Phosphorus sources and availability modify growth and distribution of root clusters and nodules of native Australian legumes

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1 Blackwell Science, LtdOxford, UK PCEPlant, Cell and Environment Blackwell Science Ltd June P availability, root clusters and legumes M. A. Adams et al /j x Original Article837850BEES SGML Plant, Cell and Environment (2002) 25, Phosphorus sources and availability modify growth and distribution of root clusters and nodules of native Australian legumes M. A. ADAMS, T. L. BELL & J. S. PATE Department of Botany, The University of Western Australia, 35 Stirling Highway, Crawley 6009, Western Australia, Australia ABSTRACT A variety of native Western Australian legumes produced root clusters in sand culture confirming field and published observations. In general, these legumes grew equally well when supplied with organic or inorganic sources of phosphorus. The nitrogen content of shoots and roots varied little among treatments for all species, however, phosphorus content was always greater in plants supplied with inositol- P. The plasticity of root growth in response to localized placement of organic and inorganic sources of phosphorus was demonstrated using a simple split root technique. Total root dry weight was, on average, more than doubled in P-amended sand when compared with non-amended sand. Root clusters tended to be produced in areas of relatively high phosphorus concentration and nodules in areas of low phosphorus concentration. Levels of phosphorus in lateral roots grown in P-amended sand were significantly different from lateral roots grown in the corresponding non-amended sand. Growth increases averaging 70% for white sand to over 100% for yellow sand indicated a large degree of plasticity in roots under conditions of heterogeneous supply of phosphorus. Spatially exclusive development of organs for the acquisition of nutrients is discussed in relation to requirements for carbon in organ production and maintenance. Key-words: inorganic P; legumes; organic P; root clusters; root plasticity. INTRODUCTION The role of root morphology, root symbioses (with mycorrhizal fungi and N-fixing bacteria), root distribution within the soil profile and exploitation of nutrient-rich patches have been identified as being of fundamental importance in the ability of Australian native plants to grow in low nutrient environments (Bowen 1981; Lamont 1982, 1993, Pate 1994; Pate & Bell 1999). Shortages of phosphorus (P) and nitrogen (N) have also been foremost in discussion on the general occurrence of sclerophylly (e.g. Turner 1994) and general distribution of Australian plants (Westoby 1988; Adams 1996) due to the demonstrably low concentrations Correspondence: T. L. Bell. tinabell@cyllene.uwa.edu.au of total P and N in soils over large areas. Plants utilizing one or more mechanisms that enable increased nutrient uptake are obviously at a distinct advantage in low nutrient environments. Lamont (1982) estimated that 88% of the 180 species investigated from highly oligotrophic heathlands near Eneabba, Western Australia, displayed some form of specialized nutrient acquisition. Pate & Bell (1999) classified 171 species found in Banksia woodlands near Perth, Western Australia into 16 trophic or nutrient-acquiring types. Proteoid or cluster roots and their homologous counterparts, root clusters (see Dinkelaker, Hengeler & Marschner 1995 for definitions of types), can be readily distinguished as groups of closely spaced lateral rootlets of limited growth along a larger lateral root axis (Purnell 1960; Lamont 1982). The main advantage of such roots was initially thought to be due to greatly increased surface area for nutrient uptake (Jeffrey 1967; Malajczuk & Bowen 1974; Dell, Kuo & Thomson 1980). More recently, the importance of exudation of organic acids that promote the solubility of chelated P has been documented (Gardner, Parbery & Barber 1981, 1982a, 1982b; Gardner, Barber & Parbery 1983; Marschner, Romheld & Cakmak 1987; Dinkelaker, Roemheld & Marschner 1989; Grierson & Attiwill 1989, Grierson 1992). In lupins, citrate production may account for 6 23% of total plant dry weight (Dinkelaker et al. 1989) and increased 40-fold in the absence of external supplies of P (Johnson, Vance & Allen 1996a). In addition, it has been suggested that root clusters enhance the uptake of Fe and Mn by reduction or acidification the rhizosphere with exudation of protons (Gardner et al. 1981, 1983; Grierson & Attiwill 1989; Dinkelaker et al. 1995). In general, a deficiency in P in the growing media promotes formation of root clusters and the proportion of the root system devoted to root clusters decreases as P availability increases (Dinkelaker et al. 1995). This is evident for members of the Proteaceae (Dell et al. 1980; Skene et al. 1996) and other diverse groups of plants (e.g. Trinick 1977; Walker & Pate 1986; Louis, Racette & Torrey 1990; Racette, Louis & Torrey 1990; Crocker & Schwintzer 1993; Hurd & Schwintzer 1996, 1997). Similarly, the availability of N affects root cluster formation such that an increase in N status also results in a reduction in proportions of root clusters (Lamont 1972a; Racette et al. 1990). The form in which N is supplied is important in root cluster formation 2002 Blackwell Science Ltd 837

2 838 M. A. Adams et al. with urea-fed plants producing a greater proportion of root clusters than nitrate-fed plants (Crocker & Schwintzer 1993) and nitrate-fed plants producing a greater proportion than ammonium-fed plants or plant relying solely on N 2 - fixation by Frankia (Racette et al. 1990). The occurrence of a subset of species forming root clusters that can also form nodules (summarized in Sprent 1995; Skene 1998) suggests an obvious hypothesis that root clusters have evolved in N-fixing species as a means of supplying the requirements of P for nodule formation and subsequent N 2 -fixation or indirectly through impacts from reduced growth (Robson 1983). Nodulation in any N-fixing species should logically be related to P-supply but experimental evidence to date is conflicting. Many studies have shown that there is no special P requirement for symbiotic N 2 -fixation in leguminous or actinorhizal plants (e.g. Robson, O Hara & Abbott 1981; Robson & Bottomley 1991; Reddell, Yun & Shipton 1997a, b). In contrast, other studies have shown high requirements for P by the host plant for nodule formation or the process of N 2 -fixation (e.g. Israel 1987; Sanginga, Danso & Bowen 1989). In specific cases involving species known to form root clusters, the above hypothesis has been variously rejected on the basis that although addition of N as nitrate suppressed formation of actinorhizally associated nodules it did not increase or decrease root cluster formation (Louis et al. 1990) and accepted on the basis of increased formation of root clusters when P-levels was varied with inorganic N-source (Racette et al. 1990). Clearly more experimental evidence is needed to explore this complex relationship. The two most ecologically important aspects of root clusters are their function and their development and distribution in heterogeneous environments. Root growth in a heterogeneous environment is frequently discussed in relation to the plasticity of plant phenotypes to variation in the availability of nutrients, especially P and N (e.g. Hackett 1972; Drew 1975; Drew & Saker 1975; Fitter 1982; Robinson & Rorison 1987; Robinson 1994, 1996; Van Vuuren, Robinson & Griffiths 1996, Kerley 2000). More specifically, under conditions of generally poor nutrient availability, zones of greater nutrient concentration promote root growth and branching (e.g. Miller & Ohlrogge 1958; Robinson & Rorison 1983; Jackson & Caldwell 1989; Pregitzer, Hendrick & Fogel 1993; Robinson 1994). For species that produce root clusters, the same types of investigations have been undertaken (see Dinkelaker et al for a review) although considerably more is known about members of the Proteaceae (Lamont 1972a, 1973, 1983; Lamont, Brown & Mitchell 1984), than non-members (Gardner et al. 1981; Louis et al. 1990; Racette et al. 1990; Reddell et al. 1997a). The aim of the series of experiments outlined in this study is to (1) confirm the presence of root clusters in a number of legumes native to Western Australia; (2) compare the relative production of root clusters and nodules of these species when grown in sand culture supplied with a range of organic and inorganic P sources; and (3) determine the plasticity of root growth in relation to heterogeneous supplies of P. MATERIALS AND METHODS In all experiments, Kennedia coccinea Vent., Kennedia prostrata R. Br., Kennedia rubicunda Vent., Bossiaea aquifolium Benth., Acacia urophylla Benth. Ex Lindley, Paraserianthes lophantha (Willd.) I. Nielsen, Daviesia cordata Smith and Viminaria juncea (Schrader & Wendl) Hoffsgg. were grown from seed after scarification by brief immersion in boiling water. All cultures were maintained in naturally lit glasshouses in Perth, Western Australia during the months of April to November Unless otherwise stated, all cultures were inoculated with Bradyrhizobium sp. at the time of sowing. Harvesting of pots involved removal of shoots from roots and separation of nodules and clusters of roots from the remainder of the root system. Each plant part was dried for 48 h at 70 C. Unless otherwise stated, all results are expressed on a pot basis and data were analysed statistically by analysis of variance and treatment means were compared using Tukey s or Student Newman Keuls tests. Experiment 1: P-sources, root cluster formation and nodulation Plants of A. urophylla, B. aquifolium, K. coccinea, K. prostrata, P. lophantha and V. juncea were grown from seed sown directly into 16-cm diameter pots containing acidwashed, white quartz sand that was free of organic matter. Germinating seedlings were thinned to three individuals per pot and were fed weekly with a modified Hoagland s nutrient solution containing balanced amounts of all essential mineral elements except P and N. Full strength stock solutions of Hoagland s nutrient solution contained: MgSO 4 7H 2 O (1 0 M), CaSO 4 (1 0 M), K 2 SO 4 (0 5 M), Na-Fe- EDTA (0 5 M), KCl (1 0 M) and the micronutrients, H 3 BO 3, MnCl 2 4H 2 O, ZnSO 4 7H 2 O, CuSO 4 5H 2 O, NaMoO 4 2H 2 O and CoCl 2 6H 2 O (Hoagland & Arnon 1950). Nutrient solutions were applied initially at one-tenth strength and increased gradually during the first 3 weeks after germination to one-quarter strength. Pots were flooded with distilled water twice weekly to prevent accumulation of salts. The full strength nutrient solution contained 0 5 mm with respect to P in the following forms: KH 2 PO 4 /K 2 HPO 4 (ortho-p), inositol phosphate (inositol-p, sodium salt, BDH), pyrophosphate (pyro-p, tetrasodium salt, BDH), choline phosphate (choline-p, Sigma-Aldrich Co.). Each treatment was replicated six times and plants were harvested after 12 weeks of growth. Shoots, nodules, root clusters and other roots including the main taproot were separated, dried and weighed. Plant parts were ground and sub-samples were digested in H 2 SO 4 /H 2 O 2 prior to automated analysis for N and P (Technicon 1977). Experiment 2: P-availability and root plasticity I Simple split root containers were assembled from four waxed cardboard cartons each of 2 L capacity and holding about 3 kg of sand (see Fig. 1A). Each of the following four

3 P availability, root clusters and legumes 839 Figure 1. Details of experimental apparatus used in experiments. (A) Cross-section of the split root containers used in experiments 2 and 3, which comprised four waxed cardboard boxes (compartments) joined after the common corner and part of the common side walls were removed to form the core. Four different soil treatments were used and the core was filled with acid-washed, white quartz sand. (B) Compartment root containers used in experiment 4 in which a flat tray was divided to form two rooting areas, each with four removable dividers spaced equally within the area. The outermost compartment was filled with acid-washed white quartz sand and alternate compartments filled with inoculated sand with or without rock phosphate. treatments were allocated randomly to one section of each container: 1 acid-washed, white quartz sand free of organic matter with no available P and no capacity for P-fixation (white sand); 2 acid-washed, white quartz sand free of organic matter to which 2 0 g of insoluble iron phytate (11 8% P, 79 µg P g 1 dry weight soil, Greaves & Webley 1969) incorporated into the surface 5 cm of sand (white sand + Fe-P o ); 3 yellow sand (ph 5 4, < 2 µg available P g 1 dry weight soil) with some capacity for P-fixation (yellow sand); and 4 yellow sand with 4 6 g of ash (0 367% P, 5 6 µg P g 1 dry weight soil, prepared by combustion of senescent foliage of Eucalyptus marginata Donn. Ex Smith and Corymbia calophylla (R. Br. Ex Lindl) K. D. Hill & L. A. S. Johnson) incorporated into the surface 5 cm of sand (yellow sand + ash-p). Several seeds of A. urophylla, K. coccinea, K. prostrata, P. lophantha and V. juncea were germinated in the core of each split root container and thinned to one plant per container approximately 10 d after germination. Plants were watered weekly for 3 weeks with one-tenth strength modified Hoagland s nutrient solution (minus P, minus N) prior to regular addition of one-quarter strength nutrient solution as detailed for experiment 1. Containers were flooded with distilled water twice weekly to prevent accumulation of salts. Each container was replicated nine times and plants were harvested 14 weeks after germination. Following removal of shoots, the lateral roots in the four treatments were separated from the taproot positioned in the centre of the split root container. Root biomass for each treatment and the core was then separated into nodules and root clusters and other roots. Dry weights of all components were measured as above. Amounts of P and N in root tissues were determined by automated analysis as detailed in experiment 1 and amounts of other elements (K, Ca and Mg) were determined using atomic absorption spectroscopy as described in Adams & Attiwill (1986). Experiment 3: P-availability and root plasticity II Split root containers as described above were used in this experiment. Three of the four treatments used in experiment 2 were repeated (i.e. white sand, yellow sand, yellow sand + ash) with North Carolina rock phosphate incorporated into white sand at the same rate of P addition ( 80 µg P g 1 dry weight soil) as used in other treatments in the fourth compartment of each container (white sand + rock- P). Three or four seeds of four species of legumes (Daviesia cordata, K. coccinea, K. prostrata, K. rubicunda) were germinated in the core of each container and thinned to a single plant approximately 10 d after germination. The addition of nutrients and watering regimes were as described for treatments in experiment 2. Each container was replicated four times and plants were harvested 24 weeks after germination. Plant parts (shoots, nodules, root clusters and other roots) were separated for the core and each treatment and dry weights determined. Experiment 4: nodule and root cluster compartmentation Compartment root containers with one removable side were constructed from flat trays divided centrally to form two rooting areas each with volumes of 33 0 cm 42 0 cm 7 5 cm (Fig. 1B). Removable dividers spaced equally within each half of the tray formed a total of 10 compartments (five compartments per half tray). Each tray therefore represented two treatment replicates. With the dividers in place, acid-washed, white quartz sand that was free of organic matter was packed into the two outermost compartments (white sand). In the remaining compartments, white quartz sand mixed with North Carolina rock phosphate at a rate of 67 µg P g 1 dry weight soil (+ P) and inoculated with Bradyrhizobium sp. or white quartz sand only ( P), also inoculated with Bradyrhizobium sp. was packed in alternating compartments (Fig. 1B). Dividers were removed from between compartments and the side-cover-

4 840 M. A. Adams et al. ing panel installed. One or two 10-day-old seedlings of each species (K. coccinea, K. prostrata, K. rubicunda and V. juncea) were transferred to the outermost white sand compartments. Seedlings were thinned to one plant per container after a further 10 d. Nutrients were added fortnightly as one-quarter strength modified Hoagland s solution (minus P, minus N) and watered with distilled water as required. Each container was replicated six times and plants harvested after 24 weeks of growth. Shoots were separated from roots and the latter divided into nodules, root clusters and other roots and dry weight of each component was determined. RESULTS Evidence of root cluster formation in native Western Australian legumes Field observations of root clusters formation were confirmed by a variety of glasshouse sand culture experiments. Root systems of all species consisted of a prominent taproot from which lateral roots branched prolifically. At irregular intervals along these lateral roots dense to medium-dense clusters of rootlets arose (Fig. 2A D & F). Dense clusters of roots were observed in V. juncea (Fig. 2B), K. prostrata (Fig. 2D) and P. lophantha, medium-dense clusters in D. cordata (Fig. 2A) and K. coccinea (Fig. 2C) and loose clusters of roots for K. rubicunda (Fig. 2F). Lengths of groups of root clusters along lateral roots were up to 3 5 cm for V. juncea and contracted to barely 1 cm for K. prostrata. Nodules displayed either a determinate (K. coccinea, K. prostrata, K. rubicunda (Fig. 2F), V. juncea) or indeterminate growth pattern (D. cordata (Fig. 2E), P. lophantha). Experiment 1: P-sources, root cluster formation and nodulation For the six species tested, inositol-p was generally as good or a better source of P for total growth as ortho-p (Fig. 3). Kennedia coccinea and A. urophylla produced significantly more total dry mass when supplied with inositol-p and ortho-p than with other sources of P (Fig. 3A & B), whereas K. prostrata and V. juncea displayed no clear preference for P source (Fig. 3C & D). Paraserianthes lophantha grew significantly larger when supplied with ortho-p (Fig. 3F) and the total biomass of B. aquifolium supplied with inositol-p was almost twice that for any other P source tested (Fig. 3E). The majority of species produced a greater mass of nodules when supplied with inositol-p and ortho-p than with other forms of P (Fig. 3). This trend was significant for A. urophylla, B. aquifolium and K. prostrata but not for K. coccinea or V. juncea. Nodulation of P. lophantha was poor, whereas an unusually large nodule mass was found for B. aquifolium supplied with inositol-p (Fig. 3E). Kennedia coccinea and V. juncea were the only two species to produce root clusters in this experiment, however, the mass of these components were small in relation to total root dry mass (10 26% and 8 10%, respectively). Greater amounts of root clusters were produced by K. coccinea when supplied with either inositol-p or ortho-p, whereas a significantly smaller amount was formed by V. juncea grown in the presence of inositol-p. Shoot : root dry weight ratios were greatest for plants grown with inositol-p (between 2 2 and 2 8) except for K. prostrata where the greatest ratio was for plants supplied with ortho-p. Nitrogen concentrations generally varied little among treatments for all species, with the exception of P. lophantha in which plants supplied with inositol-p had significantly greater concentrations of N in shoots than comparable plants supplied with other sources of P (Table 1). Nitrogen concentrations in shoots and roots were two to three times greater in P. lophantha and V. juncea than other species. Phosphorus concentrations in both shoots and roots were always significantly greater in plants supplied with inositol- P compared to plants supplied with any other source of P (Table 1). Such differences in P concentrations were marked with up to six-fold more P in shoots and five-fold more P in roots of plants supplied with inositol-p (Table 1). Paraserianthes lophantha had P concentrations in shoot and root tissue double those found in corresponding tissues of any other species. Despite large differences in concentrations among species, N content (a product of plant mass and nutrient concentration) of plants generally followed changes in mass (results not shown). The increase in concentration of P in plant tissue supplied with inositol-p translated to at least three-fold increases in P content of plants. Experiment 2: P-availability and root plasticity I For each of the five legumes grown in the first of two experiments using split root cultures, total root growth was always greatest in one of the two P-amended compartments (Fig. 4). The yellow sand + ash treatment produced greatest root growth in K. coccinea, K. prostrata, and V. juncea (Fig. 4A C), whereas the white sand + Fe-Po treatment produced greatest root growth in A. urophylla and P. lophantha (Fig. 4D & E). Paraserianthes lophantha, K. coccinea, K. prostrata, and V. juncea all produced surface mats of densely clustered roots which were morphologically different to other roots in compartments treated with ash as a source of P. Kennedia coccinea, K. prostrata, and V. juncea also produced similar but smaller mats of root clusters in compartments with white sand amended with Fe-Po. Acacia urophylla produced little in the way of root clusters or nodules in any of the treatments. Relative to the other species and treatments, P. lophantha and A. urophylla produced a considerably greater mass of roots other than the mat of roots in the Fe-Po treatments (Fig. 4D & E). Nodule biomass was generally greatest in the white sand treatment. In both of the Kennedia species the sum of the nodule weight in the white and yellow sand treatments was more than 30 times that in P-enriched treatments, whereas in V. juncea and P. lophantha it was about double. Root mass in the core of each replicated container was associated

5 P availability, root clusters and legumes 841 Figure 2. Root clusters and nodules from native south-western Australian legumes. (A) Root cluster with medium dense arrangement of rootlets from Daviesia cordata; (B) mid-portion of a dense root cluster from Viminaria juncea; (C) medium-dense root cluster from Kennedia coccinea; (D) short dense root cluster from Kennedia prostrata; (E) nodules with indeterminate growth patterning from Daviesia cordata; and (F) a nodule with determinate growth from Kennedia rubicunda. Bars are 10 mm. with the taproot. This root grew vertically and therefore did not contribute significantly to the root or nodule mass recorded for each of the four surrounding treatments. Nutrient (N, P, K, Ca, Mg) concentrations in nodules and root clusters were not significantly different among treatments for any species (Table 2). Nitrogen concentrations were always at least two to three times greater in nodules than root clusters or mats and lateral roots. Nitrogen concentration in lateral roots did not vary significantly among treatments for any species but other measured nutrients did vary significantly in certain species (Table 2). Phosphorus concentrations in lateral roots were significantly greater in P-amended white and yellow sand (i.e. white sand + Fe-Po, yellow sand + ash) for all species except A. urophylla and

6 842 M. A. Adams et al. Figure 3. Biomass of plant components of leguminous species grown with organic and inorganic sources of P. For each component, treatment means marked with the same letter are not significantly different (P < 0 05, Student Newman Keuls test). K. prostrata (Table 2). Potassium concentrations in lateral roots were generally greater in compartments filled with yellow sand than any other treatment. Calcium and Mg concentrations only varied significantly among treatments in P. lophantha, K. prostrata, and V. juncea and for these three species, Mg concentrations were generally greatest for yellow sand + ash treatments, whereas Ca concentrations were greatest for the white sand treatments. Calculation of P-contents (results not shown) clearly showed that the patterns of increased root growth in P- amended compartments were complemented by increased P concentrations in roots in those compartments and thus had greatly increased P contents. Experiment 3: P-availability and root plasticity II The much longer period of growth in this experiment confirmed the results from experiment 2. All species in all treatments produced mats of root clusters, the greatest weight of which was in produced in compartments with white or yellow sand with some form of additional P (Fig. 5). There was a less clear distinction in nodule production among treatments than found in experiment 2, although for two of the four species (K. coccinea and K. prostrata), nodule production was significantly greater in compartments without additional P than in the corresponding P-amended compartments (Fig. 5B & C). Total root mass was always greatest in one of the two P treatments and was often threeto five-fold greater than in the matching compartments containing sand only. For the three species of Kennedia, the greatest root mass was produced in the yellow sand + ash compartment, whereas for D. cordata the greatest root mass was produced in the white sand + rock phosphate compartment (Fig. 5A). Examining the cumulative data from experiments 2 and 3 (Table 3), it is evident that there is a large degree of plasticity of root growth to localized sources of P. In all instances except one, root mass was increased by more than 100% in P-amended yellow sand compared to similar unamended sand. Results were more variable for the white sand, including two instances of reduced growth in P-

7 P availability, root clusters and legumes 843 Species nutrient Shoot Root Inositol-P Other-P Inositol-P Other-P Nitrogen (µg N g 1 dry weight) Acacia urophylla Bossiaea aquifolium Kennedia coccinea Kennedia prostrata Paraserianthes lophantha 38 2* Viminaria juncea Phosphorus (µg P g 1 dry weight) Acacia urophylla 1 5* * 1 1 Bossiaea aquifolium 3 5* * 0 7 Kennedia coccinea 2 2* * 2.0 Kennedia prostrata 1 6* Paraserianthes lophantha 4 9* * 1 2 Viminaria juncea 2 5* * 0 8 Table 1. Mean N and P concentrations (µg g dry weight 1 ) in shoots (leaves plus stems) and roots (excluding nodules and clusters) of native legumes grown in sand culture with inositol-p or other sources of P. N and P concentrations in plants grown with choline- P, pyro-p and ortho-p, were not significantly different from one another and thus mean values for these sources combined are given (Other-P). Concentrations of nutrients in plants supplied with inositol-p which were significantly different are indicated by an asterisk (P < 0 05, Tukey s test) amended sand, but plasticity still averages an increase of close to 70%. When the data for both experiments 2 and 3 are combined (Fig. 6A), there is a strong linear relationship (r 2 = 0 816) between the biomass of root clusters or root mats and the total biomass of roots for each treatment. An obvious inverse relationship (r 2 = 0 468) is evident between the production of nodules and the production of a mat of root clusters (Fig. 6B). Figure 4. Biomass of root components of native legumes in compartments treated with different P-sources in split root cultures. Corresponding dry weights of shoot components (g dry wt treatment 1 ) were: Kennedia coccinea = 5 84, Kennedia prostrata = 3 42, Viminaria juncea = 2 40, Paraserianthes lophantha = 1 28, Acacia urophylla = For each root component, treatment means marked with the same letter are not significantly different (P < 0 05, Student Newman Keuls test).

8 844 M. A. Adams et al. Table 2. Nutrient concentrations (mean ± standard error) in root tissues (% dry weight) of native legumes. For each species, treatment means followed by no letter or the same letter were not significantly different (P < 0 05, Tukey s test). Nodules and root clusters/root mats were only present in some treatments and thus mean concentrations for all four treatments combined are presented Species/treatment N P K Ca Mg Nodules Acacia urophylla 4 03 ± ± ± ± ± 0 11 Kennedia coccinea 4 56 ± ± ± ± ± 0 18 Kennedia prostrata 3 51 ± ± ± ± ± 0 05 Paraserianthes lophantha 5 27 ± ± ± ± ± 0 05 Viminaria juncea 5 10 ± ± ± ± ± 0 09 Root clusters/root mats Acacia urophylla NA 1 NA NA NA NA Kennedia coccinea 1 16 ± ± ± ± ± 0 20 Kennedia prostrata 1 60 ± ± ± ± ± 0 18 Paraserianthes lophantha 1 35 ± ± ± ± ± 0 07 Viminaria juncea 1 57 ± ± ± ± ± 0 02 Lateral roots Acacia urophylla White sand 1 54 ± ± ± ± ± 0 02 White sand + Fe-Po 1 75 ± ± ± ± ± 0 03 Yellow sand 1 43 ± ± ± ± ± 0 11 Yellow sand + ash 1 53 ± ± ± ± ± 0 16 Kennedia coccinea White sand 1 21 ± ± 0 01 a 0 79 ± 0 08 a 0 48 ± ± 0 07 White sand + Fe-Po 1 40 ± ± 0 01 b 0 69 ± 0 09 a 0 56 ± ± 0 05 Yellow sand 1 22 ± ± 0 01 a 1 06 ± 0 10 b 0 43 ± ± 0 04 Yellow sand + ash 1 14 ± ± 0 01 b 0 76 ± 0 09 a 0 54 ± ± 0 12 Kennedia prostrata White sand 1 64 ± ± ± 0 25 a 0 68 ± ± 0 17 ab White sand + Fe-Po 1 09 ± ± ± 0 21 b 0 38 ± ± 0 30 a Yellow sand 1 23 ± ± ± 0 27 a 0 29 ± ± 0 05 a Yellow sand + ash 1 17 ± ± ± 0 32 ab 0 86 ± ± 0 47 b Paraserianthes lophantha White sand 1 22 ± ± 0 01 a 0 83 ± ± 0 09 a 0 84 ± 0 17 White sand + Fe-Po 1 51 ± ± 0 01 b 0 80 ± ± 0 05 ab 0 97 ± 0 13 Yellow sand 1 24 ± ± 0 01 a 1 31 ± ± 0 06 ab 1 22 ± 0 10 Yellow sand + ash 1 33 ± ± 0 01 b 1 21 ± ± 0 08 b 0 12 ± 0 11 Viminaria juncea White sand 1 92 ± ± 0 01 a 0 88 ± 0 01 ab 0 36 ± 0 04 a 0 23 ± 0 02 ab White sand + Fe-Po 2 07 ± ± 0 01 b 0 63 ± 0 06 a 0 36 ± 0 02 a 0 16 ± 0 01 b Yellow sand 2 02 ± ± 0 02 b 1 21 ± 0 18 b 0 21 ± 0 06 b 0 20 ± 0 03 a Yellow sand + ash 1 86 ± ± 0 01 b 1 00 ± 0 08 b 0 19 ± 0 06 b 0 30 ± 0 05 a 1 NA denotes data were not available. Experiment 4: Nodule and root cluster compartmentation When four species (K. coccinea, K. prostrata, K. rubicunda and V. juncea) were grown in containers with alternating bands of sand with and without a source of inorganic P, the proportion of root biomass devoted to root clusters was always highest in the bands with additional P (Fig. 7). The proportion of biomass devoted to nodules was similar in all bands of sand regardless of the P-status of the growing medium ( %). Given that + P1 denotes the band of P- amended white sand closest to the taproot and + P4 is the furthest (Fig. 7B), V. juncea was the only species to display gradually declining amounts of root clusters with distance of P-source from the taproot. Kennedia coccinea and K. prostrata showed greater root cluster formation when the band of sand containing rock phosphate was not immediately adjacent to the taproot but a little further away (i.e. bands designated + P2 or + P3). Kennedia rubicunda had similar high percentages of the root system devoted to root clusters in all P-amended bands. Root cluster formation was low in all bands of sand without additional P in all species (ranging from 0 6 to 5 4%) except for K. rubicunda ( %).

9 P availability, root clusters and legumes 845 Figure 5. Biomass of components of root systems of native legumes grown in split root cultures treated with two different P-sources in white and yellow sand. Corresponding dry weights of shoot components (g dry wt treatment 1 ) were: Daviesia cordata = 34 36, Kennedia coccinea = 24 59, Kennedia prostrata = 26 32, Kennedia rubicunda = For each root component, treatment means marked with the same letter are not significantly different (P < 0 05, Student Newman Keuls test). DISCUSSION The legumes investigated here are native to the nutrientpoor soils of the jarrah and karri forests and Banksia woodlands of south-western Australia. Our investigations provide the first reports of formation of obviously clustered roots by a number of the leguminous species and confirm field observations and earlier studies for others (e.g. Lamont 1972b; Walker & Pate 1986; Brundrett & Abbott 1991). Previously, only a single species of Acacia had been confirmed to form root clusters (Sward 1978) and although the genus Kennedia had been reported to bear root clusters (Trinick 1977), the species involved and evidence of root cluster morphology has never been published. Anatomical studies demonstrating arrangement and origin of rootlets are only available for Viminaria juncea (Lamont 1972b) and are clearly needed for all putative root-cluster-forming species. Root cluster morphology varied with species from relatively loose to very dense arrangements of determinate rootlets and from short to lengthy groupings along lateral roots. The root plasticity observed in this study, particularly the extensive formation of root clusters or root mats in P- amended sands, matches field observations of increased abundance of root clusters in microsites rich in P in an environment that is generally poor in P (e.g. immediately below the litter layer or in organically rich pockets deeper in the soil profile; see Lamont (1982), or after fire in pockets of nutrient-rich ash). Plasticity of root growth is a significant factor in acquisition of resources in natural ecosystems. Jackson & Caldwell (1996) used a modelling approach to synthesize some 5 years of intensive study of root plasticity in response to heterogeneity in resource availability. Their model suggested that a 40 80% increase in root growth (root length density) in zones of greater nutrient availability increased P-uptake by 28% and nitrate uptake by 61% relative to the case where root growth was not plastic. The remarkable plasticity of the legumes with root clusters in the present study (Table 3), averaging an increase in root mass in P-enriched compartments in excess of 100%, plus the substantial

10 846 M. A. Adams et al. Species Experiment 2 Experiment 3 White sand Yellow sand White sand Yellow sand Acacia urophylla Kennedia coccinea Kennedia prostrata Kennedia rubicunda Daviesia cordata Paraserianthes lophantha Viminaria juncea Table 3. Indices of root plasticity for legume species grown in sand culture in split roots containers in experiments 2 and 3 investigating P-availability and root plasticity. Each index is calculated as follows: [(total root wt in P-amended sand total root wt in matching non-amended sand) 100] / (total root wt in matching nonamended sand) increases in P-concentration of roots in those compartments (lateral roots increased in P concentration in every instance, sometimes by as much as three-fold), suggest that targeted plastic root growth results in substantial increases in P uptake. Proteoid roots are well known to be concentrated in the upper 5 20 cm of soil and often form dense mats (Lamont 1973; Low & Lamont 1990; Dinkelaker et al. 1995). Recent studies of the spatial and temporal demography of root growth in jarrah forests underscore the general importance of root plasticity to nutrient cycling. The mass of fine roots, and their carbon and nutrient content, in the surface horizons of soil varies by several-fold between wet and dry seasons, and several-fold again between stands with and without a Banksia understorey (Grierson & Adams 1999, 2000). Both spatial and temporal root plasticity clearly play major roles in nutrient acquisition and turnover in these nutrient poor systems. In general, root formation is regulated by a fine balance between the internal nutritional status of the plant and external nutrient status of the soil (Hutchings & de Kroon 1994, Robinson 1994). Evidence for internal nutrition control of root cluster formation comes from several sources. Firstly, P-deficiencies in plants have been found to induce citrate and malate exudation by cluster roots (Gardner et al. 1983; Marschner et al. 1987; Dinkelaker et al. 1995), secondly, foliar-applied P produced similar levels of cluster root formation as root-applied P (Louis et al. 1990; Racette et al. 1990) and, thirdly, cluster root production occurs even in hydroponic or vermiculite culture completely devoid of P (Skene et al. 1996). Despite this, as we have demonstrated here and as it has been shown elsewhere, localized P-rich patches in a low P environment result in proliferation of lateral roots and root clusters. It has been suggested that the lateral roots supporting root clusters will proliferate in high nutrient patches and as a consequence, the proportion of root clusters present in the patch will also increase (Skene et al. 1996). In a more homogeneous growing medium, lateral roots and therefore root clusters, will be more evenly distributed. In Arabidopsis, external nutrient levels have been demonstrated to be the stimulus for lateral root proliferation using a NO 3 inducible gene (Zhang & Forde 1998). Although it is obvious that nutrient availability is involved in dictating the prevalence of root clusters, additional research is needed to determine the regulating mechanisms of their formation. Although other factors may be involved in the production of nodules in unaltered white or yellow sand, the production of root clusters and nodules is probably best viewed in light of the allocation of carbon (e.g. Mooney 1972; Nielsen et al. 1994). Both organs are carbon sinks, requir- Figure 6. Relationships between various components of root systems of plants from experiments 2 and 3. (A) Positive relationship between the mass of root clusters and the mass of total roots (root clusters plus lateral roots) of plants grown in P-amended sand culture in split root containers, and (B) negative relationship between proportion of total root mass present as nodules and root clusters. Data for Acacia urophylla were omitted from this analysis as this species failed to produce root clusters in experiment 2. Results of linear regression analyses are shown on each graph.

11 P availability, root clusters and legumes 847 Figure 7. Proportion of total root biomass as either nodules or root clusters for selected native legumes grown in compartment root containers containing alternate bands of acid-washed white quartz sand and white sand combined with rock phosphate. Note that + P1 denotes the band of P-amended white sand closest to the taproot and + P4 is the furthest band and P1 denotes the band of non-amended white sand closest to the taproot and P4 is the furthest band. ing considerable transport of carbon as an energy source and as a substrate for diazotrophy in the case of nodules (e.g. in Lupinus albus L. Pate, Layzell & Atkins 1979; Layzell et al. 1981), and for exudation (notwithstanding exudation of the products of non-photosynthetic C-fixation, Johnson et al. 1996b) in the case of root clusters (Dinkelaker et al. 1995). We suggest that, in part, the distribution of nodules and clusters may be related to sink strength and, in turn, to internal requirements for N and P. Thus, when legumes are not supplied with P or N, nodules are first formed and become functional (and a carbon sink) on the primary root and, later, on lateral roots in accordance with the requirement of N for growth. Root clusters are formed and become functional on lateral roots but perhaps not where the transport of carbon is allocated to nodule formation (particularly in species possessing nodules with indeterminate growth, e.g. D. cordata) and function. Certainly there are some strong indications in the experiments presented here that this is likely to be the case and it will be interesting in future studies to examine in more detail the importance of relative strengths of root clusters and nodules as carbon sinks to their spatial distribution and function. The increased growth and uptake of P by native legumes when supplied with inositol-p in experiment 1 supports our earlier studies with lupins (Adams & Pate 1992). In particular, we noted that for the cluster-root-forming white lupin (Lupinus albus), inositol-p was the best source of P for increased growth, whereas for the non-cluster-rooted, narrow leaf lupin (L. angustifolius L.), ortho-p and inositol-p produced similar growth responses. The conclusion that inositol-p is at least as reactive as ortho-p might now be extended to the native legumes studied in experiment 1, with the caution that, this hypothesis has not yet been tested in soils with greater capacity for P-fixation than the sands used here (Adams & Pate 1992). When grown with inositol-p, uptake of P by the experimental legumes was probably well in excess of requirements, as shown by the two- to three-fold increase in P content over those grown with inorganic ortho-p. The phosphorus content of forest and woodland soils of south-western Australia is dominated by organic forms of P (Adams & Byrne 1989; Adams 1992) and given the above growth responses, it is tempting to deduce that native legumes and other species are well equipped to access sources of organic-p. However, utilization of readily available sources of organic P in solution and sand-culture has been reported on many occasions, and for plant species not specifically adapted to low nutrient soils. In habitat soil, and particularly in soils with a high capacity for P-fixation, utilization of organic P seems to be strongly limited by low solubility (Adams & Pate 1992). The adaptive potential of root clusters in highly oligotrophic soils may be related to an ability to mobilize sparingly soluble sources of P rather than to the capacity for hydrolysis of soluble organic P- esters as featured by many other plant species. Results from experiment 2 further supports this view, as increased concentrations of P were recorded in roots supplied with insoluble iron phytate, another source of organic P, but not in roots grown in white sand. The low nutrient soils of Australia in general, and the sandy soils of south-western Australia in particular, con-

12 848 M. A. Adams et al. tinue to present outstanding opportunities for integrated studies of functional, morphological and anatomical adaptations of plants for nutrient acquisition. The legumes studied here have been shown to have considerable morphological and anatomical plasticity in relation to the external availability of P as well as the capacity to use organic forms of P. Similar plasticity in nodule formation is also suggested for the acquisition of N. Physiological, morphological and anatomical plasticity in accessing and utilizing organic and inorganic sources of P and N is likely to exist in cohabiting species of legumes and non-legumes (e.g. Stewart, Pate & Unkovich 1993; Pate 1994) and symbiotic relationships with mycorrhizal fungi add a further dimension to nutrient acquisition (e.g. Bell & Pate 1995; Turnball et al. 1996) in nutrient-poor environments. 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