ENVIRONMENTAL MICROBIOLOGY

Genetic Diversity of Endophytic Diazotrophs of the WildRice, Oryza alta and Identification of the New Diazotroph,Acinetobacter oryzae sp. nov.

Hassan Javed Chaudhary & Guixiang Peng & Mei Hu &

Yumei He & Lijuan Yang & Yan Luo & Zhiyuan Tan

Received: 21 March 2011 /Accepted: 27 October 2011 /Published online: 22 November 2011# Springer Science+Business Media, LLC 2011

Abstract Thirty-three endophytic diazotrophs were isolatedfrom surface-sterilized leaves, stem, and roots of wild riceOryza alta. The SDS–PAGE profile of total protein andinsertion sequence-based polymerase chain reaction (IS-PCR) fingerprinting grouped the isolates into fourclusters (I–IV). The 16S rRNA gene sequence homologyof the representative strains B21, B31, B1, and B23 ofclusters I, II, III, and IV were assigned to Pseudomonasoleovorans (99.2% similarity), Burkholderia fungorum(99.4% similarity), Enterobacter cloacae (98.9% similarity),and Acinetobacter johnsonii (98.4% similarity), respectively.The results showed wide genetic diversity of the putativediazotrophic strains of the wild rice, O. alta, and thestrains of cluster IV are the first report of nitrogen-fixingAcinetobacter species. The cell size, phenotypic charac-ters, total protein profile, genomic DNA fingerprinting,

DNA–DNA hybridization, and antibiotic resistance differen-tiated strain B23T from its closest relatives A. johnsoniiLMG999T and Acinetobacter haemolyticus LMG996T. TheDNA–DNA hybridization also distinguished the strain B23T

from the closely related Acinetobacter species. Based onthese data, a novel species, Acinetobacter oryzae sp. nov.,and strain B23T (=LMG25575T=CGMCC1.10689T) as thetype strain were proposed.

Introduction

Rice (Oryza sativa) is the most important cereal crop andstaple food for more than 50% of the world’s population[1]. Nitrogen is the most frequent limiting nutrient in riceproduction which needs 1 kg per 15–20 kg of grain yield[2]. Therefore, exploitation of biological nitrogen fixationwould significantly contribute to long-term nitrogenavailability to the rice crop.

A variety of endophytic bacteria live, reproduce, andperpetuate generation to generation of the plants [3], butlittle is known about their significance on metabolism ofthe plants. Some endophytes, however, fix N2 which isused by the plants [4]. Diverse endophytes including thediazotrophs Klebsiella oxytoca, Enterobacter cloacae,Bradyrhizobium spp., Alcaligenes spp. [5], Azospirillumspp., Acetobacter diazotrophicus, Ideonella, and Herbas-pirillum [6, 7] have been isolated from different parts ofthe wild rice.

Acinetobacter spp. was mainly isolated from clinicalspecimens and is one of the important nosocomialpathogens, which has been known to cause different kindsof opportunistic infections [8]. At present, the genusAcinetobacter included 26 species with validly publishednames and the type species is Acinetobacter calcoaceticus

GenBank accession numbers: The 16S rDNA sequences of theorganisms have been deposited in the GenBank database underaccession numbers HQ697329, HQ697330, GU954428, andHQ697331 for strains B1, B21, B23T, and B31, respectively.

Electronic supplementary material The online version of this article(doi:10.1007/s00248-011-9978-5) contains supplementary material,which is available to authorized users.

H. J. Chaudhary :Y. He : L. Yang :Y. Luo : Z. Tan (*)Provincial Key Laboratory of Plant Molecular Breeding,College of Agriculture, South China Agricultural University,Guangzhou 510642, Chinae-mail: zytan@scau.edu.cn

G. PengCollege of Resources and Environment,South China Agricultural University,Guangzhou 510642, China

M. HuAgro-Environment Protection Institute, Ministry of Agriculture,Tianjin 300191, China

Microb Ecol (2012) 63:813–821DOI 10.1007/s00248-011-9978-5

(http://www.bacterio.cict.fr/a/acinetobacter.html). However,some species with validly published names were isolatedfrom environmental sources, such as four speciesisolated from the soil (Acinetobacter radioresistens [9],A. calcoaceticus [10], Acinetobacter brisouii [11] andAcinetobacter soli [12]), seven species isolated from theactivated sludge [13], and A. venetianus isolated fromsurface water [14]. Among the Acinetobacter spp. withvalidly published names, only A. radioresistens [9]reported the origin from cotton crops.

The southern region of China has more genetic diversityof wild rice, and endophytic bacteria would be expected inthe wild rice species. The cultivated rice Oryza sativa isclosely related to wild rice. The study was, therefore,aimed to search and explore the potent novel endophyticnitrogen-fixing bacteria in the wild rice species whichwould be helpful to develop crop improvement strategiesfor sustainable agriculture. Furthermore, a novel Acine-tobacter sp. was isolated from the wild rice O. alta andwas characterized thoroughly.

Materials and Methods

Isolates and Medium

Young roots of O. alta were collected from the WildRice Core Collection Nursery, South China AgriculturalUniversity. Different plant parts (roots or leaves) were cutinto pieces and surface sterilized according to thestandard protocol [15, 16]. Modified Döbereiner medium(MDM medium, 1 l)—sucrose 10 g, malic acid 5 g,K2HPO4·H2O 0.2 g, KH2PO4·H2O 0.4 g, NaCl 0.1 g,FeCl3 0.01 g, Na2MoO4 0.002 g, MgSO4·7H2O 0.2 g(sterilized separately), and CaCl2·H2O 0.02 g (sterilizedseparately), pH 7.0±0.2—was used for the study. Toprepare solid medium, 1.7% (w/v) agar and for semisolidmedium 0.2% (w/v) agar was added into the medium.Small vessels (approximately 10 ml) containing 5 mlMDM semi-solid nitrogen-free medium were inoculatedwith serial dilutions of root extracts. After incubation at37°C for 3–5 days, one loop of pellicle-forming culturewas transferred into fresh semi-solid medium for furtherincubation and observation of mobility. Further purifica-tion was done by repeatedly streaking the isolates onplates of solid MDM medium. Single colonies werepicked for further study. The bacteria were grown onsemisolid MDM, incubated at 37°C and 85% humidityfor 24 h, and assayed for acetylene reduction. Referencestrains A. haemolyticus LMG996T and A. johnsoniiLMG999T were purchased from BCCM/LMG (BelgianCoordinated Collections of Microorganisms, Laboratoriumvoor Microbiologie, Universiteit Gent, Belgium).

Acetylene-Reduction Assay (ARA)

The acetylene reduction assay was used to examine andscreen the nitrogen-fixing activity of the bacterial strainsfollowing Eckert et al. [17].The isolates were transferredseparately to test tubes containing 5 ml semisolid MDMand grown at 37°C for 24 h. Ethylene was measured using aSP-2100 gas chromatograph equipped with a flame ionizationdetector and a packed column (2.0-m long, 2.0 mm i.d.,stainless steel, packedwith GDX-502). The temperature of thecolumn oven was 50°C while for injection and detectortemperature was set at 180°C. The velocity of flow of N2, H2,and dried air was 30, 30, and 300 ml/min, respectively. Thenitrogenase activity was calculated from ethylene productionaccording to Baldani et al. [18].

Total Genomic DNA Extraction and PCR Amplificationof nifH Gene Fragments

Total genomic DNA was extracted and purified using thenormally small-scale preparation protocol [19]. Presence ofthe nifH gene, which codes for nitrogenase reductase, wasdetected by PCR amplification using the degenerate primersZehrf (5′-TGY GAY CCN AAR GCN GA-3′) and Zehrr(5′-ADN GCC ATC ATY TCN CC-3′) [20]. The PCRconditions were as follows: initial denaturation at 97°C for3 min, 97°C for 1 min, 55°C for 50 s, 72°C for 35 s, 32cycles, and final extension at 72°C for 5 min. The PCRproducts were separated by electrophoresis on 1.2% (w/v)agarose gel.

SDS–PAGE of Whole-Cell Protein

The methods of cell preparation and protein extraction ofTan et al. [21] were used to group the novel putativelydiazotrophic isolates using UPGMA cluster analysis [22].

Insertion Sequence-Based PCR (IS-PCR) Fingerprinting

The IS-PCR was performed to amplify the IS regionsfollowing PCR amplification and electrophoresis conditionsof Peng et al. [23].

16S rRNA Gene Sequencing and Analysis

Fragments of the 16S rRNA gene were amplified fromgenomic DNA of the isolates. The amplification andPCR-based direct sequencing procedures were followedaccording to Tan et al. [21]. The acquired sequences,together with some related sequences, were aligned withFASTA3 program package [24]. Ambiguous bases wereexcluded from the calculation of similarity. The treetopology was inferred by the neighbor-joining method,

814 H. J. Chaudhary et al.

and the phylogenetic tree was visualized and bootstrapped1,000 times of re-sampling by using the TREECONsoftware package [25].

DNA Base Composition and DNA–DNA Hybridization

DNA was isolated and purified as described by Marmur[26] and DNA base composition was determined spectro-photometrically [27]. DNA from Escherichia coli K-12 wasused as the standard for estimation of G+C (%) content.DNA–DNA relatedness was determined by the initialrenaturation rate method in 2× SSC [28].

Tests of Physiological and Biochemical Characters

The phenotypic features were used to characterize theisolates [29, 30]. These features covered the utilization ofsugars, amino acids, alcohol, and organic acids as solecarbon sources. The representative strain, B23T, was alsocharacterized by using Biolog GN2 MicroPlates (Hayward)simultaneously with the Acinetobacter species. Overnightcultures were used to inoculate the GN2 microplatesaccording to the manufacturer’s instructions. An antibioticresistance test, NaCl tolerance and pH for growth wereperformed in YMA following Tan et al. [31]. Somephysiological properties and substrate utilization weredetermined by means of the API 20 NE (bioMerieux,Montakieu Vercieu, France), in accordance with themanufacturer’s instructions. A. johnsonii LMG999T and A.haemolyticus LMG996T were used as reference strains.

Microscopy of the Bacteria

Bacterial strains were prepared and analyzed according toPei et al. [32]. Cell morphology was observed by usinglight microscopy and scanning electron microscope(SEM, FEI, XL-30) operated at 20.0 kV and a samplechamber pressure of 2.0 Torr.

Results

Bacterial Isolation, Acetylene-Reduction Assay (ARA)and nifH Gene Amplification

Altogether 33 putatively endophytic nitrogen-fixing bacteriawere isolated from the samples of O. alta (Table 1). All ofthem were Gram-negative, facultative anaerobic, straight orcurved rods. All strains reduced 70–105 nmol ethylene perhour per 108 cells at 28°C without yeast extract supplement(Table 1). The PCR amplification of the nifH gene producedthe expected fragment of about 360 bp from all isolates andthe positive control strain Azospirillum brasilense Sp7T.

SDS–PAGE of Whole-Cell Proteins and IS-PCRFingerprinting

The 33 novel diazotrophic isolates of whole-cell proteinswere grouped into four clusters (clusters I to IV) based onabove 82% similarity (Fig. 1). IS-PCR were obtained toevaluate the genotypic diversity of the isolates. The 33isolates showed very similar DNA fingerprinting patternswithin cluster, but different from each cluster (Fig. 2).

Sequencing of 16S rDNA and Phylogenetic Analysis

The 16S rRNA gene sequence analysis showed that therepresentative strain B21, B31, B1, and B23 of clusters I,II, III, and IV, respectively, was closely related to

Table 1 The origin and nitrogenase activity of putative endophyticdiazotrophs isolated from wild rice Oryza alta

Cluster Strain no. Cells Origin Nitrogenase activity(nmol ml−1 h−1 C2H4)

I B14 Curved rods Stem 70.0

I B15 Curved rods Leaf 75.5

I B16 Curved rods Stem 78.1

I B17 Straight Root 35.5

I B18 Straight Stem 80.5

I B19 Straight Stem 73.4

I B20 Curved rods Stem 79.7

I B21T Curved rods Root 99.0

I B22 Straight Stem 78.1

II B24 Straight Leaf 76.2

II B25 Straight Leaf 75.8

II B26 Curved rods Root 80.7

II B27 Curved rods Leaf 95.4

II B28 Straight Leaf 90.2

II B29 Straight Root 83.3

II B30 Straight Root 85.6

II B31T Straight Root 72.3

III B1T Curved rods Stem 92.4

III B2 Curved rods Stem 89.9

III B3 Curved rods Root 75.3

III B4 Straight Root 91.7

III B5 Straight Root 92.4

III B6 Straight Leaf 101.6

III B7 Curved rods Root 93.2

III B8 Curved rods Root 73.1

III B9 Curved rods Stem 77.9

III B10 Straight Root 105.0

III B11 Straight Stem 84.7

III B12 Straight Stem 93.1

III B13 Curved rods Leaf 97.1

IV B23T Straight Leaf 92.4

IV B32 Curved rods Root 70.8

IV B33 Straight Root 98.2

Acinetobacter oryzae sp. nov. 815

Pseudomonas oleovorans (99.2% similarity), Burkholderiafungorum (99.4% similarity), Enterobacter cloacae (98.9%similarity), and Acinetobacter johnsonii (98.4% similarity),respectively (Table 2). Cluster II was related to the class of

β-Proteobacteria and clusters I, III, and IV to the class ofγ-Proteobacteria, which showed the genetic diversity ofthe putative diazotrophic strains isolated from wild rice O.alta.

B22B19

B26

B31

B7

B16

B23

B14B15

B20B21B17B18B25B27

B24B28

B29B30B12B13B3B10B8B9B6B11

B1B2B4B5

B32B33

90807060 100Similarity %

90807060 100

III

I

II

IV

ClusterStrains

Figure 1 Dendrogram basedon SDS–PAGE of total proteinprofile of the diazotrophicstrains B1–B33 of O. alta

B15 B16 B17 B18 B19 B20 B21 B22 M bp

200

400

6008001000

1500B14 B24 B25 B26 B27 B28 B29 B30 B31 M bp

200

400

6008001000

1500

Cluster I Cluster II

M B1 B2 B3 B4 B5 B6 B7 B8 B12 B13 B9 B11 B10 M bp

200

400

6008001000

1500

bp

200

400

6008001000

1500

B33B23 MB32M 996 999LMG LMG

TTT

Cluster III Cluster IV with reference strains

Figure 2 IS-PCR fingerprinting of the diazotrophic isolates B1–B33 of clusters I–IV of O. alta and the reference strains A. haemolyticusLMG996T and A. johnsonii LMG999T. M standard DNA marker

816 H. J. Chaudhary et al.

Nitrogen fixation by Acinetobacter strains of cluster IVwas the first report. The phylogram of strain B23T of clusterIV based on 16S rRNA gene explained that its closestrelatives were A. johnsonii LMG999T (Z93440) and A.haemolyticus LMG996T (Z93437) with 79% and 61% ofbootstrap confidence. Also, the relationships to otherspecies within the genera Acinetobacter species do not getthe support from bootstrap confidence (less than 55%)(Fig. 3), which promotes further characterization of thenovel strains of cluster IV.

The total protein electrophoresis patterns of cluster IVstrains were almost identical but differed from those of theclose relatives (A. johnsonii LMG999T and A. haemolyticus

LMG996T) (Fig. 4). The IS-PCR also showed that thestrains of cluster IV shared almost identical DNA finger-printing patterns, but different from that of the referencestrains of A. johnsonii LMG999T and A. haemolyticusLMG996T (Fig. 2). The amplified fragments of the clusterIV strains ranged between 180 bp and 1,200 bp, but theAcinetobacter reference species were 200 bp to 1,700 bp inlength (Fig. 2).

DNA Base Composition and DNA–DNA Hybridization

The G+C content of the genomic DNA of strain B23T was44.6±0.4%, which was comparable to those of Acineto-

Table 2 16S rDNA sequence similarity of the representative strains of cluster I to IV isolates of O. alta

Cluster no. Representative strains(GenBank)

Numberof bases

Closest species (GenBank accession no.) 16S rDNAhomology (%)

I B21 (HQ697330) 1,423 Pseudomonas oleovorans strain DT4 (GQ387664) 99.2

II B31 (HQ697331) 1,442 Burkholderia fungorum strain DBT1 (HM113360) 99.4

III B1 (HQ697329) 1,402 Enterobacter cloacae strain ATCC13047T 98.9

IV B23 (GU954428) 1,396 Acinetobacter johnsonii strain LMG 999 T (Z93440) 98.4

A. bouvetii DSM 14964 (AF509827)

Acinetobacter johnsonii LMG 999 (Z93440)

A. towneri DSM 14962 (AF509823)

A. haemolyticus LMG 996 (Z93437)

A. schindleri LMG 19576 (AJ278311)

A. parvus LMG 21765 (AJ293691)

A. calcoaceticus LMG 1046 (AJ888983)

A. lwoffii LMG 1029 (X81665)

A. ursingii LMG 19575 (AJ275038)

A. junii LMG 998 (X81664)

A. baumannii LMG 1041 (X81660)

A. radioresistens DSM 6976 (X81666)

A. tandoii DSM 14970 (AF509830)

A. tjernbergiae DSM 14971 (AF509825)

A. baylyi DSM 14961 (AF509820)

A. gerneri DSM 14967 (AF509829)

A. grimontii 17A04 (AF509828)

85

60

57

79

63

100

63

84

81

85

61

A. beijerinckii CCM 7266 (AJ626712)

A. gyllenbergii CCM 7267 (AJ293694)

A. guillouiae LMG 988 (X81659)

A. bereziniae LMG 1003 (Z93443)

A. soli JCM 15062 (EU290155)

A. venetianus LMG 19082 (AJ295007)

A. brisouii 5YN5-8 (DQ832256)

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T

Strain B23 (GU954428)T

Figure 3 Phylogenetictree based on the 16S rRNAgene sequence of the strainB23T and A. johnsoniiLMG999T. Numbers (>55%)at nodes indicated occurrence(%) in 1,000 bootstrapped trees.Scale bar represents 2%nucleotide substitutions

Acinetobacter oryzae sp. nov. 817

bacter species (range 40–46%) [10]. The DNA–DNArelatedness among the cluster IV strains varied from 86%to 100%, indicating that they were the same genomicspecies. DNA–DNA relatedness among B23T and thereference strains of A. johnsonii LMG999T and A. haemo-lyticus LMG996T ranged from 32% to 41%, which arelower than the 70% DNA–DNA relatedness recommendedfor species delineation [33]. These results indicated that thestrains of cluster IV do not belong to either of therecognized species mentioned above.

Evaluation of Physiological and BiochemicalCharacteristics

The novel strains of cluster IV utilized a wide range ofcarbon sources like carbohydrates, organic acids, sugar,alcohols, amino acids, etc., grew at 10–41°C (optimum28°C-35°C) temperature, 4–10 pH, low (5%) NaCltolerance and resistant to ampicillin (300 μg ml−1), andstreptomycin, chloramphenicol, neomycin, and erythromycin(5 μg ml−1) (Supplementary Table 1). Strain B23T waspositive for gelatinase and catalase, and was negative forindole production, nitrate reduction, and urease activity. They

differed from A. johnsonii LMG999T and A. haemolyticusLMG996T for growth at 41°C in MDM medium, resistant tochloramphenicol (5 μg ml−1) and ampicillin (300 μg ml−1),and utilization of β-alanine, 4-hydroxybenzoate, L-ornithine,

D-mannose, D-fructose, D-melibiose, gentiobiose, D-cellobiose,

L-rhamnose, D-mannitol, xylitol, glycerol, trans-aconitate,glutarate, 2,3-butanediol, D-glucose, adipate, L-proline,and L-serine as sole carbon sources. Both A. johnsoniiLMG999T and A. haemolyticus LMG996T can utilize 4-aminobutyrate, citrate, Tween 40, Tween 80, D-tagatose,galactitol, ethabolamine, sodium citrate, sodium hippunate,urea, L-glutamine, and L-glycine as sole carbon sources, butstrain B23T cannot (Supplementary Table 1). Besides, A.johnsonii and A. haemolyticus have been isolated fromhuman specimens, but cluster IV strains were isolated fromwild rice.

Microscope

Scanning electron microscope (SEM) analysis showed thedistinctive features in cell length and diameter of B23T

(average) (0.91–1.05×0.45–0.54 μm), which is shorterthan A. johnsonii LMG999T (1.03–1.2×0.64–0.72 μm)and longer as compared to A. haemolyticus LMG996T

(0.82–0.90×0.45–0.55 μm) (Fig. 5).

Discussion

Diazotrophic bacteria have been isolated from a wide rangeof plants, and they are evidenced as able to supply fixednitrogen to their host plants. According to the concept ofHallmann et al. definition [34], the diazotrophic bacteriaisolated from O. alta could be defined as endophytesbecause they were isolated from surface-disinfected tissuesand they did not visibly harm the plant. The acetylenereduction assay is also prerequisite for screening N2-fixingendophytes [35].

Growth of the 33 bacteria, isolated from surface-sterilized roots of O. alta, on nitrogen-free semisolid mediasuggested that they would be diazotrophic bacteria endo-phytes [34–36]. Both the ability of ARA and amplificationof the nifH gene proved the proposition that the 33 strainswere diazotrophs [37, 38]. In clusters I, II, and III, groups

B23 B32 B33 M996 999LMG LMG

116.0

66.2

45.0

25.0

14.4

KDa

18.4

35.0

TTT

Figure 4 SDS–PAGE whole-cell protein profile of the diazotrophicstrains (B23T, B32, B33) of cluster IV of O. alta and reference speciesA. haemolyticus LMG996T and A. johnsonii LMG999T. M proteinmolecular mass standards

Figure 5 Scanning electronmicrographs of strain B23T ofO. alta and reference strains A.johnsonii LMG999T and A.haemolyticus LMG996T

818 H. J. Chaudhary et al.

of these strains were closely related to Pseudomonasoleovorans, Burkholderia fungorum, and Enterobactercloacae, which were established nitrogen-fixing endo-phytes, suggesting their wide distribution in nature [36, 39].

Acinetobacter species were non-motile, non-fermentative,aerobic, Gram-negative coccobacilli or bacilli [8].Currently, the genus Acinetobacter constituted 26 species,and among them, 14 species (A. baumannii, A. beijerinckii, A.bereziniae, A. calcoaceticus, A. gyllenbergii, A. guillouiae,A. haemolyticus, A. johnsonii, A. junii, A. lwoffii, A. parvus,A. radioresistens, A. schindleri, and A. ursingii) wereanthropogenic which formed a monophylic group basedon 16S rRNA gene sequence [10, 13, 14, 34, 36, 40, 42].Twelve species were isolated from environmental sources,such as soil and cotton [9–12], activated sludge [13], and

surface water [14]. Except the A. radioresistens [9] thatreported the origin from soil and cotton crops, cluster IVstrains were the first report of the nitrogen-fixing abilityand isolation from wild rice O. alta.

The benefits of Acinetobacter species were reported inthe field of environmental bioremediation, and clinical andindustrial microbiology [43]. Acinetobacter calcoaceticusstrain is tolerant to the presence of 400 ppm lead. ThePb-binding ability of inactivated cells was compared withthat of a commercial ion-exchange resin. The metal-binding ability of A. calcoaceticus followed the sequencePb greater than or equal to Cu greater than or equal to Crgreater than or equal to (Cd, Ni, and Zn) greater than orequal to Co [44]. A. johnsonii have the ability ofbiodegradation of pesticide named as malathion, which

Table 3 Carbon utilization of Acinetobacter oryzae sp. nov. B23T and reference strains of Acinetobacter species

Characteristic 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Growth at 38°C + − V + 6 + + + V+ + V + ND + + ND − +

Acid from D-glucose + V − 88 + + − V− − − − − + − − + −Utilization of:

4-Aminobutyrate − + V + 88 + + V V+ + − − − + − − + +

Trans-aconitate + − − 38 12 + + − − − − − − + − − − +

Citrate (Simmons) − + + V+ + + + V V− − V+ V − + − + + +

Glutarate + − − + + + + − − + V+ V+ − V − + + −L-Aspartate + + V + + + + V − − V+ − − + − − + +

Azelate 0 − − 63 + + + − + + + V − + − − + −β-Alanine + − − + 94 + + − − − − − − − − − + −L-Histidine − + − 94 94 + + V − − − − − − − + − +

Malonate − − V − 18 + + − − + − − − + − − − +

Histamine V − − 63 65 − − − − − − − − − − − − −L-Phenylalanine − − − − − + + − − V − − − − − − + +

Phenylacetate − − − 25 65 + + − V+ + − − − − − − + +

4-Hydroxybenzoate + − V− 88 88 + + − − − V+ V − + − − + +

L-Ornithine + − V− − − V + − − − − − V− − − − − +

L-Arginine − − V − − + + + − − − − − + − − − +

L-Leucine − + − − − + + V − + − − − − − − − +

L-Arabinose − − − − − + V − − − − − − − − − − −2,3-Butanediol + − V + + + + − − + − − − + V − + +

D-Glucose + − − − − − − − − − − − − + − − − −Trigonelline ND − − + 59 + V − − − − − − + − − − −Tricarballylate ND − − 38 12 + + − − − − V − V − − − +

Putrescine − − − − − + + − − − − − − − − − − +

Adipate + − − 63 + + + − V+ V + ND − + − − + −

Strain designations and number of evaluated strains (in parentheses) are as follows: 1 Acinetobacter oryzae sp. nov. (n=3 strains), 2 A.haemolyticus (n=5), 3 A. johnsonii (n=8), 4 A. bereziniae (n=16), 5 A. guillouiae (n=17), 6 A. baumannii (n=8), 7 A. calcoaceticus (n=3), 8 A.junii (n=4), 9 A. lwoffii (n=8), 10 A. radioresistens (n=6), 11 A. ursingii (n=15), 12 A. schindleri (n=13), 13 A. parvus (n=10), 14 A. baylyi(n=5), 15 A. towneri (n=2), 16 A. bouvetii (n=1), 17 A. gerneri (n=1), 18 A. tandoii (n=1). Data for reference species compiled from Nemec etal. [41, 42]. + all strains positive within 4 days of incubation, − all strains negative after 10 days of incubation; V+85–99% strains positive within6 days of incubation, V 16–84% strains positive within 6 days of incubation, V−1–15% strains positive within 6 days of incubation, ND notdetermined

Acinetobacter oryzae sp. nov. 819

at high levels can be toxic to wildlife and even to publichealth if not properly disposed [45]. A. haemolyticuscould be used in Cr (VI) reduction from waste watersreleased in industrial processes [32]. The benefits to theenvironment of A. oryzae sp. nov., which are the closestrelatives of A. johnsonii and A. haemolyticus, need furtherstudy, but at least they supply the nitrogen for the growthof rice plants.

The phenotyping and partial 16S rDNA sequenceanalysis showed the diversity of the diazotrophic bacteriaisolated from O. alta (Fig. 1 and Table 2), but those of thecluster IV revealed homology of proteins, IS fingerprintings,16S rDNA sequences, and DNA–DNA hybridizationresults (Figs. 1 and 2). The low DNA relatedness (rangedfrom 32% to 41%) between A. johnsonii LMG999T and A.haemolyticus LMG996T, and the differences in IS-PCRfingerprinting, SDS–PAGE of proteins, and physiologicaland biological tests indicated that cluster IV strainswere distinct from their closest relatives (Table 3).Based on these results and the definition of bacterialspecies, we suggest Acinetobacter oryzae sp. nov., a noveldiazotrophic bacterial cluster IV colonizing the wild riceO. alta.

Description of Acinetobacter oryzae sp. nov.

Acinetobacter oryzae (o.ry´zae. L. gen. n. oryzae of rice,the origin of the first strains).

The characters of the representative strain B23T, theendophytic diazotrophs of O. alta, are given in Table 3. Thebacterial colonies were circular, convex, smooth, andslightly opaque with entire margins and cells (n=3) wereGram-negative, non-motile bacilli/coccobacilli (0.91–1.05×0.45–0.54 μm). The colonies (n=3) were 1.5 to 2.0 mm indiameter after 24 h and 2.5 to 3.0 mm in diameter after 48 hat 30±1°C. The pH range for growth was between 4 and10, and 5% NaCl tolerance. The organisms were antibioticresistant (5 μg ml−1) to chloramphenicol, streptomycin,neomycin, erythromycin, and ampicillin, but sensitive tokanamycin (Supplementary Table 1). The strain B23T waspositive for gelatinase and catalase, and negative forindole production, and urease and nitrate reduction. Theorganisms used a wide range of carbon sources. Dextrin,β-alanine, 4-hydroxybenzoate, L-ornithine, D-mannose,

D-galactose, D-fructose, D-melibiose, gentiobiose, D-cellobiose, L-rhamnose, D-xylose, D-ribose, D-mannitol,xylitol, D,L-lacatate, L-malate, uridine, glycerol, trans-aconitate, glutarate, 2,3-butanediol, D-glucose, D-gluco-nate, adipate, L-proline, and L-serine can be used as solecarbon sources. Nevertheless, phenotypic characters ofB32T differed from the close relatives from a large numberof characters (Table 3).

The G+C content of genomic DNA of the type strain(B23T) was 44.6±0.4 mol%. The closest phylogeneticallyrelated species, according to 16S rRNA sequence data,was A. johnsonii and A. haemolyticus. Strain B23T

(=LMG25575T=CGMCC 1.10689T) is designated as thetype strain for the species.

Acknowledgments This work was supported by National BasicResearch Program of China (973 Program, 2010CB126502), NationalNatural Science Foundation of China (NSFC, 30770001), DoctoralFund of Ministry of Education of China (20094404110006), Scienceand Technology Program of Guangdong Province (2010B060200017,2010B090400450), and Program for New Century Excellent Talentsin University (NCET-07-0315).

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