Munusamy Madhaiyan

Growth promotion and induction of systemic resistance in rice cultivar Co-47 (Oryza sativa L.) by Methylobacterium spp.

Pink-pigmented facultatively methylotrophic bacteria (PPFMs), persistent colonizers of plant leaf surfaces, belong to the genus Methrlobacterium and are mostly transmitted through seeds. Plant growth-promoting activity of methylotrophic bacteria and their effects on disease suppression were evaluated on rice under greenhouse conditions. Rice seeds were inoculated with Methylobacterium sp. strain PPFM-Os-07 and seed germination was evaluated in terms of morphometric measurements, seedling growth, rate of germination (R(subscript G)), and seedling vigour index (SVI). Another experiment was carried out to study the induction of pathogenesis-related proteins (PR-proteins) in rice plants that were inoculated with methylotrophic bacteria by seed imbibition or foliar spray. In the third experiment, sixty-day-old rice plants grown in pots were challenge inoculated with Rhizoctonia solani strain TNAU-01. Methylobacterium inoculation promoted seed germination and plant growth. Increased plant height, number of tillers, plant biomass, and grain yield were observed. The average yield increases for seed imbibition and phyllosphere spray were, respectively, 22.1% and 24.3% greater than control. The bacteria also significantly reduced the sheath blight incidence when applied as either bacterial culture through seed imbibition and or phyllosphere spray. The percent disease reduction recorded for seed imbibition alone and for combined applications of seed imbibition and phyllosphere spray were 17.8% and 23.5%. Rice plants sprayed with PPFM-Os-07 strain showed increased presence of PR-proteins and phenolic contents on day 1 after application. Maximum phenylalanine ammonia lyase (PAL) and peroxidase activity on day 4 and β-1,3-glucanase and chitinase activity on day 5 were recorded. The results suggest that Methylobacterium inoculation may alter rice susceptibility to R. solani. This work emphasizes the importance of evaluating induced systemic resistance while studying plant-associated growth promoting bacteria.

Bacillus methylotrophicus sp. nov., a methanol-utilizing, plant-growth-promoting bacterium isolated from rice rhizosphere soil.

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°C and pH 7.0 in ammonium mineral salts (AMS) medium containing 600 mM methanol. A comparative 16S rRNA gene sequence-based 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 C₁₅:₀ anteiso, C₁₅:₀ iso, C₁₆:₀ iso and C₁₇:₀ 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) (=KACC 13105(T)=NCCB 100236(T)).

Effect of co-inoculation of methylotrophic Methylobacterium oryzae with Azospirillum brasilense and Burkholderia pyrrocinia on the growth and nutrient uptake of tomato, red pepper and rice

The present greenhouse study was undertaken to evaluate the effects of co-inoculating methylotrophic Methylobacterium oryzae CBMB20 along with nitrogen-fixing Azospirillum brasilense CW903 or a phosphate solubilizing bacterium Burkholderia pyrrocinia CBPB-HOD on the growth and nutrient uptake of tomato, red pepper and rice. Seed inoculation and soil/foliar application of the bacterial strains alone or under dual inoculation increased the plant growth in terms of shoot or root length and increased the nutrient uptake in the plants studied compared to uninoculated control plants. Co-inoculation of M. oryzae CBMB20 with A. brasilense CW903 or B. pyrrocinia CBPB-HOD improved the N and P concentration of plants, while the results varied among the plant species tested. Also, co-inoculation of the bacterial strains increased the activity of nitrogenase, urease and phosphatase enzymes in soil when compared to uninoculated control or individual inoculations. Though the inoculation effects were analyzed at an early stage of plant growth, the results conclusively suggest that M. oryzae being compatible with other microorganisms in the rhizosphere can potentially be used as individual inoculant or co-inoculated with other plant growth promoting bacteria to increase the production in sustainable agricultural systems.

Influence of outer membrane c-type cytochromes on particle size and activity of extracellular nanoparticles produced by Shewanella oneidensis

The metal‐reducing bacterium Shewanella oneidensis is capable of reducing various metal(loid)s and produces nanoparticles (NPs) extracellularly, in which outer membrane c‐type cytochromes (OMCs) have been suggested to play important roles. The objective of this study was to investigate the influence of the OMCs, that is, MtrC and OmcA, on the size and activity of the extracellular silver NPs (AgNPs) and silver sulfide NPs (Ag2S NPs) produced by S. oneidensis MR‐1. We found that (i) the lack of OMCs on S. oneidensis cell surface decreased the particle size of the extracellular biogenic AgNPs and Ag2S NPs; (ii) the biogenic AgNPs from the mutant lacking OMCs showed higher antibacterial activity; and (iii) the biogenic Ag2S NPs from the mutant lacking OMCs exhibited higher catalytic activity in methylviologen reduction. The results suggest that it may be possible to control particle size and activity of the extracellular biogenic NPs via controlled expression of the genes encoding surface proteins. In addition, we also reveal that in extracellular biosynthesis of NPs the usually neglected non‐cell‐associated NPs could have high catalytic activity, highlighting the need of novel methods that can efficiently retain extracellular NPs in the biosynthesis processes. Biotechnol. Bioeng. 2013; 110: 1831–1837.

Enterobacter arachidis sp. nov., a plant-growth-promoting diazotrophic bacterium isolated from rhizosphere soil of groundnut.

A methylotrophic nitrogen-fixing bacterial strain, Ah-143(T), isolated from the rhizosphere soil of field-grown groundnut was analysed by a polyphasic taxonomic approach. Comparative 16S rRNA gene sequence analysis combined with rpoB gene sequence analysis allocated strain Ah-143(T) to the family Enterobacteriaceae, with Enterobacter radicincitans and Enterobacter cowanii as the closest relatives. The strain is Gram-stain-negative, non-spore-forming, aerobic and motile, having straight rod-shaped cells with a DNA G+C content of approximately 53.2 mol%. The strain utilizes methanol as a carbon source and the mxaF gene was closely related to the mxaF gene of members of the genus Methylobacterium. The fatty acid profile consisted of C(16 : 0), C(17 : 0) cyclo, C(18 : 1)omega7c, summed feature 2 (iso-C(16 : 1) I and/or C(14 : 0) 3-OH) and summed feature 3 (iso-C(15 : 0) 2-OH and/or C(16 : 1)omega7c) as the major components. DNA-DNA relatedness of strain Ah-143(T) with its close relatives was less than 20 %. On the basis of the phylogenetic analyses, DNA-DNA hybridization data, and unique physiological and biochemical characteristics, it is proposed that the strain represents a novel species of the genus Enterobacter and should be named Enterobacter arachidis sp. nov. The type strain is Ah-143(T) (=NCIMB 14469(T) =KCTC 22375(T)).

Flavobacterium glycines sp. nov., a facultative methylotroph isolated from the rhizosphere of soybean.

An aerobic, yellow-pigmented, facultatively methylotrophic, Gram-staining-negative, non-spore-forming bacterium, designated strain Gm-149(T), was isolated from the rhizosphere of cultivated soybean in India. Cells were motile by gliding. The predominant cellular fatty acids were iso-C(15 : 0), summed feature 3 (comprising iso-C(15 : 0) 2-OH and/or C(16 : 1) ω 7c), C(16 : 0) 3-OH and anteiso-C(15 : 0), and the major isoprenoid quinone was MK-6. The G+C content of the genomic DNA of strain Gm-149(T) was 35.6 mol%. Comparative 16S rRNA gene sequence analysis showed that strain Gm-149(T) formed a distinct phyletic line within the genus Flavobacterium. Based on levels of pairwise 16S rRNA gene sequence similarity, strain Gm-149(T) was related most closely to the type strain of Flavobacterium daejeonense (97.1 %), but the level of DNA-DNA relatedness between these two strains was about 11.2 %. On the basis of phenotypic and genotypic data, strain Gm-149(T) is considered to represent a novel species of the genus Flavobacterium, for which the name Flavobacterium glycines sp. nov. is proposed. The type strain is Gm-149(T) (=ICMP 17618(T)=NBRC 105008(T)).

Genome Information of Methylobacterium oryzae, a Plant-Probiotic Methylotroph in the Phyllosphere

Pink-pigmented facultative methylotrophs in the Rhizobiales are widespread in the environment, and many Methylobacterium species associated with plants produce plant growth-promoting substances. To gain insights into the life style at the phyllosphere and the genetic bases of plant growth promotion, we determined and analyzed the complete genome sequence of Methylobacterium oryzae CBMB20T, a strain isolated from rice stem. The genome consists of a 6.29-Mb chromosome and four plasmids, designated as pMOC1 to pMOC4. Among the 6,274 coding sequences in the chromosome, the bacterium has, besides most of the genes for the central metabolism, all of the essential genes for the assimilation and dissimilation of methanol that are either located in methylotrophy islands or dispersed. M. oryzae is equipped with several kinds of genes for adaptation to plant surfaces such as defense against UV radiation, oxidative stress, desiccation, or nutrient deficiency, as well as high proportion of genes related to motility and signaling. Moreover, it has an array of genes involved in metabolic pathways that may contribute to promotion of plant growth; they include auxin biosynthesis, cytokine biosynthesis, vitamin B12 biosynthesis, urea metabolism, biosorption of heavy metals or decrease of metal toxicity, pyrroloquinoline quinone biosynthesis, 1-aminocyclopropane-1-carboxylate deamination, phosphate solubilization, and thiosulfate oxidation. Through the genome analysis of M. oryzae, we provide information on the full gene complement of M. oryzae that resides in the aerial parts of plants and enhances plant growth. The plant-associated lifestyle of M. oryzae pertaining to methylotrophy and plant growth promotion, and its potential as a candidate for a bioinoculant targeted to the phyllosphere and focused on phytostimulation are illuminated.

Pleomorphomonas diazotrophica sp. nov., an endophytic N-fixing bacterium isolated from root tissue of Jatropha curcas L.

A novel aerobic, non-motile, pleomorphic, Gram-negative and nitrogen-fixing bacterial strain, designated R5-392(T), was isolated from surface-sterilized root tissue of Jatropha curcas. The organism grew optimally at 30 °C in media containing 1 % (w/v) NaCl and at pH 6.0-8.0. The predominant ubiquinone was Q-10 and the major fatty acids were C18 : 1ω7c/C18 : 1ω6c, C16 : 0 and C19 : 0 cyclo ω8c. The DNA G+C content was 63.2 mol%. Analysis of the 16S rRNA gene sequence suggested that strain R5-392(T) is affiliated with the order Rhizobiales within the class Alphaproteobacteria and is most closely related to Pleomorphomonas oryzae F-7(T) (98.8 % similarity) and Pleomorphomonas koreensis Y9(T) (98.3 % similarity). Analysis of partial nifH gene sequences also revealed a monophyletic lineage within the class Alphaproteobacteria, and strain R5-392(T) was most closely related to P. oryzae F-7(T) (98 %). Highest nitrogenase activity was detected in the presence of low-level organic nitrogen or in the presence of nitrogenase co-factors (Fe/Mo) in N-free media. Phenotypic and chemotaxonomic data suggest that strain R5-392(T) represents a novel species within the genus Pleomorphomonas, for which the name Pleomorphomonas diazotrophica sp. nov. is proposed. The type strain is R5-392(T) ( = KACC 16233(T) = DSM 25022(T)).

Arachidicoccus rhizosphaerae gen. nov., sp. nov., a plant-growth-promoting bacterium in the family Chitinophagaceae isolated from rhizosphere soil

Three novel bacterial strains, designated Vu-144(T), Vu-7 and Vu-35, were isolated on minimal medium from rhizosphere soil of field-grown cowpea and subjected to a taxonomic study using a polyphasic approach. Cells of the strains were Gram-stain-negative, non-motile, non-spore-forming, coccoid rods, and formed non-pigmented colonies. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain Vu-144(T) was affiliated with an uncultivated lineage of the phylum Bacteroidetes. Its closest phylogenetic neighbour was the recently described species Niastella populi, a member of the family Chitinophagaceae, with just 90.7 % sequence similarity to the type strain. The only isoprenoid quinone detected was menaquinone 7 (MK-7). The fatty acid profiles showed large amounts of iso-C15 : 0, iso-C17 : 0 3-OH and iso-C15 : 1 G and minor amounts of summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH), C16 : 0 and other fatty acids, allowing the differentiation of the strains from other genera. The G+C content of the genomic DNA of the three strains ranged from 43.1 to 44.3 mol%. In addition to phosphatidylethanolamine, the major polar lipids were three unidentified aminophospholipids (APL1-APL3), two unidentified phospholipids (PL1, PL2) and three unidentified lipids (UL1-UL3). Biochemical test patterns also differed from those of Niastella populi and members of other genera. All three isolates showed plant-growth-promoting properties, e.g. the ability to produce indole-3-acetic acid and NH3 and to solubilize phosphate, utilized 1-aminocyclopropane 1-carboxylate (ACC) as a sole source of nitrogen and possessed the ACC deaminase enzyme. The novel isolates readily colonized roots and stimulated growth of tomato and cowpea under glasshouse conditions. Inoculated plants showed a 45-60 % increase in dry matter weight with respect to uninoculated controls. On the basis of the evidence from our polyphasic study, isolate Vu-144(T) represents a novel genus and species in the family Chitinophagaceae, for which the name Arachidicoccus rhizosphaerae gen. nov., sp. nov. is proposed. The type strain of Arachidicoccus rhizosphaerae is Vu-144(T) ( = KCTC 22378(T) = NCIMB 14473(T)).

Jatrophihabitans endophyticus gen. nov., sp. nov., an endophytic actinobacterium isolated from a surface-sterilized stem of Jatropha curcas L.

A short rod-shaped Gram-stain-positive actinobacterium was isolated as an endophyte from the tissues of Jatropha curcas cv. KB27 and was investigated by means of a polyphasic taxonomic approach. An analysis of its 16S rRNA gene sequence indicated that strain S9-650(T) forms an individual line of descent and is related to certain members of the suborder Frankineae, order Actinomycetales (<95 % sequence similarity). Distance-matrix and neighbour-joining analyses set the branching point of the novel isolate between two clades, one being represented by members of the genera Frankia (family Frankiaceae) and Acidothermus (family Acidothermaceae) and the other by members of the genera Geodermatophilus, Blastococcus and Modestobacter (family Geodermatophilaceae). The organism had meso-diaminopimelic acid as the diagnostic diamino acid in the cell-wall peptidoglycan. The acyl type was found to be N-glycolylated. The major menaquinone was MK-9(H4) and the fatty acid profile was characterized by the predominance of iso-C16 : 0, C18 : 1ω9c, anteiso-C17 : 0 and C17 : 1ω8c. The polar lipids comprised diphosphatidylglycerol, an unidentified glycolipid, phospholipids and aminolipids. The G+C content of the genomic DNA was 71.2 mol%. The distinct phylogenetic position and the phenotypic markers that clearly separate the novel organism from all other members of the suborder Frankineae indicate that strain S9-650(T) represents a novel species in a new genus, for which the name Jatrophihabitans endophyticus gen. nov., sp. nov. is proposed. The type strain of the type species is S9-650(T) ( = DSM 45627(T) = KACC 16232(T)).