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REVIEW ARTICLE
Acquisition of phosphorus and nitrogenin the rhizosphere and
plant growth promotionby microorganisms
Alan E. Richardson & Jos-Miguel Barea &Ann M. McNeill
& Claire Prigent-Combaret
Received: 24 May 2008 /Accepted: 5 January 2009 /Published
online: 27 February 2009# Springer Science + Business Media B.V.
2009
Abstract The rhizosphere is a complex environmentwhere roots
interact with physical, chemical andbiological properties of soil.
Structural and functionalcharacteristics of roots contribute to
rhizosphereprocesses and both have significant influence on the
capacity of roots to acquire nutrients. Roots alsointeract
extensively with soil microorganisms whichfurther impact on plant
nutrition either directly, byinfluencing nutrient availability and
uptake, or indi-rectly through plant (root) growth promotion. In
thispaper, features of the rhizosphere that are importantfor
nutrient acquisition from soil are reviewed, withspecific emphasis
on the characteristics of roots thatinfluence the availability and
uptake of phosphorusand nitrogen. The interaction of roots with
soilmicroorganisms, in particular with mycorrhizal fungiand
non-symbiotic plant growth promoting rhizobac-teria, is also
considered in relation to nutrientavailability and through the
mechanisms that areassociated with plant growth promotion.
Keywords Soil microorganisms . PGPR .
Mycorrhizal fungi . Exudate . Phosphorus . Nitrogen .
Uptake .Mineralization
Introduction
The rhizosphere can be defined as the zone of soilaround plant
roots whereby soil properties areinfluenced by the presence and
activity of the root.Changes to the physical, chemical and
biologicalproperties of rhizosphere soil has significant
influenceon the subsequent growth and health of plants. Interms of
nutrient acquisition, both the structural and
Plant Soil (2009) 321:305339DOI 10.1007/s11104-009-9895-2
Responsible Editor: Philippe Hinsinger.
A. E. Richardson (*)CSIRO Plant Industry,PO Box 1600, Canberra
2601, Australiae-mail: [email protected]
J.-M. BareaDepartamento de Microbiologa del Suelo y
SistemasSimbiticos, Estacin Experimental del Zaidn, CSIC,Prof.
Albareda 1,Granada 18008, Spain
A. M. McNeillUniversity of Adelaide, Soil and Land Systems,Waite
Campus,Adelaide 5005, Australia
C. Prigent-CombaretUniversit de Lyon,Lyon, France
C. Prigent-CombaretUniversit Lyon 1,Villeurbanne, France
C. Prigent-CombaretCNRS, UMR 5557, Ecologie
Microbienne,Villeurbanne, France
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functional characteristics of roots have long beenrecognized as
being important in determining thecapacity for plants to access and
mediate theavailability of essential nutrients in soil and
toalleviate against those that are toxic (Darrah 1993;Hinsinger
1998; Marschner 1995). Furthermore, rootsinteract with diverse
populations of soil microorgan-isms which have significant
implication for growthand nutrition (Curl and Truelove 1986; Bowen
andRovira 1999; Mukerji et al. 2006; Brimecombe et al.2007). Soil
nutrients are transferred towards the rootsurface through the
rhizosphere or, in the case of rootsassociated with mycorrhizal
fungi, through themycorrhizosphere, prior to acquisition.
Plants modify the physico-chemical properties andbiological
composition of the rhizosphere through arange of mechanisms, which
include acidificationthrough proton extrusion and the release of
rootexudates. Along with changes to soil pH, root exudatesdirectly
influence nutrient availability or have indirecteffects through
interaction with soil microorganisms.An outstanding feature of the
rhizosphere is thatrhizodeposition and root turnover account for up
to40% of the carbon input into soil and clearly is the majordriver
for soil microbiological processes (Grayston et al.1996; Jones et
al. 2009). Interactions between plantroots and soil microorganisms
are ubiquitous acrossvarious trophic levels and are an essential
componentof ecosystem function. It has become increasinglyevident
that root interactions with soil microorganismsare intricate and
involve highly complex communitiesthat function in very
heterogeneous environments(Giri et al. 2005). Microbial
interactions with rootsmay involve either endophytic or free living
micro-organisms and can be symbiotic, associative or casualin
nature. Beneficial symbionts include N2-fixingbacteria (e.g.
rhizobia) in association with legumesand interaction of roots with
mycorrhizal fungi, withthe later being particularly important in
relation to plantP uptake. Associative and free-living
microorganismsmay also contribute to the nutrition of plants
through avariety of mechanisms including direct effects onnutrient
availability (e.g. N2-fixation by diazotrophsand P-mobilization by
many microorganisms), en-hancement of root growth (i.e. through
plant growthpromoting rhizobacteria, or PGPR), as antagonists
ofroot pathogens (Raaijmakers et al. 2009) or assaphrophytes that
decompose soil detritus and subse-quently increase nutrient
availability through mineral-
ization and microbial turnover. Such processes arelikely to be
of greater significance for nutrientavailability in the rhizosphere
where there is increasedsupply of readily metabolizable carbon and
wheremobilized nutrients can be more easily captured byroots.
In this review we address the acquisition of nutrientsfrom soil
by plants with specific emphasis on thestructural and functional
characteristics of roots thatinfluence the availability and uptake
of P and N. Inparticular, the importance of soil microorganisms
andtheir interactions with roots in relation to
nutrientavailability is considered, along with their
associatedmechanisms of plant growth promotion. The
reviewcomplements previous reviews that have specificallyfocused on
either plant-based traits or mechanisticprocesses associated with P
and N uptake (Raghothama1999; Vance et al. 2003; Bucher 2007,
Miller andCramer 2004; Jackson et al. 2008). Although
therhizosphere is important for the efficient uptake of awider
range of macro and micronutrients (including Fe;Lemanceau et al.
2009), the review specifically focuseson N and P which are key
nutrients that limitsustainable agricultural production across much
of theglobe (Tilman et al. 2002).
Mechanisms of nutrient acquisition by plant roots
Efficient capture of nutrients from soil by roots is acritical
issue for plants given that in many environmentsnutrients have poor
availability and may be deficient foroptimal growth. Whilst
nutrient supply in soil is oftenaugmented by the application of
fertilizers, the avail-ability of nutrients is governed by a wide
range ofphysico-chemical parameters, environmental and sea-sonal
factors and biological interactions. Competitionfor nutrient uptake
across different plant species,between different roots and with
microorganisms is alsosignificant (Hodge 2004). The rate of root
growth andthe plasticity of root architecture along with
develop-ment of the rhizosphere, through either root growth
orextension of root hairs, are clearly important foreffective
exploration of soil and interception ofnutrients (Lynch 1995).
Biochemical changes in therhizosphere and interaction with
microorganisms arealso significant. However, the importance of
differentroot traits and rhizosphere-mediated processes isdependent
on the nutrient in question and other factors
306 Plant Soil (2009) 321:305339
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that include plant species and soil type (Tinker andNye 2000).
For example, for nutrients present at lowconcentrations in soil
solution and/or with poordiffusivity (e.g. P as either HPO4
2 or H2PO4, and
micronutrients, such as Fe and Zn), root growth andproliferation
into new regions of soil and release ofroot exudates are of
particular importance (Barber1995; Darrah 1993). In contrast,
nutrients present ineither higher concentrations (e.g. K+, NH4
+), or withgreater diffusion coefficients (e.g. NO3
, SO4 and
Ca2+), are able to move more freely toward the rootthrough mass
flow, where root distribution andarchitectural characteristics that
facilitate water uptakeare of greater relative significance (Barber
1995;Tinker and Nye, 2000; Lynch 2005). The relativesignificance of
such factors in the acquisition anduptake of P and N is therefore
considered in moredetail below.
Acquisition of phosphorus by plants
Phosphorus availability and uptake
Although soils generally contain a large amount of totalP only a
small proportion is immediately available forplant uptake. Plants
obtain P as orthophosphate anions(predominantly as HPO4
2 and H2PO41) from the soil
solution. In most soils the concentration of orthophos-
phate in solution is low (typically 1 to 5 M; Bieleski1973) and
must therefore be replenished from otherpools of soil P to satisfy
plant requirements. Ortho-phosphate is rapidly depleted in the
immediate vicinityof plant roots, and as such a large
concentrationgradient occurs across the rhizosphere between
bulksoil and the root surface (Gahoonia and Nielsen 1997;Tinker and
Nye 2000; Fig. 1). However, for most soilsthe rate of diffusion of
orthophosphate is insufficient toovercome localized gradients,
which in most caseslimits the uptake of sufficient P. Evidence from
bothmodelling and empirical studies also suggests thatactual P
uptake capacity at the root surface is unlikelyto be limiting for
plant growth (Barber 1995). This issupported by more recent studies
on the expression ofgenes that encode for transport proteins with
highaffinity for uptake of orthophosphate (e.g. Km ~3 M),which are
predominantly expressed in root hair cells onthe epidermis
(Mitsukawa et al. 1997; Mudge et al.2002; Schnmann et al, 2004).
Whilst expression ofthese genes has been shown to facilitate the P
uptakecapacity of cells in suspension culture (Mitsukawa etal.
1997), their over-expression in transgenic plants didnot result in
increased P uptake by barley (Hordeumvulgare L.) when grown at a
range of P concentrationsin either solution or soil (Rae et al.
2004). This isconsistent with the view that plants are well
adaptedfor uptake of P from the low concentrations that are
Root
Root exudates
Sugars
Organic anions
H+ ions
Siderophores
Phosphatases
10 mM
Desorption
Solubilization
Solubilization
Mineralization
ADSORBED P (Pi)
ORGANIC P (Po)
SOIL SOLUTION P(Pi and Po)
MINERAL P (Pi)
Root hairs
Rhizosphere Soil
~0 M1 M Diffusion
Rhizospheremicroorganisms
Organic anions
H+ ions
Siderophores
Phosphatases
Mycorrhizal fungi
Mycorrhizosphere
P uptake
Fig. 1 Physiological andchemical processes that in-fluence the
availability andtransformation of phospho-rus in the
rhizosphere(adapted from Richardson1994; Richardson 2001)
Plant Soil (2009) 321:305339 307
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typical of soil solutions as indicated by minimumuptake
concentrations (Clim values) of 0.01 to 0.1 Mfor different species
(Jungk 2001). On this basis it issuggested that the supply of P to
the root surface, andits availability as influenced by root and
microbialprocesses (as outlined below), or the capacity of rootsto
exploit new regions of soil are of greater importancefor P
acquisition than the kinetics associated with itsuptake.
The importance of root growth and architecture forthe efficient
capture of P is well documented and inmany cases is a specific
response of plants to Pdeficiency (reviewed by Hodge 2004; Lynch
2005;Raghothama 1999; Richardson et al. 2009; Vance et al.2003).
Characteristics of roots that facilitate soilexploration and hence
P uptake include; rapid rate ofroot elongation and high root to
shoot biomass ratio(Hill et al. 2006), increased root branching and
rootangle particularly in surface soils and nutrient richregions
(Lynch and Brown 2001; Manske et al. 2000;Rubio et al. 2003), high
specific root length (i.e. lengthper unit mass or root fineness;
Silberbush and Barber,1983), the presence of root hairs (Fhse et
al. 1991;Gahoonia and Nielsen 1997; Gahoonia and Nielsen2003) and,
in some species, the formation of special-ized root structures such
as aerenchyma (Fan et al.2003), dauciform roots in the Cyperaceae
(Shane et al.2006) and proteoid roots (or cluster roots) in
theProteaceae and certain Lupinus spp. (Dinkelaker et al.1995;
Gardner et al. 1981; Roelof et al. 2001; Lamberset al. 2006).
Depletion of phosphorus in the rhizosphere
The extent of P depletion in both the rhizosphere
andmycorrhizosphere has been highlighted in a number ofstudies.
Root hairs in particular contribute to increasedroot volume and can
constitute up to 70% of the totalroot surface area (Itoh and Barber
1983; Jungk 2001;Fig. 1). As such they are the major site for
nutrientacquisition and can account for up to 80% of total Puptake
in non-mycorrhizal plants (Fhse et al. 1991).Variation in length
and density of root hairs isimportant particularly under conditions
of low P andtheir contribution to P uptake has been verified
throughmodelling studies and the use of root-hairless
mutants(Gahoonia and Nielsen 2003; Ma et al. 2001).Mycorrhizal
colonization of roots (as discussed below)similarly provides a
significant increase in the effective
volume of soil explored with associated depletion ofsoil P
(Tarafdar and Marschner 1994 and as modelledby Schnepf et al.
2008). Interestingly, the benefitderived from mycorrhizal fungi has
been shown to beinversely associated with root hair length across a
rangeof plant species, suggesting a complementary functionof these
traits (Baon et al. 1994; Schweiger et al. 1995).Reduced P
acquisition by a root-hairless mutant ofbarley at low soil P was
similarly compensated for bythe presence of mycorrhiza (Jakobsen et
al. 2005a).
Depletion of P in the rhizosphere occurs from bothinorganic and
organic P which includes soluble ortho-phosphate and various forms
of extractable P (bothinorganic and organic) that are widely
considered to belabile. In addition, it is evident that pools of P
that aremore recalcitrant to extraction (e.g. NaOH-extractableP;
Fig. 2), and thus previously considered to be only ofpoor
availability to plants, can also be depleted in therhizosphere.
Such studies have used a range ofdifferent plants species and soil
types whereby roots,root hairs and mycorrhizas are separated from
bulk soilusing meshed-compartments in rhizobox systems (e.g.Chen et
al. 2002; Gahoonia and Nielsen 1997; Georgeet al. 2002; Morel and
Hinsinger 1999; Nuruzzaman etal. 2006; Tarafdar and Jungk 1987).
Such approachesare useful in identifying different processes
thatcontribute to P depletion even if they may
exaggeraterhizosphere effects. For example, Chen et al.
(2002)investigated P dynamics around the roots of ryegrass(Lolium
perenne L.) and radiata pine (Pinus radiata D.Don) and showed a
significant depletion of variouspools of P at distances of up to 2
and 5 mm from theroot surface of the two species respectively,
which wasrelated to different rhizosphere properties (Fig. 2).
Bothspecies also showed a significant increase in microbialbiomass
around the roots and associated increases inbicarbonate-extractable
organic P may be a consequenceof microbial-mediated immobilization
of orthophos-phate within the rhizosphere (Richardson et al.
2005).Further work to elucidate the role of microorganisms
ininfluencing P availability within the rhizosphere andthe extent
to which they either complement or competewith plant processes in P
acquisition is required(Jakobsen et al. 2005b).
Role of root exudates in phosphorus mobilization
The availability of P in the rhizosphere is
influencedsignificantly by changes in pH and root exudates
which
308 Plant Soil (2009) 321:305339
-
can either directly or indirectly affect nutrient availabil-ity
and/or microbial activity (Fig. 1; Richardson 1994).Acidification
of the rhizosphere in response to Pdeficiency has been demonstrated
for a number ofspecies (see review by Hinsinger et al. 2003) and
canalter the solubility of sparingly-soluble inorganic Pcompounds
(particularly Ca-phosphates in alkalinesoils), or affect the
kinetics of orthophosphateadsorption-desorption reactions in soil
and thesubsequent availability of orthophosphate and vari-ous
micronutrients in soil solution (Hinsinger andGilkes 1996; Gahoonia
and Nielsen 1992; Hinsinger2001; Neumann and Rmheld 2007).
Organic anions are released into the rhizosphere inresponse to
various nutritional stresses including P, Feand micronutrient
deficiency and Al toxicity (seereviews by Hocking 2001; Neumann and
Rmheld2007; Ryan et al. 2001). The concentration ofdifferent
organic anions is typically greater in therhizosphere (around
10-fold) compared with that inbulk soil (Jones et al. 2003).
Organic anions are
commonly released from roots in association withprotons which
results in an acidification of therhizosphere (Dinkelaker et al.
1989; Hoffland et al.1989; Neumann and Rmheld 2002). In addition
tothis change in rhizosphere pH, organic anions canalso directly
facilitate the mobilization of P throughreduced sorption of P by
alteration of the surfacecharacteristics of soil particles,
desorption of ortho-phosphate from adsorption sites (ligand
exchange andligand-promoted dissolution reactions), and
throughchelation of cations (e.g. Al and Fe in acidic soils orCa in
alkaline soils) that are commonly associatedwith orthophosphate in
soil (Bar-Yosef 1991; Jones1998; Jones and Darrah 1994b). Organic
anions alsomobilise P bound in humic-metal complexes (Gerke1993)
and have been shown to increase both theavailability of organic P
and its amenability todephosphorylation by phosphatases (Hayes et
al.2000). However, the effectiveness of different organicanions in
nutrient mobilization depends on variousfactors including; the form
and amount of the
Distance from root surface (mm)
0 2 4 6 8 10 12 14
Soil pH
5.5
6.0
6.5= 0.09LSD0.05
z
900
1000
1100
1200
1300LSD 0.05 = 64.1
Acid phosphatase
270
275
280
285
290
LSD 0.05 = 7.0
NaOH-extractable organic P
(mg P
o kg-1
)
(mg C
kg-1 )
(g p-
NP g-
1h-
1 )
850
900
950
1000
1050
1100 LSD 0.05 = 56.1
200
250
300
350
400LSD 0.05 = 28.5
Microbial biomass carbon
(pH)
Water soluble organic carbon
0 2 4 6 8 10 12 14
130
140
150
LSD 0.05 = 4.3
(mg P
i kg-1
)
NaOH-extractable inorganic P(m
g C kg
-1 )
0 2 4 6 8 10 12 14
Fig. 2 Features of rhizosphere soil from perennial
ryegrass(Lolium perenne L.; ..) and radiata pine (Pinus radiata
D.Don; - -) when grown in a rhizobox system and comparedto an
unplanted (control; __). Shown are changes inmicrobial biomass C,
water-soluble C, pH, acid phosphataseactivity and NaOH-extractable
inorganic and organic P contents
of the soil at various distances from the root surface. The
soil(orthic brown soil; Dystrochrept; Hurunui, New Zealand) had
atotal P content of 958 mg P kg1 soil. For each panel the errorbar
(LSD P=0.05) shows least significant difference (data takenfrom
Chen et al. 2002)
Plant Soil (2009) 321:305339 309
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particular anion released, with citrate and oxalatebeing more
effective relative to others (e.g. malate,malonate and tartrate
followed by succinate, fumarate,acetate and lactate; Bar-Yosef
1991) and interactionsof the anion within the soil environment,
including itseffective concentration in soil solution and
relativeturnover rate (Jones 1998). The presence of micro-organisms
is of further importance because of theircapacity to either rapidly
metabolize different organicanions within the rhizosphere or
through their ownability to release anions (Jones et al. 2003).
Forexample, in a calcareous soil, Strm et al. (2001)showed greater
stability and resistance to microbialdegradation of oxalate
compared with citrate andmalate which resulted in greater
mobilization of Pwithin localized regions of soil with a
subsequentincrease in P uptake by maize (Zea mays L.) roots(Strm et
al. 2002).
The effectiveness of organic anions in mobilizing Pfrom soil is
highlighted by studies with white lupin(Lupinus albus L.) which
exudes significant amountsof citrate (and to some extent malate)
from cluster rootsthat are formed in response to P deficiency
(Dinlelakeret al. 1989; Gardner et al. 1983; Keerthisinghe et
al.1998; Neumann and Martinoia 2002; Vance et al.2003). Citrate is
effective in mobilizing orthophosphatefrom pools of soil P that are
otherwise not available toplants that either do not exude, or show
limited releaseof organic anions, such as soybean (Glycine max
(L.)Merr.) and wheat (Triticum aestivum L.) (Braum andHelmke 1995;
Hocking et al. 1997). In addition, andanalogous to the role of root
hairs, cluster roots have arelatively short life span and form on
lateral roots asclosely packed tertiary roots with a dense covering
ofroot hairs. This provides a zone for both concentratedrelease of
organic anions and high surface area for theuptake of mobilized P
(Dinkelaker et al. 1995,Neumann and Martinoia 2002). Interestingly,
clusterroots form on plant species that are essentially
non-mycorrhizal and therefore appear to provide animportant
alternative strategy for plant acquisition ofsoil P (Shane and
Lambers 2005). Indeed, theformation of cluster roots and high rates
of organicanion release are reported for various native
Australianspecies (e.g. the Proteaceae and Casuarinaceae fami-lies)
which have evolved on low P soils (Roelofs et al.2001; Shane and
Lambers 2005). Increased organicanion efflux from roots in response
to P-deficiency alsooccurs in other species including chickpea
(Cicer
arietinum L.) and pigeon pea (Cajanus cajan L.) andto a lesser
extent in lucerne (alfalfa; Medicago sativaL.), canola (oil seed
rape; Brassica napus L.) and rice(Oryza sativa L.) (Ae et al. 1991;
Hedley et al. 1982;Hoffland et al. 1989; Lipton et al. 1987; Otani
et al.1996; Pearse et al. 2006a; Veneklaas et al. 2003;Wouterlood
et al. 2004). The increase in organic anionefflux by these species
in response to P deficiencyhowever, is considerably less than for
the Proteaceae orLupinus spp, and in many cases the
agronomicsignificance of organic anion release remains to
beverified in soil environments, as does the role ofvarious organic
anions in mobilizing P from differentforms of soil P (Pearse et al.
2006b).
Activity of phosphatases is significantly greater inthe
rhizosphere and is considered to be a generalresponse of plants to
mobilize P from organic formsin response to P deficiency
(Richardson et al. 2005).Phosphatases are required for the
hydrolysis (miner-alization) of organic P, and in bulk soil
microbial-mediated mineralization of organic P
contributessignificantly to plant availability (Frossard et
al.2000; Oehl et al. 2004). Depending on soil type andland
management, organic forms of P commonlyconstitute around 50% of the
total P in soil and is thepredominant form of P found in soil
solutions (RonVaz et al. 1993; Shand et al. 1994). Dissolved
organicP is derived largely from the turnover of soil
micro-organisms and, relative to orthophosphate, has
greatermobility in solution (Helal and Dressler 1989; Seelingand
Zasoski 1993) and is therefore of criticalimportance to the
dynamics and subsequent availabil-ity of P within the rhizosphere
(Jakobsen et al. 2005a;Richardson et al. 2005).
Extracellular phosphatases released from rootshave been
characterized for a wide range of plantspecies and been shown to be
effective for the in vitrohydrolysis of various organic P
substrates (Georgeet al. 2008; Hayes et al. 1999; Tadano et al.
1993;Tarafdar and Claassen 1988). Products of microbialturnover
also contain high amounts of dissolvedorganic P (>80%)
(primarily as nucleic acids andphospholipids), and are rapidly
mineralized in soiland as such are of high availability to plants
(Macklonet al. 1997). Direct hydrolysis of organic P andsubsequent
utilization of released orthophosphate byroots has been
demonstrated in soil using both wholeplant systems (e.g. McLaughlin
et al. 1988) and inrhizobox studies. In the later case, depletion
of
310 Plant Soil (2009) 321:305339
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various pools of extractable organic P from therhizosphere is
associated with higher activities ofphosphatases around plant roots
(Chen et al. 2002;Tarafdar and Claassen 1988; Fig. 2). In the study
byChen et al. (2002), greater net depletion of organic Pby radiata
pine (compared to ryegrass) occurreddespite similar increase in
phosphatase activity forboth species and the development of a
lesser rootmat at the soil interface for pine roots, whichsuggests
the possible involvement of other mecha-nisms. Indeed pine roots
acidified the rhizosphere to agreater extent and had higher water
soluble carbonand microbial biomass (Chen et al. 2002). Higherwater
soluble C suggests either greater root exudationor higher turnover
of microbial biomass which couldresult in increased P mobilization.
Alternatively,greater radial depletion of P by pine roots
maybeassociated with the presence of longer root hairs ormore
likely by association with ectomycorrhizalfungi. This latter
possibility is suggested by theauthors and is supported by
observations from morerecent studies where mycorrhizal fungi have
beenshown to be particular effective for the capture of Pby pine
roots (Liu et al. 2005; Scott and Condron2004). Casarin et al.
(2004) similarly showed theimportance of ectomycorrhizas for
mobilization of poorlyavailable soil P around roots of maritime
pine (Pinuspiaster Ait.) and that this benefit was from
bothincreased soil exploration and, depending on the speciesof
mycorrhizal fungi, due to the release of oxalate andprotons.
However, the relative importance of suchmicrobial processes as
compared to direct plant mech-anisms and other processes remains to
be fullyestablished. In the study by Chen et al. (2002), numbersand
activities of free-living and root-associated micro-organisms were
also enhanced significantly within therhizosphere (e.g. as shown by
increased microbialbiomass; Fig. 2) and these may also
contributesubstantially to the mechanisms of P depletion
andsolubilization.
Acquisition of nitrogen by plants
Nitrogen availability and uptake
Nitrogen occurs in soil in both organic and inorganicforms and
in addition to marked seasonal changes ischaracterised by a
heterogeneous distribution within thesoil. Nitrogen inputs through
fixation reactions (by
either symbiotic microorganisms or potentially
throughfree-living diazotrophs, as discussed below) and
trans-formations of N between different pools have
importantimplications for plant growth and for the loss of N
fromsoil systems (Jackson et al. 2008).
Microbial-mediatedmineralization of organic forms of N to
ammonium(NH4
+) and its subsequent nitrification to nitrate(NO3
) is of major significance to N availability(Fig. 3) and has
influence on root behaviour andrhizosphere dynamics. Although
mineral forms of Nhave classically been considered to dominate
plantuptake (see review by Miller and Cramer 2004), thereis
evidence that soluble organic forms of N (e.g. lowmolecular weight
compounds such as amino acids)may also play a significant role
(Chapin et al. 1993),but few studies have quantified the relative
importanceof each (Leadley et al. 1997; Schimel and Bennett2004).
Of particular significance in the rhizosphere isthe effect that
uptake of different N forms has on soilpH in the immediate vicinity
of the root and subsequentinfluence of this on nutrient
acquisition, especially inrelation to the availability of P and
various micro-nutrients (e.g. Zn, Mn and Fe) (Marschner
1995).Changes in rhizosphere pH, caused by the influx ofprotons
that occurs with uptake of NO3
, or the netrelease of protons for NH4
+ uptake, can also bringabout changes in the nature of
substrates exuded fromroots or the quantities of exudates released,
andconsequently may have major impact on the structureof microbial
communities around the root (Bowen andRovira 1991; Meharg and
Killham 1990; Smiley andCook 1983).
Both NO3 and NH4
+ reach the root surface via acombination of mass flow and
diffusion (De Willigen1986). Nitrate is typically present in soil
solution atmM concentrations and, relative to orthophosphate,
ismore mobile (Tinker and Nye 2000) and thus, ispotentially able to
move in soil by up to several mmper day (Gregory 2006). Ammonium is
less mobilesince it readily adsorbs to the cation exchange sitesin
soil and has lower rates for both mass flow anddiffusion.
Nevertheless, diffusion and mass flow isthe major pathway for
inorganic N uptake and,although it is difficult to differentiate
diffusion fromroot interception, it is generally considered
thatinterception of N in soil solution following rootextension
accounts for a small percentage only of Ntaken up by plants (Barber
1995; Miller and Cramer2004).
Plant Soil (2009) 321:305339 311
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Plant uptake of NH4+ and NO3
is a function oftheir concentrations in soil and soil solution,
rootdistribution, soil water content and plant growth rate.The
latter is most important under conditions ofliberal N supply,
whereas mineral N concentrationand root distribution are more
critical under Nlimiting conditions. Whilst some plant species
showa preference for either NH4
+ or NO3 uptake, the
significance of this for N uptake at the field level isusually
less than the abovementioned factors, espe-cially in
agro-ecosystems (McNeill and Unkovich2007). Indeed, NH4
+ tends to dominate in manynatural ecosystems for reasons which
may include asuppression of microbial-mediated
nitrification(reviewed by Subbarao et al. 2006). In addition
anddepending on soil type, non-exchangeable forms ofNH4
+ may contribute significantly to crop nutrition(Scherer and
Ahrens 1996; Mengel et al. 1990),especially for lowland rice grown
under floodedconditions (Keerthisinghe et al. 1985). Althoughmany
crop plants differ in their sensitivity to toxiceffects of NH4
+, the crucial factor seems to be therelative concentration of
the two ions, with theoptimal mix being dependent on factors such
as plantspecies, age and soil pH (Britto and Kronzucker
2002;Badalucco and Nannipieri 2007). Furthermore, uptakeof NO3
across the plasma membrane is more costly
in terms of energy expenditure but nonetheless occurseffectively
over almost the entire range of NO3
concentrations found in soil solutions (Forde andClarkson 1999).
However, for crop production sys-tems the regulation of whole plant
N uptake remainsrelatively poorly understood (Gastal and
Lemaire2002; Jackson et al. 2008), although there is evidencethat
the concentrations of both NO3
and NH4+ in soil
solution, as well as plant N status are involved (Aslamet al.
1996; Devienne-Barret et al. 2000).
The significance of soluble organic N for plantnutrition was
first highlighted in solution culturestudies and has since been
demonstrated for soils ina range of different ecosystems (Jones and
Darrah1994a; Schimel and Bennett 2004). Amino acidstypically
constitute up to half of the total soluble N insoil solution
(concentrations ranging from 0.1 to50 mM) and thus comprise a
significant part of thepotentially plant available N pool (Christou
et al.2005; Jones et al. 2002). Amino acids in soil solutionoccur
as a result of either direct exudation by roots orfrom the
breakdown of proteins and peptides fromsoil organic matter and
microbial biomass turnover asa result of microbial-derived
proteases (Jaeger et al.1999; Owen and Jones 2001). In addition
theinvolvement of plant-exuded proteases in digestionof proteins at
the root surface has been reported along
diazotrophs
soil solution
DON organic N
NH3NH4+
NO3-
NH4+
NO3-
mineralization
immobilization
soil N
soil microorganisms
nitrification
mineral N
NO2- NO2-
NON2O
N2
plant N
N fertilizerplant residues, compost, animal excreta
soil organic matter
rootexudates
NH4+
NO3-
NO2-
uptake
uptake
organic N
assimilation
uptake
legumes
symbiotic N fixation
leaching
N2N2
NH3
denitrification
Fig. 3 The plant-soil Ncycle and pathways for Ntransformation
mediated byphysiological processes(DON = dissolved organicnitrogen;
redrawn fromMcNeill andUnkovich, 2007)
312 Plant Soil (2009) 321:305339
-
with the possibility of direct uptake of proteins byroots
through endocytosis (Paungfoo-Lonhienne et al.2008). Uptake of
organic N compounds by plantsmay also be facilitated by association
with ectomy-corrhizal fungi (Chalot and Brun 1998; Nasholm et
al.1998). However, the relative importance of suchmechanisms in
many ecosystems remains debatablesince the diffusion rates of amino
acids and proteinsare typically orders of magnitude lower than for
NO3
(Kuzyakov et al. 2003), and thus mycorrhizas mayoffer distinct
advantages, whereas microorganisms inthe rhizosphere are likely to
compete more stronglyfor these compounds (Hodge et al. 2000a).
Althoughdirect evidence that plant roots can
out-competemicroorganisms for N is limited to a few studies(Hu et
al. 2001; Jingguo and Bakken 1997), there issome evidence from 15N
time-course studies whereplants accumulate 15N and thus benefit
over thelonger term which may be associated with microbialturnover
(Hodge et al. 2000b; Kaye and Hart 1997;Yevdokimov and Blagodatsky
1994).
Given the heterogeneous distribution of N in soil interms of
chemical form, temporal dynamics andspatial distribution, plants
adopt one or more of threemain strategies to optimize their
acquisition of N.Broadly these are; (i) to explore greater volume
of soiland/or soil solution by extending root length andbranching
or increasing root surface area via changesin root diameter or root
hair morphology, (ii) specificadaptive response mechanisms in order
to exploitspatial and temporal niches such as N-rich patchesor due
to the presence of particular forms of N (aminoacids, NH4
+ or NO3), and (iii) influences on plant
available N in the rhizosphere through
plant-microbialinteractions.
Root growth and morphological responses to nitrogen
The size and architecture of the root system is animportant
feature for ensuring adequate access to soilN, and root system size
(relative to shoot growth) hasgenerally been shown to increase when
N is limiting(Chapin 1980; Ericsson 1995). However, changes
tobiomass alone are not necessarily indicative of thetotal
absorptive area of a root system and morpho-logical changes can
occur without change in biomass.Although the architecture of root
systems is intrinsi-cally determined by genotype and the pattern of
rootbranching, species specific attributes related to size
and architecture are also strongly determined byexternal
physical, chemical and biological factors(Miller and Cramer 2004).
Whilst primary rootgrowth is generally less sensitive to
nutritional effectsthan is the growth of secondary or higher root
orders(Forde and Lorenzo 2001), the diameter of first andsecond
order laterals were significantly thicker incereals grown at high
concentrations of NO3
(Drewet al. 1973). Thicker roots may be more costly toproduce
but have greater capacity for the transport ofwater and nutrients
and are less vulnerable to adverseedaphic conditions (Fitter 1987).
Conversely, fineroots allow greater exploration of soil and
plantsappear to accommodate trade-off between the two byexhibiting
plasticity in root diameter (and morpholo-gy) according to the
environmental conditions (Fordeand Lorenzo 2001). Root angle, an
important com-ponent of root architecture in soil in relation to
Pdeficiency (Rubio et al. 2003), appears to beunaffected by N
deficiency.
Apart from the size and depth of root systems otherattributes
may also influence the capacity for efficientcapture of N. Only a
limited portion of the root mayactually be effective in the uptake
of N (Robinson2001) and thus the spatial localization of roots
isimportant when nutrient is distributed heterogeneous-ly (Ho et
al. 2005). Electrophysiological and molec-ular evidence supports a
role for root hairs in theuptake and transport of both NO3
and NH4+ (Gilroy
and Jones 2002) and proliferation of fine roots androot hairs in
response to localized patches of N hasbeen demonstrated (Jackson et
al., 2008).
Rooting depth, which varies greatly betweenspecies, influences
the capture of N by plants,particularly of NO3
during periods of leaching, andis clearly an important
characteristic for manyperennial agricultural species and tree
crops (Gastaland Lemaire 2002). However, a dimorphic rootsystem,
having both shallow and deep roots to enableacquisition of
mineralized N in the topsoil as well asleached N at depth, is
considered to be important (Hoet al. 2005). Indeed, vigorous wheat
lines with fastervertical root growth and more extensive
horizontalroot development have been shown to take upsignificantly
more N (Liao et al. 2006).
Roots exhibit high plasticity as a physiologicalresponse to
localized patches of organic and inorganicnutrients in soil,
including proliferation in N-richzones (Hodge et al. 1999a;
Robinson and van Vuuren
Plant Soil (2009) 321:305339 313
-
1998). This proliferation essentially involves theinitiation of
new laterals, but may also includeincreases in the elongation rate
of individual rootsand expansion of the rhizosphere through root
hairs.Roots can also enhance their physiological
ion-uptakecapacities in localized nutrient-rich zones. This
rootforaging capability is considered to be an importantplant
response to optimize resource allocation inregard to N capture and
is of particular ecologicalimportance in situations where there is
competitionwith neighbouring roots for limited resources (Hodgeet
al. 1999b; Robinson et al. 1999). However,foraging is not a fixed
property but varies withinspecies in response to different
environmental con-ditions (Wijesinghe et al. 2001), indicating that
theenvironmental context in which the root response isexpressed is
as important as the response itself.However, most studies
investigating root growth inresponse to patches of N have largely
ignored theattributes of the patch itself despite the fact that
thedynamics of nutrient transformation within the patchand
microbial interactions are of major importance.There is need for
research to follow both together,including consideration of the
complexity of inter-actions with other root systems, soil
microorganismsand fauna, and physical/chemical interactions in
thesoil (Hodge 2004).
Association with microorganisms and plantgrowth promotion
Microbial associations with roots are complex in soiland can
enhance the ability of plants to acquirenutrients from soil through
a number of mechanisms.These include; i) an increase in the surface
area ofroots by either a direct extension of existing rootsystems
(e.g. mycorrhizal associations) or ii) byenhancement of root
growth, branching and/or roothair development (e.g. through plant
growth promot-ing rhizobacteria), (iii) a direct contribution to
nutrientavailability though either N fixation (e.g. rhizobia
anddiazotrophs) or by stimulation and/or contribution tometabolic
processes that mobilize nutrients frompoorly available sources
(e.g. organic anions) or, anindirect effect on nutrient
availability by (iv) dis-placement of sorption equilibrium that
results inincreased net transfer of nutrients into solution and/or
as the mediators of transformation of nutrients
between different pools (e.g. nitrification inhibitorsand
microbial-mediated processes that alter thedistribution of
nutrients between inorganic and organ-ic forms) or v) through the
turnover of microbialbiomass within rhizosphere (Gyaneshwar et al.
2002;Jakobsen et al. 2005a; Kucey et al. 1989; Richardson2007;
Tinker 1980).
In this respect, mycorrhizal fungi, rhizobia andFrankia
microsymbionts, and plant growth promoting(PGP) microorganisms are
of particular importanceand as such have been studied most widely.
PGPmicroorganisms represent a wide diversity of bacteriaand fungi
that typically colonize the rhizosphere andare able to stimulate
plant growth through either abiofertilizing (direct) effect or
through mechanismsof biocontrol (indirect effect; Bashan and
Holguin1997; and see Raaijmakers et al. 2009; Harman et al.2004;
Fig. 4). Biofertilizing-PGPR (considered inmore detail below)
specifically refers to rhizobacteriathat are able to promote growth
by enhancing thesupply of nutrients to plants (Vessey 2003).
Forrhizobia and Frankia this involves symbiotic relation-ships with
host legume and actinorhizal plants,respectively (see review by
Franche et al. 2009), andare therefore not considered here.
Rhizobiaceae(rhizobia) can also develop non-specific
associativeinteractions with roots and promote the growth
ofnon-legumes, and as such are also commonly consid-ered as PGPR
(Sessitsch et al. 2002). However, PGPis a complex phenomenon that
often cannot beattributed to a single mechanism and, as
outlinedbelow, PGPR may typically display a combination
ofmechanisms (Ahmad et al. 2008; Kuklinsky-Sobral etal. 2004). In
addition, the effects of individual PGPRmay not occur alone but
through synergistic inter-actions between different
microorganisms.
Association with mycorrhizal fungi
Mycorrhizal symbioses are found in almost allecosystems and can
enhance plant growth througha number of processes which include
improvementof plant establishment, increased nutrient
uptake(particularly P and essential micronutrients such asZn and
Cu, but also N and, depending on soil pH,may enhance the uptake of
K, Ca and Mg; Clarkand Zeto 2000), protection against biotic and
abioticstresses and improved soil structure (Buscot 2005;Smith and
Read 2008). Mycorrhizal fungi typically
314 Plant Soil (2009) 321:305339
-
colonize the root cortex biotrophically and developexternal
hyphae (or extra-radical mycelia) whichconnect the root with the
surrounding soil. All butfew vascular plant species are able to
associate withmycorrhizal fungi. The universality of this
symbiosisimplies a great diversity in the taxonomic features ofthe
fungi and the plants involved. At least five types
of mycorrhizas are recognized, the structural andfunctional
features of which are reviewed in detailelsewhere (Smith and Read
2008; Brundrett, 2002)and are thus only considered briefly
here.
Higher plants commonly form associations withectomycorrhizas,
mainly forest trees in the Fagaceae,Betulaceae, Pinaceae,
Eucalyptus, and some woody
NO
ACC deamination
Pathogens
AM fungi
Biocontrol-PGPR
AntagonismCompetition
Biofertilizing-PGPR
Denitrification
Free N2fixationNO
N2ON2
NH4+
N03-NO2 -
Nitrification
Production of phytohormones,
siderophores, vitaminsorganic P
inorganic P
Mineralizationphosphate
Solubilization
Ethylene
IAA
ACC ACC NH3+ -KB
Biofertilizing-PGPR
Biofertilizing-PGPR
P nutrition
N nutritionBNI
Elicitingplant defense responses
positive effects of plant regulators on root development or on
hormonal pathways
negative effects of plant regulators on root development or on
hormonal pathway
positive effects of microorganisms on plant nutrition and/or
root development
plant exudates
microbial enzymatic processes
negative effects of PGPR or plants on detrimental rhizospheric
microorganisms or microbial processes
negative effects of microorganisms on plant nutrition and/or
root development
Interactions of plant growthpromoting rhizobacteria
(PGPR)
Fig. 4 Plant growth promotion mechanisms (positive andnegative
effects) associated with soil and rhizosphere (PGPR)microorganisms.
Biofertilizing-PGPR and arbuscular mycorrhi-zal (AM) fungi
stimulate plant nutrition by directly increasingthe supply of
nutrients to plants (e.g. through N fixation, Psolubilization
and/or mineralization, vitamin and siderophoreproduction) or by
increasing the plants access to nutrients due
to enhancement of root volume. Promotion of root growth islinked
to the ability of PGPR to produce phytohormones (e.g.IAA, ethylene,
NO) or by direct influences on plant hormonelevels (e.g.
deamination of ACC precursor to plant ethylene).Biocontrol-PGPR
improve plant heath by inhibiting the growthof plant pathogens or
by eliciting plant defense responses
Plant Soil (2009) 321:305339 315
-
legumes. The fungi involved are usually Basidiomy-cetes and
Ascomycetes which colonize cortical roottissues but without
intracellular penetration (Smithand Read 2008). Three other types
of mycorrhizas canbe grouped as endomycorrhizas, in which the
funguscolonizes the root cortex intercellularly. One of theseis
restricted to species within the Ericaceae (ericoidmycorrhizas),
the second to the Orchidaceae (orchidmycorrhizas) and the third,
which is by far the mostwidespread (and therefore considered in
most detailhere), are the arbuscular mycorrhizas (AM). A
fifthgroup, the ectendomycorrhizas, are associated withplant
species in families other than Ericaceae,including the Ericaless
and the Monotropaceae (arbu-toid and monotropoid mycorrhizas). The
majority ofplant families form arbuscular associations, with theAM
fungi being an obligate symbiont that is unable tocomplete its life
cycle without colonization of a hostplant. The AM fungi were
formerly included in theorder Glomales, Zygomycota, but are now
consideredas new phylum, the Glomeromycota (Redecker 2002,Schbler
et al. 2001).
The establishment of mycorrhizal fungi in rootschanges key
aspects of plant physiology, includingmineral nutrient composition
in tissues, plant hor-monal balance and patterns of C allocation.
The fungimay also alter the chemical composition of rootexudates,
whilst the development of mycelium in soilcan act as a C source for
microbial communities andintroduce physical modifications to the
soil environ-ment (Gryndler 2000). Such changes in the rhizo-sphere
can affect microbial populations bothquantitatively and
qualitatively such that the rhizo-sphere of mycorrhizal plants
(known as the mycor-rhizosphere) generally has features that
differsubstantially from those of non-mycorrhizal plants(Barea et
al. 2002; Johansson et al. 2004; Offre et al.2007). As discussed in
more detail below, a widerange of bacteria (including actinomycetes
and vari-ous PGPR) associate with mycorrhizas within
themycorrhizosphere (Rillig et al. 2006; Toljander et al.2007).
Contribution of mycorrhizas to the phosphorusnutrition of
plants
It is well established that mycorrhizal fungi
contributesignificantly to the P nutrition of plants,
particularlyunder low P conditions (Barea et al. 2008). This is
most evident for the ectomycorrhizal fungi which arelargely
associated with non-agricultural plants andappear to show greater
functional diversity than theAM fungi (Brundrett 2002). Whilst it
is generallyaccepted that mycorrhizal fungi have similar access
tosources of P in soil solution that are also directlyavailable to
plants (reviewed by Bolan 1991) thereis some evidence to suggest
that both AM andectomycorrhizal fungi have enhanced ability to
usealternative sources of P (Bolan et al. 1984; Casarin etal.
2004). For example, for AM fungi, Tawaraya et al.(2006) showed that
exudates from fungal hyphaesolubilized more P than root exudates
alone, suggest-ing that the mycorrhiza contribute to increased
Puptake through solubilization. The extra-radical my-celium of AM
fungi have also been shown to excretephosphatases which could
potentially enhance themineralization and utilization of organic P
(Koide andKabir 2000; Joner and Johansen 2000). However, it
isgenerally considered that this is unlikely to be ofmajor
significance to the overall contribution of AMfungi to plant P
nutrition (Joner et al. 2000,Richardson et al. 2007).
Alternatively, indirect effectsof mycorrhizal fungi on P mobility
may occurthrough changes in soil microbial communities withinthe
mycorrhizosphere (Barea et al. 2005b).
The increased efficiency of P acquisition bymycorrhizal plants
is based mainly on the existenceof the extra-radical mycelia which
develop into soiland allow P to be accessed by the mycorrhiza
fromsoil solution at distances up to several cm away fromthe root
and then subsequently transferred to the plant(Jakobsen et al.
1992). High mycorrhizal hyphaedensity also provides considerably
greater surfacearea for the absorption of orthophosphate by
plantsand, due to the smaller size of hyphae in relation toroots
and root hairs and their greater length relative toroot hairs,
hyphae are also most effective in exploitingsoil pores and nutrient
patches that may not bedirectly accessible to roots (Jakobsen et
al. 2005a).Thus, higher uptake of P by mycorrhizal plants
(bothectomycorrhizas and AM) can generally be explainedin term of
increased hyphal exploitation of the soil asmodelled by Schneph et
al. (2008) and the compet-itive ability of the hyphae to absorb
localized sourcesof orthophosphate and organic nutrient
patches(Tibbett and Sanders 2002; Cavagnaro et al. 2005).In this
context, numerous studies have shown positivecorrelations between
fungal variables, such as hyphal
316 Plant Soil (2009) 321:305339
-
length or hyphal density, with growth responsevariables of
colonized plants such as shoot biomass,P uptake and total P content
(Avio et al. 2006;Jakobsen et al. 2001). However, this cannot be
takenas a general conclusion since high hyphal develop-ment does
not always correlate with plant growthresponses (Smith et al. 2004)
and in some situations,particularly under fertilized field
conditions, thepresence of AM mycorrhizal fungi appears to
providelittle or no benefit in terms of plant P nutrition (Ryanand
Angus 2003, Ryan et al. 2005).
Apart from the physical extension of root systems,mycorrhizal
fungi may also acquire orthophosphatefrom soil solution at lower
concentrations than roots,but whether this contributes significant
advantage tothe P nutrition of plants remains uncertain.
Somestudies report higher affinity for orthophosphateuptake by
mycorrhizal plants (i.e. lower Km valuesthan for non-mycorrhizal
roots; Cardoso et al. 2006).Genes encoding for the high-affinity
phosphatetransport in AM fungi have been identified andshown to be
preferentially expressed in the extra-radical mycelium (Benedetto
et al. 2005; Maldonado-Mendoza et al. 2001). Mycorrhizal plants
thereforehave two pathways for P uptake, the directpathway via the
plant-soil interface through roothairs, and the mycorrhizal pathway
via the fungalmycelium (Smith et al. 2003). Interestingly,
severalstudies have shown that expression of plant epidermalP
transporters is reduced in roots that are colonized byAM fungi, and
that under these circumstances Puptake proceeds predominantly via
fungal transport-ers with subsequent transfer of P to plants at
thearbuscular-root interface (Burleigh et al. 2002; Chiouet al.
2001; Liu et al. 1998; Rausch et al. 2001). Insome cases AM
colonization results in a completeinactivation of the direct P
uptake pathway via roothairs with essentially all of the P in plant
tissues beingprovided through the mycorrhizal route (Smith et
al.2004).
Contribution of mycorrhizas to nitrogen nutrition
Several studies have also shown increased N uptakefrom soil by
roots associated with mycorrhizal fungi(Barea et al. 2005a). For
example, Ames et al. (1983)first showed that mycorrhizal hyphae
were able toabsorb, transport and utilize NH4
+ and, using 15N-based techniques, Barea et al. (1987)
demonstrated
that mycorrhizal plants under field conditions hadincreased N
uptake. Further studies using 15N andcompartmented rhizobox systems
verified that my-corrhizal hyphae in root-free compartments were
ableto access 15N (Tobar et al. 1994b). To investigatewhether the
mycorrhizal contribution to N acquisitionwas from pools of N that
were unavailable to nonmycorrhizal plants, the apparent pool size
of plantavailable N has been determined by isotopic dilution(i.e.
the AN value of the soil; Zapata 1990). Using thisapproach, higher
AN values for plants inoculated withAM fungi compared to
non-inoculated controls wereobtained, suggesting that the AM
mycelium were ableto access N from forms that were otherwise
lessavailable than that for non-mycorrhizal plants (Bareaet al.
1991). Indeed the presence of a functionaltransporter for
high-affinity uptake of NH4
+ hasrecently been identified in the extraradical myceliumof
Glomus sp. (Lpez-Pedrosa et al. 2006).
Association with free-living microorganisms
Phosphate-mobilizing microorganisms
Free-living bacteria and fungi that are able to
mobilizeorthophosphate from different forms of organic andinorganic
P have commonly been isolated from soil andin particular from the
rhizosphere of plants (Kucey et al.1989; Barea et al. 2005b).
Phosphate-solubilizingmicroorganisms (PSM) are characterized by
theircapacity to solubilize precipitated forms of P whencultured in
laboratory media and include a wide rangeof both symbiotic and
non-symbiotic organisms, suchas Pseudomonas, Bacillus and Rhizobium
spp., actino-mycetes and various fungi such as Aspergillus
andPenicillium spp. (see reviews by Gyaneshwar et al.2002; Kucey et
al. 1989; Rodriguez and Frago 1999;Subba-Rao 1982; Whitelaw 2000).
Selection of PSM isroutinely based on the solubilization of
sparinglysoluble Ca phosphates (typically, tri-calcium
phosphate[Ca3(PO4)2] and rock phosphates containing hydroxy-and
fluor-apatites [Ca5(PO4)3OH and Ca10(PO4)6F2])and Fe and Al
phosphates such as strengite(FePO4.2H2O) and variscite
(AlPO4.2H2O). Theamount of P solubilized is highly dependent on
thesource (solubility) of the P and, for different micro-organisms,
is influenced to a large extent by the cultureconditions. For
example, fungi are commonly reportedto be more effective at
solubilization of Fe and Al
Plant Soil (2009) 321:305339 317
-
phosphates, whereas the ability of different organismsto
solubilize Ca-phosphates is influenced by the sourceof carbon and
nitrogen in the media, by the bufferingcapacity of the media and
the stage at which culturesare sampled (Kucey 1983; Illmer and
Schinner 1995;Nahas 2007; Whitelaw et al. 1999). From
variousstudies it is evident that change in pH of the mediais
particularly important for solubilization of Ca-phosphates, whereby
cultures supplied with NH4
+ aremore effective than those with NO3
- due to associatedproton release and acidification of the
media. Acidifi-cation is also commonly associated with the release
oforganic anions which have been widely reported forvarious
microorganisms (i.e. with citrate, oxalate,lactate, and gluconate
being most common). Organicanions themselves may further increase
the mobiliza-tion of particular forms of poorly soluble P (e.g.
Al-Pand Fe-P) through chelation reactions (Whitelaw2000).
Under controlled growth conditions various studieshave
demonstrated enhanced growth and P nutritionof plants inoculated
with PSM which is oftenattributed to the P-solubilizing activity of
the micro-organisms involved (see reviews by Gyaneshwar et al.2002;
Kucey et al. 1989; Rodriguez and Fraga 1999;Whitelaw 2000).
However, clear effect of PSM inmore complex soil environments and
in field con-ditions, have proved more difficult to demonstrate
andinconsistent response of plants and performance ofdifferent
microorganisms have been observed. Asdiscussed by Richardson (2001)
this may be due toa range of factors that include, insufficient
knowledgefor introducing and understanding the dynamics
ofmicroorganisms and their interaction with complexmicrobial
communities in soil, the apparent lack ofany specific association
between partners, and poorunderstanding of the actual mechanisms
involved,both for the microorganisms and their interaction
andefficacy within different soil environments. Forexample, whilst
Penicillium radicum effectively sol-ubilized P in laboratory media
and was able topromote the growth of wheat, evidence for improvedP
uptake in response to inoculation was only evidentin glasshouse
trials particularly where fertilizer P wasapplied (Whitelaw et al.
1997). However, growthpromotion may not necessarily be directly
associatedwith P solubilization and production of phytohor-mones is
likely to be involved (Wakelin et al. 2006).Similarly, promotion of
root growth and enhanced P
nutrition of plants inoculated with P. bilaii has beenshown to
be primarily associated with increased rootgrowth, including
greater specific root length andproduction of longer root hairs
(Gulden and Vessey2000). Such studies highlight the difficulties
indetermining the actual mechanism associated withgrowth promotion,
as stimulation of root growth alsocontributes to greater potential
for P acquisition. It isimportant therefore that experiments
directed atdemonstrating the benefits of PSM be conductedacross a
range of P supplies, whereby benefits ofinoculation should be
negated at higher levels ofapplied P, and that specific measures of
P acquisition(e.g. by isotopic dilution) be made to confirm
Pmobilization from pools of soil that are otherwisepoorly available
to roots.
The mineralization of organic P in soil is largelymediated by
microbial processes and as such micro-organisms play a significant
role in maintaining plantavailable P. Microorganisms are able to
hydrolyze awide range of organic P substrates when grown inculture
and, when added to soil, different forms oforganic P have been
shown to be rapidly mineralized(Adams and Pate 1992; Macklon et al.
1997; and seereview by Richardson et al. 2005). Indeed benefits
ofmicrobial inoculation for the utilization of organic Pby plants
under controlled growth conditions havebeen demonstrated
(Richardson et al. 2001a). Inaddition, the microbial biomass is
important formaintaining both inorganic and organic P in
soilsolution (Seeling and Zososki 1993) and turnover ofthe biomass
represents an important potential supplyof P to plants (Oberson and
Joner 2005). Thiscontribution is likely to be of greater
significance inthe rhizosphere where there is increased amount
ofreadily metabolizable carbon and higher density ofmicroorganisms
(Brimecombe et al. 2007; Jakobsenet al. 2005b). However, the
relative importance ofmicrobial mineralization relative to the
short-termimmobilization of P by microorganisms in therhizosphere
and its impact on the availability oforthophosphate to plants
requires more detailedinvestigation.
Whilst it is evident that microbial-mediated solu-bilization and
mineralization of inorganic and organicP are important processes
whereby microorganismsare able to acquire P from soil, it has been
argued thatthey are unlikely to mobilize sufficient P above
theirown requirements to meet plant demand (Tinker
318 Plant Soil (2009) 321:305339
-
1980). Indeed, few studies have unequivocally dem-onstrated a
direct release of P by microorganisms insoil and benefits to plant
nutrition are therefore ofteninferred. Nevertheless, the cycling of
P within themicrobial biomass and its subsequent release
isparamount to the P cycle in soil and represents animportant
pathway for movement of P from varioussoil pools into
plant-available forms and may alsoserve to protect orthophosphate
from becomingunavailable in soil due to various
physicochemicalreactions (Magid et al. 1996; Oberson et al.
2001).The significance of this in the rhizosphere warrantsfurther
research.
Microbial interactions and nitrogen availability
Root exudation also has important implications forN
availability. Although the chemical compositionof exudates varies
widely for different species androot types and is primarily
comprised of Ccompounds, exudates can also contain
significantquantities of N, which is either available
tomicroorganisms in the rhizosphere or can berecaptured by plants
(Bertin et al. 2003; Uren2007). In addition, root exudates are the
majorenergy supply for the soil food web and play asignificant role
in the turnover of soil organic matterand associated nutrients.
However, as suggested byJones et al. (2004), although root exudates
have beenhypothesized to be involved in the enhanced mobi-lization
and acquisition for many nutrients in soil,there is little
mechanistic evidence from soil-basedsystems to verify this, which
is further highlightedby a recent analysis of the literature
concerningrhizodeposition by maize (Amos and Walters 2006).Despite
this, some studies have demonstrated en-hanced N cycling in the
vicinity of plant roots(Jackson et al. 2008). For example,
following theapplication of 15N labelled fertilizer, the excess
of15N in the microbial biomass increased significantlyin both
planted and control soils at up to 8 weeksafter plant emergence,
but then declined in controlsoils only (Qian et al. 1997).
Retention of 15N in themicrobial biomass in planted soil was
attributed tothe release of root-derived C from maize as estimat-ed
using 13C abundance methodology. This releaseof exudate was
suggested to promote microbialimmobilization of the N and is
consistent withgreater microbial biomass in the rhizosphere.
Increased rates of N mineralization have similarlybeen
demonstrated in the rhizosphere of slender wildoats (Avena barbata
Pott ex Link), where N mineral-ization was 10 times higher than in
bulk soil, but thiswas highly dependent on location along the
root(Herman et al. 2006). In addition, a rhizospherepriming effect
has been suggested to be involved inthe decomposition of native
soil organic matter aroundroots (Cheng et al. 2003; Kuzyakov 2002).
However,stimulation of mineralization may be dependent onplant
species and on the C:N ratio of substrates ashighlighted in a study
of peas (Pisum sativum L.)inoculated with Pseudomonas fluorescens,
whereincreased uptake of N from 15N enriched organicresidues
occurred, whereas decreased uptake wasobserved for wheat
(Brimecombe et al. 1999). Addi-tional work demonstrated that
microbial-microfaunalinteractions in the rhizosphere were also
involved inthis differential response, with lower numbers
ofnematodes and protozoa being present in the rhizo-sphere of
uninoculated peas which appeared to exert anematicidal effect
(Brimecombe et al. 2000). On thecontrary, higher numbers of
nematodes and protozoa(e.g. up to 6-fold) have generally been
reported in therhizosphere where they specifically feed on
bacteria,fungi and yeasts (Zwart et al. 1994). These
interactionsenhance N flows in the rhizosphere both directly,
viathe excretion of consumed nutrients and mineralizationof
nutrients on death (Griffiths 1989), and indirectlyvia changes to
the composition and activity of themicrobial community (Griffiths
et al. 1999). Forexample, bacteriophagous nematodes mineralized
upto six times more N than an equivalent biomass ofprotozoa grazing
on bacteria (Griffiths 1990). Netmineralization is due to
differences in C:N ratiobetween the protozoan (or nematode)
predator andthe bacterial prey and their relatively low
assimilationefficiency, whereby ~60% of ingested nutrients
aretypically excreted and thus potentially available forplant or
microbial uptake (Bonkowski 2004). More-over, plants grown under
controlled conditions havebeen observed to develop more highly
branched rootsystems in the presence of protozoa which may partlybe
explained as a response to NO3
formed frommineralization of NH4
+ excreted by protozoa (Forde2002). However, other work has
suggested that rootresponses are due to a direct phytohormone
effect bythe presence of either protozoa or a consequence ofauxin
producing bacteria stimulated by the presence of
Plant Soil (2009) 321:305339 319
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the protozoa (Bonkowski 2004; Bonkowski and Brandt2002). The
consequences of soil organisms promotinga mutually beneficial
relationship between plant rootsand bacteria in the rhizosphere on
root architecture,nutrient uptake and plant productivity is
therefore ofcurrent research interest (Mantelin and Touraine
2004).For example, in an experiment using 15N/13Clabelled organic
nutrient sources combined withmanipulation of the composition of
microfaunal pop-ulations, Bonkowski et al. (2000) observed effects
onryegrass growth and concluded that microfaunalgrazing increased
the temporal coupling of nutrientrelease and plant uptake, whereas
root foraging inorganic nutrient-rich zones enhanced the spatial
cou-pling of mineralization and plant uptake. More recentwork has
shown interaction at higher trophic levelswhere collembola that
feed on bacteria, fungi, nemat-odes and protozoa further enhanced N
mineralizationwithout alteration to the microbial biomass C
(Kanedaand Kaneko 2008).
At a larger scale there is increasing evidence fromusing in situ
15N labelling of plant root systems of theimportance of N
rhizodeposition in sustaining the Ncycle of agro-ecosystems
(Hogh-Jensen 2006; Mayeret al. 2004; McNeill and Fillery 2008). A
recent review(Wichern et al. 2008) highlights the wide variability
inresults and suggests there is need for more inves-tigations on
key environmental factors influencing theamounts of N released
under field conditions fromdifferent species. Apart from a direct
influence of therhizosphere on N deposition, there is also need to
morefully understand the role that plant roots have ininteracting
with soil microorganisms and influencingother parts of the soil N
cycle. For example, thepresence of plant-derived biological
nitrification inhib-itors (BNI) in the root zone can influence
theconversion of NH4
+ to NO3- (nitrification) and subse-
quently to the potential for gaseous losses of N
throughdenitrification (Fig. 3). Indeed nitrification
inhibitorshave been recognized for some time in native
plantecosystems, but more recently have been shown to beeffective
as root exudates from brachiaria (Brachiariahumidicola (Rendle)
Schweick) a tropical grass species(Subbarao et al. 2006). In these
systems there is greaterretention of NH4
+ in soil which has importantimplication for improving N-use
efficiency by reducingpotential N losses through NO3
leaching and/or itsconversion (denitrification) to N2O gas (via
NO),which contributes significantly to greenhouse-gas
emissions (Fillery 2007; Subbarao et al. 2006).However,
presently it appears that BNI is poorlyexpressed in many
agricultural crop species, althoughactivities has been reported for
sorghum (Sorghumbicolour (L.) Moench.), pearl millet
(Pennisetumglaucum (L.) R.Br.) and peanut (Arachis hypogaeaL.)
(Subbarao et al. 2007a) and more recently BNI hasbeen shown to be
effective in wild rye (Leymusracemosus (Lam.) Tzvelev), a wild
relative of wheat(Subbarao et al. 2007b).
Nitrogen fixation by diazotrophs
Diazotrophs (i.e. N2-fixing bacteria) are classified asbeing
either symbiotic (rhizobia and Frankia species)or as free-living
(associative) and/or root endophyticmicroorganisms (Cocking 2003).
Rhizobia developsymbiotic relationships with host legumes
andthrough atmospheric N2 fixation within nodules canprovide up to
90% of the N requirements of the plant(see Franche et al. 2009;
Hflich et al. 1994). Free-living N2-fixers also have the potential
for providingN to host plants but so far, the direct contribution
ofN-fixation by diazotrophs to the N nutrition of plantsand
subsequent growth promotion remains in ques-tion. Free-living
diazotrophs have been identified inseveral genera of common
rhizosphere-inhabitingmicroorganisms such as Acetobacter, Azoarcus,
Azo-spirillum, Azotobacter, Burkholeria,
Enterobacter,Herbaspirillum, Gluconobacter and Pseudomonas(Baldani
et al. 1997; Mirza et al. 2006; Vessey2003), with some being
recognized as endophytes.Endophytic diazotrophs may have advantage
overroot-surface associated organisms, as they can colo-nize the
interior of plant roots and establish them-selves within niches
that are more conducive toeffective N2 fixation and subsequent
transfer of thefixed N to host plants (Baldani et al. 1997;
Reinhold-Hurek and Hurek 1998).
Mutants deficient in nitrogenase activity (i.e. Nif-
mutants) have been constructed in various PGPR,including
Azospirillum brasilense, Azoarcus sp. andPseudomonas putida, and
importantly, have beenshown in several cases to retain their
ability to promoteplant growth of certain crops (Hurek et al.
1994;Lifshitz et al. 1987). This questions the relativecontribution
of N2 fixation to the growth promotioneffect. On the contrary,
Hurek et al. (2002) showed thatan endophytic strain of Azoarcus sp.
was able to fix
320 Plant Soil (2009) 321:305339
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and transfer N when associated with kallar grass(Leptochloa
fusca (L.) Kunth), as Nif- mutants gavelower plant growth
stimulation than the wild-typestrain. Similar results were obtained
in the case of theassociative symbiosis between sugar-cane
(Saccharumofficinarum L.) and the endophytic diazotroph
Aceto-bacter diazotrophicus (renamed Gluconobacterdiazotrophicus)
(Sevilla et al. 2001). Furthermore, N-balance, 15N natural
abundance and 15N dilutionstudies, performed in either pot
experiments or in thefield, have provided clear evidence of the
ability ofendophytic N2-fixing bacteria to supply significantinputs
of nitrogen to some grasses and cereals (Boddeyet al. 2001;
Oliveira et al. 2002).
In contrast to symbiotic N2 fixation, where there isdirect
transfer of N across the symbiotic interface, it isevident that
root surface associated diazotrophs seemnot able to readily release
fixed N to the host plant andthat this occurs only through
microbial turnover(Lethbridge and Davidson 1983; Rao et al.
1998).This may account for inconsistent response of
plantsinoculated with diazotrophs and indicates that there isneed
for better understanding of the potential for free-living and
endophytic diazotrophs to supply N to hostplants. In addition to
their potential for supplyingplants with N, free-living diazotrophs
may alsopromote plant growth and nutrition through variousother
mechanisms.
Other mechanisms of PGPR to enhance plantnutrition
Microbial production of phytohormones
Many PGPR produce phytohormones that areconsidered to enhance
root growth and greatersurface area (e.g. bigger roots, more
lateral rootsand root hairs) leading to an increase in exploredsoil
volume and thus plant nutrition (Fig. 4). Suchmicroorganisms,
commonly termed phytostimula-tors, include a wide range of soil
bacteria and fungi.The most common phytohormones produced byPGPR
are auxins, cytokinins, giberellins and to alesser extent ethylene,
with auxins being the mostwell characterized (Khalid et al. 2004;
Patten andGlick 1996; and see review by Arshad andFrankenberger
1998). Indeed in Azospirillum, auxin(IAA) production, rather than
N2 fixation, is gener-ally considered to be the major factor
responsible for
the PGPR response through stimulation of rootgrowth (Dobbelaere
et al. 1999).
Indole-3-acetic acid (IAA) controls a wide varietyof processes
in plant development and plant growthand plays a key role in
shaping plant root architecturesuch as regulation of lateral root
initiation, rootvascular tissue differentiation, polar root hair
posi-tioning, root meristem maintenance and root gravitro-phism
(Aloni et al. 2006; Fukaki et al. 2007).Production of IAA is
widespread among rhizobacteria(Khalid et al 2004; Patten and Glick
1996; Spaepen etal. 2007), with increasing numbers of
endophyticIAA-producing PGPR being reported (Tan and Zou2001). For
example, Kuklinsky-Sobral et al. (2004)screened a collection of
root-associated bacteria fromsoybean for their ability to produce
IAA and showedthat it was present in 28% of isolates. More
recently,the distribution of IAA biosynthetic pathways
amongannotated bacterial genomes suggests that 15% (from369
analysed) contain genes necessary for synthesisof IAA (Spaepen et
al. 2007). IAA production hasalso been observed in rhizobia and in
phytopathogen-ic bacteria, although the amount of auxins producedby
different rhizobacteria seems to differ according totheir mode of
interaction with plants (Kawaguchi andSyno 1996). Several IAA
biosynthetic pathways,classified according to their intermediates,
exist inbacteria and for most, tryptophan has been identifiedas the
precursor of IAA (Patten and Glick 1996;Spaepen et al. 2007).
However, only few specific genesand proteins involved in IAA
biosynthesis have beencharacterized to date, and only in a small
number ofPGPR (e.g. Azospirillum brasilense, Enterobactercloacae,
Pantoea agglomerans and Pseudomonasputida; Koga et al. 1991; Patten
and Glick 2002;Zimmer et al. 1998). In phytopathogenic bacteria,
IAAseems to be mainly produced from tryptophan via theintermediate
indole-3-acetamide (IAM pathway),whilst in beneficial
phytostimulatory bacteria, IAAappears to be synthesized
predominantly via indole-3-pyruvic acid (IPyA pathway) (Manulis et
al. 1998;Patten and Glick 1996; 2002; Zimmer et al. 1998).
In A. brasilense, inactivation of a key enzyme inthe IPyA
pathway (the ipdC gene, encoding anindole-3-pyruvate decarboxylase)
resulted in up to90% reduction of IAA production (Dobbelaere et
al.1999), but mutants were not completely abolished inIAA
biosynthesis, suggesting some redundancy inpathways. Irrespective
of this, various ipdC mutants
Plant Soil (2009) 321:305339 321
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displayed altered phenotypes compared to the wild-type strains
in their ability to alter wheat rootmorphology (i.e. either no
increase in root hair andlateral root formation and no decrease in
root length;Malhotra and Srivastava 2008; Dobbelaere et al1999).
The impact of exogenous auxin on plantdevelopment ranges from
positive to negative effects,and occurs as a function of the amount
of IAA produced,the cell number of auxin-producing rhizobacteria
and onthe sensitivity of the host plant to changes in
IAAconcentration (Dobbelaere et al 1999; Spaepen et al2008; Xie et
al. 1996). For example, Remans et al.(2008) highlighted cultivar
specificity in the responseof plants to auxin-producing bacterial
strains. In manyPGPR, genes involved in IAA production are
fine-regulated by stress factors that commonly occur in soiland
potentially within the rhizosphere (e.g. acidic pHand osmotic
stress), and in some cases have beenshown to be activated by plant
extracts (e.g. aminoacids such as tryptophan, tyrosine and
phenylalanine,auxins and flavonoids; Ona et al. 2005; Prinsen et
al.1991; Vande Broek et al. 1999; Zimmer et al. 1998).
Cytokinins stimulate plant cell division, control rootmeristem
differentiation, inhibit primary root elongationand lateral root
formation but can promote root hairdevelopment (Riefler et al.
2006; Silverman et al.1998). Cytokinin production has been reported
invarious PGPR including, Arthrobacter spp., Azospir-illum spp.,
Pseudomonas fluorescens, and Paenibacil-lus polymyxa (Cacciari et
al. 1989; de Salamone et al.2001; Perrig et al. 2007; Timmusk et
al. 1999).However, genes involved in the biosynthesis ofbacterial
cytokinins have not yet been characterizedin PGPR and therefore
their involvement in plantgrowth promotion largely remains
speculative.
Gibberellins enhance the development of planttissues
particularly stem tissue and promote rootelongation and lateral
root extension (Barlow et al.1991; Yaxley et al. 2001). Production
of gibberellinshave been documented in several PGPR such
asAzospirillum spp., Azotobacter spp., Bacillus pumilus,B.
licheniformis, Herbaspirillum seropedicae, Gluco-nobacter
diazotrophicus and rhizobia (Bottini et al.2004; Gutirrez-Maero et
al. 2001). Some Azospir-illum strains are also able to hydrolyze,
both in vitro(Piccoli et al. 1997) and in vivo (Cassn et al.
2001),glucosyl-conjugates of gibberellic acid, which corre-spond to
reserve or transport forms of gibberellic acidproduced by plants
(Schneider and Schliemann 1994).
This activity leads to an increase in the release ofactive forms
of the phytohormone into the rhizosphere.However, the bacterial
genetic determinants involvedin this mechanism remain to be
identified as does theprecise role of gibberellins in plant growth
promotionby PGPR.
Ethylene is a key phytohormone that can inhibitroot elongation,
nodulation and auxin transport, andpromotes seed germination,
senescence and abscis-sion of various organs and fruit ripening
(Bleeckerand Kende 2000; Glick et al. 2007b). Ethylene isrequired
for the induction of systemic resistance inplants during
associative and symbiotic plant-bacteriainteractions and, at higher
concentrations, is involvedin plant defence pathways induced in
response topathogen infection (Broekaert et al. 2006; Glick et
al.2007a). Certain PGPR such as A. brasilense havebeen shown to
produce small amounts of ethylenefrom methionine as a precursor
(Perrig et al. 2007;Thuler et al. 2003), and this ability seems to
promoteroot hair development in tomato plants (Ribaudo et al.2006).
However, a better knowledge (i.e. character-ization of bacterial
biosynthesis pathway and geneticdeterminants involved) has to be
gained in order todetermine the role of the production of this
plantgrowth regulator in the growth promoting effect ofPGPR.
Some plant-associated bacteria such as A. brasi-lense (strain
Sp245) are able to produce nitric oxide(NO) due to the activity of
nitrite reductases (Creus etal. 2005; Pothier et al. 2007). The
formation of NO isan intermediate in the denitrification pathway,
duringwhich nitrate (or nitrite) is converted to nitrogenoxides
(N2O) and to N2 (Zumft 1997; Fig. 3). Thispathway is utilized by
soil bacteria to gain energyunder oxygen-limited conditions that
may occur in therhizosphere (Hjberg et al. 1999). Although
denitri-fication by rhizobacteria diminishes the amount ofNO3
available for plant nutrition, it may havepositive effects on
root development by means ofNO production, which is a key signal
molecule thatcontrols root growth and nodulation, stimulates
seedgermination and is involved in plant defenceresponses against
pathogens (Lamattina et al. 2003;Pii et al. 2007). Furthermore, NO
can interact withother plant hormone signalling networks
includingthat for IAA (Lamattina et al 2003; Fig. 4).
Bacterialdenitrification and production of NO by A. brasilensehas
been demonstrated on wheat roots (Creus et al.
322 Plant Soil (2009) 321:305339
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2005; Neuer et al. 1985; Pothier et al. 2007) and NOproduced
during tomato root colonization stimulatedthe formation of lateral
roots (Creus et al. 2005).Nitrous oxide production therefore is
potentiallyanother plant-beneficial trait displayed by
Azospiril-lum PGPR.
Microbial enzymatic activities influencing planthormone
levels
Certain PGPR are able to stimulate plant growth bydirectly
lowering plant ethylene levels through theaction of
1-aminocyclopropane-1-carboxylic acid(ACC) deaminase (i.e.
deamination of the plant ethyleneprecursor; Fig. 4). ACC deaminase
(encoded by theacdS gene) catalyses the conversion of ACC,
theimmediate plant precursor for ethylene, into NH3
and-ketobutyrate and is widely distributed in soil fungiand
bacteria, especially the Proteobacteria (Blaha et al.2006;
Prigent-Combaret et al. 2008). Whilst theecological significance of
ACC-deaminase in soilmicroorganisms is largely unknown, in
plant-beneficialbacteria it may serve to diminish the amount of
ACCavailable for production of ethylene (Glick et al.2007a). Since
ethylene inhibits growth and elongationof root, this may lead to
enhanced root systemdevelopment (Glick et al. 2007a). Indeed, this
modelhas been validated by analysis of the root growthpromoting
effect of a Pseudomonas putida PGPR,where acdS was inactivated
(Glick et al. 1994; Li et al.2000). In the case of Azospirillum,
complementation ofAcdS- strains with an acdS gene from P.
putidaenhanced the plant-beneficial effects of these PGPRon both
tomato (Lycopersicon esculentum Mill.) andcanola (Holguin and Glick
2001; Holguin and Glick2003). Similar results were obtained
following theintroduction of the Pseudomonas acdS gene into
AcdS-
Escherichia coli, Agrobacterium tumefaciens andbiocontrol
strains of Pseudomonas spp., where expres-sion of ACC deaminase
promoted root elongation,inhibition of crown gall development and
improvedprotection against phytopathogens, respectively (Hao etal.
2007; Shah et al. 1998; Wang et al. 2000). From anumber of studies
it appears that the growth promotioneffect of ACC deaminase in
rhizobacteria is mosteffective in stress environments such as in
flooded,heavy-metal contaminated or saline soils (Cheng et al.2007;
Farwell et al. 2007) and in response tophytopathogens (Wang et al.
2000).
Cross-talk between plant-growth promoting pathwaysin plants
It is evident that plant regulatory molecules (e.g.auxin,
ethylene, NO, gibberellin etc) do not act alonebut interact with
one another in a variety of complexways (Fu and Harberd 2003; Glick
et al. 2007a;Lamattina et al. 2003). Moreover, it is clear that
thePGPR effect occurs as a result of a combination ofdifferent
mechanisms (additive hypothesis). For ex-ample, a model has been
proposed by Glick et al.2007a to describe cross-talk between auxin
andethylene in both PGPR and plants. In response toroot exudates
containing tryptophan, PGPR produceIAA that can be taken up by
plant cells. Besides thedirect effect of IAA on plant cell
proliferation andelongation, it also induces the synthesis of
ACCsynthase in plants (Abel et al. 1995) and thereby theproduction
of ethylene. A negative feedback loop,involving inhibition by
ethylene of the transcriptionof auxin response factors, would lead
in fine to a slowdown of ACC synthase activity and decrease of
ACCand ethylene biosynthesis (Glick et al. 2007a). Otherclose
interactions between IAA and ethylene path-ways have been recently
reported. It appears thatethylene triggers the accumulation of
auxin in the rootapex (Stepanova et al. 2005) and that the
transport ofauxin from the apex to the elongation zone of roots
isrequired for ethylene to inhibit root growth (Swarupet al. 2007).
Overall, the molecular mechanisms bywhich ethylene and auxin
interact to competitivelyregulate root development remain largely
unknownand future prospects will aim to clarify their respec-tive
contribution. AcdS- and IAA-producing PGPRmight promote root growth
by both a lowering ofplant ethylene production and
ethylene-dependentsignalling pathways, and through an increase in
anethylene-independent manner, of the content of IAAin roots.
Facilitating plant iron and vitamins absorption
In addition to phytohormones, PGPR may influence thegrowth of
plant roots through the production of side-rophores and vitamins.
Roots of strategy II type plants(e.g. the Graminaceae; Marschner
1995; Robin et al.2008) secrete phytosiderophores (Fe-chelators)
whichbind Fe3+ and maintain its concentration in soilsolution (see
Lemanceau et al. 2009). At the root
Plant Soil (2009) 321:305339 323
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surface chelated Fe3+ can then be directly taken upas a
phytosiderophore-Fe complex (strategy II; seeLemanceau et al.
2009), whereas in non-graminaceousspecies (strategy I type plants;
Marschner 1995; Robinet al. 2008) Fe3+ must first be reduced to
Fe2+ prior tobeing absorbed by the plant (Lemanceau et al.
2009).Rhizobacteria (and fungi) also produce siderophoresand it has
been shown that plants can absorb bacterial-Fe3+ complexes, which
includes strategy I speciespossibly by endocytosis (Bar-Ness et al.
1991; Vansuytet al. 2007). The capture of these bacterial
complexesby plants may play a significant role in nutrition
andgrowth, especially in alkaline and calcareous soilswhere Fe
availability is low (Bar-Ness et al. 1991;Masalha et al. 2000).
Moreover, this mechanism isinvolved in biocontrol activities of
PGPR and has beenlinked to competitive effects with phytopathogens
andother detrimental rhizosphere microorganisms (Duijffet al. 1993;
Longxian et al. 2005; Robin et al. 2008).
Plants under optimal growing conditions synthesizevitamins but
when grown in stressed environments,vitamin-producing rhizobacteria
may stimulate plantgrowth and yield. In particular, production of
vitaminsof the B group (e.g. thiamine, biotin, riboflavine,
niacin)has been documented in some Azospirillum, Azotobac-ter,
Pseudomonas fluorescens and Rhizobium strains(Marek-Kozaczuk and
Skorupska 2001; Revillas et al.2000; Rodelas et al. 1993; Sierra et
al. 1999). There isevidence that exogenous supply of B-group
vitamins toplants favours root development (Mozafar and
Oertli1992), but there is presently no direct evidence (e.g.using
mutants) that PGPR can stimulate plant growththrough this mechanism
(Marek-Kozaczuk andSkorupska 2001), although further work is
warranted.
Interactions between plant growth promotingmicroorganisms
Mycorrhizal associations
Microbial populations in the rhizosphere, includingknown PGPR,
can either interfere with or benefit theformation and function of
mycorrhizal symbioses(Gryndler 2000). A typical beneficial effect
is thatexerted by mycorrhizal-helper-bacteria (MHB)which stimulate
mycelial growth and/or enhancemycorrhizal formation (Garbaye 1994).
This appliesboth to ectomycorrhizal fungi (Frey-Klett et al.
2005)and to AM fungi (Barea et al. 2005b; Johansson et al.
2004) and involves a range of bacterial species,commonly
including Bacillus and Pseudomonas.Responses to MHB are associated
with both theproduction of compounds that increase root
cellpermeability and rates of root exudation, which eitherstimulate
AM fungal mycelia in the rhizosphere orfacilitate root penetration
by the fungus, and theproduction of phytohormones that influence
AMestablishment (Barea et al. 2005b). Specific rhizobac-teria are
also known to affect the pre-symbiotic stagesof AM development,
such as spore germination andrate of mycelial growth (Barea et al.
2005b). Recently,Frey-Klett et al. (2007) revisited the
significance ofMHB and differentiated the effects based on eitherAM
formation or AM function, including nutrientmobilization, N2
fixation and protection of plantsagainst root pathogens.
Given that the external mycelium of mycorrhizasact as a link
between roots and the surrounding soil,the fungus can also
synergistically interact with soilmicroorganisms that mobilize soil
P, through eithersolubilization or mineralization (Azcon et al.
1976;Barea 1991; Barea et al. 2005a; Kucey 1987; Tarafdarand
Marschner 1995). Such interactions have beeninvestigated with
32P-based methodologies usingreactive rock phosphate in a
non-acidic soil (Toro etal. 1997) and in an experiment conducted by
Barea etal. (2002) using various treatments that included i)AM
inoculation, ii) PSB inoculation, iii) AM plusPSB dual inoculation
and iv) non-inoculated controlsin a soil containing natural
populations of both AMfungi and PSB (Fig. 5). Soils were either
un-amended(without P application) or fertilized with rock
phos-phate. Both rock phosphate addition and microbialinoculation
improved biomass production and Paccumulation in plants, with dual
inoculation beingthe most effective (Barea et al. 2002; Fig.
5).Independent of rock phosphate addition, AM-inoculatedplants
showed lower specific activity for 32P thancompared to non-AM
inoculated controls, particularlywhen inoculated with PSB,
suggesting that the PSBwere effective in releasing P from sparingly
solublesources either directly from the soil or from added
rockphosphate. Other studies have similarly showed apositive
interaction between ectomycorrhizal fungiand the presence of
bacterial isolates that showpotential for the weathering of soil
minerals and itsassociated release of nutrients for plant uptake
(Uroz etal. 2007). On such evidence it suggested that mycor-
324 Plant Soil (2009) 321:305339
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rhizas are highly effective for improving the capture
ofmobilized nutrients in soil, especially in relation to
themobilization and capture of soil P.
Microbial interactions and enhanced nitrogen fixation
Interaction between mycorrhizal fungi, PGPR anddiazotrophs,
including both rhizobia and associativeN-fixers, has also received
considerable attention. In
legumes it is evident that AM can improve bothnodulation and N2
fixation within nodules (Barea etal. 2005a; Barea et al. 2005b).
Co-inoculation ofRhizobium sp. with AM fungi gave greater
growthpromotion in lucerne (alfalfa) and pea than inocula-tion of
either symbiont alone (Hflich et al. 1994).The physiological and
biochemical basis of AMfungal x Rhizobium interactions in improving
legumeproductivity suggests that the main effect of AM inenhancing
nodule activity is through a generalizedstimulation of host
nutrition, but specific hormonaleffects on root and nodule
development may alsooccur (Barea et al. 2005a).
Several re