Nitrogen fixation by intact grass-soil cores using 15N2 and acetylene reduction

Soil Biol. Biochem.

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0038-0717/85 $3.00+ 0.00 Copyright0 1985PergamonPressLtd

Vol. 17,No. I, pp. 87-91, 1985 All rights reserved

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NITROGEN FIXATION BY INTACT CORES USING 15N2 AND ACETYLENE

GRASS-SOIL REDUCTION

D. R. MORRIS, D. A. ZUBERERand R. W. WEAVER Department of Soil and Crop Sciences, Texas A&M University, College Station, TX 77843, U.S.A. (Accepted

25 July 1984)

Summary-To determine N, fixation by intact grass-soil cores, samples were collected from 25 sites in central Texas during the summer. Three cores (32 cm2 each) were extracted immediately adjacent to one another from single grass clumps or sods. Two of these cores were incubated under lOokC,H, in air and the third core was incubated for 12 h in an atmosphere with 10% “N, enrichment. Following incubation with 15N, the same core was assayed for rate of C,H, reduction (AR). Rates of AR were generally low and quite variable (O-7.6 pmol CzH4 core-’ day-‘). 15N, was incorporated into root and shoot tissues within 12-24 h. Extrapolated values of N, fixation based on “N, incorporation ranged from 0 to 20 kg N ha-’ 100 day-‘. The ratio of C,H, reduced (pmol C,H, core-’ day-‘) to N, fixed @mol N, fixed core-’ day-‘) was highly variable ranging from 0 to 12. This study confirmed that N, is fixed in the rhizosphere of grasses grown in Texas through the use of 15N, and demonstrated that incorporation of fixed N into shoots was-relatively rapid.

INTRODUCTION

search using 15N, is needed to more accurately define the actual rates of N, fixation and the fate of the fixed N. There is little information regarding measurement of rates of N, fixation by grasses using C,H, reduction and 15N2.Our objective was to determine the rate of N, fixation by intact grass-soil cores using both “N, and C2H2 reduction.

Within the past decade, considerable attention has been focused on the potential of a wide variety of tropical and temperate grasses to support active populations of N,-fixing bacteria on their root systems (Neyra and Dobereiner, 1977; van Berkum and Bohlool, 1980). Numerous investigations have led to the accumulation of records of the rates of Nz fixation for various associative symbioses (Knowles, 1977; van Berkum and Bohlool, 1980). Most of the reports are based on measurements of N, fixation made through the use of the C,H, reduction (AR) assay and by applying calculations to extrapolate these values to seasonal rates of Nz fixation. The theoretical ratio of 3 moles of C,H2 reduced for each mole of Nz reduced is used in extrapolating quantities of N, fixed based on AR. Evidence indicates that this assumption is generally not acceptable for legumes (Hardy et al., 1973; Bergersen, 1970), and even less acceptable for associative systems. Steyn and Delwiche (1970) found AR to “N, fixation ratios to vary from 3 to greater than 8 in non-symbiotic N,-fixing microorganisms. To be used with confidence the AR assay should be calibrated with careful experimentation employing the use of 15N, incorporation under the same conditions for which the AR assay is to be used. In this manner, a more acceptable C,Hz-to-N, molar ratio can be determined and used in the conversion of AR rates to Nz fixation rates. De-Polli et al. (1977) reported that incorporation of N from 15N, into two tropical grasses (Paspalum notatum and Digitaria decumbens) was slow but progressive over 24 h and translocation to rhizomes and leaves ceased after 17 h. Matsui et al. (1981), working with sugar cane in Brazil, reported that “Nz incorporated into a plant exposed to 15N, for 48 h remained in the roots and was not detected in the leaves. They suggested that a sink existed in the root system and that a time-course evaluation would be needed to detect movement to leaves. Further re-

MATERIALSAND

METHODS

Soil cores containing grasses indigenous to central Texas were obtained from 25 sites in and around College Station, Texas, during mid-summer. Cores with intact plants were extracted using a steel coring cylinder (33 x 6.4 cm dia). The coring device was placed over the above-ground portion of the grass plants and driven into the soil to a depth of 15 cm and the core extracted. In a few instances, plant tops were trimmed to a 15 cm height to facilitate taking the core sample. In order to compare C,H, reduction rates to 15N2incorporation values, triplicate cores were collected adjacent to one another and treated similarly. Soil samples were taken from around the root zone at each site. Part of the sample was used to determine gravimetric water by drying at 105°C overnight. The other part of the sample was used to obtain a curve of water potential vs gravimetric water with a pressure plate apparatus (Richards, 1965). From these curves an extrapolation of the soil core water potential was obtained. Upon return to the laboratory Plexiglass cylinders (25 x 7 cm dia) were placed over the top of the cores. The cylinder top was permanently sealed with a plastic plate and the bottom was sealed with a rubber stopper. Serum stoppers for gas displacement were located in holes drilled in the side of the cylinder near the top and bottom. Cores were incubated under 10% C,H2 in air to determine rates of CzH2 reduction (AR). To determine “Nz incorporation, 50 ml of air was removed from the cylinder head space and 87

D. R. MORRISet al

88

replaced with 50 ml of 15N2(99 atom%). Gas mixing was facilitated by repeatedly filling and emptying the syringe used to inject the desired gases. Incubation times for AR and “N, fixation were 12-15 h. Air volume in each Plexiglass cylinder plus grass-soil core was determined by displacing the air with water. The %“N enrichment for each core was calculated based on its volume and quantity of 15N, added. Concentrations ranged from 8.4 to 12.2 atom%i5N. For each site, one core was assayed for “Nz incorporation while the two companion cores were simultaneously assayed for AR. Following incubation of the 15N2soil core, the cylinder was opened and air was allowed to exchange after which it was resealed and AR assayed. All cores were held at 2%28°C in the dark. Experiments indicated that ~0, within the closed cylinder was reduced by 15520% during a 12 h incubation and CrH, production was found to be linear between 3 and 24 h using this system. After volume determinations, plant tops were cut off at the soil level and roots were washed free of soil. All plant materials were dried at 65°C for 3 days, ground three times (< 1 mm) using a cyclone mill, and analyzed for total N (Nelson and Sommers, 1973) and “N content (Porter and O’Dean, 1977). Gas chromatography and mass spectrometry

Acetylene reduction was measured by gas chromatography (Hardy et al., 1973). The GC was equipped with a hydrogen flame ionization detector and a stainless steel column (183 x 0.3 cm dia) Table 1. Soil moisture

content

packed with Porapak N (Waters Assoc., Framingham, Massachusetts). “N content was measured using a Micromass 602-D (VG Micromass, Winsford, U.K.) mass spectrometer equipped with twin Faraday bucket collectors for simultaneous mass 28 and mass 29 collection. Plant tissues from the cores not used for exposure to 15N2were used to determine 15N natural abundance. The amount of N fixed was determined using the calculations of Vose et al. (1981). RESULTS AND DISCUSSION

Acetylene reduction was highly variable between plant species and within the same plant species (Table 1). Highest rates of AR were associated with plants from soils with higher moisture contents (Table 1), although it is of interest to note that measurable AR was also associated with cores having water potentials less than - 1.5 MPa. The higher rates of AR associated with the wetter cores are most likely due to decreased aeration and the better moisture status for both plant and microbial growth. It could also be due to enhanced exudation of utilizable C sources from roots under conditions of Or depletion (Wiedenroth, 198 1). With few exceptions the three cores from the same grass clump gave reasonably consistent rates of AR. Rates of AR by the cores previously exposed to 15N, for 12-15 h were not markedly different than those for cores not treated in this way (Table 1). The rates of AR we measured are similar to those observed by

and acetylene reduction

activities

of grass-soil

cores

C,H,

Site

4 6 I

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Plant” Vaseygrass Bermudagrass Switchgrass Switchgrass Kleingrass Kleingrass Bermudagrass Vaseygrass Vaseygrass Rush Johnsongrass Crabgrass Bermudagrass Barnyardgrass Bermudagrass Bermudagrass Bermudagrass Buffelgrass BulTelgrass Kleingrass Kleingrass Switchgrass Switchgrass Johnsongrass Green Soraneletou

Soil moisture (MPa)

Core 1 (urn01 core-’

-0.03 -0.09 -0.05 -0.06 -0.22 -0.11 -1.1 - l.SDTC -0.13 - 1.5DT -0.61 -0.71 -0.91 -0.03WT -0.03WT -0.03WT -0.03WT -1.3 - 1.5DT - 1SDT - 1.5DT - 1.5DT - 1.5DT - 1SDT -0.70

0.66 0.00 0.00 0.00 0.00 0.00 0.22 0.00 1.22 0.06 0.18 0.04 0.12 1.71 0.10 0.08 0.08 0.10 0.12 0.10 0.22 0.24 0.52 0.56 0.16

Core 2 day-‘) 1.31 0.00 0.00 0.00 0.00 0.00 0.40 0.00 0.48 0.00 0.10 0.12 0.24 2.53 0.06 0.12 0.10 0.12 0.06 0.08 0.10 0.32 0.34 0.12 0.00

Core 3b 7.61 0.66 1.31 0.00 0.00 0.00 0.30 0.02 0.10 0.00 0.07 0.05 0.12 2.60 1.73 0.12 0.10 0.07 0.08 0.07 0.12 0.26 0.24 0.07 0.00

“Latin names are as follows: barnvarderass-Echinochloa crusmfli var. cruwalfi: bermudaerass-Cvnodon da&n, CL.1 Pers.: buffelnrass-&nchrus cilia& L.: southern crabgrass--Digitaria ciliaris (Retz.) Keel.; green sprangletop Leprochloa dubia (H.B.K.) Nees; Johnsongrass-Sorghum halapense (L.) Pers.; kleingrass-Panicum coloratwn L.; switchgrass-Panicurn oirgarum L.; vaseygrass-Pmpalum uroillei Steud. bValues for the third core were obtained following its exposure to ISN, for 12 h. To convert to kg N fixed ha/l00 days multiply by 2.9 (assuming C,H,/N, ratio of 3: 1). ‘DT = drier than, WT = wetter than. ,I

I

“N, incorporation by grasses Table 2. Atom%“N

excess in shoots and roots of plants from grass-soil ‘sN, or only C,H,” Shoot

Site

I 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19 20 21 22 23 24 25

89 cores exposed

to

Root

‘SN,

C,Hzb

Enrichment’

‘SN,

C,HTb

EnrichmenF

0.0036 0.0035 0.0024 0.0032 0.0034 0.0038 0.0017 0.0018 0.0025 0.0033 0.0020 0.0073 0.0029 0.0077 0.0025 0.005 I 0.0040 0.0020 0.0011 0.0014 0.0019 0.0022 0.0018 0.0018 0.0029

0.0018 0.0016 0.0016 0.0020 0.0014 0.0029 0.0016 0.0018 0.0014 0.002 1 0.0021 0.0022 0.0008 0.0030 0.0018 0.0022 0.0030 0.0020 0.0016 0.0011 0.0018 0.0009 0.0009 0.0013 0.0019

0.0018d 0.0019* 0.0008d 0.0012* 0.0020d 0.0009d 0.0001 0.0000 0.0011~ 0.0012d 0.0000 0.005ld 0.002ld 0.0047d 0.0007d 0.0029d 0.0010~ 0.0000 - 0.0005 0.0003 0.0001 0.00136 c.0009d 0.0005 0.0010~

0.0024 0.0027 0.0026 0.0027 0.0030 0.0032 0.0020 0.0024 0.0028 0.0050 0.0020 0.0053 0.0036 0.0045 0.0028 0.0055 0.0095 0.0018 0.0018 0.0020 0.0021 0.0028 0.0026 0.0022 0.0038

0.0017 0.0018 0.0018 0.0022 0.0016 0.0022 0.0015 0.0024 0.0019 0.0024 0.001 I 0.0018 0.0024 0.0028 0.0020 0.0030 0.0024 0.0015 0.0013 0.0016 0.0018 0.0018 0.0017 0.0018 0.0019

0.0007d 0.0009d 0.0008* 0.0005 0.0014d 0.0010~ 0.0005 0.0000 0.0009d 0.0026’ 0.0009* 0.0035d 0.00 12d 0.0017d 0.0008~ 0.0022d 0.007ld 0.0003 0.0005 0.0004 0.0003 0.0010~ 0.0009* 0.0004 0.0019d

aAtom%“N excess is equal to the atom%“N in the plant sample less the atom%“N in atmospheric N, (0.3663 atom%15N, Junk and Svec, 1958). qhe C,H, core value is the mean of the two crass-soil cores collected from each site. that we;e &posed to C,H,. ‘Enrichment is the value for the “N, core less the value for the C,H, core. dThe degree of enrichment exceeded two times the standard deviation (+ 0.0006) of the cores not exposed to “N,.

numerous investigators in many different geographic locales (Knowles, 1977; Dart and Day, 1975), and are lower than those reported in an earlier survey of Texas grasses made when moisture conditions were more favorable for plant growth (Weaver et al., 1980). If the theoretical 3-to-1 ratio of C,H2 reduced to N2 fixed is assumed, then on a core area basis the kg N fixed ha-’ 100 day-’ ranged from 0 to 22. Most of the rates of fixation were less than 5 kg N fixed ha-’ 100 day-‘. Measurement of Nz fixation by use of “N2 is more complicated than by AR because the gaseous N is incorporated into the plant organic matter. The degree of enrichment is dependent on the rate of Nz fixation and the quantity of N contained in the plant which dilutes the fixed N. Also, a complication arises because the plant may contain 15N in excess of that in the air due to soil enrichment with “N (Shearer et al., 1974). Control grass-soil cores not exposed to “N, were used to determine the natural abundance levels of 15N to be employed as correction factors (Table 2). The natural abundance levels of “N in the control grass-soil cores were subtracted from the 15N, exposed companion-cores to determine the degree of enrichment due to N2 fixation. Natural abundance levels in unexposed cores varied from site to site and ranged from a high of 0.0030 atom%‘5N excess in grass shoots at site 17 to 0.0009 atom”k’5N in shoots at sites 22 and 23. Discrimination of 15N causes differences in 15N content between plant tops and roots (Shearer et al., 1980). Therefore, separate analyses were made on shoots and roots. Generally there was close agreement between shoots and roots but analysis of variance

indicated a significant site by plant part interaction (P = 0.05). Most of the grass-soil cores exposed to “N, were enriched with respect to companion cores at the same location. Analytical error associated with repeated sample 15N determinations was only +O.OOOl atom%15N. Thus, precision of the values for individual exposed cores is quite high. The standard deviation for the mean value of the two plant-soil cores at each location not exposed to “N2 enrichment was + 0.003 atom%“N. If it is considered that a difference of two standard deviations from the mean atom%15N excess in the shoots or roots of the unexposed grass-soil cores is required for a significant difference, then 18 of the 25 grass-soil cores exposed to a “N, enriched atmosphere were active in N, fixation. Only for site 4 was N, fixation indicated to have occurred in shoots but not in roots. For roots from this location “N content was higher than the control roots but not enough to exceed two standard deviations from the control. Roots at site 11 indicated N, fixation occurred but there was no 15Nenrichment in shoots. The quantity of N, fixed ranged from 0 to 2.29 Fmol N core-’ day-’ for site 12 (Table 3). The highest rate extrapolated to approximately 20 kg N ha-’ assuming a constant rate of fixation for 100 days. The second highest rate was 1.19 pmol N core-’ day-’ for site 16 and it extrapolated to approximately 10 kg N ha-’ for 100 days. The same cores high in “N, fixation were not very active in AR activity (Table 1). Core 3 for sites 1, 3, 14 and 15 showed the greatest AR activity (Table 1) and all indicated significant “N, incorporation (Table 2).

90

D. R. MORRIS et al. Table 3. Dry weight and nitrogen content to “N? and the auantitv

Site I 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 aTo convert

of shoots and roots of grasses in cores exposed of N, fixed in each olant Dart N, fixed”

Dry weight

Total N

Shoots Roots (r: core-‘)

Shoots Roots (me, core-‘)

8.00

1.62 6.85 9.20 1.50 3.16 6.34 I .45 5.81 3.30 2.63 0.98 1.26 1.86 2.52 5.33 1.62 4.81 5.90 2.31 3.06 6.33 4.21 10.28 1.78

7.50 I .46 9.16 11.26 2.04 3.40 13.09 4.92 4.78 5.38 1.86 4.87

1.69 1.15 1.16 2.49 1.24 55.46 12.38 4.68 4.99 13.45 5.42 23.96 1.52

40 12 39 46 I1 20 93 26 26 43 12 61 15 4.9 14 30 7.3 350 110 21 19 36 13 110 7.3

38 9.6 21 19 5.7 20 32 7.8 14 34 15 21 24 14 33 46 15 18 25 8.1 8.6 7.0 8.4 18.5 13.5

to kg N fixed ha 100 days-’

Shoots Roots (urn01 N, cores’

multiply

The rate of AR for core 3 (Table l), site 1, extrapolated to 22 kg N ha-’ assuming a conversion ratio of C,H,:N, of 3: 1 and 100 days of activity. For core 3 at site 1, the “N2 incorporation rate was only 5 kg N ha-‘. The results indicate inconsistency between rates of AR and “N, incorporation. Cores demonstrating AR activity but not “N, incorporation would be expected if the activity was due to microorganisms in the rhizosphere. The C,H, produced by the microorganisms would be detected by GC but incorporated “N2 would not be detected in the plant if the N was not transferred from the and the microorganisms were microorganisms washed from the root surface during preparatory procedures for N analysis. No explanation can be given as to why some sites exhibited relatively high rates of “N, fixation but relatively low rates of AR. The dry weight of the shoots or roots in the cores did not correlate significantly (P = 0.05) with the quantity of N fixed in either shoots or roots (Table 3). For instance, site 18 (Table 3) contained the most roots but 15N, was not incorporated into shoots or roots (Table 2) and AR activity was low (Table 1). The quantity of shoots and roots was also relatively high for site 24 and again N, fixation was not evident (Table 2). By contrast, site 12 contained a moderate quantity of roots and the lowest amount of shoots but N2 fixation was relatively high (Table 3). The data clearly indicate that 15Nwas incorporated into plant constituents fairly rapidly (12-24 h) and that fixed “N was apparently translocated from the roots to the shoots of some but not all plants within 24 h. This implies that mineralization of ‘*N incorporated into microbial cells associated with the roots of grasses can be quite rapid as well as the subsequent assimilation and translocation of the fixed N. The data do not rule out the possibility of N, fixation occurring in the phyllosphere as a mechanism For the

0.43 0.10 0.08 0.12 0.06 0.09 0.02 0.00 0.09 0.25 0.00 0.76 0.38 0.37 0.14 0.81 0.11 0.00 0.02 0.02 0.00 0.06 0.05 0.05 0.09

0.15 0.05 0.18 0.11 0.08 0.11 0.26 0.00 0.14 0.68 0.07 1.53 0.13 0.05 0.07 0.38 0.08 0.67 0.34 0.05 0.03 0.22 0.06 0.29 0.10

Total dav-‘) 0.58 0.15 0.26 0.23 0.14 0.20 0.28 0.00 0.23 0.93 0.07 2.29 0.51 0.42 0.21 1.19 0.19 0.67 0.36 0.07 0.03 0.28 0.11 0.34 0.19

by 8.7.

rapid incorporation of 15Ninto shoot tissues. Further research is necessary to resolve this point. One problem of major concern in using the AR assay to determine rates of N, fixation by excised roots and cores is the proper conversion factor to use for converting rates of AR to rates of Nz fixation. Most investigators have simply used the theoretical molar ratio of 3 moles C2H, reduced per mole of Nz fixed. The data obtained using cores subjected first to incubation with “N, and then with 0.11 atm C2H, indicated considerable variation in C,H,-to-N, ratios (Tables 1 and 3). However, the C,H,-to-N, ratios were in the range reported for other Nz fixing systems (Bergersen, 1970; Steyn and Delwiche, 1970) and ranged from 0 to 12 moles of C,H, per mole N2 fixed. These results further emphasize the need for caution in interpretation of AR data for studies of this nature and they demonstrate that “N, incorporation studies can be of considerable value in the quantification of associative N, fixation during shortterm incubation of grass-soil cores. The incorporation of 2.29 pmol of N, in 24 h by crabgrass (Table 3) indicate agronomically-significant rates of fixation were occurring and that the plant was incorporating the fixed N. These results provide encouragement to survey additional systems using 15N, to determine if higher rates of Nz fixation are occurring and to determine the environmental constraints. Acknowledgements-We thank the Sid W. Richardson Foundation for support of this research. The technical assistance of Mrs Lisa Lucia and Mrs Mallika Rajakulendran in sample preparation and mass spectromeiric analyses is gratefully acknowledged. REFERENCES

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