Changes to mineral N cycling and microbial communities in
black spruce humus after additions of (NH
4
)
2
SO
4
and condensed
tannins extracted from
Kalmia angustifolia
and balsam ®r
R.L. Bradley
a,*, B.D. Titus
b, C.P. Preston
ba
DeÂpartement de Biologie, Faculte des Sciences, Universite de Sherbrooke, Sherbrooke, Que., Canada J1K 2R1
b
Paci®c Forestry Centre, 506 West Burnside Road, Victoria, BC, Canada V8Z 1M5
Received 22 June 1999; received in revised form 17 December 1999; accepted 18 February 2000
Abstract
Mechanisms responsible for conifer growth ``check'' on cutovers invaded by Kalmia angustifoliaL. in central Newfoundland were studied by examining eects of addedKalmiaand balsam ®r (Abies balsamea(L.) Mill) condensed tannins on black spruce humus N dynamics and microbial community development over 10 weeks using microcosms. Because of the silvicultural implications, interactions of tannins with fertiliser N, applied as (NH4)2SO4, were also studied. Both tannin types signi®cantly reduced NH4±N leaching, whereas only Kalmia tannins reduced NOÿ3±N leaching, and then only from non-fertilised humus. Tannins did not signi®cantly aect mineral N leaching from fertilised humus. Fertiliser N increased gross N mineralisation rates such that the increase in actively cycling N was many times greater than the increase in N leaching due to fertiliser N addition. Gross N mineralisation rates were higher in fertilised humus amended with tannins, suggesting possible toxicity of tannins on microbes at high N concentrations. Recovery of added tannins in leachate and in post-treatment humus samples was low. Net anaerobic N mineralisation decreased with tannin additions but increased with fertiliser N additions. There were few signi®cant treatment eects on microbial properties derived from humus respirometry. Microbial biomass and basal respiration rates of all treatments declined by 30 and 37% respectively, indicating a general loss of available C during the experiment. The ratio of Cmic-to-N mineralised as well as the nutrient de®ciency index was lowest in humus amended with Kalmia tannins, suggesting higher microbial N de®ciency in this treatment. Utilisation rates of various C sources by microbial communities showed distinctive patterns between pre-treatment and post-treatment humus samples, but did not reveal distinctive patterns among dierent treatments. Overall, results suggested that (1) condensed tannins decreased mineral N cycling abiotically by binding to and sequestering organic N sources, (2) fertiliser N counteracted negative eects of condensed tannins on humus N cycling, (3) microbial communities were N limited, which prevented abundant leaching of fertiliser N while maintaining fertiliser N in an active pool, and (4) the physiology and functional diversity of soil heterotrophic communities were controlled by C availability but were unaected by tannin or fertiliser N additions. Further work is needed to determine the ecological importance ofKalmia tannins, relative to tannins produced by other plants, in reducing humus N availability on spruce cutovers. 7 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Kalmia angustifolia; Condensed tannins; Humus N cycling; Microbial community; Microbial biomass;15Nisotope dilution;
Respiro-metry; Biolog microplate
1. Introduction
Since the 1960s, the role of the ericaceous shrub Kal-mia angustifoliaL. (hereafter referred to as Kalmia) in reducing growth of regenerating black spruce (Picea mariana (Mill.) B.S.P.) seedlings following stand
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* Corresponding author. Tel.: 8000; fax: +1-819-821-8049.
turbance in central Newfoundland, Canada, has been investigated (e.g., Peterson, 1965). A number of mech-anisms, other than competitive uptake of primary resources such as water and nutrients, may be respon-sible for Kalmia-induced growth ``check'' of spruce seedlings.
Firstly, Kalmia may suppress primary root growth of young black spruce seedlings by producing phyto-toxic compounds (Peterson, 1965; Zhu and Mallik, 1994). Leaf and litter extracts of other ericaceous and related forest plants have also been shown to have in-hibitory eects on germination and early development of conifer seedlings in other parts of the world (e.g.,
Calluna vulgaris(L.) Hull (Read, 1984),Empetrum her-maphroditumHagerup (Nilsson et al., 1993) and Vacci-nium myrtillusL. (Gallet, 1994)).
Secondly, Kalmia may cause spruce growth check abiotically through in¯uences on humus nutrient cycling by production of tannins (Kuiters, 1990). Tan-nins are phenolic compounds with the ability to form stable cross-links with proteins and other compounds. Many forest plants produce foliar tannins that are exported in litterfall to the forest ¯oor where they may in¯uence litter decomposition rates, humus formation, N cycling and ultimately plant nutrition (Handley, 1954). Tannin-induced N de®ciency has been suggested, although never proven, as a cause of conifer seedling growth check on sites dominated by Kalmia
(Bradley et al., 1997a).
Thirdly,Kalmia may produce substances that inhibit microbial or mycorrhizal communities responsible for soil nutrient cycling and plant uptake (Bradley et al., 1997c; Yamasaki et al., 1998). However, the evidence for this is contradictory and mechanisms by which plant secondary metabolites control microbial dynamics are unclear. For example, Boufalis and Pel-lissier (1994) applied a ``phytotoxic'' phenolic mixture to soil and found that O2 consumption by free-living
ectomycorrhizal fungi either increased or decreased, depending on the concentration of the mixture. Shafer and Blum (1991) found that phenolic acids, which sup-pressed cucumber seedling growth, were readily metab-olised by soil microorganisms, sometimes without detectable changes in microbial community structures. Schimel et al. (1996) separated secondary metabolites from Populus balsamifera L. into two fractions, low molecular weight phenolics and tannins, and found that the former stimulated soil respiration whereas the latter inhibited it.
Kalmia-induced growth check of conifer seedlings is site-speci®c and appears to be inversely related to site fertility (Bradley et al., 1997c). Damman (1971) reported that Kalmia humus did not mineralise detect-able amounts (i.e., <1 mg 100 gÿ1 dry wt.) of N after incubation at 218C for 100 days. Field trials have shown that N fertilisation reduced the competitive
ability of Kalmia in a jack pine (Pinus banksiana
Lamb.) forest (Prescott et al., 1995). Titus et al. (1993) have shown that Kalmia leaf tannin concentrations decreased with N fertilisation. However, the relative importance of fertiliser N and Kalmia tannins in con-trolling humus N availability and microbial dynamics is largely unknown.
Our aims were to examine the isolated eects of Kal-mia tannins on N dynamics and microbial community development. Condensed tannins from leaves of Kal-miawere isolated, puri®ed and added to humus from a black spruce cutover that had not yet been invaded by
Kalmia. The eects of Kalmia tannins were compared with those isolated from twigs and foliage of balsam ®r (Abies balsamia (L.) Mill), a conifer commonly found in association with black spruce in Newfound-land. Interactions between Kalmia or ®r tannins and fertiliser N were also tested by addition of fertiliser N applied as (NH4)2SO4.
2. Materials and methods
2.1. Forest ¯oor material
A large (ca. 20 kg fresh weight) bulked sample of humus was collected in the fall of 1996 from a 5-year old black spruce cutover near Middleton Lake (49803' N, 55859' W) in central Newfoundland where Kalmia -induced growth check of black spruce seedlings is com-mon. Humus was taken from the area on a cutover where Kalmia was not yet established, but was expected to invade based on rapid vegetative spread of the shrub from surrounding areas. The humus was sieved (6 mm) to remove roots and coarse debris and stored at 28C until used. Initial chemical characteristics of humus subsamples are presented in Table 1.
2.2. Preparation of Kalmia and balsam ®r tannins
Puri®ed condensed tannins were prepared from
Table 1
Selected chemical properties of black spruce humus collected in cen-tral Newfoundland from a recent cutover with noKalmia angustifolia
present
pH (humus:water, 1:10) 3.8
KCl extractableNH4±N (mg g
ÿ1) 4.6 KCl extractableNOÿ
3±N (mg g
ÿ1) 0.0 Bray extractable P (mg gÿ1) 20.1 Total N (mg gÿ1
) 9.0
Total P (mg gÿ1) 0.6
Total K (mg gÿ1) 0.6
Total Ca (mg gÿ1) 2.4
Total Mg (mg gÿ1) 1.0
green foliage of Kalmia and from tips (ca. 5 cm in length) of twigs plus needles of balsam ®r collected in central Newfoundland, as outlined in Preston et al. (1997). General structural information on the tannins was obtained from solution 13C NMR spectroscopy (Czochanska et al., 1980; Ayres et al., 1997). The Kal-miatannin was almost all procyanidin units, with over 90% cis stereochemistry and a very short average chain length of 2±3 units. The balsam ®r tannin was almost completely prodelphinidin units, with 75% cis
stereochemistry and an average chain length of 3±4 units. The smaller chain length forKalmia was consist-ent with its small response to the proanthocyanidin assay described below, which does not detect the chain-terminating units.
2.3. Experimental units, tannin and fertiliser N additions, and periodic leaching of humus
Experimental units consisted of 30 1-l plastic Buch-ner funnels (Bel-Art Products, Pequanoc,) equipped with fritted (`Fritware') plastic ®lter plates. Humus (72 g dry wt. equivalent) was gently tamped into each fun-nel, the tops were sealed with polyethylene ®lm to pre-vent desiccation and the funnels were incubated in the dark at 258C for 14 wk. At the same time as humus was added to the funnels, ®ve subsamples (ca. 500 g fresh wt.) of bulked humus were set aside in covered containers for determination of pre-treatment mi-crobial community characteristics (described below). Funnels were leached every 2 weeks for 14 weeks with two consecutive 200 ml aliquots of 10 mM CaCl2
sol-ution (Stanford and Smith, 1972). After addition of each aliquot, humus was left for 5 min to ensure ionic equilibrium, and then suction ®ltered at ÿ60 kPa. Humus in each funnel was then re-soaked for 5 min with 200 ml of N-free mineral nutrient solution (Stan-ford and Smith, 1972) to displace excess CaCl2and
re-store favourable microbial growth conditions. Excess nutrient solution was again removed after 5 min by suction ®ltration.
The 14-wk incubation began with a 4-wk condition-ing period to allow microbial communities to stabilise, followed by a 10-wk experimental period (referred to as T = 0±10 wk). Treatments were applied to humus ®ve times during the experiment, on the day following bi-weekly CaCl2leaching events (i.e., atT = 0, 2, 4, 6
and 8 wk). The six treatments were: (1) control (50 ml H2O), (2) Kalmia tannins (431 mg in 50 ml H2O), (3)
balsam ®r tannins (431 mg in 50 ml H2O), (4) fertiliser
N (678.5 mg (NH4)2SO4 in 50 ml H2O), (5) Kalmia
tannins + fertiliser N (431 mg tannins + 678.5 mg (NH4)2SO4 in 50 ml H2O), and (6) balsam ®r tannins
+ fertiliser N (431 mg tannins + 678.5 mg (NH4)2SO4
in 50 ml H2O). The six treatment solutions were each
applied to ®ve replicate funnels by making numerous
small injections in the top half of the humus with a large-tipped Eppendorf pipette. The cumulative treat-ment applications were equivalent to a total addition of 3% tannins and/or 1% mineral N (wt/wt) to humus in each funnel, except in the control treatment. Howard and Howard (1993) found that the initial N content of various tannin±protein complexes was ap-proximately 10%, of which a highly variable amount (6±85%) could be re-mineralised during a subsequent 1- to 2- week incubation. Based on these stoichiometric relationships, NH4±N was added to humus at a rate equivalent to 33% of added tannin weight to ensure that eective N binding would likely be compensated byNH4±N additions in ammonium sulfate fertiliser.
2.4. Chemical analyses of leachates and humus
Subsamples of leachate from each funnel were retained at T = 2, 4, 6, 8 and 10 wk. An aliquot was analysed colorimetrically for NH4±N and NO3ÿ±N concentrations using a Technicon auto-analyser, while another aliquot was analysed for total dissolved N (TDN) concentration after oxidation with persulfate (D'Elia et al., 1976). Dissolved organic N (DON) con-centration was then calculated as the dierence between TDN and DIN (i.e.,DINNH4±NNO3ÿ±N concentrations.
Condensed tannins in leachates (at T = 2, 4, 6, 8 and 10 weeks) and humus (at T = 10 wk) were ana-lysed colorimetrically after hydrolysis with butanol/ HCl using the proanthocyanidin assay (Preston, 1999; Lorenz et al., in press).
2.5. Post-treatment humus N dynamics
Two days following the ®nal leaching date (i.e.,T= 10 wk, hereafter referred to as post-treatment), a sub-sample of humus (5±8 g) was removed from each fun-nel to determine moisture content, and a second subsample (3±4 g fresh wt.) was analysed for minerali-sable N by anaerobic incubation (Waring and Brem-ner, 1964).
Post-treatment gross NH4±N transformation rates were measured by isotope dilution (Hart et al., 1995). Four humus subsamples (ca. 8 g dry wt.) from each funnel were placed in 500 ml Mason jars and 3 ml of aqueous 15NH
42SO4 solution (282.9 mg lÿ1 at 99
atom% 15N was uniformly distributed through the
25 ml25 ml) of each extract were diused onto acidi-®ed glass micro-®bre disks (Brooks et al., 1989, as modi®ed by Bradley and Fyles, 1996) and encapsulated in Sn sleeves (Europa Scienti®c, Franklin, OH). Dif-fused samples were analysed for atom% 15N analysis by continuous ¯ow mass spectrometry. Gross pro-duction and consumption rates ofNH4±N were calcu-lated using zero-order equations derived by Kirkham and Bartholemew (1954). It was assumed that isotope addition would not bias ammoni®cation rates (here-after referred to as gross mineralisation rates) in the short-term but might cause an overestimation of gross NH4±N consumption rates due to enrichment of reac-tant pools (Hart et al., 1995).
2.6. Soil respirometry
Basal respiration rate (B) and microbial biomass (MB) of the ®ve pre-treatment humus samples were determined prior to application of treatments (T = 0 wk). B and MB of the 30 treated samples were deter-mined post-treatment at T = 10 wk. A second set of post-treatment humus samples from each funnel was kept for 4 wk at 258C, and MBwas re-measured at T
= 10 + 4 wk. A third set of post-treatment humus samples from each funnel was used to determine the energy de®ciency index (EDI) and the nutrient de-®ciency index (NDI) of microbial communities (Brad-ley and Fyles, 1995a).
Bwas determined by weighing humus samples (ca. 8 g dry wt.) in 190 ml gas sampling jars, ¯ushing the headspace with ambient air for 5 min, sealing jars with air-tight lids equipped with rubber septa and sampling aliquots of air in the headspace with a needle and syr-inge after 2 h. Air samples were analysed for CO2
con-centrations using a Hewlett±Packard model 5890 GC (Hewlett±Packard, Avondale, PA) equipped with an FID and methanizer, and using N2as carrier gas.
MB was estimated by converting substrate-induced respiration rates to microbial biomass of organic resi-dues using equations in Beare et al. (1990). Ground (65 mm) glucose, Difco nutrient broth powder, yeast extracts and talc (as inert ®ller) were mixed in a ratio of 5:7:3:86 (Bradley and Fyles, 1995a). This glucose + nutrient mixture (250 mg) was dispersed into tared humus samples (ca. 8 g dry wt.) using a kitchen hand-mixer with one beater. Samples were then transferred into 190 ml gas sampling jars and left uncovered for 100 min. After reaching maximum initial substrate-induced respiration rates (Anderson and Domsch, 1978), each sample was ¯ushed for 5 min with ambient air, sealed for 30 min, and air from the head space was analysed for CO2concentration using a GC.
EDI and NDI were calculated from respiration rates induced by addition of glucose only (G, as glucose:talc = 4:25), basal respiration rates of humus samples (B,
as above), and respiration rates induced by addition of glucose + nutrients (GN, as above):
EDI GÿB=GN100% unitless 1
NDI GNÿG=GN100% unitless 2
Ambient temperature and atmospheric pressure were recorded at each measurement, and ambient CO2
con-centration was measured several times daily. For each sample, ambient CO2 concentration was subtracted
from sampled CO2 concentration and the dierence
was adjusted according to Ideal Gas Laws and cen-tered at 228C usingQ10= 2.
2.7. Potential C source utilisation patterns
The functional diversity of microbial communities was characterised in pre-treatment bulk humus (n = 5) and in post-treatment humus samples from each funnel using the Biolog GN microplating system (Bio-log, Hayward, CA). Moist humus samples (ca. 10 g) were weighed (21 mg) into polyethylene bottles con-taining glass beads (3-mm in diameter), mixed with 100 ml of 0.1% Na-pyrophosphate solution and sha-ken at high speed on a benchtop reciprocating shaker for 15 min. The resulting suspensions were diluted 100-fold in deionized water as initial tests had shown optimal colour development occurring at this dilution. Aliquots (150 ml) of each diluted suspension were added to each of 96 wells in duplicate Biolog plates. Ninety-®ve of the wells contained redox-sensitive tetra-zolium dye and a unique C source, whereas one con-trol well contained dye only. Biolog plates were incubated at 258C and colour formation in each well was read as light absorbance (590 nm) after 20, 24, 28, 44, 48, 52, 68, 72 and 76 h using an automated plate-reader (Biolog Microstation and software; Biolog, Hayward, CA).
Well absorbance values of duplicate plates were averaged. At each reading, absorbance value of control wells were subtracted from absorbance values of 95 wells containing C substrates. Average well color development (AWCD) of each plate was then calcu-lated at each reading to determine the incubation time (T0.75) corresponding to AWCD = 0.75 absorbance
units. The average number of substrates used at T0.75
SD ÿXpi lnpi 3
where pi is the ratio of colour development of the ith well to the sum of colour development in the plate. The evenness of substrate use across positive wells (i.e., substrate evenness, or SE) was calculated using the relationship:
SESD=log SR 4
2.8. Statistical analyses
The eects of tannin or fertiliser N additions on cumulative leaching of NH4±N, NO3ÿ±N, DON and tannins, on post-treatment humus N dynamics, on humus residual tannin concentrations and on humus respiration measurements were tested statistically using one-way ANOVA. Signi®cantly dierent treatment means were evaluated using Duncan's multiple range test.
The eects of treatments on NH4±N and NO3ÿ±N concentrations in leachates over the treatment period were analysed using repeated measures ANOVA (T= 2±10 wk). Both multivariate and univariate models were used to test the eect of time and its interaction with treatments (i.e., within subject dierences). To attain sphericity of variance±covariance matrices, the six treatments were divided into non-fertilised or ferti-lised groups, which were then analysed separately for tannin eects. This division of treatments was also necessary to obtain fewer treatments than repeated measurements within each statistical test, which is a criterion for repeated measures ANOVA (Potvin et al., 1990). The univariate approach used the adjusted
Huynh and Feldt (1976) F-statistic to test within-sub-ject hypotheses. The eects of tannins within each date were tested using Duncan's multiple range test.
Corrected absorbance values (T0.75) of 95 substrate
wells in each Biolog plate were centered and normal-ised [i.e., (Absorbance ÿ AWCD)/s], and principal component analysis (PCA) was performed on trans-formed data to explore eects of treatments on pat-terns of substrate use by microbial communities.
Unless otherwise stated, P values of 0.05 or less were considered statistically signi®cant.
3. Results
Results of one-way ANOVA tests for the eect of treatments on leachate N concentrations, post-treat-ment humus N dynamics, post-treatpost-treat-ment respiration measurements, and post-treatment humus tannin con-centrations are given in Table 2, and details are pre-sented below.
3.1. Nitrogen and tannins in leachate
Both Kalmiaand ®r tannins signi®cantly (P< 0.001) reduced total extractable NH4±N among non-fertilised treatments (Table 3).Kalmia tannins signi®cantly (P< 0.01) reduced total extractable NO3ÿ±N among non-fertilised humus as well as total extractable NH4±N and NO3ÿ±N among fertilised treatments. The magni-tude of tannin eects is re¯ected by between subjects
P-values given in Table 4. Tannins did not aect DON concentrations.
Adding fertiliser N to spruce humus increased
cumu-Fig. 1. Changes toNH4±N andNO3ÿ±N concentrations in leachates
from unfertilised [(a) and (b)] and fertilised [(c) and (d)] treatments; data points represent control (w),Kalmiatannin (t), ®r tannin (q), fertiliser N (*),Kalmiatannin + fertiliser N (T), and ®r tannin + fertiliser N (Q) treatments; treatment means, on the same graph within single dates, denoted by dierent lowercase letters dier sig-ni®cantly (Duncan's multiple range test,P< 0.001,P< 0.01,
P< 0.05, ns = not signi®cant). Table 2
Results of one-way ANOVAs testing treatment eects on various measurements of microbial and N dynamics in black spruce humus
Measurement df F-value Prob >F
Cumulative leachate N
NH4±N 5 351.0 0.0001
NO3ÿ±N 5 482.4 0.0001
DON 5 4.9 0.0032
Post-treatment humus N
Gross N mineralisation rate 5 27.4 0.0001 GrossNH4±N consumption rate 5 2.9 0.0335
Anaerobic mineralisation rate 5 144.3 0.0001
Post-treatment respirometry
Basal respiration rate 5 1.6 0.2029
EDI 5 1.2 0.3519
NDI 5 1.3 0.3180
lative leaching of NH4±N by an order of magnitude and cumulative leaching of NO3ÿ±N by a factor of 2 (Table 3). The N-fertilisation induced increase in extractable NH4±N and NO3ÿ±N began at T = 2 wk and remained throughout the trial (Fig. 1). Lower amounts of DON leached from humus amended with fertiliser N, but DON extracted from all treatments was low relative to DIN (Table 3).
Repeated measures ANOVA revealed signi®cant (P
< 0.001) time eects in mean extractableNH4±N
con-centrations in non-fertilised humus (Table 4 Ð within subjects). Fig. 1(a) shows a decreasing trend in NH4± N leaching from the three non-fertilised treatments. Mean extractable NO3ÿ±N concentrations also varied signi®cantly (P< 0.001) over time in both non-ferti-lised and fertinon-ferti-lised humus (Table 4 Ð within subjects), but there was no clear trend (Fig. 1(b) and (d)). The eect of tannin additions on extractable NH4±N and NO3ÿ±N concentrations also varied signi®cantly (P< 0.05) over time in non-fertilised humus (Table 4 Ð Table 3
Eect of treatments on mean cumulative leaching ofNH
4±N,NO3ÿ±N and DON over 10 weeks (SE in parentheses,n= 5)
Treatment NH
4±N (mg N g ÿ1
soil) NOÿ
3±N (mg N g ÿ1
soil) DON (ng N gÿ1soil)
Control 75.1 (3.3) 2.51 (0.06) 196 (9)
Kalmiatannins 51.2 (1.6) 2.21 (0.04) 173 (9)
Fir tannins 51.8 (3.1) 2.40 (0.04) 184 (17)
Fertiliser N 652.7 (19.6) 5.09 (0.05) 121 (8)
Kalmiatannins + fertiliser N 565.1 (14.4) 4.75 (0.10) 198 (18)
Fir tannins + fertiliser N 610.8 (32.7) 4.96 (0.09) 157 (14)
Table 4
Repeated measures ANOVAs correlating tannin additions toNH
4±N andNO3ÿ±N concentrations in leachate from non-fertilised (ÿF) and
ferti-lised (+F) humus
Ionic species Fertiliser Source of variation df MS F-value Prob > F
Between subjects
NH4 ÿF Tannins 2 23.93 24.06 0.0001
Error 12 0.99
NH4 +F Tannins 2 246.6 3.71 0.0558
Error 12 66.5
NO3ÿ ÿF Tannins 2 0.0031 11.22 0.0018
Error 12 0.0002
NOÿ
3 +F Tannins 2 0.0036 4.62 0.0326
Error 12 0.0008
Within subjects (univariate test)
NH
4 ÿF Time 4 251.7 501.86 0.0001
Timetannins 8 1.55 3.08 0.0208
Error (time) 48 0.50
NH4 +F Time 4 94.6 1.50 0.2164
Timetannins 8 96.2 1.53 0.1728
Error (time) 48 63.0
NO3ÿ ÿF Time 4 0.0176 56.68 0.0001
Timetannins 8 0.0007 2.28 0.0375
Error (time) 48 0.0003
NO3ÿ +F Time 4 0.0416 51.71 0.0001
Timetannins 8 0.0014 1.71 0.1193
Error (time) 48 0.0008
Within subjects (multivariate test) df Wilk'sl F-value Prob >F
NH
4 ÿF Time 4, 9 0.0106 210.05 0.0001
Timetannins 8, 18 0.2074 2.6905 0.0387
NH
4 +F Time 4, 9 0.5508 1.84 0.2063
Timetannins 8, 18 0.3377 1.62 0.1874
NOÿ
3 ÿF Time 4, 9 0.0234 94.07 0.0001
Timetannins 8, 18 0.2515 2.24 0.0745
NOÿ
3 +F Time 4, 9 0.0444 48.42 0.0001
univariate test). The multivariate approach gave a more conservative estimate (i.e., P = 0.0387 and 0.0745, respectively) of these same interactions. More speci®cally,Kalmia and ®r tannins both reducedNH4± N leaching from non-fertilised humus during T = 4± 10 wk (Fig. 1(a)). Kalmia tannins signi®cantly reduced NO3ÿ±N leaching from non-fertilised humus atT= 10 wk (Fig. 1(b)).
Tannins were not detected in any of the leachate samples.
3.2. Post-treatment humus N dynamics
Addition of fertiliser N increased gross N mineralis-ation rates by 1±2 orders of magnitude (Fig. 2(a)).
Gross N mineralisation rates were signi®cantly higher in fertilised humus amended with Kalmiaor ®r tannins than in fertilised humus without added tannins.
Gross N mineralisation rates of all treatments were higher than corresponding gross NH4±N consumption rates. These dierences were large in fertilised treat-ments (from 701 mgÿ1 dÿ1 for fertilised treatment to 1277mgÿ1dÿ1for the fertilised + ®r tannin treatment) and small in non-fertilised treatments (from 7mgÿ1dÿ1 for Kalmia tannin treatment to 15 mgÿ1 dÿ1 for the control). Gross NH4±N consumption rate was signi®-cantly higher in fertilised humus amended with ®r tan-nins than in other treatments (Fig. 2(b)).
Anaerobic N mineralisation rates were signi®cantly higher (ca. 4) in humus from fertilised treatments than in humus from non-fertilised treatments (Fig. 2(c)). Both Kalmia and ®r tannins signi®cantly reduced anaerobic N mineralisation rates within non-fertilised treatments.
3.3. Humus tannins
Post-treatment condensed tannin concentrations were signi®cantly (P< 0.05) higher in humus that had been amended with Kalmia tannins, either with or without fertiliser N (Table 5). However, only 1% of the amount of tannin added was recovered in these two treatments.
Fig. 2. Eect of tannins and fertiliser N additions on post-treatment measurements of (a) gross N mineralisation rate, (b) grossNH4±N
consumption rate, and (c) anaerobic net N mineralisation (vertical bars = 1 SE,n= 5).
Fig. 3. Eect of tannins and fertiliser N additions on post-treatment microbial biomass (vertical bars =1 SE,n= 5).
Table 5
Average post-treatment condensed tannin concentrations in black spruce humus amended with condensed tannins or fertiliser N; means followed by a dierent lowercase letter dier signi®cantly by Duncan's multiple range test (P< 0.05,n= 5); the percent recovery is based on control treatment value subtracted from average concentration of each treatment
Treatment mg tannins gÿ1humus % recovery
Control 385 b
Kalmiatannins (3%) 734 a 1.16%
Fir tannins (3%) 480 b 0.32%
Fertiliser N (1%) 389 b 0.01%
Kalmiatannins (3%) + fertiliser N (1%) 690 a 1.02%
3.4. Respirometry and C source utilisation patterns
There were no signi®cant treatment eects on post-treatment microbial measurements derived from humus respirometry except for a relatively weak (P= 0.02) treatment eect on MB (Table 2). Duncan's mul-tiple range test revealed higher MB in humus amended with Kalmia tannins than in other treatments (Fig. 3). Dierences in MB among all treatments were small, however, in comparison to the 30% decline in average
MB of all treatments between T = 0 and 10 wk (Table 6). Similarly, the average B of all treatments declined by 37% between T = 0 and 10 wk. There was no dierence between average MB of all treat-ments at T= 10 wk and 14 wk.
There was a large error term associated with NDI measurements. Consequently, NDI did not dier sig-ni®cantly among treatments, although treatment averages varied by as much as 100%. The highest NDI value was for humus amended with Kalmia tannins. NDI was consistently higher in non-fertilised treat-ments compared to corresponding (i.e., paired) ferti-lised treatments (data not shown). In contrast, average EDI values of all treatments varied by less than 10%.
PCA ordination based on C source utilisation terns by microbial communities showed distinctive pat-terns between pre-treatment and post-treatment humus samples (Fig. 4). Post-treatment samples had above-median scores for PCA axis 1, in contrast to pre-treat-ment samples that had below-median scores. PCA did not reveal distinctive patterns in post-treatment sub-strate use among dierent treatments. PCA axes 1 and 2 explained 31% and 8%, respectively, of the variance in the data set.
Substrate richness (SR) and substrate diversity (SD) were signi®cantly higher in pre-treatment samples than in post-treatment samples (Table 7). The average decrease in positive wells from pre-treatment to post-treatment was due to lower utilisation rates of carbo-hydrates (12 fewer wells), amino acids (8 fewer wells), amines and amides (4 fewer wells) and carboxylic acids (4 fewer wells). Utilisation rates of other classes of C Table 6
Eect of treatment duration on average basal respiration rate and microbial biomass of all treatments (SE in parentheses,n= 30; ND = not determined)
Measurement Pre-treatment (T= 0 week) Post-treatment (T= 10 weeks) Post-treatment (T= 14 weeks) Basal respiration (mg CO2±C gÿ1hÿ1) 4.66 (0.13) 3.25 (0.26) ND
Microbial biomass (mg Cmicgÿ1) 2.72 (0.08) 1.71 (0.07) 1.72 (0.07)
Fig. 4. Principal component analysis of C source utilisation patterns by microbial communities in black spruce humus prior to, and after, tannin and fertiliser N additions; PCA axes 1 plus 2 accounted for 39% of variance in data set; data points are same as described in Fig. 1; ®ve pre-treatment samples denoted by `P'.
Table 7
Eect of treatments on average number of positive wells in Biolog plates (SR), on the equivalency of colour development among positive wells (SE), and on the calculated index of functional diversity (SD) of microbial communities; all values measured at AWCD0.75are unitless; values in the same column followed by dierent lowercase letters are signi®cantly dierent (Duncan's multiple range test,P< 0.05,n= 5)
Treatment Substrate richness (SR) Substrate evenness (SE) Substrate diversity (SD)
Pre-treatment 75 a 0.88 a 4.03 a
Post-treatment
Control 54 b 0.78 a 3.57 b
Kalmiatannins 55 b 0.78 a 3.56 b
Fir tannins 54 b 0.77 a 3.50 b
Fertiliser N 58 b 0.79 a 3.62 b
Kalmiatannins + fertiliser N 58 b 0.79 a 3.61 b
compounds (esters, polymers, alcohols, aromatics, bro-minated and phosphorylated chemicals) were identical in all pre- and post-treatment plates.
4. Discussion
4.1. Leachate chemistry
The addition of fertiliser N to spruce humus resulted in a marked and sustained increase in extractable NH4±N and NO3ÿ±N during the 10-wk study. How-ever, the net increase in DIN leached was less than 10% of the fertiliser N added. Therefore, the spruce humus had a high potential for rapid immobilisation and subsequent slow release of added mineral N. Although a basic tenet of soil ecology is that microbial communities are primarily limited by available C (Smith and Paul, 1990), Hart and Stark (1997) have shown that heterotrophic communities in some mature conifer forest soils can be N-limited as well. Such a feature would predispose the black spruce ecosystem from which humus was gathered in the present study to mitigate losses of mineral N following fertiliser N application.
Addition of Kalmia and ®r tannins signi®cantly reduced NH4±N leaching from non-fertilised humus. However, such eects were not detected in fertilised treatments, probably because positive eects of fertili-ser N additions were greater than (and therefore over-whelmed) negative eects of tannin additions. In this study, fertiliser N was applied at a rate much higher than prescribed by current silvicultural practices whereas tannin addition rates were comparable to tan-nin concentrations commonly measured in litter and humus (Lorenz et al., in press). If it is assumed that the principal mode of action of tannins was to bind abiotically with nitrogenous substrates in humus, then there would be a threshold in N fertility beyond which the sequestering of potentially mineralisable N by tan-nins becomes insigni®cant. This is supported by ®eld observations that Kalmia-induced growth check of black spruce seedlings is greater on lower quality sites (Titus et al., 1995), and that application of mineral N fertilisers alleviates growth check in many problematic forest sites dominated by ericaceous shrubs such as
Calluna vulgaris(Taylor, 1991),Gaultheria shallon (Pre-scott et al., 1996) and Kalmia angustifolia (Prescott et al., 1995).
Factors controllingNO3ÿ±N dynamics depend on the nature of microbial communities involved in producing and consuming this anion. While nitri®cation in agri-cultural soils is primarily attributed to autotrophic micro-organisms, heterotrophic nitri®ers may be more prevalent in some forest soils (Hart et al., 1997; Papen and von Berg, 1998). The immediate and sustained
increase in NO3ÿ±N leaching after fertiliser-NH4 ad-dition suggests that the spruce humus we studied had a signi®cant potential for autotrophic nitri®cation. However, high net nitri®cation rates in fertilised humus could also have resulted from lower microbial assimilation rates of NO3ÿ±N due to higher NH4±N pools (Hart et al., 1994). Determining whetherNH4±N pools increased gross NO3ÿ±N production rates or decreased gross NO3ÿ±N consumption rates was beyond the scope of our study, but there is evidence from non-fertilised treatments thatNH4±N supply was not the sole factor controlling NO3ÿ±N production. The fact that only Kalmiatannins signi®cantly reduced NO3ÿ±N production in non-fertilised humus, and that this eect occurred only on the ®nal leaching date (T
= 10 wk) suggests (1) that nitri®cation was not con-trolled by NH4±N concentrations alone, and (2) that
Kalmia and ®r tannins did not aect nitri®cation path-ways in a similar manner. The possibility that Kalmia
tannins inhibited nitri®er populations cannot be excluded since a decreasing trend in NO3ÿ±N leaching was observed in the Kalmia tannin treatment, relative to other treatments, during the 10-wk study. For example, some studies have suggested that phenolic compounds including tannins, phenols and volatile ter-penoids are capable of inhibiting soil nitri®er popu-lations (e.g., Lohdi and Killingbeck, 1980; White, 1988; Paavolainen et al., 1998). However, the higher post-treatment MB measured in the Kalmia tannin treatment suggests that Kalmia tannins may have been a growth substrate for some microbes. Therefore, the apparent inhibitory action ofKalmia tannins on nitri®-cation may have been caused by increased NO3ÿ±N consumption due to higher available C in this treat-ment. Future studies should include 15NO3ÿ±N dilution assays to verify these hypotheses regarding factors con-trolling net production of NO3ÿ±N in tannin-rich humus.
Northup et al. (1995) proposed that tannin-rich plant communities conserve N by decreasing soil min-eral N pools and minimising N leaching. Based on cor-relative evidence, Northup et al. (1995) hypothesised that the preponderance of DON in soil solution is at-tributable to the presence of tannin-protein complexes which delay the onset of N saturation. However, this was not supported by our study, as the addition of fer-tiliser N decreased DON leaching, and addition of tan-nins had little eect on DON.
replenish these DON pools than to replenish DIN pools; or (2) the 10 mM CaCl2 extractant, proven to
be ecient in removing mineral N, was too weak to displace large molecular weight DON molecules of higher adsorptive capacity.
4.2. Humus N dynamics
Although the increase in mineral N leaching was modest relative to fertiliser N additions, the corre-sponding increase in gross N mineralisation rates in post-treatment humus was relatively high. Dierences between daily gross N mineralisation and daily gross NH4 consumption rates in fertilised treatments, ex-trapolated over 2 wk, were 10 times greater than bi-weekly fertiliser N addition rates. However, such extra-polation is tenuous since gross NH4±N consumption probably obeyed ®rst order kinetics and were implicitly related to the size of NH4±N pools at the time of measurement (Bradley et al., 1997c). It is therefore likely that grossNH4±N consumption rates were high-est immediately after fertiliser N addition and lowhigh-est on the date coinciding with measurement, which would explain the large dierences between the two process rates. Regardless of time-scale bias, the results of
15NH
4±N dilution assays strongly supported the
assumption that microbial communities were N de-®cient and that a major portion of addedNH4±N was used to increase and maintain actively cycling mi-crobial N pools.
One of our aims was to determine the eect of Kal-mia and ®r tannins on microbial development. Results from the few studies that have examined the eects of tannins (as opposed to other phenolic compounds) on microbial growth are contradictory, perhaps because tannins from dierent origins can vary in molecular size and structure. For example, Benoit et al. (1968) studied tannins extracted from wattle (species not speci®ed, but likely Acacia spp. or mimosa) and con-cluded that ``the principal eect of tannins on mi-crobial development is not that of toxicity'', whereas Schimel et al. (1996), using tannins extracted from bal-sam poplar, concluded that ``tannins act as a general microbial inhibitor''. Some authors reported no evi-dence for decomposition of tannins during leaf decay (Scho®eld et al., 1998) whereas others reported that certain soil organisms preferentially degrade condensed leaf tannins (Gamble et al., 1996). Thus, it is dicult to speculate whether Kalmia and ®r tannins might be toxic compounds or growth substrates for soil micro-organisms. However, it is known that toxic compounds can increase gross N mineralisation rates in humus, whereas readily metabolisable substrates such as starch can reduce gross N mineralisation rates (Schimel et al., 1992). In our study, tannins did not signi®cantly aect gross N mineralisation rates in non-fertilised humus,
and therefore it can be inferred that tannins were neither toxic nor easily metabolised by soil microbes when NH4±N concentrations were low. However, tan-nins did increase gross N mineralisation rates in ferti-lised humus, suggesting a toxic eect to microbes when NH4±N concentrations are high. This observation is reminiscent of results of Bradley et al. (1997c) in which signi®cantly higher gross N mineralisation rates occurred in fertile humus planted with Kalmia than with other seedlings, but there were no signi®cant changes in gross N mineralisation rates in poor humus planted with the same species. Thus, a parallel may be drawn between the presence of Kalmia root systems and the addition of Kalmia tannins to humus. Their similar eects suggest that Kalmia root systems release tannins or related compounds into soil, and that these may be toxic to some micro-organisms.
High mineral N fertility, and by implication high actively cycling microbial N pools, appear necessary for tannins to signi®cantly increase gross N mineralis-ation rates. Tannins possibly increase microbial death and turnover rates, with a portion of microbial N that is released being prone to form recalcitrant complexes with tannins. Howard and Howard (1993) found sub-stantial binding between protein and polyphenols released from freshly fallen litter of various tree and shrub species, and that these stable complexes signi®-cantly reduced subsequent N mineralisation rates. Thus, tannin±protein binding may oset the potential for higher N leaching due to higher gross N mineralis-ation rates.
As with NH4±N concentrations in leachates, anaero-bically mineralised N (AMN) rates were signi®cantly higher in fertilised humus than in non-fertilised humus, and addition of tannins signi®cantly reduced AMN rates within non-fertilised humus. AMN rates have been correlated with soil N availability in forest eco-systems (Keeney, 1980; Powers, 1980) and are thought to re¯ect the activity of microbial N pools (Myrold, 1987). AMN rates therefore provided further evidence that fertiliser N alleviated microbial N de®ciencies whereas tannins limited microbial access to organic N.
4.3. Tannins
Little is known about the fate of litter tannins, except that there is a large and rapid decline (around 80%) in the ®rst year of decomposition (reviewed in Lorenz et al., in press). However, tannin concen-trations in humus are generally less than 0.5%. Where an unusually high amount was found (3±4%), such as in the black spruce sites studied by Lorenz et al. (in press), it appeared to be caused more by environmen-tal factors (climate, fauna) rather than by unusually high amounts of tannin in the litter.
humus after incubation (ca. 1%), and no detectable tannin in leachates. There are no reports of similar recovery data in the literature, although Scho®eld et al. (1998) indicated that tannins leached from willow leaves were tightly sorbed to mineral soil and could not be detected by a variety of approaches. It is poss-ible that the tannins may have become tightly bound to organic matter, including proteins, or had under-gone chemical reactions with organic matter, or had been microbially metabolised. The last possibility seems less likely, based on our other data from this study.
4.4. Microbial communities
If readily-metabolisable C substrates were applied to humus at a rate comparable to the rate of tannin ad-ditions used in this experiment (i.e., 3% wt/wt over 10 wk), MB would probably increase (Bradley and Fyles, 1995b; Bradley et al., 1997b). In our study, the average post-treatment MB of all treatments declined signi®-cantly from pre-treatment values, therefore ®r and Kal-mia tannins were probably not readily metabolised by soil microbes. Experimental conditions likely decreased available C in humus because (1) the humus was main-tained at a higher and more constant temperature than in the ®eld, thus favouring rapid consumption of avail-able C sources, and (2) there were no chronic inputs of organic substrates, such as in throughfall and rhizode-position under ®eld conditions, to replenish labile C pools. Therefore, lower mineral N leaching from non-fertilised humus treated with tannins was most likely due to biochemical interference of tannins with poten-tially-mineralisable N sources as opposed to higher mi-crobial immobilisation rates induced by higher C supply.
Kalmia tannins had a lower molecular weight than ®r tannins which may have resulted in their being par-tially metabolised, which would explain why post-treatment MB was slightly higher in humus treated with Kalmia than with ®r tannins. However, this may not be of importance for two reasons. Firstly, the dierence between post-treatment MB in humus amended with Kalmia tannins and the average MB of the other ®ve treatments was about one-third of the average decline in MB of all six treatments over the 10-wk treatment period (i.e., 0.32 vs. 1.01 mg Cmic
gÿ1). Secondly, average MB of the six treatments remained constant for 4 wk following post-treatment (i.e.,T = 14 wk), and there were no signi®cant dier-ences in MB among treatments at this later date. The eect of Kalmia tannins on MB was therefore rela-tively weak and ephemeral. Benoit et al. (1968) demon-strated that considerable amounts of readily-decomposable carbohydrates were contained in crude tannin preparations and that tannin decomposition
rates decreased markedly as the purity of these tannins increased. However, use of NMR to characterise tan-nins before and after preparation ensured that the Kal-miaand ®r tannins we used were free of impurities.
Dierences in MB among treatments were small relative to dierences in N cycling rates, suggesting that MB was controlled by C availability whereas N cycling rates depended on microbial N pools and poss-ibly rates of microbial death and turnover. MB is a static measurement, which does not re¯ect microbial turnover rates. An apparent increase in microbial N with fertiliser addition without a concomitant increase in MB corroborates the premise that microbial com-munities were initially N de®cient. The ratio of Cmic
-to-N mineralised was lowest in humus amended with
Kalmia tannins, suggesting higher microbial N de-®ciency in this treatment. Thus, dierences in NDI among treatments, although statistically non-signi®-cant, were nonetheless consistent with other observed eects of fertiliser N and tannins on N cycling and mi-crobial dynamics.
Respirometry measurements are useful indicators of the ecophysiological status of microbial communities, but do not indicate whether treatments caused funda-mental changes to community structure. Biolog GN microplates, on the other hand, are commonly used as proximate indicators of the functional diversity within microbial communities, based on utilisation patterns of dierent C sources (Garland, 1996a, 1996b). Although Biolog assays do not identify or distinguish taxonomic groups within microbial communities, C source utilis-ation patterns re¯ect dierences in their metabolic ca-pacities. Since the number of viable cells in each well aects colour development (Garland, 1996a), adjust-ment of samples to equivalent inoculum cell densities has been recommended (Haack et al., 1995). The de-cision to use equivalent cell densities should, however, be determined by the nature and scope of the investi-gation. In this study, inoculum cell density was not standardised because (1) such a manoeuvre can bring about its own experimental artefact, (2) identical inoculum cell densities can result in 20% dierences in AWCD (Garland, 1996a), and more importantly (3) the aim was to determine changes in metabolic ca-pacity of microbial communities within a single humus type, on a per unit humus weight basis, and therefore changes in microbial densities as well as in community structure were implicit in the treatment eects that were investigated. For these reasons, plates were com-pared by adjusting the activity in each well to AWCD0.75, as other studies have done (Garland and
Mills, 1991; Zak et al., 1994; Garland, 1996b; Grays-ton et al., 1998).
mi-crobial communities. The decrease of SD and SR were due to lower capacities to metabolise simple as opposed to more complex C substrates. One possible explanation is that labile C, which decreased due to ex-perimental conditions (as con®rmed by MB data), cre-ated a selective pressure favouring microbial communities that metabolised more resistant C sub-strates. Fungi specialised in the breakdown of complex substrates may have increased relative to bacteria during the study period. Since Biolog assays strongly underestimate fungal activity because fungal hyphae are extracted ineciently from humus (Zak et al., 1994), lower values of SR and SD at post-treatment could therefore re¯ect an increase in fungal-to-bacterial ratios during the 10-wk experiment.
Our results emphasise the importance of distinguish-ing between factors that aect C supply, and conse-quently N cycling, and exogenous factors such as fertiliser N or some condensed tannins, that aect N cycling only. The experimental period resulted in a lower C supply which, in turn, had more eect on functional diversity of microbial communities than did the experimental treatments. Other Biolog studies have shown that microbial communities can change after ex-posure to plant roots (Garland, 1996b), perhaps because of C that is supplied by rhizodeposition. In our study, tannin and fertiliser N additions signi®-cantly modi®ed humus N cycling but did not aect C source utilisation patterns by microbial communities during the experiment. This gives rise to an important question regarding changes in site quality due to tan-nin or fertiliser N additions: To what extent does func-tional diversity of microbial communities play a role in controlling humus N dynamics? Since soil organic N is thought to mineralise stoichiometrically with soil or-ganic C (McGill and Cole, 1981), and since individual microbial populations metabolise speci®c substrates according to speci®c kinetic pro®les, the microbial functional diversity should, in theory, be linked to humus N cycling characteristics. However, the com-plexity of microbial communities probably ensures that similar substrates can be metabolised at similar rates by dierent taxa of micro-organisms, or that many micro-organisms can subsist in the absence of optimal nutrient supply.
4.5. Implications of ®ndings
We have demonstrated that, under laboratory con-ditions, condensed tannins isolated from Kalmia and balsam ®r can reduce mineral N availability in black spruce humus. The lack of recovery of tannins in lea-chate and treated humus, the general decrease in MB over the treatment period and the lack of change in C source utilisation patterns by microbial communities suggest that tannins decreased mineral N cycling by
binding to and sequestering organic N sources rather than because they were toxic to or easily metabolised by microbial communities. In order to assess the eco-logical importance of Kalmia tannins, relative to tan-nins produced by ®r or other plants, in reducing humus N availability on spruce cutovers, further work is needed to determine (1) how tannins move from
Kalmia to humus, (2) the exact nature and persistence of Kalmia tannin±protein complexes, and (3) possible uptake of tanned substrates by Kalmia and other plants.
Since the experiment was performed on humus taken from a single site, the eects of tannins and fer-tiliser N should be tested over a wide range of humus forms and site qualities before drawing general con-clusions. Nevertheless, the formation of Kalmia tan-nin±protein complexes in humus may explain the large reductions in net N mineralisation on some Kalmia
heaths (Damman, 1971) and the reduction of foliar N concentrations in black spruce seedlings growing in proximity to Kalmia on some cutovers (Yamasaki et al., 1998). Where microbial communities occupying black spruce humus are also N limited, adding fertili-ser N should not only reduce spruce growth check in the short term, but should also mitigate microbial N de®ciencies, increase active N pools and prevent rapid leaching loss of N. The importance of increased mi-crobial N uptake independent of C-supply needs further evaluation in terms of restoring site fertility.
Acknowledgements
The authors wish to thank Karen Hogg and Kevin McCullough for co-ordinating laboratory operations as well as Sarah Riecken, Carol Cutworth and Martin Smith for technical assistance. We are thankful to Dr. Bernard Colin for advice on statistical analyses. Iso-tope enrichment analyses were carried out by continu-ous ¯ow mass spectrometry at the Stable Isotope Laboratory, University of Saskatchewan; chemical analysis of the initial bulked humus was carried out at the MacMillan Bloedel Laboratory, Nanaimo, BC; all other analyses were carried out at the Paci®c Forestry Centre, Victoria, BC. The study was funded by a grant from the Science Council, Forest Renewal of British Columbia as well as Post-Doctoral Fellowship from the Natural Sciences and Engineering Science Council of Canada.
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