REGULAR ARTICLE

Functionally diverse rhizobacteria of Saccharum munja(a native wild grass) colonizing abandoned morrummine in Aravalli hills (Delhi)

Meenakshi Sharma & Vandana Mishra &

Nupur Rau & Radhey Shyam Sharma

Received: 23 August 2010 /Accepted: 10 November 2010 /Published online: 27 November 2010# Springer Science+Business Media B.V. 2010

Abstract Characterization of the rhizobacteria ofnative grasses naturally colonizing abandoned minesites may help in identification of microbial inocu-lants for ecological-restoration programmes. Eightyone strains of Saccharum munja rhizobacteria isolatedfrom an abandoned mine located on Aravalli moun-tain and 50 from bulk-region were identified using16S rRNA sequence analyses. Based on chemical-and biological-assays they were categorized intoecologically diverse functional groups (siderophore-,IAA-, ACC-deaminase-, HCN-, polyphosphate-producers; phosphate-solubilizer; antagonistic). Eightgenera, 25 species from rhizosphere and 2 genera, 5species from bulk-region were dominated by Bacillusspp. (B. barbaricus, B. cereus, B. firmus, B. flexus, B.foraminis, B. licheniformis, B. megaterium, B. pum-ilus, B. subtilis, B. thuringiensis) and Paenibacillusspp. (P. alvei, P. apiarius, P. lautus, P. lentimorbus, P.polymyxa, P. popillae). Siderophore-producers were

common in rhizosphere and bulk soil, whereas IAA-producers, N2-fixers and FePO4-solubilizers dominatedrhizosphere samples. During the reproductive phase(winter) of S. munja, siderophore-, ACC-deaminase-and polyP-producers were predominant; howeverdominance of HCN-producers in summer might beassociated with termite-infestation. In vivo ability ofselected rhizobacteria (B. megaterium BOSm201, B.subtilis BGSm253, B. pumilus BGSm157, P. alveiBGSm255, P. putida BOSm217, P. aeruginosa BGSm306) to enhance seed-germination and seedling-growthof S. munja in mine-spoil suggest their significance innatural colonization and potential for ecological-restoration of Bhatti mine.

Keywords Abandoned mine . Functional diversity .

Plant growth promoting rhizobacteria . Saccharummunja . Ecological restoration .Bacillus

Introduction

Open cast mining leads to extinction of localecosystems and results in a heterogeneous denudedlandscape subjected to a multitude of abiotic stresses(Boyer and Wratten 2010). Consequent loss of above-and below-ground biodiversity creates an extremelyharsh environment which is non-conducible forregeneration of the native ecosystem (Requena et al.2001). It remains highly denuded for decades andbecomes a favourite place for weedy exotic species

Plant Soil (2011) 341:447–459DOI 10.1007/s11104-010-0657-y

Responsible Editor: Peter A. H. Bakker.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11104-010-0657-y) containssupplementary material, which is available to authorized users.

M. Sharma :V. Mishra :N. Rau : R. S. Sharma (*)Bioresources and Environmental BiotechnologyLaboratory, Department of Environmental Biology andCentre for Environmental Management of DegradedEcosystems (CEMDE), University of Delhi,Delhi 110 007, Indiae-mail: rads26@hotmail.come-mail: rssharma@cemde.du.ac.in

which further adds to the stress on the habitat. Lack ofvegetation enhances the surface albedo and increasesthe ambient temperature. Such changes affect thenative biodiversity which has high socio-economicvalue and makes the area unusable for the localcommunities. Therefore, abandoned mines are recog-nized as man-made barren sites which have becomeboth an ecological and economic burden on society.

Bhatti mines in the Delhi-Haryana region forms thelast spur of the Aravalli range (ancient foldedmountains in northern India) and occupies an over-all area of ∼6,200 ha. Bhatti area has undergonemassive open cast mining of feldspar (for preparationof high grade pottery) and subsequently for red sandstone or morrum (building material). The site isheterogeneous with intermixing of hard stable rocksand red sand mounds prone to erosion. The area islargely denuded after 50 years of abandonment andonce served as a watershed region for Delhi(Chatterjee et al. 2009). Due to the lack ofvegetation aquifers became choked and surfacerunoff is very high. This has caused extreme waterscarcity in South Delhi. To reverse these adverseeffects, restoration of abandoned mines into biolog-ically productive habitats using native species is amatter of prime concern for ecologists and urbanplanners. Selection of appropriate biological inputs(plants and microbes) has been central to suchefforts. Wild grasses have been considered ‘nursespecies’, which can colonize extreme of habitats(Rau et al. 2009; Shu et al. 2005). Establishment ofwild grasses having economic and ecological signif-icance would be a viable strategy to convert thebarren mines into biologically and sociologicallysignificant habitats. This could be facilitated byusing diverse functional groups of rhizobacteria,which contribute to ecological success of the wildgrasses (Zhuang et al. 2007). Such bacteria canbenefit the host plant and directly and indirectlycontribute to the transformation of the habitat by: (i)release of nutrients from insoluble mineral complexes(phosphate, iron, etc.) and fixation of atmosphericnitrogen, (ii) accumulation of polyphosphate and hemo-phores, (iii) transfer of nutrients to the host plant viachelating agents (siderophore), (iv) enhancement ofnutrient acquisition by the host plant and its habitatcolonization, by production of phytohormones andreduction of the stress hormone ethylene, and (v)protecting the host plant from biotic stresses such as

pests and pathogens (production of HCN, siderophoresand antibiotics) (Rau et al. 2009; Sharma et al. 2005b).

Saccharum munja,- a perennial wild grass, is oneof the ecologically successful native colonizers of theabandoned mine in the Bhatti region. It forms purepatches on rocky habitats with skeletal soils. It formsextensive root network that binds the soil/pebbles andforms tall thick clumps with high biomass tufts. It isused by low income locals for making ropes, handfans, baskets, brooms, mats, huts, and shields for cropprotection (Sharma et al. 2005a). In summary, S.munja is a choice species for vegetation and stabili-zation of erosion prone rugged slopes and theirconversion into biologically productive sites of highsocio-economic value. However, the grass spreadsvery slowly leaving the abandoned mines barren evenafter several decades. Our research goal is to identifyand characterize plant growth stimulating rhizobac-teria associated with S. munja that can potentiallyincrease its rate of colonization. As the functionaldiversity among the rhizobacteria may vary with thespecific requirements (nutrient etc.) of the host plantduring different growth phases (vegetative and repro-ductive) emerging in different seasons, the presentstudy was undertaken to assess: (i) the functional andtaxonomic diversity of rhizobacteria in S. munjacolonizing the abandoned mine; (ii) the variations inplant growth promoting traits of rhizobacteria indifferent seasons; and (iii) the significance of rhizo-bacteria in the establishment of S. munja at the minesite.

Material and methods

Study site, soil analyses and sample collection

The Bhatti sampling site represents a 50-year oldabandoned mine located at the south eastern part ofAravalli mountains (28°24.2′N–77°13.6′E) in Delhiregion. Rhizosphere red sand samples (goethite) of anative wild grass (Saccharum munja) colonizing thesite were collected and analyzed for moisture content,pH, PO4-P, NO3-N and organic matter (%). Thecontents of Fe2+ and Fe3+ were determined bypermanganate method (Bureau of Indian Standards,IS:1493–1959, 2001). APHA protocols were followedfor soil analyses (Eaton et al. 2005; Sharma et al.2002). Standards were obtained from E. Merck

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(Darmstadt, Germany). The glasswares used wereacid washed.

Rhizobacterial isolation

Bacteria were isolated from the rhizosphere of redsand on nutrient agar medium ((g/l) peptic digest ofanimal tissue 5.0, beef extract 1.5, yeast extract 1.5,sodium chloride 5.0, agar-agar 15.0, final pH 7.4±0.2) (HiMedia Laboratories Pvt. Ltd., Mumbai, India)using the serial dilution method (Rau et al. 2009).Morphologically distinct bacterial colonies developedfor 3 days of incubation at 28°C were purified(Maullu et al. 1998). Total bacterial count per gramof the red sand was determined. The isolates wereassigned a four letter code (site and host speciesrepresented by two letters each with an arbitrarynumeral as suffix).

Bacterial identification using 16S rRNA sequenceanalyses

All the rhizobacterial isolates were identified by 16Sribosomal RNA gene sequence analyses (Sharma etal. 2008). Genomic DNA was isolated using guani-dium thiocyanate method (Pitcher et al. 1989).Approximately 1,500 bp of the 16S rDNA gene wasamplified by PCR using the following primers: 27f(5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492r(5′-TACGGYTACCTTGTTACG ACTT-3′). Ampli-fied PCR fragments were purified and sequencedusing primers 357f, 519r, and 1492r. 16S rRNA genesequences were used to search for homologoussequences in the GenBank database.

Plant growth promoting traits of the rhizobacteria

Tests were carried out in the incubator maintained at28°C. Three replicates were maintained for eachassay. The siderophore production was determinedin terms of change in colour of Chrome azurol S(CAS) dye (Schwyn and Neilands 1987). Ability tosolubilize phosphate was determined as change incolour of the bromocresol purple dye and reduction inthe turbidity of the phosphate solubilizing medium(Wenzel et al. 1994). Growth of bacteria in DF saltsminimal medium (supplemented with 3 mM ACC)and Dobereiner nitrogen-free culture (DNFC) mediumwas taken as the presence of aminocyclopropane-1-

carboxylate (ACC) deaminase activity (Penrose andGlick 2003) and nitrogen fixation ability (Han et al.2005), respectively. The HCN production was deter-mined by change in colour of the picrate paperindicator (Bakker and Schippers 1987). Based on theactivity in the assay, the rhizobacteria were groupedinto five arbitrary classes (1+, 2+, 3+, 4+, 5+). Eachtest is briefly explained below:

Siderophore production

The rhizobacterial’s ability to produce siderophoresunder Fe3+ limiting conditions was evaluated byuniversal CAS (chrome azurol S) assay (Schwynand Neilands 1987). The composition of CASmedium for a litre of overlay was as follows: Chromeazurol S (CAS) 60.5 mg, hexadecyltrimethylammo-nium bromide (HDTMA) 72.9 mg, Piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES) 30.24 g, and 1 mMFeCl3.6H2O in 10 mM HCl 10 ml. Rhizobacteriaplated on blue agar CAS medium (deferrated MM9medium supplemented with CAS) were incubated at28°C. The change in colour of the media (blue toyellow–orange) was taken as siderophore activity.Based on the diameter of the yellow-orange colourzone developed around the colony the activity wascategorized into 1+ (1.0–2.5 mm), 2+ (2.6–5.0 mm),3+ (5.1–7.5 mm), 4+ (7.6–10 mm), 5+ (>10 mm).

Phosphate solubilization ability

Rhizobacterial’s ability to solubilize different phos-phate complexes (Ca3(PO4)2, CaHPO4, FePO4,AlPO4) was assessed using phosphate solubilizingmedium ((g/l) sucrose 5.13, MgSO4.7H2O 0.49, KCl0.52, FeSO4 0.011, phosphate complex 20 mM,bromocresol purple dye 0.108, agar-agar 15.0, pH7.0) (Wenzel et al. 1994). Measurements on thechange in color and turbidity were taken after 3 days.Based on the diameter of the yellow colour zonedeveloped around the colony the activity was catego-rized into 1+ (1.0–4.0 mm), 2+ (4.1–8.0 mm), 3+(8.1–12.0 mm), 4+ (12.1–16.0 mm), 5+ (>16.0 mm).

Indole-3-acetic acid (IAA) assay

Bacterial cultures were inoculated on tryptophan(5 mM) supplemented LB agar plate ((g/l) tryptophan10.0, yeast extract 5.0, NaCl 10.0, pH 7.5) (Bric et al.

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1991). The bacteria were grown for 24 h till thecolony diameter reached 0.5–2.0 mm and thenoverlaid with a nitrocellulose membrane. After incu-bation, the membrane was removed from the plateand treated with Salkowski reagent (reagent A: 2%0.5 M FeCl3 in 35% perchloric acid; reagent B: FeCl3in 37% sulfuric acid). Bacteria showing developmentof the characteristic red or purple halo surroundingthe colony were taken as IAA production. Based onthe diameter of the halo developed on the membranethe activity was categorized into 1+ (1.0–2.0 mm), 2+(2.1–4.0 mm), 3+ (4.1–6.0 mm), 4+ (6.1–8.0 mm), 5+(>8.0 mm).

1-Aminocyclopropane-1-carboxylate (ACC)deaminase activity

ACC deaminase activity was determined by assessingthe ability of rhizobacterial isolates to utilize 1-aminocyclopropane-1-carboxylate (ACC) as solesource of N (Penrose and Glick 2003). Isolates wereinoculated in DF salts minimal medium ((g/l)KH2PO4 4.0, NaH2PO4 6.0, MgSO4·7H2O 0.2,glucose 20.0, gluconic acid 2.0, citric acid 2.0, traceelement solution 0.1 ml, FeSO4·7H2O 0.1 ml) con-taining 3 mM ACC. Medium supplemented with(NH4)2SO4 served as positive control and devoid ofnitrogen source was kept as negative control. Bacte-rial growth in ACC supplemented plate in comparisonto the control was taken as ACC deaminase activity.Based on the extent of bacterial growth (%) incomparison to the control, the activity was catego-rized into 1+ (1–20%), 2+ (21–40%), 3+ (41–60%), 4+(61–80%) and 5+ (81–100%).

Ability to grow on nitrogen free medium

Ability of rhizobacterial isolates to grow on Dober-einer nitrogen-free culture (DNFC) medium wastaken as a measure of their non-symbiotic N2-fixation ability (Han et al. 2005). The DNFCconsisted of (g/l): sucrose 10, malic acid 5,K2HPO4 0.1, KH2PO4 0.4, MgSO4·7H2O 0.2, FeCl30.03, NaCl 0.1, CaCl2·2H2O 0.02, Na2MoO4·2H2O0.02, agar-agar 3.0. Observations were recorded after48 h. Growth of rhizobacterial isolates on (NH4)2SO4

supplemented (2.0 g/l) medium was taken as a positivecontrol. Based on the extent of bacterial growth (%) incomparison to the control, the activity was categorized

into 1+ (1–20%), 2+ (21–40%), 3+ (41–60%), 4+(61–80%) and 5+ (81–100%).

Polyphosphate accumulation

Polyphosphate (Poly-P) accumulators were isolated(Jorgensen and Pauli 1995) by plating the bacteria onselective acetate minimal medium (per litre:CH3COOONa.3H2O 3.68 g, NaHPO4.2H2O28.73 mg, NH4Cl 57.27 mg, MgSO4.7H2O131.82 mg, K2SO4 26.74 mg, CaCl2.2H2O 17.2 mg,HEPES buffer 12 g, agar-agar 15 g) and 2 ml of tracemineral solution (per litre: EDTA 50 g, FeSO4.7H2O5 g, and CuSO4.5H2O 1.6 g, MnCl2.4H2O 5 g,(NH4)6Mo7O24.4H2O 1.1 g, H3BO3 50 mg, KI 10 mgand CoCl2.6H2O 50 mg). The pH was adjusted to 7.0with 1 M NaOH. To avoid any traces of phosphatemolecules, glassware were soaked overnight in 6 NHCl and rinsed 6 times with autoclaved distilled waterbefore use. After 24–48 h, growth was measured indiameter (mm). Growth on the selective medium wastaken as positive result. Based on the diameter of thecolony the activity was categorized into 1+ (1.0–4.0 mm), 2+ (4.1–8.0 mm), 3+ (8.1–12.0 mm), 4+(12.1–16.0 mm) and 5+ (>16.0 mm).

HCN production ability

Rhizobacterial isolates were grown on Kings Bmedium ((g/l) bacto-peptone 20.0, K2HPO4 1.5,MgSO4.7H2O 1.5, glycerol 5.0 ml, pH 7.2) supple-mented with glycine (4.4 g/l) (Bakker and Schippers1987). Picrate paper (Whatman paper No. 1 impreg-nated with 0.5% picric acid in 2% sodium carbonatesolution) was placed inside the lid of each Petri plate.Color change of picrate paper (yellow to brown) wastaken as HCN production. Based on the intensity ofcolour change the activity was categorized into 1+(light orange), 2+ (orange), 3+ (orangish-brown), 4+(brown), 5+ (intense brown).

Antagonistic activity

Antagonistic potency of rhizobacterial isolates wastested against 8 soil-borne plant pathogenic fungi(Fusarium graminearum F. moniliforme, F. oxyspo-rum, F. semitectum, F. udum, Aspergillus flavus,Sclerotium rolfsii, and Pythium debaryanum) usingdouble layer method (Comitini et al. 2005). An

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aliquot (100 μl) of fungal spore suspension (105–106) was spread on PDA (20 ml) plate. A secondlayer (10 ml) of PDA was overlaid and bacterialisolates were spot inoculated for growth at 28°C.Interaction between the fungal and bacterial strain(appearance of zone of inhibition) was observedperiodically for 5–7d. Based on the diameter of zoneof inhibition, antagonistic activity was grouped intodifferent classes viz. 1+ (1.0–2.0 mm), 2+ (2.1–4.0 mm), 3+ (4.1–6.0 mm), 4+ (6.1–8.0 mm) and 5+(>8.0 mm).

Seed bacterization and pot experiments with selectedsiderophore producing and phosphate solubilizingrhizobacteria

Five replicates and 10 seeds per replicate were keptfor each treatment. Seeds of S. munja were collectedfrom the Bhatti site. Seeds were surface sterilizedwith 0.1% HgCl2 solution (1 min) followed by 10%sodium hypochlorite (10 min). Surface sterilizedseeds were soaked overnight in bacterial inoculum(∼108 cfu) prepared by suspending selected rhizobac-teria (Bacillus megaterium BOSm201, B. subtilisBGSm253, B. pumilus BGSm157, Paenibacillus alveiBGSm255, Pseudomonas putida BOSm217, P. aeru-ginosa BGSm306) in sterile distilled water. In onetreatment, seeds were sown in pots (250 cc) filledwith 200 g of sterile mine spoil. In the secondtreatment, seeds were incubated in humidity chamberprepared using leachate of sterile mine spoil (Rau etal. 2009). Deferrated sterile distilled water was usedfor irrigation daily. Effect of rhizobacterial inoculationon seed germination and seedling growth wasdetermined after 10th day of planting.

Statistical analysis

Variations in proportions of plant growth promotingtraits among: (i) bacteria from rhizosphere vs bulkregions; and (ii) rhizobacteria in different samplingseason (summer vs winter), were subjected tostatistical analyses using Kruskal Wallis test (Sokaland Rohlf 1994). One way ANOVA (orthogonalanalyses) was used to test the significance of theeffect of rhizobacterial inoculation on plant growthresponses. Means of seed germination and seedlinglength in different treatments were compared byTukey’s test.

Results

Habitat characteristics

The study area (Bhatti) represents degraded land as aresult of extensive open cast mining for feldspar (usedfor preparation of high grade pottery) and subse-quently for red sand stone or morrum (constructionmaterial). The landscape was heterogeneous varyingfrom hard stable rocks to red sand mounds prone toerosion. The site is characterized by lack of soil layerand organic matter (0.1±0.009%), poor moisturecontent (0.79±0.03%) and low levels of N and P(0.01±0.006 ppm NO3-N; 0.16±0.008 ppm PO4-P).The pH was slightly alkaline (7.94±0.06). Ironcontent of the mine spoil (red sand) was quite high(2.47±0.12%) and predominantly (84.2±1.32%) oxi-dized (Supplementary Table 1). Even after 50 years ofdiscontinuation of mining activities, the area supportspatchy vegetation primarily composed of weedyspecies (Prosopis juliflora, Lantana camara, Parthe-nium hyterophorus, Cassia tora, Argemone mexicana,Tragus biflorus etc.). Among the few native grasses(Saccharum munja, Cenchrus ciliaris, Bothriochloapertusa, Chrysopogon fulvus, Dichanthium annula-tum, Eremopogon foveolatus) naturally colonizing thesite, S. munja is a high biomass producer and freefrom termite infestation. Severe infestation by subter-ranean termites is common at the site.

Species diversity among the rhizobacteria of S. munja

The total culturable bacterial count in the rhizo-sphere (1.36×108 cfu/g) of S. munja was ∼170 timeshigher than that from non-rhizosphere mine spoil(0.008×108 cfu/g). However, rhizosphere bacterialcount showed seasonal variation and it was found tobe ∼4.5 times higher in winter (1.11×108 cfu/g) ascompared to summer (0.25×108 cfu/g). The winterseason is characterized by an average high and lowtemperatures of 18°C and 7°C, respectively and anaverage precipitation of 20.3 mm, whereas insummer the average high and low temperatures are34°C and 27°C, respectively, with an averageprecipitation of 236.2 mm. Eighty one morphotypesof culturable bacteria were purified from the rhizo-sphere and fifty from non-rhizosphere (bulk). Mostof the culturable bacteria purified from rhizosphere(97.6%) and bulk region (100%) were Gram positive

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(Supplementary Table 1). Taxonomic diversity inculturable bacteria from rhizosphere (8 genera, 25species) was considerably higher than the bulkregion (2 genera, 5 species). Bacilli cell shape wasdominant among the bacteria. The rhizobacteriaharbored 10 species of Bacillus and 6 species ofPaenibacillus. A large proportion of bacteriabelonged to Bacillus spp. (rhizosphere: 69.13%;non-rhizosphere: 64.0%) and Paenibacillus spp.(rhizosphere: 17.28%; non-rhizosphere: 36.0%). Inbulk region, B. cereus, B. pumilus, B. subtilis, P.alvei and P. turicensis were common. In rhizosphere,B. flexus, B. megaterium, B. pumilus, B. subtilis,Paenibacillus lautus and P. popilliae were commonboth in summer and winter, whereas, B. cereus waspredominant in winter but absent in summer. B.barbaricus and P. lentimorbus were exclusivelyfound in summer while B. foraminis, P. alvei, P.apiarius and P. polymyxa were found only in winter.Similarly, Arthrobacter globiformis, Micrococcusluteus and Microbacterium spp. (M. barkeri, M.resistens and M. testaceum) were found only insummer while Lysinibacillus spaericus, Pseudomo-nas aeruginosa, P. putida and Streptomyces radio-pugnans were exclusively found in winter.

Diversity in plant growth promoting traitsof the rhizobacteria

The rhizobacteria were functionally diverse and pos-sessed more than one plant growth promoting trait.

Siderophore production

Siderophore production was common among bacteriafrom rhizosphere and bulk region (Fig. 1). Among therhizobacteria, the proportion of siderophore producerswas significantly higher (p<0.001) in the winter(97%) than summer (64%) (Fig. 2). In fact, rhizobac-teria with highest siderophore activity (viz. B. cereusBGSm245, B. cereus BOSm226, B. cereusBOSm227, B. megaterium BOSm201, B. pumilusBGSm251 and P. aeruginosa BGSm306) were foundin winter only (Supplementary Table 1).

Phosphate solubilization

The proportion of CaHPO4 solubilizers in rhizosphereand non-rhizosphere was higher than Ca3PO4-, AlPO4-

and FePO4-solubilizers (Fig. 1). Interestingly, none ofthe bulk isolates could solubilize FePO4. However, asignificantly (p<0.05) high proportion (27%) of therhizobacterial isolates were FePO4-solubilizers. All thephosphate-solubilizers were acid producers.

IAA production

A significantly (p<0.001) high proportion of IAA-producers were present in the rhizosphere (Fig. 1). A.globiformis BGSm142, Bacillus spp. (B. cereusBGSm247, B. firmus BGSm249, B. megateriumBOSm201, B. pumilus BOSm212, B. subtilisBOSm211), P. lautus (BGSm150, BOSm204) and S.radiopugnans BOSm214, were among the high IAA-producers from rhizosphere, whereas B. subtilis(BNRS424, BNRS610) and P. turicensis (BNRS436,BNRS620) were from non-rhizosphere (SupplementaryTable 1).

ACCD activity

The difference in proportion of ACCD-producersfrom rhizosphere (47%) and bulk (36%) regions wasnot statistically significant. However, the difference inrhizobacterial isolates from winter (63%) and summer(12%) was significant (p<0.001) (Fig. 2). None of theACCD producers from bulk region showed highactivity (Fig. 1). Among the rhizobacteria, only twoisolates (B. cereus BGSm237 and B. pumilusBGSm242) from winter showed highest ACCDactivity (Supplementary Table 1).

Nitrogen fixation

The proportion of nitrogen-fixing bacteria (38%) wassignificantly high (p<0.001) in the rhizosphere,whereas none of the isolates from bulk region showedthis activity (Fig. 1). B. pumilus was the common N2-fixer among rhizobacteria. However, other N2-fixersinclude different strains of B. cereus, B. firmus, B.licheniformis, B. megaterium, B. subtilis, M. resistens,P. alvei, P. lautus and S. radiopugnans (Supplemen-tary Table 1).

Polyphosphate accumulation

The proportion of polyphosphate producers wasmarginally higher (not significant at p<0.05) in

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Fig. 1 Proportion of different functional groups of bacteria colonizing rhizosphere of S. munja and bulk soil

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Fig. 2 Proportion of different functional groups of rhizobacteria of S. munja purified in summer and winter season

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rhizosphere than bulk region (Fig. 1). However,among the rhizobacterial isolates it was significantlyhigher (p<0.05) in winter (54%) than summer (28%)(Fig. 2). Interestingly, all the polyphosphate-producers (40%) from non-rhizosphere showed high-est activity (5+). Some of the rhizobacterial isolateswith highest activity (5+) were B. cereus (BGSm235,BGSm236, BOSm227, BOSm228, BOSm229), B.flexus (BOSm203, BOSm241), B. pumilus BGSm138,B. megaterium BOSm202, B. subtilis (BOSm211,BGSm242), B. thuringiensis BGSm238, L. sphaer-icus BOSm230, P. aeruginosa BGSm306, P. putidaBOSm217 etc. (Supplementary Table 1). However, inbulk region they were represented only by Bacillusspp. viz. B. cereus (BNRS427, BNRS28, BNRS431,BNRS435, BNRS603, BNRS606, BNRS609,BNRS611), B. pumilus (BNRS430, BNRS433,BNRS602, BNRS621), B. subtilis (BNRS416,BNRS17, BNRS18, BNRS420, BNRS604,BNRS607, BNRS608, BNRS615).

HCN production

The difference in proportion of HCN producers fromrhizosphere (15%) and bulk region (20%) was notsignificant (Fig. 1). However, in summer the propor-tion (36%) was ∼5.0 times higher (p<0.001) than thewinter (7.14%) (Fig. 2). B. pumilus (BGSm138,BGSm151, BGSm52, BGSm53, BGSm157,BGSm158, BOSm208, BOSm223, BNRS419) wasmost predominant cyanogenic bacteria. In fact, somestrains of B. pumilus (BGSm152 and BOSm208) were

the highest HCN-producers among the rhizobacteria.Other HCN producers included B. flexus BGSm160, B.megaterium BGSm141, M. barkeri BGSm148, P.popilliae (BGSm147, BOSm220). In the bulk region,strains of B. subtilis (BNRS416–418, BNRS420) werethe dominant cyanogenic bacteria.

Antagonistic activity

The proportion of rhizobacteria showing antagonisticactivity against different fungal pathogens was higherin the rhizosphere than bulk soil. A. globiformisBGSm140, B. subtilis BGSm242 and S. radiopugnans(BOSm214, BOSm215) showed antagonistic activityagainst multiple fungal pathogens (Fusarium oxyspo-rum, F. udum, F. semitectum, F. graminearum. F.moniliforme). A. globiformis BGSm140, B. subtilisBGSm242 and S. radiopugnans BOSm214 also showedhighest antagonistic activity against Sclerotium rolfsii.

Effect of rhizobacterial inoculation on seedgermination and seedling length of S. munja

Some of the most efficient siderophore producers andphosphate solubilizers were tested for their in vivoability to promote the growth of S. munja in minespoil. The rhizobacterial inoculation increased theseed germination and seedling growth in mine spoil(up to 1.77; 1.44 times) (p<0.05), and leachatechamber (up to 1.55; 1.86 times) (p<0.05) (Table 1).Inoculation with P. putida BOSm217 and P. aerugi-nosa BGSm306 significantly (p<0.05) improved the

Table 1 Details of effect of rhizobacteria inoculation on seed germination and seedling length of S. munja

Treatment Seed germination (%) Seedling length (cm)

Mine spoil Moist chamber (minespoil leachate)

Mine spoil Moist chamber (minespoil leachate)

Uninoculated (control) 27.5±2.57 26.25±6.29 2.37±0.15 3.07±0.92

Pseudomonas putida BOSm217 35.0±3.77* 41.25±6.29* 3.42±0.62* 4.65±0.19*

P. aeruginosa BGSm306 47.5±9.57* 48.75±7.50* 3.3±0.29* 4.57±0.37**

Bacillus megaterium BOSm201 38.75±4.29* 35.0±5.77 3.2±0.47* 4.45±0.51**

B. subtilis BGSm253 30.0±8.16 42.5±5.0* 2.5±0.47 3.47±1.28

B. pumilus BGSm157 40.0±5.16* 30.0±7.07 2.50±0.37 4.17±0.50

Paenibacillus alvei BGSm255 37.5±3.0* 43.75±4.78* 2.72±0.12* 3.12±0.28

*statistically significant at p<0.05

**statistically significant at p<0.10

Plant Soil (2011) 341:447–459 455

percent seed germination and seedling length in bothmine spoil and the leachate chamber. Inoculation of B.pumilus BGSm157 and B. megaterium BOSm201enhanced seed germination in mine spoil whereas B.subtilis BGSm253 enhanced seed germination inleachate chamber. P. alvei BGSm255 inoculationsignificantly (p<0.05) enhanced seed germination inboth mine spoil and leachate chamber but seedlinglength in mine spoil only, whereas B. megateriumBOSm201 significantly enhanced the seedling growthboth in mine spoil and leachate chamber.

Discussion

S. munja is a native perennial grass which producesdense erect tufts of 4–8 ft. Poor contents of moisture(0.79±0.03%), NO3-N (0.01±0.006 ppm), PO4-P(0.16±0.008 ppm), organic matter (0.1±0.009%),high level of biologically unavailable form of iron(84.2±1.32%) and lack of soil cover (∼1%) suggestthat the abandoned Bhatti mine is characterized with awide range of abiotic stresses. Sparse vegetationprimarily comprised of weedy invasive species (likeProsopis juliflora) and heavy termite infestationconstitutes the biotic stress (Sharma and Dakshini1998). Therefore, the physico-chemical and biologicalcharacteristics of the habitat make it non-amenable fornatural colonization by plants.

Occurrence of high number of rhizobacteria, withdominance of Gram-positive bacilli (Bacillus andPaenibacillus) having multiple plant growth promot-ing traits might enhance the nutrient supply to hostplant under a high stress environment. Bacilli shapeowing to high surface area (Young 2006) maximizesthe nutrient uptake. Further, Bacillus spp. are knownfor their tolerance to a wide range of abiotic stresses(Earl et al. 2008). Presence of some rhizobacterialspecies exclusively in winter (B. cereus, B. foraminis,P. alvei, P. apiarius, P. polymyxa, L. sphaericus, P.aeruginosa, P. putida and S. radiopugnans) andsummer (B. barbaricus, P. lentimorbus, A. globifor-mis, M. luteus, M. barkeri, M. resistens, M. testa-ceum) (Supplementary Table 1) could have linkageswith variations in requirement of S. munja at differentgrowth stages. Cyanogenic potential of Pseudomonasspp. has been well established (Bakker and Schippers1987). However, the absence of cyanogenic Pseudo-monas spp., and selection of cyanogenic strains of

Bacillus pumilus, B. flexus, B. megaterium andPaenibacillus popilliae at the mine site needs furtherinvestigation. Mine spoils contain high levels of toxicheavy metals which are known to induce cyanideproduction in some microbes (Faramarzi et al. 2004).Cyanogenic bacteria control the pests and weeds(Devi et al. 2007; Kremer and Souissi 2001).However, grasses have mechanisms to combat thecyanide toxicity (Sirikantaramas et al. 2008). There-fore cyanogenic rhizobacteria may contribute torelatively high colonization and spread of host plant.

Predominance of siderophore producers in rhizo-sphere and bulk region, indicate that the siderophore-mediated iron acquisition could be the primarystrategy for their survival under extreme iron stress.Iron deficiency leads to chlorosis in plants. Not even asingle individual of S. munja in natural populations atthe site showed any symptom of chlorosis. Utilizationof iron by plants from rhizobacterial siderophore-ironcomplexes has been shown in grasses (Crowley et al.1988). Significantly high proportion of IAA-producingrhizobacteria might contribute to the extensive rootnetwork of S. munja. IAA producing rhizobacteria havebeen shown to promote the root growth and increasethe root surface (lateral roots and root hairs) which inturn has positive effects on water acquisition andnutrient uptake (Dimkpa et al. 2009). For example,inoculation of Pseudomonas alcaligenes PsA15 andBacillus polymyxa BcP26 promoted growth and nutri-ent uptake in maize (Egamberdiyeva 2007). Extensiveroot network of plant is also a function of ACCdeaminase activity. ACC-deaminase producers cleaveACC (precursor of ethylene), lower the levels of stressethylene and promote the root and shoot growth. Thesignificantly high proportion of ACCD producers (47%)as compared to that reported for other plant species (4–7%) (Burd et al. 1998; Cattelan et al. 1999), suggesttheir selective accumulation in S. munja rhizosphere,which could be associated with the multitude ofstresses present at the site.

Microbial-fixed nitrogen serves as a significantsource of N for the plant growth in harsh environ-ments (Sharma et al. 2005b). Saccharum spp. (S.officinarum and S. spontaneum) are known to fix freeN to meet up to 70% of their requirement (Boddey1995). However, the contribution of rhizobacteriamay not be ignored. In fact, the importance of N2-fixing rhizobacteria in the colonization potential of S.ravennae to a N-deficient site has been emphasized

456 Plant Soil (2011) 341:447–459

(Rau et al. 2009). Studies on other Saccharum spp.revealed Nitrosomonas sp., Azotobacter sp., Azospir-illum sp., Burkholderia sp. as the dominant nitrogen-fixing rhizobacteria (Raol 1991; Tejera et al. 2005;Perin et al. 2006). However, we have not detected thesebacterial species in the abandoned Bhatti mine. As inthe case of S. munja, dominance of N2-fixing Bacillusspp. has also been reported in S. ravennae rhizosphere(Rau et al. 2009). Sclerotiium rolfsii is an importantpathogen causing rot and blight disease in Saccharumspp. and Fusarium spp. has been implicated in seedlingmortality in nursery, garden and forests worldwide(Navas-Cortés et al. 2000). Therefore, occurrence ofrhizobacteria (A. globiformis BGSm140, B. subtilisBGSm242, S. radiopugnans BOSm214, BOSm215)antagonistic to multiple fungal pathogens includingFusarium spp. and Sclerotium rolfsii may contribute tothe success of S. munja at the site.

Predominance of iron- and aluminium-phosphatesolubilizers among the rhizobacteria could beassociated with the acidic environment in therhizosphere. It may be noted that iron- andaluminium-phosphate complexes are formed inacidic conditions. Besides phosphate solubilization,accumulation of Pi as polyphosphates (poly-P)(Ohtake et al. 1998) is another bacterial strategy toensure the phosphate supply and overcome otherenvironmental stresses in low-phosphate environ-ment (Jahid et al. 2006). Therefore, a high propor-tion of poly-P producers (rhizosphere and bulkregion) may also be associated with the phosphatestress at the site.

Significant increase in total rhizobacterial counts(culturable) and dominance of siderophore-,polyphosphate- and ACCD-producers during wintermight be associated with the onset of flowering andfruiting (January–March) of S. munja. At this devel-opmental stage nutrient assimilation and other phys-iological activities are very high. On the other hand,the five fold increase in the proportion of cyanogenicrhizobacteria (HCN producers) in summer might beassociated with onset of termite infestation at Bhattisite. Subterranean termites ubiquitous in the areaseverely damage the roots and lower part of the stem.They multiply, spread and infect during June–August.A cyanogenic rhizobacterium isolated from the Bhattimine has been shown to kill subterranean termiteOdontotermes obesus in vitro (Devi et al. 2007). Itmay be noted that S. munja was resistant to these

termites. In vivo studies on cyanogenic rhizobacteriaand termite infestation may further enhance ourunderstanding on their ecological significance.

Enhanced percent seed germination and seedlinglength by the rhizobacterial inoculation (Table 1)both in seeds sown in mine spoil and moist chamberprepared using mine spoil leachate point toward thesignificance of rhizobacteria in the colonization of S.munja at the abandoned mine. As abandoned minesare characterized by a multitude of abiotic stresses,therefore rhizobacteria with multiple plant growthpromoting traits might help the host plant inalleviating the stresses. In our study, siderophoreand IAA production seems to play important role inenhancement of seed germination and seedlinggrowth. For example, isolates showing significantin vivo plant growth promotion (except P. putidaBOSm217), were siderophore and IAA producers,whereas B. subtilis BGSm253 and B. pumilusBGSm157 showing relatively low plant growthpromotion were characterized with relatively lowIAA production. The rhizobacteria of other Saccha-rum sp. have been shown to facilitate its colonizationat metal stress site (Rau et al. 2009).

Conclusion

In conclusion, the rhizosphere of S. munja harboursfunctionally diverse bacteria dominated by Bacillusspp. and Paenibacillus spp. Abundance of the mostefficient PGPR during the reproductive phase of theplant, indicate the significance of analyzing seasonalvariation among the rhizobacteria. The plant growthpromoting traits of the rhizobacteria and their abilityto enhance seed germination and seedling growth inmine spoil and leachate chamber suggest theirsignificance in the natural colonization of S. munjaat abandoned mine site. These PGPR have potentialusefulness for the development of inoculation tech-nologies for ecological restoration of barren aban-doned mines.

Acknowledgements We thank Dr Asif Mohmmed, ICGEB,New Delhi for critical comments and constructive suggestionson MS; and Professor D.M. Banerjee, University of Delhi, foruseful discussion on geology of Bhatti mine. RSS and VMthank the Department of Biotechnology (Government of India),Department of Science & Technology (Government of India) andthe University of Delhi for research grants.

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