. 9V
.3.
3%)? Ave
, Ti...

3.1.
i ...

.41.:-
A. I

«1.3,
. . H.773, Pf}...
hie. guy? .6
umﬁwﬂﬂﬁ .

L x
in? .5...

n1 :1.
.3!

.
095.09....
:3..»%

2-90.. .
.1, .
his

.. )1
‘1'}:
.55.:

haravhaawmsun

. 3.6:...
9 .v .3153]

I, . ‘c
vi... . .. .
x 900133115 t. 2

‘3‘...

3r}.
$36
5.

s‘llu'

I“ ,. .iik.

n 1.3.. v If..."

. Exgut

It Silvatvlkut
x...

Lac-£11.!

$1

4J1: K Ltmiliasl
.‘3

 

 

:3... kuﬂﬂd‘l
‘ I 5“ ll..,\.-.)Iu

Lt .0...

$0.3.(2. 3...

. )1). xI‘Qws Ill
. 5- ‘03.? «:5.

. ... . II a

 

l‘fl
3.3.0:?)
111.36.

This is to certify that the
thesis entitled

TREE VIGOR AND ITS RELATION TO EMERALD ASH BORER
(AGRILUS PLANIPENNIS FAIRMAIRE) ADULT HOST
PREFERENCE AND LARVAL DEVELOPMENT ON GREEN AND
WHITE ASH TREES

presented by

Chenin Kathleen Limback

has been accepted towards fulﬁllment
of the requirements for the

MS. degree in Entomologx

 

 

 

 

MSU Is on Minnow. We! Opportunity Employer

 

LIBRARY
Michigan State
University

 

 

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

5/08 KIProVAodPros/CIRCIDateDueJndd

 

TREE VIGOR AND ITS RELATION TO EMERALD ASH BORER (AGRILUS
PLAN/PENNIS FAIRMAIRE) ADULT HOST PREFERENCE AND LARVAL
DEVELOPMENT ON GREEN AND WHITE ASH TREES
By

Chenin Kathleen Limback

A THESIS
Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of
MASTER OF SCIENCE
Entomology

2010

 

ABSTRACT

TREE VIGOR AND ITS RELATION TO EMERALD ASH BORER (AGRIL US
PLANIPENNIS FAIRMAIRE) ADULT HOST PREFERENCE AND LARVAL
DEVELOPMENT ON GREEN AND WHITE ASH TREES

By
Chenin Kathleen Limback

Host preference of Agrilus planipennis Fairrnaire (Coleoptera: Buprestidae) was
compared between green and white ash trees subjected to four treatments (girdling, fertilization,
fertilization + Agri-Fos fungicide, untreated controls) in 2008 and 2009. Girdling reduced foliar
chlorophyll content, nitrogen concentration, and photosynthesis rates and increased foliar
carbohydrates. Effects of fertilization were minimal. Signiﬁcantly more A. planipennis adults
were captured on sticky bands on girdled trees than on other trees. The highest numbers of larvae
per m2 were found under the bark of girdled trees and green ash trees. Larval mortality caused by
intraspeciﬁc competition was higher in girdled trees than other trees. Most larvae under the bark
of girdled trees would have developed in one year. Approximately 30% of larvae on non-girdled
green ash trees would have developed in one year in 2009. In contrast, nearly all larvae on white
ash trees which were not girdled would have required two years for development. Non-girdled
white ash trees were generally not attractive to A. planipennis adults and had the lowest larval
densities. When girdled, however, white ash trees were more attractive than green ash trees which
were not girdled.

Adult A. planipennis foliar feeding preferences were evaluated in 2009 on green ash
seedlings which were girdled, fertilized, or leﬁ as untreated controls. Foliage from girdled
seedlings had lower nitrogen concentrations and lower photosynthesis rates than foliage from
trees of other treatments. Adult A. planipennis consumed more leaf area on girdled seedlings than
on fertilized or untreated seedlings in no-choice bioassays. In an outdoor choice assay, adults

appeared to preferentially feed on fertilized over untreated seedlings.

DEDICATION
This thesis is dedicated to the memory of my two great-grandmothers, Lillian
Bishop and Jessie James. These two women, each in their own way, set early examples
for me about what it means to be an intelligent, independent woman. They were

wonderful role models and human beings and always remain fondly in my memory.

iii

ACKNOWLEDGEMENTS

First and foremost I would like to thank my advisor, Dr. Deborah G. McCullough,
for her guidance and support. None of this would have been possible without her
encouragement and dedication to my project and my career. She was always available to
answer any questions or give advice, while at the same time encouraging me to become
an independent and competent researcher. Along with Deb I would like to thank my
committee members, Dr. Therese M. Poland and Dr. Bert M. Cregg, for their assistance
in developing my project and their willingness to help any time I needed advice. I would
also like to thank my undergraduate biology professor, Dr. James B. Martin at Adrian
College, for introducing me to the exciting ﬁeld of entomology and inspiring me to
become a scientist.

I would have been unable to complete my research if not for the help of all of the
undergraduate workers who were willing to assist me in my many projects. I would like
to thank Joanna Bloese, Tara Dell, James Wieferich, Jacob Bournay, Emily Pastula, Erin
Burkett, Keali Chambers, Ben Schmitt, Alison Barc, and Andrew Krieger. I would also
like to thank the lab technicians Andrea Anulewicz, David Cappaert, Bob MacDonald,
Jacob Baker, and Nick Gooch, as well as post-doctorates Nathan Siegert and Rodrigo
Mercader. Their assistance in plantation set-up and maintenance, insecticide treatment,
and help with any research questions I may have had was invaluable. Advice and support
from other graduate students in the lab including Andrew Tluczek, Sara Tanis, and

Stephen Burr have also been indispensable.

iv

I thank Yigen Chen, Monica Hufnagel, Debbie Miller, Tina Ciaramitaro, and the
rest of the workers and post-doctorates from the USDA Forest Service Northern Research
Station for their help with projects, including volatile collections and nutrient analyses. I
also thank Amanda Taylor from Bert Cregg’s lab for her help with chlorophyll and
photosynthesis testing.

I would like to thank Paul Bloese, Randy Klevickas, and the rest of the staff of the
Tree Research Center for the use of their facilities and for their advice and support with
regards to maintaining my plants under optimal conditions.

Finally, I thank my parents, Glenn and Nancy Limback, and my sister Kristin

Limback for their support and understanding throughout my graduate school experience.

TABLE OF CONTENTS

LIST OF TABLES ........................................................................................................... viii
LIST OF FIGURES ............................................................................................................ ix
CHAPTER 1

Density and Development of Emerald Ash Borer (Agrilus plam'pennis F airmaire)

Larvae on Stressed and Vigorous Green and White Ash Trees ........................................... 1
Introduction .............................................................................................................. 1
Materials and Methods ............................................................................................. 5

Study Sites ................................................................................................... 5
Plantation Study ........................................................................................... 6
Treatment Analyses ..................................................................................... 8
Adult Feeding Bioassays ............................................................................ 10
Adult A. planipennis attraction .................................................................. 12
Progeny of Caged Adults ........................................................................... 12
Volatile Production .................................................................................... l4
Larval Density and Development — Wild A. planipennis .......................... 16
Statistical Analysis ..................................................................................... 17
Results .................................................................................................................... 19
Tree Diameter ............................................................................................ 19
F oliar Nutrients and Photosynthesis — 2008 .............................................. 20
F oliar Nutrients and Photosynthesis — 2009 .............................................. 21
Volatile Production — 2009 ........................................................................ 24
A. planipennis Leaf Feeding — 2009 .......................................................... 26
Beetle Capture — 2009 ................................................................................ 27
Larval Density and Development — Progeny of Caged Adults 2009 ......... 27
Larval Density and Development — 2008 .................................................. 28
Larval Density and Development - 2009 .................................................. 29
Discussion .............................................................................................................. 30
Tables ..................................................................................................................... 39
Figures .................................................................................................................... 45

CHAPTER 2

Feeding Efﬁciency and Host Preference of Emerald Ash Borer (Agrilus plam'pennis

F airmaire) Adults on Stressed and Vigorous Green Ash Seedlings .................................. 60
Introduction ............................................................................................................ 60
Materials and Methods ........................................................................................... 64

Seedling Establishment .............................................................................. 64
Treatment Applications .............................................................................. 65
Foliar Nutrients and Photosynthesis .......................................................... 66
No-Choice Bioassays ................................................................................. 68
Choice Assay ............................................................................................. 69

vi

Statistical Analysis ..................................................................................... 69

Results .................................................................................................................... 71
F oliar Nutrients and Photosynthesis — Group 1 ......................................... 71
F oliar Nutrients and Photosynthesis — Group 2 ......................................... 72
No-Choice Bioassays ................................................................................. 73
Choice Assay ............................................................................................. 74
Discussion .................................................................................................. 75
Tables ......................................................................................................... 79
Figures ........................................................................................................ 80
APPENDIX 1
Record of Deposition of Voucher Specimens .................................................................... 90
LITERATURE CITED ...................................................................................................... 92

vii

LIST OF TABLES

Table 1.1: Mean (:tSE) nutrient concentration of foliage from

green ash and white ash trees in 2008. Values followed by different

letters within a column by species or treatment are signiﬁcantly

different ﬁom each other (Kruskal-Wallis test, 2-way ANOVA, p<0.05) ........................ 39

Table 1.2: Mean (iSE) nutrient concentration of foliage from green

ash and white ash trees in 2009. Values followed by different letters

within a column by species or treatment are signiﬁcantly different

from each other (2-way ANOVA, p<0.05) ........................................................................ 40

Table 1.3: Mean (iSE) nutrient concentration of phloem from green

ash and white ash trees in 2009. Values followed by different letters

within a column by species, treatment, or location are signiﬁcantly

different from each other (Friedman’s 3-way nonparametric ANOVA,

3-way ANOVA, p<0.05) ................................................................................................... 41

Table 1.4 Mean (iSE) volatile compound production (ng/ g dry leaf
mass) from foliage of green ash and white ash trees in 2009. Values

followed by different letters within rows and dates are signiﬁcantly
different from each other (2—way ANOVA, p<0.05) ......................................................... 42

Table 1.5 Mean (:I:SE) volatile compound production (ng) ﬁ'om bark

and phloem of green ash and white ash trees in 2009. Values followed

by different letters within rows and dates are signiﬁcantly different

from each other (2-way AN OVA, p<0.05) ........................................................................ 43

Table 1.6 Mean (iSE) (a) foliage area consumed (cmz) per beetle per

day, (b) weight of frass produced (mg) per beetle per day, (c) weight of

foliage consumed (mg) per beetle per day, and (d) fiass weight

produced: foliage weight consumed ratio (mg/mg) in no-choice

bioassays. Values followed by different letters within columns by

species or treatment are signiﬁcantly different from each other (3-way

AN OVA, Kruskal-Wallis test, p<0.05) ............................................................................. 44

Table 2.1: Mean (iSE) nutrient concentration of foliage from green ash
seedlings in 2009. Values followed by different letters within columns
are signiﬁcantly different from each other (2-way ANOVA, p<0.05) .............................. 79

viii

LIST OF FIGURES

Figure 1.1: Mean (:t SE) chlorophyll index in green and white ash

foliage by (a) treatment and (b) species in 2008. Points with different

letters are signiﬁcantly different from each other within each date

(2-way ANOVA, p<0.05) .................................................................................................. 45

Figure 1.2: Mean (i SE) photosynthesis rates (umol C02 - m-2 - 5.1)

for green and white ash trees by (a) species - treatment,

(b) species - aspect, and (c) treatment - aspect in 2008. Bars with

different letters are signiﬁcantly different from each other (3-way

ANOVA, p<0.05) .............................................................................................................. 46

Figure 1.3: Mean (3: SE) transpiration rates (mmol H20 ° m.2 ~ 5-1)
by treatment in 2008. Bars with different letters are signiﬁcantly
different from each other (3-way ANOVA, p<0.05) ......................................................... 47

Figure 1.4: Mean (:t SE) chlorophyll levels in green and white ash

foliage by treatment in 2009. Points with different letters are

signiﬁcantly different from each other within each date (2-way

ANOVA, p<0.05) .............................................................................................................. 48

Figure 1.5: Mean (:l: SE) photosynthesis rates (umol C02 - rn'2 ° s-l)

for green and white ash trees by (a) treatment on June 15, (b) aspect

on June 15, and (c) treatment on August 3 in 2009. Bars with

different letters are signiﬁcantly different from each other (3-way

ANOVA, p<0.05) .............................................................................................................. 49

Figure 1.6: Mean (i SE) transpiration rates (mmol H20 - m.2 - 8-1)

for green and white ash trees by (a) treatment on June 15, (b) species

on June 15, and (0) treatment on August 3 in 2009. Bars with different

letters are signiﬁcantly different ﬁ'om each other (3-way AN OVA, p<0.05) ................... 50

Figure 1.7: Mean (i SE) number of adult A. planipennis captured on
sticky hands by treatment in 2009. Bars with different letters are
signiﬁcantly different from each other (Kruskal-Wallis test, p<0.05) ............................... 51

Figure 1.8: Mean (i SE) percentage total larvae grouped by instar

(1st and 2nd instars, 3rd instars, 4th instars and prepupal larvae)

which hatched from eggs laid by caged adults in 2009. Bars with

different letters are signiﬁcantly different from each other within

instar group (Kruskal-Wallis test, p<0.05) ........................................................................ 52

ix

Figure 1.9: Mean (:1: SE) larval density for species and treatment in

2008. Bars with different letters are signiﬁcantly different from each

other within treatment (p<0.05). Asterisks indicate a signiﬁcant

difference between species (2-way AN OVA, p<0.05) ...................................................... 53

Figure 1.10: Mean (i SE) percentage of larvae that died for species
and treatment in 2008. Bars with different letters are signiﬁcantly
different from each other (Kruskal-Wallis test, p<0.05) ................................................... 54

Figure 1.11: Mean (3: SE) percentage late instar larvae (fourth instars -
prepupal larvae) for species and treatment in 2008. Bars with different
letters are signiﬁcantly different from each other (Kruskal-Wallis test, p<0.05) .............. 55

Figure 1.12: Mean (3: SE) larval density for species by treatment in
2009. Bars with different letters are signiﬁcantly different from each
other (2-way AN OVA, p<0.05) ......................................................................................... 56

Figure 1.13: Regression of total wild larvae found per tree on total
adults captured on sticky bands per tree in 2009 (Regression analysis, p<0.05) .............. 57

Figure 1.14: Mean (i SE) percentage of larvae that died for species
and treatment in 2009. Bars with different letters are signiﬁcantly
different from each other (Kruskal-Wallis test, p<0.05) ................................................... 58

Figure 1.15: Mean (i SE) percentage late instar larvae (fourth instars -
prepupal larvae) for species by treatment in 2009. Bars with different
letters are signiﬁcantly different from each other (2-way AN OVA, p<0.05) ................... 59

Figure 2.1: Mean (i SE) foliar chlorophyll index by treatment over
time for Group 1 seedlings. Points with different letters are signiﬁcantly
different from each other within each date (ANOVA, p<0.05) ......................................... 80

Figure 2.2: Mean (:t SE) photosynthesis rates (prnol C02 - rn—2 ' $4) of

Group 1 green ash seedlings by treatment (a) and leaf location (b) in

2009. Bars with different letters are signiﬁcantly different from each

other (2-way AN OVA, p<0.05) ......................................................................................... 81

Figure 2.3: Mean (:1: SE) transpiration rates (mmol H20 ' m-2 - 8-1) of
Groupl green ash seedlings by treatment in 2009. Bars with different
letters are signiﬁcantly different from each other (2-way ANOVA, p<0.05) ................... 82

Figure 2.4: Mean (3: SE) foliar chlorophyll index by treatment over
time for Group 2 seedlings. Points with different letters are
signiﬁcantly different from each other within each date (ANOVA, p<0.05) .................... 83

Figure 2.5: Mean (:1: SE) photosynthesis rates (umol C02 - m.2 - s-l) of

Group 2 green ash seedlings by (a) treatment and (b) and leaf location

in 2009. Bars with different letters are signiﬁcantly different from each

other (2-way AN OVA, p<0.05) ......................................................................................... 84

Figure 2.6: Mean (:l: SE) transpiration rates (mmol H20 - rn-2 ' s'l) of

Group 2 green ash seedlings by treatment in 2009. Bars with different

letters are signiﬁcantly different from each other (2-way AN OVA,

Friedman’s 2-way nonparametric ANOVA, p<0.05) ........................................................ 85

Figure 2.7: Mean (i SE) leaf area consumed (cmz) per beetle per day
in no-choice bioassays in 2009. Bars with different letters are

signiﬁcantly different ﬁom each other (Friedman’s 2-way nonparametric
ANOVA, p<0.05) .............................................................................................................. 86

Figure 2.8: Mean (i SE) weight of frass produced (mg) per beetle per
day in no-choice bioassays in 2009. Bars with different letters are
signiﬁcantly different from each other (2-way AN OVA p<0.05) ..................................... 87

Figure 2.9: Mean (:1: SE) feeding rank by treatment on Group 2

seedlings. Points with different letters are signiﬁcantly different from
each other (AN OVA, p<0.05) ............................................................................................ 88

Figure 2.10: Mean (:1: SE) percentage leaves consumed at each rank
level by treatment on Group 2 seedlings. Bars with different letters are
signiﬁcantly different from each other (Kruskal-Wallis test, p<0.05) ............................... 89

xi

CHAPTER 1
Density and Development of Emerald Ash Borer (Agrilus planipennis Fairmaire)
Larvae on Stressed and Vigorous Green and White Ash Trees.
Introduction
The invasive emerald ash borer (Agrilus planipennis Fairmaire) (Coleoptera:
Buprestidae) was ﬁrst discovered in Michigan and Windsor, Ontario, Canada in 2002 and
has become a devastating pest of ash (F raxinus spp.) in the eastern United States (Poland
and McCullough 2006). This phloem feeding beetle is native to Asia, including eastern
Russia, northeastern China, Japan, Korea, Taiwan, and Mongolia (J endek 1994, Haack et
al. 2002). An estimated 7 million ash trees were already dead or dying in Michigan when
A. planipennis was identiﬁed in 2002 (Poland and McCullough 2006). It has now killed
more than 40 million ash trees in southeast Michigan alone (www.cmeraldashborer.info
2010). Additional populations have been found in at least 14 other states and Quebec
(www.cmeraldashborer.info 2010). There are more than 800 million ash trees in
Michigan forests (USDA-FS FIA 2006, Liu and Bauer 2006). Ash is also a popularly
planted street tree in urban areas. Death of these trees caused by A. planipennis could
result in signiﬁcant economic losses (Kovas et al. 2010). There is concern that this
invasive pest, if not controlled, could spread throughout North America, largely
eliminating the ash resource.
A. planipennis adults feed on leaves of ash trees, but do not harm the tree

(Cappaert et al. 2005b). Damage is caused by larvae feeding in the cambial region.
Galleries in the phloem disrupt nutrient ﬂow and may also score the outer sapwood,

affecting water conduction (Cappaert et al. 2005b). Adult beetle emergence begins in late

spring and continues through early summer. Adult female A. planipennis require 5-7 d of
feeding before mating, and 5-7 (1 more before beginning oviposition (Bauer et al. 2004,
Lyons et al. 2004). Beetles continue to feed and oviposit throughout their 3—6 wk lifespan
(Bauer et al. 2004, Cappaert et al. 2005b). Females deposit their eggs in bark crevices or
under ﬂaps of bark (Bauer et al. 2004). After eclosion, most larvae complete four instars,
overwinter as prepupae, then pupate and emerge in late spring (Cappaert et al. 2005b,
Bauer et al. 2004). However, some A. planipennis overwinter as early instars and require
a second year of development (2-yr larvae) (Cappaert et al. 2005a). Higher proportions of
2-yr larvae occur on healthy trees that are lightly infested than on stressed trees, such as
girdled trees (Cappaert et al. 2005a, Tluczek 2009).

Five native ash tree species in the eastern United States are threatened by A.
planipennis: green ash (F raxinus pennsylvanica Marsh), white ash (F raxinus americana
L.), blue ash (Fraxinus quadrangulata Michx.), black ash (Fraxinus nigra Marsh), and
pumpkin ash (F raxinus proﬁmda Michx.) (Cappaert et al. 2005b, Poland and
McCullough 2006). A non-native ash species, European ash (F raxinus excelsior L.) is
occasionally planted as a street tree and may also be threatened by A. planipennis.
Previous research indicated that, when growing together, A. planipennis preferentially
colonized green ash trees over white ash trees, and green ash trees succumbed to attack
ﬁrst (Anulewicz et al. 2007a). The same study also revealed that white ash was colonized
by A. planipennis over blue ash. Blue ash trees may produce callous tissue over galleries,
a possible defense mechanism (Anulewicz et al. 2007a).

Like other Agrilus spp., A. planipennis prefer to oviposit on stressed trees

(McCullough et al. 2009a, 2009b). Native A grilus spp. including the bronze birch borer,

Agrilus anxius Gory, and the two-lined chestnut borer, A grilus bilineatus (Weber), are
secondary pests that feed on stressed and dying trees (Anderson 1944, Haack and
Benjamin 1982, Dunn et al. 1986). Likewise, A. planipennis is a secondary pest
throughout its native range, presumably because it shares an evolutionary history with its
host trees, which have greater defenses against it than do North American ash species

(Y n 1992; Akiyama and Ohmomo 2000; Gould et al. 2005; Herms et al. 2005; Schaefer
2005; Williams et al. 2005, 2006, Eyles et al. 2007).

Although A. planipennis will attack and kill healthy trees in the United States,
adult beetles display a stronger attraction to stressed trees, such as girdled trees (Cappaert
et al. 2005b, Tluczek 2009). Girdled trees have higher larval densities than trees stressed
by herbicide, wounding, or exposure to the stress elicitor methyl jasmonate (McCullough
et al. 2009a, 2009b; Tluczek 2009). Higher proportions of l-yr larvae on stressed trees
suggest that stressed trees have less resistance to A. planipennis than vigorous trees,
while larvae in more vigorous trees go through a second year of feeding, possibly to
compensate for the trees’ defenses.

Although much research has focused on A. planipennis and other buprestids’
attraction to stressed and dying trees, few studies have examined whether cultural
practices to improve tree vigor will have an opposite effect on A. planipennis host
preference or development. One way of increasing tree vigor may be through fertilization

to increase nutrient availability. Another option to increase tree vigor is the application of
a phosphite fungicide. Fungicides such as Agri-Fos® (Agrichem, Liquid Fertiliser Pty.
Ltd., Loganholme, Queensland, Australia) combined with the bark-penetrating surfactant

Pentra-barkTM (Quest Products Corp., Louisburg, KA, USA) induced defense

mechanisms in some hardwood trees (Garbelotto et al. 2007). Defenses induced from
phosphate fungicides can include cell wall thickening as well as enhanced ligniﬁcation
and secondary metabolite production (Guest and Grant 1991, Garbelotto et al. 2007).
Applying this treatment to the trunks of coast live oak (Quercus agrifolia Née) trees
decreased the growth of lesions attributed to the fungal pathogen Phytophthora ramorum,
which causes sudden oak death (Garbelotto et al. 2007). I hypothesized that application
of Agri-Fos + Pentra-bark to green ash and white ash trees could induce a defensive
response to A. planipennis larval feeding through callous tissue production. Callous tissue
defense responses occur in birch (Betula spp.) trees as a defense against the bronze birch
borer (Barter 1957).

Volatile emissions attract some phytophagous insects to their host plants and
these volatiles can be induced when plants are stressed (Bemays and Chapman 1994;
Dicke 2000, Rodriguez-Saona et a1. 2006, de Groot et al. 2008). A. planipennis displayed
attraction to volatiles emitted from ash foliage damaged by beetle feeding damage or the
stress elicitor methyl-jasmonate. Linalool elicited the strongest antennal responses in
females and the compound (Z)-3-hexen-1-ol resulted in the strongest antennal responses
in males (Rodriguez-Saona et al. 2006). Further studies revealed A. planipennis attraction
to lures with (Z)-3-hexenol, particularly in males (de Groot et al. 2008). Two compounds,
Manuka oil and Phoebe oil, extracted from the New Zealand tea tree (Leptospermum
scoparium J .R. and G.) and the Brazilian walnut (Phoebe porosa Mez.) trees,
respectively, contain high amounts of certain ash bark volatiles, and were attractive to A.
planipennis (Crook et al. 2008). Phloem volatiles may be partially responsible for the

strong attraction of A. planipennis to girdled trees.

I examined effects of stress and vigor treatments, including girdling, fertilization,
and fertilization + Agri-Fos, on adult A. planipennis attraction and foliar feeding
preferences and larval density, survival and development in white ash and green ash
trees. I hypothesized that (1) total larval density would be highest in green ash trees and
girdled trees, and lowest in white ash trees and trees treated with fertilizer or fertilizer +
Agri-Fos; (2) larval mortality rate would be highest and development fastest in girdled
trees; (3) girdling trees would decrease foliar nutrient concentration and photosynthesis
rates while increasing volatile productions from foliage and phloem; (4) fertilization and
enhanced fertilization treatments would increase foliar nutrient concentration and
photosynthesis rates; (5) vigorous trees would be less attractive to adult A. planipennis,
and (6) larvae feeding on trees treated with fertilizer or fertilizer plus Agri-Fos would
have lower survival or would be more likely to require a second year for development.

Study Objectives to test these hypotheses were to (1) assess effects of girdling,
fertilization and enhanced fertilization on the foliar chlorophyll, photosynthesis rates, and
nutrient concentration of green and white ash trees; (2) compare the effects of the three
treatments on volatiles produced by green and white ash foliage and phloem; and (3)
determine whether A. planipennis larval density, survival, or development rate differed

between green and white ash or among treatments.

Materials and Methods
Study Sites
Forty F raxinus americana ‘Autumn Purple’ and forty Fraxinus pennsylvanica

‘Patmore’ were planted in April 2007 at Michigan State University’s Tree Research

Center (East Lansing, MI). Diameter at breast height (dbh) averaged (:tSE) 5.24 i 0.07
cm for green ash and 5.65 :I: 0.17 cm for white ash at time of planting. Trees were
obtained from Poplar Farms Nursery (Waterman, IL) and arrived balled and burlapped in
mid-April 2007. Planting occurred from 21 April to 23 April 2007. Green and white ash
trees were planted in randomized blocks. Installation of a drip irrigation system was
completed on 12 June 2007. Irrigation ran approximately 7 h/d, 2 d/wk at 3.8 liter/h ﬁ'om
each of two emitters per tree.

The plantation was regularly mowed and weeded. In 2007, glyphosate (Round-up
herbicide, 41% active ingredient (ai) per 3.8 liter water, Monsanto Technology, L.L.C.,
St. Louis, MO, USA) was applied every 2-3 wk to minimize competing herbaceous
vegetation. Foliage on the ash trees was sprayed to runoff with cyﬂuthrin (Tempo, Do It

Yourself Pest Control, Atlanta, GA, USA) to protect trees from colonization by local A.
planipennis populations. Sprays were applied with a C02 backpack sprayer on 5 June

2007 (11.8 ml ai per 378.5 liter of water at 206.8 kpa).

Plantation Stuajz

In 2007, we assigned the 80 trees to 10 blocks. Each block consisted of a two row
planting of four green ash and four white ash trees. Within each block, a green ash and a
white ash were randomly assigned to one of four treatments: fertilization, Agri-Fos,
fertilization + Agri-Fos, and untreated control. Treatments were applied on 6 July 2007
under sunny conditions. Applications of Agri-Fos plus the surfactant Pentra-bark were
mixed as 1.4 liter Agri-Fos + 0.1 liter Pentra-bark + 2.4 liter deionized water per 3.9 liter

spray. The lower 1.5 m of each trunk was sprayed with a 7.6 liter garden sprayer at a rate

of 24.4 ml ai (mono- and di-potassium salts of phosphorous acid) per tree (approximately
15.81 ml phosphorous acid). Harrell’s Pro-Blend with Micronutrients custom-mixed 19-
5-10, granular, controlled-release fertilizer (Harrell’s, Inc., Sylacauga, AL, USA) was
applied around the base of the trees to dripline at a rate of 40 g N per tree (approximately
599 kg per ha).

In 2008, the fertilization and fertilization + Agri-Fos treatments were reapplied to
the same trees as in 2007 using the same methods. Trees that were treated with Agri-Fos
plus Pentra-bark in 2007, however, were left untreated. Fertilization and Agri-Fos
treatments were applied on 15 May 2008 under sunny conditions following the same
methods and rates as in 2007. Fertilization and fertilization + Agri-Fos trees had been
exposed to two years of treatment at the time of this study. The plantation was
subsequently divided in half, with ﬁve blocks (40 trees) in each half.

The westernmost ﬁve blocks were set aside for study in 2009. Foliage on these
trees was sprayed with cyﬂuthrin via the same methods as in 2007 to protect them from
colonization by local A. planipennis populations in 2008. Tree wrap (Jobe’s Tree Care
Products, Easy Gardener Products, Inc., Waco, TX, USA) and screening secured with
zip-ties were also placed around the trunks of these trees to exclude oviposition by local
A. planipennis.

In the remaining (easternmost) ﬁve blocks, trees which had been treated with
Agri-Fos alone in 2007 were girdled by removing the phloem and cambium ﬁ'om the
circumference of the trunk. A 20 cm wide band of bark and phloem was removed 1 m
aboveground on the trunk of each assigned tree with drawknives on 28 May 2008. These

ﬁve easternmost blocks were not sprayed with cyﬂuthrin or wrapped, permitting

colonization by wild A. planipennis during the summer. These trees were felled in
autumn to quantify larval density.

In April 2009, tree wrap and screening were removed ﬁ'om the remaining trees.
The fertilization and fertilization + Agri-Fos treatments were reapplied in mid-May 2009
under sunny conditions to the same trees as in 2007 and 2008 via the same methods and
rates. Fertilization and fertilization + Agri-Fos treatments were thus applied for three
consecutive years by summer 2009. Trees which had been previously treated with Agri-
Fos plus Pentra-bark in 2007 and left untreated in 2008 were girdled on 21 May 2009,

following the same methods as in 2008.

Treatment Analyses

Effects of the four treatments on foliar nutrient concentration, chlorophyll content,
and photosynthesis and transpiration rates were quantiﬁed in green and white ash trees in
2008 and 2009. To assess foliar nutrients, one leaf ﬁom the northern aspect of the lower
canopy of each tree was removed on 16 July 2008, 10 July 2009, and 12 August 2009.
Leaves were ﬂash frozen in liquid nitrogen and stored at -20 °C until processing. If leaves
were so small that one leaf would not provide enough leaf tissue for analysis, two
Opposite leaves on the same shoot (assumed to be the same age) were selected. Leaf
tissue was ﬁnely ground in liquid nitrogen and approximately 50 mg was extracted for
analysis. Protein (mg/ g fresh weight) was determined via Bradford protein assay and
amino acid concentration (umol/g fresh weight) was determined colorimetrically via
cadmium-ninhydrin procedure (Doi et al. 1981, Kyto 1996, Fisher et al. 2001, Bi et al.

2003, Chen et al. 2009). Total non-structural carbohydrates (mg/ g fresh weight),

calculated as the sum of glucose and starch, were determined using the glucose (HK)
assay kit (Sigma-Aldrich, St. Louis, MO, USA) and methods ﬁ'om Jones (1979). Starch
was estimated as glucose equivalents (Marquis et al. 1997, Chen et al. 2009).

From 12-14 August 2009, phloem samples were colleCted for nutrient analysis.
Four cores (10 mm diam, appx 2 m depth) of outer bark, phloem, and cambium were
obtained from the north aspect of each tree using cork borers. Two samples above 1.3 m
(above the girdle on girdled trees) and two below 1 m (below the girdle on girdled trees)
were collected. Cambium and outer bark was scraped from each sample so only phloem
remained. Samples were ﬂash frozen in liquid nitrogen and stored at -20 °C until
processing. The two cores above 1.3 meters were combined for analysis as well as the
two cores below 1 m. Samples were ground in a mill and approximately 50 mg extracted
for analysis. Phloem samples were tested for protein, amino acids, total non-structural
carbohydrate, and protein:carbohydrate ratio using the same methods applied for foliage
analyses.

On 16 July 2008 and 28 August 2009, one leaf from the lower northern aspect of
the canopy of each plantation tree was removed for total nitrogen determination. If leaves
were so small that one leaf would not provide enough leaf tissue for analysis, two
opposite leaves on the same shoot (assumed to be the same age) were selected to obtain
21 g leaf material after oven drying. Leaves were oven dried at 655°C for 72 hr in a
model 30 GC lab oven (Quincy Lab, Inc., Chicago, IL, USA). Samples were sent for total
nitrogen analysis via micro-Kjeldahl digestion procedure at Michigan State University’s

Soil and Plant Nutrient Laboratory.

F oliar chlorophyll content was analyzed weekly in 2008 and 2009 using a Minolta
SPAD 502 Chlorophyll Meter (Spectrum Technologies, Inc.,.Plainiﬁeld, IL, USA). Four
readings were taken per tree (one on each cardinal aspect of the lower canopy) and
averaged each week. The Li-Cor LI-6400 (Li-Cor Biosciences, Lincoln, NE, USA)
system was used to measure photosynthesis and transpiration rates on 24 trees on 30
August 2008. The trees were randomly selected from four of the ﬁve blocks to represent
at least two of each species by treatment combination. In 2009, photosynthesis and

transpiration rates were measured on 15 June and 3 August. Photosynthesis rates were

measured via foliage gas exchange rates (Amax) (umol C02 ' rn-2 - 5.1) with a ﬂuorescent

. . -2 -1
leaf chamber. Transprratron rates were measured as mmol H20 - m ° 3 . Rates were

assessed on a leaf from the northern lower canopy and a leaf from the southern lower
canopy of all selected trees in both years. Leaves from each aspect were analyzed

independently and not averaged per tree. Trees were analyzed with the Li-Cor in mid-

afternoon in sunny conditions in both years. Quantum ﬂux was set at 1500 umols/mz/s

for use as a light source and machine temperature was set at that of the average daily

temperature in °C. Flow was set to 500 urns and the mixer set at 400 urns COzR.

Adult Feeding Bioassays

In 2009, I conducted three no-choice bioassays with adult A. planipennis beetles.
Bioassays began on 23 June, 29 June, and 28 July 2009. The 2009 bioassays and caged
adult studies discussed below required the use of laboratory-reared A. planipennis adults.

Beetles were reared from logs which were harvested from naturally invested ash trees

10

near Lansing, MI in fall 2008 and maintained in cold storage at 39°C. After removing
logs ﬁom cold storage, they were placed in 76.2 cm long, 15.2-30.5 cm diam cardboard
tubes (Saginaw Tube Co., Saginaw, MI, USA) with plastic and in a rearing room
maintained at 267° C. Adult beetle emergence from logs followed in approximately 21
days. Emerging adults were collected daily and immediately transferred to bioassay
material for no-choice bioassays. Two leaves opposite each other on the same whorl of a
lower canopy branch of each tree were collected and scanned in a ﬂatbed scanner to
determine leaf area using WinFOLIA software (Regent Instruments, Inc.; Quebec, Qc,
Canada). After scanning, the petiole of each leaf was cut on a slant to provide a fresh
surface area for water uptake. Leaves were inserted into water-ﬁlled microcentrifuge
tubes to maintain moisture and placed individually in 150 mm diam Petri dishes. Two
newly-emerged male A. planipennis were placed in a dish with one of the leaves from
each tree, and two newly-emerged female beetles were placed in a dish with the second
leaf. Beetles were allowed to feed for three days. Petri dishes were checked daily to
record beetle mortality. After feeding, leaves were re-scanned and total leaf area
consumed was determined by comparing original leaf area to the remaining area. Leaf
area consumed was divided by total “beetle days” (sum of the number of days, to 0.5 d,
that each beetle survived) to obtain a value for total leaf area fed per beetle per day. To
account for differences in speciﬁc leaf weight between species, areas fed on white and
green ash were converted to weight of foliage consumed values using speciﬁc leaf
weights determined by Chen and Poland (2010). Fresh frass was collected from each dish

and weighed to the nearest mg to estimate feeding efﬁciency on the leaves. A frass

11

weight: foliage consumed weight ratio was analyzed for species variables to compare A.

planipennis utilization of nutrients on green and white ash foliage.

Adult A. planipennis Attraction

To assess beetle attraction to trees, sticky bands were placed on the trunk and
branches of each tree in 2009. On 3 June 2009, the trunk of each plantation tree was
wrapped with 20 cm wide bands of plastic wrap from 1.3 m to 1.5 m aboveground. Bands
were covered with Tangletrap Insect Trap Coating (Gempler’s; GHC Specialty Brands,
LLC; Madison, WI, USA). On 6-7 June 2009, two branches (aspects random) in the mid
to lower canopy of each tree were also wrapped with 16 cm long bands of plastic wrap,
just below a branch with leaves, and coated with Tangletrap. Sticky bands on the trunks
and branches were checked weekly for beetle presence through the week of 13 July 2009,

after which sticky bands were removed.

Progeny of Caged Adults

In the week of 13 July 2009, a study was initiated to examine larval development
on the study trees. After emergence from rearing logs as described above, beetles used in
caged adult studies were reared for approximately one week on either leaves from
Fraxinus uhdei (W cm), a tropical, evergreen ash grown in a polyhouse, or from one of
the plantation trees. Two plastic 118.3 ml cup cages (Kendall, Tyco Healthcare Group,
Mansﬁeld, MA, USA) were attached to each plantation tree with silicone caulking, one

on the northern and one on the southern aspect of the trunk between 1.3 and 1.5 m, in the

12

area where the sticky band was previously located. One male and one female laboratory-
reared adult A. planipennis were introduced to each cup cage.

Each cup cage included an ash leaf placed in a water-ﬁlled microcentrifuge tube
for adult feeding. Leaves were replaced as they dried out, approximately every other day.
One cup cage per tree had foliage from that tree while the other cage contained leaves
from F. uhdei. Male and female beetles that died in the ﬁrst three days were replaced, and
female beetles were allowed to oviposit through mid-August. I expected that removal of
occasional leaves from the plantation trees to feed the beetles would not cause an
excessive amount of stress to the trees, although it is possible that other studies may have
been mildly affected.

In mid-September, cup cages were careﬁrlly removed and bark in the area
immediately underneath and surrounding the cages was peeled using draw-knives.
Number and instar of A. planipennis larvae were recorded. Larval galleries which began
beneath the cages were assumed to have hatched from eggs laid by caged females, while
galleries initiated outside the cages were assumed to be wild. Larvae that hatched ﬁ'om
eggs laid by caged females were counted and development stage recorded. First and
second instars at this time of year were expected to require a second year of development
(2-yr larvae), while fourth instars and prepupae were expected to emerge the following
summer (l-yr larvae). Because it was possible that some third instar larvae in September
may have developed to fourth instars by October, third instar larvae were analyzed
separately from other instars. Wild larvae were also recorded and added to the total when

trees were felled and peeled in their entirety (see below).

13

Volatile Production

F oliar volatile emissions were collected from all plantation trees on 9-12 June, 14-
16 July, and 11-13 August in 2009. Emissions were collected by enclosing foliage on one
branch of each tree in bags (Reynolds® Oven Bags, Reynolds Consumer Products
Company, Richmond, VA, USA). Lower branches with foliage which were in close
proximity to larger branches were selected, as these would most conveniently lend
themselves to volatile collection. Because of this, no consistent aspect was used for all
trees. I inserted an activated carbon ﬁlter into the opening of each bag along with a
Super-Q Volatile collection trap (Analytical Research Systems, Inc.; Gainesville, FL,
USA) with 30 mg Alltech Super-Q adsorbent material for volatile collection. These were
tightly ﬁxed into the bag on a single branch using twist-ties. The other end of the Super-
Q trap was attached to a long tube inserted over a suction pump (Sensidyne, Clearwater,
FL) attached to a 9-volt battery. Batteries were suspended from the nearby large branches
on each tree. The volatile collection period was 24 hr. Super-Q traps were returned to the
lab and volatiles extracted with 150 LIL pentane-hexane extraction solvent to which 1.0
ng/ul of heptyl acetate was added as an internal standard with a stream of nitrogen gas.
Extracts were topped off with 100 uL hexane to minimize volatization during storage in a
-20°C freezer until processing. Processing was conducted by condensing volatiles under a
stream of pure gaseous nitrogen, followed with analysis by gas chromatography-mass
spectrometry. Two ul of each sample was injected into a Thermo Scientiﬁc Trace GC
equipped with a DSQ-II MS and a 30 m by 0.25 mm id, 0.25 pm thick TR-lMS column
using splitless injections, with the carrier gas being helium. The oven temperature cycled

beginning at 1 min at 60°C, then increased to 190°C at 10°C /min, then increased to

14

300°C at 35°C/min and was subsequently held for 5 min. Volatile compounds were
identiﬁed by comparing spectral data with spectra from Wiley 275.L and Nist98.L
libraries and spectral data ﬁ'om commercially available standards. These were conﬁrmed
by retention times of authentic standards, if available. Comparison of peak areas with that
of the internal standard and calibration curves generated with synthetic standards were
used to quantify compounds. The value of volatile compounds in ng was divided by the
dry weight of foliage used for collection to obtain a value for ng/ g dry leaf tissue of each
compound of interest. All reagents and solvents used for volatile compound analysis were
purchased from Sigma—Aldrich (St. Louis, MO, USA) or Fisher Scientiﬁc (Pittsburgh,
PA, USA).

Phloem volatiles were collected on 13-15 August and 7-9 October 2009. Bags

(Reynolds® Oven Bags, Reynolds Consumer Products Company, Richmond, VA, USA)

were wrapped around the trunk of each tree ﬁom approximately 0.7 m to 1.3 m height (1
m to 1.3 m on girdled trees to avoid epicorrnic shoots on lower trunk) and secured with
duct tape. Bags were loose to allow space inside for air movement. A small hole was cut
into each bag and an activated carbon ﬁlter and Super-Q Volatile collection trap
(Analytical Research Systems, Inc.; Gainesville, FL, USA) were inserted inside. Holes
were duct-taped shut around the ﬁlter and Super-Q trap. The other end of each Super-Q
trap was attached to a long tube inserted over a suction-pump (Sensidyne, Clearwater,
FL) attached to a 9-volt battery. The volatile collection period was 24 hr. Super-Q traps
were returned to the lab and volatiles extracted and analyzed via the same methods used

for the foliar volatiles. Total ng of each compound of interest were standardized by

15

multiplying values ﬁ'om girdled trees by two to account for the half-length of the

collection bag.

Larval Density and Development — Wild A. planipennis

Beginning in September 2008 and again in 2009, plantation trees were felled one
block at a time. F elled trees were cut into two sections in 2008 (base of the trunk to 2 m
above ground, above 2 m) and three sections in 2009 (base of the trunk to 1 m above
ground, 1 m to 2 m, above 2 m). The trunk and all branches greater than 3 cm diam were
debarked using drawknives. Bark thickness at the north and south aspect of the top and
bottom of each half of the trunk was also measured. Log diam in both years was
measured at 1 m above ground, and these measurements were compared between species
and among treatments to assess any differences in tree size. Number, stage, and condition
(live, dead) of A. planipennis larvae were recorded. First to third instars were expected to
require a second summer of development before pupation, and fourth instars and
prepupae were expected to emerge the following spring.

Area exposed on each tree was quantiﬁed using the formula for calculating

surface area of a conical ﬁ'ustum:
S = 71: ( R1 + R2 ) s

S = the surface area of the sides of the frustum not including top and bottom

circles

R1 and R2 = the radii of the top and bottom circles

s = the length up the side of the frustum.

l6

Area was calculated for each section and branch and summed to estimate total

area exposed on each tree (m2). Number of larvae was divided by the total area exposed

to standardize larval density (larvae per m2 of surface area) for each tree. Total larvae in

2009 were also regressed on the total number of adults captured on sticky bands on the

trunks to compare landing rates with egg deposition.

Statistical Analysis

All data were tested for normality using the Shapiro-Wilk test (Shapiro and Wilk
1965) and residual plots (PROC GLIMMIX, SAS Institute 2001). All parametric
statistics were run using SAS 9.1 software (PROC GLIMMIX, SAS Institute 2001). Two-
way, three-way, or four-way analysis of variance (ANOVA) were applied to data that
followed a normal distribution (SAS Institute 2001). If AN OVA results were signiﬁcant
(p<0.05), Tukey’s honestly signiﬁcant difference (HSD) multiple comparison test was
used to assess differences among treatments (Tukey 1953, SAS Institute 2001).

Foliar nutrient concentrations, volatile compound productions, chlorophyll values,
and larval density and development rates were tested using two-way AN OVA to assess
effects of tree species and treatment. Phloem nutrient concentrations were tested using
three-way ANOVA to assess effects of tree species, tree treatment, and beetle sex.
Photosynthesis and transpiration rates were tested using three-way AN OVA to assess
effects of tree species, tree treatment, and leaf aspect. Four-way AN OVA was used to
assess effects of tree species, tree treatment, cup cage aspect, and foliage type on total

larval densities from caged adults. Regression analysis (PROC REG, SAS Institute 2001)

17

was used to examine the relationship between adult A. planipennis captured on sticky
bands and total larvae discovered when peeling logs.

Foliar protein and protein:carbohydrate ratios in 2008 were normalized by log
(x+1) transformation. F oliar protein in July 2009 and protein: carbohydrate ratios in both
months in 2009 were also normalized by log (x+1) transformation. TNC and protein:
TNC ratios in phloem in 2009, mean leaf area fed by adult beetles in no-choice bioassay
round 2, larval density from caged adults, wild larval density in 2009, and several volatile
compounds in 2009 [(Z)-3-hexenyl acetate, trans-ocimene and tetradecane in June, cis-
ocimene and a—cubebene in July, a—cubebene and a—famesene in August, and 4-methyl-
dodecane and bomeol acetate for trunks in October] were also normalized by log (x+1)
transformation. Photosynthesis values in 2008 were normalized by log (x) transformation.
The October 2009 volatile compound tetracosane was normalized by inverse (l/x)
transformation. The Kenward and Roger (1997) method was used to approximate
denominator degrees of freedom when data were unbalanced (SAS Institute 2001).

If transformations did not normalize variables, nonparametric tests were used.
Friedman’s two-way or three-way nonparametric AN OVA (SAS Institute 2001) was used
to test non-normal data involving potential interactions. When signiﬁcant, nonparametric
multiple comparison tests were applied following methods from Conover (1971) and Zar
(1984). If the interaction term in Friedman’s test was p2 0.50 or no potential interactions
existed, Wilcoxon Rank Sum tests (Ott and Longnecker 2001) and Kruskal-Wallis tests
(Kruskal and Wallis 1952) were used. When signiﬁcant, multiple comparison tests were
applied following the same methods (Conover 1971, Zar 1984). Friedman’s three-way

nonparametric ANOVA was used to test protein and amino acids in phloem in 2009.

18

Kruskal-Wallis tests were used to analyze mean leaf weight fed per beetle per day and
ratio of frass produced: leaf weight fed in no-choice bioassays, adult beetle capture on
sticky bands, larval development rate from caged adults, larval mortality in both years,
and larval density in 2008. All analyses were conducted at the p<0.05 level of

signiﬁcance.

Results
Tree Diameter

Tree diam in 2008 (measured at 1 m aboveground) 1 m differed between species
species (F =1 12.27; df=l,27.44; p<0.001) and among trees in different treatments
(F=25.77; df=3,27.47; p<0.001) (N=39). Green ash trees were slightly larger (6.1 :I: 0.13
cm) than white ash trees (7.1 i 0.10 cm) and trees which had been girdled that spring (5.9
i 0.26 cm) were somewhat smaller than trees assigned to the other treatments (untreated:
6.8 :t 0.20 cm, fertilized: 6.8 i 0.16 cm, fertilizer + Agri-Fos: 6.9 :l: 0.16 cm).

In contrast to 2008, tree diam (measured at l m aboveground) did not differ
between species (p=0.37) or among treatments (p=0.50) in 2009. Mean species diameters
were 6.3 i 0.16 cm for green ash trees and 6.1 i 0.32 cm for white ash trees. Mean
diameters for treatments were 5.8 :t 0.30 cm for trees which had been girdled that spring,
6.1 :I: 0.41 cm for untreated trees, 6.5 :I: 0.41 cm for fertilized trees, and 6.3 :t 0.32 cm for

trees treated with fertilizer + Agri-Fos.

l9

F oliar Nutrients and Photosynthesis - 2008

Nitrogen concentration (ppm NH4 N) was lowest in foliage from girdled trees

(x2==11.89, df=3,35, p=0.008) but did not differ between species (p=0.07) (Table 1.1).

Protein concentration (mg/ g fresh weight) differed among treatments (F =3.1 1; df=3,28;
p=0.042) but the conservative Tukey’s HSD test did not identify which treatments
differed signiﬁcantly (Table 1.1). No protein differences were found between species
(p=0.69). Foliage from girdled trees had lower total amino acid concentration (umol/g
fresh weight) than foliage from trees of other treatments, where amino acid concentration
was almost nine-fold higher (F =5.06; dﬁ3,30; p=0.006), but amino acids did not differ
between species (p=0.87) (Table 1.1). Total foliar non-structural carbohydrate
concentration (mg/g fresh weight) displayed an opposite trend; foliage from girdled trees
had higher levels than foliage from all other treatments (F=10.00; df=3,30; p<0.001).
Total non-structural carbohydrates also differed between species, with white ash trees
having higher carbohydrate concentrations than green ash (F =9.96; df=l ,30; p=0.004)
(Table 1.1). The ratio of protein: total non-structural carbohydrates was lower in girdled
trees than in untreated or fertilized trees, with trees treated with fertilizer + Agri-Fos
being intermediate (F =8.26; df=3,24, p<0.001) (Table 1.1). No clear differences between
green ash and white ash were observed for protein:carbohydrate ratios (p=0. 17).
Chlorophyll content ﬂuctuated over the course of the summer. Girdled trees had
less chlorophyll than trees of other treatments throughout the study (p<0.001 for all
dates). White ash foliage had lower chlorophyll levels than green ash foliage on most

dates during mid-summer (Fig 1.1).

20

4

 

([3.

H

III:-

3111

Photosynthesis rates were affected by interactions between species and treatment
(F=6.47; df=3,30.13, p=0.002), treatment and aspect (F=4.78; dﬁ3,29.02; p=0.008) and

species by aspect (F =5.03; df=l,29.02, p=0.033) (Fig. 1.2). Foliage from girdled trees of

both species had lower photosynthesis rates (approximately 4 umol C02 - m—2 - s'l) than

foliage from other trees (over 12 umol C02 - m-z - s-l), regardless of aspect or species

(Fig. 1.2a, c). For green ash, fertilized trees exhibited lower photosynthesis rates than
untreated trees or trees treated with fertilizer + Agri-Fos (Fig. 1.23). Fertilizer + Agri-Fos
treated trees had higher photosynthesis rates in green ash than in white ash (Fig. 1.2a).
Foliage from the north aspect on untreated trees exhibited higher photosynthesis rates
than foliage from the north aspect of fertilized trees (Fig. 1.2c). Trees treated with
fertilizer had higher photosynthesis rates on the south aspect of the crown than on the

north aspect (Fig. 1.30). Within white ash trees, leaves on the south aspect had

approximately 2 umol C02 - m-2 - 5'1 higher photosynthesis rates than leaves on the north

aspect (Fig. 1.2b). Transpiration rates were signiﬁcantly affected by treatment (F =7 1 .09;
df=3,30.48; p<0.001) and were over twice as high on non-girdled trees as they were on
girdled trees (Fig. 1.3). Species (p=0.09) and aspect (p=0.08) did not affect transpiration

rates.

F oliar Nutrients and Photosynthesis — 2009
In July 2009, protein concentration was over 4 mg/g (fresh weight) higher in
white ash foliage than in green ash foliage (F =9.01; dﬁ1,30; p=0.005) but did not differ

among treatments (p=0.60) (Table 1.2). Total non-structural carbohydrates (F =7.89;

21

 

df=3,25.95; p<0.001) and the protein: carbohydrate ratio (F =3.93; df=3,27.23; p=0.019)
differed among treatments. Foliage from girdled trees had approximately two times
higher carbohydrate concentration than trees of all other treatments, but the foliar
protein:carbohydrate ratio of these trees was half that of trees treated with fertilizer +
Agri-Fos (Table 1.2). Treatment (p=0.08) and species (p=0.64) had no signiﬁcant effects
on amino acid concentrations. Neither total non-structural carbohydrate concentration
(p=0.98) nor protein: carbohydrate ratios (p=0.11) differed between species in July
(Table 1.2)

As in 2008, nitrogen concentration (ppm NH4 N) in 2009 was loweSt in foliage
from girdled trees (F=5.81, df=3,28, p=0.003) but did not differ between species (p=0.21)
(Table 1.2). In August, treatment and species had no effect on foliar protein (treatment
p=0.59, species p=0.33) or amino acid concentration (treatment p=0.68, species p=0.84).
Treatment affected total non-structural carbohydrate concentration (F =14.33; df=3,24.96;
p<0.001) and protein:carbohydrate ratios (F =3.53, df=3,25.27; p=0.029), but neither of
these were affected by species (total non-structural carbohydrate p=0.76, protein:
carbohydrate ratio p=0.60) (Table 1.2). Similar to July, foliage from girdled trees had
carbohydrate levels that were at nearly two times higher than in trees of all other
treatments, but foliar protein:carbohydrate ratios were less than half those of other
treatments (Table 1.2).

Species (p=0.85), treatments (p=0.98), and location (above or below the girdle,
p=0.08) did not affect total protein concentration in phloem of plantation trees in 2009
(Table 1.3). There were also no differences in amino acid concentrations between species

(p=0.84), between phloem from above or below the girdle (p=0.15), or among treatments

22

 

(p=0.88) in tree phloem (Table 1.3). Total non-structural carbohydrates in phloem were
affected by interactions between treatment and location and between species and location
(Table 1.3). Carbohydrate concentration below 1 m in girdled trees was at least three
times lower than any other location by treatment combination (F =7.57; df=3,54;
p<0.001). Phloem carbohydrate concentration below Im on white ash trees was lower
than that of either species above 1.3 m, with green ash phloem below 1 m intermediate
(F =4.07; d%1,54.02; p=0.049). Protein: carbohydrate ratios were highest below 1 m in
girdled trees, and different from either fertilized or untreated trees above 1.3 m, and
untreated trees below 1 m, with the remaining treatment by location combinations as
intermediates (F=4.46; df=3,51.04; p=0.007) (Table 1.3).

Chlorophyll content ﬂuctuated over the course of the summer; the mean content
of girdled trees dropped by late August (Fig 1.4). As in 2008, girdled trees had less
chlorophyll than trees of other treatments for most of the study; however, no clear
differences between species were seen (Fig. 1.4). Photosynthesis rates on 15 June were
affected by both treatment (F=27.29; df=3,28; p<0.001) and aspect (F=4.45; df=l,28;
p=0.044), but not by species (p=0.45) (Fig 1.5). Foliage from girdled trees had lower
photosynthesis rates than foliage from trees of other treatments (Fig. 1.5a). Foliage on the
southern aspect of trees had higher photosynthesis rates than foliage from the northern
aspect (Fig. 1.5b). No differences between species were observed. Photosynthesis rates
on 3 August were affected by treatment (F=30.93; df=3,28.36; p<0.001), but not species
(p=0. 19) or aspect (p=0.23). Girdled tree foliage again had lower photosynthesis rates
than foliage from other treatments (Fig. 1.5c), but no species or aspect effects were

observed in August. Overall, photosynthesis rates were six to eight times higher on

23

untreated trees, fertilized trees, and trees treated with fertilizer + Agri-Fos than on girdled
trees in both July and August. Transpiration rates were affected by both treatment
(F=18.08; df=3,29.47; p<0.001) and species (F=4.72; df=l,28.29; p=0.038) on 15 June
and by treatment (F=24.86; df=3,27.76; p<0.001) on 3 August (Fig. 1.6). Mirroring
photosynthesis rates, transpiration rates were lowest in girdled trees and highest in green
ash trees (Fig. 1.6). Aspect (p=0.08) on 15 June and aspect (p=0.49) and species (p=0.68)

on 3 August did not affect transpiration rates.

Volatile Production - 2009

Foliar volatile compounds varied between species and among treatments. In June,
three foliar volatile compounds were more abundant in emissions from green ash foliage
than from white ash foliage. These compounds included (Z)-3-hexenyl acetate (F=4.34;
df=l,26.29; p=0.047), trans-ocimene (F=14.45; df=l,30; p=0.001), and tetradecane
(F=3.37; df=3,26.39; p=0.034). Trans-ocimene was also over two times higher in girdled
tree foliage than in foliage from untreated trees, while fertilized trees and trees treated
with fertilizer + Agri-Fos were intermediate (F =3.32; df=3,30; p=0.033) (Table 1.4).

In July, two foliar volatile compounds were affected by either species or
treatment. Cis-ocimene was higher in green ash foliage than in white ash foliage (F =7.49;
df=1,15.91; p=0.015) (Table 1.4). The volatile compound a-cubebene was affected by a
species by treatment interaction. Foliage from girdled green ash trees contained at least
eight times higher concentration of the compound (8.60 :I: 2.31 ng/ g dry leaf tissue) than
any other species by treatment combinations (untreated green ash: 0.55 :t 0.16 ng/ g;

fertilized green ash: 0.42 i 0.13 ng/g; fertilized + Agri-Fos green ash: 0.25 i 0.16 ng/g;

24

girdled white ash: 1.27 :h 0.56 ng/g; untreated white ash: 0.13 :l: 0.01 ng/g; fertilized
white ash: 0.96 i 0.87 ng/g; fertilized + Agri-Fos white ash: 0.37 i 0.21 ng/g) (F=7.45;
df=3,16.89; p=0.002) (N=27).

The compound a—cubebene was also signiﬁcant in August foliar volatile analyses.
Over twice as much was found in green ash than in white ash (F=5.32; df=l,26; p=0.030)
Girdled tree foliage also had higher a-cubebene content than untreated or fertilized tree
foliage, with foliage treated with fertilizer + Agri-Fos as an intermediate (F =4.70;
df=3,26; p=0.009). The compound a-famesene was found in much higher quantities in
girdled tree foliage than in fertilized or untreated tree foliage. Foliage treated with
fertilizer + Agri-Fos was again intermediate (F=6.28; df=3,25; p=0.003) (Table 1.4).

For volatiles collected from tree trunks in August, only nonanal was signiﬁcant;
higher amounts were found in girdled trees than in untreated trees, and fertilized trees and
trees treated with fertilizer + Agri-Fos were intermediate (F =3.17; df=3,26; p=0.041)
(Table 1.5). For volatiles collected from tree trunks in October, the compound 4-methyl-
dodecane was over two times higher in girdled trees than in fertilized trees; untreated
trees and trees treated with fertilizer + Agri-Fos were intermediate (F = 3.65; df=3,30;
p=0.023). Both species (F=11.81; d%1,4.863; p=0.019) and treatment (F=12.82;
dﬁ3,5.141; p=0.008) were signiﬁcant for the compound borneol acetate. White ash trees
had higher bomeol acetate content than green ash trees and girdled trees had higher
bomeol acetate content than untreated trees, fertilized trees, or trees treated with fertilizer
+ Agri-Fos. Finally, the compound tetracosane was over two times higher in girdled trees

than in untreated trees, with fertilized trees and trees treated with fertilizer + Agri-Fos as

25

intermediates (F =4.98; df=3,28; p=0.007) (Table 1.5).

A. planipennis Leaf Feeding — 2009

All three rounds of adult A. planipennis no—choice feeding bioassays in 2009 had
similar results (Table 1.6). Treatment did not affect leaf area consumed (Round 1:
p=0.41, Round 2: p=0.77, Round 3 p=0.l 1) or frass production (Round 1: p=0.54, Round
2: p=0.43, Round 3 p=0.17). Values varied among rounds and no treatment was
consistently highest or lowest (Table 1.6a,b). However, species effects were signiﬁcant
for both factors. In round 1, leaf area consumed per beetle per day was about 1.5 times
higher on white ash foliage than on green ash foliage (F =7.51; df=1,64; p=0.008) and
results were similar in round 3 (F =8.96; df=1,56; p=0.004). These differences were not
signiﬁcant in round 2 (p=0.56), although the overall mean was still higher for white ash
(Table 1.6a). When species means were converted to leaf weight, however, no differences
in adult beetle consumption were detected (Round 1 p=0. 14, Round 2 p=0. 12, Round 3
p=0.67) (Table 1.60). In all three rounds, weight of frass produced per beetle per day was
1-2 mg higher when beetles fed on white ash foliage compared with green ash foliage
(Round 1:F=11.32; dﬁ1,58; p=0.001; Round 2: F=5.58; df=1,64; p=0.021; Round 3:
F=11.98; df=1,64; p=0.001) (Table 1.6b). When the ratio of frass produced to leaf weight

consumed was analyzed for species, ratios were consistently highest when beetles fed on

white ash foliage (Round 1 78:26.17, df=1,37, p<0.001, Round 2 78:23.47, df=1,40,

p<0.001, Round 3 78:14.98, df=1,36, p<0.001) (Table 1.6d). Sex of beetles had no effect

on total leaf area fed (Round 1 p=0.18, Round 2 p=0.51, Round 3 p=0.89) or total frass

produced (Round 1 p=0.07, Round 2 p=0.76, Round 3 p=0.48).

26

Beetle Capture — 2009

A. planipennis adult captures on sticky bands on the trunks of trees were affected

2
by treatment (X =23.99, df=3,37, p<0.001) but not species (p=0.07) (Fig 1.7). For both

species combined, beetle capture on girdled trees was up to eight times higher than on
trees of any of the other three treatments (Fig 1.7). Very few beetles were captured on the
sticky bands placed in the canopy, and statistical analysis was not performed on these
results. In total, one beetle was captured in the canopy of girdled green ash trees, two in
untreated green ash trees, and two in fertilized green ash trees. For white ash trees, four
beetles were captured on sticky bands in the canopy of untreated trees, two in fertilized
trees, and one in the canopy of trees treated with fertilizer + Agri-Fos . In total, beetles
captured on sticky bands in the canopy accounted for approximately 10.4% of beetles

caught on all sticky bands.

Larval Density and Development — Progeny of Caged Adults 2009

Larval density in the caged adult study was affected by a three-way interaction
between species, treatment, and foliage type (F =3.30; df=3,48; p=0.028). Mean densities
of larval progeny from caged beetles were highest on the northern aspect of untreated
trees where the caged beetles fed on F raxinus uhdei (5.2 d: 1.6 larvae) and on the northern
aspect of girdled trees where the beetles fed on foliage from the tree on which they
oviposited (4.0 :I: 0.9 larvae). Both of these means were higher than on the northern
aspect of untreated trees where beetles fed on foliage from the tree on which they

OViposited, and where zero larvae were found.

27

Development rate of larvae under cup cages was different among treatments (Fig.

1.8). Girdled trees had a higher percentage of fourth instars and prepupal larvae
(x2=45.15, df=3,20, p<0.001) and third instars (78:14.30, df=3,20, p=0.003) than any of
the three other treatments. Conversely, girdled trees had the lowest numbers of ﬁrst and

second instar larvae (x2=10.32, df=3,20, p=0.016) (Fig. 1.8). Approximately 50% of all

larvae found under cup cages on girdled trees were third instars or higher, whereas on all
other treatments, nearly 100% of larvae found were ﬁrst and second instars and likely
would have required two years for development. No signiﬁcant differences in larval
development rates between ash species were observed (ﬁrst and second instars p=0.91,

third instars p=0.l4, fourth instars and prepupal larvae p=0.42).

Larval Density and Development - 2008

Larval density was affected by both species (F =22.67; df=l,4; p=0.009) and
treatment (F =44.l4; df=3,10; p<0.001) (Fig 1.9). For both species combined, larval
densities were over twice as high on girdled trees than on trees of other treatments. For
all treatments combined, larval densities were higher on green ash trees than on white ash
trees. Larval densities on green ash trees were upwards of 300 larvae per m2. Girdled

white ash trees had at least 100 more larvae per m2 than green ash trees which were not
girdled. Very few larvae (< 7/m2) were found on white ash trees which were not girdled
(Fig 1.9).

Larval mortality from intraspeciﬁc competition was signiﬁcantly affected by

treatment (78:19.84, df=3,35, p<0.001) but not by species (p=0.21) (Fig. 1.10). A higher

28

percentage (nearly 40% on green ash trees) of larvae were found dead on girdled trees
than on trees of all other treatments (Fig. 1.10). Overall, approximately 70.0% of dead
larvae were early (lst-3rd) instars. For development rate of larval progeny of wild adults,

approximately twice as many larvae on girdled trees were late instars than on trees of any

other treatment (x2=11.038; df=3; p=0.012) (Fig. 1.11) Larval developmental rates did

not differ between species (p=0.34) (Fig. 1.11).

Larval Density and Development— 2009

Larval density in 2009 was affected by a species by treatment interaction

(F =17.57; df=3,28; p<0.001) (Fig. 1.12). Total larvae per 1112 was highest in girdled green
ash trees (over 300 larvae per m2), followed by girdled white ash trees (nearly 200 larvae

2 . . 2 . .
per m ). The lowest densrtres (less than 50 larvae per m ) were on untreated, fertrlrzer,

and fertilizer + Agri-Fos trees regardless of species, and there were no signiﬁcant
differences among these treatments (Fig 1.12). There was a signiﬁcant linear relationship
between larval density and adult beetles captured on the sticky bands on the trees
(p<0.001). The predictor coefﬁcient of 10.1 indicates that on average, roughly ten larvae

per m2 were present for every adult beetle captured (Fig. 1.13).

Larval mortality was affected by treatment (x2=18.45; df=3, 37; p=<0.001) but
not species (p=0.31) (Fig. 1.14). Mortality caused by intraspeciﬁc competition was more
than four times higher on girdled trees when compared with trees of the other three

treatments (Fig 1.14). Overall, approximately 58.1% of dead larvae were early (lst-3 rd)

instars. Larval development rate was affected by a species by treatment interaction

29

(F =4.04; df=3,32; p=0.015) (Fig. 1.15). A higher percentage of larvae were late instars on
girdled trees of both species than on other trees (Fig. 1.15). Between 70-90% of all larvae
found on girdled green and white ash trees were fourth instars or prepupae, whereas on
untreated trees, fertilized trees, or trees treated with fertilizer + Agri-Fos, more than 60%

of all larvae had reached only the ﬁrst, second, or third instar (Fig. 1.15).

Discussion

Overall, there were clear differences in the way that white and green ash trees
responded to stress- and vigor-inducing treatments. Consistent with previous observations
(Anulewicz et al. 2007a), green ash trees were more attractive to A. planipennis than
white ash trees, supporting my initial hypothesis. Higher numbers of larvae on green ash
trees also appeared to die due to intraspeciﬁc competition, but these data were not
signiﬁcant. The reasons for these differences between species may reﬂect differences in
tree vigor or nutrient composition. Differences such as the lower chlorophyll content in
white ash foliage in 2008 may be part of the reason why other studies have shown white
ash to be less attractive to A. planipennis than green ash (Anulewicz et al. 2007a).
However, this difference was not present in 2009. Additionally, none of the phloem
nutrients we measured differed between ash tree species. The lower photosynthesis rates
on the northern aspect of the canopy on only white ash trees in 2008 may reﬂect leaf
architecture. White ash leaves are larger and droopier than green ash leaves, and may thus
have less of their surface exposed to sunlight at any given time. White ash leaves were
also consumed with less efﬁciency by A. planipennis adults, consistent with observations

by Chen and Poland (2010). Speciﬁc leaf weight is also lower in white ash (0.0116 3:

30

0.0004 g/cmz) than in green ash (0.0165 i 0.0005 g/cmz), and white ash foliage has been

shown to have lower levels of some nutrients (nitrogen, phosphorous, sulfur) than green
ash foliage in other studies (Chen and Poland 2010). The lower speciﬁc leaf weight of
white ash likely explains why beetles needed to feed on a greater leaf area on foliage
from these trees, especially considering that no differences in leaf weight consumed were
observed.

Higher volatile production from green ash trees may also help to explain why they
are generally more attractive than white ash trees (Anulewicz et al. 2007a). (Z)-3-hexenyl
acetate was previously identiﬁed as comprising a majority of the volatiles found in green
and white ash foliage, along with (Z)-3-hexenol (de Groot et al. 2008). It is an antenally
active compound for A. planipennis, but antennal responses are not as strong compared
with other compounds such as (Z)-3-hexenol (Rodriguez-Saona et al. 2006, de Groot et
al. 2008). However, antennal responses do not necessarily mirror beetle attraction in the
ﬁeld. The higher production of (Z)-3-hexenyl acetate in green ash foliage which was
observed in June 2009 may be partially responsible for high A. planipennis attraction to
these trees (Rodriguez-Saona et al. 2006).

The volatile compound a—cubebene was also highest girdled green ash trees in
July and in girdled trees in August. This is a bark and foliar volatile found in high
concentration in Manuka and Phobe oil, two components of lures used for A. planipennis
attraction (Crook et al. 2008). The higher production of the essential oil bomeol acetate in
white ash over green ash foliage in October is the only case in which volatile production

was higher in white ash.

31

It is possible that different cultivars of white and green ash may respond
differently to treatments or A. planipennis attack than the ‘Autumn Purple’ and ‘Patmore’
varieties studied here. In another study, the ‘Marshall’s Seedless’ cultivar of green ash
was more resistant to A. planipennis than ‘Patmore’, exhibiting traits more similar to
‘Autumn Purple’ white ash (Rebek et al. 2008). However, those results provide insight
into these particular varieties of green and white ash and their response to A. planipennis
colonization. A 2003 survey in Iowa, a state which has recently been invaded by A.
planipennis (www.cmeraldashborer.info), identiﬁed ‘Autumn Purple’ and ‘Patmore’ as
the most popularly grown cultivars of white ash and green ash respectively among
growers (Iles and Vold 2003). Overall, many dead green ash trees have been found in the
wild and in urban environments, regardless of cultivar. It is likely that any true
differences between cultivars will be so subtle as to have a minimal effect on overall A.
planipennis choice between white and green ash trees.

All evidence thus far suggests that green ash trees are more attractive to A.
planipennis than white ash trees. Indeed, white ash trees only became as attractive as
green ash trees when the white ash trees were girdled. Girdling is therefore an efﬁcient
stressor of ash trees regardless of species, and appears to effectively eliminate whatever
inherent defenses untreated or vigorous white ash trees may possess. Girdled white ash
trees even became more attractive to A. planipennis than untreated green ash trees, which
are normally more attractive than white ash trees (Anulewicz et al. 2007a). This, as well
as the high adult beetle captures on sticky bands on girdled trees in 2009, is consistent
With my initial hypothesis and mirrors the results of previous studies indicating that

girdled ash trees are highly attractive to adult A. planipennis (Anulewicz et al. 2007b,

32

McCullough et al. 2009a, 2009b; Tluczek 2009). The signiﬁcant positive regression
relationship between adult capture rates and larval densities in ash trees suggest that most
of the adults landing on these trees are choosing to oviposit on them. In one study that
examined 23 sites across four years, A. planipennis larval density and adult capture was
consistently higher on girdled trees than healthy trees or trees stressed by herbicide or
methyl jasmonate (McCullough et al. 2009a). This is likely because A. planipennis is a
secondary pest in its native Asia and is adapted to attack stressed trees (Yu 1992;
Akiyama and Ohmomo 2000; Gould et al. 2005; Herms et al. 2005; Schaefer 2005;
Williams et al. 2005, 2006; Eyles et al. 2007).

Also consistent with my hypotheses was the fact that nearly 100% of larvae on
girdled trees of either species in 2008 and 2009 were fourth instars or prepupae and
would have developed in one year. This faster development rate on girdled trees is
consistent with past research (Cappaert et al. 2005b, Tluczek 2009). Additionally, most
larvae from caged adults would have developed in 2 years on all trees except girdled
trees, where around half would have developed in 1 year. Differences in development
rates may result from decreased defenses caused by the girdling.

The extremely high mortality (nearly 40%) on girdled green ash trees was a result

of intraspeciﬁc competition and supports my hypothesis. Girdled green ash trees had an

average of over 300 larvae per m2 in both years. This is much higher than the average
emergence density per m2 in trees killed by A. planipennis, identiﬁed by McCullough and

Siegert (2007) as 88.9 i 4.6 adults emerged per m2 of phloem over a range of size classes

for both green and white ash trees. Although larval densities on the trees they evaluated

33

were much higher in some cases, competition for food led to mortality and thus prevented
most of these larvae from developing and emerging (McCullough and Siegert (2007).
The high larval mortality on girdled trees in this study lend further support to these
conclusions.

Analyses of nutrient concentration, chlorophyll content, and photosynthesis and
transpiration rates of foliage in 2008 and 2009 further revealed clear differences among
treatments. Girdled trees were consistently different from other trees. They had the lowest
photosynthesis rates, transpiration rates, chlorophyll levels, and the lowest levels of
nitrogen, protein, and amino acids. Nitrogen is a limiting factor in ecological systems and
an important source of energy for herbivores (Mattson 1980). However, foliar nitrogen
values were only consistently below reported average summer values for ash trees (%)
(Chen and Poland 2010) on girdled trees. Total non-structural carbohydrate levels were
higher in girdled trees, but the lower ratio of protein to total non-structural carbohydrates
is likely a more important factor in nutritional beneﬁts from host material (Simpson and
Raubenheimer 1993, Lee et al. 2002, Bede et al. 2007, Chen et al. 2009). These results
together ﬁrrther emphasize that girdling is an effective stressor of host trees, and are
consistent with initial hypotheses. In girdled tree phloem, the lower carbohydrate levels
and higher protein:carbohydrate ratios below the girdle are consistent with previous
research in that carbohydrate concentration accumulates above the girdle (Noel 1970,
Roper and Williams 1989, Li et al. 2003, Mostafa and Saleh 2006, Chen and Poland
2009)

Photosynthesis rates varied greatly in 2008 due to interactions between the

species, treatment, and aspect variables, but results consistently showed over both years

34

that girdled trees exhibited very low photosynthesis rates. Water loss via transpiration
was also lowest on girdled trees, consistent with low photosynthesis rates as stomatal
closure to reduce water loss will also decrease photosynthesis (Chaves et al. 2003). The
additional fact that leaves on the south aspect of the canopy seem to display higher
photosynthesis rates than those on the north aspect may be due to greater sun exposure on
the south side of the plantation. Lower photosynthesis in girdled trees is a result of
feedback inhibition (Foyer 1987), as accumulation of sucrose changes tree chemistry,
causing a shortage of ATP and ADP available for photosynthesis. This is also consistent
with the high carbohydrate concentration of girdled foliage.

Volatile production was also higher in girdled tree foliage and phloem than in
trees of other treatments. This is consistent with previous evidence of effects of tree stress
and volatiles on A. planipennis attraction to stressed trees (Rodriguez-Saona et al. 2006).
The volatile compound previously described as an important component in some A.
planipennis lures, d-cubebene, was highest in foliage of girdled green ash trees just as it
was found highest in green ash. This likely has a large impact on the attraction of A .
planipennis to girdled and green ash trees for oviposition. Other volatile compounds
found in high quantities in girdled trees in this study which were proven to be antenally
active to A. planipennis in previous research include a-farnesene in foliage and nonanal
in phloem (Rodriguez-Saona et al. 2006).

In contrast to girdling, the results of fertilization were less dramatic. No consistent
differences were seen between trees treated with fertilizer or fertilizer + Agri-Fos and
untreated trees in any of the studies. This does not support my initial hypotheses. I

expected that fertilized trees or trees treated with fertilizer + Agri-Fos would increase tree

35

vigor but no clear effects on chlorophyll, photosynthesis, or nutrient concentration were
seen. No consistent differences in tree size between treatments were observed over the
course of the study, although girdled trees seemed small in 2008 after girdling occurred.
Also inconsistent with my hypotheses, these trees showed no indication of being less
attractive to A. planipennis than untreated trees, nor was there consistently lower survival
or slower development rates of larvae feedingon these trees. It is possible that trees may
have already had ample nutrients for these ﬁrst few years, being planted balled and
burlap with nursery soil. In this case, any fertilizers applied may have had no effect if
nitrogen was stored the leaves via luxury consumption rather than being applied to
growth (Chapin et al. 1980; Chabot and Hicks 1982).

Callous tissue, which was reportedly induced around lesions caused by the sudden
oak death pathogen on other tree species (Garbelotto et al. 2007), was never observed
around larval galleries on any trees, including those treated with Fertilizer + Agri-Fos,
regardless of species. It is therefore likely that adding Agri-Fos to fertilization treatments
has no additional effect on green and white ash tree vigor or resistance to A. planipennis.
Phosphorous concentration was not examined in foliage so it is possible that not enough
got into trees to see an effect. However, previous studies have shown trunk sprays to be
effective in applications of Agri-Fos or insecticides (Garbelotto et al. 2007, McCullough
et al. 2007, 2008). Blue ash trees have occasionally produced callous tissue around larval
galleries (Anulewicz et al. 2007a). Therefore it is possible that a treatment of Agri-Fos
may help enhance defenses in that particular species of F raxinus, but that would require

further research.

36

 

Although it is unlikely that fertilization or enhanced fertilization had any
important effects on the ash trees in this study, one interesting point may still be of
consideration. Green ash trees had higher larval densities than white ash trees across all
treatments in 2008, but there were no differences between green and white ash larval
densities for non-girdled treatments in 2009. This may be due to an increase in total
beetle population in the area over the previous year. It is also possible that after three
years of application of the fertilizer treatments, green ash trees may become less
attractive to A. planipennis, and mirror the attraction of white ash trees, but support for
this was not very conclusive. It is also possible that older trees may respond differently
to fertilizer or Agri-Fos treatments than the young trees used in this study. Whereas
fertilization did not seem to have much affect alone, it may perhaps be effective in
conjunction with applications of insecticides such as imidacloprid or dinotefuron. These
insecticides do not cause 100% larval mortality on their own (McCullough et al. 2007,
2008), but adding fertilizer may help to increase their effectiveness, either through a
simple increase in tree vigor or to help increase root uptake of the insecticides, as has
been shown in some studies (DePew 1971, Wilde et a1. 1984, DePew and Hooker 1987).

Overall, results of this study indicate A. planipennis prefer green ash over white
ash trees and girdled trees over ungirdled trees. Ungirdled green ash trees were colonized
at higher rates than ungirdled white ash trees, despite the proximity of trees of both
species to each other. Girdling white ash trees increased their attractiveness to A.
planipennis to the point where they were more attractive than untreated green ash trees,
and nearly as attractive as girdled green ash trees. Girdled trees could be useful as sinks

to attract A. planipennis, and these trees could then be removed or destroyed before beetle

37

emergence, which could help to protect other trees from this pest. Understanding the
effects various treatments have on different ash species can help homeowners, arborists,
municipalities, and other resource managers weigh options when it comes to caring for
their home or street trees. Further, understanding the mechanisms of host preference or
resistance may assist in developing cultivars that are more resistant to A. planipennis, or

enhancing survey methods for this pest.

38

 

 

Sign. 3.33. 3.1; «33.2 «33% “2+5“. -
m on a No a .1 a 9m o E a to o 3 a 02 o N; a awn Beacon -
o 3.. a ram o ad a «N o 3 a 3 o ma « 3: co m... a «.3 38:5 -
3.33 353.2 a E «3 o E “3.? 36.3.8 8.25 -
o. no a 3 o E a «A o m; a 3 o 5 a 3: o hm « o9 - :2 £55
.34 «3 no; am...” on. «3. «3 :2 2.334 - :2 820

ﬁn « £22., 3 i Em a £22, 52. mag Go a £22: soot 2.2.3 am « 32o; soot 295 A2 4:2 ES. Eoeaoﬁ 3.8%

oztsaoi oz» 23 8:5 So» 532.. cooegz .33

 

.Aoﬁaanoﬁao _ansbm-coc .98. H 02.5 .3838 .3 bo>cooam8

: .mm .mm on .wmuzv .GedvS .<>Oz< has .3 e=§-_e_m_5c aso nos aoa aorta bases?

8a “5:58.: .8 880% .3 5:28 a GEES EBB— Eouotzv .3 330:8 823/ dos—«5 wogotcmb Sta w2 no

3me 08 £36— oogomawﬁ 0:8 UZH ”£28m can £88m #:8308888: BEA—ESE 083 some?" com 82?

oogomiﬂm .33. 5 much :3 BE? 98 :3 Guam Sch 0328 no nocmbcooaOO EDP—z: Ammhv :82 H: 2an

39

 

 

o No A «a o no a 3 o E a on o E h was o 3 « own “2 + to“. - 92
o no A ma 9 no a E o 3 A Rm o. 5 H 2: o 3 A 0.2 85:5“. - 92
o Re A mm o 3 a 3 o S h 3 o 3 A no no 3 A Now Baas: - 92
3.3 E o 33.: 3.33 o 23.» 343.8 8.26 - 93
9.2.
o No a «N o 3 o as o S o oo o 3 h 2: o 3 H 3N - co< 922, one.
o to a RN o no a as o Re A 3 o 3 a mm o. 3 a 3m - co< :85 92
o no H mm o to H 3 o 3 a a.” o N... u a? - o< + to". - .2.
no 3 h mm o no a no o ON a E o am « RE - oo~__Eon_ - .2,
no 3 A an a we a oo o 3 H 5 o 3 a 3: - Base: - .2.
o no a 3 o Rm H to o no h 3 o A...” h R: - 8.30 - .3
.2.
o to h S o 3 a 3 o 5 « Sm o 3 a mop - - ﬁe. SE; .2.
8.33 3.33 2: «an a E H w: - - :2 520 .2.
Em a ﬁn u am « Em a £22, A: «:2 as: .558: 8.88 £8:
£225 3 i 29o; goo... 995 £22, 52. 9.2.3 goo... 295 5?ng .33
02:532.". ozh 23 oEE< .58 £39:

 

ASSESSED _8305bm-:oa 30% n 02.3 .cmswssas

an gas 3 boéooae R .3 .2. .9. 5th 5.: so. 5:28 E boéooome om .mm .mm .316 aeovo. .<>Oz< as
.3 3:8 :23 88m Enactmo bane—mama 2m “co—Each DO 860% 3 5:28 a £53» £88— Eouotmo .3 330:8 won—Er
.82? 8.8888“ Ste wo_ no woman one 2:88 Son 5 macro— ooaaongﬁ O38 07E. ”£88.“ 98 «292. 8 E032

oogouiwﬁ £88m doom E 80.5 2mm 853 28 can 50% Bob 03:8 mo scumbag—SO Santana Ammi .822 ”N; 2an

4O

 

 

41

- a nd H dd - - Er v - cm< 0:55

- 0 3 H 0.0 - - E3 A - :2. 9...;

- 00 Nd H .20 - - E.. v .. cw< 000.0

- 0 ad H Nd - - Em; A - zm< 000.0
00 ...FH...m moéHmd - - va u.<+...0u. ..
00 m... H ed 0 ad H «.0 - - E? v 03.5.0". -
n «.0 H 0.0 0 0.. H 3. - - E. v 02000:: -
0 Em H 0.0.. a md H o; - - E? v 00.2.0 -
00 ...m H rd 0 2 H dd - - Emé A “.4. + :0“. -
0 oé H Nd 0 m... H md - - Em... A 00.0.3.5". -
0 fr H dd 0 ad H 0.0 - .. Em... A 00.00.05 -
0 wd H 0N 0 03 H ed - - Em... A 00.8.0 -
- - mwded mndHNNP E. v - -
- - HQOHwe mdePNP 203A - -
- - 0 0d H hm 0 m... H «.3 - n.< + to“. -
- .. 0 md H dd 0 Nd H 0.9 - 005.80". -
- - 0 md H nd 0 N... H m. 3 - 00.0000: -
- - 0 ad H o... 0 0.? H m.N.. - 022.0 -

- - 0 md H «.0 0 md H o. E. - - cm< 0....>>

- - 0 md H v...” 0 Nd H v.2. - - 20.0. 000.0

.00 H .00 H 2.0.25 .00 H .mm H 8.008.. 05.58; 8.03.0

0.0.03 >0 5 :00...— 995 0.0.0; p.00...— 9.0E=. £0.03 :00...- 995
oz...nc.0~0._n. uz... 0.9.x 0:.E< .000... 0.22.".

 

.080sbm-000 .000... n 02.5 x0838 .3 $038003. . N. .3 .3. .dnuzv .Amddvm .<>OZ< >027m .<>OZ< 0508800000 503%
900060.05 0050 0000 800.. 8000...... >.0000....0w.m 0000 00.0000. .8 00000000 $0.800 .3 080.00 0 0.5.3 8000. 880%.... .3 0032.0. m00.0>
.m00.0> 008030000 2+5 mo. 00 00000 000 8000. 00000....0w.m 0.000 020.8380 .000 OZ... 5.005080000000 000800000 0003 m00.0>

000000.080 0.00 00.80 .000 0.20.5 doom 0. 0.000 000 00.0.3 .000 000 000% 80m. 800.00 .0 00000000000 000.000 Amman. 000.). 2a... 030...

 

 

 

 

 

 

 

 

 

 

:m 3.: H 8.: : 8.: H :3 : mad H 8N m 5.3 H 8.3 2383:...
:m «No H 5.: : :d H Rd : 8.: H 2.: a 8.: H 3.. 2.33:0...
u< + 5“. 25:5". 833:: 3.25 355 2:858
3:92 - aoocaBEo 2.253....
:m 8.: H «.5 :m 8.8 H 8.9. : 8...... H 5.2 m 8.2 H 9.? 23560. 2.2.
“.2 + to". 85:5". 382.5 8.26 335 23:28
2...... . 32.2an 2253....
a 8.: H Rd m 8.: H Ed - - - - 2333.5
- - : a: H 53 m 3.: H 8% - - 235.00. «.6
- - - - : 8N H No: a t: H 2.8 2882.3
- - - - m B: H 8.3 a ~92 H 8.3 22:60. «ES
- - - - : 8.: H a. 2 m 2.2 H 8% £88: gcaxozéi
:2 322. - :2 :85 :2 SE; :2 :85 :2 222, :2 :36 635 2:858
.392 22. 0:2.

 

30:2an @20on

 

$28 55.3 $9.235“. %: €33.58. mm em .8 .3. .3 .omnzv .322 .<>oz< 5.3.3 5:8 :23 82.: E226

358$:me 0.8 8.3: was £58 £53» 98:2 Santa: 3 330:8 mo=~a> .33? cog—8mg 2+5 wo_ no v83 08 30:2 commowEwmm

doom 3 much :8 833 was am: 5on 90 09:28 8on G38 .32 S w\w8 nonesvoa 25388 2:23 Amway 502 .1 2an

42

 

 

 

 

 

 

8 8.3 H 5.02 :m 2.8 H E. H z : 8.9 H 8.8 a 8. 3. H 8.8 8882.3
: 8.8 H 8.8m : 8.8 H 8.8 : 8.8 H 8. E m 8.888 H E. :88 888m .083:
8 8.8 H 8.8 : 3.8. H 8.8 :m 8.8 H 8. 8 a 8.8. H 8.88 8888.882...

“2 + to. 85:5. 8822: 3.2.0 as 2.89:8
.3080 . 30:22.5 E253...
8 82.2 H 8.28 : 8.8 H 8. x: : 3.8 H 86: a 288 H 8.2... .2202
"2 + .3. 85:5. 8.83:: 8.26 as 2.39:8
.m:m:< . moo—8.2.5 «ca—5.3....
: 8.88. H S. 85 : 8.8: H :8. - - 338: .858
:2 2.55 :2 :85 :2 8:3 :2 :85 as 8.69:8

 

 

.30qu

3:92

 

woo—.2220 3325

A30. .3 303.8%... 3 .5 .mm .mm .SHZV Amodvm .<>OZ< 827$ .05: :80 802 582...: 3:805:30. 08 88.6 was 8.6.

2:23 822 382.2: 3 @2523. 833/ .mo:_:> 38.888. CQC 8.02: a: woman 0.: 0:88:22 .8 822 8:85:85

.825, 35888... Sta mo. no :83 08 .3800 E 223: 32:3 28 8802.2. 352:2 .8 82.2 858$:me

.38 E 80.. :8 233 9:. :8 50% mo Eco—am can 2.3 So... $5 53860.: 25888 2:29» Ammi :82 m2 2an

43

 

 

 

 

 

m 00.0 H 50.0 m 00.0 H N10 0 N...0 H 50.0 m P... H 0.0.. m. 5.0 H 0.0 m 0.? H F.0N - 00< 0:55
0 _.0.0 H 0N0 0 0...0 H 3.0 0 No.0 H .00 m 0... H 0.0.. 0 N0 H 0.0 m 0.N H 0.0m - 00< c020
0 0.50m. N 0:001 w 0.500 0 0.500 N 0.50: .. 0:001 .00—500.... 00.0000

m 0.0 H 0.0 0 0.0 H 0.0 m 5.0 H 0.5 m 00.0 H 00.0 m :20 H 05.0 m 00.0 H N0.N n.< + to". -

m 0.0 H 0.0 m 0.0 H 0.0 m 0.0 H 0.0 m 09.0 H 00... m 00.0 H 00.0 m 3.0 H F52. 0055.0... -

m 0.0 H N... m 0.0 H v.0 m 5.0 H 0.0 m 5v.0 H 0N... m :20 H 55.0 m NN.0 H 00... 00.00003 -

m 0.0 H N0 0 0.0 H 0.0 m 0.0 H 0.0 m 00.0 H 00.0 m 50.0 H 55.0 m Fwd H 504 00.020 -

m 0.0 H 0.0 m 0.0 H 0.0 m 0.0 H 0.5 m 00.0 H 59... m 00.0 H 55.0 m 0.20 H 00.N - 00¢. 2.23

0 N0 H N0 0 0.0 H 0.0 0 0.0 H 5.0 0 00.0 H 00.0 m 50.0 H N50 0 Nwd H 00% - cm< 000.0
a 0.50: N 00:01 _. 00:01 0 0.50.... N 05.01 P 0:000 2.05.00... 00.0000

 

00H E

 

Fl

  

  

00

 

r

-2300 00. 00.0 .02 000.2 0

 

 

3.8032... 5:28 B 002.8%... N. .8 .E .0. .8 .8 .8 .8 .E .2 .8 .8”... 08.8. .8. 203-305.

.<>OZ< 00340 .200 0000 0.0... 2.0.2.00 00.000.00.000 0... E00000... .0 020000 00 000.200 20.05 0.2.2 80.0.0.0

>0 003200.. 00:03 .00—0050820000.. 0000.20.00 0.03 A00 000 A00 .00 0020.» 0000050000 .002? 008.0005...

2+0 02 00 00000 0.0 0.2.2 00000500.. N 0000. 0. 00.. 00.0 000..— .0008005 0200000 0. $000.00 00... 02.52.00

.0203 00000.. ”0000080 .0000? 80... A00 000 £00 .00 2.000 .00 A080 00800000 00000.. 00 .0000? A00 .000 .00 2.000

.00 A000 03:00.0 80... 00 £0.03 A00 .000 .00 2.000 .00 $0.00 000.3000 00.0 002—0.. A3 50.0 0.002 0.. 2000.

44

 

a) by treatm ent —<>— Untreated -a-— Fertilized
55 “r """"""""""""""""""""""""""""" + Fert + AF -I— Girdled

 

 

 

 

 

55 -.-b.)-hy_.s.p_e.eie_s_ ________________________________________ + Green Ash
-°-Wh'rte Ash

Foliar Chlorophyll (SPAD Value) 1: SE

 

 

 

25W

 

 

 

0 ' Jun '— """""""" JUI 5 Aug 3 Sept I
Figure 1.1: Mean (d: SE) chlorophyll index in green and white ash foliage by (a)
treatment and (b) species in 2008. Signiﬁcance letters are based on log (x+1) transformed
values. Points with different letters are signiﬁcantly different from each other within each
date (2-way ANOVA, p<0.05). Species p-values by date: 23 Jun p=0.71, 30 Jun p=0.030,
17 Jul p<0.001, 24 Jul p=0.003, 1 Aug p=0.014, 8 Aug p<0.001, 15 Aug p=0.004, 25

Aug p=0.l3, 2 Sept p=0.1 1. (N=38 for Jun, Jul, Aug dates; N=36 for 2 Sept).

45

a) species by treatment

 

 

 

 

 

 

 

 

 

 

 

    

 

 

 

20 ‘ " ’ a" ‘ ’5' u Untreated 9 Fert+AF a: Fertilized n Girdled '
ab
16 r _____________ a b _________________
b b i
~ 3 ~~~~~~~~
a .7 ~ ---------- 5: ————————
c __ c _
4 ~ 333 """ :: 31:1:
33’. :3 1:1 11’
0 _ 9.39 t‘: om
Green Ash White Ash
'” 22
to
31 b) specles by aspect I North
'9 18 a“ ---------------------------- ' ESouth “
"I
E
N
O
o
'o
E
3
.2
0
0
i
5- Green Ash White Ash
% c) treatment by aspect
f t 77777777 Untreated : Fert + AF n Fertilized n Girdled
a

      

 

 

North South

Figure 1.2: Mean (i SE) photosynthesis rates (pmol C02 - m-2 - s4) for green and white ash

trees by (a) species - treatment, (b) species - aspect, and (c) treatment - aspect in 2008.
Signiﬁcance letters are based on log (x) transformed values. Bars with different letters are

signiﬁcantly different from each other (3-way ANOVA, p<0.05). (N=48).

46

 

-1

-s )tSE
oo

-2

 

 

 

Total Follar Transplratlon (mmol H 20- m

Girdled Untreated Fertilized Fert + AF

Figure 1.3: Mean (:1: SE) transpiration rates (mmol H20 - m-2 - s'l) by treatment in 2008.

Bars with different letters are signiﬁcantly different from each other (3-way ANOVA,

p<0.05). (N=48).

47

50

 

+ Girdled —<>— Untreated
45 ,1 __________ , .................... -°— Fertillzed + Fert + AF

 

 

 

 

40*

35*

 

30 -

25*

 

 

 

20 /‘
0
1 May '|-—— Jun ---|-— Jul 1 Aug

 

Foliar Chlorophyll Index (SPAD Value) :1: SE

|- Sept -|

 

 

Figure 1.4: Mean (i SE) chlorophyll levels in green and white ash foliage by
treatment in 2009. Signiﬁcance letters are based on log (x+1) transformed values.
Points with different letters are signiﬁcantly different from each other within each
date (2-way ANOVA, p<0.05). Species p—values by date: 26 May p=0.03 1 , 9 Jun
p=0.82, 24 Jun p=0.004, 7 Jul p=0.97, 21 Jul p=0.31, 4 Aug p=0.76, 18 Aug
p=0.78, 1 Sept p=0.97, 15 Sept p=0.15. Treatment p-values by date: 26 May
p=0.93, 9 Jun p=0.004, 24 Jun — 15 Sept p<0.001. (N=38, 40, 39, 40, 39, 39, 39,

39, 37 by date).

48

10 a) By Treatment (June 15)
b

 

 

 

 

Girdled Untreated Ferh’lized Fert + AF
8 b) By Aspect (June 15)

 

 

 

 

O

 

North South
o) By Treatment (August 3)

b - b

12

 

10“

Photosynthesis (umol C02 - rn'2 - 8'1) :l: SE
N

 

 

 

 

 

Girdled Untreated Fertilized Fert + AF

Figure 1.5: Mean (:1: SE) photosynthesis rates (umol C02 . m.2 ° s-l) for green and white

ash trees by (a) treatment on June 15, (b) aspect on June 15, and (0) treatment on August
3 in 2009. Bars with different letters are signiﬁcantly different from each other (3-way

ANOVA, p<0.05). (N=47, 46 by date).

49

4 a) By Treatment (June 15)
a

 

 

 

 

Girdled Untreated F ertilized Fert + AF
a) By Specles (June 15)

 

 

 

 

 

Green Ash White Ash
c) By Treatnent (August 3)

 

Total Foliar Transpiration (mmol H 20- m'2 - 3'1) 1 SE

 

 

 

Girdled Untreated Fertilized Fert + AF

Figure 1.6: Mean (i SE) transpiration rates (mmol H20 - m.2 - 3.1) for green and white

ash trees by (a) treatment on June 15, (b) species on June 15, and (c) treatment on August
3 in 2009. Bars with different letters are signiﬁcantly different from each other (3-way

ANOVA, p<0.05). (N=47, 46 by date).

50

 

12

1o ——————————————————————— e ---------------------------------

 

 

 

 

 

Girdled Untreated Fertilized Fert+AF

Mean Adult Beetle Capture on Sticky Bands I tree (1 SE)

Figure 1.7: Mean (:1: SE) number of adult A. planipennis captured on sticky bands by
treatment in 2009. Signiﬁcance values were determined nonparametrically. Bars with

different letters are signiﬁcantly different from each other (Kruskal-Wallis test, p<0.05).

(N=40).

51

.0
co

l1st& 2nd
E 3rd
EB 4th 8. PP

0.6 -

0.4 -

0.2 -

 

Percentage Total Larvae by Instar Group (:I: SE)

O

Girdled Untreated Fertilized Fert + AF

Figure 1.8: Mean (1 SE) percentage total larvae grouped by instar (lst and 2nd

instars, 3rd instars, 4th instars and prepupal larvae) which hatched from eggs laid
by caged adults in 2009. Signiﬁcance values were determined nonparametrically.
Bars with different letters are signiﬁcantly different from each other within instar

group (Kruskal-Wallis test, p<0.05). (N=80).

52

400 -

White Ash

300 g E Green Ash

Mean larvae I m2 (1: SE)
N
8

 

Girdled Untreated Fertilized Fert + AF

Figure 1.9: Mean (i SE) larval density (m2) for species and treatment in 2008. Bars with

different letters are signiﬁcantly different from each other within treatment. Asterisks

indicate a signiﬁcant difference between species (2-way AN OVA, p<0.05) (N=3 8).

53

50%

\ White Ash
5 Green Ash .

40%

30%

20%

10%

Percentage dead larvae 1: SE

 

0%
Girdled Untreated Fertilized Fert + N:

Figure 1.10: Mean (i SE) percentage of larvae that died for species and treatment in
2008. Signiﬁcance values were determined nonparametrically. Bars with different letters
are signiﬁcantly different from each other by treatment (Kruskal-Wallis test, p<0.05).
(N =3 8).

54

120% -

E White Ash

90% _ . BGreen Ash

60%

30%

 

0%

Percentage late instar larvae :1: SE

Girdled Untreated Fertilized Fert + AF

Figure 1.11: Mean (:1: SE) percentage late instar larvae (fourth instars — prepupal
larvae) for species and treatment in 2008. Signiﬁcance values were determined
nonparametrically. Bars with different letters are signiﬁcantly different from each

other by treatment (Kruskal-Wallis test, p<0.05). (N=38.)

55

400
I White Ash

300 ——————— , ...... . ......... a Green Ash-

Mean larvae I m2 :l: SE
N
o
o

 

Girdled Untreated Fertilized Fert+AF

Figure 1.12: Mean (t SE) larval density (m2) for species by treatment in 2009.

Signiﬁcance letters are based on log (x+1) transformed values. Bars with different letters

are signiﬁcantly different from each other (2-way ANOVA, p<0.05). (N =40).

56

 

250

y=10.11x+17.06

200 —
150 -
1001

50-

 

Total Larvae Under Bark I Tree

 

 

 

O 5 10 15 20
Total Adults Captured on Sticky Bands ITree

Figure 1.13: Regression of total wild larvae found per tree on total adults captured on

sticky bands per tree in 2009 (Regression analysis, p<0.05). (N=40).

57

50%

I White Ash
________________________ a Green Ash —

40%

30%

20%

10%

Percentage dead larvae :1: SE

 

0%
Girdled Untreated Fertilized Fert+AF

Figure 1.14: Mean (:1: SE) percentage of larvae that died for species and treatment in
2009. Signiﬁcance values were determined nonparametrically. Bars with different letters
are signiﬁcantly different from each other by treatment (Kruskal-Wallis test, p<0.05).
(N =40).

58

100%

Ill

‘3 lWhite Ash
g 80% —- —————————————————————————— EGreen Ash
2

E 60%

t!)

.5

¢

:5 40%

0

U,

‘2 20%

¢

0

3

°- 0%

 

Girdled Untreated Fertilized Fert + AF

Figure 1.15: Mean (d: SE) percentage late instar larvae (fourth instars — prepupal
larvae) for species by treatment in 2009. Bars with different letters are

signiﬁcantly different from each other (2-way ANOVA, p<0.05). (N=40).

59

CHAPTER 2
Feeding Efficiency and Host Preference of Emerald Ash Borer (Agrilus planipennis
Fairmaire) Adults on Stressed and Vigorous Green Ash Seedlings.
Introduction

The emerald ash borer (A grilus planipennis F ainnaire), a devastating invasive
pest of ash (Fraxinus spp.) trees in the United States and Canada, was ﬁrst discovered in
Michigan and Windsor, Ontario, Canada in 2002. It has since been found in 14 other
states and Quebec (Poland and McCullough 2006, www.cmeraldashborer.info 2010).
More than 40 million ash (Fraxinus spp.) trees in Michigan have been killed to date
(www.cmeraldashborer.info 2010) and, if not controlled, the ash resource in the
northeastern US may be largely eliminated.

Tree injury to F raxinus spp. is caused by A. planipennis larvae feeding in the
cambial region, creating galleries in the phloem and scoring the outer sapwood, which
disrupts nutrient ﬂow and water conduction, respectively (Cappaert et al. 2005b). Adult
beetle emergence begins in late spring and continues through much of early summer.
Adult female A. planipennis require 5-7 d of feeding before mating, and 5-7 d more
before beginning oviposition (Bauer et al. 2004, Lyons et al. 2004). Beetles will continue
to feed and oviposit during the remainder of their 3-6 wk lifespan (Bauer et al. 2004,
Cappaert et al. 2005b). A. planipennis adults feed on ash leaves, but cause no signiﬁcant
defoliation (Cappaert et al. 2005b).

Like other Agrilus spp., A. planipennis prefer to oviposit on stressed trees
(McCullough et al. 2009a). Native Agrilus spp. including the bronze birch borer, A grilus

anxius Gory, and the two-lined chestnut borer, A grilus bilineatus (Weber), are secondary

60

pests that feed on stressed and dying trees (Anderson 1944, Haack and Benjamin 1982,
Dunn et al. 1986). Likewise, A. planipennis is a secondary pest throughout its native
range, presumably because it shares an evolutionary history with its host trees, which
have greater defenses against it than do North American ash species (Yu 1992; Akiyama
and Ohmomo 2000; Gould et al. 2005; Herms et al. 2005; Schaefer 2005; Williams et al.
2005, 2006, Eyles et al. 2007). Although healthy North American ash trees can succumb
to high densities of A. planipennis, adult beetles display a stronger attraction to trees
stressed by girdling, and this attraction has even affected dispersal habits as beetles are
more likely to ﬂy to areas occupied by girdled trees McCullough et al. 2009a, Mercader
et al. 2009, Siegert et al. 2010). In previous studies, girdled trees had higher larval
densities than healthy trees or trees stressed by herbicide, wounding, or exposure to the
stress elicitor methyl jasmonate (McCullough et al. 2009a, 2009b; Tluczek 2009).
Although adult foliage feeding causes little damage to the trees, examining these
feeding habits could provide insight to the basic biology of A. planipennis and the
relationship between host selection for feeding and oviposition. Historically, F raxinus
spp. has been free from major damage caused by defoliating pests (Solomon et al. 1993).
In fact, one of the most devastating invasive generalist defoliators in the northeastern
USA, the gypsy moth (Lymantria dispar L.), does not use ash as a host tree likely due to
chemical deterrents in the foliage (Markovic et al. 1997). Little research to date has
focused on adult A. planipennis host selection for foliage feeding. Adult beetles may be
more likely to oviposit on trees where they choose to feed. One study suggested that
beetles preferentially fed on clipped green, white, and black ash leaves over blue,

European (F rarinus excelsior L. ), and Manchurian (F raxinus mandshurica Rupr. ) ash

61

leaves, possibly due to differences in volatiles produced by these species (Pureswaran
and Poland 2009a). However, volatile production may differ between clipped and intact
ash leaves. A similar study reported that lower feeding on Manchurian ash may reﬂect
higher nutritional quality or stronger defenses in foliage from those trees. In comparison,
greater feeding on green ash foliage may be compensatory (Pureswaran and Poland
2009b). Other phytophagous insects beneﬁt from feeding on plants with high levels of
nutrients, particularly nitrogen, amino acids, and protein: carbohydrate ratios (Mattson
1980, Doi et al. 1981, Kyto 1996, Fisher et al. 2001, Bi et al. 2003, Chen et al. 2009,
Chen and Poland 2009). High chlorophyll content or photosynthesis rates are functions of
high nitrogen levels, and may also contribute to increased foliar nutrition and better
beetle success on these trees. Leaves with lower nutritional quality may require more
compensatory feeding by adults, while leaves with higher nutrition (e. g. foliage from
fertilized trees) may be consumed in smaller quantities (Mattson 1980, Scriber and
Slansky 1981, Chen et al. 2009, Chen and Poland 2009, Pureswaran and Poland 2009b).
Other studies suggest that phytophagous insects may feed more on fertilized
plants due to a preference for hosts with more nutrients (Kyto 1996, Glynn et al. 2003).
The Growth/Differentiation Balance Hypothesis suggests that under certain conditions,
allocation of nutrients to foliage for growth in fertilized trees may reduce the energy
available for use as secondary defense metabolites. This would cause foliage with more
nutrients to be less resistant to insect feeding damage (Loomis 1932, Lorio 1986, Herms
and Mattson 1992). However, this hypothesis has had mixed support in studies (Kyto

1996, Glynn et al. 2003). Similarly, feeding on leaves with lower nutrition may prove less

62

efﬁcient and result in increased frass production from undigested leaf material (Mattson
1980, Scriber and Slansky 1981, Chen and Poland 2009, Pureswaran and Poland 2009b).

Chen and Poland (2009) compared foliar nutrients on green ash seedlings.
Variables considered included age of leaves, leaves grown in sun vs. shade, and girdled
vs. ungirdled seedlings. The study revealed an increase in non-structural carbohydrates in
girdled seedlings but a decrease in protein:carbohydrate ratios. This is consistent with the
observation that girdling causes an accumulation of carbohydrates above the girdle while
the trunk below the girdle receives none (Noel 1970, Roper and Williams 1989, Li et al.
2003, Mostafa and Saleh 2006, Chen and Poland 2009). Results of Chen and Poland’s
(2009) study suggest that younger leaves may contain more nitrogen and other nutrients
than older leaves, as these nutrients are necessary for growth and expansion (Mattson
1980, Harper 1989, Chen et al. 2009, Chen and Poland 2009). Chen and Poland (2009)
did not report that any of the factors they studied had an effect on beetle survival, and
they did not test for differences in amount of leaf material consumed.

I examined the effects of girdling and fertilization on green ash seedlings and on
A. planipennis foliage feeding behavior on these seedlings. I hypothesized that (l) A.
planipennis would be attracted to girdled seedlings and would spend more time feeding
on foliage from these seedlings when given a choice; (2) girdled seedlings would have
lower chlorophyll, photosynthesis, and nutrition than untreated seedlings; (3) fertilized
seedlings would have higher foliar chlorophyll, photosynthesis, and nutrition than
untreated seedlings; and (4) A. planipennis would require extra feeding on girdled foliage
to compensate for its lower nutritional value. Study objectives to test these hypotheses

were to (1) assess effects of girdling and fertilization on the foliar chlorophyll,

63

photosynthesis rates, and nutrient concentration of leaves of green ash seedlings; and (2)
evaluate adult A. planipennis feeding behavior on the green ash seedlings in choice and

no-choice bioassays.

Materials and Methods
Seedling Establishment

Green ash seedlings (45—61 cm) were acquired on 30 January 2009 from Lawyer
Nursery in Plains, MT. Seedlings were potted in l-gallon containers with F afard
Heavyweight Mix #52, consisting of processed pine bark, peat moss, vermiculite, and
perlite (Conrad Fafard, Inc; Agawam, MA, USA). The mix contained a water-soluble
nutrient starter charge which was leached out by the time of treatment applications, as
veriﬁed by lower readings on an electric conductivity meter in comparison to newly
fertilized seedlings. Seedlings were stored in a polyhouse at Michigan State University’s
Tree Research Center in East Lansing, MI. Conditions in the polyhouse were
approximately 15.5-21°C and 50-70% RH. Seedlings were watered twice weekly and
grown for eight weeks before treatments began. Seedlings were randomly assigned to two
groups comprised of 48 and 36 seedlings each. The 48 seedlings in Group 1 were used
for no-choice bioassays and were maintained in the polyhouse throughout the study. The
36 seedlings in Group 2 were used for the choice assay and were moved on 15 May 2009
to an outdoor plot. These seedlings were planted pot in pot and provided with drip

irrigation.

64

 

Treatment Applications

Group 1 seedlings were randomly assigned to one of three treatments: girdling,
fertilization, and untreated control (16 seedlings per treatment). Seedlings assigned to the
fertilization treatment were fertilized weekly beginning on 31 March 2009 via a liquid
feed with 200 ppm nitrogen, 60 ppm phosphorous, 150 ppm potassium, and pH 6.5
(Peters 20-10-20 Peat Lite Special, Scotts, Marysville, OH, USA). On girdled seedlings, a
pocket knife was used to remove a 5 cm length of outer bark and phloem on the main
stem below branches on 31 March 2009. Untreated seedlings were irrigated throughout
the study, but received no nutrient supplementation.

Group 2 seedlings were randomly assigned to one of three treatments: girdling,
fertilization, and untreated control (12 seedlings per treatment). Seedlings assigned to the
fertilization treatment were fertilized weekly beginning on 28 April 2009 via the same
liquid feed applied to Group 1 seedlings. This liquid fertilizer was no longer used after
seedlings were moved outdoors. A one-time granular application of Harrell’s Pro-Blend
with Micronutrients custom-mixed 19-4—8 controlled-release fertilizer (Harrell’s, Inc.,
Sylacauga, AL, USA) was applied on 22 May 2009 to the pot around the base of each
seedling at a rate of 5.6 g N per seedling (approximately 599 kg per ha). Seedlings were
girdled on 5 May 2009 as described above. Untreated seedlings were irrigated throughout

the study, but received no nutrient supplementation.

65

F oliar Nutrients and Photosynthesis

Chlorophyll content of Group 1 seedling foliage was analyzed weekly from 7
April to 12 May 2009 using the Minolta SPAD 502 Chlorophyll Meter (Spectrum
Technologies, Inc., Plainiﬁeld, IL, USA). Four readings were taken per seedling; two
from leaves on opposite sides of the lower portion of the seedling and two from leaves on
opposite sides of the upper portion of the seedlings. These readings were averaged to
obtain a mean value for each seedling. The Li-Cor LI-64OO (Li-Cor Biosciences, Lincoln,
NE, USA) portable photosynthesis system was used to measure photosynthesis and
transpiration rates on an upper and lower leaf from ten randomly selected Group 1

seedlings per treatment on 9, 16, and 30 April 2009. Photosynthesis rates were measured

via foliage gas exchange rates (A max) (umol C02 - rn-2 - s-l). Transpiration rates were

measured as mmoles H20 - m-2 - s-l. Seedlings were analyzed with the Li-Cor in the

polyhouse in mid-aftemoon in sunny conditions. Quantum ﬂux was set at 1500

2 . .
umols/m /s for use as a light source and machine temperature was set at that of the

average daily temperature in °C. Flow was set to 500 urns and the mixer set at 400 urns

COzR.

One leaf from each of ten randomly selected Group 1 seedlings per treatment was
removed on 1 May 2009, ﬂash frozen in liquid nitrogen, and stored at -20 °C until
processing. If leaves were so small that one leaf would not provide enough leaf tissue for
processing, two opposite leaves of the same age were selected. Leaf tissue was ﬁnely
ground in liquid nitrogen and approximately 50 mg was extracted for analysis. Protein

(mg/ g fresh weight) was determined via Bradford protein assay and amino acid

66

concentration (umol/g ﬁ'esh weight) was determined colorimetrically via cadmium-
ninhydrin procedure (Doi et al. 1981, Fisher et al. 2001, Bi et al. 2003, Chen et al. 2009).
Total non-structural carbohydrates (mg/ g ﬁ'esh weight), calculated as the sum of glucose
and starch, were determined using the glucose (HK) assay kit (Sigma-Aldrich, St. Louis,
MO, USA) and methods from Jones (1979). Starch was estimated as glucose equivalents
(Marquis et al. 1997, Chen et al. 2009).

On 19 May 2009, an upper and lower leaf on each of six, randomly selected no-
choice seedlings per treatment were removed for total nitrogen determination. If leaves
were small, two opposite leaves of the same age were selected to obtain 21 g leaf
material after oven drying. Leaves were oven dried at 655°C for 72 hr in a model 30 GC
lab oven (Quincy Lab, Inc., Chicago, IL, USA). Samples were sent for total nitrogen
analysis via micro-Kjeldahl digestion procedure at Michigan State University’s Soil and
Plant Nutrient Laboratory.

Foliar chlorophyll content of Group 2 seedlings was analyzed weekly from 28
April to 28 July 2009 using the Minolta SPAD 502 Chlorophyll Meter (Spectrum
Technologies, Inc., Plainiﬁeld, IL, USA) and methods described above. The Li-Cor LI-
6400 (Li-Cor Biosciences, Lincoln, NE, USA) portable photosynthesis system was used
to measure photosynthesis and transporation rates on an upper and lower leaf of ten and
ﬁve randomly selected Group 2 seedlings per treatment on 21 May 2009 and 16 June
2009 respectively. Number of seedlings was reduced on the second date due to many

seedlings having lost their leaves by that time. Photosynthesis rates were measured via

foliage gas exchange rates (Amax) (umol C02 - m-2 - 3.1) with a ﬂuorescent leaf chamber.

. . -2 -l .
Transpiration rates were measured as mmoles H20 - m - s . Group 2 seedlings were

67

analyzed with the Li-Cor outdoors in mid-afternoon in sunny conditions via the same

methods as Group 1 seedlings.

No-Choice Bioassays

I conducted four no-choice bioassays with adult A. planipennis beetles on Group
1 seedling foliage. Bioassays began on 12 April, 20 April, 4 May, and 7 May 2009.
Beetles were reared from logs which were harvested in fall 2008 from naturally infested
trees near Lansing, MI and maintained in cold storage at 39°C. After removing logs from
cold storage, they were placed in 76.2 cm long, 15.2-30.5 cm diam cardboard tubes
(Saginaw Tube Co., Saginaw, MI, USA) in a rearing room maintained at 267° C. Adult
beetle emergence from logs followed in approximately 21 days. Emerging adults were
collected daily and immediately transferred to bioassay material. Four bioassays were
conducted to evaluate possible changes as the seedlings aged and continued to be affected
by the fertilization and girdling treatments. Two leaves opposite each other on the same
whorl of each seedling were collected and scanned in a ﬂatbed scanner to determine leaf
area using WinFOLIA software (Regent Instruments, Inc.; Quebec, Qc, Canada). After
scanning, the petiole of each leaf was cut on a slant to provide a fresh surface area for
water uptake. Leaves were inserted into water-ﬁlled microcentrifuge tubes to maintain
moisture and placed individually in 150 mm diam Petri dishes. Two newly-emerged male
A. planipennis were placed in a dish with one of the leaves from each seedling, and two
newly-emerged female beetles were placed in a separate dish with the second leaf.
Beetles were allowed to feed for three days. Petri dishes were checked daily for beetle

mortality. After feeding, leaves were re-scanned and total leaf area consumed was

68

determined by comparing original leaf area to the remaining area. Leaf area consumed
was divided by total “beetle days” (sum of the number of days, to 0.5 d, that each beetle
survived) to obtain a value for total leaf area consumed per beetle per day. Frass was
collected from each dish and weighed to the nearest mg to estimate feeding efﬁciency on

the leaves.

Choice Assay

A choice assay was conducted using the 36 outdoor seedlings from 4 June
through 13 August 2009. Seedlings used in the choice assay were exposed to feeding by
the wild A. planipennis population at the Tree Research Center. Each week, total leaves
were counted per seedling. Any live adult beetles were noted and, whenever possible,
sexed. Feeding on each leaf of a given seedling was visually examined and recorded on a
1 (very little feeding) to 5 (extensive feeding) scale. Ratings for each leaf were summed
to obtain a feeding estimate per seedling for each date. These values were compared by
treatment. The cumulative number of leaves assigned to each feeding rank was recorded
weekly. At the end of the study period, the proportion of total leaves per seedling
assigned to each feeding rank was recorded and the means were compared by treatment

across all dates.

Statistical Analysis
All data were tested for normality using the Shapiro-Wilk test (Shapiro and Wilk
1965) and residual plots (PROC GLIMMIX, SAS Institute 2001). All parametric

statistics were run using SAS 9.1 software (PROC GLIMMIX, SAS Institute 2001). One-

69

way or two-way analysis of variance (ANOVA) were applied to data that followed a
normal distribution (SAS Institute 2001). If ANOVA results were signiﬁcant (p<0.05),
Tukey’s honestly signiﬁcant difference (HSD) multiple comparison test was used to
assess differences among treatments (Tukey 1953, SAS Institute 2001).

Percentage foliar nitrogen was tested using two-way ANOVA to assess effects of
tree treatment and leaf location. Remaining foliar nutrient concentrations, chlorophyll
content in both seedling groups, and mean feeding ranks in Group 2 seedlings were tested 1
using one-way AN OVA to assess the effects of seedling treatment. Photosynthesis rates
in both groups and transpiration rates in Group 1 and in Group 2 on 21 May were tested
using two-way AN OVA to assess effects of seedling treatment and leaf location. Total
frass consumed per beetle per day was tested using two-way ANOVA to assess effects of
tree treatment and beetle sex.

Protein concentration and Group 2 seedling photosynthesis rates on 16 June were
normalized by log (x+1) transformation. Protein: carbohydrate ratios, Group 1 seedling
photosynthesis rates on 9 and 16 April, and mean feeding ranks in Group 2 seedlings on
25 June, 2 July, 9 July, and 16 July were normalized by log (x) transformation. Group 1
seedling photosynthesis rates on 30 April and mean frass produced per beetle per day in
no-choice bioassays were normalized by square root (x) transformation.

If transformations did not normalize variables, nonparametric tests were used.
Friedman’s two-way nonparametric ANOVA (SAS Institute 2001) was used to test non-
norrnal data involving potential interactions. When signiﬁcant, nonparametric multiple
comparison tests were applied following methods from Conover (1971) and Zar (1984).

If the interaction term in Friedman’s test was p2 0.50 or no potential interactions existed,

70

Wilcoxon Rank Sum tests (Ott and Longnecker 2001) and Kruskal-Wallis tests (Kruskal
and Wallis 1952) were used. When signiﬁcant, multiple comparison tests were applied
following the same methods (Conover 1971, Zar 1984). Friedman’s two-way
nonparametric ANOVA was used to test Group 2 seedling transpiration rates on 16 June
and mean leaf area consumed per beetle per day in no—choice bioassays. Kruskal-Wallis
tests were used to analyze percentage leaves consumed at each feeding rank on Group 2

seedlings. All analyses were conducted at the p<0.05 level of signiﬁcance.

Results
F oliar Nutrients and Photosynthesis — Group 1

Total percentage foliar nitrogen was higher in fertilized seedlings than in
untreated seedlings and over two times higher than in girdled seedlings (F =29.70; df = 2,
28; p<0.001) (Table 2.1). No signiﬁcant differences in nitrogen between upper and lower
leaves were observed (p=0.82). Protein concentration of foliage from fertilized seedlings
was approximately half that of girdled or untreated seedlings (F=5.27; df=2, 27; p=0.012)
(Table 2.1). Total amino acid concentration was nearly twice as high in foliage from
fertilized seedlings when compared to foliage from untreated seedlings (F =3.77, df=2,
27, p=0.036) (Table 2.1). Total foliar non-structural carbohydrates were higher in foliage
from girdled seedings than from fertilized seedlings (F =24.1 1, df=2, 27, p<0.001) (Table
2.1). There were no signiﬁcant differences in protein:carbohydrate ratios among
treatments (p=0.72) (Table 2.1).

ChlorOphyll content of Group 1 seedlings ﬂuctuated over the course of the study.

F ertilized and untreated seedlings had consistently higher chlorophyll levels than girdled

71

seedlings by mid-May, approximately one month after treatments began (Fig. 2.1). By the
end of May the average chlorophyll content of fertilized seedlings had peaked and the
chlorophyll content of girdled seedlings was at its lowest (Fig. 2.1). Photosynthesis rates
were consistently lower on girdled seedlings than on seedlings of other treatments, which
were at least twice as photosynthetically active (9 April: F =64.00; df=2,47; p<0.001; 16
April: F=74.06; df=2,52; p<0.001; 30 April: F=73.79; df=2,51; p<0.001) (Fig 2.2a). On
16 April, photosynthesis rates on fertilized seedlings were also higher than those on
untreated seedlings, but this was not the case on 9 or 30 April (Fig 2.2a). Lower, older
leaves were more photosynthetically active than upper leaves on 9 April ( =4.07;
df=l,47; p=0.049), 16 April (F=l 1.21; df=l,51; p=0.002), and 30 April (F=5.28;
df=l,51; p=0.026) (Fig 2.2b). Transpiration rates on girdled seedlings were also at least
half that of transpiration rates on other seedlings (9 April: F =8.97; df=2,45; p<0.001, 16
April: F=59.34; df=2,51; p<0.001, 30 April: F=95.16; df=2,51; p<0.001) (Fig. 2.3).
There were no transpiration differences between upper and lower leaves on 9 April

(p=0.47) or 16 April (p=0.06). On 30 April transpiration was slightly higher on lower

leaves (3.30 i 0.36 mmol H20 - m-2 . 8-1) than on upper leaves (2.97 i 0.28 mmol H20 -

m“2 - s'1)(F=4.81; df=l,51;p=0.033).

F oliar Nutrients and Photosynthesis — Group 2

The chlorophyll content of Group 2 seedlings was affected by an interaction
between date and treatment (F =6.9l; df=14,271; p<0.001) (Fig. 2.4). Chlorophyll levels
peaked in fertilized seedlings and were higher than in seedlings of other treatments by

mid-May (Fig. 2.4). By mid-June, all of the girdled seedlings had lost their leaves and all

72

untreated seedlings lost their leaves by the end of July (Fig. 2.4). Photosynthesis was
lower on girdled seedlings compared with seedlings of other treatments on 21 May
(F=34.73; df=2,53; p<0.001) and 16 June (F=19.51; df=2,19; p<0.001) (Fig 2.5a). On 21
May, photosynthesis rates of fertilized seedlings were also higher than those on untreated
seedlings. Differences were more difﬁcult to detect on 16 June due to high variability
(Fig 2.5a), and by this time average photosynthesis rates of all treatments had decreased
by half due to seedling stress. As on no-choice seedlings, photosynthesis rates on
seedlings in the choice assay were lower on upper leaves than on lower leaves on 21 May
(F=4.26; df=l,53; p=0.044). This difference was not signiﬁcant on 16 June (p=0.81) (Fig
2.5b). Transpiration rates on girdled trees were at least half that of rates on other trees on
21 May (F=32.11; df=2,51; p<0.001) but high variability obscured signiﬁcance on 16

June (p=0.77) (Fig 2.6). Transpiration rates were higher on lower leaves (2.46 i 0.30
mmol H20 - III-2 - s'l) than on upper leaves (1.70 i 0.22 mmol H20 - rn'2 - s") on 21

May, but high variability again obscured signiﬁcance on 16 June (p=0.006).

No-choice bioassays

No signiﬁcant differences in total leaf area consumed by A. planipennis were
apparent in rounds I (p=0. l4) and 4 (p=0.41). In rounds 2 and 3, beetles consumed more
leaf material from girdled seedlings than from fertilized seedlings (Round 2 F =9.65;
df=2,89; p<0.001; Round 3 F=8.79; df=2,83; p<0.001). Feeding was also higher on
foliage from untreated seedlings than from fertilized seedlings in round 3 (Fig. 2.7). F rass
production did not differ among treatments in rounds I (p=0.40) and 4 (p=0.27). In

rounds 2 and 3, beetles produced over twice as much frass when feeding on girdled

73

seedlings than they did when feeding on fertilized seedlings (Round 2 F =23.27; df=2,90;
p<0.001; Round 3 F =1 1.86; df=2,86, p<0.001). In round 2, beetles also produced more
frass when feeding on untreated seedlings than they did when feeding on fertilized
seedlings (Fig. 2.8). No differences between male and female beetles were ever observed
for total leaf area consumption (Round 1 p=0.85; Round 2 p=0.88; Round 3 p=0.06;
Round 4 p=0.42) or frass production (p=0.32; Round 2 p=0.71; Round 3 p=0. 14, Round 4

p=0.37).

Choice assay

Girdled seedlings lost their leaves soon after pots were moved outdoors and were
not included in the study. Beetles from wild A. planipennis populations in the area began
feeding on leaves of the seedlings in mid-June. Examination of the seedlings revealed
over twice as much feeding on fertilized seedlings than on untreated seedlings through
most of the summer (2 July: F=6.23; df=2,24; p =0.007; 8 July: F=7.93; df=l,22;
p=0.010; 16 July: F=6.45; df=l,20; p=0.020; 23 July: F=6.37; dﬁl,l9; p=0.021; 30 July:

F=10.37; df=1,17; p=0.005) (Fig. 2.9). Similarly, percentage of leaves fed at any rank

was either not affected by species (Rank 1 x2=0.30, df=l,l2, p=0.58, Rank 4 x2=1.49,
dﬁl,12, p=0.22, Rank 5 x2=2.29, df=1,12, p=0. 13) or was highest in fertilized seedlings

(Rank 2 x2=5.42, df=l,12, p=0.02, Rank 3 x2=5.74, df=1,12, p=0.017). Most leaves had

senesced by mid-August 2009. F ertilized seedlings maintained their leaves the longest
(Fig. 2.10). Beetles were occasionally observed on seedlings but not often enough for

statistical analysis, although beetles were usually found on fertilized seedlings. On 25

74

June three beetles were on fertilized seedlings and one beetle was ﬂying in the vicinity of
an untreated seedling; on 2 July two beetles were on fertilized seedlings; and on 16 July

one beetle was on a fertilized seedling.

Discussion

In general, girdling caused nutrient stress while fertilization increased foliar
nutrition. Girdling reduced the levels of nitrogen and chlorophyll as well as
photosynthesis and transpiration rates of foliage. Girdling also increased foliar protein
and carbohydrate levels, probably due to the inability of these nutrients to move down
through the phloem to the roots (Noel 1970, Chen and Poland 2009). However, protein:
carbohydrate ratios in girdled foliage were unaffected. Girdled seedling nutrition was
consistent with observations made by Chen and Poland (2009) on girdled seedlings,
which showed an increase in carbohydrate concentration of foliage but a decrease in
protein: carbohydrate ratios. This protein: carbohydrate ratio seems to have an effect on
the quality of nutrition beetles can obtain from leaves. No signiﬁcant differences in
protein: carbohydrate ratios were observed among treatments in this study. In contrast to
girdling, fertilization increased foliar nitrogen and amino acid concentrations in
comparison to foliage from girdled or untreated seedlings. It is unclear why protein levels
were lowest in fertilized seedling foliage when amino acids were highest; perhaps there
was a lower rate of conversion of amino acids to proteins in these seedlings, but no tests
were run to conﬁrm this.

Fertilization increased chlorophyll content in foliage of green ash seedlings in

both the polyhouse and outdoors compared with girdled seedlings. Higher photosynthesis

75

rates on the lower leaves are probably due to greater leaf age. These results are consistent
with observations made by Chen and Poland (2009) that older leaves had higher
chlorophyll content, which should correlate with photosynthetic capabilities. Water loss
via transpiration was also highest on girdled seedlings, suggesting seedlings were stressed
and photosynthesis rates were low (Chaves et al. 2003). Overall, girdling appears to be an

efficient stressor of green ash seedlings, while fertilization may serve to increase seedling

h;

vigor.

The higher consumption of leaf material on girdled seedlings by A. planipennis
when compared with fertilized seedlings in two of four no-choice bioassays may reﬂect
compensatory feeding to make up for lower levels of nitrogen or amino acids in foliage
from girdled seedlings (Mattson 1980, Scriber and Slansky 1981). Compensatory feeding
to make up for nutrition differences have also been observed in green ash in comparison
to Manchurian ash (Pureswaran and Poland 2009b, Chen and Poland 2009).

F rass production was also highest when beetles fed on foliage from girdled
seedlings. In general, relative frass amounts compared among treatments appeared to
closely match the relationships observed in amount of leaf material fed. Lower feeding
and frass production on fertilized seedlings may indicate beetles feeding on these
seedlings better utilized water and nutrients. Sex of beetles had no discernible effect on
adult feeding habits. The lack of signiﬁcant differences in feeding or frass production in
no-choice bioassay round 1 may be due to treatments not yet having affected seedling
vigor at such an early date. Reasons for the lack of any signiﬁcant differences in round 4

are unclear.

76

The amount of A. planipennis adult feeding damage was greater on fertilized
seedlings over untreated seedlings in outdoor choice tests, indicating that adults preferred
to feed on foliage with higher nutrient levels. Girdled seedlings did not tolerate exposure
to outdoor conditions, probably due to their weakened state. The fact that fertilized
seedlings maintained their leaves the longest is probably a result of their enhanced vigor.
No-choice bioassays with Group 1 seedlings revealed that compensatory feeding is
necessary on girdled foliage. If compensatory feeding makes up for the effects of lower
foliar nutritional quality, this may explain Chen and Poland’s (2009) observation that no
signiﬁcant differences exist in adult A. planipennis survival on foliage from girdled vs.
ungirdled seedlings in the laboratory. In wild conditions, however, compensatory feeding
may decrease survival by increasing the amount of time during which beetles must feed
and therefore also be exposed to predation or severe weather. Preferential feeding on
fertilized foliage is likely because feeding on these seedlings is more energy efﬁcient.
Optimal foraging theory states that animals will choose feeding habits which maximize
energy obtained from food and reduces time spent obtaining that food (MacArthur and
Pianka 1996). Therefore, feeding on girdled foliage which is both less energy efﬁcient
and increases time spent feeding should not be adaptive for these beetles. It would appear
to be more beneﬁcial in the wild to feed on healthy foliage.

Choice assay results appear to contradict the observation that adult beetles are
most attracted to stressed or girdled trees for mating and oviposition (Cappaert et al.
2005b, McCullough et al. 2009a, 2009b; Tluczek 2009). It is worthwhile to note,
however, that even the fertilized seedlings were stressed in these outdoor planting

conditions when compared to their vigor in the greenhouse in Group 1. It is possible that

77

beetles exhibited no preference on which plants to land, but simply fed longer on
fertilized seedlings after landing due to better food quality. However, this has not been
tested for and beetles observed in the plantation were almost always on fertilized
seedlings. It may be beneﬁcial to repeat these studies on larger, more mature trees that
are suitable for oviposition. Chapter I brieﬂy touches on this issue, but more research is
needed to compare adult A. planipennis foliage feeding choices with oviposition
preferences. It is unclear what the association may be between adult host choices where
both leaf feeding and oviposition are concerned. Understanding the relationship between
host choice for oviposition and host choice for foliage feeding may shed light on A.

planipennis adult behavior and host relations.

78

 

 

m md H EH 9 ed H «N m ad H 06 B NF H md m 3.. H Nam toughen.
w No H vs no oé H 09 a No H Om m o4 H Q? n mN H New 68.35::
m ed H A... m m; H «NH an ad H me o oN H 0: a w... H mdm 3.9.0
.mm H .mm H £92, an H Ems; .mm H .2 £2 5%.. 23.8:
2.203 .3 can... see...— 995 such 2.08.... 2.203 new: 53E. conciz .30..
02..."...82n. oz... 2o< o:.E< .50... £32.".

 

essences 5303.38 .95 u ozb 3:28 .3 bosses cm .8 .8 .2 .emuz. $9on .<>oz<

433% 850 :08 Bob Beanbag baneoﬁcwmm 8a .8828 55.3 ﬂute. 38056 3 332.0,. mo:.m> .829

cogommcmb 0c mo. no women 8e mate. oogomawﬁ 058 oz... ”£88m .3913 testament 2+5 mo. no women one

H.530. oogomeB 580$ doom E 35:53 awe 5on 89a amazom mo 858.588 305:: Amman. H82). ”mm 2an

79

01
O

 

+ Girdled
+ Untreated
- -------------------------------------- - ~ ‘- -- —°—Fertilized -

A
01

A
O

 

 

l
H
l
l
l
l
l
l
I
l

I
l
l
l
I
l
I
l
l
l
l
l
l
l
l
I
l
|
l
l
l
|
I
I
l
l
l
l
I
l
l
1

 

 

 

 

I Mar 1 Apr 1 May 1

 

 

Foliar Chlorophyll Index (SPAD Value) 1 SE
or
\m
w \
m V
a
or

Figure 2.1: Mean (d: SE) foliar chlorophyll index by treatment over time for Group 1
seedlings. Points with different letters are signiﬁcantly different from each other within
each date (AN OVA, p<0.05). Treatment p-values by date: 31 Mar p=0.025, 7 Apr
p=0.07, 14 Apr p=0.24, 21 Apr p=0.23, 28 Apr p=0.07, 5 May p=0.020, 12 May p<0.001,

19 May p<0.001. (N=48 for 31 Mar-12 May dates, N=47 for 19 May).

80

6 a) by treatment

 

I Girdled

E Untreated

En Fertilized
b

     
   

 

 

 

 

30 April

 
 
 

 

I Upper
E Lower
a

Photosynthesis lumol C02 - m'2 - 3'1) 1: SE

 

 

9 April 16 April 30 April

Figure 2.2: Mean (:1: SE) photosynthesis rates (umol C02 - m-2 - 5‘1) of Group 1 green

ash seedlings by treatment (a) and leaf location (b) in 2009. Signiﬁcance letters for 9 and
16 April are based on log (x+1) transformation. Signiﬁcance letters for 30 April are based
on square root (x) transformed values. Bars with different letters are signiﬁcantly

different from each other (2-way ANOVA, p<0.05). (N=59, 57, 57 by date).

81

A
O)

I Girdled
E Untreated
En Fertilized

.3
N

on

 

Transpiration (mmol H20 - rn'2 - 5'1) 1: SE
A

.L

Figure 2.3: Mean (i SE) transpiration rates (mmol H20 - rn-2 - s-l) of Group 1 green ash

seedlings by treatment in 2009. Bars with different letters are signiﬁcantly different from

each other (2-way ANOVA, p<0.05). (N=59, 57, 57 by date).

82

Foliar Chlorophyll Index (SPAD Value) :l: SE

 

35-
30*

25-

20 -

15-

10~

a a +Girdled

a ' b _ a a +Untreated

\l. __b _____ _ ___--.a. ———————————————— —o—Fertilized
a i \ " a a

___—_‘_____m_-__.______ _.—_____________----__---___________ ______

___. .--___m_------m___m-ﬂ.77-__-_..-_-_-_-._.______ ._.._.______-____.._...

 

5/t’

| Apr -|— May —-|

0

 

 

 

 

Figure 2.4: Mean (i SE) foliar chlorophyll index by treatment over time for Group 2

seedlings. Points with different letters are signiﬁcantly different from each other within

each date (AN OVA, p<0.05). Treatment p-values by date: 28 Apr p=0.636, 5 May

p=0.005, 12 Mayp=0.014, 19 Mayp=0.257, 2 Jun p<0.001, 16 Jun p<0.001, 30 Jun

p<0.001, 14 Jul p<0.001, 28 Jul p<0.001. (N=36 for 28 Apr-2 Jun, N=33 for 16 Jun,

N=24 for 30 Jun, N=23 for 14 Jul, N=19 for 28 Jul).

83

a treatment

16
I Girdled
E Untreated
12 Ha Fertilized

 

21 May 6 June

b) by locatlon

I Upper
a Lower

Photosynthesls (umol 002 - rn'2 - 3'1) 1 SE
m

 

21 May 6 June

Figure 2.5: Mean (i SE) photosynthesis rates (umol CO 2 - rrr'2 - 3-1) of Group 2 green

ash seedlings by (a) treatment and (b) and leaf location in 2009. Signiﬁcance letters on 16
June are based on log(x+1) transformed values. Bars with different letters are

signiﬁcantly different from each other (2-way ANOVA, p<0.05). (N= 57, 23 by date).

84

on

I Girdled

a Untreated
EEI Fertilized

O)

k

 

Transpiration (mmol H20 - m.2 - 5'1) 1: SE

21 May 16 June

Figure 2.6: Mean (i SE) transpiration rates (mmol H20 ° m.2 ° $4) of Group 2 green ash

seedlings by treatment in 2009. Signiﬁcance values on 16 June were determined
nonparametrically. Bars with different letters are signiﬁcantly different from each other
(2-way ANOVA, Friedman’s 2-way nonparametric ANOVA, p<0.05). (N= 57, 25 by
date).

85

2
)

o

on

E

3 I Girdled
‘7). B Untreated

3 0.6 EB Fertilized

-1

9
.rs

.0
N

 

Mean leaf area fed beetle
0
'0

Round 1 Round 2 Round 3 Round 4

Figure 2.7: Mean (i SE) leaf area consumed (cmz) per beetle per day in no-choice

bioassays in 2009. Signiﬁcance values were determined nonparametrically. Bars with
different letters are signiﬁcantly different from each other (Friedman’s 2-way

nonparametric ANOVA, p<0.05). (N=84, 95, 89, 86 respectively by round).

86

I Girdled
4 - _, , f . _ a Untreated .
Ell Fertilized

 

Mean frass produced beetle'1 day-1 (mg)
N

Round 1 Round 2 Round 3 Round 4

Figure 2.8: Mean (:1: SE) weight of frass produced (mg) per beetle per day in no-choice
bioassays in 2009. Significance letters are based on square root (x) transformed values.
Bars with different letters are signiﬁcantly different from each other (2-way AN OVA

p<0.05). (N =96, 96, 92, 88 respectively by round).

87

22% 3 2 .w. .2 2H .8
.E «H .X. «a .em .VH .eNuz. .Gosve .<>oz$ see 58 82.. seems 2282:? ea 8:2
Eobbﬁ 5.3 ﬁEom .33.? 38.28285 Cc we. no women 8a 2.3. c. 98 .23.. a. .23.. N .25..

mm co auto. 858..“in .mwagoom N 96.5 no 25828.5 .3 6.28 maven... Amm H. 882 5N 8:3“.

22. 2 o=< o 22. om 22. mm 22. S 22. m 22. N 82. mm 83H 8 92. S 82. e

_ . . _ . _ . . l . P .

 

 

 

1

 

e e 8225......
._ ............................... .--.uo.mo.:::.r

 

 

or

ON

on

oe

as r xueu Burpaaa mean

88

0.20

I Fertilized

0.16
E Untreated
0.12

0.08

0.04

 

Mean Percentage Leaves Consumed at
Rank :t SE

0.00

Feeding Rank

Figure 2.10: Mean (:t SE) percentage leaves consumed at each rank level by treatment on
Group 2 seedlings. Signiﬁcance values were determined nonparametrically. Bars with

different letters are signiﬁcantly different from each other (Kruskal-Wallis test, p<0.05).

(N=24).

89

Appendix 1

Record of Deposition of Voucher Specimens*
The specimens listed on the following sheet(s) have been deposited in the named museum(s) as
samples of those species or other taxa, which were used in this research. Voucher recognition
labels bearing the Voucher No. have been attached or included in ﬂuid-preserved specimens.
Voucher No.: 2010-05
Title of thesis or dissertation (or other research projects):
TREE VIGOR AND ITS RELATION TO EMERALD ASH BORER (AGRILUS PLANIPENNIS

FAIRMAIRE) ADULT HOST PREFERENCE AND LARVAL DEVELOPMENT ON GREEN AND
WHITE ASH TREES

Museum(s) where deposited and abbreviations for table on following sheets:

Entomology Museum, Michigan State University (MSU)

Other Museums:

Investigator’s Name(s) (typed)
Chenin Kathleen Limback

 

 

Date 06 August 2010

*Reference: Yoshimoto, C. M. 1978. Voucher Specimens for Entomology in North America.
Bull. Entomol. Soc. Amer. 24: 141-42.

Deposit as follows:
Original: Include as Appendix 1 in ribbon copy of thesis or dissertation.

Copies: Include as Appendix 1 in copies of thesis or dissertation.
Museum(s) ﬁles.
Research project ﬁles.

This form is available from and the Voucher No. is assigned by the Curator, Michigan State
University Entomology Museum.

90

 

Appendix 1.1

Voucher Specimen Data

Pages

 
 

 

 

o Ewan/$9. mama

 

 

is... “$2.2...

 

2.22.5 . ... c8225. 2.. c. 2888

 

Lo.— mcoEBoam ooﬁ: o>onm o5 oozooom
mod SN .02 Locoso>

 

2035... .5253. £550
.828 .332 $28.82.
33800: h. 3005 _mcozﬁom $3

 

 

Page 1 of 1

.__oo £035... 2.0

ghoul;

ion. .22..

A... memotoEm

macxxﬂmv can 832. So {on Love: em.
2:2. can 508%... So .oEoO

.250 2288”. 8.... :ws.

.8 522.. Hz<o_:o_.2

0.03:... 2.0 .2. .3...

 

 

 

 

 

 

 

 

 

 

 

 

 

0757mm
32 5mm :85 So: 399cm
:92 m N. .v . .00 0039.625 H246.10.5— o.._mc.:_Mu_ MECREEQ mascot.
m w 6 0.. s
new r s s e h 9 8.883589
mm m. m m m... m m m ﬁ .0 028:8 mcoEBoam .2 Son _onﬁ coxmtoﬁo .5 863m
h e t d d u V. 9
M w d O A A P N m E
co .3832

91'

LITERATURE CITED

Akiyama, K. and S. Ohmomo. 2000. The Buprestid beetles of the world. Iconographic
Series of Insects 4. Gekkan-Mushi Co., Ltd. 341 pp.

Anderson, R.F. 1944. The relation between host condition and attacks by the bronzed
birch borer. Journal of Economic Entomology 37: 588-596.

Anonymous. 2009. Cooperative Emerald Ash Borer Project
http://www. emeraldashborer. info. Accessed August 2010.

Anulewicz, A.C., D.G. McCullough, and D.L. Cappaert. 2007a. Emerald ash borer
(Agrilus planipennis) density and canopy dieback in three North American ash
species. Arboriculture and Urban Forestry 33 (5): 338-349.

Anulewicz, A.C., D.G. McCullough, T.M. Poland, and D.L. Cappaert. 2007b. Attraction
of emerald ash borer to trap trees: Can MeJA or manuka oil compete with
girdling? p. 83 in Emerald Ash Borer Research and Technology Development
Meeting, Mastro, V., and R. Reardon (comps).

Barter, G.W. 1957. Studies of the bronze birch borer, Agrilus anxius Gory, in New
Brunswick. The Canadian Entomologist 89: 12-36.

Bauer, L.S., R.A. Haack, D.L. Miller, T.R. Petrice, and H. Liu. 2004. Emerald ash borer
life cycle. p. 8 in Emerald Ash Borer Research and Technology Development
Meeting, Mastro, V., and R. Reardon (comps).

Bede, J .C., IN. McNeil, and SS. Tobe. 2007. The role of neuropeptides in caterpillar
nutritional ecology. Peptides 28: 185-196.

Bemays, EA. and R.L. Chapman. 1994. Host-plant selection by phytophagous insects.
Chapman and Hall, New York, NY.

Bi, J .L., N.C. Toscano, and MA. Madore. 2003. Effect of urea fertilizer application on
soluble protein and free amino acid contgent of cotton petioles in relation to
silverleaf whitefly (Memisia argentifolii) populations. .1. Chem. Ecol. 29: 747-
761.

Cappaert, D., D. McCullough, and T. Poland. 20053. Emerald ash borer life cycle: a
reassessment. P. 19 in Emerald Ash Borer Research and Technology
Development Meeting, Mastro, V., and R. Reardon (comps).

Cappaert, D., D.G. McCullough, T.M. Poland, and NW. Siegert. 2005b. Emerald ash
borer in North America: A research and regulatory challenge. American
Entomologist 51(3): 152—165.

92

 

Chabot, BF. and DJ. Hicks. 1982. The ecology of leaf life spans. Ann. Rev. Eco. Syst.
13: 229-259.

Chaves, M.M., J .P. Maroco, and J .S. Pereira. 2003. Understanding plant response to
drought: from genes to the whole plant. Emotional Plant Biology 30: 239-264.

Chapin, F. S. 111, DA. Johnson, and J .D. McKendrick. Seasonal movement of nutrients in

plants of differing growth form in an Alaskan tundra ecosystem: implications for
herbivory. J. Ecol 68: 189-209.

Chen, Y. and T.M. Poland. 2009. Interactive inﬂuence of leaf age, light intensity, and

girdling on green ash foliar chemistry and emerald ash borer development.
Journal of Chemical Ecology 35: 806-815.

Chen, Y. and T.M. Poland. 2010. Nutritional and defensive chemistry of three North
American ash species: possible roles in host performance and preference by
emerald ash borer adults. The Great Lakes Entomologist. (In press).

Chen, Y., X. Ni, and GD. Buntin. 2009. Physiological, nutritional, and biochemical

bases of corn resistance to foliage-feeding fall armyworm. Journal of Chemical
Ecology 35: 297-306.

Conover, W.J. 1971. Practical nonparametric statistics. Texas Technical University, New
York.

Crook, D.J., A. Khrimian, 1. Fraser, J .A. Francese, T.M. Poland, and V.C. Mastro. 2008.
Electrophysiological and behavioral responses of Agrilus planipennis
(Coleoptera: Buprestidae) to host bark volatiles. Environ. Entomol. 37 :356—365.

de Groot, P., G.G. Grant, T.M. Poland, R. Scharbach, L. Buchan, R.W. Nott, L.
Macdonald, and D. Pitt. 2008. Electrophysiological response and attraction of

emerald ash borer to green leaf volatiles (GLVs) emitted by Host Foliage. J.
Chem. Ecol. 34: 1170-1179.

DePew, L.J. 1971. Effect of infurrow treatments of three systemic insecticides on grain
sorghum emergence. J. Econ. Entomol. 64: 1321-1322.

DePew, L.J. and ML. Hooker. Effect of insecticide placement at planting for control of
greenbug (Homoptera, Aphididae) on grain-sorghum. J. Econ. Entomol. 80: 490-
493.

Dicke, M. 2000. Chemical ecology of host-plant selection by herbivorous arthropods: a
multitrophic perspective. Bioch Syst & Ecol. 28: 601-617.

Doi, E., D. Shibata, and T. Matoba. 1981. Modiﬁed colorimetric ninhydrin methods for
peptidase assay. Anal. Biochem. 118: 173-184.

93

Dunn, J .P., D.W. Kimmerer, G.L. Nordin. 1986. The role of host tree condition in attack
of white oaks by the twolined chestnut borer, Agrilus bilineatus (Weber)
(Coleoptera: Buprestidae). Oecologia 70: 596-600.

Eyles, A., W. Jones, K. Riedl, D. Cipolline, S. Schwartz, K. Chan, D.A. Herms, and P.
Bonello. 2007. Comparative phloem chemistry of Manchurian (F raxinus
mandshurica) and two North American ash species (F raxinus americana and
Fraxinus pennsylvanica). J. Chem. Ecol. 33: 1430-1448.

Fisher, G.H., I. Arias, I. Quesada, S. D’aniello, F. Errico, M.M. Di Fiore, and A.
D’aniello. 2001. A fast and sensitive method for measuring picomole levels of

total free amino acids in very small amounts of biological tissues. Amino Acids
20: 163-173.

Foyer, CH. 1987. The basis for source-sink interaction in leaves. Plant Physiology and
Biochemistry 25: 649-657.

Garbelotto, M., D]. Schmidt, and T.Y. Hamik. 2007. Phosphite injections and bark
application of phosphite + PentrabarkTM control sudden oak death in coast live
oak. Arboriculture and Urban Forestry 33 (5): 309-317.

Gould, J ., J. Tanner, D. Winograd,and S. Lane. 2005. Initial studies on the laboratory
rearing of emerald ash borer and foreign exploration for natural enemies. p. 73-74
in Emerald Ash Borer Research and Technology Development Meeting. Mastro,
V., and R. Reardon (comps).

Glynn, C., D.A. Herms, M. Agawa, R. Hansen, and W.J. Mattson. 2003. Effects of
nutrient availability on biomass allocation as well as constitutive and rapid
induced herbivore resistance in poplar. Oikos 101: 385-397.

Guest, DI. and B.R. Grant. 1991. The complex action of phosphonates as antifungal
agents. Biological Reviews of the Cambridge Philosophical Society 66: 159-187.

Haack, RA. and D.M. Benjamin. 1982. The biology and ecology of the twolined chestnut
borer, Agrilus bilineatus (Coleoptera: Buprestidae), on oaks, Quercus spp., in
Wisconsin. The Canadian Entomologist 114: 385-396.

Haack, R.A., E. Jendek, H. Liu, K.R. Marchant, T.R. Petrice, T.M. Poland, and H. Ye.
2002. The emerald ash borer: a new exotic pest in North America. Newsletter
Mich. Entomol. Soc. 47 (3and4): 1-5.

Harper, J .L. 1989. The value of a leaf. Oecologia 80: 53-58.

Herms, DA. and W.J. Mattson. 1992. The dilemma of plants: to grow or defend. Q. Rev.
Biol. 67: 283-335.

94

Herms, D.A., E. Rebek, D. Smitley, P. Bonello, and D. Cipollini. 2005. Interspeciﬁc
variation in ash resistance to emerald ash borer. P. 33 in Emerald Ash Borer
Research and Technology Development Meeting. Mastro, V., and R. Reardon
(comps).

Iles, J .K. and A.M. Vold. 2003. Landscape tree cultivar preferences in Iowa, US. Journal
of Arboriculture 29: 331-336.

Jendek, E. 1994. Studies in the East Palearctic species of the genus Agrilus Dahl, 1823
(Coleoptera: Buprestidae). Part 1. Entomol. Prob. 25: 9-25.

Jones, M.G.K. 1979. An enzymic microassay for starch. Plant, Cell and Environ. 2: 227-
234.

Kenward, MG. and J .H. Roger. 1997. Small Sample Inference for Fixed Effects from
Restricted Maximum Likelihood. Biometrics 53: 983-997.

Kovas, K.F., R.G. Haight, D.G. McCullough, R.J. Mercader, N.W. Siegert, and A.M.
Liebhold. Cost of potential emerald ash borer damage in US. communities,
2009—2019. Ecological Economics 69: 569-578.

Kruskal, W.H. and WA. Wallis. 1952. Use of Ranks in One-Criterion Variance Analysis.
Journal of the American Statistical Association 47: 583-621.

Kytti, M., P. Niemeléi, and S. Larsson. 1996. Insects on trees: population and individual
response to fertilization. Oikos 75: 148-159.

Lee, K.P., S.T. Behmer, S.J. Sempson, and DA. Raubenheimer. 2002. A geometric
analysis of nutrient regulation in the generalist caterpillar Spodoptera littoralis
(Boisduval). J. Insect Physiol. 48: 655-665.

Li, C., D. Weiss, and BE. Goldschmidt. 2003. Girdling affects carbohydrate-related gene
expression in leaves, bark and roots of alternate-bearing citrus trees. Ann. Bot. 92:
137-143.

Liu, H., and LS. Bauer. 2006. Susceptibility of Agrilus planipennis (Coleoptera:
Buprestidae) to Beauveria bassiana and Metarhizium anisopliae. J. Econ.

Entomol. 99: 1096-1 103.

Loomis, W.E. 1932. Growth-differentiation balance vs. carbohydrate-nitrogen ratio. Proc.
Am. Soc. Hort. Sci. 29: 240-245.

Lorio Jr., PL. 1986. Growth-differentiation balance: a basis for understanding southern
pine beetle-tree interactions. For Ecol. Manage. 14: 259-273.

95

 

Lyons, D.B., G.C. Jones, and K. Wainio-Keizer. 2004. The biology and phenology of the
emerald ash borer, Agrilus planipennis. p. 5 in Emerald Ash Borer Research and
Technology Development Meeting, Mastro, V., and R. Reardon (comps).

MacArthur, RH. and ER. Pianka. 1966. On Optimal Use of a Patchy Environment. The
American Naturalist 100: 603-609.

Markovic, I., D.M. Norris, and E.V. Nordhiem. 1997. Gypsy moth (Lymantria dispar)
larval development and survival to pupation on diet plus extractables from green
ash foliage). Entomologia Experimentalis et Applicata 84: 247-254.

Marquis, R.J., E.A. Newell, and AC. Villegas. 1997. Nonstructural carbohydrate
accumulation and use in an understory rain-forest shrub and relevance for the
impact of leaf herbivory. Funct. Ecol. 11: 636-643.

Mattson, W.J. Jr. 1980. Herbivory in relation to plant nitrogen content. Annu. Rev. Ecol.
Syst.11: 119-161.

McCullough, D.G., D.A. Cappaert, T.M. Poland, P. Lewis, and J. Molongoski. 2007.
Evaluation of neo-nicotinoid insectidies applied as non-invasive trunk sprays. pp.
52-54 in Emerald Ash Borer Research and Technology Development Meeting,
Mastro, V., and R. Reardon (comps).

McCullough, D.G., D.L. Cappaert, T.M. Poland, A.C. Anulewicz, P. Lewis, and J.
Molongoski. 2008. Evaluation of non-invasive trunk sprays and trunk-injected
Emamectin Benzoate. pp. 40-42 in Emerald Ash Borer Research and Technology
Development Meeting, Mastro, V., and R. Reardon (comps).

McCullough, D.G. and NW. Siegert. 2007. Estimating potential emerald ash borer
(Coleoptera : Buprestidae) populations using ash inventory data. Journal of
Economic Entomology 100: 1577-1586.

McCullough, D.G., T.M. Poland, and D. Cappaert. 2009a. Attraction of the emerald ash
borer to ash trees stressed by girdling, herbicide treatment, or wounding. Can. J.
For. Res. 39: 1331-1345.

McCullough, D.G., T.M. Poland, A.C. Anulewicz, and D. Cappaert. 2009b. Emerald ash
borer (Coleoptera: Buprestidae) attraction to stressed or baited ash trees.

Environmental Entomology 38: 1668-1679.
Mercader, R.J., N.W. Siegert, A.M. Liebhold, and D.G. McCullough. 2009. Dispersal of

the emerald ash borer, Agrilus planipennis, in newly-colonized sites. Agricultural
and Forest Entomology 11: 421-425.

96

Mostafa, E.A.M. and M.M.S. Saleh. 2006. Response of balady Mandarin trees to girdling
and potassium sprays under sandy soil conditions. Res. J. Agric. Biol. Sci. 2: 137-
141.

Noel, A.R.A. 1970. The girdled tree. Botanical Review 36: 162-195.

Ott, R.L., and M. Longnecker. 2001. An Introduction to Statistical Methods and Data
Analysis. Duxbury Thomson Learning, Inc.

Poland, T.M. and D.G. McCullough. 2006. Emerald ash borer: Invasion of the urban
forest and the threat to North America’s ash resource. Journal of Forestry 104 (3):
118-124.

Pureswaran, D.S. and T.M. Poland. 2009a. Host selection and feeding preference of
Agrilus planipennis (Coleoptera: Buprestidae) on ash (F raxinus spp.).
Environmental Entomology 38: 7 57-765.

Pureswaran, D.S. and T.M. Poland. 2009b. Effects of visual silhouette, leaf size and host
species on feeding preference by adult emerald ash borer, Agrilus planipennis
F airrnaire (Coleoptera: Buprestidae). The Great Lakes Entomologist 42: 62-71.

Rebek, E.J., D.A. Herms, and DR. Smitley. 2008. Interspeciﬁc variation in resistance to
emerald ash borer (Coleoptera: Buprestidae) among North American and Asian
ash (Fraxinus spp.). Environ. Entom. 37 : 242-246.

Rodriguez-Saona, C., T.M. Poland, J .R. Miller, L.L. Stelinski, G.G. Grant, P. de Groot,
L. Buchan, and L. Macdonald. 2006. Behavioral and electrophysiological
responses of the emerald ash borer, Agrilus planipennis, to induced volatiles of
Manchurian ash, Fraxinus mandshurica. Chemoecology 16: 75-86.

Roper, TR. and LE. Williams. 1989. Net C02 assimilation and carbohydrate partitioning

of grapevine leaves in response to trunk girdling and gibberellic acid application.
Plant Physiol. 89: 1136-1140.

SAS Institute. 2001. PROC user’s manual, version 6th ed. SAS Institute, Cary, NC.

Schaefer, PW. 2005. Foreign exploration for emerald ash borer and its natural enemies.
P. 67-68 in Emerald Ash Borer Research and Technology Development Meeting,
Mastro, V., and R. Reardon (comps).

Scriber, J .M. and F. Slansky, Jr. 1981. The nutritional ecology of immature insects.
Annu. Rev. Entomol. 26: 183-211.

Siegert, N.W., D.G. McCullough, D.W. Williams, 1. Fraser, T.M. Poland, and 8.]. Pierce.

2010. Dispersal of Agrilus planipennis (Coleoptera: Buprestidae) from discrete
epicenters in two outlier sites. Environmental Entomology 39: 253-265.

97

Simpson, SJ. and D. Raubenheimer. 1993. The central role of the haemolymph in the
regulation of nutrient intake in insects. Physiol. Entomol. 18: 395-403.

Shapiro, SS. and MB. Wilk. 1965. An analysis of variance test for normality (complete
samples). Biometrika 52: 591-611.

Solomon, J .D., T.D. Leininger, AD. Wilson, R.L. Anderson, L.C. Thompson, and F .I.
McCracken. 1993. Ash pests: a guide to major insects, diseases, air pollution
injury and chemical injury. Gen. Tech. Rep. SO-96. New Orleans, LA: US.
Department of Agriculture, Forest Service, Southern Forest Experiment Station.
45 p.

Tluczek, AR. 2009. Inﬂuence of host vigor on larval distribution, development, and
mortality of Agrilus planipennis Fairmaire (Coleoptera: Buprestidae) in North
America. Masters Thesis, Michigan State University. 132p.

T'ukey, J .W. 1953. Multiple Comparisons. Journal of the American Statistical Association
48: 624—625.

Wilde, G., T. Mize, J. Stuart, J. Whitworth, and R. Kinsinger. 1984. Comparison of
planting-time applications of granular or liquid insecticides and liquid fertilizer
plus insecticide combinations for control of chinch bugs (Heteroptera: Lygaeidae)

and greenbugs (Homoptera: Aphididae) on seedling sorghum. J. Econ. Entomol.
77: 706-708.

Williams, D., H.P. Lee, and Y.S. Jo. 2005. Explorations for natural enemies of emerald
ash borer in South Korea during 2004. p. 66 in Emerald Ash Borer Research and
Technology Development Meeting, Mastro, V., and R. Reardon (comps).

Williams, D., H.P. Lee, and Y.S. J o. 2006. Exploration for emerald ash borer and its
natural enemies in South Korea during May-June 2005. p. 52 in Emerald Ash
Borer Research and Technology Development Meeting, Mastro, V., and R.
Reardon (comps).

[USDA—PS F IA] USDA—Forest Service, North Central Research Station, Forest
Inventory and Analysis. 2006. http://www.ncrs.fs.fed.us/4801/.

Yu, C. 1992. Agrilus marcopoli Obenberger pp. 400-401 in G. Xiao (ed.) Forest insects
of China, 2nd ed. China Forestry Publishing, Beijing, China.

Zar, J .H. 1984. Biostatistical analysis. Prentice Hall, Englewood Cliffs, NJ.

98