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DISTRIBUTION OF TRUNK-INJECTED ”c
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TEST OF THE SECTORED FLOW HYPOTHESIS.

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Sara Rose Tanis

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5/08 K IProj/AccE-Pres/ClRC/DaieDue Indd

DISTRIBUTION OF TRUNK-INJECTED l4C IMIDACLOPRID IN FRAXINUS AND
ACER TREES: A TEST OF THE SECTORED FLOW HYPOTHESIS.

By

Sara Rose Tanis

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

2008

ABSTRACT
DISTRIBUTION OF TRUNK-INJECTED l4C IMIDACLOPRID IN FRAXINUS AND
ACER TREES: A TEST OF THE SECTORED—FLOW HYPOTHESIS.
By
Sara Rose Tanis
Emerald ash borer (Agrilus planipennis Fairmore, Coleoptera: Buprestidae) and

Asian longhomed beetle (Anoplophora glabripennis, Motschulsky, Coleoptera,
Cerambycidae) are invasive exotic insect pests that have destroyed millions Of trees in the
United States and Canada. Imidacloprid applied as a trunk injection is often used as a
means of control, but efficacy is variable. The Objectives of this study were to determine
the (1) spatial movement of imidacloprid in ash (F raxinus spp.) and maple (Acer spp.)
trees (2) concentration of imidacloprid in tree tissues over two growing seasons and (3)

variability Of imidacloprid concentration in trees trunk-injected in either spring or fall.

Thirty-two ash trees were trunk-injected with 14C imidacloprid in June 2006, eight

additional ash and eight maple trees were trunk-injected in September 2006. Leaves,
trunk cores, fine roots, and stems were sampled over two growing seasons. Imidacloprid
equivalent concentration (IEC) in ash leaves varied with time, and orientation to the
injection point. Leaves from trees injected in the June had higher IEC than leaves fi'om
trees injected in September 21 DAT. During the year after injection, IEC in leaves from
both sets of trees were similar, though some patterns of sectoriality were still present.
Imidacloprid equivalent concentration in maple leaves was relatively constant and did not
vary between Opposite branches or over time. We conclude that ash trees have zigzag

xylem architecture patterns and maple trees have integrated xylem architecture patterns.

DEDICATION

I would like to dedicate this thesis to my parents: Dr. Dale and Rose Marie Sutton.

2g! — you gave me an undeniable love and fascination for plants from the
moment I could follow you around in the yard. Through the years you taught me
everything from plant identification to the ups and downs of xylem and phloem. You
always pushed me because you knew I could do better — it’s funny how I see that now. It
was always nice to know that YOU survived this crazy process and that you understood

how I was feeling when I was both struggling and triumphant.

My — thank you for being such a strong emotional support. I know for a fact
that neither Dad nor I could have done this without you. You have always been there to
listen to me complain about long days and short nights and you always knew when I
needed the mint chocolate chip cookies. Thanks for letting me be me, and for knowing

when I just need tO talk. Thanks mom, for being my mom.

You have both been a wonderful inspiration to me as a professional, as a teacher,
and as a person. I could not ask for more loving or more understanding parents. Thank

you for helping me see this through.

With all my love and deepest gratitude,

Sara

In Memory of Mapel Russell Sutton and Roland L. Cregg.

iii

ACKNOWLEDGMENTS

I would like to thank the following people for their support:

Dr. Bert Cregg , my major advisor. Thank you for agreeing to take me on as a graduate
student. Your patience and knowledge helped to make this challenge enjoyable. Without
your guidance as an undergraduate I would not have embarked on this task. Thanks for
helping in the field, for making me step outside of my comfort zone, and for letting me

take control. You have been a true mentor to me.

Dr. Deb McCullough, graduate committee member. Thank you for always answering my
crazy questions, for being a great teacher, and for your dedication. I learned a great deal
from you about the “insect side” of this project and look forward to working with you in

the future.

Dr. Frank Telewski — graduate committee member. Thanks for being so energetic and
eager to help us solve this portion of the EAB/Ash puzzle. Your knowledge of plant

anatomy and physiology were a true asset.

Dr. David Mota-Sanchez, thank you for all of your help with the 14Carbon work. Your

patience and guidance on injection day were much appreciated as were all those times
that you helped me with the scintillation counter, oxidizer, and calculations. Thank you

for the support.

iv

Dr. Therese Poland, thank you for all of your help with the injection tubes and ALB

information. It was truly a pleasure working with you.

Wendy Sue Klooster, thank you for your camaraderie, your support, and for helping me
take samples in extreme heat and pouring rain. I could not have completed this project

and/or stayed sane without you. Keep running.

Anna Arend, Dan Hess, Darren Gladstone, and Jennifer Hunnel — the undergraduate

student workers who put time and effort into this project.

Marlene Cameron — For the Ash Drawings

HOGS — Horticulture Organization of Graduate Students — For support, fun, friends, and

pizza once a month!

GPC — Department of Horticulture, Graduate Programs Committee — For their undying
support of the education of all Horticulture graduate students and for their commitment to

excellence.

Last but certainly not least,

My Family.

Dave Tanis, for support, a roof over my head, and for taking care of the girls.

Laurie Sutton, for a good sense of humor, a great sense of music, and finals week care
packages.

Dr. Norman and Bonnie Letsinger, for all the fun emails, late night UNO games, and
for treating me as a daughter and friend.

Amy and Dave Cronk, for support and friendship even when you are a thousand miles
away.

Amy and Matt Tanis for support, understanding, and genuine interest in what I do every
day.

The Girls, Timber and Sunshine for unconditional love and an unending desire to play.
The boys — Gabriel, Fatman, and Obi for “helping” me type.

Cheri, Bo, Sam, Haley, Gracie, Sammi, and Cierra Layton, for making Missouri a

place I call home.

Friends — To all my friends who stood by me and understood when I wasn’t able to call

back, go to movies, or meet for coffee. It’s hard to be a graduate student’s friend. Thank

you for being mine.

vi

TABLE OF CONTENTS

LIST OF TABLES ................................................................................. viii
LIST OF FIGURES .................................................................................. ix
CHAPTER 1
LITERATURE REVIEW
Introduction ................................................................................. 1
Emerald Ash Borer .......................................................................... 2
Asian Longhorned Beetle .................................................................. 4
Fraxinus spp ................................................................................. 6
Acer spp ...................................................................................... 7
Ash and Maple in Urban Forests .......................................................... 8
Insect Impacts .............................................................................. 10
Insect Control ................................................................................ 11
Containment/Eradication ......................................................... 12
Bio-Control ........................................................................ 12
Chemical Control ................................................................. 14
Trunk Injection Application ............................................................... 15
Efficacy Variability ......................................................................... 17
Sectored Flow ............................................................................... 20
Radio-labeled Tracers ..................................................................... 24
Summary .................................................................................... 27
Bibliography ................................................................................ 34
CHAPTER 2

DISTRIBUTION OF TRUNK-INJECTED l4C IMIDACLOPRID IN FRAXINUS SPP.
TREES: A TEST OF THE SECTORED FLOW HYPOTHESIS.

Abstract ....................................................................................... 45
Introduction ................................................................................. 47
Materials and Methods ................................................................... .52
Spring Injection .................................................................... 52
Fall Injection ....................................................................... 54
Sampling ........................................................................... 54
Statistical Analysis ................................................................ 57
Results ....................................................................................... 59
Spring Injection .................................................................... 59
Fall Injection ....................................................................... 61
Spring vs. Fall Injections ......................................................... 62
Observations .................................................................................. 62

vii

Discussion ................................................................................... 64

Objective One ...................................................................... 64
Objective Two ..................................................................... 68
Objective Three .................................................................... 69
Bibliography ................................................................................... 93

CHAPTER 3

DISTRIBUTION OF TRUNK-INJECTED 14C IMIDACLOPRID IN ACER SPP. TREES:
A TEST OF THE SECTORED FLOW HYPOTHESIS.

Abstract ..................................................................................... 98
Introduction ................................................................................ 100
Materials and Methods ................................................................... 104
Trunk Injection .................................................................... 104
Sampling ........................................................................... 105
Statistical Analysis ............................................................... 108
Results ...................................................................................... 109
Discussion .................................................................................. 1 10
Objective One ..................................................................... 110
Objective Two ...................................................................... 113
Bibliography ................................................................................ 127
APPENDICES ..................................................................................... 130

viii

Table

Table 2.1

Table A.1

Table A2

Table A3

LIST OF TABLES

CHAPTER TWO

Total number of emerald ash borer (A grilus planipennis) ............

larval galleries in ash trees (Fraxinus americana and F.
pennsylvanica) and gallery density (number of larvae
galleries per meter2 trunk surface) in trees trunk-injected
in either spring or fall with ”C-imidacloprid

APPENDIX

Analysis of variance of imidacloprid equivalent concentration ......

in F raxinus americana and F. pennsylvanica trees trunk-injected
with 14c — imidacloprid 28 June 2006.

Analysis of variance of imidacloprid equivalent concentration ......

in Fraxinus americana and F pennsylvanica trees trunk-injected
with 14c — imidacloprid 5 September 2006.

Analysis of variance of Imidacloprid equivalent concentration ......

in Acer platanoides and A. x. fieemanii trees trunk-injected with
14C — imidacloprid 5 September 2006.

ix

Page

....... 71

..... 130

..... 131

....132

Figure

Figure 1.1

Figure 1.2

Figure 1.3

Figure 1.4

Figure 1.5

Figure 2.1a

Figure 2.1b

Figure 2.2

Figure 2.3a

LIST OF FIGURES

Page
CHAPTER 1

Distribution of Emerald Ash Borer (Agrilus planipennis .................... 29
Fairrnaire, Coleoptera: Buprestidae) in the North America
(www.emeraldashborer.info 2008).

Distribution Of ash trees (Fraxinus spp, Oleaceae) in North ............... 30
America (USDA NRCS, 2008)

Distribution of native white ash (Fraxinus americana L., ................... 31
Oleaceae) in North America (Burns and Honkala, 1990).

Distribution of native green ash (F raxinus pennsylvanica, .................. 32
Marsh. Oleaceae) in North America (Burns and Honkala, 2008).

Distribution Of maple trees (Acer spp. Aceraceae) in North ................ 33
America (USDA NRCS, 2008).

CHAPTER 2

Treatment schematic, trees were injected at either 0° or 90° to ............. 72
the first whorl of branches. Mean distance from injection point

to the first whorl = 1.28 m. Mean distance between whorls =

0.18 m.

Sampling schematic of trees injected at either 0° or 90° to the ............. 73
first whorl of branches. Branches Of the first three whorls were

labeled 0°, 180°, L90° or R90° in relation to the injection point.

Mean distance from injection point to the first whorl = 1.28 m.

Mean distance between whorls = 0.18 m.

Mean l4C-Imidacloprid equivalent concentration i SE. in whorl... .......74
and terminal leader leaves from 0° or 90° (in relation to the first

whorl Of branches) spring-trunk-inj ected F raxinus americana and

F. pennsylvanica trees. Braches were labeled 0°, 180°, L90° or

R90° (in relation to the injection point. An * denotes leaves

collected afier abscission.

Mean l4C-Imidacloprid equivalent concentration :1: SE. in whorl... ......76
one stems and leaves (0°, 180°, L90° or R90°-in relation to the
injection point), trunk cores (0° or 180° in relation to the injection

point) and roots of 0° and 90° (in relation to the first whorl of
branches) spring-trunk-injected F raxinus americana and F.
pennsylvanica trees, samples taken 60 days after treatment.
Inset graphs have leaf data bars removed.

Figure 2.3b Mean l4C-Imidacloprid equivalent concentration :t SE. in stem ........... 78
samples taken from the first three branch whorls of 0° and 90°
(in relation to the first whorl of branches) spring-trunk-injected
F raxinus americana and F. pennsy/vanica trees 60 days after
treatment. Stem samples were labeled 0°, 180°, L90° or R90°
in relation to the injection point.

Figure 2.4 Mean 14C-imidacloprid equivalent concentration i SE. in .................. 80
leaves and stems from first whorl branches (0°, 180°, L90° or
R90° in relation to the injection point), coarse and fine roots.
Samples taken from 0° or 90° (in relation to the first whorl of
branches) spring-trunk-injected F raxinus americana and F.
pennsylvanica trees 60 days after treatment. Inset graphs have
leaf data bars removed.

Figure 2.5 Mean 14C-Imidacloprid equivalent concentration 3: SE. in 2006 ........... 82
and 2007 stem sections from 0° and 90° (in relation to the first
whorl of branches) spring-trunk-injected F raxinus americana
and F. pennsylvanica trees. Stems were harvested in October
2007 from the first six branch whorls (where available) and
labeled 0°, 180°, L90° or R90° in relation to the injection point.

Figure 2.6 Mean l4C-imidacloprid equivalent concentration 3: SE. in ................. 84
stained and unstained trunk tissue taken at varying heights
above the center of the injection scar from 0° or 90° (in relation
to the first whorl of branches) spring-trunk-injected Fraxinus
americana and F. pennsylvanica trees harvested in October, 2007.
Inset graphs have the stained cross section data bars removed.

Figure 2.7 Mean l4C«-imidacloprid equivalent concentration :1: SE. in whorl ......... 86
and terminal leader leaves from 0° or 90° (in relation to the first
whorl of branches) fall-trunk-injected F raxinus americana and F.
pennsylvam'ca trees. Braches were labeled 0°, 180°, L90° or R90°
(in relation to the injection point. An * denotes leaves collected
after abscission.

Figure 2.8 Mean l4C-imidacloprid equivalent concentration t SE. in leaves ......... 88
and stems from first whorl branches (0°, 180°, L90°, or R90° in
relation to the injection point) and coarse and fine roots. Samples
were taken during destructive harvest from 0° or 90° (in relation
to the first whorl of branches) fall-trunk-injected Fraxinus

xi

Figure 2.9

Figure 2.10

Figure 2.11

Figure 3.1a

Figure 3.1b

Figure 3.2

americana or F. pennsylvanica.

Mean l"C-imidacloprid equivalent concentration :1: SE. in ................. 90
stained and unstained trunk tissue taken at varying heights

above the center for the injection scar from 0° and 90°

(in relation to the first whorl of branches) fall-trunk-injected

F raxinus americana and F. pennsylvanica trees harvest in

October, 2007. Inset graph has the stained data bar removed.

Mean l4C-imidacloprid equivalent concentration 3: SE. in leaves .........91
from the first three branch whorls of 0° and 90° (in relation to

the first whorl of branches) of spring- and fall-trunk-injected

F raxinus americana and F. pennsylvanica trees. Values were

averaged across whorls. Branches labeled 0°, 180°, L90° or R90°

in relation to the injection point. Samples were taken 21 days

after treatment (19 July 2006 and 26 September 2006).

Mean 14C-imidacloprid equivalent concentration i SE. in ................. 92
leaves from the first three branch whorls of 0° and 90° (in

relation to the first whorl of branches) of Spring- and fall-trunk-

injected F raxinus americana and F. pennsylvanica trees. Values

were averaged across whorls. Branches labeled 0°, 180°, L90° or

R90° in relation to the injection point. Samples were harvested on

11 June 2007.

CHAPTER 3

Treatment schematic, trees were injected at either 0° or 90° to ........... 116
the first whorl of branches. Mean distance from injection point

tO the first whorl = 1.43 m. Mean distance between whorls =

0.18 m.

Sampling schematic of trees injected at either 0° or 90° to the ........... 117
first whorl of branches. Branches of the first three whorls were

labeled 0°, 180°, L90° or R90° in relation to the injection point.

Mean distance from injection point to the first whorl = 1.43 m.

Mean distance between whorls = 0.18 m.

Mean l4C-Imidacloprid equivalent concentration i SE. in whorl ....... 1 18
and terminal leader leaves from 0° or 90° (in relation to the first
whorl of branches) trunk-injected Acer x freemanii and Acer

platanoides trees. Braches were labeled 0°, 180°, L90° or R90°
(in relation to the injection point).

xii

Figure 3.3

Figure 3.4a

Figure 3.4b

Figure 3.5

Mean l4C-Imidacloprid equivalent concentration t SE. in whorl ....... 120
one leaves and stems (0°, 180°, L90° or R90°-in relation to the

injection point), trunk cores (0° or 180° in relation to the injection

point) and roots Of 0° and 90° (in relation to the first whorl of

branches) trunk-inj ected Acer x fieemanii and Acer platanoides

trees, samples collected during destructive harvest. Inset graph

has leaf data bars removed.

Mean l4C-Imidacloprid equivalent concentration :I: SE. in 2006 - ........ 121
and 2007 stem sections from 0° (in relation to the first whorl of
branches) trunk-injected Acer x fieemanii and Acer platanoides

trees. Stems were harvested in October 2007 from the first three

branch whorls and labeled 0°, 180°, L90° or R90° in relation to the
injection point.

Mean l4C-Imidacloprid equivalent concentration t SE. in 2006 .........123
and 2007 stem sections from 90° (in relation to the first whorl of
branches) trunk-inj ected Acer x freemanii and Acer platanoides

trees. Stems were harvested in October 2007 from the first

three branch whorls and labeled 0°, 180°, L90° or R90° in relation

to the injection point.

Mean l4C-imidacloprid equivalent concentration i SE. in leaves ....... 125
from the first three branch whorls of 0° and 90° (in relation to the

first whorl of branches) of trunk-injected Acer x. freemanii and

Acer platanoides trees. Branches were labeled 0°, 180°, L90° or

R90° in relation to the injection point. Samples were taken 21 and

280 days after treatment.

xiii

CHAPTER 1

Literature Review

Introduction

Emerald ash borer (EAB, A grilus planipennis Fairmore) (Coleoptera:
Buprestidae) and Asian longhomed beetle (ALB, Anoplophora glabripennis Motschulsky)
(Coleoptera: Cerambycidae) are exotic insects from Asia. They arrived in the United
States on separate occasions in solid wood packaging material (Cappaert et al., 2005b,
Smith et al., 2001). Emerald ash borer was first identified near Detroit, Michigan in 2002
(Siegert et al., 2007). Asian longhomed beetle was first discovered in New York City,
New York in 1996 (Haack, 1997) and Chicago, Illinois in 1998 (Poland et al., 1998).
They have similar life cycles and tree damage results from larval feeding in the phloem
(Hajek and Bauer, 2007). As the larvae feed, they create galleries beneath the bark of the
tree that interrupt water and nutrient flow. In high density populations, these galleries
effectively girdle the infested tree, causing death. Since its arrival, EAB has killed over
25 million ash trees in Southeast Michigan, seven additional states, and Ontario, Canada
(www.cmeraldashborer.info, 2008). Asian longhomed beetle eradication efforts have
resulted in the removal of almost 30,000 trees of various species in Chicago, New York
City (Haack, 2003), and New Jersey (Richmond, 2008). Together, these two exotic
beetles have caused millions of dollars of damage for homeowners and local, state, and

federal governments. Federal quarantines are in effect which restrict the movement of

nursery stock, logs, chips and firewood from infested areas. If these insects are not
immediately and successfully controlled, there will be irreparable damage to forest and
urban forest ecosystems, not to mention billions of dollars in economic losses.
Unfortunately, there are currently no insecticides that are 100% effective against
either ALB or EAB. Imidacloprid applied as a trunk injection provides high levels of
control but efficacy against both of these insects is variable (Harrell, 2006, McCullough
et a1, 2004, Wang et al., 2004). Trunk-injected insecticide variability could be a result of

various physical and environmental factors.

This study used 14c-Ieheied imidacloprid applied as a trunk injection to track the

movement Of insecticide within Fraxinus spp. and Acer spp. trees over time. The
objectives were to determine the (1.) spatial movement Of imidacloprid in trees; (2.)
concentration of imidacloprid in tree tissues over two growing seasons; (3.) variability Of
imidacloprid concentration in trees trunk-injected in either spring or fall.
1. Emerald Ash Borer:

Emerald ash borer is a metallic phloem-boring beetle from Asia. It arrived in the
United States near Detroit, Michigan in the early 1990’s (Siegert et al., 2007) and was
first identified in 2002 after reports from homeowners of dead and dying ash trees.
Emerald ash borer is native to China, Japan, Taiwan, Korea, Mongolia and Eastern
Russia (Cappaert et al., 2005b). It appears to be host specific and thus far has attacked
only trees in the genus F raxinus in North America (Anulewicz et al., 2008). Emerald ash
borer is not considered a pest in its native range and very little was known about the
beetle before it gained notoriety in the United States (Liu et al., 2003). Current

distribution of EAB in North America covers over 10,000,000 hectares (40,000 sq miles)

and includes nine states and parts of Ontario, Canada (Figure 1.1)
(www.cmeraldashborer.info, 2008).

In Michigan, EAB usually has a one year life cycle, although in some outlier
populations, larvae have been known to develop over two or even three years
(McCullough et al., 2005b). Adult EAB emerge in late May through June and feed on
the leaves of ash trees, rarely causing noticeable damage. Adults mate one week after
emergence and females lay between 60 and 90 eggs approximately one week later
(McCullough and Katovich, 2004). The female EAB oviposits individual eggs on the
rough bark of one or more ash trees. During the first years Of infestation, most eggs are
laid in the upper canopy of trees but as population densities build and canopy dieback
occurs, the trunk of the tree is colonized. Adults generally live six to eight weeks. Eggs
hatch beginning in July and larvae develop through four instars. They feed on the
nutrient-rich tree phloem from late summer through fall, and create serpentine galleries
throughout the cambium and phloem of the tree. During feeding they also score the
outermost layer Of sapwood. Larvae over-winter as non-feeding pre-pupae in the xylem
or in bark. Pupation occurs in mid-April through May, and adult emergence takes place
approximately three weeks later. Emerald ash borers produce one generation of offspring
per year except in the cases where it takes two years for larvae to develop (Cappaert et al.,
2005b)

Emerald ash borer dispersal takes place at two levels: natural and human assisted.
Natural dispersal capabilities are strongest in mated females which were capable of flying
up to 1.7 km/yr, in flight mill studies, two times farther than unmated females (Taylor et

al., 2007). In low density outlier populations, most EAB rarely travel more than 1 km/yr.

Human assisted dispersal occurs when people move infested firewood, nursery stock, saw
logs, or other wood products into previously un-infested areas. Humans have been the
main cause Of EAB spread across the US (Cappaert et al., 2005b)

Since its arrival, EAB has decimated forest and urban landscapes. In low
population densities, trees can withstand minor infestations and exhibit few external
symptoms (McCullough and Siegert, 2007). However, in high density populations,
heavily infested trees may exhibit one or more Of the following symptoms: epicormic
shoots, wood pecker holes along the trunk, “D” shaped exit holes, and longitudinal bark
cracking and blistering. Canopy die-back can be as high as 30% after only two years and
death can occur, even in mature healthy trees, within three to four years of initial
infestation (McCullough and Katovich, 2004). Emerald ash borers preferentially attack
stressed trees (Poland et al., 2004) but also readily colonize healthy trees, 2.5 to 200 cm
in diameter (McCullough and Seigert, 2007).

2. Asian Longhorned Beetle:

The Asian longhomed beetle is a wood-boring invasive pest from Asia. It was
first identified in New York City (Haack et al., 1997) and populations were subsequently
discovered in Chicago (Poland et al., 1998) Jersey City, New Jersey, Toronto, Ontario,
Canada (Wang et al., 2005) and in 26 warehouses in 14 states across the US (Nowak et
al., 2001 ). The ALB is native to SE Asia and China where it is rated as one of the top ten
economic insect pests because of the damage it causes to Populus plantations (Li and Wu,
1993)

In contrast to EAB, ALB has an extremely wide host range including Acer,

Populus, Prunus, Malus, Salix, Ulmus and 21 other tree species (MacLeOd et al., 2002).

The economic implications Of widespread dispersal are potentially devastating. Apple,
cherry, maple syrup, and nursery production industries might all become victims of this
invasive insect, not to mention millions of residential and urban forest trees (Ludwig et
al,2002)

In urban landscapes, ALB prefers host trees in the genus Acer and will attack both
healthy and stressed trees (Nowak et al., 2001). Asian Longhomed Beetle can produce
one or two generations per year, depending on the time of adult emergence (Yang, 2005).
Adults usually emerge June through August but timing is variable. They feed on bark,
young stems, and occasionally leaves (Dubois et al., 2004a). In most cases, beetles stay
on the tree from which they emerge but they are capable of flying up to 1,000 m to find a
suitable host tree (Wang et al., 2005). Human assisted dispersal is also a problem (Nowak
et al., 2002). Adults mate a few days after emergence and eggs are laid one week later.
The female chews a small slit into the tree bark and oviposits a single egg underneath.
Females can lay eggs up to 32 eggs during a period lasting about five weeks (MacLeOd et
al., 2002). Oviposition location on trees varies, but normally females will lay eggs in tree
crowns during initial infestation and as trees becomes more heavily populated, eggs will
be deposited further down the trunk (Dubois et al., 2004a). Eggs hatch in 15 days (Keena,
2000). The first, second, and third instar larvae feed on nutrient rich phloem just beneath
the surface of the bark and create galleries that disrupt nutrient and water translocation
within the tree. In areas with high density ALB populations, this can cause tree mortality
within two to four years. AS larvae grow and develop, they tunnel deeper into the xylem
heartwood to feed and then over winter. In doing so, they create weak spots in the

branches. Weakened branches may succumb to wind or snow load and cause injury to

pedestrians or damage vehicles, houses, and other property (MacLeod et al., 2002).
Larvae pupate just under the bark in early spring (Poland et al., 2006a)
3. Fraxinus spp.

Fraxinus spp. trees are widely distributed across Michigan and the United States
in both urban and forest settings (Figure 1.2) The USDA Forest Service estimates that
there are over 804 million ash trees in Michigan’s forests (Poland, 2007). There are at
least 16 species of ash in North America (Poland and McCullough, 2006), and all are
likely susceptible to EAB infestation (Herms et. al, 2003). For this study, we used the
two most widely distributed and commonly occurring ash in the US; F raxinus americana
L. and F raxinus pennsylvanica Marsh, white and green ash.

White ash is the most commercially important native ash tree in North America
(Cappaert et al., 2005b). Its wood is used for tool handles, baseball bats, furniture, and
flooring (MacFarlane and Meyer, 2005). White ash is usually found on moderately moist
uplands in the northeast and north central parts of the United States (F arrer, 1995) (Figure
1.3). Though not a dominant tree in the forest it is an important species in several forest
ecosystems (MacFarlane and Meyer, 2005).

Green ash is the most widely distributed ash species (Figure 1.4) and also of
economic importance. Its wood is generally of lower quality than white ash but it is also
used for tool handles, furniture and flooring. Its seeds are a source Of food for many
types Of wildlife. It is also a popular ornamental tree. Green ash grows best on fertile,
moist, well drained soil, but it is also successful in a variety of less than ideal

environments (Farrer, 1995) and is probably the most adaptable ash species. Green ash is

widely used in the landscape because Of its adaptability, quick growth rate, and beautiful
form.
4. Acer spp;

There are approximately 100 species of maple (Acer spp.) trees and shrubs
worldwide, 13 of which are native to North America (Figure 1.5). Numerous maple
cultivars exhibit a vast array of sought after aesthetic and physiological traits. Maple
trees are common in the landscape and are Often planted for their beautiful fall color
(F arrar, 1995). Maple is also an important forest products tree. Its wood is used for a
variety of commodities including flooring, furniture, musical instrtunent panels, and
baseball bats. A unique product of maple trees is syrup and confections that are produced
from the sap that is collected in early spring (van Gelderen et al., 1994).

Norway maple (Acer platanoides L.), is native to Eastern Europe but is a popular
tree in North American landscapes (Farrer, 1995). It is one of the top five shade trees
produced annually. Its hardy nature, ease of transplanting, and diverse cultivars have
made it an over-used favorite of homeowners and city planners (Dirr, 1998). Norway
maple grows best in moist, well-drained soil but it too is highly adaptable and can
overcome a wide variety of stresses including: soil compaction, heat, and air pollution.
Norway maple is a non—native tree with prolific habits and has been described as invasive
in some areas. (N owak and Rowntree, 1990).

Freeman maple (Acer x fi‘eemanii E. Murray) is found throughout the United
States; it is a hybrid cross between A. rubrum L. and A. saccharinum L., red maple and
silver maple. It was hybridized by Oliver Freeman but was later discovered to be

occurring naturally in forests where both parents are present (van Gelderen et al., 1994).

Its cultivars are said to combine the beauty of red maple with the hardiness of silver
maple. It is more drought tolerant and can grow up to four times faster than the species
from which it is derived (Dirr, 1998). These traits, in addition to its vibrant fall color,
make it a popular ornamental tree.

5. Ash and Maple in Urba_n Forests:

Approximately 3.8 billion trees (Nowak et al., 2002) make up the urban forests
that cover approximately 3% of the United States (Dreistadt et al., 1990). These trees are
an Often overlooked resource valued at $2.4 trillion (Nowak et al., 2002). In addition to
monetary value, urban forests also provide elements that positively affect human health,
biodiversity, and wildlife habitat (Schwaab et al., 1995). They regulate urban heat effects,
sequester carbon, and remove pollutants from the atmosphere (Galvin, 1999). Urban
forests are highly vulnerable to invasive forest insect pests and pathogens because
diversity generally decreases as urbanization increases and trees in urban environments
are Often under high degrees of environmental stress (Dreistadt et al, 1990).

The over use of a single tree species and the consequences of doing so are not
new concerns to urban foresters. In 1930, Dutch elm disease (Ceratocystis ulmi
Buismann) was introduced to the US. At the time, over 60% of urban trees were
American elms (Ulmus americana L.) (Kamosky, 1979). The disease swept through the
United States and led to the removal of over 100 million trees (Carley, 2008). This
disease changed the face Of landscapes throughout the country (Karnosky, 1979), but for
a variety of reasons, only a few tree species were utilized to replace elms. Of the 3.8

billion trees currently found in urban forests, 75% represent only nine genera. Trees in

the Acer and Fraxinus genera comprise five Of the ten most common urban trees
(Dreistadt et al., 1990).

F raxinus trees were an easy choice for elm replacement, because of their ability to
tolerate a wide variety Of stresses in urban environments. For nursery growers, ash was
an easy tree to produce. It was also easy to sell to homeowners because it requires little
maintenance, has relatively few pests, and a very aesthetic growth habit. New cultivars
were introduced that were easy to maintain and aesthetically attractive. Examples
include: ‘Marshall’s Seedless’ green ash, a cloned seedless cultivar that reduced fall
cleanup, ‘Autumn Applause’, and ‘Autumn Purple” white ash cultivars with striking
purple fall foliage. With the introduction of new cultivars, ash quickly gained popularity
and became one of the most dependable and widely over-planted standards in the
landscaping industry. Between 2000 and 2003, ash trees comprised 70% of the canopy
trees bought from Ohio nurseries (Sydnor et al., 2007). Conservative estimates predict
that 12-14% of urban and suburban landscape trees in the United States are in the genus
Fraxinus. In Michigan cities and Chicago, ash trees comprise an average of 15—20% of
the urban street trees (BenDor, 2006). Today, as a result of increased ash in urban
landscapes, an epidemic equal to or greater than Dutch elm disease could very well take
place throughout the United States and Canada as a result of the EAB infestation.

The heavy reliance on maple trees in urban landscapes is also a concern. In many
communities maple trees make up 50% or more of the tree population. Commonly used
species include: A. platanoides L., A. saccharum Marsh, A. saccharinum L., and A.
rubrum L. (Bassuk, 1990). Four Of the top ten trees used in landscapes are in the genus

Acer (Dreistadt et al., 1990), a genus highly preferred by ALB (Nowak et al., 2001).

Unfortunately, maple is not the only susceptible tree; ALB has a host range of
over 24 species. In some cities, these species may make up as much as 62% of the urban
tree landscape (Antipin and Dilley, 2004). If all ALB susceptible trees were infested as
much as 68% canopy loss could occur in some cities (Jersey City, New Jersey) (Nowak et
aL,2002)

6. Insect Impacts:

Emerald ash borer likely attacks all North American ash species (F raxz‘nus spp.)
which have not evolved resistance against it. If left unchecked, EAB could be
responsible for virtually eliminating all native ash in both urban and forest environments.

Ash is an important natural resource in Michigan and North America. Of the 16
ash species native to North America, six have economic significance (Poland and
McCullough, 2006) and are used for products as diverse as Native American baskets and
major league baseball bats. The USDA. Forest Service estimates that 8 billion ash trees
in the US. are threatened by EAB, representing 7.5% of US. hardwood saw timber. If
lost, there would be at least $25.1 billion of economic loss with a compensatory value of
$282.3 billion dollars. In addition to forest products, loss of landscape trees is of equal
concern. The estimated value of ash in the landscape is $565 million with potential loss
estimated at $20-60 billion, a figure that includes tree removal but not replacement
(Poland and McCullough, 2006).

In Chicago and New York City cost of removing ALB infested trees was over $25
million (Nowak etal., 2002). Economically, ALB could represent $669 billion in
compensatory damage throughout the 48 contiguous states. The national tree impact in

urban areas could reach 34.9% canopy cover loss and 30.3% tree loss if all potential hosts

10

were attacked (Nowak et al., 2001). In addition, urban forests sequester 700 million
metric tons of carbon a figure equal to $460 million per year (Nowak et al., 2002). Asian
longhomed beetle could also wreak havoc on forest, fruit tree, maple syrup, and seed
production industries, in addition to forest ecosystems (Ludwig et al., 2002).

7. Irgect Control:

In its native range, EAB is considered only a minor pest. When EAB was first
discovered very little literature existed to guide researchers in areas of control or even life
history (Poland, 2007). In contrast, ALB was a well researched pest in China, so aspects
Of its life history had already been documented before its US. invasion (MacLeod et al.,
2002)

WOOd- and phloem-boring insects are often difficult to detect in low level
populations because their larvae are cryptic and inaccessible (Frank and Mizell, III., 2006)
and the insects can exist in low density populations for several years before outbreak
levels are reached (Shigesada and Kawasaki, 1997). Frequently, outbreaks are not
discovered until it is too late to eradicate the pest because humans have inadvertently
transported infested materials away from the site of initial invasion. In addition, exotic
wood- and phloem-boring pests are particularly dangerous because their new host trees
have no co-evolved resistance against them. For example, healthy, native birch trees
(Betula spp.) have evolved mechanisms to compartmentalize native bronze birch borer
(BBB, Agrilus anxius, Gory) larvae, while European birch used in the landscape trade do
not have the mechanism tO compartmentalize to defend against BBB and therefore must
receive insecticide treatments to survive (Muilenburg et al., 2007). The same situation is

true for ash trees. In the EAB’s native range, ash trees have evolved resistance against

11

the beetle, but N. American trees have little or no co-evolved defense mechanisms
(Herms et al., 2003). This, in addition to the lack of natural predators, can lead to
devastating outbreaks (Hajek and Bauer, 2007).

Currently, three management practices are being utilized in the United States to
control EAB and ALB populations, they are: containment/eradication, bio-control, and
chemical control.

7.a. Containment/Eradication:

The current strategy of USDA-APHIS for ALB is complete eradication.
Eradication efforts can be effective when populations are discovered and acted upon
immediately and/or before human assisted dispersal have taken place at a large scale.
Thus far, ALB populations have been discovered relatively quickly and have been kept
somewhat isolated. Chicago has not removed an ALB infested tree since 2003 (Antipin
and Dilley, 2004). New York City and Jersey City however, are still infested (Richmond,
2008).

In contrast, initial attempts to isolate and eradicate EAB infestations were often
unsuccessful. This practice would remove and destroy non-symptomatic trees that may
be current or future hosts to EAB larvae (McCullough and Seigert 2007). Currently,
eradication practices are used only in a few unique situations. Eradication Of established
EAB infestations is no longer a viable option.

7. b. Bio-Control:

In Michigan, bio-control efforts are being explored as a method of controlling

EAB populations particularly in forests. In 2007, Spathius agrili Yang (Hymenoptera:

Braconidae) was released by USDA-APHIS and 00bius agrili Zhang and Huang

12

(Hymenoptera: Encyrtidae) and T etrastichus planipennisi Yang (Hymenoptera:
Eulophidae) were released by the USDA Forest Service to aid in the control of EAB
(Federal Register, 2007, Bauer et al., 2008). These insects are parasitoid wasps that were
discovered in China (Yang et al., 2005, Strazanac and Zhong-qi, 2005, Gould et al.,
2005). In their native range, these three wasps may parasitize 50-90% of the EAB
present (Gould et al., 2005). After their discovery, the wasps were brought to the US,
reared in laboratories and tested extensively to determine host preference, ability to
disperse, and survivability in North American EAB infestations (Gould et al., 2005). In
addition, various political hurdles were cleared before the insect was released in July
2007 (Bauer et al., 2008).

Microbial control agents like entomopathogenic fungi and nematodes may also a
means of bio-control for ALB and EAB in forested areas where chemical application and
containment/eradication efforts are not physically or economically feasible (Hajek and
Bauer, 2007). Strains of Beauveria brongniartii, Sacc. applied via fungal bands have
shown to be effective against ALB for up to 31 days. The bands specifically target
cerambycid beetles because of their post-emergent wandering habits. The beetles travel
across the bands, become infected with the fungus, and die (Dubois et al., 2004a, 2004b).

They might also spread the fungi to other beetles during the mating process (Hajek and
Bauer, 2007). For control of EAB, B. bassiana strain GHA labeled as BotaniGard® was
highly virulent against adults when compared to other bacterial agents. Fungal treatment
is applied as a pre -emergent broadcast spray and is ingested by EAB adults as they chew

their way out of the tree (Liu and Bauer, 2006). Short persistence Of the fungicide limits

its effectiveness.

13

7. c. Chemical Control:

The most common treatment for landscape trees with wood- or phloem-borer
infestations is chemical control (Frank and Mizell, III., 2006). The threat of ALB
infestations continues to loom in the US and chemical treatments have been field-tested
in its native range so that researchers will be prepared in case beetle populations re-
emerge. It is currently recommended that all susceptible trees within 0.2 km (1/8 mile) of
known infested trees be either treated or removed. In general, healthy tree removal is
very unpopular with residents. As an alternative, trees are chemically treated as a
precaution (Antipin and Dilley, 2004).

Chemical control is used to treat infested high-value ash trees, primarily in urban
areas. Since the discovery of EAB in 2002, researchers have tested several methods of
chemical control including: broadcast sprays, soil drenches, trunk applications, and trunk
injections (Cappaert et al., 2005a, Cregg et al., 2006, Harrell, 2006, McCullough et al.,
2003, 2004, 2005a, 2005b, Poland and McCullough, 2006, Smitley et al, 2004, 2005).

One of the most common and effective active ingredients in these chemical
control products is imidacloprid, a highly effective, neO-nicotinoid insecticide. It can be
applied as a foliar spray, soil injection or drench, or as a trunk injection (N auen et a1,
2001, Tatter et al., 1998). It is used to control a wide variety of landscape insect pests
including leaf feeding beetles, lace bugs, aphids, scales, psyllids, adelgids leaf-miners and
wood-borers (Szczepaniec and Raupp, 2007). Imidacloprid may affect both larval and
adult stages of ALB and EAB (Poland et al., 2006a, McCullough et al., 2005b).

Imidacloprid is most effective when it is ingested by the insect (Gill et al., 1999).

NeO-nicotinoid insecticides act on insects by blocking their nicotinic acetylcholine

14

receptors. When ingested, imidacloprid is bound to the receptor site, but is only slowly
degraded, so death usually results (Gill et al., 1999). It has a chemical structure and
mode of action similar to nicotine, but mammalian toxicity is approximately 700 times
less (Sur and Stork, 2003). Imidacloprid may also act as an anti-feedant. In less than
lethal doses, it causes insects to stop feeding leading to death by starvation (Lawson and
Dahlsten, 2003, Castle et al., 2004, Wang et al., 2005). However it is possible that if
sustained levels of insecticide are not available, larvae may eventually resume feeding
(Poland et al., 2006a).

When applied as a trunk injection, imidacloprid moves rapidly up the tree and into
the canopy leaves, in some cases in as little as two days (Cregg etal., 2006). When
applied as a soil drench, it may take three to four weeks to reach the leaves (Tatter et al.,
1998).

8. Trunk Injection Application:

Imidacloprid applied as a trunk-injection has several advantages over traditional
applications including: efficiency, avoiding excess environmental contamination, few
non-target impacts, and acceptance by the public (Sanchez-Zamora and Escobar, 2000,
Sur and Stork, 2003). During application, chemical treatments using crown and/or trunk
sprays in high traffic areas can pose both perceived and real hazards. Drift is of
particular concern (Solar and Cranshaw, 1996). When applied as a trunk injection,
imidacloprid enters the tree directly, which eliminates potential drift and over application.
In addition, trunk injection applications can reduce insecticide rate, number of
applications necessary (Poland et al., 2006a), and provide a high level of efficacy for a

longer period of time than externally applied pesticide (Sanchez-Zamora and Escobar,

15

2000). Insecticide is wholly contained within the tree and not merely on the surface of
leaves or trunk where rain or irrigation water might wash it Off or dilute it. Systemically
injected insecticide treatments are also safer for applicators than other conventional
application methods (Poland et al., 2006a) and have fewer negative effects on populations
of naturally occurring insect predators (Cowles et al., 2006).

One consequence of the trunk injection process is that the tree is physically
wounded in the process of insecticide application. Trunk injections scar the conductive
tissues (Lawon and Dahlsten, 2003) and in many cases wounding takes place on an
annual basis (Cappaert et al., 2005a). The drilling process can also create entry ports for
bacterial and fungal pathogens (Lawon and Dahlsten, 2003). For example, horse-
chestnut (Aesculus hippocastaneum L.) trees being treated for horse-chestnut leaf-miner
(Cameraria ohridella, Deschka and Dimic, Lepidoptera: Gracillariidae), may receive as
many as 25 injection holes. Unfortunately, horse chestnut is limited in its ability to
compartmentalize wounds and weakened trees are even more limited. The wounds often
become infected with bacteria and trees produce an abundance of sap to flush the bacteria
from its vascular system. Wound response may discolor 30-50% Of the white wood in
the first two years, resulting in tree depreciation (Oszako et al., 2007). Although serious
wounding takes place in some species, trees such as avocado seem to overcome drill
lesions by growing new tissue over the top. However, even in trees thatare able to heal
relatively quickly after injection, recommendations still caution against performing
“insurance” trunk injections, as these excess events could lead to permanent damage

caused by the injection process (Robbertse and Duvenhage, 1999). In addition to the

16

wounds caused from drilling, bark cracking, splitting and separation can also result from
the trunk injection process (Lewis et al., 2004).
9. Efficacy Variabilig:

Trunk injection success can depend on many environmental and physical
variables (Sanchez-Zamora and Escobar, 2000). With the possible exception of a new
product containing emamectin benzoate (McCullough etal., 2008) there are no
insecticides that are 100% effective against ALB or EAB (Cappaert et al., 2005b, Poland,
2007). However, 100% insect control may not be necessary because of the tree’s
resilience to low level infestation (McCullough et al., 2004, Poland et al., 2006a). One
potential cause for variability in efficacy is that the uptake of trunk-injected insecticides
can be affected by a wide range of physical parameters including tree health, previous
injury, (Smith and Lewis, 2005) and size (McCullough eta1., 2005a,b). It can also be
affected by environmental factors such as temperature and water availability or by
cultural practices like timing of application and delivery method (Smith and Lewis, 2005).
Variability also occurs among and between tree species (Tatter et al., 1998). Variability
between trees of the same species suggests that root and crown structure, crowding, and
nutrient or water stress could all affect homogeneity of trunk-inj ected insecticides
(Young, 2002).

Efficacy variability could also be affected by the length of time between
treatments. In China, efficacy of imidacloprid against ALB in Populus spp. trees was
highest in the weeks immediately following trunk injection but was greatly reduced over
time (Poland et al., 2006b). The same was found in pin oak (Quercus palustris,

Muenchh.) and eastern hemlock (Tsuga canadensis, L.) where imidacloprid concentration

17

in sap began to decrease after approximately eight weeks (Tatter etal., 1998).
Imidacloprid provided 70% control against avocado thrips (Scirtothrips perseae
Nakahara) for the first 14 weeks in all leaves present at the time Of application but
efficacy steadily decreased thereafter and levels were reduced in newly formed leaves
(Byme etal., 2007). Additional studies on red gum eucalyptus (Eucalyptus
camaldulensis, Schlecht.) (Young, 2002) and green ash trees also showed reduced
imidacloprid residue levels one year after treatment (Lewis et al., 2004).

Most researchers agree that one year of control can be expected from trunk-
injected imidacloprid, but there are conflicting reports of efficacy beyond one year, both
among and between species. Laboratory bioassays performed to test the efficacy Of
imidacloprid against elm leaf beetle (Xanthogaleruca luteola, Miiller) found the
insecticide to be ineffective 382 days after application in one study (Lawon and Dalhsten,
2003) but another, effective control of the same insect persisted for more than one year
(Solar and Cranshaw, 1996). Differences in this instance could be a result of different
insecticide rates. Similarly, a 5% imidacloprid trunk-injection in green ash was
successful against EAB for two years in one study (Doccola et al., 2006), but in another,
a 10% imidacloprid trunk injection was successful for only one year (Cregg et al., 2006).

Poland et al. (2006b) suggest that as imidacloprid concentration increases, translocation
capability decreases. Examination of commercial formulations of imidacloprid (Merit®,
Pointer®, and Imicide®) insecticide labels contain only the percentages of active and non-

active ingredients, therefore no comparisons of adjuvants could be made.
One possible explanation for lack of extended efficacy in most cases is that

imidacloprid is not translocated from leaves back into the tree before leaf abscission. In

18

fact, a large concentration of insecticide is lost from the tree after litterfall (Cregg et al.,
2006). And so the question remains, is there enough insecticide left in the tree after
litterfall to provide a second season of effective control? Ash are resilient trees, a trait
that helps them to recover from the trunk injection process, but if two years of control can
be gained from a single injection, damage to the tree that occurs during the injection
process and overall cost Of treatment could be reduced. Better understanding of the fate
Of insecticides in plants could also reduce the number of “insurance” treatments that are
used in cases where efficacy is questionable (Castle et al., 2004).

Another factor influencing trunk-injected insecticide efficacy is the timing of the
injection process. Environmental conditions such as temperature, humidity level, and
cloud cover can all play an important part in ensuring that the maximum amount of
insecticide is moving from the injection site to the leaves (Smith and Lewis, 2005). The
timing of injection in relation to tree phenology is critical in order to attain and maintain
effective insecticide levels (Giblin et al., 2007). Trunk injections in the tree maintenance
trade usually take place in either the spring or fall of the year. Spring injections are
advantageous because the insecticide is being injected into the tree during a period of
maximum translocation. Fall injections are popular among arborists because there is
Often a lag in business Opportunities during this time. Dye studies performed by Tatter
and Tatter (1999) suggest that injected materials move downward as well as upward
within trees. This could be a result Of pressure from the injection process or wounding
events which interrupt xylem flow. With the lack of a water potential gradient which

normally moves liquid upward, downward movement into the roots can take place.

19

Tatter and Tatter (1999) hypothesized that this process could provide the mechanism that
makes fall injected materials efficacious the following spring.
10. Sectorem

Another possible explanation for efficacy variability might be that trunk-injected
insecticide is not distributed evenly within the tree (Cregg et al., 2006) which suggests
that the xylem anatomy of the tree is a highly influential factor on insecticide movement.
In addition to resource availability gradients, the xylem architecture of a tree can have a
direct effect on disease and insect control because many systemic insecticides move
along the same xylem pathways as water. (Kozlowski and Winget, 1963). If this is the
case, insecticide efficacy could be influenced by the location on the tree trunk where the
insecticide is injected. Efficacy variability could result because the insecticide is being
translocated within the tree in such a way that it never reaches insect feeding sites
(Poland et al., 2006b).

Sectoriality is a trait most Often found in trees and dicotyledonous herbs. Since it
was first identified, the theory of sectoriality has been used to describe differences in
resource allocation, plant growth, and plant-herbivore interactions (V uorisalo and
Hutchings, 1996). Resource movement in plants is accomplished and limited by the
vascular system, which is either integrated or sectored to varying degrees. Integrated
vascular systems have highly interconnected networks of xylem cells. Sectored vascular
systems have highly Specific xylem cell pathways. Sectored cell paths restrict the
transport of nutrients and water to specific parts of the plant. Orians et al., (2004)
demonstrated that sap flow in trees may be sectored or integrated depending upon tree

species. Sectoriality might be specific to different parts Of a plant, branches of a tree, or

20

even leaves on a branch (Orians et al, 2005), the term “integrated physiological units”
(IPUs) is used to describe these distinct vascular sections (Orians et al., 2002).

The ability to isolate stresses and increase exploitation efficiency (example, light)
are two advantages of sectoriality (Orians and Jones, 2001). In fact, sectored species may
perform best in variable environments (Zanne et al., 2006), which might explain the
ability of green ash to tolerate ever-changing urban environments. In sectored trees,
stress to a single branch seldom affects the other branches. Different sectors with very
different chemical and physical characteristics may co-exist on the same plant for a very
long time. Sectored vascular systems create spatial variability of resources that could
result in non-uniform plant growth and plant chemistry (Orians et al., 2004); conditions
that might effect herbivory (Orians et al., 2002).

Some patterns of sectoriality can be visually observed such as when a branch of a
tree exhibits bright fall foliage while the rest of the tree is still lush and green. The
brightly colored branch is likely being supplied by its own IPU with resources that differ
from the rest of the tree. Another visual indication might be patchy dieback patterns in
the tree crown (Elhnore et al., 2006). The degree of sectoriality probably changes
continuously within a plant over time. New connections form as new leaves, roots and
shoots are produced (Orians and Jones, 2001). But even though new connections are
formed, the patterns of sectoriality are probably consistent between saplings and mature
trees (Ellmore et al., 2006).

Sectoriality differs'within and among species (Orians et al., 2004) because Of
differences in anatomical traits (Zanne et al., 2006). It is generally accepted that ring

porous species (e. g. ash) are more highly sectored than diffuse porous species (e. g. maple)

21

(Zanne et al., 2006, Ellmore et al., 2006, Fararr, 1995). This could be a result of
differences in vessel diameter size throughout early and late wood. Ring porous species
have variable vessel diameters while diffuse porous species have smaller, more constant
vessel diameters (Shigo, 1988). Although these differences can provide some indication
as to whether a tree will be sectored or integrated, they are not absolute. Some diffuse
porous hardwood species, even some conifers that lack vessels, exhibit varying degrees
of sectoriality (Zanne et al., 2006). In some cases, sectoriality could be considered a
comparative anatomical trait (Ellmore et al., 2006). Despite some variation between
species, closely related species Often have similar IPU patterns (Orians et al., 2004).
Sectored IPUs may exhibit spiraling patterns. Helical ascent patterns are common in both
hard- and soft-wood trees (Erdesi, 1957). The pattern may turn either to the left or right
depending on tree species, age, or height. Ascent may begin vertically near the base Of
the tree, spiral with increasing height and then finally become integrated in the tree crown
(Kozlowski and Winget, 1963). Ash trees may exhibit this tendency to spiral (Erdesi,

195 7). Maple trees, on the other hand, have ascent patterns that vary between straight,
highly irregular, or spiraling. The marked differences in ascent patterns between and
within species are correlated to xylem structure (Kozlowski and Winget, 1963), which
could be a product Of phylogeny or current environment (Orians et al., 2004).

Several anatomical traits combine to determine levels of integration and
sectoriality including, vessel arrangement and size, pit structure, density, and resistance.
Zanne et a1. (2006) suggest that this is the most important trait when determining the
degree of sectoriality for tree species. Pit densities might also be an integration indicator

but Ellmore et al. (2006) determined that pit densities alone are not an absolute predictor

22

of sectoriality. Percentage of vessel wall area as inter-vessel pits could also effect levels
Of integration. For example, Betula papyrifera Marsh, 3 highly integrated species, had
26% vessel wall as pits when compared to A. saccharum Marsh, an intermediate species,
which had 9%. Tree species that have a high percentage of pits in the vessel walls could
have decreased pit resistance. In vertical pathways, this could lead to increases in lateral
movement and as a result, greater degrees of integration (Orians et al., 2005, Ellmore et
al., 2006). Kitin et al. (2004) suggest that Fraxinus trees have individual vessels that
deviate from straight longitudinal along a more tangential direction and that these vessels
are part of an interconnected group that touches and interacts with one another. This
interaction could result in a helical ascentpattem as shown in some dye experiments
(Chaney and Kozlowski, 1977). In general, species that have large early-wood areas, low
vessel density, high variation of vessel area, and greater distance between neighboring
vessels have greater degrees Of sectoriality (Zanne, et al., 2006).

In a previous study at Michigan State University, researchers used l4C labeled
imidacloprid to track trunk-injected insecticide movement in ash trees. The study
demonstrated that imidacloprid moves slowly but steadily through the tree over time and
that it accumulates in the leaves. However, the sampling protocol did not permit a
thorough examination of the spatial variability of imidacloprid distribution in the trees
(Cregg et al., 2006). We hypothesized a priori that ash trees have sectored patterns that
might cause uneven distribution Of insecticide in the tree crown leading to variable
efficacy Of trunk-injected imidacloprid. We further hypothesized that maple trees have
more integrated insecticide ascent patterns. Depending on injection location and spacing,

there could be portions of the tree that receive little if any insecticide.

23

Overall, the efficacy of trunk-injected insecticides might be influenced by how
often trunk injections take place, amount of insecticide injected, time of year that trees
are injected, and tree sectoriality. Understanding the spatial and temporal flow Of
insecticide through F raxinus spp. and Acer spp. trees will enable us to improve the
efficacy and persistence of treatments and better understand the potential for extended
EAB and ALB control.

11. Radio-labeled Tracers:

To track insecticide movement within the tree, we incorporated the use Of

4 . . . . . . .
1 Carbon (14C) labeled Imidacloprid. The use Of radioactive Isotopes as tracers 18 not a

recent development. In 1923, Hevesy used a naturally occurring lead isotope to study
mineral transport in plants. Radioactive isotopes are more accurate and take less time

than plant dyes (Jenkins, 1957) and are often used by scientists to follow the chemical

and biological pathways of plants. To use 14C as a tracer, a carbon (C) molecule Of the

compound being traced must be replaced by a 14C isotope. The replacement of C by 14C

does not alter the formula Of the compound. Radio-labeled compounds are Often applied
systemically through injection (Domir, 1978), by applying radio-labeled solutions to
plant parts (Weichel and Nauen, 2003) or by placing the radio-labeled compound in
solution and allowing the plant to translocate it through vascular tissues (N ir and Lavee,

1981).

An advantage Of using 14C is that it is readily detected by Geiger counter,

methanol extraction, or by processing samples through a biological tissue oxidizer and

then a liquid scintillation counter (LSC). However, in some cases methanol extraction

24

can be inconclusive. In a study by Domir, (1978), American elm trees (Ulmus americana
L.) were injected with 14C daminozide to determine translocation patterns. The
researchers used a methanol extraction technique and after repeated attempts, they

Observed that significant amounts of labeled compound were un-extractable from plant
stem tissues. Results from biological tissue oxidizers are more accurate because the
entire tissue sample is burned and resulting 14C02 is trapped in a liquid scintillation
cocktail (Thomson, 2002). After oxidation, the resulting cocktail is processed through a
LSC which converts the radioactivity present in the cocktail to light. The light events are
recorded as counts per minute which can then be extrapolated backward to determine the
amount of labeled compound in the tissue (Packard Tri Carb LSC homepage, 2006). Use
Of the biological tissue oxidizers eliminates the need for extraction techniques, enabling
researchers to trap nearly the entire amount of tracer compound.

Sectoriality patterns in plants can be determined using isotope labels. Peppermint

(Mentha pepper/ta L.) sectoriality was determined by applying 32P to two different

sections of root. Results showed that the radio-labeled compound was confined to leaves

with the same vascular pathway as labeled roots, indicating that peppermint xylem

anatomy is sectored in nature (Rinne and Langston, 1960). Another study using 15N

supplied as a fertilizer to lateral roots ofAcer spp. and Populas spp. saplings showed

greater degrees Of sectoriality in A cer spp. than Populus tremuloides Michx. (Orians et al.,

2004). In a study designed to understand the fate of 14C maleic hydrazide, a plant

growth inhibitor, a radio-labeled isotope was used to track the compound through maple

(A. saccharinum L.) and sycamore (Platanus occidentalis L.) trees. Samples of plant

25

tissues were taken over time to determine the rate of chemical uptake. The presence of

the 14C tracer was tracked and it was concluded that the compound was translocated to all

parts of the plant within 24 hours. In addition, the radio-labeled tracer enabled the

. . . . 14 . . .
researcher tO determine dIfferences 1n the concentration Of C maleic hydrazrde In

various plant parts. It was also observed that there was a difference in the rate of tracer
movement between the two tree species (Domir, 1978).

Use of radio-labeled tracers can also aid in the study of the metabolites that form
after trunk injection (Domir, 1978). To determine which metabolites are formed,
scientists examine plant tissues for “new” compounds that contain radio-labeled
molecules. It is well established that imidacloprid is transformed into at least three
metabolites after injection into various plants (Sur and Stork, 2003). Upon discovery and
identification, metabolites as well as pure compounds, can be tested for efficacy against
insect pests. In ash trees, metabolites can be as toxic to EAB as pure imidacloprid
(Mota—Sanchez, 2007).

Radio-labeled compounds can also be used to determine insect lethal doses in

insect bioassays. Knockdown and mortality rates of male and female EAB were

determined by using leaves from 14C imidacloprid trunk-inj ected F raxinus trees. Radio-

labeled leaves were placed in enclosed containers with EAB adults. The insects fed on
the leaves, weakened, and finally died. The leaves were removed at various stages and
scanned with a digital scanner to determine the amount Of tissue consumed by the insect.
The weight Of tissue consumed was then translated into the amount of insecticide

consumed based on the amount of radioactivity within the leaf. In other words, the

26

amount of insecticide necessary to cause insect death can be discovered by using radio-
labeled compounds (Mota—Sanchez et al, 2005).

Understanding where insecticides are within a plant at a given time is important
when making recommendations for insecticide application. Radio-labeled compounds
can be used to do this because they allow researchers to see where, when, and how

insecticides travel within the plant and what concentrations are necessary within leaves to

cause insect death. Use of 14C labeled imidacloprid in various plants has shown that it

moves primarily to shoots and leaves via the xylem (Bromilow and Chamberlain, 1989,
Sur and Stork, 2003, Tanis et al., 2007). Concentrations in leaves are usually higher than
concentrations in stems, trunk, or roots (Botts et al., 2006, Mota-Sanchez et al., 2005,
Tanis et al., 2007) and highest concentrations are expected in Older leaves (Sur and Stork,
2003). Imidacloprid generally increases in leaves and stems during the first growing
season after trunk injection. At the end Of the growing season, concentrations in leaves at
the time of abscission are as high as or higher than concentrations 60 days after injection,
which suggests that imidacloprid is not translocated back into the plant (Cregg et al.,
2006). In addition, a high amount Of compound Often remains around the injection Site
(Arron et al., 1992, Tanis et al., 2008) which may serve as the source of compound that
could provide a second year of insect control (Tanis et al., 2007).
Summag:

In summary, ALB and EAB are invasive exotic beetles. Imidacloprid, a neo-
nicotinoid insecticide applied as a soil drench or trunk injection, is an effective but
variable means of control for both insects. Efficacy of trunk-injected insecticides could

be affected by one or more Of the following factors including: (1.) length of time that the

27

insecticide has been inside the plant; (2.) time of year that trunk injections take place and
(3.) location of the injection Site. All Of these factors might further be influenced by tree
internal structure, health, and phenology. The xylem architecture of trees can play a
major role in insecticide translocation and efficacy depending on whether or not the tree

is sectored or integrated. We hypothesize that ash trees have sectored xylem structure

and that maple trees have more integrated xylem anatomy. This study will use 14C

labeled imidacloprid to determine which of the above is influential on insecticide
translocation. We will use the results to develop recommendations for arborists that will

enable them to provide the best possible means for EAB and ALB control.

28

 

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noted?» 5.82 05 E Augumofism ”833200 .gm finneewnfim $25.3 cocom :2 3808m— mo Sangria A.“ Snug

 

    
 
 

 

 

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up? .3535 £53353 .030 6:2wa
.cewEomE JESUS £95: E 20:33 gm

 

 

89.95 .525 Am< Eanofim 255.8 80

 

 

 

29

 

Figure 1.2: Distribution of ash trees (Fraxinus spp, Oleaceae) in North America (USDA

NRCS, 2008).

30

 

Distribution of native white ash (Fraxinus americana L..Oleaceae) in North

America (Burns and Honkala, 1990).

Figure 1.3

31

 

‘ I T" . . (“Eli : «9‘-.. . 'u 1 j
k._ ' - w, » _- r r ,1 --'~*'~ -—‘-——-- "‘ ~I - ‘ _____ ' ‘1' 9 A. n
Er -.___~ I l' {I} '- 51*“;‘1 _ 43:: L r _ - _ _ _ L 1.3-5.; 1‘"th ' g - L...-"'
r 'f-- r i ”no - -. I T ' ”Ti " T M F ”.1. 1"“l—-~ "‘4- L..-"'".~ .

Figure 1.4: Distribution of native green ash (Fraxinus pennsylvanica, Marsh. Oleaceae)
in North America (Burns and Honkala, 2008).

32

 

Figure 1.5: Distribution of maple trees (Acer spp, Aceraceae) in North America (USDA

NRCS, 2008).

33

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Distribution of Trunk-Injected l4C Imidacloprid in Fraxinus Trees: A Test of the
Sectored—flow Hypothesis.

ABSTRACT

The emerald ash borer (A grilus planipennis Fairmore, Coleoptera: Buprestidae)
is an invasive exotic insect pest that has destroyed millions of Ash (F raxinus spp.) trees
in Michigan, surrounding states, and Canada. Imidacloprid applied as a trunk injection
is Often used as a means of control, but effectiveness can be variable. The objective of
this study was to determine the extent to which movement of imidacloprid is sectored
within ash trees. Highly sectored flow could result in uneven distribution of compound
within trees and result in variable efficacy Of pesticide. One set Of 32 trees (16 F raxinus
americana, 16 F. pennsylvanica) was injected during the spring; a second set of 8 trees (4
F raxinus americana, 4 F. pennsylvanica) was injected during the fall. This allowed for
comparison between two injection times. We injected the trees with 14C labeled
imidacloprid either directly below a branch in the first whorl or at a right angle to
branches of the first whorl. Each branch of the first three whorls was labeled 0° /180°, or
L90° / R90° depending on the location of the branch in relation to the injection point.
Leaves and fine roots were sampled through two growing seasons. Trunk cores were
sampled during 2006. Tree stems were sampled 60 days after treatment (DAT) (spring-
injected trees only) and final destructive harvest. Litterfall was collected at the end of
both growing seasons. Imidacloprid equivalent concentration (IEC) in leaves varied with
time, and orientation to the injection point. Results are consistent with the sectored flow
hypothesis. Imidacloprid equivalent concentrations were higher in leaves of branches in
the plane of the injection point (0°) as opposed to leaves of branches on the Opposite side

Of the injection point (180°). Leaves from R90° branches had higher IEC than leaves

45

from L90° branches. We conclude that ash trees have a zigzag xylem architecture pattern
which could lead to variable IEC in the tree crown. Leaves from trees injected in the
spring had higher IEC than trees injected during the fall 21 DAT. During 2007, the year
after injection, IEC in leaves from both sets of trees were basically the same, some
patterns of sectoriality were still present, and IEC in leaves from spring-injected trees was
greatly reduced. Imidacloprid equivalent concentrations in leaves, stems, and litterfall
were higher than IEC in roots, trunk cores, and stems, indicating that imidacloprid moves

primarily through the xylem.

46

CHAPTER 2

Distribution of Trunk-Injected 14C-Imidacloprid in Fraxinus spp. Trees:

A Test of the Sectored Flow Hypothesis.

INTRODUCTION:

Emerald ash borer (EAB, Agrilus planipennis Fairmaire) (Coleoptera:
Buprestidae) is a phloem-boring insect native to Asia. The invasive metallic phloem-
borer arrived in the United States near Detroit, Michigan in the early 1990’s and was first
identified in 2002 afler reports from homeowners of dead and dying ash (F raxr'nus spp.)
trees (Siegert et al., 2007). Since its arrival, it has killed over 25 million ash trees in
Michigan and is now found in seven states in the US and Ontario, Canada. Infested trees
exhibit a variety Of symptoms including epiconnic shoots, blistered and discolored bark,
D-shaped adult exit holes, wood pecker holes and crown death (Cappaert et al., 2005b).
Economic loss of urban ash trees in the United States was estimated at $20-60 billion, a
figure that includes tree removal but not tree replacement. Economic loss of forest ash
trees in the United States is estimated at $282.3 billion (Federal Register, 2003). Emerald
ash borer is expected to attack all North American ash species (Fraxinus spp.) which
have not evolved resistance against it (Herms et al., 2004). If left unchecked, EAB could
be responsible for virtually eliminating native ash in urban and forest environments in

North America.

47

In Michigan, EAB usually has a one year life cycle, although in some outlier Sites,
larvae have been known to develop over two years (McCullough et al., 2005b). The
larvae are phloem feeders and develop through four instars; it is these larval stages that
cause the most tree damage. Larvae feed on nutrient rich phloem and create serpentine
galleries throughout the cambium of trees. Afier three to five years, multiple galleries in
a tree interrupt the translocation of nutrients and water and eventually kill it. Adults feed
on leaves but rarely cause more than noticeable damage. (Cappaert et al., 2005b).

Since the discovery of EAB in 2002, researchers have tested several methods of
chemical control including broadcast sprays, soil drenches, trunk applications, and trunk
injections ((Cappaert et al., 2005a,b, Cregg et al., 2006, Harrell, 2006, McCullough et al.,
2003, 2004, 2005a, 2005b, Smitley et al, 2004, 2005, Poland and McCullough, 2006).
One of the most common active ingredients in these applications is the neo-nicotinoid
compound imidacloprid. Imidacloprid acts on the acetylcholine receptor sites of the
emerald ash borer (Gill et al., 1999). It has very low mammalian toxicity and can be
applied systemically as a soil drench or trunk injection, which makes it acceptable in
urban settings where drift is both a real and perceived concern (Sur and Stork, 2003).
Systemic insecticides reduce the effects of drifi on humans, wildlife, and non target
insects because all of the insecticide applied is directly taken up by the plant. In addition,
trunk-injected insecticides like imidacloprid are applied at lower rates, applied less Often,
and are safer for applicators than other forms of insecticide application (Poland et al.,
2006a)

With the possible exception Of a new product containing emamectin benzoate

(McCullough et al., 2008), there are no insecticides that are 100% effective against EAB

48

(Poland, 2007). Although researchers have determined that imidacloprid is lethal to EAB
adults, efficacy is highly variable (McCullough et al., 2005a and Harrell, 2006).
Variability Of trunk-injected insecticides could be due to a variety Of parameters
including air temperature, water availability, tree health, tree injury, and tree anatomy
(Smith and Lewis, 2005). Poland et al. (2006a) suggest that the point of trunk injection
also influences insecticide efficacy and that insecticides might be translocated within
trees in such a way that they would have no effect on target insects.

Sectoriality might also affect the translocation of insecticides within trees. Orians
et a1. (2004) demonstrated that sap flow in trees may be sectored or integrated depending
upon tree species. Trees with greater degrees of sectoriality have specific translocation
paths that move resources primarily through a longitudinal plane. Trees with greater
degrees of integration have broader translocation paths that move resources in both
longitudinal and radial planes (Orians et al., 2005). Degrees of sectoriality are influenced
by the extent Of vessel to vessel contact within xylem tissue and the ratio of vessel walls
that contain bordered pits. Though not an absolute rule, ring porous trees (e. g. ash) are
generally considered sectored while diffuse porous trees (e. g. maple) are considered more
integrated. Increased vessel to vessel contact and higher pit ratio in integrated species
usually equates to higher degrees of integration, in contrast, sectored trees have less
contact between vessels and lower bordered pit ratios (Zanne et al., 2006). Highly
sectored xylem anatomy could result in uneven distribution and variable efficacy of

trunk-injected insecticide.

In a previous study at Michigan State University, researchers used l4C labeled

imidacloprid to track trunk-injected insecticide movement in ash trees. The study

49

demonstrated that imidacloprid moves slowly and steadily through trees over time and
accumulates in leaves. However, the sampling protocol did not permit a thorough
examination of spatial variability of imidacloprid distribution (Cregg et al., 2006). If ash
trees have straight sectored xylem anatomy, trunk-injected insecticides may move in a
straight line up to the canopy. Therefore, depending on injection location and spacing,
there could be areas of the crown that have reduced insecticide concentrations. If we
better understand the translocation path within trees, we can make recommendations to
arborists that might improve efficacy of trunk-injected insecticides.

Efficacy variability could also be affected by the length of time that imidacloprid
has been in the tree. Currently there are conflicting Opinions about the persistence Of
imidacloprid after trunk injection. Some research suggests that products can achieve two
years Of insect control from a single injection (Doccola et al., 2006), but other studies
have shown that only one season of control can be achieved (McCullough et al., 2005a,b,
Poland et al., 2006a,b, Tatter et al., 1998). The differences in these reports could be a
result of this percentage of active ingredient, application rates, or other differences in
trunk-injection procedures. Poland et al. (2006a,b) found that efficacy of imidacloprid
against Asian longhomed beetle (Anoplophora glabripennis Motschulsky) in Populus spp.
was highest in weeks immediately following trunk injection and then was diminished
over time. Tatter et a1. (1998), also found that imidacloprid concentration increased in
eastern hemlock (T suga canadensis, L.) in weeks immediately following injection but
began to decrease after approximately eight weeks. Cregg et a1. (2006) concluded that
imidacloprid is not translocated from leaves back into the tree before abscission and that

a large concentration Of imidacloprid is lost through litterfall. The question remains — is

50

there enough compound left in the tree after litterfall to provide a second season of
effective EAB control and if so, where is the compound stored? Ash are resilient trees, a
trait that helps them to recover from the trunk injection process; but if two years of
control can be gained from a Single injection, damage to trees that occurs during the trunk
injection process could be reduced as well as the cost Of treatment.

Another factor affecting insecticide efficacy could be the timing of imidacloprid
trunk injections. Trunk injections in the tree maintenance trade typically occur in either
spring or fall. Spring injections are advantageous because insecticide is being injected
into trees during a period of maximum translocation. Fall injections are popular because
there is Often a lag in business Opportunities during this time. Whether one application
time is more effective against EAB than the other is a question that has not been
answered.

Overall, the efficacy of trunk-injected inridacloprid might be influenced by tree
sectoriality, how Often trunk injections take place, and the time of year that trees are
injected. Understanding the spatial and temporal flow of insecticide through Fraxinus
spp. trees will enable us to Optimize trunk injection treatments and better understand the
persistence and potential for extended EAB control.

The Objectives of this study are to determine the (1) spatial movement of
imidacloprid in Fraxinus spp. trees; (2) concentration Of imidacloprid in Fraxinus spp.
trees over two growing seasons and (3) variability between spring and fall imidacloprid

trunk injections in Fraxinus spp. trees.

51

MATERIALS AND METHODS

This experiment consisted of 40 total trees, 20 F. americana (‘Autumn Applause’
or ‘Junginger’) and 20 F. pennsylvanica (‘Patmore’) (J. Frank Schmidt and Son Company,
Boring, Oregon). Trees were 4.0 to 5.0 cm (1 .5- 2.0”) caliper bare root liners planted in
pure sand in 95.0 L (25 gallon) plastic containers at the Horticulture Teaching and
Research Center, Michigan State University, Holt, Ingham County, Michigan.

Containers were spaced 2.5 m (8 ft) on center. After planting, pots were placed inside 1.2
m plastic wading pools and elevated on a single layer of brick pavers. This allowed for
drainage but kept leachate from entering the environment.

The experiment included eight replicates for the spring injection and two

replicates for the fall injection. Each replicate consisted Of four randomly placed trees,

two of each Species. Trees were fertilized with Osmocote® Plus (The Scott’s Company,

Marysville, Ohio) controlled release fertilizer at a rate Of 314 grams per pot and watered
to runoff with drip irrigation two to three times per week. Trees for this study were
individually selected and/or pruned to ensure that the first three branch whorls were
oriented at 90° angles to one another.
Spring Injection:

On 28 June 2006, 32 trees (16 F. americana, 16 F. pennsylvanica) were trunk-
injected at a Single injection point. It is important to note that this date is approximately
three weeks later than we had originally planned due tO complications related to

importing the radio-labeled imidacloprid into the United States. Each tree was injected

with 25 uCi of '4C labeled imidacloprid mixed with 3.1 ml lmicide® (Mauget®, Arcadia,

52

California, 10% active ingredient) in a ratio of 1:1300 labeled to unlabeled compound.

F raxinus americana trees were injected at 0° to the first whorl of branches (i.e. injection
port was directly below one Of the branches in the first whorl, Figure 2.1a). F raxinus
pennsylvanica trees were injected at either 0° or 90° to the first whorl of branches (Figure
2.2a). Injection holes were drilled 10.0 cm above the graft union with a cordless drill
and an 8.0 mm (5/16”) drill bit. Holes were drilled to a depth of approximately 1.5 cm and
systemic tree injection tubes (STIT) were inserted and gently tapped in with a hammer
(Helson et al., 2001). After tubes were inserted, 3.1 ml of imidacloprid mixture was
added through the top of the lower half of the STIT and 2.0 bar pressure was applied
using a hand Operated bicycle pump. All lower components of the STIT were securely
wrapped with black electrical tape to ensure that leakage was contained and to provide
stability. The upper half Of the STITs were secured to trees with tape. The STITs were
monitored for pressure, re-inflated as needed, and kept in position 2-48 hours until all
liquid was taken up by the tree. After all liquid was translocated into the tree, tape and
STIT were carefully removed. The area of the tree that contained the drill hole was
double-wrapped with electrical tape to prevent leakage or leaching of radioactive material.
Tape remained in place throughout the experiment. Three trees were removed from the
experiment due to excessive leakage during the injection process. All handling of
radioactive material complied with Michigan State University’s Office of Radiation,
Chemical, and Biological Safety (ORCBS) standards. All areas were surveyed
periodically using a Geiger counter (Ludlums Measurement Inc., Survey Meter Model
44-9, Sweetwater, TX) to ensure that no radioactive material leached into the

environment.

53

Fall Injection:

On 5 September 2006 eight additional trees, four F. americana, four F.
pennsylvanica were injected with 2.95 ml or 14(2 labeled imidacloprid mixture in the same
manner as previously described for the spring injection. The tree canopies were fully
flushed but not yet senescing. Trees were fertilized immediately after planting in the
spring and were kept well watered throughout the summer prior to injection.

Sampling:

Branches from the first three branch whorls of each tree were labeled either 0°,
180°, Right 90° (R90°) or Left 90° (L90°) depending on the position of the branch in
relation tO the injection point (Figure 2.2b). Leaf samples were Obtained by pinching Off
individual leaflets from proximal, middle and distal portions of each of the six branches.
Leaf samples from the terminal leader were removed with a pole pruner. Trunk cores
were taken 1 m above the injection point at 0° and 180° (in relation to the injection point)
using a #1 cork borer. The borer was pushed into the tree to remove approximately 2 m
depth Of tissue (bark, phloem, and sapwood). Root samples were obtained by digging
into the sand until a portion of the root system was exposed. Approximately 1 g dry
weight of fine roots were removed from coarse roots and rinsed with tap water. Whole
stem samples approximately 2 cm in length were taken from branch tips of the first three
whorls using hand pruners. All samples were placed in small paper bags, labeled and
placed in a 70° C drying oven for at least 72 hr. After drying, all leaf samples were
removed from the oven, ground to a fine powder using a mortar and pestle and placed

into individually labeled paper envelopes. Root, core, and stem samples were left whole.

54

A 150.0 — 180.0 mg sub-sample of tissue was processed in a biological tissue
Oxidizer (OX300 R. J. Harvey Instrument Corporation, Tappan, NY, USA). Stem

samples were burned on a four minute cycle, leaf samples on a three minute cycle, and

root and core samples on a two minute cycle. Resulting 14C02 was trapped in

scintillation cocktails and processed through a Packard Tri Carb liquid scintillation
counter (LSC, Packard Bioscience, Currently PerkinElmer Life and Analytical Science,
Waltham, Massachusetts, USA) on a one and one half minute cycle. Resulting counts per
minute (CPM) were recorded for each sample and converted to imidacloprid equivalents
after accounting for background activity and oxidizer and LSC efficiencies.
Imidacloprid injected into trunks of Fraxinus trees is broken-down into a series of
metabolites (Mota-Sanchez et al., 2007a), many of which exhibit insecticidal properties;
therefore we refer to the concentration of insecticide in the tree in terms Of imidacloprid
equivalents. All results are presented in pg of imidacloprid equivalents per g dry weight
of sample.

For the 2006 growing season, spring injected trees were sampled at 0, 2, 7, 21, 60,
and 98 days after treatment (DAT). Fall injected trees were sampled at 0, 2, 7, and 21
DAT. The sampling protocol included leaves from the first three branch whorls, terminal
leader leaves, trunk cores, and fine roots. Stem samples were taken from spring injected
trees only at 60 DAT to prevent excessive tree damage. In late September, all trees were
netted with bird netting tO collect litterfall. After abscission, all leaves were removed
from nets, placed in paper bags and oven dried. Sub-samples of five to seven leaves from
each tree were ground, weighed, oxidized, and processed through the scintillation counter.

After nets and leaves were removed, trees were lifted with a sling and skid steer and

55

placed pot to pot in a holding area. A double layer of hay bales was placed around the
holding area to protect the roots from winter frost. In April 2007, trees were lifted with
the sling and skid steer and placed back in their original configuration. Fertilizer and
irrigation rates in 2007 were the same as used in 2006.

For the 2007 growing season, both spring- and fall-injected trees were sampled to
collect whorl and leader leaves, and fine roots on 29 May. Additional leaf and root
samples were taken on 12 June (the week roughly corresponding to peak EAB flight in
Michigan, Anulewicz et al., 2008b), and again on 31 August. Trunk cores were not taken .
in 2007 to prevent excessive damage to the trees. At the end of the 2007 growing season,
all leaves were removed from the trees by hand. Leaves from the first three whorl
branches were kept separate from the rest of the canopy leaves. Stern samples were
removed during destructive harvest.

Fifteen trees (five F. americana/0° injection, five F. pennsylvanica/0° injection,
and five F. pennsylvanica/90 ° injection) from spring injected trees and one replication of
fall injected trees were destructively harvested in October and November, 2007. Trees
were lifted out of pots using a skid steer and chain. Sand was rinsed off roots using a
garden hose and spray nozzle. Roots were removed from the trunk using hand pnmers
and loppers and classified as either coarse or fine. Roots that were smaller than 5.0 mm
diameter were considered fine roots, all others were considered coarse roots. Branches
from whorls one through six (where applicable) were labeled (0°, 180°, R90° or L90°,
based on orientation to the injection point) and removed from the bole Of the tree with
loppers. Stem sections were removed from tips of branches and separated into 2006 and

2007 growth sections as determined by the position of the axillary leaf scars. A sub-

56

sample from each tree of five to seven leaves each from terminal leader and whorl
branches, approximately 3 g each Of fine and coarse roots, and stems from harvested trees
were dried, ground (leaves only), weighed, oxidized and processed through the LSC. The
bole of the tree was cut into sections at 10.0 cm, 1.0 m and 2.0 m above the center of the
injection scar, and a 2.0 cm thick cross-section was removed. Cross sections were split
into quarters using a hammer and chisel. One of these sections was further divided into
three pieces and a single piece was dried, weighed, oxidized, and processed through the
LSC. The cross sections cut at 10.0 cm above the injection scar contained portions of
xylem that were stained by the initial imidacloprid injection; a portion of this stained area
was also chiseled out, dried, weighed, oxidized, and processed through the LSC. All
branches, fine and coarse roots, and the tree bole were cut into manageable sections using
hand pruners, loppers, and a pruning saw, dried, and weighed.

Statistical Analysis:

Data were tested for normality using residual plots. Variables were not normal.
Imidacloprid equivalent concentrations for spring-injected ash trees were transformed
using a log (x+2) transformation. Imidacloprid equivalent concentration for fall-injected
ash trees was transformed using the log (x+2) transformation. Data were analyzed using
the Mixed procedure for mixed models in SAS statistical software (SAS Institute, Inc.
1989) with whorl nested in tree, branch position nested in whorl, tree species and day as
fixed effects and tree number as a random effect. The models for repeated measures were
examined using CS covariance structure. Analysis Of variance (ANOVA) was used to
determine which effects and interactions were significant (p<0.05) for whorl, branch

position, tree species, and day. Different ANOVAS were produced for each injection date

57

(spring vs. fall) by injection position (0 or 90). If effects were significant, LSMeans

procedures were used for mean separation (Littell et al., 1996).

58

RESULTS

Spring Injection:

Imidacloprid equivalent concentration (IEC) in leaves varied (p<0.05) with time
and orientation to the injection point. Species or branch whorl position did not affect IEC
(p<0.05). Imidacloprid equivalent concentration increased rapidly following trunk
injection throughout the 2006 growing season until 21 days after treatment (DAT). For a
given whorl, IEC was greater (p<0.05) in leaves from branches in the plane of injection
(0°) than leaves from branches Opposite the plane of injection (180°) throughout the 2006
growing season. However, the difference was less pronounced in whorl three, suggesting
that flow becomes more integrated with plant height. In general, branch position did not
affect (p>0.05) IEC in leaves from 0° or 180° branches in 2007 (Figure 2.2).

Leaves Of L90° branches had lower IEC (p<0.05) than leaves on R90° branches
throughout the 2006 growing season and were different only in leaves fiom whorl three
in 2007. Similar to leaves on 0° and 180° branches, IEC in 2007 leaves from R90° and
L90° branches was reduced by 20-fold when compared to IEC in leaves sampled in 2006
(Figure 2.2).

Imidacloprid equivalent concentration in leaves collected from the terminal leader
increased (p<0.05) through 60 DAT in 2006, but IEC levels were greatly reduced in 2007.
Imidacloprid equivalent concentration increased (p<0.05) in trees injected at 0° to the first
whorl Of branches the year after “injection (Figure 2.2).

Roots sampled in 2006 and 2007 and trunk cores (0° and 180°) collected in 2006

did not have detectable IEC. Root and trunk core samples collected 60 DAT had lower

59

(p<0.05) IEC than leaves collected from whorl and stems from 0° and R90° branches.
Roots, trunk cores, and 180° stems were not different at 60 DAT. For samples collected
60 DAT, stems had lower (p<0.05) IEC than leaves from corresponding branches and
whorls (Figure 2.3a). Stems from 0° and R90° branches had higher (p<0.05) IEC than
stems from 180° and L90° branches respectively. Overall, IEC in stems from 0° and R90°
branches were not different (p<0.05) and stems from 180° and L90° branches were not
different (p<0.05) (Figure 2.3b).

Leaves from 0°, R90° and 180° branches had higher IEC values than stems Of
corresponding branches. Stems from branches in whorl one had higher (p<0.05) IEC than
either fine or coarse roots. Fine root IEC was not different (p<0.05) fi'om coarse root IEC
(Figure 2.4). Stem samples taken from 2006 growth from 0° and R90° branches had
higher (p<0.05) IEC than stems from 180° and L90° branches consistently through whorl
four. Beyond whorl four IECS were similar between branches of the same whorl. Stem
samples taken from 2007 growth had higher (p<0.05) IEC in leaves from 0° branches
than leaves from 180° branches in whorls one and three but no other differences occurred
between branches Of the same whorl. There was no difference (p>0.05) in IEC between
2006 and 2007 stem samples for any whorl (Figure 2.5).

Wood samples taken from the stained area at 10.0 cm Of the trunk cross-sections
had IEC approximately 75 times higher than all other wood samples taken from the bole
of the tree. Imidacloprid equivalent concentrations were the same for wood cross
sections taken from 10.0 cm (no stain) and 1.0 m and samples taken from 1.0 m and 2.0

m for 0 injected trees (Figure 2.6). Samples taken from 90° injected trees had the same

60

IEC in 0.10 m (no stain) and 2.0 m sections, but sections taken from 1.0 m were higher
than both 0.10 m (no stain) and 2.0 m sections (Figure 2.6 inset).
Fall Injection:

For trees that were trunk-inj ected at 0° and 90° to the first whorl of branches in the
fall, IEC did not differ between leaves from 0° and 180° degree branches in whorl one.
Imidacloprid equivalent concentration in leaves from 0° branches did not increase beyond
7 DAT and IEC in leaves from 180° branches did not increase over time. The year after
injection, leaves from 0° branches had higher (p<0.05) IEC than leaves from 180°
branches. In addition, leaves from 0° branches had similar (p>0.05) IEC as leaves from
the corresponding branch sampled 21 DAT in 2006. Irrridacloprid equivalent
concentration in leaves from 180° branches did not increase over time during 2007
(Figure 2.7).

The general trend of IEC in R90° branches indicates some increase in the year
after injection. However, in most cases the increase was not statistically different among
sampling dates or branches. No IEC increase occurred in leaves from L90° branches
(Figure 2.6). Imidacloprid equivalent concentration was lower (p<0.05) in stems than
leaves. Roots from 2006 and 2007 and trunk cores from 2006 contained no measurable
IECs (Figure 2.8).

Wood samples taken from the 10.0 cm stained area of trunk cross sections had
IEC approximately 136 times higher than all other wood samples taken from the bole Of
the tree. Imidacloprid equivalent concentrations were the same for wood cross sections
taken from 10.0 cm (no stain) and 1.0 m which were higher than IEC in 2.0 m trunk cross

sections (Figure 2.9).

61

Spring vs Fall Injected Leaves:

Leaves harvested 21 DAT (19 July 2006 — spring injection, 26 September 2006 —
fall injection) from 0°, R90° and L90° branches on trees injected in spring had higher
IEC than leaves taken from corresponding branches of trees injected in fall (Figure 2.10).
In the year following injection, leaves from 0° branches of fall injected trees had higher
(p<0.05) IEC than leaves from 0° branches of spring injected trees. Leaves from 180°,
R90° and L90° branches did not differ among corresponding branches of trees injected in
spring or fall (Figure 2.11).

Observations:
On June 18, 2007 EAB larval galleries were positively identified in several trees

used in this study. Although not part Of our original objectives, the presence Of larvae on

l4C-imidacloprid trunk-injected trees was noteworthy. Un-infested trees were purchased

from a nursery which is well beyond the known EAB infestation. Therefore EAB larvae
present on trees were from eggs deposited in summer 2006 by local Michigan beetles.
After initial discovery, all trees were examined carefully and the presence of visible
galleries was recorded. Trees were not treated with additional insecticide. In some trees,
canopy dieback and branch damage did occur; one trunk-injected tree was completely
girdled and did not leaf out in 2007.

On trees where galleries were exposed, frass samples were removed, dried,
oxidized, and processed through the LSC. NO detectable imidacloprid equivalents were
present (data not shown). In addition, 16 dead beetles found in 12 of the pots containing
injected trees were dried, oxidized and processed through the LSC. Two beetles

contained detectable IEC (data not shown).

62

At the conclusion Of the 2007 growing season, 15 trees (see Materials and
Methods) were destructively harvested. During that time, number of galleries per tree
was recorded and galleries per m2 of tree bark was calculated (Table 2.1). In addition 12
additional trees were examined for galleries during spring, 2008. There did not appear to
be any patterns in the position Of the galleries in relation to the injection point, in fact,
galleries appeared in both the plane of and the Opposite plane Of the injection site. In

this particular case, imidacloprid trunk-injection did not prevent larval development.

63

DISCUSSION

Variability Of trunk-injected insecticide translocation can be affected by a variety
Of parameters including xylem anatomy, time of injection, and time after injection.
Although literature does exist pertaining to translocation of imidacloprid in hemlock,
avocado, and citrus (Castle et al., 2004, Cowles et al., 2006, Lawson etal., 2003, Sclar
and Cranshaw, 1996)) trees, information on the specific translocation paths of
imidacloprid in ash trees is not available. Efficacy of imidacloprid in several trials has
been variable (McCullough et al., 2005a and Harrel, 2006). This study attempts to
explain some Of that variability. Our primary objectives were to determine the (1) spatial
movement of imidacloprid in ash trees, (2) imidacloprid equivalent concentration in ash
trees over two growing seasons and (3) variability of imidacloprid equivalent
concentration between spring and fall trunk-inj ected ash trees.

Objective One-

We found that imidacloprid equivalent concentration varied in leaves from spring
and fall injected trees and orientation of branches in relation to the injection point.
During 2006, IEC increased in leaves from all six branches of both 0° and 90° spring-
injected trees. Leaves from branches in the plane Of injection (0°) had greater IEC than
leaves on the Opposite side of the injection site (180°) in both the 0° and 90° injected trees.
In addition, leaves from R90° branches had higher IEC than L90° branches. Movement
Of labeled compound was very ordered during the first growing season. Insecticide
moved quickly in the longitudinal direction (as seen in leaves from branches in the plane

of injection (0°)) but very Slowly in the radial plane (as seen in leaves from branches

64

Opposite the injection point (180°)). This is consistent with other studies that have
shown that F. americana has longitudinal movement Six orders of magnitude higher than
radial movement (Zwieniecki et al., 2001). No deviations from the pattern of ascent
occurred in any of the whorls in either injection over time. The consistent, ordered
pattern of IEC in leaves from specific branches suggests that Fraxinus spp. trees have
sectored xylem pathways. The sectored patterns of the xylem architecture create uneven
distribution of insecticide within the tree crown. Although the trees used for this study
were small (due to containment restraints), we believe that the patterns of sectoriality
would be similar in larger trees (Orians et al., 2004). AS seen here, unbalanced injection
sites could lead to uneven insecticide distribution and variable insecticide efficacy.

Ash is a ring porous species and is generally described as straight grained. Ring
porous and straight grained species usually have sectored patterns of xylem architecture.
However, patterns of sap ascent in trees normally deviate from purely straight and are
Often described as helical (Tyree and Zimmerman, 2002). If translocation patterns in ash
xylem were purely helical, we would expect to see continued spiral ascent and higher
IEC in leaves from third whorl 180° branches when compared to leaves from 0° branches,
which was not the case in this study. If the xylem architecture was straight sectored,
leaves from second whorl branches would have intermediate IECS to those of both whorls
one and three, this did not occur either. Instead we determined that ash xylem architecture
has a zigzag pattern of ascent. Starting at whorl one, the highest concentration of
insecticide appears in branches in the plane Of injection (0°). Moving up the tree to whorl
two, the highest levels occur in leaves from R90° branches. Finally, in whorl three, the

highest IEC is in leaves from branches within the plane Of injection (0°). A similar

65

zigzag pattern occurs on the opposite branches Of the tree. Again beginning at whorl one,
insecticide levels in leaves on the opposite side Of the injection site (180°) are low
compared to leaves from 0° branches. In whorl two, only small amounts Of insecticide
are present in leaves from L90° branches. Finally in whorl three, leaves from branches
opposite the injection point have low levels of insecticide. High variability in 90°
injected trees could be attributed to low sample size. Kiten et a1. (2004), propose that sap
ascent patterns of F. lanuginosa KOidz. are influenced by vessel to vessel contact, and
even though ash is considered a straight grained Wood, tangential drift in vessel position
does occur. Burggraaff (1972) describes a zigzag pattern in the xylem architecture Of F.
excelsior L. which has vessels that slant toward the right. In addition, he states that
vessels are in contact with one another in both the tangential and radial directions but that
no perforations exist that allow open contact between vessels.

In addition to leaves, imidacloprid equivalents were also present in stems. We did
not sample stems more than once per year to prevent excessive tree damage. Stem
sections harvested from 0° injected trees 60 DAT (2006) had IEC patterns similar to
leaves. Concentrations were highest in stems from 0° and R90° branches when compared
to stems from 180° and L90° branches. The IEC pattern in stems also supports the theory
of zigzag ascent. Imidacloprid equivalent concentrations in stems from 90° injected
trees were more similar between branches, but the same pattern was still present.

Patterns of sectoriality were much less pronounced in trees that were trunk-
injected during the fall. In fact, no difference in IEC in leaves between opposing
branches occurred during 2006 for trees that were injected at 0° to the first whorl of

branches or trees injected at 90° to the first whorl of branches. In 2007, however, IEC in

66

leaves from 0° branches were higher than leaves from 180° branches Of 0° injected trees
and leaves from R90° branches had higher IEC than leaves from L90° branches of 90°
injected trees. Even though these patterns were not as obvious in fall-injected trees
because of low IEC, patterns of sectoriality were consistent with those of spring injected
trees and support the zigzag ascent theory.

Several studies have demonstrated that imidacloprid movement in plants occurs
via the xylem (Bromilow and Chamberlain, 1989, Sur and Stork, 2003, Tanis et al.,
2007a). To determine whether this was the case in ash, we harvested fine roots and trunk
cores (2006 only to prevent excess tree damage) and performed leachate tests. As
previously noted, imidacloprid was present in measurable concentrations in leaves and
stems. In contrast, no measurable IEC was present in roots or trunk cores. If
imidacloprid movement was occurring in the phloem, these tissues would have IEC > 0.
TO further investigate fine roots, we also performed pour through leachate tests to
determine if fine root turnover from trunk-injected trees could contribute to imidacloprid
residues in soil. NO detectable imidacloprid equivalents were present in leachate

collected in pour through samples (Tanis et al., 2007b).

In a previous study that used 14C to examine imidacloprid movement in ash trees,

Cregg et a1. (2006) demonstrated that canopy leaves collected after abscission contained
IEC as high as those sampled before abscission. In other words, imidacloprid was not
translocated from leaves back into the trunk before litterfall. Post harvest analysis of
trunk segments of trees used in that study had very high IEC in stained areas around the
injection Site (Tanis et al., 2007b). We hypothesize that trunk tissue in this area serves as

a reservoir that supplies imidacloprid to leaves during subsequent growing seasons. TO

67

further investigate, trees from the present study were destructively harvested at the
conclusion Of the 2007 growing season. Cross sections of wood were taken from the bole
Of trees at different heights above the injection point to determine if imidacloprid was
present in woody tissue. Similar to the cross sections taken from trees in the previous
study, cross sections taken 0.10 m above the injection point had stained areas (from the
initial imidacloprid injection) that contained extremely high IECS when compared to all
other plant parts. Samples Of non-stained wood taken from the same area and points
higher on the tree had lower IEC concentrations than stained wood. These results support
the reservoir hypothesis that imidacloprid is pooled in woody tissue around the injection
point and gradually dissipates as the tree gets taller. High IEC in woody tissue and zero
IEC in roots and trunk cores support previous evidence that states that imidacloprid
moves primarily through the xylem.

Objective Two:

Our second Obj ective was to determine how much imidacloprid was present in
trees after two growing seasons. Previous studies have shown that high levels of efficacy
can be Obtained from a single injection over two growing seasons, but others have shown
that levels of insecticide are greatly reduced during the second year after injection.

In this study, spring-injected insecticide would be providing a second season of
EAB control in 2007 while fall-injected insecticide would be providing its first year of
control. In leaves from 0° and 90° spring-injected trees, IEC on 11 June 2007 was less
than 12% of levels found at the end of the growing season in 2006. In a parallel study,

bioassays conducted on adult beetles using leaves from these trees indicated that 50% of

68

A. planipennis beetles were affected, down from 80% the previous year (Mota-Sanchez et
al., 2007b).

Imidacloprid equivalent concentration in leaves from fall injected trees were
generally not different between growing seasons even though percent increase was over
75% in all branches of 0 injected trees. The question remains, is there enough compound
in the leaves to be efficacious?

Objective Three:

Our third Objective was to determine differences in IEC in spring-injected trees
compared to fall-injected trees. Here we compare trees from each injection at two
sampling dates. First, we compare trees from spring and fall injections at 21 DAT (19
July 2006 and 26 September 2006, respectively). We chose this date because it was the
last date that fall-injected trees were sampled in 2006. Leaves from spring-injected trees
had higher concentrations 21 DAT than leaves from fall-injected trees in all branch
positions. This is probably due to decreases in temperature during the fall, the length of
time before abscission, and decreased transpiration rates.

We also compared the two injections on 11 June 2007. This sampling date
roughly corresponds to 2007 EAB peak flight in Michigan (Anulewicz et al., 2008b).
Imidacloprid equivalent concentrations in leaves from spring-injected trees were lower in
2007 than 2006. Imidacloprid equivalent concentrations in leaves from fall-injected trees
were lower initially and remained relatively constant. Since less imidacloprid was

present in leaves from fall-injected trees in 2006 than spring-injected trees, less

insecticide was lost during litterfall. This would suggest that there would be more 14C-

labeled compound available for translocation in fall injected trees than spring-injected

69

trees in 2007. However, on 11 June 2007, only leaves from 0° branches from fall-
injected trees had higher IEC than leaves from corresponding branches of spring-injected
trees. Therefore, even though larger reservoirs of imidacloprid are available to fall-
injected trees than spring-injected trees, higher levels of imidacloprid are not translocated
to leaves of 180°, R90°, or L90° branches. These patterns of ascent could affect the
efficacy Of imidacloprid against EAB. If only one third of the branches receive adequate
quantities of imidacloprid during the second season (spring-injected), or following fall
injection, many EAB could feed on trees and not be affected. Results from efficacy trials
for fall-inj ected trees are not available at this time but if levels from spring-injected trees
were <50% efficacious and spring and fall injected trees only differed in two branches,
one can assume that fall-injected insecticide might be <50% efficacious as well.

In summary, we have shown that imidacloprid equivalent concentration varies in
leaves of ash trees based on the position of branches in relation to injection points and
time that trees were injected. Our results indicate that efficacy Of trunk-injected
imidacloprid could be reduced in trees injected in fall when compared to trees injected
during spring. In addition, after over-wintering, new leaves from spring-injected trees do
not have adequate levels of insecticide to provide effective EAB control based on the
rates applied. We also observed that larval galleries were present in the injected trees,
and damage from galleries in some cases was quite severe. Finally we conclude that
xylem anatomy in ash trees is such that trunk-injected imidacloprid travels through the
tree in a zigzag xylem ascent pattern. This zigzag pattern results in variable levels of

insecticide in the tree crown.

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72

 

 

 

 

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Figure 2.1b: Sampling schematic of trees injected at either 0° or 90° to the first whorl of
branches. Branches of the first three whorls were labeled 0°, 180°, L90° or R90° in
relation to the injection point. Mean distance from injection point to the first whorl =

1.28 m. Mean distance between whorls = 0.18 m.

73

Figure 2.2: Mean l4C-Imidacloprid equivalent concentration :t SE. in whorl and

terminal leader leaves from 0° or 90° (in relation to the first whorl of branches) spring-
trunk-injected Fraxinus americana and F. pennsylvanica trees. Branches were labeled 0°,
180°, L90° or R90° (in relation to the injection point. An * denotes leaves collected after

abscission.

74

Imidacloprid Equivalents jig-9'1

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75

Figure 2.3a: Mean l4C-Imidacloprid equivalent concentration :1: SE. in whorl one stems
and leaves (0°, 180°, L90° or R90°~in relation to the injection point), trunk cores (0° or
180° in relation to the injection point) and roots of 0° and 90° (in relation to the first whorl
of branches) spring-trunk-injected F raxinus americana and F. pennsylvanica trees,

samples taken 60 days after treatment. Inset graphs have leaf data bars removed.

76

 

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Figure 2.3b: Mean l4C-Imidacloprid equivalent concentration :1: SE. in stem samples
taken from the first three branch whorls of 0° and 90° (in relation to the first whorl of
branches) spring-trunk-injected F raxinus americana and F. pennsylvanica trees 60 days
after treatment. Stem samples were labeled 0°, 180°, L90° or R90° in relation to the

injection point.

78

Imidacloprid equivalents ug-g'1

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Figure 2.4: Mean MC-imidacloprid equivalent concentration d: S.E. in leaves and stems
from first whorl branches (0°, 180°, L90° or R90° in relation to the injection point), coarse
and fine roots. Samples taken from 0° or 90° (in relation to the first whorl of branches)
spring-trunk-injected F raxinus americana and F. pennsylvanica trees 60 days after

treatment. Inset graphs have leaf data bars removed.

80

Imidacloprid equivalents ug-g‘1

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81

Figure 2.5: Mean l4C-Imidacloprid equivalent concentration i S.E. in 2006 and 2007
stem sections from 0° and 90° (in relation to the first whorl of branches) spring-trunk-
injected F raxinus americana and F. pennsylvanica trees. Stems were harvested in
October 2007 from the first six branch whorls (where available) and labeled 0°, 180°,

L90° or R90° in relation to the injection point.

82

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83

Figure 2.6: Mean l4C-imidacloprid equivalent concentration i S.E. in stained and
unstained trunk tissue taken at varying heights above the center of the injection scar from
0° or 90° (in relation to the first whorl of branches) spring-trunk-injected Fraxinus
americana and F. pennsylvanica trees harvested in October, 2007. Inset graphs have the

stained cross section data bars removed.

84

Imidacloprid equivalents jig-9'1

Imidacloprid equivalents ug-g'1

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85

 

Figure 2.7: Mean 14C-imidacloprid equivalent concentration i S.E. in whorl and

terminal leader leaves from 0° or 90° (in relation to the first whorl of branches) fall-trunk-
injected F raxinus americana and F. pennsylvanica trees. Braches were labeled 0°, 180°,
L90° or R90° (in relation to the injection point). An * denotes leaves collected after

abscission.

86

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Figure 2.8: Mean C-1m1daclopr1d equlvalent concentratlon :t S.E. 1n leaves and stems

from first whorl branches (0°, 180°, L90°, or R90° in relation to the injection point) and
coarse and fine roots. Samples were taken during destructive harvest from 0° or 90° (in
relation to the first whorl of branches) fall-trunk-injected F raxinus americana or F.

pennsylvanica.

88

Imidacloprid equivalents jug-9'1

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Figure 2.9: Mean 14C-imidacloprid equivalent concentration i S.E. in stained and

unstained trunk tissue taken at varying heights above the center for the injection scar
from 0° and 90° (in relation to the first whorl of branches) fall-trunk-injected Fraxinus
americana and F. pennsylvanica trees harvested in October, 2007. Inset graph has the

stained data bar removed.

90

 

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8 E3 Fall Injection
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Figure 2.10: Mean 14C-imidacloprid equivalent concentration i S.E. in leaves from the

first three branch whorls of 0° and 90° (in relation to the first whorl of branches) of
spring- and fall-trunk-injected Fraxinus americana and F. pennsylvanica trees. Values
were averaged across whorls. Branches labeled 0°, 180°, L90° or R90° in relation to the
injection point. Samples were taken 21 days after treatment (19 July 2006 and 26

September 2006).

91

 

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- Spring Injection

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00

 

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on
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20 30 40 50 6O

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Figure 2.11: Mean l4C-imidacloprid equivalent concentration i S.E. in leaves from the

first three branch whorls of 0° and 90° (in relation to the first whorl of branches) of
spring- and fall-trunk-injected Fraxinus americana and F pennsylvanica trees. Values
were averaged across whorls. Branches labeled 0°, 180°, L90° or R90° in relation to the

injection point. Samples were harvested on 11 June 2007.

92

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McCullough, D.G., D. Cappaert, T. Poland, and DR. Smitley. 2004. Control of emerald
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McCullough, D.G., T.M. Poland, D.L.Cappaert, P. Lewis, and J. Molongowski. 2005b.
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McCullough, D.G., T.M. Poland, and P. Lewis. 2008. Emamectin Benzoate Pesticide
Now Registered for Use in Treating Ash Trees for Emerald Ash Borer (EAB). Online at:
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McCullough, D. G, D. R. Smitley, and T. M. Poland. 2003. Evaluation of insecticides to
control emerald ash borer adults and larvae. Online at
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Michigan State University. 2008. Emerald Ash Borer Information.
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Mota-Sanchez, D., B.M. Cregg, D.G. McCullough, T.M. Poland, and R.M. Hollingworth.
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female of emerald ash borer adults, Agrilus planipenm's Fairmaire (Coleoptera: Buprestidae.
Entomological Society of America. 11 December 2007. San Diego, CA.

Mota-Sanchez, D., B.M. Cregg, S.R. Tanis, D.G. McCullough, T.M. Poland, and R.M.
Hollingworth. 2007b. Acute toxicity and feeding reduction effects of imidacloprid and its
metabolites in emerald ash borer adults. Oral Presentation. Emerald Ash Borer Research
and Technology Development Meeting. 24 October 2007. Pittsburgh, PA.

Orians C.M., S.D.P. Smith, and L. Sack. 2005. How are leaves plumbed inside a branch?
Differences in leaf-to-leaf hydraulic sectoriality among six temperate tree species.
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Orians, C. M, M. M..I vanVuuren, N. L. Harris, B. A. Babst, and G. S. Ellmore. 2004.

Differential sectoriality 1n long-distance transport in temperate tree species: evidence
from dye flow, l5N transport, and vessel element pitting. Trees 18: 501- 509.

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Poland, T.M. 2007. Twenty million ash trees later: current status of emerald ash borer
in Michigan. Newsletter of the Michigan Entomological Society 52 (l and 2): 10-13.

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Poland, T.M., R.A. Haack, T.R. Petrice, D.L.Miller, and LS. Bauer, and R. Gao. 2006b.
Field evaluation of systemic insecticides for control of Anoplophora glabripennis
(Coleoptera: Cerambycidae) in China. Journal of Economic Entomology 99(2): 383-392.

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 April-May:
118-124.

Sclar, DC. and W.S. Cranshaw. 1996. Evaluation of new systemic insecticides for elm
insect pest control. Journal of Environmental Horticulture 14(1): 22-26.

Siegert, N.W., D.G. McCullough, A.M. Liebhold, and F.W. Telewski. 2007.
Resurrected from the ashes: a historical reconstruction of emerald ash borer dynamics
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injection. Third Symposium on Hemlock Woolly Adelgid in the Eastern United States.
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Smitley, D.R., T.W. Davis, K.F. Newhouse, and E. Rebek. 2005. Troy EAB test results.
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Smitley, D., T. Davis, E. Rebek, and K. Newhouse. 2004. Imidacloprid soil drench test
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Sur, R., and A. Stork. 2003. Uptake, translocation, and metabolism of imidacloprid in
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Tanis, S. R., B.M. Cregg, D. Mota—Sanchez, D.G. McCullough, T.M. Poland, and R.M.

Hollingworth. 2007a. Distribution of trunk-injected l4C imidacloprid in Fraxinus trees:
a test of the sectored flow hypothesis. In Mastro et al., 2007: 34-3 8.

Tanis, S. R., B. M. Cregg, D. Mota-Sanchez, D. G. McCullough, T. M. Poland, and R. M.
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variation between spring and fall injections. Emerald Ash Borer Research and
Technology Development Meeting, Pittsburgh, PA. Oral Presentation. 24 October 2007.

96

Tatter, T.A., J .A. Dotson, M.S. Ruizzo, and VB. Steward. 1998. Translocation of
imidacloprid in three tree species when trunk- and soil-injected. Journal of Arboriculture
24(1): 54-56.

Tyree, MT and M.H. Zimmerman. 2002. Xylem structure and the ascent of sap.
Second Edition. Springer-Verlag, Berlin, Germany. 250 pp.

Zanne, A.E., K. Sweeney, M. Sharma, and CM. Orians. 2006. Patterns and
consequences of differential vascular sectoriality in 18 temperate tree and shrub species.
Functional Ecology 20: 200-206.

Zwieniecki, M.A., P.J. Melcher, and NM. Holbrook. 2001. Hydraulic properties of

individual xylem vessels of Fraxinus americana. Journal of Experimental Botany 52
(355): 257-264.

97

Distribution of Trunk-Injected 14C Imidacloprid in Acer Trees: A Test of the
Integrated—flow Hypothesis.

ABSTRACT

The Asian Longhomed beetle (ALB, Anoplophora glabripennis Motschulsky,
Coleoptera, Cerambycidae) is an invasive exotic insect pest. In the United States, ALB
prefers host trees in the genus Acer. Imidacloprid applied as a trunk injection is often
used as a means of control, but effectiveness can be variable. The objective of this study
was to determine the extent to which movement of imidacloprid is sectored within the
trunk of maple trees. Highly sectored flow could result in uneven distribution of the
compound within the tree and result in variable efficacy of pesticide. One set of eight

trees (four Acer x fieemanii, four A. platanoides) was trurik-injected during the fall with

14C labeled imidacloprid either directly below a branch in the first whorl or at a right

angle to branches of the first whorl. Each branch of the first three whorls was labeled 0°
/180°, or L90° / R90° depending on the location of the branch in relation to the injection
point. Leaves and fine roots were sampled through two growing seasons. Trunk cores
were sampled during 2006. Tree stems were sampled during destructive harvest, fall
2007. Litterfall was collected at the end of both growing seasons. Imidacloprid
equivalent concentration (IEC) was generally constant over time and between leaves
from opposing branches of the same whorl. As opposed to ash, a sectored species,
branch position did not affect IEC in maples. We conclude that maples trees have an
integrated xylem architecture pattern which should allow for even distribution of trunk-
injected imidacloprid in the tree crown. During 2007, the year after injection, IEC in

leaves from corresponding branches were similar to levels observed in 2006.

98

Imidacloprid equivalent concentrations in leaves, stems and litterfall were higher than
IEC in roots, trunk cores, and stems, indicating that imidacloprid moves primarily

through the xylem.

99

CHAPTER 3

Distribution of Trunk-Injected 14C-Imidacloprid in Acer spp. Trees:

A Test of the Sectored Flow Hypothesis.

INTRODUCTION

The Asian longhomed beetle (ALB, Anoplophora glabripennis Motschulsky
Family: Cerambycidae) is an invasive wood-boring exotic insect from Asia. It was first
identified in the United States in New York City, New York in 1996 (Haack et al., 1997)
and later in Chicago, Illinois in 1998 (Poland et al., 1998). Since that time ALB has been
identified in other major cities including Jersey City, New Jersey and Toronto, Ontario,
Canada (Wang et al., 2005). Additional beetles have been found in 26 warehouses in
several states across the United States (Nowak et al., 2001). The ALB is native to
Southeast Asia and China where it is one of the top ten economic pests due to the damage
it causes to Populus spp. plantations (MacLeod et al., 2002).

Asian Longhomed Beetle has a wide host range including Acer, Populus, Prunus,
Malus, Salix, Ulmus and at least 21 other tree species (MacLeod et al., 2002). In urban
landscapes in the United States, ALB prefers host trees in the genus Acer (Nowak et al.,
2001)). Susceptible trees make up as much as 62% of urban tree landscapes in some
cities (Chicago, Illinois) and canopy loss in urban areas may reach as high as 68% (Jersey
City, New Jersey) if all susceptible trees were to become infested. Chicago and New

York City alone cost of removing infested trees was $25 million (Nowak et al., 2002).

100

Economically, ALB could represent $669 billion in compensatory damage throughout the
48 contiguous states. Fruit tree, maple syrup, lumber, and seed production industries
could also be severely impacted (Ludwig et al., 2002).

Human assisted dispersal has led to ALB establishment in the United States,
Canada, and Europe (Haack, 2003). Normally, beetles stay on trees from which they
emerge or travel short distances to find suitable host trees (Wang et al., 2005). Adult
beetles feed on bark, young stems and occasionally leaves, but larvae cause the majority
of structural tree damage. First, second, and third instar larvae feed on nutrient-rich
phloem just beneath the bark surface and create galleries that disrupt nutrient and water .
translocation paths within the tree. In areas with high ALB populations, this can lead to
tree mortality within two to four years. As larvae grow and develop, they tunnel deeper
into the xylem heartwood of trees and create weak spots in branches or trunks.
Weakened branches may succumb to wind and snow load and cause injury to pedestrians
or damage vehicles, houses, and other property (MacLeod et al., 2002).

Because ALB larvae bore deep into the xylem, it is difficult to kill them using
chemical control (N owak et al, 2001). Imidacloprid, a neo-nicotinoid insecticide that
targets acetylcholine receptor sites in insects, is currently being applied as a trunk
injection to kill adults and larvae (Poland eta1., 2006a). Imidacloprid is a systemic
insecticide that has a high translocation rate (Wang et al., 2005). Thus far, ALB
infestations in the US are in urban areas. Imidacloprid applied as a systemic trunk-
injected insecticide has a higher degree of public acceptance than conventionally applied
cover sprays (Smith and Lewis, 2005) because it has very low mammalian toxicity and

few non-target affects (Poland et al., 2006b, Sur and Stork, 2003).

101

Imidacloprid acts initially as an anti-feedant. It is crucial to deliver a strong and
sustained dose of imidacloprid to successfully kill larvae and adults. Poland et al (2006a)
showed that larvae can survive and pupate under sub-lethal concentrations. Adults could
exhibit the same responses, or they might fly to an untreated tree and resume feeding
(Poland et al., 2006a).

To ensure that the maximum amount of insecticide is arriving at the feeding sites
of the larvae and adult beetles it is important to understand that translocation (and
therefore efficacy) of trunk-inj ected compounds is affected by a variety of factors
including temperature, tree species, tree injury, and water availability (Poland et al.,
2006a). Efficacy of trunk-injected imidacloprid is variable against ALB (Wang et al.,
2005). The amount of time that elapses between injection events might also affect
efficacy. Several studies have shown that imidacloprid concentrations increase during
the first year after treatment and then gradually decline (Tanis, this volume, chapter two).
The point on trees where injection takes place might also influence efficacy. Poland et al.
(2006b) suggest that xylem architecture influences translocation patterns and that
insecticides might be injected and translocated in such a way that it would have no effect

on developing larvae tunneling within branches. In a previous study at Michigan State
University, researchers used l4C labeled imidacloprid to track trunk-injected insecticide

movement in F raxinus spp. trees. The study demonstrated that imidacloprid moves
slowly but steadily through the tree over time and accumulates in the leaves (Cregg et al.,
2006). Variability in efficacy in ash could be due to uneven distribution of insecticide in
the canopy due to tree anatomy. Orians et a1. (2004), reported that trees can have varying

degrees of sectored or integrated flow. Sectored trees have highly ordered, specific paths

102

of translocation, while integrated trees have less specific paths of translocation. In
general, diffuse porous trees (e. g. maple) are considered more integrated than ring porous
trees (e. g. ash) (Zanne et al., 2006). Diffuse porous trees have evenly sized vessels. Ring
porous trees have variable sized vessels (Shigo, 1998). The difference results in
increased vessel to vessel contact in diffuse porous trees that allow resource movement to
occur in both radial and tangential planes (Zanne et al., 2006). This results in even
distribution of resources within the plant. Information about tree xylem anatomy would
be helpful to ensure that injection events are taking place on the tree in such a way that
would maximize the translocation of insecticide. Spatial variability in trunk-inj ected
integrated tree species should be less than in sectored species. We hypothesize that
maple trees have integrated xylem anatomy. .

Currently there are no insecticides that are 100% effective against ALB (Poland
2007). But if we can better understand the translocation of insecticide within trees we
might increase the efficacy of trunk-injected imidacloprid. Increased efficacy would
enhance ALB eradication efforts by ensuring that maximum amounts of insecticide are
reaching ALB feeding sites. Overall, understanding spatial and temporal flow of
insecticide through Acer spp. trees will enable us to improve efficacy of trunk-injection
treatments and better understand the persistence and potential for ALB control.

The objectives of this study were to determine the spatial movement of trunk-
injected imidacloprid in Acer spp. (maple) trees and concentration of imidacloprid in

maple trees the spring after fall-trunk-injection.

103

MATERIALS AND METHODS

This experiment was performed at the Horticulture Teaching and Research Center,
Michigan State University, Holt, Ingham County, Michigan. Trees were purchased from
Weigand’s Nursery and Garden Center, Macomb, Michigan.

The eight maple trees, four Acer x fi'eemanii ‘Jeffersred’ Autumn Blaze® and four
A. platanoides ‘Princeton Gold’ used for this study were 5 cm (2”) caliper, container-
grown specimens planted in a light weight pine bark media in 75 L (15 gallon) (A. x
freemam'i) or 95 L (25 gallon) (A. platanoides) containers. They were individually
selected to ensure that the first three branch whorls were oriented at 90° angles to one
another. Containers were spaced 2.5 m (8 ft) on center, placed inside 1.2 m plastic
wading pools and elevated on a single layer of brick pavers. This allowed for drainage

and kept leachate from entering into the environment. Trees were staked to prevent

wind-throw. Trees were fertilized with Osmocote® Plus (The Scott’s Company,

Marysville, Ohio) controlled release fertilizer at a rate of 314 grams per pot and watered
to runoff with drip irrigation two to three times per week.
Trunk Injection:

On 5 September 2006, trees were trunk-injected at a single injection point with
2.94 ml of 25 uCi of 14c labeled imidacloprid mixed with rmicide® (Mauget®, Arcadia,
California, 10% active ingredient) in a ratio of 1:1300 labeled to unlabeled compound.
Half of the trees were injected at 0° to the first whorl of branches (i.e. the injection port

was directly below one of the branches on the first whorl) the other half was injected at

90° to the first whorl of branches (Figure 3.1a.). Injection positions were assigned

104

randomly within species. Injection holes were drilled 10.0 cm above the graft union with
a cordless drill and an 8 mm (5/ 16”) drill bit. Holes were drilled to a depth of
approximately 1.5 cm and systemic tree injection tubes (STIT) were inserted and gently
tapped in with a hammer (Helson et al., 2001). After tubes were inserted, 2.94 ml of
imidacloprid mixture was added through the top of the lower half of the STIT and 2.0 bar
pressure was applied using a hand operated bicycle pump. All lower components of the
STIT were securely wrapped with black electrical tape to ensure that leakage was
contained and to provide stability. The upper halves of the STITs were also secured to
trees with tape. The STITs were monitored for pressure, re-inflated as needed and kept in
position 2-48 hours until all liquid was taken up by the tree. After all liquid was
translocated into the tree, tape and STIT were carefully removed. The area of the tree that
contained the drill hole was double-wrapped with electrical tape to prevent leakage or
leaching of radioactive material. Tape remained in place throughout the experiment.
Two trees were removed from the experiment due to excessive leakage and bark cracking
during the injection process. All handling of radioactive material complied with
Michigan State University’s Office of Radiation, Chemical, and Biological Safety
(ORCBS) standards. All areas were surveyed periodically using a Geiger counter
(Ludlums Measurement Inc., Survey Meter Model 44-9, Sweetwater, TX) to ensure that
no radioactive material leached into the environment.
Sampling:

Branches from the first three branch whorls of each tree were labeled either 0°,
180°, Right 90° (R90°) or Left 90° (L90°) depending on the position of the branch in

relation to the injection point (Figure 3.1b). Leaf samples were obtained by pinching off

105

individual leaves from proximal, middle and distal portions of each of the six branches.
Terminal leader leaf samples were removed with a pole pruner. Trunk cores were taken 1
m above the injection point at 0° and 180° (in relation to the injection point) using a #1
cork borer. The borer was pushed into the tree to remove approximately 2 m depth of
tissue (bark, phloem, and sapwood). Root samples were obtained by digging into the soil
media until a portion of the root system was exposed, and then approximately 1 g dry
weight of fine roots were removed from coarse roots and rinsed with tap water. Stem
samples approximately 3 cm in length were taken from branch tips of the first three
whorls using hand pruners. All samples were placed in small paper bags, labeled and
placed in a 70° C drying oven for at least 72 hours. After drying, all leaf samples were
removed from the oven, ground to a fine powder using a mortar and pestle and placed
into individually labeled paper envelopes. Root, trunk core, and stem samples were left
whole.

A 150.0 - 180.0 mg sub-sample of tissue was burned in a R]. Harvey OX300
biological tissue oxidizer (R. J. Harvey Instrument Corporation, Tappan, NY, USA).

Stern samples were burned on a four minute cycle, leaf samples on a three minute cycle,

and root and trunk core samples on a two minute cycle. The resultant l4C02 was trapped

in scintillation cocktail and processed through a Packard Tri Carb scintillation counter
(LSC, Packard Bioscience, Currently PerkinElmer Life and Analytical Science, Waltham,
Massachusetts, USA) on a one and one half minute cycle. Resulting counts per minute
(CPM) were recorded for each sample and converted to imidacloprid equivalents after
accounting for background activity and oxidizer and LSC efficiencies. Imidacloprid

trunk-injected into plants is broken-down into a series of metabolites many of which

106

exhibit insecticidal properties (Sur and Stork, 2003); therefore we refer to the
concentration of insecticide in trees in terms of imidacloprid equivalents. All results are
presented in ug of imidacloprid equivalents per g dry weight of sample.

For the 2006 growing season, trees were sampled at 0, 2, 7, and 21 days after
treatment. The sampling protocol included leaves from the first three branch whorls,
terminal leader leaves, trunk cores, and fine roots. In late September, all trees were
netted with bird netting to collect litterfall. After abscission, all leaves were removed
from nets, placed in paper bags, and oven dried. A sub-sample of five to seven leaves
were ground, weighed, oxidized, and processed through the LSC. After nets and leaves
were removed, trees were lifted with a sling and skid steer and placed pot to pot in a
holding area. A double layer of hay bales was placed around the holding area to protect
roots from freezing temperatures over winter. In April 2007, trees were transferred into
new 95 L (25 gallon) pots, and topped off with pure sand to add weight to help prevent
wind—throw. After transplanting, they were lifted with the sling and skid steer and
placed back in their original configuration. Trees were fertilized and watered at the same
rates in 2007 as in 2006.

For the 2007 growing season, trees were sampled for whorl and terminal leader
leaves, and fine roots on 29 May, 12 June, and 31 August. Trunk cores were not taken in
2007 to prevent excessive damage to the trees. At the end of the 2007 growing season,
all leaves were removed from trees by hand. Leaves from the first three whorl branches
were kept separate from the rest of the canopy leaves.

Stem sections from branches in whorls one through three and terminal leaders

were removed in late October 2007 from branch tips and separated into 2006 and 2007

107

growth sections as determined by the position of axillary leaf scars. A sub-sample of five
to seven leaves from canopies, terminal leaders, and whorl branches were dried, ground,
weighed, oxidized, and processed through the LSC.

Pour through leachate tests were performed on 4 June, 5 July, 15 August, and 15
September 2007. Pot media containing the maple trees described above were saturated
with water and allowed to drain for ten minutes. After drainage, 2 L of fresh water was
added to the media and allowed to drain. Leachate was captured through drainage holes
at the bottom of pots with a Petri dish, poured into 15 ml scintillation vials, sealed, and

brought back to the laboratory. Immediately, 300 11L of leachate was added to 15 ml of

scintillation cocktail (Safety-Solv® Scintillation Cocktail, Research Products

lntemational Corporation, Chicago, IL). Samples were hand shaken and processed
through the LSC.
Statistical Analysis:

Data were tested for normality using residual plots. Variables were not normal.
Imidacloprid equivalent concentrations were transformed using a log (x+2)
transformation. Differences were analyzed using the Mixed procedure for mixed models
in SAS statistical software (SAS Institute, Inc. 1989) with whorl nested in tree, branch
position nested in whorl, tree species, and day as fixed effects and tree number as a
random effect. The models for repeated measures were examined using CS covariance
structure. Analysis of variance (ANOVA) was used to determine which effects and
interactions were significant (p<0.05) for whorl, branch position, tree species and day.
Different ANOVAS were produced for each injection position (0° or 90°). If effects were

significant, LSMeans procedures were used for mean separation (Littell et al., 1996).

108

RESULTS

Imidacloprid equivalent concentration in Acer spp trees was not affected by whorl
height, branch position, days after treatment, or species. In general, leaf IEC from 0°
branches did not increase over time with the exception of 2007 litterfall. Leaves from
branches opposite the injection point (180°) in the first whorl had increasing IEC through
21 DAT, while leaves from other 180° branches did not increase or decrease. Leaves
from 0° and 180° branches in whorl one had similar IEC, although IEC in 2007 litterfall
from 0° branches was higher than litterfall from 180° branches. Imidacloprid
concentrations did not vary between leaves from R90° or L90° branches during 2006 or
2007. Leaves from terminal leader branches had increasing IEC through 7 DAT but no
other variation in IEC occurred in 2006 or 2007 (Figure 3.2).

Roots from 2006 and 2007 and trunk cores from 2006 (Figure 3.3) and leachate
samples (data not shown) contained no measurable IECS. Stem samples taken from the
first three branch whorls at the end of the 2007 growing season did not exhibit any
systematic pattern of variation in IEC among branches although IEC of stems from 0°
branches were generally higher (p<0.05) than terminal leader stems (Figures 3.4a and
3.4b). Imidacloprid equivalent concentrations were higher (p<0.05) in leaves than in

stems at a given branch position.

109

DISCUSSION

In the United States, trunk-injected imidacloprid is used to treat several species of
trees against Asian longhomed beetle. We chose to use maple trees in this study because
they are planted extensively in urban areas, are a preferred ALB host tree in the US, and
have opposite branches which aids in determining sectorial xylem patterns. The primary
objectives of our study were to determine patterns of imidacloprid movement within
maple trees and imidacloprid levels in leaves from fall-injected maple trees the spring
after injection.

Objective One:

Patterns of sap ascent in trees normally deviate from purely straight and are often
described as helical (Tyree and Zimmerman, 2002). In the case of maples however, we
did not see any patterns of helical or straight ascent. Imidacloprid equivalent
concentration in leaves did not vary between opposite branches from the same whorl
throughout either growing season although some isolated incidences of IEC difference
were apparent at 2007 litterfall.

Ring porous trees (e. g. ash) are generally considered sectored while diffuse
porous trees (e. g. maple) have greater integration in their xylem anatomy due to more
contact between vessels (Shigo, 1988). Most trees in the Acer genus are classified as
diffuse porous (Zanne et al., 2006). The results of this study also suggest that Acer x.
freemanii and Acer platanoides have integrated xylem pathways.

Poland et al (2006a) suggest that xylem architecture could affect the amount of

insecticide moving through a tree and whether insecticide is translocated to ALB feeding

110

sites. As seen in a related study on- ash trees (Tanis, chapter two, this volume), sectored
xylem anatomy can cause uneven distribution of insecticide in the tree crown. Leaves
from branches opposite the injection point (180°) had much lower IEC than leaves from
branches in the plane of the injection point (0°). Patterns of sectoriality were also evident
in stems. Integrated xylem anatomy on the other hand, may contribute to more even
distribution of insecticide within the tree. Integrated xylem pathways are more
interconnected than sectored pathways so injection point and branch position may not
affect overall distribution of insecticide within the tree because movement is occurring in
both longitudinal and radial planes. This could be a result of the area of vessel walls as
bordered pits (Ellmore et al., 2006). In this study, imidacloprid equivalents moved in
integrated patterns throughout 2006 and 2007 growing seasons in trees injected at both 0°
and 90° degrees. This suggests that insecticide was evenly distributed throughout the tree
canopy. In contrast, imidacloprid moved through ash trees in a sectored zigzag pattern.
There were no patterns of sectored flow in maples during either growing season. As
seen here, injection site and branch position might not be as critical when performing
trunk injections on maples as they are when trunk-injecting ash trees or other strongly
sectored species.

In general, we found that imidacloprid equivalent concentration appears to
increase over time. However, differences in IEC from date to date were not statistically
different due to high variation among trees that is probably a result of small sample size.

In addition to leaves, imidacloprid equivalents were also present in stems. We did
not sample stems more than once to prevent excess tree damage. Like ash, the leaves and

stems of maple trees shared similar integration patterns. The lack of pattern supports our

111

theory that maple trees have integrated ascent. It is important to understand imidacloprid
movement within stems because late instar ALB larvae feed and tunnel in the xylem. In
this study we determined that IEC in maple stems is not affected by species, whorl height,
or branch position and therefore were evenly distributed throughout the tree. In ash trees,
sectoriality patterns were evident in both leaves and stems (Tanis, Chapter two, this
volume).

Several studies have demonstrated that imidacloprid movement in plants occurs
via the xylem (Bromilow and Chamberlain, 1989, Sur and Stork, 2003, Tanis et al., 2007).
Imidacloprid was present in measurable concentrations in leaves and stems but no
measurable IEC was present in roots or trunk cores. If imidacloprid movement was
occurring in phloem, these tissues would have IEC > 0. To further investigate IEC in fine
roots, we also performed pour-through leachate tests to determine if fine root turnover
from trunk-injected trees could contribute to imidacloprid residues in soil. No detectable
imidacloprid equivalents were present in collected leachate samples. The absence of
detectable IEC in fine roots, trunk cores, and water collected fi'om leachate tests suggests
that imidacloprid equivalents are not moving via the phloem from the injection point
down to roots or upward through bark. Instead, the presence of IEC in leaves and stems
supports previous studies that indicate imidacloprid moves through trees primarily in the

xylem.

. 4 . . . . .
In a prewous study that used 1 C to examine 1m1daclopr1d movement in ash trees,

Cregg et a1. (2006) demonstrated that canopy leaves collected after abscission contained
IEC as high as those sampled before abscission. In other words, imidacloprid was not

translocated from leaves back into the trunk before litterfall. This trend was also

112

apparent in maple trees used in this study. Generally, IEC in leaves from 2007 litterfall
were not different from IEC in leaves from other sample dates indicating that
imidacloprid is not translocated back into the tree before litterfall.

Objective Two:

Our second objective was to determine how much imidacloprid was present in
leaves from fall injected trees the year following injection. Some questions remain
regarding the timing of imidacloprid trunk-injections. Trunk-injections performed in
spring are advantageous because temperatures and transpiration rates are generally higher
than in the fall. These parameters promote quick translocation of insecticides to the tree
canopy. Fall injections are popular among tree care professionals because there is often a
lag in business opportunities during this time. Unfortunately, decreased temperatures and
transpiration rates during the injection process might hinder overall imidacloprid uptake
into stems and canopy leaves. One might argue that if less insecticide is lost during
litterfall, more insecticide might be available for translocation the spring after treatment.

In leaves from 0° and 90° injected trees, IEC on 11 June 2007 was approximately
35% less than levels found at the end of the growing season in 2006 (Figure 3.5). In
general, leaf IEC in maple trees was higher than IEC in ash leaves on both the final
sample date in the year of injection (26 September 2006), and the sample date that might .
correspond to periods when both insects are in active adult stages (11 June 2007).
Although the focus of our study was not to compare these two trees, it is helpfiil to create
a visual illustration of differences between sectored species (e. g. ash) and more integrated
species (e.g.maple). These differences were particularly evident on 11 June 2006 when

patterns of sectoriality were visible in ash trees and clear patterns of integration are

113

visible in maple trees. Degrees of integration/sectoriality are a result of xylem anatomy,
most likely the amount of vessel to vessel contact and/or area of walls as bordered pits.
Higher IEC in maple leaves when compared to ash leaves might be due in part to more
rapid insecticide movement. Integrated trees might be able to move imidacloprid more
efficiently from reservoirs at the base of trees up to canopy leaves when compared to
sectored trees, again probably as a result of differences in xylem architecture. Another
possibility is that increased movement of imidacloprid in maple trees might be due to
differences in leaf biomass between species. Orians et al., (2004) showed that dye ascent
can be affected by the amount of biomass associated with particular branches or sections
of trees. He also stated that differences in biomass effect on movement were different
between species. Highly integrated species like Betula papyrzfera Marsh were greatly
influenced by biomass partitioning. It is possible that there might be enough difference
between maple and ash leaf area to account for some of the differences in IEC but we
were unable to make that comparison in this study.

In summary, we have shown that imidacloprid equivalent concentration does not
vary in leaves or stems of maple trees based on the position of branches in relation to
injection points, species, or length of time after injection. The pattern of integration was
consistent through time. We were also able to show that imidacloprid is moving
primarily through the xylem and that translocation of insecticide does not occur from
leaves back into the trunk before litterfall.

An interesting follow up study might further examine the possible difference in
IEC of leaves and stems between spring and fall injections or mortality rates of live

larvae or adult beetles using parallel bioassays. Finally, we conclude that xylem

114

anatomy of Acer x. freemam'i and Acer platanoides allows for insecticide movement in
longitudinal, radial, and circumferential planes. This pattern of ascent allows for
integrated movement within the plant and even distribution of insecticide in the tree
crown at the rates applied. Based on this information, the position of trunk-injection is

not as important in integrated species as it is in sectored species.

115

 

 

Whorl 2

 

Whorl l

 

 

 

 

 

 

 

 

 

   

    

Injection Injection

Figure 3.1a: Treatment schematic, trees were injected at either 0° or 90° to the first
whorl of branches. Mean distance from injection point to the first whorl = 1.43 m.

Mean distance between whorls = 0.18 m.

116

 

 

 

. / Injection Points \ .-

    

 

 

 

 

    

Injection Injection

Figure 3.1b: Sampling schematic of trees injected at either 0° or 90° to the first
whorl of branches. Branches of the first three whorls were labeled 0°, 180°, L90° or
R90° in relation to the injection point. Mean distance from injection point to the

first whorl = 1.43 m. Mean distance between whorls = 0.18 m.

117

Figure 3.2: Mean l4C-Imidacloprid equivalent concentration i S.E. in whorl and

terminal leader leaves from 0° or 90° (in relation to the first whorl of branches) trunk-
injected Acer x fieemam’i and Acer platanoides trees. Branches were labeled 0°, 180°,

L90° or R90° (in relation to the injection point).

118

Imidacloprid equivalents rig-9'1

Acer spp. fall-trunk-injected at 0°

to the first whorl of branches.

 

 

 

 

 

 

 

 

 

 

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119

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Figure 3.3: Mean l4C-Imidacloprid equivalent concentration is S.E. in whorl one leaves

and stems (0°, 180°, L90° or R90°, in relation to the injection point), trunk cores (0° or
180°, in relation to the injection point) and roots of 0° and 90° (in relation to the first
whorl of branches) trunk-injected Acer x fieemanii and Acer platanoides trees, samples

collected during destructive harvest. Inset graph has leaf data bars removed.

120

Figure 3.4a: Mean l4C-Imidacloprid equivalent concentration :t S.E. in 2006 and 2007

stem sections from 0° (in relation to the first whorl of branches) trunk-injected Acer x
fleemanii and Acer platanoides trees. Stems were harvested in October 2007 from the
first three branch whorls and labeled 0°, 180°, L90° or R90° in relation to the injection

point.

121

 

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122

 

Figure 3.4b: Mean 14C-Imidacloprid equivalent concentration :t S.E. in 2006 and 2007

stem sections from 90° (in relation to the first whorl of branches) trunk-injected Acer x
freemanii and Acer platanoides trees. Stems were harvested in October 2007 from the
first three branch whorls and labeled 0°, 180°, L90° or R90° in relation to the injection

point.

123

 

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2006 stem sections

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124

Figure 3.5: Mean I4C-imidacloprid equivalent concentration :t S.E. in leaves from the

first three branch whorls of 0° and 90° (in relation to the first whorl of branches) of trunk-
injected Acer x. fieemanii and Acer platanoides trees. Branches were labeled 0°, 180°,
L90° or R90° in relation to the injection point. Samples were taken 21 and 280 days after

treatment.

125

Whorl 3 - 180°

Whorl 3 - 0°

Whorl 2 - R90°
Whorl 2 - L90°

Whorl 1 - 180°

Whorl 1 — 0°

Whorl 3 - 180°

Whorl 3 - 0°
Whorl 2 - R90°

Whorl 2 - L90o
Whor|1 -1 80°

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26 September 2006
21 Days after Treatment

..'t"-..-_ Ash
— Maple

 

 

 

I

100

200 300 400

Imidacloprid Equivalents (pg 9'1)

 

 

 

11 June 2007
280 Days after treatment

c.5135? ‘ Ash
— Maple

 

 

 

100

200 300 400

Imidacloprid Equivalents (pg 9’1)

126

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