Abstract: We mainly aimed at investigating symbiotic interactions of 4-year-old Acacia cyanophylla Lindl. (blue-leafed wattle) with soil indigenous rhizobia in a semiarid Mediterranean site, in terms of nodulation and N2 fixation. Secondary, we measured the density of indigenous A. cyanophylla-compatible rhizobia into the soil, in parallel with the biomass, nitrogen and 15N natural abundance (δ15N) in the shoot components. A small indigenous population of Acacia-compatible rhizobia was detected. Concurrently, there were scarce perennial nodules on A. cyanophylla. The species produced small vegetative biomass and had low N content. The biomass was more allocated to stems than to phyllodes, whereas N content was more allocated to latter component. Acacia cyanophylla and its paired non-N2-fixing Olea oleaster Hoffmgg. et Link. (wild olive tree) showed striking intraplant variation in δ15N, which suggested marked isotopic discrimination during N re-allocation among plant components. The measured N2 fixation in phyllodes of A. cyanophylla was low. It was however not possible to measure N2 fixation in total shoots, because of similar δ15N values in shoots of O. oleaster and in fully N2-dependent A. cyanophylla. Present results indicated no positive symbiotic interactions between A. cyanophylla and the indigenous population of rhizobia in semiarid Tunisia.
Introduction
The N2-fixing tree symbioses are largely used to manage nutrient-stressed soils, mainly in semiarid and arid zones, characterized by sparse and low-productive plant cover. Sustainable use of these symbioses however relies on available information of their actual N2 fixation capacities (Dommergues et al., 1999). The 15N natural abundance (δ15N) method, one of the more reliable techniques for measuring N2 fixation (Unkovich and Pate, 2001), has been widely used to measure N2 fixation in acacia symbioses under field conditions. Nevertheless, measurements based on the sampling of whole tree were poorly documented and have been made for only young tree (Muofhe and Dakora, 1999), because of the huge amount of time and labour required for harvesting aged trees. Alternatively, N2 fixation in acacia symbioses, as in other N2-fixing tree symbioses, has generally been measured using foliar δ15N (Polley et al., 1997; Galiana et al., 2002; Chikowo et al., 2004). The latter approach may provide helpful information on actual N2 fixation when δ15N values do not differ between plant components, nevertheless values can be component-linked and thus the sampling should include whole plant or total shoot rather than individual components (Peoples et al., 1991).
Within acacias, the blue-leafed wattle Acacia cyanophylla Lindl., syn. A. saligna (Labill.) H. L. Wendl. (Fabaceae/Mimosoideae) can have great potential in producing biomass in degraded areas (Nasr et al., 1986). A. cyanophylla is originated from south-western Australia and has been successfully introduced and naturalised in a wide range of contrasting environments, mainly in southern and northern regions in Africa. This is most likely because of its striking symbiotic promiscuity in nodulating and fixing N2 in symbiosis with either slow or fast-growing rhizobia, as reported by Nasr et al. (1999). The N2-fixing capacity of A. cyanophylla is largely improved by dual inoculation with compatible rhizobium and arbuscular-mycorrhizal fungus in addition to appropriate P supply (Nasr and Diem, 1987). The species however has poor growth when unable to fix N2 (Stock et al., 1995). There is little information on the nodulation patterns of A. cyanophylla symbiosis in semiarid Mediterranean environments (Nasr et al., 1995), whereas there is no available data on its N2 fixation ability in these environments.
An important consideration in introducing legumes is their symbiotic interaction with soil-resident rhizobium strains (Yates et al., 2004) and successful establishment of introduced legumes generally relies on their ability to nodulate with the soil-resident rhizobia (Parker, 1962). In Australia, indigenous rhizobia nodulating acacias are generally widespread in arid zones, where the host plants are commonly nodulated (Beadle, 1964); nodules are however most often ineffective (Lawrie, 1983). This strengthens that acacias symbioses have generally low N2-fixing capacities (Roughley, 1987; Danso et al., 1991). At our knowledge, there is no available information on long-term interactions between A. cyanophylla and soil indigenous rhizobia in the Mediterranean region. We carried out an experimental plantation in a semiarid Mediterranean site, to mainly investigate symbiotic interactions of 4-year-old A. cyanophylla with putative soil indigenous rhizobia, in terms of nodulation and N2 fixation. In parallel, we measured the density of indigenous A. cyanophylla-compatible rhizobia into the soil and the biomass, nitrogen content and δ15N in the shoot components. Implications of intraplant variation in 15N natural abundance on N2 fixation measurements are discussed.
Materials and Methods
Experimental design
The experimental site was located at the Kondar region in the semiarid central
Tunisia. The soil had poor natural plant cover and low fertility. Soil characteristics
averaged 2 mg C g-1 soil, 0.5 mg N g-1 soil, 0.9 mg P2O5
g-1 soil and 0.3 mg K2O g-1 soil. Seeds of
A. cyanophylla Lindl. (seedlot KL086) were disinfected and scarified
with 96% (v/v) H2SO4. The disinfected seeds were sown
in a tyndallized sandy clay loam soil in plastic growth bags, which were placed
in nursery benches and watered daily. No rhizobial inoculum was added to saplings.
Three-month-old saplings were outplanted to 0.5 ha plot. Wild olive tree Olea
oleaster Hoffmgg. et Link. (Oleaceae), one of the dominant native species
in the experimental site, was planted between A. cyanophylla hedgerows,
as a reference plant to measure putative N2 fixation in A.
cyanophylla. Planting was carried out at the rainy season (late fall).
Spacing between plants was 4.5 m (i.e., 494 plant ha-1). Both species
are evergreen woody plants and have a mesic origin.
Plant Sampling
When trees were 4-year-old, six randomly-selected replicates of A.
cyanophylla were harvested. Phyllodes were mixed and repetitively quartered
to generate a representative sample. Branches with different diameters were
cut into small portions and pooled in a representative sample of branch diameters.
Stems were sawn at different heights to make a composite sawdust sample. Concurrently,
the stems, branches and leaves of six O. oleaster replicates were
randomly selected, harvested and sampled separately. This sampling method can
minimize 15N measurement errors due to the nitrogen cycling in woody
plants. For each sampled A. cyanophylla tree, the ground area
of potentially nodulating roots, as defined by Nasr et al. (1995), was
dug out to 50 cm depth. Then, roots were excavated and examined for the presence
of nodules. When present, nodules were excised, gently washed, dried and weighed.
Fresh fine root samples were fixed in a solution of formalin-acetic acid-ethanol
(65-25-910, v/v/v) for mycorrhizal analysis. The fresh samples of shoot components
were separately oven-dried at 70°C to a constant weight, weighed and the
total biomass of each component was calculated. Dried samples were finely ground
for nitrogen analysis.
Analytical and Calculation Procedures
Density of rhizobia in the soil nearby A. cyanophylla was
calculated, using the most probable number method (Brockwell, 1963). The fine
root samples were cleared with 10% (w/v) KOH, stained with fuchsin-lactic acid
solution (Kormanik and McGraw, 1982) and scanned by light microscopy for the
presence of arbuscular-mycorrhizal structures.
For each shoot sample, N concentration on a dry matter basis (mg N g-1) and δ15N expressed in ‰ were measured with a CHN elemental analyser (SCA, CNRS Vernaison, France) connected to a mass spectrometer (Funnigan Mat Delta S, Bremen, Germany).
Where,
The weighted δ15N in total shoots was calculated using the following equation:
Where, TN represents the N content and L, B and S denote photosynthetic components (phyllodes for A. cyanophylla and leaves for O. oleaster), branches and stems, respectively.
The fraction of plant N derived from atmospheric N2 (%Ndfa) was measured according to the equation of Shearer and Kohl (1986), as follows:
Where,
| δ15NF | = | δ15N‰ in N2-fixing plant (A. cyanophylla) |
| δ15NNF | = | δ15N‰ in non-N2-fixing reference plant (O. oleaster) |
| B | = | δ15N‰ during N2 fixation, also named "B value" |
Based on the general equation of the standard error of %Ndfa obtained by Shearer and Kohl (1986), we calculated and used the standard error with null covariance, as follows:
Statistical Analysis
A one-way ANOVA with a plant component factor was carried out. When significant
differences were found at p<0.05, means were compared with Duncan's Multiple-Range
Test. The biomass and nitrogen content data were ln-transformed, whilst δ15N‰
data were arcsine-transformed prior to analysis. All data presented are untransformed
means.
Results and Discussion
Indigenous Rhizobia and Root Specialisations
The density of indigenous Acacia-compatible rhizobia at the experimental
site was equal to 65 infective cells g-1 soil, indicative of a very
small rhizobial population. This density was markedly lower than that nodulating
Acacia spp. in other African regions (Odee et al., 1995). Survival
of indigenous rhizobia was likely to be limited by the dry soil conditions,
which resulted from long and recurrent drought periods prevailing in the semiarid
Mediterranean zones. In parallel, roots of A. cyanophylla showed sparse
nodules, which averaged 20 g nodule (dry weight) tree-1. Root nodules
of A. cyanophylla were perennials, dichotomously branched and showed
a fresh and healthy live component, which covered a dark-coloured and suberized
dead component (Fig. 1). This indicated that nodule growth
was indeterminate and cyclic with a senescence phase, most probably at the dry
season, followed by a regrowth phase during the relatively less dry season;
thus underlying that nodule growth can cease during dry periods and resume at
relatively moist periods.
| Fig. 1: | Root nodule on Acacia cyanophylla with a Dead Component (DC) covered by a Live Component (LC). Bar is 1 cm |
| Fig. 2: | Biomass and its distribution in Phyllodes (Ph), Branches (Br) and Stems (St) of Acacia cyanophylla. Error bars indicate SE of the mean; n = 6. Bars with the same case letter are not significantly different at p<0.05 |
At senescence phase, nodules can release large numbers of viable cells, which constitute an inoculant source of new roots (Brockwell et al., 2005). Thus renewed nodule growth with seasonal mortality may be a strategy that helps nodule persistence and expansion on roots of perennial N2-fixing plants in dry environments. Analysis of the collected fine-root samples showed that both A. cyanophylla and O. oleaster were devoid of arbuscular mycorrhizal structures, which may decrease plant δ15N (Spriggs et al., 2003).
Absence of arbuscular mycorrhizal fungi could be attributed to the lack of appropriate soil moisture, concomitant to poor native-plant cover at the study site.
Distribution of Biomass, Nitrogen Content and δ15N
Biomass of stems, expressed as dry weight, of A. cyanophylla
was significantly higher than each of the two other components (Fig.
2). Within shoots, biomass was similarly distributed between phyllodes and
branches. Total biomass, on per hectare basis, was equal to 6 Mg ha-1,
strongly lower than that reported for Acacia spp. growing in other regions
(Shanmughavel and Francis, 2001; Harmand et al., 2004), probably because
performance of the nodulating rhizobium strains and environmental conditions
differed. The N content in phyllodes was low (Fig. 3) and
in the range of non-nodulated woody legumes growing on low-fertile Sahelian
soils (Breman and Kessler, 1995), a fact most likely due to very low available
soil N concomitant with no or low N2 fixation. In contrast to biomass
partitioning, N content was more allocated to phyllodes than to stems. Total
N accumulated by A. cyanophylla plantation was equal to 57 kg
N ha-1. The δ15N values in phyllodes of A. cyanophylla
and in leaves of O. oleaster were positive, whereas those in branches
and stems were negative (Fig. 4). The magnitude of intraplant
variation in δ15N, as expressed by differences in δ15N
between shoot components, varied from 1.27 to 2.81‰ for A. cyanophylla
and from 0.31 to 3.39‰ for O. oleaster. This intraplant variation
was comparable to that obtained for other woody N2-fixing and non-N2-fixing
trees (Yoneyama, 1984; Boddey et al., 2000; Schmidt and Stewart, 2003)
and indicated that 14N was preferentially re-allocated from N-enriched
photosynthetic components to woody components.
| Fig. 3: | Nitrogen content and its distribution in Phyllodes (Ph), Branches (Br) and Stems (St) of Acacia cyanophylla. Error bars indicate SE of the mean; n = 6. Bars with the same case letter are not significantly different at p<0.05 |
| Fig. 4: | 15N natural abundance (δ15N) in Photosynthetic components (Pc) (i.e., phyllodes for Acacia cyanophylla and leaves for Olea oleaster), Branches (Br), Stems (St) and Shoots (Sh) of A. cyanophylla and O. oleaster. Error bars indicate SE of the mean; n = 6. Bars with the same letter are not significantly different at p<0.05 |
This is commonly attributed to the mobilization of N from young leaves to later-formed woody tissues (Unkovich et al., 2000). Intraplant variation in δ15N obviously indicated that δ15N in phyllodes of A. cyanophylla and that in leaves of O. oleaster were not representative of the other plant components. In spite that roots were not investigated here, ideally, wherever feasible, δ15N in roots should also be measured to get further information on the distribution of δ15N within the whole plant.
Nitrogen Fixation
It is noteworthy that there was no significant difference in δ15N
between phyllodes of A. cyanophylla and leaves of O. oleaster
(Fig. 4), suggesting little or no contribution of N2
fixation to phyllode N. For measuring %Ndfa, Unkovich and Pate (2001) proposed
to derive "B value" from plants established under similar conditions to those
of the investigated ones, because several factors other than N2 fixation
per se can discriminate against 15N under fully symbiotic
conditions. Nevertheless, B values derived from host plants grown under controlled
and optimal conditions have been commonly used to measure field N2
fixation in acacias (Galiana et al., 2002; May and Attiwill, 2003; Chikowo
et al., 2004). Moreover, B values of Prosopis sp. have been widely
used to measure N2 fixation in field-growing acacias (Shearer
et al., 1983; Shearer and Kohl, 1986; Yoneyama et al., 1990; Schulze
et al., 1991; Handley et al., 1994; Polley et al., 1997).
We however used the B value derived from Acacia saligna (syn. A.
cyanophylla), as reported by Stock et al. (1995), because at
low %Ndfa values, errors associated with an inaccurate B value are small (Unkovich
et al., 1994). As expected, measured %Ndfa in phyllodes of A. cyanophylla,
using leaves of O. oleaster as a reference, resulted in a low value,
which was equal to 6.2±0.1% (Mean±SE). This value was however
in the range reported for Acacia spp. growing in other African dry zones
(Schulze et al., 1991; Handley et al., 1994; Stock et al.,
1995; Lehmann et al., 2002), thus supporting little contribution of soil-resident
rhizobia in such zones to N nutrition of acacias. The weighted δ15N
in total shoots of A. cyanophylla was equal to -0.69±0.13‰
(Mean±SE), whereas that in total shoots of O. oleaster was equal
to -1.25±0.14‰. Magnitude of difference in δ15N
between shoots (0. 65‰) of the two paired plants was higher than that between
photosynthetic components (0.12‰), underlying that %Ndfa value may be higher
when pairing shoots relative to photosynthetic components. Interestingly, there
was no significant difference (p<0.05) in δ15N between shoots
of O. oleaster, which is fully soil-N-dependent and those of fully N2-dependent
A. cyanophylla (-1.27±0.10‰). Thus, it is not possible to
measure N2 fixation in total shoots of A. cyanophylla based
on the δ15N method.
Present results showed that A. cyanophylla and its neighbouring non-N2-fixing O. oleaster had negative shoot δ15N values, whereas in contrast, Nasr et al. (2005) reported that Casuarina glauca Sieber ex. Spreng. and its neighbouring non-N2-fixing Stipa tenacissima L., in plantations in an another experimental site located at the study area, had positive shoot δ15N values. The δ15N values in the two plant pairs were however in the same range as reported in other N-limited areas (Bustamante et al., 2004). The δ15N in a particular species of plant reflects interaction of many soil and plant processes (Stewart, 2001); we however assumed that particularly root patterns should have no significant implications on δ15N of the two plant pairs because N leaching is trivial in soils in Mediterranean-type climate (Fillery, 2001) and additionally roots of all studied species lacked mycorrhizal structures, which may differently affect plant δ15N. Nevertheless, we suggested at least two possible explanations for such differences in δ15N between the two plant pairs. Firstly, these differences may reflect horizontal variation in δ15N of plant-available soil N between the two experimental sites. Secondly, each of the two plant pairs may have same patterns in N physiology, which differed from the other pair. Implications of horizontal variation in δ15N on the measurements of N2 fixation can be alleviated in planting the reference plant adjacent to its paired N2-fixing plant (Shearer and Kohl, 1986; Peoples et al., 2001), as the hedgerow planting system we used. Further detailed investigations are needed to precisely identify factors that triggered differences in δ15N between A. cyanophylla and O. oleaster pair and C. glauca and S. tenacissima pair.
It is concluded that trivial and sparse root nodulation, concurrent with low N2 fixation rate and poor plant growth indicated no positive symbiotic interactions between A. cyanophylla and the indigenous rhizobium strains. However, small population of indigenous strains in the study site commonly implies successful establishment of introduced strains. Thus, inoculating saplings with selected host-compatible strains, prior to be outplanted, can potentially stimulate growth of A. cyanophylla in semiarid Tunisia.
Acknowledgements
We thank Dr. Murray Unkovich (Soil and Land Systems, School of Earth and Environmental Sciences, University of Adelaide, Australia) for critical review of the manuscript. We also thank Dr. David Carty (NyPa-Greenbridge, Flenniken, El Dorado, USA) for helpful comments on an earlier draft of the manuscript. Thanks to Hervé Casabianca (CNRS, Solaize, France) for assistance on mass spectrometric analysis and to the Regional Department of Agriculture in Sousse (Tunisia) for field support.