Skip to main content
NCBI home page
Annals of Botany logoLink to Annals of Botany
. 2013 Mar 1;111(5):801–809. doi: 10.1093/aob/mct035

Viminaria juncea does not vary its shoot phosphorus concentration and only marginally decreases its mycorrhizal colonization and cluster-root dry weight under a wide range of phosphorus supplies

Mariana C R de Campos 1, Stuart J Pearse 1, Rafael S Oliveira 1,2, Hans Lambers 1,*
PMCID: PMC3631325  PMID: 23456689

Abstract

Background and Aims

The Australian legume species Viminaria juncea forms both cluster roots and mycorrhizal associations. The aim of this study was to identify if these root specializations are expressed at differential supplies of phosphorus (P) and at different shoot P concentrations [P].

Methods

Seedlings were planted in sand and provided with a mycorrhizal inoculum and basal nutrients plus one of 21 P treatments, ranging from 0 to 50 mg P kg−1 dry soil. Plants were harvested after 12 weeks, and roots, shoots and cluster roots were measured for length and fresh and dry weight. The number of cluster roots, the percentage of mycorrhizal colonization, and shoot [P] were determined.

Key Results

Shoot biomass accumulation increased with increasing P supply until a shoot dry weight of 3 g was reached at a P supply of approx. 27·5 mg P kg−1 dry soil. Neither cluster-root formation nor mycorrhizal colonization was fully suppressed at the highest P supply. Most intriguingly, shoot [P] did not differ across treatments, with an average of 1·4 mg P kg−1 shoot dry weight.

Conclusions

The almost constant shoot [P] in V. juncea over the very wide range of P supplies is, to our knowledge, unprecedented. To maintain these stable values, this species down-regulates its growth rate when no P is supplied; conversely, it down-regulates its P-uptake capacity very tightly at the highest P supplies, when its maximum growth rate has been reached. It is proposed that the persistence of cluster roots and mycorrhizal colonization up to the highest P treatments is a consequence of its tightly controlled shoot [P]. This unusual P physiology of V. juncea is surmised to be related to the habitat of this N2-fixing species. Water and nutrients are available at a low but steady supply for most of the year, negating the need for storage of P which would be metabolically costly and be at the expense of metabolic energy and P available for symbiotic N2 fixation.

Keywords: Cluster roots, legume, shoot phosphorus concentration, mycorrhiza, phosphorus supply, Viminaria juncea

INTRODUCTION

Soils impoverished in phosphorus (P) are very common in ancient, highly weathered landscapes (Beadle, 1966; Lambers et al., 2008). South-western Australia is one of the most P-impoverished regions on Earth (Lambers et al., 2010; Laliberté et al., 2012), but also harbours one of the world's hotspots of biodiversity (Myers et al., 2000; Hopper and Gioia, 2004). Many soils in south-western Australia typically have <1 mg of readily available P kg−1 dry soil (Singh and Gilkes, 1991; Lambers et al., 2010).

Native species in south-western Australia often exhibit a variety of P-conservation mechanisms, including long leaf longevity, high degree of sclerophylly, high photosynthetic P-use efficiency and high P-resorption efficiency (Veneklaas and Poot, 2003; Lambers et al., 2010). Mechanisms that enhance P uptake are common in the most abundant taxa in south-western Australia (Pate and Dell, 1984; Lamont, 1982). These root specializations include ‘scavenging’ mycorrhizal associations on somewhat less P-impoverished soils, and ‘mining’ cluster roots on the most P-impoverished soils (Lambers et al., 2006, 2010).

Cluster roots (also known as proteoid roots, after the Proteaceae family in which they were first discovered) are dense clusters of rootlets of limited growth (Purnell, 1960; Lamont, 2003). They exude carboxylates, which allow the plant to mine small patches of P from soil in which P is not readily available to most plants (Lambers et al., 2006). Cluster roots can be simple or compound and vary greatly in size (Skene, 1998; Shane and Lambers, 2005). They have been reported in a wide range of species, genera and families (Lambers et al., 2006), but have been researched most extensively in Proteaceae native to Australia and South Africa (Shane and Lambers, 2005; Lamont, 2003) and in Lupinus (Fabaceae), particularly Lupinus albus (Gardner et al., 1982; Watt and Evans, 1999).

Increasing P supply decreases the formation of cluster roots in the field and in glasshouse experiments (Lamont, 1972a; Crocker and Schwintzer, 1994) in most species from different families on different continents. The systemic signal leading to the suppression of cluster roots originates from the leaves, as evidenced using foliar application of P and split-root systems (Dinkelaker et al., 1995; Shane et al., 2003b).

Arbuscular mycorrhizas represent a very common symbiosis in terrestrial plants; these associations between fungi and higher plants occur in approx. 73 % of all terrestrial plant species (Brundrett, 2009). Mycorrhizal plants are considered P ‘scavengers’, accessing P that is in the soil solution beyond the zone that can be accessed by roots and root hairs (Lambers et al., 2008). The hyphae effectively increase the nutrient-acquiring surface of the roots in return for plant-derived carbon; they allow for faster and more comprehensive exploitation of the soil, accessing pools of nutrients that roots would be otherwise unable to access (Smith and Read, 2008).

Although both mycorrhizal and cluster-root strategies increase P uptake by plants, there is often a prevalence of one or the other, according to the level of P in the soil. Extremely P-impoverished soils in south-western Australia are most commonly inhabited by non-mycorrhizal species with cluster roots, which occur in most Proteaceae, and dauciform roots, which are common in some Cyperaceae (Lambers et al., 2010). As the soil P levels increase slightly, mycorrhizal species such as Myrtaceae become more abundant (Lambers et al., 2006).

Some cluster-root forming species also form mycorrhizal associations (Skene, 1998), but there is very limited information on the ecophysiology of species capable of a mycorrhizal symbiosis as well as cluster-root formation. These species provide the opportunity to explore the relationship between P supply, tissue-P dynamics and P acquisition of both root specializations within the same species, as dependent on P supply. Reddell et al. (1997) investigated Casuarina cunninghamiana, a species that produces both cluster roots and mycorrhizas, in an experiment with six levels of P (0·1–250 mg P kg−1 soil). The authors found both mycorrhizal colonization and cluster roots at a P supply between 0·1 and 10 mg P kg−1 soil, but at higher P levels both colonization and cluster-root formation decreased. Because there were only six levels of P supply, it was not possible to decide if suppression of cluster roots and mycorrhizas exhibited a differential sensitivity to P supply.

Viminaria juncea (Fabaceae) is an Australian native species that forms both cluster roots (Lamont, 1972b; Walker and Pate, 1986) and mycorrhizas (Hayman, 1986; Brundrett and Abbott, 1991). Cluster roots of V. juncea are simple cluster roots, with only first-order determinate rootlets emerging from a lateral root axis (Lamont, 1972b). Due to its multiple P-acquisition root specializations, V. juncea was chosen as the experimental species to test the hypothesis that cluster roots are expressed at the very lowest P supply, to be replaced by mycorrhizal associations at slightly higher soil P levels. Both P-acquisition mechanisms are expected to be suppressed at even higher P supply, as generally found for mycorrhizal associations (Smith and Read, 2008) and cluster roots (Shane and Lambers, 2005). We further hypothesized that as P supply in soil is increased, shoot [P] will increase, providing the systemic signal to produce fewer cluster roots and greater mycorrhizal colonization. By providing a much finer range of soil P supplies than used for C. cunninghamiana (Reddell et al., 1997), we aimed to determine a ‘turning point’ where the carbon-demanding cluster-root strategy is replaced by the strategy of scavenging mycorrhizal association, similar to what has been observed for carboxylate-exuding Kennedia species (Fabaceae) that do not form cluster roots (Ryan et al., 2012).

MATERIALS AND METHODS

Species characterization

Viminaria juncea (Schrad.) Hoffmanns. (Fabaceae) is an erect, often weeping shrub that may reach up to 6 m in height, and occurs in forest ecosystems, open woodlands and heathlands near lakes and swamps, river banks and winter-wet depressions (Walker and Pate, 1986). It is endemic to southern Australia (Walsh and Entwisle, 1996). In its early development, V. juncea seedlings lose their leaves and the modified petioles (phyllodes) replace them as the photosynthesising structures in the adult plants. The germination of its seeds is facilitated by heat shock (Bell, 1999). It is commonly found in disturbed areas, at a lower level in the landscape (Fig. 1A), suggesting it may have higher nutrient requirements and/or tolerance than, for instance, P-sensitive Banksia species or that it is less capable of coping with water stress (Fig. 1B).

Fig. 1.

Fig. 1.

Open in a new tab

Two locations at Yule Brook Reserve (Goble-Garratt et al., 1981). Left: lower elevation, where Viminaria juncea is abundant and where invasive species occur. Note Banksia stand in the background, higher in the landscape. Right: area where Banksia attenuata and B. menziesii are common, as well as several other native species, but with no V. juncea specimens and no invasive species. The Banksia area is approx. 2 m higher in the landscape than the area where V. juncea occurs. Photographs: Marion Cambridge.

Field collections

Soil collections were made in Yule Brook Reserve 32°S, 115°W (Goble-Garratt et al., 1981) to determine soil [P], both at locations low in the landscape where V. juncea occurs naturally (Fig. 1A) and at locations higher in the landscape, where V. juncea is absent and Banksia attenuata and B. menziesii (Proteaceae) trees are common (Fig. 1B). Healthy looking plants V. juncea typically produce root nodules containing pink leghaemoglobin in this environment (pers. obs.). Three samples were collected (0–5 cm depth) at three locations where V. juncea occurs and another three where Banksia trees are common. Soil samples were sieved and analysed for bicarbonate-extractable P and total P (Colwell, 1963).

Glasshouse experiments

Seeds of V. juncea were purchased from a local supplier (Nindethana Australian Seeds). The seeds had their dormancy broken through hot-water treatment prior to germination. The hot-water treatment consisted of placing the seeds in a sieve and dipping them in boiling water for 10 s. They were distributed on Petri dishes with filter paper immediately afterwards, and placed in a 15 °C chamber in May 2010. The Petri dishes were kept moist until germination.

To expose the young experimental seedlings to mycorrhizal fungal spores found in their natural habitat, the germinated seeds were planted into seedling trays filled with field soil. This soil was collected from Yule Brook reserve (Goble-Garratt et al., 1981), adjacent to the roots of naturally growing V. juncea individuals.

After 12 weeks, most of the seedlings had reached 4 cm in height and had at least 4 phyllodes. At this stage (1 October 2009) they were removed from the seedlings trays. The roots were shaken to remove excess field soil, but not washed, and the seedlings were transplanted into individual 2-L pots. These sealed pots were lined with clear plastic to prevent any leaching and filled with 1·5 kg of sterilized, washed river sand. Nutrient-impoverished sand with a very low buffer capacity was chosen as the medium, because this is natural substrate for this species (McArthur, 1991).

Each pot received 1 g of Glomus sp. inoculum at the tip of the root, and a basal nutrient solution without phosphorus, containing (per kilogram of soil): 100 mg CaCl2.2H2O; 140 mg K2SO4; 70 mg NH4NO3; 80 mg MgSO4.7H2O; 15 mg MnSO4.4H2O; 10 mg ZnSO4.7H2O; 5 mg CuSO4.5H2O; 0·7 mg H3BO3; 0·5 mg CoSO4.7H2O; 0·4 mg Na2MoO4.2H2O; 20 mg FeNaEDTA. Additional doses of NH4NO3 (70 mg) were supplied after 4, 6, 8 and 10 weeks. Plants were exposed to 21 P treatments ranging from no P to 50 mg P per kg−1 dry soil in increments of 2·5 mg P per kg−1 dry soil. Each treatment had seven replicates, and the nutrients were added by preparing a solution with deionized water and applying it to the pots. Phosphate was added as potassium phosphate. All of the nutrients were prepared in solutions and then diluted to 500 mL to be added to the soil in sealed pots. The soil in each of the pots was mixed with a trowel and left to dry for 2 d. The seedlings were transplanted into the soil after it had been turned again. Since there was sufficient potassium supplied as K2SO4, potassium deficiency or excess was not an issue.

The pots were kept in a root-cooling tank and watered with deionized water when required for the 12-week duration of the P treatment. The temperature in the glasshouse varied between 22·5 and 28·5 °C during the day and between 9·5 and 21·0 °C at night. The relative humidity was 30–65 % during the day and 46–73 % at night. The glasshouse allowed approx. 70 % of ambient light to penetrate, and the peak of light intensity at plant level varied between 1450 and 2010 µmol m−2 s−1. Plants were harvested on 5 February 2010, after 12 weeks of treatment.

Harvest

All plants were harvested on the same day (5 February 2010) and had the length of their longest phyllodes measured. The plant material was separated into shoots, non-cluster roots and cluster roots. The number of cluster roots per individual was recorded, and the fresh weight of cluster roots, roots and shoots was also measured. Nodules were not taken into account in this study.

A root sample was removed from each individual and placed in ethanol for the mycorrhizal colonization count. The remaining material was placed in an oven at 60 °C for 3 d, after which the dry weight (d. wt) of cluster roots, roots and shoots was also recorded.

To determine shoot [P], the dried shoots were ground with mortar and pestle, digested with nitric and perchloric acid and phosphate concentrations were determined using the malachite green method (Motomizu et al., 1983) using a spectrophotometer (Multiskan Spectrum, Thermo Fischer Scientific, MA, USA).

Colonization counts

The roots set aside for mycorrhizal analyses were prepared following the protocol used by Brundrett et al. (1996). Roots were cleared (diaphanized) with 10 % (w/v) KOH in an autoclave for 20 min at 121 °C and then stained with 0·05 % (w/v) Trypan Blue and preserved in lactoglycerol. Arbuscular mycorrhizal colonization counts were performed under a stereomicroscope (SteREO DiscoveryV8; Carl Zeiss MicroImaging, Göttingen, Germany) with a ×5 objective lens and an 8:1 zoom. A Petri dish marked with a quarter-inch grid was used to count the root intersections and the colonized root intersections. Colonization was considered as the number of colonized roots that intersected the grid divided by the total number of roots that intersected the grid. Hyphae, arbuscules and vesicles were all considered as evidence of colonization and were all counted together.

Statistical analyses

Analyses of variance were performed, considering the P treatment as the factor and each of the other measurements as the variables, all in one-way analyses of variance (ANOVA), using Genstat 12th Edition, 2009 (VSN International Ltd). Data with non-homogenous residual plots were log-transformed. Trend lines were checked against the data for their fit using an ANOVA on the linear regression between the original data and the predicted values of the trend line.

RESULTS

Growth response to P supply

Plant growth responded positively to increased P supply. Shoot dry weight was significantly higher (P < 0·001) for the plants with greater P supply (Fig. 2A). Average values for the different treatments ranged from 0·5 g to 3·5 g of shoot d. wt. Figure 2A shows a steady increase in shoot dry weight up to a supply of 27·5 mg P kg−1 soil; at higher P supply, the values stabilized at around 3 g.

Fig. 2.

Fig. 2.

Open in a new tab

Growth of Viminaria juncea as dependent on P supply: (A) shoot dry weight, root dry weight and cluster-root dry weight (as indicated in the key) – stacked columns give total dry weight; (B) root weight ratio; (C) average of total shoot length. No root results are significantly different (roots P = 0·06 and cluster roots P = 0·23) between P treatments. Bars indicate s.e. (n = 7) and letters above columns indicate significant differences observed in the HSD Tukey test with a significance value of 0·05.

The root dry weight did not vary significantly between treatments (P = 0·061) (Fig. 2A). Values of root dry weight varied from an average of 0·5 g to 1·8 mg, and represented between 25 and 50 % of the total plant dry weight. Significantly different values of root weight ratio (root dry weight divided by total plant dry weight) (P < 0·001) were found between treatments, but the trend was weak, as the root weight ratio decreased significantly until a P supply of 15 mg P kg−1 soil, but beyond that values were not significantly different from the no-P treatment (Fig. 2B).

The lengths of the longest phyllode at harvest were significantly different (P < 0·05) for only two of the 21 P treatments. There was an increase in shoot length from approx. 291 mm in the no-P treatment to a maximum of 700 mm, in the 37·5 mg P kg−1 soil treatment. The HSD Tukey Test revealed that, with the exception of the 37·5 mg P kg−1 soil treatment, all results were not significantly different from each other, and the additional P did not lead to a greater length of the longest phyllode (Fig. 2C).

Shoot phosphorus

Plants of all treatments showed remarkably little variation in their shoot phosphorus concentration, despite the very wide range of P supply; the average [P] was 1·4 mg P kg−1 shoot d. wt, and the range was from 1·19 to 1·68 mg P kg−1 shoot d. wt (Fig. 3). The best-fitted model was a linear curve with a negative slope, which would indicate that the plants being supplied with the highest P levels are the ones with the lowest shoot [P]. However, these results are not statistically different from each other (P = 0·173), and the shoot [P] must, therefore, be considered constant, which is a remarkable result, which we have not seen reported in the literature before.

Fig. 3.

Fig. 3.

Open in a new tab

Shoot P concentration and total shoot P content (as indicated in the key) as dependent on P supply. Phosphorus concentration: y = –0·01x + 1·53; R2 = 0·35; P = 0·17; P content: y = 1·14ln(x) + 0·65; R2 = 0·68, curve fit P < 0·005. Bars indicate s.e. (n = 7). Letters above bars indicate the results of a Tukey test on total shoot P content (i.e. white bars). Shoot P concentration showed no significant variation (P = 0·05).

The total plant P content increased with increasing P supply (Fig. 3), due to the fact that the plants accumulated more biomass with increasing P supply. The logarithmic curve fitted to the model illustrates how total plant P content increased at low to moderate P supplies, and then, from moderate to high supplies, the increase in total P saturated (Fig. 2B), in agreement with the P saturation of plant growth.

Mycorrhizal colonization and cluster-root formation

Plants in 17 out of 21 treatments showed some degree of arbuscular mycorrhizal colonization. Vesicles and few hyphae were observed in these specimens, but no arbuscules. Other fungi (most likely saprophytic) were also present in the roots of V. juncea.

Higher percentage mycorrhizal colonization occurred in the lower P treatments and a significant decreasing linear trend (R2 = 0·28; P < 0·001) was observed with increasing P supply (Fig. 4A). Similarly, there was a significant linear trend for decrease in the number of cluster roots per plant with an increase in P supply (R2 = 0·14; P < 0·05; Fig. 4A). The size of the cluster roots ranged from 3 to 20 mm.

Fig. 4.

Fig. 4.

Open in a new tab

Cluster-root formation and mycorrhizal colonization as dependent on P supply during growth. (A) Number of cluster roots per plant (y = –0·59x + 34·07; R2 = 0·14; P < 0·05) and percentage of mycorrhizal colonization (y = –0·45x + 9·72; R2 = 0·28; P < 0·001) as dependent on P supply. (B) Ratio of cluster-root weight to total root dry weight; y = –2·86ln(x) + 12·85; R2 = 0·50; P < 0·001. Bars indicate s.e. (n = 7).

No significant difference was found in the dry weight of cluster roots per plant, as dependent on P supply (Fig. 2B); the values ranged from 28 to 153 mg, without any pattern or trend, not even at the lowest levels of the P treatments. However, there were significant differences between the ratio of cluster roots to total root dry weight. This ratio followed a logarithmic downward trend (R2 = 0·50; P < 0·001), with similar ratios for most treatments above 10 mg P g−1 soil (Fig. 4B). The average size of cluster root per individual plant (data not shown) was also not significantly different between treatments (P = 0·12). No senesced cluster roots were observed in any of the samples.

Soil analyses of field-collected material

Soil analyses from Yule Brook collected near V. juncea plants showed total P values between 18 and 29 (average 23·4) mg P kg−1 dry soil; bicarbonate-extractable results varied between 1·7 and 3·1 (average 2·3) mg P kg−1 dry soil. For the soils near the Banksia trees, these values were ranged from 21 to 33 (average 25·7) mg total P kg−1 dry soil and 1·1 to 1·9 (average 1·5) mg bicarbonate-extractable P kg−1 dry soil for the V. juncea and Banksia site, respectively. This shows the soil P levels at the V. juncea site were low, but not quite as low as typically found for sites dominated by Proteaceae species (Lambers et al., 2010).

DISCUSSION

Viminaria juncea, an Australian legume species, which can produce both cluster roots and mycorrhizas, showed remarkably little response in the expression of these adaptations to an increasing P supply. Viminaria juncea increased its shoot growth at higher P supply, confirming previous findings (Adams et al., 2002). Unlike other species, however, it did not exhibit suppression of cluster-root production when supplied with P at concentrations that exceeded the requirement for maximum biomass production (up to 50 mg P kg−1 dry soil), and nor was there evidence for suppression of its mycorrhizal associations. Even more remarkable, an increasing supply of P did not affect the shoot [P] of V. juncea. We are unaware of any higher plant species or any algae that show a similar constancy of [P] with such a large variation in P supply. Below, we discuss some of the possible reasons and mechanisms for these unanticipated ecophysiological responses.

Comparison of glasshouse and field results

Soil results from Yule Brook reserve where a large population of V. juncea occurs showed ‘available P’ values of 2·3 mg P kg−1 dry soil. This is in the range of typical values for the older sand dunes on the Swan coastal plain (McArthur, 1991; Laliberté et al., 2012), but well above the level that is typical for most Proteaceae species in south-western Australia (Lambers et al., 2006, 2012). Total P values ranged between 18 and 29 mg total P kg−1 dry soil, whereas the experimental treatments ranged between 0 and 50 mg added P kg−1 dry soil. Given that V. juncea accesses sources of P beyond what is readily available (Adams et al., 2002), the range of P treatments selected for the present experiment was appropriate.

P supply affects shoot growth, but not shoot [P]

The growth of V. juncea responded to increasing P supply until saturation was reached at around 27·5 mg P kg−1 dry soil; from this growth curve we infer that the range of P treatments applied was adequate and that the experiment ran for an appropriate time period. The differences in growth were observed solely in the biomass of shoots, but not in the total biomass of roots or solely cluster roots. This agrees with data on the same species from Walker and Pate (1986), although they found a difference among accessions. The typical plant response to an increase in P supply is to show greater accumulation of biomass, but in some cases the effect on root growth is minor, as in L. albus (Pearse et al., 2006b; Keerthisinghe et al., 1998). A pronounced differential growth between shoots and roots would be evident in the root weight ratio, such as shown for Triticum aestivum, Brassica napus and Pisum sativum (Pearse et al., 2007). However, only a small increase in shoot growth, and no significant increase in root weight was observed in this study. The consequence was a non-significant decrease in root weight ratio with increasing P supply; this agrees with findings by Walker and Pate (1986) on V. juncea.

The V. juncea shoot [P] results are extraordinary. At a very low availability of P, plants may increase their growth at a slightly higher P supply without a significant increase of leaf [P]; however, they typically increase leaf or shoot [P] in response to a further increase of the P supply (Keerthisinghe et al., 1998; De Groot et al., 2003). The very tight control V. juncea shows over shoot [P] is unprecedented and implies both a very fine adjustment of plant growth when P supply is scarce, and a very strong down-regulation of P-uptake capacity at the highest levels of P supply. We surmise that the strong down-regulation of P-uptake capacity in V. juncea is related to its functioning in a symbiotic association with rhizobium in a niche where soil [P] is slightly higher than typically found in the characteristically P-impoverished landscape. Lower in the landscape, where V. juncea occurs (Fig. 1A), the supply of nutrients and water is relatively stable, compared with higher in the landscape, outside its distribution (Fig. 1B). Consequently, there might be little advantage in accumulating P reserves which would be at the expense of metabolic energy for transport of P into and within the plants. This metabolic energy could be conserved for other purposes and at times may be vital, for example when seasonal flooding may curtail the rate of root respiration of V. juncea, despite its pneumatophores and aerenchyma (Fraser, 1932; Walker et al., 1983). The relatively low requirements of metabolic energy for storage of P might sustain symbiotic N2 fixation in this legume, whose degree of nodulation decreases sharply with decreasing P supply (Walker and Pate, 1986). Symbiotic N2 fixation is a costly process, in terms of both metabolic energy and P, which amounts to 1–10 % of total plant P content (Raven, 2012).

Root adaptations for P uptake decrease marginally with increasing P supply, and remain present even at the highest treatments

Several studies have shown that mycorrhizal colonization enhances P uptake of plants (Koide, 1991; Jakobsen et al., 1992; Ryan et al., 2012), and when P supply increases the percentage of root colonization typically decreases (Pairunan et al., 1980; Bougher et al., 1990; Reddell et al., 1997).

In the present experiment with V. juncea, an increasing P supply did affect mycorrhizal colonization, but not as expected. Colonization decreased particularly between the P treatments of 2·5–12·5 mg P kg−1 dry soil; however, at higher P supply, percentage colonization was variable, though with a minute decreasing overall trend. Most importantly, at the highest levels of P, mycorrhizal colonization was still approx. 8 %, instead of being totally suppressed as would typically be expected. A lack of suppression of mycorrhizal colonization is likely to be due to tight internal control of shoot [P]. Because no sharp increase in shoot [P] was observed, systemic control of mycorrhizal formation was not exerted and hence there was no suppression colonization.

The response of cluster roots was similar to that of mycorrhizal colonization such that there was also only a very slight decreasing trend in the number of root clusters per plant, and no significant P-treatment dependent difference in cluster-root dry weight was observed. This agrees with data of Walker and Pate (1986), although they found some variation in the extent of cluster-roots suppression among V. juncea accessions. Similar to mycorrhizal infection, cluster roots were presumably not suppressed at the highest levels of P because of the lack of a systemic signal, i.e. the stable values of shoot [P] explain why there was no suppression of the production of cluster root clusters at a high P supply.

Our findings differ from those for every other species known in the literature, including the cluster-root and mycorrhizal species C. cunninghamiana (Reddell et al., 1997). In stark contrast to our findings for V. juncea, Reddell et al. (1997) determined that the number of cluster roots and percentage of colonization of C. cunninghamiana declined sharply with increasing P supply.

The role of V. juncea cluster roots has not been extensively studied. The plants, grown for 12 weeks in the glasshouse, had fairly small clusters, in size resembling those of L. albus, also a legume (Watt and Evans, 1999). The resemblance extends to the fact that cluster roots of L. albus are only partly suppressed by the addition of P in solution cultures, and only at fairly high P supply (Shane et al., 2003a). In most other studies, suppression of cluster roots at elevated P supply is very strong (Lamont, 1972a; Crocker and Schwintzer, 1993; Shane et al., 2003c; Zúñiga-Feest et al., 2010). In P-impoverished soils, where very little P is in solution, carboxylate-releasing cluster roots are a common and effective P-acquisition strategy, whereas mycorrhizas are more common and effective at slightly higher soil P concentrations (Lambers et al., 2008). We hypothesize that, as is the case in some Lupinus species (Pearse et al., 2006a), the functioning (carboxylate exudation) of root clusters in V. juncea may respond more strongly to P supply than their formation. The constant presence of cluster roots may act as a mechanism to respond quickly to demand and availability of P and negate the need for building P reserves.

Viminaria juncea is unique in its shoot [P] control and an exception to Australian native species

To our knowledge, the tight control of shoot [P] such as found for V. juncea has not been found for any other species studied thus far. The constant shoot [P] is likely to prevent the suppression of cluster-root formation and mycorrhizal structures. The discovery of the present tight control of shoot [P] in this legume leads to new questions on the regulation of P uptake: if shoot P is constant, what triggers the systemic signal leading to down-regulation of P uptake? The present findings are further evidence of the high diversity of physiological mechanisms that can be encountered in a biodiversity hotspot (Hopper and Gioia, 2004). Tight control of growth and P uptake by V. juncea provides an alternative to ‘shutting down’ mycorrhizal associations and cluster-root formation when P supply is no longer restrictive to growth.

Concluding remarks

Neither cluster-root formation nor mycorrhizal colonization was suppressed in V. juncea over a wide range of P treatments; therefore, the present results did not allow us to test our original hypothesis. Instead, we found that leaf [P] in V. juncea is tightly controlled, at a low P supply by a strong suppression of growth and at a high P supply by a strong down-regulation of the P-uptake capacity. This finding offers an explanation for the lack of suppression of cluster roots and mycorrhizal structures. We surmise that tight control of V. juncea shoot [P] reduces the energy requirement for storage of P; however, as a consequence this species requires a steady supply of nutrients. In part, this may explain why V. juncea does not occur higher in the landscape, where the nutrient supply is typically seasonal.

ACKNOWLEDGEMENTS

Thanks are due to the University of Western Australia for the IPRS and SIRF scholarships and to the School of Plant Biology for research funding and the use of infrastructure; to ANZ's Holsworth Wildlife Foundation Grant and The Mary Janet Lindsay of Yanchep Memorial Fund for additional grants. This research was supported by the Australian Research Council (ARC). We are grateful for help and advice from Erik Veneklaas and Ana Luíza Muler in germinating and cultivating the seedlings for the initial phase of the experiment and Honghua He for dealing with the soil P analyses. We thank Michelle Watt, Rebecca Ostertag and John Raven for their valuable comments on an earlier version of this manuscript, which was part of the PhD thesis of M.C.R.dC.

LITERATURE CITED

  1. Adams MA, Bell TL, Pate JS. Phosphorus sources and availability modify growth and distribution of root clusters and nodules of native Australian legumes. Plant, Cell & Environment. 2002;25:837–850. [Google Scholar]
  2. Beadle NCW. Soil phosphate and its role in molding segments of the Australian flora and vegetation, with special reference to xeromorphy and sclerophylly. Ecology. 1966;47:992–1007. [Google Scholar]
  3. Bell DT. The process of germination in Australian species. Australian Journal of Botany. 1999;47:475–517. [Google Scholar]
  4. Bougher NL, Grove TS, Malajczuk N. Growth and phosphorus acquisition of karri (Eucalyptus diversicolor F. Muell.) seedlings inoculated with ectomycorrhizal fungi in relation to phosphorus supply. New Phytologist. 1990;114:77–85. doi: 10.1111/j.1469-8137.1990.tb00376.x. [DOI] [PubMed] [Google Scholar]
  5. Brundrett MC. Mycorrhizal associations and other means of nutrition of vascular plants: understanding the global diversity of host plants by resolving conflicting information and developing reliable means of diagnosis. Plant and Soil. 2009;320:37–77. [Google Scholar]
  6. Brundrett MC, Abbott LK. Roots of jarrah forest plants. I. Mycorrhizal associations of shrubs and herbaceous plants. Australian Journal of Botany. 1991;39:445–457. [Google Scholar]
  7. Brundrett MC, Bougher N, Dell B, Grove T, Malajczuk N. Working with mycorrhizas in forestry and agriculture. Canberra: Australian Centre for International Agricultural Research; 1996. [Google Scholar]
  8. Colwell JD. The estimation of the phosphorus fertiliser requirements of wheat in southern NSW by soil analysis. Australian Journal of Experimental Agriculture and Animal Husbandry. 1963;3:190–197. [Google Scholar]
  9. Crocker LJ, Schwintzer CR. Factors affecting formation of cluster roots in Myrica gale seedlings in water culture. Plant and Soil. 1993;152:287–298. [Google Scholar]
  10. Crocker LJ, Schwintzer CR. Soil conditions affect the occurrence of cluster roots in Myrica gale L. in the field. Soil Biology and Biochemistry. 1994;26:615–622. [Google Scholar]
  11. De Groot CC, Marcelis LFM, van den Boogaard R, Kaiser WM, Lambers H. Interaction of nitrogen and phosphorus nutrition in determining growth. Plant and Soil. 2003;248:257–268. [Google Scholar]
  12. Dinkelaker B, Hengeler C, Marschner H. Distribution and function of proteoid roots and other root clusters. Botanica Acta. 1995;108:193–200. [Google Scholar]
  13. Fraser L. Reaction of Viminaria denudata to increased water content of soil. Proceedings of the Linnean Society of New South Wales. 1932;56:391–406. [Google Scholar]
  14. Gardner WK, Parbery DG, Barber DA. The acquisition of phosphorus by Lupinus albus L. I. Some characteristics of the soil/root interface. Plant and Soil. 1982;68:19–32. [Google Scholar]
  15. Goble-Garratt E, Bell D, Loneragan W. Floristic and leaf structure patterns along a shallow elevational gradient. Australian Journal of Botany. 1981;29:329–347. [Google Scholar]
  16. Hayman DS. Mycorrhizae of nitrogen-fixing legumes. World Journal of Microbiology and Biotechnology. 1986;2:121–145. [Google Scholar]
  17. Hopper SD, Gioia P. The Southwest Australian Floristic Region: evolution and conservation of a global hotspot of biodiversity. Annual Review of Ecology, Evolution and Systematics. 2004;35:623–650. [Google Scholar]
  18. Jakobsen I, Abbott LK, Robson AD. External hyphae of vesicular-arbuscular mycorrhizal fungi associated with Trifolium subterraneum L. 1. Spread of hyphae and phosphorus inflow into roots. New Phytologist. 1992;120:371–379. [Google Scholar]
  19. Keerthisinghe G, Hocking PJ, Ryan PR, Delhaize E. Effect of phosphorus supply on the formation and function of proteoid roots of white lupin (Lupinus albus L.) Plant, Cell & Environment. 1998;21:467–478. [Google Scholar]
  20. Koide RT. Nutrient supply, nutrient demand and plant response to mycorrhizal infection. New Phytologist. 1991;117:365–386. doi: 10.1111/j.1469-8137.1991.tb00001.x. [DOI] [PubMed] [Google Scholar]
  21. Laliberté E, Turner BL, Costes T, et al. Experimental assessment of nutrient limitation along a 2-million year dune chronosequence in the south-western Australia biodiversity hotspot. Journal of Ecology. 2012;100:631–642. [Google Scholar]
  22. Lambers H, Brundrett MC, Raven JA, Hopper SD. Plant mineral nutrition in ancient landscapes: high plant species diversity on infertile soils is linked to functional diversity for nutritional strategies. Plant and Soil. 2010;334:11–31. [Google Scholar]
  23. Lambers H, Cawthray GR, Giavalisco P, et al. Proteaceae from severely phosphorus-impoverished soils extensively replace phospholipids with galactolipids and sulfolipids during leaf development to achieve a high photosynthetic phosphorus-use efficiency. New Phytologist. 2012;196:1098–1108. doi: 10.1111/j.1469-8137.2012.04285.x. [DOI] [PubMed] [Google Scholar]
  24. Lambers H, Raven JA, Shaver GR, Smith SE. Plant nutrient-acquisition strategies change with soil age. Trends in Ecology and Evolution. 2008;23:95–103. doi: 10.1016/j.tree.2007.10.008. [DOI] [PubMed] [Google Scholar]
  25. Lambers H, Shane MW, Cramer MD, Pearse SJ, Veneklaas EJ. Root structure and functioning for efficient acquisition of phosphorus: matching morphological and physiological traits. Annals of Botany. 2006;98:693–713. doi: 10.1093/aob/mcl114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lamont B. The effect of soil nutrients on the production of proteoid roots by Hakea species. Australian Journal of Botany. 1972;20:27–40. [Google Scholar]
  27. Lamont BB. ‘Proteoid’ roots in the legume Viminaria juncea. Search. 1972;3:90–91. [Google Scholar]
  28. Lamont BB. Mechanisms for enhancing nutrient uptake in plants, with particular reference to mediterranean South Africa and Western Australia. Botanical Review. 1982;48:597–689. [Google Scholar]
  29. Lamont BB. Structure, ecology and physiology of root clusters – a review. Plant and Soil. 2003;248:1–19. [Google Scholar]
  30. McArthur WM. Reference soils of south-western Australia. South Perth: Department of Agriculture Western Australia; 1991. [Google Scholar]
  31. Motomizu S, Wakimoto T, Toei K. Spectrophotometric determination of phosphate in river waters with molybdate and malachite green. Analyst. 1983;108:361–367. doi: 10.1016/0039-9140(84)80269-6. [DOI] [PubMed] [Google Scholar]
  32. Myers N, Mittermeier RA, Mittermeier CG, da Fonseca GAB, Kent J. Biodiversity hotspots for conservation priorities. Nature. 2000;403:853–858. doi: 10.1038/35002501. [DOI] [PubMed] [Google Scholar]
  33. Pairunan AK, Robson AD, Abbott LK. The effectiveness of vesicular-arbuscular mycorrhizas in increasing growth and phosphorus uptake of subterranean clover from phosphorus sources of different solubilities. New Phytologist. 1980;84:327–338. [Google Scholar]
  34. Pate JS, Dell B. Kwongan: plant life of the sandplain. In Nedlands: Pate JS, Beard JS, University of Western Australia Press; 1984. Economy of mineral nutrients in sandplain species. [Google Scholar]
  35. Pearse SJ, Veneklaas EJ, Cawthray G, Bolland MDA, Lambers H. Carboxylate composition of root exudates does not relate consistently to a crop species' ability to use phosphorus from aluminium, iron or calcium phosphate sources. New Phytologist. 2007;173:181–190. doi: 10.1111/j.1469-8137.2006.01897.x. [DOI] [PubMed] [Google Scholar]
  36. Pearse SJ, Veneklaas EJ, Cawthray GR, Bolland MDA, Lambers H. Carboxylate release of wheat, canola and 11 grain legume species as affected by phosphorus status. Plant and Soil. 2006;288:127–139. [Google Scholar]
  37. Pearse SJ, Veneklaas EJ, Cawthray GR, Bolland MDA, Lambers H. Triticum aestivum shows a greater biomass response to a supply of aluminium phosphate than Lupinus albus, despite releasing fewer carboxylates into the rhizosphere. New Phytologist. 2006;169:515–524. doi: 10.1111/j.1469-8137.2005.01614.x. [DOI] [PubMed] [Google Scholar]
  38. Purnell H. Studies of the family Proteaceae. I. Anatomy and morphology of the roots of some Victorian species. Australian Journal of Botany. 1960;8:38–50. [Google Scholar]
  39. Raven JA. Protein turnover and plant RNA and phosphorus requirements in relation to nitrogen fixation. Plant Science. 2012;188/189:25–35. doi: 10.1016/j.plantsci.2012.02.010. [DOI] [PubMed] [Google Scholar]
  40. Reddell P, Yun Y, Shipton WA. Cluster roots and mycorrhizae in Casuarina cunninghamiana: their occurrence and formation in relation to phosphorus supply. Australian Journal of Botany. 1997;45:41–51. [Google Scholar]
  41. Ryan MH, Tibbett M, Edmonds-Tibbett T, et al. Carbon trading for phosphorus gain: the balance between rhizosphere carboxylates and mycorrhizal symbiosis in plant phosphorus acquisition. Plant, Cell & Environment. 2012;35:2170–2180. doi: 10.1111/j.1365-3040.2012.02547.x. [DOI] [PubMed] [Google Scholar]
  42. Shane MW, De Vos, De Roock, Lambers H. Shoot P status regulates cluster-root growth and citrate exudation in Lupinus albus grown with a divided root system. Plant, Cell & Environment. 2003;26:265–273. [Google Scholar]
  43. Shane MW, De Vos M, De Roock S, Cawthray GR, Lambers H. Effect of external phosphorus supply on internal phosphorus concentration and the initiation, growth and exudation of cluster roots in Hakea prostrata R.Br. Plant and Soil. 2003;248:209–219. [Google Scholar]
  44. Shane MW, De Vos M, De Roock S, Cawthray GR, Lambers H. Effects of external phosphorus supply on internal phosphorus concentration and the initiation, growth and exudation of cluster roots in Hakea prostrata R.Br. Plant and Soil. 2003;248:209–219. [Google Scholar]
  45. Shane MW, Lambers H. Cluster roots: a curiosity in context. Plant and Soil. 2005;274:101–125. [Google Scholar]
  46. Singh B, Gilkes RJ. Phosphorus sorption in relation to soil properties for the major soil types of south-western Australia. Australian Journal of Soil Research. 1991;29:603–618. [Google Scholar]
  47. Skene KR. Cluster roots: some ecological considerations. Journal of Ecology. 1998;86:1060–1064. [Google Scholar]
  48. Smith SE, Read DJ. Mycorrhizal symbiosis. London: Academic Press and Elsevier; 2008. [Google Scholar]
  49. Veneklaas EJ, Poot P. Seasonal patterns in water use and leaf turnover of different plant functional types in a species-rich woodland, south-western Australia. Plant and Soil. 2003;257:295–304. [Google Scholar]
  50. Walker BA, Pate JS. Progenies of Viminaria juncea (Schrad. & Wendl.) Hoffmans. (Fabaceae) and its physiological significance. Australian Journal of Plant Physiology. 1986;13:305–319. [Google Scholar]
  51. Walker BA, Pate JS, Kuo J. Nitrogen fixation by nodulated roots of Viminaria juncea (Schrad. & Wendl.) Hoffmans (Fabaceae) when submerged in water. Functional Plant Biology. 1983;10:409–421. [Google Scholar]
  52. Walsh NG, Entwisle TJ. Flora of Victoria. Vol. 3. Melbourne: Inkata Press; 1996. Dicotyledons: Winteraceae to Myrtaceae. [Google Scholar]
  53. Watt M, Evans JR. Proteoid roots: physiology and development. Plant Physiology. 1999;121:317–323. doi: 10.1104/pp.121.2.317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Zúñiga-Feest A, Delgado M, Alberdi M. The effect of phosphorus on growth and cluster-root formation in the Chilean Proteaceae: Embothrium coccineum (R. et J. Forst.) Plant and Soil. 2010;334:113–121. [Google Scholar]

Articles from Annals of Botany are provided here courtesy of Oxford University Press

RESOURCES