Nitrogenase activity associated with three tropical grasses growing in undisturbed soil cores

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So11Bwl. Biochem. Vol I.?. pp. 131 to I34 0 Pergamon Presr Ltd 1480. Prmted in Great Britam

NITROGENASE ACTIVITY ASSOCIATED WITH THREE TROPICAL GRASSES GROWING IN UNDISTURBED SOIL CORES K. L. Cunningham

Division Laboratory,

WEIER

of Tropical Crops and Pastures, Mill Road, St. Lucia, Brisbane, (nccqted

6

Azdgusf

C.S.I.R.O., Queensland

4067, Australia

1979)

Summary-Nitrogenase activity associated with the root system of three tropical grasses Axonopus compressus, Digifario decumLwns var. pungola and Pa.spalum notatum was measured by CZH2 reduction assay of soil-plant cores. The cores were incubated in perspex chambers in which 10Yb of the air was replaced with CZH2. Gas samples were taken at 7, 24, 31, 48, 55 and 72 h. No lag before onset of C2H4 production was evident and good agreement was obtained between replicates. Cumulative C2H4 produetion maintained a linear trend during the 72 h incubation. The largest increase in N,-ase activity was detected in the A. compressus-gleyed podzohc system while D. decumhens (lateritic podzolic) and P. noratum (sandy yellow podzolic) had smaller activities. Variation between sampling sites in the second year of sampling of the experiment was associated with large variations in soil moisture. NZ fixation estimated from N,-ase activity in soil-plant cores was similar to the amount of N accumulated in the above-ground herbage in the field during 12 weeks. Response curves relating N,-ase activity to soil moisture and soil temperature were established for ail species. P. notatum and D. dew&ens responded similarly to changes in both soil temperature and soil moisture while A. compressus contrasted sharply to the other two speoies in its reaction to both.

INTRODtiCTfON

The &Hz reduction assay for estimating N,-ase activity has shown that many tropical grasses in different parts of the world have active N,-fixing bacteria associated with their root systems (Dobereiner et al., 1972; Day et al., 1975a; Dobereiner and Day. 1975; Dommergues et al., 1973). Rapid progress has been made in elucidating the effects of factors such as soil water potential, pH, partial pressure of oxygen, nutritional status and temperature on N,-ase activity associated with roots under laboratory conditions (Dobereiner et al., 1976: Okon et al., 1977; Neyra and van Berkum, 1977; Day and Dobereiner, 1976). Less is known, however, of the amounts of Nz fixed under field conditions. This is primarily because of the uncertainty in extrapolating from laboratory assays of N,-ase activity, particularly from assays made with excised roots (Eskew and Ting, 1977; Tjepkema and van Berkum, 1977). The most reliable quantitative results appear to have been obtained by incubation of intact soil-plant cores IOcm in dia (Dart et al., 1972; Day er nl., 197Sb). We used this technique to assess the importance of N, fixation associated with three tropical grasses in south-east Queensland. Because soil moisture and temperature were expected to have a strong influence on the results (Vtassak eb al., 1973; Day et ul., 1975b; Balandreau ef ul., 1978) the effects of these factors were also studied. MATERIALS

AND

METHODS

The three grass species, Axonopus c~mp~esst~s, D&iclecumhens var. pang& and Paspalum notutum, were located in separate areas of N-deficient pasture turiu

at the Beerwah Pasture Research Station, 72 km north of Brisbane and 6 km ENE of the township of Beerwah (26’52’S, 15Z05S’E). The climate of the area is humid-subtropical with hot wet summers and cool to warm winters. Mean annual rainfall is 16% mm. The soil type underlying each species is shown in Table 1. The first of two experiments was designed to estimate N,-ase activity and N-uptake by the grasses during the summer. Experiment 2 was to study the effects of soil water content and temperature on N,-ase activity. Experiment

I

Measurements were made during two 12-week periods in consecutive summers: 13 December 1974 to 7 March 1975 and 30 January to 23 April 1976. Both times, a 5 x 5 m area was selected in each pasture. The area was divided into five blocks, each with five 1 m square plots. Soil-plant cores were collected five times, every 3 weeks. Each time, one core was taken from one plot in each block; each plot was sampled once only. Four cores were assayed for C2H2 reduction and one (from a different block each time) for endogenous CzH4 production. At the 0 and 12 week samplings in the second summer, in addition to taking soil-plant cores for estimates of N,-ase activity, above-ground herbage was harvested from a 0.5 x 0.5 m quadrat in each plot so that N uptake could be determined. Soil-plant cores were obtained by driving 16.5 x 12cm dia steel tubes into the soil. The tubes plus cores were taken to the laboratory, where they were weighed and the plants allowed a recovery period of 2 days (Abrantes et al., 1975) during which they

131

were watered

to their

original

weight.

K. L. WEIER

132 Table 1. Soils underlying

Grass

species

Avonopus compressus Diqircrriu drcumbens Paspcrh notutum

the three grass species at the Beerwah Pasture Research Station

Beerwah Beerwah Beerwah

Factual: key

Australian greatf soil group

Soil type* 5 3 2

Gleyed podzolic Lateritic podzolic Sandy yellow podzohc

Total N (“J

Dg 4.41 Dy 5.41

0.095 0.09 I

Gn

0.057

2.14

pedal clay * Thompson (1957). t Stephens (1962). : Northcote (I 965).

After 2 days, the cores were incubated for 72 h at 25°C in sealed perspex chambers in which 10% of the air was replaced by C2H2. At fixed times (Fig. l), 0.5 ml was removed from each chamber with a gastight syringe. C2H4 production was measured on a Varian 14OOg.c. with a 0.3cm x 1 m stainless-steel column packed with 8G-100 mesh Chromosorb 104 and maintained at EO’C; Hz f.i.d.; N2 carrier gas. Concentrations were read from a standard curve prepared by injecting known volumes of C2H4 (Hardy et tJ/.. 1968). After incubation, grass tops were removed at ground level and the dry weight of soil plus roots determined after drying at 60°C (second summer only). Where required, total N was determined on ground plant material or soil ( < 2 mm) by Kjeldahl digestion after pre-treatment with salicylic acid and sodium thiosulphate (Bremner, 1965). E.speriment

2

N,-ase activity was measured at six soil moisture contents and four soil temperatures on 12 soil-plant cores of each species, the moisture study being done first and then the temperature study, re-using the same cores. The cores were placed in a controlled environment room with a I4 h photoperiod and a light intensity of 325 ~Ern-‘s-r. The soil moisture contents were established by saturating the cores for 24 h and then allowing them to drain for 24 h. The cores were then weighed. Dupli-

cate cores for each species were maintained at this weight and a further set maintained at a higher saturated value. Other sets of duplicate cores were allowed to dry to 15, 50, 25 and ca. 15”, of the recorded weight. Ten days were required to establish the differential moisture contents. N,-ase activity was measured during 24 h at a constant room temperature of 3OC. The effect of temperature was measured at soil moisture contents of 25y;, (D. decumbens and I-‘. JlOfiJturn) and 409: (A. compressus). These moisture COIItents gave about the same relative Nz-ase activity at 30°C for each species. The sequence of temperatures used was 20, 30, 35 and 25°C. the room being maintained at each one of these for 5 days and N,-asc activity measured during 24 h. RESC’LTS

Experiment

I

For D. decumhens and P. rwtatum N,-ase activity was constant throughout 72 h, as cumulative C2H, production did not deviate significantly from a linear trend (Fig. I). Although a statistically-significant quadratic component was detected in the trend of cumulative CzH4 production with ,4. ~~mpress~.s, the magnitude of the deviation from linearity was quite small. On all but two occasions the greatest hu,-ase activity was detected in cores of the A. compressors gleyed podzolic system (Table 2). Variation between

Bdecumbens

Fig. 1. Time-course studies on soil-plant cores containing Axonopus comprrssus, and Pospalum notatum. Values plotted are those obtained for the fourth harvest content was 36.1. 26.2 and 1X.8”,,, respectively.

Digitariu tlrcumhens when soil moisture

* Average

of 5 cores.

9.5 8.2 7.6 6.9 10.1 8.5 0.95

4.3 9.7 8.3 3.9 8.2 6.9 1.35

7.0 9.1 7.1 3.9 9.8 7.4 0.8 1

P. not&urn

2 days after sampling

PM&H, core-l day-’ A. D. c0mprcssu.s decumbens

daily rates of CzH4 production

13 Dec. 1974 3 Jan. 1975 24 Jan. t975 14 Feb. 1975 7 Mar. 1975 Mean SED between times

Sampling date

Table 2. Average

30 Jan. 1976 20 Feb. 1976 12 Mar. 1976 2 Apr. 1976 23 Apr. 1976

Sampling date 6.0 11.9 39.9 14.5 13.9 17.2 3.03

3.6 5.7 23.7 6.2 4.4 8.7 2.39

@ZZH4 core-’ day-’ A. D. compressus decumhens

content

4.1 4.0 12.6 3.0 2.9 5.3 1.22

P. not&urn

for the three species in 1975 and 1976 and field moisture

22.5 31.9 42.5 36.1 36.4 33.9 2.9

A. compressus

3 months

16.9 23.8 28. I 26.2 24.1 23.8 1.15

Soil moisture (“A)* D. decumbens

for the species during

12.5 19.3 27.9 18.8 20.9 19.9 1.04

P. notatum

in 1976

K. L. WEIIX

134

4

I3

12

16

20

24

xl

32

Soil moisture I% 0 D basis) Fig. 2. Effect of field soil moisture

on nitrogen

fixation by soil-plant (r = 0.81).

sampling dates was more marked in the second year than in the first. Much of the variation in N,-ase activity in the second year could be explained by variation in soil moisture, the coefficient of correlation between log (N,-ase activity) and soil moisture being 0.81 (Fig. 2). Nz fixation in the second year estimated using the 3 : 1 ratio (C2HZ :N2) was about the same as N accumulated in the above-ground herbage (Table 3). No significant amount of endogenous C2H4 production was detected in ungassed soil-plant cores during 3 days.

The three soil--plant systems showed a similar pattern of response to variation in soil moisture content, but there were quantitative differences (Fig. 3). N,-ase activity increased steadily with moisture content until a critical moisture content was reached, and then rose very rapidly thereafter. The critical moisture contents for D. decumhens and P. notatum occurred at soil moisture potentials of - 50 and - 20 kPa compared with greater than -0.5 kPa for A. compressus. D. decumhens and P. notatum responded similarly to changes in temperatures, with a 5@60% increase in N,-ase activity between 20 and 25°C and only a slight increase with further increase in temperature (Fig. 4). The response of A. compressus was in sharp contrast to that of the other species. Table 3. Comparison

Grass

species

A. compres.sus D. decumhe,l.F P. notatum

of estimated

Dry matter 0 weeks 2773 1111 1322

nitrogen

yield (kg ha- I) 12 weeks 3959 1303 1682

fixation

correlation

DISCLSSION

The technique used in this study appears to be quite suitable for assessing the N,-ase activity of grasses in relatively undisturbed soil-plant systems. The responses observed for soil temperature and soil moisture verify this and future measurements will be made using the intact core system. The major uncertainty involved is in assuming that rates of N2 fixation can be calculated by using a ratio of 3 moles C,H2 reduced per mole of Nz fixed. Observed ratios have varied from 0.75-3.6 (Brouzes and Knowles, 1973) to 3-15 (Rice and Paul, 1971). The latter, however, did find that C2Hz to C,H4 reduction was similar where incubation periods were the same. Hardy et al. (1973) found that, when assessing the results available, the average ratios were about 3. Bergersen (1970) recommends this ratio is confirmed for each system by checking with 15N2 ; this is planned in a future phase of the work. The Nz-ase activities observed here for these three tropical grasses are comparable with those reported for other grasses in North America (Barber and Evans, 1976; Nelson er ul., 1976; Tjepkema and Evans, 1976), but less than that reported for Digitgrass and Bermuda grass (Schank et nl.. 1977) in Brazil. In the latter study, grass tops were cut and the plants allowed to grow for 2 weeks before N,-ase activity was measured. I have found that cutting plant

with nitrogen

accumulated

N yield (kg N ha- ‘) 0 weeks 12 weeks 18.6 4.8 1.1

cores giving a positive

34.4 9.x 13.5

by the grass tops m the second N accumulated* by tops (kg ha-‘) 15.X & 2.7 5.0 i 1.6 5.x f I.0

year

Estimatedt N fixation [CzH2] (kg ha-‘) 12.7 6.4 3.9

* Mrun k SE -F Estimated from assays of cores at 0, 3. 6, 9 and 12 weeks (Table 2) using a ratio of 1 mole of N, fixed: 3 moles of C2H, produced and interpolating linearly between sampling dates.

Nitrogenase

activity

in undisturbed

soil cores

135

Soil water potential (kl%) 1420 -I3 (a)

-'5

-0 5

-03

-01

A=O.378 8=0.082 60.84

34

10

30

20

40

50

SolI moistureWOD.basls) (a) Soil water potential -

I

/

25

30

Temperature

35

(“C)

Fig. 4. Relationship between soil temperature and rate of nitrogen fixation when the soil-plant cores were held at the second highest soil moisture level.

Soil moislureWOD bcws) (b) Sdl Waler pcdenllal

L__,___ 0

564

e

126

Soil molsture W&D

_ _,315

25

b&s

I

Cd Fig. 3. The relationship between soil moisture and rate of nitrogen fixation when the soil-plant cores were held at a constant temperature of 30 k I‘C. The fitted curve is Y = A* exp(B*X) (a) A. compressus (b) D. decumbens (c) P.

Ilotlztum. tops may enhance N,-ase activity in certain grass species and hence lead to higher fixation rates. Using the estimated rates of N, fixation, it appears that N accumulated by the plant tops is comparable with the N fixed by the plant. If we assume that the N fixed is immediately available to the plant (Dobereiner and Day, 1976) then this suggests that the contribution of soil N to plant growth was minimal and the plant was depending almost entirely on fixed N for its supply. The Beerwah soils are inherently infertile, low N soils and this assumption may well be true. Field conditions vary greatly from the controlled conditions of these experiments. In particular, tem-

perature and moisture fluctuate greatly and, for this reason, temperature and moisture responses were determined in order to specify what environmental conditions are necessary for rapid fixation. There were large increases in N,-ase activity when soil temperature was >25”C and when soil moisture ranged from -0.5 to -50 kPa. Soil temperatures >25”C occur sporadically between December and March each year. The response of A. compressus to temperature change differed from the other two species. This may be a species effect in that its fixation may simply be adapted to a warmer range of temperatures indicating the possibility of a different organism existing on the root system. Soil moisture will be more limiting, as moisture tensions of -0.5 to -50 kPa will only occur immediately after rain, although during the wet season lengthy periods of waterlogging may occur. The more important source of N2 fixation may well prove to be the slower, more prolonged activity between -5 and - 15 kPa for D. decumbens and P. notutum and -0.5 and - 10 kPa for A. compressus. Use of a suitable water balance model may help to elucidate this problem of assessing the input of N during all seasons of the year. Acknowledgements-1 thank Messrs D. Radke and J. E. Stewart for their assistance with the experimental work and Dr G. Dolby for assistance with statistical analyses. REFERENCES ARRANTESG. T. V., DAY J. M. and DOBEREINER J. (1975) Methods for the study of nitrogenase activity in field

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