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Seasonal variation in C2H2 reduction (N2-fixation) in the litter layer of eucalypt forests of South-Western Australia
0038-0717,87 S3.00 + 0.00
Soil Biol. Biochem. Vol. 19. No. 2, pp. 135-142. 1987 Printed in Great Britain. All rights reserved
Copyright C 1987 PergamonJournals Ltd
SEASONAL VARIATION IN C2H, REDUCTION (N,-FIXATION) IN THE LITTER LAYER OF EUCALYPT FORESTS OF SOUTH-WESTERN AUSTRALIA A. M. O’CONNELL and T. S. GROVE Division of Forest Research, CSIRO,
Private Bag, P.O. Wembley 6014, Australia
(Accepted 20 August 1986)
Summary-Seasonal
variation in CrHr reduction by forest litter was determined at four sites in janah Donn ex Sm.) and karri (E. diuersicolor F. Muell.) forests in south-western Australia. Rates of CrHr reduction and microbial activity (CO? respiration) were measured every 4 weeks for I4 months on IO intact litter mats at each site. Mean monthly rates of C,H, reduction ranged from 0 to 24 nmol C,H, g-r litter day-’ in jarrah forest and from 0 to 56 nmol CrHr g-t litter day-’ in karri forest. Seasonal variation in rates of both C,H, reduction and CO2 respiration was related to variations in moisture content and temperature of the litter. C,H, reduction and CO, respiration were highest during the cool, moist winter and decreased to zero during the warm dry summer. Functions relating CrHr reduction to temperature and moisture content were derived from laboratory incubations of leaf litter under controlled conditions. These were used to develop models of the seasonal variation in Nrase activity based on field measurements of CrHr reduction, litter moisture content and temperature. The models demonstrate that moisture content of litter is the factor contributing most to seasonal variation in N,ase activity in jarrah and karri forests. Approximate amounts of NZ fixed annually in the litter layer were 38 and 49 mg N m-r yr-’ in jarrah forest and 149 and 257 mg N m-r yr-’ in karri forest. These are within the range of values reported for other forest ecosystems. (Eucalyprus
marginara
INTRODUCTION
Nz-fixation makes small but significant contributions to the N economy of many forest ecosystems. Accretions of up to 5 g N rn-’ yr-’ have been estimated (Richards, 1964) although in most ecosystems rates of non-symbiotic fixation are at least an order of magnitude less than this. Nitrogenase (N:ase) activity is frequently associated with microorganisms participating in the decay of tree boles and coarse woody material (Cornaby and Waide, 1973; Jurgensen ef al., 1984; Larsen et al., 1982) or with finer components of forest floor litter (Baker and Attiwill, 1984; Dierberg and Brezonik, 1981; Nioh, 1980; O’Connell ef al., 1979). N,-fixation by freeliving bacteria adds to the pool of N accumulated in the ecosystem, and probably also facilitates the breakdown of these nutrient-poor substrates by wood-rotting fungi (Silvester er al., 1982). Wet sclerophyll karri (Euculyptus diversicolor F. Much.) and dry sclerophyll jarrah (E. marginafo Donn ex Sm.) are two major commercial eucalypt forests occurring in south-western Australia. O’Connell et al. (1979) demonstrated significant N2ase activity in a number of components of the litter layer of these forests. To estimate annual inputs of non-symbiotically fixed N from such data requires, among other things, an understanding of the seasonal variation in NLase activity. The south-western region of Australia has a Mediterranean climate with cool wet winters and hot dry summers. This climate leads to marked seasonal fluctuations in forest floor temperature and moisture content. Such fluctuations are likely to have a significant influence on microbial Non-symbiotic
snn 191-a
activity (Flanagan and Bunnell, 1976) including the activity of free-living Nz-fixing organisms in the litter layer. Our aims were to: (i) Determine the seasonal variation in Nzase activity in the litter layer of jarrah and karri forest and relate this to total microbial activity in the same samples. (ii) Model the response of Nzase activity to changes in litter moisture content and temperature in order to explain the seasonal variation in enzyme activity. (iii) Compare the seasonal variation and annual rates of Nzase activity at four contrasting sites in jarrah and karri forest. (iv) Estimate approximate annual inputs of N to jarrah and karri forests from Nzase activity in the litter layer. ,WATERIALS AND METHODS
Site description
Experimental areas were located in jarrah and karri forests near the townships of Dwellingup (lat 32”47’S., long ll6’2’E.) and Manjimup (lat 34”14’S., long 116’9’E.) in south-western Australia (Table I). Two sites were situated in pole stand jarrah forest about 60-yr old. One of these sites had remained unburnt for 45 yr. The second site in adjacent forest had been regularly burnt by prescribed fires, the most recent of these being 6 yr before our study began. Karri forest sites were located in a mature I:35
136
A.
M. O’CONXZLL and T. S. GROVE
Table 1. Vegetation and litter characteristics of the four experimental sites Ovcrstorey Forest Jarrah Karri
Understorcy
Site
Density (stems ha-‘)
Basal area (m: ha-‘)
Density (stems ha-‘)
BUrlI Unburnt Regrowth Mature
1370 970 5400 47
25 49 I5 41
20 100 45.ooo 48.000
Basal area (m: ha-‘) Cl
cl I4 I5
Litter mass (t ha-‘) 14’ 31 I5 35
‘Estimated from O’Connell et al. (1978).
forest stand probably several hundred yr old and in regrowth forest Il-yr old. The mature forest was burnt 13 yr before the study. No fire had occurred in the regrowth forest since the burning of logging debris remaining when the site was clearfelled before establishment of the present stand. The two jarrah forest sites contained only a sparse understorey consisting mainly of Banksia grandis Willd. and Persoonia longifolia R. Br. In contrast there was a dense understorey of shrub species at both the mature and regrowth karri forest sites dominated in each case by Trymalium spathularum (Labill.) Ostf. and the legume Bossiaea luidfuniana Tovey and Morris. Field measurements
At each site I2 litter mats (area 0.07 m’) randomly located within a 50 x 50 m study plot were cut from the forest with a rectangular metal cutter. A metal plate was forced between the cutting edge of the sampler and the soil surface and the undisturbed litter mats were removed and placed in baskets 01 the same size made with stainless steel mesh (3 x 3 mm) sides and a terylene mesh (3 x 4 mm) base. Ttie baskets were placed on the forest floor with the litter mats in their original positions and the terylene mesh base in contact with the mineral soil. They were allowed to equilibrate for 6 weeks to reduce any effects of disturbance. Thereafter, every 4 weeks for I4 months, 10 of the baskets at each site were exposed in the field to C,H2 for 24 h in 8.61 gas tight boxes (pC2H2 = 0.2 atm). Two boxes, not exposed to C2H1, were used to determine endogenous C2H, productlon. Two additional boxes without litter were exposed to C,H, to determine background C2H, present in C2H,. Gas sub-samples were returned to the laboratory for C2H, and CO? analysis using gas chromatography. No significant endogenous CLH, production was detected. Rates of C2H, production reported for samples exposed to C,H2 are net rates after subtracting background CIH,. During exposure, sample boxes were protected from direct sunlight with covers made from aluminium insulation material. Between each sampling period the stainless steel baskets containing the litter mats were replaced in their original positions on the forest floor with their terylene mesh base in contact with the mineral soil. Litter maximum and minimum temperatures were measured at three locations at each site during the 24 h exposure. Litter moisture content was determined from two bulk samples each consisting of five litter cores (area 78.5 cm?) randomly located at each site. Dry weight of litter in each basket at the two karri sites and at the unburnt jarrah site was determined after the final measurements. Litter samples at the regularly burnt jarrah forest site were destroyed by fire after the last exposure. Litter weight at this site
was assumed to be the same as that found at a nearby area with similar fire history (O’Connell et al.. 1978). Effect of repeated exposure to C,H,
Since the measurement of seasonal variation in C2H2 reduction by jarrah and karri forest litter required incubation of the same litter samples each month, an experiment was performed to assess the effect of this procedure on N:ase activity in litter. A bulk sample of partially decomposed karri leaf litter was prepared by cutting leaves in pieces approximately I x I cm. The equivalent of 2 g dry wt of this litter was placed in each of 39 cylindrical fibreglass mesh containers (2 cm dia. 8cm long, mesh size 1.5 mm). The containers were inserted vertically into the forest floor at the mature forest site on a regular grid within an area measuring I x I m so that the top of each container just protruded from the litter layer. On the first sampling date three containers were selected randomly and exposed in 50ml tubes with C2HZ for 24 h (pC2Ht = 0.2 atm). Subsequently, once a month, previously exposed containers were exposed again. together with a further three randomly selected new containers. Sample containers were replaced in the forest floor following each measurement. After I3 months, when samples had been exposed from I to I3 times, there was no significant difference in the rate at which the sets of samples reduced C,H,. Laboratory studies
Grove et al. (1981) reported the effect of litter moisture content and temperature on rates of N,ase activity in jarrah and karri forest litter. Samples of leaf litter were exposed to C,H, in the laboratory over a range of moisture contents (10 to 210% oven dry wt) and temperatures (4-43°C) and the amounts of C2H, produced were determined. These data were used in the present study to develop models of the response of N,ase activity to changing moisture content and temperature. Statistical analysis
Rates of C,H, reduction and litter respiration were examined by analysis of variance of data stratified according to sites and sampling times. The relation of moisture content and temperature of litter to rates of C-H2 reduction (Grove er al., 1981) was examined king non-linear optimization curve fitting procedures. RESULTS Seasonal oariation in C,H, reduction and respiration
Seasonal variation in microbial activity (respiration) and N2ase activity (C2H2 reduction) were closely related to variation in litter moisture content and
Acetylene reduction in eucalypt forest litter
dOOr
dOr
Regrowlh
katri
Mature
kam
Fig. I. Seasonal variation in litter CJ4, reduction (I), respiration ([7), moisture content (A), maximum temperature (e) and minimum temperature (0) at the four experimental sites.
temperature (Fig. I ). Temperatures were high during summer (December-March) and low during winter (June-August) while litter moisture content was high in winter and low in summer. The range of minimum temperatures in litter was similar at jarrah and karri forest sites (5-WC) but maximum temperatures were lower in karri forest (13-36°C) than in jarrah forest (15-44X), probably partly because of shading of the forest floor by the dense understorey at the karri forest sites. Litter moisture content was generally higher in karri forest (maximum 238% oven dry wt) then in jarrah forest (maximum 172% oven dry wt) and the litter remained moist longer in karri forest. Litter moisture content was less than 50% oven dry
wt on half of the 14 sample periods at the jarrah forest sites but was less than 50% on only 2 and 5 sample periods at the mature and regrowth karri forest sites, respectively. These differences between the two forests are probably due to differences in the seasonal pattern of rainfall at the two locations and to the effects of understorey shading at the karri forest sites. Acetylene reduction was lowest during summer when moisture limited microbial activity and highest during spring when moisture contents were high and litter temperatures were rising (Fig. I). Microbial respiration followed similar seasonal patterns to C,H, reduction at each of the experimental sites.
A. M.~‘CONNELL and T. S. GROVE
138
Table’. .Mean rates ofacetylcnereduction (pmolm-*day-’ and nmoig-’ li!terday-‘)and respiration (gCO:m-‘day-’ and mg CO:g-’ titter day-‘) by litter at jarrah and karri forest sites Acetylene reduction Forest Jarrah Karri
Site
(pmol m-‘day-‘)
Burnt Unburnt Regrowth Mature
SE&l
Litter resoiration
(nmol g-‘day-‘)
11.4’ 14.5’ 44.3b 76.5~ 8.2
8.1”’ 2% 20.4b 2.7
(gCO:m-*day-‘) 0.97’ 1.3db I .3db 2.26’ 0.09
(mg CO? g-’ day-‘) 0.W 0.U 0.W 0.6Sb O.ti
‘Calculated assuming forest floor litter weight = I400 g m-’ (O’Connell et al., 1978). ‘Within each column. sites with the same superscript letter do not differ significantly (P < 0.05) by the Newman-Ktuls multiple range lest.
Significant (P c 0.001) site x time interactions for both microbial activity and Nrase activity was due primarily to the longer period during summer of limited respiration and C,H2 reduction at jarrah than karri forest sites. Differences between forest types in this period of limited activity are determined by the periods during which moisture deficit limits microbial activity. Mean daily areal rates of C,H, reduction (Jtmol m-‘day-‘) calculated for the period May f979-April 1980 decreased in the order mature karri forest > regrowth karri forest > unburnt jarrah forest > burnt jarrah forest (Table 2). When expressed per unit weight of litter (nmol C2Hz g-’ day-‘) N,ase activity decreased in the order regrowth karri > mature karri > burnt jarrah = unburnt jarrah. Similarly mean daily areal rates of microbial respiration (g COZ ms2 day-‘) decreased in
.
the order mature karri > regrowth karri = unburnt jarrah > burnt jarrah (Table 2). However when expressed per unit weight of litter (mg CO, g-’ day-‘) microbial respiration was greatest in litter at the regrowth karri forest site and lowest in litter at the unburnt jarrah forest site. Model of C2H2 reduction
Grove et al. (1981) established relationships between C2H2 reduction and the moisture content and temperature of litter in laboratory studies of Nzase activity of jarrah and karri forest litter. These data, together with monthly field measurements of maximum and minimum temperatures and moisture levels in the litter layer at the four study sites, were used in the present study to develop models of the seasonal variation in Nzase activity in the litter layer at each site. The relationship between litter moisture content and rate of CLH, reduction during laboratory incubation (Fig. 2) was described by the Gompertz function, Nzase activity = A exp[ B( I - e-C3’)/C]
(I) where A, B and C are constants and M is litter moisture content. This relationship explained 95 and 97% of the data variation for karri and jarrah litter, respectively (Table 3). Equation (I) was used to establish a moisture factor in the range 0.0-1.0 for each monthly field sampling period at each site from the relation I
250
M F = exp[B( 1 - e-‘,“‘)/C]/exp( B/C)
(2)
where M’ is the measured forest floor litter moisture content, and B and C are constants from equation (I). Variation of C,H, reduction with temperature during laboratory exposure of litter (Fig. 3) was modeiled with the function P-eQr Nzase activity = l+ReS’
100 140 Molstuce (I)
I
200
250
Fig. 2. Response of C$i, reduction by (a) jarrah and (b) karri leaf litter to variation in moisture content in labora-
tory incubation at 20°C. Fitted curves are Gompertz functions of the form N+asc activity = A exp[B(l - c-~“)/C] where kf = litter moikre content and A, B and C arc constants.
where P, Q, R and S are constants and 7’ is litter temperature. The denominator models the N,ase logistic function response curve and the numerator is a modifying factor which accounts for decrease in Nzase activity due to death of organisms or enzyme deactivation with increasing temperatures. P is the maximum substrate-limited N,ase activity for the litter sample in the absence of temperature constraints. The model expfained 93 and 97% of the data variation in the Nzase-temperature response
Acetylene
reduction
in eucalypt
forest litter
139
Table 3. Relation between (a) N,asc activity and moisture content and (b) N,a.w activity and temperature derived from laboratory incubations of jarrah and kani forest leaf litter (a) Moisture content’ Litter
type
Jarrah Karri
(b) Tcmperaturcz
A
B
C
12
P
Q
R
0.004 (0.005) 0.033 (0.075)
0.41 (0.07) 0.17 (0.07)
0.040 (0.002) 0.022 (0.004)
0.97 (n = 30) 0.96 (n = 30)
233.6 (8.3) 244.9 (14.5)
0.158 (0.002) 0.156 (0.002)
476.8 (270.3) 823.0 (761.7)
Standard errors in parentheses. ‘N,ase activity = A cxp[B(l - c-~“)/C],
quation
6
S -0.35 (0.03) -n 15 ---(0.05)
0.97 (n = 27) 0 93 (a -..; = -7)
(I)
P-CQ’
activity = -,
‘N,ax
+ RcSrsequation(3).
curves for karri and jarrah forest litter, respectively (Table 3). Approximate hourly field temperatures for each sample day at each site were generated from maximum and minimum litter temperatures using the relation
for i = I,. . . ,24 (France and Thornley, 1984). Hourly temperatures were used with equation (3) to establish a mean daily Nzase temperature factor in the range 0.0-l .O for each sample period at each site from the relation
7 5 loor
Jarrah
:.
.
A. :
0
where A_ is the maximum C2Hz reduction rate from the temperature-NZase response curve (Fig. 3). Variation in field measurements of C2H, reduction at each site in relation to measured environmental factors was examined through relationships of field Nzase activity and the moisture and temperature factors (Table 3) derived from equations (2) and (5), according to the relations
20 30 Temoerature 1-C)
i
40
50
.
Fig. 3. Response of CrHz reduction by (a) jarrah and (b) karri leaf litter to variation in temperature in laboratory incubations. Leaf moisture content c. 200%. Fitted curves are of the form Npse activity = (P - eQr)/(l + ReSr) where T = litter temperature and P. Q, R, S are constants.
(6)
N2ase activity = KL. MF’.TFfl
(7)
and
where K,, K1, a and b are constants. The linear model involving temperature and moisture content [equation (6)] explained from 38 to 88% of the seasonal variation in CrH, reduction at the four experimental sites (Table 4). The power function [equation (7)], in which data were grouped according to forest type, explained 91% of the seasonal variation in N,ase activity in both jarrah and karri forest litter. When TF was excluded from the relationships both the linear and power models still explained a large proportion of the data variance. The linear function involving MF only explained from 73 to 9 I % and the power function involving MF only explained from 87 to 90% of the seasonal variation in CzHL reduction.
ll!_l10
N,ase activity = K, *MF.TF
DISCUSSION
The experimental procedures we used were chosen to minimize spatial variability and errors due to disturbance of the litter. Although repeated exposure of NJlxing organisms to CrH, can influence measured rates of N,ase activity (David and Fay, 1977) we found no such effect in field assays of re-exposed leaf litter. The duration of assay can also affect C2H, reduction rates due to N-depletion in the bacteria (David and Fay, 1977) and changes in O2 concentrations during exposure (Grove ef nl.. 1981; Silvester et of., 1982). In the present study CO, concentrations in the incubation vessels (mostly < 1.0%) indicated that N,ase enhancement through Or depletion was likely to be minimal during the assays (Grove et al., 1981). We chose 24 h exposure periods because this allowed field assays to be conducted over the full diurnal temperature cycle. The time course of C2H, reduction by eucalypt litter is approximately linear over this period (Grove et al., 1981). Seasonal variation in N,ase activity of free-living microorganisms in decaying wood and litter has been
A. M.-GCO~XLL and T. S. GROM
140
Table 4. Relation between variation in monthly rates of N,asc activity and litter temperature factors and (b) and moisture factors according to the functions (a) N_+se= K,.MF.TF Npse = K,.MF’.TF? (b)
(a) Forest
Site
K,
r2
K,
B
12
0.47 (0.09)
0.34 (0.18)
0.91 (n = 28)
0.78 (0.13)
0.21 (0.07)
0.91 (n = 28)
0.82 (n - 14)
Burnt Jarrah Unburnt
z
IO5 (7)
0.88 (n = 14)
(l”o:
:4”;:
0.51 (n = 14)
118 (18)
;?)
0.38 (n = 14)
180 (25)
Regrowth Karri Mature
In the power model, data were grouped according to forest type. Standard errors in parentheses.
reported by Todd ef al. (1978), Roskoski (1980) and Baker and Attiwill(l984). These variations are generally associated with seasonal changes in substrate temperature or moisture content. Such is the case in the eucalypt forests of south-western Australia where the seasonal patterns of temperature and rainfall associated with the Mediterranean climate have a marked effect on Nrase activity in forest litter. Because of the climate, conditions for non-symbiotic C2H, reduction in the litter layer are sub-optimal at most times of the year. During winter, when litter moisture contents are high, activity is limited by low temperatures, while in summer when temperatures favour activity, low moisture conditions linilt rates of C,H2 reduction. The similarity of the patterns of CzHL reductions and CO, respiration (Fig. 1) indicates that the activity of Nz-fixing organisms reflects the activity of the total microbial population. Models relating field rates of CrH, reduction to litter temperature and moisture content demonstrate that moisture is the factor which contributes most to seasonal variation in N,ase activity of jarrah and karri forest litter. The effect of temperature on field measurements of CrH, reduction is smaller, probably because during the summer period when high temperatures favour N,ase activity, microbial activity is minimal because of the low moisture status of the litter. For forest systems which are more moist during the summer season, temperature conditions in the litter layer are likely to be more important in determining rates of N,ase activity. The linear model relating moisture and temperature factors to rate of C,HI reduction provided an adequate description of the seasonal variation in Nrase activity in jarrah forest. In this ecosystem, the marked seasonal pattern of litter moisture content resulted in clearly defined periods during the year when moisture was either limiting or adequate for C2Hz reduction. Thus measured rates of N,ase activity were either close to zero or to the maximum rate of activity. Few values were recorded between these extremes. In karri forest there was significant Nrase activity over longer periods during the year and measured rates of C,H, reduction covered the range of values from zero through to the maximum rates. For these data the linear model explained a lower proportion of the seasonal variation in Nzase activity than it did for jarrah forest sites. The more complex model involving power functions of the moisture and
temperature factors provided a good description of the variation in both jarrah and karri forest data. In this model the power function effectively modifies the shapes of the temperature and moisture response curves. Exponents less than unity increase the weighting given to the lower moisture and temperature factors compared to higher factors. In both the simple and power models, moisture factors were derived from laboratory measurements (Grove et al., 1981) of the response of N,ase activity to changing litter moisture content at a single temperature close to the optimum for C,H, reduction. Similarly, temperature factors were derived from the response of N,ase activity to changing temperature for litter with a moisture content close to the optimum for C2H, reduction. Results from the fit of the power function to field CrH, reduction data indicate that the shape of the temperature response curve may vary depending on the moisture content of the litter. Likewise, the shape of the moisture response curve may vary depending on the temperature of the litter. Such an interdependence of moisture and temperature has been found for microbial respiration in tundra decomposer systems (Flanagan and Bunnell, 1976). Mean monthly rates of CIH, reduction for jarrah forest litter ranged from 0 to 24 and 0 to I3 nmol g-r day-’ for the burnt and unburnt sites, respectively. These rates are similar to those reported for litter from Pinus rudiuta (O-25 nmol g-’ day-‘) and E. oblique (O-13 nmol g-‘day-‘) growing in eastern Australia (Baker and Attiwill, 1984) and for mixed hardwood litter (24 nmol g-’ day-‘) from North America (Comaby and Waide, 1973). Rates for regrowth karri (O-56 nmol g-’ day-‘) and mature karri (G-39 nmol g-l day-‘) forest litter are higher than for jarrah forest, probably because of the more favourable microclimate for microbial activity in the forest floor of the wet sclerophyll forest as compared to the more arid conditions in the litter layer of the dry sclerophyll forest. Differences in N,ase activity between sites within each forest type are probably due partly to differences in the weight and composition of the litter layers. Studies of eucalypt litter have shown that highest N,ase activities are associated with leaf components of litter and lowest activities with woody tissues and more highly decomposed fine litter (O’Connell et al., 1979). In jarrah forest the proportion of leaf litter in
Acetylene reduction in eucalypt forest litter the forest floor decreases with time of litter accumu-
cool prescribed
lation while the proportion of twigs and fine litter increases (O’Connell et al., 1978; O’Connell et al., 1979). In karri forest the proportion of woody material in annual litterfall increases with age of the stand (O’Connell and Menage, 1982), and the proportion of leaf litter in the forest floor decreases as the litter layer develops (O’Connell, 1987). Thus, in karri forest there is a greater proportion of leaf residues in litter in regrowth forest than in mature forest and this explains the higher Nzase activity per unit weight of litter in the regrowth forest. The higher N:ase activities per unit weight of litter at the regrowth karri site as compared to the mature karri site, and at the regularly burnt jarrah site as compared to the unburnt jarrah site, are compensated by differences in the mass of the litter layer at each site. The conversion factor of C2HL. reduction to N, fixation in studies of the N,ase activity of free-living microorganisms is uncertain. Comparison between studies are usually made on the basis of the theoretical conversion ratio of 3:l although, depending on the assay conditions, this can lead to an overestimate of rates of Nz-fixation (Jurgensen et al., 1984; Silvester et al., 1982). Annual rates of Nz fixation calculated assuming this ratio are 38 and 49 mg N m-2yr-1 for the regularly burnt and unburnt jarrah forest sites, respectively, and 149 and 257 mg N m-' yr-' for the regrowth and mature karri forest sites, respectively. Jarrah forest rates are similar to those estimated for litter&of P. rudiara (52 mg Nm-: yr-‘) and E. obliqua (19 mg N me2 yr-I) growing in eastern Australia (Baker and Attiwill, 1984) and for mixed hardwood litter (63 mg N m-? yr-‘) in North America (Todd er al., 1978). Rates of non-symbiotic Nz-fixation in karri forest litter are comparable with the highest values reported for conifers (216 mg N m-l yr-I) in Sweden (Granhall and Lindbcrg, 1980) but are lower than for
forests.
Japanese
cedar
(1205 mg N m-’ yr-‘;
Nioh,
1980).
However the latter result is probably an overestimate since assays were conducted at 30°C. The estimated rates of non-symbiotic N,-fixation in the litter layers of jarrah
(c. 40 mg N m-‘ yr-‘)
and
141
fires which periodically
bum these
REFERENCES
Baker T. Cl. and Attiwill P. M. (1984) Acetylene reduction in soil and litter from pine and eucalypt forests of south-eastern Australia. Soil Biology & Biochemistry 16, 241-245. Comaby B. W. and Waide J. B. (1973) Nitrogen fixation in decaying chestnut logs. Plum and Soil 39, 445-448. David K. A. V. and Fay P. (1977) Effects of long-term treatment with acetylene on nitrogen-fixing microorganisms. Applied and Environmental Microbiology 34, 640-646.
Die&erg F. E. and Brezonik P. L. (1981) Nitrogen fixation (acetylene reduction) associated with decaying leaves of pond cypress (Taxodium disrichum var. nu~ans) in a natural and a sewage-enriched cypress dome. Applied and Environmenfal Microbiology 41, 1413-1418.
Flanagan P. W. and Bunnell F. L. (1976) Decomposition models based on climatic variables, substrate variables, microbial respiration and production. In The Role of Terrestrial and Aquatic Organisms in Decomposition Pro cesses (J. M. Anderson and A. Macfadyen. Eds), pp.
437-457. Blackwell, Oxford. France J. and Thornley J. H. M. (1984) Mathematical Models in Agriculrure. Butterworths. London. Granhall U. and Lindberg T. (1980) Nitrogen input through biological nitrogen fixation. In Srrucrure, Conrenf and Function of Northern Coni/erous Forests-An Ecosystem Study (T. Persson. Ed). pp. 333-340. Ecological Bulletins 32, Stockholm.
Grove T. S., O’Connell A. M. and Malajczuk N. (1980) Effects of fire on the growth, nutrient content and rate of nitrogen fixation of the cycad Macrozamia riedlei. Ausrralian Journal of Botany 28, 271-28 I. Grove T. S. and Malajczuk N. (1981) Nitrogen inputs to Eucalyptus marginala and E. diuersicolor forests. In Managing the Nitrogen Economies of Natural and Man Made Forest Ecosystems (R. A. Rummery and F. J. Hingston,
Eds), pp. 199-204. CSIRO Division of Land Resources Management, Perth. Grove T. S., O’Connell A. M. and Malajczuk N. (1981) Effects of environmental factors and assay procedures on rates of acetylene reduction in eucalypt litter. In Managing the Nitrogen Economies of Natural and Man Made Forest Ecosystems (R. A. Rummery and F. J. Hingston,
karri (c. 200 mg N m-* yr-‘) forest are small relative for 6 yr jarrah forest litter contains about 5700 mg N me2 (O’Connell er al., 1978) and karri forest litter contains about 22,000 mg N m-l (Hingston ef ul., 1979). The rates of N,-fixation are also low compared to rates estimated for symbiotic N,-fixing plants. In jarrah forest Acacia pufcheflu (Hingston ef al., 1982) and Mucrozumiu riedlei (Grove et al., 1980) have been reported to each fix up to 600 mg N me2 yr-‘. In karri forest estimates of rates of N,-fixation by the leguminous understorey range from 600 to 1400 mg N me2 yr-’ (Grove and Malajczuk, 1981). However, although the amounts of N fixed nonsymbiotically in jarrah and karri forest litter are small they may be ecologically important in facilitating the decay of N-poor substrates such as occur in the litter layer and in logging debris. Non-symbiotically fixed N may be particularly important in the bio-
Hingston F. J., Dimmock G. M. and Turton A. G. (1981) Nutrient distribution in jarrah (Eucalyprus marginara Donn ex Sm.) ecosystems in south-west Western Australia. Forest Ecology and Management 3, 183-207. Hingston F. J., Malajczuk N. and Grove T. S. (1982) Acetylene reduction (N,-fixation) by jarrah forest legumes following fire and phosphate application. Journal of Applied Ecology 19, 63 l-645. Jurgensen M. F.. Larsen M. J., Spano S. D., Harvey A. E. and Gale M. R. (1984) Nitrogen fixation associated with increased wood decay in Douglas-fir residue. Forest Science 30, 1038-1044. Larsen M. J., Jurgensen M. F. and Harvey A. E. (1982) N,-fixation in brown-rotted soil wood in an intermountain cedar-hemlock ecosystem. Foresr Science 2.8,
degradation by fungi of large woody residues which contain N at very low concentrations (Hingston et al., 1981) and which are probably little affected by the
Nioh I. (1980) Nitrogen fixation associated with leaf litter of Japanese cedar (Crypfomeria japonica) of various
to the pool ot N tn Ittter. Atter accumulation
Eds), pp. 179-188. CSIRO Division of Land Resources Management, Perth. Hingston F. J., Turton A. G. and Dimmock G. M. (1979) Nutrient distribution in karri (Eucalypfus dicersicolor F. Muell.) ecosystems in south-west Western Australia. Fores1 Ecology and Management 2. 133-158.
292-296.
I42
A. M. O’CONNELLand T. S. GROM
decomposition stages. Soil Science and Plant Nutrition 26, 117-126.
O’Connell A. M., Grove T. S. and Dimmock G. M. (1978) Nutrients in litter on jarrah forest soils. Austrulian Journal of Ecology 3, W-260.
O’Connell A. M., Grove T. S. and Malajczuk N. (1979) Nitrogen fixation in the litter layer of eucalypt forests. Soil Biology ck Biochemistry 11, 681-682. O’Connell A. M. and Menage P. M. A. (1982) Litter fall and nutrient cycling in karri (Eucalyptus dirersicolor F. Muell.) forest in relation to stand age. Austroliun Journal of Ecology 7, 49-62.
O’Connell A. M. (1987) Litter dynamics in karri (Euculyptus dicersicolor F. Muell.) forests of south-western Australia. Journal of Ecology. In press.
Richards B. N. (1964) Fixation of atmospheric nitrogen in coniferous forests. Austruliun Forestry 28, 68-74. Roskoski J. P. (1980) Nitrogen fixation in hardwood forests in north-eastern United States. Plant and Soil 54. 33-44. S&ester W. B.. Sollins P.. Verhoeven T. and Cline S. P. (1982) Nitrogen fixation and acetylene reduction in decay ing conifer boles: effects of incubation time, aeration, and moisture content. Cunudiun Journul of Forest Reseurch 12, 646-652.
Todd R. L.. Meyer R. D. and Waide J. B. (1978) Nitrogen fixation in a deciduous forest in the south-eastern United States. In Enuironmentul Role of Nitrogenfixing Blue-green Algae und Asymbiotic Bucteriu (U. Granhall, Ed.), pp. 172477. Ecologicul Bulletins 26, Stockholm.
Soil Biol. Biochem. Vol. 19. No. 2, pp. 135-142. 1987 Printed in Great Britain. All rights reserved
Copyright C 1987 PergamonJournals Ltd
SEASONAL VARIATION IN C2H, REDUCTION (N,-FIXATION) IN THE LITTER LAYER OF EUCALYPT FORESTS OF SOUTH-WESTERN AUSTRALIA A. M. O’CONNELL and T. S. GROVE Division of Forest Research, CSIRO,
Private Bag, P.O. Wembley 6014, Australia
(Accepted 20 August 1986)
Summary-Seasonal
variation in CrHr reduction by forest litter was determined at four sites in janah Donn ex Sm.) and karri (E. diuersicolor F. Muell.) forests in south-western Australia. Rates of CrHr reduction and microbial activity (CO? respiration) were measured every 4 weeks for I4 months on IO intact litter mats at each site. Mean monthly rates of C,H, reduction ranged from 0 to 24 nmol C,H, g-r litter day-’ in jarrah forest and from 0 to 56 nmol CrHr g-t litter day-’ in karri forest. Seasonal variation in rates of both C,H, reduction and CO2 respiration was related to variations in moisture content and temperature of the litter. C,H, reduction and CO, respiration were highest during the cool, moist winter and decreased to zero during the warm dry summer. Functions relating CrHr reduction to temperature and moisture content were derived from laboratory incubations of leaf litter under controlled conditions. These were used to develop models of the seasonal variation in Nrase activity based on field measurements of CrHr reduction, litter moisture content and temperature. The models demonstrate that moisture content of litter is the factor contributing most to seasonal variation in N,ase activity in jarrah and karri forests. Approximate amounts of NZ fixed annually in the litter layer were 38 and 49 mg N m-r yr-’ in jarrah forest and 149 and 257 mg N m-r yr-’ in karri forest. These are within the range of values reported for other forest ecosystems. (Eucalyprus
marginara
INTRODUCTION
Nz-fixation makes small but significant contributions to the N economy of many forest ecosystems. Accretions of up to 5 g N rn-’ yr-’ have been estimated (Richards, 1964) although in most ecosystems rates of non-symbiotic fixation are at least an order of magnitude less than this. Nitrogenase (N:ase) activity is frequently associated with microorganisms participating in the decay of tree boles and coarse woody material (Cornaby and Waide, 1973; Jurgensen ef al., 1984; Larsen et al., 1982) or with finer components of forest floor litter (Baker and Attiwill, 1984; Dierberg and Brezonik, 1981; Nioh, 1980; O’Connell ef al., 1979). N,-fixation by freeliving bacteria adds to the pool of N accumulated in the ecosystem, and probably also facilitates the breakdown of these nutrient-poor substrates by wood-rotting fungi (Silvester er al., 1982). Wet sclerophyll karri (Euculyptus diversicolor F. Much.) and dry sclerophyll jarrah (E. marginafo Donn ex Sm.) are two major commercial eucalypt forests occurring in south-western Australia. O’Connell et al. (1979) demonstrated significant N2ase activity in a number of components of the litter layer of these forests. To estimate annual inputs of non-symbiotically fixed N from such data requires, among other things, an understanding of the seasonal variation in NLase activity. The south-western region of Australia has a Mediterranean climate with cool wet winters and hot dry summers. This climate leads to marked seasonal fluctuations in forest floor temperature and moisture content. Such fluctuations are likely to have a significant influence on microbial Non-symbiotic
snn 191-a
activity (Flanagan and Bunnell, 1976) including the activity of free-living Nz-fixing organisms in the litter layer. Our aims were to: (i) Determine the seasonal variation in Nzase activity in the litter layer of jarrah and karri forest and relate this to total microbial activity in the same samples. (ii) Model the response of Nzase activity to changes in litter moisture content and temperature in order to explain the seasonal variation in enzyme activity. (iii) Compare the seasonal variation and annual rates of Nzase activity at four contrasting sites in jarrah and karri forest. (iv) Estimate approximate annual inputs of N to jarrah and karri forests from Nzase activity in the litter layer. ,WATERIALS AND METHODS
Site description
Experimental areas were located in jarrah and karri forests near the townships of Dwellingup (lat 32”47’S., long ll6’2’E.) and Manjimup (lat 34”14’S., long 116’9’E.) in south-western Australia (Table I). Two sites were situated in pole stand jarrah forest about 60-yr old. One of these sites had remained unburnt for 45 yr. The second site in adjacent forest had been regularly burnt by prescribed fires, the most recent of these being 6 yr before our study began. Karri forest sites were located in a mature I:35
136
A.
M. O’CONXZLL and T. S. GROVE
Table 1. Vegetation and litter characteristics of the four experimental sites Ovcrstorey Forest Jarrah Karri
Understorcy
Site
Density (stems ha-‘)
Basal area (m: ha-‘)
Density (stems ha-‘)
BUrlI Unburnt Regrowth Mature
1370 970 5400 47
25 49 I5 41
20 100 45.ooo 48.000
Basal area (m: ha-‘) Cl
cl I4 I5
Litter mass (t ha-‘) 14’ 31 I5 35
‘Estimated from O’Connell et al. (1978).
forest stand probably several hundred yr old and in regrowth forest Il-yr old. The mature forest was burnt 13 yr before the study. No fire had occurred in the regrowth forest since the burning of logging debris remaining when the site was clearfelled before establishment of the present stand. The two jarrah forest sites contained only a sparse understorey consisting mainly of Banksia grandis Willd. and Persoonia longifolia R. Br. In contrast there was a dense understorey of shrub species at both the mature and regrowth karri forest sites dominated in each case by Trymalium spathularum (Labill.) Ostf. and the legume Bossiaea luidfuniana Tovey and Morris. Field measurements
At each site I2 litter mats (area 0.07 m’) randomly located within a 50 x 50 m study plot were cut from the forest with a rectangular metal cutter. A metal plate was forced between the cutting edge of the sampler and the soil surface and the undisturbed litter mats were removed and placed in baskets 01 the same size made with stainless steel mesh (3 x 3 mm) sides and a terylene mesh (3 x 4 mm) base. Ttie baskets were placed on the forest floor with the litter mats in their original positions and the terylene mesh base in contact with the mineral soil. They were allowed to equilibrate for 6 weeks to reduce any effects of disturbance. Thereafter, every 4 weeks for I4 months, 10 of the baskets at each site were exposed in the field to C,H2 for 24 h in 8.61 gas tight boxes (pC2H2 = 0.2 atm). Two boxes, not exposed to C2H1, were used to determine endogenous C2H, productlon. Two additional boxes without litter were exposed to C,H, to determine background C2H, present in C2H,. Gas sub-samples were returned to the laboratory for C2H, and CO? analysis using gas chromatography. No significant endogenous CLH, production was detected. Rates of C2H, production reported for samples exposed to C,H2 are net rates after subtracting background CIH,. During exposure, sample boxes were protected from direct sunlight with covers made from aluminium insulation material. Between each sampling period the stainless steel baskets containing the litter mats were replaced in their original positions on the forest floor with their terylene mesh base in contact with the mineral soil. Litter maximum and minimum temperatures were measured at three locations at each site during the 24 h exposure. Litter moisture content was determined from two bulk samples each consisting of five litter cores (area 78.5 cm?) randomly located at each site. Dry weight of litter in each basket at the two karri sites and at the unburnt jarrah site was determined after the final measurements. Litter samples at the regularly burnt jarrah forest site were destroyed by fire after the last exposure. Litter weight at this site
was assumed to be the same as that found at a nearby area with similar fire history (O’Connell et al.. 1978). Effect of repeated exposure to C,H,
Since the measurement of seasonal variation in C2H2 reduction by jarrah and karri forest litter required incubation of the same litter samples each month, an experiment was performed to assess the effect of this procedure on N:ase activity in litter. A bulk sample of partially decomposed karri leaf litter was prepared by cutting leaves in pieces approximately I x I cm. The equivalent of 2 g dry wt of this litter was placed in each of 39 cylindrical fibreglass mesh containers (2 cm dia. 8cm long, mesh size 1.5 mm). The containers were inserted vertically into the forest floor at the mature forest site on a regular grid within an area measuring I x I m so that the top of each container just protruded from the litter layer. On the first sampling date three containers were selected randomly and exposed in 50ml tubes with C2HZ for 24 h (pC2Ht = 0.2 atm). Subsequently, once a month, previously exposed containers were exposed again. together with a further three randomly selected new containers. Sample containers were replaced in the forest floor following each measurement. After I3 months, when samples had been exposed from I to I3 times, there was no significant difference in the rate at which the sets of samples reduced C,H,. Laboratory studies
Grove et al. (1981) reported the effect of litter moisture content and temperature on rates of N,ase activity in jarrah and karri forest litter. Samples of leaf litter were exposed to C,H, in the laboratory over a range of moisture contents (10 to 210% oven dry wt) and temperatures (4-43°C) and the amounts of C2H, produced were determined. These data were used in the present study to develop models of the response of N,ase activity to changing moisture content and temperature. Statistical analysis
Rates of C,H, reduction and litter respiration were examined by analysis of variance of data stratified according to sites and sampling times. The relation of moisture content and temperature of litter to rates of C-H2 reduction (Grove er al., 1981) was examined king non-linear optimization curve fitting procedures. RESULTS Seasonal oariation in C,H, reduction and respiration
Seasonal variation in microbial activity (respiration) and N2ase activity (C2H2 reduction) were closely related to variation in litter moisture content and
Acetylene reduction in eucalypt forest litter
dOOr
dOr
Regrowlh
katri
Mature
kam
Fig. I. Seasonal variation in litter CJ4, reduction (I), respiration ([7), moisture content (A), maximum temperature (e) and minimum temperature (0) at the four experimental sites.
temperature (Fig. I ). Temperatures were high during summer (December-March) and low during winter (June-August) while litter moisture content was high in winter and low in summer. The range of minimum temperatures in litter was similar at jarrah and karri forest sites (5-WC) but maximum temperatures were lower in karri forest (13-36°C) than in jarrah forest (15-44X), probably partly because of shading of the forest floor by the dense understorey at the karri forest sites. Litter moisture content was generally higher in karri forest (maximum 238% oven dry wt) then in jarrah forest (maximum 172% oven dry wt) and the litter remained moist longer in karri forest. Litter moisture content was less than 50% oven dry
wt on half of the 14 sample periods at the jarrah forest sites but was less than 50% on only 2 and 5 sample periods at the mature and regrowth karri forest sites, respectively. These differences between the two forests are probably due to differences in the seasonal pattern of rainfall at the two locations and to the effects of understorey shading at the karri forest sites. Acetylene reduction was lowest during summer when moisture limited microbial activity and highest during spring when moisture contents were high and litter temperatures were rising (Fig. I). Microbial respiration followed similar seasonal patterns to C,H, reduction at each of the experimental sites.
A. M.~‘CONNELL and T. S. GROVE
138
Table’. .Mean rates ofacetylcnereduction (pmolm-*day-’ and nmoig-’ li!terday-‘)and respiration (gCO:m-‘day-’ and mg CO:g-’ titter day-‘) by litter at jarrah and karri forest sites Acetylene reduction Forest Jarrah Karri
Site
(pmol m-‘day-‘)
Burnt Unburnt Regrowth Mature
SE&l
Litter resoiration
(nmol g-‘day-‘)
11.4’ 14.5’ 44.3b 76.5~ 8.2
8.1”’ 2% 20.4b 2.7
(gCO:m-*day-‘) 0.97’ 1.3db I .3db 2.26’ 0.09
(mg CO? g-’ day-‘) 0.W 0.U 0.W 0.6Sb O.ti
‘Calculated assuming forest floor litter weight = I400 g m-’ (O’Connell et al., 1978). ‘Within each column. sites with the same superscript letter do not differ significantly (P < 0.05) by the Newman-Ktuls multiple range lest.
Significant (P c 0.001) site x time interactions for both microbial activity and Nrase activity was due primarily to the longer period during summer of limited respiration and C,H2 reduction at jarrah than karri forest sites. Differences between forest types in this period of limited activity are determined by the periods during which moisture deficit limits microbial activity. Mean daily areal rates of C,H, reduction (Jtmol m-‘day-‘) calculated for the period May f979-April 1980 decreased in the order mature karri forest > regrowth karri forest > unburnt jarrah forest > burnt jarrah forest (Table 2). When expressed per unit weight of litter (nmol C2Hz g-’ day-‘) N,ase activity decreased in the order regrowth karri > mature karri > burnt jarrah = unburnt jarrah. Similarly mean daily areal rates of microbial respiration (g COZ ms2 day-‘) decreased in
.
the order mature karri > regrowth karri = unburnt jarrah > burnt jarrah (Table 2). However when expressed per unit weight of litter (mg CO, g-’ day-‘) microbial respiration was greatest in litter at the regrowth karri forest site and lowest in litter at the unburnt jarrah forest site. Model of C2H2 reduction
Grove et al. (1981) established relationships between C2H2 reduction and the moisture content and temperature of litter in laboratory studies of Nzase activity of jarrah and karri forest litter. These data, together with monthly field measurements of maximum and minimum temperatures and moisture levels in the litter layer at the four study sites, were used in the present study to develop models of the seasonal variation in Nzase activity in the litter layer at each site. The relationship between litter moisture content and rate of CLH, reduction during laboratory incubation (Fig. 2) was described by the Gompertz function, Nzase activity = A exp[ B( I - e-C3’)/C]
(I) where A, B and C are constants and M is litter moisture content. This relationship explained 95 and 97% of the data variation for karri and jarrah litter, respectively (Table 3). Equation (I) was used to establish a moisture factor in the range 0.0-1.0 for each monthly field sampling period at each site from the relation I
250
M F = exp[B( 1 - e-‘,“‘)/C]/exp( B/C)
(2)
where M’ is the measured forest floor litter moisture content, and B and C are constants from equation (I). Variation of C,H, reduction with temperature during laboratory exposure of litter (Fig. 3) was modeiled with the function P-eQr Nzase activity = l+ReS’
100 140 Molstuce (I)
I
200
250
Fig. 2. Response of C$i, reduction by (a) jarrah and (b) karri leaf litter to variation in moisture content in labora-
tory incubation at 20°C. Fitted curves are Gompertz functions of the form N+asc activity = A exp[B(l - c-~“)/C] where kf = litter moikre content and A, B and C arc constants.
where P, Q, R and S are constants and 7’ is litter temperature. The denominator models the N,ase logistic function response curve and the numerator is a modifying factor which accounts for decrease in Nzase activity due to death of organisms or enzyme deactivation with increasing temperatures. P is the maximum substrate-limited N,ase activity for the litter sample in the absence of temperature constraints. The model expfained 93 and 97% of the data variation in the Nzase-temperature response
Acetylene
reduction
in eucalypt
forest litter
139
Table 3. Relation between (a) N,asc activity and moisture content and (b) N,a.w activity and temperature derived from laboratory incubations of jarrah and kani forest leaf litter (a) Moisture content’ Litter
type
Jarrah Karri
(b) Tcmperaturcz
A
B
C
12
P
Q
R
0.004 (0.005) 0.033 (0.075)
0.41 (0.07) 0.17 (0.07)
0.040 (0.002) 0.022 (0.004)
0.97 (n = 30) 0.96 (n = 30)
233.6 (8.3) 244.9 (14.5)
0.158 (0.002) 0.156 (0.002)
476.8 (270.3) 823.0 (761.7)
Standard errors in parentheses. ‘N,ase activity = A cxp[B(l - c-~“)/C],
quation
6
S -0.35 (0.03) -n 15 ---(0.05)
0.97 (n = 27) 0 93 (a -..; = -7)
(I)
P-CQ’
activity = -,
‘N,ax
+ RcSrsequation(3).
curves for karri and jarrah forest litter, respectively (Table 3). Approximate hourly field temperatures for each sample day at each site were generated from maximum and minimum litter temperatures using the relation
for i = I,. . . ,24 (France and Thornley, 1984). Hourly temperatures were used with equation (3) to establish a mean daily Nzase temperature factor in the range 0.0-l .O for each sample period at each site from the relation
7 5 loor
Jarrah
:.
.
A. :
0
where A_ is the maximum C2Hz reduction rate from the temperature-NZase response curve (Fig. 3). Variation in field measurements of C2H, reduction at each site in relation to measured environmental factors was examined through relationships of field Nzase activity and the moisture and temperature factors (Table 3) derived from equations (2) and (5), according to the relations
20 30 Temoerature 1-C)
i
40
50
.
Fig. 3. Response of CrHz reduction by (a) jarrah and (b) karri leaf litter to variation in temperature in laboratory incubations. Leaf moisture content c. 200%. Fitted curves are of the form Npse activity = (P - eQr)/(l + ReSr) where T = litter temperature and P. Q, R, S are constants.
(6)
N2ase activity = KL. MF’.TFfl
(7)
and
where K,, K1, a and b are constants. The linear model involving temperature and moisture content [equation (6)] explained from 38 to 88% of the seasonal variation in CrH, reduction at the four experimental sites (Table 4). The power function [equation (7)], in which data were grouped according to forest type, explained 91% of the seasonal variation in N,ase activity in both jarrah and karri forest litter. When TF was excluded from the relationships both the linear and power models still explained a large proportion of the data variance. The linear function involving MF only explained from 73 to 9 I % and the power function involving MF only explained from 87 to 90% of the seasonal variation in CzHL reduction.
ll!_l10
N,ase activity = K, *MF.TF
DISCUSSION
The experimental procedures we used were chosen to minimize spatial variability and errors due to disturbance of the litter. Although repeated exposure of NJlxing organisms to CrH, can influence measured rates of N,ase activity (David and Fay, 1977) we found no such effect in field assays of re-exposed leaf litter. The duration of assay can also affect C2H, reduction rates due to N-depletion in the bacteria (David and Fay, 1977) and changes in O2 concentrations during exposure (Grove ef nl.. 1981; Silvester et of., 1982). In the present study CO, concentrations in the incubation vessels (mostly < 1.0%) indicated that N,ase enhancement through Or depletion was likely to be minimal during the assays (Grove et al., 1981). We chose 24 h exposure periods because this allowed field assays to be conducted over the full diurnal temperature cycle. The time course of C2H, reduction by eucalypt litter is approximately linear over this period (Grove et al., 1981). Seasonal variation in N,ase activity of free-living microorganisms in decaying wood and litter has been
A. M.-GCO~XLL and T. S. GROM
140
Table 4. Relation between variation in monthly rates of N,asc activity and litter temperature factors and (b) and moisture factors according to the functions (a) N_+se= K,.MF.TF Npse = K,.MF’.TF? (b)
(a) Forest
Site
K,
r2
K,
B
12
0.47 (0.09)
0.34 (0.18)
0.91 (n = 28)
0.78 (0.13)
0.21 (0.07)
0.91 (n = 28)
0.82 (n - 14)
Burnt Jarrah Unburnt
z
IO5 (7)
0.88 (n = 14)
(l”o:
:4”;:
0.51 (n = 14)
118 (18)
;?)
0.38 (n = 14)
180 (25)
Regrowth Karri Mature
In the power model, data were grouped according to forest type. Standard errors in parentheses.
reported by Todd ef al. (1978), Roskoski (1980) and Baker and Attiwill(l984). These variations are generally associated with seasonal changes in substrate temperature or moisture content. Such is the case in the eucalypt forests of south-western Australia where the seasonal patterns of temperature and rainfall associated with the Mediterranean climate have a marked effect on Nrase activity in forest litter. Because of the climate, conditions for non-symbiotic C2H, reduction in the litter layer are sub-optimal at most times of the year. During winter, when litter moisture contents are high, activity is limited by low temperatures, while in summer when temperatures favour activity, low moisture conditions linilt rates of C,H2 reduction. The similarity of the patterns of CzHL reductions and CO, respiration (Fig. 1) indicates that the activity of Nz-fixing organisms reflects the activity of the total microbial population. Models relating field rates of CrH, reduction to litter temperature and moisture content demonstrate that moisture is the factor which contributes most to seasonal variation in N,ase activity of jarrah and karri forest litter. The effect of temperature on field measurements of CrH, reduction is smaller, probably because during the summer period when high temperatures favour N,ase activity, microbial activity is minimal because of the low moisture status of the litter. For forest systems which are more moist during the summer season, temperature conditions in the litter layer are likely to be more important in determining rates of N,ase activity. The linear model relating moisture and temperature factors to rate of C,HI reduction provided an adequate description of the seasonal variation in Nrase activity in jarrah forest. In this ecosystem, the marked seasonal pattern of litter moisture content resulted in clearly defined periods during the year when moisture was either limiting or adequate for C2Hz reduction. Thus measured rates of N,ase activity were either close to zero or to the maximum rate of activity. Few values were recorded between these extremes. In karri forest there was significant Nrase activity over longer periods during the year and measured rates of C,H, reduction covered the range of values from zero through to the maximum rates. For these data the linear model explained a lower proportion of the seasonal variation in Nzase activity than it did for jarrah forest sites. The more complex model involving power functions of the moisture and
temperature factors provided a good description of the variation in both jarrah and karri forest data. In this model the power function effectively modifies the shapes of the temperature and moisture response curves. Exponents less than unity increase the weighting given to the lower moisture and temperature factors compared to higher factors. In both the simple and power models, moisture factors were derived from laboratory measurements (Grove et al., 1981) of the response of N,ase activity to changing litter moisture content at a single temperature close to the optimum for C,H, reduction. Similarly, temperature factors were derived from the response of N,ase activity to changing temperature for litter with a moisture content close to the optimum for C2H, reduction. Results from the fit of the power function to field CrH, reduction data indicate that the shape of the temperature response curve may vary depending on the moisture content of the litter. Likewise, the shape of the moisture response curve may vary depending on the temperature of the litter. Such an interdependence of moisture and temperature has been found for microbial respiration in tundra decomposer systems (Flanagan and Bunnell, 1976). Mean monthly rates of CIH, reduction for jarrah forest litter ranged from 0 to 24 and 0 to I3 nmol g-r day-’ for the burnt and unburnt sites, respectively. These rates are similar to those reported for litter from Pinus rudiuta (O-25 nmol g-’ day-‘) and E. oblique (O-13 nmol g-‘day-‘) growing in eastern Australia (Baker and Attiwill, 1984) and for mixed hardwood litter (24 nmol g-’ day-‘) from North America (Comaby and Waide, 1973). Rates for regrowth karri (O-56 nmol g-’ day-‘) and mature karri (G-39 nmol g-l day-‘) forest litter are higher than for jarrah forest, probably because of the more favourable microclimate for microbial activity in the forest floor of the wet sclerophyll forest as compared to the more arid conditions in the litter layer of the dry sclerophyll forest. Differences in N,ase activity between sites within each forest type are probably due partly to differences in the weight and composition of the litter layers. Studies of eucalypt litter have shown that highest N,ase activities are associated with leaf components of litter and lowest activities with woody tissues and more highly decomposed fine litter (O’Connell et al., 1979). In jarrah forest the proportion of leaf litter in
Acetylene reduction in eucalypt forest litter the forest floor decreases with time of litter accumu-
cool prescribed
lation while the proportion of twigs and fine litter increases (O’Connell et al., 1978; O’Connell et al., 1979). In karri forest the proportion of woody material in annual litterfall increases with age of the stand (O’Connell and Menage, 1982), and the proportion of leaf litter in the forest floor decreases as the litter layer develops (O’Connell, 1987). Thus, in karri forest there is a greater proportion of leaf residues in litter in regrowth forest than in mature forest and this explains the higher Nzase activity per unit weight of litter in the regrowth forest. The higher N:ase activities per unit weight of litter at the regrowth karri site as compared to the mature karri site, and at the regularly burnt jarrah site as compared to the unburnt jarrah site, are compensated by differences in the mass of the litter layer at each site. The conversion factor of C2HL. reduction to N, fixation in studies of the N,ase activity of free-living microorganisms is uncertain. Comparison between studies are usually made on the basis of the theoretical conversion ratio of 3:l although, depending on the assay conditions, this can lead to an overestimate of rates of Nz-fixation (Jurgensen et al., 1984; Silvester et al., 1982). Annual rates of Nz fixation calculated assuming this ratio are 38 and 49 mg N m-2yr-1 for the regularly burnt and unburnt jarrah forest sites, respectively, and 149 and 257 mg N m-' yr-' for the regrowth and mature karri forest sites, respectively. Jarrah forest rates are similar to those estimated for litter&of P. rudiara (52 mg Nm-: yr-‘) and E. obliqua (19 mg N me2 yr-I) growing in eastern Australia (Baker and Attiwill, 1984) and for mixed hardwood litter (63 mg N m-? yr-‘) in North America (Todd er al., 1978). Rates of non-symbiotic Nz-fixation in karri forest litter are comparable with the highest values reported for conifers (216 mg N m-l yr-I) in Sweden (Granhall and Lindbcrg, 1980) but are lower than for
forests.
Japanese
cedar
(1205 mg N m-’ yr-‘;
Nioh,
1980).
However the latter result is probably an overestimate since assays were conducted at 30°C. The estimated rates of non-symbiotic N,-fixation in the litter layers of jarrah
(c. 40 mg N m-‘ yr-‘)
and
141
fires which periodically
bum these
REFERENCES
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Die&erg F. E. and Brezonik P. L. (1981) Nitrogen fixation (acetylene reduction) associated with decaying leaves of pond cypress (Taxodium disrichum var. nu~ans) in a natural and a sewage-enriched cypress dome. Applied and Environmenfal Microbiology 41, 1413-1418.
Flanagan P. W. and Bunnell F. L. (1976) Decomposition models based on climatic variables, substrate variables, microbial respiration and production. In The Role of Terrestrial and Aquatic Organisms in Decomposition Pro cesses (J. M. Anderson and A. Macfadyen. Eds), pp.
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Grove T. S., O’Connell A. M. and Malajczuk N. (1980) Effects of fire on the growth, nutrient content and rate of nitrogen fixation of the cycad Macrozamia riedlei. Ausrralian Journal of Botany 28, 271-28 I. Grove T. S. and Malajczuk N. (1981) Nitrogen inputs to Eucalyptus marginala and E. diuersicolor forests. In Managing the Nitrogen Economies of Natural and Man Made Forest Ecosystems (R. A. Rummery and F. J. Hingston,
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karri (c. 200 mg N m-* yr-‘) forest are small relative for 6 yr jarrah forest litter contains about 5700 mg N me2 (O’Connell er al., 1978) and karri forest litter contains about 22,000 mg N m-l (Hingston ef ul., 1979). The rates of N,-fixation are also low compared to rates estimated for symbiotic N,-fixing plants. In jarrah forest Acacia pufcheflu (Hingston ef al., 1982) and Mucrozumiu riedlei (Grove et al., 1980) have been reported to each fix up to 600 mg N me2 yr-‘. In karri forest estimates of rates of N,-fixation by the leguminous understorey range from 600 to 1400 mg N me2 yr-’ (Grove and Malajczuk, 1981). However, although the amounts of N fixed nonsymbiotically in jarrah and karri forest litter are small they may be ecologically important in facilitating the decay of N-poor substrates such as occur in the litter layer and in logging debris. Non-symbiotically fixed N may be particularly important in the bio-
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A. M. O’CONNELLand T. S. GROM
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