Herbaspirillum frisingense sp nov., a new nitrogen-fixing bacterial species that occurs in C4-fibre plants

The enrichment of nitrogen-fixing bacteria from the C4-fibre plants, Sparti na pectinata, Miscanthus sinensis, Miscanthus sacchariflorus and Pennisetum purpureum, with nitrogen-free semi-solid media led to the isolation of Her baspirillum-like strains among other diazotrophic bacteria. On the basis of physiological properties, phylogenetic analysis comparing 16S rDNA sequenc es and DNA-DNA hybridization experiments of chromosomal DNA the new isolate s could be grouped together in a new species with the proposed name Herbasp irillum frisingense sp. nov. Morphological characteristics, such as cell si ze and shape, colony appearance, motility and flagellation are largely iden tical to the known species Herbaspirillum rubrisubalbicans and Herbaspirill um seropedicae. On the basis of utilization of adipate (-), N-acetyl-D-gluc osamine (+), meso-erythritol (-), L-rhamnose (-) and meso-inositol (-) Herb aspirillum frisingense sp. nov, can be distinguished from other known Herba spirillum spp. Nitrogen-fixing capability was examined by PCR amplification of the nifD gene and an acetylene reduction assay, and was found with all isolates tested. 16S rDNA sequence similarity to the other Herbaspirillum s pp. is 98.5-99.1 %. In genomic DNA-DNA hybridization experiments Herbaspiri llum frisingense sp. nov. forms a homogeneous group with 70-100 +/- 10 % si milarity, clearly distinct from Herbaspirillum seropedicae and Herbaspirill um rubrisubalbicans with 1-34 % similarity. 16S rRNA-targeted oligonucleoti de probes, specific for the whole genus Herbaspirillum and for three Herbas pirillum species were designed and are suitable for fluorescence in situ hy bridization. The DNA G+C content of Herbaspirillum frisingense sp. nov. is 63 +/- 2 mol%, in agreement with the values of 61-65 % for the genus. PCR f ingerprinting exhibits a consistent pattern for groups of strains isolated from the same plant, suggesting a low genomic diversity among bacteria inha biting C4-gramineous plant tissues. Low genetic DNA diversity seems to be c ommon between probable endophytic bacterial isolates of the same taxon. The type strain of Herbaspirillum frisingense sp. nov. is GSF30(T) (= DSM 1312 8(T)).