José Ivo Baldani

Characterization of Herbaspirillum seropedicae gen. nov., sp. nov., a Root-Associated Nitrogen-Fixing Bacterium

During a survey of the occurrence of Azospirillum spp. in cereal roots, we obtained 119 isolates which could not be identified as members of one of the three previously described Azospirillum species. These strains formed a very homogeneous group of N2-fixing, microaerobic, motile, vibrioid, gram-negative rod-shaped organisms which formed a veillike pellicle in semisolid medium similar to that of Azospirillum spp. However, the new isolates differed from Azospirillum spp. by their smaller cell width (0.6 to 0.7 μm), variable flagellation (one to three flagella on one or both poles), moist brownish colonies, and broader pH and oxygen tolerance for nitrogenase activity. Organic acids were the preferred carbon sources, but glucose, galactose, L-arabinose, mannitol, sorbitol, and glycerol were also used. The guanine-plus-cytosine content of the deoxyribonucleic acid was slightly lower than the guanine-plus-cytosine contents of Azospirillum spp. (66 to 67 mol%). Deoxyribonucleic acid hybridization experiments with 17 strains of the group showed 50 to 100% complementarity, while the levels of hybridization with the type strains of Azospirillum brasilense, Azospirillum lipoferum, and Azospirillum amazonense were 23, 15, and 6%, respectively. For these new isolates we propose a new genus, Herbaspirillum (the name refers to the habitat of the organisms, the roots of cereals, which are herbaceous seed-bearing plants). The type species is named Herbaspirillum seropedicae after the place where it was first isolated. The type strain is strain Z67, which has been deposited in the American Type Culture Collection as strain ATCC 35892.

Occurrence of the endophytic diazotrophs Herbaspirillum spp. in roots, stems, and leaves, predominantly of Gramineae

Since the first description of Herbaspirillum seropedicae in 1986, few data have been published on this diazotroph, possibly due to difficulties in isolating it from soil. In the present study we found that this bacterium seems to be an obligate endophyte which has been isolated from roots, stems, and leaves of a large number of samples of more than 10 different species of the Gramineae family, but only exceptionally from other plants. H. rubrisubalbicans, previously misnamed as “Pseudomonas” rubrisubalbicans, and known as a mild pathogen of sugarcane causing mottled stripe disease, confirms the endophytie habitat of this genus. This species occurs in roots, stems, and leaves of sugarcane and seems to be restricted to this crop. Inoculation of strains from both species into soil in high numbers resulted in a rapid decline in their numbers. In only 30 days the population of Herbaspirillum spp. in soil decreased below detection limits (<100 cells g-1). When sorghum was planted in this soil, the bacteria reappeared and multiplied within the plant tissues.

Establishment of inoculated Azospirillum spp. in the rhizosphere and in roots of field grown wheat and sorghum

SummaryFour field experiments were carried out with wheat or sorghum in different regions of Brazil. The aim was to study the establishment of inoculatedAzospirillum strains, marked with resistance to various antibiotics, in the rhizosphere and in roots. The levels of the various antibiotics were chosen according to the resistance of the indigenousAzospirillum population.Azospirillum brasilense strains Sp 107 and Sp 245 could be established in all three wheat experiments and predominated within theAzospirillum population in washed, and especially in surface sterilized, roots. Strains Sp 7 and Cd established poorly in wheat roots.Azospirillum lipoferum Sp S82 represented 72% of the root isolates from sorghum inoculated with this strain. This strain and naturalAzospirillum infection became concentrated in the upper parts of the root system. Improved methods for root surface sterilization in which the absence ofAzospirillum on the root surface was established by pre-incubating roots with paraffin-capped ends in NFb medium confirmed the establishment of inoculatedAzospirillum strains within sorghum roots in the field.

Inoculation of rice plants with the endophytic diazotrophs Herbaspirillum seropedicae and Burkholderia spp.

Abstract Four experiments were performed under gnotobiotic conditions to select strains of the endophytic diazotrophs Herbaspirillumseropedicae and Burkholderia spp. as inocula of rice plants. Eighty strains of H. seropedicae originally isolated from rice, sorghum and maize plants, were tested in test tube cultures with N-free agar as the substrate. Rice plants showed medium and high increases in their fresh weight in response to inoculation with nineteen strains. These strains were tested again, and six strains were then selected to evaluate their contribution to the N of the plant via biological N2 fixation (BNF) using an agar growth medium containing 5 mg N l–1of 15N-labelled (NH4)2SO4. The contribution of the strains to plant N via BNF varied from 54% when rice plants were inoculated with strain ZAE94, to 31% when strain ZAE67 was used. These results were confirmed in the fourth gnotobiotic experiment, which also included strains of the new N-fixing bacteria belonging to the genus Burkholderia, isolated from rice, as well as a strain of Burkholderia vietnamiensis, isolated from rice rhizosphere. Burkholderia spp. strains showed similar effects to those observed for H. seropedicae strains, while B. vietnamiensis fixed only 19% of plant total N. The best four strains were tested in a pot experiment where pre-germinated, inoculated rice seedlings were grown in soil labelled with 15N. The results confirmed the gnotobiotic experiments, although the levels of N in the rice plants derived from BNF of the selected H. seropedicae and Burkholderia spp. strains were lower. Nevertheless, there was an increase in N content in grains of inoculated plants, and the results showed that the method used for strain selection is very useful and can be applied to other strains of N2-fixing bacteria and plants.

The effect of inoculating endophytic N2-fixing bacteria on micropropagated sugarcane plants

The aim of this work was to evaluate the effect of the inoculation of endophytic N2-fixing bacteria on the development of micropropagated sugarcane plants. The endophytic population of each inoculated species was monitored during the growth period, and biological nitrogen fixation (BNF) contribution of each inoculation treatment was assessed using the 15N-isotope dilution technique. Seven different combinations of inoculum were used, using five endophytic diazotrophic species (Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae, Herbaspirillum rubrisubalbicans, Azospirillum amazonense and Burkholderia sp.), originally isolated from sugarcane plants. The results showed a clear physiological effect on the development of the inoculated plants, resulting in alteration of the dry matter-partitioning pattern and increase on root dry matter as compared to uninoculated plants. Indeed, all inoculated diazotrophic species could be reisolated in high numbers from the rhizomes of the inoculated plants, even 400 days after inoculation (DAI), suggesting the establishment of the inoculated bacteria. However, a negative effect of the mixture of all five species on the survival of plantlets was observed 45 days after inoculation, just after acclimatization. The analysis of the BNF contribution using the 15N-isotope dilution technique showed that inoculation promoted some increase in the BNF contribution to the plant tissues. The best treatment was the mixture of all five strains, followed by the treatment with a mixture of Herbaspirillum spp. The contribution was much lower when the plants were inoculated with a mixture of G. diazotrophicus with A. amazonense and Burkholderia sp. A BNF contribution around 30% of total nitrogen accumulated was observed in micropropagated plants inoculated with the mixture of strains, suggesting that the combination of species in the inocula is the best strategy to improve sugarcane crops dependent on the biological nitrogen fixation process.

History on the biological nitrogen fixation research in graminaceous plants: special emphasis on the Brazilian experience.

This review covers the history on Biological Nitrogen Fixation (BNF) in Graminaceous plants grown in Brazil, and describes research progress made over the last 40 years, most of which was coordinated by Johanna Döbereiner. One notable accomplishment during this period was the discovery of several nitrogen-fixing bacteria such as the rhizospheric (Beijerinckia fluminensis and Azotobacter paspali), associative (Azospirillum lipoferum, A. brasilense, A. amazonense) and the endophytic (Herbaspirillum seropedicae, H. rubrisubalbicans, Gluconacetobacter diazotrophicus, Burkholderia brasilensis and B. tropica). The role of these diazotrophs in association with grasses, mainly with cereal plants, has been studied and a lot of progress has been achieved in the ecological, physiological, biochemical, and genetic aspects. The mechanisms of colonization and infection of the plant tissues are better understood, and the BNF contribution to the soil/plant system has been determined. Inoculation studies with diazotrophs showed that endophytic bacteria have a much higher BNF contribution potential than associative diazotrophs. In addition, it was found that the plant genotype influences the plant/bacteria association. Recent data suggest that more studies should be conducted on the endophytic association to strengthen the BNF potential. The ongoing genome sequencing programs: RIOGENE (Gluconacetobacter diazotrophicus) and GENOPAR (Herbaspirillum seropedicae) reflect the commitment to the BNF study in Brazil and should allow the country to continue in the forefront of research related to the BNF process in Graminaceous plants.

Complete genome sequence of the sugarcane nitrogen-fixing endophyte Gluconacetobacter diazotrophicus Pal5

BackgroundGluconacetobacter diazotrophicus Pal5 is an endophytic diazotrophic bacterium that lives in association with sugarcane plants. It has important biotechnological features such as nitrogen fixation, plant growth promotion, sugar metabolism pathways, secretion of organic acids, synthesis of auxin and the occurrence of bacteriocins.ResultsGluconacetobacter diazotrophicus Pal5 is the third diazotrophic endophytic bacterium to be completely sequenced. Its genome is composed of a 3.9 Mb chromosome and 2 plasmids of 16.6 and 38.8 kb, respectively. We annotated 3,938 coding sequences which reveal several characteristics related to the endophytic lifestyle such as nitrogen fixation, plant growth promotion, sugar metabolism, transport systems, synthesis of auxin and the occurrence of bacteriocins. Genomic analysis identified a core component of 894 genes shared with phylogenetically related bacteria. Gene clusters for gum-like polysaccharide biosynthesis, tad pilus, quorum sensing, for modulation of plant growth by indole acetic acid and mechanisms involved in tolerance to acidic conditions were identified and may be related to the sugarcane endophytic and plant-growth promoting traits of G. diazotrophicus. An accessory component of at least 851 genes distributed in genome islands was identified, and was most likely acquired by horizontal gene transfer. This portion of the genome has likely contributed to adaptation to the plant habitat.ConclusionThe genome data offer an important resource of information that can be used to manipulate plant/bacterium interactions with the aim of improving sugarcane crop production and other biotechnological applications.

Effect of inoculation ofAzospirillum spp. on nitrogen accumulation by field-grown wheat

SummaryTwo experiments were performed to examine the effects of inoculation of field grown wheat with various Azospirillum strains. In the first experiment the soil was sterilized with methyl bromide to reduce the Azospirillum population and15N labelled fertilizer was added to all treatments. Two strains ofAzospirillum brasilense isolated from surface sterilized wheat roots and theA. brasilense type strain Sp7 all produced similar increases in grain yield and N content. From the15N and acetylene reduction data it was apparent that these increases were not due to N2 fixation.In the second experiment performed in the same (unsterilized) soil, twoA. brasilense strains (Sp245, Sp246) and oneA. amazonense strain (Am YTr), all isolated from wheat roots, produced responses of dry matter and N content while the response to the strain Sp7 was much smaller. These data confirm earlier results which indicate that if natural Azospirillum populations in the soil are high (the normal situation under Brazilian conditions), strains which are isolated from wheat roots are better able to produce inoculation responses than strains isolated from other sources.The inoculation of a nitrate reductase negative mutant of the strain Sp245 produced only a very small inoculation response in wheat. This suggests that the much greater inoculation response of the original strain was not due to N2 fixation but to an increased nitrate assimilation due to the nitrate reductase activity of the bacteria in the roots.

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, Spartina pectinata, Miscanthus sinensis, Miscanthus sacchariflorus and Pennisetum purpureum, with nitrogen-free semi-solid media led to the isolation of Herbaspirillum-like strains among other diazotrophic bacteria. On the basis of physiological properties, phylogenetic analysis comparing 16S rDNA sequences and DNA-DNA hybridization experiments of chromosomal DNA the new isolates could be grouped together in a new species with the proposed name Herbaspirillum frisingense sp. nov. Morphological characteristics, such as cell size and shape, colony appearance, motility and flagellation are largely identical to the known species Herbaspirillum rubrisubalbicans and Herbaspirillum seropedicae. On the basis of utilization of adipate (-), N-acetyl-D-glucosamine (+), meso-erythritol (-), L-rhamnose (-) and meso-inositol (-) Herbaspirillum frisingense sp. nov. can be distinguished from other known Herbaspirillum 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 spp. is 98.5-99.1%. In genomic DNA-DNA hybridization experiments Herbaspirillum frisingense sp. nov. forms a homogeneous group with 70-100+/-10% similarity, clearly distinct from Herbaspirillum seropedicae and Herbaspirillum rubrisubalbicans with 1-34% similarity. 16S rRNA-targeted oligonucleotide probes, specific for the whole genus Herbaspirillum and for three Herbaspirillum species were designed and are suitable for fluorescence in situ hybridization. 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 fingerprinting exhibits a consistent pattern for groups of strains isolated from the same plant, suggesting a low genomic diversity among bacteria inhabiting C4-gramineous plant tissues. Low genetic DNA diversity seems to be common between probable endophytic bacterial isolates of the same taxon. The type strain of Herbaspirillum frisingense sp. nov. is GSF30T (= DSM 13128T).

Occurrence, physiological and molecular analysis of endophytic diazotrophic bacteria in gramineous energy plants

Endophytic diazotrophic bacteria could be isolated from the energy plants Pennisetum purpureum, Miscanthus sinensis, Miscanthus sacchariflorus and Spartina pectinata using semisolid nitrogen free media. Higher levels of diazotrophic bacteria were found if no nitrogen fertilizer was applied. The bacteria were characterized on the basis of typical morphology, physiological tests, and the use of phylogenetic oligonucleotide probes. They belong partially to the species Azospirillum lipoferum and Herbaspirillum seropedicae while others supposedly represent a new species of Herbaspirillum. Using PCR-fingerprinting techniques a limited genetic diversity of these isolates was found which may indicate an adaptation to the specific conditions of the interior of these plants.