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pp:1210ðcol:fig::NILÞED:Ashim

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0723-2020/$ - se

doi:10.1016/j.sy

�CorrespondE-mail addr

Systematic and Applied Microbiology ] (]]]]) ]]]–]]]

www.elsevier.de/syapm

OOF

Natural association of Gluconacetobacter diazotrophicus and diazotrophic

Acetobacter peroxydans with wetland rice

Ramachandran Muthukumarasamya,�, Ilse Cleenwerckb, Gopalakrishnan Revathia,Muthaiyan Vadivelua, D. Janssensb, B. Hostea, Kang Ui Gumc, Ki-Do Parkc,Cho Young Sonc, Tongmin Sad, Jesus Caballero-Melladoe

aMain Bio-control Research Laboratory (Unit of Tamilnadu Co-operative Sugar Federation), Good Will Avenue, Venpakkam,

Chengalpattu 603 111, Tamilnadu, IndiabBCCM/LMG Bacteria Collection, Laboratory for Microbiology, Ghent University, BelgiumcNational Yeongnam Agricultural Experiment Station, Milyang 627-130, Republic of KoreadDepartment of Agricultural Chemistry, Chungbuk National University, Cheongju, Chungbuk 361-763, Republic of KoreaeCentro de Investigacion sobre Fijacion de Nitrogeno, Universidad Nacional Autonoma de Mexico, Cuernavaca, Morelos, Mexico

Received 3 November 2004

R

ORRECTED PAbstract

The family Acetobacteraceae currently includes three known nitrogen-fixing species, Gluconacetobacter diazotro-

phicus, G. johannae and G. azotocaptans. In the present study, acetic acid-producing nitrogen-fixing bacteria wereisolated from four different wetland rice varieties cultivated in the state of Tamilnadu, India. Most of these isolateswere identified as G. diazotrophicus on the basis of their phenotypic characteristics and PCR assays using specificprimers for that species. Based on 16S rDNA partial sequence analysis and DNA: DNA reassociation experiments theremaining isolates were identified as Acetobacter peroxydans, another species of the Acetobacteraceae family, thus farnever reported as diazotrophic. The presence of nifH genes in A. peroxydans was confirmed by PCR amplification withnifH specific primers.

Scope for the findings: This is the first report of the occurrence and association of N2-fixing Gluconacetobacter

diazotrophicus and Acetobacter peroxydans with wetland rice varieties. This is the first report of diazotrophic nature ofA. peroxydans.r 2005 Published by Elsevier GmbH.

Keywords: Wetland rice; Isolation; N2 fixation; Gluconacetobacter diazotrophicus; Acetobacter peroxydans; nifH; 16S rDNA;

DNA–DNA hybridization

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UNIntroduction

Nitrogen is known as an important element in plantgrowth since it is often the primary nutrient limiting the

e front matter r 2005 Published by Elsevier GmbH.

apm.2005.01.006

ing author. +91 4114 231393; fax: +9144 24348024.

ess: mutindia@yahoo.com (R. Muthukumarasamy).

plant growth in many ecosystems. In agriculture, cropproductivity is mainly supported by the use of inorganicN fertilizers, which are expensive and damaging for theenvironment. From ecological and economical perspec-tives plant-associated biological nitrogen fixation isdesirable especially for economically important crops

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R. Muthukumarasamy et al. / Systematic and Applied Microbiology ] (]]]]) ]]]–]]]2

ECT

such as rice, which is the staple diet for more than two-fifths of the world’s population.

There are several reports on the association ofnitrogen-fixing bacteria such as Azospirillum sp. withwheat [2], Herbaspirillum seropedicae with maize [3],Gluconacetobacter diazotrophicus with sugarcane [6,13]and G. johannae and G. azotocaptans with coffee plants[11]. Also H. seropedicae [3], Burkholderia vietnamiensis

[14], Rhizobium leguminosarum bv. trifolii [45], Azoarcus[9], Serratia marcescens [16] and innumerable species ofPseudomonas [39,41] have been found in associationwith rice plants. In the family Acetobacteraceae, threenitrogen-fixing species have been described: G. diazo-

trophicus, G. johannae and G. azotocaptans [13,11,21]. G.diazotrophicus, originally described as Acetobacter dia-

zotrophicus [13] and later transferred to the genusGluconoacetobacter [43], which was subsequently cor-rected to Gluconacetobacter [44], was the first nitrogen-fixing Acetobacteraceae species described and it hasbeen investigated thoroughly with regard to its nitrogen-fixing ability [19,33]. Yet it is only since recently thatthere is evidence that it is a nitrogen contributor forcrops.

Many reports on acetic acid bacteria mention thatthese strains are mainly associated with sugar orethanol-rich environments [36,40], but recent studiesshow that their distribution is wider [21]. G. diazotro-phicus was originally isolated from sugarcane [13], butrecent reports show that this species is also associatedwith other sugar-rich plants as sweet sorghum, sweetpotato and pineapple plants [30,37] and with sugar-poorplants as coffee and ragi [21,24]. G. johannae and G.

azotocaptans were isolated from coffee [11].The present study describes the identification and

characterization of nitrogen-fixing acetic acid bacteriaG. diazotrophicus and unreported A. peroxydans recov-ered from wetland rice cultivated in India.

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UNCORMaterials and methods

Isolation of diazotrophic acetic acid bacteria from

rice

Samples were collected in triplicate from rhizospheresoil, roots and stems from four different rice varieties(Oryza sativa cv. Co 36, cv. Co 39, cv. Ponni and cv. IR50) cultivated in flooded soils of Tamilnadu, a Southernstate in India, during either flowering stage or justbefore flowering stage. The rhizosphere soil sampleswere serially diluted and 0.1ml aliquots were inoculatedinto vials containing 5ml of N-free semisolid LGImedium with an initial pH of 6.0 with 1, 10 or 30% canesugar (w/v) and 0.005% yeast extract (w/v) [31] andincubated at 32 1C for 5 days. The roots were surface

PROOF

sterilized with 70% ethanol for 5min and 0.2% mercuricchloride for 30 s and then washed several times withsterile water. Basal stem portions were cut into 5 cmpieces, surface sterilized, by dipping in 95% ethanol andthen washed six times with sterile water. From each endof the stem pieces 1 cm portions were removed bycutting with a sterile blade [16]. The outer sterility of theroot and stem samples was verified by rolling them onTryptic Soy agar (Sigma). Afterwards the samples werehomogenized with a mortar and pestle in sterilephosphate-buffered saline (PBS) and serially diluted.0.1ml aliquots were inoculated into vials containing thesemisolid LGI medium described for the rhizospheresoil.

Vials with a white to yellow surface pellicle wereassayed for acetylene reduction activity as described byHardy et al. [17]. Pellicles of the nitrogenase positivevials were sub-cultured in fresh medium before theywere streaked on LGI agar plates with 10% cane sugar(w/v) and incubated at 32 1C for 10 days. Acid-producing deep yellow and yellowish orange colonieswith dark center were picked up for further analysis.The isolates and their sources are presented in Table 1.Strains TNCSF 42, TNCSF 47, TNCSF 36 and TNCSF49 are deposited in the BCCM/LMG Bacteria Collec-tion as LMG 22174, LMG 22175, LMG 21769 andLMG 21770, respectively.

EDReference strains

The reference strains used in this study were G.

diazotrophicus LMG 7603T, G. johannae ATCC700987T, G. azotocaptans ATCC 700988T and A.

peroxydans LMG 1635T.

Phenotypic characterization

Colony morphology and pigmentation were observedon LGI agar with 10% (w/v) cane sugar and potato agarwith 10% (w/v) cane sugar [6]. Pigmentation was alsochecked on GYC agar, pH 4.5 [26]. Oxidase and catalasetests were determined using commercially available discs(Hi media). The ability to utilize various carbonsubstrates was assayed in LGI medium with NH4Cl(0.1%) and supplemented with 0.5% (w/v) of theappropriate filter sterilized carbon compound insteadof cane sugar. The ability to utilize various amino acidswas tested in LGI medium with sorbitol (0.5%) insteadof cane sugar, and supplemented with 0.1% of anappropriate filter sterilized L-amino acid. LGI mediumwith both NH4Cl (0.1%) and sorbitol (0.5%), and LGImedium lacking both, was used as positive and negativecontrols, respectively.

OOF

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Table 1. Source of diazotrophic acetic acid bacteria isolates from wetland rice (Oryza sativa)

Strain Rice variety Soil pH Source Region

Gluconacetobacter diazotrophicus

TNCSF 1 Co 39 7.4 Stem tissue Madhuranthagam, Kanchipuram

TNCSF 2 Ponni 7.2 Root tissue Nugumbal, Kanchipuram

TNCSF 21 Ponni 6.5 Root tissue Chidambaram, Cuddalore

TNCSF 41 Ponni 7.0 Rhizosphere soil Villupuram, Villupuram

TNCSF 42 IR 50 7.3 Root tissue Thirumangalam, Madurai

TNCSF 44 Co 36 6.2 Root tissue Thirukazhuakundram, Kanchipuram

TNCSF 45 Co 39 7.0 Stem tissue Thanjore, Thanjore

TNCSF 46 Ponni 7.6 Root tissue Chengalpattu, Kanchipuram

TNCSF 47 Ponni 7.2 Stem tissue Valajabad, Kanchipuram

Acetobacter peroxydans

TNCSF 20 Co 36 7.7 Root tissue Nugumbal, Kanchipuram

TNCSF 36 IR 50 7.3 Root tissue Thirumangalam, Madurai

TNCSF 37 Ponni 6.5 Root tissue Virudunagar, Tirunelveli

TNCSF 38 Co36 6.3 Stem tissue Chidambaram, Cuddalore

TNCSF 49 Ponni 7.0 Root tissue Villupuram, Villupuram

TNCSF 50 Co 36 6.2 Stem tissue Thozhudhur, Trichy

Strains TNCSF 42, TNCSF 47, TNCSF 36 and TNCSF 49 were deposited in the BCCM/LMG Bacteria Collection as LMG 22174, LMG 22175,

LMG 21769 and LMG 21770, respectively.

R. Muthukumarasamy et al. / Systematic and Applied Microbiology ] (]]]]) ]]]–]]] 3

CORRECT

Species-specific PCR for G. diazotrophicus, G.azotocaptans and G. johannae

The isolates and the type strains of the nitrogen-fixingacetic acid bacteria G. diazotrophicus LMG 7603T, G.johannae ATCC 700987T and G. azotocaptans ATCC700988T were grown in SYP [4] for 48 h at 32 1C. PCRamplifications were performed with supernatant of asingle colony that was suspended in 50 ml sterile waterand boiled for 5min at 95 1C [22]. The PCR for thedetection of G. diazotrophicus was performed by geneticmethod based on 16S rRNA gene sequence with thespecies-specific primers AC (50-CTGTTTCCCGCAAGGGAC-30) and DI (50-GCGCCCCATTGCTGGGTT-30) [35]. The species-specific PCR for G. johannae and G. azotocaptans wereperformed with the universal primer U475 (50-AAT-GACTGGGCGTAAAG-30) and with one specific pri-mer: L927Gj (50-GAAATGAACATCTCTGCT-30) forG. johannae, and L923Ga (50-AATGCTCAATCTGAA-CA-30) for G. azotocaptans according with the condi-tions described previously [11].

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U16S rDNA sequencing and phylogenetic analysis

DNA for 16S rDNA sequencing was prepared byalkalic extraction from cells grown on medium 13 fromthe Catalogue of cultures of the BCCM/LMG BacteriaCollection [20] at 28 1C. A fragment of the 16S rRNAgene of strains TNCSF 36 and TNCSF 49 was amplifiedusing oligonucleotide primers complementary to highly

ED PRconserved regions of the 16S bacterial rRNA genes. The

forward primer was 50-AGAGTTTGATCCTGGCT-CAG-30 (positions 8-27, according to the Escherichia

coli numbering system), the reverse primer 50-AAG-GAGGTGATCCAGCCGCA-30 (positions 1541-1522).PCR products were purified using a QIA quick PCRPurification Kit (Qiagen), according to the manufac-turer’s instructions. Purified PCR products were par-tially sequenced by using the ABI Prism Big DyeTerminator Cycle Sequencing Ready Reaction Kit andan Applied Biosystems 377 DNA sequencer, using theprotocols of the manufacturer (Applied Biosystems).The sequencing primers used were *Gamma, Gammaand PD [8]. Sequence assembly was performed using theprogram Auto Assembler (Applied Biosystems). Thepartial 16S rRNA gene sequences determined andsequences of strains belonging to the same phylogeneticgroup, retrieved from the EMBL library, were alignedand a phylogenetic tree was constructed by theneighbor-joining method using the Bio Numerics 1.0software package (Applied Maths). The comparison ofthe sequence data was based on 900 bases of the 16SrRNA genes. Unknown bases were discarded from thecalculations. Bootstrapping analysis was undertaken totest the statistical reliability of the topology of the treeusing 1000 bootstrap resamlings of the data. The strainnumbers, species names and accession numbers of the16S rDNA sequences retrieved from EMBL for use inthe phylogenetic analysis are presented in Fig. 1.

RECTED PROOF

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Gluconacetobacter liquefaciens LMG 1382T (X75617)

Kozakia baliensis NRIC 0488T (AB056321)

Acidomonas methanolica LMG 1668T (X77468)

Gluconobacter oxydans DSM 3503T (X73820)

Acetobacter pasteurianus LMG 1262T (X71863)

Acetobacter pomorum LTH 2458T (AJ001632)

Acetobacter peroxydans IFO 13755T (AB032352)

Acetobacter lovaniensis IFO 13753T (AB032351)

Acetobacter syzygii NRIC 0483T (AB052712)

Acetobacter tropicalis NRIC 0312T (AB032354)

Acetobacter malorum LMG 1746T (AJ419844)

Acetobacter cerevisiae LMG 1625T (AJ419843)

Acetobacter orleanensis IFO 13752T (AB032350)

Acetobacter indonesiensis NRIC 0313T (AB032356)

Acetobacter estunensis IFO 13751T (AB032349)

Acetobacter aceti NCIB 8621T (X74066)

Rhodopila globiformis DSM 161T (D86513)

Acetobacter peroxydans TNCSF 36 (AJ517177)

Asaia bogorensis NRIC 0311T (AB025928)

Acetobacter orientalis NRIC 0481T (AB052706)

Acetobacter cibinongensis NRIC 0482T (AB052710)

Acetobacter peroxydans TNCSF 49 (AJ517178)

1

Fig. 1. Neighbor-joining tree (based on 900 bases of the 16S rRNA gene sequences) reflecting the phylogenetic positions of TNCSF

36 and TNCSF 49 within the acetic acid bacteria. Rhodopila globiformisDSM 161T was used as an outgroup in this analysis. The bar

indicates 1% sequence dissimilarity. Numbers at branching points indicate bootstrap percentages derived from 1000 samples.

R. Muthukumarasamy et al. / Systematic and Applied Microbiology ] (]]]]) ]]]–]]]4

UNCORDNA preparation for G+C content determination

and for DNA–DNA hybridizations

High-molecular-weight DNA for determination of theG+C content and for DNA–DNA hybridizations wasprepared from cells grown aerobically on Z1 medium(2.0% yeast extract, 2.0% calcium lactate and 1.5%agar) at 281C by the method of Wilson [42] with minormodifications [7].

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Determination of the DNA G+C content

The G+C content of the DNA was determined byHPLC according to the method of Mesbah et al. [25].

Non-methylated phage lambda DNA (Sigma) was usedas the calibration reference.

DNA–DNA hybridizations

DNA–DNA hybridizations were performed using amodification of the microplate method described byEzaki et al. [10] and Goris et al. [15]. Hybridizationswere performed under stringent conditions at 47 1C in ahybridization solution containing 50% formamide(2� SSC, 5�Denhardt’s solution, 50% formamide,2.5% dextran sulfate, low-molecular-mass denaturedsalmon sperm DNA to a final concentration of100 mgml�1, 1.25 mg biotinylated probe DNA per milli-

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liter). The DNA relatedness percentages presented aremeans based on at least two hybridization experiments.Reciprocal reactions (e.g. A�B and B�A) wereperformed and the variation between them was withinthe limit of this method [15].

nifH PCR

The nifH gene was amplified by PCR using the primerset PolF/PolR and the conditions described previously[31].

Nucleotide sequence accession numbers

The partial 16S rDNA sequences of strains TNCSF36 and TNCSF 49 determined in this study weredeposited in EMBL under the accession numbersAJ517177 and AJ517178, respectively.

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T

Results

Isolation of diazotrophic acetic acid bacteria from

rice

Fifteen Gram-negative, acid-producing, nitrogen-fix-ing isolates were recovered from rhizosphere soil, rootsand stems of rice cultivated in India (Table 1). Thephenotypic characteristics of the isolates were deter-mined and compared with those of the known nitrogen-fixing acetic acid bacteria G. diazotrophicus, G. azoto-captans and G. johannae (Table 2). One group of 9

UNCORRECTable 2. Characteristic features of acetobacteria isolates from rice

Determinative charactersa G. d (LMG 7603T) G. j (ATCC

700987)

Size and shape of the colony

on LGI (10% sugar, after 10

days)

1.5–3mm, convex,

round, smooth

1.5–3mm, convex

round, smooth

Pigmentation on LGI (10%

sugar, after 10 days)

Yellow Yellow

Pigmentation on potato agar

(10% sugar, 10 days)

Chocolate brown pale brown

Gas production during

isolation process

� �

Successive sub-culturing is

essential (�60 times)

� �

Growth on methanol � �

Organic acids � +b

150mM of nitrate � �

G. d — G. diazotrophicus, G. j — G. johannae, G. a — G. azotocaptans, A. paAll the isolates including the type stains were Gram negative, oxidase posit

positive in the presence of 30% sucrose and possessed acetylene reduction abGrowth positive with malate.

OF

strains showed phenotypic characteristics typical of G.diazotrophicus. They formed after 10 days of incubationyellow colonies (1.5–3.0mm) on LGI agar with 10%cane sugar (w/v) and chocolate brown colonies onpotato agar with 10% cane sugar (w/v). On GYC solidmedium they produced water-soluble brown pigments.They also utilized the same carbon and nitrogensubstrates as G. diazotrophicus (Table 3). The othergroup with the remaining 6 strains differed from theknown nitrogen-fixing acetic acid bacteria. They formedafter 10 days of incubation small (0.8–1.5mm) yellow-ish-orange colonies with a dark center on LGI agar with10% cane sugar (w/v) and pale brown colonies with adark center on potato agar with 10% cane sugar (w/v).On GYC solid medium they did not produce the water-soluble brown pigments. They also differed in theirability to utilize some carbon and nitrogen substrates(Table 3). The isolation of these strains requiredsuccessive sub-culturing of the pellicles up to 50–60times in fresh medium. Gas production was noticedduring the isolation process. These strains were onlyisolated from roots and stems but not from rhizospheresoil.

ED PR

PCR specific for G. diazotrophicus, G. johannae andG. azotocaptans

Recently, clear and fast PCR methods were developedfor the identification of G. diazotrophicus [35], G.

johannae and G. azotocaptans [11]. To examine whetherthe rice isolates could be classified as any of the knownnitrogen-fixing acetic acid bacteria, PCR reactions with

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G. a (ATCC

700988)

G. d (This study) A. p (This study)

, 1.5–3mm, convex,

round, smooth

1.5–3mm, convex,

round, smooth

0.8–1.5mm, flat,

dry

Yellow Yellow Yellowish orange,

dark centered

brown (water

soluble), agar

diffusing

chocolate brown pale brown, dark

centered

� � +

� � +

� � +

� � +

� � +

— A. peroxydans.

ive, catalase negative. Oxidized ethanol and glucose and the growth was

ctivity.

UNCORRECT

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Table 3. Growth of Gluconacetobacter species and Acetobac-

ter peroxydans (A.p) on different carbon sources

Carbon substratesa G. d G. j G. a G. d

(This

study)

A. p

(This

study)

Sugars (0.5%)

Sorbitol ++ ++ + ++ ++

Meso-inositol + + � + ++

Sodium pyruvate + + � + ++

Glycerol ++ + � ++ ++

L-Rhamnose + + � + +

Ribose + + + + ++

Lactose � � � � ++

Maltose + + + + ++

Fructose + ++ ++ + +

Arabinose ++ ++ � ++ ++

Trehalose + + � + ++

Raffinose ++ + � ++ ++

Meso-erythritol ++ ++ + ++ ++

Galactose ++ ++ + ++ ++

Cello-biose + + � + +

Sodium acetate + + � + ++

Xylose ++ ++ � ++ ++

Starch � � � � +

Alcoholic sugars

(0.5%)

Ethanol + + � + ++

Methanol � � � � ++

n-Butanol � + � � ++

Organic acids (0.5%)

Succinic � � � � ++

Adipic � � � � ++

Malonic + + � + ++

Valeric � � � � ++

Fumaric � � � � ++

Acetic � � � � ++

Hippuric � � � � ++

Malic � + � � ++

Citric � � � � ++

Growth on L-

aminoacids in the

presence of sorbitol

as carbon source

L-Cysteine ++ � ++ ++ �

L-Glutamic ++ � ++ ++ ++

L-Proline ++ � � ++ ++

L-Tryptophan ++ ++ � ++ �

++, good growth,+, medium growth, �, no growth.aAll the test isolates including the type strains showed good growth

on sugars like glucose, mannose and amino acid like L-Aspartic acid.

Growth was negative in oxalic acid and amino acid like L-Glycine. See

Table 2 for other abbreviations.

R. Muthukumarasamy et al. / Systematic and Applied Microbiology ] (]]]]) ]]]–]]]6

the described species-specific primers were performed.The 9 strains forming the yellow colonies on LGImedium reacted positive in the PCR assay specific for G.

ED PROOF

diazotrophicus (based on the amplification of a specific16S rRNA gene fragment) giving a PCR product of 445bp, the size expected for this species. The results showedthat these isolates can be assigned to the species G.

diazotrophicus. The 6 strains forming the yellowish-orange colonies on LGI medium did not yield any PCRproduct in any of the species-specific PCR tests. Theseresults together with the phenotypic properties of thesestrains indicate that these isolates belong within theAcetobacteraceae family but not to any of the knownnitrogen-fixing acetic acid bacterial species thus fardescribed in this family. To identify these isolates furthertests were performed.

Phylogenetic positions of TNCSF 36 and TNCSF 49

The 16S rRNA gene of two representative strains ofthe group forming the yellowish orange colonies on LGImedium, TNCSF 36 and TNCSF 49, was partiallysequenced (729 and 948 nucleotides, respectively, weredetermined) in order to determine the phylogeneticpositions of the strains. Comparison of the partial 16SrDNA sequences of both strains revealed 99.7%sequence similarity and comparison of the partial 16SrDNA sequences determined with sequences of strainsbelonging to the same phylogenetic group, retrievedfrom the EMBL library, indicated that both strainsbelonged to the genus Acetobacter (Fig. 1). The 16SrDNA sequence of TNCSF 49 (948 nucleotides) showedsignificant sequence similarity for possible relatedness atthe species level (497%) with A. peroxydans IFO13755T (100%), A. pomorum LTH 2458T (97.9%), A.pasteurianus LMG 1262T (97.5%), A. lovaniensis IFO13753T (97.4%) and A. syzygiiNRIC 0483T (97.4%). Tothe other species of Acetobacter the similarity was below97%.

DNA relatedness and DNA base composition

TNCSF 36 and TNCSF 49 were hybridized againsteach other and against the type strain of A. peroxydansLMG 1635T their closest relative (100% 16S rDNAsequence similarity). Both strains had a high value ofDNA binding to one another (99%) and to LMG 1635T

(87–89%). The DNA G+C content of TNCSF 36 andTNCSF 49 for both strains was 60.5mol% which iscomparable to the G+C content of A. peroxydans LMG1635T (59.7mol%). These data show that both strainsbelong to the species A. peroxydans.

Presence of nif genes

A. peroxydans belongs to the Acetobacteraceae familyas G. diazotrophicus. Hitherto, A. peroxydans has neverbeen described as a diazotrophic species. Because nif

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Fig. 2. Agarose gel electrophoresis of nifH PCR amplification

products of A. peroxydans LMG 1635T (lane 1), TNCSF 36

(lane 3) and TNCSF 49 (lane 4). Lane 3: molecular weight

standard (100-bp ladder).

R. Muthukumarasamy et al. / Systematic and Applied Microbiology ] (]]]]) ]]]–]]] 7

genes are necessary for nitrogen fixation, a PCR withnifH specific primers was performed with the A.

peroxydans isolates TNCSF 36 and TNCSF 49 andwith the type strain of this species LMG 1635T. Thestrains yielded a PCR product of about 360 bp confirm-ing the presence of nif genes (Fig. 2).

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CORRECTAcetylene reduction of A. peroxydans LMG 1635

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The acetylene reduction (nitrogenase) activity ofstrain LMG 1635T was examined by the acetylenereduction assay and compared to that of the novel A.peroxydans isolates. While strain LMG 1635T showedan acetylene reduction activity lower than 10 nmol h–1

per culture (5ml), the A. peroxydans isolates TNCSF 36and TNCSF 49 produced 30 and 35 nmol of ethylene h–1

per culture, respectively. It is noteworthy that the typestrain of A. peroxydans as well as the novel isolates ofthis species showed an inconsistent nitrogenase activityeven among replicates from the same assay.

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Several studies have focused on nitrogen-fixingbacteria associated with rice [9,14,16,38,41,45] but theassociation of nitrogen-fixing acetic acid bacteria withrice cultivated under field conditions has never beenreported. The present study shows that nitrogen-fixing

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acetic acid bacteria are found in natural association withrice plants.

The acetic acid bacteria isolates recovered from riceand capable of fixing-nitrogen were assigned to thespecies G. diazotrophicus and A. peroxydans. Theidentification of the latter as a nitrogen-fixing speciesis surprising as this species was described already in 1925[36,40] and was thus far never reported as diazotrophic.Although type strain and novel isolates of A. peroxydansshowed low and inconsistent acetylene reduction activ-ities, compared to G. diazotrophicus [3], their diazo-trophic ability was confirmed by the presence of nifH

genes. On this basis, the present study shows thatnitrogen-fixing acetic acid bacteria are not restricted tothe genus Gluconacetobacter.

The isolation of G. diazotrophicus from wetland rice isnot so surprising. It is conceivable that the occurrence ofthis species, reported as a frequent colonizer of sugar-rich plants [6,30,37], in the rhizosphere as well as insiderice plants, originates from sugarcane residues left overduring prior cropping. Some rice varieties sampled inMadhuranthagam and Kanchipuram (Table 1) werecultivated as a rotation crop in sugarcane fields.However, G. diazotrophicus was also recovered fromthe inside of rice plants cultivated in some other fieldswhere there was no report of earlier sugarcane cultiva-tion (e.g., strain TNCSF 42–LMG 21767 isolated fromthe root tissue of variety IR 50 cultivated at Thiruman-galam, Madurai; Table 1). The mechanism of dispersionof G. diazotrophicus among non-vegetatively propagatedplants such as rice remains to be revealed. The presenceof VAM fungi spores, which can carry G. diazotrophicus

[30], in soils and the presence of mealy bugs, known ashost of G. diazotrophicus [1,5] and commonly occurringpests in sugarcane as well as in rice crops, could play arole.

In the present study the number of colonies of both G.

diazotrophicus and A. peroxydans, isolated from thesurface sterilized roots and stems of rice varietiescultivated in flooded fields in South India, was in therange of 102–103CFUg�1 fresh weight. Previously, ithas been reported that the number of G. diazotrophicusfound in adult sugarcane plants were in the range of105–107CFUg�1 fresh tissues [6,32], but in maturesugarcane cultivated in Brazil, the number of G.

diazotrophicus found, was only 10 to 102CFUg�1 freshweight [32]. It has been suggested that nitrogenfertilization in high levels promotes the growth of non-nitrogen-fixing bacteria and simultaneously inhibits themultiplication rate of diazotrophic bacteria [12,22,29].So the isolation of A. peroxydans from high N fertilizedsamples might have been necessitated a specific enrich-ment of this bacterium with successive sub-culturing inthe medium with a low pH and high sugar as suggestedearlier [23,12,28,29]. Although, no exact data areavailable about the levels of nitrogen fertilization

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applied in the fields where the samples were collected,generally 100–125 kg nitrogen ha–1 is recommended inthose areas. The low prevalence of acetic acid bacteria inassociation with rice can also be due to the low amountof their preferential sugar substrates present in theenvironment of this plant. Recently, it has beendemonstrated that prevalence and persistence of G.

diazotrophicus populations also differ according to thesugarcane varieties they harbor [27]. Because this studyis the first report about the natural association of twonitrogen-fixing acetic acid bacterial species with riceplants, it would be of great interest to determine theoccurrence and the persistence of these species in otherrice varieties cultivated in different regions of India andworldwide. A similar study in South Korea (manuscriptin preparation) indicates that G. diazotrophicus isnaturally associated with South Korean rice varietyHwsanbyeo in low numbers (104 g�1 fresh tissue).

The association of nitrogen-fixing G. diazotrophicus

and A. peroxydans with rice may be important foragriculture as both species may be supplying a part ofthe nitrogen that rice requires. In the case of sugarcane ithas already been proven that G. diazotrophicus is anitrogen contributor [34] and also could even morebeneficial for sugarcane plant growth by mechanismsother than nitrogen fixation [27]. But in the case of riceplants though there was a report on the BNF activity[18], there is no evidence that any one of the associatedbacterium/ or a group of bacteria reported so far beresponsible for this observed BNF. Experiment on thebeneficial effects of the association of G. diazotrophicusand A. peroxydans with rice plants are under progresswhich may throw some light on the role of these twobacteria with the rice plants.

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ORREAcknowledgements

We sincerely thank Dr. Helen Grant, TWAS, Italy forher constant encouragement. The Third World Acad-emy of Sciences, Italy is acknowledged for its fellowshipto Dr. R. Muthukumarasamy. BCCM/LMG is sup-ported by the Belgian Office for Scientific, Technical andCultural Affairs.

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