1. 1
Analysis of Litter Quality in Transgenic Chestnut Trees
Abstract
The American chestnut was a valuable species in an economic, ecological, and cultural
sense. Establishment of the Chestnut blight resulted in a major change in forest dynamics due to
the loss of the American chestnut’s functional role as a mast producer. Modern approaches for
re-establishment of the American chestnut in the United States involve both hybridization as well
as gene insertion in the form of transgenic American chestnuts. The goal of this study is to
analyze the differences between litter nutrition of two transgenic, a hybrid and a native wild-type
American chestnut. This was accomplished by assessing the carbon to nitrogen ratios. The
carbon to nitrogen ratios were analyzed using a Thermo Flash EA 1112 CHN/S/O Analyzer by
Mr. Chuck Schirmer after 6-month in situ incubation. Results showed no significant differences
between the carbon to nitrogen ratios observed with respect to genome. Individual carbon and
nitrogen concentrations did however differ among genomes, the biological significance of these
differences might be of importance in later research.
Introduction
The native geographic range of the American chestnut extended from the southern tip of
Maine as far south as Georgia where it existed as a dominant tree able to grow as tall as 100 feet
tall and up to 10 feet in dbh. Due to this species rot resistance, its lumber was largely exported
from these regions for the use of outdoor structures such as telephone poles, log cabins, flooring,
and furniture. It has been estimated that American chestnut made up approximately ¼ of all
lumber cut in the Southern Appalachians (Hepting, 1974). These sales had implications for both
local and regional economies. In addition to its economic importance, it was estimated that a

2. 2
single tree had the capacity to produce up to 6000 chestnuts which provided ample nutrients to
both local wild life and settlers inhabiting these locations (Horton, 2010). This species hard mast,
often roasted, was exported largely during the holiday seasons for consumption by settlers. For
this reason, the American chestnut was known as a heritage tree due to its cultural significance.
The introduction of the chestnut blight, caused by the fungus Cryphonectria parasitica,
resulted in the loss of an estimated 4 billion American chestnut trees from the upper canopy
throughout its native range in the span of 50 years (Vandermast, 2008). Due to this species
sprouting ability, American chestnut still exists throughout the understory in its native range in
the Northeast United states. However, due to this forced low forest reproduction, its functional
role in the upper canopy as a mast producer and timber source has become extinct.
The fungus C. parasitica invades the cambium of American chestnut through infection
courts provided by local insects and wild life. Once established, this fungus produces oxalic acid
which lowers the pH in the tissues to toxic levels. This lowered pH triggers an immune response
from the American chestnut in the form of a diffuse canker. A diffuse canker differs from a
normal canker in that the fungus is able to outgrow the trees immune response that attempts to
compartmentalize the infection from advancing to additional tissue. As canker formation
continues, nutrient and water transport from the roots to the shoots becomes hindered. It is
thought that this additional stress to the tree results in an increased inability to resist infection
(Conolly, 2007). The fungus will continue to grow until the canker spans the entirety of the tree’s
diameter, resulting in necrosis of vegetative tissues above the canker due to insufficient nutrient
and water transport. This necrosis of above ground vegetative tissue ultimately results in the
death of the tree.

3. 3
Modern approaches for re-establishment of the American chestnut throughout its native
range involve the utilization of either hybrid or transgenic species. For hybridization, Chinese
chestnuts, which are naturally resistant to the blight, are crossed with the American chestnut in
order to improve resistance to the pathogen. This method of hybridization may offer a sufficient
disease resistance for the future. However, these hybrids lack many of the desired physical
characteristics of the historic American chestnut, namely its ability to grow tall and straight
(American Chestnut Foundation). Recent advances in the development of a disease resistant
American chestnut through gene insertion have been made through the work of Dr. Powell and
numerous graduate students at SUNY ESF.
In 2013, SUNY ESF planted its first successfully resistant transgenic American chestnut
tree in an experimental forest (Powell, 2014). This tree was inserted with a gene derived from a
wheat plant which codes for the production of oxalate oxidase. This enzyme provides improved
blight resistance by its ability to break down oxalic acid (Zhang, 2013). The ability to hinder
oxalic acid production results in decreased mobility of the fungus’s mycelial fan, inhibiting the
spread to additional portions of the tree’s cambium. Today, New York state is the home of over
1000 transgenic chestnut species (Powell, 2014).
With the recent advancement of these genetically modified chestnuts comes increased
discussion of these transgenic species impact on native ecosystems. Maladaptive genes due to
gene insertion can result in decreased fitness of an organism as well as undesired phenotypic
responses. For these reasons, in order to be out planted for commercial operations, transgenic
species must undergo rigorous testing to prove ecological equivalence to the native American
chestnut species (Sedjo, 2010). The goal of this study is to compare carbon to nitrogen ratios in
leaf litter among two transgenic, a hybrid, and a wild type chestnut genome.

4. 4
Carbon to nitrogen ratios are correlated with decomposition rate, specifically
decomposition rates tend to increase with decreasing C/N ratios (Heal et al., 1997).
Decomposition is defined as the transformation of organic substances into bioavailable
compounds through biological, chemical, and mechanical weathering. Therefore, the rate at
which organic matter decomposes determines the rate at which nutrients such as nitrogen,
phosphorus, and potassium become available for uptake by plants and microorganisms.
Rates of biological and chemical reactions are influenced by a variety of abiotic factors
such as temperature and available moisture. Microbial activity in most environments is assumed
to occur at maximum rate under temperatures around 30 degrees Celsius (Paul, 2001). Microbial
response to soil moisture is less predictably correlated. Factors influencing the variability in
microbial response range from soil type, structure, and depth in the soil column. However, field
capacity moisture levels appear to optimize microbial activity (Barros et al., 1995).
Methods
The field site for this study was located at Lafayette field station, Syracuse, New York.
This one-acre plot is located in a 100-year-old oak-hickory stand with 20 percent canopy cover
which was naturally regenerated from an old agriculture field, therefore there is a presence of a
plowed A-horizon. Due to rapid decomposition on-site there is an absence of a defined organic
layer.
Senescent leaves were collected from each of the 4 chestnut genotypes: Zoar (wild-type),
Hinchee-1 (transgenic), darling-4 (transgenic), and GR86 (3/4 American hybrid). 10 grams of
leaf litter were then placed inside of 22cm by 22cm mesh bags and placed directly on top of the
Ap horizon in the field plot during November of 2012. After 6 months of in situ incubation,

5. 5
during the month of May 2013, 16 samples were removed from the field site and placed inside of
an incubator in the basement of Marshall hall. Two years later, in the month of May 2015, these
samples were removed from the incubator and oven dried at 60 degrees Celsius for 48 hours.
The litter bags were then weighed, cut open, and chestnut litter and other organic matter
and soil were placed into two separate containers. The contents in each container were re-
weighed and transferred into glass vials for storage. To ensure a consistent sample for analysis,
litter samples were ground in a Wiley Mill and then pulverized using a SPEX CertiPrep 8000
Mixer/Mill. 0.025 grams of each litter sample were weighed, transferred into tin boats, loaded
onto a carrousel, and analyzed using a Thermo Flash EA 1112 CHN/S/O Analyzer by Mr. Chuck
Schirmer.
Carbon to nitrogen ratios were calculated for each individual sample and averaged with
respect to genome. Genome effect on C/N ratios as well as individual carbon and nitrogen
concentrations were analyzed using ANOVA via SAS 9.4 at a level of significance of 0.05. The
null hypothesis of these tests being there is no significant difference in C, N, or C/N between
genomes. The alternative hypothesis being at least one mean C, N, C/N value differs relative to
genome. If a significant difference was found among genomes, they were examined using
Tukey’s tests at a level of significance of 0.05.
In order to examine the effect of abiotic factors on the decomposition rate, observed
monthly precipitation and temperature were data obtained for the city of Syracuse, New York
from UsClimateData.com. This data was then compared to 30-year normal values using percent
deviations. In addition to the data obtained for the city of Syracuse, hourly temperature data was
obtained from Mr. Gordon Heisler, a research scientist with USDA Forest Service.

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Results
Figure 1. Carbon Vs. Nitrogen values for transgenic (Darling-4, Hinchee-1) and Zoar (wild type) leaf litter
for a 6-month in situ incubation.
There was a relative dramatic decrease in C/N ratios with respect to genome for this 6 month in
situ incubation period in comparison to initial values. Mean C/N ratios ranged from 16:1 in the
hybrid species to 19:1 in the zoar (wild type) genome when comparing the ratio of means. This
compares to initial values of 29:1 in the zoar (wild type) genome and 22.5:1 in the hybrid
species.

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Figure 2. Carbon Vs. Nitrogen ratio of means for transgenic (Darling-4, Hinchee-1), Zoar (wild type) and
hybrid leaf litter for a 30 month in situ incubation period. Initial and post 6 month values were data
obtained from Amanda Gray’s thesis project.
During this 6-month decomposition period, C/N ratios were not significantly different
among litter types. There was no significant difference between the two transgenic genomes,
Hinchee-1 and Darling-4 with respect to individual carbon and nitrogen concentrations, however,
the Hinchee-1 transgenic was found to be significantly different from the wild-type Zoar genome
(Table 2). Due to a lack of replications for the hybrid chestnut species (n=2), comparisons using
ANOVA for this genome were not calculated.
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
0 6 12 18 24 30
C/Nratio
Time (months)
Darling 4
Hinchee 1
hybrid
Zoar

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Table 1. ANOVA test comparing genome effect on C/N ratio at a level of significance of 0.05.
C/N Ratio
Source DF SS MS F p
Model 2 4.856 2.428 0.720 0.509
Error 11 37.116 3.374
Corrected
Total
13 41.972
Table 2. ANOVA test comparing genome effect on individual carbon and nitrogen concentrations at a
level of significance of 0.05.
Carbon Nitrogen
Source DF SS MS F p Source DF SS MS F p
Model 2 122.690 61.345 7.520 0.009 Model 2 0.894 0.447 9.190 0.005
Error 11 89.765 8.160 Error 11 0.535 0.049
Corrected
Total
13 212.454
Corrected
Total
13 1.429
Table 3. Tukey’s HSD tests comparing genome effects on carbon and nitrogen concentrations as well as
the C/N ratios in chestnut litter samples at a level of significance of 0.05. Genomes sharing letters are
not significantly different with respect to the given response variable.
Carbon Nitrogen
Genome Tukey’s Genome Tukey’s
Hinchee-1 A Hinchee-1 A
Darling-4 A B Darling-4 A B
Zoar B Zoar B
Carbon Nitrogen
Genome C.I. Genome C.I.
Hinchee - Darling4 -0.944,9.408 Hinchee - Darling4 -0.004,0.795
Hinchee - Zoar 2.254,12.65 Hinchee - Zoar 0.232,1.032
Darling4 - Zoar -1.682,8.077 Darling4 - Zoar -0.14,0.613

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Throughout the period of November 2012 to May 2013, Syracuse experienced moderate
monthly deviations in average monthly low temperatures (Figure 3). The largest deviations in
average low temperatures for 30 year normal values were observed for the months of December
and January where Syracuse was recorded of having 20 to 30 percent warmer low temperatures.
Negligible deviations in average monthly high temperatures were observed for this period,
ranging from 2 to 12 percent.
Figure 3. Percent deviations of observed monthly temperatures from recorded 30-year normal values
for the 2013 water year in the city of Syracuse New York.
30.0
40.0
50.0
60.0
70.0
80.0
Monthlyaverage
temperature(F)
Monthly Avg (high) Lafayette
Monthly Avg (low) Lafayette

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Figure 4. Average monthly high and low temperatures observed at Lafayette field station, Syracuse, New
York through the months of March to September 2013.
Observed average monthly high and low temperatures recorded at Lafayette field station
deviate from those found for the city of Syracuse, New York obtained from Usclimatedata.com.
In addition, a buffered effect was observed between high and low temperatures on-site. The
largest observed difference between high and low temperatures was 1.9 degrees Fahrenheit for
the month of May 2013 (Figure 4).
Figure 5. Percent deviations of observed total monthly Precipitation from recorded 30-year normal
values for the 2013 water year in the city of Syracuse New York.
Throughout the 2013 water year moderate to high deviations of observed average
monthly precipitation from 30-year normal values were recorded for the city of Syracuse. The
highest deviation during this experiment was observed for the month of November 2012 with a
deviation of 75 percent.

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Discussion
[C], [N], and C/N ratios
C/N ratios at this 6-month period did not differ significantly among genomes and ranged
from 17:1 in Hinchee-1 to 18:1 in both Darling-4 and Zoar. This indicates the transgenic
genomes do not differ in terms of chemical composition of their litter. Further studies should
compare C/N ratios of transgenic species from the wild type genome at this period of
decomposition.
Lignin is a major contributing factor to the rate at which leaf litter decomposes; higher
lignin concentrations are strongly correlated with slow decomposition rates (Rahman, 2013).
Increased lignin concentrations might result in the increase of humic matter at the forest floor.
This build-up of organic matter would result in a change in soil characteristics such as increased
structure, water holding capacity, and cation exchange capacity (Ontl, 2012). Increased soil
structure and water retention are correlated with decreased erosion. This decrease in erosive
potential of upper soil layers would lead to increased surface water quality of surrounding
streams.
Soil and vegetation are major sinks for carbon in the carbon cycle. The amount of carbon
found in soil is estimated to be approximately 3170 billion metric tons (Ontl, 2012). It was found
in this study that carbon concentrations were statistically different between Hinchee-1 and our
wild-type American chestnut litter sample at a level of significance of .05. This change in carbon
storage could be of importance for carbon sequestration. However, there appears to be a
relatively large amount of variation within the wild-type genome with respect of carbon
concentrations (13.8 mg/g). It is possible with the relatively small sample size of this experiment,

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the sample mean calculated does not accurately describe the central tendency of this species
carbon concentration.
Significance tests only calculate the probability of observing the data in our sample given
the null hypothesis is true. In the field of ecology and natural sciences, a shift in statistical
analysis is pushing towards effect size and confidence intervals for newly published research
(Nakagawa, 2007). Effect size calculations deal with the magnitude of the significance observed
between treatments and are therefore a better means of discussing biological importance. Further
studies should adjust to this shift in statistical analysis.
Due to the lack of replications in our hybrid species, ANOVA and Tukey’s tests were not
performed on these samples. These hybrids are not held to the same biological testing standards
of their transgenic counterpart’s, therefore hybrid chestnut species have been outplanted by the
American Chestnut Foundation as early as 2009 (Thompson 2012). Future studies should look at
biological differences between hybrid species and transgenic species at this period of
decomposition.
Temperature and precipitation
The rate at which organic matter decomposes is determined by microorganisms,
temperature, moisture, and soil characteristics (Cortez et al, 1996). In this study, monthly
average precipitation and temperature values were recorded for the city of Syracuse and
compared to 30-year normal values. July 2013 was observed to have the highest percent
deviation in average precipitation with a value of 7.1 inches compared to the 30-year normal
value of 4.06 inches, representing a 75 percent deviation. Excess soil moisture decreases the rate
of mineralization and nitrification of organic nitrogen to form ammonium and nitrate,

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respectively. However, soil moisture was not analyzed for this study and therefore future studies
should include this covariate.
Soil respiration is strongly and positively correlated with soil temperatures. The use of
exponential models to relate the two variables illustrates the sensitivity of soil respiration to
changes in temperature (Xiao et al, 2014). The recorded average monthly temperature recordings
for the city of Syracuse did not differ largely from the 30-year normal values with the exception
of December and January. Both these months’ experience relatively warmer average high and
low temperatures when compared to the 30-year normal values. However, the temperatures
recorded at Lafayette field station illustrated a strong buffering effect with respect to observed
temperatures at the forest floor, likely due to canopy cover. For this reason, it is likely that these
deviations in air temperatures observed for the city of Syracuse would not have had a
tremendous effect on soil temperatures at the Lafayette field station.
Conclusion
There was no significant difference between C/N ratios with respect to genome.
However, significant differences for individual concentrations for carbon as well as nitrogen
were found between zoar the wild type chestnut and the hinchee-1 transgenic. Due to a lack of
replications, genome effect on C/N ratio was not analyzed for the hybrid species. Additional
research should better analyze the effects of soil temperature and moisture as a covariate on the
decomposition rates of these species. Additionally, effect size should be a focus for future
research due to its relationship to biological significance.

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