ABSTRACT
PAYNE, JASON ALLEN. The effects of fertilization and light availability on early survival
and growth of backcross hybrid American chestnuts. (Under the direction of Howard Allen).
The American chestnut (Castanea dentata Marsh. Borkh.) was once a dominant tree
species throughout much of its native range in eastern North America until the introduction
of a blight fungus (Cryphonectria parasitica Murr. Barr) decimated the population during the
first half of the 20th century. Today, American chestnut is all but extinct, persisting almost
exclusively in the forest understory as sprouts originating from blight stricken trees. In the
early 1980's, a tree improvement program was initiated by The American Chestnut
Foundation (TACF) to produce backcross hybrids using American and Chinese chestnuts
(Castanea mollissima). Presently, little is known of how TACF's backcross hybrids will
respond when exposed to traditional silvicultural treatments. First and second-year survival
and growth of 22 families from TACF's backcross breeding program were evaluated under
two silvicultural systems (clear-cut and shelterwood) and two levels of nutrition (64.2 kg
N/ha as diammonium phosphate and no fertilization). Study-wide first and second-year
survival was greater than 90%. Two-year cumulative height growth was greater in the clearcut treatment (89.5 cm) than the shelterwood treatment (60.0 cm). Fertilized seedlings had
greater first-year mean height growth (26.2 cm) than non-fertilized seedlings (19.3 cm). After
two growing seasons, cumulative height growth response to fertilization was greater only in
the clear-cut treatment (103.6 cm for fertilized vs. 75.3 cm for non-fertilized seedlings).
Genotype by environment interactions were observed for the fertilization treatment, where
the rankings of family means for cumulative height growth changed in the presence of added
nutrients. Growth and survival for hybrid families in this study are consistent with or better
than published reports for American chestnut grown under similar conditions.
© Copyright 2016 Jason Allen Payne
All Rights Reserved
2
The effects of fertilization and light availability on early survival and growth of backcross
hybrid American chestnuts
by
Jason Allen Payne
A thesis submitted to the Graduate Faculty of
North Carolina State University
in partial fulfillment of the
requirements for the degree of
Master of Science
Forestry
Raleigh, North Carolina
2016
APPROVED BY:
_______________________________
Howard Allen
Committee Chair
_______________________________
Steven E. McKeand
_______________________________
Thomas R. Wentworth
3
BIOGRAPHY
Jason Allen Payne was the second son born to Allen Payne and Yvonne Dew in March, 1977.
He was raised in Bolton, North Carolina. His interests in natural resource management began
early in life, gardening with his parents, following his father at the farm and exploring
wooded areas around his rural home. His love and appreciation for the outdoors is rooted in
experiences with his father and grandfather, Alvy D. Payne. Both taught Jason basic skills in
gardening. The elder Payne taught Jason many lessons in camping, fly fishing, quail hunting
and hard work. Allen shared his love of hunting and fishing, offering Jason a childhood filled
with opportunities to learn about whitetail deer, raccoon, quail and bluegill fishing.
Jason attended East Columbus High School in Lake Waccamaw, NC. After completing
high school in 1995, Jason attended Southeastern Community College, where he earned an
A.A.S. in Forest Management Technology in 1997. Jason transferred to North Carolina State
University (NCSU), where he graduated with a B.S. degree in forest management in the
spring of 2000. Jason began work as an environmental scientist in 2003. He returned to
forest management in 2009, when he founded Woodland Environmental Consultants, PA. In
2011, Jason accepted a land steward position with Unique Places, LLC. He was promoted to
Director of Conservation Forestry in 2013, and continues to serve in that capacity today. In
his career, Jason has managed more than 100,000-acres of natural resource management
projects across North Carolina, South Carolina and Virginia.
The most rewarding part of Jason's life is at home with his family. He married Angela
Kaye Griner in July, 2008. He is father to seven children: Matthew, Gabriel, Julie, Ashleigh,
Lauren, Olivia and Bristol.
ii
ACKNOWLEDGMENTS
I would like to thank Dr. H. Lee Allen for his support and encouragement through the years. I
am grateful for the support of Jeffrey Fisher and Tim Sweeney throughout this project. The
American Chestnut Foundation provided seedlings for the project. I am thankful for the
counsel of research scientists Jeff Donahue and Tom Saielli of The American Chestnut
Foundation for their support and collaboration throughout the project. I would like to thank
Dr. John Frampton, who was always willing to help with the statistical analysis. I would like
to thank Pat Dale for his direction in site selection and site preparation. I would like to thank
Morgan Wixted, Barry Ellenburg, Brian Johnson, Richard Carpenter and other members of
the Unique Places' staff that participated in this project. Their assistance in site preparation,
monitoring and management were essential for success. I would like to thank my sons,
Matthew and Gabriel, for their help in the field. Finally, I would like to thank my wife,
Angela, for her encouragement, support and belief from the moment she learned of my
interest in returning to school in pursuit of my graduate degree.
iii
TABLE OF CONTENTS
LIST OF TABLES................................................................................................................... v
LIST OF FIGURES ............................................................................................................... vi
CHAPTER 1 – Literature synthesis ...................................................................................... 1
Introduction ........................................................................................................................... 1
Habitat ................................................................................................................................... 2
Growth & Management ........................................................................................................ 3
CHAPTER 2 – Manuscript for Publication ......................................................................... 9
Introduction ........................................................................................................................... 9
Methods............................................................................................................................... 13
Results and Discussion ....................................................................................................... 17
Conclusion .......................................................................................................................... 22
iv
LIST OF TABLES
Table 1. P-value summary for the different analysis of variance (ANOVA) results............... 26
Table 2. Mean height and diameter growth (± SE) estimates for seedlings grown under
different silviculture and nutrition.. ........................................................................................ 26
v
LIST OF FIGURES
Figure 1. Mean two-year height growth for all families included in the study ...................... 27
Figure 2. Family means for cumulative height growth under different levels of nutrition. ... 28
vi
CHAPTER 1 – Literature synthesis
Introduction
The demise of American chestnut occurred during the first half of the 20th century before an
extensive body of published research related to the species' growth and development was
established in scientific literature (Paillet, 2002 and Jacobs, 2010). Recent advances in the
backcross breeding program of The American Chestnut Foundation (TACF) have given hope
to forest managers and restoration ecologists that the species may be able to be returned to its
historic place within the forest.
As TACF makes gain through its breeding program, researchers have become increasingly
interested in studying American chestnut with the hope that learning may be applied to
restoration strategies using backcross hybrids. Successful restoration of American chestnut
depends on a thorough understanding of strategies for deployment and management options
of backcross hybrids. Therefore, managers must be able to predict hybrid growth and survival
responses associated with environmental differences, site conditions, plant community
dynamics, management practices and selective pressures.
Unfortunately, the genus Castanea was historically omitted or only briefly mentioned in past
manuscripts detailing the silvics of North American trees (Wang et al., 2013). To address this
deficiency, the US Forest Service published "The Silvics of Castanea dentata (Marsh.)
Borkh., American Chestnut, Fagaceae (Beech Family)" (Wang et al., 2013). This manuscript
compiles current knowledge of habitat requirements, life history, special uses, genetics and
1
restoration for American chestnut in a summary publication. Jacobs (2007) also summarized
relevant information in a similar fashion; however, his work discusses silvical characteristics
of American chestnut in a more focused context of restoration.
Habitat
American chestnut is native to eastern deciduous forests of North America. Before its decline
in the 20th century, American chestnut was a component of forests from central Mississippi
and Alabama and extended northward through the Appalachians into Maine, New Hampshire
and Vermont (Wang et al., 2013). The range extended west, into Indiana and Ohio, occupying
more than 80-million hectares (Wang et al., 2013). Throughout its extent, American chestnut
naturally existed on a wide range of soils and topographies (Wang et al., 2013). Elliot and
Swank (2008) report that, in 1934, American chestnut was the dominant species across the
entire range of forest communities of varying landscape positions in the Coweeta Basin of
western North Carolina. Many reports characterize chestnut as a plastic species, and a
generalist that is adapted to a wide range of sites (Jacobs, 2007). McCament and McCarthy
(2005) found that early growth of chestnut seedlings was negatively correlated with coarse
textures (percent sand). This observation corroborated the findings of Ashe (1912), where
American chestnut grew best in rich coves of Tennessee. Considering the many and varied
recently published reports of American chestnut occurring across a wide range of sites, it is
reasonable for managers to pursue restoration projects at nearly any landscape position,
throughout the species' range. However, knowledge of chestnut growth and development
across the range of sites, within the various communities is critical for success.
2
Growth & Management
Many authors report that American chestnut is fast-growing and extremely competitive
during early development (Jacobs, 2010). A review of one and two-year growth in three
different studies with diverse treatments and site conditions indicates that American chestnut
seedlings can be expected to grow an average of 16 cm/yr, with ranges from 2 cm/yr to 37
cm/yr (Clark et al., 2012, Rhoades et al., 2009 and McCament & McCarthy, 2005). Jacobs
and Severeid (2004) report annual growth rates of 84 cm/yr for seven-year old, plantation
grown, American chestnut interplanted with black walnut and northern red oak in Wisconsin.
Jacobs et al. (2009) also reports similar, respective growth rates of 79 and 72 cm/yr for 12
and 19-year old American chestnuts.
A number of factors contribute to survival, growth and development of American chestnut
seedlings. Clark (2013) proposed three elements for successful establishment of backcross
hybrids. These include 1) the ability of seedlings to compete, 2) the ability of seedlings to
overcome deer browse and 3) having enough resistance to survive blight. The American
Chestnut Foundation’s backcross breeding program is focused on addressing the latter.
Therefore, integrating their advanced genetic material with management practices that attend
to the first two obstacles should greatly increase restoration success.
The competitive abilities of American chestnut and backcross hybrids depend in part on
shade tolerance and growth relative to other species. For example, in full sun, chestnut
seedlings or sprouts must be able to compete with fast growing pioneer species such as
yellow-poplar (Liriodendron tulipifera). Significant growth responses to competition control
3
have been observed by implementing cultural practices such as burning or herbicide
treatments (McCament & McCarthy, 2005). However, the mechanics of these treatments
come into question when examining growth responses to competition control in the presence
of other treatments, such as thinning, that increase light and nutrient availability.
Characterizing the level of shade tolerance for American chestnut will help address the
importance of light availability for the species. Joesting (2009) investigated American
chestnut growth under varying light regimes, and concluded that the species is intermediate
in shade tolerance. This is consistent with other intermediate shade tolerant species that
persist in the understory until a canopy gap opens and the individual responds quickly in
growth to occupy the dominant crown class (Joesting, 2009). Concluding her study, Joesting
suggests restorationists plant hybrids in the shaded understory and maintain the plantings
through the sapling stage in stand development before creating canopy gaps or openings for
continued growth and development. Despite these recommendations, Joesting does not offer
target light levels to optimize survival and growth under this strategy.
In an outdoor rainout shelter pot study of American chestnut grown from seed, Wang et al.
found significant responses in height and diameter at increasing light levels, up to 32% of full
light; however, no significant differences were observed in either height or diameter above
32% of full light. While the light saturation point for American chestnut was not addressed in
Wang's study, the diminished response at full light, coupled with a lack of response in height
and diameter between 32% and full light may indicate plant growth limitations due to
something other than light, such as water or nutrient availability. Wang et al. did not address
4
nutrient availability in their study; however quantifying and understanding any interaction
between light and nutrient availability would be quite valuable in developing management
protocols for chestnut restoration.
Rhoades et al. evaluated two-year growth and development of American chestnut seedlings
across two site types, under two silvicultural systems, in eastern Kentucky. The two
contrasting site types were described as "mesic" and "xeric" for the purposes of the study.
Two silvicultural treatments were imposed at each site, and consisted of a two-age
shelterwood and a midstory removal. Under the shelterwood system, 65-70% of total stand
basal area was removed, compared to only 30% of the basal area within the midstory removal
treatment. Differences in basal area removal created differences in light availability in the
forest understory. Rhoades et al. reported significant differences in fertility among sites, with
greater values of N, P, K+, Mg2+ and Ca2+ observed at the mesic sites. Soil texture differed
little between sites. Two-year height growth was 118.1 cm in the mesic shelterwood, 86.1 cm
in the mesic midstory, 90.9 cm in the xeric shelterwood and 81 cm in the xeric midstory. The
shelterwood treatments created a significant growth response as compared with the midstory
removal treatments across both sites. Interestingly, the midstory removal treatment did not
create a significant difference in chestnut growth between sites. Specifically, seedling growth
under the midstory treatment at the xeric site (low light & low fertility) was not significantly
different from chestnut growth under the midstory treatment at the mesic site (low light and
high fertility). At greater levels of light (shelterwood), significant growth responses were
observed between the two contrasting sites (mesic > xeric). One inference from this study is
5
that fertilization may be a useful management tool to increase early growth and development
for American chestnut on low fertility sites, where light is not a limiting factor.
A similar study was conducted by McNab (2003), with American chestnut seedlings
deployed across both shelterwood and midstory removal areas near Asheville, NC. The focus
of McNab's study was assessing survival and growth of bare-root seedlings when planted at
different levels of light using different combinations of tree shelters and fertilizer. This study
reports a negative relationship between fertilization and survival. However, no significant
differences in height growth were found between any of the cultural treatments. The author
admits that the small sample size made it difficult to detect any real differences.
Due to the limited availability of advanced generations of backcross hybrid material,
American chestnut has been studied to make inferences about management strategies for
deploying blight resistant backcross hybrids. No equivalence studies comparing growth
characteristics between the two can be found to substantiate similarities in predicted
responses. Therefore, studies evaluating the effects of forest practices on growth and
development of both backcross hybrids and pure American chestnuts are needed in order to
apply findings from prior research and ensure successful restoration of the species.
6
REFERENCES
Ashe, W.W., 1912. Chestnut in Tennessee. Tennessee Geological Survey Bulletin 10-B.
Nashville, TN.
Burnham, Charles R. 1981. Blight-Resistant American Chestnut: There's Hope. Plant
Disease. 65:459-460.
Clark, Stacy; Schlarbaum, Scott; Saxton, John; Hebard, Fred; Blanton, John; Casey, David ;
Crane, Barbara; MacFarlane, Russ; Rodrigue, Jason; Jim, Stelick. 2012. Lessons from the
field: The first tests of restoration American chestnut (Castanea dentata) seedlings planted in
the Southern Region. In: Butnor, John R., ed. 2012. Proceedings of the 16th biennial southern
silvicultural research conference. e-Gen. Tech. Rep. SRS-156. Asheville, NC: U.S.
Department of Agriculture Forest Service, Southern Research Station. 69-70.
Elliott, Katherine J.; Swank, Wayne T. 2008. Long-term changes in forest composition and
diversity following early logging (1919-1923) and the decline of American chestnut
(Castanea dentata). Plant Ecol. 197: 155-172.
Jacobs, Douglass F. 2010. Silvicultural and logistical considerations associated with the
pending reintroduction of American chestnut. In: Stanturf, John A., ed. 2010. Proceedings of
the 14th biennial southern silvicultural research conference. Gen. Tech. Rep. SRS–121.
Asheville, NC: U.S. Department of Agriculture, Forest Service, Southern Research Station.
207-209.
Jacobs, D.F.; Selig, M.F.; Severeid, L.R. 2009. Aboveground carbon biomass of plantationgrown American chestnut (Castanea dentata) in absence of blight. Forest Ecology and
Management. 258: 288-294.
Jacobs, Douglass F. 2007. Toward development of silvical strategies for forest restoration of
American chestnut (Castanea dentata) using blight-resistant hybrids. Biological
Conservation. 137:497-506.
Jacobs, D.F.; Severeid, L.R. 2004. Dominance of interplanted American chestnut (Castanea
dentata) in southwestern Wisconsin, USA. Forest Ecology and Management. 191: 111-120.
Joesting, H.M.; McCarthy, B.C.; Brown, K.J. 2009. Determining the shade tolerance of
American chestnut using morphological and physiological leaf parameters. Forest Ecology
and Management. 257: 280-286.
McCament, C.L.; McCarthy, B.C. 2005. Two-year response of American chestnut (Castanea
dentata) seedlings to shelterwood harvesting and fire in a mixed-oak forest ecosystem. Can.
J. For. Res., 35, 740–749.
7
McNab HW (2003) Early results from a pilot test of C. dentata seedlings under a forest
canopy. J Am Chestnut Found 16:32–41.
McWiliams, William H.; Lister, Tonya W.; LaPoint, Elizabeth B.; Rose, Anita K.; Vissage,
John S. 2006. Current status of chestnut in eastern US forests. In: Restoration of American
Chestnut to Forest Lands: proceedings of a conference and workshop held at The North
Carolina Arboretum, Asheville, North Carolina, U. S. A., May 4 6, 2004. Natural Resources
Report NPS/NCR/CUE/NRR 2006/001. [Washington, D.C.]: U.S. Dept. of the Interior,
National Park Service, National Capital Region, Center for Urban Ecology, 2006: 31-39.
Paillet, Frederick L. 2002. Chestnut: history and ecology of a transformed species. Journal of
Biogeography. 29:1517-1530.
Paillet, Frederick L. and Philip A. Rutter. 1989. Replacement of native oak and hickory tree
species by the introduced American chestnut (Castanea dentata) in southwestern Wisconsin.
Can. J. Bot. 67:3457-3469.
Rea, Glen. 2010. A New Era for TACF. J. of American Chestnut Foundation. 24:5.
Rhoades, Chuck.; David Loftis, Jeffrey Lewis and Stacy Clark. 2009. The influence of
silvicultural treatments and site conditions on American chestnut (Castanea dentata) seedling
establishment in eastern Kentucky, USA. Forest Ecology and Management. 258: 1211-1218.
Wang, G. Geoff; Knapp, Benjamin O.; Clark, Stacy L.; Mudder, Bryan T. 2013. The Silvics
of Castanea dentata (Marsh.) Borkh., American chestnut, Fagaceae (Beech Family). Gen.
Tech. Rep. SRS-GTR-173. Asheville, NC: U.S. Department of Agriculture Forest Service,
Southern Research Station. 18 p.
Wang, G. Geoff; Bauerle, William L.; Mudder, Bryan T. 2006. Effects of light acclimation on
photosynthesis, growth, and biomass allocation in American chestnut seedlings. Gen. Tech.
Rep. SRS-92. Asheville, NC: U.S. Department of Agriculture, Forest Service, Southern
Research Station. pp. 261.
8
CHAPTER 2 – Manuscript for Publication
Introduction
The demise of American chestnut (Castanea dentata Marsh. Borkh.) occurred during the first
half of the 20th century before an extensive body of published research related to the species'
growth and development was established in scientific literature (Paillet, 2002 and Jacobs,
2010). Recent advances in the backcross breeding program of The American Chestnut
Foundation (TACF) have prompted researchers to become increasingly interested in studying
American chestnut with the hope that restoration strategies can be developed for backcross
hybrids. Successful restoration requires an understanding of hybrid responses to
environmental changes, site conditions, plant community dynamics, management practices
and selective pressures.
Many studies report American chestnut as a fast-growing and extremely competitive species
during early development (Jacobs, 2010). A review of one and two-year growth in three
different studies with diverse treatment and site conditions indicates that American chestnut
can be expected to grow an average of 16 cm/yr, with ranges from 2 cm/yr to 37 cm/yr (Clark
et al., 2012, Rhoades et al., 2009 and McCament & McCarthy, 2005).
A number of factors contribute to survival, growth and development of American chestnut
seedlings. Clark (2012) proposed three elements for successful establishment of backcross
hybrids. These include 1) the ability of seedlings to compete, 2) the ability of seedlings to
overcome deer browse and 3) having enough resistance to survive blight. While the
American Chestnut Foundation’s backcross breeding program is focused on addressing the
9
latter, studies evaluating family performance under different environmental and site
conditions could prove useful in developing site specific management recommendations for
restoration plantings. Subsequently, integrating advanced genetic material with management
practices that attend to increasing early growth and survival should greatly increase
restoration success.
The competitive abilities of American chestnut and backcross hybrids depend on shade
tolerance and growth relative to other species. For example, in full sun, chestnut seedlings or
sprouts must be able to compete with fast growing pioneer species such as yellow-poplar
(Liriodendron tulipifera). Significant growth responses to competition control have been
observed by implementing cultural practices such as burning or herbicide treatments
(McCament & McCarthy, 2005). Joesting (2009) investigated shade tolerance for American
chestnut and concluded that the species is intermediate in shade tolerance. This is consistent
with other intermediate shade tolerant species that persist in the understory until a canopy
gap opens and the individual responds quickly in growth to occupy the dominant crown class
(Joesting, 2009). Concluding her study, Joesting suggests restorationists plant hybrids in the
shaded understory and maintain the plantings through the sapling stage in stand development
before creating canopy gaps or openings for continued growth and development. Despite
these recommendations, Joesting does not offer target light levels to optimize survival and
growth under this strategy.
In an outdoor rainout shelter pot study of American chestnut grown from seed, Wang et al.
(2006) found significant responses in height and diameter at increasing light levels, up to
10
32% of full light; however, no significant differences were observed in either height or
diameter above 32% of full light. The diminished response at full light, coupled with lack of
response in height and diameter between 32% and full light may indicate that, above 32%
full sun, growth was limited by something other than light, such as water or nutrient
availability. Wang's study did not address either water or nutrient availability; however,
quantifying and understanding any interactions with water, nutrients and light would be quite
valuable in developing management protocols for chestnut restoration.
Rhoades et al. (2009) evaluated two-year growth and development of American chestnut
seedlings across two site types, under two silvicultural systems, in eastern Kentucky. The two
contrasting site types were described as "mesic" and "xeric" while the two silvicultural
treatments were described as a two-age shelterwood and a midstory removal. Under the
shelterwood system, 65-70% of total stand basal area was removed, compared to only 30% of
the basal area within the midstory removal treatment. Differences in basal area removal
created differences in light availability in the forest understory, but were not quantified in the
study. Rhoades et al. (2009) reported significant differences in fertility among sites, with
greater values of N, P, K+, Mg2+ and Ca2+ observed at the mesic sites. Soil texture differed
little between sites. Shelterwood treatments were associated with a significant growth
response across sites, and height growth in the shelterwood treatments was significantly
different from the midstory treatment. Two-year height growth was 36.98 cm in mesic
shelterwood, 10.9 cm in the mesic midstory, 19.48 cm in the xeric shelterwood and 9.70 cm
in the xeric midstory. Interestingly, despite greater levels of fertility at the mesic site, there
11
was no significant difference in chestnut growth between the midstory treatments across
sites. However, at greater levels of light (Shelterwood), significant growth responses were
observed between the two contrasting sites (mesic > xeric). The results of this study highlight
the need to quantify differences in site resources in order to propose management strategies
for restoration.
A similar study was conducted by McNab et al. (2003) with American chestnut seedlings
deployed across both shelterwood and midstory removal areas near Asheville, NC. McNab's
study was focused on assessing survival and growth of bare-root seedlings when planted at
different levels of light using different combinations of tree shelters and fertilizer. This study
reports a negative relationship between fertilization and survival. However, no significant
differences in height growth were found among any of the cultural treatments. The author
admits that the small sample size made it difficult to detect any, real differences.
Due to the limited availability of advanced generations of backcross hybrid material, few
seedlings of American chestnut have been studied to make inferences about management
strategies for deploying blight resistant backcross hybrids. No equivalence studies comparing
growth characteristics between the two can be found to substantiate similarities in predicted
responses. Therefore, studies evaluating the effects of forest practices on growth and
development of both backcross hybrids and pure American chestnuts are needed in order to
apply findings from prior research and ensure successful restoration of the species. Here, we
report results from a study evaluating early growth and development of 22 families from
12
TACF's backcross breeding program under two silvicultural systems and two levels of
fertilization as compared to Chinese and American chestnut controls.
Methods
The study site was located in the Southern Appalachian Mountains, within the natural range
of American chestnut, in the Toe River Township of Avery County, North Carolina. Forest
communities supported mixed-mesophytic overstory species ranging from 55 to 80 years old.
American chestnut sprouts occurred naturally throughout the area. During the course of the
study, annual precipitation and temperature averaged 134.6 cm and 11.7°C, respectively
(State Climate Office of North Carolina, 2016). Soils across the site formed from biotite
gneiss or colluvium derived from igneous and metamorphic rock. The USDA Natural
Resources Conservation Service (NRCS) identified soils across this site as Cullasaja
(Loamy-skeletal, isotic, mesic Typic Humudepts), Unaka (Fine-loamy, isotic, mesic Humic
Dystrudepts) and Porters series (Fine-loamy, isotic, mesic Humic Dystrudepts) (Avery
County Soil Survey, 2014).
Two harvest treatments were implemented across the study site, with the shelterwood harvest
occurring in 2007 and the clear-cut harvest occurring in 2010. Site preparation for planting
occurred in July 2013; in both treatment plots, all understory vegetation (except American
chestnut in the shelterwood) was hand felled with brush saws and removed. At both
locations, immediately following felling, stumps were treated with a 25% solution of Garlon
4 (triclopyr) at a rate of less than 10.4 liters per hectare to control resprouting.
13
The study was initially intended as a TACF progeny test to evaluate blight resistance of the
most advanced genetic material in their breeding program. However, the design was revised
to further evaluate seedling performance under varying levels of resources availability. The
study was a split-plot design with silvicultural treatment (clear-cut vs. shelterwood) as the
whole plot factor, and nutrition as the sub-plot factor. Six whole plots were established in
each of the two silvicultural treatments at the same site; however, whole plots were not
replicated across multiple sites. Sub-plots were randomly assigned a nutrition treatment of
either no nutrient addition (0) or 64 kg-N +72 kg-P per hectare. Within each sub-plot, genetic
entries were randomly assigned to one of twenty-four planting locations on a 2.4m x 2.4m
spacing (four rows and six columns in each sub-plot). Genetic material consisted of twentyfour replicates of twenty-two families from TACF's tree breeding program plus Chinese
(Castanea mollissima) and American chestnut controls. The 22 hybrid families in the study
were derived from 6 common maternal grandparents, and all trace their lineage back to one
Chinese x American hybrid cross. Chinese and American controls were included as a
barometer of blight resistance to evaluate hybrid resistance over time. Chinese chestnut
seedlings were derived from two sources with unknown provenance and American chestnuts
were derived from either an eastern Kentucky or Pennsylvania provenance. Families were
represented equally among treatments (24 families per sub-plot X 24 sub-plots).
On 17 September 2013, 576, 1-0 containerized seedlings produced in 655.5 cm3 containers
(D40 Deepot™, Stuewe & Sons, 2016) were planted at an approximate density of 1736
trees/ha. Deer exclusion cages were erected within one-week following planting. Fertilizer
14
amendments were applied to sub-plot treatments as diammonium phosphate (DAP, 18-46-0)
at a rate of 5.04 g-N and 5.62 g-P per tree (64.2 kg-N and 71.6 kg-P /ha) on a single-tree
basis within a 1/2-meter radius of each seedling in May 2014. Seedlings were covered with
plastic drums and competing vegetation was controlled in June 2014 with a foliar application
of Accord© XRT II at 4% plus 1% PHASE™ surfactant.
Initial measures of seedling height and survival were collected in May 2014. Measures of
photosynthetically active radiation (PAR) were collected using an AccuPAR LP-80, at an
elevation of one-meter (1-m) above each seedling on 5 July 2014. Light measurements were
collected between the hours of 1100 and 1300 on a cloudless day with measures taken in full
sun before and after data collection to ensure consistent background conditions. Seedling
survival and height were measured at the end of the first and second growing seasons, in
October 2014 and October 2015. Seedling root collar diameter was measured at
approximately 1.5 cm above ground level on the uphill side of each seedling at the end of the
first growing season in October 2014, and again following the second growing season in
October 2015. Individual trees were survey-located in October 2014 to determine location
and elevation. Species, basal area, trees/ha, average diameter and individual tree locations of
residual overwood in the shelterwood area were also collected in October 2014.
Silvicultural treatment and fertilizer treatment were treated as fixed effects. Due to the large
number of families in this study, family would typically be treated as a random effect.
However, pedigree information obtained from TACF indicates that these families are closely
related. Therefore, family was also treated as a fixed effect in the analysis.
15
Height and diameter growth responses were evaluated by an analysis of variance (ANOVA).
The model used for the analysis is given by:
Yijkl = µ + β(Xijkl – ....)+ Si + Nj + (SN)ij + Gk + εijkl
where Y is the response variable (height or diameter growth), µ is the base level of growth
(grand mean), β is the vector of coefficients for the vector of differences between the lth
observation of initial seedling height or diameter under the kth group (Xijkl) and the mean
height or diameter of seedlings in the kth group (....), Si is the fixed effect of the ith
silvicultural treatment, Nj is the fixed effect of the jth nutrition treatment, (SN)ij is the twofactor interaction of the ith cultural treatment and jth nutrition treatment, Gk is the fixed effect
of the kth family and ε represents random or unexplained error. Height and diameter growth
were analyzed using a mixed model analysis of variance to determine significant effects of
cultural treatment, nutrition, their interaction, and family effects, while Tukey-Kramer tests
were utilized to detect significant differences among pairwise comparisons of least squared
means (PROC GLIMMIX, SAS Enterprise Guide version 6.100, SAS Institute, Cary, NC).
This analysis assumes data are derived from independent observations, data are normally
distributed for each group, and variances are equal for each group. Data were checked for
normality by examining the Kolmogorov-Smirnov statistic, the Anderson-Darling statistic,
and the Cramér-von Mises statistic (PROC UNIVARIATE, Goodness-of-Fit Tests, SAS
Enterprise Guide version 6.100, SAS Institute, Cary, NC).
16
Light availability was evaluated by an analysis of variance (ANOVA). The model used for
the analysis is given by:
Yi = µ + Si + εi
where Y is the response variable (mmol light), µ is the base level of light (grand mean), Si is
the fixed effect of the ith silvicultural treatment, and ε represents random or unexplained
error. The effect of cultural treatment on light availability was also analyzed using PROC
GLIMMIX (SAS Enterprise Guide version 6.100, SAS Institute, Cary, NC).
Results and Discussion
Light availability was significantly less in the shelterwood treatment (p < 0.0001), with the
shelterwood seedlings receiving, on average, 28.3% of the total PAR observed for seedlings
growing in the clear-cut (full sun). While mean light was lower in the shelterwood, light
availability varied greatly, ranging from 15.4 mmol to 2,022.1 mmol for individual trees.
Light availability from 4% up to 32% of full sun has been shown to significantly increase
American chestnut seedling height growth (Wang et al., 2006). Therefore, our findings are
consistent with these published results, as increasing light levels also significantly improved
seedling height growth in this study (Table 1).
Survival was exceptionally high, with more than 90% of seedlings still living in October
2015 after the first and second growing seasons. Since survival is a binomial trait that must
be transformed for analysis, and significant differences are difficult to detect above 70%,
survival analysis was not appropriate. Another study of American chestnut seedlings planted
17
in the central Appalachians reports similar survival after two growing seasons (Phelps et al.,
2005). However, other studies report lower survival rates ranging from 66% in southeastern
Kentucky (Pinchot et al., 2012) to 79% in the southern Appalachians (Clark et al., 2012). We
attribute the exceptional survival to the use of containerized seedlings. Within the clear-cut
treatment, 4.5% of seedlings were killed by voles early in the first growing season. As vole
damage occurred only in the clear-cut, and this study lacks replication of clear-cut treatments
across multiple sites, it isn't clear whether voles prefer clear-cuts in general, or just this clearcut site. Therefore, we excluded vole killed trees from the survival analysis. Vole damage is
strongly related to cover, and clear-cut sites tend to have a higher percent ground-cover than
shelterwood sites (Pusenius, 2002). Therefore, one might expect voles to prefer clear-cuts to
shelterwood areas. Still, survival differences between treatments were negligible. Silviculture
treatments and nutrition treatments had similar survival rates. These results contrast to those
where fertilization of American chestnut seedlings in the southern Appalachians was
associated with increased levels of mortality after five growing seasons (McNab et al., 2003).
It will be interesting to follow survival in the future to determine if treatments begin to have
an effect as the stand ages. Survival due to blight resistance is a principal interest of this
study. While a small number of seedlings have already died as a result of chestnut blight,
susceptible individuals are from different families, and it is still too early to know which
families in the study will survive to maturity.
One-year height growth averaged 23.0 cm across all treatments. Study-wide second-year
height growth was more than double the first-year growth, averaging 52.5 cm across all
18
treatments. Cumulative height growth after two growing seasons was 73.9 cm. These
observations are substantially greater than the first-year height growth (9.29 cm) observed for
BC2F3 hybrid chestnut seedlings in southeastern Kentucky (Pinchot et al., 2012) and slightly
greater than first-year height growth (19.27 cm) observed for American chestnut seedlings
growing in eastern Kentucky (Rhoades et al., 2009). Silviculture and nutrition treatments
had a significant effect on height growth during both years of the study (Table 1). Clear-cut
grown seedlings grew 24% more than shelterwood grown seedlings during the first growing
season (25.4 cm and 20.7 cm, respectively). These findings are consistent with other
published studies where American chestnut seedling growth increased with increasing light
availability (Rhoades et al. 2009 and Wang et al. 2006). Fertilized seedlings grew 36% more
than non-fertilized seedlings during the first growing season (26.2 cm and 19.9 cm,
respectively). There were no silviculture by nutrition treatment interactions during the first
growing season (Table 1). Therefore, initial seedling growth response to fertilization was the
same across silvicultural treatments (6.3 cm). While no interactive effects were observed
during the first year (p = 0.45), there was a significant silviculture by nutrition treatment
interaction during the second year of the study (p < 0.0001). This interaction is also evident
in the results of the analysis of cumulative height growth (p < 0.0003). Tukey-Kramer least
square means analysis reveals the source of the interactive effect (Table 2). Specifically,
fertilization continued to improve seedling height growth by 35% in the clear-cut treatment in
year two, but had no effect on seedling growth in the shelterwood areas. The same interactive
effect is evident in the total or cumulative growth response, where fertilization improved
seedlings height growth by 38% in the clear-cut, but had no significant effect in the
19
shelterwood (Table 2). These findings are consistent with a published report of American
chestnut seedlings growing in eastern Kentucky, where no difference was found in two-year
height growth between high and low fertility sites at low light levels (Rhoades et al., 2009).
Overall, seedling height growth within the best treatment combination (clear-cut/fertilization)
was 83% more than seedling height growth in the lowest treatment combination
(shelterwood/no fertilization).
First-year diameter growth was not available for analysis. Silviculture treatment, nutrition,
and genotype (family) all had significant effects on diameter growth during the secondgrowing season (Table 1). Second-year diameter growth was significantly greater in the
clear-cut compared to the shelterwood (p < 0.0001), averaging 7.2-mm and 5.3-mm,
respectively. Similarly, fertilized seedlings grew significantly more than non-fertilized
seedlings (p = 0.01), averaging 6.5-mm and 5.9-mm, respectively. There were no silviculture
by nutrition treatment interactions during the second growing season (Table 1). Seedling
growth increased similarly from shelterwood to clear-cut treatments, irrespective of nutrient
additions. On average, seedlings from all treatments in this study exhibited greater secondyear diameter growth than those reported for American chestnut growing in eastern Kentucky
under similar contrasting treatments of light and nutrition (Rhoades et al., 2009).
Although this study was established with an equal number of individuals from twenty-two
different families of TACF’s backcross breeding program, 45% of hybrid individuals within
the study share identical second generation maternal ancestry, and all trace their lineage back
to the original Graves tree used as a cornerstone in TACF's breeding program (Hebard,
20
2012). Most researchers would agree that twenty-two open pollinated families form a diverse
genetic base. However, the interrelatedness of families in this study presents limitations in
establishing a breeding population on this site. Despite the lack of genetic diversity, genotype
or family differences had a significant effect on all growth analyses in the study (Table 1).
Study-wide cumulative mean height growth by family ranged from 53 cm to 105 cm (Figure
1). Interestingly, the seedlings from two highest ranking families in the study (in terms of
height growth) are offspring from two half-sib mothers (W1-32-69 and W6-32-92). The
study contains but one other family with a similar genetic background (W4-32-87), and all
three rank in the top one-third for height growth in this study. While treatment effects such as
fertilization may become less dramatic over time, genetic differences in height growth may
continue to become more pronounced. Family means for diameter growth ranged from 4.6mm to 8.5-mm. One family (W-6-32-92) ranked in the top three for both second-year
diameter growth and cumulative height growth.
A notable result of this study is the pronounced genotype by environment interaction that
exists for families grown under different levels of nutrition (Table 1). Family rankings for
cumulative height growth are different for the two levels of nutrition (Figure 2). Specifically,
some families respond to nutrient amendments with substantially greater height growth while
other families respond very little. While it is early, if such findings persist, this information
would prove very useful in developing site specific restoration plans that include the
selection of a suite of families tailored to a site’s fertility or for management regimes that
include fertilization.
21
Conclusion
Increases in seedling growth due to silvicultural treatments that manipulate light and nutrition
have tremendous implications for American chestnut restoration projects. Clear-cut grown
seedlings with nutrient amendments can grow more than 100 cm in two-years following
planting. This treatment combination is a viable option for establishing restoration plantings
and quickly growing seedlings above the browse line for deer. Shelterwood treatments that
are often used in hardwood management to protect seedlings from frost damage may be
employed for American chestnut restoration, as well. Survival is excellent in both instances.
However, a better understanding of resource limitations within the shelterwood system is
required in order to develop strategies that increase seedling height growth to a rate closer to
that of the clear-cut. Exploratory analysis did not find a relationship between the one-time
measure of light and growth of individual trees within the shelterwood. Further investigation
is needed to better quantify the factors that play a role in height growth of seedlings growing
in the understory. Treatment effects on height growth should be monitored during
subsequent growing seasons to determine whether or not the growth response to fertilization
continues in the clear-cut or becomes insignificant as we observed in the shelterwood.
While silviculture and nutrition play an important role, early results of family and nutrition
interactions could prove monumental. Five families in this study had greater than average
cumulative mean height growth in the presence of added nutrients; however, these same
families were below average performers with respect to mean height growth in the absence of
nutrient amendments. Restorationists may find that a suite of families exist for high fertility
22
sites such as rich coves, and that a different suite of families exist for nutrient deficient sites
such as dry ridges. Further evaluation is necessary to determine whether or not the trend
holds true across multiple sites and over time.
American chestnut is a dynamic species with the ability to grow quickly in full sun and
survive years in a shaded forest understory. In the past, researchers have used American
chestnut to study management systems for restoration using backcross hybrids with little
evidence to substantiate the similarities. After two growing seasons, we see that hybrids do
not perform differently from Americans. Therefore, land managers are fortunate to have
many options for successfully deploying backcross hybrids in restoration plantings for the
future.
23
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Ecology Will Aid Restoration Efforts. J. of American Chestnut Foundation. 26, 4:27.
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Crane, Barbara; MacFarlane, Russ; Rodrigue, Jason; Jim, Stelick. 2012. Lessons from the
field: The first tests of restoration American chestnut (Castanea dentata) seedlings planted in
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canopy. J Am Chestnut Found 16:32–41.
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Stacy L.; Schweitzer, Callie J., Saxton, Arnold M.; Hebard, Frederick V. 2012. Early results
of a chestnut planting in eastern Kentucky illustrate reintroduction challenges. In: Butnor,
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25
Table 1. P-value summary for the different analysis of variance (ANOVA) results.
Treatment
Light
Availability
Yr 1 Mean
Height
Growth
Yr 2 Mean
Height
Growth
Total
Height
Growth
Yr 2 Mean
Diameter
Growth
Silviculture
<.0001
<.0001
<.0001
<.0001
<.0001
Nutrition
-
<.0001
<.0001
<.0001
0.01
Family
-
0.01
0.01
0.00
<.0001
Silviculture X Nutrition
-
0.45
<.0001
0.0004
0.33
Silviculture X Family
-
0.03
0.05
0.14
0.38
Nutrition X Family
-
0.14
0.03
0.01
0.59
Silviculture X Nutrition X Family
-
0.19
0.90
0.50
0.49
Table 2. Mean height and diameter growth (± SE) estimates for seedlings grown under different silviculture
and nutrition. Within columns, similar letters indicate that means are not significantly different based on
Tukey-Kramer least square means comparisons (α = 0.05).
Yr 1 Mean
Height Growth
(cm)
Yr 2 Mean
Height Growth
(cm)
Total Height
Growth (cm)
Yr 2 Mean
Diameter
Growth (mm)
Fertilization
28.9a (1.0)
75.1a (2.4)
103.0a (2.9)
7.6a (0.3)
No Fertilization
21.9b (1.0)
55.5b (2.4)
75.0b (2.9)
6.7a (0.3)
Fertilization
23.5b (1.0)
41.0c (2.4)
64.3c (2.9)
5.5b (0.3)
No Fertilization
18.0c (1.0)
40.7c (2.4)
56.8c (2.9)
5.1b (0.3)
Treatment
Clear-cut
Shelterwood
26
120
100
Total Height Growth (cm)
80
60
40
20
0
TACF Families
Figure 1. Mean total height growth after two years for all families included in the study.
*Note: The first family is significantly different from the last five families based on Tukey-Kramer least square means
comparisons (α = 0.05). Similarly, the second family is significantly different from the last four.
27
140
Fert
No Fert
Fert Mean
No Fert Mean
Total Height Growth (cm)
120
100
80
60
40
20
0
TACF Families
Figure 2. Family means for cumulative height growth under different levels of nutrition.
28