Chapter 93

Abiotic Stress Remediation by theArbuscular Mycorrhizal Symbiosisand Rhizosphere Bacteria/YeastInteractions

ROSARIO AZCÓN, ALMUDENA MEDINA, RICARDO AROCA andJUAN M. RUIZ-LoZANO

Departamento de Microbiología del Suelo y Sistemas Simbióticos, EstaciónExperimental del Zaidín (CS/C), Spain

93.1 INTRODUCTION

The root-soil interfaces constitute a dynamic microcosmknown as the rhizosphere where rnicroorganisms, plantroots, and soil constituents interact. The rhizosphere is aphysical, chemical, and biological environment, where analtered microbial activity is characteristic (see Chapter 4).Moreover, rhizosphere functioning is known to markedlyinftuence plant fitness and soil quality, because micro-bial developments in such environment can help the hostplant to respond to both biotic and abiotic stress conditions(Barea et al., 2005).

Drought and heavy metal contaminations are impor-tant abiotic constraints for plant development and survival.Drought has a major impact on plant growth and develop-ment, lirniting crop production worldwide (Bray, 2004).The severity of drought is unpredictable as it dependson many factors such as occurrence and distributionof rainfall, evaporative demands, and moisture-storingcapacity of soils (Farooq et al., 2009). In addition, theglobal climate change is contributing toward spreadingthe problem of water deficit to regions where drought wasnegligible in the past (see Chapters 98, 99). On the otherhand, industrial and anthropogenic activities ha ve causedpollution by toxic metals in agricultural soils, water, and

the atmosphere (Amoozegar et al., 2005). Unfortunately,metal s cannot be biodegraded. However, microorganismscan interact with these contaminants and transform themfram one chemical forrn to another by changing their oxi-dation state (Tabak et al., 2005). Accumulation of metalsby plants (phytoremediation) is becoming an additionalestablished route for the bioremediation of metal contam-ination (Tabak et al., 2005; see Chapters 94, 95). Thus,the use of beneficial rhizosphere microorganisms thatenhance plant drought tolerance and/or protect the plantsagainst heavy metal toxicity is key to improve crops andto guaranty world food production (Chaves and Oliveira,2004) Plants usually interact with soil microorganismsthat make the plants more efficient in coping withenvironmental stresses such as drought or heavy metalcontamination. These include arbuscular mycorrhizalfungi (AMF; see Chapter 43), plant-growth-promotingrhizobacteria (PGPR; see Chapter 53), and yeasts.

Several ecophysiological studies have demonstratedthat AM (arbuscular mycorrhizal) symbiosis (Chapter 4)improves plant tissue hydration and physiology underdrought stress conditions, this effect being the result ofaccumulative physical, nutritional, physiological, andcellular effects (reviewed by Augé, 2001; Aroca et al.,2011; Ruíz-Lozano et al., 2006). In the same way, it has

Molecular Microbial Ecology of (he Rhizosphere, Volume 2, First Edition. Edited by Frans J. de Bruijn.© 2013 John Wiley & Sons, Ine. Published 2013 by John Wiley & Sons, Ine.

991

992 Chapter 93 Abiotic Stress Remediation by the Arbuscular Mycorrhizal Symbiosis

been shown that AM symbiosis improves plant toleranceto heavy metal contamination (Hildebrandt et al., 2007;Joner et al., 2000b). PGPR have also been reported toenhance plant tolerance to drought (Arkhipova et al.,2007; Zahir et al., 2008; see Chapters 54, 55), as wellas to ameliorate heavy metal stress (Khan et al., 2009;Tabak et al., 2005). There is no information about thedirect effects of yeasts on plant tolerance against theseabiotic stresses. However, there are some reports showingenhanced plant biomass production after inoculationof AMF and yeasts (Gollner et al., 2006) the stimula-tion of AM root colonization by yeasts (Singh et al.,1991; Vassilev et al., 2001), or the improvement ofsoil characteristics after inoculation with a yeast strain(Medina et al., 2004a,b). Finally, a recent report showedalleviation of metal toxicity in plants by coinoculationwith AMF, Bacillus cereus and the yeast Candidaparapsilosis (Azcón et al., 2010). Furthermore, there isample evidence that direct interactions occurring amongmycorrhizal fungi and other soil microorganisms oftenresult in the promotion of key processes benefitingplant growth and health (Aroca and Ruíz-Lozano,2009; Barea et al., 2005). Thus, this chapter focuses onrecent advances in the combined use of AMF and otherbeneficial rhizosphere bacteria or yeast to improve planttolerance to drought or heavy metal contamination.

93.2 DROUGHT STRESSREMEDIATION BY AM SYMBIOSISAND RHIZOSPHEREBACTERIAlVEAST INTERACTIONS

There is ample literature showing a positive effect onplant drought tolerance when AMF and PGPR are inoc-ulated together (Kohler et al., 2009; Marulanda et al.,2009; Ruíz-Sánchez et al., 2011; Valdenegro et al., 2001).The mechanisms related to this effect can be several, butimprovements in plant root development and water uptakecapacity, alteration of plant hormonal balance, and protec-tion against the oxidative stress generated by drought seemto be involved. There are also several reports emphasizingthe importance of the origin and activities of microorgan-isms used as inoculants for the final outcome of the inter-action. In the case of coinoculation of AMF and yeasts,the stimulation of mycorrhization may be the main benefitof the interaction.

93.2.1 Importance of the Originand Activities of the Microbes in theInoculantsThe effects of interactions between a drought-adaptedstrain of Bacillus thuringiensis and two isolates of the

AMF Glomus intraradices Can indigenous drought-tolerant and a nonindigenous drought-sensitive isolate) onthe legume Retama sphaerocarpa have been investigated(Marulanda et al., 2006). The beneficial microbial effectson Retama plants were more relevant when indigenousmicroorganisms were involved. Thus, Retama plants col-onized by G. intraradices plus Bacillus possessed similarshoot length after 30 days from sowing compared withnoninoculated Retama plants after 150 days. Inoculationwith drought-adapted bacteria increased root growth by201 %, but maximum root development was obtainedby coinoculation of B. thuringiensis and the indigenousG. intraradices. The relative water uptake was higher ininoculated than in noninoculated Retama plants, and theseinoculants depleted soil water content concomitantly.Indigenous G. intraradices-colonized roots (evaluatedby functional alkaline phosphatase staining) showed thehighest intensity and arbuscule richness when associatedwith B. thuringiensis. Coinoculation of autochthonousmicroorganisms reduced by 42% the water requiredto produce 1 mg of shoot biomass. These findingsevidence the effectiveness of a rhizosphere bacterium toincrease plant water uptake when inoculated singly or incombination with an AMF.

In the same way, Marulanda et al. (2008) studiedhow the interaction between three different AMF isolates(Glomus constrictum autochthonous, GcA; G. constrictumfrom collection, GcC; and a commercial G. intraradices,Gi) and a Bacillus megaterium strain isolated from aMediterranean ca1careous soil affected Lactuca sativa L.plant growth. Inoculation with B. megaterium increasedplant growth when in combination with two of theAMF isolates (GcA and Gi), but decreased it whenin combination with Gcc. At the same time, plantsinoculated with the GcC fungus alone or in combinationwith B. megaterium (GcC + Bm) showed symptoms ofleaf stress injury with the accumulation of proline andreduction in the amount of photosynthetíc pigrnents. Theopposite occurred in plants coinoculated with Gi fungusand B. megaterium (Gi +Bm). GcC +Bm leaves alsopresented the highest glucose-6-phosphate dehydrogenase(G6PDH) and the lowest glutamine synthetase (GS)enzymatic activities, whereas Gi + Bm leaves showed thehighest GS activity.

Subsequently, Marulanda et al. (2009) conducted teststo see whether rhizosphere microorganisms could increasedrought tolerance in plants growing under water-limitedconditions. Three indigenous bacterial strains isolatedfrom drought-exposed soil and identified as Pseudomonasputida, Pseudomonas sp., and B. megaterium were ableto stimulate plant growth under dry conditions. Whenthe bacteria were grown in axenic culture at increasingosmotic stress caused by polyethylene glycol (PEG)levels (from 0% to 60%) they showed osmotic tolerance.

93.2 Drought Stress Remediation by AM Symbiosis and Rhizosphere Bacteria/Yeast Interactions 993

P. putida and B. megaterium exhibited the highestosmotic tolerance and both strains also showed increasedproline contento These bacteria seem to have developedmechanisms to cope with drought stress. The increasein IAA production by P. putida and B. megaterium at aPEG concentration of 60% is an indication of bacterialresistance to drought. Their inoculation increased shootand root biomass and water content under droughtconditions, which may be explained by bacterial IAAproduction (see also Chapter 27). B. megaterium was themost efficient bacteria under drought either applied aloneor associated with the autochthonous AMF Glomus coro-natum, G. constrictum, or Glomus claroideum (Fig. 93.1).The authors proposed that microbial activities of adaptedstrains represent a positive effect on plant developmentunder drought conditions.

Genetically modified (GM) strains of PGPR have alsobeen tested in combination with AMF. Vázquez et al.(2001) studied the effects of double inoculation ofMedicago plants with two strains of Sinorhizobiummeliloti (the wild-type (WT) strain GR4 and its GMderivative GR4(pCK3), which constitutively expressesthe nifA gene), and two species of AMF. The efficiencyof each AMF in increasing plant growth, nutrient content,nodulation, and water stress tolerance was related to theSinorhizobium strains and Medicago species. Mycorrhizalplants nodulated by the WT strain accumulated moreproline in Medicago plants under water stress thannonrnycorrhizal plants. Conversely, mycorrhizal plantsnodulated by the GM strain accumulated less proline inresponse to both AM colonization and drought. However,mycorrhizal plants nodulated by the GM Sinorhizobiumstrain suffered less from the detrimental effect of waterstress, since these plants maintained unaItered the relativeplant growth, percentage of AM colonization, root dryweight, and the rootlshoot ratio.

4000

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Figure 93.1 Shoot dry weight (mg) of Trifolium plants inoculatedwith AM only (control) or coinoculated with each of theautochthonous drought-adapted bacterial strains (Pseudomonas sp.(Pseud.), P. putida (P. put) or B. megaterium (B. meg)) and AMfungus. Adapted from Marulanda et al. (2009).

In another study, the effects of single and combinedinoculations of Medicago arborea with three Glomusspecies, the same WT and GM S. meliloti strains men-tioned in the preceding text and a PGPR (Enterobactersp.) were evaluated by Valdenegro et al. (2001). Mycor-rhizal fungi were effective in increasing plant growth inall cases, but PGPR inoculation was only effective whenassociated with specific mycorrhizal endophytes. Thus,inoculation with Enterobacter sp. increased growth ofGlomus mosseae-colonized plants associated with the WTstrain by 36% and those infected by Glomus deserticolawhen associated with the GM rhizobial strain by 40%.The authors also found that some combinations of AMF,Enterobacter, and rhizobia increased the nodule numberpresent in the roots.

93.2.2 Influence on Root WaterUptake and Hydraulic PropertiesIt is well documented that PGPR and AMF can induceplant tolerance to osmotic stresses, such as drought orsalinity (Kohler et al., 2009; Liddycoat et al., 2009; Ruíz-Lozano et al., 2006). The beneficial effects of PGPR andAMF inoculation under osmotic stress conditions are notonly recorded as an increment of plant biomass, but alsoas an improvement in water status (Aroca et al., 2007;Kohler et al., 2009; Ruíz-Lozano et al., 2009). Thus, thereare reports showing that inoculation with PGPR and/orAMF can modulate aquaporins and root hydraulic con-ductivity (Marulanda et al., 2010; Ruíz-Lozano and Aroca,2010).

Recently, there has been increasing interest in plantaquaporin regulation by AM symbiosis because it maybe involved in the modulation of root hydraulic proper-ties under drought conditions (Ruíz-Lozano and Aroca,2010). The results obtained so far on the regulation of PIPaquaporins by the AM symbiosis show that the effects ofthe symbiosis on aquaporin genes depend on the intrinsicproperties of the osmotic stress, on the plant species stud-ied, and on the specific aquaporin gene analyzed. In anycase, the induction or inhibition of particular aquaporinsby AM symbiosis should result in a better regulation ofplant water status and contribute to the global plant resis-tance to the stressful conditions (Jang et al., 2004), asevidenced by their better growth and water status underconditions of water deficit. In addition, the resuIts obtainedby Uehlein et al. (2007) suggest that the role of aquaporinsin the AM symbiosis could be more complex than simplyregulating plant water status. They described the inductionby the AM symbiosis of specific PIP and NIP aquaporinisoforms exhibiting permeability to water and arnmonia,respectively. The authors suggested that these aquaporinscould be involved in the symbiotic exchange processesbetween the fungus and plant. Indeed, the importance of

994 Chapter 93 Abiotic Stress Remediation by the Arbuscular Mycorrhizal Symbiosis

aquaporins for both nutrient and water exchanges dur-ing mycorrhizal symbioses has been recognized recently(Maurel and Plassard, 2011).

Marulanda et al. (2010) conducted an experiment withmaize plants inoculated with a B. megaterium strain pre-viously isolated from a degraded soil and characterized asa PGPR. Inoculated plants exhibited higher root hydraulicconductance (L) values under both unstressed and salt-stressed conditions (see Chapter 55). These higher L val-ues in inoculated plants correlated with higher PIP2 aqua-porin amounts in their roots under salt-stressed conditions.Also, ZmPIP1;1 protein amount under salt-stressed con-ditions was higher in inoculated leaves than in noninocu-lated ones. Hence, the authors proposed that the differentregulation of PIP aquaporin expression and abundance bythe inoculation of maize plants with the B. megateriumstrain could be one of the causes of the enhanced salttolerance in the plants.

Alguacil et al. (2009) analyzed how inoculation withthe AMF G. intraradices or with the PGPR Pseudomonasmendocina modulated the expression of a PIP2 gene in let-tuce plants subjected to drought. Results showed that bothmicrobial treatments alleviated drought in lettuce plants.However, the PIP2 gene expression was increased onlyby the AMF but not by the PGPR under these condi-tions. Unfortunately, no measurement of root hydraulicconductivity was carried out in this study. Thus, to ourknowledge, no studies have analyzed the effect of com-bined inoculation with AMF and PGPR on aquaporins androot hydraulic properties under drought conditions and thisaspect should be evaluated in the near future.

93.2.3 Evidence for Altered PlantHormonal BalanceSoil biota may improve crop drought tolerance via severaldiverse mechanisms, including the production or degrada-tion of the major groups of plant hormones that regulateplant growth and development (Dodd et al., 2010). Planthormones (abscisic acid (ABA), auxins, cytokinins (CKs),ethylene, gibberellins (GAs), jasmonic acid (JA), salicylicacid (SA» regulate multiple physiological processes in theplant, including root initiation, elongation, architecture,and root hair formation. They typically operate in complexnetworks involving cross talk and feedback (Dodd et al.,2010).

Although microorganisms can directIy affect rhizo-sphere hormone concentrations (by uptake of hormones ortheir precursors as carbon and nitrogen sources and effiuxof the synthesized hormones), there is increasing evidencethat microorganisms affect root hormone concentrationsand can also alter root-to-shoot long-distance signaling(Belimov et al., 2009), thus altering the plant hormonestatus (Dodd et al., 2010). For instance, Arkhipova et al.

(2007) found that the beneficial effect on lettuce plantgrowth under water-limited conditions caused by theinoculation with a Bacillus sp. strain was related to anincreased level of CKs in these plants. Arkhipova et al.(2007) also found that Bacillus sp.-inoculated lettuceplants had also increased amounts of ABA when com-pared with noninoculated plants. Moreover, Cohen et al.(2008) recentIy found that the PGPR Azospirillumbrasilense is able to synthesize ABA in vitro and increaseits production in the presence of NaCl. Arabidopsis plantsinoculated with Azospirillum had higher ABA contentthan noninoculated ones (Cohen et al. 2008).

One of the mechanisms by which ABA enhancesplant drought tolerance is via regulation of leaf tran-spiration and root hydraulic conductivity (Aroca et al.2006). However, although Arkhipova et al. (2007) foundan increase of ABA levels in lettuce plants inoculatedwith Bacillus sp., the authors did not find any differencein stomatal aperture between inoculated and noninoc-ulated plants. This behavior could be caused by thecounterbalance of higher levels of CKs, since the ratiobetween ABA and CKs determines stomatal aperture(Arkhipova et al., 2007). Regarding root hydraulicconductivity, Sarig et al. (1992) showed a positive effectof a PGPR (A. brasilense) inoculation on root hydraulicconductivity under control and osmotic stress conditions.More recentIy, Marulanda et al. (2010) also showedhigher root hydraulic conductivity in maize plants inoc-ulated with a B. megaterium strain previously isolatedfrom a degraded soil and characterized as a PGPR.

Another mechanism involved in plant drought toler-ance induction by PGPR is the dirninution of ethylenepraduction. PGPR containing l-aminocyclopropane-l-carboxylate (ACC) dearninase enzyme, which catabolyzesthe ethylene precursor, are able to diminish the ethylenecontent in plant tissues under drought stress, favoring theplant growth and improving water status (Mayak et al.,2004). Thus, decreased ethylene levels increase planttolerance to environmental stresses and promote legumenodulation, but the role of ethylene in mycorrhizal sym-biosis establishment and functioning is still controversial.Gamalero et al. (2008) inoculated cucumber plantswith the ACC deaminase-producing strain P. putidaUW4 Acdx" and its mutant Acd'S"; impaired in ACCdeaminase synthesis, alone or in combination with theAMF Gigaspora rosea. The AcdS+ strain, but not theAcdS- mutant, increased AM colonization and arbusculeabundance. The mycorrhizal fungus, but not the bacterialstrains, pramoted plant growth. However, the AcdS+strain, inoculated with G. rosea, induced synergisticeffects on plant biomass (Fig. 93.2), total root length,and total leaf area. These results suggest a key role ofthis enzyme in the establishment and activity of AMsymbiosis. These results have been confirmed recently by

93.2 Drought Stress Remediation by AM Symbiosis and Rhizosphere BacteriaNeast Interactions 995

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Figure 93.2 Shoot dry weight of cucumber plants inoculated ornot inoculated with the ACC deaminase-producing Pseudomonasputida UW4 Acd'S", its defective mutant Acds:', and G. rosea, aloneand in cornbination. Adapted from Gamalero et al. (2008).

Martín-Rodríguez et al. (2011), who have found that adual ethylene-dependentethylene-independent mechanismis associated with ABA regulation of the AM formationand functioning.

93.2.4 Evidence for ReducedOxidative DamageDuring drought stress, different metabolic pathways areuncoupled and electrons, which have a high energy state,are transferred to molecular oxygen to form reactive oxy-gen species (ROS). ROS, such as 102, H202, O2-, andHO·, are toxic molecules capable of causing oxidativedamage to proteins, DNA, and lipids (Miller et al., 2010).

Some studies have shown a substantial reductionin oxidative damage to lipids and proteins in nodulesof mycorrhizal soybean plants subjected to drought ascompared to the nodules of nonmycorrhizal plants (Ruíz-Lozano et al., 2001). Such reduction in oxidative damageto biomolecules was considered the main mechanism bywhich the AM symbiosis protected root nodules in legumeplants against premature nodule senescence induced bydrought stress. This could be caused by a higher activityof the antioxidant enzyme glutathione reductase (GR) inthe nodules of the AMF-colonized roots (Porcel et al.,2003) (Fig. 93.3). The GR is an important component ofthe ascorbate-glutathione cycle since it is the enzymethat regenerates oxidized glutathione into its reduced form(Noctor and Foyer, 1998). However, this beneficial effectcouId also be related to the lower water deficit sufferedby the nodules of the AMF-colonized plants (Porcel et al.,2003) or to alterations in carbon metabolism of nodules byAM symbiosis, as was observed in roots of Anthyllis cyti-soides under drought conditions (Goicoechea et al., 2005).

Kohler et al. (2008) exarnined the effects of inocula-tion with the PGPR P. mendocina alone or in combinationwith two AMF on antioxidant enzyme activities (superox-ide dismutase, catalase (CAT), and total peroxidase (POX)

activities, phosphatase and nitrate reductase activities, andsolute accumulation in lea ves of L. sativa subjected to waterstress. At moderate drought, bacterial inoculation and myc-orrhizal inoculation with G. intraradices, alone or com-bined, stimulated nitrate reductase activity significantly.At severe drought, P. mendocina alone or in combinationwith either of the selected AMF, significantly increasedphosphatase activity in lettuce roots and proline accumula-tion in leaves. Total POX and CAT activities increased inresponse to drought, whereas superoxide dismutase activitydecreased. The combined inocu1ation of PGPR and AMFshowed the highest values ofleafPOX activity under severedrought, supporting the idea of the potential use of com-bined PGPRlAMF as inoculant to alleviate the oxidativedamage produced under water stress.

The same rnicrobia1 combination (P. mendocinaand/or G. intraradices) was also tested under saltstress (Kohler et al., 2009). The plants inoculated withP. mendocina had significantly greater shoot biomassthan the control plants at both salinity levels, whereas themycorrhizal inoculation treatments were only effectivein increasing shoot biomass at the lowest salinity level.Increasing salinity stress raised significantly the totalPOX and CAT activities of lettuce leaves. However, thePGPR strain induced a higher increase in antioxidantenzyme activities in response to severe salinity thaninoculation with the AMF. Thus, the author concludedthat the induction of antioxidant enzyme activities madeP. mendocina more effective in alleviating the negativeeffect of salinity than the AMF.

There is a recent report showing regulation ofhost antioxidant compounds by the AM symbiosis in

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Figure 93.3 Glutathione reductase (GR) activity in raots andnodules of soybean plants cultivated under well-watered (WW) ordrought stress (DS) conditions. Br, Bradyrhizabium japonicum;Gm + Br, Glomus mosseae plus B. japonicum. Reproduced framPorce! et al. (2003) with kind permission from John Wiley and Sons,

996 Chapter 93

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Abiotic Stress Remediation by the Arbuscular Mycorrhizal Symbiosis

A Control

Figure 93.4 Shoot ascorbate content in rice plants inoculated withArospirillum brasilense (A) and/or with the AM fungus Glomusintraradices (M) or remaining as uninoculated controls. Plants werecultivated under well-watered conditions (black columns) orsubjected to drought (white columns). Reproduced fromRuíz-Sánchez et al. (2011) with kind permission from EIsevier.

combination with the PGPR A. brasilense under droughtstress. Indeed, Ruíz-Sánchez et al. (2011) found anincrease of ascorbic acid (AsA) content in AM plantscoinoculated with A. brasilense (Fig. 93.4). Ascorbicacid is an important nonenzymatic antioxidant compoundsince it is involved in the removal of H202 by ascorbateperoxidases, which use AsA as an electron donor, and isclosely related to glutathione in the ascorbate-glutathionecycle (Noctor and Foyer, 1998).

93.2.5 Interactions between AMFungi and YeastsThere are a few reports of combined inoculation withAMF and beneficial yeasts. The first one was provided bySingh et al. (1991), who found that inoculation of severallegume plants with a commercial yeast (Saccharomycescerevisiae¡ enhanced the AM root colonization. Subse-quently, Vassilev et al. (2001) tested the applicability ofmicrobial inoculants entrapped in alginate gel. G. deser-ticola was inoculated into soil alone or in combinationwith the P-solubilizing yeast Yarowia lipolytica. Dualinoculation with entrapped G. deserticola and Y. lipoly-tica significantly increased plant dry weight, soluble Pacquisition, and mycorrhizal index. Moreover, the yeastculture behaved as a "mycorrhiza helper microorganism,"enhancing mycorrhization of tomato roots.

The same yeast (Y. lipolyticai in combination withan AMF was used by Medina et al. (2004a, 2004b) toassess the effectiveness of two microbiologically treatedagrowastes (dry olive cake (DOC) and/or sugar beet(SB» on plant growth, soil enzymatic activities, and othersoil characteristics in a natural soil from a desertifiedarea. Dorycnium pentaphyllum, a legume plant adapted tostress situations, was the test plant to evaluate the effect

of inoculation of a native AMF and/or Y. lipolytica onamended and nonamended soils. The effectiveness andperformance of the inocula applied was only evident inamended soils. AM colonization and spore number innatural soil were increased by amendments and the inoc-ulation with Y. lipolytica promoted this value. Enzymaticactivities such as urease, ~-glucosidase, phosphatase,and protease activities were particularly increased inDOC-amended soil, while dehydrogenase activity wasgreatest in treatments inoculated with Y. Iipolytica inSB-added soil. The authors concluded that the biologicalactivities in rhizosphere of agrowaste-amended soil werepositively affected not only by the nature of amendments,but also bythe inoculant consortium applied.

More recently, Gollner et al. (2006) examinedthe interactions between the AMF G. mosseae andG. intraradices and three soil yeast species (Candidasake, Cryptococcus aerius, and Williopsis californica).When the yeasts were applied alone, only C. sakeincreased maize growth, while dual inoculation withG. intraradices and either of the three yeasts showedpositive effects on plant biomass production.

Thus, it is clear that the result of the interactionsbetween AMF and yeasts depends on the combinationof these microorganisms and that the potential beneficialeffects of this microbial consortium to cope with abioticstresses may be the results of reciprocal microbial influ-ences and deserves further investigation.

93.3 HEAVY METAL STRESSREMEOIATION BY AM SYMBIOSISANO RHIZOSPHEREBACTERIAlYEAST INTERACTIONS

The main source of metal s in the environment is derivedfrom human activities, such as mining, agriculture, anddomes tic wastes. The lithogenic source represents onlya small fraction of the total input. The high metal con-centrations cause irreversible soil degradation, affectingits physical, chemical, and biological properties, whichlimit the vegetation establishment (Navarro et al., 2008).Heavy metal s at elevated levels are also toxic to mostplants, reducing their metabolism and plant growth.Metals cannot be degraded by biological, chemical, orphysical processes; however, they can be immobilizedor sequestered by soil microorganisms and higher plantsinvolving different metabolic mechanisms (Home, 2000;see also Chapters 94, 95, 96).

Phytoremediations are technologies that use plants(and their associated microorganisms) to remove, trans-fer, stabilize, decrease, and/or decompose pollutants in theenvironment (Denton, 2007; Lombi et al., 2001).

93.3 Heavy Metal Stress Remediation by AM Symbiosis and Rhizosphere BacteriaIYeast Interactions 997

Plants used for phytoremediation are those that aregenetically capable of growing in soils with a high metalconcentration (Marchiol et al., 2004). Their root activitiescan support a ftourishing microbial consortium includingfree-living as well as symbiotic rhizobacteria and mycor-rhizal fungi assisting phytoremediation in the rhizosphere(Wenzel et al., 2008). Soil microorganisms are often welladapted to survival in the presence of heavy metals andthey can interact and transform them by changing theiroxidation state (van Hullebusch et al., 2005).

Thus, the interactions between plant roots andmicrobes in the rhizosphere may have a great inftuenceboth on the increase in nutrient uptake and on the decreasein metal toxicity (Azcón et al., 2009a; Rajkumar et al.,2008).

93.3.1 Relevance of RhizosphereMicroorganisms inPhytoremediation of Metal-PollutedSoilsRhizosphere soil microorganisms are involved in diversebiochemical processes that enhance or accelerate reveg-etation, increasing the stability of polluted ecosystems(Moynahan et al., 2002). Soil microorganisms are alsoaffected by metals; however, continuous exposure tohigh concentrations of metals can induce tolerance andpromote the development of some specialized microbialpopulations, eventually positively affecting the wholesoil-plant-microbe ecosystem (de la Iglesia et al., 2006;Ramsey et al., 2005).

AMF association contributes to the establishmentand growth of plants, particularly under adverse condi-tions such as in sites highly polluted by metals. Thesefungi have developed several strategies to cope withthese adverse conditions and to confer resistance toplants against metals (Gohre and Paszkowski, 2006;Hildebrandt et al., 2007). Thus, the application of AMFfor remediation purposes is of great biotechnologicalinterest (Hildebrandt et al., 2007; Janousková et al., 2005;see Chapter 96).

On the other hand, heavy metal-resistant bacteria(from metal-polluted soil) are able to improve plantgrowth as they may reduce metal availability in themedium and many of them are PGPR (Sheng and Xia,2006; Zaidi et al., 2006). For instance, Brevibacillus sp.was able to produce in vitro 3.8 mg/l IAA, a mechanismfor plant growth promotion under heavy metals stress(Pishchik et al., 2002) and also induced a bigger rootsystem. Consequently, the amount of root exudates alsoincreased.

The mechanisms by which AMF and/or heavy metal-resistant bacteria may reduce heavy metal availability in

the plants are several: immobilization by chelating sub-stances secreted to soil; binding of metals to biopolymersin the cell wall, such as chitin and glomalin; superficialimmobilization in the plasmatic membrane once it crossesthe cell wall; membrane transporter that mobilizes metalsfrorn the soil to the cytosol; intracellular chelation thraughmetallothioneins (MTs; González-Guerrero et al., 2006),organic acids and arnino acids (Clemens, 2001); export ofmetals from the cytosol by membrane transporters; andconfinement of metals into the vacuoles. The storage ofmetals in spores has also been described in monoxenicculture (Ferrol et al., 2009). Vivas et al. (2003a) showedan important ability of Brevibacillus sp. for Pb biosorption(26% of the biomass weight) that may have contributedto Pb removal from soil, to alleviate Pb toxicity (see alsoChapter 95).

It is widely recognized that microorganisms can pos-itively interact in the soil and that these interactions canlead to an improvement of plant development and toler-ance to stress conditions such as those produce by heavymetal contamination. Autochthonous Brevibacillus brevisand G. mosseae isolated from Cd-polluted soil achievedfurther plant growth and nutrition and less Cd concen-tration (decreasing by 2.8-fold the percentage of Cd) atthe highest Cd level when they were coinoculated in thesystem. Much of the Cd was retained in the mycorrhizalroots and thus the translocation to the shoots was inhib-ited (Weissenhorn and Leyval 1995). Cd biosorption byB. brevis also seems to contribute to the effects describedin this study (Vivas et al., 2003b).

Different strategies might be involved in preventingplant toxicity damage. Changes in metal uptake and/orinternal transportation storage can confer metal toleranceto the host plant (Scheloske et al., 2004). The microbialinocula used (AMF and/or B. brevis) seem also to confertolerance to Ni by affecting the metal's availability anduptake, showing a lower amount of Ni absorbed per unitof root mass (Vivas et al., 2006a).

Zinc is a component in a number of enzymes andDNA-binding prateins, for example zinc-finger prateins,which exist in bacteria. As this metal only occurs as thedivalent cation Zn2+, it does not undergo redox changesunder biological conditions. When AMF colonize plants,they accumulate Zn in the roots such as observed byChen et al. (2003). On the other hand, bacteria canalso bind some amounts (5.6%) of this metal, and apartfrom being beneficial to plant growth and nutrition,the additive effects can explain the alleviation of thedetrimental effects caused by Zn in dually inoculatedplants (Vivas et al., 2006b).

G. mosseae isolated from polluted sites required bac-terial inftuence to achieve greater development. Spores ofG. mosseae demonstrated increased mycelial growth by56% (without Zn) and by 133% (with 200 u.g/Zn ml),

998 Chapter 93 Abiotic Stress Remediation by the Arbuscular Mycorrhizal Symbiosis

when inoculated with the bacterium as compared withuninoculated spores (Vivas et al., 2005).

Metal-adapted Brevibacillus strains have demon-strated metal biosorption ability, plant-growth- andmycorrhizae-helper activities. Thus, dual inoculation ofplants with native Brevibacillus strains and AMF seemsto be a strategy that can be recommended for promotingplant growth in heavy metal-polluted soils.

93.3.2 Microbial Metal Toleranceand Diversity in Metal-Polluted SoilsOne of the main mechanisms involved in the bacterialadaptation to heavy metals is the adsorption of metalsto the cell wall surface (biosorption). Bacteria in soil,with 106_109 viable cells per cubic centimeter have ahigh surface area-to-volume ratio and a high capacityfor capturing metals from solutions. The negative chargeof the wall cell caused by compounds such as freeamino acids together with hydroxylic, carboxylic, andother functional groups allows microorganisms to bindand to accumulate metal cations from the environment(Haferburg and Kothe, 2007); other important mecha-nisms are transportation and cellular incorporation of themetals (bioaccumulation) and transformation of metalsthrough reduction, oxidation, and methylation reactions.Microbial inoculation using strains adapted to the highheavy metal concentrations can restore the plant biomassvalues.

The occurrence of AMF and an effective mycorrhizasymbiosis in metal-polluted soils have been extensivelyreported (Chen et al., 2005; da Silva et al., 2005). Geneticadaptation has been observed in autochthonous AMFpopulations from polluted soils, which showed a highermetal tolerance than those isolated from nonpollutedsoils (Gildon and Tinker, 1983). Metal tolerance inAMF can be achieved through the action of severalhomeostatic mechanisms such as metal chelation atan intracellular level (González-Chávez et al., 2002;González-Guerrero et al., 2005, 2009). The best charac-terized metal chelators in AMF are the MTs (Vasak andHasler, 2000) that are involved in maintaining cellularhomeostasis against high metal concentrations (Cobbettand Goldsbrough 2002). Four AM fungal genes involvedin maintaining cellular homeostasis against metals thathave been characterized are (i) GrosMTl in G. rosea(Stornmel et al., 2001); (ii) the Zn transporter GinZnTl inG. intraradices involved in vacuolar Zn compartrnental-ization (González-Guerrero et al. 2005); (iii) GmarMTlin Gigaspora margarita (BEG 34), which codes for MTsthat regulate the fungal redox potential and protect itagainst the oxidative stress produced by some metals,such as Zn or Cd (González-Guerrero et al., 2006, 2007);and (iv) GintABC1, which codes for a polypeptide of

434 amino acids that participates actively in Cu and Zndetoxification (González-Guerrero et al., 2006).

In AM-colonized plants, the expression of genesencoding plasma membrane transporters affecting elementaccumulation by plants has been reported (Burleigh andBechmann, 2002). The expression of a Zn transportergene (MtZl P2) was decreased in roots of mycorrhizalplants at a high Zn concentration of 100mg/g asdescribed by Burleigh et al. (2003). The outer surface ofthe mycelium has a larger capacity for adsorbing metalsthan root cells. The presence of negatively chargedhydroxyl, carboxyl, and other functional groups capableof binding metallic ions, such as Cu2+ and Zn", has beenreported in AM mycelia (Joner et al., 2000a; Zhou, 1999).

The metal-binding capacity of glomalin, a proteinproduced by AMF, varies with soil type and some physic-ochemical parameters, such as pH and redox potentiaLCornejo et al. (2008) observed a high correlation betweenthe glomalin concentration and the content of Cu and Znin some Cu-polluted soils (r=0.89 and 0.76 for Cu andZn, respectively, P < 0.001). The metallinked to glomalincorresponded to 1.44-27.5% of the total Cu content and5.8% of the total Zn content in the soils. This evidencesuggests an active role of this protein's efficiency in miti-gating metal stress of microorganisms and plants growingin metal-polluted soils.

The diversity of AMF in metal-polluted soils mustbe examined to identify the suitable species that areeffective in remediation processes. The diversity of AMFecotypes in metal-polluted soils is low (Pawlowska et al.,1996), being mainly Glomus and Gigaspora species(da Silva et al., 2005, 2006). AMF would promotephytoextraction when the metal concentration in thesoil is low, while phytostabilization is enhanced at highmetal concentrations developing mechanisms that allowthe metal accumulation in plant roots and prevent itstranslocation to the shoot (Audet and Charest, 2006;Citterio et al., 2005; Janousková et al., 2005).

93.4 APPLlCATION OF SOILORGANIC AMENDMENTSTO ENHANCE PLANT STRESSALLEVIATION MEDIATEDBY SOIL-BENEFICIALMICROORGANISMS

Plant productivity can be seriously limited under harshenvironmental conditions derived from soil heavy metalcontamination and/or drought stress. To assure plantestablishment and survival of a stable plant cover in theseadverse conditions, application of organic amendmentsto the soil, before the inoculation of plant-beneficialmicroorganisms, has been recornmended (Vassilev et al.,

References

2006, 2007). The utilization of biologically transforrnedagrowastes, such as Aspergillus niger-treated SB wasteand DOC has been shown to improve the physicochemi-cal and biological properties of heavy metal contaminatedand/or desertified soils (Vassileva et al., 2010). Anincrease of soil enzymatic activities, aggregate stability,water-soluble C, and water-soluble carbohydrates hasbeen reported in soils amended with these biotransforrnedagrowastes (Medina et al. 2006). Moreover, a positiveeffect of these amendments on symbiotic parameters hasbeen observed, namely, an increase in the percentageof AM root colonization and diversity (Alguacil et al.,2011), enhancement of externa! AM hyphal development(Medina et al., 2007), increase of AM inoculum potential(Medina et al. 201Ob), and nodule formation (Med-ina et al., 2004a,b). Azcón et al. (2009b) also reported apositive interaction between A. niger-treated SB and AMFin increasing bacterial rnicrobial diversity of a heavymetal-contaminated soil. All these beneficial effects havebeen proposed to be related to the improvement of plantperformance observed in mycorrhizal plants grown inamended soils. Besides an increase of plant nutrition andgrowth, an enhancement of plant tolerance to drought orheavy metal stress has also been reported. Some of themechanisms involved in that tolerance are reduction ofheavy metal translocation to the shoots (Medina et al.,2006), an increase of the osmolytes proline and sugarcontent in plant tissues, and an increase of antioxidantenzyme activity (Caravaca et al., 2005; Medina et al.,2010a).

The combined treatments involving AMF inoculationand addition of organic amendments can be regarded asa successful biotechnological strategy of reclamation andreforestation of degraded and/or contarninated soils.

ACKNOWLEDGMENTS

Parts of this work were financed by MICINN-FEDERprojects (AGL2008-00898 and AGL2009-12530-C02-02).

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