The Effect of Inoculation of a Diazotrophic Bacterial Consortium on the Indigenous Bacterial Community Structure of Sugarcane Apoplast Fluid.

The extracellular space in plants, termed the apoplast, has a pH and sugar content that enables bacterial growth and represents a possible niche for the establishment of endophytic bacteria. Previous studies have investigated the effects of diazotrophic bacteria inoculation in sugarcane varieties, but it has not yet been analyzed how the microbial community of apoplast fluid of sugarcane is affected. High-throughput next generation sequencing of the 16S rRNA gene was used throughout this study to determine the effect of inoculation with a diazotrophic bacteria consortium, previously isolated from sugarcane, on the native bacterial population of sugarcane variety RB867515 grown in the field. The analyses were carried out 450 days after inoculation. The results revealed the presence of 22 phyla, with predominance of Proteobacteria phylum. It was observed that the inoculated consortium changed the indigenous bacterial community structure of sugarcane apoplast fluid by decreasing diversity and evenness, interfering in the composition of rare species. Microbial community composition analysis revealed differences between treatments. The differential abundance test showed there were 43 amplicon sequence variants (ASVs) which were relatively more abundant in the inoculated treatment, with predominance of the Sphingomonas genus. The predicted functions of the most abundant ASVs revealed the presence of genera related to plant growth promotion and protection against phytopathogens. Analysis to evaluate the occurrence of inoculated strains in the recovered data was not conclusive since the ASVs taxonomically close to the inoculated bacteria were observed in low abundance. The present study is the first report to elucidate the bacterial community in sugarcane apoplast fluid using a culture-independent approach. It demonstrated that the diazotrophic bacterial consortium interferes in the natural bacterial community in sugarcane variety RB867515.

Temporal assessment of root and shoot colonization of elephant grass (Pennisetum purpureum Schum.) host seedlings by Gluconacetobacter diazotrophicus strain LP343.

Gluconacetobacter diazotrophicus is a species of great agronomic potential due to its growth-promotion traits. Its colonization process in different plants has been reported. However, there have been no studies regarding its structural colonization in elephant grass. This is a fast-growing C4-Poaceae plant, and its application in Brazil is mainly aimed at feeding dairy cattle, due to its high nutritional value. Also, in the last decade, this grass has been applied in the production of biofuels. The present study aimed to monitor the colonization process of strain LP343 of G. diazotrophicus inoculated in elephant grass seedlings of PCEA genotype, by using a mCherry-tagged bacterium. Samples of roots and shoots collected at different periods were visualized by confocal laser-scanning microscopy. The colony-counting assay was used to compare the number of cells recovered in different niches and a qPCR was performed for the quantification of endophytic cells in root and shoot tissues. Results suggested that the strain LP343 quickly recognized the PCEA roots as host, attached to the elephant grass roots at 6 h, and 7 days after inoculation were able to colonize the xylem vessels of roots and shoots of elephant grass. This study advances our knowledge about the colonization process of G. diazotrophicus species in elephant grass, contributing to future studies involving the plant-bacteria interaction cultivated under gnotobiotic conditions.

A Culture-Independent Approach to Enrich Endophytic Bacterial Cells from Sugarcane Stems for Community Characterization.

Bacterial endophytes constitute a very diverse community and they confer important benefits which help to improve agricultural yield. Some of these benefits remain underexplored or little understood, mainly due to the bottlenecks associated with the plant feature, a low number of endophytic bacterial cells in relation to the plant, and difficulties in accessing these bacteria using cultivation-independent methods. Enriching endophytic bacterial cells from plant tissues, based on a non-biased, cultivation-independent physical enrichment method, may help to circumvent those problems, especially in the case of sugarcane stems, which have a high degree of interfering factors, such as polysaccharides, phenolic compounds, nucleases, and fibers. In the present study, an enrichment approach for endophytic bacterial cells from sugarcane lower stems is described. The results demonstrate that the enriched bacterial cells are suitable for endophytic community characterization. A community analysis revealed the presence of previously well-described but also novel endophytic bacteria in sugarcane tissues which may exert functions such as plant growth promotion and biological control, with a predominance of the Proteobacterial phylum, but also Actinobacteria, Bacteroidetes, and Firmicutes, among others. In addition, by comparing the present and literature data, it was possible to list the most frequently detected bacterial endophyte genera in sugarcane tissues. The presented enrichment approach paves the way for improved future research toward the assessment of endophytic bacterial community in sugarcane and other biofuel crops.

Identification of Genes Involved in Indole-3-Acetic Acid Biosynthesis by Gluconacetobacter diazotrophicus PAL5 Strain Using Transposon Mutagenesis.

Gluconacetobacter diazotrophicus is a beneficial nitrogen-fixing endophyte found in association with sugarcane plants and other important crops. Beneficial effects of G. diazotrophicus on sugarcane growth and productivity have been attributed to biological nitrogen fixation process and production of phytohormones especially indole-3-acetic acid (IAA); however, information about the biosynthesis and function of IAA in G. diazotrophicus is still scarce. Therefore, the aim of this work was to identify genes and pathways involved in IAA biosynthesis in this bacterium. In our study, the screening of two independent Tn5 mutant libraries of PAL5T strain using the Salkowski colorimetric assay revealed two mutants (Gdiaa34 and Gdiaa01), which exhibited 95% less indolic compounds than the parental strain when grown in LGIP medium supplemented with L-tryptophan. HPLC chromatograms of the wild-type strain revealed the presence of IAA and of the biosynthetic intermediates indole-3-pyruvic acid (IPyA) and indole-3-lactate (ILA). In contrast, the HPLC profiles of both mutants showed no IAA but only a large peak of non-metabolized tryptophan and low levels of IPyA and ILA were detected. Molecular characterization revealed that Gdiaa01 and Gdiaa34 mutants had unique Tn5 insertions at different sites within the GDI2456 open read frame, which is predicted to encode a L-amino acid oxidase (LAAO). GDI2456 (lao gene) forms a cluster with GDI2455 and GDI2454 ORFs, which are predicted to encode a cytochrome C and an RidA protein, respectively. RT-qPCR showed that transcript levels of lao. cccA, and ridA genes were reduced in the Gdiaa01 as compared to PAL5T. In addition, rice plants inoculated with Gdiaa01 showed significantly smaller root development (length, surface area, number of forks and tips) than those plants inoculated with PAL5T. In conclusion, our study demonstrated that G. diazotrophicus PAL5T produces IAA via the IPyA pathway in cultures supplemented with tryptophan and provides evidence for the involvement of an L-amino acid oxidase gene cluster in the biosynthesis of IAA. Furthermore, we showed that the mutant strains with reduction in IAA biosynthesis ability, in consequence of the lower transcription levels of genes of the lao cluster, had remarkable effects on development of rice roots.

Structural studies of an exopolysaccharide produced by Gluconacetobacter diazotrophicus Pal5.

Gluconacetobacter diazotrophicus is a nitrogen-fixing bacterium that has been found colonizing several plants. This acid-tolerant bacterium produces phytohormones that promote plant growth and is also able to grow in high-sugar concentrations. It has been demonstrated that exopolysaccharides (EPS), which are produced by strain Pal5 of G. diazotrophicus, play an important role in plant infection. We have investigated the structure of the EPS, which was produced by a strain of Pal5 grown in liquid medium containing mannitol as the sole carbon source. The results reveal an EPS with Glc, Gal, Man in a molar ratio of 6:3:1, respectively. NMR spectroscopy and chemical derivatization have revealed that the EPS structure has 4-O-substituted units of ß-glucose, 3-O-substituted units of ß-galactose and 2-O-substituted units of α-mannose. Glucose and galactose units linked at C6 were also found. The structure proposed herein is different from EPS produced by other species of Gluconacetobacter published to date.

Identification and validation of reference genes to study the gene expression in Gluconacetobacter diazotrophicus grown in different carbon sources using RT-qPCR.

Gluconacetobacter diazotrophicus strain PAL5 is a nitrogen-fixing endophytic bacterium originally isolated from sugarcane and later on was found to colonize other plants such as rice, elephant grass, sweet potato, coffee, and pineapple. Currently, G. diazotrophicus has been considered a plant growth-promoting bacterium due to its characteristics of biological nitrogen fixation, phytohormone secretion, solubilization of mineral nutrients and antagonism to phytopathogens. Reverse transcription followed by quantitative real-time polymerase chain reaction (RT-qPCR) is a method applied for the quantification of nucleic acids because of its specificity and high sensitivity. However, the decision about the reference genes suitable for data validation is still a major issue, especially for nitrogen-fixing bacteria. To evaluate and identify suitable reference genes for gene expression normalization in the diazotrophic G. diazotrophicus, mRNA levels of fourteen candidate genes (rpoA, rpoC, recA, rpoD, fabD, gmk, recF, rho, ldhD, gyrB, gyrBC, dnaG, lpxC and 23SrRNA) and three target genes (matE, omp16 and sucA) were quantified by RT-qPCR after growing the bacteria in different carbon sources. The geNorm and Normfinder programs were used to calculate the expression stabilities. The analyses identified three genes, rho, 23SrRNA and rpoD, whose expressions were stable throughout the growth of strain PAL5 in the chosen carbon sources. In conclusion our results strongly suggest that these three genes are suitable to be used as reference genes for real-time RT-qPCR data normalization in G. diazotrophicus.

Antioxidant pathways are up-regulated during biological nitrogen fixation to prevent ROS-induced nitrogenase inhibition in Gluconacetobacter diazotrophicus.

Gluconacetobacter diazotrophicus, an endophyte isolated from sugarcane, is a strict aerobe that fixates N(2). This process is catalyzed by nitrogenase and requires copious amounts of ATP. Nitrogenase activity is extremely sensitive to inhibition by oxygen and reactive oxygen species (ROS). However, the elevated oxidative metabolic rates required to sustain biological nitrogen fixation (BNF) may favor an increased production of ROS. Here, we explored this paradox and observed that ROS levels are, in fact, decreased in nitrogen-fixing cells due to the up-regulation of transcript levels of six ROS-detoxifying genes. A cluster analyses based on common expression patterns revealed the existence of a stable cluster with 99.8% similarity made up of the genes encoding the α-subunit of nitrogenase Mo-Fe protein (nifD), superoxide dismutase (sodA) and catalase type E (katE). Finally, nitrogenase activity was inhibited in a dose-dependent manner by paraquat, a redox cycler that increases cellular ROS levels. Our data revealed that ROS can strongly inhibit nitrogenase activity, and G. diazotrophicus alters its redox metabolism during BNF by increasing antioxidant transcript levels resulting in a lower ROS generation. We suggest that careful controlled ROS production during this critical phase is an adaptive mechanism to allow nitrogen fixation.

Polyphasic characterization of Gluconacetobacterdiazotrophicus isolates obtained from different sugarcane varieties / Caracterização polifásica de isolados de Gluconacetobacterdiazotrophicus obtidos de diferentes variedades de cana-de-açúcar

A polyphasic approach was applied to characterize 35 G. diazotrophicus isolates obtained from sugarcane varieties cultivated in Brazil. The isolates were analyzed by phenotypic (use of different carbon sources) and genotypic tests (ARDRA and RISARFLP techniques). Variability among the isolates was observed in relation to the carbon source use preference. Glucose and sucrose were used by all isolates in contrast to myo-inositol, galactose and ribose that were not metabolized. The results of the analysis showed the presence of two groups clustered at 68 percent of similarity. The genetic distance was higher when RISA-RFLP analysis was used. Analysis of 16S rDNA sequences from isolates showed that all of them belonged to the G. diazotrophicus species. Neither effect of the plant part nor sugarcane variety was observed during the cluster analysis. The observed metabolic and genetic variability will be helpful during the strain selection studies for sugarcane inoculation in association with sugarcane breeding programs.

Foi realizado a caracterização polifásica de 35 isolados obtidos de variedades de cana-de-açúcar cultivadas no Brasil, através de testes fenotípicos (uso de fontes diferentes de carbono) e genotípicos (técnicas de ARDRA e RISA-RFLP). Houve variação entre os isolados com relação à utilização de fontes de carbono. Glicose e sacarose foram usadas por todos isolados, diferentemente de mio-inositol, galactose e ribose que não foram metabolizados. Os resultados da análise polifásica dos dados confirmam a formação de dois grupos, que apresentaram 68 por cento de similaridade. Observou-se maior distância genética entre os isolados quando a técnica de RISA-RFLP foi aplicada. O sequênciamento da região 16S do rDNA mostrou que todos os isolados pertencem à espécie G. diazotrophicus. Não foi observado efeito da parte da planta ou variedade de cana-de-açúcar no agrupamento dos isolados. Em conjunto, esses resultados poderão auxiliar no estudo de seleção de estirpes para inoculação em cana-de-açúcar, orientando programas de melhoramento vegetal.

Validation of a Tn5 transposon mutagenesis system for Gluconacetobacter diazotrophicus through characterization of a flagellar mutant.

Gluconacetobacter diazotrophicus is a nitrogen-fixing bacterium, which was originally isolated from the interior of sugarcane plants. The genome of strain PAL5 of G. diazotrophicus has been completely sequenced and a next step is the functional characterization of its genes. The aim of this study was to establish an efficient mutagenesis method, using the commercial Tn5 transposon EZ::Tn5Tnp Transposome (Epicentre). Up to 1 x 10(6) mutants per microgram of transposome were generated in a single electroporation experiment. Insertion-site flanking sequences were amplified by inverse PCR and sequenced for 31 mutants. For ten of these mutants, both insertion flanks could be identified, confirming the 9 bp duplication that is typical for Tn5 transposition. Insertions occurred in a random fashion and were genetically stable for at least 50 generations. One mutant had an insertion in a homolog of the flagellar gene flgA, and was therefore predicted to be affected in flagella-dependent traits and used to validate the applied mutagenesis methodology. This mutant lacked flagella and was non-motile on soft agar. Interestingly, it was also strongly affected in the ability to form biofilm on glass wool.

Polyphasic characterization of Gluconacetobacter diazotrophicus isolates obtained from different sugarcane varieties.

A polyphasic approach was applied to characterize 35 G. diazotrophicus isolates obtained from sugarcane varieties cultivated in Brazil. The isolates were analyzed by phenotypic (use of different carbon sources) and genotypic tests (ARDRA and RISA-RFLP techniques). Variability among the isolates was observed in relation to the carbon source use preference. Glucose and sucrose were used by all isolates in contrast to myo-inositol, galactose and ribose that were not metabolized. The results of the analysis showed the presence of two groups clustered at 68% of similarity. The genetic distance was higher when RISA-RFLP analysis was used. Analysis of 16S rDNA sequences from isolates showed that all of them belonged to the G. diazotrophicus species. Neither effect of the plant part nor sugarcane variety was observed during the cluster analysis. The observed metabolic and genetic variability will be helpful during the strain selection studies for sugarcane inoculation in association with sugarcane breeding programs.

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

A presente revisão aborda a história da Fixação Biológica de Nitrogênio (FBN) em Gramíneas no Brasil, procurando mostrar a evolução da pesquisa na área iniciada a mais de 40 anos sob a liderança da pesquisadora Johanna Döbereiner. Um aspecto marcante deste período foi a descoberta de diversas bactérias fixadoras de nitrogênio atmosférico tais com as rizosféricas (Beijerinckia fluminensis e Azotobacter paspali), associativas (Azospirillum lipoferum, A. brasilense, A. amazonense) e as endofíticas (Herbaspirillum seropedicae, H. rubrisubalbicans, Gluconacetobacter diazotrophicus, Burkholderia brasilensis e B. tropica). O papel destas bactérias diazotróficas em associação com as gramíneas, especialmente os cereais, tem sido estudado e muito se avançou sobre os aspectos ecológicos, fisiológicos, bioquímicos e genéticos. Os mecanismos de colonização e infecção dos tecidos das plantas foram melhor entendidos e a contribuição da FBN para o sistema solo-planta foi determinado. Estudos de inoculação de cereais com bactérias diazotróficas, têm mostrado que as endofíticas têm um maior potencial de contribuição da FBN e que o genótipo da planta influencia na associação da planta/bactéria. Os avanços alcançados apontam para uma maior exploração e entendimento desta associação endofítica. Os programas de sequenciamento do genoma: RIOGENE (Gluconacetobacter diazotrophicus) e GENOPAR (Herbaspirillum seropedicae) mostram a importância da FBN no Brasil e devem permitir que o país continue na fronteira do conhecimento em relação ao processo de FBN em gramíneas.

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.

Survival of endophytic diazotrophic bacteria in soil under different moisture levels

Neste trabalho foi avaliado o efeito da umidade do solo na sobrevivência de três espécies de bactérias diazotróficas (Azospirillum amazonense, Gluconacetobacter diazotrophicus e Azospirillum brasilense). O teor de umidade apresentou pouca influência na sobrevivência de A. brasilense, considerada uma espécie cosmopolita, enquanto A. amazonense e G. diazotrophicus, consideradas endofíticas, aumentaram o período de culturabilidade na presença de umidade no solo. Os resultados demonstram que o teor de umidade do solo possui maior influência nas espécies endofíticas, em comparação às espécies associativas.

Review: A brief story of nitrogen fixation in sugarcane - reasons for success in Brazil.

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.

Herbaspirillum-like bacteria in banana plants

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 31§C. 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.