Nitrogenase Activity Associated with some Grass Species in Czechoslovakia

Zentralbl. Mikrobiol. 143 (1988), 441 -446 VEB Gustav Fischer Verlag lena

[Institute of Soil Biology, Czechoslovak Academy of Sciences, Cesk6 Budejovice, Czechoslovakia]

Nitrogenase Activity Associated with some Grass Species in Czechoslovakia!) M,

SIMEK

With 2 Figures

Summary Nitrogenase activity associated with 5 grass species was measured on 2 sites differing in climate and soil type in Czechoslovakia. Intact grass-soil cores were calJected at intervals throughout the growing seasons 1984 and 1985 from the top soil layer (0- JOcm). In this layer rates of nitrogenase activity were generalJy low and variable within the range ofO.01-7.55nmol C2H4 x g-l (dry wt) x day-I. Great variability and some differences of nitrogenase activity were found not only throughout the growing season, but also between grass species and between sites. Most of the nitrogenase activity in the soil profile ofO-30cm was concentrated in the top 0--10cm, e.g. under a meadow grass (Poa pratensis) sward it was approximately 67 %. Higher nitrogenase activity was recorded at 0 % than at either 5 % or 21 % oxygen levels. Estimated amounts of N2 fixed during the growing season on the soil profile of 0- 30cm under individual grass species were within the range of 0.5- 3.82kg N x ha- I

Zusammenfassung Auf 2 in Klima und Bodentyp unterschiedlichen Standorten wurde die Nitrogenaseaktivitat bei Monokultur von 5 Grasarten gemessen. Intakte Gras-Boden-Schichten (0- JOcm) wurden wahrend der Vegetationsperiode in den Jahren 1984 und 1985 in bestimmten IntervalJen entnommen. In dieser oberen Bodenschicht war die Nitrogenaseaktivitat im allgemeinen gering und nahm Werte zwischen 0,01 und 7,55 nmol C2H4 x g-I (trockener Boden) x d- 1 ein. Gr6Bere Variabilitat in der Nitrogenaseaktivitat war nicht nur innerhalb der Vegetationsperiode zu verzeichnen, sondem auch zwischen den einzelnen Grasarten und Standorten. lnnerhalb des Bodenprofils 0-30cm entfielen auf die Schicht 0- JOcm die h6chsten Nitrogenaseaktivitaten, bei Poa pratensis zum Beispiel 67 %. Ein h6heres Niveau der Nitrogenaseaktivitat wurde eher bei 0 % Oxygenkonzentration als bei 5 % oder 21 % erreicht. Wiihrend der Vegetationsperiode wurde bei den verschiedenen Grasarten in der Bodenschicht von 0-30cm die N-Fixierung auf 0,5-3,82kg N/ha- 1 geschatzt.

Non-symbiotic and associative Nrfixation can make small but significant contributions to the N economy of a lot of ecosystems. This is one reason why within the past decade great attention has been paid to research into nitrogenase activity and nitrogen fixation associated with various grasses. Knowledge was reviewed recently, for instance, by BODDEY and DOBEREINER (1984). High values of nitrogenase activity have sometimes been reported from tropical or similar environments, e.g. by DAY et al. (1975), BORS et al. (1982), and also from northern Europe by HAAHTELA et al. (1981). In this paper, some results of a study of nitrogenase activity under swards of some grasses in central Europe (Czechoslovakia) are presented.

I) Presented at the Reinhardsbrunn Symposium, May 1987.

442

M. SIMEK

Material and Methods Sites description Nitrogenase activity was studied in grass monocultures in 2 different (with regard to climate and soil) sites in Czechoslovakia (Table I). The study area near Roznov p. R. is in North Moravia while the study area in Ceske Budejovice is in South Bohemia. Grasses were sown in Roznov p.R. in April 1981 and in Ceske Budejovice in April 1984. Table I. Some characteristics of the sites and grasses under study Year Site Height above sea level

Mean values of annual temperature and precipitation

Soil

1984 Rozov p. R. 570m

7.5°C 903mm

1985 Ceske Budejovice 380m

8.2°C 690mm

Mean content of nutrients (KCl) in the soil (mglkg)

Grass species

pH

p

K

podzolic 6.5 brown soil loamy sand

32

132 130

3.0

1.3

Phleurn pratense L. Festuca pratensis Hudson Paa pratensis L. Latium perenne L. Dactytis glarnerata L.

brown soil 5.9 sandy loam

65

121

ND

ND

Phleurn pratense L. Festuca pratensis Hudson Paa pratensis L.

Mg

99

ND = not determined

Field sampling and sample preparation The overground parts of plants were detached, and then soil cores containing roots were removed. Cores were extracted using a steel coring cylinder (33 x 3 .Ocm dial from the top soil layer of 0-1 0 cm. The coring device was placed into the centre of the grass plant. In a few instances cores were also extracted from the soil layers 1O-20cm and 20- 30 em. On the same day (in 1985) or on the next day (in 1984) samples were transported to the laboratory. Cores were weighed and placed into 300ml gas-tight screw capped bottles with rubber stoppers. Cores were incubated at 0 % (in argon or helium) and 5 % (in argon with oxygen) oxygen levels or in air (21 % O2), Those samples incubated at 0 % and 5 % oxygen levels were flushed 4 times (evacuated and re-filled) with the appropriate gas or gas mixture. Then 25 ml of the internal gas atmosphere was removed and the same volumes of acetylene were added. Control core samples were also set up without acetylene addition. All samples were incubated in the dark at 22°C.

Acetylene reduction activity measurement Nitrogenase activity was measured as nitrogenase acetylene reduction activity (SKRDLETA et al. 1976, SIMEK et al. 1987). After the incubation period of 66-70 h, I m1 gas samples were withdrawn using plastic (Plastipak) syringes, and analysed for ethylene/acetylene content using gas chromatograph equipped with a Porapak N column (180 x 0.3 em) and a flame ionization detector.

Calculation of rates of N 2 -fixation Since the acetylene reduction activity method is an indirect method, recalculation of the results obtained to give the amount of fixed nitrogen is not commonly recommended because of great variability of the ratio C2H2/N 2 (MORRIS et al. 1985). Estimation of nitrogen fixation was therefore performed, using a C2 H2 -to-N 2 molar ratio of four.

Nitrogenase Activity

443

Results and Discussion Studies of seasonal nitrogenase activity Nitrogenase activity was measured throughout the growing season, 6 times and 8 times in 1984 and 1985, respectively. Nitrogenase activity was highly variable not only throughout the growing season, but also between grass species in the same area. There were also some differences in nitrogenase activity of individual grass species between both the areas (Table 2). The highest mean rate of nitrogenase activity was associated with timothy (Phleum pratense) in 1984 and with meadow grass (Poa pratensis) in 1985, the lowest one with meadow fescue (Festuca pratensis). The amount of C2H2 reduced in the rhizosphere of our grasses was similar to that reported by BARBER et al. (1978). Their studies involved some grasses and other non-leguminous plants in Oregon, USA. Table 2. Nitrogenase activity in the soil under individual grass species on both sites. Samples from the soil layer of O-lOcm incubated in argon (in 1984) and/or in helium. Expressed as nanomoles of CZH4 per gram (dry weight) of sample per day. Minimum and maximum values, means and SD based on: a - 30 measurements (6 sampling dates, 5 replicates for each grass species), b - 40 measurements (8 sampling dates, 5 replicates) 1984 - Roznov p.R."

Grass species

Ph/eum pratense Poa pratensis Lolium perenne Dactylis glomerata Festuca pratensis

Nd

=

1985 - C. Budejoviceb

Xmin -

Xmax

X± SD

Xmin -

0.51 0.01 0.01 0.03 0.04 -

5.54 3.58 2.56 0.95 0.58

1.45 ± I. 16 1.16 0.57 ± 0.65 0.32 ± 0.27 0.17 ± 0.15

0.24-3.11 0.28 - 7.55 ND ND 0.23 - 3.08

1. 12 ±

Xmax

X± SD 0.80 ± 0.79 1.67 ± 1.89 ND ND 0.74 ± 0.67

not determined

Nitrogenase activity in soil profile The results presented in Table 2 are based on data from the top soil layer ofO-lOcm. However, nitrogenase activity was also measured in the soil profile to a depth of30cm. In Table 3 the results of nitrogenase activity measurements in the soil profile of meadow grass (Poa pratensis) are presented. It is clear that most nitrogenase activity (about 67 % from the soil layer of 0- 30cm) was in the top 0- 10 cm. Similar distribution of nitrogenase activity down the soil profile was also found under the other grasses. SINCLAIR et al. (1976) reported at least 80 % nitrogenase activity in the top 0-7.5 cm of some New Zealand pastures. Table 3. Nitrogenase activity in the soil profile under meadow grass (Poa pratensis) sward. Samples were incubated in argon. a - expressed as nanomoles ofC 2H4 per gram (dry weight) of soil per day based on 5 replicates for each soil layer. b - expressed as micromoles of C2H4 per soil layer per square meter per day and in % Soil layer

Xmin -

O-lOcm lO-20cm 20-30cm 0-30cm

Nitrogenase activityb

Nitrogenase activity" Xmax

0.095 - O. 165 0.016 - 0.047 0.012 - 0.029

X± SD

x

%

0.123 ± 0.082 0.037 ± 0.020 0.023 ± O.OlO

15.20 4.57 2.85 22.62

67.2 20.2 12.6 100.0

444

M.

SIMEK

Effect of oxygen level in the incubation atmosphere on nitrogenase activity According to the results of some authors (e.g. SCHANK et al. 1985, MWAURA and GRANHALL 1986, ZUBERER and ALEXANDER 1986) the composition of the gas mixture in which samples are incubated can exert an influence on the measured nitrogenase activity. Therefore we paid considerable attention to this problem. The specific group of diazotrophs is being favoured by the incubation atmosphere and oxygen partial pressure in the incubation atmosphere is perhaps of most significance in this context. In this study 3 different levels of oxygen in the incubation atmosphere were used: 0, 5 % and ambient (about 21 %) O2 concentration.

0-.--<>

---...

-

2.5

100Y. A. 95"/. A•• 5%°2 2.5

air

'l"

"a

'L

2.0

7"

.~

"2

1.5

>-

"

"It

:z:N u "0

E

~

9S'l.A •. S'l.02

0

100% A•

1.5

.!!

"It

... u

1.0

:J:

c

1.0

0

E

.;(

Z

air

'I...

~

"a

~

2.0

~

r:

0.5

« z

0

0

V

VI

VII

VIII

IX

X

:"

0.5

III Fp

~

~

El9 Lp

Php

~ Pp

(month)

Fig. 1. Nitrogenase activity in the soil under meadow grass (Poa pratensis) sward during the growing season. Samples were incubated in 3 different gas mixtures. Each point represents mean of 5 replicates. Fig. 2. Nitrogenase activity in the soil under grass swards. Samples were incubated in 3 different gas mixtures. Each column represents mean of 5 replicates. Fp = Festuca pratensis, Dg = Dactyli.l· glomerata, Lp = Lolium perenne, Php = Phleum pratense, Pp = Poa pratensis.

Fig. 1 shows the results of measurement of nitrogenase activity in the soil under meadow grass (Paa pratensis) sward during the growing season. Obviously, nitrogenase activity depended

heavily on the gas mixture composition, especially in spring and autumn. In summer the nitrogenase activity level was very low and no substantial differences in nitrogenase activity between various incubation gas mixtures were found. We ascribe the summer fall of nitrogenase activity to the reduced soil moisture owing to high temperature. A similar positive correlation between acetylene reduction and soil moisture has previously been reported in a number of occasions (e.g. ROPER 1983, BAKER and ATTIWILL 1984, MORRIS et al. 1985). Furthermore, the reduced growth of grasses in this season may result in lower production of the root exudates, which are a source of energy for soil microorganisms in general and for nitrogen fixing microorganisms in particular. The level of nitrogenase activity depended on the composition of the incubation atmosphere for meadow grass (Paa pratensis) as well as for other grasses (Fig. 2). Generally, nitrogenase activity increased with decreasing oxygen concentration.

Nitrogenase Activity

445

These results confirm that it is necessary to pay great attention to the composition of the incubation gas mixture in similar studies of non-symbiotic and associative nitrogen fixation. The problem is to decide which oxygen concentration gives a close correlation between the measured nitrogenase activity and the nitrogenase activity level in field conditions. Gas mixture with lower oxygen content seems to be more appropriate since it is well known that oxygen concentration in soil air is lower than in free air (see e.g. BUYANOVSKY and WAGNER 1983). Oxygen concentration in soil air, however, is not constant throughout the growing season and throughout the soil profile, it depends also on the type of tillage. In the light of these considerations we can say that finding a suitable oxygen concentration in the incubation atmosphere during the measurement of nonsymbiotic and associative nitrogen fixation represents a difficult methodological problem. Table 4. Estimation of biological nitrogen fixation in the soil under individual grass species on both sites. Expressed as kilogram of nitrogen fixed per hectar per 210 days of growing season and soil profile of 0- 30cm Grass species

1984 - Roznov p.R.

1985 -

Phleum pratense Poa pratensis Lalium perenne Dactylis glomerata Festuca pratensis

3.82 2.92 1.65 0.88

1.94 3.68

0.50

1.94

C.

Budejovice

Estimation of nitrogen fixation On the basis of measured values of nitrogenase activity an estimation of nitrogen fixation was carried out (Table 4). Data indicate a low agronomic significance of non-symbiotic and associative nitrogen fixation under swards in our conditions. However, the accuracy of these estimations is open to question as they depend on the unconfirmed ratio of 4 for CzHz-to-N z. In conclusion, more knowledge about nitrogenase activity in the presence of CzH 2 is necessary before it will be possible to define the levels of nitrogen fixation under these or similar grass swards.

References BAKER, T. G., ATTIWILL, P. M.: Acetylene reduction in soil and litter from pine and eucalypt forests in southeastern Australia. Soil BioI. Biochem. 16 (1984),241-245. BARBER, L. E., TJEPKEMA, J. D., EVANS, H. J.: Acetylene reduction in the root environment of some grasses and other plants in Oregon. In: Environmental Role of Nitrogen Fixing Blue-green Algae and Asymbiotic Bacteria. GRAN HALL, U. (ed.). Eco!. Bull. 26 (1978),366-372. BODDEY, R. M., DOBEREINER, J. : Nitrogen fixation associated with grasses and cereals. In: Current Developments in Biological Nitrogen Fixation. SVBBA RAo, N. S. (ed.). London (1984), 277-313. BORs, J., KLOSS, M., ZELLES, L., FENDRIK, 1.: Nitrogen fixation and nitrogen-fixing microorganisms from the rhizosphere of Diplachnefusca Lim. (Beauv.). 1. Gen. Appl. Microbiol. 28 (1982), 111-118. BVYANOVSKY, G. A., WAGNER, G. H.: Annual cycles of carbon dioxide level in soil air. Soil Sci. Soc. Am. 1. 47 (1983),1139-1145. DAY, 1. M., NEVES, M. C. P., DOBEREINER, 1.: Nitrogenase activity on roots of tropical grasses. Soil BioI. Biochem. 7 (1975), 107-112. HAAHTELA, K., WARTlOVAARA, T., SVNDMAN, V., SKVJINS, J. : Root-associated N2 fixation (acetylene reduction) by Enterobacteriaceae and Azospirillum strains in cold-climate spodosols. App!. Environ. Microbia!. 41 (1981), 203-206. MORRIS, D. R., ZVBERER, D. A., WEAVER, R. W.: Nitrogen fixation by intact grass-soil cores using lSNz and acetylene reduction. Soil BioI. Biochem. 17 (1985),87-91. MWAvRA, F., GRANHALL. U.: Nitrogen fixation (C ZH2 reduction) associated with maize (Zea mays L.) in a Swedish soil. Swedish J. agric. Res. 16 (l986), 49-56. 30

Zenlralbl. Mikrobiol., Bd. 143

446

M. SIMEK, Nitrogenase Activity

ROPER, M. M.: Field measurement of nitrogenase activity in soils amended with wheat straw. Aust. J. Agric. Res. 34 (1983),725-739. SCHANK, S. c., SMITH, R. L., MILAM, J. R., LITTELL, R. C. : Testing grass-bacteria combinations for associative N2 fixation potential. Soil and Crop Sci. Soc. Fla. Proc. 45 (1986), 179-184. SINCLAIR, A. R., HANNAGAN, R. B., RISK, W. H. : Evaluation of the acetylene reduction assay of nitrogen fixation in pastures using small core samples. N. Z. Agric. Res. 19 (1976), 451-458. SIMEK, M., VACEK, V., Ul.EHLOV A, B. : Study on nitrogen fixation by stands of white clover (Trifolium repens L.). Rostl. VYr. 33 (1987), 279- 292 (in Czech). SKRDLETA, V., NASINEC, Y., HAVLOV A, M.: Relationship between acetylene reduction and some parameters of symbiotic nitrogen fixation in pea (Pisum smivum L.). Rost!. Vyr. 22 (1976), 88! -888 (in Czech). ZuBERER, D. A., ALEXANDER, D. B. : Effects of oxygen partial pressure and combined nitrogen on Nrfixation (C 2H2 ) associated with Zea mays and other gramineous species. PI. Soil 90 (1986), 47-58. Author's address: lng. M. Simek, Institute of Soil Biology, Czechoslovak Academy of Sciences, Na sadkach 7, CS - 37005 Ceske Budejovice, Czechoslovakia.