Effects of Long Term Nitrogen Deposition on ...Effects of Long Term Nitrogen Deposition on...

Effects of Long Term Nitrogen Deposition on Ectomycorrhizal Fungal

Communities in Forests of the Northeastern United States

Alexandrea Mae Rice

Allegheny College

Meadville, PA 16337

Mentors: John Hobbie and Jerry Melillo

Ecosystems Center, Marine Biological Laboratory

Woods Hole, MA 02543

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Abstract:

Nitrogen (N) deposition has more than doubled since the industrial revolution and this is

affecting terrestrial ecosystems in a variety of ways. Nutrient cycling processes in these systems

are being altered by increased N deposition, although our understanding of many of the effects is

limited. For example, we know little about how N deposition affects mycorrhizal fungi and their

symbiotic interactions with forest trees. This issue became the topic of my research. I made

measurements to explore how long-term N additions affect species composition and abundance

as well as enzymatic activity of ectomycorrhizal fungi communities at two sites with different

nitrogen deposition rates associated with air pollution from the burning of fossil fuels for

industry and transportation, and from the fertilizer in agriculture - Harvard Forest in central

Massachusetts receiving 8 kg N ha-1

yr-1

and Bousson Forest in western Pennsylvania receiving

13 kg N ha-1

yr-1

. Both of these sites are part of long-term ecological research projects in which

large plots within the study areas have been receiving additional nitrogen inputs for over 20

years. Harvard Forest has two N addition plots, one receiving 50 kg N ha-1

yr-1

and the other 150

kg N ha-1

yr-1

. Bousson Forest has only one nitrogen addition plot receiving 100 kg N ha-1

yr-1

.

From these fertilized and adjacent control plots, I identified and counted a series of root tip fungi

and evaluated enzyme activity for phosphatase and cellulase. I observed a shift in

ectomycorrhizal fungal composition with increased N deposition and N fertilization. Generally,

Russula is favored by the higher N inputs. There was an increase in Russula abundance and a

decrease in Cenococcum at Harvard Forest. At Bousson Forest, Russula was dominant and

remained dominant with fertilization, while Cenococcum disappeared. I also saw differences in

enzymatic activity potentials between my two sites. Phosphatase activity was higher at Bousson

Forest than at Harvard Forest, indicating that Bousson Forest could be potentially becoming a

phosphorous limiting ecosystem as a result of long-term N deposition. Harvard Forest on the

other hand had higher rates of cellulase, indicating that it could be becoming a carbon limited

system. The species shift that I found generally match the findings of Sadowsky and Frey (2013),

with the exception that they found Cortinarius to decrease with fertilization, not Cenococcum.

Key Words: mycorrhizae, ectomycorrhizae, nitrogen saturation, nitrogen deposition gradient

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Introduction:

As a result of anthropogenic activities such as fertilization and fossil fuel combustion,

nitrogen deposition has nearly doubled in the last century (Vitousek et al. 1997). These

anthropogenic activities have caused a deposition gradient to occur in the northeastern United

States. The higher end of the gradient is near Pittsburgh, receiving 13 kg N ha-1

yr-1

. As we move

more northwards the nitrogen deposition lessens due to the prevailing winds. The further

northeast we go, less nitrogen is deposited because the area is downwind from the source. The

nitrogen deposited in these areas poses a major threat to ecosystems due to the fact that soils

become more fertile, changing the species compositions of the forest (Tilman 1993; Willems et

al. 1993). The species composition could be changing due to a shift in nutrient limitation in the

ecosystem. Increasing the nitrogen in a system, can shift it to a phosphorous or carbon limiting

system. Mycorrhizal fungi are greatly affected by this change in nutrient limitation because of

their role in the ecosystem. Ectomycorrhizal fungi form a symbiosis with many dominant

temperate forest trees and supply these hosts with important nutrients such as nitrogen and

phosphorus that are derived from the organic compounds in the surrounding soil. In return for

these nutrients, the plant provides the fungi with photosynthetically derived carbohydrates in the

form of simple sugars it needs to grow (Brundrett et al. 1996; Hobbie 2006; Smith and Read

1997; Read 1991). These fungi form hyphal sheaths around the root tips of the trees and by

measuring the quantity and kind of enzymes that the sheaths produce the nutrient status of the

forest can be investigated (Allen et al. 2003). It is suggested that with these anthropogenic inputs,

this role of mycorrhizae in the nitrogen cycle has been altered in soils (Sadowsky and Frey

2013).

Because of an interest in studying the effects of increased nitrogen deposition, scientists

established the Chronic Nitrogen Amendment Study at Harvard Forest. This study was

established to evaluate the impact of long term nitrogen deposition on the carbon and nitrogen

cycles in hardwood forests (Frey et al. 2004). These plots are referred to as nitrogen saturation

plots which is a term that is applied when the addition of nitrogen relieves the nitrogen limitation

in an ecosystem (Agren and Bosatta 1988; Aber 1992). It is important to study the projected

changes in the mycorrhizal community with increased nitrogen deposition and ask the question:

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Do changes in the fungi give us a clue about the changes in the nutrient cycles and limitations of

the trees?

When looking at the local and regional scale in the northeastern United States,

industrialized and agricultural areas exceed unpopulated areas in annual nitrogen deposition

(Figure 1). Toward Maine, there are fewer fossil fuel emissions due to the lack of large cities and

agricultural lands. The northeastern winds also are responsible for this gradient. As deposition

occurs, there is less to be carried towards the northeast, therefore the nitrogen deposition is much

less than areas in the south. Pennsylvania is one of those states on the southern end of this

gradient and also contains one of the most polluted cities, Pittsburgh (ALA 2014). This is the

reason for the higher nitrogen deposition to soils in Pennsylvania, 13 kg N ha-1

yr-1

, opposed to

the mere 8 kg N ha-1

yr-1

deposited on the Harvard Forest (Holland et al. 2004; Magil et al.

2004). This gradient is the reason why I chose my two sites, Bousson Experimental Forest and

Harvard Forest. Harvard Forest is on the lower end of this spectrum located near Maine, whereas

Bousson Experimental Forest is only a few hours away from Pittsburgh.

With respect to ectomycorrhizal fungi, I expected that there would be a shift in fungi

communities within the two sites as well as within experimental plots at each site receiving

different levels of nitrogen fertilizer. More specifically, there would be a decrease in species

richness as nitrogen fertilization increases due to the fact that the plants will have access to the

nitrogen for which they form some of their symbiosis with fungi. Without the need of certain

fungi, the mycorrhizae that are still needed by some plants will become more abundant because

they will not be competing with the other species. I also used isotopic analysis to try and

determine whether the nitrogen addition is breaking the one type of symbiosis between fungi and

plants. This could show whether the trees are obtaining their nitrogen from soil or

ectomycorrhizal fungi. With further additions of nitrogen in a nitrogen limited system, there is

potential for the limiting nutrient to shift to carbon or phosphorous. This is why I also analyzed

enzymatic activity on root tips for cellulase, endopeptidase, and phosphatase using fluorogenic

substrates.

Methods:

Harvard Forest

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Harvard Forest is located in north-central Massachusetts. The Chronic Nitrogen

Amendment study in Harvard Forest was begun in 1988. The 55 year old hardwood forest is

mostly dominated by Black Oak (Quercus velutina) and Red Oak (Quercus rubra). Also present

are Black Birch (Betula lenta), Paper Birch (Betula papyrifera), Red Maple (Acer rubrum), and

American Beech (Fagus grandifolia) trees (Bowden 2004). Soils are stony-sandy loams due to

their glacier origin. Agriculture was once practiced where the forest is today. The nitrogen

saturation experiment contains 3 plots that are 30 x 30 m receiving the following treatments:

control (no nitrogen added), low nitrogen (50 kg N ha-1

yr-1

), high nitrogen (150 kg N ha-1

yr-1

).

These plots are fertilized with ammonium nitrate (NH4NO3), distributed into six monthly doses .

Bousson Experimental Forest:

Bousson Experimental Research Reserve is located in northwestern Pennsylvania. The

Bousson Experimental Forest established the high nitrogen cycling site in 1994. The 80 year old

mixed deciduous forest is dominated 60% by Black Cherry (Prunus serotina Ehrh), 28% Sugar

Maple (Acer saccharum Marsh), and 12% a mixture of American beech (Fagus grandifolia) and

Red Oak (Quercus ruba). Soils are alfisols that are sandy and poorly drained. Agriculture was

once practiced on this site. The nitrogen addition plots at Bousson contain only control and

fertilized plots. There are 3 controls and 3 fertilized (100 kg N ha-1

yr-1

) 15 x 15 m plots. These

plots are fertilized with ammonium nitrate (NH4NO3) in six monthly applications from May to

October each year. Nitrogen is applied currently in a liquid form using a backpack sprayer, but in

earlier years was applied in a pellet form.

Field Sampling:

In Harvard Forest the three plots are further separated into 5 x 5 m plots, for a total of 36

plots. I selected four out of these 36 subplots for sampling. Samples were taken within the

middle 16 subplots to ensure there was no leaf litter contamination from outside the plot (Brant

et al. 2006). These were random collections with the focus on tree species that favor

ectomycorrhizal fungi, such as Red Oak (Quercus rubra) and American Beech (Fagus

grandifolia). I avoided sampling in close proximity to Acer saccharum Marsh because they are

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known to favor arbuscular mycorrhizal fungi, not ectomycorrhizal fungi. In each of these four

subplots, I collected one soil core that was 7.5 cm in diameter and 10 cm deep. I took these

samples as close to the base of the tree as possible, < 30 cm away, in an attempt to collect

ectomycorrhizal species.

In addition to the soil cores, I also collected leaf samples and mushrooms found in the

plots. The leaves were gathered from the innermost plots so that the possibility of the leaves

being from tree species within the plot was high. Five leaves of each tree species present,

American Beech (Fagus grandifolia), Red Maple (Acer rubrum), Black Oak (Quercus velutina),

and Red Oak (Quercus rubra) were gathered for isotopic analysis. The mushrooms were also

collected for isotopic analysis (Image 1and 2).

I followed the same techniques at the Bousson Experimental Forest as I did at the

Harvard Forest. Being in Woods Hole, MA I did not personally collect these samples. Dr. Rich

Bowden, a professor at Allegheny College, sent them to me. He took three soil cores from each

of the two plots in Bousson. Since Sugar Maple (Acer saccharum Marsh) is one of the dominant

species in the forest, it was hard to avoid taking cores around this species. The same size tulip

corer, 7.5 cm in diameter and 10 cm deep, was used to extract the samples less than 30 cm away

from the base of the tree. There were no mushrooms found within the plots therefore no isotopic

analysis could be done. Five leaf samples from each tree species within the plots, Sugar Maple

(Acer saccharum Marsh), Black Cherry (Prunus serotina Ehrh), American Beech (Fagus

grandifolia), and Red Oak (Quercus ruba), were collected for isotopic analysis.

Laboratory Analysis:

Identification of Fungal Hyphae on Root Tips

My soil cores, leaf samples, and mushrooms were stored on ice and brought back to

Woods Hole, MA. In the lab, I separated the tree roots from the soil. This was done by hand, not

by the use of a sieve, to protect the roots and their attached fungi from being damaged. The roots

were stored at 4 ⁰C in plastic bags with moist paper towels until they could be sorted. Within a

week after collection, the roots were separated by mycorrhizal type according to the pictures

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taken by Jesse Sadowsky, who used genetic sequencing to identify the species shown (Sadowsky

and Frey 2014). It is possible that there are tens to hundreds of additional types of fungi present

but the types of ectomycorrhizal hyphae or type of sheath described in these pictures are those

that are easily identifiable.

After identification, root tips with identified fungi were counted to calculate relative

abundance of ectomycorrhizal species. These were then stored in plastic bags with soil until

enzyme analysis could be preformed.

pH

I measured pH in the treatment plots for both Harvard Forest and Bousson Experimental

Forest. Approximately 5 g of moist soil from each soil core was placed into a urine cup. I mixed

in 50 mL of deionized water to create a 1:10 soil to water ratio. After eight hours, the pH of the

soil slurry was measured with a glass electrode.

Enzyme Activity in Root Tips

To assess the enzyme activity in the mycorrhizal fungi that I collected, I adapted methods

from Pritsch et al. (2011), Alana Thurston (2014), and Courty et al. (2005).

After 29 days of storage, the root tips and the fungal sheath from my soil cores were

cleaned with deionized water and severed with a scalpel so that they were all approximately the

same length, 0.3 mm to 1.5 mm. The fungal sheath length and widths were measured using a

Zeiss SteREO Discovery.V12 microscope and the software Axio Vision to make specific

measurements. These root tips were severed one day before being measured and two days before

enzyme assays were performed on them. Each treatment at Bousson Experimental Forest and

Harvard Forest was analyzed separately. Seven root tips and their attached hyphae for each

species per treatment, a total of 112 root tips, were used. Using a Greiner Bio-One 96-well

microplate, one root tip was placed into an individual well with 100 µL of an incubation buffer.

The incubation buffer contained 50 mL of 75 mM maleic acid with 18 mL of 200 mM glycine

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buffer to achieve a pH of 4.40. The roots were incubated for 2 hours before the enzyme

substrates were added in order to buffer the roots. After 2 hours, 50 µL of the substrates were

added to their corresponding wells. The substrates used to test enzymatic activity were MUF-

phosphate (2.4 mM) for phosphatase, MUF-D-glucoside (1.5 mM) for cellulase, and L-Leucine-

7-amido-4-methylcoumarin HCl (0.6 mM) for endopeptidase.

The analysis was performed using a Synergy H1 Hybrid plate reader. The microplates

were set to shake continuously after the initial reading. Readings were taken every minute for 5

minutes and then every 5 minutes for a total of 30 minutes. A final read was taken 10 minutes

after the 30 minute read to test further activity. The plates were not shaken for those 10 minutes.

Both of the well plates were read at wavelengths of 364 nm excitation and 445 nm emission.

A series of wells contained my standards of 0.01, 0.05, 0.1, 0.3, and 0.5 µM of the MUF

substrate as well as enzyme substrates and deionized water used as controls. Using the equation

derived from the standard curve (R2

=0.9467), I was able to calculate enzymatic activity through

the absorbance values. I also accounted for the surface area of the hyphae on the root tips for

each well by dividing the calculated enzyme activity by this area. The surface areas of each

hyphal sheath on the root were based of the equation for the surface area of a cylinder.

Isotope Analysis

The leaves, entire roots including the fungal sheaths, and mushrooms collected from

Bousson Experimental Forest and Harvard forest were placed in 20 mL scintillation vials and

dried in the drying oven at 62 ⁰C for a week. After a week, I ground them up using a mortar and

pestle. Between 3.5 and 4.5 mg of each sample was ran for δ15

N analysis by Marshall Otter at the

Ecosystems Center Stable Isotope facility. Due to limited resources, no duplicates were run, and

the different leaf species were combined into one scintillation vial for one analysis per treatment.

Results:

Analysis of Fungal Types on Root Tips

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The quantification and identification of fungi on root tips show a shift in species relative

abundance with increased nitrogen fertilization of the soils. At the Harvard Forest the most

abundant species, Cenococcum and Russula, show different trends between the treatments. Three

mycorrhizal species increase: Russula by 35%, Cortinarius by 10%, and Tomentella by 4%,

while the other two species decrease, most notable is the decrease of Cenococcum by 45%

(Figures 2, 3, 4).

In the Bousson Experimental Forest there are three species present: Russula, Tomentella,

and Cenococcum (Figure 5). Cenococcum is eliminated in the fertilized plot, and Tomentella

takes over. Russula, the dominant, remains relatively the same between treatments but with a

negligible decrease of 4% (Figure 6).

pH

Overall, the soil at both of my sites, Bousson and Harvard Forest are acidic. The Bousson

plots on average are more acidic than the Harvard Forest plots but they are not statistically

different (Figure 7).

Enzyme Activity in Hyphae on Root Tips

Overall, out of the four fungal species that I tested for potential enzyme activity, only

Russula and Cenococcum increase in enzymatic activity with increased fertilization. The

fertilized plots at Bousson have higher activity in phosphatase and cellulase than any other plots

in Bousson or Harvard Forest. For all of the plots tested, there is no endopeptidase activity

present.

The two species found in the Bousson Experiential Forest, Russula and Tomentella,

increase in phosphatase activity with nitrogen fertilization. Cellulase activity with fertilization

increases in Russula and decreases in Tomentella (Table 1).

In the Harvard Forest plots, Russula shows no difference in phosphatase activity when

fertilized, but cellulose activity increases slightly. There is also no change in phosphatase activity

in Tomentella. Phosphatase activity in Tomentella increases from the control to the low

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fertilization plots but decreases from the low fertilization to high fertilization plots. In

Cenococcum, there is overall an increase in both phosphatase and cellulase activity with

fertilization. Cortinarius shows the opposite trends. Both the phosphatase and cellulase activity

in Cortinarius decreases with fertilization (Table 1).

Isotope Analysis

Between the three treatments at Harvard Forest and the two treatments at Bousson, the

δ15

N values decrease with increased fertilization in both the root tip and leaf samples. The two

mushrooms express the same trend (Table 2). The control plot values are always higher than the

fertilized plots in both areas.

Discussion:

My study revealed several effects of nitrogen deposition and fertilizer addition on

ectomycorrhizal fungi communities. At Harvard Forest, in response to nitrogen fertilization, the

proportion of Russula increased as Cenococcum decreased. The number of species found

decreased as deposition increased. This trend was also observed at Bousson Forest. In the control

plot at Bousson, only three ectomycorrhizal species were found, Russula, Cenococcum, and

Cortinarius. Russula was over 90% dominant in the control plot and stayed that way in the

fertilized plot. My findings are similar to Sadowsky and Frey's (2013) findings at Harvard Forest

in the nitrogen fertilization plots. Their study found that Cortinarius decreased as Russula

increased. Cortinarius was not a key player in my study, and that could be due to my sample

size. I took only four soil cores in each plot. Russula on the other hand was dominant in both

studies and is known to catalyze the breakdown of cellulose (Sadowsky and Frey 2013). It has

also been found that increased nitrogen deposition increased carbon availability in a system

(Magil 2004). At Harvard Forest, the increase in Russula could possibly be explained by the

increase of carbon available to the system. With more carbon available, Russula is able to thrive,

therefore it becomes more abundant.

Ecological theory suggests that with increased nitrogen, ectomycorrhizal fungi will no

longer be needed by plants since nitrogen will be readily available to the roots (Frey 2004). To

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test this, I took samples of roots, tree leaves, and mushrooms and ran 15

N analysis on them. If

this statement was true I would expect to see the 15

N values in the tree leaves to increase as

deposition increases. In my analysis, no interpretations were able to be made due to not having

the 15

N values of the fertilizer used at both sites.

The difference that I observed between the fertilized plots at Bousson and Harvard Forest

suggest that increased nitrogen deposition impacts soil fungi enzymatic production. No

endopeptidase activity was noticed in any of the plots. This could be because enzymes are

energetically expensive to produce therefore there is no need to expend the extra energy

producing endopeptidase when nitrogen is readily available to the roots. Phosphatase and

cellulase did show activity in both Bousson and Harvard Forest. At Harvard Forest, the cellulase

rates are higher than the phosphatase rates. This could indicate that Harvard Forest is becoming a

more carbon limited system with increased nitrogen deposition. The opposite is true at Bousson

Forest where the phosphatase rates are higher than the cellulase rates. This could indicate that

Bousson Forest is becoming a more phosphorous limited system as the nitrogen inputs increase.

Further studies would need to be done in order to determine if these systems are in fact

becoming more carbon and phosphorous limited. Also, enzyme assays would need to be

conducted soon after core collection times to ensure the rates have not changed from their values

in their natural environment. To test that these rates do not change over time, I would perform

enzyme assays weekly over a period of several months time after the samples were collected. To

better understand the species composition in these sites, more soil cores would need to be taken

in each plot as well. My sample size was small, and could have missed important species. In

order to determine if the nitrogen gradient in the northeastern United States is in fact affecting

the ectomycorrhizal species composition in the forests, more sites would need to be examined

along the gradient. A site in Maine, West Virginia, Ohio, and Vermont would be distributed

along this gradient. If trends followed the ones found in my study and others, then we would be

able to deduce if nitrogen deposition is in fact affecting the ectomycorrhizal fungal communities.

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Acknowledgements:

I would like to thank my mentors John Hobbie and Jerry Melillo for their continuous

guidance through this project and for their knowledge and expertise on ectomycorrhizal fungi. I

would also like to thank Thomas Parker for his advice on how to execute this study various

times. Rich Bowden, who collected my samples from the Bousson Experimental Forest is owed a

huge thanks, because without him this study would not have been possible. The same

appreciation goes to Elena Lopez Peredo for giving me guidance on the Biotek reader and

enzyme work. Louie Kerr gave me guidance on using the dissecting microscope and additional

equipment to measure my root tips. I thank Melissa Knorr and William Werner for guiding me

through Harvard Forest, and taking time out of their day to help me gather my samples. I would

also like to thank Ken Foreman for his continuous help through this project. I would not have

know what equipment to use without him. I also would like to thank the lab TA's - Rich

McHorney, Brecia Douglas, Aliza Ray, and Hannah Kuhns for all their time and effort put into

my learning and project this semester.

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References:

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Trends Ecological Evolution 7: 220–223.

Agren, G., and E. Bosatta. 1988. Nitrogen saturation of terrestrial ecosystems. Environmental

Pollution 54: 185-197.

Allen, E.B., M.F. Allen, L. Egerton-Warburton, L. Corkidi, and A. Gomez-Pompa. 2003.

Impacts of early-and late-seral mycorrhizae during restoration in seasonal tropical

forest, Mexico. Ecological Applications 13:1701-1717.

American Lung Association (ALA). 2014. Most Polluted Cities. State of the Air 2014. Web.

Bowden, R.D., E. Davidson, and K. Savage. 2004. Chronic nitrogen additions reduce total oil

respiration and microbial respiration in temperate forest soils at the Harvard Forest.

Forest Ecology and Management 196: 43-56.

Brant, J.B., D.D. Myrold, and E.W. Sulzman. 2004. Root controls on soil microbial community

structure in forest soils. Community Ecology 148: 650-659.

Brundrett, M., N. Bougher , B. Dell, T. Grove, and N. Malajczuk. 1996. Working with

mycorrhizas in forestry and agriculture. Australian Centre for International Agricultural

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Brundrett M., N. Bougher, B. Dell, T. Grove, and N. Malajczuk. 1995. Working with

mycorrhizas in forestry and agriculture. Canberra, ACT: Australian Centre for

International Agricultural Research, 1996. Print.

Courty, P.E., K. Pritsch, M. Schloter, A. Hartmann, and J. Garbaye. 2005. Activity profiling of

ectomycorrhiza communities in two forests soils using multiple enzymatic tests. New

Phytologist 167: 309-319.

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Frey, S., M. Knorr, J.L. Parrent, and R.T. Simpson. 2004. Chronic nitrogen enrichment affects

the structure and function of the soil microbial community in temperate hardwood and

pine forests. Forest Ecology and Management 196: 159-171.

Hobbie E.A. and R. Agerer. 2010. Nitrogen isotopes in ectomycorrhizal sporocarps correspond

to belowground exploration types. Plant and Soil 327:71-83.

Hobbie, E.A. 2006. Carbon allocation to ectomycorrhizal fungi correlated with belowground

allocation in culture studies. Ecology 87: 563-569.

Holland E.A, B.H. Braswell, J.M. Sulzman, J.F. Lamarque. 2004. Nitrogen deposition onto the

United States and Western Europe. In. ORNL DAAC, Oak Ridge.

http://www.daac.ornl.gov

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Melillo, and P. Steudler. 2004. Ecosystem response to 15 years of chronic nitrogen

additions at the Harvard Forest LTER, Massachusetts, USA. Forest Ecology and

Management 196: 7-28.

Pritsch, K., P.E. Courty, J. Churin, B. Cloutier-Hurteau, M. Ali, C. Damon, M. Duchemin,

S. Egli , J. Ernst, L. Fraissinet-Tachet, F. Kuhar, E. Legname, R. Marmeisse, A. Müller,

P. Nikolova, M. Peter, C. Plassard, F. Richard, M. Schloter, M. Selosse, A. Franc, and

J. Garbaye. 2011. Optimized assay and storage conditions for enzyme activity profiling

of ectomycorrhizae. Mycorrhiza 21: 589-600.

Read, D.J. 1991. Mycorrhizas in ecosystems. Experientia 47: 376-391.

Sadowsky J. and S. Frey. 2014. Repertoire of secreted enzymes underlies a shift in abundance of

ectomycorrhizal fungal lineages due to long-term nitrogen enrichment. Unpublished

manuscript.

Sadowsky, J., and S. Frey. 2013. Harvard Forest Nitrogen Saturation Experiment uncovers

microbial response to long-term nitrogen additions. Network News, Fall 26.

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Skujins, J.J., L. Braal, and A.D. McLaren. 1962.Characterization of phosphatase in a

terrestrial soil sterilized with an electron beam. Enzymologia 25: 125- 133

Smith, S. E., D.J. Read, and F.T. Last. 1997. Mycorrhizal symbiosis. Annals of Botany 80:

701.

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enzyme activity of mycorrhizae in heated and unheated forest soils at Harvard Forest,

Petersham MA. SES at MBL Woods Hole, MA.

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colonization limitation. Ecology 74: 2179–2191.

Vitousek, P.M., J.D. Aber, R.W. Howarth, G.E. Likens, P.A. Matson, D.W. Schindler, W.H.

Schlesinger, and D.G. Tilman. 1997. Human alteration of the global nitrogen cycle:

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Figures, Tables, and Images:

Image 1: Mushroom collected from Harvard Forest. The species was not identified, and was

collected from the Low fertilized plot.

Image 2: Mushroom collected from Harvard Forest. No species identification was made. This

mushroom was collected from the High fertilized plot.

Figure 1. The nitrogen deposition gradient in northeastern United States. This gradient was

presented by J. Aber (2003) to show the difference in nitrogen deposition in the New England

area. The highest rates (10-12 kg N ha-1

yr-1

) are in southern New York and Pennsylvania due to

the presence of big cities. The low depositions are toward Maine with <4 kg N ha-1

yr-1

. Harvard

Forest is located in the light green area on this map where Massachusetts is. Bousson Forest is

located left of the red area on the map.

Figure 2. Ectomycorrhizal fungal species found in Harvard Forest. The species composition is in

relative abundance through identification and fungal counts in four soil core samples taken in the

control plot. The plot is part of the nitrogen saturation experiment at Harvard Forest.

Figure 3. Ectomycorrhizal fungal species found in the Harvard Forest low fertilization plot. The

species composition is in relative abundance through identification and fungal counts in four soil

core samples taken in this plot. The plot is part of the nitrogen saturation experiment at Harvard

Forest.

Figure 4. Ectomycorrhizal fungal species found in the Harvard Forest high fertilization plot. The



Forest.

Figure 5. Ectomycorrhizal fungal species found in the Bousson Experimental Forest control plot.

The species composition is in relative abundance through identification and fungal counts in

three soil core samples taken in this plot. The plot is part of the nitrogen saturation experiment at

Bousson Experimental Forest.

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Figure 6. Ectomycorrhizal fungal species found in the Bousson Experimental Forest fertilized

plot. The species composition is in relative abundance through identification and fungal counts in



Figure 7. The pH in the 5 fertilized plots. In Harvard Forest the sites are more acidic on average

than in Bousson. There is no statistical difference shown.

Table 1. The rate of potential enzyme activities in hyphae on root tips from the plots at Bousson

Forest and Harvard Forest. They are organized according to species found in the plots. All three

enzymes tested for are shown. The enzyme Endopeptidase was not active in any of the plots.

Table 2. The isotopic data from the leaves, mushrooms, and roots plus hyphal sheaths collected

from both Bousson Experimental Forest and Harvard Forest.

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Image 1: Mushroom collected from Harvard Forest. The species was not identified, and was

collected from the Low fertilized plot.

Image 2: Mushroom collected from Harvard Forest. No species identification was made. This

mushroom was collected from the High fertilized plot.

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Figure 1. The nitrogen deposition gradient in northeastern United States. This gradient was

presented by J. Aber (2003) to show the difference in nitrogen deposition in the New England

area. The highest rates (10-12 kg N ha-1

yr-1

) are in southern New York and Pennsylvania due to

the presence of big cities. The low depositions are toward Maine with <4 kg N ha-1

yr-1

. Harvard

Forest is located in the light green area on this map where Massachusetts is. Bousson Forest is

located left of the red area on the map.

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Figure 2. Ectomycorrhizal fungal species found in Harvard Forest. The species composition is in

relative abundance through identification and fungal counts in four soil core samples taken in the

control plot. The plot is part of the nitrogen saturation experiment at Harvard Forest.

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Figure 3. Ectomycorrhizal fungal species found in the Harvard Forest low fertilization plot. The



Forest.

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Figure 4. Ectomycorrhizal Fungi species found in the Harvard Forest high fertilization plot. The



Forest.

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Figure 5. Ectomycorrhizal fungal species found in the Bousson Experimental Forest control plot.

The species composition is in relative abundance through identification and fungal counts in



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Figure 6. Ectomycorrhizal fungal species found in the Bousson Experimental Forest fertilized

plot. The species composition is in relative abundance through identification and fungal counts in



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Figure 7. The pH in the 5 fertilized plots. In Harvard Forest the sites are more acidic on average

than in Bousson. There is no statistical difference shown.

3.7

3.8

3.9

4

4.1

4.2

4.3

4.4

4.5

4.6

HF Control HF Low Fert HF High Fert Bou Control Bou Fert

pH

Location

pH

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ECM Species Plot

Phosphatase

Enzyme activity

(µmol min-1

mm-2

)

Cellulase

Enzyme

Activity

(µmol min-1

mm-2

)

Endopeptidase

Enzyme Activity

(µmol min-1

mm-2

)

Russula

Bousson Control 0.006 0.0052 -0.0221

Bousson Fert 0.0991 0.0295 -0.0082

Harvard Forest Control 0.0057 0.002 -0.0423

Harvard Foest Low Fert 0.0043 0.0046 -0.0204

Harvard Forest High Fert 0.0073 0.0097 -0.0174

Tomentella

Bousson Control 0.0049 0.0015 -0.005

Bousson Fert 0.0142 0.0001 -0.029


Harvard Forest Low Fert 0.015 0.0272 -0.0491


Cenococcum




Cortinarius




Table 1. The rate of potential enzyme activities in hyphae on root tips from the plots at Bousson

Forest and Harvard Forest. They are organized according to species found in the plots. All three

enzymes tested for are shown. The enzyme Endopeptidase was not active in any of the plots.

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Site Sample

δ15

N

(0/00 vs. AIR)

Harvard Forest C Root 8.7

LF Root 4.3

HF Root 1.1

C Leaf 5

LF Leaf 3.9

HF Leaf 0.5

LF Mush 3.6

HF

Mush 6.7

Bousson C Root 4.2

F Root 1.2

C Leaf 0

F Leaf -1

Table 2. The isotopic data from the leaves, mushrooms, and roots plus hyphal sheaths collected

from both Bousson Experimental Forest and Harvard Forest.

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