ORIGINAL PAPER

Influence of management practices on the diversityof pseudomonads in rhizosphere soil of wheatcropping system

Shilpi Mittal & Bhavdish N. Johri

Received: 22 May 2007 /Revised: 20 November 2007 /Accepted: 6 December 2007 /Published online: 14 March 2008# Springer-Verlag 2007

Abstract The composition and diversity of the bacterialcommunities associated with plant root is influenced byvarious biotic and abiotic factors. In this study, effect of soilmanagement practices, conventional (pf) and raised bed (rb),are compared for bacterial diversity in rhizosphere of wheat(Triticum aestivum) var. UP2338, taking pseudomonads asa biomarker. Microbial diversity was assessed employingculturable and unculturable approaches. Amplified rDNArestriction analysis showed that soil management practicesand site of sampling (rhizosphere-RS and rhizoplane-RP)affected the composition of microbial communities. Sam-ple from conventional management showed a higherPseudomonas diversity (H′=3.7) than that from raised bed(H′=2.78). However, total bacterial diversity (SSCP data)was more complex in rb than pf management. Dominantmembers of both pf and rb were γ-proteobacteria asPseudomonas was dominant over other forms, whileBacillus species were only present as low numbers in rb.

Keywords Management practice . Diversity .

Pseudomonas . Raised bed . Conventional system .

Soil health

Introduction

Sustainable cropping practices with economic returns andenhancement and maintenance of soil quality are preferredover those that degrade soil. Different tillage systems affectsoil sustainability. For example, conventional tillage, withthe traditional practice of ploughing and disking to prepareland, reduces soil organic matter content and increaseserosion (Ferreira et al. 2000). As an alternative, no-tillagemanagement, with sowing directly through mulch, protectsthe soil against erosion by water, improves soil structureand size of microbial population and organic matter content(Alvarez et al. 1995). Soil management practices may affectmicrobial community structure of rhizosphere (RS) andbulk soil; soil microbial communities mediate processesessential to the productivity of soil and to the completion ofbiogeochemical cycles. However, it is not known howmanagement practices affect the composition and structureof RS microbial communities (Alvey et al. 2003).

Rice followed by wheat, has been one of the majorcropping sequences in most of the wheat-growing regions.However, cultivation practices of these two food graincrops are completely different with rice requiring water-logged conditions that create microaerophilic or anaerobicenvironment, with predominance of methanogens andsulfate reducers, whereas wheat is sown after rice andcropped under aerobic condition. To attain sustainability,rice–wheat cropping based on raised bed is being testedwhere the RS communities are not disturbed passing fromwheat to rice because aerobic conditions are maintainedthroughout. No information is currently available onmicrobial diversity of RS soil under this cropping system.

Most of the studies on RS microbial communities areconcentrated on genus Pseudomonas (Griffith et al. 1999;Heuer et al. 2001); these bacteria are widely distributed in

Biol Fertil Soils (2008) 44:823–831DOI 10.1007/s00374-007-0264-0

S. Mittal :B. JohriDepartment of Microbiology, College of Basic Sciences &Humanities, G.B. Pant University of Agriculture & Technology,Pantnagar 263 145 Uttaranchal, India

S. MittalDepartment of Microbiology, University of Delhi South Campus,Benito Juarez Road,New Delhi 110 021, India

B. Johri (*)Department of Biotechnology, Barkatullah University,Bhopal 462 026 MP, Indiae-mail: bhavdishnjohri@rediffmail.com

soil and RS because of great competitiveness and coloni-zation ability. Considerable diversity and ubiquity has beenshown in this genus (Spiers et al. 2000).

The aim of this research was to determine the diversityof pseudomonads in the RS of the wheat grown undernormal practice (pf) and as raised bed (rb) system. Toinvestigate the effect of management practice on microbialdiversity, an experimental site was selected where wheatand rice was cultivated for nearly a century. To achievesustainable system, wheat was also cultivated by raised bed(rb) cropping system.

Materials and methods

Field site, design and sampling

The field site selected for this experiment is located in thevillage Bhawanipur, Dist. Badaun (longitude: ×79.5°,latitude: ×28.1°, altitude: 150–300), India; wheat has beencultivated for nearly a century along with rice rotationfertilized by urea and diammonium phosphate. The studysite is rain-fed, and irrigation is largely dependent on themonsoon rain. Wheat was sown in plain field (conventionalpractice of wheat cultivation, var. UP2338-pf) and in raisedbeds (UP2338-rb) by leaving narrow trenches for flow ofwater (Fig. 1). Except for the management practice, allother factors, that is, plant type, soil type, irrigationschedule and fertilizer inputs, were kept constant. Standardagronomic practices of sowing, irrigation and weeding werefollowed. Soils have a sandy loam texture, and according tothe USDA classification, they were classified as: Entisoil(order), Psamment (suborder), Doarse (Family), Hyperther-mic (Regime). Soil characteristics are presented in Table 1.

Ten randomly chosen plants (ten replicates) at theflowering stage (90 days; plant size, 80 cm) were uprootedfrom pf and rb under semi-sterile conditions using sterileforceps. This sampling was done from three different fields(4,000 m2) separated by less than 2 km. Ten plants wereremoved from five different locations and pooled togetherfor each management system to make a composite sample;the plant samples were transported to the laboratory inpolybags and were processed within 6 h of sampling. Roots(10 g) with closely adhered soil were suspended in 90 mlphosphate buffer saline and vigorously shaken at 150 rpmat 15°C for 1 h. Soil suspension of RS soil was seriallydiluted and plated in three replicates on King’s B (KB) agar(10−5 to 10−6). In the case of rhizoplane (RP) fraction, rootswere removed from suspension, dried on a tissue paper andcrushed in 10 ml SDW using sterile pestle and mortar. Onemillilitre of this suspension was plated on KB agar (10−4 to10−6). Plates were incubated at 30°C in a BOD incubatorwith RH 90% for 72 h. All different morphotypes were

recovered and maintained as pure culture stabs of KB at 4°Cfor further studies. As reference strains, we used thefollowing 14 reference fluorescent Pseudomonas: Pseudo-monas fluorescens bv I, II, III, IV and V, Pseudomonaschlororaphis, P. fluorescens strain CHA0, Pseudomonasaeruginosa strain PRS9, Pseudomonas maltophila, Pseudo-monas pseudomali and P. fluorescens strains TMA3,PGNL1, PGNR1 and P1LH1 (2,4-diacetyl phloroglucinolproducers).

Assessment of genetic diversity by ARDRA

Isolates were genetically characterized employing amplifiedrDNA restriction analysis (ARDRA). Bacterial genomicDNA was extracted by cetrimonium bromide (CTAB)method (Bazzicalupo and Fani 1994). Overnight-grownbacterial culture was pelleted by centrifugation (8,000×g)and then treated with 10% SDS, proteinase K and CTAB.Protein was precipitated by phenol: Chloroform and DNAwas precipitated by centrifugation at 12,000×g for 20 minusing chilled absolute ethanol. DNAwas dissolved in 50 μlTE buffer and visualised in 0.8% agarose gel.

Polymerase chain reaction (PCR) on 16S rDNA wascarried out according to Pramanik et al. (2003) usingbacterial specific universal primers for 16S rDNA, Gm3f(5′GGT CTG AGA GGATGATCA AGT 3′) correspondingto positions 8–23 of Escherichia coli 16S rRNA and Gm4r(5′TTA GCT CCA CCT CGC GGC 3′) corresponding topositions 1492–1507 of E. coli. The 16S rDNA ampliconwas digested with restriction endonucleases TaqI, RsaI andHaeIII (MBI Fermentas) as reported by manufacturer’sinstructions. Digested 16S rDNA was separated by electro-phoresis on 2.5% agarose gel (Q-Biogen) in 1× Tris–acetate–EDTA buffer (pH 8.0) at 8 V cm−1. Similarityamong the isolates was determined by constructing adendrogram from restriction profile of 16S rDNA employ-ing the NTSYSpc 2.02i analysis package.

Phenotypic and metabolic characterization

Bacterial strains of RS and RP samples from eachmanagement practices (pf and rb) were analyzed forphenotypic characters, such as morphology, gram reactionand production of diffusible pigments, and biochemicalproperties, such as catalase and oxidase activities, levansu-crase production from sucrose, denitrification and argininedihydrolase activities. Some most distantly related isolatesin the ARDRA grouping, like RP11 and RP3, were alsoincluded in the study. Levansucrase production fromsucrose was performed by spot inoculation on sucrose agarplates (gram per litre; peptone, 10.0; meet extract, 3.0;NaCl, 5.0; agar, 15.0; phenol red aqueous solution 10.0 ml).Levansucrase producing colonies appeared large and

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mucoid. Denitrification was performed as described byJacques (1994). Arginine dihydrolase activity was deter-mined by inoculating test culture with arginine broth (5 ml);tubes were incubated at 28°C under shaking conditions(140 rpm) for 24–30 h. Freshly prepared Nessler’s reagent(250 μl) was added after incubation. Development of abrown colour within 30 s was an indication of the positivetest. Utilization of amino acids and carbohydrates as a

carbon source was tested in a basal medium whichcontained (gram per litre; (NH4)2PO4, 1.0; KCl, 0.2;MgSO4·7H2O, 0.2; agar, 10.0; bromothymol green 0.08;C-source, 0.5%. Production of extracellular hydrolase wasassayed qualitatively for amylase, cellulase, pectinase,gelatinase, lipase and protease in a plate assay. In brief,plates of basal agar medium supplemented with thesubstrate (1% starch for amylase, 0.7% carboxymethyl

Table 1 Physico-chemical properties and enzymatic activity of soil*

Physico-chemicalparameters

P total(kg/ha)

AverageP (kg/ha)

N total(kg/ha)

Exchange ofK (kg/ha)

Mn(μg/g)

Zn(μg/g)

Fe(μg/g)

TPF (g dm16 h−1)a

Alc. P (μg NP/gdm h−1)b

Ac. P (μg NP/gdm h−1)c

UP2338-pf 1,672.7 28.5 1,581.4 170.2 19.3 0.6 38.3 18.4 181.3 221.7UP2338-rb 1,686.7 30.6 1,602.3 183.8 20.0 1.1 42.7 19.5 193.5 235.1

The difference in soil characteristics of pf and rb is, however, non-significant, t=≥0.5.Available N, organic carbon, organic matter and Cu of pf, rb system were also estimated, but no differences were observed, so data was notincluded in the table.a Dehydrogenase activityb Alkaline phosphatasec Acid phosphatase

Fig. 1 Raised beds of rice–wheat cropping system showingcomplete rotation of wheat andrice cultivation suggesting raisedbed as no-tillage system

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cellulose for cellulase, 1% pectin for pectinase, 3% gelatinfor gelatinase, 1% Tween 80 for lipase and 1% skim milkfor protease) were prepared, spot inoculated with overnight-grown cultures and incubated at 28°C for 24–72 h (Bradneret al. 1999). In the case of pectin hydrolysis, plates wereoverlaid with 2% CTAB and left at room temperature for30 min followed by washing with 1 M NaCl. A clear zonearound the colonies suggested a positive test. In the case ofcellulase, after 72 h, plates were flooded with 0.1% congored solution for 10 min followed by washing with 1 MNaCl. Positive isolates showed a light yellow-orange colouraround the bacterial colony. Amylase plates were floodedwith freshly prepared Lugol’s iodine, and clear zone aroundthe colonies was indicative of positive isolates. For lipase,plates were flooded with 30% tricarboxylic acetic acid;within a few minutes, a clear zone around the bacterialcolonies was formed, which suggested positive action.

A similarity matrix between the isolates was calculatedusing Jaccard’s coefficient on the data derived from thephenotypic characters.

Sequencing of 16S rDNA

The 16S rDNA sequence was determined by the dideoxychain termination method using big dye terminator readyreaction mix (Applied Biosystems) according to themanufacturer’s instructions. Sequencing reaction productswere analyzed by capillary electrophoresis on an ABIPRISM 310 Genetic Analyzer (Applied Biosystems). TheBlast database (Altschul et al. 1997) of the National Centrefor Biotechnology Information (NCBI) was used tocompare the sequence of isolates with known 16S sequen-ces in the existing database.

Total in situ bacterial and Pseudomonas communityanalyzed by SSCP analysis

Soil (0.5 g) adhering to the root surface was taken as a RSsample, whereas the chopped roots (0.5 g) without theadhered soil was considered as RP sample. Rhizosphereand RP samples were placed in a bead-beater tube (BIO101, Inc., CA, USA) and shaken in a fastprep FP120 beadbeater (BIO 101, Inc., CA, USA) at 5.5 m s−1 for 30 s. TotalDNA from RS and RP samples was extracted by the fast-DNA extraction kit (BIO 101, Inc., CA, USA) as reportedby the manufacturers.

In the case of total bacterial community analysis, com1and com2 primers (Schwieger and Tebbe 1998) were used.The PCR reaction mixture contained 1× PCR buffer,250 μM dNTPs, 0.25 μM primers each of forward andreverse, 1 U Taq DNA polymerase (Genei) and soil DNAas a template in a total volume of 50 μl. The PCRamplification for bacterial community was performed at an

annealing temperature of 56°C for 1 min (Schwieger andTebbe 1998).

For Pseudomonas community analysis, standard PCRwas conducted using Ps-for and Ps-rev primers according toWidmer et al. (1998). The PCR product was purified byQIAquick column according to the manufacturer’s direc-tions (Qiagen, Crawley, UK). The purified product wasused as a template for nested PCR with primers Ps-for(Widmer et al. 1998) and com2 (Schwieger and Tebbe1998) using a ‘touchdown’ programme as described byStach et al. (2001).

The PCR amplification product was resolved on single-strand-conformation polymorphism (SSCP) gel; 50 μl ofPCR product and 50 μl of denaturing buffer (95%formamide, 10 mM NaOH, 20 mM EDTA, 0.2% bromo-phenol blue, 0.2% xylene cyanol) were mixed together.Sample was denatured at 95°C for 5 min and immediatelychilled on ice for 5 min. Samples were loaded on 10%(com1/com2 PCR product representing total eubacterialcommunity) and 12% (Ps-for/com2 PCR product represent-ing pseudomonads community) polyacrylamide gel and runin 1× TBE at 250 V for 6 h under cold temperature. Gelswere silver stained according to Bassam et al. (1991).

Data analysis and evaluation of diversity

The Shannon’s diversity index H' ¼ � Pni=Nð Þ ln ni=Nð Þ½ �f g,

Simpson’s diversity index l ¼ Pni=Nð Þ2

n oand evenness

index (E=H′/ln S) were calculated in a system afterARDRA, phenotypic characters, sequence data and commu-nity analysis (Ludwig and Reynolds 1988). In the formula, n,N and S described the number of species in a population,total number of species in a population and total number ofspecies in a community, respectively. For the calculation ofShannon’s diversity index with ARDRA and communityanalysis, the presence of each unique band in each profilewas considered as a single species and taken as anoperational taxonomic unit.

Nucleotide sequence accession numbers

The 16S rDNA nucleotide sequences obtained in this studyare deposited with the GenBank database under theaccession numbers AY677123–AY677127 and AY682627–AY682640.

Results

Bacterial strains and genetic diversity

A total of 53 (22 from pf and 31 from rb) differentmorphotypes were recovered from KB medium. Selection

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of bacterial isolates was based on both macroscopically andmicroscopically morphological characters.

The PCR amplification of 16S rDNA revealed aconspicuous band of 1.5 kb in all isolates. The restrictionpattern with RsaI, HaeIII and TaqI was represented by 2–3,3–5 and 2–3 bands, respectively. Concatenated unweightedpair group method with arithmetic mean (UPGMA)dendrogram showed that isolates belonging to RS and RPfraction were clearly distinct from each other and clusteredseparately into groups V and VII. Cluster II of pf wassubdivided into two subgroups; IIa contained isolates fromRS and IIb contained isolates from RP (data not shown).Phylogenetic analysis of 31 isolates from rb showed thatisolates from RS and RP were taxonomically distant;cluster II of rb alone contained isolates from both RS(57%) and RP (42%).

Concatenated dendrogram of ARDRA profiles of strainsfrom wheat var. UP2338-pf (53 isolates) and rb (45isolates) resulted in five major clusters (Fig. 2). AllPseudomonas reference strains grouped together; only30% isolates (cluster I) belonging to pf and rb showedsimilarity with reference strains. Most isolates from the twomanagement systems, pf or rb, were placed together. Basedon ARDRA, diversity indices were measured. Plain fieldwas slightly more diverse (H′, 4.305; l, 0.017) than rb (H′,4.055; l, 0.021); in terms of evenness (E; pf, 0.94 and rb,0.92), the two systems appeared nearly identical.

Phenotypic and metabolic characterization of bacterialisolates

Because recovery of bacterial isolates was performed onKB medium, tests specific to genus Pseudomonas wereperformed. Isolates PS6, PS7, PS8, PP12, PS4 fromUP2338-pf and RS16, RP13 and RP17 from UP338-rbwere closely related (85–99% similarity) with Pseudomo-nas standards (Fig. 3). Isolates PS6 and PS7 were 90%similar with P. fluorescens bv I and isolate RS16 (rb), 92%similar with P. fluorescens bv IV. According to phenotypicdiversity index (H′), isolates from rb were more diverse (H′,14.67) than those from pf (H′, 10.19).

Taxonomic characterization by partial sequencingof 16S rDNA

A total of 61% isolates from pf and rb were characterizedby employing partial sequencing of 16S rDNA. Mostisolates (73%) exhibited 80–100% similarity with γ-proteobacteria; genera Pseudomonas and Stenotrophomo-nas were predominant. Low occurrence of genera, Bacillus,Flavobacterium and Rhizobium was observed only in rbmanagement. Shannon diversity index (H′) at genus levelwas 1.21 and 1.54 for pf and rb management, respectively;evenness (E) values were similar for pf (0.88) and rb (0.86).The 16S rDNA sequences were used to construct a

Fig. 2 Combined UPGMA den-drogram showing phylogeneticrelationship in bacterial isolatesfrom UP2338-pf and UP2338-rbbased on the restriction profileof 16S rDNA with HaeIII, RsaIand TaqI

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phylogenetic tree (Fig. 4) along with that of availablesequences in the NCBI GenBank. Most of the micro-organisms from one management practice were clusteredtogether with the standard strains even at the separatebranches with close evolutionary relationship. In thisphylogram, genus Pseudomonas of γ-proteobacteria wasplaced distantly from others.

A comparison of the results of ARDRA and 16S rDNAsequencing revealed that conventional system (pf) wasmore diverse than raised bed (rb) cropping system.

Total bacterial and pseudomonads community diversity

In SSCP, cluster analysis revealed that communities fromRS or RP fraction of pf and rb management practicesgrouped together, although these fractions were individual-ly quite distinct. There was greater similarity in RS fractionof rb and pf (70%) than RP fraction (32%). If oneconsidered RS and RP fraction without taking into accountthe management practice, these two clusters showed only20% similarity (Fig. 5a).

Pseudomonads community dynamics revealed that RSsamples were richer in species composition than RPsamples. Cluster analysis of SSCP profiles generated bytwo-step PCR using Pseudomonas-specific primers showed50% similarity in RS communities and 42% in RPcommunities (Fig. 5b). Likewise, total community, Pseu-domonas community of RS and RP fractions, was groupedtogether.

Discussion

Agricultural management practices have definite impact onsoil microbial diversity, soil quality, crop health and cropyield (Feng et al. 2002). Microbial characteristics of soilsare increasingly being evaluated as sensitive indicators ofsoil quality (Nannipieri et al. 2003), because they are moresensitive than other soil properties to natural and anthropo-genic effects (Doran and Parkin 1994). In this study,microbial diversity of RS soil and RP was evaluated intwo management practices, the ‘conventional’ and ‘raisedbed’ practices with wheat var. UP2338 as covering crop.For the study, culturable (phenotypic and molecularcharacterization) and unculturable approaches (SSCP) ofdiversity were used. Cluster analysis of ARDRA profileplaced bacterial communities of pf and rb in separateclusters. Diversity indices data showed that samples fromconventional practice maintained more diverse bacterialprofile than samples from the raised bed practice. Lupwai etal. (1998) have reported that crop rotation and tillage canaffect the RS associated microbial communities, whereasdifferences in microbial communities in conventionaltillage and no-tillage system of cotton were observed byFeng et al. (2002). Change in structure of microbialcommunities because of conventional and raised bedpractices might be related to change in the physical andchemical properties of soil (Kennedy 1999).

Numerical characterization of phenotypic data showedslightly greater diversity in the two systems compared to

Fig. 3 Numerical analysisbased on phenotypic charactersof bacterial isolates fromUP2338-pf and UP2338-rb

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the genetic diversity. Phenotypic diversity, a reflection ofmetabolic activity of the microbial cell, was greater inraised bed than in conventional cropping system. Raisedbed management practice can be considered as no-till,

because soil is remixed only after 5 years. Under theseconditions, microbial communities are under less stress thanthat under conventional cropping system, because there wasno change from anaerobic to aerobic conditions when wheat

Fig. 4 Unrooted phylogeneticdendrogram (phylogram) con-structed using 16S rDNAsequences of the rhizobacteriarepresenting two managementsystems. The strains from thepresent study are in boldface.Isolates from one group wereclustered together despite varia-tion in their origin. The percen-tages of 100 bootstrap values areindicated. Scale bar=0.05estimated base change pernucleotide

Fig. 5 Cluster analysis of SSCPprofiles of, A total bacterial andB pseudomonads community ofUP2338-pf and UP2338-rb. RSrhizosphere, RP rhizoplane

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was cropped after rice. Earlier reports show that no-tillmanagement practice results in greater microbial activitythan conventional plain field cultivation practice (Valpassoset al. 2001).

Diversity of pseudomonads population has been studiedas a marker to assess changes in community structure of anecosystem (Latour et al. 1996; Gyamfi et al. 2002). Mostisolates from plain field and raised bed belonged to γ-proteobacteria with prevalence of Pseudomonas and Sten-otrophomonas. Bacillus pumilus was present in pf andBacillus licheniformis in rb, both belonging to the broadsubgroup of Bacillus subtilis; B. pumilus is mesophilic andaerobic, whereas B. licheniformis can grow up to 55°C andis a facultative anaerobe (Fritze 2004). Dominance ofpseudomonads and Bacillus species in wheat RS is welldocumented (Ross et al. 2000; Germida and Siciliano2001). Pseudomonas stutzeri was recovered only from rband not from pf. Some other genera belonged to α-proteobacteria (Agrobacterium) and F–C–B cluster (Flavo-bacterium and Sphingobacterium). Presence of Bacillus,Bosea, Sphingobacterium and Flavobacterium only in theRS of raised bed system may reflect on the alteredsuccessional pattern.

Because culturable diversity represents only a smallfraction of the existing diversity, culture-independent SSCPtool was used to analyze the total and pseudomonadcommunity structure in plain field and raised bed wheatagroecosystem. Total eubacterial community was onlymarginally higher in raised bed (H′=2.81) compared toconventional system (H′=2.7). This is in agreement withthe sequencing data, which showed that raised bed systemwas richer in terms of diversity spectrum. However, thenumber of bacterial species showed by the number of 16SrDNA bands was considerably lower than that reported byMahaffee and Kloepper (1997) and Yang and Crowley(2000) but similar to the data of Alvey et al. (2003). Suchcontrasting observations can be explained by plant species,which can affect root exudation with a different impact onthe microbial community structure (Germida et al. 1998;Miethling et al. 2000). However, use of moleculartechnique to resolve 16S rDNA fragments can also explainthe nature of such contradictory reports. Indeed, DGGE canproduce a band that is representative of more than onespecies, or a single bacterial species can be represented bymore than one band (Yang and Crowley 2000; Alvey et al.2003). The SSCP is based on the electrophoretic mobilityof the folded ssDNA, and the separation of DNA moleculedepends on the type of sequence and mass of the molecule;thus, PCR products of the same size but with differentsequences can be separated by different mobility of theirfolded structure (Stach et al. 2001). Results of SSCPprofiles of 16S rDNA of Pseudomonas community con-firmed that observed by the diversity indices and by the

sequencing with more abundance of Pseudomonas in pfthan in rb. Change in bacterial diversity depends onenvironmental conditions, such as soil temperature (Zogget al. 1997), soil moisture (Wilkinson et al. 2002) and soildepth (Brady and Weil 2002). Soil texture influences theRS microflora by limiting the availability of root exudatesto microorganisms (Chiarini et al. 1998).

Raised bed practice of rice–wheat cropping system is anew practice to improve soil quality, because conventionalpractice of wheat cultivation utilizes puddling of land, withdestruction of micro- and macroaggregates. It has earlierbeen reported that macroaggregates (>200 μM) are formedby an interaction of primary particles with microorganisms,plant roots, fungal hyphae, polysaccharides and humicmaterials and are destroyed by conventional soil manage-ment practices (Sessitsch et al. 2002), whereas this does notoccur in raised bed (no-till) management system.

In conclusion, the bacterial communities recovered fromconventional and raised bed system were distinctly placedin the UPGMA dendrogram. Based on ARDRA, sequenc-ing and SSCP, genus Pseudomonas was more diverse inconventional cropping system, but diversity of totaleubacterial community of raised bed system was higherthan in conventional system.

Acknowledgements Soil samples used in this study were derivedfrom the sites being investigated under the Indo-Swiss Collaborationin Biotechnology, Project SA7, operative between the University ofNeuchatel (PI: Prof. M. Aragno) and G B P U A & T, Pantnagar (PI:Prof. B N Johri). The grants provided by the Ministry of Environmentand Forest, Govt. of India, through project ‘Centre for Research onBacteria & Archaea’, J-22018/54/99-CSC (BC), an All India Coordi-nated Project on Taxonomy, to BNJ is gratefully acknowledged.

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