Azospirillum Melinis Sp. Nov., a Group of Diazotrophs Isolated from Tropical Molasses Grass

中国科技论文在线 http://www.paper.edu.cn

International Journal of Systematic and Evolutionary Microbiology (2006), 56, 1263–1271 DOI 10.1099/ijs.0.64025-0

Azospirillum melinis sp. nov., a group of diazotrophs isolated from tropical molasses grass

Guixiang Peng,1 Huarong Wang,2 Guoxia Zhang,2 Wei Hou,2 Yang Liu,3 En Tao Wang4 and Zhiyuan Tan2

Correspondence 1,2,3College of Resources and Environment1, Provincial Key Lab of Plant Molecular Breeding, 2 3 Zhiyuan Tan College of Agriculture and College of Life Science , South China Agricultural University, [email protected] Guangzhou 510642, China 4Departamento de Microbiologı´a, Escuela Nacional de Ciencias Biolo´gicas, Instituto Polite´cnico Nacional, Me´xico, D. F. 11340, Mexico

Fifteen bacterial strains isolated from molasses grass (Melinis minutiflora Beauv.) were identified as nitrogen-fixers by using the acetylene-reduction assay and PCR amplification of nifH gene fragments. These strains were classified as a unique group by insertion sequence-PCR fingerprinting, SDS-PAGE protein patterns, DNA–DNA hybridization, 16S rRNA gene sequencing and morphological characterization. Phylogenetic analysis of the 16S rRNA gene indicated that these diazotrophic strains belonged to the genus Azospirillum and were closely related to Azospirillum lipoferum (with 97?5 % similarity). In all the analyses, including in addition phenotypic characterization using Biolog MicroPlates and comparison of cellular fatty acids, this novel group was found to be different from the most closely related species, Azospirillum lipoferum. Based on these data, a novel species, Azospirillum melinis sp. nov., is proposed for these endophytic diazotrophs of M. minutiflora, with TMCY 0552T (=CCBAU 5106001T=LMG 23364T=CGMCC 1.5340T) as the type strain.

INTRODUCTION To date, diverse nitrogen-fixing bacteria, including Azo- spirillum lipoferum, Azospirillum brasilense, Azospirillum Association of nitrogen-fixing bacteria and herbaceous halopraeferens, Azoarcus indigens, Azoarcus communis, plants is a common phenomenon in nature. From this Azovibrio restrictus, Azospira oryzae and Burkholderia association, wild grasses can obtain nitrogen fixed by the tropica, have been isolated from the roots of numerous bacteria and grow in nitrogen-deficient soils. Diverse endo- wild and cultivated grasses grown in tropical, subtropical phytic diazotrophs have been isolated from maize, rice, and temperate regions all over the world (Kirchhof et al., sorghum, sugar cane, cameroon grass and other gramineous 1997; Reinhold et al., 1986, 1987; Reinhold & Hurek, 1988; plants (Baldani et al., 1986, 1997; Olivares et al., 1996; Reis Reinhold-Hurek et al., 1993; Reinhold-Hurek & Hurek, et al., 2004). Some of these plants could associate with a 2000; Reis et al., 2004; Tarrand et al., 1978). Among these wide range of bacteria, such as in the case of Kallar grass bacteria, Azospirillum species have been isolated from roots [Leptochloa fusca (L.) Kunth], a pioneer plant grown on salt-affected, often flooded, low-fertility soils in the Punjab of numerous wild and cultivated grasses, cereals, food crops of Pakistan, which has been found to be associated with and soils in various regions. Based on their microaerophilic five nitrogen-fixing endophytic bacterial species (Reinhold- and nitrogen-fixing characteristics, semi-solid nitrogen-free ¨ Hurek et al., 1993; Reinhold-Hurek & Hurek, 2000; Tan & medium (Dobereiner, 1980) was the key to the successful Reinhold-Hurek, 2003). isolation of these bacteria. At present, eight species have been described within this genus, including the two original species, Azospirillum lipoferum and Azospirillum brasilense Abbreviations: ARA, acetylene-reduction assay; IS-PCR, insertion (Tarrand et al., 1978), and the later-described species Azo- sequence-PCR. spirillum amazonense (Magalha˜es et al., 1983), Azospirillum The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA halopraeferens (Reinhold et al., 1987), Azospirillum irakense gene sequences of strains TMCY 243, TMCY 244, TMCY 0552T and TMCY 41 are DQ022956–DQ022959, respectively. (Khammas et al., 1989), Azospirillum largimobile (Sly & Stackebrandt, 1999), Azospirillum doebereinerae (Eckert IS-PCR fingerprinting patterns of the diazotrophic isolates from et al., 2001) and Azospirillum oryzae (Xie & Yokota, molasses grass and Azospirillum lipoferum DSM 1691T, and cellular fatty acid profiles of and data on the utilization of carbon sources by 2005). It has been reported that Azospirillum strains can Azospirillum melinis sp. nov. TMCY 0552T and Azospirillum lipoferum enhance the growth of plants by the production of phyto- DSM 1691T are available as supplementary material in IJSEM Online. hormones (Bashan & Holguin, 1997). They are also possible

64025 G 2006 IUMS Printed in Great Britain 1263 转载中国科技论文在线 http://www.paper.edu.cn

G. Peng and others

suppliers of nitrogen to their host plants (Do¨bereiner, 1983; (2001). Each bacterial isolate was inoculated into 10 ml vials con- Okon, 1985). taining 5 ml semi-solid NFB or LGI medium, which were then sealed with rubber septa. All isolates were incubated at 28 uC in the Molasses grass (Melinis minutiflora Beauv.) is a well-known dark. After 36 h, 1 % (v/v) of the air phase was replaced with acety- pasture and fodder grass in various tropical countries. The lene (Burris, 1972) and the vials were reincubated. After the addition of acetylene, three vials were sampled every 4 h for a total of 24 h leaves are covered with hairs that exude a sticky secretion to determine the amount of ethylene. Ethylene was measured using a and contain a volatile oil that gives the grass a strong and SP-2100 gas chromatograph equipped with a flame-ionization detec- distinctive odour. The odour of fresh grass is believed to tor and a packed column (2?0m62?0 mm i.d., stainless steel, repel insects, snakes and ticks. The entire plant has insec- packed with GDX-502). The velocity of flow of N2,H2 and dried air ticidal properties and has been cultivated in Brazil and was 30, 30 and 300 ml min21, respectively. Central Africa for this purpose (Thompson et al., 1978). Amplification of nifH gene fragments. To confirm further Khan et al. (1997) reported that molasses grass planted in the nitrogen-fixing capability of the isolates, DNA was extracted as corn fields significantly reduces crop damage by repelling described previously (Tan et al., 2001b) from each isolate and used destructive moths and summoning the pests’ insect enemies. as a template for amplification of the nifH gene fragment by PCR. Although molasses grass has a foul odour at certain stages, The degenerate primers Zehrf (59-TGYGAYCCNAARGCNGA-39) dried grass is free of this odour and serves as foliage. and Zehrr (59-ADNGCCATCATYTCNCC-39) (Zehr & McReynolds, Molasses grass is also drought-resistant and tolerant of soils 1989) were used. The PCR conditions were as follows: initial denaturation at 97 uC for 3 min, 30 cycles at 95 uC for 50 s, 57 uC for of fairly low fertility, and has been used as a pioneer species 35 s and 72 uC for 8 s, and final extension at 72 uC for 5 min. The after clearing of poor soils. These features indicate that products were separated by electrophoresis in 1?2 % agarose gels, molasses grass may have obtained nitrogen as a nutrient which were stained with ethidium bromide (0?5mgl21) for 30 min. from some kind of nitrogen-fixing bacteria associated with Patterns were visualized and photographed using a digital camera it, although no relevant information is available at present. (GIS-1000; Tanon) under UV light.

In the present study, we isolated and characterized some Insertion sequence-based PCR (IS-PCR) fingerprinting. This was performed to evaluate the genotypic diversity of the isolates. The nitrogen-fixing bacteria from molasses grass grown as IS-primer J3 (59-GCTCAGGTCAGGTGGCCTGG-39) was chosen pioneer plants in poor soils in a tropical region of China. according to previous studies (Adhikari et al., 1999; Weiner et al., The aims were to verify the association of this grass with 2004). All amplifications were carried out in a final volume of 50 ml diazotrophs and to classify the nitrogen-fixing bacteria and were performed using a programmable thermal cycler (model isolated from the plants. PTC-100; MJ Research). The reaction mixtures for IS-PCR con- tained (final concentrations): 50 pmol primer, 30 ng template DNA extracted from the isolate (Tan et al., 2001b), 200 mM of each dNTP METHODS (Sigma) and 3 U Taq polymerase. PCR began with a denaturation step at 97 uC for 5 min followed by 30 cycles of denaturation at 97 uC Bacterial isolation, quantification and growth media. Stems for 50 s, annealing at 60 uC for 50 s and extension at 72 uC for 2 min and roots of molasses grass were collected from Zhuhai city in each. The final extension cycle was at 72 uC for 5 min. After comple- Guangdong Province, China. Plant roots and stems were washed with tion of the PCR, samples were stored at 4 uC until gel electrophoresis. sterilized distilled water and rinsed with 96 % ethanol for 30 s, then The PCR products (10 ml aliquots) were separated in 6 % non- submerged in 0?1 % HgCl2 for 5 min and washed six times with steri- denatured polyacrylamide/bisacrylamide gels (19 : 1) in 0?56 TBE lized distilled water. The sterilized samples were then ground in 0?9% buffer (89 mM Tris, 89 mM boric acid and 0?5 M EDTA, pH 8?0). NaCl solution (1 : 5, w/v). Various carbon sources in semi-solid LGI The gels were stained with ethidium bromide and photographed as medium (Cavalcante & Do¨bereiner, 1988; Martıńez et al., 2003; Salles described for PCR of nifH. A dendrogram was created from a matrix et al., 2000) and semi-solid NFB medium (Eckert et al., 2001) were of band matching by using the unweighted pair group method with used to screen for the maximum number of putatively endophytic arithmetic means (UPGMA) (Adhikari et al., 1999; Weiner et al., diazotrophs. Small vessels (approx. 10 ml) containing 5 ml NFB or 2004). LGI semi-solid nitrogen-free medium were inoculated with serial dilutions of root extracts. With the aim of estimating the bacterial SDS-PAGE of whole-cell protein patterns. Methods of cell pre- content in the plant tissues by using the most-probable number paration, protein extraction and data analysis described previously (MPN) method, each dilution was inoculated into three vessels. After (Tan et al., 2001a) were used to estimate the diversity of the bacter- incubation at 28 uC for 3–5 days, one loop of pellicle-forming culture ial isolates. was transferred into fresh semi-solid medium for further incubation 16S rRNA gene sequencing and phylogenetic analysis. Frag- and observation of mobility. Further purification was done by repeat- ments of the 16S rRNA gene were amplified from genomic DNA of the edly streaking the isolates on plates of solid LGI or NFB medium. isolates by using the forward primer 25f (59-AACTKAAGAGTTTGAT- Single colonies were picked for further study. CCTGGCTC-39) and reverse primer 1492r (59-TACGGYTACCTTGT- Phenotypic characterization. The oxygen requirement of the iso- TACGACTT-39), as described by Hurek et al. (1997). The purified lates was determined by incubating the bacteria under aerobic and PCR products were sequenced directly as reported by Hurek et al. anaerobic conditions on solid NFB and LGI media. Colony mor- (1997), by using the sequencing primers 35f (59-CTKAAGAGTTTGA- phology was recorded on NFB medium. Cellular morphology was TCMTGGCTCAGATTGAACG-39), 342f (59-CTCCTACGGGAGGCA- examined by Gram-staining. Temperature, pH and NaCl concentra- G-39) and 930f (59-GGTTAAAACTYAAAKGAATTGACGGGGAC-39). tion ranges were determined in NFB medium using routine micro- The sequences determined, together with some related sequences biological methods. selected from GenBank with the BLAST program, were aligned by using the RDP program (Maidak et al., 1999). Alignment gaps and ambiguous Acetylene-reduction assay (ARA). The nitrogen-fixing ability of bases were excluded from the calculation of similarity. The tree topol- each isolate was tested by using the ARA, as described by Eckert et al. ogy was inferred by using the neighbour-joining method (Saitou &

1264 International Journal of Systematic and Evolutionary Microbiology 56 中国科技论文在线 http://www.paper.edu.cn

Azospirillum melinis sp. nov.

Nei, 1987) and the phylogenetic tree was visualized and bootstrapped TMCY 24 and TMCY 025. These isolates were obtained by using the TREECON software package (Van de Peer & De Wachter, from both NFB and LGI media with DL-malic acid and 1994). sucrose as the sole carbon source, respectively. The MPN 4 7 DNA base composition and DNA–DNA hybridization. DNA varied from 6610 to 2610 per gram fresh plant tissue. was isolated and purified as described by Marmur (1961) and the The bacteria were all Gram-negative, facultatively anaero- DNA base composition was determined spectrophotometrically (De bic, non-motile, straight or curved rods. Acetylene reduc- Ley et al., 1970). DNA from Escherichia coli K-12 was used as a stan- + tion was detected in all isolates 4 h after the acetylene dard for estimation of G C content. DNA–DNA relatedness was injection. Strain TMCY 0552T was able to reduce acetylene determined by using the initial renaturation rate method (De Ley 21 et al., 1970) in 26 SSC, as modified by Tan et al. (2001c). to ethylene with a mean value of 90 nmol ethylene h (108 cells)21 at 28 uC, without addition of yeast extract. This Cellular fatty acid analysis. The bacteria were incubated for 36 h amount is comparable with values obtained for other in NFB medium as mentioned above. Methods described by Sasser Azospirillum species (Eckert et al., 2001; Reinhold et al., (1990) were used to harvest the cells, extract the fatty acids and to 1987). In the PCR of the nifH gene, the expected fragment of transform the fatty acids into methyl esters. Fatty acid analysis was about 360 bp was obtained from all isolates and the positive performed using a SP-2100 (Tanon) gas chromatograph equipped T with a fused silica capillary column, SE-54 (20 m60?22 mm i.d.). control strain Azospirillum brasilense Sp 7 (Fig. 1), further Cellular fatty acid profiles were analysed by comparing the quantity confirming the nitrogen-fixing capacity of these isolates. of each compound determined as a percentage of the total fatty acids and the different kinds of fatty acid. IS-PCR fingerprinting Physiological characterization using the Biolog system. A representative strain, TMCY 0552T, and Azospirillum lipoferum T All 15 diazotrophic isolates showed very similar patterns, DSM 1691 were comparatively characterized by using Biolog GN2 but were different from that for the reference strain Azo- MicroPlates (Hayward). Overnight cultures were used to inoculate T the GN2 MicroPlates, according to the manufacturer’s instructions. spirillum lipoferum DSM 1691 (see Supplementary Fig. S1 The GN2 MicroPlates were incubated in the dark at 28 uC for 24 h in IJSEM Online). Most of the amplified fragments were and the development of colour in each test well was measured using between 240 and 1700 bp. All the isolates and the reference an automated plate reader (Benchmark; Bio-Rad). Increases in strain showed identical patterns between 339 and 1700 bp. absorbance were recorded, relative to a negative control. Similarity The novel isolates had three or four fragments between between the two strains was calculated using the formula SSM=2a/ + + 240 and 339 bp, whereas the reference strain Azospirillum (2a b c), where SSM is the simple matching coefficient, a is the lipoferum DSM 1691T showed only one fragment of 339 bp. number of positive characteristics shared by the two strains, and b and c the number of positive characteristics unique to each strain. The presence of these small fragments resulted in the novel isolates clustering together and they could be clearly Perchlorate reduction. As a phenotypic characteristic, perchlorate differentiated from Azospirillum lipoferum in the clustering reduction was determined for all the isolates and related refer- analysis (Fig. 2). The novel diazotrophic isolates were ence strains by using anaerobic culturing techniques as described by grouped together at a similarity level of 78 % and were Bruce et al. (1999), Coates et al. (1999) and Tan & Reinhold-Hurek T (2003). separated from Azospirillum lipoferum DSM 1691 at a level of 67 % similarity. RESULTS SDS-PAGE of whole-cell proteins Bacterial isolation and ARA All 15 diazotrophic isolates had almost identical protein A total of 15 endophytic bacteria were isolated from the patterns (data not shown). Combined with the results of IS- root and stem samples of molasses grass in this study and PCR fingerprinting, five diazotrophic isolates were selected numbered TMCY 41, TMCY 055, TMCY 243, TMCY 244, as representatives for further comparison with Azospirillum TMCY 255, TMCY 0256, TMCY 023, TMCY 0551, TMCY lipoferum DSM 1691T by SDS-PAGE (Fig. 3). The five 0832, TMCY 066, TMCY 225, TMCY 0552T, TMCY 0831, diazotrophic isolates had similar protein patterns, and with

Fig. 1. Electrophoretic patterns of PCR products of the nifH gene showing that all of the 15 strains isolated from molasses grass harbour the nitrogen-ﬁxing gene. Lanes: 1, genomic DNA from Azospirillum brasilense Sp 7T (positive control); 2–16, TMCY 0256, TMCY 41, TMCY 0552T, TMCY 244, TMCY 066, TMCY 225, TMCY 243, TMCY 055, TMCY 023, TMCY 255, TMCY 0551, TMCY 0831, TMCY 025, TMCY 24 and TMCY 0832, respectively; M, lambda DNA/ PstI marker.

http://ijs.sgmjournals.org 1265 中国科技论文在线 http://www.paper.edu.cn

G. Peng and others

Fig. 2. Dendrogram generated from IS-PCR ﬁngerprinting patterns showing the similarity of diazotrophs isolated from molasses grass and Azospirillum lipoferum DSM 1691T. Fig. 3. SDS-PAGE protein patterns showing the similarity among the diazotrophic strains isolated from molasses grass at least eight protein bands that were different from those of and their difference from Azospirillum lipoferum. Lanes: 1, T Azospirillum lipoferum DSM 1691 (Fig. 3). TMCY 41; 2, TMCY 0552T; 3, TMCY 243; 4, TMCY 244; 5, TMCY 066; 6, Azospirillum lipoferum DSM 1691T; M, marker. Sequencing of the 16S rRNA gene and Arrows indicate the main protein bands that are different phylogenetic analysis between the novel isolates and Azospirillum lipoferum. Four strains, TMCY 41, TMCY 0552T, TMCY 243 and TMCY 244, which were randomly selected to represent the components at retention times of 9?0, 9?8, 12?6 and diazotrophs isolated from molasses grass, were sequenced. 20?3 min (see Supplementary Fig. S2 in IJSEM Online). They had almost identical 16S rRNA gene sequences. In the These main common components made up 94 and 84?6%, reconstructed phylogenetic tree (Fig. 4), these four strains respectively, of the detected components of TMCY 0552T formed a group within the genus Azospirillum, indicating and Azospirillum lipoferum DSM 1691T (Table 2). Strain that they were a monophylic group. These four strains were T T TMCY 0552 had two components at retention times of 8 more closely related to Azospirillum lipoferum DSM 1691 and 10?8 min that were absent from Azospirillum lipoferum (97?5 % similarity) than to Azospirillum largimobile ACM T T DSM 1691 . In addition, minor components at retention 2041 and other species (similarity of 97?4 % or less). times of 13?1, 18?0, 20?5, 21?4 and 22?4 min were clear in Azospirillum lipoferum DSM 1691T, but appeared as trace DNA base composition and DNA–DNA peaks (less than 2 %) in TMCY 0552T. The relative abund- hybridization ance of each compound was also different in the two strains. The G+C content of the genomic DNA of strain TMCY T 0552 was 68?7±0?6 mol%, which is in the range for the Physiological characterization genus Azospirillum (64–71 mol%). Results of the DNA– Using the Biolog MicroPlates, we found that strain TMCY DNA hybridization are presented in Table 1. DNA–DNA T T relatedness among the strains isolated from molasses grass 0552 and Azospirillum lipoferum DSM 1691 both could utilize 42 compounds, but both could not use 17 com- varied from 81 to 95 %, with a mean of 88?7 %, indicating T that they represented the same genomic species. In contrast, pounds. TMCY 0552 could utilize 31 compounds, but not the DNA–DNA relatedness was 54–57 %, with a mean of ﬁve other compounds that could be used by Azospirillum T lipoferum (see Supplementary Table S1 in IJSEM Online). 55?6 %, among Azospirillum lipoferum DSM 1691 and three T The SSM between strain TMCY 0552 and Azospirillum strains isolated from molasses grass. The corresponding data T were 30–34 % (mean 32 %) among Azospirillum brasilense lipoferum DSM 1691 was 70 %. Sp 7T and three strains isolated from molasses grass. Perchlorate reduction Cellular fatty acid analysis As reported previously (Tan & Reinhold-Hurek, 2003), The cellular fatty acids of strain TMCY 0552T, represent- some endophytic diazotrophs have the ability of dissimi- ing the diazotrophs isolated from molasses grass, and latory (per)chloride reduction. In this study, none of the Azospirillum lipoferum DSM 1691T had four common main diazotrophic strains isolated from molasses grass could grow

1266 International Journal of Systematic and Evolutionary Microbiology 56 中国科技论文在线 http://www.paper.edu.cn

Azospirillum melinis sp. nov.

Fig. 4. Phylogenetic tree, based on 16S rRNA gene sequences constructed using the neighbour-joining method, showing the relationships of the diazotrophic bacteria isolated from molasses grass and related species. The sequence of Alcaligenes faecalis subsp. parafaecalis was used as an out- group. Bootstrap values greater than 60 % are indicated at nodes. Bar, 5 % nucleotide substitutions.

with perchlorate as the terminal electron acceptor, whereas plants. The vigorous growth of molasses grass in non- Azospirillum lipoferum DSM 1691T could. fertilized soils may also be related to this association. In the present study, we isolated 15 bacterial strains from surface- sterilized roots and stems by using nitrogen-free semi-solid DISCUSSION media. All of the strains were identiﬁed as diazotrophic Endophytic diazotrophic bacteria have been isolated from bacteria based upon the results of ARA and PCR of the nifH a wide range of plants grown in poor soils and have been gene. In an inoculation test, these strains could promote the shown to be able to supply ﬁxed nitrogen to their host growth of molasses grass and increase tolerance to acid soil

Table 1. DNA–DNA relatedness (%) among the novel Azospirillum strains isolated from molasses grass and some reference strains

E. coli K-12 was used as a negative control.

Strain TMCY TMCY TMCY TMCY TMCY TMCY TMCY TMCY 243 244 41 066 0831 24 0832 0552T

TMCY 0552T 90 86 95 84 92 93 81 100 Azospirillum lipoferum DSM 1691T 57 54 56 Azospirillum brasilense Sp 7T 32 30 34 E. coli K-12 15

http://ijs.sgmjournals.org 1267 中国科技论文在线 http://www.paper.edu.cn

G. Peng and others

Table 2. Comparison of the cellular fatty acid composition within the genus Azospirillum. The small differences among of Azospirillum melinis sp. nov. TMCY 0552T and them in IS-PCR fingerprinting and SDS-PAGE of whole-cell T Azospirillum lipoferum DSM 1691 , determined as methyl proteins showed that they were from a diverse population. esters by gas chromatography The 97?5 % and lower similarities of the 16S rRNA gene Fatty acid values are given as a percentage of the total peak area. sequence to other defined Azospirillum species, the low-to- 2, Component is missing; tr, trace peak (area less than 2 %). medium DNA–DNA relatedness with Azospirillum lipoferum and Azospirillum brasilense, and the differences in Fatty acid Strain Retention time IS-PCR fingerprinting, SDS-PAGE of proteins, lipid acids (min) and Biolog MicroPlate tests from the most closely related TMCY DSM T T species, Azospirillum lipoferum, indicated that the new 0552 1691 strains were distinct from this recognized species. In addi- Main common 71?443?69?0 tion, the group of new strains could also be distinguished 4?87?79?8 from other recognized Azospirillum species (Table 3). Tak- 5?816?712?6 ing together all the results and the current definition of 12?016?620?3 bacterial species, we propose that the novel group of endo- Total 94 84?6 phytic diazotrophic bacteria isolated from molasses grass Different 2?7 2 8 represents a novel species, Azospirillum melinis sp. nov. 3?3 2 10?8 tr 3?813?1 In previous studies, nitrogen-fixing strains belonging to the tr 2?418?0 genera Azospirillum, Azotobacter, Bacillus, Derxia, Entero- tr 2?720?5 bacter, Burkholderia, Herbaspirillum and Erwinia were tr 4?121?4 isolated from sugar cane (Boddey et al., 2003). Kallar tr 2?422?4 grass as a wild plant (Reinhold-Hurek et al., 1993; Reinhold- Total 6?015?4 Hurek & Hurek, 2000; Tan & Reinhold-Hurek, 2003) and rice as a cultivated plant (Engelhard et al., 2000) also harboured various species of the genus Azoarcus. Compared with previously reported results, molasses grass showed and they were reisolated from inoculated plants (data not a specific association with Azospirillum melinis sp. nov. shown). According to the concept of Hallmann et al. (1997), Although various culture media and aerobic and anaerobic the diazotrophic bacteria isolated from molasses grass could incubation conditions were used, only strains representing be defined as endophytes, because they were isolated from Azospirillum melinis were isolated from molasses grass in surface-disinfected tissues and did not visibly harm the this study. As there is no related information available, we plant. The density of these bacteria was also in the range for could only hypothesize that this limited diversity might be endophytic bacteria [103–106 (g fresh tissue)21] (Hallmann linked to the specificity of the association between molasses et al., 1997; McInroy & Kloepper, 1995). These results grass and its endophytes, or to the soil characteristics. More studies are needed to verify these hypotheses. demonstrated that the endophytic diazotrophic strains are potential biofertilizers for molasses grass, similar to some Azospirillum lipoferum strains (Vessey, 2003). Description of Azospirillum melinis sp. nov. 9 At present, the taxonomy of Azospirillum species is mainly Azospirillum melinis (me.lin is. N.L. fem. n. melinis genus based on a polyphasic approach including 16S rRNA gene name of stinkgrass, Melinis minutiflora Beauv.; N.L. gen. n. sequencing, DNA–DNA hybridization, fatty acid composi- melinis from stinkgrass, referring to its frequent occurrence in association with molasses grass). tion, DNA G+C content and phenotypic characterization (Eckert et al., 2001; Sly & Stackebrandt, 1999; Xie & Yokota, Cells are straight or slightly curved rods, measuring 0?7–0?8 2005). This polyphasic approach is necessary because the 61?0–1?5 mm. Gram-negative and non-motile. Facultatively consensus among different analyses can offer stable classi- anaerobic and chemo-organotrophic. Colonies on NFB fication results, whereas a single analysis may be not able medium are circular, convex and translucent, with a diameter to differentiate the species correctly. For example, Schenk of 3 mm within 3 days at 28 uC. Growth occurs at 5–37 uC & Werner (1988) reported that Azospirillum lipoferum (optimum 20–33 uC). pH range for growth is between 4 and and Azospirillum brasilense had similar fatty acid profiles, 8. Growth is inhibited by >5 % NaCl, but at concentrations and therefore it was difficult to distinguish these two long- <3 % shows ARA activity. Arabinose, D-fructose, gluconate, established species by using fatty acid profiles. In the pre- glycerol, malate, mannitol, maltose and sorbitol can be used sent study, high similarity was observed among the 15 as sole carbon sources. Does not grow with perchlorate as diazotrophic strains isolated from molasses grass in SDS- the terminal electron acceptor under anaerobic conditions. PAGE of proteins, IS-PCR fingerprinting patterns, 16S Isolated at a high frequency as endophytes from molasses rRNA gene sequencing and DNA–DNA hybridization, as grass. The G+C content of genomic DNA of the type well as in cellular and colony morphology. These data clearly strain is 68?7 mol%. The closest phylogenetic related spe- indicated that the strains represented the same species cies, according to 16S rRNA gene sequence data, are

1268 International Journal of Systematic and Evolutionary Microbiology 56 中国科技论文在线 http://www.paper.edu.cn

Azospirillum melinis sp. nov.

Table 3. Characteristics that differentiate Azospirillum melinis sp. nov. from other Azospirillum species

Species: 1, Azospirillum melinis;2,Azospirillum lipoferum;3,Azospirillum largimobile;4,Azospirillum oryzae;5,Azospirillum brasilense;6, Azospirillum amazonense;7,Azospirillum halopraeferens;8,Azospirillum irakense;9,Azospirillum doebereinerae. Data from Eckert et al. (2001), Sly & Stackebrandt (1999), Ben Dekhil et al. (1997), Khammas et al. (1989), Reinhold et al. (1987), Krieg & Do¨bereiner (1984) and Xie & Yokota (2005). +, Positive; 2, negative; +(2), positive, type strain negative; V, variable or inconsistent; V(+), variable, type strain positive; ND, not determined.

Characteristic 1 2 3 4 5 6 7 8 9

Biotin requirement 2 + 2 + 22+ 22 Growth in 3 % NaCl + 222V 2 ++2 Carbon source: N-Acetylglucosamine +++ND 2 VND+ 2 L-Arabinose ++++V + V + ND D-Cellobiose + 2222 + ND + 2 D-Fructose +++++++ V + L-Fucose + V (+)* 2 ND 2 + ND + ND D-Galactose ++++V + 2 + V Gentiobiose + 2 + ND 2 + ND + 2 D-Gluconate ++(2)* 2 ND + 2 ND 2 V D-Glucose ++++V + 2 + V Glycerol +++ND + 2 + 2 + myo-Inositol + V (+)* 22 2 + ND 22 Lactose + 2222 VND+ 2 Maltose + 2222 + ND + 2 D-Mannitol +++22 2 + 2 + D-Mannose + V (+)* 2 ND 2 +++2 L-Rhamnose + 22+ 2 VND+ 2 D-Ribose ND +++2 ++ V 2 D-Sorbitol +++22222+ Sucrose + 2 ND 22 + 2 + 2 D-Trehalose + 22ND 2 + ND + 2 DNA G+C content (mol%) 68?7 69–70 70 66?8 69–71 66–68 68–70 64–67 70?7

*Data for the type strains from Ben Dekhil et al. (1997) and Sly & Stackebrandt (1999).

Azospirillum lipoferum, Azospirillum oryzae and Azospirillum a root-associated nitrogen-fixing bacterium. Int J Syst Bacteriol 36, largimobile. The type strain is TMCY 0552T (=CCBAU 86–93. 5106001T=LMG 23364T=CGMCC 1.5340T), which was Baldani, J. I., Caruso, L., Baldani, V. L. D., Goi, S. R. & Do¨ bereiner, J. isolated from subtropical molasses grass grown in China. (1997). Recent advances in BNF with non-legume plants. Soil Biol Biochem 29, 911–922. Bashan, Y. & Holguin, G. (1997). Azospirillum-plant relationships: environmental and physiological advances (1990–1996). Can J Micro- ACKNOWLEDGEMENTS biol 43, 103–121. Ben Dekhil, S., Cahill, M., Stackebrandt, E. & Sly, L. I. (1997). This work was supported by the National Natural Science Foundation Transfer of Conglomeromonas largomobilis subsp. largomobilis to of China (30300001, 30470002) and by the Presidential Foundation the genus Azospirillum as Azospirillum largomobile comb. nov., and of South China Agricultural University. We thank Dr J. Euze´by for elevation of Conglomeromonas largomobilis subsp. parooensis to the checking the Latin name. new type species of Conglomeromonas, Conglomeromonas parooensis sp. nov. Syst Appl Microbiol 20, 72–77. Boddey, R. M., Urquiaga, S., Alves, B. J. R. & Reis, V. (2003). REFERENCES Endophytic nitrogen fixation in sugarcane: present knowledge and future applications. Plant Soil 252, 139–149. Adhikari, T. B., Mew, T. W. & Leach, J. E. (1999). Genotypic and Bruce, R. A., Achenbach, L. A. & Coates, J. D. (1999). Reduction of pathotypic diversity in Xanthomonas oryzae pv. oryzae in Nepal. (per)chlorate by a novel organism isolated from paper mill waste. Phytopathology 89, 687–694. Environ Microbiol 1, 319–329. Baldani, J. I., Baldani, V. L. D., Seldin, L. & Do¨ bereiner, J. (1986). Burris, R. H. (1972). Nitrogen fixation – assay methods and tech- Characterization of Herbaspirillum seropedicae gen. nov., sp. nov., niques. Methods Enzymol 24B, 415–431.

http://ijs.sgmjournals.org 1269 中国科技论文在线 http://www.paper.edu.cn

G. Peng and others

Cavalcante, V. A. & Do¨ bereiner, J. (1988). A new acid-tolerant Olivares, F. L., Baldani, V. L. D., Reis, V. M., Baldani, J. I. & nitrogen-fixing bacterium associated with sugarcane. Plant Soil 108, Dobereiner, J. (1996). Occurrence of the endophytic diazotrophs 23–32. Herbaspirillum spp. in roots, stems, and leaves, predominantly of Coates, J. D., Michaelidou, U., Bruce, R. A., O’Connor, S. M., Crespi, Gramineae. Biol Fertil Soils 21, 197–200. J. N. & Achenbach, L. A. (1999). Ubiquity and diversity of dissi- Reinhold, B. & Hurek, T. (1988). Location of diazotrophs in the root milatory (per)chlorate-reducing bacteria. Appl Environ Microbiol 65, interior with special attention to the Kallar grass association. Plant 5234–5241. Soil 110, 259–268. De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative Reinhold, B., Hurek, T., Niemann, E.-G. & Fendrik, I. (1986). Close measurement of DNA hybridization from renaturation rates. Eur association of Azospirillum and diazotrophic rods with different root J Biochem 12, 133–142. zones of Kallar grass. Applied Environ Microbiol 52, 520–526. Do¨ bereiner, J. (1980). Forage grasses and grain crops. In Methods Reinhold, B., Hurek, T., Fendrik, I., Pot, B., Gillis, M., Kersters, K., for Evaluation of Biological Nitrogen Fixation, pp. 535–555. Edited by Thielemans, S. & De Ley, J. (1987). Azospirillum halopraeferens sp. J. F. J. Bergersen. Chichester: Wiley. nov., a nitrogen-fixing organism associated with roots of Kallar grass Do¨ bereiner, J. (1983). Ten years of Azospirillum.InAzospirillum II: (Leptochloa fusca (L.) Kunth). Int J Syst Bacteriol 37, 43–51. Genetics, Physiology, Ecology, pp. 9–23. Edited by W. Klingmueller. Reinhold-Hurek, B. & Hurek, T. (2000). Reassessment of the taxo- Basel: Birkhaeuser. nomic structure of the diazotrophic genus Azoarcus sensu lato and Eckert, B., Weber, O. B., Kirchhof, G., Halbritter, A., Stoffels, M. & description of three new genera and new species, Azovibrio restrictus Hartmann, A. (2001). Azospirillum doebereinerae sp. nov., a nitrogen- gen. nov., sp. nov., Azospira oryzae gen. nov., sp. nov. and Azonexus fixing bacterium associated with the C4-grass Miscanthus. Int J Syst fungiphilus gen. nov., sp. nov. Int J Syst Evol Microbiol 50, 649–659. Evol Microbiol 51, 17–26. Reinhold-Hurek, B., Hurek, T., Gillis, M., Hoste, B., Vancanneyt, M., Kersters, K. & De Ley, J. (1993). Azoarcus gen. nov., nitrogen- Engelhard, M., Hurek, T. & Reinhold-Hurek, B. (2000). Preferential fixing proteobacteria associated with roots of Kallar grass (Leptochloa occurrence of diazotrophic endophytes, Azoarcus spp., in wild rice fusca (L.) Kunth) and description of two species, Azoarcus indigens species and land races of Oryza sativa in comparison with modern sp. nov. and Azoarcus communis sp. nov. Int J Syst Bacteriol 43, races. Environ Microbiol 2, 131–141. 574–584. Hallmann, J., Quadt-Hallmann, A., Mahaffee, W. F. & Kloepper, J. W. Reis, V. M., Estrada-de los Santos, P., Tenorio-Salgado, S. & (1997). Bacterial endophytes in agricultural crops. Can J Microbiol 10 other authors (2004). Burkholderia tropica sp. nov., a novel 43, 895–914. nitrogen-fixing, plant-associated bacterium. Int J Syst Evol Microbiol Hurek, T., Egener, T. & Reinhold-Hurek, B. (1997). Divergence 54, 2155–2162. in nitrogenases of Azoarcus spp., Proteobacteria of the b subclass. Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new J Bacteriol 179, 4172–4178. method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425. Khammas, K. M., Ageron, E., Grimont, P. A. D. & Kaiser, P. (1989). Salles, J. F., Gitahy, P. D. M., Skot, L. & Baldani, J. I. (2000). Use Azospirillum irakense sp. nov., a new nitrogen-fixing bacterium of endophytic diazotrophic bacteria as a vector to express the cry3A associated with rice roots and rhizosphere soil. Res Microbiol 140, gene from Bacillus thuringiensis. Braz J Microbiol 31, 154–160. 679–693. Sasser, M. (1990). Identification of bacteria by gas chromatography of Khan, Z. R., Ampong-Nyarko, K., Chiliswa, P. & 8 other authors (1997). cellular fatty acids, MIDI Technical Note 101. Newark, DE: MIDI Intercropping increases parasitism of pests. Nature 388, 631–632. Inc. Kirchhof, G., Reis, V. M., Baldani, J. I., Eckert, B., Do¨ bereiner, J. Schenk, S. U. & Werner, D. (1988). Fatty acid analysis of four Azos- & Hartmann, A. (1997). Occurrence, physiological and molecular pirillum species reveals three groups. Arch Microbiol 149, 580–582. analysis of endophytic diazotrophic bacteria in gramineous energy plants. Plant Soil 194, 45–55. Sly, L. I. & Stackebrandt, E. (1999). Description of Skermanella parooensis gen. nov., sp. nov. to accommodate Conglomeromonas Krieg, N. R. & Do¨ bereiner, J. (1984). Genus Azospirillum Tarrand, largomobilis subsp. parooensis following the transfer of Conglomero- ¨ AL Krieg and Dobereiner 1979, 79 (effective publication: Tarrand, monas largomobilis subsp. largomobilis to the genus Azospirillum. Int ¨ Krieg and Dobereiner 1978, 978). In Bergey’s Manual of Systematic J Syst Bacteriol 49, 541–544. Bacteriology, vol. 1, pp. 94–104. Edited by N. R. Krieg & J. G. Holt. Baltimore: Williams & Wilkins. Tan, Z. & Reinhold-Hurek, B. (2003). Dechlorosoma suillum Achenbach et al. 2001 is a later subjective synonym of Azospira oryzae Reinhold- Magalha˜ es, F. M., Baldani, J. I., Souto, S. M., Kuykendall, J. R. & Hurek and Hurek 2000. Int J Syst Evol Microbiol 53, 1139–1142. Do¨ bereiner, J. (1983). A new acid-tolerant Azospirillum species. Ann Acad Bras Cienc 55, 417–430. Tan, Z., Hurek, T., Gyaneshwar, P., Ladha, J. K. & Reinhold-Hurek, B. (2001a). Novel endophytes of rice form a taxonomically distinct sub- Maidak, B. L., Cole, J. R., Parker, C. T., Jr & 11 other authors (1999). group of Serratia marcescens. Syst Appl Microbiol 24, 245–251. A new version of the RDP (Ribosomal Database Project). Nucleic Tan, Z., Hurek, T., Vinuesa, P., Mu¨ ller, P., Ladha, J. K. & Reinhold- Acids Res 27, 171–173. Hurek, B. (2001b). Specific detection of Bradyrhizobium and Rhizo- Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic bium strains colonizing rice (Oryza sativa) roots by 16S-23S ribo- acid from microorganisms. J Mol Biol 3, 208–218. somal DNA intergenic spacer-targeted PCR. Appl Environ Microbiol Martıńez, L., Caballero-Mellado, J., Orozco, J. & Martıńez-Romero, E. 67, 3655–3664. (2003). Diazotrophic bacteria associated with banana (Musa spp.). Tan, Z. Y., Kan, F. L., Peng, G. X., Wang, E. T., Reinhold-Hurek, B. & Plant Soil 257, 35–47. Chen, W. X. (2001c). Rhizobium yanglingense sp. nov., isolated from McInroy, J. A. & Kloepper, J. W. (1995). Population dynamics of endo- arid and semi-arid regions in China. Int J Syst Evol Microbiol 51, phytic bacteria in field-grown sweet corn and cotton. Can J Microbiol 909–914. 41, 895–901. Tarrand, J. J., Krieg, N. R. & Do¨ bereiner, J. (1978). A taxonomic Okon, Y. (1985). Azospirillum as a potential inoculant for agriculture. study of the Spirillum lipoferum group, with description of a new Trends Biotechnol 3, 223–228. genus, Azospirillum gen. nov. and two species, Azospirillum lipoferum

1270 International Journal of Systematic and Evolutionary Microbiology 56 中国科技论文在线 http://www.paper.edu.cn

Azospirillum melinis sp. nov.

(Beijerinck) comb. nov. and Azospirillum brasilense sp. nov. Can Weiner, M., Dacko, J. & Osek, J. (2004). Insertion element IS3 PCR- J Microbiol 24, 967–980. based method for molecular analysis of enterotoxigenic Escherichia Thompson, K. C., Roa, E. J. & Romero, N. T. (1978). Anti-tick grasses coli isolated from sucking piglets with diarrhoea. Bull Vet Inst Pulawy as the basis for developing practical tropical tick control packages. 48, 241–246. Trop Anim Health Prod 10, 179–182. Xie, C.-H. & Yokota, A. (2005). Azospirillum oryzae sp. nov., a Van de Peer, Y. & De Wachter, R. (1994). TREECON for Windows: a nitrogen-ﬁxing bacterium isolated from the roots of the rice plant software package for the construction and drawing of evolutionary Oryza sativa. Int J Syst Evol Microbiol 55, 1435–1438. trees for the Microsoft Windows environment. Comput Appl Biosci Zehr, J. P. & McReynolds, L. A. (1989). Use of degenerate oligo- 10, 569–570. nucleotides for ampliﬁcation of the nifH gene from the marine Vessey, J. K. (2003). Plant growth promoting rhizobacteria as bio- cyanobacterium Trichodesmium thiebautii. Appl Environ Microbiol fertilizers. Plant Soil 255, 571–586. 55, 2522–2526.

http://ijs.sgmjournals.org 1271