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Diazotrophic bacteria isolated from banana plants were characterized by morphological and physiological aspects. Three different groups of these plant-bacteria could be established. Two of them showed similarity to species of the Herbaspirillum genus. The third one was different because used only a few carbon substrates and produced water diffusible compounds that fluoresced under UV light. All three bacterial groups were thin rods with mono or bipolar flagella, presented negative reaction in Gram stain, showed catalase activity, were able to reduce nitrate and grew better in semi-solid JNFb medium at 31ºC. The nitrogenase activity was detected in semi-solid N-free JNFb medium and expressed higher values when pH ranged from 6.5 to 7.0 (groups I and II) and 6.0 to 6.5 (group III). The diazotrophs isolated from banana plants were distinct from species of Herbaspirillum previously identified in gramineous plants. Bactérias diazotróficas do tipo Herbaspirillum isoladas de bananeiras foram avaliadas pelas características morfológicas e fisiológicas de crescimento. Três grupos de bactérias foram estabelecidos, sendo dois relacionados às espécies de Herbaspirillum e diferentemente o terceiro grupo apresentou habilidade em crescer com poucos substratos orgânicos e produziu substância fluorescente em meio B de King. As bactérias dos três grupos eram bastonetes com flagelos mono ou bipolares, apresentaram reação negativa na coloração de Gram, expressaram atividade de catalase e oxidade, foram capazes de reduzir o nitrato e cresceram melhor em meio JNFb semi-sólido incubado a 31ºC. A atividade da nitrogenase, medida através da atividade de redução de acetileno, foi máxima em meio JNFb semi-sólido, após o ajuste de pH na faixa de 6,0 a 7,0 (grupos I e II) e 5,5 a 6,5 (grupo III). As bactérias diazotróficas associadas às bananeiras são diferentes das espécies de Herbaspirillum anteriormente identificadas em gramíneas.

Diazotrophic bacteria isolated from banana plants were characterized by morphological and physiological aspects. Three different groups of these plant-bacteria could be established. Two of them showed similarity to species of the Herbaspirillum genus. The third one was different because used only a few carbon substrates and produced water diffusible compounds that fluoresced under UV light. All three bacterial groups were thin rods with mono or bipolar flagella, presented negative reaction in Gram stain, showed catalase activity, were able to reduce nitrate and grew better in semi-solid JNFb medium at 31ºC. The nitrogenase activity was detected in semi-solid N-free JNFb medium and expressed higher values when pH ranged from 6.5 to 7.0 (groups I and II) and 6.0 to 6.5 (group III). The diazotrophs isolated from banana plants were distinct from species of Herbaspirillum previously identified in gramineous plants.Bactérias diazotróficas do tipo Herbaspirillum isoladas de bananeiras foram avaliadas pelas características morfológicas e fisiológicas de crescimento. Três grupos de bactérias foram estabelecidos, sendo dois relacionados às espécies de Herbaspirillum e diferentemente o terceiro grupo apresentou habilidade em crescer com poucos substratos orgânicos e produziu substância fluorescente em meio B de King. As bactérias dos três grupos eram bastonetes com flagelos mono ou bipolares, apresentaram reação negativa na coloração de Gram, expressaram atividade de catalase e oxidade, foram capazes de reduzir o nitrato e cresceram melhor em meio JNFb semi-sólido incubado a 31ºC. A atividade da nitrogenase, medida através da atividade de redução de acetileno, foi máxima em meio JNFb semi-sólido, após o ajuste de pH na faixa de 6,0 a 7,0 (grupos I e II) e 5,5 a 6,5 (grupo III). As bactérias diazotróficas associadas às bananeiras são diferentes das espécies de Herbaspirillum anteriormente identificadas em gramíneas.

Acetobacter diazotrophicus is a nitrogen-fixing bacterium that growth inside sugar cane plant tissue where the sucrose concentration is approximately 10%. The influence of high sugar content on nitrogenase was measured in the presence of oxygen and of nitrogen added in the form of ammonium and amino acids. In all parameters analyzed, 10% sucrose protected nitrogenase against inhibition by oxygen, ammonium, some amino acids, and also to some extent by salt stress. The oxygen concentration at which inhibition occurred increased from 2 kPa in 1% glucose or gluconic acid, to 4 kPa (0.4 atm) in 10% sucrose. Nitrogenase activity was partially inhibited by increased ammonium levels (2.0, 5.0, and 10.0 mM) in the presence of 1% sucrose, but the cells maintained their nitrogenase activity at 10% sucrose. This could be explained by the slow ammonium assimilation by the cells in the presence of high sucrose concentrations, i.e., independent of its concentration between 2 and 10 mM, the assimilation of ammonium was reduced to one-third in cells grown with 10% sucrose. Some amino acids were also tested in the presence of 1 and 10% sucrose. Cells grown in 1% sucrose had their nitrogenase activity reduced by 50-98% in the presence of glutamic acid, glutamine, alanine, asparagine, or threonine, whereas with 10% sucrose, nitrogenase activity was increased by glutamic acid and was reduced by only 61-73% by the other amino acids. The effect of NaCl concentrations (0.0, 0.25, 0.5, 0.75, or 1.0%) was also studied at the two concentrations of sucrose. Nitrogenase activity and growth of A. diazotrophicus, which was visualized by the pellicle formation in semi-solid medium, showed sensitivity even to low NaCl concentrations, which was somewhat relieved at the higher sucrose level. These observations indicate different osmotolerance mechanisms for sucrose and salt.

Acetobacter diazotrophicus and Herbaspirillum seropedicae were identified by genetic methods based on 16S rRNA sequences. A specific PCR method in combination with probing was developed for A. diazotrophicus. The PCR system includes four primers, of which the primers named AC (CTGTTTCCCGCAAGGGAC) and DI (GCGCCCCATTGCTGGGTT) generated an 445 bp amplicon in all of the 11 A. diazotrophicus strains tested. The phylogenetic position of H. seropedicae was determined. H. seropedicae forms with Oxalobacter formigenes a separate lineage in the beta-subclass of Proteobacteria.

Micropropagated plantlets of sugar cane were inoculated with the N2-fixing bacterium Acetobacter diazotrophicus. Various modifications on the basic plant culture medium MS were made for the plant/bacteria association. The protocol required the inoculation of the bacteria at the end of the rooting period in a medium without hormones or vitamins, and with the concentration of sugar and mineral nutrients reduced by a factor of 10. Individual plants were inoculated with A. diazotrophicus and maintained under the appropriate light and temperature condition used for micropropagation up to 7 days. The system favored the infection and the establishment of the bacteria within the plant tissue. Bacteria colonized the plant tissue and accumulated in inter-cellular cavities and the region of lateral root emergence and also colonizes the xylem vessels. The inoculated plantlets were subsequently transferred to the acclimatization phase and after 30 days it was possible to isolate the bacteria from plant tissue. This protocol permitted studies of infection and comparison among strains.

The soybean Bradyrhizobium strain Semia 566 was introduced into soils of the Cerrados (Brazilian edaphic savannas) in the late 1960s. Then, nodule occupancy by this strain was not greater than 2%. Recently, this serogroup has been found in approximately 60% of nodules formed on soybeans cultivated in the Cerrados, replacing the strains 29W and Semia 587, the Brazilian commercial inoculant for soybean. Although some re-isolates of Semia 566, adapted to Cerrado soils, were more competitive than 29W under both field and aseptic conditions, they did not differ from the parental strain, based on their lipopolysaccharide (LPS) electrophoretic profile. The only exceptions were the isolates 4A-5 and CPAC-15 which presented an additional polysaccharidic band of low molecular weight or higher mobility. On the other hand, this same band may be induced and intensified in LPS extracted from competitive strains (29W, 220, 204, 370, 372, 516, 122 and CPAC-15) after bacterial contact with soybean roots for 6 or 12 h. In addition, a 29W Tn5 mutant with a phenotype of delayed nodulation showed a delayed induction of this polysaccharidic band. Conversely, the LPS of less competitive strains was not modified or showed a weak intensification of this band. As this band alteration was correlated with the concurrent elevation of dominance in nodules, it may be suggested that LPS plays a role in the competitive ability of rhizobia strains for nodulation.

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.

Brazil has become the world leader in replacing N fertilisers by biological N2 fixation (BNF). Even though agriculture in Brazil is one of the main export-producing activities, it has become the country which uses the lowest N applications (mean 10 kg ha−1). Soybeans selected with zero N applications have become the country's largest export product. Cereals, also selected with N supplies much below their need, unknowingly were selected for associations with diazotrophic bacteria and can obtain up to 30% of their N from BNF when fertilised with ample PK and minor elements. The largest effect in this group was obtained with sugar cane, which can obtain up to 150 kg N ha−1 from BNF. This has become the key to the success of the Brazilian Bio-fuel programme where because of increases in the energy balance when the cane is planted without any N fertiliser and without burning the leaves, the energy produced is more than 5 times that required to produce it. Recently possibilities of expanding this biofuel programme to diesel oil are arising from the isolation of probably new diazotrophs which colonise the stems of oil palms, of the Brazilian palm genotypes which usually are not fertilised with N.

SUMMARY Leaves of a mottled stripe disease-susceptible cultivar (B-4362) and of a mottled stripe disease-resistant cultivar (SP 70–1143) of sugar cane (interspecific hybrids of Saccharum) were inoculated with the diazotrophic endophytes, Herbaspirillum rubrisubalbicans or Herbaspirillum seropedicae, via injection into the apex of the stem. At 7 and 20 d.a.i., H. seropedicae could be isolated only from a small necrotic area around the point of inoculation, where there was considerable degradation of host cells, and was not detected in any other part of the leaves. This suggested a hypersensitive response by the host to this bacterium, and no disease symptoms formed on either cultivar. By contrast, H. rubrisubalbicans could be re-isolated from throughout the infected leaves of both cultivars at both harvests and produced widespread disease symptoms on the leaves of cv. B-4362. Symptoms consisted of necrotic regions near the point of inoculation, and red stripes and red patches along the vertical axis of the leaves, where the bacteria had spread in the primary and secondary veins. The xylem–conducting elements in diseased regions of leaves were filled with bacteria and, at the edges of disease symptoms, the vessels were filled with a gum which stained blue-green with toluidine blue. This material probably contained phenolic compounds, and was produced as a host defence response. Leaves of cv. SP 70–1143 only developed small red stripes near the point of inoculation. These symptoms did not spread along the leaves, and the infected xylem vessels were never seen to be completely full of bacteria. Instead, the vessels contained encapsulated bacterial colonies attached to secondary wall deposition; these colonies were surrounded by blue-green material that might have been host-defence gums. In cv. B-4362, bacteria were abundant in the intercellular spaces of mesophyll adjacent to infected xylem, and also filled sub–stomatal cavities. Immunogold labelling using polyclonal antisera raised against H. rubrisubalbicans gave a weak signal with the bacteria in cv. SP 70–1143, showing that few binding sites were available to the antibodies. By contrast, bacteria in cv. B-4362 reacted strongly with the antibody, suggesting that they had a denser coating of immunoreactive mucus. In the later stages of infection of cv. B–4362, lysed bacteria were seen within degraded plant cells surrounded by a matrix of plant gums and bacterial mucus. This matrix reacted strongly to the H. rubrisubalbicans antibodies. Immunogold labelling using antibodies against nitrogenase component II showed that nitrogenase was expressed by bacteria in the early stages of infection of cv. B–362, but not in later stages, or by bacteria infecting cv. SP 70–1143.

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 (

Two field experiments were carried out in Northern Argentina, during the 1989–1990 and 1990–1991 growing seasons, on Argentinian and Brazilian maize genotypes. The inoculant consisted of a mixture of four Azospirillum brasilense strain isolated from surface-sterilized maize roots in Argentina and three A. lipoferum strains isolated from surface-sterilized maize or sorghum roots in Brazil. Establishment of the inoculated strains was confirmed by the antibiotic resistance of the strains in the highest dilution vials. In all treatments, numbers of Azospirillum spp. were increased and the inoculated strains were found in the highest dilutions. While grain yields of the different genotypes varied between 1700 and 7300 kg ha-1, total N accumulation was much less variable. Significant inoculation effects on total N accumulation and on grain yields were consistently negative with one Argentinian genotype and positive with four Argentinian and two Brazilian genotypes. Significant inoculation effects on leaf nitrate reductase activity at the flowering stage, observed in the range-55% to +176%, indicated the presence of various interactions between the plant NO inf3 sup- metabolism and Azospirillum spp. Three Brazilian and one Argentinian maize genotype showed significant decreases in leaf nitrate reductase due to inoculation while four Argentinian genotypes showed significant increases in leaf nitrate reductase activity. The results of the present study, were consistent over the two field experiments and strongly indicate that more detailed plant genotype-Azospirillum spp. strain interaction studies, taking the entire N metabolism in the plant into account, are needed to allow better inoculation results of cereal crops.

The development of sustainable agricultural systems for the tropics requires among other technologies, alternatives for nitrogen fertilizers which are often limited in availability for financial reasons and also represent a major source of groundwater and air pollution. There are many new alternatives for the development of agricultural systems which make use of biological processes in soil. Biological nitrogen fixation (BNF), that is, the biological conversion of atmospheric dinitrogen into mineral N, is the most important alternative among them. Examples are given of the impact of various technologies used in Brazil. Soybean, introduced into the country 30 years ago, is now the second most important export crop, reaching 24 million tons annually with no N fertilizer application. Consequently, Brazil today is the country in the world which uses the lowest amounts of nitrogen fertilizers in relation to phosphate. Alternatives for crop rotations and pastures are also discussed. Possibilities of expanding BNF to cereals and other non-legume crops are gaining new credibility due to the identification of endophytic associations with diazotropic bacteria. The definite proof of substantial BNF in sugar cane with N balance and 15N methods in certain genotypes selected under low N fertilizer applications opens up new alternatives for sustainable agriculture and will be the key to viable bio-fuel programmes.

Nitrogen-free, semi-solid defined medium with crystallized cane sugar (100 g/l) supplemented with cane juice (5 ml/l) was the most selective for isolating Acetobacter diazotrophicus. Surveys of A. diazotrophicus using this medium showed that10(3) cells/g fresh wt were present at all sites in all parts of the sugar cane plant and in all trash samples examined, reaching up to 10(7)/g. Additional samples, from forage grasses and cereals and from weed species collected within the sugar cane fields, were all negative. Heat treatment (50°C for 30 min) of the sugar cane setts did not affect A. diazotrophicus numbers within the plant. Nitrogenase activity of intact soil-plant systems in pots planted with heat-treated setts did not respond to inoculation with A. diazotrophicus. The endophytic habitat of this diazotroph and its propagation within the stem cuttings was confirmed.

Significant nitrogen fixation has recently been demonstrated in Brazilian sugar cane (Saccharum officinarum) cultivars known to form associations with a number of diazotrophs, including Acetobacter diazotrophicus, an acid-tolerant endophytic bacterium which grows best on a sucrose-rich medium. In a series of experiments, aseptically-grown sugar cane plantlets were rooted in a liquid medium and inoculated with A. diazotrophicus originally isolated from field-grown sugar cane. After 4, 7, 9, and 15 d, plants were examined under light, scanning and transmission electron microscopes and the presence of A. diazotrophicus on and within plant tissues was confirmed by immunogold labelling

Mixed cultures of the nitrogen-fixing bacterium Acetobacter diazotrophicus with yeast are proposed as a model system for plant-bacteria interactions to evaluate dinitrogen fixation and excretion of combined nitrogen under conditions where available nitrogen is immediately assimilated. Growth, protein accumulation, and nitrogenase activity (ARA) of A. diazotrophicus, either in pure culture or in mixed cultures with Lipomyces kononenkoae (non-fermenting, amylolytic yeast) were studied in semi-solid nitrogen-free mineral medium. When sucrose was used as the carbon source, ARA and protein accumulation in mixed culture with L. kononenkoae were 25–32% higher than in pure cultures of A. diazotrophicus and specific nitrogenase activity of the bacterial cells was twice that observed in pure cultures. The protein accumulation in the yeast in mixed cultures was 47–51% of the total protein of the culture, irrespective of culture age or sucrose concentration in the medium. Using starch as the carbon source, no growth was observed of either the yeast strain or the bacterium in pure culture, whereas in mixed culture, protein accumulation was close to that found with sucrose.

Sweet potatoes were micropropagated and then transplanted from axnic conditions to fumigated soil in pots in the greenhouse. Spores of Glomus clarum were obtained from Brachiaria decumbens or from sweet potatoes grown in soil infected with this fungus and with an enrichment culture of Acetobacter diazotrophicus. Three experiments were carried out to measure the beneficial effects of vesicular-arbuscular mycorrhizal (VAM) fungi-diazotroph interactions on growth, nutrition, and infection of sweet potato by A. diazotrophicus and other diazotrophs obtained from sweet potato roots. In two of these experiments the soils had been mixed with 15N-containing organic matter. The greatest effects of mycorrhizal inoculation were observed with co-inoculation of A. diazotrophicus and/or mixed cultures of diazotrophs containing A. diazotrophicus and Klebsiella sp. The tuber production was dependent on mycorrhization, and total N and P accumulation were increased when diazotrophs and G. clarum were applied together with VAM fungal spores. A. diazotrophicus infected aerial plant parts only when inoculated together with VAM fungi or when present within G. clarum spores. More pronounced effects on root colonization and intraradical sporulation of G. clarum were observed when A. diazotrophicus was co-inoculated. In non-fumigated soil, dual inoculation effects, however, were of lower magnitude. 15N analysis of the aerial parts and roots and tubers at the early growth stage (70 days) showed no statistical differences between treatments except for the VAM+Klebsiella sp. treatment. This indicates that the effects of A. diazotrophicus and other diazotrophs on sweet potato growth were caused by enhanced mycorrhization and, consequently, a more efficient assimilation of nutrients from the soil than by N2 fixation. The possible interactions between these effects are discussed.

The recently described species Acetobacter diazotrophicus isolated from sugar cane roots and stems was found capable of growth at pH 3.0 and showed high nitrogenase activity even at pH 2.5. No growth occurred at pH 7.5. Extracellular oxidation of glucose followed by gluconic acid formation was necessary for initiation of logarithmic growth, which proceeded with N2 as the sole nitrogen source. N2-dependent growth did not occur in N-free liquid medium under air, but starter doses of 0.6 to 1 mM (NH4)2SO4 led to active N2-fixing cultures after 34 h. Nitrogenase activity was only partially inhibited by 20 mM (NH4+)2SO4 and several amino acids showed similar effects. However, NO3− did not inhibit or repress nitrogenase activity.

Nitrogen-fixing bacteria isolated from banana ( Musa spp.) and pineapple ( Ananas comosus (L.) Merril) were characterized by amplified 16S ribosomal DNA restriction analysis and 16S rRNA sequence analysis. Herbaspirillum seropedicae , Herbaspirillum rubrisubalbicans , Burkholderia brasilensis, and Burkholderia tropicalis were identified. Eight other types were placed in close proximity to these genera and other alpha and beta Proteobacteria .

Este trabalho objetivou selecionar bactérias diazotróficas isoladas de bananeira (Musa spp.) e avaliar sua influência no crescimento de mudas micropropagadas. Bactérias do tipo Herbaspirillum e relacionadas a Burkholderia cepacia foram inoculadas em plântulas de banana cv. Prata Anã e cv. Caipira. As bananeiras cv. Prata Anã, cultivadas in vitro com substrato pobre em N, apresentaram maior crescimento na presença de bactérias do tipo Herbaspirillum, ao passo que as bananeiras cv. Caipira cresceram melhor com o inóculo contendo bactérias do gênero Burkholderia. Em casa de vegetação, as bananeiras cv. Caipira crescidas em sacolas de plástico contendo areia e vermiculita (1:2), suplementada com a solução de Hoagland contendo 5 mg L-1 de N, apresentaram maior crescimento quando da inoculação simultânea dos dois gêneros de bactérias, em comparação à inoculação individual. A inoculação simultânea proporcionou um crescimento nas bananeiras equivalente ao observado nas plantas-controles adubadas com 50 mg L-1 de N no substrato, porém o teor e o acúmulo de N na parte aérea das bananeiras foram menores. A contribuição de bactérias diazotróficas no crescimento de bananeiras é demonstrada pela primeira vez. The objective of this work was to select strains of diazotrophic bacteria associated with banana plants and to determine micropropagated plantlet-growth-promotion by their inoculation with diazotrophs. Isolates of Herbaspirillum-like and Burkholderia cepacia related bacteria were inoculated in plantlets of banana cv. Prata Anã and the cultivar Caipira. Plantlets of banana cv. Prata Anã grown in vitro on a poor nitrogen substrate presented higher fresh weight when Herbaspirillum-like bacteria were used as inoculum, while the banana cv. Caipira grew better with the inoculation of Burkholderia cepacia related bacteria. Nevertheless, plants of the cultivar Caipira cultivated in plastic bags filled with sand and vermiculite (1:2) and supplied with Hoagland's solution containing 5 mg L-1 of N grew faster with the inoculation of Herbaspirillum-like and Burkholderia cepacia related bacteria. This effect was similar to 50 mg L-1 of N fertilizer added to the positive control. However, the control plants showed higher N content in their shoots when compared to bacterial inoculated plants. This work constitutes the first announcement of banana plant-growth-promotion by diazotrophic bacteria.

Foi avaliada a ocorrência e a distribuição de espécies de fungos micorrízicos arbusculares e A. diazotrophicus em plantios de cana-de-açúcar em diferentes tipos de manejo nos Estados do Rio de Janeiro e Pernambuco. Foram feitas 35 coletas de amostras de solo da rizosfera e de raízes de 14 variedades de cana-de-açúcar para extração de esporos e isolamento da bactéria. O numero de esporos variou de 18 a 2.070/ 100 mL de solo, e os maiores numero e diversidade de espécies foram verificados nos canaviais de Campos, RJ, especialmente naqueles que não adotam a queima de palhico. As espécies predominantes nas três localidades amostradas foram: Acaulospora sp., Scutellospora heterogama, Glomus etunicatum, Glomus occultum e Gigaspora margarita. A. diazotrophicus estava presente nas amostras de raízes colhidas em canaviais de Campos, com exceção de uma coleta de cana-de-açúcar plantada num solo usado como bacia de sedimentação de vinhaça. Não foi possível isolar essa bactéria a partir de esporos desinfestados dos FMAs nativos, apenas dos esporos lavados com agua estéril The occurrence and distribution of species of arbuscular mycorrhizae fungi and Acetobacter diazotrophicus in sugar cane (Saccharum officinarum) grown in different regimes of crop management in the States of Rio de Janeiro and Pernambuco were studied. Thirty five samples of the rhizosphere soil and roots were collected from 14 varieties of sugar cane for the extraction of spores and isolation of the bacterium. The number of spores varied from 18 to 2.070 per 100 mL of soil, and the greatest diversity of fungal species was found in the sugarcane fields of Campos (Rio de Janeiro State), especially in those where the sugarcane trash was not burned at harvest. The predominant species found in the three localities sampled were: Scutellospora heterogama, Glomus etunicatum, Glomus occultum, Glomus macrocarpum, Acaulospora sp. and Gigaspora margarita. A. diazotrophicus was present in almost all samples of root with the exception of one harvest of sugar cane taken from an area used for the sedimentation of vinasse (distillery waste). It was not possible to detect the bacterium from surface sterilised spores of native arbuscular mycorrhizal fungi (AMF), only from washed ones using sterile water. Made available in DSpace on 2016-09-30T22:36:26Z (GMT). No. of bitstreams: 1 OcorrenciademicorrizasarbuscularesedabacteriadiazotroficaAcetobacterdiazotrophicusemcanadeacucar.pdf: 290143 bytes, checksum: 2b241bcfb03cca02dddadc434072c06e (MD5) Previous issue date: 1999-05-14

The phylogenetic relationships between diazotrophic rhizosphere bacteria were studied using 16S and 23S ribosomal RNA sequences. Certain stretches of the rRNA sequences are variable enough to provide information for the development of diagnostic probes at the species level for the five Azospirillum spp., Acetobacter diazotrophicus, Herbaspirillum seropedica, H. rubrisubalbicans and „isolate E“-bacteria. The brasilian „isolate E“- type bacteria, which reside inside roots, stems and leaves of several plants, could be grouped into the beta-subgroup of proteobacteria. According to their 16S rRNA sequences they belong to the genus Burkholderia. Recently isolated diazotrophic bacteria from the C4-plants Miscanthus sinensis and Spartina pectinata were identified as Azospirillum lipoferum and Herbaspirillum-like bacteria.

15N isotope and N balance studies performed over the last few years have shown that several Brazilian varieties of sugarcane are capable of obtaining over 60% of their nitrogen (

The recent discovery of obligate endophytic diazotrophs, like Acetobacter diazotrophicus, Herbaspirillum seropedicae and H. rubrisubalbicans colonizing sugar cane roots, stems and leaves, may finally explain the high contributions of BNF to this crop. A. diazotrophicus so far has only been isolated from sugar cane, sweet potatoe and Cameroon grass, all three with high sugar contents and propagated by stem cuttings. Azoarcus spp., another obligate endophytic genus, isolated from Kallar grass (Leptochloa fusca), has been found to establish within wetland rice roots when inoculated. Herbaspirillum seropedicae has been isolated from many graminaceous plants, forage grasses as well as from maize, sorghum and rice, but it does not seem to occur in other plant families. H. rubrisubalbicans so far has only been isolated from sugar cane roots, stems and leaves. These two species, even though clearly identified as two distinct species by DNA homology, are extremely similar and can only be distinguished by growth on n-acetyl-glucosamine (H. seropedicae) and that on meso-erythritol (H. rubrisubalbicans). Transmission of H. seropedicae may occur within seeds or via graminaceous weed plants because even high numbers of this organism inoculated into soil, do not survive for more than one month. Inoculation of endophytic diazotrophs, especially A. diazotrophicus is better achieved by the use of VA mycorrhizae together with bacterial cultures or with spores containing the bacteria. Spores of various VAM species were found to contain this organism and also A. lipoferum and Klebsiella sp. The infection of the root cap zone in micropropagated sugar cane plants with A.diazotrophicus and its colonization of xylem, has been confirmed by optical and electron microscope observations with immunogold-silver-enhanced techniques. Also Herbaspirillum spp. have been localized in the metaxylem after artificial inoculation of sugar cane and sorghum, using the same technique.

Over the last 20 years many new species of N2-fixing bacteria have been discovered in association with grasses, cereals and other non-nodulating crops. Virtually all of these bacteria are microaerophylic, fixing N2 only in the presence of low partial pressures of oxygen. Until a few years ago much attention was focussed on members the genus Azospirillum and it was assumed that N2 fixation was restricted to the rhizosphere or rhizoplane of the host plants. Through the use of N balance and 15N techniques it has been shown that in the case of lowland rice, several tropical pasture grasses and especially sugar cane, the contributions of biological N2 fixation (BNF) are of agronomic significance.

Bacteria causing mottled stripe disease in sugar cane, known as Pseudomonas rubrisubalbicans, were shown to be able to fix molecular N2 and to grow on it. The root associated diazotroph known as Herbaspirillum seropedicae, after artificial inoculation caused mottled stripe disease symptoms on sorghum and Napier grass but not on sugar cane. Both bacteria could be reisolated from leaves even 60 days after. Sugar cane leaves contained large numbers of these bacteria even in the uninoculated controls. Additional physiological characteristics of six strains of P. rubrisubalbicans were compared with those of two H. seropedicae strains and were shown to be very similar.

A recent 15N 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).

By a polyphasic approach (DNA:rRNA and DNA:DNA hybridizations, and computer assisted analysis of auxanographic results) it was shown that Herbaspirillum seropedicae, EF group 1 strains and the generically misnamed [Pseudomonas] rubrisubalbicans and [Aquaspirillum] autotrophicum constitute one genus. Within this genus we delineated four groups that each deserve species rank. Species 1 contains EF group 1 strains and [Pseudomonas] rubrisubalbicans strains; species 2 is composed only of [Pseudomonas] rubrisubalbicans strains including the type strain. Species 3 contains Herbaspirillum seropedicae and one [Pseudomonas] rubrisubalbicans strain. Species 4 corresponds to [Aquaspirillum] autotrophicum.

Sugarcane was first introduced into Brazil in 1532, in São Vicente (São Paulo State) by the Portuguese. Since the first cane selection and breeding programs started in Brazil, both local and introduced material were used. In none of the breeding programs were large amounts of nitrogen fertilizer utilized, and this may be the reason why today the best materials have little demand for nitrogen fertilizer, and an effective association has developed between endophytic nitrogen-fixing bacteria and the plant. In some cases high inputs of associated biological nitrogen fixation have been observed. The oil crisis also played a role in the sugarcane story, since the alcohol-from-cane-juice (PRO-ÁLCOOL) program installed to find a substitute for gasoline in cars, stimulated the selection of highly efficient varieties with low nitrogen fertilizer input. The recent promising results involving the inoculation of micropropagated sugarcane plants with endophytic diazotrophic bacteria, along with the ongoing Brazilian sugarcane plant and bacterial genome programs, suggest that the success of the Brazilian sugarcane business may continue for many years to come, considering the potential to be exploited.

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.

The interaction between nitrate respiration and nitrogen fixation inAzospirillum lipoferum andA. brasilense was studied. All strains examined were capable of nitrogen fixation (acetylene reduction) under conditions of severe oxygen limitation in the presence of nitrate. A lag phase of about 1 h was observed for both nitrate reduction and nitrogenase activity corresponding to the period of induction of the dissimilatory nitrate reductase. Nitrogenase activity ceased when nitrate was exhausted suggesting that the reduction of nitrate to nitrite, rather than denitrification (the further reduction of nitrite to gas) is coupled to nitrogen fixation. The addition of nitrate to nitrate reductase negative mutants (nr-) ofAzospirillum did not stimulate nitrogenase activity. Under oxygen-limited conditionsA. brasilense andA. lipoferum were also shown to reduce nitrate to ammonia, which accumulated in the medium. Both species, including strains ofA. brasilense which do not possess a dissimilatory nitrite reductase (nir-) were also capable of reducing nitrous oxide to N2.

SUMMARY: N2-fixing Azospirillum brasilense possesses an H2-uptake hydrogenase activity capable of supporting H2-dependent acetylene reduction by whole cells starved of carbon metabolites. H2 did not support acetylene reduction in carbon-sufficient bacteria. although carbon substrates did not inhibit H2-dependent respiration. H2-dependent respiration was extremely O2 sensitive and could not usually protect nitrogenase against inhibition by O2. H2 partly inhibited nitrogenase activity at sub-optimal O2 concentrations: this may be because H2-dependent respiration has a greater affinity for O2 but is less efficiently coupled to ATP production than is carbon-dependent respiration.

The results of the cross reactions of the 27 strains of Azospirillum spp. with 4 fluorescent antibodies (FA) show a neat differentiation between the two species. A. lipoferum represents a more homogenous group in respect to FA reactions and highly fluorescent preparations were obtained with strains from a large scope origin against Sp59 FA, the type strain. In contrast A. brasilense contains at least three sub groups in respect to FA reactions. The first includes all denitrifing strains (nir+) which react with FA from Sp7 the type strain. None of the nir- strains reacted strongly with Sp7 FA. One part of the A. brasilense nir- group which includes the strains isolated from well sterilized rice and wheat roots (Sp 107, 107 st, 106 and 109 st) reacts with FA of their reference strain Sp107 but not with that of Sp28 FA. The strains isolated from unsterilized roots and soils reacted with SP28 FA and not with that of Sp107 FA. In addition there were 3 strains (Sp A4, 34 and 67) which reacted with neither of the FAs.

Root segments obtained from field-grown corn plants were divided into small sections and assayed for acetylene (C2H2) reduction. After overnight preincubation, C2H2 reduction started immediately and linear rates were observed for several hours. Addition of NO3− (1 mM) delayed the start of C2H2 reduction but linear rates followed thereafter. A similar pattern was observed with excised roots inoculated with Azospirillum lipoferum.Addition of malate did not eliminate the NO3− effect. Inoculation with A. lipoferum resulted in relatively large rates of C2H2 reduction and a shorter lag than normal (4 to 8 h). A transient accumulation of NO2− accompanied the disappearance of NO3− and C2H2 reduction started only after all the NO2− had been reduced. A very sizable fraction of the reduced NO3− and NO2− was accounted for as N2O accumulation. Thus, the results provided support for the in situ operation of both N2 fixation and denitrification in A. lipoferum – corn root associations and it is proposed that mature corn roots are able to provide sufficient carbon to support both processes.

Nitrogen fixation in the rhizospheres of field grown tropical forage grasses was studied by the acetylene reduction method. Values varied considerably between sites but indicate the possible economic importance of several of the species studied. Maximal nitrogenase activity measured (nmoles C 2 H 4 g −1 dry roots h −1 ) was 754 for Pennisetum purpureum , 750 for Brachiaria mutica , 341 for Digitaria decumbens , 299 for Panicum maximum , 283 for Paspalum notatum , 269 for Cynodon dactylon , 41 for Melinis minutiflora and 29 for Hyparrhenia rufa . Nitrogenase activity varied considerably with season and was maximal during active vegetative growth of two of the grasses. Significant differences between Paspalum notatum ecotypes and cultivars. in Azotohacter paspali occurrence and nitrogen fixation, indicate the possibility of plant breeding to enhance nitrogen fixation in grass rhizospherc associations. Other research lines of agronomic importance are fertilizer effects. In intact soil plant cores with the Paspalum system 10 parts/10 6 NH 4 + J-N inhibited nitrogenase activity within 2 h and 10 parts/10 6 NO − 3 -N within 4 h. but after 1 week these effects were negligible. In the field, nitrogenase activity on roots of P. purpureum and D. decumbens , assayed 2 weeks after top dressings of 20 kg N ha −1 as NH 4 NO 3 . was not affected even after eight such dressings.

Azospirillum spp. participate in all steps of the nitrogen cycle except nitrification. They can fix molecular nitrogen and perform assimilatory nitrate reduction and nitrate respiration. Culture conditions have been defined under which nitrate is used both as terminal respiratory electron acceptor and as nitrogen source for growth. Nitrate and, possibly to a very limited extent, nitrite, but not sulfate, iron or fumarate support anaerobic respiration. Under anaerobic conditions, nitrate can also supply energy for nitrogen fixation but without supporting growth. Nitrate-dependent nitrogenase activity lasts only for 3–4 h until the enzymes of assimilatory nitrate reduction are synthesized. Nitrite accumulates during this period and inhibits nitrogenase activity at concentrations of about 1 mM.

Studies of the physiology of the Spirillum lipoferum recognized as the major organism responsible for N2-fixation in the roots of Digitaria decumbens cv transvala were performed in order to improve the methods of culture and help to explain the physiology of this N2-fixing grass bacterial association. Methods for isolation, purification and N2-fixation assays are described. Acetylene concentrations used for N2-ase activity measurements should be at least 12%. the Vmax of cultures in the log phase being at a pC2H2 of 0.12 atm and the apparent Km 0.022 atm. Optimal temperatures for N2-dependent growth are between 32 and 40°C. and little N2-fixation is observed below 24°C. At 42°C the N2-ase is inactivated. When cultures grown at 28 or 36°C are transferred to lower temperatures nitrogenase activity declines rapidly. One hour after transfer to 17°C activity is about half that before transfer and is maintained at this level for at least 8 h. After transfer to 10°C activity ceases after 1 h. Growth is very pH dependent, optimal growth on N2 occurring only between pH 6.8 and 7.8. Nitrogen fixation below pH 5.5 and above 8.0 is less than one-quarter of the optimal. No N2-fixation occurs in the absence of O2 and maximal N2-dependent growth is reached at 1.5% O2 in the gas mixture bubbled through liquid cultures. In contrast to previous reports, several sugars including glucose can be used by the Spirillum for N2-fixation, but only when small amounts of starter nitrogen or organic acids are added to the medium. Efficiencies of N2-fixation on malate and glucose are similar and about 60% of that of cells incorporating NH+4-N. Efficiency of NO-3 incorporation is 74% of that of NH+4-N grown cultures. High observed efficiencies (52 mg N2 fixed g−1 malate or glucose) are attributed to carbon limited growth at optimum or O2 limited conditions, both facilitated by slow diffusion rates through the semi-solid agar medium used.

Intact soil cores containing plants of Paspalum notatum or Digitaria decumbens were selected with the acetylene reduction method, and then exposed to 15N15 to confirm nitrogen fixation in tropical grass-bacteria associations. In a preliminary experiment with P. notatum15N2 incorporation was slow but progressive during 24 h in roots but translocation to rhizomes and leaves ceased after 17h. With improved assay chambers, enrichments of 0.151 and 0.563 15N atom % excess were obtained in roots of D. decumbens cv transvala and P. notatum systems respectively, after 3 days. Enrichments in rhizomes were similar to those of roots; however in the leaves only 8% of root enrichment was observed. The addition of sucrose to the soil doubled N2-fixation in roots in both grass species studied, but did not result in increased incorporation into the leaves of P. notatum.

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 NO 3 − . 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.

A nitrogen balance study was performed on four commercial sugar cane cultivars grown in pots containing 64 kg of soil from a sugar cane growing area, with the objective of quantifying possible contributions of biological nitrogen fixation (BNF) to the plants. Distillery (stillage) waste from an alcohol distillery was added to half of the pots, and all treatments were replicated 10 times. The pots were maintained in the field and amended with the equivalent of 80 kg N ha−1 of 15N-labelled urea fertilizer. After 12 months of growth the plants accumulated the equivalent of between 10 and 24% of the total N in the soil and added fertilizer. In the next 9 months of growth, with no more N additions, the plants further accumulated the equivalent of between 8.5 and 19% of the original soil + fertilizer N. The cultivar CB 47–89 accumulated significantly more N than the other cultivars. This difference was greatest in the absence of distillery waste, and in this treatment the 15N enrichment of this cultivar was almost half that of the others, which suggests that there was a contribution of unlabelled N from the air to this cultivar via BNF. After 21 months of growth the N content of the soil and roots was determined on 5 replicates. These analyses revealed that the planted plots lost between 7.5 and 12.5 g of N from soil + fertilizer, but all accumulated more than this in plant tissue. In view of the fact that unplanted plots lost between 10 and 14 g of N during this period, the data suggest that all of the cultivars received some N from associated BNF. In the case of the cultivar CB 47–89, in the absence of distillary waste the plants removed approx. 10 g of N from the soil and fertilizer but accumulated almost 35 g of N. These data constitute the first direct evidence of very significant contributions of plant-associated BNF to a sugar cane cultivar although further experiments must be performed to determine whether such large contributions would be encountered under field conditions.

We have summarized information in four areas of the broad topic of legume-Rhizobiumsymbiosis. These include: carbon substrates provided to nodule bacteroids by the host, assimilation of fixed nitrogen by the host, O2metabolism in legume nodules and involvement of H2in nodule metabolism. Although nodules contain a variety of carbon substrates, both biochemical and genetic evidence indicate that C4 dicarboxylates are the major carbon substrates that support N2fixation in nodules. The biochemical pathways for utilization of products of N2fixation are fairly well understood but relatively little is known about the regulation of the assimilation of fixed nitrogenous compounds at the gene level. Ureides are primary nitrogenous compounds exported from nodules of the tropical legumes. Because the catabolism of these products may involve the hydrolysis of urea by nickel-dependent urease, the possible importance of nickel as a trace element in the nutrition of legumes is raised. The O2supply to nodule bacteroids is regulated by a barrier to free-O2diffusion and by leghaemoglobin. Progress has been made in understanding of the molecular genetics and biochemistry of leghaemoglobin but little is known about the mechanisms that control the physical barrier to O2diffusion. Legume nodules contain mechanisms for the disposition of peroxide and free radicals of oxygen. The importance of these systems as protective mechanisms for the O2-labile nitrogenase is discussed. Some strains ofRhizobiumform nodules which recycle the H2produced as a byproduct of N2fixation. The genes necessary for H2oxidation have been cloned and transferred within and among species ofRhizobium. The advantages and disadvantages of H2recycling in legume nodules are discussed.

The percentage of low-level streptomycin-resistant (20 μg/mL) bacteria in surface-sterilized or washed maize roots was more than a thousand times higher than that in soil populations. There was also a higher incidence of resistant bacteria in rhizosphere as compared with non-rhizosphere soil and bacteria isolated from maize roots were relatively tolerant to several other antibiotics. Azospirillum lipoferum was predominant in surface-sterilized roots of field-grown maize and was low-level streptomycin-resistant while most soil isolates were sensitive. Inoculation with A. brasilense isolated from wheat roots was unsuccessful in terms of establishment even when streptomycin-resistant strains were used. Unidentified causes of specific plant–bacteria affinities therefore transcend the role of antibiotic resistance in maize root infection.

In two field experiments, wheat was inoculated with various strains of Azospirillum spp. The two A. brasilense nir− strains isolated from surface-sterilized wheat roots increased the number of Azospirillum in surface-sterilized roots, plant dry matter, and percent N. The total N accumulated in plant tops at heading stage was increased by 30% in the first experiment (strain Sp 107 st) and by 51 and 89% (strains Sp 107 st and Sp 245, respectively) in the second experiment. The Azospirillum numbers (MPN) in chloramine-t treated roots were correlated with total nitrogen accumulation in plant tops (r = 0.92**). Numbers of Azospirillum in nonsterilized roots did not correlate with total plant N accumulation.