Sphingomonas palmae sp. nov. and Sphingomonas gellani sp. nov., endophytically associated phyllosphere bacteria isolated from economically important crop plants.

In this study, two endophytic bacterial strains designated JS21-1T and S6-262T isolated from leaves of Elaeis guineensis and stem tissues of Jatropha curcas respectively, were subjected for polyphasic taxonomic approach. On R2A medium, colonies of strains JS21-1T and S6-262T are orange and yellow, respectively. Phylogenetic analyses using 16S rRNA gene sequencing and whole-genome sequences placed the strains in distinct clades but within the genus Sphingomonas. The DNA G + C content of JS21-1T and S6-262T were 67.31 and 66.95%, respectively. Furthermore, the average nucleotide identity and digital DNA-DNA hybridization values of strains JS21-1T and S6-262T with phylogenetically related Sphingomonas species were lower than 95% and 70% respectively. The chemotaxonomic studies indicated that the major cellular fatty acids of the strain JS21-1T were summed feature 8 (C18:1 ω7c and/or C18:1 ω6c), C16:0, and C14:0 2OH; strain S6-262T possessed summed feature 3 (C16:1 ω7c and/or iso-C15:0 2-OH) and summed feature 8 (C18:1 ω6c and/or C18:1 ω7c). The major quinone was Q10, and the unique polyamine observed was homospermidine. The polar lipid profile comprised of mixture of sphingoglycolipid, phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and certain uncharacterised phospholipids and lipids. Based on this polyphasic evidence, strains JS21-1T and S6-262T represent two novel species of the genus Sphingomonas, for which the names Sphingomonas palmae sp. nov. and Sphingomonas gellani sp. nov. are proposed, respectively. The type strain of Sphingomonas palmae sp. nov. is JS21-1T (= DSM 27348T = KACC 17591T) and the type strain of Sphingomonas gellani sp. nov. is S6-262T (= DSM 27346T =  KACC 17594T).

Chitinasiproducens palmae gen. nov., sp. nov., a new member of the family Burkholderiaceae isolated from leaf tissues of oil palm (Elaeis guineensis Jacq.).

A Gram-stain-negative, aerobic, rod-shaped, leaf-associated bacterium, designated JS23T, was isolated from surface-sterilized leaf tissue of an oil palm grown in Singapore and was investigated by polyphasic taxonomy. Phylogenetic analyses based on 16S rRNA gene sequences and 180 conserved genes in the genome of several members of Burkholderiaceae revealed that strain JS23T formed a distinct evolutionary lineage independent of other taxa within the family Burkholderiaceae. The predominant ubiquinone was Q-8. The primary polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, and an unidentified aminophospholipid. The major fatty acids were C16 : 0, summed feature 3 (C16 : 1 ω7c /C16 : 1 ω6c) and summed feature 8 (C18 : 1 ω7c /C18 : 1 ω6c). The size of the genome is 5.36 Mbp with a DNA G+C content of 66.2 mol%. Genomic relatedness measurements such as average nucleotide identity, genome-to-genome distance and digital DNA-DNA hybridization clearly distinguished strain JS23T from the closely related genera Burkholderia, Caballeronia, Mycetohabitans, Mycoavidus, Pandoraea, Paraburkholderia, Robbsia and Trinickia. Furthermore, average amino acid identity values and the percentages of conserved proteins, 56.0-68.4 and 28.2-45.5, respectively, were well below threshold values for genus delineation and supported the assignment of JS23T to a novel genus. On the basis of the phylogenetic, biochemical, chemotaxonomic and phylogenomic evidence, strain JS23T is proposed to represent a novel species of a new genus within the family Burkholderiaceae, for which the name Chitinasiproducens palmae gen. nov., sp. nov., is proposed with the type strain of JS23T (= DSM 27307T=KACC 17592T).

Sphingomonas jatrophae sp. nov. and Sphingomonas carotinifaciens sp. nov., two yellow-pigmented endophytes isolated from stem tissues of Jatropha curcas L.

Two yellow-pigmented isolates, S5-249T and L9-754T, originating from surface-sterilized plant tissues of Jatropha curcas L. (Jatropha) cultivars were characterized using a polyphasic taxonomic approach. Strains S5-249T and L9-754T had 16S rRNA genes sharing 94.2 % sequence similarity with each other and 91.6-97.2 % sequence similarity with those of other species in the genus Sphingomonas, suggesting that they represent two potentially novel species. The 16S rRNA gene sequences of strains S5-249T and L9-754T shared the highest similarity to that of Sphingomonas sanguinis NBRC 13937T (96.1 and 97.2 %, respectively). The genomic DNA G+C contents of strains S5-249T and L9-754T were 66.9 and 68.5 mol%, respectively. The respiratory quinone was determined to be Q-10, and the major polyamine was homospermidine. Strains S5-249T and L9-754T contained summed feature 7 (comprising C18 : 1ω7c, C18 : 1ω9t and/or C18 : 1ω12t), C16 : 1, C14 : 0 2-OH and summed feature 4 (C16 : 1ω7t, iso-C15 : 0 2-OH and C16 : 1ω7c) as the major cellular fatty acids. The predominant polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and sphingoglycolipid. The average nucleotide identity (ANI) values between S. sanguinis NBRC 13937T and the two type strains (S5-249T and L9-754T) were 72.31 and 77.73 %, respectively. Digital DNA-DNA hybridization (dDDH) studies between the novel strains (S5-249T and L9-754T) and other species of the genus Sphingomonas were well below the thresholds used to discriminate between bacterial species. The results of dDDH and physiological tests allowed genotypic and phenotypic differentiation of the strains from each other as well as from the species of the genus Sphingomonas with validly published names. These data strongly support the classification of the strains as representatives of novel species, for which we propose the names Sphingomonas jatrophae sp. nov. (type strain S5-249T=DSM 27345T=KACC 17593T) and Sphingomonas carotinifaciens sp. nov. (type strain L9-754T=DSM 27347T=KACC 17595T).

Leaf-residing Methylobacterium species fix nitrogen and promote biomass and seed production in Jatropha curcas.

BACKGROUND: Jatropha curcas L. (Jatropha) is a potential biodiesel crop that can be cultivated on marginal land because of its strong tolerance to drought and low soil nutrient content. However, seed yield remains low. To enhance the commercial viability and green index of Jatropha biofuel, a systemic and coordinated approach must be adopted to improve seed oil and biomass productivity. Here, we present our investigations on the Jatropha-associated nitrogen-fixing bacteria with an aim to understand and exploit the unique biology of this plant from the perspective of plant-microbe interactions. RESULTS: An analysis of 1017 endophytic bacterial isolates derived from different parts of Jatropha revealed that diazotrophs were abundant and diversely distributed into five classes belonging to α, ß, γ-Proteobacteria, Actinobacteria and Firmicutes. Methylobacterium species accounted for 69.1 % of endophytic bacterial isolates in leaves and surprisingly, 30.2 % which were able to fix nitrogen that inhabit in leaves. Among the Methylobacterium isolates, strain L2-4 was characterized in detail. Phylogenetically, strain L2-4 is closely related to M. radiotolerans and showed strong molybdenum-iron dependent acetylene reduction (AR) activity in vitro and in planta. Foliar spray of L2-4 led to successful colonization on both leaf surface and in internal tissues of systemic leaves and significantly improved plant height, leaf number, chlorophyll content and stem volume. Importantly, seed production was improved by 222.2 and 96.3 % in plants potted in sterilized and non-sterilized soil, respectively. Seed yield increase was associated with an increase in female-male flower ratio. CONCLUSION: The ability of Methylobacterium to fix nitrogen and colonize leaf tissues serves as an important trait for Jatropha. This bacteria-plant interaction may significantly contribute to Jatropha's tolerance to low soil nutrient content. Strain L2-4 opens a new possibility to improve plant's nitrogen supply from the leaves and may be exploited to significantly improve the productivity and Green Index of Jatropha biofuel.