Health and mycorrhizal colonization response of sugar maple (Acer saccharum) seedlings to calcium addition in Watershed 1 at the Hubbard Brook Experimental Forest
TITLE
Health and mycorrhizal colonization response of sugar maple (Acer saccharum) seedlings to calcium addition in Watershed 1 at the Hubbard Brook Experimental Forest
PRINCIPAL INVESTIGATOR(s)
Tim Fahey
Department of Natural Resources, Cornell University
12 Fernow Hall
Ithaca, NY 14853
USA

Phone: (607)-255-5470
OTHERS INVOLVED
Stephanie Juice
ABSTRACT:
We examined the health and mycorrhizal colonization rates of sugar maple germinants on a Calcium (Ca)- treated watershed at Hubbard Brook Experimental Forest and compared their status to the health of seedlings growing on reference sites. We quantified general sugar maple germinant health by height, leaf area, biomass, and chlorophyll content. To determine vesicular-arbuscular mycorrhizal colonization rates, we first cleared and stained germinant roots and then examined their structures microscopically. We found that sugar maple seedlings on the Ca-addition site were taller, had larger leaves, more root, leaf, and stem biomass, and more chlorophyll per unit leaf area. These seedlings also had a significantly larger percentage of their roots colonized by mycorrhizal fungi. Our results indicate a correlation between soil Ca availability, mycorrhizal colonization rates, and overall seedling health.
KEYWORD SET: Hubbard Brook Ecosystem Study LTER
Acer saccharum, acid deposition, calcium, HBEF Watershed 1, HBR, Hubbard Brook LTER, seedlings, sugar maple decline, vesicular-arbuscular mycorrhizae.
KEYWORD SET: LTER Core Research Areas
disturbance, inorganic nutrients, primary production.
BEGIN DATE
2003
END DATE
2004
LOCATION
Watershed 1 is a 11.8 ha watershed extending from the weir at 488 m to the ridge at 747. More decsription of the study is included in the methodsStep section m
West bounding coordinate: -71.730866
East bounding coordinate: -71.725832
North bounding coordinate: 43.959356
South bounding coordinate: 43.952121
Elevation
Minimum: 488
Maximum: 747
(Unit: meter)
Bear Brook Watershed (west of Watershed 6)
West bounding coordinate: -71.743462
East bounding coordinate: -71.735649
North bounding coordinate: 43.957001
South bounding coordinate: 43.949928
SITE DESCRIPTION
Most of our research was conducted within and around Watershed 1 (W1), which is an 11.8 ha south-facing watershed in the Hubbard Brook Experimental Forest. Elevations range from 488 m at the lower boundary to 747 m at the ridgeline that forms the upper boundary.Seedlings used in this study were taken from transects within low (520-560 m) and mid (600-650 m) elevation sites. Samples were not collected from high (725-750 m) elevation sites due to low seedling densities, but nondestructive greenness measurements were taken there with a hand-held chlorophyll meter. Adjacent control areas occur outside of the topographical boundaries of W1 at the same elevations.
Seed samples and greenness measurements were also collected from the watershed to the west of Watershed 6 (WW6) to avoid foot traffic in W6 which is the biogeochemical reference watershed for HBEF. Additionally, greenness measurements were made on seedlings in the Sleeper's River watershed, which is a naturally calcium-rich site in northeastern Vermont.
SAMPLE COLLECTION
Sugar maple seedlings were collected from low and mid elevation sites in W1 and adjacent control areas during August 2003 and June 2004. In all cases, seedlings were collected every ten paces in transects up the South-facing slope of the site. Two seedlings were harvested from each node on the transects; all were used for leaf and shoot analyses, one from each pair was used for mycorrhizal assessment and the other for root length and area analysis.
LABORATORY PROCEDURES
Shoot Health Determinants.
Reported leaf areas include both leaves and petioles, and were measured using a Li-3100 Area Meter (Li-Cor Inc., Lincoln, Nebraska). Biomass samples were dried at 60oC until constant mass was reached and then weighed.
Chlorophyll was extracted from 3-cm2 of leaf tissue using a method described by Richardson et al. (2002). Glass test tubes containing 7-mL Dimethyl Sulfoxide (DMSO) were pre-heated to 65oC in a water bath. Leaf tissue was added to pre-heated DMSO and kept at 65oC. After 30 minutes, test tubes were removed from the water bath and 3-mL DMSO was added to each tube. 3-mL of each extract were then transferred to disposable polystyrene cuvettes. Samples were analyzed using a 20 Genesys portable spectrophotometer (Spectronic, Leeds UK), which was calibrated to zero using a cuvette containing pure DMSO. Sample absorbance was analyzed at 645 and 663-nm. Chl a, chl b, and total chlorophyll (g l-1) values were calculated using equations described by Richardson et al. (2002). These values were then converted to express the chlorophyll content of each sample's leaves (mg cm-2).
A hand-held chlorophyll meter, the SPAD-502 (Minolta Camera Co., Osaka, Japan), was also used to estimate leaf chlorophyll content. SPAD measures absorbance at both 650 and 940-nm over a 0.06-cm2 area. The values are then calculated as a greenness index and expressed in “SPAD units” (Richardson et al. 2002). Two measurements were taken per leaf (totaling four per seedling) from which the arithmetic mean was calculated and used for all analyses.
To measure seedling height, the shoot was separated from the roots above the first lateral root and measured from this point to the end of the bud using digital calipers. After measuring height, samples were dried at 60oC and dry masses were then added to calculations for shoot biomass as well as total seedling biomass.
Root Length and Mycorrhizal Colonization.
Root length and area were measured using a flatbed scanner and analyzed with Delta-T SCAN Software (Delta-T Devices Ltd., Cambridge UK). Lightly pigmented roots were first stained with methyl violet for 24-hours and rinsed with 95% EtOH to remove non-adhering stain. Lateral roots were separated from the primary root, and overlapping roots or those not lying in the same plane were cut into segments appropriate for analysis. Roots of similar diameter were placed in a transparent tray containing a thin layer of 95% EtOH. Roots were scanned using HP PrecisionScan Pro software and analyzed using Delta T-Scan. After analysis, roots were dried at 60oC until constant mass was reached and then weighed.
The procedure used to assess the mycorrhizal colonization of seedlings involved first clearing and then staining roots using a modified version of the method described by Brundrett et al. (1994). Live lateral roots were cut into 2-cm segments, and 20-cm were randomly sub-sampled for analysis. Samples were rinsed over a fine mesh screen to remove soil particles and stored in 95% EtOH. Roots were cleared using 10-mL 10% KOH in a 90oC water bath. KOH was changed every 30-minutes until pigments were no longer visible in the solution. Roots were then rinsed with nanopure water and bleached with 10-mL alkaline hydrogen peroxide (0.5% NH4O4 and 0.5% H2O2 in nanopure water) in the 90oC water bath for 30 minutes. Roots were rinsed twice to remove all bleach and stained with 0.05% Parker Quink black ink (Parker Pen Products, Newhaven UK) in 1:1 glycerol-vinegar (5% CH3COOH) for 9-12 hours. Excess stain was rinsed off and roots were de-stained in a 1:1 glycerol-vinegar solution for 2-3 days.
Root colonization percentages were also estimated using a modified version of the method described by Brundrett et al. (1994). Roots were wet-mounted and examined under 200x magnification. 100 observations of stained fungal structures were taken per sample at 1-mm intervals. Structures that intersected the ocular crosshair were counted for each observation. The following categories were used: no structures, arbuscules, vesicles, coils, and mycorrhizal hyphae. Mycorrhizal hyphae were those observed to be connected to other VAM structures, and the total quantity includes values for the other three categories because they originate from hyphae. Hyphae percentages therefore represent total percent colonization. The number of observations for each category was divided by the total number of observations in order to determine percent colonization by each structure.
REFERENCES
  • Brundrett, M.C., Melville, L., and Peterson, L. 1994. Practical methods in mycorrhizal research. Ninth North American Conference on Mycorrhizae.
  • Juice, S.M., Fahey, T.J., Siccama, T.G., Driscoll, C.T., Denny, E.G., Eagar, C., Cleavitt, N.L., Minocha, R., and Richardson, A.D. 2006. Response of sugar maple to calcium addition to northern hardwood forest at Hubbard Brook, NH. Ecology 87(5):1267-1280.
  • Richardson, A.D., Duigan, S.P., and Berlyn, G.P. 2002. An evaluation of noninvasive methods to estimate foliar chlorophyll content. New Phytologist 153:185-194.
DATA ACCESS GUIDELINES
Data Use Policy



The re-use of scientific data has the potential to greatly increase communication, collaboration and synthesis within and among disciplines, and thus is fostered, supported and encouraged. Permission to use this dataset is granted to the Data User free of charge subject to the following terms:

1) Acceptable use. Use of the dataset will be restricted to academic, research, government or other not-for-profit professional purposes.

2) Redistribution. The data and metadata are provided for use by the Data User. The Data User will not redistribute the original Data Set or metadata to others without the explicit permission of the Principal Investigator.

3) Citation. It is considered a matter of professional ethics to acknowledge the work of other scientists. Thus, the Data User will properly attribute the Data Set in any publications or in the metadata of any derived data products that were produced using the Data Set. Citation should take the following general form: Creator, Year of Data Publication, Title of Dataset, Publisher, Dataset identifier.

Citation example: Holmes, R.T. 2012. Bird Abundances at Hubbard Brook (1969-2010) and on three replicate plots (1986-2000) in the White Mountain National Forest. Durham, NH. Hubbard Brook Data Archive [Database]. http://hubbardbrook.org/data/dataset.php?id=81 (23 July 2012)

4) Acknowledgment: The Data User should acknowledge any institutional support or specific funding awards referenced in the metadata accompanying this dataset in any publications where the Data Set contributed to its content. Acknowledgments should identify the supporting party, the party that received the support, and any identifying information such as grant numbers.

Acknowledgment example: Data on [topic] were provided by [name of PI] on [date]. These data were gathered as part of the Hubbard Brook Ecosystem Study (HBES). The HBES is a collaborative effort at the Hubbard Brook Experimental Forest, which is operated and maintained by the USDA Forest Service, Northern Research Station, Newtown Square, PA. Significant funding for collection of these data was provided by [agency]-[grant number], [agency]-[grant number], etc.

5) Consultation and questions. Data users are strongly encouraged to consult with the Principal Investigator(s) who collected these data for further information. Also, when appropriate, Data Users should consider including the Principal Investigator as a collaborator and/or co-author in the use of these data.

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CONTACT PERSON

Information Manager, Hubbard Brook LTER
234 Mirror Lake Road
North Woodstock, NH 03262
USA

Phone: (603) 726-8902
Email: hbr-im@lternet.edu

Data file: w1_acsa_seed_physical.txt
Description: Height, leaf and root area, root length, and biomass from reference and Ca-treated seedlings at the Hubbard Brook LTER. Sample data collected in 2003 and 2004.
Notes on Data: Original data sheets are located at the Fahey Laboratory, Connell University. Data were last updated in 2014.
ColumnVariableDescriptionUnitsCoded?Missing value label
1YearSampling yearYYYYnnone
2WatershedWatershed namenoneynone
3ElevationElevation of transectnoneynone
4TransectTransect number within watershednoneynone
5SampleSample number within transectnoneynone
6StemLengthHeight of seedlingmillimeternnone
7Leaf1AreaArea of first leaf, including petiolecentimeterSquaredn
-999.999
8Leaf2AreaArea of second leaf, including petiole centimeterSquaredn
-999.999
9LeafDryMassDry mass of both leaves, includes petiolesgramn
-999.9999
10StemDryMassDry mass of stemgramn
-999.9999
11CorrectedLeafAreaCorrected total leaf area of seedling. Area of both leaves plus area removed for chlorophyll extraction (3.137 square centimeters)centimeterSquaredn
-999.999
12RootAreaArea of seedling root systemmillimeterSquaredn
-999.999
13RootLengthCumulative length of seedling root systemmillimetern
-999.999
14RootDryMassDry mass of seedling root systemgramn
-999.9999

CODES

Variable: Watershed
Code
Description
W1
Watershed 1 site (calcium treated)
Reference
Untreated site adjacent to Watershed 1
Variable: Elevation
Code
Description
Low
Low elevation between 500 - 550 meters
Mid
Mid elevation between 550 - 600 meters
NA
Elevation not determined for the reference site
Variable: Transect
Description:
Transects are coded as the watershed code followed by the transect number.
Variable: Sample
Description:
Each transect contains sample numbers 1-20.

MISSING VALUE CODES
Variable
Missing Value Code
Code Explanation
Leaf1Area
-999.999
Analysis not performed
Leaf2Area
-999.999
Analysis not performed
LeafDryMass
-999.9999
Analysis not performed
StemDryMass
-999.9999
Analysis not performed
CorrectedLeafArea
-999.999
Analysis not performed
RootArea
-999.999
Analysis not performed
RootLength
-999.999
Analysis not performed
RootDryMass
-999.9999
Analysis not performed

Data file: w1_acsa_seed_biomass.txt
Description: Height, leaf and root area, root length, and biomass from reference and Ca-treated seedlings at the Hubbard Brook LTER. Sample data collected in 2003 and 2004.
Notes on Data: Original data sheets are located at the Fahey Laboratory, Connell University. Data were last updated in 2014.
ColumnVariableDescriptionUnitsCoded?Missing value label
1YearSampling year.YYYYnnone
2WatershedWatershed name.noneynone
3ElevationElevation of transect.noneynone
4SampleSample number within transect. Samples are numbered between 1-30.noneynone
5DryMassDry mass of sugar maple seed.gramn
-999.9999

CODES

Variable: Watershed
Code
Description
W1
Watershed 1 site (calcium treated).
Reference
Untreated site adjacent to Watershed 1.
Variable: Elevation
Code
Description
Low
Low elevation between 500 - 550 meters.
Mid
Mid elevation between 550 - 600 meters.
NA
Elevation not determined for the reference site.
Variable: Sample
Description:
Samples are numbered between 1-30.

MISSING VALUE CODES
Variable
Missing Value Code
Code Explanation
DryMass
-999.9999
Analysis not performed

Data file: w1_acsa_seed_chl.txt
Description: Chlorophyll content and greenness measurements from seedlings collected in three areas of HBEF and Sleeper's River, VT. Samples collected in 2003.
Notes on Data: Original data sheets are located at the Fahey Laboratory, Connell University. Data were last updated in 2014.
ColumnVariableDescriptionUnitsCoded?Missing value label
1YearSampling yearYYYYnnone
2WatershedWatershed namenoneynone
3ElevationElevation of transectnoneynone
4TransectTransect number within watershednoneynone
5SampleSample number within transectnoneynone
6SPAD_greennessAn indexed chlorophyll content reading of leaf greenness measured with a SPAD-502 handheld chrolophyll meterdimensionlessnnone
7abs645Leaf aborbance at 645 nanometer wavelengthnanometern
-999.999
8abs663Leaf aborbance at 663 nanometer wavelengthnanometern
-999.999
9ChlAChlorophyll A per unit leaf areamilligramPerCentimeterSquaredn
-999.999
10ChlBChlorophyll B per unit leaf areamilligramPerCentimeterSquaredn
-999.999
11TotChlTotal chlorophyll (A+B) per unit leaf area milligrams per square centimeter.milligramPerCentimeterSquaredn
-999.999

CODES

Variable: Watershed
Code
Description
W1
Watershed 1 site (calcium treated).
Reference
Untreated site adjacent to Watershed 1.
WW6
Bear Brook reference watershed west of Watershed 6.
Sleepers
Sleepers River Watershed, VT.
Variable: Elevation
Code
Description
Low
Low elevation between 500 - 550 meters
Mid
Mid elevation between 550 - 600 meters
High
High elevation between 600 - 700 meters
NA
Elevation not determined for the reference site
Variable: Transect
Description:
Transects are coded as the watershed code followed by the transect number.
Variable: Sample
Description:
Each transect contains sample numbers 1-20.

MISSING VALUE CODES
Variable
Missing Value Code
Code Explanation
abs645
-999.999
Analysis not performed
abs663
-999.999
Analysis not performed
ChlA
-999.999
Analysis not performed
ChlB
-999.999
Analysis not performed
TotChl
-999.999
Analysis not performed

Data file: w1_acsa_seed_myco.txt
Description: Mycorrhizal colonization of sugar maple seedlings collected in reference and Ca-treated watersheds at the Hubbard Brook LTER. Sample data collected in 2003 and 2004.
Notes on Data: Original data sheets are located at the Fahey Laboratory, Connell University. Data were last updated in 2014.
ColumnVariableDescriptionUnitsCoded?Missing value label
1YearSampling yearYYYYnnone
2WatershedWatershed namenoneynone
3ElevationElevation of transectnoneynone
4TransectTransect number within watershednoneynone
5SampleA unique number assigned to each seedling.noneynone
6Total_ObsTotal number of observationsnumbernnone
7NS_ObsNumber of observations with no structuresnumbernnone
8A_ObsNumber of observations with arbusculesnumbernnone
9V_ObsNumber of observations with vesiclesnumbernnone
10C_ObsNumber of observations with coilsnumbernnone
11MH_ObsNumber of observations with mycorrhizal hyphaenumbernnone
12U1_ObsNumber of observations with non-mycorrhizal hyphaenumbernnone
13U2_ObsNumber of observations with possibly mycorrhizal hyphaenumbernnone
14Arb_ColonizationPercentage of observations with arbusculespercentnnone
15Ves_ColinizationPercentage of observations with vesiclespercentnnone
16Coil_ColonizationPercentage of observations with coilspercentnnone
17Hyphal_ColonizationPercentage of observations with hyphaepercentnnone

CODES

Variable: Watershed
Code
Description
W1
Watershed 1 site (calcium treated)
Reference
Untreated site adjacent to Watershed 1
Variable: Elevation
Code
Description
Low
Low elevation between 500 - 550 meters
Mid
Mid elevation between 550 - 600 meters
NA
Elevation not determined for the specific sample site
Variable: Transect
Description:
Transects are coded as the watershed code followed by the transect number. SPAD samples are from another collection.
Variable: Sample
Description:
Each seedling received a unique number within the transect or sample set.