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Molybdenum limitation of asymbiotic nitrogen fixation in forests of Pacific Northwest America
Soil Bid. Biochem. Vol. ?I, No. 2. pp. ?83-289, Printed in Great Britain. All rights reserved
1989
0038-0717 89 S3.00 + 0.00 Copyright C 1989 Pergamoo Press pk
MOLYBDENUM LIMITATION OF ASYMBIOTIC NITROGEN FIXATION IN FORESTS OF PACIFIC NORTHWEST AMERICA w.
B.
SILVESTER*
Department of Forest Science, Oregon State University, Corvallis. OR 97331, U.S.A. 27 June 1988)
(Accepted
Summary-Nitrogenase activity as measured by acetylene reduction and nitrogen fixation as measured by 15N, uptake is widespread in forest litter, decaying wood and the lichen Lobaria in Pacific Northwest forests. In all cases the activity of nitrogenase is enhanced by addition of plant nutrient solutions and this is shown to be a specific molybdenum effect. A wide range of forest species have litter which supports nitrogen fixation during decay and the majority show molybdenum stimulation. The effect is most prominent in the acid soils situated between the Coast Range and the Cascade Range and extends from
Oregon through Washington to British Columbia. Nitrogenase is increased quantitatively by MO additions and the effect, though best seen in the laboratory, is also measurable in the field. The results are discussed on the basis of geographical distribution of the effect, plant species and the likely role of asymbiotic nitrogen fixation in contributing to long-term nutrient cycling.
INTRODUCTION Asymbiotic N2 fixation in forests was first reported by Henry (1897) who showed oak and hornbeam leaves accumulated considerable nitrogen during incubation in the laboratory. This work attracted little attention, but was finally confirmed by Olsen (1932) whose careful experimental work proved that decaying leaves may support significant N, fixation as evidenced by a very real increase in total nitrogen. Since then a considerable body of work has shown that asymbiotic Nz fixation is commonly associated with the decay of a variety of natural organic substrates in forests (Tjepkema, 1979; Roskoski, 1980; Silvester et al., 1982; Todd er al., 1975). The lowland and mid-altitude forests of the Pacific Northwest are dominated by Douglas-fir [Pseudorsuga menziesii (Mirb) France] which forms an extensive natural feature of the vegetation. Net N accumulation in these forests has been estimated in long term chronosequence studies as 0.24.4 kgN ha-’ yr-’ (Gessel et nf., 1973) and, while some of this N input is due to nodulated plants such as Alms. a large proportion of the N accumulation occurs in the absence of obvious N, fixing plants. Asymbiotic N, fixation has been identified in decaying wood and litter on the forest floor (Silvester et al., 1982) and symbiotic nitrogen fixation in species of the lichen Loburiu in the forest canopy (Horstmann et uf.. 1982). Detailed analysis of N cycling in old growth Douglas-fir forests (Sollins ef al., 1980) shows an annual loss of 1.5 kg N ha-’ yr-’ and a net N accumulation of 5.0 kg N ha-’ yr-‘. While a slight deficiency of MO has been reported for the Pacific Northwest (Rubins, 1956) it has gener-
*Present address: Department of Biological Sciences, University of Waikato, Hamilton, New Zealand. 283
ally been in the eastern states where dramatic MO responses have been achieved. In Oregon, Dawson and Bhella (1972) reported a legume response to MO addition in western hill soils with an increase in clover production of l-20%. The present work had, as its major objective, the identification of major sites of asymbiotic nitrogen fixation and an investigation of the factors limiting this activity. MATERIALS AND METHODS
The major study site was located at 12OOm elevation along Woods Creek Road on Mary’s Peak in the Oregon Coast Range about IS km southwest of Corvallis, Oregon. The site contains stands of old growth (300-500 yr) Douglas-fir and varying amounts of western hemlock [Tsugu heferuphyllu (Raf.) Sarg.]. Second growth Douglas-fir, c. 30 years old, was also sampled in this area. Unless otherwise stated all test material came from these sites. Other species were collected in this area and from throughout Oregon and Washington States and into British Columbia. Litter or wood was normally added to septum capped bottles in the field, acetylene added to give 0.1 atm. maintained at field temperature in a polystyrene box, and returned to the laboratory for analysis. Materials for nutrient incubation were returned to the laboratory and placed in large polythene bags, loosely tied to allow air circulation but maintain high moisture, and incubated in the laboratory at 22 + 3°C. Nutrient solutions (Bollard, 1966) were added at half normal concentration either as a complete sohrtion but lacking N (-N). or as individual elements. Solutions or water were sprayed into each bag to saturate the litter and incubated as above.
284
w.
8.
SILVZSTER
Table I. Nitrogenar activity in decaying litter of various Iret species in Pacific Northwest forests. In all cases F layer only was sampled (for common names see Table 7)
Species
Sample number
Mean nitrogenam activity (nmolC,H,g-‘h-i)
3 I
15.2 13.0
4
9.2
Thuja pktta
ACW mocrophyilum Abier
grandis
Pseudotsuga caxijolia
ta
Jwlipenicoccidentalir
5.6
Abies omabilis
I
2.7
Pbn8.s pomierosa
4
2.0
Picea engelmrurnii
I
1.6
L.&x
2
0.6
Tsuga hcterophylla
4
0.6
Tsuga mertensiano
I
0.3
Pinur edulif
I
0.3
Pinus contorta
1
0
Afnur n&a
3
0
Pseudotsugat Toga PseudotsugalP.
7
heterophy{~a
Bark SaP Heart Branch Snag Sap Heart Heart Humus
111 IV Hemlock
Y;
Sap Heart
09s 0.5-2.8 0 0.4 0.4 0 W.1 O-o.2 0 O-Q.2 0
‘Decay class; in which class I is recently fallen and class V is an ill-defined humic mound. “A zero result indicates levels below an arbitrary detection limit of 0.1 nmol g-’ h“.
2.2
:
0
Acetylene reduction activity (ARA) was measured by adding 0.1 atm C,H, (generated from CaC,) to septum top bottles, incubating at room temperature and analysing for C,H, using a Hewlett Packard 5830A gas chromatograph. Acetylene was used as an internal standard in all calculations and time courses were run over a 6 h period. Endogenous C2H, production was regularly checked, as were background CrH, levels; rates reported here are net CZH, reduction after subtraction of background CzH, m all cases and based on dry weight. Nitrogen fixation was measured by “N, uptake and used to calibrate ARA. Purified “N, gas (95 atom% 15N) excess was obtained from Hornet Co. and mixed with 0.2 atm Oz. This gas was added to duplicate vials of MO treated and control leaf material and incubated for 8 h. Parallel triplicate sampies were analysed for ARA over the initial 3 h period. Gas samples were removed from the ‘jN, incubations and analysed for ambient “N, levels so that an absolute measure of activity could be obtained. Details of techniques used and final analytical procedures have been published previously (Silvester et al., 1982; S&ester, 1983). RESULTS
of asymbiotic
Typt
II
Douglas-fir
Nitrogenam activity (nmolcs CrH,g-’ h-‘1
0.2
contorta
PseudotsugajAbtus
Extent
Species
D=aY class’
7.7
2
occidentaL
Table 2. Nitrogenam activity of decaying wood. The results show the range of ARA based on at least IO samnla oer tyw
nitrogen
Jixation
Within mature forest systems of the Pacific North-
west three major sites of N2 fixation have been identified. Firstly, many tests have shown that a variety of species produce litter which supports nitrogen fixation during the decay process. Average levels of Nzase activity in forest litter range from zero in several species including the pines and alder to over I5 nmol C2HI g-’ h-’ in the F layer of red cedar litter (Table 1). The major forest type of this area is Douglas-fir and litter sampled from this forest invariably shows N,ase activity. Activity was greatest in the top F layer and in moiest material natural N,ase activity ranged from 1.0-36 nmol C2HIg-’ h-‘. When other, low activity, species are associated with Douglas-fir, the nitrogenase activities are significantly reduced (Table 1). A second important site of nitrogen fixation is in decaying wood. Considerable activity is found in lower decay classes of Douglas-fir wood with negligible activity in hemlock (Table 2). These results are considered in greater detail elsewhere (Silvester et at., 1982). Finally, species of the lichen Lobaria are widespread in Douglas-fir forest and are active in N2 fixation (Horstmann ef al., 1982). Effect
of added nutrients
Douglas-fir F litter was treated with -N (complete nutrients minus nitrogen) and phosphate (+P) SO~Utions and N,ase activity tested after 48 h (Table 3). A significant stimulation was recorded with addition of -N solution to both replicates. Replicate 2 in each case was reincubated for 48 h with -N solution giving a similar stimulation of N,ase (Table 3).
Table 3. E&IX of added nutrients on nitrogenax activity of Douglas-fir leaf litter. Two rcplicatcr each weft treated (Treated 1) with water (control). phosphate (+ P) and full nutrients (-N) solutions, incubated for 48 h and nitrogenax rates (nmol CrH,g-’ h-‘) measured for 3 h. All replicates were then incubated for 48 h after retreatment BJ shown in Treatment 2 and further tested for nitrogenax Treatment 2
Treatment I Rtulicate
Treatment
C
N.art
Treatment
Ntam
c
C
1.6 1.0
-N
1.3 3.8
+P +P
1.1 I.2
C -N
I.1 4.0
-N -N
4.6 5.1
C -N
4.0 4.5
Molybdenum
limitation
of nitrogen fixation
2as
Table 4. Effect on nitrogenax activity of various S salts and a comolete micronutrient mix (S at . 32 mg’l-’ in each case) Nitrogenax activity (nmolC,H.n-’ he’)
Treatment
2.1 a.2 2.2 2.3 1.9 9.1
Control -N MgSO, &SO, CaSO, Micronutrients
Table 5. E&t of various micronutrients in nitrogcnase activity of decaying Douglas-fir litter Nitrogenax activity (nmolC,H.g-’ h-‘1
Treatment
4.1 IS.2 5.2 4.9 4.8 19.6 3.2 5.1 3.3 4.2
Control Micronutrients Zn co B MO Fe CU c”
111 38 16
11
33 L2
I
I
0L
170
MO addition pg kg-’
It was confirmed (Table 4) that all of the N2ase enhancement was due to micronutrient additions. Neither sulphur, a known deficiency in Oregon, nor cations provided any stimulation over control. Of the micronutrients in the complete micronutrient solution, only MO had any effect (Table 5) and all nutrient stimulation of Nrase is due to MO. In general, enhancement of Nrase activity by MO addition, for the litter site studied, was consistently 3to 4-fold (Tables 3.4 and 5) regardless of the control rate. Time course and quantitative effects
Nitrogenase activity rises to a maximum at 30-40 h after MO addition, while control material retains a constant Nrase activity in the laboratory (Fig. I). In this experiment an extremely high stimulation ratio of 6.8 was achieved. N,ase activity shows a quantitative linear response to added MO (Fig. 2). Three days after MO addition a straight line relationship is shown between Nzase
Fig. 2. Response of nitrogenase activity in decaying Douglas-fir litter to addition of different amounts of MO. MO addition in pg element kg-’ dry wt of litter.
and added MO over the range 0-6Opg kg-‘. The distinct change in slope of this line after 25 days indicates that MO is still being taken up over this period and the saturation value drops correspondingly to c. 3Opg kg-‘. In the laboratory the enhanced N,ase activity is maintained over a long period. When Nrase was assayed at 3 and 25 days after MO addition a similar stimulation ratio of 4.5-4.6 was achieved (Fig. 2). In this case both control Nrase and MO treated N,ase had increased between 3 and 25 days but the stimulation ratio was maintained.
“N, uptake The effect of MO addition was tested using isN, uptake with parallel acetylene reduction assays. “N, confirmed that N, fixation is stimulated by MO addition (Table 6). While the stimulation of N, fixation was small in this particular experiment this was also mirrored in the acetylene reduction assay. The molar ratio figures fall within a normal range of values and no particular significance should be attached to the fact that values for + MO appear higher than controls as it is unlikely that the greater stimulation of acetylene reduction over Nr fixation is significant. Species, site and jield results
OO
I
I
I
1
10
16
22
50
Hours after MO addition Fig. I. Time course of nitrogenase activity in decaying Douglas-fir litter addition of MO.
Litter from a wide range of habitats, of mainly conifer species was tested for Nrase activity and MO stimulation. Both Nrase activity, and MO enhancement, was shown to be widespread (Table 7). Stimulation ratios up to 6.26 have beeen recorded from field material and even where control rates are high, such as the eastern slopes of the Coast Range in
W.
286
B. SIL~EXER
Table 6. Effect of MO addition to decaying Douglas-fir litter on ‘rN, uptake and ARA. Duplicate samples for “Nt uptake are shown and mean of three samples for ARA “N, uptake (nmol;L’g-’ h -‘)
Treatment Control +M0
I
2.74
2
2.86
I 2
of nitrogenax
Molar ratio
IO.I
3.69 3.53
3.17 4.15
17.2
4.56 4.14
1.5
1.7
Oregon, MO enhancement is significant. These results also show nitrogenase rate for an incubation in full nutrient -N solution, in some cases this incubation gave higher results than MO alone, whereas earlier experiments (Tables 4 and 5) indicated that all enhancement was due to MO. Further work is needed Table 7. MO enhancement
C:Ht reduction (nmolC,H,g-‘h-l)
to determine whether other inorganic nutrients may be limiting Nrase and over what time course. The geographic range of MO stimulation is generally confined to the area between the peaks of the Coast range and the Cascade Range (Fig. 3, Table 7). Decaying Douglas-fir wood was also tested for
activity in litter of various species at different See Fig. 3 for approximate locations
locations
in the Pacific Northwest.
Nitrogenase (nmolC,H,g-‘h-‘) Location
E. Slopes Coast Range, Oregon
w. slopes cascades Oregon
E. slopes cascades Oregon Southern Oregon
Eastern
Oregon
Washington
Vancouver Vancouver ‘Ag.
1d.E. 1d.W.
Litter’ species
Age (yr)
PC Pt Pt Pt Pt Pt A mat TP Pt (wood) Pt&Th Pt&Th Pt
60 30 30 200+ 200 400+ IS0 200+ 400 400 + 200+ :8
TP Pt+Th Pe Am Pl Pl Pt + Pp Pt Pt PI PC Lo Ag PP Jo Ag Pp+Ag Jo Pl Pr+Pp Pl Pt Pt Pl PI PttTp PI Pt Tp. Pt mixed
:: 100 moo+ 75+ 100 80 80 80 200+ 200+ 100 I50 4Of 50+ 40+
I 50 200 200 loo+ 4(M 200+ 35 40 40 40 30 I20 80 800 400
Elevation (m) 20 450 450 3M) 500 350 1150 500 450 600 850 700 1000 850 900 900 II00 I200 I200 650 650 650 I300 I300 IO00 800
IO00 IO00 850 I200 I600 I600 400 I600 1000 22 600 50 50 50 80 130 I20 30 30
+Mo %N
Control
+Mo
1.27 I.51 I.14 I .72 I .48 I.54 1.57 I.53 0.04 1.39 ND I.54 1.23 1.04 I.20 0.66 I.19 I .04 1.28 I.22 0.83 I .43 I .36 I.17 I .44 0.79 0.63 I .29 0.90 I.32 I .30 I.13 0.98 I .43 1.44 1.62 I.13 I.31 1.79 I .39 I .36 I.13 1.10 I.17 0.86
14.9 8.5 15.7 26.7 7.3 27.7 13.0 18.6 I .46 6.64 9.03 I.5 0.89 4.3 52 IS.2 0.95 2.6 3.0 5.6 8.83 I.9 2.9 2.7 2.7 4.0 0.6 14.7 1.3 9.8 7.3 Il.7 1.3 7.2 0.41 12.6 6.2 7. I 6.3 1.7 2.3 7.7 19.5 2.5 1.0
42.9 23.7 55.0 46.5 22.4 48.8 18.2 62.8 2.60 259 9.34 4.30 5.57 21.0 14.7 22.9 1.10 0.9 1.4 12.6 Il.3 5.2 2.0 4.6 2.2 6.2 0.6 23. I I.3 10.9 8.1 10.7 0.9 7.2 0.40 22.9 7.6 10.8 8.2 9.2 II.5 20.5 38.9 2.5 1.0
-N 40.7 22. I 42.8 58.6 21.9 65. I 12.2 69. I ND 18.3 6.3 4.13 7.02 30.8 27.3 33.6 I .20 1.5 I.6 Il.3 I I.3 3.1 3.2 4.3 I.8 5.1 0.6 18.4 1.0 18.1 IO.9 Il.2 I.1 Il.8 0.40 Il.4 4.8 Il.5 3.7 2.2 13.8 8.2 31.5 2.5 I .o
-MO 2.88 2.79 3.50 I .74 3.07 1.76 I .40 3.38 I .80 3.90 I .03 2.86 6.26 4.88 2.83 1.26 I.16 0.35 0.50 2.25 1.28 2.74 0.70 1.70 0.80 I.55 1.00 1.57 1.00 I.11 I.11 0.9 I 0.70 1.00 1.00 I .82 I .23 I.52 I .30 5.4 5.0 2.66 I .99 1.00 1.00
grand fir; Am. Abies magnifica red fir; Amac. Act-r mocrophyllum big leaf maple: Jo. Juniperus occidenralis juniper; Lo. Lcrix occidenrolis western larch; Pe. Picecr engelmannii Engelmann spruce; Pp. Pinus ponderoso ponderosa pine: Pt. Pseudorsugo taxfolia Douglas fir; Tp, Thuja plicoto Western red cedar; Th, Tsugo hererophylla Western hemlock. Abies grandis
western
Molybdenum
BRITISH COLUMBIA
Vancouver I.
+
\
Vancouver
J’
?5aik
------;-Seattle
#
I
I
WASHINGTON
\
OREGON
/
9
-a_______________
t
I
287
limitation of nitrogen fixation
: ---
+ Positive Mo response o No MO response
Fig. 3. Distribution of MO response on nitrogenase activity across northwest America.
MO enhancement. Eight replicates of control and MO treated wood were incubated for 48 h and assayed for N,ase showing a stimulation from 1.46 to 2.60 nmol C,H,g-’ h-’ significant at 1% level (Table 7). All the above results were obtained in the laboratory under conditions in which moisture and temperature were optimal. To test whether there is a real field MO deficiency a field trial was conducted in which 1 mz areas were pegged out under old growth Douglas-fir. MO solution. (500 pg I-‘) or distilled water (control) was sprayed to give a final MO concentration in the litter layer of 25Opg kg-‘. Material sampled 30 and 40 days after MO addition and incubated in the field showed statistically significant stimulation ratios of 1.65 and 2.48 (Table 8). It appears that MO deficiency is a real field phenomenon and as in the laboratory, a MO stimulation of N,ase can be sustained over a long period. DISCUSSION
The results produced in this study rely heavily on acetylene reduction as the assay for nitrogenase and this reliance has come under some criticism in recent years (Giller, 1987). The technique has a variety of limitations including possible spurious ethylene production from soil or organic matter and derepression of nitrogenase in the presence of the alternative substrate acetylene. We have previously addressed the problem of derepression which presents considerable complications when measuring low ARA over long periods (Silvester et al., 1982) and have shown
that incubations up to I2 h are reasonable and have limited ours in this work to 6 h. Spurious ethylene production in the presence of acetylene presents more difficulties because of the variable effects that can be obtained. While a variety of methods have been tried to assess this effect (Giller, 1987) I contend that the ultimate test of nitrogenase is either increase in total N or “N? uptake. We have verified ARA with “N, uptake from a variety of similar substrates, e.g. decaying Agathis leaves (Silvester, 1978) decaying wood (Silvester et al., 1982) and decaying mangrove leaves (Hicks and Silvester, 1985) along with the present work. I am confident the results presented represent realistic measures of nitrogenase. Further confirmation of this lies in the specific stimulation of activity by MO for which there is a specific and well acknowledged role in nitrogenase. Thus while it may be argued that a proportion, albeit a very small proportion, of measure activity may be due to non-specific ethylene production the “N, uptake and specific MO response confirm that there is a very real effect on nitrogenase. Mature conifer forests show significant adaptation to low nutrient regimes, generally requiring fewer nutrient inputs per unit wood production than do hardwood species (Waring and Franklin 1979. Gosz, 1981). This is achieved by efficient internal recycling of elements in conifers, retention of foliage and low nutrient concentrations in foliage. Overall nutrient budgets for old-growth Douglas-fir have been calculated in detail (Sollins et al., 1980) and estimated as a net increase in nitrogen of 5.0 kg N ha-’ yr-‘. Over much longer periods, which include several generations of forest, a net nitrogen accumulation is still struck, of 0.2-0.4 kg N ha-’ yr-’ (Gessel et al., 1973). Nitrogen inputs into the system include precipitation 2.0, nitrogen fixing lichens 2.8 and an unknown input of 1.7 kg N ha-’ yr-’ (Sollins et al., 1980). At least a proportion of this unknown input is accounted for by nitrogen fixation in decaying wood (Silvester et al., 1982) while a proportion is due to nitrogen fixation in litter. While it is not possible at this stage to accurately estimate the likely contribution of litter N2 fixation to total ecosystem N, it is likely to be of the same order as coming from decaying wood, i.e. c. I kg N ha-’ yr-‘. The absence of nitrogen fixation in the litter of some species, notably the pines and the relatively low levels recorded here for hemlock raises some interesting questions on the long term nitrogen status of these forests. It is notable also that N2 fixation in
Table 8. Stimulation of nitrogenase in the field. MO solution (500 cg I -‘) warsprayed onto Douglas-fir litterin the field to a final conccnrralion of 250 pg MO kg-’ litter dry wt. Core samplesof litter wre collecwd al various times after MO addition and incubated in C:H, immediaaly at field wmperawe and moisture. Differences significant at 1% level Days after MO addition 30 40 Litter cemperawe (^C) N:asc activity control +Mo +Mo -MO
9 15.3 25.3
I .65
I2 5.2 12.9 2.48
288
W.
B.
decaying wood of hemlock is also low (Silvester et al., 1982; Table 2). From the limited field surveys of natural N2 fixation in litter, three groups of activity stand out. Those species with consistently high rates, e.g. 2X&r pficatu. those with inte~~iate rates typified by Douglas-fir and those with low or zero rates, typified by the pines and hemiocks. In terms of nutrient requirements this grouping exactly matches the nutrient requirements independently assigned to these species by Gost (1981). In his review Gosz states “pines were distinguished from the rest of the conifers as a group having low requirements , , . Hemlocks have some of the lowest known nutrient requirements.. . while members of the Cupressaceae (Chumaecyparis norfhafensis and Thuju plicafu) have very high requirements, . . A number of species are intermediate (e.g. Douglas-fir)“. The above statements refer generally to nutrient requirement, more particularly to nitrogen requirement and also mirror the preference of the plant for NO; or NH,’ sources of N with the high N requiring species preferring NOT-the low N requiring species preferring NH: and the intermediate species using both forms. Any relationship between nutrient requirements, site requirements, general nutrition and N, fixation in plant residues can only remain speculative at the moment. However a working hypothesis is likely to revolve around the presence of inhibitory substances in the residues of species in poor nutrition. The links in this chain are reviewed by Gosz (1981) and in essence relate to the production of leaf polyphenols which both inhibit microbial activity in decay and also serve to lock up plant nutrients, particularly nitrogen. bringing about mor formation. Another factor can now be added to this complex array in conifer organic cycling. Molybdenum is obviously strongly limiting the rate of nitrogen fixation on some sites and these are identified as the more acid sites which are also associated with strong mor formation. From the work described above it appears that molybdenum availability is yet another factor in the complex of events relating to organic cycling in conifer forests. The question of micronutrient deficiencies in forests has seldom been raised but moly~enum deficiency, once identi~ed in crops and pasture has been one of the more easily defined and treated micronutrient limitations of plant production. Specific requirement for the element in NO> reduction and nitrogen fixation have long been established and crop limitations are readily rectified by addition of MO salts to other fettiiisers. The role of MO in nitrogen fixation was first identified by Bortels (1930) when he showed stimulation of growth of A~tobucrer chroococcum in culture by addition of trace sodium molybdate. It is interesting, in the context of the present study, that the first recorded field deficiency of MO was in asymbiotic NZ fixation on the West Coast of U.S.A. In this study Van Niel(1935) reported that soilsfrom the Monterey Peninsula would yield Azofobucfer isolates only after addition of molybdenum to the soil. Since that time many examples of MO limitation of legume production have been studied and rectified. but very little work on asymbiotic nitrogen fixing organisms has been reported.
S~~as'rm
Within North America there is a trend in reported MO requirement from mild deficiencies in the west to sometimes acute deficiencies in the eastern states (Rubins. 19%). Despite this, a number of reports. cited by Rubins (1956), report significant MO deficiencies in California, Oregon and Washington and early work in Oregon (Anon., 19%) showed that coastal and Eastern Oregon alkaline soils showed no MO response, while in the central valley, between the Coast and Cascade Ranges. MO responses were reported for hi11 soils and acid valley tfoor soils. The responses to MO reported in the present paper are consistent with the known and putative distribution of MO deficiency, i.e. all soils on which MO deficiency is found are acid and, in general, under conifer forest which enhances this acidity. It has been pointed out by Rubins (1956) that the total MO concentration in soils is relatively uniform at 2 ppm but that the level of available MO varies with pH. Thus under acid conditions free MO is low while under alkaline conditions it is possible for MO toxicity to be expressed in grazing and foraging animals. In the absence of any measured values for molybdenum in conifer leaves it is difficult to speculate as to whether it is merely the Iack of available MO in leaves which is reducing NL fixation or whether total MO is low. Walker (personal communication) using the Aspergillus assay has shown that a number of soils under Douglas-fir forest are deficient or slightly deficient in molybdenum but that none of the soils tested are in the very deficient range (i.e.
Molybdenum limitation 1of nitrogen fixation Acknowledgements-This
work was supported by grants from the National Science Foundation. The work was greatly facilitated by discussions with Drs K. Cromack, J. C. Gordon, P. Sollins and R. H. Waring. Laboratory assistance from Jan Silvester and Tom Verhoeven is gratefully acknowledged. The Pacific Northwest Forest and Range Experiment Station kindly supplied laboratory space and facilities. Isotopic nitrogen analyses were carried out by Dr B. B. McInteer, Los Alamos Scientific Laboratories. REFERENCES Anon. (1958) The Molybdenum Problem in Oregon. Miscellaneous Paper 64. Agriculture Experiment Station, Oregon State University, Corvallis. Oregon, U.S.A. Bollard E. G. (1966) A comparative study of the ability of organic nitrogenous compounds to serve as sole sources of nitrogen for the growth of plants. Planr and Soil 25, 153-166.
Bortels H. (1930) Molybdenum as a catalyst in the biological fixation of nitrogen. Archive fir Mikrobiologie I, 333-342.
Dawson M. D. and Bhella H. S. (1972) Subterranean clover (Tri/olium subterranean) yield and nutrient content as influenced by soil molybdenum status. Agronomy Journal 64, 308-31 I. Gessel S. P.. Cole D. W. and Steinbrenner E. C. (1973) Nitrogen balances in forest ecosystems of the Pacific Northwest. Soil Biology & Biochemistry 5, 19-34. Giller K. E. (1987) Use and abuse of acetylene reduction assay for measurement of associative nitrogen fixation. Soil Biology & Biochemisrry 19, 783-784. Gosz J. R. (1981) Nitrogen cycling in coniferous ecosystems. In Terrestrial Nitrogen Cycles: Processes, Ecosystem Strategies and Management Impam (Mark F. E. and Roswall T., Eds). Ecological Bulletins Stockholm 33, 405426. Henry E. (1897) L’azote et la vegetation forestiere. Revue dtaux et firers 36, 64 I.
Hicks B. J. and Silvester W. B. (1985) Nitrogen fixation associated with the New Zealand mangrove (Auicennia marina (Forsk.) Vierh. var. resini/era (F0rst.f.) Bakh) Applied and Encironmental Microbiology 49. 955-959.
Horstmann
J. C., Dennison W. C. and Silvester W. B.
289
(1982) “Nt fixation and molybdenum enhancement of acetylene reduction by Labaria spp. New Phyrologirr 92, 235-242.
Olsen C. (1932) Studies of nitrogen fixation I. Nitrogen fixation in the dead leaves of forest beds. Comptes-rendus du laboratoire Carlsberg 19, l-36.
Roskoski J. P. (1980) Nitrogen fixation in hardwood forests of the northeastern United States. Plum and Soil 54, 33-34.
Rubins E. J. (1956) Molybdenum deficiencies in the United States. Soil Science 81, 191-197. Silvester W. B. (1978) Nitrogen fixation and mineralisation in kauri (Agothis austrolis) forest in New Zealand. In Microbial Ecology (W. M. Loutit and J. A. R. Miles, Eds), pp. 138-143. Springer, Berlin. Silvester W. B. (1983) Analysis of nitrogen fixation in forest ecosystems. In Biological Nitrogen Fixation in Forest Ecosystems. Foundations and Applicafions (J. C. Gordon and C. T. Wheeler, Eds), pp. 173-212. Nijhoff/Junk, The Hague. Silvester W. B., Sollins P., Verhoeven T. and Cline S. P. (1982) Nitrogen fixation and acetylene reduction in decaying conifer boles: effects of incubation time aeration, and moisture content. Canadian Journal of Forest Research 12. 646-652.
Sollins P., Grier C. C.. McCorison F. M.. Cromack K., Fogel R. and Fredericksen R. L. (1980) The internal element cycles of an old-growth Douglas-fir ecosystem in western Oregon. Ecological Monographs SO, 261-285. Tjepkema J. (1979) Nitrogen fixation in forests of Central Massachusetts. Canadian Journal of Botany 57, I I-16. Todd R. L., Waide J. B. and Cornaby B. W. (1975) Significance of biological nitrogen fixation and denitrification in a deciduous forest ecosystem. In Mineral Cycling in Southeastern Ecosystems (F. G. Howell, J. B. Gentry and M. H. Smith, Eds). pp. 729-735. U.S. Energy Research and Development Symposium CONF-740513. National Technical Information Services...-.Sorinelield. Vermont, U.S.A. Van Niel C. B. (1935) A note on the apparent absence of Azofobacrer in soils. Archive/ur Mikrobiologie 6,2 15-2 18. Waring R. H. and Franklin J. F. (1979) Evergreen coniferous forests of the Pacific northwest. Science 204, 1380-1386.
1989
0038-0717 89 S3.00 + 0.00 Copyright C 1989 Pergamoo Press pk
MOLYBDENUM LIMITATION OF ASYMBIOTIC NITROGEN FIXATION IN FORESTS OF PACIFIC NORTHWEST AMERICA w.
B.
SILVESTER*
Department of Forest Science, Oregon State University, Corvallis. OR 97331, U.S.A. 27 June 1988)
(Accepted
Summary-Nitrogenase activity as measured by acetylene reduction and nitrogen fixation as measured by 15N, uptake is widespread in forest litter, decaying wood and the lichen Lobaria in Pacific Northwest forests. In all cases the activity of nitrogenase is enhanced by addition of plant nutrient solutions and this is shown to be a specific molybdenum effect. A wide range of forest species have litter which supports nitrogen fixation during decay and the majority show molybdenum stimulation. The effect is most prominent in the acid soils situated between the Coast Range and the Cascade Range and extends from
Oregon through Washington to British Columbia. Nitrogenase is increased quantitatively by MO additions and the effect, though best seen in the laboratory, is also measurable in the field. The results are discussed on the basis of geographical distribution of the effect, plant species and the likely role of asymbiotic nitrogen fixation in contributing to long-term nutrient cycling.
INTRODUCTION Asymbiotic N2 fixation in forests was first reported by Henry (1897) who showed oak and hornbeam leaves accumulated considerable nitrogen during incubation in the laboratory. This work attracted little attention, but was finally confirmed by Olsen (1932) whose careful experimental work proved that decaying leaves may support significant N, fixation as evidenced by a very real increase in total nitrogen. Since then a considerable body of work has shown that asymbiotic Nz fixation is commonly associated with the decay of a variety of natural organic substrates in forests (Tjepkema, 1979; Roskoski, 1980; Silvester et al., 1982; Todd er al., 1975). The lowland and mid-altitude forests of the Pacific Northwest are dominated by Douglas-fir [Pseudorsuga menziesii (Mirb) France] which forms an extensive natural feature of the vegetation. Net N accumulation in these forests has been estimated in long term chronosequence studies as 0.24.4 kgN ha-’ yr-’ (Gessel et nf., 1973) and, while some of this N input is due to nodulated plants such as Alms. a large proportion of the N accumulation occurs in the absence of obvious N, fixing plants. Asymbiotic N, fixation has been identified in decaying wood and litter on the forest floor (Silvester et al., 1982) and symbiotic nitrogen fixation in species of the lichen Loburiu in the forest canopy (Horstmann et uf.. 1982). Detailed analysis of N cycling in old growth Douglas-fir forests (Sollins ef al., 1980) shows an annual loss of 1.5 kg N ha-’ yr-’ and a net N accumulation of 5.0 kg N ha-’ yr-‘. While a slight deficiency of MO has been reported for the Pacific Northwest (Rubins, 1956) it has gener-
*Present address: Department of Biological Sciences, University of Waikato, Hamilton, New Zealand. 283
ally been in the eastern states where dramatic MO responses have been achieved. In Oregon, Dawson and Bhella (1972) reported a legume response to MO addition in western hill soils with an increase in clover production of l-20%. The present work had, as its major objective, the identification of major sites of asymbiotic nitrogen fixation and an investigation of the factors limiting this activity. MATERIALS AND METHODS
The major study site was located at 12OOm elevation along Woods Creek Road on Mary’s Peak in the Oregon Coast Range about IS km southwest of Corvallis, Oregon. The site contains stands of old growth (300-500 yr) Douglas-fir and varying amounts of western hemlock [Tsugu heferuphyllu (Raf.) Sarg.]. Second growth Douglas-fir, c. 30 years old, was also sampled in this area. Unless otherwise stated all test material came from these sites. Other species were collected in this area and from throughout Oregon and Washington States and into British Columbia. Litter or wood was normally added to septum capped bottles in the field, acetylene added to give 0.1 atm. maintained at field temperature in a polystyrene box, and returned to the laboratory for analysis. Materials for nutrient incubation were returned to the laboratory and placed in large polythene bags, loosely tied to allow air circulation but maintain high moisture, and incubated in the laboratory at 22 + 3°C. Nutrient solutions (Bollard, 1966) were added at half normal concentration either as a complete sohrtion but lacking N (-N). or as individual elements. Solutions or water were sprayed into each bag to saturate the litter and incubated as above.
284
w.
8.
SILVZSTER
Table I. Nitrogenar activity in decaying litter of various Iret species in Pacific Northwest forests. In all cases F layer only was sampled (for common names see Table 7)
Species
Sample number
Mean nitrogenam activity (nmolC,H,g-‘h-i)
3 I
15.2 13.0
4
9.2
Thuja pktta
ACW mocrophyilum Abier
grandis
Pseudotsuga caxijolia
ta
Jwlipenicoccidentalir
5.6
Abies omabilis
I
2.7
Pbn8.s pomierosa
4
2.0
Picea engelmrurnii
I
1.6
L.&x
2
0.6
Tsuga hcterophylla
4
0.6
Tsuga mertensiano
I
0.3
Pinur edulif
I
0.3
Pinus contorta
1
0
Afnur n&a
3
0
Pseudotsugat Toga PseudotsugalP.
7
heterophy{~a
Bark SaP Heart Branch Snag Sap Heart Heart Humus
111 IV Hemlock
Y;
Sap Heart
09s 0.5-2.8 0 0.4 0.4 0 W.1 O-o.2 0 O-Q.2 0
‘Decay class; in which class I is recently fallen and class V is an ill-defined humic mound. “A zero result indicates levels below an arbitrary detection limit of 0.1 nmol g-’ h“.
2.2
:
0
Acetylene reduction activity (ARA) was measured by adding 0.1 atm C,H, (generated from CaC,) to septum top bottles, incubating at room temperature and analysing for C,H, using a Hewlett Packard 5830A gas chromatograph. Acetylene was used as an internal standard in all calculations and time courses were run over a 6 h period. Endogenous C2H, production was regularly checked, as were background CrH, levels; rates reported here are net CZH, reduction after subtraction of background CzH, m all cases and based on dry weight. Nitrogen fixation was measured by “N, uptake and used to calibrate ARA. Purified “N, gas (95 atom% 15N) excess was obtained from Hornet Co. and mixed with 0.2 atm Oz. This gas was added to duplicate vials of MO treated and control leaf material and incubated for 8 h. Parallel triplicate sampies were analysed for ARA over the initial 3 h period. Gas samples were removed from the ‘jN, incubations and analysed for ambient “N, levels so that an absolute measure of activity could be obtained. Details of techniques used and final analytical procedures have been published previously (Silvester et al., 1982; S&ester, 1983). RESULTS
of asymbiotic
Typt
II
Douglas-fir
Nitrogenam activity (nmolcs CrH,g-’ h-‘1
0.2
contorta
PseudotsugajAbtus
Extent
Species
D=aY class’
7.7
2
occidentaL
Table 2. Nitrogenam activity of decaying wood. The results show the range of ARA based on at least IO samnla oer tyw
nitrogen
Jixation
Within mature forest systems of the Pacific North-
west three major sites of N2 fixation have been identified. Firstly, many tests have shown that a variety of species produce litter which supports nitrogen fixation during the decay process. Average levels of Nzase activity in forest litter range from zero in several species including the pines and alder to over I5 nmol C2HI g-’ h-’ in the F layer of red cedar litter (Table 1). The major forest type of this area is Douglas-fir and litter sampled from this forest invariably shows N,ase activity. Activity was greatest in the top F layer and in moiest material natural N,ase activity ranged from 1.0-36 nmol C2HIg-’ h-‘. When other, low activity, species are associated with Douglas-fir, the nitrogenase activities are significantly reduced (Table 1). A second important site of nitrogen fixation is in decaying wood. Considerable activity is found in lower decay classes of Douglas-fir wood with negligible activity in hemlock (Table 2). These results are considered in greater detail elsewhere (Silvester et at., 1982). Finally, species of the lichen Lobaria are widespread in Douglas-fir forest and are active in N2 fixation (Horstmann ef al., 1982). Effect
of added nutrients
Douglas-fir F litter was treated with -N (complete nutrients minus nitrogen) and phosphate (+P) SO~Utions and N,ase activity tested after 48 h (Table 3). A significant stimulation was recorded with addition of -N solution to both replicates. Replicate 2 in each case was reincubated for 48 h with -N solution giving a similar stimulation of N,ase (Table 3).
Table 3. E&IX of added nutrients on nitrogenax activity of Douglas-fir leaf litter. Two rcplicatcr each weft treated (Treated 1) with water (control). phosphate (+ P) and full nutrients (-N) solutions, incubated for 48 h and nitrogenax rates (nmol CrH,g-’ h-‘) measured for 3 h. All replicates were then incubated for 48 h after retreatment BJ shown in Treatment 2 and further tested for nitrogenax Treatment 2
Treatment I Rtulicate
Treatment
C
N.art
Treatment
Ntam
c
C
1.6 1.0
-N
1.3 3.8
+P +P
1.1 I.2
C -N
I.1 4.0
-N -N
4.6 5.1
C -N
4.0 4.5
Molybdenum
limitation
of nitrogen fixation
2as
Table 4. Effect on nitrogenax activity of various S salts and a comolete micronutrient mix (S at . 32 mg’l-’ in each case) Nitrogenax activity (nmolC,H.n-’ he’)
Treatment
2.1 a.2 2.2 2.3 1.9 9.1
Control -N MgSO, &SO, CaSO, Micronutrients
Table 5. E&t of various micronutrients in nitrogcnase activity of decaying Douglas-fir litter Nitrogenax activity (nmolC,H.g-’ h-‘1
Treatment
4.1 IS.2 5.2 4.9 4.8 19.6 3.2 5.1 3.3 4.2
Control Micronutrients Zn co B MO Fe CU c”
111 38 16
11
33 L2
I
I
0L
170
MO addition pg kg-’
It was confirmed (Table 4) that all of the N2ase enhancement was due to micronutrient additions. Neither sulphur, a known deficiency in Oregon, nor cations provided any stimulation over control. Of the micronutrients in the complete micronutrient solution, only MO had any effect (Table 5) and all nutrient stimulation of Nrase is due to MO. In general, enhancement of Nrase activity by MO addition, for the litter site studied, was consistently 3to 4-fold (Tables 3.4 and 5) regardless of the control rate. Time course and quantitative effects
Nitrogenase activity rises to a maximum at 30-40 h after MO addition, while control material retains a constant Nrase activity in the laboratory (Fig. I). In this experiment an extremely high stimulation ratio of 6.8 was achieved. N,ase activity shows a quantitative linear response to added MO (Fig. 2). Three days after MO addition a straight line relationship is shown between Nzase
Fig. 2. Response of nitrogenase activity in decaying Douglas-fir litter to addition of different amounts of MO. MO addition in pg element kg-’ dry wt of litter.
and added MO over the range 0-6Opg kg-‘. The distinct change in slope of this line after 25 days indicates that MO is still being taken up over this period and the saturation value drops correspondingly to c. 3Opg kg-‘. In the laboratory the enhanced N,ase activity is maintained over a long period. When Nrase was assayed at 3 and 25 days after MO addition a similar stimulation ratio of 4.5-4.6 was achieved (Fig. 2). In this case both control Nrase and MO treated N,ase had increased between 3 and 25 days but the stimulation ratio was maintained.
“N, uptake The effect of MO addition was tested using isN, uptake with parallel acetylene reduction assays. “N, confirmed that N, fixation is stimulated by MO addition (Table 6). While the stimulation of N, fixation was small in this particular experiment this was also mirrored in the acetylene reduction assay. The molar ratio figures fall within a normal range of values and no particular significance should be attached to the fact that values for + MO appear higher than controls as it is unlikely that the greater stimulation of acetylene reduction over Nr fixation is significant. Species, site and jield results
OO
I
I
I
1
10
16
22
50
Hours after MO addition Fig. I. Time course of nitrogenase activity in decaying Douglas-fir litter addition of MO.
Litter from a wide range of habitats, of mainly conifer species was tested for Nrase activity and MO stimulation. Both Nrase activity, and MO enhancement, was shown to be widespread (Table 7). Stimulation ratios up to 6.26 have beeen recorded from field material and even where control rates are high, such as the eastern slopes of the Coast Range in
W.
286
B. SIL~EXER
Table 6. Effect of MO addition to decaying Douglas-fir litter on ‘rN, uptake and ARA. Duplicate samples for “Nt uptake are shown and mean of three samples for ARA “N, uptake (nmol;L’g-’ h -‘)
Treatment Control +M0
I
2.74
2
2.86
I 2
of nitrogenax
Molar ratio
IO.I
3.69 3.53
3.17 4.15
17.2
4.56 4.14
1.5
1.7
Oregon, MO enhancement is significant. These results also show nitrogenase rate for an incubation in full nutrient -N solution, in some cases this incubation gave higher results than MO alone, whereas earlier experiments (Tables 4 and 5) indicated that all enhancement was due to MO. Further work is needed Table 7. MO enhancement
C:Ht reduction (nmolC,H,g-‘h-l)
to determine whether other inorganic nutrients may be limiting Nrase and over what time course. The geographic range of MO stimulation is generally confined to the area between the peaks of the Coast range and the Cascade Range (Fig. 3, Table 7). Decaying Douglas-fir wood was also tested for
activity in litter of various species at different See Fig. 3 for approximate locations
locations
in the Pacific Northwest.
Nitrogenase (nmolC,H,g-‘h-‘) Location
E. Slopes Coast Range, Oregon
w. slopes cascades Oregon
E. slopes cascades Oregon Southern Oregon
Eastern
Oregon
Washington
Vancouver Vancouver ‘Ag.
1d.E. 1d.W.
Litter’ species
Age (yr)
PC Pt Pt Pt Pt Pt A mat TP Pt (wood) Pt&Th Pt&Th Pt
60 30 30 200+ 200 400+ IS0 200+ 400 400 + 200+ :8
TP Pt+Th Pe Am Pl Pl Pt + Pp Pt Pt PI PC Lo Ag PP Jo Ag Pp+Ag Jo Pl Pr+Pp Pl Pt Pt Pl PI PttTp PI Pt Tp. Pt mixed
:: 100 moo+ 75+ 100 80 80 80 200+ 200+ 100 I50 4Of 50+ 40+
I 50 200 200 loo+ 4(M 200+ 35 40 40 40 30 I20 80 800 400
Elevation (m) 20 450 450 3M) 500 350 1150 500 450 600 850 700 1000 850 900 900 II00 I200 I200 650 650 650 I300 I300 IO00 800
IO00 IO00 850 I200 I600 I600 400 I600 1000 22 600 50 50 50 80 130 I20 30 30
+Mo %N
Control
+Mo
1.27 I.51 I.14 I .72 I .48 I.54 1.57 I.53 0.04 1.39 ND I.54 1.23 1.04 I.20 0.66 I.19 I .04 1.28 I.22 0.83 I .43 I .36 I.17 I .44 0.79 0.63 I .29 0.90 I.32 I .30 I.13 0.98 I .43 1.44 1.62 I.13 I.31 1.79 I .39 I .36 I.13 1.10 I.17 0.86
14.9 8.5 15.7 26.7 7.3 27.7 13.0 18.6 I .46 6.64 9.03 I.5 0.89 4.3 52 IS.2 0.95 2.6 3.0 5.6 8.83 I.9 2.9 2.7 2.7 4.0 0.6 14.7 1.3 9.8 7.3 Il.7 1.3 7.2 0.41 12.6 6.2 7. I 6.3 1.7 2.3 7.7 19.5 2.5 1.0
42.9 23.7 55.0 46.5 22.4 48.8 18.2 62.8 2.60 259 9.34 4.30 5.57 21.0 14.7 22.9 1.10 0.9 1.4 12.6 Il.3 5.2 2.0 4.6 2.2 6.2 0.6 23. I I.3 10.9 8.1 10.7 0.9 7.2 0.40 22.9 7.6 10.8 8.2 9.2 II.5 20.5 38.9 2.5 1.0
-N 40.7 22. I 42.8 58.6 21.9 65. I 12.2 69. I ND 18.3 6.3 4.13 7.02 30.8 27.3 33.6 I .20 1.5 I.6 Il.3 I I.3 3.1 3.2 4.3 I.8 5.1 0.6 18.4 1.0 18.1 IO.9 Il.2 I.1 Il.8 0.40 Il.4 4.8 Il.5 3.7 2.2 13.8 8.2 31.5 2.5 I .o
-MO 2.88 2.79 3.50 I .74 3.07 1.76 I .40 3.38 I .80 3.90 I .03 2.86 6.26 4.88 2.83 1.26 I.16 0.35 0.50 2.25 1.28 2.74 0.70 1.70 0.80 I.55 1.00 1.57 1.00 I.11 I.11 0.9 I 0.70 1.00 1.00 I .82 I .23 I.52 I .30 5.4 5.0 2.66 I .99 1.00 1.00
grand fir; Am. Abies magnifica red fir; Amac. Act-r mocrophyllum big leaf maple: Jo. Juniperus occidenralis juniper; Lo. Lcrix occidenrolis western larch; Pe. Picecr engelmannii Engelmann spruce; Pp. Pinus ponderoso ponderosa pine: Pt. Pseudorsugo taxfolia Douglas fir; Tp, Thuja plicoto Western red cedar; Th, Tsugo hererophylla Western hemlock. Abies grandis
western
Molybdenum
BRITISH COLUMBIA
Vancouver I.
+
\
Vancouver
J’
?5aik
------;-Seattle
#
I
I
WASHINGTON
\
OREGON
/
9
-a_______________
t
I
287
limitation of nitrogen fixation
: ---
+ Positive Mo response o No MO response
Fig. 3. Distribution of MO response on nitrogenase activity across northwest America.
MO enhancement. Eight replicates of control and MO treated wood were incubated for 48 h and assayed for N,ase showing a stimulation from 1.46 to 2.60 nmol C,H,g-’ h-’ significant at 1% level (Table 7). All the above results were obtained in the laboratory under conditions in which moisture and temperature were optimal. To test whether there is a real field MO deficiency a field trial was conducted in which 1 mz areas were pegged out under old growth Douglas-fir. MO solution. (500 pg I-‘) or distilled water (control) was sprayed to give a final MO concentration in the litter layer of 25Opg kg-‘. Material sampled 30 and 40 days after MO addition and incubated in the field showed statistically significant stimulation ratios of 1.65 and 2.48 (Table 8). It appears that MO deficiency is a real field phenomenon and as in the laboratory, a MO stimulation of N,ase can be sustained over a long period. DISCUSSION
The results produced in this study rely heavily on acetylene reduction as the assay for nitrogenase and this reliance has come under some criticism in recent years (Giller, 1987). The technique has a variety of limitations including possible spurious ethylene production from soil or organic matter and derepression of nitrogenase in the presence of the alternative substrate acetylene. We have previously addressed the problem of derepression which presents considerable complications when measuring low ARA over long periods (Silvester et al., 1982) and have shown
that incubations up to I2 h are reasonable and have limited ours in this work to 6 h. Spurious ethylene production in the presence of acetylene presents more difficulties because of the variable effects that can be obtained. While a variety of methods have been tried to assess this effect (Giller, 1987) I contend that the ultimate test of nitrogenase is either increase in total N or “N? uptake. We have verified ARA with “N, uptake from a variety of similar substrates, e.g. decaying Agathis leaves (Silvester, 1978) decaying wood (Silvester et al., 1982) and decaying mangrove leaves (Hicks and Silvester, 1985) along with the present work. I am confident the results presented represent realistic measures of nitrogenase. Further confirmation of this lies in the specific stimulation of activity by MO for which there is a specific and well acknowledged role in nitrogenase. Thus while it may be argued that a proportion, albeit a very small proportion, of measure activity may be due to non-specific ethylene production the “N, uptake and specific MO response confirm that there is a very real effect on nitrogenase. Mature conifer forests show significant adaptation to low nutrient regimes, generally requiring fewer nutrient inputs per unit wood production than do hardwood species (Waring and Franklin 1979. Gosz, 1981). This is achieved by efficient internal recycling of elements in conifers, retention of foliage and low nutrient concentrations in foliage. Overall nutrient budgets for old-growth Douglas-fir have been calculated in detail (Sollins et al., 1980) and estimated as a net increase in nitrogen of 5.0 kg N ha-’ yr-‘. Over much longer periods, which include several generations of forest, a net nitrogen accumulation is still struck, of 0.2-0.4 kg N ha-’ yr-’ (Gessel et al., 1973). Nitrogen inputs into the system include precipitation 2.0, nitrogen fixing lichens 2.8 and an unknown input of 1.7 kg N ha-’ yr-’ (Sollins et al., 1980). At least a proportion of this unknown input is accounted for by nitrogen fixation in decaying wood (Silvester et al., 1982) while a proportion is due to nitrogen fixation in litter. While it is not possible at this stage to accurately estimate the likely contribution of litter N2 fixation to total ecosystem N, it is likely to be of the same order as coming from decaying wood, i.e. c. I kg N ha-’ yr-‘. The absence of nitrogen fixation in the litter of some species, notably the pines and the relatively low levels recorded here for hemlock raises some interesting questions on the long term nitrogen status of these forests. It is notable also that N2 fixation in
Table 8. Stimulation of nitrogenase in the field. MO solution (500 cg I -‘) warsprayed onto Douglas-fir litterin the field to a final conccnrralion of 250 pg MO kg-’ litter dry wt. Core samplesof litter wre collecwd al various times after MO addition and incubated in C:H, immediaaly at field wmperawe and moisture. Differences significant at 1% level Days after MO addition 30 40 Litter cemperawe (^C) N:asc activity control +Mo +Mo -MO
9 15.3 25.3
I .65
I2 5.2 12.9 2.48
288
W.
B.
decaying wood of hemlock is also low (Silvester et al., 1982; Table 2). From the limited field surveys of natural N2 fixation in litter, three groups of activity stand out. Those species with consistently high rates, e.g. 2X&r pficatu. those with inte~~iate rates typified by Douglas-fir and those with low or zero rates, typified by the pines and hemiocks. In terms of nutrient requirements this grouping exactly matches the nutrient requirements independently assigned to these species by Gost (1981). In his review Gosz states “pines were distinguished from the rest of the conifers as a group having low requirements , , . Hemlocks have some of the lowest known nutrient requirements.. . while members of the Cupressaceae (Chumaecyparis norfhafensis and Thuju plicafu) have very high requirements, . . A number of species are intermediate (e.g. Douglas-fir)“. The above statements refer generally to nutrient requirement, more particularly to nitrogen requirement and also mirror the preference of the plant for NO; or NH,’ sources of N with the high N requiring species preferring NOT-the low N requiring species preferring NH: and the intermediate species using both forms. Any relationship between nutrient requirements, site requirements, general nutrition and N, fixation in plant residues can only remain speculative at the moment. However a working hypothesis is likely to revolve around the presence of inhibitory substances in the residues of species in poor nutrition. The links in this chain are reviewed by Gosz (1981) and in essence relate to the production of leaf polyphenols which both inhibit microbial activity in decay and also serve to lock up plant nutrients, particularly nitrogen. bringing about mor formation. Another factor can now be added to this complex array in conifer organic cycling. Molybdenum is obviously strongly limiting the rate of nitrogen fixation on some sites and these are identified as the more acid sites which are also associated with strong mor formation. From the work described above it appears that molybdenum availability is yet another factor in the complex of events relating to organic cycling in conifer forests. The question of micronutrient deficiencies in forests has seldom been raised but moly~enum deficiency, once identi~ed in crops and pasture has been one of the more easily defined and treated micronutrient limitations of plant production. Specific requirement for the element in NO> reduction and nitrogen fixation have long been established and crop limitations are readily rectified by addition of MO salts to other fettiiisers. The role of MO in nitrogen fixation was first identified by Bortels (1930) when he showed stimulation of growth of A~tobucrer chroococcum in culture by addition of trace sodium molybdate. It is interesting, in the context of the present study, that the first recorded field deficiency of MO was in asymbiotic NZ fixation on the West Coast of U.S.A. In this study Van Niel(1935) reported that soilsfrom the Monterey Peninsula would yield Azofobucfer isolates only after addition of molybdenum to the soil. Since that time many examples of MO limitation of legume production have been studied and rectified. but very little work on asymbiotic nitrogen fixing organisms has been reported.
S~~as'rm
Within North America there is a trend in reported MO requirement from mild deficiencies in the west to sometimes acute deficiencies in the eastern states (Rubins. 19%). Despite this, a number of reports. cited by Rubins (1956), report significant MO deficiencies in California, Oregon and Washington and early work in Oregon (Anon., 19%) showed that coastal and Eastern Oregon alkaline soils showed no MO response, while in the central valley, between the Coast and Cascade Ranges. MO responses were reported for hi11 soils and acid valley tfoor soils. The responses to MO reported in the present paper are consistent with the known and putative distribution of MO deficiency, i.e. all soils on which MO deficiency is found are acid and, in general, under conifer forest which enhances this acidity. It has been pointed out by Rubins (1956) that the total MO concentration in soils is relatively uniform at 2 ppm but that the level of available MO varies with pH. Thus under acid conditions free MO is low while under alkaline conditions it is possible for MO toxicity to be expressed in grazing and foraging animals. In the absence of any measured values for molybdenum in conifer leaves it is difficult to speculate as to whether it is merely the Iack of available MO in leaves which is reducing NL fixation or whether total MO is low. Walker (personal communication) using the Aspergillus assay has shown that a number of soils under Douglas-fir forest are deficient or slightly deficient in molybdenum but that none of the soils tested are in the very deficient range (i.e.
Molybdenum limitation 1of nitrogen fixation Acknowledgements-This
work was supported by grants from the National Science Foundation. The work was greatly facilitated by discussions with Drs K. Cromack, J. C. Gordon, P. Sollins and R. H. Waring. Laboratory assistance from Jan Silvester and Tom Verhoeven is gratefully acknowledged. The Pacific Northwest Forest and Range Experiment Station kindly supplied laboratory space and facilities. Isotopic nitrogen analyses were carried out by Dr B. B. McInteer, Los Alamos Scientific Laboratories. REFERENCES Anon. (1958) The Molybdenum Problem in Oregon. Miscellaneous Paper 64. Agriculture Experiment Station, Oregon State University, Corvallis. Oregon, U.S.A. Bollard E. G. (1966) A comparative study of the ability of organic nitrogenous compounds to serve as sole sources of nitrogen for the growth of plants. Planr and Soil 25, 153-166.
Bortels H. (1930) Molybdenum as a catalyst in the biological fixation of nitrogen. Archive fir Mikrobiologie I, 333-342.
Dawson M. D. and Bhella H. S. (1972) Subterranean clover (Tri/olium subterranean) yield and nutrient content as influenced by soil molybdenum status. Agronomy Journal 64, 308-31 I. Gessel S. P.. Cole D. W. and Steinbrenner E. C. (1973) Nitrogen balances in forest ecosystems of the Pacific Northwest. Soil Biology & Biochemistry 5, 19-34. Giller K. E. (1987) Use and abuse of acetylene reduction assay for measurement of associative nitrogen fixation. Soil Biology & Biochemisrry 19, 783-784. Gosz J. R. (1981) Nitrogen cycling in coniferous ecosystems. In Terrestrial Nitrogen Cycles: Processes, Ecosystem Strategies and Management Impam (Mark F. E. and Roswall T., Eds). Ecological Bulletins Stockholm 33, 405426. Henry E. (1897) L’azote et la vegetation forestiere. Revue dtaux et firers 36, 64 I.
Hicks B. J. and Silvester W. B. (1985) Nitrogen fixation associated with the New Zealand mangrove (Auicennia marina (Forsk.) Vierh. var. resini/era (F0rst.f.) Bakh) Applied and Encironmental Microbiology 49. 955-959.
Horstmann
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