So far, the occurrence of nitrogen-fixing Sphingomonas bacteria has been restricted to three strains of Sphingomonas azotifigens. In this work, a group of 46 Sphingomonas-like isolates, which originated from two rice varieties grown in two soils in Brazil, were characterized based on morphological, physiological and genetic analyses. The PCR genus specifically applied indicated that all 46 isolates belonged to the Sphingomonas genus and confirmed the results based on the yellow pigment of the colonies grown on potato agar medium and the BIOLOG data. It was also observed that 22 isolates are nitrogen-fixing bacteria as determined by the acetylene reduction method and confirmed by nifH gene detection. The genetic diversity based on the 16S rRNA analysis (amplified rDNA restriction analysis) showed that the isolates formed two distinct groups at a similarity value of 60%. Furthermore, five clusters at 60% similarity were observed with the 16S-23S intergenic space (ribosomal intergenic space analysis) analysis. Sequencing of the 16S rRNA gene and nifH fragments showed that most of the 22 nitrogen-fixing isolates formed clusters apart from that of the S. azotifigens. This is the first report on the occurrence of nitrogen-fixing Sphingomonas bacteria associated with rice grown in Brazil.

Bacteria of the genus Azospirillum are considered to be plant-growth promoting bacteria (PGPR) and stimulate plant growth directly either by synthesising phyto-hormones or by promoting nutrition by the process of biological nitrogen fixation (BNF). Although this genus extensively studied, the effects of inoculation and the possible BNF contribution of the Azospirillum amazonense specie are very scarce. The aim of this study was to isolate, characterise and evaluate auxin production and nitrogenase activity of this species and to select, by inoculation of rice plants, promising isolates based on their ability to improve plant growth, yield and the BNF contribution. One hundred and ten isolates obtained from rice were characterised and grouped according to colony features.Forty-two isolates, confirmed as A. amazonense by the fluorescent in situ hybridization (FISH) technique, were tested for auxin production and nitrogenase activity in vitro. Subsequently plant growth promotion related to plant nutrition effect was evaluated, in vitro and in greenhouse experiments. The BNF contribution to plant growth was evaluated using the 15 N isotope dilution technique. All A. amazonense strains tested produced indoles, but only 10% of them showed high production, above 1.33 μM mg protein −1 . The nitrogenase activity also was variable and only 9% of isolates showed high nitrogenase activity and the majority (54%) exhibited a low potential. The inoculation of selected strains in rice under gnotobiotic conditions reduced the growth of root and aerial part when compared to the control, showing the negative effects of excess of phytohormone accumulation in the medium. However, in the greenhouse experiment, inoculation of strains of A. amazonense increased grain dry matter accumulation (7 to 11.6%), the number of panicles (3 to 18.6%) and nitrogen accumulation at grain maturation (3.5 to 18.5%). BNF contributions up to 27% were observed for A. amazonense Y2 (wild type strain). The data presented here is the first report that the PGPR effect of A. amazonense for rice plants grown under greenhouse conditions is mainly due the BNF contribution as measured by 15 N isotope dilution technique, in contrast to the hormonal effect observed with other Azospirillum species studied. Plant Soil (

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O objetivo deste trabalho foi avaliar a diversidade de bactérias diazotróficas endofíticas, dos gêneros Herbaspirillum e Burkholderia, em duas variedades de arroz, consideradas de alta (IR 42) e baixa (IAC 4440) eficiência de fixação biológica de nitrogênio. Foram realizados dois experimentos em casa de vegetação, em vasos com dois tipos de solos, provenientes dos Estados de Goiás e do Rio de Janeiro. Foi feita a contagem do número de bactérias e o isolamento em diferentes partes e estágios de desenvolvimento das plantas, mediante o uso de meios de cultivo JNFb e JMV. Os isolados bacterianos foram caracterizados a partir de aspectos morfológicos das colônias, com o crescimento em meios de cultivo, e de testes fisiológicos (uso de fontes de carbono e atividade de redução de acetileno). A contagem revelou grande número de bactérias diazotróficas (10(6) células g-1 matéria fresca), presentes em ambas as variedades de arroz, principalmente nas amostras radiculares. Os dados, obtidos na matriz de similaridade, mostram a presença de representantes da espécie Herbaspirillum seropedicae, bem como a diversidade entre isolados pertencentes ao gênero Burkholderia.