Johanna Döbereiner

A new acid-tolerant nitrogen-fixing bacterium associated with sugarcane

During surveys of bacteria possibly responsible for N2 fixation in sugarcane, root and stem samples were collected in four sugarcane-growing regions in Brazil. A new microaerobic N2-fixing bacterium was isolated from most samples of washed roots and stems from all regions. Isolation procedures were based on semisolid diluted sugarcane juice medium followed by replication to N-free 10% sugar medium acidified with acetic acid to pH 4.5. The new bacterium is an aerobic rod, motile by 1 to 3 lateral flagella, fixes N2 in semisolid media under air but not in liquid media except when a starter dose of N is added. It has no nitrate, reductase and N2 fixation proceeds in the presence of 10mM NO3−. Best growth occurs with high sucrose concentrations (10%). Growth occurs up to 30% sucrose but not at 35%. Acid is formed reaching a final pH of below 3.0. Growth and N2 fixation proceed at this acidity. Ethanol is used for growth and is “overoxidised” (oxidized to CO2 and H2O). Acetic and lactic acids are also oxidized to CO2 and H2O. Acids produced from glucose are consumed with precipitation of CaCO3. Dark brown colonies are formed on potato agar with 10% sugar and dark orange colonies on N poor agar (20 mg yeast extract per 1) containing bromothymol blue. In view of the distinct characteristics which do not allow identification within either Frateuria, Gluconobacter, Acetobacter or any known N2-fixing bacterium a new genus and species are proposed and namedSaccharobacter nitrocaptans.

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.

Recent advances in BNF with non-legume plants

Abstract It is now well accepted that nitrogen-fixing bacteria colonising graminaceous plants can be grouped into three categories: 1, rhizosphere organisms; 2, facultative endophytes and 3, obligate endophytes. In the first category are included all species that colonise the root surface such as Azotobacter paspali, Beijerinkia spp. Facultative endophytes are these nitrogen-fixing bacteria that can colonise the surface and interior of the roots principally the four species of Azospirillum , except Azospirillum halopraeferans . The third category is constituted mainly by diazotrophs isolated more recently such as Acetobacter diazotrophicus, Herbaspirillum spp and Azoarcus spp which are able to colonise the root interior and aerial tissues of the plants. Although most of the studies related to nitrogen fixing bacteria have been concentrated on Azospirillum spp, it is the obligate endophytes, isolated more recently, that have attracted the attention of scientists working in this field. The ability to colonise the entire plant interior and locate themselves within niches protected from oxygen or other factors make them the most promising group of diazotrophs associated with graminaceous and other non-leguminous plants. In this review we compare these three groups of nitrogen-fixing bacteria, their interaction with the host plants and discuss the potential of their use in agriculture.

Host-plant specificity in the infection of cereals with Azospirillum spp

Abstract The specificity of the infection of maize, wheat and rice roots by N2-fixing Azospirillum spp was studied in four greenhouse experiments using pots with unsterilized soil and in two field experiments. In all experiments A. lipoferum was most frequently isolated from externally sterilized roots of maize, and A. brasilense nir− (nitrite reductase negative) from wheat and rice. In pot experiments, A. brasilense nir+ was isolated with moderate frequency from within maize roots but rarely from within wheat or rice roots. Inoculation of the pots with a mixture of representative strains of the three Azospirillum groups had no effect on the proportion of strains recovered from each plant species. In the field experiments, inoculation with spontaneous streptomycin-resistant mutants of two of the representative strains confirmed the apparent specificity of A. lipoferum for maize roots and of A. brasilense for wheat but the results were partially obscured by the unexpectedly high proportion of streptomycin-resistant strains isolated from within the roots of uninoculated plants.

Acetobacter diazotrophicus sp. nov., a Nitrogen-Fixing Acetic Acid Bacterium Associated with Sugarcane

Results of deoxyribonucleic acid (DNA)-ribosomal ribonucleic acid and DNA-DNA hybridizations, together with a phenotypic and chemotaxonomic analysis, revealed that nitrogen-fixing bacteria isolated from roots and stems of sugarcane belong to a new species in the genus Acetobacter, for which the name Acetobacter diazotrophicus sp. nov. is proposed. Strain LMG 7603 (= Dobereiner PAI 5 = ATCC 49037) is the type strain.

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.

Emended description of Herbaspirillum; inclusion of [Pseudomonas] rubrisubalbicans, a milk plant pathogen, as Herbaspirillum rubrisubalbicans comb. nov.; and classification of a group of clinical isolates (EF group 1) as Herbaspirillum species 3.

[Pseudomonas] rubrisubalbicans, a mild plant pathogen. Herbaspirillum seropedicae, and EF group 1 strains (clustered by an immunological method) were investigated by a polyphasic approach with DNA-rRNA and DNA-DNA hybridizations and auxanography on 147 substrates. Our results show that they all belong to the genus Herbaspirillum. In addition to H. seropedicae, two other species are described: Herbaspirillum rubrisubalbicans and a new unnamed species, Herbaspirillum species 3, containing mainly strains of clinical origin. The three species can be differentiated on the basis of their auxanographic features and DNA-DNA similarities. The type strain of H. rubrisubalbicans is NCPPB 1027 (=LMG 2286); representative strains of the third Herbaspirillum species are strains CCUG 189 (=LMG 5523), CCUG 10263 (=LMG 5934), and CCUG 11060 (=LMG 5321). It has been confirmed that H. rubrisubalbicans is an endophytic diazotroph. It colonizes the roots, the stems, and predominantly the leaves of sugarcane (Saccharum spp.), while Herbaspirillum seropedicae colonizes in large numbers many different species of the Gramineae. Both diazotrophic Herbaspirillum species could be differentiated with meso-erythritol and N-acetylglucosamine. Oligonucleotide probes based on partial sequences of the 23S rRNA of H. seropedicae and H. rubrisubalbicans (HS and HR probes, respectively), were constructed and used as diagnostic probes.

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.

Biological nitrogen fixation associated with sugar cane

A recent15N dilution/N balance study confirmed that certain sugar cane varieties are capable of obtaining large contributions of nitrogen from plant-associated N2 fixation. It was estimated that up to 60 to 80% of plant N could be derived from this source, and under good conditions of water and mineral nutrient supply, it may be possible to dispense with N fertilization of these varieties altogether. The recently discovered bacterium,Acetobacter diazotrophicus, apparently responsible for this N2 fixation associated with the plants, has unique physiological properties for a diazotroph, such as tolerance to low pH, and high sugar and salt concentrations, lack of nitrate reductase, and nitrogenase activity which tolerates short-term exposure to ammonium. Furthermore, it also behaves as an endophyte, in that it is unable to infect sugar cane plants unless through damaged tissue or by means of VA mycorrhizae and is propagated via the planting material (stem pieces).