Influence of Nutrient Additions on Fungal Community Structure
and Root Nutrient Uptake in Northern Harwood Forests
Final Report to the Edna Bailey Sussman Foundation
By
Jerome C. Barner
12/11/2015
M.S. Candidate
Department of Environmental and Forest Biology
State University of New York College of Environmental Science and Forestry
Background
Human activities have vastly increased nitrogen delivery to ecosystems, and nitrogen deposition has depleted calcium from forest soils. Temperate forests evolved under conditions of nitrogen limitation, thus nitrogen addition via air pollution is likely to cause some other element to become limiting, most likely phosphorus or calcium.
Implications of this changing nutrient environment extend to the symbiotic fungi associated with tree roots, or mycorrhizae, which are known to decrease in both species diversity and abundance in response to nitrogen additions. The effect of changes in phosphorus and calcium availability on the function of these fungi, including their role in nutrient uptake, is less well known. Tree species in northern hardwood forests form associations with different types of mycorrhizal fungi. Ectomycorrhizal fungi (EM), as opposed to Arbuscular mycorrhizae (AM, i.e. red maple), exhibit high species diversity as well as extremely different morphological and functional features between species, which has large impacts on forest nutrient cycling. Understanding these role fungi play in nutrient uptake and how these essential symbionts respond to a changing nutrient environment is critical for informing forest management decisions and sustaining forest production into the future. The objectives of this study were to examine differences in nutrient uptake in roots of Betula alleghniensis (EM) and Acer rubrum (AM), and to quantify impacts on EM fungal community composition in response to N, P, and Ca.
Introduction
1

Northern hardwood forests have been historically characterized as exhibiting nitrogen limitation, but forest harvest and base cation depletion due to acid deposition may lead to the gradual shift to a phosphorus limited state. Although N and P are generally considered most important growth limiting nutrients in forested ecosystems, plants can vary their allocation effort for multiple resources to efficiently balance their needs in a species-specific resource limited environment (Chapin et al., 2002).
Mycorrhizal fungi form symbiotic associations with over 85% of terrestrial land plants and mediate interactions between soils and vegetation (Smith and Read 2008). These obligate biotrophic partners and known to increases a plants access to spatially and temporally heterogeneous soil minerals, be essential for sustained growth and competitive ability of plants across different ecosystems, modify water relations, increase drought resistance, and provide access to nutrients not available directly to plant roots
(Brundrett 2008).
Ectomycorrhizal fungi (EM) form relationships with a wide variety of commercially important tree species in northern hardwood ecosystems, including many co-dominant species, such as Betula alleghniensis . These fungi are known to modify associated plant root morphologies, enveloping the root tip in a fungal sheath, with hyphae both internally colonized intercellular spaces between root epidermal and cortical cells (Hartig net) and growing outwardly into the soil matrix (van der Heijdin et al.
2015). The ability of tree roots associated with EM fungi to acquire nutrients is a reflection of the nutrient environment (externally in the soil, and internally within fungus and root), belowground competition, and the spatial extent of hyphal proliferation outward into the soil matrix (Brundrett 2008). Extraradical hyphae have been repeatedly shown to transport large quantities of N and P to associated plant hosts (van der Heijdin et al. 2015), but the extent of hyphal proliferation can be highly variable and exhibits specific characteristics (Agerer 2001). Additionally, species level diversity of mycorrhizal hyphal exploration types are known to decrease in response to fertilization treatments with entire functional classes disappearing as a result of N deposition
(Lilleskov et al. 2002). Little is known about the functional relationships between mycorrhizal species and nutrient uptake processes, but these interactions can dictate the ability of plants to access nutrient pools and compete both above and below ground.
2

Characterizing the mycorrhizal communities associated with fine roots of host tree species in response to nutrient manipulations can yield valuable insights into the mycorrhizal mediation of root nutrient uptake, and refine the scope of current nutrient cycling modeling efforts. This project will examine nutrient uptake between two tree species, one EM and one AM, and analyze the genetic composition of the EM community.
Methods
Study Site
Root nutrient uptake capacity experiments took place in replicated N, P, N and P,
Ca, and control treatment plots established in 2011 by the Multiple Element Limitation in
Northern Hardwood Ecosystems study (MELNHE) at the Bartlett Experimental Forest in
Bartlett, NH.
Root samples will be taken from two co-dominant hardwood tree species, yellow birch ( Betula alleghniensis ) and red maple ( Acer rubrum ), at one early successional (aged 25 - 35 yrs) stand, site C2. Soils are spodosols (typic and aquic
Haplorthods) that developed on granite and gneiss derived glacial tills. Forest composition is typical of northern hardwood forests, mature forests having an overstory dominated by sugar maple ( Acer Saccharum ), American beech ( Fagus grandifolia ) and yellow birch ( Betula alleghniensis ), with occasional presence of white ash ( Fraxinus americana ), white birch ( Betula papyrifera ) and red maple ( Acer rubrum ). Young stands are dominated by a complex of pin cherry ( Prunus pensylvanica ), red maple, yellow birch, and white birch.
Each stand consists of four treatment plots, with 50m x 50m inner plots that have been maintained with annual fertilizer treatments of nitrogen (30 kg N/ha/yr as NH
4
NO
3
) and phosphorus (10 kg P/ha/yr as NaH
2
PO
4
), since 2011; a one time calcium treatment
(wollastonite) at a rate of 1150 kg Ca/ha was applied in 2010. MELNHE treatment plots consist of: N, P, NP, Ca and a control treatment (sites C1 and C8). Plots are subdivided into 30m x 30m inner plots with a surrounding 10 m buffer zone. Sampling will take place within inner treatment areas and the buffer zone in order to sample eight yellow birch trees per treatment plot, with a total sample size of 40 root branches per stand.
3

Root Nutrient Uptake Capacity Measurements
Nutrient uptake capacity will be measured via the depletion method as described by Lucash et al. 2005, 2007 . Roots are gently extracted by hand from the organic/upper mineral soil horizons and morphologically identified using a hand lense based on prominent root characteristics (color, morphology, spatial extent) (Yanai et al. 2009).
Excavated fine roots (<2mm in diameter) are left attached to the host tree, rinsed with distilled water to remove lose soil particles, and covered in a moist paper towel until the experiment begins. When all roots are excavated, root branches are placed in 27 mL nutrient solution, in a 50 mL centrifuge tube. Soil solution concentrations are based on site-specific nutrient availabilities, determined via soil paste slurry extracts from each site. Tubes will be sealed over with Parafilm, covered with tarps to prevent evaporation, and aerated with oxygen via tubing and fish pumps, to prevent formation of depletion zones around roots. In order to ensure nutrient concentrations are available enough for measurable uptake, solutions consisting of 1x, 5x, and 10x initial solution concentrations will be utilized for each subplot. An aerated, control nutrient solution consisting of no roots will be utilized for comparative purposes. Solutions will be collected after a twohour period, this should provide sufficient time for roots to recover from the shock of excavation and provide more reliable estimates of nutrient uptake capacity (Lucash et al.
2007). Samples will be immediately filtered utilizing a 0.4 micron polycarbonate filter, kept on ice in the field, and frozen until processing. Solutions will be analyzed for ammonium and nitrate utilizing a continuous flow analyzer. Phosphorus, calcium, magnesium, potassium, and sulfur will be analyzed utilizing ICP-OES mass spectroscopy; samples will be analyzed within a QC threshold of +/- 5%.
Mycorrhizal Identification
Upon completion of the nutrient uptake experiment in the field and the laboratory
(seedlings), roots utilized for uptake will be excised and placed in CTAB
(cetyltrimetylammonium bromide) solution for preservation for DNA extraction. These roots will first be analyzed for root length, functional order, and diameter by scanning the
4

roots into WinRhizio identification software. Following methodology in Horton and
Bruns 2001 , a subsample of the EM root tips will used for DNA extraction after qualitatively assessing morphological composition of the root system. DNA will be extracted from a subsample of EM root tips, amplifying the ITS region of the nuclear rDNA genes, BLAST searching, and matching clone sequences to available databases.
Statistics
Data collected will be analyzed using a one-way ANOVA with fertilizer treatments (five levels) as treatment factors at each level of nutrient solution concentration. A repeated measures analysis will analyze the influence of increasing nutrient solution concentrations on root nutrient uptake. Information attained from increasing nutrient solution concentrations will be used to inform uptake curves utilized in terrestrial nutrient cycling models. Mycorrhizal species composition will be assessed using mean species richness and diversity index values, and visualized using Bray Curtis ordination and Non-Metric Multidimensional Scaling (NMDS) techniques . Species level composition will be tested against fertilization treatment (one way ANOVA), and across successional age and fertilization treatment (two way ANOVA). All analyses will be conducted using Statistical Analysis Software (SAS) package (SAS Institute, Raleigh,
NC).
Results
For all macronutrients tested (Al, Ca, K, P, NH
4
+
, NO
3
-
, S) significant efflux of solution concentration was seen at the 1x concentration level. This was defined as a higher concentration of nutrients in the soil solution following treatment. This could be resultant from leakiness of the roots associated with disturbance and severing the fungal hyphae, which are the water and nutrient uptake mechanisms for tree roots in the northern hardwood ecosystem. Nutrient uptake responded disparately based on increasing soil solution. For Al, K, and S, uptake increased significantly with increasing soil solution concentration. Ca, NH
4
+
, and NO
3
-
responded similarly, with uptake increasing with concentration for the 1x and 5x solutions, but leveling off at the 10x concentration. For these three nutrients, a threshold of tolerance or biotic demand may have been reached
5

for uptake, with any greater uptake resulting in negative impacts on tree health. P did not show any preference for uptake based on concentration but was very tightly grouped, with uptake responding similarly to concentration, suggesting limitation by P.
Nitrate Depletion umol/L
1000
800
600
400
200
0
-200
YB: 1x
YB: 5x
YB: 10x
RM: 1x
RM: 5x
RM: 10x
NP Control
Treatment
P N
Figure 1: Nitrate uptake by yellow birch and red maple roots in response to increasing soil solution concentration.
Pronounced differences in uptake between the two tree species were not seen for any macronutrient. Both tree species took up nutrients similarly over the given time interval, suggesting species preferences for particular nutrients is absent or poorly represented, or both species are equivocal competitors with similar rates of uptake of
6

macronutrients. Nutrient uptake between treatment plots revealed some interesting differences in this relatively young stand. For Al, significant differences between uptake in the control and P plot were seen, with higher uptake in the control plot. No treatment differences were seen for Ca, K, NH
4
+
, NO
3
-
, and S, suggesting uptake for these nutrients on this site was not influenced by nutrient treatment. P showed a slightly significant difference in uptake (p = 0.058) between the control and P plot, with greater P uptake in the control plot. Positive uptake for macronutrients seemed relatively consistent across soil solution concentrations. The majority of uptake rates at the 1x concentration exhibited efflux of nutrients, while 5x and 10x solution concentrations resulted in positive uptake of nutrients.
Phosphorus Depletion umol/L
1000
500
0
-500
-1000
-1500
-2000
YB: 1x
YB: 5x
YB: 10x
RM: 1x
RM: 5x
RM: 10x
NP
Control
Treatment
P
N
Figure 2: Phosphorus uptake by fine roots in response to increasing soil solution concentration.
7

Future Work
Continued analysis and development of regression models based on nutrient solutions will be sustained into next year. Analysis of fungal DNA collected from root tips will continue in the laboratory into next year, with the hope of building a community analysis of the EM fungal communities response to nutrient additions. Furthermore, morphotyping into hyphal exploration types and cross referencing with known exploration types will continue. Also, a new study consisting of incubating hyphal ingrowth bags filled with autoclaved sand in our study plots is currently taking place.
This will be a bioassay of EM fungal mycelium present in the soil, which can be more reliable in building community diversity estimates compared to the limited study of root tips.
Acknowlegements
I would first like to thank the Edna Bailey Foundation for the support in this research, without which would not have been possible. I would like to thank Dr. Tom
Horton for all of his help in the laboratory, all of my lab mates, including Yang Yang and
Kara Phelps. Also, I would like to thank previous graduate students, particularly Melissa
Lucash, for pioneering this method and laying the important groundwork for future study.
Literature Cited
Agerer, R. 2001. Exploration types of ectomycorrhizae. Mycorrhiza 11: 107-114.
Brundrett MC. 2008. Mycorrhizal Associations: The Web Resource. March 21 2015.
‹mycorrhizas.info›.
Chapin, F.S. III, Matson, P. A., Mooney, H. A. 2002. Principles of Terrestrial Ecosystem
Ecology. Springer Verlag, New York.
Horton, T. R., T.D. Bruns. 2001. The molecular revolution in ectomycorrhizal ecology: peeking into the black-box. Molecular Ecology 10 (8): 1855-1871.
8

Lilleskov E.A., Fahey T.J., Horton T.R., Lovett G.M. 2002. Nitrogen deposition and ectomycorrhizal fungal communities: a belowground view from Alaska. Ecology
83: 104 - 115.
Lucash, M.S., R.D. Yanai, and J.D. Joslin. 2008. Nutrient uptake by intact and disturbed roots of loblolly pine seedlings. Environmental and Experimental
Botany 64: 15-20.
Lucash, M.S., D.M. Eissenstat, J.D. Joslin, K.J. McFarlane and R.D. Yanai. 2007.
Estimating nutrient uptake by mature tree roots under field conditions: challenges and opportunities. Trees - Structure and Function 21(6):593-603.
Lucash, M.S., R.D. Yanai, and J.D. Joslin. 2005. Temporal variation in nutrient uptake capacity by intact roots of mature loblolly pine. Plant and Soil
272:253-262.
Smith SE, Read DJ. 2008. Mycorrhizal Symbiosis. Third Edition. Academic Press
Oxford, UK.
Yanai, R.D, K.J. McFarlane, M.S. Lucash, J.D. Joslin, and S.E.
Kulpa. 2009. Nutrient uptake by Engelmann spruce and subalpine fir at two Colorado subalpine forests. Forest Ecology and
Management 258(10): 2233-2241.
Van der Heijdin, M.G.A., F. Martin, M.A. Selosse, I.R. Sanders. 2014. Tansley review:
Mycorrhizal ecology and evolution: past, present, and future. New Phytologist
205: 1406-1423.
9