Plant–rhizobacteria interactions alleviate abioticstress conditionspce_2028 1682..1694

CHRISTIAN DIMKPA1, TANJA WEINAND2 & FOLKARD ASCH2

1Institute of Microbiology, Friedrich Schiller University, Neugasse 25, 07743, Jena, Germany, and 2Institute of CropProduction and Agroecology in the Tropics and Subtropics, University of Hohenheim, Garbenstr. 13, 70599 Stuttgart,Germany

ABSTRACT

Root-colonizing non-pathogenic bacteria can increase plantresistance to biotic and abiotic stress factors. Bacterialinoculates have been applied as biofertilizers and canincrease the effectiveness of phytoremediation. Inoculatingplants with non-pathogenic bacteria can provide ‘bioprotec-tion’ against biotic stresses, and some root-colonizingbacteria increase tolerance against abiotic stresses such asdrought, salinity and metal toxicity. Systematic identifica-tion of bacterial strains providing cross-protection againstmultiple stressors would be highly valuable for agriculturalproduction in changing environmental conditions. Forbacterial cross-protection to be an effective tool, a betterunderstanding of the underlying morphological, physiologi-cal and molecular mechanisms of bacterially mediatedstress tolerance, and the phenomenon of cross-protection iscritical. Beneficial bacteria-mediated plant gene expressionstudies under non-stress conditions or during pathogenicrhizobacteria–plant interactions are plentiful, but only fewmolecular studies on beneficial interactions under abioticstress situations have been reported. Thus, here we attemptan overview of current knowledge on physiological impactsand modes of action of bacterial mitigation of abiotic stresssymptoms in plants. Where available, molecular data will beprovided to support physiological or morphological obser-vations. We indicate further research avenues to enablebetter use of cross-protection capacities of root-colonizingnon-pathogenic bacteria in agricultural production systemsaffected by a changing climate.

Key-words: Bacillus spp.; ACC, auxins; cross-protection;PGPRs; plant hormones.

INTRODUCTION

In the early 1990s, interest in exploiting bacterial endo-phytes for agricultural applications increased followingresults from both field and laboratory studies which con-firmed that inoculation with non-pathogenic bacteria canhave positive effects on plant health and growth, resultingin increased yields (reviewed in Sturz, Christie & Nowak

2000). Currently, a gamut of bacterial inoculates arecommercially available for use as ‘biofertilizers’ or for ‘bio-protection’ against biotic stresses.

Considerable progress has been made in understandingthe molecular, physiological and morphological mecha-nisms underlying bacterially mediated tolerance to bioticstresses (Van Loon, Bakker & Pieterse 1998). However,although a range of examples of bacterially mediated toler-ance to abiotic stresses can be found in the literature, themodes of action largely remain elusive, because most ofthe studies focus on merely evaluating the plant growth-promoting effects. Some of the bacterial strains mitigatingabiotic stress symptoms were also shown to protect plantsagainst biotic stresses.Thus, for environmentally sustainableagricultural systems, bacterial inoculates providing cross-protection against different types of stress – biotic andabiotic – would be highly preferable. In order for it tobecome an effective tool, however, in-depth knowledge onthe phenomenon of cross-protection is required. Given thecomprehensive reviews already published summarizing themechanisms of enhanced resistance to biotic stresses, thisreview will focus on what is known about the mechanismsunderlying increased tolerance to abiotic stress as an effectof bacterial colonization.

BENEFICIAL RHIZOBACTERIA

Dense populations of microorganisms colonize the rootzone of plants. The main reason the rhizosphere is a farmore attractive habitat than bulk soil is the organic carbonprovided by plant roots. More than 85% of the total organiccarbon in the rhizosphere can originate from sloughed-offroot cells and tissues (Barber & Martin 1976). Moreover,plants supply organic carbon to their surroundings in theform of root exudates. Rhizobacteria respond to root exu-dates by means of chemotaxis towards the exudate source;and in such scenario, competent bacteria tend to modulatetheir metabolism towards optimizing nutrient acquisition(Hardoim, Van Overbeek & Van Elsas 2008 and referencestherein). In this regard, the role of bacterial motility in theirinteraction with plants has been demonstrated (Lugtenberget al. 1996). At the molecular level, signal-based communi-cations take place through plant perception of eubacterialflagellins (Gomez-Gomez & Boller 2002; Navarro et al.2006), leading to the down-regulation of genes involved in

Correspondence: F. Asch. Fax: +4971145924207; e-mail: fa@uni-hohenheim.de

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auxin signalling, thereby restricting bacterial growth in theplant (Navarro et al. 2006). However, it has also been indi-cated that flagella synthesis is an energy-consuming eventwhich,consequently, induces a rapid metabolic switch from amotile to non-motile form, upon bacteria reaching the rootepidermis. Moreover, aside from locomotion, adhesiveproperties have also been attributed to bacterial flagella(Persello-Cartieeaux, Nussaume & Robaglia 2003 and refer-ences therein). In contrast to the notion of flagella motility,non-flagella-producing rhizobacteria such as Streptomycesspp. establish beneficial interactions with plants via develop-ment of hyphae in plant tissues (Tokala et al. 2002).

Soil conditions, and the amount and composition of rootexudates play important roles in the specificity of thoseinteractions. Root zone bacteria that have been found tohave beneficial effects on various plants include species ofthe genera Arthrobacter, Azotobacter, Azospirillum, Bacil-lus, Enterobacter, Pseudomonas and Serratia (reviewed e.g.in Gray & Smith 2005), as well as Streptomyces spp. (Tokalaet al. 2002; Dimkpa et al. 2008, 2009a). Although the exactmechanisms of plant growth stimulation remain largelyspeculative, it is known that they differ between bacterialstrains and most certainly depend on the various com-pounds released by the different microorganisms. The lit-erature is replete with reports describing the production ofthe main phytohormone classes – auxins, cytokinins, gibber-ellins, abscisic acid (ABA) and ethylene – by plant growth-promoting rhizobacteria (PGPRs) (see e.g. Patten & Glick1996; Dobbelaere, Vanderleyden & Okon 2003; Arkhipovaet al. 2007; Forchetti et al. 2007; Perrig et al. 2007). Thesehormones can directly, or, in concert with other bacterialsecondary metabolites, stimulate plant growth (Patten &Glick 2002; Joo et al. 2005; Ryu et al. 2005; Aslantas, Cak-makci & Sahin 2007; Dimkpa et al. 2009a) usually, in aconcentration-dependent manner.

Other compounds produced by root zone bacteria includeenzymes, nitric oxide, osmolytes, siderophores, organic acidsand antibiotics.These may be responsible for indirect stimu-lation which includes the suppression of pathogens (e.g.Kloepper et al. 1999; Chakraborty, Chakraborty & Basnet2006; Sikora, Schafer & Dababat 2007), enhancement ofthe bioavailability of minerals in the soil (e.g. Carrillo-Castaneda et al. 2003, 2005; Dimkpa et al. 2009a), fixation ofatmospheric nitrogen (Dobbelaere et al. 2003), enhance-ment of tolerance to abiotic stresses (Grichko & Glick 2001;Yuwono, Handayani & Soedarsono 2005; Egamberdiyeva2007; Sziderics et al. 2007; Belimov et al. 2009; Dimkpa et al.2009a) or stimulation of phytohormone production by theplant (Lazarovits & Nowak 1997).

Conventionally, rhizobacteria are defined as soil-bornebacteria inhabiting the rhizosphere (Schroth & Hancock1982), but many of those colonizing the surroundings andthe surface of the root (exo-root) also penetrate into theroot cortex (endo-root). Nehl,Allen & Brown (1996), there-fore, included both ecto- and endobacteria in the termrhizobacteria. In fact, many of the root zone bacteria thathave been found to possess plant growth-promoting prop-erties are endophytic (e.g. Kloepper & Beauchamp 1992;

Sessitsch, Reiter & Berg 2004; Long, Schmidt & Baldwin2008). Bacillus, Pseudomonas, Enterobacter, Klebsiella,Serratia and Streptomyces are among the most commonlyisolated genera of endophytic bacteria. Endophytes arenot only found within the roots, but also in other parts ofthe plant, such as stems, seeds, tubers or unopened flowers(Hallmann et al. 1997 and references therein; Long et al.2008).

Gray & Smith (2005) further differentiated betweenextracellular and intracellular endophytic PGPR (ePGPRsand iPGPRs). iPGPRs can enter plant cells and are ableto produce specialized structures, the so-called nodules.ePGPRs, on the other hand, are found in the rhizosphere,on the rhizoplane or within the apoplast of the root cortex,but not inside the cells. According to their proximity to theplant root, ePGPRs can be divided into three classes: thoseliving near, but not in contact with the roots; those coloniz-ing the root surface; and those living in the spaces betweencells of the root cortex (Gray & Smith 2005). Given thedetailed reviews that already exist on nodule-forming rhizo-bia (e.g. Stougaard 2000; Hirsch, Lum & Downie 2001;Broughton et al. 2003), they will not be considered in thisreview.

MECHANISMS OF BACTERIA-MEDIATEDSTRESS TOLERANCE

Table 1 summarizes the studies published, to date, on bac-terial effects on plants under abiotic stress in relation tostress type, bacteria involved and the plant species to whichthey were applied. Based on Table 1, what is known to dateon abiotic stress resistance induced or mediated by bacteriawill be elucidated. Common adaptation mechanisms ofplants exposed to environmental stresses, such as water andnutrient deficiency, or heavy metal toxicity, include changesin root morphology, a process in which phytohormones areknown to play a key role (Potters et al. 2007, and referencestherein). Auxins, specifically indole acetic acid (IAA), areproduced in the plant shoot and transported basipetally tothe root tips (Martin & Elliott 1984), where, in low concen-trations, they enhance cell elongation, resulting in enhancedroot growth. Furthermore, auxin promotes the initiation oflateral roots. Higher concentrations of auxin in the root tips,however, have an inhibitory effect on root growth. Thisinhibition can be either direct or indirect through promot-ing the synthesis of ethylene (Eliasson, Bertell & Bolander1989; Jackson 1991), considering the relationship betweenIAA and the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC) (Glick 2003). Interestingly, bacteriacolonizing the root zone can also influence such modifica-tions in root growth (Schönwitz & Ziegler 1986; Bowen &Rovira 1991; Timmusk & Wagner 1999; German et al. 2000;Timmusk, Grantcharova & Wagner 2005; Belimov et al.2007).As indicated, the majority of root-associated bacteriathat display beneficial effects on plant growth have beenshown to produce IAA, and inoculation of various plantspecies with such bacteria has resulted in increased rootgrowth and/or enhanced formation of lateral roots and root

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hairs (see e.g. Martin et al. 1989; Patten & Glick 2002;Rajkumar et al. 2005; Chakraborty et al. 2006; Long et al.2008). Promotion of root growth results in a larger rootsurface, and can, therefore, have positive effects on wateracquisition and nutrient uptake. The availability of specificsubstrates as precursors for phytohormones, such asl-tryptophan for IAA, therefore, is a major factor determin-ing the degree of bacterial stimulation of plant growth.

Another widespread characteristic among endophyticand rhizosphere bacteria is ACC deaminase activity, andregulation of ACC is a principal mechanism by which bac-teria exert beneficial effects on abiotically stressed plants(Saleem et al. 2007). Bacteria possessing this enzyme canuse the immediate ethylene precursor ACC as a source ofnitrogen. Bacterial hydrolysis of ACC leads to a decrease inplant ethylene level, which, in turn, results in increased root

growth (Glick, Penrose & Li 1998; Burd, Dixon & Glick2000; Belimov et al. 2007, 2009; Long et al. 2008). Neverthe-less, changes in root morphology are not the only conse-quence of bacterial ACC deaminase activity, as bacterialnitric oxide has also been implicated, for instance, inAzospirillum-mediated changes in root morphology (Creuset al. 2005; Molina-Favero et al. 2008); however, decreasingthe level of ethylene alters the general stress status of theplant, as ethylene plays a key role in stress-related signaltransduction pathways. Its synthesis increases when theplant is exposed to different types of stress. For a detailedreview on the so-called stress ethylene and the alterationof plant ethylene levels by bacterial ACC deaminase, referto Glick (2005). Like ethylene, proline is often synthesizedby plants in response to various abiotic, as well as biotic,stresses, mediating osmotic adjustment, free radical

Table 1. Bacterially mediated plant tolerance to abiotic stress

Stress type Bacterial inoculate Plant species Reference

Salt Azospirillum brasilense Pea (Phaseolus vulgaris) Dardanelli et al. (2008)Salt Pseudomonas syringae,

Pseudomonas fluorescens,Enterobacter aerogenes

Maize (Zea mays) Nadeem et al. (2007)

Salt P. fluorescens Groundnut (Arachis hypogaea) Saravanakumar & Samiyappan (2007)Salt Azospirillum Lettuce (Lactuca sativa) Barassi et al. (2006)Salt Achromobacter piechaudii Tomato (Lycopersicon esculentum) Mayak, Tirosh & Glick (2004a)Salt Aeromonas hydrophila/caviae

Bacillus insolitus Bacillus sp.Wheat (Triticum aestivum) Ashraf et al. (2004)

Salt Azospirillum Maize (Z. mays) Hamdia, Shaddad & Doaa (2004)Salt A. brasilense Chickpeas (Cicer arietinum),

faba beans (Vicia faba L.)Hamaoui et al. (2001)

Drought Osmotolerant bacteria (notcompletely characterized)

Rice (Oryza sativa) Yuwono et al. (2005)

Drought Achromobacter piechaudii Tomato (L. esculentum), pepper(Capsicum annuum)

Mayak, Tirosh & Glick (2004b)

Drought Azospirillum Wheat (T. aestivum) Creus, Sueldo & Barassi (2004),Creus et al. (2005)

Drought A. brasilense Maize (Z. mays) Casanovas, Barassi & Sueldo (2002)Drought A. brasilense Common bean (P. vulgaris) German et al. (2000)Drying soil Variovorax paradoxus Pea (Pisum sativum) Belimov et al. (2009)Drying soil Bacillus Lettuce (L. sativa) Arkhipova et al. (2007)Osmotic stress

(45% PEG)Arthrobacter sp., Bacillus sp. Pepper (C. annuum) Sziderics et al. (2007)

Osmotic stress(20% PEG)in the dark

Azospirillum Wheat (T. aestivum) Pereyra, Zalazar & Barassi (2006)

Osmotic stress(20% PEG)

A. brasilense Wheat (T. aestivum) Creus, Sueldo & Barassi (1998)

Flooding Enterobacter cloacae, Pseudomonas putida Tomato (L. esculentum) Grichko & Glick (2001)Temperature Burkholderia phytofirmans Grapevine (Vitis vinifera) Barka, Nowak & Clement (2006)Temperature B. phytofirmans Potato (Solanum tuberosum) Bensalim, Nowak & Asiedu (1998)Temperature Aeromonas hydrophila, Serratia liquefaciens,

Serratia proteamaculansSoy bean (Glycine max) Zhang et al. (1997)

Nutrientdeficiency

Bacillus polymyxa, Mycobacterium phlei,Pseudomonas alcaligenes

Maize (Z. mays) Egamberdiyeva (2007)

Iron toxicity Bacillus subtilis, Bacillus megaterium,Bacillus sp.

Rice (O. sativa) Asch & Padham (2005),Terré et al. (2007)

Selected representation of studies published on beneficial effects of bacterial inoculation on plant physiology and growth under abiotic stressconditions. Because of the large number of studies on the effects of bacterial inoculation on plants grown on contaminated soils, they are notincluded in this table. For an overview of those, refer to reviews on phytoremediation (e.g. Glick 2003).

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scavenging and subcellular structure stabilization (Hare& Cress 1997). Proline synthesis has been shown to beincreased in abiotically stressed plants in the presence ofbeneficial bacteria such as Burkholderia (Barka et al. 2006),as well as Arthrobacter and Bacillus (Sziderics et al. 2007);however, in pepper, proline was also accumulated in theabsence of abiotic stress in Arthrobacter- and Bacillus-treated plants, suggesting that these bacteria could causesome biotic stress on the pepper (Sziderics et al. 2007). Atthe molecular level, gene expression changes related toethylene production have been reported in abioticallystressed plants treated with beneficial bacteria (see e.g.Timmusk & Wagner 1999; Sziderics et al. 2007). An over-view of mechanisms proposed to enhance stress toleranceis given in Figs 1 and 2, and the following paragraphs willdescribe them in more detail.

Cross-protection against abiotic andbiotic stress

The non-specificity of plant perception of abiotic stress canlead to a general response, the basis of cross-protection. Forexample, an increase in the synthesis of quaternary amines,such as glycine betaine, increases plant resistance to waterdeficiency, but also provides protection against frost andsalinity. Likewise, the up-regulation of anti-oxidativeenzymes, for example, superoxide dismutases (SODs), is ageneral response to different abiotic stress conditions. Obvi-ously, plant enzymatic anti-oxidative response to abioticstress is by no means limited to SODs. For a more detailedreview of abiotic stress and plant enzymatic responses,readers are directed to Alscher, Erturk & Heath (2002) andGratão et al. (2005), among other publications. For thisreview, we focus on the numerous studies undertaken toelucidate the plant beneficial effects of non-pathogenicrhizobacteria under different abiotic stress conditions.Interestingly, the beneficial effects of inoculation with plantgrowth-promoting root zone bacteria have been reported tobe most significant under unfavourable conditions such asflooding (Grichko & Glick 2001), drought (Mayak et al.2004a; Yuwono et al. 2005; Belimov et al. 2009), metal tox-icity (Belimov et al. 2005) or nutrient deficiency (Egamber-diyeva 2007).This is consistent with results from a computersimulation showing that the survival rate of introducedrhizobacteria in any given microbial community is animportant factor determining the degree of plant stimula-tion (Strigul & Kravchenko 2006). Because competition forlimited resources is crucial, and bacteria are also susceptibleto environmental stressors, the most prominent beneficialeffects of inoculation with a potential PGPR is to beexpected in poor soils (Ramos Solana et al. 2006), whenthe development of the indigenous microbial community isinhibited.

Inoculation with non-pathogenic root zone bacteria cantrigger signalling pathways that lead to higher pathogenresistance of the host – the so-called induced systemic resis-tance (ISR; Fig. 1e) (reviewed in Van Loon et al. 1998;Choudhary & Johri 2008;Walters and Fountaine 2009). This

is possible because a plant’s immune system consists of twobranches: one responding to pathogen virulence factors,and the other recognizing and responding to elicitor mol-ecules that are also typical for many non-pathogenic bacte-ria (Jones & Dangl 2006). Some of the bacteria that havebeen used to study beneficial effects under abiotic stressconditions, such as Bacillus sp., have been shown to induceISR (see e.g. Chakraborty et al. 2006; Barriuso et al. 2008),

Osmoticadjustment

IAA

ACCdeaminase

Ammonia & a-ketobutyrate

ISREnhancedsensitivity to JA and ethylene

Induction of signallingcascades

Nitric oxideand IAA

Enhancedlateral rootdevelopment

Decrease inthe plant‘sethylene level

ACCsynthase

S-adenosyl-methionine

ACC

Bacterial glycine betaineand/or other osmolytes

Reduction in membranepotential

Changes in saturation pattern of membrane phospholipids

Inoculation with bacteria

Changes in selectivityfor Na+, K+, Ca2+

HigherK+/Na+ ratios

(b)(a)

(c)

(d)

(e)

(f)

Po

ssib

leco

nse

qu

ence

sw

ith

inth

ep

lan

t

Figure 1. Proposed mechanisms underlying enhanced abioticstress tolerance within the plant. Inoculation with non-pathogenicroot zone bacteria can have various consequences within theplant as well as in the rhizosphere. (a) Upon bacterialinoculation, the selectivity for Na+, K+ and Ca2+ is altered,resulting in higher K+/Na+ ratios. (b) Inoculation withrhizobacteria can lead to changes in membrane phospholipidcontent and alterations in the saturation pattern of the lipids.Membrane potential is, thus, reduced. (c) Nitric oxide and indoleacetic acid (IAA) produced by bacteria promote lateral rootdevelopment in the host plant, resulting in increased root surfacearea. (d) Bacteria-produced osmolytes, such as glycine betaine,can act synergistically with plant osmolytes, accelerating osmoticadjustment. (e) Inoculation with non-pathogenic rhizobacteriacan induce signalling cascades that put the host plant in a‘primed’ physiological state as part of a phenomenon of inducedsystemic resistance (ISR). (f) Bacterial 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity reduces ‘stressethylene’ levels within the plant.

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and the primed physiological state of an inoculated plantcould, therefore, be one explanation for increased toleranceagainst abiotic stresses. Indeed, the phenomenon ofpriming, although not yet fully understood at the molecularlevel, is thought to be associated with an accumulation ofinactive signalling proteins which become activated andtransduced, upon subsequent exposure of the plant tosimilar stresses (Conrath et al. 2006). In addition, observedchanges in gene expression in Arabidopsis thaliana, firstinoculated with Paenibacillus polymyxa and then exposedto drought or infected with the pathogenic bacteriumErwinia carotovora, support the conclusion that genes

involved in plant response to biotic and abiotic stresses maybe coregulated (Timmusk & Wagner 1999). Consistent withthis, constitutive expression of the rice Osmyb4 gene encod-ing a transcription factor involved in cold acclimation,resulted in elevated tolerance of transgenic A. thaliana toboth abiotic (salt, UV, ozone, drought) and biotic (viruses,bacteria, fungi) stresses (Vannini et al. 2006). Studies usingthe promoter:GUS (beta-glucuronidase) reporter systemhave shown that the promoter of osmotins, a family of small(25–50 kDa) protective proteins that accumulate under saltstress, is responsive to the phytohormones ABA and ethyl-ene; to viral and fungal infections; to wounding; as well as tothe abiotic stresses salinity, drought and UV radiation (Liuet al. 1995). However, the accumulation of osmitine pro-teins, which leads to salt tolerance, only occurs upon saltstress and fungal infection, indicating that the regulatorymechanisms of cross-protection are very complex and occurnot only on the level of gene expression, but also on thetranslational and post-translational levels (La Rosa et al.1992). Consistent with this, Xiong & Yang (2003) showedthat disease resistance and abiotic stress tolerance in riceare inversely modulated by an ABA-inducible mitogen-activated protein kinase (MAPK). This MAPK is inducedby biotic (pathogen infection), as well as abiotic (wounding,drought, salt and cold), stresses, and increases toleranceto drought, salinity and cold stress when over-expressed.Suppression of the MAPK gene, however, significantlyenhances resistance to fungal (Magnapothe grisea) and bac-terial (Burkholderia glumae) pathogens, whereas toleranceto drought, salinity and low temperature was significantlyreduced.

Drought tolerance

As a response to water deficit, plants increase the synthesisof osmolytes, thus increasing the osmotic potential withincells (Farooq et al. 2009). Incidentally, compounds exudatedby root zone bacteria also include such osmolytes. Glycinebetaine produced by osmo-tolerant bacteria can possiblyact synergistically with plant-produced glycine betaine inresponse to the stress, and this way, increase drought toler-ance (Fig. 1d). Consistent with this, the beneficial effects ofosmolyte-producing rhizobacteria on rice were more sig-nificant when the stress conditions were more severe: dif-ferences in shoot dry weight, root dry weight and number oftillers between inoculated rice plants and non-inoculatedcontrols were more prominent under severe drought(Yuwono et al. 2005). Another important factor in thegrowth stimulation of these osmo-tolerant bacteria is theirability to produce IAA (Fig. 1c). Improvement in rootproliferation in inoculated drought-stressed rice plants islikely to be induced by this hormone (Yuwono et al. 2005),apparently for enhanced water uptake.

Bacteria occurring on root surfaces containing ACCdeaminase have been shown to modify the sensitivity ofroot and leaf growth to soil drying, apparently by influenc-ing ethylene signalling. The ACC deaminase activity ofAchromobacter piechaudi was shown to confer tolerance to

Soil sheath

Root

Bacterial EPS

Decreased flow of sodium into thestele

Binding of Cd ions in biologically unavailable complex forms

Bacterial migrationfrom the rhizoplaneto the rhizosphere

Increase in availability of nutrients

Po

ssib

leco

nse

qu

ence

sin

th

erh

izo

sph

ere

Bacterial nitrogenfixation

Organic acids

Changes in rhizospheric pH

Better iron nutrition of the plant

Iron-chelatingsiderophores

Higherfitness

Precipitation as insoluble compounds

Sorption to cell components

Intracellular sequestration

Increasednitrogenavailabilityfor theplant

(a)

(c)

(d)

(e) (f)

Heavy metals (b)

Figure 2. Proposed rhizosphere-dependent mechanismsunderlying enhanced abiotic stress tolerance by plants. (a) Hostplant nitrogen uptake can be positively influenced by bacterialnitrogen fixation. (b) The mobility of heavy metals incontaminated soils can be significantly reduced through rootzone bacteria. (c) Migration of bacteria from the rhizoplane tothe rhizosphere plays a role in reducing plant uptake of Cd.(d) Iron–siderophores complexes can be taken up by thehost plant, resulting in a higher fitness. (e) Bacterial exo-polysaccharides (EPS) lead to the development of soil sheathsaround the plant root, which reduces the flow of sodium into thestele. (f) Root zone bacteria can influence pH and redoxpotential in the rhizosphere, for instance, through the release oforganic acids. This can have positive effects on the availability ofnutrients for the plant.

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water deficit in tomato and pepper, resulting in significantincreases in fresh and dry weights. Ethylene production wasreduced in inoculated plants compared to non-inoculatedcontrols, with improved recovery from water deficiency,although inoculation did not influence relative water con-tents (Mayak et al. 2004b).

Under water deficiency, maize seedlings inoculated withAzospirillum brasilense, however, displayed improved rela-tive and absolute water contents, in comparison to non-inoculated treatments. Bacterial inoculation has also beenshown to prevent a significant drop in water potential, inparallel with a concomitant increase in root growth, totalaerial biomass and foliar area, as well as proline accumula-tion in leaves and roots. These effects were more significantat 75% reduction in water supply, compared to 50% reduc-tion (Casanovas et al. 2002). Creus et al. (2004) reported onreduced grain yield losses and higher Mg, K and Ca contentsin grains of Azospirillum-inoculated wheat exposed towater deficit. Increases in water content, relative watercontent, water potential and apoplastic water fraction wereobserved. Furthermore, lower volumetric cell wall moduli ofelasticity values were measured. Therefore, in addition to abetter water status, an ‘elastic adjustment’ is hypothesized tobe crucial in increased drought tolerance (Creus et al. 2004).Alterations in the host plant’s root morphology uponAzospirillum inoculation are widely believed to mainlyattribute to the plant’s enhanced tolerance to drought.Exactly how the bacterium induces changes in plant rootmorphology is not clear yet. Bacterial production ofhormone-like substances and their ability to stimulateendogenous hormone levels were believed to play the keyrole in this process (Dobbelaere et al. 1999; Cassan et al.2001). However, more recently, it has been found that,under aerobic conditions, A. brasilense produces significantamounts of the small diffusible gas, nitric oxide, which hasbeen shown to act as a signalling molecule in an IAA-induced pathway involved in adventitious root development(Creus et al. 2005; Molina-Favero et al. 2008).These workerssuggested that bacterial nitric oxide is involved in theenhancement of lateral root and root hair development inAzospirillum-inoculated tomato plants (Fig. 1c). As withAzospirillum, recent studies on droughted pea inoculatedwith ACC deaminase activity-containing Variovorax para-doxus, as against an ACC deaminase mutant strain, showedhormone signalling-mediated plant growth improvement,yield and water-use efficiency (Fig. 1e) (Belimov et al. 2009).

Susceptibility to water deficiency has been shown to becorrelated with membrane damage and lipid composition(Wilson, Burke & Quisenberry 1987; Moran et al. 1994).Cell membranes constitute important interfaces within acomplex system regulating a plant’s physiological status,and rhizobacteria can influence processes taking place atthese sites. Changes in proton efflux activities in wheat andcowpea, reduced membrane potentials in wheat seedlings,as well as changes in phospholipid content in the cell mem-branes of cowpea, have been observed upon inoculationwith Azospirillum (Bashan, Alcaraz-Menéndez & Toledo1992). Furthermore, in wheat seedlings, water deficit led to

changes in phospholipid composition in the root; increase inphosphatidylcholine content and a reduction of phosphati-dylethanolamine occurred (Sueldo et al. 1996), whereasinoculation with Azospirillum prevented these changes.However, compared to well-irrigated control plants, higherphosphatidylcholine and lower phosphatidylethanolamineunsaturation was observed in water-deficient inoculatedseedlings (Pereyra et al. 2006). These results suggest thatbacterially mediated changes in the elasticity of the root cellmembranes could be one of the first steps towards anenhanced tolerance to water deficiency (Fig. 1b).

At the transcriptional level, the bacterium P. polymyxaengendered the induction of a drought-responsive gene,ERD15, isolated from drought-stressed A. thaliana(Timmusk & Wagner 1999). Similarly, Rocha et al. (2007)reported the differential expression by drought treatment,of as many as 93 sugarcane genes, including well-knowndrought-responsive genes such as MRB and WRKY tran-scription factors; however, treatment of the same plant withbeneficial endophytic bacteria (Herbaspirillum spp. andGluconacetobacter diazotrophicus) resulted in the induc-tion of resistance (R) and salicylic acid biosynthesis genes.Salicylic acid production is known to be stimulated as aplant protective response to biotic stress. Thus, the findingsindicate that plants may not recognize beneficial bacteria assoon as bacterial contact is initiated. Instead a beneficialassociation is established in a later wave of activated genes.Nevertheless, in this study, drought-treated plants were notsubjected to treatment with the endophytes to evaluatetheir beneficial effect under an abiotic stress condition.

Tolerance to high soil salinity

Studies have shown that inoculation with endophytic bac-teria can mitigate the effects of salt stress in different plantspecies. Azospirillum-inoculated seeds of lettuce (Lactucasativa L., cv. Mantecosa), for instance, showed better germi-nation rates and vegetative growth than non-inoculatedcontrol plants when exposed to NaCl (Barassi et al. 2006).The relevance of decreased endogenous ethylene levels inbacterially mediated tolerance to salt stress has been high-lighted by some studies (Mayak et al. 2004a; Saravanaku-mar & Samiyappan 2007). In groundnut grown under salinefield conditions, the plant growth-promoting effects ofACC deaminase possessing Pseudomonas fluorescensTDK1 were more pronounced, compared to strains lackingthe enzyme (Saravanakumar & Samiyappan 2007). Consis-tent with this, transgenic canola expressing a bacterial ACCdeaminase gene was shown to be more tolerant to highconcentrations of salt than non-transformed control plants(Sergeeva, Shah & Glick 2006).

It has been reported that sodium uptake by plants is notaltered upon inoculation with rhizobacteria (Mayak et al.2004a). Yet, the suppression of photosynthesis observed intomato plants exposed to salt stress was less severe in plantsinoculated with Achromobacter. While the exact mecha-nisms remain unclear, it has been suggested that apart frombacterial ACC deaminase activity, elevated phosphorous

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and potassium uptake might play a role in this process(Mayak et al. 2004a). Nadeem et al. (2007) found that inocu-lation of salt-stressed maize with ACC deaminase contain-ing Pseudomonas syringae, Enterobacter aerogenes and P.fluorescens resulted in higher K+/Na+ ratios (Fig. 1a) in com-bination with high relative water, chlorophyll and lowproline contents. In this case, salt tolerance appears to bedependent on different mechanisms. High K+/Na+ ratioswere also found in salt-stressed maize in which selectivityfor Na+, K+ and Ca2+ was altered upon inoculation withAzospirillum (Hamdia et al. 2004). Furthermore, a decreaseof Na+ uptake was observed in wheat seedlings inoculatedwith strains of exopolysaccharide-producing bacteria(Ashraf et al. 2004). The authors suggested that a higherproportion of the root zone of inoculated seedlings wascovered in soil sheaths, which caused a reduced apoplasticflow of sodium ions into the stele.

Similarly, inoculation of pepper with Bacillus sp.TW4 ledto relief from osmotic stress, which is often manifested assalinity (and/or drought) stress. In these plants, genes linkedwith ethylene metabolism under abiotic stress such ascaACCO (encoding ACC oxidase) and caLTPI (an abioticstress-inducible gene encoding a lipid transfer protein; Jung,Kim & Hwang 2003) were down-regulated (Sziderics et al.2007). Because Bacillus sp. TW4 showed ACC deaminaseactivity, the authors speculated that the enzyme may beinvolved in the lower expression of these genes (Fig. 1f).Vinagre et al. (2006) reported the induction of a novelprotein kinase (SHR2) in sugarcane in the presence ofHerbaspirillum spp. and G. diazotrophicus. In contrast toendophytic bacteria inoculation, however, expression of thegene was unaffected by salt stress. This suggests a variancein plant gene expression patterns under biotic or abioticstress conditions, relative to their coregulation as previouslyobserved (Timmusk & Wagner 1999). Salt stress has alsobeen shown to affect nodulation during Phaseolus–Rhizobium interaction. However, secondary inoculationof the salt-stressed plants with Azospirillum caused anextended exudation of plant flavonoids compared to Rhizo-bium alone, implying an induction of flavonoid genes in thepresence of Azospirillum (Dardanelli et al. 2008). Thus, theco-inoculation of plants with different bacterial species maycontribute to relieving abiotic stress.

Tolerance to extreme temperatures

Temperature extremes present a stress condition for plants.For example, root elongation only occurs above a certain,species-dependent minimum temperature, and increasesalmost linearly with increasing temperature up to specificmaximum temperature when elongation rates rapidlydecrease (Erickson 1959). Zhang et al. (1997) tested thebeneficial effects of various bacterial strains on soy beangrowth and physiology under suboptimal root zone tem-peratures, and found that bacterial stimulation is interac-tively dependent on temperature. It has often been claimedthat growth-promoting effects are caused by the bacterialnitrogen-fixing activities, but in this case, positive effects

on the plant’s physiology were detected before start ofnitrogen fixation, indicating that mechanisms independentof nitrogen status are involved.

Bensalim et al. (1998) studied the effects of inoculationwith Burkholderia phytofirmans PsJN on 18 clones of potatogrown under two different temperatures (20 °C day, 15 °Cnight; 33 °C day, 25 °C night). Results from measurements ofstem length, shoot and root biomass at high temperatureindicate that colonization of the potato by rhizobacteriamight play a role in their adaptation to heat. Tuberizationwas enhanced by as much as 63% in bacteria-treated clones.Moreover, inoculation of grapevine (Vitis vinifera) with thesame bacterium, that is, B. phytofirmans PsJN, lowered therate of biomass reduction and electrolyte leakage – an indi-cator of cell membrane injury – during cold treatment (4 °C),and promoted post-chilling recovery (Barka et al. 2006).Theability of plants to withstand cold can be enhanced uponexposure to low, non-freezing temperatures. Among otherphysiological changes, increases in sugar, proline and antho-cyanin contents can be observed during this so-called coldacclimation or hardening. Grapevine plants inoculated withB.phytofirmans accumulated significantly higher amounts ofcarbohydrates compared to control plants.In addition, levelsof proline and phenols, rates of photosynthesis and starchdeposition were enhanced (Barka et al. 2006). Such physi-ological changes are also typical indicators for ISR, and theauthors, therefore, suggested that bacterially mediated tol-erance to low temperatures is positively correlated with theinduction of ISR.

Tolerance to nutrient deficiency and heavymetal toxicity

Nutrient elements, such as phosphorus, potassium, iron, zincand copper, possess limited mobility in the soil. In the caseof phosphorus, its insoluble form can be mobilized by plantexudates such as phosphatases and organic acids (Fig. 2f).Exuded carbohydrates also indirectly contribute to phos-phorus mobilization by serving as a carbon source forP-solubilizing microorganisms. Release of carbohydratesby P-deficient plants increased by 52% upon treatmentwith IAA, whereas no significant change was observedin plants sufficiently supplied with P (Wittenmayer &Merbach 2005). This led to the speculation that bacteriallyproduced IAA could also provoke higher amounts of plantcarbohydrate exudates, and, therefore, result in a betternutrient status of the bacteria. In turn, the bacteria are ableto mobilize more P; thus, plant growth-promoting effects ofP-solubilizing bacteria would be more pronounced underP-deficient conditions.

Inoculation with Pseudomonas alcaligenes PsA15,Bacillus polymyxa BcP26 and Mycobacterium phleiMbP18, respectively, promoted growth and nutrient uptakein maize. In nutrient-deficient calcisols, however, promotionof nitrogen, phosphorus and potassium uptake was found tobe much greater than on rich loamy sand soil, where stimu-lation was only detected in root growth, as well as in N andK uptake (Egamberdiyeva 2007).

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Many root zone bacteria release into the rhizospheremetal-chelating substances, such as iron-chelating sidero-phores (Fig. 2d). Siderophore-producing bacteria havebeen shown to influence plant uptake of various metals,including iron, zinc and copper (Carrillo-Castaneda et al.2003, 2005; Egamberdiyeva 2007; Dimkpa et al. 2009b). Thisway, rhizobacteria can also impact on the bioavailability ofheavy metals that can be toxic to plants in low concentra-tions. Because soil conditions influence metal valencies,microorganisms also affect metal bioavailability by acidifi-cation of the micro-environment (Fig. 2f), and by influenc-ing changes in redox potential (Smith & Read 1997; Gadd2004). Autotrophic and heterotrophic leaching, volatiliza-tion through methylation and release of chelators can mobi-lize metals, whereas sorption to cell components followedby intracellular sequestration or precipitation as insolubleorganic or inorganic compounds reduces heavy metalmobility (Fig. 2c) (White, Sayer & Gadd 1997; Gadd2004). Barley plants grown on cadmium-contaminated soilobtained 120% higher grain yield and twofold decreasedCd contents in grains when inoculated with the commer-cially available PGPR Klebsiella mobilis CIAM 880. Simu-lation of these effects with a mathematical model showedthat it is a complex process, with one of the underlyingmechanisms being bacterial migration from rhizoplane torhizosphere. Here, free Cd ions can be bound by bacteriainto complex forms that cannot be taken up by the plant(Fig. 2c) (Pishchik et al. 2002). Moreover, all bacterialstrains tested in the study possessed nitrogen-fixing ability,and produced IAA or ethylene, characteristics, which, mostlikely, played key roles in mediating stress tolerance.Indeed, the possession by some bacteria of cell wall com-ponents with metal-binding properties (Beveridge, Fors-berg & Doyle 1982) may help in bacterial accumulation ofmetals such as Cd. In turn, this feature can contribute in thereduction of Cd uptake by plants with which such bacteriaare associated (Ganesan 2008; Sinha & Mukherjee 2008).In contrast, in Brassica juncea inoculated with IAA- andsiderophore-producing bacteria, chromium uptake was notaltered, whereas chromium tolerance was enhanced (Raj-kumar et al. 2005). Indeed, the ability of some bacteria toprotect plants against either nickel, lead or zinc toxicity haspreviously been shown to be related to the production ofsiderophores (Burd et al. 2000; Dimkpa et al. 2008). Becausemicrobial iron–siderophore complexes serve as an ironsource for both monocot and dicot plants (Reid, Szaniszlo& Crowley 1986; Bar-Ness et al. 1991; Dimkpa et al.2008), iron deficiency symptoms, genuine or metal induced,common in plants grown under high heavy metal concen-trations can be prevented (Burd et al. 2000; Dimkpa et al.2008, 2009a). However, iron, although essential, can causeoxidative stress in plants when absorbed in high concentra-tions. Indeed, iron intoxication, similar to other metals, wasshown to cause the modification of enzymes related to oxi-dative stress (Sinha & Saxena 2006; Stobrawa & Lorec-Plucinska 2007; Dimkpa et al. 2009a). However, for the firsttime, microbial siderophores were shown to be able to alle-viate metal-induced oxidative stress in plants. Apparently,

by chelating and reducing toxic metal concentrations inthe root zone, siderophores exerted a bioprotective effectby lowering the formation of cell-damaging free radicals,thereby enabling a microbial IAA-mediated plant biomassincrease, which contributed in the dilution of toxic metaleffects on the plants (Dimkpa et al. 2009a). Iron toxicity is aserious constraint in wetland rice production. Preliminaryfindings, however, indicate that several Bacillus strainspossess the ability to mitigate the symptoms of iron toxicityin rice (Asch & Padham 2005; Terré et al. 2007). The under-lying mechanisms still remain elusive, although they seemto depend on the Bacillus strain and on the rice genotype.Bacillus sp., as with most other bacteria, possess SODswhich, as mentioned previously, are key enzymes involvedin the alleviation of oxidative stress in living systems.Similar to plants, bacterial SODs play an important role intheir survival in the rhizosphere, which, as noted (Wanget al. 2007), is an environment characterized by free radical-generating activities. As such, bacterial survival ensurestheir contribution to the amelioration of abiotic stress, andhence, to plant growth promotion.

APPLICATIONS

Biofertilization

Because of their ability to enhance the nutrient status oftheir host plants, the term ‘biofertilizer’ is often applied.According to Vessey (2003), biofertilizers are defined assubstances containing living organisms which, when appliedto seeds, plant surfaces or soil, colonize the rhizosphere orthe interior of the plant and promote growth by increasingthe supply or availability of primary nutrients to the host.The author points out that all PGPRs promoting plantgrowth by positively affecting the nutrient status of theirhosts should be referred to as biofertilizers; although it hadbeen previously proposed that PGPRs which do not replacethe nutrients in the soil should not be included in thisterm (Okon & Labanderagonzalez 1994). Most commonly,bacteria such as Azospirillum, Herbaspirrilum, Acetobacter,Azotobacter and Azoarocus are used as biofertilizers,mainly because of their ability to fix atmospheric nitrogen.Various inoculant mixtures from these bacteria are cur-rently commercially available for agricultural applications.Other species of bacteria such as Bacillus, Streptomyces andPseudomonas which have shown a positive effect on plantbiofertilization could be developed for commercialization.

Phytoremediation

Anthropogenic pollution of soils began with the industrialrevolution, and has since become a major issue in globaleconomy, public health and environmental conservation(White et al. 1997). Industrial waste, traffic, mining, agricul-tural and military activities are some of the major sourcesfor the release of xenobiotic organic contaminants(fuel, solvents, explosives, chemical weapons, pesticides,herbicides, etc.) or inorganic pollutants (heavy metals or

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radioactive isotopes) into the environment (reviewed inPilon-Smits 2005). Phytoremediation is a method to cleanup contaminated soils based on the ability of certain plantsto stabilize, extract, degrade or volatilize pollutants (Pilon-Smits 2005). Despite this ability, plants often suffer fromthe toxic effect of metals which affects their phytoremedia-tion potential (Dimkpa et al. 2009a,b) However, supple-mentation of such plants with soil bacteria can enhancephytoremediation, hence the term microbe-assisted phy-toremediation (White 2001; Glick 2003). In comparison toconventional clean-up methods where the soil is removedand cleaned in complicated technical processes, phytoreme-diation is very cost-effective and non-destructive to the soilstructure. Ideally, plants used for phytoremediation shouldproduce high amounts of biomass and/or hyper-accumulatethe contaminant. However, although some plants may tol-erate high amounts of a pollutant, their growth might stillbe constrained by such pollutants (Dimkpa et al. 2009a).Thus, rhizobacteria enhancing the tolerance of plants tohigh concentrations of a pollutant and/or promoting plantgrowth could provide a useful tool for making the processof phytoremediation more efficient (Glick 2003; Dimkpaet al. 2009a,b).

Commonly with metal-induced oxidative stress, ACC-induced ethylene production is a major factor affectingplant growth in metal-polluted environments, leading toreduced plant biomass and, therefore, lowered phytoreme-diation efficiency (Arshad, Saleem & Hussain 2007 and ref-erences therein). As plants do not possess ACC deaminaseactivity, transgenic plants have been developed with thiscapability, based on bacterial ACC deaminase genes. Suchapplications have contributed in enhancing plant growth,tolerance to metal stress and ultimately, to augmentingmetal accumulation for phytoremediation (Arshad et al.2007; Zhang et al. 2008).

Bioprotection

Some of the root-colonizing bacteria which have beenshown to provide protection against different types of bioticstresses can also enhance a plant’s tolerance to abioticstresses. For example, P. fluorescens TDK1, Pseudomonasputida UW4, Bacillus sp. and Arthrobacter sp. have beenshown to enhance resistance against various soil-bornepathogens, and also mitigate salt, as well as drought, stressin different plants (Mayak et al. 2004a,b; Haas & Defago2005; Saravanakumar & Samiyappan 2007; Barriuso et al.2008).

Bacillus subtilis is widely known for its biocontrol prop-erties (reviewed in Kloepper, Ryu & Zhang 2004) and hasrecently been shown to enhance tolerance to iron toxicity(Asch & Padham 2005; Terré et al. 2007). Similarly, sidero-phore production by beneficial bacteria, which protectsplants against pathogenic bacteria through better competi-tion for iron,has also been shown to be able to protect plantsfrom metal-induced oxidative stress (Dimkpa et al. 2009a).Ideally, however, future commercially available biocontrolagents should simultaneously provide cross-protection

against various stress factors, making agricultural systemsenvironmentally and economically more sustainable byreducing the need for pesticides, irrigation and otherecologically problematic and costly crop managementstrategies.

OUTLOOK

In times of global warming, agricultural production systemsare liable to changing environmental conditions. Root-colonizing bacteria have been shown to be able to mediateenhanced resistance to biotic stressors, as well as increasetolerance to abiotic stresses in host plants. Thus, specificidentification of bacterial strains that have the potential ofsimultaneously providing cross-protection against multiplestress factors would be highly valuable. Induction of ISR inthe host plants may be critical for bacteria’s ability to miti-gate both biotic and abiotic stress effects. Thus, knowledgegenerated from studies on ISR against plant pathogenswill be very useful in decoding signalling cascades inducedby root zone bacteria, resulting in enhanced tolerance toabiotic stresses. It is important to investigate in more detailif and how mechanisms triggering ISR and those leading toenhanced tolerance against abiotic stresses overlap. To thisend, research should concentrate on bacterial species thatare already known to be useful antagonists to biotic stres-sors, to investigate their capacity to mitigate abiotic stress.Many Bacillus strains, for instance, have been shown toprovide protection against a broad range of biotic stressors,such as pathogenic fungi, bacteria or insects and nematodes(see e.g. Kloepper et al. 2004). However, research onBacillus-mediated tolerance to abiotic stresses is stilllimited.

This review has shown that among abiotic stresses, thereare some common principles at work, particularly withregard to root/soil interactions and the physiologicalchanges in the root system induced by microorganisms,namely:

• Stimulation of root growth through bacterially producedIAA (and/or nitric oxide) under drought, nutrient defi-ciency, salinity and metal toxicity stress;

• Decrease in host plant stress ethylene level by bacterialACC deaminase activity under drought, salinity, heavymetal toxicity and flooding conditions; and

• Induced changes in cell wall/cell membrane underdrought and suboptimal temperature conditions.

Based on the knowledge generated up to now, we suggestseveral future avenues of research approaches: (1) investi-gating whether the underlying mechanisms of bacterialeffects are the same for different stress situations; (2) iden-tification of the physiologically active substances providingprotection against both biotic and abiotic stresses, oragainst two or more abiotic stresses, and, for those metabo-lites that are already known to be effective, to elucidatetheir modes of action in the plant’s physiology; and (3)identification of the traits, and thus the genetic backbone,that are responsive to bacterial effects across stresses.

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The successful use of rhizobacteria in applications, suchas phytoremediation, biofertilization or cross-protectionwill much depend on the ability to establish the desiredstrains in the already existing community of soil microor-ganisms. Certain rhizobacteria may show beneficial effectson plant growth and or a plant’s tolerance to biotic orabiotic stresses in greenhouse experiments under con-trolled conditions; however, these effects may not be detect-able in a natural environment.There are, for instance, manyreports on in vitro solubilization of phosphorus that couldnot be validated under field conditions (see e.g. Gyanesh-war et al. 2002). Variables such as soil type and chemicalproperties, natural selection, as well as agricultural manage-ment practices such as crop rotation or application ofpesticides are among the important factors influencing colo-nization with pre-selected beneficial microorganisms. Suc-cessful establishment of a certain inoculate might follow thefirst-come-first-served principle. The earlier in the plant’sdevelopment a bacterium can be established, the betterthe chances that it might out-compete others. Some plantspecies, however, contain bacteria under the seedcoat or within the embryo. Such bacteria will still have tocompete with beneficial bacteria, even when the latter areapplied as early as seed stage. Thus, providing a continuoussupply of carbon and energy sources will contribute to asuccessful establishment of the beneficial bacteria (Zahir,Arshad & Frankenberger 2004). Moreover, breeding effortsshould consider the abilities of plants to interact withbeneficial bacteria in order to promote specific beneficialplant–bacteria combinations. Studies on the practicabilityof the application of beneficial microorganisms in real-lifeagricultural systems are, thus, urgently needed.

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Received 31 May 2009; received in revised form 15 July 2009;accepted for publication 15 July 2009

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