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Evaluating the importance of a potential source of error when applying shoot 15N labelling techniques to legumes to quantify the below-ground transfer of nitrogen to other species

Matthew Gardner1,4, Mark Peoples3,4, Jason Condon2,4, Guangdi Li2,4, Mark Conyers2,4 and Brian Dear2,4

1NSW Department of Primary Industries, Tamworth Agricultural Institute, 4 Marsden Park Road, Tamworth, NSW 2340, matthew.gardner@dpi.nsw.gov.au
2
EH Graham Centre for Agricultural Innovation (an alliance between NSW Department of Primary Industries and Charles Sturt University), Wagga Wagga Agricultural Institute, Pine Gully Rd, Wagga Wagga NSW 2650,
3
CSIRO Plant Industry, Canberra ACT 2601
4
CRC for Future Farm Industries, 35 Stirling Highway, Crawley, WA 6009, Australia

Abstractf

Different 15N-based shoot-labelling protocols have been utilised to quantify the below-ground transfer of N between pasture or crop species. The most straight forward and effective 15N-labelling technique appears to be shoot labelling via leaflets or petioles. This paper reports a time-course study following 15N-shoot labelling of pasture legumes to ascertain whether a pulse-feeding of 15N labelled urea to the leaf resulted in induced exudation of highly 15N enriched compounds from the roots into the surrounding soil. Such an occurrence could confound interpretation of 15N data used to measure transfer of N between legume and non legume species. Small quantities of highly enriched 15N (99.8 atom% 15N) urea were fed to leaves of subterranean clover (Trifolium subteraneum; 0.22ml/plant of 1% urea solution) and lucerne (Medicago sativa; 0.45mL of 0.5% urea solution) over a period of 3 days. On days 1, 2, 4, 8, 16 and 32 after labelling, the plants were harvested and the 15N enrichment of shoots, roots and soil was determined. Soil analyses showed that the 15N enrichment of soil N was significantly higher in the lucerne treatment (12.3 ‰; parts per thousand 15N relative to the 15N content of atmospheric N2, 0.3663 atom% 15N) than subclover (11.3 ‰) (P<0.05), and both levels were significantly higher than, but very close to, the background level in the soil (10.7 ‰). The results could be explained if <10% of the lucerne root N or 12-27% of the subclover roots remained unrecovered in the soil following the sieving process. It was concluded that pulse-feeding of high concentrations of 15N labelled urea in the leaf did not induce substantial exudation of highly 15N enriched compounds from the roots into the surrounding soil.

Key Words

Subterranean Clover, Lucerne, N transfer, 15N shoot labelling

Introduction

Different 15N-based shoot-labelling protocols have been used to study the deposition of N-rich compounds and decaying root material in the plant’s rhizophere (often described as rhizodeposition). Usually plants are 15N-labelled during early growth and the label (and distribution of N) quantified later in shoots, roots and root-zone soil (e.g. McNeill et al. 1997; Khan et al. 2002). A key requirement of this approach is that the root-zone soil should not be directly labelled with 15N. In other words, any 15N in the soil should be of plant origin.

Shoots have been labelled by exposure to an atmosphere containing 15NH3 or 15N2 (single or multiple pulses) However, there is a level of sophistication and complexity in the equipment used in atmospheric labelling studies that detract from using this approach, particularly for field research (Russell and Fillery 1996). Another approach to 15N labelling involves exposing part of the root system to a 15N solution and recovering the label in the shoot and the other non-exposed part of the root (i.e. split root system) (Jensen 1996). Direct shoot labelling has also been used with the 15N being applied either as a foliar spray, by immersing attached leaflets or petioles in vials containing 15N enriched solutions, or by introducing enriched solution into the plant’s stem (Hgh-Jensen and Schjoerring 2000; Khan et al. 2002; Russell and Fillery 1996; Zhou et al. 1998).

All three approaches, and the various labelling techniques within those approaches, have been developed for particular purposes and have their own distinct advantages and limitations. It is unlikely, therefore, that any one technique will be broadly applicable to all plant species. Some of the techniques require complex and/or expensive equipment (e.g. gas-tight enclosures), some have specific plant morphological requirements (e.g. woody or hollow stems), while others result in the substantial disturbance of the plant-soil system (split-root technique) or pose potential risks of soil contamination via run-off from foliage of applied 15N (spray application). The simplest technique appears to be 15N-labelling via leaflets or petioles. It has been used in both glasshouse and field studies involving both large crop plants and small pasture species (Jensen 1996; Khan et al. 2002; McNeill et al. 1997). The objective of this study was to examine the time-course of 15N-shoot labelling to ascertain i) whether a pulse loading of high concentrations of 15N labelled urea in the leaves of subclover (Trifolium subteraneum) and lucerne (Medicago sativa) resulted in any potential artefacts such as induced exudation of highly 15N enriched compounds from the roots into the surrounding soil and ii) whether 15N shoot labelling is a viable technique for estimating the direct N transfer between legumes and non-legumes.

Methods

Soil was collected from the 0 – 15 cm depth interval of a Red Chromosol soil (Isbell 1996) derived from granite located on the Charles Sturt University farm, Wagga Wagga. The equivalent of 2.15 kg of oven dry soil was added to 3 L PVC pots by a standardised packing procedure to give an approximate bulk density of 1.3 Mg/m3. Pots were watered to 90 % field capacity and 120 mL of basal fertiliser treatments were applied evenly to the soil surface. The basal fertiliser treatment ensured all nutrients, except N, were not limiting. Three plants of lucerne and subclover were established and maintained until shoot labelling, 5 weeks after emergence.

An earlier experiment was used to determine optimum urea concentration for subclover (1% w/w) and lucerne (0.5% w/w), which also revealed that lucerne absorbed approximately double the solution compared to subclover (0.25 vs 0.50 mL/leaf). The shoot labelling technique described by Khan et al., (2002) was carried out on 1 leaf from each plant (3 leaves/pot). In brief, the selected leaflets were fully submerged in deionised water and the tips cut off with a razor. Excess water was removed with a tissue from the leaflets and the entire leaf was immediately inserted into a 2 mL Eppendorf vial containing 0.25 mL (1% w/w 15N labelled urea solution 99.8 atom % 15N excess) and 0.50 mL (0.5% w/w 15N labelled urea solution 99.8 atom % 15N excess) for subclover and lucerne, respectively. Blu-Tack was used to keep the vial in place while also sealing the top of the vial to prevent evaporative losses and attach the vial to a small wooden stake placed adjacent to the leaf. Tin-foil was placed on the soil surface beneath the fed-leaves to prevent any accidental spills of 15N-enriched urea from contaminating the soil and remained in place to protect the soil surface until leaf feeding was complete. All Eppendorf vials were removed after 72 hours and weighed to determine the quantity of solution absorbed. Fed leaves and petioles were removed 24 hours after feeding to minimise risk of directly enriching the soil and were not included in any plant analysis.

The duration of the experiment was 32 days with a total of 6 sampling times, each sampling time had 4 replicates. Sampling of pots occurred at 1 day after 15N labelling, 2 days later, 4 days later, 8 days later, 16 days later and at the completion of the experiment, 32 days after 15N labelling. Throughout the experiment all fallen leaves were collected during plant growth to minimise the direct 15N contamination of the soil, and later included in shoot analysis.

Sampling occurred by removing plant shoots at the soil surface. Root and soil material was passed through a series of sieves down to 1 mm. The collected root material was the gently washed free of remaining soil. All plant material (root and shoot) for each species was dried individually in a plant dehydrator at 80C for 48 hours and dry matter yield (DMY) determined. Plant samples were then coarsely ground using a Cyclotec sample mill, before, being finely ground to a powder with a puck and ring grinder. The total N content and 15N enrichment of the dried samples were determined by combustion using an automatic N and C analyser interfaced with a 20-20 stable isotope mass spectrometer. A representative sub sample of soil collected for total N content and 15N enrichment and dried at 40oC for 48 hours before being finely ground to a powder with a puck and ring grinder. Soil samples were analysed as per the plant samples.

Results

Subclover had significantly (P<0.05) higher (approximately 2 to 3 times more) shoot dry matter yield than lucerne at each sampling time (Table 1). Lucerne had significantly (P<0.05) greater root biomass than subclover at each of the sampling times (Table 1). Shoot dry matter accumulated exponentially for both lucerne and subclover from 1 to 32 days after shoot labelling, whereas the increase in root dry matter was much more gradual for both species (Table 1).

Table 1. Shoot and root dry matter yield (g) for lucerne and subclover 1, 2, 4, 8, 16 and 32 days following shoot labelling with 15N (99.8 atom% 15N) urea. Shoot or root data designated with a different letter represents a significant difference (P<0.05).

Days after labelling

Shoot Dry Matter (g)

Root Dry Matter (g)

Subclover

Lucerne

Subclover

Lucerne

1

22.4 cd

7.3 f

13.5 d

17.8 c

2

20.3 d

8.1 ef

13.9 d

18.4 c

4

22.1 cd

8.4 ef

13.9 d

21.2 bc

8

25.2 c

8.9 ef

15.5 cd

22.1 bc

16

31.1 b

11.8 e

17.0 cd

24.8 b

32

52.2 a

22.7 cd

19.3 c

27.3 a

lsd (P=0.05)

4.3

3.9

The 15N enrichment of lucerne shoots was significantly (P<0.05) greater than the 15N enrichment of subclover shoots, ranging from approximately 350 to 2110 ‰ higher (Table 2). Furthermore, 15N enrichment of lucerne shoots declined significantly (P<0.05) at every sample time beyond 2 days after shoot labelling, whereas enrichment of subclover shoots remained the same across all sampling times (Table 2). The 15N enrichment of roots was approximately 20 % of what was measured in the shoots (Table 2). The 15N enrichment of lucerne roots was also significantly greater than the 15N enrichment of subclover roots but unlike the lucerne shoots, the 15N enrichment remained similar up until 8 days after shoot labelling before significantly (P<0.05) declining (Table 2). Root 15N enrichment of subclover roots remained unchanged at all sampling times (Table 2).

Table 2. Shoot and root 15N enrichment (‰) for lucerne and subclover 1, 2, 4, 8, 16 and 32 days following shoot labelling with 15N (99.8 atom% 15N) urea. Shoot or root data designated with a different letter represents a significant difference (P<0.05).

Sampling Time (days)

Shoot 15N Enrichment (‰)

Root 15N Enrichment (‰)

Subclover

Lucerne

Subclover

Lucerne

1

315.4 e

2624.6 a

54.1 c

321.9 ab

2

342.7 e

2514.3 ab

60.4 c

326.9 a

4

358.5 e

2093.4 b

95.3 c

336.8 a

8

412.0 e

1581.2 c

86.9 c

340.0 a

16

258.4 e

878.3 d

91.2 c

262.4 b

32

101.4 e

450.7 de

74.0 c

220.6 b

lsd (P=0.05)

428.3

62.9

There was no significant (P<0.05) change in 15N enrichment of soil between any of the sampling times after shoot labelling. However, when averaged across all sampling times 15N enrichment of soil from lucerne (12.3 ‰) pots was significantly (P<0.05) greater than subclover soil (11.2 ‰), while both were significantly (P<0.05) greater than the soil where no enriched plants were grown (10.6 ‰) (Figure 1).

Figure 1. The 15N enrichment (‰) of soil either unenriched or with subclover or lucerne grown. lsd bar (0.4) represents a significant difference where P<0.05.

Discussion

The large increases in shoot dry matter over the 32 day period for lucerne resulted in a significant dilution of the 15N enrichment. Dilution of 15N enrichment was also observed for subclover shoots, but not to the same extent. There was little change in the 15N enrichment for either lucerne or subclover roots, which is most likely a reflection of the minor changes in root dry matter during the 32 days of the experiment. Despite the subclover and lucerne receiving equivalent quantities highly enriched 15N (99.8 atom% 15N) urea (2.9 and 3.0 mg/plot) the enrichment levels varied between the 2 species. Recovery of fed 15N was 87 and 34 % for lucerne and subclover, respectively. The 15N recovery observed for the lucerne was typical of the results reported by others (Khan et al. 2002; McNeill et al. 1997) but the recoveries for subclover were low. For both lucerne and subclover the labelled leaves and petiole (3 per pot) were not included in the 15N analysis of shoots. Therefore, it is possible that a large majority of the unrecovered 15N was not translocated from the labelled leaves and petiole. However, it was recognised that there was potential for losses associated with shoot labelling of urea as previous studies have identified that hydrolysis in the fed leaf can be exacerbated by high concentrations of urea-N. The subclover treatments in this experiment were labelled with 1 % (w/w) urea solution, which was double that of the lucerne treatments and may have an increased risk of hydrolysis losses in the fed leaves. The 1 % (w/w) urea solution concentration was selected following a preliminary study which indicated that subclover was not as sensitive to urea concentration as lucerne, but tended to take up only half the volume of solution. Ammonia could also be lost from the shoots which could be a source of error, although this ammonia might subsequently be reabsorbed by adjacent leaves or plants which could negate or create another source of error. The potential for these error sources in this study cannot be discounted.

Despite the low 15N recovery for subclover and the potential for some losses there was still sufficient enrichment of both lucerne and subclover plant material to determine whether a pulse-feeding of 15N labelled urea to the leaf resulted in induced exudation of highly 15N enriched compounds from the roots into the surrounding soil. Soil analyses showed that the 15N enrichment of soil N was significantly higher in the lucerne treatment (12.3 ‰) than subclover (11.3 ‰), and both levels were significantly higher than, but very close to, the background level in the soil (10.6 ‰). Based on the 15N enrichment achieved the results could be explained if <10% of the lucerne root N or 12-27% of the subclover roots remained unrecovered in the soil following the series of sieving process that occurred at harvest to separate soil from roots. If it is assumed that 100 % of root material was recovered, an almost impossible task, then 1.3 and 1.4 % of recovered 15N (0.4 and 1.1 % of total fed 15N urea) was potentially exuded into the soil from subclover and lucerne, respectively.

Conclusion

It was concluded that pulse-feeding of high concentrations of 15N labelled urea in the leaf did not induce substantial exudation of highly 15N enriched compounds from the roots into the surrounding soil. The most probable explanation for the presence of small quantities 15N in the soil would be associated with difficulty of completely removing root material from soil, particularly when roots are highly enriched. Therefore, shoot labelling via leaflets or petioles, which is a straight forward and effective 15N-labelling technique, appears to be a suitable method to measure transfer of N between legume and non legume species.

References

Hgh-Jensen H, Schjoerring J (2000) Below-ground nitrogen transfer between different grassland species: Direct quantification by 15N leaf feeding compared with indirect dilution of soil 15N. Plant and Soil 227, 171-183.

Isbell R (1996) 'The Australian soil classification.' (CSIRO Publishing: Melbourne).

Jensen ES (1996) Grain yield, symbiotic N fixation and interspecific competition for inorganic N in pea-barley intercrops. Plant and Soil 182, 25-38.

Khan DF, Peoples MB, Chalk PM, Herridge DF (2002) Quantifying below-ground nitrogen of legumes. 2. A comparison of 15N and non isotopic methods. Plant and Soil 239, 277-289.

McNeill AM, Zhu C, Fillery IRP (1997) Use of in situ 15N-labelling to estimate the total below-ground nitrogen of pasture legumes in intact soil and plant systems. Australian Journal of Agricultural Research 48, 295-304.

Russell C, Fillery I (1996) In situ 15N labelling of lupin below-ground biomass. Australian Journal of Agricultural Research 47, 1035-1046.

Zhou X, Madrmootoo CA, MacKenzie AF, Smith DL (1998) Distribution of 15N labeled urea injected into field grown corn plants. Journal of Plant Nutrition 21, 63-73.

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