Journal of Experimental Botany, Vol. 51, No. 343, pp. 287-297,
February 2000
© 2000 Oxford University Press
Effects of symbiosis with Frankia and arbuscular mycorrhizal fungus on the natural abundance of 15N in four species of Casuarina
1 Plant Science Group,Division of Biochemistry and Molecular Biology, Bower Building, Glasgow University, Glasgow G12 8QQ, UK
2 Forest College and Research Institute, TamilNadu Agricultural University, Mettupalayam 641 301, India
3 Department of Chemistry, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK
4 School of Applied Sciences, University of Glamorgan, Pontypridd, Mid-Glamorgan CF37 1DL, UK
5 Department of Cellular and Environmental Physiology, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK
Received 9 August 1999; Accepted 1 October 1999
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Abstract |
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The effect of interactions between Casuarina species, Frankia strains and AMF on nitrogen isotope fractionation within the plant were determined under conditions where changes in source nitrogen were minimized by growing plants in mineral nitrogen-deficient conditions and without added organic N. Casuarina cunninghamiana, C. equisetifolia, C. glauca, and C. junghuniana were inoculated singly with three Frankia strains or were dual inoculated with Frankia and Glomus fasciculatum. The %N and
15N of separated parts of plants inoculated with the three Frankia strains or with Frankia+Glomus were not significantly different within Casuarina species. However, the slow-growing C. junghuniana differed in several variables from the other three species. There was a highly significant, linear relationship between the natural logarithms of cladode N content and 15N of plants of the four Casuarina species when inoculated with Frankia or with Frankia+Glomus, showing that nitrogen supply and the correlated variable, plant growth rate, were major determinants of 15N. Provision of small quantities of (NH4)2SO4 or KNO3 increased several-fold the growth of three of the Casuarinaspecies when inoculated with Frankia alone or with Frankia+Glomus. Within species, mycorrhizal and non-mycorrhizal plants receiving supplementary soluble phosphate were of similar dry weights at harvest. 15N values for cladodes of C. cunninghamiana, C. equisetifolia and C. glauca were similar, but values for the poor growing C. junghuniana were more variable and, with the exception of plants receiving KNO3, were lower than those of the other three species. Reduced growth due to suboptimal availability of N or P had a major influence on 15N and, in these conditions where plants could not access significant amounts of organic N, outweighed any effects on cladode 15N of colonization by Glomus. 15N values of nodules were higher than other parts of Frankia or Frankia+Glomus inoculated Casuarinas, conceivably due to retention in nodules of fixed N, with 15N close to zero. Key words: Arbuscular mycorrhizas, Casuarina, Frankia, nitrogen fixation, nodules
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Introduction |
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Casuarina equisetifolia is a tropical tree, nodulated by the nitrogen-fixing actinomycete Frankia and forming symbiotic associations with both ecto- and endomycorrhizal fungi (Cervantes and Rodriguez Barrueco, 1992
). It is used widely for the stabilization and restoration of soils, as an amenity tree species and as a source of timber and fuel (Diem and Dommergues, 1990). Several studies have shown that growth, nitrogen and phosphorus accumulation in seedlings are enhanced significantly by inoculation with effective symbionts and there has been considerable interest in quantifying such effects, both in pot experiments and in the field (Daft et al., 1985; Cervantes and Rodriguez Barrueco, 1992; Sempavalan et al., 1995; SubbaRao and Rodriguez Barrueco, 1995; Vasanthrakrishna and Bagyaraj, 1993).
15N isotope dilution methods, using either 15N enriched fertilizer or 15N natural abundance (15N), have been used by several investigators to estimate nitrogen fixation in Casuarinas. Sanginga et al., using 15N-enriched fertilizer,showed wide variation in the proportion of nitrogen obtained from fixation by different provenances of C. equisetifolia and C. cunninghamiana (Sanginga et al., 1990). Foliar analysis of 3.5-year-old mixed plantations of C. equisetifolia with Eucalyptusxrobusta, periodically labelled with 15N enriched ammonium sulphate, indicated that 50–60% of the nitrogen in the trees was derived from nitrogen fixation (Parrota et al., 1994, 1996). Using 15N assays, it has been estimated that nitrogen fixation provided 38% of the nitrogen of a 3-year-old C. equisetifolia plantation on dunes in Senegal (Mariotti et al., 1992). 15N values have also been used as an indicator of nitrogen fixation by C. equisetifolia, in various habitats (Yoneyama et al., 1990, 1993). The similarity of data obtained by 15N and by difference of total nitrogen gave confidence to these estimates of nitrogen fixation (Mariotti et al., 1992) while similar values for amounts of nitrogen fixed have also been obtained from 15N and from isotope dilution of 15N-enriched fertilizer in Frankia nodulated alders (Domenach et al., 1989).
The 15N technique compares the slight depletion of 15N (-0.2 to -2.0
, Yoneyama et al., 1993) that occurs naturally in plants dependent solely on nitrogen fixation with values obtained for reference plants that are assumed to be assimilating similar sources of reduced nitrogen from the soil as the nitrogen-fixing species. Notionally, the technique is very attractive for use in the field to identify and to quantify nitrogen fixation since it avoids disturbance of the ecosystem under study, either by addition of fertilizer or through excavation of nodulated roots. However, it is now known that many internal and external factors affect plant 15N and that these can render the technique as currently practised unsuitable for field investigation of nitrogen fixation. For example, differences between the reference and the nitrogen-fixing plant species in root distribution, N transformation processes in the rhizosphere, multiple N sources and their uptake from soil, physiological processes that affect nitrogen assimilation, and associations with other soil organisms such as ecto- and endomycorrhizal fungi can all affect 15N and estimates of N fixation that may be derived from such data (Handley et al., 1993; Handley and Scrimgeour, 1997; Redecker et al., 1997; Azcon et al., 1998). Recently, it has been shown that N deficiency may affect significantly foliar and whole plant 15N in barley and that the specific effect (15N enrichment or depletion) was dependent on genotype (Robinson et al., 2000). Handley et al. demonstrated similar effects for salt-stressed barley (Handley et al., 1997).
Symbioses with mycorrhizal fungi may affect plant 15N in several ways, for example, by changing the chemical forms of soil N available to the host plant (Azcon-Aguilar et al., 1998) or by effects on external or internal partitioning of 15N. In Ricinus communis, it was found that inoculation with arbuscular mycorrhizal fungi (AMF) decreased significantly both whole plant 15N and the distribution of 15N between roots and shoots (Handley et al., 1993), although these effects could have been due to phosphorus deficiency in the uninoculated plants (Handley and Scrimgeour, 1997). In lettuce and barley, species varying in their susceptibility to infection by AMF, it was found that only in the former, more susceptible species was whole plant 15N affected significantly by AMF and by interactions of AMF with N supply (Azcon-Aguilar et al., 1998). In nitrogen-fixing species, it has been suggested that the generally lower 15N values of AMF inoculated, nodulated acacias might be due to promotion of N fixation by AMF (Michelsen and Sprent, 1994); 15N of non-nodulated Acacia nilotica was not affected significantly by inoculation with AMF. In Phaseolus vulgaris, an effect of inoculation with AMF on shoot 15N was dependent on the strain of Rhizobium with which plants were inoculated (Redecker et al., 1997). However, in many such studies, plants were grown in non-sterile soil containing unknown sources of nitrogen, thus leaving open the possibility that treatment differences were due to access to different forms of nitrogen.
The effects of mycorrhizal associations on 15N of actinorhizal plants have not been investigated in any detail. The purpose of the current investigation was to determine for Casuarinas, grown under controlled conditions, the effect of inoculation with AMF on 15N of young plants nodulated by Frankia. Plant species and Frankia strain effects have been examined. Changes in source nitrogen during the experiment were minimized by growing plants in perlite, devoid of organic nitrogen and virtually all mineral N. Consequently, it is possible to determine more precisely than hitherto whether interactions between plant species, Frankia strains and AMF affect isotope fractionation within the plant.
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Materials and methods |
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Germination of seed
Seed of Casuarina equisetifolia ssp. equisetifolia (L.) Johnson, C. cunninghamiana Miq., C. glauca Sieber ex. Spreng., and C. junghuniana Miq. of Australian origin was purchased from CSIRO, Canberra. Seed was germinated in a controlled environment chamber (dark/light regime, 24/26±2 °C; 16 h photoperiod; irradiance 270 mmol m-2 s-1 from 80 W warm white fluorescent tubes (Omega, UK) in trays of perlite/sterile quartz sand (2 : 1, v/v) mix, Silvaperl, UK, with Crone's N nutrient salts and Hoagland's A–Z micronutrients (Hooker and Wheeler, 1987
). Seedlings were used in experiments after 6 weeks (experiments 1 and 3) or 7 weeks (experiment 2) growth when they were still green in colour and approximately 2 cm high.
Culture of Frankia
Frankia strains ORS020607 (Diem and Dommergues, 1983), UGL020604 and UGL020605, all highly effective on C. equisetifolia, C. cunninghamiana and C. glauca (Sempavalan et al., 1996), were grown in static culture at 28 °C in propionate medium containing casamino acids as the N source (Hooker and Wheeler, 1987) for 6–8 weeks before use. Mycelium was harvested by centrifugation, washed with distilled water and homogenized lightly in a Potter Elvehjem homogenizer. The packed cell volume of cultures was determined by centifugation in blood cell sedimentation tubes at 350 g for 10 min. Seedlings were inoculated with the equivalent of 20 µl packed cell volume per seedling.
Culture and inoculation of AMF
Glomus fasciculatum E3 (Rothamsted Experimental Station) was maintained on roots of plantain (Plantago lanceolata L.) grown in pot culture and infection ascertained by light microscopy (Sempavalan et al., 1995). Casuarina seedlings were inoculated with finely chopped roots that were infected or uninfected with Glomus (Frankia-only plants) and mixed thoroughly with the potting medium (perlite : washed, sterile quartz sand, 2 : 1 v/v) at the rate of 0.7 g fresh weight roots seedling-1. Plantain roots were checked for infection by light microscopy prior to use.
Inoculation with Frankia (Experiment 1)
Inoculated seedlings were transplanted into pots containing 180 ml perlite/sand medium. Three seedlings were planted initially per pot and were thinned to one per pot after 10 d to remove dead, unusually small or large seedlings. Pots (6 per Frankia strain or uninoculated control) were placed on trays and watered two to three times weekly to field capacity with tap water. Pots containing uninoculated seedlings or seedlings inoculated with different strains of Frankia were separated from each other by Perspex divides. None of the uninoculated seedlings were nodulated or mycorrhizal at harvest. Each pot received a small amount of mineral N (0.4 mg NH4NO3) after transplanting the seedlings. Because of etiolation and necrosis, control seedlings were harvested after 90 d, the root systems washed with distilled water and plants dried at 60 °C to constant weight. Inoculated seedlings were harvested 134 d after inoculation, divided into nodules, roots, stems, and cladodes and dried as above. Dried plant material was ground to a fine powder using a ball mill and analysed for %N and 15N using a Europa Scientific Tracermass continuous flow isotope ratio mass spectrometer, as described previously (Handley et al., 1993).
Inoculation with Frankia and Glomus (Experiment 2)
Seedlings were inoculated with Frankia as above on transplanting to pots and received the same amount of ‘starter’ mineral N. Seedlings inoculated with the same strain of Frankia were carefully removed from the pots after 85 d, when nodules were well developed. The perlite/sand growth medium was mixed thoroughly with chopped plantain roots that were infected with Glomus. The seedlings were repotted into medium containing 0.7 g fresh weight mycorrhizal plantain roots per 180 ml pot. Great care was exercised to avoid cross-contamination of pots and control seedlings remained uninfected with either symbiont on harvest. Plants were grown for a further 87 d before harvest, drying, milling and analysis for %N and 15N as above.
A portion of the fresh roots of each plant was weighed and preserved in alcohol for qualitative determination of mycorrhizal colonization by light microscopy (Sempavalan et al., 1995).
Supply of ammonium or nitrate to mycorrhizal and non-mycorrhizal, Frankia-nodulated seedlings (Experiment 3)
Seedlings of the four Casuarina species were inoculated with Frankia UGL020605, as in Experiment 1, and transplanted into perlite/sand medium containing 4 g l-1 uninfected plantain roots, or medium containing 4 g l-1 plantain roots colonized by Glomus. Care was exercised to avoid cross-contamination of pots and infection of roots of uninoculated control seedlings with Glomus could not be detected by light microscopy at harvest. Each plant received a supplement in 50 ml water of 4 mg N from either KNO3 or (NH4)2SO4 6 d after inoculation of the 6-week-old seedlings. In addition, plants inoculated with Frankia alone received two supplements of soluble phosphate, applied as NaH2PO4/Na2HPO4 buffer, pH 6.8 at a rate of 13.5 mg P per pot 6 d after inoculation and again after 45 d. Plants were harvested 143 d after inoculation, for analysis as in Experiment 2.
Statistical analyses were carried out using Excel (Microsoft) or Statmost (DataMost Corporation)
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Results |
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Experiment 1: effects of Frankia strain on growth, %N and
15N of four species of CasuarinaDifferences between the dry weights of plants within each species when inoculated with one of three Frankia strains were significant only for C. equisetifolia inoculated with ORS020607, which showed decreased growth, and for increased growth of C. junghuniana with UGL020605 (Fig. 1
). There were substantial and significant differences between the dry weights of the four plant species, however. Mean dry weight ±SE (n=6) of C. cunninghamiana, inoculated individually with the three strains of Frankia, was 1.16±0.106 g; C. equisetifolia 0.793±0.071 g; C. glauca 0.37±0.139 g; and C. junghuniana 0.20±0.066 g. There was no significant difference between the dry weights of uninoculated control seedlings of the four species for which the mean dry weight overall was 0.025±0.0018 g.
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) with Frankia UGL020604, UGL020605 or ORS020607 (labelled 4, 5 and 7, respectively) and in Experiment 2 (
) dual inoculated with one of these Frankia strains and Glomus E3. Plant dry weights which are significantly different (P<0.05) from the weights of other Frankia strain combinations within each Casuarina species (n=6) in a particular experiment are indicated by the letter (a) in Experiment 1 and (b) in Experiment 2.The effectivity of nitrogen fixation (total plant N mg-1 nodule dry weight) varied significantly between species with C. equisetifolia showing greatest effectivity and C. junghuniana the least (Fig. 2
). Significant differences in nodule effectivity between strains when inoculated on to a single Casuarina species were limited to lower effectivity of C. equisetifolia inoculated with ORS020607, as reported previously (Sempavalan et al., 1996), and the greater effectivity of nodules on C. junghuniana inoculated with UGL020605.
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The %N and
15N values for separated parts of plants inoculated with the three Frankia strains were not significantly different within the four Casuarina species. These data are combined for each species in Fig. 3. 15N values for control (non-nodulated) seedlings were all positive, while 15N values for both cladodes and nodules of inoculated plants were all negative. 15N values and %N of both cladodes and nodules of the poor growing C.junghuniana were significantly lower than in the other species. There was a significant, positive, linear correlation between %N and 15N for nodules (r=0.73) and for cladodes (r=0.78) from plants of all four species.
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Experiment 2: effects of inoculation with Frankia and AMF
Plants in this experiment were grown for 38 d longer than in Experiment 1 (172 d compared with 134 d). At harvest, the mean dry weight of C. cunninghamiana was 4.5 times; C. equisetifolia 2.25 times; C. glauca 3.6 times and C. junghuniana 4.6 times that of plants of these species in Experiment 1 (Fig. 1). Microscopic examination showed colonization of roots of all plants inoculated with Glomus.
The effectivity of nitrogen fixation was similar to that for plants inoculated with Frankia alone, with the exception of C. equisetifolia inoculated with Frankia UGL020604 and UGL020605, where effectivity of N fixation in dual inoculated plants was three times that of plants inoculated only with Frankia (Fig. 2). However, higher effectivity was not reflected in greater growth of C. equisetifolia because a smaller weight of nodules was produced per plant. The greatly improved growth of dual inoculated C. junghuniana in Experiment 2 was accompanied by the production of a greater mass of nodules per plant rather than increased effectivity in nitrogen fixation.
As in Experiment 1, analysis of variance showed that %N and 15N of separated parts of plants inoculated with the three Frankia strains were not significantly different within the four Casuarina species. These data are combined for each species in Fig. 4. Growth of C. junghuniana was much greater in Experiment 2 and was similar to C. glauca. However, C. junghuniana still showed the lowest%N and greatest depletion of 15N in cladodes and nodules of the four species studied.
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The %N contents of cladodes and nodules of all four species in Experiment 2 were on average 18% higher and 17% lower, respectively, than in Experiment 1.
15N values for cladodes of C. cunninghamiana, C. glauca and C. junghuniana in Experiment 2 were 29% higher (less negative) than in Experiment 1, but only 9% higher in C. equisetifolia. 15N values of nodules were not significantly different in the two experiments. Regression analysis did not show a significant, linear correlation between %N and 15N for nodules or for cladodes of plants of all four species in this experiment.
Regression analysis of the natural logarithm (logn) of the combined data for cladode N content per plant and for cladode 15N of inoculated plants of all four Casuarina species in Experiments 1 and 2 showed a highly significant, exponential relationship (r=0.789; P<0.001) between these two parameters (Fig. 5).
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Experiment 3: effects of mineral nitrogen supplements on 15N of mycorrhizal and non-mycorrhizal, Frankia-nodulated seedlings
The supply of mineral nitrogen increased dry weight of plants at harvest by factors of between 2- and 5-fold compared with plants in Experiment 2, despite the growth period in Experiment 3 being 29 d less (143 d compared with 172 d) than in Experiment 2 (Fig. 6). The effect of inoculation with Glomus on growth was variable. Variability masked the significance of differences between the majority of mycorrhizal and non-mycorrhizal plants. However, mycorrhizal+NH4 plants of C. cumminghamiana and C. equisetifolia showed significantly greater growth than plants of the same species inoculated with Frankia alone+NH4 and receiving phosphate supplements.
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The data for P content of cladodes (Fig. 6) show that all non- mycorrhizal Casuarinas receiving phosphate supplements accumulated greater quantities of P than mycorrhizal plants of the same species, even though the latter showed equal or better growth overall. The same pattern of accumulation of P was found also in stems and roots (data not shown)
Average values for 15N of cladodes of all species were higher in Experiment 3 than in Experiments 1 and 2 (Fig. 7). However, the most striking feature of the data presented is the relative uniformity of 15N values for cladodes of three of the different Casuarina species, irrespective of inoculation practice or supplementary mineral nitrogen and despite the differences in 15N of the ammonium sulphate (15N=-4.43±0.099; n=4) or potassium nitrate (15N=+3.72±0.364; n=4) supplied to mycorrhizal and non-mycorrhizal plants. There were no significant differences in 15N between cladodes of non- mycorrhizal and mycorrhizal plants of all four species, but it is notable that the lowest mean value for 15N overall was shown by cladodes of the poorest growing plants, non-mycorrhizal C. junghuniana receiving NH4.
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In nodules, the %N content was similar to that in Experiments 1 and 2, with the exception of a low value for Frankia-inoculated C. equisetifolia receiving ammonium sulphate and supplemental P (Fig. 8
). However, in contrast to Experiment 1 and 2, 15N values were mostly positive for C. cunninghamiana, C. equisetifolia and C. glauca. Values of 15N for nodules of non-mycorrhizal and mycorrhizal plants receiving ammonium sulphate tended to be lower than for nitrate plants and this is most evident for C. junghuniana receiving ammonium sulphate and supplemental P.
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Discussion |
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Inoculation of four Casuarina species with three strains of Frankia gave few significant differences in growth while those differences which were observed were affected by the presence or absence of Glomus (Fig. 1). For example, in C. glauca inoculated with Frankia ORS020607 alone, dry weight at harvest was significantly lower than in plants inoculated with the other two Frankia strains. When C. glauca was inoculated with both ORS020607 and Glomus, however, plants showed a significantly higher dry weight at harvest than other dual inoculated plants of this species. Strain-dependent effects on plant growth have been demonstrated previously for Casuarina species inoculated with Frankia and AMF (Diem and Dommergues, 1990
; Sempervalan et al., 1995) and for other actinorhizal plants (for example, Isopi et al., 1994) and must arise from a wide range of interacting effects.
It is of interest in this respect that the effectivity in N2 fixation of ORS020607 when in symbiosis alone with C. glauca was significantly less than the other two strains (Fig. 2). However, when plants were also inoculated with Glomus, additional nodule material was formed that provided sufficient fixed N2 to support higher growth rates than plants inoculated with the other two strains. It has been suggested that phosphate may play an important role in the regulation of nodulation in Alnus (Wall and Huss Danell, 1996) and it is conceivable that nodulation of C. glauca by ORS020607 may be particularly susceptible to the improvements in P nutrition which would follow colonization by AMF.
As found earlier for C. equisetifolia (Mariotti et al., 1992) the data from this study did not show a statistically significant effect of Frankia strain on 15N for any of the Casuarina species studied. These results contrast with those of Bergersen et al. who found that Rhizobium strain affected the distribution of 15N in soybean (Bergersen et al., 1986). The values for 15N for cladodes and nodules of C. equisetifolia in Experiments 1 and 2 were much lower than reported previously. Thus, it was found that 15N of C. equisetifolia grown on sterile sand in mineral N-free culture averaged -0.64 (Mariotti et al., 1992). In field studies, 15N for cladodes of C. equisetifolia was -1.4 and for nodules between +0.8 and +1.1 (Yoneyama et al., 1990). These data compare with the 15N values reported here of -4.5 and -0.66 for C. equisetifolia cladodes and nodules, respectively, from plants inoculated only with Frankia (Fig. 3) and of -4.19 and -1.14, respectively, for dual inoculated plant (Fig. 4). In experiments of others, the soil contained some organic N (in Mariotti's experiments 0.026%) and uptake of this nitrogen may have contributed to the smaller depletion of 15N apparent in samples of plant tissue.
The dry weight at harvest of dual inoculated plants was several times higher than of plants inoculated with Frankia alone, due at least in part to the longer period of growth provided for the latter plants before harvest, but also conceivably through better phosphorus nutrition of mycorrhizal plants since these plants should be able to access more readily the sparingly soluble ferrous phosphate in Crone's -N nutrient medium. Although the contribution of dual inoculation to this improved growth cannot be quantified, previous studies suggest that this could be substantial. Thus, it was shown that after 60 d growth in the same environment cabinets, the fresh weight of C. equisetifolia seedlings growing in a peat/sand/perlite mix and inoculated with Frankia ORS020607 alone was less than 10% of the weight of seedlings which grew rapidly following inoculation simultaneously with Frankia and Glomus (Sempavalan et al., 1995).
Dual inoculated plants generally showed less depletion of 15N in both cladodes and nodules than those inoculated with Frankia alone. This could conceivably be due to a specific effect of mycorrhizal colonization. However, the exponential relationship demonstrated between logn N content and logn 15N of cladodes from all four Casuarina species from Experiments 1 (Frankia inoculated) and 2 (Frankia+Glomus inoculated) suggests that a higher growth rate, rather than mycorrhizal colonization per se, is the main factor influencing isotopic fractionation (Fig. 5). It is of interest in this context to consider earlier data for the non-N2-fixing plant, Ricinus communis, supplied with urea as the N source (Handley et al., 1993). It was found that colonization of roots by the arbuscular mycorrhizal fungus Glomus clarum increased values for 15N of whole plants by as much as 2. However, deficiency of P reduced the growth of non-mycorrhizal plants by 20% so that the differences in 15N could not be ascribed definitively to effects of the mycorrhizal fungus on fractionation of nitrogen isotopes (Handley and Scrimgeour, 1997). In a later experiment, (Azcon-Aguilar et al., 1998) it was shown that P deficiency had no significant effect on plant 15N while nitrogen supply did. These data strongly support the possibility that growth differences made a significant contribution to the increase in 15N of mycorrhizal compared with non-mycorrhizal plants. Growth was, in turn, related to the amount of N each plant accumulated.
Experiment 3 was designed to determine whether inoculation with Glomus affects 15N isotope fractionation in Frankia-inoculated Casuarinas when provided with either NH4 or NO3, at concentrations which would not inhibit N2 fixation. In this experiment, non-mycorrhizal plants were provided with supplementary P at rates and with a frequency that reduced greatly the growth differences between plants in Experiments 1 and 2 (Fig. 6). However, although plant weight differences were minimized successfully, the P content of non-mycorrhizal plants supplied soluble P was several-fold that of mycorrhizal plants at harvest (Fig. 6). There was no obvious correlation between plant P content and 15N, however, findings that are consistent with those of Azcon-Aguilar et al. (Azcon-Aguilar et al., 1998).
Provision of mineral N in small quantities in Experiment 3 increased seedling growth several fold compared to plants in Experiments 1 and 2. Such ‘starter nitrogen’ effects on seedling growth following inoculation have been noted previously for a range of actinorhizal species, even when inoculated with preparations of crushed field nodules (for example, Bond and Mackintosh, 1975). It is notable that the relatively high growth rate and the similarity of plant dry weights of C. cunninghamiana, C. equisetifolia and C. glauca at harvest in all treatments was paralleled by uniformity in values for 15N averaging -2.53 for these three species (Figs 6, 7). In contrast, in C. junghuniana, where plant dry weight was less than one-third of the other species (Fig. 6), cladode 15N was significantly lower than for the other three species with the exception of mycorrhizal C. junghuniana receiving NO3, in which 15N was similar to the other species (Fig. 7). This result is presumably due to a greater contribution of added NO3 (15N=+3.72) to cladode N, although variability masked the significance of the apparently better growth of plants in this treatment.
With the exception of the very low value for nodules from C. junghuniana supplied with 15NH4 (Fig. 8), depletion of 15N in nodules from seedlings in all three experiments was significantly less than in cladodes. This is in keeping with the higher 15N values of nodules compared with leaves reported previously (Yoneyama et al., 1990) for a range of shrub legumes and for C. equisetifolia. Values of 15N closer to atmospheric N2 could result from the retention in the nodules of substantial fixed nitrogen, which would be subject to fewer metabolic and transport fractionations than N in other plant parts. Data supporting this possibility were presented by Bond and Mackintosh who showed for nodulated Coriaria arborea and Hippophae rhamnoides, supplied with 15NO3, that 80% of the nodule N was derived from fixation (Bond and Mackintosh, 1975). In nodulated Alnus glutinosa supplied with 15NO3 for 7 d, it was found that whereas 75% of root N came from NO3, only 3.5% of nodule N was from this source (Baker et al., 1997).
Again with the exception of the low value for nodules from C. junghuniana supplied with 15NH4 (Fig. 8), nodule 15N in Experiment 3 was generally higher than in Experiments 1 and 2, irrespective of whether plants were mycorrhizal or non-mycorrhizal and supplied with NO3 (15N=+3.72) or NH4 (15N=-4.43). The reasons for this are unknown. However, it is notable that plant growth in Experiment 3 was improved by provision of small amounts of mineral nitrogen and that the very low value of 15N for nodules from C. junghuniana supplied with 15NH4 was for plants which showed poor growth (Fig. 6). Recycling of assimilated N from shoot to nodules has been demonstrated (Baker and Parsons, 1997; Baker et al., 1997) and it is conceivable that these last plants may have assimilated, and translocated to the nodules, a higher proportion of 15N derived from NH4. However, the relative contributions of the processes of metabolism and transport to the fractionation of N isotopes are not known.
In conclusion, the data presented does not provide evidence for a specific effect on 15N of colonization of Casuarina species by AMF when plants rely solely or mainly on N2 fixation for growth. Poor growth is likely to be a major influence in determining the extent to which 15N is depleted. In these experiments, reduced growth could be due to growth conditions that were suboptimal for a particular species (this probably explains at least in part the poor growth of C. junghuniana), or to reduced availability of phosphate in plants inoculated with Frankia alone, which was alleviated by mycorrhizal inoculation.
The data do demonstrate that final plant 15N need not be related to the initial 15N of source N, while being highly correlated with N supply and plant taxon. Given the present results and those of Robinson et al. (Robinson et al., 2000) and Handley et al. (Handley et al., 1997), it is likely that the low 15N values formerly ascribed to N2-fixation may be partially attributable to abiotic stresses interacting with plant genotype.
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Acknowledgments |
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This work was supported by European Commission contract TS3*-CT92–0090, awarded under the Science for Technology and Development programme STD3. Scottish Crop Research Institute is grant-aided by the Scottish Office Agriculture, Environment and Fisheries Department.
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Notes |
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6 To whom correspondence should be addressed. Fax: +44 141 330 4447.
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References |
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