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Nutrition, moisture and rhizobial strain influence isotopic fractionation during N2, fixation in pasture legumes
Soil
Biol.
Biochem.
Vol.
21. No.
I. pp.
6S-60.
1989
0038-0717!89
Printed in Great Britain. All rights reserved
53.00 + 0.00
Copyright Q 1989 Pergamon Press plc
NUTRITION, MOISTURE AND RHIZOBIAL STRAIN INFLUENCE ISOTOPIC FRACTIONATION DURING N2 FIXATION IN PASTURE LEGUMES F.
LEDGARD
MAF,
Private
S. Ruakura
Agricultural
Centre.
Bag, Hamilton,
New Zealand
(Accepted 27 June 1988) Summary-White clover (Tri/olium repens L.) and red clover (T. prarense L.) were grown in a medium free of combined nitrogen (N-free) with fii~obium leguminosarum bv. frijolii strain PDD 2668 or a mixture of field isolates of rhizobia. The natural abundance of tJN was always lower in shoots than in roots and nodules. Whole plant N was depleted in lJN (-0.44 and -0.1% for white and red clovers, respectively) and unaffected by inoculation treatment. However, shoots were more depleted in “N and roots and nodules were more enriched with the field isolates. With white clover, other factors examined were molybdenum (MO) and phosphorus nutrition (low vs high levels), watering (regular vs less frequent) and water-logging. Whole plant N was more depleted in tJN with low MO nutrition or with less frequent watering. Although these effects were small, they have not been accounted for in the past and indicate reduced precision in estimates of isotopic fractionation, and therefore of Nr fixation using natural “N abundance.
INTRODtiCTlON
There is increasing use of the natural IsN abundance method for measuring Nz fixation by legumes (Shearer and Kohl, 1986). This method relies on legume N assimilated from atmospheric N, having a significantly different “N abundance from that assimilated from soil. Although the natural abundance of “N in atmospheric N2 is constant (Mariotti, 1983) there can be significant isotopic fractionation during N, fixation (Kohl and Shearer 1980). Shearer and Kohl (I 986) and Ledgard and Peoples (1988) suggested that isotopic fractionation during N2 fixation by legumes should be assessed before using the natural “N abundance method to measure N2 fixation in the field. Isotopic fractionation is commonly assessed by growing the nodulated legume in N-free solution in a glasshouse and by measuring
the lJN concentration in the legume. Published estimates of isotopic fractionation during N, fixation by pasture legumes are given in Table I. Most estimates are significantly different from the “N abundance of atmospheric N:, although there are relatively large differences between laboratories. Studies have also shown differences in rJN abundance between plant parts and this is influenced by the rhizobial strain used (Steele et al., 1983; Bergersen et al., 1986; Yoneyama er al., 1986). However, there have been no reports comparing the influence of other factors (e.g. nutrition or environment) on isotopic fractionation. Such factors may have influenced the values shown in Table I. In calculating the proportion of legume N fixed using natural 15N abundance, it is common to use a single estimate of isotopic fractionation during N2 fixation (from glasshouse-grown plants) and apply it
Table I. The natural abundance of “N in nodulated pasture legumes grown in N-free medium Legume Jledicago
Trijolium
Tri/olium
Tri/olium
Cultivar sarica
pmrense
subrerraneum
repens
Lotus peduncularus
Mircille ? Hunrcr River Humer River Du puits Alpilles ? Kenland Ml Barker MI Barker Bacchus Marsh Grassl. Huia ? Common Grassl. Maku
J’sN(%o) -0.92b O.Ob 0.97 3S6 -0.92 -0.88b I .88 - I .07 2.S8 0.59 2.48 0.58 - I .9k -0.98 -0.F
‘With respect to atmospheric N,, or to a standard of -0.55% bPlanl shoots only. ‘Mean for several di!Terenr Rhizobium strains. dValues are SD. ‘Unpublished Ph.D. thesis, Australian National Umversity.
SE 0.2 0. I3 0.65’
0.14
0.11 0.91’ 0.12 0.2 0.3
Source Marioui er af. (I 980) Steele cf al. (1983) Ledgard ( 1984)’ Turner and Bergersen (1983) Yoneyama er al. (1986) Mariotti cf of. (1980) Kohl and Shearer (1980) Yoneyama cf al. (1986) Bergersen and Turner (1983) Ledgard (1984)* Turner and Bergersen (1983) Ledgard (1984r Steele ef al. (1983) Yoneyama 41 al. (1986) Smelt er al. (1983)
for Turner and Bergersen (1983).
F.
S.
66
LEDGARD
al., 1984) which was converted
to NZ (Ross and Martin, 1970) and the iSN concentration was determined using a model 602E isotope ratio mass spectrometer (VG Isogas, Middlewich, Cheshire, U.K.). Results are expressed as 61SN (in ‘XW) where:
to a range of field conditions. Thus, it is important to examine the factors that may affect this estimate. I have examined some effects of mineral nutrition, moisture and rhizobial strain on isotopic fractionation during NZ fixation by two pasture legumes.
6 IsN = 1000 x (Rump,c- Rrcrerrnc. )/Rrercrtnce
.MATERIALS AND METHODS
and
Plants and growth conditions
R = mass 29:mass 28.
Seeds of white clover (Trrfolium repens L. “Grasslands Huia”) and red clover (T. pratense L. “Grasslands Pawera”) were surface-sterilized using H,SO, and grown in 15 cm dia pots (NaOCI treated) containing I kg acid-washed quartz sand (Steele et al., 1983). After sowing and addition of inoculum, a 2 cm layer of wax-coated sand was placed on the surface of each pot to act as a dust filter. A glass tube filled with glass wool was placed in the centre of each pot for introduction of N-free nutrient solution (Smith et al., 1983) below the wax-coated sand. Each pot contained I2 plants and these were maintained in a glasshouse at temperatures between 1I and 24’C (average 17°C).
The reference was O,-free atmospheric Nz. RESULTS
E#ect of cloter and rhkobia
There was no difference between white and red clover in N accumulation when compared under similar growth conditions (Table 2). However, the N yield of shoots was larger (P < 0.05) when either clover was inoculated with rhizobial strain PDD 2668 than when inoculated with the mixed field isolates. The 6”N of white clover shoots was lower (P c 0.05) using the field isolates of rhizobia than strain PDD 2668, whereas the reverse occurred for roots. The net effect was similar ij”N values on a whole plant basis. A similar trend was observed with red clover. However, the ii”N of whole red clover plants was less negative (P < 0.05) than that of white clover. In all cases. shoots had much lower S”N values than roots. This was a mirrored relationship in that the more negative 6 “N values for clover shoots corresponded with roots having a higher positive 6”N value. The largest difference between shoots and roots occurred with the field isolates and with white clover. Seed N represented < I% of total plant N at harvest. The 5 I5N of seed N was 0.03 and I .730/wfor white and red clovers. respectively. Thus, there was no need to adjust the bi5N values of harvested white clover for that in the initial seed N of white clover. For red clover, adjustment for the S”N of seed N resulted in the S”N for treatments with PDD 2668 or field isolates changing from -0.14 and -0.1 I (Table 2) to -0.16 and -0.14%, respectively.
Treatments
White clover was used in a 2’ factorial with 2 levels of added molybdenum (MO, 0 or 0.02pg ml-‘), phosphorus (P, 5 or 4Ojlg ml-‘), watering (every day or once every 2-4 days) and Rhkobium feguminosarum bv. triJolii (strain PDD 2668 which is used for seed inoculation in New Zealand or a mixture of 20 cultures of rhizobia isolated from clover root nodules from an experimental field site). There were 2 replicates. In addition, there were 6 replicates of a water-logging treatment: white clover inoculated with rhizobial strain PDD 2668 was grown at high MO and P and subject to water-logging for 2 days in every 3. Treatments examining host effects involved comparison of white clover from the factorial outlined above; with red clover grown with either rhizobial strain PDD 2668 or the field isolates at high MO and P, and watered daily. There were 4 replicates. All treatments (except rhizobia) were imposed 2 weeks after sowing. Plunt analysis
Effect of nutrition and watering
Plants were harvested 109 days after sowing and washed free of adhering growth medium. After separation into shoots and roots and nodules, plant material was dried at 65’C, weighed and ground. Plant samples were analysed for total N (Ledgard er Table
2. Effects of host plant and rhizobia
Plants
were inoculated
with
rhizobial ootimum
on N accumulation
strain
PDD2668
nutrition White
2668 N yield
The N yield of shoots and roots was approximately halved by reduced MO, P or watering (Table 3). This was associated with lower N concentrations in shoots and roots with low P and less frequent watering, and and on the natural
or a mixture
and waterine
(Fl)
of “N.
and given
III = 4)
clover FI
abundance
of Reid isolates
Red clover SED
2668
FI
SED
(mg par’)
Shoots Roots
+ nodules
To:al
193
I53
I2
I82
139
55
54
5
64
57
248
207
246
196
-0.82
- I .41
0.27
-0.38
- 0.69
0.26
2.54
0.35
I .30
0.32
I
0.21
I6
II 5 I4
6”N (X) Shoots Roars Total
+ nodules
0.75 -0.46
-0.42
0.23
0.58 -0.14
-0.1
Isotopic f~ctionation during N2 fixation
$7
Table 3. EiTecrof optimaland sub-optimallevelsof molybdenum,phosphorus and watering on N accumulation and on Ihe natural abundance of “N in white clover (n = 16) Shoots Qximai N yield (mg pot ’ ) MO P Watering 6”N (460) MO P Watering
Roots + nodules
Sub-optimal
SED
Optimal
Subspcimal
88.7 84.6 82.2
37.1 41.2 43.6
2.9 2.9 2.9
31.5 28.5 27.9
12.7 IS.7 16.3
- 1.17 -1.24 -1.16
-1.36 -1.30 - 1.37
0.08 0.08 0.08
1.70 1.55 1.36
I .56 I .?2 1.90
with a higher N concentration with nil MO addition (data not presented). In the nil MO treatments, clovers obtained some MO as a contaminant in the water and nutrient solution. Optimum MO addition resulted in less negative 61SN values (P < 0.05) in shoots and whole plants than with no added MO. Similarly, optimum watering resulted in higher 6”N values for shoots, roots and whole plants compared with less frequent watering (when wilting of plants sometimes occurred). There was no effect of rate of P addition on 61sN of shoots, roots or whole plants. There was a significant interaction (P < 0.05) between the level of watering and the amounts of MO or P on the clover N yields, with the best nutritional response occurring under regular watering (data not presented). Watering regime also interacted with inoculation (P < 0.01). with clover N yields being higher from strain PDD 2668 than from the field isolates only with regular watering. However, 615N values showed no treatment interactions. An exception is the MO x P interaction for the roots and nodules, and whole plants (P c 0.05) (Table 4). Differences in 61sN of whole plants between the two MO levels were only present at low P status.
Water-logging reduced N accumulation in white clover by 29% in shoots and by 51% in roots (Table 5). However, there was no significant effect on 61SN, ahhough there was a trend similar to that for stress
TOtd
SED
by sub-optimal and 5).
Optimal
Sub-optimal
SED
1.3 1.3 I.3
120.2 113.1 110.1
49.8 56.9 59.9
4.1 4.1 4.1
0.16 0.16 0.16
-0.39 -0.50 -0.38
-0.62 -0.50 -0.63
0.09 0.09 0.09
MO or watering (compare Tables 3
DISCL’SSION
The S”N of shoots of white and red clover was always less than that of roots and nod&s as observed by others (Shearer er al., 1980; Steele et al., 1983; Yoneyama er al., 1986). In my study, shoots were O&4.0%0 lower than that in roots and nodules and this difference was generally larger than that due to treatments. The difference between the 6”N of shoots, and roots and nodules was in~uenced by rhizobiaf strain, being much larger for the rhizobial isolate from the field than for strain PDD 2668. Thus, if results from strain PDD 2668 were used in natural “N abundance calculations on white clover shoots from the field site (from which the field isolates were collected), NI fixation would be over-estimated. For example, the proportion of N fixed by white clover shoots at the field site averages about 75% (Ledgard et al., 1988) whereas an equivalent estimate based on the 6”N of air-derived N from strain PDD 2668 would be 84% [the 6 “N of soil-derived N at the site is about 3.8%0}. Pastoral soils can contain many strains of rhizobia and the strains that infect Trifolium species may vary with a number of factors including environmental conditions (Brockwelf et al., 1968). Consequently, it is difficult to obtain an appropriate value for the 6 15N of air-derived N in clover shoots for the rhizobial
Table 4. lnteraclion of molybdenum and phosphorus nutrition on N accumulation and on natural abundance of “N in white cfover In = 8) Shoots
--.. N yield (mg W MO 0.02 MO S&D &‘“N 0 MO 0.02 MO SED
Total
Roots + nodules
SP
4O’P
SF
4OP
SP
4OP
32.7 49.1
41.4 127.7
Il.1 20.3
14.3 42.8
43.8 70.0
55.1 170.5
pot-‘) 4.1 -1.43 -1.17
1.9 -1.30 -1.17
1.33 1.66
0.11
5.8 2.07 1.56
-0.72 -0.31
0.23
-0.49 -0.52 0.13
1Values are pg ml-‘. Table 5. Effect of water-logging on N accumulation and on Ihe natural abundance of “N in while clover. Plants were grown with oplimum amounts of all nucricnrs but with no added N (n = 4 or 6 for regular watering or water-lo~ng, respcetively) N yield (mg pot-‘)
Regular watering Water-logging SED
6’sN (%)
Shoots
Roots
Total
Shoots
Roots
Total
192.8 137.0 9.0
55.4 26.9 4.0
248.2 163.9 12.1
-0.82 -1.04 0.22
0.75 1.18 0.27
-0.46 -0.65 0.22
68
S. F.
LEDGARD
strains infectinp; clover at a field site. Caution must also be placed &I interpreting results from studies in rhizobia-free soils comparing N. fixation by legumes inoculated with different rhizodial strains using the natural abundance of “N in legume shoots (e.g. Rennie and Kemp, 1983). unless estimates of isotopic fractionation are obtained for each host-Rhicobium combination. Despite the differences in “N abundance between plant parts. similar values were obtained for whole plants infected by the field isolates and PDD 2668. Bergersen et al. (1986) obtained similar results with Lupinus spp and various strains of Rhizobium lupini. Thus. estimates of N, fixation using natural r5N abundance will not be affected by rhizobial strain provided they are based on using whole plant material. Host plants can influence isotopic fractionation during N2 fixation. Comparative values of Sr5N for whole plants were -0.44% for white clover and -0.15%~ for red clover. This indicates that isotopic fractionation during N2 fixation should be determined for each legume species being studied (Shearer and Kohl. 1986). Differences in either MO or P nutrition caused Z-fold differences in N yield. However, only MO influenced the 61SN, with whole plants having more negative values under MO limitation (-0.62 vs -0.39%). This was most prominent at low P status, for unknown reasons. The influence of MO nutrition on isotopic fractionation may be related to the significant MO requirement for nodule function (Dilworth, 1974). In most N? fixation studies, nutrient limitations (other than N) have been overcome by addition of fertilizer. In contrast, fluctuations in soil moisture are common in many field studies and the effects of watering regime on isotopic fractionation were similar to those for MO nutrition. The 615N of whole plants with less frequent watering was more negative than that for regular watering (-0.63 vs -0.38%0, respectively). This increase in isotopic fractionation from water stress may be due to increased resistance to diffusion of N2 in root nodule tissue (Pankhurst and Sprent, 1975). In practice. the treatment differences in isotopic fractionation are relatively small and will only have a minor effect on estimates of N2 fixation using natural “N abundance (e.g. about 5% error in the field study described earlier). Nevertheless, this experiment does indicate that the true error associated with the estimate of isotopic fractionation during Nz fixation in the field is greater than that recorded from a single glasshouse study. The 615N of white clover plants grown under optimum nutrition and watering ranged between -0.28 and -0.59% (average -0.45%) whereas the range over different levels of nutrition and watering was 0.00 to - I. 19% (average -0.59%). Thus, the precision in estimating NZ in published fixation using natural lSN abundance field studies may have been over-estimated.
REFERENCES
Bergersen F. J. and Turner G. L. (1983) An evaluation of lJN methods for estimating nitrogen fixation in a subterranean clover-perennial ryegrass sward. Aus~raliun Journal
of Agricultural
Research 34, 391-401.
Bergersen F. J.. Turner G. L.. Amarger N., Mariotti F. and Mariotti A. (1986) Strain of Rhkobium harini determines natural abundance of “N in root nodules of Lupinus spp. Soil Riology
& Biochemisrry
_
18, 97-101.
__
Brockwell J.. Dudman W. F.. Gibson A. H.. Helv F. W. and Robinson A. C. (1968) An integrated programme for the improvement of legume inoculant strains. Transaclions of rhe 9th Inrernutional Congress of Soil Science 2, 103-t 14. Dilworth M. J. (1974) Dinitrogen fixation. Annual Review of Plant
Physiology
25, 81-l
14.
Kohl D. H. and Shearer G. (1980) Isotopic fractionation associated with symbiotic N, fixation and uptake of NO, by plants. Plant Physiology 66, 51-56. Ledgard S. F. and Peoples M. 8. (1988) Measurement of nitrogen fixation in the field. In Advances in Nitrogen Cycling in Agriculrural Ecosystems (J. R. Wilson, Ed.), pp. 351-367. C.A.B. International. Wallingford. U.K. Ledgard S. F.. Brier G. J. and Watson R. N. (1988) New clover cultivars for Waikato dairy pasture: establishment, production and nitrogen fixation during the first year. Proceedings 207-211.
of the New Zenlund
Grassland
Associarion
49,
Ledgard S. F.. Freney J. R. and Simpson J. R. (1984) Variations in natural enrichment of “N in the profiles of some Australian pasture soils. Ausfraliun Journal of Soil Research 22. 155-164. Mariotti A. (1983) Atmospheric nitrogen is a reliable standard for natural lSN abundance measurements. Nature 303. 685-687. Mariotti A., Mariotti F.. Amarger N., Pizelle G.. Ngambi J. hl., Champigny M. L. and Moyse A. (1980) Fractionnements isotopiques de I’azote lors des processus d’adsorption des nitrates et de fixation de I’azote atmospherique par les plantes. Physiologie Vegerale 18, 163-181. Pankhurst C. E. and Sprent J. E. (1975)The effects of water stress on the respiratory and nitrogen-fixing activity of soybean root nodules. Journal of Experimenral Botany 26, 287-304.
Rennie R. J. and Kemp G. A. (1983) N2 fixation in field beans quantified by “N isotope dilution. I. Effect of strains of Rhisobium phaseoli. Agronomy Journal 75, 640-644.
Ross P. J. and Martin A. E. (1970) A rapid procedure for preparing gas samples for nitrogen- I5 determination. Analyst 95, 8 17-822. Shearer G. and Kohl D. H. (1986) NL-fixation in field settings: estimations based on natural “N abundance. Ausrralian
Journal
of Plam
Physio1pg.y
13, 699-756.
Shearer G., Kohl D. H. and Harper J. E. (1980) Distribution of ‘>N among plant parts of nodulating and nonnodulating isolines of soybeans. Plunr Physiology 66, 57-60.
Smith G. S., Johnston C. M. and Cornforth I. S. (1983) Comparison of nutrient solutions for growth of plants in sand culture. Netc Phyrologisr 94, 537-548. Steele K. W., Bonish P. M.. Daniel R. M. and O’Hara G. W. (1983) Effect of rhizobial strain and host plant on nitrogen isotopic fractionation in legumes. Plant Physiology 72. 1001-1004. Turner G. L. and Bergersen F. J. (1983) Natural abundance of “N in root nodules of soybean. lupin, subterranean clover and lucerne. Soil Biology & Biochemistry 15, 525-530.
Ackno~ledgemenfs--I thank G. J. Brier and M. S. Sprosen for skilful technical assistance, P. M. Bonish and F. J. Neville for isolation of field rhizobia and supplying strain PDD 2668 and R. A. Littler and M. S. Sprosen for statistical analyses.
Yoneyama T., Fujita K.. Yoshida T.. Matsumoto T., Kambayashi I. and Yazaki J. (1986) Variation in natural abundance of “N among plant parts and in “N/“N fractionation during N? fixation in the legume-rhizobia symbiotic system. Planr and Cell Physiology 27, 791-799.
Biol.
Biochem.
Vol.
21. No.
I. pp.
6S-60.
1989
0038-0717!89
Printed in Great Britain. All rights reserved
53.00 + 0.00
Copyright Q 1989 Pergamon Press plc
NUTRITION, MOISTURE AND RHIZOBIAL STRAIN INFLUENCE ISOTOPIC FRACTIONATION DURING N2 FIXATION IN PASTURE LEGUMES F.
LEDGARD
MAF,
Private
S. Ruakura
Agricultural
Centre.
Bag, Hamilton,
New Zealand
(Accepted 27 June 1988) Summary-White clover (Tri/olium repens L.) and red clover (T. prarense L.) were grown in a medium free of combined nitrogen (N-free) with fii~obium leguminosarum bv. frijolii strain PDD 2668 or a mixture of field isolates of rhizobia. The natural abundance of tJN was always lower in shoots than in roots and nodules. Whole plant N was depleted in lJN (-0.44 and -0.1% for white and red clovers, respectively) and unaffected by inoculation treatment. However, shoots were more depleted in “N and roots and nodules were more enriched with the field isolates. With white clover, other factors examined were molybdenum (MO) and phosphorus nutrition (low vs high levels), watering (regular vs less frequent) and water-logging. Whole plant N was more depleted in tJN with low MO nutrition or with less frequent watering. Although these effects were small, they have not been accounted for in the past and indicate reduced precision in estimates of isotopic fractionation, and therefore of Nr fixation using natural “N abundance.
INTRODtiCTlON
There is increasing use of the natural IsN abundance method for measuring Nz fixation by legumes (Shearer and Kohl, 1986). This method relies on legume N assimilated from atmospheric N, having a significantly different “N abundance from that assimilated from soil. Although the natural abundance of “N in atmospheric N2 is constant (Mariotti, 1983) there can be significant isotopic fractionation during N, fixation (Kohl and Shearer 1980). Shearer and Kohl (I 986) and Ledgard and Peoples (1988) suggested that isotopic fractionation during N2 fixation by legumes should be assessed before using the natural “N abundance method to measure N2 fixation in the field. Isotopic fractionation is commonly assessed by growing the nodulated legume in N-free solution in a glasshouse and by measuring
the lJN concentration in the legume. Published estimates of isotopic fractionation during N, fixation by pasture legumes are given in Table I. Most estimates are significantly different from the “N abundance of atmospheric N:, although there are relatively large differences between laboratories. Studies have also shown differences in rJN abundance between plant parts and this is influenced by the rhizobial strain used (Steele et al., 1983; Bergersen et al., 1986; Yoneyama er al., 1986). However, there have been no reports comparing the influence of other factors (e.g. nutrition or environment) on isotopic fractionation. Such factors may have influenced the values shown in Table I. In calculating the proportion of legume N fixed using natural 15N abundance, it is common to use a single estimate of isotopic fractionation during N2 fixation (from glasshouse-grown plants) and apply it
Table I. The natural abundance of “N in nodulated pasture legumes grown in N-free medium Legume Jledicago
Trijolium
Tri/olium
Tri/olium
Cultivar sarica
pmrense
subrerraneum
repens
Lotus peduncularus
Mircille ? Hunrcr River Humer River Du puits Alpilles ? Kenland Ml Barker MI Barker Bacchus Marsh Grassl. Huia ? Common Grassl. Maku
J’sN(%o) -0.92b O.Ob 0.97 3S6 -0.92 -0.88b I .88 - I .07 2.S8 0.59 2.48 0.58 - I .9k -0.98 -0.F
‘With respect to atmospheric N,, or to a standard of -0.55% bPlanl shoots only. ‘Mean for several di!Terenr Rhizobium strains. dValues are SD. ‘Unpublished Ph.D. thesis, Australian National Umversity.
SE 0.2 0. I3 0.65’
0.14
0.11 0.91’ 0.12 0.2 0.3
Source Marioui er af. (I 980) Steele cf al. (1983) Ledgard ( 1984)’ Turner and Bergersen (1983) Yoneyama er al. (1986) Mariotti cf of. (1980) Kohl and Shearer (1980) Yoneyama cf al. (1986) Bergersen and Turner (1983) Ledgard (1984)* Turner and Bergersen (1983) Ledgard (1984r Steele ef al. (1983) Yoneyama 41 al. (1986) Smelt er al. (1983)
for Turner and Bergersen (1983).
F.
S.
66
LEDGARD
al., 1984) which was converted
to NZ (Ross and Martin, 1970) and the iSN concentration was determined using a model 602E isotope ratio mass spectrometer (VG Isogas, Middlewich, Cheshire, U.K.). Results are expressed as 61SN (in ‘XW) where:
to a range of field conditions. Thus, it is important to examine the factors that may affect this estimate. I have examined some effects of mineral nutrition, moisture and rhizobial strain on isotopic fractionation during NZ fixation by two pasture legumes.
6 IsN = 1000 x (Rump,c- Rrcrerrnc. )/Rrercrtnce
.MATERIALS AND METHODS
and
Plants and growth conditions
R = mass 29:mass 28.
Seeds of white clover (Trrfolium repens L. “Grasslands Huia”) and red clover (T. pratense L. “Grasslands Pawera”) were surface-sterilized using H,SO, and grown in 15 cm dia pots (NaOCI treated) containing I kg acid-washed quartz sand (Steele et al., 1983). After sowing and addition of inoculum, a 2 cm layer of wax-coated sand was placed on the surface of each pot to act as a dust filter. A glass tube filled with glass wool was placed in the centre of each pot for introduction of N-free nutrient solution (Smith et al., 1983) below the wax-coated sand. Each pot contained I2 plants and these were maintained in a glasshouse at temperatures between 1I and 24’C (average 17°C).
The reference was O,-free atmospheric Nz. RESULTS
E#ect of cloter and rhkobia
There was no difference between white and red clover in N accumulation when compared under similar growth conditions (Table 2). However, the N yield of shoots was larger (P < 0.05) when either clover was inoculated with rhizobial strain PDD 2668 than when inoculated with the mixed field isolates. The 6”N of white clover shoots was lower (P c 0.05) using the field isolates of rhizobia than strain PDD 2668, whereas the reverse occurred for roots. The net effect was similar ij”N values on a whole plant basis. A similar trend was observed with red clover. However, the ii”N of whole red clover plants was less negative (P < 0.05) than that of white clover. In all cases. shoots had much lower S”N values than roots. This was a mirrored relationship in that the more negative 6 “N values for clover shoots corresponded with roots having a higher positive 6”N value. The largest difference between shoots and roots occurred with the field isolates and with white clover. Seed N represented < I% of total plant N at harvest. The 5 I5N of seed N was 0.03 and I .730/wfor white and red clovers. respectively. Thus, there was no need to adjust the bi5N values of harvested white clover for that in the initial seed N of white clover. For red clover, adjustment for the S”N of seed N resulted in the S”N for treatments with PDD 2668 or field isolates changing from -0.14 and -0.1 I (Table 2) to -0.16 and -0.14%, respectively.
Treatments
White clover was used in a 2’ factorial with 2 levels of added molybdenum (MO, 0 or 0.02pg ml-‘), phosphorus (P, 5 or 4Ojlg ml-‘), watering (every day or once every 2-4 days) and Rhkobium feguminosarum bv. triJolii (strain PDD 2668 which is used for seed inoculation in New Zealand or a mixture of 20 cultures of rhizobia isolated from clover root nodules from an experimental field site). There were 2 replicates. In addition, there were 6 replicates of a water-logging treatment: white clover inoculated with rhizobial strain PDD 2668 was grown at high MO and P and subject to water-logging for 2 days in every 3. Treatments examining host effects involved comparison of white clover from the factorial outlined above; with red clover grown with either rhizobial strain PDD 2668 or the field isolates at high MO and P, and watered daily. There were 4 replicates. All treatments (except rhizobia) were imposed 2 weeks after sowing. Plunt analysis
Effect of nutrition and watering
Plants were harvested 109 days after sowing and washed free of adhering growth medium. After separation into shoots and roots and nodules, plant material was dried at 65’C, weighed and ground. Plant samples were analysed for total N (Ledgard er Table
2. Effects of host plant and rhizobia
Plants
were inoculated
with
rhizobial ootimum
on N accumulation
strain
PDD2668
nutrition White
2668 N yield
The N yield of shoots and roots was approximately halved by reduced MO, P or watering (Table 3). This was associated with lower N concentrations in shoots and roots with low P and less frequent watering, and and on the natural
or a mixture
and waterine
(Fl)
of “N.
and given
III = 4)
clover FI
abundance
of Reid isolates
Red clover SED
2668
FI
SED
(mg par’)
Shoots Roots
+ nodules
To:al
193
I53
I2
I82
139
55
54
5
64
57
248
207
246
196
-0.82
- I .41
0.27
-0.38
- 0.69
0.26
2.54
0.35
I .30
0.32
I
0.21
I6
II 5 I4
6”N (X) Shoots Roars Total
+ nodules
0.75 -0.46
-0.42
0.23
0.58 -0.14
-0.1
Isotopic f~ctionation during N2 fixation
$7
Table 3. EiTecrof optimaland sub-optimallevelsof molybdenum,phosphorus and watering on N accumulation and on Ihe natural abundance of “N in white clover (n = 16) Shoots Qximai N yield (mg pot ’ ) MO P Watering 6”N (460) MO P Watering
Roots + nodules
Sub-optimal
SED
Optimal
Subspcimal
88.7 84.6 82.2
37.1 41.2 43.6
2.9 2.9 2.9
31.5 28.5 27.9
12.7 IS.7 16.3
- 1.17 -1.24 -1.16
-1.36 -1.30 - 1.37
0.08 0.08 0.08
1.70 1.55 1.36
I .56 I .?2 1.90
with a higher N concentration with nil MO addition (data not presented). In the nil MO treatments, clovers obtained some MO as a contaminant in the water and nutrient solution. Optimum MO addition resulted in less negative 61SN values (P < 0.05) in shoots and whole plants than with no added MO. Similarly, optimum watering resulted in higher 6”N values for shoots, roots and whole plants compared with less frequent watering (when wilting of plants sometimes occurred). There was no effect of rate of P addition on 61sN of shoots, roots or whole plants. There was a significant interaction (P < 0.05) between the level of watering and the amounts of MO or P on the clover N yields, with the best nutritional response occurring under regular watering (data not presented). Watering regime also interacted with inoculation (P < 0.01). with clover N yields being higher from strain PDD 2668 than from the field isolates only with regular watering. However, 615N values showed no treatment interactions. An exception is the MO x P interaction for the roots and nodules, and whole plants (P c 0.05) (Table 4). Differences in 61sN of whole plants between the two MO levels were only present at low P status.
Water-logging reduced N accumulation in white clover by 29% in shoots and by 51% in roots (Table 5). However, there was no significant effect on 61SN, ahhough there was a trend similar to that for stress
TOtd
SED
by sub-optimal and 5).
Optimal
Sub-optimal
SED
1.3 1.3 I.3
120.2 113.1 110.1
49.8 56.9 59.9
4.1 4.1 4.1
0.16 0.16 0.16
-0.39 -0.50 -0.38
-0.62 -0.50 -0.63
0.09 0.09 0.09
MO or watering (compare Tables 3
DISCL’SSION
The S”N of shoots of white and red clover was always less than that of roots and nod&s as observed by others (Shearer er al., 1980; Steele et al., 1983; Yoneyama er al., 1986). In my study, shoots were O&4.0%0 lower than that in roots and nodules and this difference was generally larger than that due to treatments. The difference between the 6”N of shoots, and roots and nodules was in~uenced by rhizobiaf strain, being much larger for the rhizobial isolate from the field than for strain PDD 2668. Thus, if results from strain PDD 2668 were used in natural “N abundance calculations on white clover shoots from the field site (from which the field isolates were collected), NI fixation would be over-estimated. For example, the proportion of N fixed by white clover shoots at the field site averages about 75% (Ledgard et al., 1988) whereas an equivalent estimate based on the 6”N of air-derived N from strain PDD 2668 would be 84% [the 6 “N of soil-derived N at the site is about 3.8%0}. Pastoral soils can contain many strains of rhizobia and the strains that infect Trifolium species may vary with a number of factors including environmental conditions (Brockwelf et al., 1968). Consequently, it is difficult to obtain an appropriate value for the 6 15N of air-derived N in clover shoots for the rhizobial
Table 4. lnteraclion of molybdenum and phosphorus nutrition on N accumulation and on natural abundance of “N in white cfover In = 8) Shoots
--.. N yield (mg W MO 0.02 MO S&D &‘“N 0 MO 0.02 MO SED
Total
Roots + nodules
SP
4O’P
SF
4OP
SP
4OP
32.7 49.1
41.4 127.7
Il.1 20.3
14.3 42.8
43.8 70.0
55.1 170.5
pot-‘) 4.1 -1.43 -1.17
1.9 -1.30 -1.17
1.33 1.66
0.11
5.8 2.07 1.56
-0.72 -0.31
0.23
-0.49 -0.52 0.13
1Values are pg ml-‘. Table 5. Effect of water-logging on N accumulation and on Ihe natural abundance of “N in while clover. Plants were grown with oplimum amounts of all nucricnrs but with no added N (n = 4 or 6 for regular watering or water-lo~ng, respcetively) N yield (mg pot-‘)
Regular watering Water-logging SED
6’sN (%)
Shoots
Roots
Total
Shoots
Roots
Total
192.8 137.0 9.0
55.4 26.9 4.0
248.2 163.9 12.1
-0.82 -1.04 0.22
0.75 1.18 0.27
-0.46 -0.65 0.22
68
S. F.
LEDGARD
strains infectinp; clover at a field site. Caution must also be placed &I interpreting results from studies in rhizobia-free soils comparing N. fixation by legumes inoculated with different rhizodial strains using the natural abundance of “N in legume shoots (e.g. Rennie and Kemp, 1983). unless estimates of isotopic fractionation are obtained for each host-Rhicobium combination. Despite the differences in “N abundance between plant parts. similar values were obtained for whole plants infected by the field isolates and PDD 2668. Bergersen et al. (1986) obtained similar results with Lupinus spp and various strains of Rhizobium lupini. Thus. estimates of N, fixation using natural r5N abundance will not be affected by rhizobial strain provided they are based on using whole plant material. Host plants can influence isotopic fractionation during N2 fixation. Comparative values of Sr5N for whole plants were -0.44% for white clover and -0.15%~ for red clover. This indicates that isotopic fractionation during N2 fixation should be determined for each legume species being studied (Shearer and Kohl. 1986). Differences in either MO or P nutrition caused Z-fold differences in N yield. However, only MO influenced the 61SN, with whole plants having more negative values under MO limitation (-0.62 vs -0.39%). This was most prominent at low P status, for unknown reasons. The influence of MO nutrition on isotopic fractionation may be related to the significant MO requirement for nodule function (Dilworth, 1974). In most N? fixation studies, nutrient limitations (other than N) have been overcome by addition of fertilizer. In contrast, fluctuations in soil moisture are common in many field studies and the effects of watering regime on isotopic fractionation were similar to those for MO nutrition. The 615N of whole plants with less frequent watering was more negative than that for regular watering (-0.63 vs -0.38%0, respectively). This increase in isotopic fractionation from water stress may be due to increased resistance to diffusion of N2 in root nodule tissue (Pankhurst and Sprent, 1975). In practice. the treatment differences in isotopic fractionation are relatively small and will only have a minor effect on estimates of N2 fixation using natural “N abundance (e.g. about 5% error in the field study described earlier). Nevertheless, this experiment does indicate that the true error associated with the estimate of isotopic fractionation during Nz fixation in the field is greater than that recorded from a single glasshouse study. The 615N of white clover plants grown under optimum nutrition and watering ranged between -0.28 and -0.59% (average -0.45%) whereas the range over different levels of nutrition and watering was 0.00 to - I. 19% (average -0.59%). Thus, the precision in estimating NZ in published fixation using natural lSN abundance field studies may have been over-estimated.
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Ackno~ledgemenfs--I thank G. J. Brier and M. S. Sprosen for skilful technical assistance, P. M. Bonish and F. J. Neville for isolation of field rhizobia and supplying strain PDD 2668 and R. A. Littler and M. S. Sprosen for statistical analyses.
Yoneyama T., Fujita K.. Yoshida T.. Matsumoto T., Kambayashi I. and Yazaki J. (1986) Variation in natural abundance of “N among plant parts and in “N/“N fractionation during N? fixation in the legume-rhizobia symbiotic system. Planr and Cell Physiology 27, 791-799.