Evaluation of the xylem ureide method for measuring N2 fixation in six tree legume species

Evaluation of the xylem ureide method for measuring N2 fixation in six tree legume species

Soil Eiol. Biochem. Vol. 28, No. 3, pp. 281-289, 1996 Pergamon 0038-0717(95)00144-1 Copyright 0 1996Elswier Science Ltd Printed in Great Britain. A...

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Soil Eiol. Biochem. Vol. 28, No. 3, pp. 281-289, 1996



Copyright 0 1996Elswier Science Ltd Printed in Great Britain. All rights reserved 0038-0717/96 SI5.00 + 0.00



and M. B. PEOPLES4

‘NSW Agriculture, The Tamworth Centre for Crop Improvement, RMB 944, Tamworth, NSW 2340, Australia, *CSIRO Division Tropical Crops and Pastures, P.M.B., P.O., Aitkenvale, Qld 4814, Australia, )Department of Agriculture, University of Queensland, St Lucia, Qld 4067, Australia and YXRO Division Plant Industry, P.O. Box 1600, Canberra, ACT 2601, Australia (Accepted I September 1995)

Summary-The xylem ureide method, based on analysis of xylem sap for N solutes associated with N: fixation (ureides) and soil-N use (NO,, a amino-N), is used for estimating Plix (proportion of plant N derived from NZ fixation) of herbaceous species of the leguminous tribes Phaseoleae and Desmodieue and may have application for tree legumes. We report experiments, involving Calliandra calothyrsus, Leucaena leucocephala, Gliricidia sepium, Sesbania grandipora, Desmodium rensonii and Codariocalyx gyroides, to: (i) determine for each species the composition of N solutes of xylem sap and to calibrate, where possible, the xylem ureide method using 15Nisotope dilution as an independent measure of Pf~x; and (ii) establish appropriate procedures for sampling xylem sap in field studies. In the first experiment, the tree legumes were grown for 55-77 weeks and sampled repeatedly (6-9 times) for vacuum-extracted xylem sap, shoot dry matter, total N and 15N.Asparagine was the predominant amino compound in xylem sap of all species. The relative abundance of ureide-N in xylem sap (ureide-N as a proportion of u&de-N + a aminoN + NO:-N) was compared with Pfix for five of the six tree legumes. Sap of S. grandiporu could not be analysed for ureides and NO, using the standard calorimetric methods because of high levels of non-specific, background colour. For D. rensonii and C. gyroides, relative ureide-N ranged from less than 20% (Pfix of zero) to around 65 and 40%, respectively (Pfix of lOO%). Functions describing the relationships between relative ureide-N 0,) and Plix (x) were: y = 18.3 + 0.446x (r = 0.91) for D. rensonii; and y = 8.49 + 0.279.~ (r = 0.92) for C. gyroides. With the other three species, relative ureide-N was not related to Pfix. We concluded that the xylem ureide technique is suitable for estimating Pfix for D. rensonii and C. gyroides only. In the second experiment, established trees of G. sepium, D. rensonii, C. calothyrsus and C. gyroides were used to assess sampling protcols. Results indicated that vacuum extraction of sap should proceed immediately the branch is cut, that plants need to be sampled around noon to eliminate diurnal effects and that branch-to-branch variation in relative ureide-N values within a single tree was minor.


The successful exploitation of the tree and shrub (woody perennial) legumes for forage, mulch in alley cropping systems, fuelwood and building materials and for the reclamation of N-depleted and degraded soils will depend ultimately upon management of NZ fixation (Blair et al., 1990; Danso et al., 1992; Ladha et al., 1993). Catchpool and Blair (1990), in a 1Cmonth study in the humid tropics of Indonesia, reported N yields of 848 kg ha- ’ for Leucaena leucocephala, 581 kg ha-’ for Calliandra calothyrsus and 700 kg ha-’ for Gliricidia sepium. These amounts include N taken up from the soil and therefore are greater than amounts resulting from NZ fixation activity. They do, however, serve to illustrate the potential for legume growth and accumulation of N, particularly in productive environments. Management practices likely to affect N? fixation through *Author for correspondence.

effects on yield and the proportion of legume-N derived from N? fixation (Pfix) include application of plant nutrients in mineral and organic forms, cutting (coppicing), intercropping and inoculation with rhizobia (Danso et al., 1992). It is important, when assessing such practices, that NZ fixation be accurately measured. Quantification of N? fixation by the tree and shrub legumes, however, has proved particularly difficult (Blair et al., 1990; Peoples and Herridge, 1990; Danso et al., 1992). The earliest estimates relied upon determinations of nodule mass, changes in soil N, measurements of dry matter and N of wood and leaf, and on acetylene reduction activity of harvested nodules (e.g. Guevarra et al., 1978; Lawrie, 1981; Hansen et al., 1987). The values thus obtained may have been inaccurate, either underestimating (nodule mass and activity) or overestimating (total N in dry matter) total N2 fixed. The more recently-developed “N methods were potentially more accurate, although errors caused by the poor matching of N2



D. F. Herridge et al.

fixing and reference plants in “N-enriched experimental systems may be large (Danso et al., 1991, 1992). Choice of reference plant is less critical when the natural 15N enrichment of the soil is used (Peoples et al., 1991b; Ladha et al., 1993; Hamilton et al., 1993). The major limitations of the latter method are the analytical constraints associated with measurements of 615N in harvested samples, and low or variable enrichments of 15N in forest and plantation soils (Hansen and Pate, 1987; Peoples et al., 1991b). The xylem ureide method (Peoples et al., 1989a; Peoples and Herridge, 1990) may find application with the tree legumes. Xylem sap is collected from the plants or trees under study and analysed for N solutes associated with either Nz fixation (ureides) and soil-N use (NO;, 51amino-N). The relative abundance of ureide-N is then calculated as ureide-N as a proportion of ureide-N + c( amino-N + NOT-N. Finally, Pfix is calculated from regression functions relating relative ureide-N and Pfix (e.g. Herridge, 1984; Peoples et al., 1989b; Herridge and Peoples, 1990). Sampling of sap is confined to the accessible aerial parts of the plant, thereby eliminating the problems associated with recovering nodules from the soil and matching the rooting and growth habits of test and reference plants. In initial evaluations, Hansen and Pate (1987) concluded that the method could not be used with the amide-exporting (non-ureide) species of Acacia but suggested that other species found in the same forests (e.g. Kennedia spp and Hardenbergia spp) may be suitable. van Kessel et al. (1988) reported a pot study of 35 species of leguminous trees, grown on N-free and N-rich media, in which Nz-dependent Acacia mearnsii alone had high proportions of ureides in both xylem sap and stem extracts. A further eight species had high ureide proportions in stem extracts but not in xylem sap. Data for Nz-dependent Gliricidia sepium and Sesbania grandtjlora were conflicting also with both showing high proportions of ureides in xylem sap but not in extracts. Yoneyama and Kondo (1990) had previously reported S. rostrata, S. cannabina and S. sesban as amide-producing plants, translocating the bulk of xylary-N as asparagine and < 10% as ureides. Two possible explanations for the inconsistencies in the van Kessel et al. (1988) study are as follows. Many of the Nz-dependent plants grew poorly, with up to lOO-fold differences in growth between the N2-dependent and NO;-dependent plants, making direct comparisons of N-solute compositions difficult. The calorimetric assay of Young and Conway (1942), used for estimating ureide concentrations, can give false positive and greatly overestimated values for some legume species because of coloration by non-ureide compounds (Atkins et al., 1991; Peoples et al., 1991a; Brown and Walsh, 1994). Thus, there is a need to further examine the suitability of the xylem ureide method for assessing

NZ fixation in species of tree legumes used in agricultural and forestry systems. We report experiments to: (i) determine the composition of N solutes in xylem saps of C. calothyrsus, L. leucocephala, G. sepium, S. grandtjlora, Desmodium rensonii and Codariocalyx gyroides, and calibrate, where possible, the xylem ureide technique as an assay of N? fixation using the “N isotope dilution technique to estimate Pfix; and (ii) establish appropriate procedures for sampling xylem sap in field studies.



Analysis of the composition of N solutes of xylem sap of six species of tree legume and calibration of the relationship between the relative abundance of ureide-N in xylem sap and the proportion of plant-N derived from Nr fixation (Pjix)

Plants of C. calothyrsus Meissn., L. leucocephala (Lam.) de Wit cv. Cunningham, G. sepium (Jacq.) Kunth ex Walp., S. grandzjlora (L.) Pers., D. rensonii and C. gyroides. (Roxb. ex Link) Hassk., were grown in a 3 : 1 (v / v) mixture of sand and vermiculite in 30-l., free-draining pots in a naturally-lit, temperature-controlled glasshouse for 55-77 weeks during 1988-1990. Temperatures ranged from 15-25°C at night to maxima of 30-35°C during the day. Twelve seeds were sown in each pot. Effective rhizobia, obtained from Dr R. A. Date (CSIRO Tropical Crops and Pastures, Brisbane, Qld) were added to each pot at sowing as peat inoculant in a water suspension. Strains used were TAL2 for C. calothyrsus, CB3060 for L. leucocephala, CB3090 for G. sepium, CB1212 and CB894 for S. grandtjlora, CB627 and CB756 for D. rensonii, and CB627 and CB2121 for C. gyroides. Seedlings were subsequently thinned to 3 pott’, resulting in overall tree densities of 12 m-’ of bench. A completely randomized design with 5 replicates was used. At 11-13 weeks after sowing, nutrient applications commenced. Pots were watered either daily, every second day or every third day, depending on demand, with 2-3 1. of an N-free nutrient solution (Herridge, 1984) supplemented with ‘5NO_; [K15N0, + Ca(NO,)?] to give concentrations of N of 0, 1, 2, 4 or 8 UIM. Enrichments of 15N ranged from 0.5 atom% excess (8 mM NO,) to 4.0 atom% excess (1 mM NO<). The size of pots, plant densities, concentrations of NO, and frequency of nutrient application were selected to provide appropriatelysized plants having a wide range of dependencies on NZ fixation. At 15-17 weeks after sowing, all plants were cut to 30-cm stem height. Thereafter, sampling (6-9, depending on species) involved cutting the plants to the original 30-cm height, vacuum-extracting xylem sap from the cut branch segments and collecting all clippings for measurements of harvested shoot dry matter, N and 15N concentrations. Sampling was

Evaluating NZ fixation in tree legumes via sap analysis

done around noon. Intervals between samplings ranged from 2 to 10 weeks. Xylem sap [vacuum-extracted sap, VES; see Herridge and Peoples (199O)j was collected by applying a mild vacuum (60-70 kPa) to the base of the cut branch and progressively cutting off small (3-5 cm) stem segments from the top of the branch (Peoples et al., 1989a). The replicate samples were immediately placed on ice, then frozen until analysed. The shoot clippings from the same plants were collected in a large container during sampling of xylem sap, dried in a forced-draft oven at 80°C for 48 h, weighed, then ground to pass through a 1 mm sieve. Concentrations of total N (%N) in 300 mg subsamples of shoots were determined by Kjeldahl digestion, followed by distillation and titration. The titrated distillates were then concentrated and analysed for “N (Peoples et al., 1989a). The proportions of plant N derived from Nz fixation (Pfix) were estimated using the “N dilution technique (Chalk, 1985). Thus: Pfix (%)= lOO[l - (atom% (atom%

“N excess plant)/

“N excess nutrients)].


Concentrations of ureides (allantoin and allantoic acid) in xylem sap were measured as the phenylhydrazone derivative of glyoxylate (Young and Conway, 1942). NO; in xylem sap was measured by the salicylic acid method (Cataldo ef al., 1975). The c[ amino-N content of sap was determined colorimetrically with ninhydrin (Yemm and Cocking, 1955; Herridge, 19841, using a 1 : 1, asparagine-glutamine standard. Details of procedures for the three analyses can be found in Peoples et al. (1989a). The percent relative abundance of ureide-N in xylem sap (RU) was calculated as RU = 400a/(4a + b + c),


where a, b and c are, respectively, the molar concentrations of ureides, NO, and CYamino-N (Herridge, 1984). Concentrations of individual amino acids and amides in xylem sap were measured by HPLC analysis (Peoples et al., 1986). The presence of ureides in sap was verified by determining absorption spectra of the final, coloured product of the Young and Conway (1942) procedure over the range 40&650 nm (Peoples et al., 1991a). Assessment of sampling procedures for collection of VES from tree legumes in the field

Experiments were conducted at field sites at Balai Penelitian Ternak (Research Institute for Animal Production, Ciawi, West Java, Indonesia; 6”4O’S., 106”55’E.), Silkwood (Qld, Australia; 17”46’S., 146”02’E.) and Utchee Creek (Qld, Australia; 17”38’S., 145”55’E.). Comprehensive descriptions of sites, climates and soils can be found in Horne and Blair (1991) and Bray et al. (1995).


Four of the six species in the pot calibration experiment featured in the field experiments. At Ciawi, 2-year-old trees of D. rensonii, established as borders of a coppicing experiment, were used. At Utchee Creek, 2-year-old G. sepium were used. The site at Silkwood provided uniform stands of l-year-old C. gyroides and 3-year-old C. calothyrsus. All trees had been inoculated at sowing with effective rhizobia and were well grown, i.e. 24 m in height (see Bray et al., 1995). The sampling procedure experiments were conducted at Utchee Creek in January 1989, at Ciawi in April 1989 and at Silkwood in March 1990. Diurnal fluctuations in relative ureide-N in xylem saps were assessed for each species by cutting triplicate 1.0-m long branches from 3 replicate plots for extraction of xylem sap. Sampling commenced at either 0600 or 0700 h, was repeated at half-hourly to 2-hourly intervals and concluded at 1700 or 1800 h. Samples were placed immediately on ice, then stabilized by diluting 1 : 1 with 100% ethanol (Herridge et al., 1988) until analysed for ureides, a amino-N and NO,. Effects of delaying extraction of xylem sap once the branch had been cut were examined for each species. Sap was vacuum-extracted from duplicate l-m lengths of branch at 3, 8, 15, 20, 30 and 60 min after cutting. There were 3 replicates, each consisting of two branches. Sap samples were treated as described above. For assessing branch-to-branch variation, nine 1-m long branches were cut from a single tree of each of the four species for extraction and analysis of xylem sap.


Analysis of the composition of N solutes of xylem sap of six species of tree legume

The tree legumes grew well at all levels of NO;, producing large amounts of dry matter and N (Table 1). The range for dry matter was 216 589 g pot-‘, equivalent to 0.862.36 kg m-’ bench (density of 4 pots m-?). Likewise, the range for shoot-N was 7.6-21.2 g pot-‘, equivalent to 3& 85 g m-’ bench. The highest values were recorded for L. leucocephalu supplied with 8 mM NO,. There were marginal increases in growth with increasing NO; supply for three of the five species (D. rensonii, G. sepium and L. leucocephala). With C. gyroides and C. calothyrsus, trees grown without NO; yielded as well as the trees supplied with NO.; at the highest concentrations. The five NO; treatments varied Pfix from around 10% (8 mM NO,) to 100% (0 mM NO,) and, at the intermediate levels (1, 2 and 4 mM), produced trees that used both Nz and NO.: for growth (Table 1). Responses in Pfix to NO.; supply were similar for the five species and were similar also to the response of


D. F. Herridge et al.

Table I. Effects of NO; supply on shoot dry matter and N, Pfix and concentrations of N solutes in xylem saps of five species of tree legume. grown in large pots in a glasshouse (data for S. gran&Jora not shown)

SjwciesNO; supply

Duration of growth (weeks)

No. of samplings”



Total shoot (g)’

Xylem-N solutes ([email protected]



Pfix (%Y


= Amino



395 498 443 420 473 (35.8)

12.2 14.2 12.9 11.8 13.7 (1.06)

100 82 60 30 13 (3.6)

0.98 1.10 0.98 0.51 0.56 (0.18)

1.76 2.00 2.52 2.02 3.32 (0.60)

0.57 1.16 2.48 2.56 4.06 (0.60)


447 524 511 414

11.7 13.2 12.4 10.6 11.5 (1.16)

100 78 57 23 16 (3.8)

0.29 0.25 0.25 0.14 0.12 (0.06)

1.30 1.79 1.64 I .65 (0.36)

0.57 0.88 I.21 I .53 1.71 (0.35)

D rensonii 0 rnM


1 mhr N

2 mt.r N 4 mhr N 8 rnhr N SE (difference) C. gyroides 0 rnM N

I rnM N 2 rnrvtN 4 mhr N 8 mhr N SE (difference) G. seprum 0 mht N 1 mhr N 2 mt.r N 4 rnt+rN 8 rnhr N SE (difference) C. calothwus 0 mht M I rnhr N



216 322 267 329 390 (31.9)

7.6 9.7 8.0 9.9 12.6 (0.95)

100 75 39 13 4 (4.8)

0.14 0.22 0.16 0.13 0.12 (0.04)

4.44 7.11 6.30 6.88 8.07 (1.55)

0.83 1.91 2.14 2.72 5.36 (0.90)



355 361 389 377 372 (45.3)

12.6 10.4 11.5 11.8 13.1 (1.29)

100 76 58 20

0.20 0.16 0.18 0.17 0.24 (0.04)

7.93 6.02 6.37 5.32 6.52 (I .49)

0.79 1.45 2.28 3.48 5.07 (0.63)

443 439 462 536 589 (43.8)

16.0 14.3 15.1 18.2 21.2 (1.32)

0.07 0.06 0.09 0.09 0.09 (0.03)

8.96 IO.14 11.48 13.88 13.06 (2.10)

0.56 0.83 1.30 1.53 1.74 (0.37)

2 rnM N 4 mhr N 8 mht N SE (difference) L. leucacephala 0 mhr N

I rnM N 2 mhr N 4 mt+ N 8 rnM N SE (difference)

I .48



(5:) 100 78 58 34 (4.:)

‘The initial sampling (coppicing) was not used for Pfix determination or for cohection of xylem sap “Mean values of 5 replicates; cumulative totals expressed on a pot ’basis. ?Mean values of 5 replicates, averaged over all samplings (i.e. 69, depending on species).

soybean (Glycine max [L.] Merrill cv. Bragg) (Herridge and Peoples, 1990). Ureide concentrations in xylem sap were highest for D. rensonii (all NO, treatments) and C. gyroides (0, 1 and 2 mM NO,). Lowest concentrations were recorded for L. leucocephala. tl Amino concentrations were almost the reverse; L. leucocephala had the highest concentrations, particularly at high NO, supply, and D. rensonii and C. gyroides the lowest. Xylary NO.; increased predictably with increasing NO, supply. Compositions and concentrations of N solutes in saps of D. rensonii and, to a lesser extent, C. gyroides were similar to those of soybean (Herridge and Peoples, 1990). Data for S. grandiJlora are not shown, even though the plants grew well, accumulating 225-385 g dry matter during the 5%week study. However, we could not determine concentrations of ureides and NO; using standard methods. When analysing for NO,;, sap samples turned black-dark purple, rather than clear, upon addition of the sulphuric acid-salicylic mixture. In the case of the ureide analysis, discoloration occurred during the first alkaline hydrolysis, resulting in a final colour of brown-

yellow, rather than pink-red. The brown-yellow colour gave an absorbance reading at 525 nm, the wavelength for measuring ureide concentrations (Young and Conway, 1942). Atkins et al. (1991) reported similar problems with Robiniu pseudoacacia and resorted to HPLC analysis of xylem sap to verify that non-ureide compounds were interfering with the analysis and to provide accurate ureide determinations. Peoples et al. (1991a), on the other hand, analysed the absorbance spectra of the final coloured products of the Young and Conway (1942) method to show that ureides were not present in xylem sap of forage peanuts (Aruchis pintoi L.) and that the colour was due to interference by non-ureide compounds. We used the Peoples et al. (1991a) protocol to confirm the presence of ureides in xylem saps of all species. Sap samples were from the calibration pot experiment (all species) and from the field diurnal experiment (G. sepium only). Xylem saps of N?-dependent plants of D. rensonii. C. gyroides, G. sepium, C. calothyrsus and L. Ieucocephafa showed spectral characteristics very similar to the ureide standard, i.e. peak absorbance at 52&525 nm and a slight shoulder at 550 nm


Evaluating NZfixation in tree legumes via sap analysis 0.6

0.6 (b)


G. sepium -0.5


600 450






Wavelength (nm) Fig. I. Absorption spectra of the coloured reaction products formed during the Young and Conway (1942) ureide analysis of xylem sap, vacuum-extracted from branches of Nl-dependent, pot-grown plants of (a) S. grandipora, D. rensonii, C. gyroides, G. sepium, C. calothyrsusand L. leucocephala,and 0.025 mM ureide standard; and (b) field-grown plants of G. sepiumsampled at 0600, 0700 and 1200 h, and 0.025mM ureide standard. D. rensonii (Table 2). Glutamine featured strongly in sap of C. gyroides only but accounted for < 10% of amino compounds in sap of other species. Other amino compounds to feature were tyrosine and y-aminobutyric acid (D. rensonii), arginine (G. sepium) and alanine (C. calothyrsus).

[Fig. l(a)], although there were large differences in absorbances which reflected the differences in measured concentrations (Table 1). The S. grandiJora sap, on the other hand, showed a decreasing absorbance through the 4.50-600 nm range without the characteristic ureide peak and shoulder. Thus, the Young and Conway (1942) method was not appropriate for S. grandiflora and this non-specific absorbance at 52.5 nm may explain the apparent presence of ureides in xylem sap, but not in stem extracts, of S. grandiflora in the van Kessel et al. (1988) study. Examination of sap of G. sepium from the diurnal study confirmed the presence of ureides in field-grown plants, and clearly showed an effect of time of sampling on absorbance [Fig. l(b)]. Examination of the compositions of amino acids and amides in xylem saps of the tree legumes indicated that asparagine was the predominant amino compound, with percent composition (molar basis) ranging from around 65% for L. leucocephala and 57% for S. grandifora to just 18% for Nz-dependent

Calibration of the relative abundance of ureide-N in sap and the proportion of plant-N derived from NJ fixation (Pjx)

Two species, D. rensonii and C. gyroides, transported significant amounts of xylary-N as ureides and varied the proportion of xylary-N as ureides as the dependence on Nz and nutrient-supplied NO; varied (Fig. 2). With the other species, the proportion of xylary-N as ureides remained low, i.e.
Table 2. Compositions of amino acids and amides in VES of six species of tree legume, grown in large pots in a glasshouse and supplied with either N-free nutrients (0)or nutrients containing 8.0 mht NO; (8) Species/NO; supply (mM) D 0

Amino compound Asparagine Aspartic acid Glutamine Glutamic caid Alanine Arginine y-Aaminobutyric Tyrosine [email protected]


rensonii 8

C. gyoides 0


G. 0

5 II

45 15 5 13 1 2 2
34 18 4 18 2 2 3
5 2




18 18 4 14 2

49 9 2 II



8 22 13


50 19 5 9


sepium 8

C. calorhwsus 0

% composition 33 21 I2 23 18 4 12 17 6 2 I I2 2 2

leucocephnla S. grandriflorn







30 8 I5 13 7

66 5 IO 9 2

63 5 7 IO 2

57 II 5 7

57 9 3



I 2 2












“Values shown are for composite samples, bulked over replicates (5) and times of sampling (2-3). bIncluded threonine, serine, proline, glycine, citrulline. valine. cysteine. methionine. isoleucine, leucine. phenylalanine. histidine and lysine.


D. F. Herridge et al.





20 .

Z 0







Co~ariocalyx gyroides

Desmodium rensonii

.‘,,,,,/,1#,,1/,1, \‘,‘,‘.‘\‘,‘\f,‘\‘,‘,‘,‘,‘,‘,’ ,\,,,\,\,\\\,,\, ,,,,,,,,,,,,,,,,

,,\,,.\.\,\\\\\\ ,,,,,,,,,,,~,,,, ,\\\,\\\\,\\.\\\ 7 'I' ;' ;. ; -1' .. 'I' ... ’ I

1 Gliricidia sebum

. Calliandra


. _

. _ . T 0













,‘,‘,‘,‘I’,‘,‘,‘,‘,‘,‘,‘,‘,‘,‘,‘, Leucaena leucacephala *‘/‘fl’~: I , , I I , , , ,.I K# , X’,‘,‘, .\.\\\.\\\\\\\\\.. ,,,,,,,,,,,I,,,,, \\.,\~-.\.\\\\.\\\’ ,,,,#,,,,,,#S,,,, .\\,\\\\\\\\\\\\\.

,,,,~~1,,,,1~,,,, \\,,,\\,\\\\\\\,,’ ,,,,,,,,,,,,,,,,, .\,\,.\\\,\\.\,\,’ ,,,,1,#,,,,~,,,,, \\.\,\\,\,.X\,\\\’ ,,,1~1,,,,,~1,,,, \\\.,.\\\\\\\\\\\. ,,,,11/#,,,11,,,, \\.,,\\\\.\\\\x\\. ,,,,1#,,,,,/1,,,, \\\\\\\\\,\k\\X\\. ,,,,1,11,,,,1#,,/ \\\\,\\.\.\k.\\\\. ,,,,,~#,,,,~1~,,, ,,\,\\\,\,\\\\\,,. ,,,,,,,,,,,,,,/,, *.*




‘.’ * 100

Pfix (%) Fig. 2. Relationships between the proportion of plant-N derived from Nz fixation (Pfix) and the proportional composition of N solutes in xylem sap of nodulated D. rensonii, C. gyroides, G. sepium, C. caloth,vrsus and L. leucocephala, grown for 55-77 weeks in large pots in a glasshouse. Data are the means of 6-9 plant samplings. Variations in Pfix, estimated by the rSNisotope dilution procedure, were achieved by supplying plants with either N-free nutrients or with nutrients supplemented with I, 2,4 or 8 mM 15NO;.

errors when using the method (Herridge et al., 1988; Peoples et al., 1989a,b). It is necessary to examine both potential sources of error with the tree legumes. Additionally, sampling tree legumes will involve cutting and extracting sap from individual branches of single trees. The branches should ideally show little variation in N-solute composition and yield sufficient xylem sap for chemical analysis. Therefore, variations in N-solute composition and volume of sap extracted among branches from single trees were examined.

Pfix. The linear relationships between proportions of ureide-N in xylem saps of D. rensonii and C. gyroides and Pfix are presented in Fig. 3. Assessment

of sampling

VES from

tree legumes





in the field

We have published procedures for sampling xylem sap from herbaceous legumes in the field and identified diurnal effects and delays between cutting of shoots and sap extraction to cause the greatest

.., Desmodium
















40 i


30 20

y=8.49 +0.279x

y = 18.3 + 0.446x

(r = 0.92)

(r = 0.91)













Pfix (%) Fig. 3. Relationships between Pfix and the relative abundance of ureide-N in VES of (a) D. rensonii and (b) C. g.woides. Functions describing the relationships are shown.


Evaluating N2 fixation in tree legumes via sap analysis *n





’c. cslo#hymJs 0 --*l-**I-**I.’ 600 400

, . . . , . . . , .-.I) 1200







Time (h) Fig. 4. Diurnal fluctuations in the relative abundance of ureide-N in VES of (a) D. rensoniiand C. gyroides and (b) G. sepiumand C. calothyrsus. Each point is the mean of 3 replicates of three l-m branches, sampled from field-grown trees at Ciawi (West Java, Indonesia) and Silkwood and Utchee Creek (Qld, Australia). Vertical bars denote LSDs (P = 0.05) for each species.

In the diurnal studies, significant (P < 0.05) fluctuations in relative ureide-N of xylem sap were recorded for each of the four legumes (Fig. 4). Values were highest during early morning and late afternoon. The period around noon was characterized by low and relatively uniform values. In the case of C. gyroides, however, this period of uniformity was only 2-h long (i.e. 1200-1400 h). Differences between highest and lowest values were marginal for D. rensonii and C. calothyrsus. For C. gyroides, differences between highest (0700 h) and lowest (1400 h) were almost 3-fold; for G. sepium, the range was even greater with the relative ureide-N value at 0600 h almost IO-fold that of the value at 1300 h [see also Fig. l(b)]. The diurnal patterns in relative ureide-N of xylem sap of the tree legumes were similar to the patterns reported for soybean and pigeonpea (Herridge et al., 1988; Peoples et al., 1989b). In all cases, variations were due to the combined effects of elevated concentrations of ureides at the beginning and end of daylight and elevated concentrations of CIamino-N around noon. NO; tended to be uniform in concentration throughout the day. The magnitude of the early-morning values for G. sepium and the large variation in values during the course of the day are puzzling. Relative ureide-N for the first four samplings (0600-0730 h) varied between 2656%. Ureide concentrations for the same samplings were 0.46-0.97 mM. Both sets of values are consistent with the values recorded for two ureide-exporting species-D. rensonii and C. gyroides (Table 1, Figs 1 and 2). By noon, however, when sampling is generally recommended (Herridge et al., 1988), ureide concentrations had fallen to 0.08 mM and relative ureide-N values to 7%, more typical of the amide-exporting L. leucocephala and C. calothyrsus. We can only conclude from these data that either G. sepium loaded and exported fixed-N as ureides

from the nodule at night and during the extremes of daylight rather than during the day, or that major synthesis and release of ureides into the xylem stream occurred at a low level which was not related to Nr-fixation activity. With the latter, concentrations would tend to be inversely related to transpiration rates, i.e high at night and low in the middle of the day. Neither hypothesis is undermined by results of the calibration study (Table 1; Fig. 2), because all samplings in the study were done around noon. Ureide production in plants is not always associated with N2 fixation. Ureides have been recovered from a wide variety of unnodulated, non Nz-fixing plants (Reinbothe and Mothes, 1962). Previous studies indicated almost linear increases in the relative abundance of ureide-N of xylem saps of soybean and pigeonpea with increasing time intervals between plant sampling and vacuum-extraction of sap (Herridge et al., 1988; Peoples et al., 1989b). Shifts in concentrations were likely due to time effects on differential release and binding of solutes, perhaps associated with wilting of the cut shoot. Identification of this potential source of error was important, particularly for the field application of the technique where extraction is more likely to be delayed than done immediately. Delaying extraction of xylem sap from cut branches of D. rensonii and G. sepium resulted in increased relative ureide-N values, related to higher concentrations of ureides (Fig. 5). There were no consistent effects of time delay on concentrations of either u amino-N or NO;. With a delay of 15 min, increases in relative ureide-N were 15% for D. rensonii and 63% for G. sepium. After 60 min, increases were 50% for D. rensonii and 200% for G. sepium. Delaying extraction had no significant effect on relative ureide-N values of C. calothyrsus and C. gyroides (P > 0.05). Cutting and extracting sap from nine branches of single trees of the same four species revealed little


D. F. Herridge et al.

> f/ ‘%


20 *:::

B 0 0

*z=I*I.r’I*I 102030405060


I 10

* I 20

Time delay before extracting


I 30


I 40


, 50

* I 60


sap (min)

Fig. 5. Effects of time delay between harvesting branch and vacuum-extracting xylem sap for (a) D. rensonii and C. gyroides and (b) G. sepium and C. calothyrsus. Each point is the mean of 3 replicates of two l-m branches, sampled from field-grown trees at Ciawi (West Java, Indonesia) and Silkwood and Utchee Creek (Qld, Australia). Vertical bars denote LSDs for D. rensonii and G. sepium where effectswere significant at P = 0.05.

variation between branches in the relative abundance of ureide-N (Table 3). There was more variation in the volume of sap extracted from individual branches, although even the lowest-yielding branches (0.21 ml for G. sepium and 0.34 ml for D. rensonii) provided sufficient sap for chemical analysis. The major impetus for this study was the need to develop a reliable, easy-to-use method for quantifying N2 fixation by tree and shrub legumes in the field. The xylem ureide method was promising but its application depended on quantitative relationships between the relative abundance of ureides in xylem sap and Mx. We found such a relationship in only two, D. rensonii and C. gyroides, of the six species examined and conclude that the xylem ureide method has restricted application for assessing N2 fixation by tree and shrub legumes. With L. leucocephala, C. calothyrsus and G. sepium, the proportion of xylary N as ureides remained low, i.e. < lo%, and constant over the range of Pfix. It is probable that the sixth tree was also a legume examined, S. grandipora, non-ureide producer. Our studies indicated also that the same sampling protocols should be applied to D. rensonii and C. gyroides that are recommended for the annual crop legumes (Herridge et al., 1988; Peoples et al., 1989a). Plants need to be sampled around noon to eliminate

Table 3. Variations in volume and relative abundance of ureide-N of xylem sap extracted from nine individual. I-m length branches of each of four tree legumes grown in the field in West Java and northern Queensland Volume of sap (ml) Species

Mean f SE



0.89 f 0.11 ND 0.43 k 0.06 ND

0.341.39 ND 0.21X1.75 ND


C. g,vroides G. sepium C. calothyrsus

ND = not determined.

Relative ureide-N


Mean * SE


64.8 f 42.4 + 11.2 + 8.6 *

59-69 30-49 IO-16 &I1

1.2 2.1 0.6 0.5

diurnal effects and vacuum extraction of sap should proceed immediately the branch or shoot is cut. Values for relative ureide-N of the sap can then be determined and related to the regression functions in Fig. 3 to determine Pfix. Thus, values for relative ureide-N in saps from the noon samplings in the diurnal studies (Fig. 4) were 57% for D. rensonii and 23% for C. gyroides, equivalent to Pfix values of 87% and 52%, respectively (Fig. 3).

Acknowledgements-We gratefully acknowledge the following: J. Betts, K. Cassin, R. Shapland, G. Turner and D. Lilley for plant culture and chemical analysis including 15N; F. J. Bergersen for spectral analysis of xylem saps; J. S. Pate and E. Raisins for analysis of saps for amino compounds; M. Fulloon, the QDPI staff at South Johnstone and Utchee Creek, and the forage group at Balai Penelitian Tarnak (BPT), Ciawi, for maintenance and provision of field sites and materials; Dr R. A. Date for provision of all rhizobial cultures. The work was funded jointly by NSW Agriculture, CSIRO and the Australian Centre for International Agricultural Research (ACIAR).

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