ABSTRACT
Title of Thesis:
NATIVE AND INDIGENOUS BIOCONTROLS FOR
AILANTHUS ALTISSIMA
Richard Thomas Gardner III, Master of Science, 2008
Thesis directed by:
Professor David Straney
Cell Biology and Molecular Genetics
Ailanthus altissima is one of the top invasive weed trees in North America. Native and
indigenous biocontrols consisting of insects and fungi were found in a unique series of
interactions in Maryland and Pennsylvania. The insects are Aculops ailanthii, an
eriophyoid mite and Atteva punctella, the Ailanthus web worm. Mimosa wilt, Fusarium
oxysporum, isolated from a mimosa tree, Albizia julibrissin, was successful in the
laboratory through two generations testing Koch’s postulates on Ailanthus seedlings.
Atteva punctella selectively feeds on male trees. Fusarium lateritium and/or Fusarium
solani may be sterilizing female trees through necrotic lesions, allowing herbivory on the
sterilized female trees by Atteva punctella. The carriers appear to be Atteva punctella and
Ambrosia beetles (Schall, 2007). Aculops ailanthii was found in the field and brought
back into the lab for a successful test of Koch’s postulates on seedlings. Using partial
biocontrol, control of Ailanthus appears to be possible.
NATIVE AND INDIGENOUS BIOCONTROLS FOR AILANHTUS ALTISSIMA
by
Richard Thomas Gardner III
Thesis submitted to the Faculty of the Graduate School of the
University of Maryland, College Park in partial fulfillment
of the requirements for the degree of
Master of Science
2008
Advisory Committee:
Professor David Straney, Chair
Professor Patrick Kangas
Marc Imlay, PhD
© Copyright by
Richard Thomas Gardner III
2008
FOREWORD
I decided to research invasive plants primarily because it is one of the few fields
in Biology that is still pure Classical Biology. With my gifts in understanding natural
systems and solving human caused problems within them, I was able to find potential
solutions for one invasive non-native plant and the underlying paradigms to potentially
solve similar problems with other invasive non-native plants. This research is a part of
my greater interest in protecting and preserving natural systems from human interference,
because the health of a nation is completely dependant on the health of its ecology as
every historian well knows. At the same time, as a parent, protecting the ecology protects
my children and their children through the many generations that lay ahead. Being an
ecologist, it is hard to separate the science from the need to act politically. Hopefully,
this research inspires not only a new approach to the problem of invasive non-native
plants, but also researchers to not only see the need to protect our ecosystems from the
ravages of intentional and unintentional human actions but also the need to act.
In the end this research is a mixture of hard work and luck. I knew the general
literature before I started. I happened to be walking one day and saw something not in
the literature and chose the plant I was going to study. Ailanthus altissima is one of the
few plants that fit into so many ecological paradigms so easily that it made studying it
straightforward. My committee could not have been better giving me the freedom I
needed to pursue the directions I saw best. So, I have been very lucky and at the same
time have worked very hard through three years in the field, the lab and the library.
Hopefully, the end product of this study was worth the effort.
ii
DEDICATION
This is dedicated to my committee who took in an orphan grad student and gave him both
the opportunity and the freedom to excel while following that which he thought was
right. Thank you to Dr. David Straney, Dr. Marc Imlay and Dr. Patrick Kangas.
To Annie, who had the faith and belief in me when I needed it throughout the past year.
A woman’s faith can make or break a man. When her faith in him is great, he cannot
help but be great. She has made a difference this past year.
iii
ACKNOWLEDGEMENTS
My many thanks to my ex-wife who gave me the financial ability to pursue grad school,
my children who have been patient with an absentee father while pursuing my MS, to
Viggi, Dr. Jay Stipes, Mark Schall, Dr. John Davidson, Dr. Doug Tallamy, Debbie
Norland, Christie Lockwood, KMTG and all the other people who were around to give
me advice, direction and support when I needed it.
iv
TABLE OF CONTENTS
List of tables
viii
List of photographs
ix
Introduction
1
Economic costs
1
Hypotheses
3
Enemy Release Hypothesis
4
Biotic Resistance Hypothesis
5
Invasional Meltdown Hypothesis
6
Novel Weapon Hypothesis
6
Invasiveness
7
Biological Control
10
Ailanthus altissima
15
Research Goals
18
Experiments and Results
23
Research Site Selection
24
v
Sample Designations
24
Sample Collection and Preparation
25
Stem inoculations
29
Root inoculations
31
Leaf inoculations
36
Web worm survey
39
Tree dissections
40
Aculops ailanthii
45
DNA analysis
49
Discussion
60
Conclusions
70
Appendix A
Major Sample Site Locations and Descriptions
72
Appendix B
Fungi Sampling Database
73
Appendix C
Medias, Buffers and Solutions
91
Appendix D
Fungi DNA Extraction and Identification
96
Appendix E
Fusarium Species Hosts
100
Appendix F
PCR Sample Identification
101
vi
Appendix G
Edited PCR Consensus Sequences
103
Appendix H
Selected Edited PCR Sequences Accession Data
109
Appendix I
NCBI Trees From Consensus Sequences
121
Bibliography
132
vii
LIST OF TABLES
Table 1. Results of stem inoculations started on
May 19/20 2007 and finished on July 14/15, 2007.
31
Table 2. Plant inoculation results from mimosa wilt collected
on July 30, 2007 near Goldsboro, MD
35
Table 3. Summary of Atteva punctella survey
on September 15, 2007
40
Table 4. Tree Dissection Data
42
Table 5. PCR data
56
viii
LIST OF PHOTOGRAPHS
Figure 1. Stand of Ailanthus altissima showing a dead tree,
Urbana Community Park, September, 2007.
19
Figure 2. Tray containing mimosa wilt inoculated plants at
the end of the experiment which ran from January 2, 2008 to
March 9, 2008.
34
Figure 3. Atteva punctella larvae.
38
Figure 4. Aculops ailanthii.
47
Figure 5. Tub setup for Aculops ailanthi experiment showing
infested leaves.
47
Figure 6. Aculops ailanthi infested seedlings in tub experiment.
48
Figure 7. Aculops ailanthi tub experiment control.
48
Figure 8. Run 10, a comparison of PCR samples 3, 12, 14
and 21 at 700 base pairs each, 3 mM MgCl2, and
annealing temperatures of 55 and 56 C.
51
Figure 9. Seed cluster showing premature seed drop.
66
ix
INTRODUCTION
“In natural areas, invasive plants reduce habitat for native and endangered species,
degrade riparian areas, create fire hazards, and interfere with recreational activities.
Aquatic invasive plants clog lakes and waterways and adversely affect fisheries, public
water supplies, irrigation, water treatment systems, recreational activities and shipping.”
(National Weed Strategy, 2000).
The health of a country depends on the health of its ecosystems. When a society
pushes the carrying capacity of their ecosystems to the limits, a small disruption is all that
is necessary for the ecosystems and immediately thereafter, the society, to collapse. In
North America, both the Mound Builders of the Ohio Valley and the Anasazi of the
Southwest can trace their decline to environmental collapse due to their ecosystems
becoming fragile from overuse and poor stewardship followed by an environmental
disruption such as a drought.
In the same way genetic diversity is essential for the health of a species,
biological diversity is essential for the health of the ecosystem. Plant and animal
invaders reduce the diversity and hence the viability of an ecosystem.
Economic costs
What are the economic costs of non-native invasive plants? The estimates are
generally based on loss of economic benefits such as the cost of agricultural chemicals,
loss of pasturage and grazing land, plugged waterways and the amount of money spent on
cleaning up major problems (Nat. Weed Strat., 2000). European purple loosestrife and
leafy spurge costs $45,000,000 and $144,000,000 per year according to an estimate found
1
in State Legislatures (2000). Spotted knapweed alone costs $40,000,000 per year in
control costs and covers 45,000,000 acres (Alper, 2004). In Idaho, $12,700,000 is lost
annually due to star thistle (Julia, et al., 2007). Pimentel et al (2000) estimate 50,000
invasive organisms which cause $137,000,000/year in damage. This estimate is probably
low. According to the National Weed Strategy (2000) and Record of Decision (2005),
over 133,000,000 acres are infested with exotic pest plants growing at over 3,000,000
acres/year. In 1993 the costs were estimated at over $6 billion/year in direct and indirect
costs (Nat. Weed Strat., 2000). At the same time the habitat of 2/3 of all endangered and
threatened species are affected.
Beyond the obvious economic costs associated with agriculture and agronomy,
there are intangible human costs such as compromised view sheds, eroded land,
destroyed recreational waters and recreational fisheries put into jeopardy. Any time
people think of kudzu (Pueraria montana var. lobata), they envision a very specific
image of a landscape covered in a suffocating green blanket. The image of hydrilla
(Hydrilla verticillata (L. f.) Royle) is the same, except of waterways totally plugged with
vegetation. The personal costs mean a landscape covered with exotics, hunting affected
by unnatural ground cover few species use, running through wooded areas where
multiflora rose (Rosa multiflora) rips at the skin and clothes, hiking with mile-a-minute
vine (Polygonum perfoliata) tearing the ankles, fishing holes plugged with weeds, the
water anoxic, rivers where the canoeing is challenged by mats of aquatic vegetation and
the long open marsh landscapes shortened by phragmites (Phragmites australis), no
longer habitable to ducks, herons, egrets and many other species of birds and animals.
2
The Hypotheses
An important starting point is the definition of a native versus non-native plant.
The generally accepted definition of a non-native organism is one that was introduced
since Columbus landed in the New World. In other areas of the world the definition
shifts to a similar significant historical time marker. A naturalized plant is a non-native
plant that has either passed through the invasive stage and is now under control of the
ecosystem or one that has lived in an ecosystem without having become invasive. This is
an important difference in that although the goal in the Americas is a pre-Colombian
ecosystem the immediate goal is to control and where possible eradicate the most
aggressive invaders. Therefore, even though every non-native plant has the potential to
become invasive, the immediate targets are the ones severely shifting the balance of the
ecosystem away from the pre-invasion conditions.
The term indigenous may mean three basic concepts; a native organism, an
organism which is a naturalized non-native or any native or non-native organism which
may be found in the local ecosystem. My use of the word is towards the third definition,
any native or non-native organism which can be found in the local ecosystem. I prefer
the latter, because it allows for non-native generalist and specialist herbivores which may
still be of specific use against the targeted exotic plant.
My work is framed by four concepts which taken together show a picture of
invasion, both from the actions of the invader and the actions of the ecosystem. Even
though I do not subscribe to the Gaia view of the world, I find in Nature that there is
balance, with often opposite and superficially conflicting ideas existing at the same time.
3
Using the Enemy Release, Biotic Resistance, Invasional Meltdown and Novel Weapon
hypotheses I was able to get a fuller understanding of the uniqueness of Ailanthus
altissima, how it became invasive, how to find potential biocontrols and a broader
understanding of how to control it. I was very fortunate in the plant I chose because all
four hypotheses relate directly to it.
Enemy Release Hypothesis
The Enemy Release Hypothesis (ERH) is the concept that when a new species
(exotic), is introduced to an ecosystem it has an advantage over the native organisms
because it does not bring its total “enemy” load with it (Clay, 2003). In the case of plants
which are primary energy producers in an ecosystem, this may mean hundreds of insects,
fungi, viruses, bacteria and animals are left behind. According to Doug Tallamy (2007)
the difference between a native and non-native plant’s insect load may be in the
hundreds. In a meta-analysis done by Mitchell and Power (2003; Clay, 2003) when
looking at viruses and fungi found that the load was 77% lower for plants in the new
ecosystem than in their original ecosystem.
This means that the energy spent fighting the diseases and herbivores which use it
as an energy source can be spent on growth and subsequently reproduction. This is a
tremendous advantage over native species in the same guild and native species it
competes with for the basic resources needed to grow and reproduce. I prefer to use the
term guild because it most accurately describes the place an organism has in the energy
flow within an ecosystem. Niche is like the term taxa, at the same too broad and too
4
narrow to be able to accurately describe relationships, in this case within an ecosystem
and the part an invader plays in it.
Biotic Resistance
Biotic resistance is the concept that native plants have defenses against
generalized herbivores and diseases in their home ecosystems. This is the other side of
the ERH. While non-native plants have fewer specialist predators and diseases in the
new ecosystem, they are more at risk to the new generalists because their defenses were
developed to combat specific enemies in their original ecosystem. At the same time,
generalist herbivores prefer plants that have not developed the physical and chemical
defenses specific to it, in other words non-native plants (Blossey, 1995; Parker and Hay,
2005; Parker, 2006). Note the difference between specialist and generalist herbivores and
diseases. Specialists, unless they have an unseen adaptation, will not usually be a
problem to new species at the time they are establishing in the new ecosystem. This is
enhanced if there are few or no close relatives to serve as a source of organisms which
may easily transfer to the exotic. So, this exotic plant may survive in the new ecosystem,
but will not flourish, leading to naturalization (Levine, 2004).
An example of biotic resistance was in an experiment by Parker and Hay (2005).
They fed a selection of native and non-native aquatic vegetation to two species of native
North American crayfish, Procambarus spiculifer and Procambarus acutus. The results
were a three times greater preference for the exotic vegetation over the native vegetation.
An interesting tug of war goes on when the ecosystem has both exotic plants and
their generalized herbivores with native plants and their generalized herbivores. The
5
native herbivores prefer the exotic plants and the non-native herbivores prefer the native
plants. (Parker, 2006) Each pair of organisms is working against the other pair.
Invasional Meltdown
People are the key to making a system invasible through what is called Invasional
Meltdown (Hobbs, 1995; Sakai et al, 2001; Hiero et al, 2005; Crawley et al, 1986).
Invasional meltdown happens when an ecosystem in disturbed to the extent that it is open
to invaders. In the past this has happened by a variety of measures. Development of
natural spaces into human spaces is the most obvious route. The landscape is full of
exotic plants introduced by scientists, horticulturalists and agriculturalists. Grasslands
and prairies are used to graze sheep, goats, horses and cattle. Woodlands are cut down.
Roads are built, railroads are laid down and dams are put cross rivers. All these make a
landscape vulnerable to exotic plants (Parker, 2006).
Novel Weapon
The next of the hypotheses framing my research is Novel Weapon. Novel
weapons are chemicals, physical strategies or physical structures which are unique to
exotic plants that in an ecosystem, giving the invader an advantage over the plants native
to an ecosystem. Ailanthus altissima has the two classic examples of novel weapons,
ailanthone, an allelopathic chemical and the ability to grow swiftly into the canopy,
shading and crowding out competitors (Heisey, 2003).
Together, these four concepts explain some of the essential dynamics in an
ecosystem when confronted by invasive plants (and animals). They are four aspects of
invasion, each with its part in how a plant enters an ecosystem, the barriers which must
6
be overcome and the strategies employed both by the invader and the ecosystem. In this
research on Ailanthus, each aspect of the plant’s relationship with the ecosystem was
examined to find a way to control it.
Invasiveness
“Invasiveness, not economic value must be the driver in determining whether a
plant is to be introduced into a new ecosystem.” (Reichard and Hamilton, 1997).
The primary premise with invasiveness is that any plant can become invasive
under the right conditions. The best way to handle an invasion is to prevent it
(Chornesky and Randall, 2003). If prevention fails, then early detection and treatment
are necessary to prevent a small problem from becoming an ecosystem disaster (Hobbs,
1995).
What makes a plant invasive? The most important trait is that it is associated with
human actions (Crawley et al., 1986; Hobbs, 1995; Sakai et al., 2001; Hiero et al., 2005).
Without human intervention of some form, in most cases, a plant does not easily spread
from its native range to a new one. The two most important human mediated ways
helping a plant to become invasive is by physically altering an ecosystem and by multiple
introductions of the plant (Sax, 2000). Nurseries, horticulturalists, farmers and scientists
have a long history of introducing plants for their own purposes and the plants either
escaping or being deliberately introduced into new ecosystems (Reichard and Hamilton,
1997).
The best chance a plant will survive to become invasive, outside of human
intervention or interference, is if the environmental conditions to which it is moving are
7
the same or similar to the conditions from which it came (Goodwin, 1999; Huston, 2004).
This allows the ideas contained in Novel Weapon and Enemy Release hypotheses to
present themselves.
The generally accepted traits associated with invasiveness are; 1) sexual and
asexual reproductive modes, 2) high propagule production and a way to spread the
propagules efficiently, 3) fast initial growth rate, 4) robustness in order to adapt to a wide
range of environmental conditions and 5) short intervals between large seed crops
(Rejmanek and Richardson, 1996; Heisey and Heisey, 2003; Sakai et al, 2001).
Rubus phoenicolasius and Ailanthus altissima have all these traits. The former,
Wineberry, has prolific clusters of fruits containing multiple seeds and can root from
runners. Ailanthus has ramets which grow from its roots. R. phoenicolasius has berries
eaten by birds, animals and people while Ailanthus has wind spread samaras. Both plants
grow fast and are found in a wide range of temperate habitats. Finally, both plants
produce heavy crops of seeds each year.
The phases of a plant becoming invasive are lag, log and control (Mack, 2000).
These phases correspond to Hobbs’ (1995) steps of the introduction, establishment and
the sudden notice ability of a plant which are similar to what Kolar (2001), Drake (2003)
and Levine (2003) saw. The first stage, lag, is low level. It is the stage where a plant
adapts genetically to a new habitat. In a sense it is the period of shake out of genotypic
traits/plants that are not fit for the new habitat and a build up of propagules to the point a
population explosion can occur. The second phase is when a plant goes from being a
small part of the ecosystem to dominating in the ecosystem. During this step people
8
generally begin to notice the change in the ecosystem. The last part is the phase when a
plant has developed enough predators and diseases that it is in control and becomes a
naturalized part of the landscape. No phase has a set time period as it differs with the
plant and environment.
The effects of invasive plants in the environment in regards to other plants are
they alter the light availability, nutrient cycling, water availability, soil chemistry and the
fire regime while co-opting pollinators and seed dispersers (Karban et al., 1989; Mack,
2000; Levine, 2003). At the same time, invasive plants limit the number of animals by
limiting the number of herbivores that use the exotic plant for food and the number of
herbivores, omnivores and predators that use the native plants for shelter. In essence,
only a small number of the specialist organisms that may use a particular plant for food or
shelter cannot use the exotic. (Parker, 2004; Tallamy, 2007) This magnifies up the
through an ecosystem limiting the number of organisms that depend on the herbivores for
food or animals that use the plant in other ways, impoverishing the biological diversity
(Mack, 2000; Levine, 2003).
Light availability is limited by plants either coming up earlier in the season,
growing faster or going into leaf earlier than other plants in the ecosystem, depriving the
natives of access to light. Ailanthus does this by having a growth exceeding its ecotone
competitors in its first few years of growth, outcompeting trees along the edges where
light is most available (Heisey and Heisey, 2003). Even though it goes into leaf later in
the growing season than most of its competitors it tends to form denser stands due to its
ramets which aid in blocking the light from slow growing trees. Honeysuckle (Lonicera
japonica) and kudzu (Pueraria lobata) overgrow the plants they use for support, cutting
9
off their access to light. Garlic mustard (Alliaria petiolata) comes up earlier in the season
and grows both higher and thicker than many native plants, preventing access to light.
Nutrient cycling is altered by the acquisition and storage of nutrients, especially
nitrogen. Non-natives either gather nutrients more efficiently, change the chemistry of
nutrients or store them in ways that make the nutrients unavailable to native plants.
Water availability is usually changed by taking in more water than the native plants or
changing the water retention qualities of the soil (Imlay, 2008), making the soil less able
to support those plants. Soil chemistry may be changed either by the removal of certain
substances from the soil, the addition of substances to the soil or the concentration of
substances in the area around the plant. Ailanthus and other allelopaths add chemicals
which either kill or limit the growth of other native plants. The fire regime is altered by
changing the intensity or frequency of fire (Karban et al., 1989). From informal
conversations with several people, Japanese stilt grass, Microstegium vimineum (Trin.)
Camus, is said to extremely flammable both living and dead. Pollinators and seed
dispersers can be co-opted by either maturing earlier or offering something other plants
do not offer such as more nectar, sweeter nectar or a scent that is more attractive than the
native plants offer (Karban et al., 1989). Taken together these changes to a native
system, move it to favor the invasive plants instead of the natives.
Biological Control (Biocontrol)
“Effective study in the native range to identify potential agents underpins all
efforts in classical biological control of weeds. Good agents that demonstrate both a high
10
degree of host specificity and the potential to be damaging are a very limited resource
and must therefore be carefully studied and considered.” (J.A. Goolsby, et al., 2006)
Biological control is a broad term that can encompass a variety of meanings. The
definition used in this research is the one by Waage and Greathead (1988) and Jutsum
(1988) as to the use of living organisms to control pests. The definition of pests is also
broad. In reference to this research it refers to invasive, non-native plants.
Partial Biocontrol is the combines use of biological and non-biological means to
control an invasive non-native plant. Tiourebaev et al. (2001) in his study used Fusarium
oxysporum f.sp. cannabis along with insects and mechanical means to gain some success
over Ditchweed, Cannabis sativa, in Kazakhstan. Hobbs (1995) suggests using
chemicals with biocontrols. Weakening the plant with either a biological control or a
chemical and then using the other to finish the process is a valid approach. The use of
biological controls to start controlling an invasive plant where there is enough plant
density to make the biological control effective with mechanical or chemical follow up is
another valid technique. This combination of methods is probably what needs to be done
with Ailanthus. Stands are ideal for biological control due to their density the near
proximity of target trees to each other and its clonal nature. Single trees need to be taken
care of with either mechanical or chemical methods.
One important distinction needs to be made. Classical or traditional biocontrol is
the introduction of a non-native species for the control of another non-native species.
Generally, this means the introduction of apparent specialists which feed specifically on
the target plant. In the past there have been mistakes. One of the most published is the
11
weevil Rhinocyllus conicus. It jumped from the non-native Canada thistle, Cirsium
arvense, it was introduced to control to native thistles in the Cirsium genus (Louda,
1997). That both the native and non-native are the same genus should have served as a
warning that the introduced R. conicus was apt to jump to a native in the same genus
instead of exclusively feeding on its intended host. The vast majority of biocontrol
attempts use specialists from the invader’s native ecosystem to control the invader. This
is where the problem has arisen with native biocontrols, to this point it has been very
difficult to find or develop either biocontrols specific to an invader or finding a generalist
which will minimize the damage to the local ecosystem while still controlling the
invader. Since the disaster with Canada Thistle, regulations have been put in place both
in the United States and Canada to minimize the possibility of another similar scenario.
In the Discussion section, I offer an effective way of finding effective native biocontrols.
When I developed my original research idea about using native or indigenous
organisms to control non-native organisms, my intent was to raise phytophagus insects
through enough generations that by slowly introducing an invasive into the diet,
eventually a generation of specialists to that invader would develop. However, this
changed as the research developed to the point that field work consisted of identifying
potential biocontrols instead of developing specialists and lab work revolved around
testing Koch’s postulates of what was found in the field. Therefore, attempts at raising
specialist insects were discarded, even though this may have been the right approach for
many situations.
Fungi used as mycoherbicides have been very successful due to their often being
host specific (Fravel, 2003). Some of the successes using fungal pathogens are: Senna
12
surattensis controlled by Acremonium sp., Ageratina riparia controlled by the white smut
Entyloma compositarumfrom, Clidemia hirta controlled by Colletotrichum
gloeosporioides f. sp. clidemiae, Passiflora tarminiana controlled by Septoria
passiflorae, Lantana camara controlled by Septoria sp., Sclerotinia sclerotiorum used to
control Cirsium arvense, Rottboelia cochinchinesis partially controlled by Sporisorium
ophiuri, Phytopthora palmivore controls Morrenia odorata, Fusarium oxysporum f.sp.
cannabis is used to control Cannabis sativa, Puccinia chondrilla controls Chondrilla
juncea, Fusarium oxysporum with Fusarium arthosporioides to control Orobanche in
Israel and Fusarium oxysporum from Striga hermonthica used to control S. hermonthica.
(Jutsum, 198; Hasan and Ayres, 1990; McEvoy et al., 1991; Smith et al., 1997; Anselm et
al., 2001; Hurrell et al., 2001; Nekouam et al., 2006). Colletotrichum, Gloeosporioides f.
sp. clidemiae, is now a commercial product called “Collego” (Hasan, 1990). Rottoboellia
cochinchinesis worked best where the density of the target plants was high (Smith et al.,
1997). S. sclerotiorum efficacy was affected by the time of the season and the leaf
moisture relative to application (Hurrell et al., 2001). Whereas Hasan and Ayres (1990)
suggest that use of locally found fungi to control an invasive weed is best when done as a
flood or inundation to completely overwhelm a pest plant’s defenses. These strategies
are also contingent on the density of the non-target plants which may be accidently
affected.
The use of both insects and fungi is another idea. When insects and fungi are
working in combination, the fungi are usually carried by the insects and deposited on or
in the target. This is suggested by Rayachhetry et al. (1996) in regards to combating
Melaleuca quinquenervia in Florida where the insect bored into the trees and left
13
Botryosphaeria ribis to cause cankers and other damage. Another possible insect
mediated way of infecting a plant with fungi is when an opportunist fungus is on a plant
at a level such that if the plant is wounded sufficiently by an insect, the fungus could
move inside the plant, causing disease. This is in line with the thinking of Hasan and
Ayres (1990) in relationship to water hyacinth control in Florida. Two weevils,
Neochetina eichornia and Neochetina bruchi are producing feeding wounds on Eichornia
crassipes that allows phytopathogenic fungi to enter. Wallin and Raffa (2001) and
Caesar (2005) suggest that the more severe the defoliation, the more severe the fungal
infection. This is a synergistic way to provide biocontrol, defoliation by the insect and
infection with the insects feeding providing the way for a fungus to enter the plant. This
increases the severity of the infection through fluid loss from the wounds and decreasing
the amount of energy available due to the loss of photosynthetic tissue. This suggests
that an herbivore and a fungus in one of several combinations may be the most effective
way to solve Ailanthus altissima.
To this point, there have been very few native biocontrols known to be successful.
Admittedly, a lot of this falls under the Biotic Resistance Hypothesis which limits the
perceived number of biocontrols. Perhaps a late developing native biocontrol is only an
example of Biotic Resistance removed in space or time from the original introduction of a
plant. This could be due to density of the biocontrol being too low to function as a
control, the lag time needed to adjust to a new food source, the biocontrol, although
native to a larger system, has not had the opportunity to migrate to the new food source or
the biocontrol being separated by seasonal time of appearance from the time needed to be
14
effective against the exotic. These concepts of space and time are explained further in the
Discussion.
One of the best examples of a native biocontrol used to control an exotic is found
on Eurasian watermilfoil (USDA, 2002). Myriophyllum spicatum L. is a swiftly
spreading aquatic weed with many traits associated with invasiveness, such as both
asexual and sexual reproduction. Euhrychiopsis lecontei, a native phytophagus weevil
has had some success in controlling this plant having moved from the native water milfoil
Myriophyllum sibiricum. Hope lies in the fact that those E. leconti larva which are
offspring of adults raised on the local version are more likely to feed on the non-native
M. spicatum than on the native (USDA 2002). A native specialist phytophagus insect
moves from one member of a family of related plants to another (Ding et al., 2006). The
problem in this case is that both plants have an overlapping range. There is the
possibility that both plants will be equally used and the invasive will not be eradicated.
However, there is also the strong possibility that Biotic Resistance will cause the native
phytophagus insect to prefer the defenseless exotic over the native.
Ailanthus altissima
Ailanthus altissima is one of if not the most invasive non-native deciduous tree
species in the United States. It was introduced on the East Coast in 1784. The West
Coast introductions are supposed to have been done by Chinese in the 1800’s as they
emigrated from China (Hu, 1979).
Hu (1979) and Kowarik and Saumel (2007) both give excellent descriptions of
Ailanthus altissima. The major physical features are that it is dioecious, clonal and an
15
ecotone plant. The last of these features means it is a plant that thrives on disturbance,
especially human mediated disturbance. It grows fast early in its life, up to 3 meters/year.
Seed production may be over 350,000 seeds/year. It is generally accepted that the seed
bank is one year under normal conditions, even though under lab conditions the seeds
may survive several years (Krussmann et al., 1981).
Seeds are samaras which can travel upwards of 450 meters in the right conditions
(Kowarik and Saumel, 2007). Highways serve as channels for air flow, due to their
smoothness and long straight uninterrupted stretches compared to the rest of the
landscape. The laminar air flow moves the samaras distances not possible in a rougher
natural landscape. A paper by Kowarik and Samuel (2007) suggests similar in reference
to railroads. While, the air flow over the rest of the landscape is much rougher and more
chaotic, not conducive for seed travel. At the same time, railroads and highways are
constantly being disturbed by maintenance, construction and accidents giving
disturbance-oriented plants the opportunities they need to colonize a new area both due to
the landscape being disrupted and as hitchhikers on the equipment and personages
involved, moving from one colonizing opportunity to another.
Ailanthus has four traits which make it an invasive threat to the local ecosystems.
First, Ailanthus is a disturbance and ecotone plant, easily colonizing the disturbed edges
of both intact and compromised ecosystems. This is in accordance with the Invasional
Meltdown Hypothesis. Second, along the lines of the Enemy Release Hypothesis, the
number of known herbivores and pathogens is much lower than in native trees such as
red oaks or sassafras, allowing it to spend less energy on defense mechanisms with more
energy on growth and reproduction. Third, it grows much faster than most native trees.
16
Lastly, Ailanthus is known for its alleopathy towards its plant competitors. These last
two traits are aspects of the Novel Weapon Hypothesis.
From a biocontrol perspective, the most important facts are that it is dioecious and
has a clonal mode of reproduction using propagative roots having ramets. Being
dioecious is the tree’s greatest weakness. Elimination of one gender, will suppress the
tree’s reproductive ability. In this case, elimination of the seed bearing trees combined
with the tree’s one year seed bank (Krussmann et al. 1981) suggests the possibility that if
a pathogen or phytophagus insect is introduced at a high enough density, a few years is
all that is required to eliminate the tree from large stands. Single trees and isolated stands
will still need to be removed by chemical or mechanical means.
The asexual reproductive mode means that the root barriers normally formed by
the roots of other tree species in heterogeneous woodlands are lacking, making the flow
of a pathogen from one tree to another possible either directly through the attached
reproductive roots or root grafts (Garrett, 1981; Burdon and Marshall, 1981). The clonal
reproduction further means that a stand of Ailanthus may be lacking in genetic diversity,
allowing a pathogen or phytophagus insect to infest a whole stand without different trees
within the stand having the ability to resist the pathogens or insects (Sakai et al., 2001).
The clonal reproduction via propagative roots, offers a large potentially dense target for
pathogens or phytophagus insects to locate. According to Redlin and Carris (1996), there
is a density dependant relationship between endophytic fungi and trees. Increased
density helps this relationship. This is further expanded by Karban et al. (1989) who
state that in cotton the induced resistance decreases as the density of cotton plants
increases. Wild conditions imitate Integrated Pest Management techniques which depend
17
on the high degree of diversity within a garden or farm to deter pests. Going in the
opposite direction, a monoculture, especially one based on interconnected clones, invites
problems.
Research Goals
There are no known biocontrols for Ailanthus altissima (Webster et al., 2006;
Blossey, private communications, 2007).
The only goal of my research and the reason it is so broad was to find at least one
biocontrol for Ailanthus altissima in the local ecosystems. In part, this research is based
on the research done by Dr. Jay Stipes at Virginia Polytechnic Institute and State
University using Fusarium oxysporum to control Ailanthus altissima. At the same time,
instead of narrowly focusing on endophytic fusarium phytopathogens, I chose a larger
focus, looking broadly for native or indigenous biocontrols for Ailanthus altissima.
Ailanthus altissima among the most invasive non-native trees in the United States.
It was introduced on the East Coast in 1784. The West Coast introductions are supposed
to have been done by Chinese in the 1800’s as they emigrated from China (Hu, 1979).
Until recently with the work of Tony Emmerich, a New York State forester and Dr. Jay
Stipes, biological control of this plant was considered impossible. More recently, Mark
Schall at Penn State and this work at the University of Maryland have investigated
potential biocontrols. Mark’s research is based on reports of massive diebacks of the
trees in state forests in Pennsylvania. This is similar to reports from the New York City
area by Tony Emmerich during the late 1990s and is reminiscent of similar in
Philadelphia during the 1920’s (Emmerich, 1998; Sinclair and Lyon, 2005). Both
18
Emmerich and Schall came to the same conclusion of the cause being either Verticillium
albo-atrum or Verticillum dahliae. Schall’s recent field work has shown that it is
possible to use V. dahliae in a direct tree injection to kill mature Ailanthus trees (Schall,
2008).
Figure 1
Stand of Ailanthus altissima showing a dead tree, Urbana Community Park, September,
2007.
19
The research looked at two basic taxa of organisms, phytophagus insects and
endophytic fungi. In the process my goal was to show that it is possible to use native
and/or indigenous organisms as biocontrols instead of importing organisms and therefore
importing potential problems from outside the local ecosystems.
Field observations over two years showed tips of saplings which were dead,
wilting, chlorosis and necrotic lesions. Originally, based on the work of prior
researchers, I thought I was seeing the results of endophytic pathogenic fungi. Therefore,
I started to culture apical necrotic sections of sapling trees and necrotic lesions from
trunks looking primarily for phytopathic fusarium fungi. In particular I was trying to find
F. oxysporum perniciosium as suggested in the work of Dr. Jay Stipes. This lead to doing
root dipping and stem inoculations on seedlings. The original round of these experiments
in May and June 2007 was quickly infested with Aculops ailanthi, a mite which was
accidently introduced from the field. The symptoms were the same as expected from a
wilt pathogen, wilting and drying of leaves leading to the death of the plants. This
invalidated the root inoculations and affected the validity of the stem inoculations.
The stem inoculations were discontinued at this point because they did not appear
to be a control. Another round of root dipping inoculations was run parallel to an
experiment involving Aculops ailanthi in a separate laboratory during the fall of 2007.
Included this time was a set of endopathic wilt fungi collected from a wilting mimosa
tree. The mimosa wilt was included because the infectious agent, F. oxysporum f. sp.
perniciosium is the same as Dr. Jay Stipes isolated and cultured from diseased Ailanthus
trees in Virginia. At the same time, after much field time collecting Ailanthus samples
and observing the trees it was determined that not all the wilting symptoms were from
20
pathogenic fungi and Aculops ailanthi. Instead a web worm, Atteva punctella was having
a significant impact on the saplings and possibly the adult trees. These observations lead
to a field survey of the web worm on September 16, 2007. At this time, it was discovered
that there appeared to be selective herbivory by the web worm, with a strong preference
for non-seed bearing trees which were originally assumed to be either female or
immature trees. At the same time, it became apparent that the sterilization of female trees
may be occurring because of a pathogen due the limited number of seed bearing (female)
trees seen in the field. Necrotic lesions were the most obvious cause since they were
almost universal across observed sites. Simultaneously, due to literature research, the
commonly held root to xylem and apical stem/leaf wilting was questioned, suggesting the
possibly the infection of the plants with pathogenic fungi could be coming from the
apical parts of the stems where the disease symptoms were most obvious and spreading
throughout the plant. This suggests that not only the xylem, but the phloem is involved in
the spread of wilt diseases.
The desire to find another mechanism for the disease entry other than through the
roots lead to the collection of webs, fecal pellets and web worms, both adults and larvae
to test them for pathogenic fusarium. Simultaneously, saplings were collected from the
field, dissected and sections cultured to determine where the fusarium was found in the
plants and the direction of pathogen flow. The assumption here was that the parts of a
plant with the highest pathogen concentration based on observation would be the entry
point for the infection. At the same time a gradient would show the direction of pathogen
flow within the plant.
21
The next round of research, testing the successful root inoculations from the prior
experiment through another generation of seedlings, was done to ensure the first round
was right and to show that it is possible to inoculate this pathogen through successive
generations. This meant reculturing only the fall seedlings infected with mimosa wilt and
inoculating another round of Ailanthus seedlings. This was run parallel with an
unsuccessful attempt to find a method of tree inoculation other than the roots. This
parallel research involved three experiments; uncut leaf inoculations, cut leaf inoculations
and cut leaf inoculations with apical stem pinching and the seedlings covered in plastic
bags. Fungi isolated from the fecal pellets and the web worms were used in this
experiment as they were the most likely sources of pathogens. Throughout this round of
experiments, I attempted to duplicate some of the conditions found in the wild, meaning
feeding wounds, fecal pellet consistency and the effects of having a web in regards to
moisture retention and infection severity. By using agar at 0.4 g/100 mL water I
attempted to have a viscosity that was closer to the fecal pellets than straight water. This
also allowed the mycelium and conidia to stick to the leaves, giving more time for the
infection to occur. For the part of the experiment with the plastic bags, I was trying to
imitate the moisture retaining trait a web produced. At the same time, this allowed a
moist gel to sit on the leaves, in an attempt to further enhance the possibility of infection
and come closer to perceived wild conditions.
During the early months of 2008, PCR was started to identify the successful
pathogens from the fall 2007 experiments, the mimosa wilt, and various fungi from
necrotic lesions on the trunks of trees and necrotic tissues from the apical ends of
22
saplings. By identifying the pathogens causing the necrotic lesions, it was hoped that
there would be insight into the cause of the apparent sterilization of female trees.
Throughout the research from the beginning, methods were borrowed from other
researchers, altered as needed and new methods invented to meet the needs of the
research. Outside of the root inoculations and PCR related methods, all the methods used
in this research are those of the researcher and those suggested by his advisor.
Experiments and Results
The experiments were of four types; tub inoculation with Aculops ailanthi, root
inoculations for wilting, stem inoculations for necrotic lesions and leaf inoculations as an
alternate route to introduce a pathogen into a tree. The field survey took place in midSeptember 2007, just before first frost at the Urbana Community Park; therefore it could
not be followed by a second survey in the same year. Field observations were continual
and general, used to reinforce the data. Additional information on materials, methods and
recipes is in Appendix C. A complete collection of all the electronic data consisting of
photographs, raw data, spreadsheets, references, resources and related is in the MEES
office at the University of Maryland College Park. To see all this material email
MEES@MEES.umd.edu or go to 0105 Cole Student Activities Building on the
University of Maryland College Park Campus. For the research notebooks, contact me at
rtgardner3@yahoo.com with “Ailanthus research” in the header or through the MEES
office.
23
In general two color morphologies, red and lavender/off purple, defined this
research when looking at cultures on PDA plates. This was done for three reasons. The
first reason is that lavender was the color to look for according to Dr. Jay Stipes when
searching cultures for F. oxysporum f. sp. perniciosium. The second reason is that
through microscopic examination, both these colors were indicative of fusarium fungi in
that they contained banana shaped macroconidia, oblong microconidia or round
chlamydospores. The third reason was to limit my data set so the research could be
finished in a timely manner. The choosing of these colors was not absolute and was at
the discretion of the researcher.
Research Site Selection
With the exception of Michaux State Forest and the Crone Farm, all the sample
sites were selected by mixture of intuition, observation and luck. I was told where to
look for the Ailanthus stand in Michaux by Pennsylvania State Forest rangers after
reading a report about it and the Crone Farm by Dr. Marc Imlay. The rest of the sample
site location choices have no logic except that borne of intuition and good eyes. Once a
potential site was located, I used the site if it had symptomatic trees.
Sample designations
The numbering system for sample designation as found in Appendix B was
loosely based on the GLP, Good Laboratory Practice, system of sample designation. This
system requires a unique designation for every sample and subsequent iteration of that
sample. It further requires that additional information such as date, location and other
24
information be added to the sample description. This allows for the tracking of each
sample and in data that results from that sample.
Each sample was numbered consecutively from the prior sample except where
otherwise noted. Two tree subsamples from the same original sample are designated for
example as 74-1 and 74-2. At the same time two samples from the same culture are
designated 74a and 74b. Every subsequent culturing from the originally cultured sample
is designated by an additional letter such as 74abba. This is sample 74, first culture, then
a second subculture, another second subculture and a fourth first subculture. This sample
has a minimum of one plated subculture in the first generation, two subcultures in the
second and third platings and one plated subculture in the fourth generation for a total of
six plates from the original tree sampled. This allowed for the separating of different
morphologies such as color and the distinguishing of different generations of cultures as
the original samples were plated and purified into single morphologies.
If there were several plates from the same isolate, such as 125bba, then a
designation such as 1/5 was used to distinguish the plates from each other. In this case, it
was plate number 1 from a set of 5 plates of sample 125bba.
Sample collection and preparation
The purpose of surface sterilization of branch, stem and root samples was to make
sure that epiphytic saprophytes did not contaminate cultures of the endophytic pathogens
I was looking for. Early on the sample collection consisted of cutting 2-4 cm sections
from apparently diseased trees with flame sterilized pruning shears and pliers to hold the
samples. The samples were then surface sterilized in the field by dipping for one minute
25
in 95% ethanol, denatured alcohol or 70% isopropanol, 3 minutes dipped in 2-4% bleach
solution and dipping again for 30 seconds to 1 minute in ethanol, denatured alcohol or
70% isopropanol. The samples were then allowed to air dry for a short period or the
alcohol was quickly burned off. Samples were then put into field flame sterilized 4 oz.
Ball canning jars. The original method came from Fisher et al., 1994.
This changed after a conversation with Mark Schall at the 2007 Potomac Division
of the American Phytopathology Society where he described his method of taking large
trunk sections of trees from the field with no special sterilization precautions or
storage/shipment protocols until the samples were back in his lab. This caused me to take
larger sized samples as conditions permitted, primarily in the fall of 2007 and January
2008 and to take fewer precautions when handling them such as using appropriately sized
Ziploc® bag instead of surface sterilizing in the field and using the canning jars to store
samples. At the same time, whenever possible, the Ziploc® bags were wrapped around
the sample which was then broken off and the bags sealed to minimize potential
contamination.
The surface sterilization method I used in the lab with non-field sterilized samples
changed as I was concerned that the bleach and ethanol when used on samples from
actively growing plants was penetrating too far into the xylem and phloem, destroying the
endophytic fungi I was trying to culture. Surface sterilization times were cut to 30
seconds/stage. In the very last part of the experiment, simply washing under running
water the surface of the samples was all that was done (Redlin and Carris, 1996). A
quick method comparison experiment was performed comparing surface sterilization
versus just scrubbing the larger samples supported the validity of just scrubbing the
26
samples under running water in that fungi colonies with the desired morphological
characteristics were evident sooner and at a greater density compared to surface sterilized
samples. This applies only to large samples from actively growing trees and needs
validation for small and dormant samples.
Flame sterilization in the lab was done with alcohol burners. In the field,
depending on what was available in my field kit at that time, flame sterilization was done
using a blow torch, cigarette lighter or by pouring alcohol (isopropanol or ethanol) over
the tools and lighting the tools with a cigarette lighter until the alcohol burned off. The
latter method was suggested by Dr. Straney.
Branch samples were taken by suing standard pruning shears which had been
flame sterilized or by hand breaking the branches. Spores and sporophytes were scraped
off of necrotic lesions using a flame sterilized knife into Ziploc® sandwich bags, sterile
vials or sterile canning jars. Usually this included a section of the underlying bark. The
sporophytes were usually gray, with some being off-white, orange or pink/red. The
pink/red color morphology was mostly collected during January 2008.
Two Atteva punctella adults were collected into small vials. The fecal pellets
from the larvae and web worms larvae were collected by breaking off stems containing
web worm webs and putting them into large Ziploc® bags, which were then stored in a
refrigerator.
The purpose of plating plant samples was to separate endophytic and epiphytic
fungi from the matrix they were growing on and to separate the endophytes from each
other to allow for testing of each isolate. The purpose of plating fecal pellets, adult web
27
worms and the larva of the web worms was determine if the insects are carriers of
pathogens. Most incubations were at least 5 days, the normal minimal time for a red
colony to begin to show color at room temperature.
Branch and root cross-sections of roots of 2 – 4 mm thick were plated
immediately after lab surface sterilization onto ¼ strength PDA. These samples were
plated within 36 hours of collection.
Larvae, an adult web worm, fecal pellets and soil were the only non-plant or fungi
sample types cultured. Larvae were taken from 5 mL collection vials or isolated from
collected webs. They were rinsed with ethanol, allowed to dry and squished on the
plates. The one adult used in this experiment was placed on a plate and allowed to die.
Colonies which spread from its body were isolated. Fecal pellets were isolated with
forceps and by shaking webs over a sterile surface. Collected fecal pellets were then
plated with no additional preparation. Standard isolation and culturing procedures were
followed once the morphology of individual colonies became obvious on the plates. The
fungi cultured from the larvae, adults and fecal pellets all had similar phenotypic traits.
Once colonies of fungi were visible on a plate, the method of colony isolation
consisted of cutting the desired fungus from a Petri dish with an Exacto® knife. The
sample was placed onto another ¼ strength PDA Petri dish and incubated at room
temperature. This procedure was done in a microbiological hood to prevent
contamination by the resident fungi in the lab.
After pure colonies of a fungus were isolated, single spore isolation was
performed. This was to minimize genetic variation during PCR (Geiser et al, 2004).
28
The original method of single spore isolation was to take a small sample of the fungus,
put it into several mL of sterilized tap water and then pour onto a ¼ strength PDA plate.
The plate was observed for several days until individual colonies were observed. Then
the individual colonies were cut out and put onto another ¼ strength PDA plate or a clean
up plate. However, this was abandoned as it was hard to get a low enough number of
conidia on a plate for good separation due to the high number of conidia produced by
fusarium. So, streaking was done using small wood splints instead. This proved better
than pouring because the number of conidia separated and distance between individual
colonies from single conidia was related to the length of the streak. The longer the
streak, the more apt the plate was to have colonies from single conidia.
Once a colony was determined to be from one spore, it was put onto a ¼ strength
PDA plate and allowed to cover the whole plate in preparation for inoculation or
lyophilization. All samples were put onto a cleanup plate at some point in the process
eliminate bacterial contamination as evident by slimy or liquid growth on the plate, often
overlaying the fungi colonies. The best time for the cleanup plates is after the single
spore isolation and before the plating of the samples for inoculation as this minimizes the
chances of inoculum contamination.
Stem inoculations
Stem inoculations were an experiment to see if it was possible to cause necrotic
lesions on the stems of seedlings with morphologies similar to what was found in the
field. The results of the experiments are in Table 1. The procedure was to slice the stem
into or through the vascular cambium with an Exacto® knife. At this point, two different
methods of inoculation were tried. In the first, a sterile cotton swab was dipped into
29
sterile tap water, rubbed across the plate and then the plant at the cut. The cut was
wrapped in parafilm at this point. The second method used a size 3 cork borer to take
plugs from the plates. The plugs were then placed next to the stem in a “cup” of
aluminum foil as in sample 5f. For the rest of the samples the stem and plug were
wrapped in parafilm at the site of the wound. My preference is to use the latter method as
it excludes environmental contamination while at the same time allowing a long contact
time of moist agar/sample with the tree. Controls were treated in the same ways as the
samples. Where the Q-tips were rubbed across plates, the same was done using
uninoculated plates. Where agar plugs were used to inoculate, plugs from uninoculated
plates were used. The controls were put in the same trays as the infected seedlings for
that particular inoculum. This may account for some of the anomalies in the data such as
sample 5f aluminum foil. At the same time, the Aculops ailanthi infestation may have
skewed the data.
Controls showed scarring. Positive inoculations showed stem scarring in addition
to brown necrotic matter. Negative samples resembled controls. Cut depth is an
important consideration as it may cause false positives if the cut is too deep. Depending
on the purpose of the experiment, the depth of the cut should be no greater than just
through vascular cambium and as narrow as possible.
Samples 5ea, 5f and 105 red were from lesions. The other samples; 16ba, 64 red,
74-1, 74-2 and 74-3 were from trees showing other symptoms of disease. Samples 64 red
and 74-3 were positive. The rest of the samples under different conditions may have
caused necrotic lesions, but this data is not clear on that point.
30
Table 1. Results of stem inoculations started on May 19/20 2007 and finished on July
14/15, 2007.
The appearance of the mite Aculops ailanthi in the lab may have skewed the
results as to the number of positives and the severity of the infection of
seedlings.
In the field, necrotic lesions may have been a contributing factor to the death
of trees but did not appear to be a direct cause.
sample
date
inoculated
date
censused
controls
positive
controls
negative
total
controls
%
positive
inoculated
positive
inoculated
negative
total
inoculated
%
positive
5ea
5/19/07
7/14/07
1
4
4
25
4
13
17
24
5f
5/20/07
7/15/07
1
5
6
17
0
17
17
0
5f al
foil
5/20/07
7/15/07
4
1
5
80
6
12
18
33
16ba
5/19/07
7/15/07
1
4
5
20
7
9
16
44
64 red
5/19/07
7/14/07
0
5
5
0
11
3
14
79
74-1
5/19/07
7/15/05
1
5
6
17
2
16
18
11
74-2
5/20/07
7/15/07
0
6
6
0
5
12
17
29
74-3
5/20/07
7/15/07
0
6
6
0
15
3
18
83
105
red
5/20/07
7/15/07
2
4
6
33
8
8
16
50
Root inoculations
This experiment was designed to test the various pathogens isolated from diseased
Ailanthus altissima trees and one mimosa tree. Two types of tissues were tested, tissue
from necrotic lesions and tissue from inside the vascular cambium and pith. Generally
31
the cambium and pith tissues were cultured from just below the necrotic tissue on the
apical branches and trunks to try to get active fungi.
Root inoculations followed a method suggested by Dr. Jay Stipes of Virginia
Polytechnic School and State University. Seedlings with at least their second leaves were
ripped out of the potting soil. The roots were scrubbed hard under running tap water,
with roots over 4 inches long cut back. The plants were then dipped into a water
suspension of conidia that were scraped from a particular plate. Inoculated plants were
then repotted. The remaining conidia solution was poured in the trays with the inoculated
plants.
There were two usable sets of root inoculations done. The root inoculations May
and June 2007 are not used due to several inoculation technique errors and the
appearance of Aculops ailanthi. The first usable set of root inoculations started on
September 30, 2007 and October 8, 2007, ending on December 4, 2007. This consisted
of samples 5ea, 139a, 150-1aa, 16z, 153a red, 146a and controls. The second set was
started on January 2, 2008 and ended on March 9, 2008. The latter set consisted solely of
mimosa wilt cultured from the September 30, 2007 inoculated plants and did not include
controls.
Root inoculations from the experiments done in the fall were labeled according to
the order of inoculation. Hence, trays 5 and 6 were the 5th and 6th trays inoculated.
Coincidently, they were also the trays with F. oxysporum from a wilted mimosa tree and
the only successful root inoculation results. Diseased seedlings from both trays were
cultured and successfully applied to a second generation of seedlings. This gave positive
32
results for both first and second generations of the F. oxysporum from the wilted mimosa
tree used for the original inoculation. Table 2 has the results from both generations of
experiments with this inoculum.
The controls for the experiments started on September 30, 2007 and October 8,
2007 were started on September 10, 2007 and October 9, 2007. The September 30, 2007
controls were removed from the seed germination tray, soil scrubbed from the roots and
repotted in a tray of 12 pots. The controls from October 9, 2007 were handled exactly the
same way as the plants inoculated on September 30, 2007 and October 8, 2007, except a
sterile plate replaced the plates with fungi. In the control set from September 10, 2007
and October 9, 007, zero plants were stunted or died. Of all the plants inoculated in this
time period, fungi from sample number 139, collected on June 16, 2007 at Urbana
Community Park had results of 3 dead out of 12 plants in tray 3. The plants from the
same sample had zero plants out of 12 dead in tray 4. This anomaly happened for
unknown reasons. Therefore, out of a total of 120 plants inoculated in ten trays,
including the 24 controls and excluding the 24 samples inoculated with mimosa wilt, only
3 died. Whereas, of the two trays containing mimosa wilt 18 out of 24 plants died and all
had symptoms. In the second round, started January 2, 2008, six out of 24 plants died
with a total of 23 showing symptoms. Figure 2 shows a tray of inoculated plants from
the end of this second round of mimosa wilt inoculations.
33
Figure 2
Tray containing mimosa wilt inoculated plants at the end of the experiment which ran
from January 2, 2008 to March 9, 2008.
34
Table 2. Plant inoculation results from mimosa wilt.
started: 9/30/07
started: 1/2/08
completed: 12/4/07
completed: 3/9/08
tray 5: 150-1aa 1/2 1/3 Y
tray 1: 5-1.2aaa 2/2
tray 6: 150-1aa 2/2 1/3 Y
tray 2: 5-1.2aaa 1/2
plant #
tray 5
tray 6
tray 1
tray 2
1
chlorosis and
stunting
chlorosis and
stunting
dead
stunted
2
chlorosis and
stunting
dead
chlorosis and
stunting
chlorosis and
stunting
3
chlorosis and
stunting
dead
chlorosis and
stunting
chlorosis and
stunting
4
chlorosis and
stunting
dead
dead
chlorosis and
stunting
5
chlorosis and
stunting
dead
chlorosis and
stunting
chlorosis and
stunting
chlorosis and
stunting
6
dead
dead
chlorosis and
stunting
7
dead
dead
no symptoms
dead
chlorosis and
stunting
8
dead
dead
chlorosis and
stunting
9
dead
dead
chlorosis and
stunting
chlorosis and
stunting
dead
chlorosis and
stunting
dead
dead
chlorosis and
stunting
10
11
dead
dead
dead
35
12
dead
dead
chlorosis and
stunting
infection
100%
100%
92%
100%
death
58%
92%
25%
17%
1.54x10^7
7.42x10^7
Not
determinable
5.04x10^6
spores/mL
chlorosis and
stunting
Leaf inoculations
Leaf inoculations were an attempt at trying to find an alternate route of infection
in the trees than from the roots for wilt causing fungi and necrotic lesion causing fungi
such as the Fusarium lateritium found in a larva. By doing this, I tried to be closer to the
field conditions where it was possible that the fungi were being introduced by the web
worms during their feeding on the leaves and stems of Ailanthus. Samples 175bb1a and
181bbaaa 1/5 were used in this experiment as they are fecal pellets and an adult web
worm respectively.
Three similar methods were attempted with leaf inoculations to find the
mechanism by which the tops of trees were being infected with fusarium fungi. The first
cut the leaves in several places with sterile scissors before applying a mycelium/conidia
mixture in sterile tap water to the leaves with a sterile cotton swab. The second sliced the
leaves with sterile scissors in several places before applying a solution of agar at 20% of
normal strength with mycelium/conidia in a slurry to them with a sterile cotton swab.
The third experiment painted a mixture of mycelium and conidia in a 20% agar solution
on leaves which had been cut as in the prior experiment, with a sterile cotton swab. In
36
this last experiment, a clear plastic sandwich bag was used to cover the leaves of each
plant. The sandwich bags were an attempt to imitate the humid conditions inside the
webs of Atteva punctella. All three experiments were set under grow lights for several
weeks. Controls using the same conditions and solutions as the tested plants were run for
each of the experiments.
The 20% normal agar/water solution is 0.4 grams agar/100 mL sterile tap water.
This was an attempt to imitate the fecal pellets of the web worms. The assumption was
that a transient dosing of the leaves with conidia and mycelium may not be the best
conditions under which the fungi enter into the leaves and stems of Ailanthus. Instead,
steady contact over a period of time that may start with either the laying of eggs and the
fecal pellets from the adult female web worm or the first feeding from hatching of the
larvae to the first hard frost. This is a period of at least several months as the web worms
are obvious beginning in mid July and the first hard frost may not happen locally until
mid October.
There were no significant symptoms on the controls and inoculated plants without
plastic bags on them. Controls and inoculated plants with plastic bags on them showed
symptoms of infection such as wilting and chlorosis while the bags were in place.
Several days after the bags were removed, the symptoms disappeared. The symptoms
may have been due to the moisture level in the plastic bags and may have been valid as
long as the moisture around the leaves remained high. This may be what is happening in
the field with the feeding cuts from the web worms providing a constant source of
moisture and the webs providing a semi-permeable membrane barrier which hinders loss
of moisture, keeping the leaves and stem wet. However, without further experimentation,
37
I am assuming that the infection mechanism looked for in these experiments was not
found.
In the field, the fungi may enter the trees through the cuts made into the trunk and
leaves by Atteva punctella. (Hasan and Ayres, 1990; Wallin and Raffa, 2001) My work
may not have duplicated closely enough the conditions found in the field such as the
intensity of the wounds inflicted as Atteva punctella fed, the depth of the wounds, the
right environmental conditions such as relative humidity and day/night length, the
physical properties of fecal pellets or the amount of time the symptoms needed to
manifest under natural conditions.
Figure 3
Atteva punctella larvae Urbana Community Park, September, 2007.
38
Web worm survey
This survey was another aspect of my research. During the previous two
summers, I noticed an insect feeding on Ailanthus and the subsequent damage. I
recognized this insect as a possible biocontrol during the late summer of 2007. I wanted
to identify the cause of the damage and the degree. Therefore, I went to a site I knew was
accessible to surveying and performed the survey. At the same time, I collected
specimens for preservation and culturing of the fungi in their digestive tracts, both
directly and through their fecal pellets from their webs.
This field survey was done on September 15, 2007 at Urbana Community Park,
Urbana, MD on I-287 just south of Frederick, MD, by selecting trees in the area
surrounding the power substation. Three areas were chosen for both their ease of access
and the distance they were separated from each other. Trees with seeds were excluded
from the survey as only two webs total were observed on all the trees with seeds in the
general area surrounding and including the survey.
The infestation rate for non-seed bearing trees was 97%. The level of defoliation
was not quantified. However, many of the smaller trees were totally defoliated while the
larger trees still showed a high degree of defoliation.
39
Table 3. Summary of Atteva punctella survey on September 15, 2007
64
96.88
9.3
83.21
total # of trees surveyed
percent of trees with webs
average webs/tree excluding trees with total
defoliation
total trunk length (m)
593
total webs counted
7.13
webs/meter excluding trees with total defoliation
Tree Dissections
This part of the research happened late in the process as the result of questions
about the mechanisms of infection, the location of the infections in the tree and the
direction the infections travelled. In other words, this was to determine if the pathogens
infecting Ailanthus were entering through the roots or the apical wilted areas with
potential web worm interaction or the necrotic lesions. This was done by collecting
saplings showing necroses at the top or with necrotic lesions along the trunk. They were
examined and cross-sections plated to determine the parts of the trees where the
endophytic fungi were located. Since red and purple colonies from earlier research
contained macro and microconidia indicative of fusarium they were the focus of the
research. At the same time, red colonies had been observed starting as white and
changing to red and then purple as the colony matures. No further identification was
performed outside of morphology observation as this experiment was only designed to
find the location of the endophytic fungi within the trees, not specifically identify the
species found.
40
Trees were collected on December 19, 2007 and cross sections were plated onto
1/4 PDA on December 20, 2007. Sample sections of 10 – 20 cm were scrubbed under
running tap water, rinsed with bleach and 190 – 195 proof ethanol. The plates were read
on January 7, 2008.
The data supports the idea that the fungus is introduced directly into the plant at
the apical end of the trunk/branches and possibly through the necrotic lesions on the
trunks and branches. Tree physiology has the vascular cambium in the center of roots and
towards the outside of branches and the trunk. If fungi had entered through the root hairs
and related structures, they would have been evident as the root samples were cultured
and shown a continuity throughout the trees. Instead fungi colonies were in the vascular
and cork cambiums of the trunks, generally towards the top and not lower on the trunk,
suggesting both xylem and phloem travel from the apical ends of branches and the trunk.
41
Table 4. Tree Dissection Data
sample
total tree/root
length
location, cm
from root/trunk
intersection
plate # if
applicable
182
root = 39 cm
root
1/2 , 2/2
Michaux
taken at 15 and 25
cm
10
x
1 red colony
30
x
several red
colonies
50
x
no red colonies
70
x
1 red colony
90
x
several red
colonies
110
x
2 red colonies
130
x
several red/purple
colonies
150
x
1 red colony
170
x
1 purple colony
tip
x
several red
colonies
from trunk (1/2,
2/2)
trunk length = 193
cm
results
no red colonies
183
201 cm total length
root
x
no red colonies
Michaux
of root and trunk
mid trunk
x
1 red colony
tip
x
many red colonies
no red colonies
184
267 cm total length
root
x
Michaux
of root and trunk
mid trunk
x
42
several red
colonies
tip
x
1 red colony
x
top inch of soil
x
no red colonies
186
root = 41 cm,
taken
root
x
no red colonies
Urbana
12 cm from trunk
10
x
no red colonies
30
x
purple colony
50
x
several red
colonies
70
x
no red colonies
90
x
no red colonies
tip
x
no red colonies
185
Urbana, soil
sample
trunk length = 115
cm
187
213 cm total length
root
x
no red colonies
Urbana
of root and trunk
mid trunk
x
1 red colony
tip
x
several red
colonies
188
259 cm total length
root
x
no red colonies
Urbana
of root and trunk
mid trunk
x
no red colonies
x
vascular
cambium
190
Urbana
43
1/3
several red
colonies
2/3
no red colonies
190
x
lesion
Urbana
190
3/3
several red
colonies
1/2
no red colonies
2/2
several red
colonies
x
yellow tissue
x
no red colonies
x
vascular
cambium
1/4
purple colonies
2/4
red colonies
3/4
no red colonies
4/4
no red colonies
1/2
purple and/or red
colonies
2/2
many red and
purple colonies
x
many red and
purple colonies
x
many red and
purple colonies
Urbana
191
Urbana
191
x
cork cambium
Urbana
192
x
Urbana
Urbana = Urbana Community Park
unwashed tip
washed tip
Michaux = Michaux State Forest
All measurements are taken from the base of the tree where the trunk meets the root.
44
Aculops ailanthii
The purpose of this experiment was to duplicate under controlled conditions what
happened during the prior summer accidently in the lab, when the research started in May
and June was wiped out due to an infestation of Aculops ailanthii, an eriophyoid mite.
Aculops ailanthii was accidently introduced into the laboratory from the field during May
or June 2007. The mite infested and killed a large number of plants across all the
experiments, before the cause was recognized and dealt with. The mites used in this
experiment were found in the Urbana Community Park on several trees using the
pathology of the lab plants to recognize infestation in the field.
This experiment started on September 16, 2007 and ran to December 5, 2007.
Three translucent 53 liter plastic tubs were filled with 3-8 cm of Metromix 360® potting
soil. Forty Ailanthus seedlings with at least secondary leaves were planted in each tub.
Ten wilted leaves with mites from Urbana Community Park were placed in the tub on the
soil on September 16. More wilted leaves were added on September 21. Silicone tub and
tile sealant was used to seal white rip stop nylon to the tops of the tubs, to prevent an
accidental escape of the mites. Tubs were then placed in a room full of natural light.
Observations were made every day or every second day. The control tub, same as the
tubs with mites, was started on September 21, 2007.
Tub 1 had 34 out of 40 plants dead for an 85% death rate. Tub 2 had 33 out of 40
plants dead for an 83% death rate. The control tub had 9 out of 41 plants dead for 22%
death rate. The dead plants were in the middle of the control tub where the watering
occurred. Thus some of these deaths may have been directly due to the watering or
overwatering. However, there was a difference of 63% and 61% between the control and
45
the infested tubs. In tubs 1 and 2, mites were found on the leaves of dead and living
plants, confirming infestation was occurring. Mites are stored in a 67.5% v/v
ethanol/water in a freezer.
46
Figure 4
Aculops ailanthi, three mites are along the central vein of this leaf going diagonally from upper
right to lower left.
Figure 5
Tub setup for Aculops ailanthi experiment showing infested leaves.
47
Figure 6
Aculops ailanthi infested seedlings in tub experiment.
Figure 7
Aculops ailanthi tub experiment control.
48
DNA Analysis
DNA analysis was used to make more definitive identifications of the fungi found
than is possible by either observation of physical traits such as colony morphology and
conidia shape or by identification with the host. Common procedures were used to isolate
and identify the fungi species from the sampling through blasting the DNA sequences.
Both the NCBI Nucleotide Blast (http://www.ncbi.nlm.nih.gov/blast/) and the Penn State
Fusarium (http://fusarium.cbio.psu.edu/) databases were used to identify the fungi.
Cultures were isolated first by culturing samples of various trees on ¼ strength
PDA plates. Colonies were then streaked on ¼ strength PDA as often as needed to
isolate individual colonies. The individual colonies were then cut from the growth
medium. Each colony was next put onto the center of separate minimal media plates
containing antibiotic with the centers cut out. The centers were replaced over the fungi.
Fungi were allowed to grow until they grew through the media in sufficient quantity to
restreak onto another plate containing ¼ strength PDA. At this time a portion of the
culture was put into Barz’s media and placed on a shaker until it was opaque or contained
“balls” of fungi. This was then filtered through 2 cm #40 paper filters under vacuum.
Collected fungi were then lyophilized. Lyophilized fungi were put in a -70C freezer until
they were extracted. Gel extractions were developed and performed to isolate the desired
sequences as shown in Figure 8. The isolated DNA was then extracted from the gels and
sent to Genewiz® to obtain the EF1-alpha and EF2-alpha sequences. These sequences of
700 base pairs were blasted both on the Penn State fusarium database and using the NCBI
database to get potential identifications. See the Appendices D, F, G, H and I for more
detailed information on the DNA extraction and identification.
49
As the data shows in Table 5, there is general agreement between the Penn State
database and the NCBI database. Fusarium solani, Fusarium oxysporum, F. fujikuroi, F.
lateritium and F. sporotrichioides are known to cause cankers or necrotic lesions if
circumstances are right, which means that there is a physical wound in the bark caused by
either biological or environmental stresses such as insect and freeze damage (Marasas et
al., 1984; Farr et al., 1989; Demirci and Maden, 2006; Sinclair and Lyon, 2005).
According to Mark Schall at Penn State (2007) and Farr et al. (1989) the most probable
cause of the cankers on Ailanthus is F. lateritium. My data suggests a variety of
fusariums, most notably F. solani and F. lateritium.
Definitive Fusarium identification is problematic due to its polyphyletic rather
than monophyletic nature. Fusarium oxysporum, according to Kistler (1997) is a species
that has numerous subgroups designated as formae speciales, further subdividing into
races according to not only the species but the strain of the plant infected. Various
methods have used vegetative compatibility groups, (VCGs), isozymes, DNA
fingerprinting, restriction fragment length polymorphisms (RFLPs), random amplified
polymorphic DNAs (RAPDs), electrophoretic karyotype of chromosome lengths (EK),
and DNA sequence analysis to classify isolates. (Kistler 1997)
Vegetative compatibility groups assume monophyletic groups based on imperfect
reproduction. (Baayen, 2000) The underlying paradigm is that every time a fungus
species, forma speciales or race moves from one species or strain of host to another it
may become a new formae speciales or race (Kistler, 1997). However, if two different
formae speciales of fungi of the same genus or species infect the same plant
50
simultaneously or are otherwise in close environmental association, will they exchange
genes? If so, this means that the offspring of both are no longer monophyletic.
Figure 8
Run 10. A comparison of PCR samples 3,12,14 and 21 of 700 base pairs each, at 1, 3 and
5 mM MgCl2 and annealing temperatures of 55 C and 56 C.
51
Fusarium oxysporum is a complex species or clade of fungi that is thought to be
entirely clonal, since the sexual state has not yet been found. Each member of this clade
is supposed to have adaptations unique to each host or host group and be host specific.
(Samson, 1996; Kistler, 1997; O’Donnell, 1998; Fravel, 2002; Abo et al., 2005) These
authors agree that the identification of formae speciales within this group appears to be at
best uncertain, possibly being an easy classification dumping ground for fusarium
pathogens, especially wilts. This conflicts with Geiser et al. (2004) who feel that this is a
large and complex group open to many fine identification possibilities from forma
speciales to race. In other words, this is an example of scientists who are comfortable
with broader classification systems and those who prefer narrower ones. I lean towards
the broader classification systems.
Host specificity is not always true with many crossovers possible. Sexual forms
may be more common than at first thought due to the advantages sexual reproduction
give in regards to genetic diversification and environmental adaptation (Abo et al., 2005).
To this is the added burden of sorting out random sequence differences and mutations
which may or may not be significant (Samson, 1996). Mimosa wilt in this research was
expected to be f.sp. perniciosium but turned out to possibly be either melonis, Penn State,
or cucumerinum, NCBI (Cappellini and Peterson, 1976; Fraedrich, 2000). The area I
gathered the mimosa wilt is full of truck farms. Two of the local crops are cucumbers
and cantaloupe, species that F. oxysporum cucumerinum and melonis infects. With these
crops being local to the area where the sample was taken, identification of F. oxysporum
melonis or cucumerinum in the mimosa tree may not be a misidentification. When a PCR
sequence for F. oxysporum melonis from the Penn State database was run against the
52
NCBI database, it came was identified as F. oxysporum cucumerinum. The reverse for an
NCBI strain of F. oxysporum cucumerinum came back as F. oxysporum melonis. This
represents the problems with identifying these forma speciales and points to the need for
further study and clarification of what constitutes a forma speciales and a race and
consistency in their naming. My perceptions when reading the literature, working with
the programs, especially MultAlin® and going through the databases is that the
differences between the forma speciales and races may be too small for accurate
identification using just the EF1 alpha gene.
The trend in pathogen identification is to assume that pathogens descend from
pathogens and non-pathogens from non-pathogens (Kistler, 1997). With a narrow
biological perspective, it is logical that the pathogenic traits in one organism are easiest to
hand down through asexual reproduction. From a broader biological perspective, this
does not make sense because of the examples of larger organisms which frequently
change feeding patterns as they evolve into different species from the same parent or are
given opportunities to exploit new energy sources. (Darwin’s finches are the classic
example.) This assumes completely asexual reproduction which may not be true for
Fusarium oxysporum, among other imperfect fusarium species. Add to this the constant
changes and adaptations every species make, it is not difficult to assume that many nonpathogenic fungi turn into pathogens and vice versa as opportunities present themselves.
F. oxysporum f. sp. albedinis consists of a single clonal lineage corresponding to a
single VCG, identifying it as descended from one parent (Kistler, 1997). However, F.
oxysporum f. sp. lycopersici has much greater genetic diversity while F. oxysporum f. sp.
cubense is even more complex (Kistler, 1997). The diversity of and within these species
53
points to the cladistic uncertainties in the fusarium genus and even more so to the
identification issues around F. oxysporum. With the concept of vegetative compatibility
groups and pathogenic races corresponding to one strain of a plant (crop) this adds even
more doubt to the idea that F. oxysporum is monophyletic, rather than a series of similar
genus types that may have descended from one or several sexual parents. There is too
much genetic diversity to assume that all the fusarium fungi pathogenic to a specific plant
species are from the same clonal lineage.
The problem with the NCBI database is it contains both the Pulhalla classification
system using number designations for fungi isolates and the more common naming
system such as Penn State uses. The NCBI database contains strains that were sequenced
in environmental surveys where the identification into genus and species is tenuous. The
most accurate identification from a database relies upon well identified strains from strain
collections. The Penn State database focuses upon these strains and so may offer a better
diagnostic ability.
In summary, the identities of the fusarium species where the Penn State and NCBI
databases agree, I accept. Where there is conflict, I prefer to use the identification given
by the Penn State database. At this point, I feel uncomfortable due to both the small size
of my database and the uncertainties of the relationships of forma speciales and the EF1
alpha gene due to genetic variation to make definitive identification to the formae
speciales or race level for any sample or series of related samples. For a more technical
explanation of the Fusarium-ID v. 1.0 database and sample preparation, read Geiser et al.
(2004).
54
Additional data may be found in Appendices E, F, G and H. These appendices
include all the samples extracted for PCR, cladistic trees from NCBI, more specific
identification data and the edited sequences.
55
Table 5. PCR Data
PCR
sample
PCR1
PCR4
PCR6
PCR8
PCR10
PCR11
original
sample
number
collection
location
details
data
base
EF1/EF2
organisms
158aa
Michaux
stem tip
NCBI
EF1, EF2
and
consensus
Fusarium sp. NRRL 43730,
98%, 98%, 98%
Penn
State
EF1, EF2
and
consensus
Fusarium pallidoroseum,
98%, 98%, 98%
NCBI
EF1, EF2
and
consensus
Fusarium solani strain
NRRL 32849, 99%, 99%,
99%
Penn
State
EF1, EF2
and
consensus
Fusarium solani strain mpVI,
97%, 97%, 97%
NCBI
EF1, EF2
and
consensus
Fusarium lateritium isolate
F0103, 97%, 97%, 96%
Penn
State
EF1, EF2
and
consensus
Fusarium lateritium ‘Clade
IIA', 95%, 95%, 95%
NCBI
EF1, EF2
and
consensus
Fusarium lateritium isolate
F0103, 97%, 97%, 97%
Penn
State
EF1, EF2
and
consensus
Fusarium lateritium ‘Clade
IIA', 96%, 95%, 95%
NCBI
EF1, EF2
and
consensus
Fusarium sporotrichioides,
99%, 99%, 98%
Penn
State
EF1, EF2
and
consensus
Fusarium sp. cf.
sporotrichioides isolate VI,
99%, 99%, 98%
NCBI
EF1
Fusarium solani f. sp. piperis
strain MAFF 236575, 99%,
99%, 98%
172-2
baaa
180bb1aa
"A" 1744a
"D" 153a
5-1.2aaa
Crone
farm
Michaux
Urbana
Park
Urbana
Park
mimosa
wilt from
Koch's
apical end of
sapling trunk
web worm
(larva), red
apical end of
sapling trunk,
red
parts from
wilted tree,
red
mimosa wilt
from F1 tree
5-1
56
PCR12
PCR14
6-6.1aaa
150-1aa
(mim2)
mimosa
wilt from
Koch's
mimosa
wilt for
Koch's
mimosa wilt
from F1 tree
6-6
cultured from
slant of
mimosa wilt
used to
inoculate tray
6 on 9/30/07
EF1
Fusarium oxysporum f. sp.
cucumerinum, 98%, 99%,
98%
Penn
State
EF1, EF2
and
consensus
Fusarium oxysporum f. sp.
melonis, 100%, 99%, 100%
NCBI
EF1, EF2
and
consensus
Fusarium solani f. sp. piperis
strain MAFF 236575, 99%,
99%, 98%
Penn
State
EF1, EF2
and
consensus
Fusarium oxysporum f. sp.
melonis, 100%, 99%, 99%
NCBI
EF1
Fusarium solani f. sp. piperis
strain MAFF 236575, 99%,
99%, 98%
Fusarium oxysporum f. sp.
cucumerinum, 99%, 99%,
98%
PCR16
PCR18
150-1aa
74-3.1a
mimosa
wilt for
Koch's
Michaux
cultured from
slant of
mimosa wilt
used to
inoculate tray
6 on 9/30/07
collected
3/11/07,
wilted branch
ends
57
Penn
State
EF1, EF2
and
consensus
Fusarium oxysporum f. sp.
melonis, 100%, 100%, 99%
NCBI
EF1, EF2
and
consensus
Fusarium solani f. sp. piperis
strain MAFF 236575, 99%,
99%, 96%
EF1, EF2
and
consensus
Fusarium oxysporum f. sp.
melonis, 99%,
cucumerinum 99%, melonis
96%
Penn
State
EF1, EF2
and
consensus
Fusarium oxysporum f. sp.
melonis, 100%, 100%, 99%
NCBI
EF1, EF2
and
consensus
Fusarium sp. NRRL 43680
haplotype FIESC 4-a, 92%,
93%, 93%
EF1, EF2
and
consensus
Fusarium equiseti isolate
SAT73, 92%, 93%, 93%
Fusarium sp. cf. bullatum
Penn
State
EF1, EF2
and
consensus
NRRL 31005, 92%,
Fusarium sp. cf. equiseti
NRRL 25795, 93%, 91%
PCR21
194
Urbana
Park
female tree
1, necrotic
lesion
NCBI
EF1, EF2
and
consensus
EF1, EF2
and
consensus
Fusarium lichenicola strain
NRRL 28019, 99%, 99%,
97%
Fusarium solani strain
NRRL 28018, 99%, 99%,
97%
Nectria haematococca
Penn
State
EF1, EF2
and
consensus
/Fusarium sp. cf. solani
mpVI isolate NRRL 22586,
97%, 97%, 22161 - 97%
PCR25
PCR27
PCR28
184mtaaaa
191aaaa
207aaa
Michaux
Urbana
Park
Michaux
middle of
dissected
tree
NCBI
cork
cambium
from tree
with necrotic
lesion
sporophytes
from necrotic
lesion
58
EF1, EF2
and
consensus
Fusarium solani f. sp. piperis
strain MAFF 236575, 99%,
99%, 98%
EF1, EF2
and
consensus
Fusarium oxysporum f. sp.
cucumerinum, 99%, 99%,
98%
Fusarium oxysporum f sp
Penn
State
EF1, EF2
and
consensus
NCBI
EF1, EF2
and
consensus
KSU 12914, 99%,99%,99%
EF1, EF2
and
consensus
Gibberella fujikuroi strain
NRRL 43470, 99%, 99%,
99%
Penn
State
EF1, EF2
and
consensus
Fusarium fujikuroi NRRL
13566, 97%, 97%, 97%
NCBI
EF1, EF2
and
consensus
Fusarium sp. NRRL 22586,
99%, 99%, x
melonis NRRL: 26173, 99%,
100%, 99%
Fusarium sp.
PCR29
PCR31
190aaaa
206aa
Urbana
Park
Michaux
vascular
cambium
from mature
dead tree
with blue
stain fungus
red fungi
from lesion
59
EF1, EF2
and
consensus
Fusarium solani strain FRC
S1124, 99%, 99%, x
Penn
State
EF1, EF2
and
consensus
Nectria
haematococca/Fusarium sp.
cf. solani mpVI, 99%, 99%,
99%
NCBI
EF1, EF2
and
consensus
Fusarium sp. KSU 12914,
99%, 99%, 98%
EF1, EF2
and
consensus
Gibberella fujikuroi strain
NRRL 43470, 99%, 99%,
98%
Penn
State
EF1, EF2
and
consensus
Fusarium fujikuroi NRRL
13566, 97%, 97%, 97%
NCBI
EF1, EF2
and
consensus
Fusarium lateritium isolate
F0103, 97%, 96%, 92%
Penn
State
EF1, EF2
and
consensus
Fusarium lateritium ‘Clade
IIA' isolate FRC L-200, 95%,
94%, 94%
Discussion
The sole purpose of my research was to find potential biocontrols for Ailanthus
altissima in the local ecosystems. I spent almost all my early research time in the field
observing, photographing and collecting samples. The lab work was based on the field
work. For fungi, it consisted of culturing field samples for endophytic fungi acting as
pathogens, followed by isolating and identifying them as Fusariums using the
microscope, running Koch’s postulates on pure cultures of the fungi found in the field
and finally performing PCR using the EF1 alpha gene to identify the pathogens. The
insect side consisted of identifying and testing potential insect biocontrols using a field
survey of Atteva punctella and running Koch’s postulates in the lab on Aculops ailanthii.
At the same time, several larval and adult Atteva punctella, including their fecal pellets
left in the webs, were cultured for potential pathogens.
Atteva punctella, the Ailanthus web worm is the best potential biocontrol
discovered. In this area, the larval stage is a specialist to only Ailanthus, with no other
close relatives found in the Western hemisphere outside of the tropics where the nearest
relative is Simarouba glauca (Ding et al., 2006). It appears to be carrying either
Fusarium lateritium, Fusarium solani or both as pathogenic fungi with a lesser
possibility of F. oxysporum. Regardless, Atteva punctella is a native, a specialist, widely
distributed, can act as a carrier for a pathogen, and is found locally and throughout the
United States (Ding et al., 2006).
The spread of A. punctella from the tropics northward parallels the spread of the
Northern Mockingbird, Mimus polyglottos, from the southern United States to the
northern states. According to Dr. Douglas Ruby (2006), the introduction of the
60
Multiflora Rose, Rosa multiflora, from China in the 1920’s and 1930’s supplied the bird
with a source of food in the northern states. This allowed the mockingbird to expand its
range northward. This is similar to what happened with A. punctella. A new food source
was introduced which allowed it to expand its range. The unique feature is that A.
punctella is a specialist insect unexpectedly adding a food source. The mockingbird on
the other hand is a generalist feeder within its clade which can be expected to easily take
advantage of a new food source.
The data on the potential diseases Atteva punctella carries are not definitive as the
sample set was limited. Both F. lateritium and F. solani are considered generalist
pathogens, with F. solani consisting of a large set of f. sp. (sub species). The unique
aspect of what was found with these pathogens is that they were found in the dieback at
the top of the plant and necrotic lesions on the trunk of the trees. Whether the F. solani
was resident or transient needs further research. F. lateritium was found in the web
worm sample run for PCR. If my observations hold up that the fungi were introduced
from the top of the plant through feeding wounds (Hasan and Ayres, 1990), it may be
reasonably assumed that the pathogen is using the phloem as an avenue to distribute
throughout the plant. This is partially confirmed in an experiment which cultured
sections from several plants; roots to the apical trunk ends. Fungi were found in the
trunks including the apical end of withered trunks. However, no fungi were found in the
roots. Further research is needed to find out how much the plants are affected and to
confirm the use of the phloem as opposed to the xylem when fungi are introduced at the
apical stem/trunk end of a tree.
61
According to Belisario, et. al. (2002) all the fusarium species, F. lateritium Nees:
Fr., F.oxysporum Schlechtend.:Fr., F. solani (Mart.) Sacc., and F. sporotrichioides Sherb.
that were identified by PCR on Ailanthus altissima associated with disease are capable of
causing cankers. F. lateritium may be airborne, a possible alternative to movement by
Atteva punctella or another insect vector from infected to non-infected plants. The fungi
then are able to enter and infect the plant through the feeding wounds caused by the web
worm larvae. Another possibility adding another level of complexity to this scenario is
that the conidia are deposited on plants that the adult web worm uses for food. If this is
the case, the adult web worm may be spreading the disease from plants it uses for nectar
to Ailanthus plants by way of fecal pellets or physical contact. The spores can then enter
Ailanthus through the feeding wounds caused by the larvae. Or, the larvae ingest the
spores left by the adult and leave them in fecal pellets on the infected trees so that the
fungi infect through the feeding wounds. This may lead to a phloem infection within the
tree.
According to Mark Schall, the cankers on the trunks and branches are caused by
non-native Ambrosia beetles Euwallacea validus and Xylosandrus germanus transmitting
F. lateritium (Davis and Schall, 2006). This being true, the other fusariums identified
may also be transmitted that way. This would account for the diversity of fusarium
species identified by PCR on and in the plants.
There is strong incidental evidence that the necrotic lesions, especially on
smaller branches may be causing wilting of leaves apical of the lesions. The trunk of a
maturing tree had the pith destroyed from the necrotic lesion for an unknown distance
toward the top of the tree. The same was true for smaller branches where the pith was
62
destroyed to the end of the branch from the necrotic lesion. An important unanswered
question is the place that the pith plays in the growth and survival of Ailanthus. Whether
pith death is enough to cause the death of a sapling or mature tree needs to be researched.
In the spring of 2007, taking samples of fungi from infected trees, I attempted to
make necrotic lesions on seedlings. Necrotic lesions did form. However, there is a
question about the cause of the lesions. Were they caused by the wounds, the depth of
the wounds or by the fungi? The data was partially compromised by the Aculops
ailanthii infestation. However, lesions definitely formed in two of the sample sets. PCR
was not run on any of these samples due to resource limitations.
Another interesting aspect of the Ailanthus web worm was found in the field
during the survey at the Urbana Community Park on September 15, 2007. This insect
had a strong preference for plants without seeds. Obviously female trees were excluded
from the survey due to the very low rate of infestation by the Atteva punctella. This may
be due to the female trees repelling or killing the web worms as suggested by Bawa and
Opler (1978) in a study of Simarouba glauca. In the study, the flowers on female trees
included two chemicals not found in the flowers of male trees that are assumed to repel
phytophagus insects.
Backing up the observations of the web worms in Urbana and the work by Bawa
and Opler are two articles on dioecious tropical trees. The research found there is
preferential predation on male trees by the weevil they studied over female trees leading
to the predominance of female trees over male trees (Wolfe, 1997; Marshall and Ganders,
2001). This may account for the behavior seen by A. punctella in the field. At the same
63
time it leads credence to the possibility that female trees may have a pathogen sterilizing
them due to the unexpectedly low numbers found. If so, this means the chemicals found
by Bawa and Opler (1978) in the flowers of female trees are not manufactured, giving
female trees the same or a very similar chemical composition to the male trees. The
conclusion drawn from these observations is that sterilization of female trees will help the
web worm to proliferate and better act as a biocontrol by selectively targeting male trees,
sexually immature trees and sterilized female trees.
The apparent sterility of female trees appears related to the cankers found on the
trees caused by F. lateritium or F. solani. The one sample run for PCR from a female
tree has F. solani as the most likely cause of the canker, but F lateritium remains a strong
possibility. Further backing up the data is the lack of trees with seeds at Michaux and
fewer female trees being found at the Urbana Community Park than expected. The
stands at Ft. Frederick and Rt. 273 were not large enough to make a definitive statement
even though the two related stands at Rt. 273 were devoid of seed bearing trees. The
Crone farm had no trees with seeds when visited, but had numerous dead and dying trees.
Rt. 273, Urbana Community Park, Crone farm and Michaux had ample evidence of web
worms and the damage they caused.
In a quick field survey done on June 17, 2008, it became apparent that there are
four potential times the seed production of a female tree can be limited, flower
production, the end of flowering, the start of seed development and during seed
maturation. If the number of flowers is limited due to disease or other reasons at the start
of flower production or early development, then there is the possibility that the chemicals
produced by the flowers may be at such a low level that herbivory may occur. If the
64
flowering is compromised by disease at the end of the bloom period, then the seeds will
not form. The third point, beginning of seed set, likewise limits the number of seeds
produced. Finally, if something happens during seed maturation which causes them to
drop off prematurely, the female trees will appear sterile. The data is inconclusive with
premature seed drop being the apparent cause of female sterility from the limited data set
as shown in Figure 9.
The question is at which point is a tree considered sterile and will the web worm
use the female trees? If seed maturation is the issue, then there may be a physical factor
such as noise, irritating vibrations or even abrasion causing Atteva punctella to prefer
non-seed bearing trees over seed bearing trees. This last argument is strengthened by the
fact that the seeds are at an advanced state of maturity by the time of the local appearance
of the web worm in July. Further research needs to be done to determine if it is physical
or chemical factors that determine why there is the apparent gender biased herbivory by
Atteva punctella and the relationship between the apparent female sterility, necrotic
lesions and premature seed drop.
65
Figure 9
Seed cluster showing premature seed drop.
Aculops ailanthi, the eriophyoid mite that infested my research during the summer
of 2007, is a potential biocontrol of Ailanthus, providing there is a heavy enough
infestation to do significant damage to mature trees. These mites were identified by Dr.
Jim Armine, West Virginia University (Armine, 2007). I was not able to reference these
mites outside of his note, Lin, Jin & Kuang, 1997. (I also queried Dr. Douglas Tallamy at
the University of Delaware about this reference.) Kowarik and Saumel’s article (2007)
hints that there may be some confusion over the identity of this species with an alternate
name of Aculus altissimae.
66
I collected the mites in the field from a mature tree, looking for similar damage in
the field to what was seen in the lab. The issues are whether the mite is specific to only
Ailanthus trees and if they can be put in the field early enough in the season and at a high
enough density to act as a biocontrol. The problems associated with the raising, timing
and critical density for the eriophyoid mite are the same for the Ailanthus web worm,
determining the critical density, reproductive rate, laboratory rearing conditions, ability to
distribute over a landscape and the length of the season that they can be used as a control
in the field. The positive side of doing Koch’s postulates in the lab is that the mites were
easy to raise in the lab. This was done twice, once accidently and once on purpose. It
appears that they can be raised in the lab on seedlings or young plants and then released
either onto individual plants or spread throughout an area. The most important remaining
issue is the mode of transmission. Lab observations saw them actively crawling across
leaves. In the field, it is logical to assume that they may be wind borne and that they may
crawl up the stems of individual plants. According to Dr. John A. Davidson, a retired
entomologist at the University of Maryland College Park, the eggs may overwinter in the
leaf debris around the Ailanthus stands (Davidson, 2007). At the same time, the
possibility of bird, mammal or another animal used as a way to hitchhike from plant to
plant needs to be investigated.
Jim Armine’s note infers that these insects are not native as does the reference. If
that is the case, they need to be tested for specificity to Ailanthus.
A wilted mimosa tree, Albizia julibrissin, was found near Goldsboro, Caroline
County, MD in late July 2007. Using information from discussions with Dr. Jay Stipes,
Virginia Polytechnic and State University, I decided to culture wilted sections of this tree.
67
The lab work afterwards confirmed that this was indeed a form of Fusarium oxysporum.
This finding is consistent with Dr. Jay Stipes work and the literature (Cappellini and
Richardson, 1976; Fraedrich, 2000; Sinclair and Lyon, 2005). The data was inconclusive
on the exact f. sp. of the fungi isolated. The infection rate was almost 100% taken
through two consecutive generations with 50% dead. Only the original inoculums and
samples cultured from infected seedlings in the fall 2007 experiment were identified by
PCR. By inference, the winter 2008 generation had the same fungi acting as a pathogen.
This confirmed the work of Dr. Jay Stipes in that a mimosa wilt was able to kill Ailanthus
seedlings.
The ambient temperature of the sites sampled may be the only difference between
the work of Mark Schall from Penn State and me. Both the Verticillum and Fusarium
genuses are world wide. However, it appears that the fusariums prefer an ambient
temperature of 25C to 35C and verticillium prefer 28C and lower (Nelson et al., 1981;
Pegg and Brady, 2002). This roughly translates into a line running either down the
Appalachian Mountains northeast to southwest or in my case, a line a few miles north of
Pennsylvania/Maryland border. Fortunately, Michaux State Forest is south of this
apparent boundary and Mark Schall’s sample site is north of it in the Tuscarora State
Forest of south-central Pennsylvania.
To conclude this discussion, we need to explore the concepts of space and time as
related to specialist insects and fungi. The reason this research was successful is that
there was a native biocontrol, Atteva punctella, already in a nearby ecosystem using
Simarouba glauca as a food source. The reason it did not control Ailanthus earlier is that
it was not in the same place (space) as the original introductions of Ailanthus. The web
68
worm’s native host is in the tropical parts of the Americas while Ailanthus was
introduced first in Philadelphia, then California. It took time for Ailanthus and the web
worm to meet. By that time the population and range of Ailanthus were too large and
spread out for control.
The concept of time is a little different. When looking for a native biocontrol, it
may be better to look at closely related native plants in the area and study their natural
history comparing it to the invasive plant’s natural history. Look for biocontrols which
are specific to the native plant and certain periods of that plant’s seasonal cycle. Then
compare it to the invasive plant’s natural history and see if there is a distinct time
difference between similar processes such as going into leaf, flowering or seed set. If
there is a temporal disjunction between two similar processes, then maybe the specialist
native biocontrol can be used for the same life cycle event, but at a distinctly different
point in seasonal time. For example, if the native plant goes into bloom the second week
of March and the invasive the middle of April, maybe an insect that feeds on the flowers
of the native can be used for a biocontrol. Since it is more probable that the native insect
will attack the defenseless exotic than other closely related natives, control and possible
eradication may be achieved. The same can be true with a fungus. If a fungus prefers
certain parts of a plant’s seasonal cycle and the processes are offset significantly between
native and exotic, perhaps introduction of the fungus at the proper part of the seasonal
cycle will be the key to control.
Admittedly, using the concepts of space and time instead of shotgunning exotic
specialists into an ecosystem requires finesse and patience. Applying Biotic Resistance
in reverse, an introduced specialist biocontrol is apt to prefer defenseless closely related
69
natives over the exotic it was brought in to control because the exotic already has
defenses against the biocontrol. Therefore, it is safer to use a conservative non-exotic
biocontrol approach than risk an ecosystem with an outside specialist as a potential
disastrous biocontrol.
Conclusions
The final analysis of how to control Ailanthus altissima in North America is
complex. First, to destroy the seed bank, all seed bearing trees as evident in June to early
July must be destroyed either mechanically or chemically. The clones are probably not
an issue since it appears that Atteva punctella will defoliate them. Next, there needs to be
a release of Atteva punctella at a density high enough to ensure that all the leaves on the
remaining tress and new clones are consumed. This needs to be repeated for several
years to ensure that the seed bank is destroyed and that the new trees are not allowed to
live due to herbivory by Atteva punctella and Aculops ailanthii. From my field work and
the literature, the introduction of pathogenic endophytic fusarium will happen
automatically as the web worms feed, adding another level to the plant’s destruction.
Alternatively, the data suggests that the seed bank can be destroyed by the
introduction of necrotic lesions caused by Fusarium lateritium or Fusarium solani which
appear to sterilize the female tree. Mark Schall did his inoculations mechanically.
However, the inoculations may happen on their own by two non-native ambrosia beetles,
Euwallacea validus and Xylosandrus germanus (Davis and Schall, 2006). Then Atteva
punctella and Aculops ailanthii can be used to consume the stand and control the clones.
70
The advantage to this is that it was observed from my field work there were not as many
saplings around the infected mature trees as would be expected of a tree making clones in
response to trees being cut down. One study in Hannover, Germany found 551 clones
from 21 cut saplings in the first year and 722 the second year (Kowarik and Saumel,
2007). Therefore, there is the possibility that the fungal infection either does not awaken
the mechanism which makes clones or inhibits the clone mechanism.
Now someone needs to finish this research as both Mark Schall and I will no
longer be working on this project, leaving no one to complete the work. Once the final
work is done, the eradication of Ailanthus altissima from North America is almost
certain. On a larger scale, applying the principles contained in this thesis can almost
guarantee the elimination of any exotic land plant.
71
APPENDIX A
Major Sample Site Locations and Descriptions
Urbana Community Park, Urbana, MD (3636 Urbana Pike, Frederick, MD 21704.) GPS
18-S-0296436/UTM4355097. Several acres including football fields, soccer fields,
tennis courts and open space. Trees were used mainly from around the electrical power
substation and in the corner of the woodlot between the power substation and I-270.
Trees of all age groups were represented.
Michaux State Forest, Biglerville, PA. South side of Route 30 @ 600m west of Pine
Grove Road. This site is several acres in the middle of a woodland on a hill slope. Trees
were generally sampled at least 10 meters from the road throughout the stand. Trees of all
age groups were represented.
Crone Farm. Second farm on west side of Indian Valley Trail when approaching from
the north, Westminster, MD. This is a private farm with mixed woodlot and fallow field.
Samples were taken mostly along the driveway to the house and barn. Trees were mostly
saplings and mature trees with many dead.
Ft. Frederick State Park, Big Pool, MD 21711. Trees are in a stand on the right side of
the road before entrance when approaching from Big Pool.
Route 273, Cecil County, MD. Trees on south side of road just west of Fair View Road,
west of Fair Hill, MD. This is an island stand on the edge of a road and another wooded
section at the edge of the field and bordering the road.
Rt. 313, 1.6 miles west of Goldsboro, Caroline County, MD. GPS 18-S0429442/UTM4322. South side of road near Castle Hall Road. Single mimosa tree along
highway right of way and a few feet from a wooded area. During June of 2008, the there
were two dead adult mimosa trees and one living with one living sapling at this location.
72
APPENDIX B
Fungi sampling database
#
Date
collected
Location
first
cultured
plate type &
date
1
10/14/2007
Urbana
Community Park
1/16/2007
PDA 1/12/07
2
10/14/2007
Urbana
Community Park
1/16/2007
PDA 1/12/07
3
1/13/2007
Urbana
Community Park
1/14/2007
PDA 1/12/07
sapling trunk
end
4
1/13/2007
1/14/2007
PDA 1/12/07
dead tree
sample
16 - out
numerical
of order
1/13/2007
1/14/2007
PDA 1/12/07
live infected
tree sample
5
1/13/2007
Tabler Road,
Urbana, MD
1/14/2007
PDA 1/12/07
branch lesion
6
1/14/2007
UMCP, lot 6
1/14/2007
PDA 1/12/07
branch lesion
1/20/2007
Wellington Rd.
near fairgrounds,
Prince William
County, VA
1/21/2007
PDA 1/21/07
1/20/2007
Wellington Rd.
near fairgrounds,
Prince William
County, VA
1/21/2007
PDA 1/21/07
1/20/2007
Wellington Rd.
near fairgrounds,
Prince William
County, VA
1/21/2007
PDA 1/21/07
7
8
9
Crone farm,
Westminster,
MD
Crone farm,
Westminster,
MD
73
notes
sample A of 2,
sapling trunk
end
sample B of 2,
sapling trunk
end
jelly jar ctl.
used to
compare to
centrifuge
tube, dead 2
year old trees
centrifuge tube
ctl. used to
compare to
jelly jar, dead
2 year old
trees
centrifuge tube
ctl. used to
compare to
jelly jar, dead
2 year old
trees
1/20/2007
Wellington Rd.
across from
Hayden Rd.,
Prince William
County, VA
1/21/2007
PDA 1/21/07
11
1/20/2007
Wellington Rd.
across from
Hayden Rd.,
Prince William
County, VA
1/21/2007
PDA 1/21/07
12
1/21/2007
Stanford Drive,
College Park,
MD
1/21/2007
PDA 1/21/07
13
1/21/2007
Stanford Drive,
College Park,
MD
1/21/2007
PDA 1/21/07
14
1/20/2007
Stanford Drive,
College Park,
MD
1/21/2007
PDA 1/21/07
15
1/20/2007
Stanford Drive,
College Park,
MD
1/21/2007
PDA 1/21/07
17
1/27/2007
UMCP, lot 6
1/27/2007
PDA from
Fisher
18
1/27/2007
UMCP, lot 6
1/27/2007
PDA from
Fisher
10
74
jelly jar ctl.
used to
compare to
centrifuge
tube, single
stand alone
tree,
apparently
uninfected
centrifuge tube
ctl. used to
compare to
jelly jar, single
stand alone
tree,
apparently
uninfected
jelly jar ctl.
used to
compare to
centrifuge
tube,
apparently
uninfected
tree, fungal
colony
growing
centrifuge tube
ctl. used to
compare to
jelly jar,
apparently
uninfected
tree, fungal
colony
growing
jelly jar ctl.
used to
compare to
centrifuge
tube, infected
tree branch
centrifuge tube
ctl. used to
compare to
jelly jar,
infected tree
branch
bark/vascular
cambium
scrapings
tissue from
lesion scar
using 3/8" drill
bit
tissue from
dead area
inside lesion
using 3/8" drill
bit
through crosssection from
above last
lesion up
using 3/8" drill
bit, @ 2.5 feet
above ground
I-95 rest stop
north of
Dumfries,
northbound
Fairfax
County, VA,
along the
entrance road
19
1/27/2007
UMCP, lot 6
1/27/2007
PDA 1/31/07
20
1/27/2007
UMCP, lot 6
1/27/2007
PDA from
Fisher
21
2/3/2007
1-95 Dumfries
2/3/2007
PDA from
Fisher
22
2/3/2007
Riverbend Park
2/3/2007
PDA 1/31/07
23
2/3/2007
Georgetown
Pike
2/3/2007
PDA 1/31/07
Georgetown
Pike, just west
of I-95
24
2/4/2007
Urbana
Community Park
2/4/2007
PDA 1/31/07
vascular
cambium from
lesion scar
tissue on a
mature tree
25
2/4/2007
Urbana
Community Park
2/4/2007
PDA 1/31/07
dead end of a
cloned sapling
26
2/4/2007
Urbana
Community Park
2/4/2007
PDA 1/31/07
bark and
vascular
cambium from
a healthy
control
27
2/4/2007
Urbana
Community Park
2/4/2007
PDA 1/31/07
dead end of 2
saplings
28
2/6/2007
Schumacher
Seed Co.
2/6/2007
PDA 2/6/07
Aa. seeds
29
2/6/2007
Schumacher
Seed Co.
2/6/2007
PDA 2/6/07
Aa. seeds
30
5/18/2006
Mill St.
Salisbury, MD
2/6/2007
PDA 2/6/07
dead wood
31
5/18/2006
South Park
Drive Salisbury,
MD
2/6/2007
PDA 2/6/07
dead wood
32
8/22/2006
Wilson St.
Salisbury, MD
2/6/2007
PDA 2/6/07
dead wood
75
2/10/2007
Mill St.
Salisbury, MD
2/10/2007
PDA 2/6/07
sapling trunk
end, exterior
of stand
34
2/10/2007
Mill St.
Salisbury, MD
2/11/2007
due to
researcher
error
PDA 2/6/07
sapling trunk
end, interior of
stand
35
2/10/2007
Mill St.
Salisbury, MD
2/10/2007
PDA 2/6/07
36
2/10/2007
Mill St.
Salisbury, MD
2/10/2007
PDA 2/6/07
37
2/10/2007
Mill St.
Salisbury, MD
2/10/2007
PDA 2/6/07
38
2/10/2007
Paint Branch
Trail
2/10/2007
PDA 2/6/07
39
2/10/2007
Paint Branch
Trail
2/10/2007
PDA 2/6/07
40
2/10/2007
Schumacher
Seed Co.
2/10/2007
PDA 2/6/07
41
2/13/2007
Schumacher
Seed Co.
2/13/2007
PDA 2/7/07
42
2/13/2007
Schumacher
Seed Co.
2/13/2007
PDA 2/7/07
43
2/18/2007
Rt. 273 west of
Fair Hill, MD
2/18/2007
PDA 2/22/07
saplings
44
2/18/2007
Rt. 273 west of
Fair Hill, MD
2/18/2007
PDA 1/12/07
branches from
mature trees
45
2/18/2007
Michaux St.
Forest, near
Gettysburg, PA
2/18/2007
PDA 1/21/07
46
2/18/2007
Michaux St.
Forest, near
Gettysburg, PA
2/18/2007
PDA 2/22/07
33
76
root of sapling
<3' tall with
dead trunk end
bark/vascular
cambium
scrapings
dead branch
end of tree in
sample 36
seeds from
Paint Branch
Trail
seeds from
Paint Branch
Trail, 30+ sec.
IPA then IPA
flamed off
seeds from
Schumacher
Seed Co., 30+
sec. IPA then
IPA flamed
off
seeds from
Schumacher
Seed Co.,
dipped in
H2O2
seeds from
Schumacher
Seed Co.,
dipped in
H2O2
infected
saplings and
branches from
mature trees
infected
saplings and
branches from
mature trees
47
2/18/2007
Michaux St.
Forest, near
Gettysburg, PA
48
2/18/2007
Rt. 15 near
Emmitsburg,
MD
2/18/2007
PDA 2/7/07
49
2/18/2007
Rt. 15 near
Emmitsburg,
MD
2/18/2007
PDA 2/6/07
50
2/18/2007
Schumacher
Seed Co.
2/18/2007
PDA 2/22/07
51
2/18/2007
Schumacher
Seed Co.
2/18/2007
PDA 2/22/07
52
2/18/2007
Schumacher
Seed Co.
2/18/2007
PDA 2/22/07
53
2/21/2007
Ft. Frederick
State Park, MD
2/23/2007
PDA 2/22/07
54
2/21/2007
Ft. Frederick
State Park, MD
2/23/2007
PDA 2/22/07
55
2/21/2007
Ft. Frederick
State Park, MD
2/23/2007
PDA 2/22/07
56
2/21/2007
Ft. Frederick
State Park, MD
2/23/2007
PDA 2/22/07
57
3/3/2007
Leesburg Pike,
Reston, VA
3/3/2007
PDA 3/2/07
58
3/3/2007
Leesburg Pike,
Reston, VA
3/3/2007
PDA 3/2/07
59
3/3/2007
Leesburg Pike,
Reston, VA
3/3/2007
PDA 3/2/07
2/18/2007
PDA 2/7/07
77
infected
saplings and
branches from
mature trees
infected
saplings and
branches from
mature trees
infected
saplings and
branches from
mature trees
seeds from
Schumacher
Seed Co., 60
sec. in 6%
bleach
seeds from
Schumacher
Seed Co., 3
min. in 6%
bleach
seeds from
Schumacher
Seed Co., 15
min. in 6%
bleach
mother tree
terminal
branches
beyond
apparent
fungal damage
saplings
terminal end
of trunk
lesion from
branch
terminal end
of branch
beyond lesion
on branch
saplings
terminal end
of trunk
vascular
cambium from
sapling
terminal end
of branch
beyond lesion
on branch
60
3/4/2007
Sligo Trail, nr. I495 and golf
course, DC
3/3/2007
PDA 3/2/07
terminal end
of saplings
61
3/4/2007
Sligo Trail, nr. I495 and golf
course, DC
3/3/2007
PDA 3/2/07
terminal of
branches from
mature tree
62
3/10/2007
West Virginia –
Shenk’s
3/12/2007
PDA 3/9/07
smutty branch
63
3/10/2007
West Virginia Shenk’s
3/12/2007
PDA 3/9/07
clone trunk
64
3/10/2007
West Virginia Shenk’s
3/12/2007
PDA 3/9/07
sm. clone
trunk
65
3/11/2007
West Virginia nr. Shenk’s
3/12/2007
PDA 3/9/07
66
3/11/2007
West Virginia nr. Shenk's
3/12/2007
PDA 3/9/07
67
3/11/2007
West Virginia nr. Shenk’s
3/12/2007
PDA 3/9/07
68
3/11/2007
West Virginia nr. Shenk’s
3/12/2007
PDA 3/9/07
69
3/11/2007
near Flintstone,
MD
3/12/2007
PDA 3/9/07
70
3/11/2007
near Flintstone,
MD
3/12/2007
PDA 3/9/07
71
3/11/2007
mislabeled
3/12/2007
PDA 3/9/07
72
3/11/2007
Michaux
3/12/2007
PDA 3/9/07
78
necrotic
lesion, not
surface
sterilized
necrotic
lesion, not
surface
sterilized
pink fungus on
bark, not
surface
sterilized
pink fungus on
bark, not
surface
sterilized
branches from
tree which had
been cut, not
surface
sterilized
branches from
tree which had
been cut,
surface
sterilized on
3/12/07 before
plating
lesion
scrapings, not
surface
sterilized
rotted pith
above
(towards the
stem) lesion,
not surface
sterilized
sm. lesion on
sm. stem, not
surface
sterilized
wilted branch
ends, not
surface
sterilized
bark from
dying mature
tree, not
surface
sterilized
73
3/11/2007
Michaux
3/12/2007
PDA 3/9/07
74
3/11/2007
Michaux
3/12/2007
PDA 3/9/07
75
3/11/2007
Urbana
Community Park
3/12/2007
PDA 3/9/07
76
3/11/2007
Urbana
Community Park
3/12/2007
PDA 3/9/07
sapling ends,
not surface
sterilized
77
3/11/2007
Urbana
Community Park
3/12/2007
PDA 3/9/07
from tree
w/large lesion,
not surface
sterilized
79
3/11/2007
Urbana
Community Park
3/12/2007
PDA 3/9/07
seeds, not
surface
sterilized
79
3/11/2007
Urbana
Community Park
not plated
not plated
not plated
3/17/2007
PDA 3/9/07
terminal end
of branches
3/17/2007
PDA 3/9/07
terminal end
of branches
3/17/2007
PDA 3/9/07
terminal end
of branches
3/17/2007
PDA 3/9/07
terminal end
of branches
80
3/16/2007
81
3/16/2007
82
3/16/2007
83
3/16/2007
I-64 between
Charlottesville
and Staunton,
VA, westbound,
rest area
I-64 between
Charlottesville
and Staunton,
VA, westbound,
rest area
I-64 between
Charlottesville
and Staunton,
VA, eastbound,
rest area
I-64 between
Charlottesville
and Staunton,
VA, eastbound,
rest area
79
84
3/17/2007
Rising Sun, DE
3/17/2007
PDA 3/9/07
terminal end
of branches
85
3/17/2007
Rising Sun, DE
3/17/2007
PDA 3/9/07
terminal end
of branches
3/17/2007
PDA 3/9/07
necrotic
lesion, not
surface
sterilized,
colony cut off
surface and
plated
3/18/2007
PDA 3/9/07
shoots from
stump
3/18/2007
PDA 3/9/07
small clones
3/18/2007
PDA 3/9/07
small trees
3/18/2007
PDA 3/9/07
young trees
and clones
not plated
not plated
mature trees
86
3/17/2007
87
3/18/2007
88
3/18/2007
89
3/18/2007
90
3/18/2007
91
3/18/2007
Rising Sun, DE
North St.,
Boonton, NJ,
alongside St.
Mary's cemetery
Sheep Hill, at
the end of Ross
St. in wooded
area, Boonton,
NJ
Sheep Hill, at
the end of Ross
St. in wooded
area, Boonton,
NJ
Rt. 46 near
Denville and
Mountain Lakes,
NJ
Rt. 46 near
Denville and
Mountain Lakes,
NJ
necrotic
lesion, not
surface
sterilized
terminal end
of branch from
tree with
necrotic lesion
92
3/21/2007
Rt. 29 north of
Sperryville, VA
93
3/21/2007
Rt. 29 north of
Sperryville, VA
3/21/2007
PDA 3/13/07
94
3/21/2007
Rt. 29 near
Madison, VA
3/21/2007
PDA 3/13/07
end of
branches on
mature trees
3/23/2007
Rt. 11, @ 4
miles south of
Salem, VA
PDA 3/13/07
end of
branches and
sapling trunks,
one necrotic
lesion
95
3/21/2007
PDA 3/13/07
3/25/2007
80
young trees in
a stand with
many dead
trees
end of
branches on
mature trees
and a lesion
96
3/23/2007
I-81, mile 169.4,
VA
97
3/23/2007
Rt. 651 at I-81,
VA
3/25/2007
PDA 3/13/07
98-1
3/23/2007
Rt. 651 at I-81,
VA
3/25/2007
PDA 3/13/07
cambium
98-2
3/23/2007
Rt. 651 at I-81,
VA
3/25/2007
PDA 3/13/07
cambium
99
3/23/2007
Rt. 647, near I66 exit 13
3/25/2007
PDA 3/13/07
branch ends
100
3/23/2007
I-55 mile 20.3
3/25/2007
PDA 3/13/07
branch ends
101
3/23/2007
3/25/2007
PDA 3/13/07
necrotic lesion
cambium
102
3/24/2007
3/25/2007
PDA 3/13/07
healthy
cambium
103
3/24/2007
3/25/2007
PDA 3/13/07
unhealthy
cambium
104
3/24/2007
UMCP, lot 6
3/25/2007
PDA 3/13/07
healthy
cambium
105
3/24/2007
UMCP, lot 6
3/25/2007
PDA 3/13/07
necrotic lesion
cambium
106
5/6/2007
Chapman State
Forest, MD
5/6/2007
1/4 PDA
4/27/07
stem from
dying clones
107
5/6/2007
Chapman State
Forest, MD
5/6/2007
1/4 PDA
4/27/07
stem from
dying clones
108
5/6/2007
Chapman State
Forest, MD
5/6/2007
1/4 PDA
4/27/07
wilted leaves
109
5/12/2007
Mill St.
Salisbury, MD
5/12/2007,
5/28/07
1/4 PDA
5/08/07, 1/8
PDA 5/07/07
110
5/12/2007
Mill St.
Salisbury, MD
5/12/2007
1/4 PDA
5/08/07, 1/8
PDA 5/07/07
111
5/12/2007
Mill St.
Salisbury, MD
5/12/2007,
5/28/07
1/4 PDA
5/08/07, 1/8
PDA 5/07/07
Sligo Trail, nr. I495 and golf
course, DC
Sligo Trail, nr. I495 and golf
course, DC
Sligo Trail, nr. I495 and golf
course, DC
3/25/2007
PDA 3/13/07
81
trunk or
branch
segments
w/leaves
trunk or
branch
segments
w/leaves
trunk or
branch
segments
w/leaves
necrotic lesion
from clone
trunk
trunk or
branch
segments
w/leaves
trunk or
branch
segments
w/leaves
trunk or
branch
segments
w/leaves
trunk or
branch
segments
w/leaves
trunk or
branch
segments
w/leaves
trunk or
branch
segments
w/leaves
trunk or
branch
segments
w/leaves
trunk or
branch
segments
w/leaves
branch
segment
w/leaves &
necrotic lesion
trunk or
branch
segments
w/leaves
trunk or
branch
segments
w/leaves
living branch
from an
infected tree
112
5/12/2007
Mill St.
Salisbury, MD
not plated
not plated
113
5/12/2007
Mill St.
Salisbury, MD
5/12/2007,
5/28/07
1/4 PDA
5/08/07, 1/8
PDA 5/07/07
114
5/12/2007
Wilson St.
Salisbury, MD
5/12/2007,
5/28/07
1/4 PDA
5/08/07, 1/8
PDA 5/07/07
115
5/13/2007
Rt. 273 west of
Fair Hill, MD
5/13/2007,
5/28/07
1/4 PDA
5/08/07, 1/8
PDA 5/07/07
116
5/13/2007
Rt. 273 west of
Fair Hill, MD
not plated
not plated
117
5/13/2007
Rt. 273 west of
Fair Hill, MD
not plated
not plated
118
5/13/2007
Rt. 273 west of
Fair Hill, MD
5/28/2007
1/8 PDA
5/07/07
119
5/13/2007
Michaux
5/28/2007
1/8 PDA
5/07/07
120
5/13/2007
Michaux
5/13/2007,
5/28/07
1/4 PDA
5/08/07, 1/8
PDA 5/07/07
121
5/13/2007
Urbana
Community Park
5/13/2007,
5/28/07
1/4 PDA
5/08/07, 1/8
PDA 5/07/07
122
5/13/2007
Urbana
Community Park
5/28/2007
1/8 PDA
5/07/07
123
5/13/2007
Urbana
Community Park
not plated
not plated
124
5/27/2007
not plated
not plated
125
5/27/2007
not plated
not plated
dead branches
126
5/27/2007
5/27/2007
1/4 PDA
5/8/07
infected
branch
Sligo Trail, nr. I495 and golf
course, DC
Sligo Trail, nr. I495 and golf
course, DC
Sligo Trail, nr. I495 and golf
course, DC
82
5/27/2007
Sligo Trail, nr. I495 and golf
course, DC
128
5/27/2007
Sligo Trail, nr. I495 and golf
course, DC
5/27/2007
1/4 PDA
5/8/07
129
5/27/2007
UMCP, lot 6
5/27/2007
1/4 PDA
5/8/07
130
5/27/2007
UMCP, lot 6
5/27/2007
1/4 PDA
5/8/07
131
6/15/2007
Sligo Trail, nr. I495 and golf
course, DC
6/15/2007
1/4 PDA
5/8/07
132
6/15/2007
Sligo Trail, nr. I495 and golf
course, DC
6/15/2007
1/4 PDA
5/8/07
133
6/15/2007
Sligo Trail, nr. I495 and golf
course, DC
6/15/2007
1/4 PDA
5/8/07
134
6/16/2007
Michaux
6/16/2007
1/4 PDA
5/8/07
wilted stem
135
6/16/2007
Michaux
6/16/2007
1/4 PDA
5/8/07
wilted stem
136
6/16/2007
Michaux
6/16/2007
1/4 PDA
5/8/07
curlicued stem
137
6/16/2007
Urbana
Community Park
6/16/2007
1/8PDA
5/8/07
wilted stem,
138
6/16/2007
Urbana
Community Park
6/16/2007
1/8PDA
5/8/07
139
6/16/2007
Urbana
Community Park
6/16/2007
1/8PDA
5/8/07
140
6/16/2007
Urbana
Community Park
6/16/2007
1/8PDA
5/8/07
127
5/27/2007
83
1/4 PDA
5/8/07
stem with
wilted leaves
scrapings from
a necrotic
lesion on a
tree with many
dead branches
vascular
cambium from
a necrotic
lesion
scrapings from
a necrotic
lesion on a
tree with few
dead branches
Tree #1
broken tree in
a group w/
diamond bark
canker
Tree #1
broken tree in
a group w/
diamond bark
canker
Tree #2
broken tree
with a necrotic
lesion
wilted leaves,
not surface
sterilized
before plating
wilted leaves,
not surface
sterilized
before plating
wilted flower
infloresence
stem end
7/9/2007
1/4 PDA
6/18/07 &
6/30/07
wilted
branch/main
stem, surface
sterilized and
not surface
sterilized
7/13/2007
Rt. 29
(Colesville
Road) at
northwest Trail,
north side of
road
7/14/2007
1/4 PDA
6/30/07
wilted mimosa
branches, not
surface
sterilized
143
7/21/2007
Michaux
7/21/2007
1/4 PDA
6/30/07
wilted leaf
stem
144
7/21/2007
Michaux
not plated
not plated
stem
145
7/21/2007
Michaux
not plated
not plated
stem
146
7/21/2007
Urbana
Community Park
7/21/2007
1/4 PDA
6/30/07
stem
147
7/21/2007
Urbana
Community Park
not plated
not plated
wilted leaves
148
7/21/2007
Urbana
Community Park
not plated
not plated
dead leaves
149
7/30/2007
7/30/2007
1/4 PDA
7/25/07
150
7/30/2007
7/30/2007
1/4 PDA
7/25/07
151
8/2/2007
Michaux
8/2/2007
1/4 PDA
7/25/07
152
8/2/2007
Michaux
8/2/2007
1/4 PDA
7/25/07
153
8/2/2007
Urbana
Community Park
8/2/2007
1/4 PDA
7/25/07
154
8/2/2007
Urbana
Community Park
8/2/2007
1/4 PDA
7/25/07
155
8/16/2007
Rt. 273 west of
Fair Hill, MD
8/17/2007
1/4 PDA
8/14/07
7/8/2007
Rt. 273 west of
Fair Hill, MD
142
141
Rt. 313, @ 1
mile west of
Goldsboro, MD
Rt. 313, @ 1
mile west of
Goldsboro, MD
84
stems from a
wilted mimosa
tree, 1 of 2
stems from a
wilted mimosa
tree, 2 of 2
wilted leaves,
stems or
branches from
wilted plants
wilted leaves,
stems or
branches from
wilted plants
wilted leaves,
stems or
branches from
wilted plants
wilted leaves,
stems or
branches from
wilted plants
dead trunk end
of sapling
8/17/2007
1/4 PDA
8/14/07
wilted leaves,
stems or
branches from
wilted plants
156
8/16/2007
Rt. 273 west of
Fair Hill, MD
157
8/19/2007
Michaux
8/19/2007
1/4 PDA
8/14/07
stems
158
8/19/2007
Michaux
8/19/2007
1/4 PDA
8/14/07
stems
159
8/19/2007
Michaux
8/19/2007
1/4 PDA
8/14/07
stems
160
8/19/2007
Urbana
Community Park
8/19/2007
1/4 PDA
8/14/07
primary mid
leaf stem
161
8/19/2007
Urbana
Community Park
8/19/2007
1/4 PDA
8/14/07
primary mid
leaf stem
162
9/15/2007
Urbana
Community Park
9/15/2007
AB 9/5/07
bark scrapings
163
9/15/2007
Urbana
Community Park
9/15/2007
AB 9/5/07
orange lesion
possibly in
sporulation
164
9/15/2007
Urbana
Community Park
9/15/2007
AB 9/5/07
lesion in
sporulation
165
9/15/2007
Urbana
Community Park
9/15/2007
AB 9/5/07
bark scrapings
166
9/15/2007
Urbana
Community Park
9/15/2007
AB 9/5/07
bark scrapings
167
9/15/2007
Urbana
Community Park
9/15/2007
AB 9/5/07
dead leaves
168
9/15/2007
Urbana
Community Park
9/15/2007
AB 9/5/07
dead leaves
169-1
10/6/2007
Rt. 273 west of
Fair Hill, MD
10/11/2007
1/4 PDA
9/21/07
169-2
10/6/2007
Rt. 273 west of
Fair Hill, MD
10/11/2007
1/4 PDA
9/21/07
85
necrotic pith
tissue at
terminal end
(0 cm) of dead
trunk with
apparent
Ailanthus web
worm damage
necrotic pith
tissue 10 cm
from terminal
end of dead
trunk with
apparent
Ailanthus web
worm damage
169-3
10/6/2007
Rt. 273 west of
Fair Hill, MD
10/11/2007
1/4 PDA
9/21/07
necrotic pith
tissue 20 cm
from terminal
end of dead
trunk with
apparent
Ailanthus web
worm damage,
a large red
fungus colony
on plate
169-4
10/6/2007
Rt. 273 west of
Fair Hill, MD
10/11/2007
1/4 PDA
9/21/07
dead leaves, at
least one red
fungus colony
169-5
10/6/2007
Rt. 273 west of
Fair Hill, MD
10/11/2007
1/4 PDA
9/21/07
cambium/bark,
apical end
170-1
10/6/2007
Rt. 273 west of
Fair Hill, MD
10/11/2007
1/4 PDA
9/21/07
170-2
10/6/2007
Rt. 273 west of
Fair Hill, MD
10/11/2007
1/4 PDA
9/21/07
170-3
10/6/2007
Rt. 273 west of
Fair Hill, MD
10/11/2007
1/4 PDA
9/21/07
171-1
10/20/2007
Michaux
10/21/2007
1/4 PDA
10/18/07
86
necrotic pith
tissue at
terminal end
(0 cm) of
green trunk
with apparent
Ailanthus web
worm damage,
one red fungus
colony
necrotic pith
tissue 10 cm
from terminal
end of green
trunk with
apparent
Ailanthus web
worm damage
necrotic pith
tissue 20 cm
from terminal
end of green
trunk with
apparent
Ailanthus web
worm damage,
5 small orange
on white
colonies
apical trunk
with web
worm damage,
surface
sterilized
171-2
10/20/2007
Michaux
10/21/2007
1/4 PDA
10/18/07
172-1
10/20/2007
Crone farm,
Westminster,
MD
10/21/2007
1/4 PDA
10/18/07
172-2
10/20/2007
Crone farm,
Westminster,
MD
10/21/2007
1/4 PDA
10/18/07
173-1
10/20/2007
Fort Frederick
10/21/2007
1/4 PDA
10/18/07
173-2
10/20/2007
Fort Frederick
10/21/2007
1/4 PDA
10/18/07
174-1
10/20/2007
Urbana
Community Park
10/21/2007
1/4 PDA
10/18/07
174-2
10/20/2007
Urbana
Community Park
10/21/2007
1/4 PDA
10/18/07
174-3
10/20/2007
Urbana
Community Park
10/21/2007
1/4 PDA
10/18/07
174-4
10/20/2007
Urbana
Community Park
10/21/2007
1/4 PDA
10/18/07
175
10/20/2007
Crone farm,
Westminster,
MD
10/22/2007
1/4 PDA
10/18/07
176
10/20/2007
Urbana
Community Park
10/22/2007
1/4 PDA
10/18/07
177
10/20/2007
Crone farm,
Westminster,
MD
10/23/2007
1/4 PDA
10/18/07
87
apical trunk
with web
worm damage,
surface
sterilized
apical trunk
with web
worm damage,
surface
sterilized
apical trunk
with web
worm damage,
surface
sterilized
apical trunk
with web
worm damage,
surface
sterilized
apical trunk
with web
worm damage,
surface
sterilized
apical trunk
with web
worm damage,
surface
sterilized
apical trunk
with web
worm damage,
surface
sterilized
apical trunk
with web
worm damage,
washed
apical trunk
with web
worm damage,
washed
sample 3/4,
fecal pellets
from web
worm web
sample 2/5,
fecal pellets
from web
worm
sample 2/4,
one web worm
178
10/20/2007
Urbana
Community Park
10/23/2007
1/4 PDA
10/18/07
sample 2/5,
web worms
sample 1/2,
fecal pellets
from web
worm
179
10/20/2007
Michaux
10/22/2007
1/4 PDA
10/18/07
180
10/20/2007
Michaux
10/23/2007
1/4 PDA
10/18/07
sample 1/2,
one web worm
181
10/20/2007
Michaux
10/23/2007
1/4 PDA
10/18/07
adult web
worm
182
12/19/2007
Michaux
12/20/2007
1/4 PDA
12/8/07
191 cm trunk
length,
sampled at
every 20 cm
and tip of
trunk
183
12/19/2007
Michaux
12/20/2007
1/4 PDA
12/8/07
dissected tree
184
12/19/2007
Michaux
12/20/2007
1/4 PDA
12/8/07
dissected tree
185
12/19/2007
Urbana
Community Park
12/20/2007
1/4 PDA
12/8/07
composite soil
from roots of
collected trees,
2 plates
186
12/19/2007
Urbana
Community Park
12/20/2007
1/4 PDA
12/8/07
dissected tree
187
12/19/2007
Urbana
Community Park
12/20/2007
1/4 PDA
12/8/07
dissected tree
188
12/19/2007
Urbana
Community Park
12/20/2007
1/4 PDA
12/8/07
259 cm tall
189
12/19/2007
Urbana
Community Park
12/20/2007
1/4 PDA
12/8/07
190
12/19/2007
Urbana
Community Park
12/20/2007
1/4 PDA
12/8/07
88
composite soil
from roots of
collected trees,
2 plates
side branch
from a mature
dead tree with
blue stain
fungus
191
12/19/2007
Urbana
Community Park
12/20/2007
1/4 PDA
12/8/07
trunk with
necrotic
lesions from
young tree,
possibly 3
years old,
inside tissue
showed
yellowing, a
general sign of
disease
192
12/19/2007
Michaux
12/21/2007
1/4 PDA
12/8/07
dead branches
from canopy
of a dead tree
drill dust from
stain in trunk,
assuming it is
fusarium wilt,
2 plates each
AB (3&4) and
PDA (1&2)
female tree 1
necrotic lesion
fungus
female tree 2
necrotic lesion
fungus
193
12/30/2007
Urbana
Community Park
1/5/2008
1/4 PDA
12/8/07 and
AB 12/24/07
194
1/14/2008
Urbana
Community Park
1/15/2008
1/4 PDA
1/12/08
195
1/14/2008
Urbana
Community Park
1/15/2008
1/4 PDA
1/12/08
196
1/14/2008
Urbana
Community Park
1/15/2008
1/4 PDA
1/12/08
necrotic lesion
wound tissue
197
1/14/2008
Urbana
Community Park
not plated
x
necrotic lesion
wound tissue
198
1/14/2008
Urbana
Community Park
1/15/2008
1/4 PDA
1/12/08
199
1/14/2008
Urbana
Community Park
1/15/2008
1/4 PDA
1/12/08
200
1/14/2008
Urbana
Community Park
not plated
x
201
1/14/2008
Michaux
1/15/2008
1/4 PDA
1/12/08
necrotic lesion
sporophytes
202
1/14/2008
Michaux
1/15/2008
1/4 PDA
1/12/08
necrotic lesion
wound tissue
1/15/2008
1/4 PDA
1/12/08
tree G1 tip,
rinsed with
ethanol before
plating
203
1/14/2008
Michaux
89
necrotic lesion
from large
dead tree
necrotic lesion
from older
tree, 1/2
necrotic lesion
from older
tree, 2/2
204
1/14/2008
Michaux
1/15/2008
1/4 PDA
1/12/08
tree G1
necrotic lesion
205
1/14/2008
Michaux
1/15/2008
1/4 PDA
1/12/08
tree G2 tip
206
1/14/2008
Michaux
1/15/2008
1/4 PDA
1/12/08
tree G2
necrotic lesion
207
1/14/2008
Michaux
1/15/2008
1/4 PDA
1/12/08
necrotic lesion
sporophytes
208
1/14/2008
Urbana
Community Park
1/15/2008
1/4 PDA
1/12/08
tip with lesion
209
1/14/2008
Urbana
Community Park
1/15/2008
1/4 PDA
1/12/08
tip with lesion
1/4 PDA
1/12/08
2 dead tips
with white and
pink (orange)
fungi, 1 plate
each white and
orange fungi
210
1/14/2008
Urbana
Community Park
1/15/2008
90
APPENDIX C
Medias, Buffers and Solutions
Growth medias consisted of ¼ strength Potato Dextrose Agar and antibiotic
“clean-up” plates.
Potato dextrose agar (PDA) was suggested by Samson et al. (1996) as one of the
media for growing Fusarium. At my advisor’s suggestion, I tried different strengths of
the PDA from full strength to ⅛, deciding to use ¼ strength. The original plates were
made from the first set of ingredients listed below. This changed during the research to
diluting the Difco PDA with straight agar to the proper strength.
Maltose agar was tried and discarded.
¼ strength PDA from full strength PDA
50g potato
5g table sugar
20g agar (Fisher Bioreagents or Difco)
1000 mL tap water
Unskinned potatoes were cut into small pieces and cooked in the appropriate
amount of tap water for the number of plates to be made. This was then strained through
either a Buchner funnel without filter paper or several layers of cheese cloth. The other
91
ingredients were added and the volume adjusted before autoclaving (Mueller et al.,
2004).
Or
10.0g potato dextrose agar (Difco)
11.25g agar (Fisher Bioreagents or Difco)
1000 mL tap water
This latter recipe was easier and apparently made no difference in the growth of
fungi.
Clean-up plates
0.100 g penicillin
0.300 g streptomycin
20 g agar
10 mL ethanol
1000 mL distilled water
Agar was sterilized in the autoclave. Antibiotics were added first to ethanol. This
slurry was then added to hot agar from the autoclave and swirled to mix. Plates were
then poured.
92
Barz’s Media
Barz Organic Stock
50 g glucose
8 g casein hydrolysate
0.5 g yeast extract
500 mL glass distilled water
Solution A
2 g MgSO4 · 7H2O
0.2 g CaCl2 · 2H2O
0.2 g FeSO4 · 7H2O
1000 mL glass distilled water
Solution B
0.19 g MnSO4
0.25 g NaMoO4
1000 mL glass distilled water
0.5 M PO4 Buffer, pH 7.5
5.44 g KH2PO4
36.58 g K2HPO4
93
500 mL glass distilled water
Barz Salts
10 ml Salts solution A
3 mL Salts solution B
50 mL 0.5M PO4 buffer at pH 7.5
437 mL glass distilled water
Put the solutions in the autoclave separately. Mix 50 mL autoclaved Barz
Organic Stock to each 50 mL of autoclaved Barz Salts.
This was altered in the autoclave step by adding the solutions together in 20 or 50
mL aliquots to Erlenmeyer flasks that were sealed with aluminum foil before autoclaving.
70% ethanol
37.5 mL 95% ethanol + 12.5 mL distilled H2O.
Isolation buffer
85 ml of 1% Sarkosyl (1g + 100 mL glass distilled H2O) + 10 mL 0.5M EDTA + 5 mL
1M pH 8.0 Tris.
Sarkosyl = n-lauroyl sarcosine
94
TE buffer
0.5 mL 1.0M Tris
10 uL EDTA
Running Buffer Solution
5 mL 50X TAE
245 mL distilled water
50X TAE buffer
242 g Tris base
57.1 mL acetic acid
100 mL 0.5M EDTA
Add glass distilled water to 1000 mL while adjusting pH to 8.5.
Agarose plates
0.5 g agarose
50 mL distilled water
1 mL 50X TAE
Add to a 200 mL Erlenmeyer flask. Put into a microwave until all it is entirely
dissolved. Bring back to 50 mL volume. Pour into a “small” tray. For larger plates,
adjust as necessary.
95
APPENDIX D
Fungi DNA Extraction and Identification
1. Pipette 5 mL from 20 mL or 50 mL Barz’ medium in 50 mL Erlenmeyer flask
onto plate containing fungi culture. Stir with sterile spatula or wood splint.
2. Pipette medium from plate back into original Erlenmeyer flask.
3. Incubate flask on shaker plate until enough mycelium are formed to give @
0.100g dry weight mycelium. For fusarium it appears to be 48-72 hours.
4. Filter through medium fine filter paper using vacuum. Separate sample from
filter paper and put sample into a 2 mL centrifuge tube.
5. Lyophilize overnight.
6. Drop centrifuge tube into liquid nitrogen.
7. Grind fungi mycelium into a powder either in the centrifuge tube or a mortar
containing liquid nitrogen. Put on wet ice while grinding other samples.
8. Suspend samples in centrifuge tubes with 0.75 mL isolation buffer.
9. Add one volume* 1:1 phenol:chloroform to the supernatant. Vortex,
centrifuge 5 minutes and save the supernatant.
10. Add one volume chloroform to the supernatant. Vortex, centrifuge 5 minutes
and save the supernatant.
11. Precipitate DNA by adding one tenth volume 3M sodium acetate and one
volume isopropanol to the sample. (This is a break point where samples can
be put into the refrigerator overnight.)
12. Centrifuge 12 minutes to form pellet of DNA.
13. Wash the sample with 70% ethanol then 95% ethanol.
96
14. Dry to dampness in hood (1-2 hours).
15. Add 100 uL TE buffer
16. Refrigerate overnight.
17. Vortex next day.
18. Put in freezer.
* One volume = the same volume as the supernatant. So if there is 0.45 mL supernatant,
add 0.45 mL of extraction solution.
PCR
Reagents were mixed per sample according to the following recipe for one sample and
adjusted according to the number of samples run in the order listed:
36uL H20, 10 uL 10X PCR Buffer, 1 uL EF1 at 40 uM, 1 uL EF2 at 40 mM, 1 uL dNTPs
at 5mM, 0.4 taq DNA polymer and 1 uL sample at 100 ug/uL as determined by a
Pharmacia Biotech Ultrospec 2000.
Samples were then run on a Biorad Genecycler:
Cycle repeats = 1, EID 94C for 3 minutes, 36 cycles, 94C for 45 seconds, EID 55C for 60
seconds, 72C for 60 seconds and 1 cycle at 72C for 60 seconds.
97
During the first EID cycle 5ul of 30mM MgCl2 was added once the Genecycler was at
temperature. For more than six samples, the process was put on pause until the MgCl2
was added to all the samples.
DNA Clean-up and Mailing for Analysis
After the DNA was extracted, 50 mL (analytical size) agarose gel plates (5g agarose/50
mL distilled water + 5uL ethidium bromide solution) were run to identify the band
desired using 12 uL sample sizes and the small combs.
100 mL agarose plates (prep size) were run to isolate the DNA band desired. The band
was cut out of the plates and frozen until the DNA was purified.
The DNA was purified according to the procedure on pages 23 and 24 of the MinElute®
Handbook December 2006 version by Qiagen®. We found that the reagents, especially
ethanol, needed to be fresh, i.e. as close to 200 proof as possible.
To guarantee the purity of the DNA, 2 uL sample +1 uL dye + 8 uL 50X TAE buffer
were run on an analytical sized agarose gel plate with the smallest well size available.
Using a UV spectrophotometer and camera, these samples were analyzed for DNA
quantities. A sample, PCR 16, was used as the basis for determining DNA concentration.
Samples were diluted to an estimated 2 ng/uL as desired by the GENEWIZ® company,
Plainfield, NJ and contained on their web page, before being sent to GENEWIZ® for
base pair analysis.
98
DNA Analysis
The identity of the fusarium pathogens in Ailanthus altissima was done using two
databases, Fusarium-ID v. 1.0 (http://fusarium.cbio.psu.edu/) maintained by Dr. David
Geiser at Penn State and the NCBI database,
(http://www.ncbi.nlm.nih.gov/blast/Blast.cgi?PAGE=Nucleotides&PROGRAM=blastn&
MEGABLAST=on&BLAST_PROGRAMS=megaBlast&PAGE_TYPE=BlastSearch&S
HOW_DEFAULTS=on). The other programs used are the Baylor College of Medicine
HGSC BCM Search Launcher Reverse Complement of Sequence,
(http://searchlauncher.bcm.tmc.edu/seq-util/Options/revcomp.html), Chromas Lite
Freeware v. 2.01, (http://www.technelysium.com.au/chromas_lite.html) and MultAlign,
Multiple sequence alignment by Florence Corpet, (http://bioinfo.genopoletoulouse.prd.fr/multalin/multalin.html).
DNA was extracted according to the methods listed above using QIAGEN®
reagents and methods. Extracted samples were sent to Genewiz, South Plainfield, NJ, for
sequencing. Sequences were then run on the BCM site to get the compliments of
sequences from EF2 to match the EF1 sequences. EF1 and EF2 sequences were then run
through Chromas Lite to clean up the uncertain bases. These were sent to MultAlin to
get consensus. Finally, both the MultAlin and individual EF1 and EF2 sequences were
run on the Penn State fusarium and NCBI databases.
99
APPENDIX E
Fusarium species hosts
Fusarium sp. cf.
bullatum
no information found, assumed to be a saprophyte
Fusarium
moniliforme/Gibberella
warm and tropical climates
fujikuroi
local hosts include Albizia, Philodendron, Pinus, Pseudotsuga and the
grasses maize and sorghum
Fusarium lateritium
wilt, dieback and cankering
hosts include Acer, Albizia, Ailanthus and Juglans
associated with ambrosia beetles
Fusarium lichenicola
Fusarium oxysporum f.
sp. vasinfectum
Fusarium
pallidoroseum
possible accidental isolation from lichen on a necrotic lesion
cotton and other plants
wilt, dieback, cankering, storage rots and damping off
hosts include Acer, Carex and Carya
Fusarium
sporotrichioides
wilt, dieback and cankering
soil and seeds
hosts include Pinus
Fusarium equiseti
Fusarium oxysporum
hosts include Ulmus and Malus clade has many f. sp..
Wide variety of hosts include many temperate trees.
causes vascular wilts.
Fusarium solani
wilt, dieback, cankering, root rots and damping off
hosts include Abies, Acer, Ilex, Juglans, Prunus, Quercus and Thuja
associated with ambrosia beetles
Sources: Farr et al., (1995), Mueller et al., (2004) and W. A. Sinclair and H. H. Lyon (2005)
100
APPENDIX F
PCR Sample Identification
number
sample
number
PCR 1
158aa
PCR 2
location
details
plate date
stem tip
11/12/2007
2/2
173-1 bb1aa
Michaux
Ft.
Frederick
apical end of sapling trunk
11/28/2007
1/2
PCR 3
175bb2aaa
Crone farm
fecal pellets, red
12/17/2007
PCR 4
172-2 baaa
Crone farm
apical end of sapling trunk
12/17/2007
PCR 5
169-3 bb2a
Rt. 273
20 cm pith
11/12/2007
PCR 6
180bb1aa
Michaux
web worm (larva), red
11/21/2007
PCR 7
181bbaaa
"A" 174-4a
PCR 9
"C" 162a
adult web worm
apical end of sapling trunk,
red
bark scrapings (from necrotic
lesion)
12/12/2007
PCR 8
PCR 10
"D" 153a
Michaux
Urbana
Park
Urbana
Park
Urbana
Park
parts from wilted tree, red
PCR 11
5-1.2aaa
mimosa wilt
from Koch's
PCR 12
6-6.1aaa
mimosa wilt
from Koch's
12/7/2007
12/16/2007
from
11/11/2007
11/12/2007
from
11/11/2007
PCR 13
150-1aa
(mim1)
mimosa wilt
for Koch's
PCR 14
150-1aa
(mim2)
mimosa wilt
for Koch's
PCR 15
150-1aa
mimosa wilt
for Koch's
PCR 16
150-1aa
mimosa wilt
for Koch's
PCR 17
179bb1a.1a
Michaux
PCR 18
74-3.1a
PCR 19
186aaa
PCR 20
191aaa
Michaux
Urbana
Park
Urbana
Park
mimosa wilt from F1 tree 5-1
mimosa wilt from F1 tree 6-6
cultured from plate of
mimosa wilt used to
inoculate tray 5 on 9/30/07
cultured from slant of
mimosa wilt used to
inoculate tray 6 on 9/30/07
cultured from slant of
mimosa wilt used to
inoculate tray 5 on 9/30/07
cultured from slant of
mimosa wilt used to
inoculate tray 6 on 9/30/07
stem from plant 5,
inoculation, fecal pellets, red
collected 3/11/07, wilted
branch ends
10 cm
cork cambium from tree with
necrotic lesion
101
12/5/2007
12/7/2007
1/2
2/2
1/2 from
2/2
1/2
2/2
7/30/2007
Y 2/2//1/2
*
7/30/2007
Y
2/2//2/2**
1/10/2008
1/10/2008
1/16/2008
1/11/2008
1/22/2008
1/24/2008
PCR 21
194
Urbana
Park
PCR 22
201aa
PCR 23
1/22/2008
Michaux
female tree 1, necrotic lesion
sporophytes from necrotic
lesion
184mt-aaaa
Michaux
middle of dissected tree
1/23/2008
PCR 24
203aa
Michaux
G1 tip red
1/25/2008
PCR 25
184mt-aaaa
Michaux
1/30/2008
PCR 26
179F1a.1b
PCR 27
191aaaa
Michaux
Urbana
Park
PCR 28
207aaa
Michaux
PCR 29
190aaaa
Urbana
Park
PCR 30
190aaa
Urbana
Park
middle of dissected tree
inoculum from infected
seedling, from Michaux fecal
pellets
cork cambium from tree with
necrotic lesion
sporophytes from necrotic
lesion
vascular cambium from
mature dead tree with blue
stain fungus
yellow tissue from dead
mature tree with blue stain
fungus
PCR 31
206aa
Michaux
red fungi from lesion
1/24/2008
PCR 32
204aa
Michaux
necrotic lesion, G1, red
1/24/2008
PCR 33
Michaux
necrotic lesion, G1, yellow
1/24/2008
PCR 34
204aa
5a mimosa
wilt slant
mimosa wilt
inoculum
9/30/2007
PCR 35
5-7.1aaa
mimosa wilt
F1 from infected plant
11/11/2007
PCR 36
mimosa wilt
F1 from infected plant
11/11/2007
PCR 37
5-7.1aaa
5-1.1aa
purple
mimosa wilt
F1 from infected plant
11/11/2007
PCR 38
5-7.2aa
mimosa wilt
F1 from infected plant
11/11/2007
PCR 39
6-3.1aa
150-1aa Y
1/2 1/3
mimosa wilt
11/11/2007
mimosa wilt
F1 from infected plant
one of plates cultured to use
as inoculum
lesion
F1 from infected plant
11/11/2007
Michaux
tree G2 tip
1/15/2008
PCR 43
74-3Xaa 1/2
205aaa B a
1/2
205aaa A a
1/2
Michaux
tree G2 tip
1/15/2008
*plate 1/2
from plate
1/3 of plate
labeled Y
PCR 40
PCR 41
PCR 42
**plate 2/2 from plate 1/3 of
plate labeled Y
102
1/23/2008
1/30/2008
1/27/2008
1/27/2008
1/25/2008
1/25/2008
9/23/2007
APPENDIX G
Edited PCR Consensus Sequences
Note: all lower case letters are the edits. All upper case letters are the original bases received
from Genewiz.
PCR1 CONSENSUS
cGaGG gaCccca CGTCagagTCa TG aTaaaATcAc GGTGACCGGG AGCGTCTGAA GTACATGTTA
GCCATGAGAA AAGTATTGAG TGTAAGTGAC GATAACGTAC CAATGACGGT GACATAGTAG
CGAGGAGTCT CGAACTTCCA GAGGGCGATA TCGATGGTGA TACCACGCTC ACGCTCAGCC
TTGAGCTTGT CAAGAACCCA GGCGTACTTG AAGGAACCCT TACCGAGCTC GGCGGCTTCC
TATTGTCGGG TGGTTAGTGG CTGATGGACA CGTGATGCAC AAGACATGAG TTTCTGGGAA
GAGGGCAAAC GTCTGTCGCT CGAGTGGCGG GGTTGAAACC CCACCAAAAA AAATTACGGT
TGAACCGCAA AATTTTGTAC TCGAGCGGGG TAACAGGCGC ATATTCAATC GTCGTAACTG
ATTCGACTGA TGGATCGGTG GGTAGAGGGC GTGCGATCGG GGAAATGGAA ACCAACCTTC
TCGAACTTCT CGATGGTTCG CTTGTCGATA CCACCGCACT GGTAGATCAA GTGACCGGTC
TATGCAATCT TGTCAGCAAA TATTCAAGTT GAAATTACCC TGCCACATCT GGCGGGGTTG
ATGACTGCTG ATAAGCAAAT CATCGTGGGT AGTACTCACA GTGGTCGACT .GCCAGAGTC
GACggGcCag AcaaacAcGa c......... AGTCTTGCCC CTTTCCCCCT AAAA
PCR 4 CONSENSUS
TTGGAAGGTA CCCCCCGATC ATGTTCTTGA TGAAATCaca cgGGcCGGGG GCGTCTGTTG
ATTGTTAGTG ATGAGACGGA AGTGGGAGAG ATGAGGGCGA CATACCAATG ACGGTGACAT
AGTAGCGGGG AGTCTCGAAC TTCCAGAGAG CAATATCGAT GGTGATACCA CGCTCACGCT
CGGCCTTGAG CTTGTCAAGG ACCCAGGCGT ACTTGAAGGA ACCCTTACCG AGCTCAGCGG
CTTCCTATTG TTGAACCTGT TAGTGTCTGT TGTGAACCAC GTGATGCGCG CCAAGAGGGT
TTGGTGTTTT TTGTGTGCAG GGTTCAGGGC TCGTCCAACG TCGCCCGAGT GGCGGGGTAA
ATGCCCCACC AAAAAAATTA CGGTCGAACC GCAAAATTTT TGGGACTCGG GAGAAGCGGG
CGCAGAGCGT GTCGCGGAAG AGGGAATTCG ACGGGGAATT CGATGTGGAA TAGCAAGGCG
CGATCGGGGG AGATGTCACC AACCTTCTCG AACTTCTCGA TGGTTCGCTT GTCGATACCA
CCGCACTGGT AGATCAAGTG ACCGGTCTGT AGATGATTGT CAGCATGAAG TGACTGATGA
GTACCCCGCC CGAGATACCA GGCGGGGTTC CACGACCCGA GATAAGCAGA TCGCGATGAG
GGCTTGACTT ACGGTGGTCG ACTTGCCAGA GTCGACggGc CagacGaaaa cGACGTTGAG
GTGAGTCTTG C
PCR6 CONSENSUS
CGACGGTGAC GGGAACGTCT GTATGAGGTG TTAGATGAGG CATGTGAATG AGAGCAGTAG
TGACAACATA CCAATGACGG TGACATAGTA GCGGGGAGTC TCGAACTTCC ACAGGGCAAT
GTCGATGGTG ATACCACGCT CACGCTCGGC TTTGAGCTTG TCAAGAACCC AGGCGTACTT
GAAAGAACCC TTTCCAAGCT CGGCGGCTTC CTATTGTCGA TGGTGGTTAG CAACTATCGG
ATCACATGAT GACGCGTGCC TGGGATGGGT ATTGAGTTTT GTGTGTAGGG ATCAGGGCAA
GCGCCCATCG CTCGAGTGGC GGGGTATGAT GCCCCACCAA AAAAAAAATT ACGGTCGCAC
CGCAAAATTT TTGAGCTCAA GCGGGGTAAT GGGCGCATTG CGAGTCGTGA GGTAGCGATT
CGAAGGACAA ATCGATGGGC AgAAGGCGCG CGATCGGGGG AGAAATGGAC CAACCTTCTC
GAACTTCTCG ATGGTTCGCT TGTCGATACC ACCGCACTGG TAGATCAAGT GACCGGTCTA
TCCAAAGCTG TTAGCACGAT GTGACTGTGA AATACCTCGC CAGTCTCCGG CAGGTTTTGA
CGTATGCAGA TAAGCACATT GTCGAAAGGG TAGTACTCAC AGTGGTCGAC T.GCCAGAGT
CGAC.TGGCC a..GACGACG aaagTAAGGT GAGTCTTGTC CTCCCTTACC CATAAA
103
PCR8 CONSENSUS
TTGGAGGTAC CCAGTGATCA TGTTCTTGAT GAAGccACGG TGACcGGGAG CGTCTGTATG
AGGTGTTAGA TGAGGCATGT GAATGAGAGC AGTAGTGACA ACATACCAAT GACGGTGACA
TAGTAGCGGG GAGTCTCGAA CTTCCACAGG GCAATGTCGA TGGTGATACC ACGCTCACGC
TCGGCTTTGA GCTTGTCAAG AACCCAGGCG TACTTGAAAG AACCCTTTCC AAGCTCGGCG
GCTTCCTATT GTCGATGGTG GTTAGCAACT ATCGGATCAC ATGATGACGC GTGCCTGGGA
TGGGTATTGA GTTTTGTGTG TAGGGATCAG GGCAAGCGCC CATCGCTCGA GTGGCGGGGT
ATGATGCCCC ACCAAAAAAA AATTACGGTC GCACCGCAAA ATTTTTGAGC TCAAGCGGGG
TAATGGGCGC ATTGCGAGTC GTGAGGTAGC GATTCGAAGG ACAAATCGAT GGGCAGAAGG
CGCGCGATCG GGGGAGAAAT GGACCAACCT TCTCGAACTT CTCGATGGTT CGCTTGTCGA
TACCACCGCA CTGGTAGATC AAGTGACCGG TCTATCCAAA GCTGTTAGCA CGATGTGACT
GTGAAATACC TCGCCAGTCT CCGGCAGGTT TTGACGTATG CAGATAAGCA CATTGTCGAA
AGGGTAGTAC TCACAGTGGT CGACT.GCCA GAGTCGACgg GcCagaacaa cACaAcGTTA
AGGTGAGTCT TGTCCCCCAT TACCCATAAA
PCR10 CONSENSUS
TTTTTTGGGG AAAAGGGGGC AAGACTCACC TTAACGccgg CGTcaacGGC CAcgccaacc
cgGGCAAGTC GACCACTGTG AGTACATCTG CATCACAACC CCGCCCAGAC TTGGCGGGGT
AGTTTCAATC ATCATTTTTA CTGACATGCT TTGACAGACC GGTCACTTGA TCTACCAGTG
CGGTGGTATC GACAAGCGAA CCATCGAGAA GTTCGAGAAG GTTGGTCTCA TTTTCCTCGA
TCGCGCGCCC TTCTTCCCAT CGACCCATCA TTCGAATCGC TCTCATACGA CGACTCGACA
AGCGCCTGTT ACCCCGCTCG AGTTCAAAAA TTTCACGGCT GTGTCGTGAT TTTTTTGATA
GTGGGGCTCA TACCCCGCCG CTCGAGTGAC AGGCGCTTTT GCCCTTCCCA CACATCCATT
TACATGGGCG CGCATCATCA CGTGTCAATC AGTCACTAAC CACCTGTCAA TAGGAAGCCG
CCGAGCTCGG TAAGGGTTCC TTCAAGTACG CCTGGGTTCT TGACAAGCTC AAAGCCGAGC
GTGAGCGTGG TATCACCATT GATATCGCTC TCTGGAAGTT CGAGACTCCT CGCTACTATG
TCACCGTCAT TGGTATGTTG TCACTATTGC CTTCATCACA TTCTCATACT AACATGCCTA
CCAGACGccC C..GTCACCG TcaTTTCATC AAGAACATGA CGGGGGGGAC CCCTCCAAA
PCR11 CONSENSUS
.TATgggTaa AGGAaGcAGg GACTCACCTT AACGTCGTCG TCATCGGCCA ....GACTCT
GGCA.GTCGA CCACTGTGAG TACTCTCCTC GACAATGAGC ATATCTGCCA TCGTCAATCC
CGACCAAGAC CTGGCGGGGT ATTTCTCAAA GTCAACATAC TGACATCGTT TCACAGACCG
GTCACTTGAT CTACCAGTGC GGTGGTATCG ACAAGCGAAC CATCGAGAAG TTCGAGAAGG
TTAGTCACTT TCCCTTCAAT CGCGCGTCCT TTGCCCATCG ATTTCCCCTA CGACTCGAAA
CGTGCCCGCT ACCCCGCTCG AGACCAAAAA TTTTGCAATA TGACCGTAAT TTTTTTGGTG
GGGCACTTAC CCCGCCACTT GAGCGACGGG AGCGTTTGCC CTCTTACCAT TCTCACAACC
TCAATGAGTG CGTCGTCACG TGTCAAGCAG TCACTAACCA TTCAACAATA GGAAGCCGCT
GAGCTCGGTA AGGGTTCCTT CAAGTACGCC TGGGTTCTTG ACAAGCTCAA GGCCGAGCGT
GAGCGTGGTA TCACCATCGA ACCGTCATTG GaATGTTGTC GCTCATGCTT CATTCTACTT
CTCTTCGTAC TAACATATCA CCCAGACGCT CCCGGcCaCa GTGATTTCAT CAAGAACATG
ATCATGGGTA CCTCCAA
104
PCR12 CONSENSUS
TTATGGGTAA AGGAGGACAA GACTCACCcT AaCgTCGgCa TCATCGGCCA .GTCGACTCT
GGCA.GTCGA CCACTGTGAG TACTCTCCTC GACAATGAGC ATATCTGCCA TCGTCAATCC
CGACCAAGAC CTGGCGGGGT ATTTCTCAAA GTCAACATAC TGACATCGTT TCACAGACCG
GTCACTTGAT CTACCAGTGC GGTGGTATCG ACAAGCGAAC CATCGAGAAG TTCGAGAAGG
TTAGTCACTT TCCCTTCAAT CGCGCGTCCT TTGCCCATCG ATTTCCCCTA CGACTCGAAA
TTTTTTGGTG GGGCACTTAC CCCGCCACTT GAGCGACGGG AGCGTTTGCC CTCTTACCAT
TCTCACAACC TCAATGAGTG CGTCGTCACG TGTCAAGCAG TCACTAACCA TTCAACAATA
GGAAGCCGCT GAGCTCGGTA AGGGTTCCTT CAAGTACGCC TGGGTTCTTG ACAAGCTCAA
GGCCGAGCGT GAGCGTGGTA TCACCATCGA TATTGCTCTC TGGAAGTTCG AGACTCCTCG
CTACTATGTC ACCGTCATTG GaATGTTGTC GCTCATGCTT CATTCTACTT CTCTTCGTAC
TAACATATCA CCCAGACGCc CCcccgcac. GTGATTTCAT CAAGAACATG ATCATGGGNT
ACCTCCAA
PCR14 CONSENSUS
TTATGGGTAA GGAGGACAAG ACTCACCTTA ACGTCGTagg caTCgGcCAc aTCGACTCTG
GCAAGTCGAC CACTGTGAGT ACTCTCCTCG ACAATGAGCA TATCTGCCAT CGTCAATCCC
GACCAAGACC TGGCGGGGTA TTTCTCAAAG TCAACATACT GACATCGTTT CACAGACCGG
TCACTTGATC TACCAGTGCG GTGGTATCGA CAAGCGAACC ATCGAGAAGT TCGAGAAGGT
TAGTCACTTT CCCTTCAATC GCGCGTCCTT TGCCCATCGA TTTCCCCTAC GACTCGAAAC
GTGCCCGCTA CCCCGCTCGA GACCAAAAAT TTTGCAATAT GACCGTAATT TTTTTGGTGG
GGCACTTACC CCGCCACTTG AGCGACGGGA GCGTTTGCCC TCTTACCATT CTCACAACCT
CAATGAGTGC GTCGTCACGT GTCAAGCAGT CACTAACCAT TCAACAATAG GAAGCCGCTG
AGCTCGGTAA GGGTTCCTTC AAGTACGCCT GGGTTCTTGA CAAGCTCAAG GCCGAGCGTG
AGCGTGGTAT CACCATCGAT ATTGCTCTCT GGAAGTTCGA GACTCCTCGC TACTATGTCA
CCGTCATTGG TATGTTGTCG CTCATGCTTC ATTCTACTTC TCTTCGTACT AACATATCAC
CCAGACGccC C..GTCACCG TaagTTCATC TCATGGGTAC
PCR16 CONSENSUS
GGCAAGACTC ACCTTAACGT CGTCGTCagC ccccaCagcG ACTCTGGCA. GTCGACCACT
GTGAGTACTC TCCTCGACAA TGAGCATATC TGCCATCGTC AATCCCGACC AAGACCTGGC
GGGGTATTTC TCAAAGTCAA CATACTGACA TCGTTTCACA GACCGGTCAC TTGATCTACC
AGTGCGGTGG TATCGACAAG CGAACCATCG AGAAGTTCGA GAAGGTTAGT CACTTTCCCT
TCAATCGCGC GTCCTTTGCC CATCGATTTC CCCTACGACT CGAAACGTGC CCGCTACCCC
GCTCGAGACC AAAAATTTTG CAATATGACC GTAATTTTTT TGGTGGGGCA CTTACCCCGC
CACTTGAGCG ACGGGAGCGT TTGCCCTCTT ACCATTCTCA GAGTGCGTCG TCACGTGTCA
AGCAGTCACT AACCATTCAA CAATAGGAAG CCGCTGAGCT CGGTAAGGGT TCCTTCAAGT
ACGCCTGGGT TCTTGACAAG CTCAAGGCCG AGCGTGAGCG TGGTATCACC ATCGATATTG
CTCTCTGGAA GTTCGAGACT CCTCGCTACT ATGTCACCGT CATTGGTATG TTGTCGCTCA
TGCTTCATTC TACTTCTCTT CGTACTAACA TATCACCCAG ACGccCCccc ccaaCGTGAT
TTCATCAAGA ACATGACG
105
PCR18 CONSENSUS
TTTATGGGTA AAGGGGAGAC AAGACTCACC TTAACGTCgg cGTCaTagGc CA.GTCGACT
CTGGCAAGTC GACCACTGTG AGTACTACCC TCAATGACCT GCTTATCAGC AGTCATCAAC
CCCCCCATAC GTGGCGGGGT AATTTCATTT TGGATATCTG CTAACAAAAT TGCATAGACC
GGTCACTTGA TCTACCAGTG CGGTGGTATC GACAAGCGAA CCATCGAGAA GTTCGAGAAG
GTTGGTTTCC ATTTTCCTCG ATCGCGCGTC CTCTGCCCAC CGATCCATCA CCCGAATCCG
TCTCACGACG ACTGAATATG CGCCTGTTAC CCCGCTCGAG TACAAAATTT TGCGGTTCAA
TCGTAATTTT TTGGTGCGGC TTCTACCCCG GACAGGTGTT TGCCCTTTCC CACAAAATCA
TCTTGCGCAT CACGTGTCAA ACAGTCACTA ACCACCCGAC AATAGGAAGC CGCCGAGCTC
GGTAAGGGTT CCTTCAAGTA CGCTTGGGTT CTTGACAAGC TCAAGGCCGA GCGTGAGCGT
GGTATCACCA TCGATATCGC CCTCTGGAAG TTCGAGACTC CCCGCTACTA TGTCACCGTC
ATTGGTATGT TGTCATCGCT TGCACTCATT ACTTTCTCAT GCTAACATGT GCT.CAGACG
CTCCCGgcaa CTCCCGGTCA CcgcGATTTC ATCAAGAACA TGATCCGGGG GTACCTCCAA
AAAATGC
PCR21 CONSENSUS
TTTTATGGTA AGGGGGACAA GACTCACCTT .aaggcGTCG TCATcGcCa. .GTCGACTCT
GGCA.GTCGA CCACCGTAAG TCAAGCCCTC ATCGCGATCT GCTTATCTCG GGTCGTGAAA
CCCCGCCTGG TATCTCGGGC GGGGTACTCA TCAGTCACTT AATGCTGACA ATCATCTACA
GACCGGTCAC TTGATCTACC AGTGCGGTGG TATCGACAAG CGAACCATCG AGAAGTTCGA
GAAGGTTGGT GACATCTCCC CGATCGCGCC TTGCTATTCC ACATCGAATT CCCCGTCGAA
TTCCCTCTTC CGCGACACGC TCTGCGCCCG CTTCTCCCGA GTCCCAAAAA TTTTGCGGTT
CGACCGTAAT TTTTTTGGTG GGGCATTTAC CCCGCCACTC GGGCGACGTC GGACGAGCCC
TGAACCCTGC ACACAAAAAA CACCAAACCC TCTTGGCGCG CATCACGTGG TTCACAACAG
ACACTAACTG GTTCAACAAT AGGAAGCCGC TGAGCTCGGT AAGGGTTCCT TCAAGTACGC
GACAAGCTCA AGGCCGAGCG TGAGCGTGGT ATCACCATCG ATATTGCTCT CTGGAAGTTC
GAGACTCCCC GCTACTATGT CACCGTCAT. TGGTATGTCG CCCTCATCTC TCTCAATCAC
GTCTCATCAC TAACAATCAA CAGACGCCCC CGcCaaCcgc GATTTCATCA AGAACATGAT
CNTGGGGTAC CTTCCAAA
PCR25 CONSENSUS
TTTTTGGGGT AAAAGGGGAC AAGACTCACC TTAACGTCGT aggcaTagGc CaaGTCGACT
CTGGCA.GTC GACCACTGTG AGTACTCTCC TCGACAATGA GCATATCTGc CATCGTCAAT
CCCGACCAAG ACCTGGCGGG GTATTTCTCA AAGTCAACAT ACTGACATCG TTTCACAGAC
CGGTCACTTG ATCTACCAGT GCGGTGGTAT CGACAAGCGA ACCATCGAGA AGTTCGAGAA
GGTTAGTCAC TTTCCCTTCA ATCGCGCGTC CTTTGCCCAT CGATTTCCCC TACGACTCGA
AACGTGCCCG CTACCCCGCT CGAGACCAAA AATTTTGCAA TATGACCGTA ATTCTCACAA
ATTTTTTTGG TGGGGCACTT ACCCCGCCAC TTGAGCGACG GGAGCGTTTG CCCTCTTACC
CCTCAATGAG TGCGTCGTCA CGTGTCAAGC AGTCACTAAC CATTCAACAA TAGGAAGCCG
CTGAGCTCGG TAAGGGTTCC TTCAAGTACG CCTGGGTTCT TGACAAGCTC AAGGCCGAGC
GTGAGCGTGG TATCACCATC GATATTGCTC TCTGGAAGTT CGAGACTCCT CGCTACTATG
TCACCGTCAT TGGTATGTTG TCGCTCATGC TTCATTCTAC TTCTCTTCGT ACTAACATAT
CACCCAGACG CTCCCGgcCa ccacGATTTC ATCAAGAACA TGATCACTGG GTACCTCCAA
AAAAA
106
PCR 27 CONSENSUS
TTTTTTTGGG GTGAAGGGGG CAGACTCACC TTAACGTCGT CGccacCagc caCGTCGACT
CTGGCA.GTC GACCACTGTG AGTACTACCC TCGACGATGA GCTTATCTGT CATCGTGATC
CTGACCAAGA TCTGGCGGGG TATATCTCAG AAGACAATAT GCTGACATCG CTTCACAGAC
CGGTCACTTG ATCTACCAGT GCGGTGGTAT CGACAAGCGA ACCATCGAGA AGTTCGAGAA
GGTTAGTCAC TTTCCCTTCG ATCGCGCGTC CTTTGCCCAC CGATTTCCCT TACGATTCGA
AACGTGCCTG CTACCCCGCT CGAGACCAAA AATTTTGCGA TATGACCGTA ATTTTTTTTG
GTGGGGCATT TACCCCGCCA CTCGAGTGAT GGGCGCGTTT TGCCCTTTCC TGTCCACAAC
CTCAATGAGC GCATTGTCAC GTGTCAAACT AACCATTCGA CAATAGGAAG CCGCTGAGCT
CGGTAAGGGT TCCTTCAAGT ACGCCTGGGT TCTTGACAAG CTCAAGGCCG AGCGTGAGCG
TGGTATCACC ATCGATATTG CTCTCTGGAA GTTCGAGACT CCTCGCTACT ATGTCACCGT
CATTGGTATG TTGTCGCTCA TGCTTCATTC TACTTATTCA TACTAACATA TCATTCAGAC
GCTCCCGGTC ACCacGATTT CATCAAGAAC AGCGGGGGGG GCCCCCCCAA AAAA
PCR28 CONSENSUS
TTTTTATGGG TAAAGGGGGA CAAGACTCAC CTCAACGcCG TcgcCATCGG CCACGTCGAC
TCTGGCA.GT CGACCACCGT AAGTCAAGCC CTCATCGCGA TCTGCTTATC TCGGGTCGTG
GAACCCCGCC TGGTATCTCG GGCGGGGTAC TCATCAGTCA CTTCATGCTG ACAATCATCT
ACAGACCGGT ACCAGTGCGG TGGTATCGAC AAGCGAACCA TCGAGAAGTT CGAGAAGGTT
GGTGATATCT CCCCCGATCG CGCCTTGCTA TTCCACATCG AATTCCCCGT CGAATTCCCT
CCTCCGCGAC ACGCTCTGCG CCCGCTTCTC CCGAGTCCCA AAAAATTTGC GGTTCGACCG
TAATTTTTTT GGTGGGGCAT TTACCCCGCC ACTCGGGCGA CGTTGGACAA AGCCCTGATC
CCTGCACACA AAAACACCAA ACCCTCTTGG CGCGCATCAC GTGGTTCACA ACAGACACTG
ACTGGTTCAA CAATAGGAAG CCGCTGAGCT CGGTAAGGGT TCCTTCAAGT ACGCCTGGGT
CCTTGACAAG CTCAAGGCCG AGCGTGAGCG TGGTATCACC ATCGATATTG CTCTCTGGAA
GTTCGAGACT CCCCGCTACT ATGTCACCGT CATTGGTATG TCGCCGTCAT CTCTCTCACT
CACGTCTCAT CACTAACAGT CAAaaaaacc CCCCGGCCAC CGcGATTTCA TCAAGAACAT
GATCGGGGGG ACCCCTTCCA
PCR29 CONSENSUS
TTTcTgncGc TAAgaaTGGc GGGAGGACAA GACTCACCTT AACGTCGTCG TCATCGGCCA
CGTCGACTCT GGCA.GTCGA CCACTGTGAG TACTACCCTC GACGATGAGC TTATCTGTCA
TCGTGATCCT GACCAAGATC TGGCGGGGTA TATCTCAGAA GACAATATGC TGACATCGCT
TCACAGACCG GTCACTTGAT CTACCAGTGC GGTGGTATCG ACAAGCGAAC CATCGAGAAG
TTCGAGAAGG TTAGTCACTT TCCCTTCGAT CGCGCGTCCT TTGCCCACCG ATTTCCCTTA
CGATTCGAAA CGTGCCTGCT ACCCCGCTCG AGACCAAAAA TTTTGCGATA CCCTTTCCTG
TGACCGTAAT TTTTTTTGGT GGGGCATTTA CCCCGCCACT CGAGTGATGG GCGCGTTTTG
TCCACAACCT CAATGAGCGC ATTGTCACGT GTCAAACTAA CCATTCGACA ATAGGAAGCC
GCTGAGCTCG GTAAGGGTTC CTTCAAGTAC GCCTGGGTTC TTGACAAGCT CAAGGCCGAG
CGTGAGCGTG GTATCACCAT CGATATTGCT CTCTGGAAGT TCGAGACTCC TCGCTACTAT
GTCACCGTCA TTGGTATGTT GTCGCTCATG CTTCATTCTA CTTATTCATA CTAACATATC
ATTCaaaacc ccCCcGTCAC CGTaagTTCA TCAAGAACAT
107
PCR31 CONSENSUS
TTGGCAAGTC GACCACTGTG AGTACTACCC TTTTCGACAA TGTGCTTATC TGCATACGTC
AAAACCTGCC GGAGACTGGC GAGGTATTTC ACAGTCACAT CGTGCTAACA GCTTTGGATA
GACCGGTCAC AGTGCGGTGG TATCGACAAG CGAACCATCG AGAAGTTCGA GAAGGTTGGT
CCATTTCTCC CCCGATCGCG CGCCTTCTGC CCATCGATTT TCGCTACCTC ACGACTCGCA
ATGCGCCCAT TACCCCGCTT GAGCTCAAAA ATTTTGCGGT GCGACCGTAA TTTTTTTTTT
GGTGGGGCAT CACTCGAGCG ATGGGCGCTT GCCCTGATCC CTACACACAA AACTCAATAC
CCATCCCAGG CACGCGTCAT CATGTGATCC GATAGTTGCT AACCACCATC GACAATAGGA
AGCCGCCGAG CTTGGAAAGG GTTCTTTCAA GTACGCCTGG GTTCTTGACA AGCTCAAAGC
CGAGCGTGAG CGTGGTATCA CCATCGACAT TGCCCTGTGG AAGTTCGAGA CTCCCCGCTA
CTATGTCACC GTCATaaGTA TGTTGTCACT ACTGCTCTCA TTCACATGCC TCATCTAACA
CCTCATACAG ACG.TCCCG. TCACCGTaC TTCATCAAGA ACATGATCAC TGGGTACCTC CAA
108
APPENDIX H
Selected Edited PCR Sequences Accession Data
This is the Accession data for a limited number of significant PCR sequence identifications.
PCR4 EF1
Penn State
>292 Nectria haematococca/Fusarium sp. cf. solani mpVI isolate NRRL
22586 translation elongation factor 1 alpha gene
Length = 677
Score = 1183 bits (597), Expect = 0.0
Identities = 655/669 (97%), Gaps = 2/669 (0%)
Strand = Plus / Minus
NCBI
gb|DQ247155.1| Fusarium solani strain NRRL 32849 translation elongation factor
1-alpha (EF1-alpha) gene, partial cds
Length=677
Score = 1236 bits (669), Expect = 0.0
Identities = 674/676 (99%), Gaps = 2/676 (0%)
PCR4 EF2
Penn State
292 Nectria haematococca/Fusarium sp. cf. solani mpVI isolate NRRL
22586 translation elongation factor 1 alpha gene
Length = 677
Score = 1168 bits (589), Expect = 0.0
Identities = 647/661 (97%), Gaps = 2/661 (0%)
NCBI
gb|DQ247155.1| Fusarium solani strain NRRL 32849 translation elongation factor
1-alpha (EF1-alpha) gene, partial cds
Length=677
Score = 1225 bits (663), Expect = 0.0
Identities = 668/670 (99%), Gaps = 2/670 (0%)
PCR 4 CONSENSUS
Penn State
>292 Nectria haematococca/Fusarium sp. cf. solani mpVI isolate NRRL
22586 translation elongation factor 1 alpha gene
Length = 677
Score = 1158 bits (584), Expect = 0.0
Identities = 642/656 (97%), Gaps = 2/656 (0%)
Strand = Plus / Minus
NCBI
RID: 3KMVJBKC015
gb|DQ247155.1| Fusarium solani strain NRRL 32849 translation elongation factor
1-alpha (EF1-alpha) gene, partial cds
Length=677
Score = 1214 bits (657), Expect = 0.0
Identities = 665/668 (99%), Gaps = 3/668 (0%)
109
PCR6 EF1
Penn State
>614 Fusarium 'Lateritium Clade IIA' isolate FRC L-200 translation
elongation factor 1-alpha gene
Length = 664
Score = 1047 bits (528), Expect = 0.0
Identities = 635/666 (95%), Gaps = 4/666 (0%)
NCBI
gb|DQ295133.1| Fusarium lateritium isolate F0103 translation elongation factor-1
alpha (tef1) gene, exons 1 through 4 and partial cds
Length=680
Score = 1155 bits (625), Expect = 0.0
Identities = 662/680 (97%), Gaps = 2/680 (0%)
PCR6 EF2
Penn State
>614 Fusarium 'Lateritium Clade IIA' isolate FRC L-200 translation
elongation factor 1-alpha gene
Length = 664
Score = 993 bits (501), Expect = 0.0
Identities = 619/651 (95%), Gaps = 5/651 (0%)
NCBI
gb|DQ295133.1| Fusarium lateritium isolate F0103 translation elongation factor-1
alpha (tef1) gene, exons 1 through 4 and partial cds
Length=680
Score = 1146 bits (620), Expect = 0.0
Identities = 661/680 (97%), Gaps = 5/680 (0%)
PCR6 CONSENSUS
Penn State
>614 Fusarium 'Lateritium Clade IIA' isolate FRC L-200 translation
elongation factor 1-alpha gene
Length = 664
Score = 993 bits (501), Expect = 0.0
Identities = 619/651 (95%), Gaps = 5/651 (0%)
NCBI
RID: 3KPNNBCB01
gb|DQ295133.1| Fusarium lateritium isolate F0103 translation elongation factor-1
alpha (tef1) gene, exons 1 through 4 and partial cds
Length=680
Score = 1134 bits (614), Expect = 0.0
Identities = 659/680 (96%), Gaps = 6/680 (0%)
PCR8 EF1
Penn State
>602 Fusarium 'Lateritium Clade IIA' isolate FRC L-81 translation
elongation factor 1-alpha gene
Length = 664
Score = 1065 bits (537), Expect = 0.0
Identities = 639/665 (96%), Gaps = 3/665 (0%)
NCBI
110
RID: 3KS13H0T015
gb|DQ295133.1| Fusarium lateritium isolate F0103 translation elongation factor-1
alpha (tef1) gene, exons 1 through 4 and partial cds
Length=680
Score = 1164 bits (630), Expect = 0.0
Identities = 663/679 (97%), Gaps = 2/679 (0%)
PCR8 EF2
Penn State
>602 Fusarium 'Lateritium Clade IIA' isolate FRC L-81 translation
elongation factor 1-alpha gene
Length = 664
Score = 1011 bits (510), Expect = 0.0
Identities = 623/650 (95%), Gaps = 4/650 (0%)
NCBI
gb|DQ295133.1| Fusarium lateritium isolate F0103 translation elongation factor-1
alpha (tef1) gene, exons 1 through 4 and partial cds
Length=680
Score = 1164 bits (630), Expect = 0.0
Identities = 663/679 (97%), Gaps = 2/679 (0%)
PCR8 CONSENSUS
Penn State
>602 Fusarium 'Lateritium Clade IIA' isolate FRC L-81 translation
elongation factor 1-alpha gene
Length = 664
Score = 1013 bits (511), Expect = 0.0
Identities = 624/651 (95%), Gaps = 4/651 (0%)
NCBI
RID: 3KSBMD8X015
gb|DQ295133.1| Fusarium lateritium isolate F0103 translation elongation factor-1
alpha (tef1) gene, exons 1 through 4 and partial cds
Length=680
Score = 1146 bits (620), Expect = 0.0
Identities = 653/669 (97%), Gaps = 2/669 (0%)
PCR11 EF1
Penn State
146 Fusarium oxysporum f sp melonis NRRL: 26173
Length = 649
Score = 1227 bits (619), Expect = 0.0
Identities = 619/619 (100%)
NCBI
gb|DQ452422.1| Fusarium solani f. sp. piperis strain MAFF 236575 translation
elongation factor gene, partial sequence
Length=712
Score = 1197 bits (648), Expect = 0.0
Identities = 658/662 (99%), Gaps = 3/662 (0%)
gb|DQ452427.1| Fusarium oxysporum f. sp. cucumerinum strain ATCC 16416 translation
elongation factor gene, partial sequence
111
Length=712
Score = 1181 bits (639), Expect = 0.0
Identities = 655/662 (98%), Gaps = 3/662 (0%)
PCR11 EF2
Penn State
>146 Fusarium oxysporum f sp melonis NRRL: 26173
Length = 649
Score = 1261 bits (636), Expect = 0.0
Identities = 639/640 (99%)
NCBI
gb|DQ452422.1| Fusarium solani f. sp. piperis strain MAFF 236575 translation
elongation factor gene, partial sequence
Length=712
Score = 1223 bits (662), Expect = 0.0
Identities = 669/672 (99%), Gaps = 1/672 (0%)
gb|DQ837696.1| Fusarium oxysporum f. sp. melonis isolate TX388 translation elongation
factor 1 alpha (EF-1alpha) gene, partial cds
Length=711
Score = 1199 bits (649), Expect = 0.0
Identities = 664/671 (98%), Gaps = 1/671 (0%)
PCR11 CONSENSUS
Penn State
>146 Fusarium oxysporum f sp melonis NRRL: 26173
Length = 649
Score = 983 bits (496), Expect = 0.0
Identities = 496/496 (100%)
NCBI
RID: 3KSGGRBN015
gb|DQ452422.1| Fusarium solani f. sp. piperis strain MAFF 236575 translation
elongation factor gene, partial sequence
Length=712
Score = 976 bits (528), Expect = 0.0
Identities = 550/559 (98%), Gaps = 8/559 (1%)
gb|DQ452427.1| Fusarium oxysporum f. sp. cucumerinum strain ATCC 16416 translation
elongation factor gene, partial sequence
Length=712
Score = 965 bits (522), Expect = 0.0
Identities = 548/559 (98%), Gaps = 8/559 (1%)
PCR12 EF1
Penn State
>146 Fusarium oxysporum f sp melonis NRRL: 26173
Length = 649
Score = 1229 bits (620), Expect = 0.0
Identities = 631/632 (99%), Gaps = 1/632 (0%)
NCBI
gb|DQ452422.1| Fusarium solani f. sp. piperis strain MAFF 236575 translation
elongation factor gene, partial sequence
Length=712
112
Score = 1203 bits (651), Expect = 0.0
Identities = 661/665 (99%), Gaps = 4/665 (0%)
gb|DQ452427.1| Fusarium oxysporum f. sp. cucumerinum strain ATCC 16416 translation
elongation factor gene, partial sequence
Length=712
Score = 1186 bits (642), Expect = 0.0
Identities = 658/665 (98%), Gaps = 4/665 (0%)
PCR12 EF2
Penn State
>146 Fusarium oxysporum f sp melonis NRRL: 26173
Length = 649
Score = 1251 bits (631), Expect = 0.0
Identities = 634/635 (99%)
NCBI
gb|DQ452422.1| Fusarium solani f. sp. piperis strain MAFF 236575 translation
elongation factor gene, partial sequence
Length=712
Score = 1218 bits (659), Expect = 0.0
Identities = 668/672 (99%), Gaps = 2/672 (0%)
Strand=Plus/Minus
gb|DQ452427.1| Fusarium oxysporum f. sp. cucumerinum strain ATCC 16416 translation
elongation factor gene, partial sequence
Length=712
Score = 1201 bits (650), Expect = 0.0
Identities = 665/672 (98%), Gaps = 2/672 (0%)
PCR12 CONSENSUS
Penn State
146 Fusarium oxysporum f sp melonis NRRL: 26173
Length = 649
Score = 624 bits (315), Expect = e-180
Identities = 318/319 (99%)
NCBI
RID: 3KSNK410013
gb|DQ452422.1| Fusarium solani f. sp. piperis strain MAFF 236575 translation
elongation factor gene, partial sequence
Length=712
Score = 621 bits (336), Expect = 1e-174
Identities = 352/359 (98%), Gaps = 4/359 (1%)
gb|DQ452427.1| Fusarium oxysporum f. sp. cucumerinum strain ATCC 16416 translation
elongation factor gene, partial sequence
Length=712
Score = 610 bits (330), Expect = 2e-171
Identities = 350/359 (97%), Gaps = 4/359 (1%)
Strand=Plus/Plus
PCR14 EF1
Penn State
>146 Fusarium oxysporum f sp melonis NRRL: 26173
113
Length = 649
Score = 1251 bits (631), Expect = 0.0
Identities = 631/631 (100%)
NCBI
gb|DQ452422.1| Fusarium solani f. sp. piperis strain MAFF 236575 translation
elongation factor gene, partial sequence
Length=712
Score = 1208 bits (654), Expect = 0.0
Identities = 661/664 (99%), Gaps = 2/664 (0%)
gb|DQ452427.1| Fusarium oxysporum f. sp. cucumerinum strain ATCC 16416 translation
elongation factor gene, partial sequence
Length=712
Score = 1192 bits (645), Expect = 0.0
Identities = 658/664 (99%), Gaps = 2/664 (0%)
PCR14 EF2
Penn State
146 Fusarium oxysporum f sp melonis NRRL: 26173
Length = 649
Score = 1257 bits (634), Expect = 0.0
Identities = 634/634 (100%)
NCBI
gb|DQ452422.1| Fusarium solani f. sp. piperis strain MAFF 236575 translation
elongation factor gene, partial sequence
Length=712
Score = 1236 bits (669), Expect = 0.0
Identities = 676/679 (99%), Gaps = 2/679 (0%)
gb|DQ452427.1| Fusarium oxysporum f. sp. cucumerinum strain ATCC 16416 translation
elongation factor gene, partial sequence
Length=712
Score = 1219 bits (660), Expect = 0.0
Identities = 673/679 (99%), Gaps = 2/679 (0%)
PCR14 CONSENSUS
Penn State
146 Fusarium oxysporum f sp melonis NRRL: 26173
Length = 649
Score = 1237 bits (624), Expect = 0.0
Identities = 627/628 (99%)
NCBI
RID: 3KSV5T7W013
gb|DQ452422.1| Fusarium solani f. sp. piperis strain MAFF 236575 translation
elongation factor gene, partial sequence
Length=712
Score = 1225 bits (663), Expect = 0.0
Identities = 680/688 (98%), Gaps = 2/688 (0%
gb|DQ452427.1| Fusarium oxysporum f. sp. cucumerinum strain ATCC 16416 translation
elongation factor gene, partial sequence
Length=712
Score = 1208 bits (654), Expect = 0.0
114
Identities = 677/688 (98%), Gaps = 2/688 (0%)
PCR16 EF1
Penn State
>146 Fusarium oxysporum f sp melonis NRRL: 26173
Length = 649
Score = 1227 bits (619), Expect = 0.0
Identities = 619/619 (100%)
NCBI
gb|DQ452422.1| Fusarium solani f. sp. piperis strain MAFF 236575 translation
elongation factor gene, partial sequence
Length=712
Score = 1188 bits (643), Expect = 0.0
Identities = 646/647 (99%), Gaps = 1/647 (0%)
gb|DQ016282.1| Fusarium oxysporum f. sp. melonis isolate 0348 translation elongation
factor EF1 alpha-like protein gene, partial cds
Length=652
Score = 1177 bits (637), Expect = 0.0
Identities = 644/647 (99%), Gaps = 1/647 (0%)
PCR16 EF2
Penn State
146 Fusarium oxysporum f sp melonis NRRL: 26173
Length = 649
Score = 1257 bits (634), Expect = 0.0
Identities = 634/634 (100%)
NCBI
gb|DQ452422.1| Fusarium solani f. sp. piperis strain MAFF 236575 translation
elongation factor gene, partial sequence
Length=712
Score = 1206 bits (653), Expect = 0.0
Identities = 656/657 (99%), Gaps = 1/657 (0%)
gb|DQ452427.1| Fusarium oxysporum f. sp. cucumerinum strain ATCC 16416 translation
elongation factor gene, partial sequence
Length=712
Score = 1190 bits (644), Expect = 0.0
Identities = 653/657 (99%), Gaps = 1/657 (0%)
PCR16 CONSENSUS
Penn State
>430 Fusarium concentricum NRRL 25181 translation elongation factor 1
alpha gene
Length = 636
Score = 745 bits (376), Expect = 0.0
Identities = 554/599 (92%), Gaps = 11/599 (1%)
NCBI
RID: 3KT2WK2N013
gb|DQ452422.1| Fusarium solani f. sp. piperis strain MAFF 236575 translation
elongation factor gene, partial sequence
Length=712
115
Score = 1136 bits (615), Expect = 0.0
Identities = 664/685 (96%), Gaps = 13/685 (1%)
Strand=Plus/Plus
gb|DQ452427.1| Fusarium oxysporum f. sp. cucumerinum strain ATCC 16416 translation
elongation factor gene, partial sequence
Length=712
Score = 1120 bits (606), Expect = 0.0
Identities = 661/685 (96%), Gaps = 13/685 (1%)
PCR21 EF1
Penn State
>292 Nectria haematococca/Fusarium sp. cf. solani mpVI isolate NRRL
22586 translation elongation factor 1 alpha gene
Length = 677
Score = 1120 bits (565), Expect = 0.0
Identities = 645/661 (97%), Gaps = 4/661 (0%)
NCBI
gb|DQ246876.1| Fusarium lichenicola strain NRRL 28019 translation elongation
factor 1-alpha (EF1-alpha) gene, partial cds
Length=675
Score = 1219 bits (660), Expect = 0.0
Identities = 670/674 (99%), Gaps = 4/674 (0%)
gb|DQ246875.1| Fusarium solani strain NRRL 28018 translation elongation factor
1-alpha (EF1-alpha) gene, partial cds
Length=675
Score = 1219 bits (660), Expect = 0.0
Identities = 670/674 (99%), Gaps = 4/674 (0%)
PCR21 EF2
Penn State
>292 Nectria haematococca/Fusarium sp. cf. solani mpVI isolate NRRL
22586 translation elongation factor 1 alpha gene
Length = 677
Score = 1138 bits (574), Expect = 0.0
Identities = 654/670 (97%), Gaps = 4/670 (0%)
NCBI
gb|DQ246876.1| Fusarium lichenicola strain NRRL 28019 translation elongation
factor 1-alpha (EF1-alpha) gene, partial cds
Length=675
Score = 1229 bits (665), Expect = 0.0
Identities = 673/676 (99%), Gaps = 3/676 (0%)
gb|DQ246875.1| Fusarium solani strain NRRL 28018 translation elongation factor
1-alpha (EF1-alpha) gene, partial cds
Length=675
Score = 1229 bits (665), Expect = 0.0
Identities = 673/676 (99%), Gaps = 3/676 (0%)
PCR21 CONSENSUS
Penn State
116
>315 Nectria haematococca/Fusarium sp. cf. solani mpVI isolate NRRL
22161 translation elongation factor 1 alpha gene
Length = 677
Score = 807 bits (407), Expect = 0.0
Identities = 478/491 (97%), Gaps = 4/491 (0%)
NCBI
RID: 3KTBST4U013
gb|DQ246876.1| Fusarium lichenicola strain NRRL 28019 translation elongation
factor 1-alpha (EF1-alpha) gene, partial cds
Length=675
Score = 1142 bits (618), Expect = 0.0
Identities = 658/674 (97%), Gaps = 16/674 (2%)
gb|DQ246875.1| Fusarium solani strain NRRL 28018 translation elongation factor
1-alpha (EF1-alpha) gene, partial cds
Length=675
Score = 1142 bits (618), Expect = 0.0
Identities = 658/674 (97%), Gaps = 16/674 (2%)
PCR25 EF1
Penn State
>146 Fusarium oxysporum f sp melonis NRRL: 26173
Length = 649
Score = 1221 bits (616), Expect = 0.0
Identities = 630/632 (99%), Gaps = 1/632 (0%)
NCBI
gb|DQ452422.1| Fusarium solani f. sp. piperis strain MAFF 236575 translation
elongation factor gene, partial sequence
Length=712
Score = 1208 bits (654), Expect = 0.0
Identities = 666/671 (99%), Gaps = 3/671 (0%)
Strand=Plus/Plus
gb|DQ452427.1| Fusarium oxysporum f. sp. cucumerinum strain ATCC 16416 translation
elongation factor gene, partial sequence
Length=712
Score = 1192 bits (645), Expect = 0.0
Identities = 663/671 (98%), Gaps = 3/671 (0%)
PCR25 EF2
Penn State
>146 Fusarium oxysporum f sp melonis NRRL: 26173
Length = 649
Score = 1269 bits (640), Expect = 0.0
Identities = 640/640 (100%)
NCBI
gb|DQ452422.1| Fusarium solani f. sp. piperis strain MAFF 236575 translation
elongation factor gene, partial sequence
Length=712
Score = 1219 bits (660), Expect = 0.0
gb|DQ452427.1| Fusarium oxysporum f. sp. cucumerinum strain ATCC 16416 translation
elongation factor gene, partial sequence
117
Length=712
Score = 1203 bits (651), Expect = 0.0
Identities = 662/667 (99%), Gaps = 2/667 (0%)
PCR25 CONSENSUS
Penn State
>458 Fusarium sp. cf. oxysporum f. sp. melonis NRRL 26406 translation
elongation factor gene
Length = 652
Score = 615 bits (310), Expect = e-177
Identities = 358/371 (96%), Gaps = 9/371 (2%)
NCBI
RID: 3KTJWF9N013
gb|DQ452422.1| Fusarium solani f. sp. piperis strain MAFF 236575 translation
elongation factor gene, partial sequence
Length=712
Score = 1127 bits (610), Expect = 0.0
Identities = 681/711 (95%), Gaps = 22/711 (3%)
gb|DQ452427.1| Fusarium oxysporum f. sp. cucumerinum strain ATCC 16416 translation
elongation factor gene, partial sequence
Length=712
Score = 1110 bits (601), Expect = 0.0
Identities = 678/711 (95%), Gaps = 22/711 (3%)
PCR28 EF1
Penn State
>292 Nectria haematococca/Fusarium sp. cf. solani mpVI isolate NRRL
22586 translation elongation factor 1 alpha gene
Length = 677
Score = 1304 bits (658), Expect = 0.0
Identities = 669/670 (99%), Gaps = 1/670 (0%)
NCBI
gb|AF178353.1| Fusarium sp. NRRL 22586 translation elongation factor 1 alpha
gene, partial cds
Length=677
Score = 1230 bits (666), Expect = 0.0
Identities = 669/670 (99%), Gaps = 1/670 (0%)
gb|DQ247436.1| Fusarium solani strain FRC S1124 translation elongation factor
1-alpha (EF1-alpha) gene, partial cds
Length=677
Score = 1219 bits (660), Expect = 0.0
Identities = 667/670 (99%), Gaps = 1/670 (0%)
PCR28 EF2
Penn State
>292 Nectria haematococca/Fusarium sp. cf. solani mpVI isolate NRRL
22586 translation elongation factor 1 alpha gene
Length = 677
Score = 1302 bits (657), Expect = 0.0
Identities = 668/669 (99%), Gaps = 1/669 (0%)
118
NCBI
gb|AF178353.1| Fusarium sp. NRRL 22586 translation elongation factor 1 alpha
gene, partial cds
Length=677
Score = 1230 bits (666), Expect = 0.0
Identities = 674/677 (99%), Gaps = 3/677 (0%)
gb|DQ247436.1| Fusarium solani strain FRC S1124 translation elongation factor
1-alpha (EF1-alpha) gene, partial cds
Length=677
Score = 1219 bits (660), Expect = 0.0
Identities = 672/677 (99%), Gaps = 3/677
PCR28 CONSENSUS
Penn State
>292 Nectria haematococca/Fusarium sp. cf. solani mpVI isolate NRRL
22586 translation elongation factor 1 alpha gene
Length = 677
Score = 985 bits (497), Expect = 0.0
Identities = 509/513 (99%)
NCBI
RID: 3KTT15TR015
gb|DQ452423.1| Nectria haematococca mpVI strain MAFF 840047 translation elongation
factor gene, partial sequence
Length=741
Score = 1170 bits (633), Expect = 0.0
Identities = 701/731 (95%), Gaps = 15/731 (2%)
gb|DQ247436.1| Fusarium solani strain FRC S1124 translation elongation factor
1-alpha (EF1-alpha) gene, partial cds
Length=677
Score = 1134 bits (614), Expect = 0.0
Identities = 657/675 (97%), Gaps = 13/675 (1%)
PCR31 EF1
Penn State
>614 Fusarium 'Lateritium Clade IIA' isolate FRC L-200 translation
elongation factor 1-alpha gene
Length = 664
Score = 993 bits (501), Expect = 0.0
Identities = 619/651 (95%), Gaps = 5/651 (0%)
NCBI
gb|DQ295133.1| Fusarium lateritium isolate F0103 translation elongation factor-1
alpha (tef1) gene, exons 1 through 4 and partial cds
Length=680
Score = 1146 bits (620), Expect = 0.0
Identities = 658/676 (97%), Gaps = 4/676 (0%)
PCR31 EF2
Penn State
>614 Fusarium 'Lateritium Clade IIA' isolate FRC L-200 translation
elongation factor 1-alpha gene
Length = 664
119
Score = 1001 bits (505), Expect = 0.0
Identities = 632/667 (94%), Gaps = 5/667 (0%)
NCBI
gb|DQ295133.1| Fusarium lateritium isolate F0103 translation elongation factor-1
alpha (tef1) gene, exons 1 through 4 and partial cds
Length=680
Score = 1138 bits (616), Expect = 0.0
Identities = 660/681 (96%), Gaps = 4/681 (0%)
PCR31 CONSENSUS
Penn State
>614 Fusarium 'Lateritium Clade IIA' isolate FRC L-200 translation
elongation factor 1-alpha gene
Length = 664
Score = 474 bits (239), Expect = e-135
Identities = 287/303 (94%)
NCBI
RID: 3KU1PWHK013
gb|DQ295133.1| Fusarium lateritium isolate F0103 translation elongation factor-1
alpha (tef1) gene, exons 1 through 4 and partial cds
Length=680
Score = 904 bits (489), Expect = 0.0
Identities = 606/656 (92%), Gaps = 34/656 (5%)
120
APPENDIX I
NCBI PCR trees from Consensus Sequences
PCR4 CONSENSUS
121
PCR6 CONSENSUS
122
PCR8 CONSENSUS
123
PCR11 CONSENSUS
124
PCR12 CONSENSUS
125
PCR14 CONSENSUS
126
PCR16 CONSENSUS
127
PCR21 CONSENSUS
128
PCR25 CONSENSUS
129
PCR28 CONSENSUS
130
PCR31 CONSENSUS
131
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