REGULAR ARTICLE

Variation in morphological and physiological parametersin herbaceous perennial legumes in response to phosphorussupply

Jiayin Pang & Megan H. Ryan & Mark Tibbett & Gregory R. Cawthray &

Kadambot H. M. Siddique & Mike D. A. Bolland & Matthew D. Denton &

Hans Lambers

Received: 18 July 2009 /Accepted: 30 November 2009 /Published online: 19 December 2009# Springer Science+Business Media B.V. 2009

Abstract Change in morphological and physiologicalparameters in response to phosphorus (P) supply wasstudied in 11 perennial herbaceous legume species, sixAustralian native (Lotus australis, Cullen australasicum,Kennedia prorepens, K. prostrata, Glycine canescens,C. tenax) and five exotic species (Medicago sativa,Lotononis bainesii, Bituminaria bituminosa varalbomarginata, Lotus corniculatus, Macroptiliumbracteatum). We aimed to identify mechanisms forP acquisition from soil. Plants were grown insterilised washed river sand; eight levels of P asKH2PO4 ranging from 0 to 384 μg P g−1 soil wereapplied. Plant growth under low-P conditions strong-ly correlated with physiological P-use efficiency and/

or P-uptake efficiency. Taking all species together, at6 μg P g−1 soil there was a good correlation betweenP uptake and both root surface area and total rootlength. All species had higher amounts of carbox-ylates in the rhizosphere under a low level of Papplication. Six of the 11 species increased thefraction of rhizosphere citrate in response to low P,which was accompanied by a reduction in malonate,except L. corniculatus. In addition, species showeddifferent plasticity in response to P-application levelsand different strategies in response to P deficiency.Our results show that many of the 11 species haveprospects for low-input agroecosystems based ontheir high P-uptake and P-use efficiency.

Plant Soil (2010) 331:241–255DOI 10.1007/s11104-009-0249-x

Responsible Editor: Tim Simon George.

J. Pang (*) :M. H. Ryan :G. R. Cawthray :M. D. A. Bolland :H. LambersSchool of Plant Biology,The University of Western Australia,35 Stirling Highway,Crawley, WA 6009, Australiae-mail: jpang@cyllene.uwa.edu.au

M. TibbettCentre for Land Rehabilitation,The University of Western Australia,35 Stirling Highway,Crawley, WA 6009, Australia

K. H. M. SiddiqueInstitute of Agriculture,The University of Western Australia,35 Stirling Highway,Crawley, WA 6009, Australia

M. D. A. BollandDepartment of Agricultural and Food Western Australia,PO Box 1231, Bunbury, WA 6231, Australia

M. D. DentonBiosciences Research Division,Department of Primary Industries,RMB1145 Chiltern Valley Road,Rutherglen, VIC 3685, Australia

Keywords Carboxylate exudation . Native Australianlegumes . Perennial herbaceous legume . Phosphorus .

Rhizosphere pH . Root diameter . Specific root length .

Root surface area . Total root length

Introduction

Phosphorus is the major limiting nutrient for crop andpasture production in many environments. P is readilysorbed by aluminium (Al) and iron (Fe) exposed atthe surfaces of soil constituents and by free limestonepresent in some alkaline soils. As a result, up to 80%of P applied as fertiliser may be unavailable foruptake by plant roots (Raghothama 1999; Vance et al.2003). In Western Australia, natural surface soils maycontain less than 5 mg kg−1 of bicarbonate-extractableP (McArthur 1991). When crops or pastures aregrown on such soils, application of large quantities offertilizers containing readily soluble P, such assuperphosphate, is needed (Bolland et al. 1997).Consequently, P concentrations in agricultural soilsin Australia have increased substantially.

A plant’s capacity for nutrient acquisition, espe-cially that of phosphorus, is closely associated withroot morphological parameters (Denton et al. 2006;Lynch 2007). For instance, barley genotypes withlong root hairs are better adapted to low-P soils andhave higher yield potential than genotypes with shortroot hairs (Gahoonia and Nielsen 2004). It has alsobeen shown that P uptake under low P application inrice (Oryza sativa) (Li et al. 2007) and Brassicaoleracea genotypes (Hammond et al. 2009) iscorrelated with root surface area, lateral root length,lateral root growth rate and the number of lateral roots.Changes in root morphology in response to changes inP availability are important for P-acquisition efficiency(Lambers et al. 2006). Typical morphologicalresponses to low-P stress include a higher root massratio (the ratio of root dry mass to total plant drymass), finer roots, more and longer root hairs, a morehighly branched root system, increased specific rootlength and formation of mycorrhizal associations, allof which are expected to enhance the total surfacearea available for soil exploration and acquisition ofnutrients at minimal energy cost (Lambers et al. 2006;Raghothama 1999; Vance et al. 2003).

Plants may also respond to a P-limiting environ-ment by altering their root physiology to increase the

rate of P absorption per unit root mass and length(Neumann and Martinoia 2002). One of the physio-logical mechanisms used by plants is the release oforganic acid exudates that mobilise P from themineral or organic fractions in soil. This mechanismhas been widely reported in Proteaceae from Aus-tralia, but is also found in crop species such asLupinus albus, B. napus, Triticum aestivum andOryza sativa (Lambers et al. 2006; Raghothama1999). Information on carboxylate exudation inperennial legume species is scarce and whether theexudation of carboxylate in perennial legume isrelated to their P nutrition is unknown.

The overall objective of this study was to identifymechanisms that account for variation among plantspecies in the efficiency of P acquisition and P use.As we were interested in the potential of novelperennial legumes as new forage pastures for low-input systems, we chose to investigate root morpho-logical and physiological responses of six Australiannative and five exotic herbaceous perennial legumes,when grown under a wide range of soil P supply. Ourspecific objective was to assess variation in rootdiameter, total root length, root surface area, specificroot length and the capacity to accumulate carbox-ylates in the rhizosphere. We investigated the plastic-ity of these characteristics and how they are linked toP-acquisition efficiency, together with P-use efficien-cy. This was achieved by growing 11 leguminousspecies under controlled glasshouse conditions with Psupply ranging from 0 to 384 μg g−1. Data on shootand root dry mass from this experiment have beenreported in a separate paper (Pang et al. 2009).

Materials and methods

Plant material and growth conditions

Eleven herbaceous perennial legume species werestudied (Table 1). Seeds were scarified and pre-germinated in Petri dishes at staggered times accord-ing to their pre-determined germination time using a1:100 dilution of smoke water (available from KingsPark and Botanic Garden, West Perth, WesternAustralia) to enhance germination (Tieu et al. 2001).Three germinated seedlings were planted in each potand thinned to one plant after 1 week. All seedlingswere inoculated with appropriate rhizobia at planting

242 Plant Soil (2010) 331:241–255

(Table 1) by applying a few drops of a mixture ofrhizobia in peat and water to seedlings using a plasticpipette. Rhizobia were obtained from the RutherglenCentre, Department of Primary Industries, Victoria,Australia and nodules were formed in all species. Theexperiment was carried out from June to August 2007in a glasshouse at The University of WesternAustralia, Perth, Australia. The temperature-controlled glasshouse had an average daytime tem-perature of 24 °C during the experiment.

Pots (8.5 cm×8.5 cm×18 cm) were filled with1.2 kg of sterilised washed coarse river sand. Soilsamples were air-dried and passed through 2 mmsieves. Sand analyses were performed by CSBPFutureFarm analytical laboratories (Bibra Lake,Australia). The washed river sand contained 1 μg g−1

nitrate-N and 4 μg g−1 ammonium-N measuredaccording to Searle (1984), 3 μg g−1 bicarbonate-extractable P (Colwell 1963), 6 μg g−1 total P (Allenand Jeffery 1990), had a pH (CaCl2) of 6.3 (Raymentand Higginson 1992) and a P-retention index of 1.5 mlP g−1 (Allen and Jeffery 1990). All essential nutrientsother than P were provided at final concentrations of(μg g−1 dry soil): N 30, S 50, Ca 24, Mg 10, Cu 0.5, Zn2, Mn 4, B 0.119, Mo 0.4, Fe 5, Cl 23. Nitrogen wasadded as a mixture of NH4NO3 and Ca(NO3)2 with amolar ratio of 1 to provide initial supply aftergermination, prior to nodulation. There were eight Ptreatments; P (KH2PO4) was added to the initial

nutrient solution to give final concentrations of 0, 6,12, 24, 48, 96, 192 and 384 μg P g−1 dry sand. K wasbalanced by supplying appropriate concentrations ofKCl to the P treatments. The sand was initiallymoistened to 50% water-holding capacity with nutrientsolution. Thereafter, soil water content was maintainedduring the experiment by watering pots to weight withdeionised water every other day. Plants were harvestedafter 8 weeks.

Root morphology

Root length and diameter were measured immediatelyafter harvest using an Epson 1680 scanner and theWinrhizo version 4.1c (Regent Instructions, Quebec,Canada) scanner program. Specific root length wascalculated as the root length per unit root dry weight(DW).

Rhizosphere carboxylates

Rhizosphere extracts to be analysed for organicanions were collected from two P application levels:6 (low) and 48 (high) μg P g−1 dry sand. The sandwas carefully tipped out of the pots and the rootsystems gently shaken to remove excess sand. Thesand still attached to the roots was defined asrhizosphere soil (Veneklaas et al. 2003). The rootswere transferred to a beaker containing a known

Table 1 Perennial legumes species used in the study

Species, authority and accession number Species origin Rhizobium strain number

Lotus australis Andrews SA33610 Australia RRI2252

Cullen australasicum (Schltdl.) J.W. Grimes SA42762 Australia RRI2780

Medicago sativa L. cv SARDI Ten Asia minor, Iran, Turkmenistan andTranscaucasia

RRI128

Lotononis bainesii Baker cv Miles southern Africa WSM2598

Kennedia prorepens F.Muell KIMS001 Australia RRI2505

Kennedia prostrata R.Br JUP001 Australia RRI2353

Glycine canescens F.J.Herm NIND001 Australia RRI2680

Cullen tenax (Lindl.) J.W.Grimes SA35778 Australia RRI2558

Bituminaria bituminosa (L.) C.H. Stirt varalbomarginata PNF23-A15 (common name — tedera)

Canary Islands WSM4083

Lotus corniculatus L. cv San Gabriel Mediterranean Basin, Eurasia andNorth Africa

SU343

Macroptilium bracteatum (Nees & Mart.) Maréchal &Baudet cv B1 Burgundy

South America CB1717

Plant Soil (2010) 331:241–255 243

volume of 0.2 mM CaCl2 to ensure cell integrity, andgently shaken to remove the rhizosphere soil. Asubsample of the rhizosphere extract was taken andfiltered through a 0.22 µm syringe filter into a 1 mLHPLC vial. HPLC samples were acidified withorthosphosphoric acid and frozen at −20°C, untilanalysis. HPLC analysis of the elution liquid wasperformed using a 600 E pump and 717 autosampler(Waters, Milford MA, USA). Working standards ofmalic, malonic, lactic, acetic, maleic, citric, succinic,cis-aconitic and trans-aconitic acid (ICN BiomedicalsInc, Aurora OH, USA) were used to identify carbox-ylates on an Alltima C-18 reverse phase column (250×4.6 mm, Alltech, Deerfield IL, USA) (Cawthray 2003).

Phosphorus analysis

To determine root P concentrations, root sampleswere dried and ground to a fine powder using astainless steel ball mill. Weighed subsamples ofapproximately 200 mg were digested using a hotconcentrated nitric-perchloric (3:1) acid mixture.Total P concentrations in root samples were deter-mined using a UV-VIS spectrophotometer (ShimadzuCorporation, Japan) by the malachite green method(Motomizu et al. 1983).

Calculation of phosphorus-uptake and phosphorus-use efficiency

P-uptake efficiency and physiological P-use efficien-cy when plants were grown at 6 μg P g−1 dry sandwas calculated as described by Hammond et al.(2009). Briefly, P-uptake efficiency=[(plant P con-centration at 6 µg P g−1 dry soil×DW at 6 µg P g−1)-(plant P concentration at 0 µg P g−1 dry soil × DW at0 µg P g−1)]/difference in amount of P appliedbetween 6 and 0 μg P g−1 treatments. PhysiologicalP use efficiency=DW at 6 μg P g−1/ plant Pconcentration at 6 μg P g−1 of whole plant.

Statistics

The experiment was a two-factorial (species and soilP application level) completely randomised blockdesign. Data for growth and other parameters wereanalysed by general analysis of variance (ANOVA) inGenstat version 10.2 (Lawes Agricultural Trust,Rothamsted Experimental Station, UK, 2007). Where

significant effects were found, the l.s.d. values at p=0.05 for figures are shown in figure legends.

Results

Root P concentration

Root P concentration increased steadily in response toincreased soil P application, with the highest root Pconcentration at the highest soil P application for all11 species (Fig. 1). At maximum plant growth (asindicated by the first arrow from the left in Fig. 1),Lotus australis had the highest root P concentrationamong all species, followed by Glycine canescensand L. corniculatus, while Medicago sativa andBituminaria bituminosa had the lowest root Pconcentration (<2 mg g−1 DW) (Fig. 1). At thehighest soil P application level the two native legumesKennedia prorepens and K. prostrata had the highestroot P concentration of 54 mg g−1 DW, followed byG. canescens (48 mg g−1) and L. australis(46 mg g−1); the lowest P concentrations were foundin B. bituminosa, Macroptilium bracteatum and M.sativa (16.3–19.1 mg g−1 DW) (Fig. 1).

Total root length

The effect of P application on total root length differedamong species. The total root length for all legumespecies was in a range of 1.6–12 m when plants weregrown without addition of P (Fig. 2), with root lengthof B. bituminosa, K. prostrata and K. prorepens beinghigher than that of the other species (Fig. 2).

Total root length initially increased for all specieswith increasing application of P (Fig. 2). Themaximum total root length was found under soil Papplication when maximum plant growth occurred(the first arrow from the left in graphs) in all speciesexcept in Cullen australasicum where it occurredearlier. Species differed greatly in maximum rootlength, with plants of M. sativa having the highestvalues, followed by K. prorepens and B. bituminosa,with M. bracteatum, L. australis and K. prostratalowest (Fig. 2).

When soil P application increased above the opti-mum, total root length either plateaued (L. australis, C.australasicum, M. sativa, B. bituminosa, C. tenax, G.canescens and M. bracteatum) or decreased in those

244 Plant Soil (2010) 331:241–255

species whose plant growth suffered P toxicity(Lotononis bainesii, K. prorepens, K. prostrata and L.corniculatus) (Fig. 2). Root length of K. prorepens andK. prostrata had the greatest sensitivity to high soil Papplication, with total root length decreasing to, oreven becoming less than, that when no P was added tothe soil (Fig. 2).

Specific root length (SRL)

The investigated legume species showed great varia-tion in SRL in response to P application (Fig. 3).When no P was added, SRL was highest in L. bainesii(552 m g−1 DW), followed by K. prorepens(333 m g−1 DW), and lowest in L. australis and B.bituminosa (175 m g−1 DW). SRL for the otherspecies ranged from 200 to 300 m g−1 DW. When

6 µg P g−1 was applied to soil, SRL was reduced inmost species, exceptions being L. australis, K. pro-repens and K. prostrata (Fig. 3). SRL of L. bainesiireached its minimum at 6 µg P g−1 soil, while in otherspecies SRL decreased further in response to increasedsoil P application. Lowest SRL was observed at thesoil P application level where maximum plant growthoccurred in all species except in L. australis,L. bainesii and K. prorepens (Fig. 3). SRL increasedfrom minimum values as soil P application levelsincreased for some species (Fig. 3), notablyK. prorepens, K. prostrata and L. bainesii. SRL athigh soil P supply under which plant growth sufferedfrom P toxicity in K. prorepens and K. prostrataincreased to close to the value when no P was added.However, there was no further change in SRL at highsoil P application levels in M. sativa, G. canescens and

P c

once

ntra

tion

in r

oots

(m

g g-1

DW

)

Soil P application (µg g-1)

L. australis

0 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 3840

15

30

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60

75C. australasicum

0 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 3840

15

30

45

60

75M. sativa

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840

15

30

45

60

75

L. bainesii

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840

15

30

45

60

75K. prorepens

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840

15

30

45

60

75K. prostrata

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840

15

30

45

60

75

G. canescens

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840

15

30

45

60

75C. tenax

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840

15

30

45

60

75

B. bituminos

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 19 8448 96 19 8448 96 19 8448 96 19 8448 96 19 8448 96 19 8448 96 19 8448 96 19 8448 96 19 8448 96 192 38448 96 192 3840

15

30

45

60

75

L. corniculatus

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840

15

30

45

60

75M. bract

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840

15

30

45

60

75

P c

once

ntra

tion

in r

oots

(m

g g-1

)

Soil P application (µg g-1)

L. australis

0 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 3840

15

30

45

60

75C. australasicum

0 6 12 240 6 12 24 48 96 192 38448 96 192 38419 840

15

30

45

60

75M. sativa

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840

15

30

45

60

75

L. bainesii

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 348 96 1948 96 1948 96 1948 96 1948 96 1948 96 1948 96 1948 96960

15

30

45

60

75K. prorepens

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840

15

30

45

60

75K. prostrata

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840

15

30

45

60

75

G. canescens

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840

15

30

45

60

75C. tenax

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840

15

30

45

60

75

B. bituminosa

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 19 8448 96 192 8448 96 192 38448 96 192 38448 96 192 38448 96 192 30

15

30

45

60

75

L. corniculatus

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840

15

30

45

60

75M. bracteatum

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840

15

30

45

60

75

Fig. 1 Root P concentration of 11 perennial legumes grown for8 weeks in sterilised washed river sand supplied with eightlevels of P, ranging from 0 to 384 μg P g−1 dry soil. Data aremeans±s.e. (n=3). The first arrow from the left in each graphindicates the soil P application level where maximum plantgrowth occurred; plants were P-deficient when grown below

this level. The second arrow indicates the soil P applicationlevel where plant growth suffered from P-toxicity and reducedgrowth. Lack of second arrow in some species indicates no Ptoxicity. There was a significant interaction between soil Papplication level and species (p<0.001, LSD0.05=5.5 mg g−1

DW). Note x-axis break

Plant Soil (2010) 331:241–255 245

M. bracteatum. L. australis had the smallest responseto P application level (Fig. 3).

The average SRL over all P-application ratesvaried greatly among species with native legumespecies, except C. tenax, having a higher average SRL(158–301 m g−1 DW) than exotic species, exceptL. bainesii (79–133 m g−1 DW) (Fig. 3).

Mean root diameter

Mean root diameter varied among species. Mean rootdiameter was lowest when no P was applied to soil forall species except M. bracteatum, C. australasicumand K. prorepens (Fig. 4). L. bainesii had the lowestmean root diameter when no P was applied(0.26 mm), followed by K. prorepens, K. prostrata,L. corniculatus, M. sativa, C. tenax and B. bituminosa(0.30–0.33mm), whileC. australasicum, G. canescens,

M. bracteatum and L. australis had the highest meanroot diameters (0.36–0.44 mm) (Fig. 4).

Mean root diameter increased in L. australis,M. sativa, L. bainesii and L. corniculatus when soilP application level increased from 0 to 6 μg g−1,while there was little change in other species (Fig. 4).The maximum mean root diameter was found atdifferent levels of soil P application in differentspecies. For C. australasicum, L. bainesii and G.canescens it occurred at the soil P application level atwhich maximum plant growth occurred (Fig. 4). Insome species (L. australis, M. sativa, L. corniculatusand M. bracteatum) maximum mean root diameteralso occurred at the soil P application level wheremaximum plant growth occurred, but it was firstreached when soil P application level was lower(Fig. 4). In other species (K. prorepens, K. prostrata,B. bituminosa and C. tenax) it occurred at a soil P

Tot

al r

oot l

engt

h (m

Soil P application (µg g-1)

L. australis

0 6 12 24 48 96 192 3840

15

30

45C. australasicum

0 6 12 24 48 96 192 3840

15

30

45M. sativa

0 6 12 24 48 96 192 3840

20

40

60

80

100

L. bainesii

0 6 12 24 48 96 192 3840

15

30

45K. prorepens

0 6 12 24 48 96 192 3840

15

30

45K. prostrata

0 6 12 24 48 96 192 3840

15

30

45

G. canescens

0 6 12 24 48 96 192 3840

15

30

45C. tenax

0 6 12 24 48 96 192 3840

15

30

45

B. bituminosa

0 6 12 24 48 96 192 3840

15

30

45

L. corniculatus

0 6 12 24 48 96 192 3840

15

30

45M. bracteatum

0 6 12 24 48 96 192 3840

15

30

45Tot

al r

oot l

engt

h (m

)

Soil P application (µg g-1)

L. australis

0 6 12 24 48 96 192 3840

15

30

45C. australasicum

0 6 12 24 48 96 192 3840

15

30

45M. sativa

0 6 12 24 48 96 192 3840

20

40

60

80

100

L. bainesii

0 6 12 24 48 96 192 3840

15

30

45K. prorepens

0 6 12 24 48 96 192 3840

15

30

45K. prostrata

0 6 12 24 48 96 192 3840

15

30

45

G. canescens

0 6 12 24 48 96 192 3840

15

30

45C. tenax

0 6 12 24 48 96 192 3840

15

30

45

B. bituminosa

0 6 12 24 48 96 19 840

15

30

45

L. corniculatus

0 6 12 24 48 96 192 3840

15

30

45M. bracteatum

0 6 12 24 48 96 19 840

15

30

45

Fig. 2 Total root length of 11 perennial legumes grown for8 weeks in sterilised washed river sand supplied with eightlevels of P ranging from 0 to 384 μg P g−1 dry soil. Data aremeans±s.e. (n=6). The first arrow from the left in each graphindicates the soil P application level where maximum plantgrowth occurred; plants were P-deficient when grown below

this level. The second arrow indicates the soil P applicationlevel where plant growth suffered from P-toxicity and reducedgrowth. Lack of second arrow in some species indicates no Ptoxicity. Note different y-axis scale for M. sativa. There was asignificant interaction between soil P application level andspecies (p<0.001, LSD0.05 =11.8 m). Note x-axis break

246 Plant Soil (2010) 331:241–255

application level higher than that when maximumplant growth occurred (Fig. 4). Mean root diameterdid not differ among treatments when soil P applica-tion levels were≥6 μg P g−1 soil for L. australis andM. sativa, while there was no difference among allsoil P application levels for M. bracteatum (Fig. 4).When the level of soil P application was high andplant growth suffered from P toxicity, the mean rootdiameter was significantly reduced in K. prorepens(soil P application level≥96 μg P g−1) (Fig. 4).

Rhizosphere carboxylates and pH

Rhizosphere carboxylic acid amount, expressed perroot dry mass, was compared between two levels ofsoil P application, 6 μg P g−1 and 48 μg P g−1.Carboxylate amount recovered relative to root dry

mass in the rhizosphere increased significantly in all11 species in response to low P (Fig. 5a). Five of thesix native perennial legumes (L. australis, C. austral-asicum, G. canescens, K. prorepens and K. prostrata)had a significantly higher carboxylate in therhizosphere than the exotic species at 6 μg P g−1

soil (Fig. 5a, Table 2), while all species, exceptL. corniculatus and M. bracteatum, had similarrhizosphere carboxylate amounts at 48 μg P g−1 soil(Fig. 5a). Although carboxylate composition variedamong species, all species predominantly released acombination of malic, malonic and citric acid, exceptK. prostrata, which also had a large fraction of aceticacid (Fig. 5b and c). All species, except C. austral-asicum, K. prostrata and L. bainesii, showed asignificant shift in exudate composition between6 μg P g−1 soil and 48 μg P g−1 soil. Six out of the

Spe

cific

roo

t len

gth

(m g

-1D

W)

Soil P application (µg g-1)

L. australis

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840

80

160

240

320C. australasicum

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840

80

160

240

320M. sativa

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840

80

160

240

320

L. bainesii

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840

200

400

600K. prorepens

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840

200

400

600 K. prostrata

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840

80

160

240

320

G. canescens

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840

80

160

240

320C. tenax

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840

80

160

240

320

B. bituminosa

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 19 8448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840

80

160

240

320

L. corniculatus

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840

80

160

240

320M. bracteatum

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840

80

160

240

320Spe

cific

roo

t len

gth

(m g

-1D

W)

Soil P application (µg g-1)

L. australis

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840

80

160

240

320C.

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840

80

160

240

320M. sativa

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 . 8448 96 19 8448 96 19 8448 96 19 8448 96 19 8448 96 19 8448 96 19 8448 96 19 8448 96 19 8448 96 19 840

80

160

240

320

L. bainesii

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840

200

400

600

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840

200

400

600

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840

80

160

240

320

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840

80

160

240

320C. tenax

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840

80

160

240

320

B.

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 19 8448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840

80

160

240

320

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840

80

160

240

320

0 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 19 8448 96 19 8448 96 19 8448 96 19 8448 96 19 8448 96 19 8448 96 19 8448 96 19 8448 96 19 8448 96 19 840

80

160

240

320

Fig. 3 Specific root length of 11 perennial legumes grown for8 weeks in sterilised washed river sand supplied with eightlevels of P ranging from 0 to 384 μg P g−1 dry soil. Data aremeans±s.e. (n=6). The first arrow from the left in each graphindicates the soil P application level where maximum plantgrowth occurred; plants were P-deficient when grown belowthis level. The second arrow indicates the soil P application

level where plant growth suffered from P-toxicity and reducedgrowth. Lack of second arrow in some species indicates no Ptoxicity. Note different y-axis scale for L. bainesii and K.prorepens. There was a significant interaction between soil Papplication level and species (p<0.001, LSD0.05=70.6 m g−1

DW). Note x-axis break

Plant Soil (2010) 331:241–255 247

11 species (L. australis, G. canescens, M. sativa, B.bituminosa, C. tenax and L. corniculatus) increasedfraction of rhizosphere citrate in response to low P.This was accompanied by a reduction of malonate,except in L. corniculatus, and a reduction of malate inL. australis, M. sativa and L. corniculatus. K. prorepensand M. bracteatum reduced citrate and increasedmalonate in response to low P (Fig. 5b and c). Othercarboxylic acids (lactic, maleic, cis-aconitic, succinic,fumaric and trans-aconitic acids) accounted for a totalof only 3.1% or less of total carboxylates in therhizosphere (Fig. 5b and c).

The pH of the rhizosphere solutions extracted with0.2 mM CaCl2 varied significantly among species andbetween two levels of soil P application (6 μg P g−1

and 48 μg P g−1) (Fig. 6). The soil of control pots(without plants) had a pH of 6.3. All 11 perenniallegume species acidified rhizosphere soil (Fig. 6). At

6 μg P g−1 soil, the rhizosphere pH was significantlydecreased compared with that at 48 μg P g−1 in L.australis, K. prostrata, K. prorepens and L. bainesii,while rhizosphere pH increased in M. sativa and C.tenax. There was no significant difference between 6and 48 μg P g−1 in C. australasicum, G. canescens, B.bituminosa, L. corniculatus and M. bracteatum.

P uptake efficiency and physiological P use efficiencyat 6 μg P g−1 soil

K. prorepens, C. australasicum, L. corniculatus andG. canescens had the highest P uptake efficiencyamong all species, followed by B. bituminosa andM. sativa (Table 2). The highest physiological P useefficiency among all species studied occurred inG. canescens and B. bituminosa, followed by M.sativa and L. corniculatus (Table 2). Some species

L. australis

6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840.2

0.4

0.6C. australasicum

6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840.2

0.4

0.6M. sativa

6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 19248 96 19248 96 19248 96 19248 96 19248 96 19248 96 19248 96 19248 96 19248 96 19248 96 1920.2

0.4

0.6

L. bainesii

6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840.2

0.4

0.6K. prorepens

6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840.2

0.4

0.6 K. prostrata

6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840.2

0.4

0.6

G. canescens

6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840.2

0.4

0.6C. tenax

6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840.2

0.4

0.6 L. corniculatus

6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840.2

0.4

0.6M. bracteatum

6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 19248 96 1948 96 1948 96 1948 96 1948 96 1948 96 1948 96 1948 96 1948 96 1948 96 190.2

0.4

0.6

B. bituminosa

6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 1948 96 1948 96 1948 96 1948 96 1948 96 1948 96 1948 96 1948 96 1948 96 1948 96 190.2

0.4

0.6

Soil P application (µg g-1)

L. australis

6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840.2

0.4

0.6C. australasicum

6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840.2

0.4

0.6M. sativa

6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 19248 96 19248 96 19248 96 19248 96 19248 96 19248 96 19248 96 19248 96 19248 96 19248 96 1920.2

0.4

0.6

L. bainesii

6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840.2

0.4

0.6K. prorepens

6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840.2

0.4

0.6 K. prostrata

6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840.2

0.4

0.6

G. canescens

6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840.2

0.4

0.6C. tenax

6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840.2

0.4

0.6 L. corniculatus

6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 38448 96 192 3840.2

0.4

0.6M. bracteatum

6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 19248 96 1948 96 1948 96 1948 96 1948 96 1948 96 1948 96 1948 96 1948 96 1948 96 190.2

0.4

0.6

B. bituminosa

6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 240 6 12 24 48 96 1948 96 1948 96 1948 96 1948 96 1948 96 1948 96 1948 96 1948 96 1948 96 1948 96 190.2

0.4

0.6

Mea

n ro

ot d

iam

eter

(m

m)

Soil P application (µg g-1)

384

384

384

2

Fig. 4 Mean root diameter of 11 perennial legumes grown for8 weeks in sterilised washed river sand supplied with eightlevels of P ranging from 0 to 384 μg P g−1 dry soil. Data aremeans±s.e. (n=6). The first arrow from the left in each graphindicates the soil P application level where maximum plantgrowth occurred; plants were P-deficient when grown below

this level. The second arrow indicates the soil P applicationlevel where plant growth suffered from P-toxicity and reducedgrowth. Lack of second arrow in some species indicates no Ptoxicity. There was a significant interaction between soil Papplication level and species (p<0.001, LSD0.05 =0.07 mm).Note x-axis break

248 Plant Soil (2010) 331:241–255

grown at 6 μg P g−1 dry soil (L. australis, L. bainesii,M. bracteatum, C. tenax and K. prostrata) showedboth low physiological P use efficiency and low Puptake efficiency (Table 2).

Discussion

This study compared morphological and physiologi-cal responses to P application among 11 perenniallegumes (six native and five exotic to Australia). A

large variation was found among species in responseto P. The implications of these results and other majorresults are now discussed.

Mechanisms accounting for a high productivity underlow-P conditions

To efficiently acquire P from soil and/or use Pefficiently for biomass production are importantcharacteristics for plants adapted to low-P soils. Whenplants were grown at 6 μg P g−1 dry soil, dry mass

malic malonic citric acetic other

0.0

0.2

0.4

0.6

0.8

1.0

LA CA GC KPA KPS MS LB BB CT LC MB

0.0

0.2

0.4

0.6

0.8

1.0

LA CA GC KPA KPS MS LB BB CT LC MB

Car

boxy

late

s in

the

rhiz

osph

ere

(µm

ol g

-1ro

ot D

W)

Fra

ctio

n of

tota

l

a

0

40

80

120

160

LA CA GC KPA KPS MS LB BB CT LC MB

6 P 48 P

malic malonic citric acetic other

0.0

0.2

0.4

0.6

0.8

1.0

LA CA GC KPA KPS MS LB BB CT LC MB

0.0

0.2

0.4

0.6

0.8

1.0

LA CA GC KPA KPS MS LB BB CT LC MB

Car

boxy

late

s in

the

rhiz

osph

ere

(µm

ol g

-1ro

ot D

W)

Fra

ctio

n of

tota

l

b

c

0

40

80

120

160

LA CA GC KPA KPS MS LB BB CT LC MB

6 P 48 PFig. 5 a Carboxylate recov-ered in rhizosphere extractsof 11 perennial legumesgrown for 8 weeks in steri-lised washed river sandsupplied with 6 or 48 μgP g−1 dry soil. Data aremeans±s.e. (n=6). Therewas a significant interactionbetween soil P applicationlevel and species (p<0.001,LSD0.05 =14.4 μmol g−1

root DW). b and c Fractionin rhizosphere extracts ofmalic, malonic, citric andother acids (including com-binations of trace amountsof maleate, cis-aconitate,fumarate, lactate, succinateand trans-aconitate) for 11perennial legumes appliedwith 6 (b) or 48 μg (c) P g−1

dry soil. Data are means(n=6). There was a signifi-cant interaction between soilP application level and spe-cies for all above parameters(p<0.001, LSD0.05 =0.08,0.10 and 0.10 for malic,malonic and citric acid, re-spectively). Species abbre-viations were used infigures: CA (C. australasi-cum), LA (L. australis),KPA (K. prostrata), GC(G. canescens), MS (M.sativa), KPS (K. prorepens),LB (L. bainesii), BB(B. bituminosa), CT(C. tenax), LC(L. corniculatus), MB(M. bracteatum)

Plant Soil (2010) 331:241–255 249

accumulation differed greatly among species (Table 2).As dry mass produced increased among species, sotoo did physiological P-use efficiency (Table 2). Thisreflects that the greater dry mass that a speciesproduced, the more the P taken up from the soil wasdiluted in the dry mass. The highest physiological P-

use efficiency among all species studied occurred inG. canescens and B. bituminosa and was related totheir high dry mass (Table 2). Although B. bituminosahad slightly lower P-uptake efficiency than G.canescens, it showed slightly higher dry massproduction, possibly due to its much higher physio-logical P-use efficiency (Table 2). Some speciesgrown at 6 μg P g−1 dry soil (L. australis, L. bainesii,M. bracteatum, C. tenax and K. prostrata) showedboth low physiological P-use efficiency and low P-uptake efficiency; this might be related to lowaccumulation of dry mass in these species (Table 2).

Factors investigated in this study contributing to P-uptake efficiency included root surface area, total rootlength, root diameter, SRL, root mass ratio andcarboxylate recovered in the rhizosphere (Table 2).For all species considered together at 6 μg P g−1 therewas a reasonably strong correlation between P uptakeand root surface area (r2=0.56, P<0.05), P uptakeand total root length (r2=0.62, P<0.05). Root surfacearea (RSA) and root length are known to play veryimportant roles in the exploitation of soil P (Vance etal. 2003). As plant species may use very differentstrategies to take up P from low P soils (Lambers etal. 2006), it was not surprising that there was poorcorrelation between P uptake and the other parametersstudied. A higher SRL may have contributed to

Table 2 Morphological and physiological characteristics that may have contributed to plant growth when legumes were grown at6 µg P g−1 soil, and P uptake efficiency. Data are means (n=3)

Species Dry weightat 6 μg P g−1

dry soil (g)a

Dry weightat criticalsoil P level(g)a

P uptakeefficiency (g Pin plants g−1

applied P)

Rootsurfacearea(cm2)

Totalrootlength(m)

Specific rootlength(m g−1 rootDW)

Rootmassratio(RMR)

Carboxylaterecovered in therhizosphre(µmol g−1

root DW)

Physiological Puse efficiency(g2 DW g−1 Pin plant)

L. australis 0.17 0.54 0.04 38 2.9 171 0.25 136 90

L. bainesii 0.18 0.87 0.09 22 2.2 128 0.17 38 50

M. bracteatum 0.23 1.18 0.07 42 3.9 99 0.31 15 100

C. tenax 0.29 1.26 0.11 129 12.0 187 0.30 28 110

K. prostrata 0.31 0.57 0.10 157 10.6 242 0.33 52 110

K. prorepens 0.55 1.41 0.22 161 18.6 304 0.20 49 190

C. australasicum 0.58 1.59 0.22 243 14.8 172 0.24 116 200

L. corniculatus 0.62 1.53 0.20 276 21.6 185 0.30 25 270

M. sativa 0.65 4.38 0.14 250 20.9 158 0.32 38 390

G. canescens 0.86 1.74 0.20 295 23.4 197 0.19 73 490

B. bituminosa 1.00 1.97 0.17 384 28.6 122 0.39 32 710

LSD0.05 among species 0.28 0.51 0.08 112 11.7 99 0.11 19 93

a Dry weight data sampled from growth data (Pang et al. 2009)

Roo

t rhi

zosp

here

pH

4.5

5.0

5.5

6.0

6.5

LA CA GC KPA KPS MS LB BB CT LC MB

6 48

Roo

t rhi

zosp

here

pH

4.5

5.0

5.5

6.0

6.5

LA CA GC KPA KPS LB BB CT LC

6 48

Fig. 6 pH of 0.02 mM CaCl2 extracts for the rhizosphere of 11perennial legumes grown for 8 weeks in sterilised washed riversand supplied with 6 or 48 μg P g−1 dry soil. Dashed line indicatespH of the river sand used to grow plants. Data are means±s.e. (n=6). There was a significant interaction between soil P applicationlevel and species (p<0.001, LSD0.05 =0.3). Species abbreviationswere used in figures: CA (C. australasicum), LA (L. australis),KPA (K. prostrata), GC (G. canescens), MS (M. sativa), KPS(K. prorepens), LB (L. bainesii), BB (B. bituminosa), CT(C. tenax), LC (L. corniculatus), MB (M. bracteatum)

250 Plant Soil (2010) 331:241–255

relatively high P-uptake efficiency in K. prorepens,L. corniculatus and G. canescens, while relativelylow P-uptake efficiency in L. bainesii and M.bracteatum might be partly due to their lower SRL(Table 2). A higher root mass ratio may also havecontributed to a high P-uptake efficiency in somespecies. For example, although SRL and rhizospherecarboxylate were lower in B. bituminosa than in M.sativa, a higher root mass ratio may have increased P-uptake efficiency to an intermediate level (Table 2);that is, the higher total root length for B. bituminosa at6 μg P g−1 than for M. sativa could be a contributingfactor (Fig. 2). The higher carboxylate recovery fromthe rhizosphere relative to root dry mass might havepartly contributed to a relatively high P-uptakeefficiency in K. prorepens, C. australasicum and G.canescens (Table 2). Conversely, a relatively lower P-uptake efficiency in M. bracteatum and C. tenaxcorresponded with lower rhizosphere carboxylates.

L. australis, C. tenax and K. prostrata exhibitedpoor P-uptake efficiency. However, the carboxylaterecovery from the rhizosphere of L. australis wasrelatively high. Other factors, e.g., root-hair lengthand frequency and mycorrhizal colonisation, whichwe did not study here, might also contribute greater toP-uptake efficiency (Lambers et al. 2006). Furtherwork is required to clarify these alternative factorscontributing to P-uptake efficiency.

P concentrations in roots

There was little difference in root P concentrationamong species at low levels of soil P application,while root P concentration increased with increasedsoil P application in most species, and reached veryhigh values at the highest soil P application (Fig. 1),accompanied by high shoot P concentration (Pang etal. 2009). Four native legumes (K. prorepens,K. prostrata, G. canescens, L. australis) exhibitedsignificantly higher root P concentrations than theother species at the highest P application (Fig. 1),while M. sativa showed the lowest root P concentra-tion, which might be partly explained by its muchlarger dry mass at high soil P application levels thanthe other species. Such high root P concentrationshave seldom been reported. Many Australian nativeplants have evolved in severely P-impoverishedenvironments (Handreck 1997) and efficient P-acquisition mechanisms appear to be poorly regulated

at high P availability in some of these species(Lambers et al. 2008b; Ryan et al. 2009; Shane etal. 2004). The ability to accumulate high concen-trations of P in roots of perennial legumes in thepresent study and in shoots (Pang et al. 2009) mightbe related to a low capacity to down-regulate P uptakeas found in Hakea prostrata (Shane et al. 2004).

P toxicity has been reported in Australian Protea-ceae and in crop plants, e.g., T. aestivum, Hordeumvulgare, S. lycopersicum (summarized in Table 2, inShane et al. 2004). Roots of H. prostrata are animportant storage organ for P, and P toxicity occurswhen the availability of P exceeds the capacity of roottissues to store it (Shane et al. 2004). In the presentstudy, root P toxicity resulting from high root Pconcentration was evidenced by reduced shoot androot dry mass (Pang et al. 2009), especially inK. prorepens and K. prostrata. Future work tounderstand P storage and seasonal P dynamics inperennial legumes is required.

Morphological adaptations in response to soil Papplication levels

When compared with plants supplied with P, almostall species increased their SRL in response to nil-Psupply (Fig. 3), confirming results of previous studies(Hill et al. 2006; Li et al. 2007). The response of SRLto the increase in soil P application varied greatlyamong species. Interestingly, all native legume spe-cies, except C. tenax, had a higher average SRLacross all levels of soil P application than the exoticspecies, except L. bainesii. Plants with a high SRLmay have an advantage over plants with a low SRL,because they have a lower resource expenditure toproduce root length (Eissenstat 1992). Relativelylarge increases in SRL occurred in temperate pasturespecies adjusting to low P as a consequence ofdecreasing root diameter and root mass density (rootmass / root volume) (Hill et al. 2006).

In our study, reduced mean root diameter wasobserved in response to low-P stress in most speciesand is a likely explanation for the increase in SRL,except for the exotic M. bracteatum (Fig. 4). Speciesdifferences in root diameter were large in response tolow-P stress (Fig. 4). When no soil P was applied, L.bainesii had the finest roots among all species, with aroot diameter of 0.26 mm. L. bainesii is native tosouthern Africa and occurs on gravelly and sandy

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soils, where the fine roots might have evolved as anadaptation to the local infertile soil conditions.However, there was little variation in root diameterin M. bracteatum in response to different soil Papplication levels. Indeed, a decrease in root diameteris by no means a universal response to a low P supply(Borch et al. 1999; Schroeder and Janos 2005). Zobelet al. (2006) also reported that less P did notnecessarily result in thinner, longer roots in threechicory (Cichorium intybus) cultivars.

Although there was little response of root diameterto soil P application level, an increase in SRL inresponse to low-P stress was observed in M. bractea-tum (Fig. 3). This indicates that factors in addition toroot diameter contributed to increase SRL. Indeed,Hill et al. (2006) found that changes in root massdensity were as important as changes in root diameterfor three pasture species responding to low-nutrientconditions. Zobel et al. (2006) also found that thedensity of the non-taproot portion of the root systemsof two cultivars of C. intybus decreased in the no-Ptreatment, while that of a third cultivar did notchange. Fan et al. (2003) attributed a decrease in rootmass density of roots grown under low-nutrientconditions to an increased production of aerenchyma.A decrease in root density at low nutrient levels hasalso been associated with thinner cell walls in thestele or a decrease in the stele to cortex diameter ratio,either of which could translate into more rapid radialtranslocation of nutrient and nutrient uptake (Wahland Ryser 2000).

Mean root diameter increased in response to Papplication, compared with no P, in most species, andeach species showed variation in mean root diameterin response to high soil P application. In some of thespecies a reduction in mean root diameter frommaximum values at intermediate P supply occurredwhen the soil P application level was high. InK. prorepens, where plants were suffered fromextreme P toxicity (Pang et al. 2009), it evendecreased to the value close to that when no P wasapplied. This is contrary to the findings of Crush et al.(2008), who reported a continuous increase in rootdiameter with increasing P application up to 1200 μgP g−1 dry soil in the perennial legume white clover(Trifolium repens). The reduction in mean rootdiameter at high P supply at least partly explainedthe increased SRL in some species, as there was acorrelation between mean root diameter and SRL

when all species and all soil P application levelswere considered together (r2=0.31, P<0.001). Sim-ilarly, in K. prorepens when soil P application levelwas high, SRL declined to a value close to that whenno P was added (Fig. 3).

Physiological adaptations to soil P application levels

All legumes studied had higher carboxylate in therhizosphere under a low level of P application (6 μg Pg−1 soil) than at a high level of P application (48 μg Pg−1 soil). Increased exudation of organic acids hasbeen observed for roots of many plants under P-limiting conditions, including L. albus and Cicerarietinum (Neumann and Römheld 1999). Increasedactivity of phosphoenolpyruvate carboxylase (PEPC)in response to P-starvation also occurs in many plantspecies, e.g., Solanum lycopersicum, L. albus, Pisumsativum, B. napus and B. nigra (Raghothama 1999).PEPC is a key enzyme facilitating synthesis andexudation of carboxylates (Ryan et al. 2001). Carbox-ylates can be a major component of exudates releasedby roots, especially under P deficiency. Carboxylatesare important in phosphate mobilisation when P isbound to Al, Fe or Ca (Lambers et al. 2008b;Lambers et al. 2006). Carboxylates mobilise bothinorganic and organic P because they complex metalcations that bind phosphate and displace the phos-phate from the soil matrix by ligand exchange(Lambers et al. 2006).

In all native legumes at 6 µg P g−1 dry soil, exceptC. tenax, carboxylate recovered in the rhizospherewas higher than in all exotic species (Fig. 5). This ismost likely related to these species’ evolution inseverely P-impoverished environments (Lambers etal. 2008b). In all of the legumes examined lesscarboxylate was recovered from the rhizosphere at48 µg P g−1 dry soil and the shift in carboxylaterecovery between low and high soil P applicationlevel did not discriminate native legumes from exoticspecies (Fig. 5a). In other species e.g. C. arietinum asimilar pattern of release has been found to beconstitutive (Wouterlood et al. 2005).

In the present study, carboxylate recovered in therhizosphere ranged between 14.7 μmol g−1 root dryweight in M. bracteatum and 136 μmol g−1 inL. australis; almost a 9-fold difference. A high degreeof variation among species in carboxylate in therhizosphere has been reported in many studies under

252 Plant Soil (2010) 331:241–255

different growth medium and duration of experiment.For instance, three chickpea (C. arietinum) cultivarshad carboxylate of approximately 50–200 μmol g−1

root dry weight in the rhizosphere (Veneklaas et al.2003). In response to five different P sources,T. aestivum showed the lowest carboxylate in itsrhizosphere, with only 13 μmol g−1 root dry weight,while L. cosentinii accumulated 253 μmol g−1 rootdry weight (Pearse et al. 2007).

Plants of all species produced a predominantcombination of malic, malonic and citric acid,although carboxylate composition varied amongspecies (Fig. 5b and c). All of the carboxylatesidentified in the present study have previously beenfound in root exudates of other plant species. Malateand citrate are common compounds in exudates fromAustralian Proteaceae (Lambers et al. 2002) and cropplants such as canola (Hoffland et al. 1992), wheat,tomato, chickpea and white lupin (Neumann andRömheld 1999). A large amount of malonic acid waspreviously reported in the root tissue and in rootexudates of chickpea (Neumann and Römheld 1999;Pearse et al. 2007) and pigeon pea (Otani et al. 1996).However, malonic acid is not commonly reported inother species. In the present study, lactate (~20%) wasfound exclusively in K. prostrata (Fig. 5b and c). Alarge fraction of lactate has been reported for otherAustralian native plants (Lambers et al. 2002; Roelofset al. 2001). There was a large shift in exudatecomposition in response to application of 6 μg P g−1

soil compared with that when 48 μg P g−1 soil wasapplied in all species except C. australasicum,K. prostrata and L. bainesii (Fig. 5b and c). Six ofthe 11 species (L. australis, G. canescens, M. sativa,B. bituminosa, C. tenax and L. corniculatus) increasedfraction of citrate in response to low P, accompaniedby a reduction in malonate except in L. corniculatus,and a reduction in malate in L. australis, M. sativaand L. corniculatus (Fig. 5b and c). Citrate is the onlytri-carboxylic acid among those presented and there-fore it is likely to be more effective in mobilising P.However, K. prorepens and M. bracteatum reducedcitrate and increased malonate when P supply waslower (Fig. 5b and c).

In our study, rhizosphere pH varied among species.Rhizosphere pH was poorly correlated with rhizo-sphere carboxylates (r2=0.11). Organic acids haveoften been referred to as a possible source ofrhizosphere acidification (Hinsinger et al. 2003).

However, the effects of carboxylate efflux on pH arecomplex and carboxylate exudation is not invariablyassociated with acidification (Roelofs et al. 2001).Carboxylate anions might act as a buffer and canaffect pH independently of proton efflux from plantroots. It was suggested that to reduce rhizosphere pHwould not be an efficient way for perennial legumesto access soil P since a soil pH below 6 inhibitsacquisition of soil P by roots (Lambers et al. 2008a).Indeed pH in our study was always below 6. It isfound that a major source of H+ fluxes into therhizosphere is the differential uptake of cations andanions by plant roots (Braschkat and Randall 2004).Legume plants obtain most of their nitrogen throughN2 fixation and take up more cations than anions,thus extrude H+ from their roots and acidifyrhizosphere (Tang et al. 1998). Whether acidificationof the rhizosphere in some species in the presentstudy is related to an imbalanced uptake of cationsand anions by plant roots under low-P stress needs tobe clarified, but large excess cation concentrations(total cations — total inorganic anions) have, indeed,been reported for perennial pasture legumes (Braschkatand Randall 2004). In our study, all legume specieswere inoculated and the contribution of N2 fixation torhizosphere acidification was not identified and needsfurther clarification.

Conclusions

The higher physiological P-use efficiency in G.canescens and B. bituminosa was related to theirhigh dry mass when grown at 6 μg P g−1.Considering all species together at 6 μg P g−1, therewas a strong correlation between P uptake and rootsurface area (r2=0.56, P<0.05), and P-uptake andtotal root length (r2=0.62, P<0.05). Six out of 11species increased the fraction of rhizosphere citratein response to low P, accompanied with the reductionof malonate, except for L. corniculatus. Plant speciesexhibited variation in other responses to P deficien-cy. A higher SRL may contribute to relatively highP-uptake efficiency in K. prorepens, L. corniculatusand G. canescens. A higher carboxylate recoveryfrom the rhizosphere relative to root dry mass mightpartly contribute to a relatively high P-uptakeefficiency in K. prorepens, C. australasicum and G.canescens.

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Acknowledgements This research was supported by theAustralian Research Council (ARC), the Department ofAgricultural and Food Western Australia, Heritage Seeds,the Chemistry Centre of Western Australia, and the FaceyGroup and Mingenew Irvin Group. We thank Dr DanielReal, Mr Richard Snowball and Dr Ron Yates (DAFWA),Mr Steve Hughes (SARDI), and Mr Leonard Song (HeritageSeeds) for providing legume seeds and rhizobia, Dr ClintonRevell and Mr Richard Bennett for technical advice, and MsTammy Edmonds-Tibbett and Mr Tim Morald for technicalhelp.

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