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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

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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.

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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.

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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,

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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/N

rat

io

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

Mo

nth

ly a

vera

ge

tem

pe

ratu

re (

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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