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During a survey of endophytic diazotrophic bacteria associated with different rice varieties in Tamilnadu, some "endophytes" were obtained. Thirteen bacterial isolates from surface-sterilized roots and shoots were obtained in pure culture, which produced indole acetic acid (IAA) and reduced acetylene to ethylene. Polymerase chain reaction (PCR) amplification confirmed the presence of nif-H gene in all the isolates. Morphological, biochemical, and molecular characteristics indicated that all of them belonged to the genus Burkholderia One of them, MGK3, was consistently more active in reducing acetylene, and 16S rDNA sequences of isolate MGK3 confirmed its identification as Burkholderia vietnamiensis. Colonization of rice root was confirmed by strain MGK3 marked with gusA gene. The inoculated roots showed a blue color, which was most intense at the points of lateral root emergence and at the root tip. Transverse sections of roots, 15 days after inoculation, revealed beta-glucuronidase (GUS) activity within many of the cortical intercellular spaces next to the stele and within the aerenchyma. Nitrogen fixation was quantified by using (15)N isotope dilution method with two different cultivars grown in pot and field experiments. Higher nitrogen fixation was observed in variety Ponni than in ADT-43, where nearly 42% (field) and 40% (pot) of the nitrogen was derived from the atmosphere (% Ndfa). Isolate MGK3 was used to inoculate rice seedlings in a comparison with four other diazotrophs, viz., Gluconacetobacter diazotrophicus LMG7603, Herbaspirillum seropedicae LMG6513, Azospirillum lipoferum 4B LMG4348, and B. vietnamiensis LMG10929. They were used to conduct two pot and four field inoculation experiments. MGK3 alone, and combined with other diazotrophs, performed best under both pot and field conditions: combined inoculation produced yield increases between 9.5 and 23.6%, while MGK3 alone increased yield by 5.6 to 12.16% over the uninoculated control treatment.

An isolate of a Gram-stain-positive, facultatively anaerobic, motile, rod-shaped, endosporeforming bacterium was recovered from soybean-based fermented paste. Phylogenetic analysis of the 16S rRNA gene indicated that the strain was most closely related to Bacillus sonorensis KCTC-13918 T (99.5 % similarity) and Bacillus licheniformis DSM 13 T (99.4 %). In phenotypic characterization, the novel strain was found to grow at 15-60 8C and to tolerate up to 10 % (w/v) NaCl. Furthermore, the strain grew in media with pH 6-11 (optimal growth at pH 7.0-8.0). The predominant cellular fatty acids were anteiso-C 15 : 0 (37.7 %) and iso-C 15 : 0 (31.5 %). The predominant isoprenoid quinone was menaquinone 7 (MK-7). The cell-wall peptidoglycan contained meso-diaminopimelic acid. A draft genome sequence of the strain was completed and used for phylogenetic analysis. Phylogenomic analysis of all published genomes of species in the B. licheniformis group revealed that strains belonging to B. licheniformis clustered into two distinct groups, with group 1 consisting of B. licheniformis DSM 13 T and 11 other strains and group 2 consisting of KJ-16 T and four other strains. The DNA G+C content of strain KJ-16 T was 45.9 % (determined from the genome sequence). Strain KJ-16 T and another strain from group 2 were subsequently characterized using a polyphasic taxonomic approach and compared with strains from group 1 and another closely related species of the genus Bacillus. Based upon the consensus of phylogenetic and phenotypic analyses, we conclude that this strain represents a novel species within the genus Bacillus, for which the name Bacillus paralicheniformis sp. nov. is proposed, with type strain KJ-16 T (5KACC T 5NRRL B-65293 T ).Cheonggukjang is a fermented soybean product in traditional Korean cuisine. It is produced by boiling soybeans and allowing them to ferment for 2-3 days in the presence of a 'natural' environmental inoculum present in the air or sometimes added through the addition of rice straw (Cho et al., 2014). The bacterial diversity of the cheonggukjang inoculum is known to be dominated by members of the genus Bacillus (Nam et al., 2012), some of which have been identified previously (e.g. Bacillus subtilis), whereas others have yet to be formally described. The purpose of this study was to describe one such species, for which we propose the name Bacillus paralicheniformis sp. nov.Strain KJ-16 T was isolated from a 3-day-old cheonggukjang fermentation on R2A agar at 28 8C. Growth was tested at 4, 10, 15, 28, 37, 45, 55 and 60 8C. The pH range for growth was determined from pH 3.0 to 12.0 in steps of 1.0 pH unit in R2A broth buffered with 0.1 M citrate/phosphate buffer (pH 3-7), Tris (pH 8, 9), NaCO 3 (pH 10, 11) or phosphate (pH 12) and adjusted with HCl or NaOH as needed (Breznak & Costilow, 1994). NaCl tolerance was investigated by using R2A broth supplemented with 0, 1, 2, 3, 4, 5, 7 and 10 % (w/v) NaCl. Growth under anaerobic conditions was determined on anaerobic agar (Difco) at 30 8C using a GasPak jar (,...

A Gram-positive bacterium, designated strain CBMB205 T , was isolated from the rhizosphere soil of traditionally cultivated, field-grown rice. Cells were strictly aerobic, motile, rod-shaped and formed endospores. The best growth was achieved at 30 6C and pH 7.0 in ammonium mineral salts (AMS) medium containing 600 mM methanol. A comparative 16S rRNA gene sequencebased phylogenetic analysis placed strain CBMB205 T in a clade with the species Bacillus amyloliquefaciens, Bacillus vallismortis, Bacillus subtilis, Bacillus atrophaeus, Bacillus mojavensis and Bacillus licheniformis and revealed pairwise similarities ranging from 98.2 to 99.2 %. DNA-DNA hybridization experiments revealed a low level (,36 %) of DNA-DNA relatedness between strain CBMB205 T and its closest relatives. The major components of the fatty acid profile were C 15 : 0 anteiso, C 15 : 0 iso, C 16 : 0 iso and C 17 : 0 anteiso. The diagnostic diamino acid of the cell wall was meso-diaminopimelic acid. The G+C content of the genomic DNA was 45.0 mol%. The lipids present in strain CBMB205 T were diphosphatidylglycerol, phosphatidylglycerol, a minor amount of phosphatidylcholine and two unknown phospholipids. The predominant respiratory quinone was MK-7. Studies of DNA-DNA relatedness, morphological, physiological and chemotaxonomic analyses and phylogenetic data based on 16S rRNA gene sequencing enabled strain CBMB205 T to be described as representing a novel species of the genus Bacillus, for which the name Bacillus methylotrophicus sp. nov. is proposed. The type strain is CBMB205 T (5KACC 13105 T 5NCCB 100236 T ).Methylotrophy has been recognized as a property of specialized bacteria that are capable of growth on C1-compounds and members of numerous genera and species have been found to possess this ability (Whittenbury et al., 1970;Arfman et al., 1992;Kalyuzhnaya et al., 2006;Lidstrom, 2006). It has been found that the occurrence of methylotrophy in prokaryotes does not correlate with traditional bacterial classification (Brautaset et al., 2004), as facultative methylotrophy has been shown to be a property of diverse typically heterotrophic genera (Boden et al., 2008). One of the earliest methylotrophic organisms to be isolated was the Gram-positive bacterium, 'Bacillus methylicus' (later renamed as 'Bacterium methylicum', but no longer available in culture). This aerobic, non-sporeforming, facultative methylotroph produced red pigment when grown on formate or methanol and also grew on formaldehyde (Loew, 1892;Migula, 1900;Bergey et al., 1939). Numerous specialized methylotrophs have since been described, including a great diversity of methanotrophs, some of which are obligate methane users, many that also use methanol and a few that are capable of growth on multicarbon compounds (Whittenbury et al., 1970;Dedysh et al., 2005). At the time of writing, the genus Bacillus, in the phylum Firmicutes, consisted of 225 species with validly published names (http://www.bacterio.cict.fr/ b/bacillus.html) with Bacillus subtilis as the type species (Cohn, 187...

Despite the importance of meju as a raw material used to make Korean soy sauce (ganjang) and soybean paste (doenjang), little is known about the bacterial diversity of Korean meju. In this study, the bacterial communities in meju were examined using both culture-dependent and independent methods in order to evaluate the diversity of the bacterial population. Analyses of the 16S rRNA gene sequences of the bacterial strains isolated from meju samples showed that the dominant species were related to members of the genera Bacillus, Enterococcus, and Pediococcus. The community DNAs extracted from nine different meju samples were analyzed by barcoded pyrosequencing method targeting of the V1 to V3 hypervariable regions of the 16S rRNA gene. In total, 132,374 sequences, with an average read length of 468 bp, were assigned to several phyla, with Firmicutes (93.6%) representing the predominant phylum, followed by Proteobacteria (4.5%) and Bacteroidetes (0.8%). Other phyla accounted for less than 1% of the total bacterial sequences. Most of the Firmicutes were Bacillus and lactic acid bacteria, mainly represented by members of the genera Enterococcus, Lactococcus, and Leuconostoc, whose ratio varied among different samples. In conclusion, this study indicated that the bacterial communities in meju were very diverse and a complex microbial consortium containing various microorganisms got involved in meju fermentation than we expected before.

A pink-pigmented, facultatively methylotrophic bacterium, strain CBMB20 T , isolated from stem tissues of rice, was analysed by a polyphasic approach. Strain CBMB20T utilized 1-aminocyclopropane 1-carboxylate (ACC) as a nitrogen source and produced ACC deaminase. It was related phylogenetically to members of the genus Methylobacterium. 16S rRNA gene sequence analysis indicated that strain CBMB20 T was most closely related to Methylobacterium fujisawaense, Methylobacterium radiotolerans and Methylobacterium mesophilicum; however, DNA-DNA hybridization values were less than 70 % with the type strains of these species. The DNA G+C content of strain CBMB20 T was 70.6 mol%. The study presents a detailed phenotypic characterization of strain CBMB20 T that allows its differentiation from other Methylobacterium species. In addition, strain CBMB20 T is the only known member of the genus Methylobacterium to be described from the phyllosphere of rice. Based on the data presented, strain CBMB20T represents a novel species in the genus Methylobacterium, for which the name Methylobacterium oryzae sp. nov. is proposed, with strain CBMB20 T (=DSM 18207 T =LMG 23582 T =KACC 11585 T ) as the type strain.The genus Methylobacterium includes a group of strictly aerobic, Gram-negative, pink-pigmented, facultatively methylotrophic (PPFM) bacteria characterized by their ability to utilize single-carbon compounds like methanol and formaldehyde via the serine pathway, as well as a wide range of multicarbon growth substrates (Green, 1992). Methylobacterium is classified in the a2 subgroup of the Proteobacteria and presently consists of 22 species with validly published names

The phylogenetic relationships of the type strains of 16 Erwinia species were investigated by performing a comparative analysis of the sequences of the 16s rRNA genes of these organisms. The sequence data were analyzed by the neighbor-joining method, and each branch was supported by moderate bootstrap values. The phylogenetic tree and sequence analyses confirmed that the genus Erwinia is composed of species that exhibit considerable heterogeneity and form four clades that are intermixed with members of other genera, such as Escherichia coli, Klebsiella pneumoniae, and Serratia marcescens. Cluster I includes the type strains of Envinia herbicola, Erwinia milletiae, Erwinia ananas, Erwinia uredovora, and Erwinia stewartii and corresponds to Dye's herbicola group. Cluster I1 consists of Erwinia persicinus, Erwinia rhapontici, Erwinia amylovora, and Erwinia cypripedii. Cluster I11 consists of Erwinia carotovora subspecies and Erwinia chrysanthemi and is characterized by the production of pectate lyases and cellulases. Envinia salicis, Erwinia rubrifaciens, and Erwinia nigrijluens form the cluster that is most distantly related to other Erwinia species. The data from the sequence analyses are discussed in the context of biochemical and DNA-DNA hybridization data.The genus Erwinia was proposed by Winslow et al. (51) for gram-negative, non-spore-forming, peritrichous, fermentative, rod-shaped bacteria, and it belongs to the family Enterobacteriaceae. This genus was proposed for plant-associated bacteria that are pathogens, saprophytes, and epiphytes.Although the heterogeneous taxonomic structure of the genus Erwinia has been discussed by using phenotypic data (13, 15-19, 31, 22, 35, 42, 44) and genotypic data (3, 6-10), the taxonomic position of this genus remains problematic (5, 32, 40, 41). Previously, Dye (15-18) classified the members of the genus Erwinia into four natural clusters. The carotovora group is characterized by soft-rot-causing and biochemically active species. Although some authors (10, 45) have proposed that this group should be designated the genus Pectobacterium and differentiated from other Erwinia species on the basis of distinct pathogenic and biochemical properties and this view was partially supported by DNA-DNA hybridization studies (9, lo), it has not been generally accepted. The amylovora group consists of pathogens that cause dry necrosis or wilt in their specific host plants, and the taxonomic position of this group as a true Erwinia group has rarely been questioned. Furthermore, Dye (15) considered members of this group subspecies (or varieties) of Erwinia amylovora. However, each species belonging to the amylovora group forms a distinct phenon, as shown in the numerical analyses of Verdonck et al. (44) and Mergaert et al. (35). In addition, DNA-DNA hybridization studies (3, 6, 7, 23, 37) indicated that Erwinia amylovora had low levels of relatedness to other species of the amylovora group, as well as other Erwinia species, and the DNA-DNA relatedness values for Erwinia salicis, Erw...

A 24-mer primer pair was generated by sequencing a URP-PCR fingerprinting-derived polymorphic band that is uniquely shared in Pectobacterium carotovorum ssp . carotovorum strains (Pcc). The primer set (EXPCCF/EXPCCR) amplified a single band of expected size (0·55 kb) from genomic DNA obtained from 29 Pcc strains and three Pectobacterium carotovorum ssp. wasabiae (Pcw) strains, but not from other P. carotovorum subspecies atrosepticum , betavasculorum or odoriferum , or from other Erwinia spp. or bacterial genera. The Rsa I digestion profile of the amplified bands divided Pcc strains into five groups with a unique profile from Pcw strains. First-round PCR detected between 5 × 10 2 and 1 × 10 3 colony forming units (CFU) mL − 1 and detection sensitivity was increased to as few as 2-4 CFU mL − 1 after second-round (nested) PCR. This PCR protocol was used directly to detect Pcc strains in infected plant tissues.