Inorganic N incorporation by coniferous forest floor material

Soil Bid. Biochem. Vol. 21. No. I. pp. 4146, Printed in Great Britain. All rights reserved

INORGANIC

0038-0717~89 53.00 + 0.00 Copyright C 1989 Pergamon Press plc

1989

N INCORPORATION BY CONIFEROUS FOREST FLOOR MATERIAL J. P. SCHIMEL' and M. K. FIREVONE

Department

of Plant and Soil Biology, University of California, Berkeley, CA 94720, U.S.A. (Accepfed 10 May 1988)

Summary-We examined the control of incorporation of N into material from a coniferous forest floor in the Sierra Nevada of California. Forest floor material was slurried in solutions containing ‘rN and the incorporation of N was measured in short-term (< 7 h) experiments. Ammonium incorporation was rapid (r IO pgg-’ h-l), but was limited by NH: at concentrations found in the soil solution. Methionine sulfoximine (MSX), a glutamine synthetase inhibitor, reduced NH: incorporation, also indicating that NH; supply to microorganisms was limiting. The half-saturation constants for NH: incorporation (K,) were much greater than published values for fungi, suggesting that diffusion into organic material limited NH: incorporation. Approx. 20% of NH,+ incorporation occurred by abiotic processes. Nitrate incorporation averaged 6% of NH: incorporation and was not inducible. Uptake rates by fungal strands and root-fungus associations were similar to those of the bulk material, suggesting that these structures are not a dominant sink for N in this system.

hlATERlAlS

INTRODCCTION

AND METHODS

Site description

The forest floor or organic (0) horizon is an extremely active component of forest N cycles; all above-ground litter enters it and root production is frequently abundant (Alexander and Fairley. 1983; Vogt et al., 1983). The 0 horizon may contain up to 21% of the total N in a forest ecosystem (Melillo, 1981). and this N may turn over 34 times as fast as N in the mineral soil (Aber et al., 1983). Despite this high turnover, N-cycling in the forest floor is generally tight; as little as 1% of the total N mineralized is leached out of the forest floor (Bringmark, 1980). Several aspects of N-dynamics in the forest floor have received extensive study and are relatively well understood. These include litter decomposition (Bosatta and Staaf, 1982; Melillo and Aber, 1984) and the patterns of net N-mineralization in the forest floor (Gosz. 1981; Fahey et al.. 1985). Other aspects of forest Boor N-cycling are less well understood. One important such aspect is the extent of inorganic N-incorporation by microorganisms. It is this that balances the rapid mineralization and maintains the tight N-cycle of the forest floor. We investigated the mechanisms and control of N-incorporation into coniferous forest floor material by measuring “N incorporation from slurries containing either lsN-labeled NH.,” or NO;, The experiments addressed questions concerning the pathways and kinetics of NH,+ assimilation, the extent the significance of abiotic NH,+ immobilization, and control of NO; incorporation, and the role of mycorrhizal and nonmycorrhizal mycelial strands in N-incorporation.

The research site was in Blodgett Forest Research Station, Georgetown, Calif., in the foothills of the Sierra Nevada mountains (38’52’N, IZO”4O’W).The forest is at 1400 m elevation. Precipitation averages 168cm. of which l/2 to 2/3 falls as snow during the winter. The summers are dry, with almost no precipitation. Average August daily minimum and maximum temperatures are I4 and 27’C. Average January daily minima and maxima are 0 and 9-C. The site was a 60-yr-old mixed conifer stand composed primarily of Abies concolor, Pinus lambertiana. Pseudotsuga menziesii, Libocedrus decurrens.

Pinus

ponderosa

and

The soil is a well-drained, sandy loam Ultic haploxeralf of the Holland series, formed on granodiorite parent material. This forest has a well-developed organic horizon (7.2 kg dry matter m-‘). which was three subhorizons described as 01, 02 and 03, respectively (R. Amundson, personal communication). The 01 is fresh litter, and ranges from I to 3 cm thick. The 02 is 2-3 cm thick and consists of partially decomposed, but still identifiable needles. White mycelia forming thick (Z I mm) rhizomorphs are frequent in this subhorizon. These hyphae entwine the organic material, binding it together into dense mats (Cromack er al., 1979). The organic material in these zones is frequently light colored, suggesting that the fungi may be white roots (Hintikka, 1982). The mycelia were never found associated with roots, which were usually absent from the 02. The 03 consists mostly of fibrous, but otherwise unidentifiable organic material. This subhorizon ranges between 2 and 4cm thick and tree roots are common. In the 03 and surface mineral soil. a yellow Although this fungus forms *Present address: Department of Crop and Soil Sciences. fungus is abundant. thick, wefty bundles of hyphae resulting in mycelial Michigan State University, East Lansing, MI 48824, mats, the mats are not as dense as those found in the U.S.A. 41

42

.I.P. SCHIMEL and M. K. FIRESTONE

02. The yellow fungus is often associated with roots, but the associations do not have the club roots characteristic of many ectomycorrhizas. Rather, they have the “Witches Broom” morphology described by Ogawa (1985). The yellow fungus is most likely either Rhizopogon truncatus or Piloderma bicolor (R. Molina, personal communication). Both of these form mycorrhizas with the dominant tree species. It is therefore likely that this fungus is mycorrhizal and that the associations are ectomycorrhizas. Field sampling Samples were collected on three dates, 27 March 1984, 30 January 1985 and I5 April 1985. On each sampling date, four samples (Z 1 kg each) of the 0 horizon were collected and separated into 01, 02 and 03 subhorizons. These separate samples were composited to provide bulk samples of each of the three subhorizons. Assays were started within 3 h of sampling. except for those in April 1985, which were stored on ice overnight and assayed the next day. Laboratory methods N incorporation was measured by slurrying organic horizon material (5-10 g wet) in nutrient media (75 ml) containing the “N labeled compound of interest. The basic medium contained: MgSO, (0.5 m&t). KH:PO, (0.25 mht). K:SO, (0.625 mM), CaCI, (I .25 mxt) and either (“NH,),SO, or K15N0, in various concentrations. The iJNHf and the “NO< were 70.5 and 98.0atm% “N, respectively (Monsanto Research Corporation, Miamisburg, Ohio). Slurries were shaken on a rotary shaker for either 6 or 7 h at approx. 2O’C (room temperature). Then, samples wcrc collected on a 0.5 mm mesh screen, washed with I st KCI to remove remaining inorganic “N. and then washed with water to remove the KCI. This may have leached soluble N, resulting in an underestimate of total “N-incorporation. The samples were dried at 7O.C, ground in a Wiley mill to pass a 0.4 mm sieve, and digested on a block digester using a Kjcldahl procedure. The digests were steam distilled (Keeney and Nelson, 1982) and the resulting (‘“NH,)ZSO, analyzed for “N enrichment by mass spectroscopy. All “N analyses were done by Isotope Services Inc., Los Alamos, NM. Incorporation rates of lSN were corrected for the lSN which would be rapidly exchanged into CEC sites or taken up into the free space. Samples to correct for this were prepared by slurrying material in the appropriate ‘IN solution for 5 min and then treating it as an experimental sample. The amount of ‘IN incorporated was subtracted from the experimental samples to determine the corrected N-incorporation. N incorporation rates were calculated by dividing the corrected “N incorporation rate by the actual “N-enrichment of the slurry. The lJN-enrichment of the N in the slurry was taken as the weighted average of the added “N and the initial NH,’ or NO; in the material. Since the actual concentration of the NH,+ or NO; did not change significantly during the assays. and only a small proportion of the N was incorporated. the initial “N-enrichment was used for the average ‘“N enrichment.

Abiotic incorporation Abiotic incorporation of NH; was examined in organic material which had been sterilized with propylene oxide (Nommik, 1970). Samples of the 02 and 03 subhorizons were spread in Petri dishes and placed in a desiccator with a beaker of propylene oxide. The desiccator was evacuated and refilled with air three times to distribute the propylene oxide through the material. These samples were fumigated for 48 h. The beaker of propylene oxide was then removed. and remaining propylene oxide was removed by repeated evacuation. Ammonium incorporation from 0.5 mM (NH,)2S0, was then assayed using the described procedures. Sterility was verified by measuring, the respiration of samples of material after fumigation. Kinetic constants The maximum possible rate of incorporation and the substrate concentration which allows incorporation at half this rate (V,,,,, and K,) were determined by using the direct linear plot method (Eisenthal and Cornish-Bowden, 1974). This technique is statistically more robust than the Lineweaver-Burke approach. A computer program was written to calculate the intersections. The incorporation rates were measured over 6 h from 0.05. 0.5 and 5.0 mat N solutions on samples from the April 1985 sampling. Use (.$ L-nletlrioninesrclj).rinline (MS,%‘) to inhibit glutamine swthetase To assess which of two biochemical pathways was involved in N-assimilation, t-methioninesulfoximine (MSX) was used to inhibit glutamine synthetasc activity. MSX was purchased from Sigma chemical St Louis, Missouri. Organic horizon company, material was assayed for NH; incorporation from 0.5 rnht (NH,)ZSO, in the presence and absence of 5 mst MSX. These samples were shaken for 6 h and analyzed as described above. Control of NO;

incorporation

The control of NO; incorporation was examined using the slurry method. Nitrate incorporation by fresh material from 0.5 IIIMNO; was measured over 6 h. Organic horizon material (20 g) was also exposed overnight to N sources in one of three different media (SOOml). These were: (a) water. (b) 0.1 mw KNO,, and (c) 0.1 rnbt KNO, + 0.1 mst (NH,)?SO,. After prior exposure, the material was assayed for NO; incorporation from 0.5 mw NO;. The concentration of the NO; solution was higher than would normally be found in the soil, and would make NO; uniformly available to all the microbes. Samples were explosed to the different N sources overnight to give the microbes sufficient time to induce enzyme synthesis and produce a full suite of the necessary enzymes (Pateman and Kinghorn, 1976). N-uptake b_vjingal material N uptake by the visible fungal structures (white and yellow) was measured by picking fungi out of samples of the organic horizon. Samples of the 0 horizon were collected as described above. Fungal

Forest floor N incorporation

43

trations varied between 0.3 and 2.3 mM; NO; ranged between 0.03 and 0.1 m&t on the various sampling dates. Rates of NH: incorporation in both subhorizons were between 3 and 20 pg-N g-l h-l, but there were a number of differences between sampling dates and subhorizons. In the 02. the January samples had a significantly lower incorporation rate than either the March or the April samples. In the 03 samples, the pattern was similar, but the incorporation rates in January and April 1985 were not significantly different. Incorporation rates in the samples from the 03 were always higher than those from the 02 but in April this difference was small and was not significant. The measured incorporation rates are potential rather than field rates; they are the maxima that could Statistics occur from the given substrate concentrations. However, they still suggest rapid turnover of inorganic N All assays were run in triplicate. Differences bein the forest floor. The turnover times in these tween treatments were tested using one-way ANOVA experiments were less than I days for NH: and only with a = 0.05 unless otherwise noted. a few days for NO;. The data indicate the extremely high capacity of the forest floor microbial community RESULTS AND DISCUSSION to incorporate N. Methodology The rates of NO; incorporation were always The experiments were designed to examine mech- significantly lower than the rates of NH.,+ incorpofrom the 0.05 my anisms and controls of N-incorporation, not to esti- ration. Nitrate incorporation mate field rates. The slurry assays allowed tight solutions was generally close to the limit of detection. The NO; concentrations found in the soil were control of substrate concentration and ensured uniform substrate distribution. The use of 15N usually close to 0.05 mkt. indicating that NO; incorporation by this organic horizon material is allowed measurement of total N-incorporation, minimal. Similar results were found by Overrein whereas nontracer techniques would have measured only net incorporation or release. The short assay (1967) who found that less NO; was incorporated than NH.,+ when added (at 50 pegg- ‘) to forest floor period was chosen to minimize microbial growth and material. changes in the physiological state of the microorganisms during the incubation. The specific assay Abiotic NH: incorporation period (67h) was the shortest time which allowed The proportion of incorporation via abiotic proaccurate measurement of “N incorporation. Nincorporation appeared to be linear during the assay cesses is shown in Table 2. In both subhorizons sterilization decreased NH; assimilation rates to period indicating that the rates did not change slightly under 20% of the rates in the unsterilized significantly over that time. samples. This indicates that a substantial portion of Incorporation rales of NH: and NO; the NH: assimilated was by abiotic processes. The extent of abiotic NH; incorporation in nature The rates of N-incorporation by material sampled is uncertain. The rates of abiotic immobilization in on the various dates are shown in Table I. The NO; laboratory studies usually correlate with soil organic and NH: concentrations were chosen to approximate C and pH; the mechanism is thought to involve those found in the soil solution. Field NH: concenmaterial was collected by careful picking with forceps. White hyphal strands which were unassociated with roots were picked from the 02 subhorizon. Yellow hyphal bundles and root-fungus associations were picked from the 03. These associations were collected by excavating portions of P. ponderosu root systems. Roots were collected from between two closely-spaced P. ponderosa to ensure that the roots were not from another species. Both the organic and mineral soil horizons were examined for infected root tips. Root tips with associated fungus were excised and stored on damp paper towels (< 1 h) until they were assayed for N-uptake using the slurry “N procedure. Remaining pieces of organic material adhering to the fungi were removed after the material had been incubated and dried.

Table

I.

Rates

of

inorganic material

Sampling

02

date

N

March

84

3.7b (0.3)

April

8.4a (0.5)

85

O&b

April

0.2b (0.02)

85

‘Rates

were

measured

error

horizon

subhorizon

0.5 nlM

0.05 InM

incorporation

NO;

19.&a (3.2)

3.4a (0.3)

7.8b (0.5)

4.3b (0.4)

8.9b (0.6)

ND 4.7a (0.5)

incorporation

c 0.2a

(0.2)

O.%(O.l)

ND


0.4b (0.4)

0.034 (0.01)

during

by organic

h- ‘)’ 03

ND’

0.7a (0.3)

Jan 85

%andard

NH.,+

solution

material

2. la (0.2)

(b) 84

from

-’

0.05 rnY

7.3a (0.S)~

Jan 85

March

in pg N g

subhorizon

0.5 nl.H (a)

‘Not

incorporation

(rates

0.4b (0.

I)

ND

O.OSa (0.01)

6-7 h.

of 3 replicates.

determined.

Values

within

the same subhorizon

letter are not significantly lCS1.

and

ditTerent

solution

concentration

at I = 0.05 by Duncan’s

with Multiple

the same Range

J. P.

44

SCHIMEL and

Table 2. NH; incorporation by sterilized material (rates in pg N I-’ material h-‘)’

M. K.

FIRESTONE

Table 3. Effects of MSX on NH; incorporation (rates in pg N g-’ material h-l)’

Subhorizon

Nonsterile

Sterile

% Abiotic

Subhorizon

02 03

8.4 (0.5)’ 8.9 (0.6)

1.2 (0.5) I .7(0.4)

18.4 19.1

02 03

‘Rates were measured during 6 h. ‘Standard error of 3 replicates.

of NH;

incorporation

Ammonium and NO; incorporation rates were strongly concentration dependent (Table I). In April 1985, we also measured NH: incorporation from 5.0 mM substrates. These rates were 16.0 + 1.3 and 23. I + I .3 /(g-N g-’ material h-’ for the 02 and 03 subhorizons, respectively. The incorporation rates from 0.05 and 0.5 mM solutions are shown in Table I. Kinetic constants were calculated from a curve of incorporation rate vs substrate concentration curve. The K, and I’,,,,, values for NH: incorporation were K, = 0.44 InM and I-‘,,,,, = 14.7 pg-N g-’ h-’ for the 02 and K, = 0.35 mM AND V,,, = 23.2 pg-N g-’ h-’ for the 03 subhorizons, respectively. In these samplcs. the 03 had a higher V,,, than the 02, even though the rates of incorporation at 0.5 mbt NH.,+ were similar. The concentration dependence of NH: incorporation is surprising considering that published uptake and growth constants for fungi in pure culture are much lower than the values we determined. Published values of K, for NH: uptake in fungi are frequently in the micromolar range (Button, 1985; Cook and Anthony, 1978; Hackette et al., 1970). From such values we would have predicted that NH.,+ incorporation in the forest floor would be NH: saturated. Ammonium incorporation kinetics relate to the mechanism of NH: assimilation. Glutamate dehydrogenase (GDH) functions when intracellular NH,’ concentrations are not limiting. At lower NH; concentrations, microbes shift to the glutamine synthetase-glutamate synthase (GS-GOGAT) pathway. Glutamine synthetase has a lower K,” than GDH but requires ATP and therefore has a higher energetic cost (Ingraham et al., 1983). The enzyme system active at any time can be used to indicate whether the microbial population is Nlimited or not. The active pathway can be determined by using MSX to inhibit GS (Genetet et a/., 1984). When we did this experiment, NHf incorporation by the 02 subhorizon was reduced by 24% by MSX (z < 0.05; Table 3). while in the 03 the reduction was only 13% (z c 0.075). These are most likely underestimates as inhibition may have been incomplete. These data indicate that a considerable

+ MSX

O/oReduction

P’

2.8 (0.1) 6.8 (0.5)

24 13

0.004 0.073

‘Rates were measured durincr 6 h %andard error of 3 replicates. ‘Probability that the null hypothesis(the two valuesare not different) is false, Type I error.

reaction of NH, with activated phenol or quinone rings (Stevenson, 1982; Nommik, 1965). The large amounts of lignin in conifer litter might be expected to lead to high concentrations of active phenols and quinones and thus to extensive chemical immobilization. Nommik (1970) found that this was SO in raw humus, though only when the pH was adjusted to near neutrality and above. As the pH of the forest floor at Blodgett is 4.34.5, it is somewhat surprising that abiotic NH: incorporation was an important process. Kinetics

-IMsx 3.7 (0.2)’ 7.8 (0.3)

portion of the

assimilated was via GS-GOGAT (Genetet et 1984) and therefore that N was limiting. Despite the high averge NH: concentrations in the soil, a portion at least of the microbial population was NH:-limited. It is likely that some microsites were depleted of NH;. causing microbes to shift from the GDH to the GS-GOGAT assimilation pathway. The presence of these zones may relate to the high values of K, we found for the forest floor material. Microorganisms living within organic fragments may have high C availability relative to N, thus providing a microsite of high N-assimilation capability. The rate of NH: diffusion into the organic particles may limit the NH: supply, causing both the partial shift to GS-GOGAT and the high K, values. The physical structure of the organic particles may possibly be an important control on microbial N-incorporation in the forest floor. NH:

al.,

Control

of NO;

Nitrate

incorporation

incorporation

was

low

in

these

experi-

ments. To determine if this was due to NH: repression, absence of NO; induction, or lack of enzyme capability, we treated samples to alter the existing conditions before measuring NO; incorporation, The results of this experiment are shown in Table 4. The treatment with water diluted the NH: so that it should have removed any NH: repression of NO; incorporation (NH: < 0.04 mM). The treatment with NO; was to induce NO; assimilation. The treatment with both NO; and NH,+ was to test the combination of these phenomena: i.e. if NO; assimilation was inducible, would NH: also repress it? None of these treatments significantly affected NO; assimilation. We could not cause either induction or repression of NO; assimilation. suggesting that the microbial populations of these forest floor subhorizons do not have the capability to assimilate NO; to a substantial degree. Fungal

up~cke

Ammonium uptake rates by excised fungal rhizomorphs and mycorrhizas were only slightly greater than the incorporation rates of the bulk organic material. which is composed primarily of decomTable 4. Conrrol of SO, incorporntton (rates in pg $ material h ‘I’ Subhorizon Treatment Fresh Prior exposure to H,O 0.1 m\r NO, 0.1 my SO, + 0.1 mst SH; ‘Rates \*ere measured durmg 6 h ‘Standtrd error of 3 repllaaws.

02

03

0.53 (0.1):

0.40 IO.?)

0.50(0.2) 0.43 (0.03) 0.50 (0. I I

0.45 (0. I) 0.40 (0.2) 0.27 (0.03)

g

’

Forest floor N incorporation Table 5. N-uptake from 0.5 rnM solutions by hyphal strands and mvcorrhizas (rata in leg N P-’ material h-‘)’ Materral

Source

White rhizomorphs Yellow fungus Yellow funnus-root association

02 03 03

NH;

NO,-

10.1(1.5): 14.0(1.8) IS.8 (2.61

ND’ ND I .? 10.5)

‘Rates were measured during 6 h. ‘Standard error of 3 replicates. ‘Not determined.

in the field. In conclusion,

we have identified

several

properties

in the forest floor that may be important in controlling forest N-cycles. A substantial portion of the N-incorporation potential resulted from abiotic processes. The indigenous microbial community had little capacity to assimilate NO;. The potential for NH,+ assimilation was extremely high but appeared to be at least partially NH,+ limited. The presence of localized zones of NH,+ depletion may be a critical characteristic of forest floor N-cycling. N-incorporation

Ackno*lnIXemenrs-We thank Paul Rygiewicz for assistance in the field. Eldor Paul. Ken Killham and an anonymous reviewer provided valuable commentary on the manuscript. This work was supported by a National Science Foundation grant and by a Mclntire-Stennis project of the University

of California

Agricultural

Experiment

Station.

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