Environmental and Experimental Botany 55 (2006) 97109
Symbiont nitrogenase, alder growth, and soil nitrate response tophosphorus addition in alder (Alnus incana ssp. rugosa) wetlands
Specklewetland tyfixer thatprocessingfixation anThe objecdeterminein atmospAugust. Nlower in rtwig and fbiomass oincrease infollowinggrowth ofby speckle 2004 El
ment, 106E-mail ad
0098-8472/doi:10.1016of the Adirondack Mountains, New York State, USAKemal Gokkayaa, Todd M. Hurdb, Dudley J. Raynalc,
a University of Florida, Institute of Food and Agricultural Sciences, Soil and Water Science Department,106 Newell Hall, PO Box 110510, Gainesville, FL 32611, USA
b Shippensburg University, Department of Biology, Shippensburg, PA 17257, USAc State University of New York, College of Environmental Science and Forestry, Faculty of Environmental
and Forest Biology, 350 Illick Hall, 1 Forestry Drive, Syracuse, NY 13210, USA
Accepted 6 October 2004
d alder (Alnus incana ssp. rugosa) is a characteristic species of scrub-shrub 1-type wetlands, the second most commonpe in major watersheds of the Adirondack Mountains in New York State. Speckled alder is an actinorhizal nitrogen
relies heavily on N2 over soil N and fixes substantial amounts of nitrogen in wetlands, resulting in little vegetationof anthropogenic N between alder-shrub wetlands and streams. Phosphorus (P) is an element that limits nitrogen
d plant growth. However, studies testing this hypothesis in the field, especially for actinorhizal plants, are very few.tives of this study were to evaluate the potential limitation of N fixation and growth in speckled alder by P, and tointeractions between P fertilization and nitrate levels in riparian alder stands in a region that receives elevated N
heric deposition. P fertilization significantly increased specific nitrogenase activity during the seasonal peak in earlyitrate concentrations were greater in reference plots compared to treatment plots, and phosphate concentrations wereeference plots compared to treatment plots over a period of 6 weeks in the growing season. There was a significantoliar biomass response to P fertilization in the second year after fertilization, but no significant change in individualr relative numbers of different sized nodules. Response of nitrogen fixation to P appears limited to a brief but significant
specific activity of nitrogenase late in the growing season, but P stimulated growth of above ground tissues 1 yearfertilizer application, and decreased resin-captured nitrate beneath riparian speckled alder. These results suggest thatalder and growth or activity of soil microbes, rather than nitrogen fixation, is P limited in riparian wetlands dominatedd alder, and that P controls nitrate leaching in these near-stream systems.sevier B.V. All rights reserved.
Alnus; Adirondack Mountains; Actinorhizal; Ion exchange resin; Nitrate; Phosphorus; Nitrogenase activity
onding author. Present address: University of Florida, Institute of Food and Agricultural Sciences, Soil and Water Science Depart-Newell Hall, PO Box 110510, Gainesville, FL 32611, USA. Tel.: +1 315 470 6782; fax: +1 315 470 6934.dress: firstname.lastname@example.org (D.J. Raynal)
$ see front matter 2004 Elsevier B.V. All rights reserved./j.envexpbot.2004.10.004
98 K. Gokkaya et al. / Environmental and Experimental Botany 55 (2006) 97109
Nitrogen (N)-fixing plants are known to have an in-herently hifixers (Ingeimental stunodulationin both leg1974; Israeal., 1989; E2000). The(2002) reveiting nodulin the succplain in intstrated thatlichens (cylitters of theDicranopteditions of PChapin et aof cyanobaditions to y(2000) foulation in Efolium pratconcentratinodulationA. incana sdemonstratnutrition oincana; Naddition whaddition ofalder and Dresulted inacetylene r(Binkley et
Despiteits N fixatiitation on nactinorhizaUliassi and215 kg P hatenuifoliacreased totcompared tThe treatm
nase activity per unit nodule dry mass, compared withnodule production response. They concluded that mul-tiple studies in a range of environments are necessary
neralial terrurd etin a d
al Adted N). Atmeasterce wa
ssingn) mayrfacefrom ds in thd nitra; Hurdce offor fuurces
). Thetial lilder btilizatirub wezal fix
e studse of Fa ssp.follow
e immss we
phates wass overion inssay
avail, whergher phosphorus (P) demand than non-Nstad, 1981; Sprent, 1988). Several exper-dies have shown significant responses of
, plant growth and fixation to P fertilizationsuminous and actinorhizal species (Gates,l, 1987; Reddell et al., 1988; Sanginga etkblad and Huss-Danell, 1995; Uliassi et al.,results of a field study by Uliassi and Ruessal that P limits N fixation primarily by lim-e production in thinleaf alder (A. tenuifolia)essional forests of the Tanana River flood-erior Alaska. Vitousek (1999) has demon-N fixation associated with liverworts and
anolichen, Pseudocyphellaria crocata), thetree Metrosideros polymorpha and the fernris linearis increased significantly with ad-
on young volcanic substrates in Hawaii.l. (1991) also found significant stimulationcterial acetylene reduction activity by P ad-oung soils in the high Arctic. Wall et al.
nd that the degree of N inhibition on nodu-uropean Alnus incana ssp. incana and Tri-ense grown in pouches depended on the Pon level, with increased N levels inhibitingat N/P ratios >7, but not at N/P ratios 7, insp. incana. Ekblad and Huss-Danell (1995)ed experimentally an interaction of N and Pn N fixation in seedlings of A. incana ssp.fixation decreased the most under high Nen P was limiting. In a bioassay study, the
P and sulphur (S) to soils from a stand of redouglas fir on Mt. Benson, British Columbiadoubling of red alder biomass and increasededuction activity per seedling by five-foldal., 1994).
the general view that low P availability lim-on, research investigating phosphorus lim-itrogen fixation in the field, especially forl plants, is very limited. In a recent study,
Ruess (2002) applied a 2-year total of1 in several separate applications to A.in Alaska and found that fertilization in-al nitrogen inputs to 140 41 kg ha1 yr1o 59 11 kg ha1 yr1 in unfertilized plots.ent appeared to have little effect on nitroge-
Hfixedcentreleva2002northsurfaprocecatioto sugas)standevate2003fluenneedbe so2002potenled aP ferin shnorhi
procephosresinmentadditthis aphosroottrienttemsze about the effects of P on N fixation inestrial ecosystems.al. (2001) estimated 3743 kg N ha1 yr1ense stand of A. incana ssp. rugosa in the
irondack Mountains, a region that receivesin atmospheric deposition (NADP/NTN,ospheric N deposition is a concern in the
n United States, due to nitrate influences onter acidity (Driscoll et al., 2001). Wetlandof excess nitrate (biotic uptake or denitrifi-result in either decreased nitrate or acidity
waters, and/or increased N2O (greenhouseenitrification. Dense A. incana ssp. rugosae Adirondacks are also associated with el-te in shallow groundwater (Kiernan et al.,and Raynal, 2004), demonstrating the in-
this species on riparian N cycling and therther studies on what causes wetlands toor sinks for nutrients (Busse and Gunkel,objectives of this study were to evaluate themitation of N fixation and growth in speck-y P, and to determine interactions betweenon and nitrate levels in riparian alder standstlands that receive N inputs from both acti-ation and anthropogenic N deposition.
ls and methods
y was designed to measure nitrogenase re-rankia actinomycetes in root nodules of A.rugosa to P addition during weeks immedi-ing treatment at the beginning of the grow-Presumably, nitrogenase activity would in-ediately upon increased P availability, if there P limited. Measurement of nitrate and
concentration in the soil using ion exchangecoupled to nitrogenase activity measure-the same time period, following fertilizerthe 2001-growing season. The purpose of
was to examine the changes in nitrate andconcentrations in the substrate, where thes occur to better understand patterns of nu-
ability and processing in these riparian sys-e especially nitrate leaching is a concern for
K. Gokkaya et al. / Environmental and Experimental Botany 55 (2006) 97109 99
acid-sensitive streams (Driscoll et al., 2001). We mea-sured twig elongation and twig, and foliage mass duringthe growing season of treatment (2001) and the sub-sequent season (2002) to determine response of aldergrowth to P addition. Nodules were harvested at theend of the study in September 2002.
2.1. Site description
The study was conducted in two alder-dominatedriparian wetlands (scrub-shrub 1-type; Cowardin etal., 1979) along Fishing Brook and Adjidaumo Flowat the Huntington Wildlife Forest (HWF) located inthe central Adirondack Mountains region within theAdirondack State Park of New York (Fig. 1). Fish-ing brook is large-order inlet to a lake, while Adji-daumo flow is a headwater stream in the same flowsystem. Total annual precipitation averages 101 cm andmean annual temperature is 4.4 C with a dormant sea-son mean of 2.8 C and a growing season mean of14.3 C (Shepard et al., 1989). Surrounding vegeta-tion is composed of mixed northern hardwood forestson the upper slopes. The lower slopes are character-ized by yellow birch (Betula alleghaniensis), balsamfir (Abies bsis), and re
Common wetland species include A. incana ssp. ru-gosa, Viburnum dentatum, Carex spp., Spiraea alba, S.tomentosa, Chamaedaphne calyculata, Osmunda re-galis, Calamagrostis Canadensis, and Myrica gale.Type SS1 wetlands in the Upper Hudson watershed, inwhich the study wetlands are located, had an averageof 10,979 speckled alder stems per hectare (Kiernan,2000). Speckled alder foliage from HWF wetlands av-erages 2.7% N with a C:N ratio of 18.7 (Hurd, 1999),and 85100% foliar N derived from fixation (Hurd etal., 2001). Greenwood mucky peats are present in val-ley bottom wetlands (Somers, 1986) and range from 1to 5 m in thickness in an adjacent catchment at HWF,overlying glacial till (McHale et al., 2002). These soilsare hydric, rich in organic matter, dark in color, andexhibit redoximorphic features (personal observation).
HWF is situated in a region, where atmospheric de-position of N is elevated. Inorganic nitrogen inputs inthe form of wet and dry deposition at HWF averaged4.7 kg N ha1 yr1 between 1978 and 2001 (NationalAtmospheric Deposition Program/National TrendsNetwork (NADP/NTN), 2002) and 2.6 kg N ha1 yr1between 1995 and 2000 (Park et al., 2003). Despitethese inputs, alders in the region continue to rely heav-ily on fixed N (Hurd et al., 2001), making uncer-
Fig. 1. Locati low at thMountains ofalsamea), eastern hemlock (Tsuga canaden-d spruce (Picea rubens) (Hurd et al., 1998). tain t
on of research wetlands (asterisks) at Fishing Brook and Adjidaumo FNew York State.gree to which alder wetlands in the region
e Huntington Wildlife Forest in the central Adirondack
100 K. Gokkaya et al. / Environmental and Experimental Botany 55 (2006) 97109
control elemental nutrient flux between hillslopes andstreams.
A randoblock wasboth wetlanjidaumo Flacross low(R) and treain each bloupstream frtreatment e5 m 5 m,allow accesence plots rplots receivlar triple suK) in the faddition ra
An annled alder aest, New Y0.15% P ctissue basered alder fHurd (unpu(0.080.15tal biomassFishing Brcroorganism5 g P m2,appropriate
A singletilizer wasof treatmeneach treatmphosphatealders androots extenders.
BlocksPre-treatmeon July 1tion on JmeasuredAugust 7
standing water in the plots during the treatmentperiod.
hibit agenaseto sixm in len theepth oal assal syrinzone
. Cooor applene gc pum-Parmomet
sure thtte andrated b(CaC2ng, thed tubigase
s at le0240errupt4) pro
se was, gas-trst, secon. Thitial ples groema,
one saimental design and fertilizer addition
mized block design with two plots in eachused. Seven blocks were established acrossds (four at Fishing Brook and three at Ad-
ow), in order to maximize spatial inferenceand high order riparian systems. Referencetment (P) plots were located systematicallyck so that the control plot was consistentlyom the treatment plot, to minimize potentialffects on the reference plots. Each plot waswith a 10 m buffer zone between plots tos and ensure independent treatment. Refer-eceived no fertilizer addition, and treatmented a single application of Hoffman granu-perphosphate fertilizer (0% N46% P0%orm of P2O5 at 5 g P m2, or 50 kg P ha1te.ual uptake of 3 kg P ha1 yr1 by speck-t Fishing Brook, Huntington Wildlife For-ork in 1998 was calculated by assuming
oncentration as average in speckled alderd on findings of Compton et al. (1997) foroliage in Washington (0.120.17% P) andblished data) for N fixing M. gale in Maine
% P) and multiplying that value by the to-increment estimated for speckled alders at
ook in 1998 by Hurd (1999). Because mi-s and other plants also compete for P, the
or 50 kg P ha1 addition rate was considered.application of granular superphosphate fer-
broadcast evenly by hand over the substratet plots on July 12, 2001. A 1-m zone aroundent plot was included in broadcast of super-
to ensure the even uptake of the fertilizer byeven effect of treatment on alders whoseded out of the original 5 m 5 m plot bor-
were established in May and June 2001.nt nitrogenase activity was measured112, 2001. Following fertilizer addi-
uly 12, 2001, nitrogenase activity wasat biweekly intervals on July 2425,8, and August 2021. There was no
Nflowniqueity u(Schwonlybecauto exnitroFour2 cing oat a dcentr70 mrootfoundules facetystalti(Colethermto encuve
genebideheatitacheof thevalue(23unint(C2Hgenaglassthe fiductithe innoduTjepkule cfromcase,ene reduction assay
nase activity was measured using an open,h acetylene reduction assay (ARA) tech-gives an estimate of nitrogenase activ-
he initial peak rate of acetylene reductionr and Tjepkema, 1994). This system is thele technique to measure nitrogenase activityeckled alder nodules in the field are known
pronounced acetylene-induced decline inactivity (Schwintzer and Tjepkema, 1997).
nodule clusters with attached root segmentsngth from two to four alder shrubs (depend-availability) were excavated from each plotf 510 cm and immediately transferred to ay location. Nodules were then placed in age barrel cuvette that was inserted into theof alders, where the nodules are typicallyled, humified air was sent over the nod-roximately 10 min and then 10% by volumeas (C2H2) introduced with a portable, peri-p operating on an internal lithium batteryer Masterflex L/S). A dual probe thermistor
er (Cole-Parmer model 8402-20) was usede temperature difference between inside thesoil was less than 2 C. Acetylene gas wasy mixing approximately 8 g of calcium car-) in 300 ml of water in a flask. To preventcooler with the air and C2H2 bags, and at-
ng were kept shaded with a cloth. Flow ratess were measured by a bubble flowmeter andast three times the volume of the cu...