., A. Ohdomari, N. Nakama, M. Shimoji, and M. Ishihara. 2005. Characterization
and antifungal activity of Gazyumaru (Ficus microcarpa) latex chitinases: both the
chitin binding and antifungal activities of class I chitinase are reinforced with
increasing ionic strength. Biolsci. Biotechnol.Biochem. 69: 811-818.

Taiz, L., and E. Zeiger. 1991. Plant physiology. The Benjamin/Cummings Publishing
Comp. Inc., Redwood City.

Takasugi, M., S. Okinaka, N. Katsui, T. Masamune, A. Shirata, and M. Chuchi. 1985.
Isolation and structure of lettucenin A, a novel guaianolide phytoalexin from
Lactuca sativa var. capitata (Compositae). J. Chem. Soc. Chem. Commun. 10: 621-
622.

Tamaki, H., R. W. Robinson, J. L. Anderson, and G. S. Stoewsand. 1995. Sesquiterpene
lactones in virus-resistant lettuce. J. Agric. Food Chem. 43: 6-8.









Tatchell, G. M., P. R. Ellis, R. H. Collier, D. Chandler, A. Mead. L. J. Wadhams, W. E.
Parker, J. A. Blood Smyth, and W. E. Vice. 1998. Integrated pest management of
aphids on outdoor lettuce crops, pp. 77. Final Rep. HDC Project No. FV 162. East
Malling, Horticulture Development Council.

Terra, W. R., C. Ferreira, and B. P. Jordao. 1996. Digestive enzymes, pp. 153-194. In M.
J. Lehane and P. F. Billinsley [eds.], The biology of insect midgut. Chapman and
Hall, London, UK.

Thaler, J. S. 1999. Jasmonate-induced plant defenses cause increased parasitism of
herbivores. Nature 399: 686-688.

Thaler, J. S., M. J. Stout, R. Karban, and S. S. Duffey. 1996. Exogenous jasmonates
simulate insect wounding in tomato plants (Lycopersicon esculentum) in the
laboratory and field. J. Chem. Ecol. 22: 1767-1781.

Thipyapong, P., D. Joel, and J. Steffens. 1997. Differential expression and turnover of the
tomato polyphenol oxidase gene family during vegetative and reproductive
development. Plant Physiol. 113: 707-718.

Thipyapong, P., and J. Steffens. 1997. Tomato polyphenol oxidase: differential response
of the polyphenol oxidase promoter to injuries and wound signals. Plant Physiol.
1152:409-418.

Thygesen, P., I. Dry, and S. Robinson. 1995. Polyphenol oxidase in potato: a multigene
family that exhibits differential expression patterns. Plant Physiol. 109: 525-531.

Todd, G. W., A. Gethahun, and D. C. Cress. 1971. Resistance in barley to the green bug,
Schizaphis graminum. Toxicity of phenolic and flavonoid compounds and related
substances. Ann. Entomol. Soc. Am. 64: 718-721.

Tomas-Barberan, F.A., M.I. Gil, M.Castafier, F.Artes, and M. Saltveit. 1997. Effects of
selective browning inhibitors on phenolic metabolism in stem tissue of harvested
lettuce. J. Agric. Food Chem. 45: 583-589.

Toscano, N. C., K. Kido, and R. M. Davis. 1990. Lettuce pest management guidelines.
UCPMG Publication 15. IPM Education and Publications, University of California,
Davis.

Tune, R., and D. E. Dussourd. 2000. Specialized generalists: constraints on host range in
some plusiine caterpillars. Oecologia 23: 543-549.

USDA (United States Department of Agriculture). 2002. Crop Production-Annual
Summary: 2002 Vegetable Crops Summary.NASS. http://www.usda.gov/nass/.

USDA (United States Department of Agriculture). 2005a. Vegetables and melons
outlook. February 23. ERS. http://www.ers.usda.gov/publications/vgs.









USDA (United States Department of Agriculture). 2005b. Agricultural chemical usage
2004 vegetables summary. July 2005. NASS.
http://usda.mannlib.cornell. edu/reports/nassr/other/pcu-bb/.

USDA (United States Department of Agriculture). 2005c. Agricultural chemical usage
2004 restricted use summary. October 2005. NASS.
http://usda.mannlib.cornell. edu/reports/nassr/other/pcu-bb/.

Vail, P. A., A. C. Pearson, V. Sevacherian, T. J. Henneberry, and H. T. Reynolds. 1989.
Seasonal incidence of Trichoplusia ni and Autographa californica (Lepidoptera:
Noctuidae) on alfalfa, cotton, and lettuce in the Imperial Valley of California.
Environ. Entomol. 18: 785-790.

Valle, M. G., G. Appendino, G. M. Nano, and V. Picci. 1987. Prenylated coumarins and
sesquiterpenoids from Ferula communis. Phytochemistry 26: 253-256.

van Beek, T. A., P. Mass, B. M. King, E. Leclercq, A. G. J. Voragen, and A. Groot. 1990.
Bitter sesquiterpene lactones from chicory roots. J. Agric. Food Chem. 38: 1035-
1038.

van der Arend, A. J. M., J. T. van Schijndel P. R. Ellis, and S. Derridj. 1999. The
making of the aphid resistant butterhead lettuce 'Dynamite'. Bull. OILB SROP 22:
35-43.

van Helden, M., and W. F. Tjallingii. 1993. Tissue localization of lettuce resistance to the
aphid Nasonovia ribisnigri using electrical penetration graphs. Entomol. Exp. Appl.
68: 269-278.

van Helden, M., W. F. Tjallingii, and F. L. Dieleman. 1993. The resistance of lettuce
(Lactuca sativa L.) to Nasonovia ribisnigri: bionomics of N. ribisnigri on near
isogenic lettuce lines. Entomol. Exp. Appl. 66: 53-58.

van Helden, M., and D. van der Wal. 1996. Isolation of allomones from phloem sap of
aphid-resistant lettuce by bioassay guided fractionation. Bull. OILB SROP 19: 62-
67.

van Helden, M., H. P. N. F. van Heest, T. A. van Beek, and W. F. Tjallingii. 1995.
Development of a bioassay to test phloem sap samples from lettuce for resistance to
Nasonovia ribisnigri (Homoptera, Aphididae). J. Chem. Ecol. 21: 761-774

van Melckebeke, J., S. Kino, L. de. Rooster, L. de Reycke, and R. Sarrazyn. 1999. Plant
protection in field vegetables. Nasonovia resistant cultivars: alternative for
chemical control of aphids in butterhead lettuce and iceberg lettuce grown in the
field. Proeftuinnieuws 9: 12-13.

Vet, L. E. M. 1999. Evolutionary aspects of plant carnivore interactions, pp. 3-20. In D.
Chadwick and J. Goode [eds.], Insect plant interactions and induced plant defense.
Novartis Foundation Symposium 223. John Wiley & Sons, Ltd., Chichester, U.K.









Vilmorin. 1883. Les Plantes Portageres. Cited from E. L. Sturtevant. 1886. A study of
Garden lettuce. Am. Nat. 20: 230-233.

Walker, A. J., L. Ford, M. E. N. Majerus, I. E. Geohegan, A. N. E. Birch, J. A.
Gatehouse, and A. M. R. Gatehouse. 1998. Characterization of the midgut digestive
proteinase activity of the two-spot ladybird beetle (Adalia bipunctata L.) and its
sensitivity to proteinase inhibitors. Insect Biochem. Mol. Biol. 28: 173-180.

Wang, J., and C. P. Constabel. 2004. Polyphenol oxidase overexpression in transgenic
Populus enhances resistance to herbivory by forest tent caterpillar (Malacosoma
disstria). Planta 220: 87-96.

Waterhouse, D. F., and K. R. Norris. 1987. Liriomyza species, Diptera: Agromyzidae,
leafminers, pp. 159-176. In Biological control, pacific prospects. Inkata Press,
Melbourne, Australia.

Wei, Y. D., E. de Neergaard, H. Thordal-Christensen, D. B. Collinge, and V. Smedegaard
Petersen. 1994. Accumulation of a putative guanidine compound in relation to
other early defense reactions in epidermal cells of barley and wheat exhibiting
resistance to Erysiphe graminis fsp. hordei. Physiol. Mol. Plant Pathol. 45: 469-
484.

Whitaker, T. W., A. N. Kishaba, H. H. Toba, R. Antoszewski, L. Harrison, and C. C.
Zych. 1974. Resistance in lettuce to the cabbage looper, Trichoplusia ni (Hubner),
pp. 721-764. Proc. XIX Intl. Hortic. Cong. I. Section VII. Vegetables.

Wink, M. 1997. Special nitrogen metabolism, pp. 439-486. In P. M. Dey and J. B.
Harborne [eds.], Plant biochemistry. Academic Press, London.

Winter, M., and K. Hermann. 1996. Esters and glucosides of hydroxyl cinnamic acid in
vegetables. J. Agric. Food Chem. 34: 616-620.

Wititsuwannakul D, Chareonthiphakorn N, Pace M, Wititsuwannakul D. 2002.
Polyphenol oxidases from Hevea brasiliensis: purification and characterization.
Phytochemistry 61: 115-121.

Yudin, L. S., J. J. Cho, and W. C. Mitchell. 1986. Host range of western flower thrips,
Frankliniella occidentalis (Thysanoptera: Thripidae), with special reference to
Leucaena glauca. Environ. Entomol. 5: 1292-1295.

Zalucki, M. P., and L. P. Brower. 1992. Survival of first instar larvae ofDanaus
plexippus (Lepidoptera: Danainae) in relation to cardiac glycoside and latex content
ofAsclepias humistrata (Asclepiadaceae). Chemoecology 3: 81-93.

Zalucki, M. P., and S. B. Malcolm. 1999. Plant latex and first-instar monarch larval
growth and survival on three North American milkweed species. J. Chem. Ecol. 25:
1827-1842.









Zalucki, M. P., S. B. Malcolm, T. P. Paine, C. C. Hanlon, L. P. Brower, and A. R. Clarke.
2001. It's the first bites that count: Survival of first-instar monarchs on milkweeds.
Austral Ecol. 26: 547-555.

Zar, J. H. 1984. Biostatistical analysis. 2nd ed. Prentice-Hall, Inc., Englewood Cliffs,
New Jersey.

Zeren, 0. 1985. Investigations on a new lettuce pest, Uroleucon cichorii (Hom.,
Aphididae), in the Cukurova region. Turkiye Bitki Koruma Dergisi. 9: 173-181.

Zeyen, R. J., W. R. Bushnell, T. L. W. Carver, M. P. Robbins, T. A. Clark, D. A. Boyles,
and C. P. Vance. 1995. Inhibiting phenylalanine ammonia-lyase and cinnamyl-
alcohol dehydrogenase suppresses mlal (HR) but not Mlo5 (Non-HR) barley
powdery mildew resistances. Physiol. Mol. Plant Pathol. 47: 119-140.









BIOGRAPHICAL SKETCH

Amit Sethi was born August 7, 1977, in Abohar, Punjab, India. He received his

bachelor's degree in agriculture with honors in plant protection from the Department of

Entomology, Punjab Agricultural University, Ludhiana, India in 1998. He also received

the merit fellowship during his bachelor's degree. He obtained his master's degree in

entomology from the same institute in 2000, and also received Novartis crop protection

fellowship. He worked as a research fellow in the same department for 3 years. While

working, he also obtained his M.B.A. in Operational Management from the Indira Gandhi

National Open University, New Delhi, India. In 2004, he began his Ph.D. program at the

University of Florida to study the biochemical basis of host plant resistance in romaine

lettuce under the supervision of Dr. Heather J. McAuslane in the Department of

Entomology and Nematology. He received many research and travel grants from the

department, university and also from various scientific societies. He won awards (eight)

for all of his poster and oral presentations at various state, regional and national scientific

meetings. He was an extremely good citizen in the department and the university

community. He served as historian for the Graduate Student Organization of the

department and he was involved in its many outreach and fundraising (snack-bar

coordinator) activities. He was active on the department's Social Committee and he had

served as coordinator of the Seminar Committee for several years. This committee was

totally responsible for organizing the weekly departmental seminars with local and

national speakers. He was Mayor of his married student housing complex and serves in a

leadership role on the Mayors' Council, a committee of the University's Student

Government. His long term goal is finding a challenging position in molecular and









chemical ecology highlighting insect-plant interactions with both teaching and research

responsibilities.





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1 BIOCHEMICAL MODE OF RESISTANCE TO MULTIPLE INSECT PESTS IN A ROMAINE LETTUCE CULTIVAR By AMIT SETHI A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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2 2007 Amit Sethi

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3 To my beloved father, Amar L. Sethi who has been my role-model for hard work, persistence and personal sacrifices, and who instilled in me the inspirat ion to set high goals and the confidence to achieve them, and his words of en couragement and push for tenacity ring in my ears.

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4 ACKNOWLEDGMENTS It gives me immense pleasure to record my thanks and sense of pr ofound gratitude to my major advisor, Dr. Heather J. McAuslane fo r her kind inspiration, constant supervision, constructive criticism and encouragement thr oughout the period of my Ph.D, especially time spent in informal discussions and training that have all been a valuable part of my learning experience. Expressing sense of gratitude and admiration for the kind help extended by Dr. Hans T. Alborn (CMAVE, USDA) and Dr. Bala Rathinasabap athi (Departemnt of Horticultural Sciences) is not mere obedience of convention, but a real appreciation. I am also highly obliged to Dr. Gregg S. Nuessly and Dr. Russell T. Nagata (Eve rglades Research and Education Center), the members of my committee for their guidance and valuable suggestions for the improvement of this dissertation project. I owe my sincere thanks to Jennifer Hogse tte, Jennifer Meyer, and Debbie Boyd for their timely help in the insect colony maintenance when I was away for the conferences. I am thankful to Dr. Peter Teal (CMAVE, USDA) for providi ng greenhouse space for growing lettuce plants and also Julia Meredith (CMAVE, USDA) for taking care of plants when I was away for scientific conferences. I am also thankful to Dr. Marty Marshall (Departement of Food Science and Food Nutrition) for use of his spectrophotometer. Words fail me to convey the depth of my feelin gs and gratitude to my lab mates Jennifer Meyer, Karla Addesso, Jennifer Hogsette and Mu rugesan Rangasamy for their encouragement, generosity and memo rable association. I seize the opportunity to express my moral obl igations to my brothers and their families, and in-laws for their encouragement and moral suppor t. My father deserves my heartiest thanks

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5 for his magnanimity, inspiration and encouragemen t at times of despair that helped me in innumerable ways in making this effort a success. No appropriate words could be traced in th e presently available lexicon to acknowledge the sacrifices, selfless devotion, love and unflin ching support extended by my beloved wife Dr. Ramandeep Kaur to complete this study. Putting it last, but feeling it first, I owe G od who has given me courage, patience and motivation from time to time in completing my degree successfully.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........9 LIST OF FIGURES................................................................................................................ .......10 ABSTRACT....................................................................................................................... ............14 CHAPTER 1 REVIEW OF LITERATURE.................................................................................................16 Introduction................................................................................................................... ..........16 Origin and History of Lettuce.................................................................................................17 Types of Lettuce............................................................................................................... ......18 Insect Pests and Lettuce....................................................................................................... ...18 Host Plant Resistance.......................................................................................................... ...21 Biochemical Basis of Host Plant Resistance..........................................................................24 Host Plant Resistance Due To Proteins...........................................................................24 Protease inhibitors....................................................................................................25 Cysteine protease......................................................................................................25 Oxidative enzymes...................................................................................................26 Proteins of the cell wall............................................................................................28 Secondary metabolism pathways.............................................................................29 Enzymes involved in secondary metabolism...........................................................29 Host Plant Resistance Due To Secondary Plant Compounds..........................................30 Phenolics..................................................................................................................31 Flavonoids................................................................................................................33 Terpenoids................................................................................................................35 Host Plant Resistance in Lettuce to Insect Pests....................................................................37 Aphids......................................................................................................................... .....37 Cabbage Looper...............................................................................................................39 Banded Cucumber Beetle................................................................................................40 Leafminer...................................................................................................................... ..41 Helicoverpa specie s .........................................................................................................42 Spodoptera species..........................................................................................................42 Bemisia species or strains................................................................................................43 Thrips......................................................................................................................... ......43 Research Goals................................................................................................................. ......44

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7 2 HOST PLANT RESISTANCE IN ROMA INE LETTUCE AFFECTS LARVAL FEEDING BEHAVIOR AND BIOLOGY OF TRICHOPLUSIA NI AND SPODOPTERA EXIGUA (LEP IDOPTERA: NOCTUIDAE)..............................................46 Introduction................................................................................................................... ..........46 Materials and Methods.......................................................................................................... .48 Plants......................................................................................................................... ......48 Insects........................................................................................................................ ......48 Neonate Survival and Development to Third Instar........................................................49 Survival and Development from Neonate to Adult Emergence......................................50 Fecundity and Longevity of Subsequent Generation......................................................51 Results........................................................................................................................ .............51 Neonate Survival and Development to Third Instar........................................................51 Larval Feeding Behavior.................................................................................................52 Survival and Development from Neonate to Adult Emergence......................................53 Fecundity and Longevity of Subsequent Generation......................................................54 Discussion..................................................................................................................... ..........54 3 ROMAINE LETTUCE LATEX DETERS FE EDING OF BANDED CUCUMBER BEETLE (COLEOPTERA: CHRYSOMELIDAE)...............................................................68 Introduction................................................................................................................... ..........68 Materials and Methods.......................................................................................................... .71 Plants and Insects............................................................................................................71 Artificial Diet Preparation...............................................................................................73 Latex Collection and Solvent Extraction.........................................................................74 Bioassay Conditions........................................................................................................75 Choice Tests and No-choice Tests with Fresh Latex......................................................76 Choice Tests Using Latex from Young and Mature Leaves...........................................76 No-Choice Tests Using Latex Extracts...........................................................................77 Beetle Behavior in Resp onse to Contacting Latex..........................................................77 Statistical Analysis..........................................................................................................78 Results........................................................................................................................ .............80 Latex Choice and No-Choice Tests.................................................................................80 Choice Tests Using Latex from Young and Mature Leaves...........................................81 No-Choice Tests Using Latex Extracts...........................................................................82 Beetle Behavior in Resp onse to Contacting Latex..........................................................83 Discussion..................................................................................................................... ..........84 4 BANDED CUCUMBER BEETLE (COL EOPTERA: CHRYSOMELIDAE) RESISTANCE IN ROMAINE LETTUCE: UNDERSTANDING LATEX CHEMISTRY...................................................................................................................... .104 Introduction................................................................................................................... ........104 Materials and Methods.........................................................................................................105 Plants and Insects..........................................................................................................105

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8 Assay for Feeding Deterrence.......................................................................................106 Latex Collection and Crude Extract Preparation...........................................................107 Fractionation of Crude Extract Usin g Reversed-Phase (C-18) Cartridge.....................107 Fractionation of Crude Extract Using C18, SAX and SCX Cartridges Connected in Series......................................................................................................................... .109 LC/MS Separation of SCX Fraction..............................................................................110 Statistical Analysis........................................................................................................111 Results........................................................................................................................ ...........112 Fractionation of Crude Ex tract Using C-18 Cartridge..................................................112 Fractionation of Crude Extract Using C18, SAX and SCX Cartridges Connected in Series......................................................................................................................... .113 Fractionation of SCX Fraction Using LC/MS...............................................................114 Discussion..................................................................................................................... ........114 5 INVESTIGATING ENZYME INDUCT ION AS A POSSIBLE REASON FOR LATEX-MEDIATED INSECT RESISTANCE IN ROMAINE LETTUCE.......................136 Introduction................................................................................................................... ........136 Materials and Methods.........................................................................................................138 Plants......................................................................................................................... ....138 Insects........................................................................................................................ ....138 Artificial Diet................................................................................................................ .139 Bioassay Conditions for Feeding Damage....................................................................139 Choice-tests Using Latex from Damaged and Undamaged Plants................................140 Enzyme Activity Assays................................................................................................140 Phenylalanine ammonia-lyase (PAL).....................................................................141 Polyphenol oxidase (PPO).....................................................................................141 Peroxidase (POX)...................................................................................................142 Statistical Analysis........................................................................................................142 Results........................................................................................................................ ...........143 Latex Characteristics from Da maged and Undamaged Plants......................................143 Choice-tests Using Latex from Damaged and Undamaged Plants................................143 Total Protein Content....................................................................................................145 Phenylalanine Ammonia Lyase.....................................................................................145 Polyphenol Oxidase.......................................................................................................146 Peroxidase..................................................................................................................... .146 Relationship between Female Weight Gain and Enzyme Activity...............................147 Discussion..................................................................................................................... ........147 6 SUMMARY........................................................................................................................ ..164 LIST OF REFERENCES.............................................................................................................173 BIOGRAPHICAL SKETCH.......................................................................................................206

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9 LIST OF TABLES Table page 2-1 Performance of cabbage looper and beet armyworm released as neonates onto Valmaine and Tall Guzmaine lettuce.................................................................................59 2-2 Fecundity and longevity of subsequent generation of cabbage looper and beet armyworm reared on Valmaine and Tall Guzmaine lettuce..............................................60 3-1 Dry weight consumption of diet disks treated with Valmaine (Val) or Tall Guzmaine (TG) latex under choice and no-choice tests by six D. balteata adults in 16 h...............101 3-2 Feeding deterrent activity of latex against D. balteata adults when artificial diet disks were treated with latex from either resistant Valmaine (Val) or susceptible Tall Guzmaine (TG) in choice and no-choice tests.................................................................102 3-3 Dry weight of diet consumed by six D. balteata adults in 16 h when given a choice between diet disks treated w ith latex from either young or mature leaves of resistant Valmaine or susceptible Tall Guzmaine lettuce cultivars................................................103 5-1 Total diet consumption by six D. balteata adults on two diet disks treated with latex from same lettuce cultivar, Valmaine or Ta ll Guzmaine after 24 h of their release........163

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10 LIST OF FIGURES Figure page 2-1 Experimental setup to study cabbage l ooper and beet armyworm neonate survival and development to third instar..........................................................................................61 2-2 Larval mortality of cabbage looper and beet armyworm after 1 wk of feeding on resistant Valmaine and suscep tible Tall Guzmaine lettuce................................................62 2-3 Instars of cabbage looper (CL) and b eet armyworm (BAW) surviving for 1 wk on resistant Valmaine and suscepti ble Tall Guzmaine lettuce................................................63 2-4 Feeding of two lepidopterans on lettuce............................................................................64 2-5 Feeding preference of cabbage looper (CL) and beet armyworm (BAW) larvae among lettuce leaves of different ages on re sistant Valmaine and susceptible Tall Guzmaine....................................................................................................................... ....65 2-6 Feeding behavior of beet armyworm.................................................................................66 2-7 Relationships between adult weight and fecundity of cabbage looper (CL) and beet armyworm (BAW) that developed from larv ae reared on resistant Valmaine (VAL) or susceptible Tall Gu zmaine (TG) lettuce........................................................................67 3-1 Wounding of lettuce releases a milky fluid called latex....................................................88 3-2 Colony rearing of D. balteata See text for descripti on of each stage of colony maintenance.................................................................................................................... ...89 3-3 Collection of latex from romaine lettu ce, application on artificial diet disk and bioassay setup................................................................................................................. ...90 3-4 Scheme of latex solvent extraction....................................................................................91 3-5 Latex dissolution in different solvents...............................................................................92 3-6 Feeding bioassays using fresh latex...................................................................................93 3-7 Mean number of D balteata adults feeding on artificial diet disks treated with latex from resistant Valmaine (Val), disks treated with latex from susceptible Tall Guzmaine (TG), and control di et disks in choice tests......................................................94 3-8 Mean number of D balteata adults feeding on two artificia l diet disks treated with latex from resistant Valmaine (Val), disks treated with latex from susceptible Tall Guzmaine (TG), and control diet disks in no-choice tests.................................................95 3-9 Choice tests using D. balteata adults on tw o artificial diet disks treated with latex from young and mature leaves of the same cultivar..........................................................96

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11 3-10 Number of D balteata adults feeding on artificial diet disks treated with latex from young or mature leaves of resistant Valmai ne (Val) and susceptible Tall Guzmaine (TG) in choice tests........................................................................................................... .97 3-11 No-choice tests using D. balteata adults when both the disks were smeared with either Valmaine latex extract or Tall Guzmaine latex extract...........................................98 3-12 Mean number of D balteata adults feeding on two artificia l diet disks treated with latex extracts from resistant Valmaine (V al) and susceptible Tall Guzmaine (TG), and controls in no-choice test............................................................................................99 3-13 Dry weight of diet consumed by six D. balteata adults in 16 h when both diet disks were treated with Valmaine (Val) or Tall Guzmaine (T G) latex extracts under nochoice situations.............................................................................................................. .100 4-1 Scheme for solid-phase extraction and fractionation of crude extract after passing through reversed-phase (C-18) cartridge.........................................................................118 4-2 Scheme for solid-phase extraction and fractionation of crude extract after passing through reversed-phase (C-18), anion (SAX) and cation (SCX) exchange cartridges connected in series...........................................................................................................119 4-3 Fractions obtained afte r HPLC analysis of cation exchange (SCX) fraction..................120 4-4 Color characteristics of fractions ob tained after passing crude extract through reversed phase C-18 cartridge..........................................................................................121 4-5 Bioassays of C-18 fractions applie d on artificial diet disks using D. balteata adults under no-choice conditions..............................................................................................122 4-6 Mean number of D balteata adults feeding after 90 min on two artificial diet disks treated with fractions obtain ed after passing crude extract at three pH levels through C-18 cartridge................................................................................................................. .123 4-7 Dry weight of diet consumed by D. balteata adults when disks were treated with fractions obtained after passing crude extr act with different pH levels through C-18 cartridge...................................................................................................................... .....124 4-8 Color characteristics of fractions obtained after passing C-18 unbound fraction through anion (SAX) and cati on (SCX) exchange cartridge s connected in series..........125 4-9 Bioassays of ion-exchange fractions applied on artificia l diet disks using D. balteata adults under no-choice conditions...................................................................................126 4-10 Mean number of D. balteata adults feeding after 90 min on diet disks treated with ion-exchange fractions obtained by pa ssing C-18 unbound fraction (original pH 6.5) through anion (SAX) and cation (SAX) excha nge cartridges connected in series..........127

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12 4-11 Dry weight of diet consumed by D. balteata adults when disks were treated with ionexchange fractions obtained after pass ing C-18 unbound fraction (original pH 6.5) through anion (SAX) and cation (SAX) excha nge cartridges connected in series..........128 4-12 Mean number of insects feeding after 90 min on diet disks treated with fractions obtained after LC/MS analysis of cation ex change fraction (SCX ) at pH 9.0 of the mobile phase................................................................................................................... .129 4-13 Dry weight of diet consumed by D. balteata adults when disks were treated with fractions obtained after LC/MS analysis of cation exchange fraction (SCX) at pH 9.0 of the mobile phase..........................................................................................................130 4-14 Mean number of insects feeding after 90 min on diet disks treated with fractions obtained after LC/MS analysis of cation ex change fraction (SCX ) at pH 10.0 of the mobile phase................................................................................................................... .131 4-15 Dry weight of diet consumed by D. balteata adults when disks were treated with fractions obtained after LC/MS analysis of cation exchange fraction (SCX) at pH 10.0 of the mobile phase..................................................................................................132 4-16 Electrospray LC/MS total negative ion tr ace of active fraction between 3 and 4 min....133 4-17 Structure of sesquiterpene la ctones characterized in lettuce............................................134 4-18 Chemical structures of flavonoids found in lettuce.........................................................135 5-1 Feeding damage caused by D. balteata adults on two lettuce cultivars, Valmaine (VAL) and Tall Guzmaine (TG)......................................................................................152 5-2 Adults of D. balteata feeding on diet disks treated with latex from damaged and undamaged plants of two lettuce cultiv ars, Valmaine and Tall Guzmaine......................153 5-3 Number of D. balteata adults feeding on artificial diet disks in a choice between latex from damaged and undamaged plants of Valmaine after 1, 2, 3 and 4 h of their release........................................................................................................................ ......154 5-4 Number of D. balteata adults feeding in a choice test using two artificial diet disks treated with damaged and undamaged plants of lettuce cultivar, Tall Guzmaine after 1, 2, 3 and 4 h of their release..........................................................................................155 5-5 Artificial diet consumption after 24 h by D. balteata adults in choice test using two diet disks treated with latex from dama ged and undamaged plants of two lettuce cultivars, Valmaine (VAL) and Tall Guzmaine (TG)......................................................156 5-6 Total protein content in two lettuce cultiv ars, Valmaine (VAL) and Tall Guzmaine at 1, 3 and 6 d after initiation of feeding damage by adults of D. balteata..........................157

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13 5-7 Activity of phenylalanine ammonia lyase (P AL) in two lettuce cultivars, Valmaine (VAL) and Tall Guzmaine at 1, 3 and 6 d afte r initiation of feeding damage by adults of D. balteata ...................................................................................................................158 5-8 Activity of polyphenol oxidase (PPO) in tw o lettuce cultivars, Valmaine (VAL) and Tall Guzmaine at 1, 3 and 6 d after initi ation of feeding damage by adults of D. balteata ............................................................................................................................159 5-9 Activity of peroxidase (POX) in two le ttuce cultivars, Valmaine (VAL) and Tall Guzmaine at 1, 3 and 6 d after initiati on of feeding damage by adults of D. balteata ....160 5-10 Gain in fresh weight of D. balteata females over a 6-d period of feeding on two romaine lettuce cultivars, Valmaine (VAL) and Tall Guzmaine (TG)............................161 5-11 Relationship between fresh weight gain ed by D. balteata females feeding on two lettuce cultivars, Valmaine (VAL) and Ta ll Guzmaine (TG) an d activity of PAL, PPO and POX enzymes after 1, 3 and 6 d of feeding damage.........................................162

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14 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy BIOCHEMICAL MODE OF RESISTANCE TO MULTIPLE INSECT PESTS IN A ROMAINE LETTUCE CULTIVAR By Amit Sethi December 2007 Chair: Heather J. McAuslane Major: Entomology and Nematology Lettuce ( Lactuca sativa L.) quality and yield can be reduc ed by feeding of several insect pests. Host plant resistance to these insects is an environmenta lly sound adjunct to conventional chemical control. In this study I compared the survival, development and feeding behavior of cabbage looper, Trichoplusia ni (Hbner) and beet armyworm Spodoptera exigua (Hbner) on two romaine lettuce cultivars, resistant Val maine and susceptible Tall Guzmaine. The survival and development of both species was significantly less on resistant Valmaine than on susceptible Tall Guzmaine. The two insect spec ies showed different feeding preference for leaves of different age groups on Valmaine and Tall Guzmaine. Latex from Valmaine strongly inhibite d feeding of banded cucumber beetle, Diabrotica balteata LeConte compared to Tall Guzmaine when appl ied to the surface of artificial diet in both choice and no-choice tests. In a choice test involvi ng diet disks treated with Valmaine latex from young leaves versus mature leaves, the beetle s consumed significantly less diet treated with latex from young than mature leaves. No signifi cance difference in feeding was found between diet disks treated with latex from young and mature Tall Guzmaine leaves in choice tests. Three solvents of differing polarity (wat er, methanol and methylene chlo ride) were tested to extract deterrent compounds from latex; Valmaine latex extracted with water:methanol (20:80) strongly

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15 inhibited beetle feeding when applie d to the surface of artificial diet. These studies suggest that moderately polar chemicals within latex may account for resistance in Valmaine to D. balteata. Further fractionation of methanolic crude extract of Valmaine latex was done using reverse phase and cation exchange solid-phase extraction to isolate the deterrent compounds. Retention of deterrent compounds on cation exchange resin sugge sts the presence of compounds with amine group in Valmaine latex. Further bi oassay directed fractionation of cation exchange extract using LC/MS indicates the presence of ten compounds in the act ive fraction between 3 and 4 min. The successful isolation of potent feeding deterrents against D. balteata adults provides convincing evidence of a chemical basi s for host plant resistance mediated through latex in this cultivar. Latex from damaged plants of Valmaine was much more deterrent to D. balteata adults than latex from undamaged plants when applied on the artificial diet under choice conditions and no such difference was found in Tall Guzmaine choice tests. The activit ies of three enzymes (phenylalanine ammonia lyase, polyphenol oxida se and peroxidase) significantly increased in Valmaine latex from damaged plants over time (i.e., 1, 3 and 6 d) after feeding initiation, but they remained the same in Tall Guzmaine latex. The constitutive level of phenylalanine ammonia lyase and polyphenol oxidase was also significantl y higher in the Valmaine latex than in Tall Guzmaine latex. These studies suggest that late x chemistry may change after damage due to increased activity of inducible enzymes and that inducible resist ance appears to act synergistically with constitutive resistance in Valmaine latex.

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16 CHAPTER 1 REVIEW OF LITERATURE Introduction Lettuce, Lactuca sativa L., a member of the Compositae (A steraceae), is a rosette plant that is grown commercially for its leaves. The fa mily Compositae includes a wide range of herbaceous plants and accounts for one tenth of known angiosperm species. Lettuce is one of the most important vegetable crops grown in the Unite d States, in terms of quality and quantity as well as its acreage (Ryder 1998). Demand for lettu ce grows yearly, probably due to its use as a healthy, low caloric, salad com ponent of meals. It requires minimal processing, and its long storage life, good quality and reput ation as healthy food contribute to its increase in salad bars and fast foods (Ferreres et al. 1997). During 2006, the United States produced 2,935 thousand metric tons of head lettuce, 857.8 thousand metric tons of leaf lettuce, and 990.3 thous and metric tons of romaine lettuce harvested over areas of 71,508, 29,056, and 24,929 hectares, resp ectively (Agricultura l Statistics 2007). Crisphead (iceberg) varieties predominate in the Unites States markets, particularly for extended transport. However, romaine (Cos), butterhead, and leaf type lettuces are also produced in considerable amounts. A number of other varieties that show variation in co lor from light green and yellow to deep green are also becoming mo re accepted. Romaine lettuce is the most common leaf lettuce grown throughout the United States. California is the major producer of lettuce (77% of total production) in the United States (Lauritzen 1999), followed by Arizona, Florid a and New Jersey (Kerns et al. 1999, USDA 2002). Lettuce production from the Everglades Agricultur al areas in southern Florida contributes 90% of the total state production (Hochmuth et al. 1994).

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17 Origin and History of Lettuce Lettuce originated in the Mediterranean regi on and its cultivation may have started in Egypt as early as 4500 years BC (Lindquist 1960) Lettuces were supposedly grown by Persians 500 years BC, and were introduced into China between the years 600 and 900 AD. Lettuces were mentioned in England in the fourteenth century and reached America with Columbus (Davis et al. 1997). In 1494, Columbus introduced a non-headi ng type of lettuce to the New World. This type quickly formed a seed stalk and in fact di d not become a stable food crop. Head lettuce in the United States was first reported in 1543 (Helm 1954). Salad lettuce was popular with the ancient Greeks and Romans and it arrived in the United States during coloni al days (Davis et al. 1997). Sturtevant (1886) studied the history of lettuce and observed th at 83 distinct varieties of lettuce were grown under nearly 200 names at th e New York Agricultural Experiment Station. These varieties were present in three distinct fo rm-species, the lanceolate-leaved, the Cos and the cabbage. The lanceolate-leaved form was represented by one variety, the deers tongue, and had a chicory-like appearance in some stages of its growth, as mentioned and illustrated by Bauhin (1671). This type of lettuce was submitted under the names Romaine asperge Lactuca angustana Hort., and L cracoviensis Hort by Vilmorin (1883). The Cos lettuce had upright growth of elongate d, spatulate leaves. The Cos form was less commonly grown in northern Europe as compared to the south and was seldom cultivated in France and Germany in the sixteenth century. Cabbage lettuce was characterized by rounded and spatulate leaves, growing less upright than the Cos lettuce. The commentators of the si xteenth and seventeenth centuries deemed this form-species to have been known to ancien t Greeks and Romans. Pliny (23-79 AD) and Columella (42 AD) referred to it as a va riety, Laconicon, and Tartesian or B tica,

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18 respectively. The cabbage lettuce was more wrinkl ed or blistered than the Cos (Sturtevant 1886). Pinaeus (1561) identified a heading lettuce that closely resembled the st one tennis ball variety of lettuce. Botanists were agreed in considering the cultivated lettuce as a modification of the wild species L. scariola (de Candolle 1885). In conclusion, thes e three form-species had different origins from different wild forms that had been cultivated in different regions of the world (Sturtevant 1886). Types of Lettuce There are five modern types of lettuce base d on morphological featur es: crisp-head, leaf, butterhead, cos or romaine, and stem (Davis et al. 1997, Ryder 1998). The crisp-head varieties with dense, firm heads and crisp leaves are the most significant commerci al types and take about 75 130 d from planting to mature. Leaf lettuce vari eties have frilled, glossy red or bright green leaves and mature in 45 d from planting. Leaf le ttuce is a good type of le ttuce for home gardens, as it matures quickly and is easy to grow. Butte rhead lettuce generates an unfastened and soft head, and inner leaves have an oily or buttery feel Butterhead varieties produce high quality lettuce for commercial purposes. Th ey mature slightly earlier than crisp-head varieties. The cos or romaine type of lettuce develops an elongated head of stiff, upright leaves about 80 d from planting. Cos lettuce is an important lettuce type in Europe and is also gaining popularity in the United States. Stem lettuce often is listed in catalogs under the name of Celtuce (CELery letTUCE). It is grown for its fleshy, el ongated stem rather than its leaves. Insect Pests and Lettuce Lettuce is vulnerable to attack by several insect pests from seedling to reproductive stages. The estimated average yield loss is 17 and 13% for fall and spring lettuce, respectively, due to attack of various insect pests (Anonym ous 2003). Seedling pests are bulb mites ( Rhizoglyphus spp., Tyrophagus spp.), black cutworm ( Agrotis ipsilon Hufnagel), variegated cutworm

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19 ( Peridroma saucia (Hbner)), granulate cutworm ( Feltia subterranean (Fabricius)), darkling beetles (tenebrionids), field cricket ( Gryllus spp.), garden symphylans ( Scutigerella immaculate (Newport)), pea leafminer ( Liriomyza huidobrensis (Blanchard)), serpentine leafminer ( L. trifolii (Burgess)), vegetable leafminer ( L. sativae Blanchard), and springtails. Lepidopterous pests are responsib le for major economic yield lo sses in lettuce, with losses reaching 100% if control measures are not followed (Inglis and Vestey 2001). Important lepidopterous pests include: armyworm ( Pseudaletia unipuncta Haworth), beet armyworm ( Spodoptera exigua (Hbner)), corn earworm ( Helicoverpa zea Boddie), tobacco budworm ( Heliothis virescens (Fabricius)), cabbage looper ( Trichoplusia ni (Hbner)), alfalfa looper ( Autographa californica Speyer), and saltmarsh caterpillar ( Estigmene acrea [Drury]) (Parenzan 1984, Toscano et al. 1990, McDougall et al. 2002, Anonymous 2003). In Florida, beet armyworm ( S. exigua ), southern armyworm ( S. eridania ), cabbage looper ( T. ni ), corn earworm ( H. zea ), black cutworm ( A. ipsilon ), variegated cutworm ( P. saucia ), and granulate cutworm ( F. subterranea ) are the major lepidopterous pests (Nuessly and Webb 2003). The coleopterous pests of lettuce incl ude western spotted cucumber beetle, Diabrotica undecimpunctata howardi Barber and banded cucumber beetle, D. balteata LeConte (Nuessly and Webb 2003). In Florida, cucumber beetle s are found throughout the state. The banded species is more common in centr al and southern Florida whereas the spotted species is more prevalent in northern Florida. Beetles may cause potential losses of 100% if not managed. Yield loss with proper management strategies is gene rally less than 2.5%. Cucumber beetles became a problem on lettuce in Washington, when peas an d cucumbers were grown in lettuce growing areas (Inglis and Vestey 2001).

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20 The homopterous pests are foxglove aphid ( Aulacorthum solani (Kaltenbach)), green peach aphid ( Myzus persicae (Sulzer)), potato aphid ( Macrosiphum euphorbiae (Thomas)), lettuce aphid ( Nasonovia ribisnigri (Mosley)), lettuce root aphid ( Pemphigus bursarius (L.)), and silverleaf whitefly ( Bemisia argentifolii Bellows & Perring). In Florida, Uroleucon pseudoambrosiae (Olive) is important (Mossler and Dunn 2005). Aphids appear annually in lettuce production fields and cause yield losses generally less than 2% under normal management with insecticides. Losses in Wash ington can range from 75 to 100% without the timely use of chemical control measures (Inglis and Vestey 2001). Tarnished bug ( Lygus lineolaris (Palisot) and L. hesperus Knight) is also a pest of lettu ce. It causes qualitative damage due to discharge of a toxin during feeding that can be sufficiently severe to make the heads unmarketable. This pest arises irregularly every few years, ofte n later in the spring and early summer. Potentially 100% of the acreage can be affected without a ppropriate management (Kurtz 2001, McDougall et al. 2002, Anonymous 2003). In the United States, about 93% of the lett uce area is highly dependent upon chemical control for management of economic pests (Agricu ltural Statistics 2001). Fl orida, in particular, ranks first among lettuce growing states in the us age of insecticides to manage insect pests. Florida growers applied insecticides on 98 to 100% of the states lettu ce acreage with a total annual usage ranging from 1,900 to 4,900 pounds of ac tive ingredient (Mossler and Dunn 2005). High dependence on chemicals poses a potential threat to farmers, the environment, and natural enemies of these insect pests. Dependence on chemi cals is also costly. Therefore, there is a need to look for alterative strategies for management of economic insect pests of lettuce. Host plant resistance should be one of the major component s of an integrated pest management (IPM) program and can sustain or improve production efficiency in ways that will maintain or enhance

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21 natural resources and the environment (Sharma and Ortiz 2002, Sadasivan and Thayumanavan 2003). Despite noticeable benefits of host plant resistance mediated through chemicals, it may reduce the competitive ability of plants, leading to a trade-off between growth and resistance (Herms and Mattson 1992). The production and maintenance of these chemicals require resources that are then not available for the growth and reproduction of plants. Therefore, metabolic costs are thought to be involved in re sistance (Agrawal 1999) and resistance is always affected by metabolic turnover of compounds (Fagerstrm 1989, Skogsmyr and Fagerstrm 1992, Gershenzon 1994). Management of insects based on host plant re sistance is more advantageous economically, ecologically and environmentally than manageme nt based on chemical measures (Sharma and Ortiz 2002, Sadasivan and Thayumanavan 2003). It is a very targeted and long-lasting approach to manage economic insect pests. Dependence on fewer chemical sprays and increased yields could provide economic benefits. Pl ant resistance increases ecosys tem stability due to conserving species diversity and maintains natural food webs by not disturbing natural enemies of insect pests. Host Plant Resistance Plants live in a world that is inhabited by numerous advers aries (biotic and abiotic), the major proportion of which belongs to plant-eating animals, including insects, called herbivores. In spite of the great variety of herbivores, only parts of plants ar e defoliated, and the majority of plant foliage and reproductive structures survives due to an innate capacity to tolerate herbivory by compensating for resource losses (Constabel 1999, Strauss and Agrawa l 1999), or to defend themselves and thus to reduce the amount of da mage (Constabel 1999). The ability of the plant to defend itself against herbivores using different strategies is known as host plant resistance. Host plant resistance is considered to be one of the most effective components of an integrated

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22 pest management program and has been expl oited to reduce the dependence on chemical insecticides (Panda and Kush 1995). Host plant resistance is usually compatible with other control measures like biological control and cultural controls, and maintains the food web by conserving the natural enemies. Plants possess a natural defensive system incorporating mechanical and chemical factors produced via tr anscriptional activation of corresponding genes. These defenses operate either constitutively or after damage due to enemy attack (induced resistance) (Vet 1999). Many studies have investigated host plan t preferences of herbivorous insects. Morphological structures like hair and waxes (Lucas et al. 2 000), hooks, spikes and trichomes (Gilbert 1971), leaf hard ness (Patanakamjorn and Pathak 1967) and physical factors, such as water content (Scriber 1977), and nutrient co ntent (Morrow and Fox 1980) are identified as important factors leading to rejection of or preference for ce rtain plant tissues by an insect. Low nutritional quality of the plants may impede the development of insect herbivores (Scriber and Slansky 1981). Plants are also known to be full of an array of secondary compounds, which may be toxic, lower the nutritional quality of the foliage, or ac t as antifeedants (Fraenkel 1959, Bernays and Chapman 1977, Rhoades 1979, Scri ber and Slansky 1981, Constabel 1999). Secondary chemicals are not evenly distributed in plant tissues. They are usually concentrated in specialized stru ctures, like vacuoles, idioblasts, glandular trichomes, cavities and canals (Esau 1965, Fahn 1979). Plants sequester secre tions within a diversity of canal systems that include laticifers, resin ducts and phloem (Fahn 1979, Metcal fe and Chalk 1983). The canals usually form a complex network and are effectivel y distributed throughout the plant. Secretions in these canals are characterist ically stored under pressure. Damage by insects causes an immediate release of fluids down a force gradient to the place of injury (Buttery and Boatman

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23 1976). Insects may get entrapped due to adhesive ness of some exudates (Farrell et al. 1991). The squirt gun defense mechanism in the forest plant, Bursera trimera Bullock is a good example; a fine spray of resins that is released just after attack by the chrysomelids Blepharida spp. causes larval mortality (Becerra et al. 2001). It has been shown that some insects on canal-bearing plants de fuse the canalicular reaction before feeding. The cabbage looper, T. ni ruptures Lactuca laticifers by maki ng a superficial trench before actual feeding. The trench drains th e latex from the distal tip and isolates that particular section from the main canal system (Dussourd and Denno 1991, 1994). Dussourd (1993) compared the survivorship of each instar of T. ni and yellow-striped armyworm, Spodoptera ornithogalli (Guenee) an insect that does not trench on canal-bearing plants, to the following instar on intact vs. detached leaves of L. serriola. The survivorship was high for each instar of T. ni on both leaf categories. In contrast, S. ornithogalli larvae survived only on detached leaves. Larvae of S. ornithogalli in the first and second inst ar often died with their mandibles glued together with latex. Older larvae tried to feed over and ov er again but invariably starved to death. Detaching leaves particularly of plant species with exudates, often modifies their palatability (Bernays a nd Lewis 1986, Huang et al. 2003c). Constitutive defense is common to all healthy plants and provides general protection against invasion by herbivores. Constitutive defense has also been referred to as natural or innate defense. On the other hand, induced defense is th e mechanism that must be induced or turned on by plant exposure to an herbivore. Unlike cons titutive defense, it is not immediately ready to come into play until after the plant is appropria tely exposed to herbivore. Constitutive defense is not specific, and is directed toward genera l strategic defense. Phenolic compounds were previously regarded as quantitative defenses that are always present at high levels in plant tissues

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24 (Feeny 1976). Recently, it has been shown that certain phenolics may increase after insect attack or mechanical wounding (Pullin 1987, Clausen et al. 1989, Ke and Saltveit 1989, Hartley and Lawton 1991, Brignolas et al. 1995, C onstabel 1999). Thus, it seems that induced defense may be more cost-effective for plants than constitu tive defense under certain conditions. In induced defense, secondary compounds are manufactured as reactions to insect attack or wounding, and there is no need to maintain th e compounds at a steady and effectiv e concentration as in the case of constitutive defense (Herms and Matts on 1992, Baldwin 1994, Gershenzon 1994). Induced defense contributes to plant resistance by enha ncing the action of natu ral enemies of insects (Thaler 1999). Biochemical Basis of Ho st Plant Resistance Both proteins and secondary plant compounds c ontribute to defense in plants. Secondary plant compounds are organic molecules that are not required for normal physiological processes in growth and development. These biochemicals ar e also called allelochemicals, because they influence the behavior and/or physiology of speci es other than their own. Generally, secondary plant compounds have been more extensively st udied than proteins, possibly due to their interesting structural variety and advanced biological activit ies (Duffey and Stout 1996). Host Plant Resistance Due To Proteins Molecular biology has proven to be a useful t ool in host plant resist ance research because plant defense responses can be st udied at the level of gene expr ession rather than simply with assays of the encoded proteins. Each protein in a plant is encoded by a single gene, which can be isolated and employed for developing geneti cally engineered crops with improved pest resistance. Regulation of gene e xpression is a principal way that defense proteins are generated in plants and has been confirmed by the inducti on of mRNA after herbi vory (Constabel et al. 2000).

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25 Protease inhibitors Protease inhibitors (PIs) are proteins that strongly bind proteolytic enzymes and thereby hinder their activity (Ryan 1990, Rich ardson 1991). PIs are classified as inhibitors of serine, cysteine, aspartic, or metallo-proteases (Ryan 19 90). These inhibitors eff ectively block the active site of proteases by binding to it and forming a complex with a low dissociation constant (Terra et al. 1996, Walker et al. 1998). PIs accumulate in tomato leaves in response to insect attack within hours of damage (Green and Ryan 1972). Lo w molecular weight protease inhibitors, such as leupeptin, calpin inhibitor I, and calpeptin are strong antifee dants for adult western corn rootworm, Diabrotica virgifera virgifera LeConte (Kim and Mullin 2003). All PIs possess a dior tripeptidyl aldehyde moiety, which binds cova lently with sulfhydral (SH) group on the taste chemoreceptors of insects (Kim and Mullin 2003). PIs cause hyper-production of insect digestive enzymes, which triggers the loss of sulfur amino acids, and also reduces th e quantity of proteins. As a result, insects become weak, exhibit stunted growth and ultimately die (Shulke and Murdock 1983). Cysteine protease Cysteine protease is a 33-kDa defense prot ein, which accumulates in resistant lines of maize ( Zea mays L.) in response to larval feeding of fall armyworm, S. frugiperda (Pechan et al. 2000). It accumulates in the mid whorl of the mai ze plant within 1 h of in festation and continues to build up for as long as 7 d. This protein hinders larval growth and is responsible for 60 to 80% weight loss (compared to control insects feeding on susceptible lines of maize) (Pechan et al. 2000), which is due to destruction of the peritrop hic matrix of the gut an d subsequent disruption of the normal digestive mechanism (Pechan et al. 2002).

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26 Oxidative enzymes Oxidative enzymes include phenol oxidases, peroxidases, and lipoxygenases. These are stress-associated enzymes synthesized in plants (Butt 1980). The oxidative enzymes are involved in anti-nutritive defense in plants against va rious insect pests (Felt on et al. 1989, Duffey and Felton 1991, Duffey and Stout 1996). Systemin, jasmonates, and the octadecanoid defense signaling pathway induce polyphenol oxidase and li poxygenase in tomato and cotton, and thus support the role of oxidative enzymes in plant defense (Constabel et al. 1995, Thaler et al. 1996, Bi et al. 1997a, Heitz et al. 1997). Bestwick et al. (2001) characterized proand antioxidant enzyme activities during the hypersensitive reacti on (HR) in lettuce, and reported a prolonged oxidative stress in lettuce cells experiencing HR This stress is chiefly through a boost in prooxidant activities primarily taking place in the absence of enhanced antioxidants. Polyphenol oxidase. Polyphenol oxidase (PPO) uses molecular oxygen to catalyze the oxidation of monophenolic and orthodiphenolic compounds, and is a ke y factor for darkening of many fruits and vegetables (Sherman et al. 1991, Steffens et al. 1994, Constabel and Ryan 1998). The expression of PPO is generally high in diseased, insect-damaged and wounded tissues (Mayer and Harel 1979, Stout et al. 1994, Constabel et al. 1995, Thaler et al. 1996). In crops, such as potato, tomato, apple and hybrid poplar wound-induction at the le vel of PPO mRNA has been confirmed due to accessibi lity of PPO cDNA probes (Constabel et al. 1996). PPO contacts its chemical substrates during in sect feeding. PPO produces reactive ortho -quinones which readily form alkylated amino acids, which ulti mately results in prot ein modification, crosslinking, and precipitation. This protein modification significan tly impacts insect pests by preventing efficient digestion and assimilation of nitrogen (Felt on et al. 1992, Duffey and Stout 1996).

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27 Wounding induces expression of PPO genes in damaged as well as undamaged (systemically wounded) leaves (Robison and Raffa 1997, Havill and Raffa 1999). Constabel et al. (2000) observed through southern blot analys is that hybrid poplar presumably possesses two PPO genes, with polymorphic allele s at each locus. Similarly, toma to and potato also have seven and six member PPO genes families, respectivel y (Hunt et al. 1993, Thyge sen et al. 1995). Out of seven PPO genes in tomato, only one gene is wound inducible, while th e others are regulated by development (Thipyapong and Steffens 1997, Thi pyapong et al. 1997). Therefore, the woundinduced increase in PPO activity is through transcri ptional activation of PPO genes and de novo enzyme synthesis, rather than enzyme activa tion (Bergey et al. 1996, C onstabel et al. 2000). Various plant PPOs require chemical activation to become active, as they are present in latent form in the plant (Jimenez and Garcia-Car mona 1996). The younger leaves show higher PPO activity than older leaves due to buildup of higher levels of PPO mRNA in respons e to restricted damage of old leaves (Constabel et al. 2000). Peroxidase. Peroxidase is a heme-containing enzy me that oxidizes a wide range of biological compounds, such as phenolics, indole acetic acid, and ascorb ate by utilizing hydrogen peroxide (Butt 1980). Peroxidase plays a key role in lignification of pl ant tissue. The cell wall peroxidases produce phenoxy radicals from hydroxyc innamyl alcohols that ultimately form lignin by non-enzymatic polymerization (Dougl as 1996). These enzymes also perform an important role in suberization of tissues (Kolattukudy 1981). In a ddition, they are also involved in the construction of cross-links between car bohydrates and proteins in cell walls (Fry 1986, Cassab and Varner 1988). In various plants, like tomato, rice, pea nut and bean, peroxidase level is increased after wounding of ti ssues (Breda et al. 1993, Felton et al. 1994a, Ito et al. 1994, Smith et al. 1994). Peroxidase is also involved in defense by means of cell wall reinforcement

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28 due to its role in lignification and cross-li nking of other cell wall components. Ultimately, amplified peroxidase level aff ects insect performance due to increased leaf toughness (Coley 1983). Bi et al. (1997a) observed induced resistance in cotton to H. zea due to increased peroxidase activity in previously damaged co tton foliage or squares. Similarly, Dowd and Lagrimini (1997) found that peroxidase-overproducing transgenic tobaccos, Nicotiana sylvestris (Spegazzini and Comes) and N tabacum L. experienced significantly less damage by H. zea than did wild plants, suggesting the contribution of peroxidase activity in leaf resistance to chewing insects. Aphid infesta tion in barley results in ethyl ene production and subsequent increase in hydrogen peroxide and total peroxida se activity. This highlight s the role of ethylene in the oxidative response of infested barley plants (Argandona et al. 2001). Lipoxygenase. Lipoxygenase employs molecular oxygen to oxygenate unsaturated fatty acids, like linoleic and linolenic acid, and produces fatty acid hydr operoxides (Galliard and Chan 1980, Siedow 1991). Lipoxygenase has a number of im portant roles in plant defense against insect pests. Lipoxygenase produces a direct antinutri tive effect on insects. Th is adverse effect is due to destruction of polyunsaturated fatty aci ds, which are key nutrients for most insects (Duffey and Stout 1996). Fatty ac id hydroperoxides (plus extr a free radicals) generated by lipoxygenase react with essentia l amino acids and modify prot eins. Therefore, lipoxygenase plays an antinutritive role in plant defense similar to PPO and peroxidase (Duffey and Felton 1991). Proteins of the cell wall Stresses, including insect pest and pathogen attack, modify ce ll wall contents of plants, such as carbohydrates, proteins, and phenolic s (Bowles 1990, Carpita and Gibeaut 1993). Cell wall proteins, such as proline-rich proteins (PRPs), hydroxyproline-rich glycoproteins (HRGPs),

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29 arabinogalactan proteins, and glycine-rich proteins (GRPs) are induced during wounding of leaves or stems (Showalter 1993). Secondary metabolism pathways Phenolics and phenylpropanoids are major classes of phytochemicals responsible for defense reactions in plants. These chemicals are synthesized and accumulated upon insect and pathogen attack, and mechanical wounding The phenylpropanoids are ma inly derivatives of phenylalanine (an aromatic amino acid). Plants possess hundreds of phenylpropanoids, with flavonoids and their derivatives constituting the major group (Heller and Forkman 1993). Phenylpropanoid synthesis is always initiated through a common phenylpropanoid pathway. Phenylalanine is converted through a number of steps to hydroxycinnamoyl coenzyme A (CoA) esters, which is a branching poi nt in phenylpropanoid biosynthesis Lignin precursors, cell wallbound hydroxycinnamoyl esters, and soluble glucosid es are possible end pr oducts of different branches and ultimately form lignin and various flavonoids. Enzymes involved in secondary metabolism Herbivory and wounding induces various phe nylpropanoid enzymes. Phenylalanine ammonia lyase (PAL) is the first enzyme of the phenylpropanoid pathway and catalyzes deamination of phenylalanine to cinnamic acid (H ahlbrock and Scheel 1989). PAL is inducible by insect and pathogen attack, mechanical woundi ng, exposure to ethylene and abiotic stresses, such as UV light (Hyodo et al. 1978, Jones 1984, Hahlbrock and Scheel 1989, Ke and Saltveit 1989). PAL induction is well documented in certain plant parts, like le ttuce leaves (Ke and Saltveit 1986), bean hypocotyls (Cramer et al. 1989), alfalfa (D ixon and Harrison 1990), tobacco leaves (Pellegrini et al. 1994, F ukasawa-Akada et al. 1996), potato tubers and leaves (Rumeau et al. 1990, Joos and Hahlbrock 1992), and parsley (L ois and Hahlbrock 1992). Induction of PAL takes place through transcription of a singl e or many genes. Endogenous phenylpropanoid

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30 pathway intermediates regulate the level of PA L transcripts by way of a feedback mechanism (Braun and Tevini 1993, Orr et al. 1993). Various other important phenylp ropanoid enzymes, in addition to PAL, such as 4-cinnamic acid hydroxylase (4-CH) in Arabidopsis thaliana (L.) and pea ( Pisum sutivum L.) (Frank et al. 1996, Mizutani et al. 1997) and caffeic acid Omethyltransferase in corn (Capella des et al. 1996), are also induced by insect and pathogen attack and/or wounding. Similarly, 4-c oumarate CoA ligase in tobacco, Arabidopsis and bean ( Phaseolus vulgaris L.) is also wound induc ible (Smith et al. 1994a, Ellard-Ivey and Douglas 1996, Lee and Douglas 1996). In addition, numerous shikimic acid pathway enzymes responsible for phenylalanine biosynthesis, like 3-deoxyD-arabino-heptulosonate-7-phosphate (DAHP) synthase in potato tubers ( Solanum tuberosum L.) (Dyer et al. 1989) and shikimate dehydrogenase from bell peppers (Diaz and Meri no 1998) are induced by w ounding. Peiser et al. (1998) reported that PAL inhibitors control browning of cut lettuce. Host Plant Resistance Due To Secondary Plant Compounds There are wide range of secondary plant compounds found in the plant kingdom (Luckner 1990, Dey and Harborne 1997). This wide diversity of compounds is hypothesized to be the outcome of co-evolution of plan ts with insects and pathogens (Harborne 1993). Secondary plant compounds are traditionally categorized into three major groups: carbon-based phenolics and terpenes, and nitrogen-containi ng compounds such as alkaloids (Taiz and Zeiger 1991). In general, carbon-based compounds have been consid ered cheaper defense tools than nitrogencontaining compounds, as nitrogen is vital and fr equently in limited supply for the growth of plants (Bryant et al. 1983). Gon zalez (1977) reported the presence of terpenes, sterols, flavonoids and other phenolics, and alkaloids in lettuce.

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31 Phenolics Phenolics constitute a diverse group of chemi cals, ranging from small phenolic acids to complex polymers such as tannins and ligni n (Dey and Harborne 1997). Most phenolic compounds are derivatives of the shikimic acid and phenylpropanoid pathways and bear aromatic rings having one or more hydroxyl grou ps. Phenolics can be divided into simple phenols and polyphenols based on the number of hydroxyl group attached. Simple phenols include the hydrobenzoic acids (e.g., vanillic acid), the hydroxycinna mic acids (e.g., caffeic acid) and the coumarins (e.g., umbelliferone). Polyphenols are a diverse group of plant phenolics, such as flavonoids (e.g., quercetin) and tannins (e.g., es ters of gallic acid) (Schoonhoven et al. 2005). Additional functional groups such as ester, methyl, acetyl or s ugar moieties are also found in some complicated phenolics. Stresses such as excessive light, UV, col d, nutrient deficiencies, and attacks by insects and pathogens are most commonly responsible for the induction of phenolics in plants (Dixon and Paiva 1995, Somssich et al. 1996). In intact plants, phenolics are stored in vacuoles in their less toxic glycoside forms as wa ter-soluble compounds (Hsel 1981). The wounding of cells (e.g., by insect attack) causes releas e of the glycosides from their storage site (Hsel 1981) and ultimately formation of compounds with t oxic, deterrent or nucleophilic, and nutritive-value-lowering properties after coming in contact with specific degradative enzymes. For example, toxic hydrogen cyanide is released due to hydrolysis of harmless cyanogenic glycosides by -glucosidase activity (Wink 1997). o -Substituted phenolic compounds (e.g., chlorogenic acid) produce o -quinones with the help of oxidative enzymes which alkylate ami no acids by binding to their nucleophilic groups (Felton et al. 1989, Constabel 1999) This binding hinders the a ssimilation of essential amino acids and lowers the quality of plant foliage for in sects (Felton et al. 1989). As Bi et al. (1997a) observed, wounding of cotton ( Gossypium hirsutum L.) foliage induced increased activity of

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32 oxidative enzymes that was associated with de creased levels of the nutritional antioxidant ascorbate and increased levels of phenolic prooxidants (i.e., chlor ogenic acid) and lipid peroxides. This significant decline in host nutri tional quality (due to accumulation of secondary compounds) is responsible for induced resistance in cotton foliage and squares to herbivory by H. zea, indicated by a decrease in larval growth wh en larvae fed on previously damaged foliage or squares compared to the cont rols. Chemicals like phenolics also play an important role in the inhibition of oviposition on the host plant in addi tion to reducing larval growth and survival (Dethier 1970, Todd et al. 1971, Chapman 1974, Ellig er et al. 1980, Corcuera 1993, Stotz et al. 1999). Simple phenols such as ferulic, caffeic and p -coumaric acids are pr ecursors of lignin. Upon wounding of plant tissue, a subsequent host respon se occurs involving an intensive accumulation of lignin-like polyphenolic s in wounded or ruptured cells This is followed by a rapid hypersensitive death of the cell, giving rise to a single cell brownish necros is (Moerschbacher et al. 1990, Nicholson and Hammerschmidt 1992, Wei et al. 1994, Zeyen et al. 1995). Wounding of potato tubers (Hahlbrock and Scheel 1989) a nd lettuce (Loaiza-Velarde et al. 1997, LoaizaVelarde and Saltveit 2001) induces an accumulation of phenolic conjugates, such as chlorogenic acid (a caffeic acid conj ugate). Hydrogen peroxide is required for the polymerization step in the formation of the poly (phenolic) domain of sube rized potato tubers (Razem and Bernards 2002). Ke and Saltveit (1989) also observed an incr ease in phenolic compounds (e.g., chlorogenic and isochlorogenic acids) and brown stain in lettuce tissue affect ed by russet spotting. Phenolics are known to play important role in the host plant resist ance (Dethier 1970, Todd et al. 1971, Chapman 1974, Elliger et al. 1980, Corcue ra 1993, Stotz et al. 1999). Ikonen et al. (2001) reported feeding de terrence in the willow ( Salix pentandra L.) to the leaf beetle

PAGE 33

33 ( Lochmaea capreae L.) due to high levels of chlorogenic acid in the leaves. Cole (1984) correlated resistance to lettuce root aphid with the presence of high amounts of isochlorogenic acid and the enzyme PAL in resistant lettuce culti vars. However, the increased concentration of phenolics in transgenic tobacco showing differe ntial expression of PAL does not substantiate their role in plant resistance agai nst the generalist tobacco hornworm ( Manduca sexta L.), and the specialist tobacco budworm ( H. virescens (Fabricius)) (Bi et al. 1997b). Similarly, Eichenseer et al. (1998) also did no t find any preference in larvae of M. sexta fed transgenic tobacco plants that either underor over-expresse d PAL and consequently with either lower or higher levels of phenolics than normal Tannins are complex polyphenols and are more prevalent in woody perennials than in herbaceous plants (Swain 1979). They are often considered as general feeding deterrents in plant-insect interactions, and th erefore, play an important role in chemical ecology and defense against insects (Swain 1979, Hagerman and Butl er 1991). Based on their structure, they are categorized as condensed tanni ns, or proanthocyanidins, and hydrolysable tannins, which are gallic acid or ellagic acid esters of various sugars. Caffeic acid derivatives (Ke and Saltveit 1988 ) and flavonoids (Hermann 1976) are the two main classes of simple phenols and complex polyphenols, respectively, which have been identified in lettuce. In particular, simple phenolics like monocaeffeoyl tartaric acid, chicoric acid, 5-caffeolyquinic acid and 3,5-diO -caffeoylquinic acid are present in lettuce (Winter and Hermann 1996, Ferreres et al. 1997). Flavonoids Plants flavonoids are a large group of phe nolic compounds produced by the shikimic acid pathway. Flavonoids are grouped under major cla sses, such as the flavanones, flavones, flavonols, and isoflavonoids (Harborne 1994). In the biosynthesis of flavonoids, chalcone

PAGE 34

34 synthase (CHS) is the first committed enzyme that catalyzes the formation of chalcone intermediate by condensing three malonyl-CoA and one hydroxycinnamoyl-CoA molecules. CHS is known to be involved in the response to many forms of stress in many plants, including to insects and pathogens (Dangl et al. 1989). Chalcone is then cat alyzed to flavanone with the help of the enzyme chalcone isomerase. In th e next step, flavonoid bios ynthesis splits into different branches. In the first branch, flavones are formed from flavanones due to the action of flavone synthase (Britsch 1990). Secondly, dihydro flavonols, which are pr ecursors of flavonols and anthocyanins, can be synthesized from fl avanones by the enzyme flavanone-3-hydroxylase. In the third branch, flavanones can be converted into isoflavanones in a reaction catalyzed by isoflavanone synthase (Dixon et al. 1995). Flavonoids are found in high concentrations in many plant species under normal conditions as sugar conjugates (Frst et al. 1977, Feng and McDonald 1989, Jhne et al. 1993, Stapleton and Walbot 1994), and over 50 di fferent glycosides have been identified among the more common-occurring flavonoids (Her mann 1976, 1988). Flavonoid accumulation in leaves is very much increased in response to illumination with the UV-B spectrum of visi ble light (Koes et al. 1994, Strid et al. 1994). Fl avonoids play a role in the protect ion of plants from the damaging effects of UV-light, as they have good light absorbing properties in the UV spectrum (Markham 1989). Red-pigmented lettuce, such as Lollo Rosso, contains high concentrations of anthocyanin with antioxidant and free-radical scavenging properties (Gil et al. 1998). Flavonols, such as kaempferol, quercetin and myricetin, and the analogous flavones, apigenin and luteolin are found in vegetables, fruits, and beverages (H ertog et al. 1992, 1993), and are also known to possess antioxidant and free radical scavenging activity in foods (Shahidi and Wanasundara 1992). Flavonols, such as quercetin 3-O-glucur onide, quercetin 3-O-glucoside and quercetin 3-

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35 O-(6-O-malonylglucoside) are al so present in lettuce (Winter and Hermann 1996, Ferreres et al. 1997). The lettuce varieties Lollo Rosso a nd Round contain high amounts of quercetin, varying from 11-911 g g-1 of fresh weight in the outer leaves to 450 mg g-1 in the inner leaves (Crozier et al. 1997). The pol yphenol compounds (caffeic acid derivatives, quercetin and kaempferol glycosides) are pres ent in higher amounts in lettuce grown in the field than in a greenhouse (Romani et al. 2002). The behavior, development, and growth of insects are influenced by plant flavonoids (Hedin and Waage 1986). Plant flavonoids act as feeding stimulants for the boll weevil ( Anthonomus grandis Boheman) in cotton (Hedin et al. 1988 ), or oviposition stimulants to the citrus-feeding swallowtail butterfly, Papilio xuthus L. (Nishida et al. 1987). Flavonoids may be antibiotic substances effectiv e against phytophagous insects (T odd et al. 1971, Chan and Waiss 1978, Chan et al. 1978, Joerdens-Roettger 1979, Elliger et al. 1980, Hanny 1980, Hedin et al. 1983, Peng and Miles 1988, Ridsdill-Smith et al. 1995). Rutherford (1998) observed the involvement of chlorogenates and flavonoids in the resistance of sugarcane to the stalk borer ( Eldana saccharina Walker). Two extreme types of flavonoid profile were found using nearinfrared spectroscopy (NIR), one coupled with susceptibility and other with resistance. Stalk borer larvae could be induced to feed by inclusio n of the susceptible-type flavonoid profile into a defined synthetic diet. Subsequent survival of fi rst instar larvae was grea ter on this diet than on diets containing the resistan t-type flavonoid profile. Terpenoids Terpenoids are a diverse group of chemical s which all originate from iospentenoid precursors. Based on the number of isoprene (five-carbon) units, terpenoids are classified as mono-, sesqui-, di, trior tetr a-terpenoids. They are known to ha ve various secondary functions,

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36 like defense against pathogens and insects as we ll as primary functions, such as membrane components, pigmentation, free radical scavengi ng, and growth regulat ors (Harborne 1993). Antibiotic, cytotoxic, and allergenic properties are also associated with terp enoids (Burnett et al. 1978). Many sesquiterpene lactones accumulate in canals (laticifers) closely associated with the vascular tissues of composit plants (Esau 1965) Damaged laticifers re lease latex containing sesquiterpene lactones which may have analgesic, antitussive and sedative properties (Gromek et al. 1992). Sesquiterpene lactones ar e extremely varied in their stru cture, properties and functions (Rees and Harborne 1985). The main bitter constitutive principles of Lactuca species are lactucin, lactucopicrin, 8-deoxylactucin and th eir derivatives, such as 11,13-dihydro-analogues (Barton and Narayanan 1958, van Beek et al. 1990) Two triterpenes, the quaianolides lactucin, and lactucopicrin have been isolated from dry latex of L. virosa. The presence of lactucin, 8deoxylactucin, and lactucopicrin in lettuce and chic ory make them intensely bitter (Price et al. 1990). Wounding of leaves or stems of Lactuca species releases a milky latex consisting of 15oxalyl and 8-sulphate conjugates of lactucopicrin, which ultimately revert to the parent lactone due to hydrolysis of unstable oxalates. The induced quaianolide sesquiterpene lactone phytoalexin, lettucenin A, is also present in Lactuca species, but not in ch icory (Sessa et al. 2000). Lettucenin A was initially characterized by Ta kasugi et al. (1985). It is one of the most toxic phytoalexins ever discovere d and provides resistance to le ttuce downy mildew in certain lettuce cultivars due to its str ong antimicrobial properties (Benne tt et al. 1994). Bestwick et al. (1995) isolated lettucenin A from lettuce seedlin gs with the red spot physiological disorder. A 15-glycososyl conjugate of 11,13-di hyrolactucopicrin is found in L. tartarica roots (Kisiel et al. 1997). Likewise, the related quaianolide sesquiterp ene lactone glycosides, such as picriside A

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37 (lactucin-15-glycoside) and cr epiaside A (8-deoxylactucin-15glycoside), are found in other members of the Lactuceae tribe (Seto et al. 1988). Host Plant Resistance in Lettuce to Insect Pests Aphids Many species of aphids are known to coloni ze lettuce, but few are responsible for transmission of viruses (Kennedy et al. 1962). Aphi ds are the most serious pests of lettuce in North America (Alleyne and Morrison 1977, Forbes and Mackenzie 1982, Toscano et al. 1990), Spain (Nebreda et al. 2004), and other areas of Eu rope (Ester et al. 1993, E llis et al. 1996, Martin et al. 1996, Monnet and Ricateau 1997, Parker et al. 2002). Reduction in yield of lettuce is due to direct damage caused by aphid feeding and indirect damage by aphid-transmitted virus infections. In addition, marketability of harves ted heads is greatly reduced by the physical presence of aphids (Dunn 1959, Rufingier et al. 1997). The lettuce root aphid, P. bursarius is one of the most important pests of lettuce in the United States (Swift and Lange 1980, Blackman a nd Eastop 2000), Western Europe, and Canada (Ellis 1991, Reinink and Dieleman 1993). It feeds on the youngest leaves and rapidly colonizes the heart of the lettuce (Forbes a nd Mackenzie 1982). The lettuce aphid, N. ribisnigri is a major pest in the United States, Czechoslova kia, UK, France, Germany, Netherlands and Switzerland (Reinink and Dielem an 1993, Mosler and Dunn 2005). Uroleucon ambrosiae is a pest of hydroponically-grown lettuce in Brazil (A uad and Moraes 2003, Miller et al. 2003) and Turkey (Zeren 1985). Green peach aphid, M. persicae (Capinera 2004), and potato aphid, M. euphorbiae (Reinink and Dieleman 1989), are activ e vectors of lettuce yellow virus. A variety of chemicals are sprayed to control a phids in lettuce. Therefore, to reduce lettuce growers dependence on in secticides for aphid cont rol, a number of alterna tive measures must be used as a part of IPM program t ogether with the use of varieties resistant to aphids (Tatchell et

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38 al. 1998). Plant resistance as one of the compone nts of IPM has been extensively studied to manage aphids on lettuce. Successful transfer of resistance from wild to cultivated lettuce has proven useful in controlling N. ribisnigri (Eenink et al. 1982). However these varieties afford only slight to no defense against M. persicae and M. euphorbiae (Reinink and Dieleman 1989, van Helden et al. 1993). Modern varieties of lettuce resistant to P. bursarius such as Avoncrisp and Lakeland, possess the dominant Lra gene (Dunn 1974, Ellis et al. 1994). The Lra gene is also linked to the downy mildew ( Bremia lactucae ) resistance gene, Dm6 (Harrewijn and Dieleman 1984, Ellis et al 1994, Ellis et al. 2002) However, the lettuce variety Grand Rapid, reported to be resistant to P. bursarius (Dunn and Kempton 1980), does not possess Dm6 (Crute and Dunn 1980). In addition, several factors whose genetic basis have not been identified, such as deficient nutritive value of the phloem sa p, phytochemicals (toxic or deterrents), and unacceptability of the plant surface for feeding, provide resistance to aphids (Harrewijn and Dieleman 1984). Wild lettuce species L virosa L., L. saligna L. and L. perennis L. are found to be resistant to M. persicae causing aphid mortality and lower nym ph production (Eenink and Dieleman 1982). This resistance (governed by additive ge nes) was transferred to cultivated lettuce by making a series of inter-specific cros ses (Eenink et al. 1982). Clones of M. persicae exhibit different intensities of aggressiveness on lett uce. The lettuce genotypes selected for partial resistance to the aggressive clone WMp1 possess complete or almost complete resistance to less aggressive clones (Reinink et al. 1989). Lactuca virosa is almost completely resistant to N. ribisnigri causing low feeding rate, adult and ny mphal mortality, and reduced reproduction (Eenink and Dieleman 1982). Complete resistance to N. ribisnigri is governed by the presence of

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39 the Nr gene in the plant, whereas the Nr gene provides only partial resistance to M. persicae, and no resistance to M. euphorbiae (Reinink and Dieleman 1989). Iceberg lettuce shows resistance towards three main aphids, N. ribisnigri M. euphorbiae and P. bursarius (Dunn and Kempton 1980). Ester (1998) observed 100% resistance against N. ribisnigri and M. euphorbiae in aphid-resistant butterhead lettuce cultivars. In Europe, the lettuce butterhead cultivar Dynamite shows high resistance against N. ribisnigri and P. bursarius some resistance to M. euphorbiae and U. sonchi but no resistance to the glasshouse-potato aphid, Aulacorthum solani (Kaltenbach) (van der Arend et al 1999, van Melckebeke et al. 1999). Butterhead cultivars are moderate ly to highly resistant to M. euphorbiae and U. sonchi whereas crisphead cultivars possess little or no resistance to either aphid species (Reinink and Dieleman 1989). The lettuce cultivar 'Charan' shows partial resistance to M. euphorbiae and U. sonchi (Reinink et al. 1995). Montllor and Tjallingii (1 989) electronically monitored the probing behavior of M. persicae and N. ribisnigri on susceptible and resistan t lettuce lines using a DC amplifier. They proposed the possible involve ment of both mesophyll and phloem factors in conferring resistance. van Helden and Tjallingii (1993) also discu ssed the role of phloem vessels in resistant lettuce. van Helden et al. (1995) compared the phloem sa p of both resistant and susceptible cultivars and found no relationship between phloem sap composition and resistance to N. ribisnigri However, later work by van Helden and van der Wal (1996) suggests the presence of a resistance factor against N. ribisnigri in lettuce phloem sap. The roots of lettuce cultivars showing resistance to P. bursarius have greater concentrations of isochlorogenic acid and PAL as compared to susceptible cultivars (Cole 1984). Cabbage Looper Cabbage looper, T. ni is a serious problem in all lettuce growing areas in the United States (Kerns and Palumbo 1996, Kern s et al. 1999, Agnew 2000, Kurtz 2001) It is the predominant

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40 pest of lettuce during autumn in California (Kishaba et al. 19 76, Vail et al. 1989). The larvae of T. ni often transect leaves with a narrow trench before eating to reduce exposure to exudates, such as latex, during feeding (Dussourd 2003). Ca bbage looper larvae develop faster on excised than on attached leaves of prickly lettuce, L. serriola signifying the suitability of these plants when canals are inactivated (Tune and Dussourd 2000). Lactucin from lettuce latex seems to act as a trenching stimulant, but other chemicals, such as phe nylpropanoids, monoterpenes, and furanocoumarins, show slight or no activity for inducing trenching (Dussourd 2003). The F2 plants derived from a cross between L. sativa lines and resistant lines of L. saligna were resistant (Whitaker et al. 1974) and s howed antixenosis toward T. ni (Kishaba et al. 1980). Banded Cucumber Beetle The banded cucumber beetle, D. balteata is a generalist feeder that feeds upon many plant species. In the early 1900s, this pest was mostly found in Centra l and South America and Mexico (Saba 1970, Krysan 1986, Bellows and Diver 2002). La ter on, it spread into the United States and is now established in Alabama, Arizona, Arka nsas, California, Florida, Georgia, Louisiana, Mississippi, New Mexico, North Carolina, South Carolina, and Texas (CABI 2006). It is also found throughout Florida but most commonly in the Lake Okeechobee area (Capinera 1999). It is an economic concern for lettuce cultivation in southern Florida (Nuessly and Nagata 1993). Diabrotica balteata has a high reproductive capacity (P itre and Kantack 1962), and many generations occur throughout the year (Schalk 1986). Romaine lettuce cultivars Valmaine and Ta ll Guzmaine were analyzed to assess the level of resistance to D. balteata (Huang et al. 2002). Valmaine was highly resistant whereas Tall Guzmaine was susceptible to D. balteata The mechanism of resistance was determined to be antixenosis and such little feed ing occurred on Valmaine that th e reproductive structures were not fully developed in adult females (Huang et al. 2002). However, la tex from both Valmaine

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41 and Tall Guzmaine showed antifeedant activities wh en applied to the surface of a preferred food, such as lima bean ( Phaseolus vulgaris L.) leaves. Valmaine plants that had been previously fed upon showed higher resistance to D. balteata than did Tall Guzmaine after previous feeding, suggesting involvement of physical factors and an induced mechanis m of resistance in Valmaine against the beetle (Huang et al. 2003b). Leafminer Plants in over 47 genera belonging to 10 fa milies have been recorded as hosts of leafminers. The principal leafmine r species affecting lettuce include L sativae L trifolii L huidobrensis and L. langei Frick. Both L. trifolii and L. sativae are native to America (Waterhouse and Norris 1987). In the United Stat es, these two species are found commonly in the southern United States from Florida to Ca lifornia and Hawaii (Capinera 1999). In Arizona, L. sativae is predominant during the period of Augu st to January, whereas during February L. trifolii prevails (Kerns and Palumbo 1996, Kerns et al. 1999, Agnew 2000). In recent years, populations of leafminers have increased in coas tal areas in California (Kurtz 2001). In central Florida, populations of leafminer are high be tween May and October, when minimum average temperatures are 25oC, whereas higher temperature in southern Florida favors leafminer populations throughout the year (Anonymous 1999). Leafminer larvae cause damage by mining the leaves, which may result in reduced photos ynthetic activity. Younger plants are more vulnerable to leafminer attack and severe da mage can kill the plants (Nuessly and Webb 2003). The romaine lettuce cultivar Valmaine was the most resistant to L. trifolii in tests involving three additional lettuce cultivars, Floricos 83, Parris Island Cos, and Tall Guzmaine. Adults on Valmaine had significantly re duced levels of feeding, longev ity, and fecundity (Nuessly and Nagata 1994). Liriomyza trifolii preferred to feed on the middle leaves of Valmaine plants in contrast to Tall Guzmaine where they preferre d to feed on the older and younger leaves. When

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42 honey was supplied as a supplement to the diet of Valmaine, female survivorship and reproductive rates increased to levels more similar to Tall Guzmaine suggesting a deficiency in a critical diet component in Va lmaine (Nagata et al. 1998). Mou and Ryder (2003) screened 48 varieties of cultivated lettuce, L sativa and the wild species, L. serriola L. saligna and L. virosa for resistance to L. langei Wild species had fewer leafminer stipples per unit area than cultivated lettuce. Iceberg experien ced the most stippling damage among the genotypes tested. The progenies of crosses between the resistant genotypes were selected to raise th e level of resistan ce (Mou et al. 2004, Mou and Ryder 2003). Helicoverpa specie s Heliothinae are very destructive pests of many crops and freque ntly shift to lettuce from surrounding crops, like cotton and corn (Kerns and Palumbo 1996, Kerns et al. 1999, Agnew 2000, Kurtz 2001). Corn earworm, H. zea, is found throughout the United States (Capinera 1999). It is found on all Florida vegetable crops (Martin et al 1976). In Australia, H. armigera (Hbner) and H. punctigera Wallengren are serious pests of lettuce and can cause extensive damage (Ridland et al. 2002, Dimsey and Vujovic 2003). In India, H armigera is found throughout the year in lettuce fiel ds, but is most active during March and April (Parihar and Singh 1992). Spodoptera species Beet armyworm, S. exigua is a polyphagous and widely di stributed insect (CABI 1972). It is the key pest of lettuce in the western Un ited States (Metcalf and Flint 1962, Kerns and Palumbo 1996, Kerns et al. 1999, Agnew 2000, Ku rtz 2001). In the early stage of crop development (between thinning and cupping stage) it does not cause any economic damage, but feeding during the heading stage makes the lettuce unmarketable (Kerns and Palumbo 1996, Kerns et al. 1999, Agnew 2000). Ghaffar et al. (2002) found the pupal period of S. exigua to be

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43 the shortest (5.8 d) on lettuce as compared to eggplant ( Solanum melongena L.) and field bindweed ( Convolvulus arvensis L.) (7.6 d). Bemisia species or strains The B strain of the cotton whitefly, Bemisia tabaci (or B. argentifolii the silverleaf whitefly), is one of the primary pe sts of fall lettuce in California and Arizona. It causes complete destruction of early fall planted lettuce due to the extraction of large amounts of phloem sap from seedlings (Kerns and Palumbo 1996, Kerns et al. 1999, Agnew 2000). It also causes yellowing and distortion of the leaves and can re duce dry mass accumulation by up to 41%, depending upon population level (Costa et al. 1993). In lettuce, whitefly styl ets penetrate epidermal cells and intercellular junctions while feeding. Arrangement of vascular bundles in lettuce affects the feeding behavior of whitefly. The length of the vascular bundle (2.8 mm per 1.0 mm2 leaf area) is tolerably acceptable to whitefly (Cohen et al. 1996). However, fewer minor veins (fewer vascular bundles) accounts for low success of whitefly on lettuce compared to preferred crops, such as cantaloupe and other cucurbits (Cohen et al. 1998). Thrips Western flower thrips, Frankliniella occidentalis (Pergande), and onion thrips, Thrips tabaci (Lindeman), are prevalent pests of lettuce in Arizona (Kerns and Palumbo 1996, Kerns et al. 1999, Agnew 2000, Kurtz 2001). Wester n flower thrips is a native of North America, and has a broad host range of more than 500 species re presenting 50 plant families (Beshear 1983, Yudin et al. 1986). It is most commonly found in California (Bryan and Smith 1956, Rob 1989) and Arizona (Bibby 1958) on lettuce. Thrips adults a nd larvae puncture and feed from epidermal cells (Nuessly and Webb 2003), and affect quality of lettuce, as they cause leaf stippling and rib discoloration (Kurtz 2001). Romaine lettuce is especially susceptible to thrips in Arizona (Kerns and Palumbo 1996, Kerns et al. 1999, Agnew 2000). In Florida, Frankliniella spp. are important

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44 carriers of tomato spotted wilt and escarole n ecrosis viruses (Nuessly and Webb 2003). Mollema and Cole (1996) found a positive correlation between amino acid concentration in lettuce leaves and western flower thrips dama ge suggesting that higher con centrations are important for successful thrips development. Research Goals Lettuce is an important leafy vegetable grown all over the world. In the United States, romaine lettuce is the most commonly grown leaf lettuce. It is vulnerabl e to attack by several insect pests during field produc tion. Chemical control measures are the main tools for management of insect pests on lettuce and about 93% of lettuce grown in the United States is under chemical management of noxious insects (A gricultural Statistics 2001). In southern Florida, vegetable farming involves high inte nsity pesticide usage (>20 pounds of active ingredient pesticide per acre/crop ), and often there is more than one crop per year, which further increases the amount of pesticides used (Agricultural Statistics Board 2001). In Florida, lettuce production is more concentrated in the southern part of the state, whic h is an ecologically sensitive area due to its proximity to the Ever glades National Park and heavy precipitation and run-off (Miles and Pfeuffer 1997). High dependen ce on chemicals can pose a threat to growers and natural enemies of insect pests as well as involves a heavy cost (Sharma and Ortiz 2002, Sadasivan and Thayumanavan 2003). Hence, there is a need to look for alterative tactics for management of economic insect pests. Host plant resistance is an important co mponent of integrated pest management. Management of insects based on host plant re sistance can reduce the sole dependence on chemical usage (Sharma and Ortiz 2002, Sadasivan and Thayumanavan 2003). Thus, it is essential to develop resistant varieties in lettuce to reduce th ese economic and environmental problems. The romaine lettuce cultivar Valmaine is known to possess a high level of resistance

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45 against D. balteata (Huang et al. 2002) and L. trifolii (Nuessly and Nagata 1994) as compared to three other cultivars, Parris White, Short Guzmaine, and Tall Guzmaine. Resistance was highest in Valmaine and lowest in Parris White in confirmation with pedigree analysis (Guzman 1986). Short Guzmaine is the product of Valmai ne and FL 1142, whereas Tall Guzmaine was selected from progeny of a cross between Shor t Guzmaine and Parris White. Guzman designed Tall Guzmaine for improvement of certain hortic ultural characters over Valmaine, such as thermodormancy, premature bolting, and resistance to lettuce mosaic virus and corky root rot. Breeders did not evaluate insect resistance when developing Tall Guzmaine. Further, previously wounded Valmaine plants showed higher resistance to D. balteata as compared to Tall Guzmaine suggesting the involvement of an i nduced mechanism of resistance in Valmaine (Huang et al. 2003b). Thus, it would be helpfu l to know the bioche mical mechanism of resistance in Valmaine to different insects to aid plant-breeding programs in development of new lettuce cultivars with both desirable horticultural characte rs and insect resistance. The objectives of this st udy were the following: 1. To compare survival, development and feedi ng behavior of cabbage looper and beet armyworm on Valmaine and Tall Guzmaine 2. To determine the potential of latex produced by Valmaine as a defense mechanism against banded cucumber b eetle using choice and no-choi ce tests and isolation of deterrent compounds from the la tex using solvent extraction 3. To further isolate deterrent compounds from Valmaine latex using bioassay-directed fractionation of Valmaine latex crude extract 4. To investigate enzyme induction as a possible reason for latex-mediated insect resistance in Valmaine

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46 CHAPTER 2 HOST PLANT RESISTANCE IN ROMA INE LETTUCE AFFECTS LARVAL FEEDING BEHAVIOR AND BIOLOGY OF TRICHOPLUSIA NI AND SPODOPTERA EXIGUA (LEP IDOPTERA: NOCTUIDAE) Introduction Over the past 15 yr, romaine lettuce, Lactuca sativa L., has been the fastest growing vegetable in terms of production, co nsumption, and exports in the United States. During the period 2002 to 2004, romaine le ttuce accounted for 22% of all lettuce produced in the United States and per capita use of romaine lettuce has tripled (3.7 kg) since 1992-94 (USDA 2005a). Lettuce is vulnerab le to attack by seve ral insects including lepidopterans that can be re sponsible for yield losses of 100% if populations are not managed (Inglis and Vestey 2001). In Florida, the cabbage looper, Trichoplusia ni (Hbner), and the beet armyworm, S podoptera exigua (Hbner) (Lepidoptera: Noctuidae), are serious pests of lettuce (Nuessly and Webb 2003). Economic pests are managed chemically on about 89% and 85% of head and other lettuce acreage, respectively, in the Unite d States (USDA 2005b). Florida ranks first among lettuce growing states in the usage of in secticides and grower s apply insecticides on 98 to 100% of the states le ttuce acreage to manage these insect pests (Mossler and Dunn 2005). For instance, restrict ed insecticides such as lambda-cyhalothrin (34% and 32% of head and other lettuce acreage, resp ectively) and methomyl (32% and 30% of head and other lettuce acreage, respectivel y) are extensively applied on lettuce (USDA 2005c). Rapid development of insectic ide resistance has been reported for Liriomyza spp. (Diptera: Agromyzidae) ag ainst chlorinated hydrocar bons, organophosphates and the pyrethroid permethrin (Genung 1957, Leibee 1981, Parrella and Keil 1984). The high dependence on chemicals poses a threat to ag ricultural workers and natural enemies of

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47 these insect pests and increases producti on cost. Therefore, the implementation of alternative strategies, such as host plant resistance, for the management of economic insect pests should be explored. Valmaine romaine lettuce (Leeper et al 1963) was the major cultivar grown in Florida before the adoption of Tall Guzmaine Tall Guzmaine was selected from a cross between Short Guzmaine and Parris Wh ite (Guzman 1986). Short Guzmaine was a selection from a cross between Valmaine a nd Florida 1142. Tall Guzmaine was selected for resistance to thermodormancy, prematur e bolting, lettuce mosaic virus and corky root rot; however, Guzman did not include insect re sistance in his selec tion criteria (Guzman 1986). Tall Guzmaine was found to be susceptible to the leafminer, Liriomyza trifolii (Burgess) (Nuessly and Nagata 1994) and the banded cucumber beetle, Diabrotica balteata LeConte (Coleoptera: Chrysomelidae) (Huang et al. 2002) compared to Valmaine. Therefore, I selected the same two cultivars to determine whether resistance in Valmaine extends to a third order containi ng economically important insect pests of lettuce, the Lepidoptera. In this study, I tested the performance of two noctuid defoliators important to Florida lettuce production, cabba ge looper and beet armyworm on Valmaine and Tall Guzmaine. I chose these two insect species b ecause I was interested in how ecologically similar but behaviorally different defoliato rs of lettuce would re spond to the selected lettuce cultivars. Cabbage loopers trench l eaves of latex-bearing plants (Dussourd and Denno 1994), whereas beet armyworms do not. In particular, cabbage looper has been shown to deactivate the canalicul ar defenses in wild lettuce, Lactuca serriola L., by making shallow trenches before actual feed ing (Dussourd 1997). The objectives of the

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48 study were to compare the survival, developm ent and feeding behavior of cabbage looper and beet armyworm on resistant Valmaine and susceptible Tall Guzmaine lettuce. Materials and Methods Plants Seeds of two romaine lettuce cultivars, Valmaine and Tall Guzmaine, were provided by R. T. Nagata (Everglades Resear ch and Education Center, University of Florida, FL). Seeds were germinated by placing them overnight in a Petri dish lined with wet filter paper in the laboratory. Germinated seeds were planted in a transplant tray filled with commercial soil mix (Metro Mix 220, Grace Sierra, Milpitas, CA) in a greenhouse with natural light at a mean temp erature of 27C (32 to 24C) and 68% mean RH (44 to 94%). After 2 wk, seedlings were tr ansplanted to plastic pots (15 cm diameter) filled with MetroMix 220. Plants were irriga ted daily and fertilized once per week with 10 ml of a 10 g/L solution of soluble fertil izer (Peters 20-20-20, N-P-K, W.R. Grace, Fogelsville, PA) from transplanting of seedli ngs to the end of the experiment. Four-weekold plants with six to seven tr ue leaves were used in all experiments that were conducted in the greenhouse under ambient light. Insects Cabbage looper eggs were supplied by G. L. Leibee (Mid-Florida Research and Education Centre, University of Florida, FL ) from a 1-yr-old colony, which was raised on mustard leaves. Eggs were sterilized with 500 ml 0.008 % sodium hypochlorite solution (Clorox, Oakland, CA) for 1 min in a cylindr ical container (18 cm diameter by 7.5 cm high). Sterilized eggs were rinsed twice with distilled water and were drained into a nylon strainer. Eggs were inverted into a 177-ml cup under running water until the cup was half filled with water. The remaining half cup was filled with neutralizer (10% sodium

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49 thiosulphate solution) and eggs were soaked in the cup for 2 min. Neutralizer and eggs were drained into a nylon strain er and eggs were rinsed twi ce with distilled water. After rinsing, eggs were placed on a paper towel in a cylindrical co ntainer with plastic screen lid and placed in an incubator at 27 2C, 70 10% RH, and a photoperiod of 14:10 (L:D) h. Neonates were used for bioassays. Egg masses of beet armyworm were collected from pepper plants in Citra, FL and the subsequent generations (F3 through F8) were used for bioassays. Eggs were sterilized in the same way as for cabbage looper. Newl y emerged larvae were transferred onto pinto bean diet (Guy et al. 1985) in a rectangular container (25 25 11 cm) with plastic screen lid in an incubator at the same conditions as fo r cabbage looper. Pupae were placed into paper cups and placed in the incubator. Beet armyworm adults were held in a screen cage (30.5 30.5 30.5 cm) in the incubator. Two cotton plants with three to four true leaves were used for oviposition and we re replaced with fresh ones every other day. Adults were fed a 20% sucrose solution disp ensed on a cotton wick. Neonates were used for bioassays. Neonate Survival and Development to Third Instar Thirty replicates of each cultivar were set up along greenhouse benches in a randomized complete block design. Experiment s on beet armyworm and cabbage looper were done separately under similar greenhous e conditions. Ten neonate s were placed in the central whorl of each plant and the plant was covered with a cylindrical screen cage (18.5 cm diameter 61.0 cm height) to confine the insects for feedi ng (Fig. 2-1). Plants were dissected 1 wk after infe station to locate the surviving larvae. Larval mortality, weight, instar and feeding beha vior were observed and record ed. Instars were determined by measuring head capsule widths (Capiner a 2005, 2006). Observations were also made

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50 on the preferred site of feedi ng on a leaf and within a plant. Larval mortality and weight for each species were analyzed using PROC GLM with cultivar as a fixed effect and replications as random effect (SAS Ins titute 1999). Tukeys honestly significant difference (HSD) test with a significance level of = 0.05 (SAS Institute 1999) was used for posthoc means separation. Log-likelihood ratio (G2-test) (Zar 1984) was used to analyze the frequency of surviving instar s using JMP release 5.1.2 (JMP Software, SAS Institute Inc., Cary, NC). Differences in the pr eferred site of feedi ng within a plant were analyzed by 2 goodness of fit tests (F reund and Wilson 1997). Survival and Development from Neonate to Adult Emergence Time of development from neonates to adults was investigated on both lettuce cultivars. Thirty replicates of each cul tivar were set up along greenhouse benches in a randomized complete block design. Experiment s on beet armyworm and cabbage looper were done separately under similar greenhous e conditions. Ten neonate s were placed in the central whorl of each plant and the plant was covered with a cylindrical screen cage (Fig. 2-1). Days required to develop from neonate to pupa and from pupa to adult emergence were recorded. Beet armyworm la rvae were provided MetroMix 220 in a 5cm-diameter Petri dish at the base of the plant as a pupation site. Cabbage looper pupated on the plant and on the walls of the container so were not supplied with MetroMix. Pupae were removed from the greenhouse, weighed an d put in individual cups in the incubator at 27 2C, 70 10% RH, and a photoperiod of 14:10 (L:D) h. Emerged adults were sexed, killed and then dried in an oven at 50 5C, for 3 d. Larval period, pupal fresh weight, pupal period, and dry weight of emer ged adults of each insect species were analyzed using PROC GLM with cultivar as a fixed effect and replications as random effect (SAS Institute1999). Tukeys honestly significant di fference (HSD) test with a

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51 significance level of = 0.05 (SAS Institute 1999) was used for posthoc means separation. Percent successf ul pupation and adult emergence were analyzed by two sample t -test using PROC TTEST (SAS Institu te1999). A binominal test (using the normal approximation with test statistic Z ) was used to determine whether the numbers of males versus females deviated from a 1:1 ratio on each cultivar. A Fisher's Exact test of independence was used to test whether the adult sex-ratio differed between the cultivars (Sokal and Rohlf 1995) using JMP release 5.1.2. Fecundity and Longevity of Subsequent Generation Fecundity and longevity were measured for nine pairs of newly emerged adults of each species that had been reared on either Ta ll Guzmaine or Valmaine as larvae. Each pair of adults was confined on a Tall Guzmai ne plant using a cylindrical screen cage (18.5 61.0 cm) in the greenhouse. Adults were supplied with 20% sucrose solution. Every other day, the lettuce plan t was replaced with a fresh plant. Eggs were counted on each plant and totaled over the life of each fe male. Fecundity and adult longevity of each insect species were analyzed using PROC GL M with cultivar as a main effect (SAS Institute 1999). A simple linear regression analysis was done to study the relationship between adult weight and fecundity us ing PROC REG (SAS Institute 1999). Results Neonate Survival and Development to Third Instar Larval mortality of cabbage looper and b eet armyworm after 1 wk of feeding was significantly higher on Valmaine than on Ta ll Guzmaine (Fig. 2-2). Cabbage looper mortality was 24 times higher on Valmaine than on Tall Guzmaine ( F = 242.82; df = 1, 29; P = 0.0001) whereas beet armyworm mortal ity was four times higher on Valmaine than on Tall Guzmaine ( F = 187.54; df = 1, 29; P = 0.0001). Average weight of cabbage

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52 looper feeding for 1 wk on Valmaine (75.4 3.7 mg, mean SEM) was significantly lower than that of larvae feeding on Tall Guzmaine (151.2 3.3 mg) ( F = 249.27; df = 1, 29; P = 0.0001). Beet armyworm weight was al so significantly lower (1.5 0.1 mg) on Valmaine than on Tall Guzmaine (8.3 0.8 mg) ( F = 68.71; df= 1, 29; P = 0.0001). The instar of the larvae surviving to pl ant dissection (1 wk after inoculation as neonates) differed significantly on the two lett uce cultivars for both species (Fig. 2-3). Cabbage looper and beet armyworm devel oped more slowly on Valmaine than on Tall Guzmaine. More of the surviving neonates of bot h insect species were in the early instars on Valmaine than on Tall Guzmaine. About 80% of cabbage looper surviving on Valmaine were in either th e first or second instar, wher eas on Tall Guzmaine about 80% of surviving larvae were in either the thir d or fourth instar (Fig. 2-3). Of the beet armyworm surviving for 1 wk on Valmaine, 57.7% were in the fi rst instar, whereas 78.8% were in the third instar on Tall Guzmaine (Fig. 2-3). Larval Feeding Behavior The insect species behaved differently on the lettuce cultivars. Cabbage looper cut narrow trenches across veins on the leaves and then fed on the area distal to the trench (Fig. 2-4A). This behavior released exudate from the laticifers of the leaves. Beet armyworm did not trench; neonates made sha llow scratches between the veins by feeding on parenchymatous tissue and second instars made holes through the leaf (Fig. 2-4B). The preferred site of feeding of cabbage looper ( 2 = 55.42, df = 2; P = 0.0001) and beet armyworm ( 2 = 35.13, df = 2; P = 0.0001) differed between the two cultivars (Fig. 2-5). Cabbage looper preferred to feed on the lo wermost fully mature leaves of Valmaine plants and on young and middle-aged leaves of Tall Guzmaine plants (rarely feeding on fully-matured leaves) (Fig. 2-6). Beet armywo rm preferred to feed on the lowermost fully

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53 mature leaves of Valmaine plants and on mi ddle-aged leaves of Tall Guzmaine plants. Both insect species preferred to feed on the di stal end of leaves. Ea rly instars of cabbage looper preferred to feed on the underside of the leaves, whereas early instars of beet armyworm fed on the upper side of the leaves. Survival and Development from Neonate to Adult Emergence Both cabbage looper ( F = 82.55; df = 1, 29; P = 0.0001) and beet armyworm ( F = 581.58; df = 1, 29; P = 0.0001) took significantly longer time to develop from neonate to pupation on Valmaine than on Tall Guzmaine (Table 2-1). Larval period of cabbage looper and beet armyworm was increased by 2.6 d and 5.9 d, respectively on Valmaine. Feeding on Valmaine resulted in a significant reduction in successful pupation of cabbage looper ( t = 9.75; df = 58; P <0.0001) and beet armyworm ( t = 13.46; df = 58; P <0.0001) (Table 2-1). Pupae of cabbage looper ( F = 41.53; df = 1, 29; P = 0.0001) and beet armyworm ( F = 63.84; df = 1, 29; P = 0.0001) weighed significan tly less when reared on Valmaine compared to Tall Guzmaine (Table 2-1). The duration of the pupal period of cabbage looper ( F = 44.53; df = 1, 29; P = 0.0001) and beet armyworm ( F = 30.79; df = 1, 29; P = 0.0001) was significantly increased on Valmaine (Table 2-1), thus delaying adult emergence. Successful emergence of a dults from pupae surviving on Valmaine was significantly reduced for cabbage looper ( t = 2.40; df = 58; P = 0.0196) but not for beet armyworm ( t = 1.40; df = 58; P = 0.1649) (Table 2-1). Adults of cabbage looper ( F = 83.02; df = 1, 29; P = 0.0001) and beet armyworm ( F = 196.34; df = 1, 29; P = 0.0001) surviving on Valmaine weighed significantly less than those surviving on Tall Guzmaine (Table 2-1). The mean adult sex-ratio of cabbage looper ( Z = 0.91, P = 0.3652) and beet armyworm ( Z = 0.59, P = 0.5529) did not deviate from a 1:1 ratio on Valmaine. The mean adult sex-ratio of cabbage looper ( Z = 1.30, P = 0.1950) and beet armyworm ( Z =

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54 1.33, P = 0.1845) also did not deviate from a 1:1 ratio on Tall Guzmaine. In addition, the sex-ratios of adult cabbage looper (Fis her's Exact test of independence, P = 0.1417) and beet armyworm (Fisher's Exact test of independence, P = 0.2077) on the two cultivars did not differ statistically (Table 2-1). Fecundity and Longevity of Subsequent Generation Fecundity of cabbage looper ( F = 109.36; df = 1, 8; P = 0.0001) and beet armyworm ( F = 149.14; df = 1, 8; P = 0.0001) on Valmaine was reduced by 62.8 and 67.9%, repectively, compared to that on Tall Gu zmaine (Table 2-2). Significant positive linear relationships were found between adult we ight and fecundity of both insect species on the two lettuce cultivars (Fig. 2-7). Howeve r, neither male nor female longevity of cabbage looper (male: F = 0.47; df = 1, 8; P = 0.5121; female: F = 0.47; df = 1, 8; P = 0.5121) nor beet armyworm (male: F = 0.31; df = 1, 8; P = 0.5943; female: F = 1.33; df = 1, 8; P = 0.2815) differed on Valmaine or Tall Guzmaine (Table 2-2). Discussion Performance of cabbage looper and b eet armyworm was greatly reduced on resistant Valmaine compared to Tall Guzmai ne. Insects surviving on poor quality hosts are expected to have reduced survival to ad ult emergence and reduced fecundity (Zalucki et al. 2001), as was shown in my study. Larval survival and development can be reduced on poor quality hosts due to nutritional com position and/or secondary plant metabolites (Scriber and Slansky 1981, Herms and Mattson 1992, Slansky 1992). Nutritional composition and secondary plan t metabolites vary among plants, plant parts and developmental stages (Nelson et al. 1981, Brower et al. 1982). Cabbage looper and beet armyworm larvae preferred to feed on mature leaves of Valmaine (Fig. 2-5). In lettuce, mature leaves are less nutritious than young and middle-aged leaves. Young and

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55 middle-aged lettuce leaves are more metabolically active than mature leaves, and therefore, contain higher amounts of dry matter, ascorbic acid and soluble solids, such as fructose, sucrose, glucose, fructans and ot her saccharides (McCabe et al. 2001, Siomos et al. 2002). Moreover, mature lettuce leaves alwa ys have higher amounts of anti-nutritional constituents, such as nitrates (Siomos et al. 2002). Leaf maturation is accompanied by a decline in the concentrations of proteins and other nutrients (Bernays and Chapman 1994). Therefore, feeding on less nutritious mature leaves of Valmaine may have affected the fitness of cabbage looper and beet armyworm. Larval avoidance of young and middle-aged l eaves of Valmaine may have been due to the presence of high amounts of latex and/or the chemical constituents of latex. Latex from young and middle-aged leaves was pure white and viscous whereas latex from mature leaves was a watery translucent flui d (A. Sethi, pers. obs.). Young leaves of the poinsettia, Euphorbia pulcherrima Wilenow contained higher amounts of latex and laticifer starch than mature leaves (Sp ilatro and Mahlberg 1986). The proportionally higher latex amount may have a specific purpos e related to plant defense. The defensive role of latex has been attri buted to its sticky nature, whic h would enable the plant to capture small insects and immobilize the mouthpa rts of larger insects (Farrell et al. 1991, Dussourd 1993, Dussourd and Denno 1991, 1994). An tiherbivore function of latex has been suggested in many plant systems (Shukla and Krishna-Murti 1971, Fahn 1979, Konno et al. 2004, 2006). The presence of high amounts of latex with its chemical components in young leaves (Kinghorn and Evans 1975, Swain 1977, Rees and Harborne 1985) may provide for their defense compared to mature leaves. In the chicory plant, Cichorium intybus L., sesquiterpene lactones were pr esent in the highe st amounts in the

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56 most actively growing regions of the plant and possessed an tifeedant properties against Schistocerca gregaria (Orthoptera: Acrididae) (Rees and Harborne 1985). Various organic compounds, like phenolics and terpenoi ds have been reported in latex of Lactuca spp. (Crosby 1963, Gonzales 1977, Cole 1984, Sessa et al. 2000) and their defensive role as phytoalexins has been reported against plan t diseases (Bennett et al. 1994, Bestwick et al. 1995). In lettuce, the density of latex is successively decreased from the base to the apex of the leaf (Small 1916). This may account for the preference of neonate caterpillars in my study to feed on the distal end (apex) of leaves. Certain plant enzymes, such as phenyla lanine ammonia lyase, polyphenol oxidase and peroxidase are known for their defensive role against insects. In lettuce, CamposVergas and Saltveit (2002) reported enhan ced activity of phenylal anine ammonia lyase upon mechanical wounding in young leaves comp ared to mature leaves. Phenylalanine ammonia lyase is also more active in aphid-resistant cultivars of L sativa than in susceptible cultivars (Cole 1984). Outer (i.e., older) leaves of head lettuce exhibit high c oncentrations of flavonoids, such as quercetin (Hohl et al. 2001). Quercet in and its derivatives are known to act as phagostimulants to many lepidopterans (S immonds 2003). Therefore, the feeding location of beet armyworm and cabbage looper may have been the end result of both antifeedant properties (either physical or chemical or both) of young and middle-aged leaves and phagostimulant properties of mature leaves. Cabbage looper exhibited greater fitness than beet armyworm on Valmaine. Larval mortality of cabbage looper was less compar ed to beet armyworm on Valmaine and other parameters, such as larval weight, pupal we ight, percent pupation and adult weight of

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57 cabbage looper were less affected compared to beet armyworm on Valmaine (Table 2-1). Moreover, larval development of cabbage l ooper was faster than beet armyworm, as cabbage looper larvae entered the fourth la rval after 1 wk on Valmaine while beet armyworm larvae were still in the third instar stage (Fig. 2-2). Survival and development of yellow-striped armyworm was also affected greatly on L. serriola compared to cabbage looper (Dussourd 1993) This superior performance of cabbage looper may be attributed to their feeding be havior (i.e., trenching on lati ciferous plants). Trenching blocks latex flow to intended feeding site s and may act as a count er-adaptation to the plants defensive secretions (Dussourd a nd Denno 1994). In spite of their behavioral counter-adaptation, cabbage looper performance was worse on Valmaine than on Tall Guzmaine. Lettuce possesses little tolerance for cosmetic damage and foliar feeding by lepidopterous pests greatly affects its market able production. The cons umer, especially in developed countries, will not accept produce unl ess it is free of all insects and blemishes at harvest. Further, lettuce is a short-s eason crop and insufficient time may be present between treatment of chemical and harvest fo r pesticide residues to decline to acceptable levels (Norris et al. 2003). This limits the us e of chemicals in lett uce production that do not break down rapidly. Therefore, host plan t resistance is an ec onomically, ecologically and environmentally advantageous method of insect management. The results of my study have confirmed that Valmaine expresse s considerable resist ance to lepidopterous pests in spite of their counter-strategies ag ainst plant resistance. In general, multipleinsect resistance is thought to be more de sirable than single-insect resistance (Smith 1989). Feeding on Valmaine resulted in reduc ed vigor of both insect species, which

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58 ultimately could make them more susceptibl e to other biotic and abiotic factors. However, additional research is required to determine the biochemical basis of multipleinsect resistance in lettuce. Understanding th e mechanism of resistance will certainly aid in the development of lettuce cultivars with improved pest resistance and may result in reduced pesticide usage.

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59Table 2-1. Performance of cabbage looper and beet armyworm rel eased as neonates onto Valmaine and Tall Guzmaine lettuce. Species Cultivar Larval period (days) % Pupation Pupal weight (mg) Pupal period (days) % Adult emergence Adult weight (mg) Sex-ratio (male: female) Cabbage looper Valmaine 11.7 0.2a 49.3 2.0b 172.2 4.5b8.7 0.2a 82.4 2.5a 23.4 0.6b 1.18 : 1a Tall Guzmaine 9.1 0.3b 79.0 2.3a 206.5 2.8a 7.8 0.1b 90.7 1.3a 29.7 0.6a 1 : 1.20a Beet armyworm Valmaine 19.3 0.3a 27.3 2.6b 51.4 1.4b 7.5 0.1a 86.6 2.7a 9.1 0.3 b 1.20 : 1a Tall Guzmaine 13.4 0.1b 65.3 3.8a 68.9 1.3a 6.7 0.1b 93.9 2.0a 14.9 0.3a 1 : 1.22a Means SEM followed by different lette rs for each parameter within insect species differed significantly (P 0.05) using ANOVA and Tukeys HSD test for larval period, pupal weight, pupal period and adult weight, two sample t-test for % pupation and % adult emergence, and Fisher' s Exact test of independence for sex-ratio .

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60Table 2-2. Fecundity and longevity of subs equent generation of cabbage looper and b eet armyworm reared on Valmaine and Tall Guzmaine lettuce. Species Cultivar Fecundity Male longevity (d) Female longevity (d) Cabbage looper Valmaine 146.4 8.4a 11.8 0.2a 10.1 0.2a Tall Guzmaine 393.3 18.1b 12.0 0.3a 10.3 0.3a Beet armyworm Valmaine 123.2 10.3a 6.4 0.2a 8.1 0.2a Tall Guzmaine 383.6 17.7b 6.3 0.2a 8.4 0.2a Means SEM followed by different lette rs for each parameter within insect species differed significantly (P 0.05) using ANOVA and Tukeys HSD test.

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61 Figure 2-1. Experimental setup to study ca bbage looper and beet armyworm neonate survival and development to third instar.

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62 Cabbage looperBeet armyworm Mortality % 0 10 20 30 40 50 60 Valmaine Tall Guzmaine Figure 2-2. Larval mortality of cabbage looper and beet armyworm after 1 wk of feeding on resistant Valmaine and susceptibl e Tall Guzmaine lettuce. Error bars indicate 1 SEM

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63 BAW Percentage of survivin g neonates in each instar 0 20 40 60 80 100 Valmaine Tall Guzmaine Instar 1st2nd3rd4th 0 20 40 60 80 100 CL Figure 2-3. Instars of cabbage looper (CL) and beet army worm (BAW) surviving for 1 wk on resistant Valmaine and sus ceptible Tall Guzmaine lettuce. G2 tests indicated that the instar distribution on Valmaine differed significantly from that on Tall Guzmaine (P < 0.05).

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64 Figure 2-4. Feeding of two lepidopterans on lettuce. A) Cabbage looper cutting narrow trenches on romaine lettuce, B) Beet armyworm damage on romaine lettuce: (a) & (b) shallow scratches, (c) holes. A B ( a ) ( b ) ( c )

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65 CL BAW 0 5 10 15 20 25 30 Valmaine Tall Guzmaine Leaf age YoungMiddleMature Number of plants 0 5 10 15 20 25 30 Figure 2-5. Feeding preference of cabbage looper (CL) and beet armyworm (BAW) larvae among lettuce leaves of diffe rent ages on resistant Valmaine and susceptible Tall Guzmaine. The y-axis de picts the total numbe r of plants (out of 30) on which at least so me feeding occurred on leaves of the specified age group.

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66 Figure 2-6. Feeding behavior of beet armyworm on (a ) Tall Guzmaine and (b) Valmaine, and of cabbage looper on (c) Tall Guzmaine and (d) Valmaine.

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67 Adult weight (mg) 08101214Fecundity 0 100 150 200 250 VAL BAW Adult weight (mg) 0202224262830Fecundity 0 100 150 200 250 VAL CL Adult weight (mg) 02830323436384042Fecundity 0 300 400 500 600 Adult weight (mg) 016182022Fecundity 0 300 400 500 600 TG CL TG BAWy = 9.7 x 95.3 R 2 = 0.82 p = 0.0007 y = 15.9 x 166.1 R 2 = 0.95 p < 0.0001 y = 20.7 x 101.5 R 2 = 0.94 p < 0.0001 y = 37.3 x 329.7 R 2 = 0.86 p = 0.0003 Figure 2-7. Relationships between adult weight and fecundity of cabbage looper (CL) and beet armyworm (BAW) that developed from larvae reared on resistant Valmaine (VAL ) or susceptible Tall Guzmaine (TG) lettuce.

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68 CHAPTER 3 ROMAINE LETTUCE LATEX DETERS FE EDING OF BANDED CUCUMBER BEETLE (COLEOPTERA: CHRYSOMELIDAE) Introduction Latex is the common term used to describe a frequently milky plant exudate which is typically stored under positive pressure within specialized vessels called laticifers (Fig. 3-1). These laticifers accompa ny the vascular bundles and ramify into the mesophyll to reach the epidermis (Hayward 1938, Es au 1965, Metcalfe 1967, Olson et al. 1969, Metcalfe and Chalk 1983, Fahn 1990, Kekwick 2001). About 12,500 to 20,000 plant species, belonging to >900 genera from about 40 families, most of which are dicotyledons, are known to exude late x (Esau 1965, Metcalf 1967, Lewinsohn 1991, Kekwick 2001, Evert 2006). Latex contributes to plant defens e in two different ways; physical properties (stickiness) and chemical properties (toxic constituents). Stickiness can result in the entrapment or gumming up of the mouthparts of herbivorous insects (Dillon et al. 1983, Dussourd 1993, 1995, Zaluck i and Malcolm 1999). Latex contains toxic constituents including alkaloids (Roberts 1987, Valle et al. 1987, Konno et al. 2006), cardiac glycosides (Zalucki and Br ower 1992, Zalucki and Malcolm 1999), and terpenoids (Evans and Schmidt 1976, Rees and Harborne 1985, Spilatro and Mahlberg 1986). Some insects circumvent the mechanical stickiness and toxic effects of latex by severing latex-bearing veins or by cutting trenches prior to consuming the distal tissue (Dussourd 1993, Zalucki and Malcolm 1999, Sethi et al. 2006). Lettuce, Lactuca sativa L., is one of the most im portant vegetable crops grown throughout the world and its production grow s annually (USDA 2005a). As a cultivated crop, lettuce is vulnerable to attack by various insect pest s including the banded cucumber beetle, Diabrotica balteata LeConte (Nuessly and Nagata 1993). This insect

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69 has a host range of >50 plant species in 23 families (Saba 1970) and a high reproductive potential of >800 eggs per female with a 2 to 8 wk oviposition period (Pitre and Kantack 1962). It can be found throughout the year in th e southern United States (Schalk 1986). In southern Florida, foliar feeding by D balteata adults leads to econom ic damage in lettuce due to reduction in stand and marketab ility, decreased photosynthetic area, frass contamination of the heads, and increased vulne rability to diseases. Chemical control of soil-borne eggs, larvae and pupae of this insect has been inef fective (Schalk et al. 1986) and control of the adult is th e sole promising option (Schalk et al. 1990). As a result, growers currently are dependent on pesticides (Nuessly and Nagata 1993) which can pose a threat to the environment, farm workers and natural enemies of insect pests, and increase production costs. Host plant resistance was explored as an alternative strategy for the management of this economic insect pest in a cos or romaine lettuce cul tivar, Valmaine (Nuessly and Nagata 1994, Huang et al. 2002, Sethi et al 2006). A high level of resistance was reported in Valmaine, compared to the cl osely related susceptible cultivar Tall Guzmaine against serpentine leafminer, Liriomyza trifolii (Burgess) (Nuessly and Nagata 1994), banded cucumber beetle (Huang et al. 2002) and two lepidopterans, Trichoplusia ni (Hbner) and Spodoptera exigua (Hbner) (Sethi et al. 2006). These studies suggested that Valmaine lacks feeding stimulants or contains feeding deterrents, either in the leaf cuticle or th e leaf interior. Huang et al. ( 2003a) reported that leaf surface chemicals were not responsible for resist ance in Valmaine and suggested chemicals inside the leaf may play a role. However, incorporation of freeze-dried leaves of Valmaine into an artificial diet did not deter feeding by D. balteata adults and neither did

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70 application of Valmaine latex on the leaf su rface of a favorite food, lima bean (Huang et al. 2003b). It is possible that the activity of physical and/or ch emical defenses in latex or leaf tissue may have been reduced or elim inated when whole leaves were dried and powdered. Furthermore, the physical and chemi cal properties of latex may have changed when applied on lima bean leaves due to dryi ng of the latex and/or oxidation of chemical constituents. In free-choice situations L trifolii (Nuessly and Nagata 1994), D. balteata (Huang et al. 2002), T ni and S exigua (Sethi et al. 2006) preferred to feed on mature leaves of Valmaine over young or middle-aged leaves The avoidance of young and middle-aged leaves of Valmaine may have been due to the presence of high amounts of latex and/or the chemical constituents of latex. The late x from young and middle-aged leaves is pure white and viscous, whereas latex from mature le aves is watery and translucent (Sethi et al. 2006). In this study, I report on the possible deterr ent role of latex ag ainst beetle feeding on artificial diet treated w ith freshly extracted latex from either Valmaine or Tall Guzmaine in choice and no-choice conditions. Additional tests were conducted using latex extracted from young versus mature leav es of these two cultivars to study the role of leaf age in the expression of latex deterr ence. Lastly, samples of supernatant material collected following dissoluti on of latex from both cult ivars in water/methanol combinations or methylene chloride and centr ifugation were applied to diet disks under no-choice situations to determine whethe r differences in latex chemistry between Valmaine and Tall Guzmaine contribute to the multiple insect resistance observed on Valmaine.

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71 Materials and Methods Plants and Insects Valmaine and Tall Guzmaine seeds were ge rminated by placing them overnight in a Petri dish lined with a wet filter paper in the laboratory. Germinated seeds were planted in a transplant tray filled with commercial soil mix (MetroMix 220, Grace Sierra, Milpitas, CA) in a greenhouse with natural light at a mean temperature of 27C (32 to 24 C) and 68% mean R.H. (44 to 94%). After 2 wk, seedlings we re transplanted into 15-cmdiameter plastic pots filled with MetroMix 220. Plants were irrigate d daily and fertilized once a week with 10 ml of a 10 g/l solution of soluble fertilizer (P eters 20-20-20, N-P-K, W.R. Grace, Fogelsville, PA). Bush lima bean ( Phaseolus lunatus L.) cultivar Fordhook 242 (Illinois Foundation Seeds, Champagne, IL) was grown as an a dult food source for the colony. Seeds were planted in a transplant tray filled with MetroMix 200. Lima bean plants were irrigated daily and fertilized once a week after the first true-leaf stage with the same solution used for lettuce plants. Adults of D. balteata were used because previously the same insect species was used by Huang et al. (2003b) as explained a bove. In addition, R. T. Nagata (Everglades Research and Education Center, University of Florida, FL) used the same insect species to track resistance in lettuce breeding lines. Further, adults of D. balteata were easy to rear and handle during bioassays. A colony of D. balteata was established in 2003 from a wild population of adults collected from spiny amaranth, Amaranthus spinosus L. and primrose willow, Ludwigia peruviana L. in Belle Glade, FL. The colony was supplemented with wild individuals to increase genetic diversity in 2005 and 2006. Adults of D. balteata were fed on lima bean leaves and sweet potato tubers, and larvae

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72 were reared on corn seedling roots (H93 FB37, Illinois Foundation Seeds Inc., IL) as per Huang et al. (2002) (Fig. 3-2). Adults of the D. balteata colony were confined in a ventilated plexiglas cage (30.5 30.5 30.5 cm) in an incubator at 27 2 C and R.H. 70 10% with a photoperiod of 14:10 (L:D) h (Fig. 3-2A). Oviposition was facilitated by providing two domed-shaped plastic containers (8.4 cm di ameter 7 cm high) with mesh-covered lids (0.1 0.2 cm). These plastic containers were covered with inverted strawberry baskets upon which lima bean leaves were placed (Fig. 3-2B). The plastic containers we re filled with upright small glass vials (20 ml) to hold tig htly one moist layer of cotton balls. Two circular pads, each containing four layers of premium paper to wels (Kimberly-Clark Co., Roswell, GA), and four layers of cheesecloth were placed betw een the layer of cotton balls and the meshed lid. The cheescloth and paper towel pads with e ggs were collected every 2 d and kept in a Petri dish with a screened lid in the same incubator. Three-day-old eggs were dipped in sodium hypochloride solution ( 15 ml Clorox in 485 ml water, The Clorox Co., CA) for 1 min and then rinsed thrice with deionized water to affect surf ace sterilization. The sterilized eggs were replaced in the incubato r and covered with a wet paper towel within a cylindrical container (18 cm diameter 7.5 cm high) with a screen ed lid (Fig. 3-2C). On the following day, 9 to 10 germinated corn seeds were put in the container as food for the emerging larvae. Larval D. balteata were raised on germinated corn seeds in containers designed to maintain sufficient moisture for seed growth without drowing the la rvae or covering them with soil. A preassembled germination paper with a wick stapled on each end was placed

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73 at the botton of a rectangular plastic cont ainer (32.5 17.2 10 cm) and covered with a single layer of pregerminated corn seeds. The seeds were covered with wet paper towel and the container was placed on the top of a wa ter-filled tray in such a way that the two wicks were suspended in the water. Pregermi nated corn seeds were prepared by soaking dried seeds overnight in a Clorox soloution ( 16 ml/L of water). They were rinsed with deionized water the following morning and st ored in a refrigerator until needed. These larval rearing containers we re covered with a screened lid. One-day-old emerged larvae on germinated corn seeds were next transfe rred to the rectangula r rearing containers stocked with 3-d-old germinated corn seedli ngs and kept at 27 2 C with a photoperiod of 14:10 (L:D) h in a rearing room (Fig. 3-2D, E). After 1 wk of larv al rearing in these containers, the larvae were tran sferred to a second containe r with germinated corn to complete their larval development (Fig. 3-2F, G, H). Two days after putting larvae in the second container, th ird instar larvae were collected into a container (18 cm 7.5 cm high) filled with moistened and autoclaved MetroMix 220 (Fig. 3-2I) to allow for pupati on and adult emergence. The container was covered with a dampened towel to retain moisture. After 10 d, the emerged adults were transferred into the screen cage mentioned above (Fig. 3-2J). Artificial Diet Preparation Dry mix for artificial diet is commercially available a nd has been shown to support the adult stage of D. balteata (Creighton and Cuthbert 1968). All materials required for preparing and dispensing the diet were t horoughly sanitized with sodium hypochlorite solution (Clorox, Oakland, CA) diluted 1:5 wi th deionized water. A 100-ml quantity of southern corn rootworm ar tificial diet (Bio-Serv, Fr enchtown, NJ) (Creighton and Cuthbert 1968) was prepared as follows. St erile deionized water (100 ml) and agar (1.74

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74 g) were heated on a hot plate to boiling. On ce the agar had cooled to approximately 40 C, KOH solution (1 ml) and diet dry mix (14.91 g) were added to it and thoroughly mixed to avoid the formation of lumps. The liq uid diet was dispensed into two glass Petri dishes (9 cm diameter). The diet was allowed to cool before the Petri dishes were covered with glass lids. The Petri dishes were wrappe d completely in plasti c wrap and aluminum foil, and stored in a refrigerator (4-6 C) for up to 3 h. Latex Collection and Solvent Extraction Latex (70 l) was collected from the bases (where leaf lamina joins the stem) of young and middle-aged leaves of individual plants, sites where there was a rapid exudation of latex upon cutting (Fig. 3-3). The cu ts were made using a disposable scalpel blade (Feather, Osaka, Japan). The late x was collected using a silanized 100l glass capillary tube inserted into a microdispen ser (Drummond Scientific Company, Broomall, PA) 60 s after the leaf base was cut. In a pilot study, I incorporated fresh latex into the artificial diet for D. balteata adults at two concentrations (0.1 and 0.2%) and recorded diet consumption by D. balteata adults to investigate the potential of Valmai ne latex as a mechanism of multiple insect resistance. Latex did not deter f eeding of D. balteata adults when presented in this manner. Therefore, in this study, I applied fres hly extracted latex from either Valmaine or Tall Guzmaine to artificial diet and confined D. balteata adults under choice and nochoice conditions to investigat e the possible deterrent role of latex against beetle feeding. A 1.5-cm-diameter cork borer was used to punc h out disks (1 cm thick) from cooled artificial diet. Late x (70 l) from an individual plan t was applied, immediately after collection, onto the top surface and sides of a diet disk using a microdispenser.

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75 I chose artificial diet as a substrate for a pplication of the latex because it kept latex moist for a longer time by providing more mo isture compared to lima bean leaves. In addition, latex treated diet disks fa cilitated the direct exposure of D. balteata adults to latex. As the diet disks were to tally covered with latex on all sides, it somewhat simulated the natural situation where an insect gnawing a lettuce plant is directly exposed to latex. Four different solvent combinations, i. e., water, water:methanol (20:80), water:methanol (50:50), and methylene ch loride were used to extract chemical constituents of latex (Fig. 3-5A). Latex (70 l) was collected from an individual plant in the same way as explained above and immedi ately dissolved in 10 times volume of the solvent (Fig. 3-4). After dissoluti on, samples were centrifuged at 1200 g for 20 min and supernatant was collected (Fig. 3-5B, C). The supernatant was reduced down to 1/10 volume by evaporating with nitrogen gas. An am ount of extract, equiva lent to 70 l latex, was applied to each diet disk for use in the following bioassays (Fig. 3-4). Bioassay Conditions For all experiments described below, an experimental unit consisted of two diet disks and three pairs of unfed D. balteata adults within a plastic ventilated container (10 10 8 cm). Unfed adults that had emerged w ithin 48 h of the start of the experiment were used in all tests. The diet disks we re placed on the bottom of the container and beetles were allowed to feed on the diet fo r 16 h. Each experimental unit was replicated 15 times. The experiments were carried out at 25 1C in a laboratory under a photoperiod of 14:10 (L:D) h. In all tests, the nu mber of adults feeding on each diet disk was recorded 15, 30, 60 and 90 min after thei r release into the bioassay chambers.

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76 Choice Tests and No-choice Tests with Fresh Latex Choice tests were conducted to determine whether D. balteata adults showed a preference between diet disks tr eated with latex from Valmai ne or Tall Guzmaine. Three treatment combinations were studied: latex from Valmaine versus latex from Tall Guzmaine, latex from Valmaine versus contro l (untreated diet wit hout latex), and latex from Tall Guzmaine versus control (Fig. 3-6A ). Three treatments (latex from Valmaine, latex from Tall Guzmaine, and control) also were studied in a no-choice situation, with each experimental unit containing two disks of the same treatment (Fig. 3-6B). Dry weight of diet consumed in a 16-h period was calculated for comparison among the treatments. To determine dry wei ght, an additional 10 diet disks from each treatment (Valmaine latex-treated, Tall Guzmaine latex-treated and control) were weighed individually (disk fresh weight) before they were put into an oven at 50 5C. After 3 d, these diet disks were reweighed i ndividually (disk dry weight). A dry/fresh weight ratio was calculated for each diet disk and averaged over the 10 disks. The diet fresh weight was determined for each disk fo r each treatment prior to the start of each experiment. After 16 h of exposure to beetle f eeding, the diet disk was dried in the oven for 3 d as above, reweighed and then multiplied by the corresponding average dry/fresh weight ratio. The dry weight of diet consum ed was calculated as the difference between initial and final dry weights. Choice Tests Using Latex from Young and Mature Leaves Choice tests were conducted to determine whether D. balteata adults exhibit any preference between diet disks treated with la tex from young or mature leaves of either Valmaine and Tall Guzmaine. Two treatment combinations were studied: latex from young leaves versus latex from mature leaves of Valmaine and latex from young leaves

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77 versus latex from mature leaves of Tall Guzmaine. The dry weight consumption of young and mature latex-treated diets of each cultivar in 16 h was recorded as described above. Total diet consumed per three pairs of adults for 16 h was calculated by adding consumption of the two diet disks in each container in each treatment. No-Choice Tests Using Latex Extracts Fifteen treatments were studied: five for Valmaine latex dissolved in water, water:methanol (20:80, % v/v), water:metha nol (50:50), methylene chloride, and fresh latex without solvent; five for Tall Guzmaine latex dissolved in water, water:methanol (20:80), water:methanol (50:50) methylene chloride, and fres h latex without solvent; and five for control the four so lvent combinations without la tex and untreated diet. Each experimental unit contained two disks of the same treatment. The dry weights of Valmaineand Tall Guzmaine-extract treated and control diets disk s consumed in 16 h were calculated as above. Beetle Behavior in Response to Contacting Latex Observations were made on beetle behavi or in response to contacting latex and latex extracts on diet disks in the choice and no-choice tests described above. In addition, freshly collected latex (1 l) from both Valmaine and Tall Guzmaine plants was applied to mouthparts of beetles (10 for each) usi ng a microdispenser. Using a microscope, salivation by treated beetles was observed immediately after latex application and mobility of mouthparts was observed after 24 h to distinguish if toxic constituents or stickiness contributed to the feeding deterrence of Valmaine lettuce. Individual beetles also were confined on young leaves of either Valmaine or Tall Guzmaine (10 plants for each cultivar) and observed for 90 min using a microscope to closely observe their feeding behavior in response to contacting latex during test bites.

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78 Statistical Analysis For all choice and no-choice tests using late x, the number of adults feeding on diet 15, 30, 60 and 90 min after release was analyzed as a repeated measures design using Proc GLIMMIX (SAS Institute 2003). In each choice test (Valmaine versus Tall Guzmaine, Valmaine versus control, and Ta ll Guzmaine versus control), data on number of adults feeding were analyzed as a 2 4 factorial design separate ly, in which latex was treated as one factor w ith two levels, and time interval after beetle release was treated as the other factor with four levels (15, 30, 60 and 90 min). In no-choice tests, data on number of adults feeding were analyzed as a 3 4 factorial desi gn, in which latex was treated as on factor with three levels (V almaine, Tall Guzmaine and control), and time interval after beetle release wa s treated as the other factor w ith four levels. Both variables (latex and time interval) were fixed. Fifteen groups of six be etles (i.e., rep lications) were randomly assigned to each level of latex, mean ing that beetles were nested within latex levels. Each group of six beetles was tested four times (levels of time interval). The model was number of beetles feeding = [latex] [replications(latex)] [time interval] [latex*time interval]. The error degree of freed om for latex effect was calculated as levels of latex(replications 1). The error degree of freedom fo r time interval effect and interaction was calculated as levels of latex(levels of time interval 1) (replications 1). In no-choice tests using late x extracts, the data on numb er of beetles feeding was analyzed using Proc GLM (SAS Institute 2003) se parately at each time interval with latex extract as a fixed effect and replications as a random effect. The error degree of freedom for latex extract effect was cal culated as (levels of latex ex tract -1)(replications -1). The dry weights of Valmaine, Tall Guzmaine and control diets consumed in 16 h were analyzed by paired t -tests using Proc MEANS (SAS Institute 2003) for all choice

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79 tests and by ANOVA using Proc GL M with latex as a fixed eff ect and replications as a random effect (SAS Institute 2003) for all no-ch oice tests. The total dry weight consumed by adding consumption of the two diet disks in each chocie test using latex from young and mature leaves including control disk s was also analysze d by ANOVA using Proc GLM with latex as a fixed effect and repli cations as a random effect (SAS Institute 2003). The error degree of freedom for latex/latex extract effect was calculated as (levels of latex/ latex extract -1)(re plications -1). Tukeys honestly significant difference (HSD) test with a significance level of = 0.05 (SAS Institute 2003) was used for post hoc means separation. Deterrence coefficients (relative and absolu te) were calculated (Nawrot et al. 1986) based on the amount of diet consumed. All th e data from both choice and no-choice tests were pooled and used to determine coe fficients using the following equations: Relative (R) = [(C T) / (C + T)] 100 (Choice Test) Absolute (A) = [(CC TT) / (CC + TT)] 100 (No-choice Test) where C and CC are the consumption of cont rol diet (without la tex) in choice and no-choice tests, respectively; and T and TT are the consumption of latex-treated diet (Valmaine or Tall Guzmaine) in choice and nochoice tests, respectively. The deterrent activity of the latex-treated diets was expressed by the total coefficient of deterrence (D = A + R). The deterrence coefficients were analyzed by two-sample t -tests using PROC TTEST (SAS Institute 2003).

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80 Results Latex Choice and No-Choice Tests Treatment of latex had significant effect on the number of insects feeding in all three choice tests, Valmaine (Val ) versus Tall Guzmaine (TG) ( F = 64.83; df = 1, 28; P = 0.0001), Valmaine versus control ( F = 99.27; df = 1, 28; P = 0.0001), and Tall Guzmaine versus control ( F = 5.68; df = 1, 28; P = 0.0241). Beetles avoided feeding on diet treated with Valmaine latex (Fig. 3-6A; 3-7A, B). Th e number of insects f eeding on diet treated with Valmaine latex was negligible compared to the number feeding on diet treated with Tall Guzmaine latex (Fig. 3-7A) and control diet (Fig. 3-7B). The number of insects feeding increased over time (i.e., 15, 30, 60 and 90 min) in all choice tests (Val vs. TG: F = 7.28; df = 3, 84: P = 0.0002, TG vs. control F = 9.83; df = 3, 84: P = 0.0001, Val vs.. control: F = 24.87; df = 3, 84: P = 0.0002) (Fig. 3-7A, C). Si gnificant interactions were found between latex treatment and time interv al in the choice tests involving Valmaine and Tall Guzmaine ( F = 8.56; df = 3, 84; P = 0.0001) and Valmaine and the control diet ( F = 28.47; df = 3, 84; P = 0.0001). In contrast, there wa s no significant interaction found in the choice test between Tall Guzmaine la tex-treated diet disks and control disks ( F = 1.44; df = 3, 84; P = 0.2374) (Fig. 3-7C). Beetles consum ed significantly less diet treated with Valmaine latex (Table 3-1). Beetles ate 2.9 times more on Tall Guzmaine latex treated diet than diet treated with Valmaine latex in a choice between Valmaine and Tall Guzmaine. Beetles also consumed 4.5 times mo re control diet than diet treated with Valmaine latex in a choice between Valmaine and control. Beetles also consumed 1.5 times less diet treated with Tall Guzmaine than control diet. In no-choice tests, latex also had significant on the number of insects feeding on diets ( F = 109.46; df = 2, 42; P = 0.0001). Significantly fewer insects fed on diet treated

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81 with Valmaine latex than on Tall Guzmaine la tex-treated and control diets (Figs. 3-6B, 38). No significant interaction was found betw een latex treatment and time interval ( F = 1.74; df = 3, 126; P = 0.1179). Beetles consumed 4.7 and 6.3 times more Tall Guzmaine latex-treated and control diets, respectively, than Valmaine latex-treated disks ( F = 168.31; df = 2, 42; P = 0.0001) (Table 3-1). Valmaine latex exhibited strong deterren ce against beetles in both choice and nochoice bioassays (Table 3-2). Bo th relative and absolute coefficients of deterrence for Valmaine latex-treated diets were significantly higher than those for Tall Guzmaine latex-treated diets. The total coefficient of deterrence of Valmaine latex was 3.9 times higher than that of Tall Guzmaine latex. Choice Tests Using Latex from Young and Mature Leaves In Valmaine choice test, latex significantly affected the number of insects feeding on diet ( F = 61.87; df = 1, 28; P = 0.0001), but it was not sign ificantly affected by latex treatment in Tall Guzmaine choice tests ( F = 1.84; df = 1, 28; P = 0.812). Significantly fewer insects fed on diet treated with latex from young leaves than on diet treated with latex from mature leaves of Valmaine (Fig s. 3-9, 3-10A). Adult preference for diet treated with latex from mature leaves of Valmaine increased significantly with time ( F = 30.95; df = 3, 84; P = 0.0001) (Fig. 3-10A). In the Ta ll Guzmaine latex choice test, the number of beetles feeding on diet disks tr eated with latex from young leaves did not differ significantly from that on disks treated w ith latex from mature leaves (Figs. 3-9, 310B). The number of beetles feeding on both Tall Guzmaine diets increased significantly with time ( F = 39.44; df = 3, 84; P = 0.0001) (Fig. 3-10B). Beetles consumed 7.2 times more diet tr eated with latex from mature Valmaine leaves than treated with latex from young Valm aine leaves (Table 3-3). Diet consumption

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82 did not differ significantly between diet disk s treated with latex from young and mature leaves of Tall Guzmaine. The total diet consum ed in the Valmaine latex choice test (sum of the consumption on the two disks) did not differ significantly from the amount eaten in the Tall Guzmaine latex choice test but wa s significantly less than the amount consumed in the control diet test. No-Choice Tests Using Latex Extracts Water extracts of both Valmaine and Tall Guzmaine were yellow in color, but the color of the Valmaine extract was more intense than that of the Tall Guzmaine extract (Fig. 3-5C). Water:methanol (20:80) extrac ts of both cultivars were colorless. The water:methanol (50:50) extract of Tall Guzmai ne was colorless, but it was yellow in the case of Valmaine. Methylene chloride extracts of both cultivars were white in color and sticky. Treatment of latex extracts had significan t on the number of insect feeding on diet after 15 min ( F = 11.97; df = 14, 196; P = 0.0001); 30 min ( F = 12.60; df = 14, 196; P = 0.0001); 60 min ( F = 24.42; df = 14, 196; P = 0.0001); and 90 min of release ( F = 31.93; df = 14, 196; P = 0.0001). Significantly fewer insects fed on diet disks treated with a water:methanol (20:80) extract of Valmaine latex than on diets treated with all other Valmaine and Tall Guzmaine latex extracts, as well as all the contro l diets (Figs. 3-11, 312). In addition, diet consumption was also significantly affected by the latex extract treatment ( F = 95.01; df = 14, 196; P = 0.0001). Beetles consumed significantly less diet treated with water:methanol (20:80) extract of Valmaine latex than diet treated with any other latex extract or contro l diet (Fig. 3-13). The number of insects feeding on disks (Fig. 3-12) and amount consumed (Fig. 3-13) on diet disks treated with the

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83 water:methanol (20:80) extract of Valmaine late x were equivalent to those values for diet treated with fresh Valmaine latex. Beetle Behavior in Response to Contacting Latex In latex choice tests, beetles flew immediately to the roof and walls of the container whenever they approached the Valm aine latex-treated diet disk, whereas the beetles started feeding on the Tall Guzmaine latex-treated diet disk whenever they approached it. In latex no-choice tests, beetle s generally returned to the roof and walls of the container after approaching several times the Valmaine latex-treated diet disks. The behavior of the beetles on diet treated with water:methanol (20:80) extracts of Valmaine latex was similar to that for diet treated with pure Valmaine latex. Before biting a latextreated diet disk, beetles inspected it at a close range, antennating and palpating it. In cases where the beetles landed directly on a di sk, they appeared to sense the deterrent with their tarsi, even before antennating a nd palpating the disk, a nd flew back to the container walls immediately. Beetles performe d frequent and more vigorous grooming of antennae and tarsi by passing th em through mouthparts after c ontact with Valmaine latex compared to Tall Guzmaine latex. Further ta rsal grooming was also done by scraping the legs on the elytra. Beetles salivated more when Valmaine latex was applied to their mouthparts with a microdispenser compared to Tall Guzmaine latex. Mandibles and maxillae were not gummed up and were moving freely 24 h after application of either Valmaine or Tall Guzmaine latex, but there were some traces of dried latex on the labium and tarsi. During test bites on a lettuce leaf and contact with the exuded latex, the beetles moved away from the feeding site very quickly and star ted test bites somewhere else. The response was very vigorous on Valmaine. On Tall Guzm aine, beetles resumed test bites in close

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84 proximity to the previous bites, but on Valm aine tests bites were much farther away from the previous bites. Discussion Evidence presented here i ndicates that resistance found in Valmaine romaine lettuce against D balteata is due to latex. Adult D balteata were found more frequently on diets treated with latex from Tall Guzmaine than on diets treated with Valmaine latex in both choice and no-choice tests. The alighti ng behavior of the beetles observed in my choice and no-choice tests suggests that c ontact chemosensilla are present on their antennae, palps and tarsi (Chapman 2003). These types of chemosensilla have been reported in other chrysomelids, such as on th e antennae of the cabbage stem flea beetle, Psylliodes chrysocephala L. (Isidoro et al. 1998), maxillary appendages of the western corn rootworm, D. virgifera virgifera LeConte (Chyb et al. 1995, Eichenseer and Mullin 1996), and tarsomeres of the Klamath beetle, Chrysolina brunsvicensis Gravenhorst (Rees 1969). Such chemsensilla have been found to discriminate between phagostimulants and phagodeterrents. Antennal and tarsal grooming, similar to that observed by us with D. balteata has been reported in the crucifer flea beetle, Phyllotreta cruciferae Goeze as an important part of the pr efeeding behavior for recognizing host and non-host crucifers (Henderson et al. 2004). Adult D balteata consumed significantly less Valmaine latex-treated diet compared to Tall Guzmaine latex-treated diet in both choice and no-choice tests. Huang et al. (2003b) reported that latex from both Valmaine a nd Tall Guzmaine was very deterrent to beetle feeding when applied on lima bean leaves. I believe that Tall Guzmaine latex in the studies of Huang et al (2003b) showed very high deterrence due to changes in its chemical propert ies (possibly oxidati on) after drying on the lima bean leaf

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85 surface. In my studies, the precisely defined quantities of latex (70 l) applied to diet disks did not dry significantly within the 16-h exposure period to beetles due to moisture from the artificial diet, which perhaps preven ted changes in the chemical properties of the latex. Both cultivars produce latex upon w ounding but the much higher coefficient of deterrence for Valmaine latex compared to Tall Guzmaine latex observed in my study argues that Valmaine latex is more deterr ent than Tall Guzmaine due to its physical or/and chemical properties. These propertie s may be due to the original chemicals produced by the plants or new chemical s produced by the acti on of certain plant enzymes, such as phenylalanine ammonia lyase, polyphenol oxida se and peroxidase. Valmaine also partially or totally lost its resistance in feeding bi oassays using detached leaves or leaf disks, where latex exudation was greatly reduced (Huang et al. 2003c). This further provided evidence about the defensiv e role of latex in resistant Valmaine. The strong deterrent activity of Valmaine latex extracted with water:methanol (20:80) provides compelling evidence for the chemical basis of resistance in Valmaine against D. balteata The ability of water:methanol (20:80) to extract deterrent constituents from Valmaine latex suggests that moderately polar compounds in Valmaine latex account for its feeding deterrence. The defensive role of latex due to it chemical constituents against insects ha s been reported in many plant systems, such as milkweed (Dussourd and Hoyle 2000), mulberry (Konno et al. 2006), papaya (Konno et al. 2004), and chicory (Rees and Harborne 1985). Va rious organic compounds, such as phenolics and terpenoids have been reported in latex of Lactuca spp. (Crosby 1963, Gonzalez 1977, Cole 1984, Sessa et al. 2000), and their defensive role as phytoalexins has been reported against plant diseases (Bennett et al. 1994, Bestwick et al. 1995).

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86 Latex from young leaves of Valmaine strongly deterred the feeding of D balteata adults in a choice between diet s treated with latex from young and mature leaves. Sethi et al. (2006) found that latex from young and matu re leaves differed in terms of milkiness and viscosity. Such difference s in milkiness arise due to differences in the refractive indices of the dispersing part icles (mainly terpenoids) and the dispersing medium (Esau 1965, Fahn 1990). Thus, latex from young leaves may be richer in dispersing particles, and the relatively higher amount of disper sing particles may have a specific purpose related to plant defense. Young leaves are ty pically better defended than mature leaves due to the presence of higher quantities of la tex and its associated chemical components (Swain 1977, Spilatro and Mahlberg 1986). In the chicory plant, Cichorium intybus L., sesquiterpene lactones were present in the hi ghest amounts in the most actively growing regions of the plant and possesse d antifeedant properties against Schistocerca gregaria (Orthoptera: Acrididae) (Rees and Har borne 1985). Young vines of sweetpotato, Ipomoea batatas (L.) Lam., possessed more latex and exhi bited less damage due to the sweetpotato weevil, Cylas formicarius (F.) (Coleoptera: Curculionidae) than mature vines (Data et al. 1996). Latex exudation is higher in growing re gions than in mature regions of great bindweed, Calystegia silvatica (Kit.) Griesb (Condon and Fineran 1989). Anatomy of laticifers changes during the course of their ontogeny (Olson et al. 1969). The number of laticifers and their conten ts decrease with increasing proximity to roots (Condon and Fineran 1989, Monacelli et al 2005). In mature leaves, the protoplast of laticifers degenerates near senescence indicating a low level of metabolism (Fineran 1982, 1983). Fusion of latex particles has also been seen in mature leaves when much of the latex has already vanished (Dickens on 1963, Heinrich 1967, Fineran 1982). Plug-like

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87 masses of callose have been reported at th e bases of mature leaves and no or much reduced amounts of latex exude when such l eaves are severed, completely or partially, from the plant (Spencer 1939). Young leaves have discrete files of la ticifers separated by end walls, while laticifers differentiate by breakdown of end walls in mature leaves (Condon and Fineran 1989). Thus, laticifers of young leaves may have more turgor pressure resulting in profuse latex exudation compared to mature leaves, making it more likely that insect mandibles will be exposed to latex during test bites on intact leaves. My data support a hypothesis that latex ha s a definite role in the expression of resistance in Valmaine lettuce to D. balteata and differences in latex chemistry between the two cultivars may account for this. Futu re research on the isolation of latex constituents and their biological activity is required to better u nderstand the mechanism of resistance in Valmaine lettuce. This knowledge may contribute to the development of new cultivars expressing insect resistance along with superior horticultural traits through conventional and transgenic approaches.

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88 Figure 3-1. Wounding of lettuce releas es a milky fluid called latex.

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89 Figure 3-2. Colony rearing of D. balteata See text for description of eac h stage of colony maintenance.

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90 Figure 3-3. Collection of latex from romaine lett uce, application on artificial diet disk and bioassay setup.

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91 Figure 3-4. Scheme of latex solvent extraction.

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92 Figure 3-5. Latex dissolution in different solvents. A) La tex dissolved in different solvents, B) pellet settled down afte r centrifugation, and C) supernatant collected after centrifugation. A B C

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93 Figure 3-6. Feeding bioassays us ing fresh latex. A) Choice test s: Valmaine (Val) versus Tall Guzmaine (TG), Valmaine versus control, Tall Guzmaine versus control. B) No-choice tests: Valmaine (Val ), Tall Guzmaine (TG), control. Choice tests No-choice test B A

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94 A C B c b c b a a b b ab a a ab ab ab 15306090 Number of insects feeding / disk 0 1 2 3 4 5 6 Val TG Time after release (min) 15306090 TG Control 15306090 Val Control c b c b c b a cc c Figure 3-7. Mean number of D balteata adults feeding on artificial diet disks treated with latex from re sistant Valmaine (Val), disks treated with latex from susceptible Tall Gu zmaine (TG), and control diet disks in choice tests. Error bars indicate SEM. Bars topped with different letters wi thin same panel differ significantly at the 0.05 level (Tukeys HSD test).

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95 Time after release (min) 15306090 Number of insects feeding / 2 disks 0 1 2 3 4 5 6 Val TG Control c b ab c b ab ab a a a c c Figure 3-8. Mean number of D balteata adults feeding on two artif icial diet disks treated with latex from resistant Valmaine (V al), disks treated with latex from susceptible Tall Guzmaine (TG), and cont rol diet disks in no-choice tests. Error bars indicate SEM. Bars topped with different letters differ significantly at the 0.05 level (Tukeys HSD test).

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96 Figure 3-9. Choice tests using D. balteata adults on two artificial diet disks treated with latex from young and mature leaves of the same cultivar.

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97 Time after release (min) A B 15306090 Number of insects feeding / disk 0 1 2 3 4 5 6 Val-Young Val-Mature 15306090 TG-Young TG-Mature d cd c b d a e de cd abc a bc ab abc dd Figure 3-10 Number of D balteata adults feeding on artificial diet disks treated with latex from young or mature leaves of resistant Valmaine (Val) (A) and susceptible Tall Guzmaine (TG) (B) in c hoice tests. Error bars indicate SEM. Bars topped with different letters within same panel differ significantly at the 0.05 level (Tukeys HSD test).

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98 Figure 3-11. No-choice tests using D. balteata adults when both the disks were smeared with either Valmaine latex extract or Tall Guzmaine latex extract. W Water, M Methanol, MeCl Methylene chloride. Re d circle indicates the most deterrent extract comparable to fresh latex.

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99 TG-latex TG-Water TG-W:M (50:50) TG-W:M (20:80) TG-MeCl Number of insects feeding / 2 disks 0123456 Val-latex Val-Water Val-W:M (50:50) Val-W:M (20:80) Val-MeCl 0123456 Control Water W:M (50:50) W:M (20:80) MeCl D abc abc abc a ab abc abc c abc abc bc d bc abc dA ab a ab abCab ab ab ab ab ab b c ab ab c 0123456 Val-latex Val-Water Val-W:M (50:50) Val-W:M (20:80) Val-MeCl TG-latex TG-Water TG-W:M (50:50) TG-W:M (20:80) TG-MeCl ab ab ab ab ab Control Water W:M (50:50) W:M (20:80) MeCl ab ab ab a ab ab b ab B 0123456 ab ab ab a a ab ab ab ab ab ab c ab b c c c Figure 3-12. Mean number of D balteata adults feeding on two artificial diet disks treated with latex extracts from resi stant Valmaine (Val) and susceptible Tall Guzmaine (TG), and controls in no-choice test. Error bars indicate SEM. Bars topped with different letter s within same panel (A, B, C, D) differ significantly at the 0.05 level (T ukeys HSD test). A) 15 min, B) 30 min, C) 60 min, and D) 90 min.

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100 Control Water W:M (50:50) W:M (20:80) MeCl TG-latex TG-water TG-W:M (50:50) TG-W:M (20:80) TG-MeCl Diet consumed (mg) 0102030405060 Val-latex Val-water Val-W:M (50:50) Val-W:M (20:80) Val-MeCl a a a ab a c cd c cd b cd f cd de ef Figure 3-13. Dry weight of diet consumed by six D. balteata adults in 16 h when both diet disks were treated with Valmaine (Val) or Tall Guzmaine (TG) latex extracts under no-choice situations. Error bars indicate SEM. Bars topped with different letters differ significa ntly at the 0.05 level (Tukeys HSD test).

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101 Table 3-1. Dry weight consumption of diet disks treated with Va lmaine (Val) or Tall Guzmaine (TG) latex under choice and no-choice tests by six D. balteata adults in 16 h. Mean diet consumed SEM (mg) Treatment Tests Val latex TG latex Control P value Choice* Val vs. TG 5.4 0.5 15.5 0.7 0.0001 Val vs. Control 5.5 0.5 24.7 0.5 0.0001 TG vs. Control 14.4 0.5 21.9 0.6 0.0001 No-Choice 7.3 0.4c 34.6 1.1b 46.2 2.4a 0.0001 P value from paired t -test. Means SEM followed by differe nt letters in no-choice test differed significantly ( P 0.05) using ANOVA ( F = 168.31; df = 2, 42; P = 0.0001) and Tukeys HSD test.

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102 Table 3-2. Feeding deterrent activity of latex against D. balteata adults when artificial diet disks were treated with latex from either resistant Valmaine (Val) or susceptible Tall Guzmaine (TG) in choice and no-choice tests. Deterrence coefficients Latex Relative Absolute Total Val 63.6 72.7 136.3 TG 20.7 14.4 35.0 P value 0.0001 0.0001 0.0001 P value from two sample t -test.

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103 Table 3-3. Dry weight of diet consumed by six D. balteata adults in 16 h when given a choice between diet disks treated with latex from either young or mature leaves of resistant Valmaine or susceptible Tall Guzmaine lettuce cultivars. Cultivar Choice Diet consumed (mg)^ Total diet consumed (mg) Valmaine Young latex-treated diet vs. mature latex-treated diet 3.7 0.6b 26.1 1.9a 29.8 2.2 b Tall Guzmaine Young latex-treated diet vs. mature latex-treated diet 18.1 2.0a 20.5 2.2a 38.7 3.9ab Control* 50.1 5.2a *Both disks were untreated in control diet. ^ Means SEM followed by different letters within cultivar differed significantly using paired t -test. Means SEM followed by different letters within column differed significantly ( P 0.05) using ANOVA ( F = 6.69; df = 2, 42; P = 0.0030) and Tukeys HSD test.

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104 CHAPTER 4 BANDED CUCUMBER BEETLE (COL EOPTERA: CHRYSOMELIDAE) RESISTANCE IN ROMAINE LETTUCE: UNDERSTANDING LATEX CHEMISTRY Introduction Host plant resistance is an important poten tial component of any integrated pest management program for a crop pest. Many pl ants produce compounds that mediate host plant suitability to insect herbivores (Ros enthal and Berenbaum 1991). These biologically active compounds are frequently present in visc ous secretions, such as latex or resin, within specialized canal systems separate from the vascular system (Fahn 1979, Metcalf and Chalk 1983, Farrell et al 1991). Thus, insect mouthpa rts get exposed to these compounds during test bites due to copious flow of latex at the damage site (Farrell et al. 1991). The common components of latex are pol yisoprene, proteins, amino acids, fatty acids, tetracyclic triterpenoids glycerides, waxes, starch, flavonoids, alkaloids, water, organic and inorganic salts and many uni dentified compounds (Nielson et al. 1977, Spilatro and Mahlberg 1986, Gazeley et al 1988). Examples of some compounds found in latex with activity against different insect pests include diterpenes (Evans and Schmidt 1976, Noack et al. 1980) and nonprotein amino acids in Euphorbia (Haupt 1976), cardenolides in milkweed (S eiber et al. 1982, Nishio et al. 1983), alkaloids in poppy (Roberts 1987, Matile 1976) a nd mulberry (Konno et al. 2006), sesquiterpene lactones in chicory (Rees and Harborne 1985) and cysteine proteases in papaya and fig (Konno et al. 2004). However, latex of most of the lati ciferous species within the Apocynaceae, Compositae, Euphorbiaceae, Musaceae, Pa paveraceae, and Urticaceae has not been chemically characterized. A cultivar of romaine lettuce ( Lactuca sativa L.), Valmaine, possesses insect resistance against leafminer, Liriomyza trifolii (Burgess) (Nuessly and Nagata 1994),

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105 banded cucumber beetle, Diabrotica balteata LeConte (Huang et al. 2002) and two lepidopterans, Trichoplusia ni (Hbner) and Spodoptera exigua (Hbner) (Chapter 2, Sethi et al. 2006). Latex from Valmaine appl ied to artificial diet deters feeding of D. balteata Further, a crude extract prepared by dissolving Valmaine latex in a water:methanol mixture (20:80, % v/v) also strongly inhibits beetle feeding when applied to the surface of artificial diet (Chapter 3, Sethi et al. 2007) This suggests that Valmaine latex contains deterrent co mpounds which are responsible for its resistance against multiple species of mandibulate insects. Here, I describe the isolation and char acterization of deterrent compounds from Valmaine latex against D. balteata adults using bioassaydirected fractionation. Materials and Methods Plants and Insects Seeds of the romaine lettuce cultivar, Va lmaine were germinated by putting them on moist filter paper in a Petri dish. Germin ated seeds were planted in soil-less media (MetroMix 220 potting mixture, Grace Sierra, M ilpitas, CA) and healthy seedlings were transplanted into 15-cm-diameter plastic po ts after 2 wk in a greenhouse with natural light at a mean temperature of 27 C (22 to 30C) and 68% mean R.H. (48 to 93%). Plants were fertilized with 10 ml of a 10 g/l solution of Peters 20-20-20 (N-P-K) (W.R. Grace, Fogelsville, PA) once a week. Bush lima bean ( Phaseolus lunatus L.) plants of the Fordhook 242 cultivar (Illin ois Foundation Seeds, Champagne, IL) were grown in transplant trays and fertilized with th e same solution used for lettuce plants. A wild population of D. balteata adults was collected from spiny amaranth, Amaranthus spinosus L. and primrose willow, Ludwigia peruviana L. in Belle Glade, FL in 2003. A colony was established by raising ad ults on lima bean leaves and slices of

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106 sweet potato tubers, and larvae were fed on corn seedling roots as per the methods of Huang et al. (2002) (Chapter 3). Wild indi viduals were added to the colony in 2005 and 2006 to increase genetic diversity. Unfed adults that had emerged within 48 h of the start of the experiment were used in all bioassays. Assay for Feeding Deterrence Extracts/fractions from latex obtained as described below were bioassayed on artificial diet for f eeding deterrence towards D. balteata adults under no-choice conditions. The southern corn rootworm artif icial diet (Bio-Serv, Frenchtown, NJ), and disks of diet for use in the assays, were prep ared as described in Ch apter 3 (Sethi et al. 2007). An experimental setup consisted of two diet disks placed on the bottom of a plastic container (10 10 8 cm) with screen lid and three male-female pairs of beetles. Both diet disks in each container were treate d with the same kind of extract/fraction. The beetles were allowed to feed on the diet fo r 16 h. The experiments we re carried out at 25 1C in a laboratory under a photoperiod of 14:10 (L:D). In all bioassays, the number of adults f eeding on two diet disks was recorded 90 min after their release into the container. Th e dry weights of diet consumed in 16 h were also recorded. To compensate for the weight associated with moisture loss during the feeding tests, individual fres h weights of 10 diet disks were recorded before they were dried in an oven at 50 5oC. Individual dry weights of th ese disks were recorded after 3 d and an average dry/fresh weight ratio was calculated. The diet disks for bioassays were weighed prior to the bioassay setup. At the e nd of the experiment, the remaining portions of the disks were reweighed after drying for 3 d in the oven. The amount of dry weight of diet consumed was calculated as the differen ce between initial and fi nal dry weights. Dry

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107 weights of diet disks consumed by the beetles were computed by multiplying fresh weight by the average dr y/fresh weight ratio. Latex Collection and Crude Extract Preparation Cuts were made near the leaf-bases of young and middle-aged leaves of lettuce plants (9-10 true-leaf stage) using a disposable scalpel bl ade (Feather, Osaka, Japan). Fresh latex (70 l) was collected from each plant using a 100l silanized glass capillary tube inserted into a microdispenser (Dru mmond Scientific Company, Broomall, PA) and immediately dissolved in 10 volume of water:methanol (20:80) mixture. After dissolution, samples were centrifuged at 1200 g for 20 min and then the supernatant was collected. The supernatant (hereafter termed crude extract) was concentrated to 0.1 volume (the original latex volume) by eva poration under a gentle stream of nitrogen (Chapter 3, Sethi et al. 2007). Fractionation of Crude Extract Usin g Reversed-Phase (C-18) Cartridge Reversed phase separations involve a polar or moderately polar sample matrix (mobile phase) and a nonpolar stationary phase. The analyte of interest is moderatelyto non-polar. Alkyl bonded silica (C-18) is the mo st commonly used stationary phase in solid-phase extraction (SPE) (Hennion 1999). The crude extract was first fractionated using a C-18 cartridge (300 mg, Alltech A ssociates, Inc., IL) (Fig. 4-1). Prior to application of the crude extract, the C18 cartridge was pre-conditioned with 10 ml methanol and then with 10 ml water. The crude extract was percolated through the C-18 cartridge at a rate of appr oximately 1 drop per 1.5 s using positive pressure, and the unbound fraction was collected. After percolation of the crude extract, the cartridge with retained compounds was washed with 10 volumes of a stepwise gradient of water-

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108 methanol mixtures [water, water:methanol (80:20, % v/v), water:methanol (40:60), and water:methanol (5:95)] to elute retained compounds. After percolation of each watermethanol mixture, subsequent fractions were collected a nd each fraction concentrated back to 0.1 volume under a nitrogen stream. In reversed-phase SPE procedures using C-18 packing, trapping of the analyte is optimized by adjusting the pH of the conditioning solution or aqueous sample, or by adding a small percentage of organic solvent to the sample before percolation (Hennion 1999, Simpson 2000). Adjustment of the sample pH greatly enhances retention of ionizable compounds under their neutral form on C-18 packing by making them sufficiently hydrophobic (Pichon 2000). The sample pH can also be adjusted for sample clean-up so that unwanted compounds in the sample are retained on the SPE packing (Hennion 1999, Len-Gona alez and Prez-Arribas 2000, S impson 2000). Therefore, the above extractions were repeated separately using crude extrac t acidified or basified to three different pH, i.e., at original (6.5), aci dic (3.0) and alkaline (9.0) pH. Acidification and basification of crude ex tract was achieved by adding 1 N phosphoric acid and 1 N ammonium hydroxide, respectively. The crude extract, unbound fraction, four el uted fractions [water, water:methanol (80:20), water:methanol (40:60) and water:methanol (5:95)] and the combination of all four eluted fractions were a pplied to artificial diet disks for deterrence bioassays under no-choice conditions. An amount of each extrac t/fraction, equivalent to 70-l latex, was applied to each diet disk. For controls, five water-methanol mixture combinations without latex extract (including water:me thanol (20:80)) and untreated diet disks were used. Each

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109 experimental unit was replicated nine times fo r extracts at the original pH and six times each for extracts acidified to pH 3.0 or basified to pH 9.0. Fractionation of Crude Extract Using C18, SAX and SCX Cartridges Connected in Series Ion-exchange SPE is also a commonly us ed method for the extraction of charged compounds. Negatively (anionic) and positivel y (cationic) charged compounds can be isolated on anion exchange (SAX) and ca tion exchange (SCX) stationary phases, respectively. Subsequently, these charged co mpounds can be eluted using a solution of high ionic strength that disp laces the absorbed compounds (Hennion 1999). The crude extract at original pH was next frac tionated using C-18, SAX (functional group: quaternary ammonium, counter ion: acetate) and SCX (func tional group: sulphonic acid, counter ion: hydrogen) ca rtridges (Alltech Associates, Inc ., IL) connected in series (Fig. 4-2). Prior to crude extract ap plication, C-18 cartridges were pre-conditioned with 10 ml methanol and then with 10 ml water; SAX and SCX cartridges were pre-conditioned with 10 ml water. The samples were passed by pos itive pressure through the cartridges at a flow rate of approximately 1 drop per 1.5 s. The crude extract at or iginal pH (6.5) was percolated through a C-18 cartr idge and the unbound fraction was collected. Then, the C18 unbound fraction was percolated through SAX a nd SCX cartridges connected in series and the unbound fraction was collected. Afte r percolation of th e C-18 unbound fraction, SAX and SCX cartridges with retained compounds were washed separately with 10 volumes of a stepwise gradient of NaCl so lutions (0.1, 0.5 and 1 M) to elute retained compounds. After percolation of each NaCl solu tion, subsequent fractions were collected and concentrated back to 0.1 volume under a nitrogen stream.

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110 An amount of each extract/fraction, equivale nt to 70 l latex, was applied to each diet disk for use in the bioassays. Nine treatments were studied: crude extract; C-18 unbound fraction; SAX and SCX unbound fraction; and 0.1, 0.5, 1M NaCl fractions from each SAX and SCX cartridge. Controls consiste d of untreated diet disks, disks treated with water:methanol (20:80) mixture, a nd disks treated with 0.1, 0.5 or 1 M NaCl solutions. Each experimental unit was repl icated nine times. The 0.5 M-NaCl SCX fraction exhibited the strongest deterrent activ ity and was termed SCX fraction in the following LC/MS separations. LC/MS Separation of SCX Fraction LC/MS helps in the fractionation of a samp le with simultaneous characterization of chemical compounds. Fractionating increases th e sample simplicity and ultimately makes the characterization of the compounds much easier. The SCX fraction was further fractionated by LC/MS. A Thermo Finniga n LCQ Deca XP Max was used employing electrospray ionization (ESI) (s heath gas, 25 arbitrary units; sweep gas, 10 arbitrary units; spray voltage, 5.00 kV; capillary temperature 2 85C; and capillary voltage, 3.0 V) with the Thermo Separations spectra HPLC syst em (quaternary pump P4000, autosampler AS 3000, and diode array detector UV6000). Sepa rations were performed on a PLRP-S column (100 3 m, 150 4.6 mm, Polymer Laboratories. Ltd., UK) with solvent A (water with 10 mM ammonium formate) a nd solvent B (90 acetonitrile:10 water with 10mM ammonium formate, v:v) as mobile phases for 40 min. Elution was performed using two solvent gradient systems with co lumn temperature maintained at 60C and a flow rate of 1.0 ml/min. The first gradient elution system employing solvent A at pH 9.0 began with 95:5 percent (A and B) and reach ed 45:55 at 25 min, followed by gradient to

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111 0:100 in 5 min. The solvent was then kept at the final composition for 5 min.. The second gradient, with solvent A at pH 10, began with 100:0 percent (A and B) and reached 0:100 at 25 min. It was then kept at that com position for 10 min. UV absorption was monitored at 190 800 nm, and a low-volume micro needle valve split ter P450 (Upchurch Scientific, Oak Harbor, WA) was used to spl it the solvent flow between the UV detector and MS electrospray interface up to 90:10, maki ng it possible to collect 90% of the eluted material in one minute fractions for bioassa y while simultaneously obtain LC/MS spectra. In the first gradient elution system at pH 9.0, fractions collected each minute were recombined into six major fractions (Fig. 4-3) and concentrated to a volume equivalent to 70 l of latex to treat one diet disk. Th en, these six fractions (#0-3, #4-7, #8-11, #12-15, #16-20, and #21-40) and the combination of elut ed fractions were applied on the surface of artificial diet disks. Each experimental unit was replicated six times and each unit had two diet disks treated with same kind of fraction under no choice conditions. Untreated diet and diets treated with crude extract a nd SCX fraction were used for the controls. In the second gradient elution system at pH 10.0, fractions were collected each minute but only eleven fractions were used for bioassays under no-choice conditions (#2, #3, #4-6, #20, #21, #22, #23, #24, #25, #26, and #27). Cont rols consisted of untreated diet disks and disks treated with crude extr act and SCX fraction. Each experimental unit was replicated three times. Statistical Analysis In all no-choice tests, number of adults feeding on two diet disks 90 min after beetle release and the dry weights of diets consumed in 16 h were analyzed using Proc GLM (SAS Institute 2003) with latex fraction as a fixed effect a nd replications as a random effect. The error degree of freedom fo r latex fraction effect was calculated as

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112 (levels of latex fraction -1)(replications -1). Tukeys honestly significant difference (HSD) test with a significance level of = 0.05 (SAS Institute 2003) was used for post hoc means separation. Results Fractionation of Crude Ex tract Using C-18 Cartridge Water and water:methanol (40:60) fractio ns were light yellow and milky white, respectively; the 80:20 and 5:95 water:metha nol fractions were co lorless (Fig. 4-4). Fractionation at original pH. Fractionation of the crude ex tract at its original pH and subsequent bioassays indi cated that the unbound fraction had activity equivalent to that of the crude extract (Fig. 4-5). Latex fr action had significant e ffect on the number of insects feeding on diet disks ( F = 12.05; df = 12, 96; P = 0.0001). Fewer insects were counted 90 min after their release on diet disks treated with the unbound fraction than on disks treated with any other C-18 water-met hanol mixture fraction or on control diet disks (Fig. 4-6A). Latex fraction also signifi cantly affected diet consumption by beetles ( F = 39.40; df = 12, 96; P = 0.0001). Beetles consumed signifi cantly less diet treated with the unbound fraction than diet tr eated with any other water-me thanol mixture fraction or control diet (Fig. 4-7A). Fractionation of crude extract at pH 3.0. Fractionation of th e crude extract acidified to pH 3.0 on the C-18 cartridge and su bsequent bioassays revealed that some of the deterrent compounds were retained on th e C-18 resin. Latex fraction significantly affected the number of beetles feeding ( F = 5.03; df = 12, 60; P = 0.0001). Significantly more beetles were counted 90 min after thei r release on diets treated with the watermethanol mixture extracts compared to diet treated with the unbound fraction (Fig. 4-6B). Latex fraction also had significan t effect on diet consumption ( F = 11.49; df = 12, 60; P =

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113 0.0001). The unbound fraction was still deterrent to beetle feeding as diet consumption was significantly less on it, sim ilar to that on the cr ude extract. But, in addition, the water fraction also had some deterr ent activity (Fig. 4-7B). Fractionation of crude extract at pH 9.0. Bioassays of fractions obtained by passing the crude extract basi fied to pH 9.0 over C-18 cartr idge identified deterrent activity again in the unbound frac tion with some deterrent ac tivity in the water fraction. Latex fraction had significant effect on th e number of insects feeding on diet ( F = 4.08; df = 12, 60; P = 0.0001). After 90 min, the number of in sects feeding on diet treated with the unbound fraction did not diffe r significantly from the numb er feeding on diet treated with the crude extract (Fig. 4-6C). Latex fraction also affected diet consumption by beetles ( F = 4.57; df = 12, 60; P = 0.0001). Beetles consumed similar amounts of diet treated with the unbound fraction and the crude extract (Fig. 4-7C). Fractionation of Crude Extract Using C18, SAX and SCX Cartridges Connected in Series The 0.1M NaCl fraction eluted from the S AX cartridge was colorless, but the other two fractions (0.5 and 1M NaCl) were yellow (Fig. 4-8). All three fr actions eluted from the SCX cartridge were colorless. The deterrent activity of the 0.5 M NaCl fraction obtained from the SCX cartridge was similar to that of the crude extract (Fi g. 4-9). Latex fraction ha d significant effect on the number of insects feeding on diet ( F = 31.75; df = 13, 104; P = 0.0001). Significantly fewer insects were counted on the diet disk s treated with the 0.5 M NaCl fraction from either the SAX or SCX cartridge 90 min after their release (Fig. 4-10) compared to all other fractions. Application of latex fraction also significantly affected diet consumption by beetles ( F = 54.67; df = 13, 104; P = 0.0001). Beetles consumed significantly less diet

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114 treated with the 0.5 M NaCl frac tion eluted from the SCX cartrid ge than diet treated with any other fraction from the SAX or SCX cartr idges (Fig. 4-11). Beetles also consumed significantly less diet treated with the 0.5 M NaCl fraction from the SAX cartridge but not as little as they did on disk s treated with the crude extract. Fractionation of SCX Fraction Using LC/MS At pH 9.0 of solvent A. Application of latex fraction significantly affected both the number of beetles counted on diet disks and the amount that they consumed (number of beetles on disks: F = 18.78; df = 9, 45; P = 0.0001; consumption: F = 88.34; df = 9, 45; P = 0.0001). Fewer beetles were counted on and c onsumed less of the diet disks treated with the crude extract, the SCX fraction, LC /MS fractions #0-3, fractions #21-40 as well as the combination of all LC/MS fractions (Figs. 4-12, 4-13). Some weak feeding deterrent activity was also found in the #4-7 fraction. At pH 10.0 of solvent A. Latex fraction treatment has significant effect on the number of insects feeding on diet ( F = 11.92; df = 13, 26; P = 0.0001). Diets treated with fraction #3 were as deterrent to feeding as were disks treated with either the crude extract or the SCX fraction (Fig. 4-14). Diet cons umption by beetles was also significantly affected due the treatment of latex fractions ( F = 26.74; df = 13, 26; P = 0.0001). Consumption was the lowest on the diet disk s treated with the crude extract, the SCX fraction and the #3 fraction (Fig. 4-15). This fraction was estimate d to contain about 10 peaks based on UV absorption (190 450 nm) and M+1 ions produced when analyzed using positive ion electrospray LC/MS (Fig. 4-16). Discussion The deterrent activity of the unbound fracti on of the reversed-phase extraction at the original pH indicates th at the deterrents compounds were not retained on C-18 resin.

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115 Reversed phase extractions using C-18 involve a polar or moderately polar sample matrix (mobile phase) and a nonpolar stationary phase. The analytes of interest retained on the cartridge are moderatelyto non-polar. So, this indicates th at the deterrent compounds in the crude extract are highly polar. Many biologically active compounds are known to exist in their glycosidic form. By binding to sugars, the toxicity of these compounds is reduced and their solubility is increased which facilitates th eir storage in large amounts. These compounds become more act ive after coming in contact with specific degradation enzymes (Harborne 1979, Schoonhoven et al. 2005). In both lettuce and chicory ( Chicorium intybus L.), most of the sesquiterpenes ar e found in glycosidic form and the bitterness of the plants is a ssociated with them (Price et al. 1990). Tamaki et al. (1995) also reported that 44, 34 and 56% of sesqui terpene lactones were in their bound form in the wild lettuce species, L. saligna and L. virosa and cultivated lettuce, respectively. These sesquiterpenes exhibited low retent ion on C-18 cartridges (Schenck 1966, Tamaki et al. 1995). Phenolic glycosides found in white grub-infested sugarcane (Nutt et al. 2004) and in white lupin ( Lupinus albus L.) (Stobiecki et al 1997) also exhibited low retention due to their high polar ity and solubility in water. The crude extract was fractionated at two ex treme pH levels with the intention of better retaining deterren t compounds with either acidic or alkaline characte ristic. In my study, diet consumption data indicate that so me of the deterrent compounds in the crude extract were retained on the C-18 packing both at acidic and basic pH. Some of the compounds with deterrent activity were eluted by water at both pH levels, but also by water:methanol (40:60) at basic pH.

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116 Ion exchange solid-phase extraction is commonly used for the extraction of compounds that are charged when in an a queous solution. In my study, the deterrent compounds were retained on the SCX packi ng after percolation of the unbound fraction from the C-18 cartridge, and were eluted with 0.5 M NaCl solution. Retention of deterrent compounds on SCX suggests a basic nature for the compounds. During the fractionation of the SCX fracti on using LC/MS with a mobile phase at pH 9.0, the deterrent activity was found in the very early fractions, between 0 and 3 min, indicating that this pH was not high enough to fully deprot onate a basic column, or that the early elution could be due to additiona l polar constituents of the molecule, for example sugars. Some deterrent activity wa s also found in the later fraction eluting between 21 and 40 min which might indicate the aglycon form of an earlier eluting glycosidic compound. When the pH of the mobile phase was raised to 10.0 and the gradient elution system slightly changed to accommodate very polar compounds the deterrent activity was retained on the colu mn and only found in the fraction eluting between 3 and 4 min and not in the later frac tions. The change in pH appears to have neutralized very basic compounds, and ultimat ely resulting in th eir retention on the column. However, the loss of activity in the later elutin fraction can for the moment not be easily explained. Based on UV absorption and MS data, ther e are more than ten compounds present in the fraction between 3 and 4 min, some of these compounds having substituted aromatic group characteristics. Substituted aromatic compounds previously were reported in lettuce, such as sesquiterpene lacton es (lactucin, molecu lar weight 276; and lactucopicrin, molecular weight 410) (Sessa et al. 2000) (Fig. 4-17) and flavonoids

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117 (flavonol glycosides, flavone glycoside and anthocyanidin glycosides) (Dupont et al 2000) (Fig. 4-18). But their biological activity against insects has not been reported in lettuce. However, sesquiterpene lactones provide resistance against lettuce downy mildew and the red spot physiological disorder in certain lettuce cultivars due to its strong antimicrobial properties (Bennett et al 1994, Bestwick et al. 1995). Sesquiterpene lactones play an antifeedant role in the closely related plant species chicory against Schistocerca gregaria (Forsk.) (Rees and Harborne 1985). The successful isolation of potent feeding deterrents for banded cucumber beetle from a crude extract of romaine lettuce latex provides convincing evidence of a chemical basis for host plant resistance in this variety. Deterrent co mpounds can be extracted using reversed-phase and cation exchange cartridges (SCX) linked in series, and their retention on cation exchange indicates th at they are basic. In addi tion, LC/MS analysis indicates the presence of substituted aromatic co mpounds. The chemical composition of the fraction between 3 to 4 min is being investig ated. Understanding th e defensive role of latex and its deterrent constituents (apart fr om physical defense due to stickiness) will help to better comprehend the mechanisms of insect-plant interactions. Furthermore, qualitative and quantitative knowledge of th ese biologically active compounds may help plant breeders select for genotypes with an inhe rently high level of re sistance using these compounds as markers. Insect-susceptible but ot herwise horticulturally superior cultivars could also be made more resi stant through genetic engineering.

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118 Figure 4-1. Scheme for solid-phase extracti on and fractionation of crude extract after passing through reversed-pha se (C-18) cartridge.

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119 Figure 4-2. Scheme for solid-phase extracti on and fractionation of crude extract after passing through reversed-phase (C18), anion (SAX) and cation (SCX) exchange cartridges connected in series.

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120 Figure 4-3. Fractions obtained after HPLC analysis of cati on exchange (SCX) fraction. RT: 0.00 39.98 SM: 5B 0 5 10 15 20 25 30 35 Time (min) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100Relative Abundance 10.16 9.42 11.32 7.56 7.30 12.38 12.69 5.91 5.52 14.58 4.67 15.91 18.12 27.09 23.38 36.42 31.11 1.92 #0-3 #4-7 #8-11 #12-15 #16-20 #21-40

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121 Figure 4-4. Color characteristics of frac tions obtained after passing crude extrac t through reversed phase C-18 cartridge.

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122 Figure 4-5. Bioassays of C-18 fractions a pplied on artificial diet disks using D. balteata adults under no-choice conditions.

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123 C18 fractions A)B)C) Crude extract 0123456 Combined (All 4) W:M (5:95) W:M (40:60) W:M (80:20) W Unbound fraction W:M (5:95) W:M (40:60) W:M (80:20) W W:M (20:80) Untreated a a a a a a b b a a a a a 0123456 a a a a a a b b a a a a a Number of insects feeding / 2 disks Controls 0123456 ab ab ab a ab a c bc abc ab ab ab abc Figure 4-6. Mean number of D balteata adults feeding after 90 min on tw o artificial diet disks treated with fractions obtained after passing crude extract at three pH levels through C-18 cartridge: A) original (pH 6.5), B) acidic (pH 3.0), and C) basic (pH 9.0). Error bars indicate SEM. Bars topped with different letters within same panel (A, B or C) differ significantly at the 0.05 level (Tukeys HSD test). (W Water, M Methanol).

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124 A)B)C) Diet consumed (mg)d 0102030405060 ab ab ab ab ab ab d cd bc ab a ab ab d 0102030405060 a a a a a ab c bc abc a ab a abc C18 fractions Controls Crude extract 0102030405060 Combined (All 4) W:M (5:95) W:M (40:60) W:M (80:20) W Unbound fraction W:M (5:95) W:M (40:60) W:M (80:20) W W:M (20:80) Untreated a a a a a a b b a a a a a Figure 4-7. Dry weight of diet consumed by D. balteata adults when disks were treated with fractions obtained after passing crude extract with different pH levels thro ugh C-18 cartridge: A) origin al (pH 6.5), B) acidic (pH 3.0), and C) basic (pH 9.0). Error bars indicate SEM. Bars topped with different letters within same panel (A B or C) differ signi ficantly at the 0.05 level (Tukeys HSD test). (W Water, M Methanol).

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125 Figure 4-8. Color characteristics of frac tions obtained after passing C-18 unbound frac tion through anion (SAX) and cation (SCX ) exchange cartridges connected in series.

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126 Figure 4-9. Bioassays of ion-exchange fractio ns applied on artificia l diet disks using D. balteata adults under no-choice conditions.Anion Exchange (SAX) Fractions Cation Exchange (SCX) Fractions

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127 SCXNumber of insects feeding / 2 disks 0123456 Unbound fraction 1 M NaCl 0.5M NaCl 0.1M NaCl 1 M NaCl 0.5M NaCl 0.1M NaCl 1 M NaCl 0.5M NaCl 0.1M NaCl W:M (20:80) Untreated C18 unbound fraction Crude extract SAX Controlsa a a a a b b a b a a b a a Figure 4-10. Mean number of D. balteata adults feeding after 90 min on diet disks treated with ion-exchange fractions obtained by passing C-18 unbound fraction (original pH 6.5) through anion (SAX) and cation (SAX) exchange cartridges connected in se ries. Error bars indicate SEM. Bars topped with different letters differ significantly at the 0.05 level (Tukeys HSD test).

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128 Figure 4-11. Dry weight of diet consumed by D. balteata adults when disks were treated with ion-exchange fractions obtai ned after passing C-18 unbound fraction (original pH 6.5) through anion (SAX) and cation (SAX) exchange cartridges connected in se ries. Error bars indicate SEM. Bars topped with different letters differ significantly at the 0.05 level (Tukeys HSD test). Diet consumed (mg) 0102030405060 Unbound fraction 1M NaCl 0.5M NaCl 0.1M NaCl 1M NaCl 0.5M NaCl 0.1M NaCl 1M NaCl 0.5M NaCl 0.1M NaCl W:M (20:80) Untreated C18 unbound fraction Crude extract SCX SAX Controlsa a a a a c c a b a a c a a

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129 SCX fraction Crude extract Untreated 0123456 Combined (all 6) # 21-40 # 16-20 # 12-15 # 8-11 # 4-7 # 0-3 a c c bc ab a a a c c Number of insects feeding / 2 disks Figure 4-12. Mean number of insects feedi ng after 90 min on diet disks treated with fractions obtained after LC/MS analys is of cation exchange fraction (SCX) at pH 9.0 of the mobile phase. Error ba rs indicate SEM. Bars topped with different letters differ significantly at the 0.05 level (Tukeys HSD test).

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130 SCX fraction Crude extract Untreated Diet consumed (mg) 0102030405060 Combined (all 6) # 21-40 # 16-20 # 12-15 # 8-11 # 4-7 # 0-3 ab de e de c ab a b d e Figure 4-13. Dry weight of diet consumed by D. balteata adults when disks were treated with fractions obtained af ter LC/MS analysis of cation exchange fraction (SCX) at pH 9.0 of the mobile phase. Error bars indicate SEM. Bars topped with different letters differ significa ntly at the 0.05 level (Tukeys HSD test).

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131 SCX fraction Crude extract Untreated 0123456 # 27 # 26 # 25 # 24 # 23 # 22 # 21 # 20 # 4-6 # 3 # 2 a b b a b a a a a a a a a a Number of insects feeding / 2 disks Figure 4-14. Mean number of insects feedi ng after 90 min on diet disks treated with fractions obtained after LC/MS analys is of cation exchange fraction (SCX) at pH 10.0 of the mobile phase. Error ba rs indicate SEM. Bars topped with different letters differ significantly at the 0.05 level (Tukeys HSD test).

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132 SCX fraction Crude extract Untreated Diet consumed (mg) 0102030405060 # 27 # 26 # 25 # 24 # 23 # 22 # 21 # 20 # 4-6 # 3 # 2 ab c c b c ab ab ab ab ab ab ab a ab Figure 4-15. Dry weight of diet consumed by D. balteata adults when disks were treated with fractions obtained af ter LC/MS analysis of cation exchange fraction (SCX) at pH 10.0 of the mobile phase. E rror bars indicate SEM. Bars topped with different letters differ significa ntly at the 0.05 level (Tukeys HSD test).

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133 Figure 4-16. Electrospray LC/MS to tal negative ion trace of activ e fraction between 3 and 4 min.

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134 Figure 4-17. Structure of sesquiterpene lact ones characterized in lettuce (Sessa et al. 2000).

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135 Figure 4-18. Chemical structures of flavonoi ds found in lettuce A) flavonol glycosides; B) flavone glycoside; and C) anthocyanidi n glycosides (kaempferol if R1 = H; quercetin if R1 = OH) (R = glycoside) (Dupont et al. 2000). A B C

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136 CHAPTER 5 INVESTIGATING ENZYME INDUCTION AS A POSSIBLE REASON FOR LATEXMEDIATED INSECT RESISTANCE IN ROMAINE LETTUCE Introduction Lettuce ( Lactuca sativa L.) is one of the most im portant vegetable crops grown throughout the world (Ryder 1998). Lettuce grow ers suffer huge economic losses due to various insect pest infestations because of the very high cosmetic standards demanded by consumers (Palumbo et al. 2006). The romaine lettuce cultivar, Val maine exhibits a high level of resistance against vari ous insects, including the leafminer, Liriomyza trifolii (Burgess) (Nuessly and Nagata 1994), banded cucumber beetle, Diabrotica balteata LeConte (Huang et al. 2002) (Fi g. 5-1), and two lepidopterans, Trichoplusia ni (Hbner) and Spodoptera exigua (Hbner) (Chapter 2, Sethi et al 2006). Valmaines resistance would be useful in an integrated pest mana gement program however this cultivar is not popular among growers because of its suscep tibility to thermodormancy, premature bolting, lettuce mosaic virus and corky root rot (Guzman 1986). Plant breeders have attempted to improve the horti cultural characteristics of Va lmaine through breeding, but unfortunately the horticulturally improved and currently used cultivar, Tall Guzmaine lost resistance to insects during the pr ocess (Chapter 2, Se thi et al. 2006). My previous research revealed that Valmai ne latex placed on arti ficial diet deterred D. balteata feeding, whereas latex from Tall Guzmai ne did not (Chapter 3, Sethi et al. 2007). I hypothesize that feeding deterrence due to constitutive leve ls of compounds in latex may explain the mechanism of multiple insect resistance in Valmaine. Furthermore, previously wounded Valmaine plants showed an increased localized resistance to feeding by D. balteata compared to unwounded plants, suggesting the involvement of an

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137 inducible mechanism of resistance (Huang et al. 2003b). Tall Guzmaine showed no such inducible resistance. Latex is an aqueous suspension or solu tion of complex mixtures of molecules found in specialized secretory cells of plants known as laticifers (Evert 2006). Laticifers possess high metabolic activity. In addition to synthesizing nu merous molecules (lipids, sugars and proteins) required to achieve thei r basic physiological functions, laticifers are also known to synthesize and store diverse se condary metabolites in appreciable amounts in latex (Moussaoui et al. 2001). Many defens ive compounds with demonstrated negative impact on insect fitness are stored in latex (Evans and Schmidt 1976, Haupt 1976, Matile 1976, Noack et al. 1980, Seiber et al. 1982, Nish io et al. 1983, Rees and Harborne 1985, Roberts 1987, Konno et al. 2004, 2006; Ramos et al. 2007). Activity of phenylalanine ammonia lyase, polyphenol oxidase and many other defense-related enzymes is much higher in the laticifers than in the leaves of rubber tree ( Hevea brasiliensis H.B.K.) (Broekaert et al. 1990, Kush et al. 1990, Ma rtin 1991, Gidrol et al. 1994, Pujade-Renaud et al. 1994, Wititsuwannakul et al 2002). Wounding of laticifers is also known to induce other defense-related enzymes in latex of papayas (Azarkana et al. 2004, Kydt et al. 2007), fig tree ( Ficus carica L.) (Kim et al. 2003, Taira et al 2005), rooster tree ( Calotropis procera Ait.) (Freitas et al. 2007) and Albanian spurge ( Euphorbia characias L.) (Mura et al. 2005, 2007; Fiorillo et al. 2007). Thus plant latex acts as a chemical defense due to alteration in its constituents upon insect damage. The purpose of this study was to investigat e the role of inducible enzymes in the latex-mediated multiple insect resistance in Valmaine. I asked the questions of whether enzyme activities changed after insect f eeding damage, how quickly this change

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138 occurred, how long the elevated levels laste d, and whether elevated enzyme activity was correlated with increased feeding deterrent ac tivity in latex. Hence, choice experiments were conducted with D. balteata adults between diets trea ted with latex from damaged and undamaged plants within the same cultiv ar (Valmaine and Tall Guzmaine) to look for changes in latex chemistry after beetle feeding. The inducti on of defense-related enzymes, in particular phenylalanine amm onia lyase, polyphenol oxi dase and peroxidase in latices of resistant Valmaine and suscep tible Tall Guzmaine was also compared with and without D. balteata feeding damage. Materials and Methods Plants The seeds of romaine lettuce cultivars Valmaine (resistant) and Tall Guzmaine (susceptible) were germinated overnight on moistened filter paper. The germinated seeds were planted in transplant trays filled with Metro Mix 200 (Grace Sierra, Milpitas, CA) and healthy seedlings were transplanted 2 wk later into 15-cm-diameter plastic pots. The plants were watered daily and fertilized with 15 ml of Peters 20-20-20 solution (W.R Grace, Fogelsville, PA) every week. Six-week-old lettuce pl ants (9-10 true-leaf stage) were used for the experiments. Bush lima bean seeds ( Phaseolus lunatus L.) of the cultivar Fordhook 242 (Illinois Foundation Seeds, Champagne, IL) were planted in transplant trays filled with Metro Mix 200 and fertilized with the same solution used for lettuce plants. Insects The colony of D. balteata was started from adults collected from weeds (spiny amaranth, Amaranthus spinosus L. and primrose willow, Ludwigia peruviana L.) in Belle Glade, Florida in 2003. New adults were a dded to the established colony in 2005 and

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139 2006 to increase genetic diversity. Larval st ages were reared on the roots of corn seedlings and adults were fed on lima bean le aves and sweet potato tubers (Chapter 3, Huang et al. 2002). Unfed adults, within 48 h of emergence, were used for the experiments. Artificial Diet Freshly-made southern corn rootworm ar tificial diet (Bio-Serv, Frenchtown, NJ) was used in all experiments. The diet wa s prepared according to methods previously described (Chapter 3, Sethi et al. 2007). One-cm-thick disks were punched out from cooled artificial diet using a 1.5-cm-diameter cork borer. Bioassay Conditions for Feeding Damage One hundred and eighty plants of each cu ltivar were placed individually in cylindrical screen cages (18.5 cm diameter 61.0 cm height) for use in collecting latex from plants after timed, continuous intervals of D. balteata feeding. Two male-female beetle pairs were placed into half (90 plants) of the cages of each cultivar, while the other 90 plants of each cultivar were used as unda maged checks. Beetles were allowed to feed on the plants for 6 d. Females were weighed individually before releasing them on the plants, and again at either 1, 3 or 6 d after they were released into the cages, to determine weight change. Latex was collected from plants 1, 3 and 6 d after they were released into the cages. Out of these 180 plants of each cul tivar, latex was collected from 60 plants (30 damaged and 30 undamaged checks) at each time interval (1, 3, and 6 d) after initiation of feeding damage. Out of each batch of 30 plants latex from 15 plants was used for diet disk choice tests and latex from the other 15 was used to assess enzyme activity, as explained below. Each group of 15 plants wa s further divided into 5 groups (replicates) of 3 plants for the collection of latex. An aloquot of 70 l of late x was collected from

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140 each group of three plants for use in the a ssays described below. Latex was collected using a silanized microdispenser (Drummond Scientific Company, Broomall, PA) from the leaf base (site of leaf lamina attachment to the stem, and of rapid latex exudation upon cutting) of young and middle-aged leaves of individual plants 60 s after cutting the tissue with a disposable scalpel blade (Feat her, Osaka, Japan). The experiments were carried out at 25 1 C in a laboratory under a photoperiod of 14:10 (L:D) h. Choice-tests Using Latex from Da maged and Undamaged Plants Latex (70 l) collected from plants as de scribed above was applied onto the top and side surfaces of a diet disk, immediately after collection. The experimental unit for the choice-test bioassay consisted of two diet disks, one treated with latex from beetledamaged plants and the other one with latex from undamaged checks within each cultivar. In the control experi mental units, two untreated diet disks were used. The diet disks were placed on the bottom of a plastic ventilated container (10 10 8 cm) and three male-female pairs of beetles were allo wed to feed on the disks for 24 h at 25 1 C in a laboratory. The number of adults feeding on each diet disk was recorded 1, 2, 3 and 4 h after their release into the bioassay units. Dr y weight of the diet consumed in 24 h was calculated as previously desc ribed in Chapter 3 and in Sethi et al. 2007. Total diet consumed per three pairs of adults in 24 h was calculated by adding the consumption of the two diet disks in each re pplicate of each treatment. Enzyme Activity Assays Activity of the enzymes phenylalanine ammonia lyase, polyphenol oxidase and peroxidase was assayed in th e latices of Valmaine and Tall Guzmaine 1, 3 and 6 d after

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141 initiation of beetle damage. Collect ed latex was dispensed into a -20 C chilled, 1.5-ml micro-centrifuge tube, on ice a nd immediately stored at -80 C until analyses. Frozen latex (70 l) was dissolved in 5 ml of 50 mM potassium phosphate buffer (pH 6.2) and centrifuged at 48,500 g for 45 min at 4 C (Model J2-HS, Beckman Instruments, Fullerton, CA). The supernat ant was collected and stored at -80 C until analyses. Total protein and enzyme activities were determined using a spectrophotometer (Model DU 640, Beckman Instruments, Fullert on, CA). Total protein was estimated according to the modified Lowrys method (Peterson 1977) using the Folin-Ciocalteau phenol reagent (Pierce Chemical, Rockford, IL ) and bovine serum albumin as a standard. Phenylalanine ammonia-lyase (PAL). PAL activity in latex was measured as described by Ke and Saltveit (1986) and Cam pos-Vergas and Saltveit (2002) with slight modifications. The supernatant was analyzed for PAL activity after addition of 200 l of supernatant to 400 l of 50 mM L-phenylal anine (dissolved in 20 mM potassium phosphate buffer, pH 8.8) and 400 l of 50 mM potassium phosphate buffer pH (8.8) and incubated at 40 C for 30 min. The absorbance was measured at 290 nm before and after incubation. PAL activity was expr essed as the amount of PAL ( mol mg-1 h-1) that produces 1 mol of cinnamic acid in 1 h. Cinnamic acid (0 400 mol at an increment of 15 mol) was used as a reference fo r quantification of PAL activity. Polyphenol oxidase (PPO). PPO activity was assayed following the methods of Sirinphanic and Kader (1985) and Loiaza-Velarde et al. (1997) with slight modifications. PPO activity was assessed by incubating 10 l of supernatant with 500 l of 1.6% catechol (Sigma, St. Louis, MO), 100 l of 50 mM potassium phos phate buffer (pH 6.2) and 390 l distilled water. Absorbance of th e mixture was read at 480 nm over a period

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142 of 2 min. One unit of PPO activity was defined as the amount of enzyme that produced an increase in absorbance of 0.1 per min at 480 nm. The linear portion of the curve was used to estimate the reaction rate. Peroxidase (POX). The activity of POX was determined using the methods of Loiaza-Velarde et al. (1997) with slight modifications. The POX activity was determined by combining 10 l of H2O2 (30%, v/v) in 50 l of supernatant, 300 l of 18 mM guaiacol, 100 l of 50 mM potassium phosphate buffer (pH 6.2) and 540 l of distilled water. Absorbance of the resu lting mixture was examined at 420 nm over a period of 2 min. The POX activity (mol mg protein-1 mi n-1) was determined using guaiacol molar absorptivity ( = 26.6 M-1 cm-1) at 420 nm. The reac tion rate was calculated using the linear portion of the curve. Statistical Analysis Data on number of insects feeding on diet disks treated with latex collected from plants with and without prio r beetle exposure were analyz ed as a repeated measures design using Proc GLIMMIX (SAS Institute 200 3). Separate analyses were run for disks from each cultivar at each prior beetle expos ure interval (1, 3 and 6 d). The variables latex (from damaged or undamaged plants) and ti me interval after be etle release (1, 2, 3 and 4 h) were fixed. Fifteen groups of six beetles (i.e., replications) were randomly assigned to each level of latex and tested four times (1, 2, 3 and 4 h). Data on dry weight of diet consumed under choice tests were analyzed using PROC GLM (SAS Institute 2003) with latex and time interval after beetle release as fixe d effects. Replications were treated as a random effect for each cultivar. Data on enzyme activities were analyzed using PROC GLM (SAS Institute 2003) with cultivar, latex treatment (damaged or undamaged), and time interval after feeding

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143 initiation on plants as fixed e ffects. Replications were agai n treated as a random effect. Data on beetle fresh weight gain were anal yzed using PROC GLM (SAS Institute 2003) with cultivar and time interval after feeding in itiation as fixed effects, and replications as a random effect. Tukeys honestly significant difference (HSD) test with a significance level of = 0.05 (SAS Institute 2003) was used for post hoc means separation. Simple regression analysis was done to study the rela tionship between beetle fresh weight gain and enzymatic activities using PR OC REG (SAS Institute 2003). Results Oberservations of Latex Characteristic s from Damaged and Undamaged Plants The latex from Valmaine plants damaged for 3 or 6 d browned faster and to a deeper hue than did latex co llected after 1 d of feeding damage. However, no such differences were noted in th e latex of Tall Guzmaine. The quantity of latex exuded by Tall Guzmaine plants decreased with the duration of feeding damage. Tall Guzmaine latex collected after 3 and 6 d of feeding damage was also le ss viscous, and more watery and translucent, while latex quality in Valmaine did not differ visually. Choice-tests Using Latex from Damaged and Undamaged Plants In case of Valmaine choice tests, type of latex 1 d after feed ing initiation did not have significant effect on the number of insects feeding on the diet disks ( F = 2.0851; df = 1, 8; P = 0.1585), but latex after 3 ( F = 18.96; df = 1, 8; P = 0.0001) and 6 d ( F = 14.43; df = 1, 8; P = 0.0005) after feeding initiation had significant effects. The number of D. balteata adults feeding on disks tr eated with latex from Valmaine plants that had been fed on for 1 d was not significantly different from the number feeding on disks treated with latex from undamaged Valmaine plants (Fig. 5-2 and 5-3). However, there were significant differences between disks trea ted with Valmaine latex from plants with

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144 and without feeding after 3 and 6 d. In Tall Guzmaine choice tests, latex anytime after feeding initiation did not have any significant effect on the number of beetle feeding on diet disks (1 d: F = 0.0753; df = 1, 8; P = 0.7855; 3 d: F = 0.800; df = 1, 8; P = 0.7791, 6 d: F = 0.0468; df = 1, 8; P = 0.8301). The number of beetles feeding on diet disks treated with latex from damaged Tall Guzmaine plants or with latex from undamaged plants did not differ significantly at any ti me after initiation of feedi ng damage (Fig. 5-2 and 5-4). In the Valmaine choice test, latex (damag ed or undamaged) had significant effect on diet consumption by the beetles ( F = 72.02; df = 1, 24; P = 0.0001). Adults of D. balteata consumed significantly less diet treated w ith latex from damaged plants than diet treated with latex from undamaged plants (Fi g. 5-5). Time interval (1, 3, and 6 d) after feeding initiation on plants did not have si gnificant effect on diet consumption by the beetles ( F = 1.08; df = 2, 24; P = 0.3548). But there was sign ificant intera ction between latex and time interval ( F = 3.67; df = 2, 24; P = 0.0406). The amount of diet eaten from disks treated with latex from damaged plants decreased with increasing duration of beetle feeding on plants, whereas the amount of diet eaten from disks treated with latex from undamaged plants was constant across the three time interval s after initiation of feeding (Fig. 5-5). In the Tall Guzmaine choice test, latex did not have any significant effect on diet consumption by beetles ( F = 0.2160; df = 1, 24; P = 0.6463). Diet consumption by D. balteata adults on diet treated with latex from damaged plants did not differ significantly than on diet trea ted with latex from undamaged plants (Fig. 5-5). Neither significant effect of time interval ( F = 0.60; df = 2, 24; P = 0.5592), nor significant interaction between latex and time interval ( F = 2.04; df = 2, 24; P = 0.1521) on diet consumption was found.

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145 Treatment of latex significantly affected the total diet consumption in choice tests ( F = 235.08; df = 2, 33; P = 0.0005). Total diet consumed by six D. balteata was significantly less on Valmaine la tex treated diet compared to Tall Guzmaine latex treated and control diets (Table 5-1). Diet consumption did not chan ge significantly when disks were treated with latex collected from damaged plants at different time intervals ( F = 1.11; df = 2, 33; P = 0.3412). No significan t interaction was found be tween type of latex and time interval after feeding initiation ( F = 0.6330; df = 4, 33; P = 0.6425). Total Protein Content Type of cultivar had significant e ffect on the total protein content ( F = 91.77; df = 1, 47; P = 0.0001). Total protein content was signif icantly higher (1.3 fold) in Valmaine latex than in Tall Guzmaine latex (Fig. 5-6). No significant effect of treatment (damaged or undamaged) was found on total protein content ( F = 1.49; df = 1, 47; P = 0.2281). But significant effect of time in terval after feeding damage (1, 3 and 6 d) was found ( F = 5.29; df = 2, 47; P = 0.0084). Significant interactions were found between cultivar and treatment (damaged or undamaged) ( F = 16.70; df = 1, 47; P = 0.0002), and between cultivar and time interval after feeding damage ( F = 7.61; df = 2, 47; P = 0.0013). Total protein content in Valmaine after 6 d of f eeding damage was 1.36 fold higher than after 1 d. There was no increase protein content of Tall Guzmaine latex through time. Phenylalanine Ammonia Lyase The effect of cultivar was significant on PAL activity ( F = 289.82; df = 1, 47; P = 0.0001). The activity of PAL was significantly hi gher (3.44 fold) in Valmaine latex than in Tall Guzmaine latex (Fig. 5-7). Both treatment ( F = 98.45; df = 1, 47; P = 0.0001) and time interval after feeding initiation ( F = 7.96; df = 2, 47; P = 0.0010) had significant effect on PAL activity. Signi ficant interactions were found between cultivar and

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146 treatment ( F = 20.96; df = 1, 47; P = 0.0001), and between cultivar and time interval after initiation of feeding damage ( F = 7.36; df = 2, 47; P = 0.0016). PAL activity in Valmaine latex was significantly increased after 3 d (1.81 fold) and 6 d (1.54 fold) of feeding damage, relative to 1 d after in itiation of feeding. No increas e was seen in PAL activity in the latex of Tall Guzmaine through time Polyphenol Oxidase Type of cultivar had signif icant effect on PPO activity ( F = 358.32; df = 1, 47; P = 0.0001). The activity of PPO was significantly hi gher (4.37 fold) in Valmaine latex than in Tall Guzmaine latex (Fig. 5-8). Both treatment ( F = 80.31; df = 1, 47; P = 0.0001) and time interval after feeding initiation ( F = 8.25; df = 2, 47; P = 0.0008) had significant effect on PPO activity. Significant interac tions were found between cultivar and treatment ( F = 74.86; df = 1, 47; P = 0.0001), and between cultivar and time interval after feeding damage ( F = 11.65; df = 2, 47; P = 0.0016). PPO activity was significantly increased 3 d (1.74 fold) and 6 d (1.78 fold) af ter feeding damage in Valmaine latex, but not in Tall Guzmaine latex. Peroxidase The POX activity was significantly affected by the type of cultivar ( F = 35.49; df = 1, 47; P = 0.0001). The activity of POX was significantly higher (2.1 fold) in Valmaine latex than in Tall Guzmaine latex (Fig. 5-9). Both treatment ( F = 39.29; df = 1, 47; P = 0.0001) and time interval af ter feeding initiation ( F = 4.92; df = 2, 47; P = 0.0113) had significant effect on POX activity. Significant interactions were f ound between cultivar and treatment ( F = 35.45; df = 1, 47; P = 0.0001), and between cultivar and time interval after feeding damage ( F = 5.16; df = 2, 47; P = 0.0094). POX activity was significantly

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147 increased 3 d (1.56 fold) and 6 d (2.1 fold) af ter feeding damage in Valmaine latex but not in Tall Guzmaine latex. Relationship between Female Weig ht Gain and Enzyme Activity Cultivar had significant effect on gain in female fresh weight ( F = 1269.92; df = 1, 23; P = 0.0001). Female beetles weighed signifi cantly less when fed on Valmaine than Tall Guzmaine (Fig. 5-10). Both time interval after feeding initiation on plants ( F = 30.42; df = 2, 23; P = 0.0001) and interaction between cultivar and time interval ( F = 161.35; df = 2, 23; P = 0.0001) had significant effect on female fresh weight gain. Females feeding on Tall Guzmaine weighed 2.2, 12.1, and 50.8 times more than the females on Valmaine after 1, 3 and 6 d of feed ing on the plants, respectively. Beetles lost weight over time on Valmaine whereas they ga ined weight on Tall Guzmaine (Fig. 5-10). Furthermore, a significant negative relations hip was found between female fresh weight gain and activities of each enzyme (PAL, PPO and POX) in latex from damaged plants of Valmaine (Fig. 5-11). No significant relations hip was found between fe male fresh weight gain and any of the enzyme activiti es of latex from Tall Guzmaine. Discussion Valmaine latex from damaged plants was more deterrent compared to latex from undamaged plants. This may be due to the change in the concentration of its constituents. Upon wounding, latex turns brow n after sometime due to the production of quinones that are catalyzed by PPO. The brow ning potential of the late x from damaged Valmaine plants was observed to increase with time after feeding damage. The browning is much darker in color in a disease-resistant clone of rubber tree than in a susceptible clone (Wititsuwannakul et al. 2002). Increased intensity of browning may be due to the higher activity of PPO. The intensity of browning was observed to remain the same in Tall

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148 Guzmaine latex after beetle damage. In fact the intensity of brow ning was less in latex from undamaged Tall Guzmaine plants than in latex from undamaged Valmaine plants. Tall Guzmaine damaged plants produced less la tex which was also less viscous, and more watery and translucent, while the amount of latex production and its viscosity and color (miliky white) remained the same in Valmaine latex even after beetle damage. Such differences in milkiness arise due to differences in the refractive indices of the dispersing particles (mainly terpenoi ds) and the dispersing medium (Esau 1965, Fahn1990). Thus, the production of these dispersing particles in Tall Guzmaine may have been reduced after feeding damage or the loss of large am ounts of latex during beetle feeding may have reduced the concentration of these compounds. The amount of total protein increased in latex from Valmaine after beetle damage while it did not change in Tall Guzmaine. Ni et al. (2001) also found a significant increase in the total protein content in wheat cultivars after damage by the Russian wheat aphid. The activities of all three enzymes, PAL, PPO and POX were increased significantly in Valmaine latex after 3 d of D. balteata feeding damage while they were same in Tall Guzmaine latex. Even the constitutive level of PAL and PPO in undamaged plants was significantly higher in Valmaine latex than in Tall Guzmaine latex. Alteration in the levels of these enzymes due to inse ct feeding has been observed by many other workers (Green and Ryan 1972, Cole 1984, H ildebrand et al. 1986, Felton 1989, Felton et al. 1994a, b; Miller et al. 1994, Rafi et al 1996, Jerez 1998, Stout et al. 1999, Constabel et al. 2000, Chaman et al. 2001, Ni et al 2001, Heng-Moss et al. 2004). The rate of secondary metabolism via the phenylpropa noid pathway, leading to production and accumulation of soluble phenolic compounds, is greatly increased after wounding of

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149 lettuce tissue (Toms-Barbern et al. 1997, Saltveit et al. 2005). The production of phenylpropanoid compounds plays an important role in plant defense (Hahlbrock and Scheel 1989). Phenylalanine ammonia lyase is the first committed enzyme in the phenylpropanoid pathway (Dixon and Paiva 1995) Its de novo synthesis and increased activity is an initia l response to wounding (Lopez-Galvez et al. 1997, Thoms-Barbern et al. 1997, Campos-Vergas and Saltveit 2002) that ultimately resu lts in increased concentrations of phenolic compounds (Loaiza-Velarde et al. 1997). The phenylpropanoid pathway starts with the deam ination of phenylalanine to cinnamic acid due to the action of PAL. The enhanced activity of PAL results in an increased production and accumulation of several phenolic compounds that are sequestered in the vacuole. These compounds can be oxidized to strong electrophillic quinones (brown substances) by PPO when membranes become disrupted. In addition, wounding also results in an increased ex pression of POX and lignin formation (Luh and Phithakpol 1972, Ribereau Gayon 1972, Robinson 1972, Hanson and Havir 1979, Rhodes et al. 1981). Higher activity of PAL was found in resist ant cultivars of lettuce infested with lettuce root aphid, Pemphigus bursarius (L.) (Cole 1984) and ba rley infested with greenbug, Schizaphis graminum (Rondani) (Chaman et al. 2003). The activity of PAL was also increased in strawberry leaves as a result of infestation by twospotted spider mite, Tetranychus urticae (Inoue et al. 1985). Insect resistance in many plant species (soybean, tomato, potato, cotton, rubber tree, poplar and barley) has been associated with higher activity of PPO (Gregory and Tingey 1981, Hedin et al. 1983, Felton et al. 1989, Duffey and Felton 1991, Steffens and

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150 Walter 1991, Bi et al. 1993, Felton et al. 1994a, Constabel et al. 1996, Wititsuwannakul et al. 2002, Wang and Constabel 2004, Chaman et al. 2001). Peroxidase activity is also known to increase in tomato and barley after infestation with corn earworm, Helicoverpa zea Boddie (Stout et al. 1999) and gr eenbug (Chaman et al. 2001), respectively. Earlier tests by Huang et al. 2003 found onl y localized induced resistance in Valmaine after 2 d of D. balteata damage. It is possible the 2 d feeding duration was not long enough to induce increased resistan ce (Schoonhoven et al. 2006). In our study, significant increases in the levels of all the three enzymes (PAL, POX and PPO) were only found at 3 and 6 d after feeding damage, but not after 1 d of feeding on Valmaine. Female beetles confined for 1 d on Valmaine plants had gained weight, lending support to the hypothesis that increased resistance is only induced after at least 2 d of feeding. Beetles were observed tunneling, and presumably feeding, in the midrib tissue near the proximal end of the leaf. However, after 3 d, beetles did not feed much and lost weight over the remaining 3 d of the experiment. S o, beetles may have stopped feeding due to induced resistance. Under these conditions, plants may have reached an equilibrium of defensive compounds concentrations and enzyme activities, and stopped further increment in the activities of these enzymes to save energy for development and growth. I also found a strong relationship between female weight gain and activit ies of all the three enzymes (PAL, PPO and POX), indicating a possible correlation between increased enzymes activities and decreased beetle fitness. Based on my results, I hypothesize that in creased levels of PAL, PPO and POX in Valmaine after D. balteata damage result in increased production of secondary metabolites and other unknown defensive compounds. Consequently, induced resistance

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151 in Valmaine acts synergistically with the constitutive resistance of latex and ultimately enhances its resistance against D. balteata Further research is re quired to characterize these damage-inducible enzymes at the mol ecular level to support breeding programs for the development of resistant cultivars with superior horticultural traits using either conventional or transgenic approaches.

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152 Figure 5-1. Feeding damage caused by D. balteata adults on two lettuce cultivars, Valmaine (VAL) and Tall Guzmaine (TG). TG VAL

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153 Figure 5-2. Adults of D. balteata feeding on diet disks treate d with latex from damaged and undamaged plants of two lettuce cultivars, Valmaine and Tall Guzmaine. Undamaged Damaged Damaged Undamaged Valmaine TallGuzmaine

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154 a a a b b b bb Day 1 Day 3 Time (h) 1234 Number of insect s feeding / disk 0 1 2 3 4 5 6 1234 Damaged Undamaged ns 1234 Day 6 b a b a a b a b Figure 5-3. Number of D. balteata adults feeding on artificial diet disks in a choice between latex from damaged and undamaged plants of Valmaine after 1, 2, 3 and 4 h of their release. Error bars indicate SEM. Bars topped with different letters within panel (day 1, 3 or 6) differ significantly at the 0.05 level (Tukeys HSD test).

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155 Day 1 Day 3 Time (h) 1234 Number of insect s feeding / disk 0 1 2 3 4 5 6 1234 Damaged Undamaged ns 1234 Day 6 ns ns Figure 5-4. Number of D. balteata adults feeding in a choice test using two artifi cial diet disks treated with damaged and undamaged plants of lettuce cultivar, Tall Guzmaine after 1, 2, 3 and 4 h of their release. Error bars indicate SEM. Bars topped with different letters within panel (day 1, 3 or 6) differ significantly at the 0.05 level (Tukeys HSD test).

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156 VALTG Days after beetle damage 136 Diet consumption (mg) 0 5 10 15 20 25 136 Damaged Undamaged b a bc a a c ns Figure 5-5. Artificial diet consumption after 24 h by D. balteata adults in choice test using two diet disks treated with latex from damaged and undamaged plants of two lettuce cultivars, Valmaine (VAL) and Tall Guzmaine (TG). Error bars indicate SEM. Bars topped with diff erent letters with panel (VAL or TG) differ significantly at the 0.05 level (Tukeys HSD test).

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157 Days after beetle damage 136 Total protein (g / l of latex) 0 15 20 25 30 35 VAL Damaged VAL Undamaged TG Damaged TG Undamaged ef f def cdef cdef cdef bcde ab a bcdef bc bcd Figure 5-6. Total protein content in two le ttuce cultivars, Valmaine (VAL) and Tall Guzmaine at 1, 3 and 6 d after initiation of feeding damage by adults of D. balteata. Error bars indicate SEM. Points topped with differe nt letters differ significantly at the 0.05 level (Tukeys HSD test).

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158 Days after beetle damage 136 PAL activity (mol cinnamic acid / mg / h) 0 2 4 6 8 10 VAL Damaged VAL Undamaged TG Damaged TG Undamaged de cde cde e e e b a a bcd bcd bc Figure 5-7. Activity of phenylalanine ammonia lyase (PAL) in two lettuce cultivars, Valmaine (VAL) and Tall Guzmaine at 1, 3 and 6 d after init iation of feeding damage by adults of D. balteata Error bars indicate SEM. Points topped with different letters differ significantly at the 0.05 level (Tukeys HSD test).

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159 Days after beetle damage 136 PPO activity (A 480 / mg protein / min ) 0 1 2 3 4 5 6 7 VAL Damaged VAL Undamaged TG Damaged TG Undamaged cd d d b a a bc b b Figure 5-8. Activity of polyphenol oxidase (PPO ) in two lettuce cultivars, Valmaine (VAL) and Tall Guzmaine at 1, 3 and 6 d after initiation of feeding damage by adults of D. balteata Error bars indicate SEM. Po ints topped with different letters differ significantly at th e 0.05 level (Tukeys HSD test).

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160 Days after beetle damage 136 POX activity (mol / mg protein / min) 0.00 0.02 0.04 0.06 0.08 VAL Damaged VAL Undamaged TG Damaged TG Undamaged bc ab a c c c Figure 5-9. Activity of peroxida se (POX) in two lettuce cultivars, Valmaine (VAL) and Tall Guzmaine at 1, 3 and 6 d after initi ation of feeding damage by adults of D. balteata Error bars indicate SE M. Points topped with different letters differ significantly at the 0.05 level (Tukeys HSD test).

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161 Days after beetle damage 136 Fresh weight gain / female (mg) -0.5 0.0 0.5 1.0 1.5 2.0 VAL TG d c e e b a Figure 5-10. Gain in fresh weight of D. balteata females over a 6-d period of feeding on two romaine lettuce cultivars, Valmaine (VAL) and Tall Guzmaine (TG). Error bars indicate SEM. Points t opped with different letters differ significantly at the 0.05 level (Tukeys HSD test).

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162 Fresh weight gain / female (mg) 0.00.51.01.52.0 0 2 4 6 8 10 0.00.51.01.52.0 0 2 4 6 8 10 0.00.51.01.52.0 0.00 0.02 0.04 0.06 0.08 0.10 0.00.51.01.52.02.5 PAL activity (mol cinnamic acid / mg / h) 0 2 4 6 8 10 0.00.51.01.52.02.5 PPO activity (A 480 / mg protein / min) 0 2 4 6 8 10 0.00.51.01.52.02.5 POX activity (mol / mg protein / min) 0.00 0.02 0.04 0.06 0.08 0.10 Fresh weight gain / female (mg) Fresh weight gain / female (mg) VAL VAL VAL TG TG TG y = 7.15 7.34x R sq = 0.57 P = 0.0012 y = 6.06 5.95x R sq = 0.54 P = 0.0019 y = 0.06 0.06x R sq = 0.38 P = 0.0151 y = 1.55 0.25x R sq = 0.10 P = 0.2621 y = 1.04 0.11x R sq = 0.04 P = 0.4701 y = 0.02 0.002x R sq = 0.15 P = 0.6646 Figure 5-11. Relationship between fresh weight gained by D. balte ata females feeding on two lettu ce cultivars, Valmaine (VAL) a nd Tall Guzmaine (TG) and activity of A) PAL, B) PPO a nd C) POX enzymes after 1, 3 and 6 d of feeding damage. A B C

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163 Table 5-1. Total diet consumption by six D. balteata adults on two diet disks treated with latex from same lettuce cultivar, Valmaine or Tall Guzmaine after 24 h of their release. Cultivar Days after Damage Total Diet Consumption (mg) Valmaine 1 15.2.8c 3 13.5.2c 6 12.4.2c Tall Guzmaine 1 36.7.9b 3 35.4.9b 6 32.1.8b Control 1 45.8.2a 3 47.3.6a 6 46.8.5a Means SEM followed by different letters within column differed significantly ( P 0.05) using ANOVA and Tukeys HSD test.

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164 CHAPTER 6 SUMMARY Lettuce, Lactuca sativa L., is one of the most im portant vegetable crops grown throughout the world, especially in the United St ates. California is the major producer of lettuce in the United States (77 % of all le ttuce harvested) followed by Arizona, Florida and New Jersey. In Florida, lettuce production from the Everglades Agricultural areas in south Florida contributes 90% of the tota l state production. Lettuce suffers economic losses due to several insect pe sts, such as cabbage lopper, Trichoplusia ni (Hubner); beet armyworm, Spodoptera exigua (Hubner); banded cucumber beetle, Diabrotica balteata Leconte; and leafminer, Liriomyza trifolii (Burgess). For the management of these pests, growers are dependent on pest icides. Approximately 93% of the lettuce acreage in the United States is treated with the insectic ides. Florida ranks first among lettuce growing states in the usage of insecticides to manage these insect pests. Therefore, there is a need to look for alterative strategies for manageme nt of economic insect pests. Management of insects with host plant resi stance is an important com ponent of integrated pest management strategies. The romaine lettuce cultivar, Valmain e is known to possess a high level of resistance to D. balteata and the leafminer. Diabrotica balteata feeding is increased after removal of leaf surface chemicals in Valmaine with methylene chloride, but these surface chemicals did not show any deterrence when applied to leaf surfaces of palatable lima bean at different concentrations. Therefore, it seems that internal factors are involved rather than external chemical factors in imparting resistance against D. balteata in Valmaine. Further, previously wounded Valmai ne plants showed an increased localized

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165 resistance to D. balteata compared to unwounded plants suggesting the involvement of an inducible mechanis m of resistance. The purpose of this research was to investigat e the extent of resistance in the lettuce cultivar Valmaine against another order of economically important lettuce pests, the Lepidoptera. The second objective was to identif y the mechanism of this multiple insect resistance. To address the first objective, I compar ed the survival, development and feeding behavior of cabbage looper and beet armyworm on two romaine lettuce cultivars, resistant Valmaine and the closely-related su sceptible Tall Guzmaine. Larval mortality of both species was significantly higher on Valmaine than on susceptible Tall Guzmaine. Significant difference between the cultivars wa s also observed in development. Larvae weighed six times (beet armyworm) and two times (cabbage looper) more after feeding for 1 wk on Tall Guzmaine than on Valmai ne. Larval period was 5.9 (beet armyworm) and 2.6 d (cabbage looper) longer on Valmaine than on Tall Guzmaine. Pupal duration of both insect species was also increased by almo st 1 d by feeding on Valmaine compared to Tall Guzmaine. Weights of the pupa and adu lt of both insect species were reduced on Valmaine compared to Tall Guzmaine. The se x ratio of progeny did not deviate from 1:1 when larvae were reared on resistant Valmaine The fecundity of cabbage looper and beet armyworm moths that developed from larv ae reared on Valmaine was about one third that of moths from Tall Guzmaine, but adul t longevity did not significantly differ on the two lettuce cultivars. Feeding behavior of these moth species was also significantly affected by lettuce cultivar. The two insect species showed di fferent feeding prefer ence for leaves of

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166 different age groups on Valmaine and Tall Gu zmaine. Cabbage looper preferred to feed on the lowermost fully mature leaves of Va lmaine plants and on young and middle-aged leaves of Tall Guzmaine plants (rarely feeding on fully-matur ed leaves). Beet armyworm preferred to feed on the lowermost fully mature leaves of Valmaine plants and on middleaged leaves of Tall Guzmaine plants. Both in sect species preferred to feed on the distal end of leaves. Early instars of cabbage loope r preferred to feed on the underside of the leaf, whereas early instars of beet armyworm fed on the upper side of the leaf. Cabbage loopers also cut narrow trenches on the leaf before actual feeding to block the flow of latex to the intended site of feeding. In contrast, beet armyworms did not trench; neonates made shallow scratches between the vein s by feeding on parenchymatous tissue and second instars made holes through the leaf. Th e different feeding behavior of the two species on Valmaine may explain the superior performance of cabbage looper compared to beet armyworm. Lettuce is a laticiferous plant, meaning that it produces a white milky fluid after tissue damage. Latex is stored under pressure in the laticif ers. Plant latex is a known defense in certain plants th rough its physical and chemical properties against several insects. Therefore to address my second object ive, i.e. identification of mechanism of resistance in Valmaine romaine lettuce, I hypothesized that latex al so plays a defensive role in lettuce. I again used two romaine lettuce cultivars, Valmaine (resistant) and Tall Guzmaine (susceptible) to study the potential of latex as a defense mechanism against D. balteata Latex from Valmaine strongly inhibited D. balteata feeding compared to Tall Guzmaine when applied to the surface of ar tificial diet. The amount of diet consumed

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167 from Valmaine latex treated disks was signifi cantly less than that consumed from diet treated with Tall Guzmaine latex, in both c hoice and no-choice te sts. The number of adults feeding on diet treated with Valmaine latex was less compared to Tall Guzmaine latex treated diet after 15, 30, 60 and 90 minutes of their release. These studies suggest that latex may account for resistance in Valmaine to D. balteata. All four species that have been te sted on Valmaine and Tall Guzmaine ( D. balteata leafminer, cabbage looper and beet armywo rm) prefer to feed on the lowermost fully matured leaves of resistant cultivar Valmaine Therefore I decided to test whether this kind of behavior is mediated through any di fferences in the properties of latex from young and mature leaves. Latex from the young l eaves is more viscous and solid white, whereas it is more watery and translucent in the mature leaves. Hence, I conducted choice tests using two artificial diet disks, one treated with latex from young leaves and the second one treated with latex from mature leaves. There was a significant interaction be tween leaf age and variety on diet consumption by the beetles. In Valmaine late x treated choice tests, the beetles consumed significantly less diet treated with latex from young leaves than that consumed from diet treated with latex from mature leaves. No significance difference in diet consumption was found between diets treated with late x from young and mature leaves in Tall Guzmaine latex treated choice tests. So, th is may explain insect avoidance of young and middle-aged leaves of Valmaine. After these studies, I was confident that the multiple insect resistance observed in Valmaine was mediated through latex. So in order to further inve stigate whether this resistance was due to physical or chemical pr operties of latex, I pr epared a crude extract

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168 by dissolving latex in different solvents. Three solvents of differing polarity (water, methanol and methylene chloride) were te sted to extract and compare deterrent compounds from Valmaine and Tall Guzmaine latex. Solvents and the interaction of solvent with lettuce cultivar had significant deterrent affects on beetle feeding. Valmaine latex extr acted with water:metha nol (20:80) strongly inhibited beetle feeding when applied to the surface of artificial diet. The percentage of beetles feeding on diet treated with Valmai ne water:methanol (20:80) extract was less compared to Tall Guzmaine water:methanol (20: 80) extract treated diet at intervals of 15, 30, 60 and 90 min after their release. The am ount of diet consumed in no-choice tests from disks treated with Valmaine water:meth anol (20:80) extract was significantly less than that consumed from diet disks treated with Tall Guzm aine methanol:water (80:20) extract. To study the role of physical properties of latex in Valmaine resistance, I conducted a small study by applying fresh latex on the mandibles of D. balteata adults. Beetles salivated more when Valmaine latex was app lied to their mouthparts compared to Tall Guzmaine latex but mandibles and maxi llae were not gummed up and were moving freely 24 h after application of either Valm aine or Tall Guzmaine latex (although there were traces of dried latex on the labium and tarsi). These studies strongly indicated a biochemical rather than physical ba sis of resistance in Valmaine to D. balteata The ability to extract deterrent compounds in water:methanol (20:80) suggested that moderately polar chemicals within late x may account for the observed resistance The next series of steps were conducted to isolate deterrent chemicals from the crude Valmaine latex extract (water:methanol 20:80). The crude extract was first passed

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169 through C-18 cartridges at thr ee different pH levels (natur al, acidic and alkaline) to evaluate its relative polarity. No significan t deterrent activity wa s found in the fraction eluted from the cartridge using a step gradie nt of water:methanol mixtures. The activity was only found in the unbound fraction eluting from the C-18 cartri dge, indicating that the deterrent compounds were highly polar. Next, the C-18 unbound fraction was passed through anion exchange and cation exchange ca rtridges connected in series. The retained compounds on both ion exchange cartridges were tested for feeding deterrence after they were eluted using a NaCl sa lt gradient. The 0.5M fraction obtained from the cation exchange cartridge possessed the highest dete rrent activity. Retention of the deterrent compounds in Valmaine latex on the cation exch ange column indicates its basic nature. A fraction eluting between 3 and 4 min exhibi ted the strongest deterrent activity during further fractionation of cati on exchange extract using HP LC-MS. UV absorption and MS data indicated the presence of ten compounds in this active fraction and some of these compounds have substituted aromatic structure. Hence, these results strongly support my hypothesis that unacceptability of Valmaine to D. balteata is primarily due to chemical constituents of latex. Previous research showed that there was a localized induced resistance in Valmaine plants after feeding by D. balteata In general, induced resistance involves changes in plant defensive chemistry due to alteration in the levels of various enzymes, such phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO) and peroxidase (POX). Therefore, my next steps were to evaluate th e potential activity of these three enzymes in Valmaine and Tall Guzmaine lettuce. The ques tions I tried to answ er were if, and how quickly such enzymes could be activated af ter beetle damage. If such enzymes were

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170 present and inducible by beetle feeding then for how long were levels increased, and did their higher activity co rrelate with feeding deterrent ac tivity in the latex. To answers these questions, I first tested for i nducible enzymatic activity by giving D. balteata adults a choice between diets treated with latex from either damaged or undamaged plants. Separate tests were run with extr acts from Valmaine and Tall Guzmaine. I investigated the expression of inducible enzymes phenylalanine ammonia lyase, polyphenol oxidase and peroxida se in the latex of both damaged and undamaged plants of Valmaine and Tall Guzmaine. Diet consum ption was significantly reduced when disks were treated with latex collected from bee tle-damaged Valmaine plants 3 and 6 d after feeding initiation. No significant difference was found in the diet consumption when disks were treated with latex from beetle-damaged Tall Guzmaine plants. Activities of all the three enzymes were significantly enhanced in Valmaine latex after 3 and 6 d of damage, whereas activity remained low in latex from damaged Tall Guzmaine plants. The constitutive levels of PAL and PPO were also significantly hi gher in latex from undamaged Valmaine compared to Tall Guzmaine plants. So, it seems that Valmaine is better defended in terms of higher expression of these enzymes both at constitutive and induced levels. On Valmaine, beetles gained weight after 1 d of feeding, but then lost weight after being confined on the plants for 3 and 6 d. Fresh weight gain of female D. balteata fed Tall Guzmaine plants increased in a linear fashion over the 6 d exposure period. However, a significant negative rela tionship was found between weight gain and activities of PAL, PPO and POX in Valmaine latex. These studies suggest that latex chemistry may change after beetle feeding da mage due to increased activity of inducible

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171 enzymes, and that inducible resistance app ears to act synergisti cally with constitutive resistance in Valmaine latex. Based on my findings, it appears that Va lmaine possesses both constitutive and induced resistance mechanisms, and both are mediated through late x. Solvent extraction studies of the deterrent compounds suggest the presence of biologically active nitrogenous compounds in Valmaine latex, wh ile enzyme induction studies after insect damage indicate an increase in the pheno lic compounds. Hence, constitutive and induced defenses in Valmaine may invol ve different biochemicals. In a situation where there is no constant insect pressure, Valmaine exhibits a constitutive defense and is a non-preferred host. However, in situations where there is prolonged insect pressure, and those insects either have no choice but to feed on Valmai ne or are not significantly deterred by the constituitive defenses, inducible enzymatic ac tivity in Valmaine may turn on the second line of defense to protect itself from furthe r damage. Therefore, both types of defenses might be acting synergistically in Valmaine. Further, Valmaine exhibited resistan ce only against insects having chewing mouthparts ( D. balteata adults, leafminer maggots a nd beet armyworm and cabbage looper caterpillars) and not against insects ha ving sucking mouthparts, such as whitefly (unpublished, Heather McAuslane), aphids (u npublished, Gregg Nuessly) and thrips (unpublished, Amit Sethi). This dichotomy ma y be an outcome of the mechanism of resistance in Valmaine. Because latex is f ound in laticifers which run parallel to the vascular system in the plant, chewing inse cts accidentally rupture the laticifers when attempting to feed on lettuce, resulting in th eir exposure to latex-borne feeding deterrents. On the other hand, most of the successf ul sucking insects are known to feed

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172 intercellularly and in this wa y avoid or reduce the frequency of rupturing laticifers. This may explain why Valmaine only possesses re sistance against chewing insects and not against sucking insects. Based on studies done so far, I propose a bi ochemical basis for host plant resistance in Valmaine. Further research is required to identify the deterrent compounds both at constitutive and induced levels and also to characterize these induc ible enzymes at the molecular level so that both can be used as selection markers during breeding programs to improve lettuce varieties.

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173 LIST OF REFERENCES Agnew, K. 2000. Crop profile for lettuce in Ar izona. Pesticide Information and Training Office. University of Arizona, Arizona. Agrawal, A. A. 1999. Induced plant defense in plants: the ecol ogy and evolution of restrained defense, pp. 137166. In A. A. Agrawal, S. Tuzun, and E. Bent [eds.], Induced plant defenses against pathogens and herbivores. Biochemistry, Ecology and Agriculture. APS Press, St. Paul. Agricultural Statistics. 2001. Statistical hi ghlights of U.S. agriculture 2000-2001. USDA, NASS. http://www.nass.usda.gov/index.asp Agricultural Statistics. 2003. Statistical hi ghlights of U.S. agriculture 2002-2003. USDA, NASS. http://www.nass.usda.gov/index.asp Agricultural Statistics. 2007. Vegetabl es 2006 Summary. January 2007. USDA, NASS. http://www.nass.usda.gov/index.asp Alleyne, E. H., and F. O. Mo rrison. 1977. The lettuce root aphid, Pemphigus bursarius (L.) (Homoptera: Aphidoidea) in Quebec, Canada. Ann. Entomol. Soc. Quebec 22: 171. Anonymous. 1999. Crop Profile for Celery in Florida. The National Science Foundation Center for Integrated Pest Management, North Carolina State University, Raleigh, NC. http://cipm.ncsu.edu/cropprofiles/docs/FLCelery.html Anonymous. 2003. Integrated Pest Manageme nt for Cole Crops and Lettuce, pp. 112. Agriculture and Natural Resources, Un iversity of California, Davis. http://www.ipm.ucdavis.edu/PM G/selectnewpest.lettuce.html Argandona, V. H., M. Chaman, L. Cardemil, O. Munoz, G. E. Zuniga, and L. J. Corcuera. 2001. Ethylene production and pe roxidase activity in aphid-infested barley. J. Chem. Ecol. 27: 53-68. Auad, A. M., and J. C. Moraes. 200 3. Biological aspects and life table of Uroleucon ambrosiae as a function of temperatur e. Sci. Agricola 60: 657-662. Azarkana, M., R. Wintjensb, Y. Loozeb, and D. Baeyens-Volant. 2004. Detection of three wound-induced proteins in pa paya latex. Phytochemistry 65: 525. Baldwin, I. T. 1994. Chemical change s rapidly induced by folivory, pp.1-23. In E. A. Bernays [ed.], Insect-plant interactio ns, vol 5. CRC Press Incorporated, Boca Raton. Barton, D. H. R., and C. R. Narayanan. 1958. Sesquiterpenoids. Part X. The constituents of lactucin. J. Chem. Soc. 1: 963-971.

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174 Bauhin. 1671. Cited from E. L. Sturtevant. 1886 A study of garden lettuce. Am. Nat. 20: 230-233. Becerra, J. X., D. L. Venable, P. H. Evan s, and W. S. Bowers. 2001. Interaction between chemical and mechanical defenses in the plant genus Bursera and their implications for herbivore. Am. Zool. 41: 865-876. Bellows, B. C., and S. Diver. 2002. Cucumber beetles: organic a nd biorational IPM. ATTRA National Sustainable Agriculture In formation Service, Fayetteville, AR. Bennett, M. H., M. Gallagher, C. Bestwic h, J. Rossiter, and J. Mansfield. 1994. The phytoalexin response of lettuce to challenge by Botrytis cinerea Bremia lactucae and Pseudomonas syringae pv. phaseolicola Physiol. Mol. Plant Pathol. 44: 321333. Bergey, D., G. Howe, and C. A. Ryan. 1996. Polypeptide signaling for plant defensive genes exhibits analogies to defense signa ling in animals. Proc. Natl. Acad. Sci. USA 93: 12053. Bernays, E. A., and R. F. Chapman. 1977. Dete rrent chemicals as a basis of oligophagy in Locusta gregaria L. Ecol. Entomol. 2: 1-18. Berneys, E. A., and R. F. Chapman. 1994. Ho st plant selection by phytophagous insects. Chapman & Hall, New York. Bernays, E. A., and A. C. Lewis. 1986. The eff ect of wilting on palatability of plants to Schistocerca gregaria the desert locust. Oecologia 70: 132-135. Beshear, R. J. 1983. New records of thrips in Georgia. J. Georgia Entomol. Soc. 18: 342344. Bestwick, L., A. L. Adam, N. Puri, and J. W. Mansfield. 2001. Characterization of and changes to proand anti-oxidant en zyme activities during the hypersensitive reaction in lettuce ( Lactuca sativa L.). Plant Sci. 161: 497-506. Bestwick, L., M. H. Bennett, J. W. Mansfi eld, and J. T. Rossiter. 1995. Accumulation of the phytoalexin lettucenin A and changes in 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in lettuce seedlings with the red spot disorder. Phytochemistry 39: 775-777. Bi, J. L., G. W. Felton, and A. J. Mueller. 1993. Induced resistance in soybean to Helicoverpa zea : Role of plant protein quali ty. J. Chem. Ecol. 20: 183-198. Bi, J. L., G. W. Felton, J. B. Murphy, P. A. Howles, R. A. Dixon, and C. J. Lamb. 1997a. Do plant phenolics confer resi stance to specialist and gene ralist insect herbivores? J. Agric. Food Chem. 45: 4500-4504.

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175 Bi, J. L., J. B. Murphy, and G. W. Felt on. 1997b. Antinutritive and oxidative components as mechanisms of induced resistance in cotton to Helicoverpa zea J. Chem. Ecol. 23: 97-117. Bibby, F. F. 1958. Notes on thrips of Arizona. J. Econ. Entomol. 51: 450-452. Blackman, R. L., and V. F. Eastop. 2000. A phids on the worlds crops. John Wiley & Sons, Chichester, UK. Bowles, D. J. 1990. Defense-related proteins in higher plants. A nnu. Rev. Biochem. 59: 873-907. Braun, J., and M. Tevini. 1993. Regulation of UV-protective pigment synthesis in the epidermal layer of rye seedlings ( Secale cereale L. cv. kustro). Photochem. Photobiol. 57: 318-323. Breda, C., D. Buffard, R. B. van Huystee, and R. Esnault. 1993. Di fferential expression of two peanut peroxidase cDNA clones in peanut plants and cells in suspension culture in response to stress. Plant Cell Rep. 12: 268-272. Brignolas, F., B. Lacroix, F. Lieutier, D. Sauva rd, A. Drouet, A. C. Claudot, A. Yart, A. A. Berryman, and E. Christiansen. 1995. Induced response in phenolic metabolism in two Norway spruce clones afte r wounding and inoculation with Ophiostoma polonicum a bark beetle-associated f ungus. Plant Physiol. 109: 821-827. Britsch, L. 1990. Purification and charac terization of flavone synthase 1, a 2oxoglutarate-dependent desaturase. Arch. Biochem. Biophys. 282: 152-160. Broekaert, W., H.-I. Lee, A. Kush, NH Chua, and N. Raikhel. 1990. Wound induced accumulation of mRNA containing a hevein se quence in lacticifers of rubber tree ( Hevea brasiliensis) Proc. Natl. Acad. .Sci. 87: 7633-7637. Brower, L. P., J. N. Seiber, C. J. Nelson, P. Tuskes, and S. P. Lynch. 1982. Plant determined variation in the cardenolide cont ent, thin layer chromatography profiles, and emetic potency of monarch butterflies, Danaus plexippus reared on milkweed, Asclepias eriocarpa in California. J. Chem. Ecol. 8: 579-633. Bryan, D. E., and R. F. Smith 1956. The Frankliniella occidentalis (Pergande) complex in California. Univ. Calif. Public Entomol. 10: 359-410. Bryant, J. B., F. S. Chapin III, and D. R. Klein. 1983. Carbon/nutrient balance of boreal plants in relation to vertebra te herbivory. Oikos 40: 357-368. Burnett, W. C., S. B. Jones, and T. J. Ma bry. 1978. The role of sesquiterpene lactones in plant and animal coevolution, pp. 233-257. In J. B. Harborne [ed.], Biochemical aspects of plant animal coevol ution. Academic Press, London.

PAGE 176

176 Butt, V. S. 1980. Direct oxidase s and related enzymes, pp. 81-123. In E. E. Conn and P.K. Stumpf [eds.], The biochemistry of plants, vol.2. Academic Press, New York. Buttery, B. R., and S. G. Boatman. 1976. Wa ter deficits and flow of latex, pp. 233-289. In T.T. Kozlowski [eds.], Water deficits a nd plant growth, vol. IV. Academic Press, New York, USA. CABI. 1972. Distributio n maps of pests. Spodoptera exigua (Hbner). Commonwealth Agricultural Bureau, London. Series A, Map 302. CABI 2006. Distribution Maps of Plant Pests. Diabrotica balteata Commonwealth Agricultural Bureau, London. Map 681. Campos-Vargas, R., and M. E. Saltveit. 2002. Involvement of putative chemical wound signals in the induction of phenolic metabo lism in wounded lettuce. Physiol. Plant. 114: 73 Capellades, M., M. A. Torres, I. Bastisch, V. Stiefel, F. Vignols, W. R. Bruce, D. Peterson, P. Puigdomenech, and J. Rigau. 1996. The maize caffeic acid Omethyltransferase gene promoter is activ e in transgenic tobacco and maize plant tissues. Plant Mol. Biol. 31: 307-322. Capinera, J. L. 1999. Banded cucumber beetle. Featured Creatures [O nline]. Publication Number: EENY-105. University of Florid a, Department of Entomology and Nematology. http://creatures.ifas.ufl.edu/ve g/bean/banded_cucumber_beetle.htm Capinera, J. L. 2004. Green peach aphid. F eatured Creatures [Online]. Publication Number: EENY-222. University of Florid a, Department of Entomology and Nematology. http://creatures.ifas.ufl.e du/veg/aphid/green_peach_aphid.htm Capinera, J. L. 2005. Cabbage looper. Featur ed Creatures [Online] Publication Number: EENY-116. University of Florida, Depart ment of Entomology and Nematology. http://creatures.ifas.ufl.edu/ veg/leaf/cabbage_looper.htm Capinera, J. L. 2006. Beet armyworm. Featured Creatures [Online]. Publication Number: EENY-105. University of Florida, Depart ment of Entomology and Nematology. http://creatures.ifas.ufl.e du/veg/leaf/beet_armyworm.htm Carpita, N. C., and D. M. Gibeaut. 1993. Stru ctural models of primary cell walls in flowering plants: consistency of molecular struct ure with the physical properties of the walls during growth. Plant J. 3: 1-30. Cassab, G. I., and J. E. Varner. 1988. Cell wa ll proteins. Annu. Rev. Plant Physiol. Plant Mol. Biol. 39: 321-353. Chaman, M. E., S. V. Copaja, and V. H. Argandon. 2003. Relationships between salicylic acid content, phenylalanine ammonia-lyase (P AL) activity, and resistance of barley to aphid infestation. J. Agric. Food Chem. 51: 2227 -2231.

PAGE 177

177 Chaman M. E., L. J. Corcuera, G. E. Zuniga, L. Cardemil, V. H. Argandona. 2001. Induction of soluble and cell wall peroxidases by aphid in festation in barley. J. Agric. Food Sci. 49: 2249-2253. Chan, B. G., and A. C. Waiss. 1978. Condens ed tannin, an antibiotic chemical from Gossypium hirsutum J. Insect Physiol. 24: 113-118. Chan, B. G., A. C. Waiss, R. G. Binder, and C. A. Elliger. 1978. Inhibition of lepidopterous larval growth by cotton c onstituents. Entomol. Exp. Appl. 24: 94100. Chapman, R. F. 1974. The chemical inhibiti on of feeding by phytophagous insects: a review. Bull. Entomol. Res. 64: 339-363. Chapman, R. F. 2003. Contact chemorecepti on in feeding by phytophagous insects. Annu. Rev. Entomol. 48: 455. Chyb, S., H. Eichenseer, B. Hollister, C. A. Mullin, and J. L. Frazier. 1995. Identification of sensilla involved in taste mediatio n in adult western corn rootworm ( Diabrotica virgifera virgifera LeConte). J. Chem. Ecol. 21: 313-329. Clausen, T. P., P. B. Reichardt, J. P. Brya nt, R. A. Werner, K. Post, and K. Frisby. 1989. Chemical model for short-term induction in quaking aspen ( Populus tremuloides ) foliage against herbivores. J. Chem. Ecol. 15: 2335-2346. Cohen, A. C., C. C. Chu, T. J. Henneberr y, T. Freeman, D. Nelson, J. Buckner, D. Margosan, P. Vail, and L. H. Aung. 1 998. Feeding biology of the silverleaf whitefly (Homoptera: Aleyrodidae) Chin. J. Entomol. 18: 65-82. Cohen, A. C., T. J. Henneberry, and C. C. Chu. 1996. Geometric re lationships between whitefly behavior and vascular bundle ar rangements. Entomol. Exp. Appl. 78: 135142. Cole R. A. 1984. Phenolic acids associated w ith the resistance of le ttuce cultivars to the lettuce root aphid. Ann. Appl. Biol. 105: 129-145. Coley, P. D. 1983. Herbivory and defensive char acteristics of tree species in a lowland tropical forest. Ecol. Monographs 53: 209-233. Condon, J. M., and B. A. Fineran. 1989. Distri bution and organizati on of articulated laticifers in Calystegia silvatica (Convolvulaceae). Bot. Gaz. 150: 289-302. Constabel, C. P. 1999. A survey of herb ivore-inducible defense proteins and phytochemicals, pp. 137-166. In A. A. Agrawal, S. Tuzun and E. Bent [eds.], Induced plant defenses against pathogens and herbivores: biochemistry, ecology and agriculture. APS Press, St. Paul.

PAGE 178

178 Constabel, C. P., D. R. Bergey, and C. A. Ryan. 1995. Systemin activates synthesis of wound inducible tomato leaf polyphenol oxidase via the octadecanoid defense signaling pathway. Proc. Natl. Acad. Sci. USA 92: 407-411. Constabel, C. P., D. R. Bergey, and C. A. Ryan. 1996. Polyphenol oxidase as a component of the inducible defense res ponse in tomato against herbivores, pp. 231252. In J. T. Romeo, J. A. Saunders, and P. Barbosa [eds.], Phytochemical diversity and redundancy in ecologi cal interactions. Plen um Press, New York. Constabel, C. P., Y. Peter, P. Lynn, J. Joseph, Christopher, M. E. 2000. Polyphenol oxidase from hybrid poplar: cloning and expression in response to wounding and herbivory. Plant Physiol. 124: 285-296. Constabel, C. P., and C. A. Ryan. 1998. A survey of wound and methyl jasmonateinduced leaf polyphenol oxidase in cr op plants. Phytochemistry 47: 507-511. Corcuera, L. J. 1993. Biochemical basis fo r the resistance of barley to aphids. Phytochemistry 33: 741-747. Costa, H. S., D. E. Ullman, M. W. J ohnson, and B. E. Tabashnik. 1993. Association between Bemisia tabaci density and reduced growth, yellowing, and stem blanching of lettuce and ka i choy. Plant Dis. 77: 969-972. Cramer, C., K. Edwards, M. Dron, X. Liang, S. L. Dildine, G. P. Bolwell, R. A. Dixon, C. J. Lamb, and W. Schuch. 1989. Phenylal anine ammonia-lyase gene organization and structure. Plant Mol. Biol. 12: 367-383. Creighton, C. S., and E. R. Cuthbert, Jr. 1968. A semisynthetic diet for adult banded cucumber beetles. J. Econ. Entomol. 61: 337-338. Crosby, D. G. 1963. The organic constituents of food. 1. Lettuce. J. Food Sci. 28: 347355. Crozier, A., M. E. J. Lean, M. S. McDona ld, and C. Black. 1997. Quantitative analysis of the flavonoid content of commercial tomatoes onions, lettuce, and celery. J. Agric. Food Chem. 45: 590-595. Crute, L. R., and J. A. Dunn. 1980. An associa tion between the resistance to root aphid ( Pemphigus bursarius ) and downy mildew ( Bremia lactuca Regel) in lettuce. Euphytica 29: 483-488. Dangl, J. L., K. Harlbrock, and J. Schell. 1989. Regulation and stru cture of chalcone synthase genes, pp. 155-173. In J. K. Vasil and J. Schell [eds.], Cell culture and somatic cell genetics of plants, Academic Press, New York. Data, E. S., S. F. Nottingham, and S. J. Kays. 1996. Effect of sweetpotato latex on sweetpotato weevil (Coleoptera: Curculioni dae) feeding and oviposition. J. Econ. Entomol. 89: 544-549.

PAGE 179

179 Davis, R. M., K. B. Subbarao, and E. A. Kurtz. 1997. Compendium of lettuce diseases. APS Press, St. Paul, MN. de Candolle. 1885. Origin of cultivated plants, p. 95. Cited from E. L. Sturtevant. 1886. A study of garden lettuce. Am. Nat. 20: 230-233. Dethier, V. G. 1970. Chemical interactions between plants and insects, pp. 83-102. In E. Sondheimer, and J. B. Simeone [eds.], Chemical ecology. Academic Press, New York. Dey, P. M., and J. B. Harborne. 1997. Pl ant biochemistry. Academic Press, London. Diaz, J., and F. Merino. 1998. Wound-induced shikimate dehydrogenase and peroxidase related to lignification in pepper ( Capsicum annuum ) leaves. J. Plant Physiol. 152: 51-57. Dickenson, P. B. 1963. Structure composition and biochemistry of Hevea latex, pp. 4351. In L. Bateman [ed.], The chemistry a nd physics of rubber-like substances. Maclaren and Sons, London. Dillon, P. M., S. Lowrie, and D. McKey. 1983. Disarming the Evil woman: petiole constriction by a sphingid larva circumvents me chanical defenses of its host plant, Cnidoscolus urens (Euphorbiaceae). Biotropica 15: 112-116. Dimsey, R., and S. Vujovic. 2003. Lettuce growing. Agriculture notes (AG1119). Department of Primary Industr ies, Victoria, Australia. Dixon, R. A., and M. J. Harrison. 1990. Activa tion, structure, and organization of genes involved in microbial defense in plants. Adv. Genet. 28: 166-217. Dixon, R. A., M. J. Harrison, and N. L. Paiva. 1995. The isoflavonoid phytoalexin pathway: from enzymes to genes to transc ription factors. Physiol. Plant. 93: 385392. Dixon, R. A., and N. L. Paiva. 1995. Stre ss induced phenylpropanoid metabolism. Plant Cell 7: 1085-1097. Douglas, C. J. 1996. Phenylpropanoid metabolis m and lignin biosynthesis: from weeds to trees. Trends Plant Sci. 1:171-178. Dowd, P. F., and L. M. Lagrimini. 1997. Examination of different tobacco ( Nicotiana spp.) type under and overproducing tobacco anionic peroxidase for their leaf resistance to Helicoverpa zea J. Chem. Ecol. 23: 2357-2370. Duffey, S. S., and G. W. Felton. 1991. Enzyma tic antinutritive defenses of the tomato plant against insects, pp. 167-197. I n Hedin PA [ed.], Naturally occurring pest bioregulators. ACS Press, Washington, DC.

PAGE 180

180 Duffey, S. S., and M. J. St out. 1996. Antinutritive and toxic components of plant defense against insects. Arch. Insect Biochem. Physiol. 32: 3-37. Dunn, J. A. 1959. The biology of the lettuce root aphid. Ann. Appl Biol. 47: 475-491. Dunn, J. A. 1974. Study on inheritance of resistance to root aphid Pemphigus bursarius in lettuce. Ann. Appl. Biol. 76: 9-18. Dunn, J. A., and D. P. H. Kempton. 1980. Suscep tibilities to attack by top aphids in varieties of lettuce. A nn. Appl. Biol. 94: 58-59. Dupont, M. S., Z. Mondin, G. Williamson, and K.R. Price. 2000. Effect of variety, processing and storage on the flavonoi d glycoside content and composition of lettuce and endive. J. Agric. Food Chem. 48: 3957-3964. Dussourd, D. E. 1993. Foraging with finesse: ca terpillar adaptations for circumventing plant defenses, pp. 92-131. In N. E. Stamp and T. Casey [eds], Ecological and evolutionary constraints on caterpillars. Chapman and Hall, New York, USA. Dussourd, D. E. 1995. Entrapment of aphids and whiteflies in lettuce latex. Ann. Entomol. Soc. Am. 88: 163-172. Dussourd, D. E. 1997. Plant exudates trigge r leaf-trenching by cabbage loppers, Trichoplusia ni (Noctuidae). Oeco logia 112: 362-369. Dussourd, D. E. 2003. Chemical stimulants of leaf-trenching by cabbage loopers: natural products, neurotransmitters, insecticides, and drugs. J. Chem. Ecol. 29: 2023-2047. Dussourd, D. E., and R. F. Denno. 1991. Deactivation of plant defense: correspondence between insect behaviour and secretar y canal architecture. Ecology 72: 1383-1396. Dussourd, D. E., and R. F. Denno. 1994. Host ra nge of generalist cate rpillars: trenching permits feeding on plants with secretary canals. Ecology 75: 69-78. Dussourd, D. E., and A. M. Hoyle. 2000. Poisoned plusiines: toxicity of milkweed latex and cardenolides to some generalis t caterpillars. Chemoecology 10: 11. Dyer, W. E., J. M. Henstrand, A. K. Handa, and K. M. Herrmann. 1989. Wounding induces the first enzyme of the shikimate pathway in Solanaceae. Proc. Natl. Acad. Sci., USA 86: 7370-7373. Eenink, A. H., and F. L. Dieleman. 1982. Resistan ce of lettuce to leaf aphids: research on components of resistance, on differential interactions between plant genotypes, aphid genotypes and the environment and on th e resistance level in the field after natural infestation. Med. Fac. La ndbouww. Rijksuniv. Gent 47: 607-615.

PAGE 181

181 Eenink, A. H., F. L. Dieleman, J. H. Vi sser, and A. K. Minks. 1982. Resistance of Lactuca accessions to leaf aphids : components of resistan ce and exploitation of wild Lactuca species as sources of resistan ce, pp. 349-355. Proc. 5th Intl. Symp. Insect Plant Relationships, Wageningen, The Netherlands. Eichenseer, H., J. L. Bi, and G. W. Felton. 1998. Indiscrimination of Manduca sexta larvae to overexpressed and underexpresse d levels of phenylal anine ammonia-lyase in tobacco leaves. Entomol. Exp. Appl. 87: 73-78. Eichenseer, H., and C. A. Mullin. 1996. Ma xillary appendages used by western corn rootworms, Diabrotica virgifera virgifera, to discriminate between a phagostimulant and ?deterrent. Entomol. Exp. Appl. 78: 237-242. Ellard-Ivey, M., and C. Douglas. 1996. Role of jasmonates in the elicitorand woundinducible expression of defense genes in parsley and transgenic tobacco. Plant Physiol. 112: 183-192. Elliger, C. A., B. C. Chan, and A. C. Waiss. 1980. Flavonoids as larval growth inhibitors. Naturwissenschaften 67: 358-359. Ellis, P. R. 1991. The root of the problem. Grower 116: 11-13. Ellis, P. R., S. J. McClement, P. L. Saw, K. Phelps, W. E. Vice, N. B. Kift, D. Astley, and D. A. C. Pink. 2002. Identification of source of resistance in lett uce to the lettuce root aphid Pemphigus bursariu s. Euphytica 125: 305-315. Ellis, P. R., D. A. C. Pink, and A. D. Ra msey. 1994. Inheritance of resistance to lettuce root aphid in the lettuce cultivars 'Avoncri sp' and 'Lakeland'. Ann. Appl. Biol. 124: 141-151. Ellis, P. R., G. M. Tatchell, R. H. Collier, and W. E. Parker. 1996. Assessment of several components that could be used in an inte grated programme for controlling aphids on field crops of lettuce, pp. 91. Integrated control of field vegetable pests, vol. 19. IOBC Bull. Esau, K. 1965. Plant anatomy. John Wiley & Sons, New York, USA. Ester, A. 1998. Aphid resistan ce of butterhead lettuce tested in practice. PAV Bull. Vollegrondsgroenteteelt No. February, 6-8. Ester, A., J. Gut, A. M. van Oosten, and H. C. H. Pijnenburg. 1993. Controlling aphids in iceberg lettuce by alarm pheromone in combin ation with an insecticide. J. Appl. Ent. 115: 432. Evans, F. J., and R. J. Schmidt. 1976. Two new toxins from the latex of Euphorbia poissonii Phytochemistry 15: 333-335.

PAGE 182

182 Evert, R. F. 2006. Esau's plant anatomy, merist ems, cells, and tissues of the plant body: their structure, function, and development. John Wiley & Sons, Inc. New Jersey. Fagerstrm, T. 1989. Anti-herbivory chemical de fense in plants: A note of the concept of cost. Am. Nat. 133: 281-287. Fahn, A. 1979. Secretory tissues in plants Academic Press, New York, USA. Fahn, A. 1990. Plant anatomy. Pergamon Press, New York. Farrell, B. D., D. E. Dussourd, and C. M itter. 1991. Escalation of plant disease: do latex/resin canals spur plant dive rsification? Am. Nat. 138: 891-900. Feeny, P. P. 1976. Plant apparency and chemical defense, pp. 1-40. In J. W. Wallace, and R. L. Mansel [eds.], Biochemical intera ction between plants and insects: recent advances in phytochemistry. Plenum Press, New York. Felton, G., J. Bi, C. B. Summers, A. J. Mu eller, and S. Duffey. 1994b. Potential role of lipoxygenases in defense against insect herbivory. J. Chem. Ecol. 20: 651-666. Felton, G. W., K. K. Donato, R. M. Br oadway, and S. S. Duffey. 1992. Impact of oxidized plant phenolics on the nutritional qua lity of dietary protein to a noctuid herbivore, Spodoptera exigua. J. Insect Physiol. 38: 277-285. Felton, G. W., K. Donato, R. J. Del Vecchi o, and S. S. Duffey. 1989. Activation of plant foliar oxidases by insect feeding reduces nut ritive quality of foliage for noctuid herbivores. J. Chem. Ecol. 15: 2667-2694. Felton, G. W., C. B. Summers, and A. J. Mu eller. 1994a. Oxidative responses in soybean foliage to herbivory by bean leaf beetle and three-cornered alfalfa hopper. J. Chem. Ecol. 20: 639-650. Feng, Y., and C. E. McDonald. 1989. Comparison of flavonoids in bran of four classes of wheat. Cereal Chem. 66: 516-518. Ferreres, F., M. I. Gil, M. Castaner, and F. A. Tomas-Barberan. 1997. Phenolic metabolites in red pigmented lettuce: changes with minimal processing and cold storage. J. Agric. Food Chem. 45: 4249-4254. Fineran, B. A. 1982. Distribution and organization of non-articulat ed laticifers in mature tissues of poinsettia ( Euphorbia pulcherrima Willd.) Ann. Bot. 50:207-220. Fineran, B. A. 1983. Differentiation of non-ar ticulated laticifers in poinsettia ( Euphorbia pulcherrima Willd.). Ann. Bot. 52: 279-293. Fiorillo, F., C. Palocci, S. Soro, and G. Pasqua. 2007. Latex lipase of Euphorbia characias L.: An aspecific acylhydrolase with several isoforms. Plant Sci. 172: 722.

PAGE 183

183 Forbes, A. R., and J. R. Mackenzie. 1982. The lettuce aphid, Nasonovia ribisnigri (Homoptera: Aphididae) damaging lettuce cr ops in British Columbia. J. Entomol. Soc. British Columbia 79: 28. Fraenkel, G. S. 1959. The raison d'tre of s econdary plant substanc es. Science 129: 14661470. Frank, M. R., J. M. Deyneka, and M. A. Schuler. 1996. Cloning of wound-induced cytochrome P450 monooxygenases expresse d in pea. Plant Physiol. 110: 10351046. Freitas, C. D. T., J. S. Oliveira, M. R. A. Miranda, N. M. R. Macedo, M. P. Sales, L. A. Villas-Boas, and M. V. Ramos. 2007. Enzy matic activities and protein profile of latex from Calotropis procera Plant Physiol. Biochem. 45: 781-789. Frst, S., J. B. Harborne, and L. King. 1977. Identification of the flavonoids in five chemical races of cultivated barley. Hereditas 85: 163-167. Freund, R. J., and W. J. Wilson. 1997. Statis tical methods. Academic Press, Inc, San Diego. Fry, S. C. 1986. Cross-linking of matrix polymers in the growing cell walls of angiosperms. Annu. Rev. Plant Phys iol. Plant Mol. Biol. 37: 165-186. Fukasawa-Akada, T., S. Kung, and J. C. Watson. 1996. Phenylalanine ammonia-lyase gene structure, expression, and evolution in Nicotiana Plant Mol. Biol. 30: 711722. Galliard, T., and H. W. S. Chan. 1980. Lipoxygenases, pp. 132-161. In E. E. Conn, and P. K. Stumpf [eds.], The biochemistry of pl ants, vol. 4. Academic Press, New York. Gazeley, K. F., A. D. T. Gorton, and T. D. Pendle. 1988. Latex concentrates; properties and composition, pp-63-98. In A. D. Roberts [ed.], Natural rubber science and technology, Oxford University Press, New York. Genung, W. G. 1957. Some possible cases of insect resistance to insecticides in Florida. Proc. Fla. State Hortic. Soc. 70: 148-152. Gershenzon, J. 1994. The cost of plant chemical defense against herbivory: a biochemical perspective, pp. 105-173. In E. A. Bernays [ed.], Insect-plant interactions, 5. CRC Press, Inc., Boca Raton. Ghaffar, A., M. R. Attique, and M. R. Na veed. 2002. Effect of different hosts on the development and survival of Spodoptera exigua (Hubner) (Noctuidae: Lepidoptera). Pakistan J. Zool. 34: 229-231

PAGE 184

184 Gidrol, X., H. Chrestin, H. L. Tan, and A. Kush. 1994. Hevein, a le ctin like protein from Hevea brasiliensis (Rubber tree) is involved in co agulation of latex. J.Biol. Chem. 269:9278-9283 Gil, M. I., M. Castaner, F. Ferreres, F. Artes, and F. A. B. Thomas. 1998. Modified atmosphere packaging of minimally processed Lollo Rosso ( Lactuca sativa ) phenolic metabolites and quality changes. Z. Lebensm. Untere. Forsch. 206: 350354. Gilbert, L. E. 1971. Butterfl y plant coevolution: has Passiflora adenopod a won the selection race with heliconiin e butterflies? Science 172: 582-586. Gonzalez, A. G. 1977. Lactuceae chemical review, pp.1081-1095. In V. H. Heywood and J. B. Harborne [eds.], The biology a nd chemistry of the Compositae. Academic Press, New York. Green, T. R., and C. A. Ryan. 1972. Wound-induced proteinase inhibito r in plant leaves: a possible defense mechanism agai nst insects. Science 175: 776-777. Gregory, P., W. M. Tingey. 1981. Chemical m echanisms of potato resistance to the leafhopper. pp 95-99. In Breeding for Resistance to Insects and Mites. Canterbury, England: Proc. 2nd Eucarpia/IOBC Meeti ng of the Working Group Breeding for Resistance to Insects and Mites. Gromek, D. W. Kisiel, A. Klodzinska, and E. Chojnacka-Wojcik. 1992. Biologically active preparations from Lactuca virosa L. Phytother. Res. 6: 285-287. Guy R. H., N. C. Leppla J. R. Rye, C. W. Green, S. L. Barrette, and K. A. Hollien. 1985. Trichoplusia ni pp. 487-494. In Pritam Singh and R. F. Moore [eds.], Handbook of insect rearing, vol. 2. Elsevi er Science Publishers, Amsterdam. Guzman, V. L. 1986. Short Guzmaine, Tall Guzm aine and Florigade, three cos lettuce cultivars resistant to lettuce mosaic virus. IFAS Univ. Fla. Agric. Exp. Stn. Circ. S326. Hagerman, A. E., and L. G. Butler. 1991. Tannins and lignins, pp. 355-388. In G. A. Rosenthal, and M. R. Berenbaum [eds.] Herbivores: their interaction with secondary plant metabolites. Academic Press, San Diego. Hahlbrock, K., and D. Scheel. 1989. P hysiology and molecular biology of phenylpropanoid metabolism. Annu. Rev. Plan t Physiol. Plant Mol. Biol. 40: 347369. Hanny, B. W. 1980. Gossypol, flavonoid and c ondensed tannin content of cream and yellow anthers of five cotton ( Gossypium hirsutum L.) cultivars. J. Agric. Food Chem. 28: 504-506.

PAGE 185

185 Hanson, K. R. and E. A. Havir. 1979. An introduction to the enzymology of phenylpropanoid biosynthesis, p-91-138. In T. Swain, J. B. Harborne and C. F. Van Sumere [eds]. Biochemistry of plan t phenolics. Plenum Press, New York. Harborne, J. B. 1979. Fla vonoid pigments, pp. 619-655. In G. A. Rosenthal and D. H. Janzen, [eds]. Herbivores: their inter action with secondary plant metabolites. Academic Press, New York. Harborne, J. B. 1993. Introduction to ecological biochemistry. Academic Press, London. Harborne, J. B. 1994. The flavonoids, advan ces in research since 1986. Chapman and Hall, London. Harrewijn, F., and F. L. Dieleman. 1984. The importance of mineral nutrition of the host plant in resistance breeding to aphids, pp.235-243. In Proc. Sixth Intl. Cong. Soilless Cult. Lunteren, The Netherlands. Hartley, S. E., and J. H. Lawton. 1991. Bioc hemical aspects and significance of the rapidly induced accumulation of phenolics in birch foliage, pp.105-132. In D. W. Tallamy, and M. J. Raupp [eds.], Phytoc hemical induction by herbivores. John Wiley & Sons Inc., New York. Haupt, I. 1976. Separation of the sites of synthesis and accumulation of 3, 4dihydroxyphenylalanine in Euphorbia lathyris L. Nova Acta Leopoldina Supplementum 7: 129-132. Havill, N. P., and K. F. Raffa. 1999. Eff ects of elicitation treatment and genotypic variation on induced resistance in Popul us: impacts on gypsy moth (Lepidoptera: Lymantriidae) development and f eeding behavior. Oecologia 120: 295 Hayward, H. E. 1938. The structure of ec onomic plants. Macmillan and Company, New York. Hedin, P. A., J. N. Jenkins, D. H. Collum, W. H. White, W. L. Parrot, and M. W. MacGown. 1983. Cyanidin-3-glucoside, a newl y recognized basis for resistance in cotton to the tobacco budworm Heliothis virescens (Fab.) (Lepidoptera: Noctuidae). Experientia 39: 799-801. Hedin, P. A., J. N. Jenkins, A. C. Thompson, J. C. McCarty, D. H. Smith, W. L. Parrot, and R. L. Shepherd. 1988. Effect of bior egulators on flavonoids insect resistance and yield of seed cotton. J. Agric. Food Chem. 36: 1055-1061. Hedin, P. A., and S. K. Waage. 1986. Roles of flavonoids in plant resi stance to insects, pp?. In V. Cody, E. Middleton, and J. B. Ha rborne [eds.], Plant flavonoids in biology and medicine: biochemical, pha rmacological, and structure-activity relationships. Liss, New York.

PAGE 186

186 Heinrich, G. 1967. Lichtund elektron enmikroskopische Unterssuchungen der Milchrhren von Taraxacum bicorne Flora (Jena) Abt. A. 158: 413-420. Heitz, T., D. R. Bergey, and C. A. Ryan. 1997. A gene encoding a chloroplast-targeted lipoxygenase in tomato leaves is tran siently induced by wounding, systemin, and methyl jasmonate. Plan t Physiol. 114: 1085-1093. Heller, W., and G. Forkman. 1993. Bi osynthesis of flavonoids. pp. 499-535. In J. B. Harborne [ed.], The flavonoids, advances in research since 1986. Chapman and Hall, London. Helm, J. 1954. Lactuca sativa in morphologisch-systematisch er Sicht. Kulturpflanze 2: 72-129. Henderson, A. E., R. H. Hallett, and J. J. Soroka. 2004. Prefeeding behavior of the crucifer flea beetle, Phyllotreta cruciferae on host and nonhost crucifers. J. Insect Behav. 17: 17-39. Heng-Moss, T. M., G. Sarath, F. Baxenda le, D. Novak, S. Bose, X. Ni, and S. Quisenberry. 2004. Characterization of oxida tive enzyme changes in buffalograsses challenged by Blissus occiduus J. Econ. Entomol. 97: 1086-1095. Hennion, M.-C. 1999. Solid-phase extraction: method development, sorbents, and coupling with liquid chromatogra phy. J. Chromatogr. A, 856: 3-54. Hermann, K. 1976. Flavonols and flavones in f ood plants: a review. J. Food Technol. 11: 433-448. Hermann, K. 1988. On the occurrence of flavonol and flavone glycosides in vegetables. Z. Lebensen Unters. Forsch. 186: 1. Herms, D. A., and W. J. Mattson. 1992. The dile mma of plants: to grow or defend. Quart. Rev. Biol. 67: 283-335. Hertog, M. G. L., E. J. M. Fesens, P. C. H. Hollman, M. B. Katan, and D. Kromhout. 1993. Dietary antioxidant flavonoids and the risk of coronary heart disease: the zutphen elderly study. Lancet 342: 1007. Hertog, M. G. L., P. C. H. Hollman, and D. P. Venema. 1992. Optimization of quantitative HPLC determination of potenti ally anticarcinogenic flavonoids in fruit and vegetables. J. Agric. Food. Chem. 40: 1591. Hildebrand, D. F., J. G. Rodriguez, G. C. Brown, K. T. Luu, C. S. Volden. 1986. Peroxidative responses of leaves in tw o soybean genotypes injured by two spotted spider mites (Acari: Tetranychi dae). J. Econ. Entomol. 79: 1459-1465.

PAGE 187

187 Hochmuth, G., E. Hanlon, R. Nagata, G. Snyder, and T. Schueneman. 1994. Crisphead lettuce: fertilization reco mmendations for crisphead lettuce grown on organic soils in Florida. Gainesville, FL. Flor ida Coop. Extn. Serv. Bull. SP-153. Hohl, U., B. Neubert, and H. Pforte I. Schonhof, and H. Bhm. 2001. Flavonoid concentrations in the inner leaves of head lettuce genotypes. Eur. Food Res. Technol. 213: 205-211. Hsel, W. 1981. Glycosylat ion and glycosides, pp. 725-755. In P. K. Stumpf, and E. E. Conn [eds.], The biochemistry of plants vol. 7. Academic Press Inc., New York. Huang, J., H. J. McAuslane, and G. S. Nuessly. 2003a. Effect of leaf surface extraction on palatability of romaine lettuce to Diabrotica balteata Entomol. Exp. Appl. 106: 227-234. Huang, J., H. J. McAuslane, and G. S. Nuessly. 2003b. Resistance in lettuce to Diabrotica balteata (Coleoptera: Chrysomelidae): th e roles of latex and inducible defense. Environ. Entomol. 32: 9-16. Huang, J., G. S. Nuessly, H. J. McAuslane, and R. Nagata. 2003c. Effect of screening methods on expression of romaine lettu ce resistance to adult banded cucumber beetle, Diabrotica balteata (Coleoptera: Chrysomelidae). Florida Entomol. 86: 194-198. Huang, J., G. S. Nuessly, H. J. McAuslane, and F. Slansky. 2002. Resistance to adult banded cucumber beetle, Diabrotica balteata (Coleoptera: Chrysomelidae), in romaine lettuce. J. Econ. Entomol. 95: 849-855. Hunt, M., N. Eannetta, H. Yu, S. Newma n, and J. Steffens. 1993. cDNA cloning and expression of potato polyphenol oxi dase. Plant Mol. Biol. 21: 59. Hyodo, H., H. Kuroda, and S. F. Yang. 1978. I nduction of phenylalanine ammonia lyase and increase in phenolics in lettuce leaves in relation to the development of russet spotting caused by ethylene. Pl ant Physiol. 2: 31-35. Ikonen, A., J. Tahvanainen, and H. Roininen. 2001. Chlorogenic acid as an antiherbivore defense of willows against leaf b eetles. Entomol. Exp. Appl. 99: 47-54. Inglis, D. A. and E. Vestey. 2001. Cr op profile for lettuce in Washington. http://mtvernon.wsu.edu/plant_pathology/plant_path.htm Inoue, M., S. Sezaki, T. Sorin, and T. Sugi ura. 1985. Change of phenylalanine ammonialyase activity in strawberry leaves infe sted with the two-spotted spider mite, Tetranychus urticae Koch (Acarina : Tetranychidae) Appl. Entomol. Zool. 20: 348-349.

PAGE 188

188 Isidoro, N. B., J. Ziesmann, and I. H. W illiams. 1998. Antennal contact chemosensilla in Psylliodes chrysocephala responding to cruciferous allelochemicals. Physiol. Entomol. 23: 131. Ito, H., F. Kimizuka, A. Ohbayashi, H. Matsui M. Honma, A. Shinmyo, Y. Ohashi, A. B. Caplan, and R. L. Rodriguez. 1994. Molecu lar cloning and characterization of two complementary DNAs encoding putative peroxidases from rice ( Oryza sativa L.) shoots. Plant Cell Rep. 13: 361-366. Jhne, A., C. Fritzen, and G. Weissenbc k. 1993. Chalcone synthase and flavonoid products in primary-leaf tissues of rye and maize. Planta 189: 39-46. Jerez, M. I. 1998. Response of two maize inbred lines to chinch bug feeding. M.S. thesis. Mississippi State University Mississippi. Jimenez, M., and F. Garcia-Carmona. 1996. The effect of sodium dodecyl sulfate on polyphenol oxidase. Phyt ochemistry 42: 1503. Joerdens-Roettger, D. 1979. The role of phenolic substances for host selection behaviour of the black bean aphid, Aphis fabae Entomol. Exp. Appl. 26: 49-54. Jones, D. H. 1984. Phenylalanine ammonia lyase: regulation of its i nduction and its role in plant development. Phytochemistry 23: 1349-1359. Joos, H. J., and K. Hahlbrock. 1992. Phe nylalanine ammonia lyase in potato ( Solanum tuberosum L.). Genomic complexity, structural comparison of two selected genes and modes of expression. Euro. J. Biochem. 204: 621-629. Ke, D., and M. E. Saltveit. 1986. Effects of calcium and auxin on russet spotting and phenylalanine ammonia-lyase activity in iceberg lettuce. HortScience 21: 11691171. Ke, D., and M. E. Saltveit. 1988. Plant horm one interaction and phenolic metabolism in the regulation of russet s potting in iceberg lettuce. Plant Physiol. 88: 1136-1140. Ke, D., and M. E. Saltveit. 1989. Developmen tal control of russet spotting, phenolics enzymes, and IAA oxidase in cultivars of iceberg lettuce. J. Am. Soc. Hortic. Sci. 114: 472-477. Kekwick, R. G. O. 2001. Latex and laticifers, pp. 1-6. In Encyclopedia of Life Sciences. John Wiley & Sons, Lt d: Chichester (http://www.els.net/ ). Kennedy, J. S., M. F. Day, and V. F. Eastop. 196 2. A conspectus of aphids as vectors of plant viruses, pp. 1. Commonwealth Institute of Entomology, London. Kerns, D. L., M. E. Methron, J. C. Palumbo, C. A. Sanchez, D. W. Still, B. R. Tickes, K. Umeda, and M. A. Wilcox. 1999. Guidelines for head lettuce production in Arizona. Univ. Ariz. Coop. Extn. IPM, Ser.12.

PAGE 189

189 Kerns, D. L., and J. C. Palumbo. 1996. Le ttuce IPM: southwestern USA. Yuma Valley Agricultural Center, Universi ty of Arizona, Yuma, Arizona. Kim, J. H., and C. A. Mullin. 2003. Antifeedan t effects of protei nase inhibitors on feeding behaviors of adult western corn rootworm ( Diabrotica virgifera virgifera ). J. Chem. Ecol. 29: 795-810. Kinghorn, A., and F. Evans. 1975. A biological sc reen of selected species of the genus Euphorbia for skin irritant effects. Planta 28: 325-335. Kishaba, A. N., J. D. McCreight, D. L. C oudriet, T. W. Whitaker, and G. R. Pesho. 1980. Studies of ovipositional pr eference of cabbage looper on progenies from a cross between cultivated lettuce and prickly lett uce. J. Am. Soc. Hortic. Sci. 105: 890892. Kishaba, A. N., T. W. Whitaker, W. Berry, and H. H. Toba. 1976. Cabbage looper oviposition and survival of progeny on l eafy vegetables. HortScience. 11: 216-217. Kisiel, W., B. Barszcz, and E. Sznele r. 1997. Sesquiterpene lactones from Lactuca tatarica Phytochemistry 45: 365-368. Koes, R. E., F. Quattrocchio, and J. N. M. Mol. 1994. The flavonoid biosynthetic pathway in plants: function and evolution. Bioessays 16: 123-132. Kolattukudy, P. E. 1981. Structure, biosynthesis, and biodegradation of cutin and suberin. Annu. Rev. Plant Physiol. Pl ant Mol. Biol. 32: 539-567. Konno, K., C. Hirayama, M. Nakamura, K. Ta teishi, Y. Tamura, M. Hattori, and K. Kohno. 2004. Papain protects papaya trees fr om herbivorous insects: role of cysteine proteases in la tex. Plant J. 37: 370-378. Konno, K., H. Ono, M. Nakamura, K. Tateishi, C. Hirayama, Y. Tamura, M. Hattori, A. Koyama, and K. Kohno. 2006. Mulberry late x rich in antidiabetic sugar-mimic alkaloids forces dieting on caterpillars. Proc. Natl. Acad. Sci. USA 103: 1337-1341. Krysan, J. L. 1986. Introduction: biology, di stribution, and identification of pest Diabrotica, pp.1-23. In J.L. Krysan and T.A. Miller [eds.], Methods for the study of pest Diabrotica. Spri nger-Verlag, New York. Kurtz, E. A. 2001. Crop profile for iceberg lettuce in California. California Lettuce Research Board, Salinas, California. Kush, A., E. Goyvarets, M. L. Chye, and N. H. Chua. 1990. Lacticifers-specific gene expression in Hevea brasiliensis (Rubber tree). Proc, Natl. Acad. Sci. 87: 17871790.

PAGE 190

190 Kyndt, T., E. J. M. V. Damme, J. V. Beeumen, and G. Gheysen. 2007. Purification and characterization of the cysteine proteinase s in the latex of Vasconcellea spp. FEBS J. 274: 451-462. Lauritzen, E. 1999. Monterey county agricu ltural commissioners crop report. p. 32. Monterey County, Salinas, CA: Monterey Agricultural Commissioner. Lee, D., and C. J. Douglas. 1996. Two diverg ent members of a tobacco 4-coumarate: coenzyme A ligase (4CL) gene family. cDNA structure, gene inheritance and expression, and properties of recombinan t proteins. Plant Physiol. 112: 193-205. Leeper, P. W., T. W. Whitaker, and G. W. Bohan. 1963. Valmaine a new cos-type lettuce. Am. Veg. Grower, September, p.716. Leibee, G. I. 1981. Insecticidal control of Liriomyza spp. on vegetables, pp. 216-220. In D. J. Schuster [ed.], Proceedings IFAS industry conference on biology and control of Liriomyza leafminers, November 3-4, 198. Lake Buena Vista, Florida. Len-Gona alez, M. E., and L. V. Prez-Arriba s. 2000. Chemically modified polymeric sorbents for sample preconcentration. J. Chromatogr. A 902: 3-16. Lewinsohn, T. M. 1991. The geographical distri bution of plant latex. Chemoecology. 2: 64-68. Lindqvist, K. 1960. On the origin of cu ltivated lettuce. Hereditas 46: 319-350. Loaiza-Velarde, J. G., and M. E. Saltveit. 2001. Heat shocks applied either before or after wounding reduce browning of lettu ce leaf tissue. J. Amer Soc. Hortic. Sci. 126: 227-234. Loaiza-Velarde, J. G., F. A. Toms-Barb ern, and M. E. Saltveit. 1997. Effect of intensity and duration of heat-shock treatments on wound-induced phenolic metabolism in iceberg lettuce. J. Amer. Soc. Hortic. Sci. 122: 873-877. Lois, R., and K. Hahlbrock. 1992. Differen tial wound activation of members of the phenylalanine ammonia-lyase and 4-coumarat e:CoA ligase gene families in various organs of parsley plants. Z. Naturforsch. 47: 90-94. Lopez-Galvez, G. M. E. Saltveit, and M. Cantwell. 1997. Wound induced phenylalanine ammonia lyase aivity: Factor s affecting it inducton and co rrelation with the quality of minimally processed lettuce. Postharvest Biol. Technol. 9: 223-233. Lucas, P. W., I. M. Turner, N. J. Dominy, and N. Yamashita. 2000. Mechanical defenses to herbivory. Ann. Bot. 86: 913-920. Luckner, M. 1990. Secondary metabolism in microorganisms, plants, and animals. Gustav Fischer Verlag, Jena.

PAGE 191

191 Luh, B. S., and B. Phithakpol. 1972. Character istics of polyphenyl oxidase related to browning in cling peaches. J. Food. Sci. 37: 264-268. Markham, K. R. 1989. Flavones, flavonols and their glucosides. Methods Plant Biochem. 1: 197-232. Martin, C., L. Schoen, C. Rufingier, and N. Pasteur. 1996. A contribution to the integrated pest management of the aphid Nasonovia ribisnigri in salad crops. Integrated Control of Field Vege table pests. IOBC Bull. 19: 98. Martin, M. N. 1991. The latex of Hevea brasiliensis contains high levels of both chitinases and chitinases/lysozy mes. Plant Physiol. 95: 465-476. Martin, P. B., P. D. Lingren, and G. L. Greene. 1976. Relative abundance and host preferences of cabbage l ooper, soybean looper, tobacco budworm, and corn ear worm on crops grown in northern Florida. Environ. Entomol. 5: 878-882. Matile, P. 1976. Localizations of alkaloid s and mechanism of their accumulation in vacuoles of Chelidonium majus laticifers. Nova Acta Leopoldina Supplementum 7: 65-73. Mayer, A. M., and E. Harel. 1979. Polyphenol oxidases in plants. Phytochemistry 18: 193-215. McCabe, M. S., Garratt, L. C., F. Schepers, W. J. R. M. Jordi, G. M. Stoopen, E. Davelaar, J. H. van Rhijn, J. B. Po wers, and M. R. Davey. 2001. Effects of PSAG12-IPT gene expression on development and sequence in transgenic lettuce. Plant Physiol. 127: 505-516. McDougall, S., T. Napier, J. Valenzisi, A. Watson, J. Duff, G. Geitz, and T. Franklin. 2002. Integrated pest management in lettuce: information guide, pp. 154. NSW Agriculture, Orange, Australia. Metcalf, C. L., and W. P. Flint. 1962. Destru ctive and useful insects, their habits and control, 4th edition, Mc Graw-Hill, San Francisco. Metcalfe, C. R. 1967. Distribution of latex in the plant kingdom. Econ. Bot. 21:115-127. Metcalfe, C. R., and L. Chalk. 1983. Anatom y of the dicotyledons, vol. II. Clarendon, Oxford, England. Miles, C. J., and R. J. Pfeuffer. 1997. Pes ticides in canals of South Florida. Arch. Environ. Contam. Toxicol. 32: 337-345. Miller, H., Porter, D. R., Burd, J. D ., Mornhinweg D. W., Burton, R. L. 1994. Physiological effects of Russian wheat a phid (Homoptera: Aphi didae) on resistant and susceptible barley. J. Econ. Entomol. 87: 493-499.

PAGE 192

192 Miller, N. J., A. J. Birley, A. D. J. Over all, and G. M. Tatchell. 2003. Population genetic structure of the lettuce root aphid, Pemphigus bursarius (L.), in relation to geographic distance, gene flow and host plant usage. Heredity 91: 217. Mizutani, M., D. Ohta, and R. Sato. 1997. Is olation of a cDNA and a genomic clone encoding cinnamate 4-hydroxylase from Arabidopsis and its expression manner in planta. Plant Physiol. 113:755-763. Moerschbacher, B. M., U. Noll, L. Go rrichon, and H. J. Reisener. 1990. Specific inhibition of lignification breaks hypersensit ive resistance of wheat to stem rust. Plant Physiol. 93: 465-470. Mollema, C., and R. A. Cole. 1996. Low aroma tic amino acid concentrations in leaf proteins determine resistance to Frankliniella occidentali s in four vegetable crops. Entomol. Exp. Appl. 78: 325-333. Monacelli, B., A. Valletta, N. Rascio, I. Moro, and G. Pasqua. 2005. Laticifers in Camptotheca acuminata Decne: distribution and st ructure. Protoplasma 226: 155 161. Monnet, Y., and J. F. Ricateau. 1997. La lutte a phicide raisone en cultures de laitues de plein champs: bilan de trois annes de pratique. Quatrime Conference Internationale sur les Ravageurs en Agricu lture. Montpellier, France, 6 January. 2: 497. Montllor, C. B., and W. F. Tjallingii. 1989. Stylet penetration by two aphid species on susceptible and resistant lettu ce. Entomol. Exp. Appl. 52: 103-111. Morrow, P. A., and L. R. Fox. 1980. Effect of variation in eucalyptus essential oil on insect growth and grazing damage. Oecologia 45: 209-219. Mossler, M. A., and E. Dunn. 2005. Florida crop /pest management profile: lettuce. Univ. Fla. IFAS Extn. http://edis.ifas.ufl.edu/PI070 Mou, B., and Y. B. Liu. 2003. Leafminer resi stance in lettuce. Ho rtScience 38: 570-572. Mou, B., and E. J. Ryder. 2003. Screening and breeding for resi stance to leafminer ( Liriomyza langei ) in lettuce a nd spinach, pp. 43-47. In Proc. Eucarpia Meeting leafy vegetables Gen. Breeding, the Ne therlands, 19-21 March, 2003. Centre for Genetic Resources, The Netherlands. Mou, B., E. J. Ryder, J. Tanaka, Y. B. Liu, and W. E. Chaney. 2004. Breeding for resistance to leafminer in lettuce. Acta Hort. 637: 57-62. Moussaoui, A. El, M. Nijs, C. Paul, R. Win tjens, J. Vincentelli, M. Azarkan and Yvan Looze. 2001. Revisiting the enzymes stored in the laticifers of Carica papaya in the context of their possible pa rticipation in the plant defense mechanism. Cell Mol. Life Sci. 58: 556.

PAGE 193

193 Mura A., R. Medda, S. Longu, G. Floris, A. C. Rinaldi, and A. Padiglia. 2005. A Ca2+/calmodulin-binding peroxidase from Euphorbia latex: nove l aspects of calcium-hydrogen peroxide cross-talk in the regulation of plant defenses. Biochemistry 44: 14120-14130. Mura, A., F. Pintus, R. Medda, G. Floris, A. C. Rinaldi, and A. Padiglia. 2007. Catalase and Antiquitin from Euphorbia characias : two proteins involved in plant defense? Biochemistry 72: 501-508. Nagata, R. T., L. M, Wilkinson, and G. S. Nuessly. 1998. Longevity, fecundity, and leaf stippling of Liriomyza trifolii (Diptera: Agromyzidae) as affected by lettuce cultivar and supplemental feeding. J. Econ. Entomol. 91: 999-1004. Nawrot, J., E. B oszyk, J. Harmatha, L. Novotn, and B. Dro d 1986. Action of antifeedants of plant origin on beetles infesting stored products. Acta Entomol. Bohemoslov. 83:327. Nebreda, M., A. Moreno, N. Prez, I. Palaci os, V. Seco-Fernndez, and A. Fereres. 2004. Activity of aphids associated with lettuce and broccoli in Spain and their efficiency as vectors of Lettuce mosaic virus. Virus Res. 100: 83. Nelson, C. J., J. N. Seiber, and L.P. Brower 1981. Seasonal and intr aplant variation of cardinolide content in California milkweed, Asclepias eriocarpa and implications for plant defense. J. Chem. Ecol. 7: 981-1010. Ni, X, S. S. Quisenberry, T. Heng-Moss, J. Markwell, G. Sarath, R. Klucas, and F. Baxendale 2001. Oxidative responses of resi stant and susceptible cereal leaves to symptomatic and nonsymptomatic cereal aphid (Hemiptera: Aphididae) feeding. J.Econ. Entomol. 94: 743-751. Nicholson, R. L., and R. Hammerschmidt. 1992. Phenolic compounds and their role in disease resistance. Annu. Rev. Phytopath. 30: 369-389. Nielson, P. E., H. Nishimura, J. W. Otvos, and M. Calvin. 1977. Plant crops as a source of fuel and hydrocarbon like ma terials. Science 198: 942-944. Nishida, R., T. Ohsugi, S. Kokubo, and H. Fukami. 1987. Oviposition stimulants of a citrus-feeding swallowtail butterfly, Papilio xuthus L. Experientia 43: 342-344. Nishio, S., M. S. Blum, and S. Takahashi. 1983. Intraplant distributi on of cardenolides in Asclepias humistrata (Asclepiadaceae) w ith additional notes on their fates in Tetraopes melanurus (Coleoptera: Cerambycidae) and Rhyssomatus lineaticollis (Coleoptera: Curculionidae). Mem. Coll. Agric. Kyoto Univ. 122: 43-52. Noack, E. A., A. E. G. Cera, and G. Fals one. 1980. Inhibition of mitochondrial oxidative phosphorylation by 4-deoxyphorbol trimester, a poisonous constituent of the latex sap of Euphorbia iglandulosa Desf. Toxicon 18: 165-174.

PAGE 194

194 Norris, R. F., E. P. Caswell-Chen, and M. Kogan. 2003. Concepts in integrated pest management. Prentice-Hall, New Jersey. Nuessly, G. S., and R. T. Nagata. 1993. Eval uation of damage by serpentine leafminer and banded cucumber beetle to cos lettuce. Everglades Res. Ed. Center Res. Rpt., EV-1993. 2: 76-77. Nuessly, G. S., and R. T. Nagata. 1994. Di fferential probing response of serpentine leafminer, Liriomyza trifolii (Burgess), on cos lettuce. J. Entomol. Sci. 29: 330-338. Nuessly, G. S., and S. E. Webb. 2003. Insect management for leafy vegetables (lettuce, endive and escarole). Univ. Fla. IFAS Extn. http://edis.ifas.ufl.edu/IG161 Nutt, K. A., M. G. OShea, and P. G. Allsopp. 2004. Feeding by sugarcane whitegrubs induces changes in the type s and amounts of phenolics in the roots of sugarcane. Environ. Exp. Bot. 51: 155-165. Olson, K. C., T. W. Tibbitts, and B. E. Struckmeyer. 1969. Leaf histogenesis in Lactuca sativa with emphasis upon laticifer ont ogeny. Amer. J. Bot. 56: 1212-1216. Orr, J. D., R. Edwards, and R. A. Dixon. 1993. Stress responses in alfalfa ( Medicago sativa L.) XIV. Changes in the levels of phenylpropanoid pathway intermediates in relation to regulation of l-phenylalanine ammonia-lyase in elicitor-treated cellsuspension cultures. Plant Physiol. 101: 847-856. Palumbo, J., A. Fournier, P. Ellsworth, K. Nolte, and P. Clay. 2006. Insect crop losses and insecticide usage for head lettuce in Arizona: 2004 2006. University of Arizona College of Agricultu re 2006 Vegetable Report. http://cals.arizona.e du/pubs/crops/az1419/ Panda, N., and G. S. Kush. 1995. Host plant re sistance to insects. CAB International, Wallingford, Oxon, U.K. Parenzan, P. 1984. Noctuidae (Lepidoptera, He terocera) of southern Italy (addenda). Entomologica 19: 97-134. Parihar, S. B. S., and O. P. Singh. 1992. Role of host plants in development and survival of Heliothis armigera (Hubner). Bull. Entomol. New Delhi. 33: 74-78. Parker, W. E., R. H. Collier, P. R. Ellis, A. Mead, D. Chandler, J. A. Blood Smyth, and G. M. Tatchell. 2002. Matching control options to a pest complex: the integrated pest management of aphids in sequentia lly-planted crops of outdoor lettuce. Crop Prot. 21: 235. Parrella, M. P., and B. C. Keil. 1984. In sect pest management: the lesson of Liriomyza Bull. Entomol. Soc. Am. 30: 22-25.

PAGE 195

195 Patanakamjorn, J. and M. D. Pathak. 1967. Vari etal resistance of ri ce to the Asiatic rice borer, Chilo suppressalis (Lepidoptera: Crambidae), and its association with various plant characters. Ann. Entomol. Soc. Am. 60: 287-292. Pechan, T., A. Cohen, W. P. Williams, and D. S. Luthe. 2002. Insect feeding mobilizes a unique plant defense protease that disrupts the peritrophic matrix of caterpillars. Proc. Natl. Acad. Sci. USA 99: 13319-13323. Pechan, T., L. Ye, Y. Chang, A. Mitra, L. Li n, F. M. Davis, W. P. Williams, and D. S. Luthe. 2000. A unique 33-kDa cysteine proteinase accumulates in response to larval feeding in maize genotypes resi stant to fall armyworm and other Lepidoptera. Plant Cell 12: 1031-1040. Peiser, G., G. Lopez-Galvez, M. Cantwe ll, and M. E. Saltveit. 1998. Phenylalanine ammonia lyase inhibitor controls browni ng of cut lettuce. Postharvest Biol. Technol. 14: 171-177. Pellegrini, L., O. Rohfritsch, B. Fritig, and M. Legrand. 1994. Phenylalanine ammonialyase in tobacco. Molecula r cloning and gene expression during the hypersensitive reaction to tobacco mosaic virus and the res ponse to a fungal elicitor. Plant Physiol. 106: 877-886. Peng, S. Z., and P. W. Miles. 1988. Accepta bility of catechin and its oxidative condensation products to the rose aphid, Macrosiphum rosae Entomol. Exp. Appl. 47: 225-265. Peterson, G. L. 1977. A simplifica tion of the protein assay method of Lowry et al. which is more generally applicable. Anal. Biochem. 83: 346-356. Pichon, V. 2000. Solid-phase extraction fo r multiresidue analysis of organic contaminants in water. J. Chromatogr. A 885: 195. Pinaeus, A. 1561. Hist. Plants. Cited from E. L. Sturtevant. 1886. A study of garden lettuce. Am. Nat. 20: 230-233. Pitre, N. H., Jr., and E. J. Kantack. 19 62. Biology of the banded cucumber beetle, Diabrotica balteata in Louisiana. J. Econ. Entomol. 55: 904-906. Price, K. R., M. S. Dupont, R. Shepherd, H. W. S. Chan, and G. R. Fenwick. 1990. Relationship between chemical and sensor y properties of exotic salad crops: colored lettuce ( Lactuca sativa ) and chicory ( Cichorium intybus ). J. Sci. Food Agric. 53: 185-192. Pujade-Renaud, V., A. Clement, C. PerrotRechenmann, J.-C.Prevt, H. Chrestin,J.-L. Jacob, and J. Cuern. 1994. Ethylene-induced increase in glutamine synthetase activity and mRNA levels in Hevea brasiliensis latex cells. Plant Physiol.105: 127132.

PAGE 196

196 Pullin, A. S. 1987. Changes in leaf quality following clipping and regrowth of Urtica dioica and consequences for a specialist herbivore, Aglais urticae Oikos 49: 39-45. Rafi, M. M., R. S. Zemetra, and S. S. Quisenberry. 1996. Interaction between Russian wheat aphid (Homoptera: Aphididae) and resistant and susceptible genotypes of wheat. J. Econ. Entomol. 89: 239-246. Ramos, M. V., C. D. T. Freitas, F. Staniscuaski, L. L. P. Macedo, M. P. Sales, D. P. Sousa, and C. R. Carlini. 2007. Performance of distinct crop pe sts reared on diets enriched with latex proteins from Calotropis procera : Role of laticifer proteins in plant defense. Plant Sci. 173: 349. Razem, F. A., and M. A. Bernards. 20 02. Hydrogen peroxide is required for poly(phenolic) domain formation during wound induced suberization. J. Agric. Food Chem. 50: 1009-1015. Rees, C. J. C. 1969. Chemoreceptor specificity a ssociated with choice of feeding site by the beetle, Chrysolina brunsvicensis on its food plant, Hypericum hirsutum Entomol. Exp. Appl. 12: 565-583. Rees, S. B., and J. B. Harborne. 1985. The ro le of sesquiterpene lactones and phenolics in the chemical defense of the chicory plant. Phytochemistry 24: 2225-2231. Reinink, K., and F. L. Dieleman. 1989. Resist ance in lettuce to the leaf aphids Macrosiphum euphorbiae and Uroleucon sonchi Ann. Appl. Biol. 115: 489-498. Reinink, K., and F. L. Dieleman. 1993. Survey of aphid species on lettuce. Bull. OILB SROP 16: 56-68. Reinink, K., F. L. Dieleman, and R. Groenwol d. 1995. Inheritance of partial resistance to the leaf aphids Macrosiphum euphorbiae and Uroleucon sonchi in lettuce. Ann. App. Biol. 127: 413-424. Reinink, K., F. L. Dieleman, J. Jansen, and A. M. Montenarie. 1989. Interactions between plant and aphid genotypes in resistance of lettuce to Myzus persicae and Macrosiphum euphorbiae Euphytica 43: 215-222. Rhoades, D. F. 1979. Evolution of plant chem icals defense against herbivores, pp. 3-54. In G. A. Rosenthal, and D. H. Janzen [e ds.], Herbivores: thei r interaction with secondary plant metabolites. Academic Press, New York. Rhodes, M. J. C., L. S. C. Wooltorton, and A. C. Hill. 1981. Changes in phenolic metabolism in fruit and vegeta ble tissue under stress. pp. 193-220. In J. Friend and M. J. C. Rhodes [eds.] Recent advances in the biochemistry of fruits and vegetables. Academic Press, London. Ribereau-Gayon, P. 1972. Plant phenolics. Oliver and Boyd, Edinburgh.

PAGE 197

197 Richardson, M. 1991. Seed storage proteins: the enzyme inhibitors. Methods Plant Biochem. 5: 259-305. Ridland, P., S. Vujovic, C. Murdoch, P. Williams, F. Goubran, R. Dimsey, and L. Zirnsak. 2002. Improving lettuce insect pest managementVictoria. Department of Natural Resources and Environm ent. Victoria, Australia. Ridsdill-Smith, T. J., Y. Jiang, and E. L. Ghisalberti. 1995. A method to test compounds for feeding deterrence towards red-legged ear th mite (Acarina: Penthaleidae). Ann. Appl. Biol. 127: 593-600. Rob, K. L. 1989. Analysis of Frankliniella occidentalis (Pergande) as a pest of floricultural crops in Cali fornia greenhouses. Ph.D. Dissertation, University of California, Riverside. Roberts, M. F. 1987. Papaver latex an d alkaloid storage vacuoles, pp. 513-528. In B. Marin [ed.], Plant vacuoles: their importance in solute compartmentation in cells and their applications in plant biotechnology. Plenum, New York. Robinson, T. 1972. The organic constituent of higher plants. Cordus Press. North Amherst, Mass. Robison, D. J., and K. F. Raffa. 1997. Effect of constitutive and inducible traits of hybrid poplars on forest tent cater pillar feeding and populati on ecology. For. Sci. 40: 686 714. Romani, A., P. Pinelli, C. Galadi, G. Sani A. Cimato, and D. Heimler. 2002. Polyphenols in green house and open air grow n lettuce. Food Chem. 79: 337-342. Rosenthal, G. A., and M. R. Berenbaum. 1991. Herbivores: their interaction with secondary plant metabolites. 2nd ed. Vol. 1. The chemical participants. Academic Press, New York. Rufingier, C., L. Schoen, C. Martin, and N. Pasteur. 1997. Resistance of Nasonovia ribisnigri (Homoptera: Aphididae) to five inse cticides. J. Econ. Entomol. 90: 14451449. Rumeau, D., E. A. Maher, A. Kelman, and A. M. Showalter. 1990. Extension and phenylalanine ammonia-lyase ge ne expression altered in po tato tubers in response to wounding, hypoxia, and Erwinia carotovora infection. Plant Physiol. 93: 11341139. Rutherford, R. S. 1998. Prediction of re sistance in sugarcane to stalk borer Eldana saccharina by near-infrared spectroscopy on cr ude budscale extracts: involvement of chlorogenates and flavonoids. J. Chem. Ecol. 24: 1447-1463. Ryan, C. A. 1990. Protease inhi bitors in plants: genes fo r improving defenses against insects and pathogens. A nnu. Rev. Phytopath. 28: 425-449.

PAGE 198

198 Ryder, E. J. 1998. Lettuce, endive and chicory. CABI Publishing Cambridge, UK. Saba, F. 1970. Host plant spectrum and temperature limitations of Diabrotica balteata Can. Entomol. 102: 684-691. Sadasivan S., and B. Thayumanavan. 2003. Mole cular host plant resistance to pests. Marcel Drekker, Inc. Basel. Saltveit M. E., Y.-J Choi, and F. A. Thom as-Barbern. 2005. Involvement of components of the phospholipids-signaling pathway in wound phenlproponoid metabolism in lettuce ( Lactuca sativa ) leaf tissue. Physiol. Plant. 125: 345-355. SAS Institute. 1999. Guide for personal computers, version 6. SAS Institute, Cary, NC. SAS Institute. 2003. Guide for personal computers, version 9.1.3. SAS Institute, Cary, NC. Schalk, J. M. 1986. Rearing and handling of Diabrotica balteata pp. 49-56. In J. L. Krysan and T. A. Miller [eds.] Methods for the study of pest Diabrotica Springer, NewYork. Schalk, J. M., J. R. McLaughlin, and J. H. Tumlinson. 1990. Field response of feral male banded cucumber beetles to the sex ph eromone 6,12-dimethylpentadecan-2-one. Fla. Entomol. 73: 292-297. Schalk, J. M., A. Jones, and P. D. Dukes. 1986. Factors associated with resistance in recently developed sweet potato cultivars and germplasm to the banded cucumber beetle, Diabrotica balteata LeConte. J. Agric. Entomol. 3: 329-334. Schenck, P. 1966. Szintigraphische Darste llung des parasternalen Lymphsystems. Strahlentherapie 130: 504. Schoonhoven, L. M., J. J. A. Loon, and M. Dicke. 2005. Insect plant biology. Oxford Press, New York. Scriber, J. M. 1977. Limiting effects of low leaf water content on nitrogen utilization, energy budget and larval growth of Hyalophora cecropia (Lepidoptera: Saturniidae). Oecologia 28: 269-287. Scriber, J. M., and F. Slansky Jr. 1981. Th e nutritional ecology of immature insects. Annu. Rev. Entomol. 26: 183-211. Seiber, J. N., C. J. Nelson, and S. M. Lee. 1982. Cardenolides in the latex and leaves of seven Ascelpes species and Calotropis procera Phytochemistry 21: 2343-2348. Sessa, R. A., M. H. Bennett, M. J. Lewis, J. W. Mansfield, and M. H. Beale. 2000. Metabolite profiling of ses quiterpene lactones from Lactuca species J. Biol. Chem. 275: 26877-26884.

PAGE 199

199 Sethi, A., H. J. McAuslane, R.T. Nagata, and G. S. Nuessly. 2006. Host plant resistance in romaine lettuce affects feeding behavior and biology of Trichoplusia ni and Spodoptera exigua (Lepidoptera: Noctuidae). J. Econ. Entomol. 99: 2156-2163. Sethi, A., H. J. McAuslane, R.T. Nagata, and G. S. Nuessly. 2007. Romaine lettuce latex deters banded cucumber beetle (Coleopt era: Chrysomelidae) feeding. Entomol. Exp. Appl. (In press). Seto, M., T. Miyase, K. Umehara, A. Uneno, Y. Hirano, and N. Otani. 1988. Sesquiterpenes lactones from Cichorium endivia L. and C. intybus L. and cytotoxic activity. Chem. Pharm. Bull. 36: 2423-2428. Shahidi, F., and P. K. Wanasundara. 1992. Ph enolic antioxidants. Crit. Rev. Food Sci. Nutr. 32: 67. Sharma, H. C., and R. Ortiz. 2002. Host plan t resistance to insects: An eco-friendly approach for pest management and envir onment conservation. J. Environ. Biol. 23: 111. Sherman, T. D., K. C. Vaughn, and S. O. Duke. 1991. A limited survey of the phylogenetic distribution of polyphenol oxidase. Phytochemistry 30: 2499-2506. Showalter, A. M. 1993. Structure and function of plant cell wall protei ns. Plant Cell 5: 923. Shukla, O. P., and C. R. Krishna-Murti. 1971. The biochemistry of plant latex. J. Sci. Indus. Res. 12: 640-662. Shulke, R. H., and L. L. Murdock. 1983. Lipoxyge nase, trypsin inhibitor and lectin from soybeans: effects on larval growth of Manduca sexta (Lepidoptera: Sphingidae). Environ. Entomol. 12: 787-791. Siedow, J. 1991. Plant lipoxygenase: structur e and function. Annu. Re v. Plant Physiol. Plant Mol. Biol. 42: 145-188. Simmonds, M. S. J. 2003. Flavonoidinsect in teractions: recent advances in our knowledge. Phytochemistry 64: 21-30. Simpson, N. J. K. 2000. Solid phase extraction principles, strategies and applications Marcel Dekker, New York. Siomos, A. S., P. P. Papadopoulou, C. C. D ogras, E. Vasiliadis, A. Dosas and N. Georgiou. 2002. Lettuce composition as affected by genotype and leaf position. Acta Hort. 579: 635-639. Sirinphanic, J. and A. A. Kader. 1985. E ffects of total CO2 on total phenolics, phenylalanine ammonia lyase and polyphenol oxidase in lettuce tissue. J. Amer. Soc. Hort. Sci. 110: 249-253.

PAGE 200

200 Skogsmyr, I., and T. Fagerstrm. 1992. The cost of anti-herbivory defense: an evaluation of some ecological and physiol ogical factors. Oikos 64: 451-457. Slansky, Jr. F. 1992. Allelochemical-nutritien t interactions in he rbivore nutritional ecology, pp. 135-174. In G. A. Rosenthal and M. R. Berenbaum (eds.), Herbivores: their interaction with secondary plant metabolites, 2nd ed. vol. 1: The chemical participants. Academic Press, New York. Small, J. 1916. The translocation of latex and the multiple razor. New Phytol. 15: 194199. Smith, C. G., M. W. Rodgers, A. Zimmer lin, D. Ferdinando, and G. P. Bolwell. 1994. Tissue and subcellular immunol ocalisation of enzymes of lignin synthesis in differentiating and wounded hypocotyl tissue of french bean (Phaseolus vulgaris L.). Planta 192: 155-164. Smith, C. M. 1989. Plant resistance to inse cts: a fundamental approach. John Wiley & Sons, Inc., New York. Sokal, R. R., and F. J. Rohlf. 1995. Bi ometry. W. H. Freeman & Co., New York. Somssich, I. E., P. Wernert, S. Kiedrowski, and K. Hahlbrock. 1996. Arabidopsis thaliana defense related protein EL13 is an aromatic alcohol: NADP+ oxidoreductase. Proc. Natl. Acad. Sci. USA 93: 14199-14203. Spencer, H. J. 1939. On the nature of the blocki ng of the lactiferous system at the leaf base of Hevea brasiliensis Ann. Bot. 3: 231-235. Spilatro, S. R., and P. G. Mahlberg 1986. La tex and lacticifer starch content of developing leaves of Euphorbia pulcherrima Amer. J. Bot. 73: 1312-1318. Stapleton, A. E., and V. Walbot. 1994. Fla vonoids can protect maize DNA from the induction of ultraviolet radiation damage. Plant Physiol. 105: 881-889. Steffens, J. C., and D. S. Walters. 1991. Biochemical aspects of glandular trichomemediated insect resistance in the Solanaceae, pp 136-149. In P. A. Hedin [ed]: Naturally occurring pest bi oregulators. ACS Symp. Ser. 489. Washington, DC: American Chemical Society. Steffens, J., E. Harrel, and M. Hunt. 1994. Polyphenol oxidase, pp. 276-304. In B. E. Ellis, G. W. Kuroki, and H. A. Stafford [eds.], Genetic engineering of plant secondary metabolism. Plenum Press, New York. Stobiecki, M., P. Wojtaszek, and K. Gu lewicz. 1997. Application of solid phase extraction for profiling quinolizidin e alkaloids and phenolic compounds in Lupinus albus Phytochem.Anal. 8: 153.

PAGE 201

201 Stotz, H. U., J. Kroymann, and T. Mitchell -Olds. 1999. Plant-insect interactions. Curr. Opin. Plant Biol. 2: 268-272. Stout, M. J., A. L. Fidantsef, S. S. Duffe y, and R. M. Bostock. 1999. Signal interactions in pathogen and insect attack: systemic plant-mediated in teractions between pathogens and herbivores of the tomato, Lycopersicon esculentum Physiol. Mol. Plant Pathol. 54: 115-130. Stout, M. J., J. Workman, and S. S. Duffe y. 1994. Differential inducti on of tomato foliar proteins by arthropod herbivores J. Chem. Ecol. 20: 2575-2594. Strauss, S. Y., and A. A. Agrawal. 1999. The ecology and evolution of plant tolerance to herbivory. Trends Ecol. Evol. 14: 179-185. Strid, A., W. S. Chow, and J. M. Anders on. 1994. UV-B damage and protection at the molecular level in plants. Photosynthesis Res. 39: 475-489. Sturtevant, E. L. 1886. A study of ga rden lettuce. Am. Nat. 20: 230-233. Swain, R. 1977. Secondary compounds as prot ective agents. Annu. Rev. Plant. Physiol. 28: 279-501. Swain, T. 1979. Tannins and lignins, pp. 657-682. In G. A. Rosenthal and D. H. Janzen [eds.], Herbivores: their interaction with secondary plant metabolites. Academic Press, San Diego. Swift, J. E., and W. H. Lange. 1980. Lettuce root aphid, p. 2. Leaflet No. 2668, University of California. Taira, T., A. Ohdomari, N. Nakama, M. Sh imoji, and M. Ishihara. 2005. Characterization and antifungal activit y of Gazyumaru ( Ficus microcarpa ) latex chitinases: both the chitin binding and antifungal activities of class I chitinase are reinforced with increasing ionic strength. Biolsci. Biotechnol.Biochem. 69: 811-818. Taiz, L., and E. Zeiger. 1991. Plant physiology. The Benjamin/Cummings Publishing Comp. Inc., Redwood City. Takasugi, M., S. Okinaka, N. Katsui, T. Masamune, A. Shirata, and M. Chuchi. 1985. Isolation and structure of lettucenin A, a novel guaianolide phytoalexin from Lactuca sativa var. capitata (Compositae). J. Chem. Soc. Chem. Commun. 10: 621622. Tamaki, H., R. W. Robinson, J. L. Anders on, and G. S. Stoewsand. 1995. Sesquiterpene lactones in virus-resistant lettu ce. J. Agric. Food Chem. 43: 6-8.

PAGE 202

202 Tatchell, G. M., P. R. Ellis, R. H. Collier, D. Chandler, A. Mead. L. J. Wadhams, W. E. Parker, J. A. Blood Smyth, and W. E. Vi ce. 1998. Integrated pest management of aphids on outdoor lettuce crops, pp. 77. Fi nal Rep. HDC Project No. FV 162. East Malling, Horticulture Development Council. Terra, W. R., C. Ferreira, and B. P. Jordao. 1996. Digestive enzymes, pp. 153-194. In M. J. Lehane and P. F. Billinsley [eds.], The biology of insect midgut. Chapman and Hall, London, UK. Thaler, J. S. 1999. Jasmonate-induced plant defenses cause increased parasitism of herbivores. Nature 399: 686-688. Thaler, J. S., M. J. Stout, R. Karba n, and S. S. Duffey. 1996. Exogenous jasmonates simulate insect wounding in tomato plants ( Lycopersicon esculentum ) in the laboratory and field. J. Chem. Ecol. 22: 1767-1781. Thipyapong, P., D. Joel, and J. Steffens. 1997. Differential expression and turnover of the tomato polyphenol oxidase gene family during vegetative and reproductive development. Plant Physiol. 113: 707. Thipyapong, P., and J. Steffens. 1997. Tomato polyphenol oxidase: differential response of the polyphenol oxidase promoter to in juries and wound signals. Plant Physiol. 1152: 409. Thygesen, P., I. Dry, and S. Robinson. 1995. Polyphenol oxidase in potato: a multigene family that exhibits differential expr ession patterns. Plant Physiol. 109: 525. Todd, G. W., A. Gethahun, and D. C. Cress. 1971. Resistance in barley to the green bug, Schizaphis graminum Toxicity of phenolic and flavonoid compounds and related substances. Ann. Entomol. Soc. Am. 64: 718-721. Toms-Barbern, F.A., M.I. Gil, M.Castaer, F.Artes, and M. Saltveit. 1997. Effects of selective browning inhibitors on phenolic metabolism in stem tissue of harvested lettuce. J. Agric. Food Chem. 45: 583-589. Toscano, N. C., K. Kido, and R. M. Davi s. 1990. Lettuce pest management guidelines. UCPMG Publication 15. IPM Education and P ublications, University of California, Davis. Tune, R., and D. E. Dussourd. 2000. Specialized generalists: constraint s on host range in some plusiine caterpillars. Oecologia 23: 543-549. USDA (United States Department of Agriculture). 2002. Crop Production-Annual Summary: 2002 Vegetable Crops Summary.NASS. http://www.usda.gov/nass/ USDA (United States Department of Ag riculture). 2005a. Vegetables and melons outlook. February 23. ERS. http://www.ers.usda.gov/publications/vgs

PAGE 203

203 USDA (United States Department of Agri culture). 2005b. Agricultural chemical usage 2004 vegetables summary. July 2005. NASS. http://usda.mannlib.cornell.edu/reports/nassr/other/pcu-bb/. USDA (United States Department of Agricu lture). 2005c. Agricultural chemical usage 2004 restricted use summary. October 2005. NASS. http://usda.mannlib.cornell.edu/reports/nassr/other/pcu-bb/. Vail, P. A., A. C. Pearson, V. Sevacheria n, T. J. Henneberry, and H. T. Reynolds. 1989. Seasonal incidence of Trichoplusia ni and Autographa californica (Lepidoptera: Noctuidae) on alfalfa, cotton, and lettuce in the Imperial Valley of California. Environ. Entomol. 18: 785-790. Valle, M. G., G. Appendino, G. M. Nano, a nd V. Picci. 1987. Prenylated coumarins and sesquiterpenoids from Ferula communis Phytochemistry 26: 253-256. van Beek, T. A., P. Mass, B. M. King, E. L eclercq, A. G. J. Voragen, and A. Groot. 1990. Bitter sesquiterpene lactones from chicory roots. J. Agric. Food Chem. 38: 10351038. van der Arend, A. J. M., J. T. van Schijndel P. R. Ellis, and S. Derridj. 1999. The making of the aphid resistant butterhead le ttuce 'Dynamite'. Bull. OILB SROP 22: 35-43. van Helden, M., and W. F. Tjallingii. 1993. Ti ssue localization of lettuce resistance to the aphid Nasonovia ribisnigri using electrical penetrati on graphs. Entomol. Exp. Appl. 68: 269-278. van Helden, M., W. F. Tjallingii, and F. L. Dieleman. 1993. The resistance of lettuce ( Lactuca sativa L.) to Nasonovia ribisnigri : bionomics of N. ribisnigri on near isogenic lettuce lines. Ento mol. Exp. Appl. 66: 53-58. van Helden, M., and D. van der Wal. 1996. Isolation of allomones from phloem sap of aphid-resistant lettuce by bioassay guided fractionation. Bull. OILB SROP 19: 6267. van Helden, M., H. P. N. F. van Heest, T. A. van Beek, and W. F. Tjallingii. 1995. Development of a bioassay to test phloem sap samples from lettuce for resistance to Nasonovia ribisnigri (Homoptera, Aphididae). J. Chem. Ecol. 21: 761-774 van Melckebeke, J., S. Kino, L. de. Rooster, L. de Reycke, and R. Sarrazyn. 1999. Plant protection in field vegetables. Nasonovia resistant cultivars: alternative for chemical control of aphids in butterhead lettuce and iceberg lettuce grown in the field. Proeftuinnieuws 9: 12-13. Vet, L. E. M. 1999. Evolutionary aspects of plant carnivore in teractions, pp. 3-20. In D. Chadwick and J. Goode [eds.], Insect plan t interactions and in duced plant defense. Novartis Foundation Symposium 223. John W iley & Sons, Ltd., Chichester, U.K.

PAGE 204

204 Vilmorin. 1883. Les Plantes Portageres. Cited from E. L. Sturtevant. 1886. A study of Garden lettuce. Am. Nat. 20: 230-233. Walker, A. J., L. Ford, M. E. N. Majerus, I. E. Geohegan, A. N. E. Birch, J. A. Gatehouse, and A. M. R. Gatehouse. 1998. Ch aracterization of the midgut digestive proteinase activity of the two-spot ladybird beetle (Adalia bipunctata L.) and its sensitivity to proteinase inhibitors. Insect Biochem. Mol. Biol. 28: 173-180. Wang, J., and C. P. Constabel. 2004. Polyphe nol oxidase overexpression in transgenic Populus enhances resistance to herbivory by forest tent caterpillar ( Malacosoma disstria ). Planta 220: 87-96. Waterhouse, D. F., and K. R. Norris. 1987. Liriomyza species, Diptera: Agromyzidae, leafminers, pp. 159-176. In Biological control, pacific prospects. Inkata Press, Melbourne, Australia. Wei, Y. D., E. de Neergaard, H. Thordal-Ch ristensen, D. B. Collinge, and V. Smedegaard Petersen. 1994. Accumulation of a putat ive guanidine compound in relation to other early defense reactions in epiderma l cells of barley and wheat exhibiting resistance to Erysiphe graminis f.sp. hordei Physiol. Mol. Plant Pathol. 45: 469484. Whitaker, T. W., A. N. Kishaba, H. H. T oba, R. Antoszewski, L. Harrison, and C. C. Zych. 1974. Resistance in lettu ce to the cabbage looper, Trichoplusia ni (Hubner), pp. 721-764. Proc. XIX Intl. Hortic. C ong. I. Section VII. Vegetables. Wink, M. 1997. Special nitrogen metabolism, pp. 439-486. In P. M. Dey and J. B. Harborne [eds.], Plant biochemistry. Academic Press, London. Winter, M., and K. Hermann. 1996. Esters a nd glucosides of hydroxyl cinnamic acid in vegetables. J. Agric. Food Chem. 34: 616-620. Wititsuwannakul D, Chareonthiphakorn N, Pace M, Wititsuwannakul D. 2002. Polyphenol oxidases from Hevea brasiliensis: purification and characterization. Phytochemistry 61: 115. Yudin, L. S., J. J. Cho, and W. C. Mitchell 1986. Host range of we stern flower thrips, Frankliniella occidentalis (Thysanoptera: Thripidae), with special reference to Leucaena glauca Environ. Entomol. 5: 1292-1295. Zalucki, M. P., and L. P. Brower. 1992. Survival of first instar larvae of Danaus plexippus (Lepidoptera: Danainae) in relation to cardiac glycoside and latex content of Asclepias humistrata (Asclepiadaceae). Chemoecology 3: 81-93. Zalucki, M. P., and S. B. Malcolm. 1999. Pl ant latex and first-instar monarch larval growth and survival on three North American milkweed species. J. Chem. Ecol. 25: 1827-1842.

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205 Zalucki, M. P., S. B. Malcolm, T. P. Paine, C. C. Hanlon, L. P. Brower, and A. R. Clarke. 2001. Its the first bites that count: Survival of first-instar monarchs on milkweeds. Austral Ecol. 26: 547. Zar, J. H. 1984. Biostatistical analysis. 2nd ed. Prentice-Hall, Inc., Englewood Cliffs, New Jersey. Zeren, O. 1985. Investigations on a new lettuce pest, Uroleucon cichorii (Hom., Aphididae), in the Cukurova region. Turk iye Bitki Koruma Dergisi. 9: 173-181. Zeyen, R. J., W. R. Bushnell, T. L. W. Carver M. P. Robbins, T. A. Clark, D. A. Boyles, and C. P. Vance. 1995. Inhibiting phenyl alanine ammonia-ly ase and cinnamylalcohol dehydrogenase suppresses mla1 (HR) but not Mlo5 (Non-HR) barley powdery mildew resistances. Physio l. Mol. Plant Pathol. 47: 119-140.

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206 BIOGRAPHICAL SKETCH Amit Sethi was born August 7, 1977, in Aboha r, Punjab, India. He received his bachelors degree in agricultu re with honors in plant protect ion from the Department of Entomology, Punjab Agricultural Universit y, Ludhiana, India in 1998. He also received the merit fellowship during his bachelors degree. He obtained hi s masters degree in entomology from the same institute in 2000, and also received Novartis crop protection fellowship. He worked as a research fellow in the same department for 3 years. While working, he also obtained his M.B.A. in Oper ational Management from the Indira Gandhi National Open University, New Delhi, India. In 2004, he began his Ph.D. program at the University of Florida to study the biochemical basis of host plant resistance in romaine lettuce under the supervision of Dr. Heathe r J. McAuslane in the Department of Entomology and Nematology. He received ma ny research and travel grants from the department, university and also from various sc ientific societies. He won awards (eight) for all of his poster and oral pr esentations at various state, regional and national scientific meetings. He was an extremely good citizen in the department and the university community. He served as historian for th e Graduate Student Organization of the department and he was involved in its many outreach and fundraising (snack-bar coordinator) activities. He was active on the departments Social Committee and he had served as coordinator of the Seminar Comm ittee for several years. This committee was totally responsible for organizing the weekly departmental seminars with local and national speakers. He was Mayor of his marri ed student housing complex and serves in a leadership role on the Mayors Council, a committee of the Universitys Student Government. His long term goal is finding a challenging position in molecular and

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207 chemical ecology highlighting insect-plant inte ractions with both teaching and research responsibilities.