This grant proposal requests $772.35 to fund an undergraduate research project investigating the allelopathic effects of hayscented ferns. The student hypothesizes that the ferns suppress competing plants either through chemicals released from roots/leaves or as leaf litter leachate, and aims to determine if suppression occurs at the seed germination or seedling growth stages. The project will expose seeds and seedlings of sugar maple, red maple, red oak, and black cherry to aqueous extracts and a leachate treatment from ferns. Effects on germination and growth will be measured over several months. The budget details supplies, equipment, and a timeline to complete the work by April 2015.

1. Undergraduate Research/ProjectGrantProposalForm
Deadline: Monday, October 6, 2014
Title of Project: An Allelopathic Study of the Hayscented Fern
Projected Completion Date: April 2015
Total Amount of Grant Requested: $772.35
Name of Student (Contact Person): Madison Stahr
______________________________
______________________________
______________________________
Major:Biology QPA: 3.873 #Credits Earned 88
E-mail address:m.n.stahr@eagle.clarion.edu
Phone number: (814) 243 7118
Mailing address: 105 B South 6th South Ave, Clarion, Pa 16214
Faculty/Staff Project Advisor :_Dr. Suzanne Boyden
Department: Biology
E-mail address: 265 Science and Technology Center, Biology Department, Clarion University, Clarion,
PA 16214
Campus phone: 814-393-2759
Signature of Student Applicant (Contact Person): ___________________________
Signature of Faculty/Staff Project Advisor ascertaining that he/she has read the proposal
and is in support: ______________________________________________________
Other students participating in project: ___________________________________
Other faculty/staff participating in project: ________________________________
Proposal Format: (Instruction details in section C)
Introduction
Materials and Methods
References
Budget
Addendum

2. Introduction
Here in Pennsylvania, forest ecosystems surround us. These areas are a wealth of nature;
they provide habitat for local wildlife, serve as natural air and water filtration systems, and are a
great source of timber. As with any ecosystem many things are interacting with one another, and
it is the compilation of these interactions that cause the forest to be as it is. When just one
interaction is changed so do many other components of the forest. Unfortunately the forests are
seeing the effects of one of these changes, and the changes are quite dramatic.
This change is the lack of predators for deer, a large herbivore. Because the deer serve as
prey to few animals, their population has increased significantly over many years. This pulls on
the next string in the ecosystem, which is what the deer are eating. Deer are selective browsers so
they are capable of picking and choosing what they want to eat. By doing so, deer have left their
mark on the forest system and are changing the forest understory. Many young tree seedlings and
other small plants are eaten to the point that their numbers are swiftly dwindling. Instead, what is
left are the species of plant that deer do not want to eat (Horsley, 2003).
One of these remaining species is the hayscented fern or Dennstaedtia punctilobula. This
species is a native fern present in many healthy forest ecosystems. However, in deer ravaged
systems, fern competitors are at such low numbers that fern cover has seen a rapid expansion.
This dense carpet of fern now replaces much of the diversity formerly found in many forest
understories (Horsley, 2003). The sugar maple is a prime example for this. While sugar maples
are a very prolific tree species, capable of re-sprouting from the stumps of cut down trees, they
are seeing a decline (Bailey, 2004). The oak family is another group with reduced numbers,
which is of greater concern considering their acorn production serves as a valuable food source
for much of the wildlife living around them (McShea, 2007). Even though the hayscented fern is
not the cause of the die back of mature trees, it can play a part in the lack of regenerating ones.
A previous field study, done in the late 1970’s, showed that the sugar maple, red maple,
and black cherry all had reduced numbers when in the presence of the hayscented fern. Another
study around the same time looked into the possibility of the fern being allelopathic, meaning it
can inhibit the growth of other plants by producing and releasing chemicals into its surroundings.
This study showed that the hayscented fern was allelopathic towards other fern species as well as
autotoxic (causing chemical harm to others of the same species), (Munther, 1980). This
information raises the question of whether or not the hayscented fern uses allelopathy to suppress
the growth of tree species growing around them.
Allelopathic plants generally work one of three ways; exuding chemicals through the
leaves or roots when the plant is living, or releasing the chemicals as fallen leaf litter decays. The
type of chemical released will determine how the affected plants are suppressed. One effect is
that the chemical will prevent germination of a seed or spore and therefore cease growth of a
competing plant before it has even begun. The other effect is that chemicals can inhibit plant
growth of the competing species, preventing them from growing or developing further
(Munther,1980; Butcko, 2002). Both ways lead to the mortality of the recipient plant.
The proposed study has two main goals. The first is to determine whether the hayscented
fern uses allelopathy to suppress its competitors, and if so by which of the three ways. The
second goal is to determine if the fern is simply preventing seed germination or is actually
inhibiting growth of established plants. It is my hypothesis that the fern will only use one mode
of allelopathy to effect either the seeds or the seedlings but not both.

3. Materials and Methods:
Our 4 study species will be Sugar Maple, Red Maple, Red Oak, and Black Cherry.
Seedlings of each species will be collected from the field, along with hay-scented fern. Seeds for
the 4 study species will also be purchased. The seeds and seedlings of each species will be
exposed to 4 treatments: an aqueous extract from the roots, an aqueous extract from the leaves
and stem, a leachate treatment, and a control.
For both aqueous extracts, 125 grams of either fern root or crushed leaf/stem tissue from
the hayscented fern will be mixed with 1600 mL of distilled water. Each mixture will then be
boiled for 3 minutes to stop enzymatic activity and hopefully kill any microorganisms that could
cause contamination. These mixtures will then be put into a blender for 5 minutes, and solids
strained out using cheesecloth (Munther, 1980).
The leachate treatment will be prepared using fresh fern fronds. A layer of fronds, 2
fronds thick, will be placed on top of a mesh screen above a sterile container. Fronds will then
be misted with 300 ml of distilled water, and the water will be collected within the container. All
three treatments will be stored in air tight containers in a refrigerator until used (Munther, 1980).
To test if the fern uses allelopathy to prevent germination 100 seeds of each of the 4
tested tree species will be divided into 4 groups, 1 control group and 3 treatment groups. Seeds
will be placed into individual petri dishes and allowed to germinate in an agar solution infused
with either distilled water or one of the 3 treatments types. Seedlings will be evaluated for 1
month to determine if germination is affected by the presence of a fern treatment.
To test if the fern uses allelopathy to inhibit growth of seedlings 20 seedlings of each of
the 4 tree species will be collected and separated into 4 groups, 1 control group and 3 treatment
groups. Seedlings will be rinsed thoroughly with distilled water to remove all original dirt which
could cause contamination. Each seedling will be planted into its own pot with a mixture of 1/3rd
vermiculate, 2/3rds Miracle-Gro Garden Soil for Flowers and Vegetables that has been
autoclaved prior to planting. Plants will be watered frequently enough to maintain field capacity
with either distilled water as the control, or one of the 3 treatments. Seedling diameter and height
growth will be measured weekly for 3 months to determine if plant growth is inhibited by fern
presence.
Timeline
October: collect specimens from forest and order all other supplies
November: make the treatment types, and begin sapling trials, prepare seeds for testing
December-January: Observe seedling trials
February: Begin seedling trials
March: Analyze data and make poster
April: Present study at CUP Undergraduate Poster Session and Commonwealth of Pennsylvania
University Biologists annual meeting

4. References
Bailey, S. W., S. B. Horsley, R. P. Long, and R. A. Hallett. "Influence of Edaphic Factors on
Sugar Maple Nutrition and Health on the Allegheny Plateau."Soil Science Society of
America Journal 68 (2004): pp. 243-252.Publisher US Forest Service. Web. 19 Sept.
2014.
Butcko, Victoria M., and Jensen, Richard J. “Evidence of Tissue-Specific Allelopathic Activity
in Euthamia Graminifolia and Solidago Canadensis (Asteraceae).” American Midland
Naturalist, Vol. 148, No. 2 (Oct., 2002), pp. 253-262. Publisher The University of Notre
Dame. http://www.jstor.org/stable/3083129 Web. 19 Sept. 2014
Horsley, Stephen B., Stout, Susan L., and DeCalesta, David S. "White-Tailed Deer Impact on the
Vegetation Dynamics of a Northern Hardwood Forest." Ecological Applications 13.1
(2003): 98-118. US Forest Service. Ecological Society of America. Web. 19 Sept. 2014.
McShea, William J., Healy, William M., Devers, Patrick, Fearer, Todd, Koch, Frank H.,
Stauffer, Dean, and Waldon, Jeff. “Forestry Matters: Decline of Oaks Will Impact
Wildlife in Hardwood Forests.” The Journal of Wildlife Management, Vol. 71, No. 5
(Jul., 2007), pp. 1717-1728. Publisher Wiley, on behalf of the Wildlife Society.
http://www.jstor.org/stable/4496256 Web, 19 Sept 2014
Munther, William E., and Faribrothers, David E.. “Allelopathy and Autotoxicity in Three Eastern
North American Ferns.” American Fern Journal, Vol. 70, No. 4 (Oct. - Dec., 1980), pp.
124-135. Publisher American Fern Society. http://www.jstor.org/stable/1546754. Web.
19 Sept. 2014

5. Undergraduate Research/Project Budget Form
Complete the following table. Be sure to include information regarding all non-standard materials and
services that are required to successfully complete the project, as well as any shipping & handlings costs.
When ordering supplies for the project, the student and mentor should work with the Dean’s Office of
their respective department.
Item* Source of item Quantity Unit cost Total cost Source of funds**
Supplies:
1. Red Oak seed Treeseeds.com 100 12.00 14.00 This grant
2. Red Maple seed Treeseeds.com 100 5.00 7.00 This grant
3. Sugar Maple seed Treeseeds.com 100 6.00 8.00 This grant
4. Black Cherry seed Treeseeds.com 100 4.00 6.00 This grant
5.Petri dishes Fisher Scientific 500 80.00 $90.00 This grant
6.Agar Fisher Scientific 500grams 131.40 140.00 This grant
7. Miracle-Gro Garden Walmart 4 7.97 31.88 This grant
Soil
8.Vermiculite Fisher Scientific 2 20.40 40.80 This grant
9.Planting Pots Walmart 40 7.50 300.00 This grant
10. Cheese Cloth Walmart 1 3.82 3.82 This grant
Equipment:
1. Blender Walmart 1 49.88 49.88 This grant
2.Gardman Mini Amazon 1 40.98 40.98 This grant
Greenhouse
3.LED Grow light Amazon 1 39.99 39.99 This grant
Grand totals: $772.35
*Grant funds cannot be used to pay salaries, stipends,or consulting fees. Grant funds cannot be used to purchase
supplies or equipment readily available in departments. Provide a brief justification for the item to be purchased
with the grant – use an additional sheet if necessary.
**The source of funds is assumed to be the maximum of $800 available; if the project costs exceed this amount,
indicate the additional source of funding and whether it is pending or secure.

6. Budget Justification
Supplies
1-4. Four different tree species seeds that will be tested in this experiment will be ordered because it is
both difficult to find and identify seeds in the field, or they have Fall germination.
5-6. Both the petri dishes and agar will be utilized for the seed germination aspect of the experiment.
7-8. Miracle Gro Garden soil and vermiculite will be combined to create a sterile but fertile growing
medium for the seedlings aspect of the experiment.
9. Planting pots will be used to contain the growing seedlings for the duration of the experiment
10. Cheesecloth is necessary to strain solid plant material out of the aqueous extracts that will become 2
of the 3 treatments.
Equipment
1. We will purchase a magic bullet, for dedicated lab use, for processing plant samples. A normal blender
can’t handle the fibrous material. No such instrument exists in the lab currently.
2-3.This greenhouse combined with a grow light will create an enclosed, moist environment for both seed
germination and seedling growth. It has been shown to improve germination success,which has proven to
be difficult in the STC labs.

7. Addendum
Currently I am a junior with a major in biology and a minor in chemistry. As a freshman I
was fortunate enough to work on a project that involved the spotted salamander and their
thermoregulation. Last year I was yet again able to work with spotted salamanders but from a
different approach. Instead I studied the effects of disinfectants on the salamanders’ endogenous
microbial communities using Ecoplates. These two projects were ecology based, but the use of
Ecoplates introduced me to more lab based techniques. It was over the summer where I received
a more thorough introduction to lab work through a Research Experience for Undergraduates
program at Johns Hopkins University. While there I used confocal microscopy to image the
brains of fluorescent immunohistochemically stained larval Zebrafish. While I excelled at my lab
work, and rather enjoyed it, I still am not convinced to give up the ecology side of research.
It is from these past experiences that I have come to the realization that a middle ground
between field studies and lab work would be the ideal place for me. Allelopathic studies are
perfect for this. The effects of allelopathy can be seen at the macroscopic level through field
studies. This is because, depending on how potent the allelotoxins involved are, the allelopathic
impact on forest composition can be seen to shape to forest communities. On top of this, lab
techniques can easily be utilized to better understand how an allelopathic plant functions. It is
through the combined efforts of lab and field work that this type of plant interaction can fully be
understood.
Having a chance to be involved in undergraduate research is one aspect of Clarion that I
will forever be grateful for. By being involved in previously established projects as early as my
freshman year I have already been able to improve my resume much more than students my age
who chose to attend other schools, which will be very advantageous when applying for grad
schools. The best part is, this is not the only benefit Clarion gives me. All of the professors in
Clarion’s Biology Department that run their own lab encourage students to think outside the box
and come up with worthwhile projects on their own. This allelopathic study is me doing just that.
This grant represents a project that I have put a lot of time and effort towards bringing it to life.
My hopes are that this study, with its combined field and lab work, will most accurately be able
to reflect my future career goals, providing me with valuable experience I can use for the rest of
my life. Upon completing this project, I plan on presenting the results at the Academic
Excellence Undergraduate Research Symposium at Clarion University, the Annual
Commonwealth of Pennsylvania University Biologist annual conference.