Journal of Experimental Botany, Vol. 51, No. 342, pp. 41-50,
January 2000
© 2000 Oxford University Press
Using stable isotope natural abundances (
15N and 13C) to integrate the stress responses of wild barley (Hordeum spontaneum C. Koch.) genotypes
Scottish Crop Research Institute, Dundee DD2 5DA, UK
Received 3 February 1999; Accepted 10 May 1999
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Abstract |
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To integrate the complex physiological responses of plants to stress, natural abundances (
) of the stable isotope pairs 15N/14N and 13C/12C were measured in 30 genotypes of wild barley (Hordeum spontaneum C. Koch.). These accessions, originating from ecologically diverse sites, were grown in a controlled environment and subjected to mild, short-term drought or N-starvation. Increases in total dry weight were paralleled by less negative 13C in shoots and, in unstressed and droughted plants, by less negative whole-plant 13C. Root 15N was correlated negatively with total dry weight, whereas shoot and whole-plant 15N were not correlated with dry weight. The difference in 15N between shoot and root varied with stress in all genotypes. Shoot–root 15N may be a more sensitive indicator of stress response than shoot, root or whole-plant 15N alone. Among the potentially most productive genotypes, the most stress-tolerant had the most negative whole-plant 15N, whether the stress was drought or N-starvation. In common, controlled experiments, genotypic differences in whole-plant 15N may reflect the extent to which N can be retained within plants when stressed.
Key words:
hordeum spontaneum, 13C, 15N, stress, drought, nitrogen
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Introduction |
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There is a continuing search for crops tolerant of harsh environments. One strategy is statistical, and involves examining plants from contrasting habitats, correlating their stress responses to habitat characteristics and—in suitable mapping populations—to molecular markers on the genome (Forster et al., 1997
; Ellis et al., 1997; Handley et al., 1997). This approach can reveal genotypic and phenotypic facets of stress tolerance, and their interactions. In principle, genes associated with specific physiological processes involved in stress tolerance can be identified and introgressed into breeding lines (Holmberg and Bülow, 1998).
‘Stress tolerance’ comprises many physiological processes which vary quantitatively rather than qualitatively (Yeo, 1998; Zhang et al., 1999). It is often impractical to measure each process individually on many plants. One solution is to measure surrogate variables which integrate many physiological processes. Some of the most useful of these surrogates are the natural abundances (denoted as ) of biologically important stable isotope pairs, e.g. 13C/12C and 15N/14N.
13C has been used to screen C3 genotypes for potential water use efficiency (Ehleringer et al., 1993). A robust theory is available (Farquhar et al., 1982) with which to interpret 13C variations among C3 plants in terms of measurable physical and physiological processes. Plant 13C reflects mainly the extent to which primary CO2 assimilation is limited by carboxylation and/or CO2 diffusion in leaves. Whole-plant 13C is dominated by these processes. Internal partitioning and metabolism of primary assimilate may produce differences in 13C among plant organs (Hubick and Gibson, 1993) and chemical groups (Gleixner et al., 1993; Brugnoli et al., 1998; Schmidt and Kexel, 1998). Environmental stresses (e.g. drought) modify 13C in largely predictable ways, explicable ultimately via effects on the balance between stomatal conductance and carboxylation.
In contrast, 15N has been used much less extensively in this way. Plant 15N reflects the potentially variable 15N values of external N sources and 15N/14N fractionations which occur during the assimilation, transport and loss of N. The use of 15N in plant ecophysiology is currently at the ‘pattern generation’ or ‘hypothesis development’ stage. Taxonomic and environmental variations in 15N are being explored and documented in natural and controlled environments (Handley and Scrimgeour, 1997; Handley et al., 1998), despite there being no theory able to explain these variations mechanistically. A theory has been proposed for 15N (Robinson et al., 1998) which, despite being restricted to 
-grown plants, still demanded information about the 15N values of external and internal N pools, information that is difficult to obtain routinely. The ‘decoding’ of plant 15N into underlying mechanisms promises to be a non-trivial problem.
An alternative is to find statistical associations between plant 15N, growth and specified environmental conditions, and to establish testable hypotheses about the main cause(s) of such associations. Genotypic and environmental variations in plant 15N exist. For example, Handley et al. (1997) showed that shoot 15N varied by up to 2.4
among wild barley (Hordeum spontaneum C. Koch.) genotypes grown on a common N source. Salinity caused shoot 15N to become, on average, 2 more negative. But to begin interpreting plant 15N physiologically, data for whole plants, not just shoots or roots, are required.
The purpose here is to explore further the utility of 15N as a physiological integrator. Specifically, the aims are to (1) measure the variations in shoot, root and whole-plant 15N in genotypes of one species (H. spontaneum) in relation to experimentally imposed environmental stresses and to site-of-origin conditions; (2) correlate these measurements with stress tolerance; and (3) assess the potential usefulness of 15N as an integrator of stress responses.
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Materials and methods |
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Caryopses of H. spontaneum plants collected from 30 sites (Table 1
) in the Fertile Crescent, the centre of diversity for this species, were bulked under glasshouse conditions. Surface-sterilized caryopses were germinated on moist filter paper in Petri dishes on 13 September 1996. Three days later, when roots were 5–6 mm long, seedlings were transplanted into open-ended Sarsted tubes filled with 0.8% agar containing 200 mg l-1 benzimidazole to suppress fungal pathogens. To minimize root damage and to prevent hypoxia around the embryo, a 3 mm diameter core of agar was removed from each tube, into which a seedling was inserted, and its roots covered immediately with cold agar extruded via a syringe. Seedlings were grown in a controlled environment (4 °C, lit by high-pressure sodium lamps at 300 µmol m-2 s-1, 8 h daylength) for 7 weeks' vernalization, during which time the base of each tube was kept in water.
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Vernalized seedlings were transferred, on 4 November 1996, into a glasshouse hydroponic system. Air temperature was maintained between 16–24 °C, and the glasshouse was ventilated with outside air to ensure steady [CO2] and
13C of source CO2 (c. -8). Plants were illuminated by natural daylight, supplemented by sodium lamps. The hydroponic system consisted of three 80 l troughs containing aerated half-strength Hewitt's nutrient solution (Hewitt, 1966) with additional NaSiO3 (Epstein, 1994), changed weekly. N, as Ca(NO3)2 and KNO3, was supplied at 6 mol m-3 with an initial mean 15N value in solution of +1.4±0.2. This value became more positive between solution changes, reaching +2.2 to +4.5. The causes of the gradual 15N enrichment are unknown, but could reflect the loss from roots of partly assimilated, 15N-enriched N (Robinson et al., 1998), partial denitrification of in the non-sterile solution (Robinson and Conroy, 1999), or both. Mean solution temperature was 16±0.1 °C; mean pH was 6.0±0.1. Solution [O2] at the end of the experiment (when O2 depletion would have been greatest) was 92±0.3% saturation. During the experiment, the mean outdoor solar radiation receipt was ~3 MJ m-2 d-1 (DKL Mackerron, personal communication); about half this amount would have reached the plants growing in the glasshouse.
Three experimental treatments were established: controls, in which plants were maintained in the nutrient solution throughout; drought, in which plants were, 7 d after transfer to the hydroponic system, raised out of the solution to expose their roots to air for 3 h daily (Hendry, 1993); and N starvation, in which plants were deprived of all external N after 7 d growth. In the N starvation treatment, Ca2+ and K+ were supplied as CaCl2.6H2O and K2SO4, respectively, giving both cations a uniform concentration of 2 mol m-3 in all treatments. The drought and N starvation treatments were deliberately mild, intended to reveal the diversity of sub-lethal stress responses among the H. spontaneum genotypes. Genotypes were replicated four times per treatment, and arranged in randomized blocks in each trough. Each trough contained 170 plants, of which 50 were guard plants of H. vulgare cv. Derkardo.
Plants were harvested 16 d after transfer to the nutrient solution, long after seed C and N (and their values) had been trivialized in the whole plants (on average, 8 and 0.4 mg C and N per seed, versus at least 90 and 7 mg C and N per harvested plant). Shoots were separated from roots. Plant material was oven-dried (60 °C for 48 h), weighed and milled. Concentrations of total C and N, and 13C and 15N were determined on subsamples (c. 1 mg dry wt) of shoots and roots using continuous-flow isotope ratio mass spectrometry (Europa Scientific Ltd., Crewe, UK), as described (Handley et al., 1993; Scrimgeour and Robinson, 1999). values () were calculated as
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Data were subjected to a two-way ANOVA with Genotype and Treatment as factors. It was unnecessary to transform data to homogenize variances. Statistical analyses were done with Genstat v. 5 (Genstat 5 Committee, 1993) and StatisticaTM v. 5.1 (StatSoft, Norman, Oklahoma) software.
Whole-plant 15N () was calculated as an average of shoot and root 15N weighted by the total N contents (mg) of shoots and roots
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 | (3) |
0 represent little stress, plants becoming increasingly stressed—in terms of the effect of the environment on growth—as SI 1.
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Results |
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Plant growth
Total dry weight per plant varied significantly (P<0.001) with both Treatment and Genotype, but there was no GenotypexTreatment interaction (Table 2
). Twelve genotypes showed significant reductions in total dry weight, relative to controls, in response to drought; five genotypes responded significantly to N starvation (Fig. 1). Of those genotypes which did not respond to either stress, almost all had relatively slow growth rates (as indicated by their ranking in Fig. 1, i.e. Genotypes 15 to 11). Exceptions to this were Genotypes 7 and 8 which, notably, were unaffected by N starvation despite being two of the most productive genotypes.
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), droughted (
) and N-starved (
) H. spontaneum genotypes. Genotypes are ranked in order of increasing total dry weight of controls. Code numbers (Table 1) of genotypes which responded significantly (P<0.05, LSD) to drought are underlined on the right-hand axis; those which responded significantly to N starvation are underlined on the left-hand axis. For clarity, error bars have been omitted from data points. SEs were, on average, 0.15, 0.11 and 0.13 g per plant for control, droughted and N-starved plants, respectively.
Whole-plant 15N
There were highly significant effects of Genotype and Treatment on whole-plant 15N (P
0.001; Table 2). Variations in whole-plant 15N were dominated by the Treatment main effect, but the interaction was significant. Whole-plant 15N in controls (Fig. 2) ranged from -0.9 (Genotype 12) to +0.3 (Genotype 3). Given that source had a 15N value >+1, the plants clearly discriminated against 15N (where discrimination ~ source 15N–whole-plant 15N). However, discrimination could not be quantified because of the temporal variability in source 15N (see Materials and methods).
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Whole-plant
15N responded significantly (P<0.05) to drought in 16 of the 30 genotypes; 10 genotypes responded significantly to N starvation (Fig. 2). When drought or N starvation had a significant (P<0.05) effect on whole-plant 15N, this almost always became more negative than in controls (Fig. 2); the only exception to this was Genotype 5. The mean difference in 15N between control and stressed plants was 0.6 (±0.1 SE) and 0.3 (±0.1 SE) for the drought and N starvation treatments, respectively. The largest such difference under drought was 1.4 in Genotype 15 and 1.1 when plants were N-starved (Genotype 26).
Shoot and root 15N
Shoot 15N, root 15N, and the difference between them, varied significantly with Genotype and Treatment, and there was a significant interaction between these (Table 2).
Drought and N starvation caused shoot 15N to become, on average, significantly more negative than in controls, N starvation having a larger effect (Table 3). Drought caused root 15N to become, on average, 2.1 more negative than controls. This trend was reflected in all genotypes except one (Genotype 2) whose root 15N was unaffected by drought (data not shown). In contrast, N starvation caused root 15N to become significantly less negative than the controls.
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All genotypes responded significantly (P<0.05) to one or both stresses in terms of the shoot–root
15N difference (Fig. 3). In most genotypes, the difference in shoot–root 15N was slightly positive in controls (mean 0.7±0.1), i.e. shoots were more 15N-enriched than roots. In response to drought, shoot–root 15N usually increased (mean 2.5±0.1) compared with the control. By contrast, shoot–root 15N usually decreased under N starvation (mean -0.5±0.1). When the absolute shoot–root difference in 15N between control and drought treatments was plotted against that between control and N starvation treatments, there was a significant, inverse relation between them (Fig. 4).
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There were no significant correlations between shoot
15N and shoot N content or concentration, nor between root 15N and root N content or concentration (data not shown).
13C
Whole-plant 13C varied significantly (P<0.001) with Genotype and Treatment, and there was a strong interaction (Table 2). As with whole-plant 15N, Treatment had the dominant effect on 13C. In controls, whole-plant 13C varied by 1.8, ranging from -32.4 (Genotype 14) to -30.6 (Genotype 6). Shoot and root 13C were both affected significantly by drought, but not N starvation (Table 3). Under drought, 13C became less negative than in controls and this effect was more pronounced in shoots than roots.
There were no significant correlations between 13C and 15N in shoots or roots (data not shown).
Correlations between plant dry weight and values
The heaviest plants had the least negative shoot 13C values (P<0.05; Table 4). Root and whole-plant 13C values varied similarly with total dry weight in control and droughted plants, but not when plants were N-starved. Shoot 15N was not correlated with total dry weight, although the heaviest plants had the most negative root 15N values (P<0.05; Table 4). Absolute shoot–root 15N differences in droughted or N-starved plants were correlated positively with total dry weight (P<0.05; Table 4).
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Whole-plant
15N was not correlated with total dry weight in any treatment (Table 4). Many genotypes, however, showed no dry weight response to either stress (Fig. 1). Excluding the least productive and least responsive genotypes (i.e. Genotypes 15 to 11 in the drought treatment, and 15 to 5 in the N starvation treatment: Fig. 1), revealed significant positive correlations between whole-plant 15N and the stress index, SI (P<0.05, n=18 for drought and n=8 for N starvation; Fig. 5). The least stressed of those plants (SI0) had the most negative whole-plant 15N. In droughted plants, this relation was due to shoot 15N, root 15N showing no correlation with the SI. In N-starved plants, however, both shoot and root 15N were significantly (P<0.05) correlated with the SI. In contrast, neither shoot nor root 13C was correlated with the SI (data not shown).
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Correlations between plant dry weight and total N concentrations in shoots and roots
Total dry weight of control plants was always correlated positively (P<0.05) with the concentration of total N in root dry matter, but not in shoots (Table 4). When plants were stressed, however, total dry weight became negatively correlated with total N concentration in shoots: the heaviest plants in the stress treatments had the smallest total N concentrations in their shoots.
Correlations between isotope natural abundances and long-term meteorological averages
Of the long-term meteorological averages available for the sites-of-origin (Pakniyat et al., 1997), only mean humidity at 14.00 h local time was correlated consistently with any isotopic data (). The more humid the site-of-origin, the less negative were the experimentally determined 13C values of genotypes from those sites.
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Absolute shoot–root
15N values of control and N-starved plants were also correlated positively with humidity at site-of-origin, as was shoot 15N under N starvation. Only in droughted plants was 13C correlated with mean annual and mean January temperatures. Mean January temperature was correlated positively with shoot, root and whole-plant 15N in the N starvation treatment. The only isotopic measurement with which mean annual rainfall was correlated was root 15N of controls.
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Discussion |
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Variations in whole-plant
15N in relation to drought and N starvationThe 1.3
range in whole-plant 15N in controls (Fig. 2) indicates the extent to which genotype may influence 15N/14N fractionations in H. spontaneum when plants have access to a common N source. By comparison, whole-plant 13C varied by 1.8 among controls, a larger range than that found for well-watered genotypes of H. vulgare (Acevedo, 1993); greater variability is to be expected among individuals from wild populations than among those from genetically narrower breeding lines. Whole-plant 15N may vary significantly, therefore, for reasons unconnected to changes in the 15N values of external N source(s).
15N/14N discriminations between whole plants and an external N source can have only one general cause: the loss from plants of some isotopically altered N. There is no experimental or theoretical evidence that N uptake itself fractionates 15N/14N. Possible mechanisms of N loss include: the shedding of senescent plant parts; the loss of N volatiles (e.g. NH3, NOx, amines, HCN: Wetselaar and Farquhar, 1980) from leaves into the atmosphere; and the loss of soluble N (e.g. , amino acids: Jones and Darrah, 1993; Van der Leij et al., 1998) from roots into the rooting medium. Each of these is considered as a possible explanation for the data in Fig. 2.
Senescence can be discounted: the young H. spontaneum plants showed little visible leaf senescence. In an annual species such as H. spontaneum, there is little root senescence until synchronous mortality occurs towards the end of an individual's life, as in Triticum aestivum L. (Van Vuuren et al., 1997).
NH3-N lost from organic matter can have 15N values down to -40 (Handley et al., 1996, 1999). Such a net loss of 14N will be reflected by an increase of whole plant 15N in proportion to the fraction of total plant N lost, is most likely to occur via stomata, and to increase with stomatal conductance (which should, in turn, cause shoot 13C to become more negative). Therefore, if N volatilization is significant, shoot 15N should increase as shoot 13C becomes more negative. No such relation was found for H. spontaneum, and N volatilization was unlikely to have caused the variations in whole-plant 15N shown in Fig. 2. A similar conclusion was reached by other authors (Bergersen et al., 1988; Robinson and Conroy, 1999) who were unable to attribute variations in whole-plant 15N of Glycine max (L.) Merrill or Panicum coloratum L., respectively, to N volatilization.
The loss of soluble N from roots does occur, as does its partial resorption (Jones and Darrah, 1993). As with gaseous N losses, little is known of their magnitude or 15N values relative to whole-plant 15N (Schmidt and Kexel, 1998). Robinson et al.'s theory suggested that the loss from roots of organic-N which was less 15N-enriched than total N was consistent with the 15N values measured for other N pools (e.g. root and shoot total N and ) (Robinson et al., 1998). This is not conclusive evidence but, pro tempore, N exudation seems the most likely determinant of variations in whole-plant 15N among the H. spontaneum genotypes under the conditions of the experiment. Direct tests of this possibility are now being done.
In a common, controlled environment in which source N is defined and for which a good estimate of its 15N value exists, genotypic differences in whole-plant 15N values reflect the extent to which plants retain N in their tissues. Agronomic interest in stress tolerance does not usually concern genotypes whose tolerance involves slow growth rates and, probably, small yields. Rather, the aim is to identify genotypes which have the potential to grow well should conditions allow, and to produce economically and nutritionally acceptable yields when conditions are unfavourable. In Fig. 5, the genotypes which were most productive and stress tolerant (i.e. were heaviest in comparison with their potential growth when unstressed, as indicated by their small SI values) were those which probably retained most N. Yet, those genotypes expressed the largest discriminations against 15N, i.e. they had the most negative whole-plant 15N values. According to isotope mass balance arguments, if those genotypes lost only small amounts of N, that N must have had ‘exotic’ 15N values significantly different from total plant N (Yoneyama, 1995; Schmidt and Kexel, 1998). It may be that such plants, when stressed, restrict the loss of N from their roots to only one or two amino acids, say, which happen to have ‘exotic’ 15N values ( exudation from the roots of N-starved barley is negligible: Van der Leij et al., 1998).
Conversely, genotypes expressing the smallest discriminations against 15N were smaller, contained less N and, perhaps, lost relatively more N from their tissues. They may have less capacity to restrict N loss from their roots when stressed than did more stress-tolerant genotypes. Lost N would then comprise a diverse mixture of N compounds with a correspondingly wide range of 15N values. The average 15N of lost N would then be closer to that of the total N, resulting in smaller whole-plant discriminations against 15N. These intriguing possibilities also require explicit testing.
Variations in values in relation to conditions at sites-of-origin
No consistent or strong associations were found between plant 15N measured under the experimental conditions and habitat characteristics (Table 1) or long-term meteorological averages (Table 5). 13C values, by contrast, varied consistently with site-of-origin humidity. The association between less negative 13C (measured at a common ambient vapour pressure deficit; vpd) and greater site-of-origin humidity is opposite to that found between ambient vpd and 13C discrimination in several C3 species (Madhavan et al., 1991; Masle et al., 1993). As commonly reported for other plant species, Handley et al. found that shoot 13C was most negative in H. spontaneum genotypes from sites receiving the least rainfall annually (Handley et al., 1994). That correlation was not found in this hydroponic experiment.
A strong inverse relation (r=-0.59, P<0.001) has been found between mean annual rainfall and site-averaged foliar 15N for a wide range of ecosystems (Handley et al., 1999). Such was the variability in 15N, however, that many samples were required to reveal significant correlations with environmental factors. Discrepancies between samples collected from the field and those produced in common, controlled environments have been well-documented for 13C (Condon and Richards, 1993); similar constraints will, no doubt, apply to research with 15N.
Some genotypes which originated from one locality expressed similar 15N responses to stress, while others from another site showed very different responses. For example, the Tabigha genotypes (2 and 27) showed opposite shoot–root 15N responses to drought and N starvation (Fig. 4), despite there being no significant difference in their growth or whole-plant 15N whether stressed or unstressed (Figs 1, 2). By contrast, the three Neve Ya'ar genotypes (1, 25 and 28) had very similar shoot–root 15N responses in the two stress treatments (Fig. 4). While Genotypes 1 and 28 grew similarly (Fig. 1) and had similar whole-plant 15N values (Fig. 2), Genotype 25 differed from these in shoot–root 15N when droughted, but not when N-starved.
The utility of plant 15N as a physiological integrator
Although the statistically significant correlations between 15N and total plant dry weight were not particularly strong (Table 4), they were of similar magnitude to some which have been measured between total dry weight and 13C (Condon and Richards, 1993). The correlations between whole-plant 15N and SI, especially for N starvation, but less convincingly for drought (Fig. 5), suggest that causal links exist between 15N and stress tolerance.
The possibility that, in controlled experiments, whole-plant 15N may reflect net N retention suggests that whole-plant 15N could be used to screen plants for this agriculturally and ecologically important trait. It will not be easy to apply such an idea in field settings because of the uncertainties which continue to surround the identity and 15N value(s) of plant-available N species in soil (Handley and Scrimgeour, 1997; Handley et al., 1999).
Whole-plant 15N in droughted or N-starved H. spontaneum was always more negative than in controls, confirming the report for shoot 15N of hydroponically grown plants in response to salinity (Handley et al., 1994). The whole-plant 15N of many genotypes did not respond to either drought or N starvation, whereas shoot–root 15N did. Shoot–root 15N may, therefore, be a more sensitive indicator of incipient stress than shoot, root or whole-plant 15N.
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Acknowledgments |
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The Scottish Crop Research Institute receives grant-in-aid from the Scottish Office Agriculture, Environment and Fisheries Department. Winnie Stein, Sigrun Holdus and Richard Keith provided technical help, Professor Eviatar Nevo the H. spontaneum genotypes, Dr Donald Mackerron solar radiation data, and two anonymous referees helpful comments.
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Notes |
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1 To whom correspondence should be addressed: Fax: +44 1 382 562426. E-mail: D .Robinson@scri.sari.ac.uk
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